First report of Neopestalotiopsis clavispora causing cashew leaf blight disease in India.

Cashew (Anacardium occidentale) is an important commercial crop and highly prone to many biotic and abiotic stress. During March 2021, severe leaf blight symptoms were observed in Priyanka variety with 25-30% incidence grown under greenhouse nursery at ICAR-Directorate of Cashew Research (ICAR-DCR), Puttur (12º74'08.92"N; 75º22'97.22"E), Karnataka. Initial symptoms include small, irregular necrotic spots and later, the spots enlarged and covered major portion of the leaf lamina. In severe infection, leaves exhibited coalescing of spots leading to blight appearance. The infected leaves were randomly collected (n=5) and surface sterilized with 1% sodium hypochlorite for 1 min followed by three washes in sterile distilled water (SDW). Samples were plated on PDA plates amended with Rifampicin (40 mg/L) and kept for incubation at 25±2 oC for 5 days (12/12 h dark light period). A white-greyish, aerial, cottony mycelium on upper side with light yellow colour on the reverse side was consistently isolated. The black viscous acervuli were observed after 10-12 days of incubation. The conidia were fusiform, five-celled, versicoloured with three olivaceous brown median cells, two terminal hyaline cells, measured 23.3±2.12 - 28.33±2.7 x 3.6±0.8 - 4.28±0.78 µm (n=30). The apical cells had two to three flexuous, unbranched appendages, and basal appendage was solitary, tubular and unbranched. Morphological and cultural characteristics confirmed the pathogen as Neopestalotiopsis sp. (Maharachchikumbura et al. 2012). Further, two representative isolates (CLB_SCN1 & CLB_SCN2) were subjected for molecular characterization selected for molecular identification based on ITS-rDNA, tef-1α and tub2 gene sequences and phylogenetic analysis. Genomic DNA was isolated from 15 days old cultures and internal transcribed spacer (ITS) of ribosomal DNA (rDNA) (White et al. 1990), translation elongation factor 1α (tef-1α) gene (O'Donnell et al. 1998) and beta tubulin (tub2) using ITS1/ITS4, TEF1/TEF2 and Bt2a/Bt2b (Carbone and Kohn 1999; Glass and Donaldson 1995) were amplified using primer pairs respectively. PCR amplicons were sequenced, and the sequences were deposited in GenBank (accession numbers: ITS: OP880881.1, OP880882.1; tef-1α: OP882579.1, OP882580.1; and tub2: OP882581., OP882582.1). The phylogeny was constructed based on combined ITS, tef-1a, and tub2 regions. Neighbour-Joining (NJ) analysis was conducted and the tree was constructed with the substitution models (branch support was evaluated by 1,000 bootstrap replications). Combined phylogeny confirmed that the sequences shared a common clade with N. clavispora. Hence, morphological, microscopic and molecular characterization confirmed the pathogen as N. clavispora. The pathogenicity test was done on six months old healthy grafts of Priyanka variety (n=9) and repeated thrice. Conidial suspension (2×106 spores/ml) of N. clavispora CLB_SCN1 (15 days old culture) was sprayed on the healthy cashew seedlings, and kept in greenhouse by covering with polythene bags for 24 h (>80 % RH) and maintained under greenhouse condition. The control grafts were inoculated with SDW. The inoculated plants showed blight symptoms after 7-10-day post inoculation and control remained heathy. Re-isolation was done from the symptomatic leaves and identity was confirmed using cultural and molecular studies. Earlier reports showed that, N. clavispora has been reported to cause cardamom leaf blight (Biju et al 2018) and leaf spot disease of plum (Banerjee and Rana 2020). To best of our knowledge, this is the first report of cashew leaf blight disease caused by N. clavispora from India (Farr and Rossman, 2022). Early detection will help farmer in better management and avoiding economic loss caused by N. clavispora.