CN103865825A - Bio-control bacterium for preventing and treating sesame wilt disease, separation method, inoculant, and application of the inoculant - Google Patents
Bio-control bacterium for preventing and treating sesame wilt disease, separation method, inoculant, and application of the inoculant Download PDFInfo
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Abstract
The invention discloses a bio-control bacterium for preventing and treating sesame wilt disease, a separation method, an inoculant, and an application of the inoculant. The bio-control bacterium is paenibacillus peoriae SF4-11; the preservation organization is China Center for Type Culture Collection (CCTCC); the address of the preservation organization is preservation center of Wuhan University, Luojia Mountain road, Wuchang district, Wuhan city, Hubei province; the preservation date is November 28th, 2014; and the preservation number is CCTCC No.M2012489. The bio-control bacterium is prepared by the following steps: carrying out a microorganism separation treatment and a confront culture treatment on soil of Chinese violet roots, and then carrying out a separation treatment so as to obtain the bio-control bacterium for preventing and treating the sesame wilt disease. The bio-control bacterium can effectively prevent and treat the sesame wilt disease. The inoculant prepared from the bio-control bacterium will not generate pollution to the environment during the application process, and thus is harmless. Moreover, the using amount of other chemical pesticides is reduced, the control cost of sesame diseases is reduced, the growth of sesame is promoted, and thus the inoculant and the bio-control bacterium are worthy to promote.
Description
Technical field
The present invention relates to a kind of biocontrol microorganisms of preventing and treating sesame blight, relate to separation method, this biocontrol microorganisms microbial inoculum and application thereof of this biocontrol microorganisms simultaneously, belong to plant protection technology field.
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.1,200 ten thousand mu of left and right of China's sesame Annual planting area, approximately 750,000 tons of ultimate productions have consequence in international oil seed trade.Sesame blight is (Sesame Fusarium wilt, SFW) a kind of silborne fungal diseases being 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 such as Pakistani producing country often have every year in various degree and occur.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, throughout the year incidence 15% left and right, can reach while seriously generation more than 30%, often causes that sesame seed is immature, seed is thin and hollow, easily before results, explode serious threat and hamper production and the development of sesame.Traditional method, mainly by chemical pesticide control farm crop blight disease, still, along with a large amount of uses of chemical pesticide, had caused serious pollution to physical environment in the past, had also endangered the mankind's health.And a large amount of uses of chemical pesticide also make Pathogen of Fusarium Wilt produce 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 of 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 of preventing and treating sesame blight, described biocontrol microorganisms is skin aobvious subclass genus bacillus (Paenibacillus peoriae) SF4-11 difficult to understand, preservation date: on November 28th, 2012, depositary institution: Chinese Typical Representative culture collection center, preservation address, Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University preservation center, preserving number: CCTCC NO:M2012489.
The present invention also aims to provide a kind of separation method of the biocontrol microorganisms of preventing and treating sesame blight.
The technical solution adopted in the present invention is also to provide a kind of separation method of the biocontrol microorganisms of preventing and treating sesame blight, comprises the following steps:
1) separation of pathogenic bacteria and soil microorganisms:
A) separation of pathogenic bacteria:
Get sesame blight disease plant, sick bar to sesame plant carries out disinfection, cleans, disease being felt to central bundle tissue puts on the PDA substratum that contains 50 μ g/mL paraxin again, under 26-28 ℃ of condition, cultivate 3-4d, then dominant colony is cultivated after 3-4d in PDA substratum, carry out monospore separation and purification, 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 ℃ of condition, be inverted and cultivate 1-2d; The colony inoculation growing is carried out to separation and purification in TSA substratum, then by the bacterium of purifying and 50% glycerine balanced mix-70 ℃ freezing saving backup;
2) Antagonistic Fungi screening:
By the cultivation that stands facing each other on PDK substratum of different soils bacterium and the fusarium oxysporum sesame cultivated, judge and have or not antagonistic action;
3) Antagonistic Fungi is identified to classification.
The cane that the sick bar of described sesame plant is the sick strong intersection of sesame stalk.
Described PDA substratum: potato 200g, glucose 20g, agar powder 15g, distilled water 1000mL, pH7.0-7.2,121 ℃ 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 ℃ 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 ℃ of moist heat sterilization 20min.
Described PDK substratum: potato 200g, glucose 20g, peptone 10g, agar 15g, distilled water 1000mL, pH7.0-7.2,121 ℃ of moist heat sterilization 20min.
The present invention also aims to provide a kind of biocontrol microorganisms microbial inoculum of preventing and treating sesame blight.
The technical solution adopted in the present invention is also to provide a kind of biocontrol microorganisms microbial inoculum of preventing and treating sesame blight, biocontrol microorganisms is seeded in LB liquid nutrient medium, and shaking culture 2-3d under 30-33 ℃ of condition, centrifugal, get precipitation and add sterilized water and get final product.
Described LB liquid nutrient medium is: yeast extract 5g, peptone 10g, sodium-chlor 10g, agar 15g, distilled water 1000mL, pH7.0-7.2,121 ℃ of moist heat sterilization 20min.
The present invention also aims to provide a kind of biocontrol microorganisms of preventing and treating sesame blight in the application promoting aspect sesame growth.
The technical solution adopted in the present invention is also to provide a kind of biocontrol microorganisms of preventing and treating sesame blight in the application promoting aspect sesame growth.
The present invention separates the bacterial classification that the biocontrol microorganisms obtaining is a kind of effective control sesame blight, adopt this biocontrol microorganisms to prepare microbial inoculum, method is simple, easy to operate, be convenient to the storage and transport of biocontrol microorganisms, the microbial inoculum making belongs to biotechnological formulation, in use can not pollute ecotope, can reach non-harmful effect.In addition, use biocontrol microorganisms of the present invention can reduce the consumption of other chemical pesticides, the prevention and control cost of reduction sesame disease promotes sesame growth simultaneously, has good promotional value.
Accompanying drawing explanation
Fig. 1 is that biocontrol microorganisms of the present invention and fusarium oxysporum sesame face-off are cultivated;
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 left side: be the design sketch of greenhouse of the present invention basin alms bowl test biocontrol microorganisms to sesame plant strain growth promoter action,
Fig. 3 right side: be the design sketch of land for growing field crops of the present invention basin alms bowl test biocontrol microorganisms to sesame plant strain growth promoter action.
Embodiment
Sesame rot pathogenic strains HSFO 09100 is that Henan Sesame Research Centre separated and obtains from Lu Gang township, Fengqiu, Henan Province He Zhai village sesame diseased plant with 2009, and is saved backup by plant pathology research department of Henan Sesame Research Centre.
Sesame rot pathogenic strains HSFO 10044 is that Henan Sesame Research Centre separated and obtains from Yuci county, Jinzhong City, Shanxi Province sesame diseased plant with 2010, and is 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 ℃ 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 ℃ of moist heat sterilization 20min.
TSA substratum is: TSB (Trypticase Soya Broth, Difco.) 30g, agar 15g, distilled water 1000mL, pH7.0-7.2,121 ℃ of moist heat sterilization 20min.
PDK substratum: potato 200g, glucose 20g, peptone 10g, agar 15g, distilled water 1000mL, pH7.0-7.2,121 ℃ of moist heat sterilization 20min.
LB liquid nutrient medium is: yeast extract 5g, peptone 10g, sodium-chlor 10g, agar 15g, distilled water 1000mL, pH7.0-7.2,121 ℃ 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 sick strong intersection of sesame stalk, 75% alcohol surface sterilization 30s, 3% clorox surface sterilization 3min, then uses aseptic water washing 4 times; Use again aseptic cutters cane segment from middle part rip cutting into two, make vascular bundle organization exposure out; Tweezer central bundle tissue gently, is put on the PDA substratum that contains 50 μ g/mL paraxin.In 26-28 ℃ of thermostat container, constant-temperature moisture-keeping is cultivated after 4d, picks out dominant colony PDA and cultivates after 4d, carries out monospore separation and purification, and saves 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 having diluted is coated on 1/10TSA culture plate equably, in 28 ℃ of constant incubators, be inverted and cultivate 2d, after bacterial colony grows, with the different bacterium of aseptic toothpick picking size, color or form in the separation and purification of ruling of TSA media surface.
Embodiment 3, screening of biocontrol agents
Screening of biocontrol agents adopts dull and stereotyped face-off culture method, connects Fusarium oxysporum bacterium piece in the dull and stereotyped central authorities of PDK, surveys bacterium from the reception of bacterium piece 25mm place, is placed in 26-28 ℃ of thermostat container and cultivates 4-6d, observes and has or not inhibition zone to produce, and measure inhibition zone size, as shown in Figure 1.
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, easily picking, as shown in Figure 1.
The evaluation classification of embodiment 5, biocontrol microorganisms
The biocontrol microorganisms filtering out is checked order, and qualification result is skin aobvious subclass genus bacillus (Paenibacillus peoriae) difficult to understand.
16SrDNA gene sequencing testing result:TgctatacatgcagtcgagctggattgcttatagaagcttgcttctaaacaacctagcggcggacgggtgagtaacacgtaggcaacctgcccacaagacagggataactaccggaaacggtagctaatacccgatacatccttttcctgcatgggagaaggaggaaagacggagcaatctgtcacttgtggatgggcctgcggcgcattagctagttggtggggtaaaggcctaccaaggcgacgatgcgtagccgacctgagagggtgatcggccacactgggactgagacacggcccagactcctacgggaggcagcagtagggaatcttccgcaatgggcgaaagcctgacggagcaacgccgcgtgagtgatgaaggttttcggatcgtaaagctctgttgccagggaagaacgtcttgtagagtaactgctacaagagtgacggtacctgagaagaaagccccggctaactacgtgccagcagccgcggtaatacgtagggggcaagcgttgtccggaattattgggcgtaaagcgcgcgcaggcggctctttaagtctggtgtttaatcccgaggctcaacttcgggtcgcactggaaactggggagcttgagtgcagaagaggagagtggaattccacgtgtagcggtgaaatgcgtagagatgtggaggaacaccagtggcgaaggcgactctctgggctgtaactgacgctgaggcgcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacgatgaatgctaggtgttaggggtttcgatacccttggtgccgaagttaacacattaagcattccgcctggggagtacggtcgcaagactgaaactcaaaggaattgacggggacccgcacaagcagtggagtatgtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatccctttgaccggtctagagatagacctttccttcgggaCagaggagacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggtt aagtcccgcaacgagcgcaacccttatgcttagttgccagcaggtcaagctgggca ctctaagcaga ctgccggtgacaaaccggaggaaggtggggatgacgtcaaatcatcatgcccctta tgacctgggctacacacgtactacaatggccggtacaacgggaagcgaaatcgcga ggtggagccaatcctagaaaagccggtctcagttcggattgtaggctgcaactcgc ctacatgaagtcggaattgctagtaatcgcggatcagcatgccgcggtgaatacgt tcccgggtcttgtacacaccgcccgtcacaccacgagagtttacaacacccgaagt cggtggggtaacccgcaagggagccagccgccgaaagtg
The preparation of embodiment 6, biocontrol microorganisms microbial inoculum
Biocontrol microorganisms is seeded in LB liquid nutrient medium, at 30 ℃, shaking culture 48h under 180rmp condition, the then centrifugal 10min of 6000rmp, gets precipitation and adds sterilized water and get final product, and in microbial inoculum finished product, viable bacteria total concn is 1 × 10
10-1 × 10
11cFU/mL.
The preventive and therapeutic effect of biocontrol microorganisms of the present invention to sesame blight:
Sesame plant selects respectively HSFO 09100 and HSFO 10044 as test plant.
Get 30 paper pots, 10 every group, 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 in the aseptic vermiculite of 2 times of volumes and mixes and mix thoroughly, then inoculates viable bacteria concentration 10
6the Fusarium oxysporum bacteria suspension 10mL of CFU/mL, after mixing, mixes by the volume ratio of 1: 3 vermiculite and the sterile soil that are mixed with biocontrol fungicide and Fusarium oxysporum pathogenic bacteria, as test group.Using clear water mix inoculation Fusarium oxysporum after soil as positive controls (guarantee morbidity), only using clear water mix soil as negative control group (plant health).The aseptic sesame seed having showed money or valuables one carries unintentionally is sowed in paper pot, and every alms bowl is evenly put 10.At temperature 25-28 ℃, relative humidity 60%, under the dark alternation condition of the 12h/ daylight, cultivates the 15 days sick progression of " Invest, Then Investigate ", calculates 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 level: plant part blade is wilted, there is slightly hanging contracting in stem, there is sorrel scab in rhizome portion epidermis; II level: the whole flavescence of blade, withered, whole strain is wilted, and occurs lodging, death.Calculate each processing disease index 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.
The prevention effect of table 1 biocontrol microorganisms of the present invention to the strain of sesame Pathogen of Fusarium Wilt
The growth effect (greenhouse basin alms bowl test) of biocontrol microorganisms of the present invention to sesame plant:
When sesame seed is unearthed, true leaf is while launching 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 to plant 10 strains.Under temperature 25-28 ℃, relative humidity 60%, the dark alternation condition of the 12h/ daylight, cultivate and observe plant growing way after 20 days, as shown in Fig. 3 left side.
The growth effect (land for growing field crops basin alms bowl test) of biocontrol microorganisms of the present invention to sesame plant
When sesame seed is unearthed, true leaf is while launching completely, by viable bacteria concentration 10
8cFU/mL biocontrol microorganisms 100mL root irrigation for the first time, as test group, fills with root as a control group with 100mL clear water; In viable bacteria concentration 10 for sesame initial bloom stage
8cFU/mL biocontrol microorganisms 100mL is to test group sesame plant root irrigation for the second time, and with 100mL clear water, to control group root irrigation for the second time, each group arranges 10 repetitions, eachly repeats to plant 10 strains.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 that promotes growth to sesame plant.
Claims (10)
1. the biocontrol microorganisms of control sesame blight, it is characterized in that, described biocontrol microorganisms is skin aobvious subclass genus bacillus (Paenibacillus peoriae) SF4-11 difficult to understand, depositary institution: Chinese Typical Representative culture collection center, preservation address: Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University preservation center, preserving number: CCTCCNO:M 2012489.
2. a separation method for control sesame blight biocontrol microorganisms as claimed in claim 1, is characterized in that, comprises the following steps:
1) separation of pathogenic bacteria and soil microorganisms:
A) separation of pathogenic bacteria:
Get sesame blight disease plant, sick bar to sesame plant carries out disinfection, cleans, disease being felt to central bundle tissue puts on the PDA substratum that contains 50 μ g/mL paraxin again, under 26-28 ℃ of condition, cultivate 3-4d, then dominant colony is cultivated after 3-4d in PDA substratum, carry out monospore separation and purification, 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 ℃ of condition, be inverted and cultivate 1-2d; The colony inoculation growing is carried out to separation and purification in TSA substratum, then by the bacterium of purifying and 50% glycerine balanced mix-70 ℃ freezing saving backup;
2) Antagonistic Fungi screening:
By the cultivation that stands facing each other on PDK substratum of different soils bacterium and the fusarium oxysporum sesame cultivated, judge and have or not antagonistic action;
3) Antagonistic Fungi is identified to classification.
3. a kind of separation method of preventing and treating sesame blight biocontrol microorganisms according to claim 2, is characterized in that, the cane that the sick bar of described sesame plant is the sick strong intersection of sesame stalk.
4. a kind of separation method of preventing and treating sesame blight biocontrol microorganisms according to claim 2, is characterized in that, described PDA substratum: potato 200g, glucose 20g, agar powder 15g, distilled water 1000mL, pH7.0-7.2,121 ℃ of moist heat sterilization 20min.
5. a kind of separation method of preventing and treating sesame blight biocontrol microorganisms according to claim 2, it is characterized in that, described 1/10TSA substratum is: TSB (Trypticase Soya Broth, Difco.) 3g, agar 15g, distilled water 1000mL, pH7.0-7.2,121 ℃ of moist heat sterilization 20min.
6. a kind of separation method of preventing and treating sesame blight biocontrol microorganisms according to claim 2, it is characterized in that, described TSA substratum is: TSB (Trypticase Soya Broth, Difco.) 30g, agar 15g, distilled water 1000mL, pH7.0-7.2,121 ℃ of moist heat sterilization 20min.
7. a kind of separation method of preventing and treating sesame blight biocontrol microorganisms according to claim 2, is characterized in that, described PDK substratum: potato 200g, glucose 20g, peptone 10g, agar 15g, distilled water 1000mL, pH7.0-7.2,121 ℃ of moist heat sterilization 20min.
8. a biocontrol microorganisms microbial inoculum for control sesame blight as claimed in claim 1, is characterized in that, biocontrol microorganisms is seeded in LB liquid nutrient medium, and shaking culture 2-3d under 30-33 ℃ of condition, centrifugal, get precipitation and add sterilized water and get final product.
9. the biocontrol microorganisms microbial inoculum of control sesame blight according to claim 8, is characterized in that, described LB liquid nutrient medium is: yeast extract 5g, peptone 10g, sodium-chlor 10g, agar 15g, distilled water 1000mL, pH7.0-7.2,121 ℃ of moist heat sterilization 20min.
10. a biocontrol microorganisms of preventing and treating sesame blight is in the application promoting aspect sesame growth.
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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 |
| 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 |
| CN103865824B (en) * | 2012-12-13 | 2015-09-30 | 河南省农业科学院 | A kind of prevent and treat sesame blight biocontrol microorganisms, separation method, microbial inoculum and application thereof |
| 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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