16SrII Phytoplasma Research Articles

Use of a real‐time LAMP isothermal assay for detecting 16SrII and XII phytoplasmas in fruit and weeds of the Ethiopian Rift Valley

A study to investigate the association of phytoplasmas with papaya dieback and citrus decline syndromes in Ethiopia was carried out between July 2009 and February 2010, with sampling performed in major papaya‐ and citrus‐growing areas of the Rift Valley. Samples of plants with symptoms were collected from papaya, citrus and suspected phytoplasma weed hosts and crops in and around the papaya and citrus fields studied. Nested polymerase chain reaction (nested‐PCR) was used for initial characterization, using primers that amplify regions of the 16S rRNA and secA genes, and results were then confirmed with rapid real‐time group‐specific LAMP (loop‐mediated isothermal amplification) assays. The results identified the occurrence of a phytoplasma belonging to the stolbur (16SrXII‐A) group in papaya plants showing dieback symptoms, whilst no phytoplasmas were found associated with citrus decline. These results contradict previous reports that a 16SrII phytoplasma was associated with both papaya dieback and citrus decline in Ethiopia, but correspond with the association of a 16SrXII phytoplasmas with Nivum‐Haamir‐Dieback of papaya in Israel and papaya dieback in Australia. No 16SrXII phytoplasmas were found in any of the weeds and potential alternative hosts studied, although 16SrII phytoplasmas were consistently found in Parthenium hysterophorus weed plants. These results indicate that a 16SrXII phytoplasma is associated with papaya dieback in Ethiopia, whilst the causal agent of citrus decline is not a phytoplasma and remains unidentified.

First Report of Crotalaria spectabilis Fasciation Associated with 'Candidatus Phytoplasma asteris' in India.

Crotalaria spectabilis Roth. (Fabaceae), commonly known as showy rattlebox, is an herbaceous legume mainly used as a green manure crop to improve soil properties and as a source of durable fiber. However, the plant is toxic to mammals and birds because of the presence of pyrrolidizine alkaloids. A native of India and the Malay Peninsula, the species has been introduced into other areas such as the United States and Pacific Islands where the plant is an invader of cultivated lands. Fasciated rattlebox plants were sighted in fields in New Delhi in February 2010, with approximately 99% of the examined plants symptomatic. Symptoms included flattening of stems in a descending gradient of severity from the apex to the base of each affected branch. Shoots showed longitudinal undulations bearing highly reduced leaves and uneven distribution of flowers and fruits. To identify the causal agent, 10 symptomatic plants and 8 asymptomatic plants (latter sampled from a field approximately 1.5 km away) were collected for nucleic acid analysis. Total genomic DNA was extracted from flattened stems as well as the roots of symptomatic plants, and from the same tissues of asymptomatic plants, and subjected to nested-PCR using phytoplasma 16S ribosomal DNA universal primer pair P1/P7, followed by R16F2/R2 (4). A known aster yellows-infected Catharanthus roseus plant was used as a control sample. Results depicted a characteristic phytoplasma amplicon of 1.25 kb in all samples from symptomatic plants and the control plant. No amplification was observed from asymptomatic plants. To obtain a full-length sequence, a representative amplicon was purified with the QIAquick gel extraction kit (QIAGEN, Valencia, CA), cloned into pGEM-T Easy vector (Promega, Madison, WI), and sequenced. Comparison of the 1,243-bp sequence (Genbank Accession No. HM137557) using BLAST analysis of the NCBI database showed 99% homology with sequences of members of 16SrI group phytoplasmas, i.e., 'Candidatus Phytoplasma asteris' such as Japanese spurge yellows (AB551736.1), Mulberry yellow dwarf (GQ249410.1), and Bamboo witches'-broom (FJ853161.1) phytoplasmas. The profiles of in vitro restriction fragment length polymorphism (RFLP) analysis obtained by digestion of the nested-PCR products with HhaI, KpnI, and AluI were similar to those of in silico RFLP analyses and coincided with the pattern of the 16SrI-B subgroup. Phylogenetic analysis of phytoplasma 16S rDNA sequences based on the maximum likelihood method using MEGA Version 4.1 also placed the Crotalaria fasciation (CF) phytoplasma within the 16SrI-B cluster. In India, C. tetragona plants infected with 16SrI phytoplasma (FJ185141) causing witches'-broom symptoms (1) showed 98% similarity with the CF phytoplasma. However, the results support previous molecular investigations associating 16SrI phytoplasma with fasciation of herbaceous plants, including Lilium spp. (2) and Asparagus officinalis (3). To our knowledge, C. spectabilis represents a new host that can be fasciated as a result of phytoplasma infection. Because of the weedy nature of C. spectabilis, this host could facilitate spread of the phytoplasma.

Mobile units of DNA in phytoplasma genomes

Phytoplasmas are obligate symbionts of plants and insects that are responsible for significant yield losses in diverse crops. Genome sequencing has revealed that many phytoplasma genomes appear to contain repeated genes organized in units of approximately 20 kb. These 'potential mobile units' (PMUs) resemble composite replicative transposons. PMUs contain several genes for recombination and some also contain putative 'virulence genes'. Genome alignments suggest that PMUs are involved in phytoplasma genome instability and recombination. In this edition of Molecular Microbiology, Hogenhout and colleagues report that one PMU from the aster yellows phytoplasma strain Witches' Broom (AY-WB) can exist as both a linear PMU within the chromosome and as an extrachromosomal circular form. The copy number of the circular form is much higher in the insect vector compared with the plant, and expression levels of genes present on the PMU are also higher in the insect. These observations suggest not only that this PMU could be a mobile element, but that it could also be involved in a phase-variation mechanism that allows the phytoplasma to adapt to its different hosts.

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.

The phytoplasma disease ‘mung bean phyllody’ is now present in Pakistan

Mung bean is an important source of food for humans and animals in tropical and subtropical countries, and is also used for green manure. During spring 2008, mung bean plants in Faisalabad, Pakistan, showed severe symptoms of phyllody, virescence and a bushy appearance. Stem, leaf and stalk phloem sections from plants with and without symptoms were examined under a light microscope (CXRII, Labomed, CA, USA) using Dienes’ stain. Regularly distributed dark blue areas were observed only in the phloem cells from plants with symptoms, and not in other plant tissues nor in the phloem of healthy plants, indicating the presence of a phytoplasma. Four week-old greenhouse-potted mung bean plants were grafted, and developed phyllody symptoms 25-30 days after grafting, similar to those observed in the field, whilst no symptoms developed in non-grafted control plants. To determine the 16Sr group of the phytoplasma associated with mung bean phyllody, total DNA was extracted from eight symptom-bearing plants, and amplified by nested PCR using universal 16S rDNA phytoplasma primers, P1 ⁄ P7 followed by R16F2n ⁄ R16R2. No symptomless plants were tested. All samples from plants with symptoms yielded a 1250bp PCR product, and identical RFLP profiles using the enzymes AluI and HpaII. Direct sequencing of the 16S rDNA of one representative PCR amplicon (GenBank Accession No. FJ410489) showed highest sequence identity (99%) with 16SrII ‘Candidatus Phytoplasma aurantifolia’ phytoplasmas, and phylogenetic analysis placed the phytoplasma in subgroup 16SrII-D. Phyllody symptoms of mung bean have been reported previously in India (Lakshmanan et al., 1988), Thailand (Chatchawankanphanich et al., 2000), and Australia (Wilson et al., 2001). In Australia, a 16SrII phytoplasma was associated with mung bean phyllody, and with sesame and peanut diseases (Wilson et al., 2001). Results described here confirm that a 16SrII-D phytoplasma is now present in mung bean in Pakistan.

Flavescence dorée phytoplasma affecting grapevine (Vitis vinifera) newly reported in Portugal

In 2007, during an official Flavescence dorée (FD) survey, typical FD symptoms, associated with desiccation of inflorescences, withering of clusters, irregular maturation of the wood, leaf rolling, typical vein banding, leaf yellowing on white grapevine variety Loureiro and leaf reddening on red variety Vinhão, were observed in vineyards from Amares region in northern Portugal. In 2002, similar but less severe symptoms occurring in the same region were associated with 16SrI phytoplasmas (‘Candidatus Phytoplasma asteris’), subgroup 16SrI-B (De Sousa et al., 2003). Cuttings and leaf samples were collected from plants with and without symptoms. Total DNA was extracted (Angelini et al., 2001) and assayed in a nested polymerase chain reaction (PCR) using primers P1/P7 (Deng & Hiruki, 1991) followed by either R16F2n/R16R2 (F2n/R2) (Gundersen & Lee, 1996) or R16F1(V)/R16V1731r (F1/V1) (Martini et al., 1999). Phytoplasma strains from group 16SrV (‘Ca. Phytoplasma ulmi’), elm yellows (EY1) and rubus stunt (RUS), were used as reference controls. Healthy C. roseus and no template DNA were used as negative controls. All plants with symptoms and reference controls showed PCR products (F2n/R2 and F1/V1), absent from samples from symptomless plants. Two representative F2n/R2 amplicons, were cloned, sequenced and deposited in GenBank (FJ611961 and FJ611962). The phytoplasma 16S rRNA sequences were identical and shared 99-100% identity with those of group 16SrV. FD phytoplasmas from Portugal exhibited the characteristic FD-D TaqI RFLP pattern. This is the first report of FD-D in grapevine in northern Portugal. Although the current FD outbreak is limited to Amares region, the high pressure of the Scaphoideus titanus vector populations imposes a threat for FD spread, and a current alert for the Ministry of Agriculture (DGADR). The implementation of strategies for effective control of S.titanus and FD have been suggested and included in the Portuguese legislation (Portaria nº 976/2008, 1 September). The authors would like to thank regional technicians (Entre-Douro-e-Minho and Trás-Montes) for supplying grapevine samples.

‘Candidatus Phytoplasma asteris’ identified in blackberry (Rubus fruticosus agg.) in the United Kingdom

Blackberries (Rubus fruticosus agg.) also known as brambles are a widespread and well-known aggregate species of several hundred micro-species native throughout the temperate Northern hemisphere. Tolerating poor soils, they are early colonizers of wasteland and building sites and grow vigorously in woods, scrubland, hedgerows and roadside verges. Plants produce many soft fruits notable for their high nutritional content, making them a popular ingredient in desserts, jams and wines. Numerous cultivars have been selected for commercial cultivation. In July 2008, a small patch of R. fruticosus growing by the roadside in Egham, UK, was observed showing unusual witches’ broom symptoms. Proliferations of shoots were seen at the branch nodes, midway along the stem. Symptoms on new growth presented as shortened, narrowed almost leafless branches with reduced, flower production or none at all. No other plants in the near vicinity appeared affected. Total DNA was extracted from plants with symptoms and from healthy looking blackberry plants and used as a template in a nested PCR assay with universal primers P1m (Hren et al., 2007) /P7 (Gundersen & Lee, 1996) and fU5/rU3 (Lorenz et al., 1995). Amplicons of expected size (∼880 bp) were obtained from plants with symptoms. Nested amplicons of a representative plant were purified, cloned (pGEM-T Easy Vector, Promega) and sequenced in both directions using M13 sequencing primers (http://www.dnaseq.co.uk). The phytoplasma 16Sr rDNA sequence (GenBank Accession No. FJ008925) was compared with those held in GenBank using blast. The greatest similarity (99%) was with phytoplasmas of group 16SrI, ‘Candidatus Phytoplasma asteris’, including those associated with gaillardia yellows (EF583066), poa stunt (DQ640502), and festuca yellows (DQ640504). The occurrence of two phytoplasmas belonging to groups 16SrV (‘Ca. P. ulmi’) and 16SrIII (X-disease) have previously been reported in Rubus species in the UK (Davies, 2000). This is the first record of a phytoplasma from 16SrI group recorded from Rubus in the UK. It is not known which insect vectors are associated with transmission.

First Report of Kidney Bean Little Leaf Disease Associated with 16SrII Group Phytoplasma in China.

Kidney bean (Phaseolus vulgaris) is an important cash crop in China. It is widely grown in the Yunnan Province in the southwest region. In November of 2008, a new disease was observed on kidney bean plants grown in Yuanmou County. Affected plants displayed symptoms that included numerous twisted lateral shoots with abundant, tiny trifoliate leaves that were approximately one-tenth the size of healthy leaves. Some affected leaves, which were slightly distorted and oblong to oval, were evident on diseased plants and appeared pale green. On plants with little leaf symptoms, flowers were poorly formed, withered or aborted, and no pods were present. Surveys conducted in areas affected by disease revealed the presence of the disease in approximately 10% of the plants. DNA was extracted from 0.1 g of petioles and midveins harvested from both diseased and symptomless plants with the Qiagen DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Plants were evaluated for phytoplasma infection by a nested PCR assay with phytoplasma specific ribosomal operon primer pair P1/Tint followed by R16F2/R16R2 (2,3). An rDNA product of approximately 1,250 bp was amplified from seven of seven diseased plants, whereas no products were amplified from symptomless plants or a negative control devoid of DNA. Digests of nested PCR products (approximately 200 ng) with endonucleases AluI, ScaI, or EcoRI revealed no differences in restriction fragment length polymorphism (RFLP) among diseased plants. The amplicon was cloned and sequenced (GenBank Accession No. GQ336993). Comparison of in silico RFLP profiles with published profiles showed that kidney bean little leaf phytoplasma is a member of peanut witches' broom group 16SrII. Blast analysis of the kidney bean little leaf phytoplasma 16S rDNA sequence revealed that this strain is most similar (99.0%) to Syringa oblata yellows phytoplasma (Accession No. FJ263629) and to other phytoplasmas classified as group 16SrII members. Previously, phytoplasmas identified as 16SrII strains have been reported as probable cause of cactus witches' broom (1) and crotalaria witches' broom (4) in China. To our knowledge, this is the first report of a 16SrII phytoplasma infecting the kidney bean in China.

First report of phytoplasmas affecting organoponic crops in central and eastern Cuba

The organic and hydroponic (‘organoponic’) system of intensive crop production in Cuba is used to supply the increasing urban and peri-urban demands. Surveys of organoponics in Matanzas, Las Tunas and Stgo de Cuba provinces revealed the occurrence of phytoplasma diseases with a 20–30% incidence, whose patchy distribution suggested an insect vector role in disease spread. Symptoms were observed in cabbage (Brassica oleracea), beetroot (Beta vulgaris) and cassava (Manihot esculenta), mostly associated with leaf yellowing, small fruits, and stunting. Radish (Raphanus sativus) plants showed leaf yellowing, mosaic, crinkling, and smaller leaves, and carrot (Daucus carota) plants exhibited yellowing and purpling of leaf tips becoming necrotic progressively with little leaves and shortening of internodes. A total of 32 and 12 plants with and without symptoms respectively were randomly surveyed for total DNA extraction and indexing in a nested PCR with generic phytoplasma 16S rRNA primers R16mF2/R1 and fU5/rU3. Nested PCR amplicons (~880 bp) were obtained for 26 of the 32 affected plant samples surveyed, with no amplicons from the symptomless plants. PCR products were purified, cloned (pGEMT-Easy Vector, Promega), and sequenced (http://www.dnaseq.co.uk). All samples showed Sau3AI, HpaII and HaeIII RFLP patterns identical to the 16SrI-C reference control ‘Candidatus Phytoplasma asteris’, except those from radish that were identical to the 16SrII-A reference control ‘Ca. Phytoplasma aurantifolia’. The phytoplasma 16S rRNA sequences scored 99% identity for carrot (GenBank Accession No. EU328254), cabbage (EU328252), beetroot (EU328251), and cassava (EU328256) with those of members of group 16SrI; and for the radish phytoplasma (EU328253) with members of group 16SrII. Both groups 16SrI and 16SrII occur in Cuba (Arocha et al., 2006a,b). However, this is the first report of both a 16SrI phytoplasma affecting carrot, cabbage, beetroot and cassava, as well as a 16SrII phytoplasma in radish, in organoponics in central and eastern Cuba, and raises concerns about the spread of phytoplasmas to crops cultivated under organoponic conditions. Work in the UK was done under Defra licence No. PHF 174D/5185(08/2005). Rothamsted Research receives grants from BBSRC in the UK.

‘Candidatus Phytoplasma asteris’ in Fraxinus excelsior and its association with ash yellows newly reported in Poland

Symptoms of growth abnormalities including shoot proliferation, leaf rosetting and malformation of two-to-three year-old ash trees (Fraxinus excelsior) used as rootstock, as well as on plants of cv. Aurea, were observed in a commercial nursery in Poland. Symptoms were similar to ash yellows disease associated with phytoplasmas of Group 16SrVII, ‘Candidatus Phytoplasma fraxini’ in the USA (Griffiths et al., 1999). Nucleic acids were extracted from shoot phloem of three trees with and two without symptoms, and healthy Catharanthus roseus plants, using a DNeasy Plant Mini Kit (Qiagen). In nested PCR the phytoplasma universal primers P1/P7 were used, followed by fA/rA, R16F2n/R16R2 or the group-16SrI (R16(I)F1/R16(I)R1) specific primers. Restriction fragment length polymorphism (RFLP) analysis of R16F2n/R16R2 PCR products was done using the endonucleases HhaI, MseI or RsaI. PCR-amplified phytoplasma 16S rRNA fragments from two F. excelsior (J1, J2) were purified, sequenced, and sequences analyzed using BLAST (http://www.ncbi.nlm.nih.gov) and Lasergene v. 7·1 (DNASTAR). Phytoplasma rDNA was amplified by nested PCR with fA/rA, R16F2n/R16R2 and R16(I)F1/R16(I)R1 primers from two out of three tested ash trees with disease symptoms, but not from the other tree with symptoms, nor the symptomless ash trees or healthy C. roseus. RFLP profiles indicated that ash trees were infected with a 16SrI phytoplasma (‘Ca. Phytoplasma asteris’). Sequence analysis for J2 (GenBank Accession No. EU219611) and J1 (EU219612) confirmed PCR-RFLP results, and showed a 99·7% of identity to each other, and more than 98% with those of phytoplasma members of 16SrI-B sub-group. Phylogeny analyses of F. excelsior phytoplasma sequences revealed their closest relation to 16SrI group, particularly with that of Accession No. M86340. 16SrI group phytoplasmas seem to be most widespread in Europe and America and they have been reported frequently from woody plants. In Poland they have been found in ornamental crops including magnolias, roses (Kamińska et al., 2001, 2003) and ashleaf maple trees (Kamińska & Śliwa, 2006), associated with symptoms of stunting, shoot proliferation and dieback. This is the first record on the occurrence of ‘Ca. Phytoplasma asteris’ in ash trees.

First Report of ‘CandidatusPhytoplasma asteris’ (Group 16SrI) Infecting Fruits and Vegetables in Islamabad, Pakistan

Nearby fruit and vegetable fields in Islamabad, Pakistan were surveyed for phytoplasma infection. ‘Candidatus Phytoplasma asteris’ (Group 16SrI) was found infecting mango, citrus, loquat, geranium, periwinkle, radish, blackberry and potato. Results suggest that a polyphagous vector may be involved in phytoplasma transmission to these plant species, which are first host records of 16SrI phytoplasma infection in Pakistan.

New plant hosts for group 16SrII, ‘Candidatus Phytoplasma aurantifolia’, in India

Lettuce (Lactuca sativa), carrot (Daucus carota) and French bean (Phaseolus vulgaris) are important staple food crops in India. Symptoms of leaf yellowing, chlorosis, and little leaf were recently observed in these species at the Vegetable Research Center, GBPUA&T, Uttarakhand, Pantnagar, India. Plants of Amaranthus sp. growing in adjacent hedges also exhibited leaf yellowing symptoms. Three leaf samples of each plant species with symptoms were collected, including healthy looking plants. Total DNA was extracted and indexed in a nested PCR assay with universal primers R16mF2/R1 and fU5/rU3 that target the phytoplasma 16S rRNA. PCR amplicons (880 bp) were produced in all samples with, but not without symptoms. PCR products were subjected to RFLP analyses with RsaI and HpaII restriction enzymes, and further purified, cloned (pGEM-T Easy Vector, Promega), and sequenced in both directions using M13 forward and reverse sequencing primers (http://www.dnaseq.co.uk). The 16S rRNA sequences were compared with those of reference strains from GenBank using blast, and aligned with clustal W (Thompson et al., 1994) to produce a phylogenetic tree using the neighbour-joining method and 1000 replicates with mega 3·1 (Kumar et al., 2004). RFLP profiles were all similar to those of group 16SrII, ‘CandidatusPhytoplasma aurantifolia’. Sequences of phytoplasmas (GenBank) identified in lettuce (EU362630), carrot (EU362628), French bean (EU362629) and Amaranthus sp. (EU362627) shared 99% identity to each other, and to a member of group 16SrII, ‘CandidatusPhytoplasma aurantifolia’ (EU099570). Phylogeny supported RFLP results and sequence comparisons, and indicates that the Pantnagar phytoplasmas cluster in the 16SrII group branch more closely related to the cactus witches’ broom phytoplasma. ‘CandidatusPhytoplasma aurantifolia’ has been previously reported in acid lime in India (Ghosh et al., 1999) and chickpea (Ghanekar et al., 1988), but this is the first record of a 16SrII phytoplasma isolate in lettuce, carrot, French bean, and Amaranthussp. in this country. Work in the UK was done under Defra licence No. PHF 174D/5185(08/2005). Sequencing was done by the Sequencing Service (School of Life Sciences, University of Dundee, Scotland, http://www.dnaseq.co.uk) using Applied Biosystems Big-Dye Ver 3·1 chemistry on an Applied Biosystems model 3730 automated capillary DNA sequencer.

Natural occurrence of phytoplasma associated with chickpea phyllody disease in Pakistan – a new record

During spring 2005–06 chickpea plants (cvs Desi and Kabuli) were found to be affected by a previously undescribed disease in Pakistan. Symptoms consisted of proliferation of branches with smaller leaflets, giving a bushy appearance to the plants. Affected plants were scattered in the field and were more easily spotted at flowering and podding time. The flowers developed abnormal green structures (phyllody) instead of normal flowers. At the time of crop maturity when the healthy plants were drying the diseased plants in the field were conspicuously green. Tissue samples from plants with and without symptoms were examined using a transmission electron microscope to ascertain if the disease was associated with a phytoplasma. Typical pleomorphic bodies (phytoplasma) mostly spherical to oval of a size ranging from 200–600 nm were observed only in the sieve elements of affected samples. The bodies had opaque, low electron density cytoplasm that contained ribosome like granules, DNA-strand-like structures and lacked nuclear membranes, similar to previously reported phytoplasmas (Ajayakumar et al., 2007). Such bodies were absent from healthy samples, from xylem cells, phloem parenchyma cells and companion cells of affected plants. In addition, DNA was extracted from plants with symptoms and amplified using the universal phytoplasma PCR primers P1/P7 followed by R16F2n/R16R2. PCR products were characterized by RFLP analysis and partial sequencing. All these samples gave a 1250 bp PCR product and the RFLP profile associated with 16SrII phytoplasmas, and partial sequencing confirmed that the phytoplasma had the greatest homology to 16SrII phytoplasmas. Koch's postulates were followed using 5-week-old chickpea plants grown in pots inoculated by grafting. A set of control plants was kept without grafting. Phyllody symptoms similar to those observed in the field started to develop after 25–35 days in graft-inoculated plants while no disease symptoms were observed on control plants. Chickpea has been grown in Pakistan for many years without any reported incidence of phyllody. The presence of phytoplasma disease in chickpea presents a new threat to the chickpea in Pakistan. Previously the disease was found in Ethiopia, India, Myanmar (Ghanekar et al., 1988); Australia (Saqib et al., 2005) and Oman (Al-Saady et al., 2006). This is the first report of chickpea phyllody disease in Pakistan.

First report on the association of a 16SrII phytoplasma with sesame phyllody in Pakistan

First report on the association of a 16SrII phytoplasma with sesame phyllody in Pakistan

Molecular Detection and Identification of Group 16SrI and 16SrXII Phytoplasmas Associated with Diseased Potatoes in Russia

Phytoplasmal diseases have long been suspected to occur in several potato-growing regions in Russia on the basis of symptoms and the presence of insect vectors. Symptoms resembling stolbur are most prevalent, but round leaf disease, potato witches'-broom, and potato purple top wilt also occur (1). The phytoplasma etiologies of these diseases have never been verified by molecular means. During the summer of 2006, 33 potato plants exhibiting various symptoms including purple top, round leaves, and stolbur-like symptoms characterized by purple top, stunting, bud proliferation, and formation of aerial tubers were randomly collected from the Volga River Region, Central Region, and Northern Caucasian Region in Russia. DNA extracts were prepared from 1.0 g of petioles and leaf mid veins according to a modified procedure with the Qiagen DNeasy Plant Mini Kit (Qiagen, Valencia, CA) as previously described (2). A nested PCR with primer pair P1/P7 in the first amplification followed by R16F2n/R16R2n in the second amplification was performed to detect phytoplasmas in infected potato samples (4). Potato plants maintained in the greenhouse were used as healthy controls. A negative control devoid of DNA templates was included in all PCR assays. One microliter of diluted (1:30) PCR product from the first amplification was used as the template in the nested PCR. Eight of 33 potato samples tested positive in the first PCR. Twelve of 33 potato samples tested positive in nested PCR. Nine of the 12 potato samples that tested positive for phytoplasma exhibited stolbur-like symptoms; the other three samples exhibited round leaves, stunting, or proliferation. The remaining symptomatic samples that exhibited nonspecific purple or yellow discoloration of terminal leaves, without other specific stolbur-like symptoms, may be infected by other pathogens. Restriction fragment length polymorphism (RFLP) analysis of nested PCR products (R16F2n/R16R2n amplicons, 1.2 kb) was performed. PCR products (6 μl) were digested singly with the restriction enzymes AluI, HaeIII, HhaI, HpaII, KpnI, MseI, RsaI, and Tsp509I. Comparison of RFLP profiles with published profiles (3) was used for identification of the putative phytoplasmas detected. Among the 12 PCR positive potato samples, 10 showed very similar or identical RFLP profiles to stolbur phytoplasma, a strain belonging to stolbur phytoplasma group (16Sr XII), subgroup 16SrXII-A and closely related strains, and two showed RFLP profiles similar to those of aster yellows phytoplasma group (16SrI). Nucleotide sequence analysis of cloned 16S rDNA (GenBank Accession Nos. EU344884-EU344890 and EU333396-EU333400) confirmed the results of the RFLP analyses and also indicated that the two samples showing 16SrI profiles were simultaneously infected with two phytoplasma strains belonging to subgroups 16SrI-A and 16SrI-B. To our knowledge, this is the first confirmation by molecular procedures that stolbur phytoplasma (16SrXII-A) is prevalent in several potato-growing regions and is the first report of 16SrI-A and 16SrI-B phytoplasmas associated with potatoes in Russia.

First report of a 16SrII group phytoplasma associated with die‐back diseases of papaya and citrus in Ethiopia

Citrus orange (Citrus aurantifolia) and papaya (Carica papaya) are amongst the most common fruits grown in Ethiopia and the area devoted to their cultivation has increased considerably in the last six decades (Seifu, 2003). Recently decline disorders of both citrus and papaya have been reported as causing significant yield losses amongst the major farms growing these crops in the country. Symptoms in citrus include leaf interveinal chlorosis, mosaic or mottling, a reduction in the size and curling of leaves. In severe cases, premature fruit fall, twig drying and die-back, and death of the tree. In papaya, the first symptom is a bright yellowing of the upper young leaves; later, symptoms of mosaic, crinkling and leaf tip necrosis develop, followed by a drying of the upper leaves, which progresses to death of the whole plant. Leaf samples showing symptoms from 12 papaya and 6 orange plants were collected from farms in the Melkassa area of east Shewa, during September 2005. Citrus tristeza virus (CTV) has been previously reported in Ethiopia (Khassaye et al., 1977), so citrus samples were tested for CTV by DAS-ELISA, yielding 4/6 positives. DNA was extracted from all plants and indexed in both a nested PCR with phytoplasma generic primers P1/P7 and R16F2n/R16R2, and a simple PCR for papaya bunchy top (PBT) rickettsia using primers PBTF1/PBTR1 (Davis et al., 1998). Nested PCR amplicons were obtained for 11/12 papaya and six citrus samples. Following RFLP analysis, all exhibited identical HaeIII, RsaI and AluI profiles, which were typical of a 16SrII phytoplasma. None of the papaya and citrus samples evaluated was positive for the PBT rickettsia. Sequencing was done by The Sequencing Service (Univ. Dundee, Scotland; http://www.dnaseq.co.uk) using Applied Biosystems Big-Dye Ver 3·1 chemistry, on an Applied Biosystems model 3730 sequencer. The 16S rDNA phytoplasma sequences of a representative sample of papaya (GenBank Acc. No. DQ285659) and citrus (GenBank Acc. No. DQ286576) were identical and their highest similarity (98%) was with papaya yellow crinkle in Australia (GenBank Acc. No. Y10097), a member of the 16SrII group (Candidatus Phytoplasma aurantifolia). This is the first record of phytoplasmas associated with die-back diseases of papaya and citrus in Ethiopia. The finding of a mixed infection of phytoplasma and CTV in some citrus samples will require further studies before any recommendations for disease management can be made. Work in the UK was done under Defra plant health license No. PHF 174D/5185(08/2005). Rothamsted Research receives grant aid from the Biotechnology and Biological Sciences Research Council of the UK.

‘Candidatus Phytoplasma lycopersici’, a phytoplasma associated with ‘hoja de perejil’ disease in Bolivia

New diseases known locally as 'hoja de perejil' of tomato (Lycopersicon esculentum Mill) and 'brotes grandes' of potato (Solanum tuberosum L.) were first recognized in surveys of production fields in Bolivia during 2000-2003. Alfalfa (Medicago sativa) witches' broom and little leaf diseases of native weeds Morrenia variegata and mora-mora (Serjania perulacea) were also identified near to production fields. Phytoplasma aetiology was attributed to each of these diseases following detection and initial identification of aster yellows group (16SrI) phytoplasmas in all five diseased plant species. While potato, alfalfa and mora-mora plants contained indistinguishable 16SrI-B strains, 'hoja de perejil' (THP) and morrenia little leaf (MVLL)-associated phytoplasma strains shared 97.5 % 16S rRNA gene sequence similarity with 'Candidatus Phytoplasma asteris' and related strains and <95 % similarity with all other 'Candidatus Phytoplasma' species. Phylogenetic analysis of 16S rRNA gene sequences indicated that the THP and MVLL phytoplasmas represent a novel lineage within the aster yellows (16SrI) group and, on the basis of unique 16S rRNA gene sequences, we propose that THP and MVLL phytoplasmas represent 'Candidatus Phytoplasma lycopersici', with THP as the reference strain.

First Report of a Natural Infection of Opuntia sp. by a 'Candidatus Phytoplasma asteris'-Related Phytoplasma in China.

Cacti (Opuntia spp.) are perennial, evergreen, succulent plants native to arid areas of the Americas. Because of their aesthetic appearance, many cacti have been cultivated and introduced to other parts of the world as ornamentals. Cacti are susceptible to phytoplasma infections and develop witches'-broom (WB) disease. Currently, all reported cactus WB cases are associated with infections by phytoplasmas in the peanut witches'-broom group (16SrII) (1,2,4). During a phytoplasma diversity survey carried out during 2004 in Yunnan, China, we collected 29 malformed and 14 healthy-looking naturally occurring cactus plants from 14 locations representing five geographical regions. Each of the 29 malformed plants exhibited stunted growth and possessed clusters of highly proliferating cladodia, typical symptoms of cactus WB disease. Nested-PCR was carried out on the DNA samples extracted from young cladodia of these plants using phytoplasma-universal 16S rDNA primers P1A/P7A and R16F2n/R16R2 (3). Results revealed that all 29 diseased plants that were examined were infected by phytoplasmas, whereas all 14 healthy-looking plants were negative for phytoplasmas. Subsequent restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 1.25-kb 16S rDNA fragments indicated that 28 diseased plants were infected by a phytoplasma of group 16SrII, whereas one plant (from Suan Village) was infected by a 'Candidatus Phytoplasma asteris'-related (group 16SrI) phytoplasma designated as strain YN26. Nucleotide sequence analysis of the strain YN26 partial rRNA operon (GenBank Accession No. EF190970), covering a near full-length 16S rRNA gene, a 16S-23S rRNA intergenic spacer, a tRNA-Ile gene, and a partial 23S rRNA gene, suggested that this phytoplasma is most closely related to an ash witches'-broom phytoplasma (GenBank Accession No. AY566302, 99.7% identity) and an epilobium phyllody phytoplasma (GenBank Accession No. AY101386, 99.7% identity), both members of subgroup16SrI-B. This YN26-infected cactus plant was transferred to a greenhouse and maintained for more than 2 years, during which time DNA samples were extracted and tested two additional times. The same 16S rDNA RFLP pattern type was consistently obtained in these tests, confirming that the plant remained infected by the 16SrI phytoplasma. To our knowledge, this is the first report of a natural infection of a cactus species by a group 16SrI phytoplasma. Since this 16SrI-cactus WB phytoplasma was found in the same geographical location where 16SrII-cactus WB phytoplasma was detected both in this and a previous study (1), the findings raised the question whether 16SrI- and 16SrII-cactus WB phytoplasmas have overlapping geo- and bioecological niches.

Broad bean and sweet pepper: two new hosts associated with Candidatus Phytoplasma asteris (16SrI phytoplasma group) in Cuba

Broad bean and sweet pepper: two new hosts associated with <i>Candidatus</i> Phytoplasma asteris (16SrI phytoplasma group) in Cuba

First Report of a Group 16SrII Phytoplasma Infecting Chickpea in Oman

Chickpea (Cicer arietinum), locally known as "Dungo", is grown for legume and animal feed mainly in the interior region of Oman. During February 2006, survey samples of chickpea leaves from plants showing yellows disease symptoms that included phyllody and little leaf were collected from the Nizwa Region (175 km south of Muscat). Total nucleic acid was extracted from asymptomatic and symptomatic chickpea leaves using a cetyltrimethylammoniumbromide method with modifications (3). All leaf samples from eight symptomatic plants consistently tested positive using a polymerase chain reaction assay (PCR) with phytoplasma universal primers (P1/P7) that amplify a 1.8-kb phytoplasma rDNA product and followed by nested PCR with R16F2n/R16R2 primers yielding a product of 1.2 kb (2). No PCR products were evident when DNA extracted from healthy plants was used as template. Restriction fragment length polymorphism analysis of nested PCR products by separate digestion with Tru9I, HaeIII, HpaII, AluI, TaqI, HhaI, and RsaI restriction enzymes revealed that a phytoplasma belonging to group 16SrII peanut witches'-broom group (2) was associated with chickpea phyllody and little leaf disease in Oman. Restriction profiles of chickpea phytoplasma were identical with those of alfalfa witches'-broom phytoplasma, a known subgroup 16SrII-B strain (3). To our knowledge, this is the first report of phytoplasma infecting chickpea crops in Oman.