Transcriptomic investigation of the interaction between a biocontrol yeast, Papiliotrema terrestris strain PT22AV, and the postharvest fungal pathogen Penicillium expansum on apple.

Biocontrol strategies offer a promising alternative to control plant pathogens achieving food safety and security. In this study we apply a RNAseq analysis during interaction between the biocontrol agent (BCA) Papiliotrema terrestris, the pathogen Penicillium expansum, and the host Malus domestica. Analysis of the BCA finds overall 802 upregulated DEGs (differentially expressed genes) when grown in apple tissue, with the majority being involved in nutrients uptake and oxidative stress response. This suggests that these processes are crucial for the BCA to colonize the fruit wounds and outcompete the pathogen. As to P. expansum analysis, 1017 DEGs are upregulated when grown in apple tissue, with the most represented GO categories being transcription, oxidation reduction process, and transmembrane transport. Analysis of the host M. domestica finds a higher number of DEGs in response to the pathogen compared to the BCA, with overexpression of genes involved in host defense signaling pathways in the presence of both of them, and a prevalence of pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) only during interaction with P. expansum. This analysis contributes to advance the knowledge on the molecular mechanisms that underlie biocontrol activity and the tritrophic interaction of the BCA with the pathogen and the host.

Exopolysaccharide from the yeast Papiliotrema terrestris PT22AV for skin wound healing.

INTRODUCTION: Exopolysaccharides (EPSs) are high-value functional biomaterials mainly produced by bacteria and fungi, with nutraceutical, therapeutic and industrial potentials. OBJECTIVES: This study sought to characterize and assess the biological properties of the EPS produced by the yeast Papiliotrema terrestris PT22AV. METHODS: After extracting the yeast's DNA and its molecular identification, the EPS from P. terrestris PT22AV strain was extracted and its physicochemical properties (structural, morphological, monosaccharide composition and molecular weight) were characterized. The EPS's in vitro biological activities and in vivo wound healing potential were also evaluated. RESULTS: The obtained EPS was water-soluble and revealed an average molecular weight (Mw) of 202 kDa. Mannose and glucose with 97% and 3% molar percentages, respectively, constituted the EPS. In vitro antibacterial activity analysis of the extracted EPS exhibited antibacterial activity (>80%) against Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis at a concentration of 2 mg/mL. The EPS showed cytocompatibility against the human fibroblast and macrophage cell lines and the animal studies showed a dose-dependent wound healing capacity of the EPS with higher wound closure at 10 mg/mL compared to negative and positive control after 14 days. CONCLUSION: The EPS from P. terrestris PT22AV could serve as a promising source of biocompatible macromolecules with potential for skin wound healing.

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.

Impairment of carotenoid biosynthesis through CAR1 gene mutation results in CoQ10, sterols, and phytoene accumulation in Rhodotorula mucilaginosa.

Red yeasts, mainly included in the genera Rhodotorula, Rhodosporidiobolus, and Sporobolomyces, are renowned biocatalysts for the production of a wide range of secondary metabolites of commercial interest, among which lipids, carotenoids, and other isoprenoids. The production of all these compounds is tightly interrelated as they share acetyl-CoA and the mevalonate pathway as common intermediates. Here, T-DNA insertional mutagenesis was applied to the wild type strain C2.5t1 of Rhodotorula mucilaginosa for the isolation of albino mutants with impaired carotenoids biosynthesis. The rationale behind this approach was that a blockage in carotenoid biosynthetic pathway could divert carbon flux toward the production of lipids and/or other molecules deriving from terpenoid precursors. One characterized albino mutant, namely, strain W4, carries a T-DNA insertion in the CAR1 gene coding for phytoene desaturase. When cultured in glycerol-containing medium, W4 strain showed significant decreases in cell density and fatty acids content in respect to the wild type strain. Conversely, it reached significantly higher productions of phytoene, CoQ10, and sterols. These were supported by an increased expression of CAR2 gene that codes for phytoene synthase/lycopene cyclase. Thus, in accordance with the starting hypothesis, the impairment of carotenoids biosynthesis can be explored to pursue the biotechnological exploitation of red yeasts for enhanced production of secondary metabolites with several commercial applications. KEY POINTS: • The production of lipids, carotenoids, and other isoprenoids is tightly interrelated. • CAR1 gene mutation results in the overproduction of phytoene, CoQ10, and sterols. • Albino mutants are promising tools for the production of secondary metabolites.

Complete genome sequence of the biocontrol yeast Papiliotrema terrestris strain LS28.

Papiliotrema terrestris strain LS28 is a biocontrol agent selected for its antagonistic activity against several plant pathogens both in the field and postharvest. The availability of a genome sequencing sets the foundation for the identification of the genetic mechanisms of its antagonistic activity. The genome size is 21.29 Mbp with a G+C content of 58.65%, and genome annotation predicts 8,626 protein-encoding genes. Phylogenetic analysis based on whole-genome data confirms that P. terrestris is a Tremellomycetes more closely related to Papiliotrema flavescens than Papiliotrema laurentii.

Securing fruit production: Opportunities from the elucidation of the molecular mechanisms of postharvest fungal infections.

Fruit-based diets have been adopted by the public worldwide because of their nutritional value. Many advances have also been made in the elucidation of host-pathogen interaction in the postharvest phase of fruits, in the hope of improving the management of diseases caused by pathogenic molds. In this study, we presented the molecular mechanisms by which pathogenic mold infects fruit in the postharvest phase, and focused on the knowledge gained from recent molecular techniques such as differential analysis of gene expression, targeted insertion, and mutagenesis. Current postharvest pathogenic fungal control strategies were then examined on the basis of their mechanisms for altering the infection process in order to explore new perspectives for securing fruit production. We found that biotechnological advances have led to an understanding of the new basic molecular processes involved in fruit fungal infection and to the identification of new genes, proteins and key factors that could serve as ideal targets for innovative antifungal strategies. In addition, the most commonly used steps to evaluate an approach to disrupt the fruit fungal infection process are mainly based on the inhibition of mycelial growth, spore germination, disruption of Adenosine triphosphate (ATP) synthesis, induction of oxidative stress, cell wall membrane damage, and inhibition of key enzymes. Finally, the alteration of the molecular mechanisms of signaling and response pathways to infection stimulation should also guide the development of effective control strategies to ensure fruit production.

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.

Leuconostoc mesenteroides subsp. mesenteroides LB7 isolated from apple surface inhibits P. expansum in vitro and reduces patulin in fruit juices.

This research aimed to isolate lactic acid bacteria (LAB) from apple surface and to reveal their potential to inhibit the growth of Penicillium expansum. Besides, their ability to detoxify fruit juices contaminated with mycotoxin patulin, produced by this fungi, was also studied. The isolation was performed on a typical MRS medium under ambient conditions. The molecular identification of the strains was done by sequencing the 16S rRNA genes. Antifungal activities of the isolated strains have been evaluated using dual agar plate assay protocol. A total of 11 LAB isolates was obtained from apples. These isolates showed phenotypic traits consistent with the genera of LAB. They have been identified as Leuconostoc mesenteroides subsp. mesenteroides and Weissella paramesenteroides. Among them, the strain LB7 showed exciting inhibitory activities in vitro against P. expansum. LB7 also successfully detoxified homemade and commercial fruit juices contaminated with patulin. Further research will bring the application prospects of these LABs in food biocontrol and biopreservation strategies.

Recent trends in detecting, controlling, and detoxifying of patulin mycotoxin using biotechnology methods.

Patulin (PAT) is a mycotoxin that can contaminate many foods and especially fruits and fruit-based products. Therefore, accurate and effective testing is necessary to enable producers to comply with regulations and promote food safety. Traditional approaches involving the use of chemical compounds or physical treatments in food have provided practical methods that have been used to date. However, growing concerns about environmental and health problems associated with these approaches call for new alternatives. In contrast, recent advances in biotechnology have revolutionized the understanding of living organisms and brought more effective biological tools. This review, therefore, focuses on the study of biotechnology approaches for the detection, control, and mitigation of PAT in food. Future aspects of biotechnology development to overcome the food safety problem posed by PAT were also examined. We find that biotechnology advances offer novel, more effective, and environmental friendly approaches for the control and elimination of PAT in food compared to traditional methods. Biosensors represent the future of PAT detection and use biological tools such as aptamer, enzyme, and antibody. PAT prevention strategies include microbial biocontrol, the use of antifungal biomolecules, and the use of microorganisms in combination with antifungal molecules. PAT detoxification aims at the breakdown and removal of PAT in food by using enzymes, microorganisms, and various adsorbent biopolymers. Finally, biotechnology advances will be dependent on the understanding of fundamental biology of living organisms regarding PAT synthesis and resistance mechanisms.

Isotopic Labeling Studies Reveal the Patulin Detoxification Pathway by the Biocontrol Yeast Rhodotorula kratochvilovae LS11.

Patulin (1) is a mycotoxin contaminant in fruit and vegetable products worldwide. Biocontrol agents, such as the yeast Rhodotorula kratochvilovae strain LS11, can reduce patulin (1) contamination in food. R. kratochvilovae LS11 converts patulin (1) into desoxypatulinic acid (DPA) (5), which is less cytotoxic than the mycotoxin (1) to in vitro human lymphocytes. In the present study, we report our investigations into the pathway of degradation of patulin (1) to DPA (5) by R. kratochvilovae. Isotopic labeling experiments revealed that 5 derives from patulin (1) through the hydrolysis of the γ-lactone ring and subsequent enzymatic modifications. The ability of patulin (1) and DPA (5) to cause genetic damage was also investigated by the cytokinesis-block micronucleus cytome assay on in vitro human lymphocytes. Patulin (1) was demonstrated to cause much higher chromosomal damage than DPA (5).

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.

Biocontrol Agents Increase the Specific Rate of Patulin Production by Penicillium expansum but Decrease the Disease and Total Patulin Contamination of Apples.

Synthetic fungicides are commonly employed for the control of postharvest diseases of fruits. However, due to health concerns about the use of these chemicals, alternative control methods including biocontrol based on antagonistic yeasts are gaining in popularity. In this study, we investigated the effects of two biocontrol yeasts, Rhodotorula mucilaginosa strain 3617 and Rhodotorula kratochvilovae strain LS11, on blue mold and patulin (PAT) contamination caused by Penicillium expansum strains PY and FS7 in artificially inoculated Fuji apples stored at 20°C for 9 days. To correlate the development of the P. expansum strains in yeast-treated and untreated apples with PAT production, we quantified their biomass in the infected fruits using a recently published quantitative real-time polymerase chain reaction method based on specific primers for patF, a gene from P. expansum that is involved in PAT biosynthesis. Both yeasts significantly reduced the disease incidence caused by the two strains of P. expansum up to 5-7 days of incubation, and lowered their biomass and the progression of symptoms up to 9 days. Interestingly, both yeasts strains increased the rate of PAT production (expressed as ng patulin/µg fungal DNA) by the two pathogenic strains. Nevertheless, both biocontrol agents reduced the total PAT contamination, especially in the case of P. expansum strain FS7, the higher PAT producer of the two tested P. expansum strains. Comparing between the yeast strains, R. kratochvilovae LS11 was more effective than R. mucilaginosa 3617 for the control of P. expansum.

Patulin Degradation by the Biocontrol Yeast Sporobolomyces sp. Is an Inducible Process.

Patulin is a mycotoxin produced by Penicillium expansum and a common contaminant of pome fruits and their derived products worldwide. It is considered to be mutagenic, genotoxic, immunotoxic, teratogenic and cytotoxic, and the development of strategies to reduce this contamination is an active field of research. We previously reported that Sporobolomyces sp. is able to degrade patulin and convert it into the breakdown products desoxypatulinic acid and ascladiol, both of which were found to be less toxic than patulin. The specific aim of this study was the evaluation of the triggering of the mechanisms involved in patulin resistance and degradation by Sporobolomyces sp. Cells pre-incubated in the presence of a low patulin concentration showed a higher resistance to patulin toxicity and a faster kinetics of degradation. Similarly, patulin degradation was faster when crude intracellular protein extracts of Sporobolomyces sp. were prepared from cells pre-treated with the mycotoxin, indicating the induction of the mechanisms involved in the resistance and degradation of the mycotoxin by Sporobolomyces sp. This study contributes to the understanding of the mechanisms of patulin resistance and degradation by Sporobolomyces sp., which is an essential prerequisite for developing an industrial approach aiming at the production of patulin-free products.

Transcriptomic responses of the basidiomycete yeast Sporobolomyces sp. to the mycotoxin patulin.

BACKGROUND: Patulin is a mycotoxin produced by Penicillium expansum, the causal agent of blue mold of stored pome fruits, and several other species of filamentous fungi. This mycotoxin has genotoxic, teratogenic and immunotoxic effects in mammals, and its presence in pome fruits and derived products represents a serious health hazard. Biocontrol agents in the Pucciniomycotina, such as the yeasts Sporobolomyces sp. strain IAM 13481 and Rhodosporidium kratochvilovae strain LS11, are able to resist patulin and degrade it into the less toxic compounds desoxypatulinic acid and ascladiol. RESULTS: In this investigation we applied a transcriptomic approach based on RNAseq to annotate the genome of Sporobolomyces sp. IAM 13481 and then study the changes of gene expression in Sporobolomyces sp. exposed to patulin. Patulin treatment leads to ROS production and oxidative stress that result in the activation of stress response mechanisms controlled by transcription factors. Upregulated Sporobolomyces genes were those involved in oxidation-reduction and transport processes, suggesting the activation of defense mechanisms to resist patulin toxicity and expel the mycotoxin out of the cells. Other upregulated genes encoded proteins involved in metabolic processes such as those of the glutathione and thioredoxin systems, which are essential to restore the cellular redox homeostasis. Conversely, patulin treatment decreased the expression of genes involved in the processes of protein synthesis and modification, such as transcription, RNA processing, translation, protein phosphorylation and biosynthesis of amino acids. Also, genes encoding proteins involved in transport of ions, cell division and cell cycle were downregulated. This indicates a reduction of metabolic activity, probably due to the high energy requirement by the cells or metabolic arrest while recovering from the insult caused by patulin toxicity. CONCLUSIONS: Complex mechanisms are activated in a biocontrol yeast in response to patulin. The genes identified in this study can pave the way to develop i) a biodetoxification process of patulin in juices and ii) a biosensor for the rapid and cost-effective detection of this mycotoxin.

Overcoming recalcitrant transformation and gene manipulation in Pucciniomycotina yeasts.

The red yeasts of the Pucciniomycotina have rarely been transformed with DNA molecules. Transformation methods were recently developed for a species of Sporobolomyces, based on selection using uracil auxotrophs and plasmids carrying the wild-type copies of the URA3 and URA5 genes. However, these plasmids were ineffective in the transformation of closely related species. Using the genome-sequenced strain of Rhodotorula graminis as a starting point, the URA3 and URA5 genes were cloned and tested for the transformation ability into different Pucciniomycotina species by biolistic and Agrobacterium-mediated transformations. Transformation success depended on the red yeast species and the origin of the URA3 or URA5 genes, which may be related to the high G + C DNA content found in several species. A new vector was generated to confer resistance to nourseothricin, using a native promoter from R. graminis and the naturally high G + C nourseothricin acetyltransferease gene. This provides a second selectable marker in these species. Targeted gene disruption was tested in Sporobolomyces sp. IAM 13481 using different lengths of homologous DNA with biolistic and Agrobacterium transformation methods. Both DNA delivery methods were effective for targeted replacement of a gene required for carotenoid pigment biosynthesis. The constructs also triggered transgene silencing. These developments open the way to identify and manipulate gene functions in a large group of basidiomycete fungi.

Conversion of the mycotoxin patulin to the less toxic desoxypatulinic acid by the biocontrol yeast Rhodosporidium kratochvilovae strain LS11.

The infection of stored apples by the fungus Penicillium expansum causes the contamination of fruits and fruit-derived products with the mycotoxin patulin, which is a major issue in food safety. Fungal attack can be prevented by beneficial microorganisms, so-called biocontrol agents. Previous time-course thin layer chromatography analyses showed that the aerobic incubation of patulin with the biocontrol yeast Rhodosporidium kratochvilovae strain LS11 leads to the disappearance of the mycotoxin spot and the parallel emergence of two new spots, one of which disappears over time. In this work, we analyzed the biodegradation of patulin effected by LS11 through HPLC. The more stable of the two compounds was purified and characterized by nuclear magnetic resonance as desoxypatulinic acid, whose formation was also quantitated in patulin degradation experiments. After R. kratochvilovae LS11 had been incubated in the presence of (13)C-labeled patulin, label was traced to desoxypatulinic acid, thus proving that this compound derives from the metabolization of patulin by the yeast. Desoxypatulinic acid was much less toxic than patulin to human lymphocytes and, in contrast to patulin, did not react in vitro with the thiol-bearing tripeptide glutathione. The lower toxicity of desoxypatulinic acid is proposed to be a consequence of the hydrolysis of the lactone ring and the loss of functional groups that react with thiol groups. The formation of desoxypatulinic acid from patulin represents a novel biodegradation pathway that is also a detoxification process.

Development of resources for the analysis of gene function in Pucciniomycotina red yeasts.

The Pucciniomycotina is an important subphylum of basidiomycete fungi but with limited tools to analyze gene functions. Transformation protocols were established for a Sporobolomyces species (strain IAM 13481), the first Pucciniomycotina species with a completed draft genome sequence, to enable assessment of gene function through phenotypic characterization of mutant strains. Transformation markers were the URA3 and URA5 genes that enable selection and counter-selection based on uracil auxotrophy and resistance to 5-fluoroorotic acid. The wild type copies of these genes were cloned into plasmids that were used for transformation of Sporobolomyces sp. by both biolistic and Agrobacterium-mediated approaches. These resources have been deposited to be available from the Fungal Genetics Stock Center. To show that these techniques could be used to elucidate gene functions, the LEU1 gene was targeted for specific homologous replacement, and also demonstrating that this gene is required for the biosynthesis of leucine in basidiomycete fungi. T-DNA insertional mutants were isolated and further characterized, revealing insertions in genes that encode the homologs of Chs7, Erg3, Kre6, Kex1, Pik1, Sad1, Ssu1 and Tlg1. Phenotypic analysis of these mutants reveals both conserved and divergent functions compared with other fungi. Some of these strains exhibit reduced resistance to detergents, the antifungal agent fluconazole or sodium sulfite, or lower recovery from heat stress. While there are current experimental limitations for Sporobolomyces sp. such as the lack of Mendelian genetics for conventional mating, these findings demonstrate the facile nature of at least one Pucciniomycotina species for genetic manipulation and the potential to develop these organisms into new models for understanding gene function and evolution in the fungi.

Occurrence of mycotoxin in Farro samples from southern Italy.

The occurrence of nine mycotoxins and of contamination by pre- and postharvest fungal pathogens of cereals was investigated in samples of stored Triticum monococcum L., Triticum dicoccon Schrank (emmer), and Triticum spelta L. (spelt). In Italy, all three species are collectively referred to as farro. The samples examined were harvested in summer 2000 from eight different sites in southern Italy. Conventional fluorimetric and diode array-based high-performance liquid chromatography (HPLC) analyses and HPLC-mass spectrometry analyses were used to identify fumonisin B1 in five samples (up to 70.00 microg/ kg), ochratoxin A in seven samples (up to 4.07 microg/kg), and beauvericin in three samples (up to 4.44 mg/kg). Enniatin B was detected in one sample (30.00 microg/kg), but no zearalenone or fusaproliferin was found. Deoxynivalenol and aflatoxins were not evaluated. The potentially mycotoxigenic fungal species detected were Alternaria alternata, Fusarium proliferatum, Fusarium tricinctum, Penicillium verrucosum, and Penicillium chrysogenum. This is the first report of the natural occurrence of mycotoxins in farro samples.

Effect of the Biocontrol Yeast Rhodotorula glutinis Strain LS11 on Patulin Accumulation in Stored Apples.

ABSTRACT Contamination of apples (Malus domestica) and derived juices with fungicide residues and patulin produced by Penicillium expansum are major issues of food safety. Biocontrol agents represent an alternative or supplement to chemicals for disease control. Our data show that these microbes could also contribute to actively decreasing patulin accumulation in apples. Three biocontrol agents, Rhodotorula glutinis LS11, Cryptococcus laurentii LS28, and Aureobasidium pullulans LS30, were examined for their in vitro growth in the presence of patulin and for their capability to decrease mycotoxin recovery from the medium. Strain LS11 yielded the highest growth rates and the greatest decrease of toxin recoveries. Further, it caused the appearance of two major spots on thin-layer chromatography (TLC) plates, suggesting possible metabolization of the mycotoxin. In vivo, i.e., in the low percentage of LS11-pretreated apples infected by P. expansum, patulin accumulation was significantly lower than in nontreated infected fruits. Yeast cells survived and increased in infected apples and, in a model system emulating decaying apple, resulted in accelerated breakdown of patulin and the production of the same TLC spots as those detected in vitro. These data suggest that biocontrol yeast cells surviving in decaying apples could metabolize patulin and/or negatively affect its accumulation or synthesis. To our knowledge, this is the first report describing the effect of a biocontrol agent on patulin accumulation in vivo.

Resistance of postharvest biocontrol yeasts to oxidative stress: a possible new mechanism of action.

ABSTRACT We detected the generation of the reactive oxygen species (ROS) superoxide anion ( O.(-) (2)) and hydrogen peroxide (H(2)O(2)) in apple wounds 2 immediately after wounding, and assessed the relationships between (i) timely colonization of apple wounds by biocontrol yeasts, (ii) resistance of these microorganisms to oxidative stress caused by ROS, and (iii) their antagonism against postharvest wound pathogens. We analyzed a model system consisting of two yeasts with higher (Cryptococcus laurentii LS-28) or lower (Rhodotorula glutinis LS-11) antagonistic activity against the postharvest pathogens Botrytis cinerea and Penicillium expansum. LS-28 exhibited faster and greater colonization of wounds than LS-11. In contrast to LS-28, the number of LS-11 cells dropped 1 and 2 h after application, and then increased only later. In vitro, LS-28 was more resistant to ROS-generated oxidative stress. The combined application of biocontrol yeasts and ROS-deactivating enzymes in apple wounds prevented the decrease in number of LS-11 cells mentioned above, and enhanced colonization and antagonistic activity of both biocontrol yeasts against B. cinerea and P. expansum. Polar lipids of LS-11 contained the more unsaturated and oxidizable alpha-linolenic acid, which was absent in LS-28. Resistance to oxidative stress could be a key mechanism of biocontrol yeasts antagonism against postharvest wound pathogens.