Group 16SrI Phytoplasmas Research Articles

Occurrence of Praxelis clematidea Witches'-Broom Disease Association with 16SrI Group 'Candidatus Phytoplasma asteris' in Hainan Island of China.

Praxelis clematidea is an invasive herbaceous plant belonging to Asteraceae family. From August to November 2020, the plants showing severe witches'-broom symptoms were found in farms and roadsides from Ding'an of Hainan Province, a tropical island of China. The disease symptoms were suggestive of phytoplasma infection. For pathogen detection, P. clematidea samples consisting of six symptomatic and three asymptomatic plants were collected from the farms and roadsites of Ding'an with 40 % incidence by conducting surveys and statistics. Total nucleic acids were extracted using 0.10 g of fresh leaf tissues of the plant through CTAB DNA extraction method. Conserved gene sequences of 16S rRNA and secA genes from phytoplasma were amplified by direct PCR using primer pairs of R16mF2/R16mR1 and secAfor1/secArev3, respectively. R16mF2/R16mR1 PCR amplicons were obtained for all symptomatic samples but not from the symptomless plants. The amplicons were purified and sequenced by Biotechnology (Shanghai) Co., Ltd. (Guangzhou, China). Sequences of 16S rRNA gene (1323 bp) and secA (732 bp) were obtained and all the gene sequences were identical, designated as PcWB (Praxelis clematidea witches'-broom)-hnda. Representative sequencs were deposited in Genbank with accession numbers of PP098736 (16S rDNA) and PP072216 (secA). Nucleotide BLAST (Basic Local Alignment Search Tool) search based on 16S rRNA gene sequences indicated that PcWB-hnda had 100% sequence identity (1323/1323) with 'Candidatus Phytoplasma asteris'-related strains belonging to 16SrI group like Waltheria indica virescence phytoplasma (MW353909) and Capsicum annuum yellow crinkle phytoplasma (MT760793); had 99.62 % sequence identity (1321/1326) with the phytoplasma strains of 16SrI group such as Oenothera phytoplasma (M30790). RFLP (Restriction Fragment Length Polymorphism) pattern derived from 16Sr RNA gene sequences by iPhyClassifier showed identical (similarity coefficient=1.00) to the reference pattern of 16SrI-B subgroup (GenBank accession number: AP006628). The results obtained demonstrate that the phytoplasma strain PcWB-hnda under study is a member of 16SrI-B subgroup. A BLAST search based on secA gene sequences indicated that PcWB-hnda shares 100% sequence identity (732/732 bp) with Pericampylus glaucus witches'-broom phytoplasma (MT875200), 99% sequence identify (728/732 bp) with onion yellows phytoplasma OY-M(AP006628), and 99% sequence identify (729/732 bp) with rapeseed phyllody phytoplasma isolate RP166 (CP055264), among other phytoplasma strains that belong to 16SrI group. Previous studies demonstrated that P. clematidea can be infected by phytoplasmas affiliate to the 16SrII group (GenBank accession number: KY568717 and EF061924) in Hainan Island of China. To our knowledge, this is the first report of a natural infection of P. clematidea by a group 16SrI phytoplasma in the Island of China. 16SrI group can infect agronomic important species such as areca palm in the island and P. clematidea can be a reservoir of 16SrI phytoplasmas. Therefore, it is necessary to search of potential vectors of the pathogens, which would contribute to epidemiological monitoring and prevention of the related diseases.

Ampelopsis grossedentata Represents a New Host of the 16SrI Group of Phytoplasma Associated with Yellow Leaf Symptoms in China.

Ampelopsis grossedentata, commonly known as "Vine Tea" and well-recognized for its rich flavonoid content, is mainly distributed in the southern regions of the Yangtze River basin in China. These regions include Hunan, Hubei, Jiangxi, and Guizhou provinces. Vine Tea is mainly consumed as an herbal tea and has garnered attention for its reported health benefits, including antioxidant, anti-inflammatory, anti-tumor, anti-diabetic, and neuroprotective properties. It has been used to alleviate coughs and sore throats (Zhang et al., 2021; Wang et al., 2017; Gao et al., 2009). In the Zhangjiajie region of Hunan province alone, the Vine Tea planting area reached 7,670.5 hectares and produced commercial goods worth 1.417 billion RMB in 2022. In May 2021, leaf margins and veins fading to yellowing mottling, and crumpling of leaf blades in the shape of a boat symptoms were found in ~16% of Vine Tea plants in the Sanjiakuan Township, Yongding District, Zhangjiajie region (29°15'E, 110°30' N) (Figure 1a, b, c). (Figure 1a, b, c). Phytoplasma-like microbial cells (small oval shaped bacterial cells, around 1000 nm in size) were observed in sieve tube cells in the phloem of diseased leaves using transmission electron microscopy. No such cell was observed in the phloem of healthy leaves (Figure 2a, b). To investigate the potential association between phytoplasma and the observed symptoms of the diseased plants, total DNA was isolated from ten diseasedeaves and compared with ten healthy leaves from the same field using SteadyPure Plant Genomic DNA Extraction Kit. The isolated DNAs were analyzed first in a direct PCR using universal phytoplasma primer pair R16mF2/R16mR1 targeting the 16S rRNA gene (Gundersen and Lee 1996) and specific pair rpF1/rpR1 (Lee et al. 1998) targeting the DNA fragment encoding partial ribosomal proteins (rp) L22 (complete) and S3 and S19 (partial). The initial amplified products were used as templates and further amplified by nested PCR respectively with primer pair R16F2n/R16R2 for the 16S rRNA gene (Lee et al. 1998) and the rpF2/rpR2 primer pair for the rp gene (Martini et al. 2007). No amplification was obtained with DNA from healthy leaf samples using any of the four primer pairs. The amplified fragments from diseased leaves by nested PCR were cloned and sequenced (Qingke Biotech, China). The obtained sequences have been deposited in GenBank with accession numbers OR282806 for the 16S rRNA gene and GenBank OR353012 for the rp gene. BLASTn analysis revealed that the partial 16S rRNA gene sequence in our sample shared 99.4% nucleotide sequence identity with 'Candidatus Phytoplasma sp.' (MW364378) and 'Peony yellows phytoplasma' (KY814723) of the 16SrI group. Similarly, our rp gene sequence shared 99.6% nucleotide identity with the rpI group of phytoplasma such as the 'Balsamine virescence phytoplasma' (JN572890) and 'Paulownia witches'-broom phytoplasma' (HM146079). Phylogenetic analysis of the 16S rRNA and rp sequences using MEGA version 7.0 revealed that the phytoplasma strain associated with A. grossedentata yellow leaf syndrome in our study site belonged to the 16SrI (Candidatus Phytoplasma asteris) group of phytoplasma (Figure 3a, b). Using the interactive online phytoplasma classification tool iPhyClassifier (Zhao et al., 2009), virtual restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene sequences showed our strain having a distinct RFLP map but was closest to that of the onion yellow phytoplasma 16SrI-B subgroup (GenBank accession number: AP006628), with a similarity coefficient of 0.94 (Figure 4a, b). To confirm phytoplasma transmission, healthy plants were inoculated with three scions of infected plants of A. grossedentata. After 16 days, the new leaves of the inoculated A. grossedentata showed yellow leaf symptoms (Figure 5a, b, c), akin to the symptoms originally observed in the field, and the outer contour of the leaf margin appeared chlorotic. After 26 days, primer pairs R16mF2/R16R1 and R16F2n/R16R2 were used for nested PCR detection of phytoplasma in symptomatic A. grossedentata leaves. Phytoplasma was detected in the first and second leaves of symptomatic branches and leaves while negative control showed no amplification. Sequencing of the amplified fragments showed 100% nucleotide identity to the strain from the grafting source. Our results indicated that the pathogen and the disease can be transmitted by tissue grafting, consistent with the biological characteristics of phytoplasma, and further confirmed that the phytoplasma was the pathogen of yellow leaf syndrome of A. grossedentata. Toour knowledge, this is the first report of phytoplasma of group 16SrI affecting A. grossedentata.

First report of ‘Candidatus Phytoplasma australasia’‐related strains associated with disease of Jasminum multiflorum in India

Jasminum multiflorum (cv. Star Jasmine; family Oleaceae) is an important ornamental shrub native to South-East Asia. It is cultivated across the tropics for its ornamental value, in particular its fragrant, beautiful white flowers. During February 2020, symptoms of leaf yellowing and the premature decline of inflorescences were observed on 6.5% of plants grown in home gardens (Figs. 1, 2) in the Uttara Kannada district of Karnataka state, India. Four symptomatic and two symptomless plants were subjected to genomic DNA extraction using the CTAB method. Nested PCR was performed with universal phytoplasma 16S rRNA primers P1/P7 (Deng & Hiruki, 2; Schneider et al., 6) and R16F2n/R16R2 (Gundersen & Lee, 4). A DNA extract from the periwinkle virescence phytoplasma of group 16SrI, ‘Candidatus Phytoplasma asteris’ (MN223677) was used as the positive control. Amplification of the 16S rRNA gene was obtained only from symptom-bearing jasmine and the positive control, but not from the symptomless plants. The nested PCR products (~1.25kb) were purified and sequenced directly. Sequences were compared to those of reference phytoplasmas and subjected to in silico RFLP with iPhyclassifier (Zhao et al., 2009) and phylogenetic analysis using the neighbour-joining method of MEGA X (Kumar et al., 2018). The R16F2n/R16R2 gene sequences of the J. multiflorum phytoplasmas (GenBank Accession Nos. MW049328-MW049329) showed 99.9-100% sequence identity with those of the reference phytoplasmas of the former Peanut witches’ broom (16SrII) group, ‘Candidatus Phytoplasma aurantifolia’, including ‘Ca. P. australasia’ (Y10097). The virtual RFLP profiles of both J. multiflorum phytoplasmas were identical to each other and to those of the ‘Ca. P. australasia’ related strain (Y10097) of the 16SrII-D subgroup with a similarity coefficient of 1.00. The phylogenetic tree confirmed that the J. multiflorum phytoplasmas were closely related to the 16SrII-D, ‘Ca. P. australasia’ strain cluster (Fig. 3). Based on the results of the 16S rDNA nucleotide sequence comparison, phylogenetic and RFLP analyses, the J. multiflorum phytoplasmas associated with inflorescence yellowing and premature decline were identified as strains of the ‘Ca. P. australasia’, 16SrII-D subgroup. Phytoplasmas of 16SrII, 16SrXI (‘Ca. P. oryzae’) and 16SrI (‘Ca. P. asteris’) groups have been reported previously affecting J. sambac in Oman and India (Al-Zadjali et al., 1; Madhupriya et al., 5; Gopala & Rao, 3). However, this is the first report of the association of ‘Ca. P. australasia’ affecting J. multiflorum in India. Considering the economic importance of jasmine for the ornamental industry, this report provides significant information to support informed phytosanitary measures to prevent the spread of ‘Ca. P. australasia’ to nearby jasmine and other important members of the Oleaceae. The authors thank Department of Science and Technology, Government of India for providing financial assistance through DST-INSPIRE Fellowship scheme (IF.160687).

Multilocus sequence analysis for detection of finer genetic variation and phylogenetic interrelatedness in 16SrI group phytoplasma strains infecting different plants in China

The group 16SrI phytoplasmas are associated with severe diseases of many cash and ecological plants throughout the world. To date, the genetic variation and population structure of very closely related phytoplasma strains are still not fully understood in China. In this study, a multilocus sequence analysis (MLSA) scheme was developed using ten housekeeping genes (rp, tuf, secA, secY, ipt, dnaK, fusA, gyrB, pyrG and rpoB) fragments compared with 16S rDNA to analyze 18 phytoplasma strains infecting chinaberry, lettuce, mulberry, paulownia and periwinkle from ten provinces in China. The nucleotide site polymorphisms resolved all strains into 15 sequence types (STs), demonstrating extensive genetic diversity among the 16SrI group strain population. All the strains, classified in 16SrI-B and-D subgroup by 16S rDNA analysis, clustered into one clade and clearly differentiated into discrete subclades by phylogenetic analysis of the concatenated gene sequences. The 10 chinaberry witches’ broom (CWB) strains that were most closely related to two mulberry dwarf (MD) and hardly distinguished with 16S rDNA, were definitely split into four distinct clusters and 8 STs apparently congruent to their geographical locations. Two lettuce yellows (LY) strains in Sanming, Fujian province, China were more closely related to the onion yellows OY-M strain in Japan than the periwinkle virescence (PeV) and paulownia witches’ broom (PaWB) strains in China. The levels of variation in dnaK gene were higher than those in 16S rDNA and other genes tested. This MLSA is a promising approach for phytoplasma differentiation as well as for in depth examination of strain diversity and evolution of various 16Sr groups or subgroups.

First Report of 'Candidatus Phytoplasma asteris' Associated with Witches' Brooms on Sharp-Flowered Rush (Juncus acutiflorus) in Poland.

Juncus acutiflorus L. (Juncaceae) is a species of rush widespread in Europe, North Africa, and Southwest Asia. The health of sharp-flowered rush is threatened by witches’ brooms occurrence. Typical symptoms are dense masses of shoots, usually growing from a single point, with the resulting structure resembling a broom. Witches’ brooms are typically formed in response to Livia junci (Liviinae, Hemiptera) feeding, oligofag, which typically feeds on Juncus sp. These symptoms are similar to those observed in Juncus articulatus L. (Juncaceae) related to phytoplasma infection in Silesia, Poland (Jarzembowski et al. 2015). In August 2017, samples were collected from nine symptomatic and four asymptomatic plants growing in Jagniątkow, Poland. DNA from 100 mg of inflorescence and leaf samples was extracted using a DNeasy Plant Mini Kit (Qiagen, Syngen Biotech, Wroclaw, Poland) according to the manufacturer’s protocol. Additionally, 10 L. junci specimens (adult and the last larva stage) were collected from symptomatic plants and preserved in ethanol (75%). DNA from the insects (treated as one sample) was extracted using a DNeasy Blood and Tissue Kit (Qiagen, Syngen Biotech). The extracts were used as the template in polymerase chain reaction (PCR) assays with the universal phytoplasma primers P1/P7 followed by R16F2/R16R2 (Lee et al. 1998). Additionally, primers rp1/rp2 followed by rp3/rp4, allowing amplification of fragments of rpl22 and rps3 genes (Nakamura et al. 1996), and primers AYsecYF1/AYsec for amplification of secY gene were applied. Water blank samples were included as negative controls. PCR products of expected size for each primer set were amplified from the insects and six symptomatic plants that were assayed. No amplification was observed in symptomless J. acutiflorus samples or in water blanks. Amplicons representing three genetic loci were sequenced at Genomed S.A., Warsaw, Poland. DNA samples were sequenced at least twice in both directions. Sequencing results of the PCR products confirmed that six symptomatic plants and the insects were infected by a phytoplasma. The obtained sequences were nearly identical, and representative sequences of 16S rDNA fragments (accession nos. MG976242 to MG976246), secY gene (MG983765 to MG983769), and ribosomal protein gene (MG983760 to MG983764) from four plants and one insect sample were deposited in GenBank. Determination of the phytoplasma classification group was based on the nucleotide sequence of the F2n/R2 PCR fragment within the 16S gene. Using iPhyClassifier, the online tool for phytoplasma classification (https://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier_legacy.cgi), the sequence of J. acutiflorus phytoplasma shared 99.9% identity with that of the ‘Candidatus Phytoplasma asteris’ reference strain (GenBank accession no. M30790). BLAST analysis performed for ribosomal proteins and secY genes confirmed the highest identity (99%) of analyzed sequences with those of ‘Ca. P. asteris’ (CP015149 and KJ394491, respectively). To our knowledge, this is the first report of a phytoplasma of group 16SrI affecting J. acutiflorus in Poland and in the world. The impact of the detected phytoplasma in the regional ecosystem is being assessed. It is highly probable that L. junci is a possible vector for ‘Ca. P. asteris’.

Identification of Nedotepa curta Dmitriev as a potential vector of the Côte d’Ivoire lethal yellowing phytoplasma in coconut palms sole or in mixed infection with a ‘Candidatus Phytoplasma asteris’-related strain

Over 360 Hemiptera specimens were collected using sweep nets and hand-made aspirators from coconut palm fronds in six villages of Grand-Lahou. Eight families were identified including Aphrophoridae, Achilidae, Derbidae, Flatidae, Membracidae, Pentatomidae, Tropiduchidae, and Cicadellidae, the latter being the most abundant throughout the surveyed villages. PCR assays with primers targeting the 16S rRNA and the secA translocation protein genes yielded PCR amplicons from 216 out of 296 (73%) of the tested specimens of a newly identified cicadellid leafhopper, Nedotepa curta Dmitriev. PCR amplicons were purified, cloned and sequenced. The 16S rDNA and secA sequences from N. curta showed a 99% of sequence identity with those of the Côte d’Ivoire lethal yellowing (CILY) phytoplasma, member of subgroup 16SrXXII-B ‘Candidatus Phytoplasma palmicola’-related strain, previously identified in coconut-growing villages of Grand-Lahou. This suggested N. curta as a potential vector for the CILY phytoplasma. Four symptomatic coconut palms (7.4%) were found infected by a phytoplasma of group 16SrI, in mixed infection with the CILY phytoplasma (16SrXXII-B) in two palms, and alone in the other two palms, where the CILY phytoplasma was not detected. The 16SrI phytoplasma was also found in two N. curta specimens, and in the weeds Dalbergia saxatilis and Baphia nitida. Results indicating that mixed infection of both 16SrXXII-B and 16SrI phytoplasmas occurs in coconut palms affected by CILY in Grand-Lahou, and may impact disease management and control.

Diverse phytoplasmas associated with leguminous crops in Russia

A large scale survey of diseased legume plants (mainly clover and alfalfa in the Fabaceae family) was conducted from 2009 to 2013 in four Economic Regions of Russia, Northern (Arkhangelsk and Vologda oblast), Central (Moscow oblast), Volga (Samara oblast) and West Siberian (Novosibirsk oblast). The majority of infected clover plants exhibited symptoms typical of clover phyllody (CPh), clover yellow edge (CYE), or clover proliferation (CP), and infected alfalfa plants exhibited symptoms typical of alfalfa witches’-broom (AWB). Of 161 symptomatic plants from 22 different legume species, 103 tested positive for phytoplasma infection. Phytoplasmas belonging to four groups and six subgroups were detected, of which 31.1% were group 16SrI, with the majority belonging to subgroup 16SrI-C- (causal agent of CPh disease), two belonging to 16SrI-B and two group 16SrI phytoplasmas not identified to the subgroup level;47.6% were group 16SrIII, with the majority belonging to subgroup 16SrIII-B or 16SrIII-B variant (causal agent of CYE disease), and one strain belonging to16SrIII-F; 8.7% were subgroup 16SrVI-A (causal agent of CP and AWB diseases); 9.7% were subgroup 16SrXII-A (causal agent of AWB disease); and 2.9% were mixed infected with subgroups 16SrIII-B and 16SrI-C. The predominant phytoplasma species detected varied by region. In the Northern and Central Regions, the majority of the phytoplasmas detected belonged to subgroups 16SrI-C and 16SrIII-B. In the West Siberian and the Volga Regions, the phytoplasmas predominately detected belonged to subgroups 16SrVI-A and 16SrXII-A, respectively. Subgroup 16SrIII-F was detected in a single plant in the West Siberian Region and a mixed infection of 16SrIII-B and 16SrI-C was detected in three plants, one in the Northern Region and two in the Central Region. Eleven species of insects of the order Hemiptera, suborder Auchenorrhyncha, were collected from leguminous plants in the Moscow oblast of the Central Region. Euscelis incisus and Aphrodes bicinctus were the most prevalent species and may be potential phytoplasma vectors in the Central Region.

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.

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.

First Report of Phytoplasmas Groups 16SrI and 16SrXV in Crotalaria juncea in Brazil.

Sunn hemp (Crotalaria juncea L., Fabaceae) is widely used as a cover crop in sugar cane and citrus plantations in Brazil. C. juncea has been reported in São Paulo State (SPS) by Wulff et al. (3) as a host of the phytoplasma associated with symptoms of huanglongbing (HLB) in citrus, a member of group 16SrIX, that induces witches'-broom in sunn hemp (3). In studying the distribution of group 16SrIX phytoplasma in C. juncea in SPS, we identified this species as a new host of two phytoplasmas. Sunn hemp fields were inspected for symptoms usually associated with phytoplasma infections, such as leaf yellowing, shoot proliferation, witches'-brooms, and virescence. Ninety-nine plant samples were collected and DNA was extracted with the CTAB protocol from stems. Nested PCR was carried out with primers P1/P7, followed by amplification with primers fU3/rU5 (2), both sets being universal for phytoplasma. Asymptomatic sunn hemp samples were used as negative controls and were negative in PCR reactions. PCR products were directly sequenced with primers P1/P7 and fU3/rU5 and phytoplasma identification was conducted with BLASTn and in silico RFLP analysis for delineation of subgroups (4). Plants showing leaf yellowing (three plants; Catanduva County), shoot proliferation (one plant; Ibirá County), or witches'-brooms (one plant; Promissão County) symptoms were found to be infected with the 16SrI phytoplasma group, subgroup S. The 16S rDNA sequence (GenBank Accession No. KF878383) showed 99% identity (E value 0.0) with Candidatus Phytoplasma asteris, Onion yellows phytoplasma OY-M (AP006628), Mulberry yellow dwarf phytoplasma (GQ249410), and Ash witches'-broom phytoplasma (AY566302), among other phytoplasmas from the same group. Sunn hemp plants with shoot proliferation (three plants) carried the 16SrXV phytoplasma group, subgroup A, found in Ibirá (two plants) and Catanduva (one plant) counties, SPS. This sequence (GenBank Accession No. KF878382) displayed 99% identity (E value 0.0) with Ca. P. brasiliense, Hibiscus witches'-broom phytoplasma (AF147708), Guazuma ulmifolia witches'-broom phytoplasma (HQ258882, HQ258883), and Cauliflower stunt phytoplasma (JN818845). Both phytoplasma groups described in this report, 16SrI and 16SrXV, were collected in May 2010 and both have limited geographic distribution and occurred at low incidence. Phytoplasma of group 16SrI (Ca. P. asteris) was identified in C. spectabilis in India (1). To our knowledge, this is the first report of phytoplasmas groups 16SrI and 16SrXV in sunn hemp.

First report of a ‘Candidatus Phytoplasma asteris’ isolate affecting macadamia nut trees in Cuba

DNA was extracted from 0.5 g of leaf midribs of twelve macadamia plants collected (Murray & Thomsom, 1980), and used as a template for a nested PCR assay. Universal primer pairs that target the phytoplasma 16S rRNA gene, P1 (Deng & Hiruki, 1991) and P7 (Schneider et al., 1995) were used for the first reaction, and R16F2n/R16R2 (Gundersen & Lee, 1996) for the nested reaction. Nested PCR products of expected size (approximately 1250 bp) were obtained from ten symptom-bearing plants. PCR products were purified (Wizard SV Gel and PCR Clean-Up System, Promega, Madison, WI, USA), cloned (pGEMT-Easy Vector, Promega), and two individual clones per infected plant were sequenced using Macrogene Inc. Sequencing Service. The R16F2n/R16R2 sequences were subjected to in silico restriction fragment length polymorphism (RFLP) analysis with endonucleases AluI, BfaI, BstUI, HaeIII, HhaI, HinfI, HpaII, MseI and Tsp509 (pDRAW32 AcaClone Software, http://www.acaclone.com). Phylogenetic relationships were established between the macadamia phytoplasma and those of 16SrI and other phytoplasma groups (Mega 5.0, USA). The R16F2n/R16R2 sequences of phytoplasmas detected in the symptom-bearing samples were 100% identical to each other. The consensus sequence (1262 nt) of the macadamia phytoplasma was deposited in GenBank (Accession No. KC513772) and showed 99% of sequence identity with those of the 16SrI group ‘Candidatus Phytoplasma asteris’, including phytoplasma strains AY-CVB (AY265212), ACLR-AY (AY265211) and Baigah periwinkle little leaf phytoplasma (DQ266089), all members of subgroup 16SrI-F. The virtual RFLP profile of the macadamia phytoplasma was identical to those of the 16SrI-F subgroup, which suggests that the phytoplasma strain detected in Cuban macadamia trees may be a member of this subgroup. The results of sequence and RFLP analyses were confirmed by phylogeny (Fig. 2) since the macadamia phytoplasma grouped within the cluster corresponding to the 16SrI group, closely related to those of subgroup 16SrI-F. The group 16SrI was first recorded in Cuba in 1999 (Arocha et al., 1999) and since then in several other plant species. However, this is the first report of a phytoplasma of group 16SrI associated with symptoms in macadamia nut trees, a possible new host for 16SrI phytoplasmas. These results have a significant impact for Cuban agriculture since phytoplasma group 16SrI is well known by its widest plant host range and the most complex epidemiology worldwide, and macadamia may become an important future export crop for the Cuban fruit industry.

First report of A group 16SrI phytoplasma infecting epazote in China.

Epazote (Chenopodium ambrosioides L.), a plant native to South America, is a serious invasive weed in China. In May 2011, epazotes exhibiting small leaves and fasciation indicative of phyto- plasma infection were found in a pepper field in Qijiang County (China), with an incidence lower than 3%. Stem and leaf samples were collected from symptomatic and symptomless plants, total DNA was extracted and nested PCR performed using primers P1/P7 (Deng and Hiruki, 1991; Schneider et al., 1995) followed by primers R16F2n/R16R2 (Lee et al., 1998). Fragments of 1.2 kb corresponding to 16S rDNA were amplified exclusively from DNA extracts from symptomatic plants. Purified PCR products were cloned in pGEM-T Easy Vector (Promega, USA), sequenced (GenBank accession No. JN836942) and analyzed using iPhyClas- sifier online tool. The sequence shared 99.4% identity with the 16S rDNA sequence of the ‘Candidatus Phytoplasma asteris’ refer- ence strain (M30790) that belongs to 16SrI-B subgroup. The vir- tual RFLP pattern of the epazote phytoplasma 16S rDNA gene se- quence showed maximum similarity with the reference pattern of the 16SrI-B subgroup (NC005303), with a similarity coefficient of 0.98. A phylogenetic tree based on the 16S rDNA phytoplasma se- quences showed that the sequences clustered with members of the 16SrI-B subgroup. Taken together, the epazote phytoplasma was classified in subgroup 16SrI-B. Phytoplasmas identified as 16SrI-B strains have previously been identified in Morus alba, Amaranthus hypochondriacus and Sasa fortunei in China. To our knowledge, this is the first report of a subgroup 16SrI-B phytoplasma associat- ed with diseased epazote in our country. As a widely distributed weed in southwest China, phytoplasma-infected epazote may be a potential source of inoculum for crops.

Detection and characterization of a phytoplasma associated with witches’-broom disease of Salvadora persica in India

Salvadora persica trees with symptoms of witches’-broom, little leaf and severe leaf curling were observed in the Biodiversity Park, North Delhi (India) during February 2010. Using a nested-PCR assay targeting two phytoplasma specific loci (16S ribosomal RNA and the ribosomal protein gene) and sequence analysis of the amplified products, we identified the associated phytopathogen as a phytoplasma of group 16SrI. RFLP-mediated characterization revealed that the phytoplasma belongs to subgroup 16SrI-B. Phylogenetic reconstruction also grouped the phytoplasma within group 16SrI. The study reports a new host of phytoplasma in India and worldwide.

First Report of a Group 16SrI Phytoplasma Associated with Amaranthus hypochondriacus Cladodes in China.

Amaranthus spp. are cultivated worldwide as leafy vegetable, cereal, and ornamentals. In China, stems and leaves of Amaranthus hypochondriacus L. are used as a vegetable (2). In July 2010, sporadic amaranth plants exhibiting symptoms of cladodes and spica proliferation were observed in a vegetable garden near Foshan, Guangdong, China. Stem samples were collected from two symptomatic and two asymptomatic plants. Total DNA was extracted with a modified cetyltrimethylammonium bromide (CTAB) method (1). Nested PCR with a combination of phytoplasma-specific universal primer pairs (P1/P7 and R16F2n/R16R2) amplified 16S rDNA sequences with the expected size of 1.2 kb from all samples of symptomatic amaranth plants, but not from the asymptomatic plants (3). Nested PCR products yielded identical AluI, HhaI, HpaII, HaeIII, KpnI, MseI, RsaI, Sau3AI, and TaqI restriction fragment length polymorphism (RFLP) profiles with chinaberry witches'-broom phytoplasma (16SrI-B subgroup), but different from peanut witches'-broom phytoplasma (16SrII group), jujube witches'-broom phytoplasma (16SrV group), and paulownia witches'-broom phytoplasma (16SrI-D subgroup). Nested PCR products were purified, cloned in pMD18-T Simple Vector (TaKaRa, Dalian, China), and sequenced. The 16S rDNA sequences were identical and deposited in GenBank (Accession No. JF323034). GenBank BLASTn analysis indicated that the amaranth extracts showed as high as 99% sequence identity with the members of 16SrI group phytoplasmas, including those associated with arecanut yellow leaf disease (FJ998269) and aster yellow AY-27 (HM467127). A polygenetic tree was constructed using MEGA 4.0 based on the 16S rDNA sequences of amaranth cladode phytoplasma and other phytoplasmas belonging to 16SrI phytoplasma group. In phylogenetic analysis, the sequences clustered on a single branch with members of 16SrI-B subgroup in the tree. Therefore, the phytoplasma was classified in subgroup 16SrI-B. To our knowledge, this is the first report of a subgroup 16SrI-B phytoplasma associated with diseased A. hypochondriacus in China.

Identification and molecular characterization of the phytoplasma associated with peach rosette-like disease at the Canadian Clonal Genebank based on the 16S rRNA gene analysis

Peach trees exhibiting peach rosette-like disease symptoms and infected by a phytoplasma of group 16SrI ‘Candidatus Phytoplasma asteris’ at the Canadian Clonal Genebank were further tested for the pathogen characterization based on the 16S rRNA gene. Nested PCR with phytoplasma universal primers R16mF2/R1 and R16F2n/R2 resulted in amplification of products of approximately 1.25 kb from all four symptomatic trees tested. Virtual RFLP of the R16F2n/R2 sequenced amplicons with selected restriction endonucleases showed unique RFLP patterns when compared to the described 16SrI phytoplasma subgroups; these data were confirmed by phylogenetic analyses. The phytoplasma was therefore assigned as a member of a new 16SrI subgroup (16SrI-W). Results represent the first report of a new phytoplasma 16SrI subgroup infecting peach in Canada, and provide a valuable tool for further epidemiological studies on this phytoplasma in peach.

Identification of Potential Vectors and Alternative Plant Hosts for the Phytoplasma Associated with Napier Grass Stunt Disease in Ethiopia

AbstractNapier grass (Pennisetum purpureum), the most important forage crop in East Africa, has recently been affected by a devastating disease named Napier Grass Stunt (NGS). A phytoplasma of group 16SrI has been associated with NGS in Kenya and Uganda, whereas in Ethiopia, group 16SrIII was previously identified in NGS affected fields. However, no insect vectors or alternative hosts have been recorded for NGS in East Africa. During 2005, surveys were conducted at NGS‐affected plantations of Debre‐Zeit and Zwai field stations in Addis Ababa. Leaf samples were collected from weeds located in and surrounding the NGS‐affected areas. Leafhopper species were also surveyed by vacuum sampling in a search for natural phytoplasma vectors. Total DNA was extracted from plants and insects, and used as a template in nested polymerase chain reaction (nPCR) with universal 16S rRNA phytoplasma primers. Restriction fragment length polymorphism (RFLP), sequencing of PCR products and phylogenetic analysis were conducted for a finer identification and characterization of the phytoplasma associated with NGS. A 16SrIII‐A phytoplasma with 100% of identity in the 16S rRNA sequence with that of the previously identified one in Napier Grass (accession no. DQ305977) was identified from alfalfa, Medicago sativa (accession no. DQ305982), Cynodon dactylon (accession no. DQ3058983), Exitianus sp. (DQ305980) and Leptodelphax dymas collected in Debre Zeit (accession no. DQ305979) and Zwai (accession no. DQ305978). These findings suggest that M. sativa and Cy. dactylon are alternative reservoirs, and Exitianus sp. and L. dymas, potential vectors of the 16SrIII‐A phytoplasma, which may have epidemiological implications in spreading NGS in Ethiopia.

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.

"Candidatus phytoplasma australiense," a new phytoplasma taxon associated with Australian grapevine yellows.

A phytoplasma was detected in naturally diseased 'Chardonnay' grapevines exhibiting symptoms of Australian grapevine yellows disease. The use of PCR designed to amplify phytoplasma DNA resulted in detection of phytoplasma DNA in all of the diseased plants examined; no phytoplasma DNA was detected in healthy seedling grapevines. The collective restriction fragment length polymorphism (RFLP) patterns of amplified 16S ribosomal DNA differed from the patterns described previously for other phytoplamas. On the basis of the RFLP patterns, Australian grapevine yellows phytoplasma was classified as a representative of a new subgroup, designated subgroup 16SrI-J, in phytoplasma 16S rRNA group 16SrI (aster yellows and related phytoplasmas). A phylogenetic analysis in which parsimony of 16S rRNA gene sequences from this and other group 16SrI phytoplasmas was used identified the Australian grapevine yellows phytoplasma as a member of a distinct subclade (subclade xii) in the phytoplasma clade of the class Mollicutes. A phylogenetic tree constructed on the basis of 16S rRNA gene sequences was consistent with the hypothesis that there was divergent evolution of Australian grapevine yellows phytoplasma and its closet known relative, European stolbur phytoplasma (subgroup 16SrI-G), from a common ancestor. The unique properties of the DNA from the Australian grapevine yellows phytoplasma clearly establish that it represents a new taxon, "Candidatus Phytoplasma australiense."