Polymerase Chain Reaction-restriction Fragment Length Polymorphism Patterns Research Articles

Simple PCR-RFLP detection method for genus- and species-authentication of four types of tuna used in canned tuna industry

A simple method for authentication of processed tuna products is not only for testing against fraudulent practices in the tuna industry but being a promising tool for enhancing traceability. Here, a method of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) had been developed using a part of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene as a key region for genus-and species-identification. The analysis was based on the different RFLP patterns of TaqI and HaeIII digested COI DNA from the four tuna species; i.e. Kasuwonus pelamis, Thunnus alalunga, Thunnus albacares, and Thunnus obesus, respectively. While the conventional PCR-RFLP worked effectively in raw tuna meat, a method of semi-nested PCR had to be developed in order to increase efficiency in the detection in cooked meat from canned product. The semi-nested PCR-RFLP pattern of COI DNA allowed the detection of 10% contamination of K. pelamis in canned tuna of other higher-valued species.

Spatio-temporal dynamics of Varroa destructor resistance to tau-fluvalinate in Czechia, associated with L925V sodium channel point mutation.

Extensive application of pyrethroids to control Varroa destructor, an invasive mite devastating bee colonies, has resulted in a global spread of resistant mite populations. In this study, we analyzed the spatio-temporal dynamics of resistant V. destructor populations in Czechia, stemming from the L925V mutation. Mites were collected during 2011-2018 directly or from winter beeswax debris, and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and densitometry was used to detect the L925V mutation. Pooled samples of 10 mites were classified, based on their PCR-RFLP patterns, as tau-fluvalinate-sensitive (56%), resistant (9%), or mixed (35%), with the latter including sensitive and resistant homo- and heterozygotes. We identified two zones with higher frequencies of resistance, one in southern Moravia and the other in Bohemia. The mutant populations were evenly distributed throughout the monitored districts, with a few temporal and spatial local fluctuations. The greatest increase in resistance was observed in 2016, following massive losses of bee colonies in the winter of 2015. This event appeared to be closely associated with fluctuations in resistant mite populations and their dispersion. Two outbreaks of resistance were detected in Czechia; however, the amount of applied tau-fluvalinate was not correlated with the frequency of resistance in mites. There was no remarkable increase in mite resistance in 2011-2018, although the use of tau-fluvalinate increased 40-fold between 2011 and 2015. PCR-RFLP analysis, performed on mites present in beeswax debris, is a suitable method for monitoring the L925V mutation in V. destructor. © 2018 Society of Chemical Industry.

Genotyping of major histocompatibility complex Class II DRB gene in Rohilkhandi goats by polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing.

Aim:To study the major histocompatibility complex (MHC) Class II DRB1 gene polymorphism in Rohilkhandi goat using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and nucleotide sequencing techniques.Materials and Methods:DNA was isolated from 127 Rohilkhandi goats maintained at sheep and goat farm, Indian Veterinary Research Institute, Izatnagar, Bareilly. A 284 bp fragment of exon 2 of DRB1 gene was amplified and digested using BsaI and TaqI restriction enzymes. Population genetic parameters were calculated using Popgene v 1.32 and SAS 9.0. The genotypes were then sequenced using Sanger dideoxy chain termination method and were compared with related breeds/species using MEGA 6.0 and Megalign (DNASTAR) software.Results:TaqI locus showed three and BsaI locus showed two genotypes. Both the loci were found to be in Hardy–Weinberg equilibrium (HWE), however, population genetic parameters suggest that heterozygosity is still maintained in the population at both loci. Percent diversity and divergence matrix, as well as phylogenetic analysis revealed that the MHC Class II DRB1 gene of Rohilkhandi goats was found to be in close cluster with Garole and Scottish blackface sheep breeds as compared to other goat breeds included in the sequence comparison.Conclusion:The PCR-RFLP patterns showed population to be in HWE and absence of one genotype at one locus (BsaI), both the loci showed excess of one or the other homozygote genotype, however, effective number of alleles showed that allelic diversity is present in the population. Sequence comparison of DRB1 gene of Rohilkhandi goat with other sheep and goat breed assigned Rohilkhandi goat in divergence with Jamanupari and Angora goats.

Identification of Clinical Corynebacterium striatum Strains by PCR-Restriction Analysis Using the RNA Polymerase β-subunit gene (rpob)

Corynebacterium striatum is frequently encountered in the routine clinical microbiology laboratory. It is widely disseminated in the environment and constitutes part of the normal micro-biota of the skin and mucous membrane. Identification of this species by biochemical methods remains difficult and several misidentifications have been reported previously. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for the identification of this microorganism was designed based on the hypervariable region of the polymorphic RNA polymerase β-subunit gene (rpoB). All available Corynebacterium rpoB sequences were analyzed by computerassisted restriction analysis. The rpoB PCR-RFLP pattern predicted by using endonucleases MseI and NlaIV clearly differentiated C. striatum from all other Corynebacterium species. This method was successfully applied for the reliable identification of 67 C. striatum clinical isolates and can be used for the timely detection of infected patients or for epidemiological studies.

Effective molecular examination of eukaryotic plankton species diversity in environmental seawater using environmental PCR, PCR-RFLP, and sequencing

Phytoplankton are primary producers and can be important indicators of environmental change. To monitor the plankton species composition of environmental seawater samples, we developed a molecular method composed of colony polymerase chain reaction (PCR), polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP), and sequencing. A clone library of the ribosomal small subunit RNA gene (18S rDNA) in the nuclear genome was constructed by environmental PCR using a newly designed primer set and clones were directly amplified by colony PCR. To select unique putative clones, we choose a PCR-RFLP method that employed two restriction enzymes (MseI and Tsp509I). After the PCR-RFLP pattern was evaluated, selected clones were sequenced and analyzed. In this study, we revealed the hidden biodiversity in environmental seawater containing a wide range of taxonomic groups in the Alveolata (Ciliphora and Dinophyceae), Euglenozoa, Stramenopiles (Bacillariophyta), and Viridiplantae (Chlorophyta) without the need to conduct extensive colony isolation techniques. Moreover, we found species of fungi and Metazoa (Arthropoda, Annelida, and Mollusca). Therefore, this improved molecular method can be used to generate a robust database describing the species diversity of environmental samples and provide useful information regarding the dynamics of the eukaryotic plankton community structure.

Detection of the mosquitocidal toxin genes encoding Cry11 proteins from Bacillus thuringiensis using a novel PCR-RFLP method.

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for detection of cry11 genes from Bacillus thuringiensis was established. Based on the analysis of conserved regions of the cry11 genes, 2 oligonucleotide primers were designed to amplify a 1459-bp fragment of the cry11Aa gene, and a 1471-bp of the cry11Ba and cry11Bb genes. The amplification products were digested with restriction endonuclease HinfI. Exotic B. thuringiensis strains and native isolates collected from soils, leaves and stored product dust of Argentina were analyzed to study the distribution of cry11 genes. The PCR-RFLP patterns revealed the detection of cry11 genes in 3 of 64 exotic strains and in 10 of 107 native B. thuringiensis isolates tested. Just the cry11Aa gene subclass was detected among these bacteria. Since the methodology was also developed to detect cry11Ba and cry11Bb genes, an experimental future confirmation will be required. Based on the results obtained, the PCR-RFLP method presented may be a valuable tool for specific detection of the mosquitocidal toxin genes encoding Cry11 proteins from B. thuringiensis.

고래회충유충증 감별 진단을 위한 18S ribosomal DNA (rDNA) PCR-RFLP 법 적용

고래회충유충증은 해산어류에 기생하는 고래회충과(family Anisakidae)에 속하는 선충류 유충에 의한 질병으로 유충의 직접적인 위장관내 침입으로 인한 병변과 더불어 유충의 분비 배설물에 의한 알레르기 질환도 유발될 수 있다. 고래회충유충증은 A. simplex를 비롯하여 Contracaecum, Pseudoterranova, Hysterothylacium 등의 유충에 의해 야기될 수 있으나 이들에 대한 형태학적 감별 진단은 유충의 형태적 유사성으로 인하여 매우 어려운 경우가 많다. 이러한 형태학적 진단의 어려움을 극복하고 분자생물학적 감별진단 방법을 확립하기 위하여 A. simplex, Contracaecum type A. type C' 및 Goezia 유충을 숭어, 도다리, 고등어, 아나고, 참돔 등 5종의 어류에서 분리하였다. 각각의 유충으로부터 분리한 18S rDNA를 PCR로 증폭한 후 Taq 1, Hinf I, Hha I, Alu 1, Dde I, Hae III, Sau 96I, Sau 3AI 등 8종의 제한효소를 사용하여 PCR-RFLP를 시행하였다. PCR product의 크기는 약 2.0 Kb였으며 Hinf l, Alu 1, Hha I, Dde 1 및 Hae III로 A. simplex와 Contracaecum type C'을 구분할 수 있었다. 그러나 Contracaecum type A의 경우에는 Taq I, Hinf I, Alu I 및 Dde I의 경우에는 2가지 패턴으로 나타났으며 이들 가운데 일부는 A. simplex, Contracaecum type C', 및 Goezia와 동일한 분석 패턴을 보이기도 하였다. Goezia는 사용한 8개의 제한 효소 모두에서 A. simplex 및 Contracaecum type A 및 type C'과 각기 다른 양상을 보였다. 이러한 결과로 18S rDNA PCR-RFLP 방법은 A. simplex와 Contracaecum type C'의 감별 진단에 유용한 것으로 밝혀졌으며, Contracaecum type A의 분류에는 제한적으로 사용되어야 함은 물론 형태학적 분류 기준에 대한 재검토가 뒤따라야 할 것으로 사료되었다. Anisakidosis is caused by anisakid nematodes (family Anisakidae) larvae which can cause not only direct tissue damage but also a severe allergic response related to excretory-secretion products. Lots of different species of anisakid larvae, including Anisakis simplex, Contracaecum, Goezia, Pseudoterranova, and Hysterothylacium, cause the anisakidosis. But it is difficult to diagnosis the species of larvae since the morphologies of larval anisakid nematodes are almost indistinguishable. In order to diagnosis the differential infections of larval anisakid nematodes, polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) of 18S rDNA - was conducted. Three major species of anisakid larvae including A. simplex, C.ontracaecum spp, and Goezia spp. were collected from mackerel (Scomber japonicus), mullet (Mugil cephalus), founder (Paralichthys olivaceus), eel (Astroconger myriaster) and red sea bream (Pagrus major). PCR amplified 18S rDNA from each species of anisakid larvae was digested with eight restriction enzymes including Taq I, Hinf I, Hha I, Alu I, Dde I, Hae III, Sau96 I, and Sau3A I. The original sizes of PCR amplified 18S rDNA were 2.0Kb in both anisakid larvaes and Goezia. Restrction enzymes including Hinf 1, Alu 1, Hha I, Dde 1 and Hae III cut differently and distinguished the A. simplex and Contracaecum type C'. However, Contracaecum type A showed two different restriction enzyme cutting patterns by Taq 1, Hinf I, Alu 1, and Dde 1. One of the patterns was the same as those of A. simplex, Contracaecum type C' and Goezia and the other was unique. These results suggest that PCR-RFLP pattern by Hinf 1, Alu 1, Hae I, Dde 1 and Hae III can be applied to differential diagnosis of human infection with A. simplex and Contracaecum type C'. Contracaecum type A needs further study of classification by morphological characteristics and genetic analysis.

MOLECULAR TYPING OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS STRAINS BY PCR-RFLP OF SPA GENE: A REFERENCE LABORATORY PERSPECTIVE

Purpose: To characterize methicillin-resistant Staphylococcus aureus (MRSA) strains by molecular typing based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of spa gene and to assess the utility of spa genotyping over bacteriophage typing in the discrimination of the strains. Materials and Methods: Studies were undertaken on 125 MRSA strains representing the most predominant phage types and the non phage typeable strains. Strains were typed by bacteriophage typing and PCR-RFLP of spa gene. DNA sequence analysis of the amplified spa gene fragment of the representative RFLP patterns was performed using standard protocols. Results: All the strains resistant to oxacillin were found to contain mec A gene. Fifty-two per cent of these strains were typeable by the international basic set of 23 phages. Five different PCR-RFLP patterns were observed among 125 MRSA strains. Non phage typeable strains were differentiated into four PCR-RFLP patterns. Sequencing of the spa gene from the representative strains of each RFLP pattern confirmed the length of these restriction fragments due to variation in the 24 bp and the 174 bp tandem repeats. It also revealed the presence of three new spa repeat patterns. Conclusion: The study demonstrates the importance of spa genotyping in the discrimination of MRSA strains, which were otherwise indistinguishable by bacteriophage typing. spa genotyping allowed differentiation of strains within a particular phage type. Nucleotide sequencing of isolates of different PCR-RFLP patterns indicated a correlation between the RFLP patterns of a variable number of tandem repeats and the phage type. The study provides valuable information on the epidemiological characterization of MRSA strains.

Isolation of 19 strains of Malassezia dermatis from healthy human skin in Korea

This research was conducted on the cultured samples of 160 healthy men and women aged 0-80 years without any skin disease. Nineteen clinical isolates of Malassezia dermatis showed positive in a catalase test and all grew in 0.5% Tween-60 and 0.1% Tween-80 added to 2% glucose/1% peptone culture medium. Round and ellipsoidal yeast cells and budding of the yeast cells were observed by microscopy, resembling Malassezia sympodialis, Malassezia furfur and Malassezia nana. The 26S rDNA polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) pattern was the same as for M. dermatis (JCM 11348), the standard strain. 26S rDNA and ITS1 sequencing were performed for exact identification, showing 99% accordance with M. dermatis (AB070361), M. dermatis (AB070356), confirming the species to be new and first to be reported in Korea. Taking a molecular biological classification approach by analyzing the 26S rDNA PCR-RFLP patterns, we have successfully isolated 19 cases of M. dermatis- the first in Korea.

PCR-RFLP Analysis of Chloroplast DNA Variations in the atpI-atpH Spacer Region of the Genus Camellia

Camellia is an ornamental tree that is grown worldwide. There are many excellent classical cultivars whose origins are unclear. Chloroplast DNA (cpDNA) is a good material for tracing maternal lineages and we therefore conducted polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the cpDNA atpI-atpH region from 162 accessions derived from 49 species of the genus Camellia. In order to specifically and accurately amplify the atpI-atpH region, camellia-specific primers were designed and utilized with some of the accessions. PCR amplification of the atpI-atpH region resulted in two major bands of approximately 800 bp and 1200 bp. TaqI digestion resulted in 3 different band patterns derived from the 800 bp fragment and 5 patterns derived from the 1200 bp fragment. Most of these eight PCR-RFLP patterns were distributed widely amongst species and sections of the genus. In particular, it was possible to use this approach to distinguish between the main species from which ornamental camellia cultivars are derived. Sequence analysis of the atpI-atpH region showed a number of mutations, which were characteristic of particular species. These results indicate that PCR-RFLP analysis of the atpI-atpH region will be very useful for investigating variations in the genus Camellia.

Divergence in symbiotic interactions between same genotypic PCR–RFLP Frankia strains and different Casuarinaceae species under natural conditions

The symbiotic interactions between Frankia strains and their associated plants from the Casuarinaceae under controlled conditions are well documented but little is known about these interactions under natural conditions. We explored the symbiotic interactions between eight genotypically characterized Frankia strains and five Casuarinaceae species in long‐term field trials. Characterization of strains was performed using the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) for the nifD–nifK intergenic transcribed spacer (ITS) and 16S–23S ITS. Assessments of the symbiotic interactions were based on nodulation patterns using nodule dry weight and viability, and on actual N2 fixation using the δ15N method. The PCR–RFLP patterns showed that the analyzed strains belonged to the same genotypic group (CeD group), regardless of the host species and environment of origin. The nodule viability index is introduced as a new tool to measure the viability of perennial nodules and to predict their effectiveness. The host Casuarinaceae species was a key factor influencing both the actual N2‐fixing activity of the associated Frankia strain and the viability of nodules within a location. This is the first study providing information on the symbiotic interactions between genotypically characterized Frankia strains and actinorhizal plants under natural conditions. The results revealed a way to improve a long‐term management of the Casuarinaceae symbiosis.

Comparison of the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Pattern of the Fiber Gene and Pathogenicity of Serotype-1 Fowl Adenovirus Isolates from Gizzard Erosions and from Feces of Clinically Healthy Chickens in Japan

The fiber gene sequence and pathogenicity of the serotype-1 fowl adenovirus (FAdV-1) isolated from gizzard erosions and from clinically normal chickens were compared among isolates. The FAdV-99ZH strain, which induced gizzard erosions, had a nucleotide sequence of the long fiber gene that was different from that of the Ote strain, which did not induce gizzard erosions. The differences could be distinguished by use of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The long fiber gene of 16 FAdV-1 isolates from gizzard erosions and 10 FAdV-1 isolates from the feces of clinically normal chickens was examined by use of PCR-RFLP analysis. All 16 FAdV-1 isolates from gizzard erosions had the same restriction patterns as those of strain 99ZH; however, 10 FAdV-1 isolates from normal chickens were classified into 3 groups. Specific-pathogen-free (SPF) chickens were inoculated orally with 2 FAdV-1 isolates from gizzard erosions or 3 FAdV-1 isolates from clinically normal chickens to determine the pathogenicity of each strain. Two of 2 FAdV-1 isolates from gizzard erosions induced gizzard erosions. Two of 3 FAdV-1 isolates from normal chickens had the same PCR-RFLP patterns as those of the Ote strain, but did not induce any gizzard erosions. However, 1 FAdV-1 isolate from clinically normal chickens had the same PCR-RFLP pattern as that of strain 99ZH and induced gizzard erosions. These results indicate that there are FAdV-1 strains that have different pathogenicity; one strain induces gizzard erosions, and the other does not. Use of PCR-RFLP analysis of long fiber genes may be able to distinguish between these two strains.

Detection and Identification of cry1I Genes in Bacillus thuringiensis Using Polymerase Chain Reaction and Restriction Fragment Length Polymorphism Analysis

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for detection and identification of cry1I genes from Bacillus thuringiensis (Bt) was established. Based on the analysis of conserved regions of the cry1I genes, 2 oligonucleotide primers were designed to amplify a 665-bp fragment of the genes. The amplification products were digested with restriction endonuclease HinfI or with RsaI in addition for specific detection of different variants from the known subclasses of cry1I genes. The PCR-RFLP pattern obtained revealed the detection of cry1I genes in 151 of 202 native Bt isolates. Furthermore, cry1I genes were detectable in 10 of 19 standard strains tested. The cry1Ia gene was the most abundant cry1I gene subclass present in 54 of 56 native Bt isolates and in 8 of 10 standard strains. Based on the results obtained, the PCR-RFLP method may be a valuable and reliable tool for specific detection and identification of cry1I genes.

The trnL-F plastid DNA characters of three Poa pratensis (Kentucky bluegrass) varieties

The characterization of crop cultivars (varieties) will come to depend increasingly on molecular characters in addition to traditional morphological and agronomic characters. Three cultivars of Kentucky bluegrass (Poa pratensis L.), developed by the Plant Breeding Station Hladké Životice (PBHŽ), were characterized using sequences and PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) patterns from the non-coding plastid trnL-F region (trnL intron, 549 bp, and trnL-trnF intergenic spacer [IGS], 344 to 364 bp). These characters could be readily and repeatably determined not only for mature plants, but also for seedlings (less than 12 weeks old), which are difficult to distinguish morphologically. The method is quick and sensitive. When restricted with a combination of BsaJ I and Bsm I, cultivar Slezanka has one major band, Moravanka has two, and Harmonie has three. When restricted with Alu I, the heaviest band migrates most slowly for Slezanka. It is expected that many Kentucky bluegrass cultivars will share the same trnL-F sequence, so these characters alone are not sufficient for variety identification.

Rapid assignment of the swine major histocompatibility complex (SLA) class I and II genotypes in Clawn miniature swine using PCR‐SSP and PCR‐RFLP methods

Inbred miniature swine with defined novel SLA haplotypes will be useful in allo- and xeno-transplantation studies, which can be carried out representing variable combinations of SLA haplotypes. In Clawn miniature swine, two haplotypes (c1 and c2) and one crossover haplotype (c3) have been assigned by nucleotide sequence determination of RT-PCR products of the three SLA classical class I genes and two SLA class II genes. To select SLA class I and II homozygotes in Clawn miniature swine individuals, we developed a rapid and simple SLA-class I- and II-DNA typing method by a combination of polymerase chain reaction-sequence specific primer (PCR-SSP) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques. Seven allele specific primer pairs were designed for amplification of the second exons of three SLA class I genes, SLA-1, SLA-2, and SLA-3, and one SLA class II gene, DRB1. Furthermore, based on PCR-RFLP patterns in the SLA-DQB1 gene, two allelic variants were recognized in the second exon in the Clawn miniature swine. Three haplotypes, c1, c2 and c3, were simply identified by the combination of PCR-SSP and PCR-RFLP methods in 22 samples from five families. A single allele at each of the class I and II genes was also observed in seven samples as SLA class I and II homozygotes with either the c1 or c2 haplotype. The combination of PCR-SSP and PCR-RFLP methods facilitate the rapid identification of the three haplotypes and SLA class I and II homozygotes in individual Clawn miniature swine.

Distribution and biodiversity of soybean rhizobia in the soils of Shennongjia forest reserve, China

Soil samples were collected at an altitude of 500, 1,060, 1,500, 1,950, 2,400 and 3,100 m, respectively, from Shennongjia, a forest reserve in Hubei province (central China). Their corresponding pHs were 5.50, 4.91, 5.64, 5.28, 5.49 and 4.60. By using a plant trap method, a total of 25 soybean rhizobia were isolated from the soil above an altitude of 1,500 m and all identified to be Sinorhizobium fredii. Their genetic biodiversity was characterized by 16S–23S rDNA internally transcribed spacer (ITS) region polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and random amplification DNA (RAPD) analysis. All the tested strains produced a 2.1 kb 16S–23S rDNA ITS fragment. After digestion with three restriction endonucleases (HaeIII, MspI and CfoI), respectively, great variations in 16S–23S rDNA ITS PCR-RFLP patterns were observed. The tested strains could be differentiated into 11 ITS genotypes. The genotypes of rhizobia were not related to geographical location. Twelve primers were applied to RAPD analysis and a dendrogram was obtained, showing that all the strains (including reference strain S. fredii USDA205) were divided into two diverging groups. Moreover, each group could be further divided into two subgroups. Both RAPD and 16S–23S rDNA ITS PCR-RFLP analysis indicated that a high degree of genetic diversity existed among S. fredii strains isolated from Shennongjia virgin soils. Since Shennongjia is an unexploited forest region in central China and the gene centre of soybean is located in China, the symbiotic genes harboured by these strains may be of great importance and the rich diversity of these strains might contribute to the adaptation of soybean to an alpine environment.

Antimicrobial susceptibility and strain prevalence of Korean vaginal Lactobacillus spp.

One hundred eight vaginal lactobacilli were isolated from Korean women and characterized in terms of their antibiotics susceptibility and PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) pattern. The in vitro antibiotics susceptibilities of 108 vaginal isolates to 13 antimicrobial agents were determined by broth dilution method based on NCCLS reference protocol. High rates of resistance were demonstrated for gentamicin, kanamycin, metronidazole, and streptomycin whereas all the isolates were susceptible to erythromycin. The concentrations of gentamicin, kanamycin, metronidazole, streptomycin, and erythromycin at which 90% of the vaginal isolates were inhibited (MIC90) were 100, 200, >200, 200 and 0.39 μg/mL, respectively. For molecular identification, PCR-RFLP analysis was employed where the 16S rDNA was amplified by PCR and the PCR products were digested with 8 different restriction endonucleases prior to being electrophoresed in agarose gels. Based on PCR-RFLP results, approximately half of the isolates were identified as Lactobacillus crispatus. Several isolates were further identified by DNA sequence analysis of their 16S rDNA.

Genetic Variation Among Isolates of the Web Blight Pathogen of Common Bean Based on PCR-RFLP of the ITS-rDNA Region.

Variability of 45 isolates of Rhizoctonia solani (teleomorph Thanatephorus cucumeris) causing web blight (WB) of common bean, Phaseolus vulgaris, was examined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the internal transcribed spacer regions (ITS1 and ITS2) and the 5.8S subunit (5.8S) of the nuclear ribosomal DNA repeat (ITS-5.8S-rDNA). Isolates were collected from diseased bean leaves from Argentina, Costa Rica, Cuba, Dominican Republic, Honduras, Panama, and Puerto Rico. These WB isolates belong to AG-1 and AG-2 based on anastomosis reaction. Isolates of AG-1 that cause WB were separated into three distinct groups of RFLP patterns from enzymatic digestion of a 740-bp PCR fragment. Microsclerotia-producing isolates (<1 mm) were differentiated from macrosclerotia-producing isolates (5 to 20 mm) based on PCR-RFLP patterns even though they are placed in the same AG1-1B subgroup by anastomosis reaction. WB isolates of AG-2 were separated into two distinct PCR-RFLP groups as previously reported. AG-1 macrosclerotial-producing isolates were the most virulent, whereas isolates of AG-2 were the least virulent. Genetic variability of the WB pathogen may have influenced the failure or success of management practices implemented in the past in Latin America.

Demonstration of interbreeding between virulent and avirulent populations of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) by PCR-RFLP method

The pinewood nematode, Bursaphelenchus xylophilus, is the causative agent of pine wilt disease. The virulence of nematodes significantly varies among pine trees, but does not vary within a single pine. To elucidate the reason for no variation in virulence within a single pine, a technique to investigate the population structure of nematodes of different virulence is needed. In this study, the demonstration of interbreeding between virulent and avirulent populations of B. xylophilus in vitro was attempted using PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method. Ribosomal DNA containing the 5.8S gene, the internal transcribed spacer regions 1 and 2, and partial regions of 18S and 28S genes were used for analysis. First, PCR-RFLP patterns of offspring produced by interbreeding between two individuals of virulent and avirulent isolates were analyzed. The offspring from a single-pair interbreeding showed 3-digested fragment patterns with the restriction enzyme HhaI, patterns the same as the virulent isolate, the avirulent isolate and a hybrid pattern containing both fragments of the virulent and avirulent isolates. The virulent population was mixed with the avirulent one in vitro and propagated nematodes were individually analyzed by PCR-RFLP method. The nematodes showed the same 3 PCR-RFLP patterns as the offspring from a single-pair interbreeding. The detection of nematodes with a hybrid pattern demonstrates the occurrence of interbreeding between virulent and avirulent populations of B. xylophilus.

Occurrence and Identification of Phytophthora spp. Pathogenic to Pear Fruit in Irrigation Water in the Wenatchee River Valley of Washington State

ABSTRACT Seven hundred forty-nine isolates of Phytophthora spp. were obtained from irrigation canals in eastern Washington State during the 1992 to 1995 and 1999 growing seasons. Isolates were retrieved using pear baiting techniques. All isolates were pathogenic to pear and were present in irrigation water beginning early in fruit development. Over the course of the 5 year study, 10 and 5% of isolates were identified as P. cactorum and P. citricola, respectively, using morphological criteria. The remaining isolates could not be identified using morphological criteria. Colony morphology of these isolates was characterized during all years of the study. In 1999, more detailed studies utilizing polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis of entire internal transcribed spacer (ITS) regions (ITS1, 5.8S, and ITS2) of ribosomal DNA for 180 isolates, and sequence analysis of ITS2 for 50 isolates, were used to investigate genetic variation and phylogenetic relationships among isolates. Isolates were divided into 12 groups based on their growth type on corn meal agar. Restriction digestion of the entire ITS region with three enzymes revealed 11 restriction digestion patterns among 180 isolates. PCR-RFLP and sequence data were obtained for 12 reference Phytophthora spp. (two species in each of Waterhouse's six morphological groups). Phylogenetic analysis of ITS2 regions revealed nine clades, each with strong bootstrap support. Molecular analyses revealed 23 isolates that were in the P. gonapodyides clade, 9 in the P. parasitica clade, 1 in the P. cactorum clade, 7 in the P. citricola/capsici clade, and 4 in the P. cambivora/pseudotsugae clade. The three isolates comprising clade 5 were significantly distinct from all other Phytophthora spp. in the databases and may represent a new Phytophthora sp. Colony morphology was not consistently correlated to PCR-RFLP pattern or ITS2 phylogeny, suggesting that the former criterion is insufficient for species identification. The results of this study indicate that at least nine phylogenetically distinct taxa of Phytophthora pathogenic to pear are present in irrigation water in North Central Washington.