First report of causing spot blight on cowpea in China.

Cowpea or black-eyed pea [Vigna unguiculata (L.) Walp.] is a dual-purpose leguminous crop grown for food and fodder. In September 2022, cowpea plants exhibiting symptoms of a leaf spot and blight were observed in Renda Town located in Jingning County of Gansu Province, China, with the disease incidence in individual cowpea fields as high as 100%. Diseased leaves showed variable-sized, nearly circular brown blotches, large blotches with dark brown margins, and the adaxial surfaces of blotches had small black dots and whorls (Fig. 1). Multiple isolates with consistent colony characteristics were obtained from cowpea leaves with typical symptoms. The isolates were transferred to fresh potato dextrose agar medium (PDA) and then purified by transferring hyphal tips to PDA. Three isolates, JNJD-1, JNJD-2, and JNJD-3, were selected for subsequent identification and pathogenicity determination. After eight days at 25℃ on PDA, the colonies appeared irregular, aerial mycelium dense, cottony, atrovirens to olive brown, with white hyphae on the undulate margin (Fig. 2A and B). The pycnidia were globose to sub-globose, brown to dark brown, with 70-110 μm diameters. Single celled hyaline conidia were ellipsoidal to oblong with obtuse ends, and measured 6.6-9.3 × 2.8-4.1 μm (x̄ = 7.8 × 3.5 μm, n = 50) (Fig. 2C). Morphological characteristics are similar to the description of the genus Boeremia (Aveskamp et al, 2010). Primer pairs ITS1/ITS4, LR0R/LR5, fRPB2-5F2/fRPB2-7cR, and TUB2FD/TUB4RD were used to amplify portions of the ITS, LSU, RPB2, and TUB genes, respectively (Chen et al, 2015). The obtained sequences (Accession numbers: PP033662 to PP033664 for ITS, PP033667 to PP033669 for LSU, PP035531 to PP035533 for RPB2, and PP035534 to PP035536 for TUB) were 97% identical to that of a B. exigua strain CBS 431.74 (accession no. FJ427001, EU754183, GU371780, and FJ427112) (Table 1). The constructed maximum likelihood tree indicated close relationships between three isolates and B. exigua, which clustered together (Fig. 3). Cowpea plants (cultivar Junlintianxia) at the three-leaf stage were inoculated by spraying a spore suspension (1×106 conidia/ml) of JNJD-1, JNJD-2, and JNJD-3 until run off and incubated at greenhouse conditions (25°C and 12 h light). Inoculations with sterile water were used as a control and each treatment was repeated 3 times with five plants per replicate. Small brown spots appeared on the infected leaves at 2 dpi, followed by the appearance of large blotches, with dark brown at the margin and grayish-white in the center at 5 dpi (Fig. 4A). These lesions gradually increase and coalesce, causing leaf chlorosis and finally defoliation in serious cases. Disease incidence in inoculated cowpea plants treated with the isolates JNJD-1, JNJD-2, and JNJD-3 reached almost 100%. In contrast, control plants developed no symptoms (Fig. 4B). The pathogens were re-isolated from the inoculated leaves and identified as B. exigua using morphological and molecular analysis, whereas no fungus was isolated from control leaves. The experiment was repeated once under the same conditions, yielding similar results. B. exigua has a broad host range, infecting 19 families and 31 genera of plant species, and causing leaf spots, leaf blight, and tuber rot (Lan and Duan 2022). To our knowledge, this is the first report of the pathogen B. exigua causing spot blight on cowpeas. It has been reported that B. exigua infects leguminous crops from multiple genera, such as field pea, soybean, white clover, and Dumasia villosa (Liu et al, 2023). This study further enriches the host range of this pathogen and the pathogen species of cowpea leaf diseases.