Papaya Ringspot Virus Type P Research Articles

Genetic characterization of a mild isolate of papaya ringspot virus type-P (PRSV-P) and assessment of its cross-protection potential under greenhouse and field conditions.

A mild isolate of Papaya ringspot virus type-P, abbreviated as PRSV-mild, from Ecuador was sequenced and characterized. The most distinguishing symptom induced by PRSV-mild was gray powder-like leaf patches radiating from secondary veins. In greenhouse experiments, PRSV-mild did not confer durable protection against a severe isolate of the virus (PRSV-sev), obtained from the same field. Furthermore, isolate specific detection in mixed-infected plants showed that PRSV-sev becomes dominant in infections, rendering PRSV-mild undetectable at 90-120 days post superinfection. Virus testing using isolate-specific primers detected PRSV-mild in two out of five surveyed provinces, with 10% and 48% of incidence in Santo Domingo and Los Ríos, respectively. Comparative genomics showed that PRSV-mild lacks two amino acids from the coat protein region, whereas amino acid determinants for asymptomatic phenotypes were not identified. Recombination events were not predicted in the genomes of the Ecuadorean isolates. Phylogenetic analyses placed both PRSV-mild and PRSV-sev in a clade that includes an additional PRSV isolate from Ecuador and others from South America.

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.

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.

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.

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.

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%.

Identification and partial characterization of a Carica papaya-infecting isolate of Alfalfa mosaic virus in Brazil

A Carica papaya plant with severe yellow leaf mosaic, leaf distortion, and systemic necrosis was found in the municipality of Piracicaba, state of Sao Paulo, Brazil. Transmission electron microscopy (TEM) analysis revealed the presence of potyvirus-like particles and bacilliform particles similar to those of the Alfamovirus genus. The potyvirus was identified as Papaya ringspot virus-type P (PRSV-P). Biological, serological, and molecular studies confirmed the bacilliform virus as an isolate of Alfalfa mosaic virus (AMV). Partial nucleotide and amino acid sequences of the coat protein gene of this AMV isolate shared 97–98% identity with the AMV isolates in the GenBank database. This report is the first of the natural infection of papaya plants by AMV.

Protein interaction matrix of Papaya ringspot virus type P based on a yeast two-hybrid system

Comprehensive analysis of the interactions between 10 mature proteins of Papaya ringspot virus type P (PRSV-P) was carried out based on a yeast two-hybrid system assay (YTHS). We detected 6 interactions between different viral proteins (VPg-P1, VPg-P3, VPg-CI, VPg-CP, NIaPro-CI, and NIb-P3) and 4 self-interactions (HC-Pro, VPg, NIaPro, and CP). These interactions did not show the same directionality as corresponding interactions detected in other potyviruses and consequently, a protein interaction matrix displayed different patterns. This initial map of the protein interactions of PRSV-P allows further study of various viral proteins in order to develop anew strategy to control PRSV-P infection.

Potential for using Vasconcellea parviflora as a bridging species in intergeneric hybridisation between V. pubescens and Carica papaya

There have been numerous attempts to transfer Papaya ringspot virus type P (PRSV-P) resistance from wild Vasconcellea relatives to Carica papaya L. Success has been limited by the high degree of genetic divergence and thus incompatibility between Vasconcellea spp. and C. papaya. In the present work, intergeneric hybrid populations of C. papaya × V. pubescens Lenne et Koch, C. papaya × V. parviflora A. DC. and interspecific populations of V. pubescens × V. parviflora were produced and evaluated for morphological characteristics and PRSV-P resistance. F2 and F3 populations were produced from the V. pubescens × V. parviflora F1 and PRSV-P resistant individuals with homozygous genotypes (RR) were selected with the aid of a co-dominant CAPS marker and backcrossed to V. parviflora or outcrossed to C. papaya. Leaf vein number (seven) from V. pubescens, flower colour (pink) from V. parviflora and petiole colour (red–green) and plant size from C. papaya were transferred as dominant traits in interspecific and intergeneric crosses. Other traits, e.g. flower shape and fruit size, were intermediate in hybrids. When the wild species or their hybrids were outcrossed to C. papaya, inheritance patterns did not always follow Mendelian ratios, suggesting abnormal pairing of chromosomes or preferential elimination of the Vasconcellea genes. For example, in crosses between C. papaya × V. pubescens, all flowers were female and in crosses between C. papaya × V. parviflora, the cream flower colour of C. papaya was dominant over the pink colour of V. parviflora. However, the PRSV-P resistance gene from V. pubescens was successfully backcrossed into V. parviflora from F2 hybrids, and in the F1 hybrids between C. papaya and V. parviflora, 45% pollen fertility was obtained thus V. parviflora is proposed as a potential bridging species between C. papaya and V. pubescens.

APPLICATION OF SCAR MARKERS LINKED TO A PUTATIVE PRSV-P RESISTANCE LOCUS FOR ASSISTED BREEDING OF RESISTANT C. PAPAYA CULTIVARS

Papaya ringspot virus type P (PRSV-P) is a serious disease of Carica papaya. Several wild relatives of the papaya, belonging to the genus Vasconcellea, exhibit complete resistance to PRSV-P, namely V. cundinamarcensis, V. cauliflora and V. quercifolia. Molecular genetic analysis of interspecific hybrids of V. cundinamarcensis and V. parviflora using Randomly Amplified DNA Fingerprinting (RAF) permitted the construction of genetic maps of these species. Identification and mapping of several DNA markers linked to a PRSV-P resistance gene locus in V. cundinamarcensis was possible, two of which (OPK41R and OPA115R), resulted from a Single Nucleotide Polymorphism (SNP), in close proximity to the PRSV-P resistance gene locus. The Sequence Characterised Amplified Regions (SCAR's), were shown to be present in both parents, and produced codominant markers, which were applied as reliable diagnostic markers for resistant and susceptible genotypes. The OPK41R SCAR was identified in 4 additional Vasconcellea species made available, including V. cauliflora and V. quercifolia, but not in C. papaya. The indel identified may be useful for the selection of PRSV-P resistant individuals in breeding programs with Vasconcellea and C. papaya to assist introgression of putative resistance genes. The markers were subsequently applied to an intergeneric hybrid population from C. papaya and V. quercifolia. Genetic characterisation of the hybrid population using RAF markers confirmed hybridity of the F1 and reduced V. quercifolia DNA content of back cross generations. The OPK41R and OPA115R SCARs were tested in the quercifolia hybrids, and did not identify linkage to PRSV-P resistance. From the results it can be inferred that Vasconcellea may contain multiple sources of resistance to PRSV-P. Overall these studies indicate that there is significant potential for the continued application of DNA markers in molecular breeding for PRSV-P resistant papaya genotypes.

THE APPLICATION OF BIOTECHNOLOGY IN AN INTEGRATED PROJECT OF CONSERVATION AND UTILIZATION OF PAPAYA AND ITS WILD RELATIVES

Aosiraci Carica papaya is an important crop species throughout tropical and subtropical regions of the world, including northern regions of Australia. Biotechnology is relatively advanced in papaya including techniques for micropropagation, embryogenesis, embryo rescue, transformation and the application of molecular markers. Plant improvement projects are underpinned by such technologies but also require a range of germplasm as a source of new and useful agronomic traits. A collection of valuable germplasm in the form of superior cropping genotypes of papaya and wild relatives of papaya, the Vasconcella spp., are being used in a range of plant improvement projects in our laboratories. The Vasconcella spp. are a particularly rich source of agronomically important traits, including resistance to papaya ringspot virus type P (PRSV-P), phytophthora, phytoplasma and blackspot. Intergeneric hybrids between wild relatives and papaya are now segregating for PVSP-P and molecular mapping and marker technologies are assisting with further selection of useful hybrid plants. The long-term conservation and availability of this range of germplasm is critical to sustainable utilization. Tropical fruits are typically difficult to store as seed and we are currently involved in international collaborations to develop alternative conservation technologies for tropical fruits, including papaya. This paper reports on advances in seed storage technologies, slow-growth storage and cryopreservation of both shoot tips and seeds as alternatives for use across a range of papaya genotypes for long-term ex situ conservation.

Development of a codominant CAPS marker linked to PRSV-P resistance in highland papaya

Development of resistant papaya varieties is widely considered the best strategy for long-term control of the papaya ringspot virus type P (PRSV-P). Several species of "highland papaya" from the related genus Vasconcellea exhibit complete resistance to PRSV-P, and present a valuable source of resistance genes with potential for application in Carica papaya. The objectives of this study were two fold; to identify molecular markers linked to a previously characterised PRSV-P resistance gene in V. cundinamarcensis (psrv-1), and to develop codominant marker based strategies for reliable selection of PRSV-P resistant genotypes. Using a bulked segregant analysis approach, dominant randomly amplified DNA fingerprint (RAF) markers linked to prsv-1 were revealed in the resistant DNA bulk, which comprised F2 progeny from a V. parviflora (susceptible) x V. cundinamarcensis (resistant) interspecific cross. One marker, Opk4_1r, mapped adjacent to the prsv-1 locus at 5.4 cM, while a second, Opa11_5r, collocated with it. Sequence characterisation of the Opk4_1r marker permitted its conversion into a codominant CAPS marker (PsiIk4), diagnostic for the resistant genotype based on digestion with the restriction endonuclease PsiI. This marker mapped within 2 cM of the prsv-1 locus. Psilk4 was shown to correctly identify resistant genotypes 99% of the time when applied to interspecific F2 progeny segregating for the resistant character, and has potential for application in breeding programs aimed to deliver the PRSV-P resistance gene from V. cundinamarcensis into C. papaya.

Progress in backcrossing between Carica papaya × Vasconcellea quercifolia intergeneric hybrids and C. papaya

Papaya is a major tropical fruit crop worldwide, however, all genotypes are susceptible to Papaya ringspot virus type P (PRSV-P). Protocols have been developed to produce large numbers of intergeneric hybrids between Carica papaya L. and Vasconcellea quercifolia, which is PRSV-P resistant. Although pollen fertility of these hybrids was low, backcross populations were produced in both Australia and the Philippines when papaya flowers were pollinated with pollen from 11 hybrid plants that had been identified as having some pollen fertility and were PRSV-P resistant. Plantlets were produced after embryo rescue and culture in vitro. Second backcross generations (BC2) were easier to produce than first backcross (BC1) generations as pollen fertility was >80% in male BC1 plants. Variation in time to PRSV-P symptom expression and severity of virus symptoms in the backcross generations was observed in both countries. Commercially acceptable fruit were produced in the BC2 generation. The results demonstrate that efforts in wide hybridisation to transfer PRSV-P resistance to C. papaya, are better directed towards crosses between C. papaya and V. quercifolia than with other Vasconcellea species.

Breeding for Papaya ringspot virus resistance in Carica papaya via hybridisation with Vasconcellea quercifolia

Papaya ringspot virus type P (PRSV-P) is a major threat to the papaya industry worldwide. F1 hybrids have been produced when Carica papaya L. female flowers have been pollinated with pollen of the PRSV-P resistant species Vasconcellea quercifolia. F1 plant production required embryo rescue 90 days post-pollination, and plantlet regeneration in vitro. Three hundred F1 hybrids were grown to maturity in the field and had morphological characteristics that were identical to 1 or both parents, were intermediate between those of the parents, or were greater than either parent. They had a sex ratio of 2 (male) : 49 (hermaphrodite) : 49(female). Eighteen plants (7 male and 11 hermaphrodite) produced some viable pollen. Pollen viability of these plants as measured by germination on agar medium varied between 1.1 and 6.1% (mean of 3.37 ± 0.18) compared with >90% for C. papaya. Cytogenetic studies showed limited homology between C. papaya and V. quercifolia genomes. The intergeneric hybrids varied in their reaction to strains of PRSV-P in Australia and the Philippines. Of the hybrids, 75% were resistant to PRSV-P while 25% produced virus symptoms.

PAPAYA RINGSPOT VIRUS IN AUSTRALIA AND THE DEVELOPMENT OF VIRUS RESISTANT PLANTS

Papaya is a popular fruit crop in many tropical and subtropical regions. It grows quickly and is capable of bearing large crops. Because of extensive monoculture and a narrow gene-pool, papaya plants are susceptible to numerous diseases. Papaya ringspot virus, type P (PRSV-P), causes the most widespread of these diseases. Although it is not currently a major problem in Australia, PRSV-P dramatically reduces yield in many countries. Vasconcellea quercifolia is one of four wild Carica species that are known to be PRSV-P resistant. Hybrids of these species grow vigorously in the field. Males, females and hermaphrodites have been identified, and a few male hybrids exhibited some pollen fertility. A backcross generation has been produced. One plant was fertile and showed resistance to PRSV-P. Transgenic plants containing viral coat protein constructs have also shown resistance to PRSV-P in field plantings. In this paper, progress toward achieving PRSV-P resistance for papaya using both approaches is examined.

Genetic mapping of a PRSV-P resistance gene in “highland papaya” based on inheritance of RAF markers

Papaya ringspot virus type P (PRSV-P) is a significant disease of Carica papaya. A major gene for PRSV-P resistance has been mapped in Vasconcellea cundinamarcensis, a distant relative of C. papaya. This was achieved by genetic mapping of the resistance phenotype and inherited, dominant, polymorphic randomly amplified DNA fingerprint (RAF) markers in F2 progenies of V. parviflora and V. cundinamarcensis. The parents of this cross confer resistance to several major diseases that affect C. papaya including PRSV-P in V. cundinamarcensis. Heredity of DNA markers and PRSV-P resistance was studied in the intrageneric population presented due to intergeneric fertility barriers between Carica and Vasconcellea. Genetic polymorphism between parents, based on RAF markers, was 75% with more than 70% of markers generated showing mendelian segregation for the expected ratios 1:3 or 1:1 (p < 0.05). Preferential inheritance of markers from either parent was not detected in the F2, indicating stable transfer of the genetic material. Discrete V. parviflora and V. cundinamarcensis linkage maps were compiled from 79 and 83 framework markers, delineating to 10 and 11 groups respectively. F1 and F2 progeny were screened for resistance to PRSV-P under controlled conditions. The resistant phenotype segregated 3:1 in the F2 and mapped to V. cundinamarcensis linkage group 7 with adjacent RAF markers within 4 cM. The framework maps of V. parviflora and V. cundinamarcensis presented cover 630.2 and 745.4 cM respectively, accounting for between 47–52 and 49–55 percent of the predicted genome lengths. These maps provide a platform for further genetic study of disease resistance characteristics identified in these species and the development of DNA markers tightly linked to these traits, which could be applied to the breeding of resistant C. papaya cultivars.

DEVELOPMENT OF CARICA INTERSPECIFIC HYBRIDS

Papaw production in Australia is limited by five major diseases: papaw dieback and yellow crinkle (caused by mycoplasmas); Phytophthora fruit, stem and root rots; black spot (Asperisporium caricae); and papaya ringspot virus-type P (PRSV-P). PRSV-P is the greatest problem for papaya production worldwide and potentially poses the greatest disease threat to the Australian industry. Procedures have been developed to hybridise papaya with related Carica species that are PRSV-P resistant (C. cauliflora, C. quercifolia, and C. pubescens). These procedures may also allow access to other characteristics of wild species eg. Phytophthora resistance (C. goudotiana), high sugar content (C. quercifolia) and cold tolerance (C. pubescens). A highly efficient protocol was developed to rescue and germinate C. papaya x C. cauliflora immature embryos (Magdalita, et ad. 1996). Germinated embryos formed embryogenic cultures on hormone free agar solidified medium and multiple hybrid plants were produced. The C. papaya x C. cauliflora hybrids lacked vigour and were generally infertile. Subsequently, the protocol has been adapted to produce hybrids between C. papaya and PRSV-P resistant species C. quercifolia and C. pubescens. Hybrid plants are growing vigorously in the field, and a few C. papaya x C. quercifolia plants have produced some viable pollen. Interspecific hybrid plants have been produced between C. papaya, and C. goudotiana and C. parviflora. C, parviflora has been hybridised with C. pubescens and C. goudotiana. Plants of all crosses are growing in the glasshouse or field.

Screening Carica papaya × C. cauliflora hybrids for resistance to papaya ringspot virus‐type P

Carica papaya, C. cauliflora and interspecific hybrids of these species were screened for resistance to two Australian isolates (338, 445) of papaya ringspot virus‐type P (PRSV‐P). Plants were manually inoculated with PRSV‐P in the glasshouse and the reaction assessed 30 days later by back‐inoculation to susceptible Cucurbita pepo and by a plate‐trapped antigen‐enzyme linked immunosorbent assay (PTA‐ELISA). Both parents and interspecific hybrids were also planted adjacent to infected C. papaya and 30 days later tested for PRSV‐P by PTA‐ELISA. All interspecific hybrid and C. cauliflora plants manually inoculated in the glasshouse or planted in the field failed to become infected, whereas C. papaya plants, in both situations, were infected by PRSV‐P. In addition, the surviving interspecific hybrid and C. cauliflora plants tested negative, while all C. papaya plants were positive for PRSV‐P in both the back‐inoculation and PTA‐ELISA tests. Thus, the interspecific hybrid and C. cauliflora plants were resistant to the Australian PRSV‐P isolates.