Metschnikowia pulcherrima synthesises the pigment pulcherrimin, from cyclodileucine (cyclo(Leu-Leu)) as a precursor, and exhibits strong antifungal activity against notorious plant pathogenic fungi. This yeast therefore has great potential for biocontrol applications against fungal diseases; particularly in the phyllosphere where this species is frequently found. To elucidate the molecular basis of the antifungal activity of M. pulcherrima, we compared a wild-type strain with a spontaneously occurring, pigmentless, weakly antagonistic mutant derivative. Whole genome sequencing of the wild-type and mutant strains identified a point mutation that creates a premature stop codon in the transcriptional regulator gene SNF2 in the mutant. Complementation of the mutant strain with the wild-type SNF2 gene restored pigmentation and recovered the strong antifungal activity. Mass spectrometry (UPLC HR HESI-MS) proved the presence of the pulcherrimin precursors cyclo(Leu-Leu) and pulcherriminic acid and identified new precursor and degradation products of pulcherriminic acid and/or pulcherrimin. All of these compounds were identified in the wild-type and complemented strain, but were undetectable in the pigmentless snf2 mutant strain. These results thus identify Snf2 as a regulator of antifungal activity and pulcherriminic acid biosynthesis in M. pulcherrima and provide a starting point for deciphering the molecular functions underlying the antagonistic activity of this yeast.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/mmi.14272

Snf2 controls pulcherriminic acid biosynthesis and antifungal activity of the biocontrol yeast Metschnikowia pulcherrima.

Successful biocontrol of major postharvest and soil-borne plant pathogenic fungi by antagonistic yeasts

Undesired fermentation of fruit-derived beverages by fungal, yeast and bacterial spoilage organisms are among the major contributors of product losses in the food industry. As an alternative to chemical preservatives, the use of Candida pyralidae and Pichia kluyveri was assessed for antimicrobial activity against several yeasts (Dekkera bruxellensis, Dekkera anomala, Zygosaccharomyces bailii) and fungi (Botrytis cinerea, Colletotrichum acutatum and Rhizopus stolonifer) associated with spoilage of fruit and fruit-derived beverages. The antagonistic properties of C. pyralidae and P. kluyveri were evaluated on cheap solidified medium (grape pomace extract) as well as on fruits (grapes and apples). Volatile organic compounds (VOCs) from C. pyralidae and P. kluyveri deemed to have antimicrobial activity were identified by gas chromatography-mass spectrometry (GC-MS). A cell suspension of C. pyralidae and P. kluyveri showed growth inhibition activity against all spoilage microorganisms studied. Direct contact and extracellular VOCs were two of the mechanisms of inhibition. Twenty-five VOCs belonging to the categories of alcohols, organic acids and esters were identified as potential sources for the biocontrol activity observed in this study. This study reports, for the first time, the ability of C. pyralidae to inhibit fungal growth and also for P. kluyveri to show growth inhibition activity against spoilage organisms (n = 6) in a single study.

The Use of Candida pyralidae and Pichia kluyveri to Control Spoilage Microorganisms of Raw Fruits Used for Beverage Production

Background Penicillium digitatum is one of the most important pathogens causing citrus green mold, leading to significant economic losses. Traditionally, synthetic fungicides are used to control diseases. However, the side effects of fungicides should not be ignored. Thus, antagonistic yeasts were proposed to be safe and effective alternatives for managing diseases. Orchards are excellent sources of naturally occurring antagonists against pathogens. Therefore, in the present study, antagonistic yeasts obtained from orchards were screened, and the possible biocontrol mechanisms of the most promising yeast were investigated. Results Seventy-eight isolates of yeasts (15 species of 10 genera) were obtained from citrus orchards. In in vitro assays, 16 strains showed antifungal activity against Pichia digitatum and 15 strains showed biocontrol potential against green mold on Olinda oranges. Pichia galeiformis (BAF03) exhibited the best antagonistic activity against P. digitatum during 6 days storage at 25 °C and a good antagonistic activity during 29 days at 4 °C. Pichia galeiformis (BAF03) could colonize and amplify quickly in wounded citrus. Scanning electron microscopy results showed that the citrus wound was colonised by the yeast. A total of eight volatile organic compounds (VOCs) were identified by gas chromatography-mass spectrometry The VOCs produced by P. galeiformis (BAF03) efficiently inhibited P. digitatum. Conclusion Pichia galeiformis (BAF03) isolated from a citrus orchard showed potential to control postharvest green mold of citrus. The possible mechanisms of action likely include competition for space and nutrients as well as production of VOCs.

Screening antagonistic yeasts against citrus green mold and the possible biocontrol mechanisms of Pichia galeiformis (BAF03)

Nano/microencapsulation of plant biocontrol agents by chitosan, alginate, and other important biopolymers as a novel strategy for alleviating plant biotic stresses.

Antagonistic yeasts suppress plant pathogenic fungi by various mechanisms, but their biocontrol efficacy also depends on the ability to compete and persist in the environment. The goal of the work presented here was to quantify the composition of synthetic yeast communities in order to determine the competitiveness of different species and identify promising candidates for plant protection. For this purpose, colony counting of distinct species and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS; MALDI biotyping) were used to distinguish different yeast species and to quantify the composition of a synthetic community of six yeasts (Aureobasidium pullulans, Candida subhashii, Cyberlindnera sargentensis, Hanseniaspora sp., Metschnikowia pulcherrima and Pichia kluyveri) over time, on apples and in soil, and in different growth media. These studies revealed important characteristics that predispose the different species for particular applications. For example, the competitiveness and antagonistic activity of C. subhashii was strongly increased in the presence of N-acetylglucosamin as the sole carbon source, M. pulcherrima and A. pullulans were the strongest competitors on apple, and C. sargentensis competed the best in soil microcosms. Based on these laboratory studies, M. pulcherrima and A. pullulans are promising candidates for biocontrol applications against fungal phyllosphere diseases, while C. sargentensis may hold potential for use against soilborne fungal pathogens. These results document the potential of MALDI-TOF MS for the quantitative analysis of synthetic yeast communities and highlight the value of studying microorganisms with relevant functions in moderately complex, synthetic communities and natural substrates rather than as individual isolates.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/yea.3321

Denise C. Müller

Papers

Characterization of antagonistic yeasts for biocontrol applications on apples or in soil by quantitative analyses of synthetic yeast communities.