First Report of Leaf Spot Disease Caused by <i>Curvularia trifolii</i> on <i>Trifolium repens</i> in China

Trifolium repens L. is widely grown in China as a lawn plant, forage and green manure crop. Investigations conducted in September 2022 revealed a leaf spot disease on T. repens with an 35% incidence (100 plants were investigated) in Qinghezhizhou Park, Haidian District, Beijing, China. Infected leaves showed water-soaked V-shaped yellow lesions that later turned brown and eventually led to the death of the affected leaves. Leaves displaying typical pathological symptoms were photographed and collected for pathogen isolation and identification. Symptomatic leaf tissues were excised from the margin between healthy and diseased areas, surface sterilized with 2% NaClO for 2 min and 75% ethanol for 30 s, rinsed three times with sterile water, and incubated on potato dextrose agar at 25℃. Six pure isolates were obtained by single spore isolation and were deposited in the Culture Collection of the Institute of Plant Protection, Beijing Academy of Agriculture and Forestry Sciences (JZB). The strain numbers of these six pure isolates are JZB3720003-JZB3720008. On potato carrot agar (pH = 6.5), colonies reached 55 mm in diameter after 7 days at 25℃ under a 12 h light/12 h dark photoperiod. Aerial hyphae were fluffy, appearing white to light gray, and the reverse side of the colony was fawn to dark. Conidiophores were brown, solitary, straight or slightly curved, separated, occasionally branched, 68 - 212 um. Conidiogenous cells were pale brown, solitary or opposite, geniculate, 12.2 - 26.7 × 4.7 - 6.8 µm (average: 19.0 × 5.6 µm, n = 20). Conidia were slightly verruculose, clustered, straight or slightly curved, or geniculate, narrowly ellipsoidal or fusiform, 2 - 3 (mostly 3) septate, basal and apical cells brown, middle cells dark brown with enlarged, 17.8 - 27.8 × 6.1 - 11.9 µm (average: 24.1 × 9.0 µm, n = 30). Morphologically these isolates resembled species belonging to genus Curvularia, especially the C. trifolii (Sivanesan. 1987). Genomic DNA of the six isolates was extracted using a fungal DNA kit (Cat No.18812; Yeasen, Shanghai, China). The internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (gapdh) and translation elongation factor-1α (tef-1α) genes were amplified using the primers and protocols described by Raza et al. (2019) The sequences obtained in this study were deposited in GenBank under accession numbers PV523576 to PV523581 and PV575979 to PV575990. Phylogenetic analysis was conducted using combined sequences of the four loci in the order of ITS-gapdh-tef and the maximum likelihood method. In the resulting phylogenetic tree, the six isolates clustered together with the representative strain CBS 173.55 of C. trifolii with high bootstrap support values (100). Additionally, all sequences of our isolates showed 100% identity to those of CBS 173.55. Meanwhile, we found that our isolates were morphologically consistent with C. trifolii described by Crous et al. (2011). Based on both morphological characteristics and phylogenetic results, the isolates were identified as C. trifolii. To assess pathogenicity, a spore suspension of isolate JZB3720003 (approximately 105 conidia/ml) was inoculated onto healthy leaves of one month old plants in a greenhouse at 18 - 28℃. Plants sprayed with sterilized water were used as blank controls. Ten randomly distributed inoculation points were set on each plant, with three plants inoculated per experiment, and the experiment was repeated three times. After 7 days, the leaves of the inoculated plants developed spot symptoms similar to those observed in the field, while the control plants remained healthy. The pathogen was reisolated and confirmed as C. trifolii, thereby fulfilling Koch's postulates. Curvularia trifolii has a wide host range, with over forty recorded host species (USDA-FUNGUS HOST DATABASE. https://fungi.ars.usda.gov/). There have been reports of C. trifolii causing leaf spot disease on T. repens in the United States, Japan, Kenya, and Australia (USDA-FUNGUS HOST DATABASE. https://fungi.ars.usda.gov/). In China, C. trifolii and its synonym, the fungus Brachysporium trifolii have been reported to infect the Trifolium sp., but there is no specific report indicating the exact host (Tai 1979). To our knowledge, this is the first report of leaf spot disease caused by C. trifolii on T. repens in China. This report expands the disease data repository for T. repens in China, which will be helpful in developing targeted management strategies for controlling the disease and reducing epidemic risks.