Modulation of extracellular Penicillium expansum-driven acidification by Papiliotrema terrestris affects biosynthesis of patulin and has a possible role in biocontrol activity.

The active regulation of extracellular pH is critical for the virulence of fungal pathogens. Penicillium expansum is the causal agent of green-blue mold on stored pome fruits and during its infection process acidifies the host tissues by secreting organic acids. P. expansum is also the main producer of patulin (PAT), a mycotoxin found in pome fruit-based products and that represents a serious health hazard for its potential carcinogenicity. While it is known that PAT biosynthesis in P. expansum is regulated by nutritional factors such as carbon and nitrogen and by the pH, the mechanistic effects of biocontrol on PAT production by P. expansum are not known. In this work, we assessed how optimal and suboptimal concentrations of the biocontrol agent (BCA) Papiliotrema terrestris LS28 affect both extracellular pH and PAT biosynthesis in P. expansum. In wounded apples, the optimal and suboptimal concentrations of the BCA provided almost complete and partial protection from P. expansum infection, respectively, and reduced PAT contamination in both cases. However, the suboptimal concentration of the BCA increased the specific mycotoxigenic activity by P. expansum. In vitro, the rate of PAT biosynthesis was strictly related to the extracellular pH, with the highest amount of PAT detected in the pH range 4-7, whereas only traces were detectable at pH 3. Moreover, both in vitro and in apple wounds the BCA counteracted the extracellular P. expansum-driven acidification maintaining extracellular pH around 4, which is within the pH range that is optimal for PAT biosynthesis. Conversely, in the absence of LS28 the pathogen-driven acidification led to rapidly achieving acidic pH values (<3) that lie outside of the optimal pH range for PAT biosynthesis. Taken together, these results suggest that pH modulation by LS28 is important to counteract the host tissue acidification and, therefore, the virulence of P. expansum. On the other hand, the buffering of P. expansum-driven acidification provided by the BCA increases the specific rate of PAT biosynthesis through the extension of the time interval at which the pH value lies within the optimal range for PAT biosynthesis. Nevertheless, the antagonistic effect provided by the BCA greatly reduced the total amount of PAT.

Molecular Tools for the Yeast Papiliotrema terrestris LS28 and Identification of Yap1 as a Transcription Factor Involved in Biocontrol Activity.

Fungal attacks on stored fruit and vegetables are responsible for losses of products. There is an active research field to develop alternative strategies for postharvest disease management, and the use of biocontrol agents represents a promising approach. Understanding the molecular bases of the biocontrol activity of these agents is crucial to potentiate their effectiveness. The yeast Papiliotrema terrestris is a biocontrol agent against postharvest pathogens. Phenotypic studies suggest that it exerts its antagonistic activity through competition for nutrients and space, which relies on its resistance to oxidative and other cellular stresses. In this study, we developed tools for genetic manipulation in P. terrestris to perform targeted gene replacement and functional complementation of the transcription factors Yap1 and Rim101. In vitro phenotypic analyses revealed a conserved role of Yap1 and Rim101 in broad resistance to oxidative stress and alkaline pH sensing, respectively. In vivo analyses revealed that P. terrestris yap1Δ and rim101Δ mutants display decreased ability to colonize wounded fruit compared to that of the parental wild-type (WT) strain; the yap1Δ mutant also displays reduced biocontrol activity against the postharvest pathogens Penicillium expansum and Monilinia fructigena, indicating an important role for resistance to oxidative stress in timely wound colonization and biocontrol activity of P. terrestris In conclusion, the availability of molecular tools developed in the present study provides a foundation to elucidate the genetic mechanisms underlying biocontrol activity of P. terrestris, with the goal of enhancing this activity for the practical use of P. terrestris in pest management programs based on biological and integrated control.IMPORTANCE The use of fungicides represents the most effective and widely used strategy for controlling postharvest diseases. However, their extensive use has raised several concerns, such as the emergence of plant pathogens' resistance as well as the health risks associated with the persistence of chemical residues in fruit, in vegetables, and in the environment. These factors have brought attention to alternative methods for controlling postharvest diseases, such as the utilization of biocontrol agents. In the present study, we developed genetic resources to investigate at the molecular level the mechanisms involved in the biocontrol activity of Papiliotrema terrestris, a basidiomycete yeast that is an effective biocontrol agent against widespread fungal pathogens, including Penicillium expansum, the etiological agent of blue mold disease of pome fruits. A deeper understanding of how postharvest biocontrol agents operate is the basic requirement to promote the utilization of biological (and integrated) control for the reduction of chemical fungicides.

Aspergillus flavus Exploits Maize Kernels Using an "Orphan" Secondary Metabolite Cluster.

Aspergillus flavus is a saprophytic cosmopolitan fungus, capable of infecting crops both pre- and post-harvest and exploiting different secondary metabolites, including aflatoxins. Aflatoxins are known carcinogens to animals and humans, but display no clear effect in host plants such as maize. In a previous study, we mined the genome of A. flavus to identify secondary metabolite clusters putatively involving the pathogenesis process in maize. We now focus on cluster 32, encoding for fungal effectors such as salicylate hydroxylase (SalOH), and necrosis- and ethylene-inducing proteins (npp1 domain protein) whose expression is triggered upon kernel contact. In order to understand the role of this genetic cluster in maize kernel infection, mutants of A. flavus, impaired or enhanced in specific functions (e.g., cluster 32 overexpression), were studied for their ability to cause disease. Within this frame, we conducted histological and histochemical experiments to verify the expression of specific genes within the cluster (e.g., SalOH, npp1), the production of salicylate, and the presence of its dehydroxylated form. Results suggest that the initial phase of fungal infection (2 days) of the living tissues of maize kernels (e.g., aleuron) coincides with a significant increase of fungal effectors such as SalOH and Npp1 that appear to be instrumental in eluding host defences and colonising the starch-enriched tissues, and therefore suggest a role of cluster 32 to the onset of infection.

Complete Genome Sequence of the Biocontrol Agent Yeast Rhodotorula kratochvilovae Strain LS11.

Rhodotorula kratochvilovae strain LS11 is a biocontrol agent (BCA) selected for its antagonistic activity against several plant pathogens both in the field and postharvest. Genome assembly includes 62 contigs for a total of 22.56 Mbp and a G+C content of 66.6%. Genome annotation predicts 7,642 protein-encoding genes.