CN102925387A - Bacillus simplex for inducing soybean to generate soybean cyst nematode resistance and application - Google Patents

Abstract

The invention relates to a cultivation method and application of a strain of Bacillus simplex Sneb545 capable of inducing soybean resistance to soybean cyst nematodes after soybean seeds are treated, and belongs to the technical field of microbial pesticides. The bacterial strain Sneb545 involved in the present invention has been preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee on October 11, 2012, and the preservation number is: CGMCC No.6650. After the bacterial strain is cultured by conventional liquid fermentation, the metabolites of the bacterial strain can be used to develop a seed treatment agent for treating soybean seeds, and the treated soybean seeds can induce soybeans to produce resistance to soybean cyst nematodes. The Bacillus simplex ( Bacillus simplex ) Sneb545 metabolite of the present invention can induce soybean to produce obvious resistance to the first-generation cyst nematode after treating soybean seeds, and has obvious inhibitory effect on the formation of soybean cyst nematode cysts, It is a biocontrol bacterium with great potential, which opens up a new idea for the control of soybean cyst nematode.

Description

A Simple Bacillus with Induced Soybean Resistance to Soybean Cyst Nematode and Its Application

technical field

The invention belongs to the technical field of microbial pesticides, in particular to a simple bacillus capable of inducing soybean resistance to soybean cyst nematodes and its application.

Background technique

Soybean Cyst Nematode (SCN) is caused by the infection of soybean cyst nematode ( Heterodera glycines ), and is one of the most serious diseases harmful to soybean production in the world. The disease has the characteristics of wide distribution, severe damage, wide host range, multiple transmission routes, and long survival time of dormant bodies (cysts). It is a soil-borne disease that is extremely difficult to control. In recent years, with the continuous expansion of soybean cultivation area in my country, the distribution of soybean cyst nematodes has become wider and wider. It mainly occurs in 14 provinces, municipalities and autonomous regions such as the Northeast and Huanghuaihai. %, in severe cases, it can reach 70% to 80%, or even no grains. Soybean cyst nematode is a soil-borne resident endoparasitic nematode that infects the root system of plants and has become a major problem in soybean production all over the world. Most nematicides used in chemical control seriously pollute the environment and destroy the ecological balance. In the past 20 years, with the development of sustainable agriculture and organic agriculture, people have shifted the focus of nematode disease control to biological control.

There are many biological control factors for soybean cyst nematodes, including fungi, bacteria, viruses and protozoa. The use of plant growth promoting rhizobacteria (plant growth promoting rhizobacteria, PGPR) to control soybean cyst nematodes has been studied. Becker et al. reported in 1988 that Pseudomonas and Bacillus were effective against root-knot nematodes. Diseases are preventive. Racke and Sikora reported in 1992 that the rhizosphere bacteria Bacillus sp . and B. cereus are effective in the control of southern root-knot nematode ( Meloidogyne incognita ), soybean cyst nematode ( Heterodera glycines ), corn cyst nematode ( Heterodera zeae ) and oat cyst nematode ( Heterodera avenae ) larvae are effective. Bacillus sp. bacteria can produce a variety of antibacterial substances, including lipopeptides, peptides, phospholipids, phage-like particles and bacteriocins, etc., which have antibacterial, fungal, virus and mycoplasma functions. Moreover, bacillus is easy to reproduce and has strong ability to adapt to the environment; most bacillus are non-toxic to humans and animals, and have strong environmental adaptability and environmental friendliness, so they play an important role in biological control and have great development potential. use value. Relevant studies have shown that Bacillus simplex has growth-promoting and biocontrol effects, and its control rate for Chinese cabbage soft rot is as high as 83%, while increasing the yield of Chinese cabbage by 50%, while the control of Bacillus simplex for soybean cyst nematode is still Not yet reported.

Aiming at this problem in production, a bacterium was obtained through systematic screening, and the bacterium or its metabolites were used to treat soybean seeds, which could induce soybean resistance to soybean cyst nematodes during seed germination and growth, thereby solving the problem problem of soybean cyst nematode.

Contents of the invention

The purpose of the present invention is to select a bacterial strain that can induce seedling soybeans to produce obvious resistance to cyst nematodes after soybean seeds are treated, and after the bacterial strain is cultured by conventional liquid fermentation, the metabolites of the bacteria can be used to develop a soybean treatment A seed treatment for seeds, soybean seeds treated to induce resistance to soybean cyst nematodes.

A bacterial strain obtained by screening in the present invention is Bacillus simplex Sneb545, depository unit: China Microbiological Culture Collection Management Committee General Microbiology Center; address: Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing; Deposit date: October 11, 2012; deposit registration number: CGMCC No.6650. Bacillus simplex ( Bacillus simplex ) Sneb545 is a bacterial strain with strong inducing activity to soybean screened from the experimental field of Shenyang Agricultural University in Shenyang City, Liaoning Province, China.

The present invention has found through research that many strains of soil bacteria have a certain ability to induce soybean resistance to cyst nematodes. From these bacteria, a strain with relatively good properties - Bacillus simplex ( Bacillus simplex ) Sneb545 is selected for conducted further research.

The present invention also relates to a bacterial metabolite for inducing soybean resistance against cyst nematodes, which is obtained from the bacterial strain Bacillus simplex ( Bacillus simplex ) Sneb545 of the present invention through conventional liquid fermentation culture.

Bacterial strain Bacillus simplex ( Bacillus simplex ) Sneb545 of the present invention is prepared by expanding fermentation culture after being cultured in a petri dish, and its specific method is as follows:

1. Petri dish culture

The medium formula is beef extract-peptone agar medium (NA): 3g of beef extract, 5g of peptone, 10g of sucrose, 17-20g of agar, and 1000mL of distilled water.

2. Liquid fermentation culture

The formula of the liquid fermentation medium is: by weight percentage, yeast extract 0.1%-0.2%, peptone 0.5%-0.6%, beef extract 0.3%-0.4%, sucrose 1%-2%, the rest is distilled water, pH It is 6.8-7.0.

Inoculate the strain into 250mL Erlenmeyer flask (100mL per bottle) liquid medium, ferment for 2-3d, preferably 3d at 25°C-28°C, shaker speed at 150r·min ^- 1-180r·min ^-1 .

The strains of liquid fermentation can be produced on a large scale through large-scale fermentation tanks, and its products are fermented liquid or finished products of fermented liquid.

The present invention also relates to a soybean seed treatment agent, which contains an effective amount of the Bacillus simplex ( Bacillus simplex ) Sneb545 itself or its metabolites as an effective active ingredient.

The Bacillus simplex ( Bacillus simplex ) Sneb545 or its metabolites of the present invention have inducing activity on soybeans and can be used for soybean seed treatment, and the treated soybeans have a good inhibitory effect on the cyst formation of soybean cyst nematodes, solving Solved the problem of soybean cyst nematode.

Detailed ways:

The following examples are given in order to further illustrate the invention in more detail and not to limit it.

Embodiment 1 : the cultivation of simple bacillus ( Bacillus simplex ) Sneb545

Firstly, the Bacillus simplex ( Bacillus simplex ) Sneb545 strain was fermented and cultured, and then the metabolites after fermentation and cultured were tested for soybean-inducing activity to determine their inducing effect on soybean.

Bacillus simplex ( Bacillus simplex ) Sneb545 cells were inoculated on the culture medium. The formula of the culture medium was beef extract-peptone medium, that is, the ingredients: 3g of beef extract, 5g of peptone, 10g of sucrose, 17-20g of agar, and distilled water to 1000mL, cultured at 25°C for 2 days.

Then inoculate the strains into a 250mL triangular flask (each bottle contains 100mL) liquid medium. The medium formula is: by weight percentage, yeast extract 0.1%-0.2%, peptone 0.5%-0.6%, beef extract 0.3% -0.4%, sucrose 1%-2%, the rest is distilled water, the pH is 6.8-7.0, the fermentation temperature is 25-28°C, the shaker speed is 150r·min ^-1 ~180r·min ^-1 , and the fermentation time is 2-3d. The resulting fermentation broth or finished product of the fermentation broth can be used for direct or indirect treatment of soybean seeds. Treated soybean seeds can reduce damage caused by soybean cyst nematode disease.

Embodiment 2 : the fermentation of simple bacillus ( Bacillus simplex ) Sneb545

Basically the same as Example 1, the difference is that the liquid-cultured strains were inoculated into a 1000L fermenter for fermentation, cultured at 25°C, pH was 7.0, and fermented for 3 days.

Example 3 : Verification of Soybean Systemic Resistance Induced by Bacillus simplex ( Bacillus simplex ) Sneb545

The induced systemic resistance of strain Sneb545 to soybean cyst nematode was explored by split-root system. In the root splitting test, the challenge root system and the response root system were completely isolated from each other in space, and the Sneb545 fermentation broth in the response root system had no direct contact with soybean cyst nematodes, and the control effect of the strain on nematodes was completely dependent on its induced resistance to plants. The assay method is reliable and has been widely used in the research of system-induced resistance of biocontrol microorganisms to soil-borne diseases.

1. Preparation of preparations for testing

The fermentation liquid of Bacillus simplex strain Sneb545 was prepared according to the aforementioned liquid fermentation culture method, and was set aside.

2. Preparation of Nematodes for Experiments

Soybean cyst nematode ( Heterodera glycines ): Race 3 of soybean cyst nematode was obtained from the experimental base of the Northern Nematode Research Institute of Shenyang Agricultural University. Cysts were separated from soil samples by improved elutriation-sieving method, and fresh, plump, mature and uniform cysts were picked under a stereoscope. The cysts were first sterilized with 0.5% sodium hypochlorite solution for 3 minutes, then rinsed with sterile water for 5 times, placed in a petri dish containing a small amount of sterile water, and cultured in a 25°C incubator. The second instar larvae were obtained after 15 days of shallow dish incubation. The 2nd instar larvae suspension was adjusted to 200/ml.

3. Preparation of Seeds for Experiments

Liaodou 15: provided by Liaoning Academy of Agricultural Sciences.

4. Seed treatment method

Soybean seeds were first sterilized with 5% sodium hypochlorite solution for 5 minutes, and then rinsed with sterile water for 5 times.

5. Test method

Use greenhouse potting treatment, take soil and sand without soybean cyst nematodes, sterilize in a dry heat sterilizer at 165°C for 2 hours, and then mix and load them with a ratio of V (soil): V (sand) of 2:1 16×16cm plastic bowl. Sow 3 Liao beans and 15 seeds in each pot, and when the bean sprouts grow to two cotyledons, leave 1 plant in each pot. The bean sprouts are taken out and washed, and the root system is divided into two equal parts. Take 3 plastic bowls of the same size and place them in the shape of "pin", and place the plastic bowls at the bottom in two trays with a diameter of 16 cm. Two strips of 1cm×2cm are opened at the bottom of the plastic pot on the top, and the seedlings are placed in the plastic pot on the top, so that the separated roots can reach the soil of the two plastic pots at the bottom through two cracks, and grow in the two plastic pots at the bottom The root systems of the three pots were the challenge root system and the response root system, respectively, and the soil matrix was loaded into the three pots to fix the plants. The system was placed in a greenhouse (25°C, 14h light), and 10ml of Sneb545 fermentation broth was inoculated below 2cm of topsoil in the plastic bowl of the challenging root system. One week after the inoculation of the Sneb545 fermentation broth, 10ml of the J2 suspension was inoculated into the responding root system. With sterile water as the control, a total of 2 treatments were repeated 3 times. After 35 days, the amount of cysts in the soil in the root pot and the total number of soybean cyst nematodes in the roots were investigated.

5.1 Separation and counting of cysts in soil: Weigh 200cc of each soil sample (i.e., fill a 1000mL beaker with 800mL of water and add soil to 1000mL). The number of cysts was recorded under a dissecting microscope.

5.2 Dyeing and counting of nematodes in the root: clean the root system with running water, weigh the fresh weight of the root, then use sodium hypochlorite-acid fuchsin staining method to stain the nematodes in the root, observe and record the number of nematodes in the root under a stereomicroscope, and Folded into the number of nematodes per gram of root fresh weight.

5.3 Calculation of control effect

Cyst inhibition rate (%) =

Nematode reduction rate (%) =

6 Test results

The assay showed that Sneb545 could induce soybean to produce strong resistance to soybean cyst nematode. After the strain Sneb545 was inoculated in the challenge root system, the invasion of soybean cyst nematode in the response root system was significantly reduced, and the number of cysts in the response root soil was significantly reduced. Response root soybean cyst nematode invasion decreased by 51.27%, soil cysts decreased by 65.82% (Table 1).

Table 1 The total number of invasive nematodes and soil cysts in the responding roots after inoculation with Sneb545

deal with The number of cysts in the soil Number of soybean cyst nematodes in roots Sneb545 9.0000±2.6458bB 32.0000±4.3589bB CK 26.3333±2.6034aA 65.6667±5.6075aA

Note: The survey results after 35 days, the letters after the numbers are the results of Duncan’s new multiple range test, English in different capitalization

Letters indicate extremely significant difference (P < 0. 01) and significant difference (P < 0. 05) respectively.

Embodiment 4 : the field control soybean cyst nematode effect test of simple bacillus ( Bacillus simplex ) Sneb545

Use the fermented liquid stock solution to treat the coated soybean seeds at a ratio of 1:100. After drying, the treated soybean seeds are normally sown on soybean cyst nematode disease plots. After the soybean seeds are treated with Sneb545 metabolites, the seedling stage can be induced Soybean has obvious resistance to cyst nematodes, and has obvious inhibitory effect on the formation of soybean cyst nematode cysts. It is a potential biocontrol bacterium and opens up new ideas for the prevention and treatment of soybean cyst nematodes.

1. Preparation of preparations for testing

The fermentation liquid of Bacillus simplex strain Sneb545 was prepared according to the aforementioned liquid fermentation culture method, and was set aside.

2. Preparation of seeds for testing

Liaodou 15: provided by Liaoning Academy of Agricultural Sciences.

Hefeng 50: provided by Daqing Branch of Heilongjiang Academy of Agricultural Sciences.

3. Seed treatment method

Soybean seeds were first sterilized with 5% sodium hypochlorite solution for 5 minutes, and then rinsed with sterile water for 5 times. The prepared fermented liquid is treated with seed coating with 1% seed amount, and it is set aside after being dried.

4. Test method

Soybean was inoculated with natural diseased soil in the field. Five repeated experiments were carried out in Kangping County, Liaoning Province and Daqing City, Heilongjiang Province. The soybean fields are all heavy-cropped soybean fields. The Kangping test base has been cropped for 3 years, and the field cyst volume is 40 cysts/100ml soil. The Daqing test base has been cropped for more than 6 years, and the field cyst volume is 55 cysts/100ml soil. Field plots were designed with 3 treatments, 5 repetitions, a total of 15 plots, arranged in random blocks, 5 ridges per plot, 5 meters long, 1 meter aisle, 100 seeds per ridge, and guard rows at the edge of the plot. Taking uncoated soybean seeds and soybean seeds coated with NA liquid culture solution as controls, 35 days after soybean sowing, after the growth and development of soybean cyst nematodes broke through the soybean root surface, 20 seedlings were randomly selected from each plot, and removed. After topsoil (0-5cm), dig out the roots of the plants to keep the root system intact, investigate the amount of cysts attached to the root system of a single soybean plant, and calculate the cyst inhibition rate.

5. Test results

For the soybean treated with Sneb545 strain, the root cyst growth was investigated during the cystogenesis stage of soybean cyst nematode, and the soybean seedlings treated with the bacterial strain Sneb545 had a significant inhibitory effect on the cyst formation of soybean cyst nematode, as shown in the table 2. It can be seen that the inhibition rate of Sneb545 in Daqing and Kangping reached 57.67% and 47.92%, respectively. The results of coated soybeans in Kangping and Daqing were somewhat different, which may be related to the physiological differences of soybean cyst nematodes in different regions. related to species differences.

Table 2 Effect of coating treatment of soybean seeds with Sneb545 strain on the formation of soybean root cysts

deal with Daqing, Heilongjiang Liaoning Kangping Sneb545 39.2±3.6387bBC 45.35±4.7315bcdB NA 93.2±5.0735aA 84.34±5.8726aA CK 92.6±3.7762aA 87.08±8.1215aA

Note: The survey results after 35 days, the letters after the numbers are the results of Duncan’s new multiple range test, English in different capitalization

Letters indicate extremely significant difference (P < 0. 01) and significant difference (P < 0. 05) respectively.

Claims (7)

1. the bacterial isolates that has inducing soybean Chinese People's Anti-Japanese Military and Political College beans Cyst nematode behind the strain processed soybeans seed is characterized in that: this bacterial strain be simple genus bacillus ( Bacillus simplex), being deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on October 11st, 2012, preserving number is CGMCC No.6650.

2. the liquid fermentation process of bacterial isolates claimed in claim 1, it is characterized in that: the liquid fermentation medium prescription is: by weight percentage, yeast extract 0.1%-0.2%, peptone 0.5%-0.6%, beef extract 0.3%-0.4%, sucrose 1%-2%, remainder is distilled water, pH is 6.8-7.0, leavening temperature is 25-28 ℃, shaking speed 150rmin ^-1～180rmin ^-1, fermentation time 2-3d.

3. liquid fermentation process according to claim 2, it is characterized in that: described fermentation time is 3d.

4. has the preparation method that inducing soybean produces the fermented liquid stoste of Chinese People's Anti-Japanese Military and Political College's beans Cyst nematode resistance, it is characterized in that: formed through behind the liquid fermenting by bacterial isolates claimed in claim 1, the liquid fermentation medium prescription is: by weight percentage, yeast extract 0.1%-0.2%, peptone 0.5%-0.6%, beef extract 0.3%-0.4%, sucrose 1%-2%, remainder is distilled water, pH is 6.8-7.0, and leavening temperature is 25-28 ℃, shaking speed 150rmin ^-1～180rmin ^-1, fermentation time 2-3d.

5. preparation method according to claim 4, it is characterized in that: described fermentation time is 3d.

6. a soybean seeds treatment agent is characterized in that, comprises formed fermented liquid stoste in the liquid fermentation process according to claim 2 of significant quantity, is used for inducing soybean and produces resistance to soybean cyst nematode Heterodera glycines.

7. the application of fermented liquid stoste in inducing soybean Chinese People's Anti-Japanese Military and Political College beans Cyst nematode that forms in the described liquid fermentation process of claim 2.