CN101984827A - Plant growth bacterial agent and preparation method thereof - Google Patents
Plant growth bacterial agent and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a plant growth bacterial agent of a microorganism and a preparation method thereof, which belong to the preparation technology of biological bacteria. The plant growth bacterial agent of the invention is mainly characterized by consisting of the following flora in percentage by weight: 25 to 40 percent of yeast fused strain, 15 to 30 percent of bacillus mucilaginosus, 15 to 30 percent of bacillus megaterium, 15 to 30 percent of azotobocter chroococcum and 15 to 30 percent of kahn trichoderma, and a liquid medium, wherein the collection number of the yeast fused strain is CGMCC 2461. The plant growth bacterial agent of the invention has the advantage that: experiments carried out by the inventor show that: the bacterial agent is applied during the growing period observation on a grey desert soil or saline alkali soil potted plant, so that the seedling emergence time, the seedling emergence rate, the trefoil stage and the seedling height of an experimental group are all obviously improved compared with those of a control group. During the observation on roots of the grey desert soil or saline alkali soil potted plant, the root length, the root factor, the root volume and the fresh weight of the experimental group are obviously different from those of the control group.
Description
Technical field:
The present invention relates generally to a kind of plant growing microbial inoculum and preparation method thereof that helps of microorganism.The technology of preparing of microorganism belonging to genus microbial inoculum.
Background technology:
The domestic existing multiple microbial bacterial agent that is used for plant growing, EM as Japan, supernatural power CM, VT etc., but also can help the technology of the microbial inoculum of the quick growth of plant still to belong to blank at present with engineering strain and colloid bacillus cereus, bacillus megaterium, azotobacter chroococcum, the compound method for preparing microbial inoculum of koning trichoderma bacterium of Protoplast Fusion Technique preparation.
Summary of the invention:
The objective of the invention is to avoid the prior art deficiency and a kind of plant growing microbial inoculum and preparation method thereof that helps is provided.Especially with the engineering strain of Protoplast Fusion Technique preparation and colloid bacillus cereus, bacillus megaterium, azotobacter chroococcum, the compound method for preparing microbial bacterial agent of koning trichoderma bacterium.
The raw material that uses genetic engineering bacterium and colloid bacillus cereus, bacillus megaterium, azotobacter chroococcum, koning trichoderma Cordycepps to learn the medium of compatibility is, with the waste water of fresh bean curd or bean vermicelli or corn starch or potato starch or wheat bran, corn steep liquor mixed liquor is raw material, adds the preparation of small amounts of inorganic salt.This method is that the waste liquid that utilizes food, light industry to produce is raw material, production process does not have " three wastes " and pollutes, production technology is simple, easy to operate, save manpower, save the energy, its microbial inoculum, can be used for liquid microbe fertilizer, also can be used as the microorganism fungus kind Inoculant of microbial solid fertilizer and organic compost.
Purpose of the present invention can be by realizing by the following technical solutions: a kind of microbial inoculum that helps plant growing, its main feature is by yeast fusion bacterium (Fusant between candida tropicalisand saccharomycescerevisiae) F306, colloid bacillus cereus (Bacillus megterium) B.me-8, bacillus megaterium (Bacillusmucilaginosus) B.mu-36, azotobacter chroococcum (Azotobacter chroococcum) A.c-6, koning trichoderma bacterium (T.reesi) T.r-12 and liquid nutrient medium are formed, the percentage by weight of its flora is 25～40% for the yeast fusion bacterium, colloid bacillus cereus is 15～30%, bacillus megaterium 15～30%, azotobacter chroococcum is 15～30%, the koning trichoderma bacterium is 15～30%, and the preserving number of described yeast fusion bacterium is CGMCC 2461.
The described microbial inoculum that helps plant growing, described liquid nutrient medium include and add ammonium sulfate 1-2g in 1 liter of fresh tofu wastewater or starch wastewater or corn starch wastewater or potato starch wastewaters, phosphoric acid 0.5-1ml, and the limewash with 1% is transferred pH 5.5-6.5.
The described preparation method who helps the microbial inoculum of plant growing, it has the following steps:
A. the preparation of agar slant culture-medium and culture of strains:
A. the preparation of agar slant culture-medium: glucose 0.8-1.2%, peptone 0.8-1.2%, agar 1.8-2.2%, yeast extract 0.3-0.8%, sodium chloride 0.8-1.2%, sterile water 80-120ml, after the heating for dissolving, sodium hydroxide adjust pH with 1% is to 6.2-7.2, through 110-114 ℃, 28-32 minute steam sterilizing, the test tube that is sub-packed in the dry sterilization of 15 * 1.5cm then under aseptic condition are put into the inclined-plane to wait to solidify the back standby;
B. culture of strains: the mother who gets yeast fusion bacterium, colloid bacillus cereus, bacillus megaterium, azotobacter chroococcum, koning trichoderma bacterium respectively plants the agar slant bacterium, under aseptic condition, transferred species is on the agar slant culture-medium of above-mentioned preparation, through 28-32 ℃, 24-48 hour cultivation, promptly get the agar slant bacterial classification, be first class inoculum;
The preparation of B liquid nutrient medium and the cultivation of liquid spawn:
A. the preparation of liquid nutrient medium: in 1 liter of fresh tofu wastewater or starch wastewater or corn starch wastewater or potato starch wastewater, add ammonium sulfate 1-2g, phosphoric acid 0.5-1ml, limewash with 1% is transferred pH 6.2-7.2, be sub-packed in the 500ml conical flask, the branch loading amount is 1/4 of a bottle capacity, through 110-112 ℃, 30-35 minute autoclaving cools off back standby then;
B. get the first class inoculum of yeast fusion bacterium, colloid bacillus cereus, bacillus megaterium, azotobacter chroococcum, koning trichoderma bacterium respectively, under aseptic condition, be inoculated in the liquid medium, through 28-32 ℃, 24-48 hour cultivation, its culture fluid is a second class inoculum;
C. the second class inoculum of getting yeast fusion bacterium, colloid bacillus cereus, bacillus megaterium, azotobacter chroococcum, koning trichoderma bacterium respectively is enlarged culture again, last shaking table shaken cultivation, cultivated in 28～32 ℃, 18～24 hours through amplitude 100～120r/min, temperature, its bacterium liquid is three-class strain;
D. be 25～40% with percentage by weight for the yeast fusion bacterium respectively, colloid bacillus cereus is 15～30%, bacillus megaterium 15～30%, azotobacter chroococcum is 15～30%, the koning trichoderma bacterium is 15～30% three-class strains enlarged culture again, five kinds of bacterium are at same fermentation tank, through 28-32 ℃, 18-24 hour air agitation fermented and cultured, its bacterium liquid is the yeast fusion bacterium, colloid bacillus cereus, bacillus megaterium, azotobacter chroococcum, koning trichoderma bacterium mix bacterium agent, be the level Four bacterial classification, can continue enlarged culture step by step as required, also can be used as product and use.The described preparation method who helps the plant growing microbial inoculum, the preparation of described yeast fusion bacterium has the following steps: alcohol yeast is done female inclined-plane transferred species and cultivation of planting, after liquid oscilaltion is cultivated 14 hours, carry out preliminary treatment with the EDTA-mercaptoethanol, the cellulase of use 1% and 1% glusulase carry out enzymolysis and take off wall, time is 2 hours, and 33 ℃ of temperature obtain the distillery yeast protoplast; The candida tropicalis bacterium is done female inclined-plane transferred species and cultivation of planting, after liquid oscilaltion is cultivated 14 hours, carry out preliminary treatment with the EDTA-mercaptoethanol, the cellulase of use 1.5% and 0.5% glusulase carry out enzymolysis and take off wall, time is 2.5 hours, 33 ℃ of temperature obtain the candida tropicalis protoplast; With the candida tropicalis protoplast through 0.1% iodoacetic acid deactivation, mix with 1: 1 with the distillery yeast protoplast, precipitation is suspended from the chaotropic agent of 35% polyethylene glycol, 30 ℃ of static processing of water-bath, pH 6.0, and the time is 40min, merging bacterium liquid washes repeatedly through high phosphatizing acid buffer (PBS), coat that height oozes basic type medium (MMS) and height oozes on perfect medium (YPDS) plating medium, cultivated through 30 ℃, 7 days, flat board grows the yeast fusion bacterium.
The described preparation method who helps the plant growing microbial inoculum, the medium of described yeast fusion bacterium is: brewer's wort 0.5-2%, glucose 0.5-2%, peptone 0.2-0.6%, agar powder 1-3%, sterile water 80-120ml, after the heating for dissolving, sodium hydroxide adjust pH with 1% is to 5.5-6.5, through 110-114 ℃, 30-35 minute steam sterilizing, the test tube that is sub-packed in the dry sterilization of 15 * 1.5cm then under aseptic condition are put into the inclined-plane, and to wait to solidify the back standby.
Beneficial effect of the present invention: the inventor is through overtesting, in sierozem and saline-alkali soil are observed potted plant vegetative period, no matter used microbial inoculum experimental group of the present invention and control group seedling-growing time, emergence rate, tri-leaf period, height of seedling all are significantly increased.In the potted plant root system of sierozem and saline-alkali soil is observed, no matter experimental group and control group root length, root system number, volume, all there were significant differences for fresh weight.
The inventor adopts advanced single parent's deactivation Protoplast Fusion Technique, candida tropicalis (Candidatropicalis) 968 and 2.399 liang of parent's bacterial strains of distillery yeast (Saecharomyces cervsiar) are done fusion, make up new genetic engineering bacterium, these merge bacterium and promptly have parents' biological nature, has the organized enzyme that is better than parents again, flocculability and resistant to elevated temperatures biological nature.Can make full use of bean curd, organic nutriment carries out growth and breeding in bean vermicelli and the corn potato starch wastewater, utilize on 04 21st, 2008, the address is in the Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number is saccharomyces cerevisiae F306 (Saccharomyces cerevisiae) the compatibility colloid bacillus cereus of CGMCC No.2461, bacillus megaterium, azotobacter chroococcum, the combination of koning trichoderma bacterium five bacterium, colloid bacillus cereus, bacillus megaterium, it is strong that azotobacter chroococcum has adaptability, better tolerance can be converted into available phosphorus and available potassium confession plant growing with insoluble phosphorus in the soil and potassium element; Bacillus has easy growth, and strong stress resistance adapts to extensively, not only can help plant growing, and can strengthen the diseases and insect pests resistance of plant; The koning trichoderma bacterium can the plain enzyme of eccrine fiber can be starch and sugar with the cellulose conversion in the soil, for plant growing provides nutrition, through scientific matching and unique zymotechnique, make the utility that its flora produces and secretory substance becomes separately or the base-material and the raw material of growth mutually in process of growth, by mutual symbiosis propagation relation, formed a complexity and stable microecosystem has been brought into play multi-functional advantage.
Yeast fusion bacterium, colloid bacillus cereus, bacillus megaterium, azotobacter chroococcum, koning trichoderma bacterium are in the waste water that fresh bean curd, bean vermicelli, corn, potato starch produce, add a spot of mineral salt, direct fermentation prepares liquid bacterial agent, both saved the raw material of medium, reduce raw material preparation and autoclaved operation, saved the energy, also solved the difficult problem of these industry pollution treatments, being one turns waste into wealth, the biotechnology of achieving many things at one stroke.
Yeast fusion bacterium, colloid bacillus cereus, bacillus megaterium, azotobacter chroococcum, koning trichoderma bacteria agent promptly can be used as plant growth substance, also can be used as the treatment of Organic Wastewater clarificant, also can be used as biological flocculant, surfactant, biological adsorption agents etc. are applied to industries such as oil exploitation, food industry, environmental project, medical treatment and fine chemistry industry, have wider using value.
Description of drawings:
Fig. 1 is preparation technology's schematic flow sheet of the present invention.
Embodiment:
The present invention is described in further detail below in conjunction with embodiment:
See Fig. 1, embodiment 1
(1) cultivation of test tube liquid spawn is got fresh tofu wastewater and is got supernatant 100ml through boiling natural sedimentation in 10 minutes, adds (NH
4)
2SO
40.1g, H
3PO
40.05ml, fully PH to 6.2～7.2 are regulated in the dissolving back, be sub-packed in the dry test-tube of 15 * 1.5cm, through 112 ℃, 30 minutes autoclavings are cooled to 32 ℃, inoculate F306 yeast fusion bacterium, colloid bacillus cereus, bacillus megaterium, azotobacter chroococcum, koning trichoderma bacterium respectively in the test tube liquid nutrient medium, through 28～32 ℃ of cultivations 24～48 hours, be test tube liquid spawn (first class inoculum)
(2) cultivation of conical flask liquid spawn
Get fresh tofu wastewater and get supernatant 1000ml, add (NH through boiling natural sedimentation in 10 minutes
4)
2SO
41g, H
3PO
40.5ml, fully PH to 6.2～7.2 are regulated in the dissolving back, be sub-packed in the 500ml conical flask, the branch loading amount is 1/4 of a bottle total amount, then through 112 ℃, and 30 minutes autoclavings, be cooled to 32 ℃, the test tube liquid-spawn inoculation in liquid conical flask medium, through 28～32 ℃ of cultivations 24～48 hours, is liquid conical flask bacterial classification (second class inoculum)
(3) fluid enlargement culture is produced bacterial classification
Get fresh tofu wastewater and get supernatant 10000ml, add (NH through boiling natural sedimentation in 10 minutes
4)
2SO
410g, H
3PO
45ml, fully PH to 6.2～7.2 are regulated in the dissolving back, are sub-packed in the 500ml conical flask, and the branch loading amount is 1/4 of a bottle total amount, then through 112 ℃, 30 minutes autoclavings are cooled to 32 ℃, and second class inoculum is seeded in the liquid conical flask medium, last shaking table shaken cultivation, amplitude is 100～120r/min, cultivates 18～24 hours fermented and cultured for 28～32 ℃ through temperature, and its culture fluid is a three-class strain.
(4) get three-class strain enlarged culture again, the top fermentation jar, used fresh tofu wastewater is unsterilised, with percentage by weight for the yeast fusion bacterium is 40%, colloid bacillus cereus is 15%, bacillus megaterium 15%, azotobacter chroococcum is 15%, the koning trichoderma bacterium is after 15% 5 bacteria culture fluid inserts, cultivated 18～24 hours through 28～32 ℃, the air agitation fermented and cultured, its zymotic fluid is F306 yeast fusion bacterium, colloid bacillus cereus, bacillus megaterium, azotobacter chroococcum, koning trichoderma bacteria liquid microbial inoculum, and viable count is 1.0～2.0 * 10
9Individual/ml;
(5) can enlarge output step by step as required.
Embodiment 2
(1) fresh starch wastewater 100ml is got in the cultivation of test tube liquid spawn, adds (NH
4)
2SO
40.1g, H
3PO
40.05ml, fully PH to 6.2～7.2 are regulated in the dissolving back, be sub-packed in the dry test-tube of 15 * 1.2cm, through 112 ℃, 30 minutes autoclavings are cooled to 32 ℃, inoculate F306 yeast fusion bacterium, colloid bacillus cereus, bacillus megaterium, azotobacter chroococcum, koning trichoderma bacterium respectively in the test tube liquid nutrient medium, through 28～32 ℃ of cultivations 24～48 hours, be test tube liquid spawn (first class inoculum).
(2) cultivation of conical flask liquid spawn
Get fresh starch wastewater 1000ml, add (NH
4)
2SO
41g, H
3PO
40.5ml, fully PH to 6.2～7.2 are regulated in the dissolving back, be sub-packed in the 500ml conical flask, the branch loading amount is 1/4 of a bottle total amount, then through 112 ℃, and 30 minutes autoclavings, be cooled to 32 ℃, the test tube liquid-spawn inoculation in liquid conical flask medium, through 28～32 ℃ of cultivations 24～48 hours, is liquid conical flask bacterial classification (second class inoculum).
(3) fluid enlargement culture is produced bacterial classification:
Get fresh starch wastewater 10000ml, add (NH
4)
2SO
410g, H
3PO
45ml, fully PH to 6.2～7.2 are regulated in the dissolving back, are sub-packed in the 500ml conical flask, and the branch loading amount is 1/4 of a bottle total amount, then through 112 ℃, 30 minutes autoclavings are cooled to 32 ℃, and second class inoculum is seeded in the liquid conical flask medium, last shaking table shaken cultivation, amplitude is 100～120r/min, cultivates 18～24 hours fermented and cultured for 28～32 ℃ through temperature, and its culture fluid is a three-class strain.
(4) get three-class strain enlarged culture again, the top fermentation jar, used fresh starch wastewater is unsterilised, with percentage by weight for the yeast fusion bacterium is 25%, colloid bacillus cereus is 30%, bacillus megaterium 15%, azotobacter chroococcum is 15%, the koning trichoderma bacterium is after 15% 5 bacteria culture fluid inserts, cultivated 18～24 hours through 28～32 ℃, the air agitation fermented and cultured, its ripe zymotic fluid is F306 yeast fusion bacterium, colloid bacillus cereus, bacillus megaterium, azotobacter chroococcum, koning trichoderma bacteria liquid microbial inoculum, and viable count is 0.8～1.8 * 10
9Individual/ml;
(5) can enlarge output step by step as required.
Embodiment 3
(1) the fresh corn starch wastewater is got in the cultivation of test tube liquid spawn, gets supernatant 100ml through boiling natural sedimentation in 10 minutes, adds (NH
4)
2SO
40.1g, H
3PO
40.05ml, fully PH to 6.2～7.2 are regulated in the dissolving back, be sub-packed in the dry test-tube of 15 * 1.2cm, through 112 ℃, 30 minutes autoclavings are cooled to 32 ℃, inoculate F306 yeast fusion bacterium, colloid bacillus cereus, bacillus megaterium, azotobacter chroococcum, koning trichoderma bacterium respectively in the test tube liquid nutrient medium, through 28～32 ℃ of cultivations 24～48 hours, be test tube liquid spawn (first class inoculum).
(2) cultivation of conical flask liquid spawn
Get the fresh corn starch wastewater, get supernatant 1000ml, add (NH through boiling natural sedimentation in 10 minutes
4)
2SO
41g, H
3PO
40.5ml, fully PH to 6.2～7.2 are regulated in the dissolving back, be sub-packed in the 500ml conical flask, the branch loading amount is 1/4 of a bottle total amount, then through 112 ℃, and 30 minutes autoclavings, be cooled to 32 ℃, the test tube liquid-spawn inoculation in liquid conical flask medium, through 28～32 ℃ of cultivations 24～48 hours, is liquid conical flask bacterial classification (second class inoculum).
(3) fluid enlargement culture is produced bacterial classification
Get the fresh corn starch wastewater, get supernatant 10000ml, add (NH through boiling natural sedimentation in 10 minutes
4)
2SO
410g, H
3PO
45ml, fully PH to 6.2～7.2 are regulated in the dissolving back, are sub-packed in the 500ml conical flask, and the branch loading amount is 1/4 of a bottle total amount, then through 112 ℃, 30 minutes autoclavings are cooled to 32 ℃, and second class inoculum is seeded in the liquid conical flask medium, last shaking table shaken cultivation, amplitude is 100～120r/min, cultivates 18～24 hours fermented and cultured for 28～32 ℃ through temperature, and its culture fluid is a three-class strain.
(4) get three-class strain enlarged culture again, the top fermentation jar, used fresh starch wastewater is unsterilised, with percentage by weight for the yeast fusion bacterium is 35%, colloid bacillus cereus is 15%, bacillus megaterium 20%, azotobacter chroococcum is 15%, the koning trichoderma bacterium is after 15% 5 bacteria culture fluid inserts, cultivated 18～24 hours through 28～32 ℃, the air agitation fermented and cultured, its ripe zymotic fluid is F306 yeast fusion bacterium, colloid bacillus cereus, bacillus megaterium, azotobacter chroococcum, koning trichoderma bacteria liquid microbial inoculum, and viable count is 1.2～2.2 * 10
9Individual/ml;
(5) can enlarge output step by step as required.
Embodiment 4
(1) fresh potato starch wastewater is got in the cultivation of test tube liquid spawn, gets supernatant 100ml through boiling natural sedimentation in 10 minutes, adds (NH
4)
2SO
40.1g, H
3PO
40.05ml, fully PH to 6.2～7.2 are regulated in the dissolving back, be sub-packed in the dry test-tube of 15 * 1.2cm, through 112 ℃, 30 minutes autoclavings are cooled to 32 ℃, inoculate F306 yeast fusion bacterium, colloid bacillus cereus, bacillus megaterium, azotobacter chroococcum, koning trichoderma bacterium respectively in the test tube liquid nutrient medium, through 28～32 ℃ of cultivations 24～48 hours, be test tube liquid spawn (first class inoculum).
(2) cultivation of conical flask liquid spawn:
Get fresh potato starch wastewater, get supernatant 1000ml, add (NH through boiling natural sedimentation in 10 minutes
4)
2SO
41g, H
3PO
40.5ml, fully PH to 6.2～7.2 are regulated in the dissolving back, be sub-packed in the 500ml conical flask, the branch loading amount is 1/4 of a bottle total amount, then through 112 ℃, and 30 minutes autoclavings, be cooled to 32 ℃, the test tube liquid-spawn inoculation in liquid conical flask medium, through 28～32 ℃ of cultivations 24～48 hours, is liquid conical flask bacterial classification (second class inoculum).
(3) fluid enlargement culture is produced bacterial classification:
Get fresh potato starch wastewater, get supernatant 10000ml, add (NH through boiling natural sedimentation in 10 minutes
4)
2SO
410g, H
3PO
45ml, fully PH to 6.2～7.2 are regulated in the dissolving back, are sub-packed in the 500ml conical flask, and the branch loading amount is 1/4 of a bottle total amount, then through 112 ℃, 30 minutes autoclavings are cooled to 32 ℃, and second class inoculum is seeded in the liquid conical flask medium, last shaking table shaken cultivation, amplitude is 100～120r/min, cultivates 18～24 hours fermented and cultured for 28～32 ℃ through temperature, and its culture fluid is a three-class strain.
(4) get three-class strain enlarged culture again, the top fermentation jar, used fresh starch wastewater is unsterilised, with percentage by weight for the yeast fusion bacterium is 25%, colloid bacillus cereus is 15%, bacillus megaterium 15%, azotobacter chroococcum is 30%, the koning trichoderma bacterium is after 15% 5 bacteria culture fluid inserts, cultivated 18～24 hours through 28～32 ℃, the air agitation fermented and cultured, its ripe zymotic fluid is F306 yeast fusion bacterium, colloid bacillus cereus, bacillus megaterium, azotobacter chroococcum, koning trichoderma bacteria liquid microbial inoculum, and viable count is 1.2～2.2 * 10
9Individual/ml;
(5) can enlarge output step by step as required.
Embodiment 5
The preparation of a kind of yeast fusion bacterium has the following steps: alcohol yeast is done female inclined-plane transferred species and cultivation of planting, after liquid oscilaltion is cultivated 14 hours, carry out preliminary treatment with the EDTA-mercaptoethanol, the cellulase of use 1% and 1% glusulase carry out enzymolysis and take off wall, time is 2 hours, 33 ℃ of temperature obtain the distillery yeast protoplast; The candida tropicalis bacterium is done female inclined-plane transferred species and cultivation of planting, after liquid oscilaltion is cultivated 14 hours, carry out preliminary treatment with the EDTA-mercaptoethanol, the cellulase of use 1.5% and 0.5% glusulase carry out enzymolysis and take off wall, time is 2.5 hours, 33 ℃ of temperature obtain the candida tropicalis protoplast; With the candida tropicalis protoplast through 0.1% iodoacetic acid deactivation, mix with 1: 1 with the distillery yeast protoplast, precipitation is suspended from the chaotropic agent of 35% polyethylene glycol (PEG), 30 ℃ of static processing of water-bath, pH 6.0, and the time is 40min, merging bacterium liquid washes repeatedly through high phosphatizing acid buffer (PBS) liquid, coat that height oozes basal medium (MMS) and height oozes on perfect medium (YPDS) plating medium, cultivated through 30 ℃, 7 days, flat board grows the yeast fusion bacterium.
The medium of described yeast fusion bacterium is: brewer's wort 0.5-2%, glucose 0.5-2%, peptone 0.2-0.6%, agar powder 1-3%, sterile water 80-120ml, after the heating for dissolving, sodium hydroxide adjust pH with 1% is to 5.5-6.5, through 110-114 ℃, 30-35 minute steam sterilizing, the test tube that is sub-packed in the dry sterilization of 15 * 1.5cm then under aseptic condition are put into the inclined-plane, and to wait to solidify the back standby.
Embodiment 6, and yeast fusion bacterium, colloid bacillus cereus, bacillus megaterium, azotobacter chroococcum, koning trichoderma bacteria liquid microbial inoculum are at the test effect that with sierozem and saline-alkali soil serves as test soil.
Do pot experiment with the clover seed, the results are shown in Table 1 through repeating it for three times, table 2, table 3, table 4 and table 5:
Table 1
| Project | Test is handled |
| Experimental group | Growing period is used microbial inoculum of the present invention |
| Contrast | Growing period is not used microbial inoculum |
Table 2 sierozem is observed potted plant vegetative period
| Project | Seedling-growing time (d) | Emergence rate (%) | Tri-leaf period (d) | Height of seedling (cm) |
| Experimental group | 3? | 94? | 18? | 6.2? |
| Contrast | 4? | 84? | 21? | 4.5? |
No matter test and contrast seedling-growing time, emergence rate, tri-leaf period, height of seedling all are significantly increased.
Table 3 saline-alkali soil is observed potted plant vegetative period
| Project | Seedling-growing time (d) | Emergence rate (%) | Tri-leaf period (d) | Height of seedling (cm) |
| Experimental group | 4? | 66? | 20? | 3.2? |
| Contrast | 6? | 20? | 25? | 2.5? |
No matter test and contrast seedling-growing time, emergence rate, tri-leaf period, height of seedling all are significantly increased.
The potted plant clover root system of table 4 sierozem is observed
| Project | Root length (cm) | Root system number (root) | Volume (cm 3)? | Fresh weight (Kg) |
| Experimental group | 7.9? | 7? | 0.00002? | 0.01314? |
| Contrast | 4.8? | 4? | 0.00001? | 0.00726? |
No matter test and contrast root length, root system number, volume, all there were significant differences for fresh weight.
The potted plant clover root system of table 5 saline-alkali soil is observed
| Project | Root length (cm) | Root system number (root) | Volume (cm 3)? | Fresh weight (Kg) |
| Experimental group | 4.2? | 7? | 0.000008? | 0.00811? |
| Contrast | 3.2? | 4? | 0.000006? | 0.00521? |
No matter test and contrast root length, root system number, volume, all there were significant differences for fresh weight.
Claims (5)
1. microbial inoculum that helps plant growing, it is characterized in that forming by yeast fusion bacterium, colloid bacillus cereus, bacillus megaterium, azotobacter chroococcum, koning trichoderma bacterium and liquid nutrient medium, the percentage by weight of its flora is for the yeast fusion bacterium is 25～40%, colloid bacillus cereus is 15～30%, bacillus megaterium 15～30%, azotobacter chroococcum is 15～30%, the koning trichoderma bacterium is 15～30%, and the preserving number of described yeast fusion bacterium is CGMCC 2461.
2. the microbial inoculum that helps plant growing as claimed in claim 1, it is characterized in that described liquid nutrient medium includes adding ammonium sulfate 1-2g in 1 liter of fresh tofu wastewater or starch wastewater or corn starch wastewater or potato starch wastewater, phosphoric acid 0.5-1ml, the limewash with 1% is transferred pH 5.5-6.5.
3. the preparation method who helps the microbial inoculum of plant growing as claimed in claim 1 is characterized in that having the following steps:
A. the preparation of agar slant culture-medium and culture of strains:
A. the preparation of agar slant culture-medium: glucose 0.8-1.2%, peptone 0.8-1.2%, agar 1.8-2.2%, yeast extract 0.3-0.8%, sodium chloride 0.8-1.2%, sterile water 80-120ml, after the heating for dissolving, sodium hydroxide adjust pH with 1% is to 6.2-7.2, through 110-114 ℃, 28-32 minute steam sterilizing, the test tube that is sub-packed in the dry sterilization of 15 * 1.5cm then under aseptic condition are put into the inclined-plane to wait to solidify the back standby;
B. culture of strains: the mother who gets yeast fusion bacterium, colloid bacillus cereus, bacillus megaterium, azotobacter chroococcum, koning trichoderma bacterium respectively plants the agar slant bacterium, under aseptic condition, transferred species is on the agar slant culture-medium of above-mentioned preparation, through 28-32 ℃, 24-48 hour cultivation, promptly get the agar slant bacterial classification, be first class inoculum;
The preparation of B liquid nutrient medium and the cultivation of liquid spawn:
A. the preparation of liquid nutrient medium: in 1 liter of fresh tofu wastewater or starch wastewater or corn starch wastewater or potato starch wastewater, add ammonium sulfate 1-2g, phosphoric acid 0.5-1ml, limewash with 1% is transferred pH 6.2-7.2, be sub-packed in the 500ml conical flask, the branch loading amount is 1/4 of a bottle capacity, through 110-112 ℃, 30-35 minute autoclaving cools off back standby then;
B. get the first class inoculum of yeast fusion bacterium, colloid bacillus cereus, bacillus megaterium, azotobacter chroococcum, koning trichoderma bacterium respectively, under aseptic condition, be inoculated in the liquid medium, through 28-32 ℃, 24-48 hour cultivation, its culture fluid is a second class inoculum;
C. the second class inoculum of getting yeast fusion bacterium, colloid bacillus cereus, bacillus megaterium, azotobacter chroococcum, koning trichoderma bacterium respectively is enlarged culture again, last shaking table shaken cultivation, cultivated in 28～32 ℃, 18～24 hours through amplitude 100～120r/min, temperature, its bacterium liquid is three-class strain;
D. respectively with percentage by weight for the yeast fusion bacterium is 25～40%, colloid bacillus cereus is 15～30%, bacillus megaterium 15～30%, azotobacter chroococcum is 15～30%, the koning trichoderma bacterium is 15～30% three-class strains enlarged culture again, five kinds of bacterium are at same fermentation tank, air agitation fermented and cultured through 28-32 ℃, 18-24 hour, its bacterium liquid is yeast fusion bacterium, colloid bacillus cereus, bacillus megaterium, azotobacter chroococcum, koning trichoderma bacterium mix bacterium agent, be the level Four bacterial classification, continue enlarged culture step by step then.
4. the preparation method who helps the plant growing microbial inoculum as claimed in claim 3, the preparation that it is characterized in that described yeast fusion bacterium has the following steps: alcohol yeast is done female inclined-plane transferred species and cultivation of planting, after liquid oscilaltion is cultivated 14 hours, carry out preliminary treatment with the EDTA-mercaptoethanol, the cellulase of use 1% and 1% glusulase carry out enzymolysis and take off wall, time is 2 hours, and 33 ℃ of temperature obtain the distillery yeast protoplast; The candida tropicalis bacterium is done female inclined-plane transferred species and cultivation of planting, after liquid oscilaltion is cultivated 14 hours, carry out preliminary treatment with the EDTA-mercaptoethanol, the cellulase of use 1.5 and 0.5% glusulase carry out enzymolysis and take off wall, time is 2.5 hours, 33 ℃ of temperature obtain the candida tropicalis protoplast; With the candida tropicalis protoplast through 0.1% iodoacetic acid deactivation, mix with 1: 1 with the distillery yeast protoplast, precipitation is suspended from the chaotropic agent of 35% polyethylene glycol, 30 ℃ of static processing of water-bath, pH 6.0, and the time is 40min, merging bacterium liquid washes repeatedly through high phosphatizing acid buffer (PBS), coat that height oozes basic type medium (MMS) and height oozes on perfect medium (YPDS) plating medium, cultivated through 30 ℃, 7 days, flat board grows the yeast fusion bacterium.
5. the preparation method who helps the plant growing microbial inoculum as claimed in claim 4, the medium that it is characterized in that described yeast fusion bacterium is: brewer's wort 0.5-2%, glucose 0.5-2%, peptone 0.2-0.6%, agar powder 1-3%, sterile water 80-120ml, after the heating for dissolving, sodium hydroxide adjust pH with 1% is to 5.5-6.5, through 110-114 ℃, 30-35 minute steam sterilizing, the test tube that is sub-packed in the dry sterilization of 15 * 1.5cm then under aseptic condition are put into the inclined-plane, and to wait to solidify the back standby.
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Cited By (7)
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| CN102504825A (en) * | 2011-11-11 | 2012-06-20 | 山东省农业科学院农产品研究所 | Ecologic conditioner for salt-affected soil by sea and preparation method and application of ecologic conditioner |
| CN103918721A (en) * | 2014-04-21 | 2014-07-16 | 毕景阳 | Plant growth regulator |
| CN105037013A (en) * | 2015-09-08 | 2015-11-11 | 南宁荣港生物科技有限公司 | Method for preparing soil moisture conservation enzyme organic fertilizer |
| CN105039194A (en) * | 2015-03-20 | 2015-11-11 | 甘肃明德伟业生物科技有限公司 | Yeast fused strain mixed bacterial agent and method of fermenting organic waste water therewith to prepare liquid fertilizer |
| CN105087421A (en) * | 2015-04-23 | 2015-11-25 | 甘肃长业生态生物科技集团有限公司 | Fusant mixture, preparation method of fusant mixture, and method for producing organic fertilizer |
| CN105483047A (en) * | 2015-12-30 | 2016-04-13 | 甘肃长业生态生物科技集团有限公司 | Yeast fused strain mixed bactericide and preparing method for liquid bactericide thereof |
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| CN102504825B (en) * | 2011-11-11 | 2013-12-18 | 山东省农业科学院农产品研究所 | Ecologic conditioner for salt-affected soil by sea and preparation method and application of ecologic conditioner |
| CN103918721A (en) * | 2014-04-21 | 2014-07-16 | 毕景阳 | Plant growth regulator |
| CN103918721B (en) * | 2014-04-21 | 2016-04-13 | 毕景阳 | One plant growth regulators |
| CN105039194A (en) * | 2015-03-20 | 2015-11-11 | 甘肃明德伟业生物科技有限公司 | Yeast fused strain mixed bacterial agent and method of fermenting organic waste water therewith to prepare liquid fertilizer |
| CN105087421A (en) * | 2015-04-23 | 2015-11-25 | 甘肃长业生态生物科技集团有限公司 | Fusant mixture, preparation method of fusant mixture, and method for producing organic fertilizer |
| CN105087421B (en) * | 2015-04-23 | 2018-04-06 | 甘肃长业生态生物科技集团有限公司 | The method that yeast fusion bacterium mixes microbial inoculum and preparation method thereof and production organic fertilizer |
| CN105037013A (en) * | 2015-09-08 | 2015-11-11 | 南宁荣港生物科技有限公司 | Method for preparing soil moisture conservation enzyme organic fertilizer |
| CN105037013B (en) * | 2015-09-08 | 2018-07-13 | 南宁荣港生物科技有限公司 | The production method of soil moisture conservation ferment organic fertilizer |
| CN105483047A (en) * | 2015-12-30 | 2016-04-13 | 甘肃长业生态生物科技集团有限公司 | Yeast fused strain mixed bactericide and preparing method for liquid bactericide thereof |
| CN106867937A (en) * | 2017-03-11 | 2017-06-20 | 鲁东大学 | With the bacterial strain of pea protein wastewater liquid fermenting and producing Bacillus subtilis natto microbial inoculum, method and application |
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