First Report of Leaf Spot of Bletilla striata Caused by Fusarium asiaticum in China

Bletilla striata is originally from East Asia but is now grown widely in temperate to tropical regions worldwide with significant medical and ornamental values. Recently, various diseases caused the decline of market supply. In early May 2017, severe leaf spots of B. striata were found in three fields of Jingzhou, Hubei Province, China (N 111° 15′, W 29° 26′). Eighty-four plants from a total of about 200 young plants in the field showed such symptoms. The incidence of the disease was 42% and continued to rise under suitable weather conditions. The disease mainly harmed the leaves. Lesions first appeared as gray dots and expanded irregularly. Finally, large gray-brown spots appeared on the leaves, and then the whole leaf turned yellow and necrotic. Leaf sections (5 × 5 mm) from symptomatic plants were surface sterilized with 75% ethanol for 30 s and 2% sodium hypochlorite for 2 min, rinsed four times with sterile water, and then incubated on potato dextrose agar (PDA) plates amended with 50 μg/ml of ampicillin and kanamycin. Twelve of these 16 fungal isolates that showed similar morphological characteristics were isolated from diseased leaves (Leslie and Summerell 2006). The colony of representative isolate XIJI on PDA was white with pink pigmentation in the center. Carnation leaf agar (CLA) was used to stimulate spore production, and although no microspores were observed, falcate macroconidia with single foot cells were found. These were hyaline, 30 to 45 × 4 to 6 µm, with three to five septa (n = 100). Total genomic DNA of isolate XIJI was extracted with the cetyltrimethylammonium bromide method (Stenglein and Balatti 2006). Internal transcribed spacer region, β-tubulin, translation elongation factor 1α (EF1α), and histone H3 genes were amplified, respectively, using primers ITS4/ITS5 (White et al. 1990), BT2-a/BT2-b (Glass and Donaldson 1995), EF-728F/EF-986R, and H3-1a/1b. All sequences (MK238756, MK238755, MK172751, and MK281132) showed 98 to 100% identity to sequences from isolates within the Fusarium graminearum species complex (FGSC). In a neighbor-joining phylogenetic analysis based on EF1α and H3 sequences from FGSC with MAFFT online service, isolate XIJI located on the same clade with all F. asiaticum. Therefore, isolate XIJI was identified as F. asiaticum belonging to FGSC based on morphological and molecular evidence. To confirm Koch’s postulates, conidia of isolate XIJI were collected by rinsing CLA plates with sterile distilled water. Twenty 1-year-old B. striata plants were inoculated with 20 µl of conidial suspension (1 × 10⁵ conidia/ml) per leaf by pipettor. Ten plants inoculated by sterile distilled water served as controls. All plants were covered with polyethylene bags and incubated in a growth chamber (25°C, 70% relative humidity, and 12-h light/dark) for 48 h, and then the bags were removed and the disease monitored daily. Seven days after inoculation, symptoms were the same as those on the original infected plants, whereas the leaves from the control group remained asymptomatic. The same fungus was successfully reisolated from inoculated leaves and reidentified based on morphology and molecular evidence. Epicoccum sorghinum was reported causing leaf spot of B. striata in China (Zhou et al. 2018). To the best of our knowledge, this is the first report of leaf spot caused by F. asiaticum from FGSC on B. striata in China.