101 Dothideomycetes genomes: A test case for predicting lifestyles and emergence of pathogens.

Dothideomycetes is the largest class of kingdom Fungi and comprises an incredible diversity of lifestyles, many of which have evolved multiple times. Plant pathogens represent a major ecological niche of the class Dothideomycetes and they are known to infect most major food crops and feedstocks for biomass and biofuel production. Studying the ecology and evolution of Dothideomycetes has significant implications for our fundamental understanding of fungal evolution, their adaptation to stress and host specificity, and practical implications with regard to the effects of climate change and on the food, feed, and livestock elements of the agro-economy. In this study, we present the first large-scale, whole-genome comparison of 101 Dothideomycetes introducing 55 newly sequenced species. The availability of whole-genome data produced a high-confidence phylogeny leading to reclassification of 25 organisms, provided a clearer picture of the relationships among the various families, and indicated that pathogenicity evolved multiple times within this class. We also identified gene family expansions and contractions across the Dothideomycetes phylogeny linked to ecological niches providing insights into genome evolution and adaptation across this group. Using machine-learning methods we classified fungi into lifestyle classes with >95 % accuracy and identified a small number of gene families that positively correlated with these distinctions. This can become a valuable tool for genome-based prediction of species lifestyle, especially for rarely seen and poorly studied species.

Variable number of tandem repeat markers in the genome sequence of Mycosphaerella fijiensis, the causal agent of black leaf streak disease of banana (Musa spp).

We searched the genome of Mycosphaerella fijiensis for molecular markers that would allow population genetics analysis of this plant pathogen. M. fijiensis, the causal agent of banana leaf streak disease, also known as black Sigatoka, is the most devastating pathogen attacking bananas (Musa spp). Recently, the entire genome sequence of M. fijiensis became available. We screened this database for VNTR markers. Forty-two primer pairs were selected for validation, based on repeat type and length and the number of repeat units. Five VNTR markers showing multiple alleles were validated with a reference set of isolates from different parts of the world and a population from a banana plantation in Costa Rica. Polymorphism information content values varied from 0.6414 to 0.7544 for the reference set and from 0.0400 and 0.7373 for the population set. Eighty percent of the polymorphism information content values were above 0.60, indicating that the markers are highly informative. These markers allowed robust scoring of agarose gels and proved to be useful for variability and population genetics studies. In conclusion, the strategy we developed to identify and validate VNTR markers is an efficient means to incorporate markers that can be used for fungicide resistance management and to develop breeding strategies to control banana black leaf streak disease. This is the first report of VNTR-minisatellites from the M. fijiensis genome sequence.

Permanent Genetic Resources added to Molecular Ecology Resources database 1 January 2009-30 April 2009.

This article documents the addition of 283 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Agalinis acuta; Ambrosia artemisiifolia; Berula erecta; Casuarius casuarius; Cercospora zeae-maydis; Chorthippus parallelus; Conyza canadensis; Cotesia sesamiae; Epinephelus acanthistius; Ficedula hypoleuca; Grindelia hirsutula; Guadua angustifolia; Leucadendron rubrum; Maritrema novaezealandensis; Meretrix meretrix; Nilaparvata lugens; Oxyeleotris marmoratus; Phoxinus neogaeus; Pristomyrmex punctatus; Pseudobagrus brevicorpus; Seiridium cardinale; Stenopsyche marmorata; Tetranychus evansi and Xerus inauris. These loci were cross-tested on the following species: Agalinis decemloba; Agalinis tenella; Agalinis obtusifolia; Agalinis setacea; Agalinis skinneriana; Cercospora zeina; Cercospora kikuchii; Cercospora sorghi; Mycosphaerella graminicola; Setosphaeria turcica; Magnaporthe oryzae; Cotesia flavipes; Cotesia marginiventris; Grindelia Xpaludosa; Grindelia chiloensis; Grindelia fastigiata; Grindelia lanceolata; Grindelia squarrosa; Leucadendron coniferum; Leucadendron salicifolium; Leucadendron tinctum; Leucadendron meridianum; Laodelphax striatellus; Sogatella furcifera; Phoxinus eos; Phoxinus rigidus; Phoxinus brevispinosus; Phoxinus bicolor; Tetranychus urticae; Tetranychus turkestani; Tetranychus ludeni; Tetranychus neocaledonicus; Tetranychus amicus; Amphitetranychus viennensis; Eotetranychus rubiphilus; Eotetranychus tiliarium; Oligonychus perseae; Panonychus citri; Bryobia rubrioculus; Schizonobia bundi; Petrobia harti; Xerus princeps; Spermophilus tridecemlineatus and Sciurus carolinensis.

Genetic structure of Phaeosphaeria nodorum populations in the north-central and midwestern United States.

Stagonospora nodorum blotch, caused by Phaeosphaeria nodorum, is considered one of the most destructive foliar diseases of wheat in the United States. However, relatively little is known about the population biology of this fungus in the major wheat-growing regions of the central United States. To rectify this situation, 308 single-spore isolates of P. nodorum were analyzed from 12 populations, five from hard red spring wheat cultivars in Minnesota and North Dakota and seven from soft red winter wheat in Indiana and Ohio. The genetic structure of the sampled populations was determined by analyzing polymorphisms at five microsatellite or simple-sequence repeat (SSR) loci and the mating type locus. Although a few clones were identified, most P. nodorum populations had high levels of gene (H(S) = 0.175 to 0.519) and genotype (D = 0.600 to 0.972) diversity. Gene diversity was higher among isolates collected from spring wheat cultivars in North Dakota and Minnesota (mean H(S) = 0.503) than in those from winter wheat cultivars in Indiana and Ohio (H(S) = 0.269). Analyses of clone-corrected data sets showed equal frequencies of both mating types in both regional and local populations, indicating that sexual recombination may occur regularly. However, significant gametic disequilibrium occurred in three of the four populations from North Dakota, and there was genetic differentiation both within and among locations. Genetic differentiation between the hard red spring and soft red winter wheat production regions was moderate (F(ST) = 0.168), but whether this is due to differences in wheat production or to geographical variation cannot be determined. These results suggest that sexual reproduction occurs in P. nodorum populations in the major wheat-growing regions of the central United States, and that geographically separated populations can be genetically differentiated, reflecting either restrictions on gene flow or selection.

Molecular mapping of Stb1, a potentially durable gene for resistance to septoria tritici blotch in wheat.

Septoria tritici blotch (STB), caused by the ascomycete Mycosphaerella graminicola (anamorph Septoria tritici), was the most destructive disease of wheat in Indiana and adjacent states before deployment of the resistance gene Stb1 during the early 1970s. Since then, Stb1 has provided durable protection against STB in widely grown wheat cultivars. However, its chromosomal location and allelic relationships to most other STB genes are not known, so the molecular mapping of Stb1 is of great interest. Genetic analyses and molecular mapping were performed for two mapping populations. A total of 148 F1 plants (mapping population I) were derived from a three-way cross between the resistant line P881072-75-1 and the susceptible lines P881072-75-2 and Monon, and 106 F6 recombinant-inbred lines (mapping population II) were developed from a cross between the resistant line 72626E2-12-9-1 and the susceptible cultivar Arthur. Bulked-segregant analysis with random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and microsatellite or simple-sequence repeat (SSR) markers was conducted to identify those that were putatively linked to the Stb1 gene. Segregation analyses confirmed that a single dominant gene controls the resistance to M. graminicola in each mapping population. Two RAPD markers, G7(1200) and H19(520), were tightly linked to Stb1 in wheat line P881072-75-1 at distances of less than 0.68 cM and 1.4 cM, respectively. In mapping population II, the most closely linked marker was SSR Xbarc74, which was 2.8 cM proximal to Stb1 on chromosome 5BL. Microsatellite loci Xgwm335 and Xgwm213 also were proximal to Stb1 at distances of 7.4 cM and 8.3 cM, respectively. The flanking AFLP marker, EcoRI-AGC/ MseI-CTA-1, was 8.4 cM distal to Stb1. The two RAPD markers, G7(1200) and H19(520), and AFLP EcoRI-AGC/ MseI-CTA-1, were cloned and sequenced for conversion into sequence-characterized amplified region (SCAR) markers. Only RAPD allele H19(520) could be converted successfully, and none of the SCAR markers was diagnostic for the Stb1 locus. Analysis of SSR and the original RAPD primers on several 5BL deletion stocks positioned the Stb1 locus in the region delineated by chromosome breakpoints at fraction lengths 0.59 and 0.75. The molecular markers tightly linked to Stb1 could be useful for marker-assisted selection and for pyramiding of Stb1 with other genes for resistance to M. graminicola in wheat.

Cloning and analysis of the mating-type idiomorphs from the barley pathogen Septoria passerinii.

The genus Septoria contains more than 1000 species of plant pathogenic fungi, most of which have no known sexual stage. Species of Septoria without a known sexual stage could be recent derivatives of sexual species that have lost the ability to mate. To test this hypothesis, the mating-type region of S. passerinii, a species with no known sexual stage, was cloned, sequenced, and compared to that of its close relative S. tritici (sexual stage: Mycosphaerella graminicola). Both of the S. passerinii mating-type idiomorphs were approximately 3 kb in size and contained a single reading frame interrupted by one (MAT-2) or two (MAT-1) putative introns. The putative products of MAT-1 and MAT-2 are characterized by alpha-box and high-mobility-group sequences, respectively, similar to those in the mating-type genes of M. graminicolaand other fungi. The mating-type genes of S. passerinii and M. graminicolaare evolving rapidly, approximately ten times faster than the internal transcribed spacer region of the ribosomal DNA, and are not closely related to those from Cochliobolusor other loculoascomycetes in the order Pleosporales. Therefore, the class Loculoascomycetes may be polyphyletic. Furthermore, differences between the phylogenetic trees may indicate separate evolutionary histories for the MAT-1 and MAT-2 idiomorphs. A three-primer multiplex-PCR technique was developed that allowed rapid identification of the mating types of isolates of S. passerinii. Both mating types were present in approximately equal frequencies and often on the same leaf in fields in Minnesota and North Dakota. Analyses with isozyme and random amplified polymorphic DNA markers revealed that each isolate had a unique genotype. The common occurrence of both mating types on the same leaf and the high levels of genotypic diversity indicate that S. passerinii is almost certainly not an asexual derivative of a sexual fungus. Instead, sexual reproduction probably plays an integral role in the life cycle of S. passerinii and may be much more important than previously believed in this (and possibly other) "asexual" species of Septoria.

Phylogenetic analysis of cercospora and mycosphaerella based on the internal transcribed spacer region of ribosomal DNA.

ABSTRACT Most of the 3,000 named species in the genus Cercospora have no known sexual stage, although a Mycosphaerella teleomorph has been identified for a few. Mycosphaerella is an extremely large and important genus of plant pathogens, with more than 1,800 named species and at least 43 associated anamorph genera. The goal of this research was to perform a large-scale phylogenetic analysis to test hypotheses about the past evolutionary history of Cercospora and Mycosphaerella. Based on the phylogenetic analysis of internal transcribed spacer (ITS) sequence data (ITS1, 5.8S rRNA gene, ITS2), the genus Mycosphaerella is monophyletic. In contrast, many anamorph genera within Mycosphaerella were polyphyletic and were not useful for grouping species. One exception was Cercospora, which formed a highly supported monophyletic group. Most Cercospora species from cereal crops formed a subgroup within the main Cercospora cluster. Only species within the Cercospora cluster produced the toxin cercosporin, suggesting that the ability to produce this compound had a single evolutionary origin. Intraspecific variation for 25 taxa in the Mycosphaerella clade averaged 1.7 nucleotides (nts) in the ITS region. Thus, isolates with ITS sequences that differ by two or more nucleotides may be distinct species. ITS sequences of groups I and II of the gray leaf spot pathogen Cercospora zeae-maydis differed by 7 nts and clearly represent different species. There were 6.5 nt differences on average between the ITS sequences of the sorghum pathogen Cercospora sorghi and the maize pathogen Cercospora sorghi var. maydis, indicating that the latter is a separate species and not simply a variety of Cercospora sorghi. The large monophyletic Mycosphaerella cluster contained a number of anamorph genera with no known teleomorph associations. Therefore, the number of anamorph genera related to Mycosphaerella may be much larger than suspected previously.

DNA Fingerprint Probe from Mycosphaerella graminicola Identifies an Active Transposable Element.

ABSTRACT DNA fingerprinting has been used extensively to characterize populations of Mycosphaerella graminicola, the Septoria tritici blotch pathogen of wheat. The highly polymorphic DNA fingerprints of Mycosphaerella graminicola were assumed to reflect the action of transposable elements. However, there was no direct evidence to support that conclusion. To test the transposable element hypothesis, the DNA fingerprint probe pSTL70 was sequenced, along with three other clones from a subgenomic library that hybridized with pSTL70. Analysis of these sequences revealed that pSTL70 contains the 3' end of a reverse transcriptase sequence plus 29- and 79-bp direct repeats. These are characteristics of transposable elements identified in other organisms. Southern analyses indicated that either the direct-repeat or reverse-transcriptase sequences by themselves essentially duplicated the original DNA fingerprint pattern, but other portions of pSTL70 contained single-copy DNA. Analysis of 60 progeny from a sexual cross between two Dutch isolates of Mycosphaerella graminicola identified several new bands that were not present in the parents. Thus, the putative transposable element probably is active during meiosis. Tests of single-spore isolates revealed gains or losses of one or more DNA fingerprint bands in 4 out of 10 asexual lines derived from isolate IPO94269. Therefore, DNA fingerprint patterns produced by the putative transposable element were capable of changes during asexual reproduction of this isolate. Probe pSTL70 did not hybridize at high stringency to genomic DNAs from other fungi related to Septoria and Mycosphaerella. These results indicate that the DNA fingerprint probe pSTL70 most likely identifies a transposable element in Mycosphaerella graminicola that may have been acquired recently, and appears to be active during both sexual and asexual reproduction.

Characterization of Phytophthora infestans Populations in Western Washington.

The first detection in the United States of isolates of Phytophthora infestans having metalaxyl insensitivity and complex pathotypes occurred in western Washington during the early 1990s. To determine the genetic structure of the current population in western Washington, a total of 115 isolates of P. infestans were obtained during 1996 from infected tubers or foliage of potato, tomato, nightshade, and bittersweet throughout the region. An additional 45 isolates were collected from a single field. Based on mating type, metalaxyl-insensitivity, and molecular markers (allozymes of glucose-6-phosphate isomerase, peptidase, and RG57 DNA fingerprint), all of the isolates were A1 mating type and had the US-11 multilocus genotype. Analyses of an additional 120 isolates collected during 1997 from potato, tomato, and nightshade were performed. As in 1996, US-11 was the predominant genotype detected on all three hosts. However, three additional A2 mating type genotypes were also detected: US-7, US-8, and US-14. These three genotypes represent the first A2 mating type isolates detected in western Washington. Most of a subset of 60 isolates infected 4 to 7 of the 10 potato differentials tested. This included 90% of the isolates collected in 1996 (all US-11), plus 72% of the US-11 and 100% of the US-8 and US-14 isolates collected during 1997. Virulence phenotypes in this region are complex even without the selection pressure of R-genes in the local commercial cultivars. The apparent increase in genetic variation observed in populations of P. infestans in western Washington from 1996 to 1997 most likely occurred by migration rather than by sexual recombination.

Genetic Change Within Populations of Phytophthora infestans in the United States and Canada During 1994 to 1996: Role of Migration and Recombination.

ABSTRACT Dramatic changes occurred within populations of Phytophthora infestans in the United States and Canada from 1994 through 1996. Occurrence of the US-8 genotype, detected rarely during 1992 and 1993, increased rapidly and predominated in most regions during 1994 through 1996. US-7, which infected both potato and tomato and made up almost 50% of the sample during 1993, was detected only rarely among 330 isolates from the United States analyzed during 1994. It was not detected at all in more limited samples from 1996. Thus, ability to infect both potato and tomato apparently did not increase the fitness of this genotype relative to US-8, as predicted previously. US-1, the previously dominant genotype throughout the United States and Canada, made up 8% or less of the samples analyzed during 1994 through 1996. A few additional genotypes were detected, which could indicate the beginnings of sexual reproduction of P. infestans within the United States and Canada. However, clonal reproduction still predominated in all locations sampled; opportunities for sexual reproduction probably were limited, because the A1 and A2 mating types usually were separated geographically. The high sensitivity of the US-1 genotype to the fungicide metalaxyl also could have reduced opportunities for contact between the mating types in fields where this compound was applied. The previous correlation between metalaxyl sensitivity and genotype was confirmed and extended to a new genotype, US-17: all US-1 isolates tested were sensitive; all isolates of the US-7, US-8, and US-17 genotypes tested to date have been resistant. Isolates of P. capsici and P. erythroseptica, two other species often found on tomato and potato, could be easily distinguished from each other and from P. infestans using a simple allozyme assay for the enzyme glucose-6-phosphate isomerase. This technique could be useful for rapid identification of species, in addition to genotype of P. infestans. It generally was not possible to predict which genotypes would be present in a location from 1 year to the next. Long-distance movement of US-8 in seed tubers was documented, and this was probably the primary means for the rapid spread of this genotype from 1993 through 1996.

Sensitivity to Protectant Fungicides and Pathogenic Fitness of Clonal Lineages of Phytophthora infestans in the United States.

ABSTRACT Since 1991, dramatic changes have occurred in the genetic composition of populations of Phytophthora infestans in the United States. Clonal lineages recently introduced into the United States (US-7 and US-8) are more common now than the previously dominant lineage (US-1). To help determine why these changes occurred, four clonal lineages of P. in-festans common during the early 1990s in the United States and Canada were evaluated for sensitivity to the protectant fungicides mancozeb and chlorothalonil using amended agar assays for isolates collected from 1990 to 1994. No isolate or lineage was resistant to either mancozeb or chlorothalonil. There were significant differences among isolates for degree of sensitivity to one fungicide individually, but there were no significant (P = 0.05) differences among the US-1, US-6, US-7, and US-8 clonal lineages for degree of sensitivity to both fungicides. Therefore, resistance to protectant fungicides cannot explain the rapid increase in frequency of the US-7 and US-8 clonal lineages. Three components of pathogenic fitness (latent period, lesion area, and sporulation after 96 h) were tested for the three clonal lineages that were detected most commonly during 1994 (US-1, US-7, and US-8). All but one of the isolates in this analysis were collected during 1994 and evaluated within 10 months of collection by inoculating detached leaflets of the susceptible potato cultivar Norchip. There were significant differences between the US-1 and US-8 clonal lineages for lesion area and sporulation, and between US-1 and US-7 for latent period. The US-6 clonal lineage was excluded from the pathogenic fitness experiments, because no isolates of this lineage were collected during 1994. Compared with US-7 and US-8, US-1 had the longest latent period and the smallest lesions with the least sporulation. Incorporation of the differences between US-1 and US-8 in computer simulation experiments revealed that significantly more protectant fungicide (e.g., 25%) would be required to suppress epidemics caused by the US-8 clonal lineage compared with US-1. These differences in pathogenic fitness components probably contribute to the general predominance of the "new" clonal lineages (especially US-8) relative to the "old" US-1 lineage.

Panglobal distribution of a single clonal lineage of the Irish potato famine fungus.

More than 300 isolates of the Irish potato famine fungus, Phytophthora infestans, collected in 20 countries on five continents, were analyzed for genetic variation at the mating type and two allozyme loci. A subset of more than 200 isolates was also analyzed for DNA "fingerprint" variation. A surprising result was that a single clonal lineage dominated most populations worldwide. All of the variation within this lineage appeared to have arisen by mitotic recombination or by mutation. In addition to the most common clonal lineage, a number of different, but apparently closely related, lineages occurred in the United States and Canada. The low levels of gene diversity in the derived populations compared to the presumed ancestral population in central Mexico indicate that P. infestans went through extreme genetic bottlenecks during its dispersal. The genetic data are consistent with the hypothesis that the initial migration of P. infestans in the 1840s was from Mexico to the United States and that only a single genetic individual was transported to Europe and subsequently to the rest of the world. If this hypothesis is correct, then the Irish potato famine was caused by a single clonal genotype of P. infestans.

Cloning and genetic analyses of two highly polymorphic, moderately repetitive nuclear DNAs from Phytophthora infestans.

Randomly selected clones from a Phytophthora infestans partial genomic library were characterized by hybridizing individual clones to Southern blots of total genomic DNA digested with the restriction enzyme EcoRI. Among 59 clones that were screened on seven different central-Mexican isolates, five revealed a unique banding pattern for each isolate tested. Two of these clones were tested further; the banding patterns produced by both were somatically stable when probed to DNA from 63 single-zoospore (asexual) progeny from five different "parent" isolates. For one probe, RG57, each band appeared to represent a unique genetic locus in three different crosses, and each locus segregated for the presence or absence of a band. No bands were found to be allelic, but two pairs of cosegregating loci were identified. Genetic analyses of the other probe (RG7) revealed many more pairs of cosegregating bands and some bands which were allelic. When these probes were hybridized to DNA from the other five species in Phytophthora group IV, probe RG57 hybridized strongly to DNA from P. colocasiae, P. phaseoli and P. mirabilis, but weakly or not at all to that of P. hibernalis and P. ilicis. Probe RG7 hybridized fairly strongly to DNA from all six species. Because the sequence recognized by probe RG57 appears to be evolutionarily conserved, and is dispersed, moderately repetitive and highly polymorphic, it could be very useful in additional studies on the genetics and population biology of P. infestans.