Detection Of Phytoplasma Research Articles

Natural Phytoplasma Infection of Four Phloem-Feeding Auchenorrhyncha Across Vineyard Agroecosystems in Central-Eastern Italy

The seasonal variations of grapevine yellow phytoplasma were investigated in four phloem-feeding planthopper and leafhopper species that are vectors of plant disease agents. In total, 1,148 wild specimens were collected from three vineyard agroecosystems in the Marche region (central-eastern Italy), from May to September 2008, and analyzed using polymerase chain reaction-restriction fragment-length polymorphism methods. Of 525 Euscelis lineolatus Brullé, 25.1% were positive for aster yellow phytoplasma (16SrI-C, 16SrI-B subgroups) and stolbur phytoplasma (16SrXII-A subgroup; Vergilbungskrankheit type I [VK-I]). Of 368 Hyalesthes obsoletus Signoret, 19.3% were positive for the 16SrXII-A subgroup (VK-I, VK-II; mainly according to their host plant). Of 146 Neoaliturus fenestratus (Herrich-Schäffer), 15.1% were positive for the 16SrI-C and 16SrI-B subgroups, and 7.3% of 109 Psammotettix alienus (Dahlbom) were positive for the 16SrI-B subgroup. The total inoculation efficiency in the feeding medium assays was 57.1% for P. alienus, 44.7% for E. lineolatus, 44.4% for N. fenestratus and 33.9% for H. obsoletus. All of the phytoplasma subgroups identified in the insect bodies were also detected in their feeding media. Detection of stolbur phytoplasma in E. lineolatus feeding media strengthens the hypothesis that it is a candidate vector of Bois noir disease causal agent. The phytoplasma subgroups detected in the Auchenorrhyncha species showed variations according to season and/or vineyard agroecosystem. This study highlights the different specificities of these phytoplasma-Auchenorrhyncha species relationships, and suggests a primary role of the entire vineyard agroecosystem in the epidemiology of grapevine yellow phytoplasma diseases.

The detection of viruses and phytoplasmas in dwarfed shoots of grapevine varieties Aurelius and Neuburger

The survey of occurrence of six chosen virus pathogens and phytoplasmas complex was done on plants of grapevine (Vitis vinifera L.) which showed symptoms of short-shoot syndrom. The results of serological and molecular tests did not confirm either virus or phytoplasma infection as the main source of short-shoot syndrom. The presence of 6 viruses in samples taken from 45 affected plants of grapevine on 4 habitats. The highest occurrence of viruses was found out on habitat Moravská Nová Ves, where all taken samples were infected by Grapevine leafroll-associated virus GLRaV-1 (100 %). In 66 % of the samples taken from that habitat were detected mixed infection of Grapevine virus A and GLRaV-1. These 2 pathogens but were not detected in samples from affected plants from the other habitats or very sporadically – just in 2 plants. Another 4 virus pathogens were detected either sporadically (Grapevine fleck virus, Grapevine leafroll-assoc. virus GLRaV-3, Arabis mosaic virus) or not at all (Grapevine fanleaf virus). From 270 tests made to 6 viruses were only 20 positive, e. g. 7.4 %. It means that from 45 plants were 15 infected at least by 1 virus (33 %). The phytoplasmas complex was tested in 28 plants. The result was positive only in 1 plant, by another test the Potato stolbur phytoplasma was confirmed.

A multiplex nested PCR assay for simultaneous detection of Corchorus golden mosaic virus and a phytoplasma in white jute (Corchorus capsularis L.)

A multiplex nested PCR assay was developed by optimizing reaction components and reaction cycling parameters for simultaneous detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma (Group 16Sr V-C) causing little leaf and bunchy top in white jute (Corchorus capsularis). Three sets of specific primers viz. a CoGMV specific (DNA-A region) primer, a 16S rDNA universal primer pair P1/P7 and nested primer pair R16F2n/R2 for phytoplasmas were used. The concentrations of the PCR components such as primers, MgCl2 , Taq DNA polymerase, dNTPs and PCR conditions including annealing temperature and amplification cycles were examined and optimized. Expected fragments of 1kb (CoGMV), 674bp (phytoplasma) and 370bp (nested R16F2n/R2) were successfully amplified by this multiplex nested PCR system ensuring simultaneous, sensitive and specific detection of the phytoplasma and the virus. The multiplex nested PCR provides a sensitive, rapid and low-cost method for simultaneous detection of jute little leaf phytoplasma and CoGMV. Based on BLASTn analyses, the phytoplasma was found to belong to the Group 16Sr V-C.

Indonesia

Witches’ broom disease caused by phytoplasma is a very serious disease on peanut (Arachis hypogaea) which may potentially lead to high yield loss. Insects are the most important agents of phytoplasma transmission in the field. The objective of this research was to examine the potential role of leafhoppers species as insect vector of phytoplasma and to determine their transmission characteristic. Two species of leafhopper i.e. O. argentatus and Empoasca sp. (both belong to Hemiptera: Cicadellidae) were chosen for this study. The methodology involved were transmission study of phytoplasma by O. argentatus and Empoasca sp., and molecular detection of phytoplasma by PCR technique to confirm the association of pathogen, insect vector and symptomatic plants. The result showed that specific symptom was observed when using O. argentatus in the transmission study with number of insect as low as 1 insect per plant, whereas Empoasca sp. was not able to transmit the disease. Incubation period of phytoplasma in the host plant was affected by the number of insect, i.e. the more insect vector the shortest incubation period. The phytoplasma was successfully detected using P1/P7 primer in symptomatic plants as well as in the insect vector.Key words: Empoasca sp., leafhoppers, polymerase chain reaction

Diagnostic techniques for detection of phytoplasma diseases: past and present

Phytoplasmas are intracellular plant pathogens originated from a single lineage derived from Gram-positive bacteria and belong to the class Mollicutes. Phytoplasmas are associated with important diseases in hundreds of economic plant species worldwide. They are also prevalent in natural forest ecosystems and wild plant species. Phytoplasmas are recalcitrant to cultivation and are often difficult to detect and identify due to their erratic distribution, low concentration, seasonal fluctuation and enzyme-inhibitory plant poly-saccharide and polyphenolic compounds especially in woody perennial plant hosts. Rapid, sensitive, accurate and early diagnosis of phytoplasma diseases is indispensable to reduce their economical impact. Several serological and molecular techniques have been developed for accurate and sensitive detection of phytoplasmas in both host plants and insect vectors.

Quantitative PCR assay for detection of bois noir phytoplasmas in grape and insect tissue

In Europe's vineyards Bois noir (BN) is an expanding yellows disease on Vitis vinifera . It is associated with phytoplasmas of the stolbur group (16SrXII-A). Two subtypes are important, one is associated with Urtica dioica and one with Convolvulus arvensis. Both phytoplasma types are transmitted by the insect Hyalesthes obsoletus . A nucleic acid extraction method for V. vinifera and H. obsoletus was developed together with a real time PCR (qPCR) assay based on a polymorphic sequence with homology to a putative dimethyladenosine transferase. The comparison of the conventional detection method with the qPCR assay of 40 insect and 40 V. vinifera samples showed a 10 % higher sensitivity of qPCR in plant samples. The titer of phytoplasmas in H. obsoletus was 2643-fold increased in the strongest infected samples compared to the lowest ones. The results suggest this real-time PCR as a valid and fast alternative procedure for the detection and quantification of BN phytoplasmas. The assay allows to discriminate the two phytoplasma types and to quantify phytoplasmas in H. obsoletus .

Detection and Identification of a 16SrII Group Phytoplasma Causing Clover Little Leaf Disease in Iran

AbstractWhite clover plants showing little leaf and leaf reddening symptoms were observed in Isfahan Province in central Iran. Restriction fragment length polymorphism analyses of nested PCR‐amplified fragments from Iranian clover little leaf phytoplasma isolates and representative phytoplasmas from other phytoplasma groups using AluI, CfoI, KpnI and RsaI restriction enzymes indicated that the clover phytoplasma isolates are related to the peanut WB group. Sequence analyses of partial 16S rRNA fragments showed that Iranian clover little leaf phytoplasma has 99% similarity with soybean witches'‐broom phytoplasma, a member of the peanut WB (16SrII) phytoplasma group. This is the first report of clover infection with a phytoplasma related to the 16SrII group.

Detection of Candidatus Phytoplasma aurantifolia with a quantum dots fret-based biosensor.

The witches’ broom disease of lime (WBDL) supposedly caused by Candidatus Phytoplasma aurantifolia (Ca. P. aurantifolia) is the most devastating disease of lime in Oman and southern parts of Iran. The present study describes developing a quantum dot (QD)-based nano-biosensor for the highly sensitive detection of phytoplasma in infected trees. The immunodominant membrane protein (IMP) expressed in the surface of phytoplasma was selected as a target protein for construction of a specific binding antibody. The antibody (anti-IMP) was effectively conjugated to tioglicolic acid-modified cadmium-telluride quantum dots (CdTe-QDs) synthesized in an aqueous solution via electrostatic interaction. Dye (rhodamine) molecules were attached to the IMP, then, the donor-acceptor complexes (QDs-Ab-IMPRhodamine) were formed based on the antigen-antibody interaction. The mutual affinity of the antigen and the antibody brought the CdTe QDs and rhodamine together close enough to allow the fluorescence resonance energy transfer (FRET) to occur. The immunosensor constructed showed a high sensitivity and specificity of 100%, a detection limit of 5 ca. P. aurantifolia/µl, and acceptable stability, so it could be used for detection with consistent results.

Molecular detection of Candidatus Phytoplasma spp. causing witches’ broom disease of acid lime (Citrus aurantifolia) in India

Phytoplasma infected acid lime plants in India develop characteristic symptoms like small chlorotic leaves, multiple sprouting and shortened internodes. Leaves drop prematurely and infected branches have distorted twigs resembling witches’ broom appearance which eventually show die-back symptoms. During its first report in 1999, witches’ broom disease identification was made on the basis of symptomatology and electron microscopy. However, molecular techniques have proved to be more accurate and reliable for phytoplasma detection than the conventional methods. During survey in the year 2010 six samples were collected from infected acid lime plants showing typical field symptoms from Vidarbha region of Maharastra. Initially, phytoplasma bodies were observed in phloem tissues of all six symptomatic samples under JEM 100S transmission electron microscope and all these six samples were subsequently screened using different set of phytoplasma specific universal primers by nested PCR, a widely recommended molecular technique for phytoplasma detection. In the present study P1/P7 “universal” phytoplasma-primer set was used for first round of PCR and amplified products were processed separately for nested PCR with three different nested primer pairs viz. R16F2n/R16R2, R16mF2/R16mR1 and fU5/rU3. The presence of phytoplasma was confirmed in all six suspected samples and one representative ~1.2 kb size amplicon was sequenced and deposited in GenBank as Candidatus Phytoplasma species AL-M (JQ808143). This is the first report of PCR based molecular detection of phytoplasma-induced witches’ broom disease of acid lime (WBDL) in India. Further molecular evaluation to determine the identity to the species level is in progress.

First Report of 'Candidatus Phytoplasma asteris' Affecting Woody Hosts (Fraxinus uhdei, Populus nigra, Pittosporum undulatum, and Croton spp.) in Colombia.

Phytoplasmas of the 16SrVII group in ornamental Fraxinus uhdei trees (1) growing in different cities of the Colombian Andes have been reported (2). In surveys made in Bogotá during March and May 2011, symptoms suggestive of phytoplasma infection were observed in ornamental woody species: Croton spp. (Euphorbiaceae), Pittosporum undulatum (Pittosporaceae) and Populus nigra (Salicaceae) trees, growing close to infected F. uhdei (Oleaceae). Symptoms included witches' broom, yellowing, dieback, epicormic sprouts, tufted foliage, abnormal elongation or shortening of internodes, and deliquescent branching leading to dramatic changes in crown architecture. P. undulatum and F. uhdei are introduced species representing the second and third most abundant trees in the city. P. nigra is an introduced species and Croton spp. is an Andean genus. In order to screen for the presence of phytoplasmas in Croton spp., P. undulatum, and P. nigra, four individuals of each species and two F. uhdei trees were sampled. For DNA extraction, 1 g of vascular tissue from young stems was used. Samples were tested by nested PCR with primers P1A/P7A (4) followed by R16F2n/R16R2 (3). The frequency of phytoplasma detection varied among species; P. undulatum and Croton spp. had three positives each, while P. nigra had one positive. Both F. uhdei were positive. Sequences from the amplicons (three reads) were aligned. BLAST analysis of 16S rDNA sequences from the four species tested had 99.2 to 99.7% similarity to 16SrI group sequences. Phylogenetic analysis further confirmed this relationship. Virtual sequence analysis using the iPhyclassifier tool ( http://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi ) showed that the sequence derived from P. undulatum (JQ730861) produced an identical RFLP pattern to group 16SrI-B (reference sequence NC_005303). RFLP similarity coefficients of the phytoplasmas from F. uhdei, Croton spp., and P. nigra (JQ730859, JQ730859 and JQ730861) were less than 0.97, suggesting the presence of a new subgroup within group 16SrI. The vectors of phytoplasmas are unknown in the region. Phytoplasma hosts previously reported in Colombia are: Solanum quitoense (16SrIII), Manihot esculenta (16SrIII), Liquidambar styraciflua (16SrI and 16SrVII), Elaeis guineensis (16SrI and 16SrIII), Coffea arabica (16SrIII), Cordia alliodora (16SrIII), Solanum tuberosum (16SrV and 16SrXII), and Zea mays (16SrI). To our knowledge, this is the first report of Croton spp. and P. undulatum as phytoplasma hosts. Phytoplasmas of group 16SrI are known to infect more than 100 species of different families worldwide. Detection of this group in several tree species and the observation of similar symptoms in other trees species raises concerns about a possible epidemic affecting plants in the Andean region. Implications are at several levels: i) epidemiological, with infected trees representing a potential inoculum source for other ornamental plants or crops growing in the agricultural surrounding areas; ii) economic, since eventually it will be necessary to replace diseased plants; and iii) environmental, because of the negative impact on the services provided by trees and green areas.

Rapid and reliable detection of phytoplasma by loop-mediated isothermal amplification targeting a housekeeping gene

Phytoplasmas are plant pathogenic bacteria that infect more than 700 plant species. Because phytoplasma-resistant cultivars are not available for the vast majority of crops, the most common practice to prevent phytoplasma diseases is to remove infected plants. Therefore, developing a rapid, accurate diagnostic method to detect a phytoplasma infection is important. Here, we developed a phytoplasma detection assay based on loop-mediated isothermal amplification (LAMP) by targeting the groEL gene and 16S rDNA. We designed 19 primer sets for the LAMP assay and evaluated their amplification efficiency, sensitivity, and spectra to select the most suitable primer sets to detect Candidatus Phytoplasma asteris. As a result, DNA was efficiently amplified by one of the primer sets targeting the groEL gene, and LAMP assay sensitivity with this primer set was 10-fold higher than that of the polymerase chain reaction. Moreover, the groEL gene was successfully amplified from several strains of Ca. Phytoplasma asteris by this primer set, indicating that the groEL gene can be used as a LAMP assay target gene for a broad range of phytoplasma strains. Additionally, a simple DNA extraction method that omits the homogenizing and phenol extraction steps was combined with the LAMP assay to develop a simple, rapid, and convenient diagnostic method for detecting phytoplasma.

Detection of the phytoplasmal agent of pear decline in Iran, Isfahan province, using nested-PCR

Pear decline is a very important phytoplasma disease that causes considerable quantitative and qualitative losses to this fruit crop. Due to economical importance of pear in Isfahan province, Iran, and the difficulty to determine the occurrence of the disease simply based on symptoms in orchards, a detection method for the phytoplasma causing disease in pears in the region was developed. Since the polymerase chain reaction (PCR) assay is a reliable and sensitive technique for identification of phytoplasma, nested-PCR method were employed which is included different sets of universal and specific primer pairs. Using P1/P7 together with fU5/rU3, or NPA2F/R in nested-PCR, products of the expected sizes were obtained from only 25% of the symptomatic samples. To examine the variation between phytoplasmal isolates from Isfahan and other countries, and to design a specific primer pair for the Iranian isolates, the PCR product from one of the samples, was sequenced. The BLASTN results showed high similarity to Knautia arvensis associated phytoplasma (99%). Significant homology also was found with phytoplasmas of almond witches' broom (96%), peach X disease (93%), pear decline (94%) and apple proliferation (92%). Having confirmed that there is a variation between the sequence of local phytoplasmas and similar pathogens deposited in the database, a pair of primers (fPD/rPD) were designed from the sequence using OLIGO software to increase the sensitivity of nested-PCR for detection of pear associated phytoplasma in Isfahan. In further experiments, using specific designed primers, the pathogen was detected in 72% of the samples. These primers are vastly introduced to improve the limit of detection and the specificity of the tests for the detection of pear phytoplasmas in the region.

Molecular detection and phylogeny of phytoplasmas affecting cycads in India

Cycads are an ancient group of gymnosperms, often considered to be living fossils. Recently, abnormal yellowing symptoms were observed on two cycad species of the order Cycadales: Cycas revoluta (family Cycadaceae), and Zamia furfuracea (family Zamiaceae), grown as ornamental plants in gardens of the North Delhi region, India. C. revoluta exhibited yellowing symptoms on fully developed leaves in all the whorls on the rachis and the leaf lamina (Fig. 1a). Z. furfuracea showed a pale yellow colour in the leaf lamina and little leaf symptoms (Fig. 1b). Genomic DNA was isolated from the leaf midribs of five symptom-bearing and two symptomless C. revoluta plants, and three symptom-bearing and one symptomless Z. furfuracea plants, using the CTAB method (Saghai-Maroof et al., 1987). DNA was used as a template in nested-PCR with phytoplasma universal 16S ribosomal DNA primers P1/P7 (Deng & Hiruki, 1991) and fU5/rU3 primers (Lorenz et al., 1995). The amplicons were resolved on a 1.2% agarose gel, extracted, purified (QIAquick Gel Extraction Kit, QIAGEN, USA) and sequenced. Phylogenetic relationships were evaluated using the 16S rDNA sequences of phytoplasmas identified from C. revoluta (Cycas yellowing phytoplasma) and Z. furfuracea (Zamia yellowing phytoplasma), and from 18 other 16S rDNA sequences from GenBank reference phytoplasmas using the maximum parsimony method of MEGA version 4.01. The bootstrap consensus tree was inferred from 1000 replicates using Acholeplasma laidlawaii as the outgroup. Amplicons of desired size (~880bp) were obtained in all symptom-bearing plants while no amplification was observed for the symptomless ones. BLAST analysis of the phytoplasma 16S rDNA sequences from both Cycas and Zamia yellowing phytoplasmas showed 99% sequence identity with those of members of phytoplasma group 16SrII (‘Candidatus Phytoplasma aurantifolia’). The phylogenetic tree supported the sequence comparison analysis since the 16S rDNA sequences of Cycas and Zamia yellowing phytoplasmas (GenBank Accession Nos. HQ625434 and HQ625433, respectively) clustered in the same phylogenetic branch of 16SrII phytoplasmas (Fig. 2). Previous reports indicate that gymnosperms in India are highly sensitive to phytoplasma infection. ‘Ca. Phytoplasma pini’ (AJ632155, AJ310849), group 16SrV (Schneider et al., 2005) and a ‘Ca. Phytoplasma phoenicium’-related phytoplasma (AF515637), group16SrIX-E (Davis et al., 2010) have been associated with leaf abnormalities. Moreover, a ‘Ca. Phytoplasma trifolii’-related strain (FJ002570), group 16SrVI (Gupta et al., 2010) has been associated with Araucaria heterophylla little leaf and stunting. This is the first record of cycads (Cycadales) being infected by phytoplasmas of group 16SrII, the most widespread phytoplasma group in the region. The fact that gymnosperms are increasingly grown as ornamentals in India and have been identified as natural hosts for phytoplasmas, including those of 16SrII, has epidemiological impact possibly important for future disease control in the country.

Detection and molecular characterization of sugarcane grassy shoot phytoplasma in Vietnam

Sugarcane is an important cash crop in Vietnam and has been widely promoted at national and provincial level. In 2006, a new disease was discovered in sugarcane in the Nghean Tate&Lyle Sugar Mill in Nghean Province in north-central Vietnam. The key symptoms were the formation of green grassy shoots around the base of mature stools. We applied nested polymerase chain reaction (PCR) using P1/P7 and R16F2n/R16R2 for detection and characterization of phytoplasma from the symptomatic tissues. PCR products of the expected size (approx. 1200 bp) were obtained from the 16S rDNA of the phytoplasma. The restriction fragment length polymorphism profiles indicated that all samples were infected by the same phytoplasma. Phylogenetic analysis confirmed that the SCGS phytoplasma from Vietnam belong to the 16SrXI group, formerly Rice Yellow Dwarf group.

Detection and molecular characterization of phytoplasma associated with chickpea phyllody disease in south India

Chickpea (Cicer arietinum L.) plants showing typical symptoms of infection by a phytoplasma that causes phyllody disease have been commonly observed in recent years in parts of south India. The symptoms included pale green leaves, bushy appearance due to excessive stunting of shoots, reduced internodal length and excessive axillary proliferation. The causal agent of the phyllody disease was identified based on symptoms, amplification of 16S rDNA of the phytoplasma by polymerase chain reaction (PCR) from infected samples, as well as by sequencing and phylogenetic analysis. First round PCR and nested-PCR protocols were standardized for improved efficiency and reliability of the diagnostic protocols. Using the primers P1/P7 and R16F2n/R16R2, 1,800 bp and 1,200 bp size products were amplified in first round PCR and nested-PCR protocols, respectively. The PCR product was cloned and sequenced and compared with the reference phytoplasma sequences from the database (NCBI). The Indian chickpea phyllody phytoplasma 16S rDNA sequences shared the highest nucleotide identity (>98%) with the 16S rII group phytoplasma candidates, also infecting chickpea from Australia and Pakistan. This is the first report of a phytoplasma of the 16SrII-group infecting chickpea from India. The genetic similarities and the potential threat of this new disease to chickpea cultivation in India are discussed.

Failure of Flower Bud Formation in Brassica Plants Associated with Phytoplasma Infection

Detection of phytoplasma classified in phylogenetic group 16SrI, ‘Ca. Phytoplasma asteris’, in the plants of interspecific genotypes of Brassica oleracea x Brassica napus and their back-cross toward B. oleracea (IW7.7xB1) is reported in Poland. The presence of phytoplasmas in leaves of plants showing failure of flower bud formation but not in the asymptomatic hybrids was demonstrated by polymerase chain reaction assay employing phytoplasma universal P1/P7 primer pair. Amplified cDNA fragments of two phytoplasma isolates were sequenced. The nucleotide sequences of the Brassica hybrids phytoplasma isolates showed more than 98,5 % similarity with the corresponding fragments of sequence of phytoplasmas belonging to group 16SrI, ‘Ca. Phytoplasma asteris’.

‘Candidatus Phytoplasmatrifolii’ associated with little leaf and witches’ broom disease ofDatura stramoniumL. in India

A disease characterized by little leaf and witches’ broom symptoms was observed on Datura stramonium L. plants growing widely along the roadsides at Gorakhpur, Uttar Pradesh, India. Phytoplasma detection and identification using universal primer pairs P1/P7 amplifying phytoplasma 16S-23S rDNA followed by R16F2n/R16R2 in nested PCR assays from nucleic acid extracted from symptomatic D. stramonium plants was performed. Neither by direct nor by nested PCR DNA from non-symptomatic samples was amplified. BLAST analysis of the 16S rRNA partial gene sequence of the phytoplasma identified in D. stramonium revealed its highest identity with several members of ‘Candidatus Phytoplasma trifolii’ group (clover proliferation, 16SrVI). The association of clover proliferation group (16SrVI) with little leaf disease of D. stramonium is reported here for the first time worldwide.

Detection and identification of the elm yellows group phytoplasma associated with Puna chicory flat stem in China

In 2010 and 2011, the Puna chicory flat stem (PCFS) symptom was observed in an experimental field around Yangling, Shaanxi Province, China. Pleomorphic phytoplasma-like bodies were observed in the phloem sieve tube elements of symptomatic plants under transmission electron microscope (TEM). The presence of phytoplasma was further confirmed by a nested polymerase chain reaction (PCR), which amplified a 1.2 kb fragment using universal primer pairs P1/P6 followed by further amplification using primer pairs R16F2n/R16R2. The 1.1 kb 16S rRNA gene fragments were obtained by nested PCR with primer pairs P1/P6 followed by 16SrV group-specific primer pairs R16(V)F1/R16(V)R1 and indicated that the PCFS phytoplasma was closely related to the elm yellows (EY) group (16SrV). Phylogenetic analysis and restriction fragment length polymorphism (RFLP) analysis demonstrated that the PCFS phytoplasma belongs to subgroup 16SrV-B. This is the first report of Puna chicory disease associated with a subgroup 16SrV-B of phytoplasma in China.

Detection and identification of phytoplasmas associated with longan witches' broom in Vietnam

Longan (Dimocarpus longan Lour.) belongs to the family Sapindaceae and is an important fruit tree grown mostly in the Southern part of Vietnam. In January 2010, plants of longan Tieudabo variety showing witches’ broom symptoms were observed at orchards in Dong Nai province, Southeast region. Nested PCR assays with universal phytoplasma primers amplifying 16S rRNA on nucleic acid extracted from symptomatic longan samples allow detecting expected length amplicons. RFLP analyses revealed similarity to the reference strains elm yellows (group 16SrV) and stolbur (group 16SrXII). The presence of a mixture of phytoplasmas was confirmed by nested PCR assays with group-specific primer pairs R16(I)F1/R1 and R16(V)F1/R1 together with sequencing of amplicons. To our knowledge, this is the first report of multiple phytoplasma infection associated with witches’ broom disease in longan in Vietnam.

Characterization of an isolate of ‘CandidatusPhytoplasma asteris’infectingZinnia elegansin India

Different gardens and nurseries were surveyed in various locations of Gorakhpur of Eastern Uttar Pradesh, India during 2008–2009 to verify incidence of phytoplasma disease. Typical little leaf, chlorosis, witches’ broom, yellowing and phyllody symptoms were observed on Zinnia elegans plants growing in gardens of Sugarcane Research Station, Kunraghat, Gorakhpur of Uttar Pradesh, India. The symptomatic plants were further processed for phytoplasma detection and characterization. Universal primer pair of P1/P7 amplified the 1.7 kb DNA fragment of phytoplasma 16S-23S rRNA from nucleic acid extracted from two symptomatic Z. elegans plants. In nested PCR assay with R16F2n/R16R2 a DNA fragment of ~1.2 kb was obtained. Neither by direct nor by nested PCR amplification was present from any non-symptomatic plant. Sequencing and BLAST analysis of 1.2 kb amplicon of the phytoplasma DNA revealed 99% sequence identity with the 16SrRNA of ‘Candidatus Phytoplasma asteris’ (16SrI) group strains and phylogenetic analysis confirmed this results. This is the first report of ‘Ca. P. asteris’ associated with yellowing, witches’ broom and little leaf disease of Zinnia elegans in India.