Starch gel electrophoresis of certain enzymes from five species of Fomes

The electrophoretic banding patterns of five enzymes (esterase, catechol oxidase, peroxidase, alkaline phosphatase, and acid phosphatase) from eleven isolates of five species of the wood-rotting basidiomycete, Fomes, were subjected to mathematical analysis in order to examine taxonomic relationships. Generally, greater similarity was observed between the banding patterns for isolates of the same species than between those for isolates of different species. The experimental and analytical technique used in this study could be an aid for the determination of taxonomic relationships among these and other fungi.     

Evaluation of Propionibacterium acnes Isolates Using Contour-clamped Homogeneous Electric Field Gel Electrophoresis

Contour-clamped homogeneous electric field electrophoresis was performed to compare strains ofPropionibacterium acnes isolated from patients with chronic postoperative endophthalmitis. Propionibacterium acnes isolates were obtained from the vitreous humor of nine patients with chronic postoperative endophthalmitis following cataract surgery. In two of the patients, P. acnes isolates were also obtained from the aqueous humor as well as from the vitreous humor. Bacterial DNA was digested using Not I and Spe I restriction endonucleases. The DNA fragments were then subjected to contour-clamped homogeneous electric field electrophoresis and the DNA banding patterns were analysed. Eight nonidentical banding patterns were identified among the nine vitreous isolates of P. acnes. In each of the two cases from which aqueous and vitreous isolates were recovered from the same eye, the banding patterns were identical. Contour-clamped homogeneous electric field electrophoresis is a powerful method to distinguish P. acnes isolates based on DNA banding patterns and could be used in the epidemiological study of clinical processes caused by this organism.     

Morphological and molecular characterisation of Pythium species causing chilli (Capsicum annuum L.) damping-off

Twenty-five Pythium isolates comprising five species viz., Pythium aphanidermatum, P. deliense, P. graminicola, P. heterothallicum and P. ultimum from different geographical locations of Tamil Nadu (Coimbatore, 4; Cuddalore, 6; Dindigul, 1; Dharmapuri, 1; Erode, 1; Madurai, 1; Namakkal, 7; Thanjavur, 1; Theni, 1; Thirunelveli, 1 and Vellore, 1) isolated from chilli crop were analysed with randomly amplified polymorphic DNA (RAPD) markers. Morphological and molecular characteristics of these different species were correlated with the RAPD. Polymerase chain reaction amplification of total genomic DNA with six random primers generated unique banding patterns depending on the primer and the isolate. The isolate I₁₇ produced identical banding patterns, while other isolates produced dissimilar bands within the particular species, indicating the genetic diversity among the isolates within a species. Morphological characters were also different from each other even in isolate I₁₇ which shared identical bands. Cluster analysis showed minimum and maximum per cent similarities among the tested Pythium species which ranged from 49 to 89%, respectively. RAPD markers were better suited for differentiating isolates within a species rather than species.     

Analysis of SDS-dissociated proteins of pathogenic and nonpathogenicFusarium species

SDS — dissociated proteins from eight isolates belonging to threeFusarium species were assessed and compared using polyacrylamide gel electrophoresis. Intra and inter specific variation in banding patterns were analyzed both qualitatively and quantitatively. Special emphasis was placed on variability of protein banding pattern between two nonpathogenic strains ofF. oxysporum (C5 and C14). Protein profiles from host-associated isolates were distinct, and each isolate showed a uniquely characteristic profile. Attempts were made to provide information on the genetic system controlling pathogenicity, compared to nonpathogenicity, at the protein level. The data obtained from electrophoresis support the potential use of this experimental approach to help distinguish between differentFusarium isolates.     

凝胶电冰与腐霉属的分类

用聚丙烯酰胺凝胶电泳技术分析27株计23种腐霉的菌体蛋白,结果表明:蛋白带型在种内表现出一致性,在种间则差异显著。     

十字花科上的链格孢属真菌同工酶凝胶电泳分析

本文报道对从国内外收集到的生于十字花科植物上的3种链格孢属真菌21个菌株进行的10种同工酶的聚丙烯酰胺凝胶电泳分析结果。这10种酶分别是:酯酶(EST)、碱性磷酸酶(AKP)、酸性磷酸酶(ACP)、多酚氧化酶(PPO)、过氧化氢酶(CAT)、谷氨酸脱氢酶(GDH)、甘露醇脱氢酶(MADH)、乙醇脱氢酶(ADH)、黄嘌呤脱氢酶(XDH)、过氧化物岐化酶(SOD)。对同工酶酶谱资料的聚类分析,在较高的相似性水平上将21个菌株归为3大类群,每一类群所包括的菌株与形态学的鉴定结果完全一致,各菌株的归类关系与其寄主植物、地理来源无关。表明同工酶凝胶电泳在Alternaria种级分类中是一个有用的工具,可以明确地将不同菌株鉴定到种级水平。     

Isozyme Variation and Genetic Diversity of the European Barley Powdery Mildew Population

Isozyme and virulence analyses of Erysiphe graminis bordei were performed with samples collected from different sites from nearly all over Europe. Isozymes and unspecific proteins extracted from conidia were separated by starch gel electrophoresis and isoelectric focusing, respectively, and the resulting isozyme banding patterns were compared with the corresponding virulence data. One isozyme phenotype prevailed in all samples. Only 7.9% of 280 isolates showed divergent banding patterns. Expected frequencies of isolates with divergent banding patterns were calculated for each subsample. In the Italian subsample, isolates with divergent banding patterns were significantly more frequent than expected. At the same time, isolates from Italy had significantly fewer virulence factors than those from N.W. Europe, indicating weaker selection by host resistance genes. It is suggested that isozyme uniformity in the homogeneous north-western European barley powdery mildew population has arisen from strong selection pressures for specific virulence genes. The direction of this selection, acting upon a mainly asexually reproducing population, has changed over space and time due to the introduction of new resistance genes, forcing local populations through bottlenecks. This may have led to random loss of genetic variation (genetic drift) in the barley powdery mildew gene pool.     

疫毒蛋白质的聚丙烯酰胺凝胶盘状电泳

对北京、河北、青海、山东、湖北、广东等地不同寄主上收集的,属于Phytophthora capsici, P. colocasiae, P. heveae, P. infestans, P. melonis P. nicotianae var. nicotianae和P.nicotianae var.parasitica 7个种和变种的17株菌的蛋白质,进行了聚丙烯酰胺凝胶盘状电泳的分析和比较,经多批次试验,7个种和变种各自恒定地呈现典型的蛋白质图谱,显示出种间的差异。其中P.infestans占11株,它们虽采自不同地区和寄主,又分属5个生理小种,经多次电泳,电泳图谱基本相同,但不同菌株存在一些差异。同一株菌,培养条件对电泳图谱影响不大,不论是培养在含苹果酸盼培养基上,还是培养在延胡索酸培养基上,电泳图谱基本一致,只是电泳材料必须新鲜。     

Genetic Diversity Among Iranian Isolates of Rhizoctonia solani Kühn Anastomosis Group1 Subgroups Based on Isozyme Analysis and Total Soluble Protein Pattern

Rhizoctonia solani is a destructive fungal pathogen with a wide host range. The R. solani complex species includes several divergent groups delimited by affinities for hyphal anastomosis. In this study, genetic variation among 20 isolates of R. solani anastomosis group 1 (AG1) subgroups (AG1‐IA and AG1‐IB) collected from Mâzandaran province, Iran, and standard isolates of these subgroups, was determined by isozyme analysis and total soluble protein profile. Mycelial protein pattern and isozyme analysis were studied using denaturing and non‐denaturing polyacrylamide gel electrophoresis, respectively. A total of 15 enzyme systems were tested, among which six enzymes including esterase, alkaline phosphatase, superoxide dismutase, octanol dehydrogenase, lactate dehydrogenase and mannitol dehydrogenase generated distinct and reproducible results. The soluble protein patterns were similar among the R. solani isolates examined; however, minor differences in banding pattern were observed between the two subgroups. In isozyme analysis, a total of 64 electrophoretic phenotypes were detected for all six enzymes used. Based on cluster analysis and similarity matrix, the fungal isolates were divided into two genetically distinct groups of I and II consistent with the previously reported AG1‐IA and AG1‐IB subgroups in AG1. Group I represented all isolates belonging to AG1‐IA subgroup, whereas group II represented all isolates belonging to AG1‐IB subgroup. Results from isozyme analysis suggest that the subgrouping concept within AGs is genetically based.     

蛋白和酯酶电泳在根霉鉴定上的应用

对根霉(Rhizopus)属的十个种或变种共二十四株菌的菌体可溶性蛋白和酯酶同工酶进行了电泳的研究得到对这个属分类上更多的依据.在严格控制培养,提取和电泳条件的情况下,同一株菌不同批次所得菌体蛋白电泳图谱有较好的重复性.在相同的条件下,每个种的根霉有各自特征性蛋白图谱,种内不同菌株的蛋白图谱和酯酶酶谱基本相同.特别是形态特征明显、分类地位明确的种,种内各株的图谱也较一致,如R. stolonifer;与R.circinans.在确定新变种R. delemar var. latoapicalis时,电泳图谱与R.delemar var.delema:有明显不同,起到了佐证作用.因此认为,蛋白图谱与酯酶酶谱相辅相成,在根霉种的分类中是一有效的辅助手段.     

Isozyme variation in Verticillium dahliae isolates from Crete

Fifteen isolates ofVerticillium dahliae (eight of race1, seven of race2; most from the island of Crete, Greece) were examined for isozyme and molecular variation. Among the isozyme banding patterns (zymograms) of six enzymes that were “activity-stained” after electrophoresis in 9% polyacrylamide gels, differences were observed in diaphorase, α-esterase, peroxidase and superoxide dismutase; 2, 2, 3 and 5 different types of zymograms were recorded, respectively. The zymograms could not be correlated with either race1 or2. However, all six isolates originating from the Oropedio (plateau) are, of Lasithi (Crete) showed an esterase zymogram clearly distinguishable from the other isolates. No differences were observed when staining for acid phosphatase or aspartate aminotransferase (‘glutamic-oxaloacetic transaminase’). Furthermore, electrophoresis of random-amplified polymorphic DNA (RAPD) in 2% agarose gels showed that three race-2 isolates from Oropedio of Lasithi could also be distinguished by the RAPD pattern generated with primer OPA-1. The variation observed possibly represents adaptation ofV. dahliae to the Oropedio environment.     

Electrophoretic protein patterns of three species ofPhytophthora associated with black pod disease of cocoa (Theobroma cacao L.)

When electrophoretic profiles of native proteins from vegetative mycelia ofPhytophthora palmivora, Phytophthora capsici and Phytophthora citrophthora causing black pod disease of cocoa in India were compared on a single Polyacrylamide gel, the isolates of same species were readily distinguished both qualitatively by visual similarity in banding patterns and quantitatively by calculating similarity coefficients. Similarity coefficients were generally much higher between isolates within a species than between isolates of different species. The dendrograms obtained after unweighted pair grouping with arithmetic averaging cluster analysis, revealed that all the isolates ofPhytophthora capsici were highly homogenous and formed a single cluster. The isolates ofPhytophthora citrophthora were resolved into two electrophoretic types which were clustered into two distinct sub groups.Phytophthora palmivora formed a separate group. Thus, the results reveal that polyacrylamide gel electrophoresis can be used successfully in distinguishing species and sub groups within a species ofPhytophthora encountered on cocoa. CPCRl contribution No. 914.     

Identification of Xanthomonas campestris pv. Juglandis from (Persian) English Walnut Nursery Trees in South Africa

Bacterial isolates producing yellowish colonies on Nutrient Agar were recovered from symptoms of suspect walnut blight disease on leaves of nursery trees in the southwestern Cape Province of South Africa. The isolates were identified by pathogenicity tests on leaves of walnut and plum trees in the greenhouse. Fifteen isolates from four cultivars at two nurseries produced typical lesions of blight on walnut and one isolate. typical lesions of bacterial spot disease on plum leaves. Cluster analysis was done on 28 characteristics recorded from colony growth. colour. form. and elevation on four different culture media, and starch hydrolysis on a semi-selective medium for the isolation of Xanthomonas campestris pv. juglandis. Total DNA of the isolates was digested with restriction endonuclease Spel and resolved by contour-clamped homogeneous electric field (CHEF) electrophoresis. Two phenotypic clusters were distinguished among the 15 South African and one reference strain of X.c.pv. juglandis at the 54%S_sm level. The isolate which induced disease symptoms on plum grouped with reference strains of Xanthomonas campestris pv. pruni in a third cluster. Two-thirds of the isolates were not characterized on the semi-selective medium for X.c. pv. juglandis. DNA restriction fragment banding patterns were similar for most isolates of X.c.juglandis in the same phenotypic cluster. However, DNA banding patterns were non-distinct for some isolates with similar phenotypic characters. Phenotypic characteristics and DNA restriction fragment banding patterns of the isolates were not correlated with geographical origin or cultivar specificity.     

Species-specific banding patterns of restriction endonuclease-digested mitochondrial DNA from the genusPythium

Restriction endonuclease-digested mitochondrial DNA from 29Pythium spp. showed distinctly different species-specific electrophoretic banding patterns. Numerical comparisons among species were conducted by calculating the percentage of restriction fragments having the same apparent molecular size. The greatest interspecific similarity in banding patterns (67%) was observed betweenHindIII digests ofPythium heterothallicum andP. sylvaticum. However, comparisons among other species generally revealed similarities of less than 50%, and often less than 30%. The lack of similarity of restriction banding patterns was observed even with several species that share many common morphological features:P. arrhenomanes vsP. graminicola (20%),P. myriotylum vsP. aristosporum (28%), andP. torulosum vsP. vanterpoolii (32%). In contrast to the fragment size heterogeneity among different species, isolates of the same species have highly conserved restriction patterns. Ten isolates ofP. oligandrum, collected from the United States, South Africa, and Czechoslovakia, had a minimum of 86% similarity inHindIII banding patterns. Similar results were observed with eight isolates ofP. ultimum, five ofP. acanthicum, six ofP. spinosum, five ofP. sylvaticum, and eight ofP. irregulare. However, two isolates ofP. irregulare exhibited a higher degree of heterogeneity and shared only 64 to 76% comigrating bands with the eight other isolates of this species.     

Restriction pattern analysis of genomic DNA ofFrankia isolates

Summary Total genomic DNAs ofFrankia isolates were subjected to restriction enzyme digestion and subsequent agarose gel electrophoresis. Restriction fragment banding patterns were unique for each isolate and may therefore be used as a method to distinguish between isolates which may be morphologically indistinguishable. This method might be useful for practical purposes such as tracing specificFrankia strains during field studies.     

Strain-specific differentiation of environmental Escherichia coli isolates via denaturing gradient gel electrophoresis (DGGE) analysis of the 16S-23S intergenic spacer region

Denaturing gradient gel electrophoresis (DGGE) was applied to the 16S-23S rRNA intergenic spacer region (ISR) as a means to evaluate strain level differences in Escherichia coli. The ISRs of 81 environmental E. coli isolates obtained from bovine, poultry, and human sources yielded a total of 41 unique DGGE banding patterns, with identical patterns and common bands within each source and no overlapping patterns among sources. An additional 51 isolates from two nearby streams yielded 45 unique banding patterns with no overlap between sites. However, two of the isolates from the streams had identical banding patterns to those from two of the source isolates, resulting in a total of 84 unique DGGE banding patterns out of 132 isolates identified in this study. These results revealed high diversity among environmental E. coli isolates, which made it difficult to unambiguously ascribe strains found in water samples to specific host organisms.     

Electrophoretic karyotypes of some related Mucor species

Contour clamped homogeneous electric field (CHEF) gel electrophoresis was used to obtain electrophoretic karyotypes from nine Mucorstrains representing five different species (M. bainieri, M. circinelloides, M. mucedo, M. plumbeus and M. racemosus). The chromosomal banding patterns revealed high variability among the isolates. The sizes of the DNA in the Mucor chromosomes were estimated to be between 2.5 and 8.7 Mb. The total genome sizes were calculated to be between 30.0 and 44.7 Mb. The applicability of these electrophoretic karyotypes for the investigation of genome structure, for strain identification and for species delimitation is considered.     

Isozyme polymorphisms within <Emphasis Type="Italic">Pyrenophora tritici-repentis</Emphasis> population in Argentina

Tan spot caused by Pyrenophora tritici-repentis is an economically important disease of wheat (Triticum aestivum L.) in Argentina. The success of local breeding programs for resistance to the disease depends to a large extent on the genetic variation within the pathogen population. To assay this variability, 155 isolates of P. tritici-repentis obtained from leaves of wheat and Festuca sp. of different localities were assayed and compared for isozyme polymorphisms. Isozyme analysis was performed with polyacrilamide gel electrophoresis in the systems estearase, phosphatase and peroxidase. Variation in the number and relative mobility of the bands by each system was detected in this collection of isolates. Intraspecific polymorphisms were identified in the three systems assayed. The cluster analysis based in bands patterns defined 86 electromorph types among the 155 isolates evaluated according with the Jaccard’s coefficient (CCC = 095). The isozymes patterns were not correlated with the geographic and/or wheat cultivars origin of the P. tritici-repentis isolates.     

Optimization of RAPD-PCR Fingerprinting to Analyse Genetic Variation Within Populations of Fusarium oxysporum f.sp. cubense

Genetic variation among 11 isolates of Fusarium oxysporum f.sp. cubense (FOC) was analysed by random amplification of polymorphic DNA using the polymerase chain reaction (RAPD-PCR). The isolates represented three of the four FOC races and the seven vegetative compatibility groups (VCGs) known to occur in Australia. Isolates of F. oxysporum f.sp. cubense were also compared to isolates of F. oxysporum f.sp. gladioli, F. oxysporum f.sp. zingiberi, F. oxysporum f.sp. lycopersici, F. moniliforme, Aspergillus niger and Colletotrichum gloeosporioides. DNA was extracted from fungal mycelium and amplified by RAPD-PCR using one of two single random 10-mer primers; the primer sequences were chosen arbitrarily. The RAPD-PCR products were separated by polyacrylamide gel electrophoresis producing a characteristic banding pattern for each isolate. The genetic relatedness of the F. oxysporum f.sp. cubense isolates was determined by comparing the banding patterns generated by RAPD-PCR. This RAPD-PCR analysis revealed variation at all five levels of possible genetic relatedness examined. F. oxysporum f.sp. cubense could very easily be distinguished from the other fungi, and the three races and five VCGs of F. oxysporum f.sp. cubense could also be differentiated. Within F. oxysporum f.sp. cubense, each isolate was scored for the presence or absence of each band (50 different bands were produced for primer SS01 and 59 different bands for primer RC09) and these data were clustered using the UPGMA method (unweighted pair-group method, arithmetic average). UPGMA cluster analysis of the data generated by primer SS01 revealed two distinct clusters. One cluster contained race 4 isolates (VCGs 0120, 0129 and 01211) and the other cluster contained both race 1 (VCGs 0124, 0124/5 and 0125) and race 2 isolates (VCG 0128). Similar results were obtained with primer RC09. The banding patterns for each isolate were reproducible between experiments. These results indicated that RAPD-PCR was a useful method for analysing genetic variation within F. oxysporum f.sp. cubense. Some of the advantages of this technique were that it was rapid, no sequence data were required to design the primers and no radioisotopes were required.     

Characterization of Pyrenophora Isolates Associated with Spot and Net Type Lesions on Barley in South Africa

Single-spored isolates of Pyrenophora associated with spot and net type net blotch of barley were compared using total DNA banding patterns, morphological and cultural characteristics, symptomatology and mating studies. Isolates of spot and net type net blotch were found to vary regarding conidium length and cultural growth rate. Mating studies among and between ascospore, spot and net type isolates proved unsuccessful under the conditions studied. Total DNA polymorphisms of the net spot and ascospore isolates digested with the restriction enzymes HpaII and HaeIII showed that the isolates have similar banding patterns. Random amplified polymorphic DNA (RAPD) showed that the banding patterns of the spot and net type isolates were again similar but were distinct from outgroups such as P. semeniperda and P. triticirepentis. The homology in DNA banding patterns of local isolates indicated that the difference in conidium length is insufficient to separate them as two species. It is concluded that spot and net type isolates occurring in South Africa belong to P. teres. Therefore spot type lesions are caused by P. teres f. sp. maculata and not by P. japonica as reported previously.