Identification of New Sources of Resistance to Septoria Tritici Blotch Caused by Zymoseptoria tritici

Twenty‐nine synthetic hexaploid wheats (SHWs) were evaluated for resistance to five isolates of Zymoseptoria tritici, a devastating wheat pathogen worldwide. The five Z. tritici isolates varied in their virulence spectra towards wheat genotypes, indicating that they have distinct set of avirulence genes. New isolate‐specific resistances were identified that could be used in wheat breeding programmes. Comparing with the previous studies, the number of specific resistances identified in this study is considerable. Among 150 interactions, 78 isolate‐specific resistances were identified. Interestingly, 21 wheat genotypes showed specific responses to one or more isolates tested. Of these, 12 genotypes were highly resistant to all isolates, indicating that they possess known or novel effective resistance genes. The Stb15 and Stb16/Stb17 are effective resistance genes towards isolates used in this study, indicating that the conferred resistance in these genotypes is due to the presence of either of these genes in combination or individually. Alternatively, they may carry novel broad‐spectrum resistance gene(s) that their identification is of interest. Our data suggest that the presence of complete resistance to various Z. tritici isolates in SHWs justifies the need for more in‐depth research to characterize the likely novel genes.     

Three LysM effectors of Zymoseptoria tritici collectively disarm chitin-triggered plant immunity

Chitin is a major structural component of fungal cell walls and acts as a microbe-associated molecular pattern (MAMP) that, on recognition by a plant host, triggers the activation of immune responses. To avoid the activation of these responses, the Septoria tritici blotch (STB) pathogen of wheat, Zymoseptoria tritici, secretes LysM effector proteins. Previously, the LysM effectors Mg1LysM and Mg3LysM were shown to protect fungal hyphae against host chitinases. Furthermore, Mg3LysM, but not Mg1LysM, was shown to suppress chitin-induced reactive oxygen species (ROS) production. Whereas initially a third LysM effector gene was disregarded as a presumed pseudogene, we now provide functional data to show that this gene also encodes a LysM effector, named Mgx1LysM, that is functional during wheat colonization. While Mg3LysM confers a major contribution to Z. tritici virulence, Mgx1LysM and Mg1LysM contribute to Z. tritici virulence with smaller effects. All three LysM effectors display partial functional redundancy. We furthermore demonstrate that Mgx1LysM binds chitin, suppresses the chitin-induced ROS burst, and is able to protect fungal hyphae against chitinase hydrolysis. Finally, we demonstrate that Mgx1LysM is able to undergo chitin-induced polymerization. Collectively, our data show that Z. tritici utilizes three LysM effectors to disarm chitin-triggered wheat immunity.     

Evolutionary analyses of the avirulence effector AvrStb6 in global populations of Zymoseptoria tritici identify candidate amino acids involved in recognition

We analysed the population genetic diversity of AvrStb6, the first avirulence gene cloned from the wheat pathogen Zymoseptoria tritici, using 142 Z. tritici strains sampled from four wheat fields growing on three continents. Although AvrStb6 was located in a recombination hotspot, it was found in every strain, with 71 polymorphic sites that produced 41 distinct DNA haplotypes encoding 30 AvrStb6 protein isoforms. An AvrStb6 homologue was found in the closest known relative, Z. pseudotritici, but not in three other closely related Zymoseptoria species, indicating that this gene has emerged in Zymoseptoria quite recently. Two AvrStb6 homologues with nucleotide similarities greater than 70% were identified on chromosome 10 in all Z. tritici isolates, suggesting that AvrStb6 belongs to a multigene family of candidate effectors that has expanded recently through gene duplication. The AvrStb6 sequences exhibited strong evidence for non‐neutral evolution, including a large number of non‐synonymous mutations, with significant positive diversifying selection operating on nine of the 82 codons. It appears that balancing selection is operating across the entire gene in natural field populations. There was also evidence for co‐evolving codons within the gene that may reflect compensatory mutations associated with the evasion of recognition by Stb6. Intragenic recombination also appears to have affected the diversity of AvrStb6.     

Remarkable recent changes in the genetic diversity of the avirulence gene AvrStb6 in global populations of the wheat pathogen Zymoseptoria tritici

Septoria tritici blotch (STB), caused by the fungus Zymoseptoria tritici, is one of the most economically important diseases of wheat. Recently, both factors of a gene-for-gene interaction between Z. tritici and wheat, the wheat receptor-like kinase Stb6 and the Z. tritici secreted effector protein AvrStb6, have been identified. Previous analyses revealed a high diversity of AvrStb6 haplotypes present in earlier Z. tritici isolate collections, with up to c.18% of analysed isolates possessing the avirulence isoform of AvrStb6 identical to that originally identified in the reference isolate IPO323. With Stb6 present in many commercial wheat cultivars globally, we aimed to assess potential changes in AvrStb6 genetic diversity and the incidence of haplotypes allowing evasion of Stb6-mediated resistance in more recent Z. tritici populations. Here we show, using targeted resequencing of AvrStb6, that this gene is universally present in field isolates sampled from major wheat-growing regions of the world in 2013–2017. However, in contrast to the data from previous AvrStb6 population studies, we report a complete absence of the originally described avirulence isoform of AvrStb6 amongst modern Z. tritici isolates. Moreover, a remarkably small number of haplotypes, each encoding AvrStb6 protein isoforms conditioning virulence on Stb6-containing wheat, were found to predominate among modern Z. tritici isolates. A single virulence isoform of AvrStb6 was found to be particularly abundant throughout the global population. These findings indicate that, despite the ability of Z. tritici to sexually reproduce on resistant hosts, AvrStb6 avirulence haplotypes tend to be eliminated in subsequent populations.     

Population genetic diversity of Puccinia striiformis f. sp. tritici on different wheat varieties in Tianshui, Gansu Province

Population genetic diversity in Tianshui city was analyzed with SSR markers in 605 single-pustule isolates of the stripe rust pathogen, Puccinia striiformis f. sp. tritici (Pst), obtained from 19 varieties of wheat. Significant differences in genetic diversity among populations were defected. Genetic diversity was highest in population on Tian 863-13, a highly resistant variety, whereas genetic diversity was lowest in population on Huixianhong, a highly susceptible variety. Seven populations from seven varieties that carried the common Yr18 resistance gene were clustered as one sub-group at 0.88 similarity coefficient, which showed that resistance gene selection had close relation with pathogen??s component. The results of present study can provide a theoretical basis for integrated management of wheat stripe rust and effective deployment of resistance genes in Pst over-summering zones in China.     

Sexual reproduction of Zymoseptoria tritici on durum wheat in Tunisia revealed by presence of airborne inoculum,fruiting bodies and high levels of genetic diversity

Septoria tritici blotch (STB) caused by the heterothallic ascomycete Zymoseptoria tritici is currently one of the most devastating diseases of wheat worldwide. The extent of sexual reproduction of this pathogen is well documented on bread wheat, but not on durum wheat. The objective of the present study was to quantify the occurrence of Z. tritici sexual reproduction on durum wheat in the Tunisian environment. The assessment was undertaken using a triple approach combining fruiting body assessment, ascospore trapping and population genetic analyses. The results highlighted the formation of pseudothecia on leaves and stubble from the autumn until the end of the growing season. Likewise, qPCR monitoring highlighted a constant release of Z. tritici airborne inoculum during the wheat-growing season, with a peak of production at the end of the season. Genetic investigations using microsatellites revealed high levels of gene and genotypic diversities, an equal distribution of mating types, and a lack of genetic clustering within and between growing seasons. Taken together, these findings indicate that Z. tritici undergoes sexual reproduction on durum wheat in Tunisia at least to the same extent than on bread wheat in Western Europe, and that the dry and warm climate does not affect the mating process of the fungus. Frequent occurrence of sexual reproduction is a valuable knowledge to take into account in STB control strategies on durum wheat.     

Tracking costs of virulence in natural populations of the wheat pathogen, Puccinia striiformis f.sp. tritici

Background  

Costs of adaptation play an important role in host-parasite coevolution. For parasites, evolving the ability to circumvent host resistance may trade off with subsequent growth or transmission. Such costs of virulence (sensu plant pathology) limit the spread of all-infectious genotypes and thus facilitate the maintenance of genetic polymorphism in both host and parasite. We investigated costs of three virulence factors in Puccinia striiformis f.sp. tritici, a fungal pathogen of wheat (Triticum aestivum).     

Mixed infections alter transmission potential in a fungal plant pathogen

Infections by more than one strain of a pathogen predominate under natural conditions. Mixed infections can have significant, though often unpredictable, consequences for overall virulence, pathogen transmission and evolution. However, effects of mixed infection on disease development in plants often remain unclear and the critical factors that determine the outcome of mixed infections remain unknown. The fungus Zymoseptoria tritici forms genetically diverse infections in wheat fields. Here, for a range of pathogen traits, we experimentally decompose the infection process to determine how the outcomes and consequences of mixed infections are mechanistically realized. Different strains of Z. tritici grow in close proximity and compete in the wheat apoplast, resulting in reductions in growth of individual strains and in pathogen reproduction. We observed different outcomes of competition at different stages of the infection. Overall, more virulent strains had higher competitive ability during host colonization, and less virulent strains had higher transmission potential. We showed that within-host competition can have a major effect on infection dynamics and pathogen population structure in a pathogen and host genotype-specific manner. Consequently, mixed infections likely have a major effect on the development of septoria tritici blotch epidemics and the evolution of virulence in Z. tritici.     

Evolution of resistance against powdery mildew in winter wheat populations conducted under dynamic management. I – Is specific seedling resistance selected?

Dynamic management has been proposed as a complementary strategy to gene banks for the conservation of genetic resources. The evolution of frequencies of genes for specific resistance towards powdery mildew (caused by Blumeria graminis f. sp. tritici) in populations of a French network for dynamic management of bread wheat genetic resources was investigated after 10 years of multiplication without human selection. The objective was to determine whether specific resistance gene diversity was maintained in the populations and whether any changes could be attributed to selection due to pathogen pressure. Seven populations, originating from four of the network sites, were characterized and compared to the initial population for six specific resistance gene frequencies detected by nine Blumeria graminis f. sp. tritici isolates. Diversity decreased at the population level, but because of a strong differentiation between the populations, this diversity was maintained at the network level. The comparison of Fst parameters estimated on neutral markers (RFLP) and on resistance gene data revealed that in two of the populations specific resistance genes had been selected by pathogen pressure, whereas evolution in two other populations seemed to be the result of genetic drift. For the three last populations, conclusions were less clear, as one had probably experienced a strong bottleneck and the other two presented intermediate Fst values. A dynamic management network with sites contrasted for pathogen pressure, allowing genetic drift in some populations and selection in others, appeared, at least on the short term, to be a good tool for maintaining the diversity of genes for specific resistance to powdery mildew. Received: 15 December 1999 / Accepted: 30 December 1999     

Virulence analysis of wheat powdery mildew population (Erysiphe graminis f.sp.tritici) from the region of Slovakia and Hungary

In the year 1992 a total of 163 isolates of wheat powdery mildew were tested. The samples of mildew isolates were obtained by means of a mobile spore catching apparatus. The populations from 4 regions of Slovakia and 3 regions of Hungary were analyzed. The resistance due toPm5, Pm8 andMl-i genes at the observed locations has already been overcome. The resistance genesPm1, Pm2 and a gene combinationPm2+Pm6 ensure the protection only against a part of the patho-types of powdery mildew population. Virulence corresponding to thePm4b gene has been low so far. The regional patterns of pathogen virulence are in good agreement with the gene resistance spectrum by the cultivars grown regionally. Little differences in virulence among the populations from the regions of Slovakia and Hungary indicate that this part of Eastern Europe should be considered as an epidemiologic unit.     

Diversity of the Fusarium pathogens associated with crown rot in the Huanghuai wheat-growing region of China

To investigate the distribution and diversity of the pathogens associated with Fusarium crown rot in the Huanghuai wheat-growing region (HHWGR) of China, we collected wheat samples with symptomatic stem bases from seven provinces in the HHWGR between 2013 and 2016. A total of 1196 isolates obtained from 222 locations were identified as 9 Fusarium species based on morphological and molecular identification. Of these pathogen species, F. pseudograminearum was the dominant species. Furthermore, F. sinensis was isolated from the disease specimens and tested for virulence to wheat. The result of the pathogenicity revealed that an intraspecific differentiation existed in F. pseudograminearum; sequence analysis of the EF-1α gene showed that 194 F. pseudograminearum isolates were differentiated into two distinct clades which closed to the strains from Australia and China respectively, but neither pathogenicity nor EF-1α sequence was related to the geographic origins of these isolates. However, universal rice primers-polymerase chain reaction showed a correlation with the geographical origins of the 194 isolates, which were divided into eight subclusters, the level of genetic diversity was higher within a geographical population than among the different populations. The results of these analyses can be directly used to facilitate disease monitoring and development of control strategies.     

Virulence Spectrum of Mycosphaerella graminicola Isolates on Wheat Genotypes Carrying Known Resistance Genes to Septoria tritici Blotch

The virulence spectrum of 23 monopycnidiospore isolates of Mycosphaerella graminicola was determined using wheat genotypes that carried different resistance genes (Stb1–Stb8 and Stb15). Disease severity was measured as the percentage of necrotic leaf area. The isolates used in the experiments were of diverse origin: eight from Poland, seven from Germany, and eight from other countries around the world. Analysis of variance revealed significant differences in the virulence of the isolates. Using multiple regression and Cook’s D statistic, 26 significant cultivar × isolate interactions were detected. The Israeli isolate IPO86036 showed the widest spectrum of specific reactions. It expressed specific virulence on at least four cultivars and specific avirulence on at least three. The other isolates showed specific interactions with 1–6 different cultivars. Despite the limited number of isolates that were tested, we recommend that a number of resistant lines, namely cultivars Veranopolis (Stb2), Cs/Synthetic 7D (Stb5), Arina (Stb15, Stb6 and partial resistance), and Liwilla (unknown resistance factors), could be incorporated into central European wheat breeding programmes that are aimed at developing resistance against septoria tritici blotch. In contrast, resistance gene Stb7, which is carried by cultivar Estanzuela Federal, was ineffective against most of the isolates that were used. These results on the virulence spectrum of M. graminicola isolates provide valuable information for effective wheat breeding programmes to develop resistance to the pathogen.     

Molecular characterization and functional analyses of ZtWor1, a transcriptional regulator of the fungal wheat pathogen Zymoseptoria tritici

Zymoseptoria tritici causes the major fungal wheat disease septoria tritici blotch, and is increasingly being used as a model for transmission and population genetics, as well as host–pathogen interactions. Here, we study the biological function of ZtWor1, the orthologue of Wor1 in the fungal human pathogen Candida albicans, as a representative of a superfamily of regulatory proteins involved in dimorphic switching. In Z. tritici, this gene is pivotal for pathogenesis, as ZtWor1 mutants were nonpathogenic and complementation restored the wild‐type phenotypes. In planta expression analyses showed that ZtWor1 is up‐regulated during the initiation of colonization and fructification, and regulates candidate effector genes, including one that was discovered after comparative proteome analysis of the Z. tritici wild‐type strain and the ZtWor1 mutant, which was particularly expressed in planta. Cell fusion and anastomosis occur frequently in ZtWor1 mutants, reminiscent of mutants of MgGpb1, the β‐subunit of the heterotrimeric G protein. Comparative expression of ZtWor1 in knock‐out strains of MgGpb1 and MgTpk2, the catalytic subunit of protein kinase A, suggests that ZtWor1 is downstream of the cyclic adenosine monophosphate (cAMP) pathway that is crucial for pathogenesis in many fungal plant pathogens.