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.

Ubiquitin ligase TRIM32 promotes dendrite arborization by mediating degradation of the epigenetic factor CDYL.

Proper dendritic morphology is fundamental to nerve signal transmission; thus, revealing the mechanism by which dendrite arborization is regulated is of great significance. Our previous studies have found that the epigenetic molecule chromodomain Y-like (CDYL) negatively regulates dendritic branching. Current research mostly focuses on the processes downstream of CDYL, whereas the upstream regulatory process has not been investigated to date. In this study, we identified an upstream regulator of CDYL, the E3 ubiquitin ligase tripartite motif-containing protein 32 (TRIM32), which promotes dendrite arborization by mediating the ubiquitylation and degradation of CDYL. By using mass spectrometry and biochemistry strategies, we proved that TRIM32 interacted with CDYL and mediated CDYL ubiquitylation modification in vivo and in vitro. Overexpressing TRIM32 decreased the protein level of CDYL, leading to an increase in the dendritic complexity of primary cultured rat neurons. In contrast, knocking down TRIM32 increased the protein level of CDYL and decreased the dendritic complexity. The truncated form of TRIM32 without E3 ligase activity (ΔRING) lost its ability to regulate dendritic complexity. Most importantly, knockdown of CDYL abolished the reduced complexity of dendrites caused by TRIM32 knockdown, indicating that the TRIM32-mediated regulation of dendritic development depends on its regulation of downstream CDYL. Hence, our findings reveal that TRIM32 could promote dendrite arborization by mediating CDYL degradation. This work initially defines a novel biological role of TRIM32 in regulating mechanisms upstream of CDYL and further presents a potential therapeutic target for the treatment of CDYL-related neurodevelopmental disorders.

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.

Investigation and Modeling of Capacitive Human Body Communication.

This paper presents a systematic investigation of the capacitive human body communication (HBC). The measurement of HBC channels is performed using a novel battery-powered system to eliminate the effects of baluns, cables and instruments. To verify the measured results, a numerical model incorporating the entire HBC system is established. Besides, it is demonstrated that both the impedance and path gain bandwidths of HBC channels is affected by the electrode configuration. Based on the analysis of the simulated electric field distribution, an equivalent circuit model is proposed and the circuit parameters are extracted using the finite element method. The transmission capability along the human body is also studied. The simulated results using the numerical and circuit models coincide very well with the measurement, which demonstrates that the proposed circuit model can effectively interpret the operation mechanism of the capacitive HBC.

[Molecular identification of Mentha haplocalyx and Mentha spicata with specific primers multi-PCR system].

OBJECTIVE: To screen specific SNPs loci of Mentha haplocalyx and Mentha spicata,and then specific primers were designed to identify the two species and their mixture rapidly. METHODS: PsbA-trnH sequences of Mentha haplocalyx and Mentha spicata were obtained by PCR product sequencing and downloading from GenBank. SNPs in the psbA-trnH sequences of Mentha haplocalyx and Mentha spicata were found by ClustulW program and Bioedit software. Primers for authentication of the two species were designed according to the SNP loci, and PCR reaction system was optimized to identify the original plants. RESULTS: Multi-PCR reaction system was constructed. The 181 bp identification band for Mentha haplocalyx or(and) 288 bp identification band for Mentha spicata could be produced by a single PCR reaction,which showed good identification ability to the two species. CONCLUSION: The multi-PCR reaction system can be applied to identify Mentha haplocalyx and Mentha spicata as well as their mixture.

[Preliminary study on research method of cell survival rate at procedure of cryopreservation of tissue engineered tendons].

OBJECTIVE: To study the research method of cell survival rate at the procedure of cryopreservation of tissue engineered tendons. METHODS: In the 4th generation of human fibroblasts, the dead cells were stained with propidium iodine (PI), while the living cells with Hoechst 33342 (Ho). The living cells and dead cells emitted fluorescence of red and blue respectively after they were stimulated by suitable ultra-violet, then flow cytometry was applied to distinguish them. The seeding cells were collected to make them to be the cell suspension of suitable concentration (6.0 x 10(5) cell/ml) before they were divided into two parts. We cryopreserved and defrosted one part three times to kill the cells and did not cryopreserve the other part, then we made cell suspension at different ratios of cryopreserved cell to non-cryopreserved cells. The fluorescence staining and flow cytometry were used to study the correlation between cell ratios of cryopreserved cell to non-cryopreserved cell and the cell survival rates. We compared the cell survival rates between immediate flow cytometry and that 2 hours after fluorescence staining. RESULTS: The results of flow cytometry showed that correlation between the ratio of cryopreservation and the cell survival rate was significant (r = 0.970, P < 0.05), image analysis study also showed the correlation was significant (r = 0.982, P < 0.05). The cell survival rate decreased by use of flow cytometry two hours after fluorescence staining, but there was no significant difference when compared with that of immediate flow cytometry (P > 0.05). We could also observe the cells on the tissue engineered tendons by fluorescence image directly. CONCLUSION: Flow cytometry and fluorescence image after PI and Ho staining is a good way in study cell survival rate at the procedure of cryopreservation of tissue engineered tendons.