Oncopsis alni (Schrank) (Auchenorrhyncha: Cicadellidae) as a Vector of the Alder Yellows Phytoplasma of Alnus glutinosa (L.) Gaertn.

Alder yellows phytoplasma was detected by PCR in Alnus glutinosa trees in the Palatine and Mosel areas of Germany. The restriction profiles obtained by TaqI and AluI digestion of a PCR amplified ribosomal DNA fragment from this phytoplasma and a periwinkle isolate of alder yellows from Italy (ALY) could not be distinguished while elm yellows isolates from Europe and North America led to different fragment patterns. Different restriction profiles for ALY and the German alder phytoplasma were obtained by TruI digestion of a non-ribosomal DNA fragment. Phloem feeding insects were collected from infected alder trees. Phytoplasmas of the elm-yellows group were detected by PCR in psyllids and the leafhopper Oncopsis alni. These pathogens were indistinguishable from the phytoplasma found in alder. Only O. alni was able to transmit the pathogen to healthy alder seedlings. Thus, it is the first insect known to transmit this phytoplasma. This leafhopper could be responsible for the ubiquitous infection of Alnus glutinosa due to its close association with alder and its wide distribution in Europe.     

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.     

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.     

海南长春花黄化病植原体的16S rDNA序列分析研究

 Periwinkle(Catharanthus roseus) yellows is a common disease in Hainan. Periwinkle's leaf tissue with symptoms was assayed for phytoplasma infection by using PCR assay employing phytoplasma universal 16S rRNA gene primers (Rl6mF2/Rl6mR1). A PCR product (about 1.4 kb) was amplified from periwinkle showed yellows. Nucleotide sequencing and phylogenetic tree analysis showed that the amplified 16S rDNA contained 1 432 nucleotides, the most homology was 98.1% with the members of elm yellows group (16S r Ⅴ) and clustered in the same clade, while it was under 96.1% with other phytoplasma groups. Our results suggested that the phytoplasma sample belonged to 16S rⅤgroup and was tentatively named as Hainan periwinkle yellows phytoplasma (PY-Hn). This is the first report of existence of 16S r Ⅴ group phytoplasma in naturally infected periwinkle.     

An Antibody Against the SecA Membrane Protein of One Phytoplasma Reacts with Those of Phylogenetically Different Phytoplasmas

ABSTRACT Antisera raised against phloem-limited phytoplasmas generally react only with the phytoplasma strain used to produce the antigen. There is a need for an antiserum that reacts with a variety of phytoplasmas. Here, we show that an antiserum raised against the SecA membrane protein of onion yellows phytoplasma, which belongs to the aster yellows 16S-group, detected eight phytoplasma strains from four distinct 16S-groups (aster yellows, western X, rice yellow dwarf, and elm yellows). In immunoblots, approximately 96-kDa SecA protein was detected in plants infected with each of the eight phytoplasmas. Immunohistochemical staining of thin sections prepared from infected plants was localized in phloem tissues. This antiserum should be useful in the detection and histopathological analysis of a wide range of phytoplasmas.     

Phylogenetic relationships among Flavescence dorée strains and related phytoplasmas determined by heteroduplex mobility assay and sequence of ribosomal and Nonribosomal DNA

Heteroduplex mobility assay (HMA) and DNA sequencing were performed on Flavescence dorée (FD) phytoplasma strains and related phytoplasmas belonging to the elm yellows group. Part of the ribosomal RNA gene operon and a nonribosomal DNA region were utilized for phylogenetic analyses. Two FD strains, FD92 and FD-D, detected in France and Italy, respectively, were identical in both DNA fragments, confirming previous results. Other FD strains were all very similar and most closely resembled ALY, an Italian alder phytoplasma. Phytoplasmas associated with German Palatinate grapevine yellows were shown to form a distinct subcluster, also different from the elm yellows phytoplasma subcluster. Strain disparities revealed by HMA and sequence data were mostly in agreement, highlighting the utility of HMA in differentiation and classification of phytoplasmas belonging to the same ribosomal RNA group.     

Nature and genetic relatedness of the mycoplasma-like organism causing rubus stunt in Europe

Cultivated red raspberries ( Rubus idaeus ), and wild blackberries ( R fruticosus, R. caesius , and Rubus hybrids) showing symptoms of rubus stunt were collected in Germany, France, and Italy, Ribosomal DNA of the mycoplasma-like organism (MLO) that causes rubus stunt was amplified by a polymerase chain reaction procedure and then digested with Atu I and Rsa I restriction endonucleases. All samples examined showed the same restriction profiles, which were similar to those of the MLOs inducing elm yellows and alder yellows. However, the rubus stunt MLO could be distinguished from the elm and alder MLOs by Southern blot analysis using DNA probes from a strain of the elm yellows MLO, A variability of the hybridization profiles, probably caused by restriction fragment length polymorphisms, was observed among the rubus stunt samples. From both rDNA restriction site and Southern blot analyses it can be concluded that the rubus stunt agent and the elm yellows and alder yellows MLOs are not identical but closely related and can be grouped in the same taxonomic cluster.     

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 .     

Role of wild Prunus species in the epidemiology of European stone fruit yellows

Several uncultivated trees of the species Prunus spinosa , P. cerasifera and P. domestica , sampled both adjacent to European stone fruit yellows (ESFY)-infected orchards and in isolation from cultivated stone fruit plants, were found to be infected by ESFY phytoplasma. These species were also colonized by Cacopsylla pruni , vector of the ESFY agent. In contrast, uncultivated species of Prunus avium , P. cerasus and P. mahaleb hosted neither the pathogen nor the vector. Insect- and graft-transmission trials of ESFY phytoplasma conducted under controlled conditions confirmed the data obtained in the field. The role played by the wild Prunus species is discussed and appears to be fundamental in the epidemic cycle of the disease.     

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.     

DNA probes and PCR primers for the detection of a phytoplasma associated with peanut witches'-broom

EcoRI restriction fragments of genomic DNA from the phytoplasma associated with peanut witches'-broom (PNWB) were cloned in plasmid pGEM-3Zf(+). Cloned inserts from seven PNWB-phytoplasma-specific recombinant plasmids and two subcloned plasmids were excised with restriction enzymes, labeled with digoxigenin, and used as probes. Probe PNWB281 and its derivative subclones PNWB281-4 and PNWB281-5 hybridized with DNA from PNWB-phytoplasma infected peanut and periwinkle specifically but not with DNA from healthy plants or plants infected with phytoplasmas associated with sweetpotato witches'-broom (SPWB), loofah, Ipomoea obscura, and paulownia witches'-broom, elm and aster yellows, rice yellow dwarf, and bamboo little leaf disease. Six other probes hybridized with DNA derived from PNWB and SPWB-phytoplasma-affected periwinkle but not with DNA from healthy plants or plants infected with other phytoplasmas mentioned. In Southern hybridizations, four of the nine cloned and subcloned probes could differentiate the PNWB-phytoplasma from SPWB-phytoplasma. Three primer pairs for PCR were synthesized according to the partial sequences at both ends of the cloned inserts and were able to distinguish PNWB-phytoplasma from SPWB-phytoplasma by using PCR for the first time. A minimum of 1 pg and 10 pg of total DNA from diseased periwinkle and peanut, respectively, was sufficient to amplify the specific PNWB-phytoplasma PCR fragments, allowing the detection of PNWB-phytoplasma DNA from healthy-looking periwinkle plants two weeks after graft inoculation.     

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 .     

Transmission of Rhus (Rhus javanica L.) Yellows by Hishimonus sellatus and Host Range of the Causal Phytoplasma

In 1998, rhus (Rhus javanica L.) yellows (RhY), caused by phytoplasma, was found in Miyagi Prefecture, Japan. In vector transmission tests, Hishimonus sellatus acquired RhY phytoplasma from diseased R. javanica and transmitted it to healthy R. javanica. Twenty-two species of herbaceous plants in 10 families were infected with RhY phytoplasma by H. sellatus. The host range and main symptoms on test plants of RhY phytoplasma differed from those of Macrosteles striifrons-transmitted phytoplasmas, which belong to the same 16Sr I group phytoplasma. Received 6 December 1999/ Accepted in revised form 14 May 2000     

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.     

Latent infection of asymptomatic Hawaiian sugarcane cultivars with 16SrI and 16SrXI phytoplasmas

Leaves from sugarcane were collected at the Hawaiian sugarcane breeding station and from recent and previous Hawaiian plantation fields and tested for phytoplasma by nested PCR, quantitative PCR and partly by the 16S/23S internal spacer sequence. Phytoplasmas were found in samples of 10 of the 11 tested cultivars from the station and identified as strains 16SrI phytoplasma (aster yellows phytoplasma) and 16SrXI phytoplasma (rice yellow dwarf phytoplasma). Hot water treatment could partially eliminate the phytoplasmas, but sugarcane plants in the Hawaiian plantations, which routinely use hot-water-treated seed cane cuttings, were nevertheless infected by 16SrXI phytoplasma. Samples from abandoned sugarcane plantations contained 16SrI phytoplasma or 16SrXI phytoplasma. The titre of phytoplasma was very low in all cases, i.e., at or below the detection threshold of quantitative PCR, and no difference in phytoplasma infection was observed between healthy-looking, green plants and plants that had YLS symptoms. Apparently the Hawaiian sugarcane cultivars have some kind of phytoplasma resistance under the growth conditions in Hawaii. The latent presence of phytoplasma strains calls for awareness and rigorous treatment of sugarcane setts even in cases, where YLS was so far exclusively related to the presence of Sugarcane yellow leaf virus.     

Detection and identification of a phytoplasma from lucerne with Australian lucerne yellows disease

Foliar and root symptoms are described for Australian lucerne yellows (ALuY), a disease common in Australian lucerne seed crops. A phytoplasma was detected in plants exhibiting symptoms, but not in symptomless lucerne plants. Oligonucleotide primers specific to the phytoplasma 16S-23S rRNA intergenic spacer region (SR) were used in polymerase chain reaction (PCR) assays on DNA extracted from lucerne plants with and without symptoms. Identical restriction fragment length polymorphism (RFLP) enzyme profiles were obtained for PCR products amplified from 10 yellows-affected lucerne samples. RFLP profiles obtained for four restriction enzymes were different from those of the tomato big bud (TBB) phytoplasma. ALuY phytoplasma PCR products were sequenced to determine phylogeny and were found to fall within the faba bean phyllody phytoplasma group, or phytoplasma group 16srII. Transmission electron microscopy revealed phytoplasmas in the phloem of yellows-affected plant samples, but not in symptomless plant samples. Fungal, bacterial and viral agents in the aetiology of Australian lucerne yellows were ruled out.     

Molecular identification of a Candidatus phytoplasma (Group16SrV-D) coding partial uvrB gene and degV gene on a new host – mesta (Hibiscus sabdariffa) – with phyllody and reddening of leaves in India

Mesta (Hibiscus sabdariffa) is an important bast fiber crop. In August 2011, there was an outbreak of a phytoplasma-like disease on H. sabdariffa in different villages of the northern coastal mesta-growing region of Andhra Pradesh, India, covering mainly two districts – Srikakulam and Vijayanagaram. The infected plants showed characteristic symptoms such as phyllody and reddening of leaves. PCR with P1/P7 universal primer pair of 16 S rDNA yielded amplicons of 1850 bp from all symptomatic mesta leaf samples similar to samples of brinjal little leaf (phytoplasma positive reference control). However, asymptomatic samples were not amplified. Multiplex nested-PCR showed simultaneous amplification of DNA fragments with phytoplasma specific primers, viz., P1/P7 universal primer pair of 16 S rDNA, nested primer pair R16F2n/R2, uvrB and DegV gene-specific uvrB-degVF/R primer generating amplicons of 1850 bp, 1200 bp and 1023bp, respectively. However, SecY-map gene specific primer SecY-mapF/R was not amplified. The 1023 bp nucleotide sequence of uvrB and DegV gene of the phytoplasma was deposited in the GenBank (NCBI) with the accession no. JX975061. NCBI BLASTn analysis of the 1023 bp products showed that the phytoplasma strain belonged to elm yellows group (16SrV-D). This is the first report that Hibiscus sabdariffa is infected by a phytoplasma and we named it mesta phyllody disease (MPD).     

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.     

榆树黄化病植原体的分子检测与鉴定

 利用植原体16SrRNA基因的通用引物R16rrLF2/R16mR1和R16F2n/R16R2对山东泰山上发生的榆树(Ulmus parvifolia)黄化病感病植株总DNA进行巢式PCR扩增,得到了约1.2kb的特异性片段,从分子水平证实了榆树黄化病的病原(EY-China)为植原体。将扩增到的片段测序,并进行一致性和系统进化树分析。结果表明,该分离物属于植原体榆树黄化组(Candidatus Phytoplasma ulmi),与该组成员16SrRNA序列的一致性均在98.2%以上,其中与16SrV-B亚组中的纸桑丛枝(Paper mulberry wiches'-broom)和枣疯病(Jujube witches'-broom)植原体一致性最高,达到99.4%,在系统进化树中与该亚组成员聚类到同一个分支,说明该分离物属于植原体16SrV-B亚组。本研究首次对在中国引致榆树黄化病的植原体进行了分子检测,并通过核酸序列分析将其鉴定到亚组水平。