The tangerine pathotype of Alternaria alternata affects many citrus cultivars, resulting in yield losses. The capability to produce the host-selective toxin and cell-wall-degrading enzymes and to mitigate toxic reactive oxygen species is crucial for A. alternata pathogenesis to citrus. Little is known about nutrient availability within citrus tissues to the fungal pathogen. In the present study, we assess the infectivity of a biotin deficiency mutant (ΔbioB) and a complementation strain (CP36) on citrus leaves to determine how biotin impacts A. alternata pathogenesis. Growth and sporulation of ΔbioB are highly dependent on biotin. ΔbioB retains its ability to acquire and transport biotin from the surrounding environment. Growth deficiency of ΔbioB can also be partially restored by the presence of oleic acid or Tween 20, suggesting the requirement of biotin in lipid metabolism. Experimental evidence indicates that de novo biotin biosynthesis is regulated by the NADPH oxidase, implicating in the production of H2O2, and is affected by the function of peroxisomes. Three genes involved in the biosynthesis of biotin are clustered and co-regulated by biotin indicating a transcriptional feedback loop activation. Infectivity assays using fungal mycelium reveal that ΔbioB cultured on medium without biotin fails to infect citrus leaves; co-inoculation with biotin fully restores infectivity. The CP36 strain re-expressing a functional copy of bioB displays wild-type growth, sporulation and virulence. Taken together, we conclude that the attainability or accessibility of biotin is extremely restricted in citrus cells. A. alternata must be able to synthesize biotin in order to utilize nutrients for growth, colonization and development within the host.
Keywords: Tween 20; citrus; oleic acid; peroxisomes; virulence.
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