Inheritance analysis and identification of SNP markers associated with ZYMV resistance in <Emphasis Type="Italic">Cucurbita pepo</Emphasis>

Cucurbit crops are economically important worldwide. One of the most serious threats to cucurbit production is Zucchini yellow mosaic virus (ZYMV). Several resistant accessions were identified in Cucurbita moschata and their resistance was introgressed into Cucurbita pepo. However, the mode of inheritance of ZYMV resistance in C. pepo presents a great challenge to attempts at introgressing resistance into elite germplasm. The main goal of this work was to analyze the inheritance of ZYMV resistance and to identify markers associated with genes conferring resistance. An Illumina GoldenGate assay allowed us to assess polymorphism among nine squash genotypes and to discover six polymorphic single-nucleotide polymorphisms (SNPs) between two near-isogenic lines, “True French” (susceptible to ZYMV) and Accession 381e (resistant to ZYMV). Two F₂ and three BC₁ populations obtained from crossing the ZYMV-resistant Accession 381e with two susceptible ones, the zucchini True French and the cocozelle “San Pasquale,” were assayed for ZYMV resistance. Molecular analysis revealed an approximately 90% association between SNP1 and resistance, which was confirmed using High Resolution Melt (HRM) and a CAPS marker. Co-segregation up to 72% in populations segregating for resistance was observed for two other SNP markers that could be potentially linked to genes involved in resistance expression. A functional prediction of proteins involved in the resistance response was performed on genome scaffolds containing the three SNPs of interest. Indeed, 16 full-length pathogen recognition genes (PRGs) were identified around the three SNP markers. In particular, we discovered that two nucleotide-binding site leucine-rich repeat (NBS-LRR) protein-encoding genes were located near the SNP1 marker. The investigation of ZYMV resistance in squash populations and the genomic analysis performed in this work could be useful for better directing the introgression of disease resistance into elite C. pepo germplasm.     

A consensus map for <Emphasis Type="Italic">Cucurbita pepo</Emphasis>

Using random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), simple sequence repeats (SSR), and morphological traits, the first genetic maps for Cucurbita pepo (2n=2x=40) were constructed and compared. The two mapping populations consisted of 92 F₂ individuals each. One map was developed from a cross between an oil-seed pumpkin breeding line and a zucchini accession, into which genes for resistance to Zucchini Yellow Mosaic Virus (ZYMV) from a related species, C. moschata, had been introgressed. The other map was developed from a cross between an oil-seed pumpkin and a crookneck variety. A total of 332 and 323 markers were mapped in the two populations. Markers were distributed in each map over 21 linkage groups and covered an average of 2,200 cM of the C. pepo genome. The two maps had 62 loci in common, which enabled identification of 14 homologous linkage groups. Polyacrylamide gel analyses allowed detection of a high number of markers suitable for mapping, 10% of which were co-dominant RAPD loci. In the Pumpkin-Zucchini population, bulked segregant analysis (BSA) identified seven markers less than 7 cM distant from the locus n, affecting lignification of the seed coat. One of these markers, linked to the recessive hull-less allele (AW11-420), was also found in the Pumpkin-Crookneck population, 4 cM from n. In the Pumpkin-Zucchini population, 24 RAPD markers, previously introduced into C. pepo from C. moschata, were mapped in two linkage groups (13 and 11 markers in LGpz1 and LGpz2, respectively), together with two sequence characterized amplified region (SCAR) markers linked to genes for resistance to ZYMV.     

Resistance to Tomato leaf curl New Delhi virus in Cucurbita spp.

Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus (family Geminiviridae) first reported in India and its neighbouring countries. ToLCNDV severely affects zucchini crop (Cucurbita pepo) in the main production areas of Southern Spain since 2012. This emerging begomovirus is a serious threat to this and other cucurbit crops. Breeding resistant cultivars is the most promising method for disease control, but requires the identification of sources of resistance in the Cucurbita genus. In this work, we screened for ToLCNDV resistance a large collection of Cucurbita spp. accessions, including landraces and commercial cultivars of the main cultivated species, C. pepo, Cucurbita moschata and Cucurbita maxima and wild species. The screening was performed using mechanical and whitefly inoculation. The level of resistance was assessed by scoring symptom severity and by measuring the virus content with quantitative polymerase chain reaction in selected genotypes. Diversity in the response was observed within and among species. Severe symptoms and high viral amounts were found at 30 days after mechanical and whitefly inoculation in C. pepo, in all accessions belonging to the Zucchini morphotype and to other morphotypes of both subspecies, pepo and ovifera, and even in the wild relative Cucurbita fraterna. C. maxima was also highly susceptible. This species showed characteristic symptoms of leaf decay and intense yellowing, different from those of mosaic, curling and internode shortening found in C. pepo. The only species showing resistance was C. moschata. Four accessions were symptomless or had some plants with only mild symptoms after three independent rounds of mechanical inoculation with different inoculum sources. Two of them also remained symptomless after virus inoculation with viruliferous whiteflies. ToLCNDV was detected in these asymptomatic accessions at 15 and 30 days post inoculation, but viral amounts were much lower than those found in susceptible genotypes, suggesting a high level of resistance. The symptoms in the susceptible accessions of this species were also different, with a characteristic leaf mottling, evolving to a severe mosaic. The newly identified C. moschata resistant accessions are good candidates for breeding programmes to avoid the damage caused by ToLCNDV.     

Biological and Serological Variability of Zucchini Yellow Mosaic Virus in Sudan

Zucchini yellow mosaic potyvirus (ZYMV) is prevalent in different cucurbit growing agro-ecosystems in Sudan. A study of the biological and serological variability of isolates originating from different regions was conducted to better understand ZYMV epidemiology and to develop adapted and durable control strategies. Variability was detected among isolates regarding symptomatology, host range and virulence towards the Zym resistance gene in melon ( Cucumis melo L.) PI 414723. Serological variability was also revealed using a set of seven differential monoclonal antibodies (mAbs) raised against a French isolate (ZYMV-E9). Six serotypes were differentiated, but a majority of isolates (88%) reacted with all the mAbs as did the reference strains from Italy and France. All isolates from Sudan were equally well controlled by the resistance genes described in squash ( Cucurhita moschata (Duchesne) Duchesne ex Poir. cvs. Menina and Nigeria) and in cucumber ( Cucumis sativus L. cv. TMG), or by cross protection with the mild ZYMV-WK strain. All isolates were transmitted in a nonpersistent manner by Aphis gossypii Glover and Myzus persicae Sulzer.     

Inheritance of resistance to watermelon mosaic virus in the cucumber line TMG-1: tissue-specific expression and relationship to zucchini yellow mosaic virus resistance

The inbred cucumber (Cucumis sativus L.) line TMG-1 is resistant to three potyviruses:zucchini yellow mosaic virus (ZYMV), watermelon mosaic virus (WMV), and the watermelon strain of papaya ringspot virus (PRSV-W). The genetics of resistance to WMV and the relationship of WMV resistance to ZYMV resistance were examined. TMG-1 was crossed with WI-2757, a susceptible inbred line. F₁, F₂ and backcross progeny populations were screened for resistance to WMV and/or ZYMV. Two independently assorting factors conferred resistance to WMV. One resistance was conferred by a single recessive gene from TMG-1 (wmv-2). The second resistance was conferred by an epistatic interaction between a second recessive gene from TMG-1 (wmv-3) and either a dominant gene from WI-2757 (Wmv-4) or a third recessive gene from TMG-1 (wmv-4) located 20–30 cM from wmv-3. The two resistances exhibited tissue-specific expression. Resistance conferred by wmv-2 was expressed in the cotyledons and throughout the plant. Resistance conferred by wmv-3 + Wmv-4 (or wmv-4) was expressed only in true leaves. The gene conferring resistance to ZYMV appeared to be the same as, or tightly linked to one of the WMV resistance genes, wmv-3.     

Sources of resistance to stem nematode, Ditylenchus dipsaci (Kühn) Filipjev, in species of Medicago and Trifolium

The putatively resistant lucerne cv. Euver was as susceptible to 11 English and 11 French populations of ‘lucerne race’ stem nematode, Ditylenchus dipsaci, as the susceptible cv. Europe. The ‘resistant’ cv. Vertus showed some resistance to five of the English and four of the French populations but was just as susceptible as Europe to three English and four French populations. Twelve annual species of Medicago were very resistant to a mixture of two English and two French populations of the nematode. None of 13 perennial spp. of Medicago was very resistant to the same mixture of nematodes but resistant plants were identified in M. borealis, M. dzhawakhetica, M. glutinosa and M. romanica. Twenty six spp. of Trifolium were resistant to a mixture of two English and two French populations of ‘red clover race’ stem nematode.     

Pathogen effects on vegetative and floral odours mediate vector attraction and host exposure in a complex pathosystem

Pathogens can alter host phenotypes in ways that influence interactions between hosts and other organisms, including insect disease vectors. Such effects have implications for pathogen transmission, as well as host exposure to secondary pathogens, but are not well studied in natural systems, particularly for plant pathogens. Here, we report that the beetle‐transmitted bacterial pathogen Erwinia tracheiphila – which causes a fatal wilt disease – alters the foliar and floral volatile emissions of its host (wild gourd, Cucurbita pepo ssp. texana) in ways that enhance both vector recruitment to infected plants and subsequent dispersal to healthy plants. Moreover, infection by Zucchini yellow mosaic virus (ZYMV), which also occurs at our study sites, reduces floral volatile emissions in a manner that discourages beetle recruitment and therefore likely reduces the exposure of virus‐infected plants to the lethal bacterial pathogen – a finding consistent with our previous observation of dramatically reduced wilt disease incidence in ZYMV‐infected plants.     

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.     

High-resolution mapping of zym, a recessive gene for Zucchini yellow mosaic virus resistance in cucumber

Key message

Using a high-resolution mapping approach, we identified a candidate gene for ZYMV resistance in cucumber. Our findings should assist the development of high-versatility molecular markers for MAS for ZYMV resistance.

Abstract

Zucchini yellow mosaic virus (ZYMV) causes significant disease, which leads to fruit yield loss in cucurbit crops. Since ZYMV resistance is often inherited recessively in cucumber, marker-assisted selection (MAS) is a useful tool for the development of resistant cucumber cultivars. Using 128 families of an F_2:3 population derived from a cross between susceptible ‘CS-PMR1’ and resistant ‘A192-18’ cucumber inbred lines, we confirmed that ZYMV resistance is conferred by a single recessive locus: zym ^A192-18 . We constructed a cucumber genetic linkage map that included 125 simple sequence repeat (SSR) markers segregating into 7 linkage groups (chromosomes). The zym ^A192-18 locus was mapped to chromosome 6, at genetic distances of 0.9 and 1.3 cM from two closely linked SSR markers. For high-resolution genetic mapping, we identified new molecular markers cosegregating with the zym ^A192-18 locus; using cucumber genomic and molecular marker resources and screening an F₂ population of 2,429 plants, we narrowed down the zym ^A192-18 locus to a <50-kb genomic region flanked by two SSR markers, which included six candidate genes. Sequence analysis of the candidate genes’ coding regions revealed that the vacuolar protein sorting-associated protein 4-like (VPS4-like) gene had two SNPs between the parental lines. Based on SNPs of the VPS-4-like gene, we developed zym ^A192-18 -linked DNA markers and found that genotypes associated with these markers were correlated with the ZYMV resistance phenotype in 48 cucumber inbred lines. According to our data, the gene encoding VPS4-like protein is a candidate for the zym ^A192-18 locus. These results may be valuable for MAS for ZYMV resistance in cucumber.     

Selection, biological properties and fitness of resistance-breaking strains of Tomato spotted wilt virus in pepper

In glasshouse tests, infective sap from plants infected with 17 different isolates of Tomato spotted wilt virus (TSWV) from four Australian states was inoculated to three Capsicum chinense accessions (PI 152225, PI 159236 and C00943) carrying single genes that confer hypersensitive resistance to TSWV. The normal response to inoculation was development of necrotic (hypersensitive) local lesions in inoculated leaves without systemic invasion, but 3/1386 infected plants also developed systemic susceptible reactions in addition to hypersensitive ones. Similarly when two isolates were inoculated to C. chinense backcross progeny plants, 1/72 developed systemic susceptible reactions in addition to localised hypersensitive ones. Using cultures from the four plants with susceptible reactions and following three to five further cycles of serial subculture in TSWV‐resistant C. chinense plants, four isolates were obtained that gave systemic susceptible type reactions in the three TSWV‐resistant accessions, and in TSWV‐resistant cultivated pepper (C. annuum). When three of these isolates were inoculated to tomato (Lycopersicon esculentum) breeding lines with single gene resistance to TSWV, resistance was not overcome. Similarly, none of the four isolates overcame partial resistance to TSWV in Lactuca virosa. When TSWV isolates were inoculated to tomato breeding lines carrying partial resistance from L. chilense, systemic infection developed which was sometimes followed by ‘recovery’. After four successive cycles of serial passage in susceptible cultivated pepper of a mixed culture of a resistance‐breaking isolate with the non resistance‐breaking isolate from which it came, the resistance‐breaking isolate remained competitive as both were still found. However, when the same resistance‐ breaking isolate was cultured alone, evidence of partial reversion to wild‐type behaviour was eventually obtained after five but not four cycles of long term serial subculture in susceptible pepper, as by then the culture had become a mixture of both types of strain. This work suggests that resistance‐breaking strains of TSWV that overcome single gene hypersensitive resistance in pepper are relatively stable. The findings have important implications for situations where resistant pepper cultivars are deployed widely in the field without taking other control measures as part of an integrated TSWV management strategy.     

Stylet penetration behavior of Myzus persicae related to transmission of Zucchini yellow mosaic virus

The direct current electrical penetration graph (DC‐EPG) technique was used to identify stylet penetration behaviors of Myzus persicae (Sulzer) (Hemiptera: Sternorrhyncha: Aphididae) that are related to successful acquisition and inoculation of the non‐persistently transmitted potyvirus, Zucchini yellow mosaic virus (ZYMV), to melon, Cucurbita pepo L. (Cucurbitaceae). Stylet penetration behaviors of two sources of M. persicae that differ in their ZYMV transmission efficiency were examined to determine if behavioral differences were related to their differing ZYMV transmission efficiencies. Successful acquisition of ZYMV by M. persicae was significantly related to longer total time in intracellular subphase II‐3 of potential drops. Successful inoculation of ZYMV by M. persicae was related to a greater number of intracellular punctures, shorter durations of intracellular punctures, and longer total time in intracellular subphase II‐1. Of these variables that were related to ZYMV transmission, the two aphid sources differed in the total time spent in subphases II‐1 and II‐3. These results suggest that the differences in ZYMV transmission efficiencies between the aphids may be at least partially caused by behavioral differences during stylet penetrations of the host plant tissue, specifically behaviors that occur during intracellular punctures.     

Epidemiology of an aphid nontransmissible potyvirus in fields of nontransgenic and coat protein transgenic squash

Spread of the aphid nontransmissible Zucchini yellow mosaicvirus virus (ZYMV) strain MV was monitored over two consecutive years in field plots of nontransgenic and transgenic squash expressing the coat protein (CP) gene of the aphid transmissible strain FL of Watermelon mosaic virus (WMV). The experimental approach was to mechanically inoculate plants with ZYMV strain MV and to assess subsequent transmissions, assumed to be vectored by aphids, of this strain to nonmechanically inoculated plants. Strain MV was distinguished from other ZYMV isolates by a threonine at position 10 of the CP or by a distinct electrophoretic pattern of a Nla IV-digested genomic cDNA fragment generated by RT-PCR. ZYMV strain MV was not detected in fields of nontransgenic plants, but was apparently aphid transmitted to 77 of 3,700 plants (2%) in transgenic fields. Despite the availability of numerous test plants and conditions of high disease pressure but low selection pressure, an epidemic of ZYMV strain MV did not develop in fields of transgenic plants. In contrast, the aphid transmissible ZYMV strain NY was aphid-transmitted to 99% (446/450) of transgenic plants under similar conditions. The relevance of these results in assessing environmental risks of transgenic plants expressing CP transgenes is discussed.     

Inheritance of resistance to groundnut rosette virus in groundnut (Arachis hypogaea L.)

A method of field screening groundnut seedlings for resistance to groundnut rosette virus (GRV), by means of which over 97% incidence was induced in rows of susceptible test plants, was developed at Chitedze Research Station in Malawi. Two GRV-resistant Virginia cultivars (RG 1 and RMP 40) were crossed with three susceptible cultivars, one from each of the Spanish (JL 24), Valencia (ICGM 48) and Virginia (Mani Pintar) botanical groups. Twelve F₁ reciprocal crosses and their F₂ and backcross generations were produced and the material screened in nurseries in 1985/86 and 1986/87. Seedlings raised from plants which did not become infected in the field were inoculated in the glasshouse in order to eliminate susceptible escapees. The numbers of diseased and healthy individuals in each population were subjected to χ² tests. In the majority of the F₂ populations a good fit was obtained for a ratio of one resistant to 15 susceptible plants, a ratio to be expected if resistance to GRV were determined by a pair of independent complementary recessive genes. This was further supported by data from backcross generations.     

Endophytic colonisation of squash by the fungal entomopathogen Beauveria bassiana (Ascomycota: Hypocreales) for managing Zucchini yellow mosaic virus in cucurbits

We investigated the potential of endophytic Beauveria bassiana to provide protection against Zucchini yellow mosaic virus (ZYMV), one of the most economically important viral diseases in cucurbits. Four selected B. bassiana strains were able to successfully colonise squash plants following foliar inoculation with the conidial suspension of each respective strain. However, no significant difference in percentage colonisation was observed among the tested B. bassiana strains. Disease incidence (percentage of plants showing ZYMV symptoms) and severity (rating based on a 5-point scale), sampled weekly for four weeks following the challenge inoculation of plants with ZYMV, were significantly lower in B. bassiana-inoculated plants as compared to control plants, regardless of the inoculated strain. This is, to our knowledge, the first report on the potential of endophytic B. bassiana to confer protection against plant viruses. Further studies should be conducted to determine whether such endophytic B. bassiana-mediated protection against ZYMV in squash extends to other cucurbits.     

Alatae production and population increase of aphid vectors on virus-infected host plants

Summary Zucchini yellow mosaic virus (ZYMV) and Aphis gossypii Glover are two components of a recently identified plant-parasite system that provides an excellent opportunity to study interrelations between a virus and a vector that share the same host, but have no direct physiological interaction. In a field experiment we documented numbers of alate and apterous A. gossypii on healthy Cucurbita pepo and on plants inoculated with virus 0, 7, 14, and 21 days before aphid infestation. When plants were inoculated and infested simultaneously, more than twice as many alatae were produced after 20 days of colony growth than on any other treatment. This indicates that properties unique to the early stages of viral infection somehow stimulated wing formation. Because it is spread by the activities of alatae, virus dispersal would be greater as a result of these properties. Developmental rate, total numbers of aphids, and numbers of alatae and apterae decreased as the time between virus inoculation and aphid colonization increased.     

Inheritance and sources of resistance to bacterial speck of tomato caused by Pseudomonas syringae pv. tomato

Inheritance of resistance to bacterial speck of tomato was determined by analysing F₁ F₂ and backcross progenies of crosses involving a susceptible (VF-198) and a resistant cultivar (Rehovot-13). The results fit the hypothesis that resistance is controlled by a single dominant gene in interaction with minor genes. Cultivar susceptibility to Pseudomonas syringae pv. tomato was tested under greenhouse conditions under high inoculum pressure using infested tomato seeds together with infested soils and spray-inoculated wounded plants. Of 21 species, cultivars and lines, Rehovot-13, Ontario 7710 and Lycopersiconpimpinellifolium P.I. 126927 were found to be resistant to the pathogen. VF-198 and Tropic-VF were the most susceptible. Extra Marmande, Saladette, Acc.339944–3 and the wild type Lycopersicon esculentum var. cerasiforme were moderately resistant.     

Stripe Rust Resistance in Roegneria kamoji (Poaceae: Triticeae) and its Genetic Analysis

The Roegneria kamoji accession ZY 1007 was resistant to the mixed predominant races of Puccinia striiformis f.sp. tritici (Pst) in China based on field tests at adult‐plant stage. The seedling resistance evaluation of ZY 1007 showed that it was resistant to stripe rust physiological strains CYR29, CYR33 and PST‐V26, which were the predominant races of Pst in China. The female parent R. kamoji cv. Gansi No.1 (susceptible to Pst) was crossed with ZY 1007 (resistant to Pst). Parents, F₁ and F₂ populations were tested in a field inoculated with the mixed urediniospores. ZY 1007 and all the observed 11 F₁ hybrid plants were resistant, while plants of Gansi No.1 were susceptible. Among the 221 F₂ plants, 168 plants were resistant and 53 were susceptible, and the segregation of resistant and susceptible plants fits 3R:1S ratio (χ² = 0.074, P > 0.75). It confirmed that the resistance of stripe rust in ZY 1007 was controlled by a single dominant gene and temporarily designated as YrK1007.     

Disease Interactions in a Shared Host Plant: Effects of Pre-Existing Viral Infection on Cucurbit Plant Defense Responses and Resistance to Bacterial Wilt Disease

Both biotic and abiotic stressors can elicit broad-spectrum plant resistance against subsequent pathogen challenges. However, we currently have little understanding of how such effects influence broader aspects of disease ecology and epidemiology in natural environments where plants interact with multiple antagonists simultaneously. In previous work, we have shown that healthy wild gourd plants (Cucurbita pepo ssp. texana) contract a fatal bacterial wilt infection (caused by Erwinia tracheiphila) at significantly higher rates than plants infected with Zucchini yellow mosaic virus (ZYMV). We recently reported evidence that this pattern is explained, at least in part, by reduced visitation of ZYMV-infected plants by the cucumber beetle vectors of E. tracheiphila. Here we examine whether ZYMV-infection may also directly elicit plant resistance to subsequent E. tracheiphila infection. In laboratory studies, we assayed the induction of key phytohormones (SA and JA) in single and mixed infections of these pathogens, as well as in response to the feeding of A. vittatum cucumber beetles on healthy and infected plants. We also tracked the incidence and progression of wilt disease symptoms in plants with prior ZYMV infections. Our results indicate that ZYMV-infection slightly delays the progression of wilt symptoms, but does not significantly reduce E. tracheiphila infection success. This observation supports the hypothesis that reduced rates of wilt disease in ZYMV-infected plants reflect reduced visitation by beetle vectors. We also documented consistently strong SA responses to ZYMV infection, but limited responses to E. tracheiphila in the absence of ZYMV, suggesting that the latter pathogen may effectively evade or suppress plant defenses, although we observed no evidence of antagonistic cross-talk between SA and JA signaling pathways. We did, however, document effects of E. tracheiphila on induced responses to herbivory that may influence host-plant quality for (and hence pathogen acquisition by) cucumber beetles.