Exploiting long read sequencing to detect azole fungicide resistance mutations in Pyrenophora teres using unique molecular identifiers.

Resistance to fungicides is a global challenge as target proteins under selection can evolve rapidly, reducing fungicide efficacy. To manage resistance, detection technologies must be fast and flexible enough to cope with a rapidly increasing number of mutations. The most important agricultural fungicides are azoles that target the ergosterol biosynthetic enzyme sterol 14α-demethylase (CYP51). Mutations associated with azole resistance in the Cyp51 promoter and coding sequence can co-occur in the same allele at different positions and codons, increasing the complexity of resistance detection. Resistance mutations arise rapidly and cannot be detected using traditional amplification-based methods if they are not known. To capture the complexity of azole resistance in two net blotch pathogens of barley we used the Oxford Nanopore MinION to sequence the promoter and coding sequence of Cyp51A. This approach detected all currently known mutations from biologically complex samples increasing the simplicity of resistance detection as multiple alleles can be profiled in a single assay. With the mobility and decreasing cost of long read sequencing, we demonstrate this approach is broadly applicable for characterizing resistance within known agrochemical target sites.

Virulence Profiles and Genome-Wide Association Study for Ascochyta lentis Isolates Collected from Australian Lentil-Growing Regions.

Ascochyta lentis, the causal organism of Ascochyta blight (AB) of lentil (Lens culinaris), has been shown to produce an avirulence effector protein that mediates AB resistance in certain lentil cultivars. The two known forms of the effector protein were identified from a biparental mapping population between isolates that have reciprocal virulence on 'PBA Hurricane XT' and 'Nipper'. The effector AlAvr1-1 was described for the PBA Hurricane XT-avirulent isolate P94-24 and AlAvr1-2 characterized in the PBA Hurricane XT-virulent isolate AlKewell. Here, we performed a genome-wide association study to identify other loci associated with AB for a differential set of lentil cultivars from a diverse panel of isolates collected in the Australian lentil-growing regions from 2013 to 2020. The chromosome 3 AlAvr1 locus was strongly associated with the PBA Hurricane XT, 'Indianhead', and Nipper disease responses, but one other genomic region on chromosome 11 was also associated with the Nipper disease trait. Our results corroborate earlier work that identified the AlAvr1 locus for field-collected isolates that span the period before release and after widespread adoption of PBA Hurricane XT. A multiplex PCR assay was developed to differentiate the genes AlAvr1-1 and AlAvr1-2 to predict PBA Hurricane XT avirulence and pathotype designation in the diversity panel. Increasing numbers of the PBA Hurricane XT-virulent pathotype 2 isolates across that time indicate strong selection for isolates with the AlAvr1-2 allele. Furthermore, one other region of the A. lentis genome may contribute to the pathogen-host interaction for lentil AB.

Field Pea (Pisum sativum) Germplasm Screening for Seedling Ascochyta Blight Resistance and Genome-Wide Association Studies Reveal Loci Associated with Resistance to Peyronellaea pinodes and Ascochyta koolunga.

Ascochyta blight is a damaging disease that affects the stems, leaves, and pods of field pea (Pisum sativum) and impacts yield and grain quality. In Australia, field pea Ascochyta blight is primarily caused by the necrotrophic fungal species Peyronellaea pinodes and Ascochyta koolunga. In this study, we screened 1,276 Pisum spp. germplasm accessions in seedling disease assays with a mix of three isolates of P. pinodes and 641 accessions with three mixed isolates of A. koolunga (513 accessions were screened with both species). A selection of three P. sativum accessions with low disease scores for either pathogen, or in some cases both, were crossed with Australian field pea varieties PBA Gunyah and PBA Oura, and recombinant inbred line populations were made. Populations at the F3:4 and F4:5 generation were phenotyped for their disease response to P. pinodes and A. koolunga, and genotypes were determined using the diversity arrays technology genotyping method. Marker-trait associations were identified using a genome-wide association study approach. Trait-associated loci were mapped to the published P. sativum genome assembly, and candidate resistance gene analogues were identified in the corresponding genomic regions. One locus on chromosome 2 (LG1) was associated with resistance to P. pinodes, and the 8 Mb genomic region contains 156 genes, two of which are serine/threonine protein kinases, putatively contributing to the resistance trait. A second locus on chromosome 5 (LG3) was associated with resistance to A. koolunga, and the 35 Mb region contains 488 genes, of which five are potential candidate resistance genes, including protein kinases, a mitogen-activated protein kinase, and an ethylene-responsive protein kinase homolog.

Phenotypic and Genotypic Diversity of Ascochyta fabae Populations in Southern Australia.

Ascochyta fabae Speg. is a serious foliar fungal disease of faba bean and a constraint to production worldwide. This study investigated the phenotypic and genotypic diversity of the A. fabae pathogen population in southern Australia and the pathogenic variability of the population was examined on a differential set of faba bean cultivars. The host set was inoculated with 154 A. fabae isolates collected from 2015 to 2018 and a range of disease reactions from high to low aggressiveness was observed. Eighty percent of isolates collected from 2015 to 2018 were categorized as pathogenicity group (PG) PG-2 (pathogenic on Farah) and were detected in every region in each year of collection. Four percent of isolates were non-pathogenic on Farah and designated as PG-1. A small group of isolates (16%) were pathogenic on the most resistant differential cultivars, PBA Samira or Nura, and these isolates were designated PG-3. Mating types of 311 isolates collected between 1991 and 2018 were determined and showed an equal ratio of MAT1-1 and MAT1-2 in the southern Australian population. The genetic diversity and population structure of 305 isolates were examined using DArTseq genotyping, and results suggest no association of genotype with any of the population descriptors viz.: collection year, region, host cultivar, mating type, or PG. A Genome-Wide Association Study (GWAS) was performed to assess genetic association with pathogenicity traits and a significant trait-associated genomic locus for disease in Farah AR and PBA Zahra, and PG was revealed. The high frequency of mating of A. fabae indicated by the wide distribution of the two mating types means changes to virulence genes would be quickly distributed to other genotypes. Continued monitoring of the A. fabae pathogen population through pathogenicity testing will be important to identify any increases in aggressiveness or emergence of novel PGs. GWAS and future genetic studies using biparental mating populations could be useful for identifying virulence genes responsible for the observed changes in pathogenicity.

The novel avirulence effector AlAvr1 from Ascochyta lentis mediates host cultivar specificity of ascochyta blight in lentil.

Ascochyta lentis is a fungal pathogen that causes ascochyta blight in the important grain legume species lentil, but little is known about the molecular mechanism of disease or host specificity. We employed a map-based cloning approach using a biparental A. lentis population to clone the gene AlAvr1-1 that encodes avirulence towards the lentil cultivar PBA Hurricane XT. The mapping population was produced by mating A. lentis isolate P94-24, which is pathogenic on the cultivar Nipper and avirulent towards Hurricane, and the isolate AlKewell, which is pathogenic towards Hurricane but not Nipper. Using agroinfiltration, we found that AlAvr1-1 from the isolate P94-24 causes necrosis in Hurricane but not in Nipper. The homologous corresponding gene in AlKewell, AlAvr1-2, encodes a protein with amino acid variation at 23 sites and four of these sites have been positively selected in the P94-24 branch of the phylogeny. Loss of AlAvr1-1 in a gene knockout experiment produced a P94-24 mutant strain that is virulent on Hurricane. Deletion of AlAvr1-2 in AlKewell led to reduced pathogenicity on Hurricane, suggesting that the gene may contribute to disease in Hurricane. Deletion of AlAvr1-2 did not affect virulence for Nipper and AlAvr1-2 is therefore not an avirulence gene for Nipper. We conclude that the hemibiotrophic pathogen A. lentis has an avirulence effector, AlAvr1-1, that triggers a hypersensitive resistance response in Hurricane. This is the first avirulence gene to be characterized in a legume pathogen from the Pleosporales and may help progress research on other damaging Ascochyta pathogens.

Current population structure and pathogenicity patterns of Ascochyta rabiei in Australia.

Ascochyta blight disease, caused by the necrotrophic fungus Ascochyta rabiei, is a major biotic constraint to chickpea production in Australia and worldwide. Detailed knowledge of the structure of the pathogen population and its potential to adapt to our farming practices is key to informing optimal management of the disease. This includes understanding the molecular diversity among isolates and the frequency and distribution of the isolates that have adapted to overcome host resistance across agroecologically distinct regions. Thanks to continuous monitoring efforts over the past 6 years, a comprehensive collection of A. rabiei isolates was collated from the major Australian chickpea production regions. To determine the molecular structure of the entire population, representative isolates from each collection year and growing region have been genetically characterized using a DArTseq genotyping-by-sequencing approach. The genotyped isolates were further phenotyped to determine their pathogenicity levels against a differential set of chickpea cultivars and genotype-phenotype associations were inferred. Overall, the Australian A. rabiei population displayed a far lower genetic diversity (average Nei's gene diversity of 0.047) than detected in other populations worldwide. This may be explained by the presence of a single mating-type in Australia, MAT1-2, limiting its reproduction to a clonal mode. Despite the low detected molecular diversity, clonal selection appears to have given rise to a subset of adapted isolates that are highly pathogenic on commonly employed resistance sources, and that are occurring at an increasing frequency. Among these, a cluster of genetically similar isolates was identified, with a higher proportion of highly aggressive isolates than in the general population. The discovery of distinct genetic clusters associated with high and low isolate pathogenicity forms the foundation for the development of a molecular pathotyping tool for the Australian A. rabiei population. Application of such a tool, along with continuous monitoring of the genetic structure of the population will provide crucial information for the screening of breeding material and integrated disease management packages.

Reference genome assembly for Australian Ascochyta lentis isolate Al4.

Ascochyta lentis causes ascochyta blight in lentil (Lens culinaris Medik.) and yield loss can be as high as 50%. With careful agronomic management practices, fungicide use, and advances in breeding resistant lentil varieties, disease severity and impact to farmers have been largely controlled. However, evidence from major lentil producing countries, Canada and Australia, suggests that A. lentis isolates can change their virulence profile and level of aggressiveness over time and under different selection pressures. In this paper, we describe the first genome assembly for A. lentis for the Australian isolate Al4, through the integration of data from Illumina and PacBio SMRT sequencing. The Al4 reference genome assembly is almost 42 Mb in size and encodes 11,638 predicted genes. The Al4 genome comprises 21 full-length and gapless chromosomal contigs and two partial chromosome contigs each with one telomere. We predicted 31 secondary metabolite clusters, and 38 putative protein effectors, many of which were classified as having an unknown function. Comparison of A. lentis genome features with the recently published reference assembly for closely related A. rabiei show that genome synteny between these species is highly conserved. However, there are several translocations and inversions of genome sequence. The location of secondary metabolite clusters near transposable element and repeat-rich genomic regions was common for A. lentis as has been reported for other fungal plant pathogens.

Reference Genome Assembly for Australian Ascochyta rabiei Isolate ArME14.

Ascochyta rabiei is the causal organism of ascochyta blight of chickpea and is present in chickpea crops worldwide. Here we report the release of a high-quality PacBio genome assembly for the Australian A. rabiei isolate ArME14. We compare the ArME14 genome assembly with an Illumina assembly for Indian A. rabiei isolate, ArD2. The ArME14 assembly has gapless sequences for nine chromosomes with telomere sequences at both ends and 13 large contig sequences that extend to one telomere. The total length of the ArME14 assembly was 40,927,385 bp, which was 6.26 Mb longer than the ArD2 assembly. Division of the genome by OcculterCut into GC-balanced and AT-dominant segments reveals 21% of the genome contains gene-sparse, AT-rich isochores. Transposable elements and repetitive DNA sequences in the ArME14 assembly made up 15% of the genome. A total of 11,257 protein-coding genes were predicted compared with 10,596 for ArD2. Many of the predicted genes missing from the ArD2 assembly were in genomic regions adjacent to AT-rich sequence. We compared the complement of predicted transcription factors and secreted proteins for the two A. rabiei genome assemblies and found that the isolates contain almost the same set of proteins. The small number of differences could represent real differences in the gene complement between isolates or possibly result from the different sequencing methods used. Prediction pipelines were applied for carbohydrate-active enzymes, secondary metabolite clusters and putative protein effectors. We predict that ArME14 contains between 450 and 650 CAZymes, 39 putative protein effectors and 26 secondary metabolite clusters.

Characterization of Growth Morphology and Pathology, and Draft Genome Sequencing of Botrytis fabae, the Causal Organism of Chocolate Spot of Faba Bean (Vicia faba L.).

Chocolate spot is a major fungal disease of faba bean caused by the ascomycete fungus, Botrytis fabae. B. fabae is also implicated in botrytis gray mold disease in lentils, along with B. cinerea. Here we have isolated and characterized two B. fabae isolates from chocolate spot lesions on faba bean leaves. In plant disease assays on faba bean and lentil, B. fabae was more aggressive than B. cinerea and we observed variation in susceptibility among a small set of cultivars for both plant hosts. Using light microscopy, we observed a spreading, generalized necrosis response in faba bean toward B. fabae. In contrast, the plant response to B. cinerea was localized to epidermal cells underlying germinated spores and appressoria. In addition to the species characterization of B. fabae, we produced genome assemblies for both B. fabae isolates using Illumina sequencing. Genome sequencing coverage and assembly size for B. fabae isolates, were 27x and 45x, and 43.2 and 44.5 Mb, respectively. Following genome assembly and annotation, carbohydrate-active enzyme (CAZymes) and effector genes were predicted. There were no major differences in the numbers of each of the major classes of CAZymes. We predicted 29 effector genes for B. fabae, and using the same selection criteria for B. cinerea, we predicted 34 putative effector genes. For five of the predicted effector genes, the pairwise dN/dS ratio between orthologs from B. fabae and B. cinerea was greater than 1.0, suggesting positive selection and the potential evolution of molecular mechanisms for host specificity in B. fabae. Furthermore, a homology search of secondary metabolite clusters revealed the absence of the B. cinerea phytotoxin botrydial and several other uncharacterized secondary metabolite biosynthesis genes from B. fabae. Although there were no obvious differences in the number or proportional representation of different transposable element classes, the overall proportion of AT-rich DNA sequence in B. fabae was double that of B. cinerea.

Agrobacterium tumefaciens-mediated transformation and expression of GFP in Ascochyta lentis to characterize ascochyta blight disease progression in lentil.

The plant immune system is made up of a complex response network that involves several lines of defense to fight invading pathogens. Fungal plant pathogens on the other hand, have evolved a range of ways to infect their host. The interaction between Ascochyta lentis and two lentil genotypes was explored to investigate the progression of ascochyta blight (AB) in lentils. In this study, we developed an Agrobacterium tumefaciens-mediated transformation system for A. lentis by constructing a new binary vector, pATMT-GpdGFP, for the constitutive expression of green fluorescent protein (EGFP). Green fluorescence was used as a highly efficient vital marker to study the developmental changes in A. lentis during AB disease progression on the susceptible and resistant lentil accessions, ILL6002 and ILL7537, respectively. The initial infection stages were similar in both the resistant and susceptible accessions where A. lentis uses infection structures such as germ tubes and appressoria to gain entry into the host while the host uses defense mechanisms to prevent pathogen entry. Penetration was observed at the junctions between neighbouring epidermal cells and occasionally, through the stomata. The pathogen attempted to penetrate and colonize ILL7537, but further fungal advancement appeared to be halted, and A. lentis did not enter the mesophyll. Successful entry and colonization of ILL6002 coincided with structural changes in A. lentis and the onset of necrotic lesions 5-7 days post inoculation. Once inside the leaf, A. lentis continued to grow, colonizing all parts of the leaf followed by plant cell collapse. Pycnidia-bearing spores appeared 14 days post inoculation, which marks the completion of the infection cycle. The use of fluorescent proteins in plant pathogenic fungi together with confocal laser scanning microscopy, provide a valuable tool to study the intracellular dynamics, colonization strategy and infection mechanisms during plant-pathogen interaction.