Development of TaqMan RT-qPCR for the detection of regulated citrus viruses and viroids in Aotearoa New Zealand.

The major citrus species include several economically important fruits, such as orange, mandarin, lemon, limes, grapefruit and pomelos. Since the 1980 s, total production and consumption of citrus has grown strongly with the current annual worldwide production at over 105 million tonnes. New Zealand's citrus exports, for instance, had an estimated worth of NZ$ 11.6 million (approx. US$ 7 million) in 2020. Citrus plants are prone to viral diseases, which can lead to substantial economic losses. In New Zealand, the citrus Import Health Standard (IHS) has identified 22 viruses and viroids that are subject to regulation and requires citrus nursery stock to be free of these pathogens. As such, there is a need for reliable, sensitive, and rapid detection methods to screen for these viruses and viroids during post entry quarantine. In this study, we developed TaqMan RT-qPCR assays for the detection of nine of these regulated viruses and viroids, namely citrus leaf rugose virus (CiLRV), citrus leprosis virus C (CiLV-C), citrus leprosis virus C2 (CiLV-C2), citrus leprosis virus N (CiLV-N), citrus psorosis virus (CPsV), citrus yellow mosaic virus (CYMV), citrus bent leaf viroid (CBLVd), citrus viroid V (CVd-V), and citrus viroid VI (CVd-VI). These assays have been validated and found to be highly sensitive, specific, and reliable. The implementation of these assays will facilitate the safe importation of citrus nursery stock, thus safeguarding the country's horticultural and economic interests.

Detection, Characterization, and Distribution of the First Case of Pepino Mosaic Virus in Aotearoa New Zealand.

Tomato (Solanum lycopersicum L., family Solanaceae) represents one of the most economically valuable horticultural crops worldwide. Tomato production is affected by numerous emerging plant viruses. We identified, for the first time in New Zealand (NZ), Pepino mosaic virus (PepMV) in greenhouse grown tomato crops using a combination of methods from electron microscopy and herbaceous indexing to RT-qPCR and high-throughput sequencing. Phylogenetic and genomic analysis of a near-complete PepMV genome determined that the detected strain belonged to the mild form of the CH2 lineage of the virus. Subsequently, a delimiting survey of PepMV was conducted, and PepMV was detected at four additional locations. PCR-derived sequences obtained from samples collected from different greenhouses and from herbaceous indicator plants were identical to the original sequence. Since PepMV has never been reported in NZ before, seed pathways are speculated to be the most likely source of entry into the country.

New Virus Diagnostic Approaches to Ensuring the Ongoing Plant Biosecurity of Aotearoa New Zealand.

To protect New Zealand's unique ecosystems and primary industries, imported plant materials must be constantly monitored at the border for high-threat pathogens. Techniques adopted for this purpose must be robust, accurate, rapid, and sufficiently agile to respond to new and emerging threats. Polymerase chain reaction (PCR), especially real-time PCR, remains an essential diagnostic tool but it is now being complemented by high-throughput sequencing using both Oxford Nanopore and Illumina technologies, allowing unbiased screening of whole populations. The demand for and value of Point-of-Use (PoU) technologies, which allow for in situ screening, are also increasing. Isothermal PoU molecular diagnostics based on recombinase polymerase amplification (RPA) and loop-mediated amplification (LAMP) do not require expensive equipment and can reach PCR-comparable levels of sensitivity. Recent advances in PoU technologies offer opportunities for increased specificity, accuracy, and sensitivities which makes them suitable for wider utilization by frontline or border staff. National and international activities and initiatives are adopted to improve both the plant virus biosecurity infrastructure and the integration, development, and harmonization of new virus diagnostic technologies.

Population genomic insights into invasion success in a polyphagous agricultural pest, Halyomorpha halys.

Invasive species are increasingly threatening ecosystems and agriculture by rapidly expanding their range and adapting to environmental and human-imposed selective pressures. The genomic mechanisms that underlie such rapid changes remain unclear, especially for agriculturally important pests. Here, we used genome-wide polymorphisms derived from native, invasive, and intercepted samples and populations of the brown marmorated stink bug (BMSB), Halyomorpha halys, to gain insights into population genomics processes that have promoted the successful global invasion of this polyphagous pest. Our analysis demonstrated that BMSB exhibits spatial structure but admixture rates are high among introduced populations, resulting in similar levels of genomic diversity across native and introduced populations. These spatial genomic patterns suggest a complex invasion scenario, potentially with multiple bridgehead events, posing a challenge for accurately assigning BMSB incursions to their source using reduced-representation genomic data. By associating allele frequencies with the invasion status of BMSB populations, we found significantly differentiated single nucleotide polymorphisms (SNPs) located in close proximity to genes for insecticide resistance and olfaction. Comparing variations in allele frequencies among populations for outlier SNPs suggests that BMSB invasion success has probably evolved from standing genetic variation. In addition to being a major nuisance of households, BMSB has caused significant economic losses to agriculture in recent years and continues to expand its range. Despite no record of BMSB insecticide resistance to date, our results show high capacity for potential evolution of such traits, highlighting the need for future sustainable and targeted management strategies.

ddRAD sequencing: an emerging technology added to the biosecurity toolbox for tracing the origin of brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae).

BACKGROUND: Brown marmorated stink bug (BMSB), Halyomorpha halys (Hemiptera: Pentatomidae) is native to East Asia but has invaded many countries in the world. BMSB is a polyphagous insect pest and causes significant economic losses to agriculture worldwide. Knowledge on the genetic diversity among BMSB populations is scarce but is essential to understand the patterns of colonization and invasion history of local populations. Efforts have been made to assess the genetic diversity of BMSB using partial mitochondrial DNA sequences but genetic divergence on mitochondria is not high enough to precisely accurately identify and distinguish various BMSB populations. Therefore, in this study, we applied a ddRAD (double digest restriction-site associated DNA) sequencing approach to ascertain the genetic diversity of BMSB populations collected from 12 countries (2 native and 10 invaded) across four continents with the ultimate aim to trace the origin of BMSBs intercepted during border inspections and post-border surveillance. RESULT: A total of 1775 high confidence single nucleotide polymorphisms (SNPs) were identified from ddRAD sequencing data collected from 389 adult BMSB individuals. Principal component analysis (PCA) of the identified SNPs indicated the existence of two main distinct genetic clusters representing individuals sampled from regions where BMSB is native to, China and Japan, respectively, and one broad cluster comprised individuals sampled from countries which have been invaded by BMSB. The population genetic structure analysis further discriminated the genetic diversity among the BMSB populations at a higher resolution and distinguished them into five potential genetic clusters. CONCLUSION: The study revealed hidden genetic diversity among the studied BMSB populations across the continents. The BMSB populations from Japan were genetically distant from the other studied populations. Similarly, the BMSB populations from China were also genetically differentiated from the Japanese and other populations. Further genetic structure analysis revealed the presence of at least three genetic clusters of BMSB in the invaded countries, possibly originating via multiple invasions. Furthermore, this study has produced novel set of SNP markers to enhance the knowledge of genetic diversity among BMSB populations and demonstrates the potential to trace the origin of BMSB individuals for future invasion events.

Genetic diversity analysis of brown marmorated stink bug, Halyomorpha halys based on mitochondrial COI and COII haplotypes.

BACKGROUND: In the past decade, the brown marmorated stink bug (BMSB), Halyomorpha halys (Hemiptera: Pentatomidae) has caused extensive damage to global agriculture. As a high-risk pest for many countries, including New Zealand, it is important to explore its genetic diversity to enhance our knowledge and devise management strategies for BMSB populations. In this study, two mitochondrial genes, Cytochrome c oxidase I (COI) and Cytochrome c oxidase II (COII) were used to explore the genetic diversity among 463 BMSB individuals collected from 12 countries. RESULT: In total, 51 COI and 29 COII haplotypes of BMSB were found, which formed 59 combined haplotypes (5 reported and 54 novel). Of these, H1h1 was the predominant haplotype. The haplotype diversity (Hd) and nucleotide diversity (π) were high while the neutrality (Fu's Fs) values were negative for the BMSB populations in the native countries, China, and Japan. For the BMSB populations from the invaded countries, the Fu's Fs values were negative for populations from Chile, Georgia, Hungary, Italy, Romania, Turkey, and USA, indicating that those populations are under demographic expansion. In comparison, the Fu's Fs values were positive for the populations from Austria, Serbia, and Slovenia, revealing a potential population bottleneck. Analysis of molecular variance (AMOVA) suggested that significant genetic difference exists among the BMSB populations from China, Japan, and the invasive countries. CONCLUSION: This study revealed that the haplotype diversity of the BMSB populations was high in those two studied countries where BMSB is native to (China and Japan) but low in those countries which have been invaded by the species. The analysis indicated that multiple invasions of BMSB occurred in Europe and the USA. The study also revealed three ancestral lines and most of the novel haplotypes were evolved from them. Moreover, we observed two genetic clusters in the invasive populations that are formed during different invasion events. Our study provided a comprehensive overview on the global haplotypes distribution thus expanding the existing knowledge on BMSB genetic diversity that potentially could play an important role in formulating feasible pest management strategies.