First report of Stenotrophomonas maltophilia causing root soft rot of Sanqi (Panax notoginseng) in China.

Sanqi (Panax notoginseng (Burk.) F. H. Chen) is a traditional Chinese medicinal plant with a long planting cycle of 2-3 years that makes it vulnerable to root diseases caused by several pathogens, including Fusarium solani, Alternaria panax, Phytophthoracactorum, and Pseudomonas sp. In April 2019, root soft rot samples of Sanqi were collected from a plantation site in Songming, southwest of China. Typical symptoms included root softening and necrosis, yellow leaf, and stem wilting. Ten diseased roots samples were collected and sterilized with 0.1% HgCl2 for 1 min, 75% ethanol for 2min, and then rinsed thrice with sterile water. Sterilized roots were cut into small pieces of 5 × 5 mm and cultured on the nutrient agar (NA) medium for 48 h at 28°C. From the root cultures, a total of thirteen bacterial strains were obtained. Three strains, SM 2-5, SM 2-13, and SM 2-14 were selected for further study. These three strains were gram-negative, short rod-shaped (1～2×0.5～1µm), non-spore-forming and had polar tufted flagella as observed under a transmission electron microscope (TEM). Also, the strains were positive for oxidase, beta-galactosidase, arginine dihydrolase, and lysine decarboxylase while negative for amylase and urease tested by biochemical methods (Wang 2017). To further determine the pathogenic species, genomic DNA of these three strains was extracted using a Genomic DNA Kit (Tsing Ke, Beijing, China), to PCR amplify 16S rDNA using universal primers 27F/1492R (Wang et al. 2017). Also, S. maltophilia 23S rDNA specific primers SM1/SM4 (Whitby et al. 2000) were used for PCR amplification to confirm the species. 16S rDNA sequence analysis showed that SM 2-5 (GenBank Accession No. MW555227), SM 2-13 (GenBank Accession No. MW555228), and SM 2-14 (GenBank Accession No. MW555229) shared the highest identity (>99.9%) with the S. maltophilia strains (GenBank Accession No. MT323142, MH669295, MN826555). Furthermore, 23S rDNA sequence analysis of SM 2-5 (GenBank Accession No. MZ707732), SM 2-13 (GenBank Accession No. MZ645941) and SM 2-14 (GenBank Accession No. MZ707733) revealed their high identity (>99.8%) with the S. maltophilia species. 16S and 23S rDNA phylogenetic analysis (Mega6.06) using the neighbor-joining (NJ) method with 1,000 bootstrap replicates revealed the three strains clustering with the other S. maltophilia strains. Therefore, based on morphology, metabolic profile, and sequence analysis, the three strains were identified as Stenotrophomonas maltophilia. To test pathogenicity, the strains were grown in the nutrient broth (NB) medium for 48h at 28°C until bacterial suspension reached to OD600≈1.0 (2.0×109CFU/mL). Then, healthy roots of one-year-old Sanqi plants, pre-washed with sterilized water and -poked with a sterilized needle, were soaked in bacterial suspension (2.0×109CFU/mL) of the three strains separately for inoculation 10min. Sterilized water treatment was used as a control. Subsequently, bacteria-inoculated plants were planted in sterile soil pots and cultured in a greenhouse at 28°C with shading rate of 70%. Each treatment group included 3 plants with 3 replicates. Ten days post inoculation, symptoms similar to the ones in natural conditions were observed in the bacteria-inoculated plants. Based on the disease index (Li et al. 2020), we found that among the three strains, SM 2-13 displayed the highest virulence, while no symptoms were observed in the control plants. The same bacterial strains were re-isolated from these inoculated roots and identified by the methods described above. Previous studies showed that some Stenotrophomonas species cause plant diseases such as rice white stripe (Singh et al. 2001), strawberry leaf black spot (Wang et al. 2017), Cyclobalanopsis patelliformis leaf spot (Bian et al. 2020), and Jatropha curcas L. seed borne and stem necrosis (Wang et al. 2018). To our knowledge, this is the first report confirming Stenotrophomonas maltophilia causing root soft rot of Panax notoginseng in China.

Complete genome sequence of a new isolate of potato virus M in Yunnan, China.

The complete genome sequence of a new potato virus M (PVM) isolate (PVM-YN), collected from potato (Solanum tuberosum) in Yunnan, China, was determined. It was 8,530 nucleotides (nt) in length, excluding the poly(A) tail at the 3' end, and shared 71.4-72.0% nucleotide sequence identity with available PVM isolates in the NCBI database. The coat proteins (CP) of PVM-YN shared 79.0-97.4% amino acid sequence identity with that of other isolates. It is the first report of the complete genomic sequence of a new PVM isolate infecting S. tuberosum in China.

Induced furoeudesmanes: a defense mechanism against stress in Laggera pterodonta, a Chinese herbal plant.

Laggera pterodonta displays different phenotypes in its natural habitat but expresses a uniform phenotype with large, broad leaves and fewer secondary metabolites when grown under optimal conditions. The production of six furoeudesmanes is only induced when L. pterodonta encounters stresses, conferring host resistance against a broad spectrum of plant invaders.

Anti-tobacco mosaic virus (TMV) Quassinoids from Brucea javanica (L.) Merr.

Two new quassinoids, javanicolide E (1) and javanicolide F (2), along with fifteen known C-20 quassinoids were isolated from the seeds of Brucea javanica (L.) Merr. The antitobacco mosaic virus (TMV) activity of these quassinoids was screened by the conventional half-leaf and leaf-disk method along with Western blot analysis. All of the seventeen quassinoids showed potent anti-TMV activity. Among them, eight compounds, brusatol (3), bruceine B (4), bruceoside B (5), yadanzioside I (6), yadanzioside L (7), bruceine D (8), yadanziolide A (9), and aglycone of yadanziolide D (17), showed strong antiviral activities, with IC(50) values in the range of 3.42-5.66 microM, and were much more effective than the positive control, ningnanmycin (IC(50) = 117.3 microM). The antiviral structure-activity relationships of quassinoids against TMV were also discussed.

Tobacco mosaic virus (TMV) inhibitors from Picrasma quassioides Benn.

To investigate natural inhibitors against tobacco mosaic virus (TMV) from plants, 10 known beta-carboline alkaloids and one quassinoid have been isolated from MeOH extract of the wood of Picrasma quassioides Benn. These compounds were screened for their inhibitory activities against tobacco mosaic virus (TMV). The activity of each compound against TMV infection and replication was tested using a half-leaf assay method, a leaf-disk method, and Western blotting analyses. All of the beta-carboline alkaloids showed moderate anti-TMV activities and exhibited synergistic effects when combined with the quassinoid nigakilactone B (11). To our knowledge, this is the first report on anti-TMV activity of beta-carbolines and their synergistic effects against TMV when combined with a quassinoid.

Benzylphenethylamine alkaloids from Hosta plantaginea with inhibitory activity against tobacco mosaic virus and acetylcholinesterase.

Five new benzylphenethylamine alkaloids, hostasine (1), 8-demethoxyhostasine, 8-demethoxy-10-O-methylhostasine, 10-O-methylhostasine, and 9-O-demethyl-7-O-methyllycorenine, along with 12 known compounds, were isolated from Hosta plantaginea by bioassay-guided fractionation. The structures of the new alkaloids were established by means of extensive spectroscopic methods, and the relative configuration of 1 was further confirmed by single-crystal X-ray diffraction. 7-Deoxy-trans-dihydronarciclasine (IC(50) = 1.80 microM), a known alkaloid, showed strong activity against tobacco mosaic virus by the half-leaf method. Some of these alkaloids were also evaluated for their inhibitory activity against acetylcholinesterase. 8-Demethoxy-10-O-methylhostasine was found to possess significant activity, with an IC(50) of 2.32 microM.

Seco-pregnane steroids target the subgenomic RNA of alphavirus-like RNA viruses.

Plants have evolved multiple mechanisms to selectively suppress pathogens by production of secondary metabolites with antimicrobial activities. Therefore, direct selections for antiviral compounds from plants can be used to identify new agents with potent antiviral activity but not toxic to hosts. Here, we provide evidence that a class of compounds, seco-pregnane steroid glaucogenin C and its monosugar-glycoside cynatratoside A of Strobilanthes cusia and three new pantasugar-glycosides of glaucogenin C of Cynanchum paniculatum, are effective and selective inhibitors to alphavirus-like positive-strand RNA viruses including plant-infecting tobacco mosaic virus (TMV) and animal-infecting Sindbis virus (SINV), eastern equine encephalitis virus, and Getah virus, but not to other RNA or DNA viruses, yet they were not toxic to host cells. In vivo administration of the compounds protected BALB/c mice from lethal SINV infection without adverse effects on the mice. Using TMV and SINV as models, studies on the action mechanism revealed that the compounds predominantly suppress the expression of viral subgenomic RNA(s) without affecting the accumulation of viral genomic RNA. Our work suggested that the viral subgenomic RNA could be a new target for the discovery of antiviral drugs, and that seco-pregnane steroid and its four glycosides found in the two medicinal herbs have the potential for further development as antiviral agents against alphavirus-like positive-strand RNA viruses.