| Plant endophytes are becoming very important in developing pesticides as some of themcan excrete novel biological active substances.In this research,endophytic fungus andactinomyces were isolated from 4 Celastraceae plants and the activities of metabolite fromthese isolates were assayed for screening valuable strains in pesticide study.Antibiotic activityand components of metabolite excreted in three strains were studied,their fermentationconditions were optimized,and the taxonomical status of these endophytes were distinguishedin the study.The main results are as follows:The slices of sample and tissue homogenate were used for isolating endophyte and 161strains of endophytic fungi and 28 strains of endophytic actinomyces were isolated from 4Celastraceae plants.Metabolites from endophytic fungus Hd3,2QR1,1F8 and endophyticactinomycete a4,a5,c4 and YDG17 showed antibiotic activity to some target bacteria andpathogenic fungi.Metabolites from Hd3 and 2QR1 presented insecticidal activity to targetinsect.All strains above mentioned could produce active products stably.Five endophytic actinomyces which didn't exhibit antibiotic activity and oneactinomycete exhibited weak antibiotic activity were induced with the streptomycin,and 301strains of resistant mutation were screened.Metabolites from YDG05-44 and YDG09-32showed antibiotic activity to target bacteria and pathogenic fungi,which were different fromtheir parent strains numbered YDG05 and YDG09 obviously.The activity expression inYDG05-44 and YDG09-32 were both stably.The components of liquid medium for stain YDG17,YDG09-32 and Hd3 were studied byresponse surface methodology and their fermentation conditions including culturetemperature,culture period,the pH value of medium,inoculate concentration and the volumeof liquid medium of strains were optimized.The studied results of strain YDG 17 showed thatthe suitable liquid medium was composed of 32.00 g·L-1 glucose,28.77 g·L-1 millet,3.00 g·L-1peptone and distilled water,and the suitable cultivated condition was 32℃,120h,6×107cfu·mL-1and 60mL liquid medium in 250mL flask,pH 7.The studied results of strainYDG09-32 showed that the suitable liquid medium was composed of 21.06 g·L-1 lactose,20.82 g·L-1 millet,4.72 g·L-1 beef extract,0.38 g·L-1 MgSO4·7H2O and distilled water,and the suitable cultivated condition was 29℃,96h,6×106cfu·mL-1and 80mL liquid medium in250mL flask,pH 7～9.The studied results of strain Hd3 showed that the suitable liquidmedium was composed of 24.71 g/L glucose,1.08 g/L MgSO4·7H2O,244.17 g/L potato anddistilled water;and the suitable cultivated condition was 31℃,9d,9×103cfu·mL-1 and 90mLliquid medium in 250mL flask,pH 6～7.The bioactivity and active component of metabolites from strain YDG17 were studied.Fermentation filtrate of strain YDG17 exhibited antibiotic activity against Bacillus cereus,Bacillus subtilis,Staphylococcus aureus,Escherichia coli,Pseudomonas aeruginosa and thediameters of inhibition zone were 23.5mm,23.0mm,15.3mm,11.3mm and 14.0mm,respectively.The mycelia growth and spore germination of target pathogenic fungus could beinhibited in vitro by fermentation filtrate of YDG17,and the value of EC50 inhibiting myceliagrowth against Fusarium graminearum,Botrytis cinerea and Alternaria solani were 259.98mg·L-1,336.13 mg·L-1 and 100.72 mg·L-1,respectively,the value of EC50 inhibiting sporegermination against B.cinerea was 87.84mg·L-1.The results also showed that the protectiveefficacy and curative efficacy of the fermentation filtrate against B.cinerea were 97.62% and79.23% on cucumber leaves,and were 71.34% and 64.23% on potted cucumber plants,respectively.Compared with the curves of classical antibiotics in Doskochilova system,theactive components in the fermentation filtrate were identified as alkaline and water solublecompounds.The active fraction numbered YA isolated from the broth of YDG17 by cationresin chromatography and the diameter of inhibition zone was 26.7mm against B.subtilis.Five compounds were identified by ESI-MS/MS as Streptothricin D,Streptothricin E-acid,Streptothricin F,N-acetyl streptothricin C and N-acetyl streptothricin C.The bioactivity and active component of metabolites from strain YDG09-32 were studied.Both fermentation filtrate and extract of mycelia from strain YDG09-32 exhibited obviouslyantibiotic activity to target bacteria and pathogenic fungi,and the active components wereactinomycin antibiotics by comparing with the curves of classical antibiotics in Doskochilovasystem.The active compounds were stable to light,heat,acidic and basic environment inwhich the value of pH was between 5 and 11.Seven fractions numbered Bh1,Bh2,Bh3,Bh4,Bh5 and Bh6 were isolated from fermentation filtrate of YDG09-32 by Macro-porousAdsorption Resin,Silica Gel Column Chromatograph and pre-TLC,and only Bh1,Bh5 andBh6 exhibited antibiotic activity against B.subtilis.All fractions weren't identified because oftheir low purity.The bioactivity and active component of metabolites from strain Hd3 were studied.Thebioassay results of strain Hd3's metabolites showed that the menthol extract of myceliumexhibited insecticidal activity and that the ethyl acetate extract of the fermentation broths exhibited antibiotic activities to target bacteria and pathogenic fungi.At the concentration of1000mg/L,the morality of menthol extract of mycelium was 100% against 3rd instars larva ofMythimna separate.The EC50 value of ethyl acetate extract of the fermentation broths againstB.cinerea,F.vasinfectum,Bipolaris sorokiniana,G.cingulata,F.graminearum and C.lunatain inhibiting the mycelia growth were 138.3,166.0,77.7,74.9,75.6 and 130.9 mg/L,respectively,and the EC50 value against F.vasinfectum,B.cinerea,B.sorokiniana and C.lunata in inhibiting spore germinations were 184.4,102.8,154.5 and 79.7 mg·L-1 respectively.At the concentration of 500 mg/L,the inhibition zone diameter of ethyl acetate extract of thefermentation broths of the strain Hd3 was 23.0,18.5 and 16.0mm against B.subtilis,B.cereusand S.aureus,respectively.The results of pot test indicated that ethyl acetate extract offermentation broths had 61.9% protective efficacy and 81.6% curative efficacy againstBlumeria graminis at the concentration of 2000 mg·L-1.By bioassay-guided,an insecticidal compound (H4.5) was isolated from the ethyl acetateextracts of the mycelium of strain Hd3 by Silica Gel Column Chromatograph and pre-HPLC.The bioassay results showed H4.5 exhibited strong stomach poison against 3rd instars larvaeof Mythimna separata Walker,and the value of LD50 was 3.78μg/larvae.Eight compoundsnumbered H03～H09 and H20 were obtained from the ethyl extract of fermentation broth bythe same methods as H4.5.The bioassay results showed that H03,H04 and H20 exhibitedantibiotic activity to B.subtilis and C.lunata.On the basis of spectral data (1HNMR,13CNMR,MS),the structure of H4.5,H03,H04,H05,H07,H08 and H20 was identified as2,3-dimethoxy-5-methyl phenol,geodin,chlorotrypacidin,methyl asterrate,methyldichloroasterrate,methyl chloroasterrate,asterric acid,respectively.The compound 2,3-dimethoxy-5-methyl phenol was isolated from microbes for the first time,and its activitywas reported firstly.The antibiotic activity of compound asterric acid to C.lunata was alsoreported firstly.Endophytic strain YDG17,YDG09 and Hd3 were identified by morphological,physiological and molecular biological methods.The strain YDG17 and YDG 09 were bothStreptomyces sp.and strain Hd3 was Aspergillus sp..The 16SrDNA sequences showed thatthe homology between YDG17 and S.vinaceusdrappuss was 99%,and the morphological,cultural,physiological or biochemical characteristics were also similar with those of themodel strain,so the strain YDG17 was identified as Streptomyces vinaceusdrappuss.Allcharacteristics of YDG09 were same to S.rubrolavendulae except for unutilizing arabinoseand mannitol,utilizing rhamnose and hard growth on Krasilnikov's No.1 synthetic medium,and the sequences homology of 16SrDNA between them was 99%.So the strain YDG09 maybe a variant of S.rubrolavendulae,and was named Streptomyces rubrolavendulae var euonymus.Morphological characteristics and ITS sequences of strain Hd3 were same to theAspergillusflavipes,so Hd3 was identified as Aspergillusflavipes. |
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