14 August 2024, Volume 44 Issue 2

    

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Original Article
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  Evaluation and analysis of pine resin components of Pinus elliottii clones

  ZHANG Wenjuan¹, DING Wei², FU Yuxin², ZHANG Zhihong³, ZHOU Guang², LI Huogen¹, YANG Chunxia^2*

  Guihaia. 2024, 44(2): 207-215. https://doi.org/10.11931/guihaia.gxzw202302037

  Abstract ( 36)   Knowledge map   Save

  In order to select superior clones with strong resin-producing capacity and high resin-quality from the existing fast-growing timber clones in seed orchard of Pinus elliottii, and to make high use of crop germplasm resource, 36 clones in the first generation of slash pine seed orchard were used as materials to determine their resin yield, resin mass flow rate and DBH growth, and to analyze their resin composition by GC-MS. Based on the above indicators, correlation analysis and cluster analysis was used to comprehensively evaluate the production and quality of resin among 36 clones. The results were as follows:(1)There were a total of 21 pine resin components, including 8 monoterpenes and 13 diterpenes.(2)Correlation analysis showed that the resin mass flow rate(RMR)had significant and positive correlation with the total content of monoterpene, weakly negatively correlates to abietic-type resin acid, and not significantly correlated to pimaric-type resin acid.(3)Based on the cluster analysis results integrating four types of indicators as the total monoterpene content, resin mass flow rate, abietic-type resin acid and pimaric-type resin acid, 36 clones could be divided into three categories and the difference between each type was significant. The performance of Class 1 was much better than that of the other two categories.(4)There were 17 high-resin yield pine clones(ERM≥15.15)among 36 clones, and on the basis of this, four clones(6-44, 4-11-1, 1-38, 3-64)display higher monoterpenes content, while four clones(4-11-1, 3-64, 2-0420, 3-468)showed higher contents of pimaric-type resin acid. And the content of abietic-type resin acid of clone 2-173 was the highest. In summary, a total of 21 pine resin components of P. elliottii were identified, and 36 clones were evaluated based on four indicators: the total monoterpene content, resin mass flow rate, abietic-type resin acid and pimaric-type resin acid. We not only analyzed qualitatively the resin composition but also evaluated quantitatively the resin-producing capacity of 36 clones in slash pine seed orchard. Our findings provide the scientific references for the targeted breeding of pine resin components and lay a foundation for subsequent heredity breeding and gene improvement of P. elliottii.
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  Cloning of HDR gene in Pinus massoniana and Cloningits response to drought and salt stresses

  WANG Jiawen, YAO Sheng, SU Huan, LIU Kexin, ZHU Peihuang, JI Kongshu^*

  Guihaia. 2024, 44(2): 216-234. https://doi.org/10.11931/guihaia.gxzw202212060

  Abstract ( 42)   Knowledge map   Save

  Drought and land salinization are inhibiting factors for the sustainable development of forestry, and the plants will release volatile substances such as terpenoids during biological or non-biological stress. The 1-hydroxy-2-methyl-2-(E)-buteny-4-pyrophosphate reductase is the terminal active enzyme of the MEP pathway, which provides precursor terpenoids and has the main rate-limiting effect. In order to investigate whether HDR gene of Pinus massoniana is involved in stress response under drought and salt stress, the open reading frame of PmHDR gene was cloned, and bioinformation, tissue specific expression and preliminary function were analyzed. The results were as follows:(1)The coding region length of PmHDR gene is 1 458 bp, encoding 485 amino acids, and its encoded protein contains the core sequence of LytB/IspH gene superfamily and PLN02821 multifunctional structural domain, which belongs to the HDR family.(2)The PmHDR codon use preference was weak, with a preference for codons ending in A/U. Nicotiana tabacum, Arabidopsis thaliana and Saccharomyces cerevisiae were more suitable as its heterologous expression receptors.(3)Results of qRT-PCR showed that the PmHDR gene was most highly expressed in old needles of Pinus massoniana, followed by young needles, young stem and old stem and least expressed in root.(4)The gene expression vector pBI121-PmHDR was constructed and transformed into Arabidopsis thaliana. The transgenic A. thaliana showed greater resistance to drought and salt stress. These results indicate that PmHDR is involved in plant response and regulation to drought and salt stress, and provide some theoretical supports for Pinus massoniana stress-resistance breeding.
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  Correlation of HrANR genes and flavonoid accumulation with drought resistance in sea buckthorn(Hippophae rhamnoides subsp. sinensis)

  LIU Rui, ZHAO Lang, YE Guisheng, MA Yuhua^*

  Guihaia. 2024, 44(2): 235-244. https://doi.org/10.11931/guihaia.gxzw202212006

  Abstract ( 57)   Knowledge map   Save

  Anthocyanidin reductase is one of the key enzyme involved in the synthesis of flavonoids. In order to explore the structure of ANR gene, ANR enzyme expression pattern and flavonoid content under drought stress and their correlation, a HrANR gene identified from RNA-seq data of sea buckthorn was screened and analyzed by bioinformatics soft, the expression patterns of HrANR gene in different tissues and of flavonoid contents in leaves were performed. The results were as follows:(1)The ORF of HrANR gene was 1 017 bp, which encoded 338 amino acids. It was a stable hydrophilic protein, and the homologous protein had significant family and genus characteristics.(2)HrANR gene was expressed in roots, stems and leaves of sea buckthorn under drought stress, but the expression trends were different, with an increase followed by a decrease and then an increase in roots, a continuous decrease in stems, and an increase followed by a continuous decrease in leaves.(3)The flavonoid contents in leaves of sea buckthorn under different levels of stress showed a trend that first increased continuously and then decreased slightly, increased to the highest point after rehydration. The above results indicated that the expression of HrANR gene and the changes of flavonoid content were closely related to the drought resistance of sea buckthorn. The flavonoid content in leaves was positively correlated with drought stress at the beginning of drought stress and negatively correlated with drought stress under severe stress.(4)Leaf expression, stem expression and flavonoid content of HrANR gene were negatively correlated(P_leaf= -0.751, P_stem= -0.934)and root expression was positively correlated with flavonoid content(P_root= 0.444). The above results indicate that the expression of HrANR genes and changes of flavonoid content in sea buckthorn are closely related to drought resistance, and it provides a reference for elucidating the drought resistance mechanism of sea buckthorn.
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  Construction of yeast two-hybrid cDNA library in cambium tissue of Hevea brasiliensis and screening of HbHDA6 interacting proteins

  ZHANG Shixin ¹, WU Shaohua ¹, YANG Shuguang¹, CHAO Jinquan¹, SHI Minjing¹, GE Lixin^1,2, JIANG Yi^1,2, TIAN Weimin^1*

  Guihaia. 2024, 44(2): 245-256. https://doi.org/10.11931/guihaia.gxzw202303052

  Abstract ( 66)   Knowledge map   Save

  The secondary laticifer is the position for synthesis and storage of natural rubber(NR), which is differentiated from the vascular cambium cells of bark in stem of rubber trees(Hevea brasiliensis). The quantity of secondary laticifer is depended on the frequency of the secondary laticifer differentiation from cambia, which is the main index of yield breeding of rubber tree. In previous studies, we found trichostatin A(TSA), an inhibitor of histone deacetylase(HDA), can also induce laticifer differentiation, and the histone deacetylase gene(HbHDA6)is a participator in laticifer differentiation. Because of the molecular mechanism of secondary laticifer differentiation regulated by histone acetylation has not been clarified. Therefore, we construct a yeast two-hybrid cDNA library used the vascular cambium tissues treatment by coronatine(COR), and screening the yeast two-hybrid library by HbHDA6 gene as the bait, for determining the proteins interacting with HbHDA6. The results were as follows:(1)The homogenized yeast two-hybrid cDNA library of vascular cambium was constructed by the technology of Gateway. The capacity of the primary library was 6.34 × 10⁶ CFU·mL^-1, the total number of clones was 1.27 × 10⁷, and the capacity of secondary library was 7.72 × 10⁶ CFU·mL^-1, the total number of clones was 1.54 × 10⁷, and the recombination rates of two libraries were 100%. The average length of inserted fragments was 1.1 kb and 1.2 kb in primary and secondary library, respectively.(2)The bait vector of pGBKT7-HbHDA6 for screening the proteins interacting with HbHDA6 was successfullyconstructed and confirmed no self-activation activity.(3)The pGBKT7-HbHDA6 bait vector was used to screen the constructed yeast two-hybrid cDNA library, and 22 proteins interacting with HbHDA6 were obtained by NCBI_BLAST comparison and removing duplicates, including CLP1, ERF3, ERF4, HSP82, LARP6a, APT5, PP2A, APT5, FBA6, etc. The results provide a theoretical basis for analyzing the molecular regulatory network of the secondary laticifer differentiation of rubber tree, and provide candidate genes for the rubber production potential of genetically modified and a new clue for the genetic improvement and breeding of high-performance NR.
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  AP3 gene cloning and gene-editing vector construction of Hydrangea macrophylla ‘Dooley'

  LI Tong, WANG Yueying, ZHAO Huien^*

  Guihaia. 2024, 44(2): 257-266. https://doi.org/10.11931/guihaia.gxzw202204002

  Abstract ( 135)   Knowledge map   Save

  Hydrangea macrophylla is a garden plant widely cultivated in Asia, America, and Europe with its inflorescence as main ornamental feature. It is commonly used in interior decoration and landscape creation. To investigate the role of AP3 gene in hydrangea during calyx formation, H. macrophylla ‘Dooley' was used as the material. The MADS-box class B gene HmAP3 was cloned, and its gene function was predicted by bioinformatics analysis. To explore methods for quicker breeding new varieties, highly-specific editing targets were screened and CRISPR/Cas9 gene-editing vectors were constructed. The vector sequence was integrated into the H. macrophylla genome by agrobacterium-mediated transformation. The results were as follows:(1)The cDNA sequence full length of HmAP3 was 546 bp, encoding 181 amino acids. Its amino acid sequence was 100% similar to the reference sequence and 58.8% similar to Arabidopsis thaliana.(2)AP3 differed greatly in different genera. Within the same genus, the main structure of AP3 protein was conserved and differed only in a few motifs.(3)There were two highly specific targets in HmAP3. Sequencing results indicated that two single-target CRISPR/Cas9 gene-editing vectors were constructed successfully.(4)There were five resistant buds with Cas9 sequences in their genomes. However, their target sequences did not change due to the absence of Cas9 expression. In this study, the potential of AP3 gene in the breeding work of double flower phenotype was investigated, and a preliminary exploration of CRISPR/Cas9 gene-editing technology for Hydrangea macrophylla was conducted. These results provide a basis for the breeding of H. macrophylla.
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  Cloning and expression analysis of BjGSTF12 in Brassica juncea

  ZHU Yunna, CHEN Fengmei, LI Zhixian, WANG Bin, FENG Huimin, HU Fang, LI Haibo^*

  Guihaia. 2024, 44(2): 267-280. https://doi.org/10.11931/guihaia.gxzw202305039

  Abstract ( 40)   Knowledge map   Save

  To explore the role of glutathione S-transferase gene(GST)in the accumulation of anthocyanin in Brassica juncea, one GST gene related to the anthocyanidin accumulation was cloned from near-isogenic lines of B. juncea with purple stalk and green stalk, and named as BjGSTF12. In this study, the bioinformatics characteristics of BjGSTF12 encoding protein and promoter were analyzed, the expression level of BjGSTF12 and the relationship with total anthocyanidin content were analyzed in B. juncea lines with purple stalk and green stalk. The results were as follow:(1)BjGSTF12 genes from B. juncea were successfully cloned, whose the full length of BjGSTF12 in genome and cDNA was 808 bp and 651 bp, encoding a protein of 216 amino acids. The BjGSTF12 contained the typical domains of GST proteins, namely GST_N and GST_C. However, their sequences of BjGSTF12 did not exhibit any differences between the two lines of B. juncea.(2)BjGSTF12 was closely related to AtGSTF12 in Arabidopsis, and both belonged to the φ subfamily of GST family.(3)The BjGSTF12 promoter sequences were cloned from two Brassica juncea strains of green stalk and purple stalk, and they exhibited four base mutations/insertions. However, the types and numbers of cis-acting elements did not show obvious differences between the two strains, including nine MYB transcription factor binding sites, one hormone response element, and three abiotic corresponding elements.(4)The total anthocyanidin content in B. juncea of purple stalk was significantly higher than that in green stalk ones, and the expression levels of BjGSTF12 in two lines were found to be similar to the total anthocyanidin content in both lines.(5)Protein interaction network analysis revealed that BjGSTF12 may interact with the key enzymes of anthocyanidin biosynthesis, glycosylation modification, and transporter proteins. In summary, BjGSTF12 is likely to play a key role in the accumulation of anthocyanidin in B. juncea by regulating its biosynthesis, modification, and transportation through interactions with other proteins. This study provides a theoretical reference for further study on the function of GST and the mechanism of anthocyanidin accumulation in B. juncea.
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  Metabolomics analysis of flower color substances in three Rosa rugosa cultivars

  WEI Liqin, CHONG Peifang^*, BAO Xinguang, HE Hailing, LI Qingqing

  Guihaia. 2024, 44(2): 281-290. https://doi.org/10.11931/guihaia.gxzw202211036

  Abstract ( 105)   Knowledge map   Save

  Rosa rugosa is a deciduous shrub belonging to Rosa L. in Rosaceae. It has a high ornamental value and commercial value, but its single color limits the development and utilization of rose and its application in landscape architecture. In order to explore the coloring substances of three different varieties of roses, ‘Rosa rugosa × Rosa sertata', ‘Rosa Crimson Glory' and ‘Rosa alba', this study used ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS)to detect the types and contents of flavonoids in petals. The KEGG database was used to enrich the differential metabolites, screen out the key metabolites, and analyze the correlation with the phenotypic value of flower color. The results were as follows:(1)A total of 58 metabolites were detected in the petals of three different color rose varieties, of which only one anthocyanin was cyanidin-3-O-glucoside, accounting for 30.45%.(2)K-means clustering analysis showed that a total of 12 key metabolites were annotated to the KEGG metabolic pathway. Among them, pinocembrin and myricetin were the main substances that determined the red color of ‘Rosa rugosa × Rosa sertata' and ‘Rosa Crimson Glory', and eriodictyol, luteolin and kaempferol were the main substances that determined the white color of ‘Rosa alba'. In conclusion, this study can provide a theoretical basis for the breeding of roses with specific colors and promote the application of roses in landscaping.
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  Genetic variation analyses of quality and agronomic traits of 26 Fagopyrum tatari-cymosum lines

  WANG Weixuan, TIAN Shuangqi, KE Jin, ZHANG Fan, CHENG Yuanzhi, CHEN Xiaoquan, LI Hongyou, SHI Taoxiong, CHEN Qingfu^*

  Guihaia. 2024, 44(2): 291-302. https://doi.org/10.11931/guihaia.gxzw202211018

  Abstract ( 37)   Knowledge map   Save

  Fagopyrum tatari-cymosum is a semi-perennial new buckwheat type developped from the hybridization between F. tataricum and F. cymosum. To explore the genetic laws of agronomic and quality traits of F. tatari-cymosum, 26 lines of F. tatari-cymosum were selected as materials, and their quality traits and agronomic traits were analyzed by variance analysis, correlation analysis and cluster analysis. The results were as follows:(1)The variation coefficient for quality traits of F. tatari-cymosum was gliadin content > glutenin content > flavonoids content > total protein content > globulin content > albumin content>starch content.(2)The variation coefficient for agronomic traits of F. tatari-cymosum was branch number of main stems > node number within 20 cm of the bases > main stem diameter > grain area > 1 000-grain weight > node number of main stems > plant height > grain length to width ratio > grain width > grain length > shell rate > grain perimeter > grain diameter.(3)In the correlation analysis, the flavonoid content was significantly positively correlated with albumin content; the gliadin content was significantly or extremely significantly positively correlated with the main stems diameter, the node number of main stems, the node number within 20 cm of the bases, and significantly or extremely significantly negatively correlated with the 1 000-grain weight, grain area, grain perimeter, grain width, and grain diameter; the starch content was positively correlated with grain area, grain length and grain diameter.(4)By cluster analysis, 26 F. tatari-cymosum lines were divided into three groups. Group I belonged to high starch, short stem, multi branched, low shell rate, large long grain lines, which could be used as parent material for breeding purposes of high starch and low shell rate; Group Ⅱ belonged to high protein, high stem, thick, small grain type lines, which could be used as a material for breeding purposes of high protein and strong stress resistance; Group Ⅲ belonged to high quality, high yield, large grain type lines. The results provide theoretical references for the breeding of F. tatari-cymosum.
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  Identification and expression analyses of O-acyltransferase WSD genes in Rehmannia glutinosa

  LI Huimin, YUAN Ping, DUAN Hongying, ZHOU Yanqing^*

  Guihaia. 2024, 44(2): 303-312. https://doi.org/10.11931/guihaia.gxzw202304008

  Abstract ( 54)   Knowledge map   Save

  Plant wax ester synthase catalyzes the synthesis of wax esters from long-chain alcohols and fatty acids, and plays very important roles in plant wax synthesis and some resistances to drought, pathogenic bacteria, ultraviolet radiation, cold and insect invasion and other environmental stresses; cadmium(Cd)is one of the toxic heavy metals with the highest content in environment, and seriously threatens plant growth, development, quality, yield, and plant food safety. In order to explore the Cd stress expressions of wax ester synthase genes in Rehmannia glutinosa, we identified its wax ester synthase genes from its full-length transcriptome sequencing data, analyzed both physiochemical properties, phytogenetic trees and conserved domains with bioinformatics methods, and tissue expressions and Cd stress expressions using qRT-PCR. The results were as follows:(1)Two wax ester synthase genes, named as RgOATWSD1 and RgOATWSD2, were identified, whose coding proteins were unstable hydrophobic proteins with amino acid lengths of 463 aa and 473 aa, isoelectric points of 8.86 and 9.34 and molecular weights of 51.31 kD and 52.49 kD, respectively.(2)Both proteins contained a conserved acyl_WS_DGAT domain and DUF1298 superfamily, in which the former accounted for 92.65% to 94.50% of the amino acid sequence.(3)Both proteins were located in the endoplasmic reticulum and both secondary structures were mainly composed of random coil and α-helix; RgOATWSD2 was not transmembrane protein but RgOATWSD1.(4)Both were differentially expressed in the roots, stems and leaves of R. glutinosa plants.(5)Both expressions were highly responsive to Cd stress, but both expression change trends were different under Cd stress. This study identifies two wax ester synthase genes in response to Cd stress, and lays a foundation for further research on Cd stress expression and other functions of RgOATWSD.
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  Qualitative analysis of chemical constituents of Aloe made from Aloe barbadensis by liquid chromatography-mass spectrometry

  ZHANG Jing¹, HAO Beiquan¹, LI Yinqing², PI Guopei², XU Feng^1*, LIU Guangxue¹, SHANG Mingying¹, CAI Shaoqing¹

  Guihaia. 2024, 44(2): 313-326. https://doi.org/10.11931/guihaia.gxzw202306005

  Abstract ( 50)   Knowledge map   Save

  To clarify the chemical constituents of the traditional Chinese medicine Aloe made from Aloe barbadensis, i.e., the concentrated dry matter of the juice of the leaves of A. barbadensis, a systematic qualitative analysis of them was conducted using the technique of HPLC-DAD-ESI-IT-TOF-MSⁿ in conjunction with the comparison of reference compounds and literature search. The gradient elution was performed with water(A)-acetonitrile(B)as mobile phase at a flow rate of 1.0 mL·min^-1. The liquid chromatography-mass spectrometry data were acquired under alternate negative ion and positive ion detection mode using an ESI ion source. The structure elucidation of the chemical constituents was mainly based on negative ion mass spectrometry data. The results were as follows:(1)For the first time, the fragmentation pathways of anthraquinones(aloe-emodin, physcion, and emodin-8-O-β-D-glucoside), anthrones(aloin A, aloinoside A), chromones(aloeresin D, 7-O-methylaloeresin A, altechromone A, aloesin, aloeresin G, and aloeresin C), and α-pyranones(aloenin A, aloenin B)in Aloe made from A. barbadensis were clarified. The fragmentation pathway of anthraquinones was dominated by loss of CO₂ and CO, and that of anthrones was dominated by cleavage of hexosides and loss of CO. The fragmentation pathways of chromones was dominated by cleavage of hexosides and hydrolysis of the ester group, and that of α-pyranones was dominated by cleavage of hexosides and loss of CO₂ and H₂O.(2)A total of 168 chemical constituents of Aloe made from A. barbadensis were detected, and 78 of them were identified on the basis of reference compound comparison, literature retrieval, and chemical database(such as SciFinder)searching. The 78 compounds included 3 anthraquinones, 29 anthrones, 35 chromones, 7 α-pyranones and 4 other constituents. Twenty-three of 78 compounds were discovered in the leaves of A. barbadensis for the first time. Fourteen of 23 were newly discovered compounds, including aloinoside D, isoeleutherin, and ethylidene-aloenin, possessed antibacterial, anti-inflammatory, or free radical scavenging activities. The results of this study further enrich the information on the chemical constituents of the traditional Chinese medicine Aloe made from A. barbadensis, and lay a foundation for the study of the therapeutic material basis and quality control methods of Aloe.
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  Diterpenoids with acetylcholinesterase inhibitory activity from Pieris japonica

  LI Huijuan^1,2, QUAN Wei³, LUO Ee², QIN Xujie², HUA Yan^1*

  Guihaia. 2024, 44(2): 327-332. https://doi.org/10.11931/guihaia.gxzw202210074

  Abstract ( 31)   Knowledge map   Save

  In order to investigate the diterpenoid components from the leaves of Pieris japonica and their acetylcholinesterase(ACHE)inhibitory activities, with the aid of thin-layer chromatography color characteristics, silica gel, MCI and semi-preparative high-performance liquid chromatography technology, the target constituents were isolated and purified. The structures of the obtained compounds were identified by analyses of their spectral data(NMR and MS)and comparison of their data with those of reported in the literature. Meanwhile, AChE inhibitory effects of obtained diterpenoids were evaluated for the first time by a ellman method. The results showed that eight diterpenoid compounds were isolated and identified from the leaves of P. japonica, namely pieriformoside F(1), 3-epi-grayanotoxin XVIII(2), 3-epi-grayanotoxin B(3), asebotoxin-X(4), pieriformosin B(5), asebotoxin Ⅲ(6), rhodojaponin Ⅲ(7), and pieriformosin C(8). Among them, Compound 1 was isolated from this plant for the first time, and Compound 8 showed AChE inhibitory activity. In conclusion, the results enriches the diterpenoids and bioactive components of P. japonica, which provides a certain theoretical basis for its further development and utilization.
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  Isolation and identification of pathogenic fungi from soft rot tissue of Amorphophallus konjac corm

  LI Zhumei¹, DONG Kun², ZHANG Yan'an¹, GAO Yong¹, CHEN Hong¹, FANG Pingping¹, LEI Hongxian¹, LU Xiaoqian¹, CHU Honglong^1,3*

  Guihaia. 2024, 44(2): 333-344. https://doi.org/10.11931/guihaia.gxzw202207042

  Abstract ( 51)   Knowledge map   Save

  Konjac(Amorphophallus konjac)is a horticultural plant with high nutritional and medicinal values. Soft rot is a severe disease in production of konjac and it is also the main factor restricting the development of the konjac industry. It has been reported that the soft rot of konjac is mainly caused by pathogenic bacteria(mainly including Pectobacterium aroidearum, P. carotovorum subsp. carotovorum, P. chrysanthemi and Enterobacter sp.), and there is rare reports on pathogenic fungi that cause konjac soft rot. In order to clarify the pathogenic types and infection characteristics of the soft rot in Qujing City, Yunnan Province, the diseased corms were collected for fungal isolation by tissue isolation methods. The isolated fungi were identified by morphological and molecular identification methods based on ITS and LSU sequence analyses, and pathogenicity was determined according to Koch's rule. The infection characteristic was analyzed by mixed inoculation using the identified pathogenic fungi and the pathogenic bacteria of konjac soft rot. The results were as follows:(1)Three species of Fusarium spp.(Fusarium concentricum, F. oxysporum and F. ambrosium), one species of Mucor sp., one species of Rhizopus sp., one species of Penicillium sp. and one species of Clonostachys sp. were identified.(2)Statistics analysis found that Fusarium concentricum had the highest relative abundance(45.45%).(3)Koch postulates tests showed that inoculation with F. concentricum caused obvious soft rot symptoms of konjac corms within three days.(4)In addition, mixed Pectobacterium aroidearum and Fusarium concentricum together inoculation promoted the disease development, and the weight of rotten tissue was significantly higher than that of single inoculation using F. concentricum or Pectobacterium aroidearum. Overall, these results indicate that konjac soft rot may be caused by a combination of fungus and bacterium infection. The results provide a theoretical reference for the prevention and management of konjac soft rot.
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  Identification and inhibiting effect of pathogens that caused tuber rot of Bletilla striata

  MA Xiaoya^1,2, LU Xi^1,2, WU Qiaofen², YANG Yanni², XIA Ke², ZHAO Zhiguo², ZHENG Wenjun^1*, QIU Shuo^2*

  Guihaia. 2024, 44(2): 345-353. https://doi.org/10.11931/guihaia.gxzw202302001

  Abstract ( 45)   Knowledge map   Save

  In order to identify the pathogens that caused tuber rot in Bletilla striata and study the inhibiting effects of herbal extracts on pathogens, the pathogens that caused tuber rot of B. striata were isolated using usual tissue isolation the strains were identified by morphological and molecular biological techniques, and inhibiting effects of seven traditional Chinese medicine extracts on pathogen were observed. The results were as follows:(1)A total of fourteen fungi and four bacteria were isolated from diseased leaves, leaf sheaths and tubers. But only strain GF-1 caused disease, whose symptoms consistent with those in the field. The incidences of GF-1 disease reinoculated in the field and laboratory were both 100%.(2)GF-1 was identified as a member of Epicoccum, and its colonial morphology was a circular form, with white mycelium, prostrate on the medium, aerial, diaphragms and branches. There were conidia and chlamydospores.(3)At last, the sequence of internal transcribed spacer(ITS)region of GF-1 were analyzed, the length was 522 bp. The sequence was compared with other species in the GenBank and reached 99.62% similarity to Epicoccum sorghinum (MN493119.1)isolated form Sorghum, which was closer than others, including Epicoccum sorghinum(MF948994.1)isolated form leaves of Bletilla striata.(4)GF-1 could be fully inhibited when the medium contained 0.1-0.2 g·mL^-1 extracts that extracted from seven traditional Chinese medicines, respectively. It also could be fully inhibited by 0.05 g·mL^-1 of Cinnamomum cassia or Syringa oblate. In summary, the pathogen that caused tuber rot in Bletilla striata was identified as Epicoccum sorghinum. And GF-1 could be fully inhibited cultivated on the medium which contained 0.1-0.2 g·mL^-1 herbal extracts, e.g.: Cinnamomum cassia, Syringa oblate, Cyclocarya paliurus, Bletilla striata, Houpoea officinalis, Illicium verum or Cnidium monnieri. The results provide theoretical references for the control of tuber rot of Bletilla striata.
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  Chemical constituents from Rubus suavissimus and their α-glucosidase inhibitory activities

  YANG Bingyuan, HE Ruijie, WANG Yafeng, YAN Xiaojie, HUANG Yonglin^*

  Guihaia. 2024, 44(2): 354-361. https://doi.org/10.11931/guihaia.gxzw202303011

  Abstract ( 66)   Knowledge map   Save

  Rubus suavissimus is mainly distributed in Guilin, Liuzhou, Wuzhou and other regions in Guangxi Zhuang Autonomous Region, so it is called as “Guangxi tiancha” in China. R. suavissimus, together with Siraitia grosvenorii and Stevia rebaudianastevia are praised as three famous sweet plants in Guangxi Zhuang Autonomous Region. Zhuang and Yao people use the leaves of R. suavissimus as a tea to treat diabetes, and it is known as the “divine tea” of Yao medicine. Therefore, R. suavissimus is a combination of sugar, tea and medicine, which has great potential for the development of food and medicine. In order to investigate the secondary metabolites with α-glucosidase inhibitory activity from R. suavissimus, herein, the extraction, separation and purification of secondary metabolites were performed on the leaves of R. suavissimus. The structures of purified compounds were determined based on the data of mass spectrometry(HR-ESI-MS)and nuclear magnetic resonance(¹H NMR and ¹³C NMR). In addition, the α-glucosidase inhibitory activity of the purified compounds were evaluated by pharmacological methods simultaneously. The results were as follows:(1)Ten compounds were purified and their structures were elucidated as rubusoside(1), kaempferol 3-O-robinobioside(2), gallic acid(3), dihydrodehydroconiferyl alcohol(4), 5-methoxydihydrodehydroconiferyl alcohol(5), brevifolincarboxylic acid(6), steviolmonoside(7), steviol(8), 16α, 17-dihydroxykaurane(9), and quercetin 3-O-β-D-galactopyranoside(10). Among them, compounds 2, 4, 5 and 9 were isolated from R. suavissimus for the first time.(2)Compounds 2, 3, 5, 6 and 10 showed strong inhibitory activity on α-glucosidase. The IC₅₀ values of compounds 2, 3, 5, 6 and 10 were(0.14 ± 0.03)mg·mL^-1,(0.36 ± 0.02)mg·mL^-1,(0.44 ± 0.01)mg·mL^-1,(0.53 ± 0.04)mg·mL^-1 and(0.14 ± 0.03)mg·mL^-1 respectively, which were stronger than the positive control acarbose with the IC₅₀ values as(0.69 ± 0.02)mg·mL^-1. Thus, compounds 2, 3, 5, 6 and 10, which were isolated from the leaves of R. suavissimus, could be a potential α-glucosidase inhibitors based on their bioactivity results. Compounds with α-glucosidase inhibitory activity from R. suavissimus will provide the basis for development of related hypoglycemic products.
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  Isolation and purification the polysaccharides and its antioxidant activity of ‘Zijia 1' novel variety of Acanthopanax senticosus

  ZHOU Jinyan, ZHANG Yun, LIU Liangyan, ZHANG Hui, RUAN Liuyang, ZENG Qianchun^*

  Guihaia. 2024, 44(2): 362-372. https://doi.org/10.11931/guihaia.gxzw202212045

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  ‘Zijia 1' is a new variety of Acanthopanax senticosus bred by our team, its tender stems and leaves characterized with gray-purple color and sweet taste. This study is aim to isolate and purify the polysaccharides from the tender stems and leaves of ‘Zijia 1', and determine the monosaccharide composition and molecular weight of different fractions obtained after separation, and the antioxidant activity of each fraction was evaluated. The crude Asenticosus polysaccharides(ASPS)were obtained from the tender stems and leaves of ‘Zijia 1' by water extraction and alcohol precipitation, which were then separated and purified by DEAE-Cellulose 52 ion column and Sephadex G-100 gel column to obtain a uniform component. The ion chromatography and gel permeation chromatography-refractive index-multi-angle laser light scattering method was exploited to determine the monosaccharide compositions and molecular weight of the polysaccharides fractions. The hydroxyl radical(·OH), superoxide radical(O₂^-·)and 1,1-diphenyl-2-picrylhydrazyl radical(DPPH)scavenging ability were determined to evaluate the antioxidant of each fraction in vitro. Four polysaccharides ASPA-1-1, ASPA-2-1, ASPA-3-1 and ASPN-1 were isolated and purified from ASPS, with molecular weight of 8.10, 26.15, 0.91, 0.89 kDa, respectively, mainly composed of arabinose, rhamnose, galactose, glucose, xylose, mannose, ribose, galacturonic acid and glucuronic acid in different proportions. The ASPA-1-1, ASPA-2-1, ASPA-3-1 and ASPN-1 from ‘Zijia 1' demonstrated significant scavenging activities on ·OH, O₂^-· and DPPH free radical, the ability of ASPA-2-1 to scavenge ·OH and DPPH is higher than ASPA-1-1, ASPA-3-1 and ASPN-1; ASPA-3-1 has the strongest ability to scavenge O₂^-·. Therefore, the purified polysaccharides from ‘Zijia 1' has obvious antioxidant activity, which provides a scientific theoretical basis for its further utilization.
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  Chemical constituents and their anti-inflammatory activities from the roots of Ardisia crenata var. bicolor

  YE Hongbo, ZHOU Yongqiang, LIAO Zhangrong, WEI Xin, YIN Xin, LI Jiaxin, ZHOU Ying^*

  Guihaia. 2024, 44(2): 373-381. https://doi.org/10.11931/guihaia.gxzw202208026

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  Ardisia crenata var. bicolor is a common medicine used by Miao Minority in Guizhou, which has the effects of clearing throat and benefiting pharynx, reducing swelling and relieving pain, dispelling wind and dehumidifying. In order to study the chemical constituents and anti-inflammatory activities of A. crenata var. bicolor roots, the 70% ethanol extact was separated and purified by silica gel chromatography, Sephadex LH-20 gel column chromatography, ODS reverse column chromatography and semi-preparative HPLC. The structures of the compounds were identified by spectral data of NMR, MS and published literatures. Using lipopolysaccharide(LPS)-activated RAW 264.7 cell line model in vitro, compounds 1-4 were evaluated for the inhibition against nitric oxide(NO)production. The results were as follows:(1)Ten compounds were isolated from the 70% ethanol extract and identified as 11-O-galloylbergenin(1), 11-O-(4-O-methylgalloyl)bergenin(2), 11-O-vanilloylbergenin(3), 6-O-(4-hydroxy benzoyl)bergenin(4), 11-syringyl bergenin(5), 11-O-(3',4'-dimethylgalloyl)-bergenin(6), demethoxybergenin(7), micractinin A(8), monomethyl olivetol(9), and dibutyl phthalate(10). Among them, compounds 4, 8, 9 were obtained from Ardisia for the first time.(2)The results of anti-inflammatory activities in vitro showed that compounds 1-4 could significantly inhibit NO release in RAW 264.7 cells(P<0.01), and the inhibition rates of compounds at the concentration of 20 μmol·mL^-1 were 67.09%, 66.50%, 59.83%, 36.47%, respectively. This study enriches the chemical constituents of A. crenata var. bicolor roots, clarifies the material basis of its anti-inflammatory activities, verifies the scientificity of its traditional usage, and provides new insight and scientific evidence for its rational development and utilization of the medicinal resources.
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  Diversity analysis of endophytic fungi and preliminary screening of antibacterial activity in Camellia luteoflora

  YI Hang, HE Jing, YANG Xi, RONG Shutian, WANG Li^*

  Guihaia. 2024, 44(2): 382-395. https://doi.org/10.11931/guihaia.gxzw202211028

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  In order to explore the species and population distribution of endophytic fungi in Camellia luteoflora, as well as their inhibitory effects on plant pathogenic fungi, this study used tissue separation method to isolate and purify endophytic fungi in C. luteoflora. These fungi were identified based on morphology and molecular biology, and their diversity was evaluated through statistical analysis. The strains with antibacterial activity were screened out by the plate confrontation method. The results were as follows:(1)A total of 261 strains of endophytic fungi were isolated from 324 C. luteoflora tissue samples, belonging to 1 phylum, 5 classes, 9 orders, and 22 genera. The dominant genera were Colletotrichum, Diaporthe and Pestalotiopsis with isolation frequencies of 21.84%, 16.86% and 10.34%, respectively.(2)The distribution of endophytic fungi in C. luteoflora varied in different seasons. The highest number of strains was isolated in winter(72 strains, accounting for 27.59%, belonging to 16 genera), 62 strains were isolated in spring(belonging to 13 genera), 59 strains were isolated in summer(belonging to 15 genera), and 68 strains were isolated in autumn(belonging to 13 genera). The Shannon-Wiener index(H'), Simpson index(D), Pielou's evenness index(E), and Margalef's richness index(M)were the highest in winter. The similarity of endophytic fungal species between spring and winter was higher, and the similarity between summer and autumn was higher.(3)The distribution of endophytic fungi also varied in different parts of C. luteoflora. The stems had the most abundant endophytic fungi, with 102 strains accounting for 39.08%(belonging to 15 genera), 61 strains were isolated from the roots(belonging to 10 genera), and 98 strains were isolated from the leaves(belonging to 15 genera). The H', D, E, and M were highest in the stems, and the endophytic fungal species in the leaves were most similar to those in the stems.(4)The plate confrontation results showed that among the 35 tested endophytic fungi, 26 endophytic fungi had inhibitory effects on at least one plant pathogenic fungi, accounting for 74.29%, among which CJ-Ⅱ-2, XY-V-3, QY-Ⅱ-4, QJ-Ⅲ-2 and DJ-I-2 had inhibitory effects on eight plant pathogen fungi to varying degrees. XY-V-3 had the best inhibitory effect on eight plant pathogenic fungi, and the inhibitory rate was higher than 50%. XY-V-3 and QJ-Ⅲ-2 have higher inhibitory rate than 50% on two strains of pathogenic fungi in C. luteoflora, which had the potential to control disease of C. luteoflora. To sum up, the diversity of endophytic fungi in C. luteoflora is rich, and some of the strains have higher effect of inhibiting plant pathogenic fungi, which lay a foundation for the research and development of biological control products and the disease control of C. luteoflora.
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  Chemical constituents of Pteris ensiformis from Guizhou

  XIAO Li¹, SHEN Linyan¹, ZHANG Jingjie¹, HE Kang¹, YE Jianghai¹, ZHAO Chenliang¹, ZHANG Qilong², ZOU Juan^1*

  Guihaia. 2024, 44(2): 396-404. https://doi.org/10.11931/guihaia.gxzw202212068

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  To study the chemical constituents of Pteris ensiformis, silica gel, gel, MCI, C₁₈ and other column chromatography were used for separation and purification, and their structures were identified by ¹H-NMR, ¹³C-NMR, MS, IR and othe spectral data; the anti-tumor and anti-coagulation activities of some monomers were screened by MTS, APTT, PT and TT. The results were as follows:(1)A total of 15 compounds were isolated from P. ensiformis, the compounds were 2-hydroxy-acetylpyrrole(1), N-(3-carboxypropyl)-2-acetylpyrrole(2), 3-hydroxy-2-methylpyridine(3), N-methylhydroxylamine(4), pterosin S 13-O-glucoside(5), obtupterosin C(6), ent-11α-hydroxy-15-oxokauran-19-oic acid(7), ent-11α-hydroxy-15-oxokaur-16-en-19-oic acid(8), β-sitosterol(9), ent-11α-hydroxy-15-oxokaur-16-en-19-oic acid-O-glucopyranoside(10), 5, 5'-dibutoxy-2, 2'-bifuran(11), 5, 5'-di(2-ethyl-hexyloxy)-2, 2'-bifuran(12),(-)-loliolide(13), succinic acid(14), fumaric acid(15). Compound 1 is a new natural product of pyrrole alkaloids. Compounds 1-7, 10-15 were isolated from P. ensiformis for the first time, and compounds 1, 3, 4 were isolated from Pteris for the first time.(2)The results of activity test showed that compounds 1, 2, 3, 5, 6 and 10 inhibited the growth of tumor cells HL-60, A549, SMMC-7721, MDA-MB-231 and SW480 in vitro at a concentration of 40 μmol·L^-1, At the concentration of 2.0 mmol·L^-1, compounds 1, 2, 3 and 6 shortened APTT and compounds 1, 5 and 6 prolonged PT. The study enriches the chemical constituents of P. ensiformis from Guizhou and provides a material basis for the development of anti-tumor drugs.