Erich Seemüller

Phylogenetic classification of phytopathogenic mollicutes by sequence analysis of 16S ribosomal DNA

The phylogenetic relationships of 17 phytopathogenic mycoplasmalike organisms (MLOs) representing seven major taxonomic groups established on the basis of MLO 16S ribosomal DNA (rDNA) restriction patterns were examined by performing a sequence analysis of the 16S rDNA gene. The sequence data showed that the MLOs which we examined are members of a relatively homogeneous group that evolved monophyletically from a common ancestor. In agreement with results obtained previously with other MLOs, our results also revealed that the organisms are more closely related to Acholeplasma laidlawii and other members of the anaeroplasma clade than to any other mollicutes. A phylogenetic tree based on 16S rDNAs showed that the MLOs which we examined can be divided into the following five primary clusters: (i) the aster yellows strain cluster; (ii) the apple proliferation strain cluster; (iii) the western-X disease strain cluster; (iv) the sugarcane white leaf strain cluster; and (v) the elm yellows strain cluster. The aster yellows, western-X disease, and elm yellows strain clusters were divided into two subgroups each. MLOs whose 16S rDNA sequences have been determined previously by other workers can be placed in one of the five groups. In addition to the overall division based on 16S rDNA sequence homology data, the primary clusters and subgroups could be further defined by a number of positions in the 16S rDNAs that exhibited characteristic compositions, especially in the variable regions of the gene.

Classification of plant-pathogenic mycoplasma-like organisms using restriction-site analysis of PCR-amplified 16s rDNA

Summary: A method has been developed to amplify the 16S rRNA gene of plant-pathogenic mycoplasma-like organisms (MLOs) from infected plant material using the polymerase chain reaction (PCR). The procedure is dependent on the presence of a BclI restriction site in the 16S rDNA of chloroplasts but not in that of the MLOs. This difference permits the specific amplification of the 16S rDNA of the MLOs from BclI-digested total DNA from infected plants using primers from conserved regions of this gene. In this study 16S rDNA was obtained from 52 MLO isolates from herbaceous dicots and monocots as well as woody plants. Digestion of the 16S rRNA genes using AluI endonuclease revealed seven restriction patterns, which were used to group the isolates examined. Group I, which is also characterized by the presence of two KpnI sites, consisted of 31 isolates, most of which are from herbaceous dicots. Isolates assigned to groups II to VI were mostly from woody plants, while the isolates of group VII were from monocots or obtained from a leafhopper. The restriction patterns varied little within groups; however, four group I isolates and one group IV isolate differed slightly from the typical patterns of these groups as a result of a deletion or a slight shift of one restriction site. The groupings uncovered by AluI restriction were also obtained by digesting the 16S rDNA with RsaI endonuclease. However, some atypical patterns were observed within group V isolates. The groups described on the basis of restriction digest data were supported by sequence analysis. With one exception, the 16S rDNA of isolates within the same group exhibited 97·8 to 99·5% homology while those of different groups showed 89·6 to 92·0% homology.

Classification of aster yellows-group phytoplasmas based on combined analyses of rRNA and tuf gene sequences

Seventy phytoplasma isolates, including 10 previously characterized reference strains, of the aster yellows group were examined by RFLP analysis of PCR-amplified rDNA and RFLP and sequence analysis of the tuf gene. On the basis of rDNA restriction profiles, seven previously proposed 16S rDNA subgroups (16SrI-A, -B, -C, -D, -E, -F and -K) were recognized in the material examined. In addition, three new subgroups that differ in the RFLP profiles were identified and designated 16SrI-L, 16SrI-M and 16SrI-N. Of the two types of rDNA sequences used, an 1800 bp fragment comprising the entire 16S rRNA gene and the 16S-23S rDNA spacer region proved more suitable for AY-group phytoplasma differentiation than a 1240 bp fragment of the 16S rRNA gene. Many differences in the rDNA profiles between the subgroups could be explained by sequence heterogeneity of the two phytoplasmal rRNA operons. The subgroups delineated by RFLP analysis of a 940 bp tuf gene fragment are consistent with subgroups defined on the basis of rDNA sequences. However, subgroups 16SrI-D, -L and -M showed the same tuf gene restriction profiles as subgroup 16SrI-B. This result was confirmed by sequence analysis in which these subgroups differed slightly in their tuf gene sequence, when compared with members of subgroup 16SrI-B. On the basis of combined analyses of rDNA and tuf gene sequences and in view of pathological aspects, the taxonomic distinction of AY-subgroups 16SrI-A, -B, -C, -D, -E, -F, -K and -N appears to be substantial.

Mycoplasmas of Plants and Insects

Plants and insects are habitats of several mollicute genera including Acholeplasma, Entomoplasma, Mesoplasma, Spiroplasma, and the provisionally classified phytoplasmas. Plant- and insect-associated acholeplasmas, entomoplasmas and mesoplasmas occur as saprophytes on plant surfaces including flowers, and in insects probably as commensals or symbionts (90, 106). They are pleomorphic, nonhelical in shape and culturable in vitro and consist of relatively few species. There is no indication that they are of economic importance, and only their phylogenetic positions will be discussed in this chapter. For more information, the reader is referred to references 89, 107, 109.

Characterization and classification of phytoplasmas from wild and cultivated plants by RFLP and sequence analysis of ribosomal DNA

Restriction fragment length polymorphism and sequence analysis of PCR-amplified ribosomal DNA were used to identify and classify phytoplasmas associated with diseases of various wild and cultivated plants. The diseases examined were either not known before or the presumable causal agents were not yet identified and characterized or were only known from other geographic areas. New diseases examined were those causing virescence and phyllody of Bunias orientalis and Cardaria draba. Both were associated with strains of the aster yellows phytoplasma. The same type of aster yellows phytoplasma was also found to be associated with yellows and phyllody diseases of Portulaca oleracea, Stellaria media, Daucus carota ssp. sativus, and Cyclamen persicum. In German and French DNA samples from diseased Trifolium repens, the clover phyllody phytoplasma was identified, which could clearly be distinguished from other phytoplasmas of the aster yellows group. Strains of the stolbur phytoplasma were detected in big bud-affected tomatoes and almost exclusively in Convolvulus arvensis. In Cirsium arvense and Picris echioides two distinct phytoplasmas were identified which showed relationship to the sugarcane white leaf phytoplasma group but may represent a new group or subgroup. In Conyza (syn.: Erigeron) canadensis a phytoplasma of the X-disease group was detected. A strain from Gossypium hirsutum showed the same restriction profiles as the faba bean phyllody phytoplasma.

A chromosome map of the European stone fruit yellows phytoplasma.

A physical map of the European stone fruit yellows phytoplasma strain GSFY1 chromosome was constructed using PFGE-purified genomic DNA from diseased tobacco and tomato plants. The map was generated with single and double digestions of the chromosome with SmaI, BssHII, ApaI, BamHI and XhoI restriction endonucleases and the fragments were resolved by PFGE. Reciprocal double digestions were used to locate 26 restriction sites on the chromosome. Southern blot analysis was also used to assist in the arrangement of the contiguous restriction fragments obtained. From the restriction fragments generated by double digestion, the circular chromosome was calculated to be approximately 635 kb. Loci of two rRNA operons, the operon containing the tuf gene, genes encoding an immunodominant membrane protein and a putative nitroreductase, and randomly cloned DNA fragments IH184 and AT67 were placed on the map. Digestion of chromosomal DNA of strain GSFY1 with MluI gave a complex restriction pattern, suggesting that this isolate consists of a population with heterogeneity with respect to MluI restriction sites. The GSFY1 physical map was different from that of the closely related apple proliferation phytoplasma but the genetic arrangement was similar.

Transcriptomics assisted proteomic analysis of Nicotiana occidentalis infected by Candidatus Phytoplasma mali strain AT

Phytoplasmas are pathogenic bacteria within the class of Mollicutes, which are associated with more than 1000 plant diseases. In this study, we applied quantitative mass spectrometry to analyse affected pathways of the model plant tobacco (Nicotiana occidentalis) upon Candidatus Phytoplasma mali strain AT infection. Using tissue obtained from leaf midribs, 1466 plant‐assigned proteins were identified. For 1019 of these proteins, we could reproducibly quantify the expression changes of infected versus noninfected plants, of which 157 proteins were up‐ and 173 proteins were downregulated. Differential expression took place in a number of pathways, among others strong downregulation of porphyrin and chlorophyll metabolism and upregulation of alpha‐linolenic acid metabolism, which was consistent with observed increased levels of jasmonic acid, a key signal molecule of plant defence. Our data shed light on the molecular networks that are involved in defence of plants against phytoplasma infection and provide a resource for further studies.

Chromosomal organization and nucleotide sequence of the genes coding for the elongation factors G and Tu of the apple proliferation phytoplasma.

Genes coding for elongation factors G (fus) and Tu (tuf) of the non-culturable apple proliferation (AP) phytoplasma were cloned and sequenced. Arrangement of these genes and identification of the ribosomal protein gene rps7 upstream of the fus gene suggest a transcriptional organization similar to that of the streptomycin operon of Escherichia coli and other bacteria. The fus and tuf genes from other tested phytoplasmas were found to be similarly linked as in the AP agent. Thus, it is likely that they show a similar chromosomal arrangement. This organization would be in contrast to that of the phylogenetically distinctly different culturable mollicutes of the genus Mycoplasma in which the tuf and fus genes are separately transcribed.

Analysis of expressed genes of the bacterium 'Candidatus phytoplasma Mali' highlights key features of virulence and metabolism.

‘Candidatus Phytoplasma mali’ is a phytopathogenic bacterium of the family Acholeplasmataceae assigned to the class Mollicutes. This causative agent of the apple proliferation colonizes in Malus domestica the sieve tubes of the plant phloem resulting in a range of symptoms such as witches’- broom formation, reduced vigor and affecting size and quality of the crop. The disease is responsible for strong economical losses in Europe. Although the genome sequence of the pathogen is available, there is only limited information on expression of selected genes and metabolic key features that have not been examined on the transcriptomic or proteomic level so far. This situation is similar to many other phytoplasmas. In the work presented here, RNA-Seq and mass spectrometry shotgun techniques were applied on tissue samples from Nicotiana occidentalis infected by ‘Ca. P. mali’ strain AT providing insights into transcriptome and proteome of the pathogen. Data analysis highlights expression of 208 genes including 14 proteins located in the terminal inverted repeats of the linear chromosome. Beside a high portion of house keeping genes, the recently discussed chaperone GroES/GroEL is expressed. Furthermore, gene expression involved in formation of a type IVB and of the Sec-dependent secretion system was identified as well as the highly expressed putative pathogenicity–related SAP11-like effector protein. Metabolism of phytoplasmas depends on the uptake of spermidine/putescine, amino acids, co-factors, carbohydrates and in particular malate/citrate. The expression of these transporters was confirmed and the analysis of the carbohydrate cycle supports the suggested alternative energy-providing pathway for phytoplasmas releasing acetate and providing ATP. The phylogenetic analyses of malate dehydrogenase and acetate kinase in phytoplasmas show a closer relatedness to the Firmicutes in comparison to Mycoplasma species indicating an early divergence of the Acholeplasmataceae from the Mollicutes.

Detection of Bermuda Grass White Leaf Disease in Italy and Characterization of the Associated Phytoplasma by RFLP Analysis

Bermuda grass showing symptoms of a white leaf disease has been observed in fruit orchards, vegetable fields, and uncultivated areas in the Latium and Campania regions of central and southern Italy. Using polymerase chain reaction (PCR) amplification with phytoplasma-specific primers, all symptomatic plants tested positively; whereas no amplification product was obtained from nonsymptomatic plants. Restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified ribosomal DNA revealed a uniform pattern that was similar to that of the Bermuda grass white leaf phytoplasma collected in Thailand, which is known to be a member of the sugarcane white leaf phytoplasma group. By RFLP analysis, the phytoplasma infecting Bermuda grass could be distinguished from other group members, including the phytoplasmas associated with sugarcane white leaf and Brachiaria white leaf. This is the first report on the presence of the Bermuda grass white leaf phytoplasma in Europe.