Jiasui Zhan

The global genetic structure of the wheat pathogen Mycosphaerella graminicola is characterized by high nuclear diversity, low mitochondrial diversity, regular recombination, and gene flow

A total of 1673 Mycosphaerella graminicola strains were assayed for DNA fingerprints and restriction fragment length polymorphism (RFLP) markers in the nuclear and mitochondrial genomes. The isolates were collected from 17 wheat fields located in 11 countries on five continents over a six year period (1989-1995). Our results indicate that genetic diversity in the nuclear genome of this fungus was high for all but three of the field populations surveyed and that populations sampled from different continents had similar frequencies for the most common RFLP alleles. Hierarchical analysis revealed that more than 90% of global gene diversity was distributed within a wheat field, while approximately 5% of gene diversity was distributed among fields within regions and approximately 3% was distributed among regions on different continents. These findings suggest that gene flow has occurred on a global scale. On average, each leaf was colonized by a different nuclear genotype. In contrast, only seven mtDNA haplotypes were detected among the 1673 isolates and the two most common mtDNA haplotypes represented approximately 93% of the world population, consistent with a selective sweep. Analysis of multilocus associations indicated that all field populations were in gametic equilibrium, suggesting that sexual recombination is a regular occurrence globally.

Population Structure of Mycosphaerella graminicola: From Lesions to Continents

ABSTRACT The genetic structure of field populations of Mycosphaerella graminicola was determined across a hierarchy of spatial scales using restriction fragment length polymorphism markers. The hierarchical gene diversity analysis included 1,098 isolates from seven field populations. Spatial scales ranged from millimeters to thousands of kilometers, including comparisons within and among lesions, within and among fields, and within and among regions and continents. At the smallest spatial scale, microtransect sampling was used to determine the spatial distribution of 15 genotypes found among 158 isolates sampled from five individual lesions. Each lesion had two to six different genotypes including both mating types in four of the five lesions, but in most cases a lesion was composed of one or two genotypes that occupied the majority of the lesion, with other rare genotypes interspersed among the common genotypes. The majority (77%) of gene diversity was distributed within plots ranging from approximately 1 to 9 m(2) in size. Genotype diversity (G / N) within fields for the Swiss, Texas, and Israeli fields was high, ranging from 79 to 100% of maximum possible values. Low population differentiation was indicated by the low G(ST) values among populations, suggesting a corresponding high degree of gene flow among these populations. At the largest spatial scale, populations from Switzerland, Israel, Oregon, and Texas were compared. Population differentiation among these populations was low (G(ST) = 0.05), and genetic identity between populations was high. A low but significant correlation between genetic and geographic distance among populations was found (r = -0.47, P = 0.012), suggesting that these populations probably have not reached an equilibrium between gene flow and genetic drift. Gene flow on a regional level can be reduced by implementing strategies, such as improved stubble management that minimize the production of ascospores. The possibility of high levels of gene flow on a regional level indicates a significant potential risk for the regional spread of mutant alleles that enable fungicide resistance or the breakdown of resistance genes.

Genetic Structure of Rhynchosporium secalis in Australia

ABSTRACT Restriction fragment length polymorphism (RFLP) markers were used to determine the genetic structure of Australian field populations of the barley scald pathogen Rhynchosporium secalis. Fungal isolates were collected by hierarchical sampling from five naturally infected barley fields in different geographic locations during a single growing season. Genetic variation was high in Australian R. secalis populations. Among the 265 fungal isolates analyzed, 214 distinct genotypes were identified. Average genotype diversity within a field population was 65% of its theoretical maximum. Neis average gene diversity across seven RFLP loci was 0.54. The majority (76%) of gene diversity was distributed within sampling site areas measuring 1 m(2); 19% of gene diversity was distributed among sampling sites within fields; and 5% of gene diversity was distributed among fields. Fungal populations from different locations differed significantly both in allele frequencies and genotype diversities. The degree of genetic differentiation was significantly correlated with geographic distance between populations. Our results suggest that the R. secalis population in Western Australia has a different genetic structure than populations in Victoria and South Australia.

Local adaptation and effect of host genotype on the rate of pathogen evolution: an experimental test in a plant pathosystem

Abstract Virulence is thought to be a driving force in host–pathogen coevolution. Theoretical models suggest that virulence is an unavoidable consequence of pathogens evolving towards a high rate of intrahost reproduction. These models predict a positive correlation between the reproductive fitness of a pathogen and its level of virulence. Theoretical models also suggest that the demography and genetic structure of a host population can influence the evolution of virulence. If evolution occurs faster in pathogen populations than in host populations, the predicted result is local adaptation of the pathogen population. In our studies, we used a combination of molecular and physiological markers to test these hypotheses in an agricultural system. We isolated five strains of the fungal pathogen Mycosphaerella graminicola from each of two wheat cultivars that differed in their level of resistance to this pathogen. Each of the 10 fungal strains had distinct genotypes as indicated by different DNA fingerprints. These fungal strains were re‐inoculated onto the same two host cultivars in a field experiment and their genotype frequencies were monitored over several generations of asexual reproduction. We also measured the virulence of these 10 fungal strains and correlated it to the reproductive fitness of each fungal strain. We found that host genotypes had a strong impact on the dynamics of the pathogen populations. The pathogen population collected from the moderately resistant cultivar Madsen showed greater stability, higher genotype diversity, and smaller selection coefficients than the pathogen populations collected from the susceptible cultivar Stephens or a mixture of the two host cultivars. The pathogen collection from the mixed host population was midway between the two pure lines for most parameters measured. Our results also revealed that the measures of reproductive fitness and virulence of a pathogen strain were not always correlated. The pathogen strains varied in their patterns of local adaptation, ranging from locally adapted to locally maladapted.

Distribution of mating type alleles in the wheat pathogen Mycosphaerella graminicola over spatial scales from lesions to continents

A total of 2035 Mycosphaerella graminicola strains collected from 16 geographic locations on four continents were assayed for the mating type locus. RFLP fingerprints were used to identify clones in each population. At the smallest spatial scale analyzed, both mating types were found among fungal strains sampled from different lesions of the same leaf as well as from different pycnidia in the same lesion. At larger spatial scales, the two mating types were found at equal frequencies across spatial scales ranging from several square meters to several thousand square kilometers. Though the absolute frequencies of the two mating types sometimes varied for different sampling units within the same spatial scale in the hierarchy (plots within a field, fields within a country, or different continents of the world), none of the differences were statistically significant from the null hypothesis of equal frequencies for the two mating types. The evolutionary forces likely to maintain the even distribution of the two mating types in this pathogen were discussed.

Measuring Immigration and Sexual Reproduction in Field Populations of Mycosphaerella graminicola

ABSTRACT A field experiment was conducted to determine the relative contributions of immigration and sexual reproduction to the genetic structure of Mycosphaerella graminicola populations during the course of an epidemic. The genetic structure of M. graminicola populations sampled from wheat plots inoculated artificially with 10 isolates was compared with control plots infected naturally by airborne ascospores. Restriction fragment length polymorphisms (RFLPs) were used to test the randomness of associations among loci, and DNA fingerprints were used to identify clones. All isolates in the control plots had unique genotypes and RFLP loci were at gametic equilibrium, findings consistent with random mating. The proportion of isolates in the inoculated plots with DNA fingerprints that differed from the 10 inoculated isolates increased from 3% in the early to 39 and 34% in the mid- and late season, respectively. The degree of gametic disequilibrium was higher in the mid-season than in the late-season population. By the end of the growing season, we estimate that 66% of the isolates in the inoculated plots were asexual progeny of the 10 inoculated isolates, 10% were immigrants, and 24% were sexual recombinants. The proportion of infections caused by ascospores increased over the growing season.

The Genetic Structure of Field Populations of Rhynchosporium secalis from Three Continents Suggests Moderate Gene Flow and Regular Recombination

ABSTRACT Restriction fragment length polymorphism (RFLP) markers were used to compare the genetic structure of field populations of Rhynchosporium secalis from barley. A total of 543 isolates representing 8 field populations were sampled from Australia, California, Finland, and Norway. Gene and genotype diversity were high in all populations. Neis average gene diversity across seven RFLP loci was 0.513. Hierarchical gene diversity analysis showed that 9% of the total genetic variability was distributed among continents, 4% was distributed among fields within continents, and 13% was distributed among collection stations within a field. The majority (74%) of genetic variability was distributed within collection areas of approximately 1 m(2) within fields. Gene flow appears to be significant on a regional scale but more restricted among continents. Allele frequencies were significantly different at several RFLP loci. Genetic distances were small among populations within regions and large between regions. Pairwise comparisons of genotype diversity in the populations revealed significant differences among populations that were related mainly to differences in sampling strategies. Isolates from Norway and Finland showed a lower copy hybridization pattern with probe pRS26. This probe functioned as a fingerprint probe for the California and Australian isolates. Seven out of the eight populations studied were at gametic equilibrium for RFLP loci, suggesting that R. secalis populations in Norway, Finland, and Australia undergo regular recombination, although a teleomorph has not yet been recognized.

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 QST than GST, consistent with selection for local adaptation, while pycnidial size, pycnidial density and colony size displayed a lower or significantly lower QST than GST, consistent with constraining selection. There was not a statistical difference between QST and GST 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.

Experimental measures of pathogen competition and relative fitness.

Competition among pathogen strains for limited host resources can have a profound effect on pathogen evolution. A better understanding of the principles and consequences of competition can be useful in designing more sustainable disease management strategies. The competitive ability and relative fitness of a pathogen strain are determined by its intrinsic biological properties, the resistance and heterogeneity of the corresponding host population, the population density and genetic relatedness of the competing strains, and the physical environment. Competitive ability can be inferred indirectly from fitness components, such as basic reproduction rate or transmission rate. However, pathogen strains that exhibit higher fitness components when they infect a host alone may not exhibit a competitive advantage when they co-infect the same host. The most comprehensive measures of competitive ability and relative fitness come from calculating selection coefficients in a mixed infection in a field setting. Mark-release-recapture experiments can be used to estimate fitness costs associated with unnecessary virulence and fungicide resistance.

Selection for increased cyproconazole tolerance in Mycosphaerella graminicola through local adaptation and in response to host resistance

SUMMARY Sterol demethylation inhibitors (DMIs) represent one of the largest groups of systemic fungicides that have been used to control agriculturally important fungal pathogens. Knowledge regarding the evolution of fungicide resistance in agricultural ecosystems is fragmentary and a better understanding of the processes driving the development of DMI resistance in populations of fungal pathogens is needed by plant pathologists and the agrochemical industry. We considered some of these processes using approaches based on molecular population and quantitative genetics. Five Mycosphaerella graminicola populations sampled from unsprayed wheat fields on four continents were assayed for eight restriction fragment length polymorphism (RFLP) markers and their level of tolerance to cyproconazole. DMI fungicides such as cyproconazole inhibit the enzyme eburicol 14-alpha-demethylase. The gene encoding this target, CYP51, was sequenced for all isolates. We found unimodal, continuous variations in cyproconazole tolerance among the M. graminicola isolates sampled from individual fields, consistent with a polygenic mode of inheritance. We also found that population differentiation for cyproconazole tolerance (Q(ST)) among the five M. graminicola populations was significantly higher than the corresponding population differentiation for neutral RFLP markers (G(ST)), suggesting that selection for cyproconazole tolerance in the Swiss population has already led to local adaptation that can be seen even in an unsprayed population. The Swiss population displayed the highest level of tolerance to cyproconazole, in addition to a lower than expected quantitative variation in fungicide tolerance and a skewed distribution, indicating that selection had increased the overall tolerance of this population. Further analysis with DNA sequencing showed that the population from Switzerland was dominated by isolates with several point mutations and a 6-bp deletion in CYP51. This deletion and one of the point mutations were previously related to increased resistance in field isolates. The fungal population from Oregon sampled from an unsprayed resistant host cultivar displayed the same gene diversity in RFLP loci but higher cyproconazole tolerance and quantitative variation in tolerance than the fungal population from the same field sampled from an unsprayed susceptible host cultivar.