RAPD Analysis of Pathogenic Variability in Ascochyta rabiei

Thirty Italian isolates of the phytopathogenic fungus Ascochyta rabiei (Pass.) Labr., the causal organism of Ascochyta blight on chickpea (Cicer arietinum L.), were analysed by a random oligonucleotide primer dependent polymerase chain, reaction (PCR) technique called random amplified polymorphic DNA analysis (RAPD) using three decamer primers. In previous investigations these isolates had been differentiated in six pathogenic groups. RAPD results were summarized in an analysis using the program PAUP. With each of the primers several amplification products were observed which were common to all isolates. The results of the RAPD analyses also showed that all isolates could be identified by a unique RAPD pattern. No correlation between RAPD patterns and the division of the isolates in pathogenic groups could be established. The application of the RAPD technique for cataloguing isolates and to obtain specific genetic markers for all isolates of the species Ascochyta rabiei is discussed.     

Cloning and characterization of the mating type (MAT) locus from Ascochyta rabiei (teleomorph: Didymella rabiei) and a MAT phylogeny of legume-associated Ascochyta spp

Degenerate primers designed to correspond to conserved regions of the high mobility group (HMG) protein encoded by the MAT1-2 gene of Cochliobolus heterostrophus, Cochliobolus sativus, and Alternaria alternata were used to amplify the portion of the sequence corresponding to the HMG box motif from Ascochyta rabiei (teleomorph: Didymella rabiei). A combination of TAIL and inverse PCR extended the MAT1-2 sequence in both directions, then primers designed to MAT1-2 flanking DNA were used to amplify the entire MAT1-1 idiomorph. MAT1-1 and MAT1-2 idiomorphs were 2294 and 2693 bp in length, respectively, and each contained a single putative open reading frame (ORF) and intron similar to MAT loci of other loculoascomycete fungi. MAT genes were expressed at high levels in rich medium. MAT-specific PCR primers were designed for use in a multiplex PCR assay and MAT-specific PCR amplicons correlated perfectly to mating phenotype of 35 ascospore progeny from a cross of MAT1-1 by MAT1-2 isolates and to the mating phenotype of field-collected isolates from diverse geographic locations. MAT-specific PCR was used to rapidly determine the mating type of isolates of A. rabiei sampled from chickpea fields in the US Pacific Northwest. Mating type ratios were not significantly different from 1:1 among isolates sampled from two commercial chickpea fields consistent with the hypothesis that these A. rabiei populations were randomly mating. The mating type ratio among isolates sampled from an experimental chickpea field where asexual reproduction was enforced differed significantly from 1:1. A phylogeny estimated among legume-associated Ascochyta spp. and related loculoascocmycete fungi using sequence data from the nuclear ribosomal internal transcribed spacer (ITS) demonstrated the monophyly of Ascochyta/Didymella spp. associated with legumes but was insufficiently variable to differentiate isolates associated with different legume hosts. In contrast, sequences of the HMG region of MAT1-2 were substantially more variable, revealing seven well-supported clades that correlated to host of isolation. A. rabiei on chickpea is phylogenetically distant from other legume-associated Ascochyta spp. and the specific status of A. rabiei, A. lentis, A. pisi, and A. fabae was confirmed by the HMG phylogeny     

Histological and Ultrastructural Changes in Leaves and Stems of Resistant and Susceptible Chickpea Cultivars to Ascochyta rabiei

The histo- and cytopathological effects in resistant (ILC-195) and susceptible (Canitez-87) chickpea cultivars were examined by light, transmission and scanning electron microscopy 3, 5 and 7 days after inoculation (d.a.i) of seedlings with Ascochyta rabiei. The fungus produced typical appressoria that penetrated both cuticle and stomata. The resistant plants had physical barriers and a cuticle layer against fungal penetration 3 d.a.i. The fungus spread intercellularly and subepidermally in the leaves and stems of susceptible plants 3 d.a.i., and was followed 5 d.a.i. by cell plasmolysis, degeneration of organelles and of cellulose, but not lignified, walls. Pycnidia formation occurred between 5 and 7 d.a.i. 7 d.a.i., organelle degeneration, pycnidia formation and symptom severity increased. Tracheidal elements, including lignified elements, were almost intact in both resistant and susceptible cultivars. In the susceptible plants, lignin cell walls were slightly degraded after 7 days. There was less cell degeneration and pycnidia formation in resistant plants. Some electron-dense large bodies and lipid granules were observed within intracellular fungal hyphae in infected cells of resistant plants 7 d.a.i.     

Reduction in solanapyrone phytotoxin production by Ascochyta rabiei transformed with Agrobacterium tumefaciens

Agrobacterium tumefaciens was used to transform Ascochyta rabiei, the causal agent of chickpea blight. Employing a T-DNA containing a hygromycin resistance gene (hph), 908 transformants were obtained from germinated pycnidiospores on a selective medium containing hygromycin. Transformants were confirmed using PCR and Southern analyses and of four of these that were tested, two had integrated multicopies of the hph gene, one had two copies and one had a single insertion. Transformants were tested for the production of solanapyrone A toxin using a microtitre plate assay. Loss of toxin production by transformants was confirmed by reversed phase high-performance liquid chromatography. Sixteen transformants out of 668 tested produced significantly less solanapyrone A than the wild-type strain.     

Virulence diversity of Ascochyta rabiei the causal agent of Ascochyta blight of chickpea in the western provinces of Iran

Chickpea is the third most important food legume in the world. The most important limiting factor for the chickpea production in the world, including Iran, has been the Ascochyta blight. The pathogenic variation of 40 Ascochyta rabiei isolates from the western provinces of Iran was assessed on eight chickpea differential lines. The results revealed that A. rabiei population is diverse in the western provinces of Iran and the virulence rating of isolates across differential lines showed a large but continuous pathogenic variability. Based on the statistical analysis and the continuous response in differential lines, it was not possible to categorise A. rabiei isolates in the present study into pathotypes or races. Information obtained from the current study can be valuable in developing quarantine methods aimed to prevent dissemination of highly virulent isolates and in the development of durable resistant cultivars against the Ascochyta blight of chickpea.     

Allelic variation at a hypervariable compound microsatellite locus in the ascomycete Ascochyta rabiei

The genome of the fungal chickpea pathogen Ascochyta rabiei was screened for polymorphisms by microsatellite-primed PCR. While ethidium-bromide staining of electrophoretically separated amplification products showed only limited polymorphism among 24 Tunisian A. rabiei isolates, Southern hybridization of purified PCR fragments to restriction digests of fungal DNA revealed polymorphic DNA fingerprints. One particular probe that gave rise to a hypervariable single-locus hybridization signal was cloned from the Syrian isolate AA6 and sequenced. It contained a large compound microsatellite harbouring the penta- and decameric repeat units (CATTT)_n, (CATTA)_n, (CATATCATTT)_n and (TATTT)_n. We call this locus ArMS1 (Ascochyta rabiei microsatellite 1). Unique flanking sequences were used to design primer pairs for locus- specific microsatellite amplification and direct sequencing of additional ArMS1 alleles from Tunisian and Pakistani isolates. A high level of sequence variation was observed, suggesting that multiple mutational mechanisms have contributed to polymorphism. Hybridization and PCR analyses were performed on the parents and 62 monoascosporic F₁ progeny derived from a cross between two different mating types of the fungus. Progeny alleles could be traced back to the parents, with one notable exception, where a longer than expected fragment was observed. Direct sequencing of this new length allele revealed an alteration in the copy number of the TATTT repeat [(TATTT)₅₃ to (TATTT)₆₅], while the remainder of the sequence was unchanged.     

Degradation of the Isoflavone Biochanin A and Its Glucoside Conjugates by Ascochyta rabiei

Strains of Ascochyta rabiei which are pathogenic to chickpea (Cicer arietinum L.) readily catabolized the main chickpea isoflavone biochanin A (5,7-dihydroxy-4′-methoxyisoflavone). 3′-Hydroxylation and O-demethylation reactions led to the isoflavones pratensein, genistein, and orobol, which were rapidly further degraded. Dihydrogenistein and p-hydroxyphenylacetic acid were also identified as catabolites. Biochanin A-7-O-glucoside was degraded, leading to aglycone and pratensein. Biochanin A-7-O-glucoside-6″-O-malonate, the main phenolic constituent of chickpea, was very slowly degraded without subsequent accumulation of catabolites.     

Studies on Cell Number and Nuclei in Spores and on Ploidy Level in Ascochyta rabiei Isolates

Isolates of the phytopathogenic ascomycete Ascochyta rabiei (Pass.) Labr. were stained with the DNA-specific fluorochrome 4',6-diamidino-2-phenylindole (DAPI) and compared for differences in number of nuclei per pycnidiospore and the ploidy level. Microscopic analyses revealed that within the examined isolates five different combinations of cell number and number of nuclei in spores exist. A one-celled spore may contain one, two and four nuclei, respectively, and in the case of two-celled spores there exist types with one and two nuclei in one cell. Microfluorometric analyses of wild types and benomyl-treated isolates revealed differences in ploidy level among the wild types.     

Role of amine oxidase expression to maintain putrescine homeostasis in Rhodococcus opacus

While applications of amine oxidases are increasing, few have been characterised and our understanding of their biological role and strategies for bacteria exploitation are limited. By altering the nitrogen source (NH₄Cl, putrescine and cadaverine (diamines) and butylamine (monoamine)) and concentration, we have identified a constitutive flavin dependent oxidase (EC 1.4.3.10) within Rhodococcus opacus. The activity of this oxidase can be increased by over two orders of magnitude in the presence of aliphatic diamines. In addition, the expression of a copper dependent diamine oxidase (EC 1.4.3.22) was observed at diamine concentrations > 1 mM or when cells were grown with butylamine, which acts to inhibit the flavin oxidase. A Michaelis–Menten kinetic treatment of the flavin oxidase delivered a Michaelis constant (K_M) = 190 μM and maximum rate (k_cat) = 21.8 s^?1 for the oxidative deamination of putrescine with a lower K_M (=60 μM) and comparable k_cat (=18.2 s^?1) for the copper oxidase. MALDI–TOF and genomic analyses have indicated a metabolic clustering of functionally related genes. From a consideration of amine oxidase specificity and sequence homology, we propose a putrescine degradation pathway within Rhodococcus that utilises oxidases in tandem with subsequent dehydrogenase and transaminase enzymes. The implications of PUT homeostasis through the action of the two oxidases are discussed with respect to stressors, evolution and application in microbe-assisted phytoremediation or bio-augmentation.     

Elicitor-induced metabolic changes in cell cultures of chickpea (Cicer arietinum L.) cultivars resistant and susceptible to Ascochyta rabiei

Cell-suspension cultures of two chickpea (Cicer arietinum L.) cultivars, resistant (ILC 3279) and susceptible (ILC 1929) to the fungus Ascochyta rabiei (Pass.) Lab., showed differential accumulation of the phytoalexins medicarpin and maackiain, and transient induction of related enzyme activities after application of an A. rabiei-derived elicitor. The chalcone-synthase (CHS) activity (EC 2.3.1.74) which is involved in the first part of phytoalexin biosynthesis exhibited a maximum 8–12 h after elicitation in the cells of both cultivars. Concomitant with the fivefold-higher phytoalexin accumulation, CHS activity increased twofold in the cells of the resistant cultivar. The maximum of the elicitor-induced CHS-mRNA activity was determined 4 h after onset of induction in the cultures of both cultivars, although in cells of cultivar ILC 3279 this mRNA activity was induced at a level twofold higher than that in cells of the susceptible race ILC 1929. Investigations of CHS isoenzymes by two-dimensional gel electrophoresis of immunoprecipitated in-vitro-translated protein indicated the presence of five proteins. In the cells of both cultivars only two of the isoenzymes were induced after elicitor treatment. Analysis of the total in-vitro-translated proteins by two-dimensional gel electrophoresis showed that the constitutively expressed patterns of mRNA activities in the cell cultures of the two cultivars were identical. After elicitation, considerably more translatable mRNAs were induced in the cells of cultivar ILC 3279. The few induced proteins, and their respective mRNA activities, which could be detected in the cells of the susceptible cultivar, all existed in the cells of the resistant cultivar, too. One highly induced protein (M_r 18 kDa) found in the cells of cultivar ILC 3279 reached its maximum mRNA activity 6 h after elicitor application. The amount of this protein was hardly increased in the cells of the susceptible cultivar. This protein appears to be excreted from the cells into the growth medium.Abbreviations CHS chalcone synthase - IEF isoelectric focussing - ILC international legume chickpea - PR-protein pathogenesis-related protein - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Financial support by Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie is gratefully acknowledged. The authors thank Dr. K. Hahlbrock (Max-Planck-Institut für Züchtungsforschung, Köln, FRG) for provision of antisera and the International Centre for Agricultural Research in the Dry Areas (Aleppo, Syria) for plant material.     

Mapping quantitative trait loci in chickpea associated with time to flowering and resistance to Didymella rabiei the causal agent of Ascochyta blight

Drought is the major constraint to chickpea (Cicer arietinum L.) productivity worldwide. Utilizing early-flowering genotypes and advancing sowing from spring to autumn have been suggested as strategies for drought avoidance. However, Ascochyta blight (causal agent: Didymella rabiei (Kov.) v. Arx.) is a major limitation for chickpea winter cultivation. Most efforts to introgress resistance to the pathogen into Kabuli germplasm resulted in relatively late flowering germplasm. With the aim to explore the feasibility of combining earliness and resistance, RILs derived from a cross between a Kabuli cultivar and a Desi accession were evaluated under field conditions and genotyped with SSR markers. Three quantitative trait loci (QTLs) with significant effects on resistance were identified: two linked loci located on LG4 in epistatic interaction and a third locus on LG8. Two QTLs were detected for time to flowering: one in LG1 and another on LG2. When resistance and time to flowering were analyzed together, the significance of the resistance estimates obtained for the LG8 locus increased and the locus effect on days to flowering, previously undetected, was significantly different from zero. The identification of a locus linked both to resistance and time to flowering may account for the correlation observed between these traits in this and other breeding attempts.