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.

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.

Cryptochrome 2 from Lilium × formolongi Regulates Photoperiodic Flowering in Transgenic Arabidopsis thaliana.

The photoperiodic flowering pathway is essential for plant reproduction. As blue and ultraviolet-A light receptors, cryptochromes play an important role in the photoperiodic regulation of flowering. Lilium × formolongi is an important cut flower that flowers within a year after seed propagation. Floral induction is highly sensitive to photoperiod. In this study, we isolated the CRYPTOCHROME2 gene (LfCRY2) from L. × formolongi. The predicted LfCRY2 protein was highly homologous to other CRY2 proteins. The transcription of LfCRY2 was induced by blue light. LfCRY2 exhibits its highest diurnal expression during the floral induction stage under both long-day and short-day photoperiods. Overexpression of LfCRY2 in Arabidopsis thaliana promoted flowering under long days but not short days, and inhibited hypocotyl elongation under blue light. Furthermore, LfCRY2 was located in the nucleus and could interact with L. × formolongi CONSTANS-like 9 (LfCOL9) and A. thaliana CRY-interacting basic-helix-loop-helix 1 (AtCIB1) in both yeast and onion cells, which supports the hypothesis that LfCRY2 hastens the floral transition via the CIB1-CO pathway in a manner similar to AtCRY2. These results provide evidence that LfCRY2 plays a vital role in promoting flowering under long days in L. × formolongi.

Molecular cloning of cryptochrome 1 from Lilium×formolongi and the characterization of its photoperiodic flowering function in Arabidopsis.

Lilium × formolongi is an important cut flower species that is able to flower within a year following seed propagation, with flower induction that is very sensitive to the photoperiod. Cryptochromes are blue/UV-A light receptors that regulate many important plant growth and development processes, including photoperiodic flowering. In this study, we isolated the cryptochrome 1 (CRY1) gene from L. × formolongi and analyzed its function in transgenic Arabidopsis. The predicted LfCRY1 protein was strongly homologous to other CRY1 proteins. The transcription of LfCRY1 was induced by blue light, with LfCRY1 exhibiting its highest expression and diurnal expression patterns during the flowering-induction stage under both long-day (LD) and short-day (SD) photoperiods. Overexpression of LfCRY1 in Arabidopsis promoted flowering under LDs but not SDs and inhibited hypocotyl elongation under blue light. The LfCRY1 protein was located in both the nucleus and cytoplasm. LfCRY1 interacted with the important flowering activator LfCOL9 in both yeast and onion cells. These results provide functional evidence for the role of LfCRY1 in controlling photoperiodic flowering under LDs and indicate that LfCRY1 may be a counterpart of AtCRY1. Understanding the role of LfCRY1 in photoperiodic flowering is beneficial for the molecular breeding of lilies with shorter vegetative stages.

ISSR variation in the endemic and endangered plant Cycas guizhouensis (Cycadaceae).

BACKGROUND AND AIMS: Cycas guizhouensis (Cycadaceae) is a rare and endangered species endemic to the southwest of China. An investigation was undertaken into the genetic variation of wild populations. METHODS: ISSR markers were used to determine the genetic variation within and between 12 extant populations of this species. KEY RESULTS: Low genetic diversity (at population level, P = 14.21 %, H(E) = 0.0597; at species level, P = 35.90 %, H(T) = 0.1082) and a high degree of differentiation among populations (G(ST) = 0.4321) were detected. CONCLUSIONS: This genetic structure is considered to be due to the combined effects of slow biochemical evolution, genetic drift, inbreeding and limited gene flow between populations. Based on these findings, strategies are proposed for the genetic conservation and management of the species.