【作者】 任莉；

【导师】 李国庆；

【作者基本信息】 华中农业大学 ， 植物病理学， 2008， 博士

【摘要】 由核盘菌[Sclerotinia sclerotiorum（Lib.）de Bary]引起的油菜菌核病是一种世界性分布的重要病害。重寄生真菌盾壳霉（Coniothyrium minitans）是核盘菌的一种具有广阔应用前景的生防菌。前人对盾壳霉防治核盘茵的机制和防病潜力已进行了深入研究。这些机制主要侧重于盾壳霉对核盘菌的重寄生作用,竞争作用和抗真菌作用,而对其降解核盘菌产生的草酸毒素及其产生的一系列生防效应则未见报道。草酸（Oxalic acid,OA）是核盘菌产生的一种毒素,在核盘菌的致病过程中起着决定因子的作用。盾壳霉可以寄生核盘菌的菌落以及核盘菌引起的病斑,可见盾壳霉可以抵抗一定浓度的草酸。这种抗性的本质是什么?本研究从盾壳霉降解草酸的角度出发,对盾壳霉降解草酸的能力和影响因子,以及由此产生的生防效应进行了研究,旨在发掘盾壳霉防治核盘菌的新因子,以及增强盾壳霉防治油菜菌核病的效果及稳定性。取得了如下研究结果:首先,对盾壳霉降解草酸的能力进行了定量分析。结果表明:在含有0.8～32mmol/L草酸的改良马铃薯葡萄糖培养液（mPDB）中培养盾壳霉15d（20℃）,培养液中的草酸含量下降85.9%～91.7%。这些盾壳霉培养物滤液的pH值从酸性水平（pH2.9～6.3）升高至弱碱性水平（pH7.7～8.6）。此外,从这些盾壳霉培养物滤液中还检测出氨。产生氨可能是引起培养液pH值升高的另一个原因。而当mPDB中的草酸浓度达到40和80mmol/L时,盾壳霉不能生长,培养液中的草酸含量没有发生明显下降。在另一个试验中,以含有16mmol/L草酸的mPDB培养盾壳霉（20℃,15d）,分别测定培养液和盾壳霉菌丝中的草酸含量,结果表明:培养液中草酸含量下降88.0%,菌丝提取物中没有检测到草酸的存在。由此证明盾壳霉具有降解草酸的能力。将油菜幼苗的根浸泡在盾壳霉培养物滤液中,测定其中的毒性。结果表明:盾壳霉在含有草酸（8、16、24、32mmol/L）的培养液中生长后（20℃,15d）,滤液的毒性显著降低,进一步证实盾壳霉具有解除草酸毒害作用的能力。进一步定量分析表明:盾壳霉降解草酸与盾壳霉菌丝有关。其证据包括:（1）盾壳霉培养物（mPDB+16mmol/L OA,20℃,9d）滤液（无菌丝细胞）对草酸（8mmol/L）没有降解作用,这说明滤液中不存在降解草酸的活性物质或活性物质的含量很低:（2）在盾壳霉培养物（mPDB+16mmol/L OA,20℃,9d）中继续添加草酸（16mmol/L）,再分成两种处理,一种处理是补充营养物质（0.1%KNO₃和0.5%葡萄糖,w/v）,另一种处理不添加任何营养物质,继续振荡培养（20℃,200r/min,9d）,结果表明:补充营养物质处理的草酸降解较不加营养物质处理明显提前。这说明盾壳霉菌丝生长与降解草酸是同步进行的。从盾壳霉菌丝中检测到草酸脱羧酶活性。其次,对影响盾壳霉降解草酸的因子进行了研究。结果表明:酸性环境对盾壳霉降解草酸根离子十分重要。在mPDB培养液中添加草酸钠（16mmol/L）后,培养液的pH值为6.4,添加草酸铵（16mmol/L）后,pH值为6.2。盾壳霉在这两种培养液中生长后对草酸根离子的降解率低于5%。在含草酸钠或草酸铵的培养液中添加草酸（16mmol/L）后,培养液的pH值下降至3.5～3.6。盾壳霉在这两种培养液中生长后,对草酸根离子的降解率上升至14.1%（草酸钠处理）或36.1%（草酸铵处理）。在含草酸钠或草酸铵的培养液中添加盐酸（0.2%,v/v）后,培养液的pH值下降至2.8～2.9。盾壳霉在这两种培养液中生长后,对草酸根离子的降解率上升至63.0%（草酸钠处理）或89.5%（草酸铵处理）。另一方面,用NaOH（1mol/L）调节含草酸（8、16、24、32mmol/L）的mPDB至pH7.0时,盾壳霉生长后降解草酸的能力显著降低。影响盾壳霉降解草酸效率的另一个因子是草酸浓度。在草酸浓度为8～32mmol/L范围内,草酸浓度愈高,降解速度愈慢,反之亦然。当浓度高于40mmol/L时,盾壳霉不能生长,因而不能降解草酸。比较了两个培养特性（色素产生、双链RNA有无和液体产孢）差别较大的盾壳霉菌株（Chy-1和ZS-1）降解草酸的能力。结果表明:这两个盾壳霉菌株降解草酸的能力没有显著差异。以查比培养液为基础培养基研究了5种碳源（葡萄糖、果糖、麦芽糖、乳糖、可溶性淀粉）和4种氮源（硝酸钾、硝酸铵、丙氨酸、谷氨酸）对盾壳霉降解草酸的影响。结果表明:供试5种碳源和3种氮源（硝酸钾、丙氨酸和谷氨酸）均有利于盾壳霉菌丝生长,因而降解草酸的效率较高（＞98%）。而硝酸铵不利于盾壳霉菌丝生长,因而草酸降解效率较低（46.1%）。在核盘菌菌核提取物或菌丝提取物中添加草酸（8mmol/L或16mmol/L）,接种盾壳霉,振荡培养（20℃,200r/min,15d）后培养液中的草酸全部被盾壳霉降解。第三,研究了盾壳霉降解草酸对其产生重寄生相关的胞外酶（β-1,3-葡聚糖酶、几丁质酶和蛋白酶）的影响、对这些胞外酶活性的影响以及对产生抗真菌物质的影响。结果表明:盾壳霉降解草酸后,引起环境pH升高,有利于盾壳霉产生这三种重寄生相关酶,并提高这些酶的活性。说明降解草酸有利于盾壳霉寄生核盘菌。另一方面,草酸可以诱导盾壳霉产生抑制核盘菌菌丝生长的抗真菌物质（Anti-fungalsubstance,简称AFS）。盾壳霉产生AFS的量与培养液（PDB）中的初始草酸浓度（8～32mmol/L）呈正相关（P＜0.05）,而与盾壳霉降解草酸的效率呈负相关（P＜0.01）。而在PDB培养液中添加草酸钠时（8～32mmol/L）,盾壳霉培养液的AFS活性低。说明草酸营造的酸性环境是诱导盾壳霉产生AFS的原因。基于上述研究结果,可以看出草酸在盾壳霉与核盘菌互作中扮演着重要角色—调控盾壳霉的抗真菌作用和重寄生作用。更多还原

【Abstract】 Sclerotinia rot of oilseed caused by Sclerotinia sclerotiorum is a widespread disease. Coniothyrium minitans is an antagonistic fungus of S.sclerotiorum.Many studies indicated that C.minitans has a great potential in controlling sclerotinia disease caused by S.sclerotiorum in greenhouses and fields.Oxalic acid(OA) is a phytotoxin produced by S.sclerotiorum.Previous studies showed that OA played a determinant role in pathogenesis of S.sclerotiorum.As a mycoparasite of S.sclerotiorum,C.minitans should be tolerant to OA through an unknown mechanism.In this study,degradation of OA by C.minitans and the effects of this process on suppression of S.sclerotiorum were investigated.Results achieved so far were summarized below:Firstly,the capacity of OA degradation by C.minitans was investigated by incubation of C.minitans in modified potato dextrose broth(mPDB) amended with OA at 0.8-80 mmol/L.Results showed that after incubation at 20℃for 15 days,C.minitans could grow in mPDB containing 0.8-32 mmol/L of OA and thus degrade OA by 85%-92%.The pH values of the cultural filtrates increased from 2.9-6.3 to 7.7-8.6. Mycelial growth by C.minitans was completely inhibited in mPDB containing 40 or 80 mmol/L of OA.Production of ammonia by C.minitans was observed in mPDB alone or mPDB amended with OA at 0.8-32 mmol/L and was partially responsible for the increase of pH in cultures of C.minitans.Further analysis showed that the element responsible for OA degradation was located within mycelia of C.minitans,as the cultural filtrate of C.minitans could not convert OA.Activity of oxalate decarboxylase was detected in extracts of mycelia incubated in mPDB alone or mPDB amended with OA at 16 mmol/L.Secondly,factors affecting OA degradation by C.minitans were characterized.The factors investigated include strains of C.minitans,ambient pH and carbon/nitrogen sources.Results showed that two different strains of C.minitans:ZS-1 and Chy-1 were not different in degradation of OA in mPDB amended OA at 32 or 40 mmol/L. The initial ambient pH greatly affected degradation of oxalate by C.minitans.In mPDB amended with ammonia oxalate(AO)(16 mmol/L) or sodium oxalate(SO)(16 mmol/L),the ambient pH of the media was neutral to slightly alkaline.The percentage of degradation of oxalate was less than 5%in the 15-day-old cultures of C.minitans in these media.In mPDB amended with ammonia oxalate(AO)(16 mmol/L) plus OA at 16 mmol/L or sodium oxalate(SO)(16 mmol/L) plus OA at 16 mmol/L,the ambient pH of the media decreased to 3.4-3.5.The percentage of degradation of oxalate increased to 14.1%-36.1%in cultures of C.minitans(15 d).In mPDB amended with ammonia oxalate (AO)(16 mmol/L) plus HCl at 0.2%(v/v) or sodium oxalate(SO)(16 mmol/L) plus HCl at 0.2%,the ambient pH of the media decreased to 2.8-2.9.The percentage of degradation of oxalate increased to 63.1%-89.5%after incubation in these media for 15 days.On the contrary,in mPDB amended with oxalic acid(8,16,24 or 32 mmol/L) and adjusted to be neutral with NaOH,the percentage of degradation of oxalate decreased from 82.8%-97.1%to 13.0%-21.5%.Degradation of OA by C.minitans was observed in Czapek mineral salts amended with each of the five carbon sources(glucose,fructose,maltose,lactose and soluble starch) with alanine as the constant nitrogen source or each of the four nitrogen sources (potassium nitrate,ammonia nitrate,alanine and glutamic acid) with glucose as the constant carbon source,indicating that carbon or nitrogen source may be not the important factors affecting OA degradation by C.minitans.In sclerotial extract or mycelial extract of S.sclerotiorum amended with OA(8 or 16 mmol/L),a complete degradation of OA by C.minitans was also recorded after incubation for 15 days.Thirdly,effects of degradation of OA by C.minitans on S.sclerotiorum were studied by analysis of production and activity of mycoparasitism-related extra-cellular enzymes (β-1,3-glucanase,chitinase and protease),and antifungal substances by this mycoparasitic fungus.Regarding the production ofβ-1,3-glucanase by C.minitans,results showed that after incubation for 9,12 and 15 days,the yield ofβ-1,3-glucanase in mPDB amended with 16 mmol/L of OA was significantly higher than that in mPDB alone.At that time, OA was degraded by 90%-94%in the treatment of mPDB+OA(16 mmol/L),thereby enhancing the ambient pH to 8.3-8.5.In the pH-buffered experiment,production ofβ-1,3-glucanase by C.minitans increased with the increase of the ambient pH from 3.0-8.0,implying that the beneficial effect of OA degradation by C.minitans lies in enhancement of ambient pH.Regarding the activity ofβ-1,3-glucanase produced by C.minitans,results showed that the optimum ambient pH for this enzyme was 4-6.In the enzymatic system ofβ-1,3-glucanase without amendment of OA(control),the ambient pH was 7.0 and the activity of this enzyme was 621.8μg glucose/mL/min.However,with the increase of OA from 4 to 32 mmol/L,the ambient pH decreased to 3.5-1.5 and the activity ofβ-1,3-glucanase decreased to 503.5-55.9μg glucose/mL/min.When sodium oxalate was amended in the enzymatic system ofβ-1,3-glucanase of C.minitans at the concentrations ranging from 2 to 32 mmol/L,the ambient pH and the activity ofβ-1,3-glucanase changed slightly,compared to the control treatment without addition of this chemical. Therefore,it is the acidic ambient pH created by OA that can inhibit the activity ofβ-1,3-glucanase produced by C.minitans.Regarding the production of chitinase by C.minitans,results showed that the yield of this enzyme in cultures of C.minitans amended with OA at 0.8-24 mmol/L was significantly higher than that in the cultures of C.minitans without amendment of OA. The ambient pH for these 15-day-old cultures of C.minitans amended with OA was 7-8.Regarding the activity of chitinase produced by C.minitans,results showed that the optimal pH for activity of this enzyme was 8.0.In the enzymatic system of chitinase of C. minitans amended with OA at 4-48 mmol/L,the activity was inhibited and the inhibitory degree was negatively correlated with the concentration of OA.Activity of protease was determined using gelatin-amended plate.Results showed that the optimal pH value for production of protease by C.minitans was 6-7.The highest activity of protease was detected at pH 8.0.Production and activity of protease were inhibited by high concentration of oxalic acid. Degradation of OA results in increase of ambient pH,which is not favorable for production of anti-fungal substance(AFS) by C.minitans.In another word,when OA is degraded partly or not degraded,ambient pH is low.Acidic environment is favorable for production of AFS.Results of this study suggest that OA plays an important role in regulation of the antagonistic mode of action against S.sclerotiorum.The presence of OA is favorable for production of AFS responsible for antibiosis,whereas the absence of OA is favorable for production and activity of mycoparasitism-related enzymes responsible for mycoparasitism.更多还原