New cryptogamic records. 16

The current classification of non-pigmented and late-pigmenting rapidly growing mycobacteria (RGM) capable of producing disease in humans and animals consists primarily of three groups, the Mycobacterium fortuitum group, the Mycobacterium chelonae-abscessus group and the Mycobacterium smegmatis group. Since 1995, eight emerging species have been tentatively assigned to these groups on the basis of their phenotypic characters and 16S rRNA gene sequence, resulting in confusing taxonomy. In order to assess further taxonomic relationships among RGM, complete sequences of the 16S rRNA gene (1483-1489 bp), rpoB (3486-3495 bp) and recA (1041-1056 bp) and partial sequences of hsp65 (420 bp) and sodA (441 bp) were determined in 19 species of RGM. Phylogenetic trees based upon each gene sequence, those based on the combined dataset of the five gene sequences and one based on the combined dataset of the rpoB and recA gene sequences were then compared using the neighbour-joining, maximum-parsimony and maximum-likelihood methods after using the incongruence length difference test. Combined datasets of the five gene sequences comprising nearly 7000 bp and of the rpoB+recA gene sequences comprising nearly 4600 bp distinguished six phylogenetic groups, the M. chelonae-abscessus group, the Mycobacterium mucogenicum group, the M. fortuitum group, the Mycobacterium mageritense group, the Mycobacterium wolinskyi group and the M. smegmatis group, respectively comprising four, three, eight, one, one and two species. The two protein-encoding genes rpoB and recA improved meaningfully the bootstrap values at the nodes of the different groups. The species M. mucogenicum, M. mageritense and M. wolinskyi formed new groups separated from the M. chelonae-abscessus, M. fortuitum and M. smegmatis groups, respectively. The M. mucogenicum group was well delineated, in contrast to the M. mageritense and M. wolinskyi groups. For phylogenetic organizations derived from the hsp65 and sodA gene sequences, the bootstrap values at the nodes of a few clusters were <70 %. In contrast, phylogenetic organizations obtained from the 16S rRNA, rpoB and recA genes were globally similar to that inferred from combined datasets, indicating that the rpoB and recA genes appeared to be useful tools in addition to the 16S rRNA gene for the investigation of evolutionary relationships among RGM species. Moreover, rpoB gene sequence analysis yielded bootstrap values higher than those observed with recA and 16S rRNA genes. Also, molecular signatures in the rpoB and 16S rRNA genes of the M. mucogenicum group showed that it was a sister group of the M. chelonae-abscessus group. In this group, M. mucogenicum ATCC 49650(T) was clearly distinguished from M. mucogenicum ATCC 49649 with regard to analysis of the five gene sequences. This was in agreement with phenotypic and biochemical characteristics and suggested that these strains are representatives of two closely related, albeit distinct species.

Bacteria in the genus Elizabethkingia have emerged as a cause of life-threatening infections in humans. However, accurate species identification of these pathogens relies on molecular techniques. We aimed to evaluate the accuracy of 16S rRNA and complete RNA polymerase β-subunit (rpoB) gene sequences in identifying Elizabethkingia species. A total of 173 Elizabethkingia strains with whole-genome sequences in GenBank were included. The 16S rRNA gene and rpoB gene sequences from the same Elizabethkingia strains were examined. Of the 41 E. meningoseptica strains, all exhibited >99.5% 16S rRNA similarity to its type strain. Only 83% of the 99 E. anophelis strains shared >99.5% 16S rRNA gene similarity with its type strain. All strains of E. meningoseptica and E. anophelis formed a cluster distinct from the other Elizabethkingia species in the 16S rRNA and rpoB gene phylogenetic trees. The polymorphisms of 16S rRNA gene sequences are not sufficient for constructing a phylogenetic tree to discriminate species in the E. miricola cluster (E. miricola, E. bruuniana, E. occulta, and E. ursingii). The complete rpoB gene phylogenetic tree clearly delineates all strains of Elizabethkingia species. The complete rpoB gene sequencing could be a useful complementary phylogenetic marker for the accurate identification of Elizabethkingia species.

Objective To analyze the characteristics of drug resistance to quinolones and erythromycin of clinical Campylobacter jejuni (C.jejuni) strains and to further investigate its molecular mechanisms. Methods A total of 193 clinical C. jejuni strains were isolated from feces of patients with diarrhea. Drug susceptibilities to ciprofloxacin (CIP), gentamycin (GEN), azithromycin (AZI), erythromycin (ERY), chloromycetin (CHL), doxycycline (DOX) and tetracycline (TET) were tested using standard agar dilution method. gyrA, gyrB and parC genes were amplified by polymerase chain reaction (RCR) and analyzed for molecular mechanisms of quinolones resistance, and 23S rRNA, rplD and rplV genes for erythromycin resistance. Chi-square test or Fisher′s exact two-tailed tests were used to perform the statistical analysis. Results A total of 193 clinical C. jejuni strains were isolated during 1994—2010, among which 43 C. jejuni strains were isolated in 1994—1999, 80 in 2000—2005 and 70 in 2006—2010. The drug resistance rates for CIP increased significantly from 55.8% in 1994—1999 to 95.0% in 2000—2005 and 94.3% in 2005—2010 (χ2=41.94, P<0.01). The drug resistance rates for GEN were 0 in 1994—1999, 11.3% in 2000—2005 and 10.0% in 2006—2010, but with no statistic difference (χ2=5.078, P=0.08). The drug resistance rates for AZI were 0 in 1994—1999, 3.8% in 2000—2005 and 4.3% in 2006—2010 (χ2=1.81, P=0.40). The drug resistance rates for ERY were 0 in 1994—1999, 1.3% in 2000—2005 and 4.3% in 2006—2010 (χ2=2.87, P=0.24). The drug resistance rates for CHL were 2.3% in 1994—1999, 11.3% in 2000—2005 and 20.0% in 2006—2010 (χ2=7.82, P=0.02). The drug resistance rates for DOX were 60.5% in 1994-1999, 86.3% in 2000—2005 and 82.9% in 2006—2010 (χ2=12.18, P<0.01). The drug resistance rates for TET were 74.4% in 1994—1999, 95.0% in 2000—2005 and 94.3% in 2006—2010 (χ2=15.46, P<0.01). The drug resistance rates for CIP-DOX-TET were 37.2% in 1994—1999, 83.8% in 2000—2005 and 80.0% in 2006—2010 (χ2=33.53, P<0.01). The drug resistance rates for CHL-CIP-DOX-TET were 0 in 1994—1999, 7.5% in 2000—2005 and 20.0% in 2006—2010 (χ2=12.68, P<0.01). The drug resistance rates for GEN-CIP-DOX-TET were 0 in 1994—1999, 7.5% in 2000—2005 and 8.6% in 2006—2010 (χ2=3.74, P=0.15). All 163 CIP-resistant C. jejuni strains had C257T mutation on gyrA gene. Mutations on gyrB gene were silent. ParC gene was absent in C. jejuni. Four ERY resistant C. jejuni strains had no mutation on rplD and rplV genes, but 3 of them had A2075G mutation on 23S rRNA gene. Conclusions The antimicrobial resistance rates for C. jejuni increase remarkably over the periods. C257T mutation on gyrA gene and A2075G mutation on 23S rRNA gene are main mechanisms for quinolones resistance and erythromycin resistance, respectively. Key words: Campylobacter jejuni; Drug resistance, microbial; Ciprofloxacin; Erythromycin; Resistance mechanisms

Partial sequences of 12S and 16S rRNA genes were collected from four thoracican barnacle genera, Capitulum, Chthamalus, Megabalanus, and Tetraclita, to investigate their phylogenetic relationships. Both neighbor joining and maximum parsimony analyses and combined data showed monophyly of the four genera with high bootstrap values, congruent with classification of barnacles based on morphology. The three genera, Chthamalus, Megabalanus and Tetraclita, formed a monophylic group, and the pedunculate genus, Capitulum, formed a paraphylic group with them. The phylogenetic relationships among the three sessile barnacles based on the 12S rRNA gene differed from those based on the 16S rRNA gene or a combination of the 12S and 16S rRNA genes. Based on the 12S rRNA gene the two sessile barnacle genera, Megabalanus and Chthamalus, formed one group, whereas Tetraclita formed another group. However, based on the 16S rRNA gene and combined data of the 12S and 16S rRNA genes, Megabalanus and Tetraclita were the closest relatives among the three sessile barnacles. This was congruent with the results based on larval characters and the nuclear 18S rRNA gene reported previously, but differed from morphological classification. Thus, the 16S rRNA gene appears to be more reliable than the 12S rRNA gene in elucidating the phylogenetic relationships of these four genera within the thoracican barnacles.

The Campylobacter genus consists of a number of important human and animal pathogens. Although the 16S rRNA gene has been used extensively for detection and identification of Campylobacter species, there is currently limited information on the 23S rRNA gene and the internal transcribed spacer (ITS) region that lies between the 16S and 23S rRNA genes. We examined the potential of the 23S rRNA gene and the ITS region to be used in species differentiation and delineation of systematic relationships for 30 taxa within the Campylobacter genus. The ITS region produced the highest mean pairwise percentage difference (35.94%) compared to the 16S (5.34%) and 23S (7.29%) rRNA genes. The discriminatory power for each region was further validated using Simpson's index of diversity (D value). The D values were 0.968, 0.995, and 0.766 for the ITS region and the 23S and 16S rRNA genes, respectively. A closer examination of the ITS region revealed that Campylobacter concisus, Campylobacter showae, and Campylobacter fetus subsp. fetus harbored tRNA configurations not previously reported for other members of the Campylobacter genus. We also observed the presence of strain-dependent intervening sequences in the 23S rRNA genes. Neighbor-joining trees using the ITS region revealed that Campylobacter jejuni and Campylobacter coli strains clustered in subgroups, which was not observed in trees derived from the 16S or 23S rRNA gene. Of the three regions examined, the ITS region is by far the most cost-effective region for the differentiation and delineation of systematic relationships within the Campylobacter genus.

A Gram-stain-positive, catalase-negative, coccus-shaped organism was isolated from the oral cavity of tufted capuchin (Cebus apella). Comparative 16S rRNA gene sequence analysis suggested classification of the organism within the genus Streptococcus. Strain M8T was related most closely to Streptococcus oralis ATCC 35037T (96.17 % similarity) followed by Streptococcus massiliensis CCUG 49690T (95.90 %) based on the 16S rRNA gene. Strain M8T was related most closely to S. massiliensis CCUG 49690T (86.58 %) based on the RNA polymerase β subunit-encoding gene (rpoB), and to Streptococcus tigurinus AZ_3aT (81.26 %) followed by S. massiliensis CCUG 49690T (80.45 %) based on the 60 kDa heat-shock protein gene (groEL). The phylogenetic trees of 16S rRNA, rpoB and groEL gene sequences showed that strain M8T was most closely related to S. massiliensis. Based on phenotypic characterization as well as 16S rRNA gene and housekeeping gene (rpoB and groEL) sequence data, a novel taxon, Streptococcus oricebi sp. nov. (type strain M8T = JCM 30719T = DSM 100101T), is proposed.

Objective To investigate the prevalence, molecular mechanism and genetic characteristics of azithromycin-resistant Neisseria gonorrhoeae (N.gonorrhoeae) strains isolated in Shenzhen. Methods N. gonorrhoeae strains were collected in Shenzhen from 2011 to 2015. Agar dilution method and E-test were used to detect the minimum inhibitory concentrations (MIC) of these strains to azithromycin. All azithromycin-resistant (AZM-R) strains (MIC≥2 μg/ml) and some azithromycin-sensitive strains (MIC≤0.25 μg/ml) which were randomly selected as the control group were screened for mutations in 23S rRNA, mtrR and erm genes and genotyped by using N. gonorrhoeae multi-antigen sequence typing (NG-MAST). Results A total of 788 N. gonorrhoeae strains were collected, 148 (18.8%) of which were AZM-R strains (MIC≥1 μg/ml). Eighteen out of 21 high-level AZM-R (AZM-HLR) strains had A2143G mutations in the four copies of the 23S rRNA gene. Twelve out of 29 middle-level AZM-R (AZ-MLR) strains had missense mutations, among which C2611T mutations in the four copies of the 23S rRNA were detected in 10 strains. Incidence of G45D/Y105H mutation in AZM-HLR strains was higher than that in AZM-MLR (χ2=12.702, P=0.000) or AZ-S (χ2=4.462, P=0.035) strains according to the analysis of the promoter and coding region of mtrR gene. PCR analysis revealed that only one strain carried ermB gene (MIC=2 μg/ml). The 788 N. gonorrhoeae strains were typed into 81 sequence types (STs) by NG-MAST, most of which were represented by one strain only. STs of ST3356 and ST1866 that were identified in the AZ-R strains in the current study had been noted in a previous report of emerging AZM-R N. gonorrhoeae strains in Nanjing, Chongqing and Guangzhou. Neighbor-joining (NJ) phylogenetic tree showed that the resistant strains did not form a separate cluster. Conclusion Currently, it is not suitable to use azithromycin as a monotherapy for gonorrhea in Shenzhen. Mutations of A2059G and C2611T in 23S rRNA of N. gonorrhoeae were respectively responsible for high-level and middle-level resistance to azithromycin. Repeated emergence of ST1866 and ST3356 will help us monitor and analyze the epidemiological characteristics of N. gonorrhoeae strains resistant to azithromycin in Shenzhen. Key words: Neisseria gonorrhoeae; Azithromycin; Resistance gene; NG-MAST

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.

In this study, we attempted species-level identification of frog specimen collected from Faridpur district of Bangladesh beyond it’s location outside Orissa, India. Specimen was identified morphologically at genus level as Fejervarya sp. belonging to the family Dicroglossidae using finger formula F3&gt;F4&gt;F1=F1 where F denote as toe finger. From the study two nucleotide sequences of 16S and 12S rRNA genes were obtained which contained 508bp and 408bp respectively. The Sequences were submitted to Gene Bank database with the accession number OQ231604 and OQ240197 for 16S and 12S rRNA gene sequences. Furthermore, the 16S rRNA gene sequence was used as molecular bar-code for the identified Orissa frog F. orissaensis species from Bangladesh. GC content of partial 12S and 16S rRNA genes have been calculated as 44% and 45% respectively. For 16S rRNA gene sequence there was no intra specific divergence. Whereas the inter specific polymorphic divergence were calculated 4.13% and 6.3% when the collected Orissa frog F. orissaensis was compared with that of F. iskandari and F. kupitzi, respectively. In case of 12S rRNA gene intra specific divergence was found 2.45% where the inter specific divergence were 4.41% and 6.86% when the collected Orissa frog F. orissaensis was compared with that of F. iskandari and F. limnocaris, respectively. Maximum likelihood tree also indicates that our sample Orissa frog formed a monophyletic group with F. orissaensis in both the cases of 16S and 12S rRNA genes and thus can be concluded as closely related. Therefore, the collected specimen was identified to be belonging to Fejervarya orissaensis which would be first report from Bangladesh outside Orissa, India. Bangladesh J. Zool. 51(3): 301-313, 2023

Due to their widespread distribution and virulence, protozoan species of the genus Perkinsus are especially worrisome parasites for shellfish farmers. In the present paper, we investigate the organization and the structural features of the nuclear ribosomal genes of Perkinsus atlanticus as well as the use of DNA sequence information from this region for phylogenetic analyses. This information has been useful, further, for the development of a diagnostic test based on the amplification by the polymerase chain reaction (PCR) technique. We have isolated a high-copy DNA sequence in this species, and, after its characterization, we have determined that it corresponds to the ribosomal RNA (rRNA) genes 28S-5S-18S and the intergenic spacers. By comparing the complete sequence of the 5S rRNA gene and a partial sequence of the 18S rRNA gene of P. atlanticus with the sequences of those genes in other Alveolates, we have found additional support for the hypothesis that Perkinsus is more closely related to species of Dinoflagellata than to species of Apicomplexa. The intergenic spacer sequence between the 5S and the 18S rRNA genes was used to design a pair of primers to be used as a PCR-based diagnostic test.

The aim of the study is to test a hypothesis for the phylogenetic relationships among mammalian hymenolepidid tapeworms, based on partial (D1-D3) nuclear 28S ribosomal RNA (rRNA) genes, by estimating new molecular phylogenies for the group based on partial mitochondrial cytochrome c oxidase I (COI) and nuclear 18S rRNA genes, as well as a combined analysis using all three genes. New sequences of COI and 18S rRNA genes were obtained for Coronacanthus integrus, C. magnihamatus, C. omissus, C. vassilevi, Ditestolepis diaphana, Lineolepis scutigera, Spasskylepis ovaluteri, Staphylocystis tiara, S. furcata, S. uncinata, Vaucherilepis trichophorus and Neoskrjabinolepis sp. The phylogenetic analyses confirmed the major clades identified by Haukisalmi et al. (Zoologica Scripta 39: 631-641, 2010): Ditestolepis clade, Hymenolepis clade, Rodentolepis clade and Arostrilepis clade. While the Ditestolepis clade is associated with soricids, the structure of the other three clades suggests multiple evolutionary events of host switching between shrews and rodents. Two of the present analyses (18S rRNA and COI genes) show that the basal relationships of the four mammalian clades are branching at the same polytomy with several hymenolepidids from birds (both terrestrial and aquatic). This may indicate a rapid radiation of the group, with multiple events of colonizations of mammalian hosts by avian parasites.

The heterocytous cyanobacteria form a monophyletic group according to 16S rRNA gene sequence data. Within this group, phylogenetic and morphological studies have shown that genera such as Anabaena and Aphanizomenon are intermixed. Moreover, the phylogeny of the genus Trichormus, which was recently separated from Anabaena, has not been investigated. The aim was to study the taxonomy of the genera Anabaena, Aphanizomenon, Nostoc and Trichormus belonging to the family Nostocaceae (subsection IV.I) by morphological and phylogenetic analyses of 16S rRNA gene, rpoB and rbcLX sequences. New strains were isolated to avoid identification problems caused by morphological changes of strains during cultivation. Morphological and phylogenetic data showed that benthic and planktic Anabaena strains were intermixed. In addition, the present study confirmed that Anabaena and Aphanizomenon strains were not monophyletic, as previously demonstrated. The evolutionary distances between the strains indicated that the planktic Anabaena and Aphanizomenon strains as well as five benthic Anabaena strains in cluster 1 could be assigned to a single genus. On the basis of the 16S rRNA, rpoB and rbcLX gene sequences, the Anabaena/Aphanizomenon strains (cluster 1) were divided into nine supported subclusters which could also be separated morphologically, and which therefore might represent different species. Trichormus strains were morphologically and phylogenetically heterogeneous and did not form a monophyletic cluster. These Trichormus strains, which were representatives of three distinct species, might actually belong to three genera according to the evolutionary distances. Nostoc strains were also heterogeneous and seemed to form a monophyletic cluster, which may contain more than one genus. It was found that certain morphological features were stable and could be used to separate different phylogenetic clusters. For example, the width and the length of akinetes were useful features for classification of the Anabaena/Aphanizomenon strains in cluster 1. This morphological and phylogenetic study with fresh isolates showed that the current classification of these anabaenoid genera needs to be revised.

Concerns regarding the status of marine ecosystems have increased in part due to traditional and emerging human activities in marine waters, driving a demand for approaches with high sample throughput capability to improve ecosystem monitoring. Nematodes are already used as indicator species in biodiversity assessments and biomonitoring of terrestrial and marine systems, with molecular approaches offering the opportunity to utilize these organisms further in large scale ecological surveys and environmental assessments. Based on an available nematode dataset for estuarine sediments of the Mira estuary (SW coast, Portugal), we evaluated the diversity of the nematode community of this system, using the molecular markers 18S rRNA and COI genes. These approaches were compared to voucher specimens from a morphological characterization of the same samples allowing validation and comparison between nematode communities. The spatial and temporal variability of the density and diversity of the nematode assemblages was analyzed based on morphological characterization to allow the validation and efficiency of the genetic characterization. A PCO ordination plot showed a distinct separation of the assemblages between sampling occasions confirmed by PERMANOVA analysis, which showed significant differences, although no significant differences were detected between sampling sites. The morphological characterization identified 50 genera of which only 26 and 25 distinct 18S rRNA and COI DNA barcodes, respectively, were obtained. 90.2% of the morphologically identified specimens representing eleven different genera, successfully generated DNA barcodes for both 18S rRNA and COI genes. This study confirmed that the success of the 18S rRNA gene PCR amplification is higher than of COI gene with 43 species amplified against 34. The study highlights a limitation of available sequences for both targets in databases when compared to the known diversity of marine nematodes. The gene sequences of this study enriched the databases, contributing gene sequences from 7 and 16 new genera for the 18S rRNA and COI genes, respectively. A robust database of gene sequences is a prerequisite for the development of robust high sample throughput techniques to be applied in marine assessing and monitoring programs.

Upland soil clusters alpha and gamma (USCα and USCγ) are considered a major biological sink of atmospheric methane and are often detected in forest and grassland soils. These clusters are phylogenetically classified using the particulate methane monooxygenase gene pmoA because of the difficulty of cultivation. Recent studies have established a direct link of pmoA genes to 16S rRNA genes based on their isolated strain or draft genomes. However, whether the results of pmoA-based assays could be largely represented by 16S rRNA gene sequencing in upland soils remains unclear. In this study, we collected 20 forest soils across China and compared methane-oxidizing bacterial (MOB) communities by high-throughput sequencing of 16S rRNA and pmoA genes using different primer sets. The results showed that 16S rRNA gene sequencing and the semi-nested polymerase chain reaction (PCR) of the pmoA gene (A189/A682r nested with a mixture of mb661 and A650) consistently revealed the dominance of USCα (accounting for more than 50% of the total MOB) in 12 forest soils. A189f/A682r successfully amplified pmoA genes (mainly RA14 of USCα) in only three forest soils. A189f/mb661 could amplify USCα (mainly JR1) in several forest soils but showed a strong preferential amplification of Methylocystis and many other type I MOB groups. A189f/A650 almost exclusively amplified USCα (mainly JR1) and largely discriminated against Methylocystis and most of the other MOB groups. The semi-nested PCR approach weakened the bias of A189f/mb661 and A189f/A650 for JR1 and balanced the coverage of all USCα members. The canonical correspondence analysis indicated that soil NH4+-N and pH were the main environmental factors affecting the MOB community of Chinese forest soils. The RA14 of the USCα group prefers to live in soils with low pH, low temperature, low elevation, high precipitation, and rich in nitrogen. JR1's preferences for temperature and elevation were opposite to RA14. Our study suggests that combining the deep sequencing of 16S rRNA and pmoA genes to characterize MOB in forest soils is the best choice.

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.