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.     

Ascochyta rabiei: A threat to global chickpea production

The necrotrophic fungus Ascochyta rabiei causes Ascochyta blight (AB) disease in chickpea. A. rabiei infects all aerial parts of the plant, which results in severe yield loss. At present, AB disease occurs in most chickpea‐growing countries. Globally increased incidences of A. rabiei infection and the emergence of new aggressive isolates directed the interest of researchers toward understanding the evolution of pathogenic determinants in this fungus. In this review, we summarize the molecular and genetic studies of the pathogen along with approaches that are helping in combating the disease. Possible areas of future research are also suggested.Taxonomykingdom Mycota, phylum Ascomycota, class Dothideomycetes, subclass Coelomycetes, order Pleosporales, family Didymellaceae, genus Ascochyta, species rabiei. Primary host A. rabiei survives primarily on Cicer species.Disease symptoms A. rabiei infects aboveground parts of the plant including leaves, petioles, stems, pods, and seeds. The disease symptoms first appear as watersoaked lesions on the leaves and stems, which turn brown or dark brown. Early symptoms include small circular necrotic lesions visible on the leaves and oval brown lesions on the stem. At later stages of infection, the lesions may girdle the stem and the region above the girdle falls off. The disease severity increases at the reproductive stage and rounded lesions with concentric rings, due to asexual structures called pycnidia, appear on leaves, stems, and pods. The infected pod becomes blighted and often results in shrivelled and infected seeds.Disease management strategiesCrop failures may be avoided by judicious practices of integrated disease management based on the use of resistant or tolerant cultivars and growing chickpea in areas where conditions are least favourable for AB disease development. Use of healthy seeds free of A. rabiei, seed treatments with fungicides, and proper destruction of diseased stubbles can also reduce the fungal inoculum load. Crop rotation with nonhost crops is critical for controlling the disease. Planting moderately resistant cultivars and prudent application of fungicides is also a way to combat AB disease. However, the scarcity of AB‐resistant accessions and the continuous evolution of the pathogen challenges the disease management process.Useful websites https://www.ndsu.edu/pubweb/pulse‐info/resourcespdf/Ascochyta%20blight%20of%20chickpea.pdf https://saskpulse.com/files/newsletters/180531_ascochyta_in_chickpeas‐compressed.pdf http://www.pulseaus.com.au/growing‐pulses/bmp/chickpea/ascochyta‐blight http://agriculture.vic.gov.au/agriculture/pests‐diseases‐and‐weeds/plant‐diseases/grains‐pulses‐and‐cereals/ascochyta‐blight‐of‐chickpea http://www.croppro.com.au/crop_disease_manual/ch05s02.php https://www.northernpulse.com/uploads/resources/722/handout‐chickpeaascochyta‐nov13‐2011.pdf http://oar.icrisat.org/184/1/24_2010_IB_no_82_Host_Plant https://www.crop.bayer.com.au/find‐crop‐solutions/by‐pest/diseases/ascochyta‐blight     

Characterisation of Ascochyta rabiei isolates and evaluation of genotypic stability in chickpea

Ascochyta rabiei isolates were characterised for their variability using a set of host differentials following cloth chamber screening technique. Sixty chickpea genotypes were evaluated against the characterised 10 individual pathotypes separately to identify genotypes with stable resistance during 2007–2008. Twenty four genotypes showed resistance to all the pathotypes; whereas 18 genotypes were resistant to moderately resistant to these pathotypes. The above genotypes can be considered good sources of stable resistance and recommended as donors or for direct cultivation in north western plain zone of India.     

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     

Copper amine oxidase: cunning cofactor and controversial copper

Copper amine oxidases have a complex reaction cycle that converts a primary amine and molecular oxygen into the aldehyde, ammonia and hydrogen peroxide. Coupling structural studies of freeze-trapped reaction intermediates in crystals with kinetic and spectroscopic experiments in solution has generated a detailed molecular picture of catalysis. Although dioxygen has been directly observed bound to the copper at a late stage in the reaction cycle, whether copper is the initial binding site remains controversial.     

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.     

鹰嘴豆种质资源对Ascochyta rabiei的抗性评价及遗传多态性分析

以25 个鹰嘴豆品系为试验材料,通过叶面喷雾的方式进行Ascochyta rabiei菌悬液室内外人工接种,评价不同鹰嘴豆种质资源的抗病性;同时利用RAPD方法进行基因型鉴定,采用NTSYSpc 2.10t软件对分子标记结果进行遗传相似性的统计分析并建立各品系间的亲缘关系聚类图,探讨不同鹰嘴豆品系对A.rabiei抗性与遗传多态性间的关系。通过室内和田间鹰嘴豆抗A.rabiei鉴定结果综合分析表明：在25个鹰嘴豆供试品系中,“系选 03”和“216”品系均表现出稳定抗性特性;北园春品系表现出稳定中抗特性。通过RAPD多态性引物对这25 个供试品系进行PCR扩增,共获得129 个扩增条带,其中多态性条带共有67 条,多态性比例达51.94%,遗传相似系数为0.3731-0.9254。结合抗病性和遗传多态性,经方差分析表明,本研究所采用的鹰嘴豆品系对A.rabiei的抗性强弱与其遗传相似性之间无显著相关性。     

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.     

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.     

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.