Macropsis mendax as a vector of elm yellows phytoplasma of Ulmus species

A 3-year study was carried out in north-east Italy, the site of recent elm yellows epidemics, to identify vectors for the elm yellows phytoplasma. Using PCR analysis, Ulmus minor and Ulmus pumila , each with and without symptoms, were positive for the elm yellows phytoplasma. Macropsis mendax , a univoltine and monophagous leafhopper, was shown to be the vector of the elm yellows-associated disease agent. PCR analyses demonstrated that the insect was infected both in natural conditions and in the screenhouse after acquisition-feeding on infected elm plants. Groups of M. mendax , collected from naturally infected elm trees, transmitted elm yellows phytoplasma to elm test plants. In nature, Alnus glutinosa trees affected by alder yellows were found in the surroundings of yellows-affected elm trees; the associated disease agent of alder yellows was transmitted under controlled conditions from alder to elm test plants by grafting.     

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.     

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.     

Association of phytoplasmas with the decline of European hazel in southern Italy

In the Campania region of southern ltaly. commercial orchards of European hazel ( Corylus avellana ) are severely affected by yellowing and decline. To determine whether phytoplasmas are associated with the disorder, stem samples from diseased trees were examined using polymerase chain reaction assays. No visible products were obtained by amplification of sample DNA with universal and group-specific phytoplasma primers. However, when the products obtained with universal primers were re-amplified with nested primers that were specific for the fruit tree phytoplasmas of the apple proliferation group, most samples tested positively. Restriction site analysis revealed that the trees were infected with the apple proliferation, pear decline, and European stone fruit yellows phytoplasmas in about the same proportion. Some of the trees were doubly infected with one of the fruit tree phytoplasmas and the aster yellows agent. Most of the infected trees were also identified by hybridization of the products obtained in the initial amplification with suitable oligonucleotide probes.     

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.     

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.     

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 identification of phytoplasmas in yellows-diseased weeds in Italy

Yellows-diseased plants of Crepis setosa (hawksbeard), Knautia arvensis (field scabious), Convolvulus arvensis (field bindweed), Picris echioides (bristly oxtongue), Echium vulgare (blueweed) and Calendula officinalis (pot marigold) collected in central and southern Italy were examined for phytoplasma infection by means of polymerase chain reaction (PCR) technology using universal phytoplasma primers directed to ribosomal sequences. The detected phytoplasmas were characterized and differentiated using restriction fragment length polymorphism analysis of PCR-amplified DNA. The phytoplasma detected in diseased pot marigold plants was identified as a member of the aster yellows group and proved indistinguishable from a strain of the American aster yellows phytoplasma. The phytoplasma identified in diseased field bindweed plants is a putative new type of the stolbur group that differed from the typical stolbur phytoplasma. Phytoplasmas detected in diseased hawksbeard, blueweed and field scabious plants are all putative new members of the sugarcane white leaf group while the phytoplasma detected in diseased bristly oxtongue plants represents a new member of the faba bean phyllody group. For hawksbeard and field scabious this is the first report on the occurrence of phytoplasma diseases, whereas phytoplasmas infecting bristly oxtongue and blueweed have never been characterized before.     

人工培养液在植原体昆虫介体筛选中的适用性

研究了一种人工培养液对各种常见的昆虫(主要是叶蝉)的亲和性和适用性.结果表明,该人工培养液适于本试验中大多数昆虫的人工饲养.用此方法,悬钩子广头叶蝉Macropsis.ftscula Zetterstedt和桤树广头叶蝉Oncopsis alniSchrank分别被再次确认为悬钩子矮化植原体和桤树黄化植原体的传播介体;田旋花麦蜡蝉Hyalesthes obsoletus Signoret再次被确认为葡萄黄化(stolbur)植原体的传播介体.此前,上述三种叶蝉已被传统的人工接种方法鉴定为相应植原体的传播介体.危害桤树的河谷树叶蝉Allygus modestus Scott尽管虫体DNA检测结果经常为阳性,但迄今其人工培养液的检测结果都是阴性,因此,我们认为河谷树叶蝉不是桤树黄化植原体的传播介体.Eppendorf管人工培养液饲养法不仅适用于潜在的植原体介体昆虫的筛选鉴定,而且可用于介体昆虫的生物防治研究.此外,本研究首次发现自然感染了葡萄上的一种被德国人称为"Vergi-lungskrankheit"植原体(AY组)的草地脊冠叶蝉Aprodes makarovi Zachvatkin.     

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.     

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.     

Use of heteroduplex mobility assay for identification and differentiation of phytoplasmas in the aster yellows group and the clover proliferation group

ABSTRACT This paper describes the identification and differentiation of phytoplasmas by a highly sensitive diagnostic technique, DNA heteroduplex mobility assay (HMA). Closely related phytoplasma isolates of clover proliferation (CP), potato witches'-broom (PWB), and alfalfa witches'-broom (AWB) were collected from the field from 1990 to 1999. The entire 16S rRNA gene and 16/23S spacer region were amplified by polymerase chain reaction (PCR) from the field samples and standard CP, PWB, and AWB phytoplasmas and were subjected to restriction fragment length polymorphism (RFLP) analysis and HMA. Two subgroups (I and II) of phytoplasmas in the CP group were identified by HMA but not by RFLP analysis. The results were confirmed by 16/23S spacer region sequence data analysis. After HMA analyses of the PCR-amplified 16/23S spacer region, 14 phytoplasma isolates from field samples were classified into two aster yellows subgroups: subgroup I, phytoplasma isolates from China aster (Callistephus chinensis) yellows, French marigold (Tagetes patula) yellows, cosmos (Cosmos bipinnatus cv. Dazzler) yellows, clarkia (Clarkia unguiculata) yellows, California poppy (Eschscholzia californica cv. Tai Silk) yellows, monarda (Monarda fistulosa) yellows, and strawflower (Helichrysum bracteatum) yellows; and subgroup II, phytoplasma isolates from zinnia (Zinnia elegans cv. Dahlia Flower) yellows, Queen-Annes-Lace (Daucus carota) yellows, scabiosa (Scabiosa atropurpurea cv. Giant Imperial) yellows, Swan River daisy (Brachycombe multifida cv. Misty Pink) yellows, pot marigold (Calendula officinalis) yellows, purple coneflower (Echinacea purpurea) yellows, and feverfew (Chrysanthemum parthenium) yellows. The results indicate that HMA is a simple, rapid, highly sensitive and accurate method not only for identifying and classifying phytoplasmas but also for studying the molecular epidemiology of phytoplasmas.     

Molecular identification of a phytoplasma associated with Russian olive witches’ broom in Iran

Russian olive trees (Elaeagnus angustifolia) showing witches’ broom symptoms typical of phytoplasma infection were observed in the Urmia region of Iran. A phytoplasma named Russian olive witches’ broom phytoplasma (ROWBp-U) was detected from all symptomatic samples by amplification of the 16S rRNA gene and 16S/23S rDNA spacer region using the polymerase chain reaction (PCR) which gave a product of expected length. DNA from symptomless plants used as a negative control yielded no product. The sequence of the 16S rRNA gene and 16S/23S rDNA spacer region of ROWBp-U showed 99% similarity with the homologous genes of members of the aster yellows group. We also detected a phytoplasma in neighboring alfalfa plants (AlWBp-U) showing severe witches’ broom symptoms. An 1107 bp PCR product from the 16S rRNA gene showed 99% homology with the corresponding product in ROWBp-U, suggesting the presence of the same phytoplasma actively vectored in the area. Further observations showed that Russian olive trees with typical ROWB symptoms were present in an orchard near Tehran which is located over 530 km south-east of the original Urmia site. The corresponding sequence of this phytoplasma (ROWBp-T) showed 99% homology to that of the ROWBp-U. A sequence homology study based on the 16S rRNA gene and 16S/23S rDNA spacer region of ROWBp-U and other phytoplasmas showed that ROWBp-U is most closely related to the 16SrI group. To our knowledge, this is the first report of a phytoplasma infection in a member of the Elaeagnaceae.     

菊花绿变病植原体在中国的发生及分子鉴定

对内蒙古农业大学校园内表现花器绿变症状的菊花样品进行采集和DNA提取,应用植原体16S rRNA基因和rp基因的引物进行巢式PCR扩增,从感病样品中分别扩增得到了长度均约为1.2 kb的片段。序列一致性分析表明,菊花绿变植原体16S rRNA基因与翠菊黄化植原体匈牙利风信子株系(GenBank登录号MN080271)、印度玉米株系(KY565571)、印度繁缕株系(KC623537)和印度马铃薯株系(KC312703)的核酸一致性最高,为99.9%,rp基因序列与翠菊黄化植原体立陶宛洋葱株系(GU228514)的核酸一致性最高,为99.8%。基于16S rRNA基因和rp基因构建系统进化树时发现,菊花绿变植原体均与16SrI-B亚组成员聚为一起。16S rRNA基因相似性系数分析表明,菊花绿变植原体与洋葱黄化植原体(AP006628)的相似性系数最高为1.00,洋葱黄化植原体(AP006628)在分类上属于16SrI-B亚组。因此,我们可以确定该菊花绿变植原体属于16SrI-B亚组。这是我国首次报道菊花绿变病的发生。     

Distinctions between phytoplasmas at the subgroup level detected by heteroduplex mobility assay

A total of 62 phytoplasma isolates were collected from North America, Europe and Asia and analysed by heteroduplex mobility assay (HMA) of the 16/23S spacer region amplified by the polymerase chain reaction. The results revealed wide genetic diversity among the phytoplasmas studied and a number of new phytoplasma strains were identified from known or new plant hosts in Alberta, Canada. Two distinctive subgroups were revealed by HMA in phytoplasmas associated with canola yellows, Chinese aster yellows, dandelion yellows and monarda yellows. In Alberta, two subgroups of the aster yellows group of phytoplasmas, I-A and I-B, were prevalent in naturally infected field crops and ornamentals in open gardens. The results indicated that HMA is a simple, but rapid and accurate, alternative method for the detection and estimation of genetic divergence of phytoplasmas when finer molecular characterization of phytoplasmas is required at the subgroup level.     

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.     

Chromosome sizes of phytoplasmas composing major phylogenetic groups and subgroups

ABSTRACT Chromosome sizes of 71 phytoplasmas belonging to 12 major phylogenetic groups including several of the aster yellows subgroups were estimated from electrophoretic mobilities of full-length chromosomes in pulsed-field gels. Considerable variation in genome size, from 660 to 1,130 kilobases (kb), was observed among aster yellows phytoplasmas. Chromosome size heterogeneity was also observed in the stolbur phytoplasma group (range 860 to 1,350 kb); in this group, isolate STOLF contains the largest chromosome found in a phytoplasma to date. A wide range of chromosome sizes, from 670 to 1,075 kb, was also identified in the X-disease group. The other phytoplasmas examined, which included members of the apple proliferation, Italian alfalfa witches' broom, faba bean phyllody, pigeon pea witches' broom, sugarcane white leaf, Bermuda grass white leaf, ash yellows, clover proliferation, and elm yellows groups, all have chromosomes smaller than 1 megabase, and the size ranges within each of these groups is narrower than in the aster yellows, stolbur, and X-disease groups. The smallest chromosome, approximately 530 kb, was found in two Bermuda grass white leaf phytoplasma isolates. This not only is the smallest mollicute chromosome found to date, but also is the smallest chromosome known for any cell. More than one large DNA band was observed in several phytoplasma preparations. Possible explanations for the occurrence of more than one band may be infection of the host plant by different phytoplasmas, the presence of more than one chromosome in the same organism, or the presence of large extrachromosomal DNA elements.     

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.     

Comparison of phytoplasmas infecting winter oilseed rape in the Czech Republic with Italian Brassica phytoplasmas and their relationship to the aster yellows group

Winter oilseed rape grown in several areas in South Bohemia showed symptoms of stunting, leaf reddening and extensive malformation of floral parts. Phytoplasmas were consistently observed by using electron microscopy only in phloem tissue of symptomatic plants. DNA isolated from infected and healthy control plants was used in PCR experiments. Primer pairs R16F2/R2, P1/P7 and rpF2/R2, amplifying, respectively, 16S rDNA, 16S rDNA plus spacer region and the beginning of the 23S and ribosomal protein gene L22 specific for phytoplasmas, were used. According to RFLP and sequence analyses of PCR products, the phytoplasma from rape was classified in the aster yellows phytoplasma group, subgroup 16SrI-B. The PCR products from the Czech phytoplasma-infected rape also had RFLP profiles identical to those of phytoplasma strains from Italian Brassica . This first molecular characterization of phytoplasmas infecting rape compared with strains from Brassica does not, however, clearly indicate differences among isolates of the same 16SrI-B subgroup. Further studies on other chromosomal DNA portions could help the research on host specificity or on geographical distribution of these phytoplasmas.     

Electron microscopy and molecular identification of phytoplasmas associated with strawberry green petals in the Czech Republic

The presence of phytoplasma inFragaria ananassa x Duch cv Senga Sengana showing strawberry green petals symptoms was observed by electron microscopy of phloem tissue. No phytoplasmas were found in asymptomatic strawberry plants used as controls. Nucleic acids extracted from these plants were used in nested-PCR assays with primers amplifying 16S rRNA sequences specifie for phytoplasmas. Bands of 1.2 kb were obtained and the subsequent nested-PCR with specific primers and RFLP analyses allowed to classify the detected phytoplasmas in the aster yellows group (16SrI). They belonged to the subgroup I-C of which type strain is clover phyllody phytoplasma.