Wang Shupei, Zhang Hongyan, Ruan Changqing, Yi Lanhua, Deng Lili, Zeng Kaifang
College of Food Science, Southwest University, Chongqing 400715, PR China; College of Environmental and Life Sciences, Nanning Normal university, Nanning 530001, PR China.
College of Food Science, Southwest University, Chongqing 400715, PR China.
Int J Food Microbiol. 2021 Nov 2;357:109384. doi: 10.1016/j.ijfoodmicro.2021.109384. Epub 2021 Sep 7.
Metschnikowia citriensis FL01 has great potential for biocontrol applications for its excellent biocontrol efficacy on postharvest diseases of citrus fruit, and the iron depletion by pulcherriminic acid (PA) and then formation of insoluble pigment pulcherrimin had been speculated as an important action mechanism. To identify the genes involved in pulcherrimin synthesis and reutilization in M. citriensis FL01, we de novo assembled the genome of M. citriensis FL01 based on long-read PacBio sequencing. The final assembled genome consisted of 12 contigs with a genome size of 25.74 Mb, G + C content of 49.16% and 9310 protein-coding genes. The genome-wide BLAST of the PUL genes of M. pulcherrima APC 1.2 showed that the four PUL genes were clustered and located on Contig 4 of M. citriensis FL01. In order to further clarify the role of pulcherrimin pigment on biocontrol of M. citriensis FL01, CRISPR/cas9 technology was used to knock out PUL2 gene that was responsible for PA synthesis and the pigmentless mutants with stable phenotype were obtained. The mutant strains of M. citriensis FL01 lost the ability to produce pulcherrimin pigment, and simultaneously lost the ability to inhibit the growth of Geotrichum citri-aurantii in vitro. Moreover, the biocontrol efficacy of pigmentless mutant strains against sour rot was about 80% lower than that of wild-type M. citriensis FL01. These results directly proved that the iron depletion was an important mechanism of M. citriensis FL01.
柑桔梅奇酵母FL01对柑桔采后病害具有优异的生防效果，在生物防治应用方面具有巨大潜力，推测其通过腐胺铁酸（PA）耗尽铁并随后形成不溶性色素腐胺铁是一种重要的作用机制。为了鉴定参与柑桔梅奇酵母FL01中腐胺铁合成和再利用的基因，我们基于长读长PacBio测序对柑桔梅奇酵母FL01的基因组进行了从头组装。最终组装的基因组由12个重叠群组成，基因组大小为25.74 Mb，G + C含量为49.16%，有9310个蛋白质编码基因。对梅奇酵母APC 1.2的PUL基因进行全基因组BLAST分析表明，这四个PUL基因成簇并位于柑桔梅奇酵母FL01的重叠群4上。为了进一步阐明腐胺铁色素在柑桔梅奇酵母FL01生物防治中的作用，利用CRISPR/cas9技术敲除了负责PA合成的PUL2基因，并获得了表型稳定的无色素突变体。柑桔梅奇酵母FL01的突变菌株失去了产生腐胺铁色素的能力，同时在体外失去了抑制柑桔地霉生长的能力。此外，无色素突变菌株对酸腐病的生防效果比野生型柑桔梅奇酵母FL01低约80%。这些结果直接证明了铁耗尽是柑桔梅奇酵母FL01的重要机制。
