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Abstract This study is the first to investigate the presence and movement of the novel Liberibacter species ‘Candidatus Liberibacter brunswickensis’ (CLbr) in eggplant, Solanum melongena. The psyllid, Acizzia solanicola can transmit CLbr to eggplant and CLbr can be acquired by CLbr-negative A. solanicola individuals from CLbr-positive eggplants. In planta, CLbr can replicate, move and persist. Investigation into the early development of eggplants showed that CLbr titres had increased at the inoculation site at 14 days post inoculation access period (DPIAP). CLbr had become systemic in the majority of plants tested by 28 DPIAP. The highest bacterial titres were recorded at 35 DPIAP in all samples of the inoculated leaf, the roots, stems and the midrib and petiole samples of the newest leaf (the top leaf). This finding strongly suggests that CLbr movement in planta follows the source to sink relationship as previously described for ‘Ca. Liberibacter asiaticus’ (CLas) and ‘Ca. Liberibacter solanacearum’ (CLso). No symptoms consistent with Liberibacter-associated diseases were noted for plants colonised by CLbr during this study, consistent with the hypothesis that CLbr does not cause disease of eggplant during the early stages of host colonisation. In addition, no significant differences in biomass were found between eggplant colonised with CLbr, compared to those that were exposed to CLbr-negative A. solanicola, and to control plants.

Tan spot (TS), known in Australia as yellow leaf spot, is caused by the fungal pathogen Pyrenophora tritici-repentis, a major foliar disease of wheat. Four experiments were conducted between 2016 and 2017 in two different climatic zones of southeastern Australia to determine the impact of TS on grain yield and quality in six wheat cultivars with different resistance ratings. In each experiment, high and low TS disease scenarios were applied to each cultivar. Disease severity was assessed as either whole-plot percentage leaf area affected (%LAA) or top three leaf (flag to flag-2) %LAA. Whole-plot %LAA was analyzed using both repeated measurements and area under the disease progress curve (AUDPC). For whole-plot %LAA, the repeated measurements identified differences in epidemic progression at key growth stages not identified through AUDPC. Both AUDPC and end-of-season flag to flag-2 %LAA were negatively correlated with grain yield and screenings. Increased rainfall impacting disease development and number of rain days above 5 mm increased TS severity across both climatic zones. Cultivar resistance ratings influenced grain yield loss, with moderately resistant, moderately susceptible, and susceptible cultivars losing up to 6, 18, and 24% grain yield, respectively. High disease significantly increased screenings by up to 1, 3, and 5% for moderately resistant, moderately susceptible, and susceptible cultivars, respectively. This study demonstrated that TS can cause significant grain yield losses in southeastern Australia, and a minimum cultivar rating of moderate resistance to moderate susceptibility was required to prevent severe TS and associated grain yield and quality losses. [Figure: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Abstract Background To date genomic studies on Map have concentrated on Type C strains with only a few Type S strains included for comparison. In this study the entire pan-genome of 261 Map genomes (205 Type C, 52 Type S and 4 Type B) and 7 Mycobacterium avium complex (Mac) genomes were analysed to identify genomic similarities and differences between the strains and provide more insight into the evolutionary relationship within this Mycobacterial species. Results Our analysis of the core genome of all the Map isolates identified two distinct lineages, Type S and Type C Map that is consistent with previous phylogenetic studies of Map. Pan-genome analysis revealed that Map has a larger accessory genome than Mycobacterium avium subsp. avium (Maa) and Type C Map has a larger accessory genome than Type S Map. In addition, we found large rearrangements within Type S strains of Map and little to none in Type C and Type B strains. There were 50 core genes identified that were unique to Type S Map and there were no unique core genes identified between Type B and Type C Map strains. In Type C Map we identified an additional CE10 CAZyme class which was identified as an alpha/beta hydrolase and an additional polyketide and non-ribosomal peptide synthetase cluster. Consistent with previous analysis no plasmids and only incomplete prophages were identified in the genomes of Map. There were 45 hypothetical CRISPR elements identified with no associated cas genes. Conclusion This is the most comprehensive comparison of the genomic content of Map isolates to date and included the closing of eight Map genomes. The analysis revealed that there is greater variation in gene synteny within Type S strains when compared to Type C indicating that the Type C Map strain emerged after Type S. Further analysis of Type C and Type B genomes revealed that they are structurally similar with little to no genetic variation and that Type B Map may be a distinct clade within Type C Map and not a different strain type of Map. The evolutionary lineage of Maa and Map was confirmed as emerging after M. hominissuis.

Abstract Grapevine Pinot gris virus (GPGV; genus Trichovirus in the family Betaflexiviridae) was detected in Australia in 2016, but its impact on the production of nursery material and fruit in Australia is still currently unknown. This study investigated the prevalence and genetic diversity of GPGV in Australia. GPGV was detected by reverse transcription-polymerase chain reaction (RT-PCR) in a range of rootstock, table and wine grape varieties from New South Wales, South Australia, and Victoria, with 473/2171 (21.8%) samples found to be infected. Genomes of 32 Australian GPGV isolates were sequenced and many of the isolates shared high nucleotide homology. Phylogenetic and haplotype analyses demonstrated that there were four distinct clades amongst the 32 Australian GPGV isolates and that there were likely to have been at least five separate introductions of the virus into Australia. Recombination and haplotype analysis indicate the emergence of new GPGV strains after introduction into Australia. When compared with 168 overseas GPGV isolates, the analyses suggest that the most likely origin of Australian GPGV isolates is from Europe. There was no correlation between specific GPGV genotypes and symptoms such as leaf mottling, leaf deformation, and shoot stunting, which were observed in some vineyards, and the virus was frequently found in symptomless grapevines.

Abstract Recent advances in the field of environmental DNA (eDNA) metabarcoding have produced a promising platform for early detection and broad‐spectrum monitoring of plant pests and pathogens. To date, the majority of fungal metabarcoding assays have relied upon universal primers amplifying segments of the ribosomal DNA, to ensure broad taxonomic coverage while studying changes in community composition, or more recently, to screen for presence/absence of target species. In a diagnostics framework however, single, universal molecular barcodes do not discriminate accurately enough for many groups of important fungal phytopathogens at the required species level. Here, a modular, multi‐barcode amplicon sequencing pipeline was established to provide rapid and reliable diagnostics targeting the Ophiostomatales, an order containing economically important phytopathogens vectored by bark and ambrosia beetles (Curculionidae). Using compositionally varied mock communities, we evaluated five barcoding loci: ITS1, ITS2, the large ribosomal sub‐unit (LSU), translation elongation factor 1‐alpha (TEF1α) and calmodulin (CAL), for their ability to provide species‐level resolution. The sensitivity and detection limit of the assay was established by spiking genomic DNA of the exotic Dutch elm disease pathogen, Ophiostoma novo‐ulmi Brasier, into both the controlled mock communities and eDNA sampled from Ips grandicollis (Eichhoff) beetles collected in New South Wales, Australia. The TEF1α barcode successfully detected the Dutch elm pathogen DNA with 100% accuracy down to approx. 5 pg/μl, while the ITS1 barcode had a 100% accuracy down to approx. 0.4 pg/μl within a given sample. Our results demonstrate that a dual‐barcode approach targeting the ITS1 and TEF1α regions simultaneously is sufficient for accurate species level detection and characterisation of the Ophiostomatales and offers confident detection of low‐abundance taxa (relative read abundances of 0.1%–0.01%). Multi‐barcode metabarcoding provides a framework which can quickly and efficiently be adapted to new targets when establishing high throughput diagnostics for post‐border biosecurity surveillance of fungal phytopathogens.

Abstract Background A zucchini disease outbreak with unusual symptoms associated with Pseudomonas syringae clade 2b was identified in Bundaberg, Australia during autumn 2016. To investigate the genetic diversity of the 11 Australian isolates obtained from the outbreak, the genomes were compared to the publicly available P. syringae strains in phylogroup 2. Results Average nucleotide identity refined the P. syringae clade 2b-a into four clusters (Cluster A, B, C1 and C2), an expansion from the previously identified A, B and C. Australian isolates were in Cluster A, C1 and C2. Genomic analyses highlighted several key factors that may contribute to the virulence of these isolates. Six orthologous groups, including three virulence factors, were associated with P. syringae phylogroup 2 cucurbit-infecting strains. A region of genome plasticity analysis identified a type VI secretion system pathway in clade 2b-a strains which could also contribute to virulence. Pathogenicity assays on isolates KL004-k1, KFR003-1 and 77-4C, as representative isolates of Cluster A, C1 and C2, respectively, determined that all three isolates can infect pumpkin, squash, watermelon and zucchini var. Eva with different levels of disease severity. Subsequently, type III effectors were investigated and four type III effectors (avrRpt2, hopZ5, hopC1 and hopH1) were associated with host range. The hopZ effector family was also predicted to be associated with disease severity. Conclusions This study refined the taxonomy of the P. syringae clade 2b-a, supported the association between effector profile and pathogenicity in cucurbits established in a previous study and provides new insight into important genomic features of these strains. This study also provided a detailed and comprehensive resource for future genomic and functional studies of these strains.

Post-border surveillance for forestry’s high priority pests and pathogens is conducted routinely through established programs focused on the main points-of-entry and across the major plantation growing regions. Currently, most diagnostic protocols used to identify fungal phytopathogens sampled during these surveys rely on traditional methods, such as morphological examination and DNA barcoding techniques. This stepwise process from isolation to species identification is often regarded as slow, expensive, and limited due to the need for disease manifestation and/or comprehensive expertise for rapid and accurate detection. In this study, we applied a recently validated high-throughput, dual-marker amplicon sequencing approach on insect surveillance traps from across Australia to assess its performance for the targeted surveillance of the Ophiostomatales, an order of fungi comprising notable phytopathogens which are vectored by bark beetles. By using a recently validated assay we were able to confidently characterize a range of Ophiostomatalean taxa known to be present in Australia, while reporting eight first detections from environmental DNA. Our study demonstrates the value of targeted multi-barcode amplicon sequencing for high-throughput screening of fungi caught in post-border surveillance traps, in addition to emphasizing research priorities that require further investigation before such methods can be implemented routinely for biosecurity.

Turnip yellows virus (TuYV) is a damaging virus that is persistently transmitted by aphids and infects a wide range of grain hosts including lentil (Lens culinaris Medik), field pea (Pisum sativum L.) and canola (Brassica napus L., oilseed rape). Although information is available about the effects of TuYV infection on grain yield in canola, data about its impact on yield in pulses is lacking. In this study, field experiments quantifying the effects of TuYV infection on the grain yield of lentil and field pea were conducted over three consecutive years (2018-2020) with varying weather conditions. Plants artificially inoculated with TuYV using viruliferous green peach aphid (Myzus persicae, Sulzer) were grown under typical field conditions in south-eastern Australia. At maturity, grain yield, along with associated grain and plant growth parameters, were measured. Compared to the non-inoculated control treatment, early TuYV infection reduced grain yield by up to 36% in lentil and 45% in field pea, while late TuYV infection had no significant impact on yield. Despite a high incidence of TuYV infection and significant yield losses recorded in inoculated plots, no obvious symptoms of virus infection were observed in the inoculated plots in any of the six experiments; this lack of visible symptoms in lentil and field pea has significant implications for crop health assessments, demonstrating the importance of testing for virus instead of relying solely on the presence of visual symptoms, and may also be leading to an underestimation of the importance of TuYV in pulses in Australia.

Abstract The ophiostomatoid fungi are an assemblage of ascomycetes which are arguably best-known for their associations with bark and ambrosia beetles (Curculonidae) and blue stain (sap stain) of many economically important tree species. These fungi are considered a significant threat to coniferous forests, which has resulted in numerous studies characterising the diversity of bark beetles and their ophiostomatoid associates globally. The diversity of ophiostomatoid fungi present in Australian pine plantations, however, remains largely undetermined. The aims of this study were therefore to reconsider the diversity of ophiostomatoid fungi associated with Pinus in Australia, and to establish the baseline of expected taxa found within these plantation ecosystems. To achieve this, we reviewed Australian plant pathogen reference collections, and analysed samples collected during forest health surveillance programs from the major pine growing regions in south-eastern Australia. In total, 135 ophiostomatoid isolates (15 from reference collections and 120 collected during the current study) were assessed using morphological identification and ITS screening which putatively distinguished 15 taxonomic groups. Whole genome sequencing (WGS) of representative isolates from each taxon was performed to obtain high-quality sequence data for multi-locus phylogenetic analysis. Our results revealed a greater than expected diversity, expanding the status of ophiostomatoid fungi associated with Pinus in Australia to include 14 species from six genera in the Ophiostomatales and a single species residing in the Microascales. While most of these were already known to science, our study includes seven first records for Australia and the description of one new species, Graphilbum ipis-grandicollis sp. nov.. This study also provides an early example of whole genome sequencing (WGS) approaches replacing traditional PCR-based methods for taxonomic surveys. This not only allowed for robust multi-locus sequence extraction during taxonomic assessment, but also permitted the rapid establishment of a curated genomic database for ophiostomatoid fungi which will continue to aid in the development of improved diagnostic resources and capabilities for Australian biosecurity.

Obtaining complete phytoplasma genomes is difficult due to the lack of a culture system for these bacteria. To improve genome assembly, a non-ionic, low- and iso-osmotic iodixanol (Optiprep™) density gradient centrifugation method was developed to enrich for phytoplasma cells and deplete plant host tissues prior to deoxyribonucleic acid (DNA) extraction and high-throughput sequencing (HTS). After density gradient enrichment, potato infected with a ‘Candidatus Phytoplasma australasia’-related strain showed a ∼14-fold increase in phytoplasma HTS reads, with a ∼1.7-fold decrease in host genomic reads compared to the DNA extracted from the same sample without density gradient centrifugation enrichment. Additionally, phytoplasma genome assemblies from libraries equalized to 5 million reads were, on average, ∼15,000 bp larger and more contiguous (N50 ∼14,800 bp larger) than assemblies from the DNA extracted from the infected potato without enrichment. The method was repeated on capsicum infected with Sweet Potato Little Leaf phytoplasma (‘Ca. Phytoplasma australasia’-related strain) with a lower phytoplasma titer than the potato. In capsicum, ∼threefold more phytoplasma reads and ∼twofold less host genomic reads were obtained, with the genome assembly size and N50 values from libraries equalized to 3.4 million reads ∼137,000 and ∼4,000 bp larger, respectively, compared to the DNA extracted from infected capsicum without enrichment. Phytoplasmas from potato and capsicum were both enriched at a density of 1.049–1.058 g/ml. Finally, we present two highly contiguous ‘Ca. Phytoplasma australasia’ phytoplasma reference genomes sequenced from naturally infected Solanaceae hosts in Australia. Obtaining high-quality phytoplasma genomes from naturally infected hosts will improve insights into phytoplasma taxonomy, which will improve their detection and disease management.

Abstract Background Mycobacterium avium subsp. paratuberculosis (Map) causes Johne’s disease (JD), a chronic enteritis widespread in ruminants, resulting in substantial economic losses, especially to the dairy industry. Understanding the genetic diversity of Map in Australia will assist epidemiological studies for tracking disease transmission and identify subtype characteristics for use in development of improved diagnostic typing methods. Here we investigated the phylogenetic relationships of 351 Map isolates and compared different subtyping methods to assess their suitability for use in diagnostics and accuracy. Results SNP-based phylogenetic analysis of 228 Australian isolates and 123 publicly available international isolates grouped Type S and Type C strains into two distinct lineages. Type C strains were highly monomorphic with only 20 SNP differences separating them. Type S strains, when aligned separately to the Telford strain, fell into two distinct clades: The first clade contained seven international isolates while the second clade contained one international isolate from Scotland and all 59 Australian isolates. The Australian Type B strain clustered with US bison strains. IS1311 PCR and Restriction Enzyme Analysis (REA) intermittently generated incorrect results when compared to Long Sequence Polymorphism (LSP) analysis, whole genome SNP-based phylogenetic analysis, IS1311 sequence alignment and average nucleotide identity (ANI). These alternative methods generated consistent Map typing results. A published SNP based assay for genotyping Map was found to be unsuitable for differentiating between Australian and international strain types of Map. Conclusion This is the first phylogenetic analysis of Australian Map isolates. The Type C lineage was highly monomorphic, and the Type S lineage clustered all Australian isolates into one clade with a single Scottish sheep strain. The Australian isolate classified as Type B by IS1311 PCR and REA is likely to be descended from bison and most closely related to US bison strains. Limitations of the current typing methods were identified in this study.

Abstract Rathayibacter toxicus is a toxigenic bacterial plant pathogen indigenous to Australia and South Africa. A threat to livestock industries globally, the bacterium was designated a U.S. Select Agent. Biosecurity and phytosanitary concerns arise due to the international trade of seed and hay that harbor the bacterium. Accurate diagnostic protocols to support phytosanitary decisions, delineate areas of freedom, and to support research are required to address those concerns. Whole genomes of three genetic populations of R. toxicus were sequenced (Illumina MiSeq platforms), assembled and genomic regions unique to each population identified. Highly sensitive and specific TaqMan qPCR and multiplex endpoint PCR assays were developed for the detection and identification of R. toxicus to the population level of discrimination. Specificity was confirmed with appropriate inclusivity and exclusivity panels; no cross reactivity was observed. The endpoint multiplex PCR and TaqMan qPCR assays detected 10 fg and 1 fg of genomic DNA, respectively. To enhance reliability and increase confidence in results, three types of internal controls with no or one extra primer were developed and incorporated into each assay to detect both plant and artificial internal controls. Assays were validated by blind ring tests with multiple operators in three international laboratories.

Lolium perenne (perennial ryegrass) is a common temperate pasture grass species and is favored by dairy farmers. Commercially, the known association with the mutualistic endophytic fungus Epichloë festucae subsp. lolii is used to enhance insect resistance of host plants. Knowledge of other members of the microbiome and their functions are limited. Sequencing of the V4 region of the 16S ribosomal RNA gene was used to examine the bacterial microbiome of perennial ryegrass (Alto) seed (generation 1 [G1]), subsequent mature plants grown in soil and sand, and seed (generation 2 [G2]) from crosses of these mature plants. The G1 microbiome was dominated by the class Gammaproteobacteria. The mature plant microbiomes were far more diverse, comprising up to 37 classes inclusive of Gammaproteobacteria. Different growth media yielded different microbiome profiles in mature plants. The G2 microbiome, similar to the G1 microbiome, was dominated by Gammaproteobacteria as the primary constituent, with additional supplementation from class Bacilli. This suggests the continuation of a core microbiome which persists from seed through plant maturation to seed. This study sheds new light on the hereditability of perennial ryegrass bacterial microbiomes and has identified some operational taxonomic units of potential commercial significance due to their seed transmissibility, and their roles are currently being explored.

Abstract Objective Dichelobacter nodosus is the primary aetiological agent of footrot in sheep. Ovine footrot causes considerable economic losses and substantial animal welfare issues in the Australian sheep industry. Current methods for detecting D. nodosus are difficult, laborious and time-consuming. Recently, we developed a robust LAMP assay (VDN LAMP) that was able to identify aprV2 positive D. nodosus in-field. A major advantage of LAMP technology is the ability of the assay to be performed by non-specialists with minimal training. We aimed to assess the performance of the VDN LAMP in-field in comparison to a laboratory-based aprV2/aprB2 rtPCR when used by secondary users after training by the authors. Results Two animal health officers (termed secondary users) from Department of Primary Industries and Regions, South Australia (PIRSA) were trained in the use of VDN LAMP, before carrying out in-field testing on several locations in South Australia. The performance of VDN LAMP assay by secondary user 1 was shown to successfully detect 73.91% (n = 53) aprV2 positive samples, while secondary user 2 detected 37.93% (n = 30) aprV2 positive samples. Overall, the ability to identify virulent D. nodosus by VDN LAMP by secondary users was mixed for various reasons, however, this could be rectified by additional training and commercial production of the LAMP kits to increase stability. We envisaged in the future VDN LAMP will able to be used by non-specialists to aid control programs.

In Australia, there is documented confusion from producers around the clinical disease of footrot, and anecdotally, knowledge of what tools are available for the diagnosis and management of footrot. When discussing footrot with producers, the authors noted a hesitation to discuss, with denial often expressed. The disease can be debilitating, both on the sheep's welfare and the producer's well-being, as it is a very difficult disease to manage and eradicate. Gaining an understanding of producer perceptions of the disease may help ensure any future actions for management and control are in-line with those identified by producers. A combination of a web-based, and manually distributed surveys of 45 sheep producers was conducted. This included closed- and open-ended questions, multi check box, and Likert scales. Responses were quantified by descriptive statistics and a thematic analysis conducted of short answers. The results of this survey indicate satisfaction with footrot diagnostics is low, while satisfaction with control methods is high. There was also a poor general understanding of footrot as a disease, and a general distrust in peers when it comes to correct management of footrot. This research addresses a gap in the literature about how sociological conditions affect diagnosis and control of footrot disease. It provides three main recommendations—simplifying the diagnostic message, encouraging a culture of trust among sheep producers and increasing governmental support—as a way to tackle this problem.

Plant viruses constitute a large group of pathogens causing damaging diseases in many agricultural and horticultural crops around the world [...]

Pseudomonas syringae causes bacterial blight (BB) disease worldwide on economically important fruit and vegetable crops including field pea (Pisum sativum L.). The two pathovars responsible for BB in field pea are Pseudomonas syringae pathovar pisi (Psp) and syringae (Pss). In the field, both pathovars cause indistinguishable symptoms on field pea and require laboratory diagnosis to determine the causal pathovar. To aid in-field and laboratory diagnosis, accurate, and robust loop-mediated isothermal amplification (LAMP) assays for Psp and Pss were developed. The assays were able to detect Psp or Pss on live or heat-killed bacterial cells, plant exudates, seeds, and DNA extracts with no inhibitory effects. The two specific LAMP assays developed detected Psp and Pss accurately in less than 20 min and no cross-reaction was observed with 18 strains of closely related species of Pseudomonas syringae. Compared to the conventional PCR assays, the two LAMP assays were equally specific but have advantages of producing quicker and visual live results, enabling early detection and differentiation of Psp and Pss. Our results suggested a potential use of LAMP assays for laboratory testing and can be applied for in-field surveys.

Rathayibacter toxicus is a toxigenic bacterial pathogen of several grass species and is responsible for massive livestock deaths in Australia and South Africa. Due to concern for animal health and livestock industries, it was designated a U.S. Select Agent. A rapid, accurate, and sensitive in-field detection method was designed to assist biosecurity surveillance surveys and to support export certification of annual ryegrass hay and seed. Complete genomes from all known R. toxicus populations were explored, unique diagnostic sequences identified, and target-specific primers and a probe for recombinase polymerase amplification (RPA) and endpoint PCR were designed. The RPA reaction ran at 37 °C and a lateral flow device (LFD) was used to visualize the amplified products. To enhance reliability and accuracy, primers and probes were also designed to detect portions of host ITS regions. RPA assay specificity and sensitivity were compared to endpoint PCR using appropriate inclusivity and exclusivity panels. The RPA assay sensitivity (10 fg) was 10 times more sensitive than endpoint PCR with and without a host DNA background. In comparative tests, the RPA assay was unaffected by plant-derived amplification inhibitors, unlike the LAMP and end-point PCR assays. In-field validation of the RPA assay at multiple sites in South Australia confirmed the efficiency, specificity, and applicability of the RPA assay. The RPA assay will support disease management and evidence-based in-field biosecurity decisions.

Dichelobacter nodosus is the primary etiological agent of footrot in sheep and has a variety of virulence factors. Of these, AprV2, an extracellular protease, has been shown to be capable of causing severe or “virulent” disease symptoms under the right conditions. Due to this, a loop-mediated isothermal amplification (LAMP) assay for the detection of aprV2-positive D. nodosus (VDN LAMP) was developed and evaluated for field use. A sample of 19 sheep flocks (309 sheep) in Victoria, Australia, were tested to determine the optimum conditions for in-field VDN LAMP assay use and sampling, for detecting aprV2-positive D. nodosus infected sheep. VDN LAMP performance was compared to a validated rtPCR that detects aprV2 and the benign strain counterpart, aprB2, using biologically duplicate samples to determine sensitivity and specificity. Flocks were sampled either in winter-spring (moist) or early summer (dry) conditions and had a range of clinical expressions of the disease ovine footrot. Variables considered for optimizing field performance were: sample collection method, sample preparation, clinical expression of disease, and nature of the feet when sampled (moist vs. dry, clean vs. soiled). The test was found to perform best when sheep were sampled with moist, clean feet, using a dry swab with the sample prepared in alkaline polyethylene glycol, pH 13.0, as the collection buffer. A sensitivity of 89% and specificity of 97% was seen when used in-field under these conditions, when compared to aprV2 detection by rtPCR, with “very good” agreement to rtPCR results. This study shows the VDN LAMP test is easy to use in-field to identify the presence of aprV2-positive D. nodosus in sheep flocks.

Research into the bacterial component of the seed microbiome has been intensifying, with the aim of understanding its structure and potential for exploitation. We previously studied the intergenerational seed microbiome of one cultivar of perennial ryegrass with and without one strain of the commercially deployed fungal endophyte Epichloë festucae var. lolii. The work described here expands on our previous study by exploring the bacterial seed microbiome of different commercial cultivar/Epichloë festucae var. lolii combinations in collections of single seeds from the harvest year 2016. In this dataset, a cultivar effect could be seen between the seed microbiomes from cultivars Alto and Trojan. The bacterial component of the seed microbiome from pooled seeds from a single cultivar/E. festucae var. lolii combination harvested from 13 seed production farms around Canterbury in the year 2018 was also studied. This dataset allows the effect of different production locations on the bacterial seed microbiome to be examined. By comparing the two sets of data, bacteria from the genera Pantoea, Pseudomonas, Duganella, Massilia, and an unknown Enterobacteriaceae were observed to be in common. This core bacterial microbiome was stable over time but could be affected by supplemental taxa derived from the growth environment of the parental plant; differing microbiomes were seen between different seed production farms. By comparison to a collection of bacterial isolates, we demonstrated that many of the members of the core microbiome were culturable. This allows for the possibility of exploiting these microbes in the future.

Barley yellow dwarf virus (BYDV) is transmitted by aphids and significantly reduces the yield and quality of cereals worldwide. Four experiments investigating the effects of barley yellow dwarf virus-PAV (BYDV-PAV) infection on either wheat or barley were conducted over three years (2015, 2017, and 2018) under typical field conditions in South-Eastern Australia. Plants inoculated with BYDV-PAV using viruliferous aphids (Rhopalosiphum padi) were harvested at maturity then grain yield and yield components were measured. Compared to the non-inoculated control, virus infection severely reduced grain yield by up to 84% (1358 kg/ha) in wheat and 64% (1456 kg/ha) in barley. The yield component most affected by virus infection was grain number, which accounted for a large proportion of the yield loss. There were no significant differences between early (seedling stage) and later (early-tillering stage) infection for any of the parameters measured (plant height, biomass, yield, grain number, 1000-grain weight or grain size) for either wheat or barley. Additionally, this study provides an estimated yield loss value, or impact factor, of 0.91% (72 kg/ha) for each one percent increase in natural BYDV-PAV background infection. Yield losses varied considerably between experiments, demonstrating the important role of cultivar and environmental factors in BYDV epidemiology and highlighting the importance of conducting these experiments under varying conditions for specific cultivar–vector–virus combinations.

Rathayibacter toxicus is a Gram-positive, nematode-vectored bacterium that infects several grass species in the family Poaceae. Unique in its genus, R. toxicus has the smallest genome, possesses a complete CRISPR-Cas system, a vancomycin-resistance cassette, produces tunicamycin, a corynetoxin responsible for livestock deaths in Australia, and is designated a Select Agent in the United States. In-depth, genome-wide analyses performed in this study support the previously designated five genetic populations, with a core genome comprising approximately 80% of the genome for all populations. Results varied as a function of the type of analysis and when using different bioinformatics tools for the same analysis; e.g., some programs failed to identify specific genomic regions that were actually present. The software variance highlights the need to verify bioinformatics results by additional methods; e.g., PCR, mapping genes to genomes, use of multiple algorithms). These analyses suggest the following relationships among populations: RT-IV ↔ RT-I ↔ RT-II ↔ RT-III ↔ RT-V, with RT-IV and RT-V being the most unrelated. This is the most comprehensive analysis of R. toxicus that included populations RT-I and RT-V. Future studies require underrepresented populations and more recent isolates from varied hosts and geographic locations.

Dichelobacter nododus is the causative agent of footrot, a major disease of sheep that creates welfare concerns and large economic loss. The virulence of D. nododus depends on the presence of extracellular proteases, AprV2 and AprB2, which differ by one amino acid. Strains possessing AprV2 can cause clinically virulent disease, while AprB2 may cause clinically benign disease. Current methods for detecting D. nodosus are difficult, laborious and time consuming. New techniques capable of rapidly detecting and typing D. nodosus are needed to aid control programs. Molecular methods, like real-time polymerase chain reaction (rtPCR) can detect aprV2 and aprB2, however, this assay is not field-deployable and cannot support local decision-making during an outbreak. Here we present a field-based molecular assay for detecting aprV2, using loop mediated isothermal amplification (LAMP). The aprV2 LAMP (VDN LAMP) assay was optimised to reliably detect aprV2 from laboratory purified genomic (gDNA) of virulent D. nodosus down to 5x10(-3) ng μL-1, with time to positive (Tp) ≤ 16 minutes, while aprB2 was unreliably detected at 5 ng μL-1 from 16-20 minutes. The use of field collected samples that were rtPCR positive for aprB2 resulted in no amplification, while aprV2 positive field samples by VDN LAMP assay are defined as having Tps' of < 20 minutes and melting temperature between 88.0-88.9°C. When compared to rtPCR, the VDN LAMP was shown to have a diagnostic specificity of 100% and sensitivity of 83.33%. As proof of concept, the VDN LAMP was taken on farm, with all processing occurring in-field. The on farm VDN LAMP successfully detected 91.67% aprV2 positive samples, no aprB2 positive samples (n = 9) or D. nodosus negative (n = 23) samples, with a kappa agreement of 'almost perfect' to rtPCR. This highlights the potential of the assay to inform local treatment decisions for management.

The distribution of Ilarvirus species populations amongst 61 Australian Prunus trees was determined by next generation sequencing (NGS) of amplicons generated using a genus-based generic RT-PCR targeting a conserved region of the Ilarvirus RNA2 component that encodes the RNA dependent RNA polymerase (RdRp) gene. Presence of Ilarvirus sequences in each positive sample was further validated by Sanger sequencing of cloned amplicons of regions of each of RNA1, RNA2 and/or RNA3 that were generated by species specific PCRs and by metagenomic NGS. Prunus necrotic ringspot virus (PNRSV) was the most frequently detected Ilarvirus, occurring in 48 of the 61 Ilarvirus-positive trees and Prune dwarf virus (PDV) and Apple mosaic virus (ApMV) were detected in three trees and one tree, respectively. American plum line pattern virus (APLPV) was detected in three trees and represents the first report of APLPV detection in Australia. Two novel and distinct groups of Ilarvirus-like RNA2 amplicon sequences were also identified in several trees by the generic amplicon NGS approach. The high read depth from the amplicon NGS of the generic PCR products allowed the detection of distinct RNA2 RdRp sequence variant populations of PNRSV, PDV, ApMV, APLPV and the two novel Ilarvirus-like sequences. Mixed infections of ilarviruses were also detected in seven Prunus trees. Sanger sequencing of specific RNA1, RNA2, and/or RNA3 genome segments of each virus and total nucleic acid metagenomics NGS confirmed the presence of PNRSV, PDV, ApMV and APLPV detected by RNA2 generic amplicon NGS. However, the two novel groups of Ilarvirus-like RNA2 amplicon sequences detected by the generic amplicon NGS could not be associated to the presence of sequence from RNA1 or RNA3 genome segments or full Ilarvirus genomes, and their origin is unclear. This work highlights the sensitivity of genus-specific amplicon NGS in detection of virus sequences and their distinct populations in multiple samples, and the need for a standardized approach to accurately determine what constitutes an active, viable virus infection after detection by molecular based methods.

PCR amplicon next generation sequencing (NGS) analysis offers a broadly applicable and targeted approach to detect populations of both high- or low-frequency virus variants in one or more plant samples. In this study, amplicon NGS was used to explore the diversity of the tripartite genome virus, Prunus necrotic ringspot virus (PNRSV) from 53 PNRSV-infected trees using amplicons from conserved gene regions of each of PNRSV RNA1, RNA2 and RNA3. Sequencing of the amplicons from 53 PNRSV-infected trees revealed differing levels of polymorphism across the three different components of the PNRSV genome with a total number of 5040, 2083 and 5486 sequence variants observed for RNA1, RNA2 and RNA3 respectively. The RNA2 had the lowest diversity of sequences compared to RNA1 and RNA3, reflecting the lack of flexibility tolerated by the replicase gene that is encoded by this RNA component. Distinct PNRSV phylo-groups, consisting of closely related clusters of sequence variants, were observed in each of PNRSV RNA1, RNA2 and RNA3. Most plant samples had a single phylo-group for each RNA component. Haplotype network analysis showed that smaller clusters of PNRSV sequence variants were genetically connected to the largest sequence variant cluster within a phylo-group of each RNA component. Some plant samples had sequence variants occurring in multiple PNRSV phylo-groups in at least one of each RNA and these phylo-groups formed distinct clades that represent PNRSV genetic strains. Variants within the same phylo-group of each Prunus plant sample had ≥97% similarity and phylo-groups within a Prunus plant sample and between samples had less ≤97% similarity. Based on the analysis of diversity, a definition of a PNRSV genetic strain was proposed. The proposed definition was applied to determine the number of PNRSV genetic strains in each of the plant samples and the complexity in defining genetic strains in multipartite genome viruses was explored.

Pospiviroid species are transmitted through capsicum and tomato seeds. Trade in these seeds represents a route for the viroids to invade new regions, but the magnitude of this hazard has not been adequately investigated. Since 2012, tomato seed lots sent to Australia have been tested for pospiviroids before they are released from border quarantine, and capsicum seed lots have been similarly tested in quarantine since 2013. Altogether, more than 2000 seed lots have been tested. Pospiviroids were detected in more than 10% of the seed lots in the first years of mandatory testing, but the proportion of lots that were infected declined in subsequent years to less than 5%. Six pospiviroid species were detected: Citrus exocortis viroid, Columnea latent viroid, Pepper chat fruit viroid, Potato spindle tuber viroid, Tomato chlorotic dwarf viroid and Tomato apical stunt viroid. They were detected in seed lots exported from 18 countries from every production region. In many seed lots, the detectable fraction (prevalence) of infected seeds was estimated to be very small, as low as 6 × 10−5 (~1 in 16,000; CI 5 × 10−6 to 2.5 × 10−4) for some lots. These findings raise questions about seed production practices, and the study indicates the geographic distributions of these pathogens are uncertain, and there is a continuing threat of invasion.

Rathayibacter toxicus is a gram-positive bacterium that infects the floral parts of several Poaceae species in Australia. Bacterial ooze is often produced on the surface of infected plants and bacterial galls are produced in place of seed. R. toxicus is a regulated plant pathogen in the U.S. yet reliable detection and diagnostic tools are lacking. To better understand this geographically-isolated plant pathogen, genetic variation as a function of geographic location, host species, and date of isolation was determined for isolates collected over a forty-year period. Discriminant analyses of recently collected and archived isolates using Multi-Locus Sequence Typing (MLST) and Inter-Simple Sequence Repeats (ISSR) identified three populations of R. toxicus; RT-I and RT-II from South Australia and RT-III from Western Australia. Population RT-I, detected in 2013 and 2014 from the Yorke Peninsula in South Australia, is a newly emerged population of R. toxicus not previously reported. Commonly used housekeeping genes failed to discriminate among the R. toxicus isolates. However, strategically selected and genome-dispersed MLST genes representing an array of cellular functions from chromosome replication, antibiotic resistance and biosynthetic pathways to bacterial acquired immunity were discriminative. Genetic variation among isolates within the RT-I population was less than the within-population variation for the previously reported RT-II and RT-III populations. The lower relative genetic variation within the RT-I population and its absence from sampling over the past 40 years suggest its recent emergence. RT-I was the dominant population on the Yorke Peninsula during the 2013-2014 sampling period perhaps indicating a competitive advantage over the previously detected RT-II population. The potential for introduction of this bacterial plant pathogen into new geographic areas provide a rationale for understanding the ecological and evolutionary trajectories of R. toxicus.

Apple mosaic virus (ApMV) and prune dwarf virus (PDV) are amongst the most common viruses infecting Prunus species worldwide but their incidence and genetic diversity in Australia is not known. In a survey of 127 Prunus tree samples collected from five states in Australia, ApMV and PDV occurred in 4 (3%) and 13 (10%) of the trees respectively. High-throughput sequencing (HTS) of amplicons from partial conserved regions of RNA1, RNA2, and RNA3, encoding the methyltransferase (MT), RNA-dependent RNA polymerase (RdRp), and the coat protein (CP) genes respectively, of ApMV and PDV was used to determine the genetic diversity of the Australian isolates of each virus. Phylogenetic comparison of Australian ApMV and PDV amplicon HTS variants and full length genomes of both viruses with isolates occurring in other countries identified genetic strains of each virus occurring in Australia. A single Australian Prunus infecting ApMV genetic strain was identified as all ApMV isolates sequence variants formed a single phylogenetic group in each of RNA1, RNA2, and RNA3. Two Australian PDV genetic strains were identified based on the combination of observed phylogenetic groups in each of RNA1, RNA2, and RNA3 and one Prunus tree had both strains. The accuracy of amplicon sequence variants phylogenetic analysis based on segments of each virus RNA were confirmed by phylogenetic analysis of full length genome sequences of Australian ApMV and PDV isolates and all published ApMV and PDV genomes from other countries.

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 HopX1(Ea) and a putative secondary metabolite pathway only present in Rubus-infecting strains.

Human activity is causing new encounters between viruses and plants. Anthropogenic interventions include changing land use, decreasing biodiversity, trade, the introduction of new plant and vector species to native landscapes, and changing atmospheric and climatic conditions. The discovery of thousands of new viruses, especially those associated with healthy-appearing native plants, is shifting the paradigm for their role within the ecosystem from foe to friend. The cost of new plant virus incursions can be high and result in the loss of trade and/or production for short or extended periods. We present and justify three recommendations for plant biosecurity to improve communication about plant viruses, assist with the identification of viruses and their impacts, and protect the high economic, social, environmental, and cultural value of our respective nations' unique flora: 1) As part of the burden of proof, countries and jurisdictions should identify what pests already exist in, and which pests pose a risk to, their native flora; 2) Plant virus sequences not associated with a recognized virus infection are designated as "uncultured virus" and tentatively named using the host plant species of greatest known prevalence, the word "virus," a general location identifier, and a serial number; and 3) Invest in basic research to determine the ecology of known and new viruses with existing and potential new plant hosts and vectors and develop host-virus pathogenicity prediction tools. These recommendations have implications for researchers, risk analysts, biosecurity authorities, and policy makers at both a national and an international level.