First report of Diaporthe ukurunduensis causing leaf spot disease of mulberry (Morus alba) in Kashmir Valley, India

Mulberry (Morus alba) is the sole food plant for the silkworm (Bombyx mori), therefore is essential for sericulture. The tree is also utilised for food, fodder, fuel and fibre. Mulberry is affected by several pathogens and fungal leaf spot is an important disease (Maqbool et al., 2021) which reduces leaf yield by 5–30% (Sarkar et al., 2020). The biochemistry of mulberry leaves infected by fungal plant pathogens is also affected which further reduces the quality of the silk produced (Shree & Nataraja, 1993). The present study was carried out to isolate and identify the pathogen responsible to cause the leaf spot disease of mulberry in Kashmir valley, India. Diseased leaves with brown necrotic patches were collected from 3 different areas of Kashmir valley in April 2022 (Figure 1). The leaves were cut into pieces and surface sterilised with 0.2% sodium hypochlorite for 3 minutes, then washed twice with double distilled water. The sterilised pieces were transferred to plates of potato dextrose agar (PDA) and kept in a biochemical oxygen demand incubator at 25±2°C (Hassan et al., 2022). After seven days, the fungal colonies were sub cultured and the isolated fungus was identified on the basis of cultural, microscopic and molecular attributes. The colonies were off-white in colour and flat becoming dark-brown to grey in the centre (Figure 2). Conidiophores were 12.5-19.3 × 1.4 μm, hyaline, smooth, aseptate, branched and slightly curved; alpha conidia were 6.5-12.3 × 1.7-2.5 μm, hyaline, smooth, aseptate, ellipsoidal to filiform, and beta conidia were absent (Figure 3). The fungal pathogen was identified as Diaporthe ukurunduensis (Yang et al., 2018). This was separated from the similar species D. citrichinensis by its longer conidiophores which are also slightly curved, and the absence of beta conidia which are prominent in D. citrichinensis. For molecular confirmation ITS1 and ITS4 primers were used (White et al., 1990). The amplified PCR product was sequenced and deposited in GenBank (Accession No. OP594614), which had 99.7% identity with a sequence of D. ukurunduensis (NR 165880). A pathogenicity test was done on healthy mulberry leaves by misting them with a spore solution of the isolate (1 × 106 spores /ml), whereas control plants were misted with distilled water. Three replicates for each treatment and control were maintained at a temperature of 27 ± 2°C and relative humidity above 80% in a greenhouse for 21 days. Leaf spots developed on all inoculated leaves which resembled the symptoms produced by the pathogen in the field, but the control plants remained healthy. The pathogen D. ukurunduensis was re-isolated successfully. Diaporthe ukurunduensis was first reported by Yang et al. (2018) on the twigs of Acer ukurunduensis. Here, we first report the involvement of D. ukurunduensis in causing the leaf spot disease of mulberry in India and worldwide.