1615

Journal of Food Protection, Vol. 70, No.7, 2007, Pages 1615-1621

Isolation of Maize Soil and Rhizosphere Bacteria with

Antagonistic Activity against Aspergillus flavus and
Fusarium verticillioides
JEFFREY D. PALUMBO,u TERESA L. O'KEEFFE,1

AND

HAMED K. ABBAS2

IPlant Mycotoxin Research Unit, U.S. Department of Agriculture, Agricultural Research Service, Albany, California 94710; and 2Crop Genetics and
Production Research Unit, U.S. Department of Agriculture, Agricultural Research Service, Stoneville, Mississippi 38776, USA
MS 06-613: Received 30 November 2006/Accepted 10 March 2007

ABSTRACT
Bacterial isolates from Mississippi maize field soil and maize rhizosphere samples were evaluated for their potential as
biological control agents against Aspergillus fiavus and Fusarium verticillioides. Isolated strains were screened for antagonistic
activities in liquid coculture against A. fiavus and on agar media against A. fiavus and F. verticillioides. We identified 221
strains that inhibited growth of both fungi. These bacteria were further differentiated by their production of extracellular
enzymes that hydrolyzed chitin and yeast cell walls and by production of antifungal metabolites. Based on molecular and
nutritional identification of the bacterial strains, the most prevalent genera isolated from rhizosphere samples were Burkholderia
and Pseudomonas, and the most prevalent genera isolated from nonrhizosphere soil were Pseudomonas and Bacillus. Less
prevalent genera included Stenotrophomonas, Agrobacterium, Variovorax, Wautersia, and several genera of coryneform and
enteric bacteria. In quantitative coculture assays, strains of P. chlororaphis and P. fiuOl:escens consistently inhibited growth
of A. fiavus and F. verticillioides in different media. These results demonstrate the potential for developing individual biocontrol
agents for simultaneous control of the mycotoxigenic A. fiavus and F. verticillioides.

Aspergillus fiavus and Fusarium verticillioides are

widespread inhabitants of agricultural soils associated with
many crops, including maize (com, Zea mays L.),'and are
two of several fungal species that cause ear rot of maize
(50). In addition to the economic losses caused by disease,
further losses may occur due to contamination of maize
with aflatoxins produced by A. fiavus and fumonisins produced by F. verticillioides (4, 43). These losses affect growers, processors, and livestock producers and are a threat to
human and animal health (16).
Current methods used to reduce aflatoxin and fumonisin contamination of maize include modification of cultural
practices to reduce preharvest and postharvest fungal infection (34) and introduction of resistance traits into agronomically valuable maize cultivars by conventional breeding (1, 32, 34) and molecular (19, 34) techniques. Introduction of a Bacillus thuringiensis Bt endotoxin transgene
into maize for insect resistance indirectly reduces mycotoxin contamination of maize (6, 48). In contrast, the use of
chemical pesticides in some cases has resulted in the increase in Fusarium and Aspergillus mycotoxins (17).
Several biological control methods for using fungi and
bacteria to limit mycotoxin contamination in maize have
been developed, such as use of nonaflatoxigenic A. fiavus
strains to displace aflatoxigenic strains (2, 11, 18), control
of A. fiavus by antagonistic strains of Kluyveromyces (30),
and control of F. verticillioides by endophytic and rhizosphere strains of Bacillus (5, 12). Wicklow et al. (51) found

* Author

for correspondence. Tel: 510-559-5876; Fax: 510-559-5777;

E-mail: palumbo@pw.usda.gov.~

that the fungal endophyte Acremonium zeae produced pyrrocidine antibiotics that were active against both A. fiavus
and F. verticillioides. In most of these cases, the goal of
mycotoxin reduction is met by limiting or delaying growth
of toxigenic fungi.
In this study, we hypothesized that single bacterial
strains with activity against both A. fiavus and F. verticillioides could be isolated from the maize environment. Using individual strains for biocontrol of multiple targets
would facilitate management of preharvest mycotoxin contamination of maize. Bacterial populations from maize soil
and rhizosphere samples were screened for phenotypes antagonistic against A. fiavus and F. verticillioides. Quantitative coculture assays in liquid media were used to determine the extent of the antifungal activity of these bacteria
and to predict the potential for their successful application
as biocontrol agents in the field.
MATERIALS AND METHODS
Sampling and isolation of bacteria. Soil and maize samples
were collected from maize fields in Stoneville and Elizabeth,
Miss. In November 2004 and April 2005, a total of 40 soil samples were taken from nine field plots. In May 2005, four maize
plants were collected from each of five separate field plots, for a
total of 20 samples. Bacteria were isolated from soil samples by
adding 1 g of soil to 10 m1 of sterile 0.05% Tween 80 solution,
vortexing for 30 s, sonicating on ice for 15 min in a Branson
model 3210 sonication bath, and vortexing for an additional 30 s.
To isolate maize rhizosphere bacteria, rOots were aseptically removed from each plant sample by cutting at the crown with a
sterilized razor blade. Each root sample was added to 50 ml of
sterile 0.05% Tween 80 in a l-qt (0.96-liter) polyethylene Ziploc

III

'II
1,1

,
1616

J. Food Prot., Vol. 70, No.7

PALUMBO ET AL.

bag. Bacteria were removed from root and soil surfaces by agitating the contents of each bag for 1 min to dislodge soil particles
from roots and sonicating on ice for 15 min. Sonicated soil and
root samples were serially diluted in 0.05% Tween 80 and spread
plated onto 0.1 X tryptic soy agar (TSA; Difco, Becton Dickinson,
Sparks, Md.) containing 100 mg liter- 1 cycloheximide. Plates
were incubated at 28C for 2 days, and individual colonies from
each sample were randomly selected to screen for antifungal phenotypes.
Screening for antifungal activity. Bacterial strains were initially screened for antifungal activity against the nor mutant A.
flavus strain Papa 827 (40) in potato dextrose broth (PDB; Difco,
Becton Dickinson). Individual bacteria were inoculated into single
wells of a 24-well microtiter plate with 1 ml of PDB containing
~ 103 A. flavus Papa 827 conidia and incubated at 28C for 7 days
without shaking. Antifungal activity was assessed visually by
comparing fungal growth in wells treated with bacteria with
growth in control wells containing only A. flavus Papa 827. Bacterial strains that inhibited A. flavus growth were screened further
for activity against A. flavus Papa 827 and the fumonisin-producing F. verticillioides strain 935 on potato dextrose agar (PDA;
Difco, Becton Dickinson). Plates were inoculated in the center
with 10 fLl of an A. flavus or F. verticillioides conidial suspension
(~10 5 conidia ml- I), and bacterial strains were inoculated around
the periphery of the plate, 3 cm from the conidia. Screen plates
were incubated for 7 days at 28C and assessed visually for antifungal activity.
Production of hydrolytic enzymes. Bacterial strains were
tested for extracellular hydrolysis of chitin and yeast cell walls in
defined minimal agar 813C and 813Y, respectively, as previously
described (39). Colloidal chitin was prepared from crab shell chitin (Sigma-Aldrich, St. Louis, Mo.) as previously described (31).
Yeast cell walls were prepared from autoclaved baker's yeast (Saccharomyces cerevisiae) as previously described (39). Chitin and
yeast cell walls were added to medium 813C and 813Y, respectively, at a concentration of 1% (wtlvol). Bacteria were spot inoculated and incubated at 28C for up to 5 days. Enzyme activity
that hydrolyzed chitin or yeast cell walls was measured qualitatively by the production of visible clearing zones surrounding the
bacterial colonies, typically > 1 mm beyond the edge of the colony.
Bacterial identification. Total DNA from bacteria was prepared from single colonies grown on O.5X TSA using the
InstaGene matrix (Bio-Rad, Hercules, Calif.) according to the
manufacturer's instructions. The 16S rRNA gene fragments were
amplified in PCRs using 1 to 5 fLl of each cell suspension as
template and primers 27F (5'-AGAGTTTGATCMTGGCTC
AG-3') and 1525R (5'-AAGGAGGTGWTCCARCC-3') (29).
PCR conditions were 95C for 5 min; 35 cycles of 94C for 30
s, 55C for 30 s, and noc for 90 s; and noc for 5 min. Amplification was confirmed by electrophoretic separation of the fragments on a 0.8% agarose gel, and amplified fragments were purified using QIAquick PCR purification columns (Qiagen, Inc.,
Valencia, Calif.). Purified fragments were sent to Davis Sequencing, Inc. (Davis, Calif.) for sequencing using the same primers.
Bacteria were identified based on similarities of sequences to homologous 16S rRNA gene sequences from bacterial strains designated as type strains in the Ribosomal Database Project database
(15).

For identification using the Biolog Microbial Identification

System (Biolog, Inc., Hayward, Calif.), bacteria were first separated into gram-positive or gram-negative strains using KOH (20).

Gram-positive strains were identified using GP2 MicroPlates

(Biolog) and gram-negative strains were identified using GN2
MicroPlates (Biolog), according to the manufacturer's instructions. Identification was based on the similarity index of carbon
source utilization by each strain relative to that of identified reference strains in the Biolog GP and GN databases.
Quantification of antifungal activity. A subset of 20 bacterial strains showing antifungal activity in preliminary screens
was tested for antagonism against A. flavus strain F34 and F.
verticillioides strain 935. A. flavus F34 is an afiatoxigenic S strain
that was isolated from almond orchard soil (7). Assays were performed in PDB and in maize kernel broth (CKB), which was
prepared by homogenizing frozen sweet maize kernels in distilled
water (100 g liter-I), removing solids by centrifugation at 16,000
X g for 15 min, and autoclaving. Bacteria were grown overnight
in 0.5X tryptic soy broth (Difco, Becton Dickinson) at 28C. Ten
microliters of each culture (~10 8 CFU ml- I) and ~ 102 A. flavus
F34 or F. verticillioides 935 conidia were added to 10 ml of PDB
or CKB in 100-mm-diameter petri dishes. Following incubation
at 28C for 7 days without shaking, fungal mycelium was recovered by filtration onto Whatman no. 4 paper filters and dried overnight. Fungal dry weights from six replicate samples (three replicates per treatment performed in two independent experiments)
were recorded for each combination of A. flavus or F. verticillioides + bacterial strain in each medium. Bacterial inhibition of
fungal growth was quantified relative to control cultures of fungi
grown for 7 days without bacteria in each medium. Student's t
test was used to determine the significance of differences between
control and treated samples.

RESULTS
Isolation and screening of bacterial antagonists.
Bacteria were isolated from maize field soil samples that
were taken after the 2004 growing season and at the beginning of the 2005 growing season and from maize rhizosphere samples taken approximately 4 weeks after planting in 2005. Isolated colonies from each sample were selected randomly and screened for fungal growth inhibition
against A. flavus Papa 827. This strain contains a mutation
in the nor gene, resulting in accumulation of norsolorinic
acid, a bright red-orange precursor in the aflatoxin biosynthetic pathway. Use of this strain allowed inhibition of fungal growth to be easily visualized in liquid coculture with
bacteria in multiwell culture plates. Bacterial strains that
showed inhibition of A. flavus growth in PDB coculture
screens were screened for antifungal phenotypes on PDA.
Of 1,018 strains screened, 443 strains had inhibitory activity against A. flavus, and 221 of those strains had inhibitory
activity against both A. flavus and F. verticillioides (Table
1).

Two types of fungal growth inhibition were observed

when screens were performed on agar media: (i) bacterial
inhibition of fungal growth in a visibly clear zone surrounding the bacterial colony or (ii) inhibition of fungal growth
upon contact of the mycelium with the bacterial colony.
Some bacterial strains also inhibited F. verticillioides aerial
mycelial growth, apart from lateral mycelial growth (data
not shown). Fifty-six strains produced a zone of fungal
growth inhibition beyond the edge of the bacterial colony,
suggesting the production of one or more diffusible anti-

J. Food Prot., Vol. 70, No.7

TABLE 1. Incidence and distribution of bacterial strains with

antifungal phenotypes
No. of strains with
activity against:

Source

Total
a

TABLE 3. Characteristics of bacterial strains selected for use in

quantitative antagonism assays

No. of active
strains producing:

Strain
no.

No. of
A. flavus Antifungal Chitin YCW
strains
A.
and F.
metabo- hydro- hydrolysis lysisa
screened flavus verticillioides lite(s)

Soil
Maize
rhizosphere

208

79

14

17

37

418

235

142

42

50

44

1,018

443

221

56

67

81

JP2170
JP1065
JP1203

YCW, yeast cell walls.

JPll07

microbial compounds. Antagonistic bacterial strains were

assayed for the production of extracellular hydrolyzing activity against chitin and yeast cell walls. Sixty-seven strains
produced clearing zones on colloidal chitin agar, and 81
strains produced clearing zones on yeast cell wall agar. Hydrolysis of yeast cell walls was generally interpreted as 13glucanase activity, because the S. cerevisiae cell wall is
composed predominantly of 13-1,3 and 13-1,6 glucans. The
number of strains with an antifungal phenotype is summarized in Table 1.

Classification and distribution of antagonistic bacteria. Bacterial strains were identified by a combination of
16S rRNA gene sequence analysis and the Biolog Microbial Identification System. The accuracy of each method is
limited by the diversity and accurate identification of the
bacterial species in each reference database. Therefore, only
16S rRNA gene sequences of type strains present in the
16S Ribosomal Database Project database were used for
classification. Because the species diversity in the Biolog
databases is limited, the carbon metabolism-based Biolog
microplate system was used to confirm presumptive classification to at least genus.
The major genera and bacterial groups identified from
soil and rhizosphere samples are listed in Table 2. The most
prevalent genera found in the soil samples were Pseudomonas and Bacillus and several coryneform genera, includTABLE 2. Frequency of isolation of major genera with antifun-

gal activity
No. of strains isolated from:
Soil

Rhizosphere

Achromobacter
Agrobacterium
Bacillus
Burkholderia
Chryseobacterium
Pseudomonas
Wautersia
Stenotrophomonas
Variovorax

0
0
10
1
2
14
4
2
1

Coryneform bacteria
Enteric bacteria

13

2
5
4
45
0
15
1
8
6
4
7

Species

JP2200 Achromobacter
JP2157

600

Genus or group

1617

BACTERIAL ANTAGONISM OF ASPERGILLUS AND FUSARIUM

JP1206
JP2163
JP2003
JP2047
JP2112
JP1015
JP1047
JP2034
JP2175
JP1035
JP2022
JP1026

JP2076
JP2093

xylosoxidans
Agrobaeterium
tumefaciens
A. tumefaciens
Arthrobacter nicotinovorans
A. histidinolovorans
Bacillus licheniformis
B. licheniformis
B. cereus/thuringiensis
Burkholderia cepacia
B. cepacia
B. cepacia
Pseudomonas
chlororaphis
P. chlororaphis
P. fluorescens
P. fluorescens
Wautersia eutropha
W. eutropha
Stenotrophomonas maltophilia
S. maltophilia
Variovorax paradoxus

Source

Antifungal Hydrolysis of:

metabolite(s)a Chitinb YCWc

Rhizosphere
Rhizosphere
Rhizosphere
Soil

Soil

Soil

Soil
Rhizosphere

Rhizosphere

Rhizosphere
Rhizosphere
Soil

Soil
Rhizosphere
Rhizosphere
Soil
Rhizosphere
Soil

Rhizosphere
Rhizosphere

+
+

+
+
+

+
+
+

+
+
+

Production of metabolites inhibitory to A. flavus and/or F. verticillioides. +, production of growth inhibition zone on PDA;

-, no zone of growth inhibition on PDA.

Chitin hydrolysis on 1% colloidal chitin agar. +, production of
visible clearing zone; -, no visible clearing zone.
C YCW, yeast cell wall. Hydrolysis on 1 % YCW agar. +, production of visible clearing zone; -, no visible clearing zone.
b

ing Arthrobacter, Brevibacterium, Corynebacterium, and

Microbacterium. In rhizosphere samples, Burkholderia was
clearly the predominant genus isolated, followed by Pseudomonas and Stenotrophomonas. Although coryneform
genera were less prevalent in the rhizosphere, enteric genera, including strains of Enterobacter, Serratia, and Pantoea, were more prevalent. Other genera isolated from soil
and rhizosphere samples in lower numbers included strains
of Agrobacterium, Wautersia, Variovorax, and Chryseobacterium.

Quantification of fungal growth inhibition. The 20

bacterial strains listed in Table 3 were selected for quantitative analysis of antifungal activity, based on their activity
against A. flavus Papa 827 and F. verticillioides in agar and
liquid media and their production of diffusible antimicro-

1618

J. Food Prot., Vol. 70, No.7

PALUMBO ET AL.

140

ec
0

120
100

'0

C
:E

Ol

"ijj

S
(ij

80
60

------~---

I--

40

Ol

:::>
LL

20
0

I T I Ii
I1m I", I

II

1.IUI.1iI

.4 Ilmlm

:r

1m

ill
o

..,0::

120

ec

100

0
0

'0

80

,g

:E

60

Ol

"ijj

40

(ij
Ol

:::>
LL

20
0

ii

!III~H

*m

:T

lili

J IldU11 J;nJJ illl

1
'"'"
.~.,

FIGURE 1. Growth of fungi in coculture with antagonistic bacteria. Bacterial strain numbers refer to the list in Table 3. (A)
Inhibition of A. flavus strain F34 (1II) and F. verticillioides strain
935 (D) in PDB. Fungal weights are relative to untreated control
cultures, where 100% = 26.7 mg of A. flavus or 30.6 mg ofF.
verticillioides. (B) Inhibition of A. flavus strain F34 (1II) and F.
verticillioides strain 935 (D) in eKB. Fungal weights are relative
to untreated. control cultures, where 100% = 26.0 mg of A. flavus
or 33.5 mg ofF. verticillioides. Error bars represent the standard
error of six replicate samples per treatment.

bial compounds and hydrolytic enzymes. Strains of the

same species that had different phenotypes were chosen to
determine whether strain differences would result in quantitatively distinct antifungal activity. The effect on fungal
growth of coculturing these strains with A. fiavus F34 (an
aflatoxin-producing field isolate) or F. verticillioides 935
was measured in PDB and CKB (Fig. 1). CKB was used
as a surrogate medium in place of intact maize to facilitate
measurements of fungal biomass and generally supported
the same level of growth of both fungi as did PDB on a
dry-weight basis.
Several of the strains tested significantly inhibited
growth of both fungi (P < 0.01) regardless of the medium
used (Fig. 1). All three Burkholderia cepacia strains reduced growth of A. fiavus by 74 to 79% in PDB and 75 to
89% in CKB and reduced growth of F. verticillioides by
61 to 87% in PDB and 73 to 89% in CKB. Pseudomonas
fiuorescens strains IP2034 and IP2175 and Pseudomonas
chlororaphis strain IP1015 reduced A. fiavus growth by 86
to 91 % and 69 to 96% and reduced F. verticillioides growth
by 83 to 89% and 54 to 91 % in PDB and CKB, respectively. Both strains of Stenotrophomonas maltophilia inhibited A. fiavus (83 to 92% reduction) and F. verticillioides

(83 to 93% reduction) in PDB but were less effective in

CKB (34 to 49% reduction of A. fiavus and 33 to 34%
reduction of F. verticillioides).
Other strains that were tested significantly inhibited
both fungi (P < 0.01) in only one test medium. For example, in CKB, A. fiavus and F. verticillioides were inhibited by Agrobacterium tumefaciens strain IP2170 (89 and
84% reduction, respectively), Arthrobacter nicotinovorans
strain IPlO65 (49 and 84% reduction, respectively), Bacillus strains IP1lO7 and JP2163 (74 to 75% and 60 to 63%
reductions, respectively), P. chlororaphis strain IP1047 (69
and 54% reduction, respectively), and Variovorax paradoxus strain IP2093 (74 and 44% reduction, respectively).
None of these strains produced significant fungal inhibition
in PDB (Fig. 1). Achromobacter xylosoxidans strain IP2200
inhibited A. fiavus by 84% and F. verticillioides by 84% in
PDB and inhibited A. fiavus by 73% in CKB but did not
inhibit F. verticillioides in CKB.
When individual strains of each bacterial genus were
compared, there was no consistent correlation between phenotypes or sources and antifungal activity. For example,
Bacillus licheniformis strain IPll07, which produces an antifungal metabolite, did not have more activity (quantitatively) than did B. licheniformis strain JP1206, which does
not produce an antifungal metabolite. B. licheniformis strain
IP1lO7 and Bacillus cereus/thuringiensis strain IP2163 differed in their production of chitinase activity but were not
significantly different (P > 0.05) in their quantitative antifungal activity. Strains of B. cepacia that differed in their
hydrolyzing activity against yeast cell walls did not differ
significantly in their antifungal activity, but B. cepacia
strain IP2112, which does not produce an antifungal metabolite, tended to be less inhibitory to the fungi than were
the B. cepacia strains that produced the antifungal metabolite. P. chlororaphis strain JPlO15, which showed hydrolyzing activity against yeast cell walls, performed better
than P. chlororaphis strain IPlO47, a nonactive strain, in
both media against both fungi, although the differences
were significant only for A. fiavus in PDB (P < 0.01).
Strain source (rhizosphere or bulk soil) and quantitative antifungal activities were not correlated among Wautersia eutropha and S. maltophilia strains.
DISCUSSION

We isolated and screened bacteria from maize soil and

rhizosphere samples to increase the probability of isolating
bacteria for use as biological control agents against A. fiavus and F. verticillioides in the maize environment. Using
the target environment as a source for biocontro1 organisms
resulted in the isolation of agents specifically adapted to
that environment and therefore more likely to be effective
(26). This approach has been used in several studies for
selection of biocontro1 agents, including selection of bacteria from yam farm soil for control of postharvest yam rot
(37), isolation of yeasts from grape for control of Aspergillus species on grape (10), isolation of groundnut-associated bacteria for control of groundnut collar rot disease
(27), and screening of canola-associated bacteria for control
of blackleg disease of canola (44). Native bacterial popu-

J. Food Prot., Vol. 70, No.7

BACTERIAL ANTAGONISM OF ASPERGILLUS AND FUSARIUM

lations also have been investigated as antagonists to A. flavus on almond (38), cotton (33), and maize (36) and to
Fusarium species on a variety of crops, including wheat
(45), banana (3), onion (46), and maize (13).
In the present study, we isolated bacteria with antifungal activities from all soil and rhizosphere samples collected, indicating that antagonistic phenotypes in these environments are not uncommon. Screening sequentially for antagonism against A. flavus and F. verticillioides allowed
identification of single bacterial strains with the potential to
control both pathogens. The majority of these strains were
isolated from the maize rhizosphere (Table 1), where the
total microbial population includes a diversity of saprophytic and pathogenic fungi. The specific antifungal activities we screened for-production of antifungal metabolites
and chitinase and l3-glucanase activity-were also more
prevalent in rhizosphere strains (Table 1), in which they
could contribute to ecological fitness in the rhizosphere.
Broad-spectrum antifungal activity that may be ecologically
advantageous also may be advantageous for use in biological control. For example, Lysobacter enzymogenes has biocontrol activity against Fusarium (24), Pythium (35),
Aphanomyces (23), Bipolaris (52), and Magnaporthe (28)
as a result of its production of multiple degradative enzymes and antimicrobial factors.
Identification of the antagonistic bacterial strains revealed that the most prevalent genus recovered from rhizosphere samples was Burkholderia (Table 2). These Burkholderia strains were identified by their 16S rRNA gene
sequences as B. cepacia, B. stabilis, B. vietnamiensis, B.
multivorans, and B. pyrrocinia, which are all members of
the B. cepacia complex (Bcc) (42). The presence of F. verticillioides increases Bcc populations in the maize rhizosphere (9). The same phenomenon may be occurring in the
Mississippi maize fields we tested, which probably have
background populations of indigenous F. verticillioides.
Bcc bacteria are resident rhizosphere bacteria in many plant
systems and have been investigated in the past for biocontrol applications against several soilborne fungal plant pathogens. However, Bcc bacteria also have been associated
with clinical infections, which has led to restrictions on the
use of these bacteria for plant disease control (41). The B.
cepacia strains that were tested quantitatively for antifungal
activity (Table 3) performed well against both A. flavus and
F. verticillioides (Fig. 1), but because of limitations on the
use of Bcc bacteria under field conditions, further investigation of these strains will be confined to the laboratory.
All Stenotrophomonas strains isolated in this study were
identified as S. maltophilia. Although the strains that were
assayed quantitatively for their antifungal activity (Table 3)
performed relatively well (Fig. 1), they are of limited usefulness in field applications because of the potential of S.
maitophilia to cause human disease and the difficulty in
differentiating environmental and clinical strains (8).
Although Bacillus and Pseudomonas were the predominant genera isolated from nonrhizosphere soil, relatively
few antagonistic Bacillus strains were isolated in this study
(Table 2). In contrast, in a previous study Bacillus was the
most prevalent genus with antifungal activity isolated from

1619

almond fruits, representing 54 of 126 isolates (38). Although Bacillus might be more likely than other genera to
persist in the harsh phyllosphere environment, there would
be no such selective pressure in agricultural soil given the
relative abundance of nutrients and moisture, especially in
the rhizosphere. Bacillus subtilis from maize has shown potential as a biocontrol agent against F. verticillioides in
greenhouse and in vitro studies (12). In our study, the Bacillus strains selected for the quantitative antifungal assays
(Table 3) significantly inhibited A. flavus and F. verticillioides growth in CKE but not in PDB (Fig. 1). Whether
activity in one medium or the other is more relevant to field
conditions, use of several media to demonstrate consistent
activity in vitro may be a better method for predicting field
efficacy. Bacteria screened for antifungal activity in this
study were inoculated at much higher concentrations than
were the fungal spores. Further quantitative studies using
progressively smaller bacterial populations will be useful
for determining the thresholds of antifungal efficacy in laboratory coculture and in situ.
The most promising bacteria isolated in this study for
potential development as biocontrol agents were Pseudomonas species. Of the Pseudomonas strains that were tested
for quantitative activity (Table 3), two strains consistently
inhibited A. flavus and F. verticillioides growth in both media (Fig. 1). Strain IPlO15, identified as P. chlororaphis,
and strain IP2l75, identified as P. fluorescens, produce antifungal metabolites and exhibited chitinase activity. P.
chlororaphis IPlOl5 also exhibited l3-glucanase activity
(Table 3). One distinguishing characteristic of P. chlororaphis is the production of phenazine-l-carboxamide
(PCN), which can be visualized on certain media as a green
crystalline precipitate and we observed in media with strain
IPlO15. PCN is essential for the biocontrol activity of P.
chlororaphis against tomato root rot caused by Fusarium
oxysporum (14). Other antifungal metabolites produced by
Pseudomonas species include phenazine-l-carboxylic acid
(47), 1,3-diacetylphloroglucinol (25), pyrrolnitrin (21), pyoluteorin (22), and hydrogen cyanide (49). Identification of
the specific antifungal metabolites produced by the Pseudomonas strains described in this study will aid in determining the mechanisms involved in their activity against A.
flavus and F. verticillioides and the. relative importance of
these mechanisms in the interaction of the bacteria with
each fungus. In situ studies will be essential for determining
the efficacy of selected Pseudomonas strains for controlling
A. flavus and F. verticillioides and thus reducing aflatoxin
and fumonisin contamination under field conditions.
ACKNOWLEDGMENTS
We thank Sui-Sheng T. Hua for providing A. flavus strain Papa 827
and James L. Baker, Bobbie J. Johnson, and Lorna M. Law for technical
assistance. This work was supported by U.S. Department of Agriculture
Agricultural Research Service CRIS project 5325-42000-036-00D.

REFERENCES
1.

Abbas, H. K., W. P. Williams, G. L. Windham, H. C. Pringle III, W.

Xie, and W. T. Shier. 2002. Aflatoxin and fumonisin contamination
of commercial corn (Zea mays) hybrids in Mississippi. J. Agric.
Food Chern. 50:5246-5254.

1620
2.

3.

4.

5.

6.

7.
8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

PALUMBO ET AL.

Abbas, H. K, R. M. Zablotowicz, H. A. Bruns, and C. A Abel.

2006. Biocontrol of aflatoxin in com by inoculation with non-aflatoxigenic Aspergillus flavus isolates. Biocontrol Sci. Technol. 16:
437-449.
Ayyadurai, N., P. Ravindra Naik, M. Sreehari Rao, R. Sunish Kumar,
S. K Samrat, M. Manohar, and N. Sakthivel. 2006. Isolation and
characterization of a novel banana rhizosphere bacterium as fungal
antagonist and microbial adjuvant in micropropagation of banana. J.
Appl. Microbiol. 100:926-937.
Bacon, C. W., and P. E. Nelson. 1994. Fumonisin production in com
by toxigenic strains of Fusarium moniliforme and Fusarium proliferatum. J. Food Prot. 57:514-521.
Bacon, C. W, I. E. Yates, D. M. Hinton, and E Meredith. 2001.
Biological control of Fusarium moniliforme in maize. Environ.
Health Perspect. 109(Suppl. 2):325-332.
Bakan, B., D. Melcion, D. Richard-Molard, and B. Cahagnier. 2002.
Fungal growth and Fusarium mycotoxin content in isogenic traditional maize and genetically modified maize grown in France and
Spain. J. Agric. Food Chem. 50:728-731.
Bayman, P., and J. Baker. Unpublished data.
Berg, G., N. Roskot, and K Smalla. 1999. Genotypic and phenotypic
relationships between clinical and environmental isolates of Stenotrophomonas maltophilia. J. Clin. Microbiol. 37:3594-3600.
Bevivino, A, V. Peggion, L. Chiarini, S. Tabacchioni, C. Cantale,
and C. Dalmastri. 2005. Effect of Fusarium verticillioides on maizeroot-associated Burkholderia cenocepacia populations. Res. MicrobioI. 156:974-983.
Bleve, G., E Grieco, G. Cozzi, A Logrieco, and A Visconti. 2006.
Isolation of epiphytic yeasts with potential for biocontrol of Aspergillus carbonarius and A. niger on grape. Int. J. Food Microbiol.
108:204-209.
Brown, R. L., P. J. Cotty, and T. E. Cleveland. 1991. Reduction in
aflatoxin content of maize by atoxigenic strains of Aspergillus flavus.
J. Food Prot. 54:623-626.
Cavaglieri, L., 1. Orlando, M. 1. Rodriguez, S. Chulze, and M. Etcheverry. 2005. Biocontrol of Bacillus subtilis against Fusarium verticillioides in vitro and at the maize root level. Res. Microbiol. 156:
748-754.
Cavaglieri, L., A Passone, and M. Etcheverry. 2004. Screening procedures for selecting rhizobacteria with biocontrol effects upon Fusarium verticillioides growth and fumonisin Bl production. Res. Microbiol. 155:747-754.
Chin-A-Woeng, T E C., G. V. Bloemberg, A J. van der Bij, K M.
G. M. van der Drift, 1. Schripsema, B. Kroon, R. J. Scheffer, C.
Keel, P. A. H. M. Bakker, H.-V. Tichy, E J. de Bruijn, J. E. ThomasOates, and B. J. J. Lugtenberg. 1998. Biocontrol by phenazine-lcarboxamide-producing Pseudomonas chlororaphis PCL1391 of tomato root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici. Mol. Plant Microbe Interact. 11:1069-1077.
Cole, J. R., B. Chai, R. 1. Farris, Q. Wang, S. A. Kulam, D. M.
McGarrell, G. M. Garrity, and J. M. Tiedje. 2005. The Ribosomal
Database Project (RDP-II): sequences and tools for high-throughput
rRNA analysis. Nucleic Acids Res. 33:D294-D296. Available at:
http://rdp.cme.msu.edu/index.jsp. Accessed November 2006.
Council for Agricultural Science and Technology. 2003. Mycotoxins:
risks in plant, animal, and human systems. Council for Agricultural
Science and Technology, Ames, Iowa.
D'Mello, J. P. E, A. M. C. Macdonald, D. Postel, W T P. Dijksma,
A Dujardin, and C. M. Placinta. 1998. Pesticide use and mycotoxin
production in Fusarium and Aspergillus phytopathogens. Eur. J.
Plant Pathol. 104:741-751.
Dorner, J. W, R. 1. Cole, and D. T Wicklow. 1999. Aflatoxin reduction in corn through field application of competitive fungi. J.
Food Prot. 62:650-656.
Duvick, J. 2001. Prospects for reducing fumonisin contamination of
maize through genetic modification. Environ. Health Perspect.
109(Suppl. 2):337-342.
Gregersen, T 1978. Rapid method for distinction of gram-negative
from gram-positive bacteria. Eur. J. Appl. Microbiol. Biotechnol. 5:
123-127.

J. Food Prot., Vol. 70, No.7

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.
32.

33.
34.
35.

36.

37.

38.

39.

40.
41.
42.

Howell, C. R., and R. D. Stipanovic. 1979. Control of Rhizoctonia

solani on cotton seedlings with Pseudomonas fluorescens and with
an antibiotic produced by the bacterium. Phytopathology 69:480482.
Howell, C. R., and R. D. Stipanovic. 1980. Suppression of Pythium
ultimum induced damping off of cotton seedlings by Pseudomonas
fluorescens and its antibiotic, pyoluteorin. Phytopathology 70:712715.
Islam, M. T., Y. Hashidoko, A. Deora, T. Ito, and S. Tahara. 2005.
Suppression of damping-off disease in host plants by the rhizoplane
bacterium Lysobacter sp. strain SB-K88 is linked to plant colonization and antibiosis against soilborne Peronosporomycetes. Appl.
Environ. Microbiol. 71:3786-3796.
Jochum, C. C., L. E. Osborne, and G. Y. Yuen. 2006. Fusarium head
blight biological control with Lysobacter enzymogenes strain C3.
BioI. Control 39:336-344.
Keel; C., U. Schnider, M. Maurhofer, C. Voisard, J. Laville, U. Burger, P. Wirthner, D. Haas, and G. Defago. 1992. Suppression of root
diseases by Pseudomonas fluorescens CHAO: importance of the bacterial secondary metabolite 2,4-diacetylphloroglucinol. Mol. plant
Microbe Interact. 5:4-13.
Kerry, B. R. 2000. Rhizosphere interactions and the exploitation of
microbial agents for the biological control of plant-parasitic nematodes. Annu. Rev. Phytopathol. 38:423-441.
Kishore, G. K, S. Pande, and A R. Podile. 2005. Biological control
of collar rot disease with broad-spectrum antifungal bacteria associated with groundnut. Can. J. Microbiol. 51:123-132.
Kobayashi, D. Y, and G. Y Yuen. 2005. The role of clp-regulated
factors in antagonism against Magnaporthe poae and biological control of summer patch disease of Kentucky bluegrass by Lysobacter
enzymogenes C3. Can. J. Microbiol. 51:719-723.
Lane, D. J. 1991. 16S/23S rRNA sequencing, p. 115-175. In E.
Stackebrandt and M. Goodfellow (ed.), Nucleic acid techniques in
bacterial systematics. John Wiley & Sons, New York.
La Penna, M., A Nesci, and M. Etcheverry. 2004. In vitro studies
on the potential for biological control on Aspergillus section Flavi
by Kluyveromyces spp. Lett. Appl. Microbiol. 38:257-264.
Lingappa, Y, and J. L. Lockwood. 1962. Chitin media for selective
isolation and culture of Actinomycetes. Phytopathology 52:317-323.
Menkir, A, R. L. Brown, R. Bandyopadhyay, Z. Y Chen, and T E.
Cleveland. 2006. A USA-Africa collaborative strategy for identifying, characterizing, and developing maize gerrnplasm with resistance
to aflatoxin contamination. Mycopathologia 162:225-232.
Misaghi, I. J., P. J. Cotty, and D. M. Decianne. 1995. Bacterial antagonists of Aspergillus flavus. Biocontrol Sci. Technol. 5:387-392.
Munkvold, G. P. 2003. Cultural and genetic approaches to managing
mycotoxins in maize. Annu. Rev. Phytopathol. 41:99-116.
Nakayama, T, Y. Homrna, Y Hashidoko, J. Mizutani, and S. Tahara.
1999. Possible role of xanthobaccins produced by Stenotrophomonas
sp. strain SB-K88 in suppression of sugar beet damping-off disease.
Appl. Environ. Microbiol. 65:4334-4339.
Nesci, A. v., R. V. Bluma, and M. G. Etcheverry. 2005. In vitro
selection of maize rhizobacteria to study potential biological control
of Aspergillus section Flavi and aflatoxin production. Eur. J. Plant
Pathol. 113:159-171.
Okigbo, R. N. 2005. Biological control of postharvest fungal rot of
yam (Diascarea spp.) with Bacillus subtilis. Mycapathalogia 159:
307-314.
Palumbo, J. D., J. L. Baker, and N. E. Mahoney. 2006. Isolation of
bacterial antagonists of Aspergillus flavus from almonds. Microb.
Col. 52:45-52.
Palumbo, J. D., R. E Sullivan, and D. Y Kobayashi. 2003. Molecular
characterization and expression in Escherichia coli of three 13-1,3glucanase genes from Lysobacter enzymagenes strain N4-7. J. Bacterial. 185:4362-4370.
Papa, K E. 1984. Genetics of Aspergillus flavus: linkage of aflatoxin
mutants. Can. J. Microbial. 30:68-73.
Parke, J. L. 2000. Burkhalderia cepacia: friend or foe? Plant Health
Instr. DOl: 10.1094/PHI-I-2000-0926-01.
Parke, J. L., and D. Gurian-Sherman. 2001. Diversity of the Burk-

J. Food Prot., Vol. 70, No.7

43.
44.

45.

46.

47.

BACTERIAL ANTAGONISM OF ASPERGILLUS AND FUSARIUM

holderia cepacia complex and implications for risk assessment of

biological control strains. Annu. Rev. Phytopathol. 39:225-258.
Payne, G. A. 1992. Aflatoxin in maize. Crit. Rev. Plant Sci. 10:423440.
Ramarathnam, R., and D. W. G. Fernando. 2006. Preliminary phenotypic and molecular screening for potential bacterial biocontrol
agents of Leptosphaeria maculans, the blackleg pathogen of canola.
Biocontrol Sci. Technol. 16:567-582.
Schisler, D. A., N. 1. Khan, and M. J. Boehm. 2002. Biological
control of Fusarium head blight of wheat and deoxynivalenollevels
in grain via use of microbial antagonists. Adv. Exp. Med. Bioi. 504:
53-69.
Sharifi Tehrani, A., and M. Ramezani. 2003. Biological control of
Fusarium oxysporum, the causal agent of onion wilt by antagonistic
bacteria. Commun. Agric. Appl. Bioi. Sci. 68:543-547.
Thomashow, L. S., and D. M. Weller. 1988. Role of phenazine an-

48.
49.

50.
51.

52.

1621

tibotic from Pseudomonas fluorescens in biological control of Gaeumannomyces graminis var. tritici. J. Bacteriol. 170:3499-3508.
Tubajika, K. M., and K. E. Damann. 2001. Sources of resistance to
aflatoxin production in maize. J. Agric. Food Chern. 49:2652-2656.
Voisard, C., C. Keel, D. Haas, and G. Defago. 1989. Cyanide production by Pseudomonas fluorescens helps suppress black root rot
of tobacco under gnotobiotic conditions. EMBO J. 8:351-358.
White, D. G. (ed.). 1999. Compendium of com diseases, 3rd ed.
American Phytopathological Society Press, St Paul, Minn.
Wicklow, D. T., S. Roth, S. T. Deyrup, and J. B. Gloer. 2005. A
protective endophyte of maize: Acremonium zeae antibiotics inhibitory to Aspergillus flavus and Fusarium verticillioides. Mycol. Res.
109:610-618.
Zhang, Z., and G. Y. Yuen. 1999. Biological control of Bipolaris
sorokiniana on tall fescue by Stenotrophomonas maltophilia strain
C3. Phytopathology 89:817-822.