Journal of Genetic Engineering and Biotechnology 16 (2018) 387–392
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Original Article
Isolation and characterization of Bacillus sp. strain BC01 from soil
displaying potent antagonistic activity against plant and fish pathogenic
fungi and bacteria
Md Javed Foysal a,b,⇑, Asura Khanam Lisa c
a
Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet 3114, Bangladesh
b
School of Molecular and Life Sciences, Curtin University, WA 6845, Australia
c
Department of Biotechnology and Genetic Engineering, Bangabandhu Sheikh Mujibur Rahman Science and Technology University, Gopalganj 8100, Bangladesh
a r t i c l e i n f o a b s t r a c t
Article history: Fungal and bacterial pathogens infect a diverse range of hosts including various plant and animal species.
Received 16 December 2017 Fungal and bacterial diseases, especially of plants and aquatic animals, such as fish, lead to significant
Received in revised form 23 January 2018 damage to crops and aquaculture, respectively, worldwide. The present study was conducted to isolate
Accepted 26 January 2018
and characterize potent Bacillus strains with significant antagonistic activity against the major plant
Available online 7 May 2018
and fish pathogenic fungi and bacteria. We randomly collected 22 isolates of Bacillus from the soil, rhizo-
sphere, and sediment from different parts of Bangladesh. Initial characterization, based on in vitro antag-
Keywords:
onistic activity on the culture plate, resulted in the selection of four gram-positive Bacillus sp. isolates.
Bacillus sp. BC01
Anti-bacterial and anti-fungal activity
Among these, the isolate BC01, obtained from soil demonstrated the highest broad-spectrum anti-
16s rRNA sequencing bacterial and anti-fungal activities. We confirmed the genus of BC01 to be Bacillus by morphological
Phylogenetic analysis and biochemical tests as well as using molecular data analysis tools, including the study of 16s rDNA,
phylogenetic relationship, and evolutionary divergence scores. The isolate significantly inhibited the
mycelial growth of the plant pathogen, Penicillium digitatum and fish pathogen, Aphanomyces invadans
in vitro. The anti-bacterial effect of the isolate was also evaluated against Pseudomonas spp. and
Xanthomonas spp., the two deadliest plant pathogens, and Aeromonas veronii, Pseudomonas fluorescens,
and Streptococcus iniae, three major fish pathogens that are primarily responsible for global aquaculture
loss. The results of the present study could pave the way for developing potent drugs to combat microbial
infection of plants and fish.
Ó 2018 Production and hosting by Elsevier B.V. on behalf of Academy of Scientific Research & Technology.
This is an open access article under the CC BY-NC-ND license ([Link]
nd/4.0/).
1. Introduction potential threat to the Rutaceae family [3]. Other bacterial species
such as those belonging to Pseudomonas cause a variety of plant
In recent years, a significant portion of agriculture and aquacul- diseases with diverse symptoms, such as cankers, spots, blight,
ture production has incurred huge losses due to a disease outbreak. rot, and galls, while Xanthomonas spp. (especially X. euvesicatoria
Diseases in plant and fish cause approximately 20% of crop damage and X. perforans) cause spots and blights of leaf, stem, and fruits
and 15% of production loss in aquaculture annually [1,2]. Various [4,5]. In fish, Aphanomyces invadans causes epizootic ulcerative
preventive measures, including heavy use of pesticides and chem- syndrome (EUS) leading to similar damage [6]. Other widely
ical compounds, pose a severe challenge to food security globally. known and potential disease-causing agents in fish include Aero-
For instance, Penicillium digitatum is the most dangerous pathogen monas veronii, Pseudomonas fluorescens, and Streptococcus iniae that
of the citrus plant; it causes rot or mould and is recognized as a are responsible for motile aeromonad septicemia (AMS), bacterial
hemorrhagic septicemia, and streptococcosis, respectively, in both
carp and catfish [7,8]. The management of crops in the field and
Peer review under responsibility of National Research Center, Egypt. fish in the culture ponds by pesticides and disinfectants has proven
⇑ Corresponding author at: Department of Genetic Engineering and Biotechnol-
to be difficult owing to their long-term adverse effects on the envi-
ogy, Shahjalal University of Science and Technology, Sylhet 3114, Bangladesh.
ronment and biogeochemical cycle, thereby compelling to opt for
E-mail addresses: mjfoysal-geb@[Link] (M.J. Foysal), aasurakhanam@gmail.
com (A.K. Lisa). more environment-friendly preventive approaches.
[Link]
1687-157X/Ó 2018 Production and hosting by Elsevier B.V. on behalf of Academy of Scientific Research & Technology.
This is an open access article under the CC BY-NC-ND license ([Link]
388 M.J. Foysal, A.K. Lisa / Journal of Genetic Engineering and Biotechnology 16 (2018) 387–392
A biological control is an approaches of controlling pest and 2.3. Biochemical identification of Bacillus species
pathogen in a natural way without affecting human and environ-
ment. An agent or organism which can kill or suppress the growth The suspected Bacillus colonies were identified on the basis of
of any plant or animal pathogen, is called the biological control morphology and Gram staining. Subsequent identification tests
agent, BCA. BCA or natural enemy of the pathogens could be included hemolysis, starch hydrolysis, gelatin hydrolysis, citrate
explored, as it either suppresses or reduces the effects of undesir- test, Voges–Proskauer (VP) test, and growth at different pH and
able microbes and favors the growth of desirable and eco-friendly temperature [23].
microbes and insects [9]. In the context of BCA application, Bacillus
species have been in use for the past many years such that the 2.4. Evaluation of antagonistic activity
genus is considered to be a prospective antibiotic-producing agent
[10,11]. The antagonistic activity of Bacillus sp. BC01 isolate was mea-
Species belonging to the genus Bacillus are gram-positive, sured by cross streak method as described by Ran et al., 2012
spore-forming, rod-shaped, motile bacteria that are present in [14]. A pure culture of Bacillus sp. BC01 isolate was first grown
diverse environmental conditions but mostly in the soil. Their on the nutrient broth (NB) and incubated for 24 h at 37 °C. From
long-term survival in different harsh conditions is attributed to the broth, 50 ml of Bacillus sp. culture was poured in 6 mm circular
the production of endospores by the simple and rapid development Whatman’s filter paper prepared before using punching machine.
of different reliable formulations [12]. These species provide pro- The fungal and bacterial isolates, i.e., Penicillium digitatum
tection to plants and fish against microbial infections through (PGT01), Aphanomyces invadans (Aph01), Pseudomonas spp.
diverse mode of actions, e.g., through secretion of antibiotics, (PSD05), Xanthomonas spp. (Xpp03), Aeromonas veronii (BN10),
enzymes, volatile compounds, etc. [13–16]. The use of Bacillus as Pseudomonas fluorescens (PUKL2), and Streptococcus iniae (S20)
a BCA has also reported to cut down the cost of agriculture by sup- were used from the stock sources of the USDA laboratory of the
pressing the need for fertilizers and pesticides [17]. In addition, Shahjalal University of Science and Technology, Sylhet. Glucose-
earlier reports reveal that antagonistic bacteria isolated from soil peptone (GP) broth and potato-dextrose broth (PDB) culture were
can be used as a source of potential antibiotics against fish diseases used to grow A. invadans and P. digitatum respectively. While all
[18]. However, not a single study has reported an isolate that can other bacteria were grown on nutrient broth (NA) culture and
potentially inhibit both plant and fish pathogens. Hence, any iso- incubated overnight at 37 °C for plant pathogenic fungi and bacte-
late that could considerably inhibit both kinds of pathogens would ria and 25 °C for fish pathogenic bacteria. Then, 50 ml of bacterial
serve as a landmark reference in the field of BCAs. culture was grown in nutrient agar (NA) medium by spread plate
Several molecular approaches are currently being utilized for method and aseptically poured the previously prepared filter paper
the isolation of microbes. For instance, polymerase chain reaction disc carefully on to the culture plate. In present study, we analyzed
(PCR) in combination with 16s rRNA sequencing tool is frequently 12 h, 18 h and 24 h of bacterial culture for inhibition assay. After
used for rapid and sensitive identification of microbes [19]. This 24 h of incubation, the zone of inhibition was measured according
technique has been successfully employed for identification of to Foysal et al., 2011 [7].
Bacillus spp. from various environments including soil [20]. More-
over, many sequence analysis tools (i.e., phylogeny, p-distance
2.5. Genomic DNA extraction and PCR preparation
analysis) enable precise determination of microbial clustering
within a taxon and its functional role as a pathogen [21]. Therefore,
The extraction of bacterial DNA was performed using a com-
the current study aimed to isolate and characterize the potent
mercial genomic DNA extraction kit (Bio Basic Inc.; Ontario,
antibiotic-producing Bacillus spp. and elucidate the antagonistic
Canada). Proteinase K and RNase A were added to remove protein
spectrum properties against the major plant and fish pathogens.
and RNA contaminations and obtain good-quality DNA according
to the manufacturer’s instructions. The extracted DNA was quanti-
fied by gel electrophoresis using lambda (k) DNA as the marker; it
2. Materials and methods
was then stored at 20 °C for further use. The PCR master mixture
was prepared as a 50 mL solution containing 25 mL of 2 concentra
2.1. Isolation of bacterial isolates
ted solution (Thermo Scientific, United States), 3 mL each of for-
ward primer (8F, 50 -AGAGTTTGATCCTGGCTCAG-30 ) and reverse
Bacterial samples were collected from four different parts of
primer (1492R, 50 -AGGAGGTGATCCAACCGCA-30 ) for amplification
Rajshahi and Sylhet, Bangladesh. These included the river Padma
of 16s rDNA universal sequence, 4 mL of template, and 15 mL of
sediment, hill soil of Sylhet, rhizosphere soil of bean root from
nuclease-free water [24].
Saheb bazaar, and sediment of Chalan Beel during April and May
2016. From each place, the sample was collected from at least
10 cm of depth with a sterile inoculating loop/spoon. The sample 2.6. Optimization of PCR
was stored in a pre-sterilized Eppendorf and transported to the
USDA-laboratory of the Shahjalal University of Science and Tech- PCR amplification condition was optimized after several exper-
nology at ambient temperature. Then, the samples were air dried imental trials using following parameters: An initial denaturation
by heating at 70 °C for 1 h in a dryer. step at 94 °C for 4 min followed by a second denaturation step at
94 °C for 1 min, annealing for 1 min at 62 °C, an extension at
72 °C for 90 s, and a final extension step of 72 °C for 10 min. A total
2.2. Culture conditions of 35 serial cycles of amplification reaction was performed.
For the isolation of Bacillus sp., serial dilution technique was 2.7. Sequencing of 16s rDNA
used considering different aqueous dilutions (101 to 104) using
phosphate buffered saline (PBS, pH 7.2). A sample from each dilu- The amplified PCR product was purified using PureLink PCR
tion was then streaked on a nutrient agar (NA) plate amended with purification kit (Thermo Scientific) according to the manufacturer’s
cycloheximide (100 mg mL1) to prevent fungal growth and incu- guidelines. The purified PCR product was then sent to the first BASE
bated at 37 °C for 24 h [22]. sequencing center, Malaysia, for 16s rDNA sequencing. The
M.J. Foysal, A.K. Lisa / Journal of Genetic Engineering and Biotechnology 16 (2018) 387–392 389
sequence received in raw format was then assembled for making mm for Xanthomonas sp. Aphanomyces invadans, Pseudomonas
contigs before further sequence analysis using MEGA 7.0. sp., and Aeromonas veronii. Streptococcus iniae and Pseudomonas flu-
orescens moderately inhibited by the study isolate and showed 10
2.8. Analysis of sequence data and phylogenetic relationship and 9 mm zone of inhibitions (Table 2).
The DNA sequences obtained from the NCBI nucleotide data- 3.3. DNA quantification and PCR product length
base were aligned using the muscle clustering program, MEGA
7.0. The phylogenetic tree was constructed using the neighbor- The amount of extracted DNA of Bacillus sp. BC01 isolate was
joining method with 1000 bootstrap replicates and using a Strepto- approximately 20 ng as compared to the lambda DNA marker,
myces sp. as the control strain. The scale bar indicated the number and the protein-DNA ratio was around 1.8 measured using a spec-
of differences in nucleotide substitutions per sequence. The Gen- trophotometer. This extracted DNA was used for PCR amplification.
Bank accession numbers of the Bacillus species most similar to The PCR product of Bacillus sp. BC01 isolate after amplification by
the bacterial isolates are indicated in parenthesis. Evolutionary universal primers resulted in a band of 1484 bp on the agarose
divergence study was performed as a measurement of scoring sim- gel (Fig. 2).
ilarity matrix; the lower score meant a close neighbor with a sim-
ilar evolutionary origin. The total sequence variability was
3.4. Sequence analysis
calculated as the average mean distance.
After receiving the 16s rDNA purified product sequence from
3. Results first BASE sequencing center, it was primarily edited using BioEdit
(version 7.2) that removed the overlapping sequences and chi-
3.1. Isolation and identification of bacteria meras. Before editing, the NCBI homology BLAST showed a 99.9%
similarity to Bacillus subtilis NBRC 13,719 and Bacillus subtilis
From NA culture, the colonies of prospective Bacillus sp. were BCRC10255. The phylogenetic tree analysis also supported the
identified according to Bergey’s manual for the identification of homology search and confirmed the closed positioning of Bacillus
Bacillus species modified by Wulff et al., 2002, a manual for the sp. BC01 to these two strains. Similarity score revealed a very
classification of Bacillus genus [23]. Based on the test results low divergence of the study strain with Bacillus subtilis NBRC
(Table 1), four Bacillus species, namely, BC01, BC02, BC03, and 13719 (0.0022) and Bacillus subtilis BCRC10255 (0.0030), indicating
BC04, were selected for the further in vitro study. toward a same evolutionary origin (Fig. 2 and Table 3). Bacillus
amyloliquefaciens BCRC 11601 also depicted a very high sequence
3.2. Antagonistic activity of Bacillus sp. BC01 species similarity (0.0052) to Bacillus subtilis BC01 strain. The sequence
had 99% homology and 100% coverage to over 100 strains of Bacil-
The Bacillus sp. BC01 reported potential inhibitory effect against lus sp. deposited in the NCBI data bank. The sequence is now avail-
all the tested microbes (plant and fish pathogens) in the in vitro able at the NCBI data bank under the accession number
antibiotic study. Study results revealed that overnight incubation MF355364.1.
culture (24 h) was most effective against tested isolates (Fig. 1). Here the numerical value corresponds to the following species:
Other three isolates, however, failed to exhibit their potency [1] Bacillus sp. BC01_(MF355364.1); [2] B. subtilis
against culture microbes. The highest zone of inhibition with Bacil- (NR_112629.1) NBRC_13719; [3] B. cereus (NR_112630.1)
lus sp. BC01 was 16 mm for Penicillium digitatum, followed by 15 NBRC_15305; [4] Bacillus sp. (AJ704827.1) P9; [5] B. filamentosus
Table 1
Biochemical characterization of Bacillus sp. isolates.
Isolate Source Shape Motility Hemolysis Starch Gelatin VP Citrate Growth at Growth at Growth at different
hydrolysis hydrolysis test test 6% NaCl different pH temperatures (°C)
2 5 8 10 10 25 45 55
BC01-BC04 Soil Rod + a + + + + + + + + +
Fig. 1. Inhibitory effects of Bacillus sp. BC01 against plant pathogen Penicillium digitatum and fish pathogen Aeromonas veronii with three different incubation culture, 12 h (1),
18 h (2), and 24 h (3). Other disc used as a control for this study.
390 M.J. Foysal, A.K. Lisa / Journal of Genetic Engineering and Biotechnology 16 (2018) 387–392
Table 2
Inhibitory effect of Bacillus sp. BC01 against various crop and fish pathogens (in mm).
Name of the tested Fungus Penicillium Aphanomyces Xanthomonas Pseudomonas Aeromonas Pseudomonas Streptococcus
and Bacterium digitatum (PGT01) invadans (Aph01) sp. (Xpp03) sp. (PSD05) veronii (BN10) fluorescens (PUKL2) iniae (S20)
Bacillus sp. BC01 16 15 15 15 15 9 10
Bacillus sp. BC02 9 8 8 – 7 – –
Bacillus sp. BC03 7 – 7 – – – –
Fig. 2. The amplified 1484 bp PCR product of Bacillus sp. BC01 strain with universal sequencing primer (left) and phylogenetic relationship of strain Bacillus sp. BC01
(MF355364.1) with other Bacillus sp. isolates characterized from the soil, sediment, and rhizosphere samples having antimicrobial activity (right). The tree was constructed
using the neighbor-joining method in MEGA7; the bootstrap consensus tree inferred from 1000 replicates was considered to analyze the taxa.
Table 3
Determining the evolutionary divergence between the sequences. The numbers of base differences per site from between sequences are shown. Much similar sequences marked
in bold.
1 2 3 4 5 6 7 8 9 10 11 12 13
1 –
2 0.0022
3 0.0582 0.0560
4 0.0866 0.0843 0.0724
5 0.0612 0.0590 0.0597 0.0799
6 0.0030 0.0007 0.0567 0.0851 0.0597
7 0.0149 0.0127 0.0552 0.0828 0.0590 0.0134
8 0.0052 0.0030 0.0582 0.0843 0.0582 0.0037 0.0112
9 0.0642 0.0619 0.0552 0.0731 0.0463 0.0627 0.0619 0.0619
10 0.0373 0.0351 0.0612 0.0866 0.0545 0.0358 0.0299 0.0531 0.0619
11 0.0530 0.0507 0.0500 0.0769 0.0455 0.0515 0.0478 0.0515 0.0448 0.0522
12 0.0575 0.0552 0.0649 0.0843 0.0493 0.0560 0.0522 0.0552 0.0336 0.0567 0.0500
13 0.1978 0.1955 0.1888 0.1985 0.1910 0.1963 0.1963 0.1933 0.1851 0.1970 0.1866 0.1873 –
(NR_134701.1) SGD14; [6] B. subtilis (NR_116017.1) BCRC_10255; the loss; one of them includes the use of natural enemies of micro-
[7] B. licheniformis (NR_116023.1) BCRC_11702; [8] B. amyloliquefa- bial pathogens. These antagonistic microbes utilize natural mech-
ciens (NR_116022.1) BCRC_11601; [9] B. cucumis (NR_148626.1) anisms, such as predation, to overcome the damage caused by
AP–6; [10] B. gobiensis (NR_147766.1) FJAT–4402; [11] B. vini microbes to plants and aquatic life. The present study focused on
(NR_146819.1) LAM0415; [12] B. stamsii (NR_145934.1) BoGlc83; screening antibiotic-producing Bacillus sp. that could be used as
[13] Streptomyces sp. (AF026080.1) CHR3. an alternative to commercial antibiotics and pesticides against a
broad range of microbes, especially those responsible for the pro-
duction losses in agriculture and fisheries.
4. Discussion The isolate, BC01, found in the present study belongs to the
genus Bacillus and was confirmed by the identification method of
The diseases of crop plants, fruits, and aquatic life, such as fish, Wang et al. 2009 (method for identification of eight Bacillus species
pose one of the greatest challenges to and threaten the food secu- and subspecies) [25]. The Bacillus sp. BC01 isolate depicted a
rity worldwide. These, in turn, have serious financial implications. sequence (>98%) similarity with two Bacillus subtilis species from
Various alternative management approaches are in use to prevent Wang’s study, namely Bacillus subtilis and B. amyloliquefaciens,
M.J. Foysal, A.K. Lisa / Journal of Genetic Engineering and Biotechnology 16 (2018) 387–392 391
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