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.

Survey, Detection, Characterization of Papaya Ringspot Virus from Southern India and Management of Papaya Ringspot Disease.

Papaya ringspot virus (PRSV) is a significant threat to global papaya cultivation, causing ringspot disease, and it belongs to the species Papaya ringspot virus, genus Potyvirus, and family Potyviridae. This study aimed to assess the occurrence and severity of papaya ringspot disease (PRSD) in major papaya-growing districts of Karnataka, India, from 2019 to 2021. The incidence of disease in the surveyed districts ranged from 50.5 to 100.0 percent, exhibiting typical PRSV symptoms. 74 PRSV infected samples were tested using specific primers in RT-PCR, confirming the presence of the virus. The complete genome sequence of a representative isolate (PRSV-BGK: OL677454) was determined, showing the highest nucleotide identity (nt) (95.8%) with the PRSV-HYD (KP743981) isolate from Telangana, India. It also shared an amino acid (aa) identity (96.5%) with the PRSV-Pune VC (MF405299) isolate from Maharashtra, India. Based on phylogenetic and species demarcation criteria, the PRSV-BGK isolate was considered a variant of the reported species and designated as PRSV-[IN:Kar:Bgk:Pap:21]. Furthermore, recombination analysis revealed four unique recombination breakpoint events in the genomic region, except for the region from HC-Pro to VPg, which is highly conserved. Interestingly, more recombination events were detected within the first 1710 nt, suggesting that the 5' UTR and P1 regions play an essential role in shaping the PRSV genome. To manage PRSD, a field experiment was conducted over two seasons, testing various treatments, including insecticides, biorationals, and a seaweed extract with micronutrients, alone or in combination. The best treatment involved eight sprays of insecticides and micronutrients at 30-day intervals, resulting in no PRSD incidence up to 180 days after transplanting (DAT). This treatment also exhibited superior growth, yield, and yield parameters, with the highest cost-benefit ratio (1:3.54) and net return. Furthermore, a module comprising 12 sprays of insecticides and micronutrients at 20-day intervals proved to be the most effective in reducing disease incidence and enhancing plant growth, flowering, and fruiting attributes, resulting in a maximized yield of 192.56 t/ha.

Modification of Papaya Ringspot Virus HC-Pro to Generate Effective Attenuated Mutants for Overcoming the Problem of Strain-Specific Cross Protection.

Cross protection application of HA5-1, an attenuated mutant of papaya ringspot virus (PRSV) HA strain from Hawaii, was withdrawn from Taiwan due to the narrow geographic strain specificity of HA5-1. Here, to overcome this problem, we created attenuated mutants of PRSV YK, a dominant severe strain from Taiwan, by mutating helper component protease (HC-Pro) at F7, R181, F206, and D397 residues critical for potyviral pathogenicity. PRSV YK HC-Pro R181I, F206L, and D397N single-mutant viruses induced mild symptoms, but their adverse effects on growth of papaya plants disqualified them as useful protective viruses. However, F7I single-mutant and F7I + F206L double-mutant viruses displayed mild symptoms followed by recovery, and they showed a zigzag pattern of accumulation in papaya plants, indicating their potential to trigger RNA silencing and retain partial antagonistic suppression of host defense. Although F7I + R181I and F7I + D397N double-mutant viruses caused symptomless infection, they accumulated barely above mock level and, thus, were not qualified as proper protective viruses. RNA silencing suppression (RSS) analysis by agroinfiltration in Nicotiana benthamiana plants revealed that the HC-Pro F7I and F7I + F206L mutant proteins were weaker in RSS ability than the wild-type protein. Under greenhouse conditions, F7I and F7I + F206L mutant viruses were genetically stable but not aphid transmissible. Compared with the HA5-1 mutant's low degree (10%) of protection to papaya plants, the F7I and F7I + F206L mutants provided complete (100%) protection to papaya and horn melon plants against strain YK. Thus, F7I and F7I + F206L mutants solve the problem of strain-specific protection and have great potential for control of PRSV in Taiwan.

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.

Generation of Mild Recombinants of Papaya Ringspot Virus to Minimize the Problem of Strain-Specific Cross-Protection.

Papaya ringspot virus (PRSV) causes severe damage to papaya (Carica papaya L.) and is the primary limiting factor for papaya production worldwide. A nitrous acid-induced mild strain, PRSV HA 5-1, derived from Hawaii strain HA, has been applied to control PRSV by cross-protection for decades. However, the problem of strain-specific protection hampers its application in Taiwan and other geographic regions outside Hawaii. Here, sequence comparison of the genomic sequence of HA 5-1 with that of HA revealed 69 nucleotide changes, resulting in 31 aa changes, of which 16 aa are structurally different. The multiple mutations of HA 5-1 are considered to result from nitrous acid induction because 86% of nucleotide changes are transition mutations. The stable HA 5-1 was used as a backbone to generate recombinants carrying individual 3' fragments of Vietnam severe strain TG5, including NIa, NIb, and CP3' regions, individually or in combination. Our results indicated that the best heterologous fragment for the recombinant is the region of CP3', with which symptom attenuation of the recombinant is like that of HA 5-1. This mild recombinant HA51/TG5-CP3' retained high levels of protection against the homologous HA in papaya plants and significantly increased the protection against the heterologous TG-5. Similarly, HA 5-1 recombinants carrying individual CP3' fragments from Thailand SMK, Taiwan YK, and Vietnam ST2 severe strains also significantly increase protection against the corresponding heterologous strains in papaya plants. Thus, our recombinant approach for mild strain generation is a fast and effective way to minimize the problem of strain-specific protection.

Interspecific Recombination Between Zucchini Tigre Mosaic Virus and Papaya Ringspot Virus Infecting Cucurbits in China.

Recombination drives evolution of single-stranded RNA viruses and contributes to virus adaptation to new hosts and environmental conditions. Intraspecific recombinants are common in potyviruses, the largest family of single-stranded RNA viruses, whereas interspecific recombinants are rare. Here, we report an interspecific recombination event between papaya ringspot potyvirus (PRSV) and zucchini tigre mosaic potyvirus (ZTMV), two potyviruses infecting cucurbit crops and sharing similar biological characteristics and close phylogenetic relationship. The PRSV-ZTMV recombinants were detected through small RNA sequencing of viruses infecting cucurbit samples from Guangxi and Henan provinces of China. The complete nucleotide (nt) sequences of the interspecific recombinant viruses were determined using overlapping RT-PCR. Multiple sequence alignment, recombination detection analysis and phylogenetic analysis confirmed the interspecific recombination event, and revealed an additional intraspecific recombination event among ZTMV populations in China. The symptoms and host ranges of two interspecific recombinant isolates, KF8 and CX1, were determined through experimental characterization using cDNA infectious clones. Surveys in 2017 and 2018 indicated that the incidences of the interspecific recombinant virus were 16 and 19.4%, respectively, in cucurbits in Kaifeng of Henan province. The identified interspecific recombinant virus between PRSV and ZTMV and the novel recombination pattern with the recombination site in HC-pro in potyvirid provide insights into the prevalence and evolution of ZTMV and PRSV in cucurbits.

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 788 nt overlapping region of nuclear inclusion B (NIb, 692 nt) and of capsid protein (CP, 96 nt), 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 788 nt 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.

Porcine circovirus type 2 protective epitope densely carried by chimeric papaya ringspot virus-like particles expressed in Escherichia coli as a cost-effective vaccine manufacture alternative.

Porcine circovirus type 2 (PCV2) still represents a major problem to the swine industry worldwide, causing high mortality rates in infected animals. Virus-like particles (VLPs) have gained attention for vaccine development, serving both as scaffolds for epitope expression and immune response enhancers. The commercial subunit vaccines against PCV2 consist of VLPs formed by the self-assembly of PCV2 capsid protein (CP) expressed in the baculovirus vector system. In this work, a PCV2 protective epitope was inserted into three different regions of papaya ringspot virus (PRSV) CP, namely, the N- and C-termini and a predicted antigenic region located near the N-terminus. Wild-type and chimeric CPs were modeled in silico, expressed in Escherichia coli, purified, and visualized by transmission electron microscopy. This is the first report that shows the formation of chimeric VLPs using PRSV as epitope-presentation scaffold. Moreover, it was found that PCV2 epitope localization strongly influences VLP length. Also, the estimated yields of the chimeric VLPs at a small-scale level ranged between 65 and 80 mg/L of culture medium. Finally, the three chimeric VLPs induced high levels of immunoglobulin G against the PCV2 epitope in immunized BALB/c mice, suggesting that these chimeric VLPs can be used for swine immunoprophylaxis against PCV2.

Evaluation of the Immunogenicity of a Potyvirus-Like Particle as an Adjuvant of a Synthetic Peptide.

Improvement of current vaccines is highly necessary to increase immunogenicity levels and protection against several pathogens. Virus-like particles (VLPs) are promising approaches for vaccines because they emulate infectious virus structure, but lack any genetic material needed for replication. Plant viruses have emerged as a potential framework for VLP design, mainly because there is no preexisting immunity in mammals. In this study, we evaluated the scaffold of the papaya ringspot virus (PRSV) as a VLP adjuvant for a short synthetic peptide derived from the Hemagglutinin protein of AH1 N1 influenza virus-hemagglutinin (VLP-HA). Our results demonstrated that the adjuvant property of this VLP is highly similar to the trivalent influenza vaccine, showing comparable levels of IgG- and IgA-specific antibodies to HA-derived peptide in serum and feces of vaccinated mice, respectively. Furthermore, VLP-HA-immunized mice showed Th1-biased immune response as suggested by measuring IgG subclasses in comparison with the predominance of Th2-biased immune response in trivalent influenza vaccine dose-vaccinated mice. VLP-HA administration in mice induced comparable levels of activated CD4+- and CD8+-specific T lymphocytes for the HA-derived peptide. These results suggest the potential adjuvant capacity of the PRSV-VLP as a carrier for short synthetic peptides.

Transcriptome Profiling Revealed Stress-Induced and Disease Resistance Genes Up-Regulated in PRSV Resistant Transgenic Papaya.

Papaya is a productive and nutritious tropical fruit. Papaya Ringspot Virus (PRSV) is the most devastating pathogen threatening papaya production worldwide. Development of transgenic resistant varieties is the most effective strategy to control this disease. However, little is known about the genome-wide functional changes induced by particle bombardment transformation. We conducted transcriptome sequencing of PRSV resistant transgenic papaya SunUp and its PRSV susceptible progenitor Sunset to compare the transcriptional changes in young healthy leaves prior to infection with PRSV. In total, 20,700 transcripts were identified, and 842 differentially expressed genes (DEGs) randomly distributed among papaya chromosomes. Gene ontology (GO) category analysis revealed that microtubule-related categories were highly enriched among these DEGs. Numerous DEGs related to various transcription factors, transporters and hormone biosynthesis showed clear differences between the two cultivars, and most were up-regulated in transgenic papaya. Many known and novel stress-induced and disease-resistance genes were most highly expressed in SunUp, including MYB, WRKY, ERF, NAC, nitrate and zinc transporters, and genes involved in the abscisic acid, salicylic acid, and ethylene signaling pathways. We also identified 67,686 alternative splicing (AS) events in Sunset and 68,455 AS events in SunUp, mapping to 10,994 and 10,995 papaya annotated genes, respectively. GO enrichment for the genes displaying AS events exclusively in Sunset was significantly different from those in SunUp. Transcriptomes in Sunset and transgenic SunUp are very similar with noteworthy differences, which increased PRSV-resistance in transgenic papaya. No detrimental pathways and allergenic or toxic proteins were induced on a genome-wide scale in transgenic SunUp. Our results provide a foundation for unraveling the mechanism of PRSV resistance in transgenic papaya.

Simultaneous detection of papaya ringspot virus, papaya leaf distortion mosaic virus, and papaya mosaic virus by multiplex real-time reverse transcription PCR.

Both the single infection of papaya ringspot virus (PRSV), papaya leaf distortion mosaic virus (PLDMV) or papaya mosaic virus (PapMV) and double infection of PRSV and PLDMV or PapMV which cause indistinguishable symptoms, threaten the papaya industry in Hainan Island, China. In this study, a multiplex real-time reverse transcription PCR (RT-PCR) was developed to detect simultaneously the three viruses based on their distinctive melting temperatures (Tms): 81.0±0.8°C for PRSV, 84.7±0.6°C for PLDMV, and 88.7±0.4°C for PapMV. The multiplex real-time RT-PCR method was specific and sensitive in detecting the three viruses, with a detection limit of 1.0×10(1), 1.0×10(2), and 1.0×10(2) copies for PRSV, PLDMV, and PapMV, respectively. Indeed, the reaction was 100 times more sensitive than the multiplex RT-PCR for PRSV, and 10 times more sensitive than multiplex RT-PCR for PLDMV. Field application of the multiplex real-time RT-PCR demonstrated that some non-symptomatic samples were positive for PLDMV by multiplex real-time RT-PCR but negative by multiplex RT-PCR, whereas some samples were positive for both PRSV and PLDMV by multiplex real-time RT-PCR assay but only positive for PLDMV by multiplex RT-PCR. Therefore, this multiplex real-time RT-PCR assay provides a more rapid, sensitive and reliable method for simultaneous detection of PRSV, PLDMV, PapMV and their mixed infections in papaya.

Differential Life History Trait Associations of Aphids with Nonpersistent Viruses in Cucurbits.

The diversity of vectors and fleeting nature of virus acquisition and transmission renders nonpersistent viruses a challenge to manage. We assessed the importance of noncolonizing versus colonizing vectors with a 2-yr survey of aphids and nonpersistent viruses on commercial pumpkin farms. We quantified aphid alightment using pan traps, while testing leaf samples with multiplex RT-PCR targeting cucumber mosaic virus (CMV), zucchini yellow mosaic virus (ZYMV), watermelon mosaic virus (WMV), and papaya ringspot virus (PRSV). Overall, we identified 53 aphid species (3,899 individuals), from which the melon aphid, Aphis gossypii Glover, a pumpkin-colonizing species, predominated (76 and 37% of samples in 2010 and 2011, respectively). CMV and ZYMV were not detected, but WMV and PRSV were prevalent, both regionally (WMV: 28/29 fields, PRSV: 21/29 fields) and within fields (infection rates = 69 and 55% for WMV in 2010 and 2011; 28 and 25% for PRSV in 2010 and 2011). However, early-season samples showed extremely low infection levels, suggesting cucurbit viruses are not seed-transmitted and implicating aphid activity as a causal factor driving virus spread. Interestingly, neither noncolonizer and colonizer alightment nor total aphid alightment were good predictors of virus presence, but community analyses revealed species-specific relationships. For example, cowpea aphid (Aphis craccivora Koch) and spotted alfalfa aphid (Therioaphis trifolii Monell f. maculata) were associated with PRSV infection, whereas the oleander aphid (Aphis nerii Bover de Fonscolombe) was associated with WMV spread within fields. These outcomes highlight the need for tailored management plans targeting key vectors of nonpersistent viruses in agricultural systems.

Functional Characterization of Cucumis metuliferus Proteinase Inhibitor Gene (CmSPI) in Potyviruses Resistance.

Proteinase inhibitors are ubiquitous proteins that block the active center or interact allosterically with proteinases and are involved in plant physiological processes and defense responses to biotic and abiotic stresses. The CmSPI gene identified from Cucumis metuliferus encodes a serine type PI (8 kDa) that belongs to potato I type family. To evaluate the effect of silencing CmSPI gene on Papaya ringspot virus resistance, RNA interference (RNAi) with an inter-space hairpin RNA (ihpRNA) construct was introduced into a PRSV-resistant C. metuliferus line. CmSPI was down-regulated in CmSPI RNAi transgenic lines in which synchronously PRSV symptoms were evident at 21 day post inoculation. Alternatively, heterogeneous expression of CmSPI in Nicotiana benthamiana was also conducted and showed that CmSPI can provide resistance to Potato virus Y, another member of Potyvirus, in transgenic N. benthamiana lines. This study demonstrated that CmSPI plays an important role in resistant function against potyviruses in C. metuliferus and N. benthamiana.

Reverse transcription loop-mediated isothermal amplification assay for rapid detection of Papaya ringspot virus.

Papaya ringspot virus (PRSV) and Papaya leaf distortion mosaic virus (PLDMV), which causes disease symptoms similar to PRSV, threaten commercial production of both non-transgenic-papaya and PRSV-resistant transgenic papaya in China. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to detect PLDMV was developed previously. In this study, the development of another RT-LAMP assay to distinguish among transgenic, PRSV-infected and PLDMV-infected papaya by detection of PRSV is reported. A set of four RT-LAMP primers was designed based on the highly conserved region of the P3 gene of PRSV. The RT-LAMP method was specific and sensitive in detecting PRSV, with a detection limit of 1.15×10(-6)µg of total RNA per reaction. Indeed, the reaction was 10 times more sensitive than one-step RT-PCR. Field application of the RT-LAMP assay demonstrated that samples positive for PRSV were detected only in non-transgenic papaya, whereas samples positive for PLDMV were detected only in commercialized PRSV-resistant transgenic papaya. This suggests that PRSV remains the major limiting factor for non-transgenic-papaya production, and the emergence of PLDMV threatens the commercial transgenic cultivar in China. However, this study, combined with the earlier development of an RT-LAMP assay for PLDMV, will provide a rapid, sensitive and cost-effective diagnostic power to distinguish virus infections in papaya.

Physicochemical and biochemical characterization of transgenic papaya modified for protection against Papaya ringspot virus.

BACKGROUND: Papaya, a nutritious tropical fruit, is consumed both in its fresh form and as a processed product worldwide. Major quality indices which include firmness, acidity, pH, colour and size, are cultivar dependent. Transgenic papayas engineered for resistance to Papaya ringspot virus were evaluated over the ripening period to address physicochemical quality attributes and food safety concerns. RESULTS: With the exception of one transgenic line, no significant differences (P > 0.05) were observed in firmness, acidity and pH. Lightness (L*) and redness (a*) of the pulps of non-transgenic and transgenic papaya were similar but varied over the ripening period (P < 0.05). Fruit mass, though non-uniform (P < 0.05) for some lines, was within the range reported for similar papaya cultivars, as were shape indices of female fruits. Transgene proteins, CP and NPTII, were not detected in fruit pulp at the table-ready stage. CONCLUSION: The findings suggest that transformation did not produce any major unintended alterations in the physicochemical attributes of the transgenic papayas. Transgene proteins in the edible fruit pulp were low or undetectable.

Genetic Diversity Based on Coat Protein of Papaya ringspot virus (Pathotype P) Isolates from Bangladesh.

The coat protein (CP) sequences of twelve Papaya ringspot virus (PRSV) (pathotype-P) isolates from six major papaya growing areas were determined and compared with those of published PRSV. The CP coding region varied in size from 846-852 nucleotides, encoding a protein of 282-284 amino acids. Comparative CP sequence analysis revealed that the PRSV-P isolates originating from Bangladesh were divergent up to 14 % at amino acids level. Further, the isolates from Bangladesh shared 86-95 % amino acid sequence identity with those reported from rest 21 of the Asia and 83-93 % amino acid sequence identity with isolates from the other parts of the world. A number of KE repeats were observed in the N terminus of the CP coding region of all Bangladesh isolates. Phylogenetic branching pattern revealed that the PRSV-P isolates originating from Bangladesh formed a distinct clade from those from the rest of the world. This forms the first report on the genetic diversity of PRSV-P isolates from Bangladesh.

Combining ability of summer-squash lines with different degrees of parthenocarpy and PRSV-W resistance.

The aim was to assess heterosis in a set of 16 summer-squash hybrids, and evaluate the combining capacity of the respective parental lines, which differed as to the degree of parthenocarpy and resistance to PRSV-W (Papaya Ringspot Virus-Watermelon strain). The hybrids were obtained using a partial diallel cross design (4 × 4). The lines of parental group I were 1 = ABX-037G-77-03-05-01-01-bulk, 2 = ABX-037G-77-03-05-03-10-bulk, 3 = ABX-037G-77-03-05-01-04-bulk and 4 = ABX-037G-77-03-05-05-01-bulk, and of group II, 1' = ABX-037G-77-03-05-04-08-bulk, 2' = ABX-037G-77-03-05-02-11-bulk, 3' = Clarice and 4' = Caserta. The 16 hybrids and eight parental lines were evaluated for PRSV-W resistance, parthenocarpic expression and yield in randomized complete-block designs, with three replications. Parthenocarpy and the resistance to PRSV-W were rated by means of a scale from 1 to 5, where 1 = non-parthenocarpic or high resistance to PRSV-W, and 5 = parthenocarpic or high susceptibility to PRSV-W. Both additive and non-additive gene effects were important in the expression of parthenocarpy and resistance to PRSV-W. Whereas estimates of heterosis in parthenocarpy usually tended towards a higher degree, resistance to PRSV-W was towards higher susceptibility. At least one F(1) hybrid was identified with a satisfactory degree of parthenocarpy, resistance to PRSV-W and high fruit-yield.

Combining ability of summer-squash lines with different degrees of parthenocarpy and PRSV-W resistance

The aim was to assess heterosis in a set of 16 summer-squash hybrids, and evaluate the combining capacity of the respective parental lines, which differed as to the degree of parthenocarpy and resistance to PRSV-W (Papaya Ringspot Virus-Watermelon strain). The hybrids were obtained using a partial diallel cross design (4 x 4). The lines of parental group I were 1 = ABX-037G-77-03-05-01-01-bulk, 2 = ABX-037G-77-03-05-03-10-bulk, 3 = ABX-037G77-03-05-01-04-bulk and 4 = ABX-037G-77-03-05-05-01-bulk, and of group II, 1' = ABX-037G-77-03-05-04-08-bulk, 2' = ABX-037G-77-03-05-02-11-bulk, 3' = Clarice and 4' = Caserta. The 16 hybrids and eight parental lines were evaluated for PRSV-W resistance, parthenocarpic expression and yield in randomized complete-block designs, with three replications. Parthenocarpy and the resistance to PRSV-W were rated by means of a scale from 1 to 5, where 1 = non-parthenocarpic or high resistance to PRSV-W, and 5 = parthenocarpic or high susceptibility to PRSV-W. Both additive and non-additive gene effects were important in the expression of parthenocarpy and resistance to PRSV-W. Whereas estimates of heterosis in parthenocarpy usually tended towards a higher degree, resistance to PRSV-W was towards higher susceptibility. At least one F1 hybrid was identified with a satisfactory degree of parthenocarpy, resistance to PRSV-W and high fruit-yield.