First Report of <i>Nigrospora oryzae</i> Causing Wilt in Summer Cypress (<i>Bassia scoparia)</i> in Pakistan

Summer Cypress (Bassia scoparia) is a large annual herb belonging to the family Amaranthaceae native to Eurasia. It has been introduced in many other countries of the world. In Pakistan, summer cypress is also known as kochia and grown as an ornamental plant for its red fall foliage for landscapes. During October, 2017 a survey was conducted in Punjab Province, Pakistan, where 100 wilted plant samples were collected from 30 different plantations of Faisalabad district. Up to 50% loss of plantation was noted in all visited locations. Lower parts of the plants were affected first presenting with necrosis of leaf tips surrounded by a chlorotic zone (Fig. 1. A). Then necrosis of apical margins of the plant parts occurred, followed by stem discoloration and wilting of entire herbaceous branches, leading to the partial wilting of the plants. Ultimately, whole plant wilted and died, (Fig. 1. B) appearing as though they had been scorched. Diseased tissues from lower stem (crown portion) were sampled, surface sterilized in 70 % ethanol for 30 s, and cultured on to Potato Dextrose Agar (PDA) medium. Petri dishes were incubated at 25 ˚C with alternating 12-hour periods of light and dark. Frequently observed, fast growing whitish grey fungal cultures with black pin head points were obtained after 7 days (Fig. 1. C). Young conidia were one-celled, yellow to orange in color and turned brown to black (Fig. 1. D & E), ranged in size from 11 μm to 16 μm x 9.5 μm to 12 μm (Fig. 1. F), and were ellipsoidal at maturity (Fig. 2. A). Hyphae were branched, septate and dark brown in color while conidiophores were flexuous, branched and ranged between 3.5 μm to 4.5 μm in diameter and 14.5 μm to 26.5 μm in length. Based on morphology (Ellis, 1971), the pathogen was identified as Nigrospora oryzae and submitted to the Westerdijk Collection of Fungi, Netherland (CBS 146145/RNOEG30). Total DNA of isolate EG30 was extracted and portions of the Internal transcribed spacer (ITS) region and beta-tubulin (βt) gene were amplified using the universal primers ITS1F and ITS4 (White et al., 1990) and βt2a and βt2b (Glass and Donaldson, 1995). The generated ITS (GenBank Accession No. MG745331.1 491 bp) and βt (GenBank Accession No. MN629896 408 bp) sequences were searched against GenBank using BLASTn and were 99% homologous to ITS (KX986074 525 bp ; MN341493 550 bp) and 100% homologous to βt (MK262852 409 bp) gene region from Nigrospora oryzae (Wang et al., 2017; Zhang, 2019). For pathogenicity tests, ten healthy two-month-old summer cypress plants were inoculated by soil drenching of a spore suspension (106-107 spores/mL) of the fungal isolate EG30 while five plants were treated with sterilized water and used as control treatments. Plants were incubated at 60 to 75% relative humidity (RH) and 25 ˚C in a greenhouse. Leaf necrosis and partial to whole plant witling (Fig. 2. B & C) were observed in the inoculated plants after 21 days. No symptoms appeared in control plants. A fungus was re-isolated from the lower stem (crown portion) parts of the inoculated plants that was identical in morphology to isolate EG30. No fungus resembling EG30 was isolated from the control plants. To the best of our knowledge, this is the first report of summer cypress wilt caused by Nigrospora oryzae (Berk. and Broome) Petch, a known pathogen of several important crops in China, Australia, India, Canada, and Pakistan (Sharma et al., 2013).

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