Major Susceptibility Gene Epistasis over Minor Gene Resistance to Spot Form Net Blotch in a Commercial Barley Cultivar.

Spot form net blotch, caused by Pyrenophora teres f. maculata, is a significant global disease of barley (Hordeum vulgare). Baudin, a barley cultivar that was until recently extensively grown in Western Australia, was reported as having minor seedling resistance. However, Baudin was highly susceptible to a local isolate, M3, suggesting that this isolate had gained virulence against a major susceptibility gene. M3 causes atypical lesions with pale centers early in the infection, with initial screens of a segregating population indicating that this was determined by a single locus in the Baudin genome. The susceptibility was semidominant in F1 progeny and the susceptibility gene, designated Spm1 (Susceptibility to P. teres f. maculata 1), mapped to a 190-kb section of the resistance gene-rich Mla region of chromosome 1H. Phenotyping with Ptm SP1, a non-M3 pathotype, identified a seedling resistance locus on 2H. Minor gene resistance is generally regarded as potentially durable, but our findings suggest the resistance to spot form net blotch in Baudin is nullified by strong susceptibility conferred by a separate locus on 1H. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Widespread genetic heterogeneity and genotypic grouping associated with fungicide resistance among barley spot form net blotch isolates in Australia.

Spot form net blotch, caused by Pyrenophora teres f. maculata, is a major foliar disease of barley worldwide. Knowledge of the pathogen's genetic diversity and population structure is critical for a better understanding of inherent evolutionary capacity and for the development of sustainable disease management strategies. Genome-wide, single nucleotide polymorphism data of 254 Australian isolates revealed genotypic diversity and an absence of population structure, either between states, or between fields and cultivars in different agro-ecological zones. This indicates there is little geographical isolation or cultivar directional selection and that the pathogen is highly mobile across the continent. However, two cryptic genotypic groups were found only in Western Australia, predominantly associated with genes involved in fungicide resistance. The findings in this study are discussed in the context of current cultivar resistance and the pathogen's adaptive potential.

Expansion and Conservation of Biosynthetic Gene Clusters in Pathogenic Pyrenophora spp.

Pyrenophora is a fungal genus responsible for a number of major cereal diseases. Although fungi produce many specialised or secondary metabolites for defence and interacting with the surrounding environment, the repertoire of specialised metabolites (SM) within Pyrenophora pathogenic species remains mostly uncharted. In this study, an in-depth comparative analysis of the P. teres f. teres, P teres f. maculata and P. tritici-repentis potential to produce SMs, based on in silico predicted biosynthetic gene clusters (BGCs), was conducted using genome assemblies from PacBio DNA reads. Conservation of BGCs between the Pyrenophora species included type I polyketide synthases, terpene synthases and the first reporting of a type III polyketide synthase in P teres f. maculata. P. teres isolates exhibited substantial expansion of non-ribosomal peptide synthases relative to P. tritici-repentis, hallmarked by the presence of tailoring cis-acting nitrogen methyltransferase domains. P. teres isolates also possessed unique non-ribosomal peptide synthase (NRPS)-indole and indole BGCs, while a P. tritici-repentis phytotoxin BGC for triticone production was absent in P. teres. These differences highlight diversification between the pathogens that reflects their different evolutionary histories, host adaption and lifestyles.

Volatile Molecules Secreted by the Wheat Pathogen Parastagonospora nodorum Are Involved in Development and Phytotoxicity.

Septoria nodorum blotch is a major disease of wheat caused by the fungus Parastagonospora nodorum. Recent studies have demonstrated that secondary metabolites, including polyketides and non-ribosomal peptides, produced by the pathogen play important roles in disease and development. However, there is currently no knowledge on the composition or biological activity of the volatile organic compounds (VOCs) secreted by P. nodorum. To address this, we undertook a series of growth and phytotoxicity assays and demonstrated that P. nodorum VOCs inhibited bacterial growth, were phytotoxic and suppressed self-growth. Mass spectrometry analysis revealed that 3-methyl-1-butanol, 2-methyl-1-butanol, 2-methyl-1-propanol, and 2-phenylethanol were dominant in the VOC mixture and phenotypic assays using these short chain alcohols confirmed that they were phytotoxic. Further analysis of the VOCs also identified the presence of multiple sesquiterpenes of which four were identified via mass spectrometry and nuclear magnetic resonance as ß-elemene, α-cyperone, eudesma-4,11-diene and acora-4,9-diene. Subsequent reverse genetics studies were able to link these molecules to corresponding sesquiterpene synthases in the P. nodorum genome. However, despite extensive testing, these molecules were not involved in either of the growth inhibition or phytotoxicity phenotypes previously observed. Plant assays using mutants of the pathogen lacking the synthetic genes revealed that the identified sesquiterpenes were not required for disease formation on wheat leaves. Collectively, these data have significantly extended our knowledge of the VOCs in fungi and provided the basis for further dissecting the roles of sesquiterpenes in plant disease.

Heterologous expression of cytotoxic sesquiterpenoids from the medicinal mushroom Lignosus rhinocerotis in yeast.

BACKGROUND: Genome mining facilitated by heterologous systems is an emerging approach to access the chemical diversity encoded in basidiomycete genomes. In this study, three sesquiterpene synthase genes, GME3634, GME3638, and GME9210, which were highly expressed in the sclerotium of the medicinal mushroom Lignosus rhinocerotis, were cloned and heterologously expressed in a yeast system. RESULTS: Metabolite profile analysis of the yeast culture extracts by GC-MS showed the production of several sesquiterpene alcohols (C15H26O), including cadinols and germacrene D-4-ol as major products. Other detected sesquiterpenes include selina-6-en-4-ol, ß-elemene, ß-cubebene, and cedrene. Two purified major compounds namely (+)-torreyol and α-cadinol synthesised by GME3638 and GME3634 respectively, are stereoisomers and their chemical structures were confirmed by 1H and 13C NMR. Phylogenetic analysis revealed that GME3638 and GME3634 are a pair of orthologues, and are grouped together with terpene synthases that synthesise cadinenes and related sesquiterpenes. (+)-Torreyol and α-cadinol were tested against a panel of human cancer cell lines and the latter was found to exhibit selective potent cytotoxicity in breast adenocarcinoma cells (MCF7) with IC50 value of 3.5 ± 0.58 µg/ml while α-cadinol is less active (IC50 = 18.0 ± 3.27 µg/ml). CONCLUSIONS: This demonstrates that yeast-based genome mining, guided by transcriptomics, is a promising approach for uncovering bioactive compounds from medicinal mushrooms.

Functional genomics-guided discovery of a light-activated phytotoxin in the wheat pathogen Parastagonospora nodorum via pathway activation.

Parastagonospora nodorum is an important pathogen of wheat. The contribution of secondary metabolites to this pathosystem is poorly understood. A biosynthetic gene cluster (SNOG_08608-08616) has been shown to be upregulated during the late stage of P. nodorum wheat leaf infection. The gene cluster shares several homologues with the Cercospora nicotianae CTB gene cluster encoding the biosynthesis of cercosporin. Activation of the gene cluster by overexpression (OE) of the transcription factor gene (SNOG_08609) in P. nodorum resulted in the production of elsinochrome C, a perelyenequinone phytotoxin structurally similar to cercosporin. Heterologous expression of the polyketide synthase gene elcA from the gene cluster in Aspergillus nidulans resulted in the production of the polyketide precursor nortoralactone common to the cercosporin pathway. Elsinochrome C could be detected on wheat leaves infected with P. nodorum, but not in the elcA disruption mutant. The compound was shown to exhibit necrotic activity on wheat leaves in a light-dependent manner. Wheat seedling infection assays showed that ΔelcA exhibited reduced virulence compared with wild type, while infection by an OE strain overproducing elsinochrome C resulted in larger lesions on leaves. These data provided evidence that elsinochrome C contributes to the virulence of P. nodorum against wheat.

SnPKS19 Encodes the Polyketide Synthase for Alternariol Mycotoxin Biosynthesis in the Wheat Pathogen Parastagonospora nodorum.

Alternariol (AOH) is an important mycotoxin from the Alternaria fungi. AOH was detected for the first time in the wheat pathogen Parastagonospora nodorum in a recent study. Here, we exploited reverse genetics to demonstrate that SNOG_15829 (SnPKS19), a close homolog of Penicillium aethiopicum norlichexanthone (NLX) synthase gene gsfA, is required for AOH production. We further validate that SnPKS19 is solely responsible for AOH production by heterologous expression in Aspergillus nidulans. The expression profile of SnPKS19 based on previous P. nodorum microarray data correlated with the presence of AOH in vitro and its absence in planta. Subsequent characterization of the ΔSnPKS19 mutants showed that SnPKS19 and AOH are not involved in virulence and oxidative stress tolerance. Identification and characterization of the P. nodorum SnPKS19 cast light on a possible alternative AOH synthase gene in Alternaria alternata and allowed us to survey the distribution of AOH synthase genes in other fungal genomes. We further demonstrate that phylogenetic analysis could be used to differentiate between AOH synthases and the closely related NLX synthases. This study provides the basis for studying the genetic regulation of AOH production and for development of molecular diagnostic methods for detecting AOH-producing fungi in the future.

The past, present and future of secondary metabolite research in the Dothideomycetes.

The Dothideomycetes represents a large and diverse array of fungi in which prominent plant pathogens are over-represented. Species within the Cochliobolus, Alternaria, Pyrenophora and Mycosphaerella (amongst others) all cause diseases that threaten food security in many parts of the world. Significant progress has been made over the past decade in understanding how some of these pathogens cause disease at a molecular level. It is reasonable to suggest that much of this progress can be attributed to the increased availability of genome sequences. However, together with revealing mechanisms of pathogenicity, these genome sequences have also highlighted the capacity of the Dothideomycetes to produce an extensive array of secondary metabolites, far greater than originally thought. Indeed, it is now clear that we appear to have only scratched the surface to date in terms of the identification of secondary metabolites produced by these fungi. In the first half of this review, we examine the current status of secondary metabolite research in the Dothideomycetes and highlight the diversity of the molecules discovered thus far, in terms of both structure and biological activity. In the second part of this review, we survey the emerging techniques and technologies that will be required to shed light on the vast array of secondary metabolite potential that is encoded within these genomes. Experimental design, analytical chemistry and synthetic biology are all discussed in the context of how they will contribute to this field.

A genome-wide survey of the secondary metabolite biosynthesis genes in the wheat pathogen Parastagonospora nodorum.

The model pathogen Parastagonospora nodorum is a necrotroph and the causal agent of the wheat disease Septoria nodorum blotch (SNB). The sequenced P. nodorum genome has revealed that the fungus harbours a large number of secondary metabolite genes. Secondary metabolites are known to play important roles in the virulence of plant pathogens, but limited knowledge is available about the SM repertoire of this wheat pathogen. Here, we review the secondary metabolites that have been isolated from P. nodorum and related species of the same genus and provide an in-depth genome-wide overview of the secondary metabolite gene clusters encoded in the P. nodorum genome. The secondary metabolite gene survey reveals that P. nodorum is capable of producing a diverse range of small molecules and exciting prospects exist for discovery of novel virulence factors and bioactive molecules.