FIRST REPORT OF NIGROSPORA SPHAERICA CAUSING LEAF SPOT OF KINNOW MANDARIN IN PAKISTAN

Abstract

A total of 139,000 hectares of Kinnow mandarin is under cultivation in Pakistan. In December 2015, a new disease characterized by leaf spots was detected on Kinnow mandarin in Punjab. The disease was widespread and severe in 40% of visited orchards. Initial symptoms on young leaves were small, semicircular to irregularly shaped brown spots surrounded by a yellow halo. Spots on affected leaves later turned dark brown, coalesced, and leaves sometimes fell off. Symptomatic leaf segments were disinfected and then plated aseptically on potato dextrose agar (PDA). After 5 days at 25±2 o C, the fungus developed flat, light to dark grey colonies with dark brown, septate, branched hyphae. Conidia were black, one-celled (12.4-18.6 µm) and borne at the tip of each conidiophore. The fungus was identified as Nigrospora sphaerica (Dutta et al., 2015). The internal transcribed spacer (ITS) region of rDNA was sequenced using ITS1/ITS4 primers and accession no. KX834821 was 99% identical to N. sphaerica accession no. HQ608063.1. For pathogenicity test, a conidial suspension (106 conidia/ml) from a 7-day-old culture of N. sphaerica was used to wound inoculate 10 seedlings of cv. Kinnow mandarin, followed by incubation in a controlled environment chamber at 25°C with 70 to 80% humidity. As a control, three seedlings were inoculated with sterile distilled water only. Two weeks after inoculation, symptoms observed only on the inoculated leaves and the fungus was consistently re-isolated. To our knowledge, this is the first report of N. sphaerica on Kinnow mandarin in Pakistan.