Background: TheFusarium oxysporumspecies 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 AustralianF. oxysporumisolates 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 theF. oxysporumspecies 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 AustralianF. oxysporumisolates have all supported the formation of three major clades which delineated into three species. Species 2 (Clade 3) may be calledF. oxysporumas it contains the neotype forF. oxysporum.

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.

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.

Pathogenic and putatively nonpathogenic isolates of Fusarium oxysporum are ubiquitously present in soils. Pathogenic isolates designated as formae speciales are very host specific. The genes that determine host‐specific pathogenicity may be expected to be similar between strains within a forma specialis . Three different pathways were used for mining putative effectors from 85 assembled genome sequences representing isolates from the agricultural and natural ecosystems. A total of 436 putative effectors were identified, of which 115 were present in all the isolates. The presence–absence phylogeny showed some clustering of isolates based on the ecosystems, while some clustered with isolates from different ecosystems. Isolates from the agricultural and natural ecosystems shared 358 putative effectors. Agricultural isolates had 78 unique putative effectors, while isolates from the natural ecosystems had no unique effectors. Isolates from the natural ecosystems had effectors that have previously been confirmed with virulence functions in other filamentous fungi, suggesting that the natural ecosystems may be acting as a reservoir for pathogenic isolates awaiting compatible hosts. They also possessed putative effectors with domains involved in polysaccharide degradation. The putative effectors in the natural ecosystem isolates may be important for both parasitic and ecological fitness. Further analysis of the three characterized ff. sp.— lycopersici , niveum , and pisi —revealed high conservation in the pathogenicity mechanism in F. oxysporum . However, each strain had unique putative effectors, which may be responsible for host specificity.

Allium spp. (alliums) are susceptible to rot-diseases caused by pathogenic Fusarium spp., including F. proliferatum (FP) and F. oxysporum (FO), which can cause severe crop losses. A series of pathogenicity tests of four FP isolates from garlic (Allium sativum), four FO isolates from garlic and three FO isolates from onion (Allium cepa var. cepa) were conducted on garlic seedlings and cloves, onion seedlings and bulbs, and shallot (Allium cepa var. aggregatum) bulbs to determine the virulence of the isolates. A combination of PCRs and whole-genome sequencing (WGS), using ONT long-read technology, was used to identify genes encoding putative effectors. The FP isolates caused moderate to severe symptoms in garlic and contained homologues of SIX2, CRX1 and CRX2, and either SIX9 or SIX13. The FOC ex onion isolates caused severe disease symptoms in all allium species tested, while FO from garlic caused moderate to severe disease in garlic but only mild symptoms in onion and shallot. Fusarium oxysporum f. sp. cepae ex onion potentially contained homologues of SIX3, SIX5, SIX7, SIX9, SIX10, SIX12, SIX14, C5, CRX1 and CRX2. The most pathogenic FO isolate to garlic was Fo_VPRI44630 ex garlic, which contained SIX9, SIX13, C5, CRX1 and CRX2. The difference in virulence and putative effector profiles suggests evidence of host-associated differentiation, and as such, the f. sp. or race designation between FO ex garlic and FO ex onion should be investigated further. This is an important finding for future research into best management practices and breeding for disease resistance to FO and FP in garlic.