Global genomic analyses of wheat powdery mildew reveal association of pathogen spread with historical human migration and trade.

The fungus Blumeria graminis f. sp. tritici causes wheat powdery mildew disease. Here, we study its spread and evolution by analyzing a global sample of 172 mildew genomes. Our analyses show that B.g. tritici emerged in the Fertile Crescent during wheat domestication. After it spread throughout Eurasia, colonization brought it to America, where it hybridized with unknown grass mildew species. Recent trade brought USA strains to Japan, and European strains to China. In both places, they hybridized with local ancestral strains. Thus, although mildew spreads by wind regionally, our results indicate that humans drove its global spread throughout history and that mildew rapidly evolved through hybridization.

Reciprocal Hosts' Responses to Powdery Mildew Isolates Originating from Domesticated Wheats and Their Wild Progenitor.

The biotroph wheat powdery mildew, Blumeria graminis (DC.) E.O. Speer, f. sp. tritici Em. Marchal (Bgt), has undergone long and dynamic co-evolution with its hosts. In the last 10,000 years, processes involved in plant evolution under domestication, altered host-population structure. Recently both virulence and genomic profiling separated Bgt into two groups based on their origin from domestic host and from wild emmer wheat. While most studies focused on the Bgt pathogen, there is significant knowledge gaps in the role of wheat host diversity in this specification. This study aimed to fill this gap by exploring qualitatively and also quantitatively the disease response of diverse host panel to powdery mildew [105 domesticated wheat genotypes (Triticum turgidum ssp. dicoccum, T. turgidum ssp. durum, and T. aestivum) and 241 accessions of its direct progenitor, wild emmer wheat (T. turgidum ssp. dicoccoides)]. A set of eight Bgt isolates, originally collected from domesticated and wild wheat was used for screening this wheat collection. The isolates from domesticated wheat elicited susceptible to moderate plant responses on domesticated wheat lines and high resistance on wild genotypes (51.7% of the tested lines were resistant). Isolates from wild emmer elicited reciprocal disease responses: high resistance of domesticated germplasm and high susceptibility of the wild material (their original host). Analysis of variance of the quantitative phenotypic responses showed a significant Isolates × Host species interaction [P(F) < 0.0001] and further supported these findings. Furthermore, analysis of the range of disease severity values showed that when the group of host genotypes was inoculated with Bgt isolate from the reciprocal host, coefficient of variation was significantly higher than when inoculated with its own isolates. This trend was attributed to the role of major resistance genes in the latter scenario (high proportion of complete resistance). By testing the association between disease severity and geographical distance from the source of inoculum, we have found higher susceptibility in wild emmer close to the source. Both qualitative and quantitative assays showed a reciprocal resistance pattern in the wheat host and are well aligned with the recent findings of significant differentiation into wild-emmer and domesticated-wheat populations in the pathogen.

Distinct domains of the AVRPM3A2/F2 avirulence protein from wheat powdery mildew are involved in immune receptor recognition and putative effector function.

Recognition of the AVRPM3A2/F2 avirulence protein from powdery mildew by the wheat PM3A/F immune receptor induces a hypersensitive response after co-expression in Nicotiana benthamiana. The molecular determinants of this interaction and how they shape natural AvrPm3a2/f2 allelic diversity are unknown. We sequenced the AvrPm3a2/f2 gene in a worldwide collection of 272 mildew isolates. Using the natural polymorphisms of AvrPm3a2/f2 as well as sequence information from related gene family members, we tested 85 single-residue-altered AVRPM3A2/F2 variants with PM3A, PM3F and PM3FL456P/Y458H (modified for improved signaling) in Nicotiana benthamiana for effects on recognition. An intact AvrPm3a2/f2 gene was found in all analyzed isolates and the protein variant recognized by PM3A/F occurred globally at high frequencies. Single-residue alterations in AVRPM3A2/F2 mostly disrupted, but occasionally enhanced, the recognition response by PM3A, PM3F and PM3FL456P/Y458H . Residues enhancing hypersensitive responses constituted a protein domain separate from both naturally occurring polymorphisms and positively selected residues of the gene family. These results demonstrate the utility of using gene family sequence diversity to screen residues for their role in recognition. This approach identified a putative interaction surface in AVRPM3A2/F2 not polymorphic in natural alleles. We conclude that molecular mechanisms besides recognition drive AvrPm3a2/f2 diversification.

Differentiation Among Blumeria graminis f. sp. tritici Isolates Originating from Wild Versus Domesticated Triticum Species in Israel.

Israel and its vicinity constitute a center of diversity of domesticated wheat species (Triticum aestivum and T. durum) and their sympatrically growing wild relatives, including wild emmer wheat (T. dicoccoides). We investigated differentiation within the forma specialis of their obligate powdery mildew pathogen, Blumeria graminis f. sp. tritici. A total of 61 B. graminis f. sp. tritici isolates were collected from the three host species in four geographic regions of Israel. Genetic relatedness of the isolates was characterized using both virulence patterns on 38 wheat lines (including 21 resistance gene differentials) and presumptively neutral molecular markers (simple-sequence repeats and single-nucleotide polymorphisms). All isolates were virulent on at least some genotypes of all three wheat species tested. All assays divided the B. graminis f. sp. tritici collection into two distinct groups, those from domesticated hosts and those from wild emmer wheat. One-way migration was detected from the domestic wheat B. graminis f. sp. tritici population to the wild emmer B. graminis f. sp. tritici population at a rate of five to six migrants per generation. This gene flow may help explain the overlap between the distinct domestic and wild B. graminis f. sp. tritici groups. Overall, B. graminis f. sp. tritici is significantly differentiated into wild-emmer and domesticated-wheat populations, although the results do not support the existence of a separate f. sp. dicocci.

Identification and characterization of a novel powdery mildew resistance gene PmG3M derived from wild emmer wheat, Triticum dicoccoides.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt) is one of the most important wheat diseases worldwide. Wild emmer wheat, Triticum turgidum ssp. dicoccoides, the tetraploid ancestor (AABB) of domesticated bread and durum wheat, harbors many important alleles for resistance to various diseases, including powdery mildew. In the current study, two tetraploid wheat mapping populations, derived from a cross between durum wheat (cv. Langdon) and wild emmer wheat (accession G-305-3M), were used to identify and map a novel powdery mildew resistance gene. Wild emmer accession G-305-3M was resistant to all 47 Bgt isolates tested, from Israel and Switzerland. Segregation ratios of F(2) progenies and F(6) recombinant inbred line (RIL) mapping populations, in their reactions to inoculation with Bgt, revealed a Mendelian pattern (3:1 and 1:1, respectively), indicating the role of a single dominant gene derived from T. dicoccoides accession G-305-3M. This gene, temporarily designated PmG3M, was mapped on chromosome 6BL and physically assigned to chromosome deletion bin 6BL-0.70-1.00. The F(2) mapping population was used to construct a genetic map of the PmG3M gene region consisted of six simple sequence repeats (SSR), 11 resistance gene analog (RGA), and two target region amplification polymorphism (TRAP) markers. A second map, constructed based on the F(6) RIL population, using a set of skeleton SSR markers, confirmed the order of loci and distances obtained for the F(2) population. The discovery and mapping of this novel powdery mildew resistance gene emphasize the importance of the wild emmer wheat gene pool as a source for crop improvement.

Comprehensive Evaluation of Virulence and Resistance Data: A New Analysis Tool.

The functioning and features of the new software package VAT (Virulence Analysis Tool) are introduced. VAT provides a range of methods for the analysis of plant pathosystems. The techniques are applicable to other binary data sets that are organized in large two-way tables, e.g., molecular marker data. The main features are data entry, descriptive tools, and inference statistics by resampling. About 50 well-established or newly developed indices allow a detailed diversity analysis of sexually and asexually reproducing populations. VAT facilitates a comprehensive, effective, and logically consistent evaluation and presentation of virulence and resistance data. A translation option simplifies the comparison of results from differently coded pathotypes. The software package comes with a detailed manual and is freely available on the internet at tau.ac.il/lifesci/departments/plant_s/members/kosman/VAT.html and at va-tipp.de .

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 (F(7)) 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.

Virulence and Diversity of Blumeria graminis f. sp. hordei in Israel and in the Czech Republic.

Three hundred and nine isolates were obtained from three natural populations of Blumeria graminis f. sp. hordei occurring on wild barley (Hordeum vulgare subsp. spontaneum) at two locations in Israel during 1997 and 1999. Their virulence frequency was determined on 32 differential lines. No isolate was virulent on the differential lines possessing the genes Mla13, mlo, Mlf1, and Mli, and conversely no isolate was avirulent on the differential lines possessing the genes MlRu2, MlLa, Mlh, Mla8, Mla25, and Mlj. The frequencies of isolates overcoming the genes Mlg, Mla7, and Mla27 were 0 to 16% at individual locations; frequencies of isolates overcoming the genes Mla9, Mla17, and Mla18 ranged from 37 to 78%, and frequencies of virulences to genes Mla1, Mla3, Mla6, Mlp1, Mlat, Mla12, Mlra, Mlk1, Mla19, Mla20, Mla26, Mla28, Mla29, Mla30, Mla32, and mlt1 were 79 to 99%. Based on examination of 376 isolates collected in the same years from the Czech Republic, these populations differed greatly from the Israeli ones. The Czech populations showed greater diversity of virulence and lower mean virulence complexity than the Israeli populations. Diversity in the Israeli populations differed also among clusters of niches at the same location.

pH regulates endoglucanase expression and virulence of Alternaria alternata in persimmon fruit.

The phytopathogenic fungus Alternaria alternata produces one endo-1,4-beta-glucanase, AaK1, which is an important factor in disease development in persimmon fruit. During growth of A. alternata in media containing acidified yeast extract or cell walls from persimmon fruit, the fungus secreted ammonia and raised the medium pH. A rise in media pH from 3.8 to 6.0 in the presence of cell walls induced the expression of AaK1, whereas a glucose-induced decline in pH to 2.5 repressed transcription and enzymatic production. Treatments with buffered solutions at pH 6.0 during growth of A. alternata in the presence of glucose derepressed AaK1 expression and endo-1,4-beta-glucanase production and enhanced decay development on the fruit. The results suggest that conditions affecting environmental pH modulate gene expression of AaK1 and virulence of A. alternata in persimmon fruit

Characterization of Alternaria alternata glucanase genes expressed during infection of resistant and susceptible persimmon fruits.

Summary Preharvest treatment with gibberellic acid (GA(3)) or its inhibitor paclobutrazol (PBZ) can reduce or increase, respectively, the susceptibility of persimmon fruits to Alternaria alternata. This was suggested to be the result of the ability of the fungus and produced endoglucanases to induce symptom development. To evaluate the importance of glucanases during A. alternata attack, five glucanase genes, corresponding to the C, F, and K families, were cloned from A. alternata using 'family-specific' oligonucleotide primers. The genes, present in a single copy, encode for exoglucanases AaC1 and AaC2, endoxylanase AaF1, endoglucanase AaK1, and the mixed-linked glucanase AaMLG1. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of RNA extracted from persimmon fruits, 2 and 4 days post-infection with A. alternata, showed the expression of all five glucanase genes in GA3- and PBZ-treated fruits. However, transcription levels and enzyme production of the endoglucanase (AaK1) and one exoglucanase (AaC1) were enhanced during A. alternata growth on cell walls from susceptible PBZ-treated fruits, whereas the expression of these genes and their enzyme production were significantly reduced in resistant GA(3)-treated fruits. The present results suggest the involvement of endo- and exoglucanase in symptom development caused by A. alternata in resistant and susceptible persimmon fruits.