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.

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

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

Occurrence of 'Candidatus Phytoplasma asteris'-Related Strains infecting Chinaberry (Melia azedarach) Showing Chlorotic Leaf Symptoms on Hainan Island of China.

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.

Universal, Rapid, and Visual Detection Methods for Phytoplasmas Associated with Coconut Lethal Yellowing Diseases Targeting 16S rRNA Gene Sequences.

Coconut lethal yellowing (LY) diseases caused by phytoplasmas are devastating diseases for coconut cultivation and seriously threaten the coconut industry around world. The phytoplasmas associated with the LY diseases belonged to six 16Sr groups containing 16SrI, 16SrIV, 16SrXI, 16SrXIV, 16SrXXII, and 16SrXXXII with comparatively higher variable levels. Conserved regions of the 16S rRNA genes of LY phytoplasmas belonging to the six 16Sr groups were obtained in the study. Based on the conserved region sequences of 16S rRNA genes, two sets of LAMP primers, Co-4 and Co-6, were designed and screened, and the rapid and visual detection methods universal for different groups LY phytoplasmas were established. The entire detection reactions of the universal detection methods could be completed with only 30 to 40 min of constant temperature amplification at 64°C, and the detection results were judged by the color changes of the reaction systems, which are convenient and quick. For the six groups of phytoplasmas, the estimated minimum detection limit range of the universal detection primers Co-4 and Co-6 were identical: 4.8 × 101 to 4.8 × 107 copies per 200 µl. The universal detection methods for the LY phytoplasmas established in the study are of great significance for the rapid diagnosis and identification and the efficient monitoring and early warning as well as the port inspection and quarantine of the LY phytoplasmas and their related diseases.

Molecular Identification of 'Candidatus Phytoplasma malaysianum'-Related Strains Associated with Areca catechu Palm Yellow Leaf Disease and Phylogenetic Diversity of the Phytoplasmas within 16SrXXXII Group.

Areca catechu palm is an important cash plant in Hainan Island of China and even tropical regions worldwide. Areca catechu palm yellow leaf (AcYL) disease caused by the phytoplasmas is a devastating disease for the plant production. In the study, the phytoplasmas associated with the AcYL diseases were identified and characterized based on the conserved genes of the phytoplasmas, and genetic variation and phylogenetic relationship of the phytoplasma strains in the 16SrXXXII group was demonstrated. The results indicated that Areca catechu palm showing yellow leaf symptoms were single infected by 'Candidatus Phytoplasma malaysianum'-related strains belonging to 16SrXXXII-D subgroup. BLAST and multiple sequence alignment analysis based on 16S rRNA and secA genes showed that the AcYL phytoplasmas shared 100% sequence identity and 100% homology with the 'Ca. Phytoplasma malaysianum'-related strains. Phylogenetic analysis indicated that the AcYL phytoplasmas and 'Ca. Phytoplasma malaysianum'-related strains belonging to 16SrXXXII group were clustered into one clade with a 100% bootstrap value. Based on computer-simulated digestions, 6 kinds of RFLP patterns within 16SrXXXII group were obtained and a novel subgroup in the 16Sr group was recommended to propose to describe the relevant strains in this 16Sr subgroup. To our knowledge, this is the first report that Areca catechu palm showing yellow leaf symptoms infected by 'Ca. Phytoplasma malaysianum'-related strains belonging to 16SrXXXII group. And a novel 16Sr subgroup 16SrXXXII-F was proposed based on the systematical analysis of genetic variation of all the phytoplasmas within 16SrXXXII group. The findings of this study would support references for monitoring the epidemiology and developing effective prevention strategies of the AcYL diseases.

Alocasia macrorrhiza Represents a New Host of 'Candidatus Phytoplasma asteris'-Related Strains Associated with Yellows Symptoms in China.

Alocasia macrorrhiza, which belongs to the Araceae family, is an important landscape plant in China, and has of significant medicinal uses. In 2022, A. macrorrhiza displaying abnormal symptoms were found in Qionghai, Hainan Island of China (110°23'3.06″，19°7'56.29″). The incidence of symptomatic plants was about 40% in the sampled areas. The abnormal symptoms included that the ovoid leaves color turned yellow from green gradually, with ovoid leaves chlorosis, mesophyll tissue yellowing, miniature leaves and systemic wilting. The diseased symptoms suspected to be associated with phytoplasma according to the protocols of phytoplasma identification. In order to identify the pathogen, eleven diseased samples and three asymptomatic samples were collected from an area of about 40 hectares. Total DNAs were extracted from 0.10 g fresh plant leaf tissues using a CTAB DNA extraction method. PCR amplifications were performed using primers R16mF2/R16mR1 and fTuf1/rTuf1 specific for the phytoplasma 16S rRNA and tuf genes. Target PCR amplicons were obtained from the DNA of 11 diseased samples, whereas not from the DNA of the asymptomatic samples. The PCR products were cloned and sequenced by Biotechnology (Shanghai) Co., Ltd. (Guangzhou, China), and the obtained sequences were assembled, edited and analyzed using the EditSeq program and DNAMAN version 6.0. The phytoplasma 16S rRNA and tuf gene amplicons were 1336 and 930 bp in length, respectively. The sequences of all 16S rRNA and tuf amplicons in this study were identical. The sequencing data were deposited in GenBank with accession numbers OR466206 (16S rDNA) and OR513090 (tuf). According to the methods and protocols of phytoplasma identified and classification, the phytoplasma strain was described as Alocasia macrorrhiza yellows (AmY) phytoplasma, AmY-hn strain. BLAST search were conducted based on 16Sr RNA and tuf genes. The results showed that the AmY-hn had 100 % 16Sr RNA sequence identity (1336 bp out of 1336 bp) with that of 16SrI-B subgroup phytoplasmas like onion yellows phytoplasma (OY-M, AP006628). The AmY-hn had 100 % tuf sequence identity (930 bp out of 930 bp) with that of 16SrI-B subgroup phytoplasmas like OY-M. RFLP profiles obtained with iPhyClassifier demonstrated that AmY-hn strain was a member of the 16SrI-B subgroup with a similarity coefficient 1.00 to the reference phytoplasma strain (AP006628). Separated phylogenetic analysis based on 16S rRNA and tuf genes obtained with MEGA 7.0 using the neighbor-joining (NJ) method with 1000 bootstrap value indicated that AmY-hn clustered into one clade with phytoplasma strains of OY-M and chinaberry witches'-broom (KP662119) with 100 % and 87 % bootstrap value respectively. To our knowledge, this is the first report that a 'Candidatus Phytoplasma asteris'-related strain belonging to 16SrI-B subgroup infects A. macrorrhiza in China. The 16SrI-B subgroup 'Candidatus Phytoplasma asteris'-related strains can spread outwards through the plant A. macrorrhiza. Thus, the findings in the study will be beneficial to the detection of phytoplasmas which parasitic in this plant and the epidemic monitoring of the related diseases.

Carica papaya Represents a New Host of 16SrI-B Subgroup Phytoplasma Associated with Yellow Symptoms in China.

Carica papaya Linn, belonging to the Caricaceae family, is an economic and medicinal plant, which is widely cultivated in tropical and subtropical countries (Soib et al., 2020). Beginning in 2021, abnormal symptoms of Carica papaya exhibiting leaf yellow, crinkle and leaflet were found in Wanning city of Hainan Province, China. The diseased symptoms of the plant, with about 20 % incidence in the sampling regions, were suspected to be induced by phytoplasma, a phloem-limited and could not be cultured in vitro prokaryotic pathogen. Total DNAs were extracted from 0.10 g fresh leaves of symptomatic or asymptomatic Carica papaya using CTAB DNA extraction method (Doyle and Doyle, 1990). PCR reactions were performed using primers R16mF2/R16mR1 (Gundersen and Lee, 1996), secAfor1/secArev3 (Hodgetts et al., 2008) and AYgroelF/AYgroelR (Mitrovic et al., 2011) specific for phytoplasma 16S rRNA, secA and groEL gene fragments. PCR products of the 16S rRNA, secA and groEL gene target fragments of phytoplasma were obtained from the DNA of eight diseased Carica papaya samples whereas not from the DNA of the asymptomatic plant samples. The PCR amplicons of the three genes were cloned and sequenced by Biotechnology (Shanghai) Co., Ltd. (Shanghai, China) and the sequences data were deposited in GenBank. The 16S rRNA, secAgroEL gene of phytoplasma was in length of 1326 (GenBank accession: OL625608), 716 (OL630087) and 1300 (OL630088) bp separately, putatively encoding 238 (secA) and 432 (groEL) amino acids sequence. The phytoplasma strain was named as Carica papaya yellow phytoplasma (CpY), CpY-hnwn strain. A blast search based on 16Sr RNA gene of CpY-hnwn showed 100 % sequence identity with that of 16SrI aster yellows group members (16SrI-B subgroup), such as Onion yellows phytoplasma strain OY-M (AP006628), Chinaberry witches'-broom phytoplasma (CWB) strain CWB-hnsy1 (KP662119) and CWB-hnsy2 (KP662120), Rapeseed phyllody phytoplasma isolate RP166 (CP055264). RFLP analysis based on the 16S rRNA gene fragment of CpY-hnwn was performed by the interactive online phytoplasma classification tool iPhyClassifier (Zhao et al., 2009) indicated that the phytoplasma strain is a member of 16SrI-B subgroup. Blast search based on secA gene of CpY-hnwn showed 100 % sequence identity with that of CWB strains CWB-gdgz (KP662182), CWB-jxnc (KP662180) and CWB-fjya (KP662178) belonging to 16SrI-B subgroup. Blast search based on groEL gene of CpY-hnwn showed 99.77 % sequence identity with that of mulberry dwarf phytoplasma (AB124809) and 99.69 % sequence identity with that of Onion yellows phytoplasma strain OY-M (AP006628) and Rapeseed phyllody phytoplasma isolate RP166 (CP055264). Phylogenetic analysis based on the 16S rRNA gene fragments performed by MEGA 7.0 employing neighbor-joining (NJ) method with 1000 bootstrap value (Kumar et al., 2016; Felsenstein, 1985) indicated that the CpY-hnwn phytoplasma strain was clustered into one clade with the phytoplasma strains of OY-M (AP006628), RP166 (CP055264), CWB-hnsy1 (KP662119), CWB-hnsy2 (KP662120) and areca palm yellow leaf (KF728948), with 100 % bootstrap value. To our knowledge, this is the first report that a 16SrI-B subgroup phytoplasma infects Carica papaya in Hainan Province, a tropical island of China. Carica papaya was previously reported to be infected by 16SrXII-O subgroup phytoplasmas in Nigeria (Kazeem et al., 2021), 16SrII-U subgroup in Hainan Province of China (Yang et al., 2016). The findings in this study indicated that one plant couldthe phytoplasmas belonging to different 16Sr groups, which would be beneficial to the specific detection of the pathogens and the epidemic monitoring of the related diseases. References: Doyle, J.J. and Doyle, J.L. 1990. Focus 12: 13-15. Felsenstein, J. 1985. Evolution 39: 783-791. Gundersen, D.E. and Lee, I.M. 1996. Phytopath. Medit. 35: 144-151. Hodgetts, J., et al. 2008. Int. J. Syst. Evol. Microbiol. 58: 1826-1837. Kazeem, S.A., et al. 2021. Crop Prot. 148: 105731. Kumar, S., et al. 2016. Mol. Biol. Evol. 33: 1870-1874. Mitrovic, J., et al. 2011. Ann. Appl. Biol. 159: 41-48. Soib, H.H., et al. 2020. Molecules, 25: 517. Yang, Y., et al. 2016. Int. J. Syst. Evol. Microbiol. 66: 3485-3491. Zhao, Y., et al. 2009. Int. J. Syst. Evol. Microbiol. 59: 2582-2593.

Occurrence of a 16SrII-V Subgroup Phytoplasma Associated with Witches'-Broom Disease in Melochia corchorifolia in China.

Melochia corchorifolia L. is a plant belonging to the family Sterculiaceae, extracts from this plant have been reported to inhibit melanogenesis (Yuan et al., 2020). During September to November 2020, the plants showing abnormal symptoms including witches'-broom, leaf chlorosis, leaflet and internode shortening (Fig.1), were found in Dingan county of Hainan province, China, with about 50% infection rates in the field. The disease symptoms were suspected to be caused by the phytoplasma, a plant pathogenic prokaryotes that could not be cultured in vitro. Aiming to confirm the pathogen causing the symptoms, total DNA of the symptomatic or asymptomatic Melochia corchorifolia samples were extracted by CTAB method (Doyle and Doyle, 1990) using 0.10 g fresh plant leaves using the rapid extraction kit for plant genomic DNA (CTAB Plant Genome DNA Rapid Extraction Kit, Aidlab Biotechnologies Co., Ltd, Beijing, China). PCR reactions were performed using primers R16mF2/R16mR1 (Gundersen and Lee, 1996) specific for phytoplasma 16S rRNA gene fragments. PCR products of phytoplasma 16S rRNA gene sequences were obtained from the ten symptomatic plant samples but not from the DNA of the asymptomatic plant samples. The PCR products were cloned and sequenced by Biotechnology (Shanghai) Co., Ltd. (Shanghai, China) and the data were deposited in GenBank. The sequences of 16S rRNA gene fragments amplified from the DNA extracted from the disease plant samples were all identical, with a length of 1336 bp for the 16S rRNA (GenBank accession: MZ353520). Nucleotide Blast search based on the 16S rRNA gene fragment of the phytoplasma strain showed 100% sequence identities with that of 16SrII peanut witches'-broom group members, such as Cassava witches'-broom phytoplasma (KM280679), Cleome sp. phytoplasma (KM280677), Tephrosia purpurea witches'-broom phytoplasma (MW616560), Desmodium triflorum little leaf phytoplasma (MT452308) and Peanut witches'-broom phytoplasma (JX403944). Analysis of the 16S rRNA gene sequence of McWB-hnda strain by interactive online phytoplasma classification tool iPhyClassifier (Zhao et al., 2009) indicated that the phytoplasma strain is a member of 16SrII-V subgroup. The phytoplasma strain was named as Melochia corchorifolia witches'-broom (McWB) phytoplasma, McWB-hnda strain. Phylogenetic analysis performed by MEGA 7.0 employing neighbor-joining (NJ) method with 1000 bootstrap value (Kumar et al., 2016) indicated that the McWB-hnda phytoplasma strain was clustered into one clade with the phytoplasma strains of Tephrosia purpurea witches'-broom, Cleome sp., Peanut witches'-broom, Cassava witches'-broom and Desmodium triflorum little leaf with 97 % bootstrap value (Fig.2); McWB-hnda phytoplasma strain identified in the study and Melochia corchorifolia phyllody phytoplasma strain (KX150461) belonging to 16SrI-B subgroup previously identified in the Hainan Island of China by Chen et al. (2017) are in two independent clades(Fig.2). To our knowledge, this is the first report of a 16SrII-V subgroup phytoplasma associated with Melochia corchorifolia witches'-broom disease in Hainan Province, a tropical island of China. The phytoplasma strain identified in the study was relatively close to 16SrII peanut witches'-broom group phytoplasma strains associated with witches'-broom or little leaf diseases in the plants like Peanut, Tephrosia purpurea, Cassava and Desmodium triflorum. Our finding in the study indicated that Melochia corchorifolia may act as an alternative natural host not only for 16SrI-B subgroup phytoplasma but also for 16SrII-V subgroup phytoplasma, which would contribute to the spreading of the related phytoplasma diseases.

Tephrosia purpurea Represents a New Host of 16SrII-V Subgroup Phytoplasma Associated with Witches'-Broom Disease in China.

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.

Occurrence of Phytoplasma Belonging to 16SrII Group Associated with Witches'-broom Symptoms in Emilia sonchifolia in Hainan Island of China.

Emilia sonchifolia is a medical plant belonging to the family of Asteraceae, mainly used as a traditional Chinese medicine with the function of anti-inflammatory, analgesic, antibacterial and so on. During October to November 2020, the plants showing abnormal symptoms including witches'-broom, internode shortening, leaf chlorosis and leaflet were found in Hainan province, a tropical island of China. The total DNA of the plant samples were extracted using 0.10 g fresh plant leaves using CTAB method. PCR reactions were performed using primers R16mF2/R16mR1 and secAfor1/secArev3 specific for phytoplasma 16S rRNA and secA gene fragments. The target productions of the two gene fragments of phytoplasma were detected in the DNA from three symptomatic plant samples whereas not in the DNA from the symptomless plant samples. The two gene fragments of the DNA extracted from the symptomatic plant samples were all identical, with the length of 1324 bp 16S rRNA and 760 bp secA gene sequence fragments, putatively encoding 253 (secA) amino acids sequence. The phytoplasma strain was named as Emilia sonchifolia witches'-broom (EsWB) phytoplasma, EsWB-hnda strain. To our knowledge, this was the first report that Emilia sonchifolia witches'-broom disease was caused by the phytoplasma belonging to16SrII-V subgroup in Hainan island of China, with close relationship to 16SrII peanut witches'-broom group phytoplasma strains infecting the plants like peanut, Desmodium ovalifolium and cleome from the same island of China and cassava from Viet Nam.

Waltheria indica is a New Host of Phytoplasma Belongs to 16SrI-B Subgroup Associated with Virescence Symptoms in China.

Waltheria indica L. is a kind of medicinal plants belonging to the family of Sterculiaceae distributed in China, which extracts with many active compounds used for treatment of rheumatism and sore pains (Hua et al., 2019). During September to November 2020, the plants showing abnormal symptoms including floral virescence, leaf chlorosis and leaflet, as shown in Fig.1, were found in Dingan county of Hainan province, China, with about 70% incidence. The disease symptoms which were suspected to be infected by the phytoplasma, a phloem-limited cell-wall-less prokaryotic pathogen could not be cultured in vitro, severely impacted Waltheria indica growth resulting in financial loss and ecological damage in the location. For identification of the causal pathogen, the total DNA of symptom or symptomless Waltheria indica samples were extracted using 0.10 g fresh plant tissues using CTAB method. PCR reactions were performed using primers R16mF2/R16mR1 (Lee et al., 1993) and AYgroelF/AYgroelR (Mitrovic et al., 2011) specific for phytoplasma 16S rRNA and groEL gene fragments. The target productions of the two gene fragments of phytoplasma were detected in the DNA from four symptomatic plant samples whereas not in the DNA from the symptomless plant samples. The PCR productions were sequenced and the data were deposited in GenBank. The two gene fragments of the DNA extracted from the symptom plant samples were all identical, with the length of 1340 bp 16S rRNA (GenBank accession: MW353909) and 1312 bp groEL (MW353709) gene sequence fragments, putatively encoding 437 (groEL) amino acids sequence. The phytoplasma strain was named as Waltheria indica virescence (WiV) phytoplasma, WiV-hnda strain. A Blast search based on the 16S rRNA gene fragment of WiV-hnda phytoplasma strain revealed the highest level of sequence identities (99.85%) with that of 16SrI aster yellows group members (16SrI-B subgroup), such as Onion yellows phytoplasma strain OY-M (AP006628) from Japan (Oshima et al., 2004); Periwinkle virescence phytoplasma strain PeV-hnhk (KP662136), Chinaberry witches'-broom phytoplasma strain CWB-hnsy1 (KP662119) and CWB-hnsy2 (KP662120), all the strains from Hainan island of China (Yu et al., 2017). A Blast search based on the groEL gene sequence fragment of WiV-hnda indicated 99.92% sequence identity with that of 16SrI aster yellows group members (16SrI-B subgroup) such as Onion yellows phytoplasma strain OY-M (AP006628). Homology and phylogenetic analysis by DNAMAN 5.0 and MEGA 7.0 software indicated that the phytoplasma strains of WiV-hnda, OY-M, PeV-hnhk, CWB-hnsy1 and CWB-hnsy2 were clustered into one clade based on the 16S rRNA gene fragments. WiV-hnda, OY-M and Aster yellow witches'-broom (AYWB) (CP000061) phytoplasma strains were clustered into one clade based on the groEL gene fragments. To our knowledge, this was the first time that Waltheria indica virescence disease induced by 16SrI-B subgroup phytoplasma strain was reported in China. Genetic analysis showed that WiV-hnda was closely related to the phytoplasma strains causing Onion yellows in Japan, Periwinkle virescence and Chinaberry witches'-broom disease in China.

First report of 16SrI-B subgroup related phytoplasma associated with witches'-broom symptoms in Pericampylus glaucus in China.

Pericampylus glaucus is an important medicinal plant resource containing active components with potential antitumor activity in China (Zhao & Cui, 2009). During July through August 2020, plants displayed disease symptoms including "witches' broom", leaf chlorosis, leaflet and internode shortening that impacted their growth (Fig. 1). These plants were first found in Dingan county of Hainan province, China. Total DNA from 12 plants were extracted using 0.10 g fresh plant leaves based on CTAB method. After amplification using primers specific for phytoplasma 16S rRNA, tuf and secA gene targets, R16mF2R16mR1 (Lee et al, 1993), fTuf1/rTuf1 (Schneider et al., 1997) and secAfor1/secArev3 (Hodgetts et al., 2008), the target bands of the three gene fragments of phytoplasma were detected in the disease sample DNA from six disease plants, and not in the healthy sample DNA from six healthy plants. Nucleotide sequences of the three genes were obtained from the PCR products sequencing and analyzed by DNAMAN 5.0 software. The three gene fragments of the DNA extracted from the disease samples were identical, with length of 1334 bp 16S rRNA (GenBank accession: MT872515), 989 bp tuf (MT755960) and 750 bp secA (MT755961) gene fragments, putatively encoding 329 (tuf) and 249 (secA) amino acids sequence separately. The phytoplasma strain was named as Pericampylus glaucus witches'-broom (PgWB) phytoplasma, PgWB-hnda strain, belonging to 16SrI-B subgroup by iPhyClassifier analysis. Homology and phylogenetic analysis indicated that based on 16S rRNA gene fragments, PgWB-hnda, pepper yellow crinkle phytoplasma PYC-hnhk (MT760793), chinaberry witches'-broom phytoplasma CWB-hnsy1 (KP662119) and CWB-hn (EF990733), periwinkle virescence phytoplasma PeV-hnhk (KP662136), with 100.0 % identity value, arecanut yellow leaf phytoplasma AYL-hnwn (FJ998269) and AYL-hn (FJ694685), with 99.8 % identity value, were clustered into one clade. Based on the analysis of tuf gene sequence fragments, PgWB was closely related to PYC-hnhk (MT755960), CWB-hnsy1 (KP662155), PeV-hnhk (KP662172) with 99.9 % identity value. Based on the analysis of secA gene sequence fragments, PgWB was closely related to CWB-hnsy1 (KP662173) with 99.7 % identity value, PYC-hnhk (MT755961), PeV-hnhk (KP662190) with 99.4 % identity value. To our knowledge, this is the first time that Pericampylus glaucus witches'-broom disease caused by 16SrI-B subgroup phytoplasma strain was found in China. Multilocus sequence analysis showed that PgWB was closely related to the phytoplasma strains causing pepper yellow crinkle, chinaberry witches'-broom, periwinkle virescence and areca palm yellow leaf diseases, all occurred in Hainan Island of China.

First report of phytoplasma belongs to 16SrXXXII group associated with witches'-broom symptoms in Trema tomentosa in China.

Trema tomentosa (Roxb.) Hara belonging to Ulmaceae displayed abnormal symptoms including witches'-broom, internode shortening, leaf chlorosis and leaflet that affected seriously their growth causing financial loss and ecological damage in China. During August through September 2020, these plants with the symptoms were first found and collected in Dingan and Qinghai counties of Hainan province, China. PCR were performed using the primers R16mF2/R16mR1 and secAfor1/secArev3 specific for phytoplasma 16S rRNA and secA gene fragments. The two gene fragments of the DNA extracted from the four disease samples were identical, with length of 1303 bp 16S rRNA and 587 bp secA gene fragments. The phytoplasma strain was named as Trema tomentosa witches'-broom (TtWB) phytoplasma, TtWB-hn strain. Phylogenetic and computer-simulated RFLP analyses based on the nearly full-length 16S rRNA gene sequence indicated that the TtWB phytoplasma strain is more closely related to the 16SrXXXII-A subgroup than to the other subgroups within 16SrXXXII group. It may represent a new subgroup, designed as 16SrXXXII-D subgroup, which is distinct from the other phytoplasma subgroups within the 16SrXXXII group. To our knowledge, this is the first report showing the occurrence of the phytoplasma strain belongs to 16SrXXXII-D subgroup associated with witches'-broom disease in Trema tomentosa in China. Genetic analysis indicated that the TtWB strain was closely related to the phytoplasma strains infecting periwinkle, oil palm, coconut palm in Malyasian, Camptotheca acuminate in Yunnan province of China and Elaeocarpus zollingeri in Japan.

Elevated serum leptin may be associated with disease activity and secondary osteoporosis in Chinese patients with rheumatoid arthritis.

INTRODUCTION: Rheumatoid arthritis (RA) is a systemic chronic autoimmune disease in adults that is associated with significant joint issues and systemic inflammation. One of the signs of bone damage in RA is osteoporosis (OP). Leptin is an inflammatory protein that has been reported to be related to RA. The potential relationships among leptin, disease activity, and OP in Chinese patients with RA are not well known. METHODS: In total, 245 patients with RA and 120 healthy controls were included in this study. Detailed data on the clinical characteristics and laboratory features were collected. Information about physical activity and functional status was recorded using specific questionnaires. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry (DXA). The MECALL castor-50-hf model X-ray scanner was used for the two-hand (including wrist) photographs. RESULTS: Serum leptin levels differed significantly between the RA group and healthy control subjects (1.27/3.29 vs. 0.17/0.24, Z=13.29, P<0.001). The positive rate of leptin protein in RA patients was 86.35%, which was higher than that in controls (19.55%) (χ2=28.51, P<0.001). Pearson's correlation test showed that morning stiffness, disease duration, joint swelling, joint tenderness, swollen joint count (SJC), tender joint count (TJC), health assessment questionnaire (HAQ) score, and Sharp-van der Heijde method (Sharp) score were positively correlated with the level of serum leptin (r=0.212, r=0.312, r=0.322, r=0.501, r=0.291, r=0.334, P<0.05). There was a clear increasing trend in the level of serum leptin according to the different disease activity scores and in the 28 joint activity (DAS28) groups (F=13.936, P<0.001). Elevated leptin was a risk factor for increased disease activity and OP according to logistic regression analysis. The median leptin level differed significantly between the normal bone mass group, osteopenia group, and OP group (P<0.001). An increased serum leptin level was a risk factor for RA-induced osteoporosis according to logistic regression analysis (P<0.001). CONCLUSION: These results suggest that the level of serum leptin is associated with disease activity and secondary OP among Chinese patients with RA. Key Points • Serum leptin levels in RA patients are higher than those in normal control group. • Leptin was associated with disease activity. • Leptin was associated with the occurrence of systemic osteoporosis and affects bone erosion in RA patients.

[Effect of storax on concentration of sulpiride in brain and blood by MD-HPLC].

OBJECTIVE: To observe the effect of traditional Chinese medicine storax on the concentration of combined western medicine sulpiride in brain and blood, discuss the effect of storax in inducing resuscitation and increasing the permeability of the gastrointestinal barrier (GB) and the blood-brain-barrier (BBB), and explore the interaction between storax and sulpiride. METHOD: Rats were orally administered with the drugs for one week, probes were implanted in their brains and necks by surgery. After balance for 60 min, brain microdialysis and blood microdialysis were adopted for collect dislysates from blood in right atrum and cerebral hippocampus at time periods of 30, 60, 90, 120, 150, 180 min. The concentration of sulpirde in the samples was detected by RP-HPLC. Statistical approaches were adopted to compare the contents of sulpirde in brain and blood of the two groups. RESULT: The sulpiride combined with storax group showed a significant higher concentration of sulpiride than the pure sulpiride group. The pure sulpiride group showed a concentration ratio between sulpiride in brain and blood of 1:0.2; while the sulpiride combined with storax group increased the concentration ratio between sulpiride in brain and blood to 1:0.3. Compared with the pure sulpiride group, the sulpiride combined with storax group showed an increase of concentration by 39% in brain and 69% in blood. CONCLUSION: Storax can notably increase the concentration of sulpiride in rat brain and blood, indicating that it can increase the permeation of sulpiride through gastrointestinal barrier and BBB. This study reveals the mechanism of storax in inducing resuscitation by promoting the permeation through gastrointestinal barrier and BBB.

Molecular Identification and Characterization of Two Groups of Phytoplasma and Candidatus Liberibacter Asiaticus in Single or Mixed Infection of Citrus maxima on Hainan Island of China.

The pathogens associated with citrus Huanglongbing symptoms, including yellowing and mottled leaves in Citrus maxima, an important economic crop on Hainan Island of China, were identified and characterized. In the study, detection, genetic variation and phylogenetic relationship analysis of the pathogens were performed based on 16S rRNA and ß-operon gene fragments specific to phytoplasma and Candidatus Liberibacter asiaticus. The results indicated that the pathogens-such as phytoplasma strains of CmPII-hn belonging to the 16SrII-V subgroup and CmPXXXII-hn belonging to the 16SrXXXII-D subgroup, as well as Candidatus Liberibacter asiaticus strains CmLas-hn-were identified in the diseased plant samples, with numbers of 12, 2 and 6 out of 54, respectively. Among them, mixed infection with the 16SrII-V subgroup phytoplasma and Candidatus Liberibacter asiaticus was found in the study, accounting for 7.4% (four samples). The phytoplasma strains of CmPII-hn-Tephrosia purpurea witches' broom, Melochia corchorifolia witches' broom and Emilia sonchifolia witches' broom-were clustered into one clade belonging to the 16SrII-V subgroup, with a 99% bootstrap value. The phytoplasma strains of CmPXXXII-hn and Trema tomentosa witches' broom belonging to 16SrXXXII-D, and the other 16SrXXXII subgroup strains were clustered into one clade belonging to the 16SrXXXII group with a 99% bootstrap value. There were 16 variable loci in the 16S rRNA gene sequences of the tested 16SrXXXII group phytoplasma strains, of which two bases had an insertion/deletion. The strains of Candidatus Liberibacter asiaticus, identified in the study and the strains that had been deposited in GenBank, were in one independent cluster with a 99% bootstrap value. To our knowledge, this is the first report showing that Citrus maxima can be infected by 16SrII-V and16SrXXXII-D subgroup phytoplasmas in China. Moreover, this is also the first report in which the plants are co-infected by 16SrII-V subgroup phytoplasmas and Candidatus Liberibacter asiaticus. More comprehensive and detailed identification and characterization of the pathogens associated with the diseased symptoms in Citrus maxima on the island in China would be beneficial for epidemic monitoring and for the effective prevention and control of related plant diseases.

Molecular Diversity and Evolutionary Relatedness of Paulownia Witches'-Broom Phytoplasma in Different Geographical Distributions in China.

To reveal the distribution and transmission pathway of Paulownia witches'-broom (PaWB) disease, which is caused by phytoplasmas related to genetic variation, and the adaptability to the hosts and environments of the pathogenic population in different geographical regions in China, in this study, we used ten housekeeping gene fragments, including rp, fusA, secY, tuf, secA, dnaK, rpoB, pyrG, gyrB, and ipt, for multilocus sequence typing (MLST). A total of 142 PaWB phytoplasma strains were collected from 18 provinces or municipalities. The results showed that the genetic diversity was comparatively higher among the PaWB phytoplasma strains, and substantially different from that of the other 16SrI subgroup strains. The number of gene variation sites for different housekeeping genes in the PaWB phytoplasma strains ranged from 1 to 14 SNPs. Among them, rpoB (1.47%) and dnaK (1.12%) had higher genetic variation, and rp (0.20%) had the least genetic variation. The tuf and rpoB genes showed the fixation of positively selected beneficial mutations in the PaWB phytoplasma populations, and all housekeeping genes except tuf followed the neutral evolutionary model. We found an absence of recombination among PaWB phytoplasma sequence types (STs) for each housekeeping gene except dnaK, and no evidence for such recombination events for concatenated sequences of PaWB phytoplasma strains. The 22 sequence types were identified among the concatenated sequences of seven housekeeping genes (rp, fusA, secY, secA, tuf, dnaK, and rpoB) from 105 representative strains. We analyzed all 22 STs by goeBURST algorithm, forming two clonal complexes (CCs) and three singletons. Among them, ST1, as the primary founder of CC1, had the widest geographical distribution, accounting for 72.38% of all strains, with a high frequency of shared sequence type. The results of phylogenetic analysis of the concatenated sequences further revealed that the 105 strains were clustered into two representative lineages of PaWB phytoplasma, with obvious geographical differentiation. The ST1 strains of highly homogeneous lineage-1 were a widespread and predominant population in diseased areas. Lineage-2 contained strains from Jiangxi, Fujian, and Shaanxi provinces, highlighting the close genetic relatedness of the strains in these regions, which was also consistent with the results of most single-gene phylogenetic analysis of each gene. We also found that the variability in the northwest China population was higher than in other geographical populations; the range of genetic differentiation between the south of the Yangtze River population and the Huang-huai-hai Plain (or southwest China) population was relatively large. The achieved diversity and evolution data, as well as the MLST technique, are helpful for epidemiological studies and guiding PaWB disease control decisions.

Rapid and Efficient Detection of 16SrI Group Areca Palm Yellow Leaf Phytoplasma in China by Loop-Mediated Isothermal Amplification.

Areca palm yellow leaf (AYL) disease caused by the 16SrI group phytoplasma is a serious threat to the development of the Areca palm industry in China. The 16S rRNA gene sequence was utilized to establish a rapid and efficient detection system efficient for the 16SrI-B subgroup AYL phytoplasma in China by loop-mediated isothermal amplification (LAMP). The results showed that two sets of LAMP detection primers, 16SrDNA-2 and 16SrDNA-3, were efficient for 16SrIB subgroup AYL phytoplasma in China, with positive results appearing under reaction conditions of 64oC for 40 min. The lowest detection limit for the two LAMP detection assays was the same at 200 ag/µl, namely approximately 53 copies/µl of the target fragments. Phytoplasma was detected in all AYL disease samples from Baoting, Tunchang, and Wanning counties in Hainan province using the two sets of LAMP primers 16SrDNA-2 and 16SrDNA-3, whereas no phytoplasma was detected in the negative control. The LAMP method established in this study with comparatively high sensitivity and stability, provides reliable results that could be visually detected, making it suitable for application and research in rapid diagnosis of AYL disease, detection of seedlings with the pathogen and breeding of disease-resistant Areca palm varieties.

[Content and distribution of active components in cultivated and wild Taxus chinensis var. mairei plants].

Taxus chinensis var. mairei is an endemic and endangered plant species in China. The resources of T. chinensis var. mairei have been excessively exploited due to its anti-cancer potential, accordingly, the extant T. chinensis var. mairei population is decreasing. In this paper, ultrasonic extraction and HPLC were adopted to determine the contents of active components paclitaxel, 7-xylosyltaxol and cephalomannine in cultivated and wild T. chinensis var. mairei plants, with the content distribution of these components in different parts of the plants having grown for different years and at different slope aspects investigated. There existed obvious differences in the contents of these active components between cultivated and wild T. chinensis var. mairei plants. The paclitaxel content in the wild plants was about 0.78 times more than that in the cultivated plants, whereas the 7-xylosyltaxol and cephalomannine contents were slishtly higher in the cultivated plants. The differences in the three active components contents between different parts and tree canopies of the plants were notable, being higher in barks and upper tree canopies. Four-year old plants had comparatively higher contents of paclitaxel, 7-xylosyltaxol and cephalomannine (0.08, 0.91 and 0.32 mg x g(-1), respectively), and the plants growing at sunny slope had higher contents of the three active components, with significant differences in the paclitaxel and 7-xylosyltaxol contents and unapparent difference in the cephalomannine content of the plants at shady slope. It was suggested that the accumulation of the three active components in T. chinensis var. mairei plants were closely related to the sunshine conditions. To appropriately increase the sunshine during the artificial cultivation of T. chinensis var. mairei would be beneficial to the accumulation of the three active components in T. chinensis var. mairei plants.