CN104273176B - A kind of plant organic matter-bacillus subtilis mix bacterium agent, preparation method and application - Google Patents
A kind of plant organic matter-bacillus subtilis mix bacterium agent, preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of plant organic matter-bacillus subtilis mix bacterium agent preparation method and application.The microbial inoculum is one or more as the culture medium primary raw material of bacillus subtilis HN09 and its absorption carrier of microbial inoculum using in plant organic matter print chinaberry slag, tea seed cake and palm kernel meal, it is prepared by secondary fermentation is decomposed, active constituent includes above-mentioned plant organic matter, bacillus subtilis HN09 and its fermentating metabolism object.The bacterial strain is preserved in Institute of Microorganism, Academia Sinica's Culture Collection Center, deposit number CGMCC7524 on April 26th, 2013.The bacterial strain and its microbial inoculum can be used for controlling plant diseases and soil pests, also can promote plant growth and absorb to nitrogen, are of great significance to expanding prevention and control of plant diseases, pest control range and agricultural wastes being promoted to recycle, have wide application prospect in production practice.
Description
Technical field
The invention belongs to biological pesticides and plant protection art, and in particular to a kind of bacillus subtilis strain HN09 and it
Plant organic matter-biocontrol microorganisms mix bacterium agent preparation method, and its application in plant pest management.
Background technique
It is the agricultural and sideline of neem seed, tea seed and palm kernel process that plant organic matter, which prints chinaberry slag, tea seed cake and palm kernel meal,
Product leftover bits and pieces is abandoned since utilization rate is low usually as waste.This does not obviously meet the purpose of agricultural sustainable development,
Therefore it needs to find a kind of method for effectively recycling these agricultural and sideline product leftover bits and pieces.Still contain in view of these plant organic matters
The a large amount of nutritional ingredients such as organic substance and vegetable protein, may be used as the culture medium raw material of microorganism, biocontrol microorganisms are added to
In these plant organic matters, it can not only accelerate plant organic matter maturity, while the nutrient in these plant organic matters can
To guarantee that Biocontrol Strain raised growth is bred, the two booster action can not only play the effect of biocontrol microorganisms controlling disease but also can benefit
The Nutrients uptake plant growth provided with plant organic matter；And print these plant organic matters such as chinaberry slag and tea seed cake itself still contain
Have has desinsection, the active constituent of antibacterial action and biocontrol microorganisms with the use of synergistic effect is played on a small quantity, can expand pest and disease damage
Prevention and treatment range；In addition, these plant organic matter fiber contents are high, the non-renewable resources such as vermiculite, perlite can also be replaced to make
For the novel plant absorption carrier of bacteria agent.
In the prior art, if plant organic matter print chinaberry slag, tea seed cake and palm kernel meal directly use, it will cause plant injury seedling
Phenomenon, therefore so that these plant organic matters is completed digest process by appropriate method and be necessary.Although having been reported life
It is fungi-proofing that example is compounded with pesticide or plant medical agent, but it is rotten to be different from the growth fermentation using above-mentioned plant organic matter as main medium
It is ripe and then prepare microbial inoculum, it screens and is not only resistant to contained unfavorable ingredient in above-mentioned plant organic matter but also can be using it as main culture
The bacterial strain of based substrate growth and breeding is particularly important.Except this, the bacteriostasis between different strains has a larger difference, different strains it
Between condition of culture and microbial inoculum preparation process be also very different, need suitable technology and be made into suitable for work
Industry metaplasia produces and the preparation of application.
Based on the above thinking and technical requirements, the present invention uses absorption carrier and production different from traditional biological microbial inoculum
Technique uses the plant organic matters such as print chinaberry slag, tea seed cake and palm kernel meal as the suction of the culture medium raw material of biocontrol microorganisms and its microbial inoculum
Appendix body, it is decomposed by secondary fermentation, it is prepared into the plant organic matter with prevention and treatment plant pest-bacillus subtilis mixing
Microbial inoculum (abbreviation plant organic matter microbial inoculum), this has no relevant report in terms of biological pesticide microbial inoculum research.
Chinese Patent Application No. 200810062559.1 (publication number CN101606520A) discloses a kind of novel biology
Locust control preparation adds using avermectin and biological insecticides nimbin as main material and adds special plant specifically for locust characteristic
Intermediate agent tea seed cake is compounded.Chinese Patent Application No. 201010572445.9 (publication number CN102038001A) discloses one kind
Nimbin and bacillus subtilis mixture water dispersible granules, the substance containing following weight percent: 0.1%~50% print chinaberry
Element, 0.1%~50% bacillus subtilis, 1%~30% wetting agent, 5~30% binders, 0.1%~30% disintegrating agent,
10%~60% carrier reaches using the synergistic effect relationship between nimbin active constituent and bacillus subtilis and overcomes pest
Drug resistance, improve application result of medicine, and a variety of agricultural diseases can be prevented and treated, expand the scope of application of medicament.And it is above-mentioned
Nimbin used in two patents is different from print chinaberry slag, and print chinaberry slag is the byproduct after extracted nimbin, and the two essence is not
Together.
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, are used for fermenting and producing Su Yun gold gemma
Bacillus, effective component are mainly carbon source material, nitrogen source and mineral element, the nitrogen source being made of at least one raw material
In there is tea seed cake powder, described tea seed cake powder in the medium content be only 0.5~2.5% (patent 200610122958.3 is 0.4~
2.7%).Condition of culture and microbial inoculum preparation process between different strains are very different, and above-mentioned Chinese patent application is only to close
In providing a kind of culture medium containing micro tea seed cake powder for fermenting and producing bacillus thuringiensis, mainly play regulatory role, and
It is not used as bacillus thuringiensis culture medium main component, microbial inoculum absorption carrier and synergistic component, this has with present subject matter
Essential distinction.
Chinese Patent Application No. 200510050575.5 (publication number CN1718003A) discloses a kind of AVM hereinafter Su Yun gold
Bacillus pulvis is compounded by the former medicine of AVM, Dipel and tea seed cake as filler, is desinsection, mite killing biology
Pesticide.Tea seed cake primarily serves Action of Surfactant, is not related to present subject matter as filler in the patent.
Chinese Patent Application No. 200810030925.5 (publication number CN101248835A) discloses that a kind of (sweet dew is poly- with enzyme
Carbohydrase) degradation palm kernel meal, palm kernel meal method, be made product as animal feed.Chinese Patent Application No.
(200910049520.0 publication number CN10186910A) disclose it is a kind of using fermentation method removal palm kernel meal in anti-nutritional factors
Method, which is characterized in that the anti-nutritional factors in palm kernel meal of being degraded using bacillus and saccharomycetes to make fermentation, improve palm fibre
Method of the palmitic acid benevolence dregs of rice as the nutritive value of feed.
The present inventor discloses a bacillus subtilis HN09, by itself and plant organic matter secondary fermentation, and with plant
Organic matter is as the absorption carrier in microbial inoculum preparation process, and exploitation is at strong Antifungal activity, prevention and treatment banana blight
Etc. plant diseases and soil pests, the novel plant organic matter microbial inoculum that promotes plant growth and nitrogen is absorbed.It is retrieved, is owned
Identified patent applications and document report are not related to present subject matter.
Summary of the invention
It is an object of the invention to provide a kind of novel plant organic matter-according to above-mentioned deficiency in the prior art
The preparation method of bacillus subtilis mix bacterium agent.
It is a further object of the present invention to provide the applications of above-mentioned bacillus subtilis strain HN09 and its microbial inoculum, can produce
The raw escaping gas with bacteriostatic activity there is prevention and treatment to make the soil pests such as the plant diseases such as banana blight and flea beetle
With to plants such as bananas with promotion growth.
Above-mentioned purpose that the invention is realized by the following technical scheme:
Bacillus subtilis HN09 of the present invention and plant organic matter microbial inoculum made of plant organic matter, the microbial inoculum is to print
The absorption of one or more culture medium primary raw materials and its microbial inoculum as biocontrol microorganisms HN09 carries in chinaberry slag, tea seed cake and palm kernel meal
Body, decomposed by secondary fermentation, the plant organic matter being prepared into-bacillus subtilis mix bacterium agent (is referred to as planted in the present invention
Object organic matter microbial inoculum).
Heretofore described plant organic matter is finger mark chinaberry slag, one or more, multiple combinations in tea seed cake and palm kernel meal
When ratio be weight arbitrarily compare, as the matrix culture medium of bacillus subtilis strain HN09, also function as the adsorbent of microbial inoculum,
It belongs to the scope of protection of the present invention.
Above-mentioned microbial inoculum active constituent includes the escaping gas of bacillus subtilis HN09 and its generation, fermented and cultured metabolism
Product and plant organic matter have synergistic function to the prevention and control of plant diseases, pest control between active constituent, expand prevention and control of plant diseases, pest control model
It encloses.
Steps are as follows for the specific preparation method of above-mentioned plant organic matter microbial inoculum:
(1) prepared by primary seed solution
Seed liquid culture medium be nutrient broth fluid nutrient medium: beef extract 0.5%, peptone 0.5%, NaCl0.5%,
Remaining is distilled water, pH6.5~7.3.121 DEG C of sterilizing 20min of culture medium, access the bacillus HN09 of activation, shaking flask culture.Training
The condition of supporting: revolving speed 180rpm, 30 DEG C, incubation time is for 24 hours.
(2) prepared by secondary seed solution
Secondary seed medium formula be corn flour 1~3%, soybean powder 0.5~2%, yeast powder 0.1~0.3%,
K2HPO40.05~0.15%, MgSO40.01~0.03%, CaCO30.1~0.2%, remaining is water, pH6.5~7.3.Culture
Base is down to 30 DEG C or so to temperature through 121 DEG C of sterilizing 20min, by primary seed solution by the access seeding tank training of 5~15% volume ratios
It supports.Ventilatory capacity 1: 1,28~32 DEG C of cultivation temperature, fills pressure and is maintained at 0.02~0.03MPa by 100~250 revs/min of mixing speed.
Quality and bacterium amount detection are carried out during culture.
(3) fermentation tank culture
Fermentation tank culture based formulas is the same as secondary seed solution culture medium prescription.Culture medium temperature is down to 30 DEG C or so, by second level
Seed liquor expands culture by 5~15% volume ratios access fermentor, while 0.005~0.015g/L being added in the medium
MnSO4 induction produces spore.Condition of culture, quality and bacterium amount detection are prepared with step secondary seed solution.
(4) secondary fermentation and microbial inoculum make
Fermentation liquid obtained by step (3) is added in the plant organic matter that crushed 200 mesh, progress secondary fermentation is decomposed, temperature
Degree is 20~37 DEG C, and fermentation time is 1~60 day.
The ratio mixing of 2~5Kg plant organic matter is added in every 1L fermentation liquid, 1~2% carboxymethyl cellulose auxiliary is added
Agent stirs evenly, manufactured plant organic matter-biocontrol microorganisms mix bacterium agent.Above-mentioned plant organic matter is print chinaberry slag, tea seed cake and palm
One or more in the benevolence dregs of rice, ratio is that weight arbitrarily compares when multiple combinations.Microbial inoculum is wettable powder.
(5) packaging and storage of microbial inoculum
Microbial inoculum packing is weighed with aluminum bag, sealing is deposited at aeration-drying.
It is withered that plant organic matter provided by the present invention-bacillus subtilis mix bacterium agent is particularly used in prevention and treatment banana
The plant diseases such as disease, tomato wilt, it is especially more effective to banana blight；It simultaneously can be used for the soil evil such as prevention and treatment flea beetle
Worm can also promote plant to absorb nitrogen, promote the application in plant growth.
Compared with prior art, the invention has the following advantages:
The invention discloses a kind of preparation methods of plant organic matter microbial inoculum, and this method is with renewable resource --- print chinaberry
Culture medium of the plant organic matters such as slag, tea seed cake and palm kernel meal as bacillus subtilis HN09, while will have with above-mentioned plant
Absorption carrier of the machine object as microbial inoculum is prepared into microbial inoculum by secondary fermentation is decomposed.Utilize plant organic matter ingredient and withered grass bud
Synergistic effect relationship between spore bacillus accelerates plant organic matter maturity, guarantees the breeding of Biocontrol Strain raised growth, and
And plurality of plant diseases can be prevented and treated, expand prevention and control of plant diseases, pest control range.Agriculture is economized on resources, turns waste into wealth and promoted in addition, having
The advantages that recycling of industry byproduct, and preparation process is simple, effect is good, at low cost, this be also the features of the present invention it
One.
By it is experimentally confirmed that bacillus subtilis strain HN09 and its plant organic matter microbial inoculum of the invention is withered to banana
Disease, tomato wilt all have good control efficiency, especially good control efficiency are shown to banana blight, to banana
The field efficacy of wilt disease reaches 94.7%, while the microbial inoculum has good control efficiency to soil pests flea beetle and chafer.
It can be seen that biocontrol microorganisms HN09 bacterial strain has huge application prospect in terms of prevention and treatment banana blight and soil pests,
Mix bacterium agent can carry out large-scale promotion application, have a vast market foreground, and will bring huge economic benefit and ecology
Benefit.
Bacillus subtilis of the present invention is bacillus subtilis (Bacillus subtilis) HN09, screening
From the soil of Guangzhou, Guangdong Agricultural University Of South China pesticide plant living collection, the experiment proved that, the bacterial strain and plant organic matter
There is good compatibility, producible escaping gas can effectively inhibit the growth of banana blight bacteria and tomato wilt bacterium,
This has not been reported in previous document, this provides a kind of new control measure for vascular bundle diseases.The bacterial strain is in 2013
It is preserved in Institute of Microorganism, Academia Sinica's Culture Collection Center on April 26, deposit number CGMCC7524, preservation address:
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
The bacterial strain be gram-positive bacteria, thallus be it is rod-shaped, produce gemma, have motility.In nutrient agar (NA) culture medium
Upper opaque in micro- Huang, dry tack free has gauffer protrusion, and edge is irregular.It can produce escaping gas, gas has antibacterial work
Property.
By sequencing, the 16SrDNA nucleotide sequence of the bacillus subtilis HN09 is as shown in SEQ ID NO:1.
Strain name: bacillus subtilis
Latin name: Bacillus subtilis
Strain number: HN09
Preservation mechanism: Institute of Microorganism, Academia Sinica's Culture Collection Center
Preservation mechanism abbreviation: CGMCC
Preservation organization address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Collection is registered on the books number: CGMCC7524
Detailed description of the invention
Fig. 1 is antagonism of the bacillus subtilis HN09 to No. 4 microspecies of banana blight bacteria.
Fig. 2 is inhibiting effect of the bacillus subtilis HN09 escaping gas to No. 4 microspecies of banana blight bacteria.
Fig. 3 is the pot experiment effect that plant organic matter microbial inoculum damages banana.It is left-to-right processing be respectively control,
Plant organic matter microbial inoculum and without decomposed plant organic matter.
Fig. 4 is that plant organic matter microbial inoculum prevents and treats banana blight bacteria and somatotrophic field test effect.A left side is control, right
For processing.
Specific embodiment
The enrichment isolation of 1 bacillus subtilis HN09 of embodiment and identification
1, the enrichment isolation of bacillus subtilis HN09
Using the soil acquired in Guangzhou, Guangdong Agricultural University Of South China pesticide plant living collection as soil sample.Weigh 10g soil
Earth, which is put into, (prints chinaberry slag 30g, tea seed cake 30g, palm kernel meal 30g, peptone 5g, yeast powder equipped with sterilized plant organic matter culture medium
3g, pH are natural) triangular flask in one week of enrichment culture.Then, 10g enrichment culture object is weighed to be put into equipped with 90mL sterile water
In triangular flask, 10min sufficiently is stood after oscillation.1mL supernatant is taken to be diluted to 10 by 10 times of gradient concentrations-4、10-5、10-6Concentration,
0.1mL dilution is taken respectively, is coated on LB culture medium (peptone 10g, yeast powder 5g, NaCl10g, agar powder 15g, distilled water
1L, pH are naturally, 121 DEG C of sterilizing 30min) on plate, 2~3d are cultivated in 28 DEG C of inversions, according to the features picking such as colonial morphology, color
Different single colonies further utilize plate streak to purify.
Using plate opposite culture method, with No. 4 microspecies (being indicated with FOC4) of banana blight bacteria and tomato wilt bacterium (with
FROL indicate) etc. pathogens be indicator bacteria, PDA plate center access diameter be 5mm pathogen bacteria cake after, away from bacteria cake
The bacterial strain of inoculation after purification at 2.5cm, 28 DEG C are continued to cultivate, and are observed continuously and are recorded the antibacterial band of each bacterial strain, are screened according to inhibiting rate
The strongest bacterial strain of bacteriostatic activity out.One plant of final acquisition all has very high inhibition effect to banana blight and tomato wilt
Bacterial strain HN09, antibacterial band respectively reach 1.3cm and 1.1cm；Bacterial strain HN09 can complete antibacterial banana in the culture of plate antagonism
Wilt FOC4 grows (such as Fig. 1), is accredited as bacillus subtilis (Bacillus subtilis) through subsequent, and be named as
Bacillus subtilis HN09.
2, the identification of bacillus subtilis HN09
(1) Morphological Identification: bacterial strain HN09 belongs to gram-positive bacteria, thallus be it is rod-shaped, produce gemma, there is motility.
The bacterium colony formed on nutrient agar is circle, is in yellowish opaque, dry tack free, has gauffer protrusion, edge is not
Neatly.
(2) it molecular biology identification: using the genomic DNA of bacterial strain HN09 as template, is expanded using universal primer
16SrDNA, the 16SrDNA complete sequence for obtaining the bacterial strain through sequencing contain the nucleotide base as shown in sequence SEQ ID NO:1,
Length is 1464bp.The sequence is subjected to Blast comparison in GenBank database, the sequence of amplification and bacillus subtilis
The homology of 16SrDNA sequence is up to 99% or more.
By morphological observation and molecular biology identification, determine that bacterial strain HN09 belongs to bacillus subtilis
(Bacillus subtilis), is named as bacillus subtilis HN09.
2 bacillus subtilis HN09 of embodiment, which is generated, has the experiment of bacteriostatic activity escaping gas
The bacillus subtilis HN09 of activation is inoculated in nutrient broth medium, 30 DEG C, 180rpm/min shaking table culture
24h.200 μ, 1 bacterial strain HN09 bacterium solution is taken to be spread evenly across preprepared production gas culture medium (peptone 10g, yeast powder 5g, ox
Meat extract 3g, glucose 15g, K2HPO42g, FeSO40.2g, agar 20g, distilled water 1L, pH6.8~7.2) on plate；Another
The FOC4 bacteria cake of diameter 5mm is accessed in plate PDA culture medium center, then tips upside down on the production gas culture medium flat plate of the HN09 containing bacterial strain
(processing group) is control with the plate without bacterium solution, with sealing film by gap sealing between upper and lower plate, is placed in 28 DEG C of dark trainings
It supports.3 repetitions are set.After compareing FOC4 mycelia and growing to away from plate edge 1cm, measurement mycelia grows distance, calculates average
Bacteriostasis rate.The result shows that bacterial strain HN09 can produce the escaping gas for inhibiting banana blight bacteria, as shown in Figure 2.
The preparation of the plant organic matter microbial inoculum of 3 bacillus subtilis HN09 of embodiment
(1) prepared by primary seed solution
Seed liquid culture medium be nutrient broth fluid nutrient medium: beef extract 0.5%, peptone 0.5%, NaCl0.5%,
Remaining is distilled water, pH6.5~7.3.121 DEG C of sterilizing 20min of culture medium, access the bacillus HN09 of activation, shaking flask culture.Training
The condition of supporting: revolving speed 180rpm, 30 DEG C, incubation time is for 24 hours.
(2) prepared by secondary seed solution
Secondary seed medium formula be corn flour 1~3%, soybean powder 0.5~2%, yeast powder 0.1~0.3%,
K2HPO40.05~0.15%, MgSO40.01~0.03%, CaCO30.1~0.2%, remaining is water, pH6.5~7.3.Culture
Base is down to 30 DEG C or so to temperature through 121 DEG C of sterilizing 20min, by primary seed solution by the access seeding tank training of 5~15% volume ratios
It supports.Ventilatory capacity 1: 1,28~32 DEG C of cultivation temperature, fills pressure and is maintained at 0.02~0.03MPa by 100~250 revs/min of mixing speed.
Quality and bacterium amount detection are carried out during culture.
(3) fermentation tank culture
Fermentation tank culture based formulas is the same as secondary seed solution culture medium prescription.Culture medium temperature is down to 30 DEG C or so, by second level
Seed liquor expands culture by 5~15% volume ratios access fermentor, while 0.005~0.015g/ being added in the medium
LMnSO4 induction produces spore.Condition of culture, quality and bacterium amount detection are prepared with step secondary seed solution.
(4) secondary fermentation and microbial inoculum make
Fermentation liquid obtained by step (3) is added in the plant organic matter that crushed 200 mesh, progress secondary fermentation is decomposed, temperature
Degree is 20~37 DEG C, and fermentation time is 1~60 day.
The ratio mixing of 2~5Kg plant organic matter is added in every 1L fermentation liquid, 1~2% carboxymethyl cellulose auxiliary is added
Agent stirs evenly, manufactured plant organic matter-biocontrol microorganisms mix bacterium agent.Above-mentioned plant organic matter is print chinaberry slag, tea seed cake and palm
One or more in the benevolence dregs of rice, ratio is that weight arbitrarily compares when multiple combinations.Microbial inoculum is wettable powder.
(5) packaging and storage of microbial inoculum
Microbial inoculum packing is weighed with aluminum bag, sealing is deposited at aeration-drying.
4 bacillus subtilis HN09 of embodiment is using plant organic matter as the growth characteristics of culture medium and compatibility
Fermentation liquid obtained by 3 step of embodiment (3) is transferred respectively by 10% inoculum concentration in equipped with 2Kg print chinaberry slag, tea seed cake, palm fibre
(combination 1 is print chinaberry slag: tea seed cake: palm kernel meal=1: 1: 1 for combination between palmitic acid benevolence dregs of rice culture medium and three；Combination 2 is print chinaberry slag:
Tea seed cake=2: 1；Combination 3 is print chinaberry slag: palm kernel meal=1: 2；Combination 4 is tea seed cake: palm kernel meal=1: the 1) container of culture medium
In, secondary fermentation is carried out under room temperature.Different plant organic matter culture mediums are measured respectively at 7d the method for plate culture count
Viable count.
Compatibility measurement: print chinaberry slag, tea seed cake and each 10g of palm kernel meal are weighed, methanol and sterile water 10mL are separately added into, is mixed
It is even, it is extracted 2 days in dark, centrifuging and taking supernatant, after 0.22 μm of sterilizing filter filtration sterilization, is measured and extracted with Odontothrips loti
Whether liquid has bacteriostatic activity to biocontrol microorganisms bacillus subtilis microbial agent HN09, the results showed that prints chinaberry slag, tea seed cake and palm kernel meal
Methanol and aqueous extract do not have bacteriostatic activity to bacillus subtilis microbial agent HN09, illustrate bacterial strain HN09 and plant organic matter
There is good compatibility.
Viable count of the 1 bacterial strain HN09 of table in different plant organic matter culture mediums
By 1 result of table as it can be seen that bacterial strain HN09 can be one or more in print chinaberry slag, tea seed cake, palm kernel meal plant organic matter
For growth and breeding in matrix culture medium, (chinaberry slag: tea seed cake: palm kernel meal=1 is printed: 1: 1) raw for culture medium to print chinaberry slag and combination 1
Length is best, and every gram of culture viable count respectively reaches 2.29 × 10 after cultivating 7d10With 2.02 × 1010It is a, tea seed cake and palm kernel meal
Culture viable count is respectively 1.87 × 1010With 1.73 × 1010A, other combinations are respectively 1.95 × 1010、1.81×1010With
1.78×1010It is a.
5 bacterial strain HN09 of embodiment is using different carriers as the microbial inoculum characteristic of adsorbent
By fermentation liquid obtained by 3 step of 1L embodiment (3) and 2Kg plant organic matter (print chinaberry slag, tea seed cake, palm kernel meal and
Combined between them, ratio is same as Example 4) absorption carrier be uniformly mixed, be prepared into wettable powder, room temperature preservation.Respectively
10g microbial inoculum is weighed in the 7th, 15,30,45,60d, (band sterile glass beads) are added into the triangular flask equipped with 90mL sterile water, fill
Divide oscillation, stands 10min.Different plant organic matter adsorbents are measured in the viable count of different time with the method for plate culture count.
Table 2 is using different plant organic matters as the viable count of the microbial inoculum of adsorbent
Table 2 the result shows that, with print chinaberry slag, tea seed cake and palm kernel meal and between them multiple combinations etc. difference plant organic matter make
Viable count for the absorption carrier of bacillus subtilis microbial agent, bacillus subtilis HN09 is more satisfactory, wherein to print chinaberry slag
Most, (print chinaberry slag: tea seed cake: palm kernel meal=1: 1: 1) and combination 2 (prints chinaberry slag: tea seed cake=2: 1) ideal for tea seed cake, combination 1.
To print, chinaberry slag is made and combination 1 be the viable count of microbial inoculum absorption carrier is 2.25 × 10 in 30d10With 2.17 × 1010It is a,
It is 2.06 × 10 when 60d10With 1.99 × 1010It is a；In 60d, tea seed cake, palm kernel meal, combination 3 and combination 4 are as suction
The viable count of appendix body is respectively 1.85 × 1010、1.59×1010、1.64×1010With 1.58 × 1010It is a.
The storage characteristic of 6 plant organic matter microbial inoculum of embodiment
After plant organic matter microbial inoculum packaging seal prepared by embodiment 3 and 4, be placed in room temperature storage, respectively at 30,60,
90d takes out microbial inoculum, measures plant organic matter microbial inoculum in the viable count of different periods of storage with the method for plate culture count.
Table 3 is the results show that print the different plant organic matters such as chinaberry slag, tea seed cake and palm kernel meal and their multiple combinations
Storage stability for the plant organic matter microbial inoculum of absorption carrier is good, and the viable count of bacillus subtilis HN09 compares reason
Think, is all larger than 14,000,000,000/g.Wherein to print chinaberry slag, tea seed cake, (the print chinaberry slag: tea seed cake: palm kernel meal=1: 1: 1) and group of combination 1
Conjunction 2 (print chinaberry slag: tea seed cake=2: 1) preferably, viable count is in 90d, and respectively 1.99 × 1010、1.84×1010、1.82×
1010With 1.85 × 1010It is a.
Table 3 is using different plant organic matters as the viable count of the microbial inoculum of adsorbent
Potting efficiency test of the 7 plant organic matter microbial inoculum of embodiment to banana blight
The microbial inoculum preventive effect that measurement embodiment 4 is prepared, test are respectively provided with 3 processing: processing 1: making with no treatment
For positive control, FOC4 is not added；Processing 2: the plant organic matter microbial inoculum by sterilization treatment adds FOC4 as negative control；Place
Reason 3: plant organic matter microbial inoculum adds FOC4；Processing 4: bacterial strain HN09 bacterium solution adds FOC4.
FOC4 pathogen is inoculated into PDA liquid medium, 28 DEG C after shaking table shaken cultivation 3~4 days, culture solution is used
Four layers of sterile gauze filtering, obtain FOC4 spore suspension, with sterile water diluted concentration to 107cfu/mL.
Every basin dress transplanting soil 3Kg (sterilized processing), microbial inoculum is mixed well withed the soil, 30 basins of each processing.To grow 5 to
The tissue culture seedlings of bananas by hardening of 6 true leaves is transplanted into seedling basin, 1 plant of Banana Seedlings of every basin kind.Then it will prepare
FOC4 spore suspension, which pours to be filled in plant, to be had in the soil of Banana Seedlings, and every basin irrigation amount is 15mL.Conventional fertilizer and water management, observation
Incidence is recorded, state of an illness grade is investigated after 45 days, calculates disease index and control efficiency.
Severity Scaling: 0 grade is no disease symptom；1 grade turns yellow for 1~25% blade；2 grades are wilted for 26~50% blades；3
Grade is that 51~75% blades are wilted；4 grades dead for the wilting of complete stool blade or plant.
Disease index=∑ (state of an illness grade × disease plant number)/(investigation total strain number × 4)
Control efficiency (%)=[(control disease index-processing disease index)/control disease index] × 100
Table 4 is the results show that print the plant organic matter microbial inoculum of chinaberry slag, tea seed cake, palm kernel meal and their multiple combinations to perfume (or spice)
Any of several broadleaf plants wilt disease control efficiency is significant, wherein print chinaberry slag, combination 1 (print chinaberry slag: tea seed cake: palm kernel meal=1: 1: 1) (are printed with combination 2
Chinaberry slag: tea seed cake=2: 1) preventive effect of microbial inoculum is best, respectively up to 96.8%, 95.0% and 97.6%；The preventive effect of tea seed cake microbial inoculum reaches
To 90.0%；The preventive effect of palm kernel meal microbial inoculum has reached 88.2%；The preventive effect of combination 3 and combination 4 has also reached 87.0% He
87.5%.
4 plant organic matter microbial inoculum of table is to banana blight potting control efficiency
The synergistic effect that 8 plant organic matter of embodiment and bacillus subtilis HN09 mix bacterium agent prevent and treat banana blight
Test
Measurement print chinaberry slag, tea seed cake, palm kernel meal and bacillus subtilis HN09 mix bacterium agent are anti-to banana blight respectively
The synergistic effect controlled.Using 7 indoor pot experiment method of embodiment, 30 basins of every processing, every basin sterile soil 3Kg.
Each monomer prints chinaberry slag, tea seed cake, the indoor toxicity test of palm kernel meal and bacillus subtilis HN09 bacterium solution: print chinaberry slag
It is 5,10,20,40,80g/Kg soil with tea seed cake experimental concentration；Palm kernel meal experimental concentration be 7,14,28,56,112g/Kg soil
Earth；Bacillus subtilis HN09 bacterium solution experimental concentration (1 × 105cfu/mL of spore count is denoted as 1) is 0.1,1,10,100,1000.
Toxicity test is carried out to banana blight using 7 test method of embodiment.According to the severity Scaling of each processing, remember by 2-4 grades
For morbidity, each processing inhibiting rate and correction inhibiting rate are calculated, virulence regression equation, EC are found out50With 95% confidence limit.
The total malicious factor determination of mix bacterium agent: referring to the method for Mansour (1966), inhibit banana withered each monomer medicament
25% (EC of disease of withering25) dosage with 1: 1 mixing post-processing, processing method is identical as the toxicity test of monomer.Calculate each processing suppression
The total malicious factor is calculated as follows, as the standard for judging mix bacterium agent synergistic effect in rate processed and correction inhibiting rate.
The poison factor=[(actual measurement inhibiting rate %- theory inhibiting rate %)/theoretical inhibiting rate %] × 100 altogether
The poison factor >=+20 indicate synergistic effect altogether, and≤- 20 indicate antagonism, and -20~+20 are expressed as being added work
With
Toxicity test of each monomer medicament of table 5 to banana blight
As seen from the results in Table 5, chinaberry slag, tea seed cake, palm kernel meal and bacillus subtilis HN09 monomer are printed to banana blight
Virulence regression equation, EC50And EC25。
Each plant organic matter of table 6 is with bacillus subtilis HN09 mix bacterium agent to the total malicious factor of banana blight
6 result of table is it is found that print chinaberry slag+bacillus subtilis HN09, tea seed cake+bacillus subtilis HN09, palm kernel meal+withered
The total malicious factor of 3 mix bacterium agents such as careless bacillus HN09 is all larger than 20, shows synergistic effect.
9 plant organic matter microbial inoculum of embodiment does not damage pot experiment to plant
In order to show the bacillus subtilis HN09 added during plant organic matter microbial inoculum prepared by the present invention by pair
Plant organic matter is decomposed, and plant organic matter (not decomposed) is avoided to damage plant, and 3 processing: processing 1 are arranged in this test:
It is used as positive control with no treatment；Processing 2: the plant organic matter microbial inoculum prepared in embodiment 3 and 4；Processing 3: without corruption
Ripe plant organic matter print chinaberry slag, tea seed cake and palm kernel meal.
Every basin dress transplanting soil 3Kg (sterilized processing) is mixed well withed the soil, usage amount 5% by above-mentioned setting processing
(150g), 30 basins of each processing.The tissue culture seedlings of bananas by hardening for growing 5 to 6 true leaves is transplanted into seedling basin, every basin kind
1 plant.Conventional fertilizer and water management observes and records Banana Growth situation.Fig. 3 the result shows that, plant organic matter microbial inoculum can prevent without
Decomposed plant organic matter damages banana.
Potting efficiency test of the 10 plant organic matter microbial inoculum of embodiment to phyllotreta striolata
Every kind of microbial inoculum efficiency test is respectively provided with 2 processing: processing 1: with no treatment as control；Processing 2: plant has
Machine object microbial inoculum.
Every basin dress transplanting soil 3Kg (sterilized processing), plant organic matter microbial inoculum 50g is mixed well withed the soil, each processing 3
Basin.By in 3 leaf phase leaf mustard transplanted seedling basins, every 3 seedling of basin.After leaf mustard length to 4 leaf phases, polypide is grown into consistent phyllotreta striolata
Adult is connected on leaf mustard leaf, 20, every basin.Each processing phyllotreta striolata adult density is investigated after 7d, is calculated according to the following formula anti-
Control effect:
Control efficiency (%)=[(processing Revision insect recluced rate-control Revision insect recluced rate)/(1- compares Revision insect recluced rate)] ×
100
The result shows that plant organic matter microbial inoculum has good control efficiency to phyllotreta striolata, wherein print chinaberry slag microbial inoculum
Preventive effect is best, and up to 86.3%；The preventive effect of tea seed cake microbial inoculum has reached 79.8%；The preventive effect of palm kernel meal microbial inoculum is 60.4%.
Controlling experiment of the 11 plant organic matter microbial inoculum of embodiment to anomala corpulenta
Every kind of microbial inoculum controlling experiment is respectively provided with 2 processing: processing 1: with no treatment as control；Processing 2: plant has
Machine object microbial inoculum.
Plant organic matter microbial inoculum 50g and 1Kg is mixed added with the sterilizing fine earth of uniform thickness cooking shredded potato, keeps soil water-containing
Amount is 20% or so.Each access polypide growth consistent anomala corpulenta first-instar young 20,3 repetitions of each processing.28℃
Each processing anomala corpulenta larval density is investigated after raising 7d, calculates control efficiency according to the following formula:
Control efficiency (%)=[(processing Revision insect recluced rate-control Revision insect recluced rate)/(1- compares Revision insect recluced rate)] ×
100
The result shows that plant organic matter microbial inoculum has good control efficiency to anomala corpulenta, wherein print chinaberry slag microbial inoculum
Preventive effect is up to 87.6%；The preventive effect of tea seed cake microbial inoculum has reached 90.5%；The preventive effect of palm kernel meal microbial inoculum is 76.7%.
The pot experiment of 12 plant organic matter microbial inoculum of embodiment promotion plant growth
Every kind of microbial inoculum efficiency test is respectively provided with 5 processing: processing 1: microbial inoculum is not added as control；Handle 2:2.5% plant
Organic matter microbial inoculum；Handle 3:5% plant organic matter microbial inoculum；Handle 4:10% plant organic matter microbial inoculum；Processing 5:20% plant has
Machine object microbial inoculum.
Every basin dress transplanting soil 3Kg (sterilized processing) is mixed well withed the soil microbial inoculum by soil weight mass ratio, each processing 30
Basin.By the banana tissue culture seedling replanting of the same size Jing Guo simple hardening into seedling basin, 1 plant of Banana Seedlings of every basin kind.60 days
Banana seedlings upgrowth situation is investigated afterwards, measures plant height.
The result shows that (table 7), plant organic matter microbial inoculum can promote the growth of banana seedlings, and the control group plant height that is averaged is
The average plant height of 21.4cm, print chinaberry slag, tea seed cake and palm kernel meal plant organic matter microbial inoculum processing group are respectively 25.6,24.8 Hes
23.5cm, wherein print chinaberry slag plant organic matter microbial inoculum growth-promoting effect is best.
7 plant organic matter microbial inoculum of table promotes Banana Growth pot experiment effect
The experiment that 13 plant organic matter microbial inoculum of embodiment promotes plant to absorb nitrogen
The banana seedling leaf in the test of embodiment 8 is collected, measures the content of the full nitrogen of banana seedlings respectively using Kjeldahl method,
Calculate the incrementss absorbed to nitrogen.Table 8 the result shows that, plant organic matter microbial inoculum can promote absorption of the banana to nitrogen, to nitrogen inhale
Receiving incrementss is respectively 36.3%, 15.5% and 16.7%.
The test that 8 plant organic matter microbial inoculum of table promotes banana to absorb nitrogen
Embodiment 14 prints chinaberry slag plant organic matter microbial inoculum prevention and treatment banana blight and promotes the field efficacy examination of Banana Growth
It tests
In order to show the plant organic matter microbial inoculum of bacterial strain HN09 of the present invention, control efficiency to banana blight and to perfume (or spice)
Any of several broadleaf plants promotes growth result, indoors on the basis of pot experiment, has carried out field control effectiveness test.In Guangzhou Fanyu District at the beginning of 2012
Carry out to banana the field trial of print chinaberry slag plant organic matter microbial inoculum, two groups of processing are arranged: processing 1: ridge 1 and ridge 2 are not administered work
For control；Processing 2: ridge 3~5 applies print chinaberry slag plant organic matter microbial inoculum.Banana seedlings are selected with growing consistent banana, using perfume (or spice)
Any of several broadleaf plants seedling base manure insecticide-applying way, usage amount is 60Kg per acre, is observed continuously after processing in October, 2012, investigates incidence, press
Field efficacy is calculated according to 7 method of embodiment, and measures plant height, log.
Test result shows that the withered and yellow blade quantity for printing chinaberry slag plant organic matter microbial inoculum processing group is considerably less than blank control
(Fig. 4), can be effectively reduced banana blight disease incidence, and field efficacy reaches 94.7% (table 9)；Meanwhile it is organic to print chinaberry slag plant
Object microbial inoculum can promote the growth of banana seedlings, and as shown in table 10, control has counted 119 plants, and processing group has counted 179 plants, control
Average plant height is 1.42~1.46m, and the processing group plant height that is averaged has reached 1.58~1.70m.
Table 9 prints chinaberry slag plant organic matter microbial inoculum to the field efficacy of banana blight
10 plant organic matter microbial inoculum of table promotes Banana Growth field test effect
Claims (5)
1. a kind of plant organic matter-bacillus subtilis HN09 mix bacterium agent, it is characterised in that: by bacillus subtilis
(Bacillus subtilis) HN09 and plant organic matter composition,
The bacillus subtilis HN09 carries out growth and breeding by main medium of the plant organic matter, is completed at the same time plant
Organic matter is decomposed；
The bacillus subtilis HN09 bacterial strain belongs to bacillus subtilis, is located away from Agricultural University Of South China's pesticide plant living collection
Soil, by Institute of Microorganism, Academia Sinica's Culture Collection Center preservation, deposit number CGMCC7524,
The plant organic matter is selected from print chinaberry slag, tea seed cake, palm kernel meal.
2. plant organic matter according to claim 1-bacillus subtilis HN09 mix bacterium agent, it is characterised in that: plant
Organic matter is print chinaberry slag, one or more in tea seed cake and palm kernel meal, and ratio is that weight arbitrarily compares when multiple combinations, through crushed
Resulting little particle fine powder, as the medium matrix of bacillus subtilis HN09, and serves as microbial inoculum after 100 or 200 meshes
Absorption carrier.
3. described in any item plant organic matters-bacillus subtilis HN09 mix bacterium agent is in phytopathy according to claim 1 ~ 2
Application in evil prevention and treatment, it is characterised in that: the plant disease are as follows: banana blight and tomato wilt.
4. described in any item plant organic matters-bacillus subtilis HN09 mix bacterium agent is in soil evil according to claim 1 ~ 2
Application in worm prevention and treatment, it is characterised in that: the soil pests are as follows: flea beetle and chafer.
5. described in any item plant organic matters-bacillus subtilis HN09 mix bacterium agent is promoting to plant according to claim 1 ~ 2
Object grows and to the application in nitrogen absorption.
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| CN110074141A (en) * | 2019-05-31 | 2019-08-02 | 昆明理工大学 | A kind of biocontrol agent and preparation method thereof for preventing and treating cacac moth |
| CN112457125A (en) * | 2020-12-22 | 2021-03-09 | 广西壮族自治区农业科学院 | Banana wilt prevention bio-organic fertilizer and preparation method and application thereof |
| CN114436705B (en) * | 2022-03-18 | 2022-10-11 | 河南省驻科生物科技有限公司 | Liquid fertilizer for increasing yield and preventing and treating plant diseases and insect pests and preparation method thereof |
| CN114560727A (en) * | 2022-04-15 | 2022-05-31 | 湖南山润油茶科技发展有限公司 | Preparation method and application of eco-friendly antibacterial disease-resistant easily-degradable compostable organic matter |
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| CN1432284A (en) * | 2002-01-18 | 2003-07-30 | 福建泰禾生化科技股份有限公司 | High-efficiency biological germicide and its prepn process |
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