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Biosci. 2015 International Journal of Biosciences | IJB | ISSN: 2220-6655 (Print), 2222-5234 (Online) http://www.innspub.net Vol. 6, No. 4, p. 30-39, 2015 RESEARCH PAPER Comparison of OPEN ACCESS Ascochyta rabiei isolates for cultural characteristics and isozyme Gharbi Samia1*, Karkachi Noureddine2, Chhiba Mostafa3, Kihal Merbrouk2, Henni Jamal Eddine2. 1 Department of Biotechnology, University of Sciences and the Technology of Oran, Algeria 2 Department of Biology, University of Oran, Algeria 3 Faculty of Sciences and Technology, University HASSAN 1st, Settat, Morocco Key words: Ascochyta raiei, Esterase, Acid phosphatase, Superoxide dismutase, Leucine aminopeptidase. http://dx.doi.org/10.12692/ijb/6.4.30-39 Article published on February 28, 2015 Abstract The comparison of our isolates on the basis of macroscopic and microscopic criteria reveals differences between 32 isolates of Ascochyta rabiei, it is distributed as follows, light brown, dark brown and green (50, 22 and 28%). Pathogenicity is performed on three varieties of chickpea (Flip 90-13, Flip 33-99 and ILC 1799), which showed no aggressiveness with the two varieties Flip 90-13, but with the variety ILC 1799 showed a pathological variability of 10 isolates of Ascochyta rabiei. Electrophoretic study of five isoenzymes esterase (Est), acid phosphatase (CAP), leucine aminopeptidase (LAP), superoxide dismutase (SOD), peroxidase (POX) showed that only Esterase and acid phosphatase isoenzyme have an polymorohisme. A dendrogram is established, integrating all the data. It reveals the importance of genome diversification within the species. * Corresponding Author: Gharbi Samia  samiakarkachi@hotmail.com 30 Samia et al. Int. J. Biosci. 2015 Introduction Morphological study Chickpea (Cicer arietinum) is the world’s third most The study of the macroscopic characters of the important pulse crop after bean (Phaseolus vulgaris) isolates is based on the morphological description and and peas (Pisum sativum), ascochyta blight caused by on the pigmentation of the colonies incubated during Ascochyta rabiei (Pass.) Labrousse, is a serious foliar 10 days on the flour medium of chickpea at a disease the infection resulting in severe yield losses in temperature of 22 C° and under a 12 hours Asian and African countries and all areas of production photoperiod. The microscopic study is carried out in a chickpea in the world (Singh et. al. 1997; Ilarslan and fresh state; fragments are taken from the superficial Dolar, 2002; Gharbi et. al. 2013). part of the 10-day old colonies grown on medium CSMA, then It has been widely studied in term of morphology, pathology, phytotoxin production and we proceeded to the optical microscopic observation. DNA fingerprints, but there is currently no information Pathogenicity test available regarding the use of isozyme analysis in This test is carried out with the varieties of chickpea the study of population structure of the fungus. (90-13 Flip, Flip 33-99 and ILC 1799), provided by the Algerian National Institute of Agronomic In order to better understand genetic diversity at Research of Sidi Bel Abbes (Algeria). The six-day old Ascochyta rabiei, it appeared necessary to us to seedlings are pulverized with 10 ml of a suspension of prospect other tools for characterization that the 105spores/ml, 10 seedlings were used for each isolate, morphological study and pathogenesis test. For this and then the seedlings are covered with a transparent purpose, we chose to study the polymorphism plastic film during 72 hours, followed by several isoenzymatic pulverizations of soluble mycelia proteins. The with water to maintain a locus, humidification, in order to facilitate the germination frequently have variable electrophoretic motilities. of the spores on the organs of the seedling. The This is due to variations in the contents in amino- degree of the pathogenicity is evaluated using 1- 9 acids of the molecules, which depend on the rating scale as suggested by Reddy et. al. 1984. enzymes, coded by different alleles or nucleotide sequence of the DNA (Micales et. al. 1986; Sample preparation Rosendahl and Sen, 1992). To obtain mycelia extracts for intracellular enzymes, The objective of this article is studied biometric five 5 mm plugs of mycelia from a 7 day old culture characterization, isozyme were transferred to 50 ml of growth medium GYP polymorphism to determine variability among various (peptone 1%, yeast extract 1%, glucose 1%) in 250 ml isolates of A. rabiei collected from different regions of Erlenmeyer flasks and incubated without agitation at West Algeria. 125 rpm at 25°C for 10 days. Mycelium was collected pathogenicity and on filter paper by filtration and washed carefully with Materials and methods distilled water then it was crushed in a mortar A total of 32 isolates of Ascochyta rabiei isolates used (maintained cold in the ice) in the presence of sand in this study were collected from tree different regions until obtaining a fine and homogeneous paste, we (Ain Temouchent, Mascara and Sidi Bel Abbes) of the added phosphate buffer (100 mM; pH 7,1) at 1ml/g, western was the samples was centrifuged at 10000 g for 20 performed from stems of plants showing symptoms of minutes at 4°C. The supernatant is distributed in Ascochyta blight (Khan et. al. 1999; Ilarslan and Eppondorf tubes by fraction of 100 µl, then, either Dolar, 2002; Chen et. al. 2004, Bayraktar et. al. used immediately for the electrophoresis or preserved 2007; Gharbi et. al. 2013). at the freezer. Algeria. Isolation 31 Samia et al. of the fungus Int. J. Biosci. 2015 Electrophoresis Protein Electrophoretic mobility or distance of migration extracts to vertical from the origin was recorded for all isozyme bands. In electrophoresis. Native this study the bands were scored 1 (same location) or polyacrylamide electrophoresis was conducted using 0 (absent). A phonetic dendrogram based on the a Bio-Rad Mini-PROTEAN II (glass plates: 8 x 10 estimated similarity coefficients was constructed by cm, combs: thickness 1,5 mm). The gel of acrylamide Xlstat (version 2013). polyacrylamide were gel subjected includes the stacking gel 5%,the gel buffer was 0.125 M Tris-HCl (pH 6.8), the separating gel 10% with Results 0.375 M Tris-HCl (pH 8.8) ), running buffer was Tris- The glycine (pH 8.3). Approximately 10 to 15 μl of protein macroscopic extract was placed in wells with sample buffer (0.5 M differences between isolates (Table 02). In general, Tris-HCl, 0.05% we recorded the color of the mycelium is very bromophenol blue). The gels were run at 120 V, 50 variable; it can be light brown, brown or green (Fig 1). mA per gel for 2 hours at 4°C. On the culture medium CSMA, mycelia colonies pH 6.8, 1% glycerol, and comparison and of isolates on microscopic the basis criteria of reveals develop slowly. They are first creamy, and then take Enzyme assay extremely After electrophoresis, the gels were stained according culturing, they generally present very characteristic to their enzyme system with the appropriate substrate concentric streaks. This character, we have chosen as and chemical solutions then incubated at room a criterion of morphological characterization. The temperature in dark for complete staining. In most phenomenon appears as zoning of sporulating cases incubation for about 1 to 2 hours is enough. The mycelia bands at alternating with bands of mycelia chemical ingredients are listed in Table (1). with low sporulating. The zonation pycnidia observed varied colors among isolates, after in all isolates. Data analysis Table 1. The staining solutions of isozymes. Enzyme Ingredients Quantity Authors Esterase (EC. 3.1.1.1) EST Fast bleu RR 100 mM Na-phosphate buffer pH 6.0 α- naphthyl acetate 2 % (acetone) β- naphthyl acetate 2 % (acetone) β-Naphtyl acide phosphate Fast Garnet GBC 50 mM Acetate buffer pH 5 L. Leucyl β-Naphtylamide HCl H2O Fast Black K Salt 20 mM Tris Maleate buffer pH 3,3 NaOH 2O mM Solution A : 50 mM Na-phosphate buffer pH 7,5 10 mg/ml MTT Solution B : 50 mM Na-phosphate buffer pH 7,5 TEMED Riboflavin 3 Amino, 9Ethyl carbazole Diméthyl formamide 50 mM acetate buffer pH 5 Ca₂Cl₂ (10 mM) H₂O₂ (30%) 20 mg 50 ml 1 ml 1 ml 50 mg 50mg 100 ml 50 mg 30 ml 50 mg 50 ml 20 ml Jonathan and Wendel, 1990 El-Sharabasy and Wanas, 2008 Shaw and Prasad ,1970 Acid phosphatase (EC. 3.1.3.2) PAC Leucine aminopeptidase (EC.3.4.1.1) LAP superoxide dismutase (EC. 1.15.1.1) SOD Perioxdase (EC.1.11.1.7) POX 32 Samia et al. Pan and Chen, 1988 Beckman et. al. 1964 Jevremović et. al. 2010 100ml 1 ml 100 ml 0,2 ml 1 mg 40mg 5ml 92ml 2ml 30µl Graham et. al. 1964 Li, 1981 Int. J. Biosci. 2015 We recorded the color of the colonies of 32 isolates of Temouchent aspect includes three aspects (light Ascochyta rabiei is distributed as follows, light brown, brown and green) the percentage is (34, 33 brown, dark brown and green (50, 22 and 28%) and 33%) respectively (Fig 2), along with the regions respectively (Fig 1) and no significant difference (P = of Mascara and Sidi Bel Abbes, with a rate of (43, 14 0.709) was recorded, distribution according to the and 43%) ; (54, 23 and 23%) respectively (Fig 3 and aspect of the regions is as follows, the region Ain Fig 4). Table 1. Morphological characters of isolates Ascochyta rabiei. N° Isolate region Morphological aspect 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 A4 A6 A32 A1 A2 A3 A8 A11 A13 A14 A18 A19 A20 A21 A25 A30 A31 A33 A34 A36 A37 A38 A39 A41 A42 A9 A10 A12 A16 A17 A24 A29 Ain Temouchent Light Brown Brown Brown Light Brown Light Brown Light Brown Light Brown Light Brown Light Brown Light Brown Light Brown Light Brown Light Brown Light Brown Light Brown Brown Brown Brown Brown Brown Green Green Green Green Green Light Brown Light Brown Light Brown Brown Green Green Green Sidi Bel Abbes Mascara Table 2. Test result of the pathogenicity of isolates Ascochyta rabiei. isolat Flip 90 A3 A6 A8 A11 A12 A25 A26 A27 A30 A31 33 Samia et al. 13 Flip 33 0 1 1 3 0 1 1 2 1 2 99c ILC 1 1 1 1 1 1 1 2 3 1 1799 8 7 7 8 6 6 6 5 4 6 Int. J. Biosci. 2015 On medium CSMA, color Pycnidia varies among pycniospores out mass pycnidia, mired in mucus. isolates. So it varies from pink-brown (Fig 6), but They are hyaline, ovate to oblong straight or slightly most of the isolates pink-brown color dominance. curved and rounded at the tip a few are celled but Pycnidia are globular, dark brown, 65 to 245 μm in most are unicellular, they measure (8,2-10 x 4,2-4,5 diameter and equipped with an ostiole. At maturity, μm). a Fig. 1. Different morphological aspects of Ascochyta rabiei isolates cultured on culture medium CSMDA (a: light rown; b: brown; c: green). Flip ILC 1799), after a few days of the inoculation of young seedlings, the isolates behave differently with these three varieties (Table 3), we have found that all isolates exhibit low aggressiveness the varieties of Flip and Flip 33-99c 90-13, we noted the presence of necrosis in leaves as well as stem lesions. The symptoms obtained during the last reading are assessed according to the scale at 9 points; it is Fig . 2. Different morphological aspects of Ascochyta rabiei isolates from western Algeria. primarily based on the response of the host and the development of symptoms on different aerial parts of the plant. Fig . 3. Different morphological aspects of Ascochyta Fig . 4. Different morphological aspects of Ascochyta rabiei isolates from Ain Temouchent. rabiei isolates from Mascara. Pathogenicity Karkachi et. al. 2014, they studied the macroscopic The study of pathogenicity is an important passage to variability confirm the isolates affiliation to species and to Fusarium oxysporum f.sp albedinis, they found that evaluate the level of virulence, this study was carried there are different aspects in the collection of Foa, on three varieties of chickpea (Flip 90-13, 33-99 and they showed that the ras type is majority, it present 34 Samia et al. and the microscopic appearance of Int. J. Biosci. 2015 50%, the downy type that has 16% other isolates have isolates (75%) and three pathotype II aggressive a compact and cottony mycelium having 33% of the isolates (18.75%) pathotype III (Benzohra et. al. 2010 collection. Sarwar et. al. ; Benzohra et. al. 2011 ; Benzohra et. al. 2013). 2013, evaluated the pathogenicity of 21 isolates of Ascochyta rabiei using three varieties of chickpea differentials, the variety (Aug424 and Pb-1) sensitive variety (Aug480 and CM72) and tolerant varieties (Paidar and CM88) resistant. The pathogenicity test on susceptible cultivars, resulting in the onset of symptoms involving lesions on leaves and stems and even in severe cases have resulted in death of plants (Sayed et. al. 2009). Elliott et. al. 2013, evaluated the degree of pathogenicity of a collection of isolates of Ascochyta rabiei on two growth stages of chickpea on Fig. 6. Microscopic observations of pynides and pycnidiospores of isolates of Ascochyta rabiei. seedlings and growth stages of maturity (P <0.000), they observed significant mean differences, although isolates are similar in degree of pathogenicity, their ranking on this principle was different, this suggests that the genotype-specific expression of genes for resistance to Ascochyta rabiei can be related to the stage of growth of chickpea. Three isolates Ascochyta rabiei are used to test their degrees of virulence with 52 varieties of chickpea. In addition, almost all chickpea genotypes revealed a high degree of sensitivity to pathogens except some genotypes were moderately resistant (Lamichhane et. al. 2012). Fig. 7. Esterase isozymes profiles of Ascochyta rabiei. Esterase Bands of different colors appear on gels, isoenzymes reacting with the beta naphthyl acetate produce a red band, and those reacting with alpha naphthyl acetate produce a purple band. The esterase zymogram of observation is performed after detection of the enzyme by the substrate α-naphthyl acetate (Fig 7). Four bands were revealed. They show the relative Fig. 5. Different morphological aspects of Ascochyta motilities of between 0.28 and 0.58, different isolates rabiei isolates from Sidi Bel Abbes. can be detected by the number and position of the bands identified. The heterogeneity of esterase The study of the pathogenicity of sixteen isolates of zymogram gives a fairly good representation of the Ascochyta rabiei from seven regions of north-western genetic complexity. Algeria was determined pathotypes and physiological races with seven differential chickpea lines. All Acid Phosphatase isolates were classified into three pathotypes and 6 Zymogram analysis reveals the anode and cathode for physiological races according to their aggressiveness a total migration of three active sites (Fig 8) is and virulence, respectively, one less aggressive isolate between 0.16 and 0.73. However, once again, the (6.25%) to pathotype I, 12 moderate aggressive band frequencies are simultaneous multiple fractions 35 Samia et al. Int. J. Biosci. 2015 containing very similar migration and non-separable two bands revealed using the substrate L-leucine-p- in the system used. No polymorphism was observed naphthylamide. Two bands of are detected different for acid phosphatase to the population concerned. migration: one corresponds to the isoenzyme 1 (0.42) The frequency bands are simultaneous multiple and the other corresponds to this low isozyme by fractions containing very similar and inseparable isolate number (Fig 11) appears indeed surprising this migration in the system used. result is compared with that of a similar analysis conducted on Colletotrichum grasses had revealed an average of four per strain LAP isozymes, one of which was common to both species Colletotrichum falcatum et C. graminicola (Huguenin Et. al.1978). Fig. 8. Acid phosphatase isozymes profiles of Ascochyta rabiei. Superoxide dismutase Superoxide dismutase in three electrophoretic bands Fig. 6. Peroxidase isozymes profiles of Ascochyta of the activity was detected in the upper region of the rabiei. gel just after their frequencies gel concentration is between 0.16 and 0.33 and is located in a lower band Interpretation and data analysis portion of the separation gel before the migration The combination of different patterns of data front in position 0.98 (Fig 9). obtained with 5 isonezymes isoenzyme systems allowed for the classification of the isolates presented as a dendrogram. This dendrogram constructed from the similarity matrix index Jaccard is shown in (Fig 12), it shows four main branches (A, B, C and D) separated at a rate of 0.49 similarities. Fig. 5. Superoxide dismutase isozymes profiles of Ascochyta rabiei. Peroxidase The electrophoretic profile of this system shows a single area of activity with dianisidine. All isolates have isoenzyme (0.14) in common system revealed no isozyme polymorphism (Fig 10). Leucine aminopeptidase The electrophoretic profile of LAP is characterized by 36 Samia et al. Fig. 11. Leucine aminiopeptidase isozymes profiles of Ascochyta rabiei. The branch A is formed a cluster of 12 isolates (31.25% of isolates), isolates the separate branch 4 branches A1, A2, A3, A4 and A5 for a rate of 0.69 similarity. The branch B groups 10 isolates, it includes Int. J. Biosci. 2015 that the isolates which come from two regions of Sidi Sidi Bel Abbes and Ain Temouchent. The branch D Bel Abbes and Ain Temouchent, on the other hand contains only isolate A19 which comes from Sidi Bel the branch C includes 09 isolates of Origin Mascara, Abbes. Fig. 12. Ascending Hierarchical Classification (AHC) Similarity: Jaccard Index. (Skovgaard and Rosendahl, 1998) Compared isolates Fusarium of Fusarium oxysporum resulting and coming from representative isolates of Fusarium oxysporum f. different regions with regard to compared with sp.melonis come from different parts of Iran, USA isoenzymes intra - and extracellular. Five intracellular and France, eight isolates of seven special form's Iran enzymes: dehydrogenase, and isolate F.oxysporum f. sp.niveum and the United dihydrolipoamide States were compared on the basis of isoenzyme esterase malate superoxidizes dehydrogenase, (Aly al. of isoenzyme of alkaline phosphatase (ALP), catalase (CAT), esterase (EST), malate dehydrogenase (MDH), superoxide culture dehydrogenase medium. Proteins were separated by dismutase (XDH) (SOD) proteins. 13 enzymes: amylase and lipase were there for obtained from the soluble total extracted from the mycelium, and oven extracellular (endoglucanase), of A analysis cellulase profile 2003) dehydrogenase and succinate dehydrogenase were protease, and et. and revealed an The xanthine isozyme intermittent or PAGE or by IEF, and isoenzyme beens polymorphism between different special form of F. are detected by Specific held (coloring). The isolates oxysporum 22 wherein phenotypes electrophoretic seeds (EP) were determined (Mohammadi et. al. 2004). (Dorrance et. al. 1999) Analyzed thesis isolates by the Polymorphisms of the esterase isozyme, hexose study kinase and the malate dehydrogenase are often of Stenocarpella of the maydis isolated polymorphism of from isozymes and limited isoenzyme profiles. 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