Centre des Etudes Doctorales Ibn Zohr

Formation doctorale : Sciences de la vie et ressources naturelles

THESE

Présentée par
Idriss TALIBI

Pour l‟obtention du grade de

Docteur de l’Université Ibn Zohr
Spécialité : Phytopathologie

Recherche de moyens alternatifs aux fongicides de

synthèse pour le contrôle de la pourriture amère des
agrumes

Soutenue le 08 janvier 2013

Devant la commission d‟examen composée de :

Mr. M.A. SERGHINI, Professeur, faculté des sciences, Agadir Président

Mr. M. ACHOURI, Professeur, I.A.V Hassan II, Complexe Horticole, Agadir Rapporteur
Mr. A. OUHAMMOU, Professeur, faculté des sciences Semlalia, Marrakech Rapporteur
Mr. A. CHIHAB EDDINE, Professeur, faculté des sciences, Agadir Rapporteur
Mme. A. AIT ALLA Professeur, faculté des sciences, Agadir Examinatrice
Mr. E.H. BOUDYACH, Professeur, faculté des sciences, Agadir Directeur de thèse
Mr. H. BOUBAKER, Professeur, faculté des sciences, Agadir Directeur de thèse
Mr. M. NOUBAA, Directeur de la station de conditionnement de Membre invité
la coopérative M‟BROUKA
Remerciements
J‟ai eu la chance et l‟honneur de travailler durant ces années de doctorat au sien de
l‟équipe de phytopathologie et lutte intégrée, Laboratoire des Biotechnologies et Valorisation
des Ressources naturelles (LBVRN). Je tiens, en tout premier lieu à remercier
chaleureusement mes directeurs de thèse, le Professeur El Hassan BOUDYACH, le
Professeur Hassan BOUBAKER et le Professeur Abdellah AIT BEN AOUMAR pour leur
confiance, leur patience, leur soutien ainsi que pour les conseils qu‟ils m‟ont prodigués tout
au long de cette thèse. J‟apprécie beaucoup leurs compétences à la fois professionnelles et
humaines. Je tiens aussi à vous remercier de m‟avoir supporté et pour l‟autonomie que vous
m‟avez octroyée durant toutes ces années.
J‟exprime ma gratitude au Professeur Fouad MSANDA et au Professeur Baha SAADI
pour leur contribution dans la récolte des échantillons de plantes et pour le partage de leurs
connaissances sur l‟identification des plantes utilisées dans cette étude.
Je remercie également le Professeur Abdelhamid EL MOUSADIK responsable du
laboratoire LBVRN pour sa disponibilité, ses remarques, ses conseils et ses encouragements.
Merci également au Professeur Fatima HAMADI et Docteur Soumaya EL ABED pour
leur collaboration dans la partie analyse phytochimique des fractions de plantes et pour leur
disponibilité.
Je remercie pareillement le Professeur Lalla Amina IDRISSI HASSANI, Directrice du
centre des études doctorales Ibn Zohr, pour sa disposition, ses conseils et pour la qualité et la
richesse des formations dont elle nous a fait profiter.
Je remercie Le Professeur Mohamed Amine SERGHINI, Professeur à la faculté des
sciences d‟Agadir de m‟avoir fait l‟honneur de présider le jury de cette thèse. Mes
remerciements vont également à Monsieur Ahmed OUHAMMOU, Professeur à la faculté des
sciences Semlalia Marrakech, et Monsieur Mohamed ACHOURI, Professeur à l‟institut
agronomique et vétérinaire (IAV) Complexe Horticole d‟Agadir et Monsieur Abderrahim
CHIHAB EDDINE, Professeur à la faculté des sciences Agadir et Madame Aicha AIT ALLA
Professeur à la faculté des sciences d‟Agadir d‟avoir accepté de juger ce travail malgré leurs
nombreuses sollicitations. C‟est un très grand honneur d‟avoir fait votre connaissance et de
pouvoir vous soumettre mes travaux de recherche.
Je remercie également Monsieur Mohamed NOUBAA, Directeur de la station de
conditionnement de la coopérative agricole M‟brouka pour la fourniture des fruits utilisés
dans ce travail.
Mes remerciements aussi à tous les professeurs que j'ai côtoyé au cours de ces années
pour leur amitié et leur gentillesse.
Merci également au Professeur Naima TAQARORT qui a contribué à mon initiation
au travail au laboratoire, qu‟elle trouve ici toute ma reconnaissance.
 Un remerciement spécial à mes amis et collègues Latifa, Aissam, Rachida, Redouan,
Hicham, Fayza, Nadia, Hasna, Meryem, Naima, Fadma pour leur soutien et leur amitié ce qui
a rendu cette expérience plus agréable.
Je tiens enfin à exprimer toute ma gratitude à ma famille et mes meilleurs amis dont le
soutien a été essentiel tout au long de mes études, et tout particulièrement au cours de ce
travail. A mes parents, qui me sont très chers.

Merci beaucoup
 Je dédie cette thèse

À mes très chers parents,

À mes frères et soeurs, Naima, Touda, Ismail, Rachida, Rachid, Essaid, Leila et Aissa
À tous mes amis
À la mémoire de Sana HADDI
AVANT PROPOS
Ce travail de thèse a été réalisé, dans le cadre des formations doctorales organisées par
le centre des études doctorales Ibn Zohr, entre mars 2009 et mars 2012, dans le laboratoire des
biotechnologies et valorisation des ressources naturelles (LBVRN), faculté des sciences,
Université Ibn Zohr, sous la direction de Messieurs les Professeurs El Hassan BOUDYACH
et Hassan BOUBAKER. Il fait parti des recherches menées par l‟équipe de phytopathologie et
lutte intégrée (EPLI), et qui ont pour point commun le développement de méthodes
biologiques de lutte, alternatives à la lutte chimique, permettant de mieux contrecarrer le
développement des maladies des plantes tout en réduisant impact sur l‟environnement. Ces
recherches tournent principalement autour de deux thématiques qui sont le fer de lance de
notre laboratoire (LBVRN), et se rapportent à deux cultures très importantes dans la région du
Souss-Massa-Draa (SMD), en particulier, et dans le Maroc en général, à savoir : les agrumes
et la tomate.

Les chapitres 1, 2, 3, 4 et 5 ont été rédigés en anglais et présentés sous forme d‟articles. Les
chapitres 2, 3 et 5 ont été publiés, respectivement, dans les revues Crop Protection, Letters in
Applied Microbiology et Plant Pathology Journal. Pour sa part, le chapitre 1 a été soumis
pour publication dans la revue Plant Pathology Journal. Le chapitre 4 sera soumis pour
publication une fois les analyses terminées. Certains aspects de la présente recherche ont été,
aussi, présentés dans des congrès internationaux sous forme de communications orales ou
affichées.

Publications dans des revues internationales :

Talibi, I., Askarne, L., Boubaker, H., Boudyach E.H., Ait Ben Aoumar A. 2011. In vitro and
in vivo Screening of organic and inorganic salts to control postharvest citrus sour rot caused
by Geotrichum candidum. Plant Pathology journal 10, 138-145.

Talibi, I., Askarne, L., Boubaker, H., Boudyach, E., Msanda, F., Saadi, B., Ait Ben Aoumar,
A. 2012. Antifungal activity of some Moroccan plants against Geotrichum candidum, the
causal agent of postharvest citrus sour rot. Crop Protection 35, 41-46.

Talibi, I., Askarne, L., Boubaker, H., Boudyach, E.H., Msanda, F., Saadi, B., Ait Ben
Aoumar, A. 2012. Antifungal activity of Moroccan medicinal plants against citrus sour rot
agent Geotrichum candidum. Letters in Applied Microbiology 55, 155-161.
Talibi, I., Boubaker, H., Boudyach, Ait Ben Aoumar, A. Alternative methods for the control
of postharvest citrus diseases. Plant Pathology Journal (2013) submitted.

Communications dans des congrés :

Talibi I., Askarne L., Boubaker H., Boudyach E. H., and Ait Ben Oumar, A., In vitro and in
vivo antifungal activity of eight medicinal plants against citrus sour rot agent Geotrichum
candidum. The international Citrus Congress (ICC 2012), Valence, Espagne. (Communication
affichée).

Talibi I., Askarne L., Boubaker H., Boudyach E. H., and Ait Ben Oumar, A., Effect of
organic and inorganic salts as alternative strategie for the control of postharvest citrus sour rot
agent Geotrichum candidum. The international Citrus Congress (ICC 2012), Valence,
Espagne. (Communication affichée).

Talibi, I., Askarne, L., Boubaker, H., Boudyach, E.H., Msanda, F., Saadi, B., Ait Ben
Aoumar, A., Antifungal activity of solvent extracts of some Moroccan aromatic and medicinal
plants against Geotrichum candidum, the causal agent of citrus sour rot. Rabat, 22-23 Juin
2012, Journées Internationales sur les Substances Naturelles et Développement Durable
(Communication orale)

Talibi, I., Askarne, L., Boubaker, H., Boudyach, E.H., Msanda, F., Saadi, B., Ait Ben
Aoumar, A., Biological control of postharvest citrus sour rot by solvent extracts of some
Moroccan aromatic and medicinal plants. The 3rd International Workshop on Industrial
Biotechnology, 23-24 Avril 2012, Sfax-Tunisie (Communication orale)

Talibi, I., Askarne, L., Boubaker, H., Msanda, F., Ait Ben Aoumar, A., et Boudyach, E.H.,
Phytochemical analysis and antifungal activity of extracts from selected south Moroccan
plants against Geotrichum candidum, 12-13 Mai 2011, Mohammedia, 4ème édition du
Symposium international sur les plantes aromatiques et médicinales (Communication
affichée).

Talibi, I., Askarne, L., Boubaker, H., Boudyach, E.H., Msanda, F., Saadi, B., Ait Ben
Aoumar, A., Etude de l‟activité antifongique de certaines plantes médicinales et aromatiques
du sud marocain sur l‟agent de la pourriture amère des agrumes en post-récolte. 14-16
Octobre 2010, Congrès international sur les plantes aromatiques et médicinales, Fès
(Communication orale)
Talibi, I., Askarne, L., Boubaker, H., Boudyach, E.H., Msanda, F., Ait Ben Aoumar, A.,
Evaluation de l‟activité antifongique des extraits aqueux de certaines plantes du sud Marocain
contre Geotrichum candidum, agent de la pourriture amère des agrumes en post-récolte. 25-26
Décembre 2009, Workshop de biotechnologies au service du développement durable, Béni
Mellal (Communication affichée).
CONTENTS
List of abreviations ...................................................................................................................... i
List of figures ............................................................................................................................. ii
List of tables .............................................................................................................................. iv
Abstract ..................................................................................................................................... vi
Résumé ..................................................................................................................................... vii
‫…………………………………………………………………………………………………م لخص‬viii

INTRODUCTION GÉNÉRALE ............................................................................................ 1

Chapter one : Literature review; Alternative methods for the control of

postharvest citrus diseases
Résumé ....................................................................................................................................... 5
Abstract ...................................................................................................................................... 6
I. Introduction ......................................................................................................................... 7
II. Postharvest citrus diseases .............................................................................................. 9
II.1 Penicillium rots .......................................................................................................... 10
II.2 Sour rot ...................................................................................................................... 11
II.2.1 Causal organism ................................................................................................. 12
II.2.2 Disease cycle and epidemiology ........................................................................ 12
II.2.3 Symptoms ........................................................................................................... 13
III. Postharvest fungicides treatments ................................................................................. 14
IV. Integrated strategies to control postharvest citrus diseases ........................................... 15
IV.1 Antagonistic microorganisms as biocontrol agents ................................................... 15
IV.1.1 Naturally occurring antagonists.......................................................................... 16
IV.1.2 Artificially introduced microbial antagonists ..................................................... 16
IV.1.2.1 Yeasts as biocontrol agents ......................................................................... 16
IV.1.2.2 Bacteria as biocontrol agents ...................................................................... 17
IV.1.2.3 Fungi as biocontrol agents .......................................................................... 18
IV.1.3 Mode of action of antagonistic microorganisms ................................................ 18
IV.1.3.1 Antibiosis .................................................................................................... 19
IV.1.3.2 Competition for nutrients and space ........................................................... 19
IV.1.3.3 Direct parasitism ......................................................................................... 20
IV.1.3.4 Induced resistance ....................................................................................... 21
IV.1.4 Application methods of microbial antagonists ................................................... 23
IV.1.5 Criteria for selecting a good antagonist .............................................................. 24
IV.1.6 Use of antagonist mixtures ................................................................................. 25
 IV.2 Biological control of citrus diseases with natural plant products .............................. 26
IV.2.1 Use of essential oils ............................................................................................ 27
IV.2.2 Use of crude plant extracts ................................................................................. 28
IV.2.3 Use of natural products extracted from plants.................................................... 31
IV.2.4 Mode of action of plant extracts ......................................................................... 32
IV.2.5 Application methods of plant extracts and criteria for selecting a good product33
IV.3 Control of citrus postharvest diseases by food additives and GRAS compounds ..... 34
IV.3.1 Use of salt compounds and food additives ......................................................... 36
IV.3.2 Mode of action of salt compounds ..................................................................... 36
IV.4 Use of combined strategy to control postharvest citrus diseases............................... 38
IV.4.1 Combination of microbial antagonists with other control methods ................... 38
IV.4.1.1 Microbial antagonists combined with low risk substances ......................... 39
IV.4.1.2 Combination of microbial antagonists with low levels
of conventional fungicides ......................................................................... 40
IV.4.1.3 Combination of microbial antagonists with physical control treatments .... 42
IV.4.2 Combination of salts and food additives with other control methods ................ 42
IV.4.2.1 Combination of salts and food additives with low levels
of conventional fungicides .......................................................................... 42
IV.4.2.2 Combination of salts and food additives with physical control treatments 43
V. Conclusion ........................................................................................................................ 43

Chapter two : Screening of the antifungal activity of forty three plant

extracts for the control of Geotrichum candidum
Résumé ..................................................................................................................................... 45
Abstract .................................................................................................................................... 46
I. Introduction ....................................................................................................................... 47
II. Materials and methods ...................................................................................................... 48
II.1 Collection of Plant Samples .......................................................................................... 48
II.2 Preparation of plant extracts .......................................................................................... 48
II.3 Pathogen ........................................................................................................................ 49
II.4 Fruit ............................................................................................................................... 49
II.5 Evaluation of antifungal activity of plant extracts ........................................................ 49
II.5.1 In vitro effect on mycelial growth of G. candidum ............................................ 49
II.5.2 Effect of aqueous extracts on arthrospores germination .................................... 50
II.5.3 Determination of Minimum inhibitory concentration (MIC) and minimum
fungicidal concentration (MFC) ....................................................................................... 50
II.5.4 Effects of aqueous extracts on sour rot development in artificially inoculated
and wounded fruit ............................................................................................................. 51
II.6 Statistical analysis ......................................................................................................... 52
III. Results ........................................................................................................................... 53
III.1 In vitro effect on mycelial growth of G. candidum ................................................... 53
III.2 Effect of aqueous extracts on arthrospores germination ........................................... 55
III.3 MIC and MFC ........................................................................................................... 56
III.4 In vivo test ................................................................................................................. 57
IV. Discussion ..................................................................................................................... 59
V. Conclusion ........................................................................................................................ 62

Chapter Three : Evaluation of the effectiveness of organics fractions of the

most active plants species, against G. candidum
Résumé ..................................................................................................................................... 63
Abstract .................................................................................................................................... 65
I. Introduction ....................................................................................................................... 66
II. Materials and methods .................................................................................................. 67
II.1 Collection of Plant Samples ...................................................................................... 67
II.2 Preparation of plant extracts ...................................................................................... 68
II.3 Pathogen .................................................................................................................... 68
II.4 Fruit ........................................................................................................................... 69
II.5 Evaluation of antifungal activity of plant extracts ..................................................... 69
II.5.1 In vitro effect of solvents extracts on mycelial growth of G. candidum ............ 69
II.5.2 Effect of solvents extracts on arthrospores germination .................................... 70
II.5.3 Determination of Minimum inhibitory concentration (MIC) and minimum
fungicidal concentration (MFC) ......................................................................... 70
II.5.4 Effects of solvent extracts on sour rot development in artificially inoculated and
wounded fruit ..................................................................................................... 71
II.6 Phytochemical analysis .............................................................................................. 72
II.7 Statistical analysis...................................................................................................... 72
III. Results ........................................................................................................................... 73
III.1 In vitro effect of solvents extracts on mycelial growth of G. candidum ................... 73
III.2 Effect of solvents extracts on arthrospores germination ........................................... 75
III.3 MIC and MFC ........................................................................................................... 77
III.4 In vivo test .................................................................................................................. 77
III.5 Phytochemical analysis .............................................................................................. 80
IV. Discussion ..................................................................................................................... 80
V. Conclusions ....................................................................................................................... 84
 Chapter four : Bioguided fractionation
Résumé ..................................................................................................................................... 85
Abstract .................................................................................................................................... 86
I. Introduction ....................................................................................................................... 87
II. Material and Methods ....................................................................................................... 88
II.1 Pathogen and arthrospores preparation...................................................................... 88
II.2 Preparation of plant extracts ...................................................................................... 88
II.3 Open column chromatography .................................................................................. 88
II.4 Antifungal assay ........................................................................................................ 90
II.5 Statistical analysis...................................................................................................... 91
III. Results ........................................................................................................................... 92
IV. Discussion ..................................................................................................................... 95
V. Conclusions ....................................................................................................................... 96
Chapter five : Screening of organic and inorganic salts against citrus sour
rot agent
Résumé ..................................................................................................................................... 97
Abstract .................................................................................................................................... 97
I. Introduction ....................................................................................................................... 99
II. Materials and Methods .................................................................................................... 100
II.1 Pathogen culture and chemicals .............................................................................. 100
II.2 Fruit ......................................................................................................................... 100
II.3 Determination of Minimum Inhibitory Concentration (MIC) ................................. 101
II.4 Effect of pH on mycelial growth of G. candidum ................................................... 102
II.5 Effect of salts on arthrospores germination ............................................................. 102
II.6 Effects of salts on sour rot development in artificially inoculated
and wounded fruit ....................................................................................................... 103
II.7 Statistical analysis.................................................................................................... 103
III. Results ......................................................................................................................... 104
III.1 Preliminary screening of salts (MICs) ..................................................................... 104
III.2 Effect of pH on mycelial growth of G. candidum ................................................... 106
III.3 Effect of salt compounds on arthrospores germination ........................................... 106
III.4 Effect of salt compounds on disease development .................................................. 108
IV. Discussion ................................................................................................................... 110
V. Conclusion ...................................................................................................................... 112
Discussion générale et conclusion ....................................................................................... 115
References ............................................................................................................................. 123
Appendix 1 : Cultur mediums ............................................................................................ 145
Appendix 2 : Bilan agrumicole de la campagne 2010/2011 ............................................. 146
List of abreviations
% Percentage
°C Degree Celsius
CHL Chloroform
DCM Dichloromethan
Et. Ac Ethyle Acetat
EDTA Ethylenediaminetetraacetic Acid
μg Microgram
FAO Food And Agriculture Organization
μL Microliter
μm Micrometer
g Gram
GI Germination Inhibition
GAE Gallic Acid Equivalent
GRAS Generally Recognized As Safe
HEX Hexane
h Hour
ha Hectare
l Liter
MGI Mycelial Growth Inhibition
MeOH Methanol
M Molar
MIC Minimal Inhibitory Concentration
MFC Minimum Fungicidal Concentration
mg Milligram
mL Millilitre
mm Millimeter
mM Millimolar
nm Nanometre
NYDA Nutrient Yeast Dextrose Agar
NYDB Nutrient Yeast Dextrose Broth
PDA Potato Dextrose Agar
RH Relative Humidity
SMD Souss Massa Draa
spp. Several Species
t Ton
TLC Thin Layer Chromatography
TPC Total Phenolic Content
UV Ultra-Violet
v/v Volume Per Volume
w/v Weight Per Volume

i
List of figures
Figure 1.1 : Green (A) and Blue (B) molds of citrus………………...…..…………… 10

Figure 1.2 : Physiology of Geotrichum candidum: a) Aspect on PDA medium and b)

Chains of arthrospores appearing dull gray white colony.....……………..…. 11
Figure 1.3 : Sour rot infection caused by G. candidum. a) Initial infection, b)
disintegrated fruit, c) white layer of mycelium appeared at high relative
humidity and d) synergism between sour rot and green mold…………………. 13
Figure 2.1 : Preparation of aqueous plant extracts for the in vitro antifungal
screening………………………………………………………….………... 46

Figure 2.2 : In vivo test of selected aqueous extracts…………………………………... 50

Figure 2.3 : Inhibition of mycelial growth of G. candidum by aqueous extracts of H.
umbellatum (H.u) and C. villosus (C.v) compared with the control (C)…….… 51
Figure 2.4 : Effect of aqueous plant extracts on sour rot incidence in mandarin
fruit………………………………………………………………………………………. 56

Figure 2.5 : Effect of aqueous plant extracts on sour rot severity in mandarin fruit…. 56

Figure 2.6 : Mandarin fruits treated with aqueous extracts of R. ulmifolius (R.u) and C.
villosus (C.v) and inoculated with arthrospores suspension of G. candidum,
after 7 days of incubation…………………………………………………….... 56
Figure 3.1 : Steps of preparation of organic fractions………………………………… 64
Figure 3.2 : In vitro effect of solvents extracts on mycelial growth of G. candidum…. 66
Figure 3.3 : Inhibition zone of G. candidum around the wells filled with the organic
fractions of C. villosus (C.v), H. umbellatum (H.u), C. siliqua (C.s), A. radiata
(A.r) and P. atlantica (P.a) compared with control………………………….... 71
Figure 3.4 : Germination of arthrospores of G. candidum 24h after treatment with
methanol extract of C. villous (a) and H. umbellatum (b) at 1.25 mg/ml
compared with control (c)………………………………….…………………… 73

Figure 3.5 : Effect of solvent extracts on sour rot incidence in mandarin wounds…….. 74
Figure 3.6 : Effect of solvent extracts on sour rot severity in mandarin wounds…….. 75
Figure 3.7 : Control of sour rot by methanol extract of C. villosus (C.v.M) compared
with ethyl acetate extract of the same plant (C.v. E) and control (C)………… 75

ii
Figure 3.8 : Levels of total phenols present in selected plant species…………………. 76

Figure 4.1 : Fractionation of methanol extract of C. villosus by the open columen

chromatography………………………………………………………………… 84
Figure 4.2 : Combination of fractions based on the similarity of their chemical profile 85
Figure 4.3 : Order of fractionation of the methanol extract of C. villosus…………….. 86
Figure 4.4 : Order of fractionation of the methanol extract of H. umbellatum………… 86
Figure 4.5 : In vitro effects of methanol oxtracts fractions of C. villosus on mycelial
growth of G. candidum…….……..………………………………………….. 87
Figure 4.6: Inhibition zone around wells inoculated with the subfractions of [Link] 87
Figure 4.7 : In vitro effects of subfractions from the F.7 fraction of C. villosus on
mycelial growth of G. candidum ………………………………………………. 88
Figure 4.8: Inhibition zone around wells inoculated with the subfractions of [Link] 88
Figure 4.9 : In vitro effects of methanol oxtracts fractions of [Link] on
mycelial growth of G. candidum………………….……………………………. 89
Figure 4.10 : Inhibition zone around wells inoculated with the subfractions of H.
umbellatum……………………………………………………………………... 89
Figure 4.11 : In vitro effects of subfractions from theF.6 fraction of [Link] on
mycelial growth of G. candidum………………………………..……………… 88
Figure 5.1 : Mycelial growth of G. candidum in test tubes containing NYDB medium
and diffrents concentrations of sodium carbonate…………………………….. 98
Figure 5.2: Effect of pH on in vitro mycelial growth of G. candidum. Medium pH was
adjusted with Hcl or NaOH. Bars represent standard deviations of the means 100
Figure 5.3 : Effect of pH on in vitro mycelial growth of G. candidum………………... 101

iii
List of tables
Table 1.1 : Major postharvest citrus diseases, Causal agent, Type and site of infection.. 9

Table 1.2 : Majors fungicides for control of citrus postharvest diseases……...………... 14

Table 1.3 : Microbial antagonists used for the successful control of citrus postharvest
diseases and their mode of action ……………………………………………... 20

Table 1.4 : Plant extracts used for the control of citrus postharvest diseases………...... 28

Table 1.5 : Natural compounds tested against citrus postharvest pathogens………..... 31

Table 1.6 : Salts and food additives used for the control of citrus postharvest diseases. 33

Table 1.7 : Combination of biocontrol antagonists with other control methods………. 40

Table 2.1 : In vitro effects of plant powders on mycelial growth (MG) of Geotrichum
candidum, agent of citrus sour rot……………………………………...……… 52

Table 2.2 : In vitro effect of some plants aqueous extracts on arthrospore germination
of Geotrichum candidum………………………………………………..…….. 54

Table 2.3 : Minimal inhibitory concentrations (MICs) and minimal fungicidal

concentrations (MFCs) for the plant extracts investigated against Geotrichum
candidum………………………………………………………………………. 55

Table 3.1 : Plants used in this study and yield extracted (%) with each solvent……….. 69

Table3.2 : In vitro effects of solvents extracts on mycelial growth of Geotrichum

candidum………………………………………………………………………. 70

Table 3.3 : In vitro effect of solvents extracts on spore germination of Geotrichum

candidum………………………………………………………………………. 72

Table 3.4 : Minimal inhibitory concentrations (MICs) and minimal fungicidal

concentrations (MFCs) of selected plant species……………………………… 74

Table 4.1 : Solvents mixtures used in the first column chromatography………………. 84

Table 4.2 : Solvents mixtures used in the second and third column chromatography…. 85

Table 5.1 : Salt compounds tested, their chemical formula and molecular weight.......... 95

iv
Table 5.2 : Minimum inhibitory concentrations of tested salts against Geotrichum
candidum........................................................................................................ 99

Table 5.3: In vitro effect of salt compounds on arthrospore germination of Geotrichum

candidum……………………………………………………………………… 102

Table 5.4 : Effect of salt compounds on sour rot incidence in infected mandarin fruits 102

Table 5.5 : Effect of salt compounds on disease severity……………………………… 103

v
Abstract
The objective of this thesis was to evaluate the antifungal potential of 43 plant extracts and 34
organic and inorganic salts in order to control Geotrichum candidum, the causal agent of
citrus sour rot.

The plant species tested are harvested in different regions in the Souss-Massa-Draa Region,
southern Morocco between 2008 and 2009. Aqueous extracts of these plants are screened both
in vitro and in vivo for their antifungal activity against G. candidum. Results showed that
among 43 plant species tested, Cistus villosus, Halimium antiatlanticum, H. umbellatum,
Inula viscosa, Anvillea radiata, Pistacia atlantica, Rubus ulmifolius and Ceratonia siliqua
were the most active against G. candidum. The eight selected plants are then subjected to
successive fractionation with organic solvents of increasing polarity (hexane, chloroform,
ethyl acetate and methanol). Results showed that methanol extract of C. villosus has
completely controlled sour rot development. Moreover, the disease incidence was lowered to
3.33 and 11.66% when fruit were treated with methanol extracts of C. siliqua and H.
umbellatum, respectively. Methanol extracts of C. villosus and H. umbellatum are then
subjected to further fractionation using the bioguided fractionation method. The subfractions
F.7 from C. villosus and F.6 from H. umbellatum are the most active against G. candidum by
inhibiting the mycelial growth of the pathogen with 40.33 and 27 mm diameter.

The assessment of the antifungal potential of 34 salts for the control of sour rot showed that
ammonium carbonate and EDTA recorded the lowest minimum inhibitory concentration of
0.1% (w/v) followed by boric acid, sodium carbonate and sodium metabisulfite with MIC of
0.25%. Also of interest, EDTA, boric acid, sodium metabisulfite, sodium carbonate, sodium
sulfate and sodium thiosulfate have completely inhibited the arthrospores germination of G.
candidum at 75 mM. On citrus fruit, sodium salicylate, boric acid and EDTA were the most
active by reducing the incidence of sour to 25.93% and 38.89%. These findings suggest that
C. villosus, C. siliqua and H. umbellatum plants as well as sodium salicylate, boric acid and
EDTA may be useful and effective agents for control of citrus sour rot. Such products
therefore represent a promising alternative to the use of synthetic fungicides.

Key words : Geotrichum candidum, Sour rot, Plant extracts, inorganic and organic Salts, Citrus fruit,
Postharvest

vi
Résumé
Le but de cette thèse était d‟évaluer le potentiel antifongique de 43 plantes et 34 sels
organiques et inorganiques pour contrôler la pourriture amère des agrumes, causée par le
champignon Geotrichum candidum. Les plantes testées ont été récoltées dans différentes
régions de la région du Souss-Massa-Draa, au sud du Maroc entre 2008 et 2009. Les extraits
aqueux de ces plantes ont été évalués, à la fois in vitro et in vivo, pour leur activité
antifongique contre G. candidum. Les résultats ont montré que, parmi les 43 espèces végétales
testées, Cistus villosus, Halimium antiatlanticum, H. umbellatum, Inula viscosa, Anvillea
radiata, Pistacia atlantica, Rubus ulmifolius et Ceratonia siliqua étaient les plus actives
contre G. candidum. Les huit plantes sélectionnées ont été ensuite soumises à un
fractionnement successif avec des solvants organiques de polarité croissante (l‟hexane, le
chloroforme, l'acétate d'éthyle et le méthanol). Les résultats ont montré que l'extrait
méthanolique de C. villosus a complètement contrôlé le développement de la pourriture
amère. En outre, l'incidence de la maladie a été abaissé à 3,33 et 11,66% lorsque les fruits ont
été traités par les extraits méthanoliques de C. siliqua et H. umbellatum, respectivement. Les
meilleures extraits ont, par la suite, fait l‟objet d‟une purification par la méthode du
fractionnement bio-guidé. La fraction F.7 de l‟extrait méthanolique de C. villosus est la plus
active in vitro contre G. candidum en inhibant la croissance mycélienne du champignon avec
un diamètre de la zone d‟inhibition de 40,33 mm. Concernant H. umbellatum, le meilleur
degré d‟inhibition est obtenu avec la fraction F.6 avec un diamètre de 27 mm. De même,
l‟évaluation du potentiel antifongique de 34 sels contre G. candidum, a révèlé que le
carbonate d‟ammonium et l‟EDTA ont enregistré la plus faible concentration minimale
inhibitrice (CMI) qui est de 0,1% (v/v) suivi par le carbonate de sodium et le métabisulfite de
sodium avec une CMI de 0,25%. L‟effet des meilleurs sels sur la germination des arthrospores
a montré que l‟EDTA, l‟acide borique, le métabisulfite de sodium, le carbonate de sodium, le
sulfate de sodium et le thiosulfate de sodium ont complètement inhibé la germination des
arthrospores, à la fois à 100 et à 75 mM. Appliqués sur les fruits, le salicylate de sodium,
l‟acide borique et l‟EDTA ont permis de réduire l‟incidence de la pourriture amère à 25,93 %
et 38,89% contre 100% chez le témoin. Cette étude démontre que les extraits de C. villosus,
C. siliqua et H. umbellatum ainsi que le salicylate de sodium, l‟acide borique et l‟EDTA
pourraient être mis à profit pour lutter contre la pourriture amère des agrumes en post-récolte
et représentent donc une alternative prometteuse à l‟utilisation des fongicides de synthèse.
Mots clés : Geotrichum candidum, pourriture amère, Extraits de plantes, Sels, agrumes, Post-récolte.

vii
 ‫م لخص‪:‬‬
‫‪َٕ 43‬عا يٍ انُثاذاخ‬ ‫فً إغاس انثحث عٍ انثذائم انصحٍح ٔانطثٍعٍح نهًثٍذاخ انفطشٌح االصطُاعٍح‪ ،‬ذًد دساسح ذأثٍش‬
‫انطثٍح ٔانعطشٌح‪َٕ 34 ٔ ،‬عا يٍ األيالح انععٌٕح ٔغٍش انععٌٕح عهى انفطش انًسثة نهعفٍ انًش عُذ انحٕايط انزي‬
‫ٌسثثّ انفطش انًعشٔف تاسى ‪ .Geotrichum candidum‬ذى جهة انُثاذاخ انًخرثشج فً ْزِ انذساسح يٍ يُاغق يخرهفح يٍ‬
‫‪ [Link]‬سٕاء‬ ‫انجُٕب انًغشتً تٍٍ عايً ‪ٔ ،2009 ٔ 2008‬ذى ذقٍى انًسرخهصاخ انًائٍح نٓزِ انُثاذاخ ظذ‬
‫‪َ 43‬ثرح انرً ذى اخرثاسْا ‪Cistus : villosus, Halimium‬‬ ‫تانرجاسب انًخرثشٌح أٔ انحٍح‪ .‬أظٓشخ انُرائج أٌ يٍ تٍٍ‬
‫‪antiatlanticum, H. umbellatum, Inula viscosa, Anvillea radiata, Pistacia atlantica, Rubus‬‬
‫‪ulmifolius,‬‬ ‫‪Ceratonia‬‬ ‫‪siliqua‬‬
‫ًْ األكثش َشاغا ظذ ْزا انفطش‪ْ .‬زِ انُثاذاخ ذى اخرثاس ذأثٍش يسرخهصاذٓا انععٌٕح عهى انفطش انًسثة نهعفٍ تاسرعًال‬
‫يزٌثاخ ععٌٕح )‪ ،(Hexane, Chloroforme, Acétate d‟éthyle, Méthanole‬أٔظحد انُرائج أٌ يسرخهص‬
‫انًٍثإَل نُثرح ‪ [Link]‬سٍطش ذًاو عهى ذطٕس انعفٍ‪ ،‬تاإلظافح إنى رنك ذى ذخفٍط يعذل اإلصاتح تانًشض إنى‬
‫‪ 11.66% ٔ 3.33%‬عُذيا ذًد يعانجح انفاكٓح تًسرخهصاخ انًٍثإَل ل ‪ [Link] ٔ [Link]‬عهى انرٕانً‪.‬‬
‫‪Salicylate sodium ; Acide‬‬ ‫ٔ تانًثم أظٓشخ دساسح ذأثٍش األيالح انععٌٕح ٔغٍش انععٌٕح انًخرثشج أٌ‬
‫‪ .38.89% ٔ 25.93%‬أثثرد ْزِ انذساسح أَّ ًٌكٍ ذسخٍش‬ ‫‪ borique ;EDTA‬حفظٕا يٍ حاالخ انعفٍ تُسثرً‬
‫يسرخهصاخ ‪ H. umbellatum ٔ C. siliqua ٔ C. villosus‬تاإلظافح إنى ‪ Acide borique ;EDTA‬نهحذ يٍ انعفٍ‬
‫انًش نهحٕايط‪ْ ،‬زِ انٕسائم ذعرثش تذائم ٔاعذج السرخذاو انًثٍذاخ انفطشٌح االصطُاعٍح‪.‬‬

‫ك ال مات م ف تاح ‪ :‬حاوامض امالح‪ ، ،‬م س تخ ل صات ال ناب ات ات ‪ ،‬ع فن مر ‪Geotrichum candidum ,‬‬

‫‪viii‬‬
 Introduction générale

INTRODUCTION GÉNÉRALE

Les agrumes comptent parmi les principales cultures fruitières, dont le fruit est
largement consommé aussi bien frais que sous forme de jus ou autre formes (conserve,
confiture…). L‟importance des fruits d‟agrumes est attribuée à leur richesse en vitamine C
(acide ascorbique), en fibres alimentaires, en composés phénoliques, en oligo-éléments, et
pour leur potentiel antioxydant (Gorinstein et al., 2001). Les agrumes sont cultivés dans plus
de 137 pays à travers le monde (Ismail and Zhang, 2004). À l‟échelle mondiale, la production
annuelle en fruit frais est d‟environ 120 millions de tonnes. Au Maroc, la culture des agrumes
couvre une superficie de 76500 hectares assurant une production annuelle d‟environ 1,5
millions de tonnes. Ce tonnage couvre à la fois les besoins du marché national en fruit frais,
assure l‟exportation d‟environ 570.000 t/an et alimente les unités de transformation. Ce
secteur génére des effets importants sur l‟emploi à travers la création de près de 21 millions
de journnées de travaille par an. De ce fait, le secteur des agrumes est classé parmi les plus
importants de l‟économie nationale. La région du Souss-Massa-Draa vient au premier rang à
l‟échelle nationale, en assurant environ 50 % de la production et de l‟exportation nationale
(Anonyme, 2011).

En raison de leur teneur élevée en eau et de leur richesse en élément nutritifs, les fruits
d‟agrumes sont très susceptibles aux attaques des champignons pathogènes (Eckert and
Ogawa, 1985; Tripathi and Dubey, 2004). Au Maroc, comme dans les pays en
développement, les pertes pendant la récolte, le transport et le stockage des fruits sont élevées.
Elles peuvent aller jusqu‟à 50%, ou plus, de la récolte (Eckert and Ogawa, 1985; Wisniewski
and Wilson, 1992). Les agrumes, comme toutes les autres cultures, sont affectés par plusieurs
maladies cryptogamiques. Ces maladies sont dues à des infections initiées soient avant la
récolte (e.g. Alternaria citri, Colletotrichum gloesporioides, Lasiodiplodia theobromae,
Trichoderma viride) ou après la récolte des fruits (e.g. Aspergillus niger, Geotrichum
candidum, Penicillium spp., Rhizopus stolonifer, Trichoderma viride) (Sommer, 1982; Eckert
and Brown, 1986; Holmes et al., 1994). Cependant, la quasi-totalité des pertes d‟origine
parasitaire, en post-récolte, sont dues aux champignons suivants : Geotrichum candidum
(agent de la pourriture amère), Penicillium digitatum (agent de la pourriture verte) et P.
italicum (agent de la pourriture bleue). En effet, plus de 90% des dégâts sur fruits d‟agrumes
en post-récolte sont dus aux champignons précités (Boubaker, 1993; Holmes et al., 1994).

1
 Introduction générale

La pourriture amère, causée par le champignon Geotrichum candidum Link ex Pers., est
l‟une des plus redoutables maladies d‟agrumes en post-récolte, notamment pendant la saison
des pluies et le déverdissage des fruits. Elle a été rapportée dans la majorité des régions
agrumicoles et cause des pertes économiques importantes (Eckert, 1978; Rippon and Morris,
1981; Brown, 1988; Hershenhorn et al., 1992;). Elle atteint toute les variétés d‟agrumes à
différents degrés d‟importance (Boubaker, 1993). L‟agent causal de la maladie est un parasite
de blessure qui pénètre dans les fruits par les blessures profondes qui peuvent se produire
pendant la récolte, le conditionnement ou lors du transport (Brown, 1979; Palou et al., 2008).
Le champignon dégrade le fruit en secrétant des enzymes pectinolytiques qui transforment le
fruit en une masse molle. Cet aspect favorise la propagation de la maladie par contact entre
fruit saint et fruit infecté. L‟infection par G. candidum dépend de plusieurs facteurs tels que la
présence de blessures fraîches et profondes, la turgescence de la peau des fruits, le stade de
maturité des fruits et les conditions de l‟environnement avec une température optimale qui se
situe entre 25 et 30°C et une humidité relative élevée (Brown, 1979; Baudoin and Eckert,
1985; Eckert and Brown, 1988; Cohen et al., 1991; Suprapta et al., 1995).

Etant donné qu‟il est plus facile d‟augmenter le volume des exportations en protégeant
mieux les fruits après leur récolte que de vouloir augmenter la production, cette démarche
s‟avère moins coûteuse et plus simple à atteindre dans un délai assez court. Dans ce contexte,
des traitements chimiques (utilisation des fongicides) et physique (stockage au froid) sont
pratiqués dans les stations de conditionnement afin de limiter les pertes dues aux pathogènes
de post-récolte (Palou et al., 2008). Néanmoins, ces traitements ont montré peu d‟efficacité
contre la pourriture amère des agrumes en post-récolte. En effet, le stockage au froid n‟a
qu‟un effet fongistatique ; la maladie se développe d‟une manière explosive lorsque le fruit
est transféré à la température ambiante. En revanche, les fongicides homologués pour
contrôler les maladies d‟agrumes en post-récolte (Imazalil, Thiabendazole) n‟ont aucun effet
sur G. candidum (Suprapta et al., 1997; Brown and Miller, 1999; Mercier and Smilanick,
2005; Liu et al., 2009b). La guazatine est le seul fongicide qui peut contrôler efficacement
l‟agent de la pourriture amère (Brown and Eckert, 1988). Cependant, ce produit n‟est pas
homologué en traitement de post-récolte dans la plupart des pays agrumicoles. En outre,
l'utilisation des fongicides est de plus en plus restreinte en raison de la réglementation qui
devient de plus en plus sévère, de leur toxicité, de leur effet sur l‟environnement et de
l'inquiétude croissante du consommateur à propos des résidus de pesticides dans les fruits
(Zhang and Swingle, 2003; Tripathi and Dubey, 2004; Palou et al., 2008). Les mesures

2
 Introduction générale

prophylactiques, telles que la minimisation des blessures des fruits, et les mesures d‟hygiènes
dans les stations de conditionnement restent donc le seul moyen pour contrôler cette maladie.

Il y a donc un besoin évident et croissant de trouver et de mettre en œuvre des méthodes

de lutte alternatives à la lutte chimique pour le contrôle de la pourriture amère. L'objectif est
d'accomplir un contrôle satisfaisant des pourritures de post-récolte en adoptant des
programmes de gestion intégrée des maladies. Au cours des deux dernières décennies, la lutte
biologique, par ses méthodes respectueuses de l‟environnement et de la santé du
consommateur, est apparue comme une stratégie efficace pour lutter contre les maladies
d'agrumes en post-récolte. Parmi les différentes approches biologiques, l'utilisation des
antagonistes microbiens et des substances naturelles, d‟origine végétale ou animale, est très
prometteuse et gagne de plus en plus la confiance des consommateurs. En effet, plusieurs
travaux ont porté sur la recherche de microorganismes antagonistes des champignons
pathogènes des agrumes en post-récolte (Eckert and Ogawa, 1988; Wisniewski and Wilson,
1992 ;Arras et al., 1998; Bull et al., 1998; Droby et al., 2002; Mercier and Smilanick, 2005;
Cañamás et al., 2008; Taqarort et al., 2008). Cependant seuls quelques uns de ces
antagonistes (Candida oleophila; Pseudomonas syringae; Cryptococcus albidus) sont utilisés
à l‟échelle commerciale (Droby et al., 1998; Palou et al., 2008). Une autre approche de la
lutte contre les maladies d‟agrumes en post-récolte est l'utilisation de substances naturelles
extraites à partir de plantes. Les plantes ont une capacité presque illimitée de synthétiser des
substances aromatiques. La plupart sont des métabolites secondaires, dont plus de 10.000 ont
été isolés, identifiés et définis pour leurs propriétés antimicrobiennes. Par ailleurs, ces
métabolites sont connus par leur caractère systémique, non phytotoxique et biodégradable
(Tripathi and Dubey, 2004; Kosalec et al., 2005). En raison de ces propriétés, les extraits de
plantes sont très attractifs comme moyen de lutte contre les pathogènes de post-récolte.

Une autre alternative prometteuse est l‟utilisation des sels et additifs alimentaires qui
sont des composés généralement reconnus comme sains (GRAS). Certains de ces sels sont
présents naturellement dans de nombreux fruits et légumes (Foegeding and Busta, 1991; El-
Mougy et al., 2008;) et sont, le plus souvent, utilisés dans l'industrie alimentaire comme
désinfectants ou pour contrôler le pH, le goût et la texture (Smilanick et al., 1999; Hervieux et
al., 2002; Arslan et al., 2009). En outre, ces composés ont un large spectre antimicrobien, une
faible toxicité et un coût relativement faible (Corral et al., 1988; Olivier et al., 1998; Hervieux
et al., 2002; Deliopoulos et al., 2010).

3
 Introduction générale

La plupart des travaux de recherches sur le contrôle des maladies de post-récolte sont
relatifs à la lutte contre les pourritures à Penicillium spp. En effet, très peu de travaux ont été
publiés sur la lutte contre la pourriture amère. L‟objectif de cette étude est d‟évaluer
l‟efficacité de deux stratégies alternatives au fongicides de synthése, à savoir : l‟utilisation des
extraits de plantes et l‟utilisation des sels organiques et inorganiques pour contrecarrer le
développement de la pourriture amère des agrumes en post-récolte. Pour atteindre cet objectif
les points suivants ont été traités :

1) Screening in vitro et in vivo de l‟activité antifongique des extraits aqueux de 43

plantes, récoltées dans différentes régions du sud du Maroc, contre G. candidum,
l‟agent de la pourriture amère des agrumes.
2) Evaluation de l‟activité antifongique des fractions organiques des plantes les plus
efficaces contre la pourriture amère et étude du mode d‟action des meilleures
fractions organiques sélectionnées.
3) Evaluation de l‟activité antifongique d‟une large gamme de sels organiques et
inorganiques, aussi bien in vitro qu‟in vivo, contre G. candidum.

4
 CHAPTER ONE

Literature review:
Alternative methods for the control of
postharvest citrus diseases
Submitted to Plant Pathology Journal (2013) as :
Idriss Talibi, Hassan Boubaker, Abdellah Ait Ben Aoumar, and El Hassane Boudyach.
Healthy alternatives for the control of postharvest diseases of citrus fruit.
 Chapter 1: Literature review

Résumé
Les maladies de post-récolte sont à l‟origine d‟énormes pertes des fruits d‟agrumes
pendant le stockage et le transport des fruits. Ces maladies sont contrôlées principalement par
l‟utilisation excessive de fongicides de synthèse. Cependant, l‟efficacité variable de ces
fongicides, la prolifération des souches résistantes et la préoccupation croissante de la
population envers les dangers liés à l‟utilisation de ces produits, tels que leurs effets néfastes
sur la santé et l‟environnement, a nécessité le développement de stratégies alternatives pour le
contrôle des maladies d‟agrumes en post-récolte. Par conséquent, la lutte biologique, par ses
méthodes respectueuses de l‟environnement et de la santé du consommateur, constitue une
alternative prometteuse à la lutte chimique. L‟utilisation des micro-organismes antagonistes,
des substances naturelles ou des sels organiques et inorganiques à effet antimicrobien sont les
moyens alternatifs les plus appropriés pour remplacer les fongicides de synthèse qui sont soit
interdits ou recommandé seulement pour une utilisation limitée. Cependant, l‟application de
ces alternatives seules ne peuvent pas assurer, toujours, des niveaux de contrôle
commercialement acceptable des maladies d‟agrumes en post-récolte en comparaison avec
ceux obtenus par les fongicides de synthèse. A cet effet, la protection des fruits d‟agrumes par
la combinaison de différentes méthodes de lutte qui satisfait, à la fois, les exigences
écologique et économiques est une bonne solution pour améliorer l‟efficacité de la lutte
biologique et confronter ces limites. Malgré les caractéristiques distinctives de ces méthodes
alternatives, plusieurs raisons empêchent l‟utilisation commerciale de ces traitements. Par
conséquent, les travaux de recherche doivent prêter plus d‟importance à s‟assurer que les
moyens de lutte alternatives efficaces répondent aux exigences commerciales.
Mots clés : Lutte biologique, Post-récote, Agrumes, Antagonistes, Extraits de plantes,
sels, Additifs.

5
 Chapter 1: Literature review

Abstract
The postharvest diseases of citrus fruit cause considerable losses during storage and
transportation. These diseases are managed principally by the excessive application of
synthetic fungicides. However, the increasing concern for health hazards and environmental
pollution due to chemical use has necessitated the development of alternative strategies for the
control of postharvest citrus diseases. Management of postharvest diseases by employing
microbial antagonists, natural plant-derived products and Generally Recognized As Safe
(GRAS) compounds has been demonstrated to be most suitable to replace the synthetic
fungicides which are either being banned or recommended for limited use. However,
application of these alternatives by itself may not always provide commercially acceptable
level of control of postharvest citrus diseases comparable to that obtained with synthetic
fungicides. To provide more effective disease control, than that possible with a single
approach, a multifaceted approach based on the combination of different postharvest
treatments has been adopted. Actually, despite the distinctive features of these alternative
methods, several reasons hinder the commercial use of such treatments. Consequently,
research should lend more importance to ensure appropriate tools to effective application of
these alternatives to commercial requirements.
Key words : Biological control, Post-harvest, Citrus, Microbial antagonists, Plant
extracts, Salt additives.

6
 Chapter 1: Literature review

I. Introduction

Citrus is one of the most widely produced fruit. It is grown commercially in more than
137 countries around the world (Ismail and Zhang, 2004). The production of citrus fruit
remains amongst the largest crops produced worldwide with almost 115.65 million tons
(FAO, 2011). The contribution of the citrus industry to the world economy is enormous and it
provides jobs to millions of people around the world in harvesting, handling, transportation,
storage, and marketing operations. The importance of citrus fruit is attributed to its diversified
use, which is widely consumed either as fresh fruit or as juice. In the world, Brazil is the
major citrus fruit producing country followed by the United States of America and China.
Citrus production in Morocco is 1.5 million tons from 76 500 ha under this crop (Anonymous,
2011). About 570 000 tons of fresh citrus fruit were exported (Anonymous, 2011).

Due to their higher water content and nutrient composition, citrus fruit are very
susceptible to infection by microbial pathogens during the period between harvest and
consumption (Eckert and Ogawa, 1985; Tripathi and Dubey, 2003). As citrus fruits have a pH
lower than 4, so fungi are often the predominant microorganisms in fruit and fruit products.
No bacterial postharvest disease of commercial importance is reported on citrus fruit.
Contamination and infection by pathogenic fungi occur at different stages in the field and
after harvest during marketing, and usually follows mechanical injury or physiological
breakdown of the fruit, which allow entry to these microorganisms. In a few cases, pathogens
can infect healthy tissues (Huang et al., 1992). Postharvest decays of fruit can also originate
from latent infections occurring in the orchard (Snowdon, 1990).

Losses, during transportation and storage of fresh citrus fruit, are estimated at around 5
and 10% in most developed countries, and from 25 to 30% or more in developing and
underdeveloped countries because of the lack of adequate protection measures and storage
facilities (Eckert and Ogawa, 1985; Wisniewski and Wilson, 1992). Citrus fruit are
susceptible to a number of postharvest diseases that cause significant losses during the
postharvest phase. Nevertheless, the most common and serious diseases that affect citrus fruit
are green and blue molds caused, respectively, by Penicillium digitatum Sacc. and Penicillium
italicum Wehmer, followed by sour rot caused by Geotrichum candidum Link ex Pers
(Caccioni et al., 1998; Palou et al., 2002; Zheng et al., 2005). These pathogens are strict
wound pathogens that can infect the fruit in the grove, in the packinghouse, or during
subsequent handling and storage (Brown, 1979; Palou et al., 2008). The fungal inoculum is

7
 Chapter 1: Literature review

practically always present on the surface of fruit during the season and after harvest can build
up high levels unless appropriate packinghouse sanitization measures are adopted (Kanetis et
al., 2007). Fruit infection by these fungi is enhanced during fruit degreening operation, and
during wet and rainfall seasons (Eckert, 1978; Eckert and Brown, 1986; Cohen et al., 1991,
Liu et al., 2009b). The decays are also more prevalent as fruit increases in maturity, and at
favorable temperatures and humidity (Brown, 1979; Baudoin and Eckert, 1985).

Currently, synthetic fungicides are the primary means of controlling post-harvest

diseases of citrus fruit, especially by imazalil (IZ), thiabendazole (TBZ), sodium ortho-phenyl
phenate (SOPP), fludioxonil (FLU), pyrimethanil or different mixtures of these compounds
(Eckert and Ogawa, 1988; Holmes and Eckert, 1999; Ismail and Zhang, 2004; Smilanick et
al., 2005; Palou et al., 2008). Continuous use of these fungicides has resulted in the
appearance of isolates of fungi with multiple fungicide resistances, which further complicate
the management of the decays (especially Penicillium rots) (Holmes and Eckert, 1999; Droby
et al., 2002; Mercier and Smilanick, 2005; Boubaker et al., 2009). In addition, theses
fungicides are not effective against all important pathogens. Indeed, sour rot is difficult to
control with IMZ and TBZ (Eckert, 1978; Brown, 1979; Suprapta et al., 1997; Mercier and
Smilanick, 2005;). The synthetic fungicide guazatine is the only commercial product that can
control sour rot (Brown, 1988). However, this fungicide is not authorized in several countries.
Furthermore, the use of fungicides is increasingly becoming restricted owing to stringent
regulation, carcinogenicity, high and acute residual toxicity, long degradation period,
environmental pollution and growing public concern about chemical residues in fruit (Tripathi
and Dubey, 2003; Zhang and Swingle, 2003; Palou et al., 2008).

Therefore, the challenge is to develop safer and eco-friendly alternative strategies of

controlling citrus postharvest diseases, which pose less risk to human health and environment.
Recently, several promising biological approaches have been proposed as potential
alternatives to synthetic fungicides for the control of postharvest citrus disease. These
biological control strategies include : (1) use of antagonistic microorganisms; (2) application
of naturally-derived bioactive compounds; and (3) induction of natural resistance. Among
these biological approaches, the use of the microbial antagonists, either alone or as part of an
integrated disease management policy, is quite promising and gaining popularity among
consumers (Eckert and Ogawa, 1988; Wisniewski and Wilson, 1992; Droby et al., 2002).
Interestingly, most of the antagonistic microorganisms are isolated from fruit surfaces as
epiphytic microbial population. According to Wilson et al. (1991), the postharvest

8
 Chapter 1: Literature review

environments present some unique advantages for the use of biocontrol methods. Indeed, the
postharvest environment provides the following advantages : (1) the partially controlled
environment in storage may result in a shift in the balance of interactions between host,
pathogen, and antagonistic microbe in favor of antagonist; (2) the efficacy of antagonist may
be enhanced because the biocontrol product can be applied directly onto the site where needed
in the harvested product; (3) the harvested commodity may be protected relatively free of
potential interfering factors; (4) protection is needed for a relatively short period as compared
with period of protection required for field crops; and (5) as the harvested fruits have high
market value, use of a relatively high-cost biocontrol product may be justified.

The second approach to disease control is the use of natural plant-derived compounds.
Indeed, these compounds have gained popularity and scientific interest for their antibacterial
and antifungal activity (Tripathi and Dubey, 2003; Liu et al., 2009b; du Plooy et al., 2009;
Gatto et al., 2011; Talibi et al., 2011a; Talibi et al., 2012a and b). Naturally occurring plant
products are very attractive as alternative or complementary control means because of their
antifungal activity, non-phytotoxicity, systemicity, and biodegradability (Tripathi and Dubey,
2003; Kosalec et al., 2005; Ameziane et al., 2007; Gatto et al., 2011; Talibi et al., 2011a).

However, application of these biological control methods by itself may not always provide
commercially acceptable level of control of postharvest citrus diseases. It is possible to
increase the efficacy of these methods by combining with other postharvest treatments.
Indeed, different disease management strategies have been integrated to provide more
effective disease control than that possible with a single approach. Low-toxicity chemicals,
particularly common food additives and GRAS (Generally Regarded as Safe) compounds,
were evaluated for their efficacy for the control of citrus pathogens. Strictly speaking, GRAS
compounds do not fall into the category of organic ingredients, but they are much less
harmful than many other inorganic chemicals.
The purpose of this paper is to provide an overview of the published data on the control
methods alternative to conventional chemical fungicides for the control of postharvest citrus
pathogens.
II. Postharvest citrus diseases

Diseases that occur after harvest can have a significant impact on keeping quality of fresh
citrus fruit. Losses from post-harvest diseases caused by various pathogens account for nearly
50 percent of the total wastage in citrus fruits (Ladaniya, 2008). Infection and contamination

9
 Chapter 1: Literature review

occur at different stages in the field, subsequent handling and storage activities. Citrus fruits
are susceptible to infection by many fungal pathogens (Table 1.1). These fungal pathogens
can be subdivided into two categories :

 The first category includes those pathogens that enter through a natural entry point and
possess a long inactive (quiescent) phase after infection, or symptoms may develop shortly
after infection. The main fungi belonging to this group are: Lasiodiplodia theobromae
(stem end rot), Phomopsis citri (stem end rot), Colletotrichum gloesporioïdes (Antracnose)
and Alternaria alternata (Black rot) (Boubaker, 1993) (Table 1.1).
 The second category includes wounds pathogens that enter through injuries. In this
case, the diseases appear in less than a week after the pathogen's activation upon the
release of moisture and nutrients at injury sites of the fruit rind (Eckert and Brown, 1986).
These include green and blue mold caused by Penicillium spp., and sour rot caused by
G. candidum. Over than 90 % of citrus fruit losses are due to the aforementioned fungi
(Boubaker, 1993).

Table 1.1 : Major postharvest citrus diseases, Causal agent, Type and site of infection
Infection
Disease Causal agent
site
Anthracnose Colletotrichum gloeosporioides Intact or injured rind
Black rot alternaria alternata Natural openings
Black spot Guignardia citricarpa Intact or injured rind
Blue mould Penicillium italicum Injured rind
Brown rot Phytophthora spp. Intact rind
Greasy spot Mycosphaerella citri Intact or injured rind
Green mould Penicillium digitatum Injured rind
Lime anthracnose Gloeosporium limetticola Intact or injured rind
Sour rot Geotrichum candidum Injured rind
Stem-end rot Lasiodiplodia theobromae Button
Stem-end rot Phomopsis citri Button
Trichoderma rot Trichoderma viride Injured rind

II.1 Penicillium rots

Penicillium digitatum Sacc. and Penicillium italicum Wehmer are the two most
significant and widely reported postharvest pathogens in citrus (Plaza et al., 2003; Ladaniya,
2008). The rots develop only if there are injuries in the rind. The major menace of these
pathogens is due to their spores, which appear as fine powder and are airborne and are

10
 Chapter 1: Literature review

produced on the surfaces of infected fruit. Initially, the decay appears as a soft, watery,
slightly discolored spot. After few days, very profuse sporulation can be seen on fruit which is
completely covered by white mycelium followed by olive-green and bluish spores of P.
digitatum and P. italicum respectively (Figure 1.1). Spores are easily dispersed if the fruits
were handled or shaken or if they were exposed to air currents. Although, blue mold develops
less rapidly than green mold at ambient temperatures, but it is more harmful because it
spreads in the box and healthy fruits are directly attacked, regardless of injury. The
development of the Penicillium rots is influenced by several factors such as temperature, and
relative humidity. Indeed, the optimum conditions for the growth of both fungi are a
temperature ranged between 20 and 25° C and high levels of relative humidity (>90%).
However, P. italicum is more adapted to low temperatures (<10°C) than P. digitatum (Eckert
and Ogawa, 1985).

A B

Figure 1.1 : Citrus Green (A) and Blue (B) mold in citrus fruit

II.2 Sour rot

Sour rot is an important postharvest disease of citrus fruit from most areas of the
world (Eckert, 1978; Rippon and Morris, 1981; Brown, 1988; Hershenhorn et al., 1992). It is
the most rapidly spreading postharvest disease (El Ghaouth et al., 2002). Geotrichum
candidum Link ex Pers., the causal agent of this rot, is responsible for economically
significant losses under favorable conditions for pathogen development, principally during
fruit degreening and wet seasons (Eckert and Brown, 1988; Cohen et al., 1991). Also, isolates
of G. candidum have been found to be pathogenic to the fruits of tomato, orange, lemon,
satsuma mandarin, grapefruit, cucumber and carrot (Suprapta et al., 1995; Wells, 1997; Palou
et al., 2009).

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 Chapter 1: Literature review

II.2.1 Causal organism

Sour rot of citrus fruit was first described in 1917 and the pathogen was designated as
Oospora citri-aurantii (Ferr.) Sacc. et Syd (Butler et al., 1965; Suprapta et al., 1995). Since
then, several different names have been used to designate the pathogen such as G. candidum
Link var. citri-aurantii (Ferr.) R. Ciferri. et F. Ciferri (Butler et al., 1965) and G. citri-aurantii
(Ferr.) Butler (asexual form) and Galactomyces citri-aurantii Butler (sexual form) (Butler et
al., 1988). However, G. candidum Link ex Pers is commonly the most cited to indicate the
citrus sour rot pathogen (Brown, 1979; Rippon and Morris, 1981; Kitagawa and Kawada,
1984; Baudoin and Eckert, 1985; Mercier and Smilanick, 2005; Smilanick et al., 2008). G.
candidum is a filamentous fungus which grows rapidly on potato dextrose agar (PDA),
producing a dull gray-white colony with chains of arthrospores. These arthrospores are
produced by the fragmentation of undifferentiated hyphae. The arthroconidia, which are quite
variable in size, may germinate and develop into a septate mycelium (Figure 1.2).

a b

Figure 1.2: Physiology of Geotrichum candidum : a) Aspect on PDA medium and b) Chains of
arthrospores appearing dull gray white colony

II.2.2 Disease cycle and epidemiology

G. candidum is a common inhabitant of citrus soils and is more prevalent on lower fruit
of the tree canopy and it is windborne from soil particles to surfaces of fruit (Brown, 1979;
Suprapta et al., 1996). The fungus can also accumulate in contaminate pallets, washer
brushes, belts, conveyors, and with dirt and debris in drenchers and soak tanks. The organism
is a wound pathogen requiring injury into the albedo for entry (Brown, 1979). These injuries

12
 Chapter 1: Literature review

may be caused by insects or mechanical means, such as thorn or stem punctures, or by

plugging at harvest. Fruit are more susceptible to sour rot as they become more mature, and if
they contain high amounts of rind moisture (Brown and Eckert, 1988; Suprapta et al., 1996).
Also, the fungus develops most rapidly at ambient temperatures of 25–30ºC. Thus, the
aggressiveness of the fungus increases especially during fruit degreening, wet and rainfall
seasons (Eckert, 1978; Eckert and Brown, 1988; Cohen et al., 1991; Liu et al., 2009b). In the
packing house, the sour odor, characteristic of sour rot, attracts fruit flies, which can
disseminate the fungus to other injured fruit (Brown and Eckert, 1988).

II.2.3 Symptoms

Citrus sour rot infection has the most unpleasant smell of all decays known (Boubaker,
1993; Sissay, 2007). The initial symptoms of sour rot infections are similar to those of
Penicillium rots. The lesion first appears water-soaked, light to dark yellow, and slightly
raised (Figure 1.3). The fruit becomes a soft, stinking, semi-solid mass as the fungi secrete an
active extracellular enzyme named polygalacturonase that rapidly degrade the tissue (Cohen
et al., 1991). Therefore, the fungus degrades the fruit thoroughly, causing it to disintegrate
into a slimy and watery mass. At high relative humidity, the lesions may be covered with a
yeasty, sometimes wrinkled layer of withe or cream-coloured mycelium (Baudoin and Eckert,
1985). Sour rot is often associated with green mold and is stimulated by its presence (Morris,
1982) (Figure 1.3).

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 Chapter 1: Literature review

a b

c d

Figure 1.3 : Sour rot infection caused by G. candidum. a) Initial infection, b) disintegrated fruit, c)
white layer of mycelium appeared at high relative humidity and d) synergism between sour rot and
green mold.

III. Postharvest fungicides treatments

The use of chemicals in citrus postharvest disease management has a history going back
100 years (Ladaniya, 2008). Presently, the chemical control of various rots is the integral part
of the postharvest handling system in citrus. The use of fungicides to control postharvest
citrus diseases can be carried out pre or post harvestly. Field use of fungicides for the control
of postharvest diseases is restricted to applications of copper or benomyl (Table 1.2). For the
postharvest application, several fungicides are used like Sodium orthophenylphenate (SOPP),
Thiabendazole, biphenyl, benomyl, thiophanate methyl, carbendazim, Imazalil and Guazatine
(Eckert and Sommer, 1967; Eckert and Ogawa, 1985) (Table 1.2). Some of these chemicals

14
 Chapter 1: Literature review

are still being used (Imazalil, Thiabendazole) and others are not longer registered in many
countries (Guazatine). These chemicals are used in various forms : in soak tanks or as water
sprays, as fumigants or in wax, or impregnated in paper for keeping in fruit layers or as wraps.
Nevertheless, the control of wounds pathogens with these fungicides depends on their prompt
application at required concentrations. If the application of fungicide is delayed, the pathogen
gets established, so the pathogen can escape the protective action of fungicide (Ladaniya,
2008). In addition, all fungicides are not effective against all important pathogens and there
are problems related to use of these fungicides. For example, sour rot is not controlled,
efficiently, by any postharvest chemical treatments except Guazatine (Brown, 1988), a
chemical that is not authorized in several countries. Also, the intensive and continuous use of
these fungicides has created serious problems of the emergence of resistant pathogens (El-
Goorani et al., 1984; Eckert, 1990; Holmes and Eckert, 1999; Kinay et al., 2007; Sánchez-
Torres and Tuset, 2011). Thus, all these problems require the screening of new antifungal
compounds.

Table 1.2 : Majors fungicides for control of citrus postharvest diseases

Treatments Diseases
Preharvest
Copper Brown rot
Benomyl Diplodia and Phomopsis SER; Penicillium rots; Anthracnose
Postharvest
SOPP Green mold; Sour rot
Thiabendazol Diplodia and Phomopsis SER; Penicillium rots; Anthracnose
Imazalil Penicillium rots; Diplodia, Phomopsis and Alternaria SER
Diphenyl Penicillium rots
Guazatine Penicillium rots, Sour rot
SER : Stem end rot

IV. Integrated strategies to control postharvest citrus diseases

IV.1 Antagonistic microorganisms as biocontrol agents

It has been demonstrated that there are natural microbial antagonists on fruit surface that
can suppress disease development (Wilson and Wisniewski, 1989). The use of antagonistic
microorganisms for controlling the postharvest diseases of citrus fruit is based on two
approaches : a) the use of natural epiphytic antagonists that already exists on fruit surface and
b) the artificial introduction of selective microbial antagonists that control postharvest
diseases.

15
 Chapter 1: Literature review

IV.1.1 Naturally occurring antagonists

One of the approaches to the isolation of antagonistic microorganisms for controlling

postharvest diseases is through the promotion and management of natural epiphytic
microflora, already present on fruit surface. The importance of naturally occurring microbial
antagonists is revealed when washed fruits develop more rot than unwashed ones (Wilson and
Wisniewski, 1989). Chalutz and Wilson (1990) found that washed, dried and stored citrus
fruit get infected much more rapidly than unwashed fruit. This proves that the resident
epiphytic microflora on the citrus fruit is capable of controlling citrus diseases. Several
antagonistic microorganisms, found to be effective to control citrus diseases in postharvest
phase, are isolated from the surface of citrus fruit (Wilson and Chalutz, 1989; Borras and
Aguilar, 1990; Chalutz and Wilson, 1990; Droby et al., 1999a; Taqarort et al., 2008; Abraham
et al., 2010; Osman et al., 2011). According to their origin, the naturally occurring antagonists
are more apt to gain public acceptance.

IV.1.2 Artificially introduced microbial antagonists

Several microbial antagonists have been identified and artificially introduced on citrus
fruit to control the postharvest diseases. Artificial introduction of microbial antagonists is
more effective in controlling postharvest diseases of fruits than other means of biological
control (Sharma et al., 2009). This success is due to the possibility of controlling the
environmental conditions in the postharvest phase and to direct application of biocontrol
agents toward effective sites. Currently, available microbial antagonists were developed for
the control of decays originating mainly from active infection of fruit wounds and not from
latent infections. Postharvest citrus diseases are controlled by a broad spectrum of
microorganisms such as yeasts, bacteria and fungi (Table 1.3).

IV.1.2.1 Yeasts as biocontrol agents

Treatment of citrus fruit by antagonistic yeasts is the most means used as alternative to
control postharvest diseases. This importance is attributed to several positive points making
yeasts recommended as potential microbial agents for controlling the postharvest diseases of
fruit. First, yeasts can colonize the surface of fruit for long period even under dry conditions
(Janisiewicz, 1987). Second, yeasts produce extra-cellular polysaccharides, which enhance
their survivability and restrict the growth of pathogen propagules. Third, yeasts can use

16
 Chapter 1: Literature review

nutrients rapidly and proliferate at a faster rate (Sharma et al., 2009). Effective control of
citrus fruit decay was observed with yeasts such as Candida guilliermondii (syn. : Pichia
guilliermondii, Debaryomyces hansenii), Pichia anomala, Candida saitoana, Candida
famata, Candida oleophila, Candida sake, Aureobasidium pullulans and Kloeckera apiculata
(Wilson and Chalutz, 1989; Chalutz and Wilson, 1990; Arras, 1996; Arras et al., 1998; El-
Ghaouth et al., 2000; Droby et al., 2002; Lahlali et al., 2004; Taqarort et al., 2008) (Table
1.3). Actually, at least three products based on antagonistic yeasts are commercially available
under the trade names Aspire (Ecogen Inc., USA, limited to the USA), Yield Plus (Anchor
Yeast, Cape Town, South Africa) and Shemer (AgroGreen Ashdod, registred in Palestine)
(Droby et al., 2002). A few others are at different stages of commercial development and
expected to reach the marketplace within future years. However, application of these
biocontrol products alone did not provide commercially acceptable control of fruit diseases.
The biocontrol ability of these antagonists could be enhanced by manipulation of the
environment, using mixtures of beneficial organisms, physiological and genetic enhancement
of the biocontrol mechanisms and integration of biocontrol with other methods such as low
doses of fungicides and controlled atmosphere storage (Spotts et al., 2002).

IV.1.2.2 Bacteria as biocontrol agents

Use of bacteria for postharvest diseases control of citrus has focused on their application
as biofungicides. Endophytic bacteria are ubiquitous in most plant species and can be isolated
from surface plant tissues, soils, roots, and the rhizosphere of various plants (Lai et al., 2012).
Moreover, bacteria as biocontrol agents have become of interest because of their rhizosphere
competence and their ability to colonize internal tissues of plants and thus, represent an
internal defense line against pathogens (Liu et al., 2009a). Antagonistic bacteria are well
known by their production of substances with antifungal and antibacterial properties
(Smilanick and Denis-Arrue, 1992; Leelasuphakul et al., 2008; Lucon et al., 2010; Yanez-
Mendizabal et al., 2011). Significant advances in control of postharvest citrus diseases have
been achieved in research by the use of bacterial antagonists such as Pseudomonas syringae,
P. fluorescens, Burkolderia (Pseudomonas) cepacia, Bacillus subtilis, B. thuringiensis,
Pantoea agglomerans, Enterobacter cloacae and Serratia polymuthica (Singh and Deverall,
1984; Wilson et al., 1987; Smilanick and Denis-Arrue, 1992; Bull et al., 1998; Meziane et al.,
2006; Canamas et al., 2008; Lucon et al., 2010) (Table 1.3). However, only one species of
bacteria have been mass-produced and commercialized, under the trade name BioSave 100
and 110 based on a strain of Pseudomonas syringae, to control postharvest citrus pathogens

17
 Chapter 1: Literature review

Although, antibiosis might be an effective tool for controlling postharvest diseases, the
importance is being given for the development of non-antibiotic producing microbial
antagonists for the control of postharvest diseases of fruits (El-Ghaouth et al., 2004).

IV.1.2.3 Fungi as biocontrol agents

Biological control of postharvest citrus diseases by fungal antagonists is less developed

compared with yeasts and bacteria. However, the most known antagonistic fungi like
Muscodor albus and Homoptera parasite are also tested on postharvest citrus diseases and
have shown a reduction of fruit decay (Borras and Aguilar, 1990; Benhamou, 2004; Mercier
and Smilanick, 2005) (Table 1.3). Antagonistic fungi exhibit broad spectrum in terms of
disease control and volatile antimicrobial compounds (Kiss, 2003; Ezra et al., 2004; Mercier
and Smilanick, 2005; Verma et al., 2007). The fungus Muscodor albus, a biofumigant that
produces certain low molecular weight volatiles, has been used to fumigate whole rooms of
lemons to control pathogens during storage. It is reported to be effective on green mold and
sour rot (Mercier and Smilanick, 2005). This fungus was reported to produce 28 organic
volatile compounds that show some inhibitory effect against pathogenic fungi and bacteria
(Strobel et al., 2001). Numerous species of Trichoderma have been widely used as
antagonistic fungal agents against several postharvest diseases (Okigbo and Ikediugwu, 2000;
Batta, 2004). The antagonism of these fungi is the result of its richness of antimicrobial
metabolites and physiological conformation (Verma et al., 2007).

IV.1.3 Mode of action of antagonistic microorganisms

Understanding the mode of action of antagonists allow to develop more reliable

procedures for the effective application of known antagonists and to provide a basis to select
more effective and desirable antagonists or strains of antagonists (Wisniewski and Wilson,
1992). Although considerable amount of research have been reviewed on the use of the
microbial antagonists, only few attempts have been made to study microbial interactions in
wounds of fruit. Probably, because of the difficulty in studying the complex interactions
occurring between the pathogen, antagonist, host, and possibly other microorganisms present
on the fruit surface. Several mechanisms, operating alone or in concert, are involved in
antagonistic interactions in the fructoplan. Nutrients and space competition, antibiosis and
parasitism are the major mechanisms. Additional mechanisms such as induced resistance,
interference with pathogen-related enzymes, and undoubtedly a number of still unknown
mechanisms may complete the microbial arsenal (Sharma et al., 2009). Often, more than one

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 Chapter 1: Literature review

mechanism was implicated, but in no case has a sole mechanism been found responsible for
biological control (Janisiewicz and Korsten, 2002).

IV.1.3.1 Antibiosis

Antibiotic production is one of the major modes of action of antagonists. This mechanism
was more found in bacteria than in yeasts and in filamentous fungi. Bull et al., (1998)
demonstrated that Syringomycin produced by Pseudomonas syringae controlled green mold
on lemons and inhibited the growth of G. candidum, P. digitatum and P. italicum. Similarly,
Pseudomonas cepacia was also found to be effective in controlling green mold in lemon by
producing antibiotics (Smilanick and Denis-Arrue, 1992). Moreover, Bacillus subtilis and its
antibiotics are considered to be potent biological control agents to suppress growth of P.
digitatum in the postharvest protection of citrus (Leelasuphakul et al., 2008). Pseudomonas
corrugate, Pseudomonas fluorescens, Bacillus thuringiensis, Aureobasidium pullulans and
Pichia pastoris were also found to control citrus postharvest diseases by production of
antibiotics (Smilanick and Denis-Arrue, 1992; Liu et al., 2007; Lucon et al., 2010; Ren et al.,
2011). However, the production of these antibiotics wasn‟t generally detected on the fruit;
raising a doubt on the role of the antibiosis in postharvest diseases control which explains the
fact that antibiosis may not comprise the entire mode of action of antagonists on citrus (Bull
et al., 1997). Although, antibiosis might be an effective tool for controlling postharvest
diseases by antagonistic bacteria, the importance is being given for the development of non-
antibiotic producing microbial antagonists for the control of postharvest diseases of fruits. The
main concern, related to the use of antibiotics into our food, is the adverse effect on the
resistance of human‟s pathogens to antibiotics, because of the possible residue left in the fruit,
and the possible development of resistance in fruit pathogens (El-Ghaouth et al., 2004).
Further search was directed to identify antagonists that did not produce antibiotics.

IV.1.3.2 Competition for nutrients and space

The nutrient sources in the peel of citrus fruit are frequently not sufficient for all
microorganisms, which makes the competition between pathogen and non-pathogens for
nutrient resources or site an important issue in Biocontrol. Many investigations on different
biocontrol systems have concluded that the successful competition of microbial antagonists
with the pathogens infecting fruits for nutrients and space may be the possible mechanism of
biocontrol (Droby et al., 1989; Wilson and Wisniewski, 1989; Wisniewski et al., 1989;
Mercier and Wilson, 1994; Arras et al., 1998). Indeed, many successful microbial antagonists

19
 Chapter 1: Literature review

that do not produce antibiotics are able to grow rapidly at the wound sites and are better
adapted to extreme nutritional and environmental conditions compared with postharvest
pathogens. The non-pathogenic microorganisms (especially yeasts) protect the surface of
citrus fruits by rapid colonization of wounds and thereby exhausting the limited available
substrates so that none are available for pathogen to grow. Results of study by Liu et al.
(2010) revealed that Cryptococcus laurentii colonized citrus wounds in a short time and
effectively exploit the available endogenous nutrient resources. Also Pichia guilliermondii,
Candida saitoana, Rhodotorula glutinis, Rhodosporidium paludigenum, Kloeckera apiculata,
Hanseniaspora guilliermondii and Metschnikowia andauensis were reported to compete citrus
postharvest pathogens for nutrients and space (Arras et al., 1998; El-Ghaouth et al., 2000;
Zheng et al., 2005; Long et al., 2006; Taqarort et al., 2008; Liu et al., 2010; Manso and
Nunes, 2011) (Table 1.3). The competition for nutrients and space is favored by the
attachment capability of antagonistic yeasts to pathogen hyphae. The attachment may enhance
nutrient competition as well as interfere with the ability of the pathogen to initiate infection
(El Ghaouth et al., 2002). The attachment of antagonists to pathogen hyphae has been
suggested as an important factor in competition for nutrients between the antagonistic yeast
Pichia guilliermondii and P. italicum on citrus fruit (Arras et al., 1998). Currently, there is
only fragmented data regarding the antagonist-pathogen interaction in terms of competitions
for limiting nutrient essential for pathogenesis. Once more information regarding the
specificity of competition between antagonistic and pathogens in fruit wounds is available and
genes responses of antagonism of biocontrol agents have been characterized, it will be
possible to develop antagonistic strains with a higher rate of transport and/or metabolism of
limiting nutrient essential for pathogenesis (El-Ghaouth et al., 2004).

IV.1.3.3 Direct parasitism

In this case, pathogen is directly attacked by a specific microbial antagonist that kills it
or its propagules. Parasitism depends on close contact and recognition between antagonist and
pathogen, on the secretion of lytic enzymes, and on the active growth of the parasite into the
host (El-Ghatouh et al., 2002). It is often referred to as hyperparasitism or mycoparasitism
when interactions involve a fungus. In the literature, very little information is available on the
role of direct parasitism of the microbial antagonists in controlling postharvest pathogens of
citrus fruit. Arras et al. (1998) showed that Pichia guilliermondii cells had the ability to attach
to the hyphae of Penicillium italicum. Also, the antagonistic yeast Candida saitoana was
found to be able to attack P. italicum by direct parasitism (El-Ghaouth et al., 2000).

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 Chapter 1: Literature review

IV.1.3.4 Induced resistance

Additional mode of action, such as induced resistance, has also been suggested and
several microbial antagonists were shown to induce a wide range of defense responses in
citrus fruit (Borras and Aguilar, 1990; Rodov et al., 1994; Arras, 1996; Droby et al., 2002;
Benhamou, 2004; Lucon et al., 2010). Droby et al. (2002) reported that the application of
Candida oleophila to surface wounds of grapefruit elicited systemic resistance against P.
digitatum. They also demonstrated that the induction of pathogen resistance required viable
yeast cells. Rodov et al. (1994) reported that Pichia guilliermondii induced resistance to green
mold by eliciting the production of phytoalexins (e.g. scoparone and scopoletin). Similarly,
Arras (1996) showed that scoparone accumulation could be 19 times higher when the
antagonist C. famata was inoculated 24 h prior to P. digitatum, and only four times higher if
inoculated 24 h after the pathogen. The accumulation of the phytoalexin scoparone was
correlated with increased antifungal activity in the flavedo, and resulted in enhanced
resistance of the fruit to infection by Penicillium digitatum. Although a causal connection
between the accumulation of the host defense responses and bioprotection by microbial
antagonists has not yet been clearly established, the occurrence of high levels of host
antifungal compounds in protected tissue suggests their implication in disease resistance (El
Ghaouth et al., 2004).

Table 1.3 : Microbial antagonists used for the successful control of citrus postharvest diseases and
their mode of action on citrus fruits
Microbial antagonist Pathogen Mode of action Reference

Bacteria :
Antibiosis, Competition for nutrient Bull et al., 1998
Pseudomonas syringae P. digitatum Wilson and Chalutz ,1989
and space
Penicillium spp ND Wilson and Chalutz , 1989
Antibiosis, Competition for nutrient Smilanick and Denis-
Pseudomonas cepacia P. digitatum Arrue, 1992
and space
Penicillium spp Antibiosis Wilson and Chalutz, 1989
Competition for nutrient and space,
Pseudomonas glathei P. digitatum Huang et al., 1995
Induction of resistance
Pseudomonas Antibiosis and competition for Smilanick and Denis-
P. digitatum Arrue, 1992
corrugata nutrients and space

Pseudomonas Antibiosis and competition for Smilanick and Denis-

P. digitatum Arrue, 1992
fluorescens nutrients and space

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 Chapter 1: Literature review

Paenibacillus Antibiosis and competition for

P. digitatum Lai et al., 2012
polymyxa nutrients and space
Entrobacter cloacae P. digitatum Competition for nutrient and space Wilson and Chalutz, 1989

P. digitatum Antibiosis Singh and Deverall, 1984

P. digitatum Antibiosis Leelasuphakul et al., 2008
Bacillus subtilis
P. digitatum Yanez-Mendizabal et al.,
Antibiosis 2011
G. candidum
Bacillus
P. crustosum Production of volatile compounds Arrebola et al., 2010
amyloliquefaciens
Guignardia
Bacillus thuringiensis Antibiosis, Induction of resistance Lucon et al., 2010
citricarpa
Bacillus pumilus P. digitatum ND Huang et al., 1992

Antibiosis and competition for

Serratia plymuthica Penicillium spp Meziane et al., 2006
nutrients
Penicillium spp ND Canamas et al., 2008
Pantoea agglomerans Triggers H2O2 production
P. digitatum Torres et al., 2011
Triggers enzymatic activities

Fungi :

Galactomyces citri-
P. digitatum Antibiosis Eayre et al., 2003
aurantii (avirulent)
G. candidum Mercier and Smilanick,
Muscodor albus Production of volatile compounds 2005
P. digitatum
Aureobasidium
P. digitatum Antibiosis Liu, et al., 2007
pullulans

Verticillium lecanii P. digitatum Induction of resistance Benhamou, 2004

Yeasts :

Competition for nutrient and space,

P. italicum Arras et al., 1998
Directly parasitizing the pathogen
Pichia guilliermondii P. digitatum Induction of resistance Rodov et al., 1994
P. italicum ND Lahlali et al., 2010
Penicillium spp ND Lahlali et al., 2004
Pichia anomala
P. digitatum Competition for nutrient and space Taqarort et al., 2008

Pichia pastoris G. candidum Antibiosis Ren et al., 2011

P. membranefaciens Penicillium spp induction of resistance Luo et al., 2012

Competition for nutrient and space,

Candida saitoana P. italicum El-Ghaouth et al., 2000
direct parasitism
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 Chapter 1: Literature review

Candida famata P. digitatum Induction of resistance Arras 1996

Candida guilliermondii P. digitatum Induction of resistance Arras 1996

P. digitatum Induction of resistance Droby et al., 2002

Candida oleophila
Penicillium spp ND Lahlali et al., 2004

Candida sake P. digitatum ND Droby et al., 1999a

Rhodotorula glutinis P. digitatum Competition for nutrient and space Zheng et al., 2005

Rhodosporidium
G. candidum Competition for nutrient and space Liu et al., 2010
paludigenum

Saccharomycopsis
schoenii Penicillium spp ND Pimenta et al., 2008

Debaryomyces
P. italicum Droby et al., 1989
hansenii Competition for nutrient and space
P. italicum Hernández-Montiel et
al., 2010
Penicillium spp Competition for nutrient and space Wilson and Chalutz
,1989
Penicillium spp ND Chalutz and Wilson,
1990
G. candidum ND
P. digitatum Competition for nutrient and space Taqarort et al., 2008
P. italicum Competition for nutrient and space
Cryptococcus laurentii Liu et al., 2010
G. candidum
Aureobasidium Competition for nutrient and space Wilson and Chalutz ,
Penicillium spp 1989
pullulans
Kloeckera apiculata P. italicum Competition for nutrient and space Long et al., 2006

Hanseniaspora
P. digitatum Competition for nutrient and space Taqarort et al., 2008
guilliermondii
Metschnikowia
Penicillium spp Competition for nutrient and space Manso and nunes , 2011
andauensis
Wickerhamomyces
P. digitatum Antibiosis Platania et al., 2012
anomalus
ND : No determined

IV.1.4 Application methods of microbial antagonists

Infection of citrus fruit by pathogens can occur in the field prior to harvest, and it could
be advantageous starting control at this point, as well as in the postharvest phase. So,
microbial antagonists can be applied by two ways : preharvest and postharvest application. An
important consideration for the application of antagonists at preharvest is their ability to

23
 Chapter 1: Literature review

colonize the surface of fruit both in the field and during storage and to persist, for as long as
possible, in sufficient numbers on the fruit surface to maintain an efficient decay control
(Wisniewski and Wilson, 1992). Furthermore, to be successful in preharvest application,
potential antagonists should be able to tolerate low-nutrient availability, UV rays, high
temperature and dry conditions (Spadaro and Gullino, 2004). A large number of studies
showed that several microbial antagonists are able to inhibit effectively postharvest diseases
in citrus fruit. For example, preharvest application of the yeast Pichia guilliermondii was
effective and controlled green mold (Droby et al., 1992). Similarly, preharvest application of
Pantoea agglomerans provided an effective control for orange fruit against natural
postharvest pathogen infections and artificial infections of Penicillium digitatum during
storage (Canamas et al., 2008). However, according to Sharma et al. (2009), this approach has
still many limitations and did not provide commercially acceptable control of fruit diseases.

Unlike preharvest application, postharvest application of microbial antagonists is the

most used and most practical method for controlling postharvest diseases of citrus fruit
(Sharma et al., 2009). In this case, microbial antagonists are applied either as postharvest
spray, dip or drench applications (El-Ghaouth et al., 2001; Mercier and Smilanick, 2005;
Canamas et al., 2008; Usall et al., 2008; Lahlali et al., 2010; Arrebola et al., 2010). An
unusual case is the control of citrus Penicillium decay by biofumigation with volatile
compounds produced by grain cultures of the fungus Muscodor albus (Mercier and Smilanick,
2005).

Postharvest application of Pseudomonas syringae, Pseudomonas cepacia, Bacillus

subtilis, Trichoderma viride and Debaryomyces hansenii resulted in control of Penicillium
digitatum in citrus fruit (Singh and Deverall, 1984; Wilson and Chalutz, 1989; Borras and
Aguilar, 1990; Smilanick and Denis-Arrue, 1992; Bull et al., 1998). A significant reduction in
storage decay of citrus, caused by P. digitatum, P, italicum and Geotrichum candidum, was
achieved by bringing several yeast species in direct contact with wounds in the peel of
harvested fruit (Chalutz and Wilson, 1990).

IV.1.5 Criteria for selecting a good antagonist

Few of the antagonists reported to control plant pathogens are successfully transferred
from the laboratory into the commercial conditions. In the case of citrus fruits, among all
tested antagonists on the control of postharvest decay, only three antagonists have been
patented and evaluated for commercial use. Aspire (Candida oleophila strain 182; Ecogen

24
 Chapter 1: Literature review

Inc., Langhorne, PA), Biosave (Pseudomonas syringae strain 10LP, 110, Eco Science
Corporation, USA) and Yield Plus (Cryptococcus albidus : Anchor Yeast, Cape Town, South
Africa) are available for the control of postharvest pathogens of citrus fruits (Droby et al.,
1998; Palou et al., 2008). This shows the difficulty of marketing Biocontrol which must
satisfy several conditions to be accepted for use in commercial conditions. For Biocontrol to
be successful, it would be : (a) genetically stabile; (b) effective at low concentrations; (c) not
fastidious in its nutrient requirements; (d) able to survive well under adverse environmental
conditions (including storage environments); (e) efficacious against a wide range of pathogens
on a variety of fruit and vegetables; (f) amenable to growth on an inexpensive medium in
fermenters; (g) preparable in a form that can be effectively stored and dispensed; (r) non-
productive of secondary metabolites that may be deleterious to humans : (i) resistant to
pesticides; (j) compatible with other chemical and physical treatment of the commodity; and
(k) non-pathogenic against the host (Wilson and Wisniewski, 1989). Additional criteria may
include the ability to colonize wounded and sound fruit surfaces under various conditions,
utilization of substrates occurring in fruit and resistance to synthetic fungicides applied
postharvest to control decay. Also the conditions that favor a potential antagonist should be
the same or similar to those that favors the pathogen (Janisiewicz and Korsten, 2002).
Nevertheless, Even if an antagonist is found with these desirable characteristics, economic
factors dictate whether it will be commercialized. If the potential market for the product is not
large enough to generate a profit, then it cannot be commercialized which presents difficulty
with antagonists for postharvest diseases of citrus fruits. However, the continuous withdrawal
of fungicides used to control postharvest diseases and the growing public concern about
chemical residues in fruit may encourage investing in the alternative of use of microbial
antagonists.

IV.1.6 Use of antagonist mixtures

In the enhancement of a biocontrol system, work could focus on a promising
approach, which is the development of antagonist mixtures. An effective biological control
based upon a mixture of several complementary and non-competitive antagonists is more
likely than a control based upon microorganism alone. Such mixtures have several
advantages (Janisiewicz, 1988) : apart from a wider spectrum of activity (different fruits,
cultivars and ripening stages), they can increase the efficacy (less biomass necessary), be
more reliable and allow a reduction in application times and treatment costs. Moreover, they
permit the combination of different genetic characteristics, minimising the need for genetic

25
 Chapter 1: Literature review

engineering. Although the use of antagonistic mixtures offers more effective control, the
economic viability of this approach appears to be a major obstacle for its adoption, as
registration of two microbial antagonists will cause additional burden for the industry
(Sharma et al., 2009). Most of the work relates to grape and apples, with limited studies on
other fruits, including citrus fruit. The enhancement of efficiency of microbial antagonists
may be due to : better utilization of substrate, resulting in acceleration of the growth rate;
removal of substances inhibitory to one organism by the other microbial antagonists;
production by one microbe of nutrients that may be used by another; and formation of more
stable microbial community that may exclude other microbes, including pathogens
(Janisiewicz, 1998).
IV.2 Biological control of citrus diseases with natural plant products

With consumer trends for natural alternatives to chemical-based fungicides and changes
in legislation, the use of natural products such as plant extracts may provide a solution for
both industry and consumers. Recently, attention has been paid towards the exploitation of
higher plant products as novel botanical fungicides in citrus diseases management. Indeed,
higher plants contain a wide spectrum of secondary metabolites, such as phenolics,
flavonoids, quinones, tannins, essential oils, alkaloids, saponins, sterols, with different
biological properties (Tripathi and Dubey 2003; Tayel et al., 2009). More than 1340 plant
species are known to be potential sources of antimicrobial compounds and about 10,000
secondary plant metabolites have been chemically defined for their role as antimicrobials
(Cowan, 1999; Tripathi and Dubey 2003). Plant extracts have the advantage of being
biodegradable, not phytotoxic, are generally regarded as safe to mammals (GRAS) by the
United States Food and Drug Administration (Ware and Whitacre, 2004), and are thought to
have novel modes of action (Regnault-Roger et al., 2005). Therefore, higher plants can be
exploited for the discovery of new natural fungicides which can replace the synthetic ones.
Some phytochemicals of plant origin have been formulated as botanical pesticides and are
used successfully in integrated pest management programs (Tripathi et al., 2004). In the
available literature, research on biological control of postharvest diseases of citrus fruit by
natural plant products focused mainly on the use of essential oils, volatile compounds,
aqueous and organic solvent extracts and other products extracted from plants (Agnioni et al.,
1998; Droby et al., 1999b; Arras and Usai, 2001; Du Plooy et al., 2009; Liu et al., 2009b;
Gatto et al., 2011; Talibi et al., 2012a; Talibi et al., 2012b).

26
 Chapter 1: Literature review

IV.2.1 Use of essential oils

Essential oils are natural, volatile, complex compounds known for their antimicrobial,
antioxidant and medicinal properties (Bakkali et al., 2008). Essential oils are mostly a mixture
of many different volatile compounds, their composition often varies between plant species
(Mishra and Dubey, 1994) and exert differential effects depending on both the mode of action
and the target organism (Liu et al., 2006). The development of resistant strains of fungi
against essential oils may not become a real issue as it is the case for many synthetic
fungicides because several active components are often present in the final product and
synergistic interactions may exist between the different components of the oils (Tripathi and
Dubey 2003; Tripathi et al., 2004; Dubey et al., 2006;). Furthermore, most of the components
of essential oils seem to have no specific cellular targets (Carson et al., 2002). The volatility,
ephemeral nature and biodegradability of essential oils compounds may be especially
advantageous for treatment of postharvest citrus diseases because only low residues can be
expected. These characteristics make them particularly suitable for application in their vapor
phase by a new process defined as „biofumigation‟. Recently, packing systems that deliver
volatile antimicrobials to the packaged product, known as „antimicrobial active packaging‟,
have been evaluated (Ayala-Zavala et al., 2008).
Plaza et al. (2004a) reported that Thyme and cinnamon essential oils significantly
reduced the incidence of green and blue molds of citrus. Also, thyme oil was reported to
control most postharvest citrus rots, such as green mold, bleu mold and sour rot (Arras and
Usai, 2001; Liu et al., 2009b). Many studies (Table 1.4) have documented the antifungal
effects of plant essential oils against citrus fruit pathogens (Chebli et al., 2003; Tripathi et al.,
2004; Alilou et al., 2008; du Plooy et al., 2009; Solaimani, et al., 2009; Badawy et al., 2011).
Treatment of oranges with the essential oils of Mentha arvensis, Ocimum canum and Zingiber
officinale has been found to control blue mold, thereby enhancing shelf life of fruit (Tripathi
and Dubey 2003). Dixit et al. (1995) demonstrated the efficacy of Ageratum conyzoides
essential oils for the protection of mandarins against blue mold. Essential oils of thyme,
oregano, cinnamon, clove, dictamus or mint were very effective in vitro against P. digitatum
and P. italicum, but results from in vivo experiments were contradictory and applications to
citrus fruit were often ineffective or phytotoxic (Arras and Usai, 2001; Tripathi et al., 2004).
Application of Mentha oil (Mentha arvensis) reduced blue mold caused by P. italicum in
orange and lime and enhanced the shelf life by 6 to 8 days (Tripathi et al., 2004). The oil from
Thymus capitatus exhibited strong fungicidal activity against Penicillium digitatum, P.

27
 Chapter 1: Literature review

italicum, and Alternaria citri in vitro at 250ppm. The fungitoxicity of T. capitatus essential oil
sprayed on healthy orange inoculated with P. digitatum was weak at atmospheric pressure, but
in vacuum conditions conidial mortality on fruit exocarp was high (90–97%). The efficacy of
this essential oil was comparable to that of thiabendazole (TBZ) at 2000 ppm concentration.
Carvacrol was found to be the predominant compound, accounting for 81 to 83% of essential
oil vapors present in T. capitatus and exhibited fungitoxicity (Arras and Usai, 2001). The
antifungal activity of the essential oils suggests that they may be considered as a potential
alternative to the synthetic fungicides for the control of postharvest citrus pathogens.
However, despite their potent antifungal activity, commercial implementation of treatments
with essential oils is strongly restricted in citrus because of problems related to potential
phytotoxicity, intense sensory attributes or technological application as fumigants or in
aqueous solutions (Palou et al., 2008).

IV.2.2 Use of crude plant extracts

The preservative nature of some plant extracts has been known for centuries and there
has been renewed interest in the antimicrobial properties of extracts from aromatic plants. In
recent years, several studies have been focused on screening of plant extracts to develop new
antifungal compounds that can be used to control postharvest citrus diseases. Aqueous or
organic solvent extracts of plants from different origins are sources of antifungal activity
against citrus postharvest pathogens under different experimental conditions (Obagwu and
Korsten, 2003; Abd-El-Khair and Hafez, 2006; Ameziane et al., 2007; Mekbib et al., 2007;
Gatto et al., 2011; Talibi et al., 2011a; Talibi et al., 2012a; Talibi et al., 2012b) (Table 1.4).
Talibi et al. (2012a) reported that aqueous extracts of Cistus villosus, Halimium
antiatlanticum and Ceratonia siliqua reduced significantly the incidence and severity of citrus
sour rot. Moreover, the aqueous extract of Halimium umbellatum and Inula viscosa were
found to be effective against citrus blue mold caused by P. italicum (Askarne et al., 2012).
The aqueous extract of Acacia nilotica has also shown pronounced antifungal activity against
P. italicum and enhanced the storage life of oranges for 6 days (Tripathi and Dubey, 2004).
Aqueous extracts from plants can both inhibit and stimulate fungal growth of postharvest
citrus pathogens (Ameziane et al., 2007; Talibi et al., 2012a).

Besides the aqueous extracts, organic solvent extracts of several plants were tested against
citrus pathogens. Treatment of mandarin fruit by methanol extracts of Cistus villosus,
Halimium umbellatum and Ceratonia siliqua successfully controlled the citrus sour rot (Talibi

28
 Chapter 1: Literature review

et al., 2012b). Also of interest, methanol extracts from Sanguisorba minor showed good
control of green mold (Gatto et al., 2011). Tayel et al. (2009) showed that methanol and
ethanol extracts of Punica granatum controlled citrus green mold and the antifungal activity
of these extracts were higher than that of standard Imazalil. Also Mekbib et al. (2007)
reported that wound inoculated fruit with P. digitatum and methanolic extract of Withania
somnifera and Acacia seyal did not develop decay symptoms for up to 21 days of storage at
25°C. Ameziane et al. (2007) showed that methanol extract of Cistus villosus is more active
against P. digitatum and G. candidum than the chloroformic extracts. Thus, the nature of
extraction influence on the antifungal activity of plants tested. This difference in biological
activity is due to the polarity of each solvent, i.e. the nature of the molecules extracted with
each solvent. Methanol is a polar solvent which can extract several compounds with
antimicrobial activities like alkaloids (Satish et al., 2008), triterpene glycoside (Ismail et al.,
2008), tannins (Askun et al., 2009), sesquiterpene lactones (Choi et al., 2004) and phenolic
compounds (Nicholson and Hammerschmidt, 1992; Tripathi and Dubey, 2004; Sisti et al.,
2008; Martini et al. 2009). Likewise, Askun et al. (2009) reported that Methanol provided
more consistent antimicrobial activity. The potential use of crude plant extracts to control
postharvest citrus diseases requires a detailed examination of their biological activity and
dispersion in fruit tissues and the development of a formula which inhibits growth of
pathogens without producing phytotoxic effects on fruit.

Table 1.4 : Plant extracts used for the control of citrus postharvest diseases

Plant specie Plant extract Pathogen Reference

Cymbopogon sp. Aqueous extract P. digitatum Abd-El-Khair and Hafez 2006

Lantana sp. Aqueous extract P. digitatum Abd-El-Khair and Hafez 2006

Eucalyptus sp. Aqueous extract P. digitatum Abd-El-Khair and Hafez 2006

P. digitatum
Sanguisorba minor Methanol extract Gatto et al., 2011
P. italicum
[Link]
Borago officinalis Methanol extract Gatto et al., 2011
[Link]
[Link]
Sonchus oleraceus Methanol extract Gatto et al., 2011
[Link]
Thymus sp Essential oil G. candidum Liu et al., 2009b

Thymus capitatus Essential oil [Link], Arras and Usai , 2001

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 Chapter 1: Literature review

[Link]
Thymus vulgaris Essential oil Fatemi et al., 2011
[Link]
A. citri
[Link]
Zataria multiflora Essential oil Solaimani et al., 2009
[Link]
G. candidum
Chrysanthemum Essential oil Chebli et al., 2003
P. digitatum,
Aqueous extract Talibi et al., 2012a
Cistus villosus [Link] Talibi et al, 2012b
Methanol extract
Halimium Aqueous extract Talibi et al., 2012a
[Link] Talibi et al, 2012b
antiatlanticum Methanol extract
[Link] Talibi et al., 2012a
Aqueous extract Askarne et al., 2012
Halimium umbellatum [Link]
Methanol extract [Link] Talibi et al., 2012b
Ceratonia siliqua Aqueous extract Talibi et al., 2012a
[Link] Talibi et al, 2012b
Methanol extract
Pistacia atlantica Aqueous extract Talibi et al., 2012a
[Link] Talibi et al, 2012b
Methanol extract
[Link] Talibi et al., 2012a
Aqueous extract Askarne et al., 2012
Inula viscosa P. italicum
Methanol extract G. candidum Talibi et al., 2012b

Mentha spicata Essential oil [Link] Du Plooy et al., 2009

Lippia scaberrima Essential oil [Link] Du Plooy et al., 2009

Aqueous and
Allium sativum [Link] Obagwu and Korsten, 2003
ethanol extracts
Mentha arvensis Essential oil P. italicum Tripathi et al., 2004

Zingiber officinale Essential oil P. italicum Tripathi et al., 2004

Ocimum canum Essential oil P. italicum Tripathi et al., 2004

Acacia nilotica Aqueous extract P. italicum Tripathi et al., 2002

Anvillea radiata Aqueous extract P. italicum Askarne et al., 2012

Punica granatum Methanol extract [Link] Tayel et al., 2009

Aloe barbadensi Gel [Link] Saks and Barkai-Golan, 1995

Withania somnifera Methanol extract [Link] Mekbib et al., 2007

Acacia seyal Methanol extract [Link] Mekbib et al., 2007

Bubonium imbricatum Essential oil [Link] Alilou et al., 2008

30
 Chapter 1: Literature review

Citrus sp. Essential oil [Link] Badawy et al., 2011

Ageratum conyzoides Essential oil [Link] Dixit et al., 1995

Simmondsia chinensis oil emulsion [Link] Ahmed et al., 2007

Foeniculum vulgare Essential oil [Link] El-Tobgy et al., 2010

Mentha piperita Essential oil [Link] El-Tobgy, 2010

Carum carvum Essential oil [Link] El-Tobgy, 2010

IV.2.3 Use of natural products extracted from plants

Higher plants contain a wide spectrum of secondary substances like as phenols,

flavonoids, quinones, tannins, essential oils, alkaloids, saponins and sterols (Tripathi et al.,
2004). Among the numerous natural plant products with potential antimicrobial activity are :
acetaldehyde, benzaldehyde, benzyl alcohol, ethanol, methyl salicylate, ethyl benzoate, ethyl
formate, hexanal, (E)-2-hexenal, lipoxygenases, jasmonates, allicin, glucosinolates and
isothiocyanates, etc (Utama et al., 2002; Tripathi and Dubey, 2003; Palou et al., 2008). Utama
et al. (2002) demonstrated the efficacy of acetaldehyde, benzaldehyde, cinnamaldehyde,
ethanol, benzyl alcohol, nerolidol and 2-nonanone as volatile fungitoxicants for the protection
of citrus fruit against P. digitatum. Citral was reported to inhibit mycelial growth and spore
germination of P. digitatum (Klieber et al., 2002). Also Citral has been highlighted as an
active compound in citrus fruit against decay caused by Penicillium digitatum (Fisher and
Phillips, 2008). Production of citral in the flavedo of citrus fruit has been described as a
preformed defense mechanism against infection by P. digitatum (Rodov et al., 1995).
Jasmonates (jasmonic acid and methyl jasmonate) has been found to be effective in
postharvest control of P. digitatum either after natural or artificial inoculation of grapefruit
(Droby et al., 1999b). Exposure of fruit to jasmonates also effectively reduced chilling injury
incidence after cold storage (Droby et al., 1999b). Since they are naturally occurring
compounds and are given in low doses, jasmonates may provide a more environment-friendly
means of reducing the current chemical usage. A naturally occurring compound isolated from
the flavedo tissue of grapefruit (Citrus paradisi) identified as 7-geranoxy coumarin exhibited
antifungal activity against P. italicum and P. digitatum during in vitro and in vivo tests
(Agnioni et al., 1998) (Table 1.5). Also of interest, Smilanick et al. (1995) showed that a
brief immersion of citrus fruit in a solution containing ethanol (10 % wt/vol) reduced green
mold incidence without significant injury to the fruit.

31
 Chapter 1: Literature review

Table 1.5 : Natural compounds tested against citrus postharvest pathogens

Compound Causal agent Reference

Benzaldehyde P. digitatum Wilson and Wisniewski, 1989

Cinnamaldehyde P. digitatum Wilson and Wisniewski, 1989

Acetaldehyde vapor P. digitatum Prasad and Stadelbacher, 1973

Heptanol G. candidum Suprapta et al., 1997

Octanol G. candidum Suprapta et al., 1997

Nonanol G. candidum Suprapta et al., 1997

Decanol G. candidum Suprapta et al., 1997

Geraniol G. candidum Suprapta et al., 1997

Citronellol G. candidum Suprapta et al., 1997

Suprapta et al., 1997

G. candidum Klieber et al., 2002
Citral
P. digitatum Fisher and Phillips, 2008

Thymol P. digitatum Jafarpour and Fatemi, 2012

Menthol P. digitatum Jafarpour and Fatemi, 2012

7-geranoxy coumarin P. digitatum

Agnioni et al., 1998
P. italicum
Jasmonic acid P. digitatum Droby et al., 1999b

Methyl jasmonate P. digitatum Droby et al., 1999b

Nerolidol P. digitatum Droby et al., 1999b

2-nonanone P. digitatum Droby et al., 1999b

Kaempferol P. italicum Tripathi et al., 2002

IV.2.4 Mode of action of plant extracts

As the exploitation of natural plant products to protect the postharvest decay of citrus
fruit is in its infancy, there is little information about their mechanism of action. Nevertheless,
few attempts have been done to explain these modes of action. Considering the large number
of bioactive chemical present in plant extracts, it is most likely that their antimicrobial activity
is not attributable to one specific mechanism but to diverse modes of action (Skandamis et al.,

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 Chapter 1: Literature review

2001; Carson et al., 2002). Droby et al. (1999b) reported that jasmonates suppress
successively the development of P. digitatum in grapefruit. This control might be due to the
induction of host resistance responses (Droby et al., 1999b). Also, Tripathi and Dubey (2004)
reported that jasmonates play an important role as signal molecules in plant defense responses
against pathogen attack. The same authors showed that essential oils play a role in plant
defense mechanisms against phytopathogenic micro-organisms and the synergism between
their different components reduces the chance of development of resistant races of fungi. Also
methanol extracts of Withania somnifera and Acacia seyal controlled green mold by
stimulatory effect on the host defense mechanism (Mekbib et al., 2007). These defense
mechanisms resulted in : a) synthesis of cell wall that could serve as a physical and biological
barrier to invading pathogens or b) the increase in the total soluble phenolics compounds
concentration of orange peels (Mekbib et al., 2007). Also of interest, phenolic compounds are
known to alter membrane functionality of pathogens (Lanciotti et al., 2004). However, more
investigations on the mode of action of such plant products are required so as to recommend
their formulation in control of citrus postharvest diseases.

IV.2.5 Application methods of plant extracts and criteria for selecting a

good product

Keeping in view the merits of the botanicals as postharvest fungitoxicants, the products
which are found efficacious during in vitro testings, should be properly tested for their
practical potency based on in vivo trials, organoleptic tests and safety limit profile (Tripathi
and Dubey, 2004). In in vivo trials, the efficacy of postharvest treatments of plant extracts on
citrus fruits depended on their method of application. The incorporation of essential oils into
fruit coatings primarily applied to retain moisture, is gaining popularity (Du Plooy et al.,
2009). Essential oil of Simmondsia chinensis (jojoba oil) was applied by Ahmed et al. (2007)
as a coating for „Valencia‟ oranges. They effectively maintained fruit quality for up to 60 days
(Ahmed, 2007). Also Du Plooy et al. (2009) showed that the advantage of using coatings
amended with essential oils, rather than vapour, is that there is closer contact between the
essential oils and fruit surfaces, allowing exposure of each fruit to similar concentrations of
inhibitor over a longer period. Another method of application of botanicals in controlling
citrus postharvest diseases is the immersion of citrus fruits in plant solutions. Essential oil of
Shiraz thyme showed antifungal activity against P. digitutum only in dipping application
(Solaimani et al., 2009). The same authors reported that dipping method was significantly
better than spray method on control of green mold. Also Tayel et al. (2009) demonstrated that

33
 Chapter 1: Literature review

the in vivo prevention and control of P. digitatum invading harvested citrus fruit was
successfully applied by immersing wounded fruit in pomegranate peel extract (Punica
granatum) solutions. Keeping in view the merits of the botanicals as postharvest
fungitoxicants and to ensure proper application of plant extracts, the products which are found
efficacious during in vivo application must meet the following conditions : a) The product
should be effective even for short duration treatment; b) The treatment should not have an
effect on quality parameters such as acidity, flavour and aroma and c) The lowest suitable
dose of the treatments for practical application should also be determined (Tripathi and
Dubey, 2004).
IV.3 Control of citrus postharvest diseases by food additives and GRAS
compounds

Antifungal compounds that leave low or non-detectable residues in the citrus fruit are
actively sought in research programs. Organic and inorganic salts are widely used in the food
industry; they are common food additives for leavening, pH control, taste and texture
modifications (Smilanick et al., 1999; Hervieux, et al., 2002; Arslan et al., 2009). These
compounds have a broad spectrum of activity against bacteria and fungi, and are generally
recognized as safe (GRAS) compounds for many applications, by European and North
America regulations. In addition to their consistent antimicrobial activity, they are
inexpensive, readily available, with favorable safety profile for humans and the environment
and suitable for the postharvest handling practices (Foegeding and Busta, 1991; Olivier et al.,
1998; Hervieux, et al., 2002; El-mougy et al., 2008; Deliopoulos et al., 2010). In recent years,
interest in salts has increased and numerous studies on the antimicrobial activity of a wide
range of organic and inorganic salts, against various pathogens of citrus fruit, have been
reported (Kitagawa and Kawada, 1984; Sholberg, 1998; Biggs, 1999; Gabler et al., 2001;
Palou et al., 2001; Hervieux, et al., 2002; Plaza et al., 2004b; Ilhan et al., 2006; El-Mougy et
al., 2008; Smilanick et al., 2008; Arslan et al., 2009; Montesionos-Herrero et al., 2010;
Askarne et al., 2011; Talibi et al., 2011b) (Table 1.6).
Table 1.6 : Salts and food additives used for the control of citrus postharvest diseases
Name of Salt Causal agent Remark with use Reference

Acetic acid P. digitatum Fumigation at 99.99% Sholberg, 1998

Formic acid P. digitatum Browning of the fruit peel Sholberg, 1998

Propionic acid P. digitatum Fumigation at 99 to 100% Sholberg, 1998

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 Chapter 1: Literature review

Sorbic acid G. candidum Good control at low pH Kitagawa and Kawada, 1984

G. candidum
Benzoic acid [Link] Coating with 4% of salt El-Mougy et al., 2008
P. digitatum
Boric acid G. candidum Effective at room temparature Talibi et al., 2011b

Smilanick et al., 2008

P. digitatum Effective when heated Montesinos-Herrero et al., 2009
Coating with 4% of salt El-Mougy et al., 2008
Effective when heated at 50°C Kitagawa and Kawada, 1984
Potassium sorbate G. candidum Smilanick et al., 2008
Partially effective when heated
Coating with 4% of salt El-Mougy et al., 2008
Effective at 60°C Montesinos-Herrero et al., 2009
[Link] El-Mougy et al., 2008
Coating with 4% of salt
Immersion at 45°C Palou et al., 2001
[Link] Plaza et al., 2004b
Dip not accepted by industry
Sodium carbonate Effective at 2% and 40°C Smilanick et al., 1999;

[Link] Effective when heated Smilanick et al., 1997

Dip not accepted by industry Plaza et al., 2004b
Effective at 2% and 40°C Smilanick et al., 1999
[Link]
Effective when heated
Sodium Smilanick et al., 2008
G. candidum Partially effective when heated
bicarbonate
Not effective at low
P. italicum Palou et al., 2001
concentrations
G. candidum
[Link] Coating with 4% of salt El-Mougy et al., 2008
Sodium benzoate
[Link]
[Link] Effective at 2% Hall, 1988
Sodium Combined with sodium
[Link] Cerioni et al., 2012
hypochlorite bicarbonate
G. candidum Effective at room temparature Talibi et al., 2011b
Sodium salicylate
Sodium P. digitatum Effective at 2% Hall, 1988
propionate
Sodium [Link] Effective when heated Eckert and Sommer, 1967
tetraborate
Effective when combined with P.
Calcium Chloride P. digitatum Droby et al., 1997
Guilliermondii
calcium P. digitatum Effective on lemon than orange Smilanick and Sorenson, 2001
polysulfide G. candidum Slightly effective
Hydrogen Combined with sodium
P. digitatum Cerioni et al., 2012
peroxide bicarbonate

35
 Chapter 1: Literature review

IV.3.1 Use of GRAS compounds and food additives

Among food preservatives, potassium sorbate has been evaluated for the control of citrus
green and blue molds and sour rot (Kitagawa and Kawada, 1984; Hall, 1988; Palou et al.,
2002; El-Mougy et al., 2008; Smilanick et al., 2008). This compound, classified as a minimal
risk active ingredient and exempt from residue tolerances, is more appropriate for application
as aqueous solutions (Montesinos-Herrero et al., 2010). However, its use did not become
popular because its efficacy was sometimes low, and it was reported to delay, rather than stop,
green mould infections (Smilanick et al., 2008). Sodium benzoate and benzoic acid are known
for their combination of bactericidal and bacteriostatic properties and their properties of being
nontoxic and tasteless (El-Mougy et al., 2008). Their effect on postharvest citrus diseases was
reported against sour rot, green and blue molds incidence of stored citrus fruit (El-Mougy et
al., 2008; Hall, 1988). A comparison among the inhibitory effects of various food additives
and low-toxicity chemicals against P. digitatum and P. italicum showed that potassium
sorbate and sodium benzoate were the most effective on oranges and lemons (Palou et al.,
2002). In order to extend the shelf life of fresh fruit, many other salt compounds are actually
used in many countries, aiming at the destruction of the pathogens or inhibition of their
growth. Certain compounds, such as sodium bicarbonate or sodium carbonate, have been
highly successful in controlling green mold (Smilanick et al., 1999). Also, Smilanick et al.
(2008) reported that sodium bicarbonate reduced the incidence of citrus sour rot. Moreover,
these treatments poses a minimal risk of phytotoxicity to the fruit, and can be a useful tool in
the management of fungicide resistant isolates, which have become particularly problematic
(Smilanick et al., 1999). A significant reduction in incidence of P. digitatum and P. italicum
was noted in the case of oranges treated with ammonium molybdate and sodium molybdate
(Palou et al., 2002). Besides these salts, other compounds such as orthophosphoric acid,
sodium propionate, calcium polysulfide, calcium chloride, EDTA, sodium salicylate and boric
acid have been evaluated for the control of citrus green or blue molds or sour rot and show
reduced incidence and severity of these diseases (Droby et al., 1997; Smilanick and Sorenson,
2001; El-Mougy et al., 2008; Hall, 1988; Askarne et al., 2011; Talibi et al., 2011b) (Table
1.6).

IV.3.2 Mode of action of salt compounds

Although many researchers have focused on the control of postharvest diseases of citrus
fruit by the application of salt compounds, the mechanisms by which salts inhibit

36
 Chapter 1: Literature review

microorganisms are not well understood. The various modes of action of salt compounds are
through membrane disruption, inhibition of essential metabolic functions, stresses on pH
homeostasis and through the accumulation of anions within the cell (Smilanick et al., 2005).
Bicarbonates are effective growth inhibitors of various phytopathogenic fungi in vitro. Most
citrus fungal pathogens grow better in acidic to neutral conditions than in alkaline conditions.
The principal mode of action of the bicarbonate ion is through its buffering capacity, whereby
an alkaline environment is sustained. When this happens, pathogens, such as P. digitatum
which require an acidic environment, expend more energy on fungal acid production than
hyphal extension and therefore growth may be inhibited (Pelser et al., 1977; Prusky and
Yakoby, 2003). According to Corral et al. (1988), the anions (HCO3)- or (CO3)2- are primarily
responsible for pathogen suppression by inorganic salts, and the cation plays only a minor role
in the interaction. Inhibition of G. candidum, P. digitatum and P. italicum by sorbic acid and
its salts may be caused by alteration of cell-membrane and cell transport functions, inhibition
of enzymes and protein synthesis, and by uncoupling of the oxidative phosphorylation in
mitochondria (Sofos 1986; El-Mougy et al., 2008). The pH of the bicarbonate and carbonate
solutions is important for the control of postharvest citrus diseases, because it directly affects
the germination of conidia (Pelser et al., 1977; Prusky et al., 2004) and influences the
virulence of pathogens through their colonization of host tissue (Prusky and Yakoby, 2003;
Smilanick et al., 2005). However, we and other workers showed that pH alone cannot explain
the inhibitory effect of these compounds (Palmer et al., 1997; Smilanick et al., 1999; Talibi et
al., 2011b).

The effectiveness of calcium against Penicillium digitatum on grapefruit could be due to

its direct effects on host tissue by making cell walls more resistant to pathogen penetration.
Most of the calcium that penetrates into the host tissue seems to accumulate in the middle
lamella region of the cell wall. The cations form bonds between adjacent pectic acids or
between pectic acids and other polysaccharides, forming cross bridges which make the cell
walls less accessible to the action of pectolytic enzymes of the pathogen (Droby et al., 1997).
Moreover some researchers reported that calcium ions enhance tolerance to abiotic diseases
(Wisniewski et al., 1995; Biggs et al., 1997; campanella et al., 2002). Ammonium molybdate
affects metabolic processes in several organisms (Wang et al., 1995; Bodart et al., 1999). The
basis of its biological activity was reported to be its ability to inhibit acid phosphatase which
interferes with phosphorylation and dephosphorylation (Glew et al., 1988), one of the most
important processes of cell regulating (Remaley et al., 1985; Hunter, 1995).

37
 Chapter 1: Literature review

IV.4 Use of combined strategy to control postharvest citrus diseases

Different disease management strategies that have to be applied, both at pre- and
postharvest stages, have been integrated to provide more effective disease control than that
possible with a single approach. As discussed in several works, the combination of microbial
antagonists with other alternative control methods can be a promising approach to overcome
some drawbacks in biocontrol activity, enhancing their efficacy (Huang et al., 1995; Stevens
et al., 1997; Droby et al., 1998; El-Ghaouth, et al., 2000; Arras et al., 2002; Janisiewicz and
Korsten, 2002; Porat et al., 2002; Plaza et al., 2004c; Zhang et al., 2004) (Table 1.7). The
combination of microbial antagonists with heat (Porat et al., 2002), GRAS compounds (Usall
et al., 2001), and UV-C (Stevens et al., 1997) produced a synergic effect and was superior to
all the treatments alone in controlling green and blue molds. Such combined treatments can be
easily implemented on a commercial scale in many citrus packinghouses because they are
compatible with existing facilities and postharvest handling practices. In general, five
objectives may be pursued by the integration of two or more treatments : additive or
synergistic effects to increase the effectiveness or the persistence of individual treatments;
complementary effects to combine preventive and curative activities; to delay the
development of fungicide-resistant isolates, to control fungicide-resistant isolates already
present within packinghouses and to facilitate a reduction in fungicide rates in order to
minimize fruit residues and chemical costs (Palou et al., 2008; Smilanick et al., 2008).
Several options of combination were mentioned such as combination of biocontrol agents
with salts or food additives, low levels of conventional fungicides or physical control
treatments.

IV.4.1 Combination of microbial antagonists with other control methods

As previously mentioned, the main shortcoming of the use of microbial antagonists has
been inconsistency in their performance, especially when used as a stand-alone product to
replace synthetic fungicides. Furthermore, as infection of citrus fruit occur either prior to
harvest or during harvesting and processing, microbial antagonists are expected to display
both a protective and curative activity comparable to that observed with synthetic fungicides.
None of the proposed biological control agents, however, have been shown to control
previously-established infections. The combination of biological control with other control
methods is one of the most promising means of establishing effective integrated diseases
management strategies (Schisler et al., 2011) (Table 1.7).

38
 Chapter 1: Literature review

IV.4.1.1 Microbial antagonists combined with low risk substances

Various additives have been shown to increase the effectiveness of some antagonistic
microorganisms in controlling postharvest decay. The addition of CaCl2 to antagonistic yeast
suspensions was found to enhance their biocontrol activity and reduce the populations of yeast
cells required to give effective control (McLaughlin et al., 1990; Droby et al., 1997). A
combination of the yeast antagonist, Pichia guilliermondii (107 cells ml-1) with CaCl2 in dip
application, significantly decreased the incidence of green mold caused by Penicillium
digitatum in grapefruit wounds (Droby et al., 1997). Both spore germination and germ tube
elongation of P. digitatum decreased with increasing CaCl2 concentration. In addition,
increased CaCl2 concentration also resulted in the inhibition of pectolytic activity of a crude
enzyme preparation of P. digitatum.
Enhancement of biocontrol of the green mold (Penicillium digitatum) on citrus has been
achieved by adding 2-deoxy-D-glucose at 0.2% to suspensions of the yeast antagonist
Candida saitoana applied to the fruits (El-Ghaouth et al., 2001). This combination is more
effective to control the disease than either 2-deoxy-D-glucose or Candida saitoana alone.
Another combination aimed at improving the biocontrol of fruit decay by C. saitoana is that
of the antagonistic yeast with glycolchitosan, a combination known as "a bioactive coating"
(El Ghaouth et al., 2000). When applied within 24 h after inoculation, the combination
Candida saitoana with 0.2% glycolchitosan was more effective than the antagonistic yeast
alone in controlling natural infection of oranges and lemons (mainly by P. digitatum) and the
control level was equivalent to that achieved with 2000 ppm of imazalil (El Ghaouth et al.,
2000). The bioactive coating and Imazalil treatments offered consistent control of decay on
Washington Navel oranges and Eureka lemons in early and late seasons.
The possibility of enhancing the antagonistic activity of the yeast Rhodotorula glutinis by
addition of calcium chloride and lower cell concentration of biocontrol agent was explored.
Calcium at 2% markedly enhanced the biocontrol potential of Rhodotorula glutinis inoculated
at 107 and 106cells/ml, compared with Rhodotorula glutinis alone (Arras et al., 1998). The
enhancement in the effectiveness of biocontrol may be due to the toxicity of calcium to P.
italicum by affecting the osmotic balance in the fungal cells and by inhibition of pectinolytic
enzymes in the wound site (Arras et al., 1998).
Others salt additives have also improved the bio-efficacy of some microbial antagonists
in controlling postharvest decay of citrus. Among different salt additives, sodium bicarbonate,
calcium propionate, sodium carbonate, potassium bicarbonate, potassium metabisulfite and
ammonium molybdate etc., have been found very successful when used with microbial

39
 Chapter 1: Literature review

antagonists for controlling postharvest diseases of fruits and vegetables more efficiently
(Smilanick et al., 1999; Teixidó et al., 2001; Porat et al., 2002; Obagwu and Korsten, 2003;
Zhang et al., 2004; Usall et al., 2008) (Table 1.7). Smilanick et al. (1999) showed that control
of green mold on oranges was maximized when dip treatments in sodium carbonate or
bicarbonate were followed by the application of P. syringae strain ESC10, the active
ingredient in the postharvest biological control BioSave™ products. Similar results were also
observed with lemon fruits pretreated with sodium carbonate prior to treatment with C.
saitoana (El Ghaouth et al., 2000).

IV.4.1.2 Combination of microbial antagonists with low levels of

conventional fungicides

Application of microbial antagonists by itself may not always provide commercially

acceptable level of control of postharvest diseases of citrus. However, it is possible to increase
the efficacy of microbial antagonists by combining with certain fungicides such as
thiabendazole (TBZ) and imazalil (IMZ). Compatibility between a microbial antagonist and a
synthetic fungicide offers the option of using the antagonists in combination with reduced
level of the fungicide (Sharma et al., 2009). For example, the combination of Aspire with
thiabendazole (200 ppm) reduces the incidence of decay caused by Penicillium digitatum and
P. italicum as effectively as a conventional fungicide treatment. Furthermore, Aspire is highly
efficacious against sour rot caused by Geotrichum candidum, a decay not controlled by
conventional treatment (Droby et al., 1998). A combination of the yeast Pichia guilliermondii
with reduced concentration of thiabendazole in dip application, significantly decreased the
incidence of green mold to a level similar to that achieved by currently recommended
concentration of TBZ application alone (Arras et al., 2002). The combination of Kloeckera
apiculata with Carbendazim at 40 ppm also resulted in improved control of citrus blue mold
to a level equal to that of the commercial treatment at 200 ppm of Carbendazin (Long et al.,
2006). Thus, such combination allows both maintaining very low level of chemical residue in
the citrus fruit and improving protective properties of the treatment.

40
 Chapter 1: Literature review

Table 1.7 : Combination of Biocontrol antagonists with other control methods

Antagonist Combined with Disease controlled Reference

Combination with physical control methods :

Hot-water Porat et al., 2002
Candida oleophila green mold D'hallewin et al., 2004
Ultraviolet light-C
Pseudomonas glathei Heat treatment green mold Huang et al., 1995

Bacillus subtilis Hot-water green mold Obagwu and Korsten, 2003

Pantoea agglomerans Heat treatment green mold Plaza et al., 2004c

Debaryomyces hansenii Ultraviolet light-C green mold Stevens et al., 1997

Combination with low levels of conventional fungicides :

Stem-end rot Brown and Chambers, 1996
Candida oleophila Thiabendazole
Penicillium rots Droby et al., 1998

Imazalil Stem-end rot

Pichia guilliermondii Arras et al., 2002
Thiabendazole Penicillium rots
Kloeckera apiculata Carbendazim blue mold Long et al., 2006

Combination with food additives and other salts :

Candida oleophila Sodium bicarbonate green mold Porat et al., 2002

Bacillus subtilis Sodium bicarbonate green mold Obagwu and Korsten, 2003

Sodium bicarbonate
Pseudomonas syringae green mold Smilanick et al., 1999
Sodium carbonate
Sodium carbonate Usall et al., 2008
Pantoea agglomerans green mold
Sodium bicarbonate Teixidó et al., 2001

Cryptococcus laurentii Sodium bicarbonate green mold Zhang et al., 2004

Pichia guilliermondii Calcium chloride green mold Droby et al., 1997

Glycolchitosan El-Ghaouth et al., 2000

Candida saitoana green mold El-Ghaouth et al., 2001
2-Deoxy-D-glucose
Kluyveromyces
Sodium bicarbonate green mold Geng et al., 2011
marxianus

41
 Chapter 1: Literature review

IV.4.1.3 Combination of microbial antagonists with physical control

treatments

Physical treatments could also be used in association with microbial antagonists,

complementing their activity. Pathogens treated with such physical means might be weakened
and become more vulnerable to the antagonist activity. Cold storage, curing, heat treatments
and ultraviolet (UV) light have been the main physical methods used to improve the
biocontrol efficacy of microbial antagonists (Table 1.7). For example, the bacterium
Pseudomonas glathei was evaluated for its efficacy in reducing green mold decay of citrus
caused by P. digitatum. The disease control was enhanced by treatment of citrus fruit with P.
glathei cell suspension after heat treatment (Huang et al., 1995). The antagonistic activity of
Pichia guilliermondii against P. digitatum was significantly increased by combining
controlled atmosphere with low storage temperature (Lurie et al., 1995). Similarly, El
Ghaouth et al. (2004) showed that the incidence of decay developed on grapefruits treated
with hot water and C. oleophila 24 h after inoculation with P. digitatum was dramatically
reduced in comparison to each treatment alone. Besides heat treatments, the ultraviolet light-C
is also applied in association with the antagonistic yeasts Candida oleophila and
Debaryomyces hansenii to control citrus green mold (Stevens et al., 1997; D'hallewin et al.,
2004).

IV.4.2 Combination of salts and food additives with other control methods

Successful commercial control of postharvest diseases of fruits and vegetables must be

extremely efficient, in the range of 95–98%, unlike the control of tree, field crop or soil borne
diseases. Consistent performance to such levels of control cannot presently be achieved by
alternatives to fungicides as stand-alone treatments, so strategies where they are combined are
needed to attain commercially acceptable performance (Palou et al., 2008). Among the
strategies evaluated during the last few years, the combination of salts and food additives with
low levels of conventional fungicides and physical control methods should be mentioned.

IV.4.2.1 Combination of salts and food additives with low levels of

conventional fungicides

Due to the lack of preventive activity to control postharvest citrus diseases of some salt
compounds, it was necessary to combine them with low levels of conventional fungicides.
Several works have demonstrated the efficiency of this practice. The combination of sodium

42
 Chapter 1: Literature review

bicarbonate with low levels of Imazalil (Smilanick et al., 2005), Thiabendazole (Smilanick et
al., 2006b) or Pyrimethanil (Smilanick et al., 2006a) improved their performance to control
citrus green mold. Also Smilanick et al. (2008) reported that Green mold caused by an isolate
of P. digitatum resistant to IMZ and TBZ was effectively controlled when potassium sorbate
was added to a heated IMZ or TBZ solutions.

IV.4.2.2 Combination of salts and food additives with physical control

treatments

Salts and food additives improve their efficacy when combined with curing and hot
water treatments. Sorbate potassium was reported to control postharvest sour rot when it was
applied as a hot water solution (Kitagawa and Kawada, 1984; Smilanick et al., 2008). Palou et
al. (2001) showed that temperature of sodium carbonate and sodium bicarbonate solutions
influenced effectiveness of control of postharvest green and blue mold more than
concentration or immersion period. Also of interest, Plaza et al.(2004b) reported that Dipping
fruits in a sodium carbonate solution following a curing treatment satisfactorily reduced green
and blue mold incidence during subsequent long-term storage. Besides heat treatments, salts
are applied with wax coatings, a critical operation in citrus fruit packinghouses. (Youssef et
al., 2012) reported that wax mixed with sodium bicarbonate, potassium carbonate and
potassium sorbate significantly reduced the incidence of postharvest green and blue molds.

V. Conclusion

From this review, it appears that some significant progress has been made toward
biological and integrated control of postharvest diseases of citrus fruit. Some biofungicides
are already on the market in a few countries, and will probably become more widely available
as they are registered in more areas. Other microbial antagonists should reach the market
soon. Postharvest conditions provide an ideal niche for microbial antagonists since they are
less subject to sudden weather changes, and are often equipped with a sophisticated climate
control system. However, so far, only a few products with high biocontrol potential have been
made available on a commercial scale. With intensive research being carried out in various
laboratories, the possibility of identifying potent microbes and developing suitable biocontrol
products for commercial marketing appears to be bright. On the other hand, it‟s unrealistic to
assume that microbial antagonists have the same fungicidal activity as fungicides. Optimized
postharvest usage strategies of microbial antagonists include integration with other low risk
treatments to optimize performance while allowing identification of methods that reduce the
43
 Chapter 1: Literature review

use of conventional synthetic fungicides for the control of postharvest diseases of citrus fruit.
In the development of these new strategies, emphasis was placed on minimizing human health
risks and environmental toxicity. Research should provide appropriate tools (microbial
antagonists, natural substances, GRAS compounds, etc.) to tailor a complete postharvest
citrus diseases management strategy.

44
 CHAPTER TWO

Screening of the antifungal activity

of 43 plant extracts for the control of
Geotrichum candidum
Publised in Crop Protection as :
Talibi, I., Askarne, L., Boubaker, H., Boudyach, E., Msanda, F., Saadi, B., Ait
Ben Aoumar, A., 2012. Antifungal activity of some Moroccan plants against
Geotrichum candidum, the causal agent of postharvest citrus sour rot. Crop
Protection 35, 41-46.
 Chapter 2: Screening of the antifungal activity of the aqueous plant extracts

Résumé
Les extraits aqueux de 43 échantillons de plantes récoltées dans différentes régions du
sud du Maroc ont été testés pour évaluer leur pouvoir antifongique, in vitro et in vivo, contre
Geotrichum candidum, agent causal de la pourriture amère des agrumes en post-récolte. Les
extraits aqueux de ces plantes ont été évalués, d‟abord, sur la croissance mycélienne de G.
candidum en incorporant celles-ci dans le milieu de culture à base d‟extrait de pomme de terre
(PDA). Les résultats obtenus ont montré que la plupart des plantes testées ont réduit la
croissance mycélienne de G. candidum avec des degrés d‟inhibition qui différent selon
l‟espèce testée. Le meilleur degré d‟inhibition de la croissance mycélienne a été obtenu avec
les extraits aqueux de Rubus ulmifolius, Ceratonia siliqua, Cistus monspeliensis et Halimium
umbellatum avec une inhibition totale de la croissance mycélienne de G. candidum. En outre,
les extraits aqueux de Cistus villosus, Pistacia atlantica, Halimium antiatlanticum, Inula
viscosa, Ighermia pinifolia et Hammada scoparia sont également efficaces contre G.
candidum avec un pourcentage d'inhibition de la croissance mycélienne supérieure à 80%.
Cependant, certains plantes testées dans cette étude ont stimulé la croissance de G. candidum,
à savoir : Mentha suaveolens, Psoralea bituminosa et Reseda alba. L‟effet des extraits aqueux
des plantes les plus efficaces a été évalué aussi sur la germination des arthrospores de G.
candidum. Les résultats obtenus ont révélé que parmi les extraits aqueux qui ont
complètement inhibé la croissance mycélienne de G. candidum, seuls ceux de C. villosus et H.
antiatlaticum ont également inhibé la germination des arthrospores à 5 et 2,5 mg/ml
respectivement. Les concentrations minimales inhibitrices (CMI) et fongicides (CMF)
relatives aux meilleures plantes ont été également déterminées. Les plus faibles CMI ont été
enregistrées avec les extraits aqueux de C. villosus et H. antiatlanticum avec une CMI égale à
0,156 mg/ml, suivie de C. siliqua, H. umbellatum et R. ulmifolius avec une CMI de 0,312
mg/ml. Sur la base du screening in vitro, seuls les extraits qui ont réduit la croissance
mycélienne de G. candidum de plus de 90% ont été retenus pour évaluer leur capacité à
contrôler la pourriture amère. Les résultats obtenus ont montré que les extraits aqueux de sept
espèces (H. umbellatum, I. viscosa, R. ulmifolius, C. villosus, C. siliqua, H. antiatlanticum et
P. atlantica) ont significativement réduit l‟incidence et la sévérité de la pourriture amère sans
avoir des effets phytotoxique sur les fruits traités.

Mots clés : Activité antifongique, pourriture amère, Extraits de plantes, Geotrichum candidu,
fruits d‟agrumes.

45
 Chapter 2: Screening of the antifungal activity of the aqueous plant extracts

Abstract

The powders and aqueous extracts of 43 plants species, harvested in different regions
of southern Morocco, were screened for their in-vitro and in-vivo antifungal activity against
Geotrichum candidum, the cause of citrus sour rot. Our results show that among the 43 plants
tested, the powders of Rubus ulmifolius, Ceratonia siliqua, Cistus monspeliensis and
Halimium umbellatum plants totally inhibited mycelial growth of G. candidum. Furthermore,
the powders of Cistus villosus, Pistacia atlantica, Halimium antiatlanticum, Inula viscosa,
Ighermia pinifolia and Hammada scoparia plants are also effective against G. candidum with
a percent of inhibition of mycelial growth higher than 80%. The effect of plant aqueous
extracts on arthrospores germination varied significantly (P<0.05) between tested plants.
Aqueous extracts from H. antiatlanticum and C. villosus plants showed the strongest activity.
The first plant has completely inhibited the arthrospores germination at 2.5 and 5 mg/ml, and
the second plant at 5 mg/ml. The most active plants in in vitro studies were tested in vivo
against sour rot on citrus fruit. Incidence of sour rot was lowered to 44.44 and 46.30 % when
mandarin fruit where treated by C. villosus and H. antiatlanticum aqueous extracts, compared
with 98.15% in the control. This study demonstrates that plants extracts have a high potential
to control sour rot of citrus. Such natural products therefore represent a sustainable alternative
to the use of chemical pesticides.

Keywords : Antifungal activity, sour rot, plant extracts, Geotrichum candidum, Citrus fruit.

46
 Chapter 2: Screening of the antifungal activity of the aqueous plant extracts

I. Introduction

Citrus fruit cultivation is very important in Morocco, being the first exporting
agricultural sector and playing a major role in the national economic development. The largest
volume of citrus fruit for fresh consumption and export in the morocco is grown and shipped
from packing houses in Souss-Massa-Draa (SMD) region (Boubaker et al., 2009). The citrus
fruits are exposed to many postharvest diseases during fruit processing, among which sour rot,
caused by the yeast like fungus Geotrichum candidum Link ex Pers., is responsible for
economically significant losses under favorable conditions for pathogen development,
principally during fruit degreening and wet seasons (Eckert, 1978; Eckert and Brown, 1988;
Cohen et al., 1991). The decay is, also, more prevalent as fruit increases in maturity (Brown,
1979; Suprapta et al., 1995). Fruits are infected by the fungus only through wounds. At
favorable temperatures of 25-30°C, fruit will rot completely in 4 or 5 days, and the disease
can spread by contact. In addition to citrus fruit, G. candidum is the causal agent of numerous
decays of other fruits and vegetables like tomato, cucumber, carrot and stone fruit (Wells,
1997; Suprapta et al., 1995; Palou et al., 2009). The isolate which exhibits pathogenicity to
citrus fruit was designated as G. candidum citrus race (Butler et al., 1965) or G. citri-aurantii
(Ferr.) Butler (Butler et al., 1988). Sour rot is not controlled by postharvest chemical
treatments, mainly imazalil and thiabendazole (Mercier and Smilanick, 2005), and only
controlled by Guazatine (Brown, 1988), a chemical that is not authorized in several countries.
The disease can be partially reduced by careful handling, harvesting of fruit before it becomes
over-mature, and reducing fruit storage temperatures after harvest (Eckert and Brown, 1988).
The SMD region is one of the richest regions in Morocco in terms of plant diversity
(Peltier, 1982; Msanda et al., 2005). This diversity in flora provides a rich source of natural
bioactive substances. Indeed, the scientific interest in these substances has increased today
with the search of new antifungal compounds from plant source, due to increased resistance of
postharvest fungal pathogens to fungicides, and growing concern for human safety and the
protection of the environment (Suhr and Nielson, 2003; Smilanick et al., 2008; Casals et al.,
2010). Plant extracts are very attractive as alternative or complementary control means
because of their antifungal activity, non-phytotoxicity, systemicity, and biodegradability
(Tripathi and Dubey, 2004; Kosalec et al., 2005; Ameziane et al., 2007; Gatto et al., 2011).
Limited information is available on the antifungal activity of plant extracts against G.
candidum.

47
 Chapter 2: Screening of the antifungal activity of the aqueous plant extracts

This study was aimed at the valorization of natural floral resources of SMD Valley, south
of Morocco, by in vitro and in vivo screening of their antifungal activity against Geotrichum
candidum, the causal agent of citrus sour rot.

II. Materials and methods

II.1 Collection of Plant Samples

Forty three fresh plant samples were collected from different locations in the Souss
Massa valley (south of Morocco) between March and April 2008 and 2009. All samples were
identified, and were deposited in the herbarium of the laboratory of Biotechnology and
Valorization of the Natural Resources, Faculty of Sciences, Ibn Zohr University, Agadir,
Morocco. Plant samples were cleaned, air dried in the shade, then grounde to a fine powder
using a laboratory grinding mill (Polymix PX-MFC 90D, Switzerland) and stored in the dark
at 4°C until use. The botanical name, family, and parts used of plant samples are summarized
in Table 2.1. The 43 vascular plant species belong to 36 genera and 16 botanical families.

II.2 Preparation of plant extracts

Preparation of the plant samples for the in vitro antifungal screening against Geotrichum
candidum was conducted as previously described by Ameziane et al. (2007). In brief, 10
grams of powders of each sample were added to 100 ml of molten Potato Dextrose Agar
(PDA) medium. The resulting suspensions were stirred for 10 min, autoclaved for 15 min and
subsequently filtered through four layers of sterile cheesecloth before being dispensed into 9-
cm diameter Petri plates (Figure 2.1).

Figure 2.1 : Preparation of aqueous plant extracts for the in vitro antifungal screening

For the other tests (germination assay, Determination of MIC and MFC and the in vivo assay),
aqueous plant extracts were used and prepared according to the method described by Abdel-
Monaim et al. (2011). Ten grams of each plant powder were extracted by soaking in 100 ml

48
 Chapter 2: Screening of the antifungal activity of the aqueous plant extracts

of boiling distilled water for 10 min in a magnetic hotplate stirrer. The aqueous extracts were
then filtered through a Whatman no.1 filter paper, lyophilized, and stored at -20°C until use.
Appropriate weights of each extracts were prepared in sterile distilled water to obtain the
various concentration used for the experiments.

II.3 Pathogen
Geotrichum candidum was isolated from a decayed mandarin fruit and has been one of
the most aggressive isolates in our collection. This isolate was deposited at the laboratory
under the code SR10. The fungus was maintained on PDA plates at 5°C, with periodic
transfers through citrus fruit to maintain its aggressiveness.

The pathogen inoculum consisted of aqueous arthrospores suspensions obtained from 7-

days-old cultures plates incubated at 25°C. Arthrospores were harvested by flooding plates
with 5 ml of sterile distilled water containing 0.05% (v/v) Tween 80, and passing the
suspension through two layers of sterile cheesecloth to remove hyphal fragments. The
arthrospore concentration was determined with the aid of a heamocytometer and adjusted to
106 arthrospores ml-1 with sterile distilled water.

II.4 Fruit
The fruit of mandarin (Citrus reticulata blanco) cv. “Clementine” was used. Fruits were
harvested from orchards of the M‟brouka cooperative, which used standard culture practices,
in Souss-Massa-Draa region, south of Morocco. Only healthy and commercially mature fruits
were used in the in vivo test. Freshly harvested or shortly stored (no longer than a week) fruits
were used in the experiment.

II.5 Evaluation of antifungal activity of plant extracts

II.5.1 In vitro effect on mycelial growth of G. candidum
The incorporation method was used for determining the inhibition of mycelial growth of
G. candidum by the plant powders. As described above, plant powders were tested at a
concentration of 10% (w/v). Plates were inoculated with G. candidum, using a 5-mm diameter
agar disk taken from one-week-old cultures, mycelium surface facing down. The agar plates
were then incubated at 25°C for seven days. Control consisted of unamended PDA medium.
Radial growth was determined by measuring colony size along two perpendicular axes. The
antifungal activity was expressed in terms of percentage of mycelial growth inhibition and
calculated according to the following formula :

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 Chapter 2: Screening of the antifungal activity of the aqueous plant extracts

% mycelial growth inhibition (MGI) = [(control diameter - plant extract diameter)/control

diameter] ×100. Three replicate plates were used for each treatment.

To determine whether plant extracts have fungistatic or fungicidal effect on G. candidum,

Plugs from treatment with no growth were transferred to unamended PDA medium, treatment
in which mycelial growth did not occur after additional seven days of incubation were
considered fungicidal (Alilou et al., 2008). The experiments were performed twice.

II.5.2 Effect of aqueous extracts on arthrospores germination

The germination of arthrospores of G. candidum was determined in concentrations of
0.625, 1.25, 2.5, and 5 mg. ml-1 of aqueous extract for the plant that allowed a higher mycelial
growth inhibition. Aliquots (40µl) of a arthrospores suspension (106 arthrospores. ml-1 ) were
aseptically transferred in triplicate to sterile depression slides containing 40 µl of 2 % sterile
orange juice amended with different concentrations of aqueous extracts. Inoculated slides
were placed on moist filter paper in Petri plates, sealed with Parafilm to avoid evaporation,
and then incubated at 25°C for 24h. Each slide was then fixed with acid fuchsine solution to
stop further germination (Smilanick et al., 1999). Arthrospores germination was estimated
under a microscopic equiped with a micrometer. At least 100 arthrospores within each
replicate were observed. An arthrospore was scored as germinated if the germ tube length was
equal or exceed to at least one time that of the spore body (Suprapta et al., 1997). The results
were expressed as percent spore germination inhibition and calculated by using the following
formula : GI (%) = [(Gc−Gt) /Gc)] ×100, Gc and Gt represent the mean number of
germinated spores in control and treated slides, respectively (Soylu et al., 2010). Each
treatment included three replicates and the experiment was conducted twice.

II.5.3 Determination of Minimum inhibitory concentration (MIC)

and minimum fungicidal concentration (MFC)
The minimum inhibitory concentrations (MICs) of the plant extracts were determined
by the agar dilution method. Aqueous sterile plants extracts were first diluted to the highest
concentration to be tested (5 mg/ml), and then serial two-fold dilution were made in a
concentration range from 5 to 0.3125 mg/ml in 10 ml test tubes containing melted PDA
medium. Culture plates with 24 microwells and a capacity of 4 ml per well were used.
Aqueous plant extracts amended medium was dispended in the wells of culture plates (2 ml
per well). Aliquots (10 µl) of an arthrospores suspension (106 spores/ ml) were then dropped
onto the surface of the agar medium. Culture plates were incubated at 24°C (±1°C) for 48 h.

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 Chapter 2: Screening of the antifungal activity of the aqueous plant extracts

The MICs were recorded by reading the lowest aqueous extract concentration that allowed no
visible growth of the pathogen (Phongpaichit et al., 2005).

The minimum fungicidal concentrations (MFCs) were determined by taking agar plugs
from well showing no visible mycelial growth and re-inoculating them on unamended PDA
medium. MFC was regarded as the lowest concentration of the aqueous plant extract that
prevented growth of the pathogen after the period of incubation, and indicating more than
99.5% killing of the original inoculums (Fabry et al., 1996; Ali-Shtayeh and Abu Ghdeib,
1999). There were three replicates for each plant aqueous extract at each concentration and
the experiment was conducted twice.

II.5.4 Effects of aqueous extracts on sour rot development in

artificially inoculated and wounded fruit
Based on in vitro screening, only plant species that reduced the mycelial growth of G.
candidum by more than 90% were retained. Mandarin fruits were washed, disinfected with
0.1% (v/v) sodium hypochlorite, rinsed three times in sterile distilled water and then air dried
before wounding. One wound (2mm deep and 4mm wide) was made per fruit using a sterile
needle at the equatorial side (Liu et al., 2009b). The wounds were treated with 30 µl of
aqueous plant extracts at a concentration of 50mg/ml. Controls were treated with the same
volume of sterile distilled water under the same conditions. After two hours incubation at
room temperature, each wound was inoculated with 20 µl of an aqueous suspension of
arthrospores of G. candidum (106 arthrospores. ml-1). Treated fruits were placed on plastic tray
in carton boxes and incubated at 26°C and 95% relative humidity (RH) (Figure 2.2). The
number of the infected wounds and the lesion diameters of the overall treated fruit were
determined daily. All treatments were arranged in a complete randomized block design.
Eighteen fruits constituted a single replicate and each treatment was replicated three times.
The experiment was conducted twice. The incidence and severity of disease were calculated
as follows : Disease incidence (%) = [(number of rotten wounds / number of total wounds)] x
100.

Disease severity (%) = [(average lesion diameter of treatment / average lesion diameter of
control)] x 100. In all experiments, the possible phytotoxic effect on mandarin fruit was
examined.

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 Chapter 2: Screening of the antifungal activity of the aqueous plant extracts

II.6 Statistical analysis

All data were subjected to statistical analysis of variance (ANOVA) using
STATISTICA software, version 6, Stat-Soft, 2001, France. Percentage values were subjected
to arcsine-square root transformation before analysis of variance. Mean separation was
performed following the Newman & Keuls test at P <0.05.

Figure 2.2 : In vivo test of selected aqueous extracts : a) Injury of Mandarin fruits, b) Inoculation and
treatment of fruits and c) incubation in growing room at 25°C and 95% HR

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 Chapter 2: Screening of the antifungal activity of the aqueous plant extracts

III. Results
III.1 In vitro effect on mycelial growth of G. candidum

Antifungal activity of 43 vascular plant species belonging to 16 botanical families

(Table 2.1) has been evaluated in vitro against G. candidum, the causal agent of sour rot of
citrus fruits. The powders of most tested plant species reduced colony growth of the fungus
(Table 2.1). However, the inhibitory effect varied between plant species. The in vitro
screening of plant powders has uncovered significant antifungal activity of R. ulmifolius, C.
siliqua, C. monspeliensis and H. umbellatum plants. Indeed, these plants had completely (100
%) inhibited the mycelial growth of G. candidum. Some other plant powders from C. villosus,
P. atlantica, H. antiatlanticum, I. viscosa, I. pinifolia and H. scoparia showed a higher degree
of control (MGI> 80%; Table 2.1 and Figure 2.3).

H.u C.v C

Figure 2.3 : Inhibition of mycelial growth of G. candium by aqueous extracts of H. umbellatum

(H.u) and C. villosus (C.v) compared with the control (C).

According to their antifungal activity the tested plants were divided into three groups :

The first group combines the best plants (7 plants) for which the MGI percentage is
greater than 95%. Among these plants, four (57%) belong to the family of Cistaceae (viz., C.
monspeliensis, C. villosus, H. umbellatum and H. antiatlanticum). The second group includes
plants for which the MGI percentage is between 87% and 52%. This group is represented by
13 plant species belonging to the families of Asteraceae (viz., I. pinifolia, I. viscosa, A.
radiata, A. graveolens), Zygophyllaceae (viz., F. harpago, Z. gaetulum, F. zilloides),
Chenopodiaceae (viz., H. scoparia), Lamiaceae (viz., T. werneri, T. antiatlanticum), Rosaceae
(viz., C. monogyna), Boraginaceae (viz., T. calcarata) and Cistaceae (viz., C. creticus). The
third group includes plants with a MGI percentage inferior to 45%, and some plants even
enhanced the mycelial growth of G. candidum (e.g. M. suaveolens, P. bituminosa, P.

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 Chapter 2: Screening of the antifungal activity of the aqueous plant extracts

scoparius, N. retusa, and R. alba). These results indicate that the plant powders contain
compounds with antifungal potential against G. candidum.

None of plants which had completely inhibited the mycelial growth of the fungus were found
to be fungicidal at 10% (w/v); since plugs transferred from plant powder amended PDA to
unamended PDA grow after additional 7 days incubation at 25°C (data not shown).

Table 2.1 : In vitro effects of plant powders on mycelial growth (MG) of Geotrichum candidum, agent
of citrus sour rot

Family Scientific name Part used M.G. I %

Rosaceae Rubus ulmifolius Schott Leaves+stem 100 ±0 a

Fabaceae Ceratonia siliqua L. Leaves 100 ±0 a
Cistaceae Cistus monspeliensis L. Leaves+stem 100 ±0 a
Cistaceae Halimium umbellatum (L.) Leaves+stem 100 ±0 a
Spach
Cistaceae Cistus villosus Auct. Leaves+stem 98.26 ±0.07 a
Anacardiaceae Pistacia atlantica Desf. Leaves 97.68 ±0.04 a
Cistaceae Halimium antiatlanticum Leaves+stem 95.94 ±0.04 a
Maire & Wilczek
Asteraceae Inula viscosa (L.) Aiton Leaves+stem 87.11 ±0.16 b
Asteraceae Ighermia pinifolia (Maire & Leaves+stem 85.49 ±0,04 b
Wilczek) Wiklund
Chenopodiaceae Hammada scoparia (Pomel) Leaves+stem 80.39 ±0,09 bc
Iljin
Asteraceae Anvillea radiata Coss. Durieu Leaves+stem 75.71±0 cd
Asteraceae Asteriscus graveolens Forssk. Leaves+stem+Flowers 71.9 ±0.11 cde
Less.
Zygophyllaceae Fagonia harpago Emb. Maire Leaves+stem 70.98 ±0.09 cde
Zygophyllaceae Zygophyllum gaetulum Emb. Leaves+stem 68.23 ±0.2 def
Maire
Lamiaceae Teucrium werneri Emb. Leaves+stem 67.55 ±0.11 def
Zygophyllaceae Fagonia zilloïdes Humbert Leaves+stem 65.78 ±0.04 def
Rosaceae Crataegus monogyna Jacq. Leaves+stem 61.96 ±0.04 efg
Lamiaceae Teucrium antiatlanticum Leaves+stem 61.78 ±0.09 ef
(Maire) Sauvage & Vindt
Boraginaceae Trichodesma calcarata Coss. Leaves+stem+Flowers 58.04 ±0.04 fg
Ex Batt.
Cistaceae Cistus creticus L. Leaves+stem 52.46 ±0.09 g
Asteraceae Artemisia inculta Delile Leaves+stem 44.44 ±0.09 h

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 Chapter 2: Screening of the antifungal activity of the aqueous plant extracts

Lamiaceae Lavandula coronopifolia Leaves+stem+Flowers 42.86 ±0.33 h

Poir.
Lamiaceae Thymus satureioides Coss. Leaves+stem+Flowers 30 ±0.2 ij
Lamiaceae Lavandula stoechas L. Leaves+stem+Flowers 28.8 ±0.34 ij
Lamiaceae Thymus leptobotrys Murb. Leaves+stem 27.84 ±0.09 ij
Lamiaceae Marrubium deserti (de Noé) Leaves+stem 20.78 ±0.22 jk
Coss.
Lamiaceae Ballota hirsuta Benth. Leaves+stem 20.44 ±0.98 jk
Plumbaginaceae Limoniastrum ifniense Leaves + stem 15.71 ±0.07 kl
(Caball.) Font Quer
Rutaceae Ruta montana L. Leaves+stem 15.11 ±0.16 kl
Anacardiaceae Rhus pentaphylla (Jacq.) Leaves+seeds 14.76 ±1.09 kl
Desf.
Rutaceae Ruta tuberculata Forssk. Leaves+stem 12.44 ±0.29klm
Fabaceae Ononis natrix L. Leaves+stem 12 ±0.07 klmn
Asteraceae Cotula cinerea Delile Leaves+stem+Flowers 8.44 ±0.16 lmno
Thymelaeaceae Thymelaea antiatlantica Leaves+stem 0 ±0 no
Maire
Fabaceae Lupinus angustifolius L. Leaves+stem 0 ±0 no
Asteraceae Pulicaria mauritanica Batt. Leaves+stem+Flowers 0 ±0 o
Capparaceae Maerua crassifolia Forssk. Leaves 0 ±0 no
Solanaceae Witania adpressa Coss. Ex Leaves 0±0 no
Batt.
Lamiaceae Mentha suaveolens Ehrh Leaves+stem -6.67±0 o
Fabaceae Psoralea bituminosa L. Leaves+stem -6.67±0 o
Apiaceae Pituranthos scoparius (Coss. Leaves+stem+Flowers -6.67±0 o
Durieu) Benth. & Hook
Zygophyllaceae Nitraria retusa Forssk. Asch Leaves+stem -19.42±0.18 p
Resedaceae Reseda alba L. Leaves+stem+Flowers -21.74±0 p
a
Values are means of three replicates ± standard deviation. Values followed by the same
letters were not significantly different (P <0.05) according to Newman and Keels test. The
negative values indicate a profungal activity against G. candidum.

III.2 Effect of aqueous extracts on arthrospores germination

The results shown in Table 2.2 indicate that of the plant species tested; only the
aqueous extracts of H. antiatlanticum and C. villosus at 5 mg/ml had the highest fungistatic
effect (100% inhibition) on arthrospores germination of G. candidum. Indeed, tested at 2.5
mg/ml, the aqueous extracts of H. antiatlanticum and C. villosus have inhibited the

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 Chapter 2: Screening of the antifungal activity of the aqueous plant extracts

arthrospore germination by 100 and 75%, respectively. At a concentration of 1.25 mg/ml, the
toxicity of aqueous extract of C. villosus to arthrospores was higher to that of the other tested
plants. Since percent inhibition of germination in aqueous extract amended with 2 % sterile
orange juice is higher than 70%. Tested at 5 mg/ml, the aqueous extract of I. viscosa reduced
the percentage of arthrospores germination by more than 50%. The results obtained showed
that the aqueous extracts of the others tested plants did not prevent or have slightly reduced
arthrospore germination of G. candidum, even at the highest concentration tested (5mg/ml).
Aqueous extracts from C. siliqua and R. ulmifolius reduced arthrospores germination of G.
candidum by 32.67 and 15.0 %, respectively (Table 2.2).

Table 2.2 : In vitro effect of some plants aqueous extracts on arthrospore germination of Geotrichum
candidum
a
Arthrospore germination inhibition (%)

Plant species Concentration of aqueous extracts (mg/ml)

0.625 1.25 2.5 5

Halimium umbellatum 0.0± 0 hi 18.0 ± 2 ef 17.67 ± 2.33 ef 30.67 ± 1.67 d

Halimium antiatlanticum 4.67 ± 2.67 fjh 20.67 ± 1.67 e 100.0 ± 0 a 100.0 ± 0 a
Pistacia atlantica 0.0 ± 0 hi 0.0 ± 0 hi 0.67 ± 0.67 hi 1.33 ± 1.33 hi
Cistus villosus 20.33 ± 1.83 e 70.67 ± 1.83 b 75.67 ± 2.17 b 100 ± 0 a
Hammada scoparia 0.0 ±0 hi 0.0 ±0 hi 2.0 ± 0 hi 2.67 ± 0.67 hi
Rubus ulmifolius 0.0 ± 0 hi 6.67 ± 1.67fjh 8.67 ± 0.83 fjh 15.0 ± 2.5 fj
Inula viscosa 1.33 ± 0.67 hi 2.67 ± 0.67 hi 20.67 ± 0.83 e 52.33 ± 0.83 c
Ceratonia siliqua 2.0 ± 1 hi 6.67 ± 1.67 fjh 9.33 ± 2.67 jh 32.67 ± 1.83 d
Control 0.0 ± 0 hi 0.0 ± 0 hi 0.0 ± 0 hi 0.0 ± 0 hi
a
Each value represents the mean of three replicates ± standard deviation . Means followed by
different letter (s) in each column are significantly different at P < 0.05.

III.3 . MIC and MFC

The MIC and MFC values of selected plant extracts are shown in Table 2.3. The MIC
value for C. villosus and H. antiatlanticum aqueous extracts against G. candidum, was
particularly low (0.156 mg/ml). On the other hand, the MIC value obtained by using aqueous
extracts of H. umbellatum, R. ulmifolius and C. siliqua was 0.3125 mg/ml. P. atlantica and H.

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 Chapter 2: Screening of the antifungal activity of the aqueous plant extracts

scoparia aqueous extracts were the least effective for restricting in vitro mycelial growth
(MIC>5). However, all tested plant aqueous extracts have a MFC value higher than 5mg/ml
(Table 2.3).

Table 2.3 : Minimal inhibitory concentrations (MICs) and minimal fungicidal concentrations (MFCs)
for the plant extracts investigated against Geotrichum candidum

Treatment MICa (mg/ml) MFCb (mg/ml)

Halimium umbellatum 0.3125 >5

Rubus ulmifolius 0.3125 >5
Ceratonia siliqua 0.3125 >5
Cistus villosus 0.156 >5
Halimium antiatlanticum 0.156 >5
Inula viscosa 2.5 >5
Anvillea radiata 5 ND
Pistacia atlantica >5 ND
Hammada scoparia >5 ND
a
Concentration that were fungistatic
b
Concentration that were fungicidal
ND : not determined

III.4 . In vivo test

Data presented in the figure 2.4 showed that all tested aqueous plant extracts
significantly reduced the incidence of sour rot caused by G. candidum under the laboratory
conditions. Percentages of rotted wounds were decreased by using all tested plant extracts
compared with control. Mandarin fruit treated 2 hours before pathogen inoculation by C.
villosus and H. antiatlanticum aqueous extracts resulted in the highest reduction in rot
incidence compared with the control. H. umbellatum, I. viscosa, C. siliqua, and P. atlantica
aqueous extracts had a moderate effect on sour rot, the percentage of rot incidence varying
between 55.56 and 61.11 %. In contrast, the aqueous extract of R. ulmifolius showed the least
effect on reduction of sour rot incidence (Figure 2.4 and Figure 2.6). Also, data indicated that
tested aqueous plant extracts exhibited significant reduction of disease severity compared with
the control (Figure 2.5 and Figure 2.6). They reduced disease severity from 98.5% in non-
treated fruit to a minimum of 32% in C. villosus treated fruit (Figure 2.5). Furthermore,
neither of the plants aqueous extracts showed any phytotoxic reaction on treated fruit, at the
concentration tested (50 mg/ml).

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 Chapter 2: Screening of the antifungal activity of the aqueous plant extracts

Figure 2.4 : Effect of aqueous plant extracts on sour rot incidence in mandarin fruit. Fruits were
treated with aqueous extracts, inoculated with G. candidum and held for 7 days at 26°C. Significant
differences (P < 0.05) between means were indicated by different letters above histogram bars.

Figure 2.5 : Effect of aqueous plant extracts on sour rot severity in mandarin fruit. Fruits were
treated with aqueous extracts, inoculated with G. candidum and held for 7 days at 26°C. Significant
differences (P < 0.05) between means were indicated by different letters above histogram bars.

R. ulmifolius C .villosus Control

Figure 2.6 : Mandarin fruits treated with aqueous extracts of R. ulmifolius (R.u) and C. villosus
(C.v) and inoculated with arthrospores suspension of G. candidum, after seven days of incubation.

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 Chapter 2: Screening of the antifungal activity of the aqueous plant extracts

IV. Discussion

The disease control strategies which have been studied as an alternative to currently
used fungicides for the control of postharvest citrus fruit rots usually include the use of new
fungicides (Smilanick et al., 2006a; Kanetis et al., 2007), antagonistic microorganisms (El-
Ghaouth et al., 2000; Taqarort et al., 2008), and plant extracts (Ameziane et al., 2007; Du
Plooy et al., 2009). In recent years, several studies have been focused on screening of plant
extracts to develop new antifungal compounds that can be used to control postharvest citrus
diseases (Arras and Usai, 2001; Soylu et al., 2005). In this study the antifungal activity of
crude and aqueous extracts of 43 plants species was evaluated against G. candidum under
both in vitro and in vivo conditions.

The findings showed that most of tested plant powders reduced in vitro mycelial growth
of G. candidum with different degrees depending on the type of plant species used. The
response of pathogen to 10% (w/v) plant powders varied from a 21.74% promotion of
mycelial growth to 100% inhibition. Several higher plants, belonging to different botanical
families, have been found to possess excellent antifungal activity against mycelial growth of
different fungal pathogens (Qasem and Abu-Blan, 1996; Arras and Usai, 2001; Wang et al.,
2004; Neri et al., 2006). Previous reports mentioned that the effect of plant extracts on fungal
pathogens may be attributed to their secondary metabolites (e.g., alkaloids, phenolic,
flavonoids and terpenoids compounds) with known antifungal activity (Alilou et al., 2008;
Mohamed and El-Hadidy, 2008;).

The present study pointed out that plant powders may have potential antifungal activity
and widened the list of allelopathic plants to G. candidum growth. Indeed, similar evaluation
of 21 aromatic and medicinal plant species from Morocco showed that 14 of them reduced in
vitro growth of G. candidum by more than 20%. Among them, powders from five plants (e.i,
Eucalyptus globulus, Juglans regia, Peganum harmala, Thymus leptobotrys and Cistus
villosus) completely inhibited mycelial growth of the pathogen (Ameziane et al., 2007). The
remainder of the plant powders tested exhibited much lower antifungal activities against G.
candidum, and some even enhanced the mycelial growth of pathogen. A stimulatory effect on
mycelial growth of G. candidum induced by some plant powders (e.g., Lavandula multifida,
Mentha pulegium, and M. rotundifolia) was also reported by Ameziane et al. (2007).

Also of interest, we found that the powders of R. ulmifolius and C. siliqua completely
inhibited the mycelial growth of G. candidum. In a previous study, Sisti et al. (2008) found

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 Chapter 2: Screening of the antifungal activity of the aqueous plant extracts

that the crude extract of R. ulmifolius possess potent antifungal activity against a wide range
of human pathogens. According to these authors, the antifungal activity of the crude R.
ulmifolius extract may be ascribed to the high content of tannins. Ben-Hsouna et al. (2011),
have demonstrated the capacity of C. siliqua essential oil to control a panel of fungal strains,
particularly, fungi responsible in biodeterioration of food during postharvest processing.
Therefore, the antifungal activity of these plants extracts against postharvest decay
microorganisms of citrus fruits needs to be investigated.

The in vitro trials in this study showed that H. umbellatum, I. viscosa, R. ulmifolius, C.
villosus, C. siliqua, H. antiatlanticum and P. atlantica aqueous extracts gave consistent
antifungal activity against G. candidum. In particular, C. villosus and H. antiatlanticum
extracts proved to be the best mycelial growth and arthrospores germination inhibitors of the
pathogen. Antifungal activity against G. candidum has previously been shown in trials in vitro
or in vivo by some plant extracts and essential oils (Suprapta et al., 1997; Liu et al., 2009b),
while as far as is known, H. umbellatum, I. viscosa, R. ulmifolius, C. siliqua, H.
antiatlanticum and P. atlantica aqueous extracts have not been tested against this pathogen.

The effect of different plant powders and aqueous extracts was different according to
the stage of development of G. candidum. Results revealed that among plants which
completely inhibited mycelial growth of G. candidum, only C. villosus and H. antiatlanticum
have inhibited strongly the arthrospores germination of the pathogen. This confirms that
arthrospores germination is less sensitive to these plant extracts compared with mycelial
growth. This finding is in agreement with that reported by Garduno-Pizana et al. (2010) for
other plant extracts against Fusarium oxysporum [Link]. gladioli.

The aqueous extracts from H. antiatlanticum and C. villosus completely inhibited G.

candidum arthrospores germination, whereas that of I. viscosa, C. siliqua, H. umbellatum, and
R. ulmifolius reduced spores germination by 52.33, 32.67, 30.67 and 15.0%, respectively.
Suprapta et al. (1997), reported that among three groups of volatile compounds tested, only
alcohols and aldehydes compounds inhibited, by more than 50%, mycelial growth and
arthrospores germination of G. candidum; while esters had no effect. These two crucial steps
of the infection cycle of G. candidum, were inhibited by C. villosus and H. antiatlanticum to a
high extent compared with others tested plants.

The MIC for H. antiatlanticum and C. villosus aqueous extracts against G. candidum
was particularly low (0.156 mg/ml), followed by C. siliqua, H. umbellatum, and R. ulmifolius

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 Chapter 2: Screening of the antifungal activity of the aqueous plant extracts

aqueous extracts (0.3125 mg/ml). This antifungal effect was comparable to that reported by
other findings. Bouamama et al. (2006) reported that leaf extracts of C. villosus and C.
monspeliensis show antimicrobial properties with MIC values ranging from 0.78 to 50 mg/ml
for bacteria (e.g., Staphylococcus aureus and Pseudomonas aeruginosa) and 0.19 to 200
mg/ml for yeasts and fungi (e.g., Aspergillus fumigatus, Candida albicans, C. krusei and C.
glabrata). In the same study, the authors showed that C. villosus extracts exhibited more
interesting antimicrobial activity than C. monspeliensis (Bouamama et al., 2006). The genus
Halimium is known to be rich in diterpene compounds (Urones et al., 1995; Rodilla et al.,
1998), with antimicrobial activities (Kuzma et al., 2007; Weckesser et al., 2007). In previous
studies, the antimicrobial active components from P. lentiscus, a species closely related to P.
atlantica, were characterized as α-Pinene (Magiatis et al., 1999), β-myrcene, carvacrol
(Djenane et al., 2011), verbenone, α-terpineol, linalool (Koutsoudaki et al., 2005) and
flavonic compounds (Benhammou et al., 2008).

Although in vitro tests of plant extracts is an important first step in selecting plants with
antifungal potential against postharvest citrus pathogens, in vivo tests are needed to check
whether the positive results of the in vitro tests can be obtained, too (Gorris and Smid 1995;
Tegegne et al., 2008). Results obtained in this study indicated that aqueous extracts of seven
plant species viz., H. umbellatum, I. viscosa, R. ulmifolius, C. villosus, C. siliqua, H.
antiatlanticum and P. atlantica significantly decreased the incidence and severity of sour rot,
without causing phytotoxic effects. In most commercial packing houses in SMD, pre-storage
chemical control with guazatine is commonly applied, as postharvest drench, to reduce the
incidence of sour rot in citrus fruit that are stored in cold before processing. However, this
fungicide is not registered in several countries. According to Hao et al. (2010), the lack of
registered fungicide in many countries for sour rot control is becoming a serious problem. In
this study, mandarins treated with aqueous extracts of C. villosus and H. antiatlanticum plants
were highly effective in reduction of sour rot incidence, compared with non-treated fruit.
Several studies have tested different plants extracts in controlling the same pathogen or others
and found similar effects (Neri et al., 2006; Ribera et al., 2008; Yahyazadeh et al., 2008; Liu
et al., 2009b).

Treating mandarins with aqueous extracts of the seven plants significantly reduced the
lesion diameters. However, none of these plant extracts have completely controlled the sour
rot development. We found no publication on the antifungal activity of the selected plant
species. Considering antifungal activity of H. umbellatum, I. viscosa, R. ulmifolius, C.

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 Chapter 2: Screening of the antifungal activity of the aqueous plant extracts

villosus, C. siliqua, H. antiatlanticum and P. atlantica against G. candidum, it is possible that

extracts of these plants can be used as natural control agents. Most of these plants are widely
distributed in SMD Valley and easy grown. Furthermore, the extraction method is simple
using the most available solvent (water). Moreover, the use of a crude plant extract to protect
citrus fruit against fungal attack may be acceptable in the organic production of citrus fruit.

V. Conclusion

This work is a part of an overall study that aims to determine the antifungal activity of
natural floral resources of south of Morocco, against major postharvest citrus fungal
pathogens. Among the 43 plants tested C. villosus, H. antiatlanticum, H. umbellatum, P.
lentiscus and I. viscosa showed high antifungal activities against Geotrichum candidum both
in-vitro and in-vivo. These plants possess potent antifungal activities with potential practical
applications in the treatment of postharvest sour rot of citrus fruits and should be tested in
future under degreening conditions. Moreover, new research should focus on the
phytochemical analysis to identify the active principles responsible for the antifungal effect of
each plant.

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 Chapter Three :

Evaluation of the effectiveness of

organics fractions of the most active
plants species, against G. candidum
Publised in Letters in applied microbiology as :
Talibi, I., Askarne, L., Boubaker, H., Boudyach, E.H., Msanda, F., Saadi, B., Ait
Ben Aoumar, A., 2012. Antifungal activity of Moroccan medicinal plants
against citrus sour rot agent Geotrichum candium. Letters in applied
microbiology 55, 155-161.
 Chapter 3: Effectiveness of organics fractions

Résumé
Les plantes qui ont montré un fort pouvoir antifongique ont été retenues pour évaluer
les effets de leurs fractions organiques contre G. candidum. Ceci est dans l‟objectif
d‟identifier les fractions les plus actives et aussi pour améliorer leur activité antifongique
contre l‟gent pathogène. Les espèces suivantes : C. villosus, H. antiatlanticum, H.
umbellatum, P. atlantica, I. viscosa, A. radiata, R. ulmifolius et C. siliqua ont été,
successivement, extraites avec l‟hexane, le chloroforme, l‟acétate d‟éthyle et le méthanol.
Ceci nous a permis d‟isoler quatre fractions organiques pour chaque plante. Ces fractions ont
été évaluées pour leur activité antifongique, in vitro et in vivo, contre G. candidum.L‟effet des
fractions a été évalué sur la croissance mycélienne du pathogène par la méthode de diffusion
au niveau des puits. Les résultats obtenus montrent que les espèces testées ont montré
différents degrés d‟inhibition de la croissance de G. candidum. En effet, le pouvoir inhibiteur
varie avec l‟espèce et le type de solvant d‟extraction. . Les extraits méthanoliques de C.
villosus, H. umbellatum, C. siliqua, R. ulmifolius, H. antiatlanticum, P. atlantica et les extraits
d‟acétate d‟éthyle de C. villosus, A. radiata et C. siliqua ont fortement inhibé la croissance
mycélienne du pathogène. En revanche, les extraits chloroformiques et hexaniques de toutes
les plantes testées n‟ont aucun effet sur la croissance mycélienne de G. candidum. De même,
les fractions polaires des plantes testées (méthanolique et d‟acétate d‟éthyle) ont fortement
inhibé la germination des arthrospores de G. candidum comparés aux fractions apolaires
(hexanique et chloroformique). En effet, l‟extrait méthanolique de C. villosus, H.
umbellatutm, H. antiatlanticum, [Link], R. ulmifolius et l‟extrait d‟acétate d‟éthyle de P.
atlantica et A .radiata ont inhibé la germination des arthrospores de G. candidum à plus de
92%. Les plus faibles concentrations inhibitrices (CMI) ont été obtenues par les extraits
méthanoliques de H. umbellatum (0,156 mg/ml), C. villosus et R. ulmifolius (0,625 mg/ml).
Les plus faibles concentrations minimales fongicides ont été enregistrées avec les extraits
méthanoliques de C. villosu, H. umbellatum (2,5mg/ml) et C. siliqua (5mg/ml). Les autres
extraits organiques (Hexanique, chloroformique et éthylique) n‟ont montré aucun caractère
fongicide aux concentrations testées. Sur la base de ces résultats, seuls les extraits
méthanoliques et d‟acétate d‟éthyle (fractions polaires) des plantes testées ont été retenus pour
évaluer leur capacité à réduire l‟incidence et la sévérité de la pourriture amère. Les résultats
obtenus ont montré que les extraits méthanoliques de C. villosus, C. siliqua, H. umbellatum,
H. antiatlanticum et R. ulmifolius ont significativement réduit l‟incidence et la sévérité de la
pourriture amère, par rapport au témoin. En effet, le traitement en préventif des fruits de
mandarine avec l‟extrait méthanolique de C. villosus a totalement inhibé le développement de

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 Chapter 3: Effectiveness of organics fractions

l‟agent pathogène. En plus, l‟extrait méthanolique de C. siliqua et H. umbellatum ont

respectivement réduit l‟incidence de la pourriture amère à seulement 3,3 et 11,66%, par
rapport au témoin (95%).

Mots clés : Activité antifongique, Extraits de plantes, pourriture amère, Geotrichum

candidum, Fruits d‟agrumes.

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 Chapter 3: Effectiveness of organics fractions

Abstract
The aim of this work was to find an alternative to the chemical fungicide currently used
in the control of Geotrichum candidum, the cause of citrus sour rot. Here we evaluated
methanol, ethyl acetate, chloroform and hexane extracts from eight plant species, harvested in
different regions of southern Morocco, for their in vitro and in vivo antifungal activities
against G. candidum. All plant species showed antifungal potential and the magnitude of their
inhibitory activities was species and solvents dependent. Methanol and ethyl acetate extracts
were found to inhibit the growth of G. candidum in a dose-dependent manner. Cistus villosus,
Ceratonia siliqua and Halimium umbellatum methanol extracts proved to be the most
effective inhibitors, totally inhibiting arthrospore germination of G. candidum at
concentrations of 2.5 mg/mL and lower. The methanol extracts of H. umbellatum, C. villosus
and C. siliqua exhibited strong antifungal activity with minimum inhibitory concentrations
values ranged between 0.156 and 1.25 mg/mL, and minimum fungicidal concentrations values
ranged between 2.5 and 5 mg/mL. The hexane and chloroform extracts of the studied species
showed the lesser activity. The most active solvent extracts (methanol and ethyl acetate) in in
vitro studies were tested in vivo against sour rot on citrus fruit. Incidence of sour rot was
lowered to 0.00, 3.33 and 11.66% when mandarin fruit were treated with C. villosus, C.
siliqua and H. umbellatum methanol extracts at 50 mg/mL, respectively, compared with 95%
in the control. These plant species reduced disease severity from 100% in non-treated fruit to a
0% with C. villosus, 1.8% with C. siliqua and 8.23% with H. umbellatum. Poor control was
recorded for ethyl acetate extracts. Phytochemical analysis of the most active fractions
showed that they contain different classes of flavonoids and possess high levels of total
phenols. These findings suggest that C. villosus, H. umbellatum and C. siliqua plants may be
useful and effective agents for control of citrus sour rot. Such natural products therefore
represent a sustainable alternative to the use of synthetic fungicides.

Keywords : Antifungal activity; Plant extracts; Sour rot; Geotrichum candidum; Citrus fruit

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 Chapter 3: Effectiveness of organics fractions

I. Introduction

Citrus is one of the most important fruit crop in Morocco, with an annual production of
about 1.5 million tons from 76.500 ha under this crop. The largest volume of citrus fruit for
fresh consumption and export in Morocco is grown and shipped from packing houses in the
Souss-Massa-Draa (SMD) region (Boubaker et al., 2009).

Fungal diseases are the major cause of postharvest rots of fresh citrus fruit during
storage and transport (Eckert and Eaks, 1989). Sour rot, caused by Geotrichum candidum
Link ex Pers. (syn. G. citri-aurantii), has been reported as an important postharvest disease of
citrus fruit from most areas of the world (Eckert, 1978; Rippon and Morris, 1981; Brown,
1988; Hershenhorn et al., 1992). In Morocco, production losses in commercial packing
houses may be high under favorable conditions for pathogen development, especially during
fruit degreening, wet and rainfall seasons. Fruit become more susceptible to sour rot as they
increase in maturity (Brown, 1979; Baudoin and Eckert, 1985) and at favorable temperatures
(25-30°C) for which fruit will rot completely in four to five days. The pathogen infects the
rind through wounds made by insects or by mechanical means and causes internal
disintegration and maceration of the whole fruit during storage (Brown, 1979). In addition to
citrus fruit, G. candidum is the causal agent of numerous decays of other fruits and vegetables
(Brown, 1979; Suprapta et al., 1995; Wells, 1997; Palou et al., 2009).

In humid conditions such as that during degreening, this fungal disease is difficult to
control, and frequent applications of fungicides are used to prevent sour rot (Brown, 1979).
The measures employed to manage postharvest citrus rot are not effective against G.
candidum. This pathogen is not controlled by any of the fungicides (e.g., Imazalil and
Thiabendazole) registered for use on citrus fruit (Brown and Miller, 1999; Liu et al., 2009b).
The disease can be partially reduced by sodium o-phenyl phenate (Rippon and Morris, 1981;
Feng et al., 2011), careful harvesting and handling of fruit, and by low-temperature storage
(Eckert and Brown, 1988). In most commercial packing houses in the SMD Valley, pre-
storage chemical control with guazatine is commonly applied as postharvest drench to reduce
the incidence of sour rot in citrus fruit that are stored in cold before processing or before
degreening operations. However, this fungicide is not registered in several countries.
Currently, there is no potentially effective natural product to serve as an alternative in
controlling sour rot. Therefore the challenge is to develop effective and safe strategies for the
control of citrus sour rot. Over the last years, biological control using naturally-occurring

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substances has emerged as an effective and safe strategy to control the citrus postharvest
diseases. Natural products such as herbal extracts have gained popularity and scientific
interest for their antibacterial and antifungal activity (Wilson et al., 1991; Tripathi and Duby,
2004; Abd-El-Khair and Hafez, 2006; Abad et al., 2007; Ameziane et al., 2007; Liu et al.,
2009b; du Plooy et al., 2009; Gatto et al., 2011). Plant extracts are very attractive as
alternative or complementary control means because of their antimicrobial activity, non-
phytotoxicity, systemicity, and biodegradability (Fawcett and Spencer, 1970; Tripathi and
Dubey, 2004; Kosalec et al., 2005; Ameziane et al., 2007; Gatto et al., 2011; Talibi et al.,
2011a). In a recent screening study (Talibi et al., 2012a), the powders and aqueous extracts of
43 plant species have been tested for their antifungal activity against G. candidum. Among
these 43 plants, eight species showed the best results against G. candidum (Talibi et al.,
2012a). Very little work has been done to investigate the use of natural plant products to
control citrus sour rot.

The aim of the present study is to determine the inhibitory effects of eight medicinal
plants, extracted in different organic solvents (hexane, chloroform, ethyl acetate and
methanol), on the growth of G. candidum under both in vitro and in vivo conditions, and to
obtain preliminary information on the chemical composition of the plant extracts.

II. Materials and methods

II.1 Collection of Plant Samples

Eight fresh plant samples were collected from different locations in the Souss valley
(Agadir, south of Morocco). The species of all samples were identified (Table 3.1), and were
deposited in the herbarium of the laboratory of Biotechnology and Valorization of the Natural
Resources, Faculty of Sciences, Ibn Zohr University, Agadir, Morocco. Plant samples were
cleaned, air dried in the shade, then grounded to a fine powder using a laboratory grinding
mill (Polymix PX-MFC 90D, Switzerland) and stored in the dark at 4°C until use. The
botanical name, family, and parts used of plant samples are summarized in Table 3.1. The
eight plant species belong to seven genera and five botanical families.

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II.2 Preparation of plant extracts

Preparation of the plant samples for the in vitro antifungal screening against G.
candidum was conducted as previously described by Duraipandiyan and Ignacimuthu (2007)
and Bajpai et al. (2008). Briefly, powder (20g) of each plant species was extracted with 100
mL of hexane by maceration with stirring at room temperature for 48 h and then filtered
through Whatman no. 1 filter paper to obtain particle free extract. The solvent of the filtrate
was removed by evaporation to dryness under reduced pressure at 40°C. The remains of the
plant material were extracted with chloroform, ethyl acetate and methanol sequentially in a
similar manner (Figure 3.1) and yield was determined for each extraction (Table 3.1). Each
dried plant extract was dissolved at a concentration of 200 mg/mL in the same solvent of
extraction. The extracts were then sterilized by passing through 0.2µm pore diameter
Millipore Swinex filters and kept at -20°C before assay. Solvents (analytical grade) for
extraction were obtained from commercial sources.

Figure 3.1 : Steps of preparation of organic fractions

II.3 Pathogen

The G. candidum isolate used in this study was obtained from a decayed mandarin fruit.
It is one of the most aggressive isolates in our collection. This isolate was deposited at the
laboratory of Biotechnology and Valorization of the Natural Resources under the code SR10.

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The fungus was maintained on potato dextrose agar (PDA) plates at 5°C, with periodic
transfers through citrus fruit to maintain its aggressiveness. The pathogen inoculum consisted
of aqueous arthrospores suspensions obtained from seven-day old culture plates incubated at
25°C. Arthrospores were harvested by flooding plates with 5 mL of sterile distilled water
containing 0.05% (v/v) Tween 80, and passing the suspension through two layers of sterile
cheesecloth to remove hyphal fragments. The arthrospores concentration was determined with
the aid of a heamacytometer and adjusted to 106 arthrospores/mL with sterile distilled water.

II.4 Fruit

Fruits of mandarin (Citrus reticulata Blanco cv. Clementine) were used. Fruits were
harvested from orchards of the M‟brouka cooperative, which used standard culture practices,
in the Souss-Massa-Draa Valley, southern Morocco. Only healthy and commercially mature
fruits were used in the in vivo test. Freshly harvested or briefly stored (no longer than a week)
fruits were used in the experiment.

II.5 Evaluation of antifungal activity of plant extracts

II.5.1 In vitro effect of solvents extracts on mycelial growth of G.

candidum
The solvent extracts were screened for their antifungal activity using the well-plate
diffusion method. Aliquots (100 µL) of a spore suspension (106 arthrospores /mL) of G.
candidum was overlaid on Petri plates (90 mm in diameters) containing 15 mL of PDA. Then
five-mm diameter wells were made at three locations per dishes. The wells are then filled with
20 µL of solvent extract at concentration of 2.5, 5 and 10 mg/mL. Control plates consisted of
wells filled with 20µL of the same solvent of extraction. The plates are stored at 4°C for 2
hours to allow the diffusion of extract, and then incubated at 26°C (Figure 3.2). After 48
hours incubation, the diameter (mm) of the inhibition zone was measured. By using eight
plant species, four solvents of increasing polarities, and three concentrations, 96 treatments
were generated. Three plates were used for each treatment as replications, and each
experiment was repeated twice.

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Figure 3.2 : In vitro effect of solvents extracts on mycelial growth of G. candidum

II.5.2 Effect of solvents extracts on arthrospores germination

The germination of arthrospores of G. candidum was determined in concentrations of

1.25, 2.5, 5, 10 and 20 mg/mL of each solvent extracts. Aliquots (40µL) of a spore suspension
(106 arthrospores/mL) were aseptically transferred in triplicate to sterile depression slides
containing 40µL of 2% sterile orange juice amended with different concentrations of solvent
extracts. Inoculated slides were placed on moist filter paper in Petri plates, sealed with
parafilm to avoid evaporation, and then incubated at 25°C for 24h. Each slide was then fixed
with acid fuchsine solution to stop further germination (Smilanick et al., 1999). Arthrospores
germination was estimated under a microscope using a micrometer. At least, 100 arthrospores
of each replicate were observed. An arthrospore was scored as germinated if the germ tube
length was equal or exceed that of the spore (Suprapta et al., 1997). The data were expressed
as percent spore germination inhibition and calculated by using the following formula :

GI (%) = [(Gc−Gt) /Gc)] ×100, where Gc and Gt represent the mean number of germinated
spores in control and treated slides, respectively (Soylu et al., 2010). Each treatment included
three replicates and the experiment was conducted twice.

II.5.3 Determination of Minimum inhibitory concentration (MIC)

and minimum fungicidal concentration (MFC)
The minimal inhibitory concentrations (MICs) of the plant extracts were determined by
the agar dilution method. Organic plants extracts were first diluted to the highest
concentration to be tested (5 mg/mL), and then serial two-fold dilution were made in a
concentration ranging from 5 to 0.3125 mg/mL in 10mL test tubes containing melted PDA
medium. Culture plates with 24 microwells and a capacity of 4 mL per well were used.

Solvent plant extracts amended medium was dispended in the wells of culture plates (2 mL
per well). Aliquots (10µL) of an arthrospores suspension (106arthrospores/mL) were then

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dropped onto the surface of the agar medium. Culture plates were incubated at 24°C (±1°C)
for 48 h. The MICs were recorded by reading the lowest solvent extract concentration that
allowed no visible growth of the pathogen (Phongpaichit et al., 2005).

The MFCs were determined by taking agar plugs from well showing no visible mycelial
growth and re-inoculating them on unamended PDA medium. MFC was regarded as the
lowest concentration of the solvent plant extract that prevented growth of the pathogen after
the period of incubation, and indicating more than 99.5% killing of the original inoculums
(Fabry et al., 1996; Ali-Shtayeh and Abu Ghdeib, 1999). There were three replicates for each
plant solvent extract at each concentration and the experiment was conducted twice.

II.5.4 Effects of solvent extracts on sour rot development in

artificially inoculated and wounded fruit
Based on in vitro screening results, only methanol and ethyl acetate extracts of the
plant species were tested. Mandarin fruits were washed, disinfected with 0.1% (v/v) sodium
hypochlorite, rinsed three times in sterile distilled water and then air dried before wounding.
One wound (2mm deep and 4mm wide) was made per fruit using a sterile needle at the
equatorial side (Liu et al., 2009b). The wounds were treated with 30 µL of solvent extracts at
a concentration of 50 mg/mL. Controls were treated with the same volume of sterile distilled
water under the same conditions. After two hours incubation at room temperature, each
wound was inoculated with 20 µL of an aqueous suspension of arthrospores of G. candidum
(106 arthrospores/mL). Treated fruits were placed on plastic tray in carton boxes and
incubated at 26°C and 95% relative humidity (RH). The number of the infected wounds and
the lesion diameters of the overall treated fruit were determined daily. All treatments were
arranged in a complete randomized block design. Eighteen fruits constituted a single replicate
and each treatment was replicated three times. The experiment was conducted twice. The
incidence and severity of disease were calculated as follows :

Disease incidence (%) = [(number of rotten wounds / number of total wounds)] x 100.

Disease severity (%) = [(average lesion diameter of treatment / average lesion diameter of
control)] x 100.

In all experiments, the possible phytotoxic effect on mandarin fruit was examined.

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II.6 Phytochemical analysis

The plant extracts who gave best control of sour rot were selected for their qualitative
phytochemical analysis to identify the presence of alkaloids and flavonoids. The analysis was
done using aluminium thin layer chromatography (TLC) plates (silica gel 60 F254 plates, 20 x
20 cm, Merck). The plates were developed with the eluent system ethyl acetate-methanol-
water (40/5.4/5, v/v/v). Visualization of alkaloids and flavonoids compounds was achieved by
spraying the plates with freshly prepared Dragendorff (alkaloids) and Neu (flavonoids)
reagents, respectively. The principal phytochemical constituents were characterized with
colorful reactions and by the establishment of their chromatographic profiles by TLC.
Detection was carried out visually under UV light at 365 nm.

Also, total phenolic content (TPC) of these extracts was measured by the Folin-
Ciocalteu assay method (Slinkard and Singleton, 1977) with slight modifications. To 0.5 mL
of a diluted methanolic extracts, 0.5 mL of Folin-Ciocalteau reagent was added and mixed
vigorously. After five minutes, 0.5 mL of 20% sodium carbonate (Na2CO3) was added and
mixed vigorously again. After 1min of shaken, the solution was brought up to 5 mL by adding
distilled water. The control reaction contained all reagents except the plant extracts. The
reaction mixture was then incubated in the dark at 25 °C for 90 min, and the absorbance of the
resulting color was measured at 760 nm against a distilled water/sodium carbonate blank. The
concentration of the TPC was estimated as mg of gallic acid equivalent (GAE) per gram of
plant extract by using a standard gallic acid calibration curve. Values presented are the
average of three measurements.

II.7 Statistical analysis

All data were subjected to statistical analysis of variance (ANOVA) using

STATISTICA software, version 6 (Stat-Soft, 2001, France). Percentage values were subjected
to arcsine square root transformation before analysis of variance. Mean separation was
performed following the Newman & Keuls test at P <0.05.

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III. Results

III.1 In vitro effect of solvents extracts on mycelial growth of G. candidum

The eight plant species were extracted by four solvents of increasing polarities: hexane,
chloroform, ethyl acetate and methanol. Methanol and ethyl acetate were the best solvents,
extracting a larger quantity of material (Table 3.1). The highest yield was obtained for
methanol extract of Halimium antiatlanticum (32.6%) and the lowest yield was recorded for
hexane extract of Rubus ulmifolius (2.5%).

Table 3.1: Plants used in this study and yield extracted (%) with each solvent
Extracted yield (%)
Family Plant species Part used
Hex Chl [Link] MeOH

Cistaceae Halimium umbellatum Leaves+stem 10.9 7 12.3 27.5

Asteraceae Anvillea radiata Leaves+stem 3.4 27 13.4 16.3

Cistaceae Cistus villosus Leaves+stem 4.4 7 12.1 22.4

Asteraceae Inula viscosa Leaves+stem 4.4 10 13.9 19.6

Cistaceae Halimium antiatlanticum Leaves+stem 1.7 2.6 14 32.6

Rosaceae Rubus ulmifolius Leaves+stem 2.5 4.1 14.7 22.1

Fabaceae Ceratonia siliqua Leaves 6.2 5.5 18.5 29

Anacardiaceae Pistacia atlantica Leaves 6.4 8.9 12.4 32.4

The results of the antifungal screening of methanol, ethyl acetate, chloroform and hexane
extracts of eight plant species at different concentrations are given in Table 3.2. As shown,
the methanol and ethyl acetate extracts were more active than the hexane and chloroform
extracts. Both methanol and ethyl acetate plant extracts showed antifungal activity against G.
candidum at concentration-related manner. At the concentration of 10 mg/mL, the diameter of
the inhibition zones produced against G. candidum was ranged from 20.67 to 24 mm for the
methanolic extracts from Halimium umbellatum, Cistus villosus and Ceratonia siliqua, and
20.7 to 22.7 mm for the ethyl acetate extracts from Anvillea radiata and Pistacia atlantica
(Figure 3.3). It was observed that the antifungal activity resided mainly in the methanol and
ethyl acetate extracts. Also, hexane and chloroform extracts from P. atlantica exhibited low
antifungal effect with diameter zones of inhibition of 13.7 and 11.7 mm, respectively.
However, no inhibitory effects of the hexane and chloroform extracts were observed against
G. candidum for the others tested plant species (Table 3.2). In this screening, methanol and

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ethyl acetate extracts were shown to exhibit, in vitro, a significant antifungal activity against
G. candidum. This indicates that the two organic extracts contain active compounds which are
responsible at least in part for the antifungal activity.

Table3.2. In vitro effects of solvents extracts on mycelial growth of Geotrichum candidum

a
Diameter of the inhibition zone (mm)
Extraction
Plant species Concentration of plant extracts (mg/mL)
solvent
2.5 5 10
Hexane 0.00a 0.00a 0.00a
Chloroform 0.00a 0.00a 0.00a
Anvillea radiata
Ethyl acetate 6.70bcde 17.70qr 22.70t
Methanol 0.00a 6.00bcd 7.30def
Hexane 0.00a 0.00a 0.00a
Chloroform 0.00a 0.00a 0.00a
Halimium umbellatum
Ethyl acetate 11.70ij 14.30lmn 17.00pqr
gh qr
Methanol 9.30 18.00 22.70t
Hexane 0.00a 0.00a 0.00a
Chloroform 0.00a 0.00a 0.00a
Ceratonia siliqua
Ethyl acetate 0.00a 11.30ij 15.70nop
Methanol 8.70fg 12.00ijk 20.67s
Hexane 0.00a 0.00a 0.00a
Chloroform 0.00a 0.00a 0.00a
Cistus villosus
Ethyl acetate 12.66jkl 15.66nop 21.00s
opq s
Methanol 16.33 20.66 24.00t
Hexane 4.30b 7.70cdef 13.70klm
Chloroform 0.00a 8.70fg 11.70ij
Pistacia atlantica
Ethyl acetate 14.30lmn 15.70nop 20.70s
Methanol 13.70klm 17.70qr 18.70r
Hexane 0.00a 0.00a 0.00a
Chloroform 0.00a 0.00a 0.00a
Rubus ulmifolius
Ethyl acetate 0.00a 6.86cde 12.23ijk
efg jkl
Methanol 8.00 12.86 17.56qr
Hexane 0.00a 0.00a 0.00a
Halimium Chloroform 0.00a 0.00a 0.00a
antiatlanticum Ethyl acetate 0.00a 10.60hi 15.00mno
Methanol 8.66fg 14.00lmn 16.66opq
Hexane 0.00a 0.00a 0.00a
Chloroform 0.00a 0.00a 0.00a
Inula viscosa
Ethyl acetate 0.00a 6.00bcd 7.00cdef
Methanol 0.00a 5.73bc 7.16bcde
a
Values are means of three replicates. Values followed by the same letters were not significantly
different (P <0.05) according to Newman and Keels test.

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Methanol fraction of C. v Methanol fraction of H.u Methanol fraction of C.s

Ethyl acetat fraction of A.r Ethyl acetat fraction of P.a Control

Figure 3.3 : Inhibition zone of G. candidum around the wells filled with the organic fractions of
Cistus villosus (C.v), Hallimium umbellatum (H.u), Ceratonia siliqua (C.s), Anvillea radiata (A.r) and
Pistacia atlantica (P.a) compared with control

III.2 Effect of solvents extracts on arthrospores germination

The susceptibility of G. candidum arthrospore towards various concentrations of the
organic extracts of eight plant species is summarized in Table 3.3. In general, methanol and
ethyl acetate plant extracts presented higher inhibition of arthrospores germination than
hexane and chloroform extracts. Indeed, methanol extracts from C. villosus and C. siliqua
induced 100% inhibition of arthrospore germination at the lowest concentration tested (1.25
mg/mL) (Figure 3.4), while ethyl acetate extracts from A. radiata and H. umbellatum, and
chloroform extract from P. atlantica required 10 mg/mL to bring the same effect. All applied
concentrations of methanol extract from H. umbellatum, except 1.25 mg/mL, completely
inhibited the arthrospores germination of G. candidum. Methanol extract from H.
antiatlanticum exhibited less toxicity and only concentrations of 5 mg/mL and above reduced
the percentage of arthrospores germination by more than 90%. For the hexane extracts, only
extract of P. atlantica reduced the percentage of arthrospores germination by more than 50 %
at 10 mg/mL (Table 3.3).

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Table 3.3 : In vitro effect of solvents extracts on spore germination of Geotrichum candidum
a
Spore germination inhibition (%)
Extraction
Plant species Concentration of plant extracts (mg/ mL)
solvent
1.25 2.5 5 10 20
Hexane 0.00z 0.00z 0.00z 0.00z 0.00z
Chloroform 0.00z 1.00yz 21.00op 39.34kl 59.34h
Anvillea radiata
E. acetate 78.67f 82.67e 93.34b 100.00a 100.00a
Methanol 0.00z 0.00z 0.00z 2.00yz 3.00yz
Hexane 0.00z 0.00z 0.00z 0.00z 0.00z
z z z z
Halimium Chloroform 0.00 0.00 0.00 0.00 0.00z
umbellatum E. acetate 0.00z 0.00z 16.67qr 100.00a 100.00a
Methanol 64.00h 100.00a 100.00a 100.00a 100.00a
Hexane 0.00z 0.00z 0.00z 0.00z 0.00z
Chloroform 0.00z 12.34stu 17.34pq 18±0pq 18.67opq
Ceratonia siliqua
E. acetate 0.00z 0.00z 2.00yz 7.67vwx 24.67n
Methanol 100.00a 100.00a 100.00a 100.00a 100.00a
Hexane 0.00z 0.00z 0.00z 0.00z 0.00z
yz wx uvw tuv
Chloroform 2.34 7.00 9.00 10.67 12.34stu
Cistus villosus
E. acetate 0.00z 0.00z 0.00z 10.00tuv 19.00n
Methanol 100.00a 100.00a 100.00a 100.00a 100.00a
Hexane 0.00z 0.00z 22.34n 57.67h 69.67g
Chloroform 87.34d 91.67b 94.67b 100.00a 100.00a
Pistacia atlantica
E. acetate 0.00z 3.00yz 31.67m 41.67±1.11k 92.00b
Methanol 0.00z 0.00z 0.00z 51.34±2.22j 69.34g
Hexane 0.00z 0.00z 0.00z 0.00z 0.00z
z z xy pq
Chloroform 0.00 0.00 5.00 17.34 36.34l
Rubus ulmifolius
E. acetate 18.67pq 36.00l 61.34h 69.34g 81.67e
Methanol 8.67uvw 38.67kl 59.00h 90.40c 97.00a
Hexane 0.00z 0.00z 0.00z 0.00z 0.00z
Halimium Chloroform 0.00z 0.00z 8.00vwx 13.34rst 30.00m
antiatlanticum E. acetate 0.00z 19.67opq 28.34m 64.67h 72.34g
Methanol 15.34qrs 50.67j 90.67cd 98.67a 99.67a
Hexane 0.00z 0.00z 0.00z 0.00z 0.00z
Chloroform 0.00z 0.00z 0.00z 0.00z 0.00z
Inula viscosa
E. acetate 0.00z 0.00z 0.00z 0.00z 0.00z
Methanol 0.00z 0.00z 1.34yz 2.00yz 4.00yz
a
Each value represents the mean of three replicates. Means followed by different letter (s) in
each column are significantly different at P < 0.05. Bold type : % inhibition >80%.

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 Chapter 3: Effectiveness of organics fractions

a b

c
Figure 3.4 : Germination of arthrospores of G. candidum 24h after treatment with methanol extract of
C. villous (a) and H. umbellatum (b) at 1.25 mg/ml compared with control (c)

III.3 MIC and MFC

The MIC values showed that the activity of the plant extracts varied depending on the
solvent used for extract preparation (Table 3.4). Methanol extracts of H. umbellatum, C.
villosus, R. ulmifolius and C. siliqua were the most active with MIC values of 0.156, 0.625,
0.625 and 1.25 mg/mL, respectively. For the others solvents, only chloroform and ethyl
acetate extracts of P. atlantica have inhibited growth of G. candidum at 5 mg/mL (Table 3.4).
On the other hand, the best MFC value was obtained by methanol extracts of H. umbellatum
and C. villosus which are fungicidal at 2.5 mg/mL (Table 3.4).

III.4 In vivo test

The data presented in Figure 3.5 showed that all tested methanol plant extracts
significantly reduced the incidence of sour rot caused by G. candidum under the laboratory
conditions. Percentages of rotted wounds were decreased by using all tested methanol extracts
compared with control. Mandarin fruit treated 2 hours before pathogen inoculation by
methanol extracts of C. villosus, H. umbellatum and C. siliqua, at 50 mg/mL, reduced the
incidence of sour rot to 0.0, 3.33 and 11.66%, respectively (Figure 3.5). Methanol extracts of
R. ulmifolius and H. antiatlanticum have also reduced the percentage of rot incidence to 30

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 Chapter 3: Effectiveness of organics fractions

and 30.66%, respectively, compared to the control (95%). I. viscosa, P. atlantica and A.
radiata methanol extracts have a moderate effect on sour rot, the percentage of rot incidence
varying between 50 and 65% (Figure 3.5).

Table 3.4 : Minimal inhibitory concentrations (MICs) and minimal fungicidal concentrations
(MFCs) of selected plant species
a
MICs and bMFCs (mg/ mL)
Treatement
Hex Chl Met E. Ac

MIC MFC MIC MFC MIC MFC MIC MFC

H. umbellatum >5 >5 >5 >5 < 0.156 2.5 >5 >5

[Link] >5 >5 >5 >5 5 >5 >5 >5

C. villosus >5 >5 >5 >5 0.625 2.5 >5 >5

I. viscosa >5 >5 >5 >5 >5 >5 >5 >5

H. antiatlanticum >5 >5 >5 >5 >5 >5 >5 >5

R. ulmifolius >5 >5 >5 >5 0.625 >5 >5 >5

C. siliqua >5 >5 >5 >5 1.25 5 >5 >5

P. atlantica >5 >5 5 >5 >5 >5 5 >5

a
Concentrations that were fungistatic and b Concentrations that were fungicidal

100 g g fg
90 fg efg
Disesae incidence (%)

efg defg
80 defg defg
70 cde cdef
60 cd c
50 MeOH extract
40 b b [Link] extract
30
20 a
10 a a
0

Figure 3.5 : Effect of solvents extracts on sour rot incidence in mandarin wounds. Fruit were treated
with aqueous extracts, inoculated with G. candidum and held for 7 days at 26°C. Significant
differences (P < 0.05) between means were indicated by different letters above histogram bars.

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 Chapter 3: Effectiveness of organics fractions

Also, the tested methanol extracts exhibited significant reduction of disease severity
compared with the control (Figure 3.6 and figure 3.7). They reduced disease severity from
100% in non-treated fruit to a 0% with C. villosus, 1.8% with C. siliqua, 8.23% with H.
umbellatum, 22.27% with R. ulmifolius, 23% with H. antiatlanticum and 31.82% with P.
atlantica. In contrast, ethyl acetate extracts of all tested plant species are less effective than
methanol ones (Figure 3.7). The percent of infected fruits varied between 60 and 90%
(Figure 3.5), and the disease severity between 38.63% (for P. atlantica) and 87.52% (for I.
viscosa) (Figure 3.6). Furthermore, neither of the plant extracts showed any phytotoxic
reaction on treated fruit, at the concentration tested (50 mg/mL).

110 j j
ij
Disease severity (%)

90 hi hi
ghi
70 fgh
ef efg
de e
50 cd MeOH extract
bc
30 b b [Link] extract
10 a a
a
-10

Figure 3.6 : Effect of solvents extracts on sour rot severity in mandarin wounds. Significant
differences (P < 0.05) between means were indicated by different letters above histogram bars.

C C.v.M C.v.E

Figure 3.7: Control of sour rot by methanol extract of Cistus villosus (C.v.M) compared with Ethyl
acetate extract of the same plant specie (C.v. E) and control (C).

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 Chapter 3: Effectiveness of organics fractions

III.5 Phytochemical analysis

The chemical screening of plant extracts, by using Neu reagent, permitted to detect
flavonoids in C. villosus, H. umbellatum, H. antiatlanticum, C. siliqua and R. ulmifolius.
However, for the alkaloids, Dragendorff reagent has revealed only minor bands with a light
orange color in all extracts analyzed.

The total phenolic contents of methanol extracts of selected plant species as determined
by Folin-Ciocalteu method are reported as gallic acid equivalent (GAE) (Figure 3.8). Among
analyzed plant extracts, methanol extracts from C. siliqua showed the highest amount of
phenolic compounds (165.2 mg GAE/g extract) followed by H. umbellatum and C. villosus
(139.46 and 136.13 mg GAE/g extract, respectively).

180 d
160 a
a
140
c
mg GAE/g extract

120

100 b
80

60

40

20

0
H. u C. s H. a C. v R. u

Figure 3.8 : Levels of total phenols present in selected plant species, H.u: Hallimium umbellatum, C.s
: Ceratonia siliqua, H.a : Hallimium antiatlanticum, C.v : Cistus villosus, and R.u : Rubus ulmifolius.
Results are expressed as mg of gallic acid per g of plant extract. The values represent the mean of
triplicates ± standard deviation.

IV. Discussion

Management of fungal diseases of citrus fruit continues to be one of the greatest disease
challenges in commercial packing houses. Currently novel tools, such as the use of new
fungicides and salts (Smilanick et al., 2006a; Kanetis et al., 2007; Talibi et al., 2011b),
antagonistic microorganisms (Leelasuphakul et al., 2008; Taqarort et al., 2008; Lahlali et al.,
2010), and plant extracts (Ameziane et al., 2007; Du Plooy et al., 2009), are tested as an
alternative to currently used fungicides for control of postharvest citrus fruit rots. In recent
years, several studies have been focused on screening of plant extracts to develop new

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 Chapter 3: Effectiveness of organics fractions

antifungal compounds that can be used to control postharvest citrus diseases (Droby et al.,
1999b; Arras and Usai, 2001; Soylu et al., 2005; Fisher and Phillips, 2008; Hao et al., 2010;
Talibi et al., 2012a). At present, only a few measures for the control of sour rot, in particular
in organic agriculture, are available. In this study, the antifungal activity of different organic
extracts from eight plant species was evaluated against G. candidum under both in vitro and in
vivo conditions.

The antifungal activity was confirmed in all the plant species tested, although the results
showed that different plant extracts varied in their effectiveness in inhibiting G. candidum
growth. Indeed, the inhibitory potential of the extracts was found to vary with the specific
plant species as well as the solvent used for extract preparation. The in vitro trials showed that
methanol extracts of C. villosus, H. umbellatum, C. siliqua, R. ulmifolius, H. antiatlanticum,
P. atlantica and ethyl acetate extracts of A. radiata, C. villosus and C. siliqua proved to be the
best mycelial growth inhibitors. These results concurred with those of Talibi et al. (2012a),
who reported that aqueous extracts of R. ulmifolius, C. siliqua, and H. umbellatum had
completely inhibited the mycelial growth of G. candidum. They also reported that aqueous
extracts of C. villosus and H. antiatlanticum showed a higher degree of control (% inhibition
> 92%). The hexane and chloroform extracts of the studied species showed the lesser
antifungal activity. These results may not be entirely surprising considering that a greater
variety of polar compounds could be included in the methanol and ethyl acetate fractions,
while hexane and chloroform fractions are mainly composed of non-polar compounds. Thus,
the antifungal bioactive components present in the polar solvents extracts need to be further
characterized.

Among the extracts prepared with the different solvents having different polarities,
methanol and ethyl acetate plant extracts exhibited higher inhibition of arthrospores
germination than hexane and chloroform extracts. Indeed, methanol extracts from C. villosus
and C. siliqua induced 100% inhibition of arthrospores germination at the lowest
concentration tested (1.25 mg/mL). Talibi et al. (2012a), reported that the aqueous extracts of
C. villosus and H. antiatlanticum had the highest fungistatic effect on arthrospores
germination of G. candidum. This suggests that the active components of the extracts may be
among the more polar compounds as they are found in both the aqueous and methanol
extracts. Methanol is a general solvent and tends to provide a more complete extraction of
polar and less polar compounds, while water may not extract some of the less polar
compounds. Besides G. candidum, these plant extracts also showed an inhibitory effect on the

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 Chapter 3: Effectiveness of organics fractions

growth of Clavibacter michiganensis subsp. michiganensis, causal agent of the tomato

bacterial canker (Talibi et al., 2011a).

The MIC for H. umbellatum methanol extract against G. candidum was particularly low
(0.156 mg/mL), followed by C. villosus and R. ulmifolius methanol extracts (0.625 mg/mL).
This antifungal effect was comparable to that reported by other findings. Bouamama et al.
(2006) reported that methanol leaf extract of C. villosus have MIC values ranging from 0.19
to 200 mg/mL for some yeasts and fungi. In the same study, the authors showed that C.
villosus methanol extract exhibited more interesting antifungal activity than ethyl acetate
extract (Bouamama et al., 2006). In a recent work, Talibi et al. (2012a) reported that aqueous
extracts of C. villosus, H. antiatlanticum, H. umbellatum, R. ulmifolius and C. siliqua, tested
against G. candidum, had MIC values ranging between 0.156 and 0.3125 mg/mL. In the
present investigation, the methanol extracts of H. umbellatum and C. villosus prove to have
fungicidal action at 2.5 mg/mL; while, aqueous extracts of these two species exhibited only a
fungistatic activity against G. candidum, even at 5 mg/mL (Talibi et al., 2012a).

Although in vitro tests of plant extracts is an important first step in selecting plants
with antifungal potential against postharvest citrus pathogens, in vivo tests are needed to
check whether the positive results of the in vitro tests can be obtained too (Gorris and Smid,
1995; Tegegne et al., 2008). Results obtained in this study indicated that methanol extracts of
five plant species, viz. C. villosus, C. siliqua, H. umbellatum, H. antiatlanticum and R.
ulmifolius, significantly decreased the incidence and severity of sour rot under in vivo
conditions. In a previous study, Kivack et al. (2001) found that the methanol extract of C.
siliqua possesses potent antifungal activity against a wide range of bacteria and yeast.
Moreover, Sisti et al. (2008) found that the methanol extract of R. ulmifolius exhibited
antimicrobial activity against a wide range of human pathogens. Also, Panizzi et al. (2002)
demonstrate that methanolic extract of R. ulmifolius showed a wide range of activity against
Gram-positive and Gram-negative bacteria and yeasts. According to these authors, the
phenolic and tannins fractions of R. ulmifolius showed a high antimicrobial activity. Aqueous
extracts of the same species tested in this study showed less control of citrus sour rot
compared with the methanol extracts (Talibi et al., 2012a). The findings of this study also
corresponded with a previous report (Haouala et al., 2008) on the antifungal activity of
methanol and aqueous extracts of Trigonella foenum-graecum against Rhizoctonia solani and
Alternaria sp.; methanol extract provided the best results. This could be due to difference in
proportion of chemicals responsible for antifungal activity in the two extract types (solvent

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 Chapter 3: Effectiveness of organics fractions

and aqueous) of the plant species. A. radiata and H. umbellatum ethyl acetate extracts, which
were active against G. candidum in the in vitro screening, did not significantly differ from the
control in the in vivo experiment. Therefore, for these two plant species, the in vitro and in
vivo tests presented opposing results. According to Gatto et al. (2011), numerous factors (e.g.
degradation, hydrolysis, polymerization, etc.) can affect the biological activity of certain
components of plant extract when in contact with fruit tissue.

In the present work, four organic solvents with increasing polarity were tested (Hexane,
choloroform, ethyl acetate and methanol). Among them, methanol fraction possesses great
control in both in vitro and in vivo tests. Askun et al. (2009) reported that methanol provided
more consistent antimicrobial activity. Methanol extracts of several plant species have been
found to possess excellent antimicrobial activity against different fungal pathogens and
bacteria (Sato et al., 2000; Choi et al., 2004; Ameziane et al., 2007; Bajpai et al., 2008; Satish
et al., 2008; Mahlo et al., 2010; Ahmadi et al., 2010; Hajji et al., 2010). These activities
might depend on the compounds being extracted by the solvent (Askun et al., 2009).The
phytochemical analysis of methanol extract of plant species that reduced the incidence and
severity of citrus sour rot showed that they contain different classes of flavonoids and possess
high levels of total phenols. This result is backed by several studies who reported that
methanol can extract several compounds with antimicrobial activities especially the phenolic
compounds (Nicholson and Hammerschmidt, 1992; Cowan et al., 1999; Tripathi and Dubey,
2004; Sisti et al., 2008; Martini et al., 2009). Also of interest, Cowan et al. (1999) reported
that plants have an almost limitless ability to synthesize aromatic substances that most of
which are phenols that play a role in plant defense mechanisms against pathogens. The
relation between composition and antifungal activity of these plant extracts may be
attributable both to their major bioactive compounds but also to that presents in small amount.
It is possible that they may act synergistically to contribute to the activity of the whole plant
extract.

The possibility of controlling sour rot with plant extracts appears of particular interest
considering the lack of valid alternatives to guazatine. Plant species tested in our study proved
to be useful for effective biocontrol of G. candidum on citrus fruit. C. villosus, C. siliqua and
H. umbellatum methanol extracts successfully reduced the disease incidence and disease
severity caused by G. candidum, and no phytotoxic effects were recorded on citrus fruit. The
antifungal activity of these plant extracts gives new opportunities to improve control against
postharvest citrus fruit pathogens, and, in particular, in organic agriculture.

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 Chapter 3: Effectiveness of organics fractions

If in vivo results can be confirmed under commercial conditions, C. villosus, C. siliqua

and H. umbellatum methanol extracts would be a viable option for controlling sour rot. Plant
products as antifungal agents present three main characters : the first is their natural origin
which means more safety to the consumer and the environment, the second is that they have
been considered at low risk for resistance development by pathogenic fungi and third, they
represent a rich source of potential bioactive compounds (Tripathi et al., 2008). It is believed
that it is difficult for the pathogens to develop resistance to such a mixture of plant extracts
components with, apparently, different modes of action (Daferera et al., 2003). Crude plant
extracts are generally a mixture of active and non-active compounds. The results obtained in
this work are the first scientific in vivo evidence referred to antifungal activity against G.
candidum of methanolic crude extracts of C. villosus, C. siliqua and H. umbellatum. However,
the potential use of these plant extracts to control sour rot requires a detailed examination of
their biological activity and the development of a formulation which inhibits the growth of G.
candidum at non-phytotoxic concentrations.

V. Conclusions

Of the eight plant species screened, three- C. villosus, C. siliqua and H. umbellatum-
demonstrated strong antifungal potential overall. Two others - H. antiatlanticum and R.
ulmifolius- showed a significant degree of sour rot control compared to the control. Recently,
there has been considerable interest in plant extracts with antifungal activity, which are more
acceptable, ecological safe and less hazardous than synthetic fungicides. Most of these plants
are widely distributed in SMD region and easy grown. Furthermore, the extraction method is
simple. Moreover, the use of a plant extract to protect citrus fruit against fungal attack may be
acceptable in the organic production of citrus fruit. Further research will explore commercial
potential of these plant species under degreening conditions.

84
 CHAPTER FOUR

Bioguided fractionation
Project of article :
Talibi, I., El Abed S., Boubaker, H., Boudyach, Ait Ben Aoumar.
 Chapter 4: Bioguided fractionation

Résumé
Dans l‟objectif de purifier les meilleurs fractions organiques et de comprendre le mode
avec lequel leurs composés agissent sur le pathogène, les extraits méthanoliques de C. villosus
et H. umbellatum ont subi un fractionnement bio-guidé. Cette méthode consiste à fractionner
l‟extrait en question sur gel de silice (par chromatographie sur colonne) en utilisant des
systèmes d‟élution de polarité croissante. Les sous-fractions issues de chaque fraction ont été
testées pour leur activité contre la croissance mycélienne de G. candidum. La sous-fraction
F.7 issue de l‟extrait méthanolique de C. villosus est la plus active contre G. candidum par
inhibition de la croissance mycélienne du champignon avec un diamètre de la zone
d‟inhibition de 40,33 mm autour des puits inoculés avec la sous-fraction. Pour H. umbellatum,
le meilleur degré d'inhibition de la croissance mycélienne est obtenu avec la sous-fraction F.6
avec un diamètre de la zone d'inhibition de 27 mm. Ces sous-fractions, les plus actives, ont
subi un autre fractionnement et les sous-fractions obtenues ont été testées pour évaluer leur
effet antifongique. Les résultats ont montré qu'il ya une diminution de l'activité antifongique
des extraits au fur et à mesure du fractionnement.
Mots clés: Fractionnement bioguidé, Chromatographie sur colonne, Synergie.

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 Chapter 4: Bioguided fractionation

Abstract
In the previous chapters we tested the antifungal activity, in vitro and in vivo, of four organic
fractions (Hexane, Chloroform, Ethyl acetate and Methanol) of the eight best plants selected
from the 43 plants tested initially. From these fractions we have selected the most active
fractions (methanol extracts of Cistus villosus and Halimium umbellatum) for further
purification by the bioguided fractionation method. Hence the objective of the present study
was to investigate the most active subfraction from C. villosus and H. umbellatum against G.
candidum. First of all, the methanolic extract of C. villosus and H. umbellatum were
fractionated by open column chromatography and eight subfractions were obtained from each
extract. The subfractions derived from each extract were tested for their antifungal activity
against G. candidum. The subfraction F.7 from C. villosus is the most active against G.
candidum by inhibiting the mycelial growth of the fungus, around wells inoculated with the
subfraction, with 40.33 mm diameter. For H. umbellatum, the best level of inhibition is
obtained with the subfraction F.6 with inhibition zone diameter of 27 mm. These subfractions
underwent another fractionation and the resulting subfractions were tested for their antifungal
effect. The results showed that there is loss of the biological activity with each fractionation.

Keywords : Bioguided fractionation, Columen chromatography, Synergism

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 Chapter 4: Bioguided fractionation

I. Introduction
Fruit and vegetables have a number of constitutive and inducible compounds that are
antimicrobial which can be used to produce safe marketable citrus fruits (Tripathi et al., 2008;
Tayel et al., 2009). Although more than 1340 plants are known to be potential sources of
antimicrobial compounds and about 10000 secondary plant metabolites have been chemically
defined for their role as anti-pathogenic chemicals, many of these substances can play a
fundamental role in biological control of postharvest disease (Cowan, 1999). Plant extracts
have the potential to be natural fungicides and can replace the synthetic ones. Some
phytochemicals of plant origin have been formulated as botanical pesticides and are used
successfully in integrated pest management programs as botanical pesticides. (Tripathi et al.,
2004). Plant products as antifungal agents present three main characters : the first is their
natural origin which means more safety to the consumer and the environment, the second is
that they have been considered at low risk for resistance development by pathogenic fungi and
third, they represent a rich source of potential bioactive compounds (Tripathi et al., 2008). It
is believed that it is difficult for the pathogens to develop resistance to such a mixture of plant
extracts components with, apparently, different modes of action (Daferera et al., 2003). In the
available literature, researches on biological control of postharvest diseases of citrus fruits by
plant extracts has explored various aspects such as essential oils, volatile compounds, aqueous
and solvent extracts and some products extracted from plants (Droby et al., 1999b; Arras and
Usai, 2001; Du Plooy et al., 2009; Liu et al., 2009b; Gatto et al., 2011; Talibi et al., 2012a;
Talibi et al., 2012b). Among the plants tested, Cistus villosus and Halimium umbellatum are
reported to control several plants pathogens (Bouamama et al., 2006; Ameziane et al., 2007;
Askarne et al., 2012; Talibi et al., 2012a; Talibi et al., 2012b).

This study is a continuity of works of Talibi et al. (2012b) who tested the effectiveness
of four organic fractions (hexane, chloroform, ethyl acetate and methanol) of eight plants
Against Geotrichum candidum, the causal agent of citrus sour rot. Among these fractions, the
methanol extracts of Cistus villosus and Halimium umbellatum were the most active against
the pathogen. Often, bioactive compounds constitute a very minor part of the crude extract;
the bioassay-guided fractionation maximizes the possibility of discovering new compounds
with biological activity. This methodology integrates the processes of compound isolation
using various analytical methods. In the present work, this strategy has been used to obtaine
compounds (s) from methanol fractions of C. villosus and H. umbellatum responsible (s) of
the inhibition of G. candidum.

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 Chapter 4: Bioguided fractionation

II. Material and Methods

II.1 Pathogen and arthrospores preparation
The Geotrichum candidum isolate used in this study was obtained from a decayed
mandarin fruit. It is one of the most aggressive isolates in our collection. This isolate was
deposited at the laboratory of Biotechnology and Valorization of the Natural Resources under
the number SR10. The fungus was maintained on potato dextrose agar (PDA) plates at 5°C,
with periodic transfers through citrus fruit to maintain its aggressiveness. The pathogen
inoculum consisted of aqueous arthrospores suspensions obtained from seven-day old culture
plates incubated at 25°C. Arthrospores were harvested by flooding plates with five mL of
sterile distilled water containing 0.05% (v/v) Tween 80, and passing the suspension through
two layers of sterile cheesecloth to remove hyphal fragments. The arthrospores concentration
was determined with the aid of a heamacytometer and adjusted to 106 arthrospores mL-1 with
sterile distilled water.

II.2 Preparation of plant extracts

Preparation of the extracts from Cistus villosus and Halimium umbellatum was
conducted as previously described by Duraipandiyan and Ignacimuthu (2007) and Bajpai et
al. (2008). Briefly, powder (20g) of each plant species was extracted with 100 mL of hexane
by maceration with stirring at room temperature for 48 h and then filtered through Whatman
no. 1 filter paper to obtain particle free extract. The solvent of the filtrate was removed by
evaporation to dryness under reduced pressure at 40°C. The remains of the plant material
were extracted with chloroform, ethyl acetate and methanol sequentially in a similar manner.
The extracts were then sterilized by passing through 0.2µm pore diameter Millipore Swinex
filters and kept at -20°C before assay. Solvents (analytical grade) for extraction were obtained
from commercial sources. The bioguided chromatographic fractionation process began with
the evaluation of the antifungal activity of these organic fractions. Methanol extracts are the
most actives fractions (Data not shown). Further fractionation was carried out with the more
active fractions and the process was repeated for subfractions until obtaining fraction with
pure compounds exhibiting antifungal activity.

II.3 Open column chromatography

In order to localize the active fraction, methanol extract of C. villosus and H.
umbellatum were purified using an open column chromatography. Silica gel 60 mixed with
Dichloromethane was poured slowly into column (2.5 cm× 70 cm). Then, cotton-wool was

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 Chapter 4: Bioguided fractionation

neatly placed on the top of gel to prevent disturbance at the surface during solvent
introduction. The extract tested (Methanol extract of C. villosus or H. umbellatum) was then
deposited neatly on top of the cotton-wool. Nine elution systems were added slowly in the
order as in table 4.1. In order to select the best mobile phase for eluting the methanol extracts,
5 µl of each sample were developed in thin layer chromatography (TLC) (Merck, silica gel 60
F254) with various combination of solvents. The solvent that exhibited the most favorable
separation of compounds was chosen. The vacuum was switched on and the solvent was
allowed to run through the column, until the elution system had been collected in the tubes
(Figure 4.1).

Figure 4.1 : Fractionation of methanol extract of C. villosus by the open columen chromatography

Table 4.1 : Solvents mixtures used in the first column chromatography (%)

System order Hexan Ethyl Acetate Dichloromethane Methanol

1 45 5 45 5
2 40 10 40 10
3 35 15 35 15
4 30 20 30 20
5 25 25 25 25
6 20 30 20 30
7 15 35 15 35
8 10 40 10 40
9 5 45 5 45

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 Chapter 4: Bioguided fractionation

Fractions were analyzed by thin layer chromatography. From TLC results, fractions were
combined, based on the similarity of their chemical profile (Figure 4.2). Combined fractions
were concentrated by removing the solvent by evaporation under reduced pressure at 40°C.
Each fraction was tested for its antifungal activity and the active fractions were subjected to
further column chromatography. The second and the third fractionations are carried out by
using another elution system as indicated in table 4.2. The fractionation order of methanol
extract of C. villosus and H. umbellatum is illustrated in Figure 4.3 and 4.4 respectively.
Table 4.2 : Solvents mixtures used in the second and third column chromatography

Portion of solvent (%)

System order Chloroform Ethyl Acetate Methanol Acitonitril

1 30 40 20 10
2 10 40 30 20
3 0 40 40 20
4 0 20 50 30
5 0 10 60 30
6 0 0 70 30
7 0 0 80 20
8 0 0 90 10
9 0 0 100 0

Figure 4.2 : Combination of fractions based on the similarity of their chemical profile (254 nm)

II.4 Antifungal assay

Each fraction was screened for their antifungal activity using the well-plate diffusion
method. Aliquots (100 µL) of arthrospores suspension (106 arthrospores /mL) of G. candidum
was overlaid on Petri plates (90 mm in diameters) containing 15 mL of PDA. The plates were

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 Chapter 4: Bioguided fractionation

incubated at room temperature for 30 minutes to allow a good diffusion of the suspension in
the culture medium. Then five-mm diameter wells were made at three locations per dishes.
The wells are then filled with 20 µL of the fraction at concentration of 5 mg/mL. Control
plates consisted of wells filled with 20µL of Methanol. The plates are stored at 4°C for 2
hours to allow the diffusion of extract, and then incubated at 26°C. After 48 hours incubation
the diameter of the inhibition zone is measured. Three plates were used for each treatment as
replications, and each experiment was repeated twice.

Figure 4.3 : Order of fractionation of the methanol extract of C. villosus. Hex : Hexan; DCM :
Dichloromethan; CHL : Chloroform; E. Ac : Ethyl acetate; MeOH : Methanol; Act : Acitonitril

Figure 4.4 : Order of fractionation of the methanol extract of H. umbellatum. Hex : Hexan; DCM :
Dichloromethan; CHL : Chloroform; E. Ac : Ethyl acetate; MeOH : Methanol; Act : Acitonitril

II.5 Statistical analysis

Results were subjected to statistical analysis of variance (ANOVA) using STATISTICA
software, version 6, Stat-Soft, 2001, France. Mean separation was performed following the
Newman & Keuls test at P<0.05.

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 Chapter 4: Bioguided fractionation

III. Results
III.1 Bioguided fractionation of Cistus Villosus
The methanol extracts of C. villosus was fractioned by open column chromatography.
These fractionations are guided by the antifungal activity assay of each fraction. Thus extract
of C. villosus is fractioned three times (Figure 4.3).
The first fractionation was iluted with different portions of Hexan/Ethyl Acetate/
Dichloromethane/Methanol and yielding eight subfractions. Each subfraction was evaluated
for their antifungal against Geotrichum candidum by the well-plate diffusion method and the
diameter of inhibition zone was determined and data are showen in figure 4.5. Among the
eight subfractions recovered, the fourth, sixth and seventh subfraction exhibited antifungal
activity by 21.33; 4 and 40.33 mm of inhibition zone respectively (Figure 4.5 and 4.6).
45
Diameter of the inhibition zone (mm)

40
35
30
25
20
15
10
5
0
F.1 F.2 F.3 F.4 F.5 F.6 F.7 F.8

Figure 4.5 : In vitro effects of methanol oxtracts fractions of C. villosus on mycelial growth of G.
candidum

F.4 F.7
Figure 4.6 : Inhibition zone of Geotrichum citri-aurantii around wells amended with the subfractions
of Cistus villosus

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 Chapter 4: Bioguided fractionation

The TLC analysis of the subfraction N° 7 (F.7) eluted with the mixture DCM/CHL/ Et.
Ac /MeOH (3/3/2/2) shows that it still contain many compounds (Data not shown). Therefore,
this subfraction was subjected to further fractionation with different portions of
Chloroform/Ethyl Acetate/ Methanol/Acitonitrile as elution system and yielding six
subfractions (Figure 4.3). Then, each subfraction was tested on the mycelial growth of G.
candidum. Results of the bioassay of thes subfractions showed that the antifungal activity was
recorded in the subfractions F.7.1; F.7.2; F.7.3 and F.7.4 by 18; 8.66; 5.66 and 8.66 mm of
inhibition zone respectively (Figure 4.7 and 4.8). The TLC analysis of the subfraction F.7.1
eluted with the mixture DCM/CHL/ Et. Ac /MeOH (3/3/2/2) shows six separated bands and
thus, underwent another purification and yielding seven subfractions (Figure 4.3).

20
Diameter of the inhibition zone (mm)

18
16
14
12
10
8
6
4
2
0
F.7.1 F.7.2 F.7.3 F.7.4 F.7.5 F.7.6

Figure 4.7 : In vitro effects of subfractions from the F.7 fraction of C. villosus on mycelial growth of
G. candidum

F.7.1 F.7.2

Figure 4.8 : Inhibition of zone around wells inoculated with the subfractions of C. villosus

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 Chapter 4: Bioguided fractionation

Subfractions derived from this fractionation (F.7.1.1 to F.7.1.7) are tested against G.
candidum and no subfraction has inhibited the mycelial growth of the fungus.

III.2 Bioguided fractionation of Halimium umbellatum

Like as C. villosus, the methanol extract of H. umbellatum was fractioned by open
column chromatography and the results of its fractionation are detailed in Figure 4.9. The
first subfractions derived from the methanol extracts of H. umbellatum have been tested on
mycelial growth of G. candidum. The results indicate that of the subfractions tested, only
fractions 6-7-8 have inhibited the mycelial growth of G. candidum by 27; 14.33 and 17 mm
respectively (Figure 4.9 and figure 4.10). The TLC analysis of the subfraction N°6 eluted
with the mixture DCM/CHL/ Et. Ac/MeOH (3/3/2/2), revealed superimposed bands.

35
Diameter of the inhibition zone (mm)

30

25

20

15

10

0
F.1 F.2 F.3 F.4 F.5 F.6 F.7 F.8

Figure 4.9 : In vitro effects of methanol extracts fractions of [Link] on mycelial growth of G.
candidum

F.6 F.7 F.8

Figure 4.10 : Inhibition zone of Geotrichum citri-aurantii around wells amended with the subfractions
of Hallimium umbellatum

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 Chapter 4: Bioguided fractionation

The subfraction F.6 is allowed to further fractionation with the ilution system composed of
different proportions of the mixture Chloroform/Ethyl Acetate/ Methanol/Acitonitrile (Figure
4.4). Eleven subfractions were obtained from this fractionation (F.6.1-11) and each
subfraction was tested for its antifungal activity against G. candidum. Results presented in
figure 4.11 showed that subfractions F.6.1, F.6.2, F.6.5 and F.6.7 reduced the mycelial
growth of G. candidum around inoculated wells by 8; 10; 6 and 6mm respectively.

12
Diameter of the inhibition zone (mm)

10

0
F.6.1 F.6.2 F.6.3 F.6.4 F.6.5 F.6.6 F.6.7 F.6.8 F.6.9 F.6.10 F.6.11

Figure 4.11 : In vitro effects of subfractions from the F.6 fraction of [Link] on mycelial
growth of G. candidum

IV. Discussion
From all of these results, we can deduce that more the extract is fractioned, the
efficiency of the subfractions to inhibit the mycelial growth of the fungus decreases. So there
is a loss of antifungal activity in the second and third fractionation compared to the first one.
This can be explained by the decrease of the concentration of the active compound (s) and/or
the loss of the synergistic action between any of the phytochemical constituents present in the
original fraction. The synergistic effect of phytochemical compounds is also reported by
several works (Ellof, 2004; Nenaah, 2010; Zakaria et al., 2010; Suleiman et al., 2012).
Nenaah (2010) reported that when Harmala alkaloids are applied individually exhibited
moderate antimicrobial effects. In contrast, the activity of the tested alkaloids was increased
when tested as binary or total (crude) alkaloidal mixture suggesting a kind of synergism
among these compounds (Nenaah, 2010). Likewise, Suleiman et al. (2012) reported the loss
of more than 85% of the antimicrobial activity of fractions from the original crude extract of
Loxostylis alata.

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V. Conclusions
Therefore, we can conclude that compounds from Cistus villosus and Halimium
umbellatum exhibit their antifungal activity by the synergism effect between their
phytochemical compounds. So, the optimal effectiveness of plants is not, always, due to one
main active constituent, but to the combined action of different compounds originally in the
plant. Moreover, further research should focus on the phytochemical analysis to identify the
chemical composition of the most actives fractions of each plant.

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 CHAPTER FIVE

Screening of organic and inorganic

salts to control postharvest citrus
sour rot
Published in Plant Pathology journal as :
Talibi, I., Askarne, L., Boubaker, H., Boudyach E.H., Ait Ben Oumar A., 2011.
In vitro and in vivo Screening of organic and inorganic salts to control of
postharvest citrus sour rot caused by Geotrichum candidum Plant Pathology
journal 10, 138-145.
 Chapter 5: Screening of salt compounds

Résumé
Dans l‟objectif de rechercher des alternatives saines et efficace aux fongicides
chimique utilisés dans la lutte contre la pourriture amère des agrumes en post-récolte, nous
avons testé l‟efficacité de 34 sels organiques et inorganiques contre G. candidum. Les
résultats du screening de l‟activité antifongique de ces sels sur la croissance mycélienne ont
montré que les concentrations minimales inhibitrices obtenues varient selon la nature du sel
testé et de sa concentration. La CMI la plus faible (0,1%) est obtenue avec le carbonate
d‟ammonium et l‟EDTA, suivie par le salicylate de sodium, le metabisulfite de sodium et
l‟acide borique avec une CMI de 0,25%. Les dix meilleurs sels, sélectionnés sur la base de
leur CMI, ont été, ensuite, testés pour leurs capacité à réduire ou à inhiber la germination des
arthrospores du pathogène. Les résultats obtenus ont montré que l'EDTA, l'acide borique, le
metabisulfite de sodium, le carbonate de sodium, le sulfate de sodium et le thiosulfate de
sodium ont totalement inhibé la germination des arthrospores à partir d‟une concentration de
75 et 100 mM. En revanche, le salicylate de sodium, le carbonate d'ammonium et le carbonate
de potassium se sont montrés peu efficaces sur la germination des arthrospores de G.
candidum. Considérant que plusieurs sels peuvent influencer le pH du milieu, l'effet de ce
dernier sur la croissance mycélienne de G. candidum a été déterminé. Les résultats obtenus
ont montré que la croissance mycélienne est faiblement affectée par les modifications du pH
dans la gamme de pH 4,0 à 12,0. Pour le test in vivo, des mandarines ont été traitées avec
différentes concentrations de sels (1, 2 et 3% w/v), inoculés avec une suspension
d‟arthrospores de G. candidum puis incubées à 26°C et à une humidité relative élevée. Les
résultats obtenus ont montré que seuls le salicylate de sodium, l'acide borique et l'EDTA ont
réduit significativement l‟incidence et la sévérité de la pourriture amère. En effet, testé à une
concentration de 3%, l'incidence de la maladie a été réduite à 25,93% chez les fruits traités
avec le salicylate de sodium ou l'acide borique et à 38,89% chez les fruits traités avec de
l'EDTA. Le carbonate d‟ammonium et le carbonate de sodium ont aussi significativement
réduit l‟incidence de la pourriture amère respectivement à 51,8 et 57,41%.

Mots clés : Agrumes, sels, Geotrichum candidum

Abstract

The aim of this study was to find an alternative to the chemical fungicide currently
used in the control of postharvest citrus sour rot. Here, we screened thirty-four salt

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compounds, considered as common food additives, for their activity against Geotrichum
candidum, causal agent of citrus sour rot. The lowest Minimum Inhibitory Concentrations
(MICs) values were obtained by ammonium carbonate and EDTA at a concentration of 0.1%
(w/v) and boric acid, sodium carbonate and sodium metabisulfite at 0.25% (w/v). Over all, the
medium-pH in the range of 4.0 to 12.0 did not influence the mycelial growth of the pathogen.
The ten best salt compounds were tested for their ability to reduce the arthrospores
germination of the [Link] effect of salts varied significantly (P<0.05) between tested
compounds and depended on their concentrations. The arthrospore germination was
completely inhibited by EDTA, boric acid, sodium metabisulfite, sodium carbonate, sodium
sulfate and sodium thiosulfate, both at 100 and 75 mM. The most active salts in in vitro
studies were tested in vivo against sour rot on citrus fruit. Incidence of sour rot was lowered to
25.93% and 38.89%, when mandarin fruit where treated by sodium salicylate, boric acid and
EDTA, compared with 100% in the control. However, only the application of boric acid at 3%
(w/v) reduced disease severity by more than 70%. These results suggest that sodium
salicylate, boric acid and EDTA may be useful and effective compounds for control of citrus
sour rot. Such healthy products therefore represent a sustainable alternative to the use of
guazatine mainly in organic production.

Key words : Citrus, salts, sour rot, Geotrichum candidum

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I. Introduction

Sour rot caused by the fungus Geotrichum candidum is one of the major postharvest
diseases of citrus fruits (Kitagawa and kawada, 1984). It is one of the most economically
important postharvest diseases of citrus in arid growing regions of the world (Smilanick and
Sorenson, 2001) and cause serious problems for harvested citrus fruits during handling,
transportation, exportation and storage (El-mougy et al., 2008). The organism is a wound
pathogen, infecting fruit during harvest and subsequent handling, and requiring injury into the
albedo for entry (Brown, 1979). Besides the injuries, the aggressiveness of the fungus
increases especially during fruit degreening, wet and rainfall seasons (Eckert, 1978; Eckert
and Brown, 1988; Cohen et al., 1991, Liu et al., 2009b).

The measures employed to manage postharvest citrus rot are not effective against
Geotrichum candidum. This pathogen is not controlled by any of the fungicides (e.g., Imazalil
and Thiabendazole) registered for use on citrus fruit (Eckert, 1978; Kitagawa and Kawada,
1984; Brown, 1988; Suprapta et al., 1997; Mercier et al., 2005; Smilanick et al., 2008; Liu et
al., 2009b; Feng et al., 2011). Guazatine is the only commercial fungicide that can control
sour rot (Rippon and Morris, 1981; Brown, 1988). However, this fungicide is not authorized
in several countries. The disease can be partially reduced by Sodium o-phenylphenate (SOPP)
(Rippon and Morris, 1981; Feng et al., 2011) which is found to be carcinogenic and has
promoting activity towards the urinary bladder (Kitagawa and Kawada, 1984). Therefore,
alternative treatments have become an essential requirement for the control of this disease.
Furthermore, concerns about public health risks associated to fungicide residues and
environmental issues have increased the need for these alternatives.

Use of organic and inorganic salts, generally recognized as safe (GRAS) compounds, is
an interesting alternative to control postharvest disease of citrus fruits. Many of these salts
have several advantages such as low mammalian toxicity, favorable safety profile for humans
and environment, and a relatively low cost (Olivier et al., 1998; Hervieux, et al., 2002;
Deliopoulos et al., 2010). Moreover, these compounds have a broad-spectrum antimicrobial
activity (Corral et al., 1988; Olivier et al., 1998; Deliopoulos et al., 2010) and are usually
used in the food industry for controlling pH, taste and texture (Smilanick et al., 1999;
Hervieux, et al., 2002; Arslan et al., 2009). Furthermore, several studies have reported the
effectiveness of salts to control various pathogens of many crops. Potassium sorbate (KS) was

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shown to reduce the incidence of sour rot under laboratory conditions (Kitagawa and Kawada,
1984; El-Mougy et al., 2008; Smilancik et al., 2008). Also, Sodium benzoate and KS were
used to control postharvest decays caused by many fungi (Al-Zaemey et al., 1993; Olivier et
al., 1998; El-Mougy et al., 2008; Palou et al., 2001). Palou et al. (2001) and smilanick et al.
(1999) demonstrated that sodium carbonate reduced the incidence of citrus green and blue
mold. Moreover, salt compounds also improve their performance when used in combination
with other treatments like microbial antagonists (El-Ghaouth et al., 2000; Nunes et al., 2002;
Zhang et al., 2008; Sharm et al., 2009), fungicides (Smilanick et al., 2008) or hot water
(Palou et al., 2001; Porat et al., 2002; Smilanick et al., 2008).

Although most researchers have focused on controlling green and blue mold, little has
been published about the control of sour rot. The present work was performed to evaluate the
efficacy of a wide range of organic acids and salts, for in vitro and in vivo control of G.
candidum, the causal agent of citrus sour rot.

II. Materials and Methods

II.1 Pathogen culture and chemicals

Geotrichum candidum was isolated from a decayed Clementine fruit and was one of the
most aggressive isolates in our collection. The fungus was maintained on PDA plates at 5°C,
with periodic transfers through citrus fruit to maintain its aggressiveness. The pathogen
inoculum that consisted of aqueous arthrospores suspensions obtained from 7-day-old culture
plates incubated at 25°C. Arthrospores were harvested by flooding plates with 5 ml of sterile
distilled water containing 0.05% (v/v) Tween 80, and passing the suspension through two
layers of sterile cheesecloth to remove hyphal fragments. The arthrospores concentration was
determined with the aid of a heamacytometer and adjusted to 106 arthrospores ml-1 with sterile
distilled water. A total of 34 salt compounds (Table 5.1) considered as common food
additives were used in this study to evaluate their effectiveness against G. candidum.
II.2 Fruit
The fruit of mandarin (Citrus reticulata blanco) cv. “Clementine” was used. Fruits were
harvested from orchards of the M‟brouka cooperative, which used standard culture practices,
in Souss-Massa-Draa region, Morocco. Only healthy and commercially mature fruits were
used in the in vivo test. Freshly harvested or briefly stored (no longer than a week) fruits were
used in the experiment.

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Table 5.1 : The tested compounds, their chemical formula and molecular weight
Compounds Chemical Formula Molecular weight

Ammonium acetate (NH4)C2H3O2 77.08

Ammonium carbonate (NH4)2CO3 96.09
Ammonium dihydrogen phosphate (NH4)H2PO4 115.03
Ammonium molybdate (NH4)6Mo7O24, 4H2O 1235.86
Ammonium sulfate (NH4)2SO4 132.14
Ascorbic acid C6H8O6 176.13
Aspartic acid C4H7O4N 133.11
Boric acide H3BO3 61.83
Calcium carbonate CaCO3 100.09
Calcium chloride CaCl2 110.98
Calcium Chloride CaCl2, 2H2O 147.02
Calcium nitrate Ca(NO3)2·4H2O 236.14
Citric acid C2H8O7, H2O 210.14
Dipotassium hydrogen phosphate K2HPO4 174.18
EDTA C 10 H 14 N 2 Na2 O 8, 2H2O 372.24
Glutaric acid C5H8O4 132.12
Magnesium chloride MgCl2 203.31
Magnesium sulfate MgSO4 246.48
Potassium acetate C2H3KO2 98.14
Potassium carbonate K2CO3 138.21
Potassium chloride KCl 74.55
Potassium phosphate dibasic K2HPO4 174.18
Potassium sodium tartrate C4H4KNaO6, 4H2O 282.23
Sodium acetate C2H3O2Na 82.03
Sodium bicarbonate NaHCO3 84.01
Sodium carbonate Na2CO3 105.99
Sodium chloride NaCl 58.44
Sodium metabisulfite Na2S2O5 190.1
Sodium molybdate Na2MoO4 241.95
Sodium salicylate C7H5NaO3 160.11
Sodium sulfate Na2SO4 142.04
Sodium thiosulfate Na2S2O3, 5H2O 248.18
Sodium salicylate CuSO4, 5H2O 249.68
Sodium thiosulfate Na2S2O3 - 5H2O 248.2

II.3 Determination of Minimum Inhibitory Concentration (MIC)

The minimal inhibitory concentrations (MICs) of the 34 salts were determined by the
broth dilution method using the Nutrient Yeast Dextrose Broth (NYDB : nutrient broth 8 g l–1;
yeast extract 5 g l–1; dextrose 10 g l–1) as culture medium. Each salt was tested at seven
concentrations : 0.1, 0.25, 0.5, 0.75, 1, 1.75 and 2% (w/v). One millilitre of each aqueous

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solution at desired concentration was transferred to test tubes containing 9 ml of NYDB. The
medium without salt compounds served as control. Each tube was inoculated with 100 µl of a
suspension of 106 arthrospores/ml of G. candidum and incubated at 25°C for 72 hours with
shaking. The MICs were recorded by reading the lowest salt concentration that allowed no
visible growth of the pathogen. There were three replicates for each salt at each concentration
and the experiment was conducted twice.

II.4 Effect of pH on mycelial growth of G. candidum

Since some of the salt compounds could affect the pH of NYDB medium, we tested the
effect of pH alone on mycelial radial growth of G. candidum. The pH tested varied from 2 to
12 and were adjusted with 1N HCl or NaOH. Hyphal plugs (5 mm diameter) were cut from
the periphery of actively growing colonies (7 to 10 day-old) and transferred aseptically,
mycelium down, to three replicate Petri plates containing NYDA at different pH. Radial
growth was determined daily, by measuring colony size along two perpendicular axes. The
experiment was performed with three replicate plates per treatment.

II.5 Effect of salts on arthrospores germination

To evaluate the impact of salt compounds on arthrospores germination, only salts with
MIC value equal or lower than 0.5 % were tested. Aqueous solution of salt compounds was
prepared in orange juice (2%) as nutrient medium. The germination of arthrospores of G.
candidum was determined in concentrations of 25 mM, 50 mM, 75 mM and 100 mM of each
salt. Aliquots (40µl) of an arthrospores suspension (106 arthrospores/mL) were aseptically
transferred in triplicate to sterile depression slides containing 40 µl of 2 % sterile orange juice
amended with different concentrations of salt (Droby et al., 2003). The pH of solution was not
modified; it was determined by the salt and its concentration. Inoculated slides were placed on
moist filter paper in Petri plates, sealed with Parafilm to avoid evaporation, and then
incubated at 25°C for 24h. Each depression slide was then fixed with acid fuchsine solution to
stop further germination (Smilanick et al., 1999). Arthrospores germination was estimated
under a microscope equipped with a micrometer. At least 100 arthrospores within each
replicate were observed. An arthrospore was scored as germinated if the germ tube length was
at least equal or superior to the length of the spore body (Suprapta et al., 1997). The results
were expressed as percent spore germination inhibition and calculated by using the following
formula : GI (%) = [(Gc−Gt) /Gc)] ×100, Gc and Gt represent the mean number of
germinated spores in control and treated slides, respectively (Soylu et al., 2010). Each
treatment included three replicates and the experiment was conducted twice.
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II.6 Effects of salts on sour rot development in artificially inoculated

and wounded fruit
Based on the in vitro antifungal activity, only salt compounds with a MIC equal or
inferior to 0.5 % were retained. Mandarin fruits were washed, disinfected with 0.1% (v/v)
sodium hypochlorite, rinsed three times in sterile distilled water and then air-dried before
wounding. One wound (2mm deep and 4mm wide) was made per fruit using a sterile needle at
the equatorial side (Liu et al., 2009b). The wounds were treated with 30 µl of salt solution at
concentrations of 1, 2 and 3% (w/v). Controls were treated with the same volume of sterile
distilled water under the same conditions. After two hours incubation at room temperature,
each wound was inoculated with 20 µl of an aqueous suspension of arthrospores of G.
candidum (106 arthrospores. ml-1). Treated fruits were placed on a plastic tray in cardboard
boxes and incubated at 26°C and 95% relative humidity (RH). The number of the infected
wounds and the lesion diameters of the overall treated fruit were determined daily. All
treatments were arranged in a complete randomized block design. Eighteen fruits constituted a
single replicate and each treatment was replicated three times. The experiment was conducted
twice. The incidence and severity of disease were calculated as follows :
Disease incidence (%) = [(number of rotten wounds / number of total wounds)] x 100.
Disease severity (%) = [(average lesion diameter of treatment / average lesion diameter of
control)] x 100. In all experiments, the possible phytotoxic effect on mandarin fruit was
examined.

II.7 Statistical analysis

All data were subjected to statistical analysis of variance (ANOVA) using
STATISTICA software, version 6, Stat-Soft, 2001, France. Percentage values were subjected
to arcsine- square root transformation before analysis of variance. Mean separation was
performed following the Newman & Keuls test at P<0.05.

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III. Results

III.1 Preliminary screening of salts (MICs)

The mycelial growth of Geotrichum candidum is visualized in test tubes by the presence
of a cloudy in the solution, which indicated the growth of the fungus (Figure 5.1). Two
control tubes were made; postivie control corresponding to fungal culture alone and negative
control corresponding to tubes with salts alone.

Figure 5.1 : Mycelial growth of G. candidum in test tubes containing NYDB medium and diffrents
concentrations of sodium carbonate

The in vitro antifungal activity of 34 organic acids and salts was first examined at different
concentrations, varying from 0.1 to 2 % (w/v), and showed a variable effects of tests
compounds on G. candidum growth (Table 5.2). It was noticed that the reduction in growth
was correlated to the increase in compounds concentration. The lowest MIC values were
recorded for ammonium carbonate and EDTA tested at 0.1%. Tested at 0.25% boric acid,
sodium carbonate and sodium metabisulfite inhibited completely the mycelial growth of G.
candidum.

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Table 5.2 : Minimum inhibitory concentrations of tested chemical compounds against Geotrichum
candidum
Compounds MIC (w/v)
Ammonium acetate >2
Ammonium carbonate 0.1
Ammonium dihydrogen phosphate >2
Ammonium molybdate 0.5
Ammonium sulfate >2
Ascorbic acid >2
Aspartic acid >2
Boric acide 0.25
Calcium carbonate >2
Calcium chloride >2
Calcium Chloride >2
Calcium nitrate >2
Citric acid 2
Dipotassium hydrogen phosphate >2
EDTA 0.1
Glutaric acid >2
Magnesium chloride >2
Magnesium sulfate >2
Potassium acetate >2
Potassium carbonate 0.5
Potassium chloride >2
Potassium phosphate dibasic >2
Potassium sodium tartrate >2
Sodium acetate >2
Sodium bicarbonate 0.75
Sodium carbonate 0.25
Sodium chloride >2
Sodium metabisulfite 0.25
Sodium molybdate 2
Sodium salicylate >2
Sodium sulfate 0.75
Sodium thiosulfate 0.5
Sodium salicylate 0.5
Sodium thiosulfate >2

The third lowest MIC value was recorded for potassium carbonate, ammonium molybdate and
sodium thiosulfate at 0.5%. At 0.75 % (w/v), only sodium sulfate and sodium bicarbonate
completely inhibited the mycelial growth of G. candidum. The highest MIC value (2%) was
obtained for citric acid and sodium molybdate. The others tested salt compounds are not
effective against G. candidum even at 2% (Table 5.2). The data show that G. candidum has
differential sensitivity to salts, as demonstrated by its varying rates for complete inhibition of
growth.
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III.2 Effect of pH on mycelial growth of G. candidum

To determine the effect of pH on the growth of G. candidum, NYDA medium at
different pH values was used. The obtained results demonstrate that the fungus grew both on
acidic and basic pH (Figure 5.2). The data indicate that pH from 4 to 12 does not significantly
affect the growth of the fungus after 10 days of incubation at 25°C. Furthermore, only pH 2
reduced significantly the growth of the fungus in comparison with the control (Figure 5.3).

100
Mycelial growth (%)

80

60

40

20

0
2 4 6 7 8 10 12

pH

Figure 5.2 : Effect of pH on in vitro mycelial growth of G. candidum. Medium pH was adjusted with
Hcl or NaOH. Bars represent standard deviations of the means.

III.3 Effect of salt compounds on arthrospores germination

Based on the previous results, only salt compounds that inhibited the growth of G.
candidum at concentrations lower than 0.5% (w/v) were selected and evaluated for their
potential to inhibit the arthrospores germination of the fungus. It is evident from the table 5.3,
that the salt compounds tested against G. candidum showed a reduction or complete inhibition
of arthrospores germination in a dose-dependent manner. The arthrospores germination was
completely inhibited by EDTA, Boric acid, sodium metabisulfite, sodium carbonate, sodium
sulfate and sodium thiosulfate both at 100 and 75 mM. Tested at 50 mM, sodium carbonate,
EDTA, sodium sulfate, sodium metabisulfite and boric acid, strongly inhibited the arthrospore
germination. At this concentration, the percentage of germination of arthrospores of G.
candidum oscillates between 1%, for sodium carbonate, and 8.33%, for boric acid. At 25 mM,
only sodium metabisulfite, boric acid and EDTA strongly inhibited arthrospores germination
of G. candidum, since percent germination ranged between 11.66 and 33.33%.

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pH 2 pH 4

pH 8 pH 10

pH 12 Control

Figure 5.3 : Effect of pH on in vitro mycelial growth of G. candidum

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In contrast, Sodium salicylate, ammonium carbonate and potassium carbonate, affects

strongly the mycelial growth of the fungus, are less effective on arthrospores germination.
The percentage of germination in salts amended medium varied between 50% for potassium
carbonate at 75 and 100mM, and 100% for sodium salicylate at 25mM (Table 5.3).

Table 5.3 : In vitro effect of different chemical compounds on arthrospore germination of Geotrichum
candidum.
Arthrospore germination (%)
Concentration (mM)
Chemical tested
25 50 75 100
EDTA 33.33±2.22g 2±1.33ab 0±0a 0±0a
Sodium salicylate 100±0p 87.33±3.56o 73±2 m 61.33±2.44k
Sodium metabisulfite 11.66±2.22d 6±1.33bc 0±0a 0±0a
e
Boric acid 22.66±2.44 8.33±2.22cd 0±0a 0±0a
Sodium carbonate 54.64±0.61ij 1±0ab 0±0a 0±0a
sodium sulfate 79.67±0.44n 4.83±0.78abc 0±0a 0±0a
Ammonium carbonate 99.23±0.51p 95.37±1.71p 86.58±2.88o 56.63±5.79j
l
Potassium carbonate 68.06±1.29 54.01±2.45ij 50±1i 50.63±1.04i
Ammonium molybdate 99.33±0.44p 80±1.33n 42±1.33h 12.66±1.55d
Sodium thiosulfate 69.33±1.55lm 28.33±3.55f 0±0a 0±0a
Control 100±0p 100±0p 100±0p 100±0p
Means followed by different letter (s) in each column are significantly different at P < 0.05

III.4 Effect of salt compounds on disease development

Data presented in Table 5.4 showed that all tested salt compounds significantly reduced
the incidence of sour rot caused by G. candidum under the laboratory conditions. Percentages
of rotted wounds were decreased by using all tested salts compared with control.

Table 5.4 : Effect of different chemical ccompounds on sour rot incidence on infected fruits

Disease incidence (%)

Chemical tested Concentration (% w/v)
1% 2% 3%
jhi c
EDTA 83.33f 50 38.89b
Sodium salicylate 72.22ef 46.3c 25.93a
ef
Sodium metabisulfite 74.07 66.67def 68.52de
Boric acid 66.67de 29.63a 25.93a
Sodium carbonate 72.22efg 68.52def 57.41cd
sodium sulfate 92.59ijk 77.78efg 66.67de
hij
Ammonium carbonate 83.33 64.81de 51.85c
Potassium carbonate 90.74hijk 83.33hij 64.81de
hijk
Ammonium molybdate 88.89 79.63ghi 72.22efg
Sodium thiosulfate 94.44jk 92.59ijk 77.78fgh
Control 100k 100k 100k

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 Chapter 5: Screening of salt compounds

Mandarin fruit treated 2 hours before pathogen inoculation by sodium salicylate and boric
acid at 3% resulted in the highest reduction in rot incidence compared with the control. EDTA
and ammonium carbonate had a moderate effect on sour rot, the percentage of rot incidence
varying between 38.89 and 51.85. In contrast, sodium thiosulfate and ammonium molybdate
showed the least effect on reduction of sour rot incidence (Table 5.4). Also, all the salts tested
did not reduce effectively the disease incidence at concentration of 1%. On the other hand,
data indicated that Boric acid, sodium salicylate and EDTA exhibited significant reduction of
disease severity at 2 and 3% compared with the control.

The disease severity for these salts ranged between 30.97% (sodium salicylate at 3%)
and 50% (EDTA at 2%) (Table 5.5). Also, there was a significant reduction of the rot severity
from 100% in non-treated fruit to 51.77 and 53.1% respectively, in citrus treated fruits, by
sodium carbonate and ammonium carbonate at 3%.

Table 5.5 : Effect of different chemical ccompounds on sour rot severity on infected fruits.

Disease severity (%)

Concentration (% w/v)
Salt 1% 2% 3%
ijk de
EDTA 80.97 50 38.5bc
Sodium salicylate 81.86ijk 45.13cd 30.97ab
Sodium metabisulfite 65.93gh 56.64efg 54.42def
Boric acid 63.72fg 37.61bc 22.574a
Sodium carbonate 59.29efg 55.75efg 51.77de
sodium sulfate 96.9lm 73.45hi 59.73efg
Ammonium carbonate 78.76ijk 59.73efg 53.1de
Potassium carbonate 88.94kl 83.19ijk 60.62efg
Ammonium molybdate 87.17k 84.07hij 74.34jk
Sodium thiosulfate 99.12m 98.67m 87.61kl
Control 100m 100m 100m
Fruit were treated with different concentrations of salt (1, 2 and 3%) , inoculated with G. candidum and held for
10 days at 26°C. Means followed by different letter (s) in each column are significantly different at P < 0.05

Although sodium salicylate was effective against citrus sour rot, it was phytotoxic to
fruit rind at the three tested concentrations. A drying of the rind around the salt treated site
was observed. The other salt compounds didn‟t lead any phytotoxic action on treated fruit at
all tested concentrations.

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 Chapter 5: Screening of salt compounds

IV. Discussion

The results showed that among the 34 salts tested for their minimum inhibitory
concentrations, ten were most active against Geotrichum candidum. EDTA, sodium salicylate,
sodium metabisulfite, boric acid, sodium carbonate, sodium sulfate, ammonium carbonate,
potassium carbonate, ammonium molybdate and sodium thiosulfate have MIC values ranging
between 0.1% and 0.5%. The present results are, therefore, consistent with those of Askarne
et al. (2011) who showed that from 28 studied compounds tested against Penicillium italicum,
causal agent of citrus blue mold, sodium metabisulfite, EDTA, ammonium carbonate, sodium
carbonate and boric acid were the most active against the fungi, with MICs values ranged
between 5 and 50 mM. Moreover, Palmer et al. (1997) showed that among 26 tested salts,
ammonium carbonate, potassium carbonate, sodium carbonate, and sodium metabisulfite were
effective against the mycelial growth of Botrytis cinerea. However, the same authors reported
that sodium sulfate and sodium thiosulfate did not affect the mycelial growth of the fungi.
Olivier et al. (1998) and Hervieux et al. (2002) showed that sodium carbonate, potassium
carbonate and sodium metabisulfite have completely inhibited the mycelial growth of
Helminthosporium solani, causal agent of silver scurf on potato tubers. Also, Droby et al.
(2003) demonstrates that EDTA has a distinct inhibitory effect on the radial growth of
Botrytis cinerea and Penicillium expansum in vitro.

The MIC test has allowed us to select the best salts that are effective against G.
candidum. The ten best salts were further tested for their ability to reduce or inhibit the
arthrospores germination of the pathogen. The results indicated that the arthrospores
germination was completely inhibited by EDTA, boric acid, sodium metabisulfite, sodium
carbonate, sodium sulfate and sodium thiosulfate both at 100 and 75mM. This finding
corroborates with those of Askarne et al. (2011) showing that EDTA, boric acid, sodium
metabisulfite and sodium carbonate completely inhibited spore germination of Penicillium
italicum. Furthermore, sodium carbonate and sodium metabisulfite were demonstrated to
inhibit completely the spore germination of Helminthosporium solani (Hervieux et al., 2002).
Also Smilanick et al. (1999) reported that sodium carbonate inhibit the spore germination of
P. digitatum, causal agent of citrus green mold. Mills et al. (2004) reported that Sodium
metabisulfite reduced significantly the spore germination of several phytopathogenic fungi.
However, among the tested salts, sodium salicylate, ammonium carbonate and potassium
carbonate are not effective on the arthrospores germination of the pathogen.

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 Chapter 5: Screening of salt compounds

Considering that several salt compounds influenced medium pH, the effect of pH on G.
candium growth was determined. The results showed that mycelial growth was not strongly
affected by pH modification in the range of 4.0 and 12.0. This result agrees with those of
Hervieux et al. (2002), and indicates that the inhibition obtained in the salt-amended medium
cannot be due only to a direct effect of pH on pathogen growth.

For the in vivo test, the citrus fruits were treated with different concentrations of salt
compounds (1, 2 and 3% w/v), inoculated with G. candidum and held for 10 days at 26°C.
The results showed variable effects of tested compounds on disease incidence and severity.
Sodium salicylate, boric acid and EDTA were the most effective against G. candidum in in-
vivo conditions. At concentration of 3%, the disease incidence was reduced to 25.93% in
fruits treated with sodium salicylate or boric acid and to 38.89% in fruits treated with EDTA.
The present results are consistent with previous studies which demonstrated that EDTA was
effective to control P. digitatum on oranges (Valencia-Chamorro et al., 2008) and B. cinerea
on apple fruit (Droby et al., 2003). Moreover, Smilanick and Sorenson (2001) reported that
immersion of citrus fruits in boric acid solution reduced significantly the incidence of citrus
green mold. Also of interest, we found that ammonium carbonate and sodium carbonate have
significantly reduced the incidence of sour rot to 51.8 and 57.41%, respectively. This result is
in agreement with the finding of Palou et al. (2009) who reported that the same salts reduced
the incidence of sour rot of stone fruits.

To understand the mechanisms by which fungi are tolerant or sensitive to salt

compounds, several studies were carried out. It was found that inhibition of microorganisms
by salts might be caused by reducing of the cell turgor pressure with collapse and shrinkage of
hyphae and spore (Fallik et al., 1997) or by alteration of cell-transport function, and inhibition
of enzymes involved in the glycolytic pathway (Sofos et al., 1986). However, the mechanisms
by which salts inhibit G. candidum are not well understood.

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 Chapter 5: Screening of salt compounds

V. Conclusion

Among the 34 compounds tested sodium salicylate, boric acid and EDTA showed high
antifungal activities against Geotrichum candidum both in-vitro and in-vivo. These
compounds possess potent antifungal activities with potential practical applications in the
treatment of postharvest sour rot of citrus fruits and should be tested in future under
degreening conditions.

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DISCUSSION GÉNÉRALE
&
CONCLUSION
Discussion générale

Les stratégies de lutte contre les maladies des agrumes en post-récolte proposées comme
alternative aux fongicides de synthèse comprennent généralement l‟utilisation de nouveaux
fongicides à faible risque (Smilanick et al., 2006a; Kanetis et al., 2007), des microorganismes
antagonistes (El-Ghaouth et al., 2000; Droby et al., 2002; Taqarort et al., 2008; Ren et al.,
2011), des extraits de plantes (Ameziane et al., 2007; Mekbib et al., 2007; Du Plooy et al.,
2009; Liu et al., 2009b; Gatto et al., 2011; Askarne et al., 2012), des sels organiques et
inorganiques (Hall, 1988; Palou et al., 2001; El-Mougy et al., 2008; Smilanick et al., 2008;
Askarne et al., 2011) ainsi que l‟induction des mécanismes de résistance. Dans cette étude,
l‟activité antifongique des extraits aqueux et organiques de certaines plantes médicinales et
aromatiques ainsi que celle d‟une gamme de sels organiques et inorganiques a été évaluée in
vitro et in vivo contre Geotrichum candidum, l‟agent de la pourriture amère des agrumes en
post-récolte.

Quarante trois échantillons de plantes fraîches ont été récoltés dans différentes régions
du sud marocain durant la période 2008-2009. Les 43 plantes appartiennent à 16 familles
botaniques et 36 genres. Les extraits aqueux de ces plantes ont été évalués, d‟abord, sur la
croissance mycélienne de G. candidum en incorporant celles-ci dans le milieu de culture à
base d‟extrait de pomme de terre (PDA). Les résultats obtenus ont montré que la plupart des
plantes testées ont réduit la croissance mycélienne de G. candidum avec des degrés
d‟inhibition qui différent selon l‟espèce testée. Le meilleur pourcentage d‟inhibition de la
croissance mycélienne a été obtenu avec les extraits aqueux de Ceratonia siliqua, Halimium
umbellatum, Pistacia atlantica, Halimium antiatlanticum, Cistus monspeliensis, Rubus
ulmifolius et Cistus villosus. En effet, les extraits de ces plantes ont montré une forte
inhibition de la croissance mycélienne avec un pourcentage supérieur à 95%. Des résultats
similaires ont été obtenus par Ameziane et al. (2007). D‟après ces auteurs, l‟extrait aqueux de
C. villosus a complètement inhibé la croissance mycélienne de G. candidum. De même,
l‟évaluation de l‟activité antibactérienne de quarante plantes du sud du Maroc a montré que
les extraits aqueux de R. ulmifolius, C. monspeliensis et P. atlantica figurent parmi les
meilleurs espèces qui ont inhibé la croissance de Clavibacter michiganensis subsp.
michiganensis, agent du chancre bactérien de la tomate (Talibi et al., 2011a). Cependant,
certains plantes testées dans cette étude ont stimulé la croissance de G. candidum, à savoir :

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Mentha suaveolens, Psoralea bituminosa et Reseda alba. Cet effet stimulant sur la croissance
du pathogène a été également rapporté par Ameziane et al. (2007).

En plus de la croissance mycélienne, la germination des arthrospores est considérée

comme étape cruciale du cycle d‟infection de G. candidum. À cet égard, l‟effet des extraits
aqueux a été également évalué sur la germination des arthrospores de G. candidum. Les
résultats obtenus ont révélé que parmi les extraits aqueux qui ont complètement inhibé la
croissance mycélienne de G. candidum, seuls ceux de C. villosus et H. antiatlaticum ont
également inhibé la germination des arthrospores. Ceci montre que l‟activité des plantes
testées différe selon le stade de développement du champignon. En effet, il ressort des
résultats obtenus que la germination des arthrospores s‟avère moins sensible aux plantes
testées que la croissance mycélienne. Le même constat a été obtenu par Garduno-Pizana et al.
(2010), qui ont testé l‟effet de quinze plantes Mexicaines sur Fusarium oxysporum.

Nous avons également déterminé les concentrations minimales inhibitrices (CMI) et

fongicides (CMF) relatives aux meilleures plantes. Les plus faibles CMI ont été enregistrées
avec les extraits aqueux de C. villosus et H. antiatlanticum avec une CMI égale à 0,156
mg/ml, suivie de C. siliqua, H. umbellatum et R. ulmifolius avec une CMI de 0,312 mg/ml.
Cet effet antifongique est comparable à celui rapporté dans d‟autres travaux. Ainsi,
Bouamama et al. (2006) ont rapporté que les extraits de C. villosus et C. monspeliensis
possèdent des propriétés antifongiques avec des CMI qui varient entre 0,19 et 200 mg/ml. Les
mêmes auteurs ont rapporté que l‟extrait de C. villosus présente une activité antimicrobienne
plus intense que celui de C. monspeliensis (Bouamama et al., 2006).

Bien que la réalisation des tests in vitro est une étape importante dans le criblage des
plantes médicinales et aromatiques sur la base de leur potentiel antifongique, les tests in vivo
sont également essentiels pour confirmer, d‟abord, les résultats obtenus in vitro et ensuite
mettre au point des préparations dérivées des plantes qui sont susceptibles d‟être appliquées à
l‟échelle semi-commerciale (Gorris and Smid, 1995; Tegegne et al., 2008). Sur la base du
screening in vitro, seuls les extraits qui ont réduit la croissance mycélienne de G. candidum de
plus de 90% ont été retenus pour évaluer leur capacité à contrôler la pourriture amère. Les
résultats obtenus ont montré que les extraits aqueux de sept espèces (H. umbellatum, I.
viscosa, R. ulmifolius, C. villosus, C. siliqua, H. antiatlanticum et P. atlantica) ont
significativement réduit l‟incidence et la sévérité de la pourriture amère sans avoir des effets
phytotoxique sur les fruits traités. A notre connaissance, il s‟agit du premier rapport qui

116
montre l‟efficacité de ces plantes à contrôler la pourriture amère des agrumes en post-récolte.
Le meilleur degré de contrôle de la pourriture amère est obtenu avec l‟extrait aqueux de C.
villosus et H. antiatlanticum, qui ont réduit l‟incidence de la maladie à respectivement 46 et
44 % par rapport aux fruits non traités (100%). Cependant, aucun des extraits aqueux des
plantes testées n‟a totalement contrôlé le développement de la pourriture amère.

Dans l‟objectif d‟identifier la fraction active des meilleures plantes et aussi pour
améliorer leur activité antifongique contre G. candidum, nous avons procédé à un
fractionnement de chaque plante en utilisant des solvants de polarité croissante. Les espèces
suivantes : C. villosus, H. antiatlanticum, H. umbellatum, P. atlantica, I. viscosa, A. radiata,
R. ulmifolius et C. siliqua ont été, successivement, extraites avec l‟hexane, le chloroforme,
l‟acétate d‟éthyle et le méthanol. Ceci nous a permis d‟isoler quatre fractions organiques pour
chaque plante. Ces fractions ont été évaluées pour leur activité antifongique, in vitro et in
vivo, contre G. candidum.

L‟effet des fractions a été évalué sur la croissance mycélienne du pathogène. Les
résultats obtenus montrent que les espèces testées ont montré différent degré d‟inhibition de la
croissance de G. candidum. En effet, le pouvoir inhibiteur varie avec l‟espèce et le type de
solvant d‟extraction. Les extraits méthanoliques de C. villosus, H. umbellatum, C. siliqua, R.
ulmifolius, H. antiatlanticum, P. atlantica et les extraits d‟acétate d‟éthyle de C. villosus, A.
radiata et C. siliqua ont fortement inhibé la croissance mycélienne du pathogène. Ces
résultats sont similaires à ceux obtenus en utilisant les extraits aqueux des mêmes espèces
(Talibi et al., 2012a). Par contre, les extraits hexaniques et chloroformiques des plantes testées
se sont révélés être moins efficaces. De même, les fractions polaires des plantes testées
(méthanolique et d‟acétate d‟éthyle) ont fortement inhibé la germination des arthrospores de
G. candidum comparés aux fractions apolaires (hexanique et chloroformique). En effet,
l‟extrait méthanolique de C. villosus, H. umbellatutm, H. antiatlanticum, [Link], R.
ulmifolius et l‟extrait d‟acétate d‟éthyle de P. atlantica et A .radiata ont inhibé la germination
des arthrospores de G. candidum à plus de 92%. Nous pouvons donc conclure que,
contrairement aux extraits aqueux, l‟extraction des plantes avec des solvants organiques a
amélioré leurs propriétés antifongiques. En effet, les extraits méthanoliques des plantes se
sont révélés être plus efficaces sur la croissance mycélienne et sur la germination des
arthrospores de l‟agent pathogène.

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 Les plus faibles concentrations inhibitrices (CMI) ont été obtenues par les extraits
méthanoliques de H. umbellatum (0,156 mg/ml), C. villosus et R. ulmifolius (0,625 mg/ml).
Des résultats comparables ont été obtenus par Bouamama et al. (2006) en testant les fractions
organiques de C. villosus et C. monspeliensis contre une gamme de pathogènes des plantes.
Dans cette même étude, l‟extrait méthanolique de C. villosus s‟est révélé plus efficace que
l‟extrait à l‟acétate d‟éthyle (Bouamama et al., 2006). Par ailleurs, l‟extrait méthanolique de
C. villosus et H. umbellatum s‟est révélé fongicide à partir d‟une concentration de 2,5mg/ml ;
alors que l‟extrait aqueux de ces deux plantes n‟a montré qu‟une activité fongistatique vis-à-
vis de G. candidum même à 5mg/ml.

À partir des résultats des tests in vitro, ce sont les fractions polaires (extraits
méthanoliques, d‟acétate d‟éthyle) qui ont montré des propriétés antifongiques intéressantes
comparés aux fractions apolaires (extraits héxaniques et chloroformiques). Ces résultats ne
sont pas tout à fait surprenants du fait qu'une grande gamme de composés dotés d‟activités
antimicrobiennes (alcaloïdes, tannins, composés phénoliques, terpénoïdes…) ont une affinité
pour les solvants polaires (Choi et al., 2004; Tripathi and Dubey, 2004; Ismail et al., 2008;
Satish et al., 2008; Askun et al., 2009; Martini et al. 2009). Sur la base de ces résultats, seuls
les extraits méthanoliques et d‟acétate d‟éthyle (fractions polaires) des huit plantes ont été
retenus pour évaluer leur capacité à réduire l‟incidence et la sévérité de la pourriture amère.
Les résultats obtenus ont montré que les extraits méthanoliques de C. villosus, C. siliqua, H.
umbellatum, H. antiatlanticum et R. ulmifolius ont significativement réduit l‟incidence et la
sévérité de la pourriture amère, par rapport au témoin. En effet, le traitement en préventif des
fruits de mandarine avec l‟extrait méthanolique de C. villosus a totalement inhibé le
développement de l‟agent pathogène. En plus, l‟extrait méthanolique de C. siliqua et H.
umbellatum ont respectivement réduit l‟incidence de la pourriture amère à seulement 3,3 et
11,66%, par rapport au témoin (95%). Kivack et al. (2001) ont montré que l'extrait
méthanolique de C. siliqua possède une puissante activité antibactérienne contre un large
éventail de bactéries et de levures. En outre, Sisti et al. (2008) et Panizzi et al. (2002) ont
montré que l'extrait méthanolique de R. ulmifolius est doté de propriétés antimicrobiennes.
Comme dans le cas des tests in vitro, les extraits méthanoliques ont montré plus d‟efficacité à
contrôler la pourriture amère que les extraits aqueux des mêmes plantes. Ces résultats sont en
concordance avec les travaux de Haouala et al. (2008), sur l‟activité antifongique des extraits
méthanoliques et aqueux de Trigonella foenum-graecum contre Rhizoctonia solani et
Alternaria sp. Ceci pourrait être expliqué par la nature et la proportion des composés

118
responsables de l‟activité antifongique dans chaque type d‟extrait (aqueux et méthanolique).
Les extraits à l‟acétate d‟éthyle de H. umbellatum et A. radiata bien qu‟efficace in vitro contre
G. candidum n‟ont, cependant, aucun effet sur le développement de la pourriture amère (in
vivo). Par conséquent, les tests réalisés in vitro et in vivo pour ces deux espèces ont montré
des résultats opposés. D‟après Gatto et al. (2011), de nombreux facteurs tel que la
dégradation, l‟hydrolyse ou la polymérisation, peuvent affecter l‟activité biologique de
certains composants de l‟extrait de plante lorsqu‟ils entrent en contact avec les tissus des
fruits.

Dans la présente étude, quatre fractions organiques de polarité croissante ont été testées
(hexane, chloroforme, acétate d'éthyle et le méthanol). Parmi elles, la fraction méthanolique
possède le meilleur contrôle du pathogène à la fois in vitro et in vivo. D‟après Askun et al.
(2009), les extraits méthanoliques des plantes possèdent des propriétés antimicrobiennes plus
consistantes. En effet, les extraits méthanoliques de plusieurs plantes ont été rapportés avoir
une excellente activité antimicrobienne contre différents agents pathogènes des plantes (Sato
et al., 2000; Choi et al., 2004; Ameziane et al., 2007; Bajpai et al., 2008; Satish et al., 2008;
Mahlo et al., 2010; Ahmadi et al., 2010; Hajji et al., 2010). L‟analyse phytochimique des
extraits méthanoliques des plantes qui ont réduit l‟incidence et la sévérité de la pourriture
amère a montré qu‟ils contiennent différents types de flavonoïdes et possèdent des niveaux
élevés des phénols totaux. Ce résultat est appuyé par plusieurs études qui ont confirmé que le
méthanol peut extraire plusieurs composés ayant des activités antimicrobiennes en particulier
les composés phénoliques (Nicholson and Hammerschmidt, 1992; Cowan et al., 1999;
Tripathi and Dubey, 2004; Sisti et al., 2008; Martini et al., 2009). En outre, Cowan et al.
(1999) ont signalé que les plantes ont une capacité presque illimitée à synthétiser des
substances aromatiques, dont la plupart sont des phénols, qui jouent un rôle dans les
mécanismes de défense des plantes contre les agents pathogènes.

Dans l‟objectif de purifier les meilleurs fractions organiques et de comprendre le mode

avec lequel leurs composés agissent sur le pathogène, les extraits méthanoliques de C. villosus
et H. umbellatum ont subi un fractionnement bio-guidé. Cette méthode consiste à fractionner
l‟extrait en question sur gel de silice (par chromatographie sur colonne) en utilisant des
systèmes d‟élution de polarité croissante. Les sous-fractions issues de chaque fraction ont été
testées pour leur activité contre la croissance mycélienne de G. candidum. La sous-fraction la
plus active a subi à son tour un autre fractionnement réalisé de la même manière. Les résultats
obtenus montrent que plus l‟extrait est fractionné, plus l‟activité antifongique des sous
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fractions diminue. Ceci pourrait être expliqué par la perte de synergie ou l‟effet additif entre
les principes actifs présents dans la fraction initiale ou par leur redistribution dans les
différentes sous fractions en fonction de leurs affinités pour les solvants utilisés. L'effet
synergique entre les composés actifs des plantes a été également signalé dans plusieurs
travaux (Ellof, 2004; Nenaah, 2010; Suleiman et al., 2012; Zakaria et al., 2010). Nenaah
(2010) a rapporté que les alcaloïdes de Peganum harmala lorsqu‟ils sont appliquées
individuellement, montrent des effets antimicrobiens modérés. En revanche, l‟activité
antimicrobienne augmente lorsqu‟ils sont combinés, suggérant donc une sorte de synergie
entre ces composés (Nenaah, 2010). De même, Soliman et al. (2012) ont montré que le
fractionnement de l'extrait brut de la plante Loxostylis alata entraine une diminution de
l'activité antimicrobienne de plus de 85%.

Face à l‟absence de moyens efficaces pour lutter contre cette maladie, et aux inquiétudes
grandissantes à propos des impacts sur l‟environnement et la santé humaine reliés à
l‟utilisation des fongicides synthétiques, il devient important de développer des composés
antifongiques, qui soient efficaces et sains. De nombreux travaux menés au cours des deux
dernières décennies ont démontré à cet effet que des sels organiques et inorganiques, utilisés
en industrie alimentaire et/ou pharmaceutique, ont un potentiel intéressant pour le contrôle des
maladies des plantes. L‟utilisation de sels organiques et inorganiques apparaît donc comme
une approche intéressante pour lutter contre la pourriture amère des agrumes. Dans ce
contexte, nous avons opté pour l‟évaluation, in vitro et in vivo, de l‟efficacité de 34 sels contre
G. candidum. Les résultats du screening de l‟activité antifongique de ces sels ont montré que
les CMIs obtenues varient selon la nature du sel testé. La CMI la plus faible (0,1%) est
obtenue avec le carbonate d‟ammonium et l‟EDTA, suivie par le salicylate de sodium, le
metabisulfite de sodium et l‟acide borique avec une CMI de 0,25%. Ces résultats concordent
avec ceux d‟Askarne et al. (2011), qui ont montré que parmi les 28 sels testés contre
Penicillium italicum, agent causal de la pourriture bleue des agrumes, le metabisulfite de
sodium, l‟EDTA, le carbonate d‟ammonium et l'acide borique ont été les plus actifs contre ce
champignon, avec des CMIs qui varient entre 5 et 50 mM. Des résultats similaires, rapportés
par Olivier et al. (1998) et Hervieux et al. (2002), ont montré que le carbonate de sodium, le
carbonate de potassium et le metabisulfite de sodium ont complètement inhibé la croissance
mycélienne de Helminthosporium solani. En outre, Droby et al. (2003) ont démontré que
l'EDTA a un effet inhibiteur sur la croissance mycélienne de Botrytis cinerea et Penicillium
expansum.

120
 Les dix meilleurs sels, sélectionnés sur la base de leur CMI, ont été, ensuite, testés pour
leurs capacité à réduire ou à inhiber la germination des arthrospores du pathogène. Les
résultats obtenus ont montré que l'EDTA, l'acide borique, le metabisulfite de sodium, le
carbonate de sodium, le sulfate de sodium et le thiosulfate de sodium ont totalement inhibé la
germination des arthrospores à partir d‟une concentration de 75 et 100 mM. Ce résultat
concorde avec ceux d‟Askarne et al. (2011) qui ont indiqué que l'EDTA, l'acide borique, le
metabisulfite de sodium et le carbonate de sodium ont complètement inhibé la germination
des spores de P. italicum à des concentrations qui oscillent entre 20 et 200 mM. Également,
Smilanick et al. (1999) ont rapporté que le carbonate de sodium a inhibé la germination des
spores de P. digitatum, agent causal de la pourriture verte des agrumes. En revanche, le
salicylate de sodium, le carbonate d'ammonium et le carbonate de potassium se sont montrés
peu efficaces sur la germination des arthrospores de G. candidum. Considérant que plusieurs
sels peuvent influencer le pH du milieu, l'effet de ce dernier sur la croissance mycélienne de
G. candidum a été déterminé. Les résultats obtenus ont montré que la croissance mycélienne
est faiblement affectée par les modifications du pH dans la gamme de pH 4,0 à 12,0. Ce
résultat est similaire à celui de Nigro et al. (2006), qui ont démontré que le pH n‟a qu‟un rôle
mineur dans le mode d‟action des sels. En outre, Hervieux et al. (2002), ont indiqué que
l‟inhibition de la croissance des agents pathogènes dans des milieux additionnés de sels ne
peut être due seulement à l‟effet direct du pH. Par conséquent, c‟est la nature même du sel qui
affecte le développement de G. candidum.

Pour le test in vivo, des mandarines ont été traitées avec différentes concentrations de
sels (1, 2 et 3% w/v), inoculés avec une suspension d‟arthrospores de G. candidum puis
incubées à 26°C et à une humidité relative élevée. Les résultats obtenus ont montré que seuls
le salicylate de sodium, l'acide borique et l'EDTA ont réduit significativement l‟incidence et la
sévérité de la pourriture amère. En effet, testé à une concentration de 3%, l'incidence de la
maladie a été réduite à 25,93% chez les fruits traités avec le salicylate de sodium ou l'acide
borique et à 38,89% chez les fruits traités avec de l'EDTA. Ces résultats concordent avec des
études antérieures qui ont montré que l'EDTA est efficace contre la pourriture verte des
agrumes, due à Penicilium digitatum (Valencia-Chamorro et al., 2008), et contre la pourriture
grise des pommes, due à Botrytis cinerea (Droby et al., 2003). Dans la présente étude, nous
avons trouvé que le carbonate d‟ammonium et le carbonate de sodium ont significativement
réduit l‟incidence de la pourriture amère respectivement à 51,8 et 57,41%. Ces résultats sont

121
en accord avec ceux de Palou et al. (2009) qui ont rapporté que les mêmes sels ont réduit
l‟incidence de la pourriture amère des fruits à noyaux.

En conclusion, nous avons démontré que les extraits aqueux et méthanoliques de Cistus
villosus, Ceratonia siliqua, Halimium umbellatum et H. antiatlanticum sont efficaces contre la
pourriture amère des agrumes, et sans causer aucune réaction phytotoxique sur les fruits
traités. Compte tenu de l'activité antifongique de ces plantes, Il est donc possible de les
utiliser, après de plus amples investigations, comme moyen de lutte alternatif à la guazatine.
Ces plantes sont vivaces et largement répandues dans la région SMD. En outre, la méthode
d‟extraction est assez simple et l‟utilisation des extraits des plantes pour protéger les agrumes
contre les maladies fongiques est hautement souhaitable dans la production biologique des
agrumes. Nous avons également démontré que parmi les sels testés, l‟EDTA, l‟acide borique
et le salicylate de sodium sont dotés de propriétés antifongiques intéressantes pouvant avoir
des applications pratiques dans le traitement de la pourriture amère. Ces produits sains
représentent donc une alternative prometteuse et durable à l'utilisation de la guazatine
principalement dans la production biologique.

Cette étude nécessite d‟être complétée par un certain nombre de travaux. Ainsi, on se
propose de :

 Effectuer des essais dans des conditions semi-commerciales en utilisant aussi bien les
extraits de plantes, les sels ainsi que leurs combinaisons ;
 Identifier la composition chimique des fractions actives des plantes ;
 Explorer, d‟une manière plus approfondie, les modes d‟action des sels et des extraits
de plantes sélectionnés ;
 Tester le pouvoir antifongique des huiles essentielles des plantes testées dans cette
étude contre G. candidum.

122
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144
APPENDICES
 Appendices

Appendix 1 : Cultur mediums

NYDA : Nutrient Yeast Dextrose Agar :

Nutrient browth 8g
Yeast extracts 5g
D-glucose 10g
Bacteriological agar 15g
Distilled water 1000ml

NYDB : Nutrient Yeast Dextrose Broth :

Nutrient browth 8g
Yeast extracts 5g
D-glucose 10g
Distilled water 1000ml

PDA : Potato Dextrose Agar :

Potato extract 4g
D-glucose 20g
Bacteriological agar 15g
Distilled water 1000ml

Fuchsine acide :

Fuchsine acide 0.2% w/v

Acetic acide 1 volume
Ethanol 95% 1 volume

145
 Appendices

Appendix 2 : Bilan agrumicole de la campagne

2010/2011

I- SUPERFICIE :

Le verger agrumicole du Souss occupe actuellement une superficie d'environ 36 285

ha. Les nouvelles plantations s‟élèvent à 637 ha et concernent surtout la Nuless (169 Ha) et
Afourar (152 Ha). La superficie réservée aux petits fruits a atteint 18 163 ha, contre 17 081 ha
pour le groupe des oranges, soit 50% du verger agrumicole régional.

SUPERFICIE ARRACHAGE NOUVELLES SUPERFICIE SUPERFICIE

TOTALE
(Ha) (Ha) PLANTATIONS ACTUELLE (Ha) PRODUCTIVE
DESIGNATION (Ha)
2009/10 2010/2011 2010/2011 (Ha)
(1) (2) (3) (1) + (3) - (2)

PETITS FRUITS 17565 0 598 18163 16201

Clémentine 8038 0 14 8052 7756
Nuless 2993 0 169 3161 2377
Nour 4440 0 64 4503 4224
Afourar 211 0 152 363 160
Nova 299 0 85 384 289
Fortune 29 0 0 29 29
Ortanique 303 0 0 303 303
Autres petits fruits 1253 0 115 1368 1063

ORANGES 17059 16 39 17081 16508

Maroc-Late 8763 8 23 8778 8499
Navel 3241 0 15 3257 3133
Navelina 15 0 0 15 15
Navel Late 162 0 0 162 161
Navel Lane Late 766 0 0 766 739
Salustiana 897 8 0 889 873
W. Sanguine 415 0 0 415 337
Autres Oranges 2800 0 0 2800 2751

AUTRES 1048 8 0 1040 1023

Citron 1033 8 0 1025 1008
Autres 15 0 0 15 15
TOTAL 35672 24 637 36285 33733

146
 Appendices

II- CLIMAT :
- Pluviométrie :
Le cumul des précipitations au cours de la campagne 2010/2011 a atteint 362 mm
contre 543 mm pour la campagne 2009/2010, soit une diminution de 33%. Ces précipitations
sont réparties selon les stations comme suit :
STATION/MOIS SEP10 OCT NOV DÉC JANV11 FEV MARS AVR MAI JUIN JUIL AOUT CUMUL

AIT MELLOUL
09/10 0 0 0 197 77 244 12 0 0 2 0 42 572
10/11 0 16 58 87 73 2 67 41 83 0 0 0 426
MASSA

09/10 0 0 0 105 62 167 25 0 0 0 0 0 358

10/11 0 21 45 23 45 0 60 17 90 0 0 0 300
OD TEIMA
09/10 0 0 0 249 37 236 0 0 0 0 0 22 544
10/11 0 5 93 96 57 0 74 42 75 0 0 0 442
TAROUDANT
09/10 0 0 0 239 28 243 81 3 0 0 0 41 633
10/11 0 4 80 108 52 0 82 35 81 0 0 0 442
AOULOUZ
09/10 0 0 0 201 26 274 30 13 0 0 0 33 577
10/11 10 6 149 42 59 0 94 19 67 0 0 0 444

MOYENNE
09/10 0 0 1 198 46 233 29 3 0 0 0 32 542
10/11 1 15 67 49 52 0 68 24 84 0 0 0 362

- Température :

STATION/MOIS SEP10 OCT NOV DÉC JANV11 FEV MARS AVR MAI JUIN

AIT MELLOUL
max absolue 38 30 28 28 24 23 29 35 37 41
min absolue 18 14 11 12 9 7 9 13 13 19
moy max 29.2 25.8 22.0 23.5 21.8 19.2 21.6 25.4 27.8 28.8
moy min 21.9 18.1 14.1 15.0 11.5 10.2 13.0 17.8 20.7 21.9

TAROUDANT
max absolue 43 37 30 30 25 27 33 37 38 44
min absolue 13 12 8 8 6 3 3 10 10 3
moy max 33.4 27.9 23.4 23.2 21.4 21 21.9 26.8 28.9 35.5
moy min 18.8 15.6 11.4 12.4 8.3 7.2 9.2 13.5 17.3 19.3

147
 Appendices

III- DEROULEMENT DE LA CAMPAGNE 2010/2011:

1- Production :

La production brute a été estimée à 698 300 T, soit une augmentation de 8% par
rapport à la campagne précédente (646 300 T).

VARIETES PRODUCTION (T) VARIATION %

2010/2011 2009/2010
- Clémentine 253 100 218 000 1,16
- Navel 93 100 85 000 1,10
- Maroc Late 161 300 170 000 0,95
- Autres 190 800 173 300 1,10

TOTAL 698 300 646 300 1,08

2- Exportation :
Les exportations totales ont atteint au 30 Juin 2011 365 629 T contre 345 924 T en
2009/2010, soit une augmentation de 6%. La répartition de ces exportations par variété se
présente comme suit:
(1) (2) (1)/(2) %
PRODUITS 30/06/2011 30/06/2010 EVOLUTION TOTAL PAR RAPPORT
(T) (T) NATIONAL AU NATIONAL
PETITS FRUITS 242 609 232 432 104 3491 39 69
. Clémentines 139735 138847 101 215209 64,9
. Nour 67496 74626 90 79151 85,3
. Mandarine 84 0,0
. Marisol 0 0 916 0,0
. Nova 5500 4591 120 6514 84,4
. Afourer 21213 10793 197 37931 55,9
. Muska 57 0,0
. Ortanique 8665 3575 242 9277 93,4
. Wilking
ORANGES 118 584 110 399 107 173 258 68
. Navel 15594 14029 111 23897 65,3
. Maroc Late 71398 71288 100 98599 72,4
. Salustiana 20465 17094 120 25172 81,3
. W. sanguine 11127 7988 139 25590 43,5

AUTRES 4436 3093 143 5080 87

. Pomelos 439 383 115 672 65,3
. Citron 3997 2710 147 4408 90,7
TOTAL 365 629 345 924 106 527 477 69

148
 Appendices

IV- PREVISION DE LA PRODUCTION DES AGRUMES 2011/2012:

Suite aux premières prospections effectuées dans les différentes zones de production , il
ressort les principaux points suivants :
 Augmentation de la production de :
- 10 à 20% pour le groupe clémentine ;
- 10% pour la Navel et la Maroc Late;
- 5% pour la salustiana ;
 Diminution de la production de :
- 30% pour la Nour ;
- 10% pour la Washington Sanguine et l‟Ortanique;
 Pour les autres variétés, le rendement à l‟hectare est supposé identique à celui de la
campagne écoulée
Les prévisions de la production agrumicole seraient de l‟ordre de 744 000T contre 698 300
T au cours de la campagne écoulée, soit une augmentation de 7%. Elles sont réparties comme
suit :

2011/2012 2010/2011

VARIETES
% (1)/ (2)
D'AGRUMES SUPERFICIE SUPERFICIE
RENDEMENT PRODUCTION RENDEMENT PRODUCTION
PRODUCTIVE PRODUCTIVE
T/Ha T (1) T/Ha T (2)
Ha Ha

Clémentine 10133 29,32 297100 9928 25,49 253100 1,17

Maroc-Late 8499 20,90 177600 8489 19,00 161300 1,10
Navel 4048 25,30 102400 4048 23,00 93100 1,10
Autres 11053 15,11 167000 10953 17,42 190800 0,88

TOTAL 33733 22,06 744100 33418 20,90 698300 1,07

149
 Appendices

La répartition par variété se présente comme suit :

2011/2012 2010/2011

VARIETES
% (1)/ (2)
D'AGRUMES SUPERFICIE SUPERFICIE
RENDEMENT PRODUCTION T RENDEMENT PRODUCTION
PRODUCTIVE PRODUCTIVE
T/Ha (3) (1) T/Ha (4) T (2)
Ha Ha

PETITS FRUITS 16201 24,2 392503 15848 23,6 373877 1,05

Clémentine 7756 29,3 227448 7623 25,5 194375 1,17
Nuless 2377 29,3 69701 2305 25,5 58782 1,19
Nour 4224 16,1 68004 4058 23,0 93342 0,73
Afourar 160 14,0 2240 6 10,0 60 37,33
Nova 289 24,3 7031 289 27,0 7813 0,90
Fortune 29 19,0 551 29 19,0 551 1,00
Ortanique 303 27,0 8171 303 30,0 9079 0,90
Autres p. fruits 1063 8,8 9357 1235 8,0 9877 0,95

ORANGES 16508 20,8 342657 16547 19,1 316289 1

Maroc-Late 8499 20,9 177631 8489 19,0 161285 1,10
Navel 3133 25,3 79268 3133 23,0 72062 1,10
Navelina 15 25,3 387 15 23,0 352 1,10
Navel Late 161 25,3 4064 161 23,0 3694 1,10
Navel Lane Late 739 25,3 18700 739 23,0 17000 1,10
Salustiana 873 34,7 30248 873 33,0 28808 1,05
W. Sanguine 337 36,0 12139 337 40,0 13488 0,90
Autres Oranges 2751 7,4 20219 2800 7,0 19599 1,03

AUTRES 1023 8,8 9006 1023 8,0 8184 1,10

Citron 1008 8,8 8873 1008 8,0 8064 1,10
Autres 15 8,8 132 15 8,0 120 1,10

TOTAL 33733 22,1 744165 33418 20,9 698351 1,07

150
 Appendices

SUPERFICIE ACTUELLE PAR VARIETE

SUPERFICIE ARRACHAGE NOUVELLES SUPERFICIE SUPERFICIE

TOTALE
(Ha) (Ha) PLANTATIONS ACTUELLE (Ha) PRODUCTIVE
DESIGNATION (Ha)
2009/10 2010/2011 2010/2011 (Ha)
(1) (2) (3) (1) + (3) - (2)

PETITS FRUITS 17565 0 598 18163 16201

Clémentine 8038 0 14 8052 7756
Nuless 2993 0 169 3161 2377
Nour 4440 0 64 4503 4224
Afourar 211 0 152 363 160
Nova 299 0 85 384 289
Fortune 29 0 0 29 29
Ortanique 303 0 0 303 303
Autres petits fruits 1253 0 115 1368 1063

ORANGES 17059 16 39 17081 16508

Maroc-Late 8763 8 23 8778 8499
Navel 3241 0 15 3257 3133
Navelina 15 0 0 15 15
Navel Late 162 0 0 162 161
Navel Lane Late 766 0 0 766 739
Salustiana 897 8 0 889 873
W. Sanguine 415 0 0 415 337
Autres Oranges 2800 0 0 2800 2751

AUTRES 1048 8 0 1040 1023

Citron 1033 8 0 1025 1008
Autres 15 0 0 15 15
TOTAL 35672 24 637 36285 33733

151