CN113545191A

CN113545191A - A method for enhancing the colonization efficiency and activity of biocontrol bacteria

Info

Publication number: CN113545191A
Application number: CN202110862150.3A
Authority: CN
Inventors: 黄新琦; 刘亮亮; 赵军; 蔡祖聪; 张金波
Original assignee: Nanjing Normal University
Current assignee: Nanjing Normal University
Priority date: 2021-07-29
Filing date: 2021-07-29
Publication date: 2021-10-26

Abstract

The invention discloses a method for enhancing the colonization efficiency and activity of biocontrol bacteria, comprising the following steps: soil strong reduction treatment: adding easily decomposable organic matter into the soil; irrigating the soil; Plant crops after they are evenly stabilized. The present invention enhances the colonization efficiency and activity of the biocontrol bacteria in the soil by combining the strong reduction treatment of the soil and the application of the biocontrol agent, thereby improving the prevention and control effect of the soil-borne diseases and ensuring the sustainability of the economic crops. The production process does not involve the use of any toxic and harmful chemicals. Compared with the chemical fumigation mainly used at present, it has the advantages of no pollution to the land and the environment, and ecological and environmental protection.

Description

Method for enhancing colonization efficiency and activity of biocontrol bacteria

Technical Field

The invention relates to the technical field of agricultural sustainable development, in particular to a method for enhancing colonization efficiency and activity of biocontrol bacteria.

Background

Most facilities for producing vegetables, field melons and fruits, tobacco, potatoes, Chinese medicinal plants and the like have serious soil-borne disease problems. The soil-borne disease refers to the phenomenon that soil-borne pathogenic bacteria and root-knot nematodes in soil propagate in a large quantity and invade plants under proper conditions, and finally cause the plants to develop badly and even die. In a modern intensive agricultural production system which is characterized by high multiple cropping index and high fertilizer input, the problem of soil-borne diseases is particularly prominent, great economic loss is caused every year, and sustainable development of modern agriculture in China is hindered.

In order to solve the problem, in the agricultural production, a soil chemical fumigation method before planting is mainly adopted to kill plant pathogenic microorganisms in the soil, however, the chemical fumigant kills harmful pathogenic bacteria and beneficial plant microorganisms in the soil, the worry of people about food safety and environmental protection is increasing at present, and the use of the chemical fumigation method is gradually limited. In recent years, research and application for protecting plant health by adding artificially screened and cultured beneficial microorganisms to soil to inhibit pathogenic bacteria have been attracting attention. A series of products containing high-efficiency biocontrol bacteria are developed, and the biocontrol bacteria mainly comprise bacillus, trichoderma and the like. However, since the biocontrol bacterial foreign species are often difficult to compete effectively with indigenous microorganisms after being added to soil, and cannot be colonized and survived effectively in soil, the ability of the biocontrol bacterial foreign species to inhibit the occurrence of crop diseases is greatly reduced. Enhancing the colonization efficiency of the biocontrol bacteria in the soil is a necessary prerequisite for fully playing the disease-resistant role.

Disclosure of Invention

The purpose of the invention is as follows: in order to overcome the defects of the background art, the invention discloses a method for enhancing the colonization efficiency and activity of biocontrol bacteria.

The technical scheme is as follows: the method for enhancing the colonization efficiency and activity of the biocontrol bacteria comprises the following steps:

s1, strong soil reduction treatment:

adding easily decomposed organic matters into soil;

irrigating the soil;

film covering and sealing;

s2, applying a biocontrol microbial inoculum;

and S3, uniformly mixing and stabilizing the soil, and planting the crops.

Wherein the easily-decomposed organic matters are bulk crop straws or green manure.

Furthermore, the easily-decomposed organic matters need to be crushed to be less than 4mm, the carbon-nitrogen ratio is adjusted to (22-28):1 by using nitrogen fertilizer, and the dosage is 800-.

Furthermore, the water capacity of the field needs to reach 100% when the soil is irrigated.

Furthermore, when the film is covered and sealed, the upper part and the periphery of the soil to be treated are covered by plastic films with the thickness of more than 0.04mm, and the oxygen exchange between the soil to be treated and the air is blocked.

When the soil is subjected to strong reduction treatment, the environmental temperature reaches more than 25 ℃, and the film can be removed in 3 weeks after the soil is covered with the film, so that the treatment is finished; and (5) when the environmental temperature reaches above 30 ℃, keeping the soil covering film for 2 weeks, and removing the film to finish the treatment.

Further, when the biocontrol microbial inoculum is applied, after the strong reduction treatment is finished, the biocontrol microbial inoculum is applied within 2 weeks after the film is uncovered and the soil is aired to 30% -50% of the field water capacity.

Furthermore, the crops need to be planted 1 week after the biocontrol microbial inoculum is applied.

The principle that this patent was realized: through strong reduction treatment of soil, the physical and chemical properties of the soil are improved to make the soil suitable for colonization of biocontrol bacteria; and secondly, soil microbial communities are reformed, living space and ecological niches required by the colonization of the biocontrol bacteria are created, and the aims of improving the colonization efficiency and activity of the biocontrol bacteria are finally fulfilled.

Has the advantages that: compared with the prior art, the invention has the advantages that: the method has the advantages that the colonization efficiency and activity of the biocontrol bacteria in the soil are enhanced by combining the strong reduction treatment of the soil and the application of the biocontrol bacteria agent, so that the prevention and control effect of the biocontrol bacteria on soil-borne diseases is improved, and the sustainable production of economic crops is guaranteed; the process does not involve the use of any toxic and harmful chemicals, and has the advantages of no pollution to land and environment and ecological environmental protection compared with the chemical fumigation mainly adopted at present.

Drawings

FIG. 1 shows the number of biocontrol bacteria N23 activated bacteria for each treatment in the examples of the present invention;

FIG. 2 is the total cell number of biocontrol bacteria N23 of each treatment in the examples of the present invention;

FIG. 3 shows the number of biocontrol bacteria T15 for each treatment in the example of the present invention;

FIG. 4 shows the number of Rhizoctonia solani, the pathogenic bacterium, in each treatment in the examples of the present invention.

Detailed Description

The technical solution of the present invention is further described below with reference to the accompanying drawings and examples.

A method of enhancing the efficiency and activity of colonization by a biocontrol bacterium, comprising the steps of:

s1, strong soil reduction treatment:

adding easily-decomposable organic matters including but not limited to straws of bulk crops such as rice, wheat and corn and green manure such as alfalfa into soil, crushing the mixture to be less than 4mm, adjusting the carbon-nitrogen ratio of the mixture to (22-28):1 by using nitrogen fertilizer, and using the amount of 800-1000 kg/mu;

irrigating the soil to reach 100% of field water capacity;

covering with a film, sealing, and covering the upper part and the periphery of the soil to be treated with a plastic film with the thickness of more than 0.04 mm;

S2, applying a biocontrol microbial inoculum;

including but not limited to bacillus and trichoderma; the applied biocontrol microbial inoculum comprises but is not limited to bacterial liquid and bacterial powder;

when the biocontrol microbial inoculum is applied, after the strong reduction treatment is finished, the biocontrol microbial inoculum is applied within 2 weeks after the film is uncovered and the soil is aired to 30-50% of the field water capacity.

S3, uniformly mixing and stabilizing soil, and planting crops;

the crops are planted after the biocontrol microbial inoculum is applied for 1 week.

The materials used

The soil is collected from Nanjing Yanxi and is the multi-year continuous cropping soil of the cucumbers, the incidence rate of damping off of the cucumbers cultivated on the soil is over 50 percent before collection, and the soil has basic physicochemical properties of pH 5.0, organic carbon 13.8g/kg, total nitrogen 2.06g/kg and nitrate nitrogen 270 mg/kg. The cucumber seed is Jinchun No. 4.

Example 1 (Strong reduction + biocontrol bacterium)

According to the method, the alfalfa powder which is equivalent to 1000 kg/mu is added into the soil, the carbon-nitrogen ratio of the alfalfa powder is 24:1, the alfalfa powder is irrigated to the field moisture capacity, the alfalfa powder is covered with a film and sealed, the alfalfa powder is cultured for 15 days at the temperature of 30 ℃, the film is uncovered and dried to 40% of the field moisture capacity, and then the biocontrol bacteria bacillus subtilis N23 and trichoderma harzianum T15 are added into the alfalfa powder. The inoculation concentrations of the biocontrol bacteria N23 and T15 are respectively 10 per gram of soil⁷cfu and 1g of fresh mycelia were thoroughly mixed with soil.

Example 2 (Strong reduction + biocontrol bacterium)

According to the method, the soil is converted into 900 kg/mu of rice straw, the carbon nitrogen ratio is adjusted to 22:1 by using ammonium bicarbonate, the rice straw is irrigated to the field water capacity, the rice straw is covered with a film and sealed, the rice straw is cultured for 15 days at the temperature of 30 ℃, the film is uncovered and dried to 30% of the field water capacity, and then biocontrol bacteria N23 and T15 are added into the rice straw. The inoculation concentrations of the biocontrol bacteria N23 and T15 are respectively 10 per gram of soil⁷cfu and 1g of fresh mycelia were thoroughly mixed with soil.

Example 3 (Strong reduction + biocontrol bacterium)

According to the method, 800 kg/mu of corn straws are added into soil, the carbon nitrogen ratio is adjusted to 28:1 by using ammonium bicarbonate, the soil is irrigated to the field water capacity, the film is covered and sealed, the soil is cultured for 15 days at the temperature of 30 ℃, the film is uncovered and dried to 50 percent of the field water capacity, and then biocontrol bacteria N23 and T15 are added into the soil. The inoculation concentrations of the biocontrol bacteria N23 and T15 are respectively 10 per gram of soil⁷cfu and 1g of fresh mycelia were thoroughly mixed with soil.

COMPARATIVE EXAMPLE 1 (No treatment control)

No soil treatment was performed.

Comparative example 2 (biocontrol bacteria)

The soil is only added with biocontrol bacteria N23 and T15.

Each treatment contained 3 replicates, each replicate being filled with 2.5kg of soil in a 25 x 5cm pot. After 15 days from the end of the above treatment, 9 germinated cucumber seeds were planted in each pot. Counting the morbidity of cucumber seedlings and the biomass of overground parts after culturing in a greenhouse for 40 days; soil samples are collected before transplanting and after harvesting, and the quantity of pathogenic rhizoctonia solani, biocontrol bacteria N23 and T15 in the soil is measured.

Determination of the number of pathogenic and biocontrol bacteria in soil

The total number of biocontrol bacteria N23 was measured by selective plate dilution coating (30. mu.g mL of solution was added)^-1Kanamycin in beef extract peptone medium), the soil suspension is heat-treated for 15 minutes at 80 ℃, and the spore number is measured; the determination of the amount of Rhizoctonia solani and biocontrol bacteria T15 was performed by fluorescence quantitative PCR method, as described in the literature [ Trichoderma harzianum strain SQR-T37 and its bio-organic transducer core control Rhizoctonia solani sampling-off diseases in culture sections mainly by the mycoparasitism ].

Test results

At two time points before and after the cucumber seedling is planted, the number and the total number of active cells (the sum of the number of the active cells and spores) of N23 in the treatment of the examples 1-3 (strong reduction and biocontrol bacteria) are significantly higher than those in the comparative example 2 (only the biocontrol bacteria treatment is applied, as shown in FIG. 1 and FIG. 2); the proportion of N23 active cells was 72.4%, 76.7% and 68.0% in examples 1-3 before planting, respectively, and was only 35.0% in comparative example 2; the ratios of N23 active cells in examples 1-3 after planting were 41.4%, 33.9%, and 48.0%, respectively, which was still higher than that in comparative example 2 (13.3%). After cucumber seedlings are harvested, the amount of T15 in examples 1-3 is 7.27, 4.41 and 8.38 times that in comparative example 2 (where only biocontrol bacteria treatment is applied) (as shown in FIG. 3). The number of the pathogenic bacterium, Rhizoctonia solani, in examples 1-3 was significantly lower than in comparative examples 1 and 2 (shown in FIG. 4), which were 0.66%, 1.09%, and 1.34% of comparative example 2, respectively. The control effect and total aerial biomass of cucumber seedling blight in examples 1 to 3 were significantly higher than those of comparative example 2 (see table 1). These results all show that the combination of strong reducing soil treatment and biocontrol bacteria application can significantly improve the quantity and activity of the biocontrol bacteria in the soil, thereby enhancing the disease control effect.

TABLE 1 cucumber seedling incidence and Biomass for each treatment

Claims

1. a method for enhancing biocontrol bacteria colonization efficiency and activity, is characterized in that, comprises the following steps:

S1. Soil strong reduction treatment:

Add easily decomposable organic matter to the soil;

irrigated soil;

film sealing;

S2, applying biocontrol agents;

S3. Plant crops after the soil is mixed and stabilized.

2 . The method for enhancing the colonization efficiency and activity of biocontrol bacteria according to claim 1 , wherein the easily decomposable organic matter is bulk crop straw or green manure. 3 .

3. the method for enhancing biocontrol bacteria colonization efficiency and activity according to claim 1, is characterized in that: described easily decomposable organic matter needs to be pulverized to below 4mm, adjust its carbon-nitrogen ratio to (22-28) with nitrogen fertilizer: 1. The dosage is 800-1000kg/mu.

4. The method for enhancing the colonization efficiency and activity of biocontrol bacteria according to claim 1, characterized in that: 100% field capacity is required when irrigating soil.

5. The method for enhancing the colonization efficiency and activity of biocontrol bacteria according to claim 1, characterized in that: the top and surrounding areas of the soil to be treated are covered with a plastic film with a thickness of more than 0.04 mm during film-covering and sealing.

6. The method for enhancing the colonization efficiency and activity of biocontrol bacteria according to claim 1, characterized in that: the soil strong reduction treatment environment temperature reaches more than 25 ℃, and the soil mulch film can be removed for 3 weeks and the treatment can be finished; When the temperature reaches above 30°C, keep the soil covered with film for 2 weeks before removing the film to end the treatment.

7. The method for enhancing the colonization efficiency and activity of biocontrol bacteria according to claim 1, is characterized in that: when applying biocontrol bacteria, the treatment of strong reduction is finished, and the film is removed to dry the soil to 30%-50% field holding time. Administer within 2 weeks after the amount of water.

8. The method for enhancing the colonization efficiency and activity of biocontrol bacteria according to claim 1, wherein the crops need to be planted after 1 week of applying the biocontrol bacteria.