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 Ssp I, Bsa AI, and Rsa I restriction endonucleases. However, the PYLR phytoplasma was indistinguishable from the pear decline agent by RFLP analysis of PCR-amplified ribosomal DNA.     

European stone fruit yellows phytoplasmas associated with a decline disease of apricot in southern England

Phytoplasmas detected by fluorescence microscopy and polymerase chain reaction (PCR) have been discovered infecting Prunus trees at a site in south-east England. The pathogens were detected in tissue samples taken in autumn and also in spring. The symptoms in infected trees varied from severe decline to absence. PCR experiments using group-specific primers to amplify regions of the 16S RNA gene indicated that the phytoplasmas are similar to European stone fruit yellows isolates occurring in southern and eastern Europe. This is the first record of phytoplasmas in Prunus species in the UK. The origin of the infection is unknown. The implications of this new disease for the fruit industry are discussed.     

Production of Monoclonal Antibodies against Apple Proliferation Phytoplasma and their Use in Serological Detection

Two monoclonal antibodies were obtained against the apple proliferation phytoplasma that provide easy, rapid, specific and sensitive serological detection. They reacted specifically by using ELISA and immunofluorescence techniques with apple proliferation-infected periwinkles and apple trees from different regions in northern Italy and Slovenia, but not with several other phytoplasma isolates. We did not observe any monoclonal antibody reaction even using phytoplasmas belonging to the same phylogenetic group such as European stone fruit yellows and pear decline. Two serological techniques, immunofluorescence and ELISA, were compared with DAPI staining and PCR. From July until leaf fall ELISA was as sensitive as PCR but was more rapid and convenient than PCR; immunofluorescence was useful for specific detection of apple proliferation phytoplasma on roots throughout the year. Serological techniques could be conveniently applied in the roots, stems and leaves of apple trees depending on specific phenological stages of the plants.     

Detection and characterization of phytoplasmas in diseased stone fruits and pear by PCR-RFLP analysis in Turkey

During the late summer-early autumn of 2002, surveys were carried out in Turkey to determine the presence of phytoplasma diseases in fruit trees. Phytoplasmas were detected and characterized by PCR-RFLP analysis and TEM technique in stone fruit and pear trees in the eastern Mediterranean region of the country. Six out of 24 samples, including almond, apricot, peach, pear and plum, gave positive results in PCR assays. RFLP analysis usingSspI andBsaAI enzymes of PCR products obtained with primer pair f01/r01 enabled identification of the phytoplasmas involved in the diseases. Stone fruit trees, including a local apricot variety (‘Sakıt’) and a pear sample, were found to be infected with European stone fruit yellows (ESFY, 16SrX-B) and pear decline (PD, 16SrX-C) phytoplasmas, respectively. This is the first report in Turkey of PD phytoplasma infecting pear and of ESFY phytoplasma infecting almond, apricot, myrobalan plum and peach; ESFY phytoplasma infecting Japanese plum was previously reported. http://www.phytoparasitica.org posting July 21, 2005.     

Range of phytoplasma concentrations in various plant hosts as determined by competitive polymerase chain reaction

ABSTRACT For competitive polymerase chain reaction (PCR), an internal standard DNA template was developed that consisted of a highly conserved, internally deleted 16S rDNA fragment of an aster yellows phytoplasma. The internal standard was calibrated using a quantified culture of Acholeplasma laidlawii. Serial dilutions of the internal standard and fixed amounts of target templates from infected plants were coamplified with the same primers, and the products obtained were quantified using an enzyme-linked immunosorbent assay procedure. Analysis of the data revealed that the phytoplasma concentration in the plants examined differed by a factor of about 4 x 10(6). Phytoplasma concentrations of 2.2 x 10(8) to 1.5 x 10(9) cells per g of tissue were identified in periwinkles infected with various phytoplasmas. High to moderate concentrations were detected in Malus domestica (apple) genotypes infected with the apple proliferation phytoplasma, Alnus glutinosa (alder) genotypes infected with the alder yellows phytoplasma, and most aster yellows-infected Populus (poplar) genotypes examined. Very low phytoplasma concentrations, ranging from 370 to 34,000 cells per g of tissue, were identified in proliferation-diseased apple trees on resistant rootstocks 4551 and 4608, yellows-diseased Quercus robur (oak) trees, and Carpinus betulus (hornbeam) trees. Such low concentrations, which corresponded to about 4 to 340 cells in the reaction mixture, could only be detected and quantified by nested PCR.     

Detection and Indentification of European Stone Fruit Yellows and Other Phytoplasmas in Wild Plants in the Surroundings of Apricot Chlorotic Leaf Roll-affected Orchards in Southern France

Between 1994 and 1998 a field study was conducted to identify plant hosts of the European stone fruit yellows (ESFY) phytoplasma in two apricot growing regions in southern and southwestern France where the incidence of apricot chlorotic leaf roll was high. A total of 431 samples from 51 different plant species were tested for the presence of phytoplasmas by PCR using universal and ESFY-specific primers. ESFY phytoplasma was detected in six different wild growing Prunus species exhibiting typical ESFY symptoms as well as in symptomless dog rose bushes (Rosa canina), ash trees (Fraxinus excelsior) and a declining hackberry (Celtis australis). The possible role of these plant species in the spread of ESFY phytoplasma is discussed. PCR-RFLP analysis of ribosomal DNA amplified with the universal primers was carried out to characterize the other phytoplasmas found. Thus, elm yellows phytoplasma, alder yellows phytoplasma and rubus stunt phytoplasma were detected in declining European field elm trees (Ulmus carpinifolia Gled), in declining European alder trees (Alnus glutinosa) and in proliferating Rubus spp. respectively. The presence of rubus stunt phytoplasma in great mallow (Malva sylvestris) and dog rose was demonstrated for the first time. Furthermore, the stolbur phytoplasma was detected in proliferating field bindweed (Convolvulus arvensis) and a previously undescribed phytoplasma type was detected in red dogwood (Cornus sanguinea). According to the 16S rDNA-RFLP pattern this new phytoplasma belongs to the stolbur phytoplasmas group.     

Mapping the spread of apricot chlorotic leaf roll (ACLR) in southern France and implication of Cacopsylla pruni as a vector of European stone fruit yellows (ESFY) phytoplasmas

An epidemiological study on European stone fruit yellows (ESFY) phytoplasmas infecting Prunus fruit trees was carried out from 1994 to 2000 in Languedoc-Roussillon (southern France). The spread of the disease was monitored for 7 years by visual observation of symptoms and by PCR detection of the phytoplasma in an experimental orchard planted with apricot hybrid seedlings. This indicated that aerial vectors were responsible for disease spread, and that transmission rates were low at the beginning of the spread. Seventy thousand homopteran insects were captured within and in the surroundings of highly ESFY-infected apricot orchards, of which about 10 000 were used in PCR and nested-PCR assays with universal ribosomal and ESFY-specific nonribosomal primers to detect ESFY phytoplasmas. The other insects were confined in cages for trials of transmission to test plants. ESFY phytoplasmas could not be detected by PCR in any of the leafhopper species captured but could be detected in the psyllid Cacopsylla pruni caught on Prunus domestica and Prunus cerasifera rootstock suckers of apricot trees and on Prunus spinosa . Nested PCR revealed ESFY phytoplasmas in one individual of the deltocephalid Synophropsis lauri captured on an apricot tree. Transmission trials confirmed the role of Cacopsylla pruni as the ESFY phytoplasma vector in France. When apricot seedlings were used as bait plants from April to November during two consecutive years, no natural transmission could be demonstrated. However, one out of 50 apricot seedlings left for the whole year in the orchard became infected. An early spring ESFY infection is in agreement with both the natural transmission results and the life cycle of Cacopsylla pruni .     

Fast and sensitive on-site isothermal assay (LAMP) for diagnosis and detection of three fruit tree phytoplasmas

Over the years, real-time PCR outflanked endpoint PCR in phytopathogen diagnostics, mainly because of the increase in sensitivity and timesaving aspects of the technique. However, a time consuming 16S rRNA-based nested PCR method is still the gold standard for phytoplasma diagnosis. This is also the case for phytoplasma detection in Malus, Pyrus and Prunus, the three main host plants of apple proliferation (AP), pear decline (PD) and European stone fruit yellows (ESFY) phytoplasma, respectively. The last decade, loop-mediated isothermal amplification (LAMP) (Notomi et al. 2000) is gaining a lot in significance and is also for phytoplasmas expected to become a widely used reliable diagnostic tool. High specificity and sensitivity which also requires a less stringent need for DNA purification, and the short analysis time and the limited equipment requirements makes the LAMP method a fast and affordable alternative with great point-of-care diagnostic potential. In this paper, we present a LAMP primer set for the ribosomal group 16SrX, containing the important fruit tree phytoplasmas AP, PD and ESFY. The primers were developed and validated for fast and sensitive detection and general use for diagnosis. We foresee that the LAMP technique will also have its application in on-site diagnosis of the fruit tree phytoplasmas during inspections and surveys.     

Note: A comparison of molecular diagnostic procedures for the detection of aster yellows phytoplasmas (16Sr-I) in leafhopper vectors

Different molecular procedures were compared for the detection of aster yellows phytoplasmas (AYP) in the leafhopper vectorsMacrosteles quadripunctulatus (Kirschbaum),Euscelidius variegatus (Kirschbaum) andEuscelis incisus (Kirschbaum). Polymerase chain reaction (PCR) with universal and group-specific primers designed on the 16S-rDNA sequence was most sensitive in nested assays. A dot-blot procedure with an oligoprobe designed on the 16S-rDNA was less sensitive and consistent to detect phytoplasmas in total insect DNA, but consistently detected amplicons from direct PCR. The dot-blot assay with a probe based on a phytoplasma plasmid sequence detected AYP in most vector specimens and did not react with DNAs from leafhoppers infected by flavescence dorée and psyllids infected by apple proliferation phytoplasmas. This last assay is almost devoid of contamination risks, faster and cheaper compared to PCR, therefore it has to be preferred for field-scale analysis of leafhopper populations. http://www.phytoparasitica.org posting Feb. 24, 2004.     

Genetic diversity of Czech ‘Candidatus Phytoplasma mali’ strains based on multilocus gene analyses

In several European countries apple trees are affected by apple proliferation disease, which is usually associated with the presence of ‘Candidatus Phytoplasma mali’. During 2010, samples from several apple trees displaying proliferation symptoms were collected throughout the Czech Republic to verify identity of phytoplasmas detected in association with the disease. The majority of the 74 apple trees examined using molecular tools were positive for ‘Ca. P. mali’ presence. The 16S–23S ribosomal genes, the ribosomal protein genes and the nitroreductase and rhodonase like genes were then studied to verify phytoplasma strain variability on multigenic bases. Two RFLP profiles and correspondingly two genetic lineages were found in the PCR-amplified fragments covering the 16S–23S rDNA spacer region. ‘Ca. P. mali’ strains belonging to rpX-A subgroup were identified in the majority of the apple tree sampled, whereas phytoplasmas belonging to the rpX-B subgroup were distributed sporadically. The apple proliferation subtypes AP-15 and AT-2 exhibited nearly equal occurrence; the AT-1 subtype and a mixture of the two or all three of the AP subtypes were infrequently found. The PCR/RFLP results were confirmed by nucleotide sequence analyses of selected ‘Ca. P. mali’ strains.     

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 .     

Electron Microscopy and Molecular Characterization of Phytoplasmas Associated with Strawflower Yellows in the Czech Republic

Strawflower (Helichrysum bracteatum) with symptoms resembling those associated to phytoplasma infection were observed in several areas in the Czech Republic during the period 1994–2001. Plants with leaf bronzing, reddening and necrosis, proliferation of secondary shoots, flower abnormalities and dwarfing died in advanced stages of the disease. The disease incidence ranged from 2% to 70% and caused significant loss to the flower and seed production. Transmission electron microscopy showed phytoplasmas in sieve cells of affected plants, but not in healthy ones. Association of phytoplasmas with the disease was confirmed by polymerase chain reaction using phytoplasma universal ribosomal primers R16F2n/R16R2. An amplification product of the expected size (1.2 kb) was observed in all samples of the symptomatic strawflowers. The restriction profiles obtained following separate digestion with three endonucleases (AluI, HhaI, MseI) showed that phytoplasmas infecting strawflowers from different localities in the Czech Republic were uniform and undistinguishable from aster yellows (subgroup 16SrI-B). Sequence analysis of 1771 bp of the ribosomal operon amplified with primers P1/U3, R16F2n/R2 and 16R758/P7 indicated that the closest related phytoplasmas were those associated with 'Rehmannia glutinosa var. purpurea', both originating from Bohemia. This is the first report on the occurrence of a phytoplasma-associated disease of strawflower in the Czech Republic.     

The occurrence of two phytoplasmas associated with stunted Rubus species in the UK

Polymerase chain reaction using universal primers to sequences in the 16S rRNA gene, and group-specific primers to sequences in the 16S/23S spacer region, revealed two distinct phytoplasmas occurring in Rubus plants showing symptoms of rubus stunt. One phytoplasma appeared similar to phytoplasmas in the elm yellows group; the other appeared to fall into the X disease group. This finding was confirmed by RFLP analysis of PCR products. This is the first identification of phytoplasmas from either of these groups occurring in the UK, and the first report of a phytoplasma belonging to the X disease group in Rubus .     

Development and evaluation of a one‐hour DNA extraction and loop‐mediated isothermal amplification assay for rapid detection of phytoplasmas

A rapid DNA extraction and loop‐mediated isothermal amplification (LAMP) procedure was developed and evaluated for the detection of two specific groups of phytoplasmas from infected plant material. Primers based upon the 16–23S intergenic spacer (IGS) region were evaluated in LAMP assays for amplification of group 16SrI (aster yellows group) and group 16SrXXII (Cape St Paul wilt group) phytoplasma strains. DNA could be extracted from leaf material (16SrI phytoplasmas) or coconut trunk borings (16SrXXII phytoplasmas) onto the membranes of lateral flow devices, and small sections of these membranes were then added directly into the LAMP reaction mixture and incubated for 45 min at 65°C. Positive reactions were detected through the hydroxyl napthol blue colorimetric assay within 1 h of the start of DNA extraction, and were confirmed by subsequent agarose gel electrophoresis of the LAMP products. The level of detection was comparable to that obtained by nested PCR using conventional 16S rDNA phytoplasma‐specific primers. Furthermore, the assays were specific for the phytoplasmas they were designed to detect – the 16SrI assay only detected 16SrI phytoplasmas and not those from any other phylogenetic groups, whilst the 16SrXXII assay only detected 16SrXXII phytoplasmas. The DNA extractions and LAMP assay are easy to perform, requiring minimal equipment, and may therefore form the basis of a rapid and reliable field‐detection system for phytoplasmas.     

Molecular Identification of a New Member of the Clover Proliferation Phytoplasma Group (16SrVI) Associated with Centaurea Solstitialis Virescence in Italy

In the United States, yellow starthistle (Centaurea solstitialis) is an annual invasive weed with Mediterranean origins. Malformed plants displaying witches' broom, fasciations, abortion of buds and flower virescence symptoms were observed in central Italy. Attempts to transmit the causal agent from the natural yellow starthistle host to periwinkle by grafting, resulted in typical symptoms of a phytoplasma, i.e. yellowing and shortening of internodes. The detection of phytoplasmas was obtained from both symptomatic yellow starthistle and periwinkle by the specific amplification of their 16S-23S rRNA genes. PCR amplification of extracted DNA from symptomatic plant samples gave a product of expected size. Asymptomatic plants did not give positive results. An amplicon obtained by direct PCR with universal primers P1/P7 was cloned and sequenced. The homology search using CLUSTALW program showed more than 99% similarity with Illinois elm yellows (ILEY) phytoplasma from Illinois (United States) and 97% with Brinjal little leaf (BLL) phytoplasma from India. Digestion of the nested-PCR products with restriction enzymes led to restriction fragment length polymorphism patterns referable to those described for phytoplasmas belonging to the clover proliferation (16S-VI) group. Since this is a previously undescribed disease, the name Centaurea solstitialis virescence has been tentatively assigned to it. This is a new phytoplasma with closest relationships to ILEY and BLL, but distinguishable from them on the basis of 16S rDNA homology, the different associated plant hosts and their geographical origin.     

Association of aster yellows subgroup 16SrI-B phytoplasmas with a disease of Rehmannia glutinosa var. purpurea

A new phytoplasma disease of Rehmannia glutinosa var. purpurea was observed in the Czech Republic in 1998. Infected plants showing severely proliferating shoots, leaves reduced in size with vein clearing and chlorosis, shortened internodes and virescent petals died in advanced stages of the disease. Electron microscopy examination of the ultra-thin sections revealed the presence of numerous polymorphic bodies in phloem tissue of leaf midribs and petioles. The disease was successfully transmitted from infected plant via a dodder bridge into periwinkle ( Catharanthus roseus ). The phytoplasma aetiology of this disease was further confirmed by polymerase chain reaction (PCR) using universal primers R16F2/R16R2. Restriction fragment length polymorphism (RFLP) analysis of amplification products indicated the presence of aster yellows related phytoplasmas (16SrI-B) in naturally infected samples of R. glutinosa var . purpurea and in symptomatic periwinkle after dodder transmission of the agent. A comparison of the amplified sequence with 17 sequences available in the GenBank confirmed the classification of the phytoplasma in the subgroup 16SrI-B. This is the first report of natural occurrence of phytoplasma-associated disease in R. glutinosa var. purpurea.     

Real-time PCR detection systems for Flavescence dorée and Bois noir phytoplasmas in grapevine: comparison with conventional PCR detection and application in diagnostics

A new real-time PCR detection system was developed for grapevine yellows (GY) using TaqMan minor groove binder probes and including two amplicons for group-specific detection of Flavescence dorée (FD) and Bois noir (BN) phytoplasmas, plus a universal phytoplasma amplicon. FD and BN amplicons were designed to amplify species-specific genomic DNA fragments and the universal amplicon to amplify the 16S ribosomal DNA region. Efficiency of PCR amplification, limit of detection, range of linearity and dynamic range were assessed for all three amplicons. The specificity of detection systems was tested on several other isolates of phytoplasmas and bacteria and on healthy field grapevine and insect samples. No cross-reactivity with other phytoplasma strains, plant or insect DNA was detected. The assay was compared with conventional PCR on more than 150 field grapevine, insect and field bindweed samples. Real-time PCR showed higher sensitivity as phytoplasmas were detected in several PCR-negative and in all PCR-positive samples. A data-mining analysis of results from both detection approaches also favoured real-time PCR over conventional PCR diagnostics. The developed procedure for detection of phytoplasmas in grapevine also included amplification of plant DNA co-extracted with phytoplasmic DNA, providing additional quality control for the DNA extraction and PCR amplification for each sample. The newly developed assay is a reliable, specific and sensitive method easily applicable to high-throughput diagnosis of GY.     

Differential detection and genetic relatedness of phytoplasmas in papaya

The genetic relatedness of phytoplasmas associated with dieback (PDB), yellow crinkle (PYC) and mosaic (PM) diseases in papaya was studied by restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene and 16S rRNA/23S rRNA spacer region (SR). RFLP and SR sequence comparisons indicated that PYC and PM phytoplasmas were identical and most closely related to members of the faba bean phyllody strain cluster. By comparison the PDB phytoplasma was most closely related to Phormium yellow leaf (PYL) phytoplasma from New Zealand and the Australian grapevine yellows (AGY) phytoplasma from Australia. These three phytoplasmas cluster with the stolbur and German grapevine yellows (VK) phytoplasmas within the aster yellows strain cluster. Primers based on the phytoplasma tuf gene, which amplify gene products from members of the AY strain cluster, also amplified a DNA product from the PDB phytoplasma but not from either the PYC or PM phytoplasmas. Primers deduced from the 16S rRNA/SR selectively amplified rDNA sequences from the PDB and AGY phytoplasmas but not from other members of the stolbur strain cluster. Similarly, primers designed from 16S rRNA/SR amplified rDNA from the PYC and PM phytoplasmas but not from the PDB phytoplasma. These primers may provide for more specific detection of these pathogens in epidemiological studies.     

Identification of a phytoplasma causing yellows of Monarda

Monarda yellows occurring in southern Alberta was found to be associated with a phytoplasma. Using two pairs of universal primers, 16S ribosomal DNA fragments (about 1.5 and 1.2 kb) were amplified separately by polymerase chain reaction (PCR) from DNA samples that had been extracted from infected monarda. No such DNA bands were observed using DNA samples from uninfected monarda. The DNA fragment (1.2 kb) amplified by nested-PCR was analysed and compared with western aster yellows (AY27, Canada), eastern aster yellows (EAY, USA), French hydrangea aster yellows (AYHF), Belgium hydrangea aster yellows (AYHB), clover proliferation (CP, Canada) and potato witches'-broom (PWB, Canada) by means of restriction fragment length polymorphism (RFLP) using endonucleases Alu I, Mse I, Hpa II, Sau 3AI, Kpn I and Rsa I. The results showed that monarda yellows phytoplasma belongs to the aster yellows subclade and is different from CP and PWB. This is the first report of aster yellows phytoplasma infecting monarda.