Variation, Categorization and Rapid detection of the Strains of Papaya Ringspot Potyvirus in Taiwan

Papaya ringspot is the most important disease of papaya in the world. It is caused by Papaya ringspot virus (PRSV, Potyvirus, Potyviridae). Compared to traditional symptoms in papayas incited by PRSV, present symptoms observed in the fields of Taiwan seem to be significantly different. It indicates that strain variation of PRSV probably occurs in recent years. In order to investigate current PRSV strains in Taiwan, disease samples with different symptom types were collected from orchards in Pingtung. Through 3-time successive isolation of single local lesions formed in Chenopodium quinoa, three distinct strains were obtained according to their induced symptom types in papayas. The SM strain, causing leaf severe mottling, was a common strain in Taiwan in the past; the DF strain, causing severe mottling with leaf-deformation, has recently become a new dominant strain; and the SMN strain, causing necrotic severe mottling and quick decline, is considered to be the most potentially destructive strain. For comparative genomic analysis, almost full lengths of genomic sequences of 3 PRSV strains have been determined. Molecular data demonstrate that genomic sequences are somewhat different among three strains. The one-step RT-PCR assay with strain-specific primers was developed for the detection and identification of various PRSV strains. These assays were also applied to monitor PRSV replicating in different papaya cultivars such as TN2, NTU10 and NTU11. The results in the inoculation test showed that PRSV could be accurately detected by RT-PCR even during the incubation period. Both symptom observation and RT-PCR detection revealed that the NTU11 cultivar showed high tolerance to PRSV (DF strain); NTU10 showed medium tolerance; and TN2 is a susceptible cultivar. In addition, the interaction tests between PRSV DF and SMN strains were also conducted in this thesis. The results indicated that the hosts infected with both DF and SMN produced more severe symptoms than those infected with individual DF or SMN. However, the RT-PCR assays with strain-specific primers demonstrated that SMN could interfere the replication of DF when they simultaneously infected papaya hosts. On the other hand, cytopathological studies in the PRSV-infected papayas (Tainoung NO.2) were further conducted by electron microscopy. DF induces pinwheel and scroll inclusions as well as few long laminated aggregates of virions in the diseased cells; SMN induces the pinwheels, scrolls, short curved laminated aggregates as well as huge black banded inclusions near nucleus；SM induces pinwheels, scrolls and short laminated aggregates. For more economic and efficient PRSV detection, the monoclonal antibody（McAb）against PRSV was also developed in the thesis. By using purified virus preparation of DF strain from Zucchini, Three stable McAb cell lines were obtained through virus purification, mouse-immunization and hybridoma technology. Positive results were obtained in the ELISA test with McAbs for detection of purified PRSV particles, and western blot also showed that the developed McAbs could react with coat protein of PRSV. However, the ELISA test in infected papaya tissues could not obtain satisfactory results. The extraction buffer of PRSV will be further improved for the adequate application of the McAbs in PRSV detection in host plants.