阅读说明：本技术 一种适合酸性土壤的硅酸盐细菌筛选方法及在马铃薯种植中的应用 (Silicate bacteria screening method suitable for acid soil and application of silicate bacteria in potato planting ) 是由 毛露甜 廖淑恩 林桂格 周星宇 陆镇樟 罗胜祥 于 2020-04-07 设计创作，主要内容包括：本发明公开了一种适合酸性土壤的含硅酸盐细菌筛选方法,其步骤包括：从自然环境中分离出硅酸盐细菌；分离出解钾能力最强的单个菌株；对硅酸盐细菌进行耐酸性培养筛选,筛选出在pH值5.0-5.5培养基中繁殖能力最强的硅酸盐细菌落；重复上述第二步和第三步步骤,直到筛选出解钾能力性状稳定的单个菌株为止。运用本发明筛选的硅酸盐细菌,可以保证所筛选的硅酸盐细菌,在pH值为5.0-5.5土壤中繁殖良好。本发明公开了运用上述方法筛选的硅酸盐细菌在马铃薯种植中的应用。(The invention discloses a method for screening silicate-containing bacteria suitable for acid soil, which comprises the following steps: separating silicate bacteria from the natural environment; separating out a single strain with the strongest potassium-resolving capability; performing acid-resistant culture screening on silicate bacteria, and screening out a silicate fine colony with the strongest reproductive capacity in a culture medium with a pH value of 5.0-5.5; repeating the second step and the third step until a single strain with stable potassium-solubilizing ability is screened out. The silicate bacteria screened by the method can ensure that the screened silicate bacteria can be well propagated in the soil with the pH value of 5.0-5.5. The invention discloses application of silicate bacteria screened by the method in potato planting.)

1. A silicate bacteria screening method suitable for acid soil is characterized in that:

a method for screening silicate-containing bacteria suitable for acid soil comprises the following steps:

the first step is as follows: separation: separating silicate bacteria from the natural environment;

step two, purification: separating out a single strain with the strongest potassium-resolving capability;

step three, acid resistance screening: performing acid-resistant culture screening on silicate bacteria, and screening out a silicate fine colony with the strongest reproductive capacity in a culture medium with a pH value of 5.0-5.5;

and fourthly, repeating the second step and the third step until a single bacterial strain with stable potassium-solubilizing ability is screened out.

2. The method for screening silicate bacteria suitable for acid soil according to claim 1, wherein:

before any step of the second, third and fourth steps, the method also comprises the step of selecting the bacterial species, wherein the selected silicate bacterial species is bacillus mucilaginosus.

3. The method for screening silicate bacteria suitable for acid soil according to claim 1, wherein:

before the fourth step, screening the extracellular polysaccharide production capacity of the silicate bacteria, and screening out silicate bacteria colonies with the strongest extracellular polysaccharide capacity.

4. The method for screening silicate bacteria suitable for acid soil according to claim 1, wherein:

and before the fourth step, screening the temperature resistance of silicate bacteria, and screening out silicate bacteria colonies with the highest reproductive capacity at 15-20 ℃.

5. The method for screening silicate bacteria suitable for acid soil according to claim 1, wherein:

and before the fourth step, screening the temperature resistance of silicate bacteria, and screening out silicate bacteria colonies with the highest reproductive capacity at 15-20 ℃.

6. The method for screening silicate bacteria suitable for acid soil according to claim 1, wherein:

before the fourth step, the method also comprises the step of screening salinity tolerance, and the silicate bacterial colony with the strongest reproductive capacity under the salinity of 0.2-0.3% is screened out.

7. The method for screening silicate bacteria suitable for acid soil according to claim 1, wherein:

before the fourth step, screening heavy metal ion resistance, and screening out silicate bacterial colonies with strongest reproductive capacity in an environment with heavy metal ions of certain concentration.

The heavy metal ion is Pb²⁺、Cd²⁺、Cr⁶⁺、Hg²⁺、Cu²⁺One or more of the above;

the concentration of the heavy metal ions is one of 5, 10, 15, 20, 30, 40, 50, 80 and 100 mg/L.

8. Use of silicate bacteria selected by the method according to any of claims 1 to 7 for potato cultivation, characterized in that:

the silicate bacteria screened by the method are used for preparing the bacterial fertilizer special for the potatoes.

9. The application of silicate bacteria obtained by screening according to the method of claim 8 in potato planting is characterized in that:

the special bacterial fertilizer carrier for the potatoes is bacterial bran.

10. The application of silicate bacteria obtained by screening according to the method of claim 8 in potato planting is characterized in that:

the special bacterial fertilizer for the potatoes comprises one or a mixture of powdery potash feldspar and powdery illite.

Technical Field

The invention relates to the technical field of microbial fertilizers, in particular to a silicate bacterium screening method suitable for acid soil and application of the silicate bacterium screening method in potato planting.

Background

The potato is a crop used as both grain and vegetable, and has wide application range, high economic benefit and rich nutrition. At present, the development of the potato industry is listed as an important content for increasing the income of farmers in many areas of China. The potato planting method is high in yield, good in fertilizer, high in fertilizer requirement, closely related to soil nutrient conditions, and particularly high in potassium requirement in the whole growth and development process. However, the average yield per unit area of domestic potatoes is lower than the average level in the world, which can be improved by applying microbial fertilizers, and the yield per unit area of domestic potatoes has a larger promotion space.

The microbial fertilizer is a product which causes crops to obtain a specific fertilizer effect due to the life activity of specific microorganisms, and can be divided into azotobacteria fertilizer, phosphorus bacteria fertilizer, silicate bacteria fertilizer, compound microbial fertilizer and the like. The microbial fertilizer is used, so that the production cost can be reduced, the utilization rate of the fertilizer can be improved, and the functional microbes contained in the fertilizer can inhibit or prevent soil-borne diseases, promote the release of insoluble mineral nutrients, promote the growth of crops, improve the quality of the crops, regulate the balance of an ecological system and the like. In recent years, the microbial fertilizer industry in China is rapidly developed, the number of the registered microbial fertilizer products reaches more than 1000, the using area is more than one hundred million acres, and the number is in an accelerated rising trend.

The microbial fertilizer applied to agricultural production has the advantages that the common chemical fertilizer cannot compare favorably: (1) the environment is not polluted, and the product is nontoxic and harmless to animals such as human beings and livestock; (2) repairing soil; (3) the yield and the quality of crops are improved; (4) the cost is low; (5) the fertilizer efficiency is durable.

At present, silicate bacteria, also called silicate potassium bacteria or potassium bacteria, which can effectively decompose silicate minerals mainly comprise Bacillus mucilaginosus, Bacillus circulans, Bacillus extorquens and the like. The silicate bacteria is a special functional bacterium in soil, can effectively decompose silicate minerals in the soil, and convert insoluble potassium in the soil into quick-acting potassium which can be directly absorbed and utilized by plants, thereby supplementing potassium in the soil and relieving the harm to the soil and the environment caused by excessive use of chemical fertilizers.

In recent years, a plurality of ecological problems appear in the application process of agricultural technology, which seriously threatens the sustainable development of agriculture, such as excessive use of pesticides and chemical fertilizers, pollution and damage of heavy metals and refractory organic matters to the ecological environment of soil, so that the severe problems of reduction of the variety and quantity of microorganisms in the soil, complete damage of the soil structure, reduction of the yield and quality of crops, increase of plant diseases and insect pests and the like are caused, the use amount of the chemical fertilizers and the pesticides is increased, the damage to the soil ecosystem is further aggravated, and a vicious circle is formed. Soil contamination can cause certain harmful components to accumulate in crops such as grains, affect the quality of grains, and harm human health through a food chain. In order to fundamentally restore the soil ecosystem, the use of the microbial fertilizer is a key measure, and the silicate bacteria are important components of the microbial fertilizer, so the research on the silicate bacteria is imperative, and the method has very important significance for green planting, soil modification, environmental protection and ecological agriculture.

According to data, the current potassium fertilizer yield of China accounts for 0.34% of the world, while the consumption amount reaches 14.7% of the world, and it is seen that the potassium fertilizer resource of China is very short, the import is mainly relied on, and the supply and demand are increasingly tense, especially in the south China. The potassium fertilizer is scientifically and reasonably applied to supplement potassium in soil, relieve the contradiction between supply and demand of the potassium fertilizer, protect the agricultural ecological environment, and maintain the biological approaches of high yield, stable yield and the like of agriculture.

The present silicate bacteria fertilizer uses silicate bacteria screening method, in the culture medium whose pH range is about 7.0, the single silicate bacteria strain with strong potassium-decomposing capacity is screened, then propagated and amplified.

However, the pH value of the soil in the planting field of most crops is not 7.0, some crops grow better in slightly acidic soil, and silicate bacteria with strong potassium-dissolving capacity screened by a culture medium with the pH range of about 7.0 are possibly poor in fertilizer effect due to poor reproduction capacity in the acidic soil.

For example, potatoes are suitable for growing in slightly acidic soils. According to the research, when the pH value of the soil is between 4.8 and 7.0, the growth and development of the potatoes are relatively normal. The pH value of the soil is 5.0-5.5, which is most suitable for the growth and development of the potatoes. However, silicate bacteria can grow at pH 5.0 to 9.0, but the optimum pH range is about 7.0 (Helinyan et al, research on biochemical characteristics and potassium-decomposing activity of silicate bacteria in different soils, soils 2004,36(4):434 to 437). As the reproductive capacity of silicate bacteria is poor in subacid soil which is most suitable for potato production, the potassium-dissolving effect of the existing silicate bacterial manure is poor when the silicate bacterial manure is applied to potato planting.

In addition, the temperature, salinity and heavy metal ion concentration of the soil also cause poor reproductive capacity or potassium-dissolving capacity of silicate bacteria, meanwhile, important elements for plant production also comprise phosphorus and nitrogen, and under the condition of unbalanced nutrition of potassium, phosphorus and nitrogen, the silicate bacteria with high potassium-dissolving capacity can not necessarily bring high yield of crops.

Disclosure of Invention

The invention aims to provide a silicate bacteria screening method suitable for acid soil and application of the method in potato planting, and the specific scheme is as follows:

a method for screening silicate-containing bacteria suitable for acid soil comprises the following steps:

first step, separation: separating silicate bacteria from the natural environment;

step two, purification: separating out a single strain with the strongest potassium-resolving capability;

step three, acid resistance screening: performing acid-resistant culture screening on silicate bacteria, and screening out a silicate fine colony with the strongest reproductive capacity in a culture medium with a pH value of 5.0-5.5;

and fourthly, repeating the second step and the third step until a single bacterial strain with stable potassium-solubilizing ability is screened out.

Optionally, before any of the second, third and fourth steps, selecting a bacterial species, wherein the selected silicate bacterial species is bacillus mucilaginosus.

Optionally, before the fourth step, screening the exopolysaccharide-producing ability of silicate bacteria, and screening out a silicate bacterial colony with the highest exopolysaccharide-producing ability.

Optionally, before the fourth step, silicate bacteria temperature resistance screening is further included, and silicate bacteria colonies with the highest reproductive capacity of 15-20 ℃ are screened out.

Optionally, before the fourth step, screening the temperature resistance of silicate bacteria, and screening out silicate bacteria colonies with the strongest reproductive capacity at 15-20 ℃.

Optionally, before the fourth step, the method further comprises a salinity tolerance screening step, and silicate bacterial colonies with the strongest reproductive capacity under the salinity of 0.2-0.3% are screened out.

Optionally, before the fourth step, heavy metal ion resistance screening is further included, and a silicate bacterial colony with the strongest reproductive capacity in an environment with a certain concentration of heavy metal ions is screened out.

The heavy metal ion is Pb²⁺、Cd²⁺、Cr⁶⁺、Hg²⁺、Cu²⁺One or more of the above;

the concentration of the heavy metal ions is one of 5, 10, 15, 20, 30, 40, 50, 80 and 100mg/L (pure Pb)²⁺、Cd²⁺、Cr⁶⁺、Hg²⁺And Cu²⁺Aggregate).

The invention also comprises the application of the silicate bacteria obtained by screening by the method in potato planting, in particular to a special bacterial fertilizer for preparing potatoes, wherein the silicate bacteria screened by the method are used for preparing the bacterial fertilizer for potatoes.

Further, the special bacterial fertilizer carrier for the potatoes is bacterial bran.

Further, the special bacterial fertilizer for potatoes comprises one or a mixture of powdery potash feldspar and powdery illite.

In conclusion, the silicate bacteria screened by the method can ensure that the screened silicate bacteria can be well propagated in the soil with the pH value of 5.0-5.5; furthermore, the bacillus mucilaginosus is selected, so that the silicate bacteria can be ensured to have strong potassium dissolving capacity and phosphorus dissolving and nitrogen fixing functions, and the crop nutrition is more balanced; further, silicate bacteria with the strongest extracellular polysaccharide producing capability are selected by screening the extracellular polysaccharide producing capability of the silicate bacteria, so that the silicate bacteria are beneficial to field planting in rhizosphere soil; further, the optional steps of screening the silicate bacteria for temperature resistance (15-20 ℃), salt resistance (0.2-0.3%) and heavy metal ion concentration resistance can be carried out, the method can be suitable for planting environments with different temperatures, salinity and heavy metal ion concentrations, and the screened silicate bacteria with high potassium-dissolving capacity has stronger environmental adaptability. The silicate bacteria screened by the invention are particularly suitable for potato planting (the optimum soil pH value is 5.0-5.5, the optimum growth temperature is 15-20 ℃), powdery potassium feldspar and powdery illite are added into the special bacterial manure for potatoes, extracellular polysaccharide bonded bacteria-mineral complex can be formed in the fertilizer stage, and the early potassium-dissolving capacity of the silicate bacteria is enhanced.

Drawings

FIG. 1 is a characteristic diagram of the colony shape of a silicate bacterium of the present invention on a silicate bacterium medium;

FIG. 2 is a graph showing the spore staining effect of silicate bacteria in example 2 of the present invention;

FIG. 3 is an electrophoretogram of PCR products of example 2 of the present invention;

FIG. 4 shows a phylogenetic tree according to example 2 of the present invention;

FIG. 5 is a genetic distance matrix chart in example 2 of the present invention;

FIG. 6 is a glucose standard curve prepared in example 4 of the present invention;

FIG. 7 is a graph showing the growth curves of silicate bacteria in different concentrations of heavy metals according to example 5 of the present invention;

FIG. 8 is a graph showing the variation of the content of available potassium in each group after application of the available potassium in example 7 of the present invention;

FIG. 9 shows the growth promoting effect of silicate bacteria 30d potato plants of example 7 of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, but the technical solutions do not limit the scope of the present invention.