Genetic variability of rice stripe virus after its pandemic in Jiangsu.

BACKGROUND: Rice stripe virus (RSV) caused a serious disease pandemic in rice in East China between 2001 and 2010. The continuous integrated managements reduced virus epidemic year by year until it was non-epidemic. As an RNA virus, its genetic variability after undergoing a long-term non-epidemic period was meaningful to study. While in 2019, the sudden occurrence of RSV in Jiangsu provided an opportunity for the study. METHODS AND RESULTS: The complete genome of JY2019, an RSV isolate from Jiangyan, was determined. A genotype profile of 22 isolates from China, Japan and Korea indicated that the isolates from Yunnan formed the subtype II, and other isolates clustered the subtype I. RNA 1-3 of JY2019 isolate well-clustered in the subtype I clade, and RNA 4 was also in subtype I, but it had a slight separation from other intra-group isolates. After phylogenetic analyses, it was considered NSvc4 gene contributed to the tendency, because it exhibited an obvious trend towards the subtype II (Yunnan) group. High sequence identity (100%) of NSvc4 between JY2019 and barnyardgrass isolate from different regions demonstrated genetic variation of NSvc4 was consistent in RSV natural populations in Jiangsu in the non-epidemic period. In the phylogenetic tree of all 74 NSvc4 genes, JY2019 belonged to a minor subtype Ib, suggesting the subtype Ib isolates might have existed in natural populations before the non-epidemic period, but not a dominant population. CONCLUSIONS: Our results suggested that NSvc4 gene was susceptible to selection pressure, and the subtype Ib might be more adaptable for the interaction between RSV and hosts in the non-epidemic ecological conditions.

First Report of Beet western yellows virus Infecting Wheat in China.

Wheat (Triticum aestivum L.) is an important cereal crop widely cultivated worldwide. Viral disease is a major threat to wheat yield. In April 2022, fifteen winter wheat plants with yellowing and stunting symptoms were collected from wheat fields in Jingjiang, Jiangsu Province. Total RNA of each sample was extracted, and RT-PCR was performed using two pairs of degenerate luteovirus primers Lu-F (5'-CCAGTGGTTRTGGTC-3') and Lu-R (5'-GTCTACCTATTTGG-3'), Leu-F (5'-GCTCTAGAATTGTTAATGARTACGGTCG-3') and Leu-R (5'-CACGCGTCN ACCTATTTNGGRTTNTG-3'). Amplicons with the expected size were obtained from 10 of the 15 samples (using primers Lu-F/Lu-R) and 3 of the 15 samples (using primers Leu-F/Leu-R), respectively. These amplicons were cloned into the pDM18-T vector (TaKaRa) for sequencing. Blastn alignment showed that 10 amplicons (531 bp) from Lu-F/Lu-R primers were essentially identical to one another, which shared 99.62% nucleotide sequence identity to barley yellow dwarf virus-PAV (BYDV-PAV) isolate GJ1 from Avena sativa in South Korea (LC550014). Three amplicons (635 bp) from Leu-F/Leu-R primers had 99.68% nucleotide identity to the corresponding part of an isolate of beet western yellows virus (BWYV) from saffron (Crocus sativus) in China (MG002646). Among the 13 virus-positive samples, none were co-infected by BYDV-PAV and BWYV. Then, amplification using BWYV-specific primers (BWYV-F: 5'-TGCTCCGGTTTTGACTGGAGTGT-3', BWYV-R: 5'-CGTCTACCT ATTTTGGGTTGTGG-3') generated a 1409 bp product, corresponding to the partial sequence of the viral RNA-dependent RNA polymerase gene and complete sequence of the coat protein (CP) gene. The sequences (GenBank accession no. ON924175) of amplicons from 3 BWYV samples were identical to one another, which shared 98.41% nucleotide identity to BWYV isolate Hs from Japanese hop (Humulus scandens) in China (KC210049). The predicted coat protein of the BWYV wheat isolate had 99.51% nucleotide and 100% amino acid identity to BWYV isolate Hs. BWYV infection on wheat samples was also verified by dot-nucleic acid hybridization using a digoxigenin-labeled cDNA probe against the CP gene, as described previously (Liu et al. 2007). Further, RNA-positive samples were tested by enzyme-linked immunosorbent assay (ELISA) using the ELISA reagent kit for BWYV (Catalog No. KS19341, Shanghai Keshun Biotech, Shanghai, China); test results were also BWYV-positive, confirming that both BWYV nucleic and coat protein are present in these wheat samples. BYDV-PAV is a common wheat virus (Chay et al. 1996), while BWYV has never been reported to infect wheat. BWYV, an aphid-transmitted virus belonging to the Polerovirus genus, has an extensive host range, including over 150 plant species from 23 dicotyledonous families, such as Beta vulgaris, Spinacia oleracea, Lactuca sativa, and Brassica oleracea var. italica (Duffus 1964, 1973; Russell 1965; Beuve et al. 2008). In addition, BWYV was reported to infect a monocotyledonous plant, Crocus sativus (Iridaceae) (Zheng et al. 2018). To our knowledge, this is the first report of BWYV in wheat or any other Gramineae crop. The result also implies that BWYV has a potential risk to cereal crops in the field.

First Report of Tomato mottle mosaic virus infecting Chinese snake gourd (Trichosanthes kirilowii) in China.

Tomato mottled mosaic virus (ToMMV) was first identified in tomato in Mexico (Li et al. 2013). It belongs to the genus Tobamovirus and family Virgaviridae, and is a positive-sense single-stranded RNA virus. The viral genome contains about 6400 nucleotides, encoding four proteins, including the 126 K protein, 183 K protein, movement protein (MP) and coat protein (CP) (Tu et al. 2021). ToMMV mainly poses a serious risk to solanaceous crops. The virus-infected plants appear stunted growth and top necrosis, and the disease leaves show mottled, shrinkage and necrosis symptoms, resulting in a significant decline in tomato fruit yield and quality (Li et al. 2017; Tu et al. 2021). Chinese snake gourd (Trichosanthes kirilowii Maxim) is a perennial climbing herb in the family Cucurbitaceae, and the fruit, seed, peel and root can all be used as traditional Chinese medicine. In May of 2021, twenty-seven symptomless seedlings (developed from tissue culture plantlets) were randomly collected from nursery in Fengyang, Anhui Province. Total RNA of each sample was extracted, and RT-PCR was performed using degenerate tobamovirus primers Tob-Uni1 (5'-ATTTAAGTGGASGGAAAAVCACT-3') and Tob-Uni2 (5'-GTYGTT GATGAGTTCRTGGA-3') (Letschert et al. 2002). Amplicons with expected size were obtained from 6 of 27 samples and sequenced. Alignment results showed that the nucleotide sequence identities ranged from 98.7 to 100% with all ToMMV isolates deposited in NCBI GenBank. Then, ToMMV coat protein (CP) gene was amplified using specific primers CP-F (5'-ATGTCTTACGCTATTACTT CTCCG-3') and CP-R (5'-TTAGGACGCTGGCGCAGAAG-3'). The CP fragment was obtained and sequenced. Sequence alignment indicated that CP sequence of isolate FY (GenBank accession no. ON924176) exhibited a 100% identity with ToMMV isolate LN (MN853592.1). The anti-ToMMV polyclonal antibody (PAb) was prepared by the author (S.L.) by immunizing rabbit with purified virus from Nicotiana benthamiana, and serological tests (dot-enzyme linked immunosorbent assay, Dot-ELISA) of RNA-positive T. kirilowii leaf samples using anti-ToMMV PAb were also positive. To fulfill a Koch's postulate, a pure culture of ToMMV was obtained from N. benthamiana using infectious cDNA clone of ToMMV (Tu et al. 2021), and then healthy T. kirilowii plants were mechanically inoculated with a prepared inoculum from ToMMV-infected N. benthamiana, as described previously (Sui et al. 2017). T. kirilowii seedlings showed chlorosis and leaf tip necrosis symptoms at 10 and 20 day post-inoculation respectively, and ToMMV infection on symptomatic plants was also verified by RT-PCR detection using primers CP-F and CP-R. These results demonstrated that T. kirilowii is a host of ToMMV under natural conditions, which might threaten the production of this medicinal plant. The seedlings from nursery appeared to be asymptomatic, but the plants showed chlorosis and necrosis symptoms after indoor inoculation. In qRT-PCR analysis, viral accumulation level in greenhouse-inoculated plants was a 25.6-fold of that in field-collected samples, which may be the reason of different symptom expression between field samples and inoculated samples. ToMMV has now been detected from the solanaceous (tomato, pepper and eggplant) and leguminous (pea) crops in the field (Li et al. 2014; Ambrós et al. 2017; Zhang et al. 2022). To our knowledge, this is the first report of natural infection of ToMMV in T. kirilowii as well as its natural infection on Cucurbitaceae plants.

A rice plant expressing viral glycoprotein NSvc2-NS reduces the transmission of rice stripe virus by the small brown planthopper.

BACKGROUND: Plant viruses transmitted by arthropod vectors threaten crop health worldwide. Rice stripe virus (RSV) is one of the most important rice viruses in East Asia and is transmitted by the small brown planthopper (SBPH). Previously, it was demonstrated that the viral glycoprotein NSvs2-N could mediate RSV infection of the vector midgut. Therefore, NSvc2-N protein could potentially be used to reduce RSV transmission by competitively blocking midgut receptors. RESULTS: Here, we report that transgenic rice plants expressing viral glycoprotein can interfere with RSV acquisition and transmission by SBPH. The soluble fraction (30-268 amino acids, designated NSvs2-NS ) of NSvs2-N was transformed into rice calli, which produced plants harboring the exogenous gene. When SBPH was fed on transgenic plants prior to RSV-infected rice (sequential feeding) and when insects were fed on RSV-infected transgenic plants (concomitant feeding), virus acquisition by the insect vector was inhibited, and subsequent viral titers were reduced. Immunofluorescence labeling also indicated that viral infection of the insect midgut was inhibited after SBPH was fed on transgenic plants. The system by which RSV infected insect cells in vitro was used to corroborate the role of NSvc2-NS in reducing viral infection. After the cells were incubated with transgenic rice sap, the virus infection rate of the cells decreased significantly, and viral accumulation in the cells was lower than that in the control group. CONCLUSION: These results demonstrated the negative effect of NSvs2-NS transgenic plants on RSV transmission by insect vectors, which provides a novel and effective way to control plant viral diseases. © 2022 Society of Chemical Industry.