Genetic diversity within pathogen populations is critically important for predicting pathogen evo... more Genetic diversity within pathogen populations is critically important for predicting pathogen evolution, disease outcomes and prevalence. However, we lack a good understanding of the processes maintaining genetic variation and constraints on pathogen life-history evolution. Here, we analyzed interactions between 12 wheat host genotypes and 145 strains of Zymoseptoria tritici from five global populations to investigate the evolution and maintenance of variation in pathogen virulence and reproduction. We found a strong positive correlation between virulence and reproduction, with substantial variation in both traits maintained within each pathogen population. On average, highly virulent isolates exhibited higher fecundity, which might increase transmission potential in agricultural fields planted to homogeneous hosts at a high density. We further showed that pathogen strains with a narrow host range (i.e. specialists) for fecundity were on average less virulent, and those with a broad...
The transposable element insertion upstream of Zmr1 in 3D1 downregulates Zmr1 expression. Mean gr... more The transposable element insertion upstream of Zmr1 in 3D1 downregulates Zmr1 expression. Mean gray values (0 = black, 255 = white) based on at least 35 colonies of the wild-types 3D1 and 3D7, three independent TE deletion mutants in the 3D1 background (3D1 ΔTE #93, #131, #239) and the two ectopic controls (3D1+ Hyg #3 and #6), 7 days post inoculation. Asterisks (*) indicate that the strains are significantly darker than the wild-type 3D1 (Kruskal-Wallis, p value ≤ 0.05). The experiment was performed three times with 3D1ΔTE #93 and twice with #131 and #239. (PDF 330 kb)
Sequence variation in Zmr1 promoter contributes to differences in melanin accumulation. Means and... more Sequence variation in Zmr1 promoter contributes to differences in melanin accumulation. Means and standard errors of gray values (0 = black, 255 = white) of 3D1, 3D7, 3D7Δzmr1, 3D7Δzmr1 + Zmr13D1, and 3D7Δzmr1 + Zmr13D7, 7 days post inoculation (dpi) based on at least 20 colonies. Asterisks (*) and pluses (+) indicate significant differences in mean gray values of each strain with respect to the mean gray value of 3D7 and 3D7Δzmr1, respectively (Kruskal-Wallis, p values ≤ 0.05). The experiment was performed twice with similar results. NA = not applicable. (PDF 335 kb)
3D7 accumulates more melanin than 3D1. Means and standard errors of gray values (0 = black, 255 =... more 3D7 accumulates more melanin than 3D1. Means and standard errors of gray values (0 = black, 255 = white) of at least 60 colonies of Z. tritici strains 3D1 and 3D7 at 7–12 days post inoculation (dpi). The experiment was performed three times with similar results. Asterisks indicate significant differences between 3D1 and 3D7 at each time point according to Kruskal-Wallis test (p values ≤ 0.05). n = number of colonies analyzed. (PDF 110 kb)
Generation of mutants (A) Schematic diagram showing the location of primers used for generating Z... more Generation of mutants (A) Schematic diagram showing the location of primers used for generating Zmr1 disruptant mutants (U2 + U3, D1 + D2) and the primers used for screening the transformants (U1 + Hyg UF, Hyg DF + D3). (B) Schematic diagram showing the location of primers used for generating the transposable element (TE) deletion mutants in 3D1 background (ΔTE; TE_U2 + TE_U3, TE_D1 + TE_D2) and the primers used for screening the transformants (TE_U1 + Hyg UF, Hyg DF + TE_D3). (C) Diagrams showing the location of primers used for generating the transformants expressing Zmr1 gene in locus in the 3D7Δzmr1 background. Amplification of Zmr1 gene from genomic DNA of 3D1 and 3D7 strains was performed using Zmr1 F and Zmr1 R primers; the geneticin resistance cassette (Gen) was amplified from pCGEN with Gen F and Gen R. Both amplicons were fused to the pES1 backbone to generate an intermediate construct, which was used to amplify Zmr1 and the geneticin resistance cassette using Zmr1 in locu...
No significant difference in the distribution of mean gray values of different Z. tritici strains... more No significant difference in the distribution of mean gray values of different Z. tritici strains belonging to four different populations. Distribution of mean gray values of different Z. tritici strains from four different populations across the world (3 strains from Australia, 8 from Switzerland, 9 from Israel, and 13 from the USA). The colors of the violin plot indicate different populations. Black dots represent individual data points, which correspond to the mean gray values of each Z. tritici strain at 7 days post inoculation. At least 100 colonies grown on five different plates were evaluated. The experiment was performed three times with similar results. No statistically significant differences were observed between the populations (Kruskal-Wallis test, p value ≤ 0.05). (TIF 172 kb)
Table S2. Short read coverage of Magnaporthe oryzae and wheat transcriptomes in Bangladeshi sampl... more Table S2. Short read coverage of Magnaporthe oryzae and wheat transcriptomes in Bangladeshi samples. (PDF 66 kb)
Number of subpopulations. The peaks represent possible numbers of differentiated populations amon... more Number of subpopulations. The peaks represent possible numbers of differentiated populations among the 213 isolates, with a highest likelihood of three. The bar plots refer to the membership coefficient for each isolate organized according to 13 field populations under the assumption of three subpopulations. An isolate was assigned to a subpopulation if its membership coefficient was ≥0.8. (PDF 382 kb)
Reactive oxygen species are toxic byproducts of aerobic respiration that are also important in me... more Reactive oxygen species are toxic byproducts of aerobic respiration that are also important in mediating a diversity of cellular functions. Reactive oxygen species form an important component of plant defenses to inhibit microbial pathogens during pathogen-plant interactions. Tolerance to oxidative stress is likely to make a significant contribution to the viability and pathogenicity of plant pathogens, but the complex network of oxidative stress responses hinders identification of the genes contributing to this trait. Here, we employed a forward genetic approach to investigate the genetic architecture of oxidative stress tolerance in the fungal wheat pathogen Zymoseptoria tritici. We used quantitative trait locus (QTL) mapping of growth and melanization under axenic conditions in two cross-populations to identify genomic regions associated with tolerance to oxidative stress. We found that QTLs associated with growth under oxidative stress as well as inherent growth can affect oxida...
ABSTRACTReactive oxygen species are toxic byproducts of aerobic respiration produced during cell ... more ABSTRACTReactive oxygen species are toxic byproducts of aerobic respiration produced during cell growth. They also are an important component of plant defenses to inhibit microbial pathogens. Tolerance to oxidative stress contributes to viability and pathogenicity of plant pathogens. However, the complex molecular network of oxidative stress responses hinders identification of the genes contributing to variation in this trait. Variation in genes affecting responses to oxidative stress is likely to affect the evolutionary potential of pathogen tolerance to host defences. Here, we employed a forward genetic approach to investigate the genetic architecture of oxidative stress tolerance in the fungal wheat pathogen Zymoseptoria tritici. By performing quantitative trait locus (QTL) mapping in two crosses, we identified several genomic regions associated with tolerance to oxidative stress, including a QTL having a large effect on growth under oxidative stress. We found evidence for a sign...
SummaryTolerance and resistance represent two strategies that hosts evolved to protect themselves... more SummaryTolerance and resistance represent two strategies that hosts evolved to protect themselves from pathogens. Tolerance alleviates the reduction in host fitness due to infection without reducing a pathogen’s growth, while resistance reduces pathogen growth. We investigated tolerance of wheat to the major fungal pathogen Zymoseptoria tritici in 335 elite wheat cultivars.We used a novel digital phenotyping approach that included 11,152 infected leaves and counted 2,069,048 pathogen fruiting bodies.We discovered a new component of tolerance that is based on the relationship between the green area remaining on a leaf and the number of pathogen fruiting bodies. We found a negative correlation between tolerance and resistance among intolerant cultivars, presenting the first compelling evidence for a tradeoff between tolerance and resistance to plant pathogens. Surprisingly, the tradeoff arises due to limits in the host resources available to the pathogen and not due to metabolic const...
The wheat blast disease has been a serious constraint for wheat production in Latin America since... more The wheat blast disease has been a serious constraint for wheat production in Latin America since the late 1980s. We used a population genomics analysis including 95 genome sequences of the wheat blast pathogen Pyricularia graminis-tritici (Pygt) and other Pyricularia species to show that Pygt is a distinct, highly diverse pathogen species with a broad host range. We assayed 11 neutral SSR loci in 526 Pygt isolates sampled from wheat and other grasses distributed across the wheat-growing region of Brazil to estimate gene flow, assess the importance of sexual reproduction, and compare the genetic structures of Pygt populations infecting wheat and nearby grasses. Our results suggest a mixed reproductive system that includes sexual recombination as well as high levels of gene flow among regions, including evidence for higher gene flow from grass-infecting populations and into wheat-infecting populations than vice versa. The most common virulence groups were shared between the grass- an...
Meiosis is one of the most conserved molecular processes in eukaryotes. The fidelity of pairing a... more Meiosis is one of the most conserved molecular processes in eukaryotes. The fidelity of pairing and segregation of homologous chromosomes has a major impact on the proper transmission of genetic information. Aberrant chromosomal transmission can have major phenotypic consequences, yet the mechanisms are poorly understood. Fungi are excellent models to investigate processes of chromosomal transmission, because many species have highly polymorphic genomes that include accessory chromosomes. Inheritance of accessory chromosomes is often unstable and chromosomal losses have little impact on fitness. We analyzed chromosomal inheritance in 477 progeny coming from two crosses of the fungal wheat pathogen Zymoseptoria tritici. For this, we developed a high-throughput screening method based on restriction site-associated DNA sequencing that generated dense coverage of genetic markers along each chromosome. We identified rare instances of chromosomal duplications (disomy) in core chromosomes....
Philosophical transactions of the Royal Society of London. Series B, Biological sciences, Dec 5, 2016
Agricultural ecosystems are composed of genetically depauperate populations of crop plants grown ... more Agricultural ecosystems are composed of genetically depauperate populations of crop plants grown at a high density and over large spatial scales, with the regional composition of crop species changing little from year to year. These environments are highly conducive for the emergence and dissemination of pathogens. The uniform host populations facilitate the specialization of pathogens to particular crop cultivars and allow the build-up of large population sizes. Population genetic and genomic studies have shed light on the evolutionary mechanisms underlying speciation processes, adaptive evolution and long-distance dispersal of highly damaging pathogens in agro-ecosystems. These studies document the speed with which pathogens evolve to overcome crop resistance genes and pesticides. They also show that crop pathogens can be disseminated very quickly across and among continents through human activities. In this review, we discuss how the peculiar architecture of agro-ecosystems facil...
We conducted a comprehensive analysis of virulence in the fungal wheat pathogen Zymoseptoria trit... more We conducted a comprehensive analysis of virulence in the fungal wheat pathogen Zymoseptoria tritici using QTL mapping. High throughput phenotyping based on automated image analysis allowed measurement of pathogen virulence on a scale and with a precision that was not previously possible. Across two mapping populations encompassing more than 520 progeny, 540,710 pycnidia were counted and their sizes and grey values were measured, yielding over 1.6 million phenotypes associated with pathogen reproduction. Large pycnidia were shown to produce more numerous and larger spores than small pycnidia. Precise measures of percent leaf area covered by lesions provided a quantitative measure of host damage. Combining these large and accurate phenotype datasets with a dense panel of RADseq genetic markers enabled us to genetically dissect pathogen virulence into components related to host damage and components related to pathogen reproduction. We show that different components of virulence can b...
CYP51 encodes the target site of the azole class of fungicides widely used in plant protection. S... more CYP51 encodes the target site of the azole class of fungicides widely used in plant protection. Some ascomycete pathogens carry two CYP51 paralogs called CYP51A and CYP51B. A recent analysis of CYP51 sequences in 14 European isolates of the barley scald pathogen Rhynchosporium commune revealed three CYP51 paralogs, CYP51A, CYP51B, and a pseudogene called CYP51A-p. The same analysis showed that CYP51A exhibits a presence/absence polymorphism, with lower sensitivity to azole fungicides associated with the presence of a functional CYP51A. We analyzed a global collection of nearly 400 R. commune isolates to determine if these findings could be extended beyond Europe. Our results strongly support the hypothesis that CYP51A played a key role in the emergence of azole resistance globally and provide new evidence that the CYP51A gene in R. commune has further evolved, presumably in response to azole exposure. We also present evidence for recent long-distance movement of evolved CYP51A allel...
Genetic diversity within pathogen populations is critically important for predicting pathogen evo... more Genetic diversity within pathogen populations is critically important for predicting pathogen evolution, disease outcomes and prevalence. However, we lack a good understanding of the processes maintaining genetic variation and constraints on pathogen life-history evolution. Here, we analyzed interactions between 12 wheat host genotypes and 145 strains of Zymoseptoria tritici from five global populations to investigate the evolution and maintenance of variation in pathogen virulence and reproduction. We found a strong positive correlation between virulence and reproduction, with substantial variation in both traits maintained within each pathogen population. On average, highly virulent isolates exhibited higher fecundity, which might increase transmission potential in agricultural fields planted to homogeneous hosts at a high density. We further showed that pathogen strains with a narrow host range (i.e. specialists) for fecundity were on average less virulent, and those with a broad...
The transposable element insertion upstream of Zmr1 in 3D1 downregulates Zmr1 expression. Mean gr... more The transposable element insertion upstream of Zmr1 in 3D1 downregulates Zmr1 expression. Mean gray values (0 = black, 255 = white) based on at least 35 colonies of the wild-types 3D1 and 3D7, three independent TE deletion mutants in the 3D1 background (3D1 ΔTE #93, #131, #239) and the two ectopic controls (3D1+ Hyg #3 and #6), 7 days post inoculation. Asterisks (*) indicate that the strains are significantly darker than the wild-type 3D1 (Kruskal-Wallis, p value ≤ 0.05). The experiment was performed three times with 3D1ΔTE #93 and twice with #131 and #239. (PDF 330 kb)
Sequence variation in Zmr1 promoter contributes to differences in melanin accumulation. Means and... more Sequence variation in Zmr1 promoter contributes to differences in melanin accumulation. Means and standard errors of gray values (0 = black, 255 = white) of 3D1, 3D7, 3D7Δzmr1, 3D7Δzmr1 + Zmr13D1, and 3D7Δzmr1 + Zmr13D7, 7 days post inoculation (dpi) based on at least 20 colonies. Asterisks (*) and pluses (+) indicate significant differences in mean gray values of each strain with respect to the mean gray value of 3D7 and 3D7Δzmr1, respectively (Kruskal-Wallis, p values ≤ 0.05). The experiment was performed twice with similar results. NA = not applicable. (PDF 335 kb)
3D7 accumulates more melanin than 3D1. Means and standard errors of gray values (0 = black, 255 =... more 3D7 accumulates more melanin than 3D1. Means and standard errors of gray values (0 = black, 255 = white) of at least 60 colonies of Z. tritici strains 3D1 and 3D7 at 7–12 days post inoculation (dpi). The experiment was performed three times with similar results. Asterisks indicate significant differences between 3D1 and 3D7 at each time point according to Kruskal-Wallis test (p values ≤ 0.05). n = number of colonies analyzed. (PDF 110 kb)
Generation of mutants (A) Schematic diagram showing the location of primers used for generating Z... more Generation of mutants (A) Schematic diagram showing the location of primers used for generating Zmr1 disruptant mutants (U2 + U3, D1 + D2) and the primers used for screening the transformants (U1 + Hyg UF, Hyg DF + D3). (B) Schematic diagram showing the location of primers used for generating the transposable element (TE) deletion mutants in 3D1 background (ΔTE; TE_U2 + TE_U3, TE_D1 + TE_D2) and the primers used for screening the transformants (TE_U1 + Hyg UF, Hyg DF + TE_D3). (C) Diagrams showing the location of primers used for generating the transformants expressing Zmr1 gene in locus in the 3D7Δzmr1 background. Amplification of Zmr1 gene from genomic DNA of 3D1 and 3D7 strains was performed using Zmr1 F and Zmr1 R primers; the geneticin resistance cassette (Gen) was amplified from pCGEN with Gen F and Gen R. Both amplicons were fused to the pES1 backbone to generate an intermediate construct, which was used to amplify Zmr1 and the geneticin resistance cassette using Zmr1 in locu...
No significant difference in the distribution of mean gray values of different Z. tritici strains... more No significant difference in the distribution of mean gray values of different Z. tritici strains belonging to four different populations. Distribution of mean gray values of different Z. tritici strains from four different populations across the world (3 strains from Australia, 8 from Switzerland, 9 from Israel, and 13 from the USA). The colors of the violin plot indicate different populations. Black dots represent individual data points, which correspond to the mean gray values of each Z. tritici strain at 7 days post inoculation. At least 100 colonies grown on five different plates were evaluated. The experiment was performed three times with similar results. No statistically significant differences were observed between the populations (Kruskal-Wallis test, p value ≤ 0.05). (TIF 172 kb)
Table S2. Short read coverage of Magnaporthe oryzae and wheat transcriptomes in Bangladeshi sampl... more Table S2. Short read coverage of Magnaporthe oryzae and wheat transcriptomes in Bangladeshi samples. (PDF 66 kb)
Number of subpopulations. The peaks represent possible numbers of differentiated populations amon... more Number of subpopulations. The peaks represent possible numbers of differentiated populations among the 213 isolates, with a highest likelihood of three. The bar plots refer to the membership coefficient for each isolate organized according to 13 field populations under the assumption of three subpopulations. An isolate was assigned to a subpopulation if its membership coefficient was ≥0.8. (PDF 382 kb)
Reactive oxygen species are toxic byproducts of aerobic respiration that are also important in me... more Reactive oxygen species are toxic byproducts of aerobic respiration that are also important in mediating a diversity of cellular functions. Reactive oxygen species form an important component of plant defenses to inhibit microbial pathogens during pathogen-plant interactions. Tolerance to oxidative stress is likely to make a significant contribution to the viability and pathogenicity of plant pathogens, but the complex network of oxidative stress responses hinders identification of the genes contributing to this trait. Here, we employed a forward genetic approach to investigate the genetic architecture of oxidative stress tolerance in the fungal wheat pathogen Zymoseptoria tritici. We used quantitative trait locus (QTL) mapping of growth and melanization under axenic conditions in two cross-populations to identify genomic regions associated with tolerance to oxidative stress. We found that QTLs associated with growth under oxidative stress as well as inherent growth can affect oxida...
ABSTRACTReactive oxygen species are toxic byproducts of aerobic respiration produced during cell ... more ABSTRACTReactive oxygen species are toxic byproducts of aerobic respiration produced during cell growth. They also are an important component of plant defenses to inhibit microbial pathogens. Tolerance to oxidative stress contributes to viability and pathogenicity of plant pathogens. However, the complex molecular network of oxidative stress responses hinders identification of the genes contributing to variation in this trait. Variation in genes affecting responses to oxidative stress is likely to affect the evolutionary potential of pathogen tolerance to host defences. Here, we employed a forward genetic approach to investigate the genetic architecture of oxidative stress tolerance in the fungal wheat pathogen Zymoseptoria tritici. By performing quantitative trait locus (QTL) mapping in two crosses, we identified several genomic regions associated with tolerance to oxidative stress, including a QTL having a large effect on growth under oxidative stress. We found evidence for a sign...
SummaryTolerance and resistance represent two strategies that hosts evolved to protect themselves... more SummaryTolerance and resistance represent two strategies that hosts evolved to protect themselves from pathogens. Tolerance alleviates the reduction in host fitness due to infection without reducing a pathogen’s growth, while resistance reduces pathogen growth. We investigated tolerance of wheat to the major fungal pathogen Zymoseptoria tritici in 335 elite wheat cultivars.We used a novel digital phenotyping approach that included 11,152 infected leaves and counted 2,069,048 pathogen fruiting bodies.We discovered a new component of tolerance that is based on the relationship between the green area remaining on a leaf and the number of pathogen fruiting bodies. We found a negative correlation between tolerance and resistance among intolerant cultivars, presenting the first compelling evidence for a tradeoff between tolerance and resistance to plant pathogens. Surprisingly, the tradeoff arises due to limits in the host resources available to the pathogen and not due to metabolic const...
The wheat blast disease has been a serious constraint for wheat production in Latin America since... more The wheat blast disease has been a serious constraint for wheat production in Latin America since the late 1980s. We used a population genomics analysis including 95 genome sequences of the wheat blast pathogen Pyricularia graminis-tritici (Pygt) and other Pyricularia species to show that Pygt is a distinct, highly diverse pathogen species with a broad host range. We assayed 11 neutral SSR loci in 526 Pygt isolates sampled from wheat and other grasses distributed across the wheat-growing region of Brazil to estimate gene flow, assess the importance of sexual reproduction, and compare the genetic structures of Pygt populations infecting wheat and nearby grasses. Our results suggest a mixed reproductive system that includes sexual recombination as well as high levels of gene flow among regions, including evidence for higher gene flow from grass-infecting populations and into wheat-infecting populations than vice versa. The most common virulence groups were shared between the grass- an...
Meiosis is one of the most conserved molecular processes in eukaryotes. The fidelity of pairing a... more Meiosis is one of the most conserved molecular processes in eukaryotes. The fidelity of pairing and segregation of homologous chromosomes has a major impact on the proper transmission of genetic information. Aberrant chromosomal transmission can have major phenotypic consequences, yet the mechanisms are poorly understood. Fungi are excellent models to investigate processes of chromosomal transmission, because many species have highly polymorphic genomes that include accessory chromosomes. Inheritance of accessory chromosomes is often unstable and chromosomal losses have little impact on fitness. We analyzed chromosomal inheritance in 477 progeny coming from two crosses of the fungal wheat pathogen Zymoseptoria tritici. For this, we developed a high-throughput screening method based on restriction site-associated DNA sequencing that generated dense coverage of genetic markers along each chromosome. We identified rare instances of chromosomal duplications (disomy) in core chromosomes....
Philosophical transactions of the Royal Society of London. Series B, Biological sciences, Dec 5, 2016
Agricultural ecosystems are composed of genetically depauperate populations of crop plants grown ... more Agricultural ecosystems are composed of genetically depauperate populations of crop plants grown at a high density and over large spatial scales, with the regional composition of crop species changing little from year to year. These environments are highly conducive for the emergence and dissemination of pathogens. The uniform host populations facilitate the specialization of pathogens to particular crop cultivars and allow the build-up of large population sizes. Population genetic and genomic studies have shed light on the evolutionary mechanisms underlying speciation processes, adaptive evolution and long-distance dispersal of highly damaging pathogens in agro-ecosystems. These studies document the speed with which pathogens evolve to overcome crop resistance genes and pesticides. They also show that crop pathogens can be disseminated very quickly across and among continents through human activities. In this review, we discuss how the peculiar architecture of agro-ecosystems facil...
We conducted a comprehensive analysis of virulence in the fungal wheat pathogen Zymoseptoria trit... more We conducted a comprehensive analysis of virulence in the fungal wheat pathogen Zymoseptoria tritici using QTL mapping. High throughput phenotyping based on automated image analysis allowed measurement of pathogen virulence on a scale and with a precision that was not previously possible. Across two mapping populations encompassing more than 520 progeny, 540,710 pycnidia were counted and their sizes and grey values were measured, yielding over 1.6 million phenotypes associated with pathogen reproduction. Large pycnidia were shown to produce more numerous and larger spores than small pycnidia. Precise measures of percent leaf area covered by lesions provided a quantitative measure of host damage. Combining these large and accurate phenotype datasets with a dense panel of RADseq genetic markers enabled us to genetically dissect pathogen virulence into components related to host damage and components related to pathogen reproduction. We show that different components of virulence can b...
CYP51 encodes the target site of the azole class of fungicides widely used in plant protection. S... more CYP51 encodes the target site of the azole class of fungicides widely used in plant protection. Some ascomycete pathogens carry two CYP51 paralogs called CYP51A and CYP51B. A recent analysis of CYP51 sequences in 14 European isolates of the barley scald pathogen Rhynchosporium commune revealed three CYP51 paralogs, CYP51A, CYP51B, and a pseudogene called CYP51A-p. The same analysis showed that CYP51A exhibits a presence/absence polymorphism, with lower sensitivity to azole fungicides associated with the presence of a functional CYP51A. We analyzed a global collection of nearly 400 R. commune isolates to determine if these findings could be extended beyond Europe. Our results strongly support the hypothesis that CYP51A played a key role in the emergence of azole resistance globally and provide new evidence that the CYP51A gene in R. commune has further evolved, presumably in response to azole exposure. We also present evidence for recent long-distance movement of evolved CYP51A allel...
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