Validation and Ecological Niche Investigation of a New Fungal Intraspecific Competitor as a Biocontrol Agent for the Sustainable Containment of Aflatoxins on Maize Fields

Abstract

Crop yield and plant products quality are directly or indirectly affected by climate alterations. Adverse climatic conditions often promote the occurrence of different abiotic stresses, which can reduce or enhance the susceptibility to pests or pathogens. Aflatoxin producing fungi, in particular, whose diffusion and deleterious consequences on cereals commodities have been demonstrated to highly depend on the temperature and humidity conditions that threaten increasingly larger areas. Biological methods using intraspecific competitors to prevent fungal development and/or toxin production at the pre-harvest level are particularly promising, even if their efficacy could be affected by the ecological interaction within the resident microbial population. A previously characterized Aspergillus flavus atoxigenic strain was applied in two maize fields to validate its effectiveness as a biocontrol agent against aflatoxin contamination. At one month post-application, at the harvest stage, its persistence within the A. flavus population colonizing the maize kernels in the treated area was assessed, and its efficacy was compared in vitro with a representation of the isolated atoxigenic population. Results proved that our fungal competitor contained the aflatoxin level on maize grains as successfully as a traditional chemical strategy, even if representing less than 30% of the atoxigenic strains re-isolated, and achieved the best performance (in terms of bio-competitive potential) concerning endogenous atoxigenic isolates.

Keywords: Aspergillus flavus; aflatoxin biocontrol; intraspecific biocompetition; maize protection; niche variation.

Figure 1

Assessment of insecticide formulations’ effect on FB growth, development, and colonization ability. (A): Conidia germination and hyphae early growth evaluation in a multi-well microculture system, in YES liquid medium, with 5 × 10² FB conidia in the presence of 50 and 100 μg/mL Coragen^® or Turex^® alternatively. Data reported as mean ± S.D. Asterisk indicates significant differences at p-value < 0.01. (B): FB radial growth visual evaluation in YES solid medium; 10 µL of a mixture of FB conidial suspensions (5 × 10² conidia) and 100 μg/mL of Coragen^® or Turex^® alternatively were point inoculated, in triplicate, in YES 5% agar plates; control (CNT) was represented by 10 µL of FB conidial suspensions (5 × 10² conidia) without insecticide amendment. (C): FB colonization of maize kernels surface-treated with FB conidial suspensions (5 × 10² conidia) amended with 100 μg/mL Coragen^® or Turex^® alternatively; control (CNT) was represented by maize kernels treated with FB conidial suspensions (5 × 10² conidia) without insecticide amendment.

Figure 2

Characterization of the A. flavus population at the moment of the harvest. (A): Relative abundance of the two chemotypes (afla+ and afla-) colonizing the maize kernels from the different treatments. Data, expressed as the percentage of A. flavus strains total number isolated in each treatment, are reported as mean ± S.D. Different letters indicate significant differences at p-value < 0.01. (B): Example of the molecular profiling of afla- isolates used to track the FB persistence on the crop. (C): FB persistence within the afla- A. flavus population. Data, expressed as the percentage of the afla- A. flavus strains total number isolated in each treatment, are reported as mean ± S.D. Different letters indicate significant differences at p-value < 0.05.

Figure 3

In vitro bio-competition challenge tests of the FB against the toxigenic A. flavus population colonizing the study site #2. (A): Heat map built with bio-competition efficacy of 20 afla- against 17 afla+ isolates. Conidia were co-inoculated in a 1:1 ratio. AT1+ strain was used as reference strain; data are expressed as a percentage of AF inhibition. Map was built with the Heatmapper online tool [27]. (B): Number of afla- strains effective in lowering AF production (inhibition > 50%) for each afla+ isolate plotted against the average of AF inhibition determined by effective bio-competitors. (C): Comparison of the bio-competitive efficacy of the FB and ten afla- isolates, pooled together, against ten afla+ strains. Data reported as mean ± S.D. Asterisk indicates significant differences at p-value < 0.01.