CN115725419A - Phosphorus-dissolving blueberry endophytic trichoderma and application thereof - Google Patents

Abstract

The invention discloses a phosphorus-solubilizing blueberry endophytic Trichoderma and its application. The phosphorus-solubilizing blueberry endophytic Trichoderma is named Trichoderma koningiopsis GL41 Trichoderma koningiopsis , and was preserved in China's Common Microbial Fungus on July 11, 2022. Species Collection Management Center, the deposit number is CGMCC No.40241. The phosphorus-solubilizing blueberry endophytic Trichoderma fungus of the present invention has the function of dissolving inorganic phosphorus. With the PKO solid medium as the basic medium, the available phosphorus content of GL41 Trichoderma koningiopsis gradually increases, and reaches a maximum value of 273.90 µg/ml on the 5th day, effectively promoting blueberry production. The growth has great potential to be developed into microbial inoculant.

Description

An Endophytic Trichoderma Strain of Phosphorus Solubilizing Blueberry and Its Application

technical field

The invention belongs to the technical field of agricultural microorganisms, and relates to a phosphorus-solubilizing blueberry endophytic Trichoderma and application thereof.

Background technique

Plant rhizosphere growth-promoting bacteria are a kind of beneficial bacteria that grow in the rhizosphere soil of plants. They have various growth-promoting mechanisms for plants, such as biological nitrogen fixation, dissolving phosphorus and potassium, producing plant hormones such as IAA, antagonizing pathogenic bacteria, and inducing systemic resistance. sex etc. In particular, the organic acids produced by these bacteria during the metabolic process can dissolve insoluble phosphorus and potassium in the soil to promote the growth and development of plants, and can also enhance plant resistance to diseases, heavy metals and salt by secreting siderophore and ACC deaminase. Resistance to adversities such as alkalis. It is of great significance to isolate and screen microbial strains with growth-promoting effects from the environment and develop microbial fertilizers for the reduction of environmental pollution and the development of the planting industry.

Blueberry ( Vacciniumuliginosum ) is a common name for the blue fruit type plant of Ericaceae ( Ericaceae ). Wild blueberries are widely distributed throughout the world. Mainly produced in the United States, it is mainly distributed in the Daxinganling and Xiaoxinganling forest areas in China. As a new fruit with high economic value and health care function, blueberry has a relatively large cultivation area. However, at present, most growers generally lack experience in planting blueberries in protected cultivation. When blueberries grow poorly or abnormally, it is often due to the lack of sufficient nitrogen, phosphorus, potassium and other large elements, but the application of large amounts of chemical fertilizers will lead to soil, water pollution and ecological problems. Environmental degradation.

Trichoderma belongs to Ascomycota, Coprophyta, Hypocreales, Hypocreataceae, Trichoderma, widely exists in soil under different environmental conditions. Since the mid-19th century, humans have had a preliminary understanding of Trichoderma, but it was not until the 1960s that the taxonomic status of Trichoderma was determined. Most Trichoderma can produce a variety of biologically active substances that have antagonistic effects on plant pathogenic fungi, bacteria and insects, such as cell wall degrading enzymes and secondary metabolites, and can improve the stress resistance of crops, promote plant growth and improve agricultural products. Therefore, it is widely used in biological control, biological fertilizer and soil conditioner.

Currently, the patent strains that have growth-promoting effects on blueberries are reported as DSE fungus (CN107083335A) and Bacillus (CN114134070A). However, there are few reports on the phosphorus-dissolving endophytic Trichoderma in blueberries, and there is no report on the growth-promoting effect of phosphorus-dissolving endophytic Trichoderma in blueberries.

Contents of the invention

The object of the present invention is to provide a strain of endophytic Trichoderma phosphate-solubilizing blueberry and its application. The phosphorus-solubilizing blueberry endophytic Trichoderma of the present invention is named Trichoderma koningiopsis GL41 ( Trichodermakoningiopsis ), and the phosphorus-solubilizing blueberry endophytic Trichoderma has the function of dissolving phosphorus in the medium and promoting the growth of the blueberry rhizosphere.

The technical scheme of the present invention: a strain of endophytic Trichoderma phosphoderma in blueberries, which is named Trichoderma koningiopsis GL41 Trichodermakoningiopsis , and was preserved in the China Common Microorganisms Collection on July 11, 2022 Management Center, the deposit number is CGMCC No.40241.

The aforementioned phosphorus-solubilizing blueberry endophyte Trichoderma is obtained from blueberry roots through a tissue separation method.

Aforesaid phosphorus-solubilizing blueberry endophytic Trichoderma, the tissue separation method of described phosphorus-solving blueberry endophytic Trichoderma is:

(1) Cleaning: select blueberry roots with healthy surface and no disease spots, wash them with water and dry them to obtain product A;

(2) Disinfection and sterilization: In the ultra-clean workbench, soak product A in ethanol solution, rinse with sterile water, transfer to NaClO solution for disinfection, then rinse with sterile water and blot the water to obtain product B;

(3) Preparation of PDA medium: Peel the potatoes and cut them into small pieces, boil them in water, filter them with gauze, add glucose and agar to dissolve in distilled water, spread them on a flat plate to make PDA medium;

(4) Culture separation and purification: put B product in PDA medium, place it in a constant temperature incubator for cultivation, use the fungal colony edge picking method, pick the typical fungi with different growth forms in the PDA medium and transfer them to a new PDA culture Based on the cultivation and purification in a constant temperature incubator, endophytic Trichoderma phosphate-solubilizing blueberries were obtained.

Specifically, the tissue separation method of the aforementioned phosphorus-solubilizing blueberry endophytic Trichoderma is:

(1) Cleaning: select blueberry roots with healthy surface and no disease spots, wash them with water and dry them to obtain product A;

(2) Disinfection and sterilization: In the ultra-clean workbench, immerse product A in 75% ethanol solution for 2-4s, rinse with sterile water for 4-6 times, and transfer to 3% NaClO solution for disinfection for 2- 4min, then rinse 4-6 times with sterile water and blot the water to obtain product B;

(3) Preparation of PDA medium: Take 150-250g of peeled potatoes and cut them into small pieces, boil them in water, filter them with gauze, add 15-25g of glucose and 15-25g of agar, dissolve them in 1000mL of distilled water, spread them on a flat plate , make PDA culture medium;

(4) Cultivation, isolation and purification: put product B in PDA medium, culture in a constant temperature incubator at 28°C for 3-5 days, and use fungal colony edge picking method to pick out typical fungi with different growth forms in PDA medium for transfer The new PDA culture is based on culturing and purifying in a constant temperature incubator at 28°C, and the purification is performed 3-5 times to obtain endophytic Trichoderma endophytes in phosphorus-solubilizing blueberries.

More specifically, the tissue separation method of the aforementioned phosphorus-solubilizing blueberry endophytic Trichoderma is:

(1) Cleaning: select blueberry roots with healthy surface and no disease spots, wash them with water and dry them to obtain product A;

(2) Disinfection and sterilization: In the ultra-clean workbench, soak product A in 75% ethanol solution for 3 seconds, rinse with sterile water for 5 times, transfer to 3% NaClO solution for disinfection for 3 minutes, and then use sterile Bacteria water was washed 5 times and its moisture was blotted to obtain product B;

(3) Preparation of PDA medium: Take 200g peeled potatoes and cut them into small pieces, boil them in water, filter them with gauze, add 20g of glucose and 20g of agar, dissolve them in 1000mL of distilled water, spread them on a flat plate, and make PDA culture medium. base;

(4) Cultivation, isolation and purification: put product B in PDA medium, culture in a constant temperature incubator at 28°C for 3-5 days, and use fungal colony edge picking method to pick out typical fungi with different growth forms in PDA medium for transfer Put it into a new PDA medium, cultivate and purify in a constant temperature incubator at 28°C, and purify for 3-5 times to obtain endophytic Trichoderma phospholyticus blueberry.

The application of the aforementioned phosphorus-solubilizing blueberry endophytic Trichoderma, the application of the phosphorus-solving blueberry endophytic Trichoderma in preparing a preparation for dissolving phosphorus in a medium.

The application of the aforementioned phosphorus-solubilizing blueberry endophytic Trichoderma, the application of the phosphorus-solubilizing blueberry endophytic Trichoderma in preparing a preparation for promoting blueberry rhizosphere growth.

A blueberry growth-promoting bacterium product, the active ingredient of the product comprises the phosphorus-solubilizing blueberry endophyte Trichoderma as claimed in claim 1 or 2.

A blueberry growth-promoting bacterium product, the active ingredient of which is the phosphorus-solubilizing blueberry endophyte Trichoderma described in claim 1 or 2.

A method for preparing a blueberry growth-promoting bacteria product, using the phosphorus-solubilizing blueberry endophyte Trichoderma as the active ingredient or one of the active ingredients for preparing the product.

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

The invention discloses an endophytic Trichoderma strain of phosphorus-solubilizing blueberry and its application. The tissue separation method is used to isolate the blueberry collected from Huaxi Gaopo Ecological Blueberry Garden (East Longitude 106°50′14″, North Latitude 26°16′46″, Guiyang City, Guizhou Province) ) of blueberry root endophytic fungi were isolated and cultured, and the DNA of the strain was extracted using the Fungal DNA Midi Kit. The sequences of the strains were obtained using universal primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′), and the DNA was amplified by polymerase chain reaction (PCR). Multiple sequence alignment analysis was performed on the ITS sequence and the sequences of the same genus and different species in the comparison results of Blastn, and the exogenous genus Nectriaberolinensis was introduced to perform multiple sequence alignment using Clustal W in the MEGA 7.0 software, using the neighbor-joining method (Neighbor -Joining) to build a phylogenetic IST tree. It was found that the strains GL41 and Trichodermakoningiopsis belonged to one branch of the phylogenetic tree, and the support degree was 100%, and they were clustered into one group; the endophytic fungi were identified as Trichodermakoningiopsis GL41 Trichodermakoningiopsis based on the characteristics of colony morphology and micromorphology.

The test results of the phosphorus-dissolving ability of blueberry endophytic Trichoderma koningiopsis showed that the strain was phosphorus-dissolving endophytic Trichoderma koningiopsis had a high ability to dissolve inorganic phosphorus. With PKO solid medium as the basic medium, the overall performance of GL41 Trichodermakoningiopsis decreased first and then increased. Then, the available phosphorus content gradually increased from the 1st day to the 5th day, reached the maximum value of 273.90 µg/ml on the 5th day, and dropped to 128.19 µg/ml on the seventh day.

In summary, the phosphorus-solubilizing blueberry endophytic Trichoderma fungus of the present invention has the function of efficiently dissolving phosphorus (the phosphorus is inorganic phosphorus), promotes the growth of blueberry, and has great potential to be developed into a microbial agent, and the bacterium is an endogenous blueberry Trichoderma spp. can be directly cultured and isolated from blueberry root tissue without introducing additional strains.

Description of drawings

Figure 1: Colony morphology and microscopic morphology of GL41 Trichodermakoningiopsis cultured for 6 days (A is the front of the colony and B is the back of the colony, C and D are the morphology of molecular spores and conidiophores);

Figure 2: ITS-based phylogenetic tree of GL41 Trichodermakoningiopsis ;

Figure 3: Phosphate-dissolving effect of GL41 Trichodermakoningiopsis on PKO solid medium;

Figure 4: Available phosphorus content of GL41 Trichodermakoningiopsis on PKO liquid medium;

Figure 5: Effect of GL41 Trichodermakoningiopsis on the growth of blueberry tissue culture seedlings;

Figure 6: Colonization of GL41 Trichodermakoningiopsis in the roots of blueberry tissue culture seedlings (CK is the control group, GL41 is the GL41 colonization group);

Figure 7: Colonization of GL41 Trichodermakoningiopsis in the roots of potted blueberry seedlings (CK is the control group, GL41 is the GL41 treatment group);

Figure 8: The effect of GL41 Trichodermakoningiopsis on the growth of potted blueberry seedlings (A, A1, A2 are the control group, B, B1, B2 are the GL41 treatment group.);

Figure 9: Effect of GL41 Trichodermakoningiopsis on dry weight of blueberry plants (CK is the control group, GL41 is the GL41 treatment group);

Figure 10: Effect of GL41 Trichodermakoningiopsis on the freshness of blueberry plants (CK is the control group, GL41 is the GL41 treatment group);

Figure 11: The effect of GL41 Trichodermakoningiopsis on the height of blueberry plants (CK is the control group, GL41 is the GL41 treatment group);

Figure 12: The effect of GL41 Trichodermakoningiopsis on the ground meridian of blueberry plants (CK is the control group, GL41 is the GL41 treatment group);

Figure 13: The effect of GL41 Trichodermakoningiopsis on the chlorophyll content of blueberry plants (CK is the control group, GL41 is the GL41 treatment group);

Figure 14: Effect of GL41 Trichodermakoningiopsis on soluble sugar in blueberry plants (CK is the control group, GL41 is the GL41 treatment group);

Figure 15: The effect of GL41 Trichodermakoningiopsis on the soluble protein of blueberry plants (CK is the control group, GL41 is the GL41 treatment group);

Figure 16: Effect of GL41 Trichodermakoningiopsis on blueberry plant SOD (CK is the control group, GL41 is the GL41 treatment group);

Figure 17: Effect of GL41 Trichodermakoningiopsis on CAT enzyme activity of blueberry plants (CK is the control group, GL41 is the GL41 treatment group);

Figure 18: Effect of GL41 Trichodermakoningiopsis on total nitrogen in blueberry plants (CK is the control group, GL41 is the GL41 treatment group);

Figure 19: Effect of GL41 Trichodermakoningiopsis on total phosphorus in blueberry plants (CK is the control group, GL41 is the GL41 treatment group);

Figure 20: The effect of GL41 Trichodermakoningiopsis on the total potassium nutrients of blueberry plants (CK is the control group, and GL41 is the GL41 treatment group).

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and embodiments, but not as a basis for limiting the present invention.

Example 1: Tissue isolation of endophytic Trichoderma in phosphorus-solubilizing blueberries

(1) Cleaning: Select blueberry roots with healthy surface and no disease spots, wash them with water and dry them;

(2) Disinfection and sterilization: In the ultra-clean workbench, soak the cleaned and dried blueberry roots in 75% ethanol solution for 3 seconds, rinse them with sterile water for 5 times, and transfer them to 3% (V/V) NaClO solution for disinfection 3 min, rinse 5 times with sterile water, disinfect with 75% ethanol for 30 seconds, rinse 5 times with sterile water and blot dry, and set aside;

(4) Preparation of PDA medium: Dissolve 200 g of potatoes, 20 g of glucose and 20 g of agar in 1000 mL of distilled water, spread them on plates to make PDA medium;

(3) Culture, separation and purification: put product B in PDA medium, culture in a constant temperature incubator at 28°C for 4 days, and use the fungal colony edge picking method to pick fungi with different growth forms in the PDA medium and transfer them to the new In the PDA medium, cultivate and purify in a constant temperature incubator at 28°C, purify 4 times, and obtain endophytic Trichoderma phosphate-solubilizing blueberry.

Example 2: Tissue isolation of endophytic Trichoderma in phosphorus-solubilizing blueberries

(1) Cleaning: Select blueberry roots with healthy surface and no disease spots, wash them with water and dry them;

(2) Disinfection and sterilization: In the ultra-clean workbench, soak the cleaned and dried blueberry roots in 75% ethanol solution for 2 seconds, rinse with sterile water for 4 times, and transfer to 3% (V/V) NaClO solution for disinfection For 4 minutes, rinse with sterile water 4 times, put in 75% ethanol for 20 seconds, then rinse 6 times with sterile water and dry the water, and set aside;

(4) Preparation of PDA medium: Dissolve 150 g of potatoes, 15 g of glucose and 15 g of agar in 1000 mL of distilled water, spread them on plates to make PDA medium;

(3) Cultivation, separation and purification: put product B in PDA medium, culture in a constant temperature incubator at 28°C for 5 days, use the fungal colony edge picking method to pick fungi with different growth forms in PDA medium and transfer them to the new cultured and purified in a constant temperature incubator at 28°C, and purified 5 times to obtain endophytic Trichoderma phosphate-solubilizing blueberry.

Example 3: Tissue isolation of endophytic Trichoderma in phosphorus-solubilizing blueberries

(1) Cleaning: Select blueberry roots with healthy surface and no disease spots, wash them with water and dry them;

(2) Disinfection and sterilization: In the ultra-clean workbench, soak the cleaned and dried blueberry roots in 75% ethanol solution for 3 seconds, rinse with sterile water for 6 times, and transfer to 3% (V/V) NaClO solution for disinfection 3min, rinse 4 times with sterile water, put in 75% ethanol to disinfect for 35s, then rinse 6 times with sterile water and dry the water, set aside;

(4) Preparation of PDA medium: Dissolve 250g of potatoes, 25g of glucose and 25g of agar in 1000mL of distilled water, spread them on a flat plate to make PDA medium;

(3) Culture, separation and purification: put product B in PDA medium, culture in a constant temperature incubator at 28°C for 4 days, and use the fungal colony edge picking method to pick fungi with different growth forms in the PDA medium and transfer them to the new cultured and purified in a constant temperature incubator at 28°C, and purified 5 times to obtain endophytic Trichoderma phosphate-solubilizing blueberry.

Embodiment 4: Tissue separation of endophytic Trichoderma in phosphorus-solubilizing blueberries

(1) Cleaning: Select blueberry roots with healthy surface and no disease spots, wash them with water and dry them;

(2) Disinfection and sterilization: In the ultra-clean workbench, soak the cleaned and dried blueberry roots in 75% ethanol solution for 3 seconds, rinse them with sterile water for 5 times, and transfer them to 3% (V/V) NaClO solution for disinfection 3 min, rinse 5 times with sterile water, disinfect with 75% ethanol for 30 seconds, rinse 5 times with sterile water and blot dry, and set aside;

(4) Preparation of PDA medium: Dissolve 220 g of potatoes, 15 g of glucose and 20 g of agar in 1000 mL of distilled water, spread them on plates to make PDA medium;

(3) Culture, separation and purification: put product B in PDA medium, culture in a constant temperature incubator at 28°C for 4 days, and use the fungal colony edge picking method to pick fungi with different growth forms in the PDA medium and transfer them to the new In the PDA medium, cultivate and purify in a constant temperature incubator at 28°C, purify 4 times, and obtain endophytic Trichoderma phosphate-solubilizing blueberry.

The present invention has done a large amount of analytical verification experiments, and the following is the result of the experimental research of the present invention:

1. Isolation and identification of blueberry endophytic Trichoderma

1.1 Method for isolation and identification of endophytic Trichoderma in blueberries

The endophytic fungus from the root of blueberries collected from Huaxi Gaopo Ecological Blueberry Garden (East Longitude 106°50′14″, North Latitude 26°16′46″) in Guiyang City, Guizhou Province was isolated and cultivated by tissue separation method, and the healthy surface without disease spots was selected The roots of the blueberries were washed with water and dried. In the ultra-clean workbench, the blueberry roots with a healthy surface and no disease spots were immersed in 75% ethanol solution for 3 s, rinsed with sterile water for 5 times, transferred to 3% (V/V) NaClO solution for disinfection for 3 min, and washed with sterile water for 3 min. Rinse with sterile water for 5 times, put in 75% ethanol for 30s, then rinse with sterile water for 5 times and blot the water. In each PDA (200 g potato, 20 g glucose, 20 g agar, 1000 mL distilled water) medium Put 5 blueberry root segments, repeat 20 PDA medium, and culture in a constant temperature incubator at 28 ℃ for 3-5 days; at the same time, use the plant tissue imprinting method to directly wipe the surface-sterilized blueberry root on the surface of the medium, under the same conditions Five PDA media were cultured as a control. Using the edge picking method of fungal colonies, pick fungi with typical differences in growth morphology from the PDA plate medium and transfer them to new PDA medium, cultivate them in a constant temperature incubator at 28 °C, and purify them for 3-5 times. The isolated and purified bacterial strains were inoculated into PDA medium and incubated at a constant temperature of 28°C.

Observe and photograph colony characteristics (colony color, colony shape, and edge features) with a camera. The hyphal morphology and spores of the strains were observed with a microscope to complete the morphological identification. Photograph colony characteristics (colony color, colony shape, and edge features) with a camera. DNA was extracted from the strains using the Fungal DNA Midi Kit. The sequences of the strains were obtained using universal primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′), and the DNA was amplified by polymerase chain reaction (PCR). The PCR amplification products were directly sent to Chongqing Qingke Biological Testing Co., Ltd. for gel cutting, purification and sequencing to complete molecular biological identification.

Isolation and identification results

On the PDA medium, the hyphae are luxuriant, and the cultured 3d colony has covered the entire PDA petri dish. There was no obvious ring pattern in the colony, and it was white at first, and green conidia were produced after 6 days (Fig. 1A, Fig. 1B). The conidia were monospora, the conidiophores were erect, and the conidiophores directly gave rise to phialides or secondary branches (Fig. 1C, Fig. 1D). Multiple sequence alignment analysis was performed on the ITS sequence and the sequences of the same genus and different species in the comparison results of Blastn, and the exogenous genus Nectriaberolinensis was introduced at the same time. Multiple sequence alignment was carried out using Clustal W in the MEGA7.0 software, and the neighbor-joining method (Neighbor -Joining) to build a phylogenetic IST tree. It was found that the strains GL41 and Trichodermakoningiopsis belonged to one branch of the phylogenetic tree, and the support degree was 100%, and they were clustered into one group (Figure 2). The endophytic fungi were identified as Trichoderma koningiopsis GL41 Trichodermakoningiopsis based on the colony morphological characteristics and microscopic morphological characteristics.

2. Testing

The phosphorus-solubilizing blueberry endophytic Trichoderma obtained after separation and identification was tested for detection, and the test process was as follows:

2.1 Determination of Phosphorus Solubilizing Ability of Blueberry Endophytic Trichoderma

Quantitative determination: Use a hole puncher to punch the activated bacterial block into a cake with a diameter of 5mm, and insert PKO inorganic phosphorus medium (Zn ₃ (PO ₄ ) 25.0g, sucrose 10.0g, NaCl 0.5g, KCl 0.2g, (NH ₄ ) 2S0 ₄ 0.1g, MgSO ₄ 7H ₂ O 0.1g, MnSO ₄ 0.004g, yeast extract 0.5g, FeSO ₄ 0.004g, distilled water 1000mL, agar 20.0g, pH natural), to be After it grows stably, observe whether there is a transparent phosphorus-dissolving circle around it.

Qualitative determination: take PKO medium without Agar as liquid medium, add 150 mL to each Erlenmeyer flask, under sterile conditions, take 5 pieces of bacteria cakes with a diameter of 8.0 mm and put them into Erlenmeyer flasks with fermentation broth in a shaker, and cultivated at 28 °C and 150 r/min for 7 days. After cultivation, samples were taken every day to measure the available phosphorus content in the fermentation broth, and the pH value was measured with a pH meter.

(1) Determination method of available phosphorus content: take 5 mL of supernatant, centrifuge at 12000 r/min for 5 min, take 3 ml of supernatant as the test solution, put it in a 50 ml Erlenmeyer flask, add 1 drop to each bottle 2, 4-dinitrophenol indicator, the solution gradually develops color, then add 4 mol/L NaOH solution to make the fermentation liquid of the Erlenmeyer flask turn yellow, then add 1 mol/L sulfuric acid solution to adjust the pH to make the liquid just slightly yellow . Add 5ml of molybdenum antimony anti-color agent, distilled water to 50ml, shake properly to make the liquid uniform, and after standing on a horizontal table for 30 minutes, set the wavelength of UV-visible spectrophotometer to 700 nm for colorimetry, and use uninoculated PKO The liquid fermentation liquid is used as a reference liquid, the absorption value is set to zero, the color values of different fermentation liquids are measured respectively, and the phosphorus concentration is calculated on the phosphorus standard curve.

(2) Drawing of phosphorus standard curve: dilute 100 mg/L phosphorus standard solution to 5 mg/L, take 0, 2, 4, 6, 8, 10 mL of 5 mg/L phosphorus standard solution to 50 mL In the Erlenmeyer flask, add distilled water to 30 mL, then add 2 drops of 2,4-dinitrophenol indicator, and then add 4 mol/L NaOH dropwise until the liquid in the Erlenmeyer flask is slightly yellow, and the concentration is 1 mol/L of H2SO4 until the yellow color fades, add 5mL molybdenum antimony anti-color developer respectively, shake quickly, add distilled water to the position of 50mL marking line, develop color at room temperature for 2 hours, and measure the absorbance value with a spectrophotometer at 700 nm wavelength. Draw a phosphorus standard curve, set the abscissa as the phosphorus concentration, and the ordinate as the absorbance value.

(3) Calculation formula: Available phosphorus content P (µg/mL) = (ρ×V×Ts)/Vo

ρ: the mass concentration of available phosphorus (p) found from the working curve (µg/mL); V: the constant volume of the chromogenic solution (mL), (50 mL here); Ts: fractionation multiple ( Here it is 17 times); Vo: Measure the volume (mL) of the fermentation broth (here it is 3mL).

Test results:

PKO solid medium was used as the basic medium, and the transparent circle was observed on the 4th day after inoculation (Figure 3). It can be seen from Figure 4 that the overall trend of GL41 Trichodermakoningiopsis first decreased and then increased, and the effective rate from the 1d to the 5th day Phosphorus content gradually increased, reached the maximum value of 273.90 µg/ml on the 5th day, and dropped to 128.19 µg/ml on the seventh day.

Backgraining Test of Endophytic Trichoderma in Blueberry on Tissue Culture Seedlings of Blueberry

The endophytic fungi were inoculated on blueberry aseptic seedlings, cultivated for one month, and the colonization of the strains was observed under a microscope. The method is as follows: put 1/3 moss substrate into the tissue culture bottle for sterilization, wash the root agar of the rooted blueberry tissue culture seedlings, transplant them into the above tissue culture bottles, and plant 2 plants in each bottle. Add 3ml of MEA culture solution with bacterial strain block in above-mentioned tissue culture bottle that sterilized seedling is housed after slowing down seedling one week. After 30 days, the colonization of the strains was observed.

Observation of the colonization rate: Wash the sterile seedling roots with distilled water, move them to a plate containing lactic acid for neutralization for 10 minutes, then transfer them to a plate containing 0.5% acid fuchsin for 30 seconds, and then transfer them to a lactic acid glycerin liquid (lactic acid: Glycerin: distilled water = 1:1:1) for decolorization, and finally put it on a glass slide to make slices, and observe the infection status of Trichoderma under a microscope.

test results:

GL41 colonized in the root tissue of blueberry tissue culture seedlings, and there was no obvious abnormality in the root tissue cells compared with the control treatment (Figure 5). GL41 Trichodermakoningiopsis hyphae were observed in tissue cultured plantlets inoculated with GL41 (Figure 6). It indicated that GL41 could successfully colonize blueberry roots.

vaccination test

Under sterile conditions, use an inoculation loop to take 3 activated endophyte cakes (diameter = 5 mm), inoculate them into a 250 mL Erlenmeyer flask containing 100 mL of PDB medium, and autoclave at 121°C for 30 minutes. And prepare the bacterial culture solution at 150 rpm for 7 days. Then, measure the number of spores in each bacterial culture solution with a hemocytometer, and adjust the spore concentration to 1×10 ⁵ CFU/mL for later use.

The inoculation test adopts the pot test, a total of 2 treatments, namely the control group (CK), were inoculated with GL41 spore suspension, 1 plant per pot, a total of 18 experimental pots.

Pot experiments were carried out in a greenhouse. Select healthy, disease-free, and consistent-growing one-year-old blueberry seedlings, pot them with sterilized humus soil, and inoculate them with root wound irrigation: insert a sterilized scalpel into the soil near the plants to cause root wounds, and use 100% water for each blueberry plant. 100 mL of spore suspension was instilled, and the seedlings inoculated with the same amount of sterile distilled water were used as the control group. No fertilization was applied during the growth period, and water was poured every three days, and harvested after 90 days of unified cultivation and management.

Growth medium: organic humus, crushed pine needles, leaves, high-quality sawdust, and imported coconut bran mixed fermentation humus, purchased from Lvyuan Meijia Agricultural Technology Co., Ltd., autoclaved at 121°C for 2 hours before use. Removes microorganisms from the substrate.

Effect of Endophytic Trichoderma in Blueberry on Promoting Blueberry Growth

The observation of colonization is the same as in 3; the root structure is scanned with a desktop scanner; the biomass is weighed (dry weight and fresh weight): the drying time is 48 hours in an oven at 45°C; each pot is measured with a ruler and a vernier caliper Plant height and ground diameter; chlorophyll, soluble protein, soluble sugar, SOD enzyme, CAT enzyme, total nitrogen, total phosphorus and total potassium content were determined by acetone extraction method, Coomassie brilliant blue G-250, visible spectrophotometry, visible spectrophotometry Photometric method, ultraviolet spectrophotometric method, alkali solution diffusion method, molybdenum antimony anti-colorimetric method, and flame method.

test results:

The result of colonization of potted seedlings was the same as that of tissue cultured seedlings (Figure 7). Compared with the control, inoculation of GL41 had a significant effect on promoting the growth of blueberry potted seedlings (Figure 8-20).

Sequencing results

Trichoderma koningiopsis GL41 ( Trichodermakoningiopsis ), GenBank accession number: MW750424

TGTGACCATACCAAACTGTTGCCTCGGCGGGGTCACGCCCCGGGTGCGTCGCAGCCCCGGAACCAGGCGC

CCGCCGGAGGGACCAACCAAACTCTTTCTGTAGTCCCTCGCGGACGTTATTTTCTTACAGCTCTGAGCAA

AAATTCAAAATGAATCAAAACTTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAA

ATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCA

GTATTCTGGCGGGCATGCCTGTCCGAGCGTCATTTCAACCCTCGAACCCCTCCGGGGGGTCGGCGTTGGG

GATCGGGAACCCCTAAGACGGGATCCCGGCCCCGAAATACAGTGGCGGTCTCGCCGCAGCCTCTCCTGCG

CAGTAGTTTGCACAACTCGCACCGGGAGCGCGGCGCGTCCACGTCCGTAAAACACCCAACTTCTGAAATG

TTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAT.

Claims (10)

1. A phosphorus-solubilizing blueberry endophytic trichoderma is characterized in that: the phosphorus-dissolving blueberry endophytic trichoderma harzianum is named as trichoderma pseudokoningii GL41Trichoderma koningiopsisAnd is preserved in China general microbiological culture collection management center at 7 month and 11 month in 2022, with the preservation number of CGMCC No.40241.

2. The phosphate-solubilizing blueberry endophytic trichoderma as claimed in claim 1, wherein: the phosphorus-dissolving blueberry endophytic trichoderma is obtained from the roots of blueberries by a tissue separation method.

3. The phosphate-solubilizing blueberry endophytic trichoderma as claimed in claim 1, wherein: the tissue separation method of the phosphate-solubilizing blueberry endophytic trichoderma comprises the following steps:

(1) Cleaning: selecting healthy and non-scab blueberry roots on the surface, cleaning the blueberry roots with clear water, and airing to obtain a product A;

(2) And (3) disinfection and sterilization: soaking product A in ethanol solution in a clean bench, washing with sterile water, sterilizing in NaClO solution, washing with sterile water, and sucking to remove water to obtain product B;

(3) Preparation of PDA culture medium: peeling potato, cutting into small pieces, decocting in water, filtering with gauze, adding glucose and agar, dissolving in distilled water, and spreading on a flat plate to obtain PDA culture medium;

(4) Culturing, separating and purifying: and placing the product B in a PDA culture medium, culturing in a constant-temperature incubator, picking out fungi with typical growth form difference in the PDA culture medium by using a fungus colony edge picking method, transferring to a new PDA culture medium, and culturing and purifying in the constant-temperature incubator to obtain the phosphorus-dissolving blueberry endophytic trichoderma.

4. The phosphorus-solubilizing blueberry endophytic trichoderma as claimed in claim 3, wherein: the tissue separation method of the phosphate-solubilizing blueberry endophytic trichoderma comprises the following steps:

(1) Cleaning: selecting healthy and non-scab blueberry roots on the surface, cleaning the blueberry roots with clear water, and airing to obtain a product A;

(2) And (3) disinfection and sterilization: soaking product A in 75% ethanol solution for 2-4s in a clean bench, washing with sterile water for 4-6 times, sterilizing in 3% NaClO solution for 2-4min, washing with sterile water for 4-6 times, and sucking to remove water to obtain product B;

(3) Preparation of PDA culture medium: cutting 150-250g of peeled potatoes into small pieces, adding water, boiling, filtering with gauze, adding 15-25g of glucose and 15-25g of agar, dissolving in 1000mL of distilled water, and spreading on a flat plate to obtain a PDA culture medium;

(4) Culturing, separating and purifying: and (3) putting the product B in a PDA culture medium, culturing for 3-5 days in a constant-temperature incubator at 28 ℃, picking out fungi with typical growth form difference in the PDA culture medium by using a fungi colony edge picking method, transferring the fungi to a new PDA culture medium, culturing and purifying in the constant-temperature incubator at 28 ℃, and purifying for 3-5 times to obtain the phosphorus-solubilizing blueberry endophytic trichoderma harzianum.

5. The phosphorus-solubilizing blueberry endophytic trichoderma as claimed in claim 3 or 4, wherein: the tissue separation method of the phosphate-solubilizing blueberry endophytic trichoderma comprises the following steps:

(1) Cleaning: selecting healthy and non-scab blueberry roots on the surface, cleaning the blueberry roots with clear water, and airing to obtain a product A;

(2) And (3) disinfection and sterilization: soaking product A in 75% ethanol solution for 3s in a clean bench, washing with sterile water for 5 times, sterilizing in 3% NaClO solution for 3min, washing with sterile water for 5 times, and sucking to remove water to obtain product B;

(3) Preparation of PDA culture medium: : cutting 200g of peeled potatoes into small pieces, adding water, boiling, filtering with gauze, adding 20g of glucose and 20g of agar, dissolving in 1000mL of distilled water, and flatly spreading on a flat plate to obtain a PDA culture medium;

(4) Culturing, separating and purifying: and (3) putting the product B in a PDA culture medium, culturing for 3-5 days in a constant-temperature culture box at 28 ℃, selecting the typical fungi with different growth forms in the PDA culture medium by using a fungus colony edge picking method, transferring the typical fungi to a new PDA culture medium, culturing and purifying in the constant-temperature culture box at 28 ℃, and purifying for 3-5 times to obtain the phosphorus-solubilizing blueberry endophytic trichoderma harzianum.

6. The application of the phosphorus-solubilizing blueberry endophytic trichoderma as claimed in claim 1 or 2, is characterized in that: the application of the phosphorus-solubilizing blueberry endophytic trichoderma in preparation of a preparation for dissolving phosphorus in a culture medium.

7. The use of the phosphate-solubilizing endophytic trichoderma serrulatum of blueberries as claimed in claim 1 or claim 2, wherein: the application of the phosphorus-dissolving blueberry endophytic trichoderma in preparing the preparation for promoting the growth of blueberry rhizosphere is disclosed.

8. A blueberry growth-promoting bacterium product is characterized in that: the active ingredient of the product comprises the phosphorus-solubilizing blueberry endophytic trichoderma harzianum of claim 1 or 2.

9. A blueberry growth-promoting bacterium product is characterized in that: the active ingredient of the product is the phosphate-solubilizing blueberry endophytic trichoderma koningii as described in claim 1 or 2.

10. A preparation method of a blueberry growth-promoting bacterium product is characterized by comprising the following steps: the phosphorus-solubilizing blueberry endophytic trichoderma harzianum as claimed in claim 1 or 2 is used as an active ingredient or one of the active ingredients for preparing the product.

Images