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.     

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.     

Effect of moisture on physical parameters of timber from Turkey oak (<Emphasis Type="Italic">Quercus cerris</Emphasis> L.) coppice in Central Italy

This study aims to investigate wood density at different levels of moisture, basic density and shrinkage of timber from Turkey oak (Quercus cerris L.) coppice forests growing in Central Italy. We also studied the variability in density in the trees within and among sites. Density shows no significant statistical differences in the tested population. A higher variability in the shrinkage than in the density was found. Wood moisture is referred to as dry mass and fresh mass, which is related to many performance characteristics of wood, i.e., energy production. Trends in moisture and water content were studied because these physical parameters play an important role in the specific area of firewood which requires an accurate estimation of mass, volume and energy content. This work is a contribution to improve xylo-energy estimates of small and medium forestry issues.     

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.     

Mutations in Cyp51 of Venturia inaequalis and their effects on DMI sensitivity

Journal of Plant Diseases and Protection - DMIs (sterol demethylation inhibitors) provide an important mode of action for control of Venturia inaequalis. They target the enzyme 14a-demethylase,...     

Stumpage value in the short wood system for the conversion into high forest of a oak coppice

This study aims to analyze the factors affecting the costs of different logging systems, in particular considering three different bunching-extraction methods. Moreover the stumpage value of each plot was calculated, and a comparison analysis of the results is presented. The work was carried out in Central Italy, in an aged Quercus cerris L. coppice in hill zones, with a 45% average slope. Short wood system harvesting was applied. Felling and processing were performed by chainsaw, while extraction with three different methods: plot No. 1 with mules, plot No. 2 with a tractor with winch, plot No. 3 with polyethylene chute line. Transport of firewood from the forest track to the landing was made by a tractor loaded as a mule. In the plot No. 1 the costs per unit mass (30.76 ? 33% -1 ) were higher despite having the lower cost per hour (39.99 ? -1 ). The reason was the lower productivity (1.3 t 33% ·h -1 ). The plot No. 2 had the highest cost per hour (66.79 ? -1 ). However, productivity was the highest (2.7 t 33% ·h -1 ). This aspect was notable from the financial point of view (24.74 ? 33% -1 ). Plot No. 3 was the less expensive (23.92 ? 33% -1 ). Comparing the three methods, a reduction of the costs through more appropriate extraction systems may increase the stumpage value from 8.3 to 9.65 ? 33% -1 .     

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.     

Reinvigoration of mature chestnut (Castanea sativa) by repeated graftings and micropropagation

Gradual reinvigoration of adult chestnut (Castanea sativa M. cv. Montemarano) shoots was obtained by serial grafting onto juvenile rootstocks. The phenomenon was evaluated on the basis of percentage of primary nodes regenerating axillary shoots and length and number of shoots (> 10 mm) per primary node. In vitro growth of explants from serially grafted shoots was significantly lower than that of explants from seedlings at the end of the establishment phase. Only microshoots from seedlings and plants that had been serially grafted four times could be subcultured on proliferation medium. Repeated subculture on medium containing a low cytokinin concentration induced progressive reinvigoration of microshoots derived from plants that had been serially grafted four times. The number of axillary shoots per explant increased significantly after six subcultures. After 12 subcultures, microshoots from serially grafted plants showed an increase in stem elongation, rooting and plantlet survival. After in vitro stabilization, there was no difference in in vitro performance between microshoots derived from seedlings and serially grafted plants. Microshoots multiplied from serially grafted plants displayed only a transitory appearance of juvenile traits.     

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.