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.     

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     

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.     

Diaporthe sclerotioides exhibits no host specificity among cucurbit species

Plant–fungal specificity between cucurbitaceous crops and Diaporthe sclerotioides, the causal agent of black root rot, was studied using cucumbers (Cucumis sativa), melons (Cucumis melo), pumpkins (Cucurbita maxima), watermelons (Citrullus lanatus) and bottlegourd (Lagenaria siceraria var. gourda). Twelve D. sclerotioides isolates from these cucurbit species were cross‐inoculated. The virulence of the isolates was evaluated as the area under the disease progress curve (AUDPC). All cucurbit species were susceptible to each isolate, but AUDPCs were significantly different among the hosts, with the order of greatest to least being melon, cucumber, watermelon, bottlegourd and finally, pumpkin. The infectiveness of isolates was assessed as the quantity of D. sclerotioides DNA detected in the hypocotyls of seedlings 2 weeks after inoculation using a real‐time PCR protocol. The fungal DNA quantities varied among the species in the same order as the AUDPCs. Whilst there were statistically significant correlations between the virulence and infectiveness of D. sclerotioides isolates in cucumbers, melons and bottlegourds, their coefficients of determination were not high (r² < 0·6). Orthogonal contrasts indicated no specificity in either the fungal virulence or infectiveness between D. sclerotioides isolates and the cucurbit hosts from which these isolates originated. Thus, although the degree of host susceptibility to D. sclerotioides varies among cucurbit species, the absence of specificity to the host species in either virulence or infectiveness suggests the pathogen may spread via various cucurbit crops, irrespective of their original host species.     

Melon chlorotic mosaic virus and associated alphasatellite from Venezuela: genetic variation and sap transmission of a begomovirus–satellite complex

Begomoviruses represent one of the most damaging virus groups on many important crops worldwide. In Venezuela, the begomovirus Melon chlorotic mosaic virus (MeCMV) is the major constraint for melon and watermelon production. MeCMV has been associated with the satellite Melon chlorotic mosaic alphasatellite (MeCMA). Full‐length genome sequencing of 20 and 35 isolates of MeCMV and MeCMA, respectively, was carried out to estimate their genetic variability. Furthermore, mechanical transmission assays of MeCMV alone, or in conjunction with MeCMA, were performed. Genetic variation was low among MeCMV isolates, which exhibited 97–100% nucleotide identity for the DNA‐A component and 95–100% for the DNA‐B component. Alphasatellite isolates were highly variable ranging from 86·5 to 100% nucleotide identity. MeCMV isolates were phylogenetically related to begomoviruses belonging to the Squash leaf curl virus (SLCV) clade, while MeCMA isolates were clustered in two subgroups related to alphasatellites from the New World (Cuba and Brazil). MeCMV has a host range restricted to cucurbit species and two experimental hosts: Nicotiana benthamiana and Nicotiana clevelandii. MeCMV can be mechanically transmitted with up to 100% efficiency in melon. The physiological stage of the inoculated organ (cotyledon or leaf) represents a key factor for inoculation efficiency. This result provides a simple and reliable inoculation method to develop extensive screening for MeCMV resistance sources. In addition, the complex MeCMV + MeCMA was mechanically transmitted to melon, N. benthamiana and N. clevelandii plantlets and successfully back‐transmitted. To the authors’ knowledge, this finding is the first evidence of sap transmission for a begomovirus–satellite complex.     

Occurrence of races and pathotypes of cucurbit powdery mildew in southeastern Spain

The causal agent of cucurbit powdery mildew in southeastern Spain has been investigated since 1996. Of the 139 single-spore isolates obtained, all were identified asSphaerotheca fusca. Four physiological races (1, 2, 4 and 5) of the pathogen were detected. During the survey, a population shift ofS. fusca was observed: race 1 was progressively replaced by other races. In addition, race coexistence was observed in several cucurbit greenhouses. Four host range patterns or pathotypes were distinguished among the isolates ofS. fusca. All isolates were highly aggressive on melon and especially on zucchini cultivars. No clear relationships between races and pathotypes could be established, although isolates virulent on watermelon were preferentially associated with race 1. These data show an apparent heterogeneity ofS. fusca populations in southeastern Spain that should be analyzed further. http://www.phytoparasitica.org posting Aug. 9, 2002.     

Importance of cucurbits in the epidemiology of Papaya ringspot virus type P

Papaya ringspot virus type P (PRSV‐P) systemically infects Carica papaya and species belonging to the family Cucurbitaceae. Attempts to recover PRSV‐P from naturally infected cucurbit plants grown near or among diseased papaya trees have shown conflicting results worldwide. This study aimed to evaluate the natural infection of cucurbit species grown among and near papaya trees infected with PRSV‐P in Brazil. Natural infection of cucurbits with PRSV‐P occurred in zucchini squash but not in watermelon and cucumber. However, several attempts to recover PRSV‐P from numerous Cucurbita pepo cv. Caserta (zucchini squash) plants grown 5–80 m from diseased papaya trees in the field failed. Mechanical inoculations of Cucurbita pepo cv. Caserta, Cucurbita maxima cv. Exposição (pumpkin), Cucumis sativus cv. Primepack Plus (cucumber) and Citrullus lanatus cv. Crimson Sweet (watermelon) with five Brazilian PRSV‐P isolates showed that zucchini squash was the most susceptible species followed by watermelon and cucumber, while pumpkin was not infected. The results confirmed the variable susceptibility of cucurbit species to experimental and natural PRSV‐P infection. Given these facts, the control of the disease through roguing should focus mainly on diseased papaya plants, as has been practised successfully in Brazil for many years, and on those cucurbits particularly known to be susceptible to natural infection with PRSV‐P.     

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.     

黄瓜绿斑驳花叶病毒基因组全序列分析及病害的田间调查

为明确近年来在浙江省葫芦科作物上发生的黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)基因组特征及其发生分布情况,从浙江省及上海地区的甜瓜、西瓜和瓠瓜上采集疑似样品进行RT-PCR鉴定,通过分段扩增测序的方法拼接获得基因组全序列并进行系统进化分析,利用特异性引物扩增获得CGMMV外壳蛋白(coat protein,CP)基因序列,制备CGMMV CP抗血清进行Western-blot和Dot-ELISA检测。结果显示,来自甜瓜、西瓜和瓠瓜的3个CGMMV分离物基因组全序列均具有烟草花叶病毒属典型基因组结构特征,全部由6 423 nt构成;3个全序列间的核苷酸同源性高达99.11%~99.67%,编码的CP氨基酸同源性为100%。系统进化分析发现,CGMMV不同分离物形成2个进化相关群体,3个浙江的CGMMV分离物均位于第I组内,与已报道的中国CGMMV分离物和韩国CGMMV分离物亲缘性较高。Western-blot检测表明CGMMV CP抗血清可以与感病植株中的病毒发生特异性反应,可用于CGMMV鉴定;Dot-ELISA检测发现CGMMV在浙江省和上海市的葫芦科作物上普遍存在。     

Penetration and reproduction of root-knot nematodes on cucurbit species

Cucurbits are often cultivated in rotation with Solanaceae in double-cropping systems. Most cucurbits have been described as susceptible to root-knot nematodes (RKN) but little is known on their relative levels of susceptibility. Because RKN species differ in rates of root invasion and reproductive traits, isolates of M. arenaria, M. incognita and M. javanica were compared on five cucurbit hosts in experiments run in a climate growth chamber. They included zucchini squash cv Amalthee, cucumber cv Dasher II, melon cv Pistolero, pumpkin cv Totanera and watermelon cv Sugar Baby. All cucurbits were susceptible to the three RKN isolates although M. javanica showed higher invasion rates, faster development and higher egg production than M. arenaria on the selected cucurbits. Apparent differences among cucurbits were primarily due to root invasion rates and formation of egg masses. Both Cucumis species (cucumber and melon) were better hosts for nematode invasion and reproduction than zucchini squash, followed by watermelon. Large invasion rates followed by small reproduction traits were linked to M. incognita on zucchini squash. Reduced invasion rates and egg mass formation along with delayed early development were shown on watermelon.     

黄瓜花叶病毒两个甜瓜分离物的基因组及其侵染性克隆

为明确黄瓜花叶病毒（cucumber mosaic virus,CMV）甜瓜分离物的分子变异情况及其侵染性,对2个甜瓜分离物CH99和XH18的基因组进行克隆、测序和分析,并通过构建全长cDNA克隆分析其侵染性。结果显示,黄瓜花叶病毒甜瓜CH99分离物3条RNA长度分别为3 356、3 049和2 211 nt,甜瓜XH18分离物3条RNA长度分别为3 381、3 048和2 217 nt。分离物CH99与XH18的核苷酸序列一致性为89.40%～95.80%,氨基酸序列一致性为90.00%～97.80%,CH99分离物与其他CMV分离物的核苷酸和氨基酸序列一致性平均值分别为79.23%～89.29%和73.52%～93.90%,XH18分离物与其他CMV分离物的核苷酸和氨基酸序列一致性平均值分别为79.81%～89.83%和74.02%～95.14%。遗传发育分析显示,这2个分离物均属于亚组IB成员。接种试验结果显示,分离物CH99和XH18的侵染性克隆构建成功,这2个分离物均能系统侵染本生烟、甜瓜和黄瓜,并在本生烟和甜瓜上引起较严重的症状,在黄瓜上引起的症状较弱,而二者均不能侵染西...     

Comparisons of ribosomal DNA-internal transcribed spacer sequences and biological features among <Emphasis Type="Italic">Olpidium bornovanus</Emphasis> isolates from Cucurbitaceae-cultivating soil in Japan

The chytrid fungus Olpidium bornovanus is an obligate plant parasite that acts as a vector to transmit Melon necrotic spot virus in cultivated soil. Here, we conducted a molecular phylogenetic analysis of 16 isolates of O. bornovanus taken from soil in which Cucurbitaceae plants had been grown in various locations throughout Japan. The ribosomal DNA and internal transcribed spacer region sequences of the 16 O. bornovanus isolates were divided into four molecular phylogenetic groups, designated O.bor-A to O.bor-D. Biological features of O. bornovanus isolates such as host specificity varied consistently with their molecular phylogenetic groups.     

Differential reproduction of Meloidogyne incognita and M. javanica in watermelon cultivars and cucurbit rootstocks

The suitability of watermelon cultivars and cucurbit rootstocks as hosts to Meloidogyne incognita and M. javanica was determined in pot and field experiments. Meloidogyne incognita showed higher reproduction than did M. javanica on watermelon and cucurbit rootstocks. The watermelon cultivars did not differ in host status when challenged with these two species and supported lower nematode reproduction than the cucurbit rootstocks. Rootstocks Lagenaria siceraria cv. Pelops and Cucurbita pepo AK15 supported lower reproduction than did the squash hybrid rootstocks (C. maxima × C. moschata). Egg production increased (P < 0·05) with a rising initial inoculum level (Pi) in the non‐grafted Sugar Baby but the reproduction factor Rf (eggs per plant/Pi) was similar at two Pi levels. The total egg production in the plants grafted onto squash hybrids RS841 and Titan was greater (P < 0·05) at the higher Pi, but the Rf values were lower. The development of field‐grown non‐grafted watermelon plants was significantly stunted in plots where nematodes were detected at planting. However, no differences were observed in plots with grafted plants. In plots with nematodes, non‐grafted and Titan‐grafted plants had similar yields that were higher than that of RS841‐grafted plants. In the commercial plastic houses with grafted watermelon, the average Rf value was 42‐fold, confirming the high susceptibility of squash hybrids as rootstocks for grafted watermelon. The Titan–Sugar Baby combination was tolerant to M. javanica.     

Molecular analysis and virus transmission tests place Olpidium virulentus, a vector of Mirafiori lettuce big-vein virus and tobacco stunt virus, as a distinct species rather than a strain of Olpidium brassicae

The rDNA-ITS sequences of ten single-sporangium isolates of Olpidium virulentus (a noncrucifer strain of Olpidium brassicae), which transmits Mirafiori lettuce big-vein virus (MLBVV) and tobacco stunt virus (TStV), were compared with those of six single-sporangium isolates of O. brassicae. The sequence similarity within isolates of O. virulentus or O. brassicae was almost identical (98.5%–100.0%), but was low between the two species (79.7%–81.8%). In a phylogenetic analysis of the rDNA-ITS region, O. virulentus and O. brassicae fell into two distinct clusters, indicating that O. virulentus, a vector of MLBVV and TStV, is a distinct species rather than a strain of O. brassicae.     

Genetic population structure and trichothecene genotypes of Fusarium graminearum isolated from wheat in southern Brazil

A sample of 140 Fusarium graminearum isolates from Rio Grande do Sul, Brazil, representing three populations at least 150 km from one another, were examined for trichothecene genotype based on PCR amplification of portions of the Tri3 and Tri12 genes and a species‐specific (Fg16F/R) primer pair. Genetic diversity was assessed in a sample of 103 F. graminearum lineage 7 (F. graminearum sensu stricto) isolates using amplified fragment length polymorphism (AFLP) markers. The 15‐ADON genotype was dominant, followed by the NIV genotype (2–18% prevalence), across all three populations. All NIV‐type isolates were in lineage 2 (F. meridionale) and all 15‐ADON‐type isolates were in lineage 7. Isolates with the same haplotype were rare and genotypic diversity was uniformly high (≥98% of the count), suggesting that recombination has played a significant role. The number of migrants (N_m) was estimated between 5 and 6 across all loci and all populations, but the high frequency of private alleles (up to 30%) suggests a historical, rather than contemporary, gene flow. Regarding linkage disequilibrium, 0·8, 1·5 and 2·2% of the locus pairs from the three populations were in disequilibrium, which is lower than values reported in other locations. Thus, Brazilian populations differ from those found in Europe, North America and most of Asia in the presence of a significant frequency (7·8%) of isolates of the NIV genotype in lineage 2.     

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.     

The recombinant isolate of cucurbit aphid-borne yellows virus from Brazil is a polerovirus transmitted by whiteflies

The severe yellowing disease (amarelão) on melon plants is a serious problem in Brazil, although the causative agent remained unknown for a long time. Recently, recombinant isolates of cucurbit aphid-borne yellows virus (CABYV) were reported as the possible causative agents of this disease on melon plants. Although aphids are known to be the vectors of the common type of CABYV isolates, almost no aphid colony was observed in the major melon fields in Brazil with high incidence of the severe yellowing disease. In contrast, whiteflies are often abundant. Based on this observation, the hypothesis of the transmission of recombinant CABYV by whiteflies was evaluated. After thorough transmission experiments, we found that this recombinant CABYV isolate was transmitted by the whitefly Bemisia tabaci MEAM1, but not by Aphis gossipii. Furthermore, the host response by whitefly-based inoculation in cucurbits and other indicator plants showed differences in host range when compared to the common type of CABYV. Due to its transmissibility by the whitefly and the distant relationship of the P3/P5 protein to CABYV, the name “cucurbit whitefly-borne yellows virus” is proposed for this recombinant CABYV. This is the second report of polerovirus transmission by the whitefly B. tabaci, following the report of pepper whitefly-borne vein yellows virus.     

Molecular,biological and serological variability of Zucchini yellow mosaic virus in Tunisia

A study was conducted to better understand the population structure of Zucchini yellow mosaic virus (ZYMV), a severe virus affecting cucurbit crops worldwide, in Tunisia and to estimate whether the use of resistant cultivars may provide durable control. Analysis of the polymerase and coat protein (NIb‐CP) partial sequences of 83 isolates collected in the three main cucurbit‐growing areas in Tunisia showed that ZYMV grouped into two distinct clusters within ZYMV molecular group A. An important variability was observed in the MREK motif of the P3 protein, a motif associated with tolerance breaking in ZYMV‐tolerant zucchini squash cultivars. Interestingly, significant differences were found in the distribution of the MREK variants in the two clusters defined by the partial NIb‐CP sequences, MREK and MKEK sequences being more common in cluster 1 and cluster 2, respectively. When combining NIb‐CP and P3 sequence information, ZYMV molecular variability was shown to be significantly higher in the Cap Bon region than in the Bizerte area. An important biological variability was observed in a subset of 23 isolates regarding symptomatology in susceptible or resistant cucurbits. Some isolates overcame ZYMV tolerance or resistance in zucchini squash and melon, but not in cucumber. Three serotypes were differentiated using a set of 13 monoclonal antibodies (MAbs). Seven parameters characterizing the 23 isolates, including molecular, serological and biological properties, were used for a multiple component analysis (MCA). This analysis revealed that symptom intensity of a given isolate was similar in different susceptible cucurbit hosts, suggesting similar degrees of aggressiveness in different hosts.