WO2022095127A1 - 一种生防菌株yw-1及其生防菌剂的制备和应用 - Google Patents

一种生防菌株yw-1及其生防菌剂的制备和应用 Download PDF

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WO2022095127A1
WO2022095127A1 PCT/CN2020/129982 CN2020129982W WO2022095127A1 WO 2022095127 A1 WO2022095127 A1 WO 2022095127A1 CN 2020129982 W CN2020129982 W CN 2020129982W WO 2022095127 A1 WO2022095127 A1 WO 2022095127A1
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biocontrol
strain
bacterial
agent
pepper
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PCT/CN2020/129982
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English (en)
French (fr)
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杨威
闫海霞
董光辉
邱雨生
朱跃龙
张孜
郭为
李师默
张磊
罗玉明
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淮阴师范学院
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Priority to US17/523,064 priority Critical patent/US11805782B2/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; 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

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  • the invention belongs to the technical field of plant protection, in particular to the preparation and application of a biocontrol strain YW-1 and a biocontrol fungicide.
  • Facility agriculture is a modern agricultural production method that comprehensively applies engineering equipment technology, biotechnology and environmental technology to create a suitable environment for the growth and development of animals and plants, and realize the production of animals and plants.
  • the development of facility vegetables not only alleviates the contradiction between production seasonality and consumption balance, but also can use facility cultivation to produce high-grade vegetables, famous and excellent vegetables, seasonal vegetables, and increase the variety of vegetables to meet the consumption of different levels of society. need. my country has become the country with the largest facility area in the world, and it is still growing at a rate of about 10% per year.
  • facility vegetable cultivation has the characteristics of frequent soil tillage, high degree of intensification, high multiple cropping index, and single species, etc., and continuous cropping obstacles such as soil deterioration, rampant pests and diseases, and quality degradation have generally appeared, which seriously affected the production of vegetables and farmers' income.
  • Many researchers at home and abroad believe that the aggravation of soil-borne diseases and insect pests is the most important problem causing continuous cropping obstacles after analyzing the facility cultivation under various conditions.
  • the pathogens of vegetable diseases and insect pests are often latent in the soil, such as fusarium wilt of cucumber and eggplant, blight and other pathogenic bacteria spores often overwinter in the soil and continue to damage in the coming year.
  • the temperature in the greenhouse is high and the humidity is high.
  • the types and quantities of harmful fungi increase, and the antagonistic bacteria of pathogenic bacteria in the soil decrease, which further aggravates soil-borne diseases.
  • people have accumulated various control methods in practice.
  • biocontrol strain YW-1 In view of the current situation of single action mode and small control range of biocontrol strains for plant soil-borne diseases, through the activity screening of various metabolites and the test of antibacterial activity, a biocontrol strain YW-1 and its biocontrol agent are provided. application.
  • the present invention provides the following scheme:
  • the present invention provides a biocontrol strain YW-1, the strain is Myroides odoratimimus, and the preservation number of the strain is CGMCC NO.20620.
  • the invention provides a biocontrol fungicide prepared by using the biocontrol strain YW-1.
  • the present invention provides a method for preparing a biocontrol agent. After the biocontrol strain YW-1 is cultured in LB medium, the bacterial cells are collected and diluted with sterilized water to prepare a biocontrol agent with a concentration of 10 7 CFU/mL. Bacterial agent.
  • the total concentration of viable bacteria in the bacterial suspension obtained after culturing the biocontrol strain YW-1 in the LB medium is 1 ⁇ 10 9 -1 ⁇ 10 10 CFU/mL.
  • the present invention also provides an application of the biocontrol fungicide prepared by the biocontrol strain in preventing and treating plant soil-borne diseases.
  • the plant soil-borne disease is one or more of melon wilt, bacterial wilt of tomato and pepper, and pepper blight.
  • the present invention is specially screened against a variety of common plant soil-borne diseases, and through the activity screening of a variety of metabolites and the bacteriostatic activity test, the common soil-borne diseases including fusarium wilt, bacterial wilt and blight all have better control effects, overcome the shortcomings of single disease control by biocontrol strains in the past, and have greater practical application potential and scope of application.
  • Figure 1 shows the screening results of the metabolite activity of strain YW-1;
  • A, B, and C are chitinase, cellulase, and siderophore activity detection plates, of which strain 21 is YW-1, and the other strains are strains that do not meet the screening conditions;
  • Figure 2 is the screening results of the antagonistic activity of the strain YW-1 plate;
  • Figure 2-A is the antagonistic screening of R. solanacearum, of which strain No. 21 is YW-1;
  • Figure 2-B and Figure 2-C are the antagonistic screening of Fusarium wilt and Phytophthora , wherein the linear colony is an antagonistic bacterial colony, the circular colony in Figure 2-B is the Fusarium wilt to be tested, and the circular colony in Figure 2-C is the Phytophthora to be tested, wherein No. 21 in the linear colony is YW-1 strains, strains in other linear colonies are strains that do not meet the screening conditions.
  • the invention relates to a biocontrol strain YW-1 for preventing and treating various plant soil-borne diseases, which was isolated from the soil of a pepper greenhouse in Wudun Town, Huaian City, Jiangsuzhou in 2015, and is Myroides odoratimimus. On September 5, 2020, it was deposited in the General Microbiology Center of the China Microorganism Culture Collection Management Committee (address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Chinese Academy of Microbiology), and the culture collection number is CGMCC NO.20620 .
  • LB medium was used to isolate strains by dilution plating method: 1 g of each soil sample was dissolved in 9 mL of sterile water, and then diluted to 10 -3 , 10 -4 , and 10 -5 for each group. Pipette 100 mL separately onto the medium, spread, and repeat each gradient 3 times. After labeling, they were cultured in a 28°C greenhouse incubator. After the colonies grew, they were picked and purified according to their morphological characteristics, and stored at -70°C for later use.
  • strain YW-1 The genomic DNA of strain YW-1 was extracted using a resin-type genome kit (Shanghai Saibaisheng Gene Technology Co., Ltd.), and the extracted genomic DNA was used as a template to amplify its 16S rDNA fragment using the 16S universal primer. Sequencing (Nanjing GenScript Biotechnology Co., Ltd.). The results were submitted to the NCBI database for comparison, and the similarity with the strain Myroides odoratimimus PR63039 (Accession: CP013690.1) was 99%. The 16S rDNA sequence of strain YW-1 is shown in SEQ ID No.1.
  • the screening of metabolite activity of the strain includes chitinase-producing enzyme activity, cellulase-producing activity, and siderophilic activity; as shown in Table 1 and Figure 1, the screening results of the metabolite activity of strain YW-1; C are chitinase, cellulase, and siderophore activity detection plates, of which strain No. 21 is YW-1; the specific screening method is as follows:
  • chitinase activity The strain was placed in a medium with colloidal chitin as the sole carbon source: (NH 4 H 2 PO 4 1.0g, KCl 0.2g, MgSO 4 ⁇ 7H 2 O 0.2g, colloid 1% (w/v) of chitin-like chitin to 1000ml, pH 7.0, agar 20g), cultured at 30°C for 3 days after inoculation, measure the size of the transparent circle, and select the radius of the hydrolysis circle (outer diameter and inner diameter). Poor) strains greater than 5 mm were considered to have significant chitinase activity.
  • Detection of cellulase activity connect the strain to a cellulase activity assay plate (10g peptone, 10g yeast powder, 10g sodium carboxymethyl cellulose, 5g sodium chloride, 1g potassium dihydrogen phosphate, 18g agar, and dilute to volume. to 1000ml, pH 7.0), incubated at 30°C for 48h, stained with 1g/L Congo red for 1h, removed the dye solution, and soaked in 1M NaCl for 1h. The size of the transparent circle was measured, and the strains with the radius of the hydrolysis circle (difference between the outer diameter and the inner diameter) greater than 5 mm were selected as having significant cellulase activity.
  • Detection of siderophil activity A: (1) Dissolve 60.5 mg of CAS (chromazurine S) in 50 ml of deionized water; (2) prepare 10 ml of ferric iron solution (1 mM FeCl 3 ⁇ 6H 2 O, 10 mM hydrochloric acid) as solvent); (3) 72.9 mg of HDTMA was dissolved in 40 ml of deionized water. The above three solutions were mixed and made up to 100ml, the pH was adjusted to neutral, and sterilized at 121°C for 20min. B: 900ml WA medium was added to 30.24g Pipes, pH was adjusted to 6.8, and sterilized at 121°C for 20min.
  • a and B liquids were mixed and poured into a plate, inoculated at 30°C for 3 days and observed, the size of the transparent circle was measured, and the strains with a radius of the hydrolysis circle (difference between the outer diameter and the inner diameter) greater than 5 mm were regarded as having significant siderophilic activity.
  • the inner diameter is the diameter of the colony formed by the growth of the strain on the plate, and the outer diameter is the diameter of the formed hydrolysis circle, all in millimeters (mm).
  • LB plate to activate the biocontrol bacterial strains, pick the colonies in the peak growth stage with toothpicks and place them on the YGPA plate containing the bacterial wilt pathogen, with 5 points per plate with equal intervals, cultivate at 30 °C for 48 hours, observe and record the results, and measure them respectively.
  • the inner diameter of the bacteria and the outer diameter of the ring, and the strains with the radius of the antagonistic ring (the difference between the outer diameter and the inner diameter) greater than 5 mm were considered to have significant inhibitory activity against R. solanacearum.
  • the bacterial suspension was 1 ⁇ 109-1 ⁇ 1010 CFU/mL, and then the bacterial suspension was centrifuged at 6000 rpm for 10 min to collect the bacterial cells, and diluted with sterilized water to prepare a biocontrol agent with a concentration of 10 7 CFU/mL.
  • Tomato seedlings and pepper seedlings were cultivated in plug trays, and transplanted when they had 3-4 true leaves.
  • the treatment group was treated with 20 mL of 10 7 CFU/mL inoculum for root irrigation, and the control group was treated with water for one week. Then inoculate 20 mL of 10 7 CFU/mL bacterial suspension of Ralstonia solanacearum 3721. 24 strains in each treatment group, repeated three times. Under the conditions of temperature of 25-28°C, relative humidity of 60%, and light of 12h/12h, the disease grades were investigated after 4 weeks of cultivation, and the disease severity and control effect were calculated.
  • Cucumber seedlings were cultivated in plug trays, and transplanted when they grew to 3-4 true leaves.
  • the treatment group was treated with 20 mL of 10 7 CFU/mL inoculum for root irrigation, and the control group was treated with clean water.
  • One week after transplanting inoculated with 10 5 sporangia/mL Fusarium wilt pathogen bacterial suspension 20mL. 24 strains in each treatment group, repeated three times. Under the conditions of temperature of 25-28°C, relative humidity of 60%, and light of 12h/12h, the disease grades were investigated after culturing for 3 weeks, and the disease severity and control effect were calculated.
  • the pepper seedlings were cultivated in plug trays, and transplanted when they grew to 3-4 true leaves.
  • the treatment group was treated with 20 mL of 10 7 CFU/mL inoculum for root irrigation, and the control group was treated with clean water.
  • One week after transplanting inoculated with 10 5 sporangia/mL Phytophthora suspension 20mL. 24 strains in each treatment group, repeated three times. Under the conditions of temperature of 25-28°C, relative humidity of 60%, and light of 12h/12h, the disease grades were investigated after 4 weeks of cultivation, and the disease severity and control effect were calculated.

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Abstract

提供一种生防菌株YW-1及其生防菌剂的制备和应用，菌种保藏号为CGMCC NO.20620；所述生防菌株能够有效防治瓜类枯萎病，番茄及辣椒青枯病，辣椒疫病等多种土传病害，相对于空白对照组，防治效果均达到50％以上。

Description

一种生防菌株YW-1及其生防菌剂的制备和应用 技术领域
本发明属于植物保护技术领域，具体涉及一种生防菌株YW-1及其生防菌剂的制备和应用。
背景技术
设施农业是综合应用工程装备技术、生物技术和环境技术，创造动植物生长发育的适宜环境，实现动植物生产的现代农业生产方式。设施蔬菜的发展不但缓解了生产季节性和消费均衡性之间的矛盾,而且可利用设施栽培生产出高档蔬菜、名特优蔬菜、时令蔬菜，增加蔬菜的花色品种，以满足社会不同层次的消费需求。我国已成为世界上设施面积最大的国家，且仍以每年10％左右的速度在增长。
但是，设施蔬菜发展至今，在迅猛增长的同时也存在着不可忽视的问题。设施蔬菜栽培具有土壤频繁耕作、集约化程度高、复种指数高、种类单一等特点，普遍出现了土壤恶化、病虫害发生猖獗、品质降低等连作障碍现象，严重影响了蔬菜的生产和农民的收益。国内外很多学者在分析多种条件下的设施栽培后认为，土传病虫害的加重是引起连作障碍的最主要问题。
设施栽培中，蔬菜病虫害的病原菌常潜伏于土壤中，如黄瓜、茄子的枯萎病，疫病等其病菌孢子常在土壤内越冬，来年继续为害。另外大棚内温度高湿度大，在这样小气候条件下，易于蔬菜病虫害的发生和相互传播，对蔬菜危害较大，容易形成恶性循环。随着连作年限的增加，有害真菌的种类和数量增加，土壤中病原菌的拮抗菌减少，进一步加重了土传病害。 对于土传病害的防治，人们在实践中积累了各种防治途径。包括嫁接、土壤消毒、增施有机肥，以及生物防治等。其中，利用土壤拮抗微生物控制土传病害不但能够原位进行，并且对人畜无害，对环境友好，已经受到越来越多学者的关注。但是，目前阻碍生物防治发展的最大障碍就是防治范围有限以及田间生防效果的不稳定。生防菌株对于病原物作用方式单一以及不能够适应田间环境是其中最主要因素。因此，筛选多种机制、作用范围广谱并且适应性强的生防菌株将具有更大的应用潜力及前景。
发明内容
针对目前植物土传病害生防菌株作用方式单一，防治范围小等现状，通过多种代谢产物活性筛选及抑菌活性检验，提供一种生防菌株YW-1及其生防菌剂的制备和应用。
为实现上述目的，本发明提供了如下方案：
本发明提供一种生防菌株YW-1，所述菌株为拟香味类香菌(Myroides odoratimimus)，所述菌株的保藏编号为CGMCC NO.20620。
本发明提供一种利用生防菌株YW-1制备的生防菌剂。
本发明提供一种生防菌剂的制备方法，将所述生防菌株YW-1在LB培养液培养后，收集菌体并用灭菌水进行稀释制成浓度为10 7CFU/mL的生防菌剂。
优选的，所述生防菌株YW-1在LB培养液培养后获得的菌悬液中活菌总浓度为1×10 9-1×10 10CFU/mL。
本发明还提供一种所述生防菌株制备的生防菌剂在防治植物土传病害中的应用。
优选的，所述植物土传病害为瓜类枯萎病、番茄和辣椒青枯病以及辣椒疫病中的一种或多种。
本发明公开了以下技术效果：
本发明的优点和积极效果表现在：本发明是专门针对多种常见植物土传病害进行筛选，通过多种代谢产物活性筛选及抑菌活性检验，对常见土传病害包括枯萎病、青枯病、疫病均具有较好防治效果，克服了以往生防菌株防治病害单一的缺点，具有更大的实际应用潜力及应用范围。
温室实验表明：在寄主植物移栽时以该菌剂灌根处理20mL/株，能够有效防治瓜类枯萎病，番茄及辣椒青枯病，辣椒疫病等多种土传病害，相对于空白对照组，防治效果均达到50％以上。
附图说明
图1是菌株YW-1代谢产物活性筛选结果；其中的A、B、C分别为几丁质酶，纤维素酶，嗜铁素活性检测平板，其中21号菌株为YW-1，其他菌株为不符合筛选条件的菌株；
图2是菌株YW-1平板拮抗活性筛选结果；图2-A青枯菌拮抗筛选，其中21号菌株为YW-1；图2-B和图2-C是枯萎病菌和疫霉菌的拮抗筛选，其中线形菌落是拮抗细菌菌落，图2-B的圆形菌落为待测的枯萎病菌，图2-C的圆形菌落为待测的疫霉菌，其中线形菌落中的21号为YW-1菌株，其他线形菌落中的菌株为不符合筛选条件的菌株。
具体实施方式
下面结合附图进一步说明本发明的实施例，该详细说明不应认为是对本发明的限制，而应理解为是对本发明的某些方面、特性和实施方案的更 详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式，并非用于限制本发明。另外，对于本发明中的数值范围，应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明，否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料，但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入，用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时，以本说明书的内容为准。
在不背离本发明的范围或精神的情况下，可对本发明说明书的具体实施方式做多种改进和变化，这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本申请说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等，均为开放性的用语，即意指包含但不限于。
实施例1
本发明涉及一种防治多种植物土传病害的生防菌株YW-1,为2015年在江苏省淮安市武墩镇辣椒大棚土壤中分离得到，为拟香味类香菌(Myroides  odoratimimus)，于2020年9月5日保藏于中国微生物菌种保藏管理委员会普通微生物中心(地址是北京市朝阳区北辰西路1号院3号，中国微生物科学院研究所)，菌种保藏号为CGMCC NO.20620。
1、生防菌株YW-1的分离与纯化
(1)首先用铁锹挖开植物根基部的土壤10cm深，然后用小刷子收集附着在植物根表层的土壤，收集后装入小号自封袋，标记编号迅速带回实验室进行下一步处理。
(2)本研究采用LB培养基利用稀释涂平板法进行菌株的分离：每份土壤样品取1g分别溶于9mL无菌水，然后每组稀释到10 -3、10 -4、10 -5。用移液器分别吸取100mL到培养基上，涂布，每个梯度3次重复。标记后置于28℃温室培养箱培养，待菌落长出后，根据形态特征挑取进行纯化，-70℃保存备用。
2、生防菌株YW-1的鉴定
(1)微生物学特性：菌株YW-1在LB培养基上28℃培养48h后，培养基表面长出直径1.5mm大小的菌落，颜色淡黄，菌落饱满，表面湿润有光泽，边缘平整。
(2)分子生物学特性：菌株YW-1基因组DNA提取采用树脂型基因组试剂盒(上海赛百盛基因技术有限公司)，以提取的基因组DNA为模板，利用16S通用引物扩增其16S rDNA片段并测序(南京金斯瑞生物科技有限公司)。结果提交NCBI数据库比对,与菌株Myroides odoratimimus PR63039(Accession：CP013690.1)相似性达99％。菌株YW-1 16S rDNA序列如SEQ ID No.1所示。
3、生防菌株YW-1代谢产物活性筛选
该菌株代谢产物活性筛选包括产几丁质酶酶活性，产纤维素酶活性，产嗜铁素活性；如表1和图1是菌株YW-1代谢产物活性筛选结果；其中的A、B、C分别为几丁质酶，纤维素酶，嗜铁素活性检测平板，其中21号菌株为YW-1；具体筛选方法如下：
产几丁质酶活性的检测：将菌株在以胶体状几丁质为唯一碳源的培养基：(NH 4H 2PO 4 1.0g，KCl 0.2g，MgSO 4·7H 2O 0.2g，胶体状几丁质1％(w/v)定容至1000ml，pH7.0，琼脂20g)上培养，接菌后30℃培养3天，测透明圈的大小，挑选水解圈半径(外径与内径差)大于5mm的菌株视为具有显著几丁质酶活性。
产纤维素酶活性的检测：把菌株接到纤维素酶活性测定平板(蛋白胨10g，酵母粉10g，羧甲基纤维素钠10g，氯化钠5g，磷酸二氢钾1g，琼脂18g，定容至1000ml，pH7.0)，30℃培养48h后，用1g/L的刚果红染1h后，去除染液后，用1M的NaCl浸泡1h。测透明圈的大小，挑选水解圈半径(外径与内径差)大于5mm的菌株视为具有显著纤维素酶活性。
产嗜铁素活性的检测：A：(1)将60.5mg CAS(铬天青S)溶于50ml去离子水；(2)配制10ml三价铁溶液(1mM FeCl 3·6H 2O，10mM盐酸为溶剂)；(3)将72.9mg HDTMA溶于40ml去离子水。上述三个溶液混合定容至100ml，pH调至中性，121℃灭菌20min。B：30.24g Pipes加入900ml WA培养基，pH调至6.8，121℃灭菌20min。A，B液混合倒平板，接菌30℃培养3天观察，测透明圈的大小，挑选水解圈半径(外径与内径差)大于5mm的菌株视为具有显著产嗜铁素活性。
表1 菌株YW-1代谢产物活性筛选结果
Figure PCTCN2020129982-appb-000001
注：内径为菌株在平板上生长所形成菌落直径，外径为所形成水解圈直径，单位均为毫米(mm)。
4、生防菌株YW-1平板抑菌活性检测
通过平板对峙生长法检测生防菌株YW-1对枯萎病、青枯病及疫病病原菌的平板抑制活性，如表2所示是菌株YW-1平板抑菌活性检测，以及图2是菌株YW-1平板拮抗活性筛选结果；图2-A青枯菌拮抗筛选，其中21号菌株为YW-1；图2-B和图2-C是枯萎病菌和疫霉菌的拮抗筛选，其中线形菌落是拮抗细菌菌落，图2-B的圆形菌落为待测的枯萎病菌，图2-C的圆形菌落为待测的疫霉菌，其中线形菌落中的21号为YW-1菌株，其他线形菌落中的菌株为不符合筛选条件的菌株；具体方法如下：
青枯菌抑菌活性检测：将青枯菌于YGPA平板上培养2天后，收集菌体，用无菌水悬浮，制备成OD 600＝0.2(2.0×10 8CFU/mL)的菌悬液。在45℃、400mL的YGPA培养基中加入5mL的菌悬液和2mL(5％)TZC，混匀，倒平板。用LB平板活化生防菌菌株，牙签挑取处于生长盛期的菌落点于含青枯病原菌的YGPA平板上，每板5点，间距相等，30℃培养48h，观察并且记录结果，分别量取颉颃菌内径以及颉颃圈外径，挑选拮抗圈半径(外径与内径差)大于5mm的菌株视为具有显著抑制青枯菌活性。
枯萎病及疫病抑菌活性检测：
采用对峙培养法，将保存于4℃中的疫霉菌、枯萎病菌接种到PDA平板上活化，待真菌长满平板后用灭菌的打孔器从菌落外边缘均匀的打成直径为8mm的圆形菌块。将菌丝块均匀接种在WA平板的四个方向，中间间 隔2cm。培养24h后利用接种环划线法将待测细菌划于两个菌丝块中间。25℃培养36-48h，记录抑菌半径的大小，挑选拮抗圈半径(外径与内径差)大于5mm的菌株视为具有显著抑菌活性。
表2 菌株YW-1平板抑菌活性检测
Figure PCTCN2020129982-appb-000002
实施例2
YW-1生防菌剂的制备
将生防菌株YW-1在LB培养液(胰蛋白胨10g/L，酵母提取物5g/L，氯化钠10g/L，pH＝7.2)28℃180rpm振荡培养12-16h后获得活菌总浓度为1×109-1×1010CFU/mL菌悬液，再将菌悬液于6000rpm离心10min收集菌体，用灭菌水进行稀释制成浓度为10 7CFU/mL的生防菌剂。
实施例3
YW-1生防菌剂对多种植物土传病害的温室盆钵效果验证
1、温室盆钵试验检测YW-1生防菌剂对番茄和辣椒青枯病的防效
利用穴盘培育番茄苗和辣椒苗，长到3-4片真叶时进行移栽，处理组移栽时采用20mL 10 7CFU/mL菌剂灌根处理，对照组采用清水处理，移栽一周后接种10 7CFU/mL青枯菌(Ralstonia solanacearum 3721)菌悬液20mL。每个处理组24株，三次重复。在温度25～28℃，相对湿度60％，光照12h/12h条件下，培养4周后调查病级数，计算病害严重度和防效。
按照Kempe和Sequeria 1983年提出的病害分级标准，病害严重度和防效的计算公式如下：
DI 0，无发病；
DI 1，≤25％的叶片萎蔫；
DI 2，25–50％的叶片萎蔫；
DI 3，50–75％的叶片萎蔫；
DI 4，75–100％的叶片萎蔫(Kempe and Sequeria,1983)。
Figure PCTCN2020129982-appb-000003
Figure PCTCN2020129982-appb-000004
在移栽4周后的调查结果显示(见表3和表4)，生防菌剂YW-1对番茄青枯病的防效达到59.68％，对辣椒青枯病的防效达到62.48％。
表3 温室中生防菌剂YW-1对番茄青枯病的生防效果
Figure PCTCN2020129982-appb-000005
表4 温室中生防菌剂YW-1对辣椒青枯病的生防效果
Figure PCTCN2020129982-appb-000006
2、温室盆钵试验检测YW-1生防菌剂对黄瓜枯萎病的防效
利用穴盘培育黄瓜苗，长到3-4片真叶时进行移栽，处理组移栽时采用20mL 10 7CFU/mL菌剂灌根处理，对照组采用清水处理，移栽一周后接种10 5孢子囊/mL枯萎病病原菌菌悬液20mL。每个处理组24株，三次重复。在温度25～28℃，相对湿度60％，光照12h/12h条件下，培养3周后调查病 级数，计算病害严重度和防效。
按照Kempe和Sequeria 1983年提出的病害分级标准，病害严重度和防效的计算公式如下：
DI 0，无发病；
DI 1，≤25％的叶片萎蔫；
DI 2，25–50％的叶片萎蔫；
DI 3，50–75％的叶片萎蔫；
DI 4，75–100％的叶片萎蔫(Kempe and Sequeria,1983)。
Figure PCTCN2020129982-appb-000007
Figure PCTCN2020129982-appb-000008
在移栽3周后的调查结果显示(见表5)，生防菌剂YW-1对黄瓜枯萎病的防效达到64.80％。
表5 温室中生防菌剂YW-1对黄瓜枯萎病的生防效果
Figure PCTCN2020129982-appb-000009
3、温室盆钵试验检测YW-1生防菌剂对辣椒疫病的防效
利用穴盘培育辣椒苗，长到3-4片真叶时进行移栽，处理组移栽时采用20mL 10 7CFU/mL菌剂灌根处理，对照组采用清水处理，移栽一周后接种10 5孢子囊/mL疫霉菌菌悬液20mL。每个处理组24株，三次重复。在温 度25～28℃，相对湿度60％，光照12h/12h条件下，培养4周后调查病级数，计算病害严重度和防效。
按照Kempe和Sequeria 1983年提出的病害分级标准，病害严重度和防效的计算公式如下：
DI 0，无发病；
DI 1，≤25％的叶片萎蔫；
DI 2，25–50％的叶片萎蔫；
DI 3，50–75％的叶片萎蔫；
DI 4，75–100％的叶片萎蔫(Kempe and Sequeria,1983)。
Figure PCTCN2020129982-appb-000010
Figure PCTCN2020129982-appb-000011
在移栽4周后的调查结果显示(见表6)，生防菌剂YW-1对辣椒疫病的防效达到71.36％。
表6 温室中生防菌剂YW-1对辣椒疫病的生防效果
Figure PCTCN2020129982-appb-000012
温室实验表明：在寄主植物移栽时以该菌剂灌根处理20mL/株，能够有效防治瓜类枯萎病，番茄及辣椒青枯病，辣椒疫病等多种土传病害，相对于空白对照组，防治效果均达到50％以上。
以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内

Claims (6)

  1. 一种生防菌株YW-1，其特征在于，所述菌株为拟香味类香菌(Myroides odoratimimus)，所述菌株的保藏编号为CGMCC NO.20620。
  2. 一种利用权利要求1所述生防菌株YW-1制备的生防菌剂。
  3. 一种制备权利要求2所述生防菌剂的方法，其特征在于，将所述生防菌株YW-1在LB培养液培养后，收集菌体并用灭菌水进行稀释制成总浓度为10 7CFU/mL的生防菌剂。
  4. 根据权利要求3所述的方法，其特征在于，所述生防菌株YW-1在LB培养液培养后获得的菌悬液中活菌总浓度为1×10 9-1×10 10CFU/mL。
  5. 一种权利要求2所述的生防菌剂在防治植物土传病害中的应用。
  6. 根据权利要求5所述的应用，其特征在于，所述植物土传病害为瓜类枯萎病、番茄和辣椒青枯病以及辣椒疫病中的一种或多种。
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