CN111826319B - Microbial growth promoter and application thereof - Google Patents
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- CN111826319B CN111826319B CN202010756994.5A CN202010756994A CN111826319B CN 111826319 B CN111826319 B CN 111826319B CN 202010756994 A CN202010756994 A CN 202010756994A CN 111826319 B CN111826319 B CN 111826319B
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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
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- A01N63/25—Paenibacillus
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- C—CHEMISTRY; METALLURGY
- 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 promoting agent 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 defuzvii) and Bacillus glucanicus (Paenibacillus glucanilinicus) can generate synergistic effect, and improve the utilization rate of plants on phosphorus and potassium 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 Arthrobacter (Arthrobacter defluvivii) used in the present scheme may be a standard strain Arthrobacter defluvii 4C1-a (KCTC 19209, korean type culture Collection); the Bacillus glucanolyticus (Paenibacillus glucanilyticus) used in the scheme can be a standard strain Bacillus glucanilyticus (CRBIP number: CIP107742T, pasteur institute strain collection center).
The inventor separates and obtains various functional strains from phosphate fertilizer factory tailing soil, potassium fertilizer factory tailing soil 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 promoting agent is widely applied to vegetables, food crops and the like, but the microbial growth promoting agent has a less obvious growth promoting effect on trees with a long time period, such as fruit trees and the like, so that the microbial growth promoting agent is less applied to the plantation of macadimia nuts. 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 glucanotrys is 0.8.
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 glucanotrys is 1.
By adopting the technical scheme, the proportion of 1.
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 Arthrobacter 600 The value is 0.8-1.5, OD of bacterial suspension of bacillus glucanolyticus 600 The 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 the conventional quantitative method in the prior art.
Further, the bacterial suspension of arthrobacter and the bacterial suspension of bacillus glucanotrys were mixed at a volume ratio of 1.
By adopting the technical scheme, the ratio of the arthrobacter to the bacillus glucanicus is 1, and the synergistic enhancement effect of the arthrobacter and the bacillus glucanicus 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.
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 bacteria 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 material of Experimental example 1 of the present invention (Bacillus amyloliquefaciens).
FIG. 3 is a graph showing the growth promoting effect of the potting product of Experimental example 1 of the present invention (microbial growth promoting agent B).
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 a standard strain Arthrobacter defluviii 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 test methods of gram staining, catalase, methyl red, phthalein methyl methanol (V-P), amylolysis, gelatin hydrolysis, nitrate reduction and the like refer to Bergey Manual of bacteria identification. The results of the experiments are shown in Table 2, wherein 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 of the coating | Bump | (Edge) | Colour(s) |
| Strain A | Circular shape | Drying | Is not transparent | Slightly convex | Is tidy | White grey |
| Strain B | Circular shape | Smooth and smooth | Is transparent | With a projection | Is neat | 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 medium and incubated at 37 ℃ for 24 hours with shaking at 160 rpm. Adjusting the cultured strain with spectrophotometerOD of inoculum solution (suspension formed by suspending bacteria using liquid nutrient agar medium) 600 The value is obtained. OD 600 The value is reflected by the density of the bacteria suspended in the bacterial suspension, and the OD for a given volume of bacterial suspension 600 The value may reflect the number of bacteria. OD of bacterial suspension of Arthrobacter 600 The value was adjusted to 0.8, OD of the bacterial suspension of Bacillus glucanolyticus 600 The 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 used 600 Value adjusted to 1, OD of bacterial suspension of Bacillus glucanolyticus 600 The 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 promoting agent C was prepared by the same method except that OD of the bacterial suspension of Arthrobacter was used 600 The value was adjusted to 1.5, OD of the bacterial suspension of Bacillus glucanolyticus 600 The 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, the microbial preparation was used to prepare organic fertilizer, and the commercial standard strains Arthrobacter defluvii (KCTC 19209, representing strain A) and Bacillus gluconobacter Paenibacillus glucanolyticus (CIP 107742T, representing strain B) were used. Arthrobacter (KCTC 19209) and Bacillus glucanotrys (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 revived, under the aseptic condition, nutrient Agar (NA) liquid culture medium is added into the freeze-dried bacteria, then the bacteria liquid is inoculated on the Nutrient Agar (NA) inclined plane for culture, and bacterial colony is grown on the inclined plane. And then picking colonies 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 added to the organic fertilizer and stirred. 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) 600 The value was adjusted to 0.8, OD of the suspension of Bacillus glucanolyticus (CIP 107742T instead of Strain B) 600 The 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: bacterial suspension =12g (variable between (8-12) g:50 ml), and after bagging and sealing, composting was carried out for 13 days (variable between 10-15 days) to obtain microbial organic fertilizer a.
OD of bacterial suspension of Arthrobacter (using KCTC19209 instead of Strain A) 600 The value was adjusted to 1, OD of the suspension of Bacillus glucanolyticus (CIP 107742T was used instead of Strain B) 600 The 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: bacterial liquid =10g, bagging, sealing, and composting for 15 days to obtain microbial organic fertilizer B.
OD of bacterial suspension of Arthrobacter (using KCTC19209 instead of Strain A) 600 The value was adjusted to 1.5, OD of the suspension of Bacillus glucanolyticus (CIP 107742T was used instead of strain B) 600 The 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 bacterial liquid =8g 50ml, bagging, sealing, and composting for 10 days to obtain 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 (3) filling the sieved soil into a seedling tray, selecting seeds with the same size and shape, sprinkling 1-2 seeds in each pot, covering a thin layer of 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 (110 ml), 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 is inoculated into liquid nutrient agar medium and cultured for 24h at 37 ℃ by 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) 600 And adjusting the value to be 1 to obtain the arthrobacter suspension. The growth promoter for Arthrobacter (110 ml) 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 is 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 CaCO 3 Grinding 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 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 leaf chlorophyll extracting solution.
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: c a ＝13.95A 665 -6.88A 649 Formula (1)
(2) Chlorophyll b concentration: c b ＝24.96A 649 -7.32A 665 Formula (2)
(3) Chlorophyll content (mg/g) = CVN/1000m equation (3)
Wherein C is pigment content (mg/L); v is the volume of the extract (mL); n is the dilution multiple; m is sample mass (g); 1000 is 1000ml.
(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 applied with only the glucan bacillus degrading 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.
The statistical results of the chlorophyll content measurements are shown in fig. 4 (p < 0.05 as compared to CK, data in the form of mean ± SD), the chlorophyll content of kohlrabi applied with the bacterial solution was significantly increased over CK in comparison to comparative group 1, comparative group 2 and experimental group, wherein comparative group 1 was significantly higher than CK in phytoalexin content, resulting in an increase of about 29% in the chlorophyll content of kohlrabi; 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. Tap water with the same volume is added into a control group, 10 bacteria liquid (four supply reagents, see the experimental grouping for details) are applied once per month in parallel for each treatment, the bacteria liquid is continuously applied for 4 months, and the growth condition of the bacteria liquid is regularly observed and recorded.
Experimental grouping conditions were:
experimental groups: the microbial growth promoting agent B prepared in example 2 was applied to 400ml of macadamia nut seedlings 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 solution (bacterial suspension prepared by suspending bacteria in liquid nutrient agar medium) 600 Adjusting the value to 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 the arthrobacter in the comparison group 1 with the bacillus glucans to obtain the bacillus glucans degradation promoter. 400ml of each macadamia nut small seedling is applied.
And (3) CK group: tap water, 400ml each time per macadamia nut plantlet.
The physical and chemical properties of the soil are measured by a nutrient rapid measuring instrument, and the measurement result is 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 0.43g/kg. The data obtained from the measurements were subjected to a one-way significance of variance test using SPSS 20.0 software and graphed Prism 8.0.2. Histogram.
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 (KCTC 19209 for strain a, comparative 1 ') was prepared according to the method of comparative 1, and a glucan-containing bacillus growth promoter (CIP 107742T for strain B, comparative 2 ') was prepared according to the method of comparative 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 detected is replaced, so as to prove that the effects of the strain A and the strain B are consistent with the effects of the standard strains of the strain A and the strain B. Although the consistency of strain A with KCTC19209 and strain B with CIP107742T in genetic material has been confirmed by experimental example 4, the present experiment demonstrates their functional consistency. 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: growth promoting experimental result of macadamia nut
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 fertilization method is different from the method in example 2, a ring-shaped pit is dug around the soil for planting the macadimia nut small sapling, 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 fertilization is finished. The amount of fertilizer applied was 100g per macadamia nut plantlet, 10 fertilizer applications were performed in parallel for each treatment, and the fertilizer 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 400 ml) 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 soils, analyzes the tailing soil and potassium of phosphate fertilizer plantAnd a plurality of soil samples such as tailing soil of a fertilizer plant, soil of a macadamia nut planting field and the like. 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 normal saline, shaking the conical flask for 30min at 160rpm to ensure that the soil sample is completely mixed with the sterile normal saline, and standing the conical flask for 10min for later use. Then sucking 1mL of soil supernatant suspension on a clean bench by a liquid-moving machine, adding the soil supernatant suspension into a glass test tube containing 9mL0.85% normal saline, fully and uniformly mixing to prepare a solution with the dilution of 10-2, sequentially and gradiently diluting the soil sample according to the same method, and respectively diluting the soil sample solution to 10 -4 ，10 -5 ，10 -6 The 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 subjected to conditional amplification 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 |
| ddH 2 O | 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 defluvii sp.nov.,4-chlorophenol-degrading bacteriologically isolated from sewage; JOURNAL int.J.Syst.Evol.Microbiol.58 (PT 8), 1916-1921 (2008); 16S rRNA sequence Genebank No.: AM409361.1. 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 above description is only an example of the present invention, and the general knowledge of the known specific technical solutions and/or characteristics and the like in the solutions is not described herein too much. 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, and these should also be considered as the protection scope of the present invention, which 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 promoting agent 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 promoter characterized by comprising Arthrobacter (Arthrobacter)Arthrobacterdefluvii) And Bacillus saccharified (Paenibacillusglycanilyticus) (ii) a Accession number of ArthrobacterIs KCTC 19209; the Bacillus depolymerizing polysaccharide is CIP 107742T.
2. A microbial growth promoting agent according to claim 1, wherein: the ratio of the number of bacteria of Arthrobacter to Paenibacillus amyloliquefaciens is 0.8.
3. A growth promoting microorganism as claimed in claim 2, wherein: the ratio of the number of bacteria of Arthrobacter to Paenibacillus glycan was 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 depolymerized saccharide bacillus; and mixing the bacterial suspension of the arthrobacter and the bacterial suspension of the depolymerized saccharide bacillus to obtain mixed bacterial liquid, and diluting with water to obtain the microbial growth promoter.
5. A growth promoting microorganism according to claim 4, wherein: OD of bacterial suspension of Arthrobacter 600 Value of 0.8-1.5, OD of the bacterial suspension of the depolymerized Bacillus saccharideans 600 The value is 1.
6. A growth promoting agent for microorganisms according to claim 5, wherein: the suspension of arthrobacter and the suspension of bacillus depolymerized saccharide were mixed at a volume ratio of 1.
7. A microbial growth promoting agent according to claim 6, wherein: each 10ml of the mixed bacterial suspension was diluted with 100ml of water.
8. The use of a microbial growth promoting agent of claim 7 for promoting the growth of macadamia nut trees.
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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