First Report of Causing Leaf Spot Disease on in Shaanxi Province of China.

Amorphophallus albus P. Y. Liu & J. F. Chen is a typical cash crop widely planted in southwest China (Gao et al., 2022). In early August of 2021, a peculiar leaf spot disease was first detected on A. albus in Ankang Academy of Agricultural Sciences manufacturing base (32°69'N, 109°02'E), Shaanxi, China. Small irregular yellow-brown spots (1 to 2 mm) were observed on the surface of A. albus leaf. Following infection of the leaf, it expanded (3 to 5 mm) and became necrotic. Nine planting bases were investigated, and approximately 75% of plants were symptomatic during the rapid expansion period of bulb growth in Hanyin, Langao and Hanbin counties, Ankang City, Shaanxi, China. Higher disease incidence was observed at temperatures above 30℃ and humidity above 80%. Twenty-seven symptomatic tissues of infected leaves were first surface sterilized by immersion in 75% ethanol for 1 minute, followed by rinsing three times in sterile distilled water. The tissues were then cut into 4-5 mm pieces, plated on 1.5% potato dextrose agar (PDA), and incubated at 28±2°C. The hyphal tip from the growing edge of colonies cultured for three days at 28±2℃ was transferred to PDA to obtain pure cultures. Fungal colonies were white, then grey to black with an unevenly distributed, fast-growing aerial mycelium covering the petri dish within five days at 28±2℃. The colony turned dark brown when maintained in the dark at 28±2℃ after seven days, then grayish brown upon sporulation after 15 days (Fig.1f-g). Conidia were brown or black, smooth, spherical to sub-spherical, single-celled (8-12 µm × 10-13µm, average 9-11.5 µm in diameter, n=5µm). The nutritional hyphae exhibited septa, and a portion of the aerial hyphae formed a long, rough conidium, giving rise to a nearly spherical apical sac (Fig.1h). The surface gave rise to several small peduncles bearing clusters of surfaced spherical conidia (Fig.1i). Surfaced spherical conidia were generated on the surface of the small peduncle (Fig.1j). These morphological features were consistent with Nigrospora oryzae (Li et al., 2017). Genomic DNA was extracted from mycelia of the pathogen using an Ezup column fungal genomic DNA extraction kit (Sangon Biotech, Shanghai, China). To confirm the identity of the pathogen, the genomic fragments for the internal transcribed spacer (ITS), LSU (28S) and BenA gene of the isolate were amplified by PCR (Wang et al., 2017) and sent for sequencing. The resultant sequence (GeneBank ID of gene ITS, LSU, BenA are OR723825, OR775345, OR277316, respectively) were compared with the voucher specimens. BLAST results showed >99% identity with those of N.oryzae (GeneBank ID of N.oryzae strain LC2707 ITS, LSU, BenA are KX985954, KY806242, KY019481, respectively). A neighbor joining phylogenetic tree with the concatenated sequences of these genes showed that A-pb169 had the closest match with N. oryzae (Fig. 2). For pathogenicity testing, fifty plants in a period of rapid expansion of bulb growth were selected. Four leaves per plant were inoculated by sprayed till runoff with a conidial suspension of the pathogen (50 µL, 1×106 conidia/ml sterile water), and incubated at 30±2℃ and 80 ± 5% humidity. Control plants received sterile water. On the third day after inoculation, a yellow-brown spot appeared on leave surfaces, the spot gradually expanded; the infection rate was 90 to 95%. Fifteen days after inoculation, infected leaves showed symptoms like those observed in the field, whereas 100 control leaves sprayed with sterile water remained symptomless (Fig.1 a-e). The pathogen was reisolated from infected leaves and confirmed as N. oryzae by morphology and molecular identification. To our knowledge, this is the first report of leaf spot disease of A. albus caused by N. oryzae in China. Since its one of the major cash crops of the southeastern China, further work is necessary to determine its spread and economic impact as well as developing sustainable disease management options.