Accurately resolving evolutionary relationships among organisms is a cornerstone of evolutionary biology, with phylogenetic trees serving as critical tools for this purpose. While single-gene phylogenies are widely used, they often fail to capture the full complexity of evolutionary processes, particularly in organisms with complex or variable genomes. Concatenated gene alignments, built from multiple orthologous genes, offer a more robust framework but are challenging to generate, especially in taxa with incomplete or poorly annotated genomes such as fungi. To address this challenge, we present Buscogeny (Busco Phylogeny), an open-source command-line tool that automates the construction of concatenated gene alignments and phylogenetic trees using BUSCO-derived single-copy orthologs. Buscogeny integrates key features including genome quality assessment, ortholog extraction, multiple sequence alignment, gap filtering, and recombination filtering, followed by automated phylogenetic inference. By allowing users to select ortholog datasets of varying resolutions and tailor filtering thresholds, Buscogeny is adaptable to both closely related and more distantly related taxa. We demonstrate the utility of Buscogeny in phylogenetic analyses of both bacterial and fungal genomes, including complex groups such as Alternaria, where annotation and genome completeness vary significantly. The streamlined, reproducible workflow provided by Buscogeny makes BUSCO-based phylogenetic inference accessible to researchers across disciplines, enabling robust evolutionary insights from whole-genome data.

Between 1976 and 2010, four bacterial isolates were collected in New South Wales and Queensland, Australia, and stored as part of routine biosecurity surveillance. Recently, these historic isolates were analysed as part of a larger project to enhance the taxonomic accuracy of our culture collection and improve Australia’s biosecurity preparedness. Three isolates were collected from Fragaria × ananassa , initially identified as Xanthomonas sp., and one from Medicago sativa , identified as Xanthomonas axonopodis subsp. alfalfae . In this study, we employed modern phenotypic and genomic techniques to further characterize these isolates. Matrix-assisted laser desorption ionization–time of flight MS biotyping and Biolog GEN III MicroPlates confirmed that they are members of the Xanthomonas genus but did not allow for species-level classification. Genome-relatedness indices and phylogenetic analysis confirmed that they were Xanthomonas and revealed that they represent three novel species. The maximum average nucleotide identity and digital DNA–DNA hybridization values observed when comparing the four isolates to all Xanthomonas type strains and each other were 93.9% and 50.7%, respectively. Pathogenesis assays confirmed that two of the isolates are not pathogenic to Fragaria , the plant from which they were isolated. Based on these findings, we propose the names Xanthomonas bundabergensis sp. nov. (DAR 80977 T =ICMP 24943), Xanthomonas medicagonis sp. nov. (DAR 35659 T =ICMP 24942) and Xanthomonas tesorieronis sp. nov. (DAR 34887 T =ICMP 24940).

Xylella fastidiosa is a devastating plant pathogenic bacteria known for its broad host range, in contrast to the related species Xylella taiwanensis, which is only known to cause disease in Asian pears. Despite the potential threats they pose to Australian agriculture, diagnostic assays capable of detecting both Xylella species are scarce. Bridging this critical gap, this study presents the development of the X-ComEC qPCR assay that targets a genus-specific DNA sequence, enabling accurate generic detection of all Xylella species. Benchmarking this novel qPCR assay against other published Xylella qPCR assays demonstrated its superior performance. The X-ComEC qPCR assay stands out as the only assay that can accurately detect both X. fastidiosa and X. taiwanensis without cross-reactivity with related bacteria. We have also carried out a comprehensive inter-laboratory test performance study, which demonstrated that the X-ComEC qPCR and the qPCR described by Harper et al. (Development of LAMP and real-time PCR methods for the rapid detection of Xylella fastidiosa for quarantine and field applications; erratum 2013) are highly robust and ready to use in Australia. Combining these two assays into a duplex qPCR enables simultaneous detection and species-level identification of X. fastidiosa and X. taiwanensis. The findings of this study have been incorporated into the Australian National Diagnostic Protocol for Xylella detection, arming diagnostic laboratories with critical knowledge to combat these globally significant pathogens. Overall, the collaborative and systematic approach employed in this study provides a model for developing and validating assays for all plant pathogens.

We report the presence of the emerging plant pathogen ‘ Xanthomonas cannabis ’ in Australia through a comprehensive analysis of five historical isolates and all publicly available genomes of the species. Using comparative genomics, we characterized four isolates collected from Zinnia spp. and one from Cucurbita pepo . Our findings show that the Zinnia isolates form a distinct phylogroup with the pathotype strain of ‘ X. cannabis ’ pv. zinniae . This group possesses genes for the type 3 secretion system (T3SS) and effectors, a variety of genes unique within the species, and nine genomic islands associated with virulence and drug resistance. In contrast, the C. pepo isolate is genetically distinct and lacks the T3SS but contains its own genes unique within the species. Hypersensitivity response assays confirmed the pathogenic potential of all five isolates in black bean, eggplant, green bean, tomato, sunflower, zinnia and zucchini plants. These results highlight the genetic diversity and evolving threat of this pathogen in Australia, underscoring the critical need for ongoing biosecurity surveillance.

Xanthomonas citri comprises phytopathogenic pathovars that can cause disease in mangoes and cashews. X. citri pv. mangiferaeindicae (Xcm) causes mango bacterial black spot and X. citri pv. anacardii (Xca) causes cashew bacterial black spot. Currently, there are a limited number of complete genomes available for these pathovars, hindering pathogenicity studies. Here, we collected 53 isolates of Xcm and Xca from mango hosts and generated 50 Xcm (18 complete and 32 scaffold level) and three complete non-pigmented Xca genome assemblies using Illumina and Nanopore sequencing. We used comparative genomics to identify virulence-associated genes of both pathovars and found that transcription activator-like effectors (TALEs), which aid in host-plant infection, were present in complete and circularised assemblies of Xcm. One to three plasmids were identified amongst the complete Xcm assemblies, while no plasmids were observed in Xca. Analysis of complete and circularised genomes revealed the presence of 14 TALE classes either in chromosome or plasmid positions. Amongst them, only the TalKC class was shared across all strains. Although no plasmid was found in Xca, the TalKS class genes were found in the bacterial chromosome. Virulence-associated genes varied at the interspecies level, with Xca and Xcm shown to have distinct sets of type III effectors. We observed that xopB, xopAG2 and xopAM were present in all Xca strains but absent in Xcm. In contrast, xopAW was present in all Xcm but absent in Xca. Further functional investigation of these genes could reveal those that play a critical role in pathogenicity and/or host specificity.

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.

Xanthomonas citri is a plant-pathogenic bacterium associated with a diverse range of host plant species. It has undergone substantial reclassification and currently consists of 14 different subspecies or pathovars that are responsible for a wide range of plant diseases. Whole-genome sequencing (WGS) provides a cutting-edge advantage over other diagnostic techniques in epidemiological and evolutionary studies of X. citri because it has a higher discriminatory power and is replicable across laboratories. WGS also allows for the improvement of multilocus sequence typing (MLST) schemes. In this study, we used genome sequences of Xanthomonas isolates from the NCBI RefSeq database to develop a seven-gene MLST scheme that yielded 19 sequence types (STs) that correlated with phylogenetic clades of X. citri subspecies or pathovars. Using this MLST scheme, we examined 2,911 Xanthomonas species assemblies from NCBI GenBank and identified 15 novel STs from 37 isolates that were misclassified in NCBI. In total, we identified 545 X. citri assemblies from GenBank with 95% average nucleotide identity to the X. citri type strain, and all were classified as one of the 34 STs. All MLST classifications correlated with a phylogenetic position inferred from alignments using 92 conserved genes. We observed several instances where strains from different pathovars formed closely related monophyletic clades and shared the same ST, indicating that further investigation of the validity of these pathovars is required. Our MLST scheme described here is a robust tool for rapid classification of X. citri pathovars using WGS and a powerful method for further comprehensive taxonomic revision of X. citri pathovars.

In this study, metagenomic sequence data was used to investigate the phytoplasma taxonomic diversity in vegetable-growing regions across Australia. Metagenomic sequencing was performed on 195 phytoplasma-positive samples, originating either from historic collections (n=46) or during collection efforts between January 2015 and June 2022 (n=149). The sampled hosts were classified as crop (n=155), weed (n=24), ornamental (n=7), native plant (n=6), and insect (n=3) species. Most samples came from Queensland (n=78), followed by Western Australia (n=46), the Northern Territory (n=32), New South Wales (n=17), and Victoria (n=10). Of the 195 draft phytoplasma genomes, 178 met our genome criteria for comparison using an average nucleotide identity approach. Ten distinct phytoplasma species were identified and could be classified within the 16SrII, 16SrXII (PCR only), 16SrXXV, and 16SrXXXVIII phytoplasma groups, which have all previously been recorded in Australia. The most commonly detected phytoplasma taxa in this study were species and subspecies classified within the 16SrII group (n=153), followed by strains within the 16SrXXXVIII group (‘Ca. Phytoplasma stylosanthis’; n=6). Several geographic- and host-range expansions were reported, as well as mixed phytoplasma infections of 16SrII taxa and ‘Ca. Phytoplasma stylosanthis’. Additionally, six previously unrecorded 16SrII taxa were identified, including five putative subspecies of ‘Ca. Phytoplasma australasiaticum’ and a new putative 16SrII species. PCR and sequencing of the 16S rRNA gene was a suitable triage tool for preliminary phytoplasma detection. Metagenomic sequencing, however, allowed for higher-resolution identification of the phytoplasmas, including mixed infections, than was afforded by only direct Sanger sequencing of the 16S rRNA gene. Since the metagenomic approach theoretically obtains sequences of all organisms in a sample, this approach was useful to confirm the host family, genus, and/or species. In addition to improving our understanding of the phytoplasma species that affect crop production in Australia, the study also significantly expands the genomic sequence data available in public sequence repositories to contribute to phytoplasma molecular epidemiology studies, revision of taxonomy, and improved diagnostics.

Five bacterial isolates were isolated from Fragaria × ananassa in 1976 in Rydalmere, Australia, during routine biosecurity surveillance. Initially, the results of biochemical characterisation indicated that these isolates represented members of the genus Xanthomonas. To determine their species, further analysis was conducted using both phenotypic and genotypic approaches. Phenotypic analysis involved using MALDI-TOF MS and BIOLOG GEN III microplates, which confirmed that the isolates represented members of the genus Xanthomonas but did not allow them to be classified with respect to species. Genome relatedness indices and the results of extensive phylogenetic analysis confirmed that the isolates were members of the genus Xanthomonas and represented a novel species. On the basis the minimal presence of virulence-associated factors typically found in genomes of members of the genus Xanthomonas, we suggest that these isolates are non-pathogenic. This conclusion was supported by the results of a pathogenicity assay. On the basis of these findings, we propose the name Xanthomonas rydalmerensis, with DAR 34855T = ICMP 24941 as the type strain.

ABSTRACTWe describe five bacterial isolates that were isolated fromFragaria x ananassain 1976 in Rydalmere, Australia, during routine biosecurity surveillance. Initially, biochemical characterisation identified these isolates as members of theXanthomonasgenus. To determine their species, we conducted further analysis using both phenotypic and genotypic approaches. Phenotypic analysis involved using MALDI-TOF MS and BIOLOG GEN III microplates, which confirmed that the isolates belonged to theXanthomonasgenus but could not classify species. Genome relatedness indices and extensive phylogenetic analysis confirmed that the isolates belonged to theXanthomonasgenus and represented a new species. Based on the absence of virulence factors typically found inXanthomonasspp. genomes, we suggest that these isolates are non-pathogenic. This conclusion was supported by a pathogenicity assay. Based on these findings, we propose the nameXanthomonas rydalmerenesis, with DAR34855 = ICMP24941 as the type strain.

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.

We describe five bacterial isolates that were isolated from Fragaria x ananassa in 1976 in Rydalmere, Australia, during routine biosecurity surveillance. Initially, biochemical characterisation identified these isolates as members of the Xanthomonas genus. To determine their species, we conducted further analysis using both phenotypic and genotypic approaches. Phenotypic analysis involved using MALDI-TOF MS and BIOLOG GEN III microplates, which confirmed that the isolates belonged to the Xanthomonas genus but could not classify species. Genome relatedness indices and extensive phylogenetic analysis confirmed that the isolates belonged to the Xanthomonas genus and represented a new species. Based on the absence of virulence factors typically found in Xanthomonas spp. genomes, we suggest that these isolates are non-pathogenic. This conclusion was supported by a pathogenicity assay. Based on these findings, we propose the name Xanthomonas rydalmerenesis , with DAR34855 = ICMP24941 as the type strain.### Competing Interest StatementThe authors have declared no competing interest.

EDITORIAL article Front. Plant Sci., 02 June 2023Sec. Plant Pathogen Interactions Volume 14 - 2023 | https://doi.org/10.3389/fpls.2023.1224293

Cucurbits are an important crop grown across peri-urban, coastal and inland regions of New South Wales, Australia. Viral infection is a persistent issue across cucurbit commodities, different production methods and production regions. In this study, 34 cucurbit growing properties across five production regions of New South Wales were surveyed and sampled repeatedly from 2018 to 2021. Samples were tested for the presence of known endemic viruses using both serological and molecular diagnostic methods. Viral pathogens were detected on 22 of the 34 properties sampled, and in 44% of samples tested. Annual disease incidence ranged from 0 to 90%, typically increasing towards the end of the summer growing season. Papaya ringspot virus, watermelon mosaic virus, and cucumber mosaic virus, were identified as the most frequently detected viruses. Melon necrotic spot virus and beet pseudo yellows virus were detected at low rates. Cases of mixed infections of papaya ringspot virus and watermelon mosaic virus were also detected in some samples. Furthermore, cucumber green mottle mosaic virus, a "notifiable disease", was detected for the first time in New South Wales. A newly described virus, watermelon crinkle leaf associated virus-1, was also detected using next- generation sequencing technology. The latter two virus records represent a geographic range expansion and first report for Australia respectively.

In Australia, recent investigations have reported the presence of a number of Verticillium dahliae pathotypes (VCG1A, VCG2A and VCG6) that cause disease in cotton and agricultural weeds. During these investigations, it was observed that a VCG2A V. dahliae displayed greater virulence than previously reported in local and international isolates. Genome sequencing of seven V. dahliae isolates was performed using a mixture of short and long reads sequencing technologies. Of these seven isolates, four were identified as D VCG1A, one ND VCG2A, a recently identified Australian VCG6 as well as a virulent VCG2A, classified as "defoliating-like". The secreted protein repertoire from the genomes of these Australian V. dahliae isolates was predicted using four separate signal prediction methods. The consensus secreted protein set of the isolates revealed the presence of 20 proteins that were present in all virulent isolates, including the virulent DL VCG2A, and absent in the nonvirulent VCG2A isolates. Ten of these proteins had a functional annotation and were identified as ligninase H8, lipolytic enzyme, laccase, amine oxidase, spherulin-1A, dipeptidyl-peptidase, monooxygenase, dienelactone hydrolase, carboxypeptidase and fibronectin by BLASTP.

Verticillium dahliae was isolated from Noogoora burr (Xanthium occidentale) seeds collected from mature asymptomatic plants in 2017 growing along the riverbank at Condobolin, NSW. V. dahliae was isolated from seeds by transferring surfaced-disinfested seed components onto 25% Potato Dextrose Agar (PDA) plus Novobiocin. Morphological and molecular analysis, as well as fulfilment of Koch's postulates, confirmed the causal pathogen was V. dahliae. Additional analysis was carried out to assign the isolates into a vegetative compatibility group (VCG) and pathotype. This is the first report of V. dahliae VCG6 in Australia infecting X. occidentale.

Intensive farming practices can increase exposure of animals to infectious agents against which antibiotics are used. Orally administered antibiotics are well known to cause dysbiosis. To counteract dysbiotic effects, numerous studies in the past two decades sought to understand whether probiotics are a valid tool to help re-establish a healthy gut microbial community after antibiotic treatment. Although dysbiotic effects of antibiotics are well investigated, little is known about the effects of intramuscular antibiotic treatment on the gut microbiome and a few studies attempted to study treatment effects using phylogenetic diversity analysis techniques. In this study we sought to determine the effects of two probiotic- and one intramuscularly administered antibiotic treatment on the developing gut microbiome of post-weaning piglets between their 3rd and 9th week of life. Shotgun metagenomic sequences from over 800 faecal time-series samples derived from 126 post-weaning piglets and 42 sows were analysed in a phylogenetic framework. Differences between individual hosts such as breed, litter, and age, were found to be important contributors to variation in the community composition. Host age was the dominant factor in shaping the gut microbiota of piglets after weaning. The post-weaning pig gut microbiome appeared to follow a highly structured developmental program with characteristic post-weaning changes that can distinguish hosts that were born as little as two days apart in the second month of life. Treatment effects of the antibiotic and probiotic treatments were found but were subtle and included a higher representation of Mollicutes associated with intramuscular antibiotic treatment, and an increase of Lactobacillus associated with probiotic treatment. The discovery of correlations between experimental factors and microbial community composition is more commonly addressed with OTU-based methods and rarely analysed via phylogenetic diversity measures. The latter method, although less intuitive than the former, suffers less from library size normalization biases, and it proved to be instrumental in this study for the discovery of correlations between microbiome composition and host-, and treatment factors.

Bactrocera tryoni (Froggatt) (Queensland fruit fly, or “Qfly”) is a highly polyphagous tephritid fruit fly and a serious economic pest in Australia. Qfly biology is intimately linked to the bacteria and fungi of its microbiome. While there are numerous studies of the microbiome in larvae and adults, the transition of the microbiome through the pupal stage remains unknown. To address this knowledge gap, we used high-throughput Next-Generation Sequencing (NGS) to examine microbial communities at each developmental stage in the Qfly life cycle, targeting the bacterial 16S rRNA and fungal ITS regions. We found that microbial communities were similar at the larval and pupal stage and were also similar between adult males and females, yet there were marked differences between the larval and adult stages. Specific bacterial and fungal taxa are present in the larvae and adults (fed hydrolyzed yeast with sugar) which is likely related to differences in nutritional biology of these life stages. We observed a significant abundance of the Acetobacteraceae at the family level, both in the larval and pupal stages. Conversely, Enterobacteriaceae was highly abundant (> 80%) only in the adults. The majority of fungal taxa present in Qfly were yeasts or yeast-like fungi. In addition to elucidating changes in the microbiome through developmental stages, this study characterizes the Qfly microbiome present at the establishment of laboratory colonies as they enter the domestication process.

The ability to swiftly respond to pathogen incursions relies heavily on fast and accurate diagnostics. Current published assays for citrus bacterial canker do not target Xanthomonas citri pv. citri, the causative agent, with high specificity when testing Australian samples. While the current diagnostics are useful in countries where canker is endemic, the detection of canker in Australia requires an emergency response. Close relatives to X. citri pv. citri found in Australia may generate false positives with the current recommended diagnostic assays. Therefore, we developed a more specific detection tool for citrus bacterial canker to provide greater diagnostic confidence for surveillance and eradication efforts. We used genomic comparisons of 161 Xanthomonad genomes and identified and confirmed genomic regions specific for X. citri pv. citri by performing local alignments of unique regions to reference genomes. We then developed loop-mediated isothermal amplification primers and validated them against a panel of 190 isolates to confirm specificity. Our diagnostic assay showed 100% corroboration with the concurrently developed multiplex primers and represents an improved diagnostic method capable of effective citrus bacterial canker identification.

Biological collections preserve our past, while helping protect our future and increase future knowledge. Plant bacterial culture collections are our security for domestic and global biosecurity. This feature article will provide an introduction to the global position of plant bacterial collections. The role of collections in monitoring plant pathogenic bacteria will be explored through the presentation of five cases studies. These case studies demonstrate why culture collections were imperative for the outcome in each situation. We discuss what we believe should be the best practices to improve microbial preservation and accessioning rates, and why plant bacterial culture collections must increase deposits to be prepared for future emerging pathogens. This is not only the case for global culture collections, but on a much bigger scale, our future scientific successes, our biosecurity decisions and responses, and our knowledge are contingent upon preserving our valuable bacterial strains. It is hoped that once you read this article, you will see the need to deposit your strains in registered public collections and make a concerted effort to build better bacterial culture collections with us.