Restriction fragment length polymorphism analysis of Cryptosporidium parvum isolates of bovine and human origin.

Cryptosporidium parvum oocysts isolated from different hosts and geographical areas were compared by restriction endonuclease analysis of repetitive DNA: Iowa (bovine), Florida (bovine), New York (bovine), Peru (human), Brazil (human), and Mexico (human). Southern blot hybridization analysis was performed using the restriction endonuclease enzyme Eco RI and the DNA probe pV47-2. The probe hybridized with 18 bands present in all the isolates. The Brazilian, Mexican, and Peruvian human isolates had an additional common band of 4.3 kbp that was absent in the bovine isolates. Two extra bands of 14 and 12 kbp were present in the Brazilian isolate whereas the Mexican isolate had an extra band of 14 kbp. When the Iowa and Peru C. parvum isolates were passed twice through calves, oocysts recovered from both passages showed identical banding patterns, suggesting that recombination of the repetitive sequences was not altered during sexual reproduction. The DNA digested with other restriction endonucleases were tested confirming differences between isolates. A genomic DNA library is currently being produced to better define isolate variation in C. parvum.     

Determination of genetic diversity among Indian isolates of Rhizoctonia bataticola causing dry root rot of chickpea

Genetic diversity among 27 isolates (23 from chickpea and 4 from other host crops) of Rhizoctonia bataticola representing 11 different states of India was determined by random amplified polymorphic DNA (RAPD), internal transcribed spacer restriction fragment length polymorphism (ITS-RFLP) and ITS sequencing. The isolates showed variability in virulence test. Unweighted paired group method with arithmetic average cluster analysis was used to group the isolates into distinct clusters. The clusters generated by RAPD grouped all the isolates into six categories at 40% genetic similarity. High level of diversity was observed among the isolates of different as well as same state. Some of the RAPD (OPN 4, OPN 12, and OPN 20) markers clearly distinguished majority of the isolates into the area specific groups. The ITS I, 5.8rDNA and ITS II regions of 11 isolates representing different RAPD groups were amplified with primers ITS 1 and ITS 4 and digested with seven restriction enzymes. The restriction enzymes DraI, MboI, RsaI, and AluI were found to be suitable for differentiating the isolates into five categories by showing isolate specific ITS-RFLP patterns. The isolates were variable in their nucleotide sequences of the ITS regions. This is the first study on genetic diversity among chickpea isolates of R. bataticola.     

Restriction Fragment Length Polymorphism Analysis of the Mitochondrial DNA of Fusarium oxysporum f. sp. ciceris

Seven isolates of Fusarium oxysporum f. sp. ciceris, representing pathogenic races 1 , 2, 3, and 4 from India and 0, 5, and 6 from Spain, were assayed for restriction fragment length polymorphisms (RFLPs) in the mitochondrial DNA,(mt DNA). The mt DNA fraction of total fungal DNA was purified and digested with the restriction endonucleases Bam HI, Bgl II, Eco RI. Kpn I, Sac I, Sal I, Sma I, and Xho I. The mt DNA is a circular molecule of 40.5 kb. No RFLP in the mt DNA was detected among the seven races of F. o. ciceris. The identical restriction patterns of mt DNA indicates an extensive conservation in the gene composition of mt DNA without sequence variation, and suggests that mt DNA of F. o. ciceris may not be responsible for pathogenic diversity. The restriction map of mt DNA from the race 6 isolate Fo 8272 was constructed by digestion of the mt DNA with five restriction enzymes: Eco RI, Kpn I, Sac I, Sal I, and Xho I, either singly or in selected pairs.     

Diversity of DNA methylation pattern and total DNA restriction pattern in symbiotic Nostoc

Attempts were made to use total DNA restriction patterns and the response of purified DNA to treatment with restriction endonucleases to characterize several symbiotic Nostoc strains which had been isolated from different host plants cultivated in Italy. Among 27 restriction endonucleases tested, several did not cut any DNA and no significant variation in the susceptibility of the genomes to DNA restriction was seen among the strains. Therefore the Nostoc strains could not be separated into groups based on their different susceptibilities to the action of restriction endonucleases. However, in studies of total DNA restriction patterns, the restriction endonucleases BfrI and HpaI gave unique band patterns for each cyanobacterial isolate. Different profiles were even found in strains isolated from host plants belonging to the same species. The results do not support any definition of symbiotic Nostoc genomic groups or species and show that a tight specificity between the host plant and the cyanobacterium might not exist in the symbiotic associations involving Nostoc.     

Genetic Variability of Antigen B among Echinococcus granulosus Egyptian Isolates

Genetic polymorphisms of encoding antigen B2 gene (AgB2) in Echinococcus granulosus were studied using PCR-RFLP and DNA sequencing among 20 Egyptian isolates. Five isolates from different host origins (humans, camels, pigs, and sheep) were collected and used. All examined isolates of each host group gave very similar patterns of PCR-RFLP after restriction enzyme digestion with AluI, with the gene size of approximately 140 bp and 240 bp for sheep and human isolates, and approximately 150 bp and 250 bp for pig and camel isolates. No digestion pattern was obtained after incubation of all studied isolates with EcoRI. These results reveal high intra-group homogeneity. DNA sequence analysis highlighted that human infecting strain showed 100% identity with respect to sheep infecting isolate, 96% and 99% with pig and camel infecting isolates, respectively.     

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.     

Isolation and characterization of bacteriophages infecting <Emphasis Type="Italic">Xanthomonas arboricola</Emphasis> pv. <Emphasis Type="Italic">juglandis</Emphasis>, the causal agent of walnut blight disease

Walnut orchards suffer from a blight caused by the bacteria Xanthomonas arboricola pv. juglandis. These bacteria can be infected by viral bacteriophages and this study was carried out to isolate and characterize bacteriophages from walnut orchards located throughout the South Island of New Zealand. Twenty six X. arboricola phages were isolated from three hundred and twenty six samples of plant material representing phyllosphere and rhizosphere ecosystems. The phage isolates were characterized by host-range, plaque and particle morphology, restriction digest and phylogenetic analysis and stability under various storage conditions. From capsid and tail dimensions the bacteriophages were considered to belong to the double-stranded DNA families Podoviridae and Siphoviridae. Of the twenty six bacteriophages, sixteen belonged to Podoviridae and were found both in the phyllosphere and rhizosphere. In contrast, Siphoviridae were present only in the rhizosphere isolates. Phage genome sizes ranged from 38.0 to 52.0 kb from a Hind III restriction digestion and had in common a 400 kb fragment that was identical at the DNA level. Despite the similar restriction patterns, maximum parsimony bootstrap analysis showed that the phage were members of different groups. Finally, we hypothesise that these phage might have use in a biocontrol strategy and therefore storage stability and efficacy was tested. Titres declined more than 50% over a 12-months storage period. Deep-freezing temperatures (−34°C) increased while chloroform decreased the stability.     

Rapid Detection and Identification of Six Tomato yellow leaf curl virus Isolates from Different Regions Using Polymerase Chain Reaction and Restriction Enzyme Analysis

The combinational analysis of polymerase chain reaction and restriction enzyme analysis (PCR‐RE) to distinguish six Tomato yellow leaf curl virus (TYLCV) isolates from five countries was developed. Tomato yellow leaf curl virus has spread from the Middle East to Western Europe, Central America and Eastern Asia, and occurs on infected crops such as tomatoes, peppers, cucurbits and beans. Tomato yellow leaf curl virus isolates from Jordan (TYLCV‐Mld[Jo:Cuc] and TYLCV‐IL[Jo:Cuc]), Israel (TYLCV‐IL[IL:Reo:86]), Spain (TYLCV‐Mld[ES72/97]), USA (TYLCV‐IL[US:F10:04]) and Korea (TYLCV‐KR) were collected, and the sequences of the six isolates were analysed to distinguish them by PCR‐RE combination analysis. Oligonucleotide primers for the six TYLCV isolates were designed to amplify approximately 740 base pairs including the intergenic region (IR) and parts of V1 and V2 ORF. Unique restriction enzyme sites were analysed to identify isolate‐specific restriction enzyme sites on the PCR products of each isolate. Three enzymes (DdeI, FauI and BssSI) were selected by in silico analysis, and then, the PCR products following the serial digestion of each restriction enzyme were separated by agarose gel electrophoresis to distinguish the TYLCV isolates. Taken together, the PCR‐RE combination analysis by serial digestion with three restriction enzymes could be a useful method for distinguishing the six isolates.     

Genetic Similarity among Isolates of Pyrenophora tritici-repentis, Causal Agent of Tan Spot of Wheat

Tan spot, a foliar disease of wheat, is caused by the fungus Pyrenophora tritici‐repentis. On susceptible wheat cultivars, P. tritici‐repentis induces two distinct symptoms: tan necrosis and extensive chlorosis. Presently isolates of P. tritici‐repentis are classified into 11 races based on their virulence on a set of wheat differential genotypes. In nature, this pathogen reproduces both sexually and asexually, but the extent of genetic variability in the P. tritici‐repentis population of western Canada is unknown. This study was conducted to assess the genetic variability among different isolates of P. tritici‐repentis and to determine if similarities among isolates are correlated with race classification or geographic origin of the isolates. Thirty‐three isolates of P. tritici‐repentis and one isolate each of P. teres f. sp. teres, P. teres f. sp. maculata, P. graminea, Helminthosporium sativum and an uncharacterized isolate were studied with 30 random amplified polymorphic DNA (RAPD) primers. Cluster analysis showed that all isolates had unique banding patterns and that clustering of isolates was independent of their race designation or geographic origin. Analysis of molecular variation (amova ) showed that 96.8% of variability occurred among isolates and among race variability accounted for only 3.2% of the total variability.     

Identification of Campylobacter coli isolates from animals and humans by bacterial restriction endonuclease DNA analysis.

Ninety-nine Campylobacter coli isolates were examined by bacterial restriction endonuclease DNA analysis (BRENDA) with HindIII. Isolates from poultry from the same environment had identical patterns, patterns of isolates carried by suckling piglets were generally the same as those of isolates recovered from their dams, and one human patient yielded the same BRENDA type when sampled 6 weeks later. The 14 human isolates examined produced 11 distinct BRENDA types. Forty-three C. coli isolates from pigs were represented by 20 BRENDA types. Ten C. coli isolates from the feces of gulls yielded five different BRENDA types. Thirty-two C. coli isolates from live chickens and processed chicken yielded five different BRENDA types. Three human isolates had identical DNA patterns; two were from brothers living in the same house, and the third was from a human with no apparent relationship to the brothers. Another human isolate was identical to a poultry isolate. None of the pig strains had DNA patterns resembling those of human strains, nor were the DNA patterns like those of any strains recovered from poultry or gulls. Four C. coli isolates were subcultured onto agar 23 times over a period of 45 days, and their BRENDA patterns were preserved. BRENDA shows great promise for use in epidemiological studies of C. coli.     

Virulence Analysis and Race Classification of Xanthomonas oryzae pv. oryzae in China

Two hundred and eighty‐five isolates of Xanthomonas oryzae pv. oryzae were randomly collected from 22 rice‐growing provinces in China. Ninety‐one representative isolates were chosen to assess the differential characteristics of 24 near‐isogenic rice lines containing a single resistance gene or two to four genes. Most isolates were avirulent on pyramided lines, except IRBB51, and hence, the pyramided lines cannot be used as differentials for the virulence analysis of X. oryzae pv. oryzae in China. The 13 rice lines with a single gene were used further to establish a system of races classification of X. oryzae pv. oryzae in China. IR24 and IRBB10 were susceptible to the isolates with several exceptions, whereas IRBB5, IRBB7 and IRBB21 were resistant. Based on the interactions between the isolates of X. oryzae pv. oryzae and the 13 near‐isogenic rice lines, six single‐gene rice cultivars (IRBB5, IRBB13, IRBB3, IRBB14, IRBB2 and IR24) were chosen as differentials, and the 285 tested isolates were classified into nine races. The reaction patterns of the nine races in order were: RRRRRR, RRRRRS, RRRRSS, RRRSSS, RRSSSS, RSRRRS, RSSRRS, RSSSSS and SSSSSS. The race frequencies were 10.18%, 10.53%, 4.91%, 10.18%, 24.21%, 5.96%, 11.23%, 22.46% and 0.35% respectively. The virulence of representative strains of eight Philippine races on 13 rice lines with a single gene was determined and compared with the Chinese races. The frequency distributions of X. oryzae pv. oryzae races were primarily described for the different regions in China.     

Differentiation of Coxiella burnetii isolates by analysis of restriction-endonuclease-digested DNA separated by SDS-PAGE.

Thirty-two isolates of Coxiella burnetii collected from various hosts ranging from arthropods to man were compared by restriction endonuclease (RE) digestion patterns of chromosomal DNA using SDS-PAGE. SDS-PAGE provided better DNA fragment separation than agarose gel electrophoresis and enabled the differentiation of these isolates into six distinct groups on the basis of DNA restriction fingerprints. Two groups of chronic disease isolates could be distinguished, each having unique RE digestion patterns of chromosomal DNA. Three similar but distinct RE digestion patterns were seen among the group of acute disease isolates. Three additional isolates included in this study exhibited a unique RE digestion pattern and also had a unique plasmid type, designated QpDG. DNA-DNA hybridization on selected isolates quantified the relatedness between several groups and supported the classification of these groups as distinct strains.     

Characterization of Fusarium oxysporum f.sp. cepae from Onion in Turkey Based on Vegetative Compatibility and rDNA RFLP Analysis

Seventy‐five isolates of Fusarium oxysporum f.sp. cepae, the causal agent of basal plate rot on onion, were obtained from seven provinces of Turkey. The isolates were characterized by vegetative compatibility grouping (VCGs) and restriction fragment length polymorphism (RFLP) analysis of the nuclear ribosomal DNA intergenic spacer region (IGS). Forty‐eight vegetative compatibility groups were found, each containing a single isolate. Only one isolate formed strong heterokaryons with the reference isolates of VCG 0423. Five isolates were heterokaryon self‐incompatible. Restriction fragment analysis with six different enzymes revealed 13 IGS types among 75 F. oxysporum isolates from Turkey as well as 16 reference isolates from Colorado, USA. The majority of single‐member VCGs produced identical RFLP banding patterns with minor deviations, considerably different from those of the reference VCG isolates. These results suggested that isolates of F. oxysporum f.sp. cepae in Turkey derived from distinct clonal lineages and mutations at one or more vegetative compatibility loci restrict heterokaryon formation.     

Characterization of Phytomonas isolated from fruits by electrophoretic isoenzymes and kinetoplast-DNA analysis

Abstract Two flagellates of the family trypanosomatidae were isolated from the fruits of Lycopersicon esculentum (tomato) and Annona cherimolia (cherimoya) in the southeastern region of Spain. The isolates were characterized by isoenzyme analysis using nine different isoenzymes and by analysis of kinetoplast DNA (kDNA) restriction fragment length polymorphism using four different restriction endonucleases. Most of the isoenzymes were unable to distinguish between the two fruit isolates, while they were all able to distinguish these two from four other Phytomonas isolates, three of which were from laticiferous plants i.e. Euphorbia characias E. hirta and E. hyssopifolia , and one was a phloem-restricted isolate associated with Hartrot disease. Only the enzyme Superoxide dismutase was able to differentiate between the two fruit isolates. Electrophoretic and restriction endonuclease analysis of kDNA minicircles, using four restriction enzymes, showed similar if not identical restriction cleavage patterns of the minicircles of the two isolates from fruits, while the patterns were different for the other isolates. These results confirm the hypothesis that the two isolates from fruits constitute a group of trypanosomatids that are the same or closely related and that this group can parasitize more than one host plant.     

Variability of Indian Isolates of Tilletia indica Assessed by Pathogenicity and Molecular Markers

Twenty isolates of Tilletia indica collected from sites in North and North‐western India showed pathogenic variation on 18 host differentials. Sixteen aggressive pathotypes were identified on the basis of percent coefficient of infection (PCI). Two major clusters were apparent in the dendrogram; cluster 1 comprised 13 isolates and cluster two consisted of seven isolates. One of the isolate Kashipur had a high PCI on most of the host differentials compared to other isolates. Polymerase chain reaction‐based random amplified polymorphic DNA (PCR – RAPD) analysis also divided isolates into two major clusters, one comprising of 5 isolates collected from hill and foot‐hill sites and another group comprising of 15 isolates collected from plain sites. Thus, the clusters identified based on PCI did not match closely with those identified by molecular analysis based on RAPD. Although diversity among the isolates of T. indica was absent in the rDNA‐ITS region, our study based on pathogenicity and molecular markers confirms the existence of great diversity in the pathogen, also shifting of ‘hot spot’ areas from one place to another within Karnal bunt prevailing areas.     

Use of Random Amplified Polymorphic DNA (RAPD) Analysis and Genetic Fingerprinting to Differentiate Isolates of Race O, C and T of Bipolaris maydis

Isolates of three races of Bipolaris maydis from China (races O, C and T) were compared using two techniques. Random Amplified Polymorphic DNA (RAPD) analysis using 24 primers indicated that race O and C isolates were more similar to one another than to the race T isolate. Twenty of the primers produced RAPD profiles that were similar for the race O and C isolates but differed for the race T isolate (four primers did not amplify products in any of the isolates). Four primers produced profiles which differed for all three races and two of these (A-09 and B-18) clearly differentiated the race O and race C isolates. Genetic fingerprinting of B. maydis using M13 DNA as a probe differentiated race O and C isolates from the race T with all four restriction enzymes used. Furthermore, when DNA was digested with Hind III, the hybridization profiles of the race O and C isolates differed from one another.     

Diversity and genetic structure of a natural population of Rhizobium leguminosarum bv. trifolii isolated from Trifolium subterraneum L.

A collection of 121 isolates of Rhizobium leguminosarum biovar (bv.) trifolii was obtained from root nodules of Trifolium subterraneum L. (subclover) plants growing in an established pasture. The collection consisted of a single isolate from each of 18 plants sampled from seven microplots. The following year, a further 28 and 27 isolates were collected from the first and seventh sampling points, respectively. Analysis of restriction fragment length polymorphisms (RFLPs) of both chromosomal and Sym (symbiotic) plasmid DNA and multilocus enzyme electrophoresis (MLEE) were used to assess the diversity, genetic relationships and structure of this population. Symbiotic effectiveness tests were used to examine the symbiotic phenotype of each isolate collected in the first year. Analysis of RFLPs of the first year isolates revealed 13 chromosomal types and 25 Sym plasmid types. Similar Sym plasmid types were grouped into 14 families containing 1–6 members. No new chromosomal types and six new Sym plasmid types were detected in the second year. The symbiotic effectiveness of the first year isolates of the same Sym plasmid type was similar. Significant differences in symbiotic effectiveness were detected between different Sym plasmid types in the same plasmid family. Representative isolates of each chromosomal type Sym plasmid type identified in the first year were analysed using multilocus enzyme electrophoresis. Mean genetic diversity per locus was high (0.559). Enzyme electrophoresis revealed 17 electrophoretic types (ETs). Ouster analysis of the enzyme data revealed large genetic diversity amongst the ETs. Strong linkage disequilibrium was observed for the population as a whole, i.e. clonal population structure, but significantly less disequilibrium was observed among a cluster of ETs suggesting that recombination occurred between ETs within the cluster. Our results revealed that a population of naturally occurring isolates of Rhizobium leguminosarum bv. trifolii can be genetically diverse and support the possibility that recombination plays a role in generating new genotypes.     

Molecular Epidemiology of Vibrio cholerae O1 Isolated from Sporadic Cholera Cases in Okinawa,Japan

In July 1994, 6 cholera cases due to Vibrio cholerae O1 El Tor Ogawa sporadically appeared in Okinawa. All 6 patients had no history of traveling abroad. In the period of this cholera outbreak, a strain of V. cholerae O1 El Tor Ogawa was detected from an imported fish at the Naha port quarantine station. The isolates were characterized to clarify whether or not, they belonged to a common clone. Phenotypes were identical except that one strain revealed cured Celebes and the others were original Celebes in kappa phage typing. The restriction fragment patterns of DNA of the isolates hybridized with an enzyme-labeled oligonucleotide probe for cholera toxin gene (ctx) were identical. Randomly amplified polymorphic DNA of the isolates were identical when a primer was used, but 2 patterns were seen when another primer was used. Pulsed-field gel electrophoresis of the chromosomal DNA digested with NotI restriction enzyme showed 3 patterns. The DNA fragment pattern of the strain isolated from the imported fish was different from the clinical isolates. These results suggested that there was no epidemiological relation among the strains of V. cholerae O1 isolated during this period.     

Differential Colonization Patterns of Bananas (Musa spp.) by Physiological Race 1 and Race 4 Isolates of Fusarium oxysporum f.sp. cubense

Fusarium oxysporum f.sp. cubense (Foc) is the causative agent of Fusarium wilt of bananas (Musa spp.). To clarify the colonization patterns of Foc in bananas, two green fluorescent protein‐tagged isolates, NT320 (race 1) and B2‐gfp (race 4), were used to follow infection of the banana varieties Pisang Awak and Brazil. Penetration and colonization of both isolates in roots of these two banana varieties were observed within 6 days, but sporulation in xylem vessels was not observed until day 30 postinoculation. Interestingly, B2‐gfp penetrated into xylem vessels of Pisang Awak banana roots more quickly than NT320, implying that the race 4 isolate is more virulent than the race 1 isolate. This result was further confirmed by comparing the disease severity of plants inoculated with NT320 with that of plants inoculated with B2‐gfp. Quantitative real‐time PCR revealed that some pathogenicity‐associated genes, including Fga1, Fhk1, Fow2 and Ste12, were upregulated by B2‐gfp during exposure to Brazil bananas, while they were either downregulated by NT320 or not significantly changed. These data might partly explain why the race 4 isolate was more virulent than the race 1 isolate.     

Analysis of cytomegalovirus genomes with restriction endonucleases Hin D III and EcoR-1.

Cleavage of genomes of eleven human, one simian, and one simian-related cytomegalovirus (CMV) isolate by the restriction endonucleases HinD III and EcoR-1 generated reproducible DNA fragments. The size range of CMV DNA fragments as estimated by contour length measurements in comparison with simian virus 40 form II DNA and by coelectrophoresis with EcoR-1 fragments of herpes simplex virus DNA varied between 15 X 10(6) and 0.5 X 10(6) daltons. Comparison of the cleavage products of each isolate in 1% agarose slab gels showed extensive comigration of fragments among the human CMV isolates. In the HinD III digests, three fragment bands comigrated among all human CMV isolates, and six fragments comigrated among most, but not all, human CMV isolates. In the EcoR-1 digests, nine fragment bands comigrated among all human CMV isolates, and five bands comigrated among most, but not all human isolates. Each isolate had a distinctive electrophoretic profile with either HinD III or EcoR-1 digests. No two isolates had identical HinD III or EcoR-1 patterns although some isolates did share more general pattern similarities than others. No clear-cut subgrouping of isolates based on cleavage pattern characteristics could be discerned. Comparison of HinD III and EcoR-1 patterns showed that human isolates differ greatly from nonhuman CMV isolates. HinD III and EcoR-1 digests of each isolate contained both major and minor molar classes of DNA fragments that ranged from about 1 and multiples of 1 M down to about 0.25 M; however, the summed molecular weights for major molar fragments resulting from HinD III or EcoR-1 digests of several isolates closely approximated the molecular weight of 10(8) of the intact genome.