Celeste C. Linde

The population genetics of plant pathogens and breeding strategies for durable resistance

The durability of disease resistance is affected by the evolutionary potential of the pathogen population. Pathogens with a high evolutionary potential are more likely to overcome genetic resistance than pathogens with a low evolutionary potential. We will propose a set of guidelines to predict the evolutionary potential of pathogen populations based on analysis of their genetic structure. Under our model of pathogen evolution, the two most important parameters to consider are reproduction/mating system and gene/genotype flow. Pathogens that pose the greatest risk of breaking down resistance genes are those that possess a mixed reproduction system, with at least one sexual cycle per growing season and asexual reproduction during the epidemic phase, and a high potential for gene flow. The lowest risk pathogens are those with strict asexual reproduction and low potential for gene flow. We will present examples of high- and low-risk pathogens. Knowledge of the population genetic structure of the pathogen may offer insight into the best breeding strategy for durable resistance. We will present broad guidelines suggesting a rational method for breeding durable resistance according to the population genetics of the pathogen.

Life history determines genetic structure and evolutionary potential of host–parasite interactions

Measures of population genetic structure and diversity of disease-causing organisms are commonly used to draw inferences regarding their evolutionary history and potential to generate new variation in traits that determine interactions with their hosts. Parasite species exhibit a range of population structures and life-history strategies, including different transmission modes, life-cycle complexity, off-host survival mechanisms and dispersal ability. These are important determinants of the frequency and predictability of interactions with host species. Yet the complex causal relationships between spatial structure, life history and the evolutionary dynamics of parasite populations are not well understood. We demonstrate that a clear picture of the evolutionary potential of parasitic organisms and their demographic and evolutionary histories can only come from understanding the role of life history and spatial structure in influencing population dynamics and epidemiological patterns.

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.

Molecular population genetic analysis differentiates two virulence mechanisms of the fungal avirulence gene NIP1

Deletion or alteration of an avirulence gene are two mechanisms that allow pathogens to escape recognition mediated by the corresponding resistance gene in the host. We studied these two mechanisms for the NIP1 avirulence gene in field populations of the fungal barley pathogen Rhynchosporium secalis. The product of the avirulence gene, NIP1, causes leaf necrosis and elicits a defense response on plants with the Rrs1 resistance gene. A high NIP1 deletion frequency (45%) was found among 614 isolates from different geographic populations on four continents. NIP1 was also sequenced for 196 isolates, to identify DNA polymorphisms and corresponding NIP1 types. Positive diversifying selection was found to act on NIP1. A total of 14 NIP1 types were found, 11 of which had not been described previously. The virulence of the NIP1 types was tested on Rrs1 and rrs1 barley lines. Isolates carrying three of these types were virulent on the Rrs1 cultivar. One type each was found in California, Western Europe, and Jordan. Additionally, a field experiment with one pair of near-isogenic lines was conducted to study the selection pressure imposed by Rrs1 on field populations of R. secalis. Deletion of NIP1 was the only mechanism used to infect the Rrs1 cultivar in the field experiment. In this first comprehensive study on the population genetics of a fungal avirulence gene, virulence to Rrs1 in R. secalis was commonly achieved through deletion of the NIP1 avirulence gene but rarely also through point mutations in NIP1.

Further evidence for sexual reproduction in Rhynchosporium secalis based on distribution and frequency of mating-type alleles

Rhynchosporium secalis, the causal agent of scald on barley, is thought to be exclusively asexual because no teleomorph has been found. Partial sequences of the HMG-box and alpha-domain of Rhynchosporium secalis isolates were identified and used to develop a PCR assay for the mating-type locus. PCR amplification of only one of these two domains was possible in each strain, suggesting that R. secalis has a MAT organization that is similar to other known heterothallic fungi. A multiplex PCR with primers amplifying either a MAT1-1- or MAT1-2-specific amplicon was used to determine the distribution of mating types in several R. secalis populations. In total, 1101 isolates from Australia, Switzerland, Ethiopia, Scandinavia, California, and South Africa were included in the analysis. Mating types occurred in equal frequencies for most of these populations, suggesting frequency-dependent selection consistent with sexual reproduction. In addition, both mating types were frequently found occupying the same lesion or leaf, providing opportunities for isolates of opposite mating type to interact and reproduce sexually. We propose that R. secalis should be considered a sexual pathogen, although the sexual cycle may occur infrequently in some populations.

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.

Rapid speciation following recent host shifts in the plant pathogenic fungus Rhynchosporium

Abstract Agriculture played a significant role in increasing the number of pathogen species and in expanding their geographic range during the last 10,000 years. We tested the hypothesis that a fungal pathogen of cereals and grasses emerged at the time of domestication of cereals in the Fertile Crescent and subsequently speciated after adaptation to its hosts. Rhynchosporium secalis, originally described from rye, causes an important disease on barley called scald, although it also infects other species of Hordeum and Agropyron. Phylogenetic analyses based on four DNA sequence loci identified three host-associated lineages that were confirmed by cross-pathogenicity tests. Bayesian analyses of divergence time suggested that the three lineages emerged between ∼1200 to 3600 years before present (B.P.) with a 95% highest posterior density ranging from 100 to 12,000 years B.P. depending on the implemented clock models. The coalescent inference of demographic history revealed a very recent population expansion for all three pathogens. We propose that Rhynchosporium on barley, rye, and Agropyron host species represent three cryptic pathogen species that underwent independent evolution and ecological divergence by host-specialization. We postulate that the recent emergence of these pathogens followed host shifts. The subsequent population expansions followed the expansion of the cultivated host populations and accompanying expansion of the weedy Agropyron spp. found in fields of cultivated cereals. Hence, agriculture played a major role in the emergence of the scald diseases, the adaptation of the pathogens to new hosts and their worldwide dissemination.

Diversity and evolution of effector loci in natural populations of the plant pathogen Melampsora lini

Genetic variation for pathogen infectivity is an important driver of disease incidence and prevalence in both natural and managed systems. Here, we use the interaction between the rust pathogen, Melampsora lini, and two host plants, Linum marginale and Linum usitatissimum, to examine how host-pathogen interactions influence the maintenance of polymorphism in genes underlying pathogen virulence. Extensive sequence variation at two effector loci (AvrP123, AvrP4) was found in M. lini isolates collected from across the native range of L. marginale in Australia, as well as in isolates collected from a second host, the cultivated species L. usitatissimum. A highly significant excess of nonsynonymous compared with synonymous polymorphism was found at both loci, suggesting that diversifying selection is important for the maintenance of the observed sequence diversity. Agrobacterium-mediated transient transformation assays were used to demonstrate that variants of both the AvrP123 and AvrP4 genes are differentially recognized by resistance genes in L. marginale. We further characterized patterns of nucleotide variation at AvrP123 and AvrP4 in 10 local populations of M. lini infecting the wild host L. marginale. Populations were significantly differentiated with respect to allelic representation at the Avr loci, suggesting the possibility of local selection maintaining distinct genetic structures between pathogen populations, whereas limited diversity may be explained via selective sweeps and demographic bottlenecks. Together, these results imply that interacting selective and nonselective factors, acting across a broad range of scales, are important for the generation and maintenance of adaptively significant variation in populations of M. lini.

Global Hierarchical Gene Diversity Analysis Suggests the Fertile Crescent Is Not the Center of Origin of the Barley Scald Pathogen Rhynchosporium secalis

A total of 1,366 Rhynchosporium secalis isolates causing scald on barley, rye, and wild barley (Hordeum spontaneum) were assayed for restriction fragment length polymorphism loci, DNA fingerprints, and mating type, to characterize global genetic structure. The isolates originated from 31 field populations on five continents. Hierarchical analysis revealed that more than 70% of the total genetic variation within regions was distributed within a barley field. At the global level, only 58% of the total genetic variation was distributed within fields, while 11% was distributed among fields within regions, and 31% was distributed among regions. A significant correlation was found between genetic and geographic distance. These findings suggest that gene flow is common at the local level while it is low between regions on the same continent, and rare between continents. Analyses of multilocus associations, genotype diversity, and mating type frequencies indicate that sexual recombination is occurring in most of the populations. We found the highest allele richness in Scandinavia followed by Switzerland. This suggests that R. secalis may not have originated at the center of origin of barley, the Fertile Crescent, nor in a secondary center of diversity of barley, Ethiopia.

Differential selection on Rhynchosporium secalis during parasitic and saprophytic phases in the barley scald disease cycle

ABSTRACT Competition among eight Rhynchosporium secalis isolates was assessed during parasitic and saprophytic phases of the disease cycle in field experiments conducted at two locations and over two growing seasons. The eight isolates were inoculated onto six barley populations exhibiting varying degrees of resistance. Microsatellite analysis of 2,866 isolates recovered from the field experiments showed significant, and sometimes opposite, changes in the frequencies of R. secalis genotypes during the growing season (parasitic phase) and between growing seasons (saprophytic phase). Isolates that showed the most complex virulence in greenhouse seedling assays had the lowest fitness in the field experiment. Significant differences in isolate fitness were found on different host populations and in different environments. Selection coefficients were large, indicating that evolution can occur rapidly in field populations. Although inoculated isolates had the lowest overall fitness on the moderately resistant landrace cv. Arabi Aswad, some isolates were more virulent and consistently increased in frequency on this landrace, suggesting a risk of directional selection and possible erosion of the resistance following its widespread deployment in monoculture. These results provide the first direct evidence that R. secalis pathogen genotypes differ in their saprophytic ability and parasitic fitness under field conditions.