First Report of Leaf Spot Disease Caused by Curvularia rouhanii on Vicia villosa in China

Vicia villosa is widely grown in China as a green manure crop. Investigations conducted in three fields in March of 2018 revealed a leaf spot disease on V. villosa with 25 to 50% incidence in Nanchong City, Sichuan Province, China. Infected leaves displayed irregularly shaped, scattered spots, and black mycelial layers grew on the spots' surfaces in humid environments. Leaves with typical pathological symptoms were photographed and sampled. To isolate the pathogens, the tissues from diseased and healthy junction areas on symptomatic leaves were excised, surface-sterilized, rinsed, and incubated on potato glucose agar (PCA) at 25 °C (Senanayake et al. 2020). Six pure isolates were obtained by single-spore isolation and deposited at the Culture Collection of JZB and CGMCC. The strain numbers of these six pure isolates are JZB3720009-JZB37200011 and CGMCC 3.25122-CGMCC 3.25124. On PCA plate, the colony reached 90 mm in diameter after 7 days at 25 ℃. Aerial hyphae were loose, appearing gray to gray-brown. Conidiophores are hyaline to pale brown, solitary, erect, separated, base with a narrowed basal cell, 22 to 100 μm. The apex of the conidiophores swelled, forming conidiogenous cells. Conidiogenous cells were brown to dark brown, solitary, smooth, slightly curved, subcylindrical, 7.1 to 17.0 × 5.1 to 6.8 µm (average: 12.7 × 5.9 µm, n = 20). Conidia are clustered, smooth-walled, straight, ellipsoid to cylindrical, tapering towards rounded ends, 2 to 3 but mostly 3 septate, basal and apical cells brown, middle cells dark brown with enlarged, 17.0 to 32.0 × 8.0 to 12.0 µm (average: 25.4 × 9.8 µm, n = 50). Morphologically, these isolates resembled species belonging to the genus Curvularia (Marin-Felix et al. 2017). Genomic DNA of the six isolates was extracted using a fungal DNA kit (Cat No.18812; Yeasen, Shanghai, China). The ITS region, gapdh and tef genes were amplified using the primers and procedures described by Raza et al. (2019) The sequences obtained in this study were deposited in GenBank with accession numbers PV608206 to PV608211 and PV615352 to PV615363. Phylogenetic analysis was conducted using combined ITS-gapdh-tef sequences and the maximum likelihood method. In the phylogenetic tree, the six isolates and type strain CBS 144674 of C. rouhanii clustered together with high bootstrap support values (94). Additionally, our isolates’ sequences showed a high degree of similarity to those of CBS 144674 (ITS: 99.4%; gapdh: 99.8%; tef: 99.9%). Meanwhile, we found that our isolates were morphologically consistent with C. rouhanii described by Mehrabi-Koushki et al (2018). Based on both morphological characteristics and phylogenetic results, our isolates were identified as C. rouhanii. To assess pathogenicity, the spore suspension of isolate JZB3720009 (approximately 10 5 conidia/ml) was sprayed onto healthy leaves of one-month-old plants in a greenhouse at 18 to 28 ℃. Plants sprayed with sterilized water were used as negative controls. The test was conducted three times, each time with 10 plants. After 7 days, the leaves showed spot symptoms similar to those observed in the field; control plants remained healthy. The pathogen was reisolated and confirmed as C. rouhanii, thereby fulfilling Koch's postulates. To our knowledge, this is the first report of leaf spot disease caused by C. rouhanii on V. villosa in China and the world. This report will inform the development of targeted management strategies to control the disease.