The biocompetitive potential of the A. flavus isolated population

During two years of experimentation, over 2000 afla+ and afla- A. flavus strains have been isolated from maize kernels sampled in the treated fields. A set of experiments were performed on a representative group belonging to this collection of strains to get hints on the BC responsiveness of the “endemic” population of A. flavus. Analyses were set following a preliminary conducted assay aimed at assessing in vitro the effectiveness of an atoxigenic strain against AFs accumulation by toxigenic isolates (Degola et al. 2012). Conidial suspensions of both afla+ and afla- strains were co-inoculated in same wells of a CCM microplate, then the production of aflatoxin was fluorimetrically recorded in the medium as for the aflatoxin accumulation assay; biocompetiton potential of afla- strain, estimated by comparison with control wells (inoculated with afla+ strain only), was expressed as percentage of inhibition (Figure 72).

114

Figure 72: Biocompetition activity of afla- strain on aflatoxin accumulation by a toxigenic strain. Conidia of afla- and afla+ strains were co-inoculated in an increasing afla-/afla+ ratio (1:1, 1:2 or 1:4) in CCM medium, and incubated at 25

°C for 6 days in the dark.

At first, a selection of 40 A. flavus strains were selected in order to perform a “global”

biocompetition assay: 20 afla+ strains were chosen among those that accumulated high amounts of aflatoxin, whereas 20 afla- strains were randomly selected. Each toxigenic strain was co-inoculated with each afla+ isolate, and compared with the BC. The rational was to ascertain if the failure or the limited efficacy of the BC, observed in some experimental condition in the treated fields, was the consequence of the specific ”virulence” of the afla+ population challenged by the BC or if the biocompetition did not occurred in the field owing to other unknown environmental factors.

An example of results is reported in Figure 73. As a general consideration, the inhibitory performance of BC was confirmed as the best, since it was effective in lowering the aflatoxins accumulation of all the afla+ strains; on the other hand, if separately considered, some afla- isolates resulted in an higher inhibition rate against specific afla+ strains, as in the case of OZ10- that lowered aflatoxin accumulation by OZ6+ more efficiently that the BC.

Figure 73: An example of global biocompetition assay of A. flavus population recovered on grains sampled in the experimental fields. Here, the aflatoxins containment efficacy of 9 afla- strain and BC were tested respect to 6 toxigenic strain. Conidia of afla- and afla+ strains were co-inoculated in an afla-/afla+ ratio of 1:1, in CCM medium, and incubated at 25 °C for 6 days in the dark. Inhibition of isolated afla- strains is compared with the BC activity, whereas the afla+

strain AT1+ is added as internal control.

0 20 40 60 80 100

1:1 1:2 1:4

Biocompetition % inhibition

afla+/afla-0 20 40 60 80 100

BC OZ4- OZ5- OZ7- OZ8- OZ9- OZ10- OZ11- OZ14-

OZ15-Biocompetition % inhibition

Biocompetition assay ^{AT1+ OZ1+ OZ2+ OZ3+ OZ6+ OZ16+}

115

A second test was conducted to compare the effectiveness of the BC in inhibiting the AF production with afla- endogenous isolates, even in a disadvantageous proportion: thus, a conidial suspension of the toxigenic isolate CR24+ was singularly co-inoculated with 10 afla- endogenous strains in an afla+/afla- 1:1 and 1:10 ratio.

We found that a ten-fold augmentation of the afla+ conidial concentration did not affect the biocompetition efficacy of the BC and of the majority of endogenous afla- isolates: in fact, as shown in Figure 74, only strain OZ18- diminished in aflatoxin containment ability when its presence in the co-culture was one-tenth of the total inoculum.

Figure 74: Dose-dependent biocompetition efficacy of endogenous A. flavus afla- and BC. Efficacy of 10 afla- strains is tested in 1:1 and 1:10 afla-/afla+ ratio against CR24+ strain. Inhibition of isolated afla- strains is compared with the BC activity.

Data evidenced a certain variability among A. flavus strains in their ability to prevent (for afla-) aflatoxin contamination or to overcome the intraspecific inhibitory effect (for afla+) of biocompetitors, underscoring the existence of different mechanisms that may or not act synergistically;

in consideration that in a cooperative mycelial network, such as in a colonizing inoculum, the efficacy of the interference on aflatoxin production could be determined by the strength of the vegetative compatibility (Wicklow et al. 2007), and that population of A. flavus colonizing a single field commonly include diverse VCGs (Bayman et al. 1991; Ehrlich 2008), we explored a multi-competition approach to investigate the efficacy of the use of a combination of afla- strains to ameliorate the interference with aflatoxin production by one or more afla+ strains. A microplate

0 20 40 60 80 100

0Z18- CR15- CR21- CR22- CR25- CR26- CSPT3- CSPT4- CSPT5- CSPT7- BC

Biocompetition % inhibition

In vitro biocompetition assay ^{1:1 1:10}

116

biocompetition assay was built, comparing the aflatoxin containment of the BC with the inhibitory effect of a pool of 10 endogenous afla- isolates (Figure 75).

Figure 75: Comparison between the biocompetitive efficacy of an endogenous afla- pool and the BC. The aflatoxin containment efficacy of a conidial suspension of pooled 10 afla- strains is tested against 9 afla+ endogenous strains.

Inhibition of the pool is compared with the BC activity, AT1+ strain is used as reference.

Results, presented in Figure 75, did not report significant differences between the two conditions:

in terms of aflatoxin inhibition, the biocompetitive advantage in using the pool of atoxigenic strains did not increase the containment obtained with the single BC. As well as confirmatory of the reliability of the latter against the resident population of A. flavus, this observation seemed to exclude the hypothesis of a synergism between afla- isolates; however, the further characterization of the VCG of atoxigenic isolates pooled would be helpful for clarify this aspect.

0 20 40 60 80 100

AT1+ OZ6+ CR19+ CR20+ CR23+ CR24+ CR27+ CSPT1+ CSPT2+ CSPT6+

Biocompetition % inhibition

Multi-biocompetition assay ^BC

POOL-117