Population Analysis and Subgroup Diagnosis of Lettuce necrotic yellows virus in New Zealand
Darling, Toni Louise
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Lettuce is a key economic crop in New Zealand (NZ). However, recently NZ has experienced increased crop losses of up to 50 %. These losses were due to necrosis symptoms on leaves, which were strongly associated with the plant virus lettuce necrotic yellows virus (LNYV). This virus has also been found in Australia where heavy lettuce crop losses have also occurred since 1954. Lettuce necrotic yellows virus is the type species of the genus Cytorhabdovirus, family Rhabdoviridae, commonly referred to as rhabdoviruses. This negative sense, single stranded RNA plant virus can be differentiated into two subgroups (I and II) based on genetic differences within its nucleoprotein (N). The LNYV subgroup population structure has shown to be dissimilar between NZ and Australia. In Australia, no new isolates belonging to subgroup I have been identified since 1993. It appears this subgroup has become extinct, possibly due to subgroup II. It has been suggested subgroup II may have supplanted subgroup I due to more optimal vector and/or host interactions. However, in NZ, both subgroups are still being identified across the country. It has been suggested that the high crop losses experienced in NZ recently could be due to an increased presence of subgroup II, or a more virulent strain of the virus has arrived here from Australia. To investigate the cause of these recent losses in NZ, a closed tube, quick, sensitive and specific diagnostic assay is required. The two LNYV subgroups have not demonstrated discriminating symptoms or detectable serological differences. This has meant subgroup identification needs to occur on a molecular level. In 2018, a molecular assay to diagnose the LNYV subgroups was developed using reverse transcription polymerase chain reaction (RT-PCR). Unfortunately, the developed assay had limitations, requiring further analysis using restriction fragment length polymorphism, or for each sample to be tested twice – once for each subgroup – for confidence in the diagnosis of LNYV infection and subgroup identification. It was suggested the developed LNYV subgroup specific primers could be suitable for use in an alternative molecular assay - multiplex reverse transcription quantitative polymerase chain reaction with high resolution melting (RT-qPCR-HRM). This study has focused on the development of such an assay. First the LNYV subgroup specific primers from the previous assay were tested for their suitability in a multiplex RT-qPCR-HRM assay. Through this analysis several qPCR fluorescent dyes were assessed. Although SYBR Green fluorescent dye is commonly used, several studies have shown it to inhibit PCR and the dye molecules to translocate during HRM analysis, affecting the accuracy of the assay. This was also indicated during this study, and eventually the BioRad Sso Fast EvaGreen dye was chosen as it demonstrated superior sensitivity. In this study it was discovered that the LNYV subgroup specific primers were unable to sufficiently discriminate between the two subgroups using HRM. This meant LNYV subgroup specific primers designed specifically for use in multiplex RT-qPCR-HRM analysis needed to be developed. Three primer sets were designed and tested for their usefulness in singleplex and multiplex assays. Two primer sets appeared suitable, so were used to diagnose the LNYV subgroup of previously untyped LNYV isolates. Through this analysis, one primer set appeared able to detect the LNYV subgroup, while also distinguishing variances in the amplified PCR product sequences. These variances fit a quasi-species model, which is common among RNA viruses. However, this is the first suggestion that LNYV exists as a quasi-species. This analysis also identified, for the first time, three samples co-infected with both LNYV subgroups. The LNYV subgroup specific RT-qPCR-HRM diagnostic assay developed in this study identified subgroup I and subgroup II samples from both the North and South Islands of NZ. However, of the 18 untyped samples, only four were identified as subgroup II, with a further three identified as co-infected. This indicated that subgroup II was not exhibiting a stronger presence in NZ at this time. To assess whether a new LNYV strain from Australia had arrived in NZ additional further analysis of these isolates was required. The entire N gene from these isolates was sequenced to characterise their taxonomic and evolutionary relationships using phylogenetics. A total of 43 LNYV isolates, consisting of published and unpublished sequences, from NZ and Australia were used to assess these relationships. This analysis was unable to identify an isolate of direct Australian lineage that would indicate a stronger strain had arrived in NZ. However, the analysis did reveal other relationships. LNYV subgroup II isolates from both Australia and NZ appeared to originate in Australia, while the origin for subgroup I isolates was unclear. However, within the NZ subgroup I isolates, geographic specific groupings were observed.