Raffaello Castoria

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.

Activity of the Yeasts Cryptococcus laurentii and Rhodotorula glutinis Against Post-harvest Rots on Different Fruits

More than 200 yeasts were selectively isolated from microbial populations on the surface of different fruits. Fifty of these isolates were tested against blue mould ( Penicillium expansum ) on wounded apples. Isolates LS-11 of Rhodotorula glutinis and LS-28 of Cryptococcus laurentii were the most effective antagonists. They were further evaluated at 20IC on different fruits (apples, pears, strawberries, kiwi fruits and table grapes) against several of the main post-harvest pathogens ( Botrytis cinerea, Penicillium expansum, Rhizopus stolonifer and Aspergillus niger ) and at 4IC on apples inoculated with P. expansum . At 20IC the antagonists significantly reduced rot incidence and showed a wide range of activity on different hostpathogen combinations; isolate LS-28 exhibited a higher and more stable activity than LS-11. Both yeasts were also effective against P. expansum in cold storage conditions. Populations of the two yeasts were assessed on wounded and unwounded surfaces of apples kept at both 20 and 4...

Fungal Invasion Enzymes and Their Inhibition

The first lines of defense of a plant against phytopathogenic fungi are the external cuticle and the polysaccharide-rich cell wall (Fig. 1). The vast majority of fungi need to breach these barriers to gain access to the plant tissue, and, once inside the tissue, to degrade the cell wall components in order to sustain their growth and to complete the invasion process. It is generally accepted that the enzymatic arsenal of the fungus contributes, together with mechanical forces (Howard et al. 1991; Chap. 3, this Vol.), to the degradation of both cuticle and cell walls.

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.

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.

Perception of fungal elicitors and signal transduction

Plants, like animals, are continually exposed to a vast array of potential fungal pathogens; in many cases, they resist attack by blocking fungal development soon after penetration. As plants lack a circulatory system and antibodies, they have evolved defense mechanisms that are distinct from the vertebrate immune system. In contrast to animal cells, each plant cell is capable of defending itself by means of a combination of constitutive mechanisms and induced responses. After the perception of the pathogen (recognition), the plant cell at the site of infection transmits the information inside the cell across the plasma membrane as well as to neighboring cells. As a consequence, a number of defense reactions are induced, which include a rapid and localized cell death (hypersensitive response), a rapid oxidative burst, cross-linking and strengthening of the plant cell wall, the induction of the phenylpropanoid pathway and synthesis of lignin, the accumulation of antimicrobial compounds named phytoalexins, the synthesis of hydroxyproline-rich glycoproteins (HRGPs) and fungal wall degrading enzymes (chitinases, glucanases), and the production of ethylene. The effectiveness of the plant defense responses against a pathogen depends both on the magnitude and on the rapidity of their onset (Dixon and Lamb, 1990).

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.

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.

METABOLISM OF ARACHIDONIC-ACID INVOLVED IN ITS ELICITING ACTIVITY IN POTATO-TUBER

A 5-lipoxygenase from potato converts arachidonic acid (AA) to the unstable and reactive intermediate 5-S-hydroperoxyeicosatetraenoic acid (5-S-HPETE) which, at a concentration of 1 μg per tuber slice, induces phytoalexin accumulation to higher levels than those induced by AA at 20 μg per slice. However the tissue browning elicited by the 5-S-HPETE was lower than that elicited by AA, indicating that 5-S-HPETE may account for phytoalexin induction but not for the whole browning process. GC-MS analyses of lipid extracts of potato tuber slices treated with 1-[14C]AA and other polyunsaturated fatty acids showed no incorporation of these acids into potato lipid fractions.

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.