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.     

Evolutionary divergence of the rye <Emphasis Type="Italic">Pm17</Emphasis> and <Emphasis Type="Italic">Pm8</Emphasis> resistance genes reveals ancient diversity

Key message

We have isolated a novel powdery mildew resistance gene in wheat that was originally introgressed from rye. Further analysis revealed evolutionary divergent history of wheat and rye orthologous resistance genes.

Abstract

Wheat production is under constant threat from a number of fungal pathogens, among them is wheat powdery mildew (Blumeria graminis f. sp. tritici). Deployment of resistance genes is the most economical and sustainable method for mildew control. However, domestication and selective breeding have narrowed genetic diversity of modern wheat germplasm, and breeders have relied on wheat relatives for enriching its gene pool through introgression. Translocations where the 1RS chromosome arm was introgressed from rye to wheat have improved yield and resistance against various pathogens. Here, we isolated the Pm17 mildew resistance gene located on the 1RS introgression in wheat cultivar ‘Amigo’ and found that it is an allele or a close paralog of the Pm8 gene isolated earlier from ‘Petkus’ rye. Functional validation using transient and stable transformation confirmed the identity of Pm17. Analysis of Pm17 and Pm8 coding regions revealed an overall identity of 82.9% at the protein level, with the LRR domains being most divergent. Our analysis also showed that the two rye genes are much more diverse compared to the variants encoded by the Pm3 gene in wheat, which is orthologous to Pm17/Pm8 as concluded from highly conserved upstream sequences in all these genes. Thus, the evolutionary history of these orthologous loci differs in the cereal species rye and wheat and demonstrates that orthologous resistance genes can take different routes towards functionally active genes. These findings suggest that the isolation of Pm3/Pm8/Pm17 orthologs from other grass species, additional alleles from the rye germplasm as well as possibly synthetic variants will result in novel resistance genes useful in wheat breeding.

     

Isolation of haustoria ofPuccinia graminis f.sp.tritici from wheat leaves

Haustoria of the wheat stem rust fungus,Puccinia graminis f.sp.tritici, race 32, were isolated from two different infected wheat cultivars. Yield of haustoria from the highly susceptible wheat cv. “Little Club” was 10 times higher than that of the resistant wheat line “Prelude × Eagle” carrying the Sr26 gene for resistance toP. graminis f.sp.tritici. Tests for the integrity of haustoria using the stain methylene blue showed that up to 91% of the haustoria were undamaged.     

Chromosomal location of a gene suppressing powdery mildew resistance genes Pm8 and Pm17 in common wheat (Triticum aestivum L. em. Thell.)

The chromosomal location of a suppressor for the powdery mildew resistance genes Pm8 and Pm17 was determined by a monosomic set of the wheat cultivar Caribo. This cultivar carries a suppressor gene inhibiting the expression of Pm8 in cv Disponent and of Pm17 in line Helami-105. In disease resistance assessments, monosomic F₁ hybrids (2n=41) of Caribo x Disponent and Caribo x Helami-105 lacking chromosome 7D were resistant, whereas monosomic F₁ hybrids involving the other 20 chromosomes, as well as disomic F₁ hybrids (2n=42) of all cross combinations, were susceptible revealing that the suppressor gene for Pm8 and Pm17 is localized on chromosome 7D. It is suggested that genotypes without the suppressor gene be used for the exploitation of genes Pm8 and Pm17 in enhancing powdery mildew resistance in common wheat.     

A new powdery mildew resistance allele at the Pm4 wheat locus transferred from einkorn (Triticum monococcum)

Powdery mildew is one of the most destructive foliar diseases of wheat. A set of differential Blumeria graminis f.sp. tritici (Bgt) isolates was used to test the powdery mildew response of a Triticum monococcum-derived resistant hexaploid line, Tm27d2. Segregation analysis of 95 F_2:3 lines from a Chinese Spring/Tm27d2 cross revealed that the resistance of Tm27d2 is controlled by a single dominant gene. Using monosomic analysis and a molecular mapping approach, the resistance gene was localized to the terminal end of chromosome 2AL. The linkage map of chromosome 2AL consisted of nine simple sequence repeat markers and one sequence-tagged site (STS) marker (ResPm4) indicative for the Pm4 locus. According to the differential reactions of 19 wheat cultivars/lines with known powdery mildew resistance genes to 13 Bgt isolates, Tm27d2 carried a new resistance specificity. The complete association of the resistance allele with STS marker ResPm4 indicated that it represented a new allele at the Pm4 locus. This new allele was designated Pm4d. The two flanking markers Xgwm526 and Xbarc122 closely linked to Pm4d at genetic distances of 3.4 and 1.0 cM, respectively, are present in chromosome bin 2AL1-0.85-1.00.     

Isolation of resistance gene analogues to powdery mildew resistance sequences in hexaploid wheat

This paper reports the characterization of the powdery mildew resistance homologous genes family of Triticum aestivum. Using degenerate primer pair for wheat resistance genes, we have cloned seven 3′ truncated powdery mildew resistance gene homologous fragments Tpc5a, Tp25a, Tp25b, Tp3a5a, Tp3a5b, Tp4b5a and Tp4b5b. These fragments were sequenced. The deduced amino acid sequences showed that six of them have premature stop codons. All these sequences had a very high level of similarity to known Pm resistance genes such as Pm3a, Pm3b, Pm3d and pm3f in hexaploid wheat. By ignoring the stop codons in the sequences, their deduced protein sequences were of coiled-coil (CC)-nucleotide binding site (NBS)-leucine repeat rich (LRR) structure. These results suggest that there are many powdery mildew resistance gene analogues in both resistant and susceptible wheat. Among them, small insertion/deletion events and point mutations can result in the diversity of wheat Pm resistance homologous genes.     

Rye Pm8 and wheat Pm3 are orthologous genes and show evolutionary conservation of resistance function against powdery mildew

The improvement of wheat through breeding has relied strongly on the use of genetic material from related wild and domesticated grass species. The 1RS chromosome arm from rye was introgressed into wheat and crossed into many wheat lines, as it improves yield and fungal disease resistance. Pm8 is a powdery mildew resistance gene on 1RS which, after widespread agricultural cultivation, is now widely overcome by adapted mildew races. Here we show by homology‐based cloning and subsequent physical and genetic mapping that Pm8 is the rye orthologue of the Pm3 allelic series of mildew resistance genes in wheat. The cloned gene was functionally validated as Pm8 by transient, single‐cell expression analysis and stable transformation. Sequence analysis revealed a complex mosaic of ancient haplotypes among Pm3‐ and Pm8‐like genes from different members of the Triticeae. These results show that the two genes have evolved independently after the divergence of the species 7.5 million years ago and kept their function in mildew resistance. During this long time span the co‐evolving pathogens have not overcome these genes, which is in strong contrast to the breakdown of Pm8 resistance since its introduction into commercial wheat 70 years ago. Sequence comparison revealed that evolutionary pressure acted on the same subdomains and sequence features of the two orthologous genes. This suggests that they recognize directly or indirectly the same pathogen effectors that have been conserved in the powdery mildews of wheat and rye.     

Chromosomal location of genes for resistance to powdery mildew in common wheat (Triticum aestivum L. em. Thell.) 4. Gene Pm 24 in Chinese landrace Chiyacao

 Chinese wheat landrace Chiyacao exhibited a response pattern different from that of the cultivars/lines possessing documented Pm genes after inoculation with 106 isolates of Erysiphe graminis f. sp. tritici. To characterize this resistance and to determine the chromosomal location of the gene or genes present, we crossed the landrace to susceptible cultivar ‘Chinese Spring’ and also to a set of 21 ‘Chinese Spring’ monosomic lines. Monosomic F₁ plants were allowed to self-pollinate and to produce F₂ seeds. Seedlings of F₂ plants and their parents were inoculated with isolates nos. 5 and 12 of Erysiphe graminis f. sp. tritici. The results revealed that one major dominant gene is located on chromosome 6D of Chinese common wheat landrace Chiyacao. The new gene is designated Pm 24. Received: 12 May 1997 / Accepted: 23 May 1997     

Virulence Structure of Blumeria graminis f. sp. tritici and Its Genetic Diversity by ISSR and SRAP Profiling Analyses

Blumeria graminis f. sp. tritici, which causes wheat powdery mildew, is an obligate biotrophic pathogen that can easily genetically adapt to its host plant. Understanding the virulence structure of and genetic variations in this pathogen is essential for disease control and for breeding resistance to wheat powdery mildew. This study investigated 17 pathogenic populations in Sichuan, China and classified 109 isolates into two distinct groups based on pathogenicity analysis: high virulence (HV, 92 isolates) and low virulence (LV, 17 isolates). Populations from Yibin (Southern region), Xichang (Western region), and Meishan (Middle region) showed lower virulence frequencies than populations from other regions. Many of the previously known resistance genes did not confer resistance in this study. The resistance gene Pm21 displayed an immune response to pathogenic challenge with all populations in Sichuan, and Pm13, Pm5b, Pm2+6, and PmXBD maintained resistance. AMOVA revealed significantly higher levels of variation within populations and lower levels of variation among populations within regions. High levels of gene flow were detected among populations in the four regions. Closely related populations within each region were distinguished by cluster analyses using ISSR and SRAP alleles. Both ISSR and SRAP allele profiling analyses revealed high levels of genetic diversity among pathogenic populations in Sichuan. Although ISSR and SRAP profiling analysis showed similar resolutions, the SRAP alleles appeared to be more informative. We did not detect any significant association between these alleles and the virulence or pathogenicity of the pathogen. Our results suggest that ISSR and SRAP alleles are more efficient for the characterization of small or closely related populations versus distantly related populations.     

Identification and development of diagnostic markers for a powdery mildew resistance gene on chromosome 2R of Chinese rye cultivar Jingzhouheimai

Chinese rye cultivar Jingzhouheimai (Secale cereale L.) shows a high level of resistance to powdery mildew. Identification, location, and mapping of the resistance gene would be helpful for developing a highly resistant germplasm or cultivar in wheat. Using sequential C-banding, GISH, and marker analysis, an addition chromosome with powdery mildew resistance was identified in a line derived from a cross between Chinese wheat landrace Huixianhong and rye cultivar Jingzhouheimai. The line, designated H-J DA2RDS1R(1D), had 44 chromosomes including two pairs of rye chromosomes, 1R and 2R, and lacked a pair of wheat chromosomes 1D, that is, it is a double disomic addition disomic substitution line. According to its reaction to different isolates of the powdery mildew pathogen, the resistance gene in H-J DA2RDS1R(1D) differed from the Pm8 and Pm7 genes located earlier on rye chromosomes 1R and 2R, respectively. In order to determine the location of the resistance gene, line H-J DA2RDS1R(1D) was crossed with wheat landrace Huixianhong and the F2 population and corresponding F2:3 families were tested for disease reaction and assessed with molecular markers. The results showed that a resistance gene, designated PmJZHM2RL, is located in rye chromosome arm 2RL.     

The powdery mildew resistance gene Pm8 derived from rye is suppressed by its wheat ortholog Pm3

The powdery mildew resistance gene Pm8 derived from rye is located on a 1BL.1RS chromosome translocation in wheat. However, some wheat lines with this translocation do not show resistance to isolates of the wheat powdery mildew pathogen avirulent to Pm8 due to an unknown genetically dominant suppression mechanism. Here we show that lines with suppressed Pm8 activity contain an intact and expressed Pm8 gene. Therefore, the absence of Pm8 function in certain 1BL.1RS‐containing wheat lines is not the result of gene loss or mutation but is based on suppression. The wheat gene Pm3, an ortholog of rye Pm8, suppressed Pm8‐mediated powdery mildew resistance in lines containing Pm8 in a transient single‐cell expression assay. This result was further confirmed in transgenic lines with combined Pm8 and Pm3 transgenes. Expression analysis revealed that suppression is not the result of gene silencing, either in wheat 1BL.1RS translocation lines carrying Pm8 or in transgenic genotypes with both Pm8 and Pm3 alleles. In addition, a similar abundance of the PM8 and PM3 proteins in single or double homozygous transgenic lines suggested that a post‐translational mechanism is involved in suppression of Pm8. Co‐expression of Pm8 and Pm3 genes in Nicotiana benthamiana leaves followed by co‐immunoprecipitation analysis showed that the two proteins interact. Therefore, the formation of a heteromeric protein complex might result in inefficient or absent signal transmission for the defense reaction. These data provide a molecular explanation for the suppression of resistance genes in certain genetic backgrounds and suggest ways to circumvent it in future plant breeding.     

Rye-derived powdery mildew resistance gene <Emphasis Type="Italic">Pm8</Emphasis> in wheat is suppressed by the <Emphasis Type="Italic">Pm3</Emphasis> locus

Genetic suppression of disease resistance is occasionally observed in hexaploid wheat or in its interspecific crosses. The phenotypic effects of genes moved to wheat from relatives with lower ploidy are often smaller than in the original sources, suggesting the presence of modifiers or partial inhibitors in wheat, especially dilution effects caused by possible variation at orthologous loci. However, there is little current understanding of the underlying genetics of suppression. The discovery of suppression in some wheat genotypes of the cereal rye chromosome 1RS-derived gene Pm8 for powdery mildew resistance offered an opportunity for analysis. A single gene for suppression was identified at or near the closely linked storage protein genes Gli-A1 and Glu-A3, which are also closely associated with the Pm3 locus on chromosome 1AS. The Pm3 locus is a complex of expressed alleles and pseudogenes embedded among Glu-A3 repeats. In the current report, we explain why earlier work indicated that the mildew suppressor was closely associated with specific Gli-A1 and Glu-A3 alleles, and predict that suppression of Pm8 involves translated gene products from the Pm3 locus.     

Genetic characterization of powdery mildew resistance in U.S. hard winter wheat

Powdery mildew significantly affects grain yield and end-use quality of winter wheat in the southern Great Plains. Employing resistance resources in locally adapted cultivars is the most effective means to control powdery mildew. Two types of powdery mildew resistance exist in wheat cultivars, i.e., qualitative and quantitative. Qualitative resistance is controlled by major genes, is race-specific, is not durable, and is effective in seedlings and in adult plants. Quantitative resistance is controlled by minor genes, is non-race-specific, is durable, and is predominantly effective in adult plants. In this study, we found that the segregation of powdery mildew resistance in a population of recombinant inbred lines developed from a cross between the susceptible cultivar Jagger and the resistant cultivar 2174 was controlled by a major QTL on the short arm of chromosome 1A and modified by four minor QTLs on chromosomes 1B, 3B, 4A, and 6D. The major QTL was mapped to the genomic region where the Pm3 gene resides. Using specific PCR markers for seven Pm3 alleles, 2174 was found to carry the Pm3a allele. Pm3a explained 61% of the total phenotypic variation in disease reaction observed among seedlings inoculated in the greenhouse and adult plants grown in the field and subjected to natural disease pressure. The resistant Pm3a allele was present among 4 of 31 cultivars currently being produced in the southern Great Plains. The genetic effects of several minor loci varied with different developmental stages and environments. Molecular markers associated with these genetic loci would facilitate incorporating genetic resistance to powdery mildew into improved winter wheat cultivars.     

Proteomic Analysis of the Defense Response of Wheat to the Powdery Mildew Fungus,Blumeria graminis f. sp. tritici

Powdery mildew of wheat is caused by Blumeria graminis f. sp. tritici (Bgt). Although many wheat cultivars resistant to this disease have been developed, little is known about their resistance mechanisms. The aim of this study was to identify proteins showing changes in abundance during the resistance response of the wheat line N0308 infected by Bgt. In two-dimensional electrophoresis analyses, 45 spots on the gels showed significant changes in abundance at 24, 48, and 72 h after inoculation, as compared to non-inoculated plants. Of these 45 proteins, 44 were identified by mass spectrometry analysis using the NCBInr database of Triticum aestivum (26 spots) and closely related species in the Triticum genus (18 spots). These proteins were associated with the defense response, photosynthesis, metabolism, and other cellular processes in wheat. Most of the up-regulated proteins were identified as stress- and defense-related proteins. In particular, the product of a specific powdery mildew resistance gene (Pm3b and its homolog) and some other defense- and pathogenesis-related proteins were overexpressed. The resistance gene product mediates the immune response and coordinates other cellular processes during the resistance response to Bgt.     

Identification and mapping of PmG16, a powdery mildew resistance gene derived from wild emmer wheat

The gene-pool of wild emmer wheat, Triticum turgidum ssp. dicoccoides, harbors a rich allelic repertoire for disease resistance. In the current study, we made use of tetraploid wheat mapping populations derived from a cross between durum wheat (cv. Langdon) and wild emmer (accession G18-16) to identify and map a new powdery mildew resistance gene derived from wild emmer wheat. Initially, the two parental lines were screened with a collection of 42 isolates of Blumeria graminis f. sp. tritici (Bgt) from Israel and 5 isolates from Switzerland. While G18-16 was resistant to 34 isolates, Langdon was resistant only to 5 isolates and susceptible to 42 isolates. Isolate Bgt#15 was selected to differentiate between the disease reactions of the two genotypes. Segregation ratio of F_2-3 and recombinant inbreed line (F₇) populations to inoculation with isolate Bgt#15 indicated the role of a single dominant gene in conferring resistance to Bgt#15. This gene, temporarily designated PmG16, was located on the distal region of chromosome arm 7AL. Genetic map of PmG16 region was assembled with 32 simple sequence repeat (SSR), sequence tag site (STS), Diversity array technology (DArT) and cleaved amplified polymorphic sequence (CAPS) markers and assigned to the 7AL physical bin map (7AL-16). Using four DNA markers we established colinearity between the genomic region spanning the PmG16 locus within the distal region of chromosome arm 7AL and the genomic regions on rice chromosome 6 and Brachypodium Bd1. A comparative analysis was carried out between PmG16 and other known Pm genes located on chromosome arm 7AL. The identified PmG16 may facilitate the use of wild alleles for improvement of powdery mildew resistance in elite wheat cultivars via marker-assisted selection.     

Pathotypes of Blumeria graminis f. sp. tritici,the causal agent of wheat powdery mildew from some regions of Iran

Wheat powdery mildew is controlled mainly by race-specific resistance. To be effective, breeding wheat for resistance to powdery mildew requires knowledge of virulence diversity in local populations of the pathogen. Isolates of Blumeria graminis, collected in 2009 and 2010 from three areas of Iranian production, were analysed for virulence using a host differential series comprised of 16 known genes conferring resistance to powdery mildew. The results showed that high-virulence frequencies to genes Pm1, Pm2, Pm4a, Pm5, Pm6, Pm7, Pm8 and Pm9 were found over both years and across all three areas. Virulence frequencies for Pm3a and Pm3b were intermediate, while virulence frequencies for Pm3a, Pm3c, Pm4a and Pm2, 6 were low. Genes Pm1, 2, 9 and Pm2, 4b, 8 were highly resistant in all regions. Virulence to Pm8 increased to high levels, while virulence to Pm4a decreased across the area surveyed from 2009 to 2010.     

Reaction of some wheat cultivars and breeding lines to Puccinia striiformis f. sp. tritici hot races in Iran

Many physiological races of Puccinia striiformis f. sp. tritici which cause stripe rust in wheat can be determined in different parts of the world. The emergence of new races with different pathogenicity which happens very quickly breaks cultivars resistant and cause disease. Therefore, breeding cultivar for resistance to different pathogenic races should be continued. In this research, pathogenicity of two isolates collected from two regions of Iran were determined by using wheat yellow rust differential lines, which indicated race 70E50A+ and 6E18A+ The responses of 30 wheat genotypes were separately evaluated in the forms of randomized complete block design with three replicates in the seedling stage under greenhouse condition. The components of resistance including latent period and infection type were recorded. Results indicated genotypes were evaluated in terms of both traits and were significant at 1% level. Also, the results from pathogenicity study indicated of effective gene/s included Yr1, Yr2+, Yr3, Yr4, Yr5, Yr10, Yr15, Yr24, Yr26, YrSP, YrND, YrSD and YrSU. From the genotypes studied in the greenhouse condition, 39% of the genotypes showed complete resistance to both races. Probably, resistance genes, Yr32 and YrCV, or the other unknown genes which are types of seedling resistance are either alone or in combination of one another cause strength in resistant genotypes.     

A “one-marker-for-two-genes” approach for efficient molecular discrimination of Pm12 and Pm21 conferring resistance to powdery mildew in wheat

Powdery mildew, caused by Blumeria graminis f. sp. tritici, is one of the most important wheat diseases worldwide. Pyramiding different resistance genes into single cultivar has been proposed as one remedy to provide durable resistance. Powdery mildew resistance genes Pm12 (T6BS-6SS.6SL), transferred from Aegilops speltoides to wheat cv. Wembley, and Pm21 (T6VS.6AL), introduced from Dasypyrum villosum to wheat cv. Yangmai5, conferred broad-spectrum resistance to B. graminis f. sp. tritici. Both Pm12 and Pm21 genes are located on the short arms of homologous group six involved translocated chromosomes 6SS.6BL and 6VS.6AL, respectively. Simple sequence repeat motifs of wheat simple sequence repeat (SSR) and expressed sequence tag (EST) sequences on the short arm of homologous group six chromosomes were analyzed to develop molecular markers for discriminating chromosome arms 6AS, 6BS, 6DS, 6VS, and 6SS. One EST–SSR marker, Xcau127, was polymorphic, and therefore can be used to distinguish the two resistance genes and the respective susceptible alleles. This marker allowed us to develop an efficient “one-marker-for-two-genes” procedure for identifying powdery mildew resistance genes Pm12 and Pm21 for marker-assisted selection and gene pyramiding in wheat breeding programs. Wei Song and Chaojie Xie contributed equally to this work