CN108504598A - A kind of bio-bacterial manure leavening improving rhizosphere of strawberry soil ecology structure - Google Patents

Abstract

The invention relates to a biological bacterial fertilizer starter for improving the ecological structure of strawberry rhizosphere soil, characterized in that: the biological bacterial fertilizer starter is 40-60 parts of Pseudomonas oryzae, 20-30 parts of saccharomyces, and Bacillus subtilis 20-30 parts of the biological agent made, the Pseudomonas oryzae, preservation unit: China Microbiological Culture Collection Management Committee General Microbiology Center, the address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, the preservation number is CGMCC No.15325, and the preservation date is January 29, 2018. The bacterial fertilizer made of the starter has good colonization ability in the strawberry rhizosphere and is safe and reliable, and has a good degrading effect on the self-toxic substances in the continuous-cropping strawberry soil, significantly improves the physical and chemical properties of the soil, and promotes the stress resistance of strawberry plants , Improve strawberry yield and fruit quality.

Description

A biological bacterial fertilizer starter for improving the ecological structure of strawberry rhizosphere soil

technical field

The invention relates to the field of repairing microorganisms and continuous cropping obstacles, in particular to a biological bacterial fertilizer starter for improving the ecological structure of strawberry rhizosphere soil.

Background technique

Strawberries contain a variety of vitamins and 17 kinds of amino acids, as well as calcium, iron, phosphorus, zinc and other minerals, which are very popular among consumers. Yield and quality have seriously affected the income of growers. Strawberry is a kind of herbaceous crop. It is susceptible to diseases during the growth period, and there are many kinds of diseases. If it is planted in the same field for a long time, harmful microorganisms and soil-borne pathogens will gradually accumulate, which will easily cause the breeding and spread of diseases and insect pests, and the more harmful the plant infection will be. If the disease becomes more and more serious, the growth of the plant will be weakened, gradually shrinking and dwarfing, and finally the whole plant will die, resulting in severe production reduction or even no harvest. The survey shows that the strawberry continuous cropping obstacles are mainly caused by soil obstacles, which are mainly manifested in the widening gap between the abundance and deficiency of soil trace elements, the decrease of organic matter content, secondary salinization and salinization of soil, especially the poor ecological structure of soil microorganisms. Balanced, the beneficial microbial flora decreases, and the composition ratio of the three major fungi, bacteria, fungi, and actinomycetes, changes. Simultaneously, due to the long-term use of the same strawberry strain, the disease resistance declines, the variety degenerates, the virus infestation is serious, and the disease incidence rate increases.

Adding beneficial microorganisms or antagonistic bacteria can effectively prevent the occurrence of continuous cropping obstacles caused by autotoxicity and root soil-borne diseases. At present, there are relatively few probiotics applied to strawberry continuous cropping obstacles. Most of them use fungicides and chemical fertilizers for soil treatment, which leads to problems such as soil compaction and acidification, nutrient imbalance, and secondary salinization. The lack of safe and effective biological bacteria treatment seriously restricts melons. The governance and sustainable development of crop continuous cropping obstacles have become one of the bottleneck problems that need to be solved urgently in the current agricultural production of melon crops.

At present, the planting area of edible fungi in my country is expanding rapidly. After the cultivation of edible fungi, a large amount of fungus residues are produced. Some mushroom farmers discard the fungus residues in the ditch and on the road at will, which not only causes waste of resources, pollutes the environment, but also It is more likely to be infected with pests and diseases and endanger the normal growth of edible fungi. Even some factory production companies pile up the fungus residue for a long time in order to sell the fungus residue at a better price, which not only pollutes the environment, but also breeds diseases and pests and spreads quickly. During the growth of edible fungi and the cultivation of bacteria It is easy to be infected with pests and diseases, and the quality of the produced fungus products will also be affected. The crop stalks and cottonseed hulls in the edible fungi cultivation materials are rich in lignin and cellulose, some of which are absorbed and utilized by mycelia. Hydrocarbons and trace elements can be used as good organic fertilizers for vegetable cultivation, but edible fungus residues cannot be completely absorbed by plants without fermentation treatment, and will instead destroy the original nutritional structure of the soil, resulting in unbalanced nutritional components, resulting in The absorption of plant roots is blocked and the phenomenon of root injury is formed.

Contents of the invention

In order to make up for the deficiencies of the above-mentioned prior art, the present invention proposes a biological bacterial fertilizer that improves the ecological structure of the strawberry rhizosphere soil, to improve the nutritional structure of the strawberry root soil, degrade the autotoxic substances in the root exudates, and solve the continuous cropping obstacles of strawberries Problems, the purpose of promoting plant growth and degrading self-toxicity, while rationally utilizing agricultural waste, improving the quality and economic benefits of protected agricultural land.

The object of the present invention is achieved by the following technical solutions: a biological fertilizer for improving the ecological structure of strawberry rhizosphere soil, its technical points are: the starter of biological bacteria fertilizer is 40-60 parts of Pseudomonas oryzae, yeast 20-30 parts of Bacillus subtilis and 20-30 parts of Bacillus subtilis.

The described Pseudomonas oryzihabitans (Pseudomonas oryzihabitans), preservation unit: China Microbiology Culture Collection Management Committee General Microbiology Center, address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, preservation The number is CGMCC No.15325, and the deposit date is January 29, 2018.

The colony and cell characteristics of the strain are as follows: the autotoxic substance-degrading bacteria were cultured on NA medium (yeast extract powder 1g; beef extract 3g; peptone 5g; sucrose 10g; agar 18g; distilled water 1000ml; sterilized at 121°C for 30min) After 48 hours, the colonies were smooth or wrinkled, with regular or irregular edges, plano-convex, and light yellow. After being cultured at 30°C for 2 days, the colony diameter was 2 mm.

The physiological and biochemical characteristics of the strain are: negative oxidase test, positive contact enzyme test, negative methyl red test, negative starch hydrolysis test, negative gelatin hydrolysis test, positive nitrate reduction test, negative H2S test, positive malonate utilization test , positive citrate utilization test, positive glucose utilization test, negative lactose utilization test, positive galactose utilization test, and positive maltose utilization test.

The most suitable medium for Pseudomonas oryzae strains is LB medium, the formula includes tryptone 10g/L, yeast extract 5g/L, sodium chloride (NaCl) 10g/L, and the inoculum size is 2% of the medium weight. %, the fermentation temperature is 28°C, the stirring speed is 120rpm, and the fermentation time is 5 days.

The concentration of Pseudomonas oryzae used to prepare the starter was 2.0-3.0×10 ⁸ cfu/ml.

Further, the biological bacterial fertilizer includes 30-50 parts of edible fungus residue, 50-70 parts of poultry manure, and 10-20 parts of straw powder;

Further, the preparation method of biological bacterial fertilizer comprises the following steps:

(1) Raw materials are calculated in parts by weight: 30-50 parts of edible mushroom residues, 50-70 parts of poultry manure, and 10-20 parts of straw powder are evenly mixed to obtain an organic mixture for subsequent use;

(2) preparation of starter: take materials according to the weight ratio of each component, wherein microbial inoculum: 40-60 parts of Pseudomonas oryzae, 20-30 parts of saccharomyces, 20-30 parts of Bacillus subtilis, and The materials are mixed evenly to obtain a starter;

(3) Mix the above-mentioned organic mixture and starter evenly, and start stacking and fermenting in the fermentation tank. The stacking height during fermentation is 1.2-1.5m. Before the fermentation, it is expected that the temperature will rise to 55-60°C and start stirring. 2 - Stir once every 3 days; in the middle and late stages of fermentation, the temperature should be controlled within 60-70°C. Whenever the temperature reaches above 60°C, it needs to be stirred once until the fermentation temperature drops to normal temperature. The whole fermentation process is: when the temperature is at 20 It takes 15-20 days when the temperature is below 20°C, and 6-10 days when the temperature is above 20°C, and the fermentation is completed to obtain biological bacterial fertilizer.

Further, the biological bacterial fertilizer contains 200-600 million cuf/g effective viable bacteria, contains 40-75% organic matter by weight, 5-15% NPK, 3-10% trace elements, humic Acid 15-30%, amino acid 2-8%.

Beneficial effects of the present invention: the bacterial fertilizer made from the starter has good colonization ability in the strawberry rhizosphere and is safe and reliable, and has a good degradation effect on the self-toxic substances in the continuous-cropping strawberry soil, obviously improves the physical and chemical properties of the soil, and promotes The stress resistance ability of strawberry plants can improve the output and fruit quality of strawberries; the preparation method is simple, the formula is reasonable, and the effect is stable, and the effect of alleviating the autotoxic effect of strawberries is obvious; The source of raw materials is wide, the cost is low, and it can also turn waste into treasure and realize the rational utilization of resources. In particular, the residue of edible fungi is rich in organic matter, nitrogen, phosphorus, potassium, etc., and can also improve soil structure, air permeability and water retention. The role of water capacity.

Detailed ways

1. Materials and Methods

(1) Raw materials are calculated in parts by weight: 40 parts of edible mushroom residues, 50 parts of poultry manure, and 10 parts of straw powder are mixed evenly to obtain an organic mixture for subsequent use;

(2) Preparation of starter: take materials according to the weight ratio of each component, starter I: 60 parts of Pseudomonas oryzae, 20 parts of saccharomyces, 20 parts of Bacillus subtilis, and mix the materials evenly; starter II: 50 parts of yeast, 50 parts of Bacillus subtilis, and mix the materials evenly.

Yeast and Bacillus subtilis were purchased from Zhengzhou Nongfukang Biotechnology Co., Ltd.

The medium used to isolate Pseudomonas oryzae is an inorganic salt medium, and the formula includes MgSO ₄ 7H ₂ O 0.2g/L, CaCl ₂ 2H ₂ O 0.02g/L, FeCl ₃ 0.05g/L, KH ₂ PO ₄ 1.0g/L, K ₂ HPO ₄ 1.0g/L, NH ₄ NO ₃ 1.0g/L, cholesterol 0.3g/L, agar 17g/L were treated with p-hydroxybenzene at a concentration of 200μg·mL ^-1 Formic acid, benzoic acid and cinnamic acid are used as the sole carbon source, and the pH value is adjusted to neutral.

The method for isolating Pseudomonas oryzae: take the muskmelon rhizome junction, wash it with running water, cut it into small pieces, soak it in 75% alcohol for 1min, rinse it with 0.1% mercury liter for 30s, and then rinse it with sterile water for 4 times. Grind it into a slurry in a bowl, dilute the grinding solution 3 times, spread it on an inorganic salt medium, and cultivate it in an incubator at 28°C for 48 hours to obtain a strain of Pseudomonas oryzae.

Pseudomonas oryzihabitans (Pseudomonas oryzihabitans), preservation unit: General Microbiology Center of China Committee for Culture Collection of Microorganisms, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, strain number: 20180101H16 , the deposit number is CGMCC No.15325, and the deposit date is January 29, 2018.

Preparation of Pseudomonas oryzae bacteria agent: suitable medium and LB medium for cultivating Pseudomonas oryzae bacterial strains, formula includes tryptone 10g/L, yeast extract 5g/L, sodium chloride (NaCl) 10g/L, the inoculum size is 2% of the medium weight, the fermentation temperature is 28°C, the stirring speed is 120rpm, and the fermentation time is 5 days to obtain a bacterial suspension. The concentration of Pseudomonas oryzae used to prepare the starter is 2.0-3.0×10 ⁸ cfu/ml.

(3) Mix the above-mentioned organic mixture and two kinds of starters evenly, and start stacking and fermenting in the fermentation tank. The height of the pile during fermentation is 1.5m. Before the fermentation, it is expected that the temperature will rise to 55-60°C and start stirring. 3 Stir once a day; in the middle and late stages of fermentation, the temperature is controlled within 70°C, and it needs to be stirred once every time the temperature reaches above 60°C until the fermentation temperature drops to normal temperature; the whole fermentation process is: when the temperature is below 20°C 20 days, 10 days when the temperature is above 20°C, the fermentation is completed to obtain two kinds of biological fertilizers.

1.2 Degradation of phenolic acid substances in soil by microbial agents fermented with starter Ⅰ Detection method:

(1) When strawberries are transplanted in spring, use biological bacterial fertilizer to treat with hole application, 20-30 grams per hole, set no biological fertilizer treatment as a control, and weigh 25 g of strawberry continuous cropping soil without biological fertilizer in autumn Put the strawberry continuous cropping soil applied with biological bacterial fertilizer in a 75mL centrifuge tube, add 25mL of 1mol L ^-1 NaOH solution to the soil, mix it with a vortex shaker, let it stand for 24h, and turn it over the next day. Mix for 1 hour, vibrate with an ultrasonic oscillator for 30 minutes, separate the supernatant after centrifugation, adjust the pH value of the soil solution to 2.5 with 12 mol L ^- 1 hydrochloric acid, let it stand for 2 hours, remove humic acid by centrifugation, and separate the supernatant Save the assay ready for analysis.

(2) HPLC separation and identification of phenolic acids

Preparation of standard solution: Accurately weigh 10mg of ferulic acid, benzoic acid, p-hydroxybenzoic acid, benzyl benzyl substrate, gallic acid, caffeic acid, sinapic acid, gentisic acid (provided by Shanghai Sigma Biotechnology Co., Ltd.) Put it into a 100mL volumetric flask, dissolve it with methanol and adjust the volume to 100mL, then the concentration of each phenolic acid in the standard solution is 100μg·mL ^-1 .

(3) Chromatographic conditions: HPLC method (Agilent LC 1100, USA) was used for the determination of different kinds of phenolic acids in soil. The sample must be filtered with a 0.22μm microporous membrane to remove impurities in the solution before injection. The chromatographic separation column is SB-C18 (Agilent 4.6mm×250mm), with a flow rate of 1mL·min ^-1 and a column temperature of 25°C. The sample detection wavelength is 280nm, each injection volume is 20μL, mobile phase A: methanol (chromatographic grade) and mobile phase B: acetic acid aqueous solution (chromatographic grade) with pH = 2.8, and the sample separation is achieved by gradient elution. The purpose of shortening the retention time of phenolic acids. Elution condition B: 70% (0min)→50% (15min)→30% (16min)→0% (30min)→end (30min). Retention time was used for qualitative analysis, and peak area external standard method was used for quantitative analysis.

(4) Data processing and statistical analysis

Each experiment was repeated 3 times, and statistical analysis was performed on the data using spss 17.0, and the significant difference analysis among the treatments was analyzed using the Duncan method.

The formula for calculating the content of phenolic acids R (mg·kg ^-1 ) in soil is as follows: R(mg·kg ^-1 )=

S _sample ×V _sample ×C _standard /S _sample ×m (R-the phenolic acid content of a certain substance in the sample; S _sample -sample peak area; V _sample -sample constant volume, mL; C _standard -standard solution concentration, mg· L ^-1 ; S _standard - standard solution peak area; m - sample weight, g.)

1.3 Effect of biological bacterial fertilizer on strawberry yield

Strawberry varieties tested: Dandong 99

Test site: Shenyang Agricultural University test base.

When transplanting strawberries, use bio-fertilizer and apply in holes, 20-30 grams per hole. Set no bio-fertilizer as a control, and investigate the yield during the harvest period. The strawberry fruits were weighed and the data were recorded to calculate the strawberry yield under different treatment conditions.

2 Results and Analysis

The results of the degradation of phenolic acids in strawberry continuous cropping soil by microbial agents showed that the agent had excellent degradation effects on the main phenolic acids in strawberry secretions, among which 4-coumaric acid, salicylic acid, cinnamic acid best degradation (Table 1).

Table 1 Degradation of phenolic acid substances in continuous cropping soil by the biological fertilizer fermented with starter Ⅰ

The biological fertilizer fermented with starter Ⅰ contains 500 million cuf/g of effective viable bacteria, 55.4% organic matter by weight, 10.1% NPK, 5.2% trace elements, 18.2% humic acid, and 5.8% amino acid .

The biological bacterial fertilizer fermented with starter II contains 430 million cuf/g of effective viable bacteria, 41.3% by weight of organic matter, 5.2% of NPK, 1.2% of trace elements, 9.7% of humic acid, and 1.2% of amino acids .

The results of the field experiment showed that exogenous application of the biological fertilizer fermented by starter Ⅰ can help enhance the growth and yield of strawberry plants, and reduce the dead seedling rate of strawberry transplanting (Table 2).

Table 2 Effects of microbial inoculants on strawberry disease and yield

deal with Mortality rate Yield (kg/mu) Biological fertilizer treatment (fermented by starter Ⅰ) 0 1086±3.7 Biological fertilizer treatment (fermented by starter II) 1.25 923±2.8 Without biological fertilizer treatment 5.13% 890±2.6

Claims (4)

1. A biological bacterial fertilizer starter for improving the ecological structure of strawberry rhizosphere soil is characterized in that: the biological bacterial fertilizer starter is 40-60 parts of Pseudomonas oryzae, 20-30 parts of saccharomycetes, and 20 parts of Bacillus subtilis -30 parts of biological agent made; The Pseudomonas oryzihabitans described above, deposit unit: China Microbiological Culture Collection Management Committee General Microbiology Center, address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, deposit The number is CGMCC No. 15325, and the deposit date is January 29, 2018; The colony and cell characteristics of the strain are as follows: the autotoxic substance-degrading bacteria was cultured on NA medium (yeast extract powder 1g; beef extract 3g; peptone 5g; sucrose 10g; agar 18g; distilled water 1000ml; sterilized at 121°C for 30min) After 48 hours, the colonies were smooth or wrinkled, with neat or irregular edges, plano-convex, and light yellow. After 2 days of culture at 30°C, the colony diameter was 2 mm, the cells were rod-shaped, flagella moved, no spores were produced, and Gram staining was negative; The physiological and biochemical characteristics of the strain are: negative oxidase test, positive contact enzyme test, negative methyl red test, negative starch hydrolysis test, negative gelatin hydrolysis test, positive nitrate reduction test, negative H2S test, positive malonate utilization test , Positive citrate utilization test, positive glucose utilization test, negative lactose utilization test, positive galactose utilization test, positive maltose utilization test; The most suitable medium for Pseudomonas oryzae strains is LB medium, the formula includes tryptone 10g/L, yeast extract 5g/L, sodium chloride (NaCl) 10g/L, and the inoculation volume is 2% of the medium weight. %, the fermentation temperature is 28°C, the stirring speed is 120rpm, and the fermentation time is 5 days; The concentration of Pseudomonas oryzae used to prepare the starter was 2.0-3.0×10 ⁸ cfu/ml. 2. A kind of biological bacterial fertilizer starter for improving the ecological structure of strawberry rhizosphere soil according to claim 1, characterized in that: said biological bacterial fertilizer comprises 30-50 parts of edible fungus slag, 50-70 parts of poultry manure , straw powder 10-20. 3. a kind of biological fertilizer starter for improving the ecological structure of strawberry rhizosphere soil according to claim 2, is characterized in that: the preparation method of biological bacterial fertilizer comprises the following steps: (1) Raw materials are calculated in parts by weight: 30-50 parts of edible fungus residue, 50-70 parts of poultry manure, 10-20 parts of straw powder are evenly mixed to obtain an organic mixture for later use; (2) Preparation of starter: Weigh the materials according to the weight ratio of each component, including microbial agents: 40-60 parts of Pseudomonas oryzae, 20-30 parts of yeast, 20-30 parts of Bacillus subtilis, and The materials are mixed evenly to obtain a starter; (3) Mix the above-mentioned organic mixture and starter evenly, and start to accumulate and ferment in the fermentation tank. The height of the pile during fermentation is 1.2-1.5m. Before the fermentation, it is expected that the temperature will rise to 55-60°C and start stirring. 2 -Stir once every 3 days; in the middle and late stage of fermentation, the temperature should be controlled within 60-70°C, and stir once every time the temperature reaches above 60°C, until the fermentation temperature drops to normal temperature, the whole fermentation process is: when the temperature is at 20°C It takes 15-20 days when the temperature is below 20°C, and 6-10 days when the temperature is above 20°C, and the fermentation is completed to obtain biological bacterial fertilizer. 4. a kind of biological fertilizer starter for improving the ecological structure of strawberry rhizosphere soil according to claim 3 is characterized in that: the effective viable count of 2-6 hundred million cuf/g is contained in the described biological bacterial fertilizer, press The weight ratio contains 40-75% of organic matter, 5-15% of NPK, 3-10% of trace elements, 15-30% of humic acid and 2-8% of amino acid.