【作者】 杨春平；

【导师】 吴文君；

【作者基本信息】 西北农林科技大学 ， 农药学， 2008， 博士

【摘要】 植物内生菌作为筛选农用抗生素的一类资源微生物，在农药的研究与开发中越来越受到重视。为了开发卫矛科植物内生菌资源，以期发现新的具有农药研究价值的微生物，本论文对4种卫矛科植物内生菌进行了研究，主要涉及菌种的分离、筛选、菌株鉴定、菌株诱变选育、代谢产物活性及活性成分分离以及发酵条件优化等的研究，取得了如下结果：（1）分别采用组织块培养法和匀浆法从4种新鲜卫矛科植物中分离到161株内生真菌和28株内生放线菌，通过杀虫活性、抑菌活性及遗传稳定性实验，从中筛选到能产生抑菌活性成分的内生真菌3株（Hd3、2QR1和1F8）和内生放线菌4株（a4、a5、c4和YDG17）。其中，菌株Hd3和2QR1的代谢产物也具有杀虫活性。（2）利用引入链霉素耐药性，分别对5株无抑菌活性和1株弱抑菌活性的卫矛科植物内生放线菌进行诱突，共获得链霉素变异株301株。对301株变异菌株发酵产物以枯草芽孢杆菌为指示菌进行活性筛选，获得具有显著抑菌活性且遗传稳定的突变株2株，即YDG05-44和YDG09-32。室内生物测定结果表明，YDG05-44和YDG09-32菌株的发酵产物对其它几种细菌及植物病原真菌也具有显著的抑菌活性。（3）对YDG17、YDG09-32和Hd3菌株的发酵条件进行了优化研究，确定了最佳培养基组成及培养条件。响应面分析及正交实验结果表明，YDG17菌株发酵的摇瓶培养基配方为：葡萄糖32．00 g／L，小米28．77 g／L，蛋白胨3．00 g／L。发酵条件为：pH 7，250mL三角瓶装60mL发酵液，接入6×10⁷cfu／mL菌量，32℃培养120h。YDG09-32菌株发酵的摇瓶培养基配方为：乳糖21．06 g／L，小米20．82 g／L，牛肉膏4．72 g／L，MgSO₄·7H₂O 0．38g／L。发酵条件为：pH 7～9，250mL三角瓶装80mL发酵液，接入6×10⁶cfu／mL菌量，29℃培养96h。Hd3菌株的摇瓶培养基配方为：葡萄糖24．71g／L，MgSO₄·7H₂O 1．08 g／L，土豆244．17 g／L。发酵条件为：pH 6～7，250mL三角瓶装90mL发酵液，接入9×10³cfu／mL菌量，31℃培养9d。（4）研究了YDG17菌株的代谢产物。室内生测结果表明，YDG17菌株的发酵液对几种供试细菌和病原真菌均有一定的抑制作用。对枯草芽孢杆菌（Bacillus subtills）、蜡状芽孢杆菌（Bacillus cereus）、金黄色葡萄球菌（Staphylococcus aureus）、大肠杆菌（Escherichis coli）、绿脓杆菌（Pseudomonas aeruginosa）的抑菌圈直径分别为27．0，27．0，15．3，11．3，14．0mm。对小麦赤霉病菌（Fusarium graminearum）、番茄灰霉病菌（Botrytis cinerea）、番茄早疫病菌（Alternaria solani）、西瓜枯萎病菌（Fusariumoxysporum f．sp．niveum）和桃腐烂病菌（Cytospora leucostoma）抑制菌丝生长的EC₅₀（以发酵液中的干物质计）值分别为259．98、336．13、100．72、470．11和198．58mg／L；对小麦赤霉病菌（F．graminearum）、西瓜枯萎病菌（F．oxysporum f．sp．niveum）和番茄灰霉病菌（B．cinerea）抑制孢子萌发的EC₅₀值为分别为151．67、220．69和87．84mg／L。离体子叶法测定结果表明，发酵液原液对番茄灰霉病的保护效果为97．62％，治疗效果为79．63％。盆栽试验结果表明，发酵液原液对番茄灰霉病的保护效果为71．34％，治疗效果为64．23％。通过捷克八溶剂系统纸色谱测定YDG17菌株发酵液中主要活性成分为碱性水溶性抗生素。采用离子交换法，通过抑菌活性追踪法，分离得到YDG17菌株发酵液的主要抑菌活性组分YA，ESI-MS／MS对YA化学成分进行分析，结果表明YA中的主要活性成分为链丝菌素类化合物，通过MS／MS谱图和母离子碎片峰信息，以及与标准化合物谱图进行对比，鉴定其中5个化合物为N-乙酰化链丝菌素D、N-乙酰化链丝菌素C、链丝菌素D、链里定酸-链丝菌素E和链丝菌素F。（5）研究了YDG09-32菌株的代谢产物。离体活性测定结果表明，YDG09-32菌株的发酵滤液和菌丝体中均有含有抑菌活性成分，不仅对供试细菌有一定的抑制作用，同时对小麦赤霉病菌（F．graminearum）、玉米弯孢菌（Curvularia lunta）、番茄灰霉病菌（B．cinerea）和苹果炭疽病菌（Colletotrichum gloeosporioides）等植物病原真菌有较强的抑制活性。通过Doskocilova八溶剂系统分析，判断YDGA09-32菌株发酵滤液和菌丝体提取物中的抑菌活性成分为同一类物质，即放线菌素类抗生素。YDG09-32菌株代谢的抑菌活性成分对光、热、酸和碱均有较好的稳定性。发酵产物在pH 5～11之间处理3h，或在90℃处理30min以及日光照射10d后，对抑菌活性均无明显影响。采用大孔吸附树脂吸附、硅胶柱层析及薄层制备等技术对YDG093-32菌株发酵滤液中抑菌成分进行分离纯化，得到Bh1～Bh7 7个组分，生测结果表明，仅组分Bh1、Bh5和Bh6对枯草芽孢杆菌具有较好的抑菌活性。Bh1～Bh7组分由于纯度不够，未能进行结构鉴定。（6）研究了Hd3菌株的代谢产物。分别对Hd3菌株菌丝体甲醇、乙酸乙酯和石油醚提取物及发酵滤液乙酸乙酯萃提物进行了杀虫、抑菌活性研究。结果表明，Hd3菌株的杀虫活性物质主要存在于菌丝体乙酸乙酯提取物中，在1000mg／L时，对粘虫3龄幼虫的24h死亡率为100％；抑菌活性物质主要在发酵液中，其乙酸乙酯萃取物对小麦根腐病菌（Bipolaris sorokiniana）、苹果炭疽病菌（C．gloeosporioides）、小麦赤霉病菌（F．graminearum）、番茄灰霉病菌（B．cinerea）、棉花枯萎病菌（Fusarium oxysporum f．sp．vasinfectum）和玉米弯孢菌（C．lunta）抑制菌丝生长的EC₅₀值分别为77．7，74．9、75．6、138．3、166．0和130．9 mg／L，对棉花枯萎病菌（F．oxysporumf．sp．vasinfectum）、番茄灰霉病菌（B．cinerea）、小麦根腐病菌（B．sorokiniana）和玉米弯孢菌（C．lunta）抑制孢子萌发EC₅₀值分别为184．4，102．8，154．5和79．7mg／L；盆栽试验结果表明，Hd3菌株发酵滤液乙酸乙酯萃取物在浓度为2000mg／L时，对小麦白粉病的保护和治疗效果分别为61．9％和81．6％。对Hd3菌株菌丝体乙酸乙酯提取物中的杀虫活性成分进行分离，得到一个纯化合物H4．5，经波谱分析，鉴定为2，3-二甲氧基-5-甲基苯酚，该化合物对3龄粘虫（平均体重3．75mg）的LD₅₀为3．78μg／头。通过活性追踪，采用硅胶柱层析、HPLC制备对菌株发酵滤液乙酸乙酯萃提物中抑菌活性成分进行分离，得到H03～H09，H20 8个化合物，生测结果表明，化合物H03、H04、H20具有较强的抑菌活性。经波谱分析，化合物H03、H04、H05、H07、H08和H20分别鉴定为geodin、chlorotrypacidin、methyl asterrate、methyl dichloroasterrate、methyl chloroasterrate和asterric acid，均为首次从内生黄柄曲霉代谢物中分离到的化合物。（7）通过对YDG17、YDG09和Hd3菌株形态学及分子生物学鉴定，确定Hd3菌株为曲霉属真菌：YDG09和YDG17菌株均为链霉菌属放线菌。YDG17菌株与菌株S．vinaceusdrappuss的16SrDNA同源性为99％，同时，菌株在形态特征、培养特征、生理生化特征等方面与模式菌株也比较相似。因此，将YDG17菌株鉴定为Streptomyces vinaceusdrappuss。YDG09菌株与模式菌株S．rubrolavendulae的16SrDNA同源性为99％。除了YDG09菌株不利用阿拉伯糖、甘露醇而利用鼠李糖及在克氏一号培养基上难于生长与S．rubrolavendulae菌株存在差异外，在生理生化及形态特征上两株菌均相似。因此，建议将YDG09菌株定为S．rubrolavendulae菌株的一个变种（Streptomycesrubrola vendulae var euonymus）。Hd3菌株与模式菌株Aspergillus flavipes的ITS区序列同源性为100％，在形态特征等方面两株菌也相似，因此，鉴定Hd3菌株为黄柄曲霉Aspergillus flavipes。更多还原

【Abstract】 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 3^rd 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 3^rd 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.更多还原