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The use of entomopathogenic fungi for biocontrol of plant pests is recently receiving an increased interest due to the need of reducing the impact of agricultural practices on the environment. Biocontrol efficacy could be improved by co-inoculation of different microorganisms. However, interactions between the fungal species can trigger or depress the biocontrol activity. Co-inoculation of two entomopathogenic fungi (Beauveria bassiana and B. brongniartii) was performed in vitro to evaluate the effects of their joint behaviour on a range of different carbon sources in comparison to single inoculation. The two species showed a very different metabolic profile by Phenotype MicroArrayTM. B. bassiana showed a broader metabolism than B. brongniartii on a range of substrates. B. brongniartii showed a greater specificity in substrate utilization. Several carbon sources (L-Asparagine, L-Aspartic Acid, L- Glutamic Acid, m- Erythritol, D-Melezitose, D-Sorbitol) triggered the fungal metabolism in the co-inoculum. SSR markers and Real Time qPCR analysis showed that different substrates promoted either the growth of one or the other species, suggesting a form of interaction between the two fungi, related to their different ecological niches. The methodological approach that combines Phenotype MicroArrayTM and SSR genotyping appeared useful to assess the performance and potential competition of co-inoculated entomopathogenic fungi.

A culture independent method based on qPCR was developed for the detection and quantification of two fungal inoculants in soil. The aim was to adapt a genotyping approach based on SSR (Simple Sequence Repeat) marker to a discriminating tracing of two different species of bioinoculants in soil, after their in-field release. Two entomopathogenic fungi, Beauveria bassiana and B. brongniartii, were traced and quantified in soil samples obtained from field trials. These two fungal species were used as biological agents in Poland to control Melolontha melolontha (European cockchafer), whose larvae live in soil menacing horticultural crops. Specificity of SSR markers was verified using controls consisting of: i) soil samples containing fungal spores of B. bassiana and B. brongniartii in known dilutions; ii) the DNA of the fungal microorganisms; iii) soil samples singly inoculated with each fungus species. An initial evaluation of the protocol was performed with analyses of soil DNA and mycelial DNA. Further, the simultaneous detection and quantification of B. bassiana and B. brongniartii in soil was achieved in field samples after application of the bio-inoculants. The protocol can be considered as a relatively low cost solution for the detection, identification and traceability of fungal bio-inoculants in soil.