Subgroup 16SrI-A Research Articles

Occurrence of Phytoplasmas Belonging to 16SrII Group Associated with Cyanthillium cinereum Witches'-Broom Diseases in China.

Cyanthillium cinereum, which belongs to the family of Asteraceae, is an annual or perennial herbaceous plant with significant medicinal uses for treating colds and fever. During September to November of 2020, C. cinereum showing symptoms of witches'-broom were found in economic forests distributed in Ding'an, Hainan Province of China, with 20% incidence. The symptoms of the plant were consistent with infections by 'Candidatus Phytoplasma' species. To identify the pathogen, five symptomatic and three asymptomatic C. cinereum samples were collected. Total DNAs were extracted using 0.10 g fresh leaf tissues of symptomatic and asymptomatic C. cinereum through a CTAB DNA extraction method according to Doyle and Doyle (1990). PCR amplification were performed employing the primer pairs of R16mF2/R16mR1 (Gundersen and Lee, 1996) and secAfor1/secArev3 (Hodgetts et al., 2008) specific for the conserved gene fragments of 16S rRNA and secA from phytoplasma. The PCR products were purified and sequenced through Biotechnology (Shanghai) Co., Ltd. (Guangzhou, China), and the obtained sequences were deposited in GenBank. The phytoplasmal 16S rRNA and secA gene fragments obtained in the study were all identical with the length of 1325 bp (GenBank accession: PP098738) and 741 bp (PP072217), respectively. The phytoplasma strain was described as CcWB-hnda. A BLAST search based on 16S rRNA genes indicated that CcWB-hnda strain was identical to phytoplasmas belonging to 16SrII group like peanut witches'-broom phytoplasma strain T48 (OR239773) and 'Ca. Phytoplasma aurantifolia' strain TB2022 (CP120449). Virtual RFLP profiles based on 16S rRNA gene fragments obtained by iPhyClassifier (Zhao et al., 2009) showed that CcWB-hnda strain was a member of 16SrII-A subgroup with 1.00 similarity coefficient to the reference phytoplasma strain (L33765). A BLAST search based on secA genes indicated that CcWB-hnda had 100% sequence identity with phytoplasmas belonging to 16SrII group such as 'Ca. Phytoplasma aurantifolia' isolate TB2022 (CP120449), Vigna unguiculata witches'-broom phytoplasma (OR661282) and Emilia sonchifolia witches'-broom phytoplasma (MW353710). Phylogenetic analysis based on 16S rRNA and secA genes by MEGA 7.0 employing Neighbor-Joining method with 1000 bootstrap value (Kumar et al., 2016; Felsenstein, 1985) demonstrated that CcWB-hnda was clustered into one clade with the phytoplasmas belonging to 16SrII group, with 98% and 100% bootstrap value respectively. To our knowledge, this is the first report of C. cinereum infected by phytoplasmas belonging to 16SrII-A subgroup in China. Identification of the vector insects of the pathogens is necessary in future, revealing the epidemiology of the related diseases. Phytoplasmas belonging to same 16Sr group or subgroup can infect different plants and spread through them in nature. The finding in this study will be beneficial to epidemic monitoring and early warning of C. cinereum witches'-broom disease and the related plant diseases caused by the phytoplasmas belonging to 16SrII group.

First Report of 'Candidatus Phytoplasma australasiaticum' Associated with Phyllody, Virescence, and Witches'-Broom Disease in Chrysanthemum morifolium in Taiwan.

Chrysanthemum morifolium (Asteraceae) is commonly grown as commercial cut flowers or pot mums worldwide. Common diseases of chrysanthemum include bacterial blight, fungal diseases, viruses, and phytoplasmas (Verma et al. 2003; Taloh et al. 2020). In June 2022, C. morifolium plants showing virescence, stunting, witches' broom, and phyllody symptoms were observed in 10 plants representing 10% of the estimated 100 plants in a field in Taichung City, Taiwan (Fig. S1). Three symptomatic samples along with three asymptomatic ones were collected for further study. Nested PCR was performed with two primer sets, P1/P7 (Deng and Hiruki 1991; Schneider et al. 1995) and R16F2n/R16R2 (Gundersen and Lee 1996) to amplify nearly full-length of 16S rDNA from the collected samples. The target 1.2-kb DNA band was only amplified from the symptomatic chrysanthemum plants. The amplicons were sequenced and a representative sequence deposited in GenBank under accession number OR501416. This sequence was used to search GenBank database by the Basic Local Alignment Search Tool (BLAST) program through the web service of National Center for Biotechnology Information (NCBI). In the 16S rDNA analyses, the three randomly picked amplicons from chrysanthemum phyllody phytoplasma (CPP) shared 100% identity with one another, and all shared 99.5% identity with the, 'Candidatus Phytoplasma australasiae' reference phytoplasma strain (Y10097). Further analysis using iPhyClassifier (Wei et al. 2007) revealed that CPP was most similar to the pattern of the peanut witches' broom phytoplasma in the 16SrII-A subgroup (GenBank Acc. No. L33765), with a pattern similarity coefficient of 1.0. For confirmation, the secY gene was amplified by secY-F/R primers (Li et al. 2014), the 1.2-kb band was sequenced and deposit in GenBank (Acc. No. OR508986). BLAST analysis showed that the secY sequence of CPP shared 99.93% of sequence identities to several 'Ca. P. australasiaticum' strains (MN543069, CP097312, CP120449, KC953013, MW085916, MW070030, CP040925). The phylogenetic tree analysis based on the secY gene by MEGA11 employing maximum-likelihood algorithm was performed and the bootstrap value was set as 1000 times for support of the stability for the clades. The result showed that CPP is closely related to other strains in 16SrII group (Fig. S2). Taken together, CPP is a 'Ca. P. australasiaticum' related-strain in 16SrII-A subgroup. This is the first report of chrysanthemum as a host of this phytoplasma in Taiwan, and might have an impact to the horticultural industry and the growers.

Ipomoea obscura Represents a New Host of Phytoplasma Belonging to 16SrII Group Associated with Witches'-Broom Symptoms in China.

Phytoplasmas are phloem-limited plant pathogenic prokaryotes which can not be cultured in vitro. The pathogens could cause various plant symptoms such as witches'-broom, virescence, and leaf yellows. Ipomoea obscura is a valuable plant species belonging to the family Convolvulaceae, mainly used as a traditional Chinese medicine used to treat diseases such as dehydration and diuresis. In western countries it is commonly referred to as 'obscure morning glory'. During 2020 to 2021, plants showing abnormal symptoms including witches'-broom, internode shortening, and small leaves were found in Hainan Province, a tropical island of China. Approximately 30 % of I. obscura plants in the sampling regions which spanned 400 acres, showed symptoms. In order to identify the associated pathogen, six symptomatic samples and three asymptomatic samples were collected and total DNA were extracted from 0.10 g fresh plant leaf tissues using CTAB DNA extraction method. 16S rRNA and secA gene fragments, specific to phytoplasmas, were PCR amplified using primers R16mF2/R16mR1 and secAfor1/secArev3. The target PCR bands were obtained from the DNA of six symptomatic samples, whereas not from the DNA of the asymptomatic samples. The PCR products of phytoplasma 16S rRNA and secA gene obtained from the diseased samples were cloned and sequenced by Biotechnology (Shanghai) Co., Ltd. (Guangzhou, China). The 16S rRNA and secA gene sequences identified in the study were all identical with the length of 1330 bp (GenBank accession: OR625212) and 720 bp (OR635662) respectively. According to methods and protocols of phytoplasma identification and classification (Wei and Zhao, 2022), the phytoplasma strain identified in the study was described as Ipomoea obscura witches'-broom (IoWB) phytoplasma, IoWB-hnld strain. The partial 16S rRNA gene sequence of IoWB showed 100 % sequence identity over the full 1330 bp sequence to phytoplasmas belonging to 16SrII group like cassava witches'-broom phytoplasma (KM280679). The BLAST search of the 720 bp partial secA gene fragment of IoWB showed 100% sequence identity for the full sequence to phytoplasmas belonging to 16SrII group like 'Sesamum indicum' phyllody phytoplasma (OQ420657). RFLP analysis based on the 16S rRNA gene using iPhyClassifier demonstrated that the IoWB strain was a member of 16SrII-A subgroup with the similarity coefficient 1.00 to the reference phytoplasma strain (L33765). Phylogenetic analysis based on 16S rRNA and secA genes by MEGA 7.0 employing neighbor-joining (NJ) method with 1000 bootstrap value indicated that IoWB-hnld was clustered into one clade with the phytoplasmas belonging to 16SrII group, with 98% and 100% bootstrap value separately. To our knowledge, this is the first report that Ipomoea obscura can be infected by phytoplasmas belonging to 16SrII-A subgroup in China. This report adds to the host range of 'Ca. Phytoplasma aurantifolia', documenting the symptoms on I. obscura which will assist in monitoring and control of the associated pathogen.

First report of a 'Candidatus Phytoplasma australasiaticum'-related phytoplasma strain associated with shoot proliferation disease of variegated croton in Taiwan.

Codiaeum variegatum (family Euphorbiaceae) is a leaf ornamental commonly known as variegated croton, which is often found in gardens or grown as indoor plants. In December of 2022, two cutting-propagated variegated croton plants exhibiting abnormal shoot proliferation and little leaf symptoms (Fig. S1) were found in a nursery owned by a private breeder in Wanluan Township, Pingtung County, Taiwan. The plants were propagated from a single stock plant, which died during transplanting from a commercial nursery in Changzhi Township, Pingtung County. To determine the potential cause of such symptoms, leaf tissues were collected from the center of the two symptomatic plants. Their DNA were extracted with a Synergy 2.0 Plant DNA Extraction Kit (OPS Diagnostics, Lebanon, NJ) and used for further testing. As controls, two symptomless stock plants were collected from the commercial nursery in Changzhi Township and used to produce cutting-propagated plants; leaf DNA was extracted from each plant as described above. The DNA samples were subjected to PCR testing using the phytoplasma-specific primer pair P1/P7 (Schneider et al. 1995), and only DNA from the symptomatic plants produced the expected 1.8-kb amplicon. The two phytoplasma isolates detected in the plants were designated as CvaA and CvaB. After sequencing and analyzing the data using the iPhyClassifier program (Zhao et al. 2009), both CvaA and CvaB were classified to subgroup 16SrII-A (GenBank accession no. L33765) with a similarity coefficient of 1.0. The near-full-length 16S rDNA fragments of the detected isolates (GenBank accession no. OR794242) were also identical (1,463/1,463 bp) to that of NCHU2014 (GenBank accession no. CP040925, bp 537768-539230), a reference 'Ca. Phytoplasma australasiaticum'-related strain (16SrII-A) found in Taiwan (Chang et al. 2015; Rodrigues Jardim et al. 2023). To validate the results, the DNA samples were also tested with 16SrII group-specific semi-nested PCR targeting the elongation factor Tu gene. The outer and inner primer pairs used were TUF-II-F1/TUF-II-R1 and TUF-II-F2/TUF-II-R1, respectively (Al-Subhi et al. 2017). An expected amplicon was detected in the symptomatic samples but not in the symptomless counterparts. The amplified fragments' sequences (GenBank accession no. OR634931) were identical to that of the elongation factor Tu gene of the 'Ca. Phytoplasma australasiaticum'-related strain NCHU2014 (989/989 bp; GenBank accession no. CP040925, bp 139344-140332). The protein translocase gene secY of the detected phytoplasma was also amplified and sequenced using semi-nested primers SecYF1(II), SecYF2(II) and SecYR1(II) (Lee et al. 2010). Again, the sequences of the detected isolates (GenBank accession no. OR862773) were identical to that of NCHU2014 (1,263/1,263; GenBank accession no. CP040925, bp 192846-194108). The quality of the DNA samples was confirmed by PCR targeting the plant host's 28S rDNA using primer pair 28KJ/28C (Cullings 1992) and all symptomatic and symptomless samples produced the target amplicon (0.7 kb). 16SrII phytoplasmas have been detected in different host plants in Taiwan (Chang et al. 2015). To our knowledge, this is the first record of this group of pathogens infecting variegated croton in Taiwan. Branch-inducing phytoplasma has been used to improve the ornamental values of poinsettias, another Euphorbiaceae species (Lee et al. 1997). Further testing is needed to determine whether the phytoplasma detected in this work could be used for similar purposes.

First Report of 16SrII Group Related Phytoplasma Associated with Areca Palm Yellow Leaf Disease on Areca catechu in China.

The areca palm, Areca catechu L., family Arecaceae is an important herbal medicine which has potential for the treatment of parasitic diseases, digestive function disorders and depression (Peng et al. 2015). Yellow leaf disease (YLD), caused by phytoplasma, is a destructive disease of Areca catechu. In 1981, the YLD was first discovered in Tunchang, Hainan, China. According to the investigation in 2020, the occurrence area of YLD was 32 102.38 hm2 in Hainan, China, resulting in 50%-60% yield loss. Previous researchers based on 16S rDNA gene PCR amplification analysis showed that YLD in Hainan was caused by 16SrI group phytoplasma (Che et al. 2010). In August, 2022, yellow leaf symptoms were observed on middle and lower leaves of Areca catechu. Forty symptomatic plants and three asymptomatic samples were collected in Wenchang, Hainan, China (19°33'9″N, 110°48'5″E). Forty-three samples (0.1g each) were used to extract total DNA (TIANGEN plant genomic DNA extraction kit). Phytoplasma universal primers named P1/P7 (Schneider et al. 1995) and R16F2n/R16R2 (Gundersen and Lee 1996) for 16Sr DNA and primers named fTuf1/rTuf1 and fTufu/rTufu (Schneider et al. 1997) for tuf genes were used for amplifying phytoplasma sequences from isolated DNA samples by nested PCR. No fragment was amplified in asymptomatic plants and four out of forty symptomatic samples could amplify target fragment. R16F2n/R16R2 amplicons (1 248 bp) and fTufu/rTufu amplicons (845 bp) from four symptomatic Areca catechu samples were sequenced in BGI (https://genomics.cn/). The 16Sr DNA GenBank accession numbers of four positive strains (named HNWC5, HNDZ1, HNDZ3 and HNDZ6) were OQ586072, OQ586085, OQ586086, OQ586087, respectively and the tuf GenBank accession numbers were OQ595209, OQ595210, OQ595211, OQ595212, respectively. Sequence alignment showed that the 16S rDNA and tuf sequence of HNDZ1, HNDZ3 and HNDZ6 were 100% consistent. 16S rDNA of HNWC5 was 99.96% consistent with HNDZ1 and tuf of HNWC5 was 98.31% consistent with HNDZ1. Interestingly, blast search based on 16S rDNA gene of HNWC5 showed 100% sequence identity with that of 16SrII group phytoplasma such as 'Eclipta prostrata' phytoplasma strain Ep1(MH144204.1), 'Aeschynomene americana' phytoplasma isolate AA1(MH231157.1) and 'Acacia confusa' witches'-broom phytoplasma isolate HK6(ON408364.1). Blast search based on tuf gene of HNWC5 showed 98.7% sequence identity with that of bamboo witches'-broom phytoplasma (FJ853160.1) and 91.02% sequence identity with that of 'podocarpus nagi' fasciation phytoplasma (KR633146) and 90.78% sequence identity with that of 'Musa acuminata' elephantiasis disease phytoplasma (MF983708). The phylogenetic tree was constructed based on 16Sr DNA gene by MEGA 7.0 employing neighbor-joining (NJ) method with 1000 bootstrap value (Kumar et al. 2016). The result indicated that the HNWC5, HNDZ1, HNDZ3 and HNDZ6 phytoplasma strains clustered a subclade in 16SrII group. The virtual RFLP analysis based on the 16Sr DNA gene sequence was performed by the online phytoplasma classification tool iPhyClassifier (Zhao et al. 2009) using restriction endonucleases of AluI, BamHI, BfaI, BstUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, Sau3AI, MseI, RsaI, SspI and TaqI. The result indicated that HNWC5 was most similar to the reference pattern of peanut witches'-broom phytoplasma (16SrII-A subgroup, GenBank accession: L33765) and the pattern similarity coefficient of HNWC5 is 1.00. However, the HpaII restriction endonuclease pattern of HNDZ1, HNDZ3 and HNDZ6 was different from L33765 and the similarity coefficient was 0.97, which indicated this strain may represent a new subgroup within the 16SrII group. To our knowledge, this is the first report of 16SrII group related phytoplasma associated with YLD on Areca catechu in China. Our study contributes to understanding the polymorphism of phytoplasma causing YLD and provides an important reference for pathogen specific detection.

Characterization of 16SrII group phytoplasma associated with sesame phyllody disease in different cropping seasons

A survey was conducted in the sesame fields of Karnataka, India during April–June 2010 (summer) and in Telangana State, Maharashtra and Karnataka during July–September 2010 (Kharif). In summer, disease incidence of 18.57–31.6% was recorded, whereas, in Kharif, incidence of 44.66–68%, 13–47.14% and 32.14% was recorded in Telangana State, Karnataka and Maharashtra respectively. Three symptomatic and two non-symptomatic samples from summer crop and sixteen symptomatic and two non symptomatic samples from Kharif season fields were collected. The disease etiology was confirmed using nested polymerase chain reaction (PCR) with phytoplasma universal primers P1/P7 followed by R16F2n/R16R2 which amplified ~ 1.8 and ~ 1.25 kb in the 16SrDNA gene. The BLAST and phylogenetic analysis of 16S rDNA gene further revealed that phytoplasma associated with phyllody disease in both the season were belonged to peanut witches’ broom group (16SrII). Further, iPhyClassifier analysis of 1.25 kb product of 16S rDNA sequence classified the isolates into 16SrII-A and 16SrII-D subgroups. All summer crop isolates were classified under 16SrII-D subgroup whereas; Kharif season isolates were grouped under 16SrII-A and 16SrII-D group. Phylogenetic analysis revealed the distinct subgroup-wise as well as season-wise clustering pattern where subgroup 16SrII-A and 16SrII-D clustered separately and summer and Kharif season isolates clustered separately. Our study reports that the sesame phyllody disease in Kharif season is caused by the taxonomically distinct strains (16SrII-A and 16SrII-D) whereas, in the summer crop, it is associated with 16SrII-D subgroup alone.

Fringed Spiderflower (Cleome rutidosperma) Is a New Host for Purple Coneflower Witches’ Broom Phytoplasma, a 16SrII-V Subgroup Strain in Taiwan

Fringed spiderflower (Cleome rutidosperma) is an annual herbaceous plant that grows as a weed. Originally from tropical Africa, fringed spiderflower has been introduced and is becoming widely distributed at low altitudes in Taiwan. In June 2019, symptoms such as small leaves, witches’ broom, floral virescence, and phyllody were observed on fringed spiderflowers grown at the agricultural experiment station in Wufeng, Taichung City, Taiwan. These disease symptoms are similar to those associated with purple coneflower (Echinacea purpurea) witches’ broom disease caused by a 16SrII-A subgroup phytoplasma identified in the same area (Chang et al. 2015; Tseng et al. 2012). Leaf samples of spiderflowers exhibiting disease symptoms were collected for western blotting and PCR assays. Total proteins were extracted from plant samples and examined by western blotting using the polyclonal antibody raised against immunodominant membrane protein (Imp, accession no. KPN61379) of purple coneflower witches’ broom phytoplasma. A specific signal of expected size (19 kDa) for Imp was detected in the symptomatic leaf samples but not in healthy leaf samples. For PCR analysis, the total DNA of each sample was extracted using the Plant Genomic DNA Purification Kit (DP022, Genemark, Taiwan) and used as a template in nested PCR with the phytoplasma universal primer pairs P1/P7 followed by R16F2n/R16R2 (Lee et al. 1993). DNA extracted from leaves sampled on healthy plants were used as a negative control. An amplified DNA fragment of 1.2 kb specific for 16S ribosomal RNA (16S rRNA) obtained from symptomatic leaf samples was gel purified and sequenced. The nucleotide sequence was deposited into GenBank (accession no. MN213634). BLAST analysis revealed that MN213634 is identical to the nucleotide sequence of GenBank accession LKAC01000001.1 (complement [31093 to 32611]) of phytoplasma associated with purple coneflower witches’ broom disease. Using iPhyClassifier, the virtual restriction fragment length polymorphism pattern of the 16S rDNA fragment (accession no. MN213634) confirmed that the phytoplasma detected in symptomatic fringed spiderflower belongs to the 16SrII-V subgroup. Subsequent PCR and DNA sequencing assays further confirmed that the genes encoding the putative secreted proteins (accession nos. WP_054598519 and KPN61138) identified in purple coneflower witches’ broom phytoplasma were also found in samples of symptomatic fringed spiderflowers. Our findings revealed that the fringed spiderflower is a new weed plant host for purple coneflower witches’ broom phytoplasma. This result expands our knowledge of the natural host range of 16SrII-V subgroup strains.

First Report of North American Grapevine Yellows (NAGY) in Kansas: Detection of ‘Candidatus Phytoplasma pruni’- and ‘Ca. Phytoplasma asteris’-Related Strains in Diseased Plants

During surveillance for possible presence of exotic phytoplasma pathogens in the United States, plants of cultivated grapevine (Vitis vinifera L.) exhibiting symptoms suggestive of grapevine yellows disease were observed in vineyards in Kansas during the 2013 and 2014 growing seasons. Symptoms observed in <1% of plants examined consisted of leaf yellowing, downward rolling of leaf margins, and shriveling of fruit clusters. Symptomatic plants of cultivars Riesling (designated KS-JO48) and Chardonnay (designated KS-DG12) were selected for further work. To detect and identify the suspected phytoplasmal pathogen strain(s), DNA was extracted from veins excised from symptomatic leaves and used as template in polymerase chain reactions (PCRs) for amplification and cloning of phytoplasma rRNA (rDNA) and secY gene sequences as previously described (Davis et al. 2015; Lee et al. 2010). Nucleotide sequences of the PCR products were deposited in the GenBank database under accession numbers MH085064 and MH085065 (for cloned rrnA and rrnB gene sequences, respectively, reflecting interoperon heterogeneity) and MG992477 (secY) for KS-JO48; and MH085066 (rrnA), MH085067 (rrnB), and MG992478 (secY) for KS-DG12. In silico analysis of 16S rRNA gene sequences, using iPhyClassifier (Zhao et al. 2009), revealed that plant KS-JO48 was infected by a ‘Candidatus Phytoplasma asteris’-related phytoplasma belonging to subgroup 16SrI-A (I-A), and that plant KS-DG12 was infected by a ‘Ca. P. pruni’-related phytoplasma belonging to subgroup variant 16SrIII-A* (III-A*). Nucleotide sequence alignment of the strain KS-DG12 16S rRNA gene sequence with those of North American grapevine yellows (NAGY) strains previously reported (Davis et al. 2015) revealed that strain KS-DG12 is a NAGYIIIβ sequevar corresponding to sequevar NAGYIIIβ strains found in Maryland, Virginia, New York, and Pennsylvania (Davis et al. 2015). Alignments and phylogenetic analyses of secY gene sequences confirmed the close relatedness of the Kansas subgroup 16SrI-A strain KS-JO48 and subgroup 16SrIII-A* (NAGYIIIβ sequevar) strain KS-DG12 phytoplasmas with the reference strains of ‘Ca. P. asteris’ and ‘Ca. P. pruni’, respectively. The results provide the first evidence for the presence of NAGY in the state of Kansas, while revealing that the two NAGY phytoplasma strain lineages reported here are closely related to NAGY phytoplasma strains detected in other U.S. states. Although further work will be necessary to assess the impacts of NAGY in Kansas, the findings reported here expand the known geographic range of NAGY disease and support the concept that the geographic distribution of NAGY in the United States is greater than currently realized.

Molecular identification and characterization of phytoplasmas in insect vectors of chickpea phyllody disease in Punjab, Pakistan

This study was conducted to identify the incidence of a chickpea disease consisting in phyllody, virescence and proliferation, its association with phytoplasmas and its insect vector(s). The presence of phytoplasmas in leafhopper species and chickpea plants was verified by nested PCR amplification of 16S rDNA. Orosius albicinctus, Circulifer haematoceps and Neoaliturus fenestratus resulted phytoplasma positive. The sequence of the detected phytoplasma strain exhibited the maximum nucleotide identity (99.9–100%) with peanut witches’ broom phytoplasmas, 16SrII-A subgroup.

Diversity of Phytoplasmas Associated with Several Plants in Western Java-Indonesia

Diseases caused by phytoplasmas have been reported in field crops, ornamentals, and weeds in Indonesia. However, most of phytoplasmas have not been subjected to further identification and thus, they remain unaffiliated with proper classification scheme. More reliable identification of phytoplasmas mostly rely on molecular methods. The aim of this study was to characterize the phytoplasma as the causal agent of naturally infected plants in western Java-Indonesia based on their 16S rRNA nucleotide sequences. Plant exhibiting phytoplasmal symptoms were observed and taken for further molecular examination. Eight plant species from three families in Bogor, Tangerang, and Bekasi, i.e. peanut, soybean, snakebean, Opuntia sp., betung bamboo, apus bamboo, Bermuda grass, and digitaria grass (Digitaria fuscescens) have been observed and taken as samples for further molecular examination. Nested-PCR with primer pairs P1/P7 followed by R16F2n/R16R2 resulted in amplification of products of approximately 1.2 kb from all symptomatic plant samples tested. BLAST analysis of the nucleotide sequences, phylogenetic analyses, and similarity coefficients derived from RFLP in silico revealed that there were association of a phytoplasma of 16SrII-A subgroup with phytoplasmas identified in peanut, soybean, and snakebean. Such an association showed witches’ broom symptoms; 16SrII-C with Opuntia sp. causing proliferation and mosaics; and phytoplasmas displaying yellowing and little leaf of two kinds of bamboos and white leaf of Bermuda grass and digitaria grass that were closely related to 16SrXIV-A subgroup. To our knowledge, this is the first report molecular identification of 16SrXIV-A associated with apus bamboo and digitaria grass in Indonesia.

Detection and identification of a ‘Candidatus phytoplasma aurantifolia’-related strain (16SrII-A subgroup) associated with corchorus aestuans phyllody in China

In September 2015, a phyllody that is typical of phytoplasma infection was observed on Corchorus aestuans plants in Haikou, Hainan Province, China. Total DNA from symptomatic and asymptomatic plants was extracted for molecular diagnosis. On the basis of sequence analysis and phylogenetic trees based on 16S rDNA and rp genes, the phyllody phytoplasma was ascertained to be related to ‘Candidatus phytoplasma aurantifolia’. To the best of our knowledge, this is the first report of a phytoplasma infecting C. aestuans in the world.

Comparing qPCR and Nested PCR Diagnostic Methods for Aster Yellows Phytoplasma in Aster Leafhoppers.

The aster yellows phytoplasma (AYp) is a wall-less bacterium that causes damage in multiple crops. They are spread primarily by the aster leafhopper, Macrosteles quadrilineatus (Hemiptera: Cicadellidae). A total of 3,156 aster leafhoppers were collected during the 2014 and 2015 growing seasons in Michigan celery and carrot fields using sweep nets. The objective of this study was to test previously developed 16S rDNA phytoplasma gene primers to find the most reliable and least time-consuming method for AYp detection in leafhoppers. Nested polymerase chain reaction (PCR) was performed with universal primers P1/P7 and R16F2n/R16R2, and then, restriction enzymes AluI, MseI, and HhaI identified the phytoplasma to subgroup. Over the two years, 2.2% of samples were phytoplasma positive with nested PCR, classified in subgroups 16SrI-A or 16SrI-B. All samples were also tested with a TaqMan quantitative qPCR assay with universal phytoplasma primers and probe and 4.6% tested positive. A subset of samples were also tested with AYp-specific SYBR green qPCR, showing a >93% similarity between SYBR green and TaqMan qPCR assay results. The qPCR assays were more than two times faster than nested PCR. However, qPCR assays likely have specificity issues that need to be addressed before they can be used as a reliable method of detection for AYp in leafhoppers.

Evidence for the role of an invasive weed in widespread occurrence of phytoplasma diseases in diverse vegetable crops: Implications from lineage-specific molecular markers

During the period from 2011 to 2013, several plant diseases repeatedly occurred in vegetable crops grown in Yuanmou County, Yunnan Province, China. Affected plants included cowpea, sword bean, string bean, tomato, lettuce, and water spinach. The diseased plants exhibited symptoms of witches'-broom growth and floral deformations, linking each disease to phytoplasmal infection. Phylogenetic and virtual RFLP analyses of the phytoplasmal 16S rRNA gene sequences amplified from DNA of diseased plants revealed that all of the individual strains present in the diverse vegetable plants were affiliated with a single ‘Candidatus Phytoplasma’ species (‘Ca. Phytoplasma aurantifolia’) and a single ribosomal subgroup (16SrII-A). While presence of subgroup 16SrII-A phytoplasma in this geographic region was reported previously, such widespread infections in diverse plant hosts are unveiled for the first time in this study. In pursuing the source of the infections, we found that areas surrounding the affected vegetable fields were extensively invaded by parthenium weeds (Parthenium hysterophorus); and many of the weed plants exhibited abnormal morphologies that were suspicious of, and later diagnosed with, phytoplasmal infections. Results from genotyping of 16S rRNA and lineage-specific immunodominant membrane protein genes revealed that the vegetable-infecting phytoplasmas and the parthenium weed phytoplasma belong to the same genetic lineage. The findings indicate that parthenium weed poses a substantial risk as a reservoir of phytoplasmal infection of nearby agricultural crops in the geographic region since the ecosystems of Yuanmou are insect-rich, and parthenium weed is known to attract diverse leafhoppers. Further studies are warranted to assess the impact of farmland invasions by the noxious weed and to devise practical measures for improved weed control.

First Report of ‘Candidatus Phytoplasma asteris’ Subgroup 16SrI-A Associated with a Disease of Potato (Solanum tuberosum) in Lithuania

First Report of ‘<i>Candidatus</i> Phytoplasma asteris’ Subgroup 16SrI-A Associated with a Disease of Potato (<i>Solanum tuberosum</i>) in Lithuania

Draft Genome Sequence of a 16SrII-A Subgroup Phytoplasma Associated with Purple Coneflower (Echinacea purpurea) Witches' Broom Disease in Taiwan.

The bacterial genus “Candidatus Phytoplasma” contains a group of insect-transmitted plant pathogens in the class Mollicutes. Here, we report a draft genome assembly and annotation of strain NCHU2014, which belongs to the 16SrII-A subgroup within this genus and is associated with purple coneflower witches’ broom disease in Taiwan.

Molecular Identification of Phytoplasmas Infecting Diseased Pine Trees in the UNESCO-Protected Curonian Spit of Lithuania

Although mainly known as pathogens that affect angiosperms, phytoplasmas have recently been detected in diseased coniferous plants. In 2008–2014, we observed, in the Curonian Spit of Western Lithuania and in forests of Southern Lithuania (Varena district), diseased trees of Scots pine (Pinus sylvestris) and mountain pine (Pinus mugo) with unusual symptoms similar to those caused by phytoplasmas. Diseased trees exhibited excessive branching, dwarfed reddish or yellow needles, dried shoots and ball-like structures. restriction fragment length polymorphism (RFLP) and nucleotide sequence analysis of 16S rRNA gene fragments revealed that individual trees were infected by Candidatus (Ca.) Phytoplasma pini-related strains (members of phytoplasma subgroup 16SrXXI-A) or by Ca. Phytoplasma asteris-related strains (subgroup 16SrI-A). Of the nearly 300 trees that were sampled, 80% were infected by phytoplasma. Ninety-eight percent of the positive samples were identified as Ca. Phytoplasma pini-related strains. Strains belonging to subgroup 16SrI-A were identified from only few trees. Use of an additional molecular marker, secA, supported the findings. This study provides evidence of large-scale infection of Pinus by Ca. Phytoplasma pini in Lithuania, and it reveals that this phytoplasma is more widespread geographically than previously appreciated. This is also the first report of phytoplasma subgroup 16SrI-A in pine trees.

Detection and molecular characterization of ‘Candidatus phytoplasma asteris’ related phytoplasmas infecting canola in North Dakota

Canola (Brassica napus) plants with Aster yellows-like symptoms were collected in 2011 and 2012 from canola fields in North Dakota, USA. The presence of phytoplasmas in the symptomatic plants was confirmed by a nested PCR assay using universal primer pairs P1/P7 and R16F2n/R2. Phytoplasmas were identified as ‘Candidatus phytoplasma asteris’ related strains with >99% nucleotide identity to the reference strain M30790 based on in silico analysis of the F2nR2 fragments using the iPhyClassifier online tool. Based on sequence homology searches, RFLP and phylogenetic analyses of 16S rDNA gene, phytoplasmas belonging to two distinct lineages, 16SrI-A and 16SrI-B subgroups, were identified infecting canola in North Dakota. Further characterization of the phytoplasmas based on ribosomal protein operon and secY genes also supported the presence of two distinct lineages. Sequence comparison and RFLP analyses based on the three regions confirmed the presence of mixed infection by phytoplasmas belonging to subgroups 16SrI-A and 16Sr1-B in two samples, one each in 2011 and 2012. To our knowledge, this is the first report of 16SrI-A subgroup phytoplasma and mixed infection by 16SrI-A and 16SrI-B phytoplasmas infecting canola in the USA.

Molecular characterization of phytoplasma associated with phyllody of Pedalium murex - a common weed in coconut plantations

Phytoplasma belonging to subgroup 16SrII-A and identified as “Candidatus Phytoplasma australasiae”- was found constantly associated with phyllody of Pedalium murex, a common medicinal cum succulent weed in coconut ecosystem prevalent in South India. Infected P. murex showed stunted growth with reduced leaf size, shortened internodes and the characteristic transformation of floral parts into leafy structures typical to that of phytoplasma disease. This weed could possibly serve as a reservoir of sesamum phyllody in this region but no link with root (wilt) disease in coconut could be established based on molecular characterization studies. This is the first report of the association of 16SrII-A group phytoplasma with pedalium phyllody from the world.

First Report on the Association of a 16SrII-A Phytoplasma with Sesame (Sesamum indicum) Exhibiting Abnormal Stem Curling and Phyllody in Taiwan.

Sesame (Sesamum indicum L.), an annual plant, is grown as an oilseed crop and the seeds are used in bakery products in Taiwan. In June 2013, plants exhibiting symptoms including phyllody and abnormal stem curling were observed in sesame fields in Pitou Township, Changhua County, Taiwan. Incidence of infected plants was estimated to be greater than 90% within a single field. Phytoplasmas associated with sesame exhibiting phyllody, witches'-broom, or virescence have been classified as strains of 16SrI-B in Myanmar (GenBank Accession No. AB558132), 16SrII-A in Thailand (JN006075), 16SrII-D in Oman (EU072505) and India (KF429486), 16SrIV-C in Iran (JF508515), and 16SrVI-A (KF156894) and 16SrIX (KC139791) in Turkey (1). Three symptomatic and four asymptomatic plants were uprooted and transplanted in a greenhouse for further study. Transmission electron microscopy (TEM) revealed clusters of phytoplasma cells ranging from 300 to 800 nm in diameter only in phloem sieve elements of stems of three symptomatic and two asymptomatic plants. Comparable tissues from two other symptomless plants were devoid of phytoplasma cells. Total DNA was extracted with a modified CTAB method (2) from plant tissues (100 mg each) including stem, leaf, petiole, and root from the same plants used for TEM work. Analyses by a nested PCR using universal primer pairs P1/P7 (5'-AAGAGTTTGATCCTGGCTCAGGATT/5'-CGTCCTTCATCGGCTCTT) followed by R16F2n/R16R2 (5'-GAAACGACTGCTAAGACTGG/5'-TGACGGGCGGTGTGTACAAACCCCG) were performed to detect putative phytoplasma DNA (3). Each primer pair amplified a single PCR product of either 1.8 or 1.2 kb, respectively, only from the three symptomatic and two asymptomatic plant tissues that had phytoplasma cells in their sieve elements. It is likely that these two asymptomatic plants were in the early stage of infection before symptoms became noticeable. The nested PCR products (1.2 kb) amplified from the symptomatic plants were cloned separately and sequenced (GenBank Accession Nos. KF923391, KF923392, and KF923393). BLAST analysis of the sequences revealed that they shared 99.2% sequence identity with strains reported from India and Thailand (KF429486 and JN006075), which were classified to the 16SrII-D and 16SrII-A subgroups, respectively. Moreover, iPhyClassifier software (4) was used to perform sequence comparison and generate a virtual restriction fragment length polymorphism (RFLP) profile. The 16S rDNA sequences shared 99.4% identity with that of the 'Candidatus Phytoplasma australasiae' (Y10097) and the RFLP patterns were identical to that of the 16SrII-A subgroup, indicating the Taiwanese strain is a 'Ca. P. australasiae'-related strain. To our knowledge, this is the first report of a 16SrII-A subgroup phytoplasma causing phyllody and abnormal stem curling on sesame in Taiwan. The occurrence of phytoplasma on sesame could have direct implications for the cultivation of this economically important oilseed plant and the bakery industry in Taiwan.

Occurrence of phytoplasmas in leafhoppers and cultivated grapevines in Canada

Following the recent detection of bois noir, X-disease and aster yellow (AY) in Canadian vineyards, a survey was conducted in 2006–2008 in vineyards located in Ontario (ON), British Columbia (BC) and Quebec (QC). PCR tests were used to detect phytoplasmas in grapevine cultivars and leafhopper species, and identify phytoplasma strains. The overall detection of phytoplasmas was <1% in BC, and ranged from 2.4% to 8.3% in ON and QC, respectively, with significant variability between years, cultivars and locations. A large proportion of phytoplasma-infected grapevines were symptomless and the cultivars merlot and pinot gris were the only two cultivars in which no phytoplasma were detected. Aster yellow phytoplasma strains belonging to the subgroups 16SrI-A, -B, and -C were detected in grapevines, with 16SrI-A being the most frequently found. Phytoplasma DNA was detected in 37 leafhopper species, out of which 11 are known vectors. Most leafhopper species in which phytoplasma DNA were detected are grass feeders, with the AY vector Macrosteles quadrilineatus constituting a strong potential candidate for AY transmission in ON and QC vineyards. Five new sequences of phytoplasma were found in grapevines, and 11 in leafhoppers: they were registered in GenBank. The possibility of phytoplasma transmission between leafhoppers and grapevines is discussed.