对褐飞虱高毒力的真菌菌株筛选及其应用研究
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摘要
褐飞虱Nilaparvata lugens St l是我国及东南亚地区一种远距离迁飞性水稻重要害虫。为了寻求对该虫生物防治的新途径，本论文在既有资料的基础上，进一步筛选对褐飞虱高毒力的真菌菌株，系统研究了所得高毒力黄绿绿僵菌Metarhiziumflavoviride（Mf）菌株的生物学特性、对褐飞虱不同虫态的毒力以及致病机理，研制了该菌株乳悬剂剂型，并对黄绿绿僵菌（Mf）菌株的固相发酵培养基组分的配方进行了优化，为防治褐飞虱真菌杀虫剂的开发与规模化生产提供有价值的技术资料。本研究主要获得以下结果：
1、从褐飞虱N. lugens罹病虫体上新分离出的一种黄绿绿僵菌菌株M. flavoviride（Mf），与实验室保存的黄绿绿僵菌、金龟子绿僵菌和白僵菌3种菌种5个菌株作对比，研究了在SDAY和PDA培养基上的培养性状，并测定了它们对褐飞虱成虫的毒力。结果表明：在SDAY培养基上，6个菌株菌落特征差异明显，Mf菌株菌落直径增长量最大，产孢量最高，分别为2.59mm，17.34×10~7孢子/cm~2。与其它菌株相比差异显著。Mf菌株在SDAY培养基上生生长优于PDA，Mf菌株对褐飞虱成虫具有很强的毒力，以1.0×10~8孢子/mL的孢子液接种到褐飞虱成虫体表上，累积校正死亡率达83.8%，LT50为4.47d，致死率明显高于其他真菌。
2、用分子生物学主方法对褐飞虱成虫高毒力真菌菌株进行了鉴定。采用氯化苄法、CTAB法以及裂解液法分别提取了该菌的基因组DNA；以ITS1和ITS4为绿僵菌通用引物，对供试菌株的rDNA ITS序列进行PCR扩增、琼脂糖凝胶电泳检测和序列分析，并在核酸序列数据库中进行同源序列对比。实验结果表明裂解液法提取的该种绿僵菌的基因组DNA纯度高且质量好，氯化苄法次之，CTAB法不适合提取此菌的基因组DNA；分子鉴定结果显示该菌为绿僵菌属黄绿绿僵菌（M. flavoviride)。明确侵染褐飞虱的绿僵菌种类，进而掌握其生物学征和对寄主的致病性。
3、对新分离鉴定黄绿绿僵菌Mf82菌株，将其与实验室保存的8株绿僵菌M. spp.一起，分别测定其对褐飞虱成虫毒力和几丁质酶活力，并用扫描电镜观察了Mf82菌株侵入昆虫表皮的过程和体表的形态变化，进而分析侵入能力与几丁质酶的相关性。结果表明：菌株Mf82累计校正死亡率和几丁质酶活力均最高，为82.1%和9.78U/mg，与其他菌株差异显著。同时，扫描电镜照片显示Mf82分生孢子既可以由褐飞虱体壁节间膜和凹陷处侵入，还可以从含几丁质较多的胸部背板侵入，可见筛选的Mf82菌株对褐飞虱成虫致病力强。比较9株绿僵菌菌株菌落生长速度、产孢初始时间、产孢量和萌发率显示，Mf82菌株具有生长速度快、产孢初始时间短、萌发率高和产孢量大等优点。
4、利用黄绿绿僵菌M. flavoviride82菌株五种浓度5296-5368、1055-1121、585-623、126-144和22-31孢子/mm~2侵染不同龄期褐飞虱成虫和若虫并以1100孢子/mm~2孢子悬浮液侵染褐飞虱怀卵雌成虫、雌成虫、雄成虫和不同龄期卵。结果表明：其不同浓度孢子液对褐飞虱3个发育阶段有不同程度的致病力，毒力大小顺序为成虫>高龄若虫>低龄若虫。同时，Mf82菌株对不同性别褐飞虱成虫的敏感性顺序为怀卵雌成虫>雌成虫>雄成虫。黄绿绿僵菌孢子液对各处理稻株褐飞虱产卵痕部位、卵粒均有侵染作用，10d侵染率分别为66.7%和51.2%，卵龄越低，侵染效果越好，卵龄为0.5d时侵染率最高。本研究表明黄绿绿僵菌Mf82菌株对褐飞虱成虫、若虫和卵均有较强的致病性，
5、利用扫描电镜观察了黄绿绿僵菌M. flavoviride菌株分生孢子对褐飞虱的侵染过程。结果表明：分生孢子多分布在褐飞虱节间膜、体表的褶皱凹陷等部位，主要以芽管或产生附着胞入侵，然后在体表长出菌丝和产孢。菌体进入寄主血腔后，利用体腔内营养大量增殖、扩散并侵染褐飞虱卵巢中的卵块。侵染卵块的菌丝虽被体液包裹但依然能继续生长，使卵粒失活，抑制害虫下代的数量。通过体表及体内的侵染过程，可直观地表现黄绿绿僵菌对褐飞虱的侵染能力和侵入方式，为评价该菌的杀虫作用和应用前景提供了证据。
6、用新分离出的黄绿绿僵菌M. flavoviride（Mf82）菌株与实验室保存的黄绿绿僵菌、金龟子绿僵菌和白僵菌3种菌种9个菌株作对比，测定了它们的悬乳剂对褐飞虱成虫的毒力。结果表明：Mf82菌株对褐飞虱成虫的毒力最高，以1.0×10~8个孢子/mL的孢子悬乳剂喷雾接种到褐飞虱成虫体表上，累积死亡率高达81.7%，LT50为4.6d，致病效果显著高于其他受测菌株。在此基础上研制了黄绿绿僵菌悬乳剂，并研究了其对褐飞虱的致病力。结果表明：随着黄绿绿僵菌浓度的增加，褐飞虱的累计死亡率增加，在浓度为1048个孢子/mm~2时，累计死亡率达到85.0%。利用时间-剂量-死亡率模型对数据进行处理，所建模型均顺利通过Hosmer-Lemeshow拟合异质性检验，表明模型拟合良好，并由模型估计出了该剂型对褐飞虱的致死剂量与致死时间。在接种后第7d和第9d，LC50值分别为2.1×103、9.9×102个孢子/mm~2, LC90分别为7.8×104、3.7×104个孢子/mm~2。黄绿绿僵菌悬乳剂对褐飞虱的致死时间与对数剂量相关，供试菌剂LT50值随着对数剂量的增加而递减，对数剂量由7.0增加到8.0时，LT50由8.9d降为5.7d。可见该黄绿绿僵菌悬乳剂对褐飞虱具有较强的毒力。
7、以大米粉、黄粉虫蛹壳、花生壳和瓜子壳为培养基组成成分，研究黄绿绿僵菌固相发酵最佳配方。在固相发酵条件下，利用Design-Expert软件分析各因素对产孢量的显著性，采用响应面法对黄绿绿僵菌发酵培养基配方进行研究和优化。结果表明：影响产孢量的显著成分为黄粉虫蛹壳；黄绿绿僵菌发酵最佳配方组合为：0.25g大米、0.14g黄粉虫蛹壳、1.00g花生壳、0.25g瓜子壳。在此最佳组合为培养基的条件下得到产孢量为100.013×10~8个/mL菌液，比初始设计提高了31.7%。使用本研究优化后的培养基配方，可以降低生产成本，得到较高产量的孢子，且在生产时无环境污染，对以后的大规模生产非常有利。
综上所述：本研究研究筛选所得黄绿绿僵菌Mf82菌株对褐飞虱成虫、若虫和卵均有较强的致病性，且生产容易、使用方便，是一株极具应用潜力的生防真菌。
The brown planthopper (BPH) Nilaparvata lugens St l is an important rice pest thatcan cause serious damage to the rice production in China and Southeast Asia. it isnecessary to change passive situation, change control strategy and find new biologicalcontrol method to manage the pest. In this paper, a number of entomopathogenic fungistrains were further tested on the basis of existing data and a new strain of Metarhiziumflavoviride (Mf), isolated from N. lugens with higher virulence to BPH was selected. Thebiological characteristics of Mf82and its verulence to different stages of N. lugens werecompleted studied. On the basis of bioassay, and its pathogenesis to BPH was also sudied,using its emulsifiable formulation. the components of solid-state fermentation medium forMf82spore production was optimized in order to lay foundation for large scal productionof fungi insectcide which is used to control N. lugens. The primary results are as follows:
1. The new strain of M. flavoviride Mf, isolated from N. lugens and fiveentomogenous fungi strains of two Beauveria bassiana and three Metarhizium kept in theLaboratory were tested for their cultural characteristics on SDAY and PDA and virulenceto the adults of N.lugens. Results indicated that6strains showed obvious difference onSDAY. Mf grew faster and produced more spores than other strains on SDAY. Its colonydiameter increasing per day was2.59mm and sporulation was17.34×10~7conidia/cm~2.SDAY was more suitable for Mf to grow than PDA. Mf obviously controlled N. lugens. ItsLT50value to the adults of N. lugens was4.47d and adjusted accumulative mortality was83.8%when concentration was1.0×10~8conidia/mL.
2. one isolated strain of M. flavoviride (Mf) which was high virulence to N. Lugenswas identified by Molecular biology method, three different methods were respectivelyused to extract genomic DNA from this fungus: benzyl chloride, CTAB and crackingsolution. Its rDNA ITS sequence was amplified with ITS1and ITS4as primers, and theamplified ITS sequence was detected by agarose gel electrophoresis and was analyzed inGenebank．The results of the experiments showed that the cracking solution method couldobtain the highest quantity of the purest DNA, benzyl chloride method took second place，but CTAB method was not suitable. Homologous sequence contrast was conducted in theDNA sequence database and the result showed that this fungus was M. flavoviride.Identifying the entomogenous fungus species which infects N. lugens would play animportant role for further studying on its biological characteristics and pathogenicity, andcontrolling N. lugens with it.
3. Nine strains of different species of Metarhizium, including strain of M. flavoviride(Mf82) newly isolated from N. lugens, and eight M. anisopliae strains from lab collection,were tested against the adults of N. lugens. The chitinase activities of these strains werealso assayed. The results showed that both the cumulative mortality (82.1%) and thechitinase activity (9.78U/mg) of strain Mf82were the highest among all tested strains.Scanning electron microscope (SEM) observations showed the germinating conidia of thestrain Mf82penetrated the cuticle not only via the intersegmental membranes and foldedregions, but also through the pronotum which contained a large quantity of chitin. Thebiological characteristics of these strains, including growth rate, sporulation, germinationrate and timing of sporulation, were further investigated. The results showed Mf82was thebest strain having strong pathogenicity of N. lugens.
4. In this study, laboratory bioassays were carried out on isolate of theentomopathogenic fungal specie, M. flavoviride82for the virulence to different stages of N.lugens at a series of spore concentration of5296-5368,1055-1121,585-623,126-144and22-31conidia/mm~2respectively and gravid female, female, male and eggs laid wereinfected by suspension of1100conidia/mm~2. The results showed that the pathogenicity ofMf82to adults, nymphs at different instars and egg stage were assessed at a series ofconidia concentration. This strain was most virulent to adults, moderate to old nymphs, andleast to young nymphs. The susceptible sequence was gravid female> female> male. Itwas also found that Mf82could infect both oviposition marks and eggs in rice seedlings,and the infection rate reached66.7%and51.2%respectively at10d after treatment. Theyounger the eggs, the higher the infection rate, with the0.5d-old eggs to be most easilyinfected.
5. The infection process of M. flavoviride conidia to N. lugens was observed usingscanning electron microscope (SEM).The results showed that most of conidia weredeposited on intersegmental membranes, folded regions on cuticle. The germinated conidiaof the isolate invaded the cuticle not only directly with germ tube but by producingappressorium during the course of infection. Then hypha and spores grew on cuticle of N.lugens. Thalluses entered into hemocoel and then made use of nutrition to reproduce inlarge quantities, diffused and infected eggmass of N. lugens. Even though covered withhumoral, hypha infecting eggmass still continued to grow and sporulate, inactivated eggsand inhibited the number of next generation. This phenomenon can show the infectiveability and invasive approach by infection process in vitro and in vivo. It also providesevidence that evaluate insecticidal effect and application prospect of M. flavoviride.
6. Ten strains of entomogenous fungi emulsifiable formulation; a new strain of M.flavoviride Mf82, isolated from N. lugens and six strains of Beauveria bassiana and four ofMetarhizium were bioassayed for its virulence against the adults of N. lugens and theirvirulence compared. The results showed that cumulative mortality (81.7%) of strain Mf82was the highest among all tested strains, the LT50value was4.6d at a concentration of1.0×10~8conidia/mL. On this basis, a kind of emulsifiable formulation of Mf82was studiedin the laboratory, and the bioassay of virulence of Mf82against the N. lugens was carriedout. The results showed that the daily cumulative mortality of N. lugens increased with theinfect concentration of conidial suspension, the highest concentration (1048conidia/mm~2)treatment caused85.0%. A time dose mortality model was used to analyze the bioassaydata and the model fitted the data well, resulting in parameters for estimating the time anddose effects. The estimated LC50values on day7and9after treatment were2.1×103,9.9×102conidia/mm~2, respectively; while LC90were7.8×104，3.7×104conidia/mm~2,respectively. The median lethal time of the emulsifiable formulation of M. flavoviride tothe N. lugens differed at various logarithmic dose, values of LT50of tested agents wereshortened with logarithmic dose, logarithmic dose increased from7.0to8.0, the LT50values reduced from8.9days to5.7days. The results showed that the emulsifiableformulation of M. flavoviride to N. lugens with high virulence.
7. The medium is filled with rice flour, pupa shell of Tenebrio molitor, shell ofgroundnut and shell of sun-flower seed, then summing up the best preservation program. Insolid-phase fermentation conditions, use Design-Expert software analyzing the factors forspores of significant，Optimize M. flavoviride by response surface. As a result, pupa shellof Tenebrio molitor is the significant influence factors. The best preservation program isthat the medium is filled with0.25g rice flour,0.14g pupa shell of Tenebrio molitor,1.00g shell of groundnut,0.25g shell of sun-flower seed. The greatest spore productionquantity is100.013×10~8conidia/mLevery. Using this optimization of the medium afterformula, reduce the production cost, produced higher yield spores. There was no pollutionto environment, so it is beneficial to mass production in future.
To sum up: The Mf82strain which had high pathogenecity to adults、nymphs andeggs of BPH, and what's more, it was easy to yield and use in this paper is a promisingapplication biocontrol agent.
1、从褐飞虱N. lugens罹病虫体上新分离出的一种黄绿绿僵菌菌株M. flavoviride（Mf），与实验室保存的黄绿绿僵菌、金龟子绿僵菌和白僵菌3种菌种5个菌株作对比，研究了在SDAY和PDA培养基上的培养性状，并测定了它们对褐飞虱成虫的毒力。结果表明：在SDAY培养基上，6个菌株菌落特征差异明显，Mf菌株菌落直径增长量最大，产孢量最高，分别为2.59mm，17.34×10~7孢子/cm~2。与其它菌株相比差异显著。Mf菌株在SDAY培养基上生生长优于PDA，Mf菌株对褐飞虱成虫具有很强的毒力，以1.0×10~8孢子/mL的孢子液接种到褐飞虱成虫体表上，累积校正死亡率达83.8%，LT50为4.47d，致死率明显高于其他真菌。
2、用分子生物学主方法对褐飞虱成虫高毒力真菌菌株进行了鉴定。采用氯化苄法、CTAB法以及裂解液法分别提取了该菌的基因组DNA；以ITS1和ITS4为绿僵菌通用引物，对供试菌株的rDNA ITS序列进行PCR扩增、琼脂糖凝胶电泳检测和序列分析，并在核酸序列数据库中进行同源序列对比。实验结果表明裂解液法提取的该种绿僵菌的基因组DNA纯度高且质量好，氯化苄法次之，CTAB法不适合提取此菌的基因组DNA；分子鉴定结果显示该菌为绿僵菌属黄绿绿僵菌（M. flavoviride)。明确侵染褐飞虱的绿僵菌种类，进而掌握其生物学征和对寄主的致病性。
3、对新分离鉴定黄绿绿僵菌Mf82菌株，将其与实验室保存的8株绿僵菌M. spp.一起，分别测定其对褐飞虱成虫毒力和几丁质酶活力，并用扫描电镜观察了Mf82菌株侵入昆虫表皮的过程和体表的形态变化，进而分析侵入能力与几丁质酶的相关性。结果表明：菌株Mf82累计校正死亡率和几丁质酶活力均最高，为82.1%和9.78U/mg，与其他菌株差异显著。同时，扫描电镜照片显示Mf82分生孢子既可以由褐飞虱体壁节间膜和凹陷处侵入，还可以从含几丁质较多的胸部背板侵入，可见筛选的Mf82菌株对褐飞虱成虫致病力强。比较9株绿僵菌菌株菌落生长速度、产孢初始时间、产孢量和萌发率显示，Mf82菌株具有生长速度快、产孢初始时间短、萌发率高和产孢量大等优点。
4、利用黄绿绿僵菌M. flavoviride82菌株五种浓度5296-5368、1055-1121、585-623、126-144和22-31孢子/mm~2侵染不同龄期褐飞虱成虫和若虫并以1100孢子/mm~2孢子悬浮液侵染褐飞虱怀卵雌成虫、雌成虫、雄成虫和不同龄期卵。结果表明：其不同浓度孢子液对褐飞虱3个发育阶段有不同程度的致病力，毒力大小顺序为成虫>高龄若虫>低龄若虫。同时，Mf82菌株对不同性别褐飞虱成虫的敏感性顺序为怀卵雌成虫>雌成虫>雄成虫。黄绿绿僵菌孢子液对各处理稻株褐飞虱产卵痕部位、卵粒均有侵染作用，10d侵染率分别为66.7%和51.2%，卵龄越低，侵染效果越好，卵龄为0.5d时侵染率最高。本研究表明黄绿绿僵菌Mf82菌株对褐飞虱成虫、若虫和卵均有较强的致病性，
5、利用扫描电镜观察了黄绿绿僵菌M. flavoviride菌株分生孢子对褐飞虱的侵染过程。结果表明：分生孢子多分布在褐飞虱节间膜、体表的褶皱凹陷等部位，主要以芽管或产生附着胞入侵，然后在体表长出菌丝和产孢。菌体进入寄主血腔后，利用体腔内营养大量增殖、扩散并侵染褐飞虱卵巢中的卵块。侵染卵块的菌丝虽被体液包裹但依然能继续生长，使卵粒失活，抑制害虫下代的数量。通过体表及体内的侵染过程，可直观地表现黄绿绿僵菌对褐飞虱的侵染能力和侵入方式，为评价该菌的杀虫作用和应用前景提供了证据。
6、用新分离出的黄绿绿僵菌M. flavoviride（Mf82）菌株与实验室保存的黄绿绿僵菌、金龟子绿僵菌和白僵菌3种菌种9个菌株作对比，测定了它们的悬乳剂对褐飞虱成虫的毒力。结果表明：Mf82菌株对褐飞虱成虫的毒力最高，以1.0×10~8个孢子/mL的孢子悬乳剂喷雾接种到褐飞虱成虫体表上，累积死亡率高达81.7%，LT50为4.6d，致病效果显著高于其他受测菌株。在此基础上研制了黄绿绿僵菌悬乳剂，并研究了其对褐飞虱的致病力。结果表明：随着黄绿绿僵菌浓度的增加，褐飞虱的累计死亡率增加，在浓度为1048个孢子/mm~2时，累计死亡率达到85.0%。利用时间-剂量-死亡率模型对数据进行处理，所建模型均顺利通过Hosmer-Lemeshow拟合异质性检验，表明模型拟合良好，并由模型估计出了该剂型对褐飞虱的致死剂量与致死时间。在接种后第7d和第9d，LC50值分别为2.1×103、9.9×102个孢子/mm~2, LC90分别为7.8×104、3.7×104个孢子/mm~2。黄绿绿僵菌悬乳剂对褐飞虱的致死时间与对数剂量相关，供试菌剂LT50值随着对数剂量的增加而递减，对数剂量由7.0增加到8.0时，LT50由8.9d降为5.7d。可见该黄绿绿僵菌悬乳剂对褐飞虱具有较强的毒力。
7、以大米粉、黄粉虫蛹壳、花生壳和瓜子壳为培养基组成成分，研究黄绿绿僵菌固相发酵最佳配方。在固相发酵条件下，利用Design-Expert软件分析各因素对产孢量的显著性，采用响应面法对黄绿绿僵菌发酵培养基配方进行研究和优化。结果表明：影响产孢量的显著成分为黄粉虫蛹壳；黄绿绿僵菌发酵最佳配方组合为：0.25g大米、0.14g黄粉虫蛹壳、1.00g花生壳、0.25g瓜子壳。在此最佳组合为培养基的条件下得到产孢量为100.013×10~8个/mL菌液，比初始设计提高了31.7%。使用本研究优化后的培养基配方，可以降低生产成本，得到较高产量的孢子，且在生产时无环境污染，对以后的大规模生产非常有利。
综上所述：本研究研究筛选所得黄绿绿僵菌Mf82菌株对褐飞虱成虫、若虫和卵均有较强的致病性，且生产容易、使用方便，是一株极具应用潜力的生防真菌。
The brown planthopper (BPH) Nilaparvata lugens St l is an important rice pest thatcan cause serious damage to the rice production in China and Southeast Asia. it isnecessary to change passive situation, change control strategy and find new biologicalcontrol method to manage the pest. In this paper, a number of entomopathogenic fungistrains were further tested on the basis of existing data and a new strain of Metarhiziumflavoviride (Mf), isolated from N. lugens with higher virulence to BPH was selected. Thebiological characteristics of Mf82and its verulence to different stages of N. lugens werecompleted studied. On the basis of bioassay, and its pathogenesis to BPH was also sudied,using its emulsifiable formulation. the components of solid-state fermentation medium forMf82spore production was optimized in order to lay foundation for large scal productionof fungi insectcide which is used to control N. lugens. The primary results are as follows:
1. The new strain of M. flavoviride Mf, isolated from N. lugens and fiveentomogenous fungi strains of two Beauveria bassiana and three Metarhizium kept in theLaboratory were tested for their cultural characteristics on SDAY and PDA and virulenceto the adults of N.lugens. Results indicated that6strains showed obvious difference onSDAY. Mf grew faster and produced more spores than other strains on SDAY. Its colonydiameter increasing per day was2.59mm and sporulation was17.34×10~7conidia/cm~2.SDAY was more suitable for Mf to grow than PDA. Mf obviously controlled N. lugens. ItsLT50value to the adults of N. lugens was4.47d and adjusted accumulative mortality was83.8%when concentration was1.0×10~8conidia/mL.
2. one isolated strain of M. flavoviride (Mf) which was high virulence to N. Lugenswas identified by Molecular biology method, three different methods were respectivelyused to extract genomic DNA from this fungus: benzyl chloride, CTAB and crackingsolution. Its rDNA ITS sequence was amplified with ITS1and ITS4as primers, and theamplified ITS sequence was detected by agarose gel electrophoresis and was analyzed inGenebank．The results of the experiments showed that the cracking solution method couldobtain the highest quantity of the purest DNA, benzyl chloride method took second place，but CTAB method was not suitable. Homologous sequence contrast was conducted in theDNA sequence database and the result showed that this fungus was M. flavoviride.Identifying the entomogenous fungus species which infects N. lugens would play animportant role for further studying on its biological characteristics and pathogenicity, andcontrolling N. lugens with it.
3. Nine strains of different species of Metarhizium, including strain of M. flavoviride(Mf82) newly isolated from N. lugens, and eight M. anisopliae strains from lab collection,were tested against the adults of N. lugens. The chitinase activities of these strains werealso assayed. The results showed that both the cumulative mortality (82.1%) and thechitinase activity (9.78U/mg) of strain Mf82were the highest among all tested strains.Scanning electron microscope (SEM) observations showed the germinating conidia of thestrain Mf82penetrated the cuticle not only via the intersegmental membranes and foldedregions, but also through the pronotum which contained a large quantity of chitin. Thebiological characteristics of these strains, including growth rate, sporulation, germinationrate and timing of sporulation, were further investigated. The results showed Mf82was thebest strain having strong pathogenicity of N. lugens.
4. In this study, laboratory bioassays were carried out on isolate of theentomopathogenic fungal specie, M. flavoviride82for the virulence to different stages of N.lugens at a series of spore concentration of5296-5368,1055-1121,585-623,126-144and22-31conidia/mm~2respectively and gravid female, female, male and eggs laid wereinfected by suspension of1100conidia/mm~2. The results showed that the pathogenicity ofMf82to adults, nymphs at different instars and egg stage were assessed at a series ofconidia concentration. This strain was most virulent to adults, moderate to old nymphs, andleast to young nymphs. The susceptible sequence was gravid female> female> male. Itwas also found that Mf82could infect both oviposition marks and eggs in rice seedlings,and the infection rate reached66.7%and51.2%respectively at10d after treatment. Theyounger the eggs, the higher the infection rate, with the0.5d-old eggs to be most easilyinfected.
5. The infection process of M. flavoviride conidia to N. lugens was observed usingscanning electron microscope (SEM).The results showed that most of conidia weredeposited on intersegmental membranes, folded regions on cuticle. The germinated conidiaof the isolate invaded the cuticle not only directly with germ tube but by producingappressorium during the course of infection. Then hypha and spores grew on cuticle of N.lugens. Thalluses entered into hemocoel and then made use of nutrition to reproduce inlarge quantities, diffused and infected eggmass of N. lugens. Even though covered withhumoral, hypha infecting eggmass still continued to grow and sporulate, inactivated eggsand inhibited the number of next generation. This phenomenon can show the infectiveability and invasive approach by infection process in vitro and in vivo. It also providesevidence that evaluate insecticidal effect and application prospect of M. flavoviride.
6. Ten strains of entomogenous fungi emulsifiable formulation; a new strain of M.flavoviride Mf82, isolated from N. lugens and six strains of Beauveria bassiana and four ofMetarhizium were bioassayed for its virulence against the adults of N. lugens and theirvirulence compared. The results showed that cumulative mortality (81.7%) of strain Mf82was the highest among all tested strains, the LT50value was4.6d at a concentration of1.0×10~8conidia/mL. On this basis, a kind of emulsifiable formulation of Mf82was studiedin the laboratory, and the bioassay of virulence of Mf82against the N. lugens was carriedout. The results showed that the daily cumulative mortality of N. lugens increased with theinfect concentration of conidial suspension, the highest concentration (1048conidia/mm~2)treatment caused85.0%. A time dose mortality model was used to analyze the bioassaydata and the model fitted the data well, resulting in parameters for estimating the time anddose effects. The estimated LC50values on day7and9after treatment were2.1×103,9.9×102conidia/mm~2, respectively; while LC90were7.8×104，3.7×104conidia/mm~2,respectively. The median lethal time of the emulsifiable formulation of M. flavoviride tothe N. lugens differed at various logarithmic dose, values of LT50of tested agents wereshortened with logarithmic dose, logarithmic dose increased from7.0to8.0, the LT50values reduced from8.9days to5.7days. The results showed that the emulsifiableformulation of M. flavoviride to N. lugens with high virulence.
7. The medium is filled with rice flour, pupa shell of Tenebrio molitor, shell ofgroundnut and shell of sun-flower seed, then summing up the best preservation program. Insolid-phase fermentation conditions, use Design-Expert software analyzing the factors forspores of significant，Optimize M. flavoviride by response surface. As a result, pupa shellof Tenebrio molitor is the significant influence factors. The best preservation program isthat the medium is filled with0.25g rice flour,0.14g pupa shell of Tenebrio molitor,1.00g shell of groundnut,0.25g shell of sun-flower seed. The greatest spore productionquantity is100.013×10~8conidia/mLevery. Using this optimization of the medium afterformula, reduce the production cost, produced higher yield spores. There was no pollutionto environment, so it is beneficial to mass production in future.
To sum up: The Mf82strain which had high pathogenecity to adults、nymphs andeggs of BPH, and what's more, it was easy to yield and use in this paper is a promisingapplication biocontrol agent.
引文
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