Detection and differentiation of African coconut phytoplasmas: RFLP analysis of PCR-amplified 16S rDNA and DNA hybridisation

Phytoplasmas associated with lethal decline diseases of the coconut palm (Cocos nucifera) in west and east Africa were detected by the specific amplification of their 16S rRNA genes. The primers used were based on conserved mollicute-specific and coconut-phytoplasma 16S rRNA gene sequences. Phytoplasma 16S rDNA was amplified from all African decline affected palms, some periwinkle maintained phytoplasmas, but not from healthy palms, infected palms from Florida or from the Cocos spiroplasma and Acholeplasma sp. mollicutes. African phytoplasmas were also detected by DNA hybridisation using two probes from the palm lethal yellowing phytoplasma from Florida. Probes hybridised at moderate stringency to dot blots of lethal decline affected palms from Africa, indicating possible genetic relationships between different coconut phytoplasmas. RFLP analysis of rDNA fragments (length c. 1.45 kbp) detected polymorphisms, indicating that the pathogens found in west and east Africa are not identical. This provides a useful tool for further epidemiological studies of African coconut phytoplasmal diseases.     

Characterization of Phytoplasmas Related to 'Candidatus Phytoplasma asteris' and Peanut WB Group Associated With Sweet Cherry Diseases in Iran

Symptoms suggestive of phytoplasma diseases were observed in infected sweet cherry trees growing in the central regions of Iran. Phytoplasmas were detected in symptomatic trees by the nested polymerase chain reaction (nested PCR) using phytoplasma universal primer pairs (P1/Tint, PA2F/R, R16F2/R2 and NPA2F/R). Restriction fragment length polymorphism analyses of 485 bp DNA fragments amplified in nested PCR revealed that different phytoplamas were associated with infected trees. Sequence analyses of phytoplasma 16S rRNA gene and 16S-23S intergenic spacer region indicated that the phytoplasmas related to ' Ca. Phytoplasma asteris ' and peanut WB group infect sweet cherry trees in these regions. This is the first report of the presence of phytoplasmas related to ' Ca. Phytoplasma asteris' and peanut WB group in sweet cherry trees.     

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.     

Discrimination of Rhizobium tropici and R. leguminosarum strains by PCR-specific amplification of 16S-23S rDNA spacer region fragments and denaturing gradient gel electrophoresis (DGGE)

With the aim of detecting Rhizobium species directly in the environment, specific PCR primers for Rh. tropici and Rh. leguminosarum were designed on the basis of sequence analysis of 16S-23S rDNA spacer regions of several Rh. tropici, Rh. leguminosarum and Agrobacterium rhizogenes strains. Primer specificity was checked by comparison with available rDNA spacer sequences in databases, and by PCR using DNA from target and reference strains. Sequence polymorphisms of rDNA spacer fragments among strains of the same species were detected by denaturing gradient gel electrophoresis (DGGE). The specific PCR primers designed in this study could be applied to evaluate the diversity of Rh. tropici and Rh. leguminosarum by analysing the polymorphisms of 16S-23S spacer rDNA amplified from either whole-cell or soil-extracted DNA.     

Diversity of phytoplasmas isolated from insects, determined by a DNA heteroduplex mobility assay and a length polymorphism of the 16S-23S rDNA spacer region analysis

Two techniques were developed for the analysis of non-cultivable mollicutes in insects. The first was aimed at detecting organisms belonging to undiscovered groups within the phytoplasma clade. After prescreening by polymerase chain reaction with phytoplasma-specific primers, nucleic acids from 54 positive samples were amplified using phytoplasma-specific fluorescein-labelled primers flanking the 16S-23S rDNA spacer region, which is variable in length among the phytoplasmas. The sizes of all the detected products were only those expected for already-described phytoplasma subclades. It was also shown that a single leafhopper might carry different phytoplasmas, at similar or very different relative concentrations. The second technique, based on the heteroduplex mobility assay, was designed for the detection of organisms phylogenetically similar to phytoplasmas but not recognized by the specific primer pair. As a result, signals generated by ribosomal DNA of organisms which appear to be closely related but not identical to phytoplasmas were detected.     

Species-specific PCR for identification of common contaminant mollicutes in cell culture.

Mycoplasma arginini, M. fermentans, M. hyorhinis, M. orale, and Acholeplasma laidlawii are the members of the class Mollicutes most commonly found in contaminated cell cultures. Previous studies have shown that the published PCR primer pairs designed to detect mollicutes in cell cultures are not entirely specific. The 16S rRNA gene, the 16S-23S rRNA intergenic spacer region, and the 5' end of the 23S rRNA gene, as a whole, are promising targets for design of mollicute species-specific primer pairs. We analyzed the 16S rRNA genes, the 16S-23S rRNA intergenic spacer regions, and the 5' end of the 23S rRNA genes of these mollicutes and developed PCR methods for species identification based on these regions. Using high melting temperatures, we developed a rapid-cycle PCR for detection and identification of contaminant mollicutes. Previously published, putative mollicute-specific primers amplified DNA from 73 contaminated cell lines, but the presence of mollicutes was confirmed by species-specific PCR in only 60. Sequences of the remaining 13 amplicons were identified as those of gram-positive bacterial species. Species-specific PCR primers are needed to confirm the presence of mollicutes in specimens and for identification, if required.     

Diversity of phytoplasmas isolated from insects, determined by a DNA heteroduplex mobility assay and a length polymorphism of the 16S−23S rDNA spacer region analysis

Two techniques were developed for the analysis of non-cultivable mollicutes in insects. The first was aimed at detecting organisms belonging to undiscovered groups within the phytoplasma clade. After prescreening by polymerase chain reaction with phytoplasma-specific primers, nucleic acids from 54 positive samples were amplified using phytoplasma-specific fluorescein-labelled primers flanking the 16S−23S rDNA spacer region, which is variable in length among the phytoplasmas. The sizes of all the detected products were only those expected for already-described phytoplasma subclades. It was also shown that a single leafhopper might carry different phytoplasmas, at similar or very different relative concentrations. The second technique, based on the heteroduplex mobility assay, was designed for the detection of organisms phylogenetically similar to phytoplasmas but not recognized by the specific primer pair. As a result, signals generated by ribosomal DNA of organisms which appear to be closely related but not identical to phytoplasmas were detected.     

Molecular characterization of phytoplasmas in lilies with fasciation in the Czech Republic

Lilium spp. with symptoms of severe fasciation were observed in Southern and central Bohemia during the period 1999-2003. Nucleic acids extracted from symptomatic and asymptomatic plants were used in nested-PCR assays with primers amplifying 16S-23S rRNA sequences specific for phytoplasmas. The subsequent nested-PCR with phytoplasma group-specific primers followed by RFLP analyses and the 16S ribosomal gene sequencing, allowed classification of the detected phytoplasmas in the aster yellows group, subgroups 16SrI-B and 16SrI-C alone, and in mixed infection. Samples infected by 16SrI-C phytoplasmas showed different overlapping RFLP profiles after TruI digestion of R16F2/R2 amplicons. Two of these amplicons were sequenced, one of them directly and the other after cloning; sequence analyses and blast alignment confirmed the presence of two different overlapping patterns in samples studied. The sequences obtained were closely related, respectively, to operon A and operon B ribosomal sequences of the clover phyllody phytoplasma. Direct PCR followed by RFLP analyses of the tuf gene with two restriction enzymes showed no differences from reference strain of subgroup 16SrI-C. Infection with aster yellows phytoplasmas of 16SrI-B subgroup in asymptomatic lilies cv. Sunray was also detected.     

Electron Microscopy and Molecular Characterization of Phytoplasmas Associated with Little Leaf Disease of Brinjal (Solanum melongena L.) and Periwinkle (Catharanthus roseus) in Bangladesh

In Bangladesh little leaf disease was observed in brinjal ( Solanum melongena L.) and in periwinkle ( Catharanthus roseus ). Phloem-inhabiting phytoplasmas were consistently detected in both species of diseased plants using transmission electron microscopy (TEM) and polymerase chain reaction (PCR) techniques. The shape, size and within-tissue distribution of phytoplasmas appears to be similar in both hosts. Furthermore, the molecular characterization and identifications of observed phytoplasmas were carried out based on restriction fragment length polymorphism (RFLP) patterns of PCR-amplified products (1200 bp) using phytoplasma-specific universal primers and sequencing analysis of both 16S ribosomal DNA (rDNA) and intergenic spacer region (ISR) of 16S-23S rDNA phytoplasma genes. The patterns of RFLP analysis with seven restriction enzymes exhibited a similar pattern for both phytoplasma strains. The sequence homology between these two strains showed 100% similarity based on 16S rDNA and 16S-23S ISR. Therefore, in Bangladesh the causal agents of brinjal little leaf (BLL-Bd) and periwinkle little leaf (PLL-Bd) are probably the same or closely related phytoplasma strains. These strains, are very close or identical to the strain of brinjal little leaf phytoplasma in India (BLL-In), belonging to the clover proliferation group (Lee et al., Int. J. Syst. Bacteriol. 48, 1153–1169, 1998; Seemuller et al., J. Plant Pathol. 80, 3–26, 1998).     

Assessment of the genetic diversity of Xylella fastidiosa isolated from citrus in Brazil by PCR-RFLP of the 16S rDNA and 16S-23S intergenic spacer and rep-PCR fingerprinting

The genetic diversity among twenty three strains of Xylella fastidiosa, isolated from sweet orange citrus, was assessed by RFLP analysis of the 16S rDNA and 16S-23S intergenic spacer and by rep-PCR fingerprinting together with strains isolated from coffee, grapevine, plum and pear. The PCR products obtained by amplification of the 16S rDNA and 16S-23S spacer region were digested with restriction enzymes and a low level of polymorphism was detected. In rep-PCR fingerprinting, a relationship between the strains and their hosts was observed by using the BOX, ERIC and REP primers. Two major groups were obtained within the citrus cluster and relationships to the geographic origin of the strains revealed. Citrus strains isolated from the States of São Paulo and Sergipe formed one group and strains from the Southern States formed another group. Distinct origins of X. fastidiosa in the Southern and Southeastern States is postulated. The pear isolate was distantly related to all of the other X. fastidiosa strains.     

Detection and Characterization of Phytoplasma and Sugarcane Yellow Leaf Virus Associated with Leaf Yellowing of Sugarcane

Leaves from sugarcane were collected from Egyptian plantation fields and tested for phytoplasma (Sugarcane yellows phytoplasma, SCYP) and Sugarcane yellow leaf virus (SCYLV) using nested PCR (with different primers) and RT‐PCR, respectively. These results showed significant differences in the amplification of the PCR assays. The primer MLO‐X/MLO‐Y, which amplified the 16S‐23S rDNA spacer region, was the most precise to detect the phytoplasma in sugarcane plants. Sequencing and restriction fragment length polymorphism analysis revealed that all tested phytoplasmas belonged to the 16SrI (aster yellows phytoplasma) group, with the exception of cultivar G84‐47 belonged to the 16SrXI (Rice yellow dwarf phytoplasma) group. Three Egyptian sugarcane cultivars were phytoplasma free. Phylogenetic analyses of 34 screened accessions of 16S ribosomal DNA gene sequences of Candidatus phytoplasma including the ones collected from Egypt used in this study and those extracted from GenBank showed that they split into two distinct clusters. The phylogenetic analyses indicated that these phytoplasmas are closely related and share a common ancestor. All tested Egyptian sugarcane plants were infected by SCYLV with the exception of cultivar Phil‐8013 which was virus free.     

Acholeplasma laidlawii has tRNA genes in the 16S-23S spacer of the rRNA operon.

We amplified the 16S-23S rRNA intergenic spacer region of Acholeplasma laidlawii PG8 by polymerase chain reaction (PCR) and obtained two specific PCR products in different sizes. We have sequenced both PCR products and found that one of them has sequence homologous to the spacer tRNA genes in Bacillus subtilis. This is the first evidence of tRNA genes between the 16S-23S rRNA intergenic spacer regions in members of the class Mollicutes.     

Molecular Characterization of Phytoplasmas Associated with Peach Diseases in Iran

Recently, peach trees showing leaf rolling, little leaf, rosetting, yellowing, bronzing of foliage and tattered and shot‐holed leaves symptoms were observed in peach growing areas in the central and north‐western regions of Iran. Polymerase chain reaction (PCR) and nested PCR using phytoplasma universal primer pairs P1/Tint, R16F2/R2, PA2F/R and NPA2F/R were employed to detect phytoplasmas. The nested PCR assays detected phytoplasma infections in 51% of symptomatic peach trees in the major peach production areas in East Azerbaijan, Isfahan, ChaharMahal‐O‐Bakhtiari and Tehran provinces. Restriction fragment length polymorphism (RFLP) analyses of 485 bp fragments amplified using primer pair NPA2F/R in nested PCR revealed that the phytoplasmas associated with infected peaches were genetically different and they were distinct from phytoplasmas that have been associated with peach and almond witches’‐broom diseases in the south of Iran. Sequence analyses of partial 16S rDNA and 16S–23S rDNA intergenic spacer regions demonstrated that ‘Candidatus Phytoplasma aurantifolia’, ‘Ca. Phytoplasma solani’ and ‘Ca. Phytoplasma trifolii’ are prevalent in peach growing areas in the central and north‐western regions of Iran.     

First Report of Aster Yellows Phytoplasma (16SrI‐B) Associated with Witches' Broom Disease of Melia azedarach var. japonica in Korea

Melia azedarach var. japonica trees with leaf yellowing, small leaves and witches' broom were observed for the first time in Korea. A phytoplasma from the symptomatic leaves was identified based on the 16Sr DNA sequence as a member of aster yellows group, ribosomal subgroup 16SrI‐B. Sequence analyses of more variable regions such as 16S–23S intergenic spacer region, secY gene, ribosomal protein (rp) operon and tuf gene showed 99.5?100% nucleotide identity to several GenBank sequences of group 16SrI phytoplasmas. Phylogenetic analysis confirmed that the Melia azedarach witches' broom phytoplasma belongs to aster yellows group.     

A new phytoplasma detected in the South Australian native perennial shrub, Allocasuarina muelleriana

Allocasuarina muelleriana shrubs growing in natural sclerophyll roadside vegetation near Willalooka in the upper south‐east of South Australia have a high incidence of a yellowing disorder in either all or part of the foliage, combined in some cases with a shortening and curling of the leaf‐bearing stems. Samples from symptomatic and adjacent asymptomatic plants were tested for phytoplasmas by the polymerase chain reaction (PCR) assay. All but one asymptomatic plant were negative for phytoplasmas, whereas about half of the symptomatic plants were positive. Restriction fragment polymorphism analysis of PCR products indicated that the phytoplasma was related to the buckthorn witches.‐broom (BWB) and apple proliferation (AP) groups of phytoplasmas, members of which have not been previously reported in Australia. Further evidence from the sequence of the 16S rRNA gene and the use of PCR primers specific to the AP and pear decline (PD) phytoplasmas confirmed the close relationship to the BWB and AP group phytoplasmas.     

Rapid identification of bacteria on the basis of polymerase chain reaction-amplified ribosomal DNA spacer polymorphisms.

To facilitate genus and species level identification of a broad range of bacteria without the requirement of presumptive identification, we have developed a unified set of primers and polymerase chain reaction conditions to amplify spacer regions between the 16S and 23S genes in the prokaryotic rRNA genetic loci. Spacer regions within these loci show a significant level of length and sequence polymorphism across both genus and species lines. A generic pair of priming sequences was selected for the amplification of these polymorphisms from highly conserved sequences in the 16S and 23S genes occurring adjacent to these polymorphic regions. This single set of primers and reaction conditions was used for the amplification of 16S-23S spacer regions for over 300 strains of bacteria belonging to eight genera and 28 species or serotypes, including Listeria, Staphylococcus, and Salmonella species and additional species related to these pathogenic organisms. When the spacer amplification products were resolved by electrophoresis, the resulting patterns could be used to distinguish all of the species of bacteria within the test group. Unique elements in the amplification product patterns generally clustered at the species level, although some genus-specific characteristics were also observed. On the basis of the results obtained with our test group of 300 bacterial strains, amplification of the 16S-23S ribosomal spacer region is a suitable process for generating a data base for use in a polymerase chain reaction-based identification method, which can be comprehensively applied to the bacterial kingdom.     

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

AIMS: 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. METHODS AND RESULTS: 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'. CONCLUSIONS: The method was specific, sensitive and accurate, providing a new technique for differentiating different strains from the same species. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provided the first molecular characterization of 16S rDNA and 16S-23S rDNA IGS from S. marcescens strains.     

Molecular Characterization of 16S Ribosomal DNA and Phylogenetic Analysis of Two X-disease Group Phytoplasmas Affecting China-tree (Melia azedarach L.) and Garlic (Allium sativum L.) in Argentina

Two phytoplasmas closely related to the X‐disease group were associated with China‐tree (Melia azedarach L.) and garlic (Allium sativum L.) decline diseases in Argentina. The present work was aimed at studying their phylogenetic relationship based on molecular characterization of the 16S ribosomal DNA sequences. Phytoplasma DNAs were obtained from naturally infected China‐tree and garlic plants from different geographical isolates. The results from analysis of restriction fragment length polymorphisms and nucleotide sequences of the 16S rDNA showed the affiliation of China‐tree and garlic decline phytoplasmas to the 16SrIII (X‐disease group), subgroups B and J, respectively. Both organisms had high sequence similarities in the 16SrDNA nucleotide sequence with the Chayote witches’ broom phytoplasma from Brazil. The phylogenetic tree, constructed by parsimony analysis, grouped the Garlic decline, China‐tree decline, Chayote witches’ broom and Clover yellow edge phytoplasmas into a cluster separated from the other phytoplasmas of the X‐disease group.