CN114107118A

CN114107118A - Actinomycete strain SCAUT009 and application thereof

Info

Publication number: CN114107118A
Application number: CN202111457057.0A
Authority: CN
Inventors: 赵珂; 陈强; 李静; 张紫薇; 张凌子; 邹立扣; 辜运富; 廖德聪; 向泉桔; 余秀梅
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2021-12-02
Filing date: 2021-12-02
Publication date: 2022-03-01
Anticipated expiration: 2041-12-02
Also published as: CN114107118B

Abstract

The invention discloses an actinomycete Microbacterium testaceum SCAUT009 and application thereof, which is preserved in Guangdong province microbial strain preservation center with the preservation number of GDMCC No: 61508. the Microbacterium testaceum SCAUT009 separated and screened by the invention has growth promoting capabilities of generating IAA, siderophores, dissolving phosphorus, degrading cellulose and chitin and the like, and also has stress resistance functions of salt resistance, acid and alkali resistance, drought resistance and the like, and the application of the Microbacterium testaceium SCAUT to the vetch with the cool mountain smooth leaves can promote the growth of the vetch with the cool mountain smooth leaves, not only increase the yield of pasture, but also improve the soil quality and improve tobacco planting soil, and has better application potential in agricultural production.

Description

Actinomycete strain SCAUT009 and application thereof

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to an actinomycete strain SCAUT009 and an application thereof.

Background

Liangshan is one of three major pasturing areas in Sichuan province, has a long history of animal husbandry, is one of the main sources of cash income of farmers and herdsmen, and particularly accounts for more than 70% of the income of rural families in high mountain areas. The problem that livestock lack of green feed during the overwintering and spring transition period is prominent because the Liangshan has the climatic characteristics of dry winter and spring climate, much rain in summer and clear dryness and wetness. However, in the mountainous area around the basin in the state of Liangshan, particularly in the middle and sub-high mountainous areas with the elevation of 1700-3200m, the land is greatly idle during the winter and spring. Vetch (Vicia villosa var. glabresens) is an annual or perennial herbaceous plant in the genus of Leguminosae, prefers warm and cool humid climate, is more cold-resistant and drought-resistant, is suitable for the area with the altitude of 1500 plus one jar of 3200m, is most suitable for the area with the altitude of 1800 plus one jar of 2500m, has the advantages of water retention, fertilizer retention, soil improvement, nitrogen fixation and fertilizer increase, ecological environment improvement and the like, and is the main pasture for popularization in high and cold mountainous areas. The Liangshan has the history of planting the smooth-leaf Chinese vetch for a long time, can plant the smooth-leaf Chinese vetch at idle rotation crops in winter, has the effects of fully utilizing photo-thermal resources and preserving soil moisture, increases the content of organic matters in soil, improves the soil quality, and simultaneously well solves the problem that sexual animals lack green feed when crossing winter and spring.

In recent years, excessive application of chemical fertilizers not only pollutes the environment, but also causes a series of problems of soil hardening, fertility reduction, agricultural product quality reduction and the like. Soil microorganisms play an important role in the processes of material circulation, organic matter decomposition and Plant nutrient utilization, and Plant growth promoting bacteria (PGPR) which naturally exist in soil and are colonized on Plant rhizosphere can improve the physical and chemical properties of the soil, promote Plant growth by generating growth hormone, nitrogen fixation and phosphorus dissolution, and promote Plant growth by generating substances such as chitinase, cellulase, antibiotics and the like to resist pathogen infection and improve the stress resistance of plants. Although the vetch with smooth leaves has the capability of growing nodules and fixing nitrogen with rhizobia, the biological nitrogen fixing process also needs other nutrients to participate in the growth process of the vetch with smooth leaves, and the rhizosphere growth-promoting bacteria PGPR can convert inorganic elements which cannot be utilized by plants in soil into organic substances which can be utilized by the plants, so that the supply and the growth of plant nutrient elements are promoted. Because the vetch is leguminous forage, the previous research mainly focuses on the symbiotic relationship between rhizobia and vetch, and the influence of rhizosphere growth-promoting bacteria on the growth of vetch is not reported yet.

The actinomycetes are an important component of a plant rhizosphere microbial community, play an important role in the soil substance transformation process, promote plant growth in the modes of dissolving phosphorus and fixing nitrogen, secreting plant growth regulating substances, synthesizing siderophores, inducing plants to generate resistance, generating antifungal metabolites and the like, and have great application potential in agricultural production. The smooth-leaf vetch collected in Liangshan is taken as a research object, high-efficiency PGPR strain resources are separated and screened, the stress resistance of the smooth-leaf vetch is improved by utilizing rhizosphere growth-promoting bacteria, excellent PGPR strain resources are collected for developing microbial fertilizers, the ecological benefit and the economic value of the smooth-leaf vetch are brought into play to the maximum extent, and the requirements of sustainable development and ecological environment protection of the Liangshan farming and animal husbandry are met.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: in order to excavate and screen PGPR strain resources capable of promoting the growth of vetch, and apply the PGPR strain resources to agricultural production, one of the purposes of the invention is to provide a rhizosphere actinomycete with stress resistance and growth promotion functions, and the other purpose is to provide application of the strain.

The technical scheme of the invention is as follows: actinomycete strain Microbacterium tescenum SCAUT009 separated from the rhizosphere of vetch seed, which is called SCAUT009 for short. The strain is preserved in Guangdong province microorganism culture collection center at 2021, 2 months and 7 days, and the preservation number is GDMCC No: 61508 at the deposit address of building No. 59, building No. 5, Guangdong institute of microbiology, Michelia furiosa, 100, Guangzhou, Guangdong province.

Contains microbial agent or microbial fertilizer of actinomycete Microbacterium testaceum SCAUT 009.

The actinomycete strain Microbacterium costatum SCAUT009 or the microbial agent or the microbial fertilizer is used for planting Vicia villosa Roth var. glabercensc v.

Further, the planting refers to planting vetch (Vicia villosa Roth var. glaberenccv. Liang shan) in Liangshan.

The strain morphological characteristics of SCAUT009 of the invention are as follows: at ISP₄After culturing for 3 days at 28 ℃ on a culture medium, the bacterial colony is small, the edge is neat, the surface is smooth, the color of the bacterial colony is light yellow, the bacterial colony is easy to pick, gram-positive, aerobic, bacillus pumilus and spore-free, and the size of the bacterial colony is (0.3-0.6) × (0.4-0.8) um.

The 16S rRNA sequence of SCAUT009 strain of the present invention was sequenced and submitted to the EzBioCloud website (www.ezbiocloud.net) for homology alignment and phylogenetic analysis using MEGA7.0 software. The results showed that the strain SCAUT009 has 100% homology with the 16S rRNA nucleotide sequence of Microbacterium testaceum. According to the results of experiments such as morphological characteristics and 16S rRNA sequence homology analysis, the SCAUT009 is identified to be Microbacterium testaceum.

The DNA of SCAUT009 strain of the invention is subjected to 16S rRNA gene phylogenetic analysis and is shown in a sequence table SEQ ID NO. 1.

The SCAUT009 strain of the invention has the growth promoting functions of producing IAA, siderophore, cellulase, chitinase and phosphorus dissolving, and has the stress resistance of salt tolerance, drought tolerance, acid and alkali resistance, etc.

The results of sand culture experiments show that after the actinomycete Microbacterium tristicum SCAUT009 microbial inoculum is inoculated, the plant height, the root length, the fresh weight and the dry weight of the overground part and the underground part and chlorophyll of the cold mountain smooth leaf vetch plant are respectively increased by 23.10 percent, 17.03 percent, 24.63 percent, 22.72 percent, 21.84 percent, 18.75 percent and 12.28 percent compared with the non-inoculated treatment (CK), which shows that the bacterial strain SCAUT009 can obviously promote the growth of the cold mountain smooth leaf vetch and has better growth promoting effect.

The field test result shows that after the actinomycete Microbacterium tristicum SCAUT009 microbial inoculum is inoculated, the plant height, the root length, the fresh weight, the dry weight, the root nodule number, the chlorophyll content and the crude protein content of the vetch with the cold mountain smooth leaves are improved by 27.42 percent, 22.05 percent, 30.99 percent, 25.77 percent, 24.14 percent, 25.72 percent, 17.97 percent and 9.82 percent compared with the control, the yield per mu is increased by 29.49 percent compared with the control, and the powdery mildew incidence rate of the vetch with the cold mountain smooth leaves is reduced by 64.16 percent compared with the incidence rate of the plants which are not inoculated; the alkaline hydrolysis nitrogen, the quick-acting potassium, the available phosphorus and the organic matters in the rhizosphere soil of the cold-mountain-light-leaf vetch are increased by 15.74%, 10.44%, 13.46% and 6.30% compared with the control, the urease, the acid phosphatase, the catalase and the sucrase in the soil are all increased by 32.48%, 25.44%, 30.37% and 24.78% compared with the control, the number of culturable bacteria, actinomycetes, fungi and rhizobia in the rhizosphere soil is respectively increased by 41.12%, 66.98%, 22.58% and 64.42% compared with the non-inoculation treatment, and the abundance of bacteria and fungi communities in the rhizosphere soil is obviously increased compared with the control group. After the actinomycete Microbacterium testaceum SCAUT009 microbial inoculum is inoculated, the growth of the vetch with the cool mountain smooth leaves is promoted, the nodulation capacity is increased, the quality of the plant is improved, the micro-ecological condition of the soil is improved, the soil quality is improved, and the strain resource is provided for the research and development of the microbial agent of the cool mountain smooth leaf vetch.

The determination results of indexes such as agronomic characters, dry matter accumulation and root system structures of flue-cured tobaccos in the mature period of the later crops indicate that after rhizobium is inoculated to the earlier crops, the plant height, stem circumference, effective leaf number, maximum leaf length, maximum leaf width and maximum leaf area of the later crops are respectively increased by 7.87%, 9.34%, 11.38%, 8.83%, 7.19% and 15.38% compared with those of the control group, the dry matter accumulation amount is increased by 7.57% compared with that of the control group, and the total root length, total root area, root average diameter, root volume and root hair number are respectively increased by 8.30%, 6.82%, 8.49%, 7.31% and 7.29% compared with those of the control group.

After the actinomycete Microbacterium testaceum SCAUT009 actinomycete agent is inoculated, the nodulation capability of the vetch seed of the Mount Liangbare leaf is promoted, the quality of the plant is improved, the yield is increased, the soil quality is also improved, the growth of the flue-cured tobacco of the later crop is promoted, and the strain resource is provided for the research and development of the microbial agent of the vetch seed of the Mount Liangbare leaf.

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

the Microbacterium testaceum SCAUT009 separated and screened by the invention has growth promoting capabilities of generating IAA, siderophores, dissolving phosphorus, degrading cellulose, chitin and the like, and simultaneously has stress resistance functions of salt resistance, acid and alkali resistance, drought resistance and the like, and the application of the Microbacterium testaceium SCAUT009 on the cold mountain smooth leaf vetch can achieve the effect of promoting the growth of the cold mountain smooth leaf vetch.

Preservation information:

actinomycete Microbacterium costatum SCAUT009, abbreviated as SCAUT 009. The strain is preserved in Guangdong province microorganism culture collection center at 2021, 2 months and 7 days, and the preservation number is GDMCC No: 61508 at the deposit address of building No. 59, building No. 5, Guangdong institute of microbiology, Michelia furiosa, 100, Guangzhou, Guangdong province.

Drawings

FIG. 1 shows a Microbacterium tescenum SCAUT009 at ISP for actinomycetes of vetch rhizosphere₄Colony morphology and thallus micrographs on the culture medium;

FIG. 2 is a phylogenetic tree of Microbacterium testaceum SCAUT009, a Microbacterium testaceatum, which is constructed based on 16S rRNA gene sequence;

FIG. 3 shows the growth of the vetch seed by the actinomycete Microbacterium testaceum SCAUT 009. Wherein the left side is an uninoculated control and the right side is an inoculated SCAUT009 treatment;

fig. 4 field experimental results, left: comparison; and (3) right: SCAUT 009;

FIG. 5 shows the bacterial Alpha diversity (A) and the fungal Alpha diversity (B) of the rhizosphere soil of the Mesochloa souliea.

Detailed Description

The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were all commercially available unless otherwise specified.

Example 1 isolation and purification of rhizosphere actinomycetes SCAUT009

1.1 isolation of Actinomyces rhizosphere of vetch

Collecting vetch seed in Yi autonomous city of Yi nationality of Liangshan province in Sichuan province in 2018, shaking off excessive soil on root system, placing root in 50mL sterilized centrifuge tube containing 25mL phosphate buffer solution, treating for 10min with ultrasonic wave (150W), taking out root under aseptic condition, centrifuging for 10min at 3000g, removing supernatant, and making 10min precipitate as rhizosphere soil^-1、10^-2、 10^-3Gradient dilution, taking 100ul suspension, uniformly coating on improved Gao's medium for separation, repeating the concentration for each dilution for 3 timesCulturing at 28 deg.C for 5-7 days. The strains with typical actinomycete colony characteristics are picked and purified by dilution plate streaking until the purified strains are obtained. Inoculation of the purified strains to ISP₄In liquid culture medium, shake culturing at 28 deg.C for 7d, preserving with 30% glycerol, and freezing preserving at-70 deg.C.

1.2 identification of the Strain

1.2.1 Strain morphology

Inoculating the strains at ISP₄The culture medium was cultured at 28 ℃ for 5 days, and the colony morphology of the strain was observed.

1.2.2 molecular characterization of the Strain

(1) Reagent: lysozyme, proteinase K, 10 xtae, 1 xte, 3mol/L sodium acetate (ph4.8-5.2), 70% ethanol, (saturated phenol: chloroform: isoamyl alcohol ═ 25:24:1), Mix.

(2) Primer and method for producing the same

Primer A: 5'-AGAGTTTGATCCTGGCTCAG-3' (same bases as positions 8-27 of the 5' end of 16S rRNA); primer B: 5'-TTAAGGTGATCCAGCCGCA-3' (identical to the base of the 1523-1504 site at the 3' end of 16S rRNA);

(3) actinomycete DNA extraction

Taking a little thallus to put into a sterile 1.5mL Eppendorf tube, adding 20 mul (50mg/mL) of lysozyme with the final concentration of 2 mg/mL, putting into a 37 ℃ shaking table, and carrying out 200rmp/min for 1-2 h; adding 20% SDS 50 μ L and proteinase K5 μ L (20mg/mL), mixing, placing in 55 deg.C shaking table, and treating for 1-2 hr at 200 rmp/min; adding 550 μ L (phenol: chloroform: isoamyl alcohol 25:24:1) for extraction, centrifuging at 12000rmp/min for 10min, and sucking the supernatant (repeating three times); adding 800 μ L of anhydrous ethanol and 80 μ L of 3mol/L sodium acetate (pH4.8-5.2), mixing, precipitating DNA at 4 deg.C for 1 hr, 12000rmp/min, 10min, and removing supernatant; adding 200 μ L of 70% ethanol, cleaning tube wall for 1-2 times at 12000rmp/min, centrifuging for 5min, and removing supernatant; after the ethanol had evaporated to dryness, 50. mu.L of 1 XTE was added to dissolve the DNA and stored at-20 ℃; and detecting by 1% agarose gel electrophoresis.

(4) Actinomycete 16S rRNA gene amplification

16S rRNA gene amplification conditions: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 1min, annealing at 56 deg.C for 1min, extension at 72 deg.C for 2min, 30 cycles, and total extension at 72 deg.C for 10 min. The PCR Product was purified by the Shanghai worker EZ Spin Column PCR Product Purification Kit UNlQ-1 Column PCR Product Purification Kit (SK1142-N) according to the instruction manual, and the purified Product was sequenced by Biotechnology Ltd.

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

And (3) carrying out similarity search on the sequence obtained after sequencing in NCBI by utilizing BLAST software, selecting a 16S rRNA gene sequence of the strain with the highest similarity as a reference sequence, carrying out multi-sequence comparison analysis by utilizing Clustal X software, constructing a phylogenetic tree by utilizing MEGA7.0 software and an N-J method, and determining the classification status of the actinomycetes.

1.3 results of the experiment

Actinomycetes SCAUT009 separated from the rhizosphere soil of Mesochlianus conduct of Liriosa paradisiai in ISP₄After culturing for 3 days at 28 ℃ on a culture medium, the strain is in a circular convex shape, neat in edge, moist and smooth in surface, light yellow, 1-1.5mm in diameter, gram-positive, immotile, aerobic, bacillus pumilus, spore-free and has a cell size of (0.3-0.6) × (0.4-0.8) um (figure 1).

The strain SCAUT009, which was similar to Microbacterium testaceum by 100% by 16S rRNA sequencing and BLAST homology alignment in NCBI database. According to the morphological characteristics and the results of experiments such as 16S rRNA sequence homology analysis, the SCAUT009 is identified as Microbacterium testaceum (figure 2).

Example 2 screening of growth promoting function of rhizosphere actinomycetes SCAUT009

2.1 growth promoting functional screening

2.1.1 measurement of IAA-producing ability

20 mu L of bacterial suspension is inoculated into ISP containing 0.5mol/L tryptophan₄Repeating the steps for 3 times in a liquid culture medium, setting a blank control, carrying out shake culture at 28 ℃ and 120r/min for 3d, centrifuging at 8000r/min for 24h, taking 1mL of bacterial liquid supernatant, adding 2mL of IAA color developing solution, carrying out dark reaction at 25 ℃ for 30min, carrying out color comparison at the wavelength of 530nm, and recording the absorbance value. The IAA concentration in the assay solution was calculated by adjusting to zero with the liquid medium without inoculation as a control and using the indoleacetic acid standard solutions of 0, 5, 20, 40, and 60mg/l as the standard.

2.1.2 determination of siderophore production Capacity

Dissolving 0.012g of azurine in 10mL of double distilled water, and mixing with 2mL of 1mmoL/L FeCl₃·6H₂Mixing the solution O uniformly to obtain a solution a; dissolving 0.015g of hexadecyl trimethyl ammonium bromide in 8mL of double distilled water to obtain a solution b; slowly adding the solution a into the solution b, and uniformly mixing to obtain a dye solution c; adding 20mL of 10 XMM 9 salt solution and 6.04g of piperazine diethylsulfoacid into a triangular flask containing 150mL of double distilled water, uniformly mixing, adjusting the pH to 6.8 by using 50% NaOH, and then adding 3.2g of agar powder to obtain a culture medium d; adding dye solution c, culture medium d and 1mmoL/L of CaCl₂1mmoL/L MgSO₄Sterilizing 20% glucose and 10% casamino acid at 115 deg.C for 20min, respectively, and collecting 200 μ L CaCl when the temperature of each solution is reduced to 50-60 deg.C₂，4mLMgSO₄6mL of casamino acid and 2mL of glucose are added into the culture medium d, then the dye solution c is added along the bottle wall, the mixture is fully mixed and does not generate bubbles, and then the blue detection culture medium is obtained, and the plate is inverted. Inoculating test strains by using sterile bamboo sticks, inoculating 5 fungus cakes in each plate at equal intervals, repeating the steps for 3 times for each strain, culturing for 3 days at 28 ℃, and observing and recording the size of an orange transparent ring.

2.1.3 measurement of phosphorus solubilizing ability

Preparing fungus cakes from the well-grown actinomycete strains by using a sterile hole puncher (D is 5mm), respectively inverting the fungus cakes on a PKO culture medium, respectively, culturing the fungus cakes in 4 directions of 2cm away from the center point of a flat plate at 28 ℃ for 3 days, respectively measuring the diameter (D) of a transparent ring and the diameter (D) of a bacterial colony, and calculating the ratio (D/D) of the diameter (D) of the transparent ring to the diameter (D) of the bacterial colony, wherein the larger the ratio is, the more obvious the phosphorus dissolving effect is.

2.1.4 measurement of carboxymethyl cellulase (CMCase) Activity

Preparing a seed suspension from the strains obtained by primary screening, inoculating the seed suspension into a triangular flask of a re-screening culture medium with a carbon source of CMC-Na according to the inoculation amount of 10%, carrying out shake culture at a constant temperature of 28 ℃, respectively measuring the activity of carboxymethyl cellulase (CMCase) of each strain of 3, 5, 7, 9 and 11 days, and setting 3 times of repetition.

(1) Drawing of standard curve

Drying anhydrous glucose at 80 ℃ until the weight is constant to prepare a 1mg/mL standard glucose solution, taking 6 test tubes, respectively adding 0, 0.2, 0.4, 0.6, 0.8 and 1.0mL of the standard glucose solution, adding distilled water to 2.0mL, adding 1.5mL of a DNS reagent, carrying out boiling water bath for 5min, cooling, fixing the volume to 25mL, measuring the OD value under the spectrophotometry of 540nm, and drawing a standard curve.

(2) Preparation of crude enzyme solution

Preparing a liquid re-screening culture medium, subpackaging in 250mL triangular flasks, inoculating 45mL of seed bacteria suspension in each flask, placing in a shaking table at 28 ℃ for culturing, respectively taking 1.5mL of fermentation liquor in a centrifuge tube after culturing for 7d, and centrifuging at 10000r/min for 10min to obtain a crude enzyme solution.

(3) Enzyme activity assay

The enzyme activity determination method comprises the following steps: 0.1mL of the crude enzyme solution was added to 1.9mL of a 1% CMC-Na solution. Hydrolyzing at 45 deg.C for 20min, adding 1.5mL DNS color developing solution, boiling in water bath for 5min, diluting to 25mL, carrying out color comparison at 540nm, measuring absorbance (OD), comparing with standard glucose curve, and calculating glucose amount (m) from OD value₁). 0.1mL of each supernatant was added with 1.9mL of water, 1.5mL of DNS was added, and the mixture was subjected to boiling water bath for 5min to obtain a volume of 25mL, and the amount of glucose (m) in the crude enzyme solution was measured by color comparison at 540nm₂). Adding glucose amount (m)₁) Minus the amount of glucose (m)₂) Obtaining the glucose amount really obtained by degrading 1% CMC solution by CMC enzyme, calculating the glucose amount A by the optical density value, and calculating the enzyme activity of the strain by a formula.

2.1.5 chitinase production assay

And (3) inoculating the test strain onto the MM-Chitin culture medium, and if a hydrolysis ring is generated, the test strain is proved to be a strain with the capacity of degrading Chitin.

2.2 screening of stress-resistant function

2.2.1 salt tolerance detection

Streaking test strains on ISP₄Culturing at 28 deg.C for 3-4 days, and making into bacterial suspension with sterile water. In terms of NaCl concentration: 0%, 1%, 3%, 5%, 7%, to make 7 ISPs₄Solid culture medium, dropping the bacterial suspension on the culture medium with sterile bamboo stick, repeating for 3 times, culturing at 28 deg.C for 3-4 days, observing the growth of each rhizosphere actinomycete, recording the result, and if the strain can normally grow under a certain salt concentration, thenIndicating that the strain has the ability to tolerate the salt concentration.

2.2.2 acid and base resistance detection

Streaking test strains on ISP₄Culturing at 28 deg.C for 3-4 days, and making into bacterial suspension with sterile water. By ISP₄Based on the formula of the culture medium, NaOH and KH are used₂PO₄、Na₂CO₃、KCl、NaHCO₃、Na₂HPO₄·10H₂Preparing pH reaction buffer solution by using O, sodium citrate and trisodium citrate, enabling the pH of the culture medium to reach 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0 respectively, dotting the bacterial suspension on the culture medium by using a sterile bamboo stick, repeating the steps for 3 times, culturing at 28 ℃ for 3-4 days, observing the growth condition of each rhizosphere actinomycete and recording the result, wherein if the bacterial strain can normally grow under a certain pH value, the bacterial strain has the capability of resisting the pH value.

2.2.3 drought tolerance assay

The preliminary screening test for drought tolerance was performed using polyethylene glycol (PEG)6000 to artificially simulate drought conditions, with the following 4 different PEG6000 levels:

(1) without PEG6000 (control), ISP alone was used₄A liquid culture medium;

(2) 15% of PEG 6000;

(3) 25% of PEG 6000;

(4) 35% of PEG 6000.

Their corresponding water potentials are: 0. -0.278, -0.699, -1.309 MPa. The test strains are respectively picked to 1 ring and inoculated to sterilized ISP₄Placing in liquid culture medium, shaking at 28 deg.C and 200r/min for 4d to obtain inoculum, and adjusting OD600 value to about 0.7. 0.1mL of the inoculation solution is sucked and is connected with ISP with different PEG6000 concentrations₄Shaking-culturing in liquid culture medium at 28 deg.C and 200r/min for 7d, mixing, sampling, measuring OD value at 600nm, and evaluating the growth and reproduction status by OD value. ISP with the corresponding concentrated PEG6000 before the determination₄The culture medium was zeroed for the instrument. With non-inoculated ISP₄Liquid medium was used as a control, and if the measured OD600 was greater than 0, it indicated that the strain had drought tolerance under drought conditions.

2.3 results of the experiment

Through determination, the actinomycete Microbacterium testaceum SCAUT009 has good growth promoting and stress resisting functions, the IAA yield is 37.37mg/mL, and the actinomycete Microbacterium testaceum SCAUT has the capacity of producing cellulose, producing chitinase and siderophore and dissolving phosphorus (Table 1); in addition, ISP with salt concentration of 0.5-1.5% and PEG6000 concentration of 15% at pH5-11₄Growth in medium (table 2). The result shows that the actinomycete SCAUT009 has good growth promoting and stress resisting functions and has the potential of further development and application.

TABLE 1 growth promoting function of SCAUT009 strain

TABLE 2 Strain SCAUT009 anti-adversity function

Example 3 hydroponic culture experiment of vetch with smooth leaves

3.1 hydroponic experiment

The test actinomycete strain is connected to ISP firstly₄Bevel activation followed by switching to ISP₄And (4) culturing the culture solution overnight to logarithmic phase, detecting the growth condition of the strain by using a plate method, and calculating the bacterium content.

Soaking the smooth-leaf vetch seeds overnight, then performing surface disinfection, clamping the seeds with sterile forceps, implanting the seeds into culture dishes filled with filter paper and sterile vermiculite, planting 10 seeds in each culture dish, adding sterile water to keep moisture, and keeping air circulation. And (3) sterilizing tweezers, filter paper, a culture dish, a plastic cup, vermiculite, quartz sand and low-nitrogen nutrient solution required by the test.

The formula of the low-nitrogen nutrient solution comprises the following components: KNO₃ 10.1g，KH₂PO₄ 2.2g，MnSO₄·H₂O 100.0mg，KCl 15.5g， ZnSO₄·7H₂O 25.0mg，MgSO₄·7H₂O 25.0mg，H₃BO₃ 25.0mg，CaCl₂·2H₂O 21.5g，CuSO₄·5H₂O 25.0mg，Na₂MoO₄·2H₂O 5.0mg，NaNO₃3.0g and ferric citrate 3.0 g.

Filling the sterilized vermiculite into a plastic cup filled with gauze strips, injecting nutrient solution into a lower layer glass bottle, and sealing an interface by using a preservative film. And clamping the seeds subjected to germination acceleration by using sterile tweezers, implanting the seeds into plastic cups filled with sterile vermiculite, planting 3 seeds in each cup, adding 2mL of the bacterial liquid along the roots of various seeds, and covering a layer of quartz sand on the surface layer after adding the bacterial liquid. Three replicates were set for each strain and a control treatment was set. Culturing for 45 days, and supplementing nutrient solution at random. After the cultivation is finished, the vetch is carefully taken out of the cup, vermiculite attached to the root is cleaned by clear water, and indexes such as plant height, root length, fresh weight, dry weight and the like are measured.

3.2 results of the experiment

The water culture experiment result shows that the plant height, the root length, the fresh weight and the dry weight of the overground part and the underground part and chlorophyll of the cold mountain smooth leaf vetch plant inoculated with the actinomycete Microbacterium tristicum SCAUT009 microbial inoculum are respectively increased by 23.10 percent, 17.03 percent, 24.63 percent, 22.72 percent, 21.84 percent, 18.75 percent and 12.28 percent compared with the non-inoculated treatment (CK) (figure 3, table 3), which shows that the SCAUT009 can obviously promote the growth of the cold mountain smooth leaf vetch and shows better growth promoting effect.

TABLE 3 SCAUT009 Water culture experiment result (45d)

Note: the different lower case letters in the same column indicate significant differences at the 0.05 level between the different treatments

Example 4 preparation of biofertilizer and field application Effect

Actinomycete Microbacterium testaceum SCAUT009 strain field application

4.1 preparation of the bacterial agent

Inoculating Microbacterium costatum SCAUT009 strain of rhizosphere actinomycete in ISP₄Culturing on slant culture medium in 28 deg.C incubator until thallus PorphyraeAfter the growth, the mixture is washed by sterile water and inoculated to actinomycete ISP₄Culturing in liquid culture medium at 28 deg.C and 160r/min with shaking until OD value of bacterial suspension (λ 600nm) reaches 0.9, and reserving (note: determining bacterial count of bacterial liquid per ml, i.e. 5 × 10)¹⁰cfu/ml). Wheat bran carrier is added according to the weight volume ratio, corn flour is 1 percent, bean cake powder is 1 percent, KH₂PO₄ 0.1％，CaCO₃0.1%, pH 7.0, adding water to adjust water content of testa Tritici to 40%, sterilizing at 121 deg.C under 0.1Mpa for 30 min; inoculating the prepared bacterial suspension into a solid culture medium according to the inoculation amount of 20% (volume ratio), keeping the water content of the carrier at 50-60%, and standing at 28 deg.C for culturing until the viable count of the bacterial agent is 2 × 10¹⁰cfu/g is the solid fermentation bacterial agent of SCAUT009, and the viable count of the bacterial agent is preserved for 6 months at 20 ℃ under the aseptic condition>2×10⁸cfu/g and does not affect the growth promoting activity.

4.2 field application of SCAUT009 microbial inoculum

Experimental materials: the Chinese vetch is a Chinese yam variety of Chinese yam.

The experimental site: liangshan Zhuitong county

Selecting shade half an hour before sowing, dressing seeds, placing the wheat bran carrier containing actinomycete agent in clear water, and stirring uniformly (bacteria count)>2×10⁸cfu/ml), cleaning seeds of the vetch with smooth leaves in a mesh nylon bag, soaking in a plastic basin containing an actinomycete microbial inoculum for 20-30min, taking out, and airing in the shade until the seeds are not sticky. The planting area is 1 mu, and the non-inoculation control test is carried out at the same time. The method is characterized in that a grass sample and a soil sample are collected in the full bloom stage of the smooth-leaf vetch, a sample with the size of 30cm multiplied by 30cm is randomly sampled in a community, and two rows of samples are not sampled at two sides of the community to eliminate marginal effect.

4.2.1 determination of growth and physiological index of vetch

The collected smooth leaf vetch samples are taken back to a laboratory, and after being cleaned and deslimed, the plant height, root length, fresh weight, dry weight, root nodule number, chlorophyll content, crude protein content and yield of the plants are respectively measured.

4.2.2 incidence survey of vetch seed

The incidence of the vetch is randomly sampled and investigated in the inoculated and non-inoculated treated districts, 50 plants are sampled and investigated for the incidence of all leaves, and the incidence of the diseases is calculated according to the following formula.

Disease incidence [ number of diseased plants (organs, leaves)/number of total investigated plants (organs, leaves) ] × 100%

4.2.3 determination of soil physicochemical Properties and enzyme Activity

The collected vetch rhizosphere soil samples are divided into 3 parts, and one part is used for measuring physicochemical indexes of alkaline hydrolysis nitrogen, quick-acting potassium, quick-acting phosphorus, organic matters and the like in the soil and measuring the soil enzyme activities of urease, acid phosphatase, catalase, sucrase and the like.

4.2.4 analysis of soil microbial population and population diversity

Collecting another two soil samples, and counting one soil sample by using a flat plate of soil microorganisms; for another part

The SPIN Kit for Soil total DNA (MP BIO Laboratories, California, USA) Kit extracts Soil total DNA, amplifies the V4-V5 region of bacterium 16S with primers 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 907R (5'-CCGTCAATTCCTTTGA GTTT-3'), and performs ITS2 sequencing amplification on fungi with primers ITS3-2024F (5'-GCA TCGATGAAGAACGCAGC-3') and ITS4-2409R (5'-TCCTCCGCTTATTGATATGC-3'). After amplification is completed, subsequent library construction, Miseq sequencing and sequence splicing are all carried out on a pennison Illumina Miseq sequencing platform. Bioinformatic scoring of high throughput sequencing data was performed in the pennison gene cloud (https:// www.genescloud.cn/home). According to the sequence similarity, the effective sequences are clustered into OTUs (97% similarity), species annotation is carried out on the OTUs to obtain taxonomic information of each sample, and the diversity of bacteria and fungi is calculated to obtain a related index.

4.2.5 field application effect of SCAUT009 microbial inoculum

(1) Influence on growth of vetch with cold mountain and smooth leaf

After actinomycete SCAUT009 is inoculated, the plant height, root length, fresh weight, dry weight, root number, chlorophyll content and crude protein content of the vetch with the cold mountain smooth leaves are improved by 27.42%, 22.05%, 30.99%, 25.77%, 24.14%, 25.72%, 17.97% and 9.82% (figure 4, table 4) compared with the control, and the yield per mu is increased by 29.49% (table 5). Researches show that a plurality of growth-promoting bacteria can regulate the life activities of plants and promote the growth of the plants by producing phytohormones such as indoleacetic acid, gibberellin and the like. The strain SCATU009 has the capability of producing IAA, dissolving phosphorus and the like, and promotes the growth of plants after inoculating the vetch with the cool mountain smooth leaf. In addition, the actinomycetes SCATU009 is inoculated to improve the nodulation rate of the vetch seed, which is probably related to the actinomycetes SCAUT009 with the capabilities of dissolving phosphorus and producing siderophores, the content of absorbable phosphorus and iron in soil is increased, the number of root nodules of the vetch seed is increased, the root system development is promoted, and the nitrogen fixation efficiency and the quality of the vetch seed are improved.

Influence of inoculating SCAUT009 microbial inoculum on growth of vetch

Influence of inoculating SCAUT009 microbial inoculum on yield of vetch

(2) Analysis of incidence of Medusa Liquidambaris

The common powdery mildew morbidity of the broomcorn vetch which is inoculated with the actinomycetes SCAUT009 and not inoculated with the broomcorn-leaf vetch is 4.82% and 13.45% respectively, and the morbidity of the plant which is not inoculated with the actinomycetes SCAUT009 is reduced by 64.16% compared with the morbidity of the plant which is not inoculated with the broomcorn-leaf vetch. Researches find that the actinomycetes are an important source for generating secondary metabolites such as antibiotics, the characteristic is favorable for the actinomycetes to become active antagonists of plant pathogenic bacteria, and the inoculation of the actinomycetes SCAUT009 obviously reduces the incidence rate of the vetch with the Liriopsis hanotiana, which is possibly related to the generation of certain bacteriostatic secondary metabolites by the actinomycetes SCAUT 009.

(3) Influence on soil physicochemical property and enzyme activity

After actinomycetes SCAUT009 is inoculated, the alkaline hydrolysis nitrogen, the quick-acting potassium, the available phosphorus and the organic matters in the rhizosphere soil of the vetch of the Liriodendron glaucocalyx are increased by 15.74 percent, 10.44 percent, 13.46 percent and 6.30 percent compared with the contrast, and the urease, the acid phosphatase, the catalase and the sucrase in the soil are all improved by 32.48 percent, 25.44 percent, 30.37 percent and 24.78 percent compared with the contrast (Table 6). The result shows that after the actinomycete SCAUT009 with the growth promoting function is inoculated, the number of the nodules and the symbiotic nitrogen fixation efficiency of the plants are increased, the content of available nitrogen in the soil is increased, and the soil microorganisms are promoted to participate in the conversion of nutrients in the soil, so that the activity of the soil enzyme is improved, the soil quality is improved, and good nutritional conditions are provided for the growth of the plants.

TABLE 6 influence of inoculation of SCAUT009 fungicide on the physicochemical properties of soil

(4) Influence on the number of microorganisms in the soil

The numbers of culturable bacteria, actinomycetes, fungi and rhizobia in rhizosphere soil treated by the inoculated actinomycetes SCAUT009 are obviously higher than those in non-inoculated treatment (Table 7), and are respectively increased by 41.12%, 66.98%, 22.58% and 64.42% compared with non-inoculated treatment, and the results show that the inoculated actinomycetes SCAUT009 stimulate the reproduction of indigenous microorganisms in the soil, increase the number of culturable microorganisms and particularly improve the microbial ecological conditions of the soil.

Table 7 inoculation of SCAUT009 microbial inoculum for root soil (unit: cfu/g) of vetch with Liangshan Guangshan leaf

(5) Analysis of diversity of soil microbial flora

Chao1, Shannon, Simpson and observer _ speces indexes were selected to evaluate and analyze the microbial diversity and abundance of rhizosphere soil of Actinomyces chinensis-inoculated SCAUT009 and uninoculated (control CK). As can be seen from FIG. 5, the indexes of the bacteria and fungi, namely Chao1, Shannon, Simpson and the observer _ speces, inoculated with the actinomycetes SCAUT009 in the rhizosphere soil of the cold-mountain-light-leaf vetch are all higher than those of the uninoculated control group (A and B in FIG. 5) (p is less than 0.05), wherein the indexes of the bacteria, namely Chao1, Shannon, Simpson, observer _ speces and the fungi, namely Chao1 and observer _ speces, are obviously higher than those of the control group, so that after the actinomycetes SCAUT009 is inoculated, the diversity and the abundance of the bacteria and the fungi in the rhizosphere soil of the cold-mountain-light-leaf vetch are obviously improved compared with those of the control group, and the actinomycetes SCAUT increases the composition of the microorganisms in the rhizosphere soil of the cold-mountain-light-leaf vetch, improves the complexity of the microbial community structure, establishes a good soil ecological environment for the healthy growth of plants, and promotes the growth of the plants.

EXAMPLE 5 Effect of Actinomycete inoculation SCAUT009 on growth of succeeding tobacco

5.1 Experimental arrangement

The test plants: flue-cured tobacco cloud 87

Test site: liangshan Zhuitong county

After the actinomycete SCAUT009 vetch is harvested and inoculated in the eastern Huidong tobacco area of Liangshan, flue-cured tobacco is continuously planted at the test point, and the fertilizing amount is reduced by 10 percent according to the normal level. Normal fertilizing amount for flue-cured tobacco planting in the east China tobacco section: 30 kg/mu (N: P) of compound fertilizer special for tobacco₂O₅:K₂O is 2: 3: 5) 50 kg/mu of commercial organic fertilizer and 20 kg/mu of oil cake. According to the agronomic character survey of YC/T142-2010 tobaccoIn the flue-cured tobacco mature period (70 d after the tobacco seedling is transplanted), 5 representative tobacco plants with uniform and consistent growth vigor are selected to respectively determine the agronomic characters of the flue-cured tobacco, such as the height, the stem circumference, the number of effective leaves and the like, and the root system structure, the vigor and the dry matter content of the flue-cured tobacco. The flue-cured tobacco root system structure adopts an EPSON1680 root system scanner (Epson, LongBeach, USA) to scan each group of sample root systems, and after scanning is completed, WinRhizo2005a root system analysis software is used for analyzing, so that morphological indexes such as total root length, total surface area, total volume, total root hair number and the like of the root system are obtained. Deactivating enzyme of tobacco root, stem and leaf at 105 deg.C for 20min, oven drying at 75 deg.C to constant weight, and determining biomass.

5.2 results of the experiment

After the vetch with the cool mountain smooth leaf inoculated with the actinomycetes SCAUT009 is harvested, the flue-cured tobacco yunyan 87 is continuously planted at a test point, and the determination result of the agronomic characters of the flue-cured tobacco in the mature period shows that the plant height, the stem circumference, the effective leaf number, the maximum leaf length, the maximum leaf width and the maximum leaf area of the flue-cured tobacco in the later period are respectively increased by 7.87%, 9.34%, 11.38%, 8.83%, 7.19% and 15.38% compared with the control group after the rhizobia is inoculated in the previous period (Table 8), the dry matter accumulation is increased by 7.57% compared with the control group (Table 9), and the total root length, the total root area, the root average diameter, the root volume and the root hair number are respectively increased by 8.30%, 6.82%, 8.49%, 7.31% and 7.29% compared with the control group (Table 10). The research results of the example 4 show that after the vetch is planted in the previous crop and the rhizobia is inoculated, the nitrogen content and other soil nutrient content in the soil are increased, the soil microbial community structure is optimized, the root system growth environment of the flue-cured tobacco in the next crop is improved, the root system activity is enhanced, the absorption of the flue-cured tobacco to nutrients is promoted, and the growth of the flue-cured tobacco and the accumulation of substances are driven. Therefore, the inoculation of the actinomycetes SCAUT009 not only increases the yield of the vetch in the current season of cool mountain smooth leaves, but also promotes the growth of the flue-cured tobacco of the succeeding crop by improving the soil quality.

TABLE 8 Effect on agronomic traits of flue-cured tobacco

TABLE 9 Effect on Dry matter accumulation of flue-cured tobacco

TABLE 10 Effect on root Structure of flue-cured tobacco

Note: the different lower case letters in the same column indicate that the difference at the 0.05 level was significant between the different treatments.

Sequence listing

<110> Sichuan university of agriculture

<120> actinomycete strain SCAUT009 and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1297

<212> DNA

<213> Microbacterium testaceum

<400> 1

gatcagtggc gaacgggtga gtaacacgtg agcaacctgc cctggactct gggataagcg 60

ctggaaacgg cgtctaatgc tggatatgag cctcttccgc atggtggggg ttggaaagat 120

tttttggtct gggatgggct cgcggcctat cagcttgttg gtgaggtaat ggctcaccaa 180

ggcgtcgacg ggtagccggc ctgagagggt gaccggccac actgggactg agacacggcc 240

cagactccta cgggaggcag cagtggggaa tattgcacaa tggcggaagc ctgatgcagc 300

aacgccgcgt gagggatgac ggccttcggg ttgtaaacct cttttagcag ggaagaagcg 360

aaagtgacgg tacctgcaga aaaagcgccg gctaactacg tgccagcagc cgcggtaata 420

cgtagggcgc aagcgttatc cggaattatt gggcgtaaag agctcgtagg cggtttgtcg 480

cgtctgctgt gaaatcccga ggctcaacct cgggcctgca gtgggtacgg gcagactaga 540

gtgcggtagg ggagattgga attcctggtg tagcggtgga atgcgcagat atcacgagga 600

acaccgatgg cgaaaggcag atctctgggc cgtaactgac gctgaggagc gaaagggtgg 660

ggagcaaaca ggcttagata ccctctagtc caccccgtaa acgttgggaa ctagtgtggg 720

accattccac ggtttcgtga cgcagctatc gcattatgct ccccgcctgg ggagtacggc 780

cgcaaggcta aaactcaaag gaattgacgg ggacccgcac aagcggcgga gcatgcggat 840

taattcgatg caacgcgaag aaccttacca aggcttgaca tacaccagaa cgggccagaa 900

atggtcaact ctttggacac tggtgaacag gtggtgcatg gttgtcgtca gctcgtgtcg 960

tgagatgttg ggttaagtcc cgcaacgagc gcaaccctcg ttctatgttg ccagcacgta 1020

atggtgggaa ctcatgggat actgccgggg tcaactcgga ggaaggtggg gatgacgtca 1080

aatcatcatg tcccttatgt cttgggcttc acgcatgcta caatggccgg tacaaagggc 1140

tgcaataccg tgaggtggag cgaatcccaa aaagccggtc ccagttcgga ttgaggtctg 1200

caactcgacc tcatgaagtc ggagtcgcta gtaatcgcag atcagcaacg ctgcggtgaa 1260

tacgttcccg ggtcttgtac acaccgcccg tcaagcc 1297

Claims

1. Actinomycete Microbacterium testaceum SCAUT009 is preserved in Guangdong province microorganism strain preservation center, and the preservation number is GDMCC No: 61508.

2. a microbial agent comprising the actinomycete strain Microbacterium testaceum SCAUT009 of claim 1.

3. A microbial fertilizer comprising the actinomycete strain Microbacterium testaceum SCAUT009 of claim 1.

4. Use of the actinomycete strain Microbacterium costatum SCAUT009 of claim 1, the microbial agent of claim 2 or the microbial fertilizer of claim 3 for planting vetch (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.