| Abstract |
Polygonatum sibiricum (Huangjing, Siberian Solomon’s seal) is a perennial plant in the Asparagaceae. Used in traditional Chinese medicine in northeast China, it has been widely cultivated in recent years. In 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. A survey found a disease incidence >50%. Symptoms initially appeared on infected leaves as necrotic spots, which grew into circular or elliptical, pale to light brown lesions with a well-defined dark brown margin. To identify the pathogen, leaves with typical symptoms were collected. Leaf samples were cut from the junction of healthy and symptomatic tissues in 5-mm diameter pieces. Pieces were surface sterilized with washes of 75% ethanol (25 s) and 0.1% mercuric chloride solution (1.5 min), rinsed three times with sterile distilled water, air dried (Fang 2007), plated on PDA, and incubated at 25°C for 5 days in the dark. Six colonies with similar morphology were isolated by transferring mycelium plugs from colony margins to new PDA dishes and incubating under the same conditions. Two representative isolates (S3-2 and S5-3) were used for morphological and molecular identification. Colonies were initially whitish, turning dark gray, and covered with aerial mycelium. On the reverse, colonies were initially whitish, turning gray to dark black. Conidia were hyaline, aseptate, slightly curved and apex acute, and 17.5 to 22.5 × 3 to 4 µm. Setae were dark brown, subuliform with two to four septa, 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 strains. The internal transcribed spacers (ITS) region, actin (ACT), chitin synthase 1 (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and betatubulin (TUB2) genes were amplified using specific primers and PCR conditions described in Ma et al. (2020). The resulting sequences were submitted to GenBank. BLASTn identified that ITS (OM019299, OM937757), ACT (OM455393, OM937756), CHS-1 (OM455391, OM937754), GAPDH (OM455392, OM937755), TUB2 (OM455394, OM937758) were C. circinans. The ITS, ACT, CHS1, GAPDH, and 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 the maximum likelihood method, with a bootstrap value of 1,000 replicates (Tamura et al. 2011). Monilochaetes infuscans (CBS:869.96) was used as an outgroup. The analysis grouped the strains with C. circinans with good bootstrap support, supporting the morphological and single-gene molecular identifications. Pathogenicity was tested by in vivo inoculation of leaves of 3-month-old healthy P. sibiricum seedlings. Before inoculation, all leaves were surface sterilized with 75% ethanol and washed with sterile water twice (Chen et al. 2021). Each plant was inoculated by placing three mycelial plugs (5 mm; taken from an actively growing isolate colony) on one healthy leaf; a second leaf of each plant was inoculated by a sterile PDA plug as a control. After inoculation, plants were kept in an incubator at 25°C, >80% relative humidity, and a 16/8 h day/night cycle. The experiment was repeated three times. Brown spots were seen after 6 days on inoculated leaves, but no disease symptoms were seen on controls. Acervuli and conidia were visible on the dorsal side of diseased leaves after 11 to 13 days. The same pathogen was reisolated and confirmed as C. circinans by morphological comparison. These results showed that C. circinans caused the anthracnose of P. sibiricum. To our knowledge, this is the first report of C. circinans causing anthracnose on P. sibiricum in Liaoning Province, China. Control methods should be adopted to reduce losses.
|
