Content uploaded by Mohammed Jawhar
Author content
All content in this area was uploaded by Mohammed Jawhar on Nov 15, 2020
Content may be subject to copyright.
1874-3315/20 Send Orders for Reprints to reprints@benthamscience.net
3
DOI: 10.2174/187433150201401????, 2020, 14, 3-9
The Open Agriculture Journal
Content list available at: https://openagriculturejournal.com
RESEARCH ARTICLE
In vitro Antagonistic Activity of Diverse Bacillus Species Against Fusarium
culmorum and F. solani Pathogens
M. Harba1,*, M. Jawhar1 and M.I.E. Arabi1
1Department of Molecular Biology and Biotechnology, AECS, P. O. Box 6091, Damascus, Syria
Abstract:
Background:
Fusarium culmorum and Fusarium solani are economically important fungal pathogens of many plant species causing significant yield losses
worldwide. Frequent uses of fungicides are hazardous to humans and the environment. Therefore, in vitro antagonistic activity of diverse Bacillus
species isolates with biological potential activity to control these both pathogens should be investigated.
Objective:
The objectives were to isolate and identify the Bacillus spp., which are potential controls of F. culmorum and F. solani, and to characterize
molecularly, at the species level, those isolates that have potential as biocontroller of the pathogens.
Methods:
The in vitro antagonistic potential of 40 Bacillus isolates against F. culmorum and F. solani was evaluated on the basis of fungal growth inhibition
on nutrient broth culture. The colony morphology and the 16S rRNA gene sequencing of Bacillus spp. were used to identify the isolates.
Results:
Bacillus sp. isolates were identified as B. atrophaeus, B. subtilis, Paenibacillus polymxa, B. amyloliquefaciens, B. simplex and B. tequilensis. They
had significant (P<0.05) antagonistic activities against F. culmorum and F. solani isolates as compared to the untreated control. The antagonistic
effects varied depending on the Fusarium sp. The bacterial B. subtilis isolates SY116C and SY SY118C provided the most noteworthy result as
both strongly inhibited mycelial growth of F. solani by 97.2%, while the B. tequilensis isolate SY145D was the most effective in the formation of
inhibition zones against F. culmorum by 75%.
Conclusion:
It is apparent that Bacillus sp. isolates play an important role in the inhibition of growth of F. culmorum and F. solani, and that the B. subtilis
isolates SY116C and SY118C had the highest biological potential activity against these fungi. These antagonistic effects may be important
contributors as a biocontrol approach that could be employed as a part of integrated soil pathogen management system.
Keywords: Antagonistic effect, Bacillus species, Fusarium species, In vitro.
Article History Received: May 10, 2019 Revised: July 16, 2020 Accepted: July 28, 2020
1. INTRODUCTION
Fusarium culmorum and Fusarium solani are important
species that cause significant yield losses in many plant species
[1, 2]. Fusarium culmorum is a soil-borne fungus distributed in
cooler temperate regions, and the causal agent of many
important diseases in cereals [3, 4]. F. solani is known to infect
many plant species, causing plant decline, wilting, and large
* Address correspondence to this author at Department of Molecular Biology and
Biotechnology, AECS, P. O. Box 6091, Damascus, Syria;
E-mail: ascientific@aec.org.sy
necrotic spots on tap roots [5, 6].
Fungicides are commonly used to control these diseases,
but frequent uses of these chemicals are hazardous to humans
and the environment. Therefore, the management of the soil-
borne pathogens has become one of the major concerns in
agriculture and focused on searching and selecting antagonist
microorganisms on diverse soil pathogens [7, 8]. However,
difficulties in controlling Fusarium sp. have stimulated
renewed interest in the application of biological control agents,
which has provided an effective and environmentally friendly
4 The Open Agriculture Journal, 2020, Volume 14 Harba et al.
means to control plant diseases. Among the most widely used
microbes for biocontrol agents are members of the genus
Bacillus, that offer advantages over other microorganisms,
tolerance to fluctuating pH, temperature and osmotic
conditions [9, 10]. Furthermore, Bacillus spp. are able to
colonize root surfaces, promoting plant growth and causing
mycelium lysis of several fungal agents [11, 12].
However, soil-borne fungal pathogens such Fusarium sp.
have been shown varying metabolic responses, growth patterns
and reproductive strategies in response to varying soil
microorganisms, therefore, measuring thein vitro growth rate
of fungi was considered as a simple and reliable method for
evaluating the effect of an environmental variable on the
growth of fungi, although this does not take into account
changes in mycelia density [13].
On the other hand, the 16S ribosomal RNA (16S rRNA)
gene has been widely used for the taxonomic classification of
bacteria by the detection of sequence differences in the
hypervariable regions of the 16S rRNA gene which is present
in all bacteria [14, 15].
During a polyphasic experiment, more than 525 bacilli
were isolated from different regions of Syria. In the present
study, forty of them were taken into the 16S rRNA gene
sequence analyses.
The objectives of this current work were to isolate and
identify the bacteria which havethe potential to control
Fusarium spp. (F. culmorum and F. solani) and molecular
characterization of species that have the potential for
biocontrol of the pathogen.
2. MATERIALS AND METHODS
2.1. Isolates of Bacillus sp.
The soil samples were collected from different regions
distributed widely from south to the north-west between
33.40°N and 37.17°N and 35.40°E and 42.30°E in Syria. They
were taken from 2-3 cm depth of field and were carried to
laboratory in sterile polythene bags. They were shaken in 9 ml
sterile water for 3 min at 160 rpm. Serial dilution was made
from 10-3 to 10-7 [16] and then 0.2 ml of each dilution was
spread onto Nutrient Agar (NA) medium and incubated
overnight, the colonies of prospective Bacillus sp. were
identified according to Wulff et al. [17], and 40 isolates were
selected for this study (Table 1). Six Bacillus species, namely,
B. atrophaeus, B. subtilis, P. polymxa, B. amyloliquefaciens, B.
simplex and B. tequilensis, were selected for the further in vitro
study. A pure culture of each Bacillus sp. isolate was first
grown on NA and incubated for 24 h at 30 °C.
2.2. Fungal Isolates
Fusarium culmorum and F. solani were isolated from
infected wheat plants growing in different locations of Syria.
The infected wheat stems were cut into small pieces of 1-1.5
cm, surfaces were sterilized with 5% sodium hypochlorite for 5
min, washed in sterile distilled water twice and cultured on
PDA (PDA, DIFCO, Detroit, MI. USA) medium amended with
13 mg/l kanamycin sulphate added after autoclaving and
incubated for 10 days, at 23 ±1ºC in the dark to allow mycelial
growth and sporulation. Species identification was based on the
morphological characteristics of single spored isolates as
described by Nelson et al. [18, 19]. According to a study [20],
the two virulent monosporic isolates of F. culmorum and F.
solani were selected for this study. The cultures were
maintained on silica gel at 4 ºC until needed.
2.3. In vitro Activity of Bacillus spp. Isolates against
Fusarium spp.
Bacterial colonies of different size, color and morphology
were streaked individually a few times until single colonies of
a single type were observed on the NA plates. Then 5 mm
diameter disc of each F. culmorum and F. solani was cut from
of an actively growing culture by a sterile cork borer and
placed onto the center of above NA plates. Where mycelia disc
on Nutrient Agar (NA) medium without bacteria was
maintained as control. Every elementary treatment was
repeated five times. The mean diameter of pathogen colonies
was measured after 4 days of incubation at 25°C and any
morphological alteration of colonies, in comparison to the
untreated control, was also noted. Damage caused by the
bacterium to the fungal mycelium, removed from the
confrontation zone of both microorganisms (pathogen and
antagonist), was observed under a light microscope, in
comparison to untreated controls. The percentage of inhibition
of fungal growth was calculated by the following formula
proposed by Rabindran and Vidyasekaran [21].
Where; I = Percent inhibition, C = Radial growth of the
pathogen in control, and T = Radial growth of the pathogen in
treatment.
Table1. Bacillus species used in the study.
Bacillus species Number of isolates Morphology
B. atrophaeus 3 Brown-black, opaque, smooth, circular
B. amyloliquefaciens 10 Creamy white with irregular margins
P. polymyxa 2 Milky white, thin often with amoeboid spreading
B. subtilis 20 Fuzzy white, opaque, rough, with jagged edges
B. simplex 1 Cream, gloss, with irregular margins slightly raised
B. tequilensis 4 Yellowish, opaque, smooth, circular
I = (C-T)/C x 100
In vitro Antagonistic Activity of Diverse The Open Agriculture Journal, 2020, Volume 14 5
2.4. 16S rRNA Gene Sequencing
Selected bacterial 16S rRNA was amplified in full length
by PCR using two pairs of primers, BacF (5’-
GTGCCTAATACATGCAAGTC-3’) and BacR (5'-
CTTTACGCCCAATAATTCC-3’) [12]. The PCR reaction
mix (50 μl) contained 2 μl (50-100 ng) of extracted genomic
DNA, 1x reaction buffer (TrisKCl-MgCl2), 2 mM MgCl2, 0.2
mM dNTP, 1 μM of each primer, and Taq polymerase (5U/μl,
Fermentas). PCR amplification condition was achieved using
the following parameters: An initial denaturation step at 95 °C
for 5 min followed by a second denaturation step at 95 °C for
1 min, annealing for 1 min at 54 °C, an extension at 72 °C for
90 s, and a final extension step of 72 °C for 10 min. A total of
30 serial cycles of amplification reaction were performed. PCR
products were separated on a 1.5% agarose gel and visualized
using UV light (302 nm) after staining with ethidium bromide.
Prior to sequencing, PCR products were purified with QIAgen
gel extraction kit (28704) according to the manufacturer´s
recommendations. Sequencing was carried out on a Genetic
Analyzer (ABI 310, Perklin-elmer, Applied Biosystems, USA).
The 16S rRNA sequences were compared with the known
sequences using the National Center for Biotechnology
Information (NCBI) database (http://www.ncbi.nlm.nih. gov).
2.5. Statistical Analyses
All experiments were performed in triplicate with ten Petri
dishes per replicate, for each bacterium-fungus in vitro
evaluation, using a completely randomized design. An F-test
was used to determine if the two runs of each experiment were
homogeneous and if the data could be pooled. The
homogeneity of variance test indicated that the data from both
runs of each experiment could be pooled, and thus all further
analyses were conducted on pooled data. Data were analyzed
using analysis of variance (ANOVA) and means were
separated by Tukey's test (P ≤ 0.05).
3. RESULTS AND DISCUSSION
In this present work, the antagonistic potential of the
Bacillus sp. isolates was concluded and validated by restriction
of the F. culmorum and F. solani pathogens growth and
showed zone of inhibition towards the antagonist as shown in
photo-plate of NA culture plate assay compared with the
control (Fig. 1).
On the other hand, PCR amplification with specific
primers Bac yielded single DNA fragments of ~ 545 bp,
present in all Bacillus sp. isolates (Fig. 2). On the basis of 16S
rRNA gene sequencing, Bacillus sp. isolates are identified as B.
atrophaeus, B. subtilis, P. polymxa, B. amyloliquefaciens, B.
simplex and B. tequilensis as their 16S rRNA gene sequences
displayed similarities ≤ 98% to their closely related type strains
(Table 2). Species belonging to the Paenibacillus genus were
previously re-classified under the genus Bacillus, based on
morphological characteristics. However, the PCR probe tests
suggested that a group of eleven species should be considered a
new genus, Paenibacillus, of which P. polymyxa is the type
strain [22]. The nucleotide sequences were deposited in
GenBank under accession numbers MT159352 to MT159391
(Table 3).
Fig. (1). Bacillus subtilis SY15B showing the zone of inhibition in the NA culture plate assay.
Fig. (2). Agarose gel electrophoresis of 16S rRNA of some Bacillus sp. isolates used in the study. M represents the 100-bp DNA marker (HinfI; MBI
Fermentas, York, UK).
6 The Open Agriculture Journal, 2020, Volume 14 Harba et al.
Table 2. 16S rRNA gene sequence similarity between Bacillus sp. used in this study and the microorganisms strains at NCBI.
Bacillus species Microorganisms (NCBI) 16S rRNA gene sequence similarity (%)
B. atrophaeus B. atrophaeus, ATCC 49337 96%
B. simplex B. simplex JP44SK12 (JX144702) 97%
B. subtilis B. subtilis subsp. Spizizenii JP44SK23 (JX144713) 99%
P. polymxa B. paenibacillus macqariensis subsp. Defensor (AB360546) 98%
B. amyloliquefaciens B. amyloliquefaciens (AF478077) 99%
B. tequilensis B. Bacillus tequilensis (KT760402) 98%
Table 3. Bacillus isolates showing antagonistic activity against Fusarium culmorum
No. Isolates Zone of Inhibition (%)* Antifungal activity GeneBank
accession number
B. atroplaeus
1 SY15B 45h* + MT159352
2 SY199A 46h + MT159353
3 SY63E 56.7e + MT159354
B. subtilis
4 SY35A 55ef + MT159355
5 Sy41B 53f + MT159356
6 SY44A 50g + MT159357
7 SY60A 47gh + MT159358
8 SY73B 38i + MT159359
9 SY113C 60d ++ MT159360
10 SY116C 58.3d + MT159361
11 SY118C 53.3f + MT159362
12 SY124B 45h + MT159363
13 SY130D 46h + MT159364
14 SY132E 60d ++ MT159365
15 SY133 63c ++ MT159366
17 SY132C 63.3c ++ MT159367
19 SY134D 61d ++ MT159368
20 SY135D 57.7e + MT159369
21 SY139D 36.7i + MT159370
22 SY151C 48.3g + MT159371
23 SY160C 66.7cd ++ MT159372
24 SY168C 66.7cd ++ MT159373
25 SY190E 43.3hi + MT159374
Paenibacillus polymyxa
24 SY53C 60d ++ MT159375
25 SY55B 70b ++ MT159376
B. amyloliquefaciens
26 SY82C 46h + MT159377
27 SY96C 66.7cd ++ MT159378
28 SY96E 61.7d ++ MT159379
29 SY123A 53.3f + MT159380
30 SY128B 63c ++ MT159381
31 SY134C 56.7e + MT159382
32 SY159D 61.7c ++ MT159383
33 SY177C 53.3f + MT159384
34 SY190D 51.7fg + MT159385
35 SY200D 57.7e + MT159386
B. tequilensis
36 SY69A 46h + MT159387
In vitro Antagonistic Activity of Diverse The Open Agriculture Journal, 2020, Volume 14 7
37 SY145D 75a +++ MT159388
38 SY149C 71.7ab ++ MT159389
39 SY150D 55ef + MT159390
B. simplex
40 SY198B 10k + MT159391
LSD 0.05
Zone of Inhibition = ( Radial growth of the pathogen in control - Radial growth of pathogen in treatment ) / C 100
Weak inhibition: + (Fungal growth was slightly inhibited by bacteria)
Average inhibition: ++ (Loosely arranged mycelial growth over the bacterial zone)
Strong inhibition: +++ (Fungal growth was completely inhibited before the bacterial zone)
*Values followed by different letters are significantly different at P<0.05 according to ANOVA test
The data showed that Bacillus sp. isolates had a significant
(P<0.05) antagonistic activity against both F. culmorum and F.
solani where the percentage of radial growth inhibition of the
fungi colonies significantly decreased on NA medium as
compared to the untreated controls (Tables 3 and 4). In
addition, the mean colony diameter of Fusarium sp., noted
after 4 days of incubation at 25°C, depends upon pathogens
tested and treatments realized, which is in agreement with a
previous study [23]. However, the antagonistic effects of the
bacterial isolates varied depending on the Fusarium spp. B.
subtilis isolates SY116C and SY SY118C inhibited the growth
of F. solani by 97.2% while the B. tequilensis isolate SY145D
was the most effective in the formation of inhibition zones
against of F. culmorum by 75% (Tables 3 and 4). Bacillus
subtilis isolates showed similar antagonism against Rhizoctonia
solani, Helminthosporium spp., Alternaria spp. and Fusarium
oxysporum [24], and B. tequilensis isolate SY145D showed
similar antagonism with B. tequilensis GYLH001 that had a
potential antagonism towards Magnaporthe oryzae of rice [25]
Table 4. Bacillus isolates showing antagonistic activity against Fusarium solani
No. Isolates Zone of Inhibition (%)* Antifungal activity
B. atroplaeus
1 SY15B 76.8d* +++
2 SY199A 73.9de ++
3 SY63E 57.8gh +
B. subtilis
4 SY35A 75.4de +++
5 Sy41B 87c +++
6 SY44A 76.8d +++
7 SY60A 100a +++
8 SY73B 65.2fg ++
9 SY113C 87.1c +++
10 SY116C 97.2a +++
11 SY118C 97.2a +++
12 SY124B 100a +++
13 SY130D 81.2cd +++
14 SY132E 78d +++
15 SY133 76.9d +++
17 SY132C 76.4d +++
19 SY134D 68f ++
20 SY135D 77d +++
21 SY139D 76.2d +++
22 SY151C 50h +
23 SY160C 71.2e ++
24 SY168C 88.4c +++
25 SY190E 71.5e ++
Paenibacillus polymyxa
24 SY53C 27i +
25 SY55B 33.6i +
B. amyloliquefaciens
26 SY82C 87c +++
27 SY96C 88.4c +++
28 SY96E 94.5b +++
(Table 3) contd .....
8 The Open Agriculture Journal, 2020, Volume 14 Harba et al.
29 SY123A 84cd +++
30 SY128B 70.2e ++
31 SY134C 71.2e ++
32 SY159D 62g ++
33 SY177C 73de ++
34 SY190D 69.3f ++
35 SY200D 74.5de ++
B. tequilensis
36 SY69A 73.9de ++
37 SY145D 72.4e ++
38 SY149C 85.3cd +++
39 SY150D 75.1de ++
B. simplex
40 SY198B 10k +
LSD 0.05
Zone of Inhibition = ( Radial growth of the pathogen in control - Radial growth of pathogen in treatment ) / C ×100
Weak inhibition: + (Fungal growth was slightly inhibited by bacteria)
Average inhibition: ++ (Loosely arranged mycelial growth over the bacterial zone)
Strong inhibition: +++ (Fungal growth was completely inhibited before the bacterial zone)
*Values followed by different letters are significantly different at P<0.05 according to ANOVA test
It is well known that B. subtilis strains produce a broad
spectrum of antimicrobial compounds, including
predominantly peptides as well as a couple of non-peptidic
compounds such as polypeptides, an aminosugar, and a
phospholipid [26], and their highly antifungal effects (97.2%)
on both F. culmorum and F. solani in this study, which might
be attributed to one or more antifungal compounds produced
by this biocontrol agent. However, the observed mycelial
growth inhibition and lysis formation among the colonies of
the both Fusarium pathogens might be due to the effect of the
bacterial diffusible inhibitory antibiosis substances, which
could have suppressed and restricted the growth of the
pathogen, which can be confirmed by the fact that most
Bacillus spp. have an ability to produce several antibiotics such
as bacillomycin, fengycin, mycosubtilin and zwittermicin,
which effectively suppress the growth of pathogens under in
vitro and/or in situ conditions [27 - 29]. This might explain the
formation of inhibition zones between the bacterial and the F.
culmorum and F. solani isolates shown in this study. Our
results are similar to those reported in a previous study [29,
30], whichshowed high capacity of some strains of Bacillus sp.
of the same species to inhibit the growth of several
phytopathogic fungi; this effect was attributed to the
production and secretion of antifungal compounds and
antibiotics belonging to the family of iturins and subtilins, that
act on the fungi's cell wall [30]. Hence the most likely
explanation for the growth reduction of F. culmorum and F.
solani by Bacillus sp. is that antifungal activity is increased by
co-culturing of different bacterial species.
Collectively, this work illustrates that two B. subtilis
isolates (SY116C and SY SY118C) provided the most
noteworthy result as both strongly inhibited mycelial growth of
F. solani by 97.2%, and that one B. tequilensis isolate
(SY145D) had a potent antagonistic activity of F. culmorum by
75%. These B. subtilis and B. tequilensis isolates, as potential
biocontrol agents, may provide an effective strategy to combat
plant pathogens. Field studies should be undertaken to confirm
the effectiveness of the isolates under natural field conditions
as a component of integrated disease management.
ETHICS APPROVAL AND CONSENT TO
PARTICIPATE
Not applicable.
HUMAN AND ANIMAL RIGHTS
No animals/humans were used for studies that are the basis
of this research.
CONSENT FOR PUBLICATION
Not applicable.
AVAILABILITY OF DATA AND MATERIALS
Not applicable.
FUNDING
This work was supported by the Atomic Energy
Commission of Syria.
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or
otherwise.
ACKNOWLEDGEMENTS
The authors wish to thank the Director General of AECS
and the Head of the Molecular Biology and Biotechnology
Department for their continuous support throughout this work.
Thanks are also extended to Dr. H. Ammouneh for his
assistance in achieving the experiments. We also thank Dr. A.
Al-Daoude for critical reading of the manuscript.
REFERENCES
Spanic V, Marcek T, Abicic I, Sarkanj B. Effects of Fusarium head[1]
blight on wheat grain and malt infected by Fusarium culmorum.
Toxins (Basel) 2017; 10(1)E17
[http://dx.doi.org/10.3390/toxins10010017] [PMID: 29280978]
(Table 4) contd .....
In vitro Antagonistic Activity of Diverse The Open Agriculture Journal, 2020, Volume 14 9
Al-Fadhal FA. Isolation and molecular identification of Rhizoctonia[2]
solani and Fusarium solani isolated from cucumber (Cucumis sativus
L.) and their control feasibility by Pseudomonas fluorescens and
Bacillus subtilis Egypt J Biol Pest Cont 2019; 29: 47.
Obanor F, Erginbas-Orakci G, Tunali B, Nicol JM, Chakraborty S.[3]
Fusarium culmorum is a single phylogenetic species based on
multilocus sequence analysis. Fungal Biol 2010; 114(9): 753-65.
[http://dx.doi.org/10.1016/j.funbio.2010.07.001] [PMID: 20943185]
Scherm B, Balmas V, Spanu F, et al. Fusarium culmorum: causal[4]
agent of foot and root rot and head blight on wheat. Mol Plant Pathol
2013; 14(4): 323-41.
[http://dx.doi.org/10.1111/mpp.12011] [PMID: 23279114]
Macedo R, Sales LP, Yoshida F, Silva-Abud LL, Lobo M. Potential[5]
worldwide distribution of Fusarium dry root rot in common beans
based on the optimal environment for disease occurrence. PLoS One
2017; 12(11)e0187770
[http://dx.doi.org/10.1371/journal.pone.0187770] [PMID: 29107985]
Villarino M, De la Lastra E, Basallote-Ureba MJ, et al.[6]
Characterization of Fusarium solani populations associated with
Spanish strawberry crops. Plant Dis 2019; 103(8): 1974-82.
[http://dx.doi.org/10.1094/PDIS-02-19-0342-RE] [PMID: 31210598]
Silva V, Mol HGJ, Zomer P, Tienstra M, Ritsema CJ, Geissen V.[7]
Pesticide residues in European agricultural soils - A hidden reality
unfolded. Sci Total Environ 2019; 653: 1532-45.
[http://dx.doi.org/10.1016/j.scitotenv.2018.10.441] [PMID: 30759587]
Panth M, Hassler SC, Baysal-Gurel F. A review: Methods for[8]
management of soilborne diseases in crop production. Agriculture
2020; 10
[http://dx.doi.org/10.3390/agriculture10010016]
Nicholson WL, Munakata N, Horneck G, Melosh HJ, Setlow P.[9]
Resistance of Bacillus endospores to extreme terrestrial and
extraterrestrial environments. Microbiol Mol Biol Rev 2000; 64(3):
548-72.
[http://dx.doi.org/10.1128/MMBR.64.3.548-572.2000] [PMID:
10974126]
Baril E, Coroller L, Couvert O, et al. Sporulation boundaries and spore[10]
formation kinetics of Bacillus spp. as a function of temperature, pH
and a(w). Food Microbiol 2012; 32(1): 79-86.
[http://dx.doi.org/10.1016/j.fm.2012.04.011] [PMID: 22850377]
Basha S, Ulaganathan K. Antagonism of Bacillus species (strain 121)[11]
towards Curvularia lunata. Curr Sci 2002; 82: 1457-63.
Shafi J, Tian H, Ji M. Bacillus species as versatile weapons for plant[12]
pathogens: a review. Biotechnol Biotechnol Equip 2017; 31(3):
446-59.
[http://dx.doi.org/10.1080/13102818.2017.1286950]
Köhl J, Kolnaar R, Ravensberg WJ. Mode of Action of Microbial[13]
Biological Control Agents Against Plant Diseases: Relevance Beyond
Efficacy. Front Plant Sci 2019; 10: 845.
[http://dx.doi.org/10.3389/fpls.2019.00845] [PMID: 31379891]
Goto K, Omura T, Hara Y, Sadaie Y. Application of the partial 16S[14]
rDNA sequence as an index for rapid identification of species in the
genus Bacillus. J Gen Appl Microbiol 2000; 46(1): 1-8.
[http://dx.doi.org/10.2323/jgam.46.1] [PMID: 12483598]
Callahan BJ, Wong J, Heiner C, et al. High-throughput amplicon[15]
sequencing of the full-length 16S rRNA gene with single-nucleotide
resolution. Nucleic Acids Res 2019; 47(18)e103
[http://dx.doi.org/10.1093/nar/gkz569] [PMID: 31269198]
Ammouneh H, Harba M, Idris E, Makee H. Isolation and[16]
characterization of native Bacillus Thuringiensis isolates from Syrian
soil and testing under insecticidal activities against some insect pests.
Turk J Agric For 2011; 35: 421-31.
Wulff EG, Mguni CM, Mansfeld-Giese K, Fels J, Lübeck M,[17]
Hockenhull J. Biochemical and molecular characterization of Bacillus
amyloliquefaciens, B. subtilis and B. pumilus isolates with distinct
antagonistic potential against Xanthomonas campestris pv. campestris.
Plant Pathol 2002; 51: 574-84.
[http://dx.doi.org/10.1046/j.1365-3059.2002.00753.x]
Nelson PE, Toussoun TA, Marasas WFO. Fusarium Species: An[18]
Illustrated Manual for Identification. University Park: The
Pennsylvania State Univ. Press 1983.
Kiprop EK, Baudoin JP, Mwangómbe AW, Kimani PM, Mergeai G,[19]
Maquet A. Characterization of Kenyan isolates of Fusarium udum
from Pigeonpea [Cajanus cajan (L.) Millsp.] by cultural
characteristics, aggressiveness and AFLP analysis. J Phytopathol
2002; 150: 517-27.
[http://dx.doi.org/10.1046/j.1439-0434.2002.00798.x]
Alazem M. Characterization of Syrian Fusarium species by cultural[20]
characteristics and aggressiveness. Thesis, University of Damascus,
Faculty of Agriculture. 2007; 72.
Rabindran R, Vidyasekaran P. Development of a formulation of[21]
Pseudomonas fluorescens PfALR2 for management of rice sheath
blight. Crop Prot 1996; 15: 715-21.
[http://dx.doi.org/10.1016/S0261-2194(96)00045-2]
Ash C, Priest FG, Collins MD. Molecular identification of rRNA[22]
group 3 bacilli (Ash, Farrow, Wallbanks and Collins) using a PCR
probe test. Proposal for the creation of a new genus Paenibacillus.
Antonie van Leeuwenhoek 1993-1994; 64(3-4): 253-60.
[http://dx.doi.org/10.1007/BF00873085] [PMID: 8085788]
Balouiri M, Sadiki M, Ibnsouda SK. Methods for in vitro evaluating[23]
antimicrobial activity: A review. J Pharm Anal 2016; 6(2): 71-9.
[http://dx.doi.org/10.1016/j.jpha.2015.11.005] [PMID: 29403965]
Matar SM, El-Kazzaz SA, Wagih EE, et al. Antagonistic and[24]
inhibitory effect of Bacillus subtilis against certain plant pathogenic
fungi. I Biotechnol 2009; 8: 53-61.
[http://dx.doi.org/10.3923/biotech.2009.53.61]
Li H, Guan Y, Dong Y, et al. Isolation and evaluation of endophytic[25]
Bacillus tequilensis GYLH001 with potential application for biological
control of Magnaporthe oryzae. PLoS One 2018; 13(10)e0203505
[http://dx.doi.org/10.1371/journal.pone.0203505] [PMID: 30379821]
Stein T. Bacillus subtilis antibiotics: structures, syntheses and specific[26]
functions. Mol Microbiol 2005; 56(4): 845-57.
[http://dx.doi.org/10.1111/j.1365-2958.2005.04587.x] [PMID:
15853875]
Fira D, Dimkić I, Berić T, Lozo J, Stanković S. Biological control of[27]
plant pathogens by Bacillus species. J Biotechnol 2018; 285: 44-55.
[http://dx.doi.org/10.1016/j.jbiotec.2018.07.044] [PMID: 30172784]
Pal KK, Gardener BM. Biological control of plant pathogens. The[28]
plant health instructor 2006.
[http://dx.doi.org/10.1094/PHI-A-2006-1117-02]
Ramarathnam R, Bo S, Chen Y, Fernando WGD, Xuewen G, de Kievit[29]
T. Molecular and biochemical detection of fengycin- and bacillomycin
D-producing Bacillus spp., antagonistic to fungal pathogens of canola
and wheat. Can J Microbiol 2007; 53(7): 901-11.
[http://dx.doi.org/10.1139/W07-049] [PMID: 17898845]
Arguelles-Arias A, Ongena M, Halimi B, et al. Bacillus[30]
amyloliquefaciens GA1 as a source of potent antibiotics and other
secondary metabolites for biocontrol of plant pathogens. Microb Cell
Fact 2009; 8: 63.
[http://dx.doi.org/10.1186/1475-2859-8-63] [PMID: 19941639]
© 2020 Harba et al.
This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is
available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
