Genetic variations in four geographical isolates of Gohieria fusca based on cytochrome b and internal transcribed spacer genes / 中国血吸虫病防治杂志

Objective To investigate the genetic diversity and genetic differentiation of different geographical isolates of Gohieria fusca.. Methods G. fusca isolates were sampled from Wuhu (WH), Bengbu (BB) and Bozhou cities (BZ) of Anhui Province and Jiaxing City of Zhejiang Province (JX). Mitochondrial cytochrome b (Cytb) and ribosomal internal transcribed spacer (ITS) genes were amplified in WH, BB, BZ and JX isolates of G. fusca using PCR assay. The gene sequences were edited and aligned using the software Chromas 2 and DNASTAR 1.00, and the haplotype, haplotype diversity (Hd) and nucleotide polymorphism (Pi) of each isolate were calculated using the software DnaSP 5.10.00. The genetic differentiation among isolates (Fst) and gene flow value (Nm) were estimated using the software MEGA 10.2, and a phylogenetic tree was built. Tests of neutrality and analysis of molecular variance (AMOVA) were performed using the software Arlequin 3.1 and a haplotype network was built based on the Median-Joining network using the software Network 10.2. Results PCR assay showed that the sizes of the Cytb and ITS genes were 372 bp and 1 301 to 1 320 bp, respectively. All four isolates of G. fusca presented high genetic diversity based on mitochondrial Cytb and ITS genes (Hd = 0.804, Pi = 0.006 91). AMOVA showed genetic differentiation among geographical isolates of G. fusca (Fst = 0.202 40, P < 0.05), and the genetic variation was mainly caused by intra-population variations (79.76%). Gene flow analysis showed a high level of gene flow among G. fusca isolates (Nm > 1). Tests of neutrality based on Cytb gene measured a Tajima’s D value of −1.796 31 (P < 0.05) and a Fu’s FS value of −3.293 98 (P < 0.05) in WH isolate of G. fusca, indicating population expansion in WH isolate of G. fusca. Haplotype network analysis and phylogenetic analysis revealed no remarkable geographical distribution pattern among different geographical isolates of G. fusca. All four isolates of G. fusca presented high genetic diversity (Hd = 0.985, Pi = 0.011 97). AMOVA showed moderate level of genetic differentiation between four isolates (Fst = 0.104 62, P < 0.05). The tests of neutrality based on ITS genes measured a Tajima’s D value of −6.088 20 and a Fu’s FS value of −1.935 99 (both P > 0.05) in the whole isolate of G. fusca, indicating no obviously population expansion. Conclusions The four geographical isolates of G. fusca have high genetic diversity and remarkable genetic differentiation. Since a high level of gene flow is detected among different geographical isolates of G. fusca, no obvious geographical distribution pattern of G. fusca is found.

Diversity of Endophytes in Panax quinquefolium Root / 中国实验方剂学杂志

ObjectiveTo analyze the community structure of endophytes in Panax quinquefolium root and explore the dominant endophytic bacteria and fungi， to provide scientific basis for the establishment of endophytic microbial bank in P. quinquefolium root. MethodInternal Transcribed Spacer （ITS） sequencing and 16S sequencing were performed on six P. quinquefolium root samples collected from Wendeng， Shandong province on PacBio Sequel Ⅱ. ResultA total of 8 phyla， 11 classes， 23 orders， 27 families and 53 genera of endophytic bacteria were identified in P. quinquefolium root， among which an unidentified Burkholderiaceae and an unidentified Rhizobiaceae were dominant. A total of 9 phyla， 23 classes， 35 orders， 43 families and 48 genera of endophytic fungi were identified in P. quinquefolium root， among which an unclassified Helotiales and Pseudogymnoascus were dominant. The community structure of endophytic bacteria revealed that the roots were selectively enriched with nitrogen-fixing bacteria such as unidentified Rhizobiaceae， Bradyrhizobium and Herbaspirillum， which suggested that nitrogen is important for the growth of P. quinquefolium root. The community structure of endophytic fungi indicated that P. quinquefolium in Shandong province might be infected by unclassified Helotiales. ConclusionThere is a rich diversity of endophytic bacteria and fungi in P. quinquefolium root， which provides scientific basis for studying the interaction of the plant with endophytic microorganisms and screening the endophytes to promote the growth of P. quinquefolium root.

Sequence analysis of Paragonimus internal transcribed spacer 2 and cyclooxygenase 1 genes in freshwater crabs in Henan Province / 中国血吸虫病防治杂志

Objective To investigate the sequences of internal transcribed spacer 2 (ITS2) and cyclooxygenase 1 (COX1) genes of Paragonimus metacercariae in freshwater crabs in Henan Province, identify the species of Paragonimus and evaluate its genetic relationships with Paragonimus isolates from other provinces in China. Methods Freshwater crabs were collected from 8 survey sites in Zhengzhou, Luoyang, Pingdingshan, Nanyang and Jiyuan cities of Henan Province from 2016 to 2021, and Paragonimus metacercariae were detected in freshwater crabs. Genomic DNA was extracted from Paragonimus metacercariae, and the ITS2 and COX1 genes were amplified using PCR assay, followed by sequencing of PCR amplification products. The gene sequences were spliced and aligned using the software DNASTAR, and aligned with the sequences of Paragonimus genes in the GenBank. Phylogenetic trees were created using the MEGA6 software with the Neighbor-Joining method based on ITS2 and COX1 gene sequences, with Fasciola hepatica as the outgroup. Results The detection rates of Paragonimus metacercariae were 6.83% (11/161), 50.82% (31/61), 18.52% (5/26), 8.76% (12/137), 14.29% (9/63), 17.76% (19/105), 18.50% (32/173) and 42.71% (41/96) in freshwater crabs from 8 survey sites in Zhengzhou, Luoyang, Pingdingshan, Nanyang and Jiyuan cities of Henan Province, with a mean detection rate of 19.46% (160/822), and a mean infection intensity of 0.57 metacercariae/g. The amplified ITS2 and COX1 gene fragments of Paragonimus were approximately 500 bp and 450 bp in lengths, respectively. The ITS2 gene sequences of Paragonimus metacercariae from 8 survey sites of Henan Province showed the highest homology (99.8% to 100.0%) with the gene sequence of P. skrjabini (GenBank accession number: MW960209.1), and phylogenetic analysis showed that the Paragonimus in this study was clustered into the same clade with P. skrjabini from Sichuan Province (GenBank accession number: AY618747.1), Guangxi Zhuang Autonomous Region (GenBank accession number: AY618729.1) and Hubei Province (GenBank accession number: AY618751.1), and P. miyazaki from Fujian Province (GenBank accession number: AY618741.1) and Japan (GenBank accession number: AB713405.1). The COX1 gene sequences of Paragonimus metacercariae from 8 survey sites of Henan Province showed the highest homology (90.0% to 100.0%) with the gene sequence of P. skrjabini (GenBank accession number: AY618798.1), and phylogenetic analysis showed that the Paragonimus in this study was clustered into the same clade with all P. skrjabini and clustered into the same sub-clade with P. skrjabini from Hubei Province (GenBank accession numbers: AY618782.1 and AY618764.1). Conclusions Paragonimus species from freshwater crabs in Henan Province were all characterized as P. skrjabini, and the ITS2 and COX1 gene sequences had the highest homology to those of P. skrjabini from Hubei Province. The results provide insights into study of Paragonimus in Henan Province and China.

Morphological and molecular identification of trematode isloates from laying ducks in Nanchang City / 中国血吸虫病防治杂志

Objective To identify the species of trematodes isolated from laying ducks in Nanchang City using morphological and molecular approaches. Methods Trematodes were isolated from the hepatobiliary duct, gallbladder and large intestine of market-sold laying ducks in Nanchang City. Following morphological characterization, total DNA was extracted from all trematode specimens, and internal transcribed spacer region (ITS) and cytochrome C oxidase subunit 1 (Cox1) genes were amplified using PCR assay and sequenced. Sequence alignment was performed using the Blast software, and homology and phylogenetic analyses were done in the trematode isolates based on ITS and Cox1 gene sequences. Results The morphological characteristics of two trematode isolates from the large intestine of laying ducks were similar to those of Echinostoma revolutum and E. miyagawai, and the morphological characteristics of eight trematode samples isolated from the hepatobiliary duct and gallbladder of laying ducks were similar to those of Amphimerus anatis. The ITS and Cox1 gene sequences of the two trematode isolates from the large intestine of laying ducks had 99.3% and 98.9%-99.4% homology with E. miyagawai, and the phylogenetic analysis showed that two trematode isolates had the closest genetic relationship with E. miyagawai based on ITS and Cox1 gene sequences. The ITS gene sequences of eight trematode isolates from the hepatobiliary duct and gallbladder of laying ducks shared 95.1%-95.5% with Opisthorchis sudarikovi and Clonorchis sinensis, while the Cox1 gene sequences of eight trematode isolates from the hepatobiliary duct and gallbladder of laying ducks shared 86.3%-86.4% and 85.5%-85.7% with O. viverrini and O. sudarikovi. ITS gene sequence-based phylogenetic analysis showed that the duck-derived trematode isolates had the closest genetic relationship with C. sinensis, and Cox1 gene sequence-based phylogenetic analysis showed that the duck-derived trematode isolates had the closest genetic relationship with Metorchis orientalis and O. viverrini. Conclusions The trematode isolates from the large intestine of laying ducts in Nanchang City may be E. miyagawai, and the trematode isolates from the hepatobiliary duct and gallbladder may be an unidentified trematode species of the family Opisthorchiidae.

Species identification and phylogenetic analysis of canine Leishmania in Diebu County, Gansu Province / 中华地方病学杂志

Objective:To investigate the species types and phylogenetic relationship of canine Leishmania in Diebu County, Gansu Province, so as to provide a basis for exploring new methods of prevention and control of canine visceral leishmaniasis. Methods:DNA was extracted from blood samples of eight asymptomatic Leishmania-infected dogs in Luoda administrative village in Diebu County, Gansu Province. Ribosomal internal transcribed spacer 1 (ITS-1) gene fragments were amplified and isolated by PCR, and then the amplified target fragments were sequenced. The MEGA 7.0 software was used for multiple sequence alignment, and a phylogenetic tree was constructed by neighbor-joining method to analyze the phylogenetic relationship of canine Leishmania in Diebu County, Gansu Province. Results:Fragments of about 320 bp corresponding in size to the target sequence ITS-1 were isolated from all of the eight asymptomatic Leishmania-infected dogs blood samples. ITS-1 sequence alignment showed that the sequence homology between 8 samples and Leishmania infantum MG969403, MN648755 strains was 99.1% - 100.0%; phylogenetic tree showed that all 8 samples were clustered into one branch with Leishmania infantum. Conclusion:Leishmania infantum is identified from all of the eight asymptomatic Leishmania-infected dogs blood samples in Diebu County, Gansu Province.

Occurrence and identification of Penicillium and Talaromyces species from beach sand

Aims@#Penicillium and Talaromyces were among the species of microfungi that inhabit beach sand in Batu Ferringhi Beach, Penang Island, Malaysia. Previously, Talaromyces was described as the sexual stage of Penicillium, but both are now accepted as separate genera based on molecular phylogeny. The aim of the present study was to identify species of Penicillium and Talaromyces that are present in beach sand in Malaysia.@*Methodology and results@#Species identities were confirmed according to similarities of the internal transcribed spacer regions and β-tubulin gene sequences and a phylogenetic analysis based on both regions/gene. Nine Penicillium spp. were identified as P. georgiense, P. chermesinum, P. pimiteouiense, P. citrinum, P. oxalicum, P. daleae, P. rolfsii and Penicillium sp. and the four Talaromyces spp. were T. siamense, T. atroroseus, T. minioluteus and T. fusiformis.@*Conclusion, significance and impact of study@#These findings showed that beach sand harboured a variety of Penicillium and Talaromyces species. The occurrence of Penicillium and Talaromyces in beach sands is associated with the organic matter in the sand, which provides suitable substrates and nutrient sources. Due to this, beach sand might harbour many potentially pathogenic or opportunistic species that may pose a health concern to immunocompromised individuals.

Identification of Medicinal Aconitum Species Based on ITS2 Sequences and Analysis of Genetic Relationship / 中国实验方剂学杂志

ObjectiveThe internal transcribed spacer （ITS） 2 region of ribosomal gene， a DNA barcode， was employed to identify 12 medicinal Aconitum species and the genetic relationship among the species was analyzed. MethodA total of 30 samples of the 12 species were collected. The DNA was extracted with spin column plant genomic DNA kit and the universal primers of ITS2 sequence were used for polymerase chain reaction （PCR） amplification， followed by electrophoresis detection and bi-directional sequencing. The yielded sequences were aligned and spliced by CodonCode Aligner 17.0 and sequence variation was analyzed by MEGA 7.0. The secondary structure was predicted by ITS2 Database and the neighbor-joining （NJ） method was applied to generate the phylogenetic tree. ResultThe ITS2 sequences of the 12 species were 220-221 bp， with the average guanine and cytosine （GC） content of 64.09%， 140 variable sites， 137 informative sites， and 81 conservative sites. The intraspecific genetic distance （K2P） was smaller than the interspecific genetic distance. According to the secondary structures of ITS2 sequences and NJ cluster analysis， A. scaposum， A. sinomontanum， and A. barbatum had close genetic relationship， while the rest nine showed close kinship， particularly A. soongaricum and A. yinschanicum. ConclusionITS2 sequence is of great value for the molecular identification and genetic relationship determination of Aconitum， which provides a new method for the study of ethnomedicine.

Phylogenetic and Mutation Points Analysis of Sophora japonica cv. jinhuai Based on DNA Barcoding Sequences / 中国实验方剂学杂志

ObjectiveTo conduct phylogenetic analysis of internal transcribed spacer 2 （ITS2） and chloroplast gene segments including psbA-trnH， rbcL， and matK of Sophora japonica cv. jinhuai resource samples from different geographical sources， and to explore the genetic diversity of S. japonica cv. jinhuai. MethodPolymerase chain reaction （PCR） method was used to amplify the nucleic acid sequences of ITS2， psbA-trnH， rbcL， and matK of S. japonica cv. jinhuai. Neighbor joining （NJ） method was used to construct phylogenetic trees， and Kimura 2-Parameter （K2P） model was used to calculate the genetic distance of different samples. MEGA and BIOEDIT softwares were applied for mutiple alignment and analysis of ITS2， psbA-trnH， rbcL， and matK sequences of S. japonica cv. jinhuai. ResultThe lengths of ITS2 sequence were 278-279 bp. The lengths of psbA-trnH were 289 bp. The lengths of rbcL sequence were 673 bp. The lengths of matK sequences were 786-792 bp. There were 3 mutation points in ITS2 and psbA-trnH， no mutation point in rbcL， and 13 mutation points in matK. The samples of S. japonica cv. jinhuai were clustered into two groups based on the phylogenetic tree constructed by ITS2 sequences. The sample of seedling tree in Baibao was clustered into one group， while the other 25 samples were clustered into another group. For the psbA-trnH sequence， the success rate of PCR amplification of 28 samples of S. japonica cv. jinhuai was 100%. The 28 samples of S. japonica cv. jinhuai were clustered into three groups based on the clustering results of psbA-trnH sequence. The sample of seedling tree in Shaoshui was clustered into one group. The five samples of grafting tree and seedling tree in Miaotou， grafting trees in Jiantang， Wenqiao， and Daxu， and seeding tree in Xianshui were clustered into one group. The other 21 samples were clustered into another group. The 26 samples of S. japonica cv. jinhuai were clustered into two groups based on the phylogenetic tree constructed by matK sequences. The sample of seedling tree in Xianshui was clustered into one group， while the other 25 samples were clustered into another group. The clustering results of the rbcL sequence of S. japonica cv. jinhuai could not distinguish 28 resource samples. The phylogenetic tree constructed by the combined sequence of ITS2+psbA-trnH+rbcL+matK divided S. japonica cv. jinhuai resource samples into 4 groups. The 13 samples of seedling trees in Qiyang， Daoxian， Miaotou， Shaoshui， Shitang， Xianshui， Jiantang， and Xiangli， and grafting trees in Qiyang， Miaotou， Yongsui， Wenqiao， and Yangtang were clustered into one group. The sample of seedling tree in Wenqiao was clustered into one group. The sample of seedling tree in Daxu was clustered into one group. The remaining samples were clustered into another group. ConclusionPhylogenetic and mutation analysis provide the theoretic foundation to investigate the evolution of the resources of S. japonica cv. jinhuai， and evaluate their genuineness. The results of mutation points can be used to identify the related S. japonica cv. jinhuai resources. The findings of this study show that the combination of different gene sequences has an optimal effect on plant identification.

Molecular Identification of Tibetan Codonopsis Plants Based on ITS2 and psbA-trnH Barcode / 中国实验方剂学杂志

ObjectiveTo identify the molecular biology of various species of Tibetan Codonopsis plants based on internal transcribed spacer（ITS）2 and psbA-trnH sequence barcode technology. MethodThe genomic DNA of 28 Tibetan Codonopsis plant samples from four species （Codonopsis canescens，C. foetens subsp. nervosa，C. pilosula， and C. thalictrifolia var. mollis） were extracted，and the ITS2 and psbA-trnH sequences were amplified and sequenced. The related sequences of 81 Tibetan Codonopsis plant samples belonging to 15 species were downloaded from GenBank， and MEGA 6.0 was used for sequence comparison and mutation site analysis. The GC content and genetic distance within and between species were calculated. Additionally， phylogenetic trees were constructed by maximum likelihood （ML） method， neighbor-joining （NJ） method，and unweighted pair-group method with arithmetic means （UPGMA） . ResultAccording to the mutation site，C. canescens， C. pilosula，C. pilosula subsp. tangshen， C. pilosula var. modesta，C. bhutanica，C. clematidea，C. lanceolata，C. subglobosa and C. foetens were distinguished. In the phylogenetic trees，the optimal clustering effects for ITS2 and psbA-trnH sequences were obtained using the ML method and the UPGMA method， respectively， and 12 species were effectively clustered. ConclusionITS2 and psbA-trnH sequences have a high identification rate for species of single origin，but there are still some limitations in identifying variants and original variants. This study provides basis for the identification of affinity relationship and clinical safety of Tibetan Codonopsis plants.

Identification of Armeniacae Semen Amarum Adulterated in Persicae Semen by Allele-specific Polymerase Chain Reaction / 中国实验方剂学杂志

Objective:Due to the limitation of traditional identification methods of Chinese medicinal materials， the study established a rapid method to identify Persicae Semen mixed with Armeniacae Semen Amarum by allele-specific polymerase chain reaction （PCR）. Method:By comparing the ribosomal DNA internal transcribed spacer （ITS） gene sequences of Persicae Semen and Armeniacae Semen Amarum， single nucleotide polymorphism （SNP） sites were searched and specific primers were designed. Different Persicae Semen and Armeniacae Semen Amarum samples were amplified by PCR， the effects of annealing temperature， primer concentration and cycle number on the PCR reaction system were optimized， and the specificity and detection limit of this method were investigated. In addition， the established PCR method was used to detect the samples of Persicae Semen mixed with different proportion of Armeniacae Semen Amarum from different sources and producing areas. Result:A specific PCR method for identifying Persicae Semen mixed with Armeniacae Semen Amarum was established. When the annealing temperature was 63 ℃ and the number of primer cycles was 30， only Armeniacae Semen Amarum could be amplified with 432 bp specific band， while Persicae Semen samples did not have this band. The minimum detection limit of this method for Armeniacae Semen Amarum was 0.2 ng， and the detection limit for Armeniacae Semen Amarum adulterated in Persicae Semen was 1%. Conclusion:The established allele-specific PCR method can accurately detect whether there is Armeniacae Semen Amarum in Persicae Semen， which can provide experimental basis for the quality control of Persicae Semen and guarantee the safety of its clinical use.

Development of a fluorescent recombinase-aided isothermal amplification-based nucleic acid assay for detection of Leishmania / 中国血吸虫病防治杂志

Objective To develop a fluorescent recombinase-aided isothermal amplification (RAA)-based nucleic acid assay for detection of Leshimania. Methods Specific primers and probes were designed targeting Leishmania internal transcribed spacer 1 (ITS1) gene for RAA assay, and a fluorescent RAA assay was developed for detection of Leishmania following screening of primer pairs and optimization of primer and probe concentrations. The sensitivity of RAA assay for detection of Leishmania was evaluated using recombinant plasmid containing Leishmania ITS1 gene sequences at different copies and Leshimania genomic DNA at different concentrations as templates, and the specificity of RAA assay for detection of Leishmania was evaluated using the genomic DNA of transfusion-transmitted parasites, including Babesia microti, Toxoplasma gondii, Plamodium vivax, P. ovale, P. falciparum, P. malariae, L. donovani and L. infantum. Results After the optimal primer pair was screened from 9 pairs of primer combinations, the final primer and probe concentrations were optimized as 0.3 μmol/L and 0.08 μmol/L, respectively. Nucleic acid detection of Leishmania was completed by the fluorescent RAA assay at an isothermal temperature of 39 °C within 20 min. Remarkable florescent signals were seen within 5 min following RAA detection of genomic DNA of L. donovani and L. infantum, and no cross-reactions were observed with B. microti, T. gondii, P. vivax, P. ovale, P. falciparum or P. malariae. The lowest limitation of detection of the fluorescent RAA assay was 10 copies/μL recombinant plasmid containing Leishmania ITS1 gene sequences and 1 fg/μL Leishmania genomic DNA. Conclusions A rapid, simple, sensitive and specific fluorescent RAA assay is successfully developed for detection of L. donovani and L. infantum, which is effective for field screening of leishmaniasis.

Morphological and molecular characterization of Fusarium spp. associated with Fusarium wilt disease of Piper nigrum L. in Northwestern region of Sarawak

Aims@#Piper nigrum L. (black pepper) is an economically important commodity plant in Malaysia, which generated RM 200.95 million from pepper export in the year of 2018. However, the increase in pepper production is restricted by diseases. Fusarium wilt is one of the major diseases of P. nigrum L. The objectives for this study were to isolate Fusarium spp. associated with Fusarium wilt of P. nigrum L. from selected pepper farms in the northwestern region of Sarawak and to characterize the Fusarium spp. isolated morphologically and molecularly.@*Methodology and results@#Fusarium spp. were isolated from diseased root samples. The pathogen was grown on potato dextrose agar (PDA) under dark condition at circa (ca.) 25 °C for morphological characterisation. Molecular characterisation was done by using internal transcribed spacer (ITS). Phylogenetic tree was constructed to study the genetic relationship of the isolates. Fusarium solani, F. oxysporum, F. proliferatum were the three Fusarium species identified. There were variations in morphological characters observed between and among the species, including the colony form, margin, elevation, surface appearance and pigmentation. No distinctive morphological characteristic was specific to a location. In addition, growth rate, macroconidia sporulation rate, and microconidia sporulation rate of the isolates were not correlated. In molecular phylogeny, the three Fusarium species were separated into three distinct clades representing the three identified species. The genetic relatedness between isolates within each species was depicted in the tree. @*Conclusion, significance and impact of study@#Variations were observed among isolates in this study based on morphological and molecular characterization. This study would contribute information on the variations of Fusarium spp. associated with Fusarium wilt of P. nigrum L. from the northwestern region of Sarawak.

Multidisciplinary approach detects speciation within the kissing bug Panstrongylus rufotuberculatus populations (Hemiptera, Heteroptera, Reduviidae)

BACKGROUND Panstrongylus rufotuberculatus (Hemiptera-Reduviidae) is a triatomine species with a wide geographic distribution and a broad phenotypic variability. In some countries, this species is found infesting and colonising domiciliary ecotopes representing an epidemiological risk factor as a vector of Trypanosoma cruzi, etiological agent of Chagas disease. In spite of this, little is known about P. rufotuberculatus genetic diversity. METHODS Cytogenetic studies and DNA sequence analyses of one nuclear (ITS-2) and two mitochondrial DNA sequences (cyt b and coI) were carried out in P. rufotuberculatus individuals collected in Bolivia, Colombia, Ecuador and Mexico. Moreover, a geometric morphometrics study was applied to Bolivian, Colombian, Ecuadorian and French Guiana samples. OBJECTIVES To explore the genetic and phenetic diversity of P. rufotuberculatus from different countries, combining chromosomal studies, DNA sequence analyses and geometric morphometric comparisons. FINDINGS We found two chromosomal groups differentiated by the number of X chromosomes and the chromosomal position of the ribosomal DNA clusters. In concordance, two main morphometric profiles were detected, clearly separating the Bolivian sample from the other ones. Phylogenetic DNA analyses showed that both chromosomal groups were closely related to each other and clearly separated from the remaining Panstrongylus species. High nucleotide divergence of cyt b and coI fragments were observed among P. rufotuberculatus samples from Bolivia, Colombia, Ecuador and Mexico (Kimura 2-parameter distances higher than 9%). MAIN CONCLUSIONS Chromosomal and molecular analyses supported that the two chromosomal groups could represent different closely related species. We propose that Bolivian individuals constitute a new Panstrongylus species, being necessary a detailed morphological study for its formal description. The clear morphometric discrimination based on the wing venation pattern suggests such morphological description might be conclusive.

Diversity and antimicrobial activity of culturable fungi associated with sea anemone Anthopleura xanthogrammica

Background: The main objective of this study was to isolate fungi associated with Anthopleura xanthogrammica and measure their antimicrobial and enzymatic activities. A total of 93 fungal strains associated with A. xanthogrammica were isolated in this study, of which 32 isolates were identified using both morphological characteristics and internal transcribed spacer (ITS) sequence analysis. The antibacterial activities of 32 fungal isolates were tested against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Edwardsiella tarda, Vibrio harveyi, Fusarium oxysporum, and Pyricularia oryzae by agar diffusion assay. Extracellular hydrolytic enzyme activities of the fungal isolates were determined by agar diffusion assays. Enzyme activities were detected from clear halo size. Results: The isolated fungi belonged to 18 genera within 7 taxonomic orders of 1 phylum. The genera Aspergillaceae were the most diverse and common. The antimicrobial activities of 32 isolates were evaluated, and 19 (59.4%) of fungi isolate displayed unique antimicrobial activities. All fungal strains displayed at least one enzyme activity. The most common enzyme activities in the fungi isolates were amylase and protease, while the least common were pectinase and xylanase. Conclusions: This is first report on the sea anemone-derived fungi with antimicrobial and enzyme activities. Results indicated that sea anemone is a hot spot of fungal diversity and a rich resource of bioactive natural products.

Identification of Bupleurum Seeds by ITS2 Genetic Barcoding and Seed Morphology / 中国实验方剂学杂志

Objective:To accurately identify Bupleurum seeds by traditional morphological identification method combined with DNA barcoding technique. Method:A total of 41 seed samples on the market were collected and 75 ribosomal DNA internal transcribed spacer 2 (ITS2) sequences of 15 varieties were downloaded from the GenBank database as experimental materials. The seeds were measured and observed by stereomicroscope and vernier caliper, and their 1 000-grain weights were calculated. Genomic DNA was extracted from the seeds and used as a template, and ITS2 sequences were amplified using polymerase chain reaction (PCR) and bidirectional sequencing. Species identification was conducted based on BLAST method, neighbor-joining (NJ) phylogenetic tree method, Kimura two-parameter model (K2P) genetic distance method, and secondary structure of ITS2 sequence. Result:There were slight differences in the length, width, cross-section, and 1 000-grain weight among Bupleurum seeds from different origins. The ITS2 sequences of B. chinense seeds had 2 intraspecific variable sites and 3 haplotypes, the maximum intraspecific genetic distance (0.009) was far smaller than the minimum interspecific genetic distance (0.032). B. chinense and B. scorzonerifolium in the NJ phylogenetic tree were clustered into independent branches with good monophyletic property. The secondary structure of ITS2 sequences could make up for the shortcomings of NJ tree in identifying variants. The collected 41 seeds included 30 B. chinense seeds, 3 B. scorzonerifolium seeds, 5 B. falcatum seeds, 2 B. marginatum var. stenophyllum seeds, and 1 B. smithii var. parvifolium seeds. Conclusion:The B. chinense seeds on the market have problems of diverse sources and chaotic origins. Based on the combination of ITS2 gentic barcoding and seed morphological identification, the Bupleurum seeds can be accurately identified, which provides scientific bases for establishing the quality standard of Bupleurum seeds, standardizing the cultivation of B. chinense, and solving the quality problems of B. chinense from the source, and provides a reference for the accurate identification of other medicinal plant seeds or seed medicinal materials.

DNA Barcoding Identification of Paecilomyces hepiali and Jinshuibao Capsules / 中国实验方剂学杂志

Objective::Because traditional methods are difficult to identify the fermentation mycelium, DNA barcoding technology was used to quickly identify the raw material strain Paecilomyces hepiali of Jinshuibao capsules and related products. Method::A total of 168 samples of 8 species of P. hepiali and its confusable species were identified by internal transcribed spacer (ITS) sequences, and based on the ITS sequences, P. hepiali specific primers were designed to quickly identify the related products. Result::The length of ITS sequences in 44 P. hepiali samples from different sources was 499 bp and there was no mutation site. It was shown that P. hepiali could be distinguished from 7 confusable species based on ITS sequences. The specific primer (ITS-BF/ITS-BR) of P. hepiali designed by ITS sequences could be amplified to obtain a short fragment of 102 bp in length, which could be used to rapidly identify P. hepiali from other confusable species, and to distinguish relevant products in the market. Conclusion::The rapid identification of P. hepiali and its related products can be achieved through the ITS sequences and specific primers, which provides a reference for the production and quality control of Jinshuibao capsules.

Molecular Identification of Bupleurum chinense Seeds Based on DNA Barcoding Technology / 中国实验方剂学杂志

Objective:To establish a molecular identification method for Bupleurum chinense seeds based on ribosomal DNA internal transcribed spacer (ITS) sequence, ensuring the species authenticity of the cultivated seeds of B. chinense. Method:A total of 59 seeds samples of B. chinense and its main cultivated species, marketed B. chinense were collected. The effect of different sampling amounts and different water bath conditions on DNA extraction quality of the seeds was investigated, a DNA extraction method for seeds of Bupleurum was established. Their ITS sequences were obtained by polymerase chain reaction (PCR) and bidirectional sequencing. In addition, 34 ITS sequences of main cultivated Bupleurum species, such as B. chinense, B. scorzonerifolium, B. falcatum and B. smithii, were downloaded from GenBank to enrich identification database of B. chinense seeds. The neighbor-joining (NJ) dendrogram were constructed by MEGA-X 10.0.5 software to investigate the the species identification ability of ITS sequences for B. chinense seeds. And DNA barcoding identification of marketed B. chinense seeds was conducted based on BLAST method and NJ dendrogram method. Result:In total, 59 ITS sequences were obtained. ITS sequences of B. chinense could be divided into six haplotypes, including seven variable sites. The NJ dendrogram indicated that all the haplotypes of B. chinense could form independent branches, which could be distinguished from other cultivated species of Bupleurum in the collected samples, and possessed the ability to identify species of B. chinense seeds. Based on ITS sequence barcoding identification, 3 of the 19 marketed B. chinense seeds were B. falcatum with a counterfeit rate of 15.8%. Conclusion:DNA barcoding technology based on ITS sequence can accurately and reliably identify B. chinense seeds and its adulterants, providing reference for the standardization construction of Chinese medicinal materials seeds.

DNA barcoding for molecular identification of Gynerium sagittatum(Poales: Poaceae): genetic diversity in savannah genotypes from Córdoba, Colombia

Introduction: The fiber of the Gynerium sagittatum Aubl. P. Beauv is raw material for the elaboration of several handcrafts, which are symbols of Colombian cultural identity. In the manufacture process, different genotypes are used according to the fiber quality and the type of craftsmanship, but it is believed that Gynerium is a complex species, and to date, there is no agreement on whether these genotypes belong to the same species or to different species. Objective: The aim of this study was to quickly and accurately identify wild cane plants using the nuclear ribosomal internal transcribed spacer (ITS1+ITS2), three chloroplast regions (matK, rbcL, ycf1), and their combinations. Methods: Different tests were used for discrimination: (1) inter and intraspecific distances, (2) Best Match (BM), Best Close Match (BCM), and tree-based method (3) Neighbor Joining (NJ) and (4) maximum likelihood and bayesian inference in molecular data. Results: The results showed that BM and BCM approaches revealed the low rate of correct species identification for ITS+matK (33.3 %) and ITS (28.6 %) loci, showing similarity among sequences. These results were further supported by tree-based analyses, where all individual regions and the different gene combinations had a zero discrimination rate. Conclusions: all genotypes belong to the same species of wild cane, therefore existing morphological differences can be related to phenotypic plasticity.

Introducción: La fibra de Gynerium sagittatum Aubl. P. Beauv, es materia prima esencial para la elaboración de varias artesanías, que son símbolos de la identidad cultural colombiana. En el proceso de fabricación, se utilizan diferentes genotipos de acuerdo con la calidad de la fibra y el tipo de artesanía, pero se cree que Gynerium es una especie compleja y hasta la fecha, no hay un consenso sobre si estos genotipos pertenecen a la misma especie o especies diferentes. Objetivo: Identificar de forma rápida y precisa plantas de caña silvestre utilizando el espaciador transcrito interno ribosomal nuclear (ITS1+ITS2), tres regiones de cloroplasto (matK, rbcL, ycf1) y sus combinaciones. Métodos: Se utilizaron diferentes pruebas para la discriminación: (1) distancias inter e intraespecíficas, (2) Prueba Best Match (BM), Best Close Match (BCM) y método basado en árboles (3) Neighbor Joining (NJ) y (4) Probabilidad de inferencia bayesiana mediante datos moleculares. Resultados: Los resultadosmostraron que los enfoques BM y BCM revelaron una baja tasa de identificación correcta de especies para los loci ITS+matK (33.3 %) e ITS (28.6 %), mostrando similitud entre las secuencias. Estos resultados fueron respaldados por análisis basados en árboles, donde todas las regiones individuales y las diferentes combinaciones de genes tuvieron una tasa de discriminación de cero (0 %). Conclusiones: los genotipos evaluados pertenecen a la misma especie de caña flecha y las diferencias morfológicas existentes pueden estar relacionadas con plasticidad fenotípica.

Systematic Identification and Quality Evaluation of Anemonis Flaccidae Rhizoma from 16 Different Places of Origin / 中国实验方剂学杂志

Objective:To construct a systematic identification system of Anemonis Flaccidae Rhizoma, and to evaluate the comprehensive quality of Anemonis Flaccidae Rhizoma from 16 regions in China, so as to lay a foundation for its origin selection and clinical medication safety. Method:The authenticity of Anemonis Flaccidae Rhizoma was quickly identified by traditional identification method and DNA barcode molecular identification technology, and HPLC-UV was used to determine the contents of 5 active ingredients in Anemonis Flaccidae Rhizoma. All high pressure chromatographic separations were performed with a Welch Ultimate XB-C18 column (4.6 mm×250 mm, 5 μm), the mobile phase consisted of acetonitrile-0.01% trifluoroacetic acid aqueous solution (30∶70) at a flow rate of 1.0 mL·min-1. The detection wavelength was set at 210 nm and the column temperature was maintained at 30 ℃. Result:The authenticity of Anemonis Flaccidae Rhizoma could be precisely and rapidly identified by ribosomal DNA internal transcribed spacer 2 (ITS2) sequence and traditional identification methods. BLAST comparative analysis found that medicinal materials from 16 areas were all Anemone flaccida. Based on the contents of multi-index components, it was shown that the total content of 5 triterpenoid saponins in Anemonis Flaccidae Rhizoma from Banqiao, Enshi, Hubei was the highest (10.59%), followed by Hezhang, Bijie, Guizhou (6.28%) and Duzhenwan, Changyang, Hubei (5.64%). Conclusion:DNA barcoding can be used as an effective supplement to the traditional identification technology, it can ensure the authenticity of Anemonis Flaccidae Rhizoma and the safety of clinical use. The comprehensive evaluation of multi-index components of HPLC and cluster analysis show that the quality of medicinal materials in Enshi, Changyang, Wufeng of Hubei, Bijie of Guizhou and Jinfoshan of Chongqing is superior, which can be considered as important origin of Anemonis Flaccidae Rhizoma.

Russian Characteristic Medicinal Plant Resources Based on DNA Barcode Technology / 中国实验方剂学杂志

Objective::To investigate the distribution status of medicinal plants in the wild areas of Russian Caucasus and Altai, and clarify the types and efficacy information of medicinal plants in the area, so as to dig deep into new resources and new functions of medicinal plants in the countries along the Belt and Road. Method::Medicinal plants in the wild were searched and collected to make waxy specimens, and sent back to the country to extract the total DNA of the leaves of the leaves. Internal Transcribed Spacer(ITS)sequence universal primers were used for Polymerase Chain Reaction (PCR)amplification. The PCR products were sent for the two-way sequencing, and the sequencing results are spliced by software according to National Center for Biotechnology Information(NCBI). The same ITS sequence of the highest similarity species obtained by database BLAST was analyzed by DNAman software to identify the ITS sequence of the species and the ITS sequence of the same species. The MEGA 7 software was used as the phylogenetic tree, and the Kimura-2 parameter genetic distance was used to construct the neighbor joining(NJ) phylogenetic tree by the neighbor-joining method. The confidence of each branch of the development tree was tested by the bootstrap test method. A total of 2 000 cycles were performed, and the results were identified based on the clustering results. On this basis, the key medicinal plants in the Russian Caucasus and Altay wild areas were summarized and analyzed. Result::After BLAST alignment in NCBI database, the ITS sequences of each specimen were clustered with the login sequences on the NCBI database, which were separated from the outer group. The species classification of the specimens to be identified was determined by combining the characteristics of the specimens. A total of 51 plants were identified from the specimens collected in the field, covering 44 genera of 17 families, and 29 plants had clear efficacy records. The National Drug List of the Russian Federation and the Chinese Pharmacopoeia were retrieved to summarize commonly used medicinal plants in China and conclude that 20 kinds of Chinese and Russian common medicinal materials have different medicinal effects in local areas. This study has a reference significance for expanding the scope and clinical experience of traditional Chinese medicines, and provides a basis for strengthened local species conservation, development and utilization of wild medicinal plant resources.