【作者】 韩立荣；

【导师】 张兴；

【作者基本信息】 西北农林科技大学 ， 农药学， 2010， 博士

【摘要】 土传病害是一类种类繁多、易于流行、危害性大、难以防治的重要植物病害,常对农业生产造成严重影响。目前对土传病害的防治多采用化学防治法,但是该方法只对少数病害有效,并且易造成农药残留、抗药性和环境污染等问题。利用微生物生防菌防治可以克服化学防治的上述弊病,且研究和实践已证明其对部分土传病害经济有效,因而成为防治土传病害研究的热点,越来越受到人们的重视。近年来,有报道指出,纤维素酶活力可以作为筛选土传植物病原生防菌株的一个参考指标,供试菌株纤维素酶活力越高,生防潜能越大。本研究对从土壤中分离得到的263株具纤维素酶活性菌株的抑菌活性进行了系统筛选和初步研究,并对筛选出的较高抑菌活性F10-2菌株从对部分土传病害的防治效果、菌种鉴定、纤维素酶的产酶特性、固体发酵条件优化和利用植物农药残渣作为发酵基质进行废物综合利用等方面进行了较为系统的研究,取得了以下主要结果:1、采用纤维素-刚果红选择性培养基,从采自陕西太白蔬菜基地、秦岭山区和新疆油菜种植区的土壤中分离得到了263株纤维素酶产生菌株。其中,放线菌181株,细菌64株,真菌18株。采用平板对峙法和菌丝生长速率法对这些菌株用7种重要土传病害病原菌(油菜菌核病菌、黄瓜枯萎病菌、茄子黄萎病菌、辣椒疫霉病菌、黄瓜立枯病菌、棉花黄萎病菌和小麦根腐病菌)进行了抑菌活性测定。平板对峙法测定结果表明,对至少一种供试病原菌菌丝生长抑制率达到60%以上的活性菌株有84株(即活性菌株),占总分离菌株株数的31.94%;其中放线菌菌株最多,有62株;其次是细菌,有15株;真菌只有7株。抑制生长速率法测定结果表明,上述84株活性菌株中,有16株菌株的发酵产物具有较强的抑菌作用。离体油菜叶片法测定结果表明,上述16株具较高抑菌活性菌株的发酵上清液和孢子悬浮液(或菌悬液)对油菜菌核病均有一定的防治效果。16株测试菌株中,F10-2菌株对油菜菌核病的防治效果最好,其上清液的防治效果为85.29%,孢子悬浮液的防治效果达到93.4%。盆栽药效试验测定结果表明,F10-2菌株发酵原液对辣椒疫霉病和茄子黄萎病均有较好的防治效果。特别是该菌发酵原液及孢子悬浮液对油菜菌核病表现出更好的防治效果。其发酵上清液喷雾处理的植株病叶率为4.75%,防治效果为86.81%;经F10-2菌株孢子悬浮液(107个孢子/mL)喷雾处理后的植株病叶率仅为6.67%,防效可达95.56%。2、对峙培养试验结果表明, F10-2菌株可有效控制油菜菌核菌菌丝的生长。3 d后F10-2菌株菌落即可与油菜核病菌菌落相接触,并开始包围、覆盖病原菌菌落,从而使油菜菌核病原菌生长受到显著抑制。菌核萌发结果发现,用F10-2菌株孢子悬浮液处理油菜菌核3 d后,整个菌核表面呈青绿色并开始腐烂崩溃,从而完全抑制了菌核的正常萌发;连续2周对被F10-2菌株侵染的菌核进行纤维素酶、葡聚糖酶和蛋白酶活性检测,结果表明,葡聚糖酶和纤维素酶在检测2周内活性相对较高,而蛋白酶活性很弱,几乎检测不到。这些酶类可能对抑制油菜菌核的萌发起着一定的作用。3、根据Pitt青霉鉴定方法,并结合分子生物学技术和比对分析,将F10-2菌株鉴定为青霉属瓦克青霉菌(Penicillium wakmanii Zalessky)。4、通过对纤维素酶液中各组分酶活力的测定,可以看出各酶组分中内切葡聚糖酶活性最高,其次为外切葡聚糖酶,而?-葡萄糖苷酶活性最低;内切葡聚糖酶在发酵第4 d,外切葡聚糖酶在第6 d,?-葡萄糖苷酶在第8 d时活性达最高,分别为8.79 U/mL、4.30 U/mL和1.71 U/mL。由F10-2菌株木聚糖酶产酶曲线可知,在发酵第6 d木聚糖酶活性达最高值为2.21 U/mL。通过F10-2菌株纤维素酶酶学特性研究结果可知,该酶作用最适pH为5~6之间,此区间酶活性较稳定;该酶活性在50~60℃比较稳定,酶反应最适温度为55℃,在60℃以上酶稳定性逐渐下降。5、为了解决植物源农药生产中的植物残渣二次污染问题,本研究以7种不同植物农药残渣作为主要发酵基质,对F10-2菌株产纤维素酶条件进行优化,发现川楝残渣为较为理想的发酵基质。通过单因子和响应面试验法获得了菌株F10-2固体发酵产纤维素酶的较佳条件为(按质量计算):川楝树皮残渣:麦麸:蛋白胨: KH2PO4为80: 20: 1.4: 0.4,水料比2:1,初始pH 6.2,在此条件下纤维素酶活力可达6.47 U/g,较原始培养条件提高了46.38%。6、以川楝残渣为主要发酵基质,研究了木质纤维素酶分泌特性及降解后残渣结构的变化情况。发现F10-2菌株可在固体川楝树皮残渣基质中生长,并可产生木质纤维素降解酶类。对川楝残渣扫描电镜和红外光谱分析表明,降解后残渣结构变得疏松且具有部分空隙,各官能团也发生了一定的变化,F10-2菌株对纤维素、半纤维素和木质素的降解率分别达到42.7%、33.96%和24.62%。由结果可推测,川楝树皮残渣的降解是纤维素酶、半纤维素酶、木素过氧化物酶和锰过氧化物酶共同作用的结果。以川楝残渣为主要培养基质进行固体发酵,7 d后对发酵产物进行浸提(产物与水质量比1:10),其浸提液纤维素酶活性(以FPase表示)可达到4.08 U/mL,孢子含量为4.3×107个/mL。对其浸提液进行简单的制剂加工,得到F10-2菌株活菌制剂,该制剂2倍稀释液对油菜苗期菌核病的防治效果仍可达91.43%,与F10-2菌株发酵原液的防效相当,说明对浸提液进行简单的制剂加工后,可以明显的提高其抑菌活性。对本试验的整体研究结果进行综合分析,可以初步说明,所筛选出的F10-2菌株可以在以纤维素为唯一碳源的培养基上正常生长,甚至可以高效利用植物农药残渣进行发酵而生产出具有较高纤维素酶活性的制剂,从而为消除植物农药残渣的二次污染及其综合利用开辟了一条新途径。F10-2菌株的产酶性能高,木质纤维素降解酶系齐全,为一株优良的纤维素酶产生菌株;由该菌株发酵产生的活菌酶制剂,可以通过降解菌体细胞壁、营养竞争和抑制菌丝生长等作用而有效的防治油菜菌核病等重要土传病害;直接利用具有产纤维素酶特性的生防菌制剂来防治可严重威胁农业生产的重要土传病害,是在IPM(有害生物综合管理)及IPP(农业综合生产与保护)理论指导下充分发挥生防菌潜能的初步尝试。更多还原

【Abstract】 Soil-borne disease, with large number of species, easily transmitted, great harm and hardly prevent, cause great effects on agriculture production. Chemical controls are effective against those diseases. However, repeated application of synthetic pesticides has resulted in a rapid increase in resistance, pesticide residue and environment pollution, so new control measure are needed. Recently, use of biocontrol microorganism to control soil-borne disease become research focuses because of its control effect and safety. Recent studies stimulated the investigation of properties of biocontrol microorganism and concluded that the enzyme activity of cellulase can be as screening index of biocontrol microorganism strain against soil-borne disease.In this paper, 236 strains of microorganisms with cellulase activity were isolated from soil samples, and their antifungal activity was systemic studied. The results showed that among these strains, the strain labeled F10-2 have higher antifungal activity. Then, the control efficacy strain F10-2 against soil-borne pathogen, strain identification, enzyme-producing character of cellulase, optimization of solid state fermentation, integrated utilization of plant pesticide residues degradation were researched systemically . The main results and conclusions were showed as follows:1. 236 strains of microorganisms with cellulase activity were isolated from soil samples, which dug from Taibai vegetable field of Shaanxi Province, Qingling mountain aera and rape field of Xinjiang uighur autonomous region, by using cellulose-congo red selective medium. There were 181 strains of actinomycetes, 64 strains of bacteria, 18 strains of fungi. The antifungal activities of those trains were tested against 7 soil-borne diseases by using method of plate test and mycelium growth rate method. Plate test showed that there are 84 strains of microorganisms with high antifungal activity, which inhibitory rate were greater than 60% against at least one plant pathogen, and possess 31.94% of the total separated microoganisms. Among them, actinomycetes strain was 62 strains (34.25% of all actinomycetes seperated), bacteria was 15 strains (23.44% of all bacteria separated), and fungi was 7 strains (38.89% of all fungi separated). Among 84 strains of microorganisms, mycelium growth rate method showed that inhibitory rates of fermentation broth of 16 strains (active strains) possess higher antifungal activity, and inhibitory rate of 5 strains of them were greater than 60% against 7 test soil-borne pathogen.Rape leaf test result showed that supernatant of fermentation broth and spore suspension (or bacterium suspension) of the above 16 active strains had control effect against Sclerotinia sclerotiorum. Among them, supernatant of fermentation broth of 11 strains, and spore suspension of 3 strains showed higher inhibitory rates, which control effects were greater than 60%. The strain labeled F10-2 have the best control effect, the fermentation broth and spore suspension possessed 85.29% and 93.4% control efficacy, respectively. Pot culture test showed fermentation broth of F10-2 strain had certain control effect against Phytophthora capsici and Verticillium dahliae. The protect effect and therapy effect were 73.82 % and 56.87 % against Phytophthora capsici respectively, and which were 65.21 % and 50.72 % against Verticillium dahliae respectively.2. Antagonistic culture test between F10-2 and Sclerotinia sclerotiorum showed the growth ratio of F10-2 was faster than that of Sclerotinia sclerotiorum. After the 3 days of antagonistic culture, colony of F10-2 began contact with, and then surrounded and covered Sclerotinia sclerotioru. Final, the growth of Sclerotinia sclerotiorum was inhibited, and the pathogen colony was wilt. The result showed the F10-2 had strong inhibitory action. The sprouting test of Sclerotinia sclerotiorum showed that surface of sclerotia was covered by turquoise spore of F10-2, and decayed after 7 days. All results showed spore liquid of F10-2 could control sprouting of sclerotia absolutely. Enzyme activity of ligninase, glucanase and protease were tested in 2 weeks, and results showed ligninase and glucanase had higher activities, while protease activity was too weak to identify. Glucanase arrived the highest activity after 8 days culture, reach to 11.2 U/mL. Ligninase activity arrived the highest activity at 6th day’s culture, reach to 7.5 U/mL. The bioassay under the room temperature showed that supernatant of fermentation broth of F 10-2 had higher inhibitory activity than spore suspension against Sclerotinia sclerotiorum. Pot culture test showed percentage of the disease leaves was 4.75%, and the control effect was 86.81% treated by fermentation broth. The inhibitory ratio was decreased following the concentration reduce of fermentation broth. Under the concentration of 10 times’dilution, the control effect was 39.44% only. Series of spore concentration treating of F10-2 spore showed the lowest percentage of the disease leaves was 6.67%, and the control effect was 95.56 % after treatment by 107 spores per micro litter.3. According to identification method of Pitt Penicillium, combined with molecular biology method and alignment analyses, the F10-2 strain was identified as Penicillium wakmanii.4. Enzyme activity test of cellulase liquid showed that endoglucanase had the highest enzyme activity, followed by exoglucanase, and activity ofβ-glucosidase was the lowest. At the 4th, 6th and 8th day of fermentation, enzyme activities of endoglucanase, exoglucanase andβ-glucosidase were up to the maximum, which was 8.79 U/mL、4.30 U/mL and 1.71 U/mL, reseparately. According the producing curve of xylanase of F10-2, the quantity of enzyme was rising rapidly after 2 days’culture. The enzyme activity was trend to stability between 6 and 8 days’culture. At the 6th day of culture, activity of xylanase reached to maximum value of 2.21 U/mL. Cellulase characters of F10-2 indicated that the optimum pH was 5～6, and the optimum temperature was 55℃for enzyme action. Enzyme stability decreased when the temperature was higher than 60℃.5. In order to solve the secondary pollution of plant extract residue, 7 plant pesticide draffs were used as fermentation substrate and the optimized fermenting conditions were determined by using single factor method and the response surface method. The results showed that chinaberry residues can be as optimum fermentation substrate, and the optimum composition of the fermentation medium (g/L) and conditions: chinaberry residues 8 g, bran 2 g, peptone 0.14 g, KH2PO4 0.04 g, initial pH 6.2, water ration 2:1. After optimization, ,the cellulose activity of strain F 10-2 was 6.47 U·g-1, which improved by 46·38% than original culture conditions.6. Chinaberry residues was used as fermentation substrate, and excrete character of cellulose and change of draff in structure after degradation were explored. The result showed that Penicillium wakmanii (F10-2) can grow in bark draff of chinaberry residues, and produce cellulose degrading enzyme. The scanning electron microscope and infrared spectrum analysis showed that the draff become loosen and have arisen interspace, the relative chemical functional group change, Degradating rate of F10-2 was 42.7%, 33.96% and 24.62% against cellulose, hemicellulose and lignin, reseparately. Those indicated that the degradating of bark draff was as a result of joint action of cellulase, hemicellulase, lignin peroxidase, manganese peroxidase.The fermentation production of chinaberry residues after 7 day fermentation was extract with water (Product:Water=1:10 in quality), and the cellulase activity can reach to 4.08 U/mL, and spore concentration can reach to 4.3×107 per micro litter. The fermentation liquid were formulated by simply formulation study, the liquid possesses excellent antifungal activities, the 2 fold dilution of fermentation liquid had control effect of 91.20% against Sclerotinia sclerotiorum, is higher than original fermentation liquid.The comprehensive analysis results indicated that separated F10-2 strain can culture cellulose as unique carbon source, and even high efficiently utilize plant pesticide draff to ferment and produce high cellulose activity formulation. Those finding provided a new approach to eliminate secondary pollution of plant pesticide draff and integrated utilization of plant draff. The enzyme activity of F10-2 strain fermentation is higher and enzyme system have completed function, can be as excellent cellulase producing strain. The formulation of the strain fermentation can control Sclerotinia sclerotiorum and other important soil-borne disease efficiently by nutrition competition and mycelium growth inhibition. Furthermore, direct utilization of bio-control strain of cellulase producing for prevention and cure of important soil-boron disease is a preliminary attempt under the guide of IPM and IPP theory.更多还原