【作者】 程国华；

【导师】 丁克坚；

【作者基本信息】 安徽农业大学 ， 植物病理学， 2006， 硕士

【摘要】 本论文是在安徽农业大学虫生真菌研究所研究的基础上，对延长球孢白僵菌油剂贮存期作进一步研究。主要从球孢白僵菌高质量孢子粉的制备与贮存、油剂配制前后进行处理及油剂与农药混配后相容性测定等方面出发，试图为常温下白僵菌油剂贮存期延长探索一些新的途径。试验主要研究途径及结果如下：1．试验从白僵菌对营养要求的角度出发，在白僵菌产孢培养基中添加不同的碳、氮源对三种菌株(Bb13、Bb380、Bb202)进行培养并制备孢子粉，检测其含孢量、萌发率、含水量。通过分析比较，筛选出了在实验条件下制备产量高、粉质优、活力强、含水量低的白僵菌孢子粉产孢培养基中可以添加的最佳碳有蔗糖、葡萄糖等，最佳氮源有硝酸钾、蛋白胨等。另外，不同的白僵菌菌株对高温、缺乏营养等不利环境的忍受力不同，本实验对三个不同寄主的菌株所产孢子粉质量进行检测，发现菌株Bb13所产的孢子粉质量明显优于其他两个菌株。2．在筛选碳、氮源的基础上，试验将白僵菌孢子粉制备过程中所需的营养需求及培养条件进行综合考虑，以不同的培养温度、pH值及培养基中所添加的不同碳、氮源，微量元素进行正交实验，以所产孢子的含孢量和萌发率为主要指标，筛选确定了适合球孢白僵菌Bb13孢子生长的最佳碳源是葡萄糖，最佳氮源是蛋白胨，微量元素铜和锌对产孢和孢子萌发有明显促进作用，最适pH值为6～7，最适培养温度是20～25℃。3．孢子粉贮存过程中，温度和含水量是影响孢子贮存寿命的重要因素。在4℃和25℃下，对五种含水量的孢子粉进行为期18个月的活孢率测定，分析结果得出，在4℃下，含水量为1.25～9.87％各处理的孢子贮存18个月，活孢率基本维持在90％以上，高含水量处理(13.89％)的孢子粉在前9个月的活孢率从97.38％缓慢下降至77.94％，此后活孢率下降加速，至420天时孢子全部失活，表明该含水量高的孢子粉即使冷贮也不宜七个月(此时活孢率为84.11％)；在25℃下贮存，含水量13.89％的孢子粉在第30天检测时孢子全部失活(未检测到萌发的孢子)；含水量9.87％、7.36％和4.28％处理的孢子全部失活时间分别发生于第195天、225天和420天；最低含水量处理(1.25％)的活孢率下降趋势显然缓于较高含水量处理，18个月时活孢率还有(15.94±1.26)％。因此，在常温下贮存孢子，含水量必须控制在5％以下，才能有5个月左右的贮存期并使活孢率达到80％。4．对白僵菌孢子进行限制营养培养和高温水浴等逆境处理并配成油剂后，限制营养所得孢子配成油剂的活孢率与对照相比，两者的活孢率存在显著差异。优化培养基制备孢子所配的油剂在贮存过程中，孢子活孢率虽然随着时间的推移在逐渐下降，但活孢率的测定值都高于同期限制营养的油剂活孢率。从第120天开始，限碳的油剂活孢率(38.3％)就明显的低于限氮的油剂活孢率(40.3％)，随后一直低，且存在差异显著性，到第240天时，限碳的油剂活孢率降至为0，而限氮的油剂贮存到第330天时，活孢率还维持在5.64％，由此可以看出，限氮培养的白僵菌孢子所配油剂的贮存期要比限碳的长。经过50℃水浴处理的孢子配成油剂后，贮存前270天，油剂中孢子活孢率与对照相比，都低于对照，均存在差异显著性，且水浴时间越长，孢子活孢率越低。但随着时间的推移，水浴后的孢子其油剂活孢率降低较对照降低缓慢，到第300天后，水浴后的孢子其油剂活孢率反而慢慢高于对照，存在差异显著性，因此可以得出适当地对白僵菌孢子进行高温处理，可以在油剂贮存后期提高其活孢率。另外，水浴30分钟的孢子和水浴60分钟的孢子其油剂在贮存300天后，活孢率也存在差异显著性，因此可以看出水浴时间适当延长可以在一定程度上延长油剂中后期孢子的活孢率。5．不同时间段对白僵菌油剂添加外源营养，与对照相比，其孢子的萌发率都有所提高，且与对照(萌发率为5.42％)基本存在差异显著性。而分别在前三个月对油剂添加外源营养其孢子萌发率比分别在后三个月添加的效果要好，且它们之间存在显著差异，尤其在第三个月添加外源营养后，孢子的活孢率达到了8.41％，明显高于对照和第三个月后添加外源营养的油剂中的孢子活孢率，分别为6.03％、5.59％、5.74％。所以对在常温贮存的白僵菌油剂适时添加外源营养，对于恢复孢子活力、提高油剂的活孢率是有一定的效果。6．球孢白僵菌孢子与色拉油、石蜡油的相容性很好，但通过耐热性测定可知，处理温度的高低、处理时间的长短，对不同制剂中的白僵菌分生孢子的萌发率和生活力都有影响。水剂和石蜡油剂中分生孢子随着处理温度的升高、处理时间的加长，其萌发率随之降低；对于色拉油剂中的分生孢子，除了在其萌发率达90％以上的速率比对照有所推迟外(约3h)，并没有影响其生活力。因此，色拉油可以作为白僵菌油剂很好的油剂载体。7．白僵菌与十种农药的相容性测定：试验所选的十种农药与球孢白僵菌孢子相容性大小顺序为：苏云金杆菌＞溴氢菊酯＞松毛虫病毒＞吡虫啉＞三唑磷＞杀虫双＞氧化乐果＞阿维菌素＞卡死克＞抑太保。就同一农药的不同浓度而言，农药对白僵菌孢子萌发率的影响与其浓度明显相关，且常用浓度(1.0×)下的白僵菌分生孢子萌发率都比稀释浓度(0.1×、0.2×)下的要低。常规使用浓度稀释5倍和10倍的8000IU苏云金杆菌WP、2.5％溴氢菊酯EC、松毛虫病毒WP、10％吡虫啉WP及稀释10倍的2％三唑磷EC、稀释5倍的18％杀虫双水剂中的孢子萌发率与对照无显著差异，可以和白僵菌油剂混用，使其互补，起到增效作用。更多还原

【Abstract】 On the foundation of the research by the Research Center for Entomogenous Fungi of Anhui Agricultural University, the thesis tries to make a further study on extending the storage life of Beauveria Bassiana oil preparation. Mainly from the aspects of the preparation and storing of high grade conidia of Beauveria Bassiana, the corresponding treatments for oil preparation, and the compatibility testing between oil preparation and pesticides, the thesis attempts to find some new ways to extend the storage life of Beauveria Bassiana oil preparation at normal temperature. The main researching ways and the results of the experiments are as follows:1. From the angle of the nutrition condition requested by Beauveria Bassiana, three different supplementary carbon and nitrogen sources are added to the conidial culture medium in Beauveria Bassiana, the experiment has raised three kind of strains (Bb13, Bb380, Bb202) and produced conidial powder, at the same time, the conidial content, percentage of conidial germination, and water content are examined. Through analysis and comparison, sucrose and glucose, the best carbon sources, and potassium nitrate and peptone, the best nitrogen sources have been screened and added to the conidial culture medium in Beauveria Bassiana which has high output, high quality, strong vigor and low water content. Moreover, different strains of Beauveria Bassiana have different endurance capacity to high temperature, undernourishment, etc. in disadvantageous environment, this experiment has tested conidial quality of strains of the three different hosts, and has discovered that the conidial quality produced by Bb13 obviously surpasses the other two strains.2. After screening carbon and nitrogen sources, synthetically considering the nutrition demand and the raising condition in the process of preparation of the conidial powder of Beauveria Bassiana, the orthogonal experiments are conducted under the different raising temperature, the pH value and the culture medium with different supplementary carbon and nitrogen sources and microelements. Taking conidial content and percentage of conidial germination as the main index, glucose, the best carbon source, and peptone, the best nitrogen source, are screened for the growth of Bb13, the microelements, copper and zinc, can greatly promote the production and germination of conidia, the most suitable pH value is 6～7, and the most suitable raising temperature is 20～25℃.3. In the course of storing conidial powder, temperature and water content are important factors to affect the storage life of conidia. At 4℃and 25℃, five kinds of water contents of conidial viability are tested for 18 months, the result shows, at 4℃, conidia treated by 1.25～9.87% water content can be stored for 18 months, conidial viability can be kept over 90%, treated by high water content(13.89%), the conidial viability of conidial powder slowly drops from 97.38% to 77.94% in the first 9 months, after that, the conidial viability will accelerate the speed, 420 days later, all the conidia will die out, which indicates that conidia power with high water content should be stored in cold places within 7 months (conidial viability at that time is 84.11%); at 25℃, conidia treated by 13.89% water content have completely deactivated when examined on the 30th day( No germinated conidia can be found); deactivation of all conidia treated by 9.87%, 7.36% and 4.28% water content occurs separately on the 195th day, 225th day and 420th day; treated by the lowest water content(1.25%), the conidial viability drops much slowly than that by high water content, after 18 months, the conidial viability will keep up (15.94±1.26)%. Therefore, to store conidia at normal temperature, the water content must be controlled below 5% so that conidia can be stored for about 5 months and the conidial viability can be kept over 80%.4. By controlling nutrition and bathing at high temperature, the conidia of Beauveria Bassiana have been confected into oil formulation, the conidial viability of oil formulation of conidia under controlled nutrition is compared with the controlled group, the remarkable difference exists in both. In the course of storage of oil formulation of the optimized culture medium, the conidial viability is gradually dropping down with time going on, but its tested value is always higher than the conidial viability of oil formulation when its nutrition is controlled at the same time. But from the 120th day on, the conidial viability of oil formulation (38.3%) with carbon controlled is obviously getting lower than the conidial viability of oil formulation (40.3%) with nitrogen controlled, afterwards it continuously low and there is obvious difference between them, on the 240th day, the conidial viability of oil formulation with carbon controlled falls to 0, but the conidial viability of oil formulation with nitrogen controlled still maintains at 5.64% when it is stored on the 330th day. From this it is concluded that the storage life of Beauveria Bassiana oil formulation with nitrogen controlled is longer than that with carbon controlled.After bathing 50℃, conidia are confected into oil formulation, 270 days before storing, the conidial viability in oil formulation is always lower than the controlled group, the difference among them is obvious, moreover, the longer the bathing time is, the lower the conidial viability is. But the conidial viability of bathed conidia in the oil formulation drops more slowly than that of the controlled group, however 300 days later, the conidial viability of bathed conidia in the oil formulation gradually becomes higher than that of the controlled one, there exists great difference among them, therefore, it is concluded that conidia of Beauveria Bassiana can be properly treated by high temperature, which will help to improve the conidial viability in oil formulation late in its storage. Furthermore, the conidial viability of conidia bathed for 30 minutes and conidia bathed for 60 minutes in oil formulation have great difference after storing for 300 days, according to this, if the bathing time is properly prolonged, the conidial viability in oil formulation will be lengthened to certain degree in mid and late periods of its storage.5. If extra nutrition is added to the oil formulation of Beauveria Bassiana at different period of time, the percentage of conidial germination will be improved compared with the controlled group, also there exists great difference compared with the controlled group (percentage of conidial germination is 5.42%). Moreover the extra nutrition added to the oil formulation separately in the first three months is more effective than that in the last three months, there exists great difference among them, especially in the third month, when extra nutrition is added to the oil formulation, the percentage of conidial viability will arrive at 8.41%, which is much higher than that of the controlled group, the percentage of conidial germination of conidia with extra nutrition in the oil formulation after the third month respectively is 6.03%, 5.59%, 5.74%. Therefore, to add extra nutrition to the oil formulation of Beauveria Bassiana stored at normal temperature at the right moment is an effective way to recover vigor of conidia and improve the percentage of conidial viability of oil formulation.6. The compatibility between Beauveria Bassiana and salad oil, paraffin oil is excellent, through thermal test, the change of processing temperature and the length of processing time take effect on the percentage of conidial germination and viability of conidia of Beauveria Bassiana for different formulations. With the processing temperature raising and the processing time lengthening, the percentage of conidial germination of conidia in the water formulation and paraffin oil formulation drops with it; which takes no effect on the viability of conidia in salad oil formulation except for its speed of over 90% conidial germination being delayed(approximately 3h) compared with the controlled group. Therefore, salad oil may be used as the best carrier for oil formulation of Beauveria Bassiana.7. Compatibility test between Beauveria Bassiana and ten kind of pesticides: The size order of compatibility from large to small is: Bt(bacillus thuringiensis) > Deltamethrin > DCP-V(dendrdimus cytophasmic polyhedrosis virus) > Imidacloprid > Triazophos > Bisultap > Omethoate > Abamectin > Flufenoxuron > Chlorfluazuron.As for different concentrations of the same pesticide, the effect that pesticide takes on the percentage of conidial germination of Beauveria Bassiana is closely related to its concentration, moreover, the percentage of conidial germination of Beauveria Bassiana under normal concentration(1.0×) is lower than that under diluted concentration (0.1×, 0.2×). The percentage of conidial germination has no obvious difference among the normally used concentration dilutes in 5 times of and 10 times of 8000IU Bt WP, 2.5% Deltamethrin EC, DCP-V WP, 10% Imidacloprid WP, 10 times of 2% Triazophos EC, and 5 times of 18% Bisultap compared with the controlled group, they may be combined with oil formulation of Beauveria Bassiana and work with each other to function better.更多还原