First report of Colletotrichum circinans causing anthracnose on Polygonatum sibiricum in Liaoning Province, China.

Abstract

Polygonatum sibiricum (Huangjing, Siberian Solomon's seal) is a perennial plant of the family Asparagaceae. Used in traditional Chinese medicine in northeast China, P. sibiricum has become widely cultivated in recent years. During late June and early July 2021, leaf fleck symptoms were observed on P. sibiricum at a plantation in Liaoning Province, a major Huangjing production region in China. According to our survey, the incidence of the disease was over 50%. Symptoms initially appeared on infected leaves as necrotic spots, which increased in size and extended to circular or elliptical, pale to light brown lesions with well-defined dark brown margin. To identify the pathogen, leaves showing typical symptoms of infected plants were collected. Leaf samples were excised from the junction of both healthy and symptomatic tissues as 5 mm diameter pieces. Tissue samples were surface-sterilized with successive washes of 75% ethanol (25 sec), 0.1% mercuric chloride solution (1.5 min), three rinses with sterile distilled water, and then left to air dry (Zhongda 2007). Finally, the tissue pieces were plated on potato dextrose agar (PDA), and incubated at 25°C for 5 days in the dark. Six individual colonies with similar cultural morphology were isolated by transferring mycelium plugs from the margins of colonies to new PDA dishes and were incubated under the same conditions. Two representative isolates (S3-2, S5-3) were used for morphological and molecular identifications. Colonies on PDA were initially whitish, then turned dark grey with age, and were covered with aerial mycelium. On the reverse side, colonies were initially whitish and turned grey to dark black with age. Conidia were hyaline, aseptate, little curved and apex acute, and with sizes of 17.5 to 22.5×3 to 4 µm. Setae were dark brown, subuliform with 2 to 4 septate, 110 to 160 µm long. The morphological and cultural characteristics are consistent with the description of Colletotrichum circinans (Damm, et al. 2009). For molecular identification, genomic DNA was extracted from mycelia of the representative fungal strains. Internal transcribed spacers (ITS) region, actin (ACT), chitin synthase 1(CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and betatubulin (TUB2) genes amplified by using specific primers and PCR conditions described in (Ji Ma, et al. 2020). The resulting sequences were submitted to the GenBank. BlastN revealed that ITS(OM019299, OM937757), ACT(OM455393, OM937756), CHS1(OM455391, OM937754), GAPDH(OM455392, OM937755), Tub2(OM455394, OM937758) were identified as C. circinans. The ITS, ACT, CHS1, GAPDH, Tub2 sequences of the strains and other Colletotrichum species were multiple aligned using ClustalW implemented in MEGA5.1 and used for phylogenetic tree construction by Maximum likelihood method, with bootstrap value of 1000 replicates (Tamura et al. 2011). Monilochaetes infuscans (CBS:869.96) was used as an outgroup. The analysis indicated that the strains grouped with C. circinans with good bootstrap support, further supporting the morphological and single-gene molecular identifications. Pathogenicity was demonstrated by in vivo inoculation of leaves of three-month-old healthy P. sibiricum seedlings. Before inoculation, all the leaves of P. sibiricum were surface-sterilized with 75% ethanol and washed with sterile water twice (Jin Chen, et al. 2021). Each plant was inoculated by placing three mycelial plugs (5mm; taken from an actively-growing colony of the isolates) on a single healthy leaf, and the second leaf of each plant was inoculated by a sterile PDA plug as a control. After inoculation, plants were placed in an incubator held at a constant 25℃ and relative humidity (>80%) with a 16h/8h day/night cycle. The experiment was repeated three times. The brown spots were observed after 6 days on inoculated leaves, while no visible disease symptoms were observed on control leaves. The acervulus and conidia were visible on the dorsal of diseased leaves 11-13 days after inoculation. The same pathogen was reisolated and confirmed as C.circinans by morphological comparison, fulfilling Koch's postulates. These results illustrated that C.circinans was responsible for the anthracnose disease of P. sibiricum. To our knowledge, this is the first report of C. circinans causing anthracnose on P. sibiricum in Liaoning Province, and an effective control method should be adopted to reduce losses.