Rp Gene Sequences Research Articles

Momordica charantia Is a Novel Host of 'Candidatus Phytoplasma malaysianum'-Related Strains Associated with Bitter Melon Stem Fasciation Disease in China.

Momordica charantia, also known as bitter melon, bitter gourd, and bitter squash, is a member of the Cucurbitaceae family and is widely grown in tropical and subtropical regions for its edible fruit and medicinal properties (Alves et al. 2017). In April 2022, bitter melon plants exhibiting stem fasciation and excessive tendril symptoms were observed in a 50-acre vegetable farm in Yijia Village, Weishan Yizu Huizu Autonomous County, Dali, Yunnan Province, China (Fig. 1). The farm primarily grew tomatoes, but around 400 bitter melon plants were planted in spots where tomatoes failed to establish. One plot had a 40% incidence rate, with four out of ten bitter melon plants showing symptoms. Scattered cases were observed in other plots, leading to an overall disease incidence rate of around 2% for the entire farm. Phytoplasma infection was suspected due to symptomatic plants growing in the same province as previously reported cases of phytoplasma diseases, such as happy tree (Camptotheca accuminata) witches'-broom disease, and the presence of phytoplasma-transmitting leafhoppers (Qiao et al. 2023). DNA was extracted from four symptomatic samples and two healthy controls collected from the abovementioned plot with a 40% disease incidence using Bioteke's Plant Genomic DNA Extraction Kit and then tested for phytoplasma infection. A nested PCR assay was conducted using primer pair P1/16S-SR followed by P1A/16S-SR to amplify the near full-length phytoplasma 16S rDNA (about 1.5kb) as previously described (Lee et al. 2004). None of the healthy controls tested positive for phytoplasma infection, while three out of four symptomatic plants showed positive results. The amplicons from the nested PCR were cloned into the pCRII-TOPO vector as previously described (Lee et al. 2004). The resulting clones were sequenced, and the representative sequence was deposited into GenBank (accession number PP489216). The iPhyClassifier (Zhao et al. 2009) was employed to determine the phytoplasma species and group/subgroup associated with the bitter melon stem fasciation (BMSF) disease. The results indicated that the diseased bitter melon plants were infected with a strain related to 'Candidatus Phytoplasma malaysianum' (EU371934), with a 98.07% sequence identity. The similarity coefficient was 1.00 compared to the reference strain of 16SrXXXII-D (GenBank accession: MW138004). The phytoplasma strain associated with BMSF disease was designated as BMSF1. In addition, the same DNA samples underwent further characterization of the BMSF strains. A nested PCR was conducted using primer pair rpL2F3/rpIR1A, followed by rp(III)-FN/rpIR1A to amplify a phytoplasma-specific rp gene segment (about 1.2 kb) (Martini et al. 2007; Davis et al. 2013). Three out of four samples tested positive, consistent with the 16S rRNA gene amplification results. Similarly, a primer pair L15F1/MapR1 followed by secYF1(III)/secYR1(III) was used to amplify a phytoplasma-specific partial spc operon (about 1.7 kb) that includes the complete secY gene and partial rpl15 and map genes, as previously described (Lee et al. 2010). The obtained rp and partial spc amplicons were cloned and sequenced (GenBank accession numbers PP464295 and PP464296). The rp and secY gene sequences were searched against the non-redundant nucleotide collection in the NCBI database using BLASTN. The top hit for the rp gene was 'Ca. Phytoplasma luffae' (CP054393), with 83.24% identity (1068/1283 base-matching). The top hit for the secY gene was also 'Ca. Phytoplasma luffae' (CP054393), with 72.53% identity (1294/1784 base-matching). The percent identity of the BMSF sequences compared to the top hit is low since no other group 16SrXXXII rp and secY gene sequences are available for comparison. A subgroup 16SrXXXII-D phytoplasma strain has been previously reported associated with Camptotheca acuminata witches'-broom (Qiao et al. 2023) and Trema tomentosa witches'-broom (Yu et al. 2021) in China. To our knowledge, bitter melon represents a new host of 'Candidatus Phytoplasma malaysianum'-related strains, and this is the first report of BMSF disease in China. The findings suggest that 'Candidatus Phytoplasma malaysianum'-related strains infect not only ornamental plants but also crops.

First report of 'Candidatus Phytoplasma asteris' associated with witches'-broom and plexus bud disease of Cerasus serrula in China.

Cherry blossoms (Cerasus serrula) are native to the temperate zone around the Himalayas in the northern hemisphere, mainly distributed in the west and southwest of China, including Yunnan, Sichuan and Tibet. Cherry has high ornamental, edible and medicinal value. In August 2022, we observed that Cherry trees exhibited witches' broom and plexus bud in Kunming City, Yunan Province, China. The symptoms consisted of many small branches with little leaves at the top of branches, stipule lobation, and clustered adventitious buds that are tumor-like on the branches that usually cannot sprout normally. As disease intensity increased, the branches dried up from the top to the bottom till the death of the whole plant. We named this disease C. serrula witches' broom disease (CsWB). We found CsWB in the areas of Panlong, Guandu, Xishan Districts in Kunming, where more than 17% of the plants we surveyed were infected. We collected 60 samples from across the three districts. These included 15 symptomatic and 5 asymptomatic plants per district. The lateral stem tissues were observed under a scanning electron microscope (Hitachi S-3000N). The nearly spherical bodies were found in the phloem cells of symptomatic plants. Total DNA extraction was conducted from 0.1 g tissue using the CTAB method (Porebski et al. 1997), ddH2O was used as the negative control, and Dodonaea viscose plants with witches' broom symptoms were used as the positive control. The nested PCR was used to amplify the 16S rRNA gene (Lee et al. 1993; Schneider et al. 1993) and PCR amplicon of 1.2 kb were amplified (GenBank accessions: OQ408098; OQ408099; OQ408100). The direct PCR specific to the ribosomal protein (rp) gene yielded amplicons of approximately 1.2 kb with primer pair rp(I)F1A and rp(I)R1A (Lee et a. 2003) (GenBank accessions: OQ410969; OQ410970; OQ410971). The fragment from 33 symptomatic samples was consistent with the positive control, and absent for asymptomatic samples, suggesting an association of phytoplasma with the disease. A BLAST analysis of the 16S rRNA sequences of CsWB phytoplasma showed that it has a 99.76% similarity with Trema laevigata witches' broom phytoplasma (GenBank accession MG755412). The rp sequence shared 99.75% identity with Cinnamomum camphora witches' broom phytoplasma (GenBank accession OP649594). An analysis with iPhyClassifier showed that the virtual RFLP pattern derived from the 16S rDNA sequence shares 99.3% similarity with that of the 'Ca. Phytoplasma asteris' reference strain (GenBank accession: M30790), and the virtual RFLP pattern derived from the fragment is identical (similarity coefficient 1.00) to the reference pattern of 16Sr group I, subgroup B (GenBank accession: AP006628). Thus, CsWB phytoplasma is identified as 'Ca. Phytoplasma asteris'-related strain belonging to sub-group 16SrI-B. The phylogenetic tree was constructed based on 16S rRNA gene and rp gene sequences by using MEGA version 6.0 (Tamura et al. 2013) with neighbor-joining (NJ) method and bootstrap support was estimated with 1000 replicates. The result indicated that the CsWB phytoplasma formed a subclade in 16SrI-B and rpI-B respectively. In addition, the clean 1-year-old C. serrula were tested positive for the phytoplasma using the nested PCR 30 days after being grafted with naturally infected twigs with CsWB symptoms. To the best of our knowledge, Cherry blossoms is a new host of 'Ca. Phytoplasma asteris'-related strains in China. The newly emerged disease is a threat to the ornamental value of cherry blossoms and the production of wood quality.

First report of 'Candidatus Phytoplasma asteris' associated with witches'-broom disease of Pinus yunnanensis in China.

Pinus yunnanensis is an evergreen tree belonging to Pinaceae. The species is distributed in the east of Tibet, southwest of Sichuan, southwest of Yunnan, southwest of Guizhou and northwest of Guangxi. It is an indigenous and pioneer tree species for barren mountain afforestation in southwest China. P. yunnanensis has important value to both the building and medicine industries (Liu et al. 2022). In May 2022, P. yunnanensis showing witches'-broom symptom were found in Panzhihua City, Sichuan Province, China. The symptomatic plants had yellow or red needle, and exhibited plexus bud and needle wither. The lateral buds of infected pines developed into twigs. Some lateral buds grew in clusters and a few sprouted needles (Fig.1). The disease was named the P. yunnanensis witches'-broom disease (PYWB) and was found in some areas of Miyi, Renhe, and Dongqu. More than 9% of the pines showed these symptoms in the three areas surveyed, and the disease was spreading. A total of 39 samples were collected from three areas, including 25 symptomatic plants and 14 asymptomatic plants, respectively. The lateral stem tissues of 18 samples were observed under a scanning electron microscope (Hitachi S-3000N). Spherical bodies were found in the phloem sieve cells of symptomatic pines (Fig.1). Total DNA was extracted from 18 plant samples using the CTAB method (Porebski et al. 1997) and subjected to nested-PCR testing. Double-distilled water and DNA extracted from asymptomatic plants were used as negative controls, and DNA extracted from Dodonaea viscosa affected by the D. viscosa witches'-broom disease was used as positive control. Nested PCR was employed to amplify the pathogen's 16S rRNA gene (Lee et al. 1993; Schneider et al. 1993) and 1.2 kb segment were produced (GenBank accessions OP646619; OP646620; OP646621). PCR specific to the ribosomal protein (rp) gene yielded segment of approximately 1.2 kb (Lee et al. 2003)(GenBank accessions OP649589; OP649590; OP649591). The fragment size from 15 samples was consistent with the positive control, confirming the association of phytoplasma with the disease. A BLAST analysis of the 16S rRNA sequences of P. yunnanensis witches'-broom phytoplasma showed that it shared 99.12% ~99.76% identity with that of Trema laevigata witches'-broom phytoplasma (GenBank accession MG755412). The rp sequence shared 99.84%~99.92% identity with that of Cinnamomum camphora witches'-broom phytoplasma (GenBank accession OP649594). An analysis with iPhyClassifier (Zhao et. 2013) showed that the virtual RFLP pattern derived from OP646621 16S rDNA fragment of PYWB phytoplasma is identical (similarity coefficient 1.00) to the reference pattern of 16Sr group I, subgroup B (OY-M, GenBank accession AP006628). The phytoplasma is identified as a 'Candidatus Phytoplasma asteris'-related strain belonging to sub-group 16SrI-B. Interestingly, compared to AP006628, the virtual RFLP pattern derived from OP646619 and OP646620 fragments exhibit differences in three and one cleavage site, with a similarity coefficient of 0.92 and 0.97, respectively (Fig.2). These strains may represent a new subgroup within the 16Sr group I. The phylogenetic tree was reconstructed based on 16S rRNA and rp gene sequences using MEGA versio6.0 (Tamura et al. 2013). The analysis was conducted using the neighbor-joining (NJ) method with 1,000 replicates of bootstrap analysis. The results indicated that the PYWB phytoplasmas grouped into clades including phytoplasmas belonging to 16SrI-B and rpI-B, respectively (Fig.3). In addition, 2-year-old P. yunnanensis were used for grafting assays in nursery, and the twigs from infected pine under natural conditions were used as a scion, and the phytoplasma were detected using nested PCR after grafting for 40 d (Fig.4). In 2008-2014, P. sylvestris and P. mugo in Lituania had excessive branching symptoms that were attributed to 'Ca. Phtyoplasma Pini' (16SrXXI-A) or asteris' (16SrI-A) strains (Valiunas et al. 2015). In 2015, P. pungens with abnormal shoot branching in Maryland were found to be infected by 'Ca. Phytoplasma pini' strain (16SrXXI-B) (Costanzo et al. 2016). To the best of our knowledge, P. yunnanensis is a new host of 'Ca. Phytoplasma asteris'-related strain (16SrI-B) in China. The newly emerged disease is a threat to pines.

First Report of 16SrI-B Subgroup Related Phytoplasma Associated with Yellows Symptoms of Rubus cochinchinensis in China.

Rubus cochinchinensis, an important traditional Chinese medicine in China is used to treat rheumatic arthralgia, bruises and lumbocrural pain (He et al.2005). In January 2022, yellow leaves of R. cochinchinensis were found in Tunchang City, Hainan Province, a tropical island in China. Chlorosis spread along the direction of vascular tissue while the leaf veins remain green (Fig. 1). In addition, the leaves were slightly shrunken and the growth vigor is poor (Fig. 1). By survey, we found the incidence of this disease was about 30%. Three etiolated samples and three healthy samples (0.1g each) were used to extract total DNA (TIANGEN plant genomic DNA extraction kit). Using nested PCR method, phytoplasma universal primers P1 / P7 (Schneider et al., 1995) and R16F2n / R16R2 (Lee et al. 1993) were used to amplified phytoplasma 16S rDNA gene. Primers rp F1 / R1 (Lee et al. 1998) and rp F2 / R2 (Martini et al. 2007) were used to amplified rp gene. 16S rDNA gene and rp gene fragments were amplified from three leaf etiolated samples, but not from healthy samples. The amplified fragments were cloned and sequenced, and the sequences were assembled by DNASTAR11. By sequence alignment, we found the obtained 16S rDNA and rp gene sequences of three leaf etiolated samples were same. The length of 16S rDNA fragment was 1237 bp (accession number: ON944105) and the length of rp gene fragment was 1212 bp (accession number: ON960069). The phytoplasma strain was named as 'R. cochinchinensis' yellows leaf phytoplasma (RcT), RcT-HN1 strain. The 16S rDNA gene sequence of RcT-HN1is 99.8% consistent with 16SrI-B subgroup members such as the 'Brassica napus' dwarf phytoplasma strain WH3 (MG599470.1), Chinaberry yellows phytoplasma strain LJM-1(KX683297.1) and Arecanut yellow leaf disease phytoplasma strain B165 (FJ694685.1). The rp gene sequence of RcT-HN1 is 100% consistent with rpI-B subgroup members such as the 'Salix tetradenia' witches'-broom phytoplasma strain YM-1 (KC117314.1) and Chinaberry witches'-broom phytoplasma strain Hainan (EU348781.1). The phylogenetic tree analysis, based on concatenated 16S rDNA-rp gene sequence of same group phytoplasma by MEGA 7.0 employing neighbor-joining (NJ) method with 1000 bootstrap value, were performed (Kumar et al., 2016). The results showed that RcT-HN1 phytoplasma strain formed a subclade in aster yellows group B subgroup (Fig. 2). The virtual RFLP analysis based on the 16S rRNA gene fragment of RcT-HN1 phytoplasma strain was performed by the interactive online phytoplasma classification tool iPhyClassifier (Zhao et al., 2009). The results showed that the phytoplasma strain was same as the reference pattern of the onion yellows phytoplasma of 16SrI-B (GenBank accession: AP006628), and the similarity coefficient was 1.00. This is the first report that 16SrI-B subgroup related phytoplasma infected R. cochinchinensis and caused yellows symptoms in China. The discovery of the disease is helpful to the study of the spread of phytoplasma-related diseases and protect R. cochinchinensis resources.

First report of 'Candidatus Phytoplasma asteris' associated with witches'-broom disease of Cinnamomum camphora in China.

Camphor tree (Cinnamomum camphora) has a wide distribution in the world and is mainly distributed in the South and southwest in China. It can be used as both a wood and a medicine, with high value in industry, medicine, and ecology. In May 2022, it was observed that the approximately ten to fifteen years old Camphor trees were exhibiting witches'-broom, small leaf morphology and chlorosis, and leaf drop in Panzhihua City, Sichuan Province, China. The witches'-broom symptoms consisted of many small branches with little leaves at the top of branches. It was named C. camphora witches'-broom disease (CCWB) and was found in some areas of Miyi, Yanbian, Renhe Xiqu and Dongqu counties. More than 28% of the plants were infected on the five areas surveyed. Total 100 samples were collected from five areas, with 15 symptomatic plants and 5 asymptomatic plants each area. The lateral stem tissues were observed under a scanning electron microscope (Hitachi S-3000N). The nearly spherical bodies were found in the phloem sieve cells of symptomatic plants. Total DNA extraction was conducted from 0.1 g tissue using the CTAB method (Porebski et al. 1997), ddH2O was used as the negative control, and Dodonaea viscose witches'-broom disease plants were used as the positive control. The nested PCR was employed to amplify the 16S rRNA gene (Lee et al. 1993; Schneider et al. 1993) and PCR amplicon of 1.2 kb were amplified (GenBank accessions OP662614; OP662615; OP662616). The direct PCR specific to the ribosomal protein (rp) gene yielded amplicons of approximately 1.2 kb with primer pair rp(I)F1A and rp(I)R1A (Lee et al. 2003) (GenBank accessions OP649592; OP649593; OP649594). The fragment from 25 symptomatic samples was consistent with the positive control, and asymptomatic plants were negative, confirming an association of a phytoplasma with the disease. A BLAST analysis of the 16S rRNA sequences of CCWB phytoplasma showed that it has a 99.44% similarity with Trema laevigata witches'-broom phytoplasma (GenBank accession MG755412). The rp sequence shared 99.59% identity with rapeseed phyllody phytoplasma (GenBank accession CP055264). An analysis with iPhyClassifier showed that the virtual RFLP pattern derived from the query 16S rDNA fragment of CCWB phytoplasma is most similar to the reference pattern of the 16Sr group I, subgroup B (OY-M, GenBank accession AP006628). The phytoplasma is identified as 'Ca. Phytoplasma asteris'-related strain belonging to sub-group 16SrI-B. The phylogenetic tree was constructed based on 16S rRNA gene and rp gene sequences by using MEGA version 6.0 (Tamura et al. 2013) with neighbor-joining (NJ) method and bootstrap support was estimated with 1000 replicates. The result indicated that the CCWB phytoplasma formed a subclade in 16SrI-B and rpI-B respectively. In addition, the plants were positive for the phytoplasma using nested PCR after grafting for 30 d in nursery conditions. It is noteworthy that the plants were seriously damaged by aphid, Psyllidae and Ceroplastes. It is speculated that the insects of Homoptera typically transmit phytoplasmas by feeding on plant sap, thus it is necessary to control aphids in order to control the C. camphora witches'-broom disease. To the best of our knowledge, Camphor tree is a new host plant of 'Ca. Phytoplasma asteris' in China. The newly emerged disease is a threat to Camphor tree production.

First report of 'Candidatus Phytoplasma asteris' associated with witches'-broom disease of Macadamia ternifolia in China.

Macadamia nut (Macadamia ternifolia) was first introduced into China from Australia in 1910, and the main cultivation areas were Yunnan and Guangxi. It can be used as both a fruit and a therapeutic drug, with high economic value. In March 2021, it was observed that the M. ternifolia was showing witches'-broom, leaf yellowing and plexus bud in Dehong Prefecture, Yunnan Province, China. The terminal buds of infected plants were inhibited and the lateral buds are stimulated to germinate into twigs in advance. It was named the M. ternifolia witches'-broom disease, and was found in urban and rural areas of Mangshi, Lianghe, Yingjiang, Mangdong, Longchuan and Longling cities and counties. More than 40% of the plants were infected on the seven areas surveyed. The lateral stems from symptomatic and asymptomatic plants were cut to small pieces. The tissues were treated by fixation, dehydration and spraying-gold. And the tissues were observed under a scanning electron microscope (Hitachi S-3000N) (Pathan et al. 2010). The nearly spherical bodies were found in the phloem sieve cells of symptomatic plants. Symptomatic and asymptomatic plants were collected from seven areas, ddH2O was used as the negative control, and Dodonaea viscose witches'-broom disease plants were used as the positive control. The total plants' DNA extraction was conducted from 0.1 g tissue using the CTAB method (Porebski et al. 1997), and were stored at -20 °C in a refrigerator in the Key Laboratory of Forest Disaster Warning and Control at the Southwest Forestry University. The nested PCR was employed to amplify the 16S rRNA gene with the primers P1/P7 and R16F2/R16R2 (Lee et al. 1993; Schneider et al. 1993). PCR amplicon of 1.8 kb and 1.2 kb were amplified (GenBank accessions MW892818, MW892819, MW892820, MW892821). The direct PCR with primer pairs rp(I)F/ rp(I)R (Lee et al. 2003) specific to the ribosomal protein (rp) gene yielded amplicons of approximately 1.2 kb (GenBank accessions MZ442600, MZ442601, MZ442602, MZ442603). The fragment from 21 samples was consistent with the positive control, confirming the association of phytoplasma with the disease. Interestingly, the phytoplasma/span>16S rRNA gene and rp gene was also amplified from the 4 samples of asymptomatic plant, we speculated that the latent infection and hidden symptoms existed in Macadamia nut (Moslemkhani and Sadeghi 2011). A BLAST analysis of the 16S rRNA sequences of MTWB phytoplasma showed that it has a 99% similarity with Trema laevigata witches'-broom phytoplasma (GenBank accession MG755412). The rp sequence shared 99% identity with 'Salix tetradenia' witches'-broom phytoplasma (GenBank accession KC117314). An analysis with iPhyClassifier showed that the virtual RFLP pattern derived from the query 16S rDNA fragment of MTWB phytoplasma is most similar to the reference pattern of the 16Sr group I, subgroup B (OY-M, GenBank accession AP006628), with a pattern similarity coefficient of 0.99. The phytoplasma is identified as 'Ca. Phytoplasma asteris'-related strain belonging to sub-group 16SrI-B. The phylogenetic tree was constructed based on 16S rRNA gene and rp gene sequences by using MEGA version 6.0 (Tamura et al. 2013) with neighbor-joining (NJ) method and bootstrap support was estimated with 1000 replicates. The result indicated that the MTWB phytoplasma formed a subclade in 16SrI-B and rpI-B respectively. In 2013, Macadamia nut showed leaf hardness phyllody and shoot proliferation caused by 'Ca. Phytoplasma asteris' in Artemisa, Cuba. The concern is that, the macadamia phytoplasma is closely related to the subgroup 16SrI-F, and it is significantly different from the Chinese strains (Pérez-López et al. 2013). In addition, the MTWB phytoplasma was graft-transmitted from infected to healthy plants in nursery conditions (Akhtar et al. 2009; Ikten et al. 2014). And the grafted plants were positive for the phytoplasma in the nested PCR assays. It is noteworthy that the plants were seriously damaged by aphid and it was speculated that the insects of Homoptera caused the spread of the disease by sucking plant sap, thus the aphids that transmits MTWB in China must be determined to control the M. ternifolia witches'-broom disease. To the best of our knowledge, Macadamia nut is a new host plant of 'Ca. Phytoplasma asteris' in China. The newly emerged disease is a threat to Macadamia nut.

Identification of 16SrXXX-A Phytoplasma Associated with Salt cedar (Tamarix chinensis Lour.) witches'-broom (SCWB) in Southern Xinjiang of China.

Salt cedar is an ornamental shrub/moderate tree species native to Asia and East Europe, and grows in salt-alkali soil, desert and other dry areas, which plays an important role in wind prevention and sand fixation as well as maintaining ecological balance. Salt cedar witches'-broom (SCWB), which was extremely pernicious to Salt cedar. It was first observed and reported in Xi'an, China in 2005 (Zhao et al.2005). Witches' broom symptoms were observed on 20 out of 150 (13.3%) salt plants surveyed from the Alar region and 10 out of 86 (11.6%) plants from the Akesu region in southern of Xinjiang in May 2020. The damaged plants compared with asymptomatic plants (Fig.1A), the major symptoms included branches clustered, intersegment shorten and coarsen, giving rise to the formation of clusters (Fig.1B). Total plant DNA was extracted from phloem tissues with asymptomatic symptoms and phloem tissues with witches'-broom symptoms by a CTAB-based DNA extraction method (Green et al.1999). The 16S rRNA gene and the phytoplasma universal primers P1/P7 and rpF1/rpR1 of the rp (ribosomal protein) gene were used for Polymerase chain reaction (PCR) amplification by using the extracted plant total DNA as the template. The PCR product was used as the template and the R16F2n/R16R2 prmer was used for nested PCR amplification of the 16S rRNA gene after the amplification was completed. The results show that no product was obtained in asymptomatic plants. When DNA samples from witches'-broom symptomatic plants were used as templates, fragments with lengths 1219 bp and 1174 bp, corresponding to 16S rRNA gene and rp gene, were obtained. 16S rRNA gene was sequenced and deposited in GenBank under accession number MW447513. BLAST analysis revealed that the partial 16S rRNA sequence of the phytoplasma associated with P. aphylla witches' broom showed highest sequence identity (99.67%) to salt cedar witches' broom phytoplasma, 'Candidatus Phytoplasma tamaricis' (Accession Number: FJ432664). Phylogenetic and molecular evolutionary analyses were conducted using MEGA-X (Kumar et al., 2018). Results showed taht the SCWB and 16S rXXX group's'Candidatus Phytoplasma tamaricis', (GenBank accession: FJ432664) have the highest affinity (Fig.2A). A virtual restriction fragment length polymorphism(RFLP) was done to determinethe subgroup ( Zhao et al. 2009). The 16S rDNA sequence from the Tamarix chinensis plant showed 99.3% similarity with that of the "Candidatus Phytoplasma tamaricis" reference strain (GenBank accession: FJ432664), suggesting that the phytoplasma in this study belongs to "Candidatus Phytoplasma tamaricis"-related strain. Therefore, it can be stated that SCWB belongs to the 16S rXXX group. The partial rp sequences only shared 84.74% sequence similarity with that of 'Candidatus Phytoplasma prunorum' (MG383523) of Apple proliferation group, a known subgroup 16S rX. Blast analysis based on the partial rp sequences showed that it shares less than 90% similarity with that of any known phytoplasma (Fig 2B), we suspect that this is due to a lack of sequenced rp gene sequences for the 16S rXXX group. To our knowledge, this is the first report of Salt Cedar Witches' Broom phytoplasma in Xinjiang province, China. As a consequence, we guess the SCWB phytoplasma rp gene belongs to 16S rXXX-rp group, which is also the first report about the 16SrXXX-rp group. Because SCWB1 is the only strain in the 16S rXXX group, and it is the representative strain of the 16S rXXX-A subgroup (Zhao et al. 2009). So, the SCWB disease we found in southern Xinjiang belongs to the 16S rXXX-A subgroup.

Utilization of rp and tuf genes in the characterisation of ‘Candidatus Phytoplasma australasia’ strains associated with witches’ broom andshoot proliferation of Dendrocalamus strictus in India

Two housekeeping genes (rp and tuf) specific primers designed for phytoplasmas enclosed in 16SrII group were employed for characterization of detected in Dendrocalamus strictus (Calcutta bamboo) showing witches’ broom and little leaf symptoms from four states of India. Amplification of about 1.3 kb products was achieved by utilising primers rp(II)F1/rpIR1A followed by rp(II)F2/rpIR1A in nested PCR assays from D. strictus phytoplasma strains from Karnataka and New Delhi. The samples from Karnataka, New Delhi, Uttar Pradesh and Tripura displayed amplifications of about 1.0 kb DNA for the tuf gene. Sequence comparison of the rp and tuf gene sequences confirmed that the phytoplasmas associated with the symptomatic bamboo samples from all the four states clusters with strain classified in the ‘Candidatus Phytoplasma australasia’ species.

Multilocus genotyping of ‘Candidatus Phytoplasma aurantifolia’ associated with witches’ broom disease in Citrus spp.

Witches’ broom disease of lime (WBDL), associated with ‘Candidatus Phytoplasma aurantifolia,’ is a serious disease of acid lime in Southern Iran. In order to characterize ‘Ca. P. aurantifolia’ strains, a multilocus sequence analysis protocol was developed based on different genomic loci, including 16S rRNA, 16S-23S rRNA intergenic spacer (IS), ribosomal protein genes rpl22 and rps3, cell division protein, and group II intron reverse transcriptase/mature gene, in 46 phytoplasma strains. Analyses of virtual RFLP, phylogenetics, and DNA sequence identity of 16S rRNA of Iranian phytoplasma strains associated with symptomatic citrus plants indicated that the Iranian strains were classified in ribosomal subgroup 16SrII-B. In total, three genotypes were recognized based on 16Sr virtual RFLP profiles. Phylogenetic analysis based on the rp and ftsH gene sequences showed clustering of all the strains as one group. Comparative sequence and phylogenetic analysis of group II intron genes indicated the highest differentiation of ‘Ca. P. aurantifolia’–related strains in four genotypes. The polymorphism of group II intron reverse transcriptase/mature genes fragmentation provides finer differentiation of closely related ‘Ca P. aurantifolia’ strains and reveals higher genetic diversity than previously reported.

Molecular Characterization and Detection of 16SrIII Group Phytoplasma Associated with Huanglongbing Symptoms.

When huanglongbing (HLB) was found in Brazil in 2004, 'Candidatus Liberibacter americanus' was infecting most of the trees while 'Ca. L. asiaticus' was present in a minor proportion. Currently, 'Ca. L. asiaticus' is the predominant bacterium associated with HLB in citrus trees in São Paulo (SP) and Minas Gerais (MG) States, the major citrus-growing regions in Brazil. A phytoplasma from the 16SrIX group was associated with HLB symptoms in Brazil in 2007, in plants free of Liberibacter spp. In this report, HLB samples testing negative for 'Ca. L. asiaticus', 'Ca. L. americanus', and 16SrIX phytoplasma were infected with 16SrIII phytoplasmas. Coinfection with 'Ca. L. asiaticus' and 16SrIII was also found. The 16S ribosomal RNA (rRNA) gene sequences from 22 samples were obtained and sequenced, confirming that the 16SrIII group phytoplasma is associated with HLB symptoms in SP and MG States. Ten single-nucleotide polymorphisms (SNPs) were found in the 1,427-bp 16S rRNA gene sequences from 16SrIII phytoplasmas from citrus, whereas none was detected in 16S rRNA gene sequences among 16SrIX phytoplasma from citrus. Ribosomal protein (rp) rpsSrplVrpsC gene sequences were amplified with 16SrIII group-specific primers, sequenced from a subset of nine samples, and assembled into three groups based on eight SNPs. SNPs in 16S rRNA gene and rp gene sequences are common in 16SrIII phytoplasmas from other hosts and this phytoplasma group is widespread in South America. 16SrIII phytoplasmas highly related are commonly found in Melia azedarach, a widespread tree in Brazil and Argentina. The finding of a new phytoplasma associated with HLB symptoms belonging to the 16SrIII group reinforces the need to develop diagnostic tools to assess HLB-associated microbiomes.

Morphological and host specificity evolution in coral symbiont barnacles (Balanomorpha: Pyrgomatidae) inferred from a multi-locus phylogeny

Coral-inhabiting barnacles (Thoracica: Pyrgomatidae) are obligatory symbionts of scleractinian and fire corals. We attempted to reconstruct the phylogeny of coral-inhabiting barnacles using a multi-locus approach (mitochondrial 12S and 16S rRNA, and nuclear EF1, H3 and RP gene sequences, total 3532bp), which recovered a paraphyletic pattern. The fire-coral inhabiting barnacle Wanella milleporae occupied a basal position with respect to the other coral inhabiting barnacles. Pyrgomatids along with the coral-inhabiting archaeobalanid Armatobalanus nested within the same clade and this clade was subdivided into two major lineages: Armatobalanus+Cantellius with species proposed to be the ancestral stock of extant coral barnacles, and the other comprising the remaining genera studied. Ancestral state reconstruction (ASR) suggested multiple independent fusions and separations of shell plates and opercular valves in coral barnacle evolution, which counters the traditional hypothesis founded on a scheme of morphological similarities. Most of the coral barnacles are restricted to one or two coral host families only, suggesting a trend toward narrow host range and more specific adaptation. Furthermore, there is a close linkage between coral host usage and phylogenetic relationships with sister taxa usually being found on the same coral host family. This suggests that symbiotic relationships in coral-inhabiting barnacles are phylogenetically conserved and that host associated specialization plays an important role in their diversification.

Salix tetradeniaHand.-Mazz: a new natural plant host of ‘Candidatusphytoplasma’

Summary This article reports Salix tetradenia Hand.-Mazz as a new host of Candidatus phytoplasma and demonstrates its association with witches' broom disease on S. tetradenia plants. Plants exhibited typical visual symptoms of phytoplasma with virescence, abnormality of flowers and witches' broom, and phytoplasma bodies were observed by transmission electron microscopy. Products of 1.2 kb were amplified by nested PCR using phytoplasma universal primer pairs R16F2n/R16R2, but no amplification products were obtained from symptomless plants. The sequence analysis of three 16S rDNA isolates showed 99.84%, 99.68% and 99.76% identify, respectively, with the homologous gene (nc_005303) of member of ‘Candidatus phytoplasma asteris’ (16SrI) group. Phylogenetic and virtual computer-simulated restriction fragment length polymorphism analysis of the 16S rRNA, tuf and rp gene sequences confirmed that this phytoplasma clustered in the 16SrI-B subgroup. These results indicated that the diseased S. tetradenia plants were infected by a phytoplasma of the 16SrI group. This is the first report on the occurrence of phytoplasma disease on S. tetradenia worldwide.

Molecular identification of a phytoplasma associated with Sophora Root yellows

SummaryA phytoplasma infecting Sophora Root (Sophora alopecuroides) was detected and identified in Alar, Xinjiang Uygur Autonomous Region of China. Typical phytoplasma bodies were observed in sieve tubes of the diseased plants by transmission electron microscopy. A partial 16S rRNA gene and ribosomal protein (rp) genes containing rpl22 (rplV) and rps3 (rpsC) were amplified by direct and nested PCR. Based on the sequence similarity of the 16S rRNA and rp genes with accompanying phylogenetic analyses, the phytoplasma associated with Sophora Root yellows belongs to the 16SrI group (aster yellows group). Virtual RFLP analysis of these 16S rRNA and rp gene sequences showed distinct differences from those of reference phytoplasma strains representing previously described subgroups of the 16SrI group. Moreover, the similarity coefficient (0.92) of the RFLP profile of this phytoplasma was less than the threshold similarity coefficient (0.97) required for subgroup classification. Thus, the phytoplasma isolate of Sophora Root plants, designated as ‘SoRY’, represents a new subgroup. Furthermore, this is the first report of phytoplasma disease associated with Sophora Root plants.

Characterization and molecular differentiation of 16SrI-E and 16SrIX-E phytoplasmas associated with blueberry stunt disease in New Jersey

A nested PCR assay was employed to detect the presence of phytoplasmas in 127 blueberry plants exhibiting typical or a portion of blueberry stunt (BBS) syndrome collected in 2010 and 2011, from 11 commercial farms predominantly located in two counties in New Jersey, USA. Ninety plants exhibiting typical stunt syndrome tested positive for phytoplasma infection. Restriction fragment length polymorphism (RFLP) analysis indicated that two distinct phytoplasmas were associated with BBS-diseased plants. About 95% of phytoplasmas detected were very closely related to BBS phytoplasma strains BBS3-AR (subgroup 16SrI-E) and BBS1-MI (unidentified) identified previously, and 4.4% of phytoplasmas detected belonged to the pigeon pea witches'-broom phytoplasma group (16SrIX). Sequence and phylogenetic analysis of cloned 16S rDNA further indicated the subgroup 16SrI-E related phytoplasmas represented a variant of 16SrI-E reference strain BBS3-AR, while the 16SrIX related phytoplasmas were closely related to juniper witches'-broom (JunWB) phytoplasma (16SrIX-E), representing a 16SrIX-E variant. Ribosomal protein (rp) and secY gene-based phylogenies revealed that BBS3-AR and BBS-NJ 16SrI-E strains belonged to a closely related lineage, while BBS-NJ 16SrIX-E strains and JunWB strains represented two distinct lineages. Single nucleotide polymorphisms (SNPs) analyses of rp and secY gene sequences further revealed that no specific rp gene SNPs and only two specific secY gene SNPS were present between BBS-NJ 16SrI-E strains and BBS3-AR. In contrast, BBS-NJ 16SrIX-E strains/clones had 15 consensus rp SNPs and 28 consensus secY SNPs that separated them from JunWB strains/clones. For the first time, two distinct phytoplasmas that cause BBS-disease in the U.S. was revealed.

‘Candidatus Phytoplasma pruni’, a novel taxon associated with X-disease of stone fruits, Prunus spp.: multilocus characterization based on 16S rRNA, secY, and ribosomal protein genes

X-disease is one of the most serious diseases known in peach (Prunus persica). Based on RFLP analysis of 16S rRNA gene sequences, peach X-disease phytoplasma strains from eastern and western United States and eastern Canada were classified in 16S rRNA gene RFLP group 16SrIII, subgroup A. Phylogenetic analyses of 16S rRNA gene sequences revealed that the X-disease phytoplasma strains formed a distinct subclade within the phytoplasma clade, supporting the hypothesis that they represented a lineage distinct from those of previously described 'Candidatus Phytoplasma' species. Nucleotide sequence alignments revealed that all studied X-disease phytoplasma strains shared less than 97.5 % 16S rRNA gene sequence similarity with previously described 'Candidatus Phytoplasma' species. Based on unique properties of the DNA, we propose recognition of X-disease phytoplasma strain PX11CT1(R) as representative of a novel taxon, 'Candidatus Phytoplasma pruni'. Results from nucleotide and phylogenetic analyses of secY and ribosomal protein (rp) gene sequences provided additional molecular markers of the 'Ca. Phytoplasma pruni' lineage. We propose that the term 'Ca. Phytoplasma pruni' be applied to phytoplasma strains whose 16S rRNA gene sequences contain the oligonucleotide sequences of unique regions that are designated in the formally published description of the taxon. Such strains include X-disease phytoplasma and--within the tolerance of a single base difference in one unique sequence--peach rosette, peach red suture, and little peach phytoplasmas. Although not employed for taxon delineation in this work, we further propose that secY, rp, and other genetic loci from the reference strain of a taxon, and where possible oligonucleotide sequences of unique regions of those genes that distinguish taxa within a given 16Sr group, be incorporated in emended descriptions and as part of future descriptions of 'Candidatus Phytoplasma' taxa.

Differentiation and classification of phytoplasmas in the pigeon pea witches’-broom group (16SrIX): an update based on multiple gene sequence analysis

The pigeon pea witches'-broom phytoplasma group (16SrIX) comprises diverse strains that cause numerous diseases in leguminous trees and herbaceous crops, vegetables, a fruit, a nut tree and a forest tree. At least 14 strains have been reported worldwide. Comparative phylogenetic analyses of the highly conserved 16S rRNA gene and the moderately conserved rplV (rpl22)-rpsC (rps3) and secY genes indicated that the 16SrIX group consists of at least six distinct genetic lineages. Some of these lineages cannot be readily differentiated based on analysis of 16S rRNA gene sequences alone. The relative genetic distances among these closely related lineages were better assessed by including more variable genes [e.g. ribosomal protein (rp) and secY genes]. The present study demonstrated that virtual RFLP analyses using rp and secY gene sequences allowed unambiguous identification of such lineages. A coding system is proposed to designate each distinct rp and secY subgroup in the 16SrIX group.

A new phytoplasma associated with little leaf disease in azalea: multilocus sequence characterization reveals a distinct lineage within the aster yellows phytoplasma group

An azalea little leaf (AzLL) disease characterised by abnormally small leaves, yellowing and witches'-broom growth symptoms was observed in suburban Kunming, southwest China. Transmission electron microscopic observations of single-membrane-bound, ovoid to spherical bodies in phloem sieve elements of diseased plants and detection of phytoplasma-characteristic 16S rRNA gene sequence in DNA samples from diseased plants provided evidence linking the disease to infection by a phytoplasma. Results from restriction fragment length polymorphism, phylogenetic and comparative structural analyses of multiple genetic loci containing 16S rRNA, rpsS, rplV, rpsC and secY genes indicated that the AzLL phytoplasma represented a distinct, new 16Sr subgroup lineage, designated as 16SrI-T, in the aster yellows phytoplasma group. The genotyping also revealed that the AzLL phytoplasma represented new rp and secY gene lineages [rp(I)-P and secY(I)-O, respectively]. Phylogenetic analyses of secY and rp gene sequences allowed clearer distinctions between AzLL and closely related strains than did analysis of 16S rDNA.

Phytoplasmas infecting sour cherry and lilac represent two distinct lineages having close evolutionary affinities with clover phyllody phytoplasma

Phytoplasmas infecting sour cherry and lilac in Lithuania were found to represent two lineages related to clover phyllody phytoplasma (CPh), a subgroup 16SrI-(R/S)C (formerly 16SrI-C) strain exhibiting rRNA interoperon sequence heterogeneity. 16S rDNAs amplified from the cherry bunchy leaf (ChBL) and lilac little leaf (LcLL) phytoplasmas were identical or nearly identical to those of operon rrnA and operon rrnB, respectively, of CPh. There was no evidence of 16S rRNA interoperon sequence heterogeneity in either LcLL or ChBL phytoplasma. Based on collective RFLP patterns of 16S rDNA, ChBL was classified in subgroup 16SrI-R, and LcLL was classified in new subgroup 16SrI-S. The ribosomal protein (rp) gene sequences from LcLL phytoplasma were identical to those of CPh, and strain LcLL was classified in rp subgroup rpI-C. By contrast, rp gene sequences from ChBL phytoplasma differed from those of subgroup rpI-C; based on RFLP patterns of rp gene sequences, ChBL was classified in new rp subgroup rpI-O. Single nucleotide polymorphisms (SNPs), designated here by a new SNP convention, marked members of rp subgroup rpI-C, and distinguished LcLL and CPh from ChBL and other non-rpI-C phytoplasmas in group 16SrI. The results raise questions concerning phytoplasma biodiversity assessment based on rRNA genes alone and encourage the supplemental use of a single copy gene in phytoplasma identification and classification, while drawing attention to a possible role of horizontal gene transfer in the evolutionary history of these lineages.

First Report of 'Candidatus Phytoplasma asteris'-Related Strains Infecting Lily in Mexico.

In recent years, lily (Lilium spp.) has become an important ornamental crop in diverse regions of Mexico. Since 2005, unusual symptoms have been observed on lily plants grown from imported bulbs in both greenhouse and production plots at San Pablo Ixayo, Boyeros, and Tequexquinauac, Mexico State. Symptoms included a zigzag line pattern on leaves, dwarfism, enlargement of stems, shortened internodes, leaves without petioles growing directly from bulbs, air bulbils, death of young roots, atrophy of flower buttons, and flower abortion. Symptoms were experimentally reproduced on healthy lily plants by graft inoculation. Total DNA was extracted from 50 diseased, 10 symptomless, and 10 graft-inoculated plants by the method of Dellaporta et al. (2). DNA samples were analyzed for phytoplasma presence by two different nested PCR assays. One assay employed ribosomal RNA gene primer pair P1/P7 followed by R16F2n/R16R2 (1), whereas ribosomal protein (rp) gene primer pairs rpF1/rpR1 and rp(I)F1A/rp(I)R1A (4) were used in a second assay. A DNA fragment approximately 1.2 kb long was consistently amplified from all symptomatic plant samples only by both assays. A comparative analysis of 16S rDNA sequences (Genbank Accession Nos. EF421158-EF421160 and EU124518-EU124520) and rp gene sequences (EU277012-EU277014), derived from PCR products, revealed that phytoplasma infecting lily were most similar (99.9% to 16S rDNA and 99.7% to rp) to carrot phytoplasma sp. ca2006/5 and also were similar (99.8% to 16SrDNA and 99.2% to rp) to broccoli phytoplasma sp. br273. Both carrot and broccoli phytoplasmas were classified as members of aster yellow 16S rDNA restriction fragment length polymorphism subgroup 16SrI-B (3). Although infection of lilies by aster yellows ('Ca. phytoplasma asteris') subgroup 16SrI-B and 16SrI-C was reported from the Czech Republic and Poland, to our knowledge, this is the first report of 'Ca. phytoplasma asteris'-related strains associated with lily plants in Mexico.

Ribosomal protein gene-based phylogeny for finer differentiation and classification of phytoplasmas

Extensive phylogenetic analyses were performed based on sequences of the 16S rRNA gene and two ribosomal protein (rp) genes, rplV (rpl22) and rpsC (rps3), from 46 phytoplasma strains representing 12 phytoplasma 16Sr groups, 16 other mollicutes and 28 Gram-positive walled bacteria. The phylogenetic tree inferred from rp genes had a similar overall topology to that inferred from the 16S rRNA gene. However, the rp gene-based tree gave a more defined phylogenetic interrelationship among mollicutes and Gram-positive walled bacteria. Both phylogenies indicated that mollicutes formed a monophyletic group. Phytoplasmas clustered with Acholeplasma species and formed one clade paraphyletic with a clade consisting of the remaining mollicutes. The closest relatives of mollicutes were low-G+C-content Gram-positive bacteria. Comparative phylogenetic analyses using the 16S rRNA gene and rp genes were performed to evaluate their efficacy in resolving distinct phytoplasma strains. A phylogenetic tree was constructed based on analysis of rp gene sequences from 87 phytoplasma strains belonging to 12 16Sr phytoplasma groups. The phylogenetic relationships among phytoplasmas were generally in agreement with those obtained on the basis of the 16S rRNA gene in the present and previous works. However, the rp gene-based phylogeny allowed for finer resolution of distinct lineages within the phytoplasma 16Sr groups. RFLP analysis of rp gene sequences permitted finer differentiation of phytoplasma strains in a given 16Sr group. In this study, we also designed several semi-universal and 16Sr group-specific rp gene-based primers that allow for the amplification of 11 16Sr group phytoplasmas.