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Mycopathologia (2009) 167:287–295 DOI 10.1007/s11046-008-9177-1

Biological Interactions to Select Biocontrol Agents Against Toxigenic Strains of Aspergillus flavus and Fusarium verticillioides from Maize Miriam G. Etcheverry Æ Andrea Scandolara Æ Andrea Nesci Æ Marta Sofia Vilas Boas Ribeiro Æ Paola Pereira Æ Paola Battilani

Received: 7 May 2008 / Accepted: 19 December 2008 / Published online: 27 February 2009 Ó Springer Science+Business Media B.V. 2009

Abstract Biological control represent an alternative to the use of pesticides in crop protection. A key to progress in biological control to protect maize against Fusarium verticillioides and Aspergillus flavus maize pathogens are, to select in vitro, the best agent to be applied in the field. The aim of this study was to examine the antagonistic activity of bacterial and yeast isolates against F.verticillioides and A. flavus toxigenic strains. The first study showed the impact of Bacillus amyloliquefaciens BA-S13, Microbacterium oleovorans DMS 16091, Enterobacter hormomaechei EM-562T, and Kluyveromyces spp. L14 and L16 isolates on mycelial growth of two strains of A. flavus MPVPA 2092, 2094 and three strains of F. verticillioides MPVPA 285, 289, and 294 on 3% maize meal extract agar at different water activities (0.99, 0.97, 0.95, and 0.93). From this first assay antagonistics

isolates M. oleovorans, B. amyloliquefaciens and Kluyveromyces sp. (L16) produced an increase of lag phase of growth and decreased a growth rate of all fungal strains. These isolates were selected for futher studies. In vitro non-rhizospheric maize soil (centrally and sprayed inoculated) and in vitro maize (ears apex and base inoculated) were treated with antagonistics and pathogenic strains alone in co-inoculated cultures. Bacillus amyloliquefaciens significantly reduced F. verticillioides and A. flavus count in maize soil inoculated centrally. Kluyveromyces sp. L16 reduced F. verticillioides and A. flavus count in maize soil inoculated by spray. Kluyveromyces sp. L16 was the most effective treatment limiting percent infections by F. verticillioides on the maize ears. Keywords Biological interactions Fusarium verticillioides Aspergillus flavus Maize Bacteria Yeast

M. G. Etcheverry A. Nesci P. Pereira CONICET, Buenos Aires, Argentina M. G. Etcheverry (&) A. Nesci P. Pereira Laboratorio de Ecologıá Microbiana, Departamento de Microbiologıá e Inmunologıá, Facultad de Ciencias Exactas Fı´sico Quı´micas y Naturales, Universidad Nacional de Rıó Cuarto, Ruta Nacional 36, km 601, Rıó Cuarto, Cordoba 5800, Argentina e-mail: [email protected] A. Scandolara M. S. Vilas Boas Ribeiro P. Battilani Istituto Di Entomologia e Patologıá Vegetale Della Facolta´ de Agraria, Universita´ Catolica del Sacro Cuore, Via Emilia Parmese 84, Piacenza 29100, Italy

Introduction Maize is the third most important cereal in terms of world yield and can be colonized by many soilborne plant pathogens that reduce the quality and quantity of grain production. This cereal is the host of various fungi that can produce mycotoxins. Among these fungi, Fusarium verticillioides produce toxins associated with harmful effects on animal and human health [1, 2]. Aspergillus flavus has the ability to

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invade maize during maturation in the field and contaminate the crop with aflatoxins. Aflatoxins are toxic secondary metabolites, and toxin-contaminated commodities destined to human or animal consumption, represent a significant health hazard and are, therefore, monitored and regulated [3]. The preharvest contamination with aflatoxigenic fungi is already documented in Italy [4]. Populations of Aspergillus section Flavi and Fusarium section Liseola are present in soil, crop residues, tissue growing plants, and the atmosphere surrounding a maize field [4]. These sources within maize fields may become potential inoculum sources during maize growth and may increase the inoculum level at harvest. Fungal diseases of crops are usually controlled by some cultural practices, fungicide applications, and selection of resistant maize cultivars. The use of fungicides is a controversial practice that entails undesirable environmental effects. A promising strategy to reduce aflatoxin and fumonisin accumulation in maize ears at harvest involved the biological interation among toxigenic fungi and natural biocompetitive agents. The use of bacteria or yeast to control pre- and post-harvest pathogens and pests of agricultural commodities has already been studied [5–8]. In previous in vitro studies, bacteria and yeast isolates were selected to control Aspergillus section Flavi or F. verticillioides [9–13]. These studies were all based on biocontrol agents to control maize pathogens applied in the agroecosystem from which they were originally isolated. However, there are no studies of the effects of selected biocontrol agents on toxigenic fungi from agroecosystem nonnative to them. There is an expectation that any organisms that escape from the protected environment will die out rapidly; however, non-native species may have the potential to control exotic pest and became partially established in a new area, thus, reducing the need for re-releases [6]. Based on the addition of relatively low numbers of non-native bacterial and yeast biocontrol agents to maize ears and nonrhizospheric soil at in vitro trials, the present study tested the ability of maize non-native biocontrol agents to reduce maize and soil colonization by italian fungal toxigenic species and its growth. The first objective of this study was to determine in culture medium the activities of Microbacterium oleovorans, Bacillus amyloliquefaciens, Enterobacter hormomaechei, and Kluyveromyces spp. (L14 and

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L16) on lag phase and growth rate of Aspergillus flavus and F. verticillioides strains. The second objective of this study was to determine the effect of biocontrol agents on count of Aspergillus section Flavi and Fusarium verticillioides strains in non-rhizospheric soil from maize field. And the last objective was to determine the effect of biological interaction on toxigenic fungal count in vitro maize ears.

Materials and Methods First Assessment: Antifungal Activities of Bacterial and Yeast Isolates on Toxigenic Fungi in Maize Meal Extract Agar Fungal Isolates Aspergillus flavus MPVPA 2092 and A. flavus MPVPA 2094 aflatoxins producers were isolated from maize kernels harvested from Italy [4]. These strains were identified according to Pitt and Hocking [14], Klich and Pitt [15]. Fusarium verticillioides MPVP 285, 289, and 294 fumonisins producers were isolates from kernels harvested from Italy (Scandolara et al., personal communication). F. verticillioides strains were identified according to Nelson et al. [16]. Antagonistic Isolates The isolates used were Bacillus amyloliquefaciens isolate 1 (GenBank accession No EU164542), Microbacterium oleovorans isolate 2 (GenBank accession No EU164543), Microbacterium oleovorans isolate 3 (GenBank accession No EU164544), and Enterobacter hormomaechei isolate 4 (GenBank accession No EU164545). These isolates were recovered from maize roots in previous works [10, 11]. The yeasts used belong to the genus Kluyveromyces (L14 and L16). These isolates were recovered from feedstuff in previous work [12]. Bacterial isolates were maintained in 30% glycerol/water and fungal strains in 15% glycerol–water at -80°C. Methodology Bacterial and yeast isolates were screened for their ability to inhibit fungal growth in maize meal extract


agar (MMEA) at four water activities (aw), 0.99, 0.97, 0.95, and 0.93. The culture medium used contained 3% maize meal and 1.5% agar. The aw of the basic medium (0.99) was adjusted to 0.97, 0.95, and 0.93 by the addition of known amounts of the non-ionic solute, glycerol, according to Dallyn and Fox [17]. About 0.1 ml of 109 CFU ml-1 from bacterial and yeast water suspensions adjusted at the same aw of the medium were centrally inoculated and spread over the surfaces of Petri plates with an alcoholflamed glass rod. The concentration of viable cells was measured by standard plate count method using nutrient agar and malt extract agar for bacteria and yeasts, respectively. After inoculum adsorption, each Petri plate was inoculated centrally with an agar disk of 0.5 mm diameter from 7 days sporulated MEA culture of each Aspergillus strain (A. flavus MPVPA 2092, A. flavus MPVPA 2094) and from 7 days sporulated carnation leaf agar culture of each Fusarium verticillioides strain (F. verticillioides MPVP 285, F. verticillioides MPVP 289, F. verticillioides MPVP 294). The plates of the same aw were incubated at 25°C in polyethylene bags. All the experiments were carried out with at least three separate replicate Petri plates per treatment. The colony diameter was daily measured. For each colony, two diameters, measured at right angles to one another, were averaged to find the mean diameters for every colony. All colony diameters were determined by using three replicates for each test interaction. The radial growth rate (mm h-1) was subsequently calculated by regression of the linear phase for growth; the time at which the line intercepted the x-axis was used to calculate the lag phase in relation to fungal strain, bacteria, yeast, and aw. The inhibition of mycelial growth was considered as indication of antagonism. The antagonistic isolates were used for the next experiments. Second Assessment: Biological Interactions in Non-Rhizospheric Soil Inoculum Preparation Bacillus amyloliquefaciens isolate 1 and Microbacterium oleovorans isolate 2 were grown for 24 h in nutrient broth with known amounts of a non-ionic solute, glycerol, in order to obtain inocula adjusted to each aw value. Serial decimal dilutions were done to

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prepare the appropriate inoculum level (109 CFU ml-1). The isolate Kluyveromyces L16 was grown in malt extract agar for 24 h at 25°C. The concentration of cells was measured with a Neubauer chamber and adjusted to 107 CFU ml-1. Aspergillus flavus MPVPA 2092 was induced to sporulate on MEA slanted tubes at 28°C for 7 days. A 5 ml aliquot of sterile water adjusted with glycerol was added to the slants and harvested by vigorous agitation. The spore suspension was prepared with glycerol in order to obtain an inoculum adjusted to each aw value. Serial decimal dilutions were done to prepare the appropriate inoculum level (104 cell ml-1). The concentration of cells was measured with a Neubauer chamber and the viability of cells was confirmed by the standard plate count method using malt extract agar. Fusarium verticillioides MPVP 294 was induced to sporulate on carnation leaf agar at 25°C for 14 days. Five milliliter aliquot of spore suspension was added to the slants and harvested by vigorous agitation. The conidia concentration was estimated. The spore suspension was prepared with water and glycerol in order to obtain an inoculum adjusted to each aw value. Serial decimal dilutions were done to prepare the appropriate inoculum level (104 spores ml-1). The concentration of cells was measured with a Neubauer chamber and the viability of cells was determined by the standard plate count method using potato dextrose agar. Table 1 show different treatments assayed. Inoculation This experiment was carried out on sterile nonrhizospheric soil from maize experimental field from Piacenza, Italy. Sterile soil samples in Petri plates (50 g) were centrally and sprayed inoculated with antagonist and pathogenic strains alone and in co-inoculated cultures. The central inoculation was made adding 5 ml of fungal inoculum. In sprayed inoculation, fungi inoculum was added by irrigation spray, making two applications with 2.6 ml of inoculum suspension. In both cases, toxigenic fungi were added first and after the antagonists by central inoculation. Count of Fungal Inoculated Strains and Biocontrol Agents After 14 days of Interaction Ten grams of non-rhizospheric soil of each treatment (by triplicate) were suspended in 90 ml of 0.1%

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Results

Table 1 Treatments assayed in non-rhizospheric soil Soil

FV

AF

BA

MO

Kl

T1

T2

T3

T4

T5

T6

FV

T7

T8

T9

AF FV ? AF

T10 T13

T11 T14

T12 T15

Soil

4

-1

Inoculum concentration: fungi = 10 CFU ml , bacteria = 109 CFU ml-1, and yeast 107 CFU ml-1 FV, Fusarium verticillioides; AF, Aspergillus flavus; BA, Bacillus amyloliquefaciens; MO, Microbacterium oleovorans; Kl, Kluyveromyces L16

peptone water solution. Serial decimal dilutions were made and 0.1 ml aliquots were inoculated by triplicate on Nash–Snyder Agar [16], dichloran rose bengal agar (DRBC) [14], malt extract agar, and nutrient agar to perform count of Fusarium, Aspergillus, yeast, and bacterial isolates, respectively. Third Assessment: Effect of Biocontrol Agents on Infection Percentage of Toxigenic Fungi In Vitro Maize Ears Forty-five maize ears (Pioneer Lolita FAO 500) of 120 days old were placed in plastic containers with 50 ml of Hogland solution. After that a spray inoculation was made according to the scheme showed in Table 2. The maize ears were incubated for 7 days at 258C in incubation chambers. To determine the infection percentage, 2 9 2 cm pieces of different place of the ear were placed onto the Petri plates culture media, DRBC, Nash–Snyder, DG18, MEA, NA, respectively.

Table 2 Treatments assayed in vitro maize ears

Ears FV

Ears

FV

AF

BA

MO

K1

T1

T2

T3

T4 T7

T5 T8

T6 T9

T10

T11

T12

T14

T15

AF FV ? AF

T13 4

-1

Inoculum concentration: fungi = 10 CFU ml , bacteria = 109 CFU ml-1, and yeast 107 CFU ml-1 FV, Fusarium verticillioides; AF, Aspergillus flavus; BA, Bacillus amyloliquefaciens; MO, Microbacterium oleovorans; Kl, Kluyveromyces L16

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First Assessment: Antifungal Activities of Bacterial and Yeast Isolates on Toxigenic Fungi in Maize Meal Extract Agar Table 3 showed the effect of antagonistics agents on Aspergillus flavus MPVPA 2094 growth rate and lag phase at different aw and 25°C. In untreated controls, similar lag phases were observed for all A. flavus strains at the same aw values (data not shown). The lag phase in the control ranged between 30 and 74 h. The lag phases were increased when aw decreased. All antagonistics agents caused increase of lag phases at aw = 0.99, 0.97, 0.95, and 0.93, respectively. Control cultures of A. flavus MPVPA 2094 grew faster at aw = 0.99 and 0.97 than 0.95 and 0.93 with values ranging from 0.2 to 0.09 mm h-1, respectively. Antagonistics agents treatments decreased the growth rate in percentages of 58, 53, 30, and 74% at aw = 0.99, 0.97, 0.95, and 0.93, respectively. Kluyveromyces L16 caused complete inhibition of A. flavus MPVPA 2094 growth at lower aw values tested. Thus, the potentials biocontrol agents M. oleovorans, B. amyloliquefaciens, and Kluyveromyces sp. L16 inhibited the growth rate and increased the lag phase of Aspergillus strains at aw = 0.99, 0.97, 0.95, and 0.93. The same results such as on Aspergillus were observed on F. verticillioides, a decrease in growth rate and an increase in lag phase for all Fusarium strains assayed. The effects of antagonistics microbial agents on F. verticillioides MPVP 285 on growth rate and lag phase at different water activities and 25°C are shown in Table 4. Antagonistics agents had significant inhibitory effects on lag phase. The mean lag phase in the control increased (45–74 h) between 0.99 and 0.93. The lag phase of F. verticillioides increased when aw decreased. The increase in lag phase were observed at all water activities, for all cultures paired. Untreated controls of F. verticillioides grew faster at 0.99 and 0.97 aw, the values observed were 0.35 and 0.32 mm h-1, respectively. The growth rate decreased (0.30–0.27 mm h-1) at aw = 0.95 and 0.93. The antagonistics treatments decreased the growth rate in percentages 30, 33, and 21% at aw = 0.99, 0.97, and 0.95, respectively.

Mycopathologia (2009) 167:287–295 Table 3 Antagonistics agents’ influence on growth rate and lag phase of Aspergillus flavus MPVPA 2094 at different water activities and 25°C

Ba, B.amyloliquefaciens; Mo, Microbacterium oleovorans; L16, Kluyveromyces spp; [7 days (168 h); mm h-1, range diameter growth value; h, range lag phase; NG, not growth Values are the mean of three replicates. Data with the same letter between control and treatments of the same water activity are not significantly different (P \ 0.05, Tukey test)

291 Growth rate (mm h-1)

Lag phase (h)

Water activity

Treatments

0.99

Aspergillus flavus

0.2a

30a

Ba–A. flavus

0.08b

39b

0.97

0.95

0.93

Mo–A. flavus

0.08b

45c

L16–A. flavus

0.09c

72d

Aspergillus flavus

0.18a

31a

Ba–A. flavus

0.08b

41b

Mo–A. flavus

0.08b

49c

L16–A. flavus Aspergillus flavus

0.09c 0.12a

68d 35a

Ba–A. flavus

0.08b

40b

Mo–A. flavus

0.08b

74c

L16–A. flavus

0.09c

[d

Aspergillus flavus

0.09a

74a

Ba–A. flavus

0.03b

82b

Mo–A. flavus

0.04c

91c

L16–A. flavus

NGc

[d

Second Assessment: Biological Interactions in Non-Rhizospheric Soil Table 5 showed the effect of biological interaction among M. oleovorans, B. amyloliquefaciens, and Kluyveromyces sp. L16 on F. verticillioides and A. flavus populations at non-rhizospheric soil level and the interaction when pathogens and antagonists were inoculated centrally and by spray. The control treatments showed F. verticillioides and A. flavus count of 1 9 104 CFU g-1 of soil. Antagonistics agents populations controls were B. amyloliquefaciens 1.5 9 108, M. oleovorans 1.5 9 103, and Kluyveromyces sp. L16 1 9 108. In treatments centrally inoculated B. amyloliquefaciens (T6) caused complete inhibition of F. verticillioides population in maize soil, while M. oleovorans (T7) increased F. verticillioides and A. flavus populations. Kluyveromyces L16 (T8) reduced the CFU count of both fungi. In treatments inoculated by spray F. verticillioides and A. flavus untreated control showed the same CFU count (1 9 104). Count of antagonistics agents populations alone were 1.5 9 108, 1 9 104, and 1 9 107 for B. amyloliquefaciens, M. oleovorans, and Kluyveromyces sp. L16, respectively (data no shown). Kluyveromyces sp. L16 decreased F. verticillioides population, while B. amyloliquefaciens (T6) and M. oleovorans (T7) stimulated A. flavus population.

Third Assessment: Effect of Biocontrol Agents on Infection Percentage of Toxigenic Fungi In Vitro Maize Ears The effect of biological interaction among B. amyloliquefaciens, M. oleovorans, and Kluyveromyces sp. L16 on F. verticillioides and A. flavus infection percentage at maize ears level is shown in Table 6. Our results showed that Fusarium and Aspergillus native infection percentage was different in base and apex maize ears. Maize ears apex had no native infection percentage of Fusarium and Aspergillus genus. However, maize ears base had natural Fusarium and Aspergillus infection (99% and 25%, respectively). An increase in F. verticillioides infection percentage was observed in maize ears apex in comparison with maize ears base. Antagonistics agents B. amyloliquefaciens and M. oleovorans did not reduce F. verticillioides and A. flavus sprayed inoculated in maize ears base and apex. Kluyveromyces sp. L16 (T7) caused a significant reduction of F. verticillioides native infection (T1) on maize ears apex.

Discussion This study was conduced to get information about the potentiality of some microbial agents on Aspergillus

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Table 4 Effect of antagonistic agents– Fusarium verticillioides MPVA 285 interaction on the growth rate and the lag phase at different water activities and 25°C

Values are the mean of three replicates. Data with the same letter between control and treatments of the same water activity are not significantly different (P \ 0.05, Tukey test)

Lag phase (h)

Treatments

0.99

Fusarium verticillioides

0.35a

45a

Ba–F. verticillioides

0.26c

72c

0.97 Ba, B.amyloliquefaciens; Mo Microbacterium oleovorans; L16 Kluyveromyces spp; mm h-1, range diameter growth value; h, range lag phase

Growth rate (mm h-1)

Water activity

0.95

0.93

Mo–F. verticillioides

0.20b

58b

L16–F. verticillioides

0.27d

90d


0.32a

55a


0.18d

80c


0.20c

66b

L16–F. verticillioides Fusarium verticillioides

0.27b 0.30a

95d 60a


0.23c

65b


0.25b

74c


0.25b

95d


0.27a

74a


0.27c

87c


0.28b

75b


0.30a

110d

Table 5 Effect of treatments on CFU count of F. verticillioides and A. flavus on non-rhizospheric maize soil centrally and sprayed inoculated Treatments

Log CFU count Non-rhizospheric maize soil centrally inoculated

Non-rhizospheric maize soil sprayed inoculated

F. verticillioides

F. verticillioides

A. flavus

A. flavus

T1

0

0

0

0

T2

4a

4a

4a

4a

T3

0

0

0

0

T4

0

0

0

0

T5

0

0

0

0

T6 T7

0d 4.5c

2.8c 4.2b

3.6c 3.9c

4.6b 4.6b

T8

3.7b

2.7c

1.8c

3.3c

T1, Soil control; T2, fungi control; T3, B. amyloliquefaciens control; T4, M. oleovorans control; T5, Kluyveromyces spp. L16 control; T6, A. flavus or F. verticillioides in fungal-B. amyloliquefaciens interaction; T7, A. flavus or F. verticillioides in fungal-M. oleovorans interaction; T8, A. flavus or F. verticillioides in fungal-Kluyveromyces spp. L16 interaction. Data with the same letter for different treatments in the same group are not significantly different (p \ 0.05; Kruskal–Wallis test)

section Flavi and Fusarium verticillioides growth in single and mixed cultures. It is important to take into account the methods applied to select the potentials biocontrol agents and the relationship among toxigenic fungi, antagonistics isolates, and environmental factors. Reduction in fungal growth by antagonistics agents in different environmental conditions is the first important information in the selection steps. To select a potential biocontrol agent, it is important to

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take into account the relationship between biological interactions and environmental stress factors [9]. Environmental stress factors are important because it has been observed that several interactions were influenced by water activity, temperature, and substrate [18]. Changes in environmental factors cause an impact that can be decisive in determining the co-existence level or dominance of species in a particular ecological niche [9]. The bacterial isolates


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Table 6 Effect of treatments on the infection percentage of F. verticillioides and/or A. flavus on maize ears apex and base sprayed inoculated Treatments

Infection percentage (%)a Maize ears apex F. verticillioides

T0 T1

Maize ears base A. flavus

0 20 ± 8.7

F. verticillioides

0 6.7 ± 5.8

A. flavus

99 ± 1.2

25 ± 3.4

13.3 ± 5.8

19.3 ± 0.6

T2

0

0

0

0

T3

0

0

0

0

T4

0

0

0

0

T5 T6

71.7 ± 30.1 43.3 ± 49.1

13.3 ± 23 66.7 ± 57.7

17 ± 4.3 9.3 ± 8.4

8.7 ± 8.1 13.3 ± 11.5

T7

8.3 ± 2.9

5±5

7.7 ± 8.0

14.7 ± 2.3

T0, Ear control; T1, Fungi control; T2, B. amyloliquefaciens control; T3, M. oleovorans control; T4, Kluyveromyces spp. L16 control; T5, A. flavus or F. verticillioides–B. amyloliquefaciens interaction; T6, A. flavus or F. verticillioides–M. oleovorans interaction; T7, A. flavus or F. verticillioides–Kluyveromyces spp. L16 interaction a

Values are the mean ± SD (standard deviation)

from the maize assayed showed good growth at the water activities and temperature that are appropriate for growth and toxin production by A. flavus and F. verticillioides [9, 11]. In other way, when these biocontrol agents will be used in a formulation, their tolerance to a range of water activity could give success during long-lasting survival of the formulation and then when the bacteria are applied in the maize ecosystem. The ability to compete and exclude toxigenic species during the colonization of soil increase the possibility to stablish the potential biocontrol agent in the specific niches in particles of soil and contribute to reduce the undesirable inoculum in the soil. In the other way to measure the effective colonization of toxigenic fungi, ‘‘in situ’’ microecosystem after the potential biocontrol inoculation could provide information aproximately about the competitiviness in real field situation. We conduced a procedure for the selection of potential biological control agents that could reduce the colonization by A. flavus and F. verticillioides in maize ears. Aflatoxin and fumonisin producing fungi share the same habitat with other microorganisms that can influence toxin production [7, 9]. The relationship between rhizobacterial and yeasts antagonists paired with fumonisin and aflatoxigenic species and the impact of changing aw on different methodologies in vitro has previously been considered [12, 19]. Moreover, the capacity of B. amyloliquefaciens and

M. oleovorans to reduce F. verticillioides count and fumonisin production at field level have been previously described [7, 8]. Fusarium is one of the major genera associated with maize [20]. These fungi do not, however, colonize maize grain in isolation. They become established by interacting with other colonizers such as A. flavus. This is the principal species of section Flavi isolated from maize in Italy [7]. Previously, in vitro studies suggest that when the maize becomes contaminated with toxigenic Aspergillus section Flavi and Fusarium section Liseola, the mycotoxigenic risk is represented by fumonisin accumulation [21]. However, it has to be considered that when field conditions favor the growth of one pathogenic fungi, Fusarium or Aspergillus toxigenic species, it is inevitable that toxins levels will also increase, and in such conditions, the application of the same biological control agents may provide a means to reduce fumonisin or aflatoxin sufficiently to be useful to growers and producers. There have been numerous reports that show growth parameters inhibition of fungal pathogens following treatment with bacterial strains. Our results, showed that, the potentials biocontrol agents M. oleovorans, B. amyloliquefaciens, and Kluyveromyces sp. L16 influenced the growth rate and increased the lag phase of Fusarium and Aspergillus strains assayed in vitro. These results are similar to

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those obtained in previous works with the same antagonistics agents but different fumonisin and aflatoxin producers [10, 13]. Other bacteria like Amphibacillus xylanus, Bacillus subtilis, and Sporolactobacillus inulinus were able to increase the lag phase and inhibit growth of different Aspergillus Section Flavi strains [9]. This work also showed that B. amyloliquefaciens and Kluyveromyces sp. L16 produced an inhibitory effect on F. verticillioides and A. flavus populations, respectively, in non-rhizospheric maize soil. While these results may suggest that maize seeds treatment with these microorganisms, before seeding in field, may represent an effective method for reducing Fusarium and Aspergillus field count. Previous results from field test showed that B. amyloliquefaciens and M. oleovorans reduced fumonisin content in the maize crop [7]. Other strategy was the application of antagonistic agents at maize ear level showed only Kluyveromyces sp. L16 has significant inhibition of F. verticillioides infection. The screening procedure used in the present work reveals that B. amyloliquefaciens and Kluyveromyces sp. L16 in nonrhizospheric maize soil and Kluyveromyces sp. L16 at maize ear level may be considered good candidates as potential biocontrol agents, as a useful tool to protect maize quality. However, before we can promote the application of these biocontrol agents, the impact of these agents on naturally occurring fungi of grain and the impact on aflatoxin and fumonisin production must be evaluated. Studies in this way are now in progress. Acknowledgments This work was carried out through grants from SECYT-MAE Argentina–Italy IT/PA 05-AIX/70862006-2007.


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