First report of Fusarium fujikuroi causes leaf spot in Polygonatum odoratum (Mill.) Druce in China.

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.

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

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.

Triterpenoids from stems of Kadsura heteroclita.

In the present work, we reported the triterpenoids isolated from n-butanol fraction of Kadsura heteroclita which is a Tujia ethnomedicine with trivial name "Xuetong". This effort resulted in the isolation of six unpresented triterpenoids xuetongsu A-F (1-6), along with five known triterpenoids (7-11). The structures of the reported compounds were established on the 1D, and 2D NMR and HRESIMS spectra, along with CD spectroscopic analysis. Moreover, the absolute stereochemistry of compound 7 was determined by X-ray diffraction analysis. Antioxidant and cytotoxic activities were evaluated for all isolated compounds, compound 7 shown weak cytotoxic activity against HL-60 with IC50 value of 50.0 µM.

[Comparative analysis and comprehensive evaluation for growth and photosynthetic characteristics of different lily species and varieties].

The biological characteristics,agronomic traits,yield traits,stress resistance,quality and photosynthetic characteristics among six lily varieties were compared in order to screen out the excellent lily varieties suitable for spread planting in Hunan province. Lilium longiflorum had the longest growth period,246 days,among these six lily varieties,while others were about 170 days. The leaves of L.longiflorum,self-selected variety,L. lancifolium and L. dauricum had higher chlorophyll content. No obvious difference was found in net photosynthetic rate,stomatal conductance,transpiration rate and intercellular CO2 concentration among all varieties. The self-selected variety had the highest theoretical and actual yield,2 543. 03,1 608. 65 kg per Mu(1 Mu≈666. 7 m2),respectively,but contents of polysaccharides and flavones in bulbs were lower. All of these six lily varieties can sowing,seedling emergence,growth,flowering,mature harvest in Hunan province. L. dauricum and L. lancifolium would be provided for edible lily. L. brownie and the self-selected variety are highly susceptible varieties. L. dauricum and L. lancifolium are suitable to plant widely in disease-prone regions,due to their strong resistance. L. brownie and L. lancifolium are preferred varieties for medicinal and food using for their good quality and higher contents of polysaccharides and flavones. L. davidii had lower theoretical and actual yield,so planting extension of it should be taken into account.