R. Castoria

Aureobasidium pullulans (LS-30) an antagonist of postharvest pathogens of fruits: study on its modes of action

In small-scale experiments Aureobasidium pullulans (isolate LS-30) displayed significant antagonistic activity against Botrytis cinerea, Penicillium expansum, Rhizopus stolonifer and Aspergillus niger on table grapes, and B. cinerea and P. expansum on apple fruit. To improve the performance of this yeast-like fungus, possible modes of action were investigated. Competition for nutrients appeared to play a role in the activity of this antagonist. Extracellular exochitinase [N-acetyl-b-D-glucosaminidase (Nagase)] and b-1-3-glucanase activities were also detected both in vitro and in apple wounds, which are the main sites of penetration of postharvest fungal pathogens, suggesting that these enzymes may actually be involved in the antagonistic activity of this microorganism. Neither antibiosis nor direct physical interaction of LS-30 cells with the hyphae of B. cinerea appeared to be involved in the activity of this antagonist.

beta-1,3-glucanase activity of two saprophytic yeasts and possible mode of action as biocontrol agents against postharvest diseases

Abstract The yeasts Rhodotorula glutinis (isolate LS-11) and Cryptococcus laurentii (isolate LS-28), showing different levels of antagonistic activity against a range of postharvest pathogens, were examined for their possible mode(s) of action, in order to highlight the reasons for the higher activity of isolate LS-28. Competition for nutrients appeared to play a role in the activity of both yeasts, especially in the case of isolate LS-11. Direct interaction with pathogen hyphae was shown only by cells of this same isolate, whereas no interaction with fungal hyphae was observed for the more active antagonist LS-28. The latter isolate was able to produce in vitro significantly higher levels of extracellular β -1,3-glucanase activity than LS-11 when grown in the presence of hyphal cell walls of the pathogens Penicillium expansum and Botrytis cinerea as sole carbon sources. In our experimental conditions, antibiosis did not appear to be involved in the activity of either antagonists.

Strains of Aureobasidium pullulans can lower ochratoxin A contamination in wine grapes.

Wine contamination with ochratoxin A (OTA) is due to the attack of wine grapes by ochratoxigenic Aspergillus carbonarius and Aspergillus spp. section Nigri. Four A. pullulans strains, AU14-3-1, AU18-3B, AU34-2, and LS30, are resistant to and actively degrade ochratoxin A in vitro. The less toxic ochratoxin alpha and the aminoacid L-beta-phenylalanine were the major degradation products, deriving from the cleavage of the amide bond linking these two moieties of OTA. The same strains were studied further as biocontrol agents of A. carbonarius on wine grapes in laboratory experiments. Three of the four strains significantly prevented infections by A. carbonarius. Berries pretreated with the biocontrol agents and infected with A. carbonarius contained lower amounts of OTA as compared to the untreated infected control berries. Two of these strains were shown to degrade OTA to ochratoxin alpha in fresh grape must, but the mechanisms of the decrease of OTA accumulation in infected berries pretreated with the biocontrol agents remain to be elucidated. Assessment of one strain carried out in the vineyard during the growing season of 2006 showed that the tested strain was an effective biocontrol agent, reducing both severity of Aspergillus rots and OTA accumulation in wine grapes. To our knowledge this is the first report describing the positive influence of biocontrol agents on OTA accumulation in this crop species.

Integration of Biocontrol agents and food-grade additives for enhancing protection of stored apples from Penicillium expansum

Forty-nine compounds currently used as additives in foods were tested in combination with three biocontrol agents, the yeasts Rhodotorula glutinis, Cryptococcus laurentii, and the yeastlike fungus Aureobasidium pullulans, to increase their antagonistic activity against Penicillium expansum, the causal agent of blue mold on apples. Twelve additives dramatically improved the antagonistic activity of one or more of the tested biocontrol agents. In a two-way factorial experiment with these selected additives the percentage of P. expansum rots on apples was significantly influenced by the antagonist and the additive as well as by their interaction. The combination of the biocontrol agents and some additives resulted in a significantly higher activity with respect to the single treatments applied separately, producing additive or synergistic effects. Some of the selected additives combined with a low yeast concentration (106 cells per ml) had comparable or higher efficacy than the biocontrol agents applied alone at a 100-fold higher concentration (10(8) cells per ml). Some organic and inorganic calcium salts, natural gums, and some antioxidants displayed the best results. In general, the effect of each additive was specific to the biocontrol isolate used in the experiments. Possible mechanisms involved in the activity of these beneficial additives and their potential application in effective formulations of postharvest biofungicides are discussed.

Integrated control of apple postharvest pathogens and survival of biocontrol yeasts in semi-commercial conditions

The biocontrol yeast isolates Rhodotorula glutinis LS11, Cryptococcus laurentii LS28 and Aureobasidium pullulans LS30 were tested against Botrytis cinerea and Penicillium expansum on apples artificially inoculated and stored at 3 and 20 °C. Isolates LS28 and LS30 were most effective, consistently resulting in high reductions of fungal decay, while isolate LS11 was effective only on apples stored at 3 °C. The yeasts showed good in vitro resistance to dicarboximides and copper fungicides, while they were inhibited by triazoles. Isolate LS11, in contrast to LS28 and LS30, was also inhibited by benzimidazoles. The yeasts were tested on naturally-infected apples in semi-commercial conditions for 2 years. They were applied twice: soon after harvesting and 20 days later, at the beginning of the cold storage. The antagonists significantly reduced fungal decay when combined with a low dosage of benomyl showing an activity comparable to that exerted by the fungicide alone at full dosage. Periodical monitoring of the epiphytic biocontrol yeast populations in both the field and cold room showed a good rate of survival of the antagonists on the skin of treated apples. Specific fingerprints relying on amplified restriction length polymorphism (AFLP) were used to integrate the morphology-based monitoring of the yeasts.

Environmental factors affect the activity of biocontrol agents against ochratoxigenic Aspergillus carbonarius on wine grape.

The influence of temperature and relative humidity (RH) on the activity of three biocontrol agents-the yeast Metschnikowia pulcherrima LS16 and two strains of the yeast-like fungus Aureobasidium pullulans LS30 and AU34-2-against infection by A. carbonarius and ochratoxin A (OTA) accumulation in wine grape berries was investigated in lab-scale experiments. The presence of wounds on grape skin dramatically favored infection of berries by A. carbonarius strain A1102, since unwounded berries showed very low levels of infection at all conditions of RH and temperature tested. Artificially wounded berries pre-treated with the biocontrol agents were inoculated with the ochratoxigenic A. carbonarius strain A1102 and were incubated for 5 days at two levels of RH (60% and 100%) and three different temperatures (20, 25 and 30 °C). The three biocontrol agents were able to prevent infections at 60% RH and 20 °C. At 60% RH and 25 °C only strain AU34-2 achieved some protection on day 5, whereas at 30 °C a limited biocontrol efficacy was evident only up to day 2. At 100% RH, LS16, LS30 and AU34-2 showed effective protection of grape berries at 20 °C until the 5th day of incubation. The three biocontrol agents achieved significant protection at higher temperatures only until the 2nd day after the beginning of the experiment: all three strains at 25 °C, and only strain LS16 at 30 °C. After 5 days, the three biocontrol agents were able to significantly reduce the level of OTA in berries at all the conditions tested. This occurred even when protection from infection was not significant, except at 30 °C and 100% of RH for all the three strains, and at 25 °C and 100% of RH for strain LS16. The biocontrol agents displayed a higher rate of colonization on grape berries at 20 and 25 °C than at 30 °C. The higher value of RH (100%) appeared to increase the rate of colonization, in particular at 20 and 25 °C. Taken together, our results emphasize the significant influence of environmental factors on the effectiveness of biocontrol against A. carbonarius as well as on OTA contamination in wine grape berries, and the need for biocontrol agents that can cope with the environmental conditions that are conducive to attack by A. carbonarius.

Searching for Genes Responsible for Patulin Degradation in a Biocontrol Yeast Provides Insight into the Basis for Resistance to This Mycotoxin

ABSTRACT Patulin is a mycotoxin that contaminates pome fruits and derived products worldwide. Basidiomycete yeasts belonging to the subphylum Pucciniomycotina have been identified to have the ability to degrade this molecule efficiently and have been explored through different approaches to understand this degradation process. In this study, Sporobolomyces sp. strain IAM 13481 was found to be able to degrade patulin to form two different breakdown products, desoxypatulinic acid and (Z)-ascladiol. To gain insight into the genetic basis of tolerance and degradation of patulin, more than 3,000 transfer DNA (T-DNA) insertional mutants were generated in strain IAM 13481 and screened for the inability to degrade patulin using a bioassay based on the sensitivity of Escherichia coli to patulin. Thirteen mutants showing reduced growth in the presence of patulin were isolated and further characterized. Genes disrupted in patulin-sensitive mutants included homologs of Saccharomyces cerevisiae YCK2, PAC2, DAL5, and VPS8. The patulin-sensitive mutants also exhibited hypersensitivity to reactive oxygen species as well as genotoxic and cell wall-destabilizing agents, suggesting that the inactivated genes are essential for tolerating and overcoming the initial toxicity of patulin. These results support a model whereby patulin degradation occurs through a multistep process that includes an initial tolerance to patulin that utilizes processes common to other external stresses, followed by two separate pathways for degradation.

Two rapid assays for screening of patulin biodegradation

The mycotoxin patulin is produced by the blue mould pathogen Penicillium expansum in rotting apples during postharvest storage. Patulin is toxic to a wide range of organisms, including humans, animals, fungi and bacteria. Wash water from apple packing and processing houses often harbours patulin and fungal spores, which can contaminate the environment. Ubiquitous epiphytic yeasts, such as Rhodosporidium kratochvilovae strain LS11 which is a biocontrol agent of P. expansum in apples, have the capacity to resist the toxicity of patulin and to biodegrade it. Two non-toxic products are formed. One is desoxypatulinic acid. The aim of the work was to develop rapid, high-throughput bioassays for monitoring patulin degradation in multiple samples. Escherichia coli was highly sensitive to patulin, but insensitive to desoxypatulinic acid. This was utilized to develop a detection test for patulin, replacing time-consuming thin layer chromatography or high-performance liquid chromatography. Two assays for patulin degradation were developed, one in liquid medium and the other in semi-solid medium. Both assays allow the contemporary screening of a large number of samples. The liquid medium assay utilizes 96-well microtiter plates and was optimized for using a minimum of patulin. The semi-solid medium assay has the added advantage of slowing down the biodegradation, which allows the study and isolation of transient degradation products. The two assays are complementary and have several areas of utilization, from screening a bank of microorganisms for biodegradation ability to the study of biodegradation pathways.