AbstractEmerging and re-emerging plant diseases continue to present multifarious threats to global food security. Considerable recent efforts are therefore being channeled towards understanding the nature of pathogen emergence, their spread and evolution.Xanthomonas euvesicatoriapv.perforans (Xep), one of the causal agents of bacterial spot of tomato, rapidly emerged and displaced other bacterial spot xanthomonads in tomato production regions around the world. In less than three decades, it has become a dominant xanthomonad pathogen in tomato production systems across the world and presents a model for understanding diversification of recently emerged bacterial plant pathogens. AlthoughXephas been continuously monitored in Florida since its discovery, the global population structure and evolution at the genome-scale is yet to be fully explored. The objectives of this work were to determine genetic diversity globally to ascertain if different tomato production regions contain genetically distinctXeppopulations, to examine genetic relatedness of strains collected in tomato seed production areas in East Asia and other production regions, and to evaluate variation in type III effectors, which are critical pathogenicity and virulence factors, in relationship to population structure. We used genome data from 270 strains from 13 countries for phylogenetic analysis and characterization ofXopeffector gene diversity among strains. Our results showed notable genetic diversity in the pathogen. We found genetically similar strains in distant tomato production regions, including seed production regions, and diversification over the past 100 years, which is consistent with intercontinental dissemination of the pathogen in hybrid tomato production chains. Evolution of theXeppangenome, including the acquisition and loss of type III secreted effectors, is apparent within and among phylogenetic lineages. The apparent long-distance movement of the pathogen, together with variants that may not yet be widely distributed, poses risks of emergence of new variants in tomato production.

Xylella fastidiosa is a plant-pathogenic bacterium that poses a serious threat to the production of economically important plant species including grapes, almonds, olives and a broad range of amenity plants, causing significant economic losses worldwide. While multiple molecular detection assays have been developed for X. fastidiosa, there is a lack of molecular tools available for detection and differentiation of the closely related pear pathogen, Xylella taiwanensis. In this study, we present a novel conventional PCR assay with primers that can amplify both Xylella species. The amplified product could be sequenced and used for discrimination between the two species and the subspecies within the fastidiosa species. This PCR assay was designed using a genome-informed approach to target the ComEC/Rec2 gene of both Xylella species, ensuring a higher specificity than other previously developed PCR assays. A test performance study across five national plant diagnostic laboratories in Australia and New Zealand demonstrated this assay's high sensitivity and specificity to all known species and subspecies within the Xylella genus. This PCR assay can be used for Xylella identification at the species and subspecies level and is compatible with Sanger sequencing and nanopore sequencing for rapid turnaround time. The newly developed conventional PCR assay presented here offers rapid detection and accurate identification of both Xylella species from plant, insect vector or bacterial samples, enabling timely implementation of biosecurity measures or disease management responses.

High-quality complete genomes of five Xylella fastidiosa strains were assembled by combining Nanopore and Illumina sequencing data. Among these, International Collection of Micro-organisms from Plants (ICMP) 8731, ICMP 8742 and ICMP 8745 belong to subspecies fastidiosa while ICMP 8739 and ICMP 8740 were determined as subspecies multiplex. The strains were further classified into sequence types.

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. 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. 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.

Zucchini plants with symptoms including twisted petioles, necrotic leaves, crown rot, and internal fruit rot were found in Bundaberg, Australia, at a commercial field for the first time during late autumn 2016, resulting in direct yield losses of 70 to 80%. Three Pseudomonas syringae strains isolated from symptomatic leaf (KL004-k1), crown (77-4C), and fruit (KFR003-1) were characterized and their pathogenicity evaluated on pumpkin, rockmelon, squash, and zucchini. Biochemical assays showed typical results for P. syringae. The three isolates differed, however, in that two produced fluorescent pigment (KFR003-1 and 77-4C) whereas the third, KL004-k1, was nonfluorescent. Multilocus sequence analysis classified the isolates to phylogroup 2b. The single-nucleotide polymorphism analysis of core genome from the Australian and closely related international isolates of P. syringae showed two separate clusters. The Australian isolates were clustered based on fluorescent phenotype. Pathogenicity tests demonstrated that all three isolates moved systemically within the inoculated plants and induced necrotic leaf symptoms in zucchini plants. Their identities were confirmed with specific PCR assays for P. syringae and phylogroup 2. Pathogenicity experiments also showed that the Eva variety of zucchini was more susceptible than the Rosa variety for all three isolates. Isolate KL004-k1 was more virulent than 77-4C on pumpkin, rockmelon, squash, and zucchini. This study expands the knowledge of P. syringae isolates that infect cucurbits and provides useful information for growers about the relative susceptibility of a range of cucurbit species.

Recent studies into the distribution of Xanthomonas species causing Bacterial Leaf Spot (BLS) in Australian solanaceous crops detail varied genomic profiles that may influence pathogenicity. These genomic studies are expanded upon here by reporting the pathogenicity, race and copper tolerance of the previously sequenced Xanthomonas strains. Capsicum (Yolo Wonder), tomato (Grosse Lisse) and differential lines of capsicum (Early Cal-Wonder) were used to determine pathogenicity and race. Copper tolerance of 44 Xanthomonas strains was measured by observing bacterial growth on copper sulphate amended media. Protein sequence associated with these traits was detected using genomic analysis and compared using protein alignments. Only strains of X. euvesicatoria (16 strains) were found to be pathogenic on both tomato and capsicum. These were determined to be race 4 and 9. High copper tolerance was detected in the majority of Xanthomonas strains tested. Multiple copper resistance and avirulence proteins were detected in genomic sequence. Relatively few of these were associated with plasmid sequences. The genomic basis for copper tolerance was determined to be complex, as the tolerance thresholds did not directly correlate with gene number or presence. Similarly, pathogenicity of the strains was also not always clearly linked with presence or absence of specific Avr genes. This study highlights the need for detailed and ongoing investigations into the function of these proteins and how they produce the phenotypes that affect crop production.

Cotton leaf curl Gezira virus (CLCuGeV), in association with a DNA-beta satellite, is reported as a causal agent of cotton leaf curl disease. This study reports CLCuGeV detection in field samples of Capsicum annuum and Cucumis melo from Egypt and represents an expansion of the virus host range beyond the Malvaceae family to the two plant families, Solanaceae and Cucurbitaceae .

Bacterial leaf spot (BLS) is a disease of tomato, chilli and capsicum caused by four species of Xanthomonas. BLS can cause severe crop damage and is a limiting factor of production across the world. The recent taxonomic reclassification of these bacterial species has made it necessary to review the species associated with BLS in Australia. Bacterial isolates associated with disease symptoms have been collected from fields in major production areas of Australia. This collection was supplemented with isolates from NSW and the Queensland herbarium culture collection. These isolates were identified to species using a variety of techniques, including biochemical tests, PCR, MLSA and comparative genomics. At least four species of BLS causing Xanthomonads have been identified in Australia. Comparative genomic studies of 44 sequenced genomes have shown that there is considerable diversity in these pathogen populations. Pathogen race determination and copper tolerance studies will provide functional information about the behaviour of these species and complement the genetic information available. The impact of these species on production and the biosecurity measures required to protect the Australian industry from exotic BLS strains will be discussed.

Current industry standards for phytobacteria control include copper protectants and plant host resistance. The effectiveness of copper to manage bacterial diseases is often poor, despite registered application rates being about ten times greater than required to inhibit bacterial growth in vitro. This discrepancy between in vitro compared to in-field bacterial suppression is related to the low availability of the bactericidal form of copper (i.e Cu2+) in field environments. This is further exacerbated by copper tolerant bacterial populations. Host resistance, although an effective strategy to control bacterial diseases, is limited by low availability of suitable resistance genes which are often also overcome by pathogen evolution. Previous research has indicated plant oils could be useful as an alternative for disease control (1-3). To identify new products for disease control, oil from clove, coriander, fennel, lavender, oregano and thyme were evaluated in vitro for efficacy to suppress Australian isolates of Pseudomonas syringae pv. porri, P. syringae pv. syringae, Xanthomonas euvesicatoria and X. vesicatoria, either as a volatile gas or through direct contact. Selected oils were then tested in pot trials to evaluate disease control in capsicum plants to X. euvesicatoria. An overview of the limitations of copper for disease control and results of potential oils for control of foliar phytobacteria will be presented.

Background The genetic diversity in Australian populations of Xanthomonas species associated with bacterial leaf spot in tomato, capsicum and chilli were compared to worldwide bacterial populations. The aim of this study was to confirm the identities of these Australian Xanthomonas species and classify them in comparison to overseas isolates. Analysis of whole genome sequence allows for the investigation of bacterial population structure, pathogenicity and gene exchange, resulting in better management strategies and biosecurity. Results Phylogenetic analysis of the core genome alignments and SNP data grouped strains in distinct clades. Patterns observed in average nucleotide identity, pan genome structure, effector and carbohydrate active enzyme profiles reflected the whole genome phylogeny and highlight taxonomic issues in X. perforans and X. euvesicatoria . Circular sequences with similarity to previously characterised plasmids were identified, and plasmids of similar sizes were isolated. Potential false positive and false negative plasmid assemblies were discussed. Effector patterns that may influence virulence on host plant species were analysed in pathogenic and non-pathogenic xanthomonads. Conclusions The phylogeny presented here confirmed X. vesicatoria , X. arboricola , X. euvesicatoria and X. perforans and a clade of an uncharacterised Xanthomonas species shown to be genetically distinct from all other strains of this study. The taxonomic status of X. perforans and X. euvesicatoria as one species is discussed in relation to whole genome phylogeny and phenotypic traits. The patterns evident in enzyme and plasmid profiles indicate worldwide exchange of genetic material with the potential to introduce new virulence elements into local bacterial populations.

Several species of Xanthomonas cause bacterial leaf spot, a disease that affects solanaceous crops worldwide. The diversity of 64 Australian isolates of Xanthomonas spp. associated with bacterial leaf spot in tomato, capsicum and chilli crops in eastern Australia was determined using multi-locus sequence analysis of atpD, dnaK, efp and gyrB genes, species-specific PCR assays and biochemical analyses. At least five species of Xanthomonas associated with bacterial leaf spot were identified in Australian tomato, capsicum and chilli crops and their pathogenicity assessed. Phylogenetic and biochemical analyses identified X. euvesicatoria, X. perforans and X. vesicatoria as the most frequently recovered pathogenic species. Non-pathogenic and weakly pathogenic species were also identified. The suitability of the identification methods used and the implications of the detection of these species will be discussed.

The first record of Pseudomonas syringae pv. porri on onion in Australia was confirmed in September 2012. This pathogen was previously described in Australia on leek, and pathogenicity tests determined it could infect other allium species Isolates were recovered from water-soaked lesions on onion leaf material collected from the Lockyer Valley, Queensland, Australia. The disease was observed on onion and shallot in the same area the previous year. The isolates were characterized as P syringae by biochemical tests and confirmed as the causal pathogen by Koch's postulates. Sequences of the CFL and 16S genes from these isolates had high identity to P. syringae pv. porri from Australian leek plants and a non-fluorescent pathovar of P syringae on onion reported from the United States. Surveys of the Lockyer Valley and preliminary epidemiology studies were conducted to determine the range and severity of field infections. The disease was widespread across the region and favoured cool, humid conditions. A period of heat may effectively halt disease development, though precise effects of temperature require further investigation. A distinct 'yellow leaf' symptom was observed in many field infections and may be a response to toxins, as toxin production has been reported in several other pathovars of P syringae. This study developed a reliable method for inoculation in brown onion, red onion, and leek. Opportunities for further study will be detailed, including seed testing methods, efficacy of control methods, pathogen survivability, and toxin production. The outcomes of this project will ultimately improve the productivity of the onion industry, and develop diagnostic and response protocols for endemic and exotic bacterial pathogens of allium crops.