16S-23S Intergenic Spacer Region Research Articles

Rapid Species Identification and Partial Strain Differentiation of Clostridium butyricum by PCR Using 16S‐23S rDNA Intergenic Spacer Regions

Some Clostridium butyricum strains have been used as probiotics for both humans and animals. Strain-specific identification is necessary for the manufacturing process of probiotics. The aim of this study was to determine whether there are sufficient genetic variations in 16S-23S intergenic spacer regions (ISRs) to discriminate C. butyricum at the biovar level. We amplified ISRs from five reference strains, a probiotic strain (MIYAIRI 588) and 22 isolates, and we classified them into four groups on the basis of amplification patterns (type A through D). However, amplification of ISRs is not sufficient for discriminating strains. Moreover, we compared genetic structures of these ISRs. Sequence analysis revealed that the size variations of ISRs were generated by the insertion of tRNA genes and unique sequences into the internal portion, while the external portions were highly conserved. On the basis of the highly conserved nucleotide sequences within the ISRs, we developed a PCR primer set specific to C. butyricum. In addition, the PCR primer designed from the unique inserted sequence in type B strain was useful to differentiate probiotic strains at the biovar level.

Analysis of 16S-23S intergenic spacer regions of the rRNA operons in Edwardsiella ictaluri and Edwardsiella tarda isolates from fish

To analyse interspecies and intraspecies differences based on the 16S-23S rRNA intergenic spacer region (ISR) sequences of the fish pathogens Edwardsiella ictaluri and Edwardsiella tarda. The 16S-23S rRNA spacer regions of 19 Edw. ictaluri and four Edw. tarda isolates from four geographical regions were amplified by PCR with primers complementary to conserved sequences within the flanking 16S-23S rRNA coding sequences. Two products were generated from all isolates, without interspecies or intraspecific size polymorphisms. Sequence analysis of the amplified fragments revealed a smaller ISR of 350 bp, which contained a gene for tRNA(Glu), and a larger ISR of 441 bp, which contained genes for tRNA(Ile) and tRNA(Ala). The sequences of the smaller ISR of different Edw. ictaluri isolates were essentially identical to each other. Partial sequences of larger ISR from several Edw. ictaluri isolates also revealed no differences from the one complete Edw. ictaluri large ISR sequence obtained. The sequences of the smaller ISR of Edw. tarda were 97% identical to the Edw. ictaluri smaller ISR and the larger ISR were 96-98% identical to the Edw. ictaluri larger ISR sequence. The Edw. tarda isolates displayed limited ISR sequence heterogeneity, with > or =97% sequence identity among isolates for both small and large ISR. There is a high degree of size and sequence similarity of 16S-23S ISR both among isolates within Edw. ictaluri and Edw. tarda species and between the two species. Our results confirm a close genetic relationship between Edw. ictaluri and Edw. tarda and the relative homogeneity of Edw. ictaluri isolates compared with Edw. tarda isolates. Because no differences were found in ISR sequences among Edw. ictaluri isolates, sequence analysis of the ISR will not be useful to distinguish isolates of Edw. ictaluri. However, we identified restriction sites that differ between ISR sequences of Edw. ictaluri and Edw. tarda, which will be useful in distinguishing the two species.

Detection ofRickettsia,Borrelia, andBartonellainCarios kelleyi(Acari: Argasidae)

Carios kelleyi (Colley & Kohls 1941), a tick associated with bats and bat habitats, has been reported to feed on humans, but there is little published data regarding the presence of vector-borne pathogens in these ticks. C. kelleyi nymphs and adults were collected from residential and community buildings in Jackson County, Iowa, and tested by polymerase chain reaction for Rickettsia, Borrelia, Bartonella, Coxiella, and Anaplasma. Rickettsia DNA was detected in 28 of 31 live ticks. Sequences of the 17-kDa and rOmpA genes suggest that this agent is a novel spotted fever group Rickettsia. Transstadial and transovarial transmission of this Rickettsia were demonstrated. The flagellin gene of a Borrelia, closely related to B. turicatae, was detected in one of 31 live ticks. The 16S-23S intergenic spacer region of Bartonella henselae also was detected in one of 31 live ticks. Coxiella or A. phagocytophilum DNA were not detected in these ticks.

Construction of a combined physical and genetic map of the chromosome of Lactobacillus acidophilus ATCC 4356 and characterization of the rRNA operons

The combination of PFGE and hybridization approaches was used to study the genome of Lactobacillus acidophilus neotype strain ATCC 4356. PFGE analysis of chromosomal DNA after digestion with each of the rare-cutting restriction enzymes I-CeuI, NotI, CspI, SmaI, ApaI and SgrAI allowed the size of the circular chromosome of L. acidophilus to be estimated at 2.061 Mbp. The physical map contained 86 restriction sites for the six enzymes employed, with intervals between the sites varying from 1 to 88 kbp (approximately 0.05-4.3 % of the chromosome). Based on the physical map, a genetic map was constructed via Southern blot analyses of L. acidophilus DNA using specific gene probes. A total of 73 probes representing key genes, including 12 rRNA (rrn) genes, were positioned on the latter map. Mapping analysis also indicated the presence of four rrn operons (rrnA-D) on the chromosome, each containing a single copy of each of the three rrn genes 16S (rrl), 23S (rrs) and 5S (rrf). Operon rrnD was inverted in orientation with respect to the others and contained a long 16S-23S intergenic spacer region with tRNAIle and tRNAAla genes, whereas the other operons contained a short spacer lacking any tRNA genes. The high-resolution physical/genetic map constructed in this study provides a platform for genomic and genetic studies of Lactobacillus species and for improving industrial and probiotic strains.

Systemic Disease in Vaal Rhebok ( Pelea capreolus ) Caused by Mycoplasmas in the Mycoides Cluster

In the winter of 2002, an outbreak of mycoplasma infection in Vaal rhebok (Pelea capreolus) originating from South Africa occurred 15 weeks after their arrival in San Diego, Calif. Three rhebok developed inappetence, weight loss, lethargy, signs related to pulmonary or arthral dysfunction, and sepsis. All three rhebok died or were euthanized. Primary postmortem findings were erosive tracheitis, pleuropneumonia, regional cellulitis, and necrotizing lymphadenitis. Mycoplasmas were detected in numerous tissues by electron microscopy, immunohistochemistry, and PCR. The three deceased rhebok were coinfected with ovine herpesvirus-2, and two animals additionally had a novel gammaherpesvirus. However, no lesions indicative of herpesvirus were seen microscopically in any animal. The rheboks' mycoplasmas were characterized at the level of the 16S rRNA gene, the 16S-23S intergenic spacer region, and the fructose biphosphate aldolase gene. Denaturing gradient gel electrophoresis was carried out to address the possibility of infection with multiple strains. Two of the deceased rhebok were infected with a single strain of Mycoplasma capricolum subsp. capricolum, and the third animal had a single, unique strain most closely related to Mycoplasma mycoides subsp. mycoides large-colony. A PCR survey of DNA samples from 46 other ruminant species demonstrated the presence of several species of mycoplasmas in the mycoides cluster, including a strain of M. capricolum subsp. capricolum identical to that found in two of the rhebok. These findings demonstrate the pervasiveness of mycoplasmas in the mycoides cluster in small ruminants and the potential for interspecies transmission and disease when different animal taxa come in contact.

Comparison of phenotypic and genotypic taxonomic methods for the identification of dairy enterococci.

The purpose of the study was to assess the phenotypic and genotypic taxonomic congruence in order to allow species allocation of dairy enterococci. A total of 364 enterococci isolated from ewes'milk and cheese from four Portuguese Registered Designation of Origin areas and 25 type and reference strains of Enterococcus spp. were characterized by a polyphasic taxonomical approach involving 40 physiological and biochemical tests, whole-cell protein profiles, amplification of 16S-23S intergenic spacer regions (ITS-PCR) and subsequent restriction analysis (ARDRA). Ribotyping was also performed with reference strains and a subset of 146 isolates. Numerical hierarchic data analysis showed that single-technique identification levels increase from the physiological and biochemical tests to the protein approach, being lower with ITS/ARDRA and ribotyping. Cross-analysis confirmed a higher unmatching level in all pairwise combinations involving physiological and biochemical data. Whole-cell protein profiles followed by ITS/ARDRA identified 89% of the enterococci. Reliable identification of enterococci from milk and cheese could be obtained by analysis of whole-cell protein profiles. ITS-PCR can be used to confirm E. durans and E. faecium and ARDRA further confirms E. faecalis. Results revealed E. faecalis, E. durans, E. hirae and E. faecium as the prevalent species, although species prevalence showed some degree of variation among the areas.

Cloning and Sequencing of Phytoplasma Ribosomal DNA (rONA) Associated with Kerala Wilt Disease of Coconut Palms

Using the polymerase chain reaction (PCR) the 165 rRNA gene of phytoplasma associated with Kerala wilt disease of coconut palm (Cocos nucifera L) was amplified from infected leaf samples. Within the three universal primer pairs P1/P6, P1/P7and P41 P7, the primer pair P4/P7 only showed an amplification of 650 bp DNA fragment. 5ince P4/P7 amplifies the 16S-23S intergenic spacer region of 165 rRNA gene, the PCR product 650 bp of Kerala wilt disease palm indicates the phytoplasma DNA. The amplified fragment was sequenced and deposited in Genbank data library (Accession No. AY158660). The absence of restriction sites for Bcll and Rsa/l in 650 bp indicates phytoplasmic nature of DNA and its strain difference. A comparison of the 650 bp sequence with other phytoplasmas and its restriction profile indicates Kerala wilt disease phytoplasma as a separate 165 rRNA group in the classification of phytoplasmas. To our knowledge, this report records the first finding of the phytoplasma DNA using universal primers and its sequence analysis in coconut palms of Kerala, south India.

Identification of Bartonella strains isolated from wild and domestic ruminants by a single-step PCR analysis of the 16S–23S intergenic spacer region

Of the 20 species or subspecies of Bartonella currently known, 7 cause various diseases in humans with many being zoonotic. However, some Bartonella species appear only to cause asymptomatic bacteraemia in their hosts. In ruminants, three Bartonella species ( B. bovis, B. capreoli and B. schoenbuchensis) have recently been described. However, limited or no information has yet been published concerning their mode of transmission and their possible pathogenicity for domestic cattle. The phylogenetic relationship of these species with other bacteria of the Bartonella genus has only been recently investigated. It is therefore necessary to develop appropriate tools that will easily allow identification of these ruminant strains for epidemiological and clinical studies. A single-step PCR assay, based on the amplification of a fragment of the 16S–23S rRNA intergenic spacer (ITS), was evaluated for identification of Bartonella isolated from domestic cattle and from free-ranging or captive cervids. For each Bartonella species tested, the PCR assay led to a product that was unique either for its length or its sequence. All ruminant isolates tested could be easily differentiated among themselves and from the other Bartonella species. Furthermore, sequence analysis of the PCR products revealed a close relationship between all ruminant Bartonella strains. Therefore, ITS PCR testing appears to be a convenient tool for a quick diagnosis of ruminant Bartonella species.

An Oligonucleotide Array for the Identification and Differentiation of Bacteria Pathogenic on Potato

ABSTRACT Oligonucleotides, 16 to 24 bases long, were selected from the 3' end of the 16S gene and the 16S-23S intergenic spacer regions of bacteria pathogenic on potato, including Clavibacter michiganensis subsp. sepedonicus, Ralstonia solanacearum, and the pectolytic erwinias, including Erwinia carotovora subsp. atroseptica and carotovora and E. chrysanthemi. Oligonucleotides were designed and formatted into an array by pin spotting on nylon membranes. Genomic DNA from bacterial cultures was amplified by polymerase chain reaction using conserved ribosomal primers and labeled simultaneously with digoxigenin-dUTP. Hybridization of amplicons to the array and subsequent serological detection of digoxigenin label revealed different hybridization patterns that were distinct for each species and subspecies tested. Hybridization of amplicons generally was restricted to appropriate homologous oligonucleotides and cross-hybridization with heterologous oligonucleotides was rare. Hybridization patterns were recorded as separate gray values for each hybridized spot and revealed a consistent pattern for multiple strains of each species or subspecies isolated from diverse geographical regions. In preliminary tests, bacteria could be correctly identified and detected by hybridizing to the array amplicons from mixed cultures and inoculated potato tissue.

Comparison of three molecular methods for typing Aeromonas popoffii isolates.

Three typing methods, restriction fragment length polymorphism (RFLP) of the 16S-23S intergenic spacer region (ISR), PCR amplification of the enterobacterial repetitive intergenic consensus (ERIC) and of the repetitive extragenic palindromic units (REP), were evaluated for typing 26 isolates of Aeromonas popoffii from different geographical origins. When the methods were independently studied, ERIC showed the highest discriminatory power. When the methods were combined, the best combination of two methods was ERIC with REP since strains showed a tendency to cluster according to their geographical origin. However, this tendency was reinforced with the addition of ISR-RFLP.

First Report of a Group 16SrI, Subgroup B, Phytoplasma in Diseased Epilobium hirsutum in the Region of Tallin, Estonia.

Symptoms of phyllody of flowers and general plant yellowing indicating possible phytoplasma infection were observed in diseased plants of hairy willow-weed (Epilobium hirsutum L., family Onagraceae) growing in a meadow at Harku Village near Tallin, Estonia. DNA was extracted from diseased E. hirsutum using a Genomic DNA Purification Kit (Fermentas AB, Vilnius, Lithuania) and used as a template in nested polymerase chain reaction (PCR). Ribosomal (r) DNA was initially amplified in PCR primed by phytoplasma universal primer pair P1/P7 (4) and reamplified in PCR primed by nested primer pair 16SF2n/16SR2 (F2n/R2) (1) as previously described (2). Products of 1.8 kbp and 1.2 kbp were obtained in PCR primed P1/P7 and F2n/R2, respectively, from all four symptomatic plants examined. These data indicated that the diseased E. hirsutum plants were infected by a phytoplasma, termed epilobium phyllody (EpPh) phytoplasma. The 16S rDNA amplified in PCR primed by nested primer pair F2n/R2 was subjected to restriction fragment length polymorphism (RFLP) analysis using restriction endonucleases AluI, MseI, HpaI, HpaII, HhaI, RsaI, HinfI, and HaeIII (Fermentas AB). On the basis of the collective RFLP profiles, EpPh phytoplasma was classified in group 16SrI (aster yellows phytoplasma group), subgroup B (aster yellows phytoplasma subgroup), according to the phytoplasma classification scheme of Lee et al. (3). The 1.8-kbp rDNA product of P1/P7-primed PCR, which included 16S rDNA, 16S-23S intergenic spacer region, and the 5' -end of 23S rDNA, was cloned in Escherichia coli using the TOPO TA Cloning Kit (Invitrogen, Carlsbad, Ca) according to manufacturer's instructions and sequenced. The sequence was deposited in the GenBank database as Accession No. AY101386. This nucleotide sequence shared 99.8% sequence similarity with a comparable rDNA sequence (GenBank Accession No. AF322644) of aster yellows phytoplasma AY1, a known subgroup 16SrI-B strain. The EpPh phytoplasma sequence was highly similar (99.9%) to operons rrnA (GenBank Accession No. AY102274) and rrnB (GenBank Accession No. AY102273) from Valeriana yellows (ValY) phytoplasma infecting Valeriana officinalis plants in Lithuania. ValY phytoplasma was found to exhibit rRNA interoperon sequence heterogeneity (D. Valiunas, unpublished data). To our knowledge, this is the first report to reveal E. hirsutum as a host of phytoplasma and to demonstrate the occurrence of a plant pathogenic mollicute in the northern Baltic region.

Characterization of bacterial and fungal soil communities by automated ribosomal intergenic spacer analysis fingerprints: biological and methodological variability.

Automated rRNA intergenic spacer analysis (ARISA) was used to characterise bacterial (B-ARISA) and fungal (F-ARISA) communities from different soil types. The 16S-23S intergenic spacer region from the bacterial rRNA operon was amplified from total soil community DNA for B-ARISA. Similarly, the two internal transcribed spacers and the 5.8S rRNA gene (ITS1-5.8S-ITS2) from the fungal rRNA operon were amplified from total soil community DNA for F-ARISA. Universal fluorescence-labeled primers were used for the PCRs, and fragments of between 200 and 1,200 bp were resolved on denaturing polyacrylamide gels by use of an automated sequencer with laser detection. Methodological (DNA extraction and PCR amplification) and biological (inter- and intrasite) variations were evaluated by comparing the number and intensity of peaks (bands) between electrophoregrams (profiles) and by multivariate analysis. Our results showed that ARISA is a high-resolution, highly reproducible technique and is a robust method for discriminating between microbial communities. To evaluate the potential biases in community description provided by ARISA, we also examined databases on length distribution of ribosomal intergenic spacers among bacteria (L. Ranjard, E. Brothier, and S. Nazaret, Appl. Environ. Microbiol. 66:5334-5339, 2000) and fungi.

Vibrio cholerae in recreational beach waters and tributaries of Southern California

Vibrio cholerae is the causative agent of the severe dehydrating diarrheal disease cholera. This bacterium has been detected in many estuaries around the world and the United States. In this study we examine the abundance and distribution of V cholerae in recreational beach waters and tributaries of Southern California. Water samples were taken from 11 beach locations adjacent to freshwater runoff sources between February 8th and March Ist, 1999. Water samples were also taken from rivers, creeks and coastal wetlands along the Southern California coast between May 19th and June 28th, 1999. In addition to the detection of V cholerae, environmental parameters including temperature, salinity, coliphage counts, viable heterotrophic plate counts and total bacterial direct counts were also determined to understand the relationships between the presence of V. cholerae and environmental conditions. A direct colony hybridization method using an oligonucleotide probe specific for the 16S-23S intergenic spacer region of V. cholerae,detected V. cholerae in 3 of the 11 beach samples with the highest concentration (60.9 per liter) at the mouth of Malibu Lagoon. V. cholerae and coliphage were not correlated for beach samples, indicating that the presence of V cholerae is independent of sewage pollution. V cholerae were detected in all samples taken from rivers, creeks and wetlands of coastal Southern California where salinities were between l to 34 parts per thousand (ppt), but was not found at a freshwater sampling site in upper San Juan Creek. The highest density of V cholerae was found in San Diego Creek with a concentration of 4.25 x 105 CFU/L. The geographical distribution of V. cholerae was inversely correlated with salinity. High concentrations of V cholerae were more frequently detected in waters with lower (but above 0) salinity. The results of this study provide insight into the ecology of this aquatic species and are potentially important to the understanding of the epidemiology of cholera on a global scale.

Coyotes (Canis latrans) as the reservoir for a human pathogenic Bartonella sp.: molecular epidemiology of Bartonella vinsonii subsp. berkhoffii infection in coyotes from central coastal California.

Bartonella vinsonii subsp. berkhoffii was originally isolated from a dog suffering infectious endocarditis and was recently identified as a zoonotic agent causing human endocarditis. Following the coyote bite of a child who developed clinical signs compatible with Bartonella infection in Santa Clara County, Calif., this epidemiological study was conducted. Among 109 coyotes (Canis latrans) from central coastal California, 31 animals (28%) were found to be bacteremic with B. vinsonii subsp. berkhoffii and 83 animals (76%) had B. vinsonii subsp. berkhoffii antibodies. These findings suggest these animals could be the wildlife reservoir of B. vinsonii subsp. berkhoffii. PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the gltA and 16S rRNA genes for these 31 isolates yielded similar profiles that were identical to those of B. vinsonii subsp. berkhoffii. Partial sequencing of the gltA and 16S rRNA genes, respectively, indicated 99.5 and 100% homology between the coyote isolate and B. vinsonii subsp. berkhoffii (ATCC 51672). PCR-RFLP analysis of the 16S-23S intergenic spacer region showed the existence of two different strain profiles, as has been reported in dogs. Six (19%) of 31 Bartonella bacteremic coyotes exhibited the strain profile that was identified in the type strain of a canine endocarditis case (B. vinsonii subsp. berkhoffii ATCC 51672). The other 25 bacteremic coyotes were infected with a strain that was similar to the strains isolated from healthy dogs. Based on whole bacterial genome analysis by pulsed-field gel electrophoresis (PFGE) with SmaI restriction endonuclease, there was more diversity in fingerprints for the coyote isolates, which had at least 10 major variants compared to the two variants described for domestic dog isolates from the eastern United States. By PFGE analysis, three Bartonella bacteremic coyotes were infected by a strain identical to the one isolated from three healthy dog carriers. Further studies are necessary to elucidate the mode of transmission of B. vinsonii subsp. berkhoffii, especially to identify potential vectors, and to determine how humans become infected.

Automated approach for ribosomal intergenic spacer analysis of microbial diversity and its application to freshwater bacterial communities.

An automated method of ribosomal intergenic spacer analysis (ARISA) was developed for the rapid estimation of microbial diversity and community composition in freshwater environments. Following isolation of total community DNA, PCR amplification of the 16S-23S intergenic spacer region in the rRNA operon was performed with a fluorescence-labeled forward primer. ARISA-PCR fragments ranging in size from 400 to 1,200 bp were next discriminated and measured by using an automated electrophoresis system. Database information on the 16S-23S intergenic spacer was also examined, to understand the potential biases in diversity estimates provided by ARISA. In the analysis of three natural freshwater bacterial communities, ARISA was rapid and sensitive and provided highly reproducible community-specific profiles at all levels of replication tested. The ARISA profiles of the freshwater communities were quantitatively compared in terms of both their relative diversity and similarity level. The three communities had distinctly different profiles but were similar in their total number of fragments (range, 34 to 41). In addition, the pattern of major amplification products in representative profiles was not significantly altered when the PCR cycle number was reduced from 30 to 15, but the number of minor products (near the limit of detection) was sensitive to changes in cycling parameters. Overall, the results suggest that ARISA is a rapid and effective community analysis technique that can be used in conjunction with more accurate but labor-intensive methods (e.g., 16S rRNA gene cloning and sequencing) when fine-scale spatial and temporal resolution is needed.

Development of a new PCR-ribotyping method for Clostridium difficile based on ribosomal RNA gene sequencing

PCR-ribotyping, a typing method based on polymorphism in the 16S-23S intergenic spacer region, has been recently used to investigate outbreaks due to Clostridium difficile. However, this method generates bands of high and close molecular masses which are difficult to separate on agarose gel electrophoresis. To improve reading of banding patterns of PCR-ribotyping applied to C. difficile, a partial sequencing of the rRNA genes (16S and 23S) and intergenic spacer region has been performed, then a new set of primers located closer to the intergenic spacer region has been defined. The new PCR gave reproducible patterns of bands easy to separate on agarose gel electrophoresis. Each of the 10 serogroups and 11 subgroups of serogroup A produced a different pattern. This typing method has evidenced major qualities such as easiness, rapidity and reproducibility. However, its discriminatory power has to be evaluated to validate its importance as a typing tool for C. difficile.

Development of a new PCR-ribotyping method for Clostridium difficile based on ribosomal RNA gene sequencing.

PCR-ribotying, a typing method based on polymorphism in the 16S-23S intergenic spacer region, has been recently used to investigate outbreaks due to Clostridium difficile. However, this method generates bands of high and close molecular masses which are difficult to separate on agarose gel electrophoresis. To improve reading of banding patterns of PCR-ribotyping applied to C. difficile, a partial sequencing of the rRNA genes (16S and 23S) and intergenic spacer region has been performed, then a new set of primers located closer to the intergenic spacer region has been defined. The new PCR gave reproducible patterns of bands easy to separate on agarose gel electrophoresis. Each of the 10 serogroups and 11 subgroups of serogroup A produced a different pattern. This typing method has evidenced major qualities such as easiness, rapidity and reproducibility. However, its discriminatory power has to be evaluated to validate its importance as a typing tool for C. difficile.

Histopathology and Within-Plant Distribution of the Phytoplasma Associated with Australian Papaya Dieback

Dieback-affected papaya plants were characterized by a discoloration of the contents of laticifers, while the anatomy of sieve elements was healthy in appearance until the necrotic stages of the disorder were reached. Laticifer discoloration was not always associated with the presence of phytoplasma in affected tissue, as judged by polymerase chain reaction (PCR) using primers based on the 16S rRNA gene and 16S-23S intergenic spacer region. Phytoplasma DNA was detected in a range of plant tissues, including roots, but not in mature leaves which would act as photoassimilate sources. As plants recovered from a dieback period, the extent of the distribution of both laticifer discoloration and phytoplasma DNA decreased. Phytoplasma cells were not observed in transmission electron microscopy studies of mature sieve elements of dieback-affected leaf, stem, or fruit tissue from plants at various stages of symptom expression, although PCR tests indicated the presence of phytoplasma DNA. Membrane-bound structures, similar in shape and size to phytoplasma cells but interpreted as autophagic vesicles or latex vesicles in immature laticifers, were observed within vacuoles of cells in phloem tissue in leaves displaying tissue breakdown in the form of a water-soaked appearance to veins ("X-Y" patterning). In contrast, phytoplasmas were readily observed in papaya leaves displaying symptoms of yellow crinkle. We conclude that phytoplasma cells are present in very low titer in dieback-affected tissues and that, while the plant appears to limit proliferation of the dieback-associated pathogen, this defense strategy is ultimately unsuccessful because it is associated with a rapid decline of the papaya plant.

Characterization of oligonucleotide probes for the identification of Acinetobacter spp., A. baumannii and Acinetobacter genomic species 3

The 16S-23S intergenic spacer regions of four Acinetobacter genomic species belonging to the A. calcoaceticus-A. baumannii (Acb) complex, i.e. genomic species 1 ( A. calcoaceticus), genomic species 2 ( A. baumannii), genomic species 3 and Tjernberg and Ursing (TU) genomic species 13, have been cloned and sequenced. Sequence analysis led to the discovery of a single copy of lle and Ala tRNA genes within each spacer. Sequence comparison allowed the identification of a 192-base-pair long highly conserved sequence between the 3′ end of the 16S rRNA and the 5′ end of the tRNA Ala genes. Moreover, two short regions, which were specific to, respectively, genomic species 2 and 3, could be identified. Oligonucleotides corresponding to these sequences were constructed and tested for the ability to hybridize with chromosomal DNA extracted from Acinetobacter belonging to different genomic species and with chromosomal DNA of other bacterial genera. One of these oligonucleotides was demonstrated to be useful as a sensitive and specific probe for A. baumannii. A less sensitive probe for Acinetobacter genomic species 3 was also developed.

Phylogenetic relationships of coconut phytoplasmas and the development of specific oligonucleotide PCR primers

SummaryUsing the polymerase chain reaction the 16S rRNA genes and the 16S‐23S spacer regions of phytoplasmas associated with lethal decline diseases of coconut palm (Cocos nucifera), were amplified from infected plants from Florida and the Yucatan region in Mexico and from east and west Africa. Following sequencing of the rDNA products, phylogenetic analysis confirmed that these coconut phytoplasmas form a separate cluster within the phytoplasma clade and that the pathogen causing diseases in west Africa formed a new sub‐clade within this cluster. Analysis of the 16S‐23S intergenic spacer regions confirmed the sequence diversity of this region and enabled two primers to be designed which were specific for the diseases found in east and west Africa. None of these specific primers, when paired with a universal primer, produced PCR amplification products from healthy coconut DNA, infected coconut DNA from the Caribbean or DNA from a variety of periwinkle (Catharanthus roseus)‐maintained phytoplasmas. These specific primers can serve as effective tools for identifying particular coconut phytoplasmas in field samples.