WO2025123647A1 - 一种具有防治甘蓝蚜虫效果的东洋芽孢杆菌 - Google Patents

一种具有防治甘蓝蚜虫效果的东洋芽孢杆菌 Download PDF

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WO2025123647A1
WO2025123647A1 PCT/CN2024/101846 CN2024101846W WO2025123647A1 WO 2025123647 A1 WO2025123647 A1 WO 2025123647A1 CN 2024101846 W CN2024101846 W CN 2024101846W WO 2025123647 A1 WO2025123647 A1 WO 2025123647A1
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aphids
hxbw
bacillus
preventing
strain
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倪海平
宗睿
刘金
刘鲁民
何月秋
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Qingdao Hexie Biotechnology Co Ltd
Shandong Baiwo Biotechnology Co Ltd
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Qingdao Hexie Biotechnology Co Ltd
Shandong Baiwo Biotechnology Co Ltd
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    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G13/00—Protection of plants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G7/00—Botany in general
    • A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/22—Bacillus
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P7/00—Arthropodicides
    • A01P7/04—Insecticides
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus

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  • Aphids are a general term for the superfamily Aphidoidea of the order Hemiptera, including all members of the superfamily Aphidoidea.
  • the present invention also provides a use of the Bacillus toyota HXBW-2 strain, which is the use in preparing products for preventing and controlling plant pests.
  • the plant pests are caused by cabbage aphids.
  • the present invention also provides a product for alleviating plant insect pests, wherein the product contains live bacteria and/or fermentation products of the HXBW-2 strain of Bacillus sp.
  • the product is a bacterial liquid product.
  • Figure 1 Colony morphology of strains
  • Figure 5 Aphid situation 3 days after using microbial agents, where A is before use and B is the result 3 days after use.
  • the present invention extracted 76 pure bacterial colonies from the rhizosphere soil of plants that often suffer from aphid diseases, one of which had a strong effect of killing cabbage aphids.
  • the strain was identified as Bacillus toyonensis by 16SrRNA sequencing.
  • the fungus has a good control effect on cabbage aphids and can be widely used in the control of cabbage aphids.
  • Mortality (%) number of dead aphids/total number of aphids ⁇ 100.
  • Corrected mortality rate (%) (% mortality rate of the treatment group - % mortality rate of the control group) / (100 - The mortality rate of the control group (%) ⁇ 100
  • NA aphidicide activity
  • a total of 76 strains were isolated from the rhizosphere soil, of which HXBW-2, HXBW-05, HXBW-27, HXBW-31 and HXBW-53, 5 strains had aphidicide activity.
  • the one with strong aphidicide activity was HXBW-2, whose fermentation liquid had a 96.3% aphid mortality rate at 2-fold dilution concentration.
  • HXBW-27 had moderate aphidicide activity, with a 56.7% aphid mortality rate.
  • HXBW-05, HXBW-31 and HXBW-53 showed weak aphidicide activity.
  • Table 1 Toxicity of bacterial fermentation broth to cabbage aphids
  • the pure cultured HXBW-2 strain was streaked on a NA medium plate and cultured in a 25°C incubator for 48 hours. After obvious colonies grew, the colony morphology, color, transparency, dryness, surface smoothness and edge morphology were observed. Fresh bacteria were taken for Gram staining and microscopic examination.
  • the strain is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No. 28561 and the deposit date September 26, 2023; the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.
  • Cabbage aphids were divided into 7 groups, namely experimental groups (high dose group 2 ⁇ 10 9 cfu/mL, medium-high dose group 1 ⁇ 10 9 cfu /mL, medium dose group 1 ⁇ 10 8 cfu/mL, medium-low dose group 4 ⁇ 10 7 cfu/mL, low dose group 2 ⁇ 10 7 cfu/mL), control group (bio group 2 ⁇ 10 9 cfu/mL) and blank control group (ddH 2 O).
  • experimental groups high dose group 2 ⁇ 10 9 cfu/mL, medium-high dose group 1 ⁇ 10 9 cfu /mL, medium dose group 1 ⁇ 10 8 cfu/mL, medium-low dose group 4 ⁇ 10 7 cfu/mL, low dose group 2 ⁇ 10 7 cfu/mL
  • control group bio group 2 ⁇ 10 9 cfu/mL
  • blank control group ddH 2 O
  • the HXBW-2 strain fermentation liquid was diluted 10 times, 20 times, 50 times, and 100 times, the original bacterial liquid and the diluted liquid were used for indoor quantitative spraying insecticidal test, and 1 ⁇ of Tween 20 was added as a dispersant.
  • a certain number of surviving aphids were placed on the leaves of indoor culture dishes to keep them moist, with 100 aphids per dish.
  • the fermentation liquid of the strains at different concentrations was evenly sprayed on the cabbage aphids until the surface of the insect body was moist.
  • the finished product of Bacillus thuringiensis was used as the control group, and water was used as the blank control group.
  • the test was repeated 3 times. After spraying, the culture dishes were sealed with plastic wrap, and a dozen small holes were poked with a needle to ensure air circulation. The aphid mortality rates at 2h, 8h, 24h, and 48h were recorded.
  • Mortality rate (%) number of dead insects (heads) ⁇ 100 / total number of insects treated (heads)
  • the 48h mortality rate was higher than 75%. The lower the concentration, the lower the mortality rate.
  • the mortality rate of the finished product control group (bio group) at 2h, 8h, 24h, and 48h was lower than that of the low-dose group of the experimental group, and the mortality rate after 48h was only 51%.
  • the HXBW-2 strain was tested for its outdoor efficacy against cabbage aphids.
  • the fermentation liquid was diluted to a concentration of 1 ⁇ 10 8 cfu/mL for outdoor field efficacy testing of aphids.
  • the insect population change was detected for 15 days and the efficacy was calculated.
  • the number of cabbage aphids on the roses was adjusted to be as close as possible.
  • the 24 roses were randomly divided into 3 groups with equal numbers and different plots. Leaves of the same size and age were selected from each pot of seedlings as the test objects, randomly sorted 1 to 24, and marked with labels. The number of aphids on each branch was counted and then treated. The number of aphids was observed and recorded regularly, and the control effect was calculated as follows:
  • Insect population reduction rate (%) (number of live insects before application - number of live insects after application) / number of live insects before application ⁇ 100%

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Abstract

一种具有防治甘蓝蚜虫作用的东洋芽孢杆菌，其保藏编号为CGMCCNo.28561。其可以缓解甘蓝蚜虫引起的植物虫害，在室内和室外均具有良好的杀虫效果。

Description

一种具有防治甘蓝蚜虫效果的东洋芽孢杆菌 技术领域
本发明属于微生物筛选技术领域，具体涉及一种具有防治甘蓝蚜虫效果的东洋芽孢杆菌。
背景技术
蚜虫，是半翅目蚜总科的统称，包括蚜总科下的所有成员，目前已经发现的蚜虫总共有10个科约4400种，其中多数属于蚜科。目前发现不同植物具有对应的蚜虫种类，例如松大蚜是松柏类植物的主要害虫；栾多态毛蚜主要危害栾树；苹果黄蚜主要危害苹果、海棠、木瓜、石楠、山楂等；桃蚜的寄主植物主要有桃、梨、李、梅、樱桃等蔷薇科果树。
甘蓝蚜虫(Brevicoryne brassicae Linnaeus)是蔬菜上一种重要的害虫，主要危害十字花科植物。一般甘蓝蚜通过吸食叶片和茎秆上的汁液为食，盗取植物生长营养从而引起植物叶片卷曲萎缩形变、减缓植物正常生长。
目前，甘蓝蚜虫病的防治仍以化学防治为主。化学药剂的大量使用对环境安全和人畜健康造成了巨大威胁，多种化学农药也因此被限用或禁用，寻找环境友好、安全、无污染的防治方法迫在眉睫。生防制剂作为一种环境友好的化学药剂替代品逐步引起人们的重视。而生防细菌由于安全性高，易培养和生产，受到广泛关注。据报道，蜡蚧轮枝菌、座壳孢菌和玫瑰色拟青霉对蚜虫有一定的防治作用，但其应用于甘蓝蚜虫效果欠佳。
发明内容
本发明的目的是提供一种具有防治甘蓝蚜虫作用的东洋芽孢杆菌， 能够生物防治甘蓝蚜虫，从而有效的防治甘蓝蚜虫所带来的病害损失。
本发明所提供的东洋芽孢杆菌HXBW-2株(Bacillus toyonensis)，其保藏编号为CGMCCNo.28561，保藏日期为2023年9月26日，保藏单位为中国微生物菌种保藏管理委员会普通微生物中心，地址为北京市朝阳区北辰西路1号院3号。
本发明还提供所述的东洋芽孢杆菌HXBW-2株的一种用途，是在制备用于防治植物虫害的制品中的应用。
所述的植物虫害，是甘蓝蚜虫导致的。
本发明还提供一种用于缓解植物虫害的制品，所述的制品中包含有东洋芽孢杆菌HXBW-2株的活菌和/或发酵产物。
所述的制品，为菌液制品。
本发明所筛选的东洋芽孢杆菌HXBW-2株可以缓解甘蓝蚜虫引起的植物虫害，在室内和室外均具有良好的杀虫效果，具有开发潜力。
附图说明
图1：菌株菌落形态特征图；
图2：菌株染色特征图；
图3：系统发育树图；
图4：菌株全基因组生成热图；
图5：用微生物菌剂3天后蚜虫情况，其中A为使用前，B为使用3天后的结果图。
具体实施方式
本发明从常发蚜虫病的植物根际土壤中提取出76个纯菌落，其中一株具有强的杀甘蓝蚜虫的效果，经16SrRNA测序鉴定，该菌株为东洋芽孢杆菌(Bacillus toyonensis)。本发明基于生物制剂的方法筛选的细 菌对甘蓝蚜虫具有良好的防治效果，可广泛应用于甘蓝蚜虫的防治。
下面结合具体实施例和附图对本发明进行详细的描述。
实施例1：菌株的分离筛选
1.1土壤中细菌菌株的分离
称取10g土样(常年发生蚜虫病的日光温室根际土壤)，放入装有90mL无菌水的250mL无菌三角瓶中，用封口膜封住瓶口。将三角瓶放置在25℃，200rpmmin-1摇床上震荡30min。震荡均匀后，在超净工作台上用无菌水将土壤悬浮液稀释后得到10-1-10-5浓度的悬液，分别取100μL不同浓度的土壤悬浮液，用涂布器均匀涂抹于NA培养基上，放置在25℃恒温培养箱里，黑暗条件下培养48h，待长出明显菌落后，取单菌落进行纯化。每个浓度的土壤悬液设3个重复。
1.2具有杀蚜虫活性的细菌筛选
挑取细菌单菌落于盛有100mLNA液体培养基的250mL三角瓶中(2×109CFUmL-1)，25℃，150rmin-1条件下在摇床上摇48h。取出后将发酵液在10000rpm离心机中离心10min后取上清液，再用0.45μm滤膜过滤，得到滤液备用。在12孔板中加入500μL的提前准备好的甘蓝蚜虫液(大约含有甘蓝蚜虫100条)，再分别加入500，200，100μL的细菌发酵液，用无菌水补至1mL，将发酵液制备成2倍，5倍，10倍稀释液后，测定对南方甘蓝蚜虫的毒力，12h后在体视镜下检查结果。蚜虫身体僵直不动，转移到清水中12h后仍不恢复视为死亡。以不含菌液的NA液体培养基为对照。试验设3次重复，每次重复包含3个孔。蚜虫死亡率与校正死亡率计算公式如下：
死亡率(％)＝死亡的蚜虫数量/蚜虫总数×100。
校正死亡率(％)＝(处理组的死亡率％-对照组的死亡率％)/(100- 对照组的死亡率％)×100
杀蚜虫活性(NA)按以下标准分类，NA≥80％，50％≤NA<80％，20％≤NA<50％和NA<20％分别视为有强杀蚜虫活性，中等杀蚜虫活性，轻杀蚜虫活性和没有杀蚜虫活性。
从根际土壤中共分离得到76个菌株，其中HXBW-2、HXBW-05、HXBW-27、HXBW-31和HXBW-53，5株具有杀蚜虫活性。具有强杀蚜虫活性的为HXBW-2发酵液在2倍稀释浓度下对蚜虫致死率均为96.3％。；HXBW-27具有中等杀蚜虫活性，对蚜虫致死率为56.7％；HXBW-05、HXBW-31和HXBW-53表现出较弱的杀蚜虫活性。
表1：细菌发酵液对甘蓝蚜虫的毒力表
实施例2：菌株的鉴定
2.1菌种形态特征
将纯培养保存的HXBW-2菌株划线在NA培养基平板上，25℃培养箱中恒温培养48h，待长出明显菌落后，观察菌落形态、颜色、透明度、干燥度、表面光滑度和边缘形态。取新鲜细菌进行革兰氏染色，进行镜检。
由图1观察到HXBW-2菌株菌落近圆形，呈现无光泽白色，且表面褶皱、边缘不整齐，随时间延长菌落变大，表现出泳动和自聚能力；染色结果表明HXBW-2菌为革兰氏阳性短杆状菌(图2)。
2.2菌株的理化性质
将细菌在培养基上培养一段时间后，进行各项生理生化指标测定，接触酶试验、普里斯考尔(V-P)测定、淀粉水解、明胶液化、硫化氢、硝酸盐还原、葡萄糖氧化发酵和蔗糖发酵试验。
由表2和表3可知，菌株在精氨酸、V-P明胶、蔗糖发酵、苦杏仁苷试验中均呈阳性反应。在碳源利用实验中甘油、核糖、葡萄糖、果糖、甘露糖、N-乙酰-葡糖胺、苦杏仁甙、熊果甙、七叶灵、柳醇、纤维二糖、麦芽糖、蔗糖、海藻糖、淀粉、糖原等结果呈阳性。根据《常见细菌系统鉴定手册》，符合芽孢杆菌的特征。
表2：菌株HXBW-2生理生化特性–酶活、碳源氧化表

+：阳性反应；-：阴性反应；
表3：菌株HXBW-2的碳源利用信息表

+：阳性反应；-：阴性反应；
2.3菌株16SrRNA基因序列
将筛选获得的HXBW-2菌株进行16SrDNA测序鉴定，测序结果通过NCBI数据中的Blast比对系统搜索相似序列并构建系统发育树。由图3系统进化树可知，菌株HXBW-2属于芽孢杆菌属。图4可知该菌株HXBW-2与Bacillustoyonensis(BCT-7112)模式菌株全基因组数据比较得出ANI值为：98.43％，结果大于ANI同种判断的阀值96％，所以，鉴定HXBW-2菌株为东洋芽孢杆菌(Bacillustoyonensis)。
该菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCCNo.28561，保藏日期为2023年9月26日；保藏地址：北京市朝阳区北辰西路1号院3号。
实施例3：HXBW-2对蚜虫室内杀虫效果
以试验室采集的甘蓝蚜作为供试害虫。将甘蓝蚜蚜虫连茎叶一并取回置于含有纸的培养皿内，计数生命力和活动正常的蚜虫并挑出死虫或弱虫，保证每皿内蚜虫数大于100。将甘蓝蚜虫分为7份，分别为实验组(高剂量组2×109cfu/mL、中高剂量组1×109cfu/mL、中剂量组1×108cfu/mL、中低剂量组4×107cfu/mL、低剂量组2×107cfu/mL)、对照组(bio组2×109cfu/mL)和空白对照组(ddH2O)。
在菌株库购买成品东洋芽孢杆菌(编号bio118045)冻干粉，按照说明书上的使用方法将其活化，30℃培养至2×109cfu/mL备用。
HXBW-2菌株发酵液原液稀释10倍、20倍、50倍、100倍后，用原菌液和稀释液进行室内定量喷菌杀虫试验，并加入总体积1‰的吐温20作为分散剂，取一定数量的存活蚜虫于室内培养皿叶片上保湿寄养，每皿100头，不同浓度的菌株发酵液均匀喷施到甘蓝蚜虫身上，至虫体表面潮湿，以成品东洋芽孢杆菌为对照组，以水为空白对照组，重复3次，喷菌后使用保鲜膜将培养皿封口之后用针戳十几个小孔以保证空气流通，记录2h，8h，24h，48h的蚜虫死亡率。
采用稀释涂布平板法计数菌液的浓度，记录原菌液和稀释液浓度分别为：2×109、1×109、1×108、4×107、2×107cfu/mL。
死亡率(％)＝死亡虫数(头)×100/处理总虫数(头)
表4：蚜虫死亡率数据统计表

HXBW-2菌株发酵液原液通过稀释涂布平板法计数，原液浓度为2×109cfu/mL。将菌液均匀喷施于甘蓝蚜体表后观察蚜虫的死亡情况，用毛笔轻触蚜虫体表，无明显生理反应则表明蚜虫死亡。随着时间增加(表4)，蚜虫死亡率越高，2×109cfu/mL浓度处理48h后死亡率达到89.08±2.53％，展现出良好的生防潜力，HXBW-2菌株浓度在1×108cfu/mL及以上时，48h死亡率均高于75％，浓度越低，死亡率越低。而成品对照组(bio组)在2h，8h，24h，48h的死亡率均低于实验组低剂量组，在48h后死亡率仅为51％。
实施例4：HXBW-2对蚜虫室外杀虫效果
对HXBW-2菌株进行甘蓝蚜进行室外防效试验，稀释发酵液浓度至1×108cfu/mL进行蚜虫室外田间防效检测，检测15d的虫量变化并计算防效。
处理设置为：处理1为菌液喷施1次；处理2为菌液喷施2次(第一次喷施5d后再次进行相同处理)，对照以无菌水等量喷施。
试验前调节月季上的甘蓝蚜数量尽可能相近，从24株月季中随机等量分为3组，设置不同小区。将每盆幼苗选取大小、叶龄尽一致的叶片作为检测对象，随机排序1～24，并使用标签牌标记后，统计每个枝子上蚜虫的数量后进行处理。定时观察记录蚜虫的数量，并计算防效，计算方法如下：
虫口减退率(％)＝(施药前活虫数－施药后活虫数)/施药前活虫数×100％
防效(％)＝(处理区虫口减退率－对照区虫口减退率)×100/(1－对照区虫口减退率)
表5：蚜虫虫口减退率与防效统计表
室外试验的结果见表5及图5，进一步检测HXBW-2菌株对甘蓝蚜虫的防治能力。对比处理1和处理2控制作用结果表明，两者均具有较高的虫口减退率，且防治效果处理2高于处理1。处理后前5d，虫口减退率呈现增长趋势，处理1在5天后防效开始下降，而处理2则在补药后呈现较好的防治效果。比较两者差异可进一步预测HXBW-2的时效性和最佳防治时间段大概为5d左右，进而田间用药时须在5天后补药以获得更高的防治效果。

Claims (6)

  1. 一种东洋芽孢杆菌，其特征在于，所述的东洋芽孢杆菌的保藏编号为CGMCC No.28561。
  2. 权利要求1所述东洋芽孢杆菌在制备用于防治植物虫害的制品中的应用。
  3. 如权利要求2所述的应用，其特征在于，所述的植物虫害是甘蓝蚜虫导致的虫害。
  4. 一种用于防治植物虫害的制品，其特征在于，所述的制品中包含有权利要求1所述东洋芽孢杆菌的活菌。
  5. 如权利要求4所述的制品，其特征在于，所述的制品中包含有权利要求1所述东洋芽孢杆菌的发酵产物。
  6. 如权利要求4所述的制品，其特征在于，所述的制品为菌液制品。
PCT/CN2024/101846 2023-12-13 2024-06-27 一种具有防治甘蓝蚜虫效果的东洋芽孢杆菌 Pending WO2025123647A1 (zh)

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