Papaya Ringspot Virus Isolates Research Articles

Complete genome sequencing and construction of full-length infectious cDNA clone of papaya ringspot virus-HYD isolate and its efficient in planta expression.

Papaya ringspot virus (PRSV) is a devastating Potyvirus that causes papaya ringspot disease in Carica papaya plantations globally. In this study, the complete genome sequence of a PRSV isolate from Shankarpalli, Telangana, India, was reported and designated as PRSV-HYD (KP743981.1). The genome is a single-stranded positive-sense RNA comprising 10,341 nucleotides. Phylogenetic analysis revealed that PRSV-HYD is closely related to PRSV Pune (Aundh) isolate with 92 and 95% nucleotide and amino acid sequence identity, respectively. To develop infectious cDNA (icDNA), the complete nucleotide sequence of PRSV-HYD was cloned between the right and left borders in the binary vector pCB301 using BglII and XmaI restriction sites. Cauliflower mosaic virus (CaMV) double promoter (35S) was fused at the 5'-end and Avocado sunblotch viroid (ASBVd) ribozyme (RZ) sequence was fused to the 3' end to generate an authentic 3' viral end in the transcribed mRNAs. The icDNA generated was mobilized into the Agrobacterium tumefaciens EHA 105, and the agrobacterial cultures were infiltrated into the natural host C. papaya and a non-host Nicotiana benthamiana plants; both did not show any symptoms. In RT-PCR analysis of RNAs isolated from N. benthamiana, we could detect viral genes as early as 3 days and continued up to 28 days post infiltration. Alternatively, virion particles were purified from agroinfiltrated N. benthamiana plants and introduced into C. papaya by mechanical inoculation as well as by pinprick method. In both cases, we could see visible systemic symptoms similar to that of wild type by 40 days. Additionally, we studied the expression patterns of the genes related to plant defense, transcription factors (TFs), and developmental aspects from both C. papaya and N. benthamiana.

Host range of three isolates of papaya ringspot virus (PRSV) found in North Sumatera and West Java

Papaya ringspot virus (PRSV) isolates collectd from North Sumatera and West Java were evaluated for host range using mechanical inoculation. PRSV is one of the dangerous diseases in papaya plants in Indonesia reported to have a limited host range and caused symptom variation on plants. The study aim to determine the host range of three PRSV isolates from North Sumatera and West Java to several crops. Incidence of PRSV symptoms on plants was observed by morphological characterization. The experimental results showed that two PRSV isolates from Medan Ampelas and Deli Serdang North Sumatera, and Cibinong West Java isolates were pathogenic and caused systemic symptoms to Carica papaya cv. MD (Caricacea), melon (C. melo) and bitter melon (Momordica charantia). The symptom of infected papaya by the three isolates are vein clearing, mosaic on leaves, leaf blanching, wilt, and mottle on leaf lamina. The isolates caused asymptomatic to eggplant (Solanum melongena) and choy sum (Brassica rapa), asparagus bean (Vigna unguiculata), tobacco (Nicotiana tabacum), and tomato (Solanum lycopersicum syn. lycopersicum esculentum). Melon plants showed symptoms of vein clearing and lamina blanching by Medan Ampelas, and Cibinong isolates respectively, and bitter melon showed symptoms of vein clearing and leaf malformation, and mottle by isolate of Medan Ampelas and Cibinong respectively. The two plant species display asymptomatic by isolate of Deli Serdang. Isolate of Deli serdang has the potential to be a mild strain of PRSV. Complete characterization of mild strains of plant virus through molecular technology approaches can enrich understanding of the potential of these organisms for cross-protection.

First report of zucchini tigre mosaic virus infecting four cucurbit crops in China.

Cucurbits including ridge gourd (Luffa acutangula), Chieh-qua (Benincasa hispida Cogn. var. Chieh-qua How), Trichosanthes anguina, and sponge gourd (L. cylindrica) are important vegetables in most Asian countries. The 90 viruses known to infect cucurbits include 15 species in genus Potyvirus. In October 2020, nine cucurbit samples with leaf distortion, blister and mottle were collected from the same field of Foshan City, Guangdong Province, China. All samples were tested by western blot with potyvirus-specific antibody (Agdia lnc., Elkhart, IN) and RT-PCR with potyvirus degenerate primers Sprimer/M4T (Chen et al. 2001). Seven out of nine samples were positive for potyvirus in both tests, including one ridge gourd, one Chieh-qua, one sponge gourd, two bottle gourd (Lagenaria siceraria) and two T. anguina. All PCR products (~700-bp) were cloned and sequenced. Sequences of seven amplicons (OM522614 to OM522618, OP090158 to OP090159) containing partial nib and cp genes shared 80.3-100% nucleotide (nt) identity among themselves, and 81.2-97.7% nt identity with ZTMV isolates from China (MN267689, LC371337, MK988416). Except for one Chieh-qua sample, papaya ringspot virus (PRSV) was detected in the same samples where ZTMV was found through sequencing of the amplicons mentioned above. The obtained sequences (OM808942 to OM808945, OP090170 to OP090171) were 95.4-100% identical with PRSV isolates from China. Further RT-PCR was conducted with ZTMV-specific primers ZTMVdF/ZTMVdR targeting partial P3 and 6K1 genes, and PRSV-specific primers PRSV3778F/PRSV4630R targeting partial P3, full-length 6K1 and partial CI genes for all nine samples. Consistently, seven samples were positive for ZTMV, among which one Chieh-qua sample was infected with only ZTMV and six samples were co-infected with ZTMV and PRSV. Interspecific recombination event has been reported for ZTMV (Peng et al. 2021), to detect the recombinants, RT-PCR was conducted for all nine samples with primers ZTMV600F/ZTMV2400R covering the interspecies recombination site (Peng et al. 2021). A fragment (~1.8 kb) was amplified from one T. anguina sample and sequenced (OP090172), which had 97.0% nt identity with the reported recombinant ZTMV-KF17 (MK988415). To fulfill Koch's postulates, a Chieh-qua sample detected with ZTMV but not PRSV, was used for mechanical inoculation on Chieh-qua seedlings. Blister and leaf distortion similar to the field symptoms were observed 21 days post-inoculation. ZTMV infection was verified by RT-PCR with primer pairs Sprimer/M4T and ZTMVdF/ZTMVdR, respectively, followed by sequencing. No amplicon was detected with primer pairs PRSV3778F/PRSV4630R and ZTMV600F/ZTMV2400R. To study the incidence of ZTMV and PRSV, 33 samples including T. anguina, ridge gourd and Chieh-qua were collected from three different fields in Foshan City in May 2022, and were tested by RT-PCR with ZTMV and PRSV primers aforementioned. 30.3% (10/33) of the samples were positive for ZTMV, 39.4% (13/33) tested positive for PRSV, and 21.2% (7/33) were co-infected with the two viruses. Amplicons of ZTMV (600 bp) from all positive samples were sequenced (OP090160 to OP090169), and were 84.8-85.5% identical with ZTMV-TW (LC371337). Recombinant of ZTMV was detected in one T. anguina with primers mentioned above and was sequenced (OP090173), which had 96.2% nt identity with ZTMV-KF17. To our knowledge, this is the first report of ZTMV infecting ridge gourd, Chieh-qua, T. anguina and sponge gourd. The results implied that ZTMV had a potential risk to more cucurbit crops in the field.

Modification of the Helper Component Proteinase of Papaya Ringspot Virus Vietnam Isolate to Generate Attenuated Mutants for Disease Management by Cross Protection.

Papaya (Carica papaya) production is seriously limited by papaya ringspot virus (PRSV) worldwide, including in Vietnam. Control of PRSV by cross protection is dependent on the availability of effective mild strains. Here, an infectious cDNA clone was constructed from PRSV isolate TG5 from South Vietnam. Site-directed mutagenesis with point mutations on the essential motifs of the helper component proteinase (HC-Pro) was performed, with or without deleting five amino acids (d5) from its N-terminal region. Mutants TG-d5, TG-d5I7, and TG-d5L206 containing d5, d5 + F7I, and d5 + F206L, respectively, induced mild mottling followed by symptomless recovery on papaya and infected Chenopodium quinoa without lesion formation. Each mutant accumulated in papaya at reduced levels with a zigzag pattern and was stable beyond six monthly passages. The cross-protection effectiveness of the three mutants in papaya against TG5 was investigated, each with 60 plants from three independent trials. The results showed that each mutant provided complete protection (100%) against TG5, 1 month after the challenge inoculation, as verified by the lack of severe symptoms and lack of local lesions in C. quinoa. Further tests revealed that TG-d5I7 also confers high levels of protection against other severe PRSV isolates from South Vietnam, including isolates DN (97%) and ST2 (50%). However, TG-d5I7 is ineffective or less effective (0 to 33%) against seven other severe PRSV strains from different geographic origins, including the isolate HN from North Vietnam. Our results indicate that the protection by the three mutants is highly strain-specific and suitable for the control of PRSV in South Vietnam.

Metagenomic Analysis of RNA Viruses in Watermelon

Watermelon is one of the most consumed cucurbits worldwide. Its production, nevertheless, is threatened by different pathogens that compromise the development of plants and, consequently, fruits. Here, plants showing typical virus-like symptoms were collected in the Tocantins State, Brazil. Aiming for the identification of viruses, viral RNA was sequenced by Illumina. Four virus genomes were sequenced and assembled, corresponding to isolates of Papaya ringspot virus (PRSV) and Zucchini Yellow Mosaic Virus (ZYMV) from the family Potyviridae, Groundnut ringspot virus (GRSV) from the family Bunyaviridae, and Citrullus lanatus partitivirus (CiLPV) from family Partitiviridae. Although these viruses have been previously reported infecting watermelon, we have sequenced the first complete genome of GRSV and the first complete genome of a ZYMV isolate from Brazil. Symptomatic watermelon leaves from the field were also used as inoculation for virus mechanical transmission to indicative plants. Despite co-infecting watermelon plants in the field, only the potyviruses were mechanically transmitted to indicative plants, in which ZYMV presented the broadest host range.

Genetic variability and recombination analysis of papaya infecting isolates of Papaya ringspot virus based on CP gene from Western India

Papaya ringspot disease is caused by Papaya ringspot virus (PRSV-P). The coat protein gene (CP) sequences of 14 isolates of PRSV-P from different papaya growing areas from Western Indian states, Maharashtra and Gujarat, were analysed and compared with previously reported CP gene sequences. The sequence length of CP gene ranged 855–861 nucleotides, encoding 285–287 amino acids. Sequence analysis of CP gene showed that the PRSV-P isolates from Maharashtra and Gujarat shared 80 to 100% and 81 to 98% nucleotide sequence identities, respectively with other Indian isolates. Nine putative recombination sites were identified in CP of PRSV-P isolates analysed. This study contributes in understanding of virus population and genetic recombination.

The complete sequence of a papaya ringspot virus (PRSV) isolate from West Bengal, India infecting papaya and study of genetic variation

Papaya ringspot virus (PRSV) is one of the important pathogens responsible for damage of the papaya crop and causes significant yield loss of fruits and vegetable worldwide and hence a serious threat. In this study, PRSV-infected plant samples collected from different locations in North 24 Parganas districts of West Bengal, were tested with potyvirus specific ELISA and further confirmed with potyvirus group specific degenerate primers by RT PCR, TEM and sequence analysis. Our result indicates that the disease symptoms occurred in 80–100% of the plant in the field surveyed. The complete sequence of PRSV isolate WB was determined from RT-PCR products with specific primer pairs designed covering the whole genome and confirmed by next-generation sequencing. The whole genome of the PRSV was determined and submitted to GenBank as accession no. LC482263. The sequence of PRSV-WB (10,340 bp) was found to share upto 90% and 93%, identity with other PRSV isolates at the nucleotide and amino acid levels respectively. Based on phylogenetic analysis, PRSV- WB was found to be most closely related to PRSV-BD2 (MH397222) from papaya in Bangladesh (geographically close).

DsRNA-mediated protection against two isolates of Papaya ringspot virus through topical application of dsRNA in papaya

Papaya ringspot virus (PRSV) infections in papaya result in heavy yield losses, severely affecting the papaya industry worldwide, and hence warranting for effective control measures. In the past, transgenic papaya cultivars were developed that overexpressed parts of the PRSV genome and exhibited high levels of virus resistance. In the present study, a non-transgenic approach was employed, in which in vitro produced dsRNA molecules derived from a PRSV isolate from South India (PRSV-Tirupati) was tested for dsRNA-mediated protection against two isolates of PRSV through topical application of the dsRNA on papaya. The results showed that the dsRNA molecules from both the coat protein (CP) and helper component-proteinase (HC-Pro) genes of the PRSV-Tirupati isolate conferred 100 % resistance against PRSV-Tirupati infection. Further, the same dsRNA molecules were highly effective against the PRSV-Delhi isolate on the papaya cv. Pusa Nanha, conferring a resistance of 94 % and 81 %, respectively. Systemic papaya leaves of the dsRNA-treated plants were virus-free at 14 days post-inoculation, confirming the robustness of this non-transgenic virus control strategy. In contrast, the control TMV dsRNA did not protect against the PRSV infection. This study on the topical application of dsRNA opened up a new avenue for the control of papaya ringspot disease worldwide.

Molecular diversity of Papaya ringspot virus in India: genetic recombination and mutations between the isolates from different hosts and geo-climatic locations are role players in virus evolution

Among 10 proteins encoded by Papaya ringspot virus (PRSV), only coat protein (CP) has been studied extensively from mainly the papaya-originated isolates i.e. PRSV-P. In this study, besides CP, the helper component proteinase (HC-pro) and nuclear inclusion protein-a (NIa-pro) coding regions from 19 PRSV-P and -W isolates originating from different geo-climatic locations and hosts were analyzed. Unlike CP, both HC-pro and NIa-pro did not reveal the variability in their length. HC-pro and NIa-pro sequences revealed less than 10% deduced amino acid (daa) diversity worldwide, the CP was found highly variable (up to 20% daa diversity). Highest variability in CP sequence was noticed among the cucurbit-originating PRSV-P Indian isolates, especially from Warm semi-arid climate, which was attributed to a large number of aa insertions/deletions. However, papaya-originating PRSV-P isolates from the Oceanic climate were less variable (up to 5% daa diversity). In NIa-pro, besides already known host/pathotype-specific switching of aa Lys/Asp27, switching of Lys27 → Arg27 and Asp27 → Glu27 was identified within P and W pathotypes. Additionally, a novel aa switching from Ser/Met134 → Thr134 between P and W pathotypes respectively was identified. The phylogeny of PRSV isolates was largely based on the geo-climatic locations and hosts. Through recombination detection analysis, three recombinant isolates were detected based on both NIa-pro and CP sequences, which possessed cucurbit-originated PRSV (either -P or -W) isolates as one of their parents (either major or minor.) Patterns of codon usage among the three protein coding sequences generated further revealed that the CP gene is under maximum selection pressure (with six sites) followed by NIa-pro gene (with one site). This investigation shows that the genetic recombination between the isolates originating from different hosts and geo-climatic locations as well as mutations are the significant mechanisms playing their role in generating diverse populations of PRSV.

Characterization of a severe isolate of papaya ringspot virus from papaya in western India

Papaya ringspot virus (PRSV), the causative agent of a devastating and widespread ringspot disease in papaya (Carica papaya L.), belongs to the family Potyviridae. Here, we report the first complete genome characterization of a severe PRSV isolate (PRSV-Pune; GenBank accession No. MH311882) from papaya in the Pune district, western India. The genome size of PRSV-Pune is 10,326 nucleotides (nt), excluding the poly-A tail, encoding a polyprotein of 3345 amino acids (aa). The PRSV-Pune showed identity ranging from 82 to 86% and 87–90% at the nucleotide and amino acid levels, respectively, with 29 complete PRSV-P genomes from all over the world. Variations in amino acid composition at and around two polyprotein cleavage sites of PRSV-Pune were observed: MEQY/S instead of MEQY/N in P1/HC-Pro and DYHQ/N instead of VYHQ/S in CI/6K2. Phylogenetic analysis revealed the clustering of PRSV-Pune, along with the majority of Indian PRSV isolates, with isolates of American lineage. Putative recombination events in P1, HC-Pro, CI, NIb and CP coding regions were detected in PRSV-Pune. Negative selection pressure predicted in all cistrons indicates purifying selection. Our results highlight unique attributes of the severe PRSV-Pune from papaya.

Studies on molecular variability of coat protein gene of Papaya ringspot virus-P isolates from India

Papaya ringspot virus (PRSV) is the key pathogen responsible for damage of the papaya plant and significant loss of fruit yield globally. Its coat protein (CP) sequences are highly variable and provide adaptability to the virus in the papaya plant. Due to the variability, the major concern facing of papaya cultivation is the breakdown of CP-transgenic PRSV resistance. In this study, 16 PRSV isolates were collected from different locations in seven districts of Uttar Pradesh, India and the variability of the CP gene of the virus was analysed. Further, it was compared with the isolates from other geographical origins. High diversity in CP sequences of our isolates was observed when compared with other Indian isolates. Phylogenetic analysis also revealed that PRSV isolates were more divergent from isolates of other countries. This study of sequence variation in the CP could be further very useful in virus management and development of broad-spectrum virus-resistant transgenic papaya.

Genetic Connectivity Between Papaya Ringspot Virus Genomes from Papua New Guinea and Northern Australia, and New Recombination Insights.

Isolates of papaya ringspot virus (PRSV) were obtained from plants of pumpkin (Cucurbita spp.) or cucumber (Cucumis sativus) showing mosaic symptoms growing at Zage in Goroka District in the Eastern Highland Province of Papua New Guinea (PNG) or Bagl in the Mount Hagen District, Western Highlands Province. The samples were sent to Australia on FTA cards where they were subjected to High Throughput Sequencing (HTS). When the coding regions of the six new PRSV genomic sequences obtained via HTS were compared with those of 54 other complete PRSV sequences from other parts of the world, all six grouped together with the 12 northern Australian sequences within major phylogroup B minor phylogroup I, the Australian sequences coming from three widely dispersed locations spanning the north of the continent. Notably, none of the PNG isolates grouped with genomic sequences from the nearby country of East Timor in phylogroup A. The closest genetic match between Australian and PNG sequences was a nucleotide (nt) sequence identity of 96.9%, whereas between PNG and East Timorese isolates it was only 83.1%. These phylogenetic and nt identity findings demonstrate genetic connectivity between PRSV populations from PNG and Australia. Recombination analysis of the 60 PRSV sequences available revealed evidence of 26 recombination events within 18 isolates, only four of which were within major phylogroup B and none of which were from PNG or Australia. Within the recombinant genomes, the P1, Cl, NIa-Pro, NIb, 6K2, and 5'UTR regions contained the highest numbers of recombination breakpoints. After removal of nonrecombinant sequences, four minor phylogroups were lost (IV, VII, VIII, XV), only one of which was in phylogroup B. When genome regions from which recombinationally derived tracts of sequence were removed from recombinants prior to alignment with nonrecombinant genomes, seven previous minor phylogroups within major phylogroup A, and two within major phylogroup B, merged either partially or entirely forming four merged minor phylogroups. The genetic connectivity between PNG and northern Australian isolates and absence of detectable recombination within either group suggests that PRSV isolates from East Timor, rather than PNG, might pose a biosecurity threat to northern Australian agriculture should they prove more virulent than those already present.

Sequence diversity studies of papaya ringspot virus isolates in South India reveal higher variability and recombination in the 5'-terminal gene sequences.

Papaya ringspot virus (PRSV) is one of the most devastating viruses which causes huge damage to papaya plantations across the globe. PRSV is a positive sense RNA virus encoding for a polyprotein that is processed into ten proteins. In this study for the first time we analyzed the variability for 15 PRSV isolates from a selected geographical region of a South Indian state Karnataka, which is under intensive papaya cultivation. Variability studies were done for two genes at the 5' end of the viral genome, namely P1 and helper component proteinase (Hc-Pro) and towards the 3' end, a 788nt overlapping region of nuclear inclusion B (NIb, 692nt) and of capsid protein (CP, 96nt), referred as NIb-CP. Our studies indicate that the P1 is most variable region with a wider range of sequence identity, followed by Hc-Pro, while the 788nt of NIb-CP was most conserved. P1 also showed maximum recombination events followed by Hc-Pro, whereas NIb-CP did not show any recombination. Further, the pattern and number of phylogenetic clusters was variable for each of the three genomic regions of PRSV isolates. Estimation of selection pressure for all the three PRSV genomic regions indicated negative and purifying selection.

Biological and molecular evidence of papaya ring spot virus pathotype P from mid-hills of Meghalaya, India

Papaya ring spot virus (PRSV) is the limiting factor of papaya production in India irrespective of agro-climatic conditions and it may cause crop losses up to 85–90%. In this study, diagnostic survey on PRSV was conducted in mid-hills of Ri-Bhoi district, Meghalaya, India during April to June, 2015. Reverse transcription polymerase chain reaction assay detected PRSV infection in 38 of 53 samples collected from surveyed locations with an average incidence of 71.7%. The representative isolate (PRSV-P:Umiam) from mid-hill Meghalaya was maintained in healthy papaya seedlings by standard sap inoculation method and typical symptoms were expressed within 21–24 days post inoculation. Studies on the physical properties revealed that the dilution end point of PRSV-P:Umiam at 10−2, longevity in vitro for 18 h and thermal inactivation point in between 50 and 55 °C. Among the cucurbitaceous hosts, pumpkin (Cucurbita pepo) alone exhibited chlorotic lesions on leaves within 20–25 days post inoculation. The partial coat protein (CP 750 bp) of PRSV-P:Umiam gene shared maximum nucleotide (~ 95%) and amino acid (~ 98%) identity with PRSV isolates reported from northern and eastern India. The CP based phylogeny clustered PRSV-P:Umiam with the PRSV isolates from north and east India.

Penggunaan Pelacak DNA untuk Deteksi Papaya ringspot virus dengan Metode Hibridisasi Asam Nukleat

Use of DNA Probe for Detection of Papaya ringspot virus Using Nucleic Acid Hybridization MethodPapaya ringspot caused by Papaya ringspot virus (PRSV) is one of the most destructive diseases of papaya. The disease had not been found in Indonesia, until disease outbreak in Nangroe Aceh Darussalam was reported in 2012. Since then, the disease spread rapidly in most papaya growing areas in Sumatera, Java and Bali. Papaya ringspot virus (PRSV) is generally detected using serological or polymerase chain reaction methods. Improvement in detection method is necessary to facilitate a more reliable tool for controlling the spread of PRSV. The aim of the research was to construct DNA probe for development of detection method based on nucleic acid hybridization. Molecular characterization based on HCPro gene sequence indicated high homology (97.88 to 99.05%) among PRSV isolates from Boyolali (Central Java), Medan (North Sumatera), Sleman (Yogyakarta) and Tabanan (Bali). Two DNA clones of HCPro gene were selected for probe construction and the probes were then labeled using PCR DIG-dioxigenin. Optimization of nucleic acid dot blot hybridization method to achieve strongest positive reaction was developed, i.e. using stringency washes at 1×SSC, 0.1% SDS, incubation at 60 oC for 15’. The DNA probe for PRSV has a high specificity and sensitifity; it could detect PRSV at the lowest concentration of nucleic acid (0.062 µg µL-1).

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.

Characterization of Papaya ringspot virus isolates infecting transgenic papaya \u2018Huanong No.1\u2019 in South China

In 2006, the release and cultivation of the genetically modified papaya cultivar ‘Huanong No.1’ successfully controlled the destructive papaya ringspot disease caused by Papaya ringspot virus (PRSV) in South China. However, some transgenic papaya plants from Guangdong and Hainan are found infected by PRSV. In this study, Field investigation was carried out and susceptible transgenic papaya samples were collected during 2012–2016. Twenty representative isolates were artificially inoculated into Cucurbita pepo and commercialised ‘Huanong No.1’ papaya, and results indicated that the plants showed obvious disease symptoms. Phylogenetic analysis of CP genes of 120 PRSV-infected isolates showed that PRSV can be divided into three groups. Isolates from Guangdong and Hainan belong to Group III, which is further divided into two subgroups. The isolates collected in this study have greatly diverged from the previously reported dominant strains Ys, Vb and Sm in South China, indicating that they belong to a new lineage. Further analysis showed a highly genetic differentiation between isolates, and 27.1% of the isolates were identified as recombinants on the basis of CP nucleotide sequences. These results indicate that the genetic variation of PRSV and the formation of the new virus lineage may explain the loss of transgenic papaya resistance in South China.

Use of RNAi technology to develop a PRSV-resistant transgenic papaya

Papaya ringspot virus (PRSV) seriously limits papaya (Carica papaya L.) production in tropical and subtropical areas throughout the world. Coat protein (CP)- transgenic papaya lines resistant to PRSV isolates in the sequence-homology-dependent manner have been developed in the U.S.A. and Taiwan. A previous investigation revealed that genetic divergence among Hainan isolates of PRSV has allowed the virus to overcome the CP-mediated transgenic resistance. In this study, we designed a comprehensive RNAi strategy targeting the conserved domain of the PRSV CP gene to develop a broader-spectrum transgenic resistance to the Hainan PRSV isolates. We used an optimized particle-bombardment transformation system to produce RNAi-CP-transgenic papaya lines. Southern blot analysis and Droplet Digital PCR revealed that line 474 contained a single transgene insert. Challenging this line with different viruses (PRSV I, II and III subgroup) under greenhouse conditions validated the transgenic resistance of line 474 to the Hainan isolates. Northern blot analysis detected the siRNAs products in virus-free transgenic papaya tissue culture seedlings. The siRNAs also accumulated in PRSV infected transgenic papaya lines. Our results indicated that this transgenic papaya line has a useful application against PRSV in the major growing area of Hainan, China.

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.

Molecular characterization of two papaya ringspot virus isolates that cause devastating symptoms in Norte de Santander, Colombia

Papaya ringspot virus is an RNA virus that belongs to the genus Potyvirus, family Potyviridae and affects both papaya and cucurbits, causing great economic losses. PRSV isolates are divided into biotypes P and W; both biotypes naturally infect plants in the family Cucurbitaceae, whereas the P type also naturally infects papaya (Carica papaya L). In the present study, we report the full-length genome sequence of two PRSV-P isolates sampled from the Campo Hermoso (PRSV-CH) and Villa del Rosario (PRSV-VR) localities in Norte de Santander, Colombia. The genomes of these PRSV isolates are 10,326 nt in length and have a predicted ORF of 3344 aa. The identity among Colombian PRSV isolates is 96.9% and 97.3% at the nucleotide and deduced amino acid levels, respectively. PRSV isolates from China had the lowest identity at 78.3% and 89.2% (nucleotide-amino acid), whereas the highest identities were detected in PRSV isolates from Mexico, Venezuela and Hawaii. At the polyprotein level, the amino acid composition surrounding the active polyprotein cleavage sites differ in the Colombian PRSV sequences. The predicted cleavage site in P1/HC-Pro is LEQY/N – LEQY/S instead of MEQY/N. Both of the Colombian PRSV isolates have a putative recombination event in the P1 coding region, which is common in all PRSV isolates from the American continent. The new full-length PRSV sequences from Colombia provide a better understanding of the dynamics of papaya ringspot virus infections in papaya in Colombia and worldwide.