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.

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

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

Highly Efficient Utilization of Ferrate(VI) Oxidation Capacity Initiated by Mn(II) for Contaminant Oxidation: Role of Manganese Species.

Manganese ion [Mn(II)] is a background constituent existing in natural waters. Herein, it was found that only 59% of bisphenol A (BPA), 47% of bisphenol F (BPF), 65% of acetaminophen (AAP), and 49% of 4-tert-butylphenol (4-tBP) were oxidized by 20 µM of Fe(VI), while 97% of BPA, 95% of BPF, 96% of AAP, and 94% of 4-tBP could be oxidized by the Fe(VI)/Mn(II) system [20 µM Fe(VI)/20 µM Mn(II)] at pH 7.0. Further investigations showed that bisphenol S (BPS) was highly reactive with reactive iron species (RFeS) but was sluggish with reactive manganese species (RMnS). By using BPS and methyl phenyl sulfoxide (PMSO) as the probe compounds, it was found that reactive iron species contributed primarily for BPA oxidation at low Mn(II)/Fe(VI) molar ratios (below 0.1), while reactive manganese species [Mn(VII)/Mn(III)] contributed increasingly for BPA oxidation with the elevation of the Mn(II)/Fe(VI) molar ratio (from 0.1 to 3.0). In the interaction of Mn(II) and Fe(VI), the transfer of oxidation capacity from Fe(VI) to Mn(III), including the formation of Mn(VII) and the inhibition of Fe(VI) self-decay, improved the amount of electron equivalents per Fe(VI) for BPA oxidation. UV-vis spectra and dominant transformation product analysis further revealed the evolution of iron and manganese species at different Mn(II)/Fe(VI) molar ratios.

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.

Approach to Predicting the Size-Dependent Inhalation Intake of Particulate Novel Brominated Flame Retardants.

The risk of human exposure to particulate novel brominated flame retardants (NBFRs) in the atmosphere has received increasing attention from scientists and the public, but currently, there is no reliable approach to predict the intake of these compounds on the basis of their size distribution. Here, we develop a reliable approach to predict the size-dependent inhalation intake of particulate NBFRs, based on the gas/particle (G/P) partitioning behavior of the NBFRs. We analyzed the concentrations of eight NBFRs in 363 size-segregated particulate samples and 99 paired samples of gaseous and bulk particles. Using these data, we developed an equation to predict the G/P partitioning quotients of NBFRs in particles in different size ranges (KPi) based on particle size. This equation was then successfully applied to predict the size-dependent inhalation intake of particulate NBFRs in combination with an inhalation exposure model. This new approach provides the first demonstration of the effects of the temperature-dependent octanol-air partitioning coefficient (KOA) and total suspended particle concentration (TSP) on the intake of particulate NBFRs by inhalation. In an illustrative case where TSP = 100 µg m-3, inhalation intake of particulate NBFRs exceeded the intake of gaseous NBFRs when log KOA > 11.4.

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.

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.

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.

Determination of Polycyclic Aromatic Hydrocarbons and Their Methylated Derivatives in Sewage Sludge from Northeastern China: Occurrence, Profiles and Toxicity Evaluation.

This paper assesses the occurrence, distribution, source, and toxicity of polycyclic aromatic hydrocarbons (PAHs), and their methylated form (Me-PAHs) in sewage sludge from 10 WWTPs in Northeastern China was noted. The concentrations of ∑PAHs, ∑Me-PAHs ranged from 567 to 5040 and 48.1 to 479 ng.g-1dw, which is greater than the safety limit for sludge in agriculture in China. High and low molecular weight 4 and 2-ring PAHs and Me-PAHs in sludge were prevalent. The flux of sludge PAHs and Me-PAHs released from ten WWTPs, in Heilongjiang province, was calculated to be over 100 kg/year. Principal component analysis (PCA), diagnostic ratios and positive matrix factorization (PMF) determined a similar mixed pyrogenic and petrogenic source of sewage sludge. The average values of Benzo[a]pyrene was below the safe value of 600 ng.g-1 dependent on an incremental lifetime cancer risk ILCR of 10-6. Sludge is an important source for the transfer of pollutants into the environment, such as PAHs and Me-PAHs. Consequently, greater consideration should be given to its widespread occurrence.

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.

Membrane-Bound CD40L Promotes Senescence and Initiates Senescence-Associated Secretory Phenotype via NF-κB Activation in Lung Adenocarcinoma.

BACKGROUND/AIMS: Cellular senescence acts as a barrier against tumorigenesis. The CD40L transgene, expressed in some tumor cells, not only becomes visible to antigen-presenting cells but also actively catalyzes its own termination. Here, we evaluated the effect of a membrane-bound mutant form of human CD40L (CD40L-M) on senescence and the senescence-associated secretory phenotype (SASP) in non-small cell lung cancer (NSCLC). METHODS: CD40 expression levels in the NSCLC cell lines A549/TR, A549/DDP and H460 were examined by flow cytometry. Senescent cells and tissues were identified via SA-ß-gal activity. Cell proliferation was visualized by EdU labeling. qRT-PCR, Western blotting, and immunofluorescence staining were conducted to assess mRNA and protein expression levels of CD40L, γ-H2A.X, p65, p-p65, IκBα, p53, p21 and p16. Cytokines secreted from transfected cells were tested by ELISA and cell migration assay. Capsid tyrosine-modified rAAV5-CD40L-M was packaged and carried out in vivo. RESULTS: Overexpression of CD40L-M promoted senescence, inhibited proliferation, increased DNA damage-associated γ-H2A.X, and initiated the SASP in CD40-positive NSCLC cells. NF-κB signaling was activated by CD40L-M overexpression in these cells. Knockdown of NF-κB partially overcame senescence and failed to induce SASP. Furthermore, increased p53 and p21 protein levels induced by CD40L-M were also reduced following NF-κB suppression. CONCLUSIONS: These data showed that the membrane-bound CD40L mutant may promote cellular senescence and initiate the SASP of NSCLC cells in an NF-κB-dependent manner. Therefore, CD40L-M-induced senescence may be a potential approach to protect against lung adenocarcinoma.

Removal characteristic of surfactants in typical industrial and domestic wastewater treatment plants in Northeast China.

Surfactants are widely used in household and industrial products for cleaning and/or solubilization in our daily life. Therefore, they are finally discharged into wastewater treatment plants (WWTPs), which may be the major point pollution source for environment if they were not completely removed during wastewater treatment. In this study, two typical industrial and domestic WWTPs with different wastewater treatment technologies were considered for the topic. Totally, two types of surfactants were analyzed in 24 h influent and each processing unit effluent. Four linear alkylbenzene sulfonates (LASs) with the alkyl chain from C10 to C13, and two benzalkonium chlorides (BACs) with the alkyl chain of C12 and C14 were selected as target compounds. The total concentrations of LASs in influent varied from 19.2 to 1889 µg/L and LAS-C11 and LAS-C12 were the predominant compounds with the concentration from 6.01 to 641 µg/L and 8.02-674 µg/L, respectively. The total concentrations of BACs were much lower than those of LASs, with the concentration ranging from 0.00935 to 1.85 µg/L. Significant positive correlations were observed between concentrations of LASs and BACs in influent, indicating their same and/or similar sources. Compared with the concentration of influent, the concentration of effluent was much lower, indicating the high removal efficiency by the two wastewater treatment processes. Biological treatment unit and cyclic activated sludge system were the main treatment units for the removing of surfactants, which suggested that these two types of surfactants can be easily degraded under aerobic condition. Seasonal variation indicated that the removal efficiencies of surfactants in autumn were a little higher than those in winter. The results of this study provided new insights into the environmental fate of surfactants in wastewater treatment system.

Occurrence and Source Effect of Novel Brominated Flame Retardants (NBFRs) in Soils from Five Asian Countries and Their Relationship with PBDEs.

This paper presents the first comprehensive survey of 19 novel brominated flame retardants (NBFRs) in soil samples collected among five Asian countries. High variability in concentrations of all NBFRs was found in soils with the geometric mean (GM) values ranging from 0.50 ng/g dry weight (dw) in Vietnam to 540 ng/g dw in the vicinity of a BFR manufacturer in China. In urban, rural, and background locations, the GM concentrations of ∑19NBFRs decreased in the order of Japan > South Korea > China > India > Vietnam. Correlations among different NBFR compounds were positive and statistically significant (p < 0.05), suggesting that they originate from similar sources. Evidence for simultaneous application between polybrominated diphenyl ethers (PBDEs) and NBFRs were also noted. Principal component analysis of NBFR concentrations revealed specific pollution sources for different NBFRs coming from urban, BFR-related industrial, and e-waste sites. For the first time, this study demonstrates a "point source fractionation effect" for NBFRs and PBDEs. The concentrations of all NBFRs and PBDEs were negatively and significantly correlated with the distance from BFR-related industrial and e-waste regions. Positive and significant correlation between population density and NBFR concentrations in soils was identified. Our study revealed that the primary sources effects were stronger than the secondary sources effects in controlling the levels and distribution of NBFRs and PBDEs in soils in these five Asian countries.