技术领域technical field
本发明属于农业科学害虫控制技术领域，具体涉及一种微生物诱捕剂，以及在诱捕雌雄果实蝇、斑潜蝇中的应用。The invention belongs to the technical field of pest control in agricultural science, and in particular relates to a microbial trapping agent and its application in trapping male and female fruit flies and Liriomyza sativae.
背景技术Background technique
双翅目(Diptera)是节肢动物门(Arthropoda)、有颚亚门(Mandibulata)、昆虫纲(Insecta)、有翅亚纲(Pterygota)的1目，是昆虫纲中仅次于鞘翅目、鳞翅目、膜翅目的第四大目。世界已知85000种，全球分布。中国已知4000余种。双翅目昆虫有一对翅膀和一对平衡棒，包括蚊、蝇、虻。常见双翅目一些类群如种蝇、叶潜蝇、果实蝇、麦瘿蚊等的幼虫，都是农业的重要害虫。潜蝇科为害多种豆科植物；实蝇科的许多种类为害柑橘、梨、桃等。Diptera is an order of Arthropoda, Mandibulata, Insecta, and Pterygota. It is second only to Coleoptera and Lepidoptera in Insecta. The fourth largest order of the order Hymenoptera. There are 85,000 known species in the world, with global distribution. More than 4000 species are known in China. Insects of the order Diptera have a pair of wings and a pair of balance bars, including mosquitoes, flies, and gadflies. The larvae of some groups of common Diptera, such as seed fly, leaf miner, fruit fly, wheat gall midge, etc., are important agricultural pests. Liriomypidae damage a variety of leguminous plants; many species of Tephritidae damage citrus, pears, peaches, etc.
实蝇类害虫属于双翅目实蝇科(Tephritidae)，是对果蔬类作物造成严重损失的一类重要害虫。实蝇科迄今为止已知约500属，4500余种，其中，果蔬类有害实蝇的类群达15属、22亚属、150余种，我国以柑橘小实蝇关[Bactrocera dorsalis(Hendel)]，瓜实蝇[Bactrocera cucurbitae(Coqil Lett)]，柑橘大实蝇[Bactrocera minax(Enderlein)]等较为常见，主要分布于湖南、湖北、四川、重庆、贵州、福建、广东、广西、海南、云南、台湾等省。有害实蝇的寄主范围很广，涉及经济水果类植物共计24科、42个属：蔬菜类植物4科、16属(野生寄主植物未包括在内)。实蝇类害虫繁殖力强、危害性大。在实蝇分布或流行区，寄主果实被害率平均为25-45％，严重时高达80％，甚至绝产，给果蔬类作物带来了重大经济损失，严重影响了农民增产创收。Tephritidae pests belong to the family Tephritidae of Diptera, and are an important class of pests that cause serious losses to fruit and vegetable crops. So far, about 500 genera and more than 4,500 species of Tephritidae have been known. Among them, there are 15 genera, 22 subgenuses, and more than 150 species of fruit and vegetable harmful fruit flies. In my country, Bactrocera dorsalis (Hendel) , Bactrocera cucurbitae (Coqil Lett)], Bactrocera minax (Enderlein)] are relatively common, mainly distributed in Hunan, Hubei, Sichuan, Chongqing, Guizhou, Fujian, Guangdong, Guangxi, Hainan, Yunnan , Taiwan and other provinces. The host range of harmful fruit flies is very wide, involving a total of 24 families and 42 genera of economic fruit plants; 4 families and 16 genera of vegetable plants (wild host plants are not included). Fruit fly pests have strong fecundity and great harm. In fruit fly distribution or endemic areas, the host fruit damage rate is 25-45% on average, up to 80% in severe cases, or even extinction, which has brought significant economic losses to fruit and vegetable crops and seriously affected farmers' increase in production and income.
目前，防治实蝇类害虫的方法主要有农业防治、化学防治、物理防治和生物防治等。其中农业防治包括定期清除种植园内的带虫果实、果实套袋和冬季翻耕灭蛹等；化学防治措施主要是喷施毒饵杀灭实蝇；物理防治措施采用实蝇诱捕剂和毒饵相结合的技术，目前，用于防治实蝇的诱捕剂主要是化学合成诱捕剂，如引诱酮(cue lure)、甲基丁香酚(methyleugenol)等。由于农业防治措施费时费工，效果不好；化学防治不仅容易使实蝇产生抗药性，而且对人、畜有害，易产生农药残留等环境污染的问题；在实际应用中性诱捕剂又存在挥发快、持效期短、用药频繁等问题，达不到控制蛀果率的防治目的，而且目前应用最广泛诱捕剂甲基丁香酚只引诱雄虫等等，其应用受到限制。At present, the methods of controlling fruit fly pests mainly include agricultural control, chemical control, physical control and biological control. Among them, agricultural control includes regular removal of insect-bearing fruits in the plantation, fruit bagging, plowing in winter to kill pupae, etc.; chemical control measures are mainly spraying poisonous baits to kill fruit flies; physical control measures use a combination of fruit fly traps and poison baits Currently, the traps used to control fruit flies are mainly chemically synthesized traps, such as cue lure and methyleugenol. Because agricultural control measures are time-consuming and labor-intensive, the effect is not good; chemical control not only easily makes fruit flies resistant to pesticides, but also is harmful to humans and animals, and is prone to environmental pollution such as pesticide residues; Fast, short duration, frequent use of drugs and other problems, can not reach the control purpose of controlling fruit moth rate, and currently the most widely used trapping agent methyl eugenol only lures males, etc., its application is limited.
国内外针对实蝇有糖醋液、雌性激素、蛋白质三大类诱剂。糖醋液费时、费力，成本较高；雌性激素只能诱雄虫，由于雄虫能行多次交配，防效常不太理想；蛋白质类诱捕剂可以引诱雌雄虫，效果好。但目前国外市场上的蛋白质诱剂为纯净动物蛋白，生产成本高。At home and abroad, there are three types of lures for fruit flies: sweet and sour liquid, estrogen, and protein. Sweet and sour liquid is time-consuming, laborious, and costly; estrogen can only attract male insects, because male insects can mate multiple times, the control effect is often not ideal; protein traps can attract both male and female insects, and the effect is good. However, the protein attractant currently on the foreign market is pure animal protein, and the production cost is high.
美洲斑潜蝇(Liriomyza sativae Blanchard)也属于双翅目潜蝇科(Agromyzidae)斑潜蝇属(Liriomyza)，俗称蔬菜斑潜蝇、地图虫、鬼画符等，是潜叶为害的危险性害虫。我国最早于1993年12月在海南省三亚市首次发现，随后美洲斑潜蝇逐渐向北扩散，1994年在云南昆明发生为害，1995年在上海、南京等地出现，1996年在山东、北京暴发成灾，1997年在河南、山西出现，1998年在东北、内蒙古成灾，除西藏外，全国均有分布。它的寄主植物多达200余种，其中蔬菜以茄果类(甜椒、辣椒除外)、豆类、瓜类、伞形花科等受害最重。Liriomyza sativae Blanchard also belongs to the genus Liriomyza of the family Agromyzidae of the order Diptera. It is commonly known as vegetable leafminer, map insect, ghost painting charm, etc. It is a dangerous pest that damages leaves. In my country, it was first discovered in Sanya City, Hainan Province in December 1993, and then gradually spread to the north. In 1994, it occurred in Kunming, Yunnan, in Shanghai, Nanjing and other places in 1995, and in Shandong and Beijing in 1996. Disasters occurred in Henan and Shanxi in 1997, and in Northeast China and Inner Mongolia in 1998. Except Tibet, they are distributed all over the country. It has more than 200 host plants, among which vegetables such as solanaceous fruits (except sweet peppers and peppers), beans, melons, and Umbelliferae are the most severely damaged.
美洲斑潜蝇成虫为小型蝇类，体黑色，体长1.3～2.3毫米，寿命8.7～12.6天，每雌一生产卵70～125枚，孵化率高达90％以上。幼虫乳白色至淡黄色，长3毫米左右，年繁殖15～20代。冬季进入日光温室内，可四季危害，主要有2次危害高峰，分别出现在5～6月和9～10月，年世代可达20代以上。成虫、幼虫均可危害，雌成虫在寄主叶片上刺孔并在其中取食和产卵，幼虫在叶片表皮下孵化后潜食叶肉或叶柄，产生不规则的蛇形白色虫道，破坏叶片叶绿素，影响光合作用，严重时叶片枯黄脱落甚至整株枯死；苗期造成毁苗。危害严重田块受害株100％，叶片受害70％。盛产期西葫芦，在发现危害后，7天左右吃尽叶肉，15天左右毁灭。幼虫和成虫通过取食还可传播某些病毒病。The adults of Liriomyza sativae are small flies with a black body, a body length of 1.3-2.3 mm, and a life span of 8.7-12.6 days. Each female produces 70-125 eggs, and the hatching rate is as high as 90%. The larvae are milky white to pale yellow, about 3 mm long, and reproduce 15 to 20 generations per year. Entering the solar greenhouse in winter, it can be harmed in four seasons. There are mainly two harm peaks, which appear in May-June and September-October respectively, and the annual generation can reach more than 20 generations. Adults and larvae can be harmful. Female adults pierce holes in the host leaves and feed and lay eggs in them. Larvae hatch under the leaf epidermis and sneak into the mesophyll or petiole, producing irregular snake-shaped white insect passages that destroy the chlorophyll of the leaves. , affect photosynthesis, and in severe cases, the leaves will wither and fall off, or even the whole plant will die; the seedlings will be destroyed at the seedling stage. 100% of the damaged plants and 70% of the leaves were damaged seriously. Zucchini in the rich production period will eat up the mesophyll in about 7 days and destroy them in about 15 days after the damage is discovered. Larvae and adults can also transmit certain viral diseases through feeding.
目前，随着耕作制度的变化，持续暖冬现象和露地及保护地蔬菜面积的不断扩大，潜叶蝇已成为多种蔬菜上危害特大、防治特困难的灾害性害虫之一，全年发生世代多而重叠混乱，传播蔓延快，大田春末夏初秋季保护地全年发生尤其严重。蔬菜的保护地栽培为蔬菜斑潜蝇提供了良好的越冬场所。该虫不但可在大棚内越冬，而且可在棚内发生危害，成为露地蔬菜田的重要虫源。因此,搞好大棚内的防治可有效地防止该虫在揭棚后向露地蔬菜扩散危害。At present, with the change of farming system, continuous warm winter phenomenon and the continuous expansion of open field and protected vegetable area, leaf miner has become one of the most harmful and difficult to control disastrous pests on various vegetables. The overlap is chaotic, and the spread spreads quickly, especially in Daejeon protected areas throughout the year in late spring, early summer and early autumn. The protected cultivation of vegetables provides a good overwintering place for Liriomyza sativae. The insect can not only survive the winter in the greenhouse, but also cause harm in the greenhouse, becoming an important source of insects in the open vegetable field. Therefore, doing a good job in the control of the greenhouse can effectively prevent the pest from spreading to the vegetables in the open field after the shed is uncovered.
因该虫世代重叠现象严重，抗药性强，生产中较难防治。目前，生产上对美洲斑潜蝇的防治仍主要是以化学防治为主，为了解决多年来化学农药带来的食品安全问题，应利用生物防治措施来控制虫害，但生物防治技术还没有真正在生产上应用和发挥效益，寻找针对性强，防效高，以及对人、畜安全，没有环境污染的一种新诱剂迫在眉睫。Due to the serious overlapping of generations of this insect and its strong drug resistance, it is difficult to control it in production. At present, the control of Liriomyza sativae in production is still mainly based on chemical control. In order to solve the food safety problems caused by chemical pesticides for many years, biological control measures should be used to control insect pests, but biological control technology has not really been used. It is imminent to find a new lure that has strong pertinence, high control efficiency, safety to humans and animals, and no environmental pollution to apply and exert benefits in production.
发明内容Contents of the invention
针对现有技术的需要，本发明的目的是提供一种适用于诱捕实蝇和果蝇类害虫、可雌雄同捕的新型微生物诱捕剂。Aiming at the needs of the prior art, the purpose of the present invention is to provide a novel microbial trap suitable for trapping fruit flies and fruit fly pests and capable of catching both sexes.
为实现上述发明目的，本发明的技术方案如下：For realizing the above-mentioned purpose of the invention, the technical scheme of the present invention is as follows:
1.一种微生物诱捕剂，其活性成分为解淀粉芽孢杆菌(Bacillusamyloliquefaciens)YN2010-KC CGMCC No.5722的发酵液。1. A microbial trapping agent, whose active ingredient is the fermented liquid of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) YN2010-KC CGMCC No.5722.
2.根据技术方案1所述的一种微生物诱捕剂，所述解淀粉芽孢杆菌(Bacillusamyloliquefaciens)YN2010-KC CGMCC No.5722的发酵液通过以下方法制备得到：2. according to a kind of microbial trap described in technical scheme 1, the fermented liquid of described bacillus amyloliquefaciens (Bacillus amyloliquefaciens) YN2010-KC CGMCC No.5722 is prepared by the following method:
(1)将解淀粉芽孢杆菌(Bacillus amyloliquefaciens)YN2010-KC CGMCCNo.5722活化后，按5～10％接种量，将活化的所述解淀粉芽孢杆菌(Bacillusamyloliquefaciens)YN2010-KC CGMCC No.5722接种到种子培养基，在培养温度35℃、转速120rpm/min条件下，摇床培养36～48h，得到一级种子液，所述种子培养基为液体LB培养基，pH 7.0～7.2；(1) After activating Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) YN2010-KC CGMCC No.5722, inoculate the activated Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) YN2010-KC CGMCC No.5722 into the The seed culture medium is cultivated on a shaking table for 36-48 hours at a culture temperature of 35°C and a rotation speed of 120 rpm/min to obtain a first-class seed liquid. The seed culture medium is a liquid LB medium with a pH of 7.0-7.2;
(2)按10～12％接种量，将一级种子液转接到种子罐内的二级种子培养基，在28～37℃，转速100rpm/min，通气量为1000L/h，罐压保持在0.02～0.03MPa条件下培养36～48h，得到二级种子液；所述二级种子培养基由以下质量分数的以下成分组成：玉米粉0.3～0.5％，黄豆粉1～1.5％，蔗糖0.2～0.5％，蛋白胨0.1～0.3％，鱼粉0.1～0.3％，MgSO4·7H2O 0.02～0.04％，CaCO3 0.4～0.8％，(NH4)2SO4 0.03～0.06％，余量为水，pH 7.0～7.2；(2) Transfer the primary seed solution to the secondary seed medium in the seed tank at 10-12% inoculum amount, at 28-37°C, with a rotation speed of 100rpm/min, an air flow of 1000L/h, and a tank pressure of 1000L/h. Cultivate for 36-48 hours under the condition of 0.02-0.03MPa to obtain the secondary seed liquid; the secondary seed medium is composed of the following components in the following mass fractions: 0.3-0.5% of corn flour, 1-1.5% of soybean flour, and 0.2% of sucrose ~0.5%, peptone 0.1~0.3%, fish meal 0.1~0.3%, MgSO4 ·7H2 O 0.02~0.04%, CaCO3 0.4~0.8%, (NH4 )2 SO4 0.03~0.06%, the balance is water , pH 7.0～7.2;
(3)按10～12％接种量，将二级种子液接种到发酵罐内的发酵培养基，在28～37℃，转速100rpm/min，通气量为10000L/h、罐压保持在0.02～0.03MPa条件下培养48～72h，培养后的发酵液中解淀粉芽孢杆菌(Bacillus amyloliquefaciens)YN2010-KC CGMCCNo.5722的有效活菌数≧3×109cfu/ml时，该发酵液即为所述解淀粉芽孢杆菌(Bacillusamyloliquefaciens)YN2010-KC CGMCC No.5722的发酵液，所述发酵培养基与步骤(2)所述二级种子培养基相同。(3) Inoculate the secondary seed liquid into the fermentation medium in the fermenter according to 10-12% inoculum amount, at 28-37°C, the rotating speed is 100rpm/min, the ventilation rate is 10000L/h, and the tank pressure is maintained at 0.02- Cultivate under the condition of 0.03MPa for 48～72h, when the number of effective viable bacteria of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens)YN2010 -KC CGMCCNo. The fermentation broth of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) YN2010-KC CGMCC No.5722 is described, and the fermentation medium is the same as the secondary seed medium described in step (2).
3.技术方案1或2所述的微生物诱捕剂在防治实蝇类害虫的应用。3. The application of the microbial trapping agent described in technical scheme 1 or 2 in the control of fruit fly pests.
4.技术方案1或2所述的微生物诱捕剂在防治美洲斑潜蝇的应用。4. The application of the microbial trap described in technical scheme 1 or 2 in the control of Liriomyza sativae.
5.根据技术方案3所述的微生物诱捕剂在防治实蝇类害虫的应用，所述的应用是将所述微生物诱捕剂装入塑料瓶内，每瓶的装入量为50～100mL，旋紧瓶盖，在距离瓶内液面1～3cm的瓶壁上均布4个孔，所述各孔的孔径为4～6mm；将塑料瓶悬挂于离地面0.5～1.5米处，每亩悬挂塑料瓶8～10个，相邻的塑料瓶间隔5～7m；每隔20～30天向塑料瓶内添加一次所述微生物诱捕剂25～50mL。5. According to the application of the microbial trapping agent described in technical scheme 3 in the prevention and control of fruit fly pests, the application is to pack the microbial trapping agent into a plastic bottle, and the loading capacity of each bottle is 50-100mL. Tighten the bottle cap, and evenly distribute 4 holes on the bottle wall 1-3cm away from the liquid surface in the bottle, the diameter of each hole is 4-6mm; hang the plastic bottle at a place 0.5-1.5 meters above the ground, There are 8-10 plastic bottles, and the interval between adjacent plastic bottles is 5-7m; add 25-50mL of the microbial trapping agent to the plastic bottle once every 20-30 days.
与现有技术相比，本发明具有以下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
1、本发明产品散发出的特殊气味能引诱实蝇和果蝇取食，可雌雄同捕，作用周期长，一次性投料可以两周内持效，且成本低，具有较强的竞争力。对柑橘小实蝇诱捕：比果瑞特果蝇引诱剂增加23.71％，比甲基丁香酚增加18.81％。比果瑞特果蝇引诱剂诱到的雌虫增加37.78％，比甲基丁香酚诱到的雌虫增加30倍。对柑橘大实蝇诱捕：比甲基丁香酚增加64.43％，诱捕的柑橘大实蝇雌虫数量比甲基丁香酚增加33.25倍。对美洲斑潜蝇的诱捕：雌虫率占55～60％，产生了预料不到的技术效果。甲基丁香酚诱不到斑潜蝇，只能用糖醋液为对照，而后者也能诱到雌雄虫，只是总虫量比本产品低很多。1. The special smell emitted by the product of the present invention can attract fruit flies and fruit flies to feed, and can be caught by both sexes. The action cycle is long, and the one-time feeding can last for two weeks, and the cost is low, so it has strong competitiveness. Trapping of Bactrocera citrus: 23.71% increase in Bigorit Drosophila attractant, 18.81% increase in Bimethyl eugenol. The number of females attracted by the fruit fly attractant increased by 37.78%, which was 30 times higher than that attracted by methyl eugenol. Trapping of Bactrocera citrus: 64.43% more than methyl eugenol, and the number of females of Bactrocera citrus trapped is 33.25 times higher than that of methyl eugenol. Trapping of Liriomyza sativae: the female rate accounts for 55-60%, which has produced unexpected technical effects. Methyl eugenol could not attract Liriomyza sativae, only sweet and sour solution could be used as a control, and the latter could also attract both male and female insects, but the total number of insects was much lower than this product.
2、本发明产品中不含有任何化学有毒药剂，不杀死和不杀伤任何其他非靶标生物，属于环境友好型果实蝇诱捕剂。2. The product of the present invention does not contain any chemical poisonous agents, does not kill or injure any other non-target organisms, and belongs to the environment-friendly trapping agent for fruit flies.
3、本发明产品完全可以取代化学杀虫药剂，大大减少化学农药用量，从而减少化学农药对天敌昆虫的负面影响，无抗药性。3. The product of the present invention can completely replace chemical insecticides, greatly reduce the amount of chemical pesticides, thereby reducing the negative impact of chemical pesticides on natural enemy insects, and has no drug resistance.
4、本发明产品可提高农产品质和产量，对生态安全，生态效益显著，使用简单。4. The product of the present invention can improve the quality and output of agricultural products, and has remarkable ecological benefits for ecological safety and is easy to use.
本发明所述的解淀粉芽孢杆菌Bacillus amyloliquefaciens YN2010-KC于2012年1月12日保藏在中国微生物菌种保藏管理委员会普通微生物中心(简称：CGMCC)，保藏号为CGMCC No.5722，并于2012年1月12日检测为存活。该普通微生物中心地址：中国北京市朝阳区北辰西路1号院3号，邮编:100101。The Bacillus amyloliquefaciens YN2010-KC described in the present invention was preserved in the General Microbiology Center (abbreviation: CGMCC) of the China Microbiological Culture Collection Management Committee on January 12, 2012, and the preservation number is CGMCC No.5722, and was deposited in 2012 It was detected as alive on January 12. The address of the General Microbiology Center: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China, postal code: 100101.
具体实施方式Detailed ways
下面结合实施例对本发明提供的一种微生物诱捕剂进行详细说明，但并不对本发明构成限制，本领域技术人员在此基础上改进工艺参数，改变具体的辅料类型等替换和改动，依旧属于本发明的保护范围。各实施例无特殊说明的为常规方法。A microbial trap provided by the present invention will be described in detail below in conjunction with the examples, but it does not constitute a limitation to the present invention. Those skilled in the art can improve the process parameters on this basis, change specific auxiliary material types and other replacements and changes, which still belong to this invention. protection scope of the invention. Those without special instructions in each embodiment are conventional methods.
实施例1 解淀粉芽孢杆菌(Bacillus amyloliquefaciens)YN2010-KC的获得、鉴定和保藏Example 1 Acquisition, Identification and Preservation of Bacillus amyloliquefaciens YN2010-KC
1、本发明解淀粉芽孢杆菌YN2010-KC的获得1. Obtaining of Bacillus amyloliquefaciens YN2010-KC of the present invention
1.1材料1.1 Materials
采自云南农业大学后山农场玉米根际周围的土壤。The soil was collected from the soil around the corn rhizosphere of the Houshan Farm of Yunnan Agricultural University.
1.2培养基制备1.2 Culture medium preparation
将LB固体培养基和PSA培养基(马铃薯蔗糖琼脂培养基)融化，分别倒入灭菌培养皿，冷却，制成LB和PSA平板。Melt LB solid medium and PSA medium (potato sucrose agar medium), pour them into sterilized petri dishes, cool down, and make LB and PSA plates.
1.3分离培养1.3 Isolation and culture
采用稀释平皿分离法和平板划线分离，将分离纯化所得的一菌株命名为YN2010-KC。A strain isolated and purified was named YN2010-KC by using dilution plate separation method and plate streak separation.
2、解淀粉芽孢杆菌YN2010-KC的鉴定2. Identification of Bacillus amyloliquefaciens YN2010-KC
2.1、形态学鉴定2.1. Morphological identification
取菌株YN2010-KC在LB培养基上培养，37℃培养36小时，进行形态观察鉴定，菌体为杆状、端圆，周生鞭毛、具运动性，产生椭圆形芽孢；在LB和PSA平板上菌落圆形、淡茶褐色、菌落中央颜色略深、隆起、表面干燥有褶皱。与《常见细菌系统鉴定手册》(东秀珠等编著，科学出版社，2001年)中描述的芽孢杆菌属形态特征基本一致，初步判断菌株YN2010-KC属于芽孢杆菌(Bacillus spp.)。The bacterial strain YN2010-KC was cultured on LB medium, cultured at 37°C for 36 hours, and then observed and identified by morphological observation. The upper colony is round, light brown, and the center of the colony is slightly darker, raised, and the surface is dry and wrinkled. It is basically consistent with the morphological characteristics of Bacillus described in "Common Bacteria System Identification Manual" (edited by Dong Xiuzhu, Science Press, 2001), and it is preliminarily judged that the bacterial strain YN2010-KC belongs to Bacillus spp.
2.2、生理生化鉴定2.2. Physiological and biochemical identification
菌株革兰氏染色、芽孢染色、淀粉水解、葡萄糖利用、柠檬酸盐利用、V-P反应、接触酶反应、硝酸盐还原、明胶液化等均呈阳性。Gram staining, spore staining, starch hydrolysis, glucose utilization, citrate utilization, V-P reaction, catalase reaction, nitrate reduction, gelatin liquefaction, etc. were all positive.
3、解淀粉芽孢杆菌YN2010-KC的保藏3. Preservation of Bacillus amyloliquefaciens YN2010-KC
经上述鉴定表明所分离获得的菌株为解淀粉芽孢杆菌(Bacillusamyloliquefaciens)，其代号为YN2010-KC，所述的解淀粉芽孢杆菌Bacillusamyloliquefaciens YN2010-KC于2012年1月12日保藏在中国微生物菌种保藏管理委员会普通微生物中心(简称：CGMCC)，保藏号为CGMCC No.5722，并于2012年1月12日检测为存活。该普通微生物中心地址：中国北京市朝阳区北辰西路1号院3号，邮编:100101。The above-mentioned identification shows that the isolated bacterial strain is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), its code name is YN2010-KC, and the described Bacillus amyloliquefaciens YN2010-KC was preserved in China Microorganism Culture Collection on January 12, 2012. General Microbiology Center of the Management Committee (abbreviation: CGMCC), the preservation number is CGMCC No.5722, and it was detected as alive on January 12, 2012. The address of the General Microbiology Center: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China, postal code: 100101.
实施例2 本发明微生物诱捕剂的制备方法，包括以下步骤：Embodiment 2 The preparation method of microbial trapping agent of the present invention comprises the following steps:
(1)将-80℃保存的解淀粉芽孢杆菌(Bacillus amyloliquefaciens)YN2010-KCCGMCC No.5722接种在液体LB培养基上，35℃培养24h，得活化的解淀粉芽孢杆菌(Bacillusamyloliquefaciens)YN2010-KC CGMCC No.5722，按10％接种量，将活化的所述解淀粉芽孢杆菌(Bacillus amyloliquefaciens)YN2010-KC CGMCC No.5722接种到种子培养基，在培养温度35℃、转速120rpm/min条件下，摇床培养36h，得到一级种子液，所述种子培养基为液体LB培养基，pH 7.0～7.2；(1) Inoculate Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) YN2010-KCCGMCC No.5722 stored at -80°C on liquid LB medium, and culture at 35°C for 24 hours to obtain activated Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) YN2010-KC CGMCC No.5722, inoculate the activated Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) YN2010-KC CGMCC No.5722 into the seed medium according to 10% inoculation amount, and shake bed culture for 36 hours to obtain a primary seed solution, the seed medium is a liquid LB medium, pH 7.0-7.2;
(2)按10％接种量，将一级种子液转接到种子罐内的二级种子培养基，在35℃，转速100rpm/min，通气量为1000L/h，罐压保持在0.02～0.03MPa条件下培养36～48h，得到二级种子液；得到二级种子液；所述二级种子培养基由以下质量分数的以下成分组成：玉米粉0.5％，黄豆粉1％，蔗糖0.5％，蛋白胨0.3％，鱼粉0.1％，MgSO4·7H2O 0.02％，CaCO30.4％，(NH4)2SO4 0.03％，余量为水，pH 7.0～7.2；所述二级种子培养基于121℃灭菌30min，待降温至30℃，接入一级种子液；(2) Transfer the primary seed solution to the secondary seed medium in the seed tank at 35°C with a rotation speed of 100 rpm/min, an air flow of 1000 L/h, and a tank pressure of 0.02 to 0.03 at 10% inoculum size. Cultivate under MPa condition for 36～48h, obtain secondary seed liquid; obtain secondary seed liquid; described secondary seed culture medium is made up of the following components of following mass fraction: corn flour 0.5%, soybean flour 1%, sucrose 0.5%, Peptone 0.3%, fish meal 0.1%, MgSO4 7H2 O 0.02%, CaCO3 0.4%, (NH4 )2 SO4 0.03%, the balance is water, pH 7.0-7.2; the secondary seed culture is based on 121 Sterilize at ℃ for 30 minutes, wait until the temperature drops to 30℃, and insert the first-grade seed solution;
(3)按10％接种量，将二级种子液接种到发酵罐内的发酵培养基，在35℃，转速100rpm/min，通气量为10000L/h、罐压保持在0.02～0.03MPa条件下培养48～72h，培养后的发酵液中解淀粉芽孢杆菌(Bacillus amyloliquefaciens)YN2010-KC CGMCC No.5722的有效活菌数≧3×109cfu/ml时，将发酵液用医用塑料瓶包装，即为所述微生物诱捕剂，所述发酵培养基与步骤(2)所述二级种子培养基相同。(3) According to 10% inoculum amount, inoculate the secondary seed liquid into the fermentation medium in the fermenter, at 35°C, the rotation speed is 100rpm/min, the ventilation rate is 10000L/h, and the tank pressure is maintained at 0.02-0.03MPa. Cultivate for 48-72 hours, when the number of effective viable bacteria of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) YN2010-KC CGMCC No.5722 in the cultured fermentation broth is ≧3×109 cfu/ml, pack the fermentation broth with a medical plastic bottle, It is the microorganism trapping agent, and the fermentation medium is the same as the secondary seed medium in step (2).
本发明微生物诱捕剂通过实施例2所述的微生物诱捕剂的制备方法制备得到。The microbial trapping agent of the present invention is prepared by the preparation method of the microbial trapping agent described in Example 2.
实施例3 本发明微生物诱捕剂与其它诱剂对柑橘小实蝇诱捕的对比试验Embodiment 3 The comparative test of microbial traps of the present invention and other attractants on the trapping of Bactrocera citrus
在云南省元江县柑橘园内进行诱捕效果试验，同时与“果瑞特”果蝇引诱剂，(属蛋白饵剂，市售)、甲基丁香酚(属性诱剂，市售)和水(空白对照)进行对比。本发明微生物诱捕剂用量50ml/瓶，果瑞特果蝇引诱剂用量50ml/瓶，甲基丁香酚用量3ml/瓶。Carry out trapping effect test in the citrus orchard of Yuanjiang County, Yunnan Province, simultaneously with " Guo Ruite " fruit fly attractant, (belong to albumen bait, commercially available), methyl eugenol (property attractant, commercially available) and water ( blank control) for comparison. The dosage of the microorganism trapping agent of the present invention is 50ml/bottle, the dosage of the fruit fly attractant is 50ml/bottle, and the dosage of methyl eugenol is 3ml/bottle.
每个塑料瓶装上述一种引诱剂，旋紧瓶盖，在距离瓶内液面2cm的瓶壁上均布4个孔，所述各孔的孔径为5mm。于2015年5月16日中午进行田间释放，将塑料瓶悬挂于离柑橘地0.5-1.5米处。两天后诱到柑橘小实蝇数量(头)见表1。可见，本发明微生物诱捕剂诱虫数量均高于果瑞特果蝇引诱剂和甲基丁香酚，且雌虫率高达51.67％。本发明微生物诱捕剂诱虫数量比果瑞特果蝇引诱剂增加23.71％，比甲基丁香酚增加18.81％。本发明微生物诱捕剂比果瑞特果蝇引诱剂诱到的雌虫增加37.78％，比甲基丁香酚诱到的雌虫增加30倍。Each plastic bottle is filled with the above-mentioned attractant, the bottle cap is tightened, and 4 holes are evenly distributed on the bottle wall 2 cm away from the liquid surface in the bottle, and the aperture of each hole is 5 mm. Field release was carried out at noon on May 16, 2015, and the plastic bottles were hung 0.5-1.5 meters away from the citrus field. See Table 1 for the number (head) of Bactrocera citrus fruit flies lured two days later. It can be seen that the number of insects attracted by the microbial trap of the present invention is higher than that of the fruit fly attractant and methyl eugenol, and the female insect rate is as high as 51.67%. The number of insects attracted by the microbial trap of the present invention is 23.71% higher than that of the fruit fly attractant and 18.81% higher than that of methyl eugenol. The microbial trapping agent of the present invention has a 37.78% increase in the number of females attracted by the fruit fly attractant and a 30-fold increase in the number of females attracted by the methyl eugenol.
表1 本发明微生物诱捕剂对柑橘小实蝇引诱试验Table 1 Microbe trapping agent of the present invention is to citrus Bactrocera lure test
实施例4 本发明微生物诱捕剂对柑橘果实蝇诱捕试验Embodiment 4 Microbial trapping agent of the present invention traps test of citrus fruit fly
在云南省河口县南溪农场进行果实蝇诱捕试验，试验试剂：本发明微生物诱捕剂。装试剂的塑料瓶及操作与实施例3相同，本发明微生物诱捕剂用量50ml/瓶，于2014年7月5日中午装有试剂的塑料瓶悬挂于离柑橘地0.5-1.5米处，自投料后1天、2天和3天调查各瓶中虫量见表2，可见，本发明诱捕剂发酵产生挥发性蛋白质类等化合物引诱果实蝇食用，效果显著，且雌雄同诱。Carry out fruit fly trapping test in Nanxi Farm, Hekou County, Yunnan Province, test reagent: microbial trapping agent of the present invention. The plastic bottle and the operation of the reagent are the same as in Example 3. The amount of microbial trapping agent of the present invention is 50ml/bottle. After 1 day, 2 days and 3 days, the amount of worms in each bottle of investigation is shown in Table 2, as seen, the compounds such as volatile proteins produced by the fermentation of the trap of the present invention attract fruit flies to eat, and the effect is remarkable, and both sexes are attracted.
表2 本发明微生物诱捕剂诱捕果实蝇(柑橘大实蝇、小实蝇、番石榴实蝇、瓜实蝇等)的效果Table 2 The effect of microbial traps of the present invention on trapping fruit flies (Bactrocera citrus, Bactrocera cerevisiae, Bactrocera japonica, Bactrocera melon, etc.)
实施例5 本发明微生物诱捕剂对柑橘大实蝇防治效果的对比试验Embodiment 5 The comparison test of microbial trapping agent of the present invention to the control effect of Bactrocera citrus
在云南省蒙自县柑橘园内进行诱捕柑橘大实蝇试验，同时与甲基丁香酚进行对比。本发明微生物诱捕剂用量50ml/瓶，甲基丁香酚用量50ml/瓶。In the citrus orchard of Mengzi County, Yunnan Province, the experiment of trapping Bacterophae citrus was carried out, and compared with methyl eugenol. The dosage of the microorganism trapping agent of the present invention is 50ml/bottle, and the dosage of methyl eugenol is 50ml/bottle.
装各试剂的塑料瓶及操作与实施例3相同，于2014年12月4日中午将装有试剂的塑料瓶悬挂于离柑橘地0.5-1.5米处，自投料后1天、2天和3天调查各瓶中虫量见表3，可见，用本发明微生物诱捕剂发酵产生挥发性蛋白质类等化合物引诱柑橘大实蝇食用，除第一天诱虫量低于性诱剂甲基丁香酚外，其它时间内诱捕大实蝇的效果均优于性诱剂甲基丁香酚，且雌雄同诱。三天内，本发明微生物诱捕剂诱捕柑橘大实蝇数量比甲基丁香酚增加64.43％，诱捕的柑橘大实蝇雌虫数量比甲基丁香酚增加33.25倍。The plastic bottles and operation of each reagent are the same as in Example 3. At noon on December 4, 2014, the plastic bottles with reagents were suspended at 0.5-1.5 meters away from the citrus field. The amount of worms in each bottle of the daily investigation is shown in Table 3. As can be seen, compounds such as volatile proteins produced by the fermentation of the microbial trap of the present invention are used to lure Bactrocera citrus to eat, except that the amount of insects attracted is lower than that of the sex attractant methyl eugenol on the first day. In addition, the effect of trapping Bactrocera is superior to the sex attractant methyl eugenol in other time periods, and the sex is lured equally. Within three days, the number of Bactrocera citrus flies trapped by the microbial trapping agent of the present invention is 64.43% higher than that of methyl eugenol, and the number of females of Bactrocera citrus trapped is 33.25 times higher than that of methyl eugenol.
表3 本发明微生物诱捕剂诱捕柑橘大实蝇的效果Table 3 Microbial trapping agent of the present invention traps the effect of Bactrocera citrus
实施例6 本发明微生物诱捕剂与黄板田间诱虫对比试验Embodiment 6 Comparison test of microbial trapping agent of the present invention and yellow board field trapping insects
在云南省蒙自县番茄蔬菜基地上进行诱捕效果试验，同田与30×30cm黄板进行了对比。每个黄色塑料瓶装50ml本发明微生物诱捕剂，旋紧瓶盖，在距离瓶内液面2cm处的瓶壁上均布4个孔，所述各孔的孔径为5mm。于2015年6月16日中午进行田间释放，将塑料瓶悬挂于距地面0.5～0.8米高，每亩悬挂塑料瓶8个，相邻的塑料瓶间隔5～10m；在同田，将黄板悬挂于距地面0.5-0.8米高，每亩悬挂黄板8个，相邻的黄板间隔5～10m。每两天后诱到美洲斑潜蝇数量(头)见表4。可见，本发明微生物诱捕剂平均每天诱虫数量均高于黄板，8天内，本发明微生物诱捕剂诱虫1988头，黄板诱虫379头，是黄板诱虫量的5.2倍。The trapping effect test was carried out on the tomato and vegetable base in Mengzi County, Yunnan Province, and the same field was compared with the 30×30cm yellow board. Each yellow plastic bottle packs 50ml of the microorganism trapping agent of the present invention, and the bottle cap is tightened, and 4 holes are evenly distributed on the bottle wall at 2cm from the liquid surface in the bottle, and the aperture of each hole is 5mm. Released in the field at noon on June 16, 2015, hanging plastic bottles at a height of 0.5-0.8 meters above the ground, hanging 8 plastic bottles per mu, with an interval of 5-10m between adjacent plastic bottles; Hang at a height of 0.5-0.8 meters from the ground, hang 8 yellow boards per mu, and the interval between adjacent yellow boards is 5-10m. See Table 4 for the number (head) of Liriomyza sativae lured after every two days. It can be seen that the average daily number of insects attracted by the microbial trap of the present invention is higher than that of the yellow board. In 8 days, the microbial trap of the present invention attracted 1988 insects and 379 insects were attracted by the yellow board, which is 5.2 times the amount of insects attracted by the yellow board.
表4 本发明微生物诱捕剂和黄板对美洲斑潜蝇引诱的对比试验Table 4 The comparative test of microbial trapping agent of the present invention and yellow plate to attracting Liriomyza sativae
实施7 本发明微生物诱捕剂对美洲斑潜蝇雌雄数量诱捕试验Implementation 7 The microbial trapping agent of the present invention traps the male and female numbers of Liriomyza sativae
在云南省江川县牛魔村蔬菜基地进行豇豆美洲斑潜蝇诱捕试验，将50ml本发明微生物诱捕剂直接加入外壁贴有黄色胶带塑料瓶中，旋紧瓶盖，在距离瓶内液面2cm处的瓶壁上均布4个孔，所述各孔的孔径为5mm。于2015年2月3日中午悬挂于距离地面0.5～0.8米高，自投料后1天、2天和3天调查各瓶中虫量见表5，可见，用本发明微生物诱捕剂发酵产生的挥发性蛋白质类等化合物引诱美洲斑潜蝇食用，效果显著，且雌雄同诱，雌虫率达55～60％。Carry out the trapping test of cowpea Liriomyza americana at the vegetable base of Niumo Village, Jiangchuan County, Yunnan Province, directly add 50ml of microbial trapping agent of the present invention in the plastic bottle with yellow adhesive tape on the outer wall, tighten the bottle cap, and place it at a distance of 2cm from the liquid surface in the bottle 4 holes are uniformly distributed on the bottle wall of the bottle, and the aperture of each hole is 5mm. At noon on February 3, 2015, it was suspended at a height of 0.5 to 0.8 meters from the ground, and the amount of insects in each bottle was investigated in Table 5 after 1 day, 2 days and 3 days after feeding. Compounds such as volatile proteins lure Liriomyza sativae to eat, the effect is remarkable, and both sexes are attracted, and the female rate reaches 55-60%.
表5 本发明微生物诱捕剂捕雌雄美洲斑潜蝇统计Table 5 Microbe trapping agent of the present invention catches statistics of male and female Liriomyza sativae
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| CN201510958305.8ACN105410032B (en) | 2015-12-21 | 2015-12-21 | A kind of microorganism trapping bait and application |
| PCT/CN2016/110474WO2017107872A1 (en) | 2015-12-21 | 2016-12-16 | Microbial attractant, and use |
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| CN201510958305.8ACN105410032B (en) | 2015-12-21 | 2015-12-21 | A kind of microorganism trapping bait and application |
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| PE01 | Entry into force of the registration of the contract for pledge of patent right | Denomination of invention:A Microbial Trapping Agent and Its Application Effective date of registration:20231120 Granted publication date:20180424 Pledgee:Kunming Dongfeng Sub branch of Bank of China Ltd. Pledgor:MICROBIAL FERMENTATION ENGINEERING RESEARCH CENTER Co.,Ltd. OF YUNNAN PROVINCE Registration number:Y2023530000066 | |
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| PE01 | Entry into force of the registration of the contract for pledge of patent right | Denomination of invention:A microbial trapping agent and its application Granted publication date:20180424 Pledgee:Kunming Dongfeng Sub branch of Bank of China Ltd. Pledgor:MICROBIAL FERMENTATION ENGINEERING RESEARCH CENTER Co.,Ltd. OF YUNNAN PROVINCE Registration number:Y2024980057335 |