Abstract [Objectives] This study was conducted to isolate and investigate the endophytic fungi with antimicrobial activity from the healthy leaves and bark of Metasequoia glyptostroboides Hu & W. C. Cheng regarded as an important medicinal plant in China.
　　[Methods] Endophytic fungi with visible antimicrobial activity were isolated from the healthy leaves and bark of M. glyptostroboides， and primarily screened using Escherichia coli and Staphylococcus aureus as test microorganisms.
　　[Results] Three isolates with relatively higher antimicrobial activity， labeled as MG-01， MG-02， MG-09， were selected for study. Molecular and morphological analysis indicated that the three isolated endophytes belong to Penicillium sp. The ethyl acetate （EtOAc） extracts of the three endophytes were obtained and subjected to the disk diffusion assay， using E. coil， S. aureus， Pseudomonas aeruginosa， Enterococcus faecalis， Monilia albicanas， and Bacillus subtilis as test microorganisms. The minimum inhibitory activity （MIC） of the endophyte with relatively highest antimicrobial activity was determined using two-fold broth micro-dilution method. All the endophytes showed a broad spectrum antimicrobial activity. Among the three endophytes， the strain MG-09 exhibited relatively highest antimicrobial activity with the zones of inhibition （ZOI） ranging from 14.15 to 24.07 mm and MIC ranging from 416.67 to 65.11 μg/ml. [Conclusions] The endophytic fungi colonized in M. glyptostroboides are capable of producing effective antimicrobial compounds which may be a potential source for drug discovery and biocide production.
　　Key words Endophytic fungi; Antimicrobial activity; Secondary metabolites; Metasequoia glyptostroboides
　　
　　Received： August 2， 2020  Accepted： October 7， 2020
　　Supported by the National Science Foundation of China （Grant No. 31970038）; the Science Public Welfare Fund Projects of Zhejiang Province （Grant No. 2017C32050）.
　　Yucheng QIAN （1997-）， female， P. R. China， master ， devoted to research about microbiology.
　　*Corresponding author. E-mail： sunliuxx@163.com; bxxbj2003@gmail.com.
　　 Endophytic fungi are microorganisms which commonly colonize the interior of the plants without causing diseases[1-2]. Instead， most of them are capable of protecting host plants from pathogens， insects as well as severe environment and enhancing the growth of the plants by producing various bioactive secondary metabolites[1，3]. Numerous studies indicate that these bioactive products have several advantages such as being abundant in natural world， environment-friendly， having highly efficient antibacterial activities， rich diversity and wide bacteriostasis spectrum[2]. Besides， some of them are considered as potential resources for novel natural products， which plays a significant role in modern agriculture and medicine[4-5]. Owing to these compounds， endophytic fungi with bioactive effects have attracted the attention of scientists who think the products can be an effective alternative to risky chemicals， such as some kinds of fertilizers， medicines， and pesticides[6-8]. For instance， the endophytic fungi isolated from wheat cultivars as Trichoderma hamatum， Penicillium sp. and Paecilomyces lilacinus have displayed great potential resistance against Pyrenophora tritici-repentis， Drechsler （anamorph Drechslera tritici-repentis） and Shoem （Dtr）， which cause the severe wheat disease， tan spot of wheat[9]. Fungal endophytes isolated from Eugenia jambolana have showed antimicrobial activities against Human pathogenic ATCC reference gram negative bacteria （Escherichia coli， Proteus mirabilis， Pseudomonas aeruginosa， Klebsiella pneumonia， Salmonella typhi， Shigella flexneri， Serratia marcescens）， gram-positive bacteria （Staphylococcus aureus， Enterococcus faecalis） and reference fungi （Candida albicans， Aspergillus niger）[10]. Thus， These findings demonstrate that endophytic fungi have a potential biocontrol effect not only on plant diseases， but also on human diseases. 　　Metasequoia glyptostroboides Hu & W. C. Cheng， a contemporary species of a genus recognized on the basis of fossil materials， also called as "living fossil"， is the only remaining representative of this genus and is regarded as a highly endangered species in earth[11]. Although there were three species in its genus， M. glyptostroboides is the only species remained from the last quaternary glaciers. Thus， M. glyptostroboides is regarded as an essential material for studying relict species[12-13]. Notably， in China， the tissues （like leaves and bark） of M. glyptostroboides are commonly used as traditional Chinese folk medicine which has significantly anti-microbial， analgesic and anti-inflammatory functions to treat dermatic diseases. It is confirmed that proanthocyanidins extracted from the bark of M. glyptostroboides have curative effect on ameliorate allergic contact dermatitis through decreasing T cells activation and Th1/Th17 specific cytokines production[14]. And the extractions of M. glyptostroboides can significantly inhibit CCl4-induced liver injury in mice as they have effective free radical removing and antioxidant activities[15].
　　Hitherto， few publications are available about the endophytes of M. glyptostroboides， especially endophytic fungi and bacterials[15]. And whether the medical functions of M. glyptostroboides is relative to its endophytic fungi is still unknown. In order to reveal the species and ecological distribution of endophytic fungi of M. glyptostroboides， twenty-five endophytic fungi have been isolated from the M. glyptostroboides in Xinyang， China， including five non-sporulated fungi and twenty sporulated fungi which belong to deuteromycetes. Among the isolates， Phomopsis sp.， Alternaria sp.， Colletotrichum sp. and Macrophoma sp. are considered as dominant fungi. Moreover， host habitat situations are considered as the most significant factor that affected the diversity and community structures of the endophytic fungi. In addition， the types of tissues where the endophytes colonize （such as leaves， twigs， bark and xylem） also influence the diversity and community structure. And their general colonization rate is 53.5%. The results demonstrate that the endophytic fungi of M. glyptostroboides have abundant biological diversity and various community structures[16]. Resent year， one endophytic fungal strain has been isolated from M. glyptostroboides， and it showed antagonistic activity against plant pathogenic fungi Fusarium proliferatum， Fusarium graminearum， Setosphaeria turcica， and Helminthosporium maydis[17]. It is necessary to exam whether endophytic fungi from M. glyptostroboides are capable of antagonism ability against human pathogenic bacteria. Thus， this study aims to isolate several endophytic fungi from the healthy leaves and bark of M. glyptostroboides， and sieve the strains with antimicrobial activity， and then to investigate the potential antimicrobial activity of the isolates， especially against human pathogenic bacteria. 　　Materials and Methods
　　Sample collection
　　Several branches and leaves samples were collected from healthy M. glyptostroboides trees in Wuxi Mey Blossom Garden （31°33′7.74″ N-120°13′21.54″ E）， Wuxi， Jiangsu， China. Samples were stored in sealing bags at 4 ℃ in an ice box during transport and processed immediately in the College of Life Sciences and Medicine， Zhejiang Sci-tech University （ZSTU）， Zhejiang， China.
　　Isolation of endophytic fungi
　　In order to estimate the endophytic fungi of the samples， the samples were gently washed under running tap water， followed by sterilizing the surface with 75% ethanol and rinsing with sterile distilled water. Samples were cut into segments after being dried on sterile filter paper. The segments were then put on potato dextrose agar （PDA） medium containing 100 mg/ml ampicillin and 50 mg/ml streptomycin. The plates were incubated at 30 ℃ for 30 d and observed periodically. All the single fungal colonies that could be observed were sub-cultured onto fresh PDA media till pure isolates were obtained[18-19].
　　Test microorganisms
　　For isolation and selection of endophytic fungi， two pathogenetic bacteria were used： E. coli ATCC8099 and S. aureus ATCC6538. And for disk diffusion assay and the determination of minimum inhibitory concentration （MIC）， four bacterial pathogenic strains to humans were used， among which were two gram negative strains （E. coli ATCC8099 and P. aeruginosa） and two gram positive strains （S. aureus ATCC6538and E. faecalis ATCC29212）. Besides， one fungal pathogenic strains to humans （M. albican ATCC10231） and one bacterial pathogenic strain to plants （B. subtilis ATCC6051） were used.
　　Screening of endophytic fungi with antimicrobial activity
　　Pathogenic bacteria E. coli and S. aureus were utilized as indicators to sieve the endophytes with antimicrobial ability.
　　Each isolated endophytic fungi was cultivated on the surface of a solid medium comprises of potato dextrose agar （PDA; containing potato 200 g/L， dextrose 20 g/L， agar 18 g/L， natural pH） at 30 ℃ for 7 d， until the lawn grew evenly on the surface. Each pathogenic bacteria was activated after being cultivated in the liquid medium consists of Luria-Bertani culture （LB; containing casein tryptone 10 g/L， yeast extract 5 g/L， NaCl 10 g/L， pH 7.0） at 37 ℃ for 24 h. Then， the bacterial suspension obtained was adjust to 0.6-0.8 OD， followed by inoculating and spreading the suspension into the prepared LB semi-solid medium at a volume ratio of 1∶10. Then， plugs （6 mm diameter） of growing culture were cut from the PDA plate and transferred to the surface of LB semi-solid medium with pathogenic bacteria. Then the plates with both bacteria and fungi were incubated at 30 ℃ for 24 h. The endophytic fungi showing antimicrobial activity were picked according to the appearance of any inhibition zones[20]. 　　Three endophytic fungi which showed stronger antimicrobial activity were selected for further study. The strains were stored in 50% glycerol tubes separately at -20 ℃ for further investigation.
　　Identification of endophytic fungi
　　For molecular analysis， the endophytic strains with stronger antimicrobial activity were cultivated in 50 ml of potato dextrose broth （PDB; containing potato 200 g/L， dextrose 20 g/L， natural pH）， and cultured in 250 ml flasks on a rotary shaker at 220 rpm， 28 ℃. After 3 dl， fungal samples were collected by sterile gauze， followed by washing in sterile water in order to remove debris. After washing， samples were blotted by pieces of sterile filter paper. The dried fungal samples were then fully ground in liquid nitrogen immediately. Every 20 mg of comminuted samples were added into a 1.5 ml eppendorf tube each. Rapid fungi genomic DNA isolation kit （Sangon Biotech） was used to extract fungal genomic DNA. Obtained DNA samples were sent to Sangon Biotech （Shanghai） Co.， Ltd. for PCR amplification， and the internal transcribed spacer （ITS） 1 and 4 regions of the isolates are primarily sequenced after PCR amplification. The sequences maintained were assembled using DNAMAN 8. According to the results of sequencing， the strains were identified and classified using blast against the NCBI nucleotide database and make a phylogenetic tree using MAGE 7.0[21].
　　The conservative calmodulin gene CamD and β-tubulin gene were then further sequenced. The strains were secondary identified and classified using blast against the NCBI nucleotide database， and a phylogenetic tree was made using MAGE 7.0. The interspecific differences among three isolates were analyzed[22].
　　For morphology analysis， the endophytic fungi were cultured on the plates containing PDA culture medium at 30 ℃ for 5 to 6 d， respectively. Morphology characteristics of the colony were observed under daylight conditions. Conidiophore and hyphae were maintained from the lawn and mounted in lactic acid. Microscopic observations were then performed under the microscope[23].
　　Fermentation and extraction of antimicrobial substances
　　Three mycelial agar plugs （6 mm of diameter） were cut from the 3-day-old culture of the endophytic fungi and added into 250 ml flacks containing 100 ml PDB culture medium. All the cultures were then inoculated on a rotary shaker at 220 rpm， 28 ℃ for one to two weeks. After liquid fermentation， the mycelia were filtered from the fermentation broth using sterile filter paper and ground in ethyl acetate （EtOAc） using mortar and pestle. The broken mycelia were filtered again and discarded， and the filter liquor was collected. Both the filtered fermentation broth and the filter liquor were gathered and mixed well in a 500 ml separatory funnel with 100 ml ethyl acetate （EtOAc）. After setting quietly for 24 h， the EtOAc extract at the lower level was separated from the broth using separatory funnel and filtered by Whatman # 1 filter paper. The broth was extracted three times as the method described before. All the EtOAc extract was concentrated in vacuo using a rotary evaporator （80 ℃， 100 rpm）， and then re-dissolved in the carbinol with a final concentration of 50 mg/ml[24-25]. 　　Disk diffusion assay
　　In order to investigate the antimicrobial activity of the extracts， disk diffusion method was used. Human pathogenic ATCC reference strains of bacteria and fungi were utilized to exam the antimicrobial activity of the extracts. M. albican ATCC10231 was used as fungal strain. S. aureus ATCC6538， E. faecalis ATCC29212 strains were used as gram positive bacterial strains， while E. coli ATCC8099 and P. aeruginosa was used as gram negative bacterial strains. And one bacterial pathogenic strain to plants （B. subtilis ATCC6051） was also used.
　　Each pathogenic microorganism was cultured in the liquid medium consists of LB culture at 37 ℃ for 24 h， respectively. The suspensions were obtained as inocula and adjust to 0.60-0.80 OD using ultraviolet spectrophotometer. Afterwards， the inoculum was homogenized in the prepared LB semi-solid medium at a ratio of 1∶10. The plates containing 10 ml LB solid medium and 10% pathogen solution （OD 0.60-0.80） were then prepared， followed by being divided into four equal regions. A piece of filter paper disks （6 mm of diameter） was then put on the surface of each plate at the center of the divided regions. Each disk was pipetted with 10 μl solution， which consisted of 50 mg/ml EtOAc extract， 100 μg/ml ampicillin， 100 μg/ml kanamycin， or carbinol， separately. Among the solutions， 100 μg/ml ampicillin and 100 μg/ml kanamycin were utilized as positive control， while ethly acetate as negative control[24-25]. Each treatment was repeated three times. The diameter （mm） of the clear inhibitory zone formed around the filter paper was measured， and the mean values were calculated and recorded.
　　Minimum inhibitory concentration （MIC） determination
　　The two-fold broth micro-dilution method， according to the standard practice for antimicrobial susceptibility test suggested by the NCCLS （National Committee for Clinical Laboratory Standards）， was used to determine the minimum inhibitory concentration （MIC） of the crude EtOAc extracts which presented the relatively highest antibacterial activity[4]. The tested crude extracts were diluted to the range of concentration between 5 mg/ml and 4.89 μg/ml. Mueller-Hinton broth （MHB; containing beef infusion solids 2.0 g/L， casein hydrolysate 17.5 g/L， starch 1.5 g/L， pH 7.4） and 96-well plates with round bottom wells were used to cultivate pathogenetic bacteria[24，26]. The turbidity of the bacterial suspensions was adjusted to 1.5×108 CFU/ml by standard of 0.5 McFarland. The inocula were then prepared by diluting the bacterial suspensions at the ratio of 1∶1 000. Medium with diluted extracts （100 μl） and prepared inoculum （100 μl） were isometrically added into 11 wells， respectively. And the twelfth well， set as the negative control， was only added with medium and inoculum without diluted extracts. The plates was then incubated at 30 ℃ for 24 h. The lowest concentration of the extract which displayed observable inhibition of microbial growth was recorded as the MIC. 　　Statistical analysis
　　Three replications were performed in all tests. The values maintained are presented as mean±standard deviation （n=3）.
　　Shapiro-Wilk test was used to determine statistical significance of the data using Rstudio. A w value closer to 1 meant that the data was closer to a random distribution. And a P value larger than 0.05 was considered to be statistically significant.
　　Yucheng QIAN et al. Three Novel Endophytic Fungi as Potential Candidates Producing Bioactive Metabolites Isolated from Metasequoia glyptostroboides
　　Results
　　Three fungal strains isolated from the branches and leaves samples of M. glyptostroboides trees， labeled as MG-01， MG-02， MG-09， were picked for their relatively bigger clear inhibitory zones in primary screening using two pathogenetic bacteria E. coli ATCC8099 and S. aureus ATCC6538 as test microorganisms， indicatING potential stronger antibacterial activies of isolates.
　　Fungal genomic DNA was extracted and the ITS 1 & 4 regions were sequenced successfully. The ITS1 and ITS4 regions were then assembled. The sequences maintained were submitted to the GenBank under the accession numbers： MK788343， MK788347， and MK788349. The conservative calmodulin gene CamD and β-tubulin gene were then further sequenced and also submitted to the GenBank undert the accession numbers： MT874980-MT874982 and MT898432-MT898434. The designed phylogenetic trees of ITS regions （Fig. 1） and the calmodulin gene CamD （Fig. 2） and β-tubulin gene （Fig. 3） indicated that three strains are different strains （Fig. 1-3）. The evolutionary distances among three strains were quite different from each other. According to the analysis with blast against the NCBI nucleotide database， all of three endophytic isolates belong to Penicillium sp. （Fig. 1）. Sequence of MG-01 had about 99% identity to Penicillium toxicarium， as well as Penicillium citreonigrum. Sequence of MG-09 had 98% to 99% identity to P. toxicarium. And sequence of MG-02 had 92% to 96% identity to P. toxicarium， indicating that the strain might belong to a new species.
　　
　　 According to morphology analysis， the characteristics of the colony and conidiophore were different among the three isolates. The colony characteristics of the strains MG-01， MG-02 and MG-09 are shown in Fig. 4[23]. Notably， the strain MG-09 was able to produce the yellow pigments during the growth period which could be extracted using organic solvents. It evidently showed that MG-09 is a quite different strain comparing with MG-01 and MG-02. 　　The temporary mounts of the conidiophores of the strains were prepared and observed under microscope （Fig. 5）. The conidiophores of the three endophytic fungi were "paintbrush" arrangement without vesicle， and the septate hyphae of the three isolates could be observed clearly[27]. These characteristics evidently indicated that the three isolated strains belong to Penicillium sp.
　　
　　 According to the molecular and morphology analysis， the three strains are different strains belonging to Penicillium sp. The strain MG-01 were primarily identified as P. toxicarium or P. citreonigrum， and the strain MG-02 and MG-09 were primarily identified as P. toxicarium.
　　The crude EtOAc （ethyl acetate） extracts of three endophytic fungi were extracted from PDB fermentation broth and re-dissolved in methyl alcohol at a concentration of 50 mg/ml. The crude extracts were then subjected to the disk diffusion assay in order to determine the antimicrobial activity of the isolates in proportion to the diameter of the zones of inhibition （ZOI）. All the isolated endophytic fungi displayed broad spectrum antimicrobial activity as they inhibited both bacterial （gram negative and positive） and fungal pathogenic strains （Table 1）. The ZOI produced by three endophytic fungi ranged from 7.66 to 24.07 mm. Among three isolated endophytic fungi， the strain MG-09 showed the best antimicrobial activity as it inhibited most of the test microorganisms except P. aeruginosa with the biggest ZOI ranging from 14.15 to 24.07 mm， which evidently bigger than the positive control 100 μg/ml ampicillin （6.95 to 15.16 mm） and 100 μg/ml kanamycin （11.48 to 16.87 mm）. Among the test pathogenic microorganisms， E. coli and M. albican were inhibited by all the isolated fungi while E. faecalis was only inhibited by the strain MG-09， and no isolated fungi could display a visible inhibitory action to P. aeruginosa （Fig. 6）. Moreover， both the strain MG-01 and MG-02 showed relatively highest antimicrobial activity against S. aureus， while the strain MG-09 inhibited M. albican mostly.
　　 Broth microdilution assay was used to determine the MIC of the crude EtOAc （ethyl acetate） extract extracted from the strain MG-09 as it showed relatively highest antimicrobial activity among the three isolated endophytic fungi （Table 2）. The results exhibited that the MIC of the ethyl acetate extract of the strain MG-09 ranges from 65.11 to 416.67 μg/ml. And the lowest MIC was observed in M. albican among five test pathogenic microorganisms， which was about 65.11 μg/ml. This result was in agreement with the result of the disk diffusion assay. 　　Discussion
　　M. glyptostroboides as a relict species remained in earth， is also regarded as a kind of traditional folk medicine in China， which has effective antimicrobial， analgesic， antioxidant and anti-inflammatory activity to treat diseases[14-15]. In previous studies， one endophytic fungal strain was isolated from M. glyptostroboides， which had showed antagonistic activity against plant pathogenic fungi F. proliferatum， F. graminearum， S. turcica， and H. maydis[17]. In this study， three endophytic fungi isolated from M. glyptostroboides had showed visible antimicrobial activity against human pathogenic bacteria （S. aureus， E. coli， E. faecalis）， human pathogenic fungi （M. albican） and plant pathogenic bacteria （B. subtilis）. This observation demonstrated that the endophytic fungi colonized in M. glyptostroboides had potential broad spectrum antimicrobial activity. And there may be a relation between the antimicrobial activity of M. glyptostroboides and the antimicrobial activity of its endophytic fungi， which needs to be further studied.
　　Three endophytic fungi isolated in this study were identified as Penicillium sp. Studies about the biological activity of Penicillium sp. had been reported extensively. Penicillium sp. had been reported to have antimicrobial activity against various kind of pathogenic microorganisms， such as Rhizoctonia solani， Fusarium oxysporium， Clanerela cinyulate， Phytophthora parasitica and Botrytis cirerea[28-29]. For instance， an important maize disease， banded leaf and sheath blight （BLSB）， were reported to be controlled by Penicillium sp. Compared with the negative control， treating with Penicillium sp. could sharply decrease the intensity of disease from 28% to 5%[30]. Penicillium sp. also displayed an inhibitory action against Phytophthora capsici （Leonian） which caused the pepper （Capsicum annuum） wilting disease leading to the losses of fruit yield from 10 to 100 %[31]. It demonstrates that Penicillium sp. can be a potential source for biocide production being applied in agricultural region.
　　Besides， it had been reported that Penicillium herquei displayed antimicrobial activity against pathogenic bacteria （S. aureus， B. subtilis， P. aeruginosa and Penicillium madriti）， and pathogenic fungi （Trichoderma viride， A. niger， Aspergillus flavus， and Alternaria alternata）. And the extracts of Penicillium herquei inhibited the tumor cell line MCF-7 effectively[32]. A active compound extracted from the strain Penicillium sp. HT-28 showed broad spectrum antimicrobial activity and was effective against methicillin-resistant S. aureus （MRSA）[33]. These studies suggest that the bioactive compounds extracted from Penicillium sp. can be a potential source for drug discovery， which may be an alternative for the drugs which has been resisted by some pathogenic microorganisms. 　　Conclusions
　　This research had demonstrated that the endophytic fungi colonized in M. glyptostroboides are capable of producing effective antimicrobial compounds which may be a potential source for drug discovery and biocide production. Among three endophytes， Penicillium sp. strain MG-09 displayed relatively highest antimicrobial activity. Further studies are needed on the purification and identification of the antimicrobial compounds produced by Penicillium sp. strain MG-09， and optimization of cultivation conditions for the production of bioactive substances. And the further identification of Penicillium sp. strain MG-09 is also needed.
　　References
　　[1] ANCHEEVA E， DALETOS G， PROKSCH P. Bioactive secondary metabolites from endophytic fungi[J]. Curr Med Chem， 2020， 27（11）： 1836-1854.
　　[2] ALVIN A， MILLER KI， NEILAN BA. Exploring the potential of endophytes from medicinal plants as sources of antimycobacterial compounds[J]. Microbiol Res， 2014， 169（7-8）： 483-495.
　　[3] RODRIGUEZ RJ， WHITE JR JF， ARNOLD AE， et al. Fungal endophytes： diversity and functional roles[J]. New Phytol， 2019， 182（2）： 314-330.
　　[4] RATNAWEERA PB， SILVA EDD， WILLIAMS DE， et al. Antimicrobial activities of endophytic fungi obtained from the arid zone invasive plant Opuntia dillenii and the isolation of equisetin， from endophytic Fusarium sp.[J]. Bmc Complementary & Alternative Medicine， 2015， 15（1）： 220-227.
　　[5] YU H， ZHANG L， LI L， et al. Recent developments and future prospects of antimicrobial metabolites produced by endophytes[J]. Microbiol Res， 2010， 165（6）： 437-449.
　　[6] NOHA MK， FATMA FAM， SOAD AEZ. Endophytic fungi from the medicinal herb Euphorbia geniculata as a potential source for bioactive metabolites[J]. Archives of Microbiology， 2020（202）： 247-255.
　　[7] WHITE JF， KINGSLEY KL， ZHANG QW， et al. Review： Endophytic microbes and their potential applications in crop management[J]. Pest Manag Sci， 2019， 75（10）： 2558-2565.
　　[8] STROBEL G， DAISY B. Bioprospecting for microbial endophytes and their natural products[J]. Microbiol Mol Biol R， 2003， 67（4）： 491-502.
　　[9] LARRAN S， SIMóN MR， MORENO MV， et al. Endophytes from wheat as biocontrol agents against tan spot disease[J]. Biol Control， 2016（92）： 17-23.
　　[10] YADAV A. Evaluation of in vitro antimicrobial potential of endophytic fungi isolated from Eugenia jambolana Lam[J]. Int J Pharm Pharm Sci， 2014， 5（6）： 208-211.
　　[11] JUVIK OJ， NGUYEN XHT， ANDERSEN HL， et al. Growing with dinosaurs： natural products from the Cretaceous relict Metasequoia glyptostroboides Hu & Cheng-a molecular reservoir from the ancient world with potential in modern medicine[J]. Phytochem Rev， 2016（15）： 161-195. 　　[12] HE Z， LI J， CAI Q， et al. Cytogenetic studies on Metasequoia glyptostroboides， a living fossil species[J]. Genetica， 2014， 122（3）： 269-276.
　　[13] MA J. The chronology of the "living fossil" Metasequoia glyptostroboides （Taxodiaceae）： a review （1943–2003）[J]. Harvard Papers in Botany， 2003， 1（8）： 9-18.
　　[14] CHEN F， YE X， YANG Y， et al. Proanthocyanins from the bark of Metasequoia glyptostroboides ameliorate allergic contact dermatitis through directly inhibiting T cells activation and Th1/Th17 responses[J]. Phytomedicine， 2015， 22（4）： 510-515.
　　[15] CHEN F， ZHANG L， ZONG S， et al. Antioxidant capacity and proanthocyanidin composition of the bark of Metasequoia glyptostroboides[J]. Evid-Based Compl Alt， 2014： 1-11.
　　[16] LU D， LU S， PAN Z， et al. Biological diversity and ecological distribution of endophytic fungi in Metasequoia glyptostroboides[J]. Journal of Nanjing Forestry University， 2013， 37（6）： 33-36.
　　[17] ZHANG Y， BAI J， XU K， et al. Isolation and screening of endophytic fungi with antimicrobial activity in Metasequoia[J]. Hubei Agricultural Sciences， 2018， 11（57）： 36-41.
　　[18] IDRIS A， AL-TAHIR I， IDRIS E. Antibacterial activity of endophytic fungi extracts from the medicinal plant Kigelia Africana[J]. Egypt Acad J Biolog Sci， 2013， 5（1）： 1-9.
　　[19] PILI NN， FRAN A SC， KYNDT T， et al. Analysis of fungal endophytes associated with rice roots from irrigated and upland ecosystems in Kenya[J]. Plant & Soil， 2016， 405（1-2）： 371-380.
　　[20] Dos SIP， da SLC， da SMV， et al. Antibacterial activity of endophytic fungi from leaves of Indigofera suffruticosa Miller （Fabaceae）[J]. Front Microbiol， 2015（6）： 350.
　　[21] QADRI M， JOHRI S， SHAH BA， et al. Identification and bioactive potential of endophytic fungi isolated from selected plants of the Western Himalayas[J]. SpringerPlus， 2013， 2（1）： 8.
　　[22] ZHENG H， ZHUANG W. Hymenoscyphus albidoides sp. nov. and H. pseudoalbidus from China[J]. Mycological Progress， 2013， 13（3）： 625-638.
　　[23] Lu Y， Chen C， Chen H， et al. Isolation and identification of endophytic fungi from Actinidia macrosperma and investigation of their bioactivities[J]. eCAM， 2011， 2012（1）： 1-8.
　　[24] MARCELLANO JP， COLLANTO AS， FUENTES RG. Antibacterial activity of endophytic fungi isolated from the bark of Cinnamomum mercadoi[J]. Pharmacogn J， 2017， 9（3）： 405-409.
　　[25] XING YM， CHEN J， CUI JL. Antimicrobial activity and biodiversity of endophytic fungi in Dendrobium  devonianum and Dendrobium thyrsiflorum from Vietman[J]. Current Microbiology， 2011， 62（4）： 1218-1224. 　　[26] RAVNIKAR M， TERCELJ M， JANES D， et al. Antimicrobial activities of endophytic fungi isolated from conifer needles[J]. Academic Journals， 2015， 14（10）： 867-871.
　　[27] YUAN L， LU L， LI S. Laboratory experiments in microbiology[M]. Chemical Industry Press， 2014， Beijing pp： 83-85.
　　[28] AN J， SHEN R. Isolation， screening and biocontrol mechanism of antagonistic Penicillium against Rhizoctonia solani[J]. Acta Agriculturae Boreali-occidentalis Sinica， 2015， 24（12）： 159-163.
　　[29] WANG H， SHI J， LUO Z， et al. Isolation and identification of Penicillium strain QMYCS-2 and its effect on tobacco black shank[J]. Tobacco Science & Technology， 2016， 49（2）： 14-20.
　　[30] KUSUMA A， WIDIASTUTI A， PRIYATMOJO A. Induction of resistance using Trichoderma spp. and Penicillium sp. against banded leaf and sheath blight （BLSB） caused by Rhizoctonia solani in maize[J]. Springer， 2017， Cham pp： 3-11.
　　[31] JIMNEZ-CAMARGO A， VALADEZ-MOCTEZUMA E， LOZOYA-SALDA A H. Antagonism by Penicillium sp. against Phytophthora capsici （Leonian）[J]. Rev Fitotec Mex， 2018， 41（2）： 137-148.
　　[32] LI H， CHEN Y， WANG L， et al. Study on antimicrobial and antitumor activities of marine-derived fungus Penicillium herquei FS83[J]. Biotechnology Bulletin， 2013（1）： 151-155.
　　[33] KAUR H， ARORA DS， SHARMA V. Isolation， purification， and characterization of antimicrobial compound 6-[1，2-dimethyl-6-（2-methyl-allyloxy）-hexyl]-3-（2-methoxy-phenyl）-chromen-4-one from Penicillium sp. HT-28[J]. Appl Biochem Biotechnol， 2014（173）： 1963-1976.
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