Papaya Ringspot Virus Type Research Articles

First detection of papaya ringspot virus-type W and zucchini yellow mosaic virus infecting Cucurbita maxima in Paraguay

First detection of papaya ringspot virus-type W and zucchini yellow mosaic virus infecting Cucurbita maxima in Paraguay

English

Watermelon plants can be naturally infected by several viruses in single or mixed infections, of which the diagnosis is difficult and require specific techniques. This study aims to detect and to verify the presence of Cucumber mosaic virus (CMV), Papaya ringspot virus type W (PRSV-W), Watermelon mosaic virus (WMV), Zucchini lethal chlorosis virus (ZLCV), Zucchini yellow mosaic virus (ZYMV), and the main cucurbit viruses in Brazil using multiplex reverse transcriptase polymerase chain reaction (RT-PCR) assay. Oligonucleotides were designed towards the conserved regions of the virus genomes. In the duplex-PCR, it was possible to detect all the virus combinations, except ZLCV with ZYMV. The amplified product sizes were 644 bp (CMV), 535 bp (WMV-2), 398 bp (PRSV-W), 244 bp (ZLCV), and 214 bp (ZYMV). To test the efficacy of the methodology, we analyzed plants with virus symptoms from four municipalities in the state of Tocantins, located at Brazilian Cerrado. Mixed infections were detected in 80% of samples in all the municipalities. The multiplex RT-PCR assay can be used to detect and differentiate watermelon viruses that affect crops in the state of Tocantins.   Key words: Cucumovirus, multiplex RT-PCR, Orthotospovirus, potyvirus, watermelon.

Karakterisasi Molekuler Papaya ringspot virus tipe P pada Tanaman Mentimun di Jawa

Moleculer Characterization of Papaya ringspot virus type P on Cucumber in JavaInfection of Papaya ringspot virus (PRSV) on cucumber plants showing mosaic symptom was detected using specific antibody. Further investigation was conducted to determine molecular characters and status of PRSV infecting cucumber in Java. Infection of PRSV was detected from leaf samples collected from the field using dot immunobinding assay (DIBA). Disease frequency caused by PRSV infection reached 81.11%, 95.86%, 91.66%, and 92.3% in East Java, Central Java, Yogyakarta, and West Java, respectively. Characterization of PRSV isolates was conducted by reverse transcription polymerase chain reaction (RT-PCR) using specific primers for PRSV-P and PRSV-W, followed by cloning, and DNA sequencing. DNA fragment of 470 bp was successfully amplified using specific primers for PRSV-P from several samples from Nganjuk, Brebes, Kulon Progo, and Subang; but no amplification was achieved using specific primers for PRSV-W. Nucleotide and amino acid analysis showed high homology among PRSV-P isolates from Nganjuk, Brebes, Kulon Progo, and Subang, i.e. 98.6%-99.7% and 99.3%-100%, respectively. This is an indication of a low genetic variation among PRSV-P from Java. Further phylogenetic analysis indicated that PRSV-P isolate cucumber is in the same cluster with PRSV-P isolate papaya from Bali, Indonesia. This is the first report of PRSV-P infecting cucumber in Indonesia.

Resistance of Cucurbita maxima ‘Exposição’ to zucchini lethal chlorosis virus is likely associated with restriction to the systemic movement of the virus

Zucchini lethal chlorosis virus (ZLCV), genus Orthotospovirus, so far was only reported in Brasil. The main natural hosts of ZLCV are in the cucurbit family. Pumpkin (Cucurbita maxima) cultivar Exposicao was previously reported as highly resistant to ZLCV infection. The objectives of this work were to better characterize the resistance of the Exposicao cultivar to ZLCV and to evaluate the interference of potyviruses (papaya ringspot virus type W, PRSV-W, and zucchini yellow mosaic virus, ZYMV) in the ZLCV resistance of this cultivar. C. maxima ‘Exposicao’ and zucchini (C. pepo) ‘Caserta’ (susceptible control) plants were mechanically inoculated with ZLCV in the cotyledons. Resistance to infection and systemic virus movement were evaluated at 7 and 20 days later. Virus detection was performed using PTA-ELISA and RT-PCR in the cotyledons and upper leaves. The interference of the potyviruses in ZLCV resistance was evaluated in greenhouse and field assays. ZLCV was detected in the inoculated cotyledons of both cultivars, but it did not move systemically in C. maxima ‘Exposicao’. The potyviruses PRSV-W and ZYMV did not interfere in the response of C. maxima ‘Exposicao’ to infection with ZLCV. The resistance of ‘Exposicao’ to ZLCV can be explored in future cucurbitaceae breeding programs.

Progress in introgression ofPapaya ringspot virusresistance fromVasconcellea pubescenstoCarica papaya

An attempt to develop Carica papaya L. resistant to Papaya ringspot virus type P (PRSV-P) has been established via an intergeneric cross using Vasconcellea pubescens as the resistance donor. V. pubescens has consistently been reported to be resistant to PRSV-P, unlike the resistance in other species (d’Eeckenbrugge et al., 2014). The immunity to PRSV-P is also controlled by a single dominant gene (Dillon et al., 2006), which should be easy to transfer across crops. Because of the genetic distance between the two genera and genetic incompatibility, Vasconcellea parviflora was introduced as a bridging species to transfer the PRSV-P resistance gene(s) to papaya (C. papaya). Backcrossing has been achieved to BC4 in order to maximize the V. parviflora genetic background to improve fertility. The PRSV-P resistance genotype of the BC4 was determined with previously published sequences linked to the prsv-1 locus (Dillon et al., 2006). One of the heterozygous lines, BC4#113, was selected to use as a resistance donor to cross with C. papaya line 2.001. Embryos were rescued, initiated and multiplied in vitro. The cross was successfully micropropagated and produced 18 F1 lines [C. papaya × (V. parviflora backcrossed to V. parviflora × V. pubescens)]. These F1s were genotyped at the prsv-1 locus as previously mentioned. They were subsequently acclimatized and planted in a field in north Queensland. True hybridity was revealed through morphological characterization, including pink flowers, which is a dominant trait from V. parviflora. The hybrid F1s carrying the PRSV-P locus were then successfully backcrossed to C. papaya to generate BC1 generations. They were also sib-crossed with the F1 to generate F1:F2 generations. These lines will be planted in a quarantine laboratory at Griffith University, Nathan, for evaluation of virus resistance. Fertile plants that carry the virus-resistance alleles will be used as parental lines in future breeding strategies.

Cucurbit-infecting viruses in Brazil

Cucurbit crops are grown throughout many Brazilian states and are affected by several viral diseases that represent one of the main constraints to cucurbit production in the country causing severe economic losses. During 2008-2015, a survey was performed in open-field of watermelon (Citrullus lanatus), melon (Cucumis melo), cucumber (C. sativus), pumpkin (Cucurbita spp.), chayote (Sechium edule), and gherkin (C. anguria) crops to determine distribution and relative incidence of six viruses. More than 1050 leaf samples were collected from plants showing virus-like symptoms in more than 20 counties of eight Brazilian states (Goias, Bahia, Sao Paulo, Alagoas, Pernambuco, Ceara, Rio Grande do Norte, and Maranhao) and the Federal District. Samples were screened by dot-ELISA or DAS-ELISA tests using polyclonal antibodies to investigate the occurrence of Papaya ringspot virus-type watermelon (PRSV-W), Watermelon mosaic virus (WMV), Zucchini yellow mosaic virus (ZYMV), Cucumber mosaic virus (CMV), Zucchini lethal chlorosis virus (ZLCV), and Melon yellowing-associated virus (MYaV). In addition, total DNA obtained from leaf samples was tested for geminivirus infections by PCR using a pair of universal primers. Serological tests results indicated that approximately 60% of the samples were infected with at least one of the viruses tested. PRSV-W (40.89%) and ZYMV (25.46%) were the most prevalent viruses detected in all cucurbit species. WMV and CMV were found at low percentage in the leaf samples, 8.76 and 5.02%, respectively. MYaV (22.46%) occurred mainly in melon, while ZLCV (18.11%) was detected mostly infecting watermelon and pumpkins. The occurrence of mixed infection was observed in 17.41% of the leaf samples, especially in combination with PRSV-W. None of the samples tested positive for geminivirus infection by PCR. These data confirm the economic importance of cucurbit-infecting viruses in different Brazilian regions.

Sources of resistance in accessions of Cucurbita spp. to virus species from the genus Potyvirus

ABSTRACT The identification of source of resistance in cultivated cucurbits species is very important for the development of resistant cultivars to control diseases caused by virus from the genus Potyvirus. The present research had the objective to evaluate the phenotypic reactions and the behavior of pumpkin (Cucurbita spp.) accessions to the virus species from the genus Potyvirus: Papaya ringspot virus type Watermelon (PRSV-W), Zucchini yellow mosaic virus (ZYMV) and Watermelon mosaic virus (WMV). Twenty-eight accessions of pumpkin from the Cucurbit Germplasm Bank from Embrapa Semiarido, Petrolina, PE, Brazil were evaluated. Twelve young plants from each pumpkin accession were inoculated with each one of the virus species and were maintained at greenhouse for their symptom reaction evaluations. All possible virus infections or absence of infection were confirmed by plate-trapped antigen enzyme linked immunosorbent assay (PTA-ELISA) against antisera specific to PRSV-W, ZYMV and WMV at the Plant Virus Laboratory, Universidade Federal do Ceara. Three pumpkin accessions showed extreme resistant to WMV and eight accessions presented mild mosaic when inoculated with PRSV-W. The most severe symptoms were observed on those accessions inoculated with ZYMV and 50% of the accessions showed to be highly susceptible. On the other hand, the pumpkin accessions inoculated with WMV presented the mildest symptoms, indicating that 39% of them were resistant, and 39% were tolerant. The Cucurbita spp. accessions BGC 518, BGC 530, BGC 567, and BGC 683 that showed resistance to one or more than one virus species constitute promising sources of resistance for developing virus resistant pumpkin cultivars or hybrids.

FIRST REPORT OF PAPAYA RINGSPOT VIRUS ON SNOW PEA IN CHINA

Papaya ringspot virus (PRSV), a species of the genus Potyvirus , family Potyviridae , is a major limiting factor for papaya and cucurbits cultivation in tropical and subtropi- cal regions worldwide (Purcifull et al ., 1984). PRSV mainly infects plants in the families Caricaceae , Cucurbitaceae and Chenopodiaceae , while few isolates have been reported on other hosts (Tripathi et al ., 2008). During a survey conducted in the summer of 2015, snow peas (Pisum sativum var. sac- charatum ) showing yellow ringspot symptoms were observed in some plantations in the Sichuan province (a main snow pea production area in China). The characteristic ringspot symptoms were also observed on cucurbit plants near the fields of snow pea. Leaf samples (4) were collected from symptomatic snow pea plants and tested by Western blotting with commercial antisera (Agdia, Elkhart, IN) against PRSV. PRSV was detected in all samples tested. Total RNA was extracted from each sample using Plant Total RNA Isolation Kit (FOREGENE, China). RT-PCR was performed using a pair of PRSV coat protein (CP)-specific primers: PRSV-F (5’-TAACACACTGATGGTTTTAATC-3’) and PRSV-R (5’- ATTGCGCATACCCAGGAGAGAG-3’). Expected ampli- cons of the complete CP (861 bp) were obtained, purified, cloned into the pMD19-T vector (TaKaRa), and sequenced. The sequences were identical and designated as PRSV-SC. Nucleotide BLAST analysis revealed that PRSV-SC (Gen- Bank Accession No. KX185713) shared 96% sequence iden- tity to the CP of PRSV-W isolate CI (AY027810). Phylogenet- ic analyses of CP gene of PRSV-SC using MEGA6 software revealed that its sequence clustered into the PRSV type W clade, which is separated from the PRSV-P cluster. To our knowledge, PRSV has been described in pumpkin, luffa, wa- termelon, winter melon, zucchini, muskmelon and Siraitia grosvenorii , and this is the first report of PRSV infecting snow pea in China. Recently, we showed that PRSV could also infect bitter gourd in China (Zhu et al ., 2016). Taken all together, these data suggested that PRSV presumably has a wider range of natural hosts than previously known. This apparent expansion of the natural host range is worthy of further attention.

First Report of Papaya ringspot virus Infecting Bitter Gourd in China

HomePlant DiseaseVol. 100, No. 11First Report of Papaya ringspot virus Infecting Bitter Gourd in China PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Papaya ringspot virus Infecting Bitter Gourd in ChinaL. Zhu, T. Yang, L. J. Chen, H. H. Lin, and D. H. XiL. ZhuSearch for more papers by this author, T. YangSearch for more papers by this author, L. J. ChenSearch for more papers by this author, H. H. LinSearch for more papers by this author, and D. H. XiSearch for more papers by this authorAffiliationsAuthors and Affiliations L. Zhu T. Yang L. J. Chen H. H. Lin D. H. Xi , Ministry of Education Key Laboratory for Bio-Resource and Eco-Environment, College of Life Science, Sichuan University, Chengdu 610064, China. Published Online:25 Aug 2016https://doi.org/10.1094/PDIS-01-16-0086-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Papaya ring spot virus (PRSV), a species of the genus Potyvirus in the family Potyviridae, is an important pathogen of papaya (Carica papaya L.) and cucurbits in tropical and subtropical regions worldwide (Bateson et al. 2002). The virus is a major limiting factor in papaya production in South China and has been described in pumpkin, luffa, watermelon, winter melon, zucchini, muskmelon, and Siraitia grosvenorii (Zhang et al. 2014; Zhu et al. 2014). During a survey conducted in the summer of 2014, bitter gourd plants showing vein clearing, leaf roll, and stunting symptoms were observed in selected vegetable greenhouses in Sichuan Province of Southwest China. The disease incidence was more than 35% in most fields (>85%), and green peach aphids were observed on some plants. Leaf samples (50) were randomly collected from 15 greenhouses in the main production areas in Chengdu, Shuangliu, and Pengzhou, and tested by Western blotting with commercial antisera (Agdia, Elkhart, IN) against TMV, Chilli veinal mottle virus (CHiVMV), and PRSV. PRSV was detected in five of the samples tested. To confirm the presence of PRSV, total RNA was extracted from the five samples and tested by RT-PCR using a pair of potyvirus group primers (5′-GTITGYGTIGAYGAYTTYAAYAA-3′ and 5′-TCIACIACIGTIGAIGGYTGNCC-3′) (Zheng et al. 2010) and a pair of PRSV-specific primers PRSV-F (5′-TAACACACTGATGGTTTTAATC-3′) and PRSV-R (5′-ATTGCGCATACCCAGGAGAGAG-3′). Expected fragments of a 350-bp region of the nuclear inclusion (NIb) and the complete coat protein (CP) (951 bp) were both obtained. The amplicons were subsequently purified, cloned into the pMD19-T vector (TaKaRa), and sequenced. Sequences of either NIb or CP from five samples, designated as PRSV-SC-J, were identical. Nucleotide BLAST analysis of the NIb (GenBank Accession KT818626), and CP (KU365987) showed that this isolate shares the highest identity (NIb: 92%, CP: 99%) with four isolates from Taiwan (JX448370.1, JX448369.1, JX448373.1, and JX448372.1). The isolate clustered with the PRSV type P isolates in the P clade in phylogenetic analyses of the CP nucleotide sequences of isolates available in GenBank. Recently, an isolate of a CMV-II (Cucumber mosaic virus subgroup II), derived from PRSV-infected bitter gourd leaves, was detected and presumably is involved in facilitative interactions. To our knowledge, this is the first report of PRSV infecting bitter gourd in China. Due to the severity of the bitter gourd disease and its continued increased planting in the areas, better management of seed, vector, and greenhouse sanitation is necessary to control the disease.

Aphid vectors of Papaya ringspot virus and their weed hosts in orchards in the major papaya producing and exporting region of Brazil

Papaya ringspot, one of the most important diseases of papaya, is caused by Papaya ringspot virus type P (PRSV-P), which is transmitted by various aphid species. Objectives of this research were to identify the aphid species and their weed hosts of papaya orchards in the major papaya producing and exporting region of Brazil in northern Espírito Santo State. Aphids were collected from weed hosts, and with Möericke-type traps in papaya orchards of twenty farms in Linhares, Sooretama and Aracruz municipalities of Espírito Santo, Brazil. A total of forty aphid species were collected, of which 22 are new records in the State. Thirty-eight aphid species were identified from traps, including Aphis craccivora Koch, A. gossypii Glover, A. spiraecola Patch, Myzus persicae (Sulzer), and Toxoptera citricidus (Kirkaldy), known vectors of PRSV-P. Cryptomyzus Oestlund (Aphididae) is recorded for the first time in Brazil. Thirteen aphid species associated with 22 weed species of 14 families were collected. Aphis gossypii, which has been reported to be one of the most efficient vectors of PRSV-P, infested the greatest number of weed hosts. Chamaesyce hyssopifolia (L.) Small, Euphorbia hirta L. (Euphorbiaceae), Commelina benghalensis L. (Commelinaceae), Lepidium virginicum L. (Brassicaceae) and Sida sp. (Malvaceae) were the most frequently infested weeds. Commelina benghalensis was infested by the greatest number of aphid species during this study. Integrated management of weed hosts of aphids to reduce aphid populations in papaya orchards may be useful for control of PRSV-P. Based on accurate knowledge of the aphid and weed species present in papaya orchards obtained in this study, integrated pest management efforts may be more efficiently directed toward eliminating the most frequently infested weed species that serve as hosts for aphid vectors of PRSV-P.

A kinase gene potentially implicated in resistance toPapaya ringspot virusinVasconcellea quercifolia

Papaya ringspot virus type P (PRSV-P) is one of the most devastating diseases of Carica papaya (papaya). No resistance has been reported within the cultivated genome; however, resistance exists within wild related species in genus Vasconcellea. Since 1998, breeding strategies have focussed on transferring resistance alleles from V. pubescens and V. quercifolia into V. parviflora and papaya. Previously, the V. pubescens resistance was characterised at the prv1 locus, located in close proximity to two kinase (VP_STK1 and VP_STK2) and one leucine rich repeat (VP_LRR) genes. Subsequently, the functional response of these genes in V. quercifolia, and potential differential response among wild and cultivated genomes, was assessed by transcriptional profiling following PRSV-P inoculation. Initially, the resistance response within V. quercifolia was confirmed through a controlled inoculation bioassay. Following inoculation with an equal viral titer, the viral coat protein (CP) level was significantly up regulated in C. papaya compared to non-significant CP expression in V. quercifolia from 20 to 30 days after inoculation (DAI). Putative resistance genes were identified in V. quercifolia using RACE-PCR and named VQ_STK2, VQ_STK2.1 and VQ_LRR. VQ_STK2 was significantly and differentially up-regulated in V. quercifolia between 0.15 and 1.5 fold from 5 to 30 DAI. In contrast, VQ_STK2 was significantly reduced after papaya plants were inoculated to the lowest point of down-regulation of 0.6 fold expression at 20 DAI. This indicated that STK2 may have a role in the sustained viral defence mechanism present in V. quercifolia and will be a target for further functional validation.

DEVELOPING DNA MARKERS FOR A WILD SOURCE OF RESISTANCE TO PAPAYA RINGSPOT VIRUS

Production of papaya (Carica papaya) is limited throughout its growing regions by the devastating disease of Papaya Ringspot Virus type P (PRSV-P). Papaya breeding programmes for PRSV-P resistance target two sources of resistance from crop wild relatives. The resistance loci from V. pubescens were identified in a segregating population resulting from an interspecific cross betweenV. pubescens and V. parviflora. The resistance loci in V. pubescens were located on supercontig (SC) 28 of the papaya whole genome sequence although SC28 is not yet anchored to any linkage group (LG). Recent investigations indicate that SC28 could be located at the end of LG5. Another species V. quercifolia also carries PRSV-P resistance, although more quantitative in action, and this species produces some fertile hybrids when crossed to C. papaya. The aim of this study was to target polymorphism at the end of LG5 and on SC28 in C. papaya and V. quercifolia. Five candidate resistant genes were used, corresponding to three nucleotide binding sites (NBS; NBS9.305, NBS9.308 and NBS18.235) at the end of LG5 and one NBS (NBS28.12) and one kinase gene (STK105) on SC28. Primers were designed for each gene and used to amplify DNA of papaya variety ‘2.001’ and an accession of V. quercifolia. Resultant DNA sequences were aligned by BLASTN to the papaya whole genome sequence, indicating 98-100% and 89-99% identities for the ‘2.001’ and V. quercifolia amplicons, respectively, and 82-93% identity between these two species.

Management of PRSV-P in papaya through time of planting and border cropping

An experiment was carried out to study the effect of aphid population on incidence of Papaya ringspot virus type papaya (PRSV-P) by planting papaya during different months of the year (2010–12) at Experimental Farm of ICAR-IARI Regional Station, Pune. The population monitoring of aphid vectors of PRSV-P indicated peak population of aphids in January and low population from March onwards till September. Papaya (Red Lady) planted from February to April showed significantly less incidence of PRSV-P compared to those planted from September to January. Virus incidence recorded at flowering time was high (100%) in September to January plantation, while it was about 50–80% when planted in February to April. PRSV-P incidence on papaya planted in different months showed positive correlation with aphid population. PRSV-P tolerant papaya line, Pune Sel.-3, planted in March with maize as border crop showed reduced virus incidence of 5 to 31 per cent among different years.

Isolation and Characterisation of PRSV-P Resistance Genes in Carica and Vasconcellea.

Papaya (Carica papaya L.) is one of the major tropical fruit crops worldwide, but it is limited throughout its range by papaya ringspot virus type P (PRSV-P). Previous genetic studies identified a functional PRSV-P resistance marker in a mapping population of F2 plants of Vasconcellea pubescens (resistant to PRSV-P) × Vasconcellea parviflora (susceptible to PRSV-P) and showed that the marker exhibited homology to a serine threonine protein kinase (STK) gene. Full length cDNAs of putative PRSV-P resistance genes designated CP_STK from C. papaya and VP_STK1 and VP_STK2 from V. pubescens were cloned by rapid amplification of cDNA ends (RACE). Due to a frame-shift mutation, the two homologous sequences are transcribed and edited differently such that the gene product in V. pubescens is two separate transcripts, whereas in C. papaya they are fused into a single message. A peroxisomal targeting signal (PTS2) present in VP_STK2 but absent in the other transcripts may be the functional source of PRSV resistance in V. pubescens. The STK gene from V. pubescens may have been derived from an alternative splicing to confer resistance. The putative resistance gene, VP_STK2, that was identified in this study is a potential new source of PRSV-P resistance for papaya genotypes.

Detection of Papaya ringspot virus Type W Infecting the Cucurbit Weed Cucumis melo var. dudaim in Florida

This is the first report of Papaya ringspot virus type W infecting Cucumis melo var. dudaim, a cucurbit weed, in Florida. It provides an overview of this virus reservoir for growers, extension workers, crop consultants, and research and regulatory scientists. Accepted for publication 21 January 2014. Published 18 March 2014.

Physiological Effects of Squash vein yellowing virus Infection on Watermelon.

Squash vein yellowing virus (SqVYV) is the cause of viral watermelon vine decline. The virus is whitefly-transmitted, induces a systemic wilt of watermelon plants, and causes necrosis and discoloration of the fruit rind. In the field, SqVYV is often detected in watermelon in mixed infections with other viruses including the aphid-transmitted Papaya ringspot virus type W (PRSV-W). In this study, watermelon plants of different ages were inoculated with SqVYV or SqVYV+PRSV-W in the greenhouse or SqVYV in the field to characterize the physiological response to infection. Symptoms of vine decline appeared about 12 to 16 days after inoculation with SqVYV regardless of plant age at time of inoculation, plant growth habit (trellised or nontrellised), and location (greenhouse or field). However, the presence of PRSV-W delayed the appearance of vine decline symptoms by 2 to 4 days, and vine decline did not develop on plants with no fruit. For all inoculation treatments, more severe symptoms were observed in younger watermelon plants. Physiological responses to SqVYV infection included reduction in plant and fruit weights, alterations in fruit rind and flesh color, reduction in fruit sucrose content, increase in fruit acid content, and changes in plant nutrient composition, particularly increases in Ca, Mg, B, Mn, and Zn and decreases in K and N. These results demonstrate wide-ranging physiological effects of SqVYV infection and provide new insights into watermelon vine decline.

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.

VIRUSES INFECTING MELON AND WATERMELON IN NORTHEASTERN BRAZIL

The Northeastern Brazil has a great potential for production of melon ( Cucumis melo ) and watermelon ( Citrullus lanatus ). More than 20 virus species have been found naturally infecting cucurbit species around the world, but only seven of them were already found infecting those cucurbit crops in Northeastern Brazil. Plant viruses have been identified by several methods involving their morphological, physical, biological, cytological, serological and molecular properties, but serology is one of the most specific and accessible methods to obtain a rapid and precise diagnosis of a plant disease caused by virus. Several serological techniques were developed and the advent of the enzyme-linked immunosorbent assay (ELISA) has facilitated the use of serology in the identification and characterization of plant viruses, including those infecting melon and watermelon. Virus infections in melon and watermelon are considered of great importance because they can seriously affect yield all over the world, mainly in developing countries. The present review describes biological, morphological, serological and molecular properties of important virus species infecting melon and watermelon in commercial fields of Northeastern Brazil. The review covers the virus species from the following family and genera: Potyviridae genus Potyvirus : Papaya ringspot virus type Watermelon; Watermelon mosaic virus and Zucchini yellow mosaic virus ; Bromoviridae genus Cucumovirus : Cucumber mosaic virus ; Secoviridae , Subfamily: Comvirinae, genus Comovirus : Squash mosaic virus and possible viruses that cause yellowing in melon. DOI: http://dx.doi.org/10.17525/vrr.v17i1-2.60

In Vitro Regeneration and Genetic Transformation of Cucumis metuliferus through Cotyledon Organogenesis

This study was undertaken to establish for the first time an efficient regeneration and transformation system for Cucumis metuliferus line PI292190, which is the source of a well-defined resistant gene, Wmv, that provides resistance against Papaya ringspot virus type P (PRSV-P) and PRSV-W (formerly known as Watermelon mosaic virus 1, WMV-1). Different combinations of growth regulators were evaluated for the regeneration of cotyledon explants. Adventitious buds or shoot primordia were obtained within 3 to 4 weeks on regeneration medium. After shoot development, adventitious buds or shoot primordia were transferred to elongation medium for 3 to 4 weeks and these shoots were subcultured onto rooting medium for another 1 to 2 weeks. Under optimal culture conditions, a total of 7 to 10 weeks was necessary to obtain C. metuliferus plantlets from cotyledons. Furthermore, transgenic plants were successfully obtained using an Agrobacterium tumefaciens-mediated transformation method as shown by polymerase chain reaction analysis and histochemical β-glucuronidase (GUS) assay. A total of nine transgenic plants were developed from 360 cotyledon explants, giving a transformation frequency of 2.5%.

Generation of transgenic watermelon resistant to Zucchini yellow mosaic virus and Papaya ringspot virus type W

Zucchini yellow mosaic virus (ZYMV) and Papaya ringspot virus type W (PRSV W) are major limiting factors for production of watermelon worldwide. For the effective control of these two viruses by transgenic resistance, an untranslatable chimeric construct containing truncated ZYMV coat protein (CP) and PRSV W CP genes was transferred to commercial watermelon cultivars by Agrobacterium-mediated transformation. Using our protocol, a total of 27 putative transgenic lines were obtained from three cultivars of 'Feeling' (23 lines), 'China baby' (3 lines), and 'Quality' (1 line). PCR and Southern blot analyses confirmed that the chimeric construct was incorporated into the genomic DNA of the transformants. Greenhouse evaluation of the selected ten transgenic lines of 'Feeling' cultivar revealed that two immune lines conferred complete resistance to ZYMV and PRSV W, from which virus accumulation were not detected by Western blotting 4 weeks after inoculation. The transgenic transcript was not detected, but small interfering RNA (siRNA) was readily detected from the two immune lines and T(1) progeny of line ZW 10 before inoculation, indicating that RNA-mediated post-transcriptional gene silencing (PTGS) is the underlying mechanism for the double-virus resistance. The segregation ratio of T(1) progeny of the immune line ZW10 indicated that the single inserted transgene is nuclearly inherited and associated with the phenotype of double-virus resistance as a dominant trait. The transgenic lines derived from the commercial watermelon cultivars have great potential for control of the two important viruses and can be implemented directly without further breeding.