Climate, host and geography shape insect and fungal communities of trees.

Non-native pests, climate change, and their interactions are likely to alter relationships between trees and tree-associated organisms with consequences for forest health. To understand and predict such changes, factors structuring tree-associated communities need to be determined. Here, we analysed the data consisting of records of insects and fungi collected from dormant twigs from 155 tree species at 51 botanical gardens or arboreta in 32 countries. Generalized dissimilarity models revealed similar relative importance of studied climatic, host-related and geographic factors on differences in tree-associated communities. Mean annual temperature, phylogenetic distance between hosts and geographic distance between locations were the major drivers of dissimilarities. The increasing importance of high temperatures on differences in studied communities indicate that climate change could affect tree-associated organisms directly and indirectly through host range shifts. Insect and fungal communities were more similar between closely related vs. distant hosts suggesting that host range shifts may facilitate the emergence of new pests. Moreover, dissimilarities among tree-associated communities increased with geographic distance indicating that human-mediated transport may serve as a pathway of the introductions of new pests. The results of this study highlight the need to limit the establishment of tree pests and increase the resilience of forest ecosystems to changes in climate.

Diagnosis of Fusarium oxysporum f. sp. ciceris causing Fusarium wilt of chickpea using loop-mediated isothermal amplification (LAMP) and conventional end-point PCR.

Fusarium oxysporum (Fo) is ubiquitous in soil and forms a species complex of pathogenic and putatively non-pathogenic strains. Pathogenic strains cause disease in over 150 plant species. Fusarium oxysporum f. sp. ciceris (Foc) is a major fungal pathogen causing Fusarium wilt in chickpeas (Cicer arietinum). In some countries such as Australia, Foc is a high-priority pest of biosecurity concern. Specific, sensitive, robust and rapid diagnostic assays are essential for effective disease management on the farm and serve as an effective biosecurity control measure. We developed and validated a novel and highly specific PCR and a LAMP assay for detecting the Indian Foc race 1 based on a putative effector gene uniquely present in its genome. These assays were assessed against 39 Fo formae speciales and found to be specific, only amplifying the target species, in a portable real-time fluorometer (Genie III) and qPCR machine in under 13 min with an anneal derivative temperature ranging from 87.7 to 88.3 °C. The LAMP assay is sensitive to low levels of target DNA (> 0.009 ng/µl). The expected PCR product size is 143 bp. The LAMP assay developed in this study was simple, fast, sensitive and specific and could be explored for other Foc races due to the uniqueness of this marker to the Foc genome.

Australian native Glycine clandestina seed microbiota hosts a more diverse bacterial community than the domesticated soybean Glycine max.

BACKGROUND: Plant microbiome composition has been demonstrated to change during the domestication of wild plants and it is suggested that this has resulted in loss of plant beneficial microbes. Recently, the seed microbiome of native plants was demonstrated to harbour a more diverse microbiota and shared a common core microbiome with modern cultivars. In this study the composition of the seed-associated bacteria of Glycine clandestina is compared to seed-associated bacteria of Glycine max (soybean). RESULTS: The seed microbiome of the native legume Glycine clandestina (crop wild relative; cwr) was more diverse than that of the domesticated Glycine max and was dominated by the bacterial class Gammaproteobacteria. Both the plant species (cwr vs domesticated) and individual seed accessions were identified as the main driver for this diversity and composition of the microbiota of all Glycine seed lots, with the effect of factor "plant species" exceeded that of "geographical location". A core microbiome was identified between the two Glycine species. A high percentage of the Glycine microbiome was unculturable [G. clandestina (80.8%) and G. max (75.5%)] with only bacteria of a high relative abundance being culturable under the conditions of this study. CONCLUSION: Our results provided novel insights into the structure and diversity of the native Glycine clandestina seed microbiome and how it compares to that of the domesticated crop Glycine max. Beyond that, it also increased our knowledge of the key microbial taxa associated with the core Glycine spp. microbiome, both wild and domesticated. The investigation of this commonality and diversity is a valuable and essential tool in understanding the use of native Glycine spp. for the discovery of new microbes that would be of benefit to domesticated Glycine max cultivars or any other economically important crops. This study has isolated microbes from a crop wild relative that are now available for testing in G. max for beneficial phenotypes.

Production of Primary Metabolites by Rhizopus stolonifer, Causal Agent of Almond Hull Rot Disease.

Species in the fungal genus Rhizopus are able to convert simple sugars into primary metabolites such as fumaric acid, lactic acid, citric acid, and, to a lesser extent, malic acid in the presence of specific carbon and nitrogen sources. This ability has been linked to plant pathogenicity. Rhizopus stolonifer causes hull rot disease in almonds, symptoms of which have been previously associated with the fungus's production of fumaric acid. Six isolates of R. stolonifer taken from infected almond hulls were grown in artificial media amended with one of four carbon sources (glucose, fructose, sucrose, and xylose) and two nitrogen sources (asparagine and ammonium sulphate) chosen based on almond hull composition and used in industry. Proton nuclear magnetic resonance (1H NMR)-based metabolomics identified that R. stolonifer could metabolise glucose, fructose, sucrose, and to a lesser extent xylose, and both nitrogen sources, to produce three metabolites, i.e., fumaric acid, lactic acid, and ethanol, under in vitro conditions. Sugar metabolisation and acid production were significantly influenced by sugar source and isolates, with five isolates depleting glucose most rapidly, followed by fructose, sucrose, and then xylose. The maximum amounts of metabolites were produced when glucose was the carbon source, with fumaric acid produced in higher amounts than lactic acid. Isolate 19A-0069, however, preferred sucrose as the carbon source, and Isolate 19A-0030 produced higher amounts of lactic acid than fumaric acid. This is the first report, to our knowledge, of R. stolonifer producing lactic acid in preference to fumaric acid. Additionally, R. stolonifer isolate 19-0030 was inoculated into Nonpareil almond fruit on trees grown under high- and low-nitrogen and water treatments, and hull compositions of infected and uninfected fruit were analysed using 1H NMR-based metabolomics. Glucose and asparagine content of uninfected hulls was influenced by the nitrogen and water treatments provided to the trees, being higher in the high-nitrogen and water treatments. In infected hulls, glucose and fructose were significantly reduced but not sucrose or xylose. Large amounts of both fumaric and lactic acid were produced, particularly under high-nitrogen treatments. Moreover, almond shoots placed in dilute solutions of fumaric acid or lactic acid developed leaf symptoms very similar to the 'strike' symptoms seen in hull rot disease in the field, suggesting both acids are involved in causing disease.

First report of ryegrass cyst nematode, Heterodera mani, in Tasmania, Australia.

Cyst nematodes of the genus Heterodera are a major group of sedentary plant parasites causing a significant economic impact, restricting production and market access globally (Moens et al. 2018). The ryegrass cyst nematode Heterodera mani is in the Avenae group and is found predominantly in pastures and grasslands in Europe, California, and South Africa. It was first described by Mathews (1971) from Northern Ireland. Known hosts are grasses (family Poaceae), principally Lolium perenne (perennial ryegrass), but also Dactylis glomerata (cat grass) and Festuca pratensis (meadow fescue) (Subbotin et al. 2010). Mowat (1974) reported that H. mani causes negligible damage to the yield of L. perenne in pot trials; however, Maas & Brinkman (1982) determined that it may cause significant damage to spring and autumn-sown perennial ryegrass in field conditions. During a routine examination for potato cyst nematode from a farm near Mawbanna in north-west Tasmania, Australia, several pale to dark brown Heterodera cysts were extracted that were lemon shaped with the presence of a small vulval cone at the posterior end and a distinct neck. The J2 (n=20) stylet length ranged from 24-26 µm with round knobs deeply concave anteriorly, hyaline tail length was 37-42 µm, true tail length ranged from 59-68 µm and total body length varied from 526-559 µm. All the above characters match those described for H. mani (Subbotin et al. 2010). To verify this identification, DNA was extracted from five individual J2 juveniles from a single cyst using QIAamp DNA micro kit (Qiagen®), and two gene regions amplified: internal transcribed spacer region of ribosomal RNA (ITS-rRNA) with primer pair AB28 and TW81 and cytochrome oxidase 1 (CO1) with primer pair JB3 and JB5 (Bowles et al. 1992; Curran et al. 1994; Derycke et al. 2005). One PCR reaction contained 10 µM (1 µl each) of each primer, 12.5 µl of OneTaq® DNA Polymerase and 5 µl of DNA template with a final volume of 25 µl. PCR products were sent for purification and Sanger sequencing at Macrogen (Seoul, Rep. of Korea). All resulting sequences were trimmed, aligned, and analysed using Geneious Prime® 2022.0.1 (www.geneious.com). Five ITS sequences (accessions ON402852-ON402856) and five CO1 sequences (accessions ON402857-ON402861) were submitted to GenBank. These ITS sequences were very similar to each other and exhibited 99.16-100% similarity with that of H. mani isolate from Hamminkeln, Germany (AY148377) (Subbotin et al. 2018). The CO1 sequences exhibited 98.96-100% similarity with that of H. mani isolate from Washington, USA (MG523097) (Subbotin et al. 2003). Obtained sequences were mapped to reference sequences downloaded from NCBI GenBank and maximum likelihood phylogenetic trees were calculated. Due to the lack of further living nematode material, pot experiments were not performed. Such experiments are not feasible in Tasmania currently and transfer of live nematode material to the Australian mainland presents logistic and legal issues. However, morphological and molecular evidence for species determination of H. mani was unequivocal and contributes to the list of cyst nematode species present in Australia. This is the first detection of H. mani in Australia and is a range extension of the species from North America, Africa, and Europe to Australia. The nematode may cause damage to perennial ryegrass in Australia, however, impact on yield still needs to be investigated.

Draft Genome Sequences for Three Ophiostoma Species Acquired during Revisions of Australian Plant Pathogen Reference Collections.

The fungal genus Ophiostoma contains numerous species that share close associations with wood-boring insects, a relationship with important consequences for global biosecurity. Here, we provide draft genomes for three Ophiostoma species within the well-known Ophiostoma ulmi complex. These resources are valuable for future research efforts related to Ophiostoma and the establishment of biosecurity-focused databases.

Corrigendum: Implications of Seed Vault Storage Strategies for Conservation of Seed Bacterial Microbiomes.

[This corrects the article DOI: 10.3389/fmicb.2021.784796.].

The Role of Soil Microbial Diversity in the Conservation of Native Seed Bacterial Microbiomes.

Research into understanding the structure, composition and vertical transmission of crop seed microbiomes has intensified, although there is much less research into the seed microbiomes of crop wild relatives. Our previous study showed that the standard seed storage procedures (e.g., seed drying and storage temperature) can influence the seed microbiome of domesticated Glycine max. In this study, we characterized the seed microbiota of Glycine clandestina, a perennial wild relative of soybean (G. max (L.) Merr.) to expand our understanding about the effect of other storage procedures such as the periodic regeneration of seed stocks to bulk up seed numbers and secure viability on the seed microbiome of said seed. The G. clandestina microbiota was analysed from Generation 1 (G1) and Generation 2 (G2) seed and from mature plant organs grown in two different soil treatments T (treatment [native soil + potting mix]) and C (control [potting mix only]). Our dataset showed that soil microbiota had a strong influence on next generation seed microbiota, with an increased contribution of root microbiota by 90% and seed transmissibility by 36.3% in G2 (T) seed. Interestingly, the G2 seed microbiota primarily consisted of an initially low abundance of taxa present in G1 seed. Overall, our results indicate that seed regeneration can affect the seed microbiome composition and using native soil from the location of the source plant can enhance the conservation of the native seed microbiota.

Worldwide diversity of endophytic fungi and insects associated with dormant tree twigs.

International trade in plants and climate change are two of the main factors causing damaging tree pests (i.e. fungi and insects) to spread into new areas. To mitigate these risks, a large-scale assessment of tree-associated fungi and insects is needed. We present records of endophytic fungi and insects in twigs of 17 angiosperm and gymnosperm genera, from 51 locations in 32 countries worldwide. Endophytic fungi were characterized by high-throughput sequencing of 352 samples from 145 tree species in 28 countries. Insects were reared from 227 samples of 109 tree species in 18 countries and sorted into taxonomic orders and feeding guilds. Herbivorous insects were grouped into morphospecies and were identified using molecular and morphological approaches. This dataset reveals the diversity of tree-associated taxa, as it contains 12,721 fungal Amplicon Sequence Variants and 208 herbivorous insect morphospecies, sampled across broad geographic and climatic gradients and for many tree species. This dataset will facilitate applied and fundamental studies on the distribution of fungal endophytes and insects in trees.

Implications of Seed Vault Storage Strategies for Conservation of Seed Bacterial Microbiomes.

Global seed vaults are important, as they conserve plant genetic resources for future breeding to improve crop yield and quality and to overcome biotic and abiotic stresses. However, little is known about the impact of standard storage procedures, such as seed drying and cold storage on the seed bacterial community, and the ability to recover seed-associated bacteria after storage. In this study, soybean [Glycine max (L.) Merr.] seeds were analyzed to characterize changes in the bacterial community composition and culturability under varying storage conditions. The G. max bacterial microbiome was analyzed from undried seed, dried seed, and seed stored for 0, 3, 6, and 14months. Storage temperatures consisted of -20°C, 4°C, and room temperature (RT), with -20°C being commonly used in seed storage vaults globally. The seed microbiome of G. max was dominated by Gammaproteobacteria under all conditions. Undried seed was dominated by Pantoea (33.9%) and Pseudomonas (51.1%); however, following drying, the abundance of Pseudomonas declined significantly (0.9%), Pantoea increased significantly (73.6%), and four genera previously identified including Pajaroellobacter, Nesterenkonia, env.OPS_17, and Acidibacter were undetectable. Subsequent storage at RT, 4, or -20°C maintained high-abundance Genera at the majority of time points, although RT caused greater fluctuations in abundances. For many of the low-abundance Genera, storage at -20°C resulted in their gradual disappearance, whereas storage at 4°C or RT resulted in their more rapid disappearance. The changes in seed bacterial composition were reflected by cultured bacterial taxa obtained from the stored G. max seed. The main taxa were largely culturable and had similar relative abundance, while many, but not all, of the low-abundance taxa were also culturable. Overall, these results indicate that the initial seed drying affects the seed bacterial composition, suggesting that microbial isolation prior to seed drying is recommended to conserve these microbes. The standard seed storage condition of -20°C is most suitable for conservation of the bacterial seed microbiome, as this storage temperature slows down the loss of seed bacterial diversity over longer time periods, particularly low-abundance taxa.

Restricted Nitrogen and Water Applications in the Orchard Modify the Carbohydrate and Amino Acid Composition of Nonpareil and Carmel Almond Hulls.

Hull rot disease of almond (Prunus dulcis), caused by the fungus Rhizopus stolonifer, is prevalent in well maintained orchards where trees are provided plenty of water and nitrogen to increase the growth and yield. The predominantly grown variety Nonpareil is considered very susceptible to hull rot, while the pollinator variety Carmel is more resistant. Reduced nitrogen rates and restricted irrigation scheduling decreased the incidence and severity of hull rot in Californian orchards. As a part of our research, the hull composition of Australian almond fruits of Nonpareil and Carmel varieties, grown under two levels of irrigation (high and low) and two levels of nitrogen (high and low), were analysed using 1H NMR-based metabolomics. Both Nonpareil and Carmel hulls contained sugars such as glucose, sucrose, fructose and xylose, and amino acids, particularly asparagine. Variety was the major factor with Nonpareil hulls significantly higher in sugars and asparagine than Carmel. Within varieties, nitrogen influenced the relative concentrations of glucose, sucrose and asparagine. In Nonpareil, high nitrogen high water (the control) had relatively high glucose and asparagine content. High nitrogen low water increased the sucrose component, low nitrogen high water increased the glucose component and low nitrogen low water increased the sucrose and asparagine components. In Carmel, however, high nitrogen low water and low nitrogen high water increased sucrose and asparagine, and low nitrogen low water increased sucrose and glucose. Hull rot symptoms are caused by fumaric acid production by R. stolonifer growing within the hull. These changes in the hull composition under different nitrogen and water scenarios have the potential to affect the growth of R. stolonifer and its metabolite production in hull rot disease.

Comparative transcriptomic analysis of races 1, 2, 5 and 6 of Fusarium oxysporum f.sp. pisi in a susceptible pea host identifies differential pathogenicity profiles.

BACKGROUND: The fungal pathogen Fusarium oxysporum f.sp. pisi (Fop) causes Fusarium wilt in peas. There are four races globally: 1, 2, 5 and 6 and all of these races are present in Australia. Molecular infection mechanisms have been studied in a few other F. oxysporum formae speciales; however, there has been no transcriptomic Fop-pea pathosystem study. RESULTS: A transcriptomic study was carried out to understand the molecular pathogenicity differences between the races. Transcriptome analysis at 20 days post-inoculation revealed differences in the differentially expressed genes (DEGs) in the Fop races potentially involved in fungal pathogenicity variations. Most of the DEGs in all the races were engaged in transportation, metabolism, oxidation-reduction, translation, biosynthetic processes, signal transduction, proteolysis, among others. Race 5 expressed the most virulence-associated genes. Most genes encoding for plant cell wall degrading enzymes, CAZymes and effector-like proteins were expressed in race 2. Race 6 expressed the least number of genes at this time point. CONCLUSION: Fop races deploy various factors and complex strategies to mitigate host defences to facilitate colonisation. This investigation provides an overview of the putative pathogenicity genes in different Fop races during the necrotrophic stage of infection. These genes need to be functionally characterised to confirm their pathogenicity/virulence roles and the race-specific genes can be further explored for molecular characterisation.

Ophiostomatoid fungi associated with pine bark beetles and infested pines in south-eastern Australia, including Graphilbum ipis-grandicollis sp. nov.

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

Re-Evaluation of the Podosphaera tridactyla Species Complex in Australia.

The Podosphaera tridactyla species complex is highly variable morphologically and causes powdery mildew on a wide range of Prunus species, including stone fruit. A taxonomic revision of the Po. tridactyla species complex in 2020 identified 12 species, seven of which were newly characterised. In order to clarify which species of this complex are present in Australia, next generation sequencing was used to isolate the fungal ITS+28S and host matK chloroplast gene regions from 56 powdery mildew specimens of stone fruit and ornamental Prunus species accessioned as Po. tridactyla or Oidium sp. in Australian reference collections. The specimens were collected in Australia, Switzerland, Italy and Korea and were collected from 1953 to 2018. Host species were confirmed using matK phylogenetic analysis, which identified that four had been misidentified as Prunus but were actually Malusprunifolia. Podosphaera species were identified using ITS+28S phylogenetic analysis, recognising three Podosphaera species on stone fruit and related ornamental Prunus hosts in Australia. These were Po.pannosa, the rose powdery mildew, and two species in the Po. tridactyla species complex: Po. ampla, which was the predominant species, and a previously unidentified species from peach, which we describe here as Po. cunningtonii.

Colletotrichum Species Causing Anthracnose of Citrus in Australia.

Colletotrichum spp. are important pathogens of citrus that cause dieback of branches and postharvest disease. Globally, several species of Colletotrichum have been identified as causing anthracnose of citrus. One hundred and sixty-eight Colletotrichum isolates were collected from anthracnose symptoms on citrus stems, leaves, and fruit from Victoria, New South Wales, and Queensland, and from State herbaria in Australia. Colletotrichum australianum sp. nov., C. fructicola, C. gloeosporioides, C. karstii, C. siamense, and C. theobromicola were identified using multi-gene phylogenetic analyses based on seven genomic loci (ITS, gapdh, act, tub2, ApMat, gs, and chs-1) in the gloeosporioides complex and five genomic loci (ITS, tub2, act, chs-1, and his3) in the boninense complex, as well as morphological characters. Several isolates pathogenic to chili (Capsicum annuum), previously identified as C. queenslandicum, formed a clade with the citrus isolates described here as C. australianum sp. nov. The spore shape and culture characteristics of the chili and citrus isolates of C. australianum were similar and differed from those of C. queenslandicum. This is the first report of C. theobromicola isolated from citrus and the first detection of C. karstii and C. siamense associated with citrus anthracnose in Australia.

Australia: A Continent Without Native Powdery Mildews? The First Comprehensive Catalog Indicates Recent Introductions and Multiple Host Range Expansion Events, and Leads to the Re-discovery of Salmonomyces as a New Lineage of the Erysiphales.

In contrast to Eurasia and North America, powdery mildews (Ascomycota, Erysiphales) are understudied in Australia. There are over 900 species known globally, with fewer than currently 60 recorded from Australia. Some of the Australian records are doubtful as the identifications were presumptive, being based on host plant-pathogen lists from overseas. The goal of this study was to provide the first comprehensive catalog of all powdery mildew species present in Australia. The project resulted in (i) an up-to-date list of all the taxa that have been identified in Australia based on published DNA barcode sequences prior to this study; (ii) the precise identification of 117 specimens freshly collected from across the country; and (iii) the precise identification of 30 herbarium specimens collected between 1975 and 2013. This study confirmed 42 species representing 10 genera, including two genera and 13 species recorded for the first time in Australia. In Eurasia and North America, the number of powdery mildew species is much higher. Phylogenetic analyses of powdery mildews collected from Acalypha spp. resulted in the transfer of Erysiphe acalyphae to Salmonomyces, a resurrected genus. Salmonomyces acalyphae comb. nov. represents a newly discovered lineage of the Erysiphales. Another taxonomic change is the transfer of Oidium ixodiae to Golovinomyces. Powdery mildew infections have been confirmed on 13 native Australian plant species in the genera Acacia, Acalypha, Cephalotus, Convolvulus, Eucalyptus, Hardenbergia, Ixodia, Jagera, Senecio, and Trema. Most of the causal agents were polyphagous species that infect many other host plants both overseas and in Australia. All powdery mildews infecting native plants in Australia were phylogenetically closely related to species known overseas. The data indicate that Australia is a continent without native powdery mildews, and most, if not all, species have been introduced since the European colonization of the continent.

Rediscovering an old foe: Optimised molecular methods for DNA extraction and sequencing applications for fungarium specimens of powdery mildew (Erysiphales).

The purpose of this study was to identify a reliable DNA extraction protocol to use on 25-year-old powdery mildew specimens from the reference collection VPRI in order to produce high quality sequences suitable to address taxonomic phylogenetic questions. We tested 13 extraction protocols and two library preparation kits and found the combination of the E.Z.N.A.® Forensic DNA kit for DNA extraction and the NuGen Ovation® Ultralow System library preparation kit was the most suitable for this purpose.

Phylogenetic relationship between Australian Fusarium oxysporum isolates and resolving the species complex using the multispecies coalescent model.

BACKGROUND: The Fusarium oxysporum species complex (FOSC) is a ubiquitous group of fungal species readily isolated from agroecosystem and natural ecosystem soils which includes important plant and human pathogens. Genetic relatedness within the complex has been studied by sequencing either the genes or the barcoding gene regions within those genes. Phylogenetic analyses have demonstrated a great deal of diversity which is reflected in the differing number of clades identified: three, five and eight. Genetic limitation within the species in the complex has been studied through Genealogical Concordance Phylogenetic Species Recognition (GCPSR) analyses with varying number of phylogenetic 'species' identified ranging from two to 21. Such differing views have continued to confuse users of these taxonomies. RESULTS: The phylogenetic relationships between Australian F. oxysporum isolates from both natural and agricultural ecosystems were determined using three datasets: whole genome, nuclear genes, and mitochondrial genome sequences. The phylogenies were concordant except for three isolates. There were three concordant clades from all the phylogenies suggesting similar evolutionary history for mitochondrial genome and nuclear genes for the isolates in these three clades. Applying a multispecies coalescent (MSC) model on the eight single copy nuclear protein coding genes from the nuclear gene dataset concluded that the three concordant clades correspond to three phylogenetic species within the FOSC. There was 100% posterior probability support for the formation of three species within the FOSC. This is the first report of using the MSC model to estimate species within the F. oxysporum species complex. The findings from this study were compared with previously published phylogenetics and species delimitation studies. CONCLUSION: Phylogenetic analyses using three different gene datasets from Australian F. oxysporum isolates have all supported the formation of three major clades which delineated into three species. Species 2 (Clade 3) may be called F. oxysporum as it contains the neotype for F. oxysporum.

Molecular Assessment of the Introduction and Spread of Potato Cyst Nematode, Globodera rostochiensis, in Victoria, Australia.

Potato cyst nematodes (PCN) are damaging soilborne quarantine pests of potato in many parts of the world. There are two recognized species, Globodera pallida and G. rostochiensis, with only the latter species-the golden cyst nematode-present in Australia. PCN was first discovered in Australia in 1986 in Western Australia, where it was subsequently eradicated and area freedom for market access was reinstated. In Victoria, PCN was first detected in 1991 east of Melbourne. Since then, it has been found in a small number of localized regions to the south and east. Strict quarantine controls have been in place since each new detection. It has previously been speculated that there were multiple separate introductions of PCN into Victoria. Our study utilized a historic (years 2001 to 2014) PCN cyst reference collection to examine genetic variability of Victorian PCN populations to investigate potential historical origins and subsequent changes in the populations that might inform patterns of spread. DNA was extracted from single larvae dissected from eggs within cysts and screened using nine previously described polymorphic microsatellite markers in two multiplex polymerase chain reaction assays. Sequence variation of the internal transcribed spacer region of the DNA was also assessed and compared with previously published data. A hierarchical sampling strategy was used, comparing variability of larvae within cysts, within paddocks, and between local regions. This sampling revealed very little differentiation between Victorian populations, which share the same microsatellite allelic variation, with differences between local regions probably reflecting changes in allele frequencies over time. Our molecular assessment supports a probable single localized introduction into Victoria followed by limited spread to nearby areas. The Australian PCN examined appear genetically distinct from populations previously sampled worldwide; thus, any new exotic incursions, potentially bringing in additional PCN pathotypes, should be easily differentiated from existing established local PCN populations.