Group 16SrI Research Articles

First report of ‘Candidatus Phytoplasma asteris’ (16SrI group) associated with yellows disease of gerbera (Gerbera jamesonii) from India

Gerbera jamesonii (Asteraceae) is an important ornamental plant grown throughout the world. It is amongst the ten most important cut flowers in floricultural trade. In 2012, severe leaf yellows, shortening of whole plant and flower deformation symptoms were observed on gerbera plants growing in a polytunnel at Lucknow, Uttar Pradesh, India. The disease incidence on gerbera was about 15-20%. Symptoms exhibited by diseased gerbera were suggestive of the presence of phytoplasma. Total DNA was extracted from leaf samples collected from symptom-bearing plants (Ahrens & Seemüller, 1) and PCRs were performed using P1/P6 universal primers (Deng & Hiruki, 2) followed by nested PCR using primers R16F2n/R16R2 (Gundersen & Lee, 3). Products of ~1.2 kb were amplified for four out of four samples from symptom-bearing plants, but not from the symptomless plants, demonstrating the association of a phytoplasma with the disease. Amplicons from two samples, Gerbera jamesonii yellows phytoplasma Lucknow 1 (GYLu1) and Gerbera jamesonii yellows phytoplasma Lucknow 2 (GYLu2), were cloned, sequenced and sequence data was deposited in GenBank, under Accession Nos. JX674049 (GYLu1) and KC880350 (GYLu2). Sequence comparison using BLASTn (http://blast.ncbi.nlm.nih.gov/Blast.cgi) revealed 99% homology between GYLu1 and GYLu2. GYLu1 and GYLu2 also shared 99% sequence identity with several strains of ‘Candidatus Phytoplasma asteris’ reported worldwide. A similarity of 98% was verified between GYLu1 and GYLu2 when compared with the sequence of 'G. jamesonii' phytoplasma (JF795864), a member of 'Ca. P. asteris' from Italy. To find out phylogenetic relationships of the gerbera phytoplasma strains under study and 16S rDNA sequences of ‘Ca. P. asteris’ strains and other phytoplasmas available in GenBank, a phylogenetic tree was generated using the neighbour-joining method (Fig. 1). The results demonstrated that GYLu1 and GYLu2 clustered with the strains of ‘Ca. P. asteris’. Based on high sequence identities (99%) and close phylogenetic relationships with Italian gerbera phytoplasma strain of ‘Ca. P. asteris’, the gerbera phytoplasmas from India were identified as ‘Ca. P. asteris‘-related strains Gerbera is reported as a host of strains of ‘Ca. P. aurantifolia’ (group 16SrII) in Australia (Siddique, 5), and ‘Ca. P. asteris’ in Italy (Spanò et al., 6). In India, ‘Ca. P. asteris’ (16SrI group) has been reported on several plant species (Rao et al., 4). However, neither ‘Ca. P. asteris’ nor any phytoplasma has been previously reported on gerbera from India. We report here the association of ‘Ca. P. asteris’ with yellows disease of gerbera for the first time in India. Since gerbera is propagated by vegetative means and mass multiplication through tissue culture, detection of phytoplasma in gerbera is of utmost importance for disease management. The authors are thankful to the Council of Scientific and Industrial Research (CSIR), New Delhi, India for funding under project BSC117.

Phytoplasma diseases on major crops in Vietnam

The nested-PCR technique was applied to detect and identify phytoplasmas associated with major crops in Vietnam including sugarcane, cassava and longan. Phylogenetic analysis confirmed that phytoplasmas associated with the sugarcane grassy shoot and white leaf diseases belong to the rice yellow dwarf group (16SrXI). In cassava showing witches’ broom symptoms a mixed infection of phytoplasmas related to ‘Candidatus Phytoplasma asteris’ (group 16SrI) and ‘Ca. P. aurantifolia’ (group 16SrII) was detected. In samples showing longan witches’ broom disease three phytoplasmas including ‘Ca. P. aurantifolia’ (group 16SrII), elm yellows (group 16SrV) and “stolbur” (group 16SrXII) were identified.

First Report on the Molecular Identification of Phytoplasma (16SrII-D) Associated with Witches' Broom of Kalmegh (Andrographis paniculata) in India.

Andrographis paniculata (family Acanthaceae), also known as "King of Bitters" or Kalmegh, is an important medicinal plant used for the treatment of various diseases. It has antimicrobial, antiviral, anti-inflammatory, hepatoprotective, antidiabetic, antihyperglycemic, and antioxidant properties (1). During June 2014, while performing a routine survey of the commercial trial fields of Kalmegh at Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow, India, typical phytoplasma disease symptoms such as virescence, proliferation, and witches' broom along with little leaf and stunted growth were observed. The disease incidence was estimated to be approximately 7 to 10%. To ascertain the presence of phytoplasma, 16 samples of leaves were collected from nine different field sites, and total genomic DNA was extracted from the symptomatic and symptomless Kalmegh plants by the CTAB method. Direct and nested PCR assays were performed targeting the 16S rDNA using generic phytoplasma primer pairs P1/P6 followed by R16F2n/R16R2 (2). Resulting bands of the expected size (1.5 kb and 1.2 kb, respectively) were amplified from symptomatic plants. No amplification was observed with DNA from asymptomatic plant samples. The purified nested PCR products were cloned into E. coli DH5α, using the pGEM-T Easy vector (Promega, United States) and sequenced with primers M13For/M13Rev using an automatic sequencer (ABI Prism, Perkin Elmer) at CIMAP. The sequence was analyzed by BLASTn and found to share 99% similarity with Echinacea witches'-broom phytoplasma and Sesame phyllody phytoplasma strain (GenBank Accession Nos. JF340080 and KF612966, respectively), which belong to the 16SrII-D group. The sequence was deposited in NCBI as GenBank Accession No. KM359410. A phylogenetic tree using MEGA v5.0 (4) was constructed with 16S rDNA; consensus sequences of phytoplasmas belonging to distinct groups revealed that the present phytoplasma clustered with the 16SrII group. iPhyClassifier software was used to perform sequence comparison and generate a virtual restriction fragment length polymorphism (RFLP) profile (5). On the basis of iPhyClassifier, the 16S rDNA sequence analysis of our isolate showed 99.2% similarity with that of the 'Candidatus Phytoplasma australasiae' reference strain (GenBank Accession No. Y10097), which belongs to 16Sr group II. The virtual RFLP pattern of F2n/R2 fragment was most similar to the 16SrII-D subgroup (similarity coefficient of 0.91) but showed a difference in profile with HpaI, HhaI, and MseI enzymes. Several bacterial/fungal and viral diseases have been reported on A. paniculata (3); however, to our knowledge, this is the first report of witches' broom disease in India and the first record of a 16SrII-D group phytoplasma on Kalmegh. Its presence in Kalmegh is of great significance due to its commercial interest.

Identification and molecular characterization of the blueberry stunt phytoplasma in Canada

Blueberry farms in Quebec, Canada, were recently surveyed for phytoplasma-associated disease presence. Farmers previously reported a number of high bush blueberries showing stunt symptoms in three blueberry farms. Bushes were tested for phytoplasma based on the 16S rRNA gene. Nested PCR with universal phytoplasma primers resulted in amplification of phytoplasma DNA from blueberry bushes in samples from two of the farms surveyed. The 16S rDNA of the blueberry stunt phytoplasma from Quebec showed a 99% sequence identity with that of the blueberry stunt phytoplasma from Michigan (AY265220) from group 16SrI ‘Candidatus Phytoplasma asteris’ subgroup 16SrI-E. Phylogeny results confirmed the clustering of the blueberry stunt phytoplasma from Quebec within the same phylogenetic branch of phytoplasmas of group 16SrI, closely related to subgroup 16SrI-E. Preliminary virtual RFLP confirmed the sequence and phylogeny classification results. Further actual and virtual analyses are in progress to verify the presence of other possible phytoplasma strains from the Quebec blueberry farms.

Status of alfalfa witches’ broom phytoplasma disease in Iran

Alfalfa witches’ broom (AWB) is one of the most important and destructive diseases of alfalfa in Iran. Based on characteristic disease symptoms and direct and nested polymerase chain reactions, the status of AWB disease was evaluated in different growing areas of Iran. Restriction fragment length polymorphism was used to identify AWB disease associated phytoplasmas. Furthermore, infection rate, disease severity, death rate of infected plant in the summer and winter and overwintering of disease vector were assessed. Based on the results, AWB disease was reported on different alfalfa cultivars in Yazd, Fars, Sistan-Va-Baluchestan, Kerman, Hormozgan, Bushehr, Esfahan, Chaharmahal-Va-Bakthiari, South Khorasan and Khuzestan provinces of Iran. Phytoplasmas associated with AWB in these areas were identified as ‘Candidatus Phytoplasma aurantifolia’, belonging to peanut witches’ broom (16SrII) group. In Abarkooh and Ashkezar (Yazd province) and Bondarooz (Bushehr province) the recorded disease incidence was up to 100%. The highest disease severity was found in Rezvan Shahr (Ashkezar, Yazd province) in 3 years old alfalfa fields. The highest death rate of infected plants in summer and winter were recorded as 26% and 13% in Ashkezar and Abarkooh in Yazd province, respectively. Different nymph stages of the insect vector, Orosius albicinctus, were identified on tamarisk (Tamarix aphylla) and saxaul (Haloxylon persicum and H. aphyllum) in the winter. The highest population of O. albicinctus, observed on tamarisk plants adjacent to the infected alfalfa fields in Milleshbar (Ardakan, Yazd province), suggested this as a possible source of natural spread of AWB.

Incidence, distribution, economic importance of alfalfa witches’ broom disease in Sistan-Baluchestan (Iran) and characterization of associated phytoplasmas

Alfalfa witches’ broom (AWB) is a devastating disease distributed mainly in tropical provinces of Iran and widespread all over the Sistan-Baluchestan province. The highest incidence was found in Rask (Sarbaz) (70%), Sefidaba (Zabol) (46.26%), Sarboog (Nikshahr) (45.4%), Mirjaveh (Zahedan) (40%), Shams Abad (Saravan) (32.2%), Namardian (Iranshahr) (27%), Kahir (Chabahar) (25.7%) and Najaf Abad (Khash) (17.8%). Dodder inoculation of periwinkle plants with AWBagents from different areas induced undistinguishable symptoms. Detection and identification of AWB associated phytoplasmas was based on characteristic disease symptoms and direct and nested polymerase chain reaction using P1/P7, R16mF2/R16mR2 and R16F2n/R16R2 primer pairs. RFLP and phylogenetic analyses of 16S rDNA showed that AWB associated phytoplasmas from Sistan-Baluchestan belong to peanut witches’ broom (16SrII) group. Virtual digestion of R16F2n/R16R2 sequence of a Nikshahr AWB strain exhibited RFLP profiles referable to those of members of the 16SrII phytoplasma group, subgroup D.

Status of sesame phyllody and its control methods in Yazd, Iran

Phyllody is one of the most important diseases of sesame in Iran. During 2008–10, to evaluate disease status, a survey was carried out in sesame fields in Yazd province. Occurrence of sesame phyllody was observed in all the sesame growing fields in Yazd, but whole infection of fields was observed in Rezvan Shahr (Ashkezar, Yazd province). On the basis of RFLP analysis sesame phyllody phytoplasma was identified as a member of peanut witches’ broom (16SrII) group. No differences were observed in plant heights of infected and healthy plants. Total seed yield was reduced by 55.07% in infected plants and 1,000 seed weight showed a 21.49% decrease in infected plants. Wrinkled seeds increased in diseased plants up to 56.07%. Results of variance analysis of control methods showed that sowing date and spraying have significant effects on infection. Mean comparison of infection percent with LSD tests showed that delay in sowing date reduced sesame phyllody up to 31%. Spraying with Confidor reduced disease incidence at the first and second sowing date by 18.8 and 7.8% respectively but no differences were observed in yields. Seed treatment with Gaucho has no effect on disease incidence. Collectively, sowing of sesame straight after wheat harvesting in May needed spraying to reduce disease incidence, but delay in the sowing date to July 5 can reduce disease incidence without any significant differences in seed yield. Delay in sowing date is dependent on weather and needs to be determined for each area.

An up to date status of alfalfa witches’ broom disease in Iran

Among the cultivated forage crops, alfalfa is very important in Iran. Nearly 28 years ago, symptoms of alfalfa witches’ broom (AWB) were reported from Jiroft, southern of Iran. Since then, the disease was reported from other parts of Iran and the epidemic of AWB was recorded in Chahgeer, Yazd in 1997. Now AWB disease is widespread all over the country especially in tropical and subtropical areas. The phytoplasma strains reported from alfalfa in Iran were classified in 16SrI, 16SrII, 16SrVI and 16SrXII groups. The major symptoms reported are little leaf, internode shortening, flower virescence, phyllody, proliferation and sterility, witches'broom, leaf yellowing, leaf curling and death. Orosius albicinctus was identified as vector of the disease in Fars and Yazd provinces. Prosopis farcta and Cardaria draba were natural host plants of the AWB. The disease reduced plant heights, leaf area, shoot dry and fresh weight and seed yields of alfalfa crops. So far no effective control strategies are in practice; however use of dense planting and field resistant varieties are recommended for prevent ing AWB disease.

Preliminary study on some ornamental plant phytoplasma diseases in north of Iran

Because of the importance of ornamental plants culture in north of Iran, and the outbreak of suspicious symptoms of phytoplasma diseases on these plants, a study was carried out in order to detect and identify phytoplasmas associated with them. The presence of phytoplasma in samples of cockscomb, tagetes and rose plants was shown by direct and nested-PCR assays using the phytoplasma-specific primers, P1/P7 and R16F2n/R2. Sequences of cockscomb, Indian marigold and rose phytoplasmas have high homology with the members of aster yellows phytoplasma group (16SrI). Phylogenetic analysis showed that cockscomb, tagetes and rose phytoplasmas are closely related to 16SrI-M the first one and 16SrI-B the latter two, respectively. To our knowledge, this is the first report of a member of 16SrI group phytoplasma infection in cockscomb in Iran. Moreover, the present study is the first report of phytoplasma disease outbreak on rose in north of Iran by subgroup classification of the associated phytoplasma.

Overview on a century progress in research on sesame phyllody disease

Sesame is the most ancient oil seed plant cultivated in the tropics of Asia and South Africa. Among the major constraints, phyllody associated with phytoplasma presence is the major limiting factor affecting its cultivation firstly described more than 100 years ago. Almost all the sesame varieties under cultivation are susceptible to the disease that continues to cause severe economic losses. Different symptomatology is present: the most common symptoms are phyllody, flower virescence and witches’ broom affecting growth and yield. Several detection methods like Diene's stains and TEM techniques were developed for the detection of phytoplasmas in phloem sieve tissues, however the PCR based assays were established for the identification of the phytoplasmas associated with the disease. After rRNA sequencing and restriction analyses, a wide genetic diversity was identified among sesame phyllody associated phytoplasmas: four ribosomal groups 16SrI, 16SrII, 16SrVI and 16SrIX were detected. The disease spreads in nature by different leafhopper species, among them Orosius orientalis was reported as the major vector. Moreover many weed species are reported hosts of the sesame phyllody-associated phytoplasmas. No effective control measures of the disease were developed, exceptresistance, management of insect vectors andaltering the dates of sowing to avoid peaks of insect vector population. So far no resistant genotype of sesame is available. However, attempts have been made to confer resistance against the phyllody disease in sesame through intra-and interspecific crosses of two wild species Sesamum mulayanum and S. alatum. The review reports uptodate information about history, geographic distribution, symptomatology, diagnosis, genetic diversity, epidemiology and management of sesame phyllody disease.

A new disease inCitrus aurantifoliain Oman, “sudden decline”, is associated with a pathogen complex including a 16SrII group phytoplasma

Witches’ broom disease of lime is a devastating disease in the Sultanate of Oman. In recent years a new disease has been observed, which promotes a sudden decline in Citrus aurantifolia (acid lime). The etiological agent of this new disease has not yet been determined. In this study the presence of phytoplasmas and RNAs was evaluated in plants showing the sudden decline symptomatology. The results reveal the presence of a complex of at least three different pathogens: 16SrII phytoplasmas, Citrus tristeza virus and Citrus exocortis viroid.

First report of phytoplasma associated withEucalyptus urophyllashowing witches’ broom in Brazil

Eucalyptus production is of great importance to Brazil's economy. Diseases affecting the growth and the architecture of these trees lead to large losses in yield and quality of the wood. The occurrence of eucalyptus trees showing witches’ broom and little leaf symptoms in different areas of Brazil is affecting seedling production. In this work, a phytoplasma was found associated with these symptomatic plants and was identified as belonging to the 16SrII group. This is the first report of a phytoplasma associated with eucalyptus in Brazil.

Identification of phytoplasmas belonging to the ribosomal groups 16SrIII and 16SrV in Chilean grapevines

Nested polymerase chain reaction assays using phytoplasma universal primer pairs P1A/P7A and R16F2n/R2 detected phytoplasmas in grapevine varieties Syrah and Superior in Chile showing yellows symptoms. Cloning, sequencing, and in silico restriction fragment length polymorphism analyses, allowed identification of phytoplasmas belonging to ribosomal subgroups 16SrV-A (‘Candidatus Phytoplasma ulmi’-related) and 16SrI II-J (‘Ca. P. pruni’-related). This is the first report of these phytoplasmas in grapevine in Chile.

Cyperus rotundusL. a new host species for ‘CandidatusPhytoplasma aurantifolia’ - related phytoplasmas in Cuba

The identification of phytoplasmas in asymptomatic plants of Cyperus rotundus growing in common bean crops cultivated in Cuba is reported and the potential r isk of infection to cultivated species is discussed. The detected phytoplasmas belong to ribosomal group 16SrII, as confirmed by homology on part of the 16S rDNA sequence obtained after sequencing amplicons obtained in nested-PCR assays. This is the first report of the presence of 16SrII phytoplasmas in C. rotundus; further studies are necessary to assess the epidemiologic aspects of this finding in Cuban hort icultural cultivations especially in common beans

First Report of Aster Yellows Phytoplasma (16SrI‐B) Associated with Witches' Broom Disease of Melia azedarach var. japonica in Korea

AbstractMelia azedarach var. japonica trees with leaf yellowing, small leaves and witches' broom were observed for the first time in Korea. A phytoplasma from the symptomatic leaves was identified based on the 16Sr DNA sequence as a member of aster yellows group, ribosomal subgroup 16SrI‐B. Sequence analyses of more variable regions such as 16S–23S intergenic spacer region, secY gene, ribosomal protein (rp) operon and tuf gene showed 99.5−100% nucleotide identity to several GenBank sequences of group 16SrI phytoplasmas. Phylogenetic analysis confirmed that the Melia azedarach witches' broom phytoplasma belongs to aster yellows group.

‘CANDIDATUS PHYTOPLASMA ASTERIS’ ASSOCIATION WITH LEAF YELLOWS AND WITCHES’ BROOM SYMPTOMS OF CODIAEUM VARIEGATUM IN INDIA

Codiaeum variegatum (garden-grown croton species), a member of the family Euphorbiaceae. is native to southern India, Sri Lanka, Indonesia, Malaysia, and the western Pacific Ocean islands, where it grows in open forests and scrub. In August 2013, leaf yellows and witches' brooms symptoms were observed on C. variegatum plants grown at Lucknow (Uttar Pradesh, India) and Sitamadhi (Bihar, India). Three symptomatic samples from each locations were tested for phytoplasma detection using the universal primer P1/P7 (Deng and Hiruki, 1991;Schneider et al., 1995) in a first round, then primers R16F2n/R16R2 in nested PCR assays (Gundersen and Lee, 1996). Products of ca. 1.8 kb and 1.2 kb, respectively, were amplified from all symptomatic samples. Six amplicons of ca. 1.2 kb were directly sequenced from both the ends and found 99% identical to each others. One representative 16Sr DNA C. variegatum phytoplasma sequence form each location was deposited in GenBank under the accession Nos KJ161308 (Lucknow) and KJ161309 (Sitamadhi). Both tphytoplasma isolates shared maximum 16S rDNA sequence identity (99%) among themselves and with several isolates of Candidatus Phytoplasma asteris (16SrI group) from different parts of the world. Results of phylogenetic analyses of 1.25 kb 16Sr DNA products from Lucknow and Sitamadhi isolates revealed that the Lucknow isolate clustered together with strains of 16SrI-D subgroup, whereas the Sitamadhi isolate clustered with 16Sr I-B member strains of reference isolates in GenBank. Candidatus Phytoplasma asteris has been associated with diseased Croton spp. from Colombia (Perilla et al., 2012; HG764351) but, to the best of our knowledge it has never been reported from India.

First Report of Aster Yellows Phytoplasma in Soybean in Michigan.

During the summer of 2012, soybean plants in a commercial field in Clinton County, Michigan, exhibited symptoms characteristic of phytoplasmal diseases (1,2). Symptoms included extensive top dieback, stunting, purple stem surfaces, internal necrosis, leaf vein discoloration, and bud proliferation. Approximately 80% of plants in a half hectare along the southern edge of the field were symptomatic, although the majority of plants in the 4-ha field appeared symptomless. Total genomic DNA was extracted from one symptomatic and one asymptomatic leaf sample using a Qiagen DNeasy Plant Mini Kit (Qiagen, Germantown, MD) according to manufacturer's instructions. The DNA was used as template in direct PCR primed by the phytoplasma-universal primers P1/P7 followed by nested PCR primed by P1/AYint (3). Reactions containing template DNA from the symptomatic plant yielded ribosomal RNA gene amplicons of 1.8 kbp (P1/P7-primed) and 1.6 kbp (P1/AYint-primed), respectively. Reactions containing template DNA from the asymptomatic plant or water did not yield amplicons. The products of PCRs primed by P1/P7 were purified using PureLink PCR Purification kit (Life Technologies, Carlsbad, CA) and cloned in a pGem T-Easy vector system (Promega, Madison, WI). Three separate clones were sequenced using the sequencing primers M13For, M13Rev, SAYF nt 755, (5'-AAAGCGTGGGGAGCAAACAG), and SAYR nt 1159, (5'-TTTGACGTCGTCCCCACCTT). The sequences of all three clones were identical. A consensus (Sequencher 4.1, Gene Codes Corporation, Ann Arbor, MI) nucleotide sequence of 1,830 bp was deposited in GenBank under the accession number KF528320. A BLASTn similarity search revealed that the sequence shared 100% identity to rDNA of aster yellows phytoplasma (AF222063). Additionally, analysis of the 16Sr group/subgroup classification, based on in silico RFLP analyses using iPhyClassifier (4), indicated that the soybean phytoplasma is a member of subgroup 16SrI-B aster yellows phytoplasma subgroup. In a phylogenic tree deduced using the neighbor joining algorithm, the phytoplasma consensus sequence obtained from soybean in Michigan clustered with other group 16SrI (aster yellows phytoplasma) strains. While aster yellows phytoplasma has been previously reported in soybean in Wisconsin (2), to our knowledge, this is the first report of aster yellows in soybean in Michigan.

Bougainvillea Shoot Proliferation, a New Disease Induced by Distinct Phytoplasmas

AbstractBougainvillea‐potted plants exhibiting typical phytoplasma‐induced symptoms, characterized by foliar chlorosis, shoot proliferation, leaf and bract deformations, and decline were observed in commercial nurseries, located in the state of São Paulo, Brazil. In this study, PCR assays using group‐specific primers revealed that phytoplasmas affiliated with the groups 16SrI and 16SrIII were associated with symptomatic plants. Molecular analysis based on conventional and virtual RFLP patterns and similarity coefficient calculations identified these phytoplasmas as belonging to subgroups 16SrI‐B and 16SrIII‐B. Phylogenetic analysis confirmed that these phytoplasmas were closely related to representatives of both subgroups. Transmission assays using dodder supported the initial evidence that the symptoms were associated with phytoplasmas.

First Report of 16SrII-D Phytoplasma 'Candidatus Phytoplasma aurantifolia' Associated with Mung Bean Phyllody in Andhra Pradesh, India.

Mung bean (Vigna radiata (L.) R. Wilczek) is an important edible legume grown in Asia, particularly in the Indian subcontinent, where it is used for human and animal consumption. In September 2013, 10% of a group of 90 mung bean breeding lines in experimental plots of S. V. Agricultural College, Tirupati, Andhra Pradesh, India, exhibited symptoms typical of a phytoplasma infection, including stunting, extensive proliferation of branches, reduction in leaf size, phyllody, and longitudinal splitting of green pods followed by germination of green seeds producing small plants. These symptoms have been associated with mung bean phyllody (1,3). While the severity of infection varied within each line, on average, 20% of symptomatic plants did not produce seeds at all. Leaf samples from two symptomatic plants and one asymptomatic plant were collected and DNA was extracted from leaves following a CTAB DNA extraction procedure (2). Direct PCR and nested PCR assay was performed using phytoplasma 16S rRNA universal primer pairs P1/P7 and R16F2n/R16R2 (4). Both of the symptomatic samples produced 1.8 kb and 1.2 kb size amplicons after direct and nested PCR cycles, respectively. No amplicons were produced with DNA from the asymptomatic sample. Nested PCR products (1.2 kb) from the two symptomatic samples corresponding to the F2nR2 region of 16S rDNA were directly sequenced on an ABI 3730 XL automated sequencer at McLab sequencing services (McLab, CA). Both samples were 100% identical and the representative sequence was designated as APMBP and deposited in GenBank with the accession number KF811205. BLAST analysis revealed a 100% sequence identity with 16SrII group 'Candidatus Phytoplasma aurantifolia' phytoplasmas that include sesame phyllody phytoplasmas isolated from sesame and tomato in India (KF429485 and JX104335, respectively). Subgroup identification was performed using the iPhyClassifier online tool (5). The samples were identified as 16SrII-D subgroup phytoplasma based on their 100% identity to the reference strain (GenBank Accession No. Y10097) and virtual RFLP profiles. Phylogenetic analysis based on the F2nR2 sequences with the representative sequences were placed the APMBP in a single distinct cluster with the 16SrII-D reference strain Y10097. Although occurrence of phyllody on mung bean in India was first reported in 1988 (3), the report was based on the appearance of symptoms. This pathogen was recently reported as associated with mung bean phyllody in Pakistan (1). To our knowledge, this is the first report of 'Ca. P. aurantifolia' strain infecting mung bean in India. Phytoplasmas belonging to subgroup 16SrII-D are known to have a wide host range, including chickpea, peanuts, sesame, and tomato, which are commonly grown in this region. Mung bean plants infected early failed to produce normal seeds indicating of the potential of this 16SrII-D phytoplasma to become a production constraint for mung bean and other host crops in the area.

First Report of Lettuce Chlorotic Leaf Rot Disease Caused by Phytoplasma in China

Lettuce (Lactuca sativa) as annual or biennial crop is an important vegetable in China. The lettuce variety Feiqiao, which is extensively cultivated in autumn and winter, is grown for its stem and is a characteristic species bred in Yong'an City. Since October 2005, a new disease of lettuce has been observed sporadically in the fields. Initially, chlorotic symptoms, or a faded red color, were observed on the inner leaves of the infected lettuce plants. Then, the inner leaves bleached and appeared pale, while the top leaves became straight and elongated, and stopped growing. Gummosis was observed at the base of young leaves, and the whole plant became stunted and died. The disease was named lettuce chlorotic leaf rot disease. In 2008, there was a disease outbreak in Yong'an City with an incidence of approximately 30%. In 2012, total DNA was extracted from 0.1 g of leaf tissue collected from 20 symptomatic and five asymptomatic lettuce plants using the CTAB method. A PCR analysis was performed using the phytoplasma-specific primer set R16mF2/R16mR1 (1). An approximately 1.4-kb amplicon was obtained from all 20 symptomatic plants, but no corresponding DNA fragment was amplified from the five asymptomatic plants. PCR products were cloned in Escherichia coli DH5α, using the pMD18-T vector (TaKaRa, Japan), and two isolates were sequenced. The two 1,431-bp sequences were identical (GenBank Accession No. KJ668578). A BLAST analysis revealed a 99% identity between lettuce chlorotic leaf rot phytoplasma and mulberry dwarf phytoplasma, a group 16SrI phytoplasma described by Win et al. in 2012 (3). After analyzing with iPhyclassifier, the virtual RFLP pattern derived from the 16S rDNA F2n/R2 fragment was most similar to the reference pattern of the 16Sr group I, subgroup B (NC_005303), with a pattern similarity coefficient of 0.99 (2). Additionally, the leaf veins and roots with symptoms were processed for ultrastructural examinations using transmission electron microscopy. Many typical phytoplasma-like bodies were observed in the sieve elements in the leaf veins and roots, and they were spherical to oval or dumbbell shaped and 200 to 800 nm in diameter. In agreement with these findings, seven strains of phytoplasma from 16SrI-A and 16SrI-B subgroups were reported in association with lettuce plants exhibiting various types of symptoms which were not completely consistent with those observed in China (4). To our knowledge, this is the first report of a phytoplasma associated with lettuce in China.