CN115725419B - A phosphorus-solubilizing blueberry endophytic Trichoderma strain and its application - Google Patents

Abstract

The invention discloses a strain of endophytic Trichoderma that solubilizes blueberries and its application. The described endophytic Trichoderma that solubilizes blueberries is named Trichoderma koningiopsis GL41 and was deposited in China General Microbiology on July 11, 2022. Species Collection Management Center, the collection number is CGMCC No.40241. The phosphorus-solubilizing blueberry endophytic Trichoderma of the present invention has the function of dissolving inorganic phosphorus. Using PKO solid culture medium as the basic culture medium, the available phosphorus content of GL41 Trichoderma koningiopsis gradually increases, reaching a maximum value of 273.90µg/ml on the 5th day, effectively promoting blueberry growth. growth, it has great potential to be developed into microbial inoculants.

Description

A phosphorus-solubilizing blueberry endophytic Trichoderma strain and its application

Technical field

The invention belongs to the technical field of agricultural microorganisms and relates to a phosphate-solubilizing blueberry endophytic Trichoderma strain and its application.

Background technique

Plant rhizosphere growth-promoting bacteria are a type of beneficial bacteria that grow in the rhizosphere soil of plants. They have a variety of growth-promoting mechanisms for plants, such as biological nitrogen fixation, solubilizing 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 insoluble potassium in the soil to promote plant growth and development. They can also enhance plant resistance to diseases, heavy metals and salts by secreting siderophores and ACC deaminase. Resistance to alkali and other adversities. Isolating and screening microbial strains with growth-promoting effects from the environment and developing microbial fertilizers for application are of great significance to reducing environmental pollution and developing the planting industry.

Blueberry ( Vacciniumuliginosum ) is the common name for blue-fruited plants of the genus Vaccinium in the Ericaceae family. Wild blueberries are widely distributed around the world. Mainly produced in the United States, it is mainly distributed in the forest areas of the Greater Khingan Mountains and Lesser Khingan Mountains in China. As an emerging fruit with high economic value and health care functions, blueberry’s cultivation area has been greatly developed. However, most current growers generally lack experience in facility cultivation of blueberries. When blueberries grow poorly or abnormally, it is often due to the lack of sufficient nitrogen, phosphorus, potassium and other large elements. However, the application of large amounts of chemical fertilizers can lead to soil and water pollution and ecological pollution. Environmental degradation.

Trichoderma belongs to the phylum Ascomycota, class Faecalia, order Hypocartes, family Hypomycota, genus Trichoderma, and 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 species can produce a variety of bioactive substances that are antagonistic to 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. yield, so it is widely used in biological control, biofertilizers and soil conditioners.

Currently, the patented strains that have a growth-promoting effect on blueberries are reported to be DSE fungus (CN107083335A) and Bacillus (CN114134070A). However, there are few reports on endophytic Trichoderma in phosphate-solubilizing blueberries, and there is no report on the growth-promoting effect of endophytic Trichoderma in phosphate-solubilizing blueberries.

Contents of the invention

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

The technical solution of the present invention: a strain of endophytic Trichoderma that solubilizes blueberries. The described endophytic Trichoderma that solubilizes blueberries is named Trichodermakoningiopsis GL41 Trichodermakoningiopsis and was deposited in the China General Microbial Culture Collection on July 11, 2022. Management Center, the preservation number is CGMCC No.40241.

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

The aforementioned endophytic Trichoderma in phosphate-solubilizing blueberries, the tissue separation method of the endophytic Trichoderma in phosphorus-solubilizing blueberries is:

(1) Cleaning: Select blueberry roots with healthy surface and no spots, wash them with clean 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 absorb the moisture to obtain product B;

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

(4) Culture, isolation and purification: Place product B in PDA culture medium and place it in a constant temperature incubator for culture. Use the fungal colony edge picking method to select fungi with typical growth morphology differences in the PDA culture medium and transfer them to new PDA cultures. Based on culture and purification in a constant-temperature incubator, endophytic Trichoderma in phosphorus-solubilizing blueberries was obtained.

Specifically, the aforementioned tissue isolation method of endophytic Trichoderma in phosphate-solubilizing blueberries is:

(1) Cleaning: Select blueberry roots with healthy surface and no spots, wash them with clean 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 2-4 seconds, rinse with sterile water 4-6 times, and transfer to a NaClO solution with a volume ratio of 3% for 2-4 seconds. 4min, then rinse with sterile water 4-6 times and absorb the water to obtain product B;

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

(4) Culture, isolation and purification: Place product B in PDA culture medium and place it in a constant temperature incubator at 28°C for 3-5 days. Use the fungal colony edge picking method to select fungi with typical growth morphological differences in the PDA culture medium. The new PDA was cultured and purified in a constant temperature incubator at 28°C, and purified 3-5 times to obtain endophytic Trichoderma in blueberries.

More specifically, the aforementioned tissue isolation method of endophytic Trichoderma in phosphate-solubilizing blueberries is:

(1) Cleaning: Select blueberry roots with healthy surface and no spots, wash them with clean 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 5 times, transfer to NaClO solution with a volume ratio of 3% for 3 minutes, and then disinfect with sterile water. Rinse the bacteria with water 5 times and absorb the water to obtain product B;

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

(4) Culture, isolation and purification: Place product B in PDA culture medium and place it in a constant temperature incubator at 28°C for 3-5 days. Use the fungal colony edge picking method to select fungi with typical growth morphological differences in the PDA culture medium. into a new PDA medium, culture and purify in a constant temperature incubator at 28°C, and purify 3-5 times to obtain endophytic Trichoderma in phosphate-solubilizing blueberries.

The application of the aforementioned phosphorus-solubilizing endophytic Trichoderma in blueberries is used in preparing preparations for dissolving phosphorus in the culture medium.

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

A blueberry growth-promoting bacteria product, the active ingredient of which includes the phosphorus-solubilizing blueberry endophytic Trichoderma as claimed in claim 1 or 2.

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

A method for preparing a blueberry growth-promoting bacteria product, using the phosphorus-solubilizing blueberry endophytic Trichoderma described in claim 1 or 2 as an active ingredient or one of the active ingredients in preparing the product.

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

The invention discloses a phosphorus-solubilizing blueberry endophytic Trichoderma strain and its application. The tissue separation method is used to collect the plant from the high-slope ecological blueberry garden of Huaxi, Guiyang City, Guizhou Province (106°50′14″ east longitude, 26°16′46″ north latitude). ) of blueberry root endophytic fungi were isolated and cultured, and the DNA of the strain was extracted using the Fungal DNA Midi Kit. The sequence of the strain was 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 conducted between the ITS sequence and sequences belonging to different species in the Blastn alignment results. At the same time, the exogenous genus Nectriaberolinensis was introduced and Clustal W in MEGA7.0 software was used for multiple sequence alignment. The neighbor-joining method (Neighbor) in MEGA 7.0 was used. -Joining) to construct a phylogenetic IST developmental tree. It was found that strains GL41 and Trichodermakoningiopsis were located on one branch of the phylogenetic tree, with a support level of 100% and clustered into one category. The endophytic fungi were identified as Trichoderma pseudokoningiopsis GL41 Trichodermakoningiopsis based on the colony morphological characteristics and microscopic morphological characteristics.

The results of the determination of the phosphorus-solubilizing ability of blueberry endophytic Trichoderma show that this strain is a phosphorus-solubilizing blueberry endophytic Trichoderma and has a high ability to dissolve inorganic phosphorus. Using PKO solid medium as the basic medium, the overall performance of GL41 Trichodermakoningiopsis first decreased and then increased. On the downward trend, the available phosphorus content gradually increased from the 1st to the 5th day, reaching the maximum value of 273.90 µg/ml on the 5th day, and dropped to 128.19µg/ml on the seventh day.

To sum up, the phosphorus-solubilizing blueberry endophytic Trichoderma of the present invention has the function of efficiently dissolving phosphorus (the phosphorus is inorganic phosphorus), promotes the growth of blueberries, and has great potential to be developed into a microbial agent, and the bacterium is an endophytic agent in blueberries. Trichoderma can be directly isolated from blueberry root tissue culture without introducing additional strains.

Description of drawings

Figure 1: Colony morphology and microscopic morphology of GL41 Trichodermakoningiopsis after 6 days of culture (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: Evolutionary tree of GL41 Trichodermakoningiopsis based on ITS;

Figure 3: Phosphorus solubilizing 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 blueberry potted seedlings (CK is the control group, GL41 is the GL41 treatment group);

Figure 8: Effect of GL41 Trichodermakoningiopsis on the growth of blueberry potted seedlings (A, A1, and A2 are the control group, and B, B1, and 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: Effect of GL41 Trichodermakoningiopsis on blueberry plant height (CK is the control group, GL41 is the GL41 treatment group);

Figure 12: Effect of GL41 Trichodermakoningiopsis on blueberry plant meridians (CK is the control group, GL41 is the GL41 treatment group);

Figure 13: Effect of GL41 Trichodermakoningiopsis on 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: Effect of GL41 Trichodermakoningiopsis on soluble protein in blueberry plants (CK is the control group, GL41 is the GL41 treatment group);

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

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

Figure 20: Effect of GL41 Trichodermakoningiopsis on total potassium nutrient content of blueberry plants (CK is the control group, GL41 is the GL41 treatment group).

Detailed ways

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

Example 1: Tissue isolation of endophytic Trichoderma in blueberries

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

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

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

(3) Culture, isolation and purification: Place product B in PDA culture medium and place it in a constant temperature incubator at 28°C for 4 days. Use the fungal colony edge picking method to select fungi with typical growth morphological differences in the PDA culture medium and transfer them to new cells. In the PDA culture medium, it was cultured and purified in a constant temperature incubator at 28°C, and purified four times to obtain endophytic Trichoderma in phosphate-solubilizing blueberries.

Example 2: Tissue isolation of endophytic Trichoderma in blueberries

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

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

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

(3) Culture, isolation and purification: Place product B in PDA culture medium and place it in a constant temperature incubator at 28°C for 5 days. Use the fungal colony edge picking method to select fungi with typical growth morphology differences in the PDA culture medium and transfer them to new cells. In the PDA culture medium, it was cultured and purified in a constant temperature incubator at 28°C, and purified five times to obtain endophytic Trichoderma in phosphorus-solubilizing blueberries.

Example 3: Tissue isolation of endophytic Trichoderma in blueberries

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

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

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

(3) Culture, isolation and purification: Place product B in PDA culture medium and place it in a constant temperature incubator at 28°C for 4 days. Use the fungal colony edge picking method to select fungi with typical growth morphological differences in the PDA culture medium and transfer them to new cells. In the PDA culture medium, it was cultured and purified in a constant temperature incubator at 28°C, and purified five times to obtain endophytic Trichoderma in phosphorus-solubilizing blueberries.

Example 4: Tissue isolation of endophytic Trichoderma in blueberries

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

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

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

(3) Culture, isolation and purification: Place product B in PDA culture medium and place it in a constant temperature incubator at 28°C for 4 days. Use the fungal colony edge picking method to select fungi with typical growth morphological differences in the PDA culture medium and transfer them to new cells. In the PDA culture medium, it was cultured and purified in a constant temperature incubator at 28°C, and purified four times to obtain endophytic Trichoderma in phosphate-solubilizing blueberries.

The present invention has done a large number of analysis and verification experiments. The following are the results of the experimental research of the present invention:

1. Isolation and identification of endophytic Trichoderma in blueberries

1.1 Methods for isolation and identification of endophytic Trichoderma in blueberries

The tissue separation method was used to isolate and culture the endophytic fungi in the roots of blueberries collected from the ecological blueberry garden on the high slope of Huaxi, Guiyang City, Guizhou Province (106°50′14″ east longitude, 26°16′46″ north latitude), and selected healthy and disease-free surfaces. Wash the blueberry roots with clean water and dry them. In the ultra-clean workbench, soak the blueberry roots with healthy and disease-free surfaces in 75% ethanol solution for 3 seconds, rinse with sterile water 5 times, transfer to 3% (V/V) NaClO solution for 3 minutes, and disinfect with Rinse 5 times with sterile water, put in 75% ethanol to disinfect for 30 seconds, then rinse 5 times with sterile water and absorb the water, in each PDA (200 g potato, 20 g glucose, 20 g agar, 1000 mL distilled water) culture medium Place 5 blueberry root segments, repeat 20 times of PDA culture medium, and place it in a constant temperature incubator at 28°C for 3-5 days. At the same time, use the plant tissue imprinting method to lightly wipe the surface-sterilized blueberry roots directly on the surface of the culture medium. Under the same conditions, Culture 5 PDA media as controls. Using the fungal colony edge picking method, fungi with typical growth morphological differences in the PDA plate culture medium were picked and transferred to new PDA culture medium, cultured in a constant temperature incubator at 28°C, and purified 3-5 times. The strains obtained were isolated and purified, inoculated into PDA culture medium, and cultured at a constant temperature of 28°C.

Use a camera to observe and photograph colony characteristics (colony color, colony shape, and edge characteristics). Use a microscope to observe the hyphal morphology and spores of the strain to complete morphological identification. Use a camera to capture colony characteristics (colony color, colony shape, and edge features). The DNA of the strains was extracted using the Fungal DNA Midi Kit. The sequence of the strain was 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 Company for gel cutting, purification and sequencing to complete molecular biology identification.

Isolation and identification results

On the PDA culture medium, the hyphae were luxuriant, and the colonies had grown to cover the entire PDA culture dish after 3 days of cultivation. The colonies had no obvious ring patterns, were white at first, and produced green conidia 6 days later (Figure 1A, Figure 1B). Conidiospores are single and the conidiophores are erect, and the conidiophores directly produce bottle stalks or produce secondary branches (Figure 1C, Figure 1D). Multiple sequence alignment analysis was conducted between the ITS sequence and sequences belonging to different species in the Blastn alignment results. At the same time, the exogenous genus Nectriaberolinensis was introduced and Clustal W in MEGA7.0 software was used for multiple sequence alignment. The neighbor-joining method (Neighbor) in MEGA 7.0 was used. -Joining) to construct a phylogenetic IST developmental tree. It was found that strains GL41 and Trichodermakoningiopsis are located on one branch of the phylogenetic tree, with a support level of 100% and clustered into one category (Figure 2). The endophytic fungi were identified as Trichoderma pseudokoningiopsis GL41 Trichodermakoningiopsis based on the colony morphological characteristics and microscopic morphological characteristics.

2. Detection test

After isolation and identification of the endophytic Trichoderma in phosphorus-solubilizing blueberries, a detection test was carried out. The test process is as follows:

2.1 Determination of phosphate-solubilizing ability of endophytic Trichoderma in blueberries

Quantitative determination: Use a hole punch to punch the activated bacterial mass into bacterial cakes with a diameter of 5 mm, and add PKO inorganic phosphorus culture medium (Zn ₃ (PO ₄ ) 25.0g, sucrose 10.0g, NaCl 0.5g) on a clean bench. KCl 0.2g, (NH ₄ ) 2S0 ₄ 0.1g, MgSO ₄ ·7H ₂ O0.1g, MnSO ₄ 0.004g, yeast extract 0.5g, FeSO ₄ 0.004g, distilled water 1000mL, agar 20.0g, pH natural), wait After it grows stably, observe whether a transparent phosphorus-dissolved ring appears on its periphery.

Qualitative determination: Use PKO culture medium without Agar as the liquid culture medium. Add 150 mL to each Erlenmeyer flask. Under sterile conditions, take 5 pieces of bacterial cakes with a diameter of 8.0 mm and put them into Erlenmeyer flasks with fermentation liquid. medium, moved to a shaker, and cultured for 7 d at 28°C and 150 r/min. After culture, 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 and place 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 each time to make the Erlenmeyer flask fermentation liquid turn yellow, then add 1 mol/L sulfuric acid solution to adjust the pH to make the liquid just slightly yellow. . Add 5 ml of molybdenum antimony anti-chromogenic agent, and adjust the volume to 50 ml with distilled water. Shake appropriately to make the liquid uniform. After letting it stand on a horizontal table for 30 minutes, set the UV-visible spectrophotometer to a wavelength of 700 nm for color comparison. Use uninoculated PKO The liquid fermentation broth is used as the reference fluid, and the absorption value is set to zero. The color values of different fermentation broths are measured respectively, and the phosphorus concentration is calculated on the phosphorus standard curve.

(2) Drawing of phosphorus standard curve: dilute the 100 mg/L phosphorus standard solution to 5 mg/L, take 0, 2, 4, 6, 8, 10 mL of the 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 drop by drop until the liquid in the Erlenmeyer flask turns slightly yellow. The added concentration is 1 mol/L H2SO4 until the yellow color fades, add 5mL of molybdenum and antimony anti-color reagent respectively, shake quickly, add distilled water to the 50mL mark position, 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, with 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), (here it is 50 mL); Ts: the fractionation multiple ( Here it is 17 times); Vo: Determine the volume of fermentation broth (mL) (here it is 3mL).

Test results:

Using PKO solid medium as the basic medium, observe the transparent circle on the 4th day after inoculation (Figure 3). From Figure 4, it can be seen that GL41 Trichodermakoningiopsis generally shows a trend of first decreasing, then increasing and then decreasing, and the effective results from the 1st to the 5th day are The phosphorus content gradually increased, reaching the maximum value of 273.90 µg/ml on the 5th day, and dropped to 128.19µg/ml on the seventh day.

Back grafting test of blueberry endophytic Trichoderma on blueberry tissue culture seedlings

Endophytic fungi were inoculated onto sterile blueberry seedlings, cultured for one month, and the colonization of the strains was observed under a microscope. The method is as follows: fill 1/3 of the moss substrate into the tissue culture bottle for sterilization, wash the root agar of the rooted blueberry seedlings, transplant them into the above tissue culture bottle, and plant 2 plants in each bottle. After one week of slowing down the seedlings, add 3 ml of MEA culture medium with strain blocks into the above-mentioned tissue culture bottle containing sterile seedlings. The colonization status of the strains was observed after 30 days.

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

test results:

GL41 colonized in the root tissue of blueberry tissue culture seedlings. Compared with the control treatment, there were no obvious abnormalities in the cells of the root tissue (Figure 5). GL41 Trichodermakoningiopsis hyphae were observed in tissue culture seedlings inoculated with GL41 (Fig. 6). It shows that GL41 can successfully colonize blueberry roots.

vaccination test

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

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

Pot experiments were conducted in a greenhouse. Select healthy, disease-free, uniformly growing one-year-old blueberry seedlings, pot them with sterilized humus soil, and use the root wound irrigation method to inoculate: insert a sterilized scalpel into the soil near the plant to cause a root wound, and use 100 mL of spore suspension was infused, and seedlings inoculated with an equal amount of sterile distilled water were used as a control group. No fertilizer is applied during the growth period, watering is done every three days, and the crops are harvested after 90 days of unified cultivation and management.

Growth medium: organic humus, crushed pine needles, leaves, high-quality sawdust, imported coconut bran mixed fermented humus soil, purchased from Luyuan Meijia Agricultural Technology Co., Ltd., autoclaved at 121°C for 2 hours before use. Remove microorganisms from substrates.

Effects of Endogenous Trichoderma in Blueberry on Promoting Blueberry Growth

The observation of colonization is the same as 3; the root system 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. The plant height and ground diameter; the contents of chlorophyll, soluble protein, soluble sugar, SOD enzyme, CAT enzyme, total nitrogen, total phosphorus and total potassium were determined by acetone extraction method, Coomassie brilliant blue G-250, visible spectrophotometry, and visible spectrophotometry. Photometry, ultraviolet spectrophotometry, alkaline hydrolysis diffusion method and molybdenum antimony colorimetric method, flame method determination.

test results:

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

Sequencing results

Trichodermakoningiopsis GL41 ( Trichodermakoningiopsis ), GenBank accession number: MW750424

TGTGACCATACCAAACTGTTGCCTCGGCGGGGTCACGCCCCGGGTGCGTCGCAGCCCCGGAACCAGGCGC

CCGCCGGAGGGACCAACCAAACTCTTTCTGTAGTCCCCTCGCGGACGTTATTTCTTACAGCTCTGAGCAA

AAATTCAAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAA

ATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCA

GTATTCTGGCGGGCATGCCTGTCCGAGCGTCATTTCAACCCTCGAACCCCTCCGGGGGGTCGGCGTTGGG

GATCGGGAACCCCTAAGACGGGATCCCGGCCCCGAAATACAGTGGCGGTCTCGCCGCAGCCTCTCCTGCG

CAGTAGTTTCACAACTCGCACCGGGAGCGCGGCGCGTCCACGTCCGTAAAACACCCAACTTCTGAAATG

TTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAT.

Claims (6)

1. A strain of endophytic Trichoderma that solubilizes blueberries, characterized in that: the described endophytic Trichoderma that solubilizes blueberries is Trichoderma koningiopsis GL41, which was deposited in China General Microbiology on July 11, 2022 Species Collection Management Center, the collection number is CGMCC No.40241. 2. Application of the phosphorus-solubilizing blueberry endophytic Trichoderma as claimed in claim 1, characterized in that: the phosphorus-solubilizing blueberry endophytic Trichoderma is used in the preparation of preparations for dissolving inorganic phosphorus in the culture medium. 3. Application of the phosphorus-solubilizing blueberry endophytic Trichoderma as claimed in claim 1, characterized in that: the phosphorus-solubilizing blueberry endophytic Trichoderma is used in the preparation of preparations for promoting blueberry rhizosphere growth. 4. A blueberry growth-promoting bacterial product, characterized in that: the active ingredient of the product includes the phosphate-solubilizing blueberry endophytic Trichoderma as claimed in claim 1. 5. A blueberry growth-promoting bacteria product, characterized in that: the active ingredient of the product is the phosphate-solubilizing blueberry endophytic Trichoderma as claimed in claim 1. 6. A method for preparing a blueberry growth-promoting bacteria product, characterized in that: the phosphorus-solubilizing blueberry endophytic Trichoderma as claimed in claim 1 is used as an active ingredient or one of the active ingredients in preparing the product.