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.

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.

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 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.

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.

Novel insights into the effect of paroxetine administration in pilocarpine­induced chronic epileptic rats.

The aim of the present study was to investigate the role of paroxetine intervention in epilepsy, and its association with the expression of serotonin transporter (SERT) and hippocampal apoptosis. Thirty adult male Sprague Dawley rats were divided into control vehicle (n=6) and epileptic (n=24) groups. Status epilepticus (SE) was induced via systemic injection of pilocarpine, and seizure activity was monitored via video electroencephalogram. The epileptic group was then randomly divided into two groups; Four weeks following SE induction, paroxetine (5 mg/kg/day; SE + paroxetine group) or normal saline (SE group) was intraperitoneally injected for 4 weeks. Brain tissue was collected to evaluate apoptosis via terminal deoxynucleotidyl transferase dUTP nick­end labeling. SERT, B­cell lymphoma­2 (Bcl­2) and brain derived neurotropic factor (BDNF) expression levels were evaluated by western blotting, and miR­16 expression was evaluated by reverse transcription­quantitative polymerase chain reaction. Paroxetine did not affect the mortality of the pilocarpine­induced chronic epileptic rats. Spontaneous recurrent seizures (SSRs) were observed 7­28 days following SE induction. The frequency and stage of the SSRs were reduced by paroxetine administration. Apoptotic cells were observed in the epileptic hippocampus. Following paroxetine intervention, the staining intensity and number of apoptotic cells were significantly decreased. Expression levels of BDNF and Bcl­2 were lower in the SE group compared with the vehicle group. The former was not altered by paroxetine injection; however, the latter was increased. In the SE group, SERT expression was not altered in the raphe nucleus but was decreased in the hippocampus. Following paroxetine administration, SERT expression was decreased in the raphe nucleus and increased in the hippocampus. In the SE group, miR­16 expression was decreased in the raphe nucleus and increased in the hippocampus. Following paroxetine administration, miR­16 expression was not altered in the raphe nucleus but was reduced in the hippocampus. In conclusion, the seizures and hippocampal apoptosis observed in chronic epileptic rats were alleviated by paroxetine treatment. This effect may be associated with the reduced Bcl­2 and BDNF expression and the modulation of SERT expression. The alterations in miR­16 expression may provide a potential explanation for the modulation of apoptosis; however, further research is required to determine the complete underlying molecular mechanism.

Seizure-induced 5-HT release and chronic impairment of serotonergic function in rats.

We analyzed the dynamic concentration change of serotonin (5-HT) and its main metabolite 5-hydroxyindoleacetic acid (5-HIAA) within the epileptic hippocampus in rats. Seizure was induced by systemic injection of pilocarpine (320mg/kg, i.p.). Using electroencephalography (EEG) recordings, we found that primary seizure discharge was induced 30min after pilocarpine administration and that recurrent discharge peaked 14d after the onset of status epilepticus (SE). The extracellular fluid in the hippocampus was sampled by microdialysis from conscious animals at various time points before and after SE. The concentrations of 5-HT and 5-HIAA in the samples were measured by high-performance liquid chromatography and electrochemical detection (HPLC-ECD). Interestingly, 5-HT levels in the hippocampus were dramatically increased within the 30min following SE. This reversed to basal level by 4d after SE and continued to drop to 48% at 7d and 28% of basal level 14d after SE. Accordingly, a marked increase of 5-HIAA in the hippocampus appeared at 2d after SE, then gradually declined to levels below baseline. To identify serotonergic neurons in the raphe nuclei (a major source of 5-HT release in the brain), brain sections were immunostained for tryptophan hydroxylase (TPH). The number of TPH positive neurons and the intensity of TPH staining significantly decreased at 28d after SE. These data suggest that pilocarpine induces depletion of 5-HT in the hippocampus and significantly compromise serotonergic neurons in the raphe nuclei. The loss of serotonergic function may play a significant role in the pathophysiology of epilepsy.

[The effect of high frequency stimulation of epileptic foci on the release of glutamate and gamma-aminobutyric acid in hippocampus of the kainic acid-kindled rats].

OBJECTIVE: To observe the dynamics of hippocampal release of glutamate (Glu) and gamma-aminobutyric acid (GABA) in epilepsy (TLE) after administration with high frequency stimulation (HFS). METHODS: The SD were divided into four groups (n =10): (1) Control group (KB) the rats were injected intraperitoneally with saline 0.9%. (2) Kainic acid (KA) group: the rats were injected with KA. (3) Pseudo-deep brain stimulation (DBS) group: the KA-induced rats were implanted with rheophores alone. (4) DBS group: KA induced-rats with DBS in hippocampal epileptic foci. We then collected hippocampal extracellular fluid by microdialysis and the levels of Glu and GABA were measured by high-performance liquid chromatography (HPLC) and fluorescence detection. RESULTS: There was no difference in the baseline of Glu and GABA in the four groups. In contrast, a significant increase in the content of Glu and GABA was shown in the three periods of KA-kindled seizures. Electrical stimulation of hippocampus resulted in a decrease of hippocampal Glu contents, while there was no change in GABA contents. Additionally, HFS of hippocampus normalized the Glu/GABA ratio in the chronic period of seizures. CONCLUSION: The high frequency stimulation of epileptic foci may protect against seizures by modulating the extracellular release of hippocampal Glu.