MAT Genes Research Articles

Cercospora zeina from Maize in South Africa Exhibits High Genetic Diversity and Lack of Regional Population Differentiation.

South Africa is one of the leading maize-producing countries in sub-Saharan Africa. Since the 1980s, Cercospora zeina, a causal agent of gray leaf spot of maize, has become endemic in South Africa, and is responsible for substantial yield reductions. To assess genetic diversity and population structure of C. zeina in South Africa, 369 isolates were collected from commercial maize farms in three provinces (KwaZulu-Natal, Mpumalanga, and North West). These isolates were evaluated with 14 microsatellite markers and species-specific mating type markers that were designed from draft genome sequences of C. zeina isolates from Africa (CMW 25467) and the United States (USPA-4). Sixty alleles were identified across 14 loci, and gene diversity values within each province ranged from 0.18 to 0.35. High levels of gene flow were observed (Nm = 5.51), and in a few cases, identical multilocus haplotypes were found in different provinces. Overall, 242 unique multilocus haplotypes were identified with a low clonal fraction of 34%. No distinct population clusters were identified using STRUCTURE, principal coordinate analysis, or Weir's theta θ statistic. The lack of population differentiation was supported by analysis of molecular variance tests, which indicated that only 2% of the variation was attributed to variability between populations from each province. Mating type ratios of MAT1-1 and MAT1-2 idiomorphs from 335 isolates were not significantly different from a 1:1 ratio in all provinces, which provided evidence for sexual reproduction. The draft genome of C. zeina CMW 25467 exhibited a complete genomic copy of the MAT1-1 idiomorph as well as exonic fragments of MAT genes from both idiomorphs. The high level of gene diversity, shared haplotypes at different geographical locations within South Africa, and presence of both MAT idiomorphs at all sites indicates widespread dispersal of C. zeina between maize fields in the country as well as evidence for sexual recombination. The outcomes of this genome-enabled study are important for disease management since the high diversity has implications for dispersal of fungicide resistance should it emerge and the need for diversified resistance breeding.

MAT gene idiomorphs suggest a heterothallic sexual cycle in the citrus pathogen Phyllosticta citricarpa

Sexual reproduction in fungi is controlled by mating type genes, which are located at the MAT locus. In this study, we investigated the structure of this locus in the phytopathogenic fungus Phyllosticta citricarpa, the causal agent of citrus black spot disease. Despite intensive study, its sexual state has never been observed in single-spore culture. Through analysis of the genome sequences of two individual P. citricarpa isolates, the sequence of the DNA lyase gene was identified and, as previously reported in the literature, the mating type genes were located in the 3′ flanking region of this gene. The results suggested that P. citricarpa is heterothallic, owing to the exclusive presence of the MAT1–1 or MAT1–2 gene in individual strains. In order to characterize the MAT locus, we designed primers to amplify this region. P. citricarpa was found to have complete and apparently functional copies of MAT genes, containing α-1 and HMG domains, present in different isolates. In addition to MAT1–2-1 and MAT1–1-1 genes, the MAT1–1-4 gene was located in the 5′ flanking region of the MAT1–1-1 gene and the MAT1–2-5 gene was located in 5′ flanking region of the MAT1–2-1 gene. A multiplex PCR protocol was also developed to differentiate P. citricarpa idiomorphs, which can be used in distribution and incidence studies of mating type strains, in order to determine the occurrence of sexual reproduction and to facility crossing studies. Furthermore, in Brazil, the two idiomorphs occur in a 1:1 ratio, which is expected in sexually reproducing populations.

Characterization of MAT gene functions in the life cycle of Sclerotinia sclerotiorum reveals a lineage-specific MAT gene functioning in apothecium morphogenesis

Sclerotinia sclerotiorum (Lib.) de Bary is a phytopathogenic fungus that relies on the completion of the sexual cycle to initiate aerial infections. The sexual cycle produces apothecia required for inoculum dispersal. In this study, insight into the regulation of apothecial multicellular development was pursued through functional characterization of mating-type genes. These genes are hypothesized to encode master regulatory proteins required for aspects of sexual development ranging from fertilization through fertile fruiting body development. Experimentally, loss-of-function mutants were created for the conserved core mating-type genes (MAT1-1-1, and MAT1-2-1), and the lineage-specific genes found only in S. sclerotiorum and closely related fungi (MAT1-1-5, and MAT1-2-4). The MAT1-1-1, MAT1-1-5, and MAT1-2-1 mutants are able to form ascogonia but are blocked in all aspects of apothecium development. These mutants also exhibit defects in secondary sexual characters including lower numbers of spermatia. The MAT1-2-4 mutants are delayed in carpogenic germination accompanied with altered disc morphogenesis and ascospore production. They too produce lower numbers of spermatia. All four MAT gene mutants showed alterations in the expression of putative pheromone precursor (Ppg-1) and pheromone receptor (PreA, PreB) genes. Our findings support the involvement of MAT genes in sexual fertility, gene regulation, meiosis, and morphogenesis in S. sclerotiorum.

Direct repeat-mediated DNA deletion of the mating type MAT1-2 genes results in unidirectional mating type switching in Sclerotinia trifoliorum.

The necrotrophic fungal pathogen Sclerotinia trifoliorum exhibits ascospore dimorphism and unidirectional mating type switching - self-fertile strains derived from large ascospores produce both self-fertile (large-spores) and self-sterile (small-spores) offsprings in a 4:4 ratio. The present study, comparing DNA sequences at MAT locus of both self-fertile and self-sterile strains, found four mating type genes (MAT1-1-1, MAT1-1-5, MAT1-2-1 and MAT1-2-4) in the self-fertile strain. However, a 2891-bp region including the entire MAT1-2-1 and MAT1-2-4 genes had been completely deleted from the MAT locus in the self-sterile strain. Meanwhile, two copies of a 146-bp direct repeat motif flanking the deleted region were found in the self-fertile strain, but only one copy of this 146-bp motif (a part of the MAT1-1-1 gene) was present in the self-sterile strain. The two direct repeats were believed to be responsible for the deletion through homologous intra-molecular recombination in meiosis. Tetrad analyses showed that all small ascospore-derived strains lacked the missing DNA between the two direct repeats that was found in all large ascospore-derived strains. In addition, heterokaryons at the MAT locus were observed in field isolates as well as in laboratory derived isolates.

The cacao pathogen Moniliophthora roreri (Marasmiaceae) possesses biallelic A and B mating loci but reproduces clonally.

The cacao pathogen Moniliophthora roreri belongs to the mushroom-forming family Marasmiaceae, but it has never been observed to produce a fruiting body, which calls to question its capacity for sexual reproduction. In this study, we identified potential A (HD1 and HD2) and B (pheromone precursors and pheromone receptors) mating genes in M. roreri. A PCR-based method was subsequently devised to determine the mating type for a set of 47 isolates from across the geographic range of the fungus. We developed and generated an 11-marker microsatellite set and conducted association and linkage disequilibrium (standardized index of association, IA(s)) analyses. We also performed an ancestral reconstruction analysis to show that the ancestor of M. roreri is predicted to be heterothallic and tetrapolar, which together with sliding window analyses support that the A and B mating loci are likely unlinked and follow a tetrapolar organization within the genome. The A locus is composed of a pair of HD1 and HD2 genes, whereas the B locus consists of a paired pheromone precursor, Mr_Ph4, and receptor, STE3_Mr4. Two A and B alleles but only two mating types were identified. Association analyses divided isolates into two well-defined genetically distinct groups that correlate with their mating type; IA(s) values show high linkage disequilibrium as is expected in clonal reproduction. Interestingly, both mating types were found in South American isolates but only one mating type was found in Central American isolates, supporting a prior hypothesis of clonal dissemination throughout Central America after a single or very few introductions of the fungus from South America.

Discovery of a sexual cycle in Talaromyces amestolkiae

Talaromyces amestolkiae is a common cosmopolitan species that has been cultured from indoor house dust, sputum and lungs from cystic fibrosis patients, indoor air, wheat, soil, pineapple, sculptures and manure. It was described as an asexual Talaromyces species and was reported to produce black sclerotia. In this study we report on the induction of sexual reproductive structures in T. amestolkiae. The mating type of 18 T. amestolkiae strains was determined with MAT-specific primers. Subsequently opposite mating types were inoculated on oatmeal agar and malt-extract agar and incubated 6–20 wk at 25 and 30 C in darkness. After incubation single ascospore isolations were made and evidence of recombination in the offspring was examined by amplified fragment length polymorphism and pairwise homoplasy index test, which is implemented in Splitstree4. The offspring displayed clear evidence of recombination on a genetic level as shown in the variations observed between banding patterns in the amplified fragment length polymorphism. Also a net-like and reticulated NeighborNet was observed and the pairwise homoplasy index test for recombination supported the presence of recombination (P = 0.003372). The distribution of MAT1-1 and MAT1-2 genes in the progeny showed a close to 1 : 1 ratio. Talaromyces amestolkiae is only the second heterothallic Talaromyces species to produce ascomata and ascospores under laboratory conditions.

Arabidopsis Infection by Haploid or Diploid Strains of Ustilago maydis Reveals its Capacity as A Necrotrophic or Biotrophic Phytopathogen

The use of experimental patho-systems is a widely used strategy for understanding the pathogenic processes developed by many infectious agents. Ustilago maydis is a Basidiomycota fungus that naturally infects only maize and teozintle. We have established that under axenic conditions, either haploid or diploid strains of U. maydis can infect Arabidopsis thaliana, and in this work we analyzed the different behaviour of U. maydis haploid, diploid, and merodiploid strains in the mating genes during Arabidopsis infection. We determined the severity of infection, cell death, and the production of phytohormones and reactive oxygen species (ROS), occurring in the invaded host. Our results indicate that whereas U. maydis diploid and merodiploid strains behave as biotrophic parasites, the behaviour of the haploid strain is necrotrophic. We suggest that this difference resides in the fact that only the former two strains, but not the haploid one, are able to synthesize the master regulator of the biotrophic pathogenic behaviour, the bE/bW heterodimer encoded by the b genes of the two mating partners.

Draft Genome Sequences of Two Closely Related Aflatoxigenic Aspergillus Species Obtained from the Ivory Coast.

Aspergillus ochraceoroseus and Aspergillus rambellii were isolated from soil detritus in Taï National Park, Ivory Coast, Africa. The Type strain for each species happens to be the only representative ever sampled. Both species secrete copious amounts of aflatoxin B1 and sterigmatocystin, because each of their genomes contains clustered genes for biosynthesis of these mycotoxins. We sequenced their genomes using a personal genome machine and found them to be smaller in size (A. ochraceoroseus = 23.9 Mb and A. rambellii = 26.1 Mb), as well as in numbers of predicted genes (7,837 and 7,807, respectively), compared to other sequenced Aspergilli. Our findings also showed that the A. ochraceoroseus Type strain contains a single MAT1-1 gene, while the Type strain of A. rambellii contains a single MAT1-2 gene, indicating that these species are heterothallic (self-infertile). These draft genomes will be useful for understanding the genes and pathways necessary for the cosynthesis of these two toxic secondary metabolites as well as the evolution of these pathways in aflatoxigenic fungi.

Genetics of mating in members of the Chaetomiaceae as revealed by experimental and genomic characterization of reproduction in Myceliophthora heterothallica

Members of the Chaetomiaceae are among the most studied fungi in industry and among the most reported in investigations of biomass degradation in both natural and laboratory settings. The family is recognized for production of carbohydrate-active enzymes and antibiotics. Thermophilic species are of special interest for their abilities to produce thermally stable enzymes and to be grown under conditions that are unsuitable for potential contaminant microorganisms. Such interests led to the recent acquisition of genome sequences from several members of the family, including thermophilic species, several of which are reported here for the first time. To date, however, thermophilic fungi in industry have served primarily as parts reservoirs and there has been no good genetic model for species in the family Chaetomiaceae or for thermophiles in general. We report here on the reproductive biology of the thermophile Myceliophthora heterothallica, which is heterothallic, unlike most described species in the family. We confirmed heterothallism genetically by following the segregation of mating type idiomorphs and other markers. We have expanded the number of known sexually-compatible individuals from the original isolates from Indiana and Germany to include several isolates from New Mexico. An interesting aspect of development in M. heterothallica is that ascocarp formation is optimal at approximately 30°C, whereas vegetative growth is optimal at 45°C. Genome sequences obtained from several strains, including isolates of each mating type, revealed mating-type regions whose genes are organized similarly to those of other members of the Sordariales, except for the presence of a truncated version of the mat A-1 (MAT1-1-1) gene in mating-type a (MAT1-2) strains. In M. heterothallica and other Chaetomiaceae, mating-type A (MAT1-1) strains have the full-length version of mat A-1 that is typical of mating-type A strains of diverse Ascomycota, whereas a strains have only the truncated version. This truncated mat A-1 has an intact open reading frame and a derived start codon that is not present in mat A-1 from A strains. The predicted protein contains a region that is conserved across diverse mat A-1 genes, but it lacks the major alpha1 domain, which characterizes proteins in this family and is known to be required for fertility in A strains from other Ascomycota. Finally, we have used genes from M. heterothallica to probe for mating genes in other homothallic and heterothallic members of the Chaetomiaceae. The majority of homothallic species examined have a typical mat A-1,2,3 (MAT1-1-1,2,3) region in addition to an unlinked mat a-1 (MAT1-2-1) gene, reflecting one type of homothallism commonly observed in diverse Ascomycota.

Region-specific variation in the properties of skeletal adipocytes reveals regulated and constitutive marrow adipose tissues.

Marrow adipose tissue (MAT) accumulates in diverse clinical conditions but remains poorly understood. Here we show region-specific variation in MAT adipocyte development, regulation, size, lipid composition, gene expression and genetic determinants. Early MAT formation in mice is conserved, whereas later development is strain dependent. Proximal, but not distal tibial, MAT is lost with 21-day cold exposure. Rat MAT adipocytes from distal sites have an increased proportion of monounsaturated fatty acids and expression of Scd1/Scd2, Cebpa and Cebpb. Humans also have increased distal marrow fat unsaturation. We define proximal ‘regulated’ MAT (rMAT) as single adipocytes interspersed with active haematopoiesis, whereas distal ‘constitutive’ MAT (cMAT) has low haematopoiesis, contains larger adipocytes, develops earlier and remains preserved upon systemic challenges. Loss of rMAT occurs in mice with congenital generalized lipodystrophy type 4, whereas both rMAT and cMAT are preserved in mice with congenital generalized lipodystrophy type 3. Consideration of these MAT subpopulations may be important for future studies linking MAT to bone biology, haematopoiesis and whole-body metabolism.

Selective Advantages of a Parasexual Cycle for the YeastCandida albicans

The yeast Candida albicans can mate. However, in the natural environment mating may generate progeny (fusants) fitter than clonal lineages too rarely to render mating biologically significant: C. albicans has never been observed to mate in its natural environment, the human host, and the population structure of the species is largely clonal. It seems incapable of meiosis, and most isolates are diploid and carry both mating-type-like (MTL) locus alleles, preventing mating. Only chromosome loss or localized loss of heterozygosity can generate mating-competent cells, and recombination of parental alleles is limited. To determine if mating is a biologically significant process, we investigated if mating is under selection. The ratio of nonsynonymous to synonymous mutations in mating genes and the frequency of mutations abolishing mating indicated that mating is under selection. The MTL locus is located on chromosome 5, and when we induced chromosome 5 loss in 10 clinical isolates, most of the resulting MTL-homozygotes could mate with each other, producing fusants. In laboratory culture, a novel environment favoring novel genotypes, some fusants grew faster than their parents, in which loss of heterozygosity had reduced growth rates, and also faster than their MTL-heterozygous ancestors-albeit often only after serial propagation. In a small number of experiments in which co-inoculation of an oral colonization model with MTL-homozygotes yielded small numbers of fusants, their numbers declined over time relative to those of the parents. Overall, our results indicate that mating generates genotypes superior to existing MTL-heterozygotes often enough to be under selection.

Experimental swap of Anopheles gambiae's assortative mating preferences demonstrates key role of X-chromosome divergence island in incipient sympatric speciation.

Although many theoretical models of sympatric speciation propose that genes responsible for assortative mating amongst incipient species should be associated with genomic regions protected from recombination, there are few data to support this theory. The malaria mosquito, Anopheles gambiae, is known for its sympatric cryptic species maintained by pre-mating reproductive isolation and its putative genomic islands of speciation, and is therefore an ideal model system for studying the genomic signature associated with incipient sympatric speciation. Here we selectively introgressed the island of divergence located in the pericentric region of the X chromosome of An. gambiae s.s. into its sister taxon An. coluzzii through 5 generations of backcrossing followed by two generations of crosses within the introgressed strains that resulted in An. coluzzii-like recombinant strains fixed for the M and S marker in the X chromosome island. The mating preference of recombinant strains was then tested by giving virgin recombinant individuals a choice of mates with X-islands matching and non-matching their own island type. We show through genetic analyses of transferred sperm that recombinant females consistently mated with matching island-type males thereby associating assortative mating genes with the X-island of divergence. Furthermore, full-genome sequencing confirmed that protein-coding differences between recombinant strains were limited to the experimentally swapped pericentromeric region. Finally, targeted-genome comparisons showed that a number of these unique differences were conserved in sympatric field populations, thereby revealing candidate speciation genes. The functional demonstration of a close association between speciation genes and the X-island of differentiation lends unprecedented support to island-of-speciation models of sympatric speciation facilitated by pericentric recombination suppression.

A novel locus for mycelial aggregation forms a gateway to improved Streptomyces cell factories.

BackgroundStreptomycetes produce a plethora of natural products including antibiotics and anticancer drugs, as well as many industrial enzymes. Their mycelial life style is a major bottleneck for industrial exploitation and over decades strain improvement programs have selected production strains with better growth properties. Uncovering the nature of the underlying mutations should allow the ready transfer of desirable traits to other production hosts.ResultsHere we report that the mat gene cluster, which was identified through reverse engineering of a non-pelleting mutant selected in a chemostat, is key to pellet formation of Streptomyces lividans. Deletion of matA or matB, which encode putative polysaccharide synthases, effects mycelial metamorphosis, with very small and open mycelia. Growth rate and productivity of the matAB null mutant were increased by over 60% as compared to the wild-type strain.ConclusionHere, we present a way to counteract pellet formation by streptomycetes, which is one of the major bottlenecks in their industrial application. The mat locus is an ideal target for rational strain design approaches aimed at improving streptomycetes as industrial production hosts.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0224-6) contains supplementary material, which is available to authorized users.

Maintenance of sex-related genes and the co-occurrence of both mating types in Verticillium dahliae.

Verticillium dahliae is a cosmopolitan, soilborne fungus that causes a significant wilt disease on a wide variety of plant hosts including economically important crops, ornamentals, and timber species. Clonal expansion through asexual reproduction plays a vital role in recurring plant epidemics caused by this pathogen. The recent discovery of recombination between clonal lineages and preliminary investigations of the meiotic gene inventory of V. dahliae suggest that cryptic sex appears to be rare in this species. Here we expanded on previous findings on the sexual nature of V. dahliae. Only 1% of isolates in a global collection of 1120 phytopathogenic V. dahliae isolates contained the MAT1-1 idiomorph, whereas 99% contained MAT1-2. Nine unique multilocus microsatellite types comprised isolates of both mating types, eight of which were collected from the same substrate at the same time. Orthologs of 88 previously characterized sex-related genes from fungal model systems in the Ascoymycota were identified in the genome of V. dahliae, out of 93 genes investigated. Results of RT-PCR experiments using both mating types revealed that 10 arbitrarily chosen sex-related genes, including MAT1-1-1 and MAT1-2-1, were constitutively expressed in V. dahliae cultures grown under laboratory conditions. Ratios of non-synonymous (amino-acid altering) to synonymous (silent) substitutions in V. dahliae MAT1-1-1 and MAT1-2-1 sequences were indistinguishable from the ratios observed in the MAT genes of sexual fungi in the Pezizomycotina. Patterns consistent with strong purifying selection were also observed in 18 other arbitrarily chosen V. dahliae sex-related genes, relative to the patterns in orthologs from fungi with known sexual stages. This study builds upon recent findings from other laboratories and mounts further evidence for an ancestral or cryptic sexual stage in V. dahliae.

Characterization of mating type (MAT) alleles differentiated by a natural inversion in Sclerotinia minor

A recent study on fungal mating type genes revealed two MAT alleles within homothallic Sclerotinia sclerotiorum differentiated by an inversion, Inv− (inversion negative) and Inv+ (inversion positive). An analysis of mating type in closely related S. minor was conducted to shed light on the evolution of this MAT inversion. Inv− and Inv+ MAT alleles were identified in S. minor and were characterized. Both MAT alleles in S. minor were flanked by APN2 and SLA2, and consisted of two idiomorphs fused as in other homothallic ascomycetes. However, in the Inv+ MAT, the 3·6 kb MAT region was inverted relative to the Inv− MAT. Except for the inversion, both Inv− and Inv+ MAT in S. minor were equal in size and identical in nucleotide sequence. The MAT inversion in Inv+ S. minor was at exactly the same place as in Inv+ S. sclerotiorum and affected three of four MAT genes: MAT1‐1‐1 was truncated and MAT1‐2‐4 and MAT1‐2‐1 were inverted. Unlike S. sclerotiorum, expression of MAT genes did not differ between Inv− and Inv+ S. minor. The 250 bp inverted repeat motif that flanked the inverted MAT region in S. sclerotiorum and believed responsible for the MAT inversion was also found in S. minor, but was 256 bp. Depending on the MAT genes, 93–96% nucleotide identity was observed between Sclerotinia species. Both Inv+ and Inv− MAT S. minor and S. sclerotiorum isolates were commonly found in lettuce fields of Arizona along with MAT heterokaryons.

Nothing special in the specialist? Draft genome sequence of Cryomyces antarcticus, the most extremophilic fungus from Antarctica.

The draft genome of the Antarctic endemic fungus Cryomyces antarcticus is presented. This rock inhabiting, microcolonial fungus is extremely stress tolerant and it is a model organism for exobiology and studies on stress resistance in Eukaryots. Since this fungus is a specialist in the most extreme environment of the Earth, the analysis of its genome is of important value for the understanding of fungal genome evolution and stress adaptation. A comparison with Neurospora crassa as well as with other microcolonial fungi shows that the fungus has a genome size of 24 Mbp, which is the average in the fungal kingdom. Although sexual reproduction was never observed in this fungus, 34 mating genes are present with protein homologs in the classes Eurotiomycetes, Sordariomycetes and Dothideomycetes. The first analysis of the draft genome did not reveal any significant deviations of this genome from comparative species and mesophilic hyphomycetes.

An alkaline pH control strategy for methionine adenosyltransferase production in Pichia pastoris fermentation

Pichia pastoris is a successful system for expressing heterologous proteins and its fermentation pH is always maintained below 7.0. However, particular proteins are unstable under acidic conditions, such as methionine adenosyltransferase (MAT), and thus fermentation under acidic pH conditions is unsuitable because protein activity is lost owing to denaturation. Here, a strategy employing alkaline pH in the late fermentation period was developed to improve MAT production. Initially, P. pastoris KM71 was transformed with the mat gene to overexpress MAT. After 72 h of in vitro incubation at different pH values, the expressed MAT displayed highest stability at pH 8.0; however, pH 8.0 inhibited cell growth and induced cell rupture, thus affecting protein production. To balance MAT stability and Pichia cell viability, different pH control strategies were compared. In strategy A (reference), the induction pH was maintained at 6.0, whereas in strategy B, it was gradually elevated to 8.0 through a 25 h transition period (80 ∼ 105 h). MAT activity was 0.86 U/mg (twofold higher than the control). However, MAT content was reduced by 50% when compared with strategy A, because of proteases released upon cell lysis. To improve cell viability under alkaline conditions, glycerol was added in addition to methanol (strategy C). When compared with strategy B, the MAT-specific activity remained nearly constant, whereas the expression level increased to 1.27 g/L. The alkaline pH control strategy presented herein for MAT production represents an excellent alternative for expressing proteins that are stable only under alkaline conditions.

Occurrence of barley pathogenicPyrenophoraspecies and their mating types in Hungary

Net blotch and leaf stripe caused by Pyrenophora teres and P. graminea, respectively, are two major foliar diseases of barley. These two species are able to infect wheat, too. The species composition of these pathogens was examined, for the first time, in four different regions of Hungary in 2006–2010. Altogether 204 isolates were obtained from 99 winter barley, 55 spring barley and 50 wheat leaf samples collected in commercial fields and experimental stations, and species assignment was carried out using species-specific PCR reactions. Most isolates belonged to P. teres f. teres (68%), 26% to P. teres f. maculata and only 6% of the isolates were assigned to P. graminea. Interestingly, all but one of the P. graminea isolates came from the western part of Hungary, while both forms of P. teres occurred in each region. The distribution of mating type genes was also examined in 144 isolates. The overall ratio of MAT1 and MAT2 genes in P. graminea, P. teres f. maculata and P. teres f. teres was 5:3, and close to 2:1 and 1:1, respectively. Both MAT1 and MAT2 isolates of each fungal species/form were distributed in almost all regions over several years, indicating a high potential for sexual outcrossing within local populations of these pathogens. Our survey may be helpful to determine priorities in disease resistance breeding programs. Further studies are in progress to examine the population structure of the most abundant pathogen P. teres f. teres.

De novo genome assembly of the soil-borne fungus and tomato pathogen Pyrenochaeta lycopersici.

BackgroundPyrenochaeta lycopersici is a soil-dwelling ascomycete pathogen that causes corky root rot disease in tomato (Solanum lycopersicum) and other Solanaceous crops, reducing fruit yields by up to 75%. Fungal pathogens that infect roots receive less attention than those infecting the aerial parts of crops despite their significant impact on plant growth and fruit production.ResultsWe assembled a 54.9Mb P. lycopersici draft genome sequence based on Illumina short reads, and annotated approximately 17,000 genes. The P. lycopersici genome is closely related to hemibiotrophs and necrotrophs, in agreement with the phenotypic characteristics of the fungus and its lifestyle. Several gene families related to host–pathogen interactions are strongly represented, including those responsible for nutrient absorption, the detoxification of fungicides and plant cell wall degradation, the latter confirming that much of the genome is devoted to the pathogenic activity of the fungus. We did not find a MAT gene, which is consistent with the classification of P. lycopersici as an imperfect fungus, but we observed a significant expansion of the gene families associated with heterokaryon incompatibility (HI).ConclusionsThe P. lycopersici draft genome sequence provided insight into the molecular and genetic basis of the fungal lifestyle, characterizing previously unknown pathogenic behaviors and defining strategies that allow this asexual fungus to increase genetic diversity and to acquire new pathogenic traits.

Microsatellite and mating type primers for the maize and sorghum pathogen, Exserohilum turcicum

Northern corn leaf blight (NCLB) is a destructive foliar disease of maize that results from infection with the fungal pathogen, Exserohilum turcicum. Annual yield losses incurred from NCLB in South Africa may exceed 50 % when environmental conditions optimal for disease development prevail. In order to study the genetic diversity of E. turcicum, 13 microsatellite markers and mating type PCR primers were developed. Thirty-two primer pairs were designed from the E. turcicum genome sequence to flank microsatellite regions. A multiplex PCR assay amplifying both mating type idiomorphs was designed from the MAT1-1 and MAT1-2 gene sequences, and a protocol for multiplex PCR amplification of MAT loci was optimized. Initial screening identified 13 microsatellite regions that were polymorphic in 9 isolates of E. turcicum. To test the efficacy of the markers, 26 isolates of E. turcicum from 6 South African provinces, including 2 isolates from sorghum, were genotyped. A total of 90 alleles across 13 loci were obtained and the gene diversity ranged from 0.074 to 0.929. Cross-species amplification with E. rostratum was obtained for one SSR marker (SSR27). The MAT markers were specific to E. turcicum and could be used to differentiate isolates of E. turcicum and E. rostratum. The markers developed in this study will be useful to elucidate the population genetic structure, genetic diversity and mode of reproduction of E. turcicum on maize and sorghum.