First Report of Botrytis cinerea Causing Gray Mold on Astragalus membranaceus in China.

Abstract

Astragalus membranaceus, known as Huang-qi, is a perennial herbal medicine plant that grows 50 to 150 cm high, with 13 to 31 small leaves (Fu et al. 2014). In September 2019, a disease investigation was conducted in Weiyuan, Gansu province, China, while the weather was rainy and cool. In 50% of the investigated fields (n=10, the average size of 10 fields is 0.14 ha), visible gray mold was observed on the stems of A. membranaceus. Approximately 30%-50% of the stems in those fields were withered and necrotic, with abundant dark brown mold due to presence of conidiophores. To further determine the causal agent of disease, 12 symptomatic samples were collected from 4 different fields (3 symptomatic samples per field). The stem samples were disinfected with 10% sodium hypochlorite for 1 minute, cut into pieces (2 to 3mm × 10 mm, n=12), rinsed 3 times with sterile water, and dried on sterile tissue. Samples were then placed on potato dextrose agar (PDA), 3 pieces per plate, which were incubated at 25℃ in the dark. After 3 days, hyphal tips growing from the disinfected tissues were individually transferred to new PDA plates and incubated at 25℃ in the dark. From the ten isolates obtained, GWT6-2 was chosen as a representative isolate for further study. Colonies of GWT6-2 had gray aerial mycelia with the margins that eventually turned taupe. In addition, some black and hard sclerotia (1.93 to 12.21 mm × 1.29 to 11.57 mm, n=20) with round or irregular shape developed on the colonies after approximately 10 days of incubation at 25℃ in the dark. Hyaline and round or elliptical conidia (7.86 to 14.53 μm × 6.81 to 11.79 μm, n=50) were attached on the top of brown conidiophores. Based on morphological characteristics, the isolate was initially identified as Botrytis sp. (Ellis 1971). For pathogenicity tests, 5 mm agar plugs prepared from 1-week-old cultures of GWT6-2 were placed on four stabbed stems of A. membranaceus (planted in flowerpots, 230 × 170 mm). A similar number of plants inoculated with PDA plugs (5 mm) were used as control, and the assay was replicated 3 times. All the treatments were kept in plastic buckets with a temperature of 20℃-25℃ and relative humidity of 80%-90%. After 48h, the agar plugs were removed, and the plants were maintained in the plastic buckets for 3-4 days. Waterlogged spots developed on the inoculated stems of A. membranaceus 3 days after inoculation, which elongated to 3-5cm after 4-5 days of inoculation, and the gray mold that appeared on the diseased lesions was similar to that observed in the fields. The pathogen was reisolated from diseased tissues, and the cultural (colonies) and microscopic characteristics (conidia and conidiophores) were similar to those of original isolate. However, controls were asymptomatic. To further identify the species, the genomic DNA of GWT6-2 was extracted, and the internal transcribed spacer (ITS), heat shock protein (HSP60) and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) genes were amplified with the primers ITS1/ITS4 (White et al. 1990), HSP60-F/HSP60-R and G3PDH-F/G3PDH-R (Staats et al. 2005), respectively. The PCR amplicons were sequenced and compared to other sequences in GenBank using BLAST. The results indicated that the ITS, HSP60 and G3PDH sequences of GWT6-2 (MT225784.1, MT230538.1, MT263015.1) were 99.08%, 100% and 99.25% identical to the sequences of B. cinerea strain GZFQ-1 (MH454037.1, MH479931.1, MH479930.1) and B. cinerea isolate 5-3 (MH718836.1, MH796663.1, MH796662.1), respectively. A multilocus phylogenetic tree was constructed with the ITS, HSP60 and G3PDH reference sequences, and the result revealed that the grouping of strain GWT6-2 was supported by 99% bootstrap value. Based on the morphological characteristics and molecular identification, the strain GWT6-2 was eventually identified as Botrytis cinerea. To our knowledge, this is the first report of B. cinerea causing gray mold on the stems of A. membranaceus in China. The occurrence of B. cinerea may affects the aerial growth of A. membranaceus resulting in economic yield losses of this important medicinal plant. Therefore, further investigations into the impact of this pathogen on A. membranaceus growth and management options are needed.