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1. Strawberry vein banding virus P6 protein intracellular transport and an important domain identification

PAN Yuan, ZHOU Xiu-hong, LI Shuai, FENG Ming-feng, SHI Man-ling, ZUO Deng-pan, JIANG Xi-zi, CHEN Jing, HU Ya-hui, ZHANG Xiang-xiang, JIANG Tong

Journal of Integrative Agriculture 2018, 17 (09): 2031-2041. DOI: 10.1016/S2095-3119(18)61978-1

摘要（379）

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Strawberry vein banding virus (SVBV)-infected strawberry cells contain cytoplasmic inclusions with isometric particles. To identify the components of the inclusions, green fluorescent protein (GFP) was fused to the carboxy-terminus (C-terminus) of SVBV open reading frames, these constructs were separately transformed into Agrobacterium tumefaciens and infiltrated into Nicotiana benthamiana leaves. Results showed that the SVBV P6 protein assembled into prominent and amorphous inclusion bodies (IBs). To investigate P6 subcellular localization, P6-GFP was ectopically expressed in N. benthamiana leaves by agroinfiltration and then stained with 4´,6-diamidino-2-phenylindole (DAPI). We found the P6 protein accumulated in the nuclei and also formed cytoplasmic IBs with different sizes. To further determine the location of P6 IBs in the cytoplasm, and explore whether the P6 IBs move freely or depend on cytoskeleton and endoplasmic reticulum (ER), the microfilament marker protein (GFP-ABD2-GFP), microtubules marker protein (mCherry-MAP65-1) and ER marker protein (mCherry-HDEL) were separately coexpressed with P6-GFP and into N. benthamiana leaves by agroinfiltration, exhibiting that P6 IBs aligned with cytoskeleton and endoplasmic reticulum. Meanwhile, coinfiltration of P1 and P6 indicated the P6 colocalized with the P1 protein at periphery of cells. The P6 protein contains one C-terminal nuclear localization signal (NLS) region, a P6 protein mutant with a deleted NLS did not localize in the nucleus, did not form IBs, and was unable to facilitate exogenous GFP expression. These results demonstrate that the deleted NLS region is an important P6 domain required for biological functions. In summary, the mobile P6 IBs are associated with ER, microfilaments and microtubules and move along microfilaments to the SVBV P1 protein in the PD.

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2. Two mutations in the truncated Rep gene RBR domain delayed the Wheat dwarf virus infection in transgenic barley plants

Pavel Cejnar, Ludmila Ohnoutková, Jan Ripl, Tomá? Vl?ko, Jiban Kumar Kundu

Journal of Integrative Agriculture 2018, 17 (11): 2492-2500. DOI: 10.1016/S2095-3119(18)62000-3

摘要（282）

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Wheat dwarf virus (WDV), an important cereal pathogen, is closely related to Maize streak virus (MSV), a model virus of the Mastrevirus genus. Based on its similarity to known MSV resistance strategies, a truncated part of the WDV replication-associated (RepA) gene (WDVRepA215) and the WDV RepA gene with a mutated retinoblastoma-related protein (RBR) interaction domain (WDVRepA215RBR^mut) were cloned into the pIPKb002 expression vector and transformed into immature embryos of spring barley cv. Golden Promise plants through Agrobacterium-mediated transformation. A detailed study of T₁-generation plants infected by leafhoppers (Psammotettix alienus) fed on infection sources of variable strength was performed over a 5-week period encompassing the initial stages of virus infection. A DNA WDV TaqMan qPCR assay normalized using the DNA puroindoline-b SYBR Green qPCR assay for samples on a per week basis revealed an approximately 2-week delay in WDVRepA215RBR^mut plants to WDVRepA215 plants before significant increases in the WDV viral levels occurred. Both WDVRepA215 and WDVRepA215RBR^mut plants showed similar levels of transgenic transcripts over the screened period; however, the transgenic plants also showed increased numbers of infected plants compared to the control plants.

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3. Distribution pattern and titer of Candidatus Liberibacter asiaticus in periwinkle (Catharanthus roseus)

LI Ya, XU Mei-rong, DAI Ze-han, DENG Xiao-ling

Journal of Integrative Agriculture 2018, 17 (11): 2501-2508. DOI: 10.1016/S2095-3119(18)61918-5

摘要（319）

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Candidatus Liberibacter asiaticus (CaLas), an uncultured Gram-negative alphaproteobacterium, is the causal agent of Huanglongbing (HLB) in citrus. CaLas resides in phloem sieve tubes and has been shown to be unequally distributed in different tissues. Although HLB is a disease of citrus plants, it has been demonstrated that periwinkle can serve as an experimental host of CaLas, which can be transmitted from citrus to periwinkle via the parasitic plant dodder (Cuscuta spp.). To investigate the distribution of CaLas in various periwinkle tissues, the bacteria were transmitted from an infected periwinkle plant to healthy periwinkles by top-grafting. The movement of the inoculum and associated titer changes were observed over time in various tissues. CaLas could be detected in the leaves, main stems, and roots of infected periwinkle by conventional PCR, and in all three tissues a clear time-dependent change in CaLas titer was observed, with titer increasing soon after inoculation and then decreasing as disease symptoms became severe. The highest titer was found at 25, 35 and 35 days after inoculation in leaves, main stems and roots, respectively. The titer in leaves was much higher than in the main stems and roots at the same time point, and the spatial distribution of CaLas in the leaves, main stems and roots of infected periwinkle was uneven, similar to what has been shown in citrus. The results provide guidance for selecting the proper periwinkle tissues and sampling times for early detection of CaLas.

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4. Limited infection by ‘Candidatus Liberibacter asiaticus’ in ‘Valencia’ sweet orange trees in the presence of Citrus tristeza virus

FU Shi-min, Cristina Gouin, ZHOU Chang-yong, John S. Hartung

Journal of Integrative Agriculture 2019, 18 (10): 2284-2293. DOI: 10.1016/S2095-3119(19)62605-5

摘要（118）

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Huanglongbing (HLB) is the most destructive disease of citrus and is associated with ‘Candidatus Liberibacter asiaticus’ (CLas), a member of the α-proteobacteria. Citrus tristeza virus (CTV) is another pathogen of citrus with very great historic as well as current importance. Both CLas and CTV are phloem-restricted pathogens. A severe CTV isolate, CTV-B6, and CLas-B232 induce a group of symptoms of phloem dysfunction that overlap, but the mild isolate CTV-B2 does not cause any loss to commercial trees. Prior inoculation and establishment of CLas-B232 did not affect subsequent establishment of either CTV-B2 or CTV-B6, while super infection by CLas-B232 was reduced by prior establishment of CTV-B2 and to a lesser extent by prior infection with CTV-B6. Trees co-infected with CTV-B6 and CLas-B232 developed more severe symptoms, typical of CTV-B6, than either of the two pathogens co-infected with CTV-B2. In this study, we confirmed that CLas established in the rootlets earlier and with higher concentration than in leaves. The distribution of CLas in the plant infected by CLas-B438 alone and with CTV-B2 fits a previously proposed model but CLas was more sporadically distributed in a plant co-infected by CLas and CTV-B2 than in a plant infected by CLas alone. These biological phenomena are aligned with previously analyzed transcriptome data and the study provides a novel idea that mild CTV strains may provide some protection against CLas by limiting its multiplication and spread. The protective effect may be due to opposite regulation of key host defense pathways in response to CTV-B2 and CLas-B438.

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5. Sugarcane mosaic virus infection of model plants Brachypodium distachyon and Nicotiana benthamiana

XU Jing-sheng, DENG Yu-qing, CHENG Guang-yuan, ZHAI Yu-shan, PENG Lei, DONG Meng, XU Qian, YANG Yong-qing

Journal of Integrative Agriculture 2019, 18 (10): 2294-2301. DOI: 10.1016/S2095-3119(19)62572-4

摘要（115）

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Sugarcane mosaic virus (SCMV; genus Potyvirus, family Potyviridae) is a causal pathogen of sugarcane mosaic disease, and it is widespread in regions where sugarcane (Saccharum spp. hybrids) is grown. It is difficult to investigate the molecular mechanism of pathogen infection in sugarcane because of limited genomic information. Here, we demonstrated that SCMV strain FZ1 can systemically infect Brachypodium distachyon inbred line Bd21 and Nicotiana benthamiana through inoculation, double antibody sandwich enzyme-linked immunosorbent, transmission electron microscopy, and reverse transcription PCR assays. The leaves of Bd21 developed mosaic symptoms, while the leaves of N. benthamiana showed no obvious symptoms under the challenge of SCMV-FZ1. We concluded that B. distachyon inbred line Bd21 is a promising experimental model plant compared with N. benthamiana for study on the infectivity of SCMV. This is the first report on the SCMV infection of model plants B. distachyon inbred line Bd21 and N. benthamiana, which will shed light on the mechanism of SCMV infection of sugarcane and benefit sugarcane breeding against sugarcane mosaic disease.

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6. Quantitative analysis of the interaction of heterologous viruses with Plum pox virus in C5 HoneySweet transgenic plums

Khushwant Singh, Tereza Neubauerová, Jiban Kumar Kundu

Journal of Integrative Agriculture 2019, 18 (10): 2302-2310. DOI: 10.1016/S2095-3119(18)62136-7

摘要（111）

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Stone fruits are an important crop in most parts of the world and are heavily challenged by several viruses including Plum pox virus (PPV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), and Apple chlorotic leaf spot virus (ACLSV). We validated the PPV resistance in C5 plum plants (commercially known as HoneySweet) grown in the Czech Republic for more than 16 years in a field trial experiment under natural environmental conditions. We quantified single (PPV-Rec) and mixed viruses (PPV-Rec+ACLSV, PPV-Rec+PDV and PPV-Rec+ACLSV+PDV) in C5 transgenic plums inoculated for the period 2016 to 2018. The accumulation of PPV-Rec was high (~5.43E+05 copies) compared with that of ACLSV (~8.70E+04 copies) in the inoculated graft of C5 transgenic plants. Leaves close to the inoculum sources showed a differential level of virus titre in single and mixed infections (~10 to ~5×10² copies). C5 plants with permanent virus pressure showed 10³- to 10⁵-fold fewer copies of viruses than those of the inoculated graft. We observed high accumulation of conserved miRNAs such as miR167, miR69 and miR396 in C5 plants co-infected with PPV, ACLSV and PDV that are associated with its resistance against viruses. Overall, i) C5 transgenic plums showed high resistance to PPV infection, and a low level (~32 copies) of PPV only accumulated in some grafted plants, ii) high accumulation of PPV was found in inoculated grafts in single PPV infection and mixed infections, iii) heterologous virus infection sustained by ACLSV or PDV did not suppress PPV resistance, and iv) high and low conserved microRNAs accumulated in C5 plants.

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7. Molecular, serological and biological characterization of a novel Apple stem pitting virus strain from a local pear variety grown in China

LI Liu, ZHENG Meng-meng, MA Xiao-fang, LI Yuan-jun, LI Qing-yu, WANG Guo-ping, HONG Ni

Journal of Integrative Agriculture 2019, 18 (11): 2549-2560. DOI: 10.1016/S2095-3119(19)62636-5

摘要（127）

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Apple stem pitting virus (ASPV) is an important causal agent of pear diseases. Nowadays, the infection status and molecular characteristics of the virus in old pear trees have never been investigated. In this study, we provide the first complete genome sequence of an ASPV isolate LYC from an over 300-year-old tree of a local Pyrus bretschneideri cultivar ‘Chili’ specifically grown at Laiyang area in China. ASPV-LYC possesses a chimeric genome consisting of 9 273 nucleotides excluding a poly(A) tail at its 3´ end and harboring a recombination region in its open reading frame (ORF1) with Aurora-1 and KL9 identified as the major and minor parents. Western blot analysis with antisera against recombinant coat proteins (CPs) of three ASPV isolates from pear indicates that ASPV-LYC is serologically related to these ASPV isolates, but with differential activities. Further biological tests on indicator plants of Pyronia veitchii show that ASPV-LYC can induce serious leaf and stem symptoms as other ASPV isolates. The results provide an important information for understanding molecular evolution of ASPV and suggest a need to prevent dissemination of the isolate among pear trees.

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8.

Evaluation of the biocontrol potential of Aspergillus welwitschiae against the root-knot nematode Meloidogyne graminicola in rice (Oryza sativa L.)

LIU Ying, DING Zhong, PENG De-liang, LIU Shi-ming, KONG Ling-an, PENG Huan, XIANG Chao, LI Zhong-cai, HUANG Wen-kun

Journal of Integrative Agriculture 2019, 18 (11): 2561-2570. DOI: 10.1016/S2095-3119(19)62610-9

摘要（110）

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The root-knot nematode Meloidogyne graminicola is considered one of the most devastating pests in rice-producing areas, and nematicides are neither ecofriendly nor cost effective. More acceptable biological agents are required for controlling this destructive pathogen. In this study, the biocontrol potential of Aspergillus welwitschiae AW2017 was investigated in laboratory and greenhouse experiments. The in vitro ovicidal and larvicidal activities of A. welwitschiae metabolites were tested on M. graminicola in laboratory experiments. The effect of A. welwitschiae on the attraction of M. graminicola to rice and the infection of rice by M. graminicola was evaluated in a greenhouse. The bioagent AW2017 displayed good nematicidal potential via its ovicidal and larvicidal action. The best larvicidal activity was observed at a concentration of 5×AW2017, which caused an 86.2% mortality rate at 48 h post inoculation. The highest ovicidal activity was recorded at a concentration of 5×AW2017, which resulted in an approximately 47.3% reduction in egg hatching after 8 d compared to the control. Under greenhouse conditions, the application of A. welwitschiae significantly reduced the root galls and nematodes in rice roots compared to the control. At a concentration of 5×AW2017, juveniles and root galls in rice roots at 14 d post inoculation (dpi) were reduced by 24.5 and 40.5%, respectively. In addition, the attraction of M. graminicola to rice roots was significantly decreased in the AW2017 treatment, and the development of nematodes in the AW2017-treated plants was slightly delayed compared with that in the PDB-treated control plants. The results indicate that A. welwitschiae is a potential biological control agent against M. graminicola in rice.

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9. Mapping subgenomic promoter of coat protein gene of Cucumber green mottle mosaic virus

LIU Mei, LIU Li-ming, WU Hui-jie, KANG Bao-shan, GU Qin-sheng

Journal of Integrative Agriculture 2020, 19 (1): 153-163. DOI: 10.1016/S2095-3119(19)62647-X

摘要（138）

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Many plant viruses utilize subgenomic RNA as gene expression strategy, therefore mapping subgenomic promoter (SGP) is extremely important for constructing viral vectors. Although Cucumber green mottle mosaic virus (CGMMV)-based virus vectors have been constructed, SGP of the coat protein (CP) has not yet mapped. To this end, we firstly presumed 13 nucleotides upstream of the start codon as the transcription starting site (TSS) as previous study identified by random amplification of cDNA ends (RACE). Secondly, the region from nucleotides –110 to +175 is the putative CP SGP, as predicted, a long stem loop structure by the secondary structure of RNA covering movement protein (MP) and CP. To map the CGMMV CP SGP, we further constructed a series of deletion mutants according to RNA secondary structure prediction. The deletion of TSS upstream significantly enhanced CP transcription when 105 nucleotides were retained before the CP TSS. For the downstream of CP TSS, we analyzed the expression of enhanced green fluorescent protein (EGFP) in a series of vectors with partial deletion of the CGMMV CP and found that the nucleotides from +71 to +91 played a key role in the EGFP expression at the transcription level, while EGFP showed the highest expression level when 160 nucleotides were retained downstream of the CP TSS. To confirm these results, we applied online software MEME to predict the motifs and cis-acting elements in the 466 nucleotides covering the sequences of deletion analysis. Conserved motifs and relative acting elements were in regions in which transcription levels were the highest or enhanced. To our best knowledge, this is the first mapping of CGMMV SGP.

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10.

Molecular and biological characterization of melon-infecting squash leaf curl China virus in China

WU Hui-jie, LI Meng, HONG Ni, PENG Bin, GU Qin-sheng

Journal of Integrative Agriculture 2020, 19 (2): 570-577. DOI: 10.1016/S2095-3119(19)62642-0

摘要（173）

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It has been reported that squash leaf curl China virus (SLCCNV) infects some Cucurbitaceae crops except for melon (Cucumis melo L.). A new disease of melon exhibiting severe leaf curl and dwarfing was observed in Hainan Province of China. In this study, the pathogen was identified as SLCCNV through biological and molecular characterization. The isolate (SLCCNV-HN) possess a bipartite genome, DNA-A (HM566112.1) with the highest nucleotide identity (99%) to SLCCNV-Hn (MF062251.1) pumpkin and SLCCNV-Hn61 (AM260205.1) squash isolates from China, whereas DNA-B (HM566113.1) with the highest nucleotide identity (99%) to SLCCNV-Hn (MF062252.1). Phylogenetic analyses based on the full-length SLCCNV-HN DNA-A and -B sequences indicated that SLCCNV-HN melon isolate is clustered with SLCCNV-Hn pumpkin, SLCCNV-Hn61 and SLCCNV-SY squash isolates from southern China, forming an independent cluster. Infectious clone of SLCCNV-HN was constructed and the melon plants were inoculated and the infection rate is 100%, the systemic symptoms in melon showed identical to those of melon plants infected in fields. Additionally, melon plants transmission of this virus by Bemisia tabaci with a transmission rate of 95% (19/20) showed leaf curl and dwarf symptoms 15 days post transmission, thereby fulfilling Koch’s postulates. Analysis of genomic organization and phylogenetic trees indicated that SLCCNV-HN melon isolate belongs to the Begomovirus genus. To the best of our knowledge, this is the first characterization of melon-infecting SLCCNV through its genome, infectious clone and transmission.

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11. Prevalence and genetic diversity of grapevine fabavirus isolates from different grapevine cultivars and regions in China

FAN Xu-dong, ZHANG meng-yan, ZHANG Zun-ping, REN Fang, HU Guo-jun, DONG Ya-feng

Journal of Integrative Agriculture 2020, 19 (3): 768-774. DOI: 10.1016/S2095-3119(19)62677-8

摘要（129）

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A total of 288 grapevine samples of 61 different grapevine cultivars, collected from 22 provinces and regions, were analyzed by reverse transcription-PCR (RT-PCR) for the presence of grapevine fabavirus (GFabV). PCR detection results showed the incidences of GFabV were 12.8% (30/235) and 48.1% (25/52) in the asymptomatic and symptomatic vines, respectively. The genetic diversity of GFabV isolates was analyzed based on partial nucleotide and encoded amino acid sequences of the RNA1 and RNA2 polyprotein genes. Phylogenetic analyses of the RNA1 and RNA2 gene sequences divided the GFabV isolates into five well-defined groups. Groups 1, 2, and 4 comprised only Chinese isolates. This article represents the first report for the prevalence and genetic diversity of GFabV in grapevines grown in China.

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12.

Monoclonal antibody-based serological detection of potato virus M in potato plants and tubers

ZHANG Yu, GAO Yan-ling, HE Wan-qin, WANG Ya-qin, QIAN Ya-juan, ZHOU Xue-ping, WU Jian-xiang

Journal of Integrative Agriculture 2020, 19 (5): 1283-1291. DOI: 10.1016/S2095-3119(19)62755-3

摘要（140）

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Potato virus M (PVM) is one of the common and economically important potato viruses in potato-growing regions worldwide. To investigate and control this viral disease, efficient and specific detection techniques are needed. In this study, PVM virions were purified from infected potato plants and used as the immunogen to produce hybridomas secreting PVM-specific monoclonal antibodies (MAbs). Four highly specific and sensitive murine MAbs, i.e., 1E1, 2A5, 8A1 and 17G8 were prepared through a conventional hybridoma technology. Using these four MAbs, we have developed an antigen-coated plate (ACP)-ELISA, a dot-ELISA and a Tissue print-ELISA for detecting PVM infection in potato plants and tubers. PVM could be detected in infected potato plant tissue crude extracts diluted at 1:10 240 (w/v, g mL^–1) by the dot-ELISA or at 1:163 840 (w/v, g mL^–1) by the ACP-ELISA. The Tissue print-ELISA is the quickest and easiest assay among the three established serological assays and is more suitable for onsite large-scale sample detection. Detection results of the field-collected samples showed that PVM is currently widespread in the Yunnan and the Heilongjiang provinces in China. The field sample test results of the developed serological assays were supported by the results from RT-PCR and DNA sequencing. We consider that the newly established ACP-ELISA, dot-ELISA and Tissue print-ELISA can benefit PVM detection in potato plant and tuber samples and field epidemiological studies of PVM. These assays can also facilitate the production of virus-free seed potatoes and breeding for PVM-resistant potato cultivars, leading to the successful prevention of this potato viral disease.

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13. Virome identification in wheat in the Czech Republic using small RNA deep sequencing

Khushwant SINGH, Jana JAROŠOVÁ, Jan FOUSEK, CHEN Huan, Jiban Kumar KUNDU

Journal of Integrative Agriculture 2020, 19 (7): 1825-1833. DOI: 10.1016/S2095-3119(19)62805-4

摘要（143）

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High-throughput deep-sequencing technology and bioinformatics analysis of the small RNA (sRNA) population isolated from plants allows universal virus detection and complete virome reconstruction for a given sample. In the present sRNA deep-sequencing analysis of virus-infected wheat samples in the Czech Republic, samples were firstly tested for barley yellow dwarf viruses (BYDVs), wheat streak mosaic virus (WSMV) and wheat dwarf virus (WDV) using ELISA, RT-PCR and PCR. Subsequent sRNA sequencing of these samples yielded more than ~60 million single-end 50-bp reads with high confidence for nine field samples of wheat. Overall, 16.5% of reads were virus-specific and 83.5% were mapped to the host. More 21-nt reads (~7.7E+06 reads) were found than 24-nt (~6.20E+06 reads) or 22-nt (~4.30E+06 reads) reads. De novo assembly of the high-quality contigs revealed the presence of three earlier reported viruses in the Czech Republic: BYDVs (31.48%), WSMV (24.23%) and WDV (26.66%). We also showed the presence of cereal yellow dwarf virus (14.33%; two species CYDV-RPS and CYDV-RPV (family Luteoviridae/Polerovirus) and wheat yellow dwarf virus (WYDV, 3.30%; Luteoviridae). Phylogenetic analysis showed CYDV and WYDV grouped separately from BYDVs. Furthermore, several recombination breakpoints were found among the groups of yellow dwarf viruses (BYDVs, CYDV, and WYDV). Using RNA deep sequencing, we confirmed the presence of the three known viruses (BYDVs, WSMV, and WDV) and the first record of two species of CYDV and WYDV in wheat in the Czech Republic.

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14. A sensitive SYBR Green RT-qPCR method for grapevine virus E and its application for virus detection in different grapevine sample types

REN Fang, ZHANG Zun-ping, FAN Xu-dong, HU Guo-jun, ZHANG Meng-yan, DONG Ya-feng

Journal of Integrative Agriculture 2020, 19 (7): 1834-1841. DOI: 10.1016/S2095-3119(19)62784-X

摘要（140）

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To develop a rapid and high-sensitivity method for detection of grapevine virus E (GVE), a SYBR Green based real-time fluorescence quantitative RT-PCR method (RT-qPCR) was established. This method could be used to detect GVE specifically, and the sensitivity was about 100 times greater than conventional RT-PCR. An excellent linear correlation (R²=0.997) and a high amplification efficiency (E=97.5%) were obtained from the standard curve of this method. Reproducibility tests revealed that the coefficients of variation in the intra- and inter-assay results were 0.31–1.03% and 0.82–2.62%, respectively, indicating a good reproducibility. The RT-qPCR method could be used to detect GVE in a wide range of grapevine sample types. The detection rates of RT-qPCR for nearly all sample types from different positions and seasons were higher than conventional RT-PCR. The detection rates in spring, summer, autumn and winter increased gradually. Samples in autumn and winter were best for detection, and the detection rates of most samples were 80–100%, which were 10 to 40% higher than conventional RT-PCR. In general, old petioles and branches were the best tissues for GVE detection. The detection rates of these samples in each season were all 100%, which were 20 to 40% higher than conventional RT-PCR. The second highest rates were in the old leaf, with detection rates for RT-qPCR of 80–100% in all seasons, which were 20 to 40% higher than conventional RT-PCR. GVE could be difficultly detected in young leaves by conventional RT-PCR, and the detection rates were only 0–50%, while by RT-qPCR the rates could increase to 0–80%. A total of 33 out of 363 samples (belonging to 68 cultivars) from 20 regions in China were detected to be positive by RT-qPCR (9.1%), which was more than twice the rate of the conventional RT-PCR (3.9%).

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15. Identification of a novel emaravirus infecting lilac through next-generation sequencing

WANG Ya-qin, SONG Yu, CAO Meng-ji, CHENG Qi, WU Jian-xiang, HU Tao

Journal of Integrative Agriculture 2020, 19 (8): 2064-2071. DOI: 10.1016/S2095-3119(19)62806-6

摘要（148）

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Emaraviruses are multipartite negative-sense single-stranded viruses that usually infect flowering plants. Using unbiased next-generation sequencing (NGS) approach, a novel virus from lilac leaves with yellow mottle symptom was identified. Five viral genomic RNA segments were detected and completely sequenced. The molecular characteristics of the viral RNAs and proteins resemble those of members of the genus Emaravirus. The virus was found to be mechanically transmissible to Nicotiana benthamiana. Sequence alignment and phylogenetic analyses of the virus support the proposal of creating a new species in the genus Emaravirus for which the name lilac chlorotic ringspot-associated virus (LiCRaV) is proposed.

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16. Decreasing detection frequency of MITE (MCLas-A) in the population of ‘Candidatus Liberibacter asiaticus’ recently collected in southern China

CUI Xue-jin, ZENG Chun-hua, LIU Ke-hong, TENG Cai-ling, ZHOU Chang-yong, WANG Xue-feng

Journal of Integrative Agriculture 2020, 19 (10): 2597-2601. DOI: 10.1016/S2095-3119(20)63217-8

摘要（122）

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An active miniature inverted-repeat transposable element (MITE), MCLas-A, was previously identified from ‘Candidatus Liberibacter asiaticus’ known to be associated with citrus Huanglongbing (HLB, yellow shoot disease). To explore the recent transposition status of MCLas-A, 389 ‘Ca. L. asiaticus’ strains collected from nine regions in China were amplified using a specific primer set and three representative ‘Ca. L. asiaticus’ strains were analyzed by next-generation sequencing (NGS) approach. PCR and genomic analysis showed that the entire MCLas-A was only present in 1.80% (7/389) and the jumping-out type of the MITE was predominant (81.23%) in samples tested, suggesting high frequency transposition occurred in ‘Ca. L. asiaticus’ strains recently collected from China. Biological roles of transposition of the active MITE remain to be determined.

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17. Molecular diversity of barley yellow dwarf virus-PAV from China and the Czech Republic

May Oo kHINE, brozenká MICHAELA, LIU Yan, Jiban kumar kUNDU, WANG Xi-feng

Journal of Integrative Agriculture 2020, 19 (11): 2736-2745. DOI: 10.1016/S2095-3119(20)63241-5

摘要（137）

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Wheat yellow dwarf disease (BYD), caused by different species of barley/cereal yellow dwarf viruses (B/CYDVs), is one of the most serious cereal diseases in China and the Czech Republic. Because genetic diversity of the virus directly influences disease epidemiology, the molecular diversity and population structure of 24 Chinese isolates and 16 the Czech Republic isolates of BYDV-PAV from different regions in two countries were analyzed by sequencing their coat protein (CP) and readthrough protein (RTP) domain (RTD) genes and comparing the sequences with six CP and 16 RTP sequences of BYDV-PAV isolates from the NCBI database based on nucleotide identity position, phylogenetic analysis and nucleotide diversity. Nucleotide identities between the Chinese and the Czech Republic isolates for the CP were 76.6–99.4%, 73.9–89.1% for RTD (ORF5), respectively. The Chinese and the other country isolates showed 74.7–99.2% nucleotide identity for RTP (ORF3+ORF5). Phylogenetic analysis of CP sequences showed that 20 Chinese isolates clustered in the same clade, but the other four Chinese isolates clustered in another clade with the isolates from the Czech Republic and other counties. The population of BYDV-PAV in China had greater nucleotide variability and was more divergent than that in the Czech Republic. Geographical and ecological factors but not hosts might contribute to the population differences in the two countries.

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18. Biological and molecular characterization of tomato brown rugose fruit virus and development of quadruplex RT-PCR detection

YAN Zhi-yong, ZHAO Mei-sheng, MA Hua-yu, LIU Ling-zhi, YANG Guang-ling, GENG Chao, TIAN Yan-ping, LI Xiang-dong

Journal of Integrative Agriculture 2021, 20 (7): 1871-1879. DOI: 10.1016/S2095-3119(20)63275-0

摘要（200）

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番茄褐色皱果病毒（tomato brown rugose fruit virus，ToBRFV）是2015年首次报道的一种烟草花叶病毒属病毒，是番茄安全生产的严重威胁。该病毒已经传播到美洲、亚洲和欧洲的十个国家。2019年，ToBRFV在中国山东发生。本论文旨在明确ToBRFV山东分离物（ToBRFV-SD）的症状、寄主范围和分子特性，并建立一种有效的检测方法。田间调查ToBRFV-SD在不同品种的症状表现。将ToBRFV-SD接种辣椒、本氏烟、马铃薯、茄子、中烟102和50个番茄品种，鉴定其寄主范围。分段克隆ToBRFV-SD基因组片段，并测定其序列；利用BioEdit version 7.2.6比对ToBRFV所有分离物的全基因组序列，分析序列一致率；利用MEGA version 10.1.5构建系统发育树。根据ToBRFV、烟草花叶病毒（tobacco mosaic virus，TMV）、番茄花叶病毒（tomato mosaic virus，ToMV）和番茄斑萎病毒（tomato spotted wilt virus，TSWV）等四种番茄重要病毒基因组的保守区域设计特异性引物，建立四重RT-PCR检测体系。ToBRFV-SD在番茄叶片引起不同程度的花叶和疱斑，在花萼和花梗上引起坏死，在番茄果实上引起畸形、黄斑和褐色皱缩坏死斑。ToBRFV-SD可侵染番茄、辣椒和本氏烟，隐症侵染马铃薯、茄子和烟草品种中烟102。测试的50个番茄品种均不抗ToBRFV-SD。ToBRFV-SD和以色列分离物ToBRFV-IL基因组的核苷酸和氨基酸一致率最高。在基于全基因组序列的系统进化树中，所有ToBRFV分离物聚集到一个分枝，与烟草花叶病毒分枝距离较近。随后，我们建立了四重RT-PCR检测体系，能够通过一个RT-PCR反应，同时检测并区分ToBRFV、TMV、ToMV和TSWV。本研究明确了ToBRFV-SD的症状、寄主范围和分子特性，建立了能区分ToBRFV、TMV、ToMV和TSWV四重RT-PCR检测体系，对指导ToBRFV的早期检测和防控有积极作用。

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19. Robust molecular detection of the new Tomato brown rugose fruit virus in infected tomato and pepper plants from Turkey

Hakan FIDAN, Pelin SARIKAYA, Kubra YILDIZ, Bengi TOPKAYA, Gozde ERKIS, Ozer CALIS

Journal of Integrative Agriculture 2021, 20 (8): 2170-2179. DOI: 10.1016/S2095-3119(20)63335-4

摘要（171）

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Tomato brown rugose fruit virus (ToBRFV) causes severe fruit loss in tomato (Solanum lycopersicum) and pepper (Capsicum annuum) plants. It is an emerging Tobamovirus that is spreading globally. The major challenge is to develop a reliable method for the detection of the virus, and to better characterize the symptoms it causes. The aims of this study, therefore, were to characterize the symptom development on tomato and pepper plants, and to establish a reliable detection method for the virus. Following infection of the tomato and pepper plants with ToBRFV, the leaves turned chlorotic, mosaic or mottled, while the fruit became rugose, necrotic and marbled, and showed discoloration with yellow or brown spots. Transmission electron microscopy (TEM) revealed single rod-like virus particles characteristic of the Tobamoviruses. Classical reverse transcription PCR (RT-PCR) and quantitative PCR (qPCR) with specific primers and probes confirmed that the virus is ToBRFV. We found that the resistance genes from tomato, Tm-22, and pepper, L1, L2, L3 and L4, did not confer resistance to ToBRFV. Here, we present a PCR-based method as a diagnostic test for detecting ToBRFV in infected seeds. This method will help to prevent further spread of the virus in commercial seeds.

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20. Elimination of grapevine fleck virus and grapevine rupestris stem pitting-associated virus from Vitis vinifera 87-1 by ribavirin combined with thermotherapy

HU Guo-jun, DONG Ya-feng, ZHANG Zun-ping, FAN Xu-dong, REN Fan

Journal of Integrative Agriculture 2021, 20 (9): 2463-2470. DOI: 10.1016/S2095-3119(20)63336-6

摘要（127）

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本研究以感染葡萄斑点病毒（grapevine fleck virus，GFkV）和沙地葡萄茎痘伴随病毒（grapevine rupestris stem pitting-associated virus，GRSPaV）的87-1葡萄试管苗作为试验材料，研究了不同浓度的病毒醚处理（15和25 μg·mL^-1; R15和R25）、热处理（37°C; T）以及病毒醚结合热处理（R15+T和R25+T）对葡萄试管苗中两种病毒的脱除效果。处理过程中发现，在R15和R25病毒醚处理的过程中，葡萄试管苗均产生了药害，主要表现为植株长势弱。热处理对葡萄植株的生长具有促进作用。T、R15+T和R25+T三个高温处理中植株的株高显著高于CK、R15和R25三个常温处理的植株株高。此外，两种方法的结合可以降低高温对植株的热损伤，处理结束时T、R15+T和R25+T三个处理的植株死亡率分别为51.4%、11.4%和8.6%。处理结束后切取一定大小的茎尖进行再生，各处理再生植株的存活率均高于68.0%。同时发现，病毒醚的处理时间与再生茎尖的成活率和两种病毒的脱除效率有关。R15+T处理30、40和50 d，植株存活率分别为97.3%、90.7%和74.4%。3个时间段中GRSPaV的脱除率分别为55.6%、84.6%和93.8%。此外，病毒醚的浓度也与两种病毒的脱除效率有关。R25(35/44)处理对GFkV的脱除率比R15(25/45)处理高23.9%，对GRSPaV的脱除率比R15处理高7.0%。对各处理的脱除效率进行分析发现，热处理结合化学处理可以明显促进GFkV和GRSPa的脱除，R25+T处理50天可完全脱除葡萄试管苗中的这两种病毒。

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21. Combining simplified DNA extraction technology and recombinase polymerase amplification assay for rapid and equipment-free detection of citrus pathogen Phytophthora parasitica

CHEN Wei-yu, YU Jia, XU Heng, LU Xin-yu, DAI Ting-ting, TIAN Yue-e, SHEN Dan-yu, DOU Dao-long

Journal of Integrative Agriculture 2021, 20 (10): 2696-2705. DOI: 10.1016/S2095-3119(20)63459-1

摘要（127）

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由寄生疫霉（Phytophthora parasitica）引起的柑橘根腐病是柑橘生产上普遍发生、对柑橘危害较为严重的一种病害。本研究的目的是建立一种简易快速的结合侧流层析技术判定结果的重组酶聚合酶扩增（lateral flow strip-based recombinase polymerase amplification, LF-RPA）方法检测寄生疫霉，为诊断和防治寄生疫霉引起的柑橘根腐病提供技术支撑。以Ypt1基因为检测的靶标序列，通过多序列比对分析，设计寄生疫霉的特异性引物和探针，建立并优化LF-RPA检测方法，并与简易核酸提取技术相结合，对该检测方法的特异性、灵敏度和实际应用效果进行评估。对LF-RPA体系中的温度、引物和探针比例、时间进行了优化，使得LF-RPA在40°C孵育温度下反应20分钟后即可肉眼判断检测结果。特异性试验中，LF-RPA能特异性地检测出不同来源的寄生疫霉，而其它亲缘关系相近的卵菌病原菌均未检测出。灵敏度试验结果显示，LF-RPA的最低检测灵敏度为1 pg。为了使LF-RPA检测方法更适宜基层使用，对四种不同的简易核酸提取技术进行了比较评估，发现基于PEG-NaOH的简易核酸提取技术更适合本研究。将LF-RPA与PEG-NaOH核酸提取技术相结合，不需要特定的仪器设备，只需要能维持40°C的保温杯，即可在30分钟内完成从发病植株的核酸提取到结果判定的整个检测过程。利用该方法，成功地从接种寄生疫霉的柑橘叶片、枝条和果实上检测出寄生疫霉；此外也从田间采集的10份柑橘发病样品中检测出其中3份样品含有寄生疫霉本研究成功建立了一种针对寄生疫霉的LF-RPA快速检测方法，该方法与简易核酸提取技术相结合，非常适宜于基层使用，为柑橘根腐病的快速诊断奠定了技术基础。

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22.

DENG Dong, SUN Su-li, DU Chen-zhang, XIANG Chao, LONG Jue-chen, CHEN Wei-dong, ZHU Zhen-dong

Journal of Integrative Agriculture 2021, 20 (11): 2957-2965. DOI: 10.1016/S2095-3119(21)63629-8

摘要（120）

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由核盘菌（Sclerotinia sclerotiorum）引起的豌豆菌核病是我国常见的豌豆病害。然而，我们近期在重庆和四川调查中发现，引起豌豆菌核病的病原菌可有多个核盘菌种。为此，我们对引起重庆和四川豌豆菌核病的病原菌种进行鉴定，以期为病害防控及抗病育种奠定基础。利用常规组织分离方法对采自重庆永川与合川、四川仪陇的病株及菌核进行病原菌分离，对获得的类似核盘菌的真菌分离物用PDA（potato dextrose agar）培养基培养，观察并测量菌落形态、生长速率与菌核大小。利用核糖体基因内转录区（rDNA-ITS）序列和种特异性分子标记进行分离物的分子鉴定。rDNA-ITS序列采用真菌通用引物ITS4/ITS5对分离物基因组DNA进行PCR扩增，种特异性分子标记鉴定分别利用核盘菌、小核盘菌（S. minor）和三叶草核盘菌（S. trifoliorum）特异性引物SMLcc2F/R、SSasprF/R与STCadF/ R对分离物基因组DNA进行PCR扩增，系统发育分析选取10个代表分离物进行ITS序列测序和采用Mega X软件中非加权组平均法（UPGMA）构建系统发育树。致病性测定采用菌丝块接种豌豆植株茎秆方法进行。菌落生长速率和菌核大小差异可将30个分离物区分为“快速生长-中菌核”、“中速生长-小菌核”和“慢速生长-大菌核”3中类型，分别有6个、7个和17个分离物，与已报道的核盘菌属中的核盘菌、小核盘菌和三叶草核盘菌的类似。ITS序列扩增片段将30个分离物分为2个带型，10个代表分离物进一步的系统发育分析表明，2个分离物与小核盘菌（KY550019、MN421822）聚在同一分支，2个分离物与核盘菌（MN216247、MK734068和MF776031）聚在同一分支，6个分离物与三叶草核盘菌（AY187070、AY547267）聚在同一分支。核盘菌种特异性引物扩增结果表明，30个分离物中分别有6、7和17个分离物扩增出小核盘菌、核盘菌和三叶草核盘菌的目标条带。综合形态和分子特征结果，我们确定30个分离物中有6为小核盘菌、7个为核盘菌和17为三叶草核盘菌。致病性测定结果表明，30个分离物对2个豌豆品种均具有致病性，病害症状与田间自然发病症状相似，且从感病植物中分离出病原菌分离物与接种分离物相同。本研究证明重庆市和四川省部分地区的豌豆菌核病有小核盘菌、核盘菌和三叶草核盘菌3种病原菌，其中小核盘菌和三叶草核盘菌引起豌豆菌核病是在我国西南地区首次报道。

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23. Three sensitive and reliable serological assays for detection of potato virus A in potato plants

WU Jia-yu, ZHANG Yu, ZHOU Xue-ping, QIAN Ya-juan

Journal of Integrative Agriculture 2021, 20 (11): 2966-2975. DOI: 10.1016/S2095-3119(20)63492-X

摘要（158）

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马铃薯A病毒（potato virus A，PVA）是马铃薯种植区域侵染马铃薯的常见且经济重要的病毒之一，建立快速、灵敏、高通量的PVA检测技术对防控该病毒病害具有重要意义。本研究利用感染PVA的马铃薯植物中提纯的PVA病毒粒子为免疫原，免疫BALB/c小鼠，经杂交瘤技术获得4株能分泌抗PVA单克隆抗体的杂交瘤细胞株(2D4、8E11、14A6和16H10)。Western blot分析发现，4株单抗均与PVA的假定外壳蛋白亚基有特异性免疫反应。以4株杂交瘤细胞分泌的单抗为核心相继建立了检测马铃薯叶片和马铃薯块茎等中PVA的抗原包被板子酶联免疫吸附试验（antigen-coated plate enzyme-linked immunosorbent assay，ACP-ELISA）、斑点酶联免疫吸附试验（Dot-ELISA）和组织印迹酶联免疫吸附试验（Tissue print-ELISA）三种血清学方法。特异性分析结果表明，ACP-ELISA和Dot-ELISA检测感染PVA马铃薯病叶的灵敏度达到1:327680倍和1:10240倍稀释 (w/v, g/mL)。Tissue print-ELISA是这三种血清学检测方法中最快、最简便的检测技术，更适合现场大规模样品检测。利用建立的三种血清学方法对2019年从云南省和浙江省田间采集的22个马铃薯样品进行检测，发现有3个样品感染PVA。进一步通过RT-PCR检测和PVA CP基因的克隆测序及核酸序列比对证实3种PVA血清学检测方法在马铃薯田间样品调查上的准确性。我们的研究结果表明，灵敏、特异PVA单抗的创制及血清学检测方法的建立在PVA田间检测、病害流行病学的研究、马铃薯无毒种薯的生产方面具有巨大应用潜力。

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24. JIA-2022-0065 一种同时检测6种侵染番茄的类病毒的通用探针的制备及马铃薯纺锤形块茎类病毒（PSTVd）在中国番茄上的首次报道

ZHANG Yu-hong, LI Zhi-xin, DU Ya-jie, LI Shi-fang, ZHANG Zhi-xiang

Journal of Integrative Agriculture 2023, 22 (3): 790-798. DOI: 10.1016/j.jia.2022.08.119

摘要（337）

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【目的】马铃薯纺锤形块茎类病毒属的多种类病毒可侵染番茄，造成严重病害，威胁产业发展。研发番茄类病毒检测技术可为病害防控提供技术支撑。此外，虽然国外有报道说我国出口的番茄种子上携带类病毒，但仍未有定论，尚需确认，并且有必要弄清楚我国番茄上类病毒的发生情况。【方法】使用序列比对的方法，比较能够侵染番茄的不同类病毒的基因组序列，找到保守序列，以此为模板，通过体外转录的方法制备RNA探针。通过斑点杂交的方法，测试该探针的检测灵敏度和特异性。另外，利用该通用探针，使用斑点杂交的方法，普查我国番茄上的类病毒发生情况。【结果】能够侵染番茄的6种类病毒：金鱼花潜隐类病毒（CLVd）、辣椒小果类病毒（PCFVd）、马铃薯纺锤块茎类病毒（PSTVd）、番茄顶缩类病毒（TASVd）、番茄褪绿矮缩类病毒（TCDVd）和番茄雄性株类病毒（TPMVd）的基因组序列含有一段长度为61 bp的保守序列。利用该序列制备的RNA探针能够同时检测这6种类病毒，其杂交检测的适宜反应温度为55℃~60℃。虽然该探针的检测灵敏度略低于每种类病毒的特异性探针，但可以满足田间大量样品的快速检测的需求。利用该探针，从我国番茄温室栽培的番茄植株上检测到了PSTVd，这是PSTVd在我国番茄上的首次报道。基因组序列比较分析发现，我国PSTVd番茄分离物的序列与国外茄科作物上PSTVd的序列最为接近，并且感染PSTVd的番茄的种子是从国外进口的。这表明我国番茄上的PSTVd应该是通过进口的番茄种子从国外传入的。【结论】研发出了一种能够同时检测侵染番茄的6种类病毒的通用探针，该探针具有快速灵敏的特点，适用于大量样品的快速检测，为番茄类病毒的监测及防控提供了技术支撑；此外，证实我国温室栽培的番茄上的确有PSTVd的发生，应该是通过种子由国外传入的，因此，需要加强进口种子及繁殖材料的检验和检疫，也要加强田间种植番茄的监测。

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25. 水稻条纹病毒胁迫下抗感品种的差异转录组研究

LIU Yu, LIU Wen-wen, LI Li, Frederic FRANCIS, WANG Xi-feng

Journal of Integrative Agriculture 2023, 22 (6): 1750-1762. DOI: 10.1016/j.jia.2022.10.010

摘要（271）

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水稻条纹叶枯病是由灰飞虱（Laodelphax striatellus Fallen）传播的水稻条纹病毒（rice stripe virus, RSV）侵染所致，因其危害性严重被称为水稻的“癌症”。目前，关于RSV侵染后，抗、感水稻品种之间的分子差异以及水稻与RSV之间的相互作用的研究仍然不够充分。本文利用转录组测序技术（RNA-sequencing, RNA-Seq）分析了在RSV侵染后不同抗性水平水稻品种在转录水平上的差异。通过GO（Gene Ontology）注释鉴定到了抗、感品种在接种后2天、10天和20天后与转录因子（transcription factor, TF）、过氧化物酶（peoxidase）和激酶（kinase）相关的差异表达基因。结果表明：抗病品种中与这三类蛋白相关的差异表达基因虽然数量上比感病品种要少，但经显著性分析，在| log2(FoldChange) | > 0 & padj < 0.05的条件下，表达量呈现显著上调或下调表达趋势。通过KEGG（Kyoto Encyclopedia of Genes and Genomes）分析，鉴定出参与抗病反应途径的差异基因，包括植物激素信号转导和植物-病原体相互作用。结果表明涉及植物激素信号转导，脱落酸（abscisic acid, ABA）负调控的抗性反应和油菜素内酯（brassinosteroids, BR）正调控的抗性反应在抗、感品种间无差异，但涉及水杨酸（salicylic acid, SA）介导和茉莉酸（jasmonic acid, JA）/乙烯（ethylene, ET）介导的抗性反应有所不同。抗、感品种在三个时间节点的差异表达基因在病原相关分子模式激发的免疫反应（PAMP-triggered immunity, PTI）和效应蛋白激发的免疫反应（Effector-triggered immunity, ETI）都存在，但感病品种的特有基因大多涉及PTI，而抗病品种涉及ETI的特有基因数量更多。以上结果揭示了RSV侵染后抗、感品种在转录水平上的差异，为阐明了水稻与RSV互作的机制奠定了基础。

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26. Characterization of volatile organic compounds in grafted tomato plants upon potyvirus necrotic infection

Roberta SPANÒ, Mariarosaria MASTROCHIRICO, Francesco LONGOBARDI, Salvatore CERVELLIERI, Vincenzo LIPPOLIS, Tiziana MASCIA

Journal of Integrative Agriculture 2023, 22 (8): 2426-2440. DOI: 10.1016/j.jia.2023.02.032

摘要（125）

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A headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME/GC-MS) method was used to study the volatile organic compounds (VOCs) associated with the differential immune response of tomato plants infected with the recombinant strain of potato virus Y (PVY^C-to), necrogenic to tomato. Analysis was carried out in UC82 (UC), a virus susceptible tomato variety, comparing the same UC plants grafted or not onto a virus tolerant tomato ecotype, Manduria (Ma); the three types of samples used for the GC-MS analysis were mock-inoculated UC/Ma plants, UC/Ma+PVY^C-to and UC+PVY^C-to plants; the VOCs obtained were 111. Results from symptomatic PVY^C-to-infected UC plants showed a VOCs composition enriched in alcohols, fatty acid derivates, benzenoids, and salicylic acid derivatives, while in mock-inoculated UC/Ma plants VOCs were mainly characterized by methyl ester compounds. The VOC profile was in line with RNAseq data analyses, denoting that PVY^C-to viral RNA accumulation and disease symptoms induce the specific transcriptional activation of genes involved in VOCs biosynthesis. Furthermore, principal component analysis highlighted that VOCs of PVY^C-to-infected and mock-inoculated grafted plants were much closer each other than that of symptomatic PVY^C-to-infected non-grafted UC plants. These results suggest that VOCs profiles of tomato plants are related to the viral RNA accumulation, disease intensity and graft-derived tolerance to PVY^C-to infection.

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27. 烟草花叶病毒双位点弱毒突变体的研发

Xiaojie Xu, Shaoyan Jiang, Chunju Liu, Xujie Sun, Qing Zhu, Xiuzhai Chen, Pengchao Jiang, Fenglong Wang, Yanping Tian, Xiangdong Li

Journal of Integrative Agriculture 2024, 23 (7): 2318-2331. DOI: 10.1016/j.jia.2024.02.019

摘要（99）

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烟草（Nicotiana tabacum）和番茄（Solanum lycopersicum）是我国两种主要的经济作物，其安全生产受到烟草花叶病毒（tobacco mosaic virus，TMV）的严重威胁。交叉保护是预防植物病毒病安全且环保的措施。然而，稳定的TMV弱毒突变体却很少。TMV基因组为单链RNA，其编码的p126蛋白参与病毒复制、细胞间运动、抑制寄主RNA沉默和病毒症状形成等过程。在本研究中我们发现，将p126蛋白中第196位的精氨酸（arginine，R¹⁹⁶）替换为天冬氨酸（aspartic acid，D），第614位的谷氨酸（glutamic acid，E⁶¹⁴）替换为甘氨酸（glycine，G），第643位的丝氨酸（serine，S⁶⁴³）替换为苯丙氨酸（phenylalanine，F），或者把第730位的D（D⁷³⁰）替换为S，均可以显著降低TMV的致病力和复制水平。然而，只有S643F突变可以降低TMV p126的RNA沉默抑制活性。由于RNA病毒的RNA聚合酶缺乏校对和修复活性，导致RNA病毒基因组易发生突变。一些携带单一致弱突变位点的病毒突变体可能会通过自发突变恢复其致病力，而在多个致弱位点有突变的突变体恢复强致病力的风险会相对较低。为了获得稳定的TMV弱毒突变体，我们在p126中两个位点同时引入突变，发现仅双位点突变体TMV-E614G-S643F可以系统侵染烟草植株，而且不引起明显的症状，因此将其作为用于交叉保护的候选突变体。我们将其在烟草植株中连续继代接种六次，发现双位点突变体TMV-E614G-S643F可以稳定遗传。交叉保护效果实验结果表明，双位点突变体TMV-E614G-S643F可以有效保护烟草和番茄植株免受野生型TMV侵染。本研究报道了一种具有应用前景的TMV双位点弱毒突变体，可用于通过交叉保护来防治烟草和番茄植株中的TMV。

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28. 尤力克柠檬锌指蛋白ClDOF3.4与柑橘黄化脉明病毒外壳蛋白互作抑制病毒侵染

Ping Liao, Ting Zeng, Mengyang Huangfu, Cairong Zheng, Jiequn Ren, Changyong Zhou, Yan Zhou

Journal of Integrative Agriculture 2024, 23 (6): 1979-1993. DOI: 10.1016/j.jia.2024.03.049

摘要（83）

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柑橘黄化脉明病毒（citrus yellow vein clearing virus，CYVCV）是一种新型柑橘病毒，该病毒已对印度、伊朗、土耳其、中国、美国和韩国的柠檬、酸橙和部分宽皮等柑橘品种的种植生产造成了严重的损失。CYVCV外壳蛋白（CP）具有RNA沉默抑制子活性，与病毒致病性和症状密切相关。DOF（DNA-binding with one finger）是高等植物特有的锌指蛋白，在植物生长发育和抗病原物侵染的过程中扮演着重要作用。然而，目前尚未开展尤力克柠檬锌指蛋白ClDOF与CYVCV的互作研究。因此，本研究通过一系列体内和体外互作验证实验，以及基因瞬时表达、基因瞬时沉默和尤力克柠檬遗传转化等手段，验证了ClDOF3.4和CYVCV CP之间的互作关系及其对尤力克柠檬响应CYVCV免疫应答的影响。试验结果表明，ClDOF3.4蛋白在体内和体外均与CP存在相互作用关系，且ClDOF3.4蛋白N端的104-280个氨基酸序列是与CP相互作用的关键区段。在尤力克柠檬叶片中瞬时表达ClDOF3.4可以诱导水杨酸通路相关基因和超敏反应标记基因的上调表达，造成接种部位活性氧爆发和离子渗透坏死、并引起H₂O₂和水杨酸积累；而ClDOF3.4基因瞬时沉默尤力克柠檬叶片中的水杨酸和超敏反应相关基因下调表达。此外，该研究进一步通过农杆菌接种分别在瞬时表达及沉默ClDOF3.4基因的尤力克柠檬叶片中瞬时表达CP，结果表明ClDOF3.4负向调控CP在尤力克柠檬中的表达和积累。接种CYVCV六个月后，ClDOF3.4转基因尤力克柠檬植株的CYVCV含量约为对照植株的69.4%，与对照植株相比，转基因植株感病后的症状明显减轻，仅出现轻度的叶片斑驳症状。上述研究表明，ClDOF3.4不仅与CYVCV-CP存在相互作用，还可以通过水杨酸信号通路诱导尤力克柠檬对CYVCV的免疫应答。这是首次关于锌指蛋白参与柑橘抗病毒病的报道，该研究结果为进一步研究柑橘抗CYVCV的分子机制奠定了基础。

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29. 细菌蛋白Rhp-PSP通过核酸内切酶活性抑制植物病毒增殖

Lijie Chen, Qianze Peng, Xiaohua Du, Weixing Zhang, Ju’e Cheng, Shu’e Sun, Deyong Zhang, Pin Su, Yong Liu

Journal of Integrative Agriculture 2024, 23 (6): 1967-1978. DOI: 10.1016/j.jia.2024.03.051

摘要（96）

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植物病毒病的发生在全球范围内造成巨大的作物损失。然而，目前没有非常有效的措施来控制植物病毒病。此前，我们鉴定了一种由沼泽红假单胞菌JSC-3b菌株产生的抗病毒蛋白Rhp-PSP。本研究中，我们发现Rhp-PSP的抗病毒活性依赖于其核酸内切酶活性。将129位的精氨酸（R）残基转化为丙氨酸（A），降低了对烟草花叶病毒（TMV）外壳蛋白（CP）RNA的核糖核酸内切酶活性，从而损害了Rhp-PSP的抗病毒活性。进一步的研究表明，突变体Rhp-PSP^R129A不能像野生型那样形成同源三聚体，这说明了四级连接对核糖核酸内酶活性的重要性。Rhp-PSP在本氏烟草中的过表达显著增强了幼苗对TMV的抗性，而Rhp-PSP^R129A过表达则没有，这证实了核糖核酸内切酶活性是Rhp-PSP抗病毒活性的原因。此外，Rhp-PSP喷施在番茄和辣椒叶面上，显著降低了病毒性疾病的发病指数，表明Rhp-PSP在实践中显示出开发抗病毒药物的潜力。

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