CN117925481A - Tu Yang Yabao bacillus WYJ-3 and application thereof - Google Patents

Abstract

Tu Yang Yabao bacillus WYJ-3 and application abstract thereof: the plant rhizosphere growth promoting bacterium is a strain of soil Yang Yabao bacillus WYJ-3 separated from green sword bean rhizosphere soil, the soil Yang Yabao bacillus WYJ-3 is named Bacillus toyonensis, and the soil Yang Yabao bacillus WYJ-3 is preserved in China general microbiological culture collection center (CGMCC) with a preservation number of 26913. Tu Yang Yabao bacillus WYJ-3 has the ability to produce ferrite and produce auxin indoleacetic acid (IAA). The soil Yang Yabao bacillus WYJ-3 can improve the root system activity, plant height, root length, plant dry/fresh weight and yield of green sword beans. Meanwhile, the soil Yang Yabao bacillus WYJ-3 can obviously increase the iron content of green sword beans, and has a good inhibition effect on Fusarium solani. The soil Yang Yabao bacillus WYJ-3 has strong defensive power against green sword bean root rot, and can also improve the yield and quality of green sword beans under the field condition.

Description

Tu Yang Yabao bacillus WYJ-3 and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a soil Yang Yabao bacillus WYJ-3 and application thereof.

Background

Green sword beans are used as important components in vegetables, have rich juice, good palatability and high yield, are deeply favored by people in China, and have a main dominant position in the economic development in northern China. In recent years, as the planting area of green sword beans is continuously increased, the demand of chemical fertilizers is also increased. However, long-term application of the fertilizer can not only affect the nutrient absorption efficiency of plants, but also have a certain negative effect on the soil structure. Meanwhile, the normal growth of green sword beans can be influenced by the continuous increase of pathogenic microorganisms in the soil. The green sword bean root rot caused by Fusarium solani is a soil-borne disease which is destructive to green sword beans, and the yield and quality of the green sword beans are seriously damaged.

At present, chemical methods such as chemical reagents, pesticides and the like can be adopted to solve the problem. However, the use of chemical pesticides has different negative effects on human beings, livestock and the environment, and causes problems of increased drug resistance of pathogenic bacteria, reduced food safety and the like. The microbial control is a disease control method for inhibiting the activity of pathogenic matters through various modes such as parasitism, bacteriolysis, resistance activation and the like under the action of microorganisms. The microbial control has the advantages of sufficient resources, low investment, no pollution and the like. At present, microbial preparations have become research hotspots, and how to effectively increase the yield of beans, improve the quality of beans and relieve the soil condition has become the focus of research of current agricultural microorganisms. The invention takes green sword beans in Heilongjiang province as main test materials, separates and screens out the product with good capacity of producing indoleacetic acid (IAA) and siderophores, and lays a theoretical and practical foundation for further development and application of the growth-promoting microbial inoculum for agriculture.

The beneficial plant bacteria and the host plant form a reciprocal symbiotic relationship through interaction in the long-term evolution process, namely, the plant provides nutrition and a relatively suitable growth environment for the bacteria, and the bacteria promote the growth of the plant through self secretion substances or promoting the plant to produce secondary metabolites. Some plant growth promoting bacteria can promote the adaptation of host plants to stress by secreting indoleacetic acid (IAA) and having the capabilities of dissolving potassium, producing ferrites, dissolving inorganic phosphorus and the like, so that the plant growth is promoted. Compared with the traditional fertilizer, the microbial agent has the advantages of safety, no pollution, soil improvement, root rot prevention and treatment, contribution to realizing sustainable development of agriculture and the like. In recent years, development and application of microbial fertilizers in China have been greatly developed, but still high-efficiency strains are required to be further screened to further expand the application range of the microbial fertilizers.

Disclosure of Invention

The invention aims to overcome the problems of serious environmental pollution caused by chemical fertilizers for promoting plant growth in the prior art, and provides a preparation method and application of a green microbial agent. The invention provides Bacillus toyonensisWYJ-3 which has good stability and capacity of producing ferrite and indoleacetic acid (IAA), and can promote the growth of green sword beans under the condition of reducing the application of chemical fertilizers.

The invention is realized by the following technical scheme:

the strain producing the ferrites is separated from green sword bean rhizosphere soil growing in farmland soil, the strain with the ferrites producing capacity is firstly obtained through preliminary screening on an LB culture medium with the pH value of 7.2, and then the strain with the highest ferrites producing capacity is screened through secondary screening of a CAS culture medium.

The strain WYJ-3 shows a strong ability to secrete indoleacetic acid (IAA) and produce ferrites. By reducing the application of chemical fertilizers, the negative effect of soil pollution on green sword beans is improved. Indoleacetic acid (IAA) can promote plant growth, and the bacterial inoculation of green sword bean can obviously promote plant growth, so that the root system activity, plant height, dry/fresh weight and yield of green sword bean are improved, and the quality of green sword bean is improved. Meanwhile, the strain also has the capability of effectively preventing and treating root rot.

Bacillus toyonensisWYJ-3 has the following characteristics:

The microbial inoculum Bacillus toyonensisWYJ-3 can synthesize indoleacetic acid (IAA) and produce ferrite, can effectively promote the absorption of plants to nutrient substances and promote the growth of plants under the condition of reducing the application of chemical fertilizers. Meanwhile, the yield of crops can be obviously improved, and the quality of the crops can be improved. Bacillus toyonensis WYJ-3 is a beneficial microbial agent, and the strain is separated from rhizosphere soil of green sword beans, so that the defect that chemical agents generate secondary harm to the environment is overcome, a series of environmental problems cannot be caused by the use of the strain, pollution-free production of green sword beans is facilitated, and the strain is suitable for development and application as an agricultural microbial agent.

The invention performs the following preservation on the strain:

Preservation time: 2023, 3, 28, deposit: chinese, beijing. The China general microbiological culture Collection center (CGMCC) of China national academy of sciences microbiological culture Collection center (CGMCC) of China, the Korean area North Star, west Lu No. 1, 3 of Beijing; the preservation number of the soil Yang Yabao bacillus Bacillus toyonensisWYJ-3 is CGMCC No. 26913.

Drawings

FIG. 1 is a ferriphilic diagram of B.terreus WYJ-3 (Bacillus toyonensis) on cromolyn CAS medium.

FIG. 2 shows the growth of Bacillus Tuberosus WYJ-3 (Bacillus toyonensis) on LB medium.

FIG. 3 shows the indoleacetic acid (IAA) synthesis content of Bacillus Tuber WYJ-3 (Bacillus toyonensis).

FIG. 4 is an effect of Bacillus terrae WYJ-3 (Bacillus toyonensis) on green sword bean root system viability.

FIG. 5 is a graph showing the effect of Bacillus terrae WYJ-3 (Bacillus toyonensis) on yield of green sword bean.

FIG. 6 is a graph showing the effect of Bacillus terrae WYJ-3 (Bacillus toyonensis) on iron content of green sword bean.

FIG. 7 shows the inhibition of Fusarium oxysporum by Bacillus terrae WYJ-3 (Bacillus toyonensis).

FIG. 8 index of disease and relative control of green sword bean after inoculation with Bacillus Tuber WYJ-3 (Bacillus toyonensis).

Description of the embodiments

The invention will now be described in further detail with reference to the specific drawings and examples, which are given by way of illustration and not limitation. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. In the following quantitative experiments, three or more repeated experiments were performed, and the results were averaged.

1. Isolation and characterization of Bacillus Tu-like WYJ-3 (Bacillus toyonensis)

1. Isolation of the Strain of Bacillus Tu-like WYJ-3 (Bacillus toyonensis)

(1) Soil sample collection

The rhizosphere soil of the green sword bean to be tested is taken from the place after Harbin university of Heilongjiang province, and the obtained plant with the rhizosphere soil is put into a fresh-keeping bag prepared in advance and is brought back to a laboratory for 4 ℃ for storage for later use.

(2) Screening and purification of strains

Cleaning rhizosphere soil of green sword bean by a brush, weighing 1 g rhizosphere soil sample, placing the rhizosphere soil sample into a conical flask containing 99 mL sterile water and glass beads, oscillating by a shaking table for 30 min, standing, transferring into an ultra-clean workbench, taking supernatant, sequentially diluting the uniformly mixed rhizosphere soil sample by a 10-time method according to 10 ^-3、10^-4、10^-5、10^-6 concentration to prepare soil suspension, sequentially mixing the diluted soil sample uniformly, sucking 0.1 mL, coating the diluted soil sample on a chromium-aohol CAS culture medium plate, repeating each dilution gradient for 3 times, and inversely culturing in a 30 ℃ constant temperature incubator for 1-3 d. Selecting single colony with orange halo, performing plate streak purification, and repeating until pure culture is obtained. And colonies were picked and inoculated on LB solid medium for preservation at 4℃and prepared glycerol bacteria were preserved in small tubes at-80℃as shown in FIG. 1.

2. Identification of Strain WYJ-3

(1) Molecular characterization

Genomic DNA extraction was performed on strain WYJ-3, and PCR amplification and sequencing of 16S rDNA were performed.

The primers used for PCR amplification were:

F8：5'-AGAGTTTGATCCTGGCTCAG-3'

R1541：5'-AAGGAGGTGATCCAGCCGCA-3'

The PCR amplification conditions were: 3min at 94 ℃;94℃30 s, 55℃30 s, 72℃90 s,30 cycles; the reaction was circulated indefinitely at 72℃for 10 min and 4 ℃. And sequencing the PCR amplification product, wherein the sequencing result is shown in a sequence table.

(2) Morphological identification

Bacterial strain WYJ-3 was colony opaque, colony flat, milky white, semi-dry, translucent, colony edge clean and pigment free on LB medium (fig. 2).

(3) The physiological and biochemical identification results are shown in table 1:

Table 1: physiological and biochemical identification results

Note that: positive +; negative.

2. Synthesis content determination of indoleacetic acid (IAA) of soil Yang Yabao bacillus WYJ-3 (Bacillus toyonensis) strain

The strain WYJ-3 bacterial suspension is inoculated into DF culture medium for culturing 2 d, 10% of the bacterial suspension is taken to be cultured 2 d in DF with three concentrations of 0, 100, 200 and 500 mug/mL respectively, each treatment is repeated for 3 times, the synthesis amount of the bacterial strain indoleacetic acid (IAA) is measured, and the absorbance value of bacterial solution OD ₆₀₀ is measured. The remaining bacterial suspension was centrifuged, and the supernatant was added with 2mL S reagents, dark reaction 20 min, and absorbance was measured 535 and nm. DF blank control (fig. 3) was used.

Different lower case letters indicate significant differences between treatments （P <0.05）.Different lowercase letters mean the significant differences among treatment（P <0.05）.

As a result, as shown in FIG. 3, the IAA synthesis amounts of the test strains all increased with the increase in the L-Trp concentration. WYJ-3 has the capacity of synthesizing indoleacetic acid (IAA) at the concentration of L-Trp of 0, and the synthesis amount is 41.58 mug/mL; at L-Trp concentrations of 100, 200 and 500. Mu.g/mL, the amount of WYJ-3 synthetic IAA was 46.98. Mu.g/mL, 51.90. Mu.g/mL and 55.86. Mu.g/mL, respectively.

3. Field inoculation test of Tu Yang Yabao bacillus WYJ-3 (Bacillus toyonensis)

Bacterial strain WYJ-3 bacterial suspension is inoculated in LB culture medium for overnight culture, bacterial cells are collected by centrifugation and resuspended in sterile water, and the bacterial concentration OD ₆₀₀ = 0.5 +/-0.04 is regulated. Selecting full healthy green sword bean seeds with consistent size and no diseases, carrying out surface disinfection, soaking the seeds in 75% ethanol for 3min times, washing the seeds with sterile water for 3 times, soaking the seeds in a 2% sodium hypochlorite solution for 30 s times, washing the seeds with sterile water for 5 times, treating the seeds with bacterial suspension, treating the seeds with a control group with sterile water only, culturing the seeds at room temperature until the seeds germinate, and sowing the seeds in a field.

The field to be tested is a farm garden of Harbin university of Heilongjiang province, 10 seedlings are planted in each ridge, and 3 germinated seeds are planted in each hole. The experimental group was poured once every 7 d bacterial suspensions, and the control group was poured with an equal amount of sterile water. And (5) culturing in a field, and properly supplementing water. The seed of the strain was evaluated for its effect on the growth of green sword bean in a field environment. Sampling after planting 30 d, measuring plant root system activity, leaf chlorophyll content and plant biomass, and measuring pod yield and quality of green Canavalia gladiata after 90: 90 d is mature.

① Determination of root Activity

Root tip samples were weighed into 10 mL beakers, added with equal amounts of the mixed solution 10 mL of the 0.4% TTC solution and phosphate buffer, fully immersed in the solution, and incubated at 37℃in the dark for 1-3 h. 1 mol/L of sulfuric acid 2mL was then added to stop the reaction. And simultaneously, a group of blank experiments are carried out, sulfuric acid is added firstly, then root samples are added, the operation method is the same as above, then the roots are taken out, the absorbed moisture is added into 3-4 mL ethyl acetate and a small amount of quartz sand, and the mixture is ground in a mortar to extract the triphenylmethyl hydrazone. The red extract was transferred into a test tube, the residue was washed three times with a small amount of ethyl acetate, the test tube was transferred into the test tube, finally ethyl acetate was added to a volume of 10 mL, the color was compared with a spectrophotometer OD ₄₈₅ wavelength, absorbance was measured by a blank experiment as a reference, and the standard curve was checked to determine the TTC reduction (FIG. 4).

* Indicating that the difference was significant at P <0.05 level. * Indicating that the difference was significant at a level of 0.01< p < 0.05. * Indicating that the difference was significant at the level of 0.001< p < 0.01.

As shown in FIG. 4, inoculation with microbial inoculum WYJ-3 increased root activity by 158.24% compared to the Control (CK) group without inoculation.

② Determination of the amount of biomass

TABLE 2 Effect of Tu Yang Yabao Bacillus WYJ-3 on green sword bean biomass

The results showed that the plant height, root length, fresh weight above ground, fresh weight below ground, dry weight above ground and dry weight below ground of green sword bean after inoculation with the microbial inoculum WYJ-3 were increased by 58.38%, 77.01%, 47.80%, 82.25%, 31.75% and 74.58%, respectively, compared to the control group (CK) without inoculation.

③ Yield of green sword bean

The results showed that inoculation with WYJ-3 inoculum increased the yield of green Canavalia by 32.57% compared to the control group (CK) without inoculation (FIG. 5).

④ Quality of green sword bean

Determination of iron content of green sword bean: oven drying, pulverizing, weighing, placing in a digestion tube, adding concentrated HNO ₃ and concentrated H ₂SO₄, and placing 12H. And (3) placing the digestion tube on a digestion furnace, boiling until the solid is completely dissolved into liquid, and cooling the solution to be light yellow. Respectively placing the clear solutions into beakers, adjusting the pH to 4-6 with NaOH solution, respectively transferring into 50mL volumetric flasks, adding water to the scale, and shaking to obtain the sample solution to be measured. Samples were accurately removed 5mL each, treated as described above, absorbance of each sample measurement was measured at OD ₅₁₀, and the iron content of the samples was calculated by the following formula.

Iron content = 10 XV/m

Wherein: x: iron content (μg/mL) in the sample solution to be measured;

m: sample mass (g);

v: total volume of sample fluid to be measured (mL)

The results showed that inoculation of the inoculum WYJ-3 increased the iron content of green Canavalia by 87.88% compared to the control group (CK) without inoculation (FIG. 6).

Inhibition of Canavalia gladiata root rot by Tu Yang Yabao bacillus WYJ-3 (Bacillus toyonensis)

1. Plate counter experiment for antagonizing plant pathogenic fungi by isolated strain

After the soil Yang Yabao bacillus WYJ-3 (Bacillus toyonensis) was activated at 37 ℃ on PDA medium, the plant pathogenic fungus Fusarium solani (Fusarium solani) was punched out of the stipe with an 8mm puncher and placed in the center of PDA medium, the strain WYJ-3 was inoculated at 2 cm from the edge of the dish, the dish was incubated at 28 ℃ for 3-5 d, and whether or not a zone of inhibition was generated around the colony was observed (fig. 7).

As can be seen from FIG. 7, soil Yang Yabao bacillus WYJ-3 (Bacillus toyonensis) acts as an inhibitor for Fusarium solani.

The calculation formula of the bacteriostasis rate is as follows:

TABLE 3 WYJ-3 inhibition of Fusarium solani growth

Pathogenic bacteria	Fusarium solani (L.) Kuntze
		Antibacterial rate	67.21%

Field test of soil Yang Yabao bacillus WYJ-3 (Bacillus toyonensis) for controlling root rot of green Canavalia

The bacterial suspension of the WYJ-3 microbial inoculum is used for soaking seeds to prevent and treat the green sword bean root rot, and the prevention and treatment effect on the green sword bean plant root rot is obvious. The control group is soaked in sterile water, and the plant planted in the mixed soil has serious infection degree. Wherein the middle stem forms a large area of decay.

Green sword bean root rot grading standard:

Level 0: healthy, root neck has no disease spots; stage I: small light brown lesions 25% of the principal root; stage II: 25% -49% of primary roots of brown lesions; class III: 50-74% of the principal root is brown spots and principal root contractions; grade IV: 75% -100% of main roots are brown lesions, few lateral roots, withered plants, short plants or dead plants.

The disease index and relative control effect of each treatment group are calculated according to the disease index grading standard.

As shown in the graph, in the green sword bean seedling stage, the roots of the control group plants treated by the sterile suspension show large-area brown lesions, and the disease degree is serious. The treated group inoculated with the isolated bacterial suspension WYJ-3 was less diseased and the relative control of WYJ-3 was 21.03% (FIG. 8).

The invention discloses a strain of soil Yang Yabao bacillus WYJ-3, belonging to the field of microorganisms. The soil Yang Yabao bacillus WYJ-3 is named Bacillus toyonensis and is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.26913. The soil Yang Yabao bacillus WYJ-3 has the capability of producing ferriphilic and indoleacetic acid (IAA). WYJ-3 can be inoculated to green sword bean to improve the biomass and the quality such as iron content. Meanwhile, the compound fertilizer has a certain effect on preventing and treating green sword bean root rot. The strain is suitable for being developed and utilized as an agricultural microbial inoculum.

Claims (8)

1. The strain of soil Yang Yabao bacillus WYJ-3 is characterized in that the strain of soil Yang Yabao bacillus WYJ-3 is named Bacillus toyonensis and is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.26913.

2. Use of the soil Yang Yabao bacillus WYJ-3 of claim 1 as a ferrite-producing agent.

3. The use according to claim 2, wherein the microbial inoculum is a microbial inoculum having a ferritic philic producing capacity and also having an indoleacetic acid (IAA) synthesis promoting capacity.

4. The use of the soil Yang Yabao bacillus WYJ-3 as claimed in claim 1 for the preparation of a fungicide for promoting the yield, improving the quality and helping the control of plant diseases caused by plant pathogenic fungi of green sword beans.

5. The method according to claim 4, wherein the bacterial preparation is synthesized by using a bacterial preparation which produces ferrite and indoleacetic acid (IAA).

6. The use of the soil Yang Yabao bacillus WYJ-3 as claimed in claim 1 for the preparation of a fungicide for promoting the yield of green sword beans, improving the quality thereof and helping the green sword beans to prevent and treat plant diseases caused by plant pathogenic fungi.

7. The use according to claim 1, characterized in that the bacterial preparation prepared is a ferrite-producing bacterial preparation having indoleacetic acid (IAA) synthesis capacity.

8. Use of the soil Yang Yabao bacillus WYJ-3 as claimed in claim 1 as a ferrite-producing microbial agent.