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Verticillium dahliae is an agricultural phytopathogen of notable importance around the world and is increasingly impacting the billion-dollar Australian cotton industry. In diseased Australian cotton, the VCG classification system of V. dahliae does not adequately reflect the disease symptoms observed. The non-defoliating VCG2A isolates have caused significant damage, a problem not readily observed overseas, and the defoliating VCG1A isolates have not always caused widespread severe symptoms as expected. This study examined a selection of V. dahliae isolates derived from diseased cotton plants in NSW, Australia, America and Israel and classified them into groups using Inter-Simple Sequence Repeats (ISSR). A subset of these isolates were tested in a pathogenicity assay using cotton plants. The combined results confirmed that Australia has VCG2A isolates capable of causing significant disease (“defoliating-like”) and that when analysed using ISSR, they are distinct from the non-virulent VCG2A populations. Additionally, there is a group of VCG1A isolates that are able to cause significant damage to cotton plants. The ISSR analysis provides a better understanding of the V. dahliae populations circulating within Australia and is a useful tool for diagnostic use, with a potential use for diagnostic confirmation of a causative strain.

Verticillium dahliae is a soil-borne phytopathogen and the causal agent of Verticillium wilt. It affects many agriculturally important crops around the world, including cotton. In Australia, the billion-dollar cotton industry is increasingly impacted by Verticillium wilt. Internationally it has been reported that the defoliating V. dahliae Vegetative Compatibility Group (VCG) 1A causes severe damage to cotton. In Australia however, the non-defoliating VCG2A is causing more severe damage to crops in fields than the defoliating VCG1A. This review examines the current research to understand the Australian V. dahliae situation, including current classification systems, genetic analyses and management strategies. It appears that virulence cannot be defined solely by VCG in Australian Verticillium dahliae isolates causing disease in cotton, and that the industry must continually adapt their practices in order to keep the disease under control.

Xanthomonas vasicola pv. vasculorum is an emerging bacterial plant pathogen that causes bacterial leaf streak on corn. First described in South Africa in 1949, reports of this pathogen have greatly increased in the past years in South America and in the United States. The rapid spread of this disease in North and South America may be due to more favorable environmental conditions, susceptible hosts and/or genomic changes that favored the spread. To understand whether genetic mechanisms exist behind the recent spread of X. vasicola pv. vasculorum, we used comparative genomics to identify gene acquisitions in X. vasicola pv. vasculorum genomes from the United States and Argentina. We sequenced 41 genomes of X. vasicola pv. vasculorum and the related sorghum-infecting X. vasicola pv. holcicola and performed comparative analyses against all available X. vasicola genomes. Time-measured phylogenetic analyses showed that X. vasicola pv. vasculorum strains from the United States and Argentina are closely related and arose from two introductions to North and South America. Gene content comparisons identified clusters of genes enriched in corn X. vasicola pv. vasculorum that showed evidence of horizontal transfer including one cluster corresponding to a prophage found in all X. vasicola pv. vasculorum strains from the United States and Argentina as well as in X. vasicola pv. holcicola strains. In this work, we explore the genomes of an emerging phytopathogen population as a first step toward identifying genetic changes associated with the emergence. The acquisitions identified may contain virulence determinants or other factors associated with the spread of X. vasicola pv. vasculorum in North and South America and will be the subject of future work.

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.

Background Mass-rearing, domestication and gamma irradiation of tephritid fruit flies used in sterile insect technique (SIT) programmes can negatively impact fly quality and performance. Symbiotic bacteria supplied as probiotics to mass-reared fruit flies may help to overcome some of these issues. However, the effects of tephritid ontogeny, sex, diet and irradiation on their microbiota are not well known. Results We have used next-generation sequencing to characterise the bacterial community composition and structure within Queensland fruit fly, Bactrocera tryoni (Froggatt), by generating 16S rRNA gene amplicon libraries derived from the guts of 58 individual teneral and mature, female and male, sterile and fertile adult flies reared on artificial larval diets in a laboratory or mass-rearing environment, and fed either a full adult diet (i.e. sugar and yeast hydrolysate) or a sugar only adult diet. Overall, the amplicon sequence read volume in tenerals was low and smaller than in mature adult flies. Operational taxonomic units (OTUs), belonging to the families Enterobacteriaceae (8 OTUs) and Acetobacteraceae (1 OTU) were most prevalent. Enterobacteriaceae dominated laboratory-reared tenerals from a colony fed a carrot-based larval diet, while Acetobacteraceae dominated mass-reared tenerals from a production facility colony fed a lucerne chaff based larval diet. As adult flies matured, Enterobacteriaceae became dominant irrespective of larval origin. The inclusion of yeast in the adult diet strengthened this shift away from Acetobacteraceae towards Enterobacteriaceae. Interestingly, irradiation increased 16S rRNA gene sequence read volume. Conclusions Our findings suggest that bacterial populations in fruit flies experience significant bottlenecks during metamorphosis. Gut bacteria in teneral flies were less abundant and less diverse, and impacted by colony origin. In contrast, mature adult flies had selectively increased abundances for some gut bacteria, or acquired these bacteria from the adult diet and environment. Furthermore, irradiation augmented bacterial abundance in mature flies. This implies that either some gut bacteria were compensating for damage caused by irradiation or irradiated flies had lost their ability to regulate bacterial load. Our findings suggest that the adult stage prior to sexual maturity may be ideal to target for probiotic manipulation of fly microbiota to increase fly performance in SIT programmes.

Background The Sterile Insect Technique (SIT) is being applied for the management of economically important pest fruit flies (Diptera: Tephritidae) in a number of countries worldwide. The success and cost effectiveness of SIT depends upon the ability of mass-reared sterilized male insects to successfully copulate with conspecific wild fertile females when released in the field. Methods We conducted a critical analysis of the literature about the tephritid gut microbiome including the advancement of methods for the identification and characterization of microbiota, particularly next generation sequencing, the impacts of irradiation (to induce sterility of flies) and fruit fly rearing, and the use of probiotics to manipulate the fruit fly gut microbiota. Results Domestication, mass-rearing, irradiation and handling, as required in SIT, may change the structure of the fruit flies’ gut microbial community compared to that of wild flies under field conditions. Gut microbiota of tephritids are important in their hosts’ development, performance and physiology. Knowledge of how mass-rearing and associated changes of the microbial community impact the functional role of the bacteria and host biology is limited. Probiotics offer potential to encourage a gut microbial community that limits pathogens, and improves the quality of fruit flies. Conclusions Advances in technologies used to identify and characterize the gut microbiota will continue to expand our understanding of tephritid gut microbial diversity and community composition. Knowledge about the functions of gut microbes will increase through the use of gnotobiotic models, genome sequencing, metagenomics, metatranscriptomics, metabolomics and metaproteomics. The use of probiotics, or manipulation of the gut microbiota, offers significant opportunities to enhance the production of high quality, performing fruit flies in operational SIT programs.

Using a previously described metagenomics dataset of 27 billion reads, we reconstructed over 50 000 metagenome-assembled genomes (MAGs) of organisms resident in the porcine gut, 46.5 % of which were classified as >70 % complete with a

Background Early weaning and intensive farming practices predispose piglets to the development of infectious and often lethal diseases, against which antibiotics are used. Besides contributing to the build-up of antimicrobial resistance, antibiotics are known to modulate the gut microbial composition. As an alternative to antibiotic treatment, studies have previously investigated the potential of probiotics for the prevention of postweaning diarrhea. In order to describe the post-weaning gut microbiota, and to study the effects of two probiotics formulations and of intramuscular antibiotic treatment on the gut microbiota, we sampled and processed over 800 faecal time-series samples from 126 piglets and 42 sows. Results Here we report on the largest shotgun metagenomic dataset of the pig gut lumen microbiome to date, consisting of >8 Tbp of shotgun metagenomic sequencing data. The animal trial, the workflow from sample collection to sample processing, and the preparation of libraries for sequencing, are described in detail. We provide a preliminary analysis of the dataset, centered on a taxonomic profiling of the samples, and a 16S-based beta diversity analysis of the mothers and the piglets in the first 5 weeks after weaning. Conclusions This study was conducted to generate a publicly available databank of the faecal metagenome of weaner piglets aged between 3 and 9 weeks old, treated with different probiotic formulations and intramuscular antibiotic treatment. Besides investigating the effects of the probiotic and intramuscular antibiotic treatment, the dataset can be explored to assess a wide range of ecological questions with regards to antimicrobial resistance, host-associated microbial and phage communities, and their dynamics during the aging of the host.

Using a previously described metagenomics dataset of 27 billion reads, we reconstructed over 50,000 metagenome-assembled genomes (MAGs) of organisms resident in the porcine gut, 46.5% of which were classified as >70% complete with a

Bactrocera tryoni (Froggatt), the Queensland fruit fly (Qfly), is a highly polyphagous tephritid fly that is widespread in Eastern Australia. Qfly physiology is closely linked with its fungal associates, with particular relationship between Qfly nutrition and yeast or yeast-like fungi. Despite animal-associated fungi typically occurring in multi-species communities, Qfly studies have predominately involved the culture and characterisation of single fungal isolates. Further, only two studies have investigated the fungal communities associated with Qfly, and both have used culture-dependant techniques that overlook non-culturable fungi and hence under-represent, and provide a biased interpretation of, the overall fungal community. In order to explore a potentially hidden fungal diversity and complexity within the Qfly mycobiome, we used culture-independent, high-throughput Illumina sequencing techniques to comprehensively, and holistically characterized the fungal community of Qfly larvae and overcome the culture bias. We collected larvae from a range of fruit hosts along the east coast of Australia, and all had a mycobiome dominated by ascomycetes. The most abundant fungal taxa belonged to the genera Pichia (43%), Candida (20%), Hanseniaspora (10%), Zygosaccharomyces (11%) and Penicillium (7%). We also characterized the fungal communities of fruit hosts, and found a strong degree of overlap between larvae and fruit host communities, suggesting that these communities are intimately inter-connected. Our data suggests that larval fungal communities are acquired from surrounding fruit flesh. It is likely that the physiological benefits of Qfly exposure to fungal communities is primarily due to consumption of these fungi, not through syntrophy/symbiosis between fungi and insect ‘host’.

BackgroundEarly weaning and intensive farming practices predispose piglets to the development of infectious and often lethal diseases, against which antibiotics are used. Besides contributing to the build-up of antimicrobial resistance, antibiotics are known to modulate the gut microbial composition. Studies have previously investigated the effects of probiotics as alternatives to antibiotic treatment for the prevention of post-weaning diarrhea. In order to describe the post-weaning gut microbiota, and the effects of two probiotics formulations and of intramuscular antibiotic treatment on the gut microbiota, we processed over 800 faecal time-series samples from 126 piglets and 42 sows, generating over 8Tbp of metagenomic shotgun sequence data. Here we describe the animal trial procedures, the generation of our metagenomic dataset and the analysis of the microbial community composition using a phylogenetic framework.ResultsFactors such as age, litter effects, and breed, by significantly correlating with gut microbial community shifts, can be major confounding factors in the assessment of treatment effects. Intramuscular antibiotic treatment and probiotic treatments were found to correlate with alpha and beta diversity, as well as with a transient establishment of Mollicutes and Lactobacillales, respectively. We found the abundance of certain taxa to correlate with weight gain.ConclusionsOur findings demonstrate that breed, litter, and age, are important contributors to variation in the community composition, and that treatment effects of the antibiotic and probiotic treatments were subtle, while host age was the dominant factor in shaping the gut microbiota of piglets after weaning. The current study shows, by means of a phylogenetic diversity framework, that the post-weaning pig gut microbiome appears 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.

BackgroundIntensive farming practices can increase exposure of animals to infectious agents against which antibiotics are used. Besides leading to antimicrobial resistance (AMR), 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. However, 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.MethodsShotgun metagenomic sequences from over 800 faecal time-series samples derived from 126 piglets and 42 sows were analysed in a phylogenetic framework to characterise the developing gut microbial community composition of post-weaning piglets. We assessed the effects of intramuscular antibiotic treatment and probiotic oral treatment on the diversity of these gut microbial communities using alpha and beta diversity measures.ResultsDifferences 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.DiscussionThe 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.

Intensive pig production systems often rely on the use of antimicrobials and heavy metal feed additives to maintain animal health and welfare. To gain insight into the carriage of antimicrobial resistance genes (ARGs) in the faecal flora of commercially reared healthy swine, we characterised the genome sequences of 117 porcine commensal E. coli that carried the class 1 integrase gene (intI1+). Isolates were sourced from 42 healthy sows and 126 of their offspring from a commercial breeding operation in Australia in 2017. intI1+ E. coli was detected in 28/42 (67%) sows and 90/126 (71%) piglets. Phylogroup A, particularly clonal complex 10, and phylogroup B1 featured prominently in the study collection. ST10, ST20, ST48 and ST361 were the dominant sequence types. Notably, 113/117 isolates (96%) carried three or more ARGs. Genes encoding resistance to -lactams, aminoglycosides, trimethoprim, sulphonamides, tetracyclines and heavy metals were dominant. ARGs encoding resistance to last-line agents, such as carbapenems and third generation cephalosporins, were not detected. IS26, an insertion sequence noted for its ability to capture and mobilise ARGs, was present in 108/117 (92%) intI1+ isolates, and it played a role in determining class 1 integron structure. Our data shows that healthy Australian pig faeces are an important reservoir of multidrug resistant E. coli that carry genes encoding resistance to multiple first-generation antibiotics and virulence-associated genes.

Verticillium dahliae, the causal agent of Verticillium wilt, is a soil-borne ascomycete that infects numerous agriculturally important crops globally, including cotton. As a billion-dollar industry, cotton is economically important to Australia and the management of disease such as Verticillium wilt is key for the success of the industry. Internationally, defoliating V. dahliae isolates belonging to Vegetative Compatibility Group (VCG) 1A cause severe damage to cotton, while non-defoliating VCG2A isolates result in significantly less disease. However, in Australia, VCG2A is causing more severe damage to crops in the field than the defoliating VCG1A. This study aimed to replicate field observations in controlled greenhouse conditions. We examined and compared disease symptoms on a range of Australian commercial cotton varieties when inoculated with different V. dahliae VCGs. Seedlings were root dipped in conidial suspensions and assessed over seven weeks. The final disease score, disease over time and root length were analysed. Plant mortality resulted from both V. dahliae VCG1A and VCG2A isolates across all cotton varieties used, confirming that there are virulent VCG2A isolates present in Australia. To our knowledge, although virulent on other plant hosts, V. dahliae VCG2A has not previously been reported to be highly virulent in cotton. We infer that virulence cannot be defined solely by VCG in Australian V. dahliae isolates causing disease in cotton.

Bactrocera tryoni (Froggatt) (Queensland fruit fly, or &ldquo, Qfly&rdquo, ) 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 (&gt, 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.

Backround Commensal microbes can promote survival and growth of developing insects, and have important fitness implications in adulthood. Insect larvae can acquire commensal microbes through two main routes: by vertical acquisition from maternal deposition of microbes on the eggshells and by horizontal acquisition from the environment where the larvae develop. To date, however, little is known about how microbes acquired through these different routes interact to shape insect development. In the present study, we investigated how vertically and horizontally acquired microbiota influence larval foraging behaviour, development time to pupation and pupal production in the Queensland fruit fly (‘Qfly’), Bactrocera tryoni. Results Both vertically and horizontally acquired microbiota were required to maximise pupal production in Qfly. Moreover, larvae exposed to both vertically and horizontally acquired microbiota pupated sooner than those exposed to no microbiota, or only to horizontally acquired microbiota. Larval foraging behaviour was also influenced by both vertically and horizontally acquired microbiota. Larvae from treatments exposed to neither vertically nor horizontally acquired microbiota spent more time overall on foraging patches than did larvae of other treatments, and most notably had greater preference for diets with extreme protein or sugar compositions. Conclusion The integrity of the microbiota early in life is important for larval foraging behaviour, development time to pupation, and pupal production in Qflies. These findings highlight the complexity of microbial relations in this species, and provide insights to the importance of exposure to microbial communities during laboratory- or mass-rearing of tephritid fruit flies.

AimsDickeya species are high consequence plant pathogenic bacteria listed among the quarantine pathogens of the European Union; associated with potato disease outbreaks and subsequent economic losses worldwide. Early, accurate, and reliable detection of Dickeya spp. is needed to prevent establishment and further dissemination of this pathogen. Therefore, a multiplex TaqMan qPCR was developed for sensitive detection of Dickeya spp. and specifically, D. dianthicola.Methods and ResultsA signature genomic region for the genus Dickeya (mglA/mglC) and unique genomic region for D. dianthicola (alcohol dehydrogenase) were identified using a whole genome based comparative genomics approach. The developed multiplex TaqMan qPCR was validated using extensive inclusivity and exclusivity panels, and naturally/artificially infected samples to confirm broad range detection capability and specificity. Both sensitivity and spiked assays showed detection limit of 10 fg DNA.ConclusionThe developed multiplex assay is sensitive and reliable to detect Dickeya spp. and D. dianthicola with no false positives or false negatives. It was able to detect mixed infection from naturally and artificially infected plant materials.Significance and ImpactThe developed assay will serve as a practical tool for screening of propagative material, monitoring the presence and distribution, and quantification of target pathogens in a breeding program. The assay also has applications in routine diagnostics, biosecurity and microbial forensics.

Gut bacteria play a key role in insect fitness, but the changes in gut microbiome profile across developmental stages of holometabolous insects remains little explored. Understanding changes in the microbiome across life stages is an important step toward understanding the associated shifts in functional relationships and trade-offs. Here, we characterised the microbiome of larvae, pupae, and adults of the highly polyphagous fruit fly Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) using next-generation sequencing. We sampled individuals from colonies that had been recently introduced to the laboratory environment from naturally infested fruits at generations one (‘G1’) and five (‘G5’). Alpha diversity increased across developmental stages at both G1 and G5, with maximum diversity in adults. Community composition changed across developmental stages and between generations. In G1, larval and pupal microbiomes were dominated by the genus Asaia whereas adult microbiomes were dominated by Enterobacter. In G5, larval and pupal microbiomes contained a high relative abundance of Asaia, but pupae also had a high relative abundance of Staphylococcus and Burkholderia, and there were no dominant patterns in adults. Our findings provide insights into the developmental stage-dependent microbiome associations of a polyphagous fly, and how host-symbiont interactions change at each life stage through the transition from nature to laboratory environments.

Insects typically host substantial microbial communities (the ‘microbiome’) that can serve as a vital source of nutrients and also acts as a modulator of immune function. While recent studies have shown that diet is an important influence on the gut microbiome, very little is known about the dynamics underpinning microbial acquisition from natural food sources. Here, we addressed this gap by comparing the microbiome of larvae of the polyphagous fruit fly Bactrocera tryoni (‘Queensland fruit fly’) that were collected from five different fruit types (sapodilla [from two different localities], hog plum, pomegranate, green apple, and quince) from North-east to South-east Australia. Using Next-Generation Sequencing on the Illumina MiSeq platform, we addressed two questions: (1) what bacterial communities are available to B. tryoni larvae from different host fruit; and (2) how does the microbiome vary between B. tryoni larvae and its host fruit? The abundant bacterial taxa were similar for B. tryoni larvae from different fruit despite significant differences in the overall microbial community compositions. Our study suggests that the bacterial community structure of B. tryoni larvae is related less to the host fruit (diet) microbiome and more to vertical transfer of the microbiome during egg laying. Our findings also suggest that geographic location may play a quite limited role in structuring of larval microbiomes. This is the first study to use Next-Generation Sequencing to analyze the microbiome of B. tryoni larvae together with the host fruit, an approach that has enabled greatly increased resolution of relationships between the insect’s microbiome and that of the surrounding host tissues.

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. Electronic supplementary material The online version of this article (10.1186/s12864-019-5600-x) contains supplementary material, which is available to authorized users.

© Copyright © 2019 Wyrsch, Reid, DeMaere, Liu, Chapman, Roy Chowdhury and Djordjevic. IncHI2 ST3 plasmids are known carriers of multiple antimicrobial resistance genes. Complete plasmid sequences from multiple drug resistant Escherichia coli circulating in Australian swine is however limited. Here we sequenced two related IncHI2 ST3 plasmids, pSDE-SvHI2, and pSDC-F2_12BHI2, from phylogenetically unrelated multiple-drug resistant Escherichia coli strains SvETEC (CC23:O157:H19) and F2_12B (ST93:O7:H4) from geographically disparate pig production operations in New South Wales, Australia. Unicycler was used to co-assemble short read (Illumina) and long read (PacBio SMRT) nucleotide sequence data. The plasmids encoded three drug-resistance loci, two of which carried class 1 integrons. One integron, hosting drfA12-orfF-aadA2, was within a hybrid Tn1721/Tn21, with the second residing within a copper/silver resistance transposon, comprising part of an atypical sul3-associated structure. The third resistance locus was flanked by IS15DI and encoded neomycin resistance (neoR). An oqx-encoding transposon (quinolone resistance), similar in structure to Tn6010, was identified only in pSDC-F2_12BHI2. Both plasmids showed high sequence identity to plasmid pSTM6-275, recently described in Salmonella enterica serotype 1,4,[5],12:i:- that has risen to prominence and become endemic in Australia. IncHI2 ST3 plasmids circulating in commensal and pathogenic E. coli from Australian swine belong to a lineage of plasmids often in association with sul3 and host multiple complex antibiotic and metal resistance structures, formed in part by IS26.

Xanthomonas vasicolapv.vasculorum (Xvv)is an emerging bacterial plant pathogen that causes bacterial leaf streak on corn. First described in South Africa in 1949, reports of this bacteria have greatly increased in the past years in South America and in the U.S., where it is now present in most of the corn producing states. Phenotypic characterization showed that the emerging U.S. and South American Xvvpopulations may have increased virulence in corn compared to older strains. To understand the genetic mechanisms behind the increased virulence in this group, we used comparative genomics to identify gene acquisitions inXvvgenomes from the U.S. and Argentina. We sequenced 41 genomes ofXvvand the related sorghum-infectingX. vasicolapv.holcicola(Xvh).A comparison of all availableX. vasicolagenomes showed the phylogenetic relationships in the group and identified clusters of genes associated with the emergingXvvpopulations. The newly acquired gene clusters showed evidence of horizontal transfer toXvvand included candidate virulence factors. One cluster, in particular, corresponded to a prophage transferred fromXvhto allXvvfrom Argentina and the U.S. The prophage contains putative secreted proteins, which represent candidates for virulence determinants in these populations and await further molecular characterization.

In holometabolous insects, adult fitness depends on the quantity and quality of resource acquired at the larval stage. Diverse ecological factors can influence larval resource acquisition, but little is known about how these factors in the larval environment interact to modulate larval development and adult traits. Here, we addressed this gap by considering how key ecological factors of larval density, diet nutritional composition, and microbial growth interact to modulate pupal and adult traits in a polyphagous tephritid fruit fly, Bactrocera tryoni (aka “Queensland fruit fly”). Larvae were allowed to develop at two larval densities (low and high), on diets that were protein‐rich, standard, or sugar‐rich and prepared with or without preservatives to inhibit or encourage microbial growth, respectively. Percentage of adult emergence and adult sex ratio were not affected by the interaction between diet composition, larval density, and preservative treatments, although low preservative content increased adult emergence in sugar‐rich diets but decreased adult emergence in protein‐rich and standard diets. Pupal weight, male and female adult dry weight, and female (but not male) body energetic reserves were affected by a strong three‐way interaction between diet composition, larval density, and preservative treatment, whereby in general, low preservative content increased pupal weight and female lipid storage in sugar‐rich diets particularly at low‐larval density and differentially modulated the decrease in adult body weight caused by larval density across diets. Our findings provide insights into the ecological factors modulating larval development of a polyphagous fly species and shed light into the ecological complexity of the larval developmental environment in frugivorous insects.

Propagating epizootics due to Pilchard herpesvirus (PHV) occurred in the Australian population of pilchard, Sardinops sagax neopilchardus (Steindachner) (Clupeidae), in 1995 and 1998-99, with up to 60% losses. No mortality events have been evident in the ensuing 7 years, one reason for which could be that PHV is now endemic. During 2004, a survey was conducted to establish if PHV was present in pilchards in Australia. The pilchard is a highly active, pelagic schooling fish which is found in subpopulations, creating difficulties for the conduct of surveys. It occurs in Australian coastal waters and embayments below about 25°S latitude, feeds on plankton and is predated by birds, mammals and larger fish. It reaches sexual maturity at 2 years of age, spawns at sea, enters embayments when about 5 months old and returns to sea when about 1 year old. It may live for 6-9 years, reaching a maximum length of 200 mm. It forms schools and may travel up to 30 km per day. Pilchards aggregate in mobile shoals of fish containing large highly mobile schools, which interact randomly and exchange individuals. Four subpopulations were defined for the purposes of this survey based on differences in biological characteristics: south-eastern Queensland/northern New South Wales (NSW), Victoria/South Australia (SA), south coast Western Australia (SWA) and west coast Western Australia (WWA). Specimens were obtained from the catch of commercial fishermen using random sampling where possible. Polymerase chain reaction (PCR) for the detection of PHV was performed after appraising the suitability of all available tests according to their impact on sample size requirements, total survey costs and logistical constraints. In the analysis, estimates of true prevalence (TP) of infection and 95% confidence limits were adjusted from the apparent prevalence estimates provided by PCR results. Percentage TP of PHV and corresponding 95% confidence intervals for the four subpopulations: NSW, SA, SWA and WWA were thus estimated as 0 (0-1.5), 31 (22-43), 42 (31-55) and 29 (20-41), respectively. PHV is now endemic in Australian populations of pilchard. Implications of the findings for fisheries management are discussed.

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.

Background Gut microbiota affects tephritid (Diptera: Tephritidae) fruit fly development, physiology, behavior, and thus the quality of flies mass-reared for the sterile insect technique (SIT), a target-specific, sustainable, environmentally benign form of pest management. The Queensland fruit fly, Bactrocera tryoni (Tephritidae), is a significant horticultural pest in Australia and can be managed with SIT. Little is known about the impacts that laboratory-adaptation (domestication) and mass-rearing have on the tephritid larval gut microbiome. Read lengths of previous fruit fly next-generation sequencing (NGS) studies have limited the resolution of microbiome studies, and the diversity within populations is often overlooked. In this study, we used a new near full-length (> 1300 nt) 16S rRNA gene amplicon NGS approach to characterize gut bacterial communities of individual B. tryoni larvae from two field populations (developing in peaches) and three domesticated populations (mass- or laboratory-reared on artificial diets). Results Near full-length 16S rRNA gene sequences were obtained for 56 B. tryoni larvae. OTU clustering at 99% similarity revealed that gut bacterial diversity was low and significantly lower in domesticated larvae. Bacteria commonly associated with fruit (Acetobacteraceae, Enterobacteriaceae, and Leuconostocaceae) were detected in wild larvae, but were largely absent from domesticated larvae. However, Asaia, an acetic acid bacterium not frequently detected within adult tephritid species, was detected in larvae of both wild and domesticated populations (55 out of 56 larval gut samples). Larvae from the same single peach shared a similar gut bacterial profile, whereas larvae from different peaches collected from the same tree had different gut bacterial profiles. Clustering of the Asaia near full-length sequences at 100% similarity showed that the wild flies from different locations had different Asaia strains. Conclusions Variation in the gut bacterial communities of B. tryoni larvae depends on diet, domestication, and horizontal acquisition. Bacterial variation in wild larvae suggests that more than one bacterial species can perform the same functional role; however, Asaia could be an important gut bacterium in larvae and warrants further study. A greater understanding of the functions of the bacteria detected in larvae could lead to increased fly quality and performance as part of the SIT. Electronic supplementary material The online version of this article (10.1186/s40168-018-0463-y) contains supplementary material, which is available to authorized users.

Porcine faecal waste is a serious environmental pollutant. Carriage of antimicrobial resistance and virulence-associated genes (VAGs) and the zoonotic potential of commensalEscherichia colifrom swine is largely unknown. Furthermore, little is known about the role of commensalE. colias contributors to the mobilisation of antimicrobial resistance genes between food animals and the environment. Here, we report whole genome sequence analysis of 141E. colifrom the faeces of healthy pigs. Most strains belonged to phylogroups A and B1 and carried i) a class 1 integron; ii) VAGs linked with extraintestinal infection in humans; iii) antimicrobial resistance genesblaTEM, aphAl, cmlA, strAB, tet(A)A,dfrA12, dfrA5, sul1, sul2, sul3; iv)IS26;and v) heavy metal resistance genes (merA, cusA, terA). Carriage of the sulphonamide resistance genesul3was notable in this study. The 141 strains belonged to 42 multilocus sequence types, but clonal complex 10 featured prominently. Structurally diverse class 1 integrons that were frequently associated with IS26 carried unique genetic features that were also identified in extraintestinal pathogenicE. coli(ExPEC) from humans. This study provides the first detailed genomic analysis and point of reference for commensalE. coliof porcine origin, facilitating tracking of specific lineages and the mobile resistance genes they carry.Conflict of Interest StatementNone to declare.

Yeasts, often in hydrolyzed form, are key ingredients in the larval and adult diets of tephritid fruit fly colonies. However, very little is known about the presence or role of yeasts in the diets of tephritid fruit flies in nature. Previous studies have identified bacteria but not detected yeasts in the gut of Queensland fruit fly, Bactrocera tryoni (Froggatt), one of Australia's most economically damaging insect pests of horticultural crops and of significant biosecurity concern domestically and internationally. Here we demonstrate that cultivable yeasts are commonly found in the gut of B. tryoni larvae from fruit hosts. Analysis of the ITS1, 5.8S rRNA gene, and ITS2 sequences of randomly selected isolates identified yeasts and yeast-like fungi of the genera Aureobasidium, Candida, Cryptococcus, Hanseniaspora, Pichia, and Starmerella. The prevalence of these yeasts in fruits suggests that larvae consume the yeasts as part of their diet. This work highlights that yeasts should be considered in future tephritid larval gut microbiota studies. Understanding tephritid-microbial symbiont interactions will lead to improvements in artificial diets and the quality of mass-reared tephritids for the sterile insect technique.

Bactrocera papayae Drew & Hancock, Bactrocera philippinensis Drew & Hancock, Bactrocera carambolae Drew & Hancock, and Bactrocera invadens Drew, Tsuruta & White are four horticultural pest tephritid fruit fly species that are highly similar, morphologically and genetically, to the destructive pest, the Oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae). This similarity has rendered the discovery of reliable diagnostic characters problematic, which, in view of the economic importance of these taxa and the international trade implications, has resulted in ongoing difficulties for many areas of plant protection and food security. Consequently, a major international collaborative and integrated multidisciplinary research effort was initiated in 2009 to build upon existing literature with the specific aim of resolving biological species limits among B. papayae, B. philippinensis, B. carambolae, B. invadens and B. dorsalis to overcome constraints to pest management and international trade. Bactrocera philippinensis has recently been synonymized with B. papayae as a result of this initiative and this review corroborates that finding; however, the other names remain in use. While consistent characters have been found to reliably distinguish B. carambolae from B. dorsalis, B. invadens and B. papayae, no such characters have been found to differentiate the latter three putative species. We conclude that B. carambolae is a valid species and that the remaining taxa, B. dorsalis, B. invadens and B. papayae, represent the same species. Thus, we consider B. dorsalis (Hendel) as the senior synonym of B. papayae Drew and Hancock syn.n. and B. invadens Drew, Tsuruta & White syn.n. A redescription of B. dorsalis is provided. Given the agricultural importance of B. dorsalis, this taxonomic decision will have significant global plant biosecurity implications, affecting pest management, quarantine, international trade, postharvest treatment and basic research. Throughout the paper, we emphasize the value of independent and multidisciplinary tools in delimiting species, particularly in complicated cases involving morphologically cryptic taxa.

This study investigated the phylogeny and molecular epidemiology of Australian porcine enterotoxigenic Escherichia coli (ETEC) isolates (n = 70) by performing multilocus sequence typing (MLST), random amplified polymorphic DNA (RAPD) analysis, virulence gene analysis, plasmid, bacteriocin, integron and antimicrobial resistance gene typing, and antimicrobial susceptibility phenotyping. Isolates of the most commonly observed O serogroup (O149) were highly clonal with a lower frequency of antimicrobial resistance compared with the less common O141 serogroup isolates, which were more genetically diverse and resistant to a greater array of antimicrobials. The O149 and O141 isolates belonged to sequence types (STs) ST100 and ST1260, respectively. A small number of new STs were identified for the least common serogroups, including O157 (ST4245), O138 (ST4244), O139 (ST4246) and O8 (ST4247). A high frequency of plasmid replicons was observed among all ETEC isolates. However, O149 isolates predominantly carried IncFIB, I1, HI1 and FIC, whereas O141 isolates carried a more varied array, including IncI1, FIB, FIC, HI1, I1, Y and, most significantly, A/C. O141 isolates also possessed a greater diversity of bacteriocins, with almost one-half of the isolates carrying colicin E3 (44.4%; 12/27) and E7 (48.1%; 13/27). This study shows that Australian porcine ETEC are distinct from isolates obtained in other parts of the world with respect to the MLST profile and the absence of resistance to critically important antimicrobials, including third-generation cephalosporins and fluoroquinolones.