CN114015614B - Actinomycete strain SCAUT013 and its application - Google Patents

Abstract

The invention discloses an actinomycete strain Micrococcus luteus SCAUT013 and application thereof, wherein the actinomycete strain Micrococcus luteus SCAUT013 is stored in Guangdong province microorganism strain collection center, and the storage number is GDMCC NO: 61507. the Micrococcus luteus SCAUT013 provided by separation and screening has the growth promoting capabilities of generating IAA, siderophores, dissolving phosphorus, degrading cellulose, chitin and the like, and simultaneously has the stress resistance functions of salt resistance, acid and alkali resistance, drought resistance and the like.

Description

Actinomycete strain SCAUT013 and its application

technical field

The invention belongs to the field of microorganisms, and in particular relates to an actinomycete strain SCAUT013 and its application.

Background technique

Liangshan Prefecture is one of the three major pastoral areas in Sichuan Province. Animal husbandry has a long history and is one of the main sources of cash income for farmers and herdsmen. Especially in high mountain areas, cash income from animal husbandry accounts for more than 70% of rural household income. Because Liangshan Prefecture has a dry climate in winter and spring, rainy summer, and distinct wet and dry climate, the problem of livestock lack of green fodder during winter and spring is very prominent. However, in the basin and surrounding mountainous areas of Liangshan Prefecture, especially in the Zhongshan and sub-alpine mountains with an altitude of 1700-3200m, the land is largely idle during winter and spring. Vicia villosa var.glabresens (Vicia villosa var. glabresens) is an annual or perennial herb of the genus Leguminosae. It is the most suitable and has the advantages of water conservation, fertilizer conservation, soil improvement, nitrogen fixation and fertilizer enhancement, and ecological environment improvement. Liangshan prefecture has a long history of planting S. japonica. It can be planted in idle stubble in winter, which can make full use of light and heat resources and preserve moisture, increase the content of organic matter in the soil, improve soil quality, and at the same time be very efficient. It solves the problem of lack of green fodder for sexual animals during winter and spring.

In recent years, excessive application of chemical fertilizers has not only polluted the environment, but also caused a series of problems such as soil compaction, decline in fertility, and decline in the quality of agricultural products. Soil microorganisms play an important role in the process of material recycling, organic matter decomposition and plant nutrient utilization. Plant growth promoting rhizobacteria (PGPR), which naturally exist in soil and colonize plant rhizosphere, can not only improve soil physical and chemical properties, but also It can also promote plant growth by producing growth hormone, nitrogen fixation, and phosphorus dissolution. Secondly, PGPR can also resist pathogen infection by producing chitinase, cellulase, antibiotics and other substances, and promote plant growth by improving the stress resistance of plants. . Although A. chinensis has the ability to nodulate and fix nitrogen in symbiosis with rhizobia, the biological nitrogen fixation process and the growth process of A. chinensis also require the participation of other nutrients. The rhizosphere growth-promoting bacteria PGPR can remove the soil that cannot be planted by plants. The utilized inorganic elements transform the organic matter utilized by plants to promote the supply and growth of plant nutrients. Since A. glabrata is a leguminous forage, previous studies have mainly focused on the symbiotic relationship between rhizobia and A. glabrata, and the effect of rhizosphere growth-promoting bacteria on the growth of A. glabrata has not yet been reported. .

Actinomycetes are an important part of the plant rhizosphere microbial community, and play an important role in the transformation of soil materials. Fungal metabolites promote plant growth and have great application potential in agricultural production. Taking the A. chinensis collected in Liangshan Prefecture as the research object, to isolate and screen high-efficiency PGPR strain resources, in order to use the rhizosphere growth-promoting bacteria to improve the stress resistance of A. chinensis, and to collect excellent PGPR strain resources for the development of microbial fertilizers, Maximize the ecological benefits and economic value of A. chinensis, which is in line with the sustainable development of agriculture and animal husbandry and the needs of ecological environmental protection in Liangshan Prefecture.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is: in order to excavate and screen the PGPR strain resources that can promote the growth of A. chinensis, and apply it in agricultural production, one of the objects of the present invention provides a strain with anti-stress and growth-promoting functions. The rhizosphere actinomycetes, another purpose is to provide the application of this strain.

The technical scheme of the present invention is as follows: an actinomycete strain Micrococcusluteus SCAUT013, referred to as SCAUT013, isolated from the rhizosphere of A. chinensis. The strain has been deposited in the Guangdong Provincial Microbial Culture Collection Center on February 7, 2021, the preservation number is GDMCC NO: 61507, and the preservation address is 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province, Guangdong Province Microorganisms graduate School.

A microbial inoculum or microbial fertilizer containing the actinomycete strain Micrococcus luteus SCAUT013.

Use of the above-mentioned actinomycete strain Micrococcus luteus SCAUT013 or microbial inoculants or microbial fertilizers on the cultivation of Vicia villosa Roth var.glabrescenscv.Liang shan.

Further, the planting refers to planting Vicia villosa Rothvar.glabrescenscv.Liang shan in Liangshan Prefecture.

The morphological characteristics of the strain of SCAUT013 of the present invention: after culturing on ISP4 medium for 3 days at 28°C, the colony is small, round and convex, with neat edges and smooth surface, the colony color is light yellow, easy to pick, and Gram staining is positive. , Aerobic, bulbous, no spores, cell diameter 0.8um.

The 16S rRNA sequence of the SCAUT013 strain of the present invention was determined, and the determined sequence was submitted to the EzBioCloud website (www.ezbiocloud.net) for homology comparison, and MEGA 7.0 software was used for phylogenetic analysis. The results showed that the 16S rRNA nucleotide sequence homology between strain SCAUT013 and Micrococcus luteus was 100%. Based on the morphological characteristics and 16S rRNA sequence homology analysis and other experimental results, SCAUT013 was identified as Micrococcus luteus.

The DNA of the SCAUT013 strain of the present invention is subjected to 16S rRNA gene phylogenetic analysis, which is shown in SEQ ID NO.1 of the sequence table.

The SCAUT013 strain of the present invention has the growth-promoting functions such as IAA production, siderophore production, cellulase production, oligoenzyme production and phosphorus dissolution, and also has stress resistance such as salt tolerance, drought tolerance, acid and alkali tolerance.

The results of sand culture experiment show that after inoculation with Actinomycetes Micrococcus luteus SCAUT013, the plant height, root length, fresh weight and dry weight of aboveground and underground parts, and chlorophyll of A. (CK), increased by 29.54%, 25.38%, 22.39%, 17.74%, 25.27%, 23.52%, and 13.44%, respectively, indicating that the strain SCAUT013 can obviously promote the growth of A. chinensis in Liangshan, and has a good growth-promoting effect. .

The field test results showed that the plant height, root length, fresh weight, dry weight, root weight, nodule number, chlorophyll content and crude protein content of A. It increased by 20.18%, 24.36%, 31.98%, 26.22%, 24.14%, 28.77%, 15.38%, 9.89%, and the yield per mu increased by 30.52% compared with the control, and the incidence of powdery mildew of A. chinensis in Liangshan was lower than that of the non-inoculated plants. The incidence rate decreased by 52.71%; the alkali-hydrolyzed nitrogen, available potassium, available phosphorus and organic matter in the rhizosphere soil of A. chinensis in Liangshan were increased by 19.81%, 12.63%, 10.24% and 5.19% compared with the control. Acid phosphatase, catalase, and sucrase were all increased by 38.60%, 23.48%, 49.07%, and 26.05% compared with the control. The number of culturable bacteria, actinomycetes, fungi and rhizobia in the rhizosphere soil was comparable to that of the uninoculated treatment. Compared with the control group, the richness of bacterial and fungal communities in the rhizosphere soil was significantly increased compared with the control group. After inoculation with actinomycetes Micrococcus luteus SCAUT013 inoculum, it promoted the growth of A. chinensis, increased the nodulation ability, improved the quality of plants, improved the micro-ecological conditions of the soil, and improved the soil quality. The research and development of A. violaceum microbial inoculum provides bacterial resources.

The results of the measurement of agronomic characters, dry matter accumulation and root structure of flue-cured tobacco at the maturity stage of the subsequent crop showed that the plant height, stem circumference, effective leaf number, maximum leaf length and maximum leaf size of the latter flue-cured tobacco were treated with rhizobium inoculation in the previous crop. Compared with the control group, the width and maximum leaf area increased by 12.88%, 17.94%, 14.81%, 16.71%, 14.17% and 20.77%, respectively, and the dry matter accumulation increased by 19.52%. Compared with the control group, the average diameter, root volume and root hair number increased by 14.08%, 20.60%, 12.61%, 18.67% and 21.69%, respectively.

After inoculation with Actinomycetes Micrococcus luteus SCAUT013 inoculum, it promoted the nodulation ability of A. glabrata, improved the quality of plants, improved soil quality, and promoted the growth of subsequent flue-cured tobacco. The research and development of sub-microbial inoculants provide bacterial resources.

Compared with the prior art, the present invention has the following beneficial effects:

The Micrococcus luteus SCAUT013 provided by the separation and screening of the present invention has the growth-promoting ability of producing IAA, siderophore, dissolving phosphorus, degrading cellulose and chitin, etc., and also has the anti-stress functions such as salt resistance, acid and alkali resistance, drought resistance, etc. The application of the Liangshan S. japonica seed can achieve the effect of promoting the nodulation and growth of the Liangshan S. japonica. The invention not only increases the yield of forage grass, but also improves the soil quality and improves the tobacco planting soil, and has the advantages of agricultural production. good application potential.

Deposit information:

Actinomycete strain Micrococcus luteus SCAUT013, referred to as SCAUT013. The strain has been deposited in the Guangdong Provincial Microbial Culture Collection Center on February 7, 2021, the preservation number is GDMCC NO: 61507, and the preservation address is 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province, Guangdong Province Microorganisms graduate School.

Description of drawings

Fig. 1 is the colony morphology and bacterial cell micrograph of Micrococcus luteus SCAUT013 on ISP4 medium;

Fig. 2 is a phylogenetic tree of Micrococcusluteus SCAUT013 phylogenetic tree constructed based on 16S rRNA gene sequence;

Figure 3 shows the effect of Micrococcus luteus SCAUT013 on the rhizosphere actinomycetes of A. chinensis by sand culture in promoting the growth of A. chinensis. The left side is the control without inoculation, and the right side is inoculated with SCAUT013;

Figure 4 Results of field experiments, left: control; right: SCAUT013;

Fig. 5 Bacterial Alpha diversity (A) and fungal Alpha diversity (B) in the rhizosphere soil of A. glabrata in Liangshan.

Detailed ways

The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from commercial channels unless otherwise specified.

Example 1 Isolation and purification of Actinomyces rhizosphere SCAUT013

1.1 Isolation of rhizobacterial actinomycetes from A. chinensis in Liangshan

In 2018, A. chinensis was collected in Huidong County, Liangshan Yi Autonomous Prefecture, Sichuan Province, and the excess soil on the roots was shaken off. The roots were placed in a 50mL sterilized centrifuge tube containing 25mL of phosphate buffer, and treated with ultrasonic waves (150W). After 10 minutes, the roots were taken out under sterile conditions, and the supernatant was removed by centrifugation at 3000g for 10 minutes. The precipitation was the rhizosphere soil, which was diluted in ^10-1 , ^10-2 , and ^10-3 gradients. Separation was carried out on Gao's medium, and three replicates were made for each dilution concentration, and cultured in a constant temperature incubator at 28°C for 5-7 days. Pick out strains with typical actinomycete colony characteristics, streak with dilution plates, and purify until purified strains are obtained. The purified strains were inoculated into ISP ₄ liquid medium, cultured with shaking at 28°C for 7 days, and preserved with 30% glycerol, and then frozen at -70°C.

1.2 Identification of strains

1.2.1 Morphology of strains

The strains were inoculated on ISP4 medium, cultured at 28°C for 5 days, and the colony morphology of the strains was observed.

1.2.2 Molecular identification of strains

(1) Reagents: lysozyme, proteinase K, 10×TAE, 1×TE, 3mol/L sodium acetate (pH4.8-5.2), 70% ethanol, (saturated phenol:chloroform:isoamyl alcohol=25:24: 1), Mix.

(2) Primers

Primer A: 5'-AGAGTTTGATCCTGGCTCAG-3' (same as the 8-27 bases at the 5' end of 16S rRNA); Primer B: 5'-TTAAGGTGATCCAGCCGCA-3' (same as the 1523-1504 bases at the 3' end of 16S rRNA) same basis);

(3) DNA extraction of actinomycetes

Take a small amount of bacteria into a sterile 1.5mL Eppendorf tube, add 20μl (50mg/mL) of lysozyme with a final concentration of 2mg/mL, put it in a 37°C shaker, 200rmp/min, 1-2h; add 20% SDS 50 μL and 5 μL of proteinase K (20 mg/mL), mix well, put into a 55°C shaker, and treat at 200 rmp/min for 1-2 h; add 550 μL (phenol:chloroform:isoamyl alcohol=25:24:1) for extraction, 12000 rmp/ Centrifuge for 10 min, aspirate the supernatant (repeated three times); add 800 μL of absolute ethanol, 80 μL of 3mol/L sodium acetate (pH 4.8-5.2), mix well, precipitate DNA at 4°C for 1 h, 12000 rmp/min, 10 min, discard the supernatant Add 200 μL 70% ethanol, wash the tube wall 1-2 times, 12000rmp/min, centrifuge for 5 min, discard the supernatant; after the ethanol evaporates, add 50 μL 1×TE to dissolve the DNA, and store at -20°C; 1% agar Glycogel electrophoresis detection.

(4) Amplification of 16S rRNA gene in Actinomycetes

16S rRNA gene amplification conditions: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 1 min, annealing at 56°C for 1 min, extension at 72°C for 2 min, 30 cycles, and total extension at 72°C for 10 min. The PCR product was purified by Shanghai Sangon EZ Spin Column PCR Product Purification Kit UN1Q-1 Column PCR Product Purification Kit (SK1142-N), and the purified product was sent to Sangon Bioengineering Co., Ltd. for sequencing.

(5) 16S rRNA gene sequence analysis and phylogenetic tree construction

The sequence obtained after sequencing was used to search for similarity in NCBI using BLAST software, and the 16S rRNA gene sequence of the published strain with the highest similarity was selected as the reference sequence, and Clustal X software was used for multiple sequence alignment analysis, and MEGA7. The phylogenetic tree was constructed by N-J method to determine the taxonomic status of actinomycetes.

1.3 Experimental results

The actinomycete SCAUT013 was isolated from the rhizosphere soil of A. chinensis in Liangshan. After culturing it on ISP ₄ medium for 3 days at 28°C, the colonies were small, round and convex, with neat edges, smooth surfaces, and pale colonies. Yellow, easy to pick, Gram-positive, aerobic, bulb-shaped, without spores, 0.8um in diameter (Figure 1).

The 16S rRNA sequence of the strain SCAUT013 was determined, and the BLAST homology alignment was performed in the NCBI database. The similarity between this strain and Micrococcus luteus was 100%. Based on the experimental results of morphological characteristics, physiological and biochemical characteristics, and 16SrRNA sequence homology analysis, SCAUT013 was identified as Micrococcus luteus (Figure 2).

Example 2 Screening of the growth-promoting function of Actinomyces rhizosphere SCAUT013

2.1 Screening of growth-promoting functions

2.1.1 Determination of ability to produce IAA

Take 20 μL of bacterial suspension into ISP4 liquid medium containing 0.5mol/L tryptophan, repeat 3 times, set blank control, culture at 28°C for 3 days at 120r/min shaker, centrifuge at 8000r/min for 24h, take 1mL of bacterial solution To the supernatant, add 2 mL of IAA color developing solution, react in the dark at 25 °C for 30 min, measure the color at a wavelength of 530 nm, and record the absorbance value. Take the uninoculated liquid medium as the control for zero adjustment, and use the indole acetic acid standard solution with concentrations of 0, 5, 20, 40, and 60 mg/l as the standard as above, and calculate the concentration of IAA in the assay solution.

2.1.2 Determination of siderophore capacity

Dissolve 0.012 g of chrome azure in 10 mL of double distilled water, and mix with 2 mL of 1 mmol/L FeCl ₃ ·6H ₂ O solution to obtain solution a; dissolve 0.015 g of cetyltrimethylammonium bromide in 8 mL Double distilled water to obtain solution b; slowly add solution a to solution b, mix well to obtain dye liquor c; add 20 mL of 10×MM9 salt solution and 6.04 g piperazine diethanesulfonic acid into a solution containing 150 mL of double distilled water Mix evenly in the Erlenmeyer flask and adjust the pH to 6.8 with 50% NaOH, then add 3.2 g of agar powder to obtain medium d; mix dye solution c, medium d, 1 mmoL/L CaCl ₂ , 1 mmoL/L MgSO ₄ , 20% glucose and 10% casein amino acids were sterilized at 115°C for 20min, respectively. When the temperature of each solution dropped to 50-60°C, 200 μL CaCl ₂ , 4 mL MgSO ₄ , 6 mL casein amino acids, and 2 mL glucose were added to the medium d. , and then add the dye solution c along the bottle wall, mix well without generating air bubbles to get the blue detection medium, and pour the plate. The test strains were inoculated with sterile bamboo sticks, each plate was inoculated with 5 bacterial cakes at equal intervals, and each strain was repeated 3 times at 28°C for 3 days, and the size of the orange transparent circle was observed and recorded.

2.1.3 Detection of phosphorus-dissolving ability

The well-grown actinomycete strains were made into bacterial cakes with a sterile hole punch (d=5mm), respectively placed on the PKO medium. After 3 days, the diameter of the transparent circle (D) and the diameter of the colony (d) were measured respectively, and the ratio (D/d) of the two was calculated. The larger the ratio, the more significant the phosphorus-dissolving effect.

2.1.4 Determination of carboxymethyl cellulase (CMCase) activity

The bacterial strain obtained from the primary screening was made into a seed suspension, inoculated in a triangular flask of a re-screening medium with a carbon source of CMC-Na according to 10% of the inoculum, and incubated at 28°C with constant temperature shaking. 9. The carboxymethyl cellulase (CMCase) activity of each strain of 11d was set up in 3 replicates.

(1) Drawing of standard curve

Dry anhydrous glucose at 80°C to constant weight to make 1mg/mL standard glucose solution, take 6 test tubes and add 0, 0.2, 0.4, 0.6, 0.8, 1.0mL of standard glucose solution respectively, add distilled water to 2.0mL, add DNS reagent 1.5mL, boiling water bath for 5min, after cooling, dilute the volume to 25mL, measure the OD value at the spectrophotometer 540nm, and draw a standard curve.

(2) Preparation of crude enzyme solution

The prepared liquid re-screening medium was divided into 250 mL conical flasks, each bottle was 45 mL, and 5 mL of seed bacterial suspension was inserted, and placed in a shaker at 28 ° C for cultivation. In the tube, centrifuge at 10000r/min for 10min to obtain crude enzyme solution.

(3) Determination of enzyme activity

Enzyme activity determination method: Take 0.1 mL of crude enzyme solution and add 1.9 mL of CMC-Na solution with a mass fraction of 1%. Hydrolyze for 20 minutes at a constant temperature of 45 °C, add 1.5 mL of DNS chromogenic solution for boiling water bath for 5 minutes, dilute the volume to 25 mL, measure the absorbance (OD) value at 540 nm, compare it with the standard glucose curve, and calculate the glucose from the OD value. Quantity (m ₁ ). In addition, add 0.1 mL of each supernatant, add 1.9 mL of water, add 1.5 mL of DNS, take a boiling water bath for 5 min, dilute to 25 mL, colorimetrically measure at 540 nm, and measure the amount of glucose (m ₂ ) in the crude enzyme solution. Subtract the amount of glucose (m ₂ ) from the amount of glucose (m ₁ ) to obtain the amount of glucose obtained by degrading 1% CMC solution by CMC enzyme, the amount of glucose A calculated from the optical density value, and calculate the enzyme activity of the strain by formula.

2.1.5 Detection of chitinase production

Using MM-Chitin medium, inoculate the tested strain on the medium, if a hydrolysis circle is generated, it is proved to be a strain with the ability to degrade chitin.

2.2 Anti-stress function screening

2.2.1 Salt tolerance test

The test strains were streaked on ISP ₄ culture at 28°C for 3-4 days, and sterile water was used to make bacterial suspension. With NaCl concentration: 0%, 1%, 3%, 5%, 7%, 7 kinds of ISP ₄ solid medium were prepared, the bacterial suspension was spotted on the medium with a sterile bamboo stick, and repeated 3 times, 28°C Cultivate for 3-4 days, observe the growth of each rhizosphere actinomycetes and record the results. If the strain can grow normally under a certain salt concentration, it means that the strain has the ability to tolerate the salt concentration.

2.2.2 Acid and alkali resistance detection

The test strains were streaked on ISP ₄ culture at 28°C for 3-4 days, and sterile water was used to make bacterial suspension. Based on the formula of ISP4 medium, the pH reaction buffer was prepared with NaOH, KH ₂ PO ₄ , Na ₂ CO ₃ , KCl, NaHCO ₃ , Na ₂ HPO ₄ ·10H ₂ O, sodium citrate, and trisodium citrate. The pH of the medium reaches 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, respectively, and the bacterial suspension is dotted on the medium with a sterile bamboo stick for 3 repetitions, and cultured at 28°C for 3-4 days , observe the growth of each rhizosphere actinomycetes and record the results. If the strain can grow normally under a certain pH, it means that the strain has the ability to resist the pH.

2.2.3 Drought tolerance test

The primary screening test of drought tolerance was conducted using polyethylene glycol (PEG) 6000 to simulate drought conditions artificially, and the test was set up with the following 4 different levels of PEG 6000:

(1) Without PEG 6000 (control), only ISP ₄ liquid medium was used;

(2) 15% PEG 6000;

(3) 25% PEG6000;

(4) 35% PEG6000.

Their corresponding water potentials are: 0, -0.278, -0.699, -1.309MPa, respectively. One loop of the tested strains was picked and inoculated into the sterilized ISP ₄ liquid medium, placed on a shaker at 28°C, 200r/min shaking culture for 4d to make an inoculum, and the OD600 value was adjusted to about 0.7. Pipette 0.1mL of the inoculum into ISP ₄ liquid medium with different PEG6000 concentrations, cultivate at 28°C, 200r/min shaker for 7 days, then mix and sample, measure its OD value at 600nm, and evaluate its growth and reproduction by the size of the OD value situation. Before the measurement, the instrument should be zero-adjusted with the ISP ₄ medium of the corresponding concentration of PEG6000. Taking the uninoculated ISP ₄ liquid medium as a control, if the measured OD 600 is greater than 0, it means that the strain has drought tolerance under drought conditions.

2.3 Experimental results

Table 1 Growth-promoting function of strain SCAUT013

Table 2 Anti-stress function of strain SCAUT013

It was determined that the actinomycete Micrococcus luteus SCAUT013 had good growth-promoting and stress-resisting functions, its IAA yield was 78.64 mg/mL, and it had the ability to produce cellulase, chitinase and phosphate (Table 1). In addition, growth was performed in ISP ₄ medium at pH 4-12, salt concentration 0.5-2.5%, and PEG6000 concentration 15%-35% (Table 2). The results showed that the actinomycetes SCAUT013 had good growth-promoting and anti-stress functions, and had the potential for further development and application.

Embodiment 3 The hydroponic experiment of A. chinensis

3.1 Hydroponics experiment

The tested actinomycete strains were first activated by ISP ₄ slant, then transferred to ISP ₄ culture medium, and cultured overnight to log phase. The growth of the strains was detected by plate method, and the bacterial content was calculated.

The seeds of A. chinensis were soaked overnight for surface disinfection. The seeds were picked up with sterile tweezers and placed in a petri dish with filter paper and sterile vermiculite. Each petri dish was planted with 10 seeds, and then sterile water was added to maintain moisture. , and maintain air circulation. Sterilize the tweezers, filter paper, petri dish, plastic cup, vermiculite, quartz sand, and low nitrogen nutrient solution required for the experiment.

Low nitrogen nutrient solution formula: KNO ₃ 10.1g, KH ₂ PO ₄ 2.2g, MnSO ₄ ·H ₂ O 100.0mg, KCl 15.5g, ZnSO ₄ ·7H ₂ O 25.0mg, MgSO ₄ ·7H ₂ O 25.0mg, H ₃ BO ₃ 25.0 mg, CaCl ₂ ·2H ₂ O 21.5 g, CuSO ₄ ·5H ₂ O 25.0 mg, Na ₂ MoO ₄ ·2H ₂ O 5.0 mg, NaNO ₃ 3.0 g, ferric citrate 3.0 g.

Put the sterilized vermiculite into a plastic cup plugged with gauze strips, inject nutrient solution into the lower glass bottle, and seal the interface with plastic wrap. Use sterile tweezers to pick up the germinated seeds and place them in plastic cups filled with sterile vermiculite. Plant 3 seeds in each cup. Then add 2 mL of the bacterial solution along the roots of various seeds. After adding the bacterial solution, cover the surface with a layer of Quartz sand. Three replicates were set for each bacteria, and a control treatment was set. Cultured for 45 days, supplemented with nutrient solution from time to time. After the cultivation, carefully take out the A. chinensis from the cup, wash the vermiculite attached to the roots with clean water, and measure the plant height, root length, fresh weight, chlorophyll and other indicators.

3.2 Experimental results

The results of hydroponics experiments showed that the plant height, root length, fresh weight and dry weight of shoots and shoots, and chlorophyll of the plants inoculated with Micrococcus luteus SCAUT013 were higher than those without inoculation (CK), respectively. 29.54%, 25.38%, 22.39%, 17.74%, 25.27%, 23.52%, 13.44% (Figure 3, Table 3), indicating that SCAUT013 can obviously promote the growth of A. ability.

Table 3SCAUT013 hydroponic experiment results (45d)

Note: Different lowercase letters in the same column indicate significant differences among different treatments at the 0.05 level

The preparation of embodiment 4 biological fertilizer and field application effect

Field application of micrococcus luteus SCAUT013 strain of actinomycete rhizosphere

4.1 Preparation of bacterial agent

The rhizosphere actinomycetes Micrococcus luteus SCAUT013 strain was inoculated on the ISP ₄ slant medium, cultivated in a 28°C incubator until the bacterial fur was covered, washed with sterile water, and inoculated in the actinomycetes ISP ₄ liquid medium. Placed at 28°C, incubated with constant temperature shaking at 160 r/min until the OD value of the bacterial suspension (λ=600 nm) reached 0.9, then used for subsequent use (Note: the number of bacteria per ml of bacterial solution is defined, that is, 5 × 10 ¹⁰ cfu/ml). Use wheat bran carrier, add by weight and volume, corn flour 1%, bean cake flour 1%, KH ₂ PO ₄ 0.1%, CaCO ₃ 0.1%, pH 7.0, add water to adjust the water content of wheat bran to 40%, 121 ° C, 0.1 Mpa steam sterilization for 30min; inoculate the prepared bacterial suspension in the solid medium according to the inoculum amount of 20% (volume ratio), keep the water content of the carrier at 50-60%, and culture at 28°C until the bacterial agent The number of viable bacteria is 2×10 ¹⁰ cfu/g, which is the solid-state fermentation inoculum of SCAUT013. The inoculum is stored at 20℃ for 6 months under aseptic conditions, and the number of viable bacteria is >2×10 ⁸ cfu/g and does not affect its Promote vitality.

4.2 Field application of bacterial agent SCAUT013

Experimental materials: Liangshan local pasture variety Liangshan Guangye Zihuazizi.

Experimental location: Huidong County, Liangshan Prefecture

Half an hour before sowing, choose a shady place for seed dressing, place the wheat husk carrier containing actinomycetes in clean water and stir evenly (the number of bacteria > 2×10 ⁸ cfu/ml), and the seeds of A. chinensis are placed in a mesh After washing in a nylon bag, put it in a plastic pot filled with actinomycetes for 20-30 minutes, then take it out, and then air-dry it in a dark place until it doesn't stick to the hands before planting. The planting area was 1 mu, and a control experiment without inoculation was carried out at the same time. Grass and soil samples were collected during the full blooming period of A. chinensis, and sampling was carried out randomly in the plot with a 30cm×30cm quadrat. In order to exclude marginal effects, two rows on each side of the plot were not sampled.

4.2.1 Determination of growth and physiological indexes of A. chinensis in Liangshan

The collected A. chinensis samples were brought back to the laboratory. After washing and removing mud, the plant height, root length, fresh weight, dry weight, root weight, number of nodules, chlorophyll content, crude protein content and yield were measured.

4.2.2 Investigation on the incidence of A. chinensis in Liangshan

Random sampling was carried out in the inoculated and uninoculated plots to investigate the incidence of A. chinensis, and 50 plants were sampled to investigate the incidence of all leaves, and the incidence rate was calculated according to the following formula.

Incidence rate = [number of diseased plants (organs, leaves) / total number of investigated plants (organs, leaves)] × 100%

4.2.3 Soil physicochemical and enzyme activity determination

The rhizosphere soil samples of A. chinensis were collected and divided into 3 parts, and one was used to determine the physical and chemical indicators such as alkali-hydrolyzed nitrogen, available potassium, available phosphorus, and organic matter in the soil, as well as urease, acid phosphatase, and catalase. , Invertase and other soil enzyme activity determination.

4.2.4 Analysis of soil microbial quantity and population structure

SPIN Kit for Soil(MP BIO Laboratories,California,USA)试剂盒提取土壤总DNA，采用引物515F(5'-GTGCCAGCMGCCGCGGTAA-3')和907R(5'-CCGTCAATTCCTTTGA GTTT-3')对细菌16S的V4-V5区进行扩增，采用引物ITS3-2024F(5'-GCA TCGATGAAGAACGCAGC-3')和ITS4-2409R(5'-TCCTCCGCTTATTGATATGC-3')对真菌进行ITS2测序扩增。扩增完成后，后续文库构建、Miseq测序、序列拼接均在派诺森Illumina MiSeq测序平台进行。高通量测序数据的生物信息学分在派诺森基因云(https://www.genescloud.cn/home)进行。根据序列相似度，将有效序列聚类为OTU(97％相似度)，对OTU进行物种注释获得各样品的分类学信息，计算细菌和真菌多样性并获得相关指数。Two other soil samples were collected, one with a plate count of soil microorganisms; the other with

Soil total DNA was extracted by the SPIN Kit for Soil (MP BIO Laboratories, California, USA), and primers 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 907R (5'-CCGTCAATTCCTTTGA GTTT-3') were used for V4- of bacterial 16S The V5 region was amplified, and the fungi were amplified by ITS2 sequencing using primers ITS3-2024F (5'-GCA TCGATGAAGAACGCAGC-3') and ITS4-2409R (5'-TCCTCCGCTTATTGATATGC-3'). After the amplification was completed, subsequent library construction, Miseq sequencing, and sequence splicing were all performed on the Parsons Illumina MiSeq sequencing platform. Bioinformatics credits for high-throughput sequencing data were performed at Pyronesen Gene Cloud (https://www.genescloud.cn/home). According to the sequence similarity, the effective sequences were clustered into OTUs (97% similarity), the species annotation was performed on the OTUs to obtain the taxonomic information of each sample, the bacterial and fungal diversity was calculated and the correlation index was obtained.

4.3 Analysis of field application effect of bacterial agent SCAUT013

4.3.1 The effect on the growth of A. chinensis in Liangshan

After inoculation with Actinomycetes SCAUT013, the plant height, root length, fresh weight, dry weight, root weight, nodule number, chlorophyll content and crude protein content of A. glabrata were increased by 20.18%, 24.36% and 31.98% compared with the control. %, 26.22%, 24.14%, 28.77%, 15.38%, 9.89% (Figure 4, Table 4), the yield per mu increased by 30.93% compared with the control (Table 5). Studies have shown that many growth-promoting bacteria can regulate plant life activities and promote plant growth by producing plant hormones such as indoleacetic acid and gibberellin. Strain SCAUT013 has the ability to produce IAA, dissolve phosphorus, etc., and promote the growth of plants after inoculation with A. In addition, we also found that the inoculation of Actinomycetes SCAUT013 increased the nodulation rate of A. glabrata in Liangshan, which may be related to the ability of Actinomycetes SCAUT013 to dissolve phosphorus and produce siderophores. The increase of the content increases the number of nodules of A. glabrata in Liangshan, promotes the development of the root system, improves the nitrogen fixation efficiency and the quality of A. glabrata in Liangshan.

Table 4 The effect of inoculating SCAUT013 inoculum on the growth of A. chinensis

Note: Different lowercase letters in the same column indicate significant differences among different treatments at the 0.05 level

Table 5 Influence of inoculation with SCAUT013 inoculum on the yield of A. chinensis

Note: Different lowercase letters in the same column indicate significant differences among different treatments at the 0.05 level

4.3.2 Analysis on the incidence rate of A. chinensis in Liangshan

The incidence of common powdery mildew in the inoculated SCAUT013 and non-inoculated plants were 5.86% and 13.45%, respectively, and the incidence of inoculated plants was reduced by 56.43% compared with non-inoculated plants. The study found that actinomycetes are an important source of secondary metabolites such as antibiotics, and this feature helps actinomycetes become active antagonists of plant pathogens. morbidity, which may be related to the production of some bacteriostatic secondary metabolites by Actinomycete SCAUT013.

4.3.3 Effects on soil physicochemical and enzymatic activities

After inoculation with Actinomycetes SCAUT013, the alkali-hydrolyzed nitrogen, available potassium, available phosphorus and organic matter in the rhizosphere soil of A. chinensis in Liangshan increased by 19.81%, 12.63%, 10.24% and 5.19% compared with the control. Phosphatase, catalase, and sucrase were all increased by 38.60%, 23.48%, 49.07%, and 26.05% compared with the control (Table 6). The results showed that the inoculation of the actinomycete SCAUT013 with the function of promoting growth not only increased the number of root nodules and the efficiency of symbiotic nitrogen fixation, but also increased the available nitrogen content in the soil, and promoted the participation of soil microorganisms in the transformation of nutrients in the soil. It improves soil enzyme activity, improves soil quality, and provides good nutrient conditions for plant growth.

Table 6 Effects of inoculation with SCAUT013 inoculum on physical and chemical properties of rhizosphere soil of A. chinensis in Liangshan

Note: Different lowercase letters in the same column indicate significant differences among different treatments at the 0.05 level

4.3.4 Effects on the number of soil microorganisms

The numbers of culturable bacteria, actinomycetes, fungi and rhizobia in the rhizosphere soil inoculated with Actinomycete SCAUT013 were significantly higher than those in the non-inoculated treatment (Table 7), which were increased by 51.69%, 66.99%, and 57.14%, respectively, compared with the non-inoculated treatment. %, 63.61%, the results showed that the inoculation of Actinomycetes SCAUT013 stimulated the reproduction of indigenous microorganisms in the soil, increased the number of culturable microorganisms, and especially improved the microecological conditions of the soil.

Table 7 is inoculated with SCAUT013 inoculum on the rhizosphere soil of A. chinensis in Liangshan (unit: cfu/g)

Note: Different lowercase letters in the same column indicate significant differences among different treatments at the 0.05 level

4.3.5 Analysis of soil microbial flora diversity

The Chao1, Shannon, Simpson and Observed_species indices were selected to evaluate and analyze the microbial diversity and richness of the rhizosphere soil of A. chinensis inoculated with Actinomycetes SCAUT013 and uninoculated (control group CK). It can be seen from Figure 5 that the Chao1, Shannon, Simpson and Observed_species indices of bacteria and fungi in the rhizosphere soil of A. chinensis inoculated with Actinomycetes SCAUT013 were higher than those of the uninoculated control group (A, B in Figure 5) ( p<0.05), indicating that after inoculation with Actinomycetes SCAUT013, the richness and diversity of bacterial and fungal communities in the rhizosphere soil of A. glabrata were significantly increased compared with the control group, indicating that Actinomycetes SCAUT013 increased the number of The composition of soil microbes in the rhizosphere of A. japonica improves the complexity of the microbial community structure, and builds a good soil ecological environment for the healthy growth of plants, which is beneficial to the growth of plants.

Example 5 Effect of inoculation with Actinomycetes SCAUT013 on the growth of subsequent tobacco

5.1 Experimental arrangement

Test plant: flue-cured tobacco cloud 87

After harvesting and inoculating Actinomycetes SCAUT013 A. chinensis in Huidong tobacco area of Liangshan Prefecture, flue-cured tobacco was continued to be planted at the experimental site, and the fertilization amount was reduced by 10% according to the normal level. The normal fertilization amount for flue-cured tobacco planting in Huidong Tobacco Area: 30kg/mu of compound fertilizer for tobacco (N: _P2O5 :K2O is ₂ :3: ₅ ), commercial organic fertilizer 50kg/mu, and oil dry 20kg/mu. According to the "YC/T 142-2010 Survey and Measurement Methods of Tobacco Agronomic Characters", at the maturity stage of flue-cured tobacco (70 days after seedling transplanting), 5 representative tobacco plants with uniform and consistent growth were selected to measure the plant height, stem circumference, and efficacy of flue-cured tobacco. The number of leaves and other agronomic characters were measured, and the root structure, vitality and dry matter content of flue-cured tobacco were determined. The root system structure of flue-cured tobacco was scanned by EPSON1680 root system scanner (Epson, LongBeach, USA). After scanning, the root system was analyzed by WinRhizo2005a root system analysis software to obtain the total root length, total surface area, total volume and total root hair number of the root system. and other morphological indicators. The roots, stems and leaves of flue-cured tobacco were respectively fixed at 105°C for 20 min, and dried at 75°C to a constant weight to determine the biomass.

5.2 Experimental results

After the Liangshan Guangye Zihuazizi inoculated with Actinomycetes SCAUT013 was harvested, the flue-cured tobacco Yunyan 87 was continued to be planted at the test site. The measurement results of the agronomic characters of the flue-cured tobacco at the mature stage showed that the treatment of the previous inoculation with Rhizobium, the plant height of the subsequent flue-cured tobacco , stem circumference, effective leaf number, maximum leaf length, maximum leaf width, and maximum leaf area increased by 12.88%, 17.94%, 14.81%, 16.71%, 14.17%, and 20.77%, respectively, compared with the control group (Table 8). The total root length, total root area, root average diameter, root volume, and root hair number increased by 14.08%, 20.60%, 12.61%, 18.67%, and 21.69%, respectively, compared with the control group. (Table 10). Based on the research results of Example 4, it shows that the previous planting of A. glabrata and rhizobia inoculation increased the content of nitrogen and other soil nutrients in the soil, optimized the soil microbial community structure, and improved the root growth environment of the subsequent flue-cured tobacco. It enhances the vitality of the root system and promotes the absorption of nutrients by flue-cured tobacco, thereby driving the growth of flue-cured tobacco and the accumulation of substances. Therefore, the inoculation of Actinomycetes SCAUT013 not only increased the yield of A. glabrata in the current season, but also promoted the growth of the subsequent crop flue-cured tobacco by improving the soil quality.

Table 8 Effects on agronomic characters of flue-cured tobacco

Note: Different lowercase letters in the same column indicate significant differences among different treatments at the 0.05 level

Table 9 Effects on dry matter accumulation of flue-cured tobacco

Note: Different lowercase letters in the same column indicate significant differences among different treatments at the 0.05 level

Table 10 Effects on root structure of flue-cured tobacco

Note: Different lowercase letters in the same column indicate significant differences among different treatments at the 0.05 level

sequence listing

<110> Sichuan Agricultural University

<120> Actinomycete strain SCAUT013 and its application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1111

<212> DNA

<213> Micrococcus luteus

<400> 1

cgtccaccgc atggtgggtg ttggaaagat ttatcggttt tggatggact cgcggcctat 60

cagcttgttg gtgaggtaat ggctcaccaa ggcgacgacg ggtagccggc ctgagagggt 120

gaccggccac actgggactg agacacggcc cagactccta cgggaggcag cagtggggaa 180

tattgcacaa tgggcgaaag cctgatgcag cgacgccgcg tgagggatga cggccttcgg 240

gttgtaaacc tctttcagta gggaagaagc gaaagtgacg gtacctgcag aagaagcacc 300

ggctaactac gtgccagcag ccgcggtaat acgtagggtg cgagcgttat ccggaattat 360

tgggcgtaaa gagctcgtag gcggtttgtc gcgtctgtcg tgaaagtccg gggcttaacc 420

ccggatctgc ggtgggtacg ggcagactag agtgcagtag ggagactgg aattcctggt 480

gtagcggtgg aatgcgcaga tatcaggagg aacaccgatg gcgaaggcag gtctctgggc 540

tgtaactgac gctgaggagc gaaagcatgg ggagcgaaca ggattagata ccctggtagt 600

ccatgccgta aacgttgggc actaggtgtg gggaccattc cacggtttcc gcgccgcagc 660

taacgcatta agtgccccgc ctggggagta cggccgcaag gctaaaactc aaaggaattg 720

acggggggccc gcacaagcgg cggagcatgc ggattaattc gatgcaacgc gaagaacctt 780

accaaggctt gacatgttcc cgatcgccgt agagatacga tttccccttt ggggcgggtt 840

cacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa 900

cgagcgcaac cctcgttcca tgttgccagc acgtaatggt ggggactcat gggagactgc 960

cggggtcaac tcggaggaag gtgaggacga cgtcaaatca tcatgcccct tatgtcttgg 1020

gcttcacgca tgctacaatg gccggtacaa tgggttgcga tactgtgagg tggagctaat 1080

cccaaaaagc cggtctcagt tcggattggg g 1111

Claims (5)

1. Actinomycete strainsMicrococcus luteusSCAUT013, stored in GuangdongThe provincial microorganism strain preservation center, the preservation number is GDMCC NO: 61507.

2. an actinomycete strain comprising the compound of claim 1Micrococcus luteusA microbial agent of SCAUT 013.

3. Comprising the actinomycete strain of claim 1Micrococcus luteusA microbial fertilizer of SCAUT 013.

4. The actinomycete strain of claim 1Micrococcus luteusUse of SCAUT013, the microbial agent of claim 2 or the microbial fertilizer of claim 3 for planting Vicia villosa (Vicia villosa Roth var. glabercensc v. liang shan).

5. The use of claim 4, wherein the planting is planting vetch (Vicia villosa Roth var. glabercensc v. liang shan) in Liangshan.

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