Trap-based surveillance strategies are widely used for monitoring of invasive insect species, aiming to detect newly arrived exotic taxa as well as track the population levels of established or endemic pests. Where these surveillance traps have low specificity and capture non-target endemic species in excess of the target pests, the need for extensive specimen sorting and identification creates a major diagnostic bottleneck. While the recent development of standardized molecular diagnostics has partly alleviated this requirement, the single specimen per reaction nature of these methods does not readily scale to the sheer number of insects trapped in surveillance programmes. Consequently, target lists are often restricted to a few high-priority pests, allowing unanticipated species to avoid detection and potentially establish populations.DNA metabarcoding has recently emerged as a method for conducting simultaneous, multi-species identification of complex mixed communities and may lend itself ideally to rapid diagnostics of bulk insect trap samples. Moreover, the high-throughput nature of recent sequencing platforms could enable the multiplexing of hundreds of diverse trap samples on a single flow cell, thereby providing the means to dramatically scale up insect surveillance in terms of both the quantity of traps that can be processed concurrently and number of pest species that can be targeted. In this review of the metabarcoding literature, we explore how DNA metabarcoding could be tailored to the detection of invasive insects in a surveillance context and highlight the unique technical and regulatory challenges that must be considered when implementing high-throughput sequencing technologies into sensitive diagnostic applications.

The plant pathogen Erwinia amylovora can be divided into two host-specific groupings; strains infecting a broad range of hosts within the Rosaceae subfamily Spiraeoideae (e.g., Malus, Pyrus, Crataegus, Sorbus) and strains infecting Rubus (raspberries and blackberries). Comparative genomic analysis of 12 strains representing distinct populations (e.g., geographic, temporal, host origin) of E. amylovora was used to describe the pan-genome of this major pathogen. The pan-genome contains 5751 coding sequences and is highly conserved relative to other phytopathogenic bacteria comprising on average 89% conserved, core genes. The chromosomes of Spiraeoideae-infecting strains were highly homogeneous, while greater genetic diversity was observed between Spiraeoideae- and Rubus-infecting strains (and among individual Rubus-infecting strains), the majority of which was attributed to variable genomic islands. Based on genomic distance scores and phylogenetic analysis, the Rubus-infecting strain ATCC BAA-2158 was genetically more closely related to the Spiraeoideae-infecting strains of E. amylovora than it was to the other Rubus-infecting strains. Analysis of the accessory genomes of Spiraeoideae- and Rubus-infecting strains has identified putative host-specific determinants including variation in the effector protein HopX1Ea and a putative secondary metabolite pathway only present in Rubus-infecting strains.

A novel pair of universal primers was developed to detect potyvirus species after conserved sites were identified using all fulllength potyvirus sequences available by 2005. The breadth of specificity of the new primers, NIb2F and NIb3R, was investigated and compared with the specificity of two routinely used primer pairs in plant virus diagnostic laboratories. RNA from 40 potyvirus isolates representing 23 recognized and three possible new species was tested. Reactions with NIb2F and NIb3R produced amplicons of 350 bp from all 40 virus isolates tested. Reactions with the previously published WCIEN and Potyvirid primers amplified cDNA from 32 and 21 isolates, representing possibly 21 and 15 species, respectively. The identity of 12 unknown potyvirus isolates was confirmed by sequencing and three were found to be potentially distinct potyvirus species. Gel banding patterns from reactions with NIb2F and NIb3R were simpler to interpret than those from reactions with the other two primer sets; fewer products were visible and the cDNA fragments were less variable in size. RT-PCR with the novel primers is predicted to be able to detect virus isolates from all major groups within the genus Potyvirus and its reliability makes it well suited for use as a routine diagnostic assay.

Eradication of plant pathogen incursions is very important for the protection of plant industries, managed gardens and natural environments worldwide. The consequence of a pathogen becoming endemic can be serious, in some cases having an impact on the national economy. The current strategy for eradication of a pathogen relies on techniques for the treatment, removal and disposal of affected host plants. There are many examples where these techniques have been successful but many where they have not. Success relies on a sound understanding of the biology and epidemiology of the pathogen and its interaction with the host. Removal and disposal of infected plant material for eradication and containment of plant and soil inhabiting fungal, bacterial and viral pathogens are reviewed by considering black Sigatoka of banana, apple scab, maize smut, fireblight, citrus canker and sharka disease of stone-fruit crops. In examining examples of dealing with plant pathogens and diseased host material around the world, particularly Australasia, various techniques including burning, burying, pruning, composting, soil-and biofumigation, solarization, steam sterilization and biological vector control are discussed. Gaps in the literature are identified and emphasize the insufficient detail of information available from past eradications. More effort is required to produce and publish scientific evidence to support the success or otherwise of techniques and suggestions for future research are proposed.

No abstract

Current atmospheric CO 2 levels are about 400 lmol mol À1 and are predicted to rise to 650 lmol mol À1 later this century. Although the positive and negative impacts of CO 2 on plants are well documented, little is known about interactions with pests and diseases. If disease severity increases under future environmental conditions, then it becomes imperative to understand the impacts of pathogens on crop production in order to minimize crop losses and maximize food production. Barley yellow dwarf virus (BYDV) adversely affects the yield and quality of economically important crops including wheat, barley and oats. It is transmitted by numerous aphid species and causes a serious disease of cereal crops worldwide. This study examined the effects of ambient (aCO 2 ; 400 lmol mol À1 ) and elevated CO 2 (eCO 2 ; 650 lmol mol À1 ) on noninfected and BYDV-infected wheat. Using a RT-qPCR technique, we measured virus titre from aCO 2 and eCO 2 treatments. BYDV titre increased significantly by 36.8% in leaves of wheat grown under eCO 2 conditions compared to aCO 2 . Plant growth parameters including height, tiller number, leaf area and biomass were generally higher in plants exposed to higher CO 2 levels but increased growth did not explain the increase in BYDV titre in these plants. High virus titre in plants has been shown to have a significant negative effect on plant yield and causes earlier and more pronounced symptom expression increasing the probability of virus spread by insects. The combination of these factors could negatively impact food production in Australia and worldwide under future climate conditions. This is the first quantitative evidence that BYDV titre increases in plants grown under elevated CO 2 levels.

High‐throughput sequencing (HTS) technologies have revolutionized plant pest research and are now raising interest for plant pest diagnostics, with plant virus diagnostics at the forefront of development. However, the application of HTS in plant pest diagnostics raises important challenges that plant health regulators will have to address. Adapted infrastructures, technical guidelines and training are pivotal for further use and adoption of the HTS technologies in the phytosanitary framework.

With its small size and low cost, the hand-held MinION sequencer is a powerful tool for in-field surveillance. Using a metagenomic approach, it allows non-targeted detection of viruses in a sample within a few hours. This study aimed to determine the ability of the MinION to metagenomically detect and characterise a virus from an infected mosquito. RNA was extracted from an Aedes notoscriptus mosquito infected with Ross River virus (RRV), converted into cDNA and sequenced on the MinION. Bioinformatic analysis of the MinION reads led to detection of full-length RRV, with reads of up to 2.5kb contributing to the assembly. The cDNA was also sequenced on the MiSeq sequencer, and both platforms recovered the RRV genome with >98% accuracy. This proof of concept study demonstrates the metagenomic detection of an arbovirus, using the MinION, directly from a mosquito with minimal sample purification.