First Report of Leaf Spot on Medicinal Herb Picrorhiza kurroa (Royle ex. Benth) Caused by Alternaria tenuissima in India.

Picrorhiza kurroa Royle ex. Benth is an herbaceous perennial plant of the Schrophulariaceae family, native to the northwestern Himalayas, and valued for its hepatoprotective, antioxidant, anti-inflammatory, and immunomodulatory activities (Banerjee et al. 2008). Its medicinal properties are attributed to iridoid glycosides, picroside I (P-I), and picroside II (P-II) (Gao and Zhou 2005). Leaf blight was observed in P. kurroa plants growing in a nursery of the Himalayan Forest Research Institute, Manali, Himachal Pradesh (20°35.6′ to 32°6.1′ N; 78°57.8′ to 77°33.7′ E; 1,900 m elevation) during July and August 2014. Symptoms were small, circular, brown necrotic spots on the abaxial and adaxial sides of the leaves. Lesions gradually increased in size and coalesced irregularly, resulting in withering, extensive drying, and shedding. The pathogen was isolated by placing the margin of surface-sterilized necrotic leaf tissue on potato dextrose agar (PDA) and the resulting hyphae were subcultured onto PDA. The fungal colony was olive-green with smooth edges. The culture was further purified to a single-spore isolate. Conidiophores were short, arising singly, measuring 81.6 to 163 μm long and 4 to 8 μm wide. Conidia varied from 26 to 48 μm long and 9.8 to 16 μm wide, with a beak length of 5 to 8 μm (n = 50). Horizontal and vertical septations of conidia varied from 1 to 6 and 0 to 2, respectively (Kou et al. 2014). On the basis of morphological characteristics of conidiophores and conidia, the pathogen was identified as Alternaria sp. (Simmons 1999). Mycelial genomic DNA was extracted and a portion of the ITS1/4 (402 bp) was amplified by PCR. Sequencing was carried out using Sanger’s method and MegaBLAST analysis of amplicon sequence with 2× coverage showed 100% similarity (e-value 0.0) with Alternaria tenuissima GenBank Accession No. KM921667.1. Sequencing information for ITS 1/4 region has been submitted to GenBank (KT823704). For species validation, gene-specific primers corresponding to A. tenuissima histone (CN-L-01), Hrip1, and LEU2 genes were amplified using genomic DNA which showed 99% similarity to A. tenuissima accessions EF371552.1, HQ713431.1, and EU294180.1, respectively. Koch’s postulates were conducted to prove pathogenicity by inoculating twelve detached, surface-sterilized healthy leaves with 5 μl (1 × 105 spores/ml) conidia suspended in sterile water. Inoculated as well as nontreated controls were incubated in a growth chamber with 80 to 90% relative humidity and 12-h photoperiod. After 72 h, spots similar to infected field plants developed on all inoculated parts of the leaves. The lesions enlarged with time whereas control leaves remained asymptomatic. The morphological traits of the fungal spores reisolated from inoculated leaves were similar to those isolated previously from the infected plants. Leaves are a major source of P-I in P. kurroa; therefore, fungal infestations can cause significant reductions in leaf biomass. This can affect supply of quality raw material for herbal preparations as the majority of plant material is collected from the wild. Proper identification of this pathogen would facilitate investigation of sustainable disease control in this pathosystem and development of efficient cultivation methods for this limited plant resource. To our knowledge, this is the first report of A. tenuissima causing leaf spot on P. kurroa. © 2016, American Phytopathological Society. All rights reserved.