Giuseppe Lima

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.

Effectiveness of Aureobasidium pullulans and Candida oleophila against postharvest strawberry rots

Abstract Many yeasts, including yeast-like fungi, were selectively isolated from fruits and vegetables. In several assays performed on strawberries, table grape berries and kiwifruit, the yeast-like fungus Aureobasidium pullulans L47 and the yeasts Candida vanderwaltii L60 and C. oleophila L66 were the most effective antagonists of Botrytis cinerea and Rhizopus stolonifer . Isolates L47 and L66 were utilized in trials on strawberries grown under plastic tunnels. They were applied at flowering (full bloom and late petal fall) and at fruit maturity (just before or after harvest). Isolate L47 was the most effective against both B. cinerea and R. stolonifer . Both antagonists were more active when applied at the flowering stage, with isolate L47 more effective than vinclozolin. The antagonist population was consistently high on flowers and developing fruits, and on cold-stored strawberries. Isolates L47 and L66 showed a low sensitivity towards some fungicides in culture and were able to grow at temperatures between those of cold storage and 33 °C. Competition for nutrients seems to be the main mode of action.

Use of UV-C light to reduce Botrytis storage rot of table grapes

Abstract Single table grape berries ( Vitis vinifera L. cv. Italia) were irradiated with ultraviolet-C (UV-C) doses ranging from 0.125 to 4 kJ m −2 and inoculated with Botrytis cinerea . The pathogen was inoculated on artificial wounds at different times (0, 24, 48, 72, 96 and 144 h) after irradiation and the berries were stored either at 21 or 3°C. To check the influence of UV-C irradiation on the wound-healing processes, trials using berries wounded just before the UV-C irradiation and inoculated at different times were also performed. Significantly lower numbers of infected berries and lesion diameter were found in berries treated with UV-C doses ranging from 0.125 to 0.5 kJ m −2 . There was also a significantly lower level of disease in berries inoculated after 24–48 h than in those inoculated just after (10–15 min) the UV-C treatment. Thus, pretreatment with low UV-C doses followed by artificial inoculation with B. cinerea reduces postharvest grey mould of table grapes, suggesting induced resistance to the disease, both in berries wounded before and after irradiation. The microbial epiphytic population on UV-C-treated berries was also monitored. Results showed a significantly higher increase in the population of yeasts (including yeast-like fungi) and bacteria on berries irradiated with 0.25 and 0.5 kJ m −2 than on unirradiated control berries.

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.

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...

Influence of antagonist, host fruit and pathogen on the biological control of postharvest fungal diseases by yeasts

The yeasts Rhodotorula glutinis (LS-11), Cryptococcus laurentii (LS-28), Candida famata (21-D) and Pichia guilliermondii (29-A) and the yeast-like fungus Aureobasidium pullulans (LS-30), previously selected and characterized for mechanisms of action and antagonistic activity against postharvest pathogens in small and large-scale experiments, were used in this study in order to assess interrelationships among the main factors (antagonist, host fruit and fungal pathogen) involved in biological control of postharvest diseases. The antagonists were evaluated for their inhibitory activity (IA) against six common postharvest fungal pathogens on six different host fruits. Artificially wounded fruits were first inoculated with the antagonist and 2 h later with the pathogen; subsequently they were kept at 20°C for 4–6 days. The IA of each antagonist was evaluated and data were submitted to factorial analysis of variance. The populations of antagonists were also monitored on wounded and unwounded fruits kept at 20°C for 7 days. Each factor examined (antagonist, host fruit and fungal pathogen) as well as their interactions significantly affected the IA. However, among the antagonists, isolates LS-28 and LS-30 were only slightly affected by both host and pathogen, showing a wide range of activity, whereas isolate LS-11 had a variable IA. All the antagonists rapidly colonized the wounds, while their population remained substantially unchanged on unwounded fruits. These results suggest that in order to select yeasts with a broad spectrum of action, more suitable for commercial development, it would be advantageous to perform preliminary assays against several pathogens and in particular on different fruit species.

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.

Biocontrol by yeasts of blue mould of citrus fruits and the mode of action of an isolate of Pichia guilliermondii

SummaryNineteen yeast isolates, from different fruits, were tested for antagonistic activity against blue mould (Penicillium italicum) on artificially wounded citrus fruits. Strains 1 A, 5A, and 29A of Pichia guilliermondii, strains 21D, 22D and 30F of Candida famata and strain 13 L of C. sake reduced infection from 86 to 98% compared with the control. Isolate 5 A of P. guilliermondii was the most effective antagonist and its mode of action was investigated further. Its activity against the pathogen declined when the number of yeast cells inoculated in the wound was reduced although it showed good ability to colonize the wound site and grow rapidly, cell numbers increasing by up to 60-fold within 24 h. Scanning electron microscopy of the wound also showed attachment of the yeast cells to the pathogen hyphae. The high antagonistic activity of isolate 5A appeared to be related to its capability to occupy space and use nutrients available in the wound, thus depriving the pathogen of them. It also induced in ...

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.

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.