Amplicon sequencing is a powerful tool for analyzing the fungal composition inside plants, whereas its application for the identification of etiology for plant diseases remains undetermined. Here, we utilize this strategy to clarify the etiology responsible for tea leaf brown-black spot disease (LBSD), a noticeable disease infecting tea plants etiology that remains controversial. Based on the ITS-based amplicon sequencing analysis, Didymella species were identified as separate from Pestalotiopsis spp. and Cercospora sp., which are concluded as the etiological agents. This was further confirmed by the fungal isolation and their specific pathogenicity on diverse tea varieties. Based on the morphologies and phylogenetic analysis constructed with multi-loci (ITS, LSU, tub2, and rpb2), two novel Didymella species-tentatively named D. theae and D. theifolia as reference to their host plants-were proposed and characterized. Here, we present an integrated approach of ITS-based amplicon sequencing in combination with fungal isolation and fulfillment of Koch's postulates for etiological identification of tea plant disease, revealing new etiology for LBSD. This contributes useful information for further etiological identification of plant disease based on amplicon sequencing, as well as understanding, prevention, and management of this economically important disease.
Keywords: Didymella; amplicon sequencing; etiological identification; fungal identification; high-throughput sequencing; two new taxa.