CN111826319A - Microbial growth promoter and application thereof - Google Patents
Microbial growth promoter and application thereof Download PDFInfo
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- CN111826319A CN111826319A CN202010756994.5A CN202010756994A CN111826319A CN 111826319 A CN111826319 A CN 111826319A CN 202010756994 A CN202010756994 A CN 202010756994A CN 111826319 A CN111826319 A CN 111826319A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/25—Paenibacillus
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- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
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Abstract
The invention relates to the technical field of planting, in particular to a microbial growth promoter and application thereof. The microbial growth promoter comprises arthrobacter and bacillus glucanotrys, and the two bacteria have synergistic effect and can generate better growth promoting effect on macadamia nut trees. The microbial growth promoter can improve the utilization rate of nutrient substances in soil, thereby achieving the purposes of improving the nutrient absorption of plants, promoting the growth of plants and improving the ecological environment of soil. The microbial growth promoting agent can be applied to practical operation of macadimia nut planting, and the economic value of the macadimia nut is further improved.
Description
Technical Field
The invention relates to the technical field of planting, in particular to a microbial growth promoter and application thereof.
Background
Macadamia nuts have high economic value, enjoy the reputation of "the king of dried fruits" and are now widely planted in China. At present, research on the planting technology of the macadimia nuts is limited in the aspects of seedling raising, field management, pest control and the like, no research on special fertilizer for the macadimia nuts exists, and the research on application of microbial fertilizer on the macadimia nut planting is lacked.
The microbial fertilizer in a broad sense means that in the process of life activities, besides nutrition supply, microorganisms can stimulate plants to produce various active substances, such as growth hormone, or can antagonize the pathogenic action of certain pathogenic microorganisms in soil on the plants, counteract the influence of chemical synthetic fertilizers, degrade harmful pollutants and the like. The microorganism is used as a growth promoter for plant growth, the fertilizer (microbial fertilizer) containing the microorganism growth promoter is comprehensive in nutrients compared with a chemical fertilizer, can generate a special application effect, is more in variety, mild in fertilizer efficiency and free of pollution, can make up for the deficiency in agricultural production, and plays an important role in a sustainable agricultural soil ecosystem. The microbial fertilizer products registered by the Ministry of agriculture of China have 9 microbial inoculum types and 2 bacterial fertilizer types, and the main functional strains comprise azotobacter, phosphate solubilizing bacteria, potassium solubilizing bacteria and the like. The existing microbial fertilizer products are not rich enough in variety, cannot meet the requirements of practical application, and have no microbial inoculum or bacterial fertilizer specially aiming at macadamia nut planting. At the present stage, the planting of the macadimia nuts still depends on chemical fertilizers and common organic fertilizers, the use of the chemical fertilizers has certain negative effects on the ecological environment, and the common organic fertilizers cannot effectively promote the growth and fruiting of the macadimia nut trees. There is a need to develop a fertilizer containing microbial growth promoting agent for macadimia nuts in the revolving door, so as to further improve the economic value of the macadimia nuts.
Disclosure of Invention
The invention aims to provide a microbial growth promoter to solve the technical problem of lack of a microbial fertilizer specially aiming at macadimia nuts.
In order to achieve the purpose, the invention adopts the following technical scheme:
a microbial growth promoter comprises Arthrobacter and Bacillus glucanicus.
The principle and the advantages of the scheme are as follows: arthrobacter (Arthrobacter defuzii) and Bacillus glucanotryticus (Paenibacillus glucanilyticus) can generate a synergistic effect, and the utilization rate of phosphorus and potassium in soil by plants is improved, so that the aims of improving the nutrient absorption of the plants, promoting the growth of the plants and improving the ecological environment of the soil are fulfilled. The Arthrobacter (Arthrobacter defluvivii) used in the present protocol may be the standard strain Arthrobacter defluvivii 4C1-a (KCTC19209, korean typical culture collection); the Bacillus glucanus (Paenibacillus glucanilyticus) used in the present protocol may be a standard strain Bacillus glucanus (CRBIP code: CIP107742T, Pasteur institute species Collection).
The inventor separates and obtains various functional strains from the tailing soil of a phosphate fertilizer plant, the tailing soil of a potassium fertilizer plant and the like, and performs functional screening, quantitative analysis and nucleic acid identification on the functional strains. The bacillus arthrobacter and bacillus glucanotrys have strong synergistic effect, and can synergistically promote the growth of the macadamia nut. The inventor further uses the standard strains of arthrobacter and bacillus glucans to confirm the synergistic effect, finds that the growth promotion effect of the arthrobacter and the bacillus glucans on the small seedling of the macadimia nut is not obvious when the arthrobacter and the bacillus glucans are used alone, but finds that the plant height and the breast diameter of the macadimia nut are obviously improved compared with a blank control when the two bacteria are used in combination, and indicates that the two functional bacteria have a good growth promotion effect on the macadimia nut when the two bacteria are used in combination.
The microbial growth promoter is widely applied to vegetables, food crops and the like, but the microbial growth promoter has less application to macadamia nut planting because the microbial growth promoter has less obvious growth promoting effect on trees with longer time period, such as fruit trees and the like. The invention widens the growth promoting substances for macadimia nuts and provides a new scheme for the planting of the macadimia nuts.
Further, the ratio of the number of bacteria of Arthrobacter to the number of bacteria of Bacillus glucanicus is 0.8:1-1.5: 1.
By adopting the technical scheme, the arthrobacter and the bacillus glucanotrys can generate stronger synergistic action in the proportion.
Further, the ratio of the number of bacteria of Arthrobacter to the number of bacteria of Bacillus glucanicus was 1: 1.
By adopting the technical scheme, the ratio of 1:1 is the optimal ratio of the arthrobacter and the bacillus glucanotrys to generate the synergistic effect.
Further, the microbial fertilizer is prepared by the following method: obtaining bacterial suspension of arthrobacter and bacterial suspension of bacillus glucanotrys; and mixing the bacterial suspension of the arthrobacter and the bacterial suspension of the bacillus glucanotus to obtain a mixed bacterial liquid, and diluting with water to obtain the microbial growth promoter.
By adopting the technical scheme, the microbial growth promoter can be prepared by adopting a method of direct mixing and water dilution, and is simple and easy to implement.
Further, OD of bacterial suspension of Arthrobacter600The value is 0.8-1.5, OD of bacterial suspension of bacillus glucanolyticus600The value is 1.
With the above technical solution, absorbance represents the concentration (number per unit volume) of bacteria, and the method of absorbance to measure the amount of bacteria is a conventional quantitative method in the prior art.
Further, the bacterial suspension of arthrobacter and the bacterial suspension of bacillus glucanotrys are mixed according to the volume ratio of 1:1 to obtain mixed bacterial liquid.
By adopting the technical scheme, the ratio of the arthrobacter to the bacillus glucanotrys is 1:1, and the synergistic enhancement effect of the arthrobacter and the bacillus glucanotrys can be better exerted.
Further, 100ml of water was used to dilute each 10ml of the mixed bacterial solution.
By adopting the technical scheme, the dilution gradient of 10:100 is adopted, so that the bacteria applied to the plants can be ensured not to be too concentrated or too dilute, the too concentrated bacteria liquid can influence the osmotic pressure balance of the plant rhizosphere, and the too dilute bacteria liquid can cause the effect of the bacteria on the plants to be unobvious.
Further, an application of the microbial growth promoting agent in macadamia nut planting.
By adopting the technical scheme, the growth promoter consisting of the arthrobacter and the bacillus glucans can promote the growth of macadimia nut trees and can be applied to the planting practice of the macadimia nuts.
Further, the microbial growth promoting agent is applied to the macadamia nut seedlings once a month.
By adopting the technical scheme, the microbial growth promoting agent is applied to the macadimia nut seedlings every month, so that the plant height and the breast diameter of the seedlings are obviously increased, and a very obvious synergistic growth promoting effect is achieved.
Further, the mixed bacterial liquid is mixed with an organic fertilizer, and the microbial organic fertilizer is obtained after anaerobic fermentation.
By adopting the technical scheme, the microbial growth promoting agent of the scheme is used for fermenting the organic fertilizer, and the obtained microbial organic fertilizer is matched with the microbial growth promoting agent for use, so that the growth promoting effect can be exerted while the nutrition of macadimia nuts is enhanced, and the microbial growth promoting agent is expected to become a new bacterial fertilizer type microbial fertilizer product which can be widely popularized and applied.
Drawings
FIG. 1 is a graph showing the growth promoting effect of a pot culture according to Experimental example 1 of the present invention (Arthrobacter).
FIG. 2 is a diagram showing the growth promoting effect of the potting (glucan degrading bacillus) according to Experimental example 1 of the present invention.
FIG. 3 is a graph showing the growth promoting effect of the potting (growth promoting agent B for microorganisms) in Experimental example 1 of the present invention.
FIG. 4 is a graph showing statistical results of chlorophyll content measurement in Experimental example 1 of the present invention.
FIG. 5 shows the statistical results of the plant heights of macadamia nut of Experimental example 2.
FIG. 6 shows the statistical results of the chest diameters of macadamia nuts in Experimental example 2.
Detailed Description
Example 1: strain acquisition and identification
Taking a nutrient agar slant containing a strain A (identified as Arthrobacter defluvii) and a nutrient agar slant containing a strain B (identified as Bacillus cereus) to carry out strain morphology and physiological property identification (slant streak culture of bacteria is a conventional means in the prior art and is not described herein again). The strain A and the strain B are dominant strains separated from soil, and the obtaining process is detailed in experimental example 4.
And (3) morphological identification of the strain: inoculating the single colony on the inclined plane onto a Nutrient Agar (NA) plate, culturing at 37 ℃ for 24h, and observing the surface morphology of the single colony, such as size, shape, transparency and the like. The colony morphology is observed by utilizing a method of streaking three zones on a culture medium plate, wherein the strain is streaked and cultured on the plate, and is cultured in a constant temperature incubator at 37 ℃ for 24 hours to observe the colony morphology. Observation of cell morphology, the isolated strain was cultured in a 37 ℃ incubator for 24 hours, gram-stained, and the morphology of the cells was observed with an optical microscope. The experimental results are shown in table 1, the colony morphology of arthrobacter is round, the surface is dry, and the colony is opaque; the bacillus glucanolyticus is round, has smooth surface and transparent bacterial colony. The colony morphology of the strain A is consistent with that of the standard strain Arthrobacter defluvivii 4C 1-a; the colony morphology of the strain B is consistent with that of a standard strain Bacillus glucanotryticus.
And (3) identifying physiological and biochemical indexes of the strain: the methods of gram staining, catalase, methyl red, phthalein methyl methanol (V-P), amylolysis, gelatin hydrolysis, nitrate reduction and the like are referred to Berger's Manual of bacteria identification. The experimental results are shown in table 2, in which arthrobacter is a gram-positive bacterium and bacillus glucanolyticus is a gram-negative bacterium. The identification result of the strain A is consistent with that of the standard strain Arthrobacter defluvivii 4C 1-a; the identification result of the strain B is consistent with that of the standard strain Bacillus glucanotryticus.
TABLE 1 bacterial colony characteristics and culture shapes
Strain numbering | Shape of | Surface of | Transparency | Bump | Edge of a container | Colour(s) |
Strain A | Circular shape | Drying | Is not transparent | Slightly convex | Is tidy | White grey |
Strain B | Circular shape | Is smooth and smooth | Is transparent | With a projection | Is tidy | Colorless and colorless |
TABLE 2 physiological and biochemical characteristics of some functional strains
Note: "+" indicates positive, and "-" indicates negative.
Example 2: preparation of microbial growth promoter
The single colonies (strain A and strain B) of example 1 were inoculated into liquid nutrient agar culturesCulturing in the culture medium at 37 ℃ for 24h by shaking at 160 rpm. Adjusting OD of two kinds of bacteria liquid (bacterial suspension formed by suspending bacteria in liquid nutrient agar culture medium) with spectrophotometer600The value is obtained. OD600The value is reflected by the density of the bacteria suspended in the bacterial suspension, and the OD for a given volume of bacterial suspension600The value may reflect the number of bacteria. OD of bacterial suspension of Arthrobacter600The value was adjusted to 0.8, OD of the bacterial suspension of Bacillus glucanolyticus600The value is adjusted to 1, 5ml of arthrobacter bacterial suspension and 5ml of glucan-degrading bacillus bacterial suspension are mixed, and then 100ml of tap water is added for dilution, so as to obtain the microbial growth promoter A.
The microbial growth promoter B was prepared by the same method except that OD of the bacterial suspension of Arthrobacter was used600The value was adjusted to 1, OD of the bacterial suspension of Bacillus glucanolyticus600The value is adjusted to 1, 5ml of arthrobacter bacterial suspension and 5ml of glucan-degrading bacillus bacterial suspension are mixed, and then 100ml of tap water is added for dilution, so as to obtain the microbial growth promoting agent B.
The microbial growth promoter C was prepared by the same method except that OD of the bacterial suspension of Arthrobacter was used600The value was adjusted to 1.5, OD of the bacterial suspension of Bacillus glucanolyticus600The value is adjusted to 1, 5ml of arthrobacter bacterial suspension and 5ml of glucan-degrading bacillus bacterial suspension are mixed, and then 100ml of tap water is added for dilution, so as to obtain the microbial growth promoting agent C.
Example 3: preparation of organic fertilizer
In this example, microbial agents were used to prepare organic fertilizers, and commercially available standard strains, Arthrobacter defluvii (KCTC19209, for strain A) and Bacillus glucanolyticus Paenibacillus glucanilyticus (CIP107742T, for strain B), were used. Arthrobacter (KCTC 19209) and Bacillus glucanicus (CIP 107742T) are all model strains, and are recorded in Experimental example 4. Two kinds of freeze-dried bacteria (dry powder) in dry powder form need to be recovered, under the aseptic condition, a Nutrient Agar (NA) liquid culture medium is added into the freeze-dried bacteria, then a bacterial liquid is inoculated on a Nutrient Agar (NA) inclined plane for culture, and a bacterial colony grows on the inclined plane. And then picking the colony on a Nutrient Agar (NA) slant for streak culture to obtain a single colony. The recovery methods of the two bacteria are consistent.
The specific scheme for preparing the organic fertilizer comprises the following steps:
two bacterial suspensions were prepared according to the method of example 2, and the two suspensions were mixed (mixed bacterial solution) and then added to the organic fertilizer and stirred uniformly. The specific preparation method of the mixed bacterial liquid comprises the following steps: OD of bacterial suspension of Arthrobacter (using KCTC19209 instead of Strain A)600The OD of the suspension of Bacillus glucanolyticus (CIP107742T instead of strain B) was adjusted to 0.8600The value was adjusted to 1, and 5ml of the suspension of arthrobacter and 5ml of the suspension of bacillus glucanolyticus were mixed to obtain a mixed bacterial solution. The organic fertilizer (adopting commercial fertilizer meeting NY525-2012 standard, the mass fraction of organic matter is 60%, the mass fraction of nitrogen, phosphorus and potassium is 15%) and the mixed bacterial liquid are used in the following proportion: the microbial organic fertilizer A is obtained by bagging and sealing 12 g-50 ml (8-12 g-50 ml) of the bacterial liquid, and then composting for 13 days (10-15 days).
OD of bacterial suspension of Arthrobacter (using KCTC19209 instead of Strain A)600The value was adjusted to 1, OD of the suspension of Bacillus glucanolyticus (CIP107742T was used instead of Strain B)600The value was adjusted to 1, and 5ml of the suspension of arthrobacter and 5ml of the suspension of bacillus glucanolyticus were mixed to obtain a mixed bacterial solution. The mixed bacteria liquid and the organic fertilizer are uniformly mixed, and the use ratio of the organic fertilizer to the mixed bacteria liquid is as follows: and (3) packaging the organic fertilizer and the bacterial liquid into bags, sealing the bags, and composting for 15 days to obtain the microbial organic fertilizer B.
OD of bacterial suspension of Arthrobacter (using KCTC19209 instead of Strain A)600The value was adjusted to 1.5, OD of the suspension of Bacillus glucanolyticus (CIP107742T was used instead of Strain B)600The value was adjusted to 1, and 5ml of the suspension of arthrobacter and 5ml of the suspension of bacillus glucanolyticus were mixed to obtain a mixed bacterial solution. The use ratio of the organic fertilizer to the mixed bacterial liquid is as follows: organic fertilizer, namely bacterial liquid 8g and 50ml, bagging, sealing and composting 10And (5) obtaining the microbial organic fertilizer C.
Experimental example 1: growth promotion experiment of kohlrabi potted plant
(1) Soil collection: the soil to be tested is waste greenhouse soil of a certain university, the soil type is yellow soil, 0-20cm of plough layer soil on the ground surface is collected for potting experiment, all the collected soil is sieved by a 2mm sieve to remove impurities in the soil, after uniform mixing, a part of the soil is taken to be measured by a nutrient quick-measuring instrument to determine the physical and chemical properties of the soil, the measurement result is that the organic matter is 10.31g/kg, the quick-acting phosphorus is 30.32mg/kg, the quick-acting potassium is 68.19mg/kg, the ammonium nitrogen is 16.78mg/kg, the total nitrogen is 1.77g/kg, the pH is 7.59, and the soil is kept in a normal-temperature shade place for later use.
(2) Seed germination: the experimental object is kohlrabi seedling, and the kohlrabi variety is 'sweet crisp big kohlrabi' provided by the company of the old culture of the chenchen table. And (2) loading the sieved soil into a seedling tray, selecting seeds with the same size and shape, sowing 1-2 seeds in each pot, covering a layer of thin soil on the seeds, uniformly and thoroughly watering, regularly watering for waiting for the seeds to germinate, transplanting 3-4 seedlings into seedling pots with the caliber of 200mm and the height of 170mm after the seedlings grow out, and treating the seedlings in parallel for 5 times.
(3) Inoculating bacterial liquid: after 7 days of rejuvenation, the microbial growth promoting agent B prepared in example 2 (experimental group) was applied to kohlrabi roots (110ml), while the control group (CK) used an equivalent amount of tap water (110 ml). Set up two single fungus contrast groups simultaneously, do respectively:
comparative group 1: the arthrobacter single colony of example 1 was inoculated into liquid nutrient agar medium and cultured at 37 ℃ for 24 hours with shaking table at 160 rpm. Adjusting OD of the cultured strain with spectrophotometer to obtain bacterial liquid (bacterial suspension prepared by suspending bacteria in liquid nutrient agar medium)600And adjusting the value to be 1 to obtain the arthrobacter suspension. The growth promoter for Arthrobacter (110ml) was obtained by diluting 10ml of the suspension of Arthrobacter with 100ml of tap water.
Comparative group 2: the Arthrobacter in the comparative group 1 was replaced with Bacillus glucanolyticus to obtain a Bacillus glucanolyticus growth promoter (110 ml).
Each treatment was repeated 5 times, watered periodically, and its growth was recorded periodically.
(4) Plant physiological index measurement
And (4) measuring plant physiological indexes of the kohlrabi seedlings after 40 days of planting, wherein the plant growth indexes comprise plant height, plant leaf chlorophyll content and the like.
The chlorophyll content was measured by the following method:
taking fresh kohlrabi leaves, removing main veins of the kohlrabi leaves, and cutting the rest parts into pieces. Weighing 0.2g of cut fresh leaves, placing the leaves into a mortar for later use, keeping out of the sun during grinding, and adding a small amount of CaCO3Grinding the powder and quartz sand, adding a small amount of 95% ethanol, grinding into homogenate, adding 10mL of 95% ethanol, and continuously grinding until the mixture turns white. And then washing the residue on the periphery of the mortar and the pestle by using 3mL of 95% ethanol, finally pouring all the washing liquid into a 10mL centrifuge tube, centrifuging for 20min at 4000r/min, then transferring to a 50mL brown volumetric flask, shaking up and fixing the volume to obtain the solution, namely the chlorophyll extracting solution of the leaves.
And calculating the contents of chlorophyll a, chlorophyll b and total chlorophyll in the kohlrabi leaves. The absorbance was measured at 665nm, 649nm and 470m using an ultraviolet spectrophotometer with 95% ethanol added as a blank control.
Calculating the content (mg/L) of chlorophyll a and chlorophyll b and the total concentration (mg/g) of chlorophyll according to a formula.
(1) Chlorophyll a concentration: ca=13.95A665-6.88A649Formula (1)
(2) Chlorophyll b concentration: cb=24.96A649-7.32A665Formula (2)
(3) Chlorophyll content (mg/g) ═ CVN/1000m formula (3)
Wherein C is the pigment content (mg/L); v is the volume of the extract (mL); n is the dilution multiple; m is the sample mass (g); 1000 is 1000 ml.
(5) Results of the experiment
The growth promoting effect of the pot culture with only the application of the growth promoting agent of the mycobacterium is shown in the figure 1; the growth promoting effect of the pot culture only applying the glucan degrading bacillus growth promoting agent is shown in a figure 2; the growth promoting effect of potted plants by applying the microbial growth promoting agent B is shown in FIG. 3. According to the experimental results, the growth promoting effect of the bacterial strains on kohlrabi can be obviously seen in the aspects of plant height, root system density, leaf size and the like through surface observation of the microbial agents of the comparison group 1, the comparison group 2 and the experimental group.
Statistical results of chlorophyll content measurements are shown in fig. 4 (p is less than 0.05 compared with CK, and data in the form of mean ± SD), the chlorophyll content of kohlrabi applied with the bacterial liquid is significantly improved compared with CK in comparison with that of comparative group 1, comparative group 2 and experimental group, wherein comparative group 1 is significantly higher than CK in chlorophyll content of plant, so that the chlorophyll content of kohlrabi is increased by about 29%; the experimental group showed significantly higher plant chlorophyll content than CK, increasing the chlorophyll content of kohlrabi by about 54%.
Experimental example 2: growth promotion experiment of macadamia nut
The test is started in 12 months in 2019, the place is a macadimia nut nursery garden in Jiangcheng county of Yunnan province, and a small seedling of the macadimia nut which is grafted for one year in the nursery garden is selected as a test material. The control group was added with tap water of equal volume, 10 treatments were set for each treatment in parallel, the bacterial solution (four supply reagents, see experimental groups for details) was applied once a month for 4 months continuously, and the growth was recorded by regular observation.
Experimental grouping conditions were:
experimental groups: the microbial growth promoting agent B prepared in example 2 was applied to 400ml of macadamia nut plantlets each time.
Comparative group 1: a single colony of Mycobacterium of example 1 was inoculated into a liquid nutrient agar medium and cultured at 37 ℃ for 24 hours with shaking 160 rpm. Adjusting OD of the cultured strain with spectrophotometer to obtain bacterial liquid (bacterial suspension prepared by suspending bacteria in liquid nutrient agar medium)600And adjusting the value to be 1 to obtain the arthrobacter suspension. The growth promoter for the arthrobacter is obtained by adding 100ml of tap water into 10ml of the bacterial suspension of the arthrobacter to dilute. 400ml of each macadamia nut small seedling (seedling) is applied.
Comparative group 2: replacing arthrobacter in the comparison group 1 with bacillus glucanolysis to obtain the bacillus glucanolysis growth promoter. 400ml of the macadamia nut plantlet is applied to each macadamia nut plantlet.
And (3) CK group: tap water was applied in an amount of 400ml per small seedling of macadamia ternifolia.
The physical and chemical properties of the soil are measured by a nutrient quick-measuring instrument, and the measurement results are that the organic matter is 7.85g/kg, the quick-acting phosphorus is 28.39mg/kg, the quick-acting potassium is 50.79mg/kg, the ammonium nitrogen is 17.81mg/kg, and the total nitrogen is 0.43 g/kg. The data obtained from the measurements were subjected to a one-way significance of variance test using SPSS20.0 software and graphed Prism 8.0.2.
The results are shown in FIGS. 5 and 6 (p < 0.05; p < 0.01; data in mean + -SD form) for CK, FIG. 5 shows the comparison of the heights of macadamia nuts, and FIG. 6 shows the comparison of the chest diameters of macadamia nuts. According to the experimental result, the increase of the plant height and the breast diameter of the macadimia nut is not obviously promoted by using the arthrobacter or the bacillus amyloliquefaciens alone, but the plant height and the breast diameter of the macadimia nut seedlings can be obviously improved after the microbial inoculum of the scheme is applied, so that the growth of the macadimia nut trees can be promoted by the two bacteria through synergistic action.
In addition to the experiments using strain a and strain B, in this case, microbial growth promoter B 'was prepared using standard strains KCTC19209 and KCTC19209 (experimental group') in a manner similar to microbial growth promoter B except that strain a was replaced with KCTC19209 and strain B was replaced with CIP 107742T. And an arthrobacter growth promoter (KCTC19209 for strain a, control 1 ') was prepared according to the method of control 1, and a glucan-containing bacillus growth promoter (CIP107742T for strain B, control 2 ') was prepared according to the method of control 2, and a blank CK ' (same CK) was set. According to the method of the experimental example, the macadimia nut growth promoting experiment is repeated, namely the experimental process is the same, and only the growth promoting agent to be tested is replaced, so that the effects of the strain A and the strain B are consistent with those of the standard strains of the strain A and the strain B. Although the identity of strain A with KCTC19209 and strain B with CIP107742T in the genetic material has been confirmed by Experimental example 4, this experiment demonstrates their functional identity. The experimental results are shown in table 3 (p is less than 0.05 compared with CK'), and the experiment proves that the standard strain also has a synergistic experimental phenomenon, and the two strains act together to promote the growth of the macadamia nut seedlings.
Table 3: results of macadamia nut growth promoting experiments
Experimental example 3
The experimental protocol of this example is substantially the same as that of example 2, except that microbial organic fertilizers A-C are used to replace microbial agents (experiment groups A, B and C, respectively; a CK group is also provided to which the common organic fertilizer of example 3 is applied and which is watered; and the reagent of the comparison group is the reagent used in the experiment group of example 2). Since the fertilizer is solid, the fertilizing method is different from the embodiment 2, a ring-shaped pit is dug around the soil for planting the macadamia nut saplings, pit application is carried out (microbial organic fertilizer is put into the pit, and then soil is backfilled), and a proper amount of water is poured after the fertilization is finished. The amount of fertilizer applied was 100g per small macadamia nut seedling, 10 treatments were performed in parallel, and the application was as shown in table 3. The microbial organic fertilizer is applied to the experimental group A, the experimental group B and the experimental group C once in 2019 for 12 months, and then the microbial growth promoting agents A-C (application amount is 400ml) in the example 1 are respectively applied to the experimental group A, the experimental group B and the experimental group C for 3 months continuously, and the growth condition of the microbial growth promoting agents is recorded by regular observation once per month. The plant height and the breast height of the small saplings are measured, the experimental results are shown in table 4, and the microbial organic fertilizer prepared by the method has a good growth promoting effect on the macadimia nut small saplings, and is superior to that of the microbial growth promoter in the embodiment 1 which is used alone.
Table 4: promoting effect of microbial organic fertilizer on plant height and breast diameter (mean plus or minus SD)
Experimental example 4: soil strain isolation test
The research separates the dominant bacteria from various types of soil and analyzes the tailing soil of phosphate fertilizer plantSoil, potash fertilizer plant tailing soil, macadamia nut planting land soil and other soil samples. The method for separating the strains from the soil sample comprises the following specific steps: taking 10g of a soil sample, putting the soil sample into a conical flask containing 90mL of physiological saline, shaking the conical flask at 160rpm for 30min to ensure that the soil sample is completely and uniformly mixed with the sterile physiological saline, and standing the conical flask for 10min for later use. Then sucking 1mL of soil supernatant suspension on a superclean bench by a liquid moving machine, adding the soil supernatant suspension into a glass test tube containing 9 mL0.85% physiological saline, fully and uniformly mixing to prepare a solution with the dilution of 10-2, sequentially diluting the soil sample in a gradient manner according to the same method, and respectively diluting the soil sample solution to 10-4,10-5,10-6The cells were spread on selective medium plates (3 dishes per gradient) using an applicator, sealed, inverted and incubated in a 37 ℃ incubator until colonies (approximately 5-7 days available) grew on the plates, and single colonies were obtained. And screening multiple aspects of phosphate and potassium dissolution, biocontrol effects, synergistic effects and the like of hundreds of single colonies obtained by separation to finally obtain two strains (A and B) with synergistic effects, performing species identification on the strains A and B respectively, and further researching the growth promotion effect of the two strains.
The isolated strains were activated on nutrient agar plates. Inoculating to nutrient agar liquid culture medium, performing amplification culture to logarithmic phase, and centrifuging; collecting thallus as DNA extraction material, extracting DNA with biological reagent kit, and extracting genome DNA of each strain of bacteria. Detecting with 1.0% agarose, and reserving after qualification. The 16S rDNA fragment is used as a PCR amplification template and is conditionally amplified according to a PCR amplification system. The PCR reaction system is shown in Table 5.
Table 5: PCR reaction System (50. mu.L)
Reagent | Volume of |
2×Taq PCR Master Mix | 25μL |
27F(10μmol/L)(5'-AGAGTTTGATCCTGGCTCAG-3',SEQ ID NO:1) | 1μL |
1492R(10μmol/L)(5'-GGTTACCTTGTTACGACTT-3',SEQ ID NO:2) | 1μL |
DNA template | 3μL |
ddH2O | 20μL |
PCR amplification conditions: 3min at 94 ℃; 94 ℃ 30s, 52 ℃ 30s, 72 ℃ 45s (35 cycles); 10min at 72 ℃. And (3) detecting the PCR product by using 1.0% agarose gel, and sending the PCR product to Shanghai biological engineering for sequencing after the PCR product is qualified. And splicing the sequencing result by using DNAMAN6.0, and then performing homologous sequence comparison on an NCBI website, and selecting a sequence with the highest homologous comparison result with the sequencing strain. The homology of the identified strain A and a standard strain Arthrobacter defluvii 4C1-a reaches more than 99 percent, and the information of the standard strain is as follows: korea Collection for Type Cultures (Korean Type culture Collection) deposit number: KCTC19209, reference: kim, k.k.; arthrobacter defluviii sp.nov.,4-chlorophenol-degrading bacterial isolated from sewage; JOURNAL int.J.Syst.Evol.Microbiol.58(PT 8),1916-1921 (2008); 16S rRNA sequence Genebank number: AM 409361.1. The homology of the strain B and a standard strain Bacillus glucanolyticus Paenibacillus is more than 99 percent, and the information of the standard strain is as follows: CRBIP number CIP107742T (Pasteur institute culture Collection).
The foregoing is merely an example of the present invention and common general knowledge in the art of designing and/or characterizing particular aspects and/or features is not described in any greater detail herein. It should be noted that, for those skilled in the art, without departing from the technical solution of the present invention, several variations and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
SEQUENCE LISTING
<110> southwest university of forestry
<120> a microbial growth promoter and use thereof
<130>2020.7.28
<160>2
<170>PatentIn version 3.5
<210>1
<211>20
<212>DNA
<213> Artificial sequence
<400>1
<210>2
<211>19
<212>DNA
<213> Artificial sequence
<400>2
ggttaccttg ttacgactt 19
Claims (10)
1. A microbial growth promoting agent comprising Arthrobacter and Bacillus amyloliquefaciens.
2. A growth promoting microorganism as claimed in claim 1, wherein: the ratio of the number of bacteria of arthrobacter to the number of bacteria of bacillus glucanicus is 0.8:1-1.5: 1.
3. A growth promoting microorganism as claimed in claim 2, wherein: the ratio of the number of bacteria of arthrobacter to the number of bacteria of bacillus glucanotrys is 1: 1.
4. A growth promoting microorganism as claimed in claim 3, wherein: the microbial growth promoter is prepared by the following method: obtaining bacterial suspension of arthrobacter and bacterial suspension of bacillus glucanotrys; and mixing the bacterial suspension of the mycobacterium and the bacterial suspension of the bacillus glucanotus to obtain a mixed bacterial liquid, and diluting with water to obtain the microbial growth promoter.
5. A growth promoting agent for microorganisms according to claim 4, wherein: OD of bacterial suspension of Arthrobacter600The value is 0.8-1.5, OD of bacterial suspension of bacillus glucanolyticus600The value is 1.
6. A growth promoting agent for microorganisms according to claim 5, wherein: and mixing the bacterial suspension of arthrobacter and the bacterial suspension of bacillus glucanotus in a volume ratio of 1:1 to obtain a mixed bacterial liquid.
7. A growth promoting microorganism in accordance with claim 6, wherein: each 10ml of the mixed bacterial solution was diluted with 100ml of water.
8. The use of a microbial growth promoting agent according to claim 7 in macadamia nut planting.
9. Use according to claim 8, characterized in that: the microbial growth promoting agent is applied to macadamia nut seedlings once a month.
10. Use according to claim 8, characterized in that: and mixing the mixed bacterial liquid with an organic fertilizer, and performing anaerobic fermentation to obtain the microbial organic fertilizer.
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