Letters in Applied Microbiology 2005, 41, 390–396 doi:10.1111/j.1472-765X.2005.01785.

In vitro influence of bacterial mixtures on Fusarium

verticillioides growth and fumonisin B1 production: effect of
seeds treatment on maize root colonization

L. Cavaglieri1, J. Orlando1 and M. Etcheverry2

1
Departamento de Microbiologı´a e Inmunologı´a, Universidad Nacional de Rı´o Cuarto, Rı´o Cuarto, Córdoba, and 2Member of the
Research Career, Comisión Nacional de Investigaciones Cientı´fica y Técnicas (CONICET), Argentina

2005/0157: received 15 February 2005, revised 24 June 2005 and accepted 27 June 2005

ABSTRACT
L . C A V A G L I E R I , J . O R L A N D O A N D M . E T C H E V E R R Y . 2005.
Aims: Enterobacter cloacae, Microbacterium oleovorans, Pseudomonas solanacearum and Bacillus subtilis were
investigated in order to evaluate: (i) the inoculum size of two bacterial mixtures on Fusarium verticillioides growth
and fumonisin B1 production in vitro at different water activities and (ii) the efficacy of a seed treatment with the
best bacterial mixture on F. verticillioides root colonization in greenhouse studies.
Methods and Results: The influence of bacterial mixtures (1 ¼ E. cloacae and M. oleovorans and 2 ¼
P. solanacearum and B. subtilis) to antagonize 13 F. verticillioides strains at different inoculum concentrations (108,
109 and 1010 cells ml)1) and water activities (0Æ937, 0Æ955 and 0Æ982 aW) were examined. Antibiosis, growth rate and
fumonisin B1 production were determined. Bacterial mixture 1 proved to exert the most effective control. Seed
treatment with mixture 1 at 108 cells ml)1 had the best inhibitory effect on F. verticillioides root colonization.
Conclusions: These results suggest that the combination E. cloacae and M. oleovorans has the potential for the
biological control of F. verticillioides as a maize seed inoculant.
Significance and Impact of the Study: The application of this knowledge contributes to prevent the vertical
transmission of F. verticillioides.

Keywords: bacterial mixtures, biocontrol, Fusarium verticillioides, inoculum size, maize, roots.

These symptomless endophytic fungi of maize and grasses can

INTRODUCTION
Fusarium verticillioides, the most prevalent Fusarium in
freshly harvested maize in Argentina (Chulze et al. 1996), is
the most commonly soil-borne fungal pathogen infecting
maize (Zea mays L.) (Bacon et al. 2001). Fusarium verticil-
lioides produces fumonisins (Nelson et al. 1993), which are
toxins affecting human and animal health, especially
fumonisin B1 (Harrison et al. 1990; Chu and Li 1994).
Microbial endophytes colonize host tissues and establish
long-term associations without doing substantive harm to the
host. Endophytic fungi colonize plants and produce toxins
without any signs that they are intimately associated and the
plants never show signs that they are internally infected.
Correspondence to: Lilia Cavaglieri, Departamento de Microbiologı́a e Inmunologı́a,
Universidad Nacional de Rı́o Cuarto Ruta 36 km, 601 (5800) Rı́o Cuarto, Córdoba,
Argentina (e-mail: lcavaglieri@exa.unrc.edu.ar).
produce toxins that affect the health of humans and domestic
animals when ingested. There are two associations, the tall
fescue–Neotyphodium Glenn, Bacon and Hanlin obligate
association and the maize–F. verticillioides facultative associ-
ation (Bacon et al. 2001; Oren et al. 2003). Several pathways
for F. verticillioides infection have been proposed. The fungus
can infect the kernels or establish a systemic infection where
the fungal conidia or mycelia can be either carried inside the
seeds or on the seed surface. In this case, F. verticillioides
moves from the roots to the stalk and arrives to the kernels
(Oren et al. 2003). Few studies have taken into account seed-
borne and roots colonization by F. verticillioides as the possible
cause of systemic infection that could result in fumonisins
beings present in kernels (Bacon et al. 2001).
Seed treatment with biocontrol agents (BCAs) is one of
the most appropriate methods for biocontrol of soil-borne
ª 2005 The Society for Applied Microbiology
 BACTERIAL MIXTURES ON FUSARIUM VERTICILLIOIDES
plant pathogens in the spermosphere and rhizosphere
(Kerry 2000). Criteria for successful biocontrol are that
the antagonists should be able to colonize and show activity
in the germinating seeds and the developing roots. Different
studies have determined the influence of BCAs to protect
seeds and roots (Lumsden et al. 1995; Steinberg et al. 1999),
but the use of combinations of multiple antagonistic micro-
organisms that might prevent F. verticillioides roots colon-
ization has not been extensively evaluated.
In previous studies we have selected M. oleovorans (initially
identified as Azotobacter armeniacus), E. cloacae (initially
identified as Arthrobacter globiformis), B. subtilis and
P. solanacearum according to exclusionary principles criteria
(Cavaglieri et al. 2004a,b). These bacteria obtained higher
niche overlap indices (NOIs) that were related to their
competitive ability to use carbon sources. NOI estimates the
ecological similarity between strains (Wilson and Lindow
1994) and is inversely correlated with the level of coexistence.
To become established, F. verticillioides needs to compete with
other colonizers including bacterial antagonists. Higher NOIs
represent low coexistence as well as higher competition by
carbon sources. Microbacterium oleovorans, E. cloacae, B. sub-
tilis and P. solanacearum demonstrated to be effective agents to
inhibit F. verticillioides growth and fumonisin B1 production
in vitro when used singly. The selection of appropriate BCAs
able to grow under a wide range of water activities (aW) was a
precondition for obtaining proper biological control. In
addition, BCAs should be capable of pre-emptively excluding
F. verticillioides under field conditions.
In the present study, isolates E. cloacae, M. oleovorans,
P. solanacearum and B. subtilis, were investigated in order to
evaluate (i) the inoculum size of two bacterial antagonistic
mixtures on F. verticillioides growth and fumonisin B1
production in vitro at different aW and (ii) the efficacy of the
seed treatment with the best antagonistic bacterial mixture
on F. verticillioides root colonization in greenhouse studies.
MATERIALS AND METHODS
Micro-organisms
All bacterial and fungal strains were isolated from endo-
rhizosphere zone of maize during the seedling stage
according to Cavaglieri et al. (2004a). A. globiformis
(UNRC5 1265 demonstrated 98Æ7% similarity with E. cloa-
cae 1360; No. 3; Y17665), A. armeniacus (UNRC1 1294
demonstrated 99Æ7% similarity with M. oleovorans 1410;
type strain, DSM 16091; BAS69Type; AJ 698723), B. subtilis
(RCRL 1289 demonstrated 100% similarity with B. subtilis
1413, MO4; AY553097). Enterobacter cloacae, M. oleovorans
and B. subtilis were identified by Macrogen (Pathfinder in
Genomics Research, Macrogen Inc., Seoul, Korea).
Pseudomonas solanacearum identification was performed
ª 2005 The Society for Applied Microbiology, Letters in Applied Microbiology, 41, 390–396, doi:10.1111/j.1472-765X.2005.01785.x
391
according to Bergey’s manual of systematic bacteriology
(Holt 1993).
Thirteen F. verticillioides strains that produce fumonisin
B1 were used (Cavaglieri et al. 2004c). They were classified
according to Nelson et al. (1993).
Bacterial inoculum
The bacteria used to make the mixtures were grouped
according to exclusionary principles criteria assayed previ-
ously (Cavaglieri et al. 2004a). Enterobacter cloacae and
M. oleovorans obtained higher NOIs than P. solanacearum
and B. subtilis, so we grouped the strains with the highest
NOIs as mixture 1 and the strains with the lowest NOIs as
mixture 2. Thus, we made two bacterial mixtures as follows:
two loop full of each bacterium, from 3-day-old cultures on
tryptic soy agar (TSA) were transferred separately to 50 ml
tryptic soy broth (TSB) and incubated overnight at 25
C.
Bacterial cells were centrifuged (4500 g, 15 min) and resus-
pended in 0Æ01% sodium laurylsulphate modified with
glycerol to 0Æ982, 0Æ955 and 0Æ937 aW (Dallyn and Fox
1980). Serial decimal dilutions were done to obtain 108, 109
and 1010 cells ml)1 from each bacterial suspension. The
concentration of cell suspension was determined by using a
haemocytometer.
Mixtures 1 and 2 were then prepared. About 1 ml of
E. cloacae and 1 ml of M. oleovorans suspensions, with the
same concentrations, were mixed into sterile tubes in order
to obtain the mixture 1 at 108, 109 and 1010 cells ml)1.
About 1 ml of B. subtilis and P. solanacearum suspensions,
with the same concentrations, were mixed into sterile
tubes in order to obtain the same concentrations of
mixture 2.
Fusarium inoculum
Each isolate of F. verticillioides was induced to sporulate on
carnation leaf agar (CLA) consisting of 2% agar plus small
pieces of sterile irradiated carnation leaf for 7 days at 25
C.
Conidia were harvested by scraping the mycelium and were
suspended in 0Æ01% sodium laurylsulphate modified with
glycerol as described above. The inoculum size was adjusted
at 109 spores ml)1. About 1 ml of each strain were mixed in
a tube in order to obtain a mixture of 13 strains of
F. erticillioides at 109 spores ml)1.
Assessment of antibiosis
Maize meal extract agar (MMEA) was adjusted at 0Æ982,
0Æ955 and 0Æ937 aWas previously described by Cavaglieri
et al. (2004a). About 10 ll of F. verticillioides mixture
(109 spores ml)1) were inoculated in the centre of each plate
separately. A volume of 10 ll of each bacterial mixture
392 L . C A V A G L I E R I ET AL.
suspensions (109, 1010 and 1011 cells ml)1 separately) were
inoculated around the fungal inocula. Treatments were
incubated at 25
C for up to 10 days in polyethylene bags.
The antibiosis zone was determined by measuring the zone
of inhibition of F. verticillioides growth (mm) and co-
inoculated treatments were compared with controls without
bacteria. This assessment was carried out with at least three
separate replicates per treatment.
Assessment of antifungal activity by bacterial
mixtures upon F. verticillioides
A volume of 100 ll of each bacterial mixture at different
inoculum sizes (108, 109 and 1010 cells ml)1) were inocu-
lated separately in Petri dishes (60 · 10 mm). Before
cooling, 10 ml of MMEA adjusted at different aW (as
previously described) were poured into the Petri dishes
containing the correspondent mixture. Then, 10 ll of
F. verticillioides mixture was inoculated in the centre of
each plate. Controls without fungal inoculum were made.
Cultures were incubated at 25
C for 20 days in polyethy-
lene bags. The diameter of F. verticillioides colonies was
measured daily with a ruler. The growing diameters of
the co-inoculated cultures were compared with the control
F. verticillioides cultures. The experiments were carried
out in triplicate and repeated three times. The slopes of
the regression lines of the linear portions of the radial
extension rates were used for calculating growth rates
(Caims and Magan 2003).
Determination of fumonisin production
All the material from each co-inoculated Petri dishes was
put in 125 ml Erlenmeyer flasks. About 10 ml of aceto-
nitrile : water (1 : 1) was added. The flasks were shaken
overnight and then filtered through Whatman No. 4 filter
paper (Etcheverry et al. 2002). The extracts were frozen at
)20
C until analyses. The diluted extracts were quanti-
tatively determined by a modified HPLC method pro-
posed by Shephard et al. (1990) as modified by Doko
et al. (1995).
Seed bacterization
The mixture E. cloacae and M. oleovorans at 108 cells ml)1
was obtained as previously described. Seventy-five maize
seeds were submerged in 100 ml of bacterial inoculum in
250 ml Erlenmeyer flasks. Control seeds were submerged
in 100 ml of TSB. Flasks were incubated at 25
C on a
rotatory shaker at 70 rev min)1 for 2 h, to allow bacte-
rization rather than adherence. After incubation, excess
inoculum was removed and seeds were immediately
planted for greenhouse studies.
ª 2005 The Society for Applied Microbiology, Letters in Applied Microbiology, 41, 390–396, doi:10.1111/j.1472-765X.2005.01785.x
Greenhouse studies
The mixture E. cloacae and M. oleovorans showed the most
uniform response at all treatments evaluated. It was tested
for the ability to inhibit the F. verticillioides root colonization
as previously described by Weller and Cook (1986) at
108 cells ml)1 (the lower inoculum size) in order to
minimize the ecosystem perturbation.
Plastic tubes 2Æ5 cm in diameter · 16Æ5 cm long were
filled with 2 cm of field maize soil. One treated seed was
placed on top of the soil, covered with 2 cm of sand–
vermiculite (1 : 1, v/v) and water was added to each tube
on alternate days. Nontreated seeds were planted as
checks in each assay. After planting racks of tubes were
covered with aluminium foil, kept in the dark for 5 days
at 20
C, then uncovered and kept at 20
C with a 14 h
photoperiod. After 20 days, all plants were collected and
CFU counts of F. verticillioides strains from rhizoplane
and endorhizosphere levels were made. To obtain bacterial
populations from the maize rhizoplane, roots were gently
washed in phosphate-buffered saline (PBS; Oxoid Ltd,
London, UK) for 2 min to remove the adhering soil, and
then shaken for 5 min in fresh PBS containing 0Æ025%
Tween 20. Threefold dilution of the suspensions was
plated on tryptic soy broth plus 2% agar (TSBA) and
King’s medium B. Plates were incubated for 24–48 h at
28
C. Total counts and counts per colony type were
made. One colony per colony type was isolated and
purified on TSBA. To quantify the endorhizosphere
populations, maize roots were washed as described earlier
and surface-dried between sheets of tissue paper, weighed
and surface-sterilized by gently shaking in 70% ethanol
(1 min), 20% household bleach (5 min) and thiosulphate
Ringer solution (5 min; Oxoid Ltd). Samples were
macerated in 90 ml of PBS with a mortar and pestle.
Threefold dilution was plated on TSBA. Petri plates were
incubated for 24–48 h at 28
C. Total counts and counts
per colony type were made. One colony per colony type
was isolated and purified on TSBA. Five replications were
made for each bacterial mixture. This assay was done
twice. Percentage of inhibited F. verticillioides was deter-
mined as the F. verticillioides CFU counts obtained from
bacterial treatment as a proportion of the total CFU
counts obtained from control without bacterial antago-
nists · 100.
% inhibited F: verticillioides
F: verticillioides CFU counts (bacterial treatment)
¼
Total F: verticillioides CFU counts (control)

100
 BACTERIAL MIXTURES ON FUSARIUM VERTICILLIOIDES 393

a
Statistical analysis 4·0 a
a
Antibiosis and growth rate data analysis were performed by 3·5
b b
ANOVA. Mean values of treatments were compared by 3·0 b
Duncan multiple range test. The antibiosis data were

Antibiosis (mm)
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2·5 c
transformed to mm þ 05: Fumonisin B1 data obtained
were transformed ppm1 þ 1 before applying the analysis of 2·0 c
variance. Afterwards, Scheffe test was used to determine the d
1·5
differences between the control and co-inoculated cultures.
1·0 e e e
Fusarium verticillioides CFU counts were transformed to
log10(x + 1) to obtain the homogeneity of variance. Mean 0·5
values of treatments were compared using Fisher’s protected 0·0
LSD test (Quinn and Keough 2002).
0·18
a
0·16 a
RESULTS b
0·14 bc

Growth rate (mm h–1)

Bacterial mixture 1 (E. cloacae and M. oleovorans) 0·12 c

Figure 1a shows the antibiosis of bacterial mixture 1 on 0·10

F. verticillioides mixture growth at different inoculum sizes 0·08 e
ef
and aW. All factors tested exert a significant fungal growth 0·06 ef
g fg
decrease (P < 0Æ05). All inocula were different with respect 0·04 g
to the control without bacteria at all water activities assayed, g
0·02
but the 108 cells ml)1 dose showed the most uniform
response throughout all the treatments. 0·00
Figure 1b shows the effect of different inoculum sizes and
1·2
aW on F. verticillioides mixtures growth rate. At the highest
aW level examined (0Æ982 aW) the 109 and 108 cells ml)1 1·0
a a a a a

doses exerted a significant decrease on growth rate, where as

Fumonisin B1 (µg g–1)

ab
all inocula exerted a significant decrease on fungal growth 0·8
rate at the aW levels 0Æ955 and 0Æ937.
bc
Mean values of fumonisin B1 production were compared 0·6
to determine the influence of the bacterial mixture 1. The cd cd cd cd
0·4
fumonisin production was not reduced at 0Æ982 and
0Æ937 aW levels, while all inocula had a significant inhibitory
0·2 d
effect at 0Æ955 aW (Fig. 1c).
0·0
0 1010 109 108
Bacterial mixture 2 (P. solanacearum Inoculum size (cell ml–1)
and B. subtilis)
Fig. 1 The effect of bacterial mixture 1 (Enterobacter cloacae and
Figure 2a shows the antibiosis activity of the bacterial
Microbacterium oleovorans) influence on Fusarium verticillioides at
mixture 2 on F. verticillioides mixture. At 0Æ982 aW, the different inoculum sizes and water activities on antibiosis (a),
inocula 109 and 1010 cells ml)1 exert a significant decrease F. verticillioides growth rate (b), lag phase (c) and fumonisin B1
on the fungal mixture, whereas the 108 and 1010 cells ml)1 production (d) when growing alone (cero represents fungi only) and in
diminished the fungal growth at 0Æ955 aW level. At 0Æ937 aW co-culture.
p ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiAntibiosis and fumonisin B1 data were transformed to
the inocula 108 and 109 cells ml)1 demonstrated the most mm þ 05 and to ðlg 1g1 Þ þ 1, respectively. Groups with different
effective antibiosis. letters are significantly different according to Fisher’s least significant
The effect of bacterial mixture 2 on F. verticillioides difference test (P < 0Æ05) for antibiosis, growth rate and lag phase and
mixture growth rate was evaluated (Fig. 2b). At 0Æ982 aW according to Scheffe test (P < 0Æ05) for fumonisin B1 production. n, aw
there was a significant reduction of the fungal growth rate at 0.982; u, aw 0.955; , aw 0.937
1010 cells ml)1 dose. The inocula 109 cells ml)1 exert the
same effect at 0Æ955 aW. The 108 cells ml)1 concentration
was not effective at any of the aW levels assayed.
ª 2005 The Society for Applied Microbiology, Letters in Applied Microbiology, 41, 390–396, doi:10.1111/j.1472-765X.2005.01785.x
394 L . C A V A G L I E R I ET AL.

7 When the mean values of fumonisin B1 production were

a compared, none of the inocula tested were able to reduce the
6 toxin concentrations (Fig. 2c).
5 b
Antibiosis (mm)

b
Greenhouse studies
4
c c
cd Table 1 shows the influence of the mixture 1, which exerted
3
the most consistent in vitro influence, at 108 cells ml)1 on
cde F. verticillioides root colonization. All Fusarium spp. were
2 cde
de isolated and the F. verticillioides percentage was determined
e e e
1 in treatments with antagonists and without bacterial antag-
onists. In the control treatment, the isolation percentages of
0
F. verticillioides ranged between 21% and 18%, respectively,
in the rhizoplane and endorhizosphere zones. In compar-
0·18
b b ison, colonization was inhibited completely when the
0·16 mixture 1 was applied with no F. verticillioides being isolated
bc
0·14 cd from either the rhizoplane or the endorhizosphere.
Growth rate (mm h–1)

d a
0·12
de
0·10 DISCUSSION
0·08 This work reveals that a mixture of rhizobacteria could be
ef
0·06 efg fg used to inhibit the F. verticillioides maize root colonization.
0·04 g The application of this knowledge contributes to prevent the
vertical transmission of F. verticillioides.
0·02 In previous studies the utility of NOI has been demon-
0·00 strated to choose the best mixture of rhizobacteria as
potential BCAs (Cavaglieri et al. 2004a,b). In the present
1·0 study, the E. cloacae and M. oleovorans mixture showed the
a most uniform response to inhibit the in vitro growth and
0·8 fumonisin B1 production by F. verticillioides strains.
Fumonisin B1 (µg g–1)

The inhibition of F. verticillioides root colonization by

0·6 ab ab individual rhizobacterial strains used in this study has
previously been demonstrated (Cavaglieri et al. 2004c). This
ab work shows an advance in the sense to prove the successful
0·4
utilization of rhizobacterial mixtures.
The interactions between the environment, the initial
0·2 concentration of the introduced BCA and the concentra-
bc
c c c c tion of the pathogen will affect the persistence of the
c
0·0 BCA, its ability to colonize the rhizosphere and its
0 1010 109 108 ultimate biocontrol efficacy (Parke 1990). Control of
Inoculum size (cell ml ) –1 F. verticillioides could be achieved at all aW and inoculum
densities assayed in vitro. In previous studies we deter-
Fig. 2 The effect of bacterial mixture 2 (Pseudomonas solanacearum mined that total endorhizosphere and rhizoplane bacterial
and Bacillus subtilis) influence on Fusariun verticillioides at different
populations ranged between 108 and 109 CFU g)1 when
inoculum sizes and water activities on antibiosis (a), F. verticillioides
the maize plant was 15 days old (maize seedling) (Cav-
growth rate (b), lag phase (c) and fumonisin B1 production (d) when
growing alone (cero represents fungi only) andpin co-culture. Antibiosis
aglieri et al. 2004c). Establishment and persistence of high
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi populations of BCAs within the rhizosphere is critical to
and fumonisin B1 data were transformed to mm þ 05 and to
ðlg 1g1 Þ þ 1, respectively. Groups with different letters are significantly achieving effective control of soil-borne diseases (Weller
different according to Fisher’s least significant difference test 1988). Here, the antagonistic inocula at the same level of
(P < 0Æ05) for antibiosis, growth rate and lag phase and according to rhizobacterial populations found in maize roots were
Scheffe test (P < 0Æ05) for fumonisin B1 production. n, aw 0.982; u, aw applied in order to enhance the level and persistence of
0.955; , aw 0.937 rhizosphere colonization. This inoculum size was the
lower one that demonstrated an effective control in vitro
ª 2005 The Society for Applied Microbiology, Letters in Applied Microbiology, 41, 390–396, doi:10.1111/j.1472-765X.2005.01785.x
 BACTERIAL MIXTURES ON FUSARIUM VERTICILLIOIDES
Table 1 Influence of the mixture Microbacterium oleovorans and
Enterobacter cloacae seed bacterization on the percentage of Fusariun
verticillioides maize roots colonization growing alone and in co-culture
C* I

Endo-rhizosphere
44Æ95 ± 2Æ32 0
Rhizoplane
36Æ70 ± 2Æ11 0
*C (control): percentage ± SD of Fusarium verticillioides counts
isolated from maize roots without bacterial treatment.

I (interaction): percentage ± SD of Fusarium verticillioides counts
isolated from maize roots with bacterial treatment at 108 cells ml)1.
F. verticillioides counts were not detected.
and has been used by other authors to evaluate bacterial
antagonists as BCAs (Larkin and Fravel 1998).
In this work E. cloacae and M. oleovorans mixture was able
to totally inhibit F. verticillioides maize roots colonization.
Other authors have reported the effects of roots colonization
by bacteria under controlled conditions (Kerry 2000; Bacon
et al. 2001). At present, the information of the mechanism of
action of bacteria on F. verticillioides is limited. We
presuppose that the bacterial mixture becomes effective by
usurping a high percentage of the resources that would
otherwise be available to the fungal pathogen. Once the
antagonists have colonized the root system, they should be
able to maintain their position by the production of
antibiotics that suppress growth of root-infecting fungi.
In the present study, a strong correlation between antag-
onism recorded in vitro and the effectiveness of the E. cloacae
and M. oleovorans mixture to inhibit F. verticillioides colon-
ization in greenhouse trials was observed. In contrast,
Bevivino et al. (1998) obtained a poor correlation between
in vitro and in greenhouse studies when B. cepacia against
F. moniliforme and F. proliferatum in maize were assayed.
Mixtures of compatible different species of BCAs may
result in better substrate colonization, may be better adapted
to environmental changes, may present different pathogen
suppressive mechanisms and may protect against a broad
range of pathogens (Duffy and Weller 1995).
The E. cloacae and M. oleovorans mix has the potential for
the biological control of F. verticillioides as maize inoculant.
Several areas need improvement if the maximum potential
of mixtures of BCAs is to be attained. Improved formula-
tions and appropriate adjuvant are now under study.
ACKNOWLEDGEMENT
This work was supported by grants from Agencia Nacional
de Promoción Cientı́fica y Tecnológica (ANPCYT) FON-
CYT – PICT 14551 granted from 2005 to 2007.
ª 2005 The Society for Applied Microbiology, Letters in Applied Microbiology, 41, 390–396, doi:10.1111/j.1472-765X.2005.01785.x
395
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