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Antagonistic Effects of Bacillus Species in Biocontrol of
Tomato Fusarium Wilt
Caroline Fadeke Ajilogba, Olubukola Oluranti Babalola* and Faheem Ahmad
Department of Biological Sciences, Faculty of Agriculture, Science and Technology
North-West University Mafikeng Campus, Private Bag X2046 Mmabatho 2735
KEYWORDS Bacillus spp. Biological Control. Fusarium solani. Inhibition. L. esculentum
ABSTRACT Fusarium solani is the causative organism of Fusarium wilt of tomato (Lycopersicon esculentum Mill).
Four Bacillus spp identified as B. amyloliquefaciens, B. cereus, B. pumilus and B. subtilis were tested for biocontrol
activities in vitro as zone of inhibition and in vivo as percent disease control and disease incidence. The result from in
vitro analysis showed that B. amyloliquefaciens inhibited the growth of F. solani the most by 95.2% while B. cereus had
the highest growth of F. solani and inhibition of 55.7%. B pumilus and B. subtilis showed inhibition of 70.46% and
82.1% respectively and were significantly lower (p=0.05) than the control with 100% F. solani growth. In vivo, B. cereus
had the least disease incidence and highest percent disease control (18.75% and 81.2%). This was significantly different
from the control (100% and 0%), B. amyloliquefaciens (25% and 75%), B. pumilus (37.5% and 62.5%) and B. subtilis
(37.5% and 62.5%). Also, growth parameters like shoot and root length from treatment with B. cereus (38 mm ±1.47 and
31 mm ±1.22) were significantly longer (p=0.05) compared to the control. This result shows that these four Bacillus spp
are very effective biocontrol agents and should be harnessed for further biocontrol applications.
*Address for correspondence:
Telephone (work): +27 183892568
Fax: +27 183892134
E-mail: olubukola.babalola@nwu.ac.za
INTRODUCTION
Tomato is one of the most important veg-
etables because of its health benefits and phy-
tochemical properties. Because of its low calo-
rie and absence of cholesterol, it is one of the
recommendations of diets needing low choles-
terol. They are quite rich in many important nu-
trients and vitamins which include phosphorus
and potassium and also vitamins B and C. They
are also very important against common cancers
like breast and prostate cancer.
As important as tomato is nutritionally and in
being an important cash crop for smallholders
and medium-scale commercial farmers in Africa,
soil-borne pathogens inflicts a lot of diseases and
infections on it (Babalola and Glick 2012). Such
diseases include Bacterial wilt, root knot nema-
todes disease, early blight, late blight and
Fusarium wilt. Fusarium wilt is a devastating dis-
ease of tomato and causes a lot of loss to farmers
worldwide. symptoms begins as gradual yellow-
ing and wilting of the lower leaves (Khan and
Khan 2002) which is brought about by the growth
of the microconidia inter-cellularly in the xylem
of the stem and root. As a result of the failure of
the infected xylem of the plant to meet the water
requirement of the plant, death of the tomato plant
is inevitable (Burgess et al. 2008). Spores from
the conidia are released into surrounding tissues
as the plant dies. They later form chlamydospores
that fall back into the soils (Jones 2000). These
spores can remain in the soil for as long as 30
years until favourable conditions are available
and they can re-infect plants (Thangavelu et al.
2004).
Several microorganisms are being used in the
control of tomato pests and diseases. Listed
among tomato pest control agents are F.
compactum and F. arthrosporioides (Babalola
2010a,b,c). Included in tomato disease control
agents are Trichoderma, Pseudomonas and Ba-
cillus species. Bacillus-based biocontrol agents
are quite important in the management of pests
and plant diseases (Jacobsen et al. 2004). Vari-
eties of Bacillus and Paenibacillus help to pro-
mote the health of crops and control diseases by
producing antibiotic metabolites, suppressing
plant pathogens, others antagonise plant patho-
gens by competing for nutrients like iron and
phosphate, others indirectly fix nitrogen which
they make available to the plants and help stimu-
late plant nutrient uptake (Gardener 2004). This
research seeks to elucidate the biocontrol abili-
ties of these four Bacillus spp in order to make
use of these abilities for further biocontrol inter-
ventions.
MATERIAL AND METHODS
Bacillus spp Inoculum Preparation
The following typed cultures and locally iso-
lated organisms (LIO) from the culture collec-
© Kamla-Raj 2013 Ethno Med, 7(3): 205-216 (2013)
tion of the Microbial Biotechnology Research
Group of the Biological Sciences department of
the North-West University Mafikeng Campus,
South Africa were used for this study: B. subtilis
(ATCC 11774), B. cereus (ATCC 11778), B.
amyloliquefaciens (LIOBac179), B. pumilus
(LIOBac269). The inoculum preparation was
carried out according to Cavaglieri et al. (2005).
Two loopfuls of each of the bacteria from 3-day
old cultures on tryptic soy agar (TSA) were trans-
ferred separately to 50 ml tryptic soy broth (TSB)
medium and incubated overnight at 28±2°C. Vi-
ability was confirmed by standard plate count
method using trytone soy broth plus 2% agar
(TSBA). These inocula were prepared in order
for them to be used in vitro for antifungal activi-
ties of the Bacillus isolates and also their
biocontrol activities in vivo.
The inocula for use in the greenhouse were
prepared from a 24 h shaken culture of each of
the Bacillus isolate incubated at 28±2°C. Ten-
fold serial dilution was carried out and a con-
centration of 5×107 was achieved Five ml of
Bacillus suspension containing 5×107 cfu/ml was
used as inoculant in the greenhouse.
Preparation of Phytopathogenic Fungi
Fusarium solani ATCC 36031 (Davies Diag-
nostic, South Africa) was used for the research.
Fungal strains and inoculum was prepared by
culturing it on Potato dextrose agar (PDA) for
10 days in Petridishes. The microconidial sus-
pension of F. solani was prepared by pouring 1
ml of sterile water in each petri dish in order to
loosen the spores from the medium. The inocu-
lum was then scrapped with the aid of a sterile
spatula from the surface of the Petri dishes in
other to be sure of the viability of the cells and 1
ml was made up to 20 ml in sterile bottles. The
bottles were properly shaken in the rotary shaker
to dislodge the spores from the mycelia of the
fungi to get a concentration of 106 spore concen-
tration. The spore concentration was adjusted to
the required concentration of 109 spores/ml by
taking 1ml of the spore suspension and diluting
with 9 ml sterile distilled water and then injected
into sterile soils in the greenhouse (Adebayo and
Ekpo 2005).
Antifungal Activities of Bacillus spp
Based on antifungal activities, the Bacillus spp
were tested for the following:
Detection of Hydrogen Cyanide (HCN)
Production
Production of HCN was detected according
to the method of Lorck (1948) freshly grown cells
were spread on a tryptone soy agar (30 g) to
which 4.5 g/l glycine had been added and a ster-
ilized filter paper saturated with 1% solution of
picric acid and 2% sodium carbonate was placed
in the upper lid of the Petridish, which was then
sealed with parafilm and incubated at 30°C for 4
days, a change in colour of the filter paper from
yellow to reddish brown was an index of cyano-
genic activity while no colour change represent
no cyanogenic activity.
Determination of Indole Acetic Acid
Production (IAA)
Eight grammes of nutrient broth (Merck) was
suspended in 500 ml of distilled water; freshly
grown cultures were inoculated into 10 ml nutri-
ent broth to which tryptophan had been added (1
mg and 3 mg tryptophan) in each test tube and
incubated at 30°C for 48 h. A 4 ml culture was
removed from each test tube and centrifuged at
10,000 rpm for 15 min. An aliquot of 1 ml of
supernatant was transferred into a fresh tube to
which 50 µl of 10 mM orthophosphoric acid and
a 2 ml of Salkowski reagent comprising of (1 ml
of 0.5 M FeCl3 in 50 ml of 35% HCIO4) were
added. The mixture was incubated at room tem-
perature for 25 minutes. The development of a
pink colour indicated the presence of indole ace-
tic acid (Brick et al. 1991). The absorbance of
the pink solution from each isolate was measured
and recorded at 530 nm using spectrophotom-
eter (Thermo Spectronic, Merck, SA).
Phosphate Solubilisation
Phosphate solubilization is a complex phe-
nomenon for selectively screening the bacteria
which have the ability to release inorganic phos-
phate from tricalcium phosphate. Pikovskaya’s
medium, is a selective medium for phosphate
solubilizing microorganisms (PSM) was used to
which tricalcium phosphate (TCP) has been
added as it will enhance formation of halo zones.
The medium was autoclaved at 121°C for 15 min
and poured into Petridishes. Isolates were
streaked on the Petridishes and incubated for 3
days at 27°C. Bacillus isolates that were able to
CAROLINE FADEKE AJILOGBA, OLUBUKOLA OLURANTI BABALOLA AND FAHEEM AHMAD
206
solubilize developed clear zones around colo-
nies (Pikovskaya 1948).
Antagonistic Activity of Bacillus spp
The test was carried out to see the effect of
the Bacillus isolates on the fungi before the
growth of the fungi in vitro. Fungal inhibition
tests were performed by plate assay. A loop of
Bacillus culture was streaked over the surface of
a PDA plate (Biolab) and after 4 days of incuba-
tion at 28°C, each Petridish was inoculated with
a loopful containing mycelia of F. solani.
Petridishes were then incubated at 25°C for 10
days and examined for inhibition of fungus
growth by the Bacillus isolates. A zone of inhi-
bition around the Bacillus isolate indicated posi-
tive response. The width of cleared zones of an-
tagonism (distances between the bacterial and
fungal growth) were measured after 10 days.
Each experiment was repeated four times. The
results were expressed as the mean values with
standard error deviation in inhibition distance
between the growths of the corresponding
Fusarium isolate and the presence of the Bacil-
lus isolate tested. Percentage inhibition was cal-
culated as follows:
% inhibition = 1-(fungal growth/control growth)
× 100
Control experiment with only growth of Ba-
cillus isolates and fungi in two separate
Petridishes were observed.
Green House Experiment
Two-week old tomato seedlings were trans-
planted into 24cm-diameter pots. Each pot con-
tained sterile vermiculite, peat, and perlite in the
ratio 3:4:1. At 5 weeks, the different treatments
with Bacillus isolates were applied as outlined
in the experimental design using 4 trials (each
trial comprised of 120 plants) for each of the
Bacillus isolates, a negative control with no F.
solani and a positive control with inoculation of
F. solani. Plants were watered daily in the green
house at 25°C at 60-90% relative humidity for
10 weeks. At the end of 10 weeks, samples were
harvested to assess the effect of the various Ba-
cillus isolates on the different growth parameters.
Significant difference was assessed from the
mean of each of the different treatments. The
experiment was repeated twice.
The growth parameters assessed include:
length of shoot and root length and randomly
selected seedlings were used to determine each
parameter per treatment.
Disease Scoring and Data Recording
Disease incidence was recorded by counting
the number of infected plants and dividing it with
the total number of plants assessed in each treat-
ment. The result obtained was converted to per-
centage using the formula:
Disease incidence = (Number of diseased plants/
number of plants assessed) x 100 (Haruna et al.
2012).
Percent disease control was obtained by the
formula below
Number of diseased plants in control = Number
of disease plant in treatment ×100 Number of
plants in control
RESULTS
In vitro Antagonistic Effects of Bacillus spp
In vitro analysis revealed that the four Bacil-
lus isolates viz B. amyloliquefaciens, B. cereus,
B. pumilus and B. subtilis inhibited the growth
of F. solani significantly. The results obtained
are presented in Figures 1, 2.
The results obtained showed that B. amylo-
liquefaciens inhibited the growth of F. solani the
most by 95.20% with only 2.50 mm growth in
diameter of F. solani while B. cereus had the high-
est growth of F. solani with 23.07 mm growth in
diameter and inhibition of 55.70%. The other
Bacillus isolates also inhibited the growth of F.
solani significantly as shown on Figure 1.
Mechanism of Biocontrol of Bacillus spp
The mechanism of antagonism of the four
Bacillus isolates was investigated and the mecha-
nism results are presented (Table 1).
Phosphate Solubilization
Bacillus isolates were examined for their abil-
ity to solubilize phosphate as an indirect mecha-
nism of biocontrol by modifying the environmen-
tal condition. In order to examine the Bacillus
isolates for their ability to solubilize phosphate,
a standard agar medium; (pH 6-7) containing 5
g of tricalcium phosphate (TCP) as a sole source
of phosphorus was prepared and used. Only B.
ANTAGONISTIC EFFECTS OF BACILLUS SPECIES IN BIOCONTROL OF TOMATO FUSARIUM WILT207
Fig. 1. Inhibitory effects of the four Bacillus spp against F. solani in vitro Petridishes containing PDA were inocu-
lated with both F. solani and the Bacillus isolates to measure mycelia growth inhibition by Bacillus isolates. (A)
show F. solani growth diameter of 52 mm (control). (B), (C), (D) and (E) shows 2.5 mm, 23.07 mm, 9.32mm and
15.36mm growth diameter respectively. Values are mean of four replicates. Bac A = B. amyloliquefaciens, Bac C =
B. cereus, Bac P = B. pumilus, Bac S = B. subtilis.
CAROLINE FADEKE AJILOGBA, OLUBUKOLA OLURANTI BABALOLA AND FAHEEM AHMAD
208
Fig. 2. Inhibitory effect of the four Bacillus isolates against F. solani in vitro at P=0.05. Bac A = B. amyloliquefaciens,
Bac C = B. cereus, Bac P = B. pumilus, Bac S = B. subtilis. Percent inhibition value for Bac A, Bac C, Bac P and Bac
S is 95.20, 55.70, 70.46 and 82.10 respectively while FSP is the growth of F. solani alone in the Petri dish. Each
value is average of four replicates.
Table 1: Mechanism of inhibition by the four Bacillus spp
Antifungal metabolite production
Treatments Phosphate HCN IAA
No 1mg 3mg
tryptophan tryptophan tryptophan
Bac A+-0.249 0.258 0.294
Bac C --0.248 0.284 0.294
Bac P --0.261 0.271 0.284
Bac S --0.226 0.247 0.263
Control (Sterile distilled water) --0.064 - -
All isolates were negative to HCN production and all were positive to the utilization of tryptophan which is a
precursor of IAA while only Bac A solubilized phosphate. Bac A = B. amyloliquefaciens, Bac C = B. cereus, Bac P =
B. pumilus, Bac S = B. subtilis.
amyloliquefaciens showed clear zones around the
streaked isolate. All the others were negative
without any clear zones (Table 1).
HCN Production
In vitro production of HCN by the four Ba-
cillus spp was carried out using the picric acid
assay. None of these isolates produced HCN
(Table 1).
IAA Production
Production of IAA by all the Bacillus isolates
was detected by the production of pink colour
by all of them. Production of IAA was not de-
pendent on the presence of tryptophan even
though highest concentration was read from Ba-
cillus isolates to which tryptophan had been
added. This also means that there is correlation
between the amount of tryptophan and amount
of IAA produced. All the Bacillus isolates pro-
duced indole acetic acid when grown in media
containing tryptophan which is obvious by the
production of pink colour by all isolates in dif-
ferent concentrations (Table 1). Using spectro-
photometer (Thermo Spectronic, Merck, SA),
absorbance at 530 nm revealed that B. amylo-
liquefaciens and B. cereus had the highest ab-
sorbance of 0.29 nm from the test tube having 3
ANTAGONISTIC EFFECTS OF BACILLUS SPECIES IN BIOCONTROL OF TOMATO FUSARIUM WILT209
solubilization
mg tryptophan while B. subtilis had the least ab-
sorbance of 0.26 nm from the 3 mg test tube. All
the Bacillus isolates inoculated with or without
tryptophan showed different levels of absorbance
but the levels of absorbance gradually increased
from isolates inoculated without tryptophan to
isolates inoculated with 3 mg tryptophan. In-
crease in the absorbance from zero tryptophan
to 1 mg tryptophan was 3% while that of 1 mg
tryptophan to 3 mg tryptophan was 13.95% in B.
amyloliquefaciens. Level of absorbance exhib-
ited by B. cereus, increased from zero tryptophan
to 1 mg tryptophan with 14.51% while from 1
mg tryptophan to 3 mg tryptophan with 3.52%.
In B. Pumilus, the increase from zero tryptophan
to 1 mg tryptophan was 3.83% while that of 1
mg tryptophan to 3mg tryptophan was 4.79%.
Absorbance level in B. subtilis, increased from
zero tryptophan to 1 mg with 9.29% while from
1 mg to 3mg with 6.47%. This shows that the
highest increase in the level of absorbance based
on the increase in tryptophan was exhibited by
B. amyloliquefaciens while B. cereus exhibited
the lowest.
Biocontrol Potentiality of Bacillus spp on
Fusarium Wilt of Tomato Plants in the
Screen House
As a result of the in vitro performance of the
four Bacillus isolates in antagonising the growth
of F. solani, greenhouse experiments were car-
ried out to analyse their biocontrol activities.
Greenhouse experiment was carried by using
completely randomized block design, with 4 main
blocks:
•Tomato planted without F. solani and
Bacillus isolates.
•Tomato planted with only the different
Bacillus isolates.
•Tomato planted with both Bacillus isolates
and F. solani.
•Tomato planted with only F. solani
The result obtained is presented in Table 2
and Figure 3a, b, c, d.
Degree of Disease Incidence in Tomato
Plants After Treatment with Bacillus spp
Incidence of disease the screen house is quite
different compared to the pattern of inhibition in
vitro. Disease incidence varied from 18.75% in
treatments with Bac C to 25% in treatments with
Table 2: Effect of various treatments on plant growth
parameters in tomato plants treated with Bacillus spp
and infected with Fusarium solani
Treatments Shoot length Root length
(mm) (mm)
Bac AF 38±1.22b 29.5±1.04b
Bac CF 38±1.47b 31±1.22b
Bac PF 27±0.70a 18.5±1.65a
Bac SF 29±2.27a 18.5±1.25a
Control 24±0.85a 16.75±1.10a
Values are mean of 4 replicates ± SE. Each replicate
had a total of 120 plants. Values with different letters
are significantly different at P= 0.05 by Duncan’s LSD.
Bac A = B. amyloliquefaciens, Bac C = B. cereus, Bac P
= B. pumilus, Bac S = B. subtilis.
Bac A and 37.5% in treatments with Bac P and
Bac S. They were significantly different from the
control which had 100% disease incidence
though there was no occurrence of wilt on plants
that were not infected with F. solani and that were
in turn not treated. In summary, all four Bacillus
spp were effective in reducing disease incidence
and thus disease control.
Degree of Disease Control Using Bacillus
spp as Treatments
Percent disease control using Bacillus spp in
the screen house followed the pattern of percent
disease incidence. The treatment with the high-
est disease control activity was Bac C with 81.2%
disease control. Others are 75% in Bac A and
62.5% in Bac P and Bac S. This is quite signifi-
cantly different from the control having 0% dis-
ease control.
Plant Growth Parameters
Plant growth parameters to be evaluated from
the tomato plants treated with the four Bacillus
spp include; shoot and root length.
Shoot Length of the tomato plants
treated with Bacillus isolates
The shoot length of the plants not infected
with FSP but treated with Bac C was 42 mm long
and the longest. It was significantly different from
the control which was 32 mm long and also from
Bac P which was 36 mm long and Bac S which
was 35 mm long but not significantly different
from Bac A which was 38 mm long. While the
plants that were infected with FSP and the treated
with Bacillus spp also had Bac A and Bac C hav-
CAROLINE FADEKE AJILOGBA, OLUBUKOLA OLURANTI BABALOLA AND FAHEEM AHMAD
210
Fig. 3. Dry roots of tomato plants that have been treated with different Bacillus isolates. Bac A = B. amyloliquefaciens,
Bac C = B. cereus, Bac P = B. pumilus, Bac S = B. subtilis
ANTAGONISTIC EFFECTS OF BACILLUS SPECIES IN BIOCONTROL OF TOMATO FUSARIUM WILT211
ing the longest shoot length of 38 mm and were
significantly different from the control which had
shoot length of 24 mm and Bac P and Bac S which
had shoot length of 27 mm and 29 mm respec-
tively (Table 2).
Root Length of the Tomato Plants
Treated with Bacillus isolates
For plants not infected with FSP but treated
with Bacillus spp, the root length of plants treated
with Bac C was the longest with 34 mm and was
significantly different from the Bac A which had
root length of 29 mm, Bac P and Bac S which
had 23.25 mm and 23.5 mm respectively. Root
lengths of plants treated with Bac C and Bac A
were significantly different from the other treat-
ments and from the control with root length of
21.5 mm. Plants infected with FSP and treated
with Bac C also had the longest root length of 31
mm and was significantly different from the con-
trol having 16.75 mm. Bac A having root length
of 29.5 mm was not significantly different from
Bac C but both of them were significantly differ-
ent from Bac P and Bac S having root length of
18.5 mm each (Table 2).
DISCUSSION
In vitro Antagonistic Effects of
Bacillus spp on Fusarium solani
Eleven Bacillus spp isolated from the rhizo-
sphere were evaluated for their PGPR and
biocontrol potential against F. solani in vitro. The
result revealed that all the Bacillus spp sup-
pressed the mycelial growth of F. solani in vary-
ing degree ranging from 55.7% by B. cereus (Bac
C) to 95.2% by B. amyloliquefaciens (Bac A).
This inhibitory activity of Bac A was reported to
be as a result of antifungal compounds or me-
tabolites released into the PDA medium (Dihazi
et al. 2012). Also it has been reported that B.
amyloliquefaciens strains have been able to in-
hibit the growth of a variety of fungal pathogens
because of their ability to produce a vast array
of antibiotics such as zwittermicin, bacillomycin,
fengycin, bacilysin and difficidin (Athukorala et
al. 2009; Chen et al. 2009). B. subtilis also in-
hibited the growth of F. solani by 82.1% in vitro.
This result agrees with Adebayo and Ekpo
(2005), because B. subtilis inhibited fungal
growth and also promoted the growth of tomato
plant in screen house trial. B. subtilis has been
shown to have a broad spectrum of antimicro-
bial activities over diverse fungal and bacteria
pathogen (Grover et al. 2009). Over 70 % of
mycelial growth of F. solani in vitro was inhib-
ited by B. pumilus (Bac P). This may be as a
result of production of antibiotic, competition
with pathogen for nutrients and direct antago-
nism (Akhtar et al. 2010). Bacillus spp are known
to reduce wilting index in F. udum, increase plant
growth and cause rapid colonization of tomato
tissue in order to induce systemic resistance
against F. oxysporum (Kloepper et al. 2004).
Mechanism of Biocontrol of
Bacillus spp against Fusarium solani
Phosphate Solubilization
B. amyloliquefaciens solubilized phosphate
out of the four Bacillus isolates used in this re-
search. Majority of the strain isolated from po-
tato crop rhizosphere that solubilizes tricalcium
phosphate (58%) belonged to B. amylolique-
faciens isolates and they also had in vitro antago-
nism against Rhizoctonia solani and Fusarium
solani (Calvo et al. 2010). B. amyloliquefaciens
sks-bnj-1 (AY 932823) possessed multiple plant
growth-promoting traits which included produc-
tion of indole-3-acetic acid (IAA), solubilization
of zinc, production of ACC deaminase, solubili-
zation of phosphate, production of phytases,
HCN and cellulases. It also improved the growth
of soybean by improving nutrient assimilation,
rhizosphere properties and yield (nutrient con-
tent of soybean) compared to unioculated con-
trol (Sharma et al. 2013a). B. amyloliquefaciens
AM1 and D29 inhibited the growth of R.
solanacearum T-91 and produced IAA, side-
rophore and solubilize phosphate (Almoneafy et
al. 2012). B. subtilis and B. cereus isolated from
groundnut rhizosphere were able to solubilize
phosphate (Maheswar and Sathiyavani 2012). B.
subtilis inhibited the growth of F. oxysporum (25-
34%) in vitro and Botryodiphodia theobromae
isolated from post-harvest rotten yam tuber
(100%). It was able to solubilise phosphate and
promote elongation of root in seedlings (70-74%)
of Cicer arietinum compared to the control
(Swain and Ray 2009). B. subtilis strain D16 in-
hibited the growth of R. solanacearum, produced
IAA and siderophore but did not solubilize phos-
phate which is similar to the result of this research
(Almoneafy et al. 2012).
CAROLINE FADEKE AJILOGBA, OLUBUKOLA OLURANTI BABALOLA AND FAHEEM AHMAD
212
Other Bacillus isolates that solubilise phos-
phate include B. thuringiensis, B. sphaericus and
B. megaterium (Akgül and Mirik 2008). Phos-
phorus is a very important macronutrient needed
for plant growth and development. Microorgan-
isms help to convert insoluble phosphorus in the
soil to soluble ones that are accessible by plants
for growth and increased yield (Saharan and
Nehra 2011). This helps to increase the uptake
of phosphorus by plants (Chen et al. 2006; Igual
et al. 2001), they are therefore quite important
to biotechnological aspect of agriculture in other
to meet the phosphorus needs of plants
HCN Production
Positive colour change of filter paper to red-
dish brown indicated the production of HCN.
None of the four Bacillus isolates produced HCN
which is similar to Singh et al. (2008) in whose
research the Bacillus isolate were all negative
for HCN. Cyanide is a toxic and dreaded chemi-
cal produced by many rhizobacteria. Some bac-
teria synthesis it, others excrete it and yet others
metabolize it in other to avoid predation and
competition (Zeller et al. 2007). Hydrogen cya-
nide is a gas that affects the metabolism of most
root especially of weeds negatively. Production
of Hydrogen cyanide in Bacillus is about 50% in
both rhizospheric soils and nodules compared to
Pseudomonas that is over 80% (Ahmad et al.
2008; Charest et al. 2005). Plant growth was
enhanced invitro by most of the rhizospheric iso-
lated that produced HCN (Wani et al. 2007).
HCN produced by rhizospheric bacteria isolated
from chickpea rhizosphere also promoted plant
growth directly, indirectly and synergistically
(Joseph et al. 2006). According to Karuppiah and
Rajaram (2011), most of the Bacillus isolates
(Bacillus BA1, BA3, BA4, BA6, BA7, and BA8)
from rhizosphere of vegetable plants produced
HCN and siderophore and so had antifungal ac-
tivity against Penicillium spp, F. oxysporum and
Cercospora spp. HCN has been reported as been
effective in the control of wilt of cucumber
caused by Pythium ultimum. It chelates metals
and upsets perspiration (Keel et al. 1996). B.
amyloliquefaciens sks-bnj-1 (AY 932823) pro-
duced HCN among other Plant growth promot-
ing characteristics; improved plant growth and
increased soybean yield (Sharma et al. 2013).
Others include B. megatatrium JUMB1, JUMB2,
JUMB3, JUMB4, JUMB5, JUMB6 and JUMB
7 which all produced HCN, IAA, Ammonia and
siderophore but were unable to solubilize phos-
phate (Shobha and Kumudimi 2012). Reports
have shown that HCN influences plant growth
indirectly especially isolates from rhizosphere of
chickpea, rice and mangrove (Joseph et al. 2007;
Samuel and Mathklaruppan 2011; Shobha and
Kumidimi 2012).
IAA Production
All the Bacillus spp produce indole acetic acid
from tryptophan to enhance plant growth as the
pink colour was produced by all isolates in dif-
ferent concentrations. This is similar to produc-
tion of auxin which is the commonest form of
IAA by B. amyloliquefaciens KPS46 which also
supported growth of soybean (Buensanteai et al.
2008). Most of the strains isolated from the rhizo-
sphere of potato crop (81%) were from the B.
amyloliquefaciens strain and they produced IAA
(Calvo et al. 2010). Others are B. amylolique-
faciens AM1 and D29 and B. subtilis strain D16
(Almoneafy et al. 2010). B. cereus RS87 signifi-
cantly promoted growth of root length, plant
height and seedling emergence over control and
produced IAA (Jetiyanon et al. 2008). The abil-
ity of B. amyloliquefaciens FZB24 to enhance
growth and control plant disease could be as a
result of production of plant hormones such as
indole-3-acetic acid (IAA) (Bottini et al. 2004;
Bloemberg and Lugtenberg 2001). B. subtilis B1,
B6, B28 and B99 significantly promoted growth
and biocontrol activity against F. oxysporum f.sp
ciceris in chickpea compared to untreated con-
trol (15.8-44.8 %). They were observed to pro-
duce IAA, HCN and antifungal volatiles among
others (Karimi et al. 2012). B. subtilis WR-W2
and B. amyloliquefaciens MR-A1 produced dif-
ferent concentration of IAA with B. subtilis pro-
ducing more compared to B. amyloliquefaciens.
Sometimes, auxins are produced when there is a
precursor such as L-tryptophan, which helps to
increase the production of IAA in Bacillus
amyloliquefaciens FZB42 (Idris et al. 2007). B.
licheniformis K11 and B. subtilis AH18 both pro-
duced antifungal β-glucanase, siderophere and
auxins. They were also involved in phosphate
solubilization. This led to up to 20% increase in
leaf, stem and root growth of red pepper and to-
mato (Lim and Kim 2009). According to Joseph
et al 2007, while working with chickpea, all Ba-
cillus isolates produced IAA. Production of IAA
ANTAGONISTIC EFFECTS OF BACILLUS SPECIES IN BIOCONTROL OF TOMATO FUSARIUM WILT213
in plants help to increase root dry weight and
thereby increase the plants’ ability to take up N,
P, K compared to non-inoculated control
(Etesami et al. 2009). It helps to stimulate plant
growth and increased the uptake of N, P, K, Ca
and Mg in sweet potato cultivar (Farzana and
Radizah 2005). It caused increase in vegetables
especially cucumber, pepper and tomato
(Kidoglu et al. 2007). It is responsible for early
growth promotion in soybean (Glycine max L)
and corn (Zea mays L) (Cassana et al. 2009).
The response of plant to different concentration
of auxin (Sarwar et al. 1994) is different and the
difference can depend on the type of microor-
ganism (Ahmad et al. 2005). Even though some
microorganisms produce high concentration of
auxin, that is, IAA and this helps to increase plant
growth and yield in wheat crop (Khalid et al.
2004), others producing low concentration of
IAA also improve plant growth (Tsavkelova et
al. 2007).
Relationship between Growth
Parameters and Disease Incidence
In this research the various treatments reduced
disease incidence and promoted growth param-
eters compared to the control in the greenhouse.
They were all effective in promoting tomato
growth which led to increase in the shoot and
root dry weight compared to the control. This is
because where Bacillus spp or and their by-prod-
ucts are applied to plants, the outcome is disease
control (Gardener 2004).
According to (Singh et al. 2008), chir-pine
seeds treated with B. subtilis BN1 demonstrated
early seed emergence, viability and increased
biomass. In comparison to uninoculated seeds
and seeds infested with M. phaseolina, disease
severity was significantly reduced. B sphaericus
and B. brevis-2 increased plant length signifi-
cantly while B. megaterium, B. polymyxa, B.
sphaericus, B. brevis -1 and B. thuringiensis in-
creased significantly by 30-54% the number of
pods. Pod weight was increased by 25% while
seed yield by 35% in plants treated with B.
thuringiensis (de Freitas et al. 1997).
Shoot and root length were enhanced as well
as increase in fresh biomass and total dry matter
using rhizospheric Bacillus spp for the biocontrol
of anthracnose caused by Colletotrichum
acutatum on pepper. AB05 (B. amylolique-
faciens) and AB12 (B. subtilis) inhibited the
growth of C. acutatum by 60% and induced in-
crease in weight of pepper fruit. In the green-
house disease was more than 30%. These
rhizobacteria solubilized phosphate and pro-
duced phytohormone IAA which are factors re-
garded as systemic acquired resistance induced
in different and diverse plants making such iso-
lates to be considered as potential biocontrol
agents (Lamsal et al. 2012).
Two strains of B. pumilus (203-6 and 203-7)
and one of B. mycoides (Strain Bac J) were able
to significantly reduce the severity of Cercospora
leaf spot of sugar beet which is caused by
Cercospora beticola Sacc. They were able to do
this by eliciting ISR (Bargabus et al. 2002;
Bargabus et al. 2004; Kloepper et al. 2004).
Growth of banana plantlets increased and
Fusarium wilt of banana caused by F. oxysporum
cubense was controlled as a result of treatment
with B. pumilus ENF24 (Figueiredo et al. 2010).
B. cereus was effective in suppressing alfalfa
diseases, enhancing the emergence of seedling
and increasing nodulation in common beans
(Camacho et al. 2001; Figueiredo et al. 2007).
B. megaterium has been found to increase growth
parameters in the root which include the length
of the root and the dry matter content of the root
(Kaymak et al. 2008).
B. subtilis FZB24 and FZB37 inhibited myce-
lial growth of F. oxysporum, R. solani and
Sclerotinia Sclerotiosum in vitro. Incidence of
F. oxysporum disease was significantly reduced
by up to 50% while plant height, root and shoot
fresh weight increased significantly compared to
the control. The result of the greenhouse was
quite different from the result in vivo which
means that antifungal activities in vitro did not
always correlate with disease reduction in vivo
(Schmledeknecht et al. 2001). This is quite simi-
lar to the result from this research. Bacteria that
antagonise soil-borne pathogen in vitro are not
necessary the most effective in vivo and vice-
versa (Chérif et al. 2002). Bacillus spp from the
rhizosphere have been reported to be effective
against a variety of soil borne pathogens. They
are able to do this using diverse mechanisms
(Choudhary and Johri 2009; Kloepper et al.
2004). Colonization of root was not inspected in
this research but from the morphology of the root
samples, those treated with Bacillus isolates had
more root hairs compared to the uninoculated
control.
CAROLINE FADEKE AJILOGBA, OLUBUKOLA OLURANTI BABALOLA AND FAHEEM AHMAD
214
CONCLUSION
This research shows that Bacillus species are
quite important and effective as biocontrol
agents. Their effectiveness is also observed in
their ability to promote growth in plants. Re-
search is continuing to be able to formulate them
into microbial agents that will be health and en-
vironmentally friendly.
ACKNOWLEDGMENTS
We gratefully acknowledge the North-West
University for bursary to the first author and the
National Research Foundation, South Africa, for
grant that supports work in our laboratory.
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