Sustainability in Plant and Crop Protection 17

Md. Aslam Khan

Wasim Ahmad Editors

Microbes
for Sustainable
lnsect Pest
Management
Hydrolytic Enzyme & Secondary
Metabolite – Volume 2
Chapter 2
Role of Bacterial and Fungal Chitinases
in Integrated Management of Pest
and Diseases of Agro-Horticultural Crops

Ajinath S. Dukare, Sangeeta Paul, A. D. Asha, N. Nivetha, Chetana Aggarwal,

and Pratap Divekar

Abstract Chitin is an important structural component of many plant pathogenic

fungi. Similarly, it is also an important part of the insect cuticle and peritrophic
matrices, which function as a permeability barrier, enhance digestive processes and
protecting the brush border from mechanical disruption as well as from attacks by
toxins and pathogens. Chitin degrading lytic enzymes (such as chitinases, and glu-
canase) produced by bacteria and other microorganisms can impede the growth of
many insect pests and fungal phytopathogens that pose a severe risk to global crop
production. Pathogenic microorganisms produce a variety of lytic enzymes such as
proteases, chitinases, lipases etc. which play an important role in the virulence of
entomopathogens. Many chitinolytic bacteria have the potential to control pests and
fungal pathogens of crops owing to their ability to disintegrate chitin containing
cellular structures. Currently, efforts are being made to discover producers of chi-
tinolytic enzymes in nature. Production of lytic enzymes has been reported in a
number of virulent pathogens such as Serratia, Pseudomonas or Bacillus spp.
Bioprospecting and exploitation of chitinolytic bacteria will help in developing bio-
control agents, which have the potential to control fungal plant pathogens and insect
pests. Thus, these bacteria-based biofungicides and biopesticides may replace or
supplement the chemical fungicides and insecticides, reducing the negative impact
of chemicals on the environment and supporting the sustainable development of
agriculture-based ecosystem. This chapter focuses on the scope and potential of

A. S. Dukare (*)
Horticultural Crop Processing Division, ICAR-CIPHET, Abohar, Punjab, India
S. Paul · A. D. Asha · N. Nivetha
Division of Microbiology, ICAR-IARI, New Delhi, India
C. Aggarwal
ICAR-National Research Center on Plant Biotechnology, New Delhi, India
P. Divekar
ICAR-Indian Institute of Vegetable Research, Varanasi, India

© Springer Nature Switzerland AG 2021 33

Md. A. Khan, W. Ahmad (eds.), Microbes for Sustainable lnsect Pest
Management, Sustainability in Plant and Crop Protection 17,
[Link]
34 A. S. Dukare et al.

chitinolytic bacterial and fungal organisms in the management of insect pests and
fungal pathogens of agricultural crops.

Keywords Chitinolytic organisms · Chitinase enzymes · Insect cuticle · Fungal

cell wall · Chitin degradation mechanisms · Pests and pathogen biocontrol

2.1 Introduction

Pathogenic microorganisms and harmful insect pests affecting plant health are a
major threat to food production and ecosystem stability, worldwide. Among the
biotic factors, fungal phytopathogens and insects pests are responsible for consider-
able economic losses in a wide variety of cultivated crops. More than 65,000 diverse
pest species are presently recognized, mainly fungi, weeds, and arthropods, causing
up to 40% preharvest- and 10% post-production losses in agro-horticulture
(Chandler et al. 2011). In India only, almost 30% of the crop yield, corresponding
to 30 million tons of food, is wasted due to attacks by various insect pests, disease,
and weeds (Koul 2011). Insects are one of the major natural hazards to any agro-­
horticultural products and encompass a remarkable group in the animal kingdom,
accounting for 70% of all animals present in the world. According to an estimate,
one third of the global agricultural production, amounting to about several billion
dollars, is damaged annually by over 20,000 species of insects in field and storage
conditions (Mariapackiam and Ignacimuthu 2008).
Similarly, pathogenic fungi are important causal agents of plant diseases of eco-
nomic importance, and more than 60% of the literature in plant diseases is devoted
to fungal infections (Hawksworth et al. 1995). In the past few decades, agricultural
production in an intensified agricultural system was more and more dependent on
agrochemicals as a relatively reliable method of crop protection. An excessive use
of synthetic chemicals caused, however, several negative effects such as the devel-
opment of fungicidal/insecticidal resistance in pathogens/pests, detrimental impacts
on environment and human health, and harmful impact to their non-target insect/
beneficial microflora, as well as possible bioaccumulation of toxic xenobiotics in
the ecosystem and food supply chain (Hardy 2014; Czaja et al. 2015). Furthermore,
higher cost of agrochemicals such as pesticides, mainly in poorer regions of the
world, demanded for alternative and ecofriendly methods of crop protection, which
have more and wider public acceptance. Among safer options, biological control
using potential antagonistic microorganisms is being considered as an alternative or
supplemental approach or method, reducing the chemicals use in sustainable agri-
culture (Gerhardson 2002; Dukare et al. 2011).
Microbe-mediated biological crop protection is an attractive and promising tech-
nology with no concern for negative impact on the environment and biodiversity
2 Role of Bacterial and Fungal Chitinases in Integrated Management of Pest… 35

(Dukare and Paul 2018). The suppression of many crop pests and diseases using
microbial biological agents holds great promise in facilitating and developing
organic agriculture. Many biocontrol agents are safe to deliver, simpler in use, non-­
polluting, compatible with the conventional and low-input agricultural practices,
induce host plant resistance and in many cases improve plant growth and yield by
acting as bio fertilizers or phyto-stimulators.
Biological control of insect pest and fungal diseases using chitinase producing
microorganisms has received a large amount of attention in recent times. Owing to
the wider distribution of chitin in the ecosystem, microbial chitinases have lately
achieved interest for their probable use as potential biopesticides in integrated pest
management (IPM) strategies for controlling fungi, insects and nematodes (Hjort
et al. 2014; Berini et al. 2016). Additionally, chitinase hydrolytic enzymes, pro-
duced by a diverse range of living organisms such as bacteria, fungi, nematodes,
snails, insects, crustaceans and plants are receiving, for the purpose of pathogenesis,
morphogenesis, parasitism, and defense, global attention with regard to their devel-
opment as chemical defense proteins in transgenic plants and as microbial biocon-
trol agents. Widely occurring chitinolytic microbes have been preferred as a source
of chitinase because of easy availability of raw materials for their cultivation and
low production cost of chitinase enzymes.
Chitinases act as plant protection agents by hydrolyzing chitin. This polymer is
an important constituent of the fungal cell wall, a structural component of insect
exoskeleton and is present in the egg shells of nematodes. Chitin is degraded into a
variety of products that include the deacylated oligomer chitosan, monomer
N-acetylglucosamine and the disaccharide chitobiose. At the same time, chitinases
are safe for plants and vertebrates as they are devoid of any chitin, therefore, holding
a larger potential for IPM than other hydrolytic enzymes (Neeraja et al. 2010). The
degradation of fungal cell wall leads to the loss of structural integrity, deformity,
and eventual cell death (El-Tarabily 2006). Bacterial chitinases have been widely
demonstrated as inhibiting fungal growth and can therefore be effective in control-
ling plant-pathogenic fungal diseases (Ordentlich et al. 1988). Similarly, extracel-
lularly produced microbial chitinases hydrolyze chitin present in the exoskeleton
and gut linings of insects, which leads to decline in the insect feeding rate and
eventually yielding its death. Due to these properties, uses of chitinolytic microor-
ganisms have stirred substantial interest in biological management of economically
important insect pests of agricultural crops, during last years (Mubarik et al. 2010;
Abdullah et al. 2014).

2.2 Occurrence of Chitin and Chitinolytic Organisms

Chitin is abundant in a broad range of earth environment and constitutes a structural

component in many living organisms, e.g., fungi, nematodes, insects or crustaceans
(Gooday 1990a, b). It is a characteristic component of the exoskeletons of arthro-
pods and insects, of the fungal cell walls, as well as the radulae of mollusks and
36 A. S. Dukare et al.

internal shells of cephalopods. In accordance with the ubiquitous presence of the

chitin polymer, chitinases, which degrade chitin, are found in numerous types of
living organisms, such as bacteria, fungi (Gooday 1990a), archaea (Gao et al. 2003),
phytoplancton (Štrojsová and Dyhrman 2008) and rotifers (Strojsova and Vrba
2005). The presence of chitinolytic microorganisms can be traced to a wide range of
environments including all the regions of the earth’s biosphere. Several microorgan-
isms are able to produce chitinase and degrade chitin under both aerobic and anaer-
obic environments. It is believed that chitin decomposition in natural habitats is
mostly and predominantly mediated by bacteria or groups of bacteria. Kielak et al.
(2013) reported that hydrolysis of the chitin polymer in soil ecosystems is corre-
lated with the abundance of bacteria. However, depending on the presence of vari-
ous environmental conditions such as temperature and pH, fungi may constitute
important agents of chitin hydrolysis in the later stages of its degradation process
(Manucharova et al. 2011).
In general, fungal chitinases have been studied more extensively than the bacte-
rial ones. However, there are several bacterial species known to produce extracel-
lular chitinase enzymes in culture (Frandberg and Schnurer 1998; Viswanathan and
Samiyappan 2001). Chitinase enzymes, which are involved in chitin degradation,
are commonly distributed in several bacterial genera such as Aeromonas,
Arthrobacter, Bacillus, Clostridium, Chromobacterium, Enterobacter, Erwinia,
Flavobacterium, Klebsiella, Pseudomonas, Streptomyces, Serratia and Vibrio etc.
In natural ecosystems, chitin is degraded mainly by the above heterotrophic chitino-
lytic bacteria. Chitin-containing cell walls of plant pathogenic fungi are effectively
hydrolyzed and ruptured down, individually or in combination, by the lytic enzymes
such as chitinases, chitosanases and glucanases, of the chitinolytic bacteria.
Bacterial chitinases have been extensively reported as fungal growth inhibitors, and
can therefore be effective in controlling fungal plant diseases.

2.3 Chitinolytic Enzymes: Nomenclature and Classification

Chitinases (CHIs) are usually extracellular inducible enzymes secreted by a variety

of microorganisms, insects, plants, and animals and even present in the blood serum
of human (Gohel et al. 2006). CHIs break down the β-1,4-glycosidic bonds that are
present between the N-acetyl-D-glucosamine residues of chitin polymer. The entire
de-polymerization of chitin to free the N-acetylglucosamine (GlcNAc) residue is
carried out by a diverse group of chitinolytic enzyme systems (Patil et al. 2000;
Gohel et al. 2006).
The nomenclature of chitinolytic enzymes is ambiguous. Based on the location
of the hydrolyzed bond, CHIs (EC [Link]) can be grouped into two classes.
Endochitinases, the first class enzymes, slice chitin polymer randomly and produce
low molecular weight oligomers such as diacetylochitobiose, chititriose and chito-
totetriose. The exochitinases generate chitobiose from either reducing or non-­
reducing sites of the polymer. In recent times, there were two other categories of
2 Role of Bacterial and Fungal Chitinases in Integrated Management of Pest… 37

these enzymes which include chitobiases, accountable for the degradation of chito-
biose, and β-N-acetylglucosaminidases that produce monomer unit of β-N-acetyl-
D-glucosamine (Saks and Jankiewicz 2010). At present, chitobiase and
β-N-acetylglucosaminidases are included in the enzyme family of β-N-­
acetylhexosaminidases (EC.[Link]), (according to the Nomenclature Committee
of the International Union of Biochemistry and Molecular Biology) (Saks and
Jankiewicz 2010). Based on the mechanism of hydrolysis, CHIs may also be classi-
fied into three types: a) β-1, 4-N-acetyl-glucosaminidases (EC [Link]), which
catalyze the cleavage of the chitin polymer from the terminal end into individual
units of GlcNAc; b) endochitinases (EC [Link]) randomly splitting at internal sites
along the total extent of the chitin microfibril chain, and c) exochitinases (EC
[Link]) that cause continuous release of diacetylchitobiose, in a stepwise manner,
such that no monosaccharides or oligosaccharides are produced.

2.4 Family of Chitinase Enzymes

The diverse configurations of chitins present in nature are degraded by the chitin-
ases produced by bacteria and related chitinase-producing organisms. Based on
similarity in amino acid sequences, chitinolytic enzymes are grouped into the fami-
lies 18, 19, and 20 of glycosyl hydrolase enzymes (Brzezinska et al. 2014). Family
18 includes CHIs enzymes obtained from bacteria, fungi, viruses, animals, and
some plant chitinases, all of which have a diverse evolutionary background.
Plant chitinases (classes I, II, and IV) and some Streptomyces chitinases (Hart
et al. 1995) are included in Family 19 of glycosyl hydrolases. Family 20 of glycosyl
hydrolases includes N-acetylglucosaminidase from Vibrio harveyi and β-N-acetyl
hexosaminidase enzymes from human and Dictyostelium discoideum (Patil et al.
2000; Duo-Chuan et al. 2005; Dahiya et al. 2006).
Based on the amino acid sequence of individual catalytic domains, bacterial chi-
tinases are further grouped into three major subfamilies: A, B and C. Bacillus
licheniformis produces five chitinases of 42, 49, 53, 62 and 66 kDa molecular
weight, while B. circulans produces six distinct chitinases of which chitinase A1 is
primarily responsible for chitin degradation. Many well known biocontrol bacteria
including Serratia marcescens, Pseudomonas aeruginosa K-187, Aeromonas spp.
and Streptomyces griseus are capable of synthesizing various CHIs (Brzezinska
et al. 2014), with molecular weight ranging from 20 to 120 kDa (Joo 2005; Kavitha
and Vijayalakshmi 2011). Serratia marcescens strain Nima produces an endochitin-
ase (Chi60), an exochitinase (Chi50), and an N-acetyl glucosaminidase that showed
a 43 fold higher chitinolytic activity than those of other strains (Ruiz-Sánchez et al.
2005). Mehmood et al. (2010) demonstrated that four chitinases secreted by
Aeromonas caviae CB101 are encoded by a single chitinase gene Chi1. Furthermore,
the well-known fungus Trichoderma harzianum secretes N-acetylglucosaminidases,
endochitinases, and chitobiosidase (Haran et al. 1995). Aggarwal et al. (2015a)
reported presence of two isoforms of chitinases in entomopathogenic S. marcescens
38 A. S. Dukare et al.

with molecular weight of ~51 kDa and 32 kDa. The optimum temperatures for CHIs
functioning are around 40 °C with a broad pH range (5–8). Based on the source of
CHIs, their activity can be stabilized, improved or inhibited in the presence of dif-
ferent metal ions.
CHIs are adaptive enzymes, produced only under definite conditions incited by
specific factors. They are regulated by inducer/repressor compounds. Chitin is
mostly an inducer molecule, while glucose or other easily storable carbon sources
may act as a repressor molecule in the medium. An analysis using several carbon
substrates have revealed the correlation between a metabolized carbon source and
the extracellular production of chitinolytic enzymes. Inducing the production of
chitinases via several chitin substances is a characteristic of a certain enzyme-­
producing species of bacteria or fungi. Many studies have used colloidal chitin to
enhance production of CHIs. For examples, chitinolytic activity was found in bac-
teria grown on media constituting glycol or colloidal chitin, N-acetylglucosamine,
chito-oligosaccarides, and/or the cell wall fractions of a few chitin containing
molds. On the contrary, almost no or minimal activity was visible when same bac-
teria were cultured on media containing glucose or laminarin as the sole carbon
source (Zhang et al. 2001; Miyamoto et al. 2007; Saks and Jankiewicz 2010).
Molecular investigations have revealed the presence, in Pseudoalteromonas pisci-
cida, Streptomyces thermoviolaceus and other bacteria, of a two-component signal
transduction system consisting of two regulatory proteins (histidine kinase and a
response regulator) that are implicated in the regulation of chitinase synthesis. In
response to external stimuli/signals, an autophosphorylation occurs at the histidine
residue of the bacterial kinase, followed by the transfer of a phosphate group to an
asparagine residue in the response regulator. Eventually, the phosphorylated
response regulator, in combination with a promoter sequence, causes the activation
of and transcription of genes encoding for chitinase enzymes (Saito et al. 1998;
Brzezinska et al. 2014).

2.5  hitin as an Important Structural Component of Insect

C
Pests and Fungal Pathogens

2.5.1 Structural and Functional Role of Chitin in Insects

The extracellular integument layer of insects, known as “cuticle”, is secreted by the

insect epidermis and forms the outer covering of insects, as well as in other arthro-
pods. Apart from the insect exterior, the cuticle covering can be observed on the
interior parts including the foregut, hindgut, and trachea. The typical components of
the cuticle layers, arranged in an exocuticle and endocuticle, include proteins, lip-
ids, and chitin in cross-linking with each other, so as to provide varying degree of
hardness and elasticity (Wigglesworth 1948). Chitin is also present in the peri-
trophic matrix (PM) of insect. This is a sleeve-like extracellular covering layer that
encircles the food bolus present in the gut of most arthropods and insects (Hegedus
2 Role of Bacterial and Fungal Chitinases in Integrated Management of Pest… 39

et al. 2009). In addition to chitin, the other important components of this matrix are
proteins and proteo-glycans. This compound acts as a physical barricade towards
invading pathogens, aids in the digestion process, protects gut epithelial cells from
the detrimental impact of food particles and prevents the entrance of the swallowed
insect pathogens and harmful toxins into the midgut epithelium (Tellam 1996). The
PM is analogous to the protective mucosal layer that lines the digestive tracts of
mammals. It also separates and organizes digestive processes within the midgut.
The constituting components of PM such as membrane protein (peritrophins) and
chitin may associate with other components such as enzymes and food molecules.
The formation of chitin is usually found at the tips of midgut microvilli. The PM
protein, peritrophins, has domains which are alike the gastrointestinal mucus pro-
teins (mucins) and some other domains that are capable of binding to the chitin
molecule. The PM formation can occur in the midgut or in the total organ (type I),
or merely at the opening of the midgut (type II). Most insects contains type I PM,
whereas type II PM is limited to larval and adult (except hematophagous) mosqui-
toes, flies (Diptera) and a few Lepidopteran adults. The PM is absent in Hemiptera
and Thysanoptera, whose cells have perimicrovillar membranes.
Chitin chemical formula is (C8H13O5N)n. It is a long-chain polymer of
N-acetylglucosamine, a derivative of glucose. It is widely distributed and the second
most abundant polysaccharide in nature, after cellulose (Fig. 2.1). Numerous bene-
fits are offered to animals possessing chitin as exoskeletons or as their structural
component. To mention, chitin gives and defines the basic shape of the arthropods
and provides protection from the probable desiccation and dehydration caused by
adverse external factors (Anderson 1997). The chitin micro fibrils of PM form

Fig. 2.1 Structure of chitin polymer found in cuticle and peritropic membrane of insects and cell
wall of certain plant pathogenic fungi
40 A. S. Dukare et al.

association with the extremely hydrated proteoglycan matrix released by the gut
cells of insects, contributing significantly in providing a tensile strength.

2.6  ode of Action of Chitinolytic Bacteria

M
in Biological Suppression

2.6.1  echanism of Action of Microbial Chitinolytic Enzymes

M
in Insect Pests Biocontrol

Hydrolytic enzymes (including chitinsases, proteases and lipases) that can degrade
and lyse the chitin-containing insect tissues can serve as an important virulence fac-
tor for the entomopathogenic microorganisms (Aggarwal et al. 2017). Chitinases
have been isolated from numerous bacterial strains and reported to be effective
against many pests and diseases (Gomaa 2012). A large number of bacteria that can
cause pathogenesis/virulence in insect have a great potential for use in biological
control of economically important pests of agricultural crops.
Chitin is insoluble polysaccharide made up of linear chains of GlcNAc residues,
cross-linked by hydrogen bonds. It is the most important structural constituent of
the external skeleton (50% of the cuticle is made up of chitin), hindgut, foregut,
midgut lining of the PM. Therefore, it is also vital for ensuring the structural integ-
rity of many insects and nematodes pests (Bhattacharya et al. 2007). The insect
exoskeleton, cuticle and PM is broken down by individual or combined actions of a
binary enzyme complex, involving chitinase and β-N-acetylglucosaminidases
(NAG) enzymes (Filho et al. 2002).
Microbially produced chitinases complex de-polymerize chitin components into
their monomeric or oligomeric units, thus degrading the foremost constituents of
the insect exoskeleton. Sometimes, extra proteins that have one or more chitin-­
binding domains (which are however deprived of a chitinolytic activity) may aug-
ment the process of chitin degradation (Vaaje-Kolstad et al. 2005). This system, that
likely operates in the gut during PM break down, increases the PM porosity, finally
causing insects’ death (Khajuria et al. 2010).
There is a correlation between virulence and chitinase production that can cause
weakening and slimming of the insect cuticular structure. In addition, if these
enzymes act on the insect intestine, a crucial damage to the PM may occur
(Chandrasekaran et al. 2014). The hydrolytic action of chitinase and the subsequent
damage caused to the gut of the larvae PM prevent the insect from feeding and,
therefore, eventually leads to its death. In alternative it damages the cuticle resulting
into abnormal molting (Suganthi et al. 2017). This reality has opened a new poten-
tiality in relation to the exploitation of chitinolytic enzymes in the biocontrol of
insect pests.
2 Role of Bacterial and Fungal Chitinases in Integrated Management of Pest… 41

[Link] Effect of Chitinase on the Peritrophic Membrane

Chitin biosynthesis and its degradation are important metabolic pathways in several
arthropods, and particularly most crucial in inhibition of the insect cuticle and PM
(Husen et al. 2015). The capability of chitinases to degrade the PM of the insect gut
has been explored for many years. In 1993, Shahabuddin et al. demonstrated that the
addition of exogenous chitinase, isolated from S. griseus in a blood meal in vivo
hampered the function of the PM in Anopheles freeborni. Likewise, feeding of the
fifth instar larvae of Spodoptera littoralis on recombinant endochitinase ChiA
caused holes and perforations in the PM (Regev et al. 1996). The excessive intake
of chitinase has been found to have serious physiological consequences with death
of exposed insects (Herrera-Estrella 1999). The midgut chitinases appear to be
implicated in the development, puncture and degradation of the peritrophic matrix
that protects the gut epithelium from destructive factors (Filho et al. 2002).
Histological study confirmed the significant negative effects of chitinases on the
treated larval gut, epithelial cells and PM. SEM studies on PM from the midgut of
Spodoptera litura larvae fed on a chitinolytic S. marcescens strain revealed consid-
erable damage to the midgut (Aggarwal et al. 2017). Chitinase isolated from B. sub-
tilis efficiently inhibited the gut enzyme activity, growth and development of
S. litura larvae (Chandrasekaran et al. 2012). Paenibacillus larvae, a bacterial
pathogen of honey bee larvae, expresses a chitin-binding and degrading protein
which successfully degrades the chitin-containing gut line of PM (Garcia et al.
2013). This study demonstrated that the break down of the PM lining the midgut
epithelium is a key step in the virulence of P. larvae. These observations confirmed
that PM is the first barrier that bacteria have to overcome when trying to infringe the
epithelium and penetrate the hemocoel. The mechanism of action of chitinase
enzymes on insects are given in Fig. 2.2.

2.6.2  echanism of Action of Chitinolytic Enzymes

M
on Fungal Pathogens

The extracellular hydrolytic enzymes of antagonistic microbes cause mycoparasit-

ism of fungal pathogens, by attaching and degrading fungal hyphae. Direct parasit-
ism or mycoparasitism is the ability of an antagonistic microorganism to attach
hyphae to produce extracellular cell wall lytic enzymes (Dukare et al. 2018).
Mycoparasitism of antagonists depends upon the sequential occurrence of the fol-
lowing events: (i) coming into close contact with the fungal pathogens, (ii) mutual
recognition between antagonist and pathogen, (iii) lytic enzymes secretion and (iv)
active growth of the antagonist inside the host (Talibi et al. 2014). The negative
effects of myco-parasitism result in the killing of the pathogen propagules, lysis
with destruction of its cellular structures and disintegration of the cellular integrity.
The fungal cell wall, composed by chitin and glucan in combination with wall
42 A. S. Dukare et al.

Fig. 2.2 Schematic representation of chitin degradation in insect by chitinase, and chitinase mode
of action in insect. (Adapted in modified from Chandrasekaran et al. 2012)

proteins, is disintegrated by an individual or collective actions of lytic enzymes such

as chitinases, glucanases, chitosanases, cellulase, and proteases produced by the
antagonists, thus contributing to the biocontrol activity (Spadaro and Droby 2016).
Figure 2.3 demonstrates the action of different chitinolytic enzymes in the complete
dissolution of chitin-containing cell wall of pathogenic fungi. Moreover, these
enzymes adversely affect pathogen’s conidial germination, germ tube elongation,
and may cause damages to the oospores (El-Tarabily 2006). Accordingly, several
reported microbial biocontrol agents of different fungal pathogens produce extra-
cellular hydrolytic enzymes. For example, the biocontrol action of the antagonist
yeast Candida oleophila against Penicillium expansum, causing blue mold decay in
apple, was mediated through an extracellularly produced β-1,3-glucanase enzyme
(Urbina et al. 2016). In the process, the action of the purified glucanase enzyme
reduced the P. expansum conidia germination and caused mycelium inhibition.
Banani et al. (2015) reported chitinase activity of the antagonistic yeast
Metschnikowia fructicola and demonstrated that chitinase gene MfChi was over
induced in the presence of the yeast Monilinia fructicola cell wall. An overexpressed
MfChi chitinase in Pichia pastoris controlled the growth of M. fructicola and
M. laxa in vitro, and in vivo on peach fruits. The antimicrobial actions of many spe-
cies of Bacillus and Pseudomonas are credited to the extracellular chitinolytic
enzymes they release (Yu et al. 2008). Some other detrimental effects of enzymatic
actions on pathogens are cellular deformities, protoplasmic damages, mycelial
2 Role of Bacterial and Fungal Chitinases in Integrated Management of Pest… 43

Fig. 2.3 Schematic diagram representing action of different chitinolytic enzymes involved in the
complete dissolution of chitin-containing pathogenic fungal cell wall. (Adapted from Dukare
et al. 2018)

distortion and lyses, leakage of cellular contents, and changes in membrane perme-
ability (Di Francesco et al. 2016). Therefore, the loss of fungal cytoplasm due to the
enzymatic cell wall disintegration is one of the key modes accountable for biocon-
trol actions of chitinase and other lytic enzymes secreted by microbial antagonists.

2.7 Chitinase Deployed as Successful Biocontrol Agents

2.7.1 Chitinases in Pest Management of Insects

Bacterial chitinases are active at an alkaline pH range. They can consequently degrade
the chitin present in the insects gut lining, which has an alkaline pH (Bhattacharya
et al. 2007; Aggarwal et al. 2015b). Chitin metabolism is an excellent target for selec-
tive pest control (Shternshis et al. 2005). Most chitinases deployed as insecticides are
isolated and purified from the bacterial genera such as Bacillus, Pseudomonas,
Serratia, Streptomyces spp. etc. In addition, Nawani and Kapadnis (2003) reported
the presence of chitin degrading actinobacterial genera including Saccharopolyspora,
Kitasatospora, Nocardiopsis, Nocardioides, Herbidospora, Microbispora,
44 A. S. Dukare et al.

Micromonospora, and Actinoplanes. Their investigation formed a comprehensive

base to study the diversity of chitinolytic systems in actinobacteria.
In recent times, microbial chitinases have been utilized as insecticide and fungi-
cide for pest and disease control (Berini et al. 2018). Chitinolytic enzymes derived
from bacteria are being used to augment the efficiency of microbial insecticides
such as B. thuringiensis and Baculoviruses. These enzymes, by disrupting the PM,
enable microbes and entomopathogens to march into the hemocoel of insects
(Hegedus et al. 2009).
The soil bacterium S. marcescens produces different extracellular enzymes
including chitinases exhibiting insecticidal effects (Petersen and Tisa 2013). This
bacterium is a well recognized soil microbe for the rapid degradation of chitin pres-
ent in the ecosystem. In a range of experimental setups, chitinases, and chitinase
genes of S. marcescens have demonstrated their potential as effective biocontrol
agents against several pests (Brurberg et al. 2000; Aggarwal et al. 2015b). Serratia
marcescens strain STS caused a 90% mortality in S. litura grown on a semi-­synthetic
diet (Aggarwal et al. 2014). Another S. marcescens strain SEN, isolated from dis-
eased insects, showed predominant chitinase activities including exochitinase,
endochitinase and chitobiosidase (Aggarwal et al. 2015b). This strain had strong
insecticidal properties against all growth stages of S. litura larvae. The intake of sub
lethal doses of this strain reduced larval and pupal weight, normal pupation, emer-
gence of adult with a delay in the larval period. In addition, fecundity and hatch-
ability was also significantly affected. Similarly, purified chitinase from
Psuedomonas fluorescens exhibited potent insecticidal activity against the tea mos-
quito bug Helopeltis theivora (Suganthi et al. 2017). The implicated mechanism of
insecticidal action was attributed to the hydrolysis of chitin.
Another bacterium, Bacillus thuringiensis, well known for its pesticidal activity,
also produces many chitinases with varying molecular weights (66, 60, 47 and
32 kDa) (Thamthiankul et al. 2001). Larvae of the spruce budworm Choristoneura
fumiferana died more quickly when exposed to a chitinase-Bacillus mixture
(Thamthiankul et al. 2001). The biocontrol potential of the soil bacterium
Paenibacillus spp. D1 against larvae of Helicoverpa armigera, commonly known as
legume pod borer, was ascribed to its high rate of chitinase production (Singh et al.
2016). The exposure of insects to this bacterium noticeably reduced their feeding
rate and larval body weight. This effect was attributed to the hydrolysis and degra-
dation of the insects’ chitinous structures. This was apparent from the decrease in
the total chitin content and increased larval mortality when treated with Paenibacillus
spp. D1 and chitinase, as compared to untreated control.
The application of specific metabolites such as chitinase is always beneficial in
comparison to the use of living microbial cell (Shternshis et al. 2005). Chitinase
functions as both a systemic toxic and contact component, causing insect death.
Therefore, this strategy is opening a new avenue in the development of chitinase-­
based biopesticidal formulations. The production of low-cost chitinases has received
interest as a potential biocontrol molecule for management of many insects. It is a
rising field of research, and has been evaluated for numerous pests of agricultural
crops. Table 2.1 shows the list of chitinolytic microorganisms (bacteria and fungi)
and their produced chitinases implicated in the biological control of insect pests.
2 Role of Bacterial and Fungal Chitinases in Integrated Management of Pest… 45

Table 2.1 Chitinolytic microorganisms (bacteria and fungi) and their chitinase enzymes
implicated in the biological control of insect pests
Chitinolytic Reported
microorganism chitinase Target insect pests References
Bacillus circulans No. Not available Lymantria dispara Lertcanawanichakul
4.1 et al. (2004)
Bacillus subtilis Not available Spodoptera litura Chandrasekaran et al.
(2012)
Bacillus licheniformis ChiBIA Spodoptera exigua Thamthiankul et al.
(2001)
Bacillus thuringiensis Chi Helicoverpa armigera Ni et al. (2015)
[Link] 960235–459
YBT-9602
Bacillus thuringiensis Chi A Spodoptera exigua and Liu et al. (2010)
subsp. colmeri Helicoverpa armigera
Brevibacillus Mixture of six Plutella xylostella Prasanna et al. (2013)
laterosporus Lak1210 chitinases
Pseudomonas Not available Helopeltis theivora Suganthi et al. (2017)
fluorescens MP-13
Serratia marcescens ChiAII Spodoptera littoralis Regev et al. (1996)
Serratia marcescens Mixture of Spodoptera litura Aggarwal et al.
SEN two chitinases (2015a, b)
Serratia marcescens Chi A, Chi B,
Malacosoma Neustria, Danişmazoğlu et al.
WW4 and Chi C Helicoverpa armigera (2015)
Serratia marcescens Chi B, Chi CGalleria mellonella Ozgen et al. (2013)
Xd1 Drosophila melanogaster
Yersinia entomophaga Chi1 and Chi2 Costelytra zealandica, Busby et al. (2012)
MH96 Adoryphorus couloni,
Acrossidius tasmaniae, and
Plutella xylostella
Trichoderma harzianum Not available Helicoverpa armigera Binod et al. (2007)
Trichoderma viride Mixture of at Bombyx mori Berini et al. (2016)
least four
chitinases
Beauveria bassiana BbChit1 Myzus persicae Fang et al. (2009)
Beauveria bassiana Chimeric Myzus persicae Fang et al. (2009)
BbChit1
Metarhizium anisopliae Chi2 Dysdercus peruvianus Boldo et al. (2009)
Isaria fumosorosea IfChit1 Plutella xylostella Huang et al. (2016)
46 A. S. Dukare et al.

2.7.2  hitinolytic Microbes and Enzymes as Biocontrol Agents

C
of Plant Fungal Diseases

Chitinolytic enzymes have received attention for the biological control of soil borne
fungal phytopathogens. Hydrolytic enzymes such as chitinases, chitosanases and
glucanases, individually or in combination, effectively hydrolyze and break down
the chitin containing cell walls of fungi. Those enzymes possessing activity of chitin
degradation are generally referred to as chitinolytic enzymes. A positive correlation
between biological control of some soil-borne fungal diseases of crop plants with
chitinase enzyme production by chitinolytic bacteria has been demonstrated. Several
studies have demonstrated in vitro lysis of fungal cell walls either by chitinase or
β-1, 3-glucanase, alone or in combination. Two chitinase enzymes from B. amylo-
liquefaciens V656 exhibited inhibitory activities on the growth of Fusarium oxyspo-
rum (Wang et al. 2002). Antifungal chitinase of B. subtilis showed strong inhibitory
activity against several phytopathogenic fungi (Chang et al. 2007; Yang et al. 2009).
Biocontrol ability of Stenotrophomonas maltophilia and S. marcescens for control-
ling growth of Rhizoctonia solani, F. oxysporum and Botrytis cinerea is mainly
attributed to their chitinase enzymes produced (Someya et al. 2000). Table 2.2 pro-
vides a partial list of chitinase-producing bacterial and fungal agents, deployed for
phytopathogens control.
El-Mougy and Abdel-Kader (2009) reported that chitinolytic bacteria such as
B. subtilis and P. fluorescens, and their chitinase enzymes (culture filtrates), exhib-
ited antagonistic activity against selected soilborne root rot pathogens (R. solani and
F. solani) in vitro. In vitro antagonistic assays were performed with dual culture and
agar diffusion techniques. Anitha and Rebeeth (2009) reported that chitinase pro-
ducing S. griseus was able to inhibit the growth of some soil borne plant pathogens
of tomato such as F. oxysporum, F. solani, R. solani and Alternaria alternata.
Fungal spore germination and hyphal growth of pathogenic F. oxysporum and
Aspergillus niger was inhibited significantly by chitinase from Serratia proteamac-
ulans (Mehmood et al. 2009). Chitinase from B. thuringiensis contributed to the
biocontrol of Sclerotium rolfsii and other phytopathogenic fungi in soybean seeds
(Reyes-Ramírez et al. 2006). Spore germination of two fungal pathogens was inhib-
ited by chitinase A produced by B. thuringiensis subsp. colmeri (Liu et al. 2010).
Gupta et al. (2006) showed that chitinase producing P. aeruginosa GRC1 was
strongly antagonistic against Sclerotinia sclerotiorum, the causal organism of stem
end rot of peanut, in vitro and in vivo. They also observed increased seed germina-
tion and reduction in stem-rot infestation when peanut seeds were treated with
P. aeruginosa GRC1 strain in vivo. High levels of chitinase production and in vitro
antifungal ability against Sclerotinia minor, a pathogen causing basal drop disease
of lettuce, was observed among 23 bacteria, 38 streptomycete and 15 non-­
streptomycete actinomycetes., In vitro assays were used to test the antifungal poten-
tial of Serratia marcescens, Streptomyces viridodiasticus and Micromonospora
carbonacea under pot conditions, based on production of β-1,3-glucanase enzyme,
antifungal activity, ability to colonize roots and rhizosphere of lettuce. Under
2 Role of Bacterial and Fungal Chitinases in Integrated Management of Pest… 47

Table 2.2 Partial list of chitinase producing bacterial and fungal agents deployed for controlling
main phytopathogens of agricultural crops
Biocontrol agent involved Target pathogens/diseases References
Species of Acinetobacter, Acremonium, Alternaria, Fusarium, Medina-de la
Bacterium, Burkholderia, Penicillium Rosa et al. (2016)
Paenibacillus,
Pseudomonas, Rhizobium,
Shewanella, Sphingomonas and
Stenotrophomonas
Bacillus licheniformis Fusarium solani, Fusarium oxysporum, Jankiewicz et al.
Rhizoctonia solani, Phytophthora (2016)
cinnamomi
Bacillus subtilis F. oxysporum f. sp. ciceris, F. oxysporum f. Basha and
sp. ricini, and R. solani Ulaganathan
(2014)
Bacillus amyloliquefaciens and Ganoderma boninense Azizah et al.
Serratia marcescens (2015)
B. thuringiensis subsp. Colletotrichium gloeosporioides Fuente-Sacido
tenebrionis DSM-2803 et al. (2016)
Bacillus licheniformis Phoma medicaginis Slimene et al.
(2015)
Stenotrophomonas maltophilia Alternaria alternata, Rhizoctonia solani, Jankiewicz et al.
Fusarium solani, F. oxysporum. (2016)
Streptomyces sp. ACT7 F. oxysporum, Alternaria sp. Thirumurugan
et al. (2015)
Bacillus subtilis, and B. Fusarium oxysporum f. sp. cubense Nawangsih, and
pumilus Purba (2013)
Streptomyces hygroscopicus Colletotrichum gloeosporioides, Prapagdee et al.
Sclerotium rolfsii (2008)
Serratia proteamaculans 3095 Fusarium oxysporum Chung and Kim
(2007)
Alcaligenes xylosoxydans Fusarium spp., Rhizoctonia bataticola Vaidya et al.
(2003)
Lactobacillus plantarum Aspergillus spp., Fusarium culmorum, Russo et al.
Penicillium spp., Cladosporium spp. (2017)
Leucosporidium scottii Penicillium expansum, Botrytis cinerea Vero et al. (2013)
Rhizobium spp. Aspergillus spp., Curvularia lunata, Sridevi and
Fusarium spp., Sclerotinia sclerotiorum Mallaiah (2008)
Enterobacter NRG4 Aspergillus niger, Fusarium moniliforme, Dahiya et al.
Mucor rouxi, Rhizopus nigricans (2006)
Bacillus cereus QQ308 Fusarium spp., Pythium ultimum Chang et al.
(2007)
Bacillus circulans GRS 243 Phaeoisariopsis personata Kishore et al.
(2005)
Pseudomonas aeruginosa GRC1 Sclerotinia sclerotiorum Gupta et al.
(2006)
Serratia proteamaculans 336x Gaeumannomyces graminis Wang et al.
(2014)
(continued)
48 A. S. Dukare et al.

Table 2.2 (continued)

Biocontrol agent involved Target pathogens/diseases References
Paenibacillus elgii HOA73 Cladosporium spp., Botrytis cinerea Kim et al. (2017)
Brevibacillus laterosporus Fusarium equiseti Prasanna et al.
Lak1210 (2013)
Trichoderma harzianum Rhizoctonia solani, Fusarium spp., Kowsari et al.
ABRIICC T8-7MK Sclerotinia sclerotiorum, Verticillium (2014)
dahliae, Alternaria brassicola
Trichoderma harzianum CECT Botrytis cinerea Limón et al.
2413 (2001)
Trichoderma atroviride Rhizoctonia solani, Sclerotinia Matroodi et al.
PTCC5220 sclerotiorum, Alternaria alternata (2013)
Talaromyces flavus CGMCC Verticillium dahliae, Sclerotinia Duo-Chuan et al.
3.4301 sclerotiorum, Rhizoctonia solani, (2005)
Alternaria alternata, Fusarium
moniliforme, Magnaporthe grisea
Clonostachys rosea Sclerotinia sclerotiorum Sun et al. (2017)
Aspergillus terreus Aspergillus spp., Penicillium oxysporium, Farag et al.
Rhizoctonia solani, Candida albicans, (2016)
Fusarium solani

controlled glasshouse conditions, all the three isolates, individually or in combina-

tion, were antagonistic to S. minor and significantly reduced incidence of basal drop
disease (El-Tarabily et al. 2000).
Quecine et al. (2008) reported that twenty five chitinase-producing endophytic
isolates of Streptomyces spp., proceeding from citrus and soybean, exhibited anti-
fungal activity in vitro against Guignardia citricarpa, Phytophthora parasitica,
Colletotrichum sublineolum, R. solani, Pythium sp. and F. oxysporum. These authors
also found a positive genetic and phenotypic correlation between chitinolytic poten-
tial and antagonistic activity against C. sublineolum, R. solani, G. citricarpa and
F. oxysporum. Yasir et al. (2009) enriched chitinolytic bacterial communities in ver-
micompost that reduced the spore germination of F. moniliforme. Two chitinolytic
bacterial strains, Paenibacillus spp. 300 and Streptomyces spp. 385, suppressed
fusarium wilt of cucumber (Cucumis sativus) caused by F. oxysporum f. sp. cuc-
umerinum in non-sterile, soil-less potting medium. A mixture of the two strains in a
ratio of 1:1 or 4:1 gave significantly better control of the disease then each of the
strains used individually or in other ratios. Malathi and Viswanathan (2013) demon-
strated the role of microbial chitinase (Trichoderma spp. and Pseudomonas spp.)
against Colletotrichum falcatum in vitro and in vivo conditions. Under in vivo con-
ditions, the native rhizospheric strains of fluorescent pseudomonads and T. harzia-
num from sugarcane were able to protect the crop from soil borne inocula of red rot,
with an efficacy positively correlated with the soil chitinase activity.
There are many reports of chitinase activity of biocontrol agents, as further influ-
enced by inoculations with one or more pathogens. Hjort et al. (2014) targeted chi-
tin degrading enzymes of an uncultured bacterial community through a functional
metagenomics approach. They identified a novel bacterial chitinase, Chi18H8 using
2 Role of Bacterial and Fungal Chitinases in Integrated Management of Pest… 49

a fosmid library of a suppressive soil metagenome. This enzyme showed antifungal

activity against several important phytopathogens such as Colletotrichum gloeospo-
rioides, F. oxysporum, Penicillium chrysogenum, A. alternata, Aspergillus niger,
and Rhizopus stolonifer. These authors also reported that this was the first chitinase
isolated from a metagenome library with a potential for controlling fungal crop
diseases. Muhammad et al. (2014) isolated a B. thuringiensis producing chitinase
with a maximum activity at 35 °C and neutral pH. In vitro and detached leaf assays
showed that chitinolytic bacteria have an antagonistic activity and fungistatic effi-
cacy against Colletotrichum gloeosporioides and Curvularia affinis. Saleem and
Kandasamy (2014) reported presence of chitinase Chi25 in B. subtilis strain BC121
and demonstrated its antagonistic activity vs F. oxysporum f. sp. ciceri through the
inhibition of spore germination and hyphal extension. Glasshouse experiment also
revealed presence of antifungal activity against pathogenic fungi whereby it reduced
incidence of fusarium wilt by 80%. These authors also reported that the fungistatic
activity of the strain against plant pathogenic fungi both in vitro and in vivo was
mediated by a chitinase.
Siti et al. (2015) reported the antifungal activity of chitinolytic B. amyloliquefa-
ciens and S. marcescens against Ganoderma boninense, the pathogen responsible
for deadly Basal Stem Rot (BSR) disease of oil palm trees. Under in vitro condi-
tions, both bacteria showed percentage inhibition of G. boninense by 68.19 and
40.29%, respectively, using a dual culture method. In vitro hyphal lysis demon-
strated the role of chitinase in growth inhibition. Thirumurugan et al. (2015) iso-
lated actinobacteria from twelve sediment samples and found them positive for
chitinase activities using enrichment chitin agar media. The most potential chitinase
producing isolate, Streptomyces spp. ACT7, showed antagonistic activity vs F. oxy-
sporum and Alternaria sp. under in vitro conditions. Imen et al. (2015) isolated nine
chitinase-producing cultures from Tunisian soil, among which culture S213, identi-
fied as B. licheniformis, exhibited strong chitinolytic activity. SDS-PAGE analysis
of the secreted colloidal chitin-induced proteins showed presence of a 65 kDa chi-
tinase. Bacterial culture supernatant containing extracellular chitinase inhibited
growth of several phytopathogenic fungi including Phoma medicaginis. The strain
was efficient in reducing the damping-off caused by P. medicaginis in Medicago
truncatula.
Out of thirteen cultures of actinobacteria isolated from mangrove soils, eleven
produced chitin hydrolysis zone on chitinase mineral agar medium. Seven isolates
showed antifungal activity against F. oxysporum on potato dextrose agar, under in
vitro conditions. Out of these seven isolates, three actinobacteria (VMK1, VMK4
and VMK9) were found as most potent and showed reduction in disease incidence
up to 80%, under in vivo conditions. Selected cultures were identified as Streptomyces
spp. based on morphological and microscopic observations (Vaijayanthi and
Vijayakumar 2016).
Fuente-Sacido et al. (2016) cloned and expressed the endochitinase chiABtt gene
of B. thuringiensis subsp. tenebrionis DSM-2803 in Escherichia coli. They also
reported, for the first time, characterization of a chitinase synthesized by B. thuring-
iensis subsp. tenebrionis DSM-2803 and inhibition effects on radial growth and
50 A. S. Dukare et al.

hyphae of C. gloeosporioides, the etiological agent of anthracnose in crops.

Jankiewicz et al. (2016) reported presence of at least two different isoforms (Chi1
and Chi2) of chitinase in B. licheniformis M3. They also reported that this bacterium
was antagonistic towards pathogenic F. solani, responsible for wilting in solanaceae
family crops. Use of formulations based on antifungal chitin-degrading enzymes
directly for the suppression of plant fungal diseases has opened a new era in biologi-
cal disease control.

2.8 Conclusions

Chitinolytic microorganisms may be produced for a potential biotechnological

application in various natural environments. Their application is not limited to deg-
radation of the waste containing chitin. Numerous studies indicated the possibility
of their use for production of chitinolytic enzymes with fungicidal or insecticidal
activity against some fungal phytopathogens and insect pests, respectively. In bio-
logical control of insect pests and crop pathogens, the application of various metab-
olites, including CHIs, appears to be more efficient, since they show stronger
pesticidal and fungicidal activity than purified enzymes. The use of agents in a con-
sortium of chitinolytic microorganisms seems to bring better results in controlling
insect pests and fungal phytopathogens of crops. Chitinases are potent weapons in
the armory of strategies developed, during time, by microorganisms for virulence.
A benefit can be obtained by exploiting microbial chitinases as biotechnological
tools for controlling different plant pests and pathogens. Increasingly stringent reg-
ulation on use of synthetic pesticides globally, and the urgent need for sustainable
IPM strategies, may impel a more systematic analysis of the potential of chitinases
as biopesticides in the coming era.

References

Abdullah, R. R., Sukar, N. A., & Ghanim, N. M. (2014). Improving the efficiency of Bacillus
thuringiensis against insects of different feeding habit by plasmid transfer technique. Life
Science Journal, 11, 308–318.
Aggarwal, C., Paul, S., Paul, B., Khan, M. A., & Tyagi, S. P. (2014). A modified semi-synthetic
diet for bioassay of non-sporeforming entomopathogenic bacteria against Spodoptera litura.
Biocontrol Science and Technology, 24, 1202–1205.
Aggarwal, C., Paul, S., Tripathi, V., Paul, B., & Khan, M. A. (2015a). Chitinase producing Serratia
marcescens for biocontrol of Spodoptera litura (Fab) and studies on its chitinolytic activities.
Annals of Agricultural Research, 36(2), 132–137.
Aggarwal, C., Paul, S., Tripathi, V., Paul, B., & Khan, M. A. (2015b). Chitinolytic activity in
Serratia marcescens (strain SEN) and potency against different instars of Spodoptera litura
(Fab.) with effect of sublethal doses on insect development. Biological Control, 60, 631–640.
2 Role of Bacterial and Fungal Chitinases in Integrated Management of Pest… 51

Aggarwal, C., Paul, S., Tripathi, V., Paul, B., & Khan, M. A. (2017). Characterization of putative
virulence factors of Serratia marcescens strain SEN for pathogenesis in Spodoptera litura.
Journal of Invertebrate Pathology, 143, 115–123.
Anderson, D. T. (1997). Atlas of invertebrate anatomy. Portland: International. Specialized Book
Service.
Anitha, A., & Rebeeth, M. (2009). In vitro antifungal activity of Streptomyces griseus against phy-
topathogenic fungi of tomato field. Academy Journal of Plant Science, 2, 119–123.
Azizah, S. N, Mubarik, N. R., & Sudirman, L. I. (2015). Potential of Chitinolytic Bacillus amy-
loliquefaciens SAHA 12.07 and Serratia marcescens KAHN 15.12 as Biocontrol Agents of
Ganoderma boninense. Research Journal of Microbiology, 10, 452465.
Banani, H., Spadaro, D., Zhang, D., Matic, S., Garibaldi, A., & Gullino, M. L. (2015). Postharvest
application of a novel chitinase cloned from Metschnikowia fructicola and over expressed in
Pichia pastoris to control brown rot of peaches. International Journal of Food Microbiology,
199, 54–61.
Basha, S., & Ulaganathan, K. (2014). Identification of a Broad-Spectrum Antifungal Chitinase
from Bacillus Subtilis Strain BC121. Research & Reviews: Journal of Microbiology and
Biotechnology.
Berini, F., Caccia, S., Franzetti, E., Congiu, T., Marinelli, F., Casartelli, M., & Tettamanti,
G. (2016). Effects of Trichoderma viride chitinases on the peritrophic matrix of Lepidoptera.
Pest Management Science, 72, 980–989.
Berini, F., Katz, C., Gruzdev, N., Casartelli, M., Tettamanti, G., & Marinelli, F. (2018). Microbial
and viral chitinases: Attractive biopesticides for integrated pest management. Biotechnology
Advances, 36, 818–838.
Bhattacharya, D., Nagpure, A., & Gupta, R. K. (2007). Bacterial chitinases: Properties and poten-
tial. Critical Reviews in Biotechnology, 27, 21–28.
Binod, P., Sukumaran, R. K., Shirke, S. V., Rajput, J. C., & Pandey, A. (2007). Evaluation of
fungal culture filtrate containing chitinase as a biocontrol agent against Helicoverpa armigera.
Journal of Applied Microbiology, 103(5), 1845–1852.
Boldo, J. T., Junges, A., do Amaral, K. B., Staats, C. C., Vainstein, M. H., & Schrank, A. (2009).
Endochitinase CHI2 of the biocontrol fungus Metarhizium anisopliae affects its virulence
toward the cotton stainer bug Dysdercus peruvianus. Current Genetics, 55(5), 551–560.
Brurberg, M. B., Synstad, B., Klemsdal, S. S., van Aalten, D. M. F., Sundheim, L., & Eijsink,
V. G. H. (2000). Chitinases from Serratia marcescens; recent development. Research in
Microbiology, 5, 187–204.
Brzezinska, M. S., Jankiewicz, U., Burkowska, A., & Walczak, M. (2014). Chitinolytic micro-
organisms and their possible application in environmental protection. Current Microbiology,
68, 71–81.
Busby, J. N., Landsberg, M. J., Simpson, R. M., Jones, S. A., Hankamer, B., Hurst, M.R., & Lott,
J.S. (2012). Structural analysis of Chi1 chitinase from Yen-Tc: the multisubunit insecticidal
ABC toxin complex of Yersinia entomophaga. Journal of Molecular Biology, 415(2), 359–371.
Chandler, D., Bailey, A. S., Tatchell, G. M., Davidson, G., Greaves, J., & Grant, W. P. (2011). The
development, regulation and use of biopesticides for integrated pest management. Philosophical
Transactions of the Royal Society, B: Biological Sciences, 366, 1987–1998.
Chandrasekaran, R., Revathi, K., Nisha, S., Kirubakaran, S. A., Sathish-Narayanan, S., & Senthil-­
Nathan, S. (2012). Physiological effect of chitinase purified from Bacillus subtilis against the
tobacco cutworm Spodoptera litura Fab. Pesticide Biochemistry and Physiology, 104, 65–71.
Chandrasekaran, R., Revathi, K., Thanigaivel, A., Kirubakaran, S. A., & Senthil-Nathan, S. (2014).
Bacillus subtilis chitinase identified by matrix-assisted laser desorption/ionization time-of
flight/time of flight mass spectrometry has insecticidal activity against Spodoptera litura Fab.
Pesticide Biochemistry and Physiology, 116, 1–12.
Chang, W. T., Chen, Y. C., & Jao, C. L. (2007). Antifungal activity and enhancement of plant growth
by Bacillus cereus grown on shellfish chitin wastes. Bioresource Technology, 98, 1224–1230.
52 A. S. Dukare et al.

Chung, S., & Kim, S. D. (2007). Escherichia coli can produce recombinant chitinase in the
soil to control the pathogenesis by Fusarium oxysporum without colonization. Journal of
Microbiology and Biotechnology, 17(3), 474–480.
Czaja, K., Góralczyk, K., Struciński, P., Hernik, A., Korcz, W., Minorczyk, M., Lyczewska, M., &
Ludwicki, J. K. (2015). Biopesticides – Towards increased consumer safety in the European
Union. Pest Management Science, 71, 3–6.
Dahiya, N., Tewari, R., & Hoondal, G. S. (2006). Biotechnological aspects of chitinolytic enzymes:
A review. Applied Microbiology and Biotechnology, 25, 1–10.
Di Francesco, A., Martini, C., & Mari, M. (2016). Biological control of postharvest diseases by
microbial antagonists: How many mechanisms of action? European Journal of Plant Pathology,
145, 711–717.
Danişmazoğlu, M., Demir, İ., Sezen, K., Muratoğlu, H., & Nalçacioğlu, R. (2015). Cloning
and expression of chitinase A, B, and C (chiA, chiB, chiC) genes from Serratia marcescens
­originating from Helicoverpa armigera and determining their activities. Turkish Journal of
Biology, 39, 78–87.
Dukare, A. S., & Paul, S. (2018). Effect of chitinolytic biocontrol bacterial inoculation on the soil
microbiological activities and fusarium population in the rhizophere of pigeon pea (Cajanus
cajan). Annals of Plant Protection Sciences, 26, 98–103.
Dukare, A. S., Prasanna, R., Dubey, S. C., Chaudhary, V., Nain, L., Singh, R., & Saxena,
A. K. (2011). Evaluating novel microbe amended composts as biocontrol agents in tomato.
Crop Protection, 30, 436–442.
Dukare, A. S., Paul, S., Nambi, V. E., Gupta, R. K., Singh, R., Sharma, K., & Vishwakarma,
R. K. (2018). Exploitation of microbial antagonists for the control of postharvest diseases of
fruits: A review. Critical Reviews in Food Science and Nutrition, 58, 1–16.
Duo-Chuan, L. I., Chen, S., & Jing, L. U. (2005). Purification and partial characterization of two
chitinases from the mycoparasitic fungus Talaromyces flavus. Mycopathologia, 159, 223–229.
El-Mougy, N. S., & Abdel-Kader, M. M. (2009). Salts application for suppressing potato early
blight disease. Journal of Plant Protection Research, 49, 353–361.
El-Tarabily, K. A. (2006). Rhizosphere-competent isolates of Streptomycete and non-­
streptomycete actinomycetes capable of producing cell-wall degrading enzymes to control
Pythium aphanidermatum damping-off disease of cucumber. Canadian Journal of Botany, 84,
211–222.
El-Tarabily, K. A., Soliman, M. H., Nassar, A. H., Al-Hassani, H. A., Sivasithamparam, K.,
McKenna, F., & Hardy, G. E. S. J. (2000). Biological control of Sclerotinia minor using a
chitinolytic bacterium and actinomycetes. Plant Pathology, 49, 573–583.
Fang, W., Feng, J., Fan, Y., Zhang, Y., Bidochka, M. J., St Leger, R. J., & Pei, Y. (2009). Expressing
a fusion protein with protease and chitinase activities increases the virulence of the insect
pathogen Beauveria bassiana. Journal of Invertebrate Pathology, 102(2), 155–159.
Farag, A. M., Abd-Elnabey, H. M., Ibrahim, H. A., & El-Shenawy, M. (2016). Purification, char-
acterization and antimicrobial activity of chitinase from marine-derived Aspergillus terreus.
Egyptian Journal of Aquatic Research, 42(2), 185–192.
Filho, B. P., Lemos, F. J., Secundino, N. F., Pascoa, V., Pereira, S. T., & Pimenta, P. F. (2002).
Presence of chitinase and beta-N-acetylglucosaminidase in the Aedes aegypti: A chitinolytic
system involving peritrophic matrix formation and degradation. Insect Biochemistry and
Molecular Biology, 32, 1723–1729.
Frandberg, E., & Schuurer, I. (1998). Antifungal activity of chitinolytic bacteria isolated from air
tight stored cereal grain. Canadian Journal of Microbiology, 44, 121–127.
Fuente-Sacido, N. M. d. l., Vazquez, L., Garcia-Pérez, A. P., Perez, U. B., Bideshi, D. K., Salcedo-­
Hernández, R., et al. (2016). The endochitinase ChiA Btt of Bacillus thuringiensis subsp.
tenebrionis DSM-2803 and its potential use to control the phytopathogen Colletotrichum
gloeosporioides. Microbiology Open, 5, 1–12.
2 Role of Bacterial and Fungal Chitinases in Integrated Management of Pest… 53

Gao, J., Bauer, M. W., Shockley, K. R., Pysz, M. A., & Kelly, R. M. (2003). Growth of hyperther-
mophilic archaeon Pyrococcus furiosus on chitin involves two family 18 chitinases. Applied
and Environmental Microbiology, 69, 3119–3128.
García-Lara, S., Ortíz-Islas, S., & Villers, P. (2013). Portable hermetic storage bag resistant to
Prostephanus truncatus, Rhyzopertha dominica, and Callosobruchus maculatus. Journal of
Stored Products Research, 54, 23–25.
Gerhardson, B. (2002). Biological substitutes for pesticides. Trends in Biotechnology, 20, 338–343.
Gohel, V., Singh, A., Vimal, M., Ashwini, P., & Chhatpar, H. S. (2006). Bioprospecting and anti-
fungal potential of chitinolytic microorganisms. African Journal of Biotechnology, 5, 54–72.
Gomaa, Z. G. (2012). Chitinase production by Bacillus thuringiensis and Bacillus lichenformis:
Their potential in antifungal biocontrol. Journal of Microbiology, 50, 103–111.
Gooday, G. W. (1990a). The ecology of chitin degradation. Advancement in Microbial Ecology,
11, 387–430.
Gooday, G. W. (1990b). Physiology of microbial degradation of chitin and chitosan. Biodegradation,
1, 177–190.
Gupta, C., Kumar, B., Dubey, R., & Maheshwari, D. (2006). Chitinase-mediated destructive antag-
onistic potential of Pseudomonas aeruginosa GRC1 against Sclerotinia sclerotiorum causing
stem rot of peanut. Biological Control, 51, 821–835.
Haran, S., Schickler, H., Oppenheim, A., & Chet, I. (1995). New components of the chitinolytic
system of Trichoderma harzianum. Nucleic Acids Research, 99, 447–450.
Hardy, M. C. (2014). Resistance is not futile: It shapes insecticide discovery. Insects, 5, 227–242.
Hart, P. J., Pfluger, H. D., Monzingo, A. F., Hollis, T., & Robertus, J. D. (1995). The refined crystal
structure of an endochitinase from Hordeum vulgare L. seeds at 1.8 A resolution. Journal of
Molecular Biology, 248, 402–413.
Hawksworth, D. L., Kirk, P. M., Sutton, B. C., & Pegler, D. N. (1995). Ainsworth and Bisby’s
dictionary of the fungi (8th ed., p. 616). Wallingford: CAB International.
Hegedus, D., Erlandson, M., Gillott, C., & Toprak, U. (2009). New insights into peritrophic matrix
synthesis, architecture and function. Annual Review of Entomology, 54, 285–302.
Herrera-Estrella, A., & Chet, I. (1999). Chitinases in biological control. EXS, 87, 171–184.
Hjort, K., Presti, I., Elväng, A., Marinelli, F., & Sjöling, S. (2014). Bacterial chitinase with phyto-
pathogen control capacity from suppressive soil revealed by functional metagenomics. Applied
Microbiology and Biotechnology, 98, 2819–2828.
Huang, Z., Hao, Y., Gao, T., Huang, Y., Ren, S., & Keyhani, N. O. (2016). The Ifchit1 chitinase
gene acts as a critical virulence factor in the insect pathogenic fungus Isaria fumosorosea.
Applied Microbiology and Biotechnology, 100(12), 5491–5503.
Husen, T. J., Kamble, S. T., & Stone, J. M. (2015). Effect of pentoxifylline on chitinolytic enzyme
activity in the eastern subterranean termite (isoptera: Rhinotermitidae). Faculty Publ., Dept of
Entomology, 426. [Link]
Imen, B. S., Olfa, T., Dorra, G., Bacem, M., Jean, M. S., et al. (2015). Isolation of a chitinolytic
Bacillus licheniformis S213 strain exerting a biological control against Phoma medicaginis
infection. Applied Microbiology and Biotechnology, 175, 3494–3506.
Jankiewicz, U., Kozłowska, M., Świontek-Brzezińska, M., Nowosielski, J., & Stępniewska-Jarosz,
S. (2016). The chitinolytic bacterium Bacillus licheniformis M3 and its potential application
in biological plant protection. Electronic Journal of Polish Agricultural Universities, 19(2), 1.
(Available online: [Link]
Joo, G. J. (2005). Purification and characterization of an extracellular chitinase from the antifungal
biocontrol agent Streptomyces halstedii. Biotechnology Letters, 27, 1483–1486.
Kavitha, A., & Vijayalakshmi, M. (2011). Partial purification and antifungal profile of chitinase
produced by Streptomyces tendae TK-VL-333. Annals of Microbiology, 61, 597–603.
Khajuria, C., Buschman, L. L., Chen, M. S., Muthukrishnan, S., & Zhu, K. Y. (2010). A gut spe-
cific chitinase gene essential for regulation of chitin content of peritrophic matrix and growth of
Ostrinia nubilalis larvae. Insect Biochemistry and Molecular Biology, 40, 621–629.
54 A. S. Dukare et al.

Kielak, A. M., Cretoiu, M. S., Semenov, A. V., Sorensen, S. J., & van Elsas, J. D. (2013). Bacterial
chitinolytic communities respond to chitin and pH alteration in soil. Applied and Environmental
Microbiology, 792, 63–272.
Kim, Y. H., Park, S. K., Hur, J. Y., & Kim, Y. C. (2017). Purification and characterization of a
major extracellular chitinase from a biocontrol bacterium, Paenibacillus elgii HOA73. Plant
Pathology Journal, 33(3), 318–328.
Kishore, G. K., Pande, S., & Podile, A. R. (2005). Biological control of late leaf spot of peanut
(Arachis hypogaea) with chitinolytic bacteria. Phytopathology, 95(10), 1157–1165.
Koul, O. (2011). Microbial biopesticides: Opportunities and challenges. CAB Reviews: Perspectives
in Agriculture, Veterinary Science, Nutrition and Natural Resources, 6, 1–26.
Kowsari, M., Motallebi, M., & Zamani, M. (2014). Protein engineering of chit42 towards improve-
ment of chitinase and antifungal activities. Current Microbiology, 68(4), 495–502.
Lertcanawanichakul, M., Wiwat, C., Bhumiratana, A., & Dean, D.H. (2004). Expression of
chitinase-­encoding genes in Bacillus thuringiensis and toxicity of engineered B. thuringiensis
subsp. aizawai toward Lymantria dispar larvae. Current Microbiology, 48, 175–181.
Limón, M. C., Margolles-Clark, E., Benitez, T., & Penttila, M. (2001). Addition of substrate
binding domains increases substrate binding capacity and specific activity of a chitinase from
Trichoderma harzianum. FEMS Microbiology Letters, 198(1), 57–63.
Liu, D., Cai, J., Xie, C. C., Lium, C., & Chen, Y. H. (2010). Purification and partial characterization
of a 36-kDa chitinase from Bacillus thuringiensis subsp. colmeri, and its biocontrol potential.
Enzyme Microbial Technology, 46, 252–256.
Malathi, P., & Viswanathan, R. (2013). Role of microbial chitinase in the biocontrol of sugarcane
red rot caused by Colletotrichum falcatum Went. European Journal of Biological Sciences,
6, 17–23.
Manucharova, N. A., Vlasenko, A. N., Meńko, E. V., & Zvyagintsev, D. G. (2011). Specificity of
the chitinolytic microbial complex of soils incubated at different temperatures. Microbiology,
80, 205–215.
Mariapackiam, S., & Ignacimuthu, S. (2008). Larvicidal & histopathological effects of oil formu-
lation on Spodoptera litura. In S. Ignacimuthu & S. Jeyaraj (Eds.), Recent trends in insect pest
management. New Delhi: Elite Publishing House Pvt. Ltd.
Matroodi, S., Motallebi, M., Zamani, M., & Moradyar, M., (2013). Designing a new chitinase with
more chitin binding and antifungal activity. World Journal of Microbiology and Biotechnology,
29(8), 1517–1523.
Medina-de la Rosa, G., López-Reyes, L., Carcaño-Montiel, M. G., López-Olguín, J. F., Hernández-­
Espinosa, M. Á., & Rivera-Tapia, J. A. (2016). Rhizosphere bacteria of maize with chitinolytic
activity and its potential in the control of phytopathogenic fungi. Archives of Phytopathology
and Plant Protection, 49(11–12), 310–321.
Mehmood, M. A., Xiao, X., Hafeez, F. Y., Gai, Y., & Wang, F. (2009). Purification and charac-
terization of a chitinase from Serratia proteamaculans. World Journal of Microbiology and
Biotechnology, 25, 1955–1961.
Mehmood, M. A., Gai, Y., Zhuang, Q., Wang, F., Xiao, X., & Wang, F. (2010). Aeromonas caviae
CB101 contains four chitinases encoded by a single gene chi1. Molecular Biotechnology, 44,
213–220.
Miyamoto, K., Okunishi, M., Nukui, E., Tsuchiya, T., Kobayashi, T., Imada, C., & Tsujibo,
H. (2007). The regulator CdsS/CdsR twocomponent system modulates expression of genes
involved in chitin degradation of Pseudoalteromonas piscicida strain O-7. Archives of
Microbiology, 188, 619–628.
Mubarik, N. R., Mahagiani, I., Anindyaputri, A., Santoso, S., & Rusmana, I. (2010). Chitinolytic
bacteria isolated from chili rhizosphere: Chitinase characterization and its application as bio-
control for whitefly (Bemisia tabaci Genn.). Journal of Agriculture and Biological Science, 5,
430–435.
Muhammad, A., Nisa, R. M., & Aris, T. W. (2014). Partial purification of bacterial chitinase as
biocontrol of leaf blight disease on oil palm. Research Journal of Microbiology, 9, 265–277.
2 Role of Bacterial and Fungal Chitinases in Integrated Management of Pest… 55

Nawangsih, A. A., & Purba, F. (2013). Isolation of fluorescent pseudomonads, heat tolerant and
Chitinolytic bacteria in banana rhizosphere with Antagonistic activities against Fusarium oxy-
sporum f. Sp. Cubense In vitro and molecular identification of selected isolates. Journal of
ISSAAS, 19(2), 30–40.
Nawani, N. N., & Kapadnis, B. P. (2003). Chitin degrading potential of bacteria from extreme and
moderate environment. Indian Journal of Experimental Biology, 41, 248–254.
Neeraja, C., Anil, K., Purushotham, P., Suma, K., Sarma, P., Moerschbacher, B. M., & Podile,
A. R. (2010). Biotechnological approaches to develop bacterial chitinases as a bioshield against
fungal diseases of plants. Critical Reviews in Biotechnology, 30, 231–241.
Ordentlich, A., Elad, Y., & Chet, I. (1988). The role of chitinase of Serratia marcescens in biocon-
trol of Sclerotium rolfsii. Phytopathology, 78, 84–88.
Ozgen, A., Sezen, K., Demir, I., Demirbag, Z., & Nalcacioglu, R. (2013). Molecular characteriza-
tion of chitinase genes from local isolate of Serratia marcescens and their contribution to the
insecticidal activity of Bacillus thuringiensis strains. Current Microbiology, 67(4), 499–504.
Patil, R. S., Ghormade, V., & Deshpande, M. V. (2000). Chitinolytic enzymes: An exploration.
Enzyme Microbial Technology, 26, 473–483.
Petersen, L. M., & Tisa, L. S. (2013). Friend or foe? A review of the mechanisms that drive Serratia
towards diverse lifestyles. Canadian Journal of Microbiology, 59, 627–640.
Prapagdee, B., Kuekulvong, C., & Mongkolsuk, S., (2008). Antifungal potential of F. Berini et al.
extracellular metabolites produced by Streptomyces hygroscopicus against phytopathogenic
fungi. International Journal of Biological Sciences, 4(5), 330–337.
Prasanna, L., Eijsink, V. G., Meadow, R., & Gåseidnes, S. (2013). A novel strain of Brevibacillus lat-
erosporus produces chitinases that contribute to its biocontrol potential. Applied Microbiology
and Biotechnology, 97 (4),1601–1611.
Quecine, M. C., Araujo, W. L., Marcon, J., Gai, C. S., Azevedo, J. L., & Pizzirani-Kleiner,
A. A. (2008). Chitinolytic activity of endophytic Streptomyces and potential for biocontrol.
Letters in Applied Microbiology, 47, 486–491.
Regev, A., Keller, M., Strizhov, N., Sneh, B., Prudovsky, E., Chet, I., et al. (1996). Synergistic activ-
ity of a Bacillus thuringiensis d-endotoxin and a bacterial endochitinase against Spodoptera lit-
toralis larvae. Applied and Environmental Microbiology, 62, 3581–3586.
Reyes-Ramírez, A., Escudero-Abarca, B. I., Aguilar-Uscanga, G., Hayward-Jones, P. M., &
Barboza-Corona, J. E. (2006). Antifungal activity of Bacillus thuringiensis chitinase and
its potential for the biocontrol of phytopathogenic fungi in soybean seeds. Journal of Food
Science, 69, 131–134.
Ruiz-Sánchez, A., Cruz-Camarillo, R., Salcedo-Hernández, R., & Barboza-Corona, J. E. (2005).
Chitinases from Serratia marcescens Nima. Biotechnology Letters, 27, 649–653.
Russo, P., Arena, M. P., Fiocco, D., Capozzi, V., Drider, D., & Spano, G. (2017). Lactobacillus
plantarum with broad antifungal activity: a promising approach to increase safety and shelf-life
of cereal-based products. International Journal of Food Microbiology, 247, 48–54.
Saito, A., Fujii, T., Yoneyama, T., & Miyashita, K. (1998). glkA is involved in glucose repression
of chitinase production in Streptomyces lividans. Journal of Bacteriology, 180, 2911–2914.
Saks, E., & Jankiewicz, U. (2010). Chitinolytic activity of bacteria. Advances in Biochemistry,
56, 1–8.
Saleem, B., & Kandasamy, U. (2014). Identification of a broad spectrum antifungal chitin-
ase from Bacillus subtilis strain BC121. Research & Reviews: Journal of Microbiology and
Biotechnology, 3, 1–7.
Shahabuddin, M., Toyoshima, T., Aikawa, M., & Maslow, D. C. (1993). Transmission-blocking
acitivity of a chitinase inhibitor activation of malarial parasite chitinase by mosquito protease.
Proceedings of National Academy of Sciences, USA, 90, 4266–4270.
Shternshis, M. V., Shpatova, T. V., Borisov, B. A., & Beljaev, A. A. (2005). The effect of entomo-
pathogenic hyphomycetes and phytoverm on raspberry cane midge and the spur blight causal
agent. Mycology and Phytopathology, 3, 21.
56 A. S. Dukare et al.

Singh, A. K., Singh, A., & Joshi, P. (2016). Combined application of chitinolytic bacterium
Paenibacillus sp. D1 with low doses of chemical pesticides for better control of Helicoverpa
armigera. International Journal of Pest Management, 62, 222–227.
Siti, N. A., Nisa, R. M., & Lisdar, I. S. (2015). Potential of chitinolytic Bacillus amyloliquefaciens
SAHA 12.07 and Serratia marcescens KAHN 15.12 as biocontrol agents of Ganoderma boni-
nense. Research Journal of Microbiology, 10, 452–465.
Slimene, I. B., Tabbene, O., Gharbi, D., Mnasri, B., Schmitter, J. M., Urdaci, M. C., & Limam,
F. (2015). Isolation of a chitinolytic Bacillus licheniformis S213 strain exerting a biological
control against phoma medicaginis infection. Applied Biochemistry and Biotechnology, 175,
3494–3506.
Someya, N., Kataoka, N., Komagata, T., Hirayae, K., Hibi, T., & Akutsu, K. (2000). Biological
control of cyclamen soil borne diseases by Serratia marcescens. Plant Disease, 84, 334–340.
Spadaro, D., & Droby, S. (2016). Development of biocontrol products for postharvest diseases of
fruit: The importance of elucidating the mechanisms of action of yeast antagonists. Trends in
Food Science and Technology, 47, 39–49.
Sridevi, M., & Mallaiah, K. V. (2008). Factors affecting chitinase activity of Rhizobium sp. from
Sesbania sesban. Biologia, 63(3), 307–312.
Sun, Z. B., Sun, M. H., Zhou, M., & Li, S. D. (2017). Transformation of the endochitinase gene
Chi67-1 in Clonostachys rosea 67-1 increases its biocontrol activity against Sclerotinia sclero-
tiorum. AMB Express 7(1), 1.
Štrojsová, A., & Dyhrman, S. T. (2008). Cell-specific β-N-acetylglucosaminidase activity in cul-
tures and field populations of eukaryotic marine phytoplankton. FEMS Microbiology Ecology,
64, 351–361.
Štrojsová, M., & Vrba, J. (2005). Direct detection of digestive enzymes in planktonic rotifers using
enzyme-labelled fluorescence (ELF). Marine and Freshwater Research, 56, 189–195.
Suganthi, M., Senthilkumar, P., Arvinth, S., & Chandrashekara, K. N. (2017). Chitinase from
Pseudomonas fluorescens and its insecticidal activity against Helopeltis theivora. Journal of
General and Applied Microbiology, 63, 222–227.
Talibi, I., Boubaker, H., Boudyach, E. H., & Ait BenAoumar, A. (2014). Alternative methods for
the control of postharvest citrus diseases. Journal of Applied Microbiology, 117, 1–17.
Tellam, R. L. (1996). The peritrophic matrix. In M. J. Lehane & P. F. Billingsley (Eds.), Biology of
the insect midgut (pp. 86–114). Cambridge: Chapman & Hall.
Thamthiankul, S., Suan-Ngay, S., Tantimavanich, S., & Panbangred, W. (2001). Chitinase from
Bacillus thuringiensis subsp. pakistani. Applied Microbiology and Biotechnology, 56, 395–401.
Thirumurugan, D., Sankari, D., & Vijayakumar, R. (2015). Screening of chitinase production and
antifungal activity of Streptomyces sp. ACT7 from east coast region, South India. International.
Journal of Pharmaceutical Sciences, 7, 38–41.
Urbina, C. T., Prieto, V. G., Lopez, C. G., Albores, F. V., Reyes, D. B., Muniz, C. A., & Barrios,
D. O. (2016). Purification and characterization of β-1, 3-glucanase from Candida oleophila for
the biocontrol of Penicillium expansum. Research & Reviews: Journal of Botanical Science,
5, 38–45.
Vaaje-Kolstad, G., Houston, D. R., Riemen, A. H., Eijsink, V. G., & van Aalten, D. M. (2005).
Crystal structure and binding properties of the Serratia marcescens chitin-binding protein
CBP21. Journal of Biological Chemistry, 280, 11313–11319.
Vaidya, R., Roy, S., Macmill, S., Gandhi, S., Vyas, P., & Chhatpar, H. S. (2003). Purification
and characterization of chitinase from Alcaligenes xylosoxydans. Biotechnology Letters, 25(9),
715–717.
Vaijayanthi, G., & Vijayakumar, R. (2016). Screening of chitinolytic actinobacteria for biological
control of Fusarium oxysporum: A pathogen causing damping-off and basal rot diseases in
onion. World Journal of Pharmacy and Pharmaceutical Sciences, 5, 940–947.
Vero, S., Garmendia, G., González, M. B., Bentancur, O., & Wisniewski, M. (2013). Evaluation
of yeasts obtained from Antarctic soil samples as biocontrol agents for the management of
postharvest diseases of apple (Malus × domestica). FEMS Yeast Research, 13(2), 189–199.
2 Role of Bacterial and Fungal Chitinases in Integrated Management of Pest… 57

Viswanathan, R., & Samiyappan, R. (2001). Antifungal activity of chitinase produced by fluores-
cent pseudomonads against Colletotrichum falcaturn went causing red root disease in sugar-
cane. Microbiological Research, 155, 305–314.
Wang, S. L., Shih, I. L., Liang, T. W., & Wang, C. H. (2002). Purification and characterization
of two antifungal chitinases extracellular produced by Bacillus amyloliquefaciens V656 in
a shrimp and crab shell powder medium. Journal of Agricultural and Food Chemistry, 50,
2241–2248.
Wang, M., Xing, Y., Wang, J., Xu, Y., & Wang, G. (2014). The role of the chi1 gene from the
endophytic bacteria Serratia proteamaculans 336x in the biological control of wheat take-all.
Canadian Journal of Microbiology, 60(8), 533–540.
Wigglesworth, V. B. (1948). The insect cuticle. Biological Reviews, 23, 408–451.
Yang, C. Y., Ho, Y. C., Pang, J. C., Huang, S. S., & Tschen, J. S. (2009). Cloning and expression
of an antifungal chitinase gene of a novel Bacillus subtilis isolate from Taiwan potato field.
Bioresource Technology, 100, 1454–1458.
Yasir, M., Aslam, Z., Kim, S. W., Lee, S. W., Jeon, C. O., & Chung, Y. R. (2009). Bacterial com-
munity composition and chitinase gene diversity of vermicompost with antifungal activity.
Bioresource Technology, 100, 4396–4403.
Yu, T., Wang, L., Yin, Y., Wang, Y., & Zheng, X. (2008). Effect of chitin on the antagonistic activity
of Cryptococcus laurentii against Penicillium expansum in pear fruit. International Journal of
Food Microbiology, 122, 44–48.
Zhang, Z., Yuen, G. Y., Sarath, G., & Penheiter, A. R. (2001). Chitinases from the plant disease
biocontrol agent Stenotrophomonas maltophilia C3. Phytopathology, 91, 204–211.