ABSTRACT
Polygonatum odoratum (Mill.) Druce, a member of Liliaceae, is one of the traditional Chinese herbal plants mainly used in Jilin, Hubei, Guangxi, Zhejiang, Liaoning, Hunan and Guangdong provinces. Leaf spot disease of P. odoratum was continuously observed in the Planting Demonstration Garden in Changsha (28 °48 N; 113° 34E), Hunan Province of China, in May 2021 and May 2022. The symptoms initially appeared as tiny reddish-brown spots and continued to expand, resulting in round, oval, or irregular tan lesions with necrotic, film-shaped, or perforated central tissues. Leaf spot disease affects approximately 60-70% of plants. For pathogen isolation, symptomatic leaf samples were collected and disinfected with 70% ethanol for 30 s and 3% sodium hypochlorite for 2 min, followed by rinsing with sterile distilled water. Subsequently, small pieces (3 × 3 mm) of diseased tissues were placed on potato dextrose agar (PDA) and incubated in the dark at 25 °C for 24 h to 36 h. The emerging fungal hyphal tips were transferred to PDA and purified by the single-spore method (Yu, et al., 202). In total, 50 disease spots were isolated, and 10 cultures with the same appearance were obtained. Two strains coded as hnxryzy and hnxryzy01 were randomly selected for identification. After 6 days of culture in PDA, dense pink colonies were observed with a mean radial growth rate of 7.5 mm/day. Strains cultured 6 days on synthetic low nutrient medium, microconidia were oval or ovate (7.5-9.67 µm × 2.49-3.57 µm(n = 50)), and macroconidia were sickle-shaped and slightly curved, gradually tapering at both ends, with 2-5 pseudoseptate (10.01-22.14 µm × 2.07-4.22 µm (n = 50)). These morphological characteristics were consistent with the description of Fusarium fujikuroi (Fang, et al., 2021). Furthermore, primers ITS1/ITS4, EF728F/EF986R, Bt2a/Bt2b, RPB1-F5/RPB1-R8 and fRPB2-5F2/fRPB2-7cR (Li, et al., 2013, Xie, et al., 2022) were used to amplify the partial region of the internal transcribed spacer (ITS) , the translation elongation factor EF-1α,ß-tubulin,polymerase II largest subunit (RPB1) and RNA polymerase II second largest subunit (RPB2) genes from strains hnxryzy and hnxryzy01, respectively. Amplicons were sequenced by Tsingke Biotechnology Co., Ltd. The expected sequences of ITS, EF-1α, ß-tubulin, RPB1 and RPB2 of hnxryzy and hnxryzy01 were obtained. The sequence alignment of hnxryzj and hnxryzj01 with the Fusarium ID databased and NCBI shows the following results: The sequences of ITS region, EF-1α, ß-tubulin , RPB1 and RPB2 of strain hnxryzy (GenBank accession nos. ON797440, ON820553, ON820554, OP413443, and OP413445, respectively) and strain hnxryzy01 (GenBank accession nos. ON965284, ON968721, ON968722, OP413444, and OP413446, respectively) were 99% to 100% identical to those of F. fujikuroi (GenBank accession numbers CP023090, KC874784, MN490089, MN193916, and MN193888, respectively). Then a phylogenetic tree based on EF-1α, RPB1, and RPB2 sequences was constructed (Torres-Cruz, et al., 2022). The strains hnxryzy and hnxryzy01 were more closely related to F. fujikuroi ( NRRL13566 GenBank accession nos. AF160279, JX171456, and JX171570, respectively), with bootstrap values of 99%. Two sets (5 plants in each set) of potted plants were used in pathogenicity assays. Wounded leaves were sprayed with conidial suspensions (100 µL, 1 × 107 spores/mL) and sterile water as control. Inoculated plants were covered with plastic bags for 24 h, and maintained at 25 ° C in 12/12 h light/dark conditions in the greenhouse (Yu, et al., 2022). Pathogenicity assays were repeated thrice. Dark brown spots identical to those seen in the field were observed 14 days after inoculation, while the control leaves did not exhibit any symptoms. In this study, the pathogen F. fujikuroi was successfully reisolated from the leaves of inoculated samples showing symptoms, thereby verifying Koch's postulate. To our knowledge, this is the first report of F. fujikuroi inducing leaf spot on P. odoratum in China. Since F. fujikuroi is a common pathogenic fungus that infects different plant species(Qiu, et al., 2020), more attention should be paid to its prevalence in P. odoratum and the potential risk of outbreak in other provinces of China.
ABSTRACT
Sedum plumbizincicola X.H. Guo et S.B. Zhou sp. nov. is a plant species of the family Crassulaceae that has the ability to hyperaccumulate cadmium and zinc in high concentrations (Liu et al. 2017). In April of 2018 and 2019, a disease of damping-off was observed on S. plumbizincicola seedlings in a nursery in Changsha (28°13'N; 112°56'E), the Hunan Province of China, in which nearly 1 million seedlings were planted. Approximately 40% of the surveyed plants were infected. The affected plants displayed water-soaking on the shoots and stems, and chlorosis on the leaves. As the disease spread upward, leaf stalks or the whole plants became wilted and collapsed. Five diseased stem and leaf samples were collected. Symptomatic tissues were excised and surface sterilized with 70% ethanol for 10 s, and 0.1% HgCl2 for 2 min, washed with sterile distilled water for three times, and then cultured on potato dextrose agar (PDA) at 26°C in darkness. Fungal colonies were similar in morphology: white, light gray to brown, with hyphae branched at nearly right angles, septa near the branching point and constrictions at the base of hyphal branches. After 10 days, white-gray to brown sclerotia were produced. The morphological characteristics were consistent with those of Rhizoctonia solani J.G. Kühn (Sneh et al. 1991). Genomic DNA of a representative isolate was extracted using the cetyltrimethylammonium bromide method. The internal transcribed spacer (ITS) region of rDNA was amplified and sequenced with the primer pairs ITS4/ITS5 (White et al. 1990). When analyzed by the BLASTn program, the ITS sequence (GenBank Accession No. MN961664) had 100% identity to the corresponding gene sequence of R. solani anastomosis group (AG) 2-1(Accession Nos: LC202869.1 and MH862641.1). In addition, primers Rhsp1/ITS4B and Rhsp2/ITS1F specific for R. solani, and AG21sp/ITS4B specific for R. solani AG 2-1 were also used (Salazar et al. 2000). Results revealed that our isolate was R. solani AG 2-1. Pathogenicity was confirmed via in vivo inoculation of one-month-old S. plumbizincicola seedlings in sterilized nursery soil with four representative isolates. For each pot, five 5-mm-diameter mycelial plugs from 7-days old colonies on PDA were placed in the soil near the base of the stems. Plants inoculated with agar plugs without mycelium served as controls. The inoculated plants were kept in a growth chamber at 25°C with a 12/12 h light/dark cycle. Pathogenicity tests were performed twice, with three replicative potted plants for each isolate in each test. Approximately 25 days after inoculation, the damping-off symptoms resembling those observed in the field were displayed on the inoculated plants, while no obvious symptoms were observed on the control plants. R. solani was re-isolated from all infected plants and molecularly characterized, thus confirming Koch's postulates. R. solani has been previously reported as the pathogen of damping-off disease in many plants, such as canola (Paulitz et al. 2006) and oat (Zhang et al. 2016). However, to the best of our knowledge, this is the first report of R. solani causing damping-off of S. plumbizincicola in China. S. plumbizincicola is widely planted for heavy metal pollution treatment in China. The occurrence of this disease could seriously affect the production of the seedlings, and management strategies should be developed.