CN114181852B - Actinomycete strain SCAUT001 and application thereof - Google Patents

Abstract

The invention discloses an actinomycete strain Williamsia herbipolensis SCAUT001, which is preserved in Guangdong province microbial strain preservation center with the preservation number of GDMCC No:61375. the Williamsia herbipolaris SCAUT001 provided by separation and screening has growth promoting capabilities of generating IAA, siderophores, dissolving phosphorus, degrading cellulose and chitin and the like, and simultaneously has stress resistance functions of salt resistance, acid and alkali resistance, drought resistance and the like, and the effect of promoting the nodulation and growth of the vetch with the cool mountain smooth leaves is obtained by applying the method to the vetch with the cool mountain smooth leaves.

Description

Actinomycetes strain SCAUT001 and its application

technical field

The invention belongs to the field of microorganisms, and in particular relates to an actinomycete strain SCAUT001 and an application thereof.

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 the climate characteristics of dry winter and spring, rainy summer, and distinct dryness and wetness, the problem of lack of green fodder for livestock during winter and spring is very prominent. However, in the mountainous areas around the basin of Liangshan Prefecture, especially in the Zhongshan and sub-alpine areas at an altitude of 1700-3200m, a large amount of land is left idle during winter and spring. Viciavillosa var. glabresens is an annual or perennial herbaceous plant belonging to the leguminous family Vegetables. It likes a cool and humid climate, and is more resistant to cold and drought. It is suitable for areas at an altitude of 1500-3200m, especially at an altitude of 1800-2500m. It is suitable, has the advantages of water retention, fertilizer retention, soil improvement, nitrogen fixation and fertilization, and improvement of ecological environment. It is the main pasture grass popularized in alpine mountainous areas. Liangshan Prefecture has a long history of planting purple vetch, which can be planted in idle stubble in winter, which can make full use of light and heat resources and conserve moisture, increase the content of organic matter in the soil, improve soil quality, and at the same time It has solved the problem of lack of green fodder for sexual animals to survive winter and spring.

In recent years, the excessive use of chemical fertilizers not only pollutes the environment, but also causes a series of problems such as soil compaction, decreased fertility, and decreased quality of agricultural products. Soil microorganisms play an important role in the process of material cycle, organic matter decomposition and plant nutrient utilization. Plant growth promotingrhizobacteria (PGPR), which naturally exist in soil and colonize plant rhizosphere, can not only improve soil physical and chemical properties, It can also promote plant growth by producing growth hormone, nitrogen fixation, and phosphorus dissolution. Secondly, PGPR can also promote plant growth by producing chitinase, cellulase, antibiotics and other substances to resist pathogen infection and improve plant stress resistance. . Although Vitonia glabra has the ability to symbiotically form nodules with rhizobia and fix nitrogen, the process of biological nitrogen fixation and the growth of Vitex glabra also require the participation of other nutrients. The inorganic elements used transform the organic substances used by plants to promote the supply and growth of plant nutrients. Since A. glabra is a leguminous forage, previous studies mainly focused on the symbiotic relationship between rhizobia and A. glabra, but there is no report on the effect of rhizosphere growth-promoting bacteria on the growth of A. glabra. .

Actinomycetes are an important part of the plant rhizosphere microbial community, and play an important role in the transformation of soil substances. They dissolve phosphorus and fix nitrogen, secrete plant growth regulators, synthesize siderophore, induce plant resistance and produce resistance. Fungal metabolites and other ways to promote plant growth have great application potential in agricultural production. Taking the purple sweet potato collected in Liangshan Prefecture as the research object, the high-efficiency PGPR strain resources were isolated and screened, in order to improve the stress resistance of purple sweet potato by using rhizosphere growth-promoting bacteria, and to collect excellent PGPR strain resources for the development of microbial fertilizers. Maximizing the ecological benefits and economic value of Vitex glabrata is in line with the sustainable development of agriculture and animal husbandry in Liangshan Prefecture and the needs of ecological environment protection.

Contents of the invention

The technical problem to be solved by the present invention is: in order to excavate and screen the PGPR bacterial strain resource that can promote the growth of Vitonia glabrata and apply it in agricultural production, one of the purposes of the present invention is to provide a strain with stress resistance and growth-promoting functions Another object of the rhizosphere actinomycete is to provide the application of the strain.

The technical solution of the present invention is: actinomycetes strain Williamsia herbipolensis SCAUT001, referred to as SCAUT001, which has been preserved in the Guangdong Microbial Culture Collection Center on December 17, 2020, with the preservation number GDMCC No: 61375, and the preservation address is Guangdong Guangdong Institute of Microbiology, 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou City, China.

Microbial agent containing the actinomycete strain Williamsia herbipolensis SCAUT001.

Microbial fertilizer containing the actinomycete strain Williamsia herbipolensis SCAUT001.

Application of actinomycete strain Williamsia herbipolensis SCAUT001 or microbial agent or microbial fertilizer containing the strain on the planting of Vicia villosa Roth var. glabrescenscv. Liang shan.

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

Morphological characteristics of the bacterial strain of SCAUT001 of the present invention: after being cultivated on TSA medium at 28°C for 3 days, the bacterial colony is small, round and convex, with neat edges and smooth surface, the colony color is orange-red, easy to pick, and Gram staining is positive , Aerobic, Brevibacterium, no spores, the cell size is 1.5×1.0um.

The 16S rRNA sequence of the SCAUT001 strain of the present invention was determined, and the measured sequence was submitted to the EzBioCloud website (www.ezbiocloud.net) for homology comparison, and the phylogenetic analysis was performed using MEGA 7.0 software. The results showed that the 16S rRNA nucleotide sequence homology between strain SCAUT001 and Williamsia herbipolensis was 100%. Based on the experimental results of morphological characteristics and 16S rRNA sequence homology analysis, SCAUT001 was identified as Williamsiaherbipolensis.

The 16S rRNA gene phylogenetic analysis of the DNA of the SCAUT001 strain of the present invention is shown in the sequence table as SEQ ID NO.1.

The SCAUT001 strain of the present invention has growth-promoting functions such as IAA production, siderophore production, cellulase production, chitosan production, and phosphorus dissolution, and also has stress resistance capabilities such as salt tolerance, drought tolerance, and acid and alkali tolerance.

The results of the sand culture experiment show that after inoculation with the actinomycete Williamsia herbipolensis SCAUT001 bacterial agent, the plant height, root length, fresh weight and dry weight of the aboveground and underground parts, and chlorophyll of the Liangshan purple vetch plants were compared with those without inoculation. , which increased by 24.32%, 23.70%, 21.61%, 19.05%, 16.05%, 13.33%, and 11.09%, respectively, indicating that the strain SCAUT001 can obviously promote the growth of Liangshan purple vetch, and has a good growth-promoting effect.

The results of field experiments showed that after inoculation with the actinomycete Williamsia herbipolensis SCAUT001, the plant height, root length, fresh weight, dry weight, root weight, number of nodules, chlorophyll content and crude protein content of Liangshan purple sweet potato were all higher than those of the control Increased by 27.83%, 30.15%, 21.26%, 20.10%, 22.5%, 26.45%, 9.27%, 10.04%, the yield per mu increased by 27.98% compared with the control, and the incidence of powdery mildew of Liangshan purple vetch was lower than that of plants treated without inoculation The incidence of the disease was reduced by 58.68%; the alkaline nitrogen, available potassium, available phosphorus and organic matter in the rhizosphere soil of Liangshan purple sweet potato increased by 23.96%, 20.96%, 17.71% and 12.73% compared with the control, and the urease, Acid phosphatase, catalase, and sucrase were all increased by 45.83%, 28.67%, 43.80%, and 32.83% compared with the control. The ratios were increased by 52.89%, 76.48%, 59.32%, and 70.40%, respectively, and the richness of bacterial and fungal communities in the rhizosphere soil was significantly higher than that of the control group.

The measurement results of the agronomic traits, dry matter accumulation and root structure of the flue-cured tobacco at the maturity stage of the subsequent stubble showed that the plant height, stem girth, effective leaf number, maximum leaf length, and maximum leaf Compared with the control group, the width and maximum leaf area increased by 9.02%, 12.91%, 17.99%, 12.09%, 13.69%, and 24.13%, respectively, and the dry matter accumulation increased by 11.43%. Compared with the control group, the average diameter, root volume, and root hair number increased by 11.77%, 8.60%, 12.35%, 14.37%, and 13.72%, respectively.

After being inoculated with the actinomycete Williamsia herbipolensis SCAUT001, it promoted the growth of Liangshan Guangye purple sweet potato, increased the ability of nodulation, improved the quality of the plant, improved the micro-ecological conditions of the soil, improved the soil quality, and promoted The growth of flue-cured tobacco provides strain resources for the research and development of microbial inoculum of Vitex glabrata.

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

The Williamsia herbipolensis SCAUT001 provided by the separation and screening of the present invention has the ability to produce IAA, siderophore, dissolve phosphorus, degrade cellulose and chitin, etc., and also has stress resistance functions such as salt tolerance, acid and alkali tolerance, and drought tolerance. The application of Liangshan Guangye Zihuazi to obtain the effect of promoting the nodulation and growth of Liangshan Guangyezihuazi. The present invention not only increases the yield of forage grass, but also improves the soil quality and improves the soil for planting tobacco. It provides guarantee for planting and stabilizing the quality of tobacco leaves, and has good application potential in agricultural production.

Preservation information:

Actinomycetes strain Williamsia herbipolensis SCAUT001, referred to as SCAUT001, was deposited in the Guangdong Microbial Culture Collection Center on December 17, 2020, with the preservation number GDMCC No: 61375, and the preservation address is No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province Guangdong Institute of Microbiology, 5th Floor, Building 59, Compound.

Description of drawings

Fig. 1 is the colony morphology and thallus micrograph of actinomycete Williamsia herbipolensis SCAUT001 on ISP ₄ medium;

Figure 2 is a phylogenetic tree of Actinomyces glabrasia herbipolensis SCAUT001 constructed based on the 16S rRNA gene sequence;

Fig. 3 is the effect of the rhizosphere actinomycete Williamsia herbipolensis SCAUT001 on promoting the growth of Viola glabrata through sand culture, wherein the left side is the control without inoculation, and the right side is the treatment of inoculation with SCAUT001;

Figure 4 Field experiment results, left: control; right: SCAUT001;

Fig. 5 Alpha diversity of bacteria (A) and alpha diversity of fungi (B) in rhizosphere soil of Vitex 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 sources unless otherwise specified.

The separation and purification of embodiment 1 rhizosphere actinomycete SCAUT001

1.1 Isolation of actinomycetes from the rhizosphere of Vitex glabrata in Liangshan

In 2018, the purple vetch was collected in Huidong County, Liangshan Yi Autonomous Prefecture, Sichuan Province. The excess soil on the root system was shaken off, and the root was placed in a 50mL sterilized centrifuge tube filled with 25mL phosphate buffer solution, 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 . Separation was carried out on Gao's medium, and each dilution concentration was repeated three times, and cultured in a constant temperature incubator at 28°C for 5-7d. Pick the strains with typical actinomycete colony characteristics, and use the dilution plate streaking method to purify until the purified strains are obtained. The purified strains were inoculated into ISP ₄ liquid medium, cultured with shaking at 28°C for 7 days, preserved with 30% glycerol, and frozen at -70°C.

1.2 Strain identification

1.2.1 Strain morphology

The strain was inoculated on ISP ₄ medium, cultured at 28°C for 5 days, and the colony morphology of the strain was observed.

1.2.2 Molecular identification of strains

(1) Reagent: 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′ (identical to the 8-27 bases at the 5′ end of 16S rRNA); same base);

(3) Actinomycetes DNA extraction

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

(4) Actinomycetes 16S rRNA gene amplification

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

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

The sequence obtained after sequencing was searched for similarity in NCBI using BLAST software, and the 16S rRNA gene sequence of the published strain with the highest similarity was selected as a reference sequence, and Clustal X software was used for multiple sequence comparison analysis, and MEGA 7.0 software was used to The phylogenetic tree was constructed by N-J method to determine the taxonomic status of Actinomycetes.

1.3 Experimental results

The actinomycete SCAUT001 was isolated from the rhizosphere soil of Vitex glabrata in Liangshan. After cultured on ISP ₄ medium at 28°C for 3 days, the colony was small, round and convex, with neat edges and smooth surface, and the color of the colony was orange. Red, easy to pick, Gram-positive, aerobic, Brevibacterium, no spores, the size of the bacteria is 1.5×1.0um (Figure 1).

The 16S rRNA sequence of the strain SCAUT001 was determined, and the BLAST homology comparison was performed in the NCBI database. The similarity between the strain and Williamsia herbipolensis was 100%. Based on the experimental results of morphological characteristics, physiological and biochemical characteristics, and 16S rRNA sequence homology analysis, SCAUT001 was identified as Williamsia herbipolensis (Figure 2).

The growth-promoting function screening of embodiment 2 rhizosphere actinomycete SCAUT001

2.1 Screening of growth-promoting function

2.1.1 Determination of IAA Production Ability

Take 20 μL of the bacterial suspension and insert it into the ISP ₄ liquid medium containing 0.5mol/L tryptophan, repeat 3 times, set up a blank control, culture on a shaker at 28°C for 3 days at 120r/min, centrifuge at 8000r/min for 24h, and take 1mL of bacteria Add 2 mL of IAA chromogenic solution to the liquid supernatant, react in the dark at 25°C for 30 min, perform colorimetry at a wavelength of 530 nm, and record the absorbance value. With the uninoculated liquid culture medium as the control zero adjustment, the indole acetic acid standard solution with the concentration of 0, 5, 20, 40, 60mg/L is the same as above to make the calibration curve, and calculate the concentration of IAA in the measurement solution.

2.1.2 Determination of siderophore production capacity

Dissolve 0.012g of chrome azure in 10mL of double distilled water, and mix with 2mL of 1mmoL/L FeCl ₃ 6H ₂ O solution to obtain solution a; dissolve 0.015g of cetyltrimethylammonium bromide in 8mL In double-distilled water, solution b was obtained; slowly add solution a to solution b, and mix evenly to obtain dye solution c; add 20 mL of 10×MM9 salt solution and 6.04 g of piperazine diethanesulfonic acid into a container filled with 150 mL of double-distilled water After mixing evenly in the conical flask, adjust the pH to 6.8 with 50% NaOH, then add 3.2g agar powder to obtain medium d; mix dye solution c, medium d, 1mmoL/L of CaCl ₂ , 1mmoL/L of MgSO ₄ , 20% glucose and 10% casamino acids were sterilized at 115°C for 20 minutes, and when the temperature of each solution dropped to 50-60°C, 200 μL CaCl ₂ , 4 mL MgSO ₄ , 6 mL casamino acids, and 2 mL glucose were added to the medium d , and then add dye solution c along the bottle wall, mix well without generating bubbles to obtain the blue detection medium, and pour the plate. Use sterile bamboo sticks to inoculate the strains to be tested, inoculate 5 bacterial cakes equidistantly on each plate, repeat 3 times for each strain, culture at 28°C for 3 days, observe and record the size of the orange transparent circle.

2.1.3 Detection of phosphorus dissolving ability

Use a sterile puncher (d=5mm) to make bacteria cakes from well-grown actinomycete strains, place them upside down on the PKO medium respectively, culture the bacteria cakes at 28°C in four directions 2 cm from the center of the plate 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 strains obtained by primary screening were made into seed suspension, inoculated in the Erlenmeyer flask of the re-screening medium whose carbon source was CMC-Na according to 10% inoculum amount, and cultured with constant temperature and vibration at 28° C., respectively measured 3, 5, 7, The activity of carboxymethyl cellulase (CMCase) of each bacterial strain in 9 and 11d, set 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, boiled water bath 5min, after cooling, dilute to 25mL, measure OD value under spectrophotometry 540nm, draw the standard curve.

(2) Preparation of crude enzyme solution

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

(3) Determination of enzyme activity

Enzyme activity assay method: take 0.1mL crude enzyme solution, add 1.9mL CMC-Na solution with a mass fraction of 1%. Hydrolyze at a constant temperature of 45°C for 20 minutes, add 1.5mL of DNS chromogenic solution for boiling water bath for 5 minutes, set the volume to 25mL, measure the color at 540 nm, measure the absorbance (OD) value, compare with the standard glucose curve, and calculate from the OD value Glucose volume (m1). Add 0.1 mL of each supernatant, add 1.9 mL of water, add 1.5 mL of DNS, boil in water for 5 minutes, and set the volume to 25 mL. Measure the glucose content (m2) of the crude enzyme solution by colorimetric at 540 nm. The glucose amount (m1) is subtracted from the glucose amount (m2) to obtain the glucose amount obtained by degrading 1% CMC solution by the CMC enzyme, and the glucose amount A calculated by the optical density value is used to calculate the enzyme activity of the bacterial strain.

2.1.5 Detection of chitinase production

Using MM-Chitin medium, inoculate the test strain on the medium, if a hydrolysis circle is produced, it proves that it is a strain with the ability to degrade chitin.

2.2 Anti-stress function screening

2.2.1 Salt tolerance test

The tested strains were streaked and cultured in ISP ₄ based on 28°C for 3-4 days, and sterile water was used to make a bacterial suspension. With NaCl concentration: 0%, 1%, 3%, 5%, 7%, 7 kinds of ISP ₄ solid medium were made, and the bacterial suspension was spotted on the medium with a sterile bamboo stick, and repeated 3 times, 28 Cultivate at ℃ for 3-4 days, observe the growth of each rhizosphere actinomycete 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 testing

The tested strains were streaked and cultured in ISP ₄ based on 28°C for 3-4 days, and sterile water was used to make a bacterial suspension. Based on the ISP ₄ medium formula, use NaOH, KH ₂ PO ₄ , Na ₂ CO ₃ , KCl, NaHCO ₃ , Na ₂ HPO ₄ 10H ₂ O, sodium citrate, and trisodium citrate to configure the pH reaction buffer, Make the pH of the culture medium reach 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0 respectively, point the bacterial suspension onto the culture medium with a sterile bamboo stick, do 3 repetitions, and culture at 28°C for 3- 4d, observe the growth of each rhizosphere actinomycete and record the results. If the strain can grow normally at a certain pH, it shows that the strain has the ability to withstand the pH.

2.2.3 Drought tolerance test

The preliminary screening test of drought tolerance was carried out using polyethylene glycol (PEG) 6000 artificially simulated drought conditions, and the following four different PEG 6000 levels were set in the test:

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

(2) 15% PEG 6000;

(3) 25% PEG6000;

(4) 35% PEG6000.

Their corresponding water potentials are: 0, -0.278, -0.699, -1.309MPa respectively. Pick one loop of the tested strains and inoculate them into the sterilized ISP ₄ liquid medium, place them on a shaker at 28°C and 200r/min for 4 days to make an inoculum, and adjust the OD600 value to about 0.7. Pipette 0.1mL inoculum into ISP ₄ liquid medium with different concentrations of PEG6000, culture on a shaker at 28°C and 200r/min for 7 days, then mix and sample, measure its OD value at 600nm, and evaluate its growth and reproduction based on the OD value situation. The instrument should be zeroed with ISP ₄ culture medium with corresponding concentration of PEG6000 before measurement. Using the uninoculated ISP4 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

It has been determined that the IAA yield of the actinomycete Williamsia herbipolensis SCAUT001 is 62.13 mg/mL, and has the ability to produce cellulase, chitinase and phosphorus solubilization (Table 1); , a salt concentration of 0.5-2%, and a PEG6000 concentration of 15%-25% in ISP ₄ medium (Table 2). The results showed that actinomycetes SCAUT001 had better growth-promoting and stress-resistance functions, and had the potential for further development and application.

Table 1 Growth-promoting function of strain SCAUT001

Table 2 Stress resistance function of strain SCAUT001

Embodiment 3 hydroponic experiment of purple sweet potato

3.1 Hydroponics experiment

The actinomycete strains to be tested were first activated on the ISP ₄ plate medium, and then transferred to the ISP ₄ liquid medium for overnight culture to the logarithmic phase. The growth of the strains was detected by the plate method, and the bacterial content was calculated.

After soaking the seeds of Viola glabrata overnight, carry out surface disinfection, use sterile tweezers to grasp the seeds and plant them in a petri dish equipped with filter paper and sterile vermiculite, plant 10 seeds in each petri dish, and then add sterile water to keep moisture , and maintain air circulation. Sterilize the tweezers, filter paper, petri dishes, plastic cups, 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, iron citrate 3.0 g.

Put the sterilized vermiculite into a plastic cup stuffed 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 implant them into plastic cups filled with sterile vermiculite. Plant 3 seeds in each cup, then add 2 mL of the bacterial solution along the roots of each seed, and cover the surface with a layer of bacterial solution after adding the bacterial solution. quartz sand. Three replicates were set up for each bacterium, and a control treatment was set up. Cultured for 45 days, supplemented with nutrient solution from time to time. After the cultivation, carefully take the purple vetch from the cup, wash the vermiculite attached to the root with clean water, and measure the plant height, root length, fresh weight, dry weight and other indicators.

3.2 Experimental results

The results of hydroponic experiments showed that the plant height, root length, fresh weight and dry weight of the aboveground and underground parts, and chlorophyll of the Liangshan purple vetch plants inoculated with the actinomycete Williamsia herbipolensis SCAUT001 were all compared with those of the non-inoculated treatment (CK) , respectively increased by 24.32%, 23.70%, 21.61%, 19.05%, 16.05%, 13.33%, 11.09% (Table 3, Fig. 3), indicating that the bacterial strain SCAUT001 can obviously promote the growth of Liangshan guangyezihuazi. Good growth-promoting ability.

Table 3 SCAUT001 hydroponic experiment results (45d)

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

Preparation of embodiment 4 biological fertilizer and field application effect

Field Application of Actinomycete Williamsia herbipolensis SCAUT001 Strain

1. Bacteria preparation

The actinomycete Williamsia herbipolensis SCAUT001 strain was inoculated on the ISP ₄ slant medium, cultivated in a 28°C incubator until the bacterial lawn was overgrown, washed with sterile water, inoculated in the actinomycetes ISP ₄ liquid medium, and placed in Shake at 28°C and 160 r/min until the OD value of the bacterial suspension (λ=600nm) reaches 0.9, then set aside (note: specify the number of bacteria per ml of bacterial solution, that is, 5×10 ¹⁰ cfu/ml). Wheat bran carrier, added by weight and volume ratio, 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 moisture content of wheat bran to 40%, 121°C, 0.1 Mpa steam sterilization for 30 min; inoculate the prepared bacterium suspension in a solid culture medium with an inoculum size of 20% (volume ratio) and mix evenly, so that the water content of the carrier is maintained at 50-60%, and cultured statically at 28°C When the number of viable bacteria in the agent reaches 2×10 ¹⁰ cfu/g, it is the solid-state fermentation agent of SCAUT001. The number of viable bacteria in the agent is stored at 20°C under sterile conditions for 6 months and the number of viable bacteria is >2×10 ⁸ cfu/g and Does not affect its life-promoting activity.

2. Field application of fungicide Williamsia herbipolensis SCAUT001

Experimental materials: Liangshan local pasture species Liangshan Guangye Zihuazi.

Experimental location: Huidong County, Liangshan Prefecture

Half an hour before sowing, choose a shaded place for seed dressing, put the wheat bark carrier containing actinomycetes in clear water and stir evenly (the number of bacteria is >2×10 ⁸ cfu/ml), and put the seeds of Viola glabra in net After cleaning in a nylon bag, place it in a plastic basin filled with actinomycete agent, soak for 20-30 minutes, take it out, and air-dry it in a dark place until it does not stick to your hands before sowing. The planting area is 1 mu, and a control test without inoculation is carried out at the same time. Grass samples and soil samples were collected during the full flowering stage of Vitex glabrata, and random samples were taken 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.

(1) Determination of Growth and Physiological Indexes of Liangshan Violet Verticillium

The collected samples of purple sweet potato were taken back to the laboratory. After cleaning and removing mud, the plant height, root length, fresh weight, dry weight, root weight, root nodule number, chlorophyll content, crude protein content and its yield were measured respectively.

(2) Investigation on the incidence of Vitiligo glabrata in Liangshan

In the inoculated and non-inoculated plots, random sampling was conducted to investigate the incidence of Vitonia glabrata, 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)/number of total investigated plants (organs, leaves)]×100%

(3) Determination of soil physicochemical and enzyme activity

The rhizosphere soil samples were collected from Viola glabrata and divided into 3 parts, one part was used to determine the physical and chemical indicators such as alkaline nitrogen, available potassium, available phosphorus and organic matter in the soil, as well as urease, acid phosphatase and catalase , sucrase and other soil enzyme activities.

(4) Analysis of soil microbial quantity and population diversity

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。The other two soil samples were collected, one was counted by the plate count of soil microorganisms;

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

3. Analysis of field application effect of bacterial agent SCAUT001

(1) Effects on the growth of Liangshan Violet vetiver

After being inoculated with actinomycete SCAUT001, the plant height, root length, fresh weight, dry weight, root weight, root nodule number, chlorophyll content and crude protein content of Liangshan purple sweet potato were all increased by 27.83%, 30.15%, 21.26% compared with the control. %, 20.10%, 22.5%, 26.45%, 9.27%, 10.04% (Figure 4, Table 4), and the yield per mu increased by 27.98% 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 indole acetic acid and gibberellin. The strain SCAUT001 has the ability to produce IAA and dissolve phosphorus, and it can promote the growth of plants after being inoculated with Liangshan guangyezihuazi. In addition, we also found that the inoculation of actinomycete SCAUT001 increased the nodulation rate of Liangshan purple vetch, which may be related to the ability of actinomycete SCAUT001 to dissolve phosphorus and produce iron carriers. The absorbable phosphorus and iron in the soil The increase of content increases the number of root nodules of Liangshan purple sweet potato, promotes the root development, improves the nitrogen fixation efficiency and the quality of Liangshan bright leaf purple sweet potato.

Table 4 Effect of inoculating SCAUT001 bacterial agent on the growth of Vitex glabrata

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

Table 5 Effect of inoculation of SCAUT001 bacterial agent on the yield of purple vetch

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

(2) Analysis of the incidence rate of Vitex glabrata in Liangshan

The common powdery mildew incidence rate of Liangshan purple sweet potato inoculated with Actinomyces SCAUT001 and not inoculated was 5.37% and 13.45%, respectively, and the incidence of inoculated plants was reduced by 58.68% compared with non-inoculated plants. The study found that actinomycetes are an important source of secondary metabolites such as antibiotics. This feature helps actinomycetes to become active antagonists of plant pathogens. This may be related to some antibacterial secondary metabolites produced by Actinomycetes SCAUT001.

(3) Effects on soil physicochemical and enzyme activity

After being inoculated with actinomycete SCAUT001, the alkaline nitrogen, available potassium, available phosphorus and organic matter in the rhizosphere soil of Vitex glabrata in Liangshan increased by 23.96%, 20.96%, 17.71%, and 12.73% compared with the control, and the urease, acidic Phosphatase, catalase, and sucrase were all increased by 45.83%, 28.67%, 43.80%, and 32.83% compared with the control (Table 6). The results showed that the inoculation of actinomycete SCAUT001 with growth-promoting function not only increased the number of root nodules and symbiotic nitrogen fixation efficiency of plants, 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, thereby Improve soil enzyme activity, improve soil quality, and provide good nutritional conditions for plant growth.

Table 6 Effects of inoculation of SCAUT001 bacterial agent on the physical and chemical properties of the rhizosphere soil of Liangshan purple sweet potato

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

(4) The impact on the number of soil microorganisms

The number of cultivable bacteria, actinomycetes, fungi and rhizobia in the rhizosphere soil inoculated with actinomycete SCAUT001 was significantly higher than that in the non-inoculated treatment (Table 7), which increased by 52.89%, 76.48%, and 59.32% respectively compared with the non-inoculated treatment. %, 70.4%, the results showed that the inoculation of actinomycetes SCAUT001 stimulated the reproduction of indigenous microorganisms in the soil, increased the number of cultivable microorganisms, especially improved the micro-ecological conditions of the soil.

Table 7 Inoculation of SCAUT001 bacterial agent on the rhizosphere soil of Liangshan purple sweet potato (unit: cfu/g)

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

(5) Analysis of the diversity of soil microbial flora

The Chao1, Shannon, Simpson and Observed_species indexes were selected to evaluate and analyze the microbial diversity and richness of the rhizosphere soil of Liangshan purple sweet potato inoculated with actinomycetes SCAUT001 and uninoculated (control group CK). It can be seen from Figure 5 that the Chao1 and Observed_species indexes of bacteria and fungi in the rhizosphere soil inoculated with actinomycete SCAUT001 Liangshan Vitiligo glabrata were higher than those of the uninoculated control group (A, B in Figure 5) (p<0.05) , indicating that after inoculation with actinomycete SCAUT001, the richness of bacterial and fungal communities in the rhizosphere soil of Liangshan purple sweet potato was significantly increased compared with the control group, indicating that actinomycete SCAUT001 increased the soil microbial The composition increases the complexity of the microbial community structure, builds a good soil ecological environment for the healthy growth of plants, and promotes the growth of plants.

Effect of Example 5 Inoculation of Actinomycetes SCAUT001 on the Growth of Later Stubble Tobacco

1. Experimental arrangement

Test plant: flue-cured tobacco cloud 87

After harvesting and inoculating the Actinomycetes SCAUT001 in the Huidong Tobacco Area of Liangshan Prefecture, the planting of flue-cured tobacco continued at the experimental site, and the amount of fertilization was reduced by 10% from the normal level. Normal fertilization amount for flue-cured tobacco planting in Huidong Tobacco Area: Tobacco-specific compound fertilizer 30kg/mu (N:P ₂ O ₅ :K ₂ O is 2:3:5), commercial organic fertilizer 50kg/mu, and oil 20kg/mu. According to "YC/T 142-2010 Tobacco Agronomic Characters Investigation and Measurement Method", five representative tobacco plants with uniform growth were selected at the maturity stage of flue-cured tobacco (70 days after transplanting) to measure the plant height, stem girth, effective Agronomic traits such as leaf number, root structure, vigor and dry matter content of flue-cured tobacco were measured. The root system structure of flue-cured tobacco is scanned by EPSON1680 root system scanner (Epson, Long Beach, USA) to scan the roots of each group of samples. After the scanning is completed, the root system analysis software WinRhizo2005a is used to analyze the total root length, total surface area, total volume and total root hair number of the root system. and other morphological indicators. Roots, stems, and leaves of flue-cured tobacco were killed at 105°C for 20 minutes, dried at 75°C to constant weight, and then the biomass was measured.

2. Experimental results

After harvesting the Liangshan purple sweet potato inoculated with actinomycete SCAUT001, flue-cured tobacco Yunyan 87 was continued to be planted at the test site, and the agronomic traits of the flue-cured tobacco at the mature stage were measured. , stem girth, effective number of leaves, maximum leaf length, maximum leaf width, and maximum leaf area increased by 9.02%, 12.91%, 17.99%, 12.09%, 13.69%, and 24.13% (Table 8) respectively compared with the matched group. Compared with the control group, the amount increased by 11.43% (Table 9), and the total root length, total root area, root average diameter, root volume, and root hair number increased by 11.77%, 8.60%, 12.35%, 14.37%, and 13.72% respectively compared with the control group (Table 10). Based on the research results of Example 4, it shows that the planting of Liangshan glabra vetch and inoculation with rhizobia in the previous crop 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 flue-cured tobacco in the subsequent crop. 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 actinomycete SCAUT001 not only increased the yield of Liangshan succulents in the current season, but also promoted the growth of flue-cured tobacco in subsequent crops by improving soil quality.

Table 8 Effects on the Agronomic Characters of Flue-cured Tobacco

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

Table 9 Effects on Dry Matter Accumulation of Flue-cured Tobacco

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

Table 10 Effects on the Root System Structure of Flue-cured Tobacco

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

sequence listing

<110> Sichuan Agricultural University

<120> Actinomycetes strain SCAUT001 and its application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 2

<211> 1265

<212>DNA

<213> Williamsia herbipolensis

<400> 2

ggtggtggaa agtttttcgg tacgagatgg gcccgcggcc tatcagcttg ttggtggggt 60

aatggcctac caaggcgacg acgggtagcc ggcctgagag ggcgaccggc cacactggga 120

ctgagacacg gcccagactc ctacgggagg cagcagtggg gaatattgca caatgggcgc 180

aagcctgatg cagcgacgcc gcgtgaggga tgacggcctt cgggttgtaa acctctttca 240

ccagggacga agagtgattg acggtacctg gagaagaagc accggccaac tacgtgccag 300

cagccgcggt aatacgtagg gtgcgagcgt tgtccggaat tactgggcgt aaagagctcg 360

taggcggttt gtcgcgtcgt tcgtgaaatc ttgatgctta acatcaagcg tgcgggcgat 420

acgggcagac ttgagtacta caggggagac tggaattcct ggtgtagcgg tgaaatgcgc 480

agatatcagg aggaacaccg gtggcgaagg cgggtctctg ggtagtaact gacgctgagg 540

agcgaaagcg tgggtagcga acaggattag atacccctggt agtccacgcc gtaaacggtg 600

ggtactaggt gtgggttcct tttcacggga tccgtgccgt agctaacgca ttaagtaccc 660

cgcctgggga gtacggccgc aaggctaaaa ctcaaaggaa ttgacggggg cccgcacaag 720

cggcggagca tgtggattaa ttcgatgcaa cgcgaagaac cttacctggg tttgacatac 780

accagaaagc tgtagagata cagcccccct tgtggttggt gtacaggtgg tgcatggctg 840

tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttgtcct 900

gtattgccag cgggttatgc cggggacttg caggagactg ccggggtcaa ctcggaggaa 960

ggtggggatg acgtcaagtc atcatgcccc ttatgtccag ggcttcacac atgctacaat 1020

ggccggtaca gagggctgcg ataccgtgag gtggagcgaa tcccttaaag ccggtctcag 1080

ttcggatcgg ggtctgcaac tcgaccccgt gaagtcggag tcgctagtaa tcgcagatca 1140

gcaacgctgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac gtcatgaaag 1200

tcggtaacac ccgaagccgg tggcctaacc cttgtgggagg gagctgtcga aggtgggatc 1260

ggcga 1265

Claims (5)

1. Actinomycetes strain Williamsia herbipolensis SCAUT001, preserved in the Guangdong Microbial Culture Collection Center with the preservation number GDMCC No: 61375. 2. the microbial bacterial agent that contains the actinomycetes bacterial strain Williamsia herbipolensis SCAUT001 described in claim 1. 3. the microbial fertilizer that contains the actinomycetes bacterial strain Williamsia herbipolensis SCAUT001 described in claim 1. 4. the actinomycete bacterial strain Williamsia herbipolensis SCAUT001 described in claim 1 or the microbial inoculum agent described in claim 2 or the microbial fertilizer described in claim 3 in Liangshan Guangye Zihuazi ( Vicia villosa Roth var.glabrescenscv.Liang shan ) for planting purposes. 5. The use according to claim 4, wherein the planting refers to the planting of Vicia villosa Roth var. glabrescenscv. Liang shan in Liangshan Prefecture.

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