Functional Variation of Plant-Pathogen Interactions: New Concept and Methods for Virulence Data Analyses.

Classical virulence analysis is based on discovering virulence phenotypes of isolates with regard to a composition of resistance genes in a differential set of host genotypes. With such a vision, virulence phenotypes are usually treated in a genetic manner as one of two possible alleles, either virulence or avirulence in a binary locus. Therefore, population genetics metrics and methods have become prevailing tools for analyzing virulence data at multiple loci. However, a basis for resolving binary virulence phenotypes is infection type (IT) data of host-pathogen interaction that express functional traits of each specific isolate in a given situation (particular host, environmental conditions, cultivation practice, and so on). IT is determined by symptoms and signs observed (e.g., lesion type, lesion size, coverage of leaf or leaf segments by mycelium, spore production and so on), and assessed by IT scores at a generally accepted scale for each plant-pathogen system. Thus, multiple IT profiles of isolates are obtained and can be subjected to analysis of functional variation within and among operational units of a pathogen. Such an approach may allow better utilization of the information available in the raw data, and reveal a functional (e.g., environmental) component of pathogen variation in addition to the genetic one. New methods for measuring functional variation of plant-pathogen interaction with IT data were developed. The methods need an appropriate assessment scale and expert estimations of dissimilarity between IT scores for each plant-pathogen system (an example is presented). Analyses of a few data sets at different hierarchical levels demonstrated discrepancies in results obtained with IT phenotypes versus binary virulence phenotypes. The ability to measure functional IT-based variation offers promise as an effective tool in the study of epidemics caused by plant pathogens.

Introgression of leaf rust and stripe rust resistance from Sharon goatgrass (Aegilops sharonensis Eig) into bread wheat (Triticum aestivum L.).

Leaf rust and stripe rust are devastating wheat diseases, causing significant yield losses in many regions of the world. The use of resistant varieties is the most efficient way to protect wheat crops from these diseases. Sharon goatgrass (Aegilops sharonensis or AES), which is a diploid wild relative of wheat, exhibits a high frequency of leaf and stripe rust resistance. We used the resistant AES accession TH548 and induced homoeologous recombination by the ph1b allele to obtain resistant wheat recombinant lines carrying AES chromosome segments in the genetic background of the spring wheat cultivar Galil. The gametocidal effect from AES was overcome by using an "anti-gametocidal" wheat mutant. These recombinant lines were found resistant to highly virulent races of the leaf and stripe rust pathogens in Israel and the United States. Molecular DArT analysis of the different recombinant lines revealed different lengths of AES segments on wheat chromosome 6B, which indicates the location of both resistance genes.

Genetic differentiation of Puccinia triticina populations in the Middle East and genetic similarity with populations in Central Asia.

Leaf rust of wheat, caused by Puccinia triticina, is a common and widespread disease in the Middle East. The objective of this study was to determine whether genetically differentiated groups of P. triticina are present in the Middle East region and to compare the population from the Middle East with the previously characterized population from Central Asia to determine whether genetically similar groups of isolates are found in the two regions. In total, 118 isolates of P. triticina collected from common wheat and durum wheat in Egypt, Israel, Turkey, Ethiopia, and Kenya were tested for virulence on 20 lines of wheat with single genes for leaf rust resistance and for molecular genotypes with 23 simple-sequence repeat (SSR) markers. After removal of isolates with identical virulence and SSR genotype in each country, 103 isolates were retained for further analysis. Clustering of SSR genotypes based on two-dimensional principal coordinates and virulence to wheat differential lines grouped the isolates into four Middle East (ME) groups. The two largest ME groups had virulence phenotypes typical of isolates collected from common wheat and two smaller ME groups had virulence typical of isolates collected from durum wheat. All pairs of ME groups were significantly differentiated for SSR genotype based on R(ST) and F(ST) statistics, and for virulence phenotype based on Φ(PT). All ME groups had observed values of heterozygosity greater than expected and significant fixation indices that indicated the clonal reproduction of urediniospores in the overall population. Linkage disequilibria for SSR genotypes was high across the entire population. The overall values of R(ST) and F(ST) were lower when isolates were grouped by country of origin that indicated the likely migration of isolates within the region. Although the two ME groups with virulence typical of isolates from common wheat were not differentiated for SSR genotype from groups of isolates from Central Asia based on R(ST), there was no direct evidence for migration between the two regions because all ME isolates differed from the Central Asia isolates for SSR genotypes.

Genome size in natural and synthetic autopolyploids and in a natural segmental allopolyploid of several Triticeae species.

Nuclear DNA amount (1C) was determined by flow cytometry in the autotetraploid cytotype of Hordeum bulbosum, in the cytologically diploidized autotetraploid cytotypes of Elymus elongatus, Hordeum murinum subsp. murinum and Hordeum murinum subsp. leporinum, in Hordeum marinum subsp. gussoneanum, in their progenitor diploid cytotypes, and in a newly synthesized autotetraploid line of E. elongatus. Several lines collected from different regions of the distribution area of every taxon, each represented by a number of plants, were analyzed in each taxon. The intracytotype variation in nuclear DNA amount of every diploid and autotetraploid cytotype was very small, indicating that no significant changes have occurred in DNA amount either after speciation or after autopolyploid formation. The autotetraploid cytotypes of H. bulbosum and the cytologically diploidized H. marinum subsp. gussoneanum had the expected additive amount of their diploid cytotypes. On the other hand, the cytologically diploidized autotetraploid cytotypes of E. elongatus and H. murinum subsp. murinum and H. murinum subsp. leporinum had considerably less nuclear DNA (10%-23%) than the expected additive value. Also, the newly synthesized autotetraploid line of E. elongatus showed similar reduction in DNA as its natural counterpart, indicating that the reduction in genome size occurred in the natural cytotype during autopolyploidization. It is suggested that the diploid-like meiotic behavior of these cytologically dipolidized autotetraploids is caused by the instantaneous elimination of a large number of DNA sequences, different sequences from different homologous pairs, leading to differentiation of the constituent genomes. The eliminated sequences are likely to include those that participate in homologous recognition and initiation of meiotic pairing. A gene system determining exclusive bivalent pairing by utilizing the differentiation between the two groups of homologues has been presumably superimposed on the DNA reduction process.

Nuclear DNA amount and genome downsizing in natural and synthetic allopolyploids of the genera Aegilops and Triticum.

Recent molecular studies in the genera Aegilops and Triticum showed that allopolyploidization (interspecific or intergeneric hybridization followed by chromosome doubling) generated rapid elimination of low-copy or high-copy, non-coding and coding DNA sequences. The aims of this work were to determine the amount of nuclear DNA in allopolyploid species of the group and to see to what extent elimination of DNA sequences affected genome size. Nuclear DNA amount was determined by the flow cytometry method in 27 natural allopolyploid species (most of which were represented by several lines and each line by several plants) as well as 14 newly synthesized allopolyploids (each represented by several plants) and their parental plants. Very small intraspecific variation in DNA amount was found between lines of allopolyploid species collected from different habitats or between wild and domesticated forms of allopolyploid wheat. In contrast to the constancy in nuclear DNA amount at the intraspecific level, there are significant differences in genome size between the various allopolyploid species, at both the tetraploid and hexaploid levels. In most allopolyploids nuclear DNA amount was significantly less than the sum of DNA amounts of the parental species. Newly synthesized allopolyploids exhibited a similar decrease in nuclear DNA amount in the first generation, indicating that genome downsizing occurs during and (or) immediately after the formation of the allopolyploids and that there are no further changes in genome size during the life of the allopolyploids. Phylogenetic considerations of the origin of the B genome of allopolyploid wheat, based on nuclear DNA amount, are discussed.

Genome size and genome evolution in diploid Triticeae species.

One of the intriguing issues concerning the dynamics of plant genomes is the occurrence of intraspecific variation in nuclear DNA amount. The aim of this work was to assess the ranges of intraspecific, interspecific, and intergeneric variation in nuclear DNA content of diploid species of the tribe Triticeae (Poaceae) and to examine the relation between life form or habitat and genome size. Altogether, 438 plants representing 272 lines that belong to 22 species were analyzed. Nuclear DNA content was estimated by flow cytometry. Very small intraspecific variation in DNA amount was found between lines of Triticeae diploid species collected from different habitats or between different morphs. In contrast to the constancy in nuclear DNA amount at the intraspecific level, there are significant differences in genome size between the various diploid species. Within the genus Aegilops, the 1C DNA amount ranged from 4.84 pg in A. caudata to 7.52 pg in A. sharonensis; among genera, the 1C DNA amount ranged from 4.18 pg in Heteranthelium piliferum to 9.45 pg in Secale montanum. No evidence was found for a smaller genome size in annual, self-pollinating species relative to perennial, cross-pollinating ones. Diploids that grow in the southern part of the group's distribution have larger genomes than those growing in other parts of the distribution. The contrast between the low variation at the intraspecific level and the high variation at the interspecific one suggests that changes in genome size originated in close temporal proximity to the speciation event, i.e., before, during, or immediately after it. The possible effects of sudden changes in genome size on speciation processes are discussed.

Virulence associations in oat crown rust.

ABSTRACT Isolates of Puccinia coronata obtained from natural populations of Avena sterilis in Israel, winter oat (A. sativa) cultivars in Texas, and spring oat cultivars in the Northern Plains states of Minnesota, North Dakota, and South Dakota were analyzed for significance of pairwise virulence associations. Isolates from all three regions were tested on 25 oat lines with single P. coronata (Pc) genes for crown rust resistance from A. sterilis and one line with a Pc gene from A. sativa. Isolates from Israel were tested also on 11 Iowa backcross lines with undesignated crown rust resistance genes from A. sterilis. Four associated virulence groups were identified from significant positive virulence associations that were consistent across all three regions. Group 38 included virulence to Pc-38, Pc-39, Pc-55, Pc-63, and Pc-71; group 45 included virulence to Pc-45, Pc-46, Pc-48, Pc-52, Pc-54, and Pc-57; group 58 included virulence to Pc-35, Pc-40, Pc-58, and Pc-59; and group 61 included virulence to Pc- 36, Pc-51, Pc-56, Pc-60, and Pc-61. Virulence to Pc-70 showed the strongest association to virulences in group 38 but also showed significant association with virulence to Pc-45, Pc-35, and Pc-58. Virulences in group 61 were consistently negatively associated with virulences in group 38 in each region. In Israel, virulences to five of the Iowa lines showed positive associations to virulences in group 61 and negative associations to virulences in groups 38 and 45. Close similarity of reactions of nearly all isolates to Pc-39, Pc-55, and Pc-71 suggest that these genes may be identical or nearly identical alleles.

Leaf Rust and Stem Rust Resistance in Triticum dicoccoides Populations in Israel.

A total of 742 single plant accessions of Triticum dicoccoides were collected from 26 locations in Israel. All accessions were evaluated for leaf rust (Puccinia triticina) resistance in field plots at Tel Aviv, and subsets of 284 and 468 accessions were tested in the greenhouse in Tel Aviv and St. Paul, MN, respectively, for seedling resistance to leaf rust; 460 accessions were also tested for seedling resistance to stem rust (Puccinia graminis f. sp. tritici) in St. Paul. One accession was highly resistant to leaf rust in seedling tests in Tel Aviv, and 21 others had moderately susceptible to moderately resistant seedling resistance. Four accessions were highly resistant to leaf rust in seedling tests in St. Paul, and 11 were resistant to at least one stem rust race. Adult resistance to leaf rust was more common than seedling resistance among the accessions; 21 accessions had less than 25% leaf rust severity in field plots compared with 80 to 90% severity for highly susceptible accessions. Most of the accessions with effective adult plant resistance came from two nearby locations in Upper Galilee, a region where populations of T. dicoccoides are most extensive and genetically diverse. These accessions may provide valuable new partial resistance genes for durable protection against leaf rust in cultivated wheat.

Resistance to Leaf Rust, Stripe Rust, and Stem Rust in Aegilops spp. in Israel.

In all, 1,323 single plant accessions of Aegilops bicornis, A. kotschyi, A. longissima, A. ovata, A. searsii, A. sharonensis, A. speltoides, and A. variabilis collected from 18 regions in Israel and 2 adjacent regions in Lebanon and Egypt were evaluated for leaf rust (Puccinia triticina) and stripe rust (P. striiformis) resistance in field plots and for seedling resistance to leaf rust and stem rust (P. graminis f. sp. tritici) in greenhouse tests. Nearly all accessions of A. speltoides were highly resistant to leaf rust, stripe rust, and stem rust. A. longissima and A. ovata were highly resistant to stripe rust, whereas A. bicornis and A. kotschyi were highly susceptible. A. searsii was highly susceptible to stem rust, but 24 to 51% of accessions of A. bicornis, A. longissima, A. ovata, and A. variabilis were resistant to stem rust. Except for A. ovata and A. speltoides, more than 95% of the Aegilops accessions were susceptible to leaf rust caused by P. recondita alternating on Anchusa spp. Only Aegilops ovata was susceptible to P. recondita from Echium spp. A. bicornis, A. koschyi, and A. searsii were highly susceptible as seedlings to common wheat leaf rust caused by P. triticina. Most accessions of A. variabilis and about half of the accessions of A. longissima had good seedling resistance to P. triticina. Few accessions of A. ovata showed seedling resistance to the P. triticina population in Israel, but 30% were resistant to U.S. isolates. In field tests, A. bicornis showed high susceptibility to common wheat leaf rust, but more than 90% of the accessions of the other Aegilops spp. developed little or no leaf rust on adult plants. The Aegilops spp. in Israel and adjoining countries provide a rich and varied source of rust resistance for wheat breeding.

Morphology, life cycle biology, and DNA sequence analysis of rust fungi on garlic and chives from california.

ABSTRACT In the late 1990s, commercial garlic fields in California (CA) were devastated by an outbreak of rust caused by Puccinia allii. We compared collections of the pathogen from garlic (Allium sativum) and chives (A. schoenoprasum) in central CA and Oregon (OR) to collections from garlic and leek (A. porrum and A. ampeloprasum) in the Middle East. Teliospores from the CA and OR collections were smaller in length, width, and projected cross-sectional area compared with collections from the Middle East. CA and OR collections had a shortened life cycle, in which pycnia and aecia were not formed. Germinating teliospores produced a two-celled promycelium, resulting in two basidiospores, each initially with two nuclei, indicating that this rust was homothallic. In addition, the morphology of the substomatal vesicles was different between the CA-OR (fusiform) and the Middle Eastern (bulbous) collections. DNA sequence analysis of the nuclear ribosomal internal transcribed spacer region showed that the CA and OR rust collections formed a well-supported cluster distinct from the Middle Eastern and European samples. These results suggest that the rust on garlic and chives in CA and OR is a different species than the rust fungus on garlic and leek in the Middle East.

Genetic Variation and Virulence on Lr26 in Puccinia triticina.

ABSTRACT The genetic relationships between isolates of Puccinia triticina virulent on wheat with the Lr26 resistance gene were studied. The diversity within and between isolates of P. triticina from Israel, Europe, and the United States was determined by virulence on near-isogenic Thatcher lines and by random amplified polymorphic DNA. According to the molecular markers, isolates that were virulent on Lr26 had diversity levels similar to those of Lr26 nonpathogenic isolates. Distances between subpopulations of isolates virulent and avirulent on Lr26 varied and were unrelated to the Lr26 virulence phenotype. Cluster analysis suggested four groups, three of which were closely associated with the geographical origin of the isolates-Israel, the United States, and Europe. All four groups included both Lr26 virulent and avirulent pathotypes. The results showed that Lr26 virulent rust pathotypes are as genetically dissimilar as the rest of the population. The cluster analysis showed that the rust population in Israel includes at least two different subpopulations, both of which contain Lr26 virulent and Lr26 avirulent isolates.

Patterns of Virulence in Natural Populations of Puccinia coronata on Wild Oat in Israel and in Agricultural Populations on Cultivated Oat in the United States.

ABSTRACT Crown rust (Puccinia coronata) in indigenous populations of Avena sterilis has been cited as an example of stability of wild pathosystems that consist of natural mixtures of resistance and virulence. This study confirmed that virulence/avirulence polymorphisms in P. coronata on A. sterilis in Israel are highly diverse and that super races do not dominate. Isolates of P. coronata from Israel in 1991 to 1996 were polymorphic for virulence to 35 of 36 differential oat lines with resistance genes from A. sterilis. On average, isolates of P. coronata were more highly virulent to differentials with Pc genes from A. sterilis accessions from Israel than to differentials with Pc genes from other countries. Isolates from Israel also were more virulent on average to 10 additional differentials with Pc genes derived from A. sativa than to differentials with Pc genes from A. sterilis. Frequencies of virulence were usually higher in collections of P. coronata from Israel than in collections from cultivated oat in the United States, even though several of the Pc genes in the differentials have been used extensively in American oat cultivars. Mean virulence complexity of P. coronata from eight regions of Israel was not correlated with the distribution of resistance among collections of A. sterilis from previous surveys in the same areas, probably because pathogen migration between regions within Israel is sufficient to obscure effects of selection locally.

Self-fertility and other distinguishing characteristics of a new morphotype of Puccinia coronata pathogenic on smooth brome grass.

A new morphotype of Puccinia coronata, pathogenic to Bromus inermis, a common roadside and pasture grass in the northern United States, was discovered in the 1990s and described as P. coronata f. sp. bromi by Delgado et al in 2001. Puccinia coronata f. sp. bromi does not require fertilization of pycnia to produce aecia on its alternate host, whereas fertilization is required in all other varieties or formae speciales of P. coronata with aecial hosts in the family Rhamnaceae and for which life cycles have been described. Promycelia of P. coronata f. sp. bromi produce only two basidiospores, and each receives a pair of nuclei from the promycelium. The nuclei divide again so that mature basidiospores each contain four nuclei. Puccinia coronata f. sp. bromi has smaller teliospores than P. coronata var. avenae, and its substomatal vesicles are non-septate and distinctly shaped compared to those of P. coronata var. avenae. In addition, nuclei of P. coronata f. sp. bromi contain less DNA than those of P. coronata var. avenae. Puccinia coronata f. sp. bromi is further distinguished from P. coronata var. avenae and P. coronata var. hordei in being avirulent on both oat and barley, whereas neither P. coronata var. avenae nor P. coronata var. hordei are virulent on Bromus inermis.

Comparative Analysis of Indices in the Study of Virulence Diversity Between and Within Populations of Puccinia recondita f. sp. tritici in Israel.

ABSTRACT Isolates of Puccinia recondita f. sp. tritici (n = 260) obtained from bread, durum, and wild emmer wheat leaf collections throughout Israel during 1993 to 1997 were analyzed for virulence on a set of wheat differentials. The overall frequency of virulence increased on differentials possessing resistance genes Lr1, Lr2a, Lr3, and Lr26 and decreased on Lr17, Lr21, and Lr30. Genes Lr9 and Lr24 were resistant, while Lr18 was susceptible (98% in 1996) to all tested leaf rust isolates and Lr10 and Lr23 were susceptible to more than 78% of the isolates. Diversity between populations (years) was assessed using Kosman's H(KB) (based on degrees of similarity among distinct phenotypes) and H(KDis) (based on frequencies of individual virulences) and Nei's and Rogers' distances. The greatest difference occurred between the 1993 and 1994 populations. Phenotypic diversity within each population (year) was analyzed using the Shannon's, Simpson's, and Kosman's indices. The highest diversity within years was recorded in 1994 and significantly increased from 1993 to 1994 for all indices. The variance in the diversity between populations can be only partially explained by differences between corresponding diversities within population. The comparative analysis of diversity between and within populations over the 5 years enabled a detailed study of changes in the pathogen population. The results show that the different measures do not yield the same rank order of diversity.