International Journal of Research Studies in Biosciences (IJRSB)

August 2015, PP 6-14

ISSN 2349-0357 (Print) & ISSN 2349-0365 (Online)
www.arcjournals.org

Isolation and Characterization of Bacterial Antagonist to Plant

Pathogenic Fungi (Fusarium Spp.) from Agro Based Area of
Bilaspur
Toppo, S.R.1, Naik, U.C.2
Department of Microbiology and Bioinformatics,
Bilaspur University, Bilaspur

Abstract: A variety of fungi are known to cause important plant diseases, resulting in a significant lost in
agricultural crops. The plant diseases need to be controlled to maintain the level of yield both quantitatively and
qualitatively. Farmers often rely heavily on the use of synthetic fungicides to control the plant diseases.
However, the environmental problems caused by excessive use and misuse of synthetic fungicide have led to
considerable changes in people’s attitudes towards the use of synthetic pesticides in agriculture. Today, there is
an increase in the awareness of the the healthy food and healthy environment. In response to this need, some
researchers have focused their effort to develop alternative measures to synthetic chemicals for controlling
plant diseases. Among these, is that people about referred to as biological control using microbial antagonists.
Many microbial antagonists have been reported to posses antagonistic activities against plant fungal pathogens,
such as Pseudomonas fluorescens, Agrobacterium radiobacter, Bacillussubtilis,B.cereus,B. amyloliquefaciens,
Trichoderma virens, Burkholderia cepacia, Saccharomyces spp., Gliocadium spp. Antagonistic bacteria
commonly inhabit soil which can be used as biological control agent for the management of soil borne diseases
of various crops. In this study a total of three (03) composite soil samples were collected from the agro-based
areas at the bank of Arpa river of Bilaspur city and 15 isolates of bacteria were isolated using Nutrient agar
medium. The morphological characterstics and bio-chemical characterstics of all bacterial isolates were
documented. The objective of this study is to isolate indigenous bacteria from agricultural soils and screen them
for their growth inhibition of phyto-pathogenic fungus isolated from infected plants and seeds.
Keywords: Antagonism, phytopathogenic fungi, soil borne bacteria.

1. INTRODUCTION
The rhizosphere is considered as the hot spot of biodiversity.Competition for nutrient sources in the
rhizosphere is very high. Therefore, different microorganisms have developed distinct strategies,
ranging from antagonistic to synergistic interactions, both among themselves and with the plant
(Perotto and Bonfante 1997).The highest portion of microorganisms which inhabit the rhizosphere are
fungi and bacteria. The most common method for isolation of soil bacteria is the serial dilution
method which allow the enumeration of only viable living cell in the soil. Bacterial population is one-
half of the total microbial biomass in the soil ranging from 1,00,000 to several hundred millions per
gram of soil, depending upon the physical chemical and biological condition of the soil. In few
decades a large number of bacteria from the rhizosphere soil have been isolated and identified among
which the common species of Pseudomonas, Azospirillum, Azotobacter, Enterobacter, Bacillus and
Serratia have reported (Kloepper et al., 1989; Okon and Labandera-Gonzalez, 1994 ;Glick, 1995).
The bacteria isolated from rhizosphere soils, have proved to be beneficial to the plants by directly
having an effect on nitrogen fixation (Han et al., 2005), solubilization of nutrients (Rodriguez and
Fraga 1999), and indirectly by antagonizing pathogenic fungi by the production of siderophores, -1,3-
glucanase, antibiotics, fluorescent pigments and cyanide (Pal et al., 2001).Agricultural crops are
exposed to approximately 70,000 species of pests, but of these only 10% are considered serious pests
(Pimentel, 1997). Classes of fungi that commonly cause diseases in agricultural crops are
Plasmodiophoromycetes(root disease of cereals, and powdery scab of potato), Oomycetes (late blight
and white rust disease), Zygomycetes (cause soft rot of fruit), Ascomycetes and Deuteromycetes
(cause leaf spots, blights, cankers, fruit spots, fruit rots, stem rots, root rots, vascular wilts, soft rot),
and Basidomycetes (cause rust and smut diseases (Agrios, 2005).
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Toppo, S.R. & Naik, U.C.

The fungi referred to as Fusarium spp. are the asexual state and are mostly known by their anamorph
name and placed in the class Deuteromycotina (syn.Fungi imperfecti) (Nath, et al.1970; Smith,
1981).The genus Fusarium includes plant pathogenic spp. such as F. avenaceum, F. culmorum,
F.equiseti, F.graminearum, F.oxysporum, F.sporotrichoides, F.verticillioides (syn.F. moniliforme).
When acting as pathogens they mainly attack immature host plants and causes seedling blight, root-
,crown- and foot-rot (Pettitt et al.1996); can penetrate only in damage tissues e.g. snow mould, leaf
and stem infections (Parry et al. 1990); or senescent, e.g. node infection, or as in the case with scab
(syn. head blight, ear blight) of wheat (Triticum aestivum) infection takes place at the stage of
anthesis. At the time of grain formation the plant are tender and relatively unprotected (Parry et
al.1995). Disease caused by Fusarium are popularly referred to as fusarioses. The objective of this
present study was to isolate and identify the diversity of bacteria from the rhizospheric soil of
agrobased area of bank of Arpa river Bilaspur and to screen its ability to antagonize phytopathogenic
fungi.

Figure01. bacterial colony

2. MATERIAL AND METHOD
2.1. Culture Media
The media used in the present study were potato dextrose agar (PDA) for fusarium cultivation and
nutrient agar medium for maintaining bacterial strains.The potato agar media consisted of a liter of
potato extract prepared in laboratory from 200 g peeled potato boiled in water and 20 g agar for The
reason behind choosing this media for the culture of Fusarium strains relies in the findings of ( Jiao et
al.2008) according to whom the expression of important trichothecene biosynthesis genes (tri4 and
tri5) were up-regulated in a high sucrose containing media but not in glucose. Also, Vujanovic and
Mansour (2011) reported that 15-ADON chemotype of Fusarium graminearum produced DON and
ZEA mycotoxins only in the presence of high sucrose concentrations (20% and above).
2.2. Soil Samples
Soil samples were collected in the month of February 2015 from agro based areas of bank of Arpa
river in Bilaspur city and brought to the laboratory. The method used was composite sampling
(Walworth 2006). Samples were collected to a depth of about 6 to 8 inches from rhizospheric area of
crops. 5-8 sub-samples were taken depending on field size and uniformity. The sub-samples were
thoroughly mixed to obtain a representative sample and analysis of this sample gave average values
for the entire area. Soil samples were immediately air-dried at room temperature for two to three days.
The desired sample amount was then removed (200gm), for microbiological analysis and the
remainder was discarded.
2.3. Analysis of Soil Samples for Microbial Content
Analysis of each soil samples was done separately, bacterial-flora were isolated by serial dilution and
plating on suitable growth media (Pandey et al., 2006). Upon incubation each cell gave rise to a
colony on the agar surface (Steubing, 1993). Nutrient agar medium used was from Hi Media,Bombay,
India.
2.4. Characterization Of Bacterial Culture
Isolated bacterial cultures were characterized by studying cultural characteristics of individual isolates
on nutrient agar and morphological characteristics of bacterial cultures were studied microscopically.
(Aneja, 2003).

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Isolation and Characterization of Bacterial Antagonist to Plant Pathogenic Fungi (Fusarium Spp.) from
Agro Based Area of Bilaspur

3. BIOCHEMICAL CHARACTERIZATION
For determination of the biochemical activity of antagonistic bacteria many biochemical tests were
carried out for both epiphytic and soil isolates. cellular characteristics such as gram stain reaction, cell
size, morphology and arrangement were observed and identified. Bacterial isolates were inoculated
with nutrient agar media of unknown (14) and incubated at 37 degrees Celsius for 48 hours. After the
incubation period, a small sample of a bacterial colony was placed on a slide, heat fixed and gram
stained. The gram stained slide was then observed under the microscope at1000x magnification. Both
Purple and pink (coccus/bacillus) shaped bacteria were observed. The bacteria appeared to be
arranged in grape-like clusters. Based on the observations, it was determined that unknown bacterium
(14) are gram(+ve & -ve)bacillus, coccus. In addition, a catalase test was also done in order to
determine the presence of the enzyme catalase. A small sample of a live bacterial colony of unknown
(14) from the NAM media was placed on a slide. Two small drops of 3% hydrogen peroxide H 2O2
were added. Gas bubbles were observed, resulting in a catalase positive test result. Unknown (14)
contains the enzyme catalase and is able to convert into water and Oxygen.Another test was for the
screening of starch hydrolysis in culture striked in starch agar plate with starch only as carbon
sources. After incubation indivisual plates were flooded with gram’s iodine to produce a deep blue
colour and in zone of degradation no blue colour forms which is the basis of detection and screening
of amylolytic strain.Urea hydrolysis is a liquid medium used to test for the presence of the urea
hydrolyzing enzyme, Urease. The enzyme hydrolyses urea to ammonia and carbon dioxide. It
differentiates rapid urease-positive bacteria from slow urease-positive bacteria and Urease-negative
bacteria. Color change to shocking pink indicates a positive test, but sometimes the incubation period
might take up to eight days to see this result.
4. ISOLATION OF FUNGI
The conventional method of isolating well separated spores streaked on the agar medium under a
stereoscopic microscope was used by Tuite (1969). fungal cultures stored in a high nutrient medium,
such as potato dextrose agar [PDA] may lose their ability to produce enzymes or metabolites, or
perform other functions and care is therefore needed in selecting a storage medium (Smith and
Onions, 1994). Some other sophisticating single spore isolation technique using micromanipulators
and a hand maid glass needle were described by Matsushima (1975), Teik-Khiang (1999). Also
preparing Fusarium cultures for identification was held by the single spore technique, devised by
Hansen (1926) and Toussoun and Nelson (1976). In fact fungal isolates grown from single spores
demonstrate species characteristics and uniform growth more clear than those of mass transfer of
inoculums (Leslie and Summerell, 2006).
5. ANTIMICROBIAL ACTIVITY BY DUAL CULTURE METHOD
The antagonistic activity of all bacteria isolates was studied on Fusarium by dual culture technique
(Francisco et al., 2011).On Petri dishes with PDA placing equidistantly a 5 mm-disk of the bacteria
and on the other side of the Petri dish, a disk of mycelium of the same diameter of Fusarium. The
plates inoculated were incubated at 25 ± 2°C until the growth of control treatment, covered the Petri
dish. The effect of bacteria on plant pathogens was determined by mycelia growth inhibition (Saeideh
et al., 2008).
6. POINT INOCULATION METHOD
Each of the bacterial isolates was point inoculated at sides 3cm from the center of the plate and after
that six day old culture of F. oxysporum was point inoculated in the center of the plate. Control plate
was only point inoculated with F. oxysporum (G.Shobha, BS.Kumudini). The plates were sealed with
parafilm and incubated at 28±2°C for 4-5 days (Kumar et al. 2002). Antagonistic activity was
investigated for four to seven days after incubation at room temperature (28±2°C).
After 5 days incubations the percent inhibition in growth of pathogen was calculated by the formula:
% inhibition = R1 – R2 X 100/ R1
r1= radial growth of Fusarium in
control

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Toppo, S.R. & Naik, U.C.

r2= radial growth of fusarium in dual

innoculation
7. RESULT AND DISCUSSION
In the present study 3 composite soil samples (A, B and C ) were obtained as representative sample
from agro based area of bank of Arpa river Bilaspur by composite sampling method. A total of 14
bacterial isolates were differentiated and maintained as pure cultures on nutrient agar. From soil
sample (A) pure culture of five bacteria were isolated, from soil sample (B) 4 bacterial culture were
isolated and from sample (C)5 bacterial isolates were obtained.

Figure02. bacterial isolates

Cultural and morphological characteristics of all 14 bacterial isolates are depicted in table- (02) Both
gram positive and negative bacilli along with gram negative cocci were isolated in the present
investigation. A total of 06 Gram positive bacilli,04 Gram negative bacilli, 04 Gram positive cocci
were isolated of the total samples used in the current study.

Gram positive

Gram negative

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Isolation and Characterization of Bacterial Antagonist to Plant Pathogenic Fungi (Fusarium Spp.) from
Agro Based Area of Bilaspur

Bacteria are the most abundant and predominant organisms found in the soil habitat. The bacterial
abundance is 2–20 times higher in the rhizosphere than in the bulk soil (Morgan et al. 2005).Kloepper
et al., (1989); Okon and Labandera-Gonzalez, (1994) ;Glick, (1995) reported that large number of
Gram positive and Gram negative bacteria are common inhabitants of rhizospheric soil including
species of
Pseudomonas,Azospirillum,Azotobactr,Klebsiella,Enterobacter,Alcaligens,Arthobacter,Burkholderia,
Bacil-lus & Serratia.

AntagonistsBU2,BU5,BU6,BU7,BU8,BU9,were aerobic Gram-positive rod-shaped bacteria, motile

and able to grow at high temperature.Catalase and starch hydrolysis tests showed variable
reaction.These biochemical tests and physical properties show that both antagonists are Bacillussp.
(Fahy and Hayward, 1983; Cappuccino and Sherman, 1996).
BU1, BU3, BU4, BU10, BU11, BU12, BU13, BU14, were short Gram-negative rods and coccus.
Catalase were positive.starch hydrolysis testes were positive in negative.Hence, these antagonists
were placed in the family Enterobacteriaceace (Fahy andHayward,1983; Cappuccino and
Sherman,1996).
Table (03) depicts biochemical characterization of 14 bacterial isolates. Screening of Amylase
Producing Bacteria was done on starch agar medium. Among 14 bacterial strains 10 bacterial strains
showed amylase activity while the rest 4 bacetrial isolates showed negative test for amylase activity.
BU1,BU2,BU3,BU6,BU7,BU9,BU10,BU11,BU12,BU13,BU14 bacterial isolate were able to
produce catalase enzyme while the BU4,BU5,BU8 shows negative catalase activity. High catalase
activity were observed in BU3,BU6,BU10,BU13,BU14 isolates. Yellow indicated in all the 14
bacterial sample the organism only has low urease activity the phosphate buffer is not neutralise and
no production of NH3 take place. Thus urease test by all the bacterial sample is negative.In the similar
studies made by Fahy and Hayward, (1983); Cappuccino and Sherman, (1996), bacterial antagonists
isolated by them were aerobic Gram-positive rod-shaped bacteria, motile and able to grow at high.

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Toppo, S.R. & Naik, U.C.

8. ISOLATION AND SCREENING OF POTENTIAL ANTAGONISTS

Screening for antagonism between the microorganisms isolated from the rhizosphere and fusarium
spicies were done by dual culture technique.The potato dextrose agar (PDA) medium in culture plates
was simultaneously seeded with actively growing 3mm mycelial blocks of fusarium and the
antagonist isolates. Four days old Fusarium point inoculated in one side of the pda plate near
centre.where as each individuals antagonists were seeded at 2cm equidistant point near the periphery
from the centre and incubated at 28± 1oC. Isolations yielded 14 different colonies of bacteria and
among which 9 colonies Of bacteria showed potential as antagonists for inhibiting fusarium
species(Table04 ). Among these, bacterial isolates ‘4’ and ‘5’ displayed a high ability to suppress
fusarium growth, with inhibition zone over 10 mm. out of 14 bacterial isolates 9 isolates smaples were
effective in tested methods (Table04 ).

Figure03. microscopic view of identified fusarium species

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Isolation and Characterization of Bacterial Antagonist to Plant Pathogenic Fungi (Fusarium Spp.) from
Agro Based Area of Bilaspur

TABLE NO.4 depictes invitro antagonism effect of 14 bacterial isolates against test pathogen
fusarium species.the result of invitro experiment revails that isolates number
BU1,BU2,BU3,BU4,BU5,BU9,BU11,BU12,BU14 these are positive for antagonistic activity against
the test pathogen.

9. DISCUSSION
Rajput et al., 2013 in their Investigation on leaf spot disease (Alternaria alternata (FR.) Keissler) of
Brinjal (Solanum melongena L.) under south Gujarat condition find out suitable management
strategies to control the pathogen alternative to chemical fungicides by using bioagents to control the
pathogen. They evaluated eight known bioagents by dual culture, pathogen at periphery and pathogen
at the centre technique to monitor antagonistic effect. Their results revealed that out of all the eight
bioagents used, three bioagents viz., Trichoderma viride (IARI isolate) (74.77%, 69.04% and
79.45%), Trichoderma viride (Navsari isolate) (74.14%, 66.08%, and 76.99%) maximum growth
inhibition in dual culture, pathogen at periphery and pathogen at the centre methods respectively), T.
harzianum (Junagadh isolate) (71.25%, 59.96% and 74.78%) maximum growth inhibition in dual
culture, pathogen at periphery and pathogen at the centre methods respectively, showed strong
antagonistic effect to inhibit the mycelia growth of the pathogen significantly.
Thirty isolates of Pseudomonas fluorescens obtained from citrus rhizosphere were tested for
antifungal activity against Phytophthora spp. P. fluorescens isolate Pf20 was found efficient in
inhibiting the mycelial growth upto 38.88%. The antifungal compounds were extracted were found
inhibitory to the growth of Rhizoctonia solani (42.79%), Phytophthora parasitica (28.57%), P.
palmivora (25.98%) and Fusarium solani (20.45%) (Koche et al., 2013). In another investigation
carried out by Dewangan et al., 2014 to screen different media for growth and colony formation of
test bacterium Pseudomonas fluorescens and to check its antagonistic activity on fungal plant
pathogens by in vitro dual culture techniques. Out of the seven different media tested, King’s ‘B’
media was best with an OD value of 2.50 and 6 X 108 cfu among the different media evaluated. The
growth was fast in King’s ‘B’ medium, fair in Sim agar medium and slow in remaining media tested.
In dual culture method, P.fluorescens on co-inoculation with fungal pathogens decreased their growth
rate. Maximum inhibition was observed in Sclerotium rolfsii (63.15%) followed by Fusarium
oxysporum (61.85%) Rhizoctonia bataticola (55.56%) and
R.solani (53.15 %). Also Bacteria and Fusarium oxysporum from the rhizoplane soil and surrounding
soil of healthy and diseased tomato plants of district regions of Karnataka were collected. The best
antagonist bacterial strains against Fusarium oxysporum isolate, were identified as BS1, BS5 and
BS18. All bacterial isolates resulted effective for the in vitro control of growth of Fusarium
oxysporum. Their results suggested that Pseudomonas fluorescens isolates had an excellent potential
to be used as biocontrol agents of Fusarium oxysporum in tomato greenhouses at the field level.
(ASHA et al., 2011). The work of Mezeal, (2014) reveals the antagonistic effect of microorganism

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Toppo, S.R. & Naik, U.C.

against tomato disease caused by Rhioctonia solani and Fusarium oxysporum. For which four
bacterial strains of, Bacillus subtilis and fife strain of Pseudomonas fluorescens were isolated from
tomato field soil. The antagonistic microorganisms against the pathogens were observed by Dual
Culture Technique. P. fluorescens 5 isolate was found to show 81.3% and 77.4 of growth inhibition
against R.solani and F. oxysporum respectively while B. subtilis177.4% and 73.2 of growth inhibition
against test pathogens respectively. Their report suggested that P. fluorescens5 isolate were more
effective than Bacillus subtilis1 might be used potential biological control agents of tomato plants in
Iraqi soil.
All the above observations reviewed supported our finding that soil rhizospheric bacteria are potential
enough to antagonize plant pathogenic fungi Fusarium spp. and these finding will help in future
endeavor on biocontrol of fungal phytopathogens in Bilaspur.
10. CONCLUSION
The results of this study proved that properly selected bacterial isolates have the potential to
significantly suppress the growth of Fusarium spp. The efficiency of the inhibition could be
influenced by the specific interactions between bacterial strains (competition, antagonism) and by the
particularities of target fungal pathogens species.
ACKNOWLEDGMENTS
The research presented here is part of M.Sc. thesis of the second author at Department Of
Microbiology And Bioinformatics, Bilaspur University, Bilaspur
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