Occurrence of leaf spot caused by Stemphylium vesicarium on Red Clover (Trifolium pratense) in China.

Abstract

Red clover (Trifolium pratense) is becoming increasingly important in grassland systems (sheep and beef) due to its high productivity, protein content and nitrogen-fixing ability which allows lower N fertilizer use (Murray et al. 2007). In June 2021, an unidentified foliar disease occurred seriously in experimental fields of red clover (25.61°N, 102.48°E) in Kunming city, Yunnan Province, China. Disease incidence was approximately 55% with an average disease severity of 41.1% in the fields (about 1.5 ha). Initially, the symptoms consisted of cicular to oval leaf spots with dark brown edges. In severely diseased plots, the diseased spots continued to expand and fuse along the leaf veins, forming long and narrow leaf spots, and the middle of the leaves were decayed and hollow. To isolate the pathogen, small pieces (3×3 mm) from the margin of infected lesions were surface-sterilized in 75% ethanol solution for 30 s, 1% NaClO solution for 90 s, rinsed three times with sterilized distilled water, air dried, and plated onto potato dextrose agar (PDA). The same fungus was isolated in 90% of the samples and purified by transferring the hyphal tip from the edge of colonies to fresh PDA plates. The colonies produced relatively dense light-grayish aerial mycelium in the early stage, which turned grayish upon mature, and then dark brown on the back of the colonies with dense small black patches. Conidiophores were unbranched, straight or flexuous, accompanied by slight or obvious swelling of its apical cells. Conidia were olive brown, irregularly round, 1 to 3 transverse septa, 0 to 2 longitudinal or oblique septa, and measured 16.0 - (24.8)- 30.1 × 13.9 - (21.4) - 28.6 µm (n = 50). Morphological characteristics were consistent with those described for Stemphylium vesicarium (Simmons, 1967). For molecular identification, total genomic DNA was isolated from mycelia collected from 6 day-old colonies of three representative isolates LZQF-1-LZQF-3 using the Omega D3195 fungal genomic DNA extraction kit. Fragments of three genes, including those encoding the ITS region of rDNA, calmodulin gene (cmdA), and glyceraldehyde-3-phosphate dehydrogenase (gpd) regions of isolates LZQF-1-LZQF-3 were amplified by the primers described previously (Woudenberg et al. 2017) and sequenced. Resulting sequences of representative strain LZQF-1 were deposited in GenBank with accession numbers of OL615093 (ITS), OL624541 (cmdA) and OL624538 (gpd), respectively. A nucleotide BLAST search revealed ITS, cmdA and gpd sequences to be 100%, 100% and 99.0% similar to the corresponding sequences (accessions numbers KU850563.1, KU850853.1 and KU850710.1) of ex-type strain CBS 370.51 of S. vesicarium. The three locus datasets were combined by SequenceMatrix 1.8 (Vaidya et al. 2011), and the strain LZQF-1-LZQF-3 and S. vesicarium (CBS 192.86, CBS 370.51 and CBS 205.82) formed a subclade with 99% bootstrap support. Pathogenicity tests were performed on LZQF-1 in a greenhouse at 18 °C-26 °C with 75% relative humidity. Three pots each containing three 50-day-old red clover were sprayed with the conidial suspension (3 × 105 conidia/ml) while another three pots were sprayed with sterile water as a control. Two weeks later, the inoculated leaves showed oval leaf spots with dark brown edges, distinctive symptoms of S. vesicarium infection. S. vesicarium was reisolated and confirmed by morphological and molecular features. There were no symptoms on the control leaves. S. vesicarium as a pathogen causing leaf spot disease has been reported on red clover in Canada (Jasalavich et al. 1995) and in Russia (Babuschkina et al. 1995). To our knowledge, this is the first report of leaf spot of red clover caused by S. vesicarium in China. The identification of Stemphylium leaf spot lays the groundwork for future investigations into epidemiology and management of S. vesicarium on red clover.