马铃薯疮痂病菌致病相关基因的克隆及表达

 A pathogenic-related gene nec1 was cloned in Streptomyces scabies CPS-1, potato scab strain. Analysis results showed that the length of open reading frame(ORF) for nec1 gene was 666 bp, and the GC content was 54.2%. Sequence alignment indicated that a 650 bp up-stream sequence shared 91% similarity with IS 256 family transposase nucleotide sequences by BLASTn searches against GenBank. The segments obtained by PCR amplification were digested by enzymes SphⅠand SacⅠ, and linked to the expression vector pIJ702. The recombinants were transformed into nonpathogenicity strain Streptomyces lividans 66 TK24. Bioas-say results suggested that the transformants possessed the same symptoms as pathogenic strain on potato tuber slices and radish seedlings, which implied that nec1 gene was associated with the pathogenicity of S. scabies CPS-1.     

侵染云南花魔芋的马铃薯Y病毒属病毒分离物的检测及分子鉴定

 YN80 was isolated from Amorphophallus rivieri Durieu showing mosaic and crinkle symptoms in Songming, Yunnan province. Flexuous filamentous particles were found in diseased leave sap and pinwheel inclusion bodies were found in the leave tissue. YN80 had positive reaction to universal antibody of Potyvirus by DAS-ELISA. 3'-terminal sequence of YN80 was cloned and sequenced. cp gene of YN80 consisted of 987 nt, encoded 328 aa (36.1 kDa). Sequence analysis showed that YN80 shared the highest identity (97.0%)with CP amino acid sequence of Dasheen mosaic virus (DsMV). These data indicated that YN80 was an isolate of DsMV. This is the first molecular identification of A. rivieri Durieu isolate of DsMV in China.     

小西葫芦黄花叶病毒山东南瓜分离物的分子特性

 Zucchini yellow mosaic virus (ZYMV) was detected by RT-PCR from pumpkin (Cucurbita moschata) plant showing yellowing and mosaic symptom from Liaocheng, Shandong Province. The 3'-termial 1 684 bp genomic sequence covered 633 bp of NIb encoding sequence, 840 bp of cp gene and 211 bp of 3'-untranslated region of the isolate ZYMV-Liaocheng was determined. The cp gene of ZYMV-Liaocheng shared identities of 81.4%-98.8% and 89.4%-99.5% at nucleotide and amino acid levels, respectively, with other ZYMV sequences available in the GenBank. Phylogenetic analysis indicated that ZYMV could be clustered to 6 genotypes. ZYMV-Liaocheng belonged to genotypeⅠ, which contained isolates from Asia, Europe and America. Genotypes Ⅲ and Ⅴ were unique and contained only isolates from East Asia. The isolates from East Asia had the highest variability.     

花生斑驳病毒青岛分离物cp基因序列克隆与分析

 Peanut mottle virus(PeMoV) was detected via RT-PCR from two peanut samples(QD5 and QD6) with mottle symptom collected from Qingdao, Shandong Province.The 3'-terminal 892 bp fragments of their genome were cloned and sequenced.The cp genes of QD5 and QD6 were 837 bp in length and encoded 278 amino acids(aa), with DAA at aa sites regulating aphid transmission.QD5 and QD6 shared nucleotide identities of 95.3%-99.4% and aa identities of 93.5%-99.6% in cp genes with other PeMoV isolates available in the GenBank.The phylogenetic results showed that PeMoV were clustered to three groups, America, Asia and Australia, which were consistent with their geographical origins.This is the first molecular evidence on the incidence of PeMoV in China.     

甘蔗黄叶病毒复制酶基因的克隆及序列分析

 The gene of RNA-dependent RNA polymerase (RdRP) was cloned by RT-PCR from Sugarcane yellow leaf virus-Fuzhou isolate (CHN-FJ1) and then cloned into pMD18-T vector. The sequence showed that the fragment comprised 1 212 nucleotides including part gene of ORF1 and ORF2. The ORF2 was involved the RdRP gene consisted of 995 nucleotides and encoded putative protein of 331 amino acids. Compared the nucleo-tide sequence and encoded putative protein of CHN-FJ1 with the other isolates from different countries, they shared the homology above 92.0%. Phylogenetic tree suggested that the sixteen isolates were classified into four types according to the amino acid sequence of the RdRP. One of the groups contained CHN-FJ1 and the other isolates from China, American, Brazil, Australia and Colombia;however, there was the closest relation between CHN-FJ1 and BRA-YL1 isolate from Brazil.     

广东番茄上检测到Tospovirus病毒

 Some tomato samples possibly infected by tospovirus in Guangdong were detected with indirect ELISA and RT-PCR. The results showed that the virus infected tomato did not react with the antiserum of Tomato spotted wilt virus (TSWV), but about 500 bp fragment of RT-PCR shared 83%-84% nucleotide identities with N gene of those reported tospoviruses. The phylogenetic tree of the N gene fragment compared with those of other tospoviruses indicated that the virus infected tomato was belonged to Tospovirus.     

地高辛标记cDNA探针检测苹果茎痘病毒

 Partial sequence(314 bp) of ASPV was cloned and used as a probe labelled with digoxigenin-11dUTP. The total RNA extracted from samples with Apple stem pitting virus and a series of dilutions of plasmid with ASPV-cDNA were detected by dot blot hybridization. The results showed that the probe was sensitive and specific. The probe couldn't hybridize with total RNA of Apple stem grooving virus, Apple mosaic virus and Apple chlorotic leaf spot virus samples as well as negative control, only hybridized with that extracted from dormant shoot infected with ASPV. The sensitivity for detection of plasmid contained ASPV-cDNA was 1.64 μg.     

进境豇豆种子携带种传病毒的检测与鉴定

 Imported cowpea seeds were detected with growing test, ELISA assay and RT-PCR method. The ELISA results showed that cowpea seedlings with symptoms reacted positively with antibody against Southern bean mosaic virus (SBMV). The 979 bp of fragment could be amplified from two positive ELISA samples using primers specific for Southern cowpea mosaic virus (SCPMV), and the sequence determination results proved that the pathogen existing in imported cowpea seeds was SCPMV. The positive ELISA results with SBMV antibody could be further confirmed by RT-PCR amplification with specific primers designed to amplify the coat protein gene and 3' noncoding region of SCPMV and SBMV. The RT-PCR method presented here was suitable for molecular identification of SBMV and SCPMV in entry-exit plant quarantine laboratories.     

马铃薯Y病毒中国分离物的分子株系组成

 马铃薯Y病毒(potato virus Y,PVY)主要侵染马铃薯和烟草等茄科作物,给世界农业造成巨大经济损失。本文对测定的23个及GenBank中注册的52个中国PVY分离物ORF序列进行了系统发育、重组和选择压等分析。系统发育分析表明,根据ORF序列可把我国75个PVY分离物和国外30个参比分离物分成O、C、E、NTN-NW(SYR-I型)、NTN-NW(SYR-II型)、NTN(NTN-a型)、NTN(NTN-b型)、NA-N/NTN、Eu-N、N-Wi(N:O型)和N-Wi(N-Wi型)等11个分子株系,其中中国PVY分离物属于除E和C株系外的9个分子株系。除ME162、guiyang、PVYzu、SD-G、WA-13和CN:JL-1:17等 6个分离物基因组中未检测到重组,其余69个分离物均存在明显重组。根据重组位点的不同,中国PVY可分为11种重组类型,其中5种为新的重组类型。选择压分析表明,中国PVY分离物的11个基因均处于负选择,其中核内含体b基因受到的选择压最大,PIPO受到的选择压最小。基因流分析表明,黑龙江、河南和山东PVY分离物间基因交流频繁,马铃薯与烟草PVY分离物之间基因交流频繁。本研究的结果明确了中国PVY分离物的分子株系组成,对指导PVY的检测和防控具有积极作用。     

Identification of the molecular make-up of the Potato virus Y strain PVY(Z): genetic typing of PVY(Z)-NTN

Potato virus Y (PVY) strains were originally defined by interactions with different resistance genes in standard potato cultivars. Five distinct strain groups are defined that cause local or systemic hypersensitive responses (HRs) in genetic background with a corresponding N gene: PVY(O), PVY(N), PVY(C), PVY(Z), and PVY(E). The nucleotide sequences of multiple isolates of PVY(O) and PVY(N) differ from each other by ≈8% along their genomes. Additionally, complete genome sequences of multiple recombinant isolates are composed of segments of parental PVY(O) and PVY(N) sequences. Here, we report that recombinant isolate PVY-L26 induces an HR in potato 'Maris Bard' carrying the putative Nz gene, and is not recognized by two other resistance genes, Nc and Ny(tbr). These genetic responses in potato, combined with the inability of PVY-L26 to induce vein necrosis in tobacco, clearly define it as an isolate from the PVY(Z) strain group and provide the first information on genome structure and sequence of PVY(Z). The genome of PVY-L26 displays typical features of European NTN-type isolates with three recombinant junctions (PVY(EU-NTN)), and the PVY-L26 is named PVY(Z)-NTN. Three typical PVY(NTN) isolates and two PVY(N) isolates, all inducing vein necrosis in tobacco, were compared with PVY-L26. One PVY(NTN) isolate elicited HR reactions in Maris Bard, similar to PVY-L26, while two induced a severe systemic HR-like reaction quite different from the quasi-symptomless reaction induced by two PVY(N) isolates. 'Yukon Gold' potato from North America produced HR against several PVY(NTN) isolates, including PVY-L26, but only late and limited systemic necrosis against one PVY(N) isolate. Consequently, according to symptoms in potato indicators, both PVY(Z) and PVY(NTN) isolates appeared biologically very close and clearly distinct from PVY(O) and PVY(N) strain groups.     

Diversity Among Potato virus Y Isolates Obtained from Potatoes Grown in the United States

ABSTRACT Potato field isolates (Solanum tuberosum) of Potato virus Y (PVY) collected from the midwestern and western United States were characterized using serological, molecular, and biological assays. PVY field isolates were grouped into the previously defined categories: PVY(O), European PVY(NTN), North American PVY(NTN), and PVY(N:O) recombinant and four previously undefined groups. Studies reported here agree with published reports from Europe and elsewhere in North America as PVY isolates capable of causing veinal necrosis in tobacco indicator plants appear in high frequency. In contrast to European experiences, PVY tuber necrosis isolates have a PVY(O) coat protein rather than that of PVY(N). Several PVY(N:O) recombinant isolates induced potato tuber necrotic ringspot disease (PTNRD) in the highly susceptible potato cv. Yukon Gold. The PTNRD symptoms produced by these PVY(N:O) recombinants were atypical compared with lesions found on the same cultivar infected with either the European or North American PVY(NTN) isolates. These PVY(N:O) isolates produced a roughly circular, sunken necrotic lesion on the surface of the tuber instead of the typical external sunken ring pattern displayed by PVY(NTN) isolates. This study establishes the complex nature of PVY populations within the U.S. potato industry and clearly demonstrates the diverse nature of PVY in the United States.     

Genetic diversity of the ordinary strain of Potato virus Y (PVY) and origin of recombinant PVY strains

The ordinary strain of Potato virus Y (PVY), PVY(O), causes mild mosaic in tobacco and induces necrosis and severe stunting in potato cultivars carrying the Ny gene. A novel substrain of PVY(O) was recently reported, PVY(O)-O5, which is spreading in the United States and is distinguished from other PVY(O) isolates serologically (i.e., reacting to the otherwise PVY(N)-specific monoclonal antibody 1F5). To characterize this new PVY(O)-O5 subgroup and address possible reasons for its continued spread, we conducted a molecular study of PVY(O) and PVY(O)-O5 isolates from a North American collection of PVY through whole-genome sequencing and phylogenetic analysis. In all, 44 PVY(O) isolates were sequenced, including 31 from the previously defined PVY(O)-O5 group, and subjected to whole-genome analysis. PVY(O)-O5 isolates formed a separate lineage within the PVY(O) genome cluster in the whole-genome phylogenetic tree and represented a novel evolutionary lineage of PVY from potato. On the other hand, the PVY(O) sequences separated into at least two distinct lineages on the whole-genome phylogenetic tree. To shed light on the origin of the three most common PVY recombinants, a more detailed phylogenetic analysis of a sequence fragment, nucleotides 2,406 to 5,821, that is present in all recombinant and nonrecombinant PVY(O) genomes was conducted. The analysis revealed that PVY(N:O) and PVY(N-Wi) recombinants acquired their PVY(O) segments from two separate PVY(O) lineages, whereas the PVY(NTN) recombinant acquired its PVY(O) segment from the same lineage as PVY(N:O). These data suggest that PVY(N:O) and PVY(N-Wi) recombinants originated from two separate recombination events involving two different PVY(O) parental genomes, whereas the PVY(NTN) recombinants likely originated from the PVY(N:O) genome via additional recombination events.     

Serological Analysis and Coat Protein Sequence Determination of Potato Virus Y (PVY) Pepper Pathotypes and Differentiation from Other PVY Strains

The serological relationships of Potato Virus Y (PVY) isolates belonging to the pepper pathotypes 0, 1 and 1-2 were established by enzyme-linked immunosorbent assay (ELISA). PVY pepper pathotypes did not react with monoclonal antibodies which typically recognize non-pepper strains within the PVY group, leading to discrimination between these two groups of strains. No serological differences were found between the three PVY pepper pathotypes. The coat protein (CP) nucleotide and predicted amino acid sequences of the three different PVY pepper pathotypes were determined. The highest sequence similarity was found between pathotypes 0 and 1 (99.2%), while the lowest occurred between these two and pathotype 1–2 (98.1%). PVY strains from potato and tobacco appeared more distantly related. Phenetic analysis of the CP amino acid sequences showed that the PVY pepper pathotypes formed a tightly clustered group separate from other PVY strains.     

Significance of weed hosts for Potato virus Y protection in Syria

A survey of Potato virus Y (PVY) was carried out on weeds growing in and around potato fields in Syria during the autumn growing seasons of 2002, 2004 and 2006. A total of 59 samples of eight weed species and three tobacco ( Nicotiana tabacum ) samples were tested by ELISA using PVY antisera. Among them 15 samples belonged to Solanum nigrum (7 samples), Physalis sp. (6 samples) and tobacco (2 samples) were PVY infected. This suggests that weed hosts and neighbouring tobacco fields are natural reservoirs of PVY in Syria. According to their biological, serological and molecular characteristics, the majority of weed PVY isolates belonged to the PVY^SYR variant indicating a possible correlation between the high incidence of PVY^SYR in potato and weed hosts. This is the first report on the occurrence and characterization of weed PVY isolates from Syria.     

马铃薯Y 病毒贵州黔西烟草分离物P1 基因序列分析

 Potato virus Y (PVY) is the type member of the genus Potyvirus and infects several important solanaceous crops, including tobacco. PVY is one of the most widespread and economically destructive viruses.The P1 region of PVY had been used to determine the diversity, evolution and the homology based on the
geographic location of the virus. The P1 region of PVY tobacco isolate from Qianxi in Guizhou Province was cloned and sequenced. Database searches and multiple sequence alignment showed subtle variation within the same strain, while apparent variation between O strain and the other three strains, N, NTN and N∶O. Moreover, variation in P1 region of Qianxi isolate derived from the gene mutation rather than the recombination among genomes. Phylogenic analysis revealed that the clustering only discriminated O strain from others. Consequently, sequence analysis in P1 region is unable to afford strain determination.     

小RNA深度测序鉴定昭通市烟草脉斑病病毒

烟草脉斑病在云南昭通市的烟草上发生危害严重,为明确昭通市烟草脉斑病的病毒种类,本研究采集昭通市的4个烟草种植区症状表现为疑似烟草脉斑病的烟草样品,采用小RNA深度测序技术对不同来源的混合样品进行小RNA测序分析。结果显示混合样品中的病毒分别属于烟草花叶病毒属Tobamovirus、马铃薯Y病毒属Potyvirus和马铃薯卷叶病毒属Polerovirus。根据不同病毒属设计通用引物,分别对不同地区的烟草样品进行RT-PCR验证,结果表明,烟草样品中病毒种类有烟草花叶病毒、烟草脉带花叶病毒、烟草脉扭病毒和马铃薯Y病毒的PVYN、PVYN-Wi和PVYNTN株系。     

Hairpin-based virus resistance depends on the sequence similarity between challenge virus and discrete,highly accumulating siRNA species

Virus resistance can be effectively generated in transgenic plants by using the plant’s silencing machinery. To study the specificity of gene-silencing-based resistance, homozygous tobacco (Nicotiana tabacum L.) plants containing a 597-nt hairpin RNA construct of the Potato Virus Y (PVY) replicase sequence were challenged with a variety of PVY strains. The transgene-carrying tobacco line was immune to five potato PVY strains with high sequence similarity (88.3–99.5%) to the transgene. Infection with more distant tomato and pepper PVY field strains (86–86.8% sequence similarity) caused delayed symptom appearance in the transgenic tobacco. Transgene production of small interfering (si) RNA was detected by northern blot and measured using a custom-designed microarray for the detection of small RNAs. siRNA accumulation peaks were observed throughout the inverted-repeat transgene. In the resistance-breaking tomato and pepper strains there were nucleotide differences in the sequences correlated to siRNA transgene accumulation, indicating the role of siRNA specificity in resistance breaking. The log of transgene siRNA signal intensity increased with probe GC content, indicating that the accumulating siRNA molecules were GC-rich. Sequence similarity of highly accumulating siRNAs with the target virus strain appears to be important for both resistance and resistance-breaking characteristics.     

A novel type of Potato virus Y recombinant genome,determined for the genetic strain PVYE

Two Potato virus Y (PVY) isolates collected in Brazil, PVY‐AGA and PVY‐MON, were identified as recombinants between two parent genomes, PVY^NTN and PVY‐NE‐11, with a novel type of genomic pattern. The new recombinants had an ordinary PVY^NTN genome structure for approximately 6·7‐kb from the 5′‐end of the genome whereas the 3′‐terminal 3·0‐kb segment had two fragments of NE‐11‐like sequence separated by another small PVY^NTN‐like fragment. PVY strains are defined based on the hypersensitive resistance (HR) response in potato indicators. Both PVY‐AGA and PVY‐MON isolates did not induce the HR in potato cultivars carrying Ny, Nc, or (putative) Nz genes and thus were able to overcome all known resistance genes to PVY. Only one of the two isolates, PVY‐AGA, induced a vein necrosis reaction in tobacco. The biological responses of the potato indicators and tobacco defined PVY‐MON as an isolate of the PVY^E strain. To distinguish PVY‐AGA and PVY‐MON from other PVY^NTN isolates, an RT‐PCR test was developed utilizing new specific primers from the capsid protein gene area and producing a characteristic 955‐bp band. Serological profiling of these PVY isolates with three monoclonal antibodies revealed an unusual reactivity, where one of the two commercial PVY^N‐specific monoclonal antibodies did not recognize PVY‐AGA. The ability of these new PVY recombinants to overcome resistance genes in potato producing mild or no symptoms, combined with the lack of serological reactivity towards at least one PVY^N‐specific antibody may present a significant threat posed by these isolates to seed potato production areas.