Summary 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.

The recently characterized strain Pseudomonas orientalis F9, an isolate from apple flowers in a Swiss orchard, exhibits antagonistic traits against phytopathogens. At high colonization densities, it exhibits phytotoxicity against apple flowers. P. orientalis F9 harbors biosynthesis genes for the siderophore pyoverdine as well as for the antibiotics safracin and phenazine. To elucidate the role of the three compounds in biocontrol, we screened a large random knockout library of P. orientalis F9 strains for lack of pyoverdine production or in vitro antagonism. Transposon mutants that lacked the ability for fluorescence carried transposons in pyoverdine production genes. Mutants unable to antagonize Erwinia amylovora in an in vitro double-layer assay carried transposon insertions in the safracin gene cluster. As no phenazine transposon mutant could be identified using the chosen selection criteria, we constructed a site-directed deletion mutant. Pyoverdine-, safracin-, and phenazine mutants were tested for their abilities to counteract the fire blight pathogen Erwinia amylovora ex vivo on apple flowers or the soilborne pathogen Pythium ultimum in vivo in a soil microcosm. In contrast to some in vitro assays, ex vivo and in vivo assays did not reveal significant differences between parental and mutant strains in their antagonistic activities. This suggests that, ex vivo and in vivo, other factors, such as competition for resources or space, are more important than the tested antibiotics or pyoverdine for successful antagonism of P. orientalis F9 against phytopathogens in the performed assays. IMPORTANCE Pseudomonas orientalis F9 is an antagonist of the economically important phytopathogen Erwinia amylovora, the causal agent of fire blight in pomme fruit. On King’s B medium, P. orientalis F9 produces a pyoverdine siderophore and the antibiotic safracin. P. orientalis F9 transposon mutants lacking these factors fail to antagonize E. amylovora, depending on the in vitro assay. On isolated flowers and in soil microcosms, however, pyoverdine, safracin, and phenazine mutants control phytopathogens as clearly as their parental strains.

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.

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Metschnikowia pulcherrima synthesizes the red pigment pulcherrimin, from cyclodileucine (cyclo(Leu-Leu)) as a precursor, and exhibits strong antifungal activity against notorious plant pathogenic fungi such as Botrytis and Gibberella (i.e., Fusarium). 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 wildtype strain with a spontaneously occurring, pigmentless, weakly antagonistic mutant derivative. Whole genome sequencing of the wildtype and mutant strains identified a point mutation that creates a premature stop codon in the transcriptional regulator SNF2 in the mutant strain. Complementation of the snf2 mutant strain with the wildtype SNF2 gene restored pigmentation and recovered the strong antifungal activity of M. pulcherrima against plant pathogens in vitro and on cherries. Ultra-performance liquid chromatography-high resolution heated electrospray ionization mass spectrometry (UPLC HR HESI-MS) proved the presence and structure of the pulcherrimin precursors cyclo(Leu-Leu) and pulcherriminic acid and also identified new compounds that likely represented an additional precursor and degradation products of pulcherriminic acid and/or pulcherrimin. All of these compounds were identified in the wildtype 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. Significance statementMetschnikowia pulcherrima is a strongly antifungal yeast and a most promising species for the control of notorious plant diseases. This multidisciplinary study on the M. pulcherrima mode of action compared a wildtype isolate with a pigmentless mutant exhibiting reduced antifungal activity. The transcriptional regulator Snf2 was identified as a "biocontrol regulator" controlling antifungal activity of M. pulcherrima via PUL gene transcription, cyclodipeptide synthesis and additional, yet uncharacterized mechanisms. The identification of cyclo(Leu-Leu), pulcherriminic acid, as well as novel precursor and degradation products of pulcherrimin, opens up new avenues for research on the metabolism and functions of pulcherrimin. Overall, this works establishes M. pulcherrima as a genetically tractable model and will benefit the development of biocontrol solutions for important plant diseases. BIOLOGICAL SCIENCES, MicrobiologyGenome sequencing & mapping of mutations Genomic DNA of the M. pulcherrima strain APC 1.2 was extracted using the Qiagen DNeasy Plant Mini Kit and sequenced on the PacBio RS II platform (performed at the Functional Genomics Center Zurich). Subsequent de novo genome assembly, polishing and resequencing were performed using PacBio SMRT Portal 2.3.0...