BOX Primers Research Articles

REP-PCR DISTINGUISHES RICE BACTERIAL BLIGHT PATHOGEN (XANTHOMONAS ORYZAE pv. ORYZAE) STRAINS OF INDIAN MAINLAND AND BAY ISLANDS

Thirty-two strains of Xanthomonas oryzae pv. oryzae (Xoo) representing 12 pathotypes from major rice growing Indian mainland states and Andaman Islands were analyzed using RepPCR and IS1112-based PCR approaches. All four PCR primers (BOX, REP, ERIC and JEL primers) confirmed the presence of high genetic variability among the Xoo strains studied. The REP primers generated more PCR fragments than other set of primers and the amplification pattern of BOX, REP, ERIC and JEL primers were 277, 171, 147 and 149 respectively with the band size varying from 200 to 3000 base pairs. Cluster analysis of 32 Xoo strains revealed that the combined dendrogram constructed using Rep–PCR analysis was effective in distinguishing Islands Xoo strains from Indian mainland strains with the similarity index of Islands clusters of about 53%. In case of IS1112 repeat element based PCR approach, the two of the mainland strains (DX_027 and DX_143) were grouped with one mainland isolate ANBB_4 in one sub cluster whereas remaining three mainland isolates grouped separately in a main cluster. This indicated that Islands strains were genetically distinct from mainland strains. Also, the Rep-PCR approach was found to be more precise and thus suitable for discriminating the variability among Xoo strains emanating from geographically diverse locations.

MOLECULAR CHARACTERIZATION OF ERWINIA AMYLOVORA STRAINS ORIGINATED FROM POME FRUIT AND INDIGENOUS PLANT IN MONTENEGRO

In the period from 2012-2015 plant samples with fireblight symptoms were collected from pome fruit and indigenous plant, in main fruitgrowing regions of Montenegro. After succesfull isolation, pathogenicity of the obtained strains was tested by artificial inoculation of immature pear fruits, variety Viljamovka. Hypersensitive reaction was tested on tobacco leaves, variety White Burley. Identification and genetic diversity studies were performed using several molecular techniques on 18 Erwinia amylovora strains originating from quince, pear, apple and hawthorn. Bacterial identity was confirmed by nested PCR in which all studied strains produced the expected amplification fragment of plasmid pEA29. To detect potential genetic variations in E. amylovora population, rep-PCR was conducted. Using REP, ERIC and BOX primers, in all three PCR reactions, differences between studied strains were detected, i.e. pear strains had different genetic profiles from all other studied strains, including reference strain. Genetic variability of selected E. amylovora strains was studied by RAPD-PCR as well. Both of the used random primers, CUGEA-3 and CUGEA-5, showed discriminatory potential by separating genetic profiles of pear strains from all other studied strains, including reference strain. This is the first study of genetic variability of E. amylovora in Montenegro.

Determination of Isolated Salmonella Bacteria from Infected Animal Husbandry of Malayer City with using Molecular Methods

Salmonella species are gram negative bacteria and members of Enterobacteriaceae family. It has a rod-shaped appearance; it is catalase positive, oxidase negative, non-spore. Salmonella classified into two species, Salmonella Enterica and Salmonella Bangori. Salmonella is now one of the main reasons of diarrhea and vomiting in humans in many countries and especially in industrialized. In a study in Japan 164 Salmonella digestions were collected during 2006 to 2008 which 81 digestions were Salmonella Infantis. Salmonella-specific characteristics are studied in the two phenotype and genotype methods. In this research, with using genotype methods based on PCR, genetic diversity was evaluated; this PCR includes rep-PCR based on repetitive sequence elements (method was done by the use of three primers ERIC, REP and BOX). Studied showed most isolated strains were relevant to Salmonella Enteritidis and dendorogram study showed that the bacteria were grouped in one cluster in dendrogram that all 37 strains were put in a large cluster of Salmonella’s type which is divided into two clusters: Salmonella Enterica and Bongori. The results also in this experiment reflect the efficiency of rep-PCR method by using three ERIC, REP and BOX primers.

Diversity of Serratia marcescens Strains Associated with Cucurbit Yellow Vine Disease in Georgia.

Cucurbit yellow vine disease (CYVD), caused by the squash bug (Anasa tristis)-transmitted bacterium Serratia marcescens, was first identified in Oklahoma and Texas in 1988 and in Georgia in 2012. S. marcescens is a highly diverse species found in many ecological niches. In previous studies, CYVD strains of S. marcescens formed a closely related group separate from non-CYVD strains based on biological and molecular characterization techniques. Multilocus sequence analysis (MLSA) of six housekeeping genes and repetitive elements-based polymerase chain reaction (rep-PCR) using the BOX and ERIC primers were used to assess the genetic diversity of CYVD strains of S. marcescens collected in Georgia together with a strain from Texas and seven non-CYVD strains of S. marcescens. rep-PCR results revealed genetic diversity among CYVD strains while MLSA results showed a 100% similarity across the six loci for all but one of the CYVD strains, which differed at the icd locus by five polymorphisms. For both methods, CYVD strains clustered separately from nonplant-pathogenic S. marcescens strains and were most similar to a rice endophyte strain. One CYVD strain isolated from a squash bug shared genetic similarities with non-CYVD strains, and may be the result of a recombination event between CYVD and non-CYVD strains.

Population features of Xanthomonas arboricola pv. pruni from Prunus spp. orchards in northern Italy

Bacterial leaf/fruit spot and canker of stone fruits, caused by Xanthomonas arboricola pv. pruni, is a recurrent disease in Italy. A set of 23 strains has been isolated in peach and plum orchards in an intensively stone fruit cultivated area located in north-eastern Italy. They were all identified as X. arboricola pv. pruni by means of phytopathological and serological features: hypersensitive reaction on bean pods, pathogenicity test on immature peach or plum fruitlets, identification by immunofluorescence assay and conventional PCR. Phylogenetic analysis based on sequencing of the gyrB housekeeping gene of the isolates showed that they formed a unique clade, well characterised and separated from other xanthomonads. An insight into the genetic population features was attempted by rep-PCR analysis, using the ERIC, REP and BOX primers. The combined rep-PCR fingerprints showed a slight intra-pathovar variation within our isolates, which grouped in five close clusters. Copper resistance has been assessed in vitro for our whole X. arboricola pv. pruni collection, highlighting that two isolates show a level of resistance in vitro up to 200 ppm of copper. Nonetheless, the copLAB gene cluster, present in many other species of Xanthomonads, was not detected in any isolate, confirming the presence of a still unknown mechanism of copper detoxification in our Xanthomonads arboricola pv. pruni tolerant/resistant strains.

Isolation and characterization of Pseudomonas syringae pv. syringae which induce leaf spot on walnut

In 2012, a leaf spot disease on walnut seedlings was observed in Hamedan province of Iran. The spots were necrotic with yellow halos. Symptomatic samples were collected and suspected bacterial agent was isolated on nutrient agar medium. Phenotypic characteristics such as production of fluorescent pigment on KB medium, LOPAT test and utilization of various carbon sources, revealed that the strains were Pseudomonas syringae pv. syringae. All tested strains were pathogenic on peach, plum and walnut seedling. To investigate the genetic diversity among the strains, rep-PCR using BOX and ERIC primers was performed. Results showed high similarity among the strains from the same region, while variation was found among those from different areas and they were divided into two groups based on geographic regions. The phylogenetic analysis based on 16S rRNA, rpoD and gyrB sequences showed that representative isolates were closely related to P. syringae pv. syringae. To the best of our knowledge, this is the first report of the presence of P. syringae pv. syringae as a causal agent of walnut seedlings leaf spot in Iran and worldwide.

English

Jatropha curcas L., a non-edible Euphorbiaceae oil-rich crop cultivated in subtropical/tropical countries, has gained global attention as a promising renewable resource for biodiesel production. Rep-polymerase chain reaction (PCR) was used to investigate the genetic diversity of 15 populations of J. curcas L. Distinct populations of the plant growing wildly in Mauritius were characterised using three molecular markers random-amplified polymorphic DNA (RAPD), repetitive extragenic palindromic (REP), and BOX. Furthermore, to confirm that the amplicons obtained with rep-PCR were derived from mitochondrial genomes, six randomly chosen bands were cloned and sequenced to demonstrate that the amplified products were mitochondrial genome-specific. The average polymorphism information content (PIC) values were 0.329 and the average percentage of polymorphic loci obtained were 89.28 for BOX primer, followed by RAPD (83.41), and REP (55.81) among the different populations with the percentage polymorphic loci ranging from 13.95 to 100. The homology recorded clearly indicated that the amplified products were mitochondrial genome-specific. Rep-PCR provides a quick and cheap method to study diversity at the mitochondrial level in plants. Key words: Genetic diversity, Jatropha curcas, rep-polymerase chain reaction (PCR).

Repetitive element palindromic PCR (rep-PCR) as a genetic tool to study interspecific diversity in Euphorbiaceae family

Background: The classification of diversity in germplasm collections is important for plant breeding. The repetitive element palindromic-polymerase chain reaction (rep-PCR) technique was used to investigate inter-specific diversity within 17 species from the Euphorbiaceae family using REP and BOX primers. Results: The agglomerative cluster analysis was used to evaluate the scoring data. BOX and REP gave amplification with polymorphism of 98.84% and 100% respectively. REP marker demarcated between the subgenus peltatae. Both markers confirmed Jatropha tanjorensis as a natural hybrid between Jatropha gossypifolia and Jatropha curcas . Five random sequences from the rep-PCR gels were chosen, cloned and sequenced. The blast results demonstrated that the amplified products were from the mitochondrial genomes. Conclusion: The rep-PCR molecular tool can be used to characterize diversity in plants as they are suitable for distinguishing eukaryotic genomes effectively.

Pseudomonas syringae pv. actinidiae strains isolated from past and current epidemics to Actinidia spp. reveal a diverse population structure of the pathogen

A total of 40 Pseudomonas syringae pv. actinidiae (Psa) strains isolated from past and current epidemics of kiwifruit bacterial canker worldwide were compared using repetitive-sequence PCR (rep-PCR) fingerprinting with BOX, ERIC and REP primer sets. The strains were also assessed for the presence of 23 type III secretion system effector genes, tolerance to sodium arsenite, the presence of coronatine and phaseolotoxin and for growth trends in Actinidia deliciosa leaves. Rep-PCR revealed the occurrence of 11 different Psa lineages among the strains and indicated a relevant genetic variability within the strains isolated in Japan during 1984–2011, whereas all strains obtained from the current pandemic showed the same banding pattern. All lineages showed the same growth trend when inoculated into A. deliciosa leaves. The difference between Psa strains from past and current epidemics was confirmed by the detection of different repertoires of type III effector protein genes. Not all Psa strains isolated in Japan during past epidemics of kiwifruit bacterial canker amplify genes of the argK-tox cluster of phaseolotoxin, suggesting their absence or gene mutation. The results of an arsenic tolerance assay indicated that almost all strains isolated in Italy during the current epidemic of kiwifruit bacterial canker were relatively tolerant to 0.15–0.35 mM sodium arsenite, whereas those isolated in Chile and New Zealand were very sensitive. Remarkably, the two strains from China were tolerant or very sensitive. Collectively, these data indicate a composite population structure of this pathogen, which was able to diversify in Japan during 27 years of recurrent infections to A. deliciosa. The current naming of Psa populations based on their numbering and presence/absence of phytotoxins should be reconsidered.

First Report of Pseudomonas syringae pv. coriandricola Causing Bacterial Leaf Spot on Carrot, Parsley, and Parsnip in Serbia.

During the spring of 2014, a severe leaf spot disease was observed on carrot (Daucus carota), parsley (Petroselinum crispum), and parsnip (Pastinaca sativa) on a 0.5-ha vegetable farm in Vojvodina Province, Serbia. The disease appeared under wet and cool conditions with 5 to 25% of plants infected for each of the three crops. Symptoms were characterized as brown angular leaf spots, ~2 mm in diameter, often limited by veins. Collected symptomatic leaves were rinsed and dried at room temperature, and leaf sections taken from the margin of necrotic tissue were macerated in sterile phosphate buffer and streaked onto nutrient agar with 5% (w/v) sucrose (NAS). After isolation, whitish, circular, dome-shaped, Levan-positive colonies consistently formed. Five strains from each host (carrot, parsley, and parsnip) were used for further study. Strains were gram-negative, aerobic, and positive for catalase and tobacco hypersensitive reaction but negative for oxidase, rot of potato slices, and arginine dihydrolase. These reactions corresponded to LOPAT group Ia, which includes Pseudomonas syringae pathovars (3). Repetitive extragenic palindromic sequence (Rep)-PCR fingerprint profiles using the REP, ERIC, and BOX primers (4) were identical for all strains. Sequence typing of the housekeeping genes gyrB and rpoD (1) was performed for three representative strains (one from each host). Sequences were deposited in the NCBI GenBank database as accessions KM979434 to KM979436 (strains from carrot, parsnip, and parsley, respectively) for the gyrB gene and KM979437 to KM979439 (strains from parsnip, parsley and carrot, respectively) for the rpoD gene. Sequences were compared with pathotype strain Pseudomonas syringae pv. coriandricola ICMP12471 deposited in the Plant Associated and Environmental Microbes Database ( http://genome.ppws.vt.edu/cgi-bin/MLST/home.pl ). BLAST analysis revealed 100% homology for gyrB and 99% homology for rpoD. Pathogenicity was tested with five representative strains from each host on four-week-old plants of carrot (cv. Nantes), parsley (cv. NS Molski), and parsnip (cv. Dugi beli glatki) using two methods: spraying the bacterial suspension (108 CFU ml-1) on the leaves until runoff (5) and injecting the bacterial suspension into leaves with a hypodermic syringe (2). Four plants were used per strain and method. Sterile distilled water was applied as a negative control treatment for each plant species. All plants were kept in a mist room with 100% humidity for 4 h, then transferred to a greenhouse at 25°C and 80% relative humidity and examined for symptom development over a period of three weeks. For all strains, inoculated leaves first developed water-soaked lesions on the leaves 5 to 7 days after inoculation (DAI); 14 DAI lesions became dark brown, often surrounded by haloes. No symptoms were observed on control plants inoculated with sterile distilled water. For fulfillment of Koch's postulates, re-isolations were done onto NAS. Re-isolated bacteria were obtained from each inoculated host and confirmed to be identical to the original isolates using the LOPAT tests and Rep-PCR fingerprinting profiles. Based on the pathogenicity test accompanied by completion of Koch's postulates, sequence analysis, and bacteriological tests, the strains were identified as P. s. pv. coriandricola. To our knowledge, this is the first report of bacterial leaf spot of carrot, parsley, and parsnip in Serbia. It may present a threat to production due to quality requirements for fresh market.

First Report of Pseudomonas syringae pv. aptata Causing Bacterial Leaf Spot on Sugar Beet in Serbia

A severe bacterial leaf spot was observed during June and July 2013 on commercial cultivars of sugar beet (Beta vulgaris var. saccharifera) in the Vojvodina Province of Serbia. Serbia is a major sugar beet production area in southeastern Europe, with 62,895 ha and 3 million tons of sugar beet yield in 2013. A foliar leaf spot observed in 25 commercial sugar beet fields surveyed ranged from 0.1 to 40% severity. Symptoms were characterized as circular or irregular, 5- to 20-mm diameter, white to light brown necrotic spots, each with a dark margin. Diseased leaves were rinsed in sterilized, distilled water (SDW) and dried at room temperature, and leaf sections taken from the margin of necrotic tissue were macerated in SDW. Isolations from 48 symptomatic leaves onto nutrient agar with 5% (w/v) sucrose (NAS) produced bacterial colonies that were whitish, circular, dome-shaped, and Levan-positive. Representative isolates (n = 105) were Gram negative; aerobic; positive for catalase, fluorescence on King's medium B, and tobacco hypersensitivity; and negative for oxidase, potato rot, and arginine dehydrolase. These reactions corresponded to LOPAT group Ia, which includes Pseudomonas syringae pathovars (2). Repetitive extragenic palindromic sequence (rep)-PCR was used for genetic fingerprinting the isolates using the REP, ERIC, and BOX primers. Twenty-five different profiles were obtained among the strains. From each profile group, one representative strain was sequenced for the gyrB gene (1). Four heterogenic groups were observed, and representative gyrB gene sequences of each group were deposited in the NCBI GenBank (Accession Nos. KJ950024 to KJ950027). The sequences were compared with those of pathotype strain P. syringae pv. aptata CFBP 1617 deposited in the PAMDB database; one strain was 100% homologous, and the other three were 99% homologous. To fulfill identification of the Serbian sugar beet isolates, gltA and rpoD partial gene sequences were determined (1), and the sequences were deposited as Accession Nos. KM386838 to KM386841 for gltA and KM386830 to KM38683033 for rpoD. The sequences were 100% homologous with those of pathotype strain CFBP 1617. Pathogenicity of each of four representative bacterial strains was tested on 3-week-old plants of the sugar beet cultivars Marinela, Serenada, and Jasmina (KWS, Belgrade, Serbia) and Lara (NS Seme, Novi Sad, Serbia) by atomizing a bacterial suspension of ~106 CFU/ml of the appropriate isolate onto the abaxial leaf surface of three plants per cultivar until water-soaking of the leaf surface was observed. Three plants of each cultivar atomized similarly with P. syringae pv. aptata CFBP 2473 and SDW served as positive and negative control treatments, respectively. Inoculated plants were kept in a clear plastic box at 80 to 100% RH and 17 ± 1°C and examined for symptom development over 3 weeks. For all test isolates and the control strain, inoculated leaves first developed water-soaked lesions 7 days after inoculation (DAI). By 10 to 14 DAI, lesions were necrotic and infection had spread to the petioles. By 21 DAI, wilting was observed on more than 50% of inoculated plants. Negative control plants were symptomless. Bacteria re-isolated onto NAS from inoculated leaves had the same colony morphology, LOPAT results, and gyrB partial gene sequences as described for the test strains. No bacteria were re-isolated from negative control plants. Based on these tests, the pathogen causing leaf spot on sugar beet in Serbia was identified as P. syringae pv. aptata.

PHENOTYPE AND GENOTYPE DIVERSITY OF IRANIAN STREPTOMYCES ISOLATES THAT CAUSE POTATO COMMON SCAB

During 2006 and 2007, 110 Streptomyces strains were isolated from potato tubers with various symptoms including erumpent, deep-pitted and superficial scab lesions from different regions of Fars and Hamedan provinces of Iran. The isolates were identified as Streptomyces scabies, S. acidiscabies, S. turgidiscabies, S. aureofaciens, S. reticuliscabies, S. griseus, S. europaeiscabies and S. stelliscabies using physiological, biochemical and morphological tests and were classified into six groups using the NTSYSpc version 2.02e software. The isolates were divided into nine clusters based on the numerical analysis of rep-PCR using Box primers. Clustering based on phenotypic features confirmed. In some cases, the clustering based on rep-PCR with Box primers. Rep-PCR DNA fingerprints of Streptomyces strains with BoxA1R primers were relatively unique, consistent, and reproducible. Our results show that Box-PCR is a very useful tool for a quick determination of strain identity.

Molecular characterization of Bacillus thuringiensis using rep-PCR

The genetic divergence of 65 strains of Bacillus thuringiensis (Bt) was determined using Rep-PCR. Based on the repetitive sequences the BOX primer was the most informative with 26 fragments, followed by ERIC (19) and REP (10), generating a total of 55 fragments. The dendogram shows that ten groups were formed when 45% was the average distance of the population: group 1 with 41,5% of the isolates, 33,8% of the isolates were distributed in other groups and 24,6% did not formed distinct group. 53,2% of the isolates from Embrapa are in the group 1, and 29,8% of the isolates are distributed in other groups. Bt strains from USDA and Institute Pasteur showed more variability.

FIRE BLIGHT: FIRST REPORT IN LATIUM, ITALY

Fire blight, caused by Erwinia amylovora, a major pathogen of pome fruits and many ornamentals, included in the EPPO A2 list of quarantine organism, was detected in 1990 in the main pome fruit-growing areas of Italy, including Emilia-Romagna where, since 1994, it spread rapidly causing epidemic outbreaks in 1997-1998. In October 2012, during the official monitoring made by the regional Plant Protection Service (PPS), a sample was collected in a nursery in Viterbo province (Latium, central Italy) from a quince plant showing shoot tips withered in the typical 'shepherd’s crook', blackish-brown discoloration of the cortex which, when removed, disclosed the typical red streaks. Following isolation on nutrient sucrose agar (NSA) levan-positive colonies were obtained and purified. The bacterial isolates did not produce fluorescent pigment on CSGA, induced hypersensitive response in tobacco leaves and were oxidase-negative. One isolate (CRA-PAV 1751) was lyophilized and conserved. Specific PCR protocols (Bereswill et al., 1992; Llop et al., 2000; Taylor et al., 2001) yielded amplicons of the expected size. Rep-PCR (using BOX and ERIC primers) fingerprinting pattern was similar to that of the reference strain OMP-BO 1077/7. Pathogenicity tests on immature pear fruits and re-isolation were performed successfully. This is the first report of E. amylovora from Latium, which was declared as "protected zone" by EU Directive 2000/29/EC. Following our finding, the regional PPS has adopted all the measures required by the Ministerial Decree 10/9/99 No. 356, to restrain the spread of the pathogen. This highlights the importance of healthy nursery productions, especially for pathogens like E. amylovora for which prevention is the main control strategy, given that no effective chemical or integrated control methods are available.

CHARACTERIZATION OF RALSTONIA SOLANACEARUM CAUSING BACTERIAL WILT IN BELL PEPPER IN THE STATE OF PERNAMBUCO, BRAZIL

Seventy-seven bacterial strains obtained from wilted bell pepper plants from the Agreste and Mata mesoregions of Pernambuco State (northeastern Brazil), were identified as Ralstonia solanacearum using multiplex PCR. The strains were further analyzed to characterize their biochemical, physiological, and molecular diversities. The biovars and biotypes were determined biochemically, and a molecular characterization of the strains was made using multiplex PCR, rep-PCR, and ISSR. The genotypic diversity was assessed, considering the number of genotypes observed and how they were distributed throughout the populations, for their differences in terms of richness, evenness and diversity. Strains of biovar 3, biotype 8 and phylotype I were predominant (97.40%), but biovar 1, biotypes 3 and 6 and phylotype II were also present. Rep-PCR analysis using REP and BOX primers, and ISSR showed similarities among the majority of the strains; however, these primers did not allow strain separation by biovars, biotypes, phylotypes or areas. Analysis of the genotypic diversity revealed a moderate diversity in the overall population, with a high variability in the strains from the same municipality.

Characterization of native fluorescent Pseudomonas isolates associated with alfalfa roots in Uruguayan agroecosystems

Establishment of alfalfa crops is continuously threatened by seedling diseases caused by soilborne pathogens. The use of plant beneficial bacteria as inoculants is a feasible and environmentally friendly means to control soil pathogens. Identifying effective plant growth-promoting strains to use on local crops under local environmental conditions requires the screening of large collections of native isolates. A collection of 738 rhizospheric fluorescent Pseudomonas isolates was obtained from alfalfa plants from three agroecological regions representative of Uruguayan agricultural systems. The isolates were evaluated for in vitro pathogen inhibition, biosurfactant production, phosphate solubilization and the presence of genes involved in antibiotic synthesis. Isolates with strong in vitro antagonistic activity toward Pythium debaryanum were more abundant in alfalfa plants established in a previously natural ecosystem while biosurfactant producers were less abundant in that location. A subset of isolates was selected for genotypic characterization by rep-PCR using BOX primers. Twenty-four genotypes were defined, sixteen from a single geographical origin and eight composed of isolates from multiple origins. Genotypic profiles correlated well with phenotypic traits. A subset of isolates was assayed to determine their ability to protect alfalfa against P. debaryanum damping-off and to promote plant growth. Five native Pseudomonas isolates showed significant effects on alfalfa by increasing plant biomass and/or protecting from pathogen infection. Plant growth promoting isolates from each location were genotypically similar. Our work contributes to the knowledge of the phenotypic and genotypic diversity of rhizospheric fluorescent pseudomonads of forage legumes and the frequency of plant growth promoting traits associated with this group of bacteria in different agricultural systems.

Genetic Differentiation of Pseudomonas syringae Pathovar tomato from Other P. syringae Pathovars using REP-PCR and URP-PCR

For the genetic differentiation of <TEX>$Pseudomonas$</TEX> <TEX>$syringae$</TEX> pathovar <TEX>$tomato$</TEX>, a total of 51 <TEX>$P.$</TEX> <TEX>$syringae$</TEX> pv. strains infecting 33 different host plants were analyzed using repetitive element PCR(REP-PCR) and universal rice primer PCR(URP-PCR). The entire DNA fingerprint profiles were analyzed using unweighted pair-group method with arithmetic averages (UPGMA). The 51 <TEX>$P.$</TEX> <TEX>$syringae$</TEX> pv. strains could be divided into five clusters based on 65% similarity by Rep-PCR using BOX, ERIC, and REP primers. <TEX>$P.$</TEX> <TEX>$syringae$</TEX> pv. <TEX>$tomato$</TEX> cluster was well separated from other 31 <TEX>$P.$</TEX> <TEX>$syringae$</TEX> pathovars. <TEX>$P.$</TEX> <TEX>$syringae$</TEX> pv. <TEX>$tomato$</TEX> cluster included only <TEX>$P.$</TEX> <TEX>$syringae$</TEX> pv. <TEX>$maculicola$</TEX> and <TEX>$P.$</TEX> <TEX>$syringae$</TEX> pv. <TEX>$tomato$</TEX>. <TEX>$P.$</TEX> <TEX>$syringae$</TEX> pv. <TEX>$tomato$</TEX> strains could be divided into two genetic groups. Meanwhile, the Pseudomonas pv. strains could be divided into four clusters based on 63% similarity by URP-PCR using 2F, 9F, and 17R primers. <TEX>$P.$</TEX> <TEX>$syringae$</TEX> pv. <TEX>$tomato$</TEX> cluster was also well separated from 30 other <TEX>$P.$</TEX> <TEX>$syringae$</TEX> pathovars. In this case, <TEX>$P.$</TEX> <TEX>$syringae$</TEX> pv. <TEX>$tomato$</TEX> cluster included <TEX>$P.$</TEX> <TEX>$syringae$</TEX> pv. <TEX>$maculicola$</TEX>, <TEX>$P.$</TEX> <TEX>$syringae$</TEX> pv. <TEX>$berberidi$</TEX>, and <TEX>$P.$</TEX> <TEX>$syringae$</TEX> pv. <TEX>$tomato$</TEX>. <TEX>$P.$</TEX> <TEX>$syringae$</TEX> pv. <TEX>$tomato$</TEX> strains was also separated into two genetic groups by URP-PCR analysis. Overall, our work revealed that <TEX>$P.$</TEX> <TEX>$syringae$</TEX> pv. <TEX>$tomato$</TEX> can be genetically differentiated from other <TEX>$P.$</TEX> <TEX>$syringae$</TEX> pathovars by the DNA fingerprint profiles of REP-PCR and URP-PCR. We first report that there are two genetically diverged groups in <TEX>$P.$</TEX> <TEX>$syringae$</TEX> pv. <TEX>$tomato$</TEX> strains.

Phenotypic and genotypic diversity of Pseudomonas aeruginosa strains isolated from hospitals in siedlce (Poland).

A total of 62 Pseudomonas aeruginosa strains isolated from two hospitals in Siedlce (Poland) were studied by repetitive element based PCR (rep-PCR) using BOX primer. BOX-PCR results revealed the presence of 7 numerous genotypes and 31 unique patterns among isolates. Generally, the strains of P. aeruginosa were characterized by resistance to many antibiotics tested and by differences in serogroups and types of growth on cetrimide agar medium. However, the P. aeruginosa strains isolated from faeces showed much lower phenotypic and genotypic variations in comparison with strains obtained from other clinical specimens. It was observed that genetic techniques supported by phenotypic tests have enabled to conduct a detailed characterization of P. aeruginosa strains isolated from a particular environment at a particular time.

Differentiation of Pseudomonas syringae pathovars originating from stone fruits

Due to an overlapping host range, similar symptomatology and many common characteristics, Pseudomonas syringae pathovars originating from stone fruits can easily be misidentified. In order to select tests for rapid and efficient differentiation of P. s. pvs. syringae, morsprunorum and persicae, we studied the suitability and differentiating potential of some standard bacteriological and molecular methods. Differentiation of the strains was performed using LOPAT, GATTa and ice nucleation tests, nutrient sucrose broth growth and utilization of various carbon sources. PCR method enabled the detection of toxin-producing genes: syrB and syrD in P. s. pv. syringae, and cfl gene in P. s. pv. morsprunorum race 1. Syringomycin production by pv. syringae was confirmed in bioassay using Geotrichum candidum, Saccharomyces cerevisiae and Rhodotorula pilimanae as indicator organisms. Pathogenicity test on lemon and immature nectarine fruits, as well as on string bean pods, showed different intensity of reaction of the inoculated material which could separate pv. syringae from the other two pathovars. PCR-based repetitive sequences, Rep-PCR with REP, ERIC and BOX primers revealed different genetic profiles within P. syringae pathovars.

Analysis of bacterial community in bulking sludge using culture-dependent and -independent approaches

The bacterial community of a bulking sludge from a municipal wastewater treatment plant with anoxic-anaerobic-oxic process was investigated by combination of cultivation and 16S rRNA gene clone library analysis for understanding the causes of bulking. A total of 28 species were obtained from 63 isolates collected from six culture media. The most cultivable species belonged to γ-Proteobacteria including Klebsiella sp., Pseudomonas sp., Aeromonas sp. and Acinetobacter sp. Further analysis of these strains by repetitive sequence based on polymerase chain reaction (rep-PCR) technology showed that rep-PCR yielded discriminatory banding patterns within the same genus using REP and BOX primer sets. While the culture-independent assessment revealed that β-Proteobacteria was the dominant group in the bulking sample. Sequence analysis revealed that the highest proportion (14.7%) of operational taxonomic units was 98% similar to Candidatus Accumulibacter phosphatis, which is used to remove phosphorous from wastewater. Our results indicated that combining différent approaches can produce complementary information, thus generate a more accurate view of microbial community in bulking sludge.