Mating type, pathotype and RAPDs analysis inDidymella rabiei, the agent of ascochyta blight of chickpea

Eleven pathotype groups (A-K), including five not previously reported, ofDidymella rabiei (anamorphAscochyta rabiei), representing isolates of the pathogen from Ascochyta blight-affected chickpeas mainly from India, Pakistan, Spain and the USA, were characterized using 44 single-spore isolates tested against seven differential chickpea lines. Of 48 isolates tested for mating type, 58% belonged to MAT 1-1 and 42% to MAT 1-2. Thirty-nineD. rabiei isolates, as well as two isolates ofAscochyta pisi and six isolates of unrelated fungi, were analyzed using Randomly Amplified Polymorphic DNAs (RAPDs) employing five primers (P2 at 40°C, and OPA3, OPC1, OPC11 and OPC20 at 35°C). Computer cluster analysis (UPGMA / NTSYS-PC) detected a relatively low level of polymorphism among all theD. rabiei isolates, although atca 7% dissimilarity,ca 10 RAPD groups [I-X] were demarcated, as well as subclustering within the larger groups. By the same criteria, the maximum dissimilarity for the whole population ofD. rabiei isolates wasca 13%. No correlation was found between different RAPD groups, pathotype, or mating type ofD. rabiei, although some evidence of clustering based on geographic origin was detected. The use of RAPDs enabled us to identify specific DNA fragments that may have a potential use as genetic markers in sexual crosses, but none which could be used as virulence markers.     

Genetic and pathogenic diversity within Ascochyta rabiei(Pass.) Lab. populations in Pakistan causing blight of chickpea (Cicer arietinum L.)

Pathogenic and genetic diversity in Ascochyta rabiei populations in Pakistan were evaluated. Biological pathotyping of 130 A. rabiei isolates (obtained from hierarchically collected samples) was conducted on a set of three chickpea differentials, i.e. ILC 1929 (susceptible), ILC 482 (tolerant) and ILC 3279 (resistant), under controlled conditions. Disease severity data were recorded 12 days after inoculation. Statistical analysis grouped the isolates into three pathotype classes. Four isolates belonged to pathotype I (least aggressive), 79 isolates to pathotype II (medium aggressive) and 47 isolates to pathotype-III (highly aggressive).Genetic analysis was performed using RAPDs and oligonucleotide fingerprinting, where Hinf I-digested DNA was hybridized to the³²P-endlabeled oligonucleotide probes (CAA)₅, (GAA)₅, (GA)₈, (CA)₈and (GATA)₄. Dendrograms produced by cluster analysis discriminated 46 genotypes in the A. rabiei population of Pakistan. Genetic distances and relatedness between isolates were calculated. At a genetic distance of 0.3, genotypes were divided into six distinct genotype groups A, B, C, D, E and F containing 16, 11, 2, 5, 5 and 7 isolates, respectively. Most of the genotypes were area specific or predominated in certain areas but did not belong to a distinct pathotype, while most of the aggressive isolates (pathotype III) occurred in Northern Punjab and in the North Western Frontier Province.     

Aggressiveness of eight <Emphasis Type="Italic">Didymella rabiei</Emphasis> isolates from domesticated and wild chickpea native to Turkey and Israel,a case study

Ascochyta blight, caused by Didymella rabiei, affects both domesticated chickpea and its congeneric wild relatives. The aim of this study was to compare the aggressiveness of D. rabiei isolates from wild and domesticated Cicer spp. in Turkey and Israel on wild and domesticated hosts from both countries. A total of eight isolates of D. rabiei sampled from C. pinnatifidum, C. judaicum and C. arietinum in Turkey and Israel was tested on two domesticated chickpea cultivars and two wild Cicer accessions from Turkey and Israel. Using cross-inoculation experiments, we compared pathogen aggressiveness across the different pathogen and host origin combinations. Two measures of aggressiveness were used, incubation period and relative area under the disease progress curve. The eight tested isolates infected all of the host plants, but were more aggressive on their original hosts with one exception; Turkish domesticated isolates were less aggressive on their domesticated host in comparison to the aggressiveness of Israeli domesticated isolates on Turkish domesticated chickpea. C. judaicum plants were highly resistant against all of the isolates from different origins except for their own isolates. Regardless of the country of origin, the wild isolates were highly aggressive on domesticated chickpea while the domesticated isolates were less aggressive on the wild hosts compared with the wild isolates. These results suggest that the aggressiveness pattern of D. rabiei on different hosts could have been shaped by adaptation to the distinct ecological niches of wild vs. domesticated chickpea.     

Characterization of chickpea differentials for pathogenicity assay of ascochyta blight and identification of chickpea accessions resistant to Didymella rabiei

Forty-eight chickpea germplasm lines, including 22 differentials used in previous studies, were characterized for disease phenotypes following inoculation with six isolates of Didymella (anamorph Ascochyta ) rabiei , representing a wide spectrum of pathogenic variation. Representative isolates were also directly compared with six previously identified races on eight chickpea genotypes. Many of the chickpea differentials reacted similarly to inoculation with each isolate of D. rabiei , and several previously identified races caused similar levels of disease on the differentials. This indicates that the number of differentials can be reduced significantly without sacrificing accuracy in describing pathogenic variation of D. rabiei on chickpea. Pathogenic variation among samples of US isolates allowed classification of the isolates into two pathotypes. The distribution of disease phenotypes of the 48 germplasm lines was bimodal after inoculation with pathotype I isolates, whereas the distribution of disease phenotypes was continuous after inoculation with pathotype II isolates. Such distinct distribution patterns suggest that chickpea plants employ different resistance mechanisms to each pathotype and that the two pathotypes may have different genetic mechanisms controlling pathogenicity. The advantages of using the two-pathotype system in assaying pathogenicity of the pathogen and in studying resistance mechanisms of the host are discussed. Three chickpea accessions, PI 559361, PI 559363 and W6 22589, showed a high level of resistance to both pathotypes, and can be employed as resistance sources in chickpea breeding programmes for resistance to ascochyta blight.     

Structure and pathogenic variability in Ascochyta rabiei populations on chickpea in the Canadian prairies

A population study of Ascochyta rabiei from the Canadian prairies was conducted to assess pathogenicity among isolates with the objectives to investigate the existence of a race or pathotype structure and to evaluate whether there had been a shift to higher aggressiveness between 1998 and 2002. Ninety-nine isolates collected in 1998, 2001 and 2002 were inoculated onto seven differential chickpea genotypes. Significant isolate × differential interactions occurred, but accounted for a small proportion of the total variability. It was found that very few interaction effects between all combinations of differentials and isolates were significant and frequency distributions of disease severity of isolates tested on the differentials revealed continuous distributions. These results suggest that no genotype-specific relationship existed between A. rabiei and its host and that Canadian populations of the pathogen cannot be objectively classified into races or pathotypes. Isolates from 2001 and 2002 caused significantly more disease than isolates from 1998, suggesting that disease epidemics encountered since 1999 were in part caused by a shift in the population to higher aggressiveness.     

华南水稻白叶枯病菌致病性分化检测与分析

为了解华南稻区水稻白叶枯病菌的致病性分化和变异动态,采集华南地区水稻白叶枯病病叶标样分离病原菌,应用中国鉴别寄主IR26、南粳15、爪哇14、特特普、金刚30和国际水稻已知抗病基因的近等基因系IRBB5、IRBB13、IRBB3、IRBB14、IRBB2、IR24两套鉴别寄主,在水稻孕穗期采用剪叶法接种,依据寄主和菌株的互作反应检测病菌的致病性分化。结果显示,参试菌株可划分为Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅸ六个致病型和R1、R2、R3、R4、R5、R8、R10七个致病小种。Ⅴ、Ⅳ致病型和R8、R5小种出现频率分别为27.40%、19.30%和44.67%、15.34%,为华南稻区优势种群。Ⅸ、Ⅴ、Ⅳ致病型和R8、R5小种对500份华南稻区品种资源的致病率依次为96.40%、95.00%、50.40%、62.00%和42.60%;Ⅸ致病型毒性最强且发展很快;强致病菌系Ⅴ型已替代Ⅳ型发展为华南优势致病菌系。     

Towards identifying pathogenic determinants of the chickpea pathogen <Emphasis Type="Italic">Ascochyta rabiei</Emphasis>

Ascochyta blight is a serious disease of cool-season grain legumes (chickpea, faba bean, lentil and pea) caused by fungal species of the anamorphic genus Ascochyta and related genera. Despite extensive studies on the biology, ecology, epidemiology and management of the disease, little is known about the pathogenic determinants of these pathogens. This research aims at using Ascochyta rabiei as a model for the genus in investigating genetic factors of pathogenicity, with the ultimate goal of elucidating pathogenic mechanisms. Three advances were made: (1) insertional mutants with altered pathogenicity were identified through in vivo screening, and genomic regions adjacent to the insertion sites in selected mutants were determined; (2) a phage library of A. rabiei genomic DNA was constructed, and the library was estimated to provide complete coverage of the A. rabiei genome. This library was used successfully to recover clones with DNA adjacent to insertional mutation sites and to isolate specific genes; (3) DNA probes specific for an acyl-CoA ligase (cps1) and a polyketide synthase gene (pks1) were developed and library clones containing the corresponding genomic regions were identified from the phage library. These advances provide the foundation and necessary tools for experimentation of ectopic complementation assays and targeted mutagenesis to elucidate the genetic mechanisms of pathogenicity of A. rabiei.     

Factors Influencing Transmission of Didymella rabiei (Ascochyta Blight) from Inoculated Seed of Chickpea Under Controlled Conditions

Ascochyta blight of chickpea (Cicer arietinum), caused by the fungus Didymella rabiei, has the potential to cause 100% crop loss in severe epiphytotics. Management of this disease often involves reducing sources of inoculum. The influence of sowing depth, host resistance, seed infection level and soil temperature on disease transmission was investigated in a series of glasshouse and growth room trials using seed artificially inoculated with D. rabiei. A positive correlation (R²=0.9992) was observed between rate of seed infection and the incidence of disease on seedlings. Disease transmission to seedlings was not significantly influenced by sowing depth (1, 3 and 6 cm) in separate trials on two cultivars. Susceptibility of the host showed no obvious influence on the frequency of disease transmission in two trials conducted using four cultivars ranging from highly susceptible to moderately susceptible/moderately resistant. Trials conducted in controlled conditions showed that there was no obvious relationship between soil temperature (5, 9, 14 and 19 °C) and the incidence of disease on seedlings.     

Airborne ascospores ofDidymella rabiei as a major primary inoculum for Ascochyta blight epidemics in chickpea crops in southern Spain

The incidence and severity of Ascochyta blight in potted chickpea trap plants exposed for 1-wk periods near infested chickpea debris in Córdoba, Spain, or in chickpea trap crops at least 100 m from infested chickpea debris in several locations in southern Spain were correlated with pseudothecial maturity and ascospore production ofDidymella rabiei from nearby chickpea debris. The period of ascospore availability varied from January to May and depended on rain and maturity of pseudothecia. The airborne concentration of ascospores ofD. rabiei was also monitored in 1988. Ascospores were trapped mostly from the beginning of January to late February; this period coincided with that of maturity of pseudothecia on the chickpea debris. Most ascospores were trapped on rainy days during daylight and 70% were trapped between 12.00 and 18.00 h. Autumn-winter sowings of chickpea were exposed longer to ascospore inoculum than the more traditional spring sowings because the autumn-winter sowings were exposed to the entire period of ascospore production on infested chickpea debris lying on the soil surface.     

Genetics of virulence in Ascochyta rabiei

In order to critically test the hypothesis that virulence variation in the Ascochyta rabiei/chickpea pathosystem is a discrete character under simple genetic control, a genetic cross was made between a highly virulent isolate of A. rabiei from Syria and a less virulent isolate from the USA. Two independent virulence assays conducted by inoculating susceptible and resistant chickpea cultivars under controlled conditions with 77 independent progeny isolates from this cross revealed a continuous distribution of disease phenotypes. Bimodality, as would be predicted for the segregation of virulence under simple genetic control, was not supported by statistical tests of the progeny phenotype distribution. anova revealed highly significant pathogen‐genotype × host‐genotype interactions demonstrating the segregation of genes controlling specialization on the two cultivars tested. These interactions could be localized to two isolates that changed virulence rank on the cultivars. It was concluded that variation in virulence to these two cultivars is under quantitative genetic control. If this conclusion applies to other cultivars, it can be speculated that the discrete categories of virulence variation identified in previous studies were probably the result of incomplete sampling of host resistance or pathogen virulence variation and/or of selection for increased virulence in contemporary A. rabiei populations.     

Alternative hosts and plant tissues for the survival,sporulation and spread of the Ascochyta blight pathogen of chickpea

Various crop and weed species were infected naturally by Didymella rabiei (anamorph: Ascochyta rabiei) in blight-affected chickpea fields in the Palouse region of eastern Washington and northern Idaho, USA. The fungus was isolated from asymptomatic plants of 16 species commonly found in commercial crops in this region. Isolates of the pathogen from crop and weed species were pathogenic to chickpea and indistinguishable in cultural and morphological characteristics from isolates of D. rabiei from chickpea. Both mating types of D. rabiei were isolated from eight naturally infected plant species. Chickpeas were infected by D. rabiei when plants emerged through infested debris of seven crop and weed species. The teleomorph developed on overwintered tissues of seven plant species infected naturally by D. rabiei in a blight screening nursery and on debris of wheat, white sweet clover and pea inoculated with ascospores of D. rabiei or conidia of two compatible isolates of the pathogen. Didymella rabiei naturally infected 31 accessions of 12 Cicer spp. and the teleomorph developed on the overwintered debris of all accessions, including those of three highly resistant perennial species. The fungus developed on the stem and leaf pieces of ten plant species common to southern Spain inoculated with conidia of two compatible isolates of D. rabiei, and formed pseudothecia with asci and viable ascospores on six of ten species and pycnidia with conidia on all plant species.     

Control of seed-borne pathogens on legumes by microbial and other alternative seed treatments

Greenhouse trials were carried out in order to test the efficacy of different seed treatments as alternatives to chemicals against Colletotrichum lindemuthianum cause of anthracnose on bean and Ascochyta spp. cause of Ascochyta blights on pea, respectively. Resistance inducers, commercially formulated microorganisms, non-formulated selected strains of different microorganisms (fungi, bacteria and yeasts) and plant extracts were applied as dry or liquid seed treatments on naturally infested seeds. Seedling emergence and disease incidence and/or severity were recorded. Almost all seed treatments turned out to be ineffective in controlling the Ascochyta infections, which is in line with the literature stating that these pathogens are difficult to control. The only alternative treatments that gave some control of Ascochyta spp. were thyme oil and a strain of Clonostachys rosea. The resistance inducers tested successfully controlled infections of bean by C. lindemuthianum. Among the formulated microorganisms, Bacillus subtilis-based formulations provided the best protection from anthracnose. Some strains of Pseudomonas putida, a disease-suppressive, saprophytic strain of Fusarium oxysporum and the mustard powder-based product Tillecur also proved to be effective against bean anthracnose. However, among the resistance inducers as well as among the other groups, certain agents caused a significant reduction of plant emergence. Different alternative seed treatments can therefore be used for the control of C. lindemuthianum on bean, while on pea only thyme oil and a strain of Clonostachys rosea showed some effectiveness against Ascochyta spp.     

Host status and reaction of <Emphasis Type="Italic">Medicago truncatula</Emphasis> accessions to infection by three major pathogens of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>) and alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis>)

Ditylenchus dipsaci, the stem nematode of alfalfa (Medicago sativa), Mycosphaerella pinodes, cause of Ascochyta blight in pea (Pisum sativum) and Aphanomyces euteiches, cause of pea root rot, result in major yield losses in French alfalfa and pea crops. These diseases are difficult to control and the partial resistances currently available are not effective enough. Medicago truncatula, the barrel medic, is the legume model for genetic studies, which should lead to the identification and characterization of new resistance genes for pathogens. We evaluated a collection of 34 accessions of M. truncatula and nine accessions from three other species (two from M. italica, six from M. littoralis and one from M. polymorpha) for resistance to these three major diseases. We developed screening tests, including standard host references, for each pathogen. Most of the accessions tested were resistant to D. dipsaci, with only three accessions classified as susceptible. A very high level of resistance to M. pinodes was observed among the accessions, none of which was susceptible to this pathogen. Conversely, a high level of variation, from resistant to susceptible accessions, was identified in response to infection by A. euteiches.     

Comparison of the epidemiology of ascochyta blights on grain legumes

Asochyta blights of grain legumes are caused by fungal pathogens in the genus Ascochyta. Different species infect the different legume species, and in pea three species including Phoma medicaginis var. pinodella have been implicated in ascochyta blight. The impact of the diseases varies between crops, countries, seasons and cropping systems, and yield loss data collected under well-defined conditions is scarce. However, ascochyta blights are considered major diseases in many areas where legumes are grown. Symptoms appear on all aerial parts of the plant, and lesions are similar for most of the species, except for M. pinodes and P. medicaginis var. pinodella. Infected seed, stubble and/or air-borne ascospores are major sources of primary inoculum. Their importance varies between species and also between regions. All Ascochyta spp. produce rain-splashed conidia during the cropping season which are responsible for the spread of the disease within the crop canopy. Only in pea are ascospores involved in secondary disease spread. Limited data suggests that Ascochyta spp. may be hemibiotrophs; however, toxins characteristic for necrotrophs have been isolated from some of the species. Modelling of ascochyta blights is still in the developmental stage and implementation of such models for disease forecasting is the exception.     

Validation of a QTL for resistance to ascochyta blight linked to resistance to fusarium wilt race 5 in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.)

Ascochyta blight caused by Ascochyta rabiei and fusarium wilt caused by Fusarium oxysporum. f. sp. ciceris are the two most serious diseases of chickpea (Cicer arietinum). Quantitative trait loci (QTL) or genes for ascochyta blight resistance and a cluster of resistance genes for several fusarium wilt races (foc1, foc3, foc4 and foc5) located on LG2 of the chickpea map have been reported independently. In order to validate these results and study the linkage relationship between the loci that confer resistance to blight and wilt, an intraspecific chickpea recombinant inbred lines (RIL) population that segregates for resistance to both diseases was studied. A new LG2 was established using sequence tagged microsatellite sites (STMS) markers selected from other chickpea maps. Resistance to race 5 of F. oxysporum (foc5) was inherited as a single gene and mapped to LG2, flanked by the STMS markers TA110 (6.5 cM apart) and TA59 (8.9 cM apart). A QTL for resistance to ascochyta blight (QTL_AR3) was also detected on LG2 using evaluation data obtained separately in two cropping seasons. This genomic region, where QTL_AR3 is located, was highly saturated with STMS markers. STMS TA194 appeared tightly linked to QTL_AR3 and was flanked by the STMS markers TR58 and TS82 (6.5 cM apart). The genetic distance between foc5 and QTL_AR3 peak was around 24 cM including six markers within this interval. The markers linked to both loci could facilitate the pyramiding of resistance genes for both diseases through MAS.     

Relation between pathogenicity and genetic variation within <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis>

The relation between diversity of pathogenicity on clubroot-resistant (CR) cultivars of Chinese cabbage (Brassica rapa subsp. pekinensis) bred in Japan and DNA polymorphisms in 17 populations of Plasmodiophora brassicae from cruciferous plants was examined by inoculation tests and random amplified polymorphic DNA (RAPD) analysis using 18 arbitrary primers. Four pathotypes (A–D) were identified after inoculation of six CR cultivars of Chinese cabbage in the 17 populations from cruciferous crops. A relatively high level of genetic diversity was also detected among these populations in the RAPD analysis. Although the four pathotypes could not be clearly differentiated using the RAPD data, most populations of three pathotypes had a consistent location on the dendrogram. All pathotype B (virulent on five cultivars except Utage 70) and D (avirulent on all cultivars) populations, which were common in incompatible interactions with cv. Utage 70, were located in a single subcluster. All five pathotype C populations (virulent only on cv. Utage 70) except for one population grouped in another single subcluster. Because four pathotype A populations (virulent on all six cultivars, races 4 and 9) fell in different subclusters, the populations may be genetically polyphyletic. Populations from cruciferous weed Cardamine flexuosa differed remarkably from those from cruciferous crops in pathogenicity on common cultivars of Chinese cabbage and turnip and C. flexuosa, but they grouped in a single cluster with all race 9 populations from crops. Race 9 populations from crops may thus be closely related to populations from the weed rather than to races 1 and 4 from crops.     

Aggressiveness and its role in the adaptation of plant pathogens

Aggressiveness, the quantitative component of pathogenicity, and its role in the adaptation of plant pathogens are still insufficiently investigated. Using mainly examples of biotrophic and necrotrophic fungal pathogens of cereals and Phytophthora infestans on potato, the empirical knowledge on the nature of aggressiveness components and their evolution in response to host and environment is reviewed. Means of measuring aggressiveness components are considered, as well as the sources of environmental variance in these traits. The adaptive potential of aggressiveness components is evaluated by reviewing evidence for their heritability, as well as for constraints on their evolution, including differential interactions between host and pathogen genotypes and trade-offs between components of pathogenicity. Adaptations of pathogen aggressiveness components to host and environment are analysed, showing that: (i) selection for aggressiveness in pathogen populations can be mediated by climatic parameters; (ii) global population changes or remarkable population structures may be explained by variation in aggressiveness; and (iii) selection for quantitative traits can influence pathogen evolution in agricultural pathosystems and can result in differential adaptation to host cultivars, sometimes leading to erosion of quantitative resistance. Possible links with concepts in evolutionary ecology are suggested.     

Intrapathotype diversity for aggressiveness and pathogen evolution in cultivar mixtures

ABSTRACT A model was developed and used to study the consequences of diversity for aggressiveness within pathotypes on pathogen evolution in two-component and four-component cultivar mixtures. It was assumed that, within a pathotype, a proportion of the isolates would have higher or lower spore efficacy than the average on a given host genetic background. Two situations were examined in which the pathogen can have either independent or negatively correlated values for spore efficacy on different cultivars. In the latter case, a pathogen genotype more aggressive than the average on a host genotype was always less aggressive on other host genotypes. In the simulations, isolates with greater aggressiveness relative to a host genotype were selected for and increased in frequency. However, because simple pathotypes always reproduced on the same host genotype whereas complex pathotypes were able to grow on several hosts, selection was faster for simple pathotypes. Pathotypes with two different levels of diversity for aggressiveness were compared with nondiversified pathotypes. In order to make comparisons, the effect of a 5 and 10% cost of virulence on the development of complex pathotypes was simulated. In general, increased diversity within pathotypes reduced the rate of increase of complex pathotypes in host mixtures, and this effect was stronger with greater frequencies of autodeposition of pathogen spores.     

Sources of resistance to ascochyta blight in wild Cicer species

Ascochyta blight [Ascochyta rabiei (Pass.) Lab.] is the major foliar disease of chickpea (Cicer arietinum L.). In search of better sources of resistance to ascochyta blight, 201 accessions of 8 annual wildCicer species were evaluated in field and greenhouse for 3 years (1988 to 1991) at Tel Hadya, Syria. One accession each ofC. judaicum Boiss (ILWC 165) andC. pinnatifidum Jaub. & Spach. (ILWC 159) were consistently rated resistant in both field and greenhouse evaluations. Another three accessions ofC. judaicum (ILWC 61, ILWC 154, ILWC 199) and six accessions ofC. pinnatifidum (ILWC 78, ILWC 88, ILWC 155, ILWC 160, ILWC 162, ILWC 203) were resistant or moderately resistant. The blight-resistant accessions ofC. judaicum originated from Jordan, Lebanon, Syria, and Turkey; and those ofC. pinnatifidum from Syria and Turkey. None of the accessions ofC. bijugum, C. chorassanicum, C. cuneatum, C. echinospermum, C. reticulatum andC. yamashitae were resistant to blight.