Evaluation of pathogenicity factors in biocontrol yeasts Kluyveromyces thermotolerans isolated from Argentinean vineyards

NALLY, M.C.; PONSONE M.L.; TORO M.E.; VAZQUEZ F.; CHULZE, S.N

Apulia, Martina Franca

Congreso; ISM-MycoRed International Conference; 2013

ISM-MycoRed

Introduction: Biological control has been proposed as a strategy to reduce the impact of ochratoxigenic fungal species in vineyards. However, numerous cases of clinical infections caused by yeasts have been reported in the literature in recent years, particularly in immunocompromised. This shows once more the importance to study human pathogenicity factors of biocontrol yeasts. Some fungal properties are frequently associated with pathogenesis, e.g. the ability to grow at high temperatures, to adhere and invade host cells and secrete degradative enzymes such as proteinases and phospholipases. Objective: To evaluate pathogenic factors related with humans in biocontrol yeasts Kluyveromyces thermotolerans. Materials and methods: 1-Yeasts strains: K. thermotolerans RCKT4 and RCKT5 isolated from vineyard (Mendoza, Argentina). These strains were able to control Aspergillus carbonarius and A. niger aggregate species growth and ochratoxin A accumulation. 2- Pathogenic Assays: a- Growth at 37 °C and 42 °C: Ten µL (106cfu/mL) of the bio-control yeast cultures were plated onto YEPD-Agar. Plates were incubated at 37 °C, 42 °C and 25 °C (control) for 3 days. b- Phospholipases: This activity was detected with Egg–Yolk medium. Plates were inoculated with 10 µL drops of suspended yeast cells (106 cfu/mL) and incubated at 30 °C for 7–10 d. c- Proteases: This activity was detected in bovine serum albumin (BSA) test medium. Each plate was inoculated with 10 µL of suspended yeast cells (106 cfu/mL) and plates were incubated at 37 °C for 4 d. d- Pseudohyphal formation: Biocontrol yeasts (10µL, 106 cfu/mL) were streaked onto SLAD-Agar, and plates were incubated at 30 °C and observed after 4 d. e- Invasive growth: Biocontrol yeasts were inoculated on YEPD-Agar, and plates were first incubated at 30 °C for 3 d and then at room temperature for an additional 2 d. f- control: Human pathogenic yeast, Candida albicans ATCC10231, was used as positive control in all pathogenicity assays. Results: The biocontrol yeasts K. thermotolerans RCKT4 and RCKT5 grew at 37 °C and not grew at 42 °C. Non pseudohyphal formation, invasive growth, phospholipase and proteolityc activities were observed in the biocontrol yeasts assayed. None of the strains assayed showed all the pathogenic characteristics at the same time Conclusions. Therefore these yeasts can be used as biofungicide in field conditions. The possibility of using biocontrol agents no pathogenic as a tool to reduce the impact of grape rot by Aspergillus and to diminish the use of chemical fungicides in vineyard is relevant.