New Results

Species hybridisation and clonal expansion as a new fungicide resistance evolutionary mechanism in Pyrenophora teres spp

Chala Turo, Wesley Mair, Anke Martin, Simon Ellwood, Richard Oliver, Francisco Lopez-Ruiz
doi: https://doi.org/10.1101/2021.07.30.454422

ABSTRACT

The barley net blotch diseases are caused by two fungal species of the Pyrenophora genus. Specifically, spot form net blotch is caused by P. teres f. sp. maculata (Ptm) whereas net form net blotch is caused by P. teres f. sp. teres (Ptt). Ptt and Ptm show high genetic diversity in the field due to intraspecific sexual recombination and hybridisation of the two species although the latter is considered rare. Here we present occurrence of a natural Ptt/Ptm hybrid with azole fungicides resistance and its implication to barley disease management in Australia. We collected and sequenced a hybrid, 3 Ptm and 10 Ptt isolates and performed recombination analyses in the intergenic and whole genome level. Eleven out of 12 chromosomes showed significant (P < 0.05) recombination events in the intergenic regions while variable recombination rate showed significant recombination across all the chromosomes. Locus specific analyses of Cyp51A1 gene showed at least four recombination breakpoints including a point mutation that alter target protein function. This point mutation did not found in Ptt and Ptm collected prior to 2013 and 2017, respectively. Further genotyping of fourteen Ptt, 48 HR Ptm, fifteen Ptm and two P. teres isolates from barley grass using Diversity Arrays Technology markers showed that all HR Ptm isolates were clonal and not clustered with Ptt or Ptm. The result confirms occurrence of natural recombination between Ptt and Ptm in Western Australia and the HR Ptm is likely acquired azole fungicide resistance through recombination and underwent recent rapid selective sweep likely within the last decade. The use of available fungicide resistance management tactics are essential to minimise and restrict further dissemination of these adaptive HR Ptm isolates.

INTRODUCTION

Spot form net blotch (SFNB) caused by Pyrenophora teres f. sp. maculata (Smedegard-Petersen, 1971) (Ptm) and net form net blotch (NFNB) caused by Pyrenophora teres f. sp. teres (anamorph Drechslera teres [Sacc.] Shoem.) (Ptt) are two closely related major fungal pathogens of barley (Hordeum vulgare L.). Worldwide importance of the two forms varies, with one or the other predominant in a particular region based on environmental, agronomic, and varietal factors (McLean et al., 2009). The pathogens can cause yield losses of 10 - 44% (Steffenson et al., 1991; Jebbouj & El Yousfi, 2009; McLean et al., 2009). In Australia, the damage inflicted by both pathogens has been estimated to be on average AUD$62 million with potential losses estimated at AUD$309 million annually in the absence of control measures (Murray & Brennan, 2010). Symptoms induced by both pathogens can be easily distinguished, with Ptm producing dark brown spots and Ptt dark net-like lesions along the leaves (Smedegard-Petersen, 1971).

Demethylase-inhibitor (DMI) fungicides are one of the primary modes of action (MOA) employed as part of the chemical management of SFNB and NFNB (Akhavan et al., 2017). However, the widespread use of DMI chemistry has led to the emergence of fungicide resistance in recent years. Mutation F489L in the DMI fungicide target gene, Cyp51A, was found to be associated with DMI resistance in Ptt isolates since 2013 in Western Australia (WA)(Mair et al., 2016). From 2016 onwards, Ptm isolates resistant to DMI fungicides were found to carry mutation F489L and/or an insertion of a 134 bp fragment of a transposable element in the Cyp51A promoter region (Mair et al., 2020). Cyp51A exists as a single copy in Ptm while two copies have been identified in Ptt (Mair et al., 2016).

Worldwide studies on population genetics of Ptt and Ptm have shown extremely high levels of genetic diversity in field populations (Rau et al., 2003; Serenius et al., 2007; Bogacki et al., 2010; McLean et al., 2010; Gupta et al., 2012; McLean et al., 2014; Akhavan et al., 2016). These genetic variations were driven by sexual reproduction, gene flow and transposable element invasion. More recently, Ptt population genetic studies using Diversity Arrays Technology (DArT) found rapid changes in population genetic structure under field conditions (Poudel et al., 2019b).

Continuous growth of barley can lead to build up of both Ptt and Ptm populations, which favours their genetic diversification and ultimately the emergence of new pathotypes through sexual recombination. Recombination can rapidly assemble new combinations of alleles to overcome selection pressure, invade novel niches or lead to emergence of new pathogenic species (McDonald & Linde, 2002; Depotter et al., 2016).

In addition to intraspecific sexual recombination, occurrence of interspecific hybridization between Ptt and Ptm has been reported by several authors. The first putative hybrid was reported from South Africa in 2002 (Campbell et al., 2002), followed by the Czech Republic (Leisova et al., 2005) and Australia (Lehmensiek et al., 2010; McLean et al., 2014). These recombinant hybrids are able to retain their genetic stability over time (Campbell & Crous, 2003; Jalli, 2011)). The symptoms induced by hybrids are indistinguishable from those produced by Ptt or Ptm, and some hybrids show more virulence than either parents (Campbell et al., 1999; Campbell et al., 2002; Campbell & Crous, 2003).

Interspecific hybridization in P. teres have previously been studied using amplified fragment length polymorphisms (AFLP), random amplified polymorphic DNA (RAPD) or form-specific PCR markers, which depend on comparatively small number of molecular markers (Campbell et al., 2002; Rau et al., 2003; Lehmensiek et al., 2010; McLean et al., 2014; Poudel et al., 2017). Population genetic analyses can now be performed using higher throughput technologies such as DArT (Ndjiondjop et al., 2017). Furthermore, the ever decreasing costs of whole genome sequencing allowed population genomic studies to uncover the level of introgression occurring across diverse microbes. Such studies were applied to detect recombination among strains or closely related species of Zymoseptoria species (Stukenbrock & Dutheil, 2017), Aspergillus fumigatus (Abdolrasouli et al., 2015), Blumeria species (Feurtey et al., 2019) and Histoplasma species (Maxwell et al., 2018). In some instances, introgressed regions have been associated with responses to stresses (Zhang et al., 2018) or adaptation to new hosts (Feurtey et al., 2019).

The almost simultaneous emergence of the same DMI resistance mutation in both Ptt and in highly resistant P. teres isolates that induced typical spot form symptoms (termed here “HR Ptm”), raises the question as to whether fungicide resistance emerged as a result of hybridization between these two pathogens. Here we present a comprehensive genetics and genomic studies that dissects the occurrence of natural hybridization between Ptt and Ptm, and determines the origin of fungicide resistant hybrids.

MATERIALS AND METHODS

Isolate collection and species identification using form-specific primers

A total of 48 HR Ptm isolates were obtained from diseased leaf and stubble samples collected in the 2017 and 2018 cropping seasons from nine locations across the barley growing regions of WA (Table 1). Fungal isolations from infected leaves were conducted as previously described (Mair et al., 2020). For isolation from infected stubble, tissue was washed with sterile water, dried on paper towel, placed in petri dishes on moist paper, and incubated at 15 °C for 5 days. A single conidium was transferred to V8PDA (10 g potato-dextrose agar, 3 g CaCO3, 15 g agar, 150 mL V8 juice in 850 mL deionized H2O) plates amended with ampicillin (100 μg mL−1), streptomycin (100 μg mL−1), and neomycin (50 μg mL−1). The isolates were grown on V8PDA for 5 days at room temperature. Genomic DNA was isolated from mycelia on a Biosprint 15 instrument using Biosprint DNA Plant Kit (QIAGEN, Hilden, Germany) as per manufacturer’s instructions. Genomic DNA was visualized by electrophoresis on 1% agarose gel, assessed for purity on a Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA USA), and quantitated using a Quantus fluoromoter with the QuantiFluor dsDNA kit (Promega, Madison, WI, USA). The isolates were identified using form-specific molecular markers and PCR conditions described in Poudel et al. (2017).

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Table 1.

Description of isolates used in the current study

Whole genome sequencing and assembling

For whole genome sequencing, isolates 9193, Ko103, 16FRG073, 17FRG026, and 17FRG089 were cultured and genomic DNA extracted as described by Syme et al. (2018). DNA was assessed for purity and quantitated as described above. Sequencing was performed by Macrogen (Seoul, South Korea) using 20 kbp SMRTbell libraries on the PacBio RSII platform (Pacific BioSciences, Menlo Park, CA, USA) as per manufacturer’s protocols. The reads were assembled into contigs using canu version 1.6 (Koren et al., 2017).

Genetic diversity analysis

SilicoDArT and SNP DArT markers were used to analyse the genetic diversity of 48 DMI resistant isolates collected from WA. 15 Ptm, 14 Ptt isolates collected between 1996 and 2019 together with two P. teres isolates obtained from barley grass grass (Hordeum leporinum) were also included in the study. DArTseq was performed by Diversity Arrays Technology Pty Ltd (Canberra, ACT, Australia) with the P. teres array using the HiSeq 2000 platform (Illumina, San Diego, CA, USA) as per manufacturer’s protocols. Only markers with sequences present in the W1-1 (GenBank BioProject PRJEB18107, assembly GCA_900232045.2) or the 0-1 (GenBank BioProject PRJNA392275, assembly GCA_006112615.1) reference genomes were used, and markers with more than 5% missing data were removed; the remaining 9,656 silicoDArT and SNP markers were used in the analysis. The similarity matrix was constructed using the DICE coefficient (Dice, 1945) in the Dissimilarity program of the Dissimilarity Analyses and Representation for Windows (DARwin) software package, version 6.0.21 (http://darwin.cirad.fr/) (Perrier & Jacquemoud-Collet, 2006). Cluster analyses of the matrix values was performed using the Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) (Sneath & Sokal, 1973) in the Hierarchical Clustering program, and the dendrogram was produced using Trees Draw program of DARwin.

DArT SNP markers generated for each fungal species were used to compute genetic diversity using R statistical software (https://www.r-project.org/)(R Core Team, 2018). The raw DArT data were imported and converted into genlight object using gl.read.dart() function implemented in dartR package (Gruber et al., 2018). Fixation indexes (FST) were computed using ‘stamppFst’ to explore genetic differentiation, as implemented in R statistical package ‘StAMPP’ (Pembleton et al., 2013). The genetic similarity of individuals was analysed and visualized using Principal Coordinates Analyses (PCoA) using dartR statistical package (Gruber et al., 2018) developed for analyses of DArT markers. The PCoA was based on standardized covariance of genetic distances calculated from DArT markers and the analyses was performed using default parameters. The Nei’s genetic distances between pairs were computed using the StAMPP package (Pembleton et al., 2013).

Analyses of recombination in the intergenic regions

The reference genome sequence of Ptt isolate W1-1 (Syme et al. (2018) was used in this analysis. Coordinates of protein coding genes were taken from the General Feature Format (GFF) file from W1-1, after adding 500 bp upstream and downstream of each gene using the slope command available in the Bedtools suite (Quinlan & Hall, 2010). Any regions that overlapped with repetitive elements were discarded and the remaining coordinates were used to extract 1 kb intergenic regions. Homologous regions were searched from other Ptt and Ptm genomes sequenced in this study (9193, Ko103, 16FRG073, 17FRG026, and 17FRG089), as well as the previously published genomes of isolates SG1 (Ellwood et al., 2012), NB29, NB85, Cad64 and Mur2 (Syme et al., 2018), and 73, 9122 and 9139 (Moolhuijzen et al., 2020) using Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990), and the best matching regions were extracted to generate multiple sequence alignments for each intergenic regions using MAFFT (Katoh, 2002). The aligned regions were subjected to recombination test using PHIPack (Bruen, 2005).

Whole genome sequence based recombination analysis

The genome sequences of nine Ptt isolates (two DMI-resistant: Ko103 and 17FRG026, and seven DMI-sensitive: 73, 9122, 9139, 9193, NB29, NB85 and W1-1), four Ptm isolates (one DMI-resistant: 16FRG073, and three DMI-sensitive: Cad64, Mur2 and SG1), and one DMI-resistant putative hybrid (“HR Ptm”, 17FRG089), were investigated to determine whether recombination events between Ptt and Ptm isolates had occurred. Genome sequences were aligned using Sibeliaz (Minkin & Medvedev, 2019) using sequence of eleven k-mers. The multiple sequence alignments were filtered (MinBlockLength of 100 bp), duplicates removed, concatenated and ordered relative to W1-1 after masking any repetitive regions. The ordered alignments were filtered using MafFilter (Dutheil et al., 2014). Only blocks where all isolates were present were retained; a window of 10 bp was slided by 1 bp, and windows containing at least two insertion and or deletion events were discarded and the containing blocks split; in addition, windows with a total of > 100 gap characters were discarded and the containing blocks were split; and all blocks were merged according to reference genome with empty positions filled by “N”. Refined multiple sequence alignments were subjected to variable recombination rate analyses using LDJump (Hermann et al., 2019) with segment length of 5 kb interval, P value of 0.05. LDJump requires other recombination analyses tools including LDhat (Auton & McVean, 2007) and PhiPack to estimate variable recombination rate and generate a recombination map in a specific DNA segment. Final recombination maps were generated for each chromosome using LDJump. The segment length was set above recommended 1 kb to avoid imputation due to low SNP density.

Phylogenetic analysis

Phylogenetic analyses was performed on selected intergenic regions and all the twelve chromosomes. In intergenic regions, multiple sequence alignment was performed using MAFFT version 5 (Katoh, 2002) with default parameters. Phylogenetic trees were generated for selected genomic regions following the neighbour-joining method with Juke-Cantor genetic distance with bootstrap sampling of 1,000 random replicate sampling and 1,000,000 Seeding in Genious version 8.0.3 (Kearse et al., 2012). Similarly, in chromosome level sequence alignments, the phylogenetic trees were constructed using modified neighbour-joining method (Gascuel, 1997) using maximum likelihood distance estimated with pairwise substitution model K80 (kappa=2) as implemented in MafFilter program and trees were visualized using FigTree V1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/).

RESULTS

Fungicide resistant isolates show evidence of hybridisation between Ptt and Ptm and recombination of the mutant Cyp51A allele

Independent repeated PCR analyses using Ptm- and Ptt- specific primers showed all HR Ptm isolates were positive for all 6 Ptm specific markers (Figure 1). Five of the Ptt-specific markers amplified only all of the Ptt isolates, while one Ptt-specific marker (PttQ4) was able to amplify both all Ptt and all HR Ptm isolates. On the other hand, all six of the Ptm-specific markers amplified only Ptm. An identical pattern was observed across all 48 HR Ptm isolates screened using the form-specific markers.

Figure 1.
Figure 1.

Amplification of using form specific markers. The lanes 1. 17FRG164 2. 17FRG165 3. 17FRG166 4. 17FRG167 5. 17FRG186 6. 17FRG187 7. 17FRG195 8. 17FRG196 9. 17FRG197 10. 17FRG198 11. SG1 (Ptm Control) 12. Ko103 (Ptt Control) 13. No Template Control. Lane 1-10 isolates carry F489L (c1467a) mutation in highly resistant Ptm isolates.

The multiple sequence alignments of 64 Cyp51A coding sequences from Ptt and Ptm indicated a preferential mutation pattern in all the HR Ptm isolates. All HR Ptm isolates carry the SNP c1467a for the amino acid substitution F489L, identical to the SNP found in all DMI resistant Ptt (Figure 2). At the proximal polymorphic site in the Cyp51A sequence (position 1488), all HR Ptm, as well as all Ptt (both DMI-sensitive and resistant) carry variant g1488; all other Ptm (both DMI-sensitive and resistant) carry c1488. Genetic recombination rate analyses using DnaSP identified at least four break within Cyp51A (Figure 2).

Figure 2.
Figure 2.

The four recombination break points predicted in CYP51A protein coding region identified using DnaSP. Position 1467 is a mutation (c1467a) that alter the protein function and observed only in HR Ptm and Ptt.

Hybrid genome shows evidence of hybridization between Ptt and Ptm

We further tested signatures of hybridization among isolates of Ptt, Ptm and HR Ptm first in the conserved intergenic regions and then extended to chromosome level. A total of 1,393 intergenic loci with 1 kb length were identified and tested for presence of recombination on the twelve Ptt chromosomes and their respective homologs in Ptm and HR Ptm. The recombination analyses using PhiPack revealed the existence of significant (P < 0.05) recombination events on 75 of the loci (Table 2). The number of significant recombination events across intergenic regions ranged from twelve on Chr02 to two on Chr04, while no significant intergenic recombination was detected on Chr12.

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Table 2.

Recombination frequency detected in the intergenic 1 kb regions across 12 chromosomes

For further large-scale recombination analysis, we generated multiple sequence alignments varying between 3.84 Mb on Chr01 and 0.94 Mb on Chr12. Occurrence of recombination was observed on all the twelve chromosomes. Chr11 had the highest recombination rate (0.0437) followed by Chr06 (0.0394) (Figure 4). As opposed to the intergenic regions, all twelve of the chromosomes showed significant evidence of recombination, which appeared to occur in clusters across certain chromosomal regions as indicated on the recombination map shown in figure 4.

Figure 3.
Figure 3.

Potential recombination regions predicted in CYP51A protein coding sequences. The red horizontal line is drawn at P value of 0.05.

Figure 4
Figure 4

Estimated recombination map for fourteen P. teres isolates (10 Ptt, 3 Ptm, HR Ptm). Each segment has 5 kb length. The peak values indicate the higher recombination rate.

Close inspection of the aligned intergenic regions where recombination was detected revealed consistent SNP patterns in the HR Ptm isolate 17FRG089 (Figure 5A). In this isolate, the sequence comprised an admixed mosaic of SNPs characteristic of either Ptt or Ptm haplotypes; the observed SNP patterns spanned short genomic regions often less than 1 kb before switching between Ptt and Ptm. An example of this can be seen in the conserved intergenic region of Chr06, where CYP51A gene resides (Figure 5A).

Figure 5.
Figure 5.

The mutation pattern observed in the HR Ptm isolate 17FRG089 in the intergenic region on Chr06 (A), partial alignment of Chr07 (B) and the corresponding Neighbour-Joining trees (C, D) constructed using Juke-Cantor genetic distance model with bootstrap sampling of 1000 replicates. Vertical lines in the sequence alignment show mismatch positions. Red and black broken boxes indicate the alternating SNP pattern of HR Ptm isolate 17FRG089 between Ptt and Ptm, respectively.

Likewise, in a 2.1 kb region from a larger alignment of Chr07 (1,565,695 – 1,567,791), conserved HR Ptm SNPs were located in short segments alternating between Ptt and Ptm sequence, with a larger proportion of SNPs corresponding to Ptm (Figure 5B). A short 573 nucleotide segment (1,565,695 – 1,566,268) contained 20 SNPs of which the first eleven consecutive ones were mapped to Ptm and the last five consecutive SNPs were mapped to Ptt. The remaining four SNPs did not exist in either Ptm or Ptt. Five more HR Ptm SNPs found in an adjacent 167 nucleotide segment (positions 1,566,345, 1,566,434, 1,566,446, 1,566,504 and 1,566,512) were unambiguously mapped to Ptt, Ptm, Ptm, Ptt and Ptm in that order. A similar pattern could be observed in further SNP positions between (1,566,513 and 1,567,791), with three consecutive SNPs are aligned to Ptm, followed by another three SNPs that aligned to Ptt and final fifteen SNPs mapped to Ptm.

Neighbour-joined phylogenetic trees constructed from subsets of the intergenic region showed deviation of the HR Ptm isolate 17FRG089 from other Ptm isolates with 100% bootstrap support, but more related to Ptm than Ptt (Figure 5 C and D). The chromosome-level phylogenetic analyses is also consistent with the phylogenies constructed from the intergenic sequences. The HR Ptm isolate 17FRG089 is closely related to Ptm with the branching pattern identical on six of the chromosomes (1 – 2 and 9 – 12) and basal to the Ptm isolates, indicating distinct genotypic composition of the HR Ptm isolates (Figure 7). Interestingly, on six of the chromosomes (Chr03 – Chr08), the HR Ptm isolate 17FRG089 is closely related to one of the DMI sensitive Ptm isolates (Mur2), with a high number of SNPs shared between these two isolates indicating a common ancestral background. However, Ptm isolate Mur2 lacks the alternating SNP signatures observed in HR Ptm isolate 17FRG089.

Figure 7.
Figure 7.

Chromosome based phylogenetic analyses of Ptt, Ptm and HR Ptm isolates. The HR Ptm isolate 17FRG089 is represented in bold font.

Genetic structure analyses reveals clonal population of HR Ptm distinct from both Ptm and Ptt

The use of silicoDArT marker analyses to determine the genetic structure of the HR Ptm isolates, allowed to distinctly group Ptt, Ptm, barley grass P. teres and HR Ptm isolates (Figure 8). The HR Ptm did not cluster with Ptt or barley grass P. teres; they also did not cluster with the Ptm isolates but were more closely related to them. A high degree of clonality was observed among the 48 HR Ptm, with all of the isolates across the nine sites, separated in some cases by more than 440 km, being genetically identical to one another.

Figure 8.
Figure 8.

Genetic relationship among Ptt, Ptm, HR Ptm and barley grass P teres

Average Nei’s genetic distance computed among each cluster using DArT SNP showed the highest genetic distance between the HR Ptm and Ptt (0.807) isolates, while the lowest genetic distance was found between Ptm and the HR Ptm isolates (0.189). Similarly, the largest (0.981) and lowest (0.842) genetic differentiation (FST) was observed between HR Ptm and barley grass P. teres, and between Ptm and HR Ptm (Table 3), respectively, indicating HR Ptm are more closely related to Ptm. Further molecular analyses of variance using Nei’s genetic distance also revealed significant differences in the extent of genetic differentiation among the observed clusters accounting for over 99% of the total variation (Table 4).

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Table 3.

Pairwise FST values (lower metrics) and Jaccard genetic distances (upper metrics) between four populations calculated from DArT SNP loci using 100 bootstrap. For FST, lower and upper bound confidence intervals calculated from bootstrapping are listed within brackets.

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Table 4

Molecular analyses of variance (MANOVA) components on metrics of genetic distance between ‘Populations’

PCoA analyses of DArT SNP showed that 55.8%, 20.9% and 11.7% of the total variations were explained by the first, second and third PCoA observed among Ptt, HR Ptm, Ptm and barley grass P. teres isolates, respectively (Figure 8A). The first PCoA clearly separated HR Ptm from Ptt and barley grass P. teres. Similarly, the second PCoA separated barley grass P. teres from Ptt, while the third PCoA distinguished barley grass P. teres from Ptm isolates. Consistent with cluster analysis, all 48 HR Ptm isolates were tightly clustered on the PCoA indicating little allele frequencies overlap between HR Ptm isolates and other Ptm, Ptt or barley grass P. teres isolates (Figure 8A). Further analyses of SNP generated from the whole genome alignment confirmed similar result. Majority of the genetic variations were captured in the first PCoA (79%) and separated Ptt from the rest while the second PCoA explained only 4% of the total variation (Figure 8B). Overall, genetic variation analyses indicated that the HR Ptm isolates collected from WA are genetically different from other Ptm, and appears clonally propagated across the collection sites.

DISCUSSION

The recent availability of genome sequences coupled with sensitive and reliable molecular detection tools have facilitated the discovery of natural interspecies hybridization between the two economically important pathogens of barley, P. teres f. teres and P. teres f. maculata. Here we employed comprehensive molecular and population genetic analyses to characterize a recently emerged population of P. teres isolates (HR Ptm) highly resistant to DMI fungicides able to induce symptoms shared by both P. teres f. teres and P. teres f. maculata characteristics.

The amplification in all the HR Ptm isolates of one Ptt-specific marker (PttQ4) provided the first indication that the HR Ptm isolates are hybrids. These markers were specifically developed to distinguish Ptt from Ptm and to identify hybrids between the two forms (Poudel et al., 2017). Recent molecular characterization using these marker set of over 300 Australian P. teres isolates collected across four decades, was unable to detect any hybrids (Poudel et al., 2019a). Poudel et al. (2019a) was also unable to detect hybrids among over 800 conidia and 200 ascospores screened from three artifical field inoculation experiments of Ptt and Ptm conducted across three seasons. A previous study by Poudel et al. (2017) using the same marker set confirmed a single field hybrid among over 200 Australian P. teres isolates tested. Rau et al. (2007) concluded that Ptt and Ptm are reproductively isolated and hybridisation between the two forms under field conditions to be either rare or absent, and furthermore that the two formae speciales are in an advanced stage of speciation and should be considered as distinct species. In contrast to these studies, we have found a relatively large number of hybrids isolates in the WA P. teres population using the form-specific markers, although samples were not collected randomly and so the true frequency of hybrids in the WA P. teres population cannot be estimated confidently. Given the lack of genetic diversity among the HR Ptm isolates, it is likely that they all derive from a single hybridisation event, in which case our findings would still accord with those from previous studies that suggest that such events are a rare occurrence in nature. A range of reproductive barriers have been hypothesized to explain the infrequent observation of Ptt and Ptm hybrids in nature (Poudel et al., 2019a), including genetic incompatibilities causing unfit hybrid progeny, or reduced fitness from intermediate traits. On the other hand, Campbell and Crous (2003) found that laboratory Ptt and Ptm hybrids remained fertile, virulent and genetically stable. It is possible that the acquisition of a DMI resistance mutation in an evolutionary landscape dominated by heavy fungicide selection is compensatory to the fitness penalties that may inhere from interspecies hybridisation.

Intergenic recombination

All the HR Ptm isolates carry the non-synonymous point mutation c1467a in the Cyp51A gene, which results in the amino acid substitution F489L in CYP51A. An identical SNP for the F489L mutation has previously been reported in Ptt, where it is associated with reduced sensitivity to DMI fungicides(Mair et al., 2016). We evaluated whether the c1467a mutation occurred de novo in HR Ptm or was acquired from Ptt through genetic recombination between the two forms. Two independent genetic recombination analyses, using LDJump and DNA sequence polymorphism analyses tool (DnaSP) (Librado & Rozas, 2009) both indicated intragenic recombination within the Cyp51A coding sequence between Ptt and Ptm, and one of the sites involved in recombination included mutation c1467a. Previous reports have shown that intragenic recombination is an important evolutionary process in populations under fungicide selection, acting as a potential source of novel fungicide resistance alleles (Brunner et al., 2008). Intragenic recombination in the Cyp51 gene has been previously reported in Z. tritici (Brunner et al., 2008; Estep et al., 2015), and has been associated with increased resistance to DMI fungicides. The mutations in the Cyp51 gene in European Z. tritici all emerged only once or twice before spreading into the broader population via gene flow, recombining to form novel combinations of resistance mutations (Brunner et al., 2008). In a North American population of Z. tritici, the G460D mutation of Cyp51 likely emerged de novo only once, before entering the broader population via at least two distinct intragenic recombination events (Estep et al., 2015). Similarly, intragenic recombination has also been detected in the Cyp51A gene of Rhynchosporium commune (Brunner et al., 2016). The current study is, to our knowledge, the first report of interspecific recombination involving a gene associated with fungicide resistance in P. teres species.

The intergenic and whole chromosome recombination analyses using de novo assembled genome sequences of each P. teres isolates provided strong evidence of genetic exchange between the two forms, consistent with analyses using form-specific markers and recombination analyses of the Cyp51A gene. Previous studies have indicated that de novo assembly-based analyses enables identification of highly variable genomic regions involved in hybridization which are missed through raw read mapping approach (Feurtey et al., 2019). Studies of Zymoseptoria ardabiliae and Z. tritici indicated high recombination rates in intergenic regions (Stukenbrock & Dutheil, 2018). Similar evidence of recent hybridization has been reported within several species of the Zymoseptoria genus (Feurtey et al., 2019). Recombination has also been detected in intergenic regions between members of the Histoplasma genus (Maxwell et al., 2018).

Population structure

The neighbour joining phylogenetic analyses using subset of intergenic sequences showed that the HR Ptm isolates (represented by 17FRG089) lie between Ptt and Ptm with 100% bootstrap support. Further chromosome-level phylogenetic tree analyses were also concordant to phylogenetic trees generated from the intergenic sequences, indicating that the HR Ptm isolates are genetically distinguishable from both Ptt and Ptm isolates. The whole genome-based PCoA also separated HR Ptm (17FRG089) as distinct from Ptt and Ptm, suggesting unique genetic constitution of the HR Ptm isolate in the current study. Since the HR Ptm was represented with single genome sequence, we utilised DArT SNP analyses with a larger number of HR Ptm isolates to capture the genetic variation that existed among Ptt, Ptm, HR Ptm and P. teres isolates from barley grasses. The smallest computed genetic distance (0.080) was between HR Ptm and other Ptm isolates, suggesting that the genetic makeup of the HR Ptm is mainly Ptm, likely resultant from multiple backcrossing of the progenies following the hybridisation event. The clustering of the HR Ptm into a single group on dendrogram and PCoA (Figure 8 and 9) showed an absence of genetic diversity among the HR Ptm isolates. The HR Ptm were sampled from sites separated by up to 440 km, from Frankland River in the Great Southern region to Gibson in the Esperance region in WA, suggesting a clonal expansion of the HR Ptm isolates as a potential explanation for their spread across a wide geographical area in a very short period of time. These results are congruent with asexual selection of progeny harbouring DMI-resistance alleles under the selective pressure of DMI use, following a single hybridisation event. DMI-resistant A. fumigatus isolates from India were highly related despite their collection points were more than 1000 km apart (Abdolrasouli et al., 2015). The authors concluded that the DMI-resistance mutations were therefore likely associated with an A. fumigatus highly fit genotype that had undergone a recent, rapid selective sweep. A similar dynamic may account for the lack of observed variation among isolates of the HR Ptm genotype, with DMI fungicide use or possibly varietal choice acting as the selective sweep within the WA Ptm population.

Figure 9.
Figure 9.

Distribution of Ptt, Ptm, HR Ptm and barley grass P. teres isolates in different quadrants based on principal coordinate analyses using SNP DArT markers (A) and whole genome SNP data (B). (A) In SNP DArT marker analysis, PCoA 1, PCoA 2 and PCoA 3 explain 55.8% and 20.9% 11.7% of the total variations, respectively. (B) In Large scale PCoA analyses using SNP generated from genome, the first PCoA alone explained 79% of the total variation.

Implications

Campbell and Crous (2003) previously raised the epidemiological implications of natural hybridisation between Ptt and Ptm, with the possibility of new genotypes thereby being introduced into populations. Interspecies recombination is increasingly acknowledged for its significant contribution to pathogen diversification (Feurtey & Stukenbrock, 2018). Genetic hybridization studies between the grass symbionts Epichloe festucae and Epichloe gansuensis indicated that genetic introgression of fungi living on the same hosts significantly contribute to their adaptive evolution (Zhang et al., 2018). In some cases hybridisation can even lead to the formation of novel species, as in the wheat pathogen Zymoseptoria pseudotritici (Stukenbrock et al., 2012), or novel formae speciales, as in the triticale pathogen Blumeria graminis f. sp. triticale (Menardo et al., 2016). Although true population frequencies cannot be estimated confidently, it is apparent that among the DMI-resistant WA Ptm isolates found so far, the clonal HR Ptm genotype was clearly predominant (Mair et al., 2020), suggesting the possibility of lineage replacement.

Historically, Ptm was reported as much less prevalent than Ptt in WA; a 1995-96 survey was the first to observe the spot form net blotch disease outside of the northern barley-growing region of WA, while it reached epidemic levels in the southern agricultural regions of WA in 1997-1998 (Gupta & Loughman, 2001). In subsequent years, Ptm became much more prevalent across WA southern barley-growing regions, aided by the wide spread adoption of susceptible varieties such as Gairdner, Hamelin, Vlamingh and Baudin (Gupta et al., 2012). Campbell et al. (2002) suggested that growing barley cultivars susceptible to both forms in very close proximity potentially favour sexual reproduction between the two forms. The lack of resistant cultivars together with the introduction of stubble retention practices and high inclusion of continuous barley, has led to an increase in disease incidence and severity in recent years (Gupta et al., 2012)(McLean et al., 2009). These conditions have likely increased the opportunities for hybridisation between the two pathogens as they co-exist infecting the same host. The HR Ptm isolates were mainly associated with barley cv. Oxford, a variety that has become increasingly susceptible to Ptt over recent years with the spread of the new, aggressive ‘Oxford virulent’ pathotype, particularly around the Esperance and Great Southern growing regions (Shackley, 2019). The other varieties with which the HR Ptm genotype was associated (albeit at lower frequencies), Planet and La Trobe, are also closely related to Oxford.

The use of DMI fungicides in barley in Australia begun in 1995 and has increased substantially in subsequent decades (Tucker et al., 2015). It appears likely that the Ptt-Ptm hybridization event took place in the last decade, because the point mutation c1467a in Cyp51A gene is absent in Ptm collections prior to 2017 (Mair et al., 2020), and the same Cyp51A point mutation in Ptt was found only from 2013 onwards (Mair et al., 2016). Analyses of samples from older collections may allow us to more accurately estimate the exact time of hybridisation using a population genomics approach.

Ptm conidia are generally understood to be dispersed primarily by wind and rain splash over relatively short distances (Poudel, 2018), so other factors, such as dispersal on hay or possibly on seed, must account for the observed wide geographical distribution of the HR Ptm genotype in WA in a relatively short period of time. Although only Ptt has been reported to be seed transmitted (McLean et al., 2009), it is not clear if this would also be true for any putative Ptt-Ptm hybrids, and it is possible that genes involved in Ptt seed transmission have introgressed during hybridisation. Additional genes affecting virulence on different host varieties may also have been acquired by the hybrid, which may help account for the observed preponderance of HR Ptm isolates being derived from cv. Oxford and a small number of closely-related cultivars. In light of these findings, there is an urgent need for the deployment of integrated disease and resistance management strategies that take into account not just the effect of fungicide use and varietal choice in both Ptt and Ptm, but also the possible exchange of fungicide resistance and virulence genes between the two forms.

ACKNOWLEDGEMENT

The authors would like to thank the sample contributions made by growers and agronomists. This study was conducted by the Centre for Crop and Disease Management, a joint initiative of Curtin University and the Grains Research and Development Corporation (research grant CUR00023). We also acknowledge the assistance of resources from the National Computational Infrastructure (NCI Australia), an NCRIS enabled capability supported by the Australian Government.

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Posted July 31, 2021.
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Species hybridisation and clonal expansion as a new fungicide resistance evolutionary mechanism in Pyrenophora teres spp
Chala Turo, Wesley Mair, Anke Martin, Simon Ellwood, Richard Oliver, Francisco Lopez-Ruiz
bioRxiv 2021.07.30.454422; doi: https://doi.org/10.1101/2021.07.30.454422
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