Papaya Ringspot Virus Research Articles

Application of plant growth-promoting rhizobacteria to control Papaya ringspot virus and Tomato chlorotic spot virus

Papaya ringspot virus (PRSV-W) and Tomato chlorotic spot virus (TCSV) are responsible for severe losses in cucurbits and tomato production in south Florida and other regions in the USA. Traditional chemicals are not effective to control these viruses. Using plant growth-promoting rhizobacteria (PGPR) may present an alternative to control these viruses. Results from this study demonstrated that applying mixtures of PGPR strains is more efficient to control PRSV-W and TCSV compared to individual PGPR strain only. The application method significantly affected the efficiency of PGPR to control PRSV-W and TCSV. The highest reduction in disease severity of both PRSV-W and TCSV occurred in case of soil drenching with PGPR, followed by root dipping and seed coating treatments. Application of PGPR mixtures of (IN937a & SE34) or (IN937a &, SE34 & T4) were the most efficient methods to control these viral diseases.

Generation of stable infectious clones of plant viruses by using Rhizobium radiobacter for both cloning and inoculation

A novel Rhizobium radiobacter (synonym Agrobacterium tumefaciens)-mediated approach was developed to generate stable infectious clones of plant viruses. This method uses R. radiobacter for both cloning and inoculation of infectious clones, bypassing the requirement of cloning in E. coli to avoid the instability. Only three steps are included in this method: (i) construct viral genome-encoding plasmids in vitro by one-step Gibson assembly; (ii) transform the assembled DNA products into R. radiobacter; (iii) inoculate plants with the R. radiobacter clones containing the viral genome. Stable infectious clones were obtained from two potyviruses papaya ringspot virus (PRSV) and papaya leaf distortion mosaic virus (PLDMV) using this method, whereas attempts utilizing "classical" E. coli cloning system failed repeatedly. This method is simple and efficient, and is promising for a wide application in generation of infectious clones of plant virus, especially for those which are instable in E. coli.

MOLECULAR EVALUATION OF POTENTIAL RESISTANCE TO PAPAYA RING SPOT VIRUS IN MOUNTAIN PAPAYA (VASCONCELLEA CUNDINAMARCENSIS)

Papaya Ring Spot virus (PRSV) is a devastating viral disease in papaya cultivation in Sri Lanka. It was reported that mountain papaya (Vasconcellea cundinamarcensis) exhibits resistance to PRSV. Therefore, this study was conducted to evaluate the potential resistance to PRSV in mountain papaya to be used in papaya improvement breeding programs. Carica papaya and V. Cundinamarcensis were inoculated with PRSV by mechanical inoculation technique and symptoms were evaluated visually. In order to detect the latent infection, a molecular method was developed. PRSV RNA genome segments were amplified using three sets of primers by RTPCR and an amplicon was sequenced to confirm the identity of PRSV. Functional ability of the cDNA was examined using SAND family protein gene (sand) as the reference gene. According to the results, visual symptoms were not observed in mountain papaya while standard visual symptoms appeared in Carica papaya. Three sets of primers were able to amplify the corresponding amplicons in inoculated Carica papaya and sequencing data confirmed that the amplicon was from PRSV. Negative amplification was observed in mountain papaya from the three sets of primers although sand gene amplification was observed, proving the correct functionality of cDNA. Therefore, it can be concluded that mountain papaya is likely to be resistant to PRSV.

Meiotic Studies in Papaya (Carica papaya (L)) infected by Papaya Ring Spot Virus (PRSV)

Meiotic Studies in Papaya (Carica papaya (L)) infected by Papaya Ring Spot Virus (PRSV)

New species in the papaya ringspot virus cluster: Insights into the evolution of the PRSV lineage

The “Papaya ringspot virus (PRSV) cluster” of cucurbit-infecting potyviruses contains five acknowledged species that have similar biological, serological and molecular properties. Additional data suggest there are other uncharacterized species from various locations in the world that likely belong to the PRSV cluster including a new PRSV-like virus reported from Sudan in 2003. Molecular and biological data indicated that the virus from Sudan belongs to a new species, tentatively named wild melon vein banding virus (WMVBV). The complete nucleotide sequence of a second virus from Sudan revealed it was a divergent relative of Moroccan watermelon mosaic virus (MWMV). Based on sequence similarity this virus was determined to be a distinct species and tentatively named Sudan watermelon mosaic virus (SuWMV). Molecular analyses indicate that SuWMV is a recombinant between WMVBV- and MWMV-related viruses. Based on surveys performed in Sudan between 1992 and 2012, SuWMV appeared 10 times more frequent than WMVBV in that country (14.6% vs. 1.5% of the samples tested). The geographic structure and molecular diversity patterns of the putative and acknowledged species suggest that the PRSV-like cluster originated in the Old World about 3600 years ago, with an important diversification in Africa.

Molecular detection of watermelon mosaic virus associated with a serious mosaic disease on Cucurbita pepo L. in Shanxi, China.

With continued expansion of Cucurbita pepo L. cultivation, viral diseases affecting the crop have become more serious in recent years, causing enormous losses in yield and quality. A virus sample was obtained from Wenshui in Shanxi province, China. Double-stranded RNA technology and sequence-independent amplification (SIA) were used to identify the virus that induced C. pepo L. mosaic disease. SIA and sequencing results showed the presence of watermelon mosaic virus (WMV) in diseased C. pepo L. leaves. The complete sequence of WMV from the Shanxi isolate (i.e., WMV-WS) was cloned and analyzed for further characterization. The genomic RNA of WMV-WS is 10,040 nucleotides in length and encodes a putative polyprotein of 3218 amino acids. Phylogenetic analysis indicate that all WMV isolates were divided into four groups and WMV-WS isolate belong to Group 4. Further analysis showed that these WMV isolates were not only to a certain degree related to the host, but also related to geographical origin of isolates. Our results provide information for a better understanding of the genetic diversity of WMV isolates infecting C. pepo L. in Shanxi, China.

Impact of Vat resistance in melon on viral epidemics and genetic structure of virus populations

Cultivar choice is at the heart of cropping systems and resistant cultivars should be at the heart of disease management strategies whenever available. They are the easiest, most efficient and environmentally friendly way of combating viral diseases at the farm level. Among the melon genetic resources, Vat is a unique gene conferring resistance to both the melon aphid Aphis gossypii and the viruses it carries. The ‘virus side’ of this pleiotropic phenotype is seldom regarded as an asset for virus control. Indeed, the effect of Vat on virus epidemics in the field is expected to vary according to the composition of aphid populations in the environment and long-term studies are needed to draw a correct trend. Therefore, the first objective of the study was to re-evaluate the potential of Vat to reduce viral diseases in melon crops. The second objective was to investigate the potential of Vat to exert a selection pressure on virus populations. We monitored the epidemics of Cucurbit aphid-borne yellows virus (CABYV), Cucumber mosaic virus (CMV), Watermelon mosaic virus (WMV) and Zucchini yellow mosaic virus (ZYMV) in two melon lines having a common genetic background, a resistant line (R) and a susceptible line (S), in eight field trials conducted in southeastern France between 2011 and 2015. Vat had limited impact if any on WMV epidemics probably because A. gossypii is not the main vector of WMV in the field, but a favorable impact on CMV, yet of variable intensity probably related to the importance of A. gossypii in the total aphid population. Vat had a significant impact on CABYV epidemics with mean incidence reduction exceeding 50% in some trials. There was no effect of Vat on the structure of virus populations, both for the non-persistent WMV transmitted by numerous aphid species and for the persistent CABYV transmitted predominantly by A. gossypii.

Molecular Evidence for Association of <I>Papaya ringspot virus</I> with Papaya from North East Hill Region of India

Molecular Evidence for Association of <I>Papaya ringspot virus</I> with Papaya from North East Hill Region of India

The occurrence and distribution of major viruses infecting cucurbits in Tamil Nadu state, India

Field surveys were conducted during 2012–14 to document the occurrence and distribution of viruses infecting cucurbits in all the seven agroclimatic zones of Tamil Nadu state, south India. Samples collected from various types of cucurbits showing virus-like symptoms were tested for Cucumber mosaic virus (CMV), Papaya ringspot virus (PRSV), Zucchini yellow mosaic virus (ZYMV), Cucumber green mottle mosaic virus (CGMMV) and begomoviruses using serological (Dot Immuno Binding Assay, DIBA) and molecular (RT-PCR for RNA viruses and PCR for DNA viruses) methods. Results indicated higher incidence (98.6%) of begomoviruses, followed by PRSV (32.1%) and CGMMV (22.2%). ZYMV and CMV were detected at a lower frequency of 7.1% and 5%, respectively. Mixed infections with two or three of these viruses were observed in more than 50% of the samples analyzed. Sequencing of begomovirus-specific amplicons indicated the presence of sequences highly similar to Tomato leaf curl New Delhi virus (ToLCNDV) and Squash leaf curl China virus (SLCCNV).

First Report of Papaya ringspot virus Associated With a Ringspot Disease of Zucchini in Northern China

First Report of <i>Papaya ringspot virus</i> Associated With a Ringspot Disease of Zucchini in Northern China

A Mixed Infection of Lettuce chlorosis virus, Papaya ringspot virus, and Tomato yellow leaf curl virus-IL Detected in a Texas Papaya Orchard Affected by a Virus-Like Disease Outbreak.

Severe virus-like symptoms consisting of mosaic, distortion, yellowing, and brittleness were observed on papaya plants in a 20-ha orchard in South Texas during the 2014-15 growing season. Incidence of symptomatic plants increased from ∼40 to 100% within 6 months of the outbreak; the most severely affected plants were stunted, and fruit yield and quality were reduced compared with asymptomatic plants. The orchard papaya plant virome was explored using the Illumina NextSeq 500 platform and results were validated by Sanger DNA sequencing of complete viral genomes obtained by PCR amplification. The combined results revealed the presence of Papaya ringspot virus (PRSV; Potyvirus), Lettuce chlorosis virus (LCV; Crinivirus), and Tomato yellow leaf curl virus-IL (TYLCV-IL; Begomovirus). The RT-PCR analyses of leaves from 51 randomly sampled papaya plants indicated the presence of PRSV, LCV, and TYLCV-IL in 100, 39.2, and 15.7% of the samples, respectively. Plants infected with PRSV, in combination with LCV and/or TYLCV-IL, exhibited more severe symptoms compared with plants infected with PRSV alone. Furthermore, successful whitefly-mediated transmission of TYLCV-IL and LCV was accomplished by exposing virus-free papaya seedlings to viruliferous Bemisia tabaci (Genn.) under greenhouse conditions. The results of this study document a new host record for LCV and the first successful whitefly-mediated transmission of TYLCV-IL and LCV to papaya. As a perennial crop, infected papaya serving as an over-seasoning reservoir for TYLCV-IL and LCV, presents a new challenge to viral disease management in papaya orchards.

Papaya ringspot virus Populations From East Timorese and Northern Australian Cucurbit Crops: Biological and Molecular Properties, and Absence of Genetic Connectivity.

To examine possible genetic connectivity between crop viruses found in Southeast Asia and Australia, Papaya ringspot virus biotype W (PRSV-W) isolates from cucurbits growing in East Timor and northern Australia were studied. East Timorese samples from cucumber (Cucumis sativus) or pumpkin (Cucurbita moschata and C. maxima) were sent to Australia on FTA cards. These samples and others of pumpkin, rockmelon, honeydew melon (Cucumis melo), or watermelon (Citrullus lanatus) growing in one location each in northwest, north, or northeast Australia were subjected to high throughput sequencing (HTS). When the 17 complete PRSV genomic sequences obtained by HTS were compared with 32 others from GenBank, the five from East Timor were in a different major phylogroup from the 12 Australian sequences. Moreover, the East Timorese and Australian sequences each formed their own minor phylogroups named VI and I, respectively. A Taiwanese sequence was closest to the East Timorese (89.6% nt dentity), and Mexican and Brazilian sequences were the closest to the Australian (92.3% nt identity). When coat protein gene (CP) sequences from the 17 new genomic sequences were compared with 126 others from GenBank, three Australian isolates sequenced more than 20 years ago grouped with the new Australian sequences, while the closest sequence to the East Timorese was from Thailand (93.1% nt identity). Recombination analysis revealed 13 recombination events among the 49 complete genomes. Two isolates from East Timor (TM50, TM32) and eight from GenBank were recombinants, but all 12 Australian isolates were non-recombinants. No evidence of genome connectivity between Australian and Southeast Asian PRSV populations was obtained. The strand-specific RNA library approach used optimized data collection for virus genome assembly. When an Australian PRSV isolate was inoculated to plants of zucchini (Cucurbita pepo), watermelon, rockmelon, and honeydew melon, they all developed systemic foliage symptoms characteristic of PRSV-W, but symptom severity varied among melon cultivars.

FIRST REPORT OF CUCURBIT APHID-BORNE YELLOWS VIRUS AFFECTING SUMMER SQUASH AND MELON IN MONTENEGRO

The phytosanitary status of cucurbit crops in the central and coastal regions of Montenegro was evaluated with a survey in 2015. Leaf samples of summer squash, zucchini squash (Cucurbita pepo L.) and melon (Cucumis melo L.) showing mosaic, interveinal mottling or yellowing, bright yellowing, blisters and distortions were collected from 101 plants. All samples were screened by DAS-ELISA (Bioreba, Switzerland) for Cucumber mosaic virus (CMV), Watermelon mosaic virus (WMV), Moroccan watermelon mosaic virus (MWMV), Zucchini yellow mosaic virus (ZYMV), Zucchini yellow fleck virus (ZYFV) and Papaya ringspot virus (PRSV). Serological analysis showed that 91.1% of the samples were infected with at least one of three viruses, the most prevalent of which was CMV (62.4%), followed by WMV (50.5%) and ZYMV (12.9%). Samples from 20 plants showing symptoms resembling those caused by poleroviruses were analyzed by RT-PCR using the coat protein (CP) gene-specific primers CE9/CE10 of Cucurbit aphid-borne yellows virus (CABYV) (Juarez et al., 2004) An amplicon of the expected size (600 bp) was generated from six samples of summer squash and one of zucchini squash. Single infections by CABYV were found in two of seven positive samples, while other five were co-infected with CMV, WMV and/or ZYMV. Nucleotide sequences of the complete CP gene from four Montenegrin isolates (accession Nos. KX398662-KX398665) were identical to one another, sharing the highest nucleotide identity (99.1%) with an isolate from France (X76931). A previous survey in the former Yugoslavia did not reveal the presence of CABYV (Dukic et al., 2002). Afterwards, the virus has been reported in several neighboring countries (Tomassoli and Meneghini, 2007; Vucurovic et al., 2011), but to our knowledge, this is the first report of CABYV infecting C. pepo in Montenegro.

Sunn hemp intercrop and mulch increases papaya growth and reduces wind speed and virus damage

Papaya (Carica papaya L.) production in south Florida is limited by trade winds, storm wind damage, and papaya ringspot virus (PRSV) infection. This study was implemented to test a system intercropping sunn hemp (Crotalaria juncea L.) with papaya during papaya establishment, then mowing the intercrop and using it as a mulch on papaya beds. The study was performed in 2014 and repeated in 2015. Treatments included no sunn hemp (No-SH), sunn hemp mown early (SH-ME) 65days after transplanting (DAT), and sunn hemp mown late (SH-ML) at 100 DAT. Wind speeds, sunn hemp growth and biomass contribution, papaya above ground growth, yield, flowering date, weeding time, and gas exchange variables, including net CO2 assimilation (A) and stomatal conductance (gs) of H2O were measured. In 2015, additional measurements included PRSV infection rates and root growth via minirhizotron imaging. Results indicated a positive establishment environment as a result of the sunn hemp intercrop-mulching system. The system reduced weed pressure, reduced wind speed within papaya rows, and increased papaya growth after mowing and mulching. In 2015, standing sunn hemp reduced PRSV infection rates and hastened flowering. Mulching increased root growth in 2015, and A and gs for 2 months after mulching. The SH-ML treatment led to elongation and reduced growth due to excessive shading. With the appropriate timing of mowing, this system promises improved production efficiency and reduced risks for south Florida papaya producers.

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.

Plant-pathogen interactions controlled by theFom-1/PrvandFom-2loci of melon

We cloned the genomic locus for melon resistance towards Fusarium oxysporum f.sp. melonis (FOM) races 0 and 1, and the Papaya ring spot virus (PRSV). The two adjacent genes encode proteins of the NBL (nucleotide binding site-leucine rich repeat) family; Prv carries an extra NB domain, and both have alternative, differentially expressed splice variants. The genes' expression patterns were studied using a promoter-reporter system in transgenic melon roots: both genes were expressed around the vascular tissue and in root tips. In our quest for possible fungal proteins that could interact with Fom-2, another resistance gene, we isolated FOM homologs of secreted in xylem (six) genes that, in Fusarium oxysporum f.sp. lycopersici, encode Avr factors. Two such homologs were found, but their distribution among FOM races did not fit the “gene for gene” model. We performed a proteomic analysis of the xylem sap of melon plants that were infected with FOM-0. A total of 513 melon protein and 9 fungal proteins could be annotated against the melon and FOM genomes, respectively. Many of the melon proteins were up-regulated by inoculation.

Papaya ringspot virus W infecting Luffa aegyptiaca in Cuba

Luffa aegyptiaca (sponge gourd) plants showing severe leaf mosaic and deformation symptoms were observed in Villa Clara, Cuba. Electron microscopy observations of leaf dip preparations revealed flexuous filamentous particles, which were identified as Papaya ringspot virus (PRSV) by ELISA test. PRSV was mechanically transmitted to healthy Cucurbita moschata plants. Sequence analysis of the RT-PCR product obtained from the capsid protein gene showed highest identity with other PRSV isolates from United States of America, Australia, Venezuela, Brazil, India and Cuba, ranging between 93.2–96.5% and 93.4–98.1% for nucleotide and amino acid sequences, respectively. Phylogenetic analysis showed two main clusters. Cluster I included isolates from the Americas-Australia group and India, including sponge gourd isolate, while the cluster II included isolates from China, Thailand and Taiwan. To our knowledge, this is the first report of PRSV-W infecting sponge gourd in Cuba.

English

Common impromptu observations between 2010 and 2012 of apparent virus-like disease symptoms in watermelons and pumpkins in Uganda prompted this study. However, there was no recorded evidence of virus infection in these crops anywhere in Uganda or eastern Africa as a region. Thus, 374 and 522 watermelon and pumpkin plants, respectively, growing in 13 fields were surveyed to record virus-like disease symptoms in Uganda’s four districts of Mbale and Kamuli (eastern region), Mpigi and Masaka (central region) during August to November 2013, and January to March 2014. Leaf samples were also collected and tested for four viruses known to commonly infect watermelons and pumpkins worldwide. Symptom severity was assessed using a scale of 0 to 5, while virus incidence was determined by serological methods. The four viruses tested were Cucumber mosaic virus (CMV), Watermelon mosaic virus (WMV), Zucchini yellow mosaic virus (ZYMV) and Cucurbit aphid borne-yellows virus (CABYV). In both crops, there was a higher incidence of virus-like diseases in central than in eastern region (P = 0.000). All the four viruses were detected with CMV as the most common, followed by WMV, ZYMV, and CABYV. Differences in virus incidences between the districts were not always significant. Single CMV, dual CMV+WMV and triple CMV+WMV+ZYMV were the most common single, and multiple infections in both crops. Watermelons contained more single virus infections than mixed infections (P < 0.001), but this difference was not significant in pumpkins (P = 0.468). In all, single virus infections were significantly higher in watermelons than pumpkins (P < 0.001). This is the first study to report incidence of viruses and virus-like diseases in watermelons and pumpkins in Uganda, and in eastern Africa as a region. The importance of these results with respect to crop production and next steps in virus disease management are discussed. Key words: Cucurbits, DAS-ELISA, multiple virus infection, TAS-ELISA, virus incidence, Uganda.

First Complete Genome Sequence of Papaya ringspot virus-W Isolated from a Gourd in the United States.

ABSTRACTIn the United States, the Papaya ringspot virus was first reported from papaya in Florida in 1949. Here, we determined the first complete genome sequence (10,302 nucleotides) of a Papaya ringspot virus-W isolate, which was collected from a commercial field of gourd in Tulsa, OK.

Use of a chimeric transgene construct to confer broad resistance in zucchini (Cucurbita pepo L.) plants against cucurbit-infecting potyviruses occurring in KwaZulu-Natal, South Africa

The frequent identification of cucurbit-infecting viruses across the province of KwaZulu-Natal (KZN) in South Africa necessitates developing effective methods of controlling these diseases. Putative transgenic baby marrow (Cucurbita pepo L.) plants resistant to Zucchini yellow mosaic virus (ZYMV), Moroccan watermelon mosaic virus (MWMV) and Zucchini shoestring virus (ZSSV), three potyviruses occurring in KZN, were developed in this study. A portion of the coat protein coding sequences of each of the selected viruses was amplified by RT-PCR and inserted into the plant expression vector pEPJ86-m/2N. The segment containing the expression cassette was subcloned into the binary vector pGA482G before being introduced into Rhizobium radiobacter (formerly Agrobacterium tumefaciens) strain LBA4404 (pAL4404)(pBI121). Baby marrow cotyledon explants, transformed using Rhizobium-mediated transformation, were regenerated in vitro under kanamycin selection. A total of 94 putative transgenic marrows lines were successfully regenerated from 250 explants. PCR results showed that 84 out of the 94 lines had the transgene. Of these 84 lines, 76 showed resistance after mechanical inoculation with the viruses under study. The preliminary results show the potential of using transgenic cucurbits with resistance to the three potyviruses as an effective strategy to control potyvirus diseases on cucurbits.