Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Growth-inhibiting effects of concentrations of fusaric acid

on the growth of Bacillus mojavensis and other biocontrol
Bacillus species
C.W. Bacon1, D.M. Hinton1 and A. Hinton Jr2
1 Toxicology & Mycotoxin Research Unit, ARS, USDA, Russell Research Center, Athens, GA 30604, USA
2 Poultry Processing & Meat Quality Research Unit, ARS, USDA, Russell Research Center, Athens, GA 30604, USA

Keywords Abstract
Bacillus amyloliquefaciens, Bacillus
atrophaeus, Bacillus licheniformis, Bacillus Aims: To determine the effects of concentrations of fusaric acid on the growth
mojavensis, Bacillus subtilis, Bacillus of several strains of the biocontrol bacterial endophyte Bacillus mojavensis and
vallismortis, biological control, bacterial other species within the Bacillus subtilis group, as well as the genetic relation-
endophyte, fumonisin, fusaric acid, Fusarium ships within this small group of Gram-positive bacteria, and their antagonisms
verticillioides, mycotoxin, Paenibacillus
to Fusarium verticillioides, which produce fusaric acid.
lentimorbus, Paenibacillus popilliae, Zea mays.
Methods and Results: The growth of 50 Bacillus strains and species were tested
Correspondence at two concentrations of fusaric acid determined in maize infected by an isolate
C.W. Bacon, USDA, ARS, Russell Research of F. verticillioides. Molecular characterizations of the strains and species of
Center, P.O. Box 5677, 950 College Station bacteria were determined with an automated ribotyper. The growth of bacteria
Road, Athens, GA 30604, USA. measured under both concentrations with an automated turbidometer, Bio-
E-mail: cbacon@saa.ars.usda.gov screen, indicated that fusaric acid was toxic to most strains of the bacterial
endophyte B. mojavensis. However, the effects of these two concentrations on
2005/0853: received 22 July 2004, revised 30
April 2005 and accepted 1 May 2005
other Bacillus species varied in that fusaric acid was either bacteriocidal or bac-
teriostatic to most species.
doi:10.1111/j.1365-2672.2005.02770.x Conclusions: These data indicate that the concentrations of fusaric acid are
inhibitory to the growth of most Bacillus species, some of which are used as
biocontrol agents. This suggests that the endophytic and saprophytic states of
F. verticillioides and other Fusarium species cannot be controlled by fusaric-
acid-sensitive Bacillus species.
Significance and Impact of Study: Mycotoxic Fusarium species, such as F. vert-
icillioides, are competitive because all produce fusaric acid, which is inhibitory
to biocontrol bacteria, and mutants tolerant to fusaric acid must be developed
in order to be effective on biocontrol bacteria.

the bacterium Bacillus mojavensis was shown to be endo-

Introduction
phytic and capable of serving as a biocontrol for the end-
Endophytic and epiphytic species of Bacillus have been ophytic state of F. verticillioides and for reducing
shown to have potential for reducing pathogenicity and fumonisin accumulation in maize (Bacon and Hinton
in planta growth and mycotoxin accumulation by Fusa- 2001, 2002). However, this species and 11 other Fusarium
rium species (Hallmann et al. 1997; Chanway 1998; Sturz species known to date have been shown to produce fusa-
et al. 2000, Bacon et al. 2001). An important endophytic ric acid (5-butylpicolinic acid) (Backmann 1956; Luz
species is Fusarium verticillioides, which produces the et al. 1990; Bacon et al. 1996; Capasso et al. 1996), a
fumonisin mycotoxins in maize, and is associated with toxin implicated in the wilt of tomato (Yabuta et al.
equine leucoencephalomalacia, pulmonary oedema syn- 1937; Gaumann 1957) and diseases of other plants (Yab-
drome in swine, liver cancer in rats and human oesopha- uta et al. 1937; Tamari and Kaji 1954; Gaumann 1957;
geal cancer (Riley et al. 1993; Marasas 2001). Recently, Davis 1969; Drysdale 1984). Fusaric acid, a common

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology 100 (2006) 185–194
No claim to original US government works 185
Fusaric acid inhibits Bacillus species C.W. Bacon et al.

contaminant of feedstuffs (Smith and Sousadias 1993; teria were stored as silica gel cultures at )20
C, and cul-
Porter et al. 1995), is also mildly toxic to mice (Yabuta tures were maintained on nutrient agar (Difco
et al. 1937; Porter et al. 1995), and has several important Laboratories). The medium used to study the effects of
pharmacological properties (Porter et al. 1995). fusaric acid on the growth of the bacteria was J-broth
In addition to its toxicity to animals, fusaric acid is (Gordon et al. 1973), distilled water: tryptone 5.0 g l)1,
toxic to several micro-organisms, as well as plant physio- yeast extract 15.0 g l)1, K2HPO 3.0 g l)1, glucose
logical processes (Maloy and Nunn 1981; Luz et al. 1990; 2.0 g l)1, pH 7.5. Inoculum of each strain was prepared
Marrèet al. 1993). It has been shown to inhibit growth from overnight cultures grown in J-broth.
and metabolism of several strains of soil-borne species of Growth rate experiments were performed with an auto-
Pseudomonas fluorescens, especially biocontrol isolates. In mated turbidometer, the Microbiology Bioscreen C
this species, fusaric acid was shown specifically to sup- Reader (Labsystems, Helsinki, Finland), in 100-well sterile
press the production of the antibiotic 2,4-diacetylphlorog- microplates. Each well contained 190 ll of J-broth, to
lucinol, which serves to control the growth of most fungi which was added 10 ll of bacterial inoculum (103 CFU
and is responsible for the biocontrol activity of soil-borne per ml) in J-broth. The cultures were incubated at 30
C
Paenibacillus fluorescens strains (Notz et al. 2002). Addi- with constant shaking and the OD600 measured at 30-min
tional studies have demonstrated the widespread but intervals over the incubation period of 48 h. The concen-
varying sensitivity of Paenibacillus fluorescens, Bacillus spe- trations of fusaric acid (Sigma Chemical) used to measure
cies and Paenibacillus macerans to fusaric acid (Schnider- the in vitro toxicity, 100 and 200 lg ml)1, were based on
Keel et al. 2000; Landa et al. 2002; Notz et al. 2002). data of the patented isolate of B. mojavensis (Bacon et al.
Bacillus mojavensis is closely related to Bacillus subtilis, 2004), which showed growth was inhibited by 30–90% of
from which it and several other species were recently the controls. The concentrations of fusaric acid for other
erected (Nakamura 1989; Roberts et al. 1994, 1996; toxicity tests were measured at the average in planta con-
Nakamura et al. 1999; Takami et al. 2000). The finding centrations of fusaric acid determined in this work.
that B. mojavensis and other closely related species within Growth curves and several other plot parameters were
the B. subtilis group can only be distinguished by differ- generated and analysed by the software package of the
ences in their DNA sequences, and that there are very lit- Bioscreen C Reader (research express, version 1.00).
tle phenotypic differences among the group (Roberts The specific rates of growth were expressed as a percent-
et al. 1994, 1996; Nakamura et al. 1999) suggest the uni- age of reduction over controls that contained no fusaric
versal sensitivities of Bacillus species to fusaric acid, which acid. Sensitivity of fusaric acid was rated at the
prompted this study. The universal production of fusaric 100-lg ml)1 concentration as tolerant, moderately toler-
acid by F. verticillioides (Bacon et al. 1996) and other Fus- ant or intolerant depending on the percentage of fusaric
arium species suggests that biocontrol of these fungi by B. acid inhibition of 0–40, 41–80 and 81–100, respectively.
mojavensis and other Bacillus species might be reduced Strains that were not sensitive at this concentration were
considerably, provided toxic concentrations of fusaric acid rated insensitive. All experiments were performed in trip-
are encountered. However, in vitro tests for antagonism licate, and were repeated independently.
to F. verticillioides by Bacillus species and the sensitivities
of the Bacillus species to fusaric acid are unknown. Thus,
Fungal culture and antagonism assay
the first objective was to examine the effects of fusaric
acid on the growth of the available strains of the endo- The strains of F. verticillioides, RRC 408 and MRC 826,
phytic species B. mojavensis, to distinguish and determine used to infect the maize cultivar P3167, are two of several
if there are any relationships at the molecular level to the strains of this fungus that infect maize and remain as a
desert origins of the strains. The second objective of this symptomless endophyte on this cultivar and produce the
study was to determine if the closely related species of fumonisin mycotoxins and fusaric acid (3Æ00 ppm) on
Bacillus, some of which are intended for use in various autoclaved corn and in planta (Bacon and Hinton 1996b;
biocontrol strategies, are affected by fusaric acid. Bacon et al. 2001). Another strain used was a UV mutant,
RRC 28-5, developed from RRC 408, which produces
only trace amounts of fusaric acid (0Æ09 ppm) on auto-
Materials and methods
claved corn and is referred to as fusaric acid-less mutant
in this study. The fungi were grown on potato dextrose
Bacterial strains and culture conditions
agar (PDA) medium for 7–14 days at room temperature.
The patented biocontrol B. mojavensis strain RRC 101 The assay for growth antagonism of strains and species
(ATCC 55732) and the other bacterial species used in this of bacteria to cultures of F. verticillioides MRC 826 was
study and their sources are presented in Table 1. The bac- determined with an agar diffusion assay on Petri plates of

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C.W. Bacon et al. Fusaric acid inhibits Bacillus species

Table 1 The origin of Bacillus species, strains, and relatives used in this study

Species Strain number* Source

Bacillus mojavensis ATCC 51516 Soil, Mojave Desert, CA, USA

ATCC 51517 Gobi Desert, People’s Republic of China, NRRL B-14708
NRRL B-14698T F.M. Cohan, soil, Mojave Desert, CA, USA
NRRL B-14699 F.M. Cohan, soil, Mojave Desert, CA, USA
NRRL B-14700 F.M. Cohan, soil, Mojave, CA, USA
NRRL B-14701 F.M. Cohan, soil, Mojave, CA, USA
NRRL B-14702 F.M. Cohan, soil, Mojave, CA, USA
NRRL B-14703 M.S. Roberts, soil, Gobi Desert, People’s Republic of China
NRRL B-14704 F.M. Cohan, soil, Gobi Desert, People’s Republic of China
NRRL B-14705 F.M. Cohan, Gobi Desert soil, People’s Republic of China
NRRL B-14706 M.S. Roberts, Gobi Desert soil, People’s Republic of China
NRRL B-14707 F.M. Cohan, Gobi Desert soil, People’s Republic of China
NRRL B-14708 F.M. Cohan, Gobi Desert soil, People’s Republic of China
NRRL B-14709 M.S. Roberts, Gobi Desert soil, People’s Republic of China
NRRL B-14710 F.M. Cohan, Gobi Desert soil, People’s Republic of China
NRRL B-14711 F.M. Cohan, Gobi Desert soil, People’s Republic of China
NRRL B-14712 F.M. Cohan, Gobi Desert soil, People’s Republic of China
NRRL B-14713 M.S. Roberts, Gobi Desert soil, People’s Republic of China
NRRL B-14714 M.S. Roberts, Sahara Desert soil, Nefta, Tunisia
NRRL B-14715 M.S. Roberts, Sahara Desert soil, Nefta, Tunisia
NRRL B-14716 M.S. Roberts, Sahara Desert soil, Nefta, Tunisia
NRRL B-14718 F.M. Cohan, Sahara Desert soil, Nefta, Tunisia
NRRL B-14719 M.S. Roberts, Sahara Desert soil, Nefta, Tunisia
NRRL B-14817 K.E. Duncan, soil, Tumamoc Hill, AZ, USA
NRRL B-14818 K.E. Duncan, soil, Tumamoc Hill, AZ, USA
NRRL B-14824 M.S. Roberts, soil, Sahara Desert, Nefta, Tunisia
RRC 101 (ATCC 55732) Maize kernels, northern Italy
RRC 112 Rifampicin mutant of RRC 101
Bacillus subtilis ATCC 6051T Type for the species, Marbury strain, H.J. Conn
BD170 D. Pubnau (transformation host)
RRC 111 BD170 transformed with B. mojavensis RRC 101DNA
RRC 113 UV mutant 12 of RRC 111
RRC 114 UV mutant 50 of RRC 111
ATCC 6633 N.R. Smith
ATCC 33608 D. Dubnau (BD170)
ATCC 49343 K.F. Anderson (S.N. McDermott)
ATCC 55422 Sandy soil, Japan
ATCC 55614 Soil, fungal antagonist (patented)
B. subtilis ATCC 55675 Transport enhancer (patented)
Bacillus amyloliquefaciens NRRL B-14393T Soil, J. Fukomoto as strain F
Bacillus atrophaeus NRRL NRS-213T Colorado soil, N.R. Smith
Bacillus licheniformis NRRL NRS-1264T Soil, R.E. Gordon
Paenibacillus popilliae NRRL B-2309T Anti-Japanese beetle spore dust
Paenibacillus lentimorbus NRRL B-2522T Diseased Japanese beetle grub
Bacillus vallismortis NRRL B-14890 Sand dune with mesquite tree, Death Valley
NRRL B-14891 Alluvial fan dune with holly bush, Death Valley
NRRL B-14892 Sand dune with mesquite tree
NRRL B-14893 Alluvial fan with creosote bush, Death Valley
NRRL B-14894 Alluvial fan arroyo with holly bush, Death Valley

*NRRL, Northern Regional Research Laboratory culture collection, Peoria, IL, USA; ATCC, American Type Culture Collection, Rockville, MD, USA;
RRC, Russell Research culture collection, Athens, GA, USA.

All B. mojavensis, B. vallismortis and B. amyloliquefaciens strains are indicated here according to Roberts et al. (1994, 1996) and Nakamura (1989).

nutrient agar (Difco Inc.) as the base agar. This assay was here as the diffusible inhibitor. Bacterial inocula and F.
based on the production of an inhibitor as the major verticillioides plug were each placed on the opposite edge
characteristic of the patented isolate, which is referred to of a Petri dish. The fungal inocula were added to the

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology 100 (2006) 185–194
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Fusaric acid inhibits Bacillus species C.W. Bacon et al.

plates 5 days after the bacterial inoculum, and plates were ChemStation (Agilent Technologies, Palo Alto, CA,
incubated at 25–27
C. The widths of cleared zones of ant- USA) that was operated by mis, version 4.5 software.
agonism (distances between the bacterial and fungal The mis software identified and quantified FAMEs of
growth) were measured after 21 days and classified as bacterial isolates and compared the FAME profiles with
previously described (Bacon and Hinton 2002): ), the TSBA 40 MIS computer library to which the type
<0Æ10 mm; +, <3 mm; ++, 3–9 mm; +++, >9–18 mm; strain B. mojavensis, NRRL B-14698 (ATCC 51516), the
++++, > 18–20 mm; +++++, >20 mm. biocontrol strain RRC 101 and other known isolates of
the B. subtilis group were added. Fatty acid analyses were
determined on the 24 strains of B. mojavensis, the closely
Ribotyping and fatty acid analysis
related strains including two strains of B. subtilis, as well
Molecular characterization and identification of B. moja- as on the type strains of Bacillus amyloliquefaciens, Bacil-
vensis and Bacillus species were performed with an auto- lus licheniformis, and Bacillus atrophaeus. Isolates were
mated ribotyping instrument (RiboPrinterTM Microbial identified as B. mojavensis when their FAME profiles
Characterization System, Qualicon/Dupont, Wilmington, matched the profile of the type and the degree of the
DE, USA), which used DNA fragments cut with the match between the unknown isolates, and the data for
restriction enzyme EcoRI prior to hybridization to a the isolate entered in the MIS library were indicated by
cloned chemiluminescently labelled rDNA probe. Bacterial the Similarity Index value assigned to unknowns. Similar-
strains were cultivated overnight in J-broth, harvested by ity Index values may range from 0Æ00 (no match) to 1Æ00
centrifugation (10 000 g) for 10 min and re-suspended in (identical match).
phosphate buffered saline, proceeding according to the
microbial characterization system ribotyping protocol.
Results
Ribotype banding profiles were developed and numerical
analyses and cluster analyses were carried out using Band pattern analysis using the restriction enzyme EcoRI
appropriate software available to the program. indicated that all B. mojavensis isolates grouped into two
Confirmation of the diversity of bacteria by the ribo- large clusters with one major conserved region similar to
typing procedure was performed with the fatty acid B. subtilis, but that these clusters were distinct and differ-
methyl esters (FAME) procedure. The fatty acid profiles ent from the other closely related reference species
of B. mojavensis isolates, along with B. subtilis strains, (Fig. 1). Ribotyping after EcoRI restriction showed that all
were performed using the MIDI Sherlock Microbial strains shared a faint band that was characteristic of the
Identification System (MIS) (MIDI Inc., Newark, DE, genus, with the exception of B. lichensformis. All strains
USA). Strains were transferred from nutrient agar to of B. mojavensis shared a band, although faint, which was
Difco tryptic soy broth (TSBA) (Becton, Dickson, and absent in the other species. However, this band was
Co., Sparks, MD, USA) supplemented with 1.5% of shared by B. subtilis and B. atrophaeus but was greatly
Difco Agar (Becton, Dickson, and Co.) and incubated reduced in the mutant strains of B. mojavensis RRC 111
at 28
C for 24 h. Following three consecutive transfer UV12. The ribotyping procedure reduced the 14 strains
and incubation periods on TSBA, each culture was har- of B. mojavensis into six ribotype clusters and separated
vested, and fatty acids were extracted from the cellular this species from the other closely related species within
membranes using a four-step extraction procedure. The the B. subtilis group (Fig. 1). The data indicated that this
first step consisted of saponification by heating the cells procedure, although not intended, is apparently useful for
at 100
C for 30 min in a methanolic sodium hydroxide strain typing of this species within the B. subtilis group.
solution composed of 15% (w/v) NaOH, dissolved in The ribotype profiles did not distinguish the strains of B.
50% (v/v) methanol. The second step consisted of mojavensis into any pattern characteristic of and unique
methylating the saponified cellular fatty acids in a solu- to the strains isolated from the four major desert regions
tion of 3Æ25 N hydrochloric acid in methanol (46% of the world (Fig. 1).
v/v) at 80
C for 10 min. During the third step, FAME The patented biocontrol endophytic strain, RRC101, its
analyses were then extracted from the aqueous phase mutants 111 and UV12, all formed a unique subcluster
following a 10-min liquid–liquid extraction with a 1 : 1 consisting of 13 bands that were distinctly different from
(v/v) solution of hexane and methyl-tert-butyl ether. the other isolates, except for strain NRRL B-14818 which
The resulting FAME extracts were washed for 5 min in also clustered within this subgroup indicating a close gen-
a sodium hydroxide solution (1Æ08%, w/v). During the etic relationship to RRC101. However, strain RRC101 ori-
fourth step, the FAME extracts were transferred to ginated from corn kernels obtained from northern Italy,
sample vials for identification and quantification by an whereas NRRL B-14818 originated from a desert soil sam-
Agilent 6890 gas chromatograph (GC) and computer ple from Arizona, USA. Strains in this subcluster showed

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C.W. Bacon et al. Fusaric acid inhibits Bacillus species

Ribotypes Strains Species Origin

% similarity
40 50 60 70 80 100

NRS1264 Bacillus licheniformis

NRRLB-14824 Bacillus mojavensis S

NRRLB-14700 Bacillus mojavensis M

ATCC51517 Bacillus mojavensis G

NRRLB-14698 Bacillus mojavensis M

NRRLB-14707 Bacillus mojavensis G

NRRLB-14711 Bacillus mojavensis G

NRRLB-14709 Bacillus mojavensis G

ATCC51516 Bacillus mojavensis M

RRC111,UV12 Bacillus mojavensis I

RRC111 Bacillus mojavensis I

NRRLB-14818 Bacillus mojavensis AZ

RRC101 Bacillus mojavensis I

NRRLB-14714 Bacillus mojavensis S

NRRLB-14817 Bacillus mojavensis S

ATCC6051 Bacillus subtilis

BD170 Bacillus subtilis

ATCC6051-2 Bacillus subtilis

NRRLB-14393 Bacillus amyloliquefaciens

NRS-213 Bacillus atrophaeus

Figure 1 Phylogenetic dendrogram and ribotype patterns obtained with EcoRI restriction enzyme of 16S rDNA sequences of 14 randomly selec-
ted Bacillus mojavensis strains and other type species of the Bacillus subtilis group. Dendrogram was calculated with Dice coefficient using UPGMA
clustering methods. Origin of strains: S, Sahara Desert; M, Mojave Desert; G, Gobi Desert; I, southern Italy; Az, Arizona.

the most fragmented polymorphisms and subspecies vari- Specific rates of growth of all strains were measured
ation. While we were successful in distinguishing several under the two concentrations. Bacillus mojavensis NRRL
selected strains from B. subtilis using the fatty acids analy- B-14708 was the only strain tolerant to fusaric acid at the
sis, this proved difficult when we analysed the other spe- lower concentration. All strains were sensitive to the
cies within the B. subtilis group (data not shown). higher concentration of fusaric acid; therefore, strains
However, the utility of analysing for specific fatty acids were rated according to their sensitivities at the lower
for species of this group was indicated as the desired pro- concentration. The largest percentage of fusaric-acid-tol-
cedure (Roberts et al. 1994), which was not utilized in erant strains was from the Gobi Desert strains, and strain
our approach but which our data indicate should not be NRRL B-14708 showed natural resistance at the low con-
the definitive analysis of this diverse but relatively small centration, i.e. not sensitive. Only one strain from the
group of species. Mojave Desert was rated moderately tolerant, NRRL B-
The diversity in strain variation is reflected in the phe- 14699. The specific rates of growth data indicated that
notypes of strains of B. mojavensis, which varied in their the remaining species of the B. subtilis group tested were
ability, both quantitatively and qualitatively, to produce also sensitive to the concentrations of fusaric acid
an agar-diffusible inhibitory substance, as well as toler- (Tables 3 and 4). Bacillus subtilis ATCC 55614 was
ances to the lower concentration of fusaric acid (Table 2). the most tolerant strain of this species (Table 3), while

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology 100 (2006) 185–194
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Fusaric acid inhibits Bacillus species C.W. Bacon et al.

Table 2 Inhibitory activity of fusaric acid on the specific rate of Table 3 Inhibitory activity of fusaric acid on the specific rate of
growth of Bacillus mojavensis strains growth of Bacillus subtilis strains and their antagonism to Fusarium
verticillioides
% Fusaric acid
inhibition* % Fusaric acid inhibition*

100 200 Strains 100 lg ml)1 200 lg ml)1 Antagonism

Strains Origin lg ml)1 lg ml)1 Antagonism

ATCC 6051 74a 95a )
ATCC 51516 Mojave 68a 92a ) BD 170 84a 93a )
ATCC 51517 Gobi 61a 88a ++ RRC 111 37b 94a +++
NRRL B-14698T Mojave 92b 93a + RRC 113 57a 77a )
NRRL B-14699 Mojave 21c 91a ) RRC 114 73a 78a ++
NRRL B-14700 Mojave 76a 83a ) ATCC 6633 84a 66b )
NRRL B-14701 Mojave 67a 83a +++ BD170 60a 96a )
NRRL B-14702 Mojave 47b 78a ) ATCC 49343 29c 66b ++
NRRL B-14703 Gobi 91a 94a ) ATCC 55422 53a 69b +++++
NRRL B-14704 Gobi 27c 97a ) ATCC 55614 25c 73a +++++
NRRL B-14705 Gobi 49b 79a ) ATCC 55675 39b 84a )
NRRL B-14706 Gobi 26c 94a )
NRRL B-14707 Gobi 62b 87a ++ *Inhibitory activity expressed as a percentage of the control values.
NRRL B-14708 Gobi 0d 93a ) Values represent the means of six replicate cultures. Means within a
NRRL B-14709 Gobi 68a 92a + column followed by a different letter were significantly different at
NRRL B-14710 Gobi 87b 92a + P £ 0.05 according to Fisher’s protected least significant difference
NRRL B-14711 Gobi 70a 92a ) test.
NRRL B-14712 Gobi 19c 90a )
Zones of antagonism were measured after 21 days following inocula-
NRRL B-14713 Gobi 61a 94a ) tion of plates with F. verticillioides MRC 826; ), <0.10 mm (not
NRRL B-14714 Sahara 64a 83a + antagonistic); +, <3 mm; ++, 3–9 mm; +++, >9–18 mm; ++++, >18–
NRRL B-14715 Sahara 82b 72b ) 20 mm; +++++, >20 mm.
NRRL B-14716 Sahara 77a 89a ++
NRRL B-14718 Sahara 33c 81a ++
NRRL B-14719 Sahara 63a 86a ++
Table 4 Inhibition by fusaric acid to the specific rates of growth of
NRRL B-14817 Sahara 62a 91a +
Bacillus species and their antagonism to Fusarium verticillioides
NRRL B-14818 Sahara 55a 86a +++
NRRL B-14824 Sahara 69a 88a )
% Fusaric acid inhibition*
RRC 101 Italy 59a 83a +++
(ATCC 55732) Species 100 lg ml)1 200 lg ml)1 Antagonism

RRC 112 Mutant of 81b 94b ++
RRC101 Bacillus amyloliquefaciensT 28a 60a C++
Bacillus atrophaeusT 33a 71a ++++
*Percentage of fusaric acid inhibition was determined within a 48-h Bacillus licheniformisT 74b 82a —
incubation period at 30
C. Values represent the means of six replicate Paenibacillus 61b 69a +
cultures. Means within a column followed by a different letter were popilliae B-2309T
significantly different at P £ 0.05 according to Fisher’s protected least Paenibacillus 81b 75a ++
significant difference test. lentimorbus B-2522T

Antagonism determined after 21 days following co-inoculations of Bacillus vallismortis 67b 75a ++
fungi with bacteria on nutrient agar plates; ), <0.10 mm (not antago- B-14890
nistic); +, <3 mm; ++, 3–9 mm; +++, >9–18 mm; ++++, >18– B. vallismortis B-14891 64b 69a ++
20 mm; +++++, >20 mm. B. vallismortis B-14892 0c 13b )
B. vallismortis B-14893 80b 72a ++
B. vallismortis B-14894 57b 70a )
Bacillus vallismortis NRRL B-14892 was the most tolerant
of the remaining species (Table 4). Indeed, this strain was *Inhibitory activity expressed as a percentage of the control values.
tolerant of the higher concentration of fusaric acid, indi- Values represent the means of six replicate cultures. Means within a
cating resistance. However, this strain was not antagonis- column followed by a different letter were significantly different at
tic to F. verticillioides. P £ 0.05 according to Fisher’s protected least significant difference
test.
The antagonistic responses varied according to the

Zones of antagonism were measured after 21 days following inocula-
strain of B. mojavensis as indicated by the zones of inhibi- tion of plates with F. verticillioides MRC 826; ), <0.10 mm (not
tion. Only 44% of the strains produced this diffusible antagonistic); +, <3 mm; ++, 3–9 mm; +++, >9–18 mm; ++++, >18–
substance on the nutrient agar culture medium. However, 20 mm; +++++, >20 mm; C++, contact kill with necrotic hyphae and
all strains were antagonistic because those strains that did an antagonism zone.

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C.W. Bacon et al. Fusaric acid inhibits Bacillus species

not produce a zone of antagonism caused necrotic hyphae The infection of hosts and biosynthesis of fusaric acid
upon contact with the bacterial colony (data not shown). by isolates of Fusarium species in the absence of disease
This suggests either that there are two active inhibitory symptoms (Bacon and Hinton 1996a) suggest that toxic
compounds or that the same inhibitory substance is not concentrations of fusaric acid are not produced in the
membrane permeable in all strains. Several other Bacillus Fusarium-infected plants, fusaric acid is not produced, or
species were antagonistic to the fungus (Tables 3 and 4). modern-day plant cultivars are resistant to the in planta
Indeed, the largest antagonistic responses were those pro- concentrations that are produced. Because all isolates of
duced by two strains of B. subtilis, ATCC 55614 and F. verticillioides examined to date produce fusaric acid
55422, followed by B. atrophaeus NRS-213. Strain ATCC (Bacon et al. 1996), and because most of these isolates
55614 was also very antagonistic to F. verticillioides. How- maintain a symptomless endophytic biotrophic or hemi-
ever, one species did not produce an agar-diffusible sub- biotrophic association with maize, the in planta interac-
stance, but was inhibitory as evidenced by the production tion of fusaric acid might be multifunctional and
of necrotic hyphae upon contact with the fungal colony therefore complex. Further, the biosynthesis of fusaric
(data not shown). acid by soil-borne nonpathogenic isolates of other Fusa-
rium species suggests a role in the interaction with other
soil-borne organisms. The values of fusaric acid used in
Discussion
this study are similar and relevant to those expected to
We have established that all the strains and species of occur in the symplasm. Indeed, the amounts of fusaric
Bacillus tested in this study were sensitive to fusaric acid. acid isolated from natural products are well within the
Several Bacillus species used in this study have been recom- range used in this study (Smith and Sousadias 1993; Por-
mended for use as biocontrol agents; however, our data ter et al. 1995). Because fusaric acid readily chelates sev-
suggest that if fusaric acid is in their environment, their eral ions, which decreases it toxicity (Backmann 1956;
effectiveness may be severely limited. Thus, the numbers of Malini 1966; Bochner et al. 1980; Duffy and Défago
bacteria that are sensitive to fusaric acid, and probably ant- 1997), the interaction of specific metal ions with the final
agonized by species of Fusarium, are larger than previously expression of toxicity should also be considered.
considered. The Fusarium species are a highly successful Recently, studies have shown that fusaric-acid-produ-
group of fungi that produce a variety of secondary meta- cing strains of Fusarium oxysporum altered the biocontrol
bolites, some of which figure into an organism’s competi- activity of a strain Paenobacillus fluorescens by repressing
tion for colonization of plants, especially in homologous the expression of genes involved in the production of the
niches such as soil, phyllosphere and endophytic spaces. In inhibitor 2,4-diacetylphloroglucinol, rendering this bacter-
addition to its arsenal of defensive secondary metabolites ium ineffective in controlling the growth of the fungus
(Thrane 2001), the Fusarium species produce fusaric acid, (Schnider-Keel et al. 2000; Notz et al. 2002). The biocon-
which historically is known as a wilt toxin. trol B. mojavensis RRC101 also produces an inhibitory
However, the toxicity of fusaric acid is wide-acting; it substance in vitro, although it has been neither chemically
is biologically active in micro-organisms, animals and identified nor established as being produced in planta.
plants. This is due to its activity as a lipophilic chelating The use of B. mojavensis as an endophytic biocontrol has
agent (Malini 1966; Maloy and Nunn 1981). Specifically, decreased the accumulation of the fumonisin mycotoxins
in plants this toxin has been shown to act as a weak un- and endophytic infection of maize by F. verticillioides
dissociated acid while in the apoplasm; however, when it under greenhouse conditions (Bacon et al. 2001). Biocon-
passes through the cell membrane and accumulates in the trol of this fungus by B. mojavensis is high when tested
symplasm, it is dissociated and the extent of dissociation against nonproducing fusaric acid mutants of F. verticil-
is Ph dependent (Marrèet al. 1993). In the symplasm it is lioides (Bacon et al. 2004).
known to inhibit mitochondrial respiration (Paquin and The eight Bacillus species used in this study are very clo-
Waygood 1957; Arias 1985), decrease ATP concentration sely related but are easily distinguished by differences in
and destroy membrane integrity (D’Alton and Etherton their DNA sequences, and cannot be distinguished by
1984; Arias 1985). In general, the activity of this toxin in phenotypic characterization in general (Roberts et al. 1994,
plants proceeds first with a nonspecific phase that 1996; Nakamura et al. 1999). Significant differences in the
involves cell membrane penetrations, followed by a sec- levels of ten fatty acids were reported by Roberts et al.
ond phase that involves specific cell targets such as mito- (1994) 1996) for distinguishing the species used in this
chondria (Marrèet al. 1993). Thus, when testing for study. We found better separation using the ribotyper,
activity at the cellular level, it is necessary to test for tox- which is in agreement with Roberts et al. (1994) 1996) and
icity with values that are anticipated to occur in the sym- Nakamura et al. (1999). All strains of B. mojavensis were
plasm (Davis 1969; Bacon et al. 2004). rated as endophytic (Bacon and Hinton 2002), although

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology 100 (2006) 185–194
No claim to original US government works 191
Fusaric acid inhibits Bacillus species C.W. Bacon et al.

not all were tested for in planta protection against infec- are current and widespread in agriculture (Hallmann et al.
tion by F. verticillioides. Because all were endophytic the 1997; Chanway 1998; Kobasyashi and Palumbo 2000; Sturz
relationship between ribotype patterns, geographic origin et al. 2000). Other uses are also anticipated, i.e. surrogate
and the endophytic trait cannot be explored. The 14 ran- transformation and paratransgenesis, two methods in
domly selected strains used for ribotyping were isolated which symbiotic micro-organisms are transformed and
from different sampling locations of the great deserts, but inserted into host plants, which leads to an indirect modifi-
many showed subspecies variation in the ribotypes, as well cation of the plant’s genome and subsequent phenotype
as subspecies variation fragment polymorphisms, inde- resulting in novel methods for designing symbionts, such
pendent of geographic origin. An analysis of the remaining as endophytic bacteria, for control of specific diseases or
13 strains would not alter the geographic pattern observed. enhanced processes. However, the ubiquitous production
Thus, there was no correlation between specific desert ori- of fusaric acid by endophytic Fusarium species, pathogenic
gin, ribotype pattern and fusaric acid sensitivity. Further, and nonpathogenic, raises the questions as a possible
the ribotype groups in the area of subspecies variation impediment to the successful use of endophytic biocontrol
indicated a considerable overlap of genetically diverse bacteria. A similar concern for epiphytic, rhizospheric and
strains and fragment polymorphisms within the world’s phyllospheric biocontrol bacteria has been expressed (Toy-
distribution, suggesting a high degree of genetic plasticity, oda et al. 1988), which together with this present study sug-
which indicates that these isolates are not clones. gests that an important role for fusaric acid is for defence
In addition to the Bacillus species studied in this work, and competition for species of Fusarium, and that fusaric-
Landa et al. (2002) determined that B. circulans and acid-tolerant mutants of endophytic biocontrol bacteria
B. megaterium were susceptible to graded concentrations should be developed.
of fusaric acid, of which 100 and 200 lg/ml showed the
most consistent inhibition as did the present study on B.
Acknowledgements
mojavensis. Several other Pseudomonas and Paenibacillus
species have now been identified as being sensitive to fus- We thank L. Nakamura for his generous support and
aric acid, and it has been shown that two species of Bacil- supply of bacterial strains used in this study. We also
lus were the most sensitive of the three genera used in thank R. Bennett for technical assistance.
their study (Landa et al. 2002). Our research indicates
that there is marked variation within the genus Bacillus
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