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Fungal plant pathogens, such as Zymoseptoria tritici (formerly known as Mycosphaerella graminicola), secrete repertoires of effectors to facilitate infection or trigger host defence mechanisms. The discovery and functional characterization of effectors provides valuable knowledge that can contribute to the design of new and effective disease management strategies. Here, we combined bioinformatics approaches with expression profiling during pathogenesis to identify candidate effectors of Z. tritici. In addition, a genetic approach was conducted to map quantitative trait loci (QTLs) carrying putative effectors, enabling the validation of both complementary strategies for effector discovery. In planta expression profiling revealed that candidate effectors were up-regulated in successive waves corresponding to consecutive stages of pathogenesis, contrary to candidates identified by QTL mapping that were, overall, expressed at low levels. Functional analyses of two top candidate effectors (SSP15 and SSP18) showed their dispensability for Z. tritici pathogenesis. These analyses reveal that generally adopted criteria, such as protein size, cysteine residues and expression during pathogenesis, may preclude an unbiased effector discovery. Indeed, genetic mapping of genomic regions involved in specificity render alternative effector candidates that do not match the aforementioned criteria, but should nevertheless be considered as promising new leads for effectors that are crucial for the Z. tritici-wheat pathosystem., (© 2015 BSPP AND JOHN WILEY & SONS LTD.)

Oomycete species occupy many different environments and many ecological niches. The genera Phytophthora and Pythium for example, contain many plant pathogens which cause enormous damage to a wide range of plant species. Proper identification to the species level is a critical first step in any investigation of oomycetes, whether it is research driven or compelled by the need for rapid and accurate diagnostics during a pathogen outbreak. The use of DNA for oomycete species identification is well established, but DNA barcoding with cytochrome c oxidase subunit I (COI) is a relatively new approach that has yet to be assessed over a significant sample of oomycete genera. In this study we have sequenced COI, from 1205 isolates representing 23 genera. A comparison to internal transcribed spacer (ITS) sequences from the same isolates showed that COI identification is a practical option; complementary because it uses the mitochondrial genome instead of nuclear DNA. In some cases COI was more discriminative than ITS at the species level. This is in contrast to the large ribosomal subunit, which showed poor species resolution when sequenced from a subset of the isolates used in this study. The results described in this paper indicate that COI sequencing and the dataset generated are a valuable addition to the currently available oomycete taxonomy resources, and that both COI, the default DNA barcode supported by GenBank, and ITS, the de facto barcode accepted by the oomycete and mycology community, are acceptable and complementary DNA barcodes to be used for identification of oomycetes., (© 2011 Blackwell Publishing Ltd and Her Majesty the Queen in Right of Canada [2011]. Reproduced with the permission of the Minister of the Department of Agriculture and Agri-Food Canada.)

The foliar disease septoria tritici blotch, caused by the fungus Mycosphaerella graminicola, is currently the most important wheat disease in Europe. Gene expression was examined under highly different conditions, using 10 expressed sequence tag libraries generated from M. graminicola isolate IPO323 using seven in vitro and three in planta growth conditions. To identify fungal clones in the interaction libraries, we developed a selection method based on hybridization with the entire genomic DNA of M. graminicola, to selectively enrich these libraries for fungal genes. Assembly of the 27,007 expressed sequence tags resulted in 9,190 unigenes, representing 5.2 Mb of the estimated 39-Mb genome size of M. graminicola. All libraries contributed significantly to the number of unigenes, especially the in planta libraries representing different stages of pathogenesis, which covered 15% of the library-specific unigenes. Even under presymptomatic conditions (5 days postinoculation), when fungal biomass is less than 5%, this method enabled us to efficiently capture fungal genes expressed during pathogenesis. Many of these genes were uniquely expressed in planta, indicating that in planta gene expression significantly differed from in vitro expression. Examples of gene discovery included a number of cell wall-degrading enzymes, a broad set of genes involved in signal transduction (n=11) and a range of ATP-binding cassette (n=20) and major facilitator superfamily transporter genes (n=12) potentially involved in protection against antifungal compounds or the secretion of pathogenicity factors. In addition, evidence is provided for a mycovirus in M. graminicola that is highly expressed under various stress conditions, in particular, under nitrogen starvation. Our analyses provide a unique window on in vitro and in planta gene expression of M. graminicola.

We studied the possibility of a teleomorph associated with the genotypically diverse septoria speckled leaf blotch (SSLB) pathogen of barley, Septoria passerinii. A teleomorph in the genus Mycosphaerella had been predicted previously based on phylogenetic analyses. This prediction was tested with experiments in the Netherlands and the United States by co-inoculating isolates with opposite mating types onto susceptible barley cultivars and monitoring leaves for sexual structures and for the discharge of ascospores. Characterization of putative hybrid progeny by both molecular (AFLP, RAPD, mating type, and ITS sequencing) and phenotypic analyses confirmed that a Mycosphaerella teleomorph of S. passerinii has been discovered approximately 125 years after the description of the anamorph. Progeny had recombinant genotypes of the molecular alleles present in the parents, and the identities of representative progeny isolates as S. passerinii were confirmed by ITS sequencing. A previously unknown sexual cycle explains the high degree of genetic variation among isolates found in nature. The experimental identification of a predicted teleomorph for S. passerinii indicates that cryptic sexual cycles may be common for many other "asexual" fungi with high levels of genotypic diversity.

ABSTRACT A new devastating disease in the United States, commonly known as Sudden Oak Death, is caused by Phytophthora ramorum. This pathogen, which previously was described attacking species of Rhododendron and Viburnum in Germany and the Netherlands, has established itself in forests on the central coast of California and is killing scores of native oak trees (Lithocarpus densiflora, Quercus agrifolia, Q. kelloggii, and Q. parvula var. shrevei). The phytosanitary authorities in the European Union consider non-European isolates of P. ramorum as a threat to forest trees in Europe. To date, almost all European isolates are mating type A1 while those from California and Oregon are type A2. The occurrence of both mating types in the same region could lead to a population capable of sexual recombination, which could generate a new source of diversity. To prevent contact between these two populations, a rapid, reliable, and discriminating diagnostic test was developed to easily distinguish the two populations. Based on a DNA sequence difference in the mitochondrial Cytochrome c oxidase subunit 1 (Cox1) gene, we developed a single-nucleotide polymorphism (SNP) protocol to distinguish between isolates of P. ramorum originating in Europe and those originating in the United States. A total of 83 isolates of P. ramorum from Europe and 51 isolates from the United States were screened and all isolates could be consistently and correctly allocated to either the European or the U.S. populations using the SNP protocol.

An F(1) mapping population of the septoria tritici blotch pathogen of wheat, Mycosphaerella graminicola, was generated by crossing the two Dutch field isolates IPO323 and IPO94269. AFLP and RAPD marker data sets were combined to produce a high-density genetic linkage map. The final map contained 223 AFLP and 57 RAPD markers, plus the biological traits mating type and avirulence, in 23 linkage groups spanning 1216 cM. Many AFLPs and some RAPD markers were clustered. When markers were reduced to 1 per cluster, 229 unique positions were mapped, with an average distance of 5.3 cM between markers. Because M. graminicola probably has 17 or 18 chromosomes, at least 5 of the 23 linkage groups probably will need to be combined with others once additional markers are added to the map. This was confirmed by pulsed-field gel analysis; probes derived from 2 of the smallest linkage groups hybridized to two of the largest chromosome-sized bands, revealing a discrepancy between physical and genetic distance. The utility of the map was demonstrated by identifying molecular markers tightly linked to two genes of biological interest, mating type and avirulence. Bulked segregant analysis was used to identify additional molecular markers closely linked to these traits. This is the first genetic linkage map for any species in the genus Mycosphaerella or the family Mycosphaerellaceae.

Both mating-type loci from the wheat septoria leaf blotch pathogen Mycosphaerella graminicola have been cloned and sequenced. The MAT1-2 gene was identified by screening a genomic library from the MAT1-2 isolate IPO94269 with a heterologous probe from Tapesia yallundae. The MAT1-2 idiomorph is 2772 bp and contains a single gene encoding a putative high-mobility-group protein of 394 amino acids. The opposite idiomorph was obtained from isolate IPO323, which has the complementary mating type, by long-range PCR using primers derived from sequences flanking the MAT1-2 idiomorph. The MAT1-1 locus is 2839 bp in size and contains a single open reading frame encoding a putative alpha1-domain protein of 297 amino acids. Within the nonidiomorphic sequences, homology was found with palI, encoding a membrane receptor from Aspergillus nidulans, and a gene encoding a putative component of the anaphase-promoting complex from Schizosaccharomyces pombe and a DNA-(apurinic or apyrimidinic) lyase from S. pombe. For each of the MAT genes specific primers were designed and tested on an F1 mapping population that was generated from a cross between IPO323 and IPO94269. An absolute correlation was found between the amplified allele-specific fragments and the mating type as determined by backcrosses of each F1 progeny isolate to the parental isolates. The primers were also used to screen a collection of field isolates in a multiplex PCR. An equal distribution of MAT1-1 and MAT1-2 alleles was found for most geographic origins examined.

ABSTRACT Specific resistances to isolates of the ascomycete fungus Mycosphaerella graminicola, which causes Septoria tritici blotch of wheat, have been detected in many cultivars. Cvs. Flame and Hereward, which have specific resistance to the isolate IPO323, were crossed with the susceptible cv. Longbow. The results of tests on F1 and F2 progeny indicated that a single semidominant gene controls resistance to IPO323 in each of the resistant cultivars. This was confirmed in F3 families of Flame x Longbow, which were either homozygous resistant, homozygous susceptible, or segregating in tests with IPO323 but were uniformly susceptible to another isolate, IPO94269. None of 100 F2 progeny of Flame x Hereward were susceptible to IPO323, indicating that the resistance genes in these two cultivars are the same, closely linked, or allelic. The resistance gene in cv. Flame was mapped to the short arm of chromosome 3A using microsatellite markers and was named Stb6. Fifty-nine progeny of a cross between IPO323 and IPO94269 were used in complementary genetic analysis of the pathogen to test a gene-for-gene relationship between Stb6 and the avirulence gene in IPO323. Avirulence to cvs. Flame, Hereward, Shafir, Bezostaya 1, and Vivant and the breeding line NSL92-5719 cosegregated, and the ratio of virulent to avirulent was close to 1:1, suggesting that these wheat lines may all recognize the same avirulence gene and may all have Stb6. Together, these data provide the first demonstration that isolate-specific resistance of wheat to Septoria tritici blotch follows a gene-for-gene relationship.

Segregation of avirulence in Mycosphaerella graminicola, a heterothallic ascomycete that causes wheat septoria tritici leaf blotch, was studied in F1, BC1, and F2 populations by inoculation assays on five wheat cultivars in the seedling stage and by amplified fragment length polymorphism and random amplified polymorphic DNA analyses. F1 was generated by crossing isolates IPO323 (avirulent) and IPO94269 (virulent). All F1, BC1, and F2 progeny isolates were virulent on the susceptible check cultivar Taichung 29 and were avirulent on the resistant check cultivar Kavkav-K4500. Avirulence segregation was observed in F1 and in several BC1 and F2 generations on the differential cultivars Shafir, Kavkaz, and Veranopolis at a 1:1 ratio. Avirulence for the three differential cultivars always cosegregated. We conclude that avirulence in isolate IPO323 is controlled by a single, seemingly complex locus.

Monospore isolates of Mycosphaerella graminicola considered to originate from one ascus were analysed by the polymerase chain reaction (PCR) with 32 RAPD primers. Eighteen of these revealed three classes of polymorphisms, which enabled a RAPD-based tetrad analysis. Four pairs of isolates resulting from a single diploid nucleus were determined. A procedure to cross these isolates was developed to investigate the mating system. Three of six crosses were successful, and the segregation of mating types in accordance with the tetrad analysis strongly points to a bipolar heterothallic mating system in M. graminicola. Random ascospore progenies from the successful crosses, each comprising 54 isolates, were studied with three primers to determine the mode of inheritance of the RAPD markers. Mendelian segregation and recombination of RAPD markers was observed in all progenies.