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Journal of Plant Pathology (2010), 92 (2), 343-347

Edizioni ETS Pisa, 2010

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CHITINOLYTIC BACILLUS spp. ISOLATES ANTAGONISTIC

TO FUSARIUM MONILIFORME IN MAIZE
W. Bressan and J.E. Fontes Figueiredo
Embrapa Milho e Sorgo, Microbiology Department, Rod. MG 424 Km 65, 35701-970, Sete Lagoas, MG, Brazil

SUMMARY

Six isolates of Bacillus spp. (BM1, BM2, BM3, BM4,

BM5 and BM6) were evaluated to determine their antagonistic activity towards Fusarium moniliforme in
maize seeds. A preliminary survey detected F. moniliforme (Giberella fujikuroi) in all subsamples of maize
seeds apparently free from disease. The fungus incidence in the seeds ranged from 26.7% to 61.4%. All
Bacillus spp. isolates suppressed the growth of F. moniliforme in vitro and in maize seed trials. The highest disease control in seeds was given by isolate BM2 (79.6%)
and the lowest by isolate BM5 (54.2%). In soil trials,
the reduction in diseased plants ranged from 65% to
78% and no significant differences (p0,05) were observed among Bacillus spp. isolates in controlling the
pathogen. All Bacillus spp. isolates produced chitinase,
but isolates BM2 and BM3 had the highest chitinase activity. These results indicate that Bacillus spp. isolates
have antagonist activity towards F. moniliforme and
have a potential to be used as a biocontrol agent. The
possible role of chitinase secreted by Bacillus spp. isolates in the suppression of the fungal infection is discussed.
Key words: Bacillus, Fusarium, chitinase, biocontrol.

INTRODUCTION

Fusarium moniliforme (Giberella fujikuroi), commonly infects a wide range of crops and is a major pathogen
of Gramineae, particularly in tropical and subtropical
regions. On maize (Zea mays L.) the fungus causes
seedling blight as well as root, stalk, ear and kernel rot
(Galperin et al., 2003). Its pathogenicity is affected by
biotic and abiotic conditions (Velluti et al., 2000). F.
moniliforme overwinters as chlamydospore-like structures and mycelium in plant debris or crop residues

Corresponding author: W. Bressan

Fax: +55.31.30271188
E-mail: bressan@cnpms.embrapa.br

(Nyvall and Kommedahl, 1970) and as mycelium on

seed. The infection may occur either at the seedling
stage, and the fungus then grows systemically producing
symptoms during the later stages of plant development,
or later in the growing season. Common points of entry
are roots and stalks at the base of leaf sheaths.
Biological control of soilborne plant pathogens is a
powerful alternative management strategy. In addition
to protecting seeds, biocontrol agents colonize the rhizoplane or rhizosphere when added as seed treatment,
and may protect the underground parts of plants from
attack (Ahmad and Baker, 1987).
Endophytic bacteria grow in different tissues of various symptomless plants and many genera have or may
have potential for biocontrol including Agrobacterium,
Arthrobacter, Bacillus, Pseudomonas, Erwinia, Streptomyces, Serratia, and Xanthomonas (Weller, 1988; Dowing and Thompson, 2000; Kawase et al., 2004; Hoster et
al., 2005). Bacillus spp. have been tested on a wide variety of plants for ability to control diseases (Paulitz and
Belanger, 2001), although relatively little is known
about the exact mechanisms of control (Huang et al.,
2005).
Many mechanisms of pathogen suppression by bacteria have been suggested: substrate competition and
niche exclusion, production of siderophores and/or
production of antibiotics and induced resistance. In addition, chitinases (EC 3.2.1.14) are present in a wide
range of microorganisms such as bacteria and fungi.
These enzymes are glycosyl hydrolases which catalyse
the degradation of chitin. Application of chitin to soils
or foliage increases numbers of chitinolitic bacteria,
providing pathogen control (Mitchell and Alexander,
1961). Aeromonas, Serratia, Myxobactyer, Vibrio, Streptomyces, and Bacillus produce chitinases (Cody et al.,
1990; Jian-Gang et al., 2008) mainly for utilizing chitin
as an energy source (Roberts and Selitrennikoff, 1988;
Helisto et al., 2001; Hoell et al., 2005). Although strains
of Serratia marcesens, Enterobacter agglomerans,
Stenotrophomonas matophilia, Bacillus chitinolyticus and
Bacillus circulans are reported to effectively control Sclerotium rolfsii and Rhizoctonia solani (Dowing and
Thomson, 2000; Hoster et al., 2005; Jian-Gang et al.,
2008), the data related to supression of Fusarium wilts

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Chitinolytic Bacillus spp. antagonisitic to Fusarium

by chitinolytic bacteria are limited.

The objective of this study was to evaluate the effectiveness of Bacillus spp. isolates as biocontrol agents of
Fusarium moniliforme in maize and to measure the production of chitinases by these isolates.

MATERIALS AND METHODS

Macroscopically disease-free maize (Zea mays) seeds

of cv. BR201 were divided into six subsamples and surveyed for Fusarium moniliforme incidence. Twenty five
maize seeds were placed in a plastic germination box
(10.5 10.5 cm) containing sterilized paper over 4%
PDA (potato-dextrose-agar). The germination boxes
were incubated for 8 days at 222C, in a 12/12 h
light/dark photoperiod. The percentage incidence of F.
moniliforme on maize seed was evaluated using a stereomicroscope at 50X magnification (Da et al., 1987).
Bacillus spp. isolates (BM1, BM2, BM3, BM4, BM5
and BM6) were selected by recovering and screening
endophytic bacteria from apparently healthy maize
plants, which were classified as nonpathogenic in tests
previously conducted in greenhouse conditions. The
isolates were grown and maintained on D2 medium
(Kado and Heskett, 1970) in the collection of Embrapa
Milho e Sorgo, Sete Lagoas, MG, Brazil
Bacillus spp. isolates were selected in vitro for chitinolytic activity on chitin-agar medium (Hsu and Lockwood, 1975). Colloidal chitin, prepared from practicalgrade chitin by the method of Roberts and Selitrennikoff
(1988), was added to the agar medium as sole carbon and
energy source. Chitinase production was determined by
plating a 20 l drop of a suspension of the isolate (107
CFU ml-1) on 0.2% chitin agar medium and incubating
the plates at 28C. Colonies were regarded as chitinaseproducing if they induced clear zones of chitin hydrolysis. For each strain there were 10 replicate plates.
Chitinase production by Bacillus spp. isolates was
measured using individual 50 ml Erlenmayer flasks containing 80 ml of minimal salt medium amended with 2
mg ml-1 colloidal chitin and inoculated with 3 ml of a
suspension (107 CFU ml-1) of each isolate grown at 28C
for 72 h in liquid medium containing (g/l): potato
starch, 10.0; yeast extract, 3.0; (NH4)2 HPO4, 2.0; and
KH2PO4, 2.0. Five replicate Erlenmeyer cultures were
used for each isolate. The flasks were incubated on a rotary shaker at 180 rpm for 7 days at 28C and the suspension from each flask was centrifuged for 30 min at
10,000g. The supernatant was filtered through sterile
Millipore membranes, collected in sterile tubes and was
used as a source of crude enzymes. Chitinase activity
was assayed by measuring the release of N-acetyl-D-glucosamine (NAG), according to Tweddell et al. (1994). A
reaction mixture containing 1 ml of the culture supernatant and 1 ml of colloidal chitin (2% wt/vol) in 45

Journal of Plant Pathology (2010), 92 (2), 343-347

mM sodium acetate buffer, pH 6.8, was incubated in a

water bath for 1 h at 36C. After incubation the reaction was terminated by heating in boiling water for 20
min and the mixture was centrifuged at 3,000g for 20
min. The concentration of NAG in the supernatant was
determined as described by Reissig et al. (1955). One
unit of chitinase activity (CHU) was defined as the
amount of enzyme that liberated 1 mol of NAG mg-1
protein per hour at 50C. Protein concentration was determined according to Bradford (1976) with bovine
serum albumin as standard. The experiment was repeated twice.
In vitro tests to detect antagonistic activity against F.
moniliforme were done in Petri dishes containing PDA
medium. The same six Bacillus spp. isolates (107 CFU
ml-1) with chitinolytic activity were selected and
streaked in the center of the Petri dishes. The agar disc
method (Hinton and Bacon, 1995) was used to evaluate
in vitro antifungal activity. One disc was cut from a F.
moniliforme culture and placed on the outer margin of
the Petri dish. Plates were incubated at 28C for 8 days
and observed for mycelial inhibition. Plates inoculated
with F. moniliforme alone served as the control.
An experiment to assess disease in maize seeds inoculated with F. moniliforme and Bacillus spp. isolates was
arranged in a completely randomized design using the F.
moniliforme isolate FMS1 and 6 Bacillus spp. isolates.
The replicates were 10 germination boxes containing 25
maize seeds for each treatment.
FMS1 was isolated from maize cultured on PDA solid media for 10 days at 222C and maintained in 15%
glycerol at -80C in the collection of Embrapa Milho e
Sorgo. Inocula consisted of both conidia and mycelium
together obtained by flooding a Petri dish with sterile
distilled water. The conidial concentrations averaged
from 106 to 109 conidia ml-1 and were brought to a standard concentration of 107 conidia ml-1. Bacterial inoculum suspensions were adjusted to 107 CFU ml-1.
Seeds of maize cv. BR 201, were disinfected prior to
treatment by dipping them in a solution of commercial
bleach of (5.15% NaClO), containing two drops of
Tween 20 for 5 min on an orbital shaker, followed by
five rinses in sterile distilled water under agitation. Samples of 25 disinfected seeds were placed in a plastic germination box containing sterilized filter paper over 4%
PDA. This medium was used to promote fungal development and to facilitate evaluation of the treatments.
The boxes were arranged in a completely randomized
design with the following treatments: surface-disinfected seed + F. moniliforme isolate, surface-disinfected
seed + F. moniliforme isolate + Bacillus spp. isolates.
The positive controls were boxes with surface-disinfected seeds inoculated with F. moniliforme and the negative controls consisted of boxes containing only disinfected seeds. The germination boxes were incubated for
8 days at 222C, in a 12/12 light/dark photoperiod.

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Evaluation of fungal infections in the seeds was made

under a stereomicroscope. The trial was repeated twice.
Effects of Bacillus isolates on Fusarium disease development in maize plants were tested in a potting soil
bioassay in a greenhouse, using pots containing 10 kg of
sterilized (120C for 3 h), sandy-loam soil arranged in a
completely randomized design experiment with the following treatments: 6 Bacillus sp. antagonist isolates, the
F. moniliforme isolate FMS1 and 6 replicates for each
treatment. Each replicate consisted of three plants. Positive controls included seeds not inoculated with Bacillus spp. planted in soil inoculated with the pathogen.
Negative controls consisted of seeds not pot-inoculated
with Bacillus spp. planted in soil not inoculated with the
pathogen. Five pots each sown with five surface-sterilized maize seeds were used per treatment.
Soil inoculum consisted of propagules of F. moniliforme on talc powder produced by the method of Locke
and Coulhoun (1974). Sterilized soil and talc powder inoculum were mixed to obtain final pathogen densities
of 1 106 propagules g soil-1.
Antagonistic Bacillus spp. isolate inocula were grown
for 24 h at 28C on KB-agar plates, and suspensions were
prepared in sterile 10 mM MgSO4. The inoculum was adjusted to 1 106 CFU ml-1 before seed inoculation.
Seeds were inoculated by placing surface-disinfected
maize seeds in beakers containing each bacterial inoculum and shaking on an orbital shaker at 100 rpm and
28C for 24 h followed by air-drying. Non-inoculated
seeds that were shaken under the same conditions but
soaked in sterile deionized water served as negative controls.
For each treatment five seeds were sown in plastic
pots containing 10 kg of a 1:1 mixture of steamed sandyloam soil inoculated with F. moniliforme strain FMS1 (1
106 propagules g soil-1) Ten days after sowing the
seedlings were thinned to three per pot. Disease was
monitored for up to 40 days and measured as the percentage of seedlings showing symptoms of Fusarium disease (yellowing, dropping of leaves or vascular discolouration). Disease incidence was confirmed by plating
slices cut from the lower stem and roots from diseased

Bressan and Fontes Figueiredo

Table 1. Percentage of Fusarium moniliforme incidence in

maize seed samples (survey trial).
F. moniliforme incidencea
36.5 c
48.2 b
29.5 c
61.4 a
30.8 c
26.7 c

Subsample number
1
2
3
4
5
6
a

Within column, values followed by the same letter do not differ

significantly at P0.05 according to Duncans multiple range test.

seedlings, surface-disinfected in 1% sodium hypochlorite, on Komada Fusarium-selective medium (Komada,

1975). The experiment was repeated twice. All data were
analysed using SAS (SAS Institute Inc., USA). Data were
arcsine-square-root transformed prior to analysis. Means
were compared by Duncans multiple-range test. Statistical significance was determined at P<0.05.

RESULTS AND DISCUSSION

In preliminary experiments, maize seeds were surveyed for F. moniliforme and its presence was confirmed
in all subsamples apparently free from disease. Fungal incidence ranged from 26.7% to 61.4% (Table 1). F. moniliforme may remain undetected in kernels until germination, when it infects the emerging seedlings. Yields are reduced in symptomless infected plants (Galperin et al.,
2003). Bacon et al. (2001) claimed that endophytic infections transmitted through systemically infected seeds are
not controlled by fungicide seed dressings. Our data indicate that symptomless seeds may in fact be latently infected and are an important source of inoculum.
The chitinase activity of a wide range of bacterial isolates has been investigated by determining the size of a
clear zone produced on chitin agar plates. This method
is widely accepted as a standard test for chitinase activity. Chitinase was produced in all Bacillus spp. isolates
studied (Table 2). Production did not differ significantly
(p0.05) among the isolates (Table 2). Bacillus species

Table 2. Chitinolysis, total protein and chitinase activity (CHU) from Bacillus spp. isolates. Values are means of two experiments containing ten replications for each treatment.
Bacillus spp.
isolates
BM1
BM2
BM3
BM4
BM5
BM6

Diameter of clear zone

(mm) *
19 a
20 a
18 a
18 a
17 a
19 a

345

Total protein
(mg)
49.7 c
69.6 a
68.1 a
51.8 bc
46.2 c
49.4 c

CHU (mol NAG

h-1 mg-1 protein)
3.89 b
5.09 a
4.95 a
3.39 b
3.26 b
3.59 b

*Clear zone of chitinolysis around colonies on chitin-agar medium.

Within column, values followed by the same letter do not differ significantly
at P0.05 according to Duncans multiple range test.

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Chitinolytic Bacillus spp. antagonisitic to Fusarium

Fig. 1. Suppression of Fusarium disease of maize plants in

potting soil by Bacillus spp. isolates. Values are means of 18
plants for each treatment. Vertical bars indicate standard errors (p0.05) by Duncans test.

have been shown to produce a high level of chitinolytic

enzymes (Cody et al., 1990; Champen et al., 1999). A
high correlation between chitinolysis and production of
bioactive compounds has been reported (Chen et al.,
1991; Pisano et al., 1992).
Total protein and chitinase activity (CHU) varied
among the isolates. Isolates BM2 and BM3 showed
highest total protein and CHU activity. The chitinolytic
activity of Bacillus spp. isolates on inhibition of conidial
germination and germtube growth may be directly correlated with disease suppression as reported by Zhang
et al. (2001). To demonstrate that antifungal activity is
caused by enzymes such as chitinase, Hoster et al.
(2005) inactivated the proteins using heat treatment or
proteases and observed a strong reduction or loss of the
antifungal properties. Our in vitro tests of the antagonistic activity of Bacillus spp. isolates showed that all
Bacillus spp. isolates suppressed growth of F. moniliforme on plates. In the inhibition zone of the six isolates
there was no physical contact between isolates and the
pathogen suggesting that the Bacillus isolates produced
antifungal metabolities. Chitinases detected in the previous trials may be involved in fungal growth inhibition
by hydrolyzing the chitin in the fungal cell wall. These
activities were also reported by Matsuda et al. (2001)
and Hoster et al. (2005). Chitinase causes the swelling
of fungal mycelium and the formation of vacuoles. This
is sometimes accompanied by the degradation of hyphal
cell walls and the release of intracellular components into the medium (Melentev et al., 2001). The capacity of
Bacillus spp. isolates to grow actively on media containing chitin or fungal biomass indicate the mycolytic potential of these strains, which determines their antagonistic activity against phytopathogenic fungi.

Journal of Plant Pathology (2010), 92 (2), 343-347

In the maize seed trials where antagonistic activity of

the Bacillus spp. isolates was evaluated, all isolates significantly reduced disease incidence. No significant difference in biocontrol activity of the F. moniliforme in the
maize seeds was observed among the isolates BM1, BM2,
BM3 and BM4. Highest disease reduction was obtained
with the Bacillus isolate BM2 (79.6%) and the lowest
with isolate BM5 (54.2%), compared to the control.
In soil treated with the pathogen, disease was effectively controlled by seed inoculation with Bacillus spp.
isolates. No significant differences were observed in the
efficacy of the Bacillus isolates in controlling the
pathogen (Fig. 1). The control treatment (pathogen only) showed 98% Fusarium disease, whereas the biocontrol treatments had only 22% to 35% diseased plants.
These data confirm the results observed in the in vitro
trial that showed the antagonist activity of Bacillus spp.
isolates to F. moniliforme. Pleban et al. (1997) found that
the crude extracellular chitinase of an endophytic bacterium B. cereus 65, decreased spore germination of F.
oxysporum and reduced disease incidence by 70%,
whereas B. cereus YQ308 inhibited the growth F. oxysporum, F. solani and Pythium ultimum (Chang et al., 2003).
The expression of antagonism by a microorganism
towards a pathogen in culture medium cannot generally
be taken as evidence of control in situ (Hoitink and
Boechm, 1999). However, results obtained in the present study showed that Bacillus isolates had the same antagonistic activities to F. moniliforme in culture medium
as on maize seeds in soil.
This work indicates that Bacillus spp. isolated from
maize have potential as biocontrol agents. The isolates
clearly produce chitinases with antifungal properties
and suppress the activity of F. moniliforme.

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