CN104273176A - Plant organic matter-bacillus subtils mixed inoculum and its preparation method and use - Google Patents
Plant organic matter-bacillus subtils mixed inoculum and its preparation method and use Download PDFInfo
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Abstract
The invention discloses a plant organic matter-bacillus subtils mixed inoculum and its preparation method and use. The preparation method comprises that one or more of plant organic matters of azadirachta residue, tea cake and palm kernel meal as bacillus subtils HN09 medium main raw materials and an inoculums adsorption carrier are subjected to two-step fermentation decomposition so that the plant organic matter-bacillus subtils mixed inoculum is obtained. Active components of the plant organic matter-bacillus subtils mixed inoculum comprise the plant organic matters, bacillus subtils HN09 and fermentation metabolite of the bacillus subtils HN09. The bacillus subtils HN09 is preserved in the China general microbiological culture collection center (CGMCC) on April 26, 2013 and has a preservation number of CGMCC NO. 7524. The bacillus subtils HN09 and the plant organic matter-bacillus subtils mixed inoculum can be used for preventing and controlling plant diseases and soil insects, can promote plant growth and nitrogen absorption of plants, have important meaning for enlarging an insect disease prevention and control scope and promoting agricultural waste recycle and have a wide application prospect in production practice.
Description
Technical field
The invention belongs to biopesticide and plant protection art, be specifically related to the preparation method of a kind of bacillus subtilis strain HN09 and its plant organic matter-biocontrol microorganisms mix bacterium agent, and the application in plant pest management.
Background technology
Withered and the palm kernel meal of plant organic matter print chinaberry slag, tea is the agricultural byproducts offcuts of neem seed, tea seed and palm kernel process, because availability is low, abandons usually used as discarded object.This does not obviously meet the object of agricultural sustainable development, therefore needs a kind of method finding these agricultural byproducts offcuts of effective recycling.In view of these plant organic matters are still containing the nutrient component such as a large amount of organic substances and vegetable protein, the culture medium raw material of microorganism can be used as, biocontrol microorganisms is added in these plant organic matters, not only can accelerate plant organic matter maturity, nutrient simultaneously in these plant organic matters can ensure that Biocontrol Strain raised growth is bred, the Nutrients uptake plant growth that both booster actions not only can play the effect of biocontrol microorganisms controlling disease but also plant organic matter can be utilized to provide; And print chinaberry slag and these plant organic matters itself such as tea is withered are still containing the active component on a small quantity with desinsection, antibacterial action, and biocontrol microorganisms is with the use of playing synergistic effect, can expand extermination of disease and insect pest scope; In addition, these plant organic matter fiber contents are high, and the non-renewable resources such as vermiculite, perlite can also be replaced as the novel plant absorption carrier of bacteria agent.
In prior art, if plant organic matter print chinaberry slag, tea are withered and palm kernel meal directly uses, plant can be caused to hinder seedling phenomenon, therefore make these plant organic matters complete digest process by appropriate method and be necessary.Although have been reported biocontrol microorganisms and agricultural chemicals or the composite example of plant medical agent, but be different from and grow fermentation maturity and then prepare microbial inoculum using above-mentioned plant organic matter as main medium, screen that can to tolerate the bacterial strain that in above-mentioned plant organic matter, contained unfavorable composition can be bred using it as main medium matrix growth again particularly important.Except this, the bacteriostasis between different strains has larger difference, and the condition of culture between different strains and microbial inoculum preparation technology also have a great difference, needs suitable technology and is made into the preparation being applicable to suitability for industrialized production and application.
Based on above thinking and technical requirement, the present invention adopts the absorption carrier and the manufacture craft that are different from traditional biological microbial inoculum, use the culture medium raw material of plant organic matter as biocontrol microorganisms and the absorption carriers of microbial inoculum thereof such as the withered and palm kernel meal of print chinaberry slag, tea, become thoroughly decomposed through secondary fermentation, be prepared into the plant organic matter-bacillus subtilis mix bacterium agent (being called for short plant organic matter microbial inoculum) with control plant pest, this has no relevant report in the research of biopesticide microbial inoculum.
Chinese Patent Application No. 200810062559.1 (publication number CN101606520A) discloses a kind of novel biological locust control preparation, with Avermectin and biological insecticides nimbin for main material, add specially for locust characteristic add special plant intermediate agent tea withered carry out composite.Chinese Patent Application No. 201010572445.9 (publication number CN102038001A) discloses a kind of nimbin and bacillus subtilis mixture water dispersible granules, material containing following percentage by weight: 0.1% ~ 50% nimbin, 0.1% ~ 50% bacillus subtilis, 1% ~ 30% wetting agent, 5 ~ 30% binding agents, 0.1% ~ 30% disintegrant, 10% ~ 60% carrier, utilize the synergistic effect relation between nimbin active component and bacillus subtilis, reach the pesticide resistance overcoming insect, improve application result of medicine, and multiple agricultural disease can be prevented and treated, expand the scope of application of medicament.And the nimbin that above-mentioned two patents use is different from print chinaberry slag, print chinaberry slag is the by product after extracting nimbin, and both essence is different.
Chinese Patent Application No. 95100402.6 (publication number CN1117080A) and Chinese Patent Application No. 200610122958.3 (publication number CN101168730) disclose a kind of bacteria culture media, for fermenting and producing bacillus thuringiensis, its active ingredient is mainly carbon source material, nitrogen source and mineral element, have the withered powder of tea in the nitrogen source be made up of at least one raw material, the withered powder of said tea in the medium content is only 0.5 ~ 2.5% (patent 200610122958.3 is 0.4 ~ 2.7%).Condition of culture between different strains and microbial inoculum preparation technology have a great difference, above-mentioned Chinese patent application is only about providing a kind of medium containing the withered powder of micro-tea for fermenting and producing bacillus thuringiensis, mainly play regulatory role, not as bacillus thuringiensis medium main component, microbial inoculum absorption carrier and synergistic component, this and present subject matter have essential distinction.
Chinese Patent Application No. 200510050575.5 (publication number CN1718003A) discloses a kind of A Wei thuringiensis bacillus powder, by the former medicine of Avermectin, bacillus thuringiensis,Bt and compositely to form as the tea of filler is withered, is desinsection, kills mite biopesticide.In this patent, tea is withered as filler, mainly plays Action of Surfactant, does not relate to present subject matter.
Chinese Patent Application No. 200810030925.5 (publication number CN101248835A) discloses one enzyme (mannase) and to degrade the method for palm kernel meal, palm kernel meal, and obtained product is as animal feed.Chinese Patent Application No. 200910049520.0 (publication number CN10186910A) discloses a kind of method utilizing fermentation method to remove antinutritional factor in palm kernel meal, it is characterized in that, adopt the antinutritional factor that bacillus and saccharomycetes to make fermentation are degraded in palm kernel meal, improve the method for palm kernel meal as the nutritive value of feed.
The present inventor discloses a bacillus subtilis HN09, by itself and plant organic matter secondary fermentation, and using plant organic matter as the absorption carrier in microbial inoculum preparation process, be developed to and there is plant disease and soil pests, Promoting plant growth and the novel plant organic matter microbial inoculum to nitrogen absorption such as strong Antifungal activity, control banana blight.Through retrieval, all patent applications of having applied for and bibliographical information all do not relate to present subject matter.
Summary of the invention
The object of the invention is to according to above-mentioned deficiency of the prior art, provide a kind of preparation method of novel plant organic matter-bacillus subtilis mix bacterium agent.
Another object of the present invention is to provide the application of above-mentioned bacillus subtilis strain HN09 and microbial inoculum thereof, it can produce the escaping gas with bacteriostatic activity, to soil pests such as plant disease and flea beetle such as banana blights, there is preventive and therapeutic effect, to plants such as bananas, there is growth promoting effects effect.
The present invention is achieved through the following technical solutions above-mentioned purpose:
The plant organic matter microbial inoculum that bacillus subtilis HN09 of the present invention and plant organic matter are made, this microbial inoculum is to print one or more medium primary raw materials as biocontrol microorganisms HN09 and the absorption carrier of microbial inoculum thereof in the withered and palm kernel meal of chinaberry slag, tea, become thoroughly decomposed through secondary fermentation, the plant organic matter be prepared into-bacillus subtilis mix bacterium agent (referred to as plant organic matter microbial inoculum in the present invention).
Plant organic matter described in the present invention be in the withered and palm kernel meal of finger mark chinaberry slag, tea one or more; during multiple combination ratio be weight arbitrarily than; as the matrix medium of bacillus subtilis strain HN09, also serve as the adsorbent of microbial inoculum, all belong to protection scope of the present invention.
Above-mentioned microbial inoculum active component comprises escaping gas, the fermented and cultured metabolite of bacillus subtilis HN09 and its generation, and plant organic matter, has synergistic function, expand extermination of disease and insect pest scope between active component to the extermination of disease and insect pest.
Concrete preparation method's step of above-mentioned plant organic matter microbial inoculum is as follows:
(1) primary seed solution preparation
Seed liquor medium is nutrient broth liquid nutrient medium: beef extract 0.5%, peptone 0.5%, NaCl0.5%, and all the other are distilled water, pH6.5 ~ 7.3.Medium 121 DEG C of sterilizing 20min, the bacillus HN09 of access activation, Shaking culture.Condition of culture: rotating speed 180rpm, 30 DEG C, incubation time 24h.
(2) secondary seed solution preparation
Secondary seed medium formula is corn flour 1 ~ 3%, analysis for soybean powder 0.5 ~ 2%, dusty yeast 0.1 ~ 0.3%, K
2hPO
40.05 ~ 0.15%, MgSO
40.01 ~ 0.03%, CaCO
40.1 ~ 0.2%, all the other are water, pH6.5 ~ 7.3.Medium, through 121 DEG C of sterilizing 20min, treats that temperature is down to about 30 DEG C, by primary seed solution by 5 ~ 15% volume ratio access seed tank culture.Throughput 1: 1, mixing speed 100 ~ 250 revs/min, cultivation temperature 28 ~ 32 DEG C, fills with pressure and remains on 0.02 ~ 0.03MPa.Quality and the detection of bacterium amount is carried out between culture period.
(3) fermentation tank culture
Fermentation tank culture based formulas is with secondary seed solution culture medium prescription.Medium temperature is down to about 30 DEG C, secondary seed solution is expanded by 5 ~ 15% volume ratio access fermentation tanks and cultivates, add 0.005 ~ 0.015g/L MnSO in the medium simultaneously
4spore is produced in induction.Condition of culture, quality and bacterium amount detect to be prepared with step secondary seed solution.
(4) secondary fermentation and microbial inoculum make
Added by step (3) gained zymotic fluid and pulverized in 200 object plant organic matters, carry out secondary fermentation and become thoroughly decomposed, temperature is 20 ~ 37 DEG C, and fermentation time is 1 ~ 60 day.
Add the ratio mixing of 2 ~ 5Kg plant organic matter in every 1L zymotic fluid, add 1 ~ 2% carboxymethyl cellulose adjuvant, stir, the plant organic matter made-biocontrol microorganisms mix bacterium agent.Above-mentioned plant organic matter be in the withered and palm kernel meal of print chinaberry slag, tea one or more, during multiple combination ratio be weight arbitrarily than.Microbial inoculum is wetting powder.
(5) packaging of microbial inoculum and storage
Take in microbial inoculum packing and aluminium matter bag, sealing, deposits in aeration-drying place.
Plant organic matter provided by the present invention-bacillus subtilis mix bacterium agent specifically can be used for plant disease such as control banana blight, tomato wilt etc., especially more effective to banana blight; Also can be used for the soil pests such as control flea beetle simultaneously, also can promote that plant is to the application in nitrogen absorption, Promoting plant growth.
Compared with prior art, the present invention has following beneficial effect:
The present invention discloses a kind of preparation method of plant organic matter microbial inoculum, the method is using renewable resources---and the plant organic matters such as the withered and palm kernel meal of print chinaberry slag, tea are as the medium of bacillus subtilis HN09, simultaneously by using above-mentioned plant organic matter as the absorption carrier of microbial inoculum, become thoroughly decomposed through secondary fermentation and be prepared into microbial inoculum.Utilize the synergistic effect relation between plant organic matter composition and bacillus subtilis, accelerate plant organic matter maturity, ensure the breeding of Biocontrol Strain raised growth, and can plurality of plant diseases be prevented and treated, expand extermination of disease and insect pest scope.In addition, have economizing on resources, turn waste into wealth and promote the advantage such as to recycle of agricultural by product, and preparation technology is simple, effective, cost is low, and this is also one of feature of the present invention.
Confirm through experiment, bacillus subtilis strain HN09 of the present invention and plant organic matter microbial inoculum thereof all have good control efficiency to banana blight, tomato wilt, especially good control efficiency is shown to banana blight, reach 94.7% to the field efficacy of banana blight, this microbial inoculum has good control efficiency to soil pests flea beetle and chafer simultaneously.As can be seen here, this biocontrol microorganisms HN09 bacterial strain has huge application prospect in control banana blight and soil pests, and its mix bacterium agent can carry out large-scale promotion application, has wide market prospects, will bring huge economic benefit and ecological benefits.
Bacillus subtilis of the present invention is bacillus subtilis (Bacillus subtilis) HN09, screen the soil from Guangzhou, Guangdong Agricultural University Of South China pesticide plant living collection, the experiment proved that, this bacterial strain and plant organic matter have good compatibility, producible escaping gas effectively can suppress the growth of banana blight bacteria and tomato wilt bacterium, this has no report in document in the past, and this is a kind of new prophylactico-therapeutic measures for vascular bundle diseases provides.This bacterial strain is preserved in DSMZ of Institute of Microorganism, Academia Sinica on April 26th, 2013, and preserving number is CGMCC7524, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
This bacterial strain is gram-positive bacteria, and thalline is shaft-like, produces gemma, has motility.Opaque in micro-Huang on nutrient agar (NA) medium, dry tack free, have gauffer projection, edge is irregular.Can produce escaping gas, gas has bacteriostatic activity.
Through sequencing, the 16SrDNA nucleotide sequence of this bacillus subtilis HN09 is as shown in SEQ ID NO:1.
Strain name: bacillus subtilis
Latin name: Bacillus subtilis
Strain number: HN09
Preservation mechanism: DSMZ of Institute of Microorganism, Academia Sinica
Preservation mechanism is called for short: CGMCC
Preservation organization address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Register on the books numbering: CGMCC7524 at preservation center
Accompanying drawing explanation
Fig. 1 is the antagonism of bacillus subtilis HN09 to banana blight bacteria No. 4 microspecies.
Fig. 2 is that bacillus subtilis HN09 escaping gas is to the inhibitory action of banana blight bacteria No. 4 microspecies.
Fig. 3 is the pot experiment effect that plant organic matter microbial inoculum damages banana.Left-to-right process is respectively contrast, plant organic matter microbial inoculum and without the plant organic matter become thoroughly decomposed.
Fig. 4 is plant organic matter microbial inoculum control banana blight bacteria and somatotrophic field test effect.Left is contrast, and right is process.
Embodiment
The enrichment isolation of embodiment 1 bacillus subtilis HN09 and qualification
1, the enrichment isolation of bacillus subtilis HN09
With the soil gathered in Guangzhou, Guangdong Agricultural University Of South China pesticide plant living collection for soil sample.Take 10g soil and put into enrichment culture one week in the triangular flask that sterilized plant organic matter medium (print chinaberry slag 30g, the withered 30g of tea, palm kernel meal 30g, peptone 5g, dusty yeast 3g, pH nature) is housed.Then, take 10g enrichment culture thing and put into the triangular flask that 90mL sterile water is housed, fully leave standstill 10min after vibration.Get 1mL supernatant and be diluted to 10 by 10 times of gradient concentrations
-4, 10
-5, 10
-6concentration, get 0.1mL dilution respectively, coat LB medium (peptone 10g, dusty yeast 5g, NaCl10g, agar powder 15g, distilled water 1L, pH nature, 121 DEG C of sterilizing 30min) on flat board, be inverted cultivation 2 ~ 3d for 28 DEG C, the single bacterium colony different according to the feature such as colonial morphology, color picking utilizes plate streak purifying further.
Adopt dull and stereotyped opposite culture method, with pathogens such as banana blight bacteria No. 4 microspecies (representing with FOC4) and tomato wilt bacterium (representing with FROL) for indicator bacteria, after PDA dull and stereotyped central authorities access diameter is the pathogen bacterium cake of 5mm, bacterial strain after inoculating purifying apart from bacterium cake 2.5cm place, 28 DEG C are continued to cultivate, the antibacterial band of each bacterial strain of Continuous Observation record, filters out the strongest bacterial strain of bacteriostatic activity according to inhibiting rate.Final acquisition one strain all has the bacterial strain HN09 of very high inhibition effect to banana blight and tomato wilt, antibacterial band reaches 1.3cm and 1.1cm respectively; This bacterial strain HN09 cultivates can grow (as Fig. 1) by complete antibacterial banana blight bacteria FOC4 at dull and stereotyped antagonism, is accredited as bacillus subtilis (Bacillus subtilis) through follow-up, and called after bacillus subtilis HN09.
2, the qualification of bacillus subtilis HN09
(1) Morphological Identification: this bacterial strain HN09 belongs to gram-positive bacteria, thalline is shaft-like, produces gemma, has motility.The bacterium colony that nutrient agar is formed is for circular, and opaque in micro-yellow, dry tack free, has gauffer projection, and edge is irregular.
(2) molecular biology identification: with the genomic DNA of bacterial strain HN09 for template, adopt universal primer amplification 16SrDNA, obtain the 16SrDNA complete sequence of this bacterial strain containing, for example nucleotide base shown in sequence SEQ ID NO:1 through order-checking, length is 1464bp.This sequence is carried out Blast comparison at GenBank database, and the autoploidy of the sequence of amplification and the 16SrDNA sequence of bacillus subtilis reaches more than 99%.
By morphological observation and molecular biology identification, determine that this bacterial strain HN09 belongs to bacillus subtilis (Bacillus subtilis), called after bacillus subtilis HN09.
Embodiment 2 bacillus subtilis HN09 produces has the experiment of bacteriostatic activity escaping gas
The bacillus subtilis HN09 of activation is inoculated in nutrient broth medium, 30 DEG C, 24h cultivated by 180rpm/min shaking table.Get 200 μ 1 bacterial strain HN09 bacterium liquid and be spread evenly across preprepared aerogenesis medium (peptone 10g, dusty yeast 5g, beef extract 3g, glucose 15g, K
2hPO
42g, FeSO
40.2g, agar 20g, distilled water 1L, pH6.8 ~ 7.2) on flat board; At the FOC4 bacterium cake of another dull and stereotyped PDA medium central authorities access diameter 5mm, then tip upside down on the aerogenesis culture medium flat plate containing bacterial strain HN09 (processed group), not contain the flat board of bacterium liquid for contrast, with diaphragm seal by gap sealing between upper and lower flat board, be placed in 28 DEG C of dark culturing.3 repetitions are set.After contrast FOC4 mycelial growth to anomaly panel edges 1cm, measure mycelial growth distance, calculate average bacteriostasis rate.Result shows, bacterial strain HN09 can produce the escaping gas suppressing banana blight bacteria, as shown in Figure 2.
The preparation of the plant organic matter microbial inoculum of embodiment 3 bacillus subtilis HN09
(1) primary seed solution preparation
Seed liquor medium is nutrient broth liquid nutrient medium: beef extract 0.5%, peptone 0.5%, NaCl0.5%, and all the other are distilled water, pH6.5 ~ 7.3.Medium 121 DEG C of sterilizing 20min, the bacillus HN09 of access activation, Shaking culture.Condition of culture: rotating speed 180rpm, 30 DEG C, incubation time 24h.
(2) secondary seed solution preparation
Secondary seed medium formula is corn flour 1 ~ 3%, analysis for soybean powder 0.5 ~ 2%, dusty yeast 0.1 ~ 0.3%, K
2hPO
40.05 ~ 0.15%, MgSO
40.01 ~ 0.03%, CaCO
40.1 ~ 0.2%, all the other are water, pH6.5 ~ 7.3.Medium, through 121 DEG C of sterilizing 20min, treats that temperature is down to about 30 DEG C, by primary seed solution by 5 ~ 15% volume ratio access seed tank culture.Throughput 1: 1, mixing speed 100 ~ 250 revs/min, cultivation temperature 28 ~ 32 DEG C, fills with pressure and remains on 0.02 ~ 0.03MPa.Quality and the detection of bacterium amount is carried out between culture period.
(3) fermentation tank culture
Fermentation tank culture based formulas is with secondary seed solution culture medium prescription.Medium temperature is down to about 30 DEG C, secondary seed solution is expanded by 5 ~ 15% volume ratio access fermentation tanks and cultivates, add 0.005 ~ 0.015g/LMnSO in the medium simultaneously
4spore is produced in induction.Condition of culture, quality and bacterium amount detect to be prepared with step secondary seed solution.
(4) secondary fermentation and microbial inoculum make
Added by step (3) gained zymotic fluid and pulverized in 200 object plant organic matters, carry out secondary fermentation and become thoroughly decomposed, temperature is 20 ~ 37 DEG C, and fermentation time is 1 ~ 60 day.
Add the ratio mixing of 2 ~ 5Kg plant organic matter in every 1L zymotic fluid, add 1 ~ 2% carboxymethyl cellulose adjuvant, stir, the plant organic matter made-biocontrol microorganisms mix bacterium agent.Above-mentioned plant organic matter be in the withered and palm kernel meal of print chinaberry slag, tea one or more, during multiple combination ratio be weight arbitrarily than.Microbial inoculum is wetting powder.
(5) packaging of microbial inoculum and storage
Take in microbial inoculum packing and aluminium matter bag, sealing, deposits in aeration-drying place.
Embodiment 4 bacillus subtilis HN09 take plant organic matter as growth characteristics and the compatibility of medium
Embodiment 3 step (3) gained zymotic fluid is transferred in being equipped with that 2Kg prints that chinaberry slag, tea are withered by 10% inoculum concentration respectively, between palm kernel meal medium and three combination (combination 1 is print chinaberry slag: tea is withered: palm kernel meal=1: 1: 1; Combination 2 is print chinaberry slag: tea is withered=and 2: 1; Combination 3 is print chinaberry slag: palm kernel meal=1: 2; Combination 4 is that tea is withered: palm kernel meal=1: in the container of 1) medium, carry out secondary fermentation under room temperature condition.The viable count of different plant organic matter medium is measured respectively at 7d the method for plate culture count.
Compatible to measure: take the withered and each 10g of palm kernel meal of print chinaberry slag, tea, add methyl alcohol and sterile water 10mL respectively, mixing, lixiviate 2 days in dark, centrifuging and taking supernatant, after 0.22 μm of sterile filter is degerming, measure extract with Odontothrips loti and whether have bacteriostatic activity to biocontrol microorganisms bacillus subtilis microbial agent HN09, result shows to print the methyl alcohol of the withered and palm kernel meal of chinaberry slag, tea and aqueous extract does not have bacteriostatic activity to bacillus subtilis microbial agent HN09, illustrates that this bacterial strain HN09 and plant organic matter have good compatibility.
Table 1 bacterial strain HN09 is at the viable count of different plant organic matter medium
From table 1 result, bacterial strain HN09 can one or more be growth and breeding in matrix medium in, palm kernel meal plant organic matter withered at print chinaberry slag, tea, to print chinaberry slag and combination 1 (print chinaberry slag: tea is withered: palm kernel meal=1: 1: 1) for medium growth is best, after cultivating 7d, every gram of culture viable count reaches 2.29 × 10 respectively
10with 2.02 × 10
10individual, tea is withered is respectively 1.87 × 10 with palm kernel meal culture viable count
10with 1.73 × 10
10individual, other combinations are respectively 1.95 × 10
10, 1.81 × 10
10with 1.78 × 10
10individual.
Embodiment 5 bacterial strain HN09 take different carriers as the microbial inoculum characteristic of adsorbent
By 1L embodiment 3 step (3) gained zymotic fluid and 2Kg plant organic matter (withered, the palm kernel meal of print chinaberry slag, tea and they between combine, ratio is identical with embodiment 4) absorption carrier mixes, be prepared into wetting powder, room temperature preservation.Respectively at the 7th, 15,30,45,60d takes 10g microbial inoculum, be added to (band sterile glass beads) in the triangular flask that 90mL sterile water is housed, fully vibrate, leave standstill 10min.The viable count of different plant organic matter adsorbent at different time is measured with the method for plate culture count.
The viable count of the microbial inoculum that table 2 is adsorbent with different plant organic matter
Table 2 result shows, to print the withered and palm kernel meal of chinaberry slag, tea, and between them the different plant organic matter such as multiple combination as the absorption carrier of bacillus subtilis microbial agent, the viable count of bacillus subtilis HN09 is all more satisfactory, wherein with print chinaberry slag, withered, the combination of tea 1 (print chinaberry slag: tea is withered: palm kernel meal=1: 1: 1) and combination 2 (print chinaberry slag: tea is withered=2: 1) ideal.With print chinaberry slag do and combination 1 be the viable count of microbial inoculum absorption carrier when 30d for 2.25 × 10
10with 2.17 × 10
10individual, be 2.06 × 10 when 60d
10with 1.99 × 10
10individual; When 60d, tea is withered, palm kernel meal, combination 3 and combination 4 are respectively 1.85 × 10 as the viable count of absorption carrier
10, 1.59 × 10
10, 1.64 × 10
10with 1.58 × 10
10individual.
The storage characteristic of embodiment 6 plant organic matter microbial inoculum
After the plant organic matter microbial inoculum packaging seal prepare embodiment 3 and 4, be placed in room temperature storage, respectively at 30,60,90d takes out microbial inoculum, with the viable count of the method for plate culture count mensuration plant organic matter microbial inoculum in different period of storage.
Table 3 result shows, to print the withered and palm kernel meal of chinaberry slag, tea, and the different plant organic matter such as their multiple combinations is that the bin stability of the plant organic matter microbial inoculum of absorption carrier is good, the viable count of bacillus subtilis HN09 is all more satisfactory, is all greater than 14,000,000,000/g.Wherein to print, chinaberry slag, tea are withered, combination 1 (print chinaberry slag: tea is withered: palm kernel meal=1: 1: 1) and combination 2 (print chinaberry slag: tea is withered=2: 1) best, viable count, when 90d, is respectively 1.99 × 10
10, 1.84 × 10
10, 1.82 × 10
10with 1.85 × 10
10individual.
The viable count of the microbial inoculum that table 3 is adsorbent with different plant organic matter
Embodiment 7 plant organic matter microbial inoculum is to the potted plant efficiency test of banana blight
Measure the microbial inoculum preventive effect that embodiment 4 prepares, test all arranges 3 process: process 1: do not do any process as positive control, do not add FOC4; Process 2: through the plant organic matter microbial inoculum of sterilization treatment as negative control, add FOC4; Process 3: plant organic matter microbial inoculum, adds FOC4; Process 4: bacterial strain HN09 bacterium liquid, adds FOC4.
Be inoculated into by FOC4 pathogen in PDA liquid nutrient medium, culture fluid four layers of sterile gauze, after 3 ~ 4 days, filter, obtain FOC4 spore suspension, by sterile water diluted concentration to 10 by 28 DEG C of shaking table shaken cultivation
7cfu/mL.
Soil 3Kg (through sterilization treatment) transplanted by every basin dress, microbial inoculum and soil mixed, each process 30 basin.The tissue culture seedlings of bananas through hardening growing 5 to 6 true leaves is transplanted in seedling basin, every basin kind 1 strain Banana Seedlings.Then being watered by the FOC4 spore suspension prepared to be filled in plant has in the soil of Banana Seedlings, and every basin irrigation amount is 15mL.Conventional fertilizer water management, observed and recorded incidence, 45 days " Invest, Then Investigate " state of an illness grades, calculate disease index and control efficiency.
Severity Scaling: 0 grade is without disease symptom; 1 grade is that 1 ~ 25% blade turns yellow; 2 grades is that 26 ~ 50% blades are wilted; 3 grades is that 51 ~ 75% blades are wilted; 4 grades is the wilting of complete stool blade or plant death.
Disease index=∑ (state of an illness grade × disease plant number)/(investigating total strain number × 4)
Control efficiency (%)=[(contrast disease index-process disease index)/contrast disease index] × 100
Table 4 result shows, print chinaberry slag, tea are withered, the plant organic matter microbial inoculum of palm kernel meal and their multiple combinations is remarkable to banana blight control efficiency, wherein print chinaberry slag, combination 1 (print chinaberry slag: tea is withered: palm kernel meal=1: 1: 1) and the preventive effect of combination 2 (print chinaberry slag: tea is withered=2: 1) microbial inoculum best, respectively up to 96.8%, 95.0% and 97.6%; The preventive effect of the withered microbial inoculum of tea reaches 90.0%; The preventive effect of palm kernel meal microbial inoculum reaches 88.2%; The preventive effect of combination 3 and combination 4 also reaches 87.0% and 87.5%.
Table 4 plant organic matter microbial inoculum is to the potted plant control efficiency of banana blight
The synergy test that embodiment 8 plant organic matter and bacillus subtilis HN09 mix bacterium agent are prevented and treated banana blight
Measure that print chinaberry slag, tea are withered respectively, synergistic effect that palm kernel meal and bacillus subtilis HN09 mix bacterium agent are prevented and treated banana blight.Adopt embodiment 7 indoor pot experiment method, often process 30 basins, every basin sterile soil 3Kg.
Each monomer print chinaberry slag, tea are withered, the Toxicity Determination of palm kernel meal and bacillus subtilis HN09 bacterium liquid: print chinaberry slag and the withered experimental concentration of tea be 5,10,20,40,80g/Kg soil; Palm kernel meal experimental concentration is 7,14,28,56,112g/Kg soil; Bacillus subtilis HN09 bacterium liquid experimental concentration (spore count 1 × 10
5cfu/mL is designated as 1) be 0.1,1,10,100,1000.Embodiment 7 test method is adopted to carry out toxicity test to banana blight.According to the severity Scaling of each process, be namely designated as morbidity by 2-4 level, calculate each process inhibiting rate and proofread inhibit rate, obtain virulence regression equation, EC
50with 95% confidence limit.
The common malicious factor determination of mix bacterium agent: with reference to the method for Mansour (1966), suppressed by each monomer medicament the dosage of banana blight 25% (EC25) with 1: 1 mixing post processing, processing method is identical with the toxicity test of monomer.Calculate each process inhibiting rate and proofread inhibit rate, be calculated as follows the common malicious factor, as the standard judging mix bacterium agent synergistic effect.
The malicious factor altogether=[(the theoretical inhibiting rate % of actual measurement inhibiting rate %-)/theoretical inhibiting rate %] × 100
The malicious factor >=+20 indicate synergistic effect altogether, and≤-20 indicate antagonism, and-20 ~+20 are expressed as summation action
The each monomer medicament of table 5 is to the toxicity test of banana blight
As seen from the results in Table 5, print that chinaberry slag, tea are withered, palm kernel meal and bacillus subtilis HN09 monomer be to virulence regression equation, the EC of banana blight
50and EC
25.
The each plant organic matter of table 6 and bacillus subtilis HN09 mix bacterium agent are to the common malicious factor of banana blight
Table 6 result is known, and print chinaberry slag+bacillus subtilis HN09, tea is withered+and the common malicious factor of 3 mix bacterium agents such as bacillus subtilis HN09, palm kernel meal+bacillus subtilis HN09 is all greater than 20, shows synergistic effect.
Embodiment 9 plant organic matter microbial inoculum does not damage pot experiment to plant
In order to show that the bacillus subtilis HN09 added in plant organic matter microbial inoculum process prepared by the present invention is by becoming thoroughly decomposed to plant organic matter, avoid plant organic matter (not becoming thoroughly decomposed) to damage plant, this test arranges 3 process: process 1: do not do any process as positive control; Process 2: the plant organic matter microbial inoculum of preparation in embodiment 3 and 4; Process 3: the withered and palm kernel meal without the plant organic matter print chinaberry slag become thoroughly decomposed, tea.
Soil 3Kg (through sterilization treatment) transplanted by every basin dress, and mix by above-mentioned set handling and soil, usage amount is 5% (150g), each process 30 basin.The tissue culture seedlings of bananas through hardening growing 5 to 6 true leaves is transplanted in seedling basin, the strain of every basin kind 1.Conventional fertilizer water management, observed and recorded Banana Growth situation.Fig. 3 result shows, plant organic matter microbial inoculum can prevent the plant organic matter without becoming thoroughly decomposed from damaging banana.
Embodiment 10 plant organic matter microbial inoculum is to the potted plant efficiency test of phyllotreta striolata
Often kind of microbial inoculum efficiency test all arranges 2 process: process 1: do not do any process in contrast; Process 2: plant organic matter microbial inoculum.
Soil 3Kg (through sterilization treatment) transplanted by every basin dress, plant organic matter microbial inoculum 50g and soil mixed, each process 3 basin.By in 3 leaf phase leaf mustard transplanted seedling basins, every basin 3 seedling.Treat that leaf mustard grows to 4 leaf after dates, polypide is grown consistent phyllotreta striolata adult and receive on leaf mustard leaf, 20, every basin.7d " Invest, Then Investigate " respectively processes phyllotreta striolata adult density, according to following formulae discovery control efficiency:
Control efficiency (%)=[(process Revision insect recluced rate-contrast Revision insect recluced rate)/(1-contrasts Revision insect recluced rate)] × 100
Result shows, plant organic matter microbial inoculum has good control efficiency to phyllotreta striolata, and the preventive effect wherein printing chinaberry slag microbial inoculum is best, up to 86.3%; The preventive effect of the withered microbial inoculum of tea reaches 79.8%; The preventive effect of palm kernel meal microbial inoculum is 60.4%.
Embodiment 12 plant organic matter microbial inoculum is to the controlling experiment of anomala corpulenta
Often kind of microbial inoculum controlling experiment all arranges 2 process: process 1: do not do any process in contrast; Process 2: plant organic matter microbial inoculum.
Plant organic matter microbial inoculum 50g and 1Kg is added with the sterilizing fine earth mixing of even thickness cooking shredded potato, keeps soil moisture content to be about 20%.Each access polypide grows consistent anomala corpulenta first-instar young 20, each process 3 repetition.Raise 7d " Invest, Then Investigate " for 28 DEG C and respectively process anomala corpulenta larval density, according to following formulae discovery control efficiency:
Control efficiency (%)=[(process Revision insect recluced rate-contrast Revision insect recluced rate)/(1-contrasts Revision insect recluced rate)] × 100
Result shows, plant organic matter microbial inoculum has good control efficiency to anomala corpulenta, wherein prints the preventive effect of chinaberry slag microbial inoculum up to 87.6%; The preventive effect of the withered microbial inoculum of tea reaches 90.5%; The preventive effect of palm kernel meal microbial inoculum is 76.7%.
The pot experiment of embodiment 13 plant organic matter microbial inoculum Promoting plant growth
Often kind of microbial inoculum efficiency test all arranges 5 process: process 1: not with microbial inoculum in contrast; Process 2:2.5% plant organic matter microbial inoculum; Process 3:5% plant organic matter microbial inoculum; Process 4:10% plant organic matter microbial inoculum; Process 5:20% plant organic matter microbial inoculum.
Soil 3Kg (through sterilization treatment) transplanted by every basin dress, microbial inoculum and soil mixed, each process 30 basin by soil heavy mass ratio.By the banana group training seedling replanting of the same size through simple hardening in seedling basin, every basin kind 1 strain Banana Seedlings.60 days " Invest, Then Investigate " banana seedlings upgrowth situations, measure plant height.
Result shows (table 7), plant organic matter microbial inoculum can promote the growth of banana seedlings, the average plant height of control group is 21.4cm, print chinaberry slag, tea average plant height that is withered and palm kernel meal plant organic matter microbial inoculum processed group are respectively 25.6,24.8 and 23.5cm, wherein print chinaberry slag plant organic matter microbial inoculum growth-promoting effect best.
Table 7 plant organic matter microbial inoculum promotes Banana Growth pot experiment effect
Embodiment 11 plant organic matter microbial inoculum promotes the experiment that plant absorbs nitrogen
Collect the banana seedlings blade in embodiment 8 test, utilize Kjeldahl method to measure the content of the full nitrogen of banana seedlings respectively, calculate the recruitment that nitrogen is absorbed.Table 8 result shows, plant organic matter microbial inoculum all can promote that banana is to the absorption of nitrogen, absorbs recruitment be respectively 36.3%, 15.5% and 16.7% to nitrogen.
Table 8 plant organic matter microbial inoculum promotes the test that banana absorbs nitrogen
Embodiment 14 prints chinaberry slag plant organic matter microbial inoculum control banana blight and promotes the field efficacy experiment of Banana Growth
In order to show the plant organic matter microbial inoculum of bacterial strain HN09 of the present invention, to the control efficiency of banana blight and to banana growth promoting effects effect, on the basis of indoor pot experiment, carry out field control effectiveness test.Carry out at Fanyu District, Guangzhou banana the field trial printing chinaberry slag plant organic matter microbial inoculum at the beginning of 2012, two groups of process are set: process 1: ridge 1 and ridge 2 not dispenser are in contrast; Process 2: ridge 3 ~ 5 applies print chinaberry slag plant organic matter microbial inoculum.Select banana seedlings to grow consistent banana ground, adopt banana seedlings base manure insecticide-applying way, every mu of usage amount is 60Kg, in Continuous Observation to 2012 year October after process, investigation incidence, calculates field efficacy according to embodiment 7 method, and measure plant height, log.
Result of the test shows, the withered and yellow blade quantity of print chinaberry slag plant organic matter microbial inoculum processed group is obviously less than blank (Fig. 4), and can effectively reduce the banana blight incidence of disease, field efficacy reaches 94.7% (table 9); Meanwhile, print chinaberry slag plant organic matter microbial inoculum can promote the growth of banana seedlings, and as shown in table 10,119 strains have been added up in contrast, and processed group has added up 179 strains, and contrasting average plant height is 1.42 ~ 1.46m, and the average plant height of processed group reaches 1.58 ~ 1.70m.
Table 9 prints chinaberry slag plant organic matter microbial inoculum to the field efficacy of banana blight
Table 10 plant organic matter microbial inoculum promotes Banana Growth field test effect
Claims (12)
1. plant organic matter-bacillus subtilis HN09 mix bacterium agent (referred to as plant organic matter microbial inoculum), is characterized in that: the novel mix bacterium agent by agricultural byproducts plant organic matter and bacillus subtilis HN09 thalline being main component.
2. plant organic matter according to claim 1, it is characterized in that: it is print chinaberry slag, tea is withered and in palm kernel meal one or more, during multiple combination ratio be weight arbitrarily than, the granule fine powder of gained after pulverizing 100 or 200 mesh sieves, as the medium matrix of bacillus subtilis HN09, and serve as the absorption carrier of microbial inoculum.
3. bacillus subtilis HN09 according to claim 1, it is characterized in that: can with plant organic matter described in claim 2 for main medium growth and breeding, can produce the escaping gas with bacteriostatic activity, its 16S rDNA nucleotide sequence is as shown in SEQ ID NO:1.
4. the plant organic matter according to claim 2 and 3 and bacillus subtilis HN09 have synergistic function on plant pest management, expand extermination of disease and insect pest scope.
5. bacillus subtilis HN09 according to claim 3 is with plant organic matter described in claim 2 in main medium growth and breeding process, completes plant organic matter simultaneously and becomes thoroughly decomposed, and avoids plant organic matter directly to use and produces injury to plant.
6. microbial inoculum according to claim 1, is characterized in that: with plant organic matter, bacillus subtilis HN09 thalline and fermentating metabolism product thereof for raw material, become thoroughly decomposed through secondary fermentation, and adds appropriate carboxymethyl cellulose adjuvant and be mixed.
7. the preparation method of plant organic matter microbial inoculum and production technology according to claim 1 or 6, comprises the following steps:
(1) primary seed solution preparation: the bacillus HN09 of activation is inoculated into nutrient broth liquid nutrient medium by 5 ~ 15% volume ratios, with 180rpm rotary speed 30 DEG C of Shaking culture 24h, obtains primary seed solution;
(2) secondary seed solution preparation: primary seed solution step (1) obtained is by 5 ~ 15% volume ratio access seed tank culture, throughput 1: 1, with 100 ~ 250 revs/min of mixing speeds, 28 ~ 32 DEG C are cultured to thalline exponential phase, obtain secondary seed solution;
(3) fermentation tank culture: secondary seed solution step (2) obtained expands by 5 ~ 15% volume ratio access fermentation tanks cultivates, and fermentation tank culture based formulas is corn flour 1 ~ 3%, analysis for soybean powder 0.5 ~ 2%, dusty yeast 0.1 ~ 0.3%, K
2hPO
40.05 ~ 0.15%, MgSO
40.01 ~ 0.03%, CaCO
40.1 ~ 0.2%, all the other are water, pH6.5 ~ 7.3.The same step of condition of culture (2), adds 0.005 ~ 0.015g/L MnSO simultaneously in the medium
4spore is produced in induction;
(4) secondary fermentation: step (3) gained zymotic fluid to be added in above-mentioned plant organic matter one or more, carry out secondary fermentation and become thoroughly decomposed, temperature is 20 ~ 37 DEG C, and fermentation time is 1 ~ 60 day.
8. the preparation method of microbial inoculum according to claim 7, is characterized in that: the ratio mixing adding 2 ~ 5Kg plant organic matter in every 1L zymotic fluid, adds 1 ~ 2% carboxymethyl cellulose adjuvant, stir, the plant organic matter microbial inoculum made.
9. the bacillus subtilis HN09 according to claim 1 or 3, it is characterized in that: HN09 bacterial strain belongs to bacillus subtilis (Bacillus subtils), be located away from the soil of Agricultural University Of South China's pesticide plant living collection, by the preservation of DSMZ of Institute of Microorganism, Academia Sinica, preserving number is CGMCC7524.
10. the bacillus subtilis HN09 according to claim 1 or 7 and its application of plant organic matter microbial inoculum in control of plant disease, is characterized in that: described plant disease comprises: banana blight and tomato wilt etc.
The application of 11. plant organic matter microbial inoculums according to claim 1 or 7 in soil pests control, is characterized in that: described soil pests comprises: flea beetle and chafer etc.
12. plant organic matter microbial inoculums according to claim 1 or 7 are in Promoting plant growth and the application in absorbing nitrogen.
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