【作者】 杨丽萍；

【导师】 林芳灿； 姜道宏；

【作者基本信息】 华中农业大学 ， 微生物学， 2007， 博士

【摘要】 核盘菌[Sclerotinia sclerotiorum（Lib．）de Bary]是一种世界性分布的重要植物病原真菌，在我国严重威胁油菜生产。利用有益微生物控制核盘菌是目前菌核病防治研究的重点方向。本研究分离到一株对核盘菌有显著拮抗活性的真菌PY-1菌株，并对其分类地位进行了鉴定、对其控制油菜菌核病生防效果进行了评估，对PY-1菌株产抗真菌物质（AFS，Antifungal substance）的条件进行了优化，初步明确了其所产AFS的特性，并对该AFS进行了初步分离纯化，结果如下：1．拮抗菌PY-1菌株的鉴定：根据形态学特征及ITS序列分析，鉴定PY-1菌株为草酸青霉菌（Penicillium oxalicum），在真菌界的分类地位上属于丝孢子真菌，丝孢纲，丝孢目，青霉属（Penicillium），而其有性代为子囊菌门，不整囊菌纲，散囊菌目，散囊菌属真菌。2．PY-1菌株对核盘菌作用机理：对峙培养试验表明PY-1菌株对核盘菌的菌丝有强烈的拮抗作用，抑菌面积达9．6cm²，2个月后观察该抑菌面积仍不变。在20ml的PDA培养基中添加1ml的发酵液抑制核盘菌生长率为62．24％，其中1ml发酵液中的AFS相当于4μg纯的农利灵。另外研究还发现：PY-1菌株不能寄生菌核，对菌核的萌发没有明显的抑制作用，但可以降低菌核的产量。而且还可抑制核盘菌子囊孢子侵染油菜叶片。3．PY-1菌株的诱导抗病性：将PY-1菌株的孢子液（10⁷个孢子／mL）和发酵液原液处理棉花根部后，分别在5d、7d、15d后接种炭疽病菌（Colletotrichum gossypii）的孢子（10⁶个／ml），12d后统计发现：处理5d后接种病原菌，对照的病情指数为30．44，而孢子和发酵液处理的病情指数分别为16．13、14．50，相对防效分别达到47．02％、52．33％；处理7d后接种病原菌，对照的病情指数为22．95，而孢子和发酵液处理的病情指数分别为11．80、13．86，相对防效分别达到48．58％、39．65％；处理15d后接种病原菌，孢子液处理后的发病率仅为53％，其它两个处理发病率为100％。对照病情指数为25．40，孢子和发酵液处理的病情指数分别为8．40、16．64，相对防效分别达到66．92％、34．21％。结果表明PY-1菌株可诱导棉花对炭疽菌产生抗性。两种诱导因子相比较而言，孢子液的诱导作用更好一些。4．PY-1菌株对油菜菌核病的控制作用：（1）室内离体叶片接种试验表明：对照平均病斑直径较大为4．07±0．68cm，用PY-1孢子（10⁷个孢子／mL）处理后叶片发病率为75％，平均病斑直径只有0．73⁰．64cm。而用发酵液原液处理后，15片叶片中只有1片叶片发病，病斑直径为1cm。（2）盆栽试验结果表明：PY-1菌株的孢子和发酵液对菌核病都有很好的控制作用，只接种核盘菌的油菜植株发病叶片数多，而且病斑直径为2．80±0．5；经PY-1菌株10⁷的孢子处理后只有2片叶片发病，而用发酵原液处理后只有1片叶片发病。（3）2007年田间试验结果表明PY-1菌株的孢子液（10⁷个孢子／mL）和发酵液（稀释5倍）对菌核病都具有一定的防效，两个处理的病情指数要低于与对照处理病情指数，也较菌核净处理的低。PY-1菌株的孢子、发酵液以及菌核净的相对防效分别为52．65％、39．48％、31．04％。5．影响PY-1菌株产AFS因子的研究：（1）PY-1菌株AFS的产量与接种量、发酵温度、培养基的起始pH值有关，最适的接种量为0．2-0．4ml（10⁷个孢子／mL!），最适的发酵温度为20℃，发酵最适的起始pH为4-5。研究还发现该菌对通气量要求不高，摇瓶中装30—100ml培养基都比较适宜AFS的产生。（2）以CMS为基本培养基进行了碳源、氮源的筛选，产生AFS最为适宜的碳源有蔗糖、甘露醇、甘油，最为适宜的氮源有精氨酸、花生饼粉、鱼粉。此外，PDB培养基适合PY-1菌株的生长和AFS的产生。（3）以PDB、CMS为基本培养基筛选到了最佳发酵培养基：KH₂PO₄，1．0g；MgSO₄．7H₂O，0．5g；CuSO₄．5H₂O，0．005g；KCL，0．5g；精氨酸，0．871g：蔗糖10．12g，蒸馏水至1000ml。同时还研究了该菌的发酵代谢过程的一些代谢参数的变化，如pH，总糖，还原糖，氨基氮，菌丝生物产量等，AFS的产量在发酵第3d即可达到最高值。6．PY-1菌株所产AFS的抗菌谱：抑菌谱测定试验结果表明PY-1菌株所产的AFS对多种重要的植物病原真菌（如稻瘟菌、灰霉菌等）的抑制作用较强，而对水稻纹枯病菌、草莓白绢病菌等病原菌没有拮抗作用，表明PY-1菌株所产生抗生物质是一种抗真菌物质，具有一定的选择性。7．PY-1菌株所产AFS的稳定性：将PY-1菌株的发酵液经高温（121℃）处理30min的时间后，仍有较高的抑菌活力，表明该AFS具耐高温特性。酸碱稳定性测定试验表明该AFS在pH3-12范围内抑菌率变化幅度为52．17％-25．6％，在酸性条件下比较稳定，在碱性条件下活性下降。将PY-1菌株发酵液的乙醚提取物经紫外线照射2h后，抑菌活性提高的幅度为58．6％。发酵液在室温存放一年后丧失抑菌活性。但在4℃存放一年后抑菌活性下降幅度为30．4％。表明PY-1菌株的AFS可在4℃长时间存放。8．PY-1菌株所产AFS初步鉴定：（1）八溶剂系统纸层析试验结果表明，PY-1菌株所产的AFS是一种脂溶性的化合物，属金色抗霉素类抗生物质。pH纸层析实验结果表明，PY-1菌株AFS在酸性区域和碱性区Rf值有两个高峰，可知该AFS可能为一两性抗生素类物质。9．PY-1菌株AFS的粗提取：（1）PY-1菌株发酵液粗提取物和菌丝提取物对核盘菌菌丝生长都有显著的抑制作用，表明PY-1菌株的AFS胞内胞外均有产生。用极性较弱的有机溶剂乙酸乙酯、氯仿、乙醚等提取后效果较好，表明该AFS极性较低。进一步研究表明最适提取比例为：发酵液：乙醚=4：3（V／V）。10．PY-1菌株AFS的分离和纯化：（1）通过薄层层析（TLC），在展开剂乙酸乙酯：甲醇：水=100：10：1中可分离到3种物质，Rf值分别约为0．16，0．76，0．89。经生物检测Rf值0．89的物质具有生物活性。（2）将经过TLC精制后的样品再利用高效液相色谱（HPLC）进行分离。经过HPLC两次分离，在流动相：甲醇：水=70：30；流速：0．8ml／min的条件下分离出两个活性峰a₁、a₂，保留时间分别为26．33min、32．12min。（3）将经过HPLC纯化得到的两个活性组分a₁、a₂的纯品，采用EI-MS测定了分子量，分别为274．2、326．1。更多还原

【Abstract】 Sclerotinia sclerotiorum is a plant pathogenic fungus found inhibiting soil ubiquitously in many parts of the world. In China, this pathogen causes stem rot on oilseed rape （Brassica napus）, and causes serious losses every year. Biological control is a useful alternative to chemical fungicides against Scleriotinia diseases. In this paper, a antagonistic fungus strain PY-1 which showed strong inhibition against S. sclerotiorum was screened out, and the taxonomy of PY-1 strain was identified. The potential for biological control of oilseed rape stem rot was evaluated, and the cultural conditions for the fermentation of antifungal substance （AFS） was optimized. The stability of AFS was examined under several conditions, the AFS also was extracted from fermentation liquid and partially separated and purified by HPLC, and two active components were primary identified. The main results were listed below.1. Identification of PY-1 strain: PY-1 strain was identified as Penicllium oxalicum based on the characteristics of the colony morphology, conidiophore, phialides and conidia, and the identification was confirmed with alignment of ITS sequence of PY-1 and that of other fungi on GneBank.2. The mechanism of antagonism on S. sclerotiorum by strain PY-1: Strain PY-1 showed strong inhibition to the mycelial growth of S. sclerotiorum on PDA plates. The inhibition zone could remain for more than two months. One milliliter of fermentation liquid in 19 ml PDA could inhibit the mycelial growth of 5. sclerotiorum significantly, the inhibition effect was 62.24 %. Comparing with Carbendazim, the relative activity of anti-fungal substance of PY-1 produced at the third day was about 4μg a.i. per millilitre. Strain PY-1 could neither parasitize the sclerotia of 5. sclerotiorum, and nor suppress the mycelial germination of the sclerotia, but it may slightly reduce the product of sclerotia in PDA plate。3. Biological control potential of oilseed rape stem rot: （1） Both spores and fermentation liquid of PY-1 could suppress the infection of S. sclerotiorum on detached oilseed rape leaves. With an application of 10⁷ spores/ml on leaves, average diameter of lesion being 0.73±0.64 cm, the size of lesions were significantly reduced comparing to water-treated control with average size of 4.07±0.68cm, and only 75% tested leaves could be successfully induced to form lesions. Only one lesion on detached leaves was induced among 15 treated leaves with the undiluted fermentation liquid, and the lesion was 1.0 cm. （2） Both spores and fermentation liquid of PY-1 could suppress the infection of S. sclerotiorum on living oilseed rape plants. With an application of 107 spores/ml on plants, the average diameter of lesion was 0.73±0.64 cm, and only two leaves produced lesions in all of treated plants, while more leaves were infected and the average size of lesion was 2.80±0.5 cm in water-treated control. Only one leaf was induced to produce a lesion among all fermentation liquid-treated plants. （3） Field experiments in 2007 showed that strain PY-1 （including spores with a concentration of 1×10⁷ spore/ml and 5 fold diluted fermentation liquid） could significantly suppress sclerotinia stem rot of rape seed, the relative biocontrol efficiency were 52.65 % and 39.48 % respectively, while the effect of dimathchlon was 31.04 %.4. Induced resistance of strain PY-1: Acquired resistance of cotton against Colletotrichum gossyoii was observed when treated cotton roots with spores （10⁷spores /ml） and fermentation liquid of strain PY-1. If cotton plants were treated with spores 15 d ahead of inoculation of Colletotrichum gossyoii, the disease index was suppressed with a rate of 66.92%, however, only 34.21% was achieved with fermentation liquid.5. Facts affecting the production of AFS of Strain PY-1: （1） Production of AFS of strain PY-1 was related with the inoculation quantity, fermentation temperature and primary pH value of medium. The optimum fermentation conditions for inoculation quantity for 0.2-0.4 ml of 1.5×10⁷ spores/ml), and primary pH of medium ranged from 4 to 5, and fermentation temperature for 20℃were benefit for the high production of AFS. Furthermore, packaging volume of medium in flask which may influence the aerial condition did not effect the production of AFS significantly. （2） Effect of carbon sources and nitrogen sources on AFS production was studied using CMS medium as basic media. In the carbon source experiment, four （sucrose, glycerol, trehalose, mannitol） out of nine carbon source could be the most optimum by PY-1 for AFS production, and three nitrogen source including L-Arg、fish powder and peanut powder were the most optimum nitrogen source. PDB medium was suit for AFS production also. （3） The integrative condition of optimum medium and fermentation conditions for production of AFS of PY-1 strain were studied, and results showed that the optimal fermentation medium was KH₂PO₄,1.0g; MgSO₄.7H₂O,0.5g; CuSO₄.5H₂O,0.005g; KCL, 0.5g; L-Arg, 0.871g ; sugar, 10.12g. The pH value, reducing sugar and total sugar, ammoniacal nitrogen and biomass were changed during process of fermentation, and AFS production could y reach a peak on the 3rd day. 6. The antagonism range of strain PY-1: Strain PY-1 showed significant suppressing the growth of many fungal pathogens, such as Alternaria alternata, Botrytis cinerea, Colletotrichum gloeosporhides, Gibberella zeae, Magnorpthe grisea, Monilinia laxa, and Verticillium dahilae. However, strain PY-1 did not show any effect the growth of Rhizoctonia solani and Sclerotium spp, and plant pathogenic bacteria.7. Some primary properties of AFS produced by strain PY-1: The extract from fermentation liquid with aether showed significant anti-fungal activity against S. clerotiorum, and was used as crude AFS for testing the stability under given conditions. The AFS was very stable to high temperature when treated with at 121℃for 30min, it was also acidic or alkaline-stable, the activity of AFS was not significantly reduced when treated in buffers with a wide range of pH values （3-12）; and interestingly, the antagonistic activity was improved when treated with UV-irradiation.8. Primary identification of the AFS produced by strain PY-1: Paper chromatogram analysis indicated that the AFS produced by strain PY-1 was a fat-soluble and ambisextrous antibiotic.9. Primary separation of the AFS produced by strain PY-1: The AFS existed both in mycelial and fermentation liquid of strain PY-1 and could be extracted with aether and chloroform, suggesting that the AFS was a low polarity substance. The optium extract proportion for the extraction of AFS was 3/4 volume of ethyl acetate.10.Primary purification of the AFS produced by strain PY-1: （1） Three substances were separated from the AFS in solvent system （Ethyl acetate: methanol: H₂O=100:10:l） though TLC, and the substance with a Rf of 0.89 showed strong antagonistic activity. （2） The activity band on the thin lay were collected as crude sample of the AFS, and further separated with HPLC, and a second round separation with HPLC was carried out to obtain two active compounds a1 and a2 using a mobile phase of 70% methanol,30% double distilled H₂O. The retention time of al was 11.038 min, and that of a2 was 13.598 min with a flow rate of 0.8 ml min-1. （3） The molecular weights of a₁ and a₂ were examined with EI-MS, and the results showed that the molecular weight of a₁ and a₂ were 274.2 D and 326.1 D respectively.更多还原