First report of Fusarium oxysporum causing stem spots on Polygonatum odoratum in China.

ABSTRACT

Polygonatum odoratum (Mill.) Druce is a perennial herb in the Liliaceae family and it is one of the traditional Chinese medicinal plants. Modern pharmaceutical studies demonstrate that P. odoratum contains polysaccharides, saponins, alkaloids, flavonoids, volatile oil, and other active components (Jiang-Nan, et al., 2018). From May to June 2022, the stem spot disease was discovered on P. odoratum in the planting demonstration garden in Changsha (28°20N; 113°07E), Hunan province of China. The disease seriously retarded plant growth and was estimated to have affected approximately 40-50% of the plants, significant economic losses to growers. Plants had oval tan spots on the stems, which were light in the center and dark at the margin. The spots in the back expanded and joined together, where the disease was severe, and chlorosis was near the stem spot, while many leaves turned completely yellow and withered before falling to the ground. Finally, the whole plant faded to light green and dried up. In order to isolate pathogens, symptomatic stem samples (5×5 mm) were collected from the edges of the lesions and excised symptomatic tissues consisting of diseased and healthy parts were surface-sterilized with 2% solution of sodium hypochlorite (0.1% active ingredient of chlorine) for 1 min and 75% ethanol for 30 s. The samples were then washed thrice with sterile distilled water, air-dried on the sterile filter papers under aseptic conditions, and finally plated onto Potato Dextrose Agar (PDA) plates, which were incubated at 25 °C for 24 h to 36 h in the dark. Additionally, the emerging fungal hyphal tips were transferred to PDA and purified by the single-spore method. Next, forty plants with stem spots were isolated, and 8 cultures with the same appearance were obtained. Two strains coded hnxryzj and hnxryzj1 were randomly selected, for identification. With a mean radial growth rate of 7.5 mm/day, white and dense colonies were observed after 6 days of culture on PDA. After hnxryzj was cultured on SNA, microconidia were oval or ovate (9.25-14.8µm × 2.18-3.76µm), macroconidia were sickle-shaped and slightly curved, with 2-5 septa (21.52-23.49µm × 2.64-4.51µm (n = 50)). These morphological characteristics were consistent with the description of Fusarium oxysporum (Mirghasempour, et al., 2022) Furthermore, we amplified the partial region of the internal transcribed spacer (ITS) region, the translation elongation factors EF-1α, ß-tubulin, polymerase II largest subunit (RPB1) and RNA polymerase II second largest subunit (RPB2) genes from strain hnxryzj and hnxryzj1, based on the primer pairs ITS1/ITS4, EF728F/EF986R, Bt2a/Bt2b, RPB1-F5/RPB1-R8 and fRPB2-5F2/fRPB2-7cR (Li, et al., 2013, Xie, et al., 2022), and amplicons were sequenced by Tsingke Biotechnology Co. Ltd. By sequence alignment, the ITS, EF-1α, ß-tubulin , RPB1 and RPB2 of hnxryzj and hnxryzj1 were identical, respectively. The sequence alignment of hnxryzj and hnxryzj1 with the Fusarium ID database and NCBI shows the following results: the ITS region, EF-1α, RPB1 and RPB2 sequences of the strain hnxryzj (GenBank accession nos. ON872218, ON897740, OP467556 and OP467557) and hnxryzj1 (GenBank accession nos. OP071248, OP087208, OP467558 and OP467559) were 100% identical to those of F. oxysporum (GenBank accession nos. MZ890536, LC469784 , MT179509 and MW368380, respectively); whereas the ß-tubulin sequences of the strain hnxryzj (GenBank accession nos. ON897741) and hnxryzj1 (GenBank accession nos. OP087207) were 96.9% identical to those of F.oxysporum (CBS144135 GenBank accession nos. MH485136). Subsequently, a phylogenetic tree was established combining EF-1α, RPB1, and RPB2. Strains hnxryzj and hnxryzj1 were F.oxysporum (JW257006 GenBank accession nos. MZ921883, MZ921657 and MZ921752)(Torres-Cruz, et al., 2022), with bootstrap values 100%. The pathogenicity test was carried out by placing mycelial discs obtained from colonies that had been actively growing on PDA for 6 days. In the pathogenicity test, two sets (5 plants in each set) of potted plants, whose stems were wounded, were taken. In one set (5 plants), the PDA cakes with F. oxysporum (d=5mm, the same below) were inoculated on the stems scratched by an inoculation needle (sterilized) (the front of the colony was close to the wound of the stem). In the other set (5 plants), potted plants inoculated with the sterile PDA cakes were served as controls. In a 25 °C greenhouse, each treatment was given a 12h/12h light/dark cycl(Nabi, et al., 2019). The symptoms were observed, and the fungus cake was removed 5 days after inoculation. Then, after 18 days, typical symptoms of oval tan spots similar to original diseased plants in the field were found on the inoculated stems, and 32 days later, the inoculated plant died, while the control stems remained asymptomatic. In addition, F. oxysporum was isolated and identified from the inoculated, symptomatic stems, verifying Koch's postulates. Based on our knowledge, this is the first report of F. oxysporum causing stem spots on P. odoratum in China. Only one other study from China that root rot of Phyllostachys officinalis also resulted from F. oxysporum (Pang, et al., 2022). Furthermore, P. odoratum is an medicinal material in Hunan province. Therefore, comprehensive prevention and control methods are required.