CN101054565A - Biological control strain capable of preventing and curing root knot nematode disease for greenhouse vegetable - Google Patents
Biological control strain capable of preventing and curing root knot nematode disease for greenhouse vegetable Download PDFInfo
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- CN101054565A CN101054565A CN 200710021376 CN200710021376A CN101054565A CN 101054565 A CN101054565 A CN 101054565A CN 200710021376 CN200710021376 CN 200710021376 CN 200710021376 A CN200710021376 A CN 200710021376A CN 101054565 A CN101054565 A CN 101054565A
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Abstract
The present invention relates to a biological control strain AR156 for preventing and treating root-knot nematodes of greenhouse vegetable, belonging to plant protection technical field. The bacteria is bacillus sp., which is cultivated in PDA culturing liquor under 37 degree and 180rpm, then is centrifuged 10min under 6000rpm. Picking wet bacteria and bacteria storage liquid is prepared into bacteria agent according to 1:40 (g/ml)proportion. The living bacteria concentration is 1*109-1*1012cfu/ml.The effective rate of the bacteria agent amount to 58-75% for preventing root-knot nematodes of cucumber, towel gourd, tomato etc, and yield increase more than 30-50%. The bacteria agent can also treat wilt, epidemic disease and soil hardening. The bacteria agent can be sprayed, composted and irrigated to root.
Description
One, technical field
The present invention relates to the anti-bacterial strain AR156 of a kind of control curing greenhouse vegetable root knot nematode diease occurrence, belong to the farm crop technical field of preventing and curing diseases, be exclusively used in the non-environmental pollution control of root knot nematode disease.
Two, technical background
Root knot nematode (Meloidogyne spp.) platymiscium root system obligate endoparasite is the main diseases original of universal threat agriculture production, also is the external Quarantine Objects of countries in the world.It not only makes crop yield reduce, and quality reduces, and can cause compound infecting with other pathogens.The root knot nematode of having put down in writing is so far had an appointment 70 kinds, wherein Meloidogyne incognita (M.incognita), javanese root knot nematode (M.javanicica) and peanut root-knot nematode (M arenaria) are most important plant nematode kinds because of its wide distributivity and many hosts property.In China, these three kinds of root knot nematodes are economized (district) at great majority all generation, infects farm crop and reaches more than hundred kinds, and field crop, vegetables, fruit tree, flowers, medicinal plant etc. almost every kind of crop all can be infected.Root knot nematode disease (the object of causing harm: nearly all vegetables such as cucumber, wax gourd, watermelon, sponge gourd, tomato, capsicum, eggplant, potato, lettuce, spinach, Radix Dauci Sativae, Chinese cabbage, leguminous vegetable) be one of domestic and international the most serious Plant diseases.Attack parasitogenic Plant diseases by plant nematode, the plant that is injured can grow because of nutrition in the intrusion nematode cartridge influences normally; Secretory product in the nematode metabolic process also can stimulate cell and the tissue of host plant, causes the plant deformity, makes the agricultural-food underproduction and downgrade; In addition; can also pass through eelworm harm; aggravate the generation of other disease; especially protect the ground vegetables; cause the long-term underproduction 15-25% of host; sometimes reach more than 70%, seriously restricted the production and the foreign exchange earning of booth vegetable, fruit tree, flowers and other crops, have in addition to the stage that can't plant.According to estimates, the financial loss that suffers because of causing harm of root knot nematode every year of the production of China farm crop reaches more than several hundred million units.
Because the root knot nematode disease belongs to soil-borne disease, so control is comparatively difficult.At present, people mainly take organic phosphates chemistry nematocides, the disease-resistant crop varieties of choosing seeds, implement crop rotation, set up anosis seedling base and carry out the generation that the root knot nematode disease is controlled in strict quarantine to importing and exporting farm crop.Though chemistry nematocides preventive effect is remarkable, its poor selectivity is destroyed the ecotope of farm crop; Be difficult for degraded, high residue is absolutely unsafe to people and animals; Destroy Soil structure, cause soil to desertify; The resistance problem becomes increasingly conspicuous.These factors make the use range of chemical insecticide be subjected to strict restriction.Therefore, property prophylactico-therapeutic measures has non-chemically become the core that the farm crop root knot nematode disease is administered at present, and biological control then is the most promising method wherein.In the prior art according to this breadboard preservative fluid for thallus patent (publication CN1358838, open day: on July 17th, 2002) method, but thalline room temperature preservation 2～3 years.
Three, summary of the invention
Technical problem: the objective of the invention is has the biological prevention and control agent of higher preventive effect mainly to concentrate on some fungi preparations, the too high defective of use cost at of the prior art to the root knot nematode disease, provide exploitation a kind of single biocontrol microorganisms bacterial strain and microbial inoculum thereof of preventing and treating the root knot nematode disease evil, the preventive effect of this microbial inoculum can reach more than 50%, and the volume increase function is arranged.
Technical scheme
A kind of biocontrol strain AR156 that prevents and treats the curing greenhouse vegetable root knot nematode disease is bacillus (Bacillus sp.), is deposited at China Committee for Culture Collection of Microorganisms common micro-organisms center on January 26th, 2007, and culture presevation number is CGMCC NO.1929.
The method that biocontrol strain AR156 is used to prepare biocontrol fungicide is:
1) this inoculation is cultivated 37 ℃ of 180rpm respectively in the PDA nutrient solution, in Bechtop, take a sample respectively after 16～20 hours and survey the OD value at 600nm place, return to zero with the nutrient solution that does not connect bacterium in the mensuration process;
2) when being cultured to the OD value between 0.5～0.8 the time, to plant daughter bacteria liquid and add in the PDA nutrient solution, 37 ℃ of 180rpm shaking culture 20～24 hours, the centrifugal 10min of 6000rpm then with 1: 100 volume ratio, every 1000ml nutrient solution obtains the wet bacterium of 13～16g, and the wet bacterium of every gram contains 4 * 10
10～10
15Individual thalline, get wet bacterium and preservative fluid for thallus by 1g: 40ml is made into microbial inoculum, and the viable bacteria total concn is 1 * 10 in the finished product
9～1 * 10
12Fu/ml.Used PDA nutrient solution compound method is:
Be cut into small pieces to be put in the water after peeling with 200 gram potatoes and boil, boil at least after the boiling more than 20 minutes, then filter with four layers of hospital gauze, filtrate adds 20 gram common sucrose, is settled to 1000 milliliters, and the pH value transfers to 7.2～7.4, sterilizes 20 minutes for 121 ℃ then.
Beneficial effect
Advantage of the present invention and positively effect show: the present invention is the biological prevention and control agent of developing at root knot nematode disease specially.Owing to be biotechnological formulation, there are not a series of problems that use brought fully because of chemical pesticide, thereby help the nuisanceless production of vegetables, the peasant can or reduce the consumption of other control root knot nematode disease chemical pesticides, this not only can be the peasant and reduces expenses, and helps the outlet of vegetables.Simultaneously, this microbial inoculum increases production function in addition, can be the peasant and increases income.
Isolated test shows, presses AR156 biocontrol fungicide 1 * 10
9Cfu/ml, this concentration is active and the hatching of ovum all had good restraining effect (table 1, table 2) to the root knot nematode second instar larvae.The greenhouse experiment shows: this bacteria preparation can significantly reduce the intrusion of root knot nematode, and has the effect that promotes tomato growth concurrently.The warmhouse booth experiment shows: use this microbial inoculum can reach more than the 58-75% the prevention effect of root knot nematode disease evil, volume increase can reach 30-50% above (table 3 ~ 4)
This microbial inoculum with wet bacterium and this breadboard preservative fluid for thallus (belong to this laboratory granted patent, patent No. ZL02112559.7, publication CN1358838, open day: on July 17th, 2002) be made into microbial inoculum by 1: 40 (g/ml), but room temperature preservation 2～3 years.
This microbial inoculum can be poured into before transplant in the greenhouse and make compost in the fertilizer, buries in the soil in whole ground process with fertilizer then, also can irritate root and use when planting seedling.
Four embodiments
The present invention relates to the anti-bacterial strain AR156 of a kind of control curing greenhouse vegetable root knot nematode diease occurrence, be genus bacillus (Bacillussp).Be deposited at China Committee for Culture Collection of Microorganisms common micro-organisms center on January 26th, 2007, culture presevation number is CGMCC NO.1929.
The method that biocontrol strain AR156 is used to prepare biocontrol fungicide is conventional bacterial cultivation:
1) this inoculation is cultivated 37 ℃ of 180rpm respectively in the PDA nutrient solution, in Bechtop, take a sample respectively after 16～20 hours and survey the OD value at 600nm place, return to zero with the nutrient solution that does not connect bacterium in the mensuration process;
2) when being cultured to the OD value between 0.5～0.8 the time, to plant daughter bacteria liquid and add in the PDA nutrient solution, 37 ℃ of 180rpm shaking culture 20～24 hours, the centrifugal 10min of 6000rpm then with 1: 100 volume ratio, every 1000ml nutrient solution obtains the wet bacterium of 13～16g, and the wet bacterium of every gram contains 4 * 10
10～10
15Individual thalline, get wet bacterium and preservative fluid for thallus and (belong to this laboratory granted patent, patent No. ZL02112559.7, publication CN1358838, open day: on July 17th, 2002) be made into the AR156 biocontrol fungicide by 1g: 40ml, the viable bacteria total concn was 1 * 10 in the finished product
9～1 * 10
12Fu/ml.Used PDA nutrient solution compound method is:
Be cut into small pieces to be put in the water after peeling with 200 gram potatoes and boil, boil at least after the boiling more than 20 minutes, then filter with four layers of hospital gauze, filtrate adds 20 gram common sucrose, is settled to 1000 milliliters, and the pH value transfers to 7.2～7.4, sterilizes 20 minutes for 121 ℃ then.
(1) indoor isolated measuring
The preparation of 1 root knot nematode second instar larvae suspension
From the fresh pieces of an egg of tomato root picking, place the graceful funnel of shellfish to hatch, begin to collect larva behind the 24h, collect once every day, collects a week altogether.The dilution of nematode liquid is 100 J2/ml, and 4 ℃ of preservations are to be used.
The preparation of 2 ovum suspension
Method (MeClure, M.A., Kruk, T.H.﹠amp according to MeClure etc.; Misaghi, I.1973.A method for obtaining quantities of clean Meloidogyne eggs.Journal ofNematology, 5:230), put into triangular flask after will having the tomato root of root knot to shred, add 0.5%NaCl0 liquid 100ml, firmly shake 1-2min after, pour into fast in the 200-500 mesh sieve group and and wash 10min with tap water, collect the ovum in 500 eye mesh screens, being diluted to concentration is the ovum suspension of 3000/ml.
The 3AR156 biocontrol fungicide is to the effect of Meloidogyne incognita second instar larvae
Average room temperature 20 ℃ ± time, bacteria suspension is drawn 2ml, adding diameter is in the sterilization culture dish of 7.5cm, adds the suspension of 2ml second instar larvae again, compares with clear water, each is handled and repeats 3 times.Behind 12h, 24h and 36h, observe the vigor of nematode respectively.
The 4AR156 biocontrol fungicide is to the effect of Meloidogyne incognita ovum hatching
Average room temperature 20 ℃ ± time, draw bacteria suspension 2ml, adding diameter is in the sterilization culture dish of 7.5cm, adds 2ml ovum suspension again, compares with clear water, each is handled and repeats 3 times.Behind 4d, 6d, 8d and 10d, observe the hatching situation of larva respectively.
(2) greenhouse basin alms bowl test
1 tomato seedling is cultivated:
Tomato seeds carries out surface sterilization to be handled: sterilized water was washed three times after the alcohol with 75% was handled 15sec, and sterilized water was washed three times after 5% chlorine bleach liquor handled 15min.The planting seed of surface sterilization is cultivated stand-by in sterilization soil.
2 sterilization soil preparations:
With sylvogenic soil, fine sand, organic, 121 ℃ of sterilization 20mins in the high-pressure sterilizing pot by behind 2: 2: 1 volume mixings.The sterilization back is divided in the basin alms bowl that installs to diameter 25cm stand-by.
3 irritate root inoculation strains tested bacterium suspension (10
9CFUmL-1) 40ml, 100 J2/ml second instar larvaes of every alms bowl inoculation Meloidogyne incognita suspension 10ml after 2 days.
4 postvaccinal basin alms bowl random alignment are on the platform in greenhouse.Cultivate the 8 slow taking-up inoculation in week back plant for 25～28 ℃.Water fine laundering root system divides another name root length, stem length and dry matter weight, record root knot number under anatomical lens.
(3) warmhouse booth test
Field test is established three processing: A, biocontrol fungicide AR156 altogether; B, Avrmectin; C, blank.Each sub-district 12m
2, the triplex row isolated area is established in the minizone, and each is handled by completely random district group and arranges, and repeats 4 times, and complete test is totally 12 sub-districts.It is even experimental field to flatten rake before the test earlier, has divided the sub-district by above-mentioned design, holds isolating trenches successfully and plants plant hole.In transplanting preceding 15 days, use biocontrol fungicide AR156, every mu is used 1500 milliliter 10
9The AR156 preparation of CFU/ml, dilute 1000 times after with the sterilization the atomizer even spraying, cover mulch film; Every mu of usefulness was 500 milliliters when 1.8% abamectin emulsifiable concentrate was transplanted, and by 1: 1000 dilution proportion, sprinkled the pouring root.
5.3.2 drug effect and output are investigated in the hazard rating of 120 days investigation root knot nematodes, calculate preventive effect, and the stimulation ratio of cucumber yield.Disease index (%)=(plant strain numbers at different levels * level value)/(investigating total strain number * superlative degree value) * 100%.
Experimental result
(1) isolated experiment shows (table 1, table 2), presses AR156 biocontrol fungicide 1 * 10
9Cfu/ml, this concentration is active and the hatching of ovum all had the good restraining effect to root knot nematode Meloidogyne incognita second instar larvae, the hatching that can reach 91.4%, 10 day root-knot nematode egg to root knot nematode second instar larvae activity inhibition after 48 hours only reaches 9.8%.
Table 1 AR156 biocontrol fungicide is to the influence of root knot nematode second instar larvae vigor
| Handle | Maximum inhibition (%) | ||||
| 12hr | 24hr | 36hr | 48hr | ||
| Bacteria suspension | AR156 CK | 45.5 1.9 | 73.6 3.53 | 87.5 5.67 | 91.4 7.89 |
Annotate: AR156: bacterial concentration 10
9Cfu/ml; CK: clear water contrast;
Table 2AR156 is to the influence of Meloidogyne incognita Meloidogyne incognita hatching rate
| Handle | Egg hatching rate (%) | ||||
| 4d | 6d | 8d | 10d | ||
| Bacteria suspension | AR156 CK | 1.50 26.3 | 3.43 33.6 | 7.48 48.4 | 9.8 66.4 |
Annotate: AR156: bacterial concentration 10
9Cfu/ml CK: clear water contrast;
(2) greenhouse basin alms bowl test (table 3): test-results shows that the AR156 biocontrol fungicide can significantly reduce the intrusion of root knot nematode, the preventive effect of tomato Meloidogyne incognita disease is reached 64%, and have the effect that promotes tomato growth concurrently.
Table 3:AR156 microbial inoculum is to the preventive effect of tomato Meloidogyne incognita disease
| Handle | Stem long (cm/ strain) | Root long (cm/ strain) | Dry weight (g/ strain) | Root knot number (piece/strain) | Preventive effect (%) |
| AR156 CK | 34.09 26.50 | 19.50 17.81 | 2.84 2.37 | 177 327 | 64 |
Annotate: AR156: bacterial concentration 10
9Cfu/ml, CK: clear water contrast;
(3) warmhouse booth test (table 4): use the AR156 biocontrol fungicide and handle after 120 days (consumption is a 1500ml/ mu), biocontrol fungicide AR156 is 64.4% to the average preventive effect of cucumber root knot nematode, and the highest preventive effect 75% is better than Avrmectin (40.1%).In addition, biocontrol fungicide AR156 has tangible promotion energy for growth, improves the output of crops such as cucumber, sponge gourd, tomato, and stimulation ratio average out to 39.8% is up to 200%.
Table 4AR156 biocontrol microorganisms preparation is used the back 120 days prevention effect to the cucumber root knot nematode
*
| The medicament title | Dispenser dosage | Average disease index (%) | Average prevention effect (%) | The highest prevention effect (%) | Average stimulation ratio (%) |
| Preparation AR156 1.8% Avrmectin blank | 1.5L/ mu 0.5L/ mu | 32.6 58.4 91.5 | 64.4a 36.2b | 75.0 40.1 | 39.8a 13.2c |
Claims (5)
1, a kind of biocontrol strain AR156 that prevents and treats the curing greenhouse vegetable root knot nematode disease, be bacillus (Bacillus sp.), be deposited at China Committee for Culture Collection of Microorganisms common micro-organisms center on January 26th, 2007, culture presevation number is CGMCC NO.1929.
2, the biocontrol fungicide of the described biocontrol strain AR156 preparation of claim 1.
3, the application of the described biocontrol fungicide of claim 2 aspect the control curing greenhouse vegetable root knot nematode.
4, the described biocontrol strain AR156 of claim 1 is used to prepare the method for biocontrol fungicide, it is characterized in that:
1) this inoculation is cultivated 37 ℃ of 180rpm respectively in the PDA nutrient solution, in Bechtop, take a sample respectively after 16～20 hours and survey the OD value at 600nm place, return to zero with the nutrient solution that does not connect bacterium in the mensuration process;
2) when being cultured to the OD value between 0.5～0.8 the time, to plant daughter bacteria liquid and add in the PDA nutrient solution, 37 ℃ of 180rpm shaking culture 20～24 hours, the centrifugal 10min of 6000rpm then with 1: 100 volume ratio, every 1000ml nutrient solution obtains the wet bacterium of 13～16g, and the wet bacterium of every gram contains 4 * 10
10～10
16Individual thalline, get wet bacterium and preservative fluid for thallus by 1g: 40ml is made into microbial inoculum, and the viable bacteria total concn is 1 * 10 in the finished product
9～1 * 10
12Cfu/ml.
5, biocontrol strain AR156 according to claim 4 is used to prepare the method for biocontrol fungicide, it is characterized in that, used PDA nutrient solution compound method is:
Be cut into small pieces to be put in the water after peeling with 200 gram potatoes and boil, boil at least after the boiling more than 20 minutes, then filter with four layers of hospital gauze, filtrate adds 20 gram common sucrose, is settled to 1000 milliliters, and the pH value transfers to 7.2～7.4, sterilizes 20 minutes for 121 ℃ then.
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| CN101550405B (en) * | 2009-03-31 | 2010-12-08 | 江苏丘陵地区镇江农业科学研究所 | Strain TX-4 used for preventing aphelenchoides besseyi or vegetable root knot nematode and preparation thereof |
| CN102154157A (en) * | 2010-12-28 | 2011-08-17 | 南京农业大学 | Bio-control strain 4AT8 capable of preventing and curing various bacterial diseases |
| CN102578152A (en) * | 2012-01-16 | 2012-07-18 | 南京农业大学 | Bacillus cereus AR156 wettable powders as well as preparation and application thereof |
| CN103305438A (en) * | 2013-05-20 | 2013-09-18 | 江苏省农业科学院 | Biocontrol strain Jdm2 for preventing and treating tomato root-knot nematode diseases and antibiological inoculant thereof |
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| CN104621184A (en) * | 2015-01-23 | 2015-05-20 | 青岛农业大学 | Composite inoculant for preventing and treating cucumber root knot nematode disease and preparation method thereof |
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| CN106399165A (en) * | 2016-09-12 | 2017-02-15 | 华中农业大学 | Bacillus atrophaeus for preventing and controlling plant root-knot nematodes and application thereof |
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| CN109207381A (en) * | 2018-11-02 | 2019-01-15 | 河南省科学院生物研究所有限责任公司 | It is a kind of prevent and treat nematodiasis biocontrol bacterial strain SFC-3 and its application |
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| CN101550405B (en) * | 2009-03-31 | 2010-12-08 | 江苏丘陵地区镇江农业科学研究所 | Strain TX-4 used for preventing aphelenchoides besseyi or vegetable root knot nematode and preparation thereof |
| CN105104433B (en) * | 2010-10-25 | 2018-04-13 | 马罗内生物创新公司 | Reduce the method that root-knot nematode encroaches in plant |
| CN105104433A (en) * | 2010-10-25 | 2015-12-02 | 马罗内生物创新公司 | Chromobacterium bioactive compositions and metabolites |
| CN102154157A (en) * | 2010-12-28 | 2011-08-17 | 南京农业大学 | Bio-control strain 4AT8 capable of preventing and curing various bacterial diseases |
| CN102154157B (en) * | 2010-12-28 | 2012-07-04 | 南京农业大学 | Bio-control strain 4AT8 capable of preventing and curing various bacterial diseases |
| CN102578152A (en) * | 2012-01-16 | 2012-07-18 | 南京农业大学 | Bacillus cereus AR156 wettable powders as well as preparation and application thereof |
| US9468215B2 (en) | 2013-01-30 | 2016-10-18 | Bio Insumos Nativa Spa | Bionematicide composition and method for controlling phytopathogenic nematodes using the same |
| CN103305438A (en) * | 2013-05-20 | 2013-09-18 | 江苏省农业科学院 | Biocontrol strain Jdm2 for preventing and treating tomato root-knot nematode diseases and antibiological inoculant thereof |
| CN103947485A (en) * | 2014-04-16 | 2014-07-30 | 安徽省农业科学院植物保护与农产品质量安全研究所 | Simple and convenient pesticide applying method for preventing and treating underground insects |
| CN104621184A (en) * | 2015-01-23 | 2015-05-20 | 青岛农业大学 | Composite inoculant for preventing and treating cucumber root knot nematode disease and preparation method thereof |
| CN106399165A (en) * | 2016-09-12 | 2017-02-15 | 华中农业大学 | Bacillus atrophaeus for preventing and controlling plant root-knot nematodes and application thereof |
| CN106399165B (en) * | 2016-09-12 | 2019-09-24 | 华中农业大学 | One plant of atrophy bacillus for preventing and treating plant root-knot nematode and its application |
| CN107254414A (en) * | 2017-07-13 | 2017-10-17 | 中国农业科学院植物保护研究所 | A kind of biocontrol bacterial strain AW2017 for preventing and treating paddy rice root-knot nematode and its application |
| CN107254414B (en) * | 2017-07-13 | 2020-05-05 | 中国农业科学院植物保护研究所 | Biocontrol strain AW2017 for preventing and treating rice root-knot nematode and application thereof |
| CN109207381A (en) * | 2018-11-02 | 2019-01-15 | 河南省科学院生物研究所有限责任公司 | It is a kind of prevent and treat nematodiasis biocontrol bacterial strain SFC-3 and its application |
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