Complete genome sequence of polygonatum kingianum mottle virus infecting Polygonatum kingianum Coll. et Hemsl in Yunnan, China.

The complete genome sequence of a putative novel potyvirus, tentatively named "polygonatum kingianum mottle virus" (PKgMV; GenBank accession no. ON428226), infecting Polygonatum kingianum in China, was obtained by next-generation sequencing (NGS), reverse transcription polymerase chain reaction (RT-PCR), and rapid amplification of cDNA ends (RACE). PKgMV exhibits the typical genome organization and characteristics of members of the genus Potyvirus, with a length of 10,002 nucleotides (nt) and a large open reading frame (nt 108 to 9,746) encoding a polyprotein of 3,212 amino acids (aa) (363.68 kDa). Pairwise comparisons revealed that the PKgMV polyprotein shares 50.5-68.6% nt and 43.1-72.2% aa sequence identity with reported members of the genus Potyvirus. Moreover, phylogenetic analysis indicated that PKgMV is closely related to polygonatum kingianum virus 1 (PKgV1; accession no. MK427056). These results suggest that the PKgMV is a novel member of the genus Potyvirus of the family Potyviridae.

Advances and Prospects of Virus-Resistant Breeding in Tomatoes.

Plant viruses are the main pathogens which cause significant quality and yield losses in tomato crops. The important viruses that infect tomatoes worldwide belong to five genera: Begomovirus, Orthotospovirus, Tobamovirus, Potyvirus, and Crinivirus. Tomato resistance genes against viruses, including Ty gene resistance against begomoviruses, Sw gene resistance against orthotospoviruses, Tm gene resistance against tobamoviruses, and Pot 1 gene resistance against potyviruses, have been identified from wild germplasm and introduced into cultivated cultivars via hybrid breeding. However, these resistance genes mainly exhibit qualitative resistance mediated by single genes, which cannot protect against virus mutations, recombination, mixed-infection, or emerging viruses, thus posing a great challenge to tomato antiviral breeding. Based on the epidemic characteristics of tomato viruses, we propose that future studies on tomato virus resistance breeding should focus on rapidly, safely, and efficiently creating broad-spectrum germplasm materials resistant to multiple viruses. Accordingly, we summarized and analyzed the advantages and characteristics of the three tomato antiviral breeding strategies, including marker-assisted selection (MAS)-based hybrid breeding, RNA interference (RNAi)-based transgenic breeding, and CRISPR/Cas-based gene editing. Finally, we highlighted the challenges and provided suggestions for improving tomato antiviral breeding in the future using the three breeding strategies.

Genomic characterization and seed transmission of a novel unclassified partitivirus infecting Polygonatum kingianum Coll. et Hemsl.

This study identified a novel virus in the family Partitiviridae infecting Polygonatum kingianum Coll. et Hemsl, which is tentatively named polygonatum kingianum cryptic virus 1 (PKCV1). PKCV1 genome has two RNA segments: dsRNA1 (1926 bp) has an open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp) of 581 amino acids (aa), and dsRNA2 (1721 bp) has an ORF encoding a capsid protein (CP) of 495 aa. The RdRp of PKCV1 shares 20.70-82.50% aa identity with known partitiviruses, and the CP of PKCV1 shares 10.70-70.80% aa identity with known partitiviruses. Moreover, PKCV1 phylogenetically clustered with unclassified members of the Partitiviridae family. Additionally, PKCV1 is common in P. kingianum planting regions and has a high infection rate in P. kingianum seeds.

Contributions of ectomycorrhizal fungi in a reclaimed poplar forest (Populus yunnanensis) in an abandoned metal mine tailings pond, southwest China.

Reclamation using fast-growing trees has great potential for agroforestry development on former non-ferrous metal mining areas. However, the functional traits of ectomycorrhizal fungi (ECMF) and the relationship between ECMF and reclaimed trees remain unknown. Here, the restoration of ECMF and their functions in reclaimed poplar (Populus yunnanensis) growing in a derelict metal mine tailings pond were investigated. We identified ECMF belonging to 15 genera in 8 families, suggesting the occurrence of spontaneous diversification as poplar reclamation progressed. We described a previously unknown ectomycorrhizal relationship between poplar roots and Bovista limosa. Our results showed that B. limosa PY5 alleviated the phytotoxicity of Cd and enhanced poplar heavy metal tolerance, resulting in increased plant growth due to reduced Cd accumulation in host tissues. As part of the improved metal tolerance mechanism, PY5 colonization activated antioxidant systems, enhanced the conversion of Cd into inactive chemical forms, and promoted the compartmentalization of Cd into host cell walls. These results suggest that introducing adaptative ECMF may be an alternative to bioaugmenting reforestation and phytomanagement programs of fast-growing native trees in the barren metal mining and smelting areas.

Harnessing an arbuscular mycorrhizal fungus to improve the adaptability of a facultative metallophytic poplar (Populus yunnanensis) to cadmium stress: Physiological and molecular responses.

Populus yunnanensis Dode, a facultative metallophytic poplar, exhibits afforestation potential in barren mine tailing areas. However, the interactions and functional roles of arbuscular mycorrhizal fungus (AMF) in P. yunnanensis adaptability to heavy metal stress remain unclear. Physiological and molecular responses of P. yunnanensis plantlets to AMF (Funneliformis mosseae) under cadmium (Cd) stress (50 mg kg-1) were investigated. Results showed attenuation of Cd phytotoxicity effects on cell organelles upon AMF inoculation, which also reduced the Cd concentration in the poplar leaves, stems, and roots. Under Cd stress, AMF-blocking of metal transporter (e.g., Ca2+ channel) activity occurred, decreasing root cell Cd influx by reducing H+ efflux. Bioaugmentation of rhizosphere sediments by AMF to stabilize metals with a decreasing DTPA-extractable Cd also occurred. The AMF inoculation promoted Cd conversion into inactive, less phytotoxic forms, and helped to maintain ion homeostasis and relieve nutritional ion (e.g., Ca, Mg) disorders caused by excessive Cd. Leaf enzyme and non-enzyme antioxidant systems were triggered. Root and leaf physiological response patterns differed. The AMF regulated the poplar functional genes, and nine metal-responsive gene clusters were identified. We suggest that AMF is a functional component of P. yunnanensis phenotype extension, contributing to strong adaptability to unfavorable mine tailings conditions.

Occurrence, Distribution, Evolutionary Relationships, Epidemiology, and Management of Orthotospoviruses in China.

Orthotospoviruses are responsible for serious crop losses worldwide. Orthotospoviral diseases have spread rapidly in China over the past 10 years and are now found in 19 provinces. Currently, 17 Orthotospovirus species have been reported in China, including eight newly identified species from this genus. The number of new highly pathogenic Orthotospovirus strains or species has increased, likely because of the virus species diversity, the wide range of available hosts, adaptation of the viruses to different climates, and multiple transmission routes. This review describes the distribution of Orthotospovirus species, host plants, typical symptoms of infection under natural conditions, the systemic infection of host plants, spatial clustering characteristics of virus particles in host cells, and the orthotospoviral infection cycle in the field. The evolutionary relationships of orthotospoviruses isolated from China and epidemiology are also discussed. In order to effectively manage orthotospoviral disease, future research needs to focus on deciphering the underlying mechanisms of systemic infection, studying complex/mixed infections involving the same or different Orthotospovirus species or other viruses, elucidating orthotospovirus adaptative mechanisms to multiple climate types, breeding virus-resistant plants, identifying new strains and species, developing early monitoring and early warning systems for plant infection, and studying infection transmission routes.

Characterization of a New Orthotospovirus from Chilli Pepper in Yunnan Province, China.

Chilli pepper (Capsicum annuum L.) is one of the most important crops in Yunnan Province, China. An orthotospovirus isolate 14YV855 was isolated from a diseased chilli pepper plant exhibiting yellow ringspots and necrosis on leaves in Shiping County, Honghe Hani and Yi Autonomous Prefecture, Yunnan Province in 2014. The complete genome sequence of 14YV855 was determined. The small, medium, and large RNAs are 3,428, 4,781, and 8,917 nucleotides long, respectively. The complete nucleocapsid (N) protein of 14YV855 shares a high amino acid identity of 84.8 to 89.9% to that of Capsicum chlorosis virus (CaCV), Groundnut bud necrosis virus (GBNV), Watermelon bud necrosis virus (WBNV), and Watermelon silver mottle virus (WSMoV), which is slightly less than the 90% identity threshold for the demarcation of new Orthotospovirus sp. Phylogenetic analyses revealed that the N protein and RNA-dependent RNA polymerase of 14YV855 are the most related to WSMoV, while the NSs, NSm, and Gn/Gc proteins are similar to those of GBNV. As expected, 14YV855 is serologically related to CaCV, GBNV, WBNV, and WSMoV when the monoclonal antibody against the N protein of WSMoV was used; however, 14YV855 can be distinguished from other orthotospoviruses by reverse-transcription PCR using the specific primers. Our results indicate that 14YV855 is a new Orthotospovirus sp. belonging to the WSMoV serogroup and is provisionally named Chilli yellow ringspot virus.

Characterization of a new isolate of pepper chlorotic spot virus from Yunnan province, China.

A new isolate, 14YV733, of pepper chlorotic spot virus (PCSV) from chili peppers in Yunnan province of China was identified. The typical tospoviral particles of 80-120 nm in diameter were observed by electron microscopy. The virus caused systemic symptoms in several solanaceous plants and the Brassica rapa L. Chinensis group with mechanical inoculation. The sap from infected leaves reacted positively to a rabbit antibody to the N protein of watermelon silver mottle virus (WSMoV) in immunoblotting. The S, M, and L RNAs of PCSV-14YV733 are 3310 nts, 4711 nts, and 8913 nts long, respectively. This is the first report of complete sequences of PCSV in mainland China. Phylogenetic analysis of all tospoviral proteins indicated that PCSV-14YV733 is closely related to members of the WSMoV serogroup.

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.

Monoclonal antibodies for differentiating infections of three serological-related tospoviruses prevalent in Southwestern China.

BACKGROUND: The thrips-borne tospoviruses Calla lily chlorotic spot virus (CCSV), Tomato zonate spot virus (TZSV) and a new species provisionally named Tomato necrotic spot associated virus (TNSaV) infect similar crops in southwestern China. The symptoms exhibiting on virus-infected crops are similar, which is difficult for distinguishing virus species by symptomatology. The sequences of nucleocapsid proteins (NPs) of CCSV, TNSaV and TZSV share high degrees of amino acid identity with each other, and their serological relationship was currently demonstrated from the responses of the previously reported monoclonal antibodies (MAbs) against the NP of CCSV (MAb-CCSV-NP) and the nonstructural NSs protein of Watermelon silver mottle virus (WSMoV) (MAb-WNSs). Therefore, the production of virus-specific antibodies for identification of CCSV, TNSaV and TZSV is demanded to improve field surveys. METHODS: The NP of TZSV-13YV639 isolated from Crinum asiaticum in Yunnan Province, China was bacterially expressed and purified for producing MAbs. Indirect enzyme-linked immunosorbent assay (ELISA) and immunoblotting were conducted to test the serological response of MAbs to 18 tospovirus species. Additionally, the virus-specific primers were designed to verify the identity of CCSV, TNSaV and TZSV in one-step reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Two MAbs, denoted MAb-TZSV-NP(S15) and MAb-TZSV-NP(S18), were screened for test. MAb-TZSV-NP(S15) reacted with CCSV and TZSV while MAb-TZSV-NP(S18) reacted specifically to TZSV in both indirect ELISA and immunoblotting. Both MAbs can be used to detect TZSV in field-collected plant samples. The epitope of MAb-TZSV-NP(S18) was further identified consisting of amino acids 78-86 (HKIVASGAD) of the TZSV-13YV639 NP that is a highly conserved region among known TZSV isolates but is distinct from TNSaV and TZSV. CONCLUSIONS: In this study, two MAbs targeting to different portions of the TZSV NP were obtained. Unlike MAb-CCSV-NP reacted with TNSaV as well as CCSV and TZSV, both TZSV MAbs can be used to differentiate CCSV, TNSaV and TZSV. The identity of CCSV, TNSaV and TZSV was proven by individual virus-specific primer pairs to indicate the correctness of serological responses. We also proposed an serological detection platform using MAb-CCSV-NP, MAb-TZSV-NP(S15) and MAb-TZSV-NP(S18) to allow researchers and quarantine staff to efficiently diagnose the infections of CCSV, TNSaV and TZSV in China and other countries.

Full-length M and L RNA sequences of tospovirus isolate 2009-GZT, which causes necrotic ringspot on tomato in China.

The virus isolate 2009-GZT, collected from tomato in Guizhou province of China, was identified as a new member of the genus Tospovirus based on its S RNA sequence. Because its provisional name, "tomato necrotic spot virus" (TNSV), was identical to an already existing member of the genus Ilarvirus, 2009-GZT was renamed "tomato necrotic spot-associated virus" (TNSaV). In this study, the full-length sequences of the genomic M and L RNAs of TNSaV were determined and analyzed. The M RNA has 4,773 nucleotides (nt), encoding the NSm protein of 309 aa (34.4 kDa) in the viral (v) strand and the glycoprotein precursor (Gn/Gc) of 1123 aa (128 kDa) in the viral complementary (vc) strand. The NSm and Gn/Gc of TNSaV share the highest aa sequence identity (86.2 % and 86.9 %, respectively) with those of tomato zonate spot virus. The L RNA contains 8,908 nt and codes for the putative RNA-dependent RNA polymerase (RdRp) of 2885 aa (332 kDa) in the vc strand. The RdRp of TNSaV shares the highest aa sequence identity (85.2 %) with that of calla lily chlorotic spot virus (CCSV). Serological assays showed that TNSaV cross-reacts with rabbit antisera against watermelon silver mottle virus (WSMoV) NP and CCSV NP, indicating that TNSaV is a member of the WSMoV serogroup.

Clustering and cellular distribution characteristics of virus particles of Tomato spotted wilt virus and Tomato zonate spot virus in different plant hosts.

BACKGROUND: Tomato spotted wilt virus (TSWV) and Tomato zonate spot virus (TZSV) are the two dominant species of thrip-transmitted tospoviruses, cause significant losses in crop yield in Yunnan and its neighboring provinces in China. TSWV and TZSV belong to different serogroup of tospoviruses but induce similar symptoms in the same host plant species, which makes diagnostic difficult. We used different electron microscopy preparing methods to investigate clustering and cellular distribution of TSWV and TZSV in the host plant species. RESULTS: Negative staining of samples infected with TSWV and TZSV revealed that particles usually clustered in the vesicles, including single particle (SP), double particles clustering (DPC), triple particles clustering (TPC). In the immunogold labeling negative staining against proteins of TZSV, the antibodies against Gn protein were stained more strongly than the N protein. Ultrathin section and high pressure freeze (HPF)-electron microscopy preparations revealed that TSWV particles were distributed in the cisternae of endoplasmic reticulum (ER), filamentous inclusions (FI) and Golgi bodies in the mesophyll cells. The TSWV particles clustered as multiple particles clustering (MPC) and distributed in globular viroplasm or cisternae of ER in the top leaf cell. TZSV particles were distributed more abundantly in the swollen membrane of ER in the mesophyll cell than those in the phloem parenchyma cells and were not observed in the top leaf cell. However, TZSV virions were mainly present as single particle in the cytoplasm, with few clustering as MPC. CONCLUSION: In this study, we identified TSWV and TZSV particles had the distinct cellular distribution patterns in the cytoplasm from different tissues and host plants. This is the first report of specific clustering characteristics of tospoviruses particles as well as the cellular distribution of TSWV particles in the FI and globular viroplasm where as TZSV particles inside the membrane of ER. These results indicated that tospoviruses particles possessed specific and similar clustering in the saps of diseased plants. Furthermore, the results of this study will also provide a basis for further study on the tospoviruses assembling, maturation and movement.

Identification of a new tospovirus causing necrotic ringspot on tomato in China.

BACKGROUND: Emerging tospoviruses cause significant yield losses and quality reduction in vegetables, ornamentals, and legumes throughout the world. So far, eight tospoviruses were reported in China. Tomato fruits displaying necrotic and concentric ringspot symptoms were found in Guizhou province of southwest China. FINDING: ELISA experiments showed that crude saps of the diseased tomato fruit samples reacted with antiserum against Tomato zonate spot virus (TZSV). Electron microscopy detected presence of quasi-spherical, enveloped particles of 80-100 nm in such saps. The putative virus isolate was designated 2009-GZT. Mechanical back-inoculation showed that 2009-GZT could infect systemically some solanaceous crop and non-crop plants including Capiscum annuum, Datura stramonium, Nicotiana benthamiana, N. rustica, N. tabacum and Solanum lycopersicum. The 3012 nt full-length sequence of 2009-GZT S RNA shared 68.2% nt identity with that of Calla lily chlorotic spot virus (CCSV), the highest among all compared viruses. This RNA was predicted to encode a non-structural protein (NSs) (459 aa, 51.7 kDa) and a nucleocapsid protein (N) (278 aa, 30.3 kDa). The N protein shared 85.8% amino acid identity with that of CCSV. The NSs protein shared 82.7% amino acid identity with that of Tomato zonate spot virus(TZSV). CONCLUSION: Our results indicate that the isolate 2009-GZT is a new species of Tospovirus, which is named Tomato necrotic spot virus (TNSV). This finding suggests that a detailed survey in China is warranted to further understand the occurrence and distribution of tospoviruses.

[Expression and subcellular location of NSm protein of Tomato spotted wilt virus in plant and insect cells].

OBJECTIVE: Expression and subcellular location of NSm protein of Tomato spotted wilt virus were studied using plant and insect cells. METHODS: First, the NSm gene, located on the ambisense M RNA segment of tomato spotted wilt virus, was cloned into the pCHF3 vector which includes a GFP gene. Agrobacterium-mediated transient expression from N. benthamiana leaves was used to study the location of NSm in plant cells. Second, to test whether plant-specific components were involved in tubule formation, the NSm gene was also expressed in a heterologous expression system, i. e., insect cells. T. ni (Tn) cells were infected with a recombinant baculovirus expressing the NSm gene. RESULTS: NSm-GFP fusion proteins diffused in the tobacco epidermal cells and were located at the edge of the cell walls. These proteins can also form discontinuous green fluorescent spots at the plasmodesmata, which were sometimes present in pairs between two neighboring cells. However, GFP proteins expressed alone distributed evenly around the cell wall and in the nucleus. In the entomic Tn cells, NSm proteins formed a large number of tubular structures extending from the surface. CONCLUSION: These findings suggest that NSm protein target the plasmodesmata specifically in plant cells, and they also could form tubular structures on the surface when expressed in entomic Tn cells.