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.     

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.     

Different ecological affinities and aggressiveness patterns among Didymella rabiei isolates from sympatric domesticated chickpea and wild Cicer judaicum

Domesticated chickpea (Cicer arietinum) and its wild relative C. judaicum grow in sympatric distribution in Israel and both are susceptible to Ascochyta blight caused by Didymella rabiei. C. arietinum was grown for millennia in drier and hotter Levantine spring conditions while C. judaicum grows in the wetter and milder winters. Accordingly, it is possible that D. rabiei isolates originated from C. arietinum are adjusted to the less favorable spring conditions. Here, 60 isolates from both origins were tested in vitro for their hyphal growth at 15 and 25 degrees C. Isolates from C. arietinum had a significantly larger colony area at 25 degrees C than at 15 degrees C (P < 0.001) while no such differences were detected between isolates from C. judaicum. D. rabiei isolates from wild and domesticated origins were used to inoculate nine C. judaicum accessions and two domesticated chickpea cultivars and their aggressiveness patterns were determined using five measures. On domesticated chickpea, isolates from domesticated origin were significantly more aggressive in four out of the five aggressiveness measures than isolates from wild origin. On C. judaicum, isolates from wild origin were generally more aggressive than isolates from domesticated origin. The results suggest that the habitat segregation between wild and domesticated Cicer influences the pathogens ecological affinities and their aggressiveness patterns.     

Distribution of Mating Types and Diversity in Virulence of <Emphasis Type="Italic">Didymella rabiei</Emphasis> in Israel

The distribution of mating types and diversity in virulence of Didymella rabiei populations were studied in Israel from 1997 to 1999. Forty-one monoconidial D. rabiei isolates from 18 commercial fields distributed among all the chickpea production areas of the country were paired with MAT1-1 and MAT1-2 mating type tester isolates of D. rabiei. Both mating types were found in all chickpea production areas of the country. Of the 18 fields sampled, MAT1-1 was observed in 44%, and MAT1-2 in 88% of the sites. In some sites both mating types were present in close proximity, suggesting that sexual reproduction of the pathogen was feasible. The contribution of sexual reproduction of the fungus to virulence diversity was tested on detached leaves of six differential chickpea cultivars. Nine isolates were derived from different well separated foci (derived from ascospores as inoculum) and eight isolates were derived from a single, well defined infection focus (derived from sister conidia). In the analyses of variance the cultivar × isolate interaction showed no significant (P of F>0.09) effect on disease incidence; the chickpea cultivars differed significantly (P of F<0.0001) in their response to D. rabiei; and the isolate effect was highly significant (P of F = 0.0007) for the conidial population, but not significant (P of F>0.1) among isolates of the ascosporic population. Nevertheless, when comparing a cultivar at a time, the ascosporic and conidial populations did not differ significantly (P of F>0.1) in their virulence diversity. Virulence of 41 isolates collected from the different chickpea fields was tested on detached leaves of four Israeli cultivars that differ in their field response to D. rabiei. The cultivar × isolate interaction showed no significant effect (P of F = 0.95) on disease incidence. The main effects of cultivar and isolate on disease incidence were highly significant (P of F<0.0001). Accordingly, our data do not support the hypothesis that there is pathogenic specialization in the D. rabiei–C. arietinum pathosystem in Israel.     

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.     

Development of the teleomorph of Ascochyta rabiei on culture media

Ascochyta blight caused by Didymella rabiei (anamorph: Ascochyta rabiei) is an important foliar disease of chickpea in many countries. The fungus is heterothallic and requires the pairing of two compatible mating types for the teleomorph to develop. In nature, the teleomorph only develops on chickpea debris that overwinters on the soil surface in the presence of both mating types. When natural and synthetic agar media were seeded with conidial suspensions of compatible isolates of D. rabiei from Spain and the United States and incubated under favourable conditions for teleomorph development, the teleomorph only developed on 2?% water agar amended with powdered chickpea stems or hot water extracts of chickpea stems, but not on 14 other natural or synthetic media. Ascospore isolates of D. rabiei from pseudothecia that developed on agar media were indistinguishable in cultural and morphological characteristics from isolates of the fungus from chickpea. Production of pseudothecia and ascospores on the best culture medium was always lower than on stem pieces of chickpea straw used as a control treatment. Ascospores discharged from pseudothecia that developed on powdered chickpea stem media onto chickpea seedlings were pathogenic, inducing symptoms identical to those caused by ascospores from chickpea stem pieces or conidia from a chickpea isolate of the fungus. This is the first report of the teleomorph of D. rabiei developing on culture media.     

Sympatric ascochyta complex of wild Cicer judaicum and domesticated chickpea

The aim of this study was to isolate, identify and characterize ascochyta blight pathogens from Cicer judaicum , a wild annual Cicer species which grows in Israel and other Mediterranean countries in sympatric distribution with legume crops, and determine their virulence and aggressiveness to other wild and domesticated legumes. Native C. judaicum plants exhibited symptoms resembling ascochyta diseases of grain legume crops. Two distinct pathogens were isolated and identified as Phoma pinodella and Didymella rabiei using morphological and molecular tools; their infectivity was verified using Koch's postulates. The virulence of these pathogens was examined on 13 legume species, of which P. pinodella was virulent to Pisum sativum , P. fulvum , C. judaicum , C. arietinum , C. reticulatum , C. pinnatifidum and C. bijugum . Didymella rabiei infected all these Cicer species, but not the other legume species tested. Aggressiveness of the pathogens was tested on wild and domesticated chickpea and pea. Didymella rabiei isolated from C. judaicum had significantly higher ( P  < 0·001) aggressiveness than P. pinodella from C. judaicum on both wild and domesticated chickpea. Disease severity on the former species ranged from 62·5% to 70% and on the latter from 41% to 56%. Phoma pinodella isolates from C. judaicum were more aggressive on C. arietinum and P. sativum than on C. judaicum and P. fulvum . Results of the current study suggest that C. judaicum may serve as an alternative host to ascochyta pathogens that endanger chickpea and possibly other crops and wild species growing in close proximity.     

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.     

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.     

Resistance to root-lesion nematode Pratylenchus neglectus identified in a new collection of two wild chickpea species (Cicer reticulatum and C. echinospermum) from Turkey

Chickpea (Cicer arietinum) is a major legume crop, with Australia being the second largest producer worldwide. Pratylenchus neglectus is a root-lesion nematode that invades, feeds and reproduces in roots of pulse and cereal crops. In Australia, chickpea and wheat (Triticum aestivum) are commonly grown in rotation and annual damage by P. neglectus accounts for large economic losses to both crops. Cultivated chickpea has narrow genetic diversity that limits the potential for improvement in resistance breeding. New collections of wild chickpea species, C. reticulatum and C. echinospermum, have substantially increased the previously limited world collection of wild Cicer germplasm and offer potential to widen the genetic diversity of cultivated chickpea through the identification of accessions with good resistance. This research assessed 243 C. reticulatum and 86 C. echinospermum accessions for response to P. neglectus in replicated experiments under controlled glasshouse conditions from 2013 and 2014 collection missions that were received, tested and analysed in two experimental sets. Multi-experiment analyses showed lower P. neglectus population densities in both sets of wild Cicer accessions tested than Australia's elite breeding cultivar PBA HatTrick at the significance level p < 0.05. Provisional resistance ratings were given to all genotypes tested in both experimental sets, with C. reticulatum accessions CudiB_008B and Kayat_066 rated as resistant in both Set 1 and Set 2. New sources of resistance to P. neglectus observed in this study can be introgressed into commercial chickpea cultivars to improve their resistance to this nematode.     

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.     

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.     

Towards the first linkage map of the<Emphasis Type="Italic">Didymella rabiei</Emphasis> genome

A genetic map was developed for the ascomyceteDidymella rabiei (Kovachevski) v. Arx (anamorph:Ascochyta rabiei Pass. Labr.), the causal agent of Ascochyta blight in chickpea (Cicer arietinum L.). The map was generated with 77 F₁ progeny derived from crossing an isolate from the U.S.A. and an isolate from Syria. A total of 232 DAF (DNA Amplification Fingerprinting) primers and 37 STMS (Sequence-Tagged Microsatellite Site) primer pairs were tested for polymorphism between the parental isolates; 50 markers were mapped, 36 DAFs and 14 STMSs. These markers cover 261.4cM in ten linkage groups. Nineteen markers remained unlinked. Significant deviation from the expected 1:1 segregation ratios was observed for only two markers (Prob. of χ²<0.05). The implications of our results on ploidy level of the asexual spores are discussed. http://www.phytoparasitica.org posting Sept. 5, 2002.     

Diagnostics,genetic diversity and pathogenic variation of ascochyta blight of cool season food and feed legumes

Molecular diagnostic techniques have been developed to differentiate the Ascochyta pathogens that infect cool season food and feed legumes, as well as to improve the sensitivity of detecting latent infection in plant tissues. A seed sampling technique was developed to detect a 1% level of infection by Ascochyta rabiei in commercial chickpea seed. The Ascochyta pathogens were shown to be genetically diverse in countries where the pathogen and host have coexisted for a long time. However, where the pathogen was recently introduced, such as A. rabiei to Australia, the level of diversity remained relatively low, even as the pathogen spread to all chickpea-growing areas. Pathogenic variability of A. rabiei and Ascochyta pinodes pathogens in chickpea and field pea respectively, appears to be quantitative, where measures of disease severity were based on aggressiveness (quantitative level of infection) rather than on true qualitative virulence. In contrast, qualitative differences in pathogenicity in lentil and faba bean genotypes indicated the existence of pathotypes of Ascochyta lentis and Ascochyta fabae. Therefore, reports of pathotype discrimination based on quantitative differences in pathogenicity in a set of specific genotypes is questionable for several of the ascochyta-legume pathosystems such as A. rabiei and A. pinodes. This is not surprising since host resistance to these pathogens has been reported to be mainly quantitative, making it difficult for the pathogen to overcome specific resistance genes and form pathotypes. For robust pathogenicity assessment, there needs to be consistency in selection of differential host genotypes, screening conditions and disease evaluation techniques for each of the Ascochyta sp. in legume-growing countries throughout the world. Nevertheless, knowledge of pathotype diversity and aggressiveness within populations is important in the selection of resistant genotypes.     

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.     

The sympatric Ascochyta pathosystems of Near Eastern legumes,a key for better understanding of pathogen biology

The primary and secondary centres of origin of domesticated plants are often also the places of origin of their pathogens. Therefore, the Near Eastern cradle of agriculture, where crop plants, their wild progenitors, and other con-generic taxa grow sympatrically, may hold some clues on the biology of the pathogens of the respective crops. Unlike the situation in the well-studied Near Eastern cereals and their important diseases, hardly any data are available on basic questions regarding grain legumes. What is the role of genetic diversity at resistance loci of the wild hosts and is it greater compared with the cultigens? Are populations of Ascochyta pathogens infecting wild legumes genetically distinct from populations infecting their domesticated counterparts, and if so, is this differentiation related to differences in host specialization or to adaptation to different ecological conditions? Do isolates originating from wild taxa exhibit a similar level of aggressiveness and have different aggressiveness alleles compared with those originating from domesticated grain legumes? In this review we propose an experimental framework aimed at gaining answers to some of the above questions. The proposed approach includes comparative epidemiology of wild vs. domesticated plant communities, co-evolutionary study of pathogens and their hosts, phenotypic and genetic characterization of fungal isolates from wild and domesticated origins, and genetic analyses of pathogenicity and parasitic fitness among progeny derived from crosses between isolates from wild and domesticated hosts.     

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.     

Variation in resistance to Orobanche crenata in species of Cicer

Crenate broomrape (Orobanche crenata) is a major constraint for legume cultivation in Mediterranean agriculture. Field trials, pot and in vitro experiments demonstrated that resistance to O. crenata is present in chickpea and wild species of Cicer. The resistance is the result of the combination of several mechanisms, including low induction of parasite seed germination and in some accessions, either a darkening at the infection site on the host root that prevents establishment, or a reduced development of established parasite tubercles.     

Climatic Zoning and Plant Disease Potential – Examples from the Near and Middle East1

Actual plant disease and pest occurrence depends on many genetic and environmental factors, and frequently obscures the basic suitability of a given location to support or prevent epidemic development. In order to allow the demarcation of climatic zones related to the potential of disease or pest occurrence, we have used long-term average climatic data, especially monthly average temperatures and monthly average rainfall. If applied to sugar beet leaf pathogens such as Cercospora beticola and Erysiphe betae in the Near and Middle Eastern region, some interesting zoning became possible, which could be verified by extended field studies. Other examples that have been analysed in the region are apple scab, Venturia inaequalis, and downy mildew of grapes, Plasmopara viticola. A recent and ongoing analysis of the factors controlling chickpea anthracnose caused by Ascochyta rabiei indicates that the same principle may be applied for very different pathogens. Large-scale planning and control strategies as tried by the International Agricultural Research Centers should therefore be based on careful climatic zoning for plant pest and disease potential, to avoid waste of the limited genetic and financial resources available.