First Report of 16SrII Group-Related Phytoplasma Associated with Phyllody and Small Leaves of Mungbean ( Vigna radiata ) and Cowpea ( Vigna unguiculata ) in Guangxi, 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.

Tephrosia purpurea is a medical plant with excellent insecticidal activity belonging to the family of Leguminosae distributed throughout southern of China (Pei et al., 2013). During January to February 2021, the plants showing abnormal symptoms including witches'-broom, internode shortening, leaf chlorosis and leaflet formation, as shown in Fig.1, were found in Ledong County of Hainan Province, a tropical island in China, with about 60 % incidence. The Tephrosia purpurea disease symptoms were suspected to be induced by phytoplasma, a phloem-limited prokaryotic pathogen which can not be cultured in vitro and which causes severe financial loss and ecological damage to the island. Total DNA from the symptomatic and asymptomatic samples of Tephrosia purpurea were extracted using 0.10 g fresh plant leaves and branches by CTAB method (Doyle and Doyle, 1990). 16S rRNA and secA gene sequence fragments of phytoplasma were detected through PCR amplification using primers R16mF2/R16mR1 (Gundersen and Lee, 1996) and secAfor1/secArev3 (Hodgetts et al., 2008). The two gene sequence fragments of phytoplasma were obtained from the DNA of six symptomatic plant samples whereas not from the DNA of six asymptomatic plant samples. These amplified products were sequenced and the data were deposited in GenBank. The two gene sequence fragments of the DNA obtained from the diseased plant samples were all identical, with a length of 1335 bp for the 16S rRNA (GenBank accession: MW616560) and 729 bp for the secA gene (MW603929). The secA gene fragment putatively encodes for 242 amino acids. The phytoplasma strain was named as Tephrosia purpurea witches'-broom (TpWB) phytoplasma, TpWB-hnld strain. 16S rRNA gene sequence fragment of TpWB-hnld was analyzed by online tool iPhyClassifier (Wei et al., 2007), indicating that the pathogen strain was a member of subgroup 16SrII-V and a 'Candidatus Phytoplasma aurantifolia'-related strain. Blast analysis based on the 16S rRNA gene sequence fragment of TpWB-hnld showed 100 % sequence identity with that of peanut witches'-broom group members (16SrII group), such as Cassava witches'-broom phytoplasma (KM280679) and Cleome sp. phytoplasma (KM280677); Blast analysis based on the secA gene sequence fragment of TpWB-hnld showed 100 % sequence identity with that of peanut witches'-broom group members (16SrII group), such as sesame phyllody phytoplasma (JN977044). Homology and phylogeny were analyzed using the software of DNAMAN 5.0 and MEGA 7.0, indicating that TpWB-hnld and other subgroup 16SrII-V phytoplasma strains, including Cassava witches'-broom phytoplasma, Cleome sp. phytoplasma, Crotalaria witches'-broom phytoplasma (EU650181) and Desmodium ovalifolium witches'-broom phytoplasma (GU113152), were clustered into one clade with 98 % bootstrap value based on the 16S rRNA gene sequence fragments; TpWB-hnld and sesame phyllody phytoplasma were clustered into one clade based on the secA gene sequence fragments. Multiple alignment based on the 16S rRNA gene sequence fragment showed that the TpWB-hnld phytoplasma strain showed 98 % sequence identity with TpWB phytoplasma strain (HG792252) belonging to 16SrII-M subgroup reported in India (Yadav et al., 2014). To our knowledge, this was the first time that 16SrII-V subgroup phytoplasma associated with Tephrosia purpurea witches'-broom disease was identified in China. Molecular analysis based on the 16S rRNA and secA gene sequence fragments indicated that TpWB-hnld phytoplasma was a member of subgroup 16SrII-V and a 'Candidatus Phytoplasma aurantifolia'-related strain.

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

Gerbera is the most popular cut flower known for its variety of colors and is grown across the world. Its production is challenged by numerous diseases affecting production and quality. During our survey, ten samples from the gerbera plants exhibiting phyllody disease symptoms were collected from Bangalore Rural District, Karnataka, India. The association of phytoplasma with the gerbera phyllody samples was confirmed by PCR using 16SrRNA, SecY, Ribosomal protein (rp) and SecA gene-specific primers. PCR products were amplified from all ten gerbera plants using phytoplasma-specific primers. The amplified PCR products were cloned and sequenced; the sequences of the ten clones were identical. Therefore, representative isolate (GePP1, Gerbera phyllody phytoplasma) was selected for further analysis. The sequence analysis showed that GePP1 shared maximum nucleotide (nt) identity of 97.1% (16SrRNA) with Eggplant big bud, 98.7 to 98.8% (SecY gene) with Tomato big bud, 99.2 to 99.6% (rp gene) with Alfalfa witches-broom (EF193371) and 99.1% (SecA gene) with Sesame phyllody phytoplasmas and that it belongs to the Ca. P. aurantifolia (16SrII) group. This result was well supported by the phylogenetic analysis showing GePP1 (16Sr RNA, SecY, rp and SecA genes) closely clustering with the Ca. P. aurantifolia 16SrII group isolates reported so far. The virtual RFLP pattern generated for the phytoplasma from gerbera was different (similarity coefficient 0.89) from the reference pattern of Ca. P. aurantifolia (16Sr II) subgroup after analysis with four enzymes (BfaI, Hha1, Sau3AI and RsaI). Based on the threshold similarity coefficient for a new subgroup (delineation should be set at 0.97), the GePP1 may be considered as new subgroup of Ca. P. aurantifolia (16SrII) group. This is the first report of Ca. P. aurantifolia belonging to 16Sr II group affecting gerbera in India. Keywords: Candidatus Phytoplasma aurantifolia; phyllody; gerbera; PCR; phylogenetic analysis.

Phytoplasmas are classified into 16Sr groups and subgroups and 'Candidatus Phytoplasma' species, largely or entirely based on analysis of 16S rRNA gene sequences. Yet, distinctions among closely related 'Ca. Phytoplasma' species and strains based on 16S rRNA genes alone have limitations imposed by the high degree of rRNA nucleotide sequence conservation across diverse phytoplasma lineages and by the presence in a phytoplasma genome of two, sometimes sequence-heterogeneous, copies of the 16S rRNA gene. Since the DNA-dependent RNA polymerase (DpRp) β-subunit gene (rpoB) exists as a single copy in the phytoplasma genome, we explored the use of rpoB for phytoplasma classification and phylogenetic analysis. We sequenced a clover phyllody (CPh) phytoplasma genetic locus containing ribosomal protein genes, a complete rpoB gene and a partial rpoC gene encoding the β'-subunit of DpRp. Primers and reaction conditions were designed for PCR-mediated amplification of rpoB gene fragments from diverse phytoplasmas. The rpoB gene sequences from phytoplasmas classified in groups 16SrI, 16SrII, 16SrIII, 16SrX and 16SrXII were subjected to sequence similarity and phylogenetic analyses. The rpoB gene sequences were more variable than 16S rRNA gene sequences, more clearly distinguishing among phytoplasma lineages. Phylogenetic trees based on 16S rRNA and rpoB gene sequences had similar topologies, and branch lengths in the rpoB tree facilitated distinctions among closely related phytoplasmas. Virtual RFLP analysis of rpoB gene sequences also improved distinctions among closely related lineages. The results indicate that the rpoB gene provides a useful additional marker for phytoplasma classification that should facilitate studies of disease aetiology and epidemiology.

In our previous study based on hsp60 PCR-restriction fragment length polymorphism and 16S rRNA gene sequencing, we stated that the bifidobacterial strains isolated from the individual faecal samples of five baby common marmosets constituted different phylogenetically isolated groups of the genus Bifidobacterium. In that study, we also proposed that these isolated groups potentially represented novel species of the genus Bifidobacterium. Out of them, Bifidobacterium aesculapii, Bifidobacterium myosotis, Bifidobacterium tissieri and Bifidobacterium hapali, have been described recently. Another strain, designated MRM 8.19T, has been classified as member of the genus Bifidobacterium on the basis of positive results for fructose-6-phosphate phosphoketolase activity and analysis of partial 16S rRNA, hsp60, clpC, dnaJ, dnaG and rpoB gene sequences. Analysis of 16S rRNA and hsp60 gene sequences revealed that strain MRM 8.19T was related to B. tissieri DSM 100201T (95.8 %) and to Bifidobacterium bifidum ATCC 29521T (93.7 %), respectively. The DNA G+C composition was 63.7 mol% and the peptidoglycan structure was l-Orn(Lys)-l-Ser. Based on the phylogenetic, genotypic and phenotypic data reported, strain MRM 8.19T represents a novel taxon within the genus Bifidobacterium for which the name Bifidobacterium catulorum sp. nov. (type strain MRM 8.19T=DSM 103154T=JCM 31794T) is proposed.

A new disease named as periwinkle leaf yellowing (PLY) was first observed in a flower production farm in Dayuan Township (Taoyuan county, Taiwan) in August 2005. Sequence analysis of 16S rDNA, 16S-23S rDNA ISR, and partial 23S rDNA sequence revealed that the causative agent of PLY was closely related to the phytoplasmas of the aster yellows (AY) group (16SrI group) which cause diseases in many horticultural and vegetable crops worldwide, and can be delineated into 10 subgroups. Six cultivated plants including periwinkle plant, chrysanthemum, cosmos, torenia, Persian violet, goosegrass and cucumber, were determined to be the host plants of PLY phytolasma. Monthly PCR detection indicated that PLY phytoplasma was detected in host plants from June to October in 2007, and from July to October in 2007. However, it can be detected earlier since April in 2009. To further clarify the phylogenetic relationship of strain PLY among 16SrI phytoplasmas, six phylogenetic trees were constructed in this study. Beside the phylogenetic trees based on the independent analysis of 16S rRNA, rplV-rpsC, secY and tuf gene sequences, two other trees based on the analysis of the comprising gene sequence of 16S rRNA, rplV-rpsC and secY, and the comprising gene sequence of rplV-rpsC and secY were also constructed. The results indicate that the strain PLY was closely related to 16SrI-B and 16SrI-D subgroup, and could be a new subgroup based on the phylogenetic tree constructed by rplV-rpsC gene sequences. In addition, the phylogenetic analysis of the comprised gene sequences showed similar tree topology when compared with sequence analysis of the rplV/ rpsC gene or of the secY gene alone. The main difference is that the branch lengths were elongated in the comprised gene tree. To further confirm the subgroup affiliation of PLY phytoplasma, the 16S rRNA gene sequences of 10 closely related phytoplasma strains were digested in silico, and the similarity coefficients were then calculated. The results also support the conclusion that PLY phytoplasma might belongs to a new 16SrI subgroup. The putative restriction site analysis can also distinguish PLY phytoplasma from other close related phytoplasma strains in phylogenetic analysis.

Fenugreek (Trigonella foenum-graecum) is a leafy vegetable and spice crop, native to Indian subcontinent and Eastern Mediterranean region. Phytoplasma infection symptoms were observed in fenugreek at ICAR-National Bureau of Plant Genetic Resources Regional Station, Jodhpur and Agricultural Research Station Mandore Jodhpur, Rajasthan, India. The first appearance of phytoplasma suspected symptoms of little leaf was recorded after 50 days of sowing in the months of January 2022. The major symptoms recorded were virescence, phyllody, shoot proliferation, witches-broom, little leaf, yellowing and overall stunted growth in 146 germplasm accessions at NBPGR research farm, Jodhpur and one major commercially cultivated variety RMT 305 at Mandore Jodhpur. Ten samples from symptomatic and five samples from asymptomatic fenugreek plants were collected and processed for total DNA extraction using the Qiagen DNeasy plant mini kit (Germany). The extracted DNA was amplified using nested PCR assays with universal phytoplasma detection primers for 16S rRNA gene (P1/P7 and R16F2n/R16R2) and secA gene specific primers (SecAfor1/SecArev3 and SecAfor2/SecArev3) (Schneider et al. 1995; Gundersen and Lee 1996; Hodgetts et al. 2008). The amplicons of ∼1.25 kb with 16S rRNA and ∼480 bp with secA gene specific primers were amplified in all symptomatic fenugreek samples. In negative control (asymptomatic plants) no amplification was observed with either of gene specific primers in gel electrophoresis. PCR amplified products from the six selected positive samples (FPP-NBPGR-J-01 to FPP-NBPGR-J-04 and FPP-MND-01 to FPP-MND-02) of 16S rRNA and secA gene, were sequenced from both ends. Sequences were deposited in the NCBI GenBank with accession numbers ON756108-ON756113 for 16S rRNA gene sequences and ON745809 to ON745814 for secA gene sequences. BLAST analysis of 16S rRNA and secA sequences revealed 100% sequence identity among themselves and 99.95 to 100% sequence identity with the earlier reported phytoplasma strains of aster yellows group related phytoplasma strains (GenBank Acc. No. MN239504, MN080270) belonging to Ca. P. asteris (16SrI group). Further analyses of the 16S rRNA and secA gene-based phylogenetic tree and the iPhyClassifier-based virtual RFLP analysis of 16S rRNA gene study demonstrated that the phytoplasma associated with fenugreek phyllody belonged to 16Sr group I ('Ca. P. asteris') and subgroup B (GenBank accession AP006628), with similarity coefficient of 1.0. Earlier association of 16Sr-II-D subgroup (Ca. P. australasiae) with fenugreek as host was reported from Pakistan (Malik et al., 2020). To the best of our knowledge, this is the first report of a 'Ca. P. asteris', 16SrI-B subgroup related phytoplasma strain associated with fenugreek phyllody in the world. The 16SrI-B phytoplasma strain is a widely distributed strain associated with several agricultural and horticultural crops of India (Rao 2021). This is not only the first instance of fenugreek phyllody disease found in India, but also the first instance of fenugreek phyllody caused by 16SrI-B subgroup phytoplasma worldwide. This report has epidemiological significance and needs immediate attention, as fenugreek is one of the most common seed spice crop being grown all over India.

Chinaberry (Melia azedarach), belonging to the family of Meliaceae, is an ornamental tree distributes across southern of China. In the autumn of 2021, In an area of 400 acres located in Wanning city of Hainan Province, a tropical island in China, with coordinates of 110°28'42.72″E, 19°2'9.96″N, about 20 % (100) of the chinaberry trees showed disease symptoms included chlorotic leaves. The disease symptoms were consistent with infections by a phloem-limited prokaryotic pathogen phytoplasma. The samples of six symptomatic and three asymptomatic were collected for pathogen detection. To identify the pathogen, total nucleic acids were extracted from 0.10 g fresh leaf tissues from the diseased and healthy plant using CTAB DNA extraction method based on Doyle and Doyle. Three primer pairs of R16mF2/R16mR1, secAfor1/secArev3 and fTuf1/rTuf1 were used for specific identification of phytoplasma conserved gene fragments of 16S rDNA, secA and tuf, PCR amplification. Target PCR bands were amplified from the DNA of six diseased chinaberry samples, but not from the DNA of the healthy samples. The products of amplified were cloned and sequenced by Biotechnology (Shanghai) Co., Ltd. (Guangzhou, China). The phytoplasma gene sequences of 16S rRNA, secA and tuf were obtained and all the sequences were identical with the length of 1336 bp, 710 bp and 955 bp, respectively. Representative sequence data for strain MaCL-hn were deposited in Genbank under accession Nos. OR438638 (16S rDNA), OR513089 (secA) and OR860415 (tuf). The phytoplasma strain identified in the study was described as chinaberry chlorotic leaf (MaCL) phytoplasma, MaCL-hn strain. BLAST search based on 16S rRNA genes showed that 43 strains in 16SrI group 'Candidatus Phytoplasma asteris' showed 100% similarity with the 16SRNA sequence of MaCL-hn. BLAST search based on secA genes showed that 9 strains in the phytoplasma group showed 100% similarity with the 16SRNA sequence of MaCL-hn. BLAST search based on tuf genes showed that 21 strains in the phytoplasma group showed 100% similarity with the 16SRNA sequence of MaCL-hn. RFLP analysis based on iPhyClassifier indicated that the MaCL-hn strain was a member of 16SrI-B subgroup with a similarity coefficient 1.00 to the reference phytoplasma strain (AP006628). Phylogenetic tree was constructed based on 16S rRNA by MEGA 11.0 using neighbor-joining (NJ) method with 1000 bootstrap value. The results showed that the MaCL-hn strains were clustered into one clade with 16SrI group 'Ca. Phytoplasma asteris' related strains with 99 % bootstrap value. Multilocus sequence analysis (MLSA) based on the concatenated sequences with the length of 3001 bp including the sequences of 16S rRNA, secA and tuf showed that the MaCL-hn strains were clustered into one clade with the phytoplasma strains in the group with 100 % bootstrap value. To our knowledge, this is the first report that chinaberry can be infected by 'Ca. Phytoplasma asteris'-related strains belonging to 16SrI-B subgroup on Hainan Island of China. This finding in the study will contribute to the epidemic monitoring and the preventive management of the phytoplasmas and their related diseases.

RFLP analyses of 16S rDNA nested PCR products from 34 phytoplasma strains with 17 restriction enzymes delineated distinct pattern types. Based on similarity coefficients derived from RFLP analyses, the 34 representative phytoplasma strains were differentiated into 14 major groups (termed 16Sr groups) and 32 sub-groups. The similarity coefficients of RFLP patterns between distinct groups were 90% or below. By including additional groups and sub-groups from which RFLP analyses were not performed but for which 16S rDNA sequence data were available to predict restriction sites, a total of 14 groups and 41 sub-groups were proposed. By combined RFLP analyses of 16S rRNA and ribosomal protein gene sequences, thus far, a total of 46 subgroups have been recognized. The phytoplasma 16Sr groups were consistent with the phylogenetic groups (subclades) defined by phylogenetic analysis of near-full-length 16S rRNA gene sequences, indicating that the RFLP-based groups are phylogenetically valid. The approach using RFLP analyses of PCR-amplified 16S rDNA (and ribosomal protein gene sequences) provides a simple, reliable and rapid means for differentiation and classification of unknown phytoplasmas.

Phytoplasmas classified in group 16SrXII infect a wide range of plants and are transmitted by polyphagous planthoppers of the family Cixiidae. Based on 16S rRNA gene sequence identity and biological properties, group 16SrXII encompasses several species, including 'Candidatus Phytoplasma australiense', 'Candidatus Phytoplasma japonicum' and 'Candidatus Phytoplasma fragariae'. Other group 16SrXII phytoplasma strains are associated with stolbur disease in wild and cultivated herbaceous and woody plants and with bois noir disease in grapevines (Vitis vinifera L.). Such latter strains have been informally proposed to represent a separate species, 'Candidatus Phytoplasma solani', but a formal description of this taxon has not previously been published. In the present work, stolbur disease strain STOL11 (STOL) was distinguished from reference strains of previously described species of the 'Candidatus Phytoplasma' genus based on 16S rRNA gene sequence similarity and a unique signature sequence in the 16S rRNA gene. Other stolbur- and bois noir-associated ('Ca. Phytoplasma solani') strains shared >99 % 16S rRNA gene sequence similarity with strain STOL11 and contained the signature sequence. 'Ca. Phytoplasma solani' is the only phytoplasma known to be transmitted by Hyalesthes obsoletus. Insect vectorship and molecular characteristics are consistent with the concept that diverse 'Ca. Phytoplasma solani' strains share common properties and represent an ecologically distinct gene pool. Phylogenetic analyses of 16S rRNA, tuf, secY and rplV-rpsC gene sequences supported this view and yielded congruent trees in which 'Ca. Phytoplasma solani' strains formed, within the group 16SrXII clade, a monophyletic subclade that was most closely related to, but distinct from, that of 'Ca. Phytoplasma australiense'-related strains. Based on distinct molecular and biological properties, stolbur- and bois noir-associated strains are proposed to represent a novel species level taxon, 'Ca. Phytoplasma solani'; STOL11 is designated the reference strain.

Peony (Paeonia suffruticosa Andr.), belonging to family Paeoniaceae, is an important medicinal and ornamental plant. During August of each year from 2016 to 2023, peony plants at Heze city were found to exhibit leaf yellows symptoms. The incidence rate of the symptomatic plant was recorded from 10% to 30% in four peony gardens with about 200 acres. Total DNA was extracted from 0.10 g fresh plant leaf tissues from 24 symptomatic and 8 asymptomatic samples using rapid plant genomic DNA isolation kit (Aidlab Biotechnology, Beijing, China). The extracted DNA was amplified by nested polymerase chain reaction using universal primers R16mF2/R16mR1 followed by R16F2/R16R2 (Lee et al., 1993; Gundersen and Lee, 1996) specific for the 16S rRNA gene and new designed tuf gene specific primers JWB-tuforfF1 (5'-ATGGCTGAAATATTTTCAAGAG-3') and JWB-tuforfR1 (5'-TTATTCTATGATTTTAATAACAG-3') followed by JWB-tuforfF2 (5'-ATGTAAACGTAGGAACTATTGG-3') and JWB-tuforfR2 (5'- TCCGATAGTTCTTCCACCTTCAC-3'). Amplicons of about 1.25 kb and 1.02 kb (16S rRNA gene and tuf gene, respectively) were obtained in 8 symptomatic samples from four peony gardens. However, no amplification was obtained in any of the asymptomatic samples. The representative amplicons of 16S rRNA and tuf genes of three positive samples (Heze-9, -18 and -27) were cloned into a zero background pLB-simple vector (Tiangen Biotechnology, Beijing, China) and sequenced by Taihe Biotechnology, Beijing, China. Sequences obtained in the study were deposited in NCBI GenBank with accession numbers PP504882, PP504883 and PP504884 for the 16S rRNA gene as well as PP530237, PP530238 and PP530239 for the tuf gene. The phytoplasma strain under the study was described as peony yellows (PeY) phytoplasma, PeY-Heze strain. Alignment analysis by DNAMAN software showed that three 16S rRNA gene sequences obtained in the study shared 99.36% to 99.60% sequence identity and three tuf gene sequences obtained in the study were identical. BLAST analysis of the 16S rRNA gene sequences of the PeY-Heze phytoplasma strains showed 99.60%-99.84% sequence identity with 'Candidatus Phytoplasma ziziphi' (GenBank accession: CP025121). And tuf sequences of the strains showed 100% similarity with 'Ca. P. ziziphi' (CP025121). Interestingly, the virtual RFLP patterns derived from three 16Sr RNA gene sequences obtained in the study by iPhyClassifier (Zhao et al., 2009) were different from the reference patterns of all previously established 16Sr groups/subgroups. The most similar are the reference pattern of the 16Sr group VII, subgroup E (AY741531), with a similarity coefficient of 0.72, which is less than 0.85. These phytoplasma strains may represent a new 16Sr group. Phylogenetic analysis based on 16S rRNA genes using MEGA 7.0 by neighbor-joining (NJ) method with 1000 bootstrap value indicated that PeY-Heze strains clustered into one clade with the phytoplasma strains of 'Ca. P. ziziphi' with 68% bootstrap value. Although there are several reports available on 'Ca. P. solani' infecting peony in Shandong Province, China (Gao et al., 2013). To our knowledge, this is the first report of 'Ca. P. ziziphi'-related strains infecting peony in China. The findings in this study will be beneficial to the detection, quarantine, and prevention of peony yellows phytoplasmas in China.

Aster yellows (AY) group (16SrI) phytoplasmas are associated with over 100 economically important diseases worldwide and represent the most diverse and widespread phytoplasma group. Strains that belong to the AY group form a phylogenetically discrete subclade within the phytoplasma clade and are related most closely to the stolbur phytoplasma subclade, based on analysis of 16S rRNA gene sequences. AY subclade strains are related more closely to their culturable relatives, Acholeplasma spp., than any other phytoplasmas known. Within the AY subclade, six distinct phylogenetic lineages were revealed. Congruent phylogenies obtained by analyses of tuf gene and ribosomal protein (rp) operon gene sequences further resolved the diversity among AY group phytoplasmas. Distinct phylogenetic lineages were identified by RFLP analysis of 16S rRNA, tuf or rp gene sequences. Ten subgroups were differentiated, based on analysis of rp gene sequences. It is proposed that AY group phytoplasmas represent at least one novel taxon. Strain OAY, which is a member of subgroups 16SrI-B, rpI-B and tufI-B and is associated with evening primrose (Oenothera hookeri) virescence in Michigan, USA, was selected as the reference strain for the novel taxon 'Candidatus Phytoplasma asteris'. A comprehensive database of diverse AY phytoplasma strains and their geographical distribution is presented.

Pea (Pisum sativum L.) is a leguminous vegetable crop, and India holds the fourth position in the production, primarily contributed by three major states: Uttar Pradesh, Madhya Pradesh, and Punjab (Anonymous, 2022). However, a survey conducted in February 2023 at the National Seed Corporation Farm (15 hectares) in Hisar, Haryana, revealed deformities in the growth of some pea plants. Approximately 10% of these plants exhibited a distinct bushy appearance, accompanied by phyllody and witches'-broom symptoms, characterized by deformed leaves and short internodes (Fig. 1). In response to these observed anomalies, a detailed molecular analysis was conducted at the Plant Pathology Laboratory, IARI, New Delhi. The investigation involved the collection of ten samples each from symptomatic and asymptomatic plants, and DNA was extracted from 100 mg leaf midribs using the CTAB method (Ahrens and Seemüller, 1992). The extracted DNA (100 ng/µl) along with one positive (Catharanthus roseus from glass house, 16SrII-D group) and one negative (without template DNA), served as a template for PCR reactions targeting the 16Sr RNA and secA genes. Universal primers P1/P7 and secAfor1/secArev3 were employed in the first round of PCR for the respective genes. Subsequently, the product from the 1st round was diluted and used as a template for the 2nd round of PCR with primers R16F2n/R16R2 for 16Sr RNA and secAfor2/secArev3 for the secA gene (Gundersen and Lee 1996; Deng and Hiruki 1991; Hodgetts et al. 2008). This nested-PCR approach yielded distinct bands, approximately 1.2 kb (16Sr RNA) and 480 bp (secA), from the DNA of all ten symptomatic plants and positive sample, while no bands were observed in any of the asymptomatic plants. The nested PCR products were sequenced by BBS labs (Barcode biosciences, Bengaluru). The 16Sr RNA gene sequences, showing 100% similarity, were submitted to NCBI as representative sequences (Acc. No. OQ690013, OQ690014, OQ709133, OQ709134). Similarly, secA gene sequences were submitted with Acc. Nos. OR604283-86. BLAST analysis revealed a maximum 99.76% identity with Onion yellows phytoplasma and 'Ca. P. asteris' reference strain for 16Sr RNA gene, and a maximum 100% identity with 'Elaeis guineensis' stunt phytoplasma for secA gene. The phylogenetic tree constructed using the 16S rRNA and secA gene sequences indicated that the pea phytoplasma strains of this study clustered with 'Ca. P. asteris' (16SrI-B) related strains (Fig 2a and 2b). Additionally, the 16S rRNA sequences from this study, when subjected to Virtual RFLP using iPhyclassifier (Zhao et al. 2009), exhibited a pattern (Fig. 3) matching the reference pattern of the 16S group I, subgroup B (GenBank accession: M30790), with a similarity coefficient of 1.0. Previously, Rao et al. (2021) reported several crops associated with the 16SrI ribosomal group, including eight sub-groups from India. However, this report represents the first instance of a phytoplasma 16SrI-B group associated with phyllody and witches'-broom symptoms in pea, both in India and globally. Considering the economic importance of pea as a vegetable crop, the observed disease incidence and affected area are significant. Urgent attention is required to conduct additional research and implement preventive measures to avert the potential outbreak of this disease in the near future.

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.