First report of Penicillium oxalicum causing Blue Mold on Astragalus membranaceus in Jilin Province, China

Astragalus membranaceus (Fisch.)Bge. is a perennial herb in the Leguminosae and distributed in northern China. Its root extracts are used against inflammation and tumors by practitioners of Chinese herbal medicine (Auyeung et al. 2016). Owing to the sharp decrease in the number of natural A. membranaceus in recent years, it is legally protected. During July to late-October 2016, blue mold was observed on A. membranaceus in Dehui city, Jilin province, China. More than 90 per cent of the 15 ha of the plants in the cultivation base were infected, and almost 50 per cent of the infected plants died. The disease mainly affected leaves. Symptoms initially were circular to irregular white patches. The lesions enlarged quickly, and thick fungal sporulation formed with yellow halos on both surfaces of leaves, causing plants to wither or eventually die. Tissues were excised from diseased and healthy leaves (each about 5 mm2), surface sterilized by soaking in 75 per cent ethyl alcohol for 30 s, rinsed three times in sterilized distilled water, plated on potato dextrose agar (PDA)medium, and incubated at 27°C. Three isolates were obtained from infected tissues by single-spore isolation method. On PDA medium, colonies were flat or nearly flat, globoid, and villous. Mycelia were initially white or pale-green and cottony, and then they grew rapidly and produced greenish blue pigments in 3 to 5 days. After culturing 6 days, conidia were examined microscopically. Conidia were unicellular, globoid or oval-shaped, and colorless. Conidial dimensions were 4.5 to 5.5 × 2.5 to 4.0 ?m. The morphological characteristics of the fungus were similar to Penicillium oxalicum. The primers ITS5 and ITS4 were used to amplify the internal transcribed spacer region of ribosomal DNA by polymerase chain reaction assay from extracted genomic DNA HQ1. The amplified DNA fragments were sequenced. The sequence (582-bp segment)of HQ1 was submitted to GenBank (accession no. KY064026)and showed 98 per cent homology with the sequence of P. oxalicum strain BGPUP4 (KP780809), confirming the species identification of the fungus (Singh et al. 2017). Pathogenicity tests were conducted using field inoculations and detached leaf assays. In each test, a conidial suspension (1 × 106 conidia/ml)was sprayed onto healthy leaves. Control plants were treated with sterilized distilled water. In field inoculation, plants were covered with polyethylene bags (Mukhtar et al. 2017). For detached leaf assays, three leaves per plate for four sealed Petri dishes were incubated at 27°C. Symptoms appeared on the detached leaves after 3 days, and after 15 days on the field-inoculated leaves. Symptoms were the same as those on the original infected plants, whereas the control plants were asymptomatic. The same fungus was successfully reisolated from inoculated leaves and was similar in morphological characteristics to P. oxalicum, completing Koch's postulates (Bozkurt et al. 2016). To our knowledge, this is the first report of P. oxalicum causing blue mold on A. membranaceus in China. The disease may cause huge losses of its yield and quality.