First Report of Botrytis deweyae Causing Gray mold on Polygonatum cyrtonema in China.

Abstract

Polygonatum cyrtonema Hua., is one of the cultivated varieties of Polygonatum sibiricum Redouté., which also an important cash crop in China (Chen, J., et al. 2021). From 2021 to 2022, symptoms resembling gray mold were observed on P. cyrtonema leaves with 30 to 45% disease incidence in Wanzhou District (30°38'1″N, 108°42'27″E) of Chongqing. The symptoms started to occur from April to June and more than 39% of leaves were infected from July to September. Symptoms started as irregular brown spots and progressed to the leaf edges or tips and stems. In dry conditions, the infected tissue appeared dry and thin, light brown in color, and became dry and cracked in the later stages of disease development. When the relative humidity was high, infected leaves developed water-soaked decay with a brown stripe around the lesion, and a gray mold layer appeared. To identify the causal agent, 8 typical diseased leaves were collected, leaf tissues were chopped into small pieces (3×5 mm), surface sterilized for 1 min in 70% ethanol and 5 minutes in 3% sodium hypochlorite, rinsed three times using sterile water, placed onto potato dextrose agar (PDA) amended with streptomycin sulfate (50 μg/ml) and incubated at 25°C for 3 days in dark conditions. Then 6 colonies (3.5 to 4 cm diameter) with similar morphology were transferred onto new plates. In the initial stage of growth of isolates, all hyphal colonies were white, dense, and clustered, and dispersed in all directions. After 21 days, brown to black-colored sclerotia (2.3 to 5.8 mm diameter) were observed embedded on the bottom of the medium. The six colonies were confirmed to be Botrytis sp. based on the morphological characteristics. The conidia were attached in branches on the conidiophores in grape-like clusters. Conidiophores were straight and 150 to 500 μm in length, and the conidia were single-celled, long ellipsoidal, or oval-like, with no septa and 7.5 to 20 × 3.5 to 14 μm (n=50). For molecular identification, DNA was extracted from representative strains 4-2 and 1-5. The internal transcribed spacer (ITS) region and sequences from the RNA polymerase II second largest subunit (RPB2), and the heat-shock protein 60 (HSP60) genes were amplified using primers ITS1/ITS4, RPB2for/RPB2rev, and HSP60for/HSP60rev, respectively (White T.J., et al.1990; Staats, M., et al. 2005). The sequences were deposited in GenBank: 4-2 [ITS; OM655229: RPB2; OM960678: HSP60; OM960679] and 1-5 [ITS; OQ160236: RPB2; OQ164790: HSP60; OQ164791]. These sequences from isolates 4-2 and 1-5 had 100% similarity to the B. deweyae CBS 134649/ MK-2013 [ITS; HG799538.1: RPB2; HG799518.1: HSP60; HG799519.1] ex-type sequences, and phylogenetic analyses based on multi-locus alignment demonstrated strains 4-2 and 1-5 as B. deweyae. Isolate 4-2 was used to verify whether B. deweyae can cause gray mold on P. cyrtonema, by conducting Koch's postulates experiments (Gradmann, C., 2014). The leaves of P. cyrtonema planted in pots were washed with sterile water, and brushed with 10 mL of hyphal tissue in 55% glycerin. Leaves of another plant were brushed with 10 mL 55% glycerin as control, and Kochs' postulates experiments were conducted three times. Inoculated plants were kept in a chamber with 80% relative humidity at 20 ± 1°C. Seven days after inoculation, disease symptoms similar to those in the field were observed on leaves, whereas control plants remained asymptomatic. The fungus was reisolated from inoculated plants and identified as B. deweyae based on multi-locus phylogenetic analysis. To our knowledge, B. deweyae is mostly found on Hemerocallis, is likely to be an important contributor to the development of 'spring sickness' symptoms (Grant-Downton, R.T., et al. 2014.), and this is the first report of B. deweyae causing gray mold on P. cyrtonema in China. Although B. deweyae has a limited host range, it might also become a potential threat to P. cyrtonema. This work will provide a basis for the prevention and treatment of the disease in the future.