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.     

Multiple alleles for zucchini yellow mosaic virus resistance at the zym locus in cucumber

 Sources of resistance to several potyviruses have been identified and characterized within the cucumber (Cucumis sativus L.) germplasm. Resistance to zucchini yellow mosaic virus (ZYMV) is present in inbred lines derived from the Dutch hybrid Dina (Dina-1) and from the Chinese cultivar ‘Taichung Mou Gua’ (TMG-1). Tests of allelism indicated that the genes for resistance to ZYMV in TMG-1 and Dina-1 are at the same locus; however, the two genotypes exhibited different phenotypes in response to cotyledon inoculation with ZYMV. Dina-1 exhibited a distinct veinal chlorosis and accumulation of virus limited to the first and/or second true leaves, while TMG-1 remained symptom-free and did not accumulate virus. The distinct veinal chlorosis phenotype in Dina-1 was dominant to the symptom-free phenotype in TMG-1 and was shown not to be due to a separate gene. These results indicate that a series of alleles differing in effectiveness and dominance relationships occurs at the zym locus such that Zym>zym ^Dina>zym ^TMG-1. In addition to ZYMV resistance, TMG-1 is also resistant to watermelon mosaic virus (WMV), the watermelon strain of papaya ringspot virus (PRSV-W) and the Moroccan watermelon mosaic virus (MWMV); the WMV and MWMV resistances are at the same locus, or tightly linked to the zym locus. Dina-1 also was found to be resistant to PRSV-W and MWMV. The gene for MWMV resistance in Dina-1 appeared to be at the same locus or tightly linked (<1% recombination) to the gene for ZYMV resistance. In contrast to the response to ZYMV inoculation, Dina-1 does not exhibit distinct veinal chlorosis when inoculated with PRSV-W or MWMV. Collectively, these observations suggest that the gene(s) conferring resistance to ZYMV, WMV, and MWMV may be part of a gene cluster for potyvirus resistance in cucumber. Received: 12 November 1996 / Accepted: 25 April 1997     

Occurrence, distribution and relative incidence of mosaic viruses infecting field--grown squash in Tehran province, Iran

Squash (Cucurbita pepo) belongs to Cucurbitaceae family. Every year Cucurbitaceae are planted world wide. They are one of the most important economic crops. Cucurbitaceae are threatened by viruses. Many viruses damage the plants of this family. Since nine viruses have been reported on squash from Iran. In this survey, during 2002--2003, to determine the distribution of Cucumber mosaic virus (CMV), Zucchini yellow mosaic virus (ZYMV) and Watermelon mosaic virus (WMV), 466 samples were collected from squash field in Tehran province. Infected plants showing symptoms such as: mosaic, yellowing, deformation, shoestring of leaves and fruit deformation and yield reduction. Distribution of CMV, ZYMV and WMV were determined by DAS-ELISA. Thepercentage of ZYMV, WMV and CMV were 35.6, 26.1 and 25.1% respectively. Triple infection (CMV+ZYMV+WMV) were found in 6.4% of samples. ZYMV were found the most frequently the viruses. This is the first report of WMV on squash in Tehran province.     

Cantaloupe line CZW-30 containing coat protein genes of cucumber mosaic virus,zucchini yellow mosaic virus,and watermelon mosaic virus-2 is resistant to these three viruses in the field

Cantaloupe line CZW-30 containing coat protein gene constructs of cucumber mosaic cucumovirus (CMV), zucchini yellow mosaic potyvirus (ZYMV), and watermelon mosaic virus 2 potyvirus (WMV-2) was investigated in the field over two consecutive years for resistance to infections by CMV, ZYMV, and/or WMV-2. Resistance was evaluated under high disease pressure achieved by mechanical inoculations and/or natural challenge inoculations by indigenous aphid vectors. Across five different trials, homozygous plants were highly resistant in that they never developed systemic symptoms as did the nontransformed plants but showed few symptomatic leaves confined close to the vine tips. Hemizygous plants exhibited a significant delay (2–3 weeks) in the onset of disease compared to control plants but had systemic symptoms 9–10 weeks after transplanting to the field. Importantly, ELISA data revealed that transgenic plants reduced the incidence of mixed infections. Only 8% of the homozygous and 33% of the hemizygous plants were infected by two or three viruses while 99% of the nontransformed plants were mixed infected. This performance is of epidemiological significance. In addition, control plants were severely stunted (44% reduction in shoot length) and had poor fruit yield (62% loss) compared to transgenic plants, and most of their fruits (60%) were unmarketable. Remarkably, hemizygous plants yielded 7.4 times more marketable fruits than control plants, thus suggesting a potential commercial performance. This is the first report on extensive field trials designed to assess the resistance to mixed infection by CMV, ZYMV, and WMV-2, and to evaluate the yield of commercial quality cantaloupes that are genetically engineered.     

Non-synonymous single nucleotide polymorphisms in the watermelon eIF4E gene are closely associated with resistance to <Emphasis Type="Italic">Zucchini yellow mosaic virus</Emphasis>

Zucchini yellow mosaic virus (ZYMV) is one of the most economically important potyviruses infecting cucurbit crops worldwide. Using a candidate gene approach, we cloned and sequenced eIF4E and eIF(iso)4E gene segments in watermelon. Analysis of the nucleotide sequences between the ZYMV-resistant watermelon plant introduction PI 595203 (Citrullus lanatus var. lanatus) and the ZYMV-susceptible watermelon cultivar ‘New Hampshire Midget’ (‘NHM’) showed the presence of single nucleotide polymorphisms (SNPs). Initial analysis of the identified SNPs in association studies indicated that SNPs in the eIF4E, but not eIF(iso)4E, were closely associated to the phenotype of ZYMV-resistance in 70 F₂ and 114 BC_1R progenies. Subsequently, we focused our efforts in obtaining the entire genomic sequence of watermelon eIF4E. Three SNPs were identified between PI 595203 and NHM. One of the SNPs (A241C) was in exon 1 and the other two SNPs (C309A and T554G) were in the first intron of the gene. SNP241 which resulted in an amino acid substitution (proline to threonine) was shown to be located in the critical cap recognition and binding area, similar to that of several plant species resistance to potyviruses. Analysis of a cleaved amplified polymorphism sequence (CAPS) marker derived from this SNP in F₂ and BC_1R populations demonstrated a cosegregation between the CAPS-2 marker and their ZYMV resistance or susceptibility phenotype. When we investigated whether such SNP mutation in the eIF4E was also conserved in several other PIs of C. lanatus var. citroides, we identified a different SNP (A171G) resulting in another amino acid substitution (D71G) from four ZYMV-resistant C. lanatus var. citroides (PI 244018, PI 482261, PI 482299, and PI 482322). Additional CAPS markers were also identified. Availability of all these CAPS markers will enable marker-aided breeding of watermelon for ZYMV resistance.     

Characterization of sources of resistance to the watermelon strain of Papaya ringspot virus in cucumber: allelism and co-segregation with other potyvirus resistances

At least three sources of resistance to the watermelon strain of Papaya ringspot virus (PRSV-W) have been identified in cucumber (Cucumis sativus L.) including: ’TMG-1’, an inbred line derived from the Taiwanese cultivar, ’Taichung Mou Gua’; ’Dina-1’, an inbred line derived from the Dutch hybrid ’Dina’; and the South American cultivar ’Surinam’. In this investigation we sought to determine the inheritance of resistance to PRSV-W in ’Dina-1’, the allelic relationships among the three sources of PRSV-W resistance, and the relationship between PRSV-W resistance and known resistances to other cucurbit potyviruses. Like ’Surinam’ and ’TMG-1’, resistance in ’Dina-1’ is controlled by a single gene. Despite differences in dominance vs recessive performance and patterns of virus accumulation, all three sources of resistance complemented each other. ’TMG-1’ and ’Dina-1’ also possess co-segregating, single-gene resistances to Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus and Moroccan watermelon mosaic virus. Sequential inoculations and F₃ family analysis indicated that resistance to PRSV-W completely co- segregated with resistance to ZYMV in ’TMG-1’. Although PRSV-W resistances are at the same locus in both ’TMG-1’ and ’Surinam’, ’Surinam’ is only resistant to PRSV-W, and progeny of ’TMG-1’×’Surinam’ were resistant to PRSV-W but susceptible to ZYMV. Susceptibility to ZYMV and resistance to PRSV-W in ’Surinam’ was not influenced by co-inoculation or sequential in- oculations of the two viruses. Collectively, the co- segregation of resistances to PRSV-W, ZYMV, WMV and MWMV in ’TMG-1’ (within 1 cM), allelism of PRSV-W resistances in ’TMG-1’ and ’Surinam’, and resistance to only PRSV-W in ’Surinam’, suggest that multiple potyvirus resistance in cucumber may be due to different alleles of a single potyvirus resistance gene with differing viral specificities, or that the multiple resistances are conferred by a tightly linked cluster of resistance genes, of which ’Surinam’ only possesses one member. Received: 22 July 1999 / Accepted: 2 December 1999     

Reactions of some cucurbitaceous species Tozucchini yellow mosaic virus (ZYMV)

Zucchini yellow mosaic virus (ZYMV) is a widespread serious pathogen of cucurbitaceous plants. ZYMV was first detected in Hungary in 1995. Since then it has become one of the most dangerous viruses of the Cucurbitaceae family causing serious epidemics. The virus has many hosts, which - particularly perennial ones - may play important role as virus reservoirs and infection sources in virus epidemiology. On the other hand wild weed species maybe sources of resistance to viruses. Our research was carried out on a total of 15 wild species from 8 genera (Cucumis, Cucurbita, Cyclanthera, Ecballium Momordica, Lagenaria, Zehneria, Bryonia). Test plants were mechanically inoculated with ZYMV. Local and systemic symptoms were determined and 5 weeks after inoculation DAS-ELISA tests were also carried out. Symptomless plants were reinoculated to Cucumis sativus cv. Accordia test plants. On the basis of the results we determined the percentages of infections and so we classified the test-plants into sensitive and resistance categories. On the basis of the results new host plants of ZYMV are the followings: Bryonia dioica, Cyclanthera pedata, Ecballium elaterium, Momordica balsamina, Momordica rostrata, and Zehneria scabra. Among them Momordica balsamina and Ecballium elaterium showed latent to ZYMV. Bryonia alba and Zehneria indica are especially remarkable, because they proved resistant to ZYMV on the basis of symptomatology and serology. Our results might have significant role in the field of research of host range, virus resistance and virus differentiation.     

Transgenic watermelon rootstock resistant to CGMMV (cucumber green mottle mosaic virus) infection

In watermelon, grafting of seedlings to rootstocks is necessary because watermelon roots are less viable than the rootstock. Moreover, commercially important watermelon varieties require disease-resistant rootstocks to reduce total watermelon yield losses due to infection with viruses such as cucumber green mottle mosaic virus (CGMMV). Therefore, we undertook to develop a CGMMV-resistant watermelon rootstock using a cDNA encoding the CGMMV coat protein gene (CGMMV-CP), and successfully transformed a watermelon rootstock named gongdae. The transformation rate was as low as 0.1–0.3%, depending on the transformation method used (ordinary co-culture vs injection, respectively). However, watermelon transformation was reproducibly and reliably achieved using these two methods. Southern blot analysis confirmed that the CGMMV-CP gene was inserted into different locations in the genome either singly or multiple copies. Resistance testing against CGMMV showed that 10 plants among 140 T₁ plants were resistant to CGMMV infection. This is the first report of the development by genetic engineering of watermelons resistant to CGMMV infection.     

Outcomes of co-infection by two potyviruses: implications for the evolution of manipulative strategies

Recent studies have documented effects of plant viruses on host plants that appear to enhance transmission by insect vectors. But, almost no empirical work has explored the implications of such apparent manipulation for interactions among co-infecting pathogens. We examined single and mixed infections of two potyviruses, watermelon mosaic virus (WMV) and zucchini yellow mosaic virus (ZYMV), that frequently co-occur in cucurbitaceae populations and share the same aphid vectors. We found that ZYMV isolates replicated at similar rates in single and mixed infections, whereas WMV strains accumulated to significantly lower levels in the presence of ZYMV. Furthermore, ZYMV induced changes in leaf colour and volatile emissions that enhanced aphid (Aphis gossypii) recruitment to infected plants. By contrast, WMV did not elicit strong effects on plant–aphid interactions. Nevertheless, WMV was still readily transmitted from mixed infections, despite fairing poorly in in-plant competition. These findings suggest that pathogen effects on host–vector interactions may well influence competition among co-infecting pathogens. For example, if non-manipulative pathogens benefit from the increased vector traffic elicited by manipulative competitors, their costs of competition may be mitigated to some extent. Conversely, the benefits of manipulation may be limited by free-rider effects in systems where there is strong competition among pathogens for host resources and/or access to vectors.     

A genetic map of cucumber composed of RAPDs, RFLPs, AFLPs, and loci conditioning resistance to papaya ringspot and zucchini yellow mosaic viruses.

The watermelon strain of papaya ringspot virus (PRSV-W) and zucchini yellow mosaic virus (ZYMV) are potyviruses that cause significant disease losses in cucumber. Resistances have been identified primarily in exotic germplasm that require transfer to elite cultivated backgrounds. To select more efficiently for virus resistances, we identified molecular markers tightly linked to PRSV-W and ZYMV resistances in cucumber. We generated F6 recombinant inbred lines (RILs) from a cross between Cucumis sativus L. 'Straight 8' and a line from 'Taichung Mou Gua', TMG1 (susceptible and resistant, respectively, to both viruses), and studied the segregations of amplified fragment length polymorphism (AFLP) markers, randomly amplified polymorphic DNAs (RAPDs), restriction fragment length polymorphisms (RFLPs), and resistances to PRSV-W and ZYMV. A 353-point map of cucumber was generated, delineating 12 linkage groups at LOD 3.5. Linkage arrangements among RFLPs were consistent with previously published maps; however linkages among RAPDs in our map did not agree with a previously published map. Resistances to PRSV-W and ZYMV were tightly linked (2.2 cM) and mapped to the end of one linkage group. One AFLP cosegregated with resistance to ZYMV.     

Melon RNA interference (RNAi) lines silenced for Cm-eIF4E show broad virus resistance

Efficient and sustainable control of plant viruses may be achieved using genetically resistant crop varieties, although resistance genes are not always available for each pathogen; in this regard, the identification of new genes that are able to confer broad-spectrum and durable resistance is highly desirable. Recently, the cloning and characterization of recessive resistance genes from different plant species has pointed towards eukaryotic translation initiation factors (eIF) of the 4E family as factors required for the multiplication of many different viruses. Thus, we hypothesized that eIF4E may control the susceptibility of melon (Cucumis melo L.) to a broad range of viruses. To test this hypothesis, Cm-eIF4E knockdown melon plants were generated by the transformation of explants with a construct that was designed to induce the silencing of this gene, and the plants from T2 generations were genetically and phenotypically characterized. In transformed plants, Cm-eIF4E was specifically silenced, as identified by the decreased accumulation of Cm-eIF4E mRNA and the appearance of small interfering RNAs derived from the transgene, whereas the Cm-eIF(iso)4E mRNA levels remained unaffected. We challenged these transgenic melon plants with eight agronomically important melon-infecting viruses, and identified that they were resistant to Cucumber vein yellowing virus (CVYV), Melon necrotic spot virus (MNSV), Moroccan watermelon mosaic virus (MWMV) and Zucchini yellow mosaic virus (ZYMV), indicating that Cm-eIF4E controls melon susceptibility to these four viruses. Therefore, Cm-eIF4E is an efficient target for the identification of new resistance alleles able to confer broad-spectrum virus resistance in melon.     

Effects of barley yellow mosaic disease resistant gene rym1 on the infection by strains of Barley yellow mosaic virus and Barley mild mosaic virus

Although a Chinese landrace of barley, Mokusekko 3, is completely resistant to all strains of Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV), and is known to have at least two resistant genes, rym1 and rym5, only rym5 has been utilized for BaYMV resistant barley breeding in Japan. In order to clarify the effect of rym1 on BaYMV and BaMMV, and to utilize the gene for resistant barley breeding, the susceptibilities of only rym1 carrying breeding lines against BaYMV and BaMMV were investigated. In the assessment of resistance to BaYMV-I, 341 F(2) populations derived from a cross between the resistant line Y4 with only rym1 and the susceptible cv Haruna Nijo shows that the segregation loosely fits a 1R:3S ratio (0.05 > P > 0.01), suggesting that the resistance is controlled by a single recessive gene, rym1. Further, none of the F(3) lines derived from the nine resistant F(2) plants showed any disease symptoms in the field infected by BaYMV-I. The same nine F(3) lines showed almost the same agronomic characters in the field infected by BaYMV-III as those in the uninfected field, apart from the symptom of showing numerous mosaics. This result indicates that the gene rym1 has an acceptable level of resistance to BaYMV-III. In the assessment of resistance to BaYMV-II, BaMMV-Ka1 and -Na1, an artificial infection method was adopted and the susceptibilities to those viruses were investigated. Although the control varieties, Ko A and Haruna Nijo, were infected with all of them, the rym1 gene carrying BC(2)F(3) lines were completely resistant to all strains. In summary, rym1 is completely resistant to BaYMV-I, -II, BaMMV-Ka1 and -Na1, and has an acceptable level of resistance to BaYMV-III. This study concludes with a discussion of the reason why the important resistance gene rym1 was eliminated along with resistant cultivars during breeding for resistance to BaYMV.     

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.