Thermal adaptation in the fungal pathogen Mycosphaerella graminicola

Genetic differentiation in thermal adaptation can result from a trade-off between the performance of organisms across different temperatures or from the accumulation of deleterious mutations. In this experiment, we assayed thermal sensitivity of 138 genetically distinct Mycosphaerella graminicola isolates sampled from five host populations in four locations under two temperature regimes (22 and 15 °C) and found significant differences in growth rate and response to temperature among populations. On average, genetic differentiation accounted for more than 50% of phenotypic variation in thermal adaptation while plasticity contributed less than a quarter of phenotypic variation. Populations originating from warm places performed better under the high-temperature regime and had a larger positive response to increasing temperature. Pairwise population differentiation (Q(ST) ) in temperature sensitivity, measured by taking the ratio of growth rates at 22 to 15 °C, was positively and significantly correlated to the pairwise difference in annual mean temperature at the collection sites. Because overall Q(ST) in temperature sensitivity was significantly higher than overall G(ST) in neutral restriction fragment length polymorphism loci, we believe that the primary mechanism underlying this thermal adaptation is antagonistic pleiotropy. Our results indicate that temperature sensitivity is a better indicator of thermal adaptation than growth rate at individual temperatures.     

Mycosphaerella graminicola: from genomics to disease control

This Mycosphaerella graminicola pathogen profile covers recent advances in the knowledge of this ascomycete fungus and of the disease it causes, septoria tritici blotch of wheat. Research on this pathogen has accelerated since publication of a previous pathogen profile in this journal in 2002. Septoria tritici blotch continues to have high economic importance and widespread global impact on wheat production. Taxonomy: Mycosphaerella graminicola (Fuckel) J. Schröt. In Cohn (anamorph: Septoria tritici Roberge in Desmaz.). Kingdom Fungi, Phylum Ascomycota, Class Loculoascomycetes (filamentous ascomycetes), Order Dothideales, Genus Mycosphaerella, Species graminicola. Host range: Bread and durum wheat (Triticum aestivum L. and T. turgidum ssp. durum L.). Disease symptoms: Initially leaves develop a chlorotic flecking, which is followed by the development of necrotic lesions which contain brown–black pycnidia. Necrosis causes a reduction in photosynthetic capacity and therefore affects grain yield. Disease control: The disease is primarily controlled by a combination of resistant cultivars and fungicides. Rapid advances in disease control, especially in resistance breeding, are opening up new opportunities for the management of the disease. Useful websites: http://genome.jgi‐psf.org/Mycgr3/Mycgr3.home.html .     

Detection of strobilurin-resistant isolates of Mycosphaerella graminicola in Morocco

Septoria tritici blotch caused by Mycosphaerella graminicola (anamorph: Septoria tritici) is nowadays one of the most frequently occurring diseases on both bread and durum wheat crops. Two hundred and thirty isolates of the fungus were sampled from six distinct wheat-producing regions of Morocco in order to investigate the resistance of M. graminicola to strobilurins in this country, where this fungicide class is increasingly used in wheat-pest management. A subset of 134 isolates was first collected in 2008 from Meknes-Tafilalet, Tadla-Azilal, Gharb and Chaouia. Furthermore, 96 additional isolates were sampled in 2010 from the fourth regions investigated in 2008 plus Fes-Boulmane and Doukkala-Abda. Sensitivity or resistance within the isolates were determined by screening the G143A cytochrome b substitution conferring resistance. We used a mismatch amplification mutation assay allowing the amplification of either G143 (sensitive) or A143 (resistant) allele. All the 2008 isolates were found to be sensitive since they carry the wild-type allele G143. However, 9 (9%) out of the 2010 isolates were found to contain the resistant allele A143 and therefore to be resistant. Four of them were from Gharb and five from Fes-Boulmane. This study highlighted for the first time the occurrence of strobilurin-resistant isolates of M. graminicola in Morocco. Further genetic investigations should determine if the resistant isolates emerged independently in Morocco or traveled by wind-migration from Europe.     

Distribution of airborne Mycosphaerella graminicola inoculum at the field scale

A network of 10 Burkard 7-day spore-recording traps was set up in the Walloon region in Belgium to monitor the airborne inoculum of wheat pathogens. Three spore traps were used to analyse the distribution of Mycosphaerella graminicola inoculum at the field scale, at 1 m above ground level. Two traps were set up in a wheat field 100 m apart. The third trap was placed 70 m away in a sugar beet field adjacent to the wheat field. Total DNA from each fragment of spore trap tape corresponding to 1 day sampling was extracted and the quantity of M. graminicola was assessed using real-time polymerase chain reaction (PCR) assay. The experiment was conducted from July to October 2009. Positive detections were obtained for between 33 and 36 days, depending on the spore traps. When detected, the daily quantities of cDNA, collected from a volume of 14.4 m3, fluctuated between 4.84E+00 and 6.10E+03. Correlation coefficients higher than 0,82 and no significant differences were observed between the quantities of M. graminicola collected by the three spore traps, indicating that, at 1 m above ground level, the distribution of inoculum can be considered as homogenous at the tested field scale. This study confirms that spore traps coupled with real-time PCR could be used to assess the airborne inoculum of M. graminicola and to understand the development of the disease at this scale.     

Molecular Variability of Mycosphaerella graminicola as Detected by RAPD Markers

A total of 90 isolates of Mycosphaerella graminicola, the cause of septoria tritici leaf blotch of wheat, were tested for DNA polymorphism using 15 decamer random primers. There was a high level of genetic variability among isolates. In 131 random amplified polymorphic DNA (RAPD) fragments, which were produced, 96% were polymorphic. Based on multilocus analysis, 40 different molecular phenotypes were detected. These molecular phenotypes were randomly distributed among sampling sites, suggesting that no clonal structure existed in the population. Cluster analysis showed that the maximum similarity value among isolates was approximately 81% and no identical isolates were detected, indicating that every isolate was a unique genotype. The high degree of DNA polymorphism, the large number of different molecular phenotypes, their random distribution and the results of the cluster analysis all suggested that sexual reproduction has a major role in the genetic structure of M. graminicola in western Canada. The presence of sexual reproduction provides the opportunity for development of new virulent genotypes in the population and suggests that the pathogen may adapt rapidly to any race‐specific sources of resistance. Therefore, when breeding for resistance to M. graminicola, emphasis should be placed on use of non‐race‐specific resistance.     

Microsatellite markers provide evidence for sexual reproduction of Mycosphaerella graminicola in Saskatchewan.

This study examined the genetic structure of a Saskatchewan population of Mycosphaerella graminicola, cause of the foliar disease Septoria tritici blotch of wheat. Such knowledge is valuable for understanding the evolutionary potential of this pathogen and for developing control strategies based on host resistance. Nine pairs of single-locus microsatellite primers were used to analyze the genomic DNA of 90 isolates of M. graminicola that were collected using a hierarchical sampling procedure from different locations, leaves, and lesions within a wheat field near Saskatoon. Allelic series at eight different loci were detected. The number of alleles per locus ranged from one to five with an average of three alleles per locus. Genetic diversity values ranged from 0.04 to 0.67. Partitioning the total genetic variability into within- and among-location components revealed that 88% of the genetic variability occurred within locations, i.e., within areas of 1 m(2), but relatively little variability occurred among locations. Low variability among locations and a high degree of variability within locations would result if the primary source of inoculum was airborne ascospores, which would be dispersed uniformly within the field. This finding was confirmed by gametic disequilibrium analysis and suggests that the sexual reproduction of M. graminicola occurs in Saskatchewan.     

Genetic diversity and population structure in French populations of Mycosphaerella graminicola

Mycosphaerella graminicola populations were examined in France with microsatellite markers and PCR-SSCP analysis of partial actin and β-tubulin encoding sequences. A total of 363 isolates was sampled in 2005 from 17 provinces, and genotypes from corresponding strains were characterized. Unique haplotypes comprised 84% of the population, and gene diversity was high nationwide (0.70) and locally. A moderate genetic differentiation (G(ST) = 0.18) was found and indicated that in France the M. graminicola population was more structured than in other previously studied European countries. Bayesian structure analysis identified three genetic clusters distributed among the 17 provinces. Our results highlighted the potential for the adaptation of the fungus to local conditions, leading to genetic clusters among the French population of the fungus as well as genotype flow between regional clusters.     

The interaction among evolutionary forces in the pathogenic fungus Mycosphaerella graminicola

The population genetic dynamic of a species is driven by interactions among mutation, migration, drift, mating system, and selection, but it is rare to have sufficient empirical data to estimate values for all of these forces and to allow comparison of the relative magnitudes of these evolutionary forces. We combined data from a mark-release-recapture experiment, extensive population surveys, and computer simulations to evaluate interactions among these evolutionary forces in the pathogenic fungus Mycosphaerella graminicola. The results from these studies showed that, on average, the immigration rate was 0.027, the fraction of outcrossing individuals was 0.035, and the selection coefficient associated with immigrants was 0.106 each generation. We also estimated that effective population sizes for this fungus were larger than 24,000 and the mutation rate for the RFLP markers used in surveys and field experiments was approximately 4 x 10(-5). Computer simulations based on these estimates indicate that, on average, the global population of M. graminicola has reached equilibrium. Population genetic parameters including number of alleles, gene diversity, and population subdivision estimated from the computer simulations were surprisingly close to empirical estimates. Simulations also revealed that random drift is the major evolutionary force decreasing genetic variation in this fungus, followed by natural selection. The major force adding to genetic variation was mutation, followed by gene flow and sexual recombination. Gene flow played the leading role in decreasing population subdivision while natural selection was the major factor increasing population subdivision.     

Migration patterns among global populations of the pathogenic fungus Mycosphaerella graminicola

DNA sequences from five nuclear loci and data from three microsatellites were collected from 360 isolates representing 14 globally distributed populations of the plant pathogenic fungus Mycosphaerella graminicola. Haplotype networks were constructed for the five sequence loci and population subdivision was assessed using Hudson's permutation test. Migration estimates were calculated using six regional populations for both the sequence and microsatellite loci. While subdivision was detected among the six regional populations, significant gene flow was indicated among some of the populations. The European and Israeli populations contributed the majority of historical immigrants to the New World. Migration estimates for microsatellite loci were used to infer more recent migration events among specific New World populations. We conclude that gene flow was an important factor in determining the demographic history of Mycosphaerella graminicola.     

Molecular modelling of the emergence of azole resistance in Mycosphaerella graminicola

A structural rationale for recent emergence of azole (imidazole and triazole) resistance associated with CYP51 mutations in the wheat pathogen Mycosphaerella graminicola is presented, attained by homology modelling of the wild type protein and 13 variant proteins. The novel molecular models of M. graminicola CYP51 are based on multiple homologues, individually identified for each variant, rather than using a single structural scaffold, providing a robust structure-function rationale for the binding of azoles, including important fungal specific regions for which no structural information is available. The wild type binding pocket reveals specific residues in close proximity to the bound azole molecules that are subject to alteration in the variants. This implicates azole ligands as important agents exerting selection on specific regions bordering the pocket, that become the focus of genetic mutation events, leading to reduced sensitivity to that group of related compounds. Collectively, the models account for several observed functional effects of specific alterations, including loss of triadimenol sensitivity in the Y137F variant, lower sensitivity to tebuconazole of I381V variants and increased resistance to prochloraz of V136A variants. Deletion of Y459 and G460, which brings about removal of that entire section of beta turn from the vicinity of the binding pocket, confers resistance to tebuconazole and epoxiconazole, but sensitivity to prochloraz in variants carrying a combination of A379G I381V ΔY459/G460. Measurements of binding pocket volume proved useful in assessment of scope for general resistance to azoles by virtue of their accommodation without bonding interaction, particularly when combined with analysis of change in positions of key amino acids. It is possible to predict the likely binding orientation of an azole molecule in any of the variant CYPs, providing potential for an in silico screening system and reliable predictive approach to assess the probability of particular variants exhibiting resistance to particular azole fungicides.     

Activity of Azole Fungicides and ABC Transporter Modulators on Mycosphaerella graminicola

The antimicrobial activity of the azole fungicides cyproconazole and propiconazole as single active ingredients and in mixtures with the ATP-Binding Cassette (ABC) transporter modulators rhodamine 6G, quercetin, quinidine, and verapamil and the strobilurin kresoxim-methyl was assessed against the wheat pathogen Mycosphaerella graminicola . Interactions amongst these compounds were evaluated on germination and germ tube growth of pycnidiospores using the Colby and Wadley method. Water agar proved to be the best test medium since all pycnidiospores germinated within 24 h of incubation and apical germ tube growth dominated over bud formation by intermediate cells. Analysis with the Colby method revealed that interactions between the compounds in all mixtures tested on germination of pycnidiospores were additive. With regard to germ tube growth, mixtures of cyproconazole and verapamil or kresoxim-methyl displayed a synergistic interaction. Analysis of mixtures of cyproconazole and kresoxim-methyl with the Wadley method revealed that the interaction between the two compounds was purely additive. These results indicate that the Colby method overestimated the interaction between these two compounds in a mixture.     

Single strand conformation polymorphism is a sensitive method for screening nucleotide variations in Mycosphaerella graminicola

Single Strand Conformation Polymorphism (SSCP) and sequencing were performed in order to assess molecular polymorphism of mating type sequences in the heterothallic ascomycete Mycosphaerella graminicola, the causal agent of Septoria tritici blotch of wheat. The screening was undertaken on mat1-1 and mat1-2 partial sequences of 341 and 657 bp, respectively, amplified with multiplex PCR from 510 French single-conidial strains plus the two reference isolates IPO323 and IPO94269 from The Netherlands. After restriction with Taq1 in order to reduce the fragment sizes, all digested amplicons were subjected to SSCP. Sequencing was then performed when a SSCP pattern deviates from the most frequently occurring profile. Among the assessed strains, 228 ones plus IPO323 were MAT1-1 and 282 ones plus IPO94269 were MAT1-2. Among the MAT1-1 strains, only a single one exhibited a SSCP profile distinct to the other MAT1-1 strains, whereas 10 MAT1-2 strains (among which 2 and 4 with same profiles, respectively) showed a SSCP profile differing to the other MAT1-2 strains. Sequencing revealed that all polymorphisms observed on SSCP gels were single nucleotide variations and all strains displaying the same SSCP profiles showed identical nucleotide sequences. Among the seven disclosed nucleotide variations, only two were non-synonymous and both were non-conservative. This study reports a high sensitivity of SSCP allowing detection of single point mutations in M. graminicola, shows a conservation of mating type idiomorphs in the fungus at both sequence and population scales, but also suggests a difference in polymorphism level between the two mating type sequences.     

Origin and domestication of the fungal wheat pathogen Mycosphaerella graminicola via sympatric speciation

The Fertile Crescent represents the center of origin and earliest known place of domestication for many cereal crops. During the transition from wild grasses to domesticated cereals, many host-specialized pathogen species are thought to have emerged. A sister population of the wheat-adapted pathogen Mycosphaerella graminicola was identified on wild grasses collected in northwest Iran. Isolates of this wild grass pathogen from 5 locations in Iran were compared with 123 M. graminicola isolates from the Middle East, Europe, and North America. DNA sequencing revealed a close phylogenetic relationship between the pathogen populations. To reconstruct the evolutionary history of M. graminicola, we sequenced 6 nuclear loci encompassing 464 polymorphic sites. Coalescence analyses indicated a relatively recent origin of M. graminicola, coinciding with the known domestication of wheat in the Fertile Crescent around 8,000-9,000 BC. The sympatric divergence of populations was accompanied by strong genetic differentiation. At the present time, no genetic exchange occurs between pathogen populations on wheat and wild grasses although we found evidence that gene flow may have occurred since genetic differentiation of the populations.     

Both mating types of the wheat pathogen Mycosphaerella graminicola are present in Morocco

Septoria tritici blotch caused by the heterothallic ascomycete Mycosphaerella graminicola is one of the most currently damaging diseases on wheat crops worldwide. So far, no information was reported about the status of sexual reproduction of this pathogen under Moroccan conditions. We investigated here for the first time the occurrence of the two mating types (MAT1-1 and MAT1-2) of M. graminicola in Morocco by sampling 141 single-conidial isolates from 4 important wheat producing regions (Gharb, Sa?s, Chaouia and Tadla). The mating type of each isolate was determined by amplification with multiplex PCR of a partial sequence from the corresponding idiomorph. Overall, 43% out of the assessed isolates were MAT1-1 and 57 % were MAT1-2. Both mating types were identified within the 3 sampled regions Gharb, Sa?s and Chaouia, but not in Tadla, where only MAT1-2 isolates were found. The presence of the two mating types highlighted here offers a suitable genetic condition for M. graminicola to occur sexual reproduction in Morocco. The potential of sexual recombination will be examined by the study of mating type frequencies using a large sample size as well as by searching and quantification of pseudothecia in the field.     

MgHog1 regulates dimorphism and pathogenicity in the fungal wheat pathogen Mycosphaerella graminicola

The dimorphic ascomycete pathogen Mycosphaerella graminicola switches from a yeastlike form to an infectious filamentous form that penetrates the host foliage through stomata. We examined the biological function of the mitogen-activated protein kinase-encoding gene MgHog1 in M. graminicola. Interestingly, MgHog1 mutants were unable to switch to filamentous growth on water agar that mimics the nutritionally poor conditions on the foliar surface and, hence, exclusively developed by a yeastlike budding process. Consequently, due to impaired initiation of infectious germ tubes, as revealed by detailed in planta cytological analyses, the MgHog1 mutants failed to infect wheat leaves. We, therefore, conclude that MgHog1 is a new pathogenicity factor involved in the regulation of dimorphism in M. graminicola. Furthermore, MgHog1 mutants are osmosensitive, resistant to phenylpyrrole and dicarboximide fungicides, and do not melanize.     

Variation for neutral markers is correlated with variation for quantitative traits in the plant pathogenic fungus Mycosphaerella graminicola

We compared genetic variation and population differentiation at RFLP marker loci with seven quantitative characters including fungicide resistance, temperature sensitivity, pycnidial size, pycnidial density, colony size, percentage of leaves covered by pycnidia (PLACP) and percentage of leaves covered by lesions (PLACL) in Mycosphaerella graminicola populations sampled from four regions. Wide variation in population differentiation was found across the quantitative traits assayed. Fungicide resistance, temperature sensitivity, and PLACP displayed a significantly higher Q(ST) than G(ST), consistent with selection for local adaptation, while pycnidial size, pycnidial density and colony size displayed a lower or significantly lower Q(ST) than G(ST), consistent with constraining selection. There was not a statistical difference between Q(ST) and G(ST) in PLACL. We also found a positive and significant correlation between genetic variation in molecular marker loci and quantitative traits at the multitrait scale, suggesting that estimates of overall genetic variation for quantitative traits in M. graminicola could be derived from analysis of the molecular genetic markers.     

Mutagenesis and functional studies with succinate dehydrogenase inhibitors in the wheat pathogen Mycosphaerella graminicola

A range of novel carboxamide fungicides, inhibitors of the succinate dehydrogenase enzyme (SDH, EC 1.3.5.1) is currently being introduced to the crop protection market. The aim of this study was to explore the impact of structurally distinct carboxamides on target site resistance development and to assess possible impact on fitness. We used a UV mutagenesis approach in Mycosphaerella graminicola, a key pathogen of wheat to compare the nature, frequencies and impact of target mutations towards five subclasses of carboxamides. From this screen we identified 27 amino acid substitutions occurring at 18 different positions on the 3 subunits constituting the ubiquinone binding (Qp) site of the enzyme. The nature of substitutions and cross resistance profiles indicated significant differences in the binding interaction to the enzyme across the different inhibitors. Pharmacophore elucidation followed by docking studies in a tridimensional SDH model allowed us to propose rational hypotheses explaining some of the differential behaviors for the first time. Interestingly all the characterized substitutions had a negative impact on enzyme efficiency, however very low levels of enzyme activity appeared to be sufficient for cell survival. In order to explore the impact of mutations on pathogen fitness in vivo and in planta, homologous recombinants were generated for a selection of mutation types. In vivo, in contrast to previous studies performed in yeast and other organisms, SDH mutations did not result in a major increase of reactive oxygen species levels and did not display any significant fitness penalty. However, a number of Qp site mutations affecting enzyme efficiency were shown to have a biological impact in planta.Using the combined approaches described here, we have significantly improved our understanding of possible resistance mechanisms to carboxamides and performed preliminary fitness penalty assessment in an economically important plant pathogen years ahead of possible resistance development in the field.