CN111134142B - Method for improving yield of dioscorea opposita by using bacillus subtilis - Google Patents

Method for improving yield of dioscorea opposita by using bacillus subtilis Download PDF

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CN111134142B
CN111134142B CN202010058101.XA CN202010058101A CN111134142B CN 111134142 B CN111134142 B CN 111134142B CN 202010058101 A CN202010058101 A CN 202010058101A CN 111134142 B CN111134142 B CN 111134142B
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bacillus subtilis
dioscorea opposita
yield
liquid
compound composition
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CN111134142A (en
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张爱中
姜俊玲
薛宝
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Henan Zhongwei Chunyu Plant Nutrition Co ltd
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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
    • 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
    • A01N31/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic oxygen or sulfur compounds
    • A01N31/08—Oxygen or sulfur directly attached to an aromatic ring system
    • A01N31/16—Oxygen or sulfur directly attached to an aromatic ring system with two or more oxygen or sulfur atoms directly attached to the same aromatic ring system
    • 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
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus

Abstract

The application belongs to the technical field of crop cultivation, and particularly relates to a method and a patent application for improving the yield of Chinese yam by using bacillus subtilis. The microbial agent compound composition for improving the yield of the dioscorea opposita comprises the components of bacillus subtilis liquid and sodium guaiacolate; the bacillus subtilis is a bacillus subtilis ZWCA02 strain, is preserved in China general microbiological culture Collection center in 6 months and 3 days in 2019, and has the preservation number as follows: CGMCC No. 17889. According to the application, a microbial agent compound composition specially used for improving the yield of the dioscorea opposita is prepared by screening and applying related microbial strains. Preliminary experiment results show that the yield of the dioscorea opposita thumb can be effectively improved, the quality of the dioscorea opposita thumb is further improved, the continuous cropping effect of crops is improved, the effect of the compound composition of the microbial inoculum is lasting, and the micro-ecological environment of soil can be continuously improved.

Description

Method for improving yield of dioscorea opposita by using bacillus subtilis
Technical Field
The application belongs to the technical field of crop cultivation, and particularly relates to a method and a patent application for improving the yield of Chinese yam by using bacillus subtilis.
Background
In the existing crop cultivation process, due to the application of a large amount of artificial compositions such as pesticides, chemical fertilizers and the like, serious negative effects are caused, for example, the quality of crops is obviously reduced, meanwhile, the growth of the crops is in a sub-health state, and the phenomena of diseased roots, diseased seedlings, diseased stems, diseased ears and the like are common; on the other hand, the use of a large amount of pesticides and chemical fertilizers has serious negative effects on the ecological environment, and the problems of soil hardening, acidification, organic matter reduction, fertilizer efficiency reduction and the like are increasingly serious. Therefore, it is imperative to update and improve agricultural cultivation methods.
In the prior art, microbial agents with various nutrient elements are matched, and the microbial agents have the advantages of environmental protection, no pollution, lasting action effect and the like, so that the microbial agents have better application prospects in cultivation of various crops such as part of field crops, fruit trees, vegetables and the like. And because part of the microbial agents have the technical effects of inhibiting pathogenic bacteria, enhancing the disease resistance of plants and the like, the application of the microbial agents has very important technical significance for reducing the using amount of pesticides and fertilizers.
Huai Yam (rhizoma Dioscoreae)Dioscorea oppositaThunb.) is a perennial twining herbaceous vine of Dioscoreaceae, Dioscorea, which is one of the four famous dioscorea opposita (rhizoma dioscoreae opposita (L.) zel of the four Huai nationality: (D. Opposita cv. tiegun) Is the best product of Chinese yam. The dioscorea opposita has wide growth distribution, but the Tiegun dioscorea opposita which is both excellent in medicine and food is mainly produced in Wen county and Wu\38495in Henan province. The Chinese yam has the characteristics of rich nutrition, high protein content and low sugar content, and has various effects of reducing blood sugar, reducing blood fat, strengthening spleen, tonifying lung and the like, so the Chinese yam belongs to one of the first foods published in China and having strong edible and medicinal functions, and is widely concerned and loved by people. In order to meet the increasing demand of people on increasing production, it is obviously necessary to make intensive research on how to improve the planting yield of dioscorea opposita.
Disclosure of Invention
The application aims to provide a method for improving the yield of Chinese yam, and in order to achieve the purpose, the application provides a microbial agent compound composition which is based on the compound composition to achieve the purpose of improving the yield of Chinese yam.
The technical solution adopted in the present application is detailed as follows.
A microbial inoculum compound composition for improving the yield of Chinese yam is in a liquid form, and comprises the components of Bacillus subtilis ZWCA02 bacterial liquid and sodium guaiacol; in terms of mass-to-volume ratio,
bacillus subtilis ZWCA02 bacterial liquid: sodium guaiacol =1000 mL: 0.1 g;
the Bacillus subtilis ZWCA02 bacterial liquid is Bacillus subtilis ZWCA02 strain fermentation liquid, and bacterial liquid OD600The value is 0.3 to 0.7; the effective viable count of the final microbial agent compound composition finished product is more than or equal to 5 multiplied by 108cfu/ml；
The bacillus subtilis ZWCA02 strain is preserved in China general microbiological culture Collection center in 6.3.2019, and the preservation numbers are as follows: CGMCC No.17889, the preservation address is: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North;
the guaiacol sodium is nitroguaiacol sodium, guaiacol is a synthetic plant growth regulator based on natural components, CAS # is 67233-85-6, chemical name is 2-methoxy-5-nitrophenol sodium, and molecular formula is C7H6NO4Na, having the English designation Sodium 5-nitroguaiacolate or 2-Methoxy-5-nitrophenol Sodium Salt; the content of the active ingredient of the guaiacol sodium adopted in the application is calculated by 98%.
The preparation method of the microbial agent compound composition for improving the yield of the dioscorea opposita comprises the following steps:
(1) preparation of Bacillus subtilis ZWCA02 bacterial liquid
Fermenting to OD by liquid culture with Bacillus subtilis ZWCA02 strain as fermentation strain600The value is 0.3-0.7 for standby;
(2) mixed preparation of microbial inoculum compound composition
And (2) adding guaiacol sodium into the bacterial liquid prepared in the step (1), and uniformly mixing to obtain the microbial agent compound composition provided by the application.
The microbial agent compound composition for improving the yield of the dioscorea opposita is applied to dioscorea opposita cultivation, and is used for improving the yield of the dioscorea opposita, and when the microbial agent compound composition is applied to soil at the root of seedlings, the amount of each seedling is 1-3 g in the seedling stage of the dioscorea opposita.
The bacillus subtilis ZWCA02 strain used in the application is separated from natural soil in natural environment. The preliminary study proves that the strain can induce plants to generate growth hormone after infecting the roots of the plants, change the relevant physiological and biochemical characteristics and genetic performance of the plants, and show that a large number of adventitious roots are generated at the roots, the plants grow rapidly, the growth quantity is increased, and therefore the nutrient absorption efficiency and the growth and development capacity of the plants are improved.
The guaiacol sodium (5-nitroguaiacol sodium and 2-methoxy-5-nitrophenol sodium) is a growth regulator and a cell activator, has strong osmosis, can activate bacillus subtilis microbial cells, and promotes protoplasm flow. Is often used for regulating the growth and development of plants, promoting the absorption of nutrients by animals, and enhancing the resistance and immunity of animals, plants and microorganisms to the environment.
The Chinese yam is used as a rhizome crop, the vigorous degree and the final yield of the Chinese yam in the whole growth period depend on the root system development condition of the Chinese yam to a great extent, so that the promotion of the growth of the root system of the Chinese yam is also the key for improving the yield of the Chinese yam. Based on the above, the specific bacillus subtilis ZWCA02 strain and guaiacol sodium are used as main regulators, and humic acid with a good soil structure improvement effect is compounded, so that the root growth of the dioscorea opposita thumb can be remarkably stimulated, the nutrient absorption efficiency and transportation and transformation of the dioscorea opposita thumb are improved, the growth and development of the dioscorea opposita thumb are promoted, and the aim of improving the yield of the dioscorea opposita thumb is fulfilled. Further, the result of the quality measurement of the dioscorea opposita thunb shows that the quality of the dioscorea opposita thunb is further improved after the microbial agent compound composition provided by the application is adopted.
In a word, the microbial agent compound composition specially used for improving the yield of the dioscorea opposita is prepared by screening and applying related microbial strains and matching with related production process technology and production system optimization. Preliminary experiment results show that after the composition is applied, the yield of the dioscorea opposita thumb can be effectively improved, the quality of the dioscorea opposita thumb can be further improved, the continuous cropping effect of crops can be improved, the effect of the microbial agent compounded composition is lasting, and the soil micro-ecological environment can be continuously improved, so that the composition has good practical value and popularization and application significance.
Drawings
FIG. 1 colony shape and color of Bacillus subtilis ZWCA02 in solid medium, wherein A is the result of 24h growth in 28 ℃ incubator; b is the result of the growth in an incubator at 28 ℃ for 72 hours;
FIG. 2 shows the statistical results of the yields of different experimental groups.
Detailed Description
The present application is further illustrated by the following examples.
Example 1
It should be first described that,the application relates to a specific beneficial microorganism strain bacillus subtilis (Bacillus subtilis)Bacillus subtilis) The experimental use of ZWCA02 strain, therefore, this example first briefly describes the process of obtaining this strain by screening as follows.
In a test base used by the applicant (a base set by Wei chemical engineering Co., Ltd. in Henan, the applicant in the New Henan, the New plain), the inventor finds that the hardening condition of part of soil in the same plot is relatively lighter when being crossed with the hardening condition of other plots, and the crop yield is relatively higher.
A dilution plate method is adopted, soil samples collected from soil of a test base are dissolved with sterile water indoors, supernatant is sequentially diluted and coated on a plate, single bacteria are selected for plate scribing purification, primary screening and secondary screening are gradually carried out, so that dominant strains are selected and identified. In the screening and culturing process of the strains, a YEPD yeast extract peptone glucose medium (the YEPD plate medium is prepared by dissolving 10g of yeast extract and 20g of peptone, adding 20g of agar powder, sterilizing at 121 ℃ for 20min, adding 100ml of 20% sterile glucose solution, pouring 10-15ml of the medium into each culture dish, cooling and solidifying, and inoculating the strains) is adopted for culturing at 28 ℃. The growth of 24h and 72h cultures is shown in panel A and B of FIG. 1, respectively.
The results of further physicochemical property determinations are summarized in the following table:
Figure DEST_PATH_IMAGE001
。
by combining the results of the morphology, the physicochemical properties and the like of the related colonies, the inventor identifies that the method is as follows: bacillus subtilis, therefore this strain was named: bacillus subtilis ZWCA 02. In view of the possible soil improvement application and crop yield increase application of the strain, the inventors further preserved it with microorganisms, that is,the inventor uses Bacillus subtilis in 2019 in 6 months and 3 days (Bacillus subtilis) The ZWCA02 strain is preserved in China general microbiological culture Collection center, and the preservation number is: CGMCC No.17889, the preservation address is: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
Example 2
Based on the strain screened in example 1, the inventors further fermented it to prepare a fermentation liquid for use, and this example is briefly described below with respect to a specific fermentation process.
Selecting a single bacillus subtilis ZWCA02 colony preserved and cultured on a solid plate, inoculating the colony into 5ml of liquid LB culture medium, and performing shaking culture on a constant temperature shaking table at the temperature of 28 ℃ and the rotating speed of 200 r/min for 18h to serve as seed liquid;
the LB culture medium is prepared by adding 5g yeast extract, 10g tryptone and 10g NaCl into 950 ml deionized water, and shaking until the solute is completely dissolved; then adjusting the pH value to 7.0 by using 5mol/L NaOH, and fixing the volume to one liter by using deionized water; finally steam sterilizing at 15psi for 21 min;
then, according to the inoculation ratio of 1% volume ratio, transferring the seed liquid after the shake flask culture into a fermentation culture medium, culturing at 28 ℃ under the aerobic condition with the ventilation ratio of 1:1 and 180 r/min to OD600The value is about 0.6 for standby.
The fermentation medium contains the following components in each kilogram of the medium:
corn flour 13g, glucose 5g, bean flour 20g, calcium carbonate CaCO37g of ammonium sulfate (NH)4)2SO41g of potassium phosphate K2HPO4 0.3g, KCl 0.4g, magnesium sulfate MgSO4·7H2O0.2 g, manganese sulfate MnSO4·H20.2g of O; the pH of the culture medium is = 7.0-7.2.
Example 3
On the basis of fermentation liquor of example 2, the inventor further prepares a microbial agent compound composition for improving the yield of the dioscorea opposita. The preparation method comprises the following steps: and (3) adding guaiacol sodium into the bacterial liquid prepared in the example 2, and uniformly mixing to obtain the microbial agent compound composition provided by the application.
It should be noted that the guaiacol sodium used in this example is a nitroguaiacol sodium product from high-tech chemical ltd, wei, and the purity is 98%.
In terms of specific dosage, the weight-volume ratio is as follows:
bacillus subtilis ZWCA02 bacterial liquid: sodium guaiacol =1000 ml: 0.1 g;
the effective viable count of the final microbial agent compound composition finished product is 5 multiplied by 108About cfu/ml.
In order to determine the specific application effect of the microbial agent compound composition, the inventor carries out further planting experiments, and the specific experimental process is briefly described as follows.
Planting place:
the planting site is located in a yam planting base in the Lulol land of Jozuo city, Wen do, Henan province, and belongs to the main production area of the Chinese yam, and the planting land is a yam planting base for continuous cropping and repeated cropping for three years;
selecting a new iron second variety of the dioscorea opposita;
before planting, the average content of organic matters in soil of a planting plot is determined to be 6.5/kg, the content of alkaline hydrolysis nitrogen is determined to be 25.3 mg/kg, the content of quick-acting phosphorus is determined to be 7.1 mg/kg, the content of quick-acting potassium is determined to be 59.8 mg/kg, and the total microbial content is determined to be 3 multiplied by 105/g。
Experimental setup:
during planting, healthy yam seedlings are selected, 200 yam seedlings are processed each time, 4 treatments are respectively arranged, and each treatment time is 20m24 repetitions:
experimental group 1: bacillus subtilis liquid + guaiacol (Bacillus subtilis ZWCA02 liquid 1000ml, guaiacol sodium 0.1 g);
experimental group 2: bacillus subtilis liquid (Bacillus subtilis ZWCA02 liquid 1000 ml);
experimental group 3: guaiacol (0.1 g sodium guaiacol/1L aqueous solution).
Blank control group: and (4) clear water.
The specific application mode is that the dosage of each seedling is 1.2ml (the planting density is 10 plants/m)2），And uniformly applying the fertilizer to a soil plough layer around the seedlings.
And (5) normal water and fertilizer management is carried out until the Chinese yam is harvested.
After the yam is planted, the growth condition of the yam root system is observed, and the result shows that the underground root system of the experiment groups (experiment group 1, experiment group 2 and experiment group 3) adopting the bacterial liquid is generally developed and is superior to that of the control group not adopting the bacterial liquid.
And (5) counting the yam yield condition of each experimental cell. The results are shown in the following table (yields in Kg per hectare):
Figure 661038DEST_PATH_IMAGE002
for ease of comparison, further drawings are shown in FIG. 2.
As can be seen from the comparison of fig. 2 and the results in the above table, both the guaiacol and the specific bacillus subtilis ZWCA02 bacterial liquid exhibit a certain yield increase effect, but after the two bacterial liquids are compounded and applied, the two bacterial liquids further exhibit a certain synergistic effect (it should be noted that, in the actual experiment process, the total yield does not have a significant difference under the condition of different dosages after compounding (1.2 ml, 2.4ml and 3ml are respectively tested), so the above experimental data is not shown), the yield of dioscorea opposita can be further increased, and a better application prospect is exhibited.

Claims (3)

1. A microbial inoculum compound composition for improving the yield of Chinese yam is characterized in that the composition is in a liquid form, and the components of the composition consist of bacillus subtilis liquid and guaiacol sodium;
the bacillus subtilis is a bacillus subtilis ZWCA02 strain, has been preserved in China general microbiological culture Collection center in 6 months and 3 days in 2019, and has the preservation numbers as follows: CGMCC No.17889, the preservation address is: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North;
in terms of mass-to-volume ratio,
b, bacillus subtilis liquid: sodium guaiacol =1000 mL: 0.1 g;
the bacillus subtilis liquid is bacillus subtilis ZWCA02 strain fermentation liquid, and the liquid OD600The value is 0.3 to 0.7; the number of effective viable bacteria in the final microbial agent compound composition finished product is more than or equal to 5 multiplied by 108cfu/ml。
2. The preparation method of the microbial inoculant compound composition for improving the yield of the dioscorea opposita thunb as claimed in claim 1, which is characterized by comprising the following steps:
(1) preparation of Bacillus subtilis liquid
Preparing a bacillus subtilis ZWCA02 strain fermentation liquid by using a bacillus subtilis ZWCA02 strain as a fermentation strain in a liquid culture mode, and fermenting until the bacillus subtilis OD600The value is 0.3 to 0.7;
(2) mixed preparation of microbial inoculum compound composition
And (2) adding sodium guaiacolate into the bacillus subtilis ZWCA02 strain fermentation liquor prepared in the step (1), and uniformly mixing.
3. The application of the microbial inoculant compound composition for improving the yield of the dioscorea opposita thunb in dioscorea opposita cultivation as claimed in claim 1, is characterized in that in the specific application, 1-3 g of each seedling of the dioscorea opposita thunb is uniformly sprayed to soil at the root of each seedling in the seedling stage of the dioscorea opposita thunb.
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