Kampmann, Maren

Kiss, Emese

Schneider, Bernd

Seemüller, Erich

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Bulletin of Insectology 64 (Supplement): S163-S164, 2011 ISSN 1721-8861    Molecular differentiation of severe and mild strains of ‘Candidatus Phytoplasma mali’ and evidence that their interaction in multiply infected trees determines disease severity  Erich SEEMÜLLER1, Maren KAMPMANN1, Emese KISS2, Bernd SCHNEIDER1 1Julius Kuehn Institute, Federal Research Centre for Cultivated Plants, Institute for Plant Protection in Fruit Crops and Viticulture, D-69221 Dossenheim, Germany 2Plant Protection Institute of the Hungarian Academy of Science, H-1525 Budapest, Hungary    Abstract  Previous work has shown that multiple infections of apple trees by distinctly different strains of ‘Candidatus Phytoplasma mali’ are widespread. In the current study, pathological data of infected trees with single or multiple phytoplasma were analyzed and compared with molecular data based on a hflB gene of the infecting phytoplasmas. Single-strand conformation polymorphism and sequence analysis of a variable hflB gene fragment revealed that mild and severe strains can be distinguished by their SSCP pro-files and their phylogenetic clustering. Analysis of cloned sequences from mild and severe single-strain accessions resulted in two groups of reads that clustered, according to their virulence, distantly in the phylogram. Based on this data, the clustering patterns of multiple-strain accession sequences indicated that nearly all of them were composed of mild and severe strains. Our data indi-cate that the virulence of multiple-strain accessions is determined by the ratio of the occurring mild and severe strains in that mild accessions were characterized by the predominance of sequences representing mild strains and vice versa. There is evidence that shifts in the population may occur that drastically alter virulence of multiple-strain accessions.  Key words: Apple proliferation, ‘Candidatus Phytoplasma mali’, virulence, multiple infections, classification.   Introduction  Apple proliferation (AP), associated with ‘Candidatus Phytoplasma mali’, is one of the most important phyto-plasma diseases in Europe. AP induces a range of symp-toms that are either specific, such as witches brooms, rosettes, and enlarged stipules, or largely nonspecific, such as foliar reddening, yellowing, growth suppression and undersized fruits. However, symptom expression is often subject to fluctuation. Diseased trees may recover and may show no or only mild symptoms for shorter or longer periods after which severe symptoms may reap-pear. Symptom development also depends on the viru-lence of the infecting AP phytoplasma strains (Seemüller and Schneider, 2007). ‘Ca. P. mali’ is, at the level of ribosomal DNA se-quences, a homogenous species throughout Europe. To elucidate the molecular diversity within this taxon, several other approaches have been employed. How-ever, the genotypes delineated in these efforts were unrelated to phenotypical traits such as virulence and other pathological characteristics. High resolution of ‘Ca. P. mali’ genotypes was achieved by single-strand conformation polymorphism (SSCP) analysis of a frag-ment of the ATP00464-type hflB gene of strain AT (Schneider and Seemüller, 2009). In continuing this work, we identified DNA samples yielding more com-plex SSCP profiles that suggested the presence of mul-tiple infections in a single tree. Cloning of PCR prod-ucts of such samples resulted in clone populations showing distinct profile polymorphisms and diverse sequences that clustered distantly when subjected to phylogenetic analysis. The collective data indicated that they were composed of two or three distinct ‘Ca. P. mali’ strains (Seemüller et al., 2010). Recent work also suggested that multiple infections are of pathological relevance, probably due to antago-nistic strain interactions leading to shifts in the popula-tions (Seemüller et al., 2010). In the research presented here we characterized the AP phytoplasmas present in singly and multiply infected trees. By linking virulence of the infecting phytoplasmas with their SSCP and se-quence data, it was possible to distinguish and classify mild and severe strains at the molecular level.   Materials and methods  Root or shoot scions from 27 donor trees naturally in-fected by ‘Ca. P. mali’ were collected in Germany, France and Italy to graft-inoculate healthy rootstocks. The recipient trees were grown in the open or in an unheated greenhouse. The occurrence of AP symptoms was annually recorded using a rating scheme from 0 (no symptom) to 3 (severe symptoms such as witches brooms, stunting or reduced fruit size). The figures obtained were used for evaluating both symptomatology and virulence. Root and/or the current season’s shoot samples were collected from infected apple trees. DNA was extracted from phloem preparations of roots or shoots using a cetyltrimethylammonium bromide procedure (Seemüller and Schneider, 2007). PCR amplification was per-formed employing primer pair fHflB3_1/rHflB3 (5’-TTCTAGCTATTCATCGTGAA-3’/5’-CGGCGCGAT TAGTAGCTCC-3’) that specifically amplifies a 528-bp Phytoplasma-plant interaction and control of phytoplasma diseases I  S164fragment of ATP00464 hflB gene homologues from all AP phytoplasma strains (Schneider and Seemüller, 2009). hflB gene fragments obtained by PCR amplification were ligated into pGEM-T Easy vector system (Prome-ga) and transformed to Escherichia coli XL1 Blue cells (Stratagene). Colony PCR was employed to amplify the inserts of recombinant plasmids using the primer pair described above. The PCR products were subjected to SSCP analysis. Selected cloned fragments were se-quenced using M13 forward and reverse primers. Alignment of sequences was performed with ClustalX2 (Thompson et al., 1997). For phylogenetic comparison a 454-bp fragment was selected and analyzed employing the MEGA4 program using Neighbor-Joining parame-ters and bootstrap analysis (Tamura et al., 2007).   Results and discussion  In the current study the virulence of 27 ‘Ca. P. mali’ accessions collected from three different groups of trees was observed over six to 17 years. These trees showed all major traits of AP disease, namely expression of severe symptoms at the onset of disease, inconsistent symptomatology, recovery, reappearance of disease and great differences in the virulence of the infecting phyto-plasmas. From our work, there is evidence that a major reason for the unusual symptomatology of AP is the obviously widespread occurrence of multiple infections. Our study revealed that about half of the accessions examined were multiply infected and were composed of two to five distinct strains. The real percentage of mul-tiple infections is probably even higher considering the fact that sometimes many cloned fragments had to be examined to identify this phenomenon.  In our attempt to molecularly characterize the viru-lence of ‘Ca. P. mali’ strains we investigated the rela-tionship between symptomatology and molecular traits of the infecting phytoplasmas. This effort was greatly facilitated by the availability of single-strain accessions. Cloning of hflB gene fragments of severe and mild strains of such accessions resulted in two types of se-quences that clustered separately in phylogenetic analy-sis. This fact and the sequence diversity among the various single-strain accessions enabled us to prove that nearly all multiple-strain accessions were composed of mild and severe strains and to classify the cloned se-quences accordingly. The separated clustering of clones representing severe and mild strains was supported by a range of molecular markers at the nucleotide and amino acid level. The molecular classification of the components of multiple-strain accessions reflects their virulence. Cloned sequences of highly virulent accessions clus-tered predominantly with sequences of severe strains whereas sequences of mild accessions clustered mainly with sequences of mild strains. However, shifts in the phytoplasma composition may occur that drastically alter virulence. There is indication that growing condi-tions trigger such shifts that seem to play an important role in the symptomatology of AP phytoplasma-infected trees. Our data also indicate that another mechanism may exist leading to low virulence. A few accessions proved to be highly virulent in the field but did not develop any symptom following graft transmission and greenhouse growing. As sequences representing mild and severe strains were present in equal numbers, it is conceivable that virulence factors were inhibited by action of the mild strain components. In this study, we succeeded to classify phytoplasma virulence at the molecular level. In this way, it was pos-sible to resolve the phytopathogenic composition of multiple-strain accessions and to estimate their impor-tance for symptom expression and course of disease. As shown for one accession, the potential of multiple-strain accessions is not restricted to symptom expression in apple alone; it also affects the host range. Following dodder transmission, two mild strains of this accession grew specifically in either periwinkle or tobacco. The early observations with aster yellows phytoplasmas (Freitag, 1964; Kunkel, 1955) indicate that multiple phytoplasma infections and strain interactions are not restricted to the AP agent but are probably widespread phenomena in phytoplasmology. They are not only im-portant to better understand symptomatology and course of disease, but they also affect detection (primer and antibody specificity), identification and classification.   References  FREITAG J. H., 1964.- Interaction and mutual suppression among three strains of aster yellows virus.- Virology, 24: 401-413. KUNKEL L. O., 1955.- Cross protection between strains of aster yellows.- Advances in Virus Research, 3: 251-273. SCHNEIDER B., SEEMÜLLER, E., 2009.- Strain differentiation of ‘Candidatus Phytoplasma mali’ by SSCP and sequence analyses of the hflB gene.- Journal of Plant Pathology, 91: 103-112. SEEMÜLLER E., SCHNEIDER B., 2007.- Differences in virulence and genomic features of strains of 'Candidatus Phytoplasma mali', the apple proliferation agent.- Phytopathology, 97: 964-970. SEEMÜLLER E., KISS E., SULE S., SCHNEIDER B., 2010.- Multi-ple infection of apple trees by distinct strains of 'Candidatus Phytoplasma mali' and its pathological relevance.- Phytopa-thology, 100: 863-870. TAMURA K., DUDLEY J., NEI M., KUMAR S., 2007.- MEGA4: Molecular evolutionary genetics analysis (MEGA) soft-ware version 4.0.- Molecular Biology and Evolution, 24: 1596-1599. THOMPSON J. D., GIBSON T. J., PLEWNIAK F., JEANMOUGIN F., HIGGINS D. G., 1997.- The ClustalX windows interface: flexi-ble strategies for multiple sequence alignment aided by qual-ity analysis tools.- Nucleic Acids Research, 24: 4876-4882.    Corresponding author: Erich SEEMÜLLER (e-mail: erich.seemueller@jki.bund.de). Julius Kuehn Institute, Insti-tute for Plant Protection in Fruit Crops and Viticulture, D-69221 Dossenheim, Germany.   

Molecular differentiation of severe and mild strains of ‘Candidatus  Phytoplasma mali’ and evidence that their interaction in multiply infected trees determines disease severity

Molecular differentiation of severe and mild strains of ‘Candidatus Phytoplasma mali’ and evidence that their interaction in multiply infected trees determines disease severity

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