Tritici Isolates Research Articles

Virulence variation and pathotypes of Zymoseptoria tritici isolates causing wheat leaf blotch in Oromia, Ethiopia

Leaf blotch, caused by Zymoseptoria tritici, is a fungal disease that poses a severe threat to wheat production worldwide. Knowledge of virulence variability is crucial in choosing effective control measures. However, there have only been a few studies of the pathogenic variability and pathotypes within Ethiopian isolates. Hence, the objective of this study was to assess the virulence spectrum and variability of Z. tritici isolates. Forty-three isolates were tested for their virulence and pathotype against 7 wheat differential lines that have different resistance genes. A pathogenicity assay detected 41 differential line-specific virulent isolates among 301 interactions between a host and pathogen based on the percentage coverage of the leaf area by pycnidia. Some isolates were virulent against 50 %–60 % of the resistant genes, but most of them were virulent against some differential lines. Isolates such as EtA-11, EtSh-1, EtSh-2, EtSh-4, and EtA-19 expressed broad-spectrum virulence, highlighting that such isolates are useful for germplasm screening. The isolates were classified into 25 pathotypes, defined by their differential virulence responses. They were also assigned to two clusters according to their mean pycnidia percent. Pathotypes and principal component analysis detected 58.1 % and 62.2 % pathogenic diversity in Ethiopian isolates, respectively. The current findings provide information that breeders can use to identify and select more resistant varieties for farmers.

Stomatal penetration: the cornerstone of plant resistance to the fungal pathogen Zymoseptoria tritici

BackgroundSeptoria tritici blotch (STB), caused by the foliar fungus Zymoseptoria tritici, is one of the most damaging disease of wheat in Europe. Genetic resistance against this fungus relies on different types of resistance from non-host resistance (NHR) and host species specific resistance (HSSR) to host resistance mediated by quantitative trait loci (QTLs) or major resistance genes (Stb). Characterizing the diversity of theses resistances is of great importance for breeding wheat cultivars with efficient and durable resistance. While the functional mechanisms underlying these resistance types are not well understood, increasing piece of evidence suggest that fungus stomatal penetration and early establishment in the apoplast are both crucial for the outcome of some interactions between Z. tritici and plants. To validate and extend these previous observations, we conducted quantitative comparative phenotypical and cytological analyses of the infection process corresponding to 22 different interactions between plant species and Z. tritici isolates. These interactions included four major bread wheat Stb genes, four bread wheat accessions with contrasting quantitative resistance, two species resistant to Z. tritici isolates from bread wheat (HSSR) and four plant species resistant to all Z. tritici isolates (NHR).ResultsInfiltration of Z. tritici spores into plant leaves allowed the partial bypass of all bread wheat resistances and durum wheat resistance, but not resistances from other plants species. Quantitative comparative cytological analysis showed that in the non-grass plant Nicotiana benthamiana, Z. tritici was stopped before stomatal penetration. By contrast, in all resistant grass plants, Z. tritici was stopped, at least partly, during stomatal penetration. The intensity of this early plant control process varied depending on resistance types, quantitative resistances being the least effective. These analyses also demonstrated that Stb-mediated resistances, HSSR and NHR, but not quantitative resistances, relied on the strong growth inhibition of the few Z. tritici penetrating hyphae at their entry point in the sub-stomatal cavity.ConclusionsIn addition to furnishing a robust quantitative cytological assessment system, our study uncovered three stopping patterns of Z. tritici by plant resistances. Stomatal resistance was found important for most resistances to Z. tritici, independently of its type (Stb, HSSR, NHR). These results provided a basis for the functional analysis of wheat resistance to Z. tritici and its improvement.

Haplotype-based association mapping of genomic regions associated with Zymoseptoria tritici resistance using 217 diverse wheat genotypes

BackgroundSeptoria tritici blotch (STB) is considered to be one of the most destructive foliar wheat diseases and is caused by Zymoseptoria tritici. The yield losses are severe and in Northwestern Europe can reach up to 50%. The efficacy of fungicides is diminishing due to changes in the genetic structure of the pathogen. Therefore, resistance breeding is the most effective strategy of disease management. Recently, genome-wide association studies (GWAS) have become more popular due to their robustness in dissecting complex traits, including STB resistance in wheat. This was made possible by the use of large mapping populations and new sequencing technologies. High-resolution mapping benefits from historical recombination and greater allele numbers in GWAS.ResultsIn our study, 217 wheat genotypes of diverse origin were phenotyped against five Z. tritici isolates (IPO323, IPO88004, IPO92004, IPO86036 and St1-03) and genotyped on the DArTseq platform. In polytunnel tests two disease parameters were evaluated: the percentage of leaf area covered by necrotic lesions (NEC) and the percentage of leaf area covered by lesions bearing pycnidia (PYC). The disease escape parameters heading date (Hd) and plant height (Ht) were also measured. Pearson’s correlation showed a positive effect between disease parameters, providing additional information. The Structure analysis indicated four subpopulations which included from 28 (subpopulation 2) to 79 genotypes (subpopulation 3). All of the subpopulations showed a relatively high degree of admixture, which ranged from 60% of genotypes with less than 80% of proportions of the genome attributed to assigned subpopulation for group 2 to 85% for group 4. Haplotype-based GWAS analysis allowed us to identify 27 haploblocks (HBs) significantly associated with analysed traits with a p-value above the genome-wide significance threshold (5%, which was –log10(p) > 3.64) and spread across the wheat genome. The explained phenotypic variation of identified significant HBs ranged from 0.2% to 21.5%. The results of the analysis showed that four haplotypes (HTs) associated with disease parameters cause a reduction in the level of leaf coverage by necrosis and pycnidia, namely: Chr3A_HB98_HT2, Chr5B_HB47_HT1, Chr7B_HB36_HT1 and Chr5D_HB10_HT3.ConclusionsGWAS analysis enabled us to identify four significant chromosomal regions associated with a reduction in STB disease parameters. The list of valuable HBs and wheat varieties possessing them provides promising material for further molecular analysis of resistance loci and development of breeding programmes.

Genetic mapping of loci affecting seedling and adult-plant resistance to powdery mildew derived from two CIMMYT wheat lines

Main conclusionPM3 and PM8 alleles carried by two CIMMYT wheat lines confer powdery mildew resistance in seedlings and/or adult plants. A stage-specific epistatic interaction was observed between PM3 and PM8.Powdery mildew is an important foliar disease of wheat. Major genes for resistance, which have been widely used in wheat breeding programs, are typically effective against only limited numbers of virulence genes of the pathogen. The main aim of this study was to map resistance loci in wheat lines 7HRWSN58 and ZWW09-149 from the International Maize and Wheat Improvement Center (CIMMYT). Doubled haploid populations (Magenta/7HRWSN58 and Emu Rock/ZWW09-149) were developed and grown in controlled environment experiments and inoculated with a composite of Blumeria graminis f.sp. tritici isolates that had been collected at various locations in Western Australia. Plants were assessed for powdery mildew symptoms (percentage leaf area diseased) on seedlings and adult plants. Populations were subjected to genotyping-by-sequencing and assayed for known SNPs in the resistance gene PM3. Linkage maps were constructed, and markers were anchored to the wheat reference genome sequence. In both populations, there were asymptomatic lines that exhibited no symptoms. Among symptomatic lines, disease severity varied widely. In the Magenta/7HRWSN58 population, most of the observed variation was attributed to the PM3 region of chromosome 1A, with the allele from 7HRWSN58 conferring resistance in seedlings and adult plants. In the Emu Rock/ZWW09-149 population, two interacting quantitative trait loci were mapped: one at PM3 and the other on chromosome 1B. The Emu Rock/ZWW09-149 population was confirmed to segregate for a 1BL·1RS translocation that carries the PM8 powdery mildew resistance gene from rye. Consistent with previous reports that PM8-derived resistance can be suppressed by PM3 alleles, the observed interaction between the quantitative trait loci on chromosomes 1A and 1B indicated that the PM3 allele carried by ZWW09-149 suppresses PM8-derived resistance from ZWW09-149, but only at the seedling stage. In adult plants, the PM8 region conferred resistance regardless of the PM3 genotype. The resistance sources and molecular markers that were investigated here could be useful in wheat breeding.

Identification of Wheat Septoria tritici Resistance Genes in Wheat Germplasm Using Molecular Markers.

Zymoseptoria tritici (Z. tritici) is the main threat to global food security; it is a fungal disease that presents one of the most serious threats to wheat crops, causing severe yield losses worldwide, including in Kazakhstan. The pathogen leads to crop losses reaching from 15 to 50%. The objectives of this study were to (1) evaluate a wheat collection for Z. tritici resistance during the adult plant and seedling growth stages, (2) identify the sources of resistance genes that provide resistance to Z. tritici using molecular markers linked to Stb genes, and (3) identify potentially useful resistant wheat genotypes among cultivars and advanced breeding lines. This study evaluated 60 winter and spring wheat genotypes for Z. tritici resistance. According to the field reactions, 22 entries (35.7%) showed ≤10% disease severity in both years. The resistant reaction to a mix of Z. tritici isolates in the seedling stage was associated with adult plant resistance to disease in four wheat entries. The resistance of Rosinka 3 was due to the presence of Stb8; Omskaya 18 showed an immune reaction in the field and a moderately susceptible reaction in the seedling stage, possibly provided by a combination of the Stb7 and Stb2 genes. The high resistance in both the adult and seedling stages of Omskaya 29 and KR11-03 was due to the Stb4 and Stb2 genes and, possibly, due to the presence of unknown genes. A linked marker analysis revealed the presence of several Stb genes. The proportion of wheat entries with Stb genes was quite high at twenty-seven of the genotypes tested (45.0%), including four from Kazakhstan, nine from Russia, nine from the CIMMYT-ICARDA-IWWIP program, and five from the CIMMYT-SEPTMON nursery. Among the sixty entries, ten (16.7%) carried the resistance genes Stb2 and Stb8, and the gene Stb4 was found in seven cultivars (11.6%). Marker-assisted selection can be efficiently applied to develop wheat cultivars with effective Stb gene combinations that would directly assist in developing durable resistance in Kazakhstan. Resistant genotypes could also be used as improved parents in crossing programs to develop new wheat cultivars.

Sensitivity of Lithuanian Zymoseptoria tritici to Quinone Outside Inhibitor and Succinate Dehydrogenase Inhibitor Fungicides

Septoria tritici blotch (STB) ais one of the most damaging winter wheat diseases worldwide, presenting a significant threat to its yields. The causal STB agent, Zymoseptoria tritici, also presents a challenge to control due to its rapid adaptation to fungicides. This requires researchers to continuously monitor the pathogen and investigate and explore strategies to manage the spread of the disease and the development of resistance in the pathogen. Therefore, this study presents the current situation and describes changes in the sensitivity of Z. tritici isolates from Lithuania to quinone outside inhibitors (QoIs) and succinate dehydrogenase inhibitors (SDHIs) for the years 2019–2022. The isolates were tested at five different concentrations of two QoI fungicides (azoxystrobin and pyraclostrobin) and three SDHI fungicides (fluxapyroxad, benzovindiflupyr, and bixafen). During the test period, the EC50 values of the tested QoIs increased, while no clear changes were observed in the SDHIs. The most pronounced shift was observed for the active QoI substance pyraclostrobin. The distribution of the EC50 values of the SDHI fungicides showcased one isolate with an outstandingly high EC50 value of 2.6 mg L−1. The results of this study did not reveal any strong patterns of cross-resistance between the fungicides tested. However, a significant positive, moderate correlation (r = 0.55) was found between fluxapyroxad and benzovindiflupyr. Overall, the results of this study contribute to the understanding of the fungicide-resistance situation of Z. tritici in Lithuania and may complement management strategies for the pathogen and its fungicide resistance.

Use of chitin:DNA ratio to assess growth form in fungal cells

BackgroundDimorphism, the ability to switch between a ‘yeast-like’ and a hyphal growth form, is an important feature of certain fungi, including important plant and human pathogens. The switch to hyphal growth is often associated with virulence, pathogenicity, biofilm formation and stress resistance. Thus, the ability to accurately and efficiently measure fungal growth form is key to research into these fungi, especially for discovery of potential drug targets. To date, fungal growth form has been assessed microscopically, a process that is both labour intensive and costly.ResultsHere, we unite quantification of the chitin in fungal cell walls and the DNA in nuclei to produce a methodology that allows fungal cell shape to be estimated by calculation of the ratio between cell wall quantity and number of nuclei present in a sample of fungus or infected host tissue. Using the wheat pathogen Zymoseptoria tritici as a test case, with confirmation in the distantly related Fusarium oxysporum, we demonstrate a close, linear relationship between the chitin:DNA ratio and the average polarity index (length/width) of fungal cells. We show the utility of the method for estimating growth form in infected wheat leaves, differentiating between the timing of germination in two different Z. tritici isolates using this ratio. We also show that the method is robust to the occurrence of thick-walled chlamydospores, which show a chitin:DNA ratio that is distinct from either ‘yeast-like’ blastospores or hyphae.ConclusionsThe chitin:DNA ratio provides a simple methodology for determining fungal growth form in bulk tissue samples, reducing the need for labour-intensive microscopic studies requiring specific staining or GFP-tags to visualise the fungus within host tissues. It is applicable to a range of dimorphic fungi under various experimental conditions.

STUDY OF PATHOGENICITY OF GAEUMANNOMYCES TRITICI - THE TAKE-ALL FUNGUS

Isolates of Gaeumannomyces spp. obtained from diseased roots of winter wheat showing take-all symptoms were characterized by pathogenicity. All isolates were more pathogenic on wheat and barley than on oat, and were identified as G. tritici. Most isolates of G. tritici were characterized as middle pathogenic, the pathogenicity of one isolate was higher than those of others, and two isolates showed the lowest pathogenicity. 30 isolates of dark-colored fungi were isolated from the affected plants of winter wheat, which according to cultural characteristics were previously assigned to the genus Gaeumannomyces. To establish the species affiliation of the selected Gaeumannomyces isolates, we investigated their pathogenicity and specialization on three grain crops: wheat, barley, and oats. All isolates affected the roots of all three crops, but they were more pathogenic on wheat and barley. Based on this feature, they were previously assigned to the subspecies G. graminis var. tritici (now the species G. tritici). The pathogenic properties of the isolates were studied on seedlings of Yelyk wheat plants. All G. tritici isolates analyzed by us showed a high level of pathogenicity. The G. tritici 9/1 isolate was characterized by significantly higher pathogenicity compared to other isolates according to the "disease development" indicator. Most of the other isolates showed medium pathogenicity (3/20, 7/20, 2/16/21, 3/16/21, 16/22), and only two isolates (1/20, 4/16/21) were low pathogenic. In our research, in addition to the "disease development" indicator in points (0-4) to assess the pathogenicity of isolates, we used indicators that characterize the growth and development of plants, namely "weight of 1 seedling" and "mass of roots from 1 seedling". Taking into account the data on these indicators, isolate 9/22 was characterized by significantly higher pathogenicity compared to other isolates: when affected by it, the mass of wheat seedlings and roots was significantly lower than in other variants. Isolates 3/20 and 7/20 were characterized by medium pathogenicity, when infected with them, a significant decrease in the mass of the roots of seedlings was noted. Isolates 16/22 and 3/16/21 showed a low level of pathogenicity - the decrease in root mass was insignificant.

Identification of a Novel Pm65 Allele Conferring a Wide Spectrum of Resistance to Powdery Mildew in Wheat Accession PI 351817.

Powdery mildew is caused by the highly adaptive biotrophic fungus Blumeria graminis f. sp. tritici infecting wheat worldwide. Novel powdery mildew resistance genes are urgently needed that can be used rapidly in wheat cultivar development with minimal disruption of trait advances elsewhere. PI 351817 is a German cultivar exhibiting a wide spectrum of resistance to B. graminis f. sp. tritici isolates collected from different wheat-growing regions of the United States. Evaluation of an F2 population and 237 F2:3 lines derived from OK1059060-2C14 × PI 351817 for responses to B. graminis f. sp. tritici isolate OKS(14)-B-3-1 identified a single dominant gene, designated Pm351817, for powdery mildew resistance in PI 351817. Using bulked segregant analysis (BSA) and simple sequence repeat (SSR) markers, Pm351817 was mapped in the terminal region of the long arm of chromosome 2A. Deep sequencing of the genotyping-by-sequencing libraries of the two parental lines identified a set of single-nucleotide polymorphism (SNP) markers in the 2AL candidate gene region. Those SNP markers was subsequently converted to Kompetitive allele-specific PCR (KASP) markers for genotyping the mapping population. Linkage analysis delimited Pm351817 to a 634-kb interval between Stars-KASP656 (771,207,512 bp) and Stars-KASP662 (771,841,609 bp) on 2AL, based on the Chinese Spring reference sequence IWGSC RefSeq v 2.1. Tests of allelism indicated that Pm351817 is located at the Pm65 locus. Pm351817 shows resistance to all B. graminis f. sp. tritici isolates used in this study and can be used to enhance powdery mildew resistance in the United States. KASP markers flanking Pm351817 can be used to select Pm351817 in wheat breeding programs after further tests for polymorphism.

QTL mapping reveals both all-stage and adult-plant resistance to powdery mildew in Chinese elite wheat cultivars.

Powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is a threat to wheat production in China. Mapping quantitative trait loci (QTL) for resistance to powdery mildew and developing breeder-friendly markers are important initial steps in breeding resistant cultivars. An all-stage resistance gene and several QTL were identified using a population of 254 recombinant inbred lines (RILs) developed from a Jingdong 8/Aikang 58 cross. The population was evaluated for powdery mildew resistance across six field environments over three consecutive growing seasons utilizing two different mixtures of Bgt isolates, named #Bgt-HB and #Bgt-BJ. Using genotypic data obtained from the Wheat TraitBreed 50K SNP array, seven stable QTL were identified on chromosome arms 1DL, 2AL, 2DS, 4DL, 5AL, 6BL.1 and 6BL.2. The QTL on 2AL conferred all-stage resistance to Bgt race E20 in greenhouse tests and explained up to 52% of the phenotypic variance in field trials but was resistant only against #Bgt-HB. The gene involved in this QTL was predicted to be Pm4a based on genome location and gene sequence. QPmja.caas-1DL, QPmja.caas-4DL and QPmja.caas-6BL.1 were identified as potentially new QTL for powdery mildew resistance. QPmja.caas-2DS and QPmja.caas-6BL.1 were effective against both Bgt mixtures indicating their probable broad-spectrum resistance. A Kompetitive Allele-Specific PCR (KASP) marker closely linked to QPmja.caas-2DS was developed and validated in a panel of 286 wheat cultivars. Since both Jingdong 8 and Aikang 58 have been leading cultivars and breeding parents, the QTL and marker reported represent valuable resources for wheat researchers and breeders.

Resistance to Powdery Mildew Is Conferred by Different Genetic Loci at the Adult-Plant and Seedling Stages in Winter Wheat Line Tianmin 668.

Winter wheat line Tianmin 668 was crossed with susceptible cultivar Jingshuang 16 to develop 216 recombinant inbred lines (RILs) for dissecting its adult-plant resistance (APR) and all-stage resistance (ASR) against powdery mildew. The RIL population was genotyped on a 16K genotyping by target sequencing single-nucleotide polymorphism array and phenotyped in six field trials and in the greenhouse. Three loci-QPmtj.caas-2BL, QPmtj.caas-2AS, and QPmtj.caas-5AL-conferring APR to powdery mildew were detected on chromosomes 2BL, 2AS, and 5AL, respectively, of Tianmin 668. The effect of resistance to powdery mildew for QPmtj.caas-2BL was greater than that of the other two loci. A Kompetitive allele-specific PCR marker specific for QPmtj.caas-2BL was developed and verified on 402 wheat cultivars or breeding lines. Results of virulence and avirulence patterns to 17 Blumeria graminis f. sp. tritici isolates, bulked segregant analysis-RNA-sequencing, and a genetic linkage mapping identified a resistance allele at locus Pm4 in Tianmin 668 based on the seedling phenotypes of the RIL population. The PCR-based DNA sequence alignment and cosegregation of the functional marker with the phenotypes of the RIL population demonstrated that Pm4d was responsible for the ASR to isolate Bgt1 in Tianmin 668. The dissection of genetic loci for APR and ASR may facilitate the application of Tianmin 668 in developing powdery mildew-resistant wheat cultivars.

Epiphytic proliferation of Zymoseptoria tritici isolates on resistant wheat leaves

The wheat pathogen Zymoseptoria tritici is capable of a long period of pre-invasive epiphytic growth. Studies have shown that virulent isolates vary in the extent, duration and growth form of this epiphytic growth, and the fungus has been observed to undergo behaviours such as asexual reproduction by budding and vegetative fusion of hyphae on the leaf surface. This epiphytic colonisation has been investigated very little during interactions in which an isolate of Z. tritici is unable to colonise the apoplast, as occurs during avirulence. However, avirulent isolates have been seen to undergo sexual crosses in the absense of leaf penetration, and it is widely accepted that the main point of distinction between virulent and avirulent isolates occurs at the point of attempted leaf penetration or attempted apoplastic growth, which fails in the avirulent case. In this work, we describe extensive epiphytic growth in three isolates which are unable or have very limited ability to invade the leaf, and show that growth form is as variable as for fully virulent isolates. We demonstrate that during certain interactions, Z. tritici isolates rarely invade the leaf and form pycnidia, but induce necrosis. These isolates are able to achieve higher epiphytic biomass than fully virulent isolates during asymptomatic growth, and may undergo very extensive asexual reproduction on the leaf surface. These findings have implications for open questions such as whether and how Z. tritici obtains nutrients on the leaf surface and the nature of its interaction with wheat defences.

Mixed infections of Zymoseptoria tritici lead to reduced Septoria tritici blotch disease symptoms

AbstractZymoseptoria tritici is the causal fungus of Septoria tritici blotch (STB), a globally important foliar disease of wheat. Infections by more than one strain of a pathogen, or co‐infections, are known to be the most prevalent form of infection in the field. The impact that co‐infection of Z. tritici has on disease severity for the plant host and its effect on plant defence are not fully understood. Here, isolates of Z. tritici were extracted from STB‐presenting leaves from UK sites. Disease assays comparing single‐isolate inocula to mixed inocula were carried out on three cultivars of wheat, Flame, Stigg and Longbow. Leaves infected with mixed inocula were found to show low disease symptoms, including lesion and pycnidial coverage, compared with those infected with single isolates. Growing mixed isolates together in liquid media did not reveal evidence of competition or decreased growth compared with single‐isolate inocula. Wheat infected with a mixture of at least two Z. tritici isolates (of differing aggressiveness) could induce Pr‐1 (Pathogenesis‐related 1) at a significantly higher level in distal uninfected leaves compared with distal leaves of plants infected with a single aggressive isolate. Expression of Pr‐1 in plants with mixed inocula was intermediate to plants infected with an aggressive isolate (lower expression) and plants infected with a less aggressive isolate (higher expression). This suggests that wheat undergoes systemic “sensitization” by the least aggressive Z. tritici isolate during co‐infection.

Effector-mediated partial and nonhost disease resistance in wheat.

Effector-mediated partial and nonhost disease resistance in wheat.

Genetic characteristics and linkage of virulence genes of the Puccinia striiformis f. sp. tritici TSA-6 isolate to Yr5 host resistance.

To understand inheritance of the TSA-6 Puccinia striiformis f. sp. tritici isolate, virulent to Yr5, that was recently detected in China, we analyzed avirulence and virulence of 120 selfed progeny lines from Berberis shensiana. The results showed that the TSA-6 isolate is virulent against the Yr5 resistance gene, and overall progeny lines were categorized into 73 virulence phenotypes (VPs), and of these, 72 VPs differed from the isolate TSA-6, and only 1 VP including 3 progeny was identical to the parental isolate. The analyses indicated that the TSA-6 isolate is homozygous for avirulence at Yr10, Yr15 and Yr26 resistance loci, and virulence at YrA resistance locus. The TSA-6 isolate is heterozygous for avirulence at the Yr2, Yr3, Yr5, Yr7 and Yr8 resistance loci, which are controlled by a dominant/recessive relationship. The Yr1, Yr6, Yr9, Yr17, Yr27, Yr25, Yr28, Yr29, Yr32, YrTr1 and YrSP resistance loci are governed by two complementary dominant/recessive genes. Avirulence against heterozygous Yr4, Yr43，Yr44, Yr76 and YrExp2 resistance loci is regulated by a dominant and recessive, or a dominant and suppressor gene pairs. Totally, 117 multi-locus genotypes (MLGs) were detected at 24 KASP-SNP marker loci among the 120 progeny. Using these marker loci, we constructed a linkage map with a genetic distance interval spanning 624.5 cM. Quantitative trait loci (QTL) corresponding to phenotypic segregation for virulence at 20 Yr resistance loci in addition to the Yr1 resistance locus were identified. These results facilitate our understanding of Pst virulence evolution, and simplify breeding of wheat cultivars with effective resistance to wheat stripe rust.

SSR variability of stem rust pathogen on spring bread wheat in Russia.

Wheat stem rust caused by Puccinia graminis f. sp. tritici Erikss. & E. Henning (Pgt), which used to be a harmful disease of winter wheats in the southern part of Russia, has been largely affecting the yield of spring bread wheat in the territories of temperate climate zone since 2009. A total of 222 Pgt isolates were obtained from samples of susceptible cultivars of spring bread wheat in Central and Volga regions, and Omsk and Novosibirsk provinces in 2019. Genotyping of the isolates was carried out at 16 SSR loci. Number of alleles, proportion of heterozygotes and deviation from Hardy-Weinberg equilibrium were determined at each SSR locus. Based on genetic variability of SSR genotypes, it was shown that the Pgt population is subdivided into two large clusters in the territory of Russian temperate climate zone: "European" population (the Central region) and "Asian" one (the Volga region and two main wheat provinces of Western Siberia). Both the Pgt populations are characterized by a mixed mode of reproduction (sexual and clonal), but different sources of inoculum seem to shape a genotype structure within them. A group of Pgt genotypes with high variability, the inbreeding coefficient closed to zero and low observed heterozygosity was revealed among samples from Omsk. Moreover, two singular SSR genotypes identified among the Asian samples of Pgt isolates should attract a special attention in monitoring of stem rust in order to disclose unexpected rapid changes of the pathogen in the corresponding regions and to prevent the disease outbreak.

Measuring resistance in durum wheat-Zymoseptoria tritici interaction using aggressiveness quantitative traits

Phenotyping for quantitative resistance to Septoria tritici blotch (STB), one of the most economically devastating diseases of durum wheat (Triticum durum Desf.) in North African region, is a limiting factor for breeding progress. Here we tested main phenotypic markers of aggressiveness affecting host damage and pathogen reproduction reported in earlier studies for measuring differences in quantitative host resistance. A collection of 11 Tunisian durum wheat genotypes varying in resistance to the pathogen was inoculated by a highly aggressive and virulent isolate of Zymoseptoria tritici in a replicated field and controlled experiments, conducted in 2017-2018 at the experimental station of the Regional Field Crops Research Center of Beja in Tunisia. The severity of infection caused by Z. tritici isolate, including percentage of leaf area covered by chlorotic lesions (PLACCL), percentage of leaf area covered by necrosis (PLACN), and percentage of leaf area covered by pycnidia (PLACP) as well as latent period (LP) and pycnidia density (PYCdens) were assessed visually and quantified. The results showed that there were significant differences in all assessed traits on the tested genotypes and significant positive correlations were obtained between PLACN, PLACP and PYCdens. Similarly, there was a significant negative correlation between LP and these last traits. The PLACP was found as the more reliable trait to discriminate genotypes to different classes of resistance, consistent with the results obtained for the remaining traits. These data suggest that using both necrotic leaf area (PLACN) bearing pycnidia and PYCdens can improve selection for quantitative STB resistance.

A large bioassay identifies Stb resistance genes that provide broad resistance against Septoria tritici blotch disease in the UK.

Septoria tritici blotch (STB) is one of the most damaging fungal diseases of wheat in Europe, largely due to the paucity of effective resistance genes against it in breeding materials. Currently dominant protection methods against this disease, e.g. fungicides and the disease resistance genes already deployed, are losing their effectiveness. Therefore, it is vital that other available disease resistance sources are identified, understood and deployed in a manner that maximises their effectiveness and durability. In this study, we assessed wheat genotypes containing nineteen known major STB resistance genes (Stb1 through to Stb19) or combinations thereof against a broad panel of 93 UK Zymoseptoria tritici isolates. Seedlings were inoculated using a cotton swab and monitored for four weeks. Four infection-related phenotypic traits were visually assessed. These were the days post infection to the development of first symptoms and pycnidia, percentage coverage of the infected leaf area with chlorosis/necrosis and percentage coverage of the infected leaf area with pycnidia. The different Stb genes were found to vary greatly in the levels of protection they provided, with pycnidia coverage at four weeks differing significantly from susceptible controls for every tested genotype. Stb10, Stb11, Stb12, Stb16q, Stb17, and Stb19 were identified as contributing broad spectrum disease resistance, and synthetic hexaploid wheat lines were identified as particularly promising sources of broadly effective STB resistances. No single Z. tritici isolate was found to be virulent against all tested resistance genes. Wheat genotypes carrying multiple Stb genes were found to provide higher levels of resistance than expected given their historical levels of use. Furthermore, it was noted that disease resistance controlled by different Stb genes was associated with different levels of chlorosis, with high levels of early chlorosis in some genotypes correlated with high resistance to fungal pycnidia development, potentially suggesting the presence of multiple resistance mechanisms.The knowledge obtained here will aid UK breeders in prioritising Stb genes for future breeding programmes, in which optimal combinations of resistance genes could be pyramided. In addition, this study identified the most interesting Stb genes for cloning and detailed functional analysis.

Shifting sensitivity of septoria tritici blotch compromises field performance and yield of main fungicides in Europe.

Septoria tritici blotch (STB; Zymoseptoria tritici) is a severe leaf disease on wheat in Northern Europe. Fungicide resistance in the populations of Z. tritici is increasingly challenging future control options. Twenty-five field trials were carried out in nine countries across Europe from 2019 to 2021 to investigate the efficacy of specific DMI and SDHI fungicides against STB. During the test period, two single DMIs (prothioconazole and mefentrifluconazole) and four different SDHIs (fluxapyroxad, bixafen, benzovindiflupyr and fluopyram) along with different co-formulations of DMIs and SDHIs applied at flag leaf emergence were tested. Across all countries, significant differences in azole performances against STB were seen; prothioconazole was outperformed in all countries by mefentrifluconazole. The effects also varied substantially between the SDHIs, with fluxapyroxad providing the best efficacy overall, while the performance of fluopyram was inferior to other SDHIs. In Ireland and the UK, the efficacy of SDHIs was significantly lower compared with results from continental Europe. This reduction in performances from both DMIs and SDHIs was reflected in yield responses and also linked to decreased sensitivity of Z. tritici isolates measured as EC50 values. A clear and significant gradient in EC50 values was seen across Europe. The lower sensitivity to SDHIs in Ireland and the UK was coincident with the prevalence of SDH-C-alterations T79N, N86S, and sporadically of H152R. The isolates' sensitivity to SDHIs showed a clear cross-resistance between fluxapyroxad, bixafen, benzovindiflupyr and fluopyram, although the links with the latter were less apparent. Co-formulations of DMIs + SDHIs performed well in all trials conducted in 2021. Only minor differences were seen between fluxapyroxad + mefentrifluconazole and bixafen + fluopyram + prothioconazole; the combination of benzovindiflupyr + prothioconazole gave an inferior performance at some sites. Fenpicoxamid performed in line with the most effective co-formulations. This investigation shows a clear link between reduced field efficacy by solo SDHIs as a result of increasing problems with sensitivity shifting and the selection of several SDH-C mutations. The presented data stress the need to practice anti-resistance strategies to delay further erosion of fungicide efficacy.

Key infection stages defending heat stress in high-temperature-resistant Blumeria graminis f. sp. tritici isolates.

With the increase of temperature in the winter wheat-growing regions in China, the high-temperature-resistant Blumeria graminis f. sp. tritici (Bgt) isolates developed in the fields. To clarify the key infection stages and the roles of heat shock protein (HSP) genes of high-temperature-resistant Bgt isolates defending high temperature, 3 high-temperature-resistant and 3 sensitive Bgt isolates were selected from 55 isolates after determination of temperature sensitivity. And then they were used to investigate the infection stages and the expression levels of HSP genes, including Bgthsp60, Bgthsp70, Bgthsp90, and Bgthsp104, at 18°C and 25°C. The formation frequency of abnormal appressoria and inhibition rate of haustoria formation of high-temperature-resistant isolates at 25°C were lower than those of high-temperature-sensitive isolates, while major axis of microcolonies of high-temperature-resistant isolates was higher than those of high-temperature-sensitive isolates at 25°C. The results indicated that haustoria formation and hyphal expansion were the key infection stages of defense against heat stress in high-temperature-resistant isolates. Further analyses of HSP genes found the expression levels of Bgthsp60 and Bgthsp70c were upregulated at 24 and 72 h post-inoculation in high-temperature-resistant isolates, while no significant difference was observed for Bgthsp90 and Bgthsp104 genes. Taken together, the basis of high-temperature-resistant Bgt isolates is associated with induced expression of Bgthsp60 and Bgthsp70c response to heat stress in haustoria formation and hyphal expansion stages.