rDNA Restriction Fragment Length Polymorphism Research Articles

Diversity of Bradyrhizobium strains isolated from root nodules of the shrubby legume Cytisus villosus growing in the Moroccan Rif

Abstract The genotypic and phenotypic diversity of nineteen bradyrhizobial strains isolated from the root nodules of wild-grown Cytisus villosus in the central-western region of the Moroccan Rif were carried out. PCR-based RFLP (restriction fragment length polymorphism) of the 16S rDNA (ARDRA) of 19 strains using the restriction enzymes AluI, CfoI, DdeI, HinfI, MspI and RsaI showed that they grouped in 3 clusters. The cluster I includes the type strain Bradyrhizobium canariense BTA-1T, the cluster II the type strain Bradyrhizobium japonicum USDA6T and the cluster III the type strains Bradyrhizobium cytisi CTAW11T and Bradyrhizobium rifense CTAW71T. The results from 63 physiological and biochemical tests confirmed the existence of the B. canariense and the B. japonicum groups but also allowed the separation of the B. cytisi and B. rifense strains in two groups. The phenotypic characteristics analysed permitted description of a wide physiological and biochemical diversity among the strains, and showed that the heavy metals resistance test was the most discriminating characteristic.

Phenotypic characterization of Astragalus glycyphyllos symbionts and their phylogeny based on the 16S rDNA sequences and RFLP of 16S rRNA gene

In this study, the nitrogen fixing Astragalus glycyphyllos symbionts were characterized by phenotypic properties, restriction fragment length polymorphism (RFLP), and sequences of 16S rDNA. The generation time of A. glycyphyllos rhizobia in yeast extract mannitol medium was in the range 4–6 h. The studied isolates exhibited a low resistance to antibiotics, a moderate tolerance to NaCl, assimilated di- and trisaccharides, and produced acid in medium containing mannitol as a sole carbon source. In the cluster analysis, based on 86 phenotypic properties of A. glycyphyllos symbionts and the reference rhizobia, examined isolates and the genus Mesorhizobium strains were placed on a single branch, clearly distinct from other lineages of rhizobial genera. By the comparative analysis of 16S rRNA gene sequences and 16S rDNA–RFLP, A. glycyphyllos nodulators were also identified as the members of the genus Mesorhizobium. On the 16S rDNA sequence phylogram, the representatives of A. glycyphyllos nodule isolates formed a robust, monophyletic cluster together with the Mesorhizobium species at 16S rDNA sequence similarity of these bacteria between 95 and 99 %. Similarly, the cluster analysis of the combined RFLP–16S rDNA patterns, obtained with seven restriction endonucleases, showed that A. glycyphyllos rhizobia are closely related to the genus Mesorhizobium bacteria. The taxonomic approaches used in this paper allowed us to classify the studied bacteria into the genus Mesorhizobium.

First Report of Elm Yellows Subgroup 16SrV-B Phytoplasma as the Cause of Rose Balsam Phyllody in China.

Rose balsam (Impatiens balsamina L.) is an ornamental species frequently cultivated in China and the red flower is often used as nail polish in rural regions. The phytoplasmas previously reported with rose balsam phyllody in China have been classified as aster yellows group (16SrI) (1). In August 2012, some rose balsams were observed with typical phytoplasma symptoms in Handan City, Hebei Province, China, with an incidence of about 70% in the fields. The flowers turned green and petals fascicled. The new leaves wrinkled and deformed and internodes shortened. Infected plants were stunted, matured prematurely, and failed to produce seeds. To confirm phytoplasma infection, 100 mg of plant tissue (leaves, petals) was collected from five symptomatic and four asymptomatic plants and total DNA was extracted using a modified cetyltrimethylammonium bromide (CTAB) method (2). The 16S rDNA gene was amplified by nested PCR using primer pair P1/P7 followed by R16F2n/R16R2 (3). No amplicons were generated with DNA from asymptomatic samples, but amplicons of approximately 1.2 kb were obtained with DNA from five symptomatic samples. The amplified products were purified with aTIANgel midi purification kit (Tiangen, Beijing) and sequenced at the Sangon Biotech facility (Shanghai, China). The sequences of the amplicons were 100% identical and deposited in NCBI GenBank (Accession No. KC993832). The 16S rDNA gene sequence from this phytoplasma was 99% similar to Jujube witches broom phytoplasma (JQ675716), Puna chicory flat stem phytoplasma (JN582266), Plum yellows phytoplasma (FJ459914), and other elm yellows group phytoplasmas by BLAST search of the NCBI database. Restriction fragment length polymorphism (RFLP) analyses were carried out by digesting the 1.2-kb R16F2n/R16R2 nested PCR product with restriction enzymes AluI, RsaI, HhaI, HpaI, Eco RI, TaqI, HaeIII, HinfI, and KpnI (Takara, Dalian). The 16S rDNA RFLP patterns matched that of Jujube witches broom phytoplasma (JWB, subgroup 16SrV-B) (4). Nucleotide sequences of rose balsam phyllody were analyzed by iPhyClassifier software, which revealed that it had maximum similarity to the reference pattern of 16Sr group V, subgroup B (AB052876). All samples were detected with transmission electron microscopy. The results showed phytoplasma-like cells in phloem sieve element of symptomatic plants, while no phytoplasma-like cells were observed in healthy phloem tissues. The phytoplasma cells ranged from 230 to 470 nm in diameter and were ellipsoidal or orbicular with visible membranes. Combining the RFLP pattern and sequence analysis by iPhyClassifier, we classified the phytoplasma causing rose balsam phyllody into subgroup 16SrV-B. To our knowledge, this is the first report of 16SrV-B group phytoplasmas infecting rose balsam in China.

Partial Characterization of Spiroplasma citri Isolates Associated with Necrotic Yellows Disease of Safflower in Iran

AbstractSpiroplasma citri was associated with a disease of safflower characterized by stunting, yellowing, phloem discoloration and local or general necrosis in the Fars province of Iran. It was identified by ELISA using a locally produced polyclonal antiserum, by PCR with specific primers and isolation in culture medium. The 16S rDNA restriction fragment length polymorphism patterns of safflower isolates were identical with those of the other S. citri isolates. A known isolate of S. citri from periwinkle induced stunting, yellowing, phloem discoloration and wilting in safflower seedlings when transmitted by dodder under greenhouse conditions. A primer pair designed on the basis of S. citri plasmid was more sensitive than those based on spiralin gene or 16S rDNA for the detection of S. citri. Based on the sequence of the spiralin gene, S. citri isolates from safflower as well as other Iranian isolates were variable and grouped into two genetic clusters with 91.9–92.9% identity between groups. This is the first report of association of S. citri with a safflower disease.

Characterization of halophilic bacteria from environmental samples from the brackish water of Pulicat Lake, India

Culture dependent phenotypic characterization and 16S rDNA based phylogenetic analyses were applied to study the aerobic halophilic bacterial population present in the Pulicat brackish-water Lake of India. Five different media were employed for isolation of bacteria. A total of 198 morphotypes were recovered, purified and screened for salt tolerance in nutrient agar medium amended with 5–25% NaCl. Based on 16S rDNA restriction fragment length polymorphism analysis with three restriction endonucleases, 51 isolates tolerant to 5% or more NaCl were grouped into 29 clusters. Phylogenetic analysis using 16S rRNA gene sequences revealed that 29 strains could further be allocated into two clades: 19 to Firmicutes and 10 to γ-Proteobacteria. Firmicutes included low G+C Gram-positive bacteria related to family Bacillaceae, which included five genera Bacillus, Virgibacillus, Rummelibacillus, Alkalibacillus and Halobacillus. Another genera included in Firmicutes was Salimicrobium halophilum. In the γ-Proteobacteria group, all the isolates belonged to one genus Halomonas, represented by six different species Halomonas salina, H. shengliensis, H. salifodinae, H. pacifica, H. aquamarina and H. halophila. Most of the isolates exhibited cellulase, xylanase, amylase and protease activities.

Polymorphism in the internal transcribed spacer (ITS) region of the ribosomal DNA among different Fusarium species

Fusarium species causing wilt diseases in different plants were characterised by comparing nonpathogenic and different pathogenic species using rDNA RFLP analysis. The ITS (internal transcribed spacer) region of 12 isolates belonging to the section Elegans, Laseola, Mortiella, Discolor, Gibbosum, Lateritium and Sporotrichiella were amplified by universal ITS primers (ITS-1 and ITS-4) using polymerase chain reaction (PCR). Amplified products, which ranged from 522 to 565 bp were obtained from all 12 Fusarium isolates. The amplified products were digested with seven restriction enzymes, and restriction fragment length polymorphism (RFLP) patterns were analysed. A dendrogram derived from PCR-RFLP analysis of the rDNA region divided the Fusarium isolates into three major groups. Assessment of molecular variability based on rDNA RFLP clearly indicated that Fusarium species are heterogeneous and most of the forma speciales have close evolutionary relationships.

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.

Culture-dependent and culture-independent diversity of Actinobacteria associated with the marine sponge Hymeniacidon perleve from the South China Sea

In this report, the diversity of Actinobacteria associated with the marine sponge Hymeniacidon perleve collected from a remote island of the South China Sea was investigated employing classical cultivation and characterization, 16S rDNA library construction, 16S rDNA-restriction fragment length polymorphism (rDNA-RFLP) and phylogenetic analysis. A total of 184 strains were isolated using seven different media and 24 isolates were selected according to their morphological characteristics for phylogenetic analysis on the basis of their 16S rRNA gene sequences. Results showed that the 24 isolates were assigned to six genera including Salinispora, Gordonia, Mycobacterium, Nocardia, Rhodococcus and Streptomyces. This is the first report that Salinispora is present in a marine sponge from the South China Sea. Subsequently, 26 rDNA clones were selected from 191 clones in an Actinobacteria-specific 16S rDNA library of the H. perleve sample, using the RFLP technique for sequencing and phylogenetic analysis. In total, 26 phylotypes were clustered in eight known genera of Actinobacteria including Mycobacterium, Amycolatopsis, Arthrobacter, Brevibacterium, Microlunatus, Nocardioides, Pseudonocardia and Streptomyces. This study contributes to our understanding of actinobacterial diversity in the marine sponge H. perleve from the South China Sea.

Occurrence and diversity of Arcobacter spp. along the Llobregat River catchment, at sewage effluents and in a drinking water treatment plant

The presence of Arcobacter species in faecally contaminated environmental waters has previously been studied. However, the ability to eliminate Arcobacter during the water treatment processes that produce drinking water has been little studied. We have investigated the prevalence and diversity of Arcobacter spp. throughout the year at 12 sampling points in the Llobregat River catchment (Catalonia, Spain) including 3 sites at a drinking water treatment plant. Positive samples for Arcobacter spp., came predominantly from the most faecally polluted sites. Recovery rates from all sites were greater in the spring (91.7%) and summer (83.3%) than in autumn and winter (75.0% in both cases), but this trend was not statistically evaluated due to the limited number of samples. Among the 339 colonies analyzed, the most prevalent species by multiplex PCR and 16S rDNA restriction fragment length polymorphism were Arcobacter butzleri (80.2%), followed by Arcobacter cryaerophilus (19.4%) and Arcobacter skirrowii (0.3%). Isolates showed a high genotype diversity as determined by the enterobacterial repetitive intergenic consensus PCR. In fact, 91.2% (309/339) of the colonies had different genotypes, i.e. 248 of them among the 275 isolates of A. butzleri and 60 among the 63 isolates of A. cryaerophilus and 1 genotype of A. skirrowii. Arcobacter was never detected or isolated from finished drinking water, demonstrating that water treatment is effective in removing Arcobacter species.

The presence of both bone sialoprotein-binding protein gene and collagen adhesin gene as a typical virulence trait of the major epidemic cluster in isolates from orthopedic implant infections

Staphylococcus aureus is a major, highly clonal, pathogen causing implant infections. This study aimed at investigating the diverse distribution of bacterial adhesins in most prevalent S. aureus strain types causing orthopaedic implant infections. 200 S. aureus isolates, categorized into ribogroups by automated ribotyping, i.e. rDNA restriction fragment length polymorphism analysis, were screened for the presence of a panel of adhesins genes. Within the collection of isolates, automated ribotyping detected 98 distinct ribogroups. For many ribogroups, characteristic tandem genes arrangements could be identified. In the predominant S. aureus cluster, enlisting 27 isolates, the bbp gene encoding bone sialoprotein-binding protein appeared a typical virulence trait, found in 93% of the isolates. Conversely, the bbp gene was identified in just 10% of the remaining isolates of the collection. In this cluster, co-presence of bbp with the cna gene encoding collagen adhesin was a pattern consistently observed. These findings indicate a crucial role of both these adhesins, able to bind the most abundant bone proteins, in the pathogenesis of orthopaedic implant infections, there where biomaterials interface bone tissues. This study suggests that specific adhesins may synergistically act in the onset of implant infections and that anti-adhesin strategies should be targeted to adhesins conjointly present.

Prevalence of Arcobacter in meat and shellfish.

Arcobacter is considered an emergent foodborne and waterborne enteropathogen. However, its prevalence in foods of animal origin is only partially known, because most studies have been concentrated on poultry, pork, and beef, and methods applied do not allow identification of all currently accepted Arcobacter species. We investigated the prevalence of Arcobacter in 203 food samples, 119 samples of seven different types of meats and 84 samples of four types of shellfish. Isolates were identified in parallel by using a published multiplex PCR method and a recently described 16S rDNA restriction fragment length polymorphism method that allows all currently accepted Arcobacter species to be characterized. The global prevalence of Arcobacter was 32%; it was highest in clams (5 of 5 samples, 100%) and chicken (9 of 14 samples, 64.3%) followed by pork (9 of 17 samples, 53.0%), mussels (23 of 56 samples, 41.1%), and duck meat (2 of 5 samples, 40.0%). Turkey meat and beef had a similar recovery rate (10 of 30 samples, 33.3%; 5 of 16 samples, 31.3%; respectively), and rabbit meat had the lowest rate (1 of 10 samples, 10.0%). No arcobacters were found in oysters, frozen shrimps, or sausages. This food survey is the first in which five of the seven accepted Arcobacter species have been isolated. Arcobacter butzleri was the most prevalent species (63.0% of isolates) followed by Arcobacter cryaerophilus (26.6%), Arcobacter mytili (4.7%), Arcobacter skirrowii (3.1%), and Arcobacter nitrofigilis (3.1%). Three (4.7%) of the isolates were classified as belonging to three potentially new phylogenetic lines. Our results indicated that Arcobacter species are widely distributed in the food products studied.

First Report of a New Phytoplasma Subgroup, 16SrIII-T, Associated with Decline Disease Affecting Sweet and Sour Cherry Trees in Lithuania

During July 2007, sweet (Prunus avium) and sour cherry (P. cerasus) trees exhibiting disease symptoms suggestive of possible phytoplasma infection were observed in a large orchard in the Kaunas Region of Lithuania. Samples of leaf tissue were collected from 13 sweet cherry trees that were affected by a decline disease (designated cherry decline, ChD) characterized by symptoms that included leaf reddening and premature leaf drop and two sour cherry trees exhibiting proliferation of branches and nonseasonal flowering. To assess the diseased trees for phytoplasma infection, DNA was extracted with a Genomic DNA Purification Kit (Fermentas, Vilnius, Lithuania) and used as template in nested PCRs, primed by phytoplasma universal primer pairs P1/P7 and R16F2n/R16R2 for amplification of 16S ribosomal (r) DNA sequences (1,2). The 1.2-kbp DNA sequences amplified from all 15 trees were subjected to restriction fragment length polymorphism (RFLP) analyses with AluI, MseI, KpnI, HhaI, HaeIII, HpaII, RsaI, HinfI, TaqI, Sau3AI, and BfaI. The collective profiles indicated that DNAs were derived from two different phytoplasmas. One of them, designated ChD phytoplasma, was found in 11 sweet cherry trees and two sour cherry trees and tentatively classified as a member of new subgroup designated 16SrIII-T in 16S rDNA RFLP group 16SrIII (X-disease phytoplasma group). It was observed that the ChD phytoplasma caused different symptoms in sweet and sour cherry. The amplified ChD phytoplasma 16S rDNA was cloned in Escherichia coli, sequenced, and the sequence deposited in the GenBank database (Accession No. FJ231728). The ChD phytoplasma 16S rDNA shared 98.4 and 98.6% sequence identity with the 16S rDNAs from stone fruit-infecting phytoplasmas associated with western X-disease (GenBank Accession No. L04682) and Canada X-disease (GenBank Accession No. L33733), respectively, indicating that the three strains are closely related. Interestingly, the ChD phytoplasma 16S rDNA shared 99.8% sequence identity with 16S rDNA from one operon (rrnB, GenBank Accession No. AF370120) from a phytoplasma previously found to be associated with dandelion virescence (DanVir) disease in Lithuania. The operon rrnA (GenBank Accession No. AF370119) shared 99.3% sequence identity (2). The high similarity of the ChD 16S rRNA gene sequence to that of DanVir rrnB suggests the possibility that ChD and DanVir may belong to a single phytoplasma species and that dandelion is possibly an alternate host of ChD phytoplasma. The other phytoplasma, found in two sweet cherry trees, was classified in subgroup 16SrI-B of 16S rDNA RFLP group 16SrI ('Candidatus Phytoplasma asteris' and related strains) and was designated cherry proliferation phytoplasma (GenBank Accession No. FJ231729). Thus, in Europe, cherry may be affected by diseases associated with phytoplasmas belonging to groups 16SrI, 16SrIII, 16SrX, and 16SrXII (3,4). The infections by diverse phytoplasma strains and species underscore the need for production of phytoplasma-free planting stock and for intensified research to reduce ecological and economic impacts of these phytoplasmas.

First Report of Syringa oblata and S. reticulata Leafroll Disease in China.

Syringa oblata is an important ornamental tree widely grown in China. In September of 2008, S. oblata plants exhibiting symptoms of leafroll and yellowing were found in a garden on the Northwest A&F University campus. Samples were collected from this site. Total DNA was extracted from 0.5 g of phloem tissue from leaf midribs and stems of each sample. DNA samples were analyzed with a nested PCR assay using phytoplasma 16S rDNA universal primers R16mF2/R16mR1 followed by specific primers R16F2n/R16R2 (1), which amplified a 1,452- and 1,246-bp product, respectively. We tested all 30 lilac samples, 20 of which had symptoms of leafroll and yellowing. These produced the expected 1,452- and 1,246-bp PCR products In contrast, the remaining 10 samples from symptomless trees yielded no PCR products. We also surveyed another lilac variety (Syringa reticulata), which is widely grown on the campus, and tested 50 samples with the above method. Again, 1.4- and 1.2-kb PCR products were amplified from all 30 trees displaying leafroll and yellowing symptoms, but not from the other 20 samples from symptomless trees. A comparative analysis of sequences derived from the two hosts showed that the phytoplasmas infecting them were most similar (>99%) to paulownia witches'-broom (PaWB) phytoplasma (GenBank Accession No. EF199937). Restriction fragment length polymorphism (RFLP) analysis of the nested 1.2-kb 16S rDNA products with endonucleases AluI and MseI indicated that all symptomatic plants were infected by the phytoplasmas belonging to aster yellow group (16SrI) subgroup D (16SrI-D) PaWB phytoplasma (2). 16S rDNA sequence comparisons and RFLP analysis of the cloned 16S rDNA from S. oblata (GenBank Accession No. FJ445224) and S. reticulate (GenBank Accession No. FJ445225) indicated that the phytoplasmas infecting them were nearly identical (99.8% identity). To our knowledge, this is the first report of the presence of the phytoplasma associated with a leafroll disease of S. oblata and S. reticulata in China.

Ditylenchus destructor and Ditylenchus dipsaci

<i>Ditylenchus destructor</i> and <i>Ditylenchus dipsaci</i>

First Report of 'Candidatus Phytoplasma asteris'-Related Strains Infecting Lily in Mexico.

In recent years, lily (Lilium spp.) has become an important ornamental crop in diverse regions of Mexico. Since 2005, unusual symptoms have been observed on lily plants grown from imported bulbs in both greenhouse and production plots at San Pablo Ixayo, Boyeros, and Tequexquinauac, Mexico State. Symptoms included a zigzag line pattern on leaves, dwarfism, enlargement of stems, shortened internodes, leaves without petioles growing directly from bulbs, air bulbils, death of young roots, atrophy of flower buttons, and flower abortion. Symptoms were experimentally reproduced on healthy lily plants by graft inoculation. Total DNA was extracted from 50 diseased, 10 symptomless, and 10 graft-inoculated plants by the method of Dellaporta et al. (2). DNA samples were analyzed for phytoplasma presence by two different nested PCR assays. One assay employed ribosomal RNA gene primer pair P1/P7 followed by R16F2n/R16R2 (1), whereas ribosomal protein (rp) gene primer pairs rpF1/rpR1 and rp(I)F1A/rp(I)R1A (4) were used in a second assay. A DNA fragment approximately 1.2 kb long was consistently amplified from all symptomatic plant samples only by both assays. A comparative analysis of 16S rDNA sequences (Genbank Accession Nos. EF421158-EF421160 and EU124518-EU124520) and rp gene sequences (EU277012-EU277014), derived from PCR products, revealed that phytoplasma infecting lily were most similar (99.9% to 16S rDNA and 99.7% to rp) to carrot phytoplasma sp. ca2006/5 and also were similar (99.8% to 16SrDNA and 99.2% to rp) to broccoli phytoplasma sp. br273. Both carrot and broccoli phytoplasmas were classified as members of aster yellow 16S rDNA restriction fragment length polymorphism subgroup 16SrI-B (3). Although infection of lilies by aster yellows ('Ca. phytoplasma asteris') subgroup 16SrI-B and 16SrI-C was reported from the Czech Republic and Poland, to our knowledge, this is the first report of 'Ca. phytoplasma asteris'-related strains associated with lily plants in Mexico.

Occurrence of a Clover Proliferation (16SrVI) Group Phytoplasma Associated with Little Leaf Disease of Portulaca grandiflora in India.

Portulaca grandiflora (family Portulacaceae), commonly known as moss rose purslane, is a popular ornamental plant widely grown in temperate climates because it blooms all summer. Portulaca is also used for medicinal purposes since it is rich in vitamins A, B1, and C and has antimicrobial and cytotoxic activity. Since March 2005, 30 to 50% of P. grandiflora plants in the ornamental gardens as well as in pots at the Central Institute of Medicinal and Aromatic Plants, Lucknow, India have displayed symptoms resembling phytoplasma infection. Disease symptoms start as a typical bud proliferation, downward curling, and diminishing size of leaves, followed by overall stunted growth and yellowing of the whole plant from April to June. Some plants also formed rosettes and a proliferation of axillary shoots resulting in a witches'-broom appearance. Typical pleomorphic bodies, mostly spherical to oval, ranging from 340 to 1,100 nm were observed only in sieve elements of infected plants by transmission electron microscopy (TEM). On the basis of symptoms, TEM observations, PCR, and response to antibiotic treatment, the causal organism was identified as phytoplasma (1). Total genomic DNA from healthy and infected plants was extracted with the CTAB buffer method (2). Of 27 suspected samples screened by PCR, 23 were phytoplasma positive. Presence of phytoplasmas in plants was demonstrated by a nested PCR assay employing primer pair P1/P6 followed by R16F2n/R16R2 that generated rDNA products of 1.5 and 1.2 kb, respectively, only from symptomatic plants. No differences among phytoplasmas in Portulaca plants were detected by restriction fragment length polymorphism (RFLP) analysis of nested rDNA (1.2 kb) products using endonucleases BamHI, RsaI, AluI, HpaII, and EcoRI. Comparative analysis of RFLP patterns with those derived from reference phytoplasmas tentatively identified the Portulaca little leaf (PLL) phytoplasma as a member of 16S rDNA RFLP group 16SrVI (3). A nested PCR product (1.25 kb) was cloned with a TOPO TA cloning kit (Invitrogen, Carlsbad, CA) and sequenced. The sequence was deposited in the GenBank database (Accession No. EF651786). Sequence analysis revealed the PLL phytoplasma to be most similar (98%) to Indian brinjal little leaf (Accession No. EF186820) and 'Candidatus Phytoplasma trifolii' (Accession No. AY390261), two 16SrVI group phytoplasmas previously reported from India and Canada, respectively. The status of PLL (EF651786) was also verified by in silico RFLP analysis (4) of the F2n/R2 sequence of six closely related strains (Accession Nos. AF228052, AY390261, AY270156, AY409070, AY409069, and EF186820) of the 16SrVI group using 17 restriction enzymes (AluI, BamHI, BfaI, BsfUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, MseI, Sau3AI, RsaI, SspI, and TaqI). In silico restriction digestion and virtual gel plotting showed similar patterns for all enzymes. To our knowledge, this is the first report of a 16SrVI group phytoplasma infecting Portulaca plants in India.

Community structure and functional diversity of indigenous fluorescent Pseudomonas of long-term swine compost applied maize rhizosphere

The community structure and catabolic functional diversity of indigenous rhizosphere fluorescent pseudomonad isolates recovered from un-amended soil (control treated) and following long-term (6 years) swine compost application (compost treated) were studied. Soils collected for the pot experiments were from a Taiwanese experimental farm established in 1995 to study the long-term effects of crop rotation and organic amendment. In the pot experiments, corn seed was cultured; after the elongation stage, 13 and 32 indigenous rhizosphere fluorescent pseudomonad isolates were collected from compost-treated ( n=13) and control-treated ( n=32) soils. All isolates were classified by 16S rDNA restriction fragment length polymorphism (RFLP) and unweighted pair-group method using arithmetic averages analyses. Most (84.4%) of the control-treated soil isolates belonged to genotype A, which corresponded to Pseudomonas putida based on 16S rDNA sequences analysis. However, only 61.5% of compost-treated soil isolates were from genotype A; 30.7% of the isolates belonged to genotype B, whose 16S rDNA profile corresponded to Stenotrophomonas maltophilia. Differing catabolic potentials of the P. putida isolates from control- and compost-treated soils were evident; isolates from control-treated soils utilized organic acids including citric acid, cis-aconitic acid, and formic acid more actively than compost-treated soil isolates. Moreover, the latter isolates more efficiently utilized carbohydrates of i-erythritol and l-rhamnose than P. putida isolates from control-treated soil, consistent with the alteration in the catabolic functions of indigenous rhizosphere P. putida by long-term compost application.

Molecular characterization of Serratia marcescens strains by RFLP and sequencing of PCR-amplified 16S rDNA and 16S?23S rDNA intergenic spacer

To establish the specific DNA patterns in 16S rDNA and 16S-23S rDNA intergenic spacer (IGS) regions from different kinds of Serratia marcescens strains using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and sequences analysis. Two pairs of primers based on the 16S rDNA and 16S-23S rDNA IGS were applied to amplify the rrn operons of two kinds of S. marcescens strains. About 1500 bp for 16S rDNA and four fragments of different sizes for 16S-23S rDNA IGS were obtained. PCR-amplified fragments were analysed by RFLP and sequence analysis. Two distinct restriction patterns revealing three to five bands between two kinds of strains were detected with each specific enzyme. According to the sequence analysis, two kinds of strains showed approximately 97% sequence homology of 16S rDNA. However, there was much difference in the sequences of IGS between the two kinds of strains. Intercistronic tRNA of strains H3010 and A3 demonstrated an order of tRNA of 5'-16S-tRNA(Ala)-tRNA(Ile)-23S-3', but strain B17 harboured the tRNA of 5'-16S-tRNA(Glu)-tRNA(Ile)-23S-3'. The method was specific, sensitive and accurate, providing a new technique for differentiating different strains from the same species. This paper provided the first molecular characterization of 16S rDNA and 16S-23S rDNA IGS from S. marcescens strains.

Intraspecific variation and population structure of the German cockroach,Blattella germanica, revealed with RFLP analysis of the non‐transcribed spacer region of ribosomal DNA

Little information is available on genetic variation within and between populations of pest cockroaches. In this study, intraspecific HindIII polymorphism was investigated in the German cockroach, Blattella germanica (Linnaeus) (Dictyoptera, Blattaria: Blattellidae), using restriction fragment length polymorphisms (RFLP) of the non-transcribed spacer (NTS) region of ribosomal DNA (rDNA). Individual male insects were collected from infestations at three different pig farms. Each population was characterized by HindIII restriction fragment frequencies and haplotype (a particular X-chromosome pattern) frequencies. The inheritance of the X-chromosome HindIII rDNA patterns over 12 generations (3 years) follows Mendelian patterns, and the stability of this polymorphic marker indicates infrequent genetic recombination of variable sites. Although pairwise genetic distance measures were uncorrelated with geographical distance, the pattern of genetic differentiation of the three cockroach populations suggests that human-mediated transport of cockroaches is an important force in shaping the population genetic structure of cockroach infestations, at least at the regional scale of 10-100 km. Sequence variation in the ribosomal NTS is a useful marker, and RFLP of rDNA is a simple, robust and reproducible technique for differentiating recently diverged cockroach populations.

First Report of a Natural Infection of Opuntia sp. by a 'Candidatus Phytoplasma asteris'-Related Phytoplasma in China.

Cacti (Opuntia spp.) are perennial, evergreen, succulent plants native to arid areas of the Americas. Because of their aesthetic appearance, many cacti have been cultivated and introduced to other parts of the world as ornamentals. Cacti are susceptible to phytoplasma infections and develop witches'-broom (WB) disease. Currently, all reported cactus WB cases are associated with infections by phytoplasmas in the peanut witches'-broom group (16SrII) (1,2,4). During a phytoplasma diversity survey carried out during 2004 in Yunnan, China, we collected 29 malformed and 14 healthy-looking naturally occurring cactus plants from 14 locations representing five geographical regions. Each of the 29 malformed plants exhibited stunted growth and possessed clusters of highly proliferating cladodia, typical symptoms of cactus WB disease. Nested-PCR was carried out on the DNA samples extracted from young cladodia of these plants using phytoplasma-universal 16S rDNA primers P1A/P7A and R16F2n/R16R2 (3). Results revealed that all 29 diseased plants that were examined were infected by phytoplasmas, whereas all 14 healthy-looking plants were negative for phytoplasmas. Subsequent restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 1.25-kb 16S rDNA fragments indicated that 28 diseased plants were infected by a phytoplasma of group 16SrII, whereas one plant (from Suan Village) was infected by a 'Candidatus Phytoplasma asteris'-related (group 16SrI) phytoplasma designated as strain YN26. Nucleotide sequence analysis of the strain YN26 partial rRNA operon (GenBank Accession No. EF190970), covering a near full-length 16S rRNA gene, a 16S-23S rRNA intergenic spacer, a tRNA-Ile gene, and a partial 23S rRNA gene, suggested that this phytoplasma is most closely related to an ash witches'-broom phytoplasma (GenBank Accession No. AY566302, 99.7% identity) and an epilobium phyllody phytoplasma (GenBank Accession No. AY101386, 99.7% identity), both members of subgroup16SrI-B. This YN26-infected cactus plant was transferred to a greenhouse and maintained for more than 2 years, during which time DNA samples were extracted and tested two additional times. The same 16S rDNA RFLP pattern type was consistently obtained in these tests, confirming that the plant remained infected by the 16SrI phytoplasma. To our knowledge, this is the first report of a natural infection of a cactus species by a group 16SrI phytoplasma. Since this 16SrI-cactus WB phytoplasma was found in the same geographical location where 16SrII-cactus WB phytoplasma was detected both in this and a previous study (1), the findings raised the question whether 16SrI- and 16SrII-cactus WB phytoplasmas have overlapping geo- and bioecological niches.