Publications by authors named "Richard P Oliver"

112 Publications

High frequency of fungicide resistance-associated mutations in the wheat yellow rust pathogen Puccinia striiformis f. sp. tritici.

Pest Manag Sci 2021 Mar 31. Epub 2021 Mar 31.

John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK.

Background: Reliance on fungicides to manage disease creates selection pressure for the evolution of resistance in fungal and oomycete pathogens. Rust fungi (Pucciniales) are major pathogens of cereals and other crops and have been classified as low-risk for developing resistance to fungicides; no cases of field failure of fungicides in a cereal rust disease has yet been recorded. Recently, the Asian soybean rust pathogen, Phakopsora pachyrhizi evolved resistance to several fungicide classes, prompting us to screen a large sample of the globally widespread wheat yellow rust pathogen, Puccinia striiformis f. sp. tritici (Pst), for mutations associated with fungicide resistance.

Results: We evaluated 363 Pst isolates from Europe, USA, Ethiopia, Chile, China and New Zealand for mutations in the target genes of demethylase inhibitor (DMI; Cyp51) and succinate dehydrogenase inhibitor (SDHI; SdhB, SdhC and SdhD) fungicides. A high proportion of Pst isolates carrying a Y134F DMI resistance-associated substitution in the Cyp51 gene was found among those from China and New Zealand. A set of geographically diverse Pst isolates were also found to display a substitution in SdhC (I85V) that is homologous to that reported recently in P. pachyrhizi and linked to SDHI resistance.

Conclusion: The identification of resistance-associated alleles confirms that cereal rusts are not immune to fungicide resistance and that selection for resistance evolution is operating at high levels in certain locations. It highlights the need to adopt fungicide resistance management practices and to monitor cereal rust species for development of resistance.
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http://dx.doi.org/10.1002/ps.6380DOI Listing
March 2021

Rapid in situ quantification of the strobilurin resistance mutation G143A in the wheat pathogen Blumeria graminis f. sp. tritici.

Sci Rep 2021 Feb 25;11(1):4526. Epub 2021 Feb 25.

Centre for Crop and Disease Management, School of Molecular and Life Sciences, Curtin University, Bentley, Perth, WA, 6102, Australia.

As the incidence of fungicide resistance in plant pathogens continues to increase, control of diseases and the management of resistance would be greatly aided by rapid diagnostic methods. Quantitative allele-specific PCR (ASqPCR) is an ideal technique for the in-field analysis of fungicide resistance as it can quantify the frequency of mutations in fungicide targets. We have applied this technique to the fungal pathogen Blumeria graminis f. sp. tritici (Bgt), the causal agent of wheat powdery mildew. In Australia, strobilurin-resistant Bgt was first discovered in 2016. Molecular analysis revealed a nucleotide transversion in the cytochrome b (cytb) gene in the cytochrome bc1 enzyme complex, resulting in a substitution of alanine for glycine at position 143 (G143A). We have developed an in-field ASqPCR assay that can quantify both the resistant (A143) and sensitive (G143) cytb alleles down to 1.67% in host and Bgt DNA mixtures, within 90 min of sample collection. The in situ analysis of samples collected during a survey in Tasmania revealed A143 frequencies ranging between 9-100%. Validation of the analysis with a newly developed laboratory based digital PCR assay found no significant differences between the two methods. We have successfully developed an in-field quantification method, for a strobilurin-resistant allele, by pairing the ASqPCR assay on a lightweight qPCR instrument with a quick DNA extraction method. The deployment of these type of methodologies in the field can contribute to the effective in-season management of fungicide resistance.
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http://dx.doi.org/10.1038/s41598-021-83981-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7907364PMC
February 2021

Parallel evolution of multiple mechanisms for demethylase inhibitor fungicide resistance in the barley pathogen Pyrenophora teres f. sp. maculata.

Fungal Genet Biol 2020 Dec 6;145:103475. Epub 2020 Oct 6.

Centre for Crop and Disease Management, Curtin University, Bentley, WA 6102, Australia.

The fungal pathogen Pyrenophora teres f. sp. maculata (Ptm), responsible for spot-form of net blotch (SFNB), is currently the most significant disease of barley in Australia and a major disease worldwide. Management of SFNB relies heavily on fungicides and in Australia the demethylase inhibitors (DMIs) predominate. There have been sporadic reports of resistance to DMIs in Ptm but the mechanisms remain obscure. Ptm isolates collected from 1996 to 2019 in Western Australia were tested for fungicide sensitivity levels. Decreased sensitivity to DMIs was observed in isolates collected after 2015. Resistance factors to tebuconazole fell into two classes; moderate resistance (MR; RF 6-11) and high resistance (HR; RFs 30-65). Mutations linked to resistance were detected in the promoter region and coding sequence of the DMI target gene Cyp51A. Solo-LTR insertion elements were found at 5 different locations in the promoter region. Three different non-synonymous mutations encoded an altered protein with a phenylalanine to leucine substitution at position 489, F489L (F495I in the archetype CYP51A of Aspergillus fumigatus). F489L mutations have also been found in DMI-resistant strains of P. teres f. sp. teres. Ptm isolates carrying either a LTR insertion element or a F489L allele displayed the MR1 or MR2 phenotypes, respectively. Isolates carrying both an insertion element and a F489L mutation displayed the HR phenotype. Multiple mechanisms acting both alone and in concert were found to contribute to DMI resistance in Ptm. Moreover, these mutations have emerged repeatedly in Western Australian Ptm populations by a process of parallel evolution.
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http://dx.doi.org/10.1016/j.fgb.2020.103475DOI Listing
December 2020

Genome-Wide Association Mapping of Resistance to Septoria Nodorum Leaf Blotch in a Nordic Spring Wheat Collection.

Plant Genome 2019 11;12(3):1-15

Dep. of Plant Sciences, Norwegian Univ. of Life Sciences, Post Box 5003, NO-1432, ÅS, Norway.

Core Ideas: First genome-wide association mapping of adult plant Septoria nodorum blotch resistance. Some adult plant resistance loci were shared with seedling resistance loci. Other adult plant resistance loci were significant across environments. Resistant haplotypes were identified, which can be used for breeding. Parastagonospora nodorum is the causal agent of Septoria nodorum leaf blotch (SNB) in wheat (Triticum aestivum L.). It is the most important leaf blotch pathogen in Norwegian spring wheat. Several quantitative trait loci (QTL) for SNB susceptibility have been identified. Some of these QTL are the result of underlying gene-for-gene interactions involving necrotrophic effectors (NEs) and corresponding sensitivity (Snn) genes. A collection of diverse spring wheat lines was evaluated for SNB resistance and susceptibility over seven growing seasons in the field. In addition, wheat seedlings were inoculated and infiltrated with culture filtrates (CFs) from four single spore isolates and infiltrated with semipurified NEs (SnToxA, SnTox1, and SnTox3) under greenhouse conditions. In adult plants, the most stable SNB resistance QTL were located on chromosomes 2B, 2D, 4A, 4B, 5A, 6B, 7A, and 7B. The QTL on chromosome 2D was effective most years in the field. At the seedling stage, the most significant QTL after inoculation were located on chromosomes 1A, 1B, 3A, 4B, 5B, 6B, 7A, and 7B. The QTL on chromosomes 3A and 6B were significant both after inoculation and CF infiltration, indicating the presence of novel NE-Snn interactions. The QTL on chromosomes 4B and 7A were significant in both seedlings and adult plants. Correlations between SnToxA sensitivity and disease severity in the field were significant. To our knowledge, this is the first genome-wide association mapping study (GWAS) to investigate SNB resistance at the adult plant stage under field conditions.
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http://dx.doi.org/10.3835/plantgenome2018.12.0105DOI Listing
November 2019

Reference Genome Assembly for Australian Isolate ArME14.

G3 (Bethesda) 2020 07 7;10(7):2131-2140. Epub 2020 Jul 7.

Centre for Crop and Disease Management, School of Molecular and Life Sciences, Curtin University, Bentley, WA, Australia,

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 isolate ArME14. We compare the ArME14 genome assembly with an Illumina assembly for Indian 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 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.
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http://dx.doi.org/10.1534/g3.120.401265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7341154PMC
July 2020

Low Amplitude Boom-and-Bust Cycles Define the Septoria Nodorum Blotch Interaction.

Front Plant Sci 2019 31;10:1785. Epub 2020 Jan 31.

Centre for Crop and Disease Management, School of Molecular and Life Sciences, Curtin University, Perth, WA, Australia.

Introduction: Septoria nodorum blotch (SNB) is a complex fungal disease of wheat caused by the Dothideomycete fungal pathogen . The fungus infects through the use of necrotrophic effectors (NEs) that cause necrosis on hosts carrying matching dominant susceptibility genes. The Western Australia (WA) wheatbelt is a SNB "hot spot" and experiences significant under favorable conditions. Consequently, SNB has been a major target for breeders in WA for many years.

Materials And Methods: In this study, we assembled a panel of 155 WA isolates collected over a 44-year period and compared them to 23 isolates from France and the USA using 28 SSR loci.

Results: The WA population was clustered into five groups with contrasting properties. 80% of the studied isolates were assigned to two core groups found throughout the collection location and time. The other three non-core groups that encompassed transient and emergent populations were found in restricted locations and time. Changes in group genotypes occurred during periods that coincided with the mass adoption of a single or a small group of widely planted wheat cultivars. When introduced, these cultivars had high scores for SNB resistance. However, the field resistance of these new cultivars often declined over subsequent seasons prompting their replacement with new, more resistant varieties. Pathogenicity assays showed that newly emerged isolates non-core are more pathogenic than old isolates. It is likely that the non-core groups were repeatedly selected for increased virulence on the contemporary popular cultivars.

Discussion: The low level of genetic diversity within the non-core groups, difference in virulence, low abundance, and restriction to limited locations suggest that these populations more vulnerable to a population crash when the cultivar was replaced by one that was genetically different and more resistant. We characterize the observed pattern as a low-amplitude boom-and-bust cycle in contrast with the classical high amplitude boom-and-bust cycles seen for biotrophic pathogens where the contrast between resistance and susceptibility is typically much greater. Implications of the results are discussed relating to breeding strategies for more sustainable SNB resistance and more generally for pathogens with NEs.
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http://dx.doi.org/10.3389/fpls.2019.01785DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005668PMC
January 2020

"CATAStrophy," a Genome-Informed Trophic Classification of Filamentous Plant Pathogens - How Many Different Types of Filamentous Plant Pathogens Are There?

Front Microbiol 2019 21;10:3088. Epub 2020 Jan 21.

Laboratory of Phytopathology, Department of Plant Sciences, Wageningen University & Research, Wageningen, Netherlands.

The traditional classification of fungal and oomycete phytopathogens into three classes - biotrophs, hemibiotrophs, or necrotrophs - is unsustainable. This study highlights multiple phytopathogen species for which these labels have been inappropriately applied. We propose a novel and reproducible classification based solely on genome-derived analysis of carbohydrate-active enzyme (CAZyme) gene content called CAZyme-Assisted Training And Sorting of -trophy (CATAStrophy). CATAStrophy defines four major divisions for species associated with living plants. These are monomertrophs (Mo) (corresponding to biotrophs), polymertrophs (P) (corresponding to necrotrophs), mesotrophs (Me) (corresponding to hemibiotrophs), and vasculartrophs (including species commonly described as wilts, rots, or anthracnoses). The Mo class encompasses symbiont, haustorial, and non-haustorial species. Me are divided into the subclasses intracellular and extracellular Me, and the P into broad and narrow host sub-classes. This gives a total of seven discrete plant-pathogenic classes. The classification provides insight into the properties of these species and offers a facile route to develop control measures for newly recognized diseases. Software for CATAStrophy is available online at https://github.com/ccdmb/catastrophy. We present the CATAStrophy method for the prediction of trophic phenotypes based on CAZyme gene content, as a complementary method to the traditional tripartite "biotroph-hemibiotroph-necrotroph" classifications that may encourage renewed investigation and revision within the fungal biology community.
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http://dx.doi.org/10.3389/fmicb.2019.03088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6986263PMC
January 2020

Genetic analysis of wheat sensitivity to the ToxB fungal effector from Pyrenophora tritici-repentis, the causal agent of tan spot.

Theor Appl Genet 2020 Mar 8;133(3):935-950. Epub 2020 Jan 8.

John Bingham Laboratory, NIAB, Huntingdon Road, Cambridge, CB3 0LE, UK.

Key Message: Genetic mapping of sensitivity to the Pyrenophora tritici-repentis effector ToxB allowed development of a diagnostic genetic marker, and investigation of wheat pedigrees allowed transmission of sensitive alleles to be tracked. Tan spot, caused by the necrotrophic fungal pathogen Pyrenophora tritici-repentis, is a major disease of wheat (Triticum aestivum). Secretion of the P. tritici-repentis effector ToxB is thought to play a part in mediating infection, causing chlorosis of plant tissue. Here, genetic analysis using an association mapping panel (n = 480) and a multiparent advanced generation intercross (MAGIC) population (n founders = 8, n progeny = 643) genotyped with a 90,000 feature single nucleotide polymorphism (SNP) array found ToxB sensitivity to be highly heritable (h ≥ 0.9), controlled predominantly by the Tsc2 locus on chromosome 2B. Genetic mapping of Tsc2 delineated a 1921-kb interval containing 104 genes in the reference genome of ToxB-insensitive variety 'Chinese Spring'. This allowed development of a co-dominant genetic marker for Tsc2 allelic state, diagnostic for ToxB sensitivity in the association mapping panel. Phenotypic and genotypic analysis in a panel of wheat varieties post-dated the association mapping panel further supported the diagnostic nature of the marker. Combining ToxB phenotype and genotypic data with wheat pedigree datasets allowed historic sources of ToxB sensitivity to be tracked, finding the variety 'Maris Dove' to likely be the historic source of sensitive Tsc2 alleles in the wheat germplasm surveyed. Exploration of the Tsc2 region gene space in the ToxB-sensitive line 'Synthetic W7984' identified candidate genes for future investigation. Additionally, a minor ToxB sensitivity QTL was identified on chromosome 2A. The resources presented here will be of immediate use for marker-assisted selection for ToxB insensitivity and the development of germplasm with additional genetic recombination within the Tsc2 region.
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http://dx.doi.org/10.1007/s00122-019-03517-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7021774PMC
March 2020

The identification and deletion of the polyketide synthase-nonribosomal peptide synthase gene responsible for the production of the phytotoxic triticone A/B in the wheat fungal pathogen Pyrenophora tritici-repentis.

Environ Microbiol 2019 12 21;21(12):4875-4886. Epub 2019 Nov 21.

Centre for Crop and Disease Management, Curtin University, Perth, 6150, Western Australia, Australia.

The economically important necrotrophic fungal pathogen, Pyrenophora tritici-repentis (Ptr), causes tan spot of wheat, a disease typified by foliar necrosis and chlorosis. The culture filtrate of an Australian Ptr isolate, M4, possesses phytotoxic activity and plant bioassay guided discovery led to the purification of necrosis inducing toxins called triticone A and B. High-resolution LC-MS/MS analysis of the culture filtrate identified an additional 37 triticone-like compounds. The biosynthetic gene cluster responsible for triticone production (the Ttc cluster) was identified and deletion of TtcA, a hybrid polyketide synthase (PKS)-nonribosomal peptide synthase (NRPS), abolished production of all triticones. The pathogenicity of mutant (ttcA) strains was not visibly affected in our assays. We hypothesize that triticones possess general antimicrobial activity important for competition in multi-microbial environments.
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http://dx.doi.org/10.1111/1462-2920.14854DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6915911PMC
December 2019

A specific fungal transcription factor controls effector gene expression and orchestrates the establishment of the necrotrophic pathogen lifestyle on wheat.

Sci Rep 2019 11 4;9(1):15884. Epub 2019 Nov 4.

School of Molecular and Life Sciences, Centre for Crop and Disease Management, Curtin University, Bentley, 6102, Perth, Western Australia, Australia.

The fungus Parastagonospora nodorum infects wheat through the use of necrotrophic effector (NE) proteins that cause host-specific tissue necrosis. The ZnCys transcription factor PnPf2 positively regulates NE gene expression and is required for virulence on wheat. Little is known about other downstream targets of PnPf2. We compared the transcriptomes of the P. nodorum wildtype and a strain deleted in PnPf2 (pf2-69) during in vitro growth and host infection to further elucidate targets of PnPf2 signalling. Gene ontology enrichment analysis of the differentially expressed (DE) genes revealed that genes associated with plant cell wall degradation and proteolysis were enriched in down-regulated DE gene sets in pf2-69 compared to SN15. In contrast, genes associated with redox control, nutrient and ion transport were up-regulated in the mutant. Further analysis of the DE gene set revealed that PnPf2 positively regulates twelve genes that encode effector-like proteins. Two of these genes encode proteins with homology to previously characterised effectors in other fungal phytopathogens. In addition to modulating effector gene expression, PnPf2 may play a broader role in the establishment of a necrotrophic lifestyle by orchestrating the expression of genes associated with plant cell wall degradation and nutrient assimilation.
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http://dx.doi.org/10.1038/s41598-019-52444-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6828707PMC
November 2019

Analysis of mutations in West Australian populations of Blumeria graminis f. sp. hordei CYP51 conferring resistance to DMI fungicides.

Pest Manag Sci 2020 Apr 21;76(4):1265-1272. Epub 2019 Nov 21.

School of Molecular and Life Sciences, Curtin University, Bentley, Australia.

Background: Powdery mildew caused by Blumeria graminis f. sp. hordei (Bgh) is a constant threat to barley production but is generally well controlled through combinations of host genetics and fungicides. An epidemic of barley powdery mildew was observed from 2007 to 2013 in the West Australian grain belt.

Results: We collected isolates across Australia, examined their sensitivity to demethylation inhibitor (DMI) fungicides and sequenced the Cyp51B target gene. Five amino acid substitutions were found, of which four were novel. The most resistant haplotypes increased in prevalence from 0% in 2009 to 16% in 2010 and 90% in 2011. Yeast strains expressing the Bgh Cyp51 haplotypes replicated the altered sensitivity to various DMIs and these results were complemented by in silico protein docking studies.

Conclusions: The planting of very susceptible cultivars and the use of a single fungicide mode of action was followed by the emergence of a major epidemic of barley powdery mildew. Widespread use of DMI fungicides led to the selection of Bgh isolates carrying both the Y137F and S524T mutations, which, as in Zymoseptoria tritici, account for resistance factors varying from 3.4 for propiconazole to 18 for tebuconazole, the major azoles used at that time in WA. © 2019 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
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http://dx.doi.org/10.1002/ps.5636DOI Listing
April 2020

Heterologous Expression of the Effector Proteins ToxA and ToxB, and the Prevalence of Effector Sensitivity in Australian Cereal Crops.

Front Microbiol 2019 12;10:182. Epub 2019 Feb 12.

Centre for Crop and Disease Management, School of Molecular and Life Sciences, Curtin University, Bentley, WA, Australia.

Here, we evaluate the expression of the proteinaceous effectors ToxA and ToxB, produced by the necrotrophic fungal pathogen , which confer tan spot disease susceptibility on wheat. These necrotrophic effectors were expressed in two heterologous systems: and . The SHuffle system was demonstrated to be superior to in generating high-levels of recombinant proteins that were soluble and stable. In addition, protein extracts from induced non-specific chlorosis on wheat, postulated to be caused by co-purified glucanases secreted by the host. Up to 79.6 μg/ml of ToxB was obtained using the SHuffle system in the absence of the native signal peptide, whilst the ToxA yield was considerably lower at 3.2 μg/ml. Results indicated that a histidine tag at the ToxA C-terminus interfered with effector functionality. Heterologously expressed ToxA and ToxB were tested on a panel of Australian cereals, including 122 varieties of bread wheat, 16 durum, 20 triticale and 5 barley varieties, as well as common plant model species including tobacco and . A varying degree of effector sensitivities was observed, with a higher ToxB sensitivity and prevalence in the durum and triticale varieties. ToxB-induced chlorosis was also detected on barley. The heterologous expression of effectors that are easily scalable, will facilitate effector-assisted selection of varieties in wheat breeding programs as well as the investigation of effectors in host and non-host interactions.
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http://dx.doi.org/10.3389/fmicb.2019.00182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379657PMC
February 2019

Genomic distribution of a novel Pyrenophora tritici-repentis ToxA insertion element.

PLoS One 2018 31;13(10):e0206586. Epub 2018 Oct 31.

Centre for Crop Disease and Management, Department of Environment and Agriculture, Curtin University, Bentley, Western Australia, Australia.

The ToxA effector is a major virulence gene of Pyrenophora tritici-repentis (Ptr), a necrotrophic fungus and the causal agent of tan spot disease of wheat. ToxA and co-located genes are believed to be the result of a recent horizontally transferred highly conserved 14kb region a major pathogenic event for Ptr. Since this event, monitoring isolates for pathogenic changes has become important to help understand the underlying mechanisms in play. Here we examined ToxA in 100 Ptr isolates from Australia, Europe, North and South America and the Middle East, and uncovered in isolates from Denmark, Germany and New Zealand a new variation, a novel 166 bp insertion element (PtrHp1) which can form a perfectly matched 59 bp inverted repeat hairpin structure located downstream of the ToxA coding sequence in the 3' UTR exon. A wider examination revealed PtrHp1 elements to be distributed throughout the genome. Analysis of genomes from Australia and North America had 50-112 perfect copies that often overlap other genes. The hairpin element appears to be unique to Ptr and the lack of ancient origins in other species suggests that PtrHp1 emerged after Ptr speciation. Furthermore, the ToxA UTR insertion site is identical for different isolates, which suggests a single insertion event occurred after the ToxA horizontal transfer. In vitro and in planta-detached leaf assays found that the PtrHp1 element insertion had no effect on ToxA expression. However, variation in the expression of ToxA was detected between the Ptr isolates from different demographic locations, which appears to be unrelated to the presence of the element. We envision that this discovery may contribute towards future understanding of the possible role of hairpin elements in Ptr.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0206586PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6209302PMC
April 2019

Correction to: Comparative genomics of the wheat fungal pathogen Pyrenophora tritici-repentis reveals chromosomal variations and genome plasticity.

BMC Genomics 2018 09 13;19(1):673. Epub 2018 Sep 13.

Centre for Crop Disease and Management, Department of Environment and Agriculture, Curtin University, Bentley, Western Australia, Australia.

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http://dx.doi.org/10.1186/s12864-018-5059-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6136185PMC
September 2018

Pan-Parastagonospora Comparative Genome Analysis-Effector Prediction and Genome Evolution.

Genome Biol Evol 2018 09 1;10(9):2443-2457. Epub 2018 Sep 1.

Centre for Crop & Disease Management, School of Molecular & Life Sciences, Curtin University, Bentley, Western Australia, Australia.

We report a fungal pan-genome study involving Parastagonospora spp., including 21 isolates of the wheat (Triticum aestivum) pathogen Parastagonospora nodorum, 10 of the grass-infecting Parastagonospora avenae, and 2 of a closely related undefined sister species. We observed substantial variation in the distribution of polymorphisms across the pan-genome, including repeat-induced point mutations, diversifying selection and gene gains and losses. We also discovered chromosome-scale inter and intraspecific presence/absence variation of some sequences, suggesting the occurrence of one or more accessory chromosomes or regions that may play a role in host-pathogen interactions. The presence of known pathogenicity effector loci SnToxA, SnTox1, and SnTox3 varied substantially among isolates. Three P. nodorum isolates lacked functional versions for all three loci, whereas three P. avenae isolates carried one or both of the SnTox1 and SnTox3 genes, indicating previously unrecognized potential for discovering additional effectors in the P. nodorum-wheat pathosystem. We utilized the pan-genomic comparative analysis to improve the prediction of pathogenicity effector candidates, recovering the three confirmed effectors among our top-ranked candidates. We propose applying this pan-genomic approach to identify the effector repertoire involved in other host-microbe interactions involving necrotrophic pathogens in the Pezizomycotina.
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http://dx.doi.org/10.1093/gbe/evy192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6152946PMC
September 2018

Assessing European Wheat Sensitivities to Necrotrophic Effectors and Fine-Mapping the Locus Conferring Sensitivity to the Effector SnTox3.

Front Plant Sci 2018 4;9:881. Epub 2018 Jul 4.

Genetics and Breeding Department, National Institute of Agricultural Botany, Cambridge, United Kingdom.

is a necrotrophic fungal pathogen of wheat ( L.), one of the world's most important crops. mediates host cell death using proteinaceous necrotrophic effectors, presumably liberating nutrients that allow the infection process to continue. The identification of pathogen effectors has allowed host genetic resistance mechanisms to be separated into their constituent parts. In , three proteinaceous effectors have been cloned: , , and . Here, we survey sensitivity to all three effectors in a panel of 480 European wheat varieties, and fine-map the wheat SnTox3 sensitivity locus using genome-wide association scans (GWAS) and an eight-founder wheat multi-parent advanced generation inter-cross (MAGIC) population. Using a Bonferroni corrected ≤ 0.05 significance threshold, GWAS identified 10 significant markers defining a single locus, , located on the short arm of chromosome 5B explaining 32% of the phenotypic variation [peak single nucleotide polymorphisms (SNPs), Excalibur_c47452_183 and GENE-3324_338, -log = 20.44]. Single marker analysis of SnTox3 sensitivity in the MAGIC population located via five significant SNPs, defining a 6.2-kb region that included the two peak SNPs identified in the association mapping panel. Accordingly, SNP Excalibur_c47452_183 was converted to the KASP genotyping system, and validated by screening a subset of 95 wheat varieties, providing a valuable resource for marker assisted breeding and for further genetic investigation. In addition, composite interval mapping in the MAGIC population identified six minor SnTox3 sensitivity quantitative trait loci, on chromosomes 2A (value = 9.17), 2B (, = 0.018), 3B (, = 48.51), 4D (, = 0.028), 6A ( = 8.51), and 7B (, = 0.020), each accounting for between 3.1 and 6.0 % of the phenotypic variance. Collectively, the outcomes of this study provides breeders with knowledge and resources regarding the sensitivity of European wheat germplasm to effectors, as well as simple diagnostic markers for determining allelic state at .
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http://dx.doi.org/10.3389/fpls.2018.00881DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039772PMC
July 2018

Improved Detection and Monitoring of Fungicide Resistance in f. sp. With High-Throughput Genotype Quantification by Digital PCR.

Front Microbiol 2018 13;9:706. Epub 2018 Apr 13.

The Fungicide Resistance Group, Centre for Crop and Disease Management, School of Molecular and Life Sciences, Curtin University, Bentley, WA, Australia.

The increased occurrence of triazole fungicide resistant strains of f. sp. () is an economic concern for the barley industry in Australia and elsewhere. High levels of resistance to triazoles in the field are caused by two separate point mutations in the gene, Y136F and S509T. Early detection of these mutations arising in pathogen field populations is important as this allows time for changes in fungicide practices to be adopted, thus mitigating potential yield losses due to fungicide failure and preventing the resistance from becoming dominant. A digital PCR (dPCR) assay has been developed for the detection and quantification of the Y136F and S509T mutations in the gene. Mutation levels were quantifiable as low as 0.2% in genomic DNA extractions and field samples. This assay was applied to the high throughput screening of field and bait trial samples from barley growing regions across Australia in the 2015 and 2016 growing seasons and identified the S509T mutation for the first time in the Eastern states of Australia. This is the first report on the use of digital PCR technology for fungicide resistance detection and monitoring in agriculture. Here we describe the potential application of dPCR for the screening of fungicide resistance mutations in a network of specifically designed bait trials. The combination of these two tools constitute an early warning system for the development of fungicide resistance that allows for the timely adjustment of management practices.
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http://dx.doi.org/10.3389/fmicb.2018.00706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5908980PMC
April 2018

Comparative genomics of the wheat fungal pathogen Pyrenophora tritici-repentis reveals chromosomal variations and genome plasticity.

BMC Genomics 2018 Apr 23;19(1):279. Epub 2018 Apr 23.

Centre for Crop Disease and Management, Department of Environment and Agriculture, Curtin University, Bentley, Western Australia, Australia.

Background: Pyrenophora tritici-repentis (Ptr) is a necrotrophic fungal pathogen that causes the major wheat disease, tan spot. We set out to provide essential genomics-based resources in order to better understand the pathogenicity mechanisms of this important pathogen.

Results: Here, we present eight new Ptr isolate genomes, assembled and annotated; representing races 1, 2 and 5, and a new race. We report a high quality Ptr reference genome, sequenced by PacBio technology with Illumina paired-end data support and optical mapping. An estimated 98% of the genome coverage was mapped to 10 chromosomal groups, using a two-enzyme hybrid approach. The final reference genome was 40.9 Mb and contained a total of 13,797 annotated genes, supported by transcriptomic and proteogenomics data sets.

Conclusions: Whole genome comparative analysis revealed major chromosomal segmental rearrangements and fusions, highlighting intraspecific genome plasticity in this species. Furthermore, the Ptr race classification was not supported at the whole genome level, as phylogenetic analysis did not cluster the ToxA producing isolates. This expansion of available Ptr genomics resources will directly facilitate research aimed at controlling tan spot disease.
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http://dx.doi.org/10.1186/s12864-018-4680-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5913888PMC
April 2018

Novel sources of resistance to Septoria nodorum blotch in the Vavilov wheat collection identified by genome-wide association studies.

Theor Appl Genet 2018 Jun 22;131(6):1223-1238. Epub 2018 Feb 22.

Centre for Crop and Disease Management, School of Molecular and Life Sciences, Curtin University, Bentley, WA, Australia.

Key Message: The fungus Parastagonospora nodorum causes Septoria nodorum blotch (SNB) of wheat. A genetically diverse wheat panel was used to dissect the complexity of SNB and identify novel sources of resistance. The fungus Parastagonospora nodorum is the causal agent of Septoria nodorum blotch (SNB) of wheat. The pathosystem is mediated by multiple fungal necrotrophic effector-host sensitivity gene interactions that include SnToxA-Tsn1, SnTox1-Snn1, and SnTox3-Snn3. A P. nodorum strain lacking SnToxA, SnTox1, and SnTox3 (toxa13) retained wild-type-like ability to infect some modern wheat cultivars, suggesting evidence of other effector-mediated susceptibility gene interactions or the lack of host resistance genes. To identify genomic regions harbouring such loci, we examined a panel of 295 historic wheat accessions from the N. I. Vavilov Institute of Plant Genetic Resources in Russia, which is comprised of genetically diverse landraces and breeding lines registered from 1920 to 1990. The wheat panel was subjected to effector bioassays, infection with P. nodorum wild type (SN15) and toxa13. In general, SN15 was more virulent than toxa13. Insensitivity to all three effectors contributed significantly to resistance against SN15, but not toxa13. Genome-wide association studies using phenotypes from SN15 infection detected quantitative trait loci (QTL) on chromosomes 1BS (Snn1), 2DS, 5AS, 5BS (Snn3), 3AL, 4AL, 4BS, and 7AS. For toxa13 infection, a QTL was detected on 5AS (similar to SN15), plus two additional QTL on 2DL and 7DL. Analysis of resistance phenotypes indicated that plant breeders may have inadvertently selected for effector insensitivity from 1940 onwards. We identify accessions that can be used to develop bi-parental mapping populations to characterise resistance-associated alleles for subsequent introgression into modern bread wheat to minimise the impact of SNB.
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http://dx.doi.org/10.1007/s00122-018-3073-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5945755PMC
June 2018

Regulation of proteinaceous effector expression in phytopathogenic fungi.

PLoS Pathog 2017 Apr 20;13(4):e1006241. Epub 2017 Apr 20.

Centre for Crop and Disease Management, Department of Environment and Agriculture, Curtin University, Bentley, Western Australia, Australia.

Effectors are molecules used by microbial pathogens to facilitate infection via effector-triggered susceptibility or tissue necrosis in their host. Much research has been focussed on the identification and elucidating the function of fungal effectors during plant pathogenesis. By comparison, knowledge of how phytopathogenic fungi regulate the expression of effector genes has been lagging. Several recent studies have illustrated the role of various transcription factors, chromosome-based control, effector epistasis, and mobilisation of endosomes within the fungal hyphae in regulating effector expression and virulence on the host plant. Improved knowledge of effector regulation is likely to assist in improving novel crop protection strategies.
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http://dx.doi.org/10.1371/journal.ppat.1006241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5398718PMC
April 2017

Codon optimization underpins generalist parasitism in fungi.

Elife 2017 02 3;6. Epub 2017 Feb 3.

LIPM, Université de Toulouse, INRA, CNRS, Castanet-Tolosan, France.

The range of hosts that parasites can infect is a key determinant of the emergence and spread of disease. Yet, the impact of host range variation on the evolution of parasite genomes remains unknown. Here, we show that codon optimization underlies genome adaptation in broad host range parasites. We found that the longer proteins encoded by broad host range fungi likely increase natural selection on codon optimization in these species. Accordingly, codon optimization correlates with host range across the fungal kingdom. At the species level, biased patterns of synonymous substitutions underpin increased codon optimization in a generalist but not a specialist fungal pathogen. Virulence genes were consistently enriched in highly codon-optimized genes of generalist but not specialist species. We conclude that codon optimization is related to the capacity of parasites to colonize multiple hosts. Our results link genome evolution and translational regulation to the long-term persistence of generalist parasitism.
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http://dx.doi.org/10.7554/eLife.22472DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5315462PMC
February 2017

Fungal Plant Pathogenesis Mediated by Effectors.

Microbiol Spectr 2016 12;4(6)

Center for Crop and Disease Management, Department of Environment and Agriculture, Curtin University, Perth, Western Australia 6102, Australia.

The interactions between fungi and plants encompass a spectrum of ecologies ranging from saprotrophy (growth on dead plant material) through pathogenesis (growth of the fungus accompanied by disease on the plant) to symbiosis (growth of the fungus with growth enhancement of the plant). We consider pathogenesis in this article and the key roles played by a range of pathogen-encoded molecules that have collectively become known as effectors.
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http://dx.doi.org/10.1128/microbiolspec.FUNK-0021-2016DOI Listing
December 2016

A functionally conserved Zn Cys binuclear cluster transcription factor class regulates necrotrophic effector gene expression and host-specific virulence of two major Pleosporales fungal pathogens of wheat.

Mol Plant Pathol 2017 04 24;18(3):420-434. Epub 2017 Jan 24.

Department of Environment & Agriculture, Centre for Crop and Disease Management, Curtin University, Bentley, 6102, Perth, Australia.

The fungus Parastagonospora nodorum is the causal agent of Septoria nodorum blotch of wheat (Triticum aestivum). The interaction is mediated by multiple fungal necrotrophic effector-dominant host sensitivity gene interactions. The three best-characterized effector-sensitivity gene systems are SnToxA-Tsn1, SnTox1-Snn1 and SnTox3-Snn3. These effector genes are highly expressed during early infection, but expression decreases as the infection progresses to tissue necrosis and sporulation. However, the mechanism of regulation is unknown. We have identified and functionally characterized a gene, referred to as PnPf2, which encodes a putative zinc finger transcription factor. PnPf2 deletion resulted in the down-regulation of SnToxA and SnTox3 expression. Virulence on Tsn1 and Snn3 wheat cultivars was strongly reduced. The SnTox1-Snn1 interaction remained unaffected. Furthermore, we have also identified and deleted an orthologous PtrPf2 from the tan spot fungus Pyrenophora tritici-repentis which possesses a near-identical ToxA that was acquired from P. nodorum via horizontal gene transfer. PtrPf2 deletion also resulted in the down-regulation of PtrToxA expression and a near-complete loss of virulence on Tsn1 wheat. We have demonstrated, for the first time, evidence for a functionally conserved signalling component that plays a role in the regulation of a common/horizontally transferred effector found in two major fungal pathogens of wheat.
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http://dx.doi.org/10.1111/mpp.12511DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6638278PMC
April 2017

Evaluation of a Multilocus Indel DNA Region for the Detection of the Wheat Tan Spot Pathogen Pyrenophora tritici-repentis.

Plant Dis 2016 Nov 8;100(11):2215-2225. Epub 2016 Sep 8.

Centre for Crop and Disease Management, Department of Environment and Agriculture, School of Science, Curtin University, Bentley, WA 6102, Australia.

Tan spot or yellow (leaf) spot disease of wheat (Triticum spp.) is caused by Pyrenophora tritici-repentis, a necrotrophic fungal pathogen that is widespread throughout the main wheat-growing regions in the world. This disease is currently the single most economically important crop disease in Australia. In this study, a real-time quantitative polymerase chain reaction (qPCR) assay was developed as a diagnostic tool to detect the pathogen on wheat foliar tissue. A multicopy locus (PtrMulti) present in the P. tritici-repentis genome was assessed for its suitability as a qPCR probe. The primer pair PtrMulti_F/R that targets the region was evaluated with respect to species specificity and sensitivity. A PtrMulti SYBR qPCR assay was developed and proved to be suitable for the identification and relative quantification of P. tritici-repentis with a detection limit of DNA levels at <0.1 pg. Variation of the PtrMulti copy number between the geographical representatives of P. tritici-repentis strains examined was minimal, with the range of 63 to 85 copies per genome. For naturally infected wheat field samples, the incidence of P. tritici-repentis DNA on leaves quantified by qPCR varied up to 1,000-fold difference in the concentration, with a higher incidence of DNA occurring on the lower canopy for most of the growth stages examined. At the early growth stages, qPCR assay was able to detect P. tritici-repentis DNA on the younger leaves in the absence of visible tan spot lesions. These results demonstrate the potential of PtrMulti probe to be used for early detection and rapid screening of tan spot disease on wheat plants.
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http://dx.doi.org/10.1094/PDIS-03-16-0262-REDOI Listing
November 2016

Demethylase Inhibitor Fungicide Resistance in Pyrenophora teres f. sp. teres Associated with Target Site Modification and Inducible Overexpression of Cyp51.

Front Microbiol 2016 19;7:1279. Epub 2016 Aug 19.

Department of Environment and Agriculture, Centre for Crop and Disease Management, Curtin University Bentley, WA, Australia.

Pyrenophora teres f. sp. teres is the cause of net form of net blotch (NFNB), an economically important foliar disease in barley (Hordeum vulgare). Net and spot forms of net blotch are widely controlled using site-specific systemic fungicides. Although resistance to succinate dehydrogenase inhibitors and quinone outside inhibitors has been addressed before in net blotches, mechanisms controlling demethylation inhibitor resistance have not yet been reported at the molecular level. Here we report the isolation of strains of NFNB in Australia since 2013 resistant to a range of demethylase inhibitor fungicides. Cyp51A:KO103-A1, an allele with the mutation F489L, corresponding to the archetype F495I in Aspergillus fumigatus, was only present in resistant strains and was correlated with resistance factors to various demethylase inhibitors ranging from 1.1 for epoxiconazole to 31.7 for prochloraz. Structural in silico modeling of the sensitive and resistant CYP51A proteins docked with different demethylase inhibitor fungicides showed how the interaction of F489L within the heme cavity produced a localized constriction of the region adjacent to the docking site that is predicted to result in lower binding affinities. Resistant strains also displayed enhanced induced expression of the two Cyp51A paralogs and of Cyp51B genes. While Cyp51B was found to be constitutively expressed in the absence of fungicide, Cyp51A was only detected at extremely low levels. Under fungicide induction, expression of Cyp51B, Cyp51A2, and Cyp51A1 was shown to be 1.6-, 3,- and 5.3-fold higher, respectively in the resistant isolate compared to the wild type. These increased levels of expression were not supported by changes in the promoters of any of the three genes. The implications of these findings on demethylase inhibitor activity will require current net blotch management strategies to be reconsidered in order to avoid the development of further resistance and preserve the lifespan of fungicides in use.
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http://dx.doi.org/10.3389/fmicb.2016.01279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4990540PMC
September 2016

Validation of Genome-Wide Association Studies as a Tool to Identify Virulence Factors in Parastagonospora nodorum.

Phytopathology 2016 10 15;106(10):1177-1185. Epub 2016 Aug 15.

First, second, fourth, fifth, and ninth authors: Department of Plant Pathology, North Dakota State University, Fargo 58108; third and ninth authors: United States Department of Agriculture-Agricultural Research Service, Northern Crop Science Laboratory, Cereal Crops Research Unit, Fargo, ND, 58102; sixth author: Department of Plant Science, North Dakota State University, Fargo; seventh author: Centre for Crop and Disease Management, Department of Environment and Agriculture, School of Science, Curtin University, Perth, WA 6102, Australia; and eighth author: Institute of Integrative Biology, Plant Pathology Group, Swiss Federal Institute of Technology, ETH Zentrum, LFW, CH-8092 Zürich, Switzerland.

Parastagonospora nodorum is a necrotrophic fungal pathogen causing Septoria nodorum blotch on wheat. We have identified nine necrotrophic effector-host dominant sensitivity gene interactions, and we have cloned three of the necrotrophic effector genes, including SnToxA, SnTox1, and SnTox3. Because sexual populations of P. nodorum are difficult to develop under lab conditions, genome-wide association study (GWAS) is the best population genomic approach to identify genomic regions associated with traits using natural populations. In this article, we used a global collection of 191 P. nodorum isolates from which we identified 2,983 single-nucleotide polymorphism (SNP) markers and gene markers for SnToxA and SnTox3 to evaluate the power of GWAS on two popular wheat breeding lines that were sensitive to SnToxA and SnTox3. Strong marker trait associations (MTA) with P. nodorum virulence that mapped to SnTox3 and SnToxA were first identified using the marker set described above. A novel locus in the P. nodorum genome associated with virulence was also identified as a result of this analysis. To evaluate whether a sufficient level of marker saturation was available, we designed a set of primers every 1 kb in the genomic regions containing SnToxA and SnTox3. Polymerase chain reaction amplification was performed across the 191 isolates and the presence/absence polymorphism was scored and used as the genotype. The marker proximity necessary to identify MTA flanking SnToxA and SnTox3 ranged from 4 to 5 and 1 to 7 kb, respectively. Similar analysis was performed on the novel locus. Using a 45% missing data threshold, two more SNP were identified spanning a 4.6-kb genomic region at the novel locus. These results showed that the rate of linkage disequilibrium (LD) decay in P. nodorum and, likely, other fungi is high compared with plants and animals. The fast LD decay in P. nodorum is an advantage only if sufficient marker density is attained. Based on our results with the SnToxA and SnTox3 regions, markers are needed every 9 or 8 kb, respectively, or in every gene, to guarantee that genes associated with a quantitative trait such as virulence are not missed.
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http://dx.doi.org/10.1094/PHYTO-02-16-0113-FIDOI Listing
October 2016

OcculterCut: A Comprehensive Survey of AT-Rich Regions in Fungal Genomes.

Genome Biol Evol 2016 07 3;8(6):2044-64. Epub 2016 Jul 3.

Department of Environment & Agriculture, Centre for Crop and Disease Management, Curtin University, Perth, Australia Curtin Institute for Computation, Curtin University, Perth, Australia.

We present a novel method to measure the local GC-content bias in genomes and a survey of published fungal species. The method, enacted as "OcculterCut" (https://sourceforge.net/projects/occultercut, last accessed April 30, 2016), identified species containing distinct AT-rich regions. In most fungal taxa, AT-rich regions are a signature of repeat-induced point mutation (RIP), which targets repetitive DNA and decreases GC-content though the conversion of cytosine to thymine bases. RIP has in turn been identified as a driver of fungal genome evolution, as RIP mutations can also occur in single-copy genes neighboring repeat-rich regions. Over time RIP perpetuates "two speeds" of gene evolution in the GC-equilibrated and AT-rich regions of fungal genomes. In this study, genomes showing evidence of this process are found to be common, particularly among the Pezizomycotina. Further analysis highlighted differences in amino acid composition and putative functions of genes from these regions, supporting the hypothesis that these regions play an important role in fungal evolution. OcculterCut can also be used to identify genes undergoing RIP-assisted diversifying selection, such as small, secreted effector proteins that mediate host-microbe disease interactions.
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http://dx.doi.org/10.1093/gbe/evw121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4943192PMC
July 2016

Proposal for a unified nomenclature for target-site mutations associated with resistance to fungicides.

Pest Manag Sci 2016 Aug 16;72(8):1449-59. Epub 2016 Jun 16.

Centre for Crop Disease Management, Department of Environment and Agriculture, Curtin University, Bentley, WA, Australia.

Evolved resistance to fungicides is a major problem limiting our ability to control agricultural, medical and veterinary pathogens and is frequently associated with substitutions in the amino acid sequence of the target protein. The convention for describing amino acid substitutions is to cite the wild-type amino acid, the codon number and the new amino acid, using the one-letter amino acid code. It has frequently been observed that orthologous amino acid mutations have been selected in different species by fungicides from the same mode of action class, but the amino acids have different numbers. These differences in numbering arise from the different lengths of the proteins in each species. The purpose of the present paper is to propose a system for unifying the labelling of amino acids in fungicide target proteins. To do this we have produced alignments between fungicide target proteins of relevant species fitted to a well-studied 'archetype' species. Orthologous amino acids in all species are then assigned numerical 'labels' based on the position of the amino acid in the archetype protein. © 2016 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
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http://dx.doi.org/10.1002/ps.4301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5094580PMC
August 2016

Differential effector gene expression underpins epistasis in a plant fungal disease.

Plant J 2016 08 7;87(4):343-54. Epub 2016 Jul 7.

Centre for Crop and Disease Management, Department of Environment and Agriculture, Curtin University, Bentley, WA, 6102, Australia.

Fungal effector-host sensitivity gene interactions play a key role in determining the outcome of septoria nodorum blotch disease (SNB) caused by Parastagonospora nodorum on wheat. The pathosystem is complex and mediated by interaction of multiple fungal necrotrophic effector-host sensitivity gene systems. Three effector sensitivity gene systems are well characterized in this pathosystem; SnToxA-Tsn1, SnTox1-Snn1 and SnTox3-Snn3. We tested a wheat mapping population that segregated for Snn1 and Snn3 with SN15, an aggressive P. nodorum isolate that produces SnToxA, SnTox1 and SnTox3, to study the inheritance of sensitivity to SnTox1 and SnTox3 and disease susceptibility. Interval quantitative trait locus (QTL) mapping showed that the SnTox1-Snn1 interaction was paramount in SNB development on both seedlings and adult plants. No effect of the SnTox3-Snn3 interaction was observed under SN15 infection. The SnTox3-Snn3 interaction was however, detected in a strain of SN15 in which SnTox1 had been deleted (tox1-6). Gene expression analysis indicates increased SnTox3 expression in tox1-6 compared with SN15. This indicates that the failure to detect the SnTox3-Snn3 interaction in SN15 is due - at least in part - to suppressed expression of SnTox3 mediated by SnTox1. Furthermore, infection of the mapping population with a strain deleted in SnToxA, SnTox1 and SnTox3 (toxa13) unmasked a significant SNB QTL on 2DS where the SnTox2 effector sensitivity gene, Snn2, is located. This QTL was not observed in SN15 and tox1-6 infections and thus suggesting that SnToxA and/or SnTox3 were epistatic. Additional QTLs responding to SNB and effectors sensitivity were detected on 2AS1 and 3AL.
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http://dx.doi.org/10.1111/tpj.13203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5053286PMC
August 2016

Dissecting the role of histidine kinase and HOG1 mitogen-activated protein kinase signalling in stress tolerance and pathogenicity of Parastagonospora nodorum on wheat.

Microbiology (Reading) 2016 06 15;162(6):1023-1036. Epub 2016 Mar 15.

Department of Environment and Agriculture, Centre for Crop and Disease Management, Curtin University, Bentley, WA 6102, Australia.

The HOG1 mitogen-activated protein kinase (MAPK) pathway is activated through two-component histidine kinase (HK) signalling. This pathway was first characterized in the budding yeast Saccharomyces cerevisiae as a regulator of osmotolerance. The fungus Parastagonospora nodorum is the causal agent of septoria nodorum blotch of wheat. This pathogen uses host-specific effectors in tandem with general pathogenicity mechanisms to carry out its infection process. Genes showing strong sequence homology to S. cerevisiae HOG1 signalling pathway genes have been identified in the genome of P. nodorum. In this study, we examined the role of the pathway in the virulence of P. nodorum on wheat by disrupting putative pathway component genes: HOG1 (SNOG_13296) MAPK and NIK1 (SNOG_11631) hybrid HK. Mutants deleted in NIK1 and HOG1 were insensitive to dicarboximide and phenylpyrrole fungicides, but not a fungicide that targets ergosterol biosynthesis. Furthermore, both Δnik1 and Δhog1 mutants showed increased sensitivity to hyperosmotic stress. However, HOG1, but not NIK1, is required for tolerance to elevated temperatures. HOG1 deletion conferred increased tolerance to 6-methoxy-2-benzoxazolinone, a cereal phytoalexin. This suggests that the HOG1 signalling pathway is not exclusively associated with NIK1. Both Δnik1 and Δhog1 mutants retained the ability to infect and cause necrotic lesions on wheat. However, we observed that the Δhog1 mutation resulted in reduced production of pycnidia, asexual fruiting bodies that facilitate spore dispersal during late infection. Our study demonstrated the overlapping and distinct roles of a HOG1 MAPK and two-component HK signalling in P. nodorum growth and pathogenicity.
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http://dx.doi.org/10.1099/mic.0.000280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5042077PMC
June 2016