CN103865825B - Prevent and treat the biocontrol microorganisms of sesame blight, separation method, microbial inoculum and application thereof - Google Patents
Prevent and treat the biocontrol microorganisms of sesame blight, separation method, microbial inoculum and application thereof Download PDFInfo
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- CN103865825B CN103865825B CN201210538699.8A CN201210538699A CN103865825B CN 103865825 B CN103865825 B CN 103865825B CN 201210538699 A CN201210538699 A CN 201210538699A CN 103865825 B CN103865825 B CN 103865825B
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Abstract
The invention discloses a kind of prevent and treat sesame blight biocontrol microorganisms, separation method, biocontrol microorganisms microbial inoculum and application thereof, wherein, this biocontrol microorganisms is aobvious subclass genus bacillus (Paenibacillus peoriae) SF4-11 of skin Austria, depositary institution: China typical culture collection center, preservation address, Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University preservation center, preservation date: on November 28th, 2012, preserving number: CCTCC NO:M2012489.The present invention, by carrying out microorganism separation, opposite culture to Philippine Violet Herb root soil, is separated the biocontrol microorganisms obtaining preventing and treating sesame blight.Biocontrol microorganisms of the present invention is a kind of bacterial classification of effective control sesame blight, adopts microbial inoculum prepared by this biocontrol microorganisms, in use can not pollute ecotope, can reach non-harmful effect.Use biocontrol microorganisms of the present invention can reduce the consumption of other chemical pesticides, reduce the prevention and control cost of sesame disease, promote sesame growth, there is good promotional value.
Description
Technical field
The present invention relates to a kind of biocontrol microorganisms preventing and treating sesame blight, relate to the separation method of this biocontrol microorganisms, this biocontrol microorganisms microbial inoculum and application thereof simultaneously, belong to technical field of plant protection.
Background technology
Plant blight is the important plant soil-borne diseases of a class, and Main Pathogenic Bacteria is Fusarium oxysporum, and this disease endangers plant from plant root, can cause the extensive underproduction of plant.Wherein, sesame is endangered the most serious a kind of farm crop.
Sesame (Sesamum indicum), is under the jurisdiction of pedaliaceae flax and belongs to, and is one of the most ancient in the world oil crops, the high-quality oil crops that Ye Shi China is important.China's sesame Annual planting area about 1,200 ten thousand mu, ultimate production about 750,000 tons, has consequence in international oil seed trade.Sesame blight is (Sesame Fusarium wilt, SFW) a kind of silborne fungal diseases caused by fusarium oxysporum sesame (Fusarium oxysporum f.sp.sesami (Zaprometoff) Castellani, FOS) infection process.Sesame all can be infected by Fusarium oxysporum in the time of infertility.This disease belongs to worldwide disease, and India, the Sudan, Egypt and many sesame producing countries such as Pakistani often have every year and occur in various degree.This disease mainly occurs in northeast, North China, northwest, the Yellow River and Huai He River and Yangze river and Huai river some areas in China, long-term incidence about 15%, can more than 30% be reached during serious generation, often cause that sesame seed is immature, seed is thin and hollow, easily explode before results, serious threat and hamper production and the development of sesame.Traditional method was mainly by chemical pesticide control farm crop blight disease in the past, but along with a large amount of uses of chemical pesticide, caused serious pollution, also compromise the health of the mankind to physical environment.And a large amount of uses of chemical pesticide also make Pathogen of Fusarium Wilt create resistance, prevent and treat blight effect and have a greatly reduced quality.
Summary of the invention
The object of this invention is to provide a kind of biocontrol microorganisms preventing and treating sesame blight.
In order to realize above object, the technical solution adopted in the present invention is to provide a kind of biocontrol microorganisms preventing and treating sesame blight, described biocontrol microorganisms is aobvious subclass genus bacillus (Paenibacillus peoriae) SF4-11 of skin Austria, preservation date: on November 28th, 2012, depositary institution: China typical culture collection center, preservation address, Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University preservation center, preserving number: CCTCC NO:M 2012489.
The present invention also aims to provide a kind of separation method preventing and treating the biocontrol microorganisms of sesame blight.
The technical solution adopted in the present invention is also to provide a kind of separation method preventing and treating the biocontrol microorganisms of sesame blight, comprises the following steps:
1) separation of pathogenic bacteria and soil microorganisms:
A) separation of pathogenic bacteria:
Get sesame blight disease plant, the sick bar of sesame plant is carried out disinfection, cleaned, again disease is felt on PDA substratum that central bundle tissue puts into containing 50 μ g/mL paraxin, 3-4d is cultivated under 26-28 DEG C of condition, then after dominant colony being cultivated 3-4d in PDA substratum, carry out single spore separation purifying, save backup;
B) separation of soil microorganisms:
Take Philippine Violet Herb root soil, add sterilized water, adopt E-test dilution soil supension, get concentration gradient soil supension and be uniformly coated on 1/10TSA culture plate, under 26-28 DEG C of condition, be inverted and cultivate 1-2d; The colony inoculation grown is carried out separation and purification in TSA substratum, then by the bacterium of purifying and 50% glycerine balanced mix-70 DEG C of freezen protective for subsequent use;
2) Antagonistic Fungi screening:
By the different soils bacterium of cultivation and fusarium oxysporum sesame opposite culture on PDK substratum, determine whether antagonistic action;
3) qualification classification is carried out to Antagonistic Fungi.
The sick bar of described sesame plant is the cane of the strong intersection of sesame stalk disease.
Described PDA substratum: potato 200g, glucose 20g, agar powder 15g, distilled water 1000mL, pH7.0-7.2,121 DEG C of moist heat sterilization 20min.
Described 1/10TSA substratum is: TSB (Trypticase Soya Broth, Difco.) 3g, agar 15g, distilled water 1000mL, pH7.0-7.2,121 DEG C of moist heat sterilization 20min.
Described TSA substratum is: TSB (Trypticase Soya Broth, Difco.) 30g, agar 15g, distilled water 1000mL, pH7.0-7.2,121 DEG C of moist heat sterilization 20min.
Described PDK substratum: potato 200g, glucose 20g, peptone 10g, agar 15g, distilled water 1000mL, pH7.0-7.2,121 DEG C of moist heat sterilization 20min.
The present invention also aims to provide a kind of biocontrol microorganisms microbial inoculum preventing and treating sesame blight.
The technical solution adopted in the present invention is also to provide a kind of biocontrol microorganisms microbial inoculum preventing and treating sesame blight, is seeded in by biocontrol microorganisms in LB liquid nutrient medium, shaking culture 2-3d under 30-33 DEG C of condition, centrifugal, gets precipitation and adds sterilized water and get final product.
Described LB liquid nutrient medium is: yeast extract 5g, peptone 10g, sodium-chlor 10g, distilled water 1000mL, pH7.0-7.2,121 DEG C of moist heat sterilization 20min.
The present invention also aims to provide a kind of biocontrol microorganisms preventing and treating sesame blight promoting the application in sesame growth.
The technical solution adopted in the present invention is also to provide a kind of biocontrol microorganisms preventing and treating sesame blight promoting the application in sesame growth.
The present invention is separated the bacterial classification that the biocontrol microorganisms obtained is a kind of effective control sesame blight, this biocontrol microorganisms is adopted to prepare microbial inoculum, method is simple, easy to operate, be convenient to the storage and transport of biocontrol microorganisms, obtained microbial inoculum belongs to biotechnological formulation, in use can not pollute ecotope, non-harmful effect can be reached.In addition, use biocontrol microorganisms of the present invention can reduce the consumption of other chemical pesticides, reduce the prevention and control cost of sesame disease, promote sesame growth simultaneously, there is good promotional value.
Accompanying drawing explanation
Fig. 1 is biocontrol microorganisms of the present invention and fusarium oxysporum sesame opposite culture;
Fig. 2 is the colonial morphology of biocontrol microorganisms of the present invention on culture plate;
Fig. 3 be biocontrol microorganisms of the present invention to sesame plant strain growth promoter action design sketch,
In figure, Fig. 3 is left: for greenhouse of the present invention basin alms bowl test biocontrol microorganisms is to the design sketch of sesame plant strain growth promoter action,
Fig. 3 is right: for land for growing field crops of the present invention basin alms bowl test biocontrol microorganisms is to the design sketch of sesame plant strain growth promoter action.
Embodiment
To be Henan Sesame Research Centre with 2009 be separated sesame Pathogen of Fusarium Wilt strain HSFO 09100 from Lu Gang township, Fengqiu, Henan Province He Zhai village sesame diseased plant obtains, and saved backup by plant pathology research department of Henan Sesame Research Centre.
To be Henan Sesame Research Centre with 2010 be separated sesame Pathogen of Fusarium Wilt strain HSFO 10044 from Yuci county, Jinzhong City, Shanxi Province sesame diseased plant obtains, and saved backup by plant pathology research department of Henan Sesame Research Centre.
Philippine Violet Herb root soil is The Cloud Terrace village, Xixia, Henan Violaceae Philippine Violet Herb root soil.
The preparation of substratum:
PDA substratum: potato 200g, glucose 20g, agar powder 15g, distilled water 1000mL, pH7.0-7.2,121 DEG C of moist heat sterilization 20min.
1/10TSA substratum is: TSB (Trypticase Soya Broth, Difco.) 3g, agar 15g, distilled water 1000mL, pH7.0-7.2,121 DEG C of moist heat sterilization 20min.
TSA substratum is: TSB (Trypticase Soya Broth, Difco.) 30g, agar 15g, distilled water 1000mL, pH7.0-7.2,121 DEG C of moist heat sterilization 20min.
PDK substratum: potato 200g, glucose 20g, peptone 10g, agar 15g, distilled water 1000mL, pH7.0-7.2,121 DEG C of moist heat sterilization 20min.
LB liquid nutrient medium is: yeast extract 5g, peptone 10g, sodium-chlor 10g, distilled water 1000mL, pH7.0-7.2,121 DEG C of moist heat sterilization 20min.
The separation of embodiment 1, pathogenic bacteria
With aseptic cutters, cane is cut into the segment of 2cm at the strong intersection of sesame stalk disease, 75% alcohol surface sterilization 30s, 3% clorox surface sterilization 3min, then uses aseptic water washing 4 times; Again with aseptic cutters cane segment from middle part rip cutting into two, vascular tissue is revealed; Tweezer central bundle tissue gently, is put on the PDA substratum containing 50 μ g/mL paraxin.After constant-temperature moisture-keeping cultivates 4d in 26-28 DEG C of thermostat container, after picking out dominant colony PDA cultivation 4d, carry out single spore separation purifying, and save backup.
The separation of embodiment 2, soil microorganisms
Get the Philippine Violet Herb root soil 1g of collection, with sterilized water gradient dilution to 10
-5-10
-3g/mL, the mixed solution diluted is coated equably on 1/10TSA culture plate, be inverted in 28 DEG C of constant incubators and cultivate 2d, after bacterial colony grows, with the different bacterium of sterile toothpick picking size, color or form in TSA media surface line separation and purification.
Embodiment 3, screening of biocontrol agents
Screening of biocontrol agents adopts dull and stereotyped opposite culture method, connects Fusarium oxysporum bacterium block, surveys bacterium from the reception of bacterium block 25mm place, be placed in 26-28 DEG C of thermostat container and cultivate 4-6d, observe and produce with or without inhibition zone, and measure inhibition zone size, as shown in Figure 1 in the dull and stereotyped central authorities of PDK.
Antagonism test result shows, finds a kind of sesame blight biocontrol microorganisms, called after SF4-11 in Philippine Violet Herb root soil.
Embodiment 4, biocontrol microorganisms morphological observation
Biocontrol microorganisms SF4-11 bacterium colony cream white, subcircular, diameter 1-2mm, surface drying gauffer, opaque, easy picking, as shown in Figure 1.
The qualification classification of embodiment 5, biocontrol microorganisms
Check order to the biocontrol microorganisms filtered out, qualification result is the aobvious subclass genus bacillus (Paenibacillus peoriae) of skin Austria.
16SrDNA gene sequencing result:
tgctatacatgcagtcgagctggattgcttatagaagcttgcttctaaacaacctagcggcggacgggtgagtaacacgtaggcaacctgcccacaagacagggataactaccggaaacggtagctaatacccgatacatccttttcctgcatgggagaaggaggaaagacggagcaatctgtcacttgtggatgggcctgcggcgcattagctagttggtggggtaaaggcctaccaaggcgacgatgcgtagccgacctgagagggtgatcggccacactgggactgagacacggcccagactcctacgggaggcagcagtagggaatcttccgcaatgggcgaaagcctgacggagcaacgccgcgtgagtgatgaaggttttcggatcgtaaagctctgttgccagggaagaacgtcttgtagagtaactgctacaagagtgacggtacctgagaagaaagccccggctaactacgtgccagcagccgcggtaatacgtagggggcaagcgttgtccggaattattgggcgtaaagcgcgcgcaggcggctctttaagtctggtgtttaatcccgaggctcaacttcgggtcgcactggaaactggggagcttgagtgcagaagaggagagtggaattccacgtgtagcggtgaaatgcgtagagatgtggaggaacaccagtggcgaaggcgactctctgggctgtaactgacgctgaggcgcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacgatgaatgctaggtgttaggggtttcgatacccttggtgccgaagttaacacattaagcattccgcctggggagtacggtcgcaagactgaaactcaaaggaattgacggggacccgcacaagcagtggagtatgtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatccctttgaccggtctagagatagacctttccttcgggacagaggagacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttatgcttagttgccagcaggtcaagctgggcactctaagcagactgccggtgacaaaccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtactacaatggccggtacaacgggaagcgaaatcgcgaggtggagccaatcctagaaaagccggtctcagttcggattgtaggctgcaactcgcctacatgaagtcggaattgctagtaatcgcggatcagcatgccgcggtgaatacgttcccgggtcttgtacacaccgcccgtcacaccacgagagtttacaacacccgaagtcggtggggtaacccgcaagggagccagccgccgaaagtg
The preparation of embodiment 6, biocontrol microorganisms microbial inoculum
Be seeded in by biocontrol microorganisms in LB liquid nutrient medium, at 30 DEG C, shaking culture 48h under 180rmp condition, the then centrifugal 10min of 6000rmp, get precipitation and add sterilized water and get final product, and in microbial inoculum finished product, viable bacteria total concn is 1 × 10
10-1 × 10
11cFU/mL.
Biocontrol microorganisms of the present invention is to the preventive and therapeutic effect of sesame blight:
Sesame plant selects HSFO 09100 and HSFO 10044 as test plant respectively.
Get 30 paper pots, often organize 10, be divided into 3 groups, be respectively test group, positive controls and negative control group.Get viable bacteria concentration 10
8the microbial inoculum 15mL of CFU/mL, pours mixing in the sterile vermiculite of 2 times of volumes into and mixes thoroughly, then inoculate viable bacteria concentration 10
6the Fusarium oxysporum bacteria suspension 10mL of CFU/mL, after mixing, mixes the vermiculite and sterile soil that are mixed with biocontrol fungicide and Fusarium oxysporum pathogenic bacteria, as test group by the volume ratio of 1:3.After mixing soil using clear water inoculation Fusarium oxysporum as positive controls (guarantee morbidity), only using clear water mix soil as negative control group (plant is healthy).Sowed in paper pot by the aseptic sesame seed showed money or valuables one carries unintentionally, every alms bowl evenly puts 10.At temperature 25-28 DEG C, under the dark alternation condition of relative humidity 60%, the 12h/ daylight, cultivate the 15 days sick progression of " Invest, Then Investigate ", calculate disease severity and preventive effect.
According to blight generation in seedling stage symptom, sesame blight in seedling stage plague grade is divided into 3 grades.0 grade: grow fine without illness, overground part cauline leaf is normal, and rhizome portion vascular bundle is normal; I grade: plant part blade is wilted, there is slightly hanging contracting in stem, sorrel scab appears in rhizome portion epidermis; II grade: blade all turns yellow, withered, whole strain is wilted, and occurs lodging, dead.Each process disease index is calculated according to disease plague grade.Severity calculation formula is as follows:
Preventive effect calculation formula is as follows:
After plantation, the investigation result of the 18th day is as table 1.
Table 1 biocontrol microorganisms of the present invention is to the prevention effect of sesame Pathogen of Fusarium Wilt strain
Biocontrol microorganisms of the present invention is to the growth effect (test of greenhouse basin alms bowl) of sesame plant:
When sesame seed is unearthed, true leaf launches completely, by viable bacteria concentration 10
8cFU/mL biocontrol microorganisms 15mL root irrigation, as test group, fills with root as a control group with 15mL clear water.Each group arranges 10 repetitions, eachly repeats plantation 10 strain.Temperature 25-28 DEG C, relative humidity 60%, under the dark alternation condition of the 12h/ daylight, cultivate and observe plant growing way afterwards, as shown in Fig. 3 left side in 20 days.
Biocontrol microorganisms of the present invention is to the growth effect (test of land for growing field crops basin alms bowl) of sesame plant
When sesame seed is unearthed, true leaf launches completely, by viable bacteria concentration 10
8cFU/mL biocontrol microorganisms 100mL first time root irrigation, as test group, fills with root as a control group with 100mL clear water; In sesame initial bloom stage viable bacteria concentration 10
8cFU/mL biocontrol microorganisms 100mL is to test group sesame plant second time root irrigation, and with 100mL clear water to control group second time root irrigation, each group arranges 10 repetitions, eachly repeats plantation 10 strain.Under the envrionment conditions of land for growing field crops, observe plant growing way in each vegetative period, as shown in Fig. 3 right side.
As can be seen from Figure 3, adopt biocontrol microorganisms microbial inoculum of the present invention to fill with the sesame plant of root, than adopting, the sesame plant strain growth situation of clear water filling root is good, and plant is more healthy and strong, and therefore, biocontrol microorganisms microbial inoculum of the present invention has the effect of growth promoting effects to sesame plant.
Claims (4)
1. prevent and treat the biocontrol microorganisms of sesame blight, it is characterized in that: described biocontrol microorganisms be skin Leah series bacillus difficult to understand (
paenibacillus peoriae) SF4-11, depositary institution: China typical culture collection center, preservation address: Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University preservation center, preserving number: CCTCC NO:M 2012489.
2. adopt a biocontrol microorganisms microbial inoculum for biocontrol microorganisms as claimed in claim 1, it is characterized in that: biocontrol microorganisms is seeded in LB liquid nutrient medium, shaking culture 2-3d under 30-33 DEG C of condition, centrifugal, get precipitation and add sterilized water and get final product.
3. biocontrol microorganisms microbial inoculum according to claim 2, is characterized in that: described LB liquid nutrient medium is: yeast extract 5g, peptone 10g, sodium-chlor 10g, distilled water 1000mL, pH7.0-7.2,121 DEG C of moist heat sterilization 20min.
4. the biocontrol microorganisms preventing and treating sesame blight is as claimed in claim 1 promoting the application in sesame growth.
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| CN113215041A (en) * | 2021-05-12 | 2021-08-06 | 广东丽豪生物农业有限公司 | Microbial agent and planting method for promoting growth of sesame |
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| CN103865824A (en) * | 2012-12-13 | 2014-06-18 | 河南省农业科学院 | Bio-control bacterium for preventing and treating sesame wilt disease, separation method, inoculant, and application of the inoculant |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103865824A (en) * | 2012-12-13 | 2014-06-18 | 河南省农业科学院 | Bio-control bacterium for preventing and treating sesame wilt disease, separation method, inoculant, and application of the inoculant |
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| Title |
|---|
| Improvement of biological control capacity of Paenibacillus polymyxa E681 by seed pelleting on sesame;Ryu Choong-Min 等;《Biological Control》;20060430;第39卷(第3期);摘要 * |
| Isolation and evaluation of protective effect against Fusarium wilt of sesame plants of antibiotic substance from Bacillus polymyxa KB-8;Huyn JaeWook 等;《Plant Pathology Journal》;19991231;第15卷(第3期);152-157 * |
| 内生细菌对芝麻枯萎病菌的拮抗作用及其特性;王俊芳 等;《安徽农学通报》;20121024;第18卷(第15期);36-38 * |
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