High temperatures activate local viral multiplication and cell-to-cell movement of Melon necrotic spot virus but restrict expression of systemic symptoms

The infection of melon plants by Melon necrotic spot virus (MNSV) and the development of necrotic disease symptoms are a seasonal occurrence in Japan, which take place between winter and early summer, but not during mid-summer. In this paper we investigate the effect of three different temperatures (15, 20, and 25 degrees C) on the local and systemic expression of MNSV in melon plants. Previously, the incidence of plants expressing systemic symptoms caused by MNSV and other viruses was found to be greater at temperatures less than 20 degrees C. In this study, our temperature-shift experiments support previous studies that found the expression of systemic symptoms increases as temperature falls from 25 to 20 degrees C and decreases as temperature rises from 20 to 25 degrees C. However, MNSV replication in melon cells and local viral movement within leaves following the inoculation of melon protoplasts or cotyledons were more frequent at 25 degrees C than at 15 or 20 degrees C.     

Watermelon necrosis caused by a strain of melon necrotic spot virus

A severe disease of watermelon (Citrullus vulgaris) grown in plastic houses in Crete, was characterized by leaf and stem necrosis followed by death of the plants. A strain of melon necrotic spot virus (MNSV) was identified as the causal agent of the disease on the basis of biological, morphological and serological properties. The watermelon strain of MNSV induced only local necrotic lesions in melon and cucumber plants and was serologically distinct from MNSV Cretan isolate. Gomphrena globosa was found a useful herbaceous host for differentiating it from common isolates of MNSV.     

New severe strains of Melon necrotic spot virus: symptomatology and sequencing

New strains of Melon necrotic spot virus (MNSV), designated MNSV-YS and MNSV-KS, caused much more severe growth retardation on melon plants than MNSV-NH, which was previously reported as the most severe strain of MNSV in Japan. MNSV-YS spread much more quickly than MNSV-NH in infected plants, and induced more severe growth retardation, even though the appearance of necrotic lesions on inoculated cotyledons was much slower. MNSV-KS had properties intermediate between those of the other two strains. The results suggest that faster-spreading strains can multiply more rapidly as a result of lower levels of activity in inducing necrotic lesions in melon plants. The complete sequences of MNSV-YS and MNSV-KS were determined, and an RT–PCR–RFLP method based on these sequences was successfully developed to detect and discriminate between the three strains.     

A new strain of Melon necrotic spot virus that is unable to systemically infect Cucumis melo

We report a new strain of Melon necrotic spot virus (MNSV) that is unable to systemically infect Cucumis melo. A spherical virus (W-isolate), about 30 nm in diameter like a carmovirus, was isolated from watermelons with necrotic symptoms. The W-isolate had little serological similarity to MNSV, and it did not cause any symptoms in six melon cultivars susceptible to MNSV; however, the host range of the W-isolate was limited exclusively to cucurbitaceous plants, and transmission by O. bornovanus was confirmed. Its genomic structure was identical to that of MNSV, and its p89 protein and coat protein (CP) showed 81.6 to 83.2% and 74.1 to 75.1% identity to those of MNSV, respectively. Analysis of protoplast showed that the W-isolate replicated in melons at the single-cell level. Furthermore, chimeric clones carrying the CP of MNSV induced necrotic spots in melons. These results suggested that the absence of symptoms in melons was due to a lack of ability of the W-isolate to move from cell to cell. In view of these findings, we propose that the new isolate should be classified as a novel MNSV watermelon strain.     

Seed transmission of Melon necrotic spot virus and efficacy of seed-disinfection treatments

Rates of seed transmission of Melon necrotic spot virus (MNSV) were estimated in seedlings grown from commercial melon ( Cucumis melo ) cv. Galia F₁ seeds. Seedlings at the cotyledon stage and adult plants were assayed for MNSV by DAS-ELISA and RT-PCR. None of the seedling groups tested positive for MNSV by ELISA. The proportion of seedlings infected with MNSV was at least 7 and 8% in seed lots 05 and 06, respectively, as estimated from RT-PCR analysis of grouped seedlings. Fourteen and eight grouped samples (10 seedlings per group), of a total of 200 and 100 seedlings, respectively, grown from infected seeds were MNSV-positive in seed lots 05 and 06, respectively, corresponding to seed-to-seedling transmission rates of 11·3 and 14·8%, respectively. Several seed-disinfection treatments were evaluated for their ability to prevent seed transmission of MNSV. The results suggest that a treatment of 144 h at 70°C can be used to eradicate MNSV in melon seeds without hindering germination.     

携带GFP的MNSV侵染性克隆载体的构建

正甜瓜坏死斑点病毒(Melon necrotic spot virus,MSNV)属于番茄丛矮病毒科(Tombusviridae)香石竹斑驳病毒属(Carmovirus),其主要寄主是葫芦科作物。MNSV首次在我国江苏海门发生[1],随后在山东寿光[2]、青州市[3]及广西南宁[4]等地均有报道。种子带毒是该病害初侵染的主要来源[5],随着种子调运、设施栽培推广和甜瓜产业的发展,该病害日趋严重。MNSV侵染初期叶片、叶柄和茎杆出现坏死斑或     

携带GFP的MNSV侵染性克隆载体的构建

Melon necrotic spot virus (MNSV) is a newly reported melon-infecting virus species in China, which is seriously terrible in melon production. The MNSV full-length cDNA of MNSV was obtained by RT-PCR and was ligated to pXT1 vector to obtain the infectious clone vector pXT1-MNSV. The melon was inoculated with pXT1-MNSV, and melon leaves infected with pXT1-MNSV were collected and inoculated into the healthy melon by sap. The results showed that the infectious clone pXT1-MNSV was well pathogenic and could be transmitted by sap, so the Koch′s law was completed. Using MNSV infectious clone, the MNSV infectious clone vector pMNSV-cp-GFP harboring full-length GFP was constructed, Because GFP fluorescence can be detected by RT-PCR and in both melon roots and leaves by confocal scanning laser microscopy. Here, We constructed MNSV infections clone and pMNSV-cp-GFP clone vectors for the first time in China. The MNSV infections clone provided a tool for the analysis of its pathogenic mechanism at the molecular level. In addition, we also constructed MNSV infectious clone vectors carrying GFP, which could be used in the fluorescent labeling of MNSV and its distribution, movement and colonization during virus infection.     

Functional degeneration of the resistance gene nsv against Melon necrotic spot virus at low temperature

The single recessive gene, nsv, which confers resistance against Melon necrotic spot virus (MNSV), has recently been used to develop virus-resistant melon cultivars in Japan. However, the Chiba isolate of MNSV, a common isolate in Japan, infected resistant cultivars when inoculated melon plants were grown at 15°C. Viral RNAs accumulated in protoplasts from resistant cultivars at both 15 and 20°C. Mechanical inoculation of the cotyledons caused MNSV to spread throughout the leaves at 15°C, but not at 20°C. These results support our novel hypothesis that a temperature-sensitive inactivation of disease resistance genes occurs at the nsv locus in melon cultivars with the resistance gene grown at temperatures below 20°C. The first and second authors contributed equally to this research.     

MNSV p42与寄主因子3-HIBADH互作

甜瓜坏死斑点病毒(melon necrotic spot virus,MNSV)是侵染甜瓜的重要病毒之一,为害严重.为了明确MNSV的致病机制,本研究以pGBK-p42为诱饵载体,利用Mating的方法从感染MNSV的甜瓜cDNA文库中筛选出6个与p42互作的寄主因子,进一步利用酵母双杂交系统、双分子荧光互补技术(Bi...     

两种甜瓜病毒寿光分离物的分子检测与鉴定

甜瓜坏死斑点病毒(Melon necrotic spot virus,MNSV)及瓜类褪绿黄化病毒(Cucurbit chlorotic yellows virus,CCYV)近年来在瓜类种植区均大面积发生,危害较为严重,已成为制约甜瓜生产的重要因素。本研究广泛收集疑似感染MNSV及CCYV的甜瓜病叶,从中提取植物总RNA进行RT-PCR扩增,将产物分别连接到pEASYT1Simple克隆载体上,对含有目的片段的重组子进行测序及比对分析。结果显示得到了与预期结果一致的DNA序列,其扩增产物大小分别为673bp(MNSV)和877bp(CCYV)。同源性分析结果表明,MNSV和CCYV寿光分离物的核苷酸序列与中国其他地区或一些国家已报道的分离物同源性达99%~100%。     

Virus transmission by host-specific strains ofOlpidium bornovanus andOlpidium brassicae

Zoospores of 12 isolatesO. bornovanus from geographically diverse sites and representing the three host specific cucurbit strains were tested as vectors for seven viruses using watermelon bait plants and the in vitro acquisition method. All isolates of the cucumber, melon, and squash strains transmitted melon necrotic spot carmovirus (MNSV) and cucumber necrosis tombusvirus (CNV) but none transmitted petunia asteroid mosaic tombusvirus (PAMV) or tobacco necrosis necrovirus (TNV). The isolates varied as vectors of three other carmoviruses: cucumber leaf spot virus (CLSV); cucumber soil borne virus (CSBV); and squash necrosis virus (SqNV). All cucumber isolates transmitted CLSV and SqNV but not CSBV. Some of the melon isolates transmitted CLSV and SqNV but none transmitted CSBV. Two squash isolates transmitted CSBV and SqNV but not CLSV. Two isolates ofO. brassicae transmitted only TNV and a third did not transmit any of the viruses. The species of bait plant sometimes affected transmission. The most efficient vector strains ofO. bornovanus, as determined by reducing zoospores and virus in the inoculum, were the cucumber strain for CLSV; the cucumber strain for CNV if cucumber was the bait plant or melon strain if watermelon was the bait plant; and the squash strain for SqNV. The plurivorous strain ofO. brassicae was the most efficient vector of TNV.Olpidium bornovanus is the first vector reported for CSBV and is confirmed as a vector of SqNV. It is proposed that all carmoviruses may have fungal vectors.Ligniera sp. did not transmit any of the viruses in one attempt.Abbreviations CLSV cucumber leaf spot virus - CNV cucumber necrosis virus - CSBV cucumber soil borne virus - MNSV melon necrotic spot virus - PAMV petunia asteroid mosaic virus - SqNV squash necrosis virus - TNV tobacco necrosis virus - TBSV tomato bushy stunt virus     

广西三种甜瓜病毒分离物的分子检测与鉴定

瓜类褪绿黄化病毒Cucurbits chlorotic yellows virus(CCYV)、甜瓜黄化斑点病毒Melon yellow spot virus(MYSV)及甜瓜坏死斑点病毒Melon necrotic spot virus(MNSV)是近年来报道侵染瓜类作物的新病毒,在个别种植区大面积发生,对生产构成严重威胁。为了解3种病毒在广西的发生情况,先后到各西甜瓜种植区进行了调查,并采集疑似CCYV、MYSV及MNSV的甜瓜病叶样品,提取病叶总RNA,通过特异性引物分别进行一步法RTPCR扩增,电泳结果显示RT-PCR产物与预期大小一致的序列条带;将扩增产物分别连接到pMD19-T克隆载体上,挑选阳性克隆子进行序列测定及比对分析。结果表明:CCYV、MYSV和MNSV广西分离物的CP基因序列与其他已报道核苷酸序列一致性分别达95.1%~100%、96.5%~99%和83.7%~92.5%。     

Genetic diversity of Melon necrotic spot virus and Olpidium isolates from different origins

The geographic incidence, genetic diversity and phylogenetic relationships of Melon necrotic spot virus (MNSV) and Olpidium isolates were studied in three cucurbit species from several Latin American and European countries on different collecting dates. Of the 112 cucurbit samples analysed, 69 from Guatemala, Honduras, Mexico, Panama and Spain were DAS‐ELISA‐positive for MNSV. Olpidium bornovanus and O. virulentus infections, and MNSV infections mixed with these Olpidium species, were observed for all these countries. Twenty‐nine MNSV isolates from all the origins where the virus was detected were selected and amplified by RT‐PCR. The resulting RT‐PCR of the p29, p89, p7A, p7B and p42 proteins was used to estimate the genetic diversity and the phylogenetic relationships of the MNSV population. The sequences obtained in this study were compared with the MNSV sequences of the NCBI database, and three groups were recovered by nucleotide composition according to geographical origins: the EU‐LA genotype group (with two subgroups: EU and LA, European and Latin American isolates, respectively), the JP melon genotype group (Japanese melon reference isolates) and the JP watermelon genotype group (Japanese watermelon reference isolates). The genetic diversity in the entire p7A and p7B proteins of MNSV suggests that these coding regions are under strong selective pressure. Additionally, the rDNA‐ITS region was analysed in 40 O. bornovanus and O. virulentus isolates associated with each geographical location and host examined. Phylogenetic analysis showed two groups for each Olpidium species, and these groupings were related to the host from which they were originally isolated.     

Fungal pathogens associated with melon collapse in Spain*

Spain produces 43 200 ha of melons with a considerable export to European markets. In the last 10 years, melon cultivation in Spain has decreased more than 40% due mainly to collapse of the vines caused by soil‐borne diseases. Serious economic losses have resulted. In order better to understand the aetiology of this disease, a survey of 217 melon fields throughout the melon production areas of Spain was conducted from 1987 to 1996 to analyse the fungal population associated with roots. In addition, the presence of melon necrotic spot carmovirus (MNSV) was studied in 93 fields. This virus is present mainly in southeastern Spain. The predominant fungal species isolated from 82.5% of sampled fields with symptoms of collapse was Acremonium cucurbitacearum. Roots affected by this fungus show corky brown areas soon after transplanting. Small secondary roots and root hairs become necrotic, although there is continuous production of new rootlets. This process continues until the late stages of the disease. As the fruits approach maturity, the entire plant wilts and dies. Other fungal species associated with melon collapse are: Monosporascus cannonballus (isolated from 29.5% of sampled fields), Macrophomina phaseolina (32.7%) and Rhizoctonia solani (31.8%). Of these, the incidence of M. cannonballus isolated from diseased melons has increased substantially over the past 10 years. Melon collapse in Spain is complex because several fungi capable of causing collapse of the vines are prevalent and often isolated from roots in the same field. In addition, other minor pathogens, such as Rhizopycnis vagum and Plectosporium tabacinum, are frequently isolated from symptomatic vines and may also contribute to the death of the plants.     

Amino acid substitution in the coat protein of Melon necrotic spot virus causes loss of binding to the surface of Olpidium bornovanus zoospores

Melon necrotic spot virus (MNSV) is transmitted by the fungus Olpidium bornovanus. In this study, we used immunofluorescence microscopy to detect MNSV particles over the entire surface of the O. bornovanus zoospore; MNSV particles were not detected on the related fungus O. virulentus, which cannot transmit MNSV. The amino acid substitution Ile → Phe at position 300 in the MNSV coat protein resulted in loss of both specific binding and fungal transmission, while virion assembly and biological aspects were unaffected. Taken together, these results suggest that the MNSV coat protein acts as a ligand to the O. bornovanus zoospore as part of a fungal-vector transmission system.     

西瓜花叶病毒2号(WMV-2)对棉蚜生物学特性的影响

通过对棉蚜寄生选择、显微测量、解剖法及室内培养，研究了西瓜花叶病毒2号对棉蚜寄生甜瓜的趋向性、体长、胚胎数量、繁殖力等的影响。结果表明，棉蚜嗜好感病的甜瓜；病株上棉蚜胚胎数目比健株多2～3个；病株上棉蚜的体长大于健康株；用Logistic曲线拟合，病株上棉蚜最大生物容纳量和瞬时增加速率大于健株；在寄主甜瓜接毒2周内，病株上棉蚜个体繁殖力大于健株，但接毒2周后，病株上棉蚜的繁殖力小于健株；病毒WMV-2对棉蚜种群结构也有影响。     

The role ofMacrophomina phaseolina in a collapse of melon plants in Israel

A collapse of melon plants (Cucumis melo L.) grown under both winter and summer conditions in Israel was shown to be incited by the soilborne pathogenMacrophomina phaseolina (Tassi) Goid. Although symptoms were noted in the crown area of the plant, results of root isolations confirmed that the damage encountered under local conditions was due primarily to a root rot caused by this pathogen. We found infection to be more prevalent in summer than in winter sowings, and collapse in the summer occurred sooner in later than in earlier sowings. Methyl bromide fumigation controlled the pathogen, prevented collapse, and increased yield as compared with the control treatment.     

Characterization and host range evaluation of Pseudomonas viridiflava from melon, blite, tomato, chrysanthemum and eggplant

The fluorescent bacterium Pseudomonas viridiflava (Burkholder) Dowson was identified as the causal agent of bacterial leaf blight of melon, tomato, blite, eggplant and of pith necrosis of chrysanthemum plants. Koch's postulates were fulfilled on greenhouse-grown or on potted plants under controlled environmental conditions. Twenty seven cultivated and one weed species, when artificially inoculated, were found to be susceptible to a strain of the bacterium isolated from cucumber. In cross-inoculation tests on potted plants, each strain induced the disease symptoms, independently of the host of origin. To our knowledge, melon and blite have not been previously reported as natural hosts of P. viridiflava. Also this is the first record of the bacterium as a foliar pathogen of melon, tomato, eggplant and blite and as a stem pathogen of chrysanthemum in Greece.     

Identification and characterization of a mechanical transmissible begomovirus causing leaf curl on oriental melon

Oriental melon plants, Cucumis melo var. makuwa cv. Silver Light, showing virus-induced symptoms of mosaic, leaf curl and puckering were observed in the fields of eastern Taiwan in 2007. A virus culture, designated as SL-1, isolated from the diseased melon was established in systemic host plants, Nicotiana benthamiana and oriental melon, by mechanical inoculation. SL-1 did not react to the antisera against common cucurbit-infecting RNA viruses. Viral DNAs extracted from the diseased plant were amplified with the degenerate primers for begomoviruses. The full-length genomic DNA-A and DNA-B of SL-1 were sequenced and found to be closest, with 97.7% and 90.6% nucleotide identity, respectively, to Tomato leaf curl New Delhi begomovirus (ToLCNDV) cucumber isolate from a group of cucurbit-infecting begomoviruses. The virus SL-1 was designated as ToLCNDV oriental melon isolate (ToLCNDV-OM). The pathogenicity of ToLCNDV-OM was confirmed by agroinfection. Progeny virus from the agroinfected N. benthamiana plants was able to infect oriental melon by mechanical inoculation and caused symptoms similar to the original diseased melon in the field. The ToLCNDV-OM also infected five other species of cucurbitaceous plants by mechanical inoculation. This is the first report of a new ToLCNDV isolate causing severe disease on oriental melon in Taiwan.     

携带eGFP的ZYMV侵染性克隆的构建及其侵染性

 小西葫芦黄花叶病毒(zucchini yellow mosaic virus,ZYMV)是瓜类作物主要病毒之一,严重威胁瓜类产业的可持续发展。本研究在ZYMV 甜瓜分离物CH-87侵染性克隆基础上,利用同源重组策略在NIb和CP编码序列之间插入了eGFP,命名为pXT1-ZYMV-eGFP。将pXT1-ZYMV-eGFP转化农杆菌并接种西瓜和甜瓜,结果发现pXT1-ZYMV-eGFP可以系统侵染西瓜和甜瓜,引起典型的花叶症状,在紫外灯下发病植株呈现绿色荧光。经摩擦接种证实,该克隆接种后所产生的病毒子代可以稳定的传播繁殖。研究表明携带eGFP的ZYMV侵染性克隆成功构建,可用于该病毒致病性及病毒与寄主互作的相关研究。