Phytoplasma 16S rDNA Sequences Research Articles

First report of ‘Candidatus Phytoplasma trifolii’ associated with little leaf disease of Capsicum chinense from the northeast of India

King chilli or bhut jolokia (Capsicum chinense, family Solanaceae), is a variety of capsicum indigenous to northeast India. It is considered one of the hottest chillies in the world and is one of the most valuable spice crops in India, being grown for culinary and medicinal purposes. In April 2022, phytoplasma-associated symptoms such as shortening of leaves, petioles and internodes, and stunting of the whole plant were observed on C. chinense plants in Jorhat, a district in Assam, India (Figure 1). To investigate the possibility of a phytoplasma being associated with the symptoms, total DNA was isolated from leaf samples collected from six diseased and six asymptomatic plants using a CTAB protocol (Kollar et al., 1990). The DNA was analysed by direct and nested PCR with universal phytoplasma 16S rRNA primers, and P1/P6 (Deng & Hiruki, 1991) and R16F2n/R16R2 primers (Gundersen & Lee, 1996), respectively. Amplicons of the expected size (1.5 Kb after direct PCR and 1.25 Kb after nested PCR) were obtained from diseased plants only. Nested PCR R16F2n/R16R2 products were Sanger sequenced in each direction, and the consensus sequence deposited in GenBank (Accession No. ON819760). BLASTn analysis revealed that the C. chinense phytoplasma 16Sr DNA sequence shared >99% sequence identity with those of the 16SrVI group including the isolate used to describe ‘Candidatus Phytoplasma trifolii’ (AY390261; Hiruki & Wang, 2004) and many strains found in India (e.g. MN861357 and MN861370). A phylogenetic tree (Figure 2) and an iPhyClassifier-based virtual RFLP analysis of the 16S rRNA gene sequence revealed that the phytoplasma-associated with little leaf of C. chinense belongs to the16SrVI-D subgroup with a similarity coefficient of 1.00. Previously, different phytoplasma subgroups have been associated with Capsicum annuum in India (‘Ca. P. asteris’, 16SrII-D; Sharma et al., 2015). In the present study, we confirmed the association of the ‘Ca. P. trifolii’ subgroup D of phytoplasmas with king chilli. The 16SrVI-D subgroup of phytoplasma has been associated with several other agricultural crops and weeds in northeast India (Dutta et al., 2022). To the best of our knowledge, this is the first report of the association of the 16SrVI-D phytoplasma subgroup with C. chinense and extends the known host range of this phytoplasma in India. In this study, affected crops had about 5–8% disease incidence based on visual observation and bearing in mind the symptoms observed, significant yield losses in commercially grown king chilli may occur. Further epidemiological and insect vector studies are needed to help formulate effective management strategies to prevent further spread.

First report of a ‘Candidatus Phytoplasma australasia’ (16SrII‐D) strain associated with witches’ broom and little leaf symptoms of Martynia annua in India

Martynia annua (Martyniaceae) is a small, herbaceous annual plant distributed across India. It is used for the treatment of epilepsy, tuberculosis, sore throats and wounds (Kaushik et al., 2021). In August 2021, symptoms of witches’ broom and little leaf were observed in M. annua in the Anantapur forest area, Andhra Pradesh. India. Leaves were collected from two diseased and two symptomless plants (Figures 1 and 2). Total DNA was extracted from 100 mg of leaf midribs using a modified CTAB DNA extraction method (Murray & Thompson, 1980). DNA extracts were analysed by direct and nested PCR with universal phytoplasma 16S rRNA primers, P1/P7 (Deng & Hiruki, 1991) and R16F2n/R16R2 (Gundersen & Lee, 1996), respectively. Amplicons of the expected size (c. 1.8 kb after direct PCR and c. 1.3 kb after nested PCR) were obtained from diseased samples but not symptomless plants. Nested PCR R16F2n/R16R2 products were directly sequenced (Eurofins Genomics India Pvt., Ltd., India) and the consensus sequence was deposited in GenBank (Accession No. OK135941). A BLASTn analysis revealed that the M. annua phytoplasma 16Sr DNA sequence shared 100% sequence identity with those of 16SrII group phytoplasmas, including ‘Candidatus Phytoplasma australasia’ (MN565885, MN565886) and ‘Ca. Phytoplasma aurantifolia’ (MK217101, MK421430). Phylogenetic analysis (Tamura et al., 2007) based on the R16F2n/R2 sequence and performed with the neighbour-joining method (MEGA software version 7.1) revealed that the Anantapur 16SrII phytoplasma had the closest relationship with 16SrII, subgroup D isolates, particularly with the strain ‘Ca. P. australasia’ (Y10096) (Figure 3). Virtual RFLP profiles using restriction enzyme AluI and the R16F2nR2 sequence were generated with iPhyClassifier (https://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi). The RFLP patterns of the Anantapur phytoplasma 16S rDNA F2nR2 sequence with AluI enzyme agreed with the phylogenetic grouping. The Anantapur phytoplasma was confirmed as a member of the 16Sr group II, subgroup D with a similarity coefficient of 1.00, compared with virtual RFLP profiles of the ‘Ca. P. australasia’ strain (Y10097). This is the first report of a ‘Ca. Phytoplasma australasia’ strain (16SrII-D) associated with witches’ broom and little leaf symptoms of M. annua in India. Since the 16SrII group has a wide host range and occurrence in India, and particularly the subgroup 16SrII-D in the south region, the present finding has a significant impact for future epidemiological studies. The authors would like to thank the support and encouragement from the Associate Director of the Research, Regional Agricultural Research Station, Tirupati.

First Report of Phytoplasma ‘Candidatus Phytoplasma aurantifolia’ Associated with Purple Top Diseased Potatoes (Solanum tuberosum) in Guangdong Province, China

China is the largest producer of potatoes in the world. In 2014, a total of 96 million metric tons of potatoes were produced on more than 5.6 million hectares. Recognizing its importance, the Chinese central government elevated potatoes in 2010 to the status of the fourth major staple crop, following rice, wheat, and corn. In recent years, phytoplasma-associated diseases are becoming increasingly important in potato production, with an incidence rate of 80 to 100% in some commercial fields, according to disease surveys conducted by our team from 2005 to 2017 in seed and commercial fields of Yunnan Province and Inner Mongolia Autonomous Region. In China, six phytoplasma 16Sr groups, associated with vegetables, cereals, flowers, shrubs, fruit, and ornamental trees, in 29 genera, have been reported (https://plantpathology.ba.ars.usda.gov/cgi-bin/​resource/phytoclass.cgi?strainsearchbox=&countrysearchbox=China&submit_country_​search=search&diseasesearchbox=&hostsearchbox=&group_popup=I&subgroup_popup=​all). We found that phytoplasma group 16SrI, 16SrVI, and 16SrXII were associated with potatoes. Information on phytoplasma associated with potatoes in many potato growing areas remains little known (Cheng et al. 2015). Guangdong Province, especially Huidong County, is one of the major winter potato production areas in China. During a potato disease survey conducted in Huidong County, Guangdong Province (22.9850°N, 114.7200°E) in March 2014, prior to harvest, eight samples collected displayed symptoms of proliferation, upright growth, purpling of apical leaves, shortened and thick-ended stolons, stolons with multiple tubers, and formation of aerial tubers—all characteristics of phytoplasma disease. The incidence of phytoplasma in those fields ranged from 20 to 35%. Four samples were collected from asymptomatic plants at the same time. Total DNA was extracted from tissues (leaves, stems, and roots) of symptomatic and asymptomatic plants using a DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. A nested polymerase chain reaction (PCR) was performed by using primer pair P1/P7, followed by P1A/16S-SR (Lee et al. 2004). A 1.5-kb PCR fragment was amplified from the DNAs of all symptomatic plants and none of the symptomless plants. The PCR products were cloned into a pCR8/GW/TOPO vector (Invitrogen, Carlsbad, CA) and sequenced by GENEWIZ (South Plainfield, NJ). The potato phytoplasma 16S rDNA sequence (GenBank accession no. KM212951) was analyzed by iPhyClassifier software (https://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi) (Zhao et al. 2013), and the results showed that the 16S rRNA gene sequence had a 99.7% sequence identity to the reference strain (GenBank accession no. Y10097) for ‘Candidatus Phytoplasma aurantifolia’ (White et al. 1998). The restriction fragment length polymorphism similarity is identical (coefficient 1.00) to the reference pattern of the 16Sr group II, subgroup A (GenBank accession no. L33765). The phylogenetic tree was constructed using representative phytoplasma strains reported from China by the neighbor-joining method with 1,000 bootstrap replicates in the program MEGA 4 (Tamura et al. 2007). The results indicated that the potato phytoplasma 16S rDNA sequence isolated in this study clustered with two 16SrII group strains reported from China (tomato yellows strain SGW and kidney bean little leaf [Dong et al. 2013]) and from Korea (pigeon pea witches’ broom). ‘Ca. P. australiense’ is on the A1 regulated organism list for Canada and Bahrain and quarantined by the United States. To our knowledge, this is the first report of ‘Ca. P. australiense’ associated with potatoes in China. Phytoplasmas from Guangdong Province may be easily spread to spring and summer potato production areas through tubers. This might be one of the factors accounting for the increasingly widespread and important of phytoplasma-associated diseases on potatoes in China. Therefore, further study on identifying alternate host plants, potential vectors, and geographic distribution will be helpful to control this phytoplasmal disease.

First Report of ‘Candidatus Phytoplasma asteris’ Related Strain Associated with Sweet Cherry Fasciation Disease in China

HomePlant DiseaseVol. 102, No. 1First Report of ‘Candidatus Phytoplasma asteris’ Related Strain Associated with Sweet Cherry Fasciation Disease in China PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of ‘Candidatus Phytoplasma asteris’ Related Strain Associated with Sweet Cherry Fasciation Disease in ChinaJ. Wang, C. X. Ai, X. M. Yu, K. P. Zhang, R. Gao, and X. D. LiJ. Wang†Corresponding author: J. Wang; E-mail: E-mail Address: jiewangsdau@163.comSearch for more papers by this author, C. X. AiSearch for more papers by this author, X. M. YuSearch for more papers by this author, K. P. ZhangSearch for more papers by this author, R. GaoSearch for more papers by this author, and X. D. LiSearch for more papers by this authorAffiliationsAuthors and Affiliations J. Wang † C. X. Ai X. M. Yu K. P. Zhang , Shandong Institute of Pomology, Taian, 271000, China R. Gao , Institute of Plant Protection, Shandong Academy of Agricultural Sciences, Jinan, Shandong, 250100, China X. D. Li , Department of Plant Pathology, College of Plant Protection, Shandong Agricultural University, Taian, Shangdong 271018, China. Published Online:24 Oct 2017https://doi.org/10.1094/PDIS-06-17-0816-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Sweet cherry (Prunus avium L.) is one of the most popular fruit crops grown worldwide as a nutritious horticultural fruit. It has high antioxidant potential because of the rich content of polyphenols, vitamin C, and other nutritious chemicals. Phytoplasmal diseases on cherry widely occur in China, Iran, Turkey, the Czech Republic, Lithuania, and Chile, with symptoms of branch proliferation, leaf yellowing, floral virescence, and so on (Wang et al. 2015). In June 2015, 2-year-old sweet cherry (Prunus avium L. cv. Brooks) trees with symptoms of typical stem fasciation and top sickle-like deformation, decline, delay in flowering, and stiff fruit were observed in a sweet cherry orchard in Zoucheng, Shandong, China (35°24′N, 116°58′E). Three diseased trees were found in 2 ha of cultivated area surveyed, indicating this disease may be sporadic and rare. To identify the causal agent, total genomic DNA was extracted from leaves and stems of three symptomatic trees and two asymptomatic trees using the SurePlant DNA Kit (CWBIO, Beijing, China). Phytoplasma specific 16SrRNA gene and tuf gene fragments were amplified using phytoplasma specific universal primers, P1/P7 and R16F2n/R16R2 (Lee et al. 1998), and fTuf1/rTuf1 and fTufu/rTufu (Schneider et al. 1997), respectively. Specific fragments, approximately 1.25 kb and 0.8 kb in length, were amplified from symptomatic plants, while no fragment was amplified from asymptomatic samples. After the fragments were cloned into the pMD18-T vector (Takara Bio Inc., Dalian, China), sequences of 1,244 bp and 841 bp were obtained (GenBank accession nos. KY828850 and MF174060). Phylogenetic analysis of phytoplasma 16S rDNA sequences or tuf gene sequences based on the maximum likelihood method using MEGA 6.0 placed the SCF phytoplasma within the 16SrI cluster. The iPhyClassifier (Zhao et al. 2009) analysis showed that the obtained 16S rDNA sequence shared 99.6% similarity with that of the ‘Candidatus Phytoplasma asteris’ reference strain (M30790), and sweet cherry fasciation (SCF) phytoplasma was almost identical (similarity coefficient of 0.98) to the 16SrI-B reference strain (NC005303). The profiles of in vitro RFLP analysis obtained by digestion of the nested-PCR products with TaqI and BfaI were similar to those of in silico RFLP analyses and confirmed that the SCF phytoplasma is a variant of 16SrI-B. Moreover, the SCF phytoplasma shared 99.1% similarity with cherry leaf bunchy phytoplasma ChBL strain (AY034089, Lithuania) and 99.0% similarity with sweet cherry virescence phytoplasmas (HQ148153, China), both of which belong to the 16SrI group. Furthermore, the SCF phytoplasma shared 90.0% similarity with sweet virescence phytoplasmas (KF148153, 16SrV-B), which have also been reported in China. As one of the most widespread phytoplasma groups, aster yellows group (16SrI) was divided into at least 20 subgroups (Fránová et al. 2016). To our knowledge, this is the first worldwide report on the 16SrI-B variant phytoplasma having a significant impact on sweet cherry production with the symptoms of stem fasciation, yellowing, and stunting.

FIRST REPORT OF A 16SrIX GROUP (PIGEON PEA WITCHES'-BROOM) PHYTOPLASMA ASSOCIATED WITH GRAPEVINE YELLOWS IN IRAN

Association of 16SrXII-A phytoplasmas with grapevine yellows (GY) has been previously reported from Fars and Lorestan provinces of Iran (Salehi et al., 2014). During 2013-2014 surveys, symptoms resembling those of GY disease including thickening and downward rolling of leaves, vein chlorosis and necrosis, shoot dieback and stunting were observed in vineyards of Shiraz area (Fars province, Iran). Total DNA samples extracted from eight symptomatic and eight symptomless vines were tested for phytoplasma presence. In direct PCR using primers P1/P7 and nested PCR using primer pairs P1/P7 followed by R216F2n/R16R2 (Lee et al., 1998), respectively amplicons of ca. 1.8 and 1.2 kb were amplified from symptomatic but not from symptomless vines. Samples that yielded P1/P7 amplicons (4/8) were cloned and sequenced; obtained sequences showed 100% identity. A BLAST search using full length 16S rRNA gene sequence of a representative of these sequences (GenBank Accession No. KX011516) revealed that the sequence shared 99% homology with Indian Brassica rapa phyllody phytoplasma (HM988986), a representative of 16SrIX-C subgroup. Computer-simulated restriction analysis using iPhyClassifier showed that the RFLP profile of the Shiraz GY (SGY) phytoplasma 16S rDNA sequence was identical (similarity coefficient 1.00) to Picris echioides yellows phytoplasmas (Y16389) representative of subgroup C of the 16SrIX group. Phylogenetic analysis revealed that SGY phytoplasma clustered with16SrIX group phytoplasmas closer to Indian B. rapa phyllody phytoplasma, a 16SrIX-C member. To our knowledge, this is the first report of natural infection of grapevine by a 16SrIX phtoplasma in Iran.

Association of pigeon pea witches’ broom phytoplasma infectingCarpobrotus edulis(L.) N.E. Br in India

Leaf yellowing and little leaf symptoms with reduced size of flowers were observed in Carpobrotus edulis at Gorakhpur, Uttar Pradesh, India in the years 2012–2013. Phytoplasma presence was confirmed in symptomatic C. edulis leaves using phytoplasma universal primers P1/P7 and R16F2n/R16R2 that produced expected size amplicons of ~1.8 kb and ~1.2kb, respectively. BLAST analysis of 16S rDNA sequence of the phytoplasma detected revealed 99% similarity with those of tomato big bud, Brassica rapa phyllody, Iranian almond witches’ broom, Phlox drummondii, sesame phyllody and Iran eggplant big bud all belonging to 16SrIX group. Phylogenetic analysis of the C. edulis phytoplasma 16S rDNA sequence confirmed its closest relationship with members of the 16SrIX group. Virtual RFLP analysis of 1.25 kb sequence of 16S rDNA identified it as a new strain in the 16SrIX-C subgroup that is for the first time reported in C. edulis species worldwide.

First Report of a 16SrIX Group (Pigeon Pea Witches'-Broom) Phytoplasma Associated with Sesame Phyllody in Turkey.

Sesame (Sesamum indicum L.) is an important oilseed crops widely grown in the southern regions of Turkey. Sesame seeds are primarily used in production of tahini as well as a garnish on sweets and bakery products in the country. Sesame plants with phyllody disease symptoms have increasingly been observed in the fields of Antalya province since 2007. The disease incidence in these fields was found to range from 37 to 62% (2). Infected plants display a variety of the disease symptoms such as virescence, asymptomatic shoot proliferation, infertile flower formation, reduced leaf size, and thin and weak capsule development. Total genomic DNA was extracted from samples collected from symptomatic (10 plants) and asymptomatic healthy-looking plants (10 plants) using a CTAB method and amplified with universal primers P1/P7 and R16F2n/R16R2 in direct and nested PCR, respectively (1,3). Amplifications of the DNA from the symptomatic plants yielded a product of 1.8 kb in direct and 1.2 kb in nested PCR assays. No amplification was observed in symptomless plants of the same age and collected from the same fields. Amplicons were purified, cloned in a pTZ57R/T Vector, and sequenced using a Beckman Coulter 8000 CEQ Genetic Analysis System. Four aligned 16S rDNA sequences (1,845 bp) were found to be all identical and belonging to one species. One sequence was deposited in GenBank under the accession number KC139791. A BLAST similarity search revealed that the sequence shared 99% homology with the sequences of the members of 16SrIX group phytoplasmas, 'Brassica rapa' phyllody phytoplasma (HM559246.1) and Iranian Almond witches'-broom phytoplasma (DQ195209.1) available in GenBank. In addition, iPhyClassifier software (4) was employed to create a virtual RFLP profile. The analysis showed that the RFLP profile of the sesame phytoplasma 16S rDNA sequence is identical (a similarity coefficient of 1.00) to the profile of the 16Sr group IX phytoplasma reference sequence (Y16389). A phylogenetic tree was also constructed using the neighbor joining plot option of the Clustal X program. The sequence clustered together with 16SrIX group phytoplasmas. To our knowledge, this is the first report of a natural infection of sesame by a new phytoplasma species from the 16SrIX group in Turkey.

Association of Pigeon pea witches' broom phytoplasma (16Sr IX) infecting Phlox drummondii in India

Phlox is a genus of 67 species of perennial and annual plants in the family Polemoniaceae. Phlox are popular ornamental plants, and traditionally used to treat aches, colds, anaemia, diarrhoea and eczema. During a survey at the Indian Agricultural Research Institute Campus, New Delhi, from 2010 to 2011, symptoms including extensive yellowing and stunting, proliferation of shoots, little leaves and reduced size of flowers were observed in approximately 17% of plants of Phlox drummondii (Fig. 1). To investigate the possibility of a phytoplasma association with the symptoms, total DNA was isolated from leaf midribs and stems of three different infected plants. Healthy-looking plants without symptoms were also collected to use as a negative control using the phytoplasma enrichment procedure (Ahrens & Seemüller, 1). The DNA isolated from periwinkle infected with toria phyllody phytoplasma (group 16SrIX, pigeon pea witches' broom phytoplasma; Azadvar et al., 2) and maintained in greenhouse was used as positive control. Total DNA was used as a template for nested PCR using universal primers that target the phytoplasma 16S rRNA gene: P1/P7 (Deng & Hiruki, 3; Schneider et al., 5) and R16F2n/R16R2 (Gundersen & Lee, 4). Expected size amplicons of ~1.8 kb and ~1.2 kb, respectively, were amplified from all the three symptomatic P. drummondii plants (Fig. 2). However, no PCR products were obtained from the healthy-looking plant samples. The amplicon of ~1.2 kb was sequenced from a representative sample, and the consensus sequence obtained was submitted to GenBank (Accession No. KC178678). BLAST analysis of the partial 16S rDNA sequence of the P. drummondii phytoplasma revealed the highest sequence identity (99%) with that of phytoplasma members of group 16SrIX, ‘Candidatus Phytoplasma phoenicium’, the former Pigeon pea witches' broom group. Phylogenetic analysis (MEGA version 4.0, USA) confirmed the closest phylogenetic relationship of the P. drummondii phytoplasma 16S rDNA sequence with those of the 16SrIX phytoplasma group (Fig. 3). Hence, the P. drummondii phytoplasma was identified as a 16SrIX isolate. Phytoplasmas of group 16SrI ‘Candidatus Phytoplasma asteris’ subgroup M (16SrI-M) have been previously associated with P. drummondii in Lithuania (Samuitienė et al., 6). However, this is the first world report of the association of the 16SrIX group with a phytoplasma disease on P. drummondii. The first author is thankful to Department of Science and Technology, New Delhi, India for providing financial assistance under WOS ‘A’ scheme. The authors wish to express sincere thanks to Head, Division of Plant Pathology and Director, Indian Agricultural Research Institute for providing laboratory facilities.

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 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.

First Report of Group 16SrXII Phytoplasma Causing Stolbur Disease in Potato Plants in the Eastern and Southern Anatolia Regions of Turkey.

In recent years, a stolbur-like disease has had devastating effects on the yield and marketable quality of potato production in Erzurum (Eastern Anatolia) and Akcakale-Sanliurfa (Southern Anatolia) regions of Turkey. Potato plants exhibited several different symptoms including stunting, upward rolling of the top leaves along with reddish or purplish coloration, chlorosis, shortened internodes, swollen nodes, proliferated axillary buds, aerial tubers, and early plant decline. An extensive survey from 2003 to 2010 was performed and diseased plant samples were collected. Total genomic DNAs were isolated from the leaf mid-veins of the six different symptomatic and two symptomless plants selected. Nested-PCRs, carried out by using phytoplasma-universal primer pair P1/P7 followed by R16F2n/R16R2 (2), amplified 16S rDNA fragments (F2nR2) from only templates derived from symptomatic plants. F2nR2 PCR products from two independent symptomatic plants were cloned and sequenced from both directions with M13 universal primers. The obtained 16S rDNA sequence (GenBank Accession No HM485579) was subjected to virtual restriction fragment length polymorphism (RFLP) analysis using iphyclassifier software (3). Results indicated that the phytoplasma, here identified in association with potato plants, shared best sequence identity (99%) with members of subgroup 16SrXII-A (e.g., GenBank Accession No. EU010006). Moreover, collective RFLP pattern of potato-associated phytoplasma differed from digestion profiles of previously described 16SrXII subgroups, sharing best similarity coefficient (0.94) with the reference phytoplasma strain of subgroup 16SrXII-A (GenBank Accession No. AJ964960). Thus, it was confirmed that potato-associated phytoplasma represents a new 16SrXII subgroup (16SrXII-N). Furthermore, a new primer set (PatsecF/PatsecR) was designed for priming specific PCR-amplification of potato-associated phytoplasma 16S rDNA sequence. PCR reaction was successfully used for specifically detecting stolbur phytoplasma in infected potato plants. The use of this method may help to determine possible alternative hosts and vectors of potato phytoplasma, which is important for development of an integrated management strategy for effective control of this disease in the future. Presence of potato stolbur diseases in the Eastern Anatolia Region of Turkey has previously been reported (1). To our knowledge, this is the first report of occurrence of a 16SrXII group phytoplasma causing potato stolbur diseases caused in the Eastern and Southern Anatolia regions of Turkey.

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

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

First report of group 16SrI, ‘Candidatus Phytoplasma asteris’ associated with Mimosa pudica yellows in Indonesia

Mimosa pudica is a perennial plant, widespread in tropical and sub-tropical regions. Known in English as the sensitive plant because of its rapid leaf movements when touched, it is a serious weed in managed grasslands and plantation crops such as coffee, tea and oil palm (Waterhouse, 1993). It is also a popular ornamental plant with medicinal properties. On Samosir Island in North Sumatera, M. pudica is a common invasive species of crops and waste areas. In October 2007, leaf yellowing, little leaf and proliferation of axillary shoots were observed in plants from roadsides and in crop fields around Pangururan village. Leaf and stem samples were collected from three plants with symptoms and one symptomless plant. Total DNA was extracted and used as a template in a nested PCR assay with phytoplasma universal 16S rRNA gene primers R16F2m/R1 and fU5/rU3. All samples with symptoms yielded PCR products of expected size (∼880 bp), and identical HaeIII, RsaI and AluI RFLP profiles. No PCR products were produced with DNA from the symptomless plant. Nested PCR amplicons were purified and directly sequenced (http://www.dnaseq.co.uk). The 16S rDNA sequences were 100% identical, and deposited in GenBank (Accession No. EU273883). BLAST comparisons of the M. pudica phytoplasma 16S rDNA sequence yielded the highest identity (99%) with those of members of group 16SrI, ‘Candidatus Phytoplasma asteris’. Phytoplasmas have been found previously in Indonesia associated with diseases such as legume witches’ broom (Roechan et al., 1978), chilli and tamarillo diseases (Harling et al., 2009) and Kalimantan coconut wilt (Warokka et al., 2006), however, none of these have been from ‘Candidatus Phytoplasma asteris’. This is the first record of a yellows disease on M. pudica worldwide, its association with a phytoplasma and identification of ‘Ca. Phytoplasma asteris’ in Indonesia. Work in the UK was done under Defra plant health licence no. PHL 174B/4612(09/20003).

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

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

Phytoplasma associated with a witches’ broom disease of Gleditsia sinensis (Fabaceae) newly reported in China

Gleditsia sinensis (Chinese honeylocust) is widely distributed in China due to its important use in traditional Chinese medicine. In August of 2008, G. sinensis trees in Shanxi Province, exhibited symptoms of witches’ broom, stunting, smaller leaves, and yellowing gradually becoming browning: a phytoplasma infection was suspected. Total DNA was extracted from 0·5 g of phloem tissue from leaf midribs and stems of eight plants with, and eight plants without symptoms (Angelini et al., 2001). DNA was analyzed for phytoplasma by a nested PCR assay using phytoplasma universal 16S rDNA primers R16mF1/R16mR1 and R16F2n/R16R2 (Gundersen & Lee, 1996). PCR products of 1·4 and 1·2 kb, respectively, were produced from all plants with symptoms, but not from symptomless plants. Restriction fragment length polymorphism (RFLP) analyses of R16F2n/R16R2 amplicons with AluI, HhaI, TaqI, EcoRI, HaeIII, RsaI and HpaII endonucleases, indicated that all plants with symptoms contained a phytoplasma strain of group 16SrV (‘Candidatus Phytoplasma ulmi’), subgroup B (16SrV-B). The G. sinensis phytoplasma 16S rDNA sequence (GenBank Accession No. FJ457095) scored the highest identity (99%) with those of group 16SrV, thereby confirming strain identity based on RFLP. Phytoplasmas of 16SrV have previously reported on jujube (Zhu et al., 1997) and paper mulberry (Liu et al., 2004) causing witches’ broom in China. This is the first report of a ‘Candidatus Phytoplasma ulmi’-related strain affecting G. sinensis in China.

New 16Sr subgroups and distinct single nucleotide polymorphism lineages among grapevine Bois noir phytoplasma populations

AbstractBois noir (BN) is an insect‐transmitted grapevine yellows disease caused by phytoplasmas belonging to the stolbur subgroup 16SrXII‐A. In Italy, increasing prevalence of stolbur phytoplasma strains in vineyards suggests progressive spread of the disease and potential for heavy impacts on the wine industry. In this study, we investigated the genetic diversity of stolbur phytoplasma strains in BN phytoplasma populations. Nucleotide sequences of16S rRNAgenes from stolbur phytoplasma strains affecting vineyards in the Lombardy region of Italy and stolbur phytoplasma 16S rDNA sequences retrieved from GenBank were subjected to virtual restriction fragment length polymorphism analysis. Calculation of virtual restriction similarity coefficients revealed the presence of new subgroups in group 16SrXII (stolbur phytoplasma group). Representative strains of confirmed new subgroups 16SrXII‐F (XII‐F) and XII‐G and tentative new subgroups XII‐A1 through XII‐A19, XII‐H, XII‐I, and XII‐J as well as known subgroup XII‐A were from grapevines; strains representing three additional tentative new subgroups (XII‐K, XII‐L and XII‐M) were from other plant hosts. Nucleotide sequence alignments identified no less than nine genetically distinct 16S rDNA single nucleotide polymorphism lineages from grapevine, indicating a high degree of genetic heterogeneity within BN phytoplasma populations. The findings open new opportunities for in‐depth studies of the distribution of grapevine‐associated 16SrXII phytoplasma strains in weeds, insect vector populations and grapevines from vineyards located in different geographic areas.

Occurrence of Petunia Flattened Stem Caused by Phytoplasma

This study describes a phytoplasmal disease occurring in Petunia leaves grown in the glasshouse of the National Horticultural Research Institute, Suwon, Korea. Abnormal growth like flattened stem with flower malformation or phyllody was observed from the plant. The DNA extracted from the diseased leaves was amplified using a universal primer pair of P1/P6 derived from the conserved 16S rRNA gene of Mollicutes giving the expected polymerase chain reaction(PCR) product of 1.5 kb. In the nested PCR assays, the expected DNA fragment of 1.1 kb was amplified with the specific primer pair R16F1/R16R1 that was designed on the basis of aster yellows(AY) phytoplasma 16S rDNA sequences. The 1.1 kb PCR products were cloned and nucleotide sequences were determined, and the sequences of the cloned 168 rRNA gene were deposited in the GenBank database under the accession no. of EU267779. Analysis of the homology percent of the 168 rDNA of PFS-K showed the closest relationship with Hydrangea phyllody phytoplasma(AY265215), Brassica napus phytoplasma(EU123466) and AY phytoplasma CHRY(AY180956). Phytoplasma isolated from the diseased Petunia was designated as Petunia flat stem phytoplasma Korean isolate(PFS-K) in this study. Flattened stem occurring in Petunia was confirmed as infection of AY group of phytoplasma by determination of 16S rRNA gene sequences of phytoplasma and microscopic observation of phytoplasma bodies. This is the first report on the phytoplasmal disease in Petunia in Korea.

First Report on the Witches' Broom in Annual Statice (Limonium sinuatum) in Korea

In 2003 typical phytoplasma symptoms of witches` broom and flower malformation were observed on statice (Limonium sinuatum) plants grown at commercial greenhouses in Busan, South Korea. The DNA extracted from the infected leaves was amplified using universal primer pair of Pl/P6 derived from conserved 16S rRNA gene of Mollicutes giving the expected Polymerase chain reaction (PCR) product of 1.5 kb. In the nested PCR assays, the expected DNA fragment of 1.1 kb was amplified with the specific primer pair 16Fl/Rl that was designed on the basis of aster yellows (AY) phytoplasma 16S rDNA sequences. The 1.1 kb PCR products were cloned and nucleotide sequences were determined. The sequences were identical to that of Onion yellows OY phytoplasma (GenBank accession no. D12569) isolated from Onion in Japan. Electron microscopy of thin sections of leaf veins showed phytoplasma bodies in the phloem. Statice witches` broom symptom occurred on statice in commercial greenhouses in Korea was confirmed as infection of AY phytoplasma by transmission electron microscopy observation, and by determination of 16S rRNA gene sequences of phytoplasma.

'Candidatus phytoplasma ziziphi', a novel phytoplasma taxon associated with jujube witches'-broom disease.

Phylogenetic relationships of five jujube witches'-broom (JWB) phytoplasma isolates from four different districts, and other phytoplasmas, were investigated by 16S rDNA PCR amplification and sequence analysis. The 16S rDNA sequences of any pair of the five isolates of JWB phytoplasmas were > 99.5% similar. The JWB phytoplasma 16S rDNA sequences were most closely related to that of the elm yellows (EY) phytoplasma in 16S-group VIII. Phylogenetic analysis of the 16S rDNA sequences from the JWB phytoplasma isolates, together with sequences from most of the phytoplasmas archived in GenBank, produced a tree in which the JWB isolates clustered as a discrete subgroup. The uniqueness of the JWB phytoplasma appears to be correlated with a specific insect vector (Hishimonus sellatus) and the host plant (Zizyphus jujuba), or with a specific geographical distribution. The unique properties of the JWB phytoplasma sequences clearly indicate that it represents a novel taxon, 'Candidatus Phytoplasma ziziphi'.

Molecular and microscopical detection of aster yellows phytoplasma associated with infected parsnip

Typical phytoplasma yellows symptoms were observed in parsnip ( Pastinaca sativa L.) plants grown around Edmonton, Alberta, Canada. Examination of ultrathin sections of leaf midribs by electron microscopy revealed numerous phytoplasma bodies localized in the phloem cells. DNA extracted from the infected leaves was amplified with a 16S rDNA universal primer pair P1/P6 giving the expected PCR product of 1.5 kb. The phytoplasma was confirmed as a member of the aster yellows (AY) group by amplification with the specific primer pair R16(1)/F1/R1 that was designed on the basis of AY phytoplasma 16S rDNA sequences. In the nested PCR assays, the expected DNA fragment of 1.1 kb was amplified with this specific primer set. Similar restriction patterns were found for the 1.1 kb PCR products of the phytoplasma isolated from parsnip and an AY phytoplasma control after digestion with restriction endonucleases AluI, HhaI, KpnI and RsaI. This is the first reported observation of aster yellows in parsnip in Canada.