Cucurbit Powdery Mildew: First Insights for the Identification of the Causal Agent and Screening for Resistance of Squash Genotypes (Cucurbita moschata (Duchesne ex Lam.) Duchesne ex Poir.) in Mendoza,Argentina

The cucurbit powdery mildew (CPM) caused by different fungal species is a major concern for cucurbit crops around the world. In Argentina CPM constitutes the most common and damaging disease for cucurbits, especially for squash crops (Cucurbita moschata). The present study displays initial insights into the knowledge of the disease in western Argentina, including the determination of the prevalent species causing CPM, as well as the evaluation of the resistance of squash cultivars and breeding lines. Fungal colonies were isolated from samples collected in Mendoza province, Argentina. A field trial was also performed to assess the resistance of five squash accessions, including commercial cultivars and breeding lines. The severity of CPM was analyzed and epidemiological models were built based on empirical data. The morphological determinations and analysis with specific molecular markers confirmed Podosphaera xanthi as the prevalent causal agent of CPM in Mendoza. The results od the field trial showed differences in the resistance trait among the squash accessions. The advanced breeding line BL717/1 showed promising results as source of CPM resistance for the future development of open pollinated resistant cultivars, a crucial tool for an integrative control of the disease.     

Relative incidence,spatial distribution and genetic diversity of cucurbit viruses in eastern Spain

Viral diseases that could cause important economic losses often affect cucurbits, but only limited information on the incidence and spatial distribution of specific viruses is currently available. During the 2005 and 2006 growing seasons, systematic surveys were carried out in open field melon (Cucumis melo), squash and pumpkin (Cucurbita pepo), watermelon (Citrullus lanatus) and cucumber (Cucumis sativus) crops of the Spanish Community of Valencia (eastern Spain), where several counties have a long standing tradition of cucurbit cultivation and production. Surveyed fields were chosen with no previous information as to their sanitation status, and samples were taken from plants that showed virus‐like symptoms. Samples were analysed using molecular hybridisation to detect Beet pseudo‐yellows virus (BPYV), Cucurbit aphid‐borne yellows virus (CABYV), Cucumber mosaic virus (CMV), Cucumber vein yellowing virus (CVYV), Cucurbit yellow stunting disorder virus (CYSDV), Melon necrotic spot virus (MNSV), Papaya ring spot virus (PRSV), Watermelon mosaic virus (WMV) and Zucchini yellow mosaic virus (ZYMV). We collected 1767 samples from 122 independent field plots; out of these, approximately 94% of the samples were infected by at least one of these viruses. Percentages for the more frequently detected viruses were 35.8%, 27.0%, 16.5% and 7.2% for CABYV, WMV, PRSV and ZYMV, respectively, and significant deviations were found on the frequency distributions based on either the area or the host sampled. The number of multiple infections was high (average 36%), particularly for squash (more than 57%), with the most frequent combination being WMV + PRSV (12%) followed by WMV + CABYV (10%). Sequencing of WMV complementary DNA suggested that ‘emerging’ isolates have replaced the ‘classic’ ones, as described in southern regions of France, leading us to believe that cucurbit cultivation could be severely affected by these new, emerging isolates.     

葫芦科蔬菜种质资源对南方根结线虫的抗性评价

根据不同种质的来源地、农艺性状等背景信息,从国家蔬菜种质资源中期库中选取具代表性的444份主要瓜类作物地方品种,分属葫芦科7个属的13个种或变种,采用病土接种法进行苗期根结线虫抗性鉴定,得出了不同葫芦科作物对南方根结线虫的抗性分布范围。忽略基因型差异,不同作物的平均病级指数从小到大的顺序为:冬瓜、西瓜、丝瓜、节瓜、苦瓜、越瓜、甜瓜、菜瓜、瓠瓜、黄瓜、中国南瓜、印度南瓜、美洲南瓜。通过抗性鉴定,共获得27份抗根结线虫种质(病级指数1~2),包括12份冬瓜、3份苦瓜、7份丝瓜和5份西瓜。     

Cotton,cowpea and sesame are alternative crops to cucurbits in soils naturally infested with Monosporascus cannonballus

Monosporascus cannonballus is an important cucurbit root pathogen, which has been reported in the main production areas of melon and watermelon in Brazil and worldwide and potentially capable to colonize roots of different species. Crop rotation is considered an effective management strategy to prevent this disease. The aim of this study was to evaluate the response of different crops, pumpkin, cotton, cowpea, sesame, watermelon, melon, corn, cucumber, sorghum and tomato, to the infection of this pathogen. Seedlings were transplanted into plastic containers with an inoculum concentration of 20 colony‐forming units (CFU) g^?1 of M. cannonballus. Fifty days after transplanting, the variables analysed were the degree of disease severity on the root system and the frequency of reisolation. On cucurbits, the results demonstrated different degrees of susceptibility among crops and cultivars, being melon and watermelon the most sensitive species. In contrast, Cucurbita cultivars were the most tolerant. Regarding non‐cucurbit crops, maize, sorghum and tomato presented root discoloration and M. cannonballus was reisolated from roots. Cotton, cowpea and sesame cultivars were not affected by the pathogen, so they can be considered as alternative crops to be cultivated, or in rotation with cucurbits, in M. cannonballus infested soils.     

黄瓜资源对黄瓜绿斑驳病毒广东分离物的抗性水平鉴定

【背景】黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)是严重威胁葫芦科作物生产的毁灭性病原之一,该病毒已入侵我国十多个省份,危害西瓜、黄瓜等作物并造成严重的经济损失。早在2009年广东即发现CGMMV为害西瓜和黄瓜,但黄瓜等葫芦科作物对其抗性情况尚不清楚。【方法】采用人工机械摩擦接种方法,测定了14份黄瓜种质资源对CGMMV广东分离物的抗性水平。【结果】从广东葫芦病样中分离获得CGMMV,该病毒分离物MP基因序列与国内报道的各分离物同源率均在99%以上;14份黄瓜种质资源对该病毒分离物均表现为感病。【结论与意义】广东主要黄瓜资源对CGMMV均表现为感病,这为我省防控该病毒病提供了科学依据,也为黄瓜抗病育种提供了指导。     

Transgenic Melon and Squash Expressing Coat Protein Genes of Aphid-borne Viruses do not Assist the Spread of an Aphid Non- transmissible Strain of Cucumber Mosaic Virus in the Field

Transgenic melon and squash containing the coat protein (CP) gene of the aphid transmissible strain WL of cucumber mosaic cucumovirus (CMV) were grown under field conditions to determine if they would assist the spread of the aphid non-transmissible strain C of CMV, possibly through heterologous encapsidation and recombination. Transgenic melon were susceptible to CMV strain C whereas transgenic squash were resistant although the latter occasionally developed chlorotic blotches on lower leaves. Transgenic squash line ZW-20, one of the parents of commercialized cultivar Freedom II, which expresses the CP genes of the aphid transmissible strains FL of zucchini yellow mosaic (ZYMV) and watermelon mosaic virus 2 (WMV 2) potyviruses was also tested. Line ZW-20 is resistant to ZYMV and WMV 2 but is susceptible to CMV. Field experiments conducted over two consecutive years showed that aphid-vectored spread of CMV strain C did not occur from any of the CMV strain C-challenge inoculated transgenic plants to any of the uninoculated CMV-susceptible non- transgenic plants. Although CMV was detected in 3% (22/764) of the uninoculated plants, several assays including ELISA, RT- PCR-RFLP, identification of CP amino acid at position 168, and aphid transmission tests demonstrated that these CMV isolates were distinct from strain C. Instead, they were non-targeted CMV isolates that came from outside the field plots. This is the first report on field experiments designed to determine the potential of transgenic plants expressing CP genes for triggering changes in virus-vector specificity. Our results indicate that transgenic plants expressing CP genes of aphid transmissible strains of CMV, ZYMV, and WMV 2 are unlikely to mediate the spread of aphid non-transmissible strains of CMV. This finding is of practical relevance because transgenic crops expressing the three CP genes are targeted for commercial release, and because CMV is economically important, has a wide host range, and is widespread worldwide.     

Biosafety considerations of RNAi-mediated virus resistance in fruit-tree cultivars and in rootstock

A major application of RNA interference (RNAi) is envisaged for the production of virus-resistant transgenic plants. For fruit trees, this remains the most, if not the only, viable option for the control of plant viral disease outbreaks in cultivated orchards, due to the difficulties associated with the use of traditional and conventional disease-control measures. The use of RNAi might provide an additional benefit for woody crops if silenced rootstock can efficiently transmit the silencing signal to non-transformed scions, as has already been demonstrated in herbaceous plants. This would provide a great opportunity to produce non-transgenic fruit from transgenic rootstock. In this review, we scrutinise some of the concerns that might arise with the use of RNAi for engineering virus-resistant plants, and we speculate that this virus resistance has fewer biosafety concerns. This is mainly because RNAi-eliciting constructs only express small RNA molecules rather than proteins, and because this technology can be applied using plant rootstock that can confer virus resistance to the scion, leaving the scion untransformed. We discuss the main biosafety concerns related to the release of new types of virus-resistant plants and the risk assessment approaches in the application of existing regulatory systems (in particular, those of the European Union, the USA, and Canada) for the evaluation and approval of RNAi-mediated virus-resistant plants, either as transgenic varieties or as plant virus resistance induced by transgenic rootstock.     

Overview of the origins and history of the five major cucurbit crops: issues for ancient DNA analysis of archaeological specimens

The gourd family, Cucurbitaceae, contains five vegetable crops of worldwide importance, the pumpkins and squash (Cucurbita spp.), watermelons (Citrullus lanatus), melons (Cucumis melo) and cucumbers (Cucumis sativus). Here is presented a synopsis of the origin and history of these cucurbit crops. Historical records of the use of cucurbits by people take the form of archaeobotanical finds, iconography and literature. The weight of the evidence indicates that Cucurbita spp. were first cultivated in the Americas at least 10,000 years ago, and that by 1492 ce a number of cultivar-groups of pumpkins and squash had been developed by indigenous American peoples. Watermelons were cultivated in northeastern Africa at least 4,000 years ago, first probably as a source of fresh water. Melons and cucumbers are native to Asia, probably initially cultivated for the use of the young fruits as vegetables. Melons spread to eastern Africa at an early date, but cucumbers are probably a more recent domesticate and spread westwards later, reaching Europe in early medieval times. Sequencing of cucurbit plant genomes and advances in ancient DNA research offer much promise for obtaining an improved assessment of cucurbit crop origins, specifically the genetic constitution and geographical home of ancestral source populations. Next-generation genomic sequencing, if applied to an appropriate array of archaeological cucurbit remains and modern germplasm, could contribute much to the understanding of the history and evolution under domestication of cucurbit crops.     

The cucurbit images (1515-1518) of the Villa Farnesina, Rome

BACKGROUND: The gorgeous frescoes organized by the master Renaissance painter Raphael Sanzio (1483-1520) and illustrating the heavenly adventures of Cupid and Psyche were painted between 1515 and 1518 to decorate the Roman villa (now known as the Villa Farnesina) of the wealthy Sienese banker Agostino Chigi (1466-1520). Surrounding these paintings are festoons of fruits, vegetables and flowers painted by Giovanni Martini da Udine (1487-1564), which include over 170 species of plants. A deconstruction and collation of the cucurbit images in the festoons makes it possible to evaluate the genetic diversity of cucurbits in Renaissance Italy 500 years ago. FINDINGS: The festoons contain six species of Old World cucurbits, Citrullus lanatus (watermelon), Cucumis melo (melon), Cucumis sativus (cucumber), Ecballium elaterium (squirting cucumber), Lagenaria siceraria (bottle gourd) and Momordica balsamina (balsam apple), and two or three species of New World cucurbits, Cucurbita maxima, C. pepo and, perhaps, C. moschata (pumpkin, squash, gourd). The images of C. maxima are the first illustrations of this species in Europe.     

转基因植物对农业生物多样性的影响

论述了近年来转基因植物对农业生态系统生物多样性影响的研究进展．主要在遗传多样性、物种多样性和生态系统多样性3个层次上予以评述．包括转基因植物对作物遗传多样性的影响;转基因植物的外源基因向杂草和近缘野生种转移;转基因抗虫植物对目标害虫的影响。抗除草剂转基因植物对作物和杂草的影响,抗病毒转基因植物对病毒的影响;转基因植物对非目标生物的影响,对土壤生态系统的影响等．     

Virus infections in wild plant populations are both frequent and often unapparent

? Premise of the study: Pathogens are thought to regulate host populations. In agricultural crops, virus infection reduces yield. However, in wild plants little is known about the spatial and temporal patterns of virus prevalence. Thus, pathogen effects on plant population dynamics are unclear. Prevalence data provide necessary background for (1) evaluating the effects of virus infection on plant population size and dynamics and (2) improving risk assessment of virus-resistant transgenic crops. ? Methods: We used ELISA and RT-PCR to survey wild Cucurbita pepo populations over 4 years for five viruses, aphid-transmitted viruses of the genus Potyvirus as a group and PCR to survey for virus-resistance transgenes. In addition, we surveyed the literature for reports of virus prevalence in wild populations. ? Key results: In 21 C. pepo populations, virus prevalence (0-74%) varied greatly among populations, years, and virus species. In samples analyzed by both ELISA and RT-PCR, RT-PCR detected 6-44% more viruses than did ELISA. Eighty percent of these infections did not cause any visually apparent symptoms. In our samples, the virus-resistance transgene was not present. In 30 published studies, 92 of 146 tested species were infected with virus, and infection rates ranged from 0.01-100%. Most published studies used ELISA, suggesting virus prevalence is higher than reported. ? Conclusions: In wild C. pepo, the demographic effects of virus are likely highly variable in space and time. Further, our literature survey suggests that such variation is probably common across plant species. Our results indicate that risk assessments for virus-resistant transgenic crops should not rely on visual symptoms or ELISA and should include data from multiple populations over multiple years.     

Engineering zucchini yellow mosaic potyvirus as a non-pathogenic vector for expression of heterologous proteins in cucurbits

Plant virus vectors provide an attractive biotechnological tool for the transient expression of foreign genes in whole plants. As yet there has been no use of recombinant viruses for the improvement of commercial crops. This is mainly because the viruses used to create vectors usually cause significant yield loss and can be transmitted in the field. A novel attenuated zucchini yellow mosaic potyvirus (AG) was used for the development of an environmentally safe non-pathogenic virus vector. The suitability of AG as an expression vector in plants was tested by analysis of two infectious viral constructs, each containing a distinct gene insertion site. Introduction of a foreign viral coat protein gene into AG genome between the P1 and HC-Pro genes, resulted in no expression in planta. In contrast, the same gene was stably expressed when inserted between NIb and CP genes, suggesting that this site is more suitable for a gene vector. Virus-mediated expression of reporter genes was observed in squash and cucumber leaves, stems, roots and edible fruit. Furthermore, AG stably expressed human interferon-alpha 2, an important human anti-viral drug, without affecting plant development and yield. Interferon biological activity was measured in cucumber and squash fruit. Together, these data corroborate a biotechnological utility of AG as a non-pathogenic vector for the expression of a foreign gene, as a benefit trait, in cucurbits and their edible fruit.     

Gaps and perspectives of pathotype and race determination in <Emphasis Type="Italic">Golovinomyces cichoracearum</Emphasis> and <Emphasis Type="Italic">Podosphaera xanthii</Emphasis>

Golovinomyces cichoracearum and Podosphaera xanthii (family Erysiphaceae) are the most important species causing cucurbit powdery mildew (CPM), a serious disease of field and greenhouse cucurbits. Both species are highly variable in their pathogenicity and virulence, as indicated by the existence of large number of different pathotypes and races. Various independent systems of CPM pathotype and race determinations and denominations are used worldwide. CPM pathotype identification is based on intergeneric and interspecific differences in host-CPM interactions. The most commonly used set of CPM pathotype differentials includes one genotype from four species representing three agriculturally important cucurbit genera plus two genotypes from a fifth species, melon Cucumis melo L. CPM races are characterized by specialization on different cultivars or lines of one host species and have, to date, been differentiated only on melon (C. melo L.). The most frequently used set of melon differentials includes 11 genotypes that can differentiate CPM races originating from melon and other cucurbits, e.g., cucumber, Cucurbita spp., and watermelon. In this paper, we critically review the current state, gaps, and perspectives in our understanding of pathogenicity variation in these two CPM pathogens at the pathotype and race levels.     

A tobacco ringspot virus-based vector system for gene and microRNA function studies in cucurbits

Cucurbits are economically important crops worldwide. The genomic data of many cucurbits are now available. However, functional analyses of cucurbit genes and noncoding RNAs have been impeded because genetic transformation is difficult for many cucurbitaceous plants. Here, we developed a set of tobacco ringspot virus (TRSV)-based vectors for gene and microRNA (miRNA) function studies in cucurbits. A TRSV-based expression vector could simultaneously express GREEN FLUORESCENT PROTEIN (GFP) and heterologous viral suppressors of RNA silencing in TRSV-infected plants, while a TRSV-based gene silencing vector could knock down endogenous genes exemplified by PHYTOENE DESATURASE (PDS) in Cucumis melo, Citrullus lanatus, Cucumis sativus, and Nicotiana benthamiana plants. We also developed a TRSV-based miRNA silencing vector to dissect the functions of endogenous miRNAs. Four representative miRNAs, namely, miR159, miR166, miR172, and miR319, from different cucurbits were inserted into the TRSV vector using a short tandem target mimic strategy and induced characteristic phenotypes in TRSV-miRNA-infected plants. This TRSV-based vector system will facilitate functional genomic studies in cucurbits.     

Toxicity of gallic acid to melon fruit fly,Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae)

Melon fruit fly, Bactrocera cucurbitae (Coquillett) is an important pest of cucurbits and other vegetable crops. It is not only a serious pest of cucurbit crops but sometimes also attacks non-host plants. In an endeavour to explore secondary metabolites as important and safe means of pest management, we investigated the effects of gallic acid, a phenolic compound, on the growth and development of melon fruit fly, B. cucurbitae. Larval survival and emergence were severely affected by gallic acid treatment. Both decreased in a concentration dependent manner with increase in concentration. Gallic acid-treated larvae took longer duration to pupate and reach the adult stage as compared to control larvae. Inhibitory effects of gallic acid were also observed on larval weight, pupal weight, mean relative growth rate and food assimilated which decreased with treatment. The ability of gallic acid to disrupt the development of B. cucurbitae suggests that the phenolic compound might have caused oxidative stress in the body of the insect.     

Dynamic Regulation of Novel and Conserved miRNAs Across Various Tissues of Diverse Cucurbit Species

MicroRNA genes (miRNAs) encoding small non-coding RNAs are abundant in plant genomes and play a key role in regulating several biological mechanisms. Five conserved miRNAs, miR156, miR168-1, miR168-2, miR164, and miR166 were selected for analysis from the 21 known plant miRNA families that were recovered from deep sequencing data of small RNA libraries of pumpkin and squash. A total of six novel miRNAs that were not reported before were found to have precursors with reliable fold-back structures and hence considered novel and were designated as cuc_nov_miRNAs. A set of five conserved, six novel miRNAs, and five uncharacterized small RNAs from the deep sequencing data were profiled for their dynamic regulation using qPCR. The miRNAs were evaluated for differential regulation across the tissues among four diverse cucurbit species, including pumpkin and squash (Cucurbita moschata Duch. Ex Poir. and Cucurbita pepo L.), bitter melon (Momordica charantia L.), and Luffa (Loofah) (Luffa acutangula Roxb.). Expression analysis revealed differential regulation of various miRNAs in leaf, stem, and fruit tissues. Importantly, differences in the expression levels were also found in the leaves and fruits of closely related C. moschata and C. pepo. Comparative miRNA profiling and expression analysis in four cucurbits led to identification of conserved miRNAs in cucurbits. Predicted targets for two of the conserved miRNAs suggested miRNAs are involved in regulating similar biological mechanisms in various species of cucurbits.     

An Integrated Genetic and Cytogenetic Map of the Cucumber Genome

The Cucurbitaceae includes important crops such as cucumber, melon, watermelon, squash and pumpkin. However, few genetic and genomic resources are available for plant improvement. Some cucurbit species such as cucumber have a narrow genetic base, which impedes construction of saturated molecular linkage maps. We report herein the development of highly polymorphic simple sequence repeat (SSR) markers originated from whole genome shotgun sequencing and the subsequent construction of a high-density genetic linkage map. This map includes 995 SSRs in seven linkage groups which spans in total 573 cM, and defines ∼680 recombination breakpoints with an average of 0.58 cM between two markers. These linkage groups were then assigned to seven corresponding chromosomes using fluorescent in situ hybridization (FISH). FISH assays also revealed a chromosomal inversion between Cucumis subspecies [C. sativus var. sativus L. and var. hardwickii (R.) Alef], which resulted in marker clustering on the genetic map. A quarter of the mapped markers showed relatively high polymorphism levels among 11 inbred lines of cucumber. Among the 995 markers, 49%, 26% and 22% were conserved in melon, watermelon and pumpkin, respectively. This map will facilitate whole genome sequencing, positional cloning, and molecular breeding in cucumber, and enable the integration of knowledge of gene and trait in cucurbits.     

Effects of Mixed Cropping on a Soil Nematode Community in Honduras

Nematode-resistant tropical legumes are effective in reducing populations of plant-parasitic nematodes when used in rotation systems. Mixed cropping is a common practice of many small farmers in Central America, but little is known about the effects of tropical legumes on nematode communities under these systems. To examine the effects of intercropping on the nematode fauna associated with squash (Cucurbita pepo) and cucumber (Cucumis sativa) in Honduras, two field experiments were conducted to compare nematode density and diversity in soil under cucurbits grown as a monocrop with that in soil under cucurbits intercropped with alfalfa (Medicago sativa) or hairy indigo (Indigofera hirsuta). A parallel series of field tests compared soil nematode communities associated with a cucurbit monocrop and a cucurbit intercropped with marigold (Tagetes patula), which may decrease nematode populations through the production of toxic root exudates. Among all four tests, over a period of 90 days, there were no consistent differences in densities of various nematode genera or trophic groups in intercropped versus monocropped plants, nor were there consistent differences in community diversities among treatments.