First Report of Bipolaris zeae Causing Leaf Spot on Setaria viridis in China.

Abstract

Green bristlegrass (Setaria viridis) is an annual dominant herb in arid sandy grasslands with strong drought resistance to water stress (Valença et al. 2020). In August 2021, brown or dark tan spots on the leaves of S. viridis were observed on natural grassland in Qingyang city, Gansu Province, China (36.55°N, 107.32°E). Leaf disease incidence was around 13%, and the infected area of each leaf was about 20%. For isolation, 30 tissue pieces (5×5 mm) from 10 symptomatic leaf samples were surface-sterilized with 70% ethanol for 30 seconds and rinsed three times with steriled distilled water. Then these tissues were placed on the potato dextrose agar (PDA) at 25 °C, and incubated in dark from 2 to 5 days. Sixteen single-spored cultures with consistent colony characteristics were obtained by single spore isolation as described in Manamgoda et al. (2012). Colonies on PDA were irregular shapes with black color and white edge; conidia were dark brown with septate, cylindrical, straight or flexuous geniculate at the upper end, 22.7 to 69.38×9.2 to 17.6 µm (n = 50; average size 40.8×13.2 µm) under microscope (40X). The internal transcribed spacer (ITS) and glyceraldehyde-3-phosphate dehydrogenase (GPD) regions of the representative isolate 83HXGWC, 84HXGWC and 85HXGWC were amplified using the primers of ITS1/ITS4 and GPD1/GPD2 (Manamgoda et al. 2012). All the amplified gene sequences were deposited into GenBank with accession numbers ITS (OP703331, OQ978850, and OQ978851) and GPD (OQ559683, OQ992505, and OQ992506). BLAST analysis of both the two segments showed 100% identity with those reported sequences of ex-type isolate BRIP 11512 (KJ415538 and KJ415408) of B. zeae Sivan (Tan et al. 2014; Bhunjun et al. 2020), respectively. In the maximum-likelihood phylogenetic tree, 83-HXGWC, 84HXGWC and 85HXGWC strains were grouped with the reference sequences of B. zeae with a high bootstrap supporting values of 100%. Five healthy green bristlegrass (6 weeks) plants in pots were sprayed with conidial suspensions (1×106 conidia ml-1). Another five pots were sprayed with sterilized distilled water (controls). Then all pots were individually covered with transparent polyethylene bags for 5 days to maintain high relative humidity and placed in a greenhouse maintained at 18 to 25 °C. After incubation for 14 days, the typical symptoms of leaf spots developed from brown to dark brown on the plants inoculated with conidial suspension, whereas no symptoms were observed on the control plants. The measurement of pathogenicity was carried out three times. The same pathogens were consistently reisolated from inoculated leaves, and was confirmed as B. zeae based on morphological and molecular analyses, fulfilling Koch's postulates. This study provides a new record of B. zeae on green bristlegrass on natural grassland in China, which can potentially impose disease challenges to other crops. Since, B. oryzae has been known to cause disease in cereal crops (Manamgoda et al. 2014), this can be a potential alternate source of survival of this pathogen.