First report of Boeremia exigua 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.

First Report of Maize Stalk Rot Caused by Fusarium nelsonii in China.

Maize (Zea Mays L.) is one of the main crops in Ningxia Province, China, and stalk rot has become a serious disease of maize in this area. Infected plants showed softening of the stalks at lower internodes, which lodged easily and died prematurely during grain filling, and the pith tissue internally appeared to be disintegrating and slightly brown to reddish. In September 2018, symptomatic tissue was collected from seventeen locations in Ningxia. The incidence ranged from 5% to 40% in surveyed fields, reaching as high as 86% in certain plots. The discolored stalk pith tissues from the lesion region were cut into small pieces (approximately 0.5 × 0.2 cm), superficially disinfected with 75% ethanol for 1 min and rinsed three times with sterile water before plating on potato dextrose agar (PDA) medium with chloromycetin. The purified strains were obtained by single-spore separation and transferred to PDA and carnation leaf agar (CLA) medium. Morphological and molecular characteristics confirmed the presence of nine Fusarium species in these samples, including Fusarium graminearum species complex and Fusarium verticillioides. Four isolates of Fusarium nelsonii were recovered from samples collected in Shizuishan and Wuzhong. On PDA plates, the floccose to powdery, white to rose-colored aerial mycelia were produced and covered plates after 8 days of incubation, producing abundant mesoconidia and chlamydospores. Mesoconidia were fusiform or lanceolate until slightly curved with 0-3 septa, and chlamydospores were initially smooth and transparent, and became verrucous and light brown. Macroconidia produced in CLA were straight or curved and falcate, usually having 3-5 septa, with beak-shaped strongly curved apical cells and foot-shaped basal cells. Two isolates (SS-1-7 and ZY-2-2) were selected for molecular identification, and the total DNA was extracted using a fungal genomic DNA separation kit (Sangon Biotechnology, Shanghai, China). Sequence comparison of EF-1α (GenBank accession numbers MW294197 and MW294198) and RPB2 (Accession MW294176 and MW294177) genes showed 97% homology with the sequences of F. nelsonii reported in GenBank (accession MN120760 for TEF and accession MN120740 for RPB2). Pathogenicity tests with two isolates (SS-1-7 and ZY-2-2) were performed by individually inoculating five 10-leaf stage maize plants at between the 2nd and 3rd stem nodes from the soil level with 20 µl conidial suspension at a concentration of 106 conidia/ml as described by Zhang et al. (2016). Five maize plants inoculated with sterile water were used as controls. The inoculated plants were kept at 25 ± 0.5°C in the greenhouse with a photoperiod of 12 h. After 30 days, all plants inoculated with the conidial suspension formed an internal dark brown necrotic area around the inoculation site, whereas the control plants showed no symptoms. The pathogen was re-isolated from the necrotic tissue of the inoculated plants and identified by morphological characteristics as F. nelsonii. This species was first described by Marasas et al. (1998), and it is expanding its host range and has been isolated from sorghum, Medicago, wheat, and cucumber (Ahmad et al. 2020). The pathogen should be paid more attention owing to a serious risk of trichothecene and aflatoxin contamination (Astoreca et al. 2019; Lincy et al. 2011). To our knowledge, this is the first report of maize stalk rot caused by F. nelsonii in China. References: Ahmad, A., et al. 2020. Plant disease.1542 https://doi.org/10.1094/PDIS-11-19-2511-PDN Astoreca, A. L., et al. 2019. Eur. J. Plant Pathol. 155:381. Lincy, S. V., et al. 2011. World J. Microbiol. Biotechnol. 27:981. Marasas, W. F. O., et al. 1998. Mycologia 90:505. Zhang, Y., et al. 2016. PLoS Pathog. 12:e1005485. Funding: This research was financially supported by National R & D Plan of China (No.2019QZKK0303); Ningxia Agriculture and Forestry Academy Science and Technology Cooperation Project (DW-X-2018019).