New Avirulence Genes in the Phytopathogenic Fungus Leptosphaeria maculans

ABSTRACT Leptosphaeria maculans, the causal agent of stem canker of oilseed rape (Brassica napus), develops gene-for-gene interactions with oilseed rape, and four L. maculans avirulence (AVR) genes (AvrLm1, AvrLm2, AvrLm4, and alm1) were previously genetically characterized. Based on the analysis of progeny of numerous in vitro crosses between L. maculans isolates showing either already characterized or new differential interactions, this work aims to provide an overview of the AVR genes that may specify incompatibility toward B. napus and the related species B. juncea and B. rapa. Two novel differential interactions were thus identified between L. maculans and B. napus genotypes, one of them corresponding to a complete resistance to European races of L. maculans. In both cases, a single gene control of avirulence was established (genes AvrLm3 and AvrLm7). Similarly, a single gene control of avirulence toward a B. rapa genotype, also resistant to European L. maculans isolates, was demonstrated (gene AvrLm8). Finally, a digenic control of avirulence toward B. juncea was established (genes AvrLm5 and AvrLm6). Linkage analyses demonstrated that at least four unlinked L. maculans genomic regions, including at least one AVR gene cluster (AvrLm1-AvrLm2-AvrLm6), are involved in host specificity. The AvrLm3-AvrLm4-AvrLm7 region may correspond either to a second AVR gene cluster or to a multiallelic AVR gene.     

Identification of Molecular Markers Linked to a Pyrenophora teres Avirulence Gene

ABSTRACT Genetic control of avirulence in the net blotch pathogen, Pyrenophora teres, was investigated. To establish an appropriate study system, a collection of 10 net form (P. teres f. teres) and spot form (P. teres f. maculata) isolates were evaluated on a set of eight barley lines to identify two isolates with differential virulence on an individual host line. Two net form isolates, WRS 1906, exhibiting avirulence on the cv. Heartland, and WRS 1607, exhibiting high virulence, were mated and 67 progeny were isolated and phenotyped for reaction on Heartland. The population segregated in a 1:1 ratio, 34 avirulent to 33 virulent (chi(2) = 0.0, P = 1.0), indicating single gene control of WRS 1906 avirulence on Heartland. Bulked segregant analysis was used to identify six amplified fragment length polymorphism markers closely linked to the avirulence gene (Avr(Heartland)). This work provides evidence that the P. teres-barley pathosystem conforms to the gene-for-gene model and represents an initial step toward map-based cloning of this gene.     

Relationship between Avirulence Genes of the Same Family in Rice Blast Fungus Magnaporthe grisea

A genetic cross between rice-field isolates of Magnaporthe grisea produced progeny segregating for avirulence/ virulence on six rice cultivars among nine race differentials, while on three other cultivars, Shin 2 (Pik-s), Aichi Asahi (Pia) and Ishikari Shiroke (Pii), parental and progeny isolates were all virulent. Based on segregation ratios in 115 progeny isolates, avirulence on Kanto 51 (Pik), Yashiro-mochi (Pita), Fukunishiki (Piz) and Toride 1 (Piz-t) is under monogenic control. On Tsuyuake (Pik-m) and Pi No. 4 (Pita-2), however, a disproportionate ratio in the segregation was observed, suggesting that avirulence on these two cultivars is controlled by two or more genes. Assuming that the avirulence gene AvrPik-m consists of at least two genes, AvrPik-m1 and AvrPik-m2, each of which functions in the whole gene AvrPik-m, and that one of AvrPik-m1 and AvrPik-m2 is AvrPik, we could account for the disproportion in the avirulence/virulence segregation of the progeny. This hypothesis would also be consistently applied for avirulence gene AvrPita-2. There seem to be two types of the avirulence genes : AvrPik-m, that is comprised of the tightly linked genes, AvrPik-ml (=AvrPik) and AvrPik-m2, and AvrPita-2, that is comprised of the loosely linked genes AvrPita-2A (=AvrPita) and AvrPita-2B. As one possible explanation of the rice resistant reaction to blast, multiple specificity was suggested for the first time for the blast fungus. On the contrary, the avirulence genes AvrPiz and AvrPiz-t were inherited independently, despite the corresponding genes for resistance (Piz and Piz-t) being located at the same locus. The cross of rice blast isolates (races 447 and 337) produced only 25 kinds of races in the progeny, although theoretically about 64 kinds of races should be produced if six avirulence genes segregated independently. Because no progeny are with AvrPik (or AvrPita) and without AvrPik-m (or AvrPita-2), the number of races theoretically should be 36 at most. A number of strains, such as races 377 and 737, with a single avirulence gene were obtained from this cross. These strains may be valuable for analysis of resistance genes in rice plant. Received 19 August 2002/ Accepted in revised form 11 November 2002     

Virulence and Molecular Polymorphism in International Collections of the Wheat Leaf Rust Fungus Puccinia triticina

ABSTRACT Collections of Puccinia triticina, the wheat leaf rust fungus, were obtained from Great Britain, Slovakia, Israel, Germany, Australia, Italy, Spain, Hungary, South Africa, Uruguay, New Zealand, Brazil, Pakistan, Nepal, and eastern and western Canada. All single-uredinial isolates derived from the collections were tested for virulence polymorphism on 22 Thatcher wheat lines that are near-isogenic for leaf rust resistance genes. Based on virulence phenotype, selected isolates were also tested for randomly amplified polymorphic DNA (RAPD) using 11 primers. The national collections were placed into 11 groups based on previously established epidemiological zones. Among the 131 single-uredinial isolates, 105 virulence phenotypes and 82 RAPD phenotypes were described. In a modified analysis of variance, 26% of the virulence variation was due to differences in isolates between groups, with the remainder attributable to differences within groups. Of the RAPD variation, 36% was due to differences in isolates between groups. Clustering based on the average virulence distance (simple distance coefficient) within and between groups resulted in eight groups that differed significantly. Collections from Australia-New Zealand, Spain, Italy, and Britain did not differ significantly for virulence. Clustering of RAPD marker differences (1 - Dice coefficient) distinguished nine groups that differed significantly. Collections from Spain and Italy did not differ significantly for RAPD variation, neither did collections from western Canada and South America. Groups of isolates distinguished by avirulent/virulent infection types to wheat lines with resistance genes Lr1, Lr2a, Lr2c, and Lr3 also differed significantly for RAPD distance, showing a general relationship between virulence and RAPD phenotype. The results indicated that on a worldwide level collections of P. triticina differ for virulence and molecular backgrounds.     

Identification of an Avirulence Gene in the Fungus Magnaporthe grisea Corresponding to a Resistance Gene at the Pik Locus

ABSTRACT A rice isolate of Magnaporthe grisea collected from China was avirulent on rice cvs. Hattan 3 and 13 other Japanese rice cultivars. The rice cv. Hattan 3 is susceptible to almost all Japanese blast fungus isolates from rice. The genetic basis of avirulence in the Chinese isolate on Japanese rice cultivars was studied using a cross between the Chinese isolate and a laboratory isolate. The segregation of avirulence or virulence was studied in 185 progeny from the cross, and monogenic control was demonstrated for avirulence to the 14 rice cultivars. The resistance gene that corresponds to the avirulence gene (Avr-Hattan 3) is thought to be located at the Pik locus. Resistance and susceptibility in response to the Chinese isolate in F(3) lines of a cross of resistant and susceptible rice cultivars were very similar to the Pik tester isolate, Ken54-20. Random amplified polymorphic DNA markers and restriction fragment length polymorphism markers from genetic maps of the fungus were used to construct a partial genetic map of Avr-Hattan 3. We obtained several flanking markers and one co-segregated marker of Avr-Hattan 3 in the 144 mapping population.     

A Study on the Phylogeny of the Dyer's Woad Rust Fungus and Other Species of Puccinia from Crucifers

ABSTRACT The identity of a Puccinia species occurring on the introduced weed dyer's woad (Isatis tinctoria) was studied using sequences from the internal transcribed spacer of the nuclear ribosomal DNA. The relationship of this fungus to other Puccinia species occurring on the family Brassicaceae in Europe and North America was examined, and we tested the hypothesis that P. thlaspeos and P. monoica are correlated species. The data suggest that the Puccinia species from dyer's woad is closely related to the North American species P. consimilis and may be derived from an indigenous strain of P. consimilis that switched hosts. Thus, the Puccinia species from dyer's woad is probably native to North America and is unlikely to cause disease epidemics on indigenous plants if used as a biological control agent against dyer's woad. P. thlaspeos appears to be polyphyletic and, therefore, P. thlaspeos and P. monoica do not appear to be correlated species. Additional DNA sequence data will be needed to clarify further the phylogeny of Puccinia species on the family Brassicaceae.     

Genetic Analysis and Identification of Amplified Fragment Length Polymorphism Markers Linked to the alm1 Avirulence Gene of Leptosphaeria maculans

ABSTRACT A gene-for-gene interaction was previously suggested by mapping of a single major locus (LEM 1) controlling cotyledon resistance to Leptosphaeria maculans isolate PHW1245 in Brassica napus cv. Major. In this study, we obtained further evidence of a gene-for-gene interaction by studying the inheritance of the corresponding avirulence gene in L. maculans isolate PHW1245. The analysis of segregating F(1) progenies and 14 test crosses suggested that a single major gene is involved in the interaction. This putative avirulence gene was designated alm1 after the resistance locus identified in B. napus. Amplified fragment length polymorphism (AFLP) markers were used to generate a rudimentary genetic linkage map of the L. maculans genome and to locate markers linked to the putative avirulence locus. Two flanking AFLP markers, AC/TCC-1 and AC/CAG-5, were linked to alm1 at 3.1 and 8.1 cM, respectively. Identification of markers linked to the avirulence gene indicated that the differential interaction is controlled by a single gene difference between parental isolates and provides further support for the gene-for-gene relationship in the Leptosphaeria-Brassica system.     

Specificity of Prehaustorial Resistance to Puccinia hordei and to Two Inappropriate Rust Fungi in Barley

ABSTRACT To elucidate the specificity of prehaustorial resistance to inappropriate rust fungi, we studied two populations of recombinant inbred lines of barley that segregated for partial resistance (PR) to Puccinia hordei and for the resistance to the inappropriate rust species P. recondita f. sp. tritici and P. hordei-murini. PR to P. hordei is prehaustorial and nonhypersensitive, and its level can be assessed accurately by measuring the latent period of the fungus. The resistance to the inappropriate rust species is a combination of prehaustorial (nonhypersensitive) and posthaustorial (hypersensitive) mechanisms. The amount of nonhypersensitive, early abortion of P. recondita f. sp. tritici and P. hordei-murini sporelings reflects the degree of prehaustorial defense to the two inappropriate rust species. All lines showing a long latent period of P. hordei also had a relatively high level of early abortion of the growth of P. recondita f. sp. tritici and P. hordei-murini. This indicates that genes for PR to P. hordei are also effective against these two inappropriate rust species. The reverse was not necessarily true; some lines showing a high level of early abortion of P. recondita f. sp. tritici and P. hordei-murini had a low level of PR to P. hordei. Moreover, lines with a similar level of prehaustorial resistance to P. recondita f. sp. tritici could differ considerably in their prehaustorial resistance to P. hordei-murini. This indicates that genes for prehaustorial resistance may exhibit rust species specificity.     

Relationship Among Genes Conferring Partial Resistance to Leaf Rust (Puccinia triticina) in Wheat Lines CI 13227 and L-574-1

ABSTRACT This study describes the segregation of genes for resistance to the fungus Puccinia triticina in a cross between partially resistant wheat lines L-574-1 and CI 13227 with two and four genes for resistance, respectively. The objectives of this study were to use parental, F(1), F(2), and backcross populations to quantify maternal effects, degree of dominance, and transgressive segregation, and to determine whether CI 13227 and L-574-1 share any resistance genes for long latent period or small uredinia. In two experiments conducted in the greenhouse, the uppermost leaf of adult wheat plants was inoculated prior to heading with P. triticina. On days 6 to 21 after inoculation, the number of uredinia that erupted from the leaf surface was counted and used to calculate the mean latent period (MLP). The length and width of five arbitrarily selected uredinia were measured and used to calculate uredinium area. Midparent values, degree of dominance, and broad-sense heritability were calculated for MLP and uredinium area. For experiment A, MLP values for CI 13227, L-574-1, F(1), and F(2) generations were 12.2, 10.5, 10.2, and 10.6 days, respectively. For experiment B, MLP values for CI 13227, L-574-1, F(1), F(2), backcross to CI 13227, and backcross to L-574-1 were 12.3, 10.0, 10.6, 10.8, 11.1, and 10.0 days, respectively. The inheritance of long latent period was partially recessive, and no maternal effect was present (P = 0.62 to 0.87 for the comparison of means in reciprocal crosses). Broad-sense heritability for MLP ranged from 0.72 to 0.74, and there was transgressive segregation in the F(2) and backcross populations. Uredinia of the F(1) generation were slightly larger than uredinia for CI 13227. The inheritance of uredinium size was partially dominant, and no maternal effect was present (P = 0.5 to 0.63). Broad-sense heritability for uredinium area ranged from 0.36 to 0.73 and transgressive segregation was present in the F(2) and backcross populations. The results for MLP indicate that lines CI 13227 and L-574 likely share one gene for resistance (based on F(1) values) but not two genes (based on the presence of transgressive segregation). CI 13227 and L 574-1 appear to have at least one gene difference for uredinium area. The linear relationship between uredinium area regressed onto MLP was significant (P < 0.001) and r(2) values ranged from 0.14 to 0.26. These results indicate that the resistance in CI 13227 and L-574-1 could be combined to create wheat cultivars with greater partial resistance than that possessed by either parent based on MLP or uredinium size.     

水稻防御反应相关基因对稻瘟病菌的响应

水稻与稻瘟病菌的互作已成为研究植物与病原真菌互作的模式系统。利用RT-PCR技术检测了6个水稻品种(分别含有抗病基因Pik-s、Pita、Pit、Pi1、Pi9的近等系及回交亲本丽江新团黑谷LTH)与稻瘟病菌互作过程中多个信号相关及PR基因的表达。结果表明带有稻瘟病抗性基因Pik-s、Pita、Pit的水稻品种和LTH对稻瘟病菌#626侵染表现为亲和互作,带Pi1和Pi9的水稻品种表现为非亲和互作;稻瘟病菌接种后,亲和互作中MAPK6和MAPK12表现为上调表达,带有抗性基因Pi9的水稻品种IRBL22中BIMK2表现为上调表达。总体来看,含有不同抗病基因的水稻近等系中的PR基因对稻瘟病菌的响应较为多样,非亲和互作中在早期或早中期表现为PR基因上调表达,而亲和互作中主要在晚期上调表达,说明这些PR基因表达的时间在植物与病原互作的不同时期发挥着不同的作用。     

Genetic variation in Puccinia graminis collected from oats, rye, and barberry

Puccinia graminis, the causal agent of stem rust, was collected from its alternate host barberry (Berberis spp.) and two different uredinial hosts, oats (Avena sativa) and rye (Secale cereale). The samples were analyzed using 11 polymorphic simple sequence repeat (SSR) markers. There were large differences between fungal populations on oats (P. graminis f. sp. avenae) and rye (P. graminis f. sp. secalis), and the genetic variation within the different formae speciales was also high. It was possible to distinguish between the two formae speciales on barberry. Additional genotypic groups not present in the field samples from oats and rye were also identified on barberry. Our results confirm the importance of barberry in maintaining the populations of P. graminis in Sweden and the importance of the sexual stage for the survival of the pathogen.     

Genes Governing Resistance to Puccinia hordei in Thirteen Spring Barley Accessions

ABSTRACT Leaf rust, caused by Puccinia hordei, is an important disease of barley in many parts of the world. In the eastern United States, this disease was effectively controlled for over 20 years through the deployment of cultivars carrying the resistance gene Rph7. Isolates of P. hordei with virulence for Rph7 appeared in this region in the early 1990s rendering barley cultivars with this gene vulnerable to leaf rust infection. From a preliminary evaluation test, 13 accessions from diverse geographic locations possessed resistance to P. hordei isolate VA90-34, which has virulence for genes Rph1, 2, 4, 6, 7, 8, and 11. Each of these 13 accessions was crossed with susceptible cvs. Moore or Larker to characterize gene number and gene action for resistance to P. hordei. Additionally, the 13 accessions were intercrossed and crossed to host differential lines possessing genes Rph3, Rph5, and Rph9 to determine allelic relationships of resistance genes. Seedlings of F(1), F(2), and BC(1)F(1) populations were evaluated in the greenhouse for their reaction to P. hordei isolate VA90-34. Leaf rust resistance in six of the accessions including Collo sib, CR270.3.2, Deir Alla 105, Giza 119, Gloria, and Lenka is governed by a single dominant gene located at or near the Rph3 locus. All accessions for which the gene Rph3 was postulated to govern leaf rust resistance, except for Deir Alla 105, likely possess an allele different than Rph3.c found in Estate based on the differential reaction to isolates of P. hordei. The resistance gene in Grit and Donan is located at or near the Rph9 locus. Alleles at both the Rph3 and Rph9 loci confer resistance in Femina and Dorina. In addition to Rph3, Caroline and CR366.13.2 likely possess a second unknown recessive gene for leaf rust resistance. Resistance in Carre 180 is governed by a recessive gene that is different from all other genes considered in this study. Identification of both known and unique genes conferring leaf rust resistance in the barley germplasm included in this study provides breeding programs with the knowledge and opportunity to assess currently used sources of leaf rust resistance and to incorporate new sources of resistance into their programs.     

Genetic Diversity in Australian Populations of Puccinia graminis f. sp. avenae

ABSTRACT Sequence-tagged microsatellite profiling was used to develop 110 microsatellites for Puccinia graminis f. sp. tritici (causal agent of wheat stem rust). Low microsatellite polymorphism was exhibited among 10 pathogenically diverse P. graminis f. sp. tritici isolates collected from Australian cereal growing regions over a period of at least 70 years, with two polymorphic loci detected, each revealing two alleles. Limited cross-species amplification was observed for the wheat rust pathogens, P. triticina (leaf rust) and P. striiformis f. sp. tritici (stripe rust). However, very high transferability was revealed with P. graminis f. sp. avenae (causal agent of oat stem rust) isolates. A genetic diversity study of 47 P. graminis f. sp. avenae isolates collected from an Australia-wide survey in 1999, and a historical group of 16 isolates collected from Australian cereal growing regions from 1971 to 1996, revealed six polymorphic microsatellite loci with a total of 15 alleles. Genetic analysis revealed the presence of several clonal lineages and subpopulations in the pathogen population, and wide dispersal of identical races and genotypes throughout Australian cereal-growing regions. These findings demonstrated the dynamic population structure of this pathogen in Australia and concur with the patterns of diversity observed in pathogenicity studies.     

Genetics of resistance to race TTKSK of Puccinia graminis f. sp. tritici in Triticum monococcum

Race TTKSK (or Ug99) of Puccinia graminis f. sp. tritici possesses virulence to several stem rust resistance genes commonly present in wheat cultivars grown worldwide. New variants detected in the race TTKSK lineage further broadened the virulence spectrum. The identification of sources of genetic resistance to race TTKSK and its relatives is necessary to enable the development and deployment of resistant varieties. Accessions of Triticum monococcum, an A-genome diploid wild and cultivated wheat, have previously been characterized as resistant to stem rust. Three resistance genes were identified and introgressed into hexaploid wheat: Sr21, Sr22, and Sr35. The objective of this study was to determine the genetic control and allelic relationships of resistance to race TTKSK in T. monococcum accessions identified through evaluations at the seedling stage. Generation F(2) progeny of 8 crosses between resistant and susceptible accessions and 13 crosses between resistant accessions of T. monococcum were evaluated with race TTKSK and often with North American races, including races QFCSC, TTTTF, and MCCFC. For a selected population segregating for three genes conferring resistance to race TTKSK, F(2:3) progeny were evaluated with races TTKSK, QFCSC, and TTTTF. In that population, we detected two genes conferring resistance to race TTKSK that are different from Sr21, Sr22, and Sr35. One of the new genes was effective to all races tested. The identification of these genes will facilitate the development of varieties with new resistance to race TTKSK.     

PCR-based Assays to Assess Wheat Varietal Resistance to Blotch (Septoria Tritici and Stagonospora Nodorum) and Rust (Puccinia Striiformis and Puccinia Recondita) Diseases

A multiplex Polymerase Chain Reaction (PCR) assay was developed to detect and quantify four fungal foliar pathogens in wheat. For Septoria tritici (leaf blotch) and Stagonospora nodorum (leaf and glume blotch), the -tubulin gene was used as the target region. Diagnostic targets for Puccinia striiformis (stripe or yellow rust) and P. recondita (brown rust) were obtained from PCR products amplified with -tubulin primer sequences. Final primer sets were designed and selected after being tested against several fungi, and against DNA of infected and healthy wheat leaves. For detection of the four pathogens, PCR products of different sizes were amplified simultaneously, whereas no products were generated from wheat DNA or other non-target fungi tested. The presence of each of the diseases was wheat tissue- and cultivar specific. Using real-time PCR measurements with the fluorescent dye SYBR Green I, PCR-amplified products could be quantified individually, by reference to a standard curve generated by adding known amounts of target DNA. Infection levels for each of the diseases were measured in the flag leaf of 19 cultivars at Growth Stage (GS) 60–64 in both 1998 and 1999. The infection levels for the cultivars were ranked, and showed, with a few exceptions, a good correlation with the NIAB Recommended List for winter wheat, which is based on visual assessment of symptoms. With PCR, the presence of the different pathogens was accurately diagnosed and quantification of pre-symptomatic infection levels was possible. Although sampling and DNA detection methods need further optimisation, the results show that multiplex PCR and quantitative real-time PCR assays can be used in resistance screening to measure the interaction between different pathogens and their hosts at different growth stages, and in specific tissues. This should enable an earlier identification of specific resistance mechanisms in both early-stage breeding material and field trials.     

我国小麦秆锈菌种群动态分析

１９９５～１９９６年间,在我国７省区６２个县点共收集标样２８３份,涉及品种１１７个,分离到菌株４５５份,鉴定出１５个致病类型,其出现类型的发生频率分别是２１Ｃ３ＣＫＲ为１７．５％,２１Ｃ３ＣＦＨ９．６％,２１Ｃ３ＣＫＨ８．８％,２１Ｃ３ＣＦＲ６．０％,２１Ｃ３ＣＴＲ２２．０％,２１Ｃ３ＣＰＲ９．４％,２１Ｃ３ＣＴＨ８．８％,２１Ｃ３ＣＰＨ１１．９％,３４ＭＫＧ１．３％,３４Ｃ２ＭＫＨ０．２％,３４Ｃ２ＭＫＫ０．１％,３４Ｃ２ＭＦＨ３．６％,３４Ｃ２ＭＦＫ０．４％,３４Ｃ１ＭＫＲ和３４Ｃ１ＭＦＲ为０．２％。１９９６年２１Ｃ３ＣＫＲ的出现频率上升为第１位,对Ｓｒ１１有毒力的致病类型出现频率有所下降。