Apple Proliferation Phytoplasma Research Articles

In vitro grafting : a new tool to transmit pome fruit phytoplasmas to non-natural fruit tree hosts.

Long-term maintained, homogeneous in vitro shoot cultures of Pyrus communis infected with pear decline (PD) phytoplasmas and Malus pumila infected with apple proliferation (AP) phytoplasmas were used to inoculate by in vitro grafting the non-natural plant hosts Pyronia veitchii and Prunus marianna. Healthy intergeneric heterografts and autografts with phytoplasma-infected graft tips were performed as controls. The transmission rate, as revealed by PCR detection of the respective phytoplasma, was 35% for the transmission of PD phytoplasmas from Pyrus to Pyronia compared to 75% for the Pyrus/Pyrus-smtograft control, and 52% for the transmission of AP phytoplasmas from Malus to Pyronia compared to 90% for the Malus/Malus-autogratt control. Only one AP-inoculated Pyronia plantlet exhibited AP-typical symptoms. No transmission of PD or AP phytoplasmas to Prunus could be achieved.

Isolation of the gene encoding an immunodominant membrane protein of the apple proliferation phytoplasma, and expression and characterization of the gene product.

An immunodominant membrane protein (IMP) of the apple proliferation (AP) phytoplasma was detected in preparations from AP-diseased periwinkle plants using monoclonal and polyclonal antibodies to the AP agent. Following isolation from Western blots and partial sequencing, degenerate oligonucleotides derived from the IMP sequence were used as probes to identify a DNA fragment containing the ORF encoding the IMP. Complete sequencing and subsequent analysis of the cloned DNA fragment revealed the presence of two ORFs, predicted to encode proteins with molecular masses of 25 kDa (P-318A) and 19 kDa (P-318B). Whilst database searches failed to assign a possible function to P-318A, analysis of P-318B predicted an amphiphilic membrane protein with a positively charged N-terminal portion, followed by a hydrophobic segment forming an alpha-helix, and a hydrophilic C-terminal part located outside of the cell. The amphiphilic nature of P-318B was confirmed by its solubility in Triton X-114. The gene encoding P-318B was expressed in Escherichia coli and the resulting protein was used to immunize rabbits. The antiserum obtained reacted specifically with P-318B. The same protein was also detected by an antiserum raised against antigen preparations from AP-diseased plants. The P-318B antiserum did not react with antigen preparations from plants infected with the closely related pear decline phytoplasma. However, in Southern hybridization studies, the gene encoding the IMP hybridized to genomic fragments of the pear decline and European stone fruit yellows phytoplasmas. It also showed significant sequence similarity to a gene encoding an antigenic membrane protein of the sweet potato witches' broom phytoplasma, but not to a gene encoding a major immunogenic membrane protein of an aster yellows group phytoplasma. Since it appears that most phytoplasmas possess a major immunogenic membrane protein which may have a function in pathogenesis, this work may be a basis for further studies on fundamental aspects of host-pathogen interactions. It also describes a new approach to obtain suitable immunogens to produce specific antibodies for detection and characterization of the non-culturable phytoplasmas.

Effect of phytoplasmal infection on concentration and translocation of carbohydrates and amino acids in periwinkle and tobacco

Source and sink tissue of Catharanthus roseus (periwinkle) infected with the grapevine yellows (GY), apple proliferation (AP), or ash yellows (ASHY) phytoplasma and of Nicotiana tabacum (tobacco) infected with the AP phytoplasma, were examined for concentrations of glucose, fructose, sucrose, starch and total amino acids. Phytoplasmal infection caused growth inhibition and severe yellowing in both plants, the GY phytoplasma being more virulent than the AP and ASHY agents in periwinkle. Leaf size in periwinkle was greatly reduced by the ASHY phytoplasma with little or no effect caused by either the AP or GY agents. Both phytoplasma strain and host plant had an influence on the observed results. In source leaves of periwinkle and tobacco, infection mostly led to a marked increase in soluble carbohydrates and starch. Increase was smallest in periwinkle affected by ASHY. During a dark period of 42 h, carbohydrate levels tended to decrease in healthy plants whereas they remained high in diseased plants. In sink leaves of periwinkle, infections by the GY and AP phytoplasmas led to a marked decrease in sugar whereas starch levels were either only slightly or not affected at all. Infection by the ASHY phytoplasma led to a smaller decrease of sugars and an increase in starch. In tobacco sink leaves, sugars remained largely unchanged whereas starch increased after infection with the AP phytoplasma. In periwinkle roots, soluble carbohydrates were not markedly altered but starch was considerably reduced, in the GY and AP phytoplasmas in particular. In tobacco roots, there was a general decrease in soluble carbohydrates and starch following infection by the AP phytoplasma. The amino acid concentrations were not altered by infection, except that source and sink leaves of ASHY-infected periwinkles and source leaves of AP-infected tobacco showed higher levels of amino acids than healthy plants. The results are discussed with respect to the effect of phytoplasmas on phloem transport, source leaf phenotype and sink growth.

Phytoplasma transmission by in vitro graft inoculation as a basis for a preliminary screening method for resistance in fruit trees

In vitro grafting was tested as a technique for inoculating Prunus rootstock Prunus marianna GF 8‐1 with European stone fruit yellows (ESFY) phytoplasmas and apple rootstock Malus pumila MM106 with apple proliferation (AP) phytoplasmas. In vitro shoot cultures of ESFY‐infected Prunus marianna GF 8‐1 and AP‐infected Malus pumila MM106 were used as graft inoculum to transmit the phytoplasmas to the respective healthy rootstock. Phytoplasma transmission was assessed after a graft contact of 1, 2 or 3 months. Healthy autografts were used as controls to monitor parameters of in vitro grafting. Successful graft union formation ranged from 58 to 79% irrespective of the plant species and the sanitary state of the graft. Pathogen‐specific polymerase chain reaction (PCR) was used to test the inoculated rootstocks for the presence of ESFY and AP phytoplasmas, respectively. The rate of ESFY phytoplasma transmission in successful Prunus‐grafts increased from 69 to 94% with the time of contact. AP phytoplasma transmission to Malus occurred in 80 to 97% of successful grafts. To our knowledge this is the first report of phytoplasma transmission by grafting in vitro. The results provide a good basis for the establishment of a preliminary in vitro screening method for phytoplasma resistance in Prunus and Malus.

OCCURRENCE OF APPLE PROLIFERATION AND PEAR DECLINE PHYTOPLASMAS IN DISEASED PEAR TREES IN HUNGARY

SUMMARY Pear trees with proliferation and decline symptoms were sampled in orchards in Hungary.We describe the results obtained with different approaches in detecting phytoplasmas associated with the symptoms.Analyses with primers designed on ribosomal and non-ribosomal DNA sequences and the restriction profiles of the amplified products revealed the presence of phytoplasmas belonging to the apple proliferation group. This appears to be the first report on the presence of mixed infections of apple proliferation and pear decline phytoplasmas in diseased pear trees in Hungary.

PCR Assay for specific detection of European stone fruit yellows phytoplasmas and its use for epidemiological studies in France

DNA amplification by polymerase chain reaction was used to specifically detect phytoplasmas associated with severe decline diseases of European stone fruits. PCR primers were designed according to the partial sequence of a nonribosomal genomic fragment of European stone fruit yellows phytoplasmas obtained by direct sequencing of a specific PCR product. A PCR assay was developed which resulted in specific amplification of a 237 bp-DNA fragment from total DNA extracts derived from over 300 stone fruit samples. No PCR product was obtained with DNA from healthy controls or plants diseased with various other phytoplasmas, e.g. the closely related apple proliferation and pear decline phytoplasmas. Phytoplasma infection was checked in all samples by PCR amplification with universal ribosomal primers. Detection rate with specific and universal primers was correlated by 97%. European stone fruit yellows phytoplasmas were detected in samples of 114 out of 139 examined orchards which represent the major stone fruit growing regions of France. Typical symptoms like chlorotic leaf roll in summer and off-season growth in winter were correlated by 95% to the presence of phytoplasmas. However, phytoplasmas were also detected in 51% of samples derived from trees showing non-specific symptoms. A comparison study including 201 samples showed that 81% of the PCR-positive samples were also tested positive using fluorescence microscopy with DAPI staining.

Genetic comparison of the peach yellow leaf roll agent with European fruit tree phytoplasmas of the apple proliferation group

Restriction fragment length polymorphism (RFLP) analysis of PCR‐amplified ribosomal DNA and Southern blot hybridization using cloned chromosomal DNA fragments from the apple proliferation (AP) phytoplasma as probes were used to investigate the genetic relationship of the California peach yellow leaf roll (PYLR) agent with phytoplasmas causing fruit tree diseases in Europe. This comparison showed that the California PYLR phytoplasma is closely related to apple proliferation (AP), pear decline, and European stone fruit yellows phytoplasmas and that it is a member of the phylogenetic AP group. The PYLR agent could clearly be distinguished from the AP and European stone fruit yellows phytoplasmas by Southern blot hybridization with DNA fragments from the AP phytoplasma and by RFLP analysis of ribosomal DNA employing SspI, BsaAI, and RsaI restriction endonucleases. However, the PYLR phytoplasma was indistinguishable from the pear decline agent by RFLP analysis of PCR‐amplified ribosomal DNA.

Long‐term maintenance of nonculturable apple‐proliferation phytoplasmas in their micropropagated natural host plant

Phytoplasmas associated with apple proliferation (AP) disease of apple trees have been maintained in their micropropagated natural host plant Malus pumila since 1985. Different isolates of these nonculturable plant pathogens could thus be studied in vitro. Amplification of a pathogen‐specific DNA fragment by polymerase chain reaction (PCR) confirmed the presence of AP phytoplasmas in the diseased plants even after 10 years of in‐vitro propagation. Restriction fragment length polymorphism analysis of the amplified chromosomal DNA fragments revealed no genetic difference between the AP phytoplasma isolates. Growth parameters, symptom expression and phytoplasma concentration were examined to compare the in‐vitro behaviour of four different AP phytoplasma isolates and to compare different subculture conditions. A comparison of these data obtained after 2 or 8 years of micropropagation revealed no essential differences. Eight years after culture initiation, diseased shoots still exhibited typical symptoms like witches’ broom, small leaves with large stipules and stunted growth. The use of phytoplasma‐diseased micropropagated plants to establish a ‘type culture collection’ of these otherwise nonculturable plant pathogens is discussed.

16S rDNA‐derived oligonucleotide probes for the differential diagnosis of plum leptonecrosis and apple proliferation phytoplasmas

The 16S rRNA gene of plum leptonecrosis phytoplasma has been PCR‐amplified, cloned and almost completely sequenced (1201 bp). The sequence analysis confirmed the close genetic relationship between plum leptonecrosis phytoplasma and the phytoplasmas associated with other stone‐fruit diseases in Europe. By comparison with the 16S rDNA sequence of apple proliferation phytoplasma, two oligonucleotides were selected, differing by two nucleotides, which were specific for apple proliferation and plum leptonecrosis phytoplasmas, respectively. The oligonucleotides were labelled with digoxigenin and hybridized, in the presence of tetramethylammonium chloride, to 16S rDNA fragments amplified from apple and plum leaf samples. The results showed that, under the described hybridization conditions, the two phytoplasmas could clearly be distinguished. The advantage of the proposed technique over 16S rDNA restriction fragment length polymorphism is discussed.

Detection of the Apple Proliferation and Pear Decline Phytoplasmas by PCR Amplification of Ribosomal and Nonribosomal DNA

Ribosomal and nonribosomal sequences were analyzed to design polymerase chain reaction (PCR) primers for detection and identification of the phytoplasmas that cause apple proliferation (AP) and pear decline (PD). A ribosomal primer pair (fU5/rU3) was developed that initiated amplification of the target DNA from all 42 samples from PD-infected pear trees and 36 samples from AP-infected apple trees. These primers also amplified rDNA in all other taxonomically different phytoplasma strains that were tested. A pair of group-specific primers (fO1/rO1) derived from the 16S rRNA gene was identified for detection of the closely related phytoplasmas associated with AP, PD, and European stone fruit yellows. PCR detection of the PD agent with primer pairs fU5/rU3 and fO1/rO1 was considerably more sensitive than microscopic detection using the 4'-6-diamidino-2-phenylindole fluorescence method. A more specific ribosomal primer pair (fPD/rO1) amplified phytoplasma rDNA in all samples from infected pear trees but showed some cross-amplification of AP rDNA. Restriction enzyme analysis of the PCR products obtained with primer pairs fO1/rO1 and fPD/rO1 distinguished the AP and PD phytoplasmas. One pair of ribosomal (fPD/rPDS) primers specifically amplified DNA of the PD phytoplasma but from only about 80% of the infected pear trees. Three pairs of nonribosomal primers amplified phytoplasmal DNA from AP- or PD-infected trees or from both phytoplasmas but failed to detect all strains of either of the two pathogens. These results show that is not possible to detect all strains of the AP and PD phytoplasmas, respectively, with pathogen-specific primers.

Maintenance of Two Types of Apple Proliferation Phytoplasmas in the Micropropagated Non-Natural Host "Pyronia veitchii"

Maintenance of Two Types of Apple Proliferation Phytoplasmas in the Micropropagated Non-Natural Host "Pyronia veitchii"