CN115725419B - Phosphorus-dissolving blueberry endophytic trichoderma and application thereof - Google Patents
Phosphorus-dissolving blueberry endophytic trichoderma and application thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
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Abstract
The invention discloses a phosphorus-dissolving blueberry endophytic trichoderma and application thereof, wherein the phosphorus-dissolving blueberry endophytic trichoderma is named as trichoderma pseudokoningii GL41Trichoderma koningiopsisThe strain is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC No.40241 at the time of 7.11 of 2022. The phosphorus-dissolving blueberry endophytic trichoderma has the function of dissolving inorganic phosphorus, and takes PKO solid culture medium as basic culture medium, GL41Trichoderma koningiopsisThe effective phosphorus content of the blueberry powder is gradually increased, the 5 th day reaches the maximum value 273.90 mug/ml, the growth of the blueberry is effectively promoted, and the blueberry powder has great potential for developing into microbial agents.
Description
Technical Field
The invention belongs to the technical field of agricultural microorganisms, and relates to a phosphorus-dissolving blueberry endophytic trichoderma and application thereof.
Background
The plant rhizosphere growth promoting bacteria are beneficial bacteria growing in plant rhizosphere soil, and have various growth promoting mechanisms for plants, such as biological nitrogen fixation, phosphorus dissolution and potassium dissolution, plant hormone production such as IAA, antagonism of pathogenic bacteria, induction of systemic resistance and the like. In particular, organic acid generated by the bacteria in the metabolic process can dissolve insoluble phosphorus and insoluble potassium in soil to promote the growth and development of plants, and can also promote the resistance of plants to diseases, heavy metals, saline-alkali and other adverse conditions by secreting siderophores and ACC deaminase. The microbial strain with growth promoting effect is separated and screened from the environment, and the microbial fertilizer is developed and applied, so that the microbial fertilizer has important significance in reducing environmental pollution and developing planting industry.
Blueberry @Vacciniumuliginosum) Is of the genus Vaccinium of the family Ericaceae (Ericaceae)Vaccinium) The common name of the blue fruit type plants. Wild blueberries are widely distributed throughout the world. Mainly produced in the united states, and is mainly distributed in the areas of great and small axies in china. The blueberry is used as an emerging fruit with higher economic value and health care function, and the cultivation area is greatly developed. However, most growers currently lack facility cultivation blueberry planting experience, and when blueberries grow poorly or abnormally, the blueberries often lack abundant nitrogen, phosphorus, potassium and other macroelements, but the application of a large amount of chemical fertilizers can cause soil and water source pollution and ecological environment deterioration.
Trichoderma ascomycetes, hypsizygus, hypocreaceae, trichoderma are widely present in soil under different environmental conditions. From the middle of the 19 th century, humans had preliminary knowledge of trichoderma, but the classification status of trichoderma was not established until the 60 th century. Most trichoderma can produce various bioactive substances with antagonism to plant pathogenic fungi, bacteria and insects, such as cell wall degrading enzymes and secondary metabolites, and can improve stress resistance of crops, promote plant growth and increase yield of agricultural products, so that the trichoderma is widely used for biological control, biofertilizer and soil conditioner.
The current patent strains with growth promoting effect on blueberries are reported as DSE fungi (CN 107083335A) and bacillus (CN 114134070 a). The report of the phosphorus-dissolving blueberry endophytic trichoderma is less, and the report of the phosphorus-dissolving blueberry endophytic trichoderma on blueberry growth promotion effect is not reported.
Disclosure of Invention
The invention aims to provide a phosphorus-dissolving blueberry endophytic trichoderma and application thereof. The phosphorus-dissolving blueberry endophytic trichoderma is named trichoderma pseudokoningii GL41Trichodermakoningiopsis) The phosphorus-dissolving blueberry endophytic trichoderma has the function of dissolving phosphorus in a culture medium and the effect of promoting the growth of blueberry rhizosphere.
The technical scheme of the invention is as follows: the phosphorus-dissolving blueberry endophytic trichoderma is named as trichoderma pseudokoningii GL41TrichodermakoningiopsisThe strain is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC No.40241 at the time of 7.11 of 2022.
The phosphorus-dissolving blueberry endophytic trichoderma is obtained from the root of the blueberry by a tissue separation method.
The tissue separation method of the phosphorus-dissolving blueberry endophytic trichoderma comprises the following steps of:
(1) Cleaning: selecting blueberry roots with healthy surfaces and no disease spots, cleaning the blueberry roots with clear water, and airing the blueberry roots to obtain a product A;
(2) Sterilizing: soaking the product A in ethanol solution in an ultra-clean workbench, washing with sterile water, transferring into NaClO solution for disinfection, washing with sterile water, and absorbing water to obtain product B;
(3) Preparation of PDA medium: peeling potato, cutting into small pieces, adding water, boiling, filtering with gauze, adding glucose and agar, dissolving in distilled water, spreading on a flat plate, and making into PDA culture medium;
(4) Culturing, separating and purifying: placing the product B in a PDA culture medium, placing the product B in a constant temperature incubator for culture, and selecting fungi with typical growth morphology differences in the PDA culture medium by using a fungus colony edge selection method, transferring the fungi to a new PDA culture medium for culture and purification based on the constant temperature incubator, thereby obtaining the phosphorus-dissolving blueberry endophytic trichoderma.
Specifically, the tissue separation method of the phosphorus-dissolving blueberry endophytic trichoderma comprises the following steps:
(1) Cleaning: selecting blueberry roots with healthy surfaces and no disease spots, cleaning the blueberry roots with clear water, and airing the blueberry roots to obtain a product A;
(2) Sterilizing: immersing the product A in 75% ethanol solution for 2-4s in an ultra-clean workbench, 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 drying to obtain product B;
(3) Preparation of PDA medium: cutting 150-250g peeled potato into small pieces, adding water, boiling, filtering with gauze, adding 15-25g glucose and 15-25g agar, dissolving in 1000mL distilled water, spreading on a flat plate, and making into PDA culture medium;
(4) Culturing, separating and purifying: placing the product B in a PDA culture medium, placing in a constant temperature incubator at 28 ℃ for culturing for 3-5d, and selecting fungi with typical growth morphology differences in the PDA culture medium by using a fungus colony edge selection method, transferring to a new PDA culture medium, culturing and purifying in the constant temperature incubator at 28 ℃ for 3-5 times, thereby obtaining the phosphorus-dissolving blueberry endophytic trichoderma.
More specifically, the tissue separation method of the phosphorus-dissolving blueberry endophytic trichoderma comprises the following steps:
(1) Cleaning: selecting blueberry roots with healthy surfaces and no disease spots, cleaning the blueberry roots with clear water, and airing the blueberry roots to obtain a product A;
(2) Sterilizing: immersing the product A in 75% ethanol solution for 3s in an ultra-clean workbench, washing with sterile water for 5 times, transferring into NaClO solution with the volume ratio of 3% for disinfection for 3min, washing with sterile water for 5 times, and absorbing water to obtain product B;
(3) Preparation of PDA medium: : cutting 200g peeled potato into small pieces, adding water, boiling, filtering with gauze, adding 20g glucose and 20g agar, dissolving in 1000mL distilled water, and spreading in flat plate to obtain PDA culture medium;
(4) Culturing, separating and purifying: placing the product B in a PDA culture medium, placing in a constant temperature incubator at 28 ℃ for culturing for 3-5d, selecting fungi with typical growth morphology differences in the PDA culture medium, transferring the fungi to a new PDA culture medium, culturing and purifying in the constant temperature incubator at 28 ℃ for 3-5 times, and obtaining the phosphorus-dissolving blueberry endophytic trichoderma.
The application of the phosphorus-dissolving blueberry endophytic trichoderma in preparing the preparation for dissolving phosphorus in the culture medium.
The application of the phosphorus-dissolving blueberry endophytic trichoderma in preparation of the preparation for promoting growth of blueberry rhizosphere.
A blueberry growth promoting bacterial product, the active ingredient of which comprises the trichoderma reesei endophyte of phosphate dissolving blueberry according to claim 1 or 2.
A blueberry growth promoting bacterial product, the active ingredient of which is the phosphorus-dissolving blueberry endophytic trichoderma reesei of claim 1 or 2.
A method for preparing a blueberry growth promoting bacterial product, which adopts the phosphorus-dissolving blueberry endophytic trichoderma as claimed in claim 1 or 2 as an active ingredient or one of active ingredients for preparing the product.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses a phosphorus-dissolving blueberry endophytic trichoderma and application thereof, which are used for separating and culturing blueberry root endophytic fungi obtained from a high-slope ecological blueberry garden (106 DEG 50 '14' east longitude and 26 DEG 16 '46' North latitude) of Guiyang city streams in Guizhou province by using a tissue separation method, and extracting DNA of a strain by using Fungal DNA Midi Kit. The sequences of the strains were obtained using the universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') and the DNA was amplified by Polymerase Chain Reaction (PCR). Multiple sequence comparison analysis is carried out on the ITS sequence and sequences of the same genus and different species in the Blastn comparison result, and simultaneously the ITS sequence and the Blastn comparison result are introducedNectriaberolinensisThe exogenous genus uses Clustal W in MEGA7.0 software for multiple sequence alignment, and a systematic IST developmental tree is constructed using the Neighbor-Joining method (MEGA 7.0). Strain GL41 was found to be identical toTrichodermakoningiopsisThe support degree of the plant is 100%, and the plant is classified into one branch of phylogenetic tree; identification of endophytic fungi by combining morphological characteristics and microscopic morphological characteristics of colonies into Trichoderma pseudokoningii GL41Trichodermakoningiopsis。
The determination result of the phosphorus-dissolving capability of the blueberry endophytic trichoderma reesei shows that the strain has higher inorganic phosphorus-dissolving capability for the phosphorus-dissolving blueberry endophytic trichoderma reesei, and takes PKO solid culture medium as basic culture medium, GL41TrichodermakoningiopsisThe general expression is a trend of descending, ascending and then descendingThe effective phosphorus content gradually rises from 1d to 5d, the 5d reaches a maximum value of 273.90 mu g/ml, and the seventh day is reduced to 128.19 mu g/ml.
In conclusion, the phosphorus-dissolving blueberry endophytic trichoderma has the function of efficiently dissolving phosphorus (the phosphorus is inorganic phosphorus), has great potential for developing a microbial agent in blueberry growth promotion, is the blueberry endophytic trichoderma, can be directly obtained by tissue culture and separation of blueberry roots, and does not need to additionally introduce strains.
Drawings
Fig. 1: GL41TrichodermakoningiopsisColony morphology and microscopic morphology after 6D culture (A is colony front and B is colony back, C and D are molecular spore and conidiophore morphology);
fig. 2: ITS-based GL41TrichodermakoningiopsisEvolving a tree;
fig. 3: GL41TrichodermakoningiopsisPhosphorus-solubilizing effect on PKO solid media;
fig. 4: GL41TrichodermakoningiopsisEffective phosphorus content on PKO broth;
fig. 5: GL41TrichodermakoningiopsisInfluence on the growth of blueberry tissue culture seedlings;
fig. 6: GL41TrichodermakoningiopsisColonization at roots of blueberry tissue culture seedlings (CK as control group, GL41 as GL41 colonization group);
fig. 7: GL41TrichodermakoningiopsisColonization at roots of blueberry potted seedlings (CK as control group, GL41 as GL41 treatment group);
fig. 8: GL41TrichodermakoningiopsisEffect on blueberry pot seedling growth (a, A1, A2 is control group, B1, B2 is GL41 treatment group.);
fig. 9: GL41TrichodermakoningiopsisEffect on blueberry plant dryness (CK as control group, GL41 as GL41 treatment group);
fig. 10: GL41TrichodermakoningiopsisEffect on fresh blueberry plants (CK as control group, GL41 as GL41 treatment group);
fig. 11: GL41TrichodermakoningiopsisInfluence on blueberry plant seedling heightCK is control group, GL41 is GL41 treatment group);
fig. 12: GL41TrichodermakoningiopsisInfluence on the ground warp of blueberry plants (CK as control group, GL41 as GL41 treatment group);
fig. 13: GL41TrichodermakoningiopsisEffect on chlorophyll content of blueberry plants (CK as control group, GL41 as GL41 treatment group);
fig. 14: GL41TrichodermakoningiopsisEffects on soluble sugars in blueberry plants (CK as control group, GL41 as GL41 treatment group);
fig. 15: GL41TrichodermakoningiopsisEffects on soluble proteins of blueberry plants (CK as control group, GL41 as GL41 treatment group);
fig. 16: GL41TrichodermakoningiopsisEffects on SOD of blueberry plants (CK as control group, GL41 as GL41 treatment group);
fig. 17: GL41TrichodermakoningiopsisInfluence on CAT enzyme activity of blueberry plants (CK is control group, GL41 is GL41 treatment group);
fig. 18: GL41TrichodermakoningiopsisEffects on nutrient total nitrogen of blueberry plants (CK as control group, GL41 as GL41 treatment group);
fig. 19: GL41TrichodermakoningiopsisEffects on the total phosphorus of blueberry plant nutrients (CK as control group, GL41 as GL41 treatment group);
fig. 20: GL41TrichodermakoningiopsisEffect on total potassium of blueberry plant nutrients (CK as control group, GL41 as GL41 treatment group).
Detailed Description
The invention is further illustrated by the following figures and examples, which are not intended to be limiting.
Example 1: tissue separation of phosphorus-dissolving blueberry endophytic trichoderma
(1) Cleaning: selecting a blueberry root with healthy surface and no disease spots, cleaning with clear water, and airing;
(2) Sterilizing: immersing the cleaned and dried blueberry root in 75% ethanol solution for 3s in an ultra-clean workbench, washing with sterile water for 5 times, transferring into 3% (V/V) NaClO solution for disinfection for 3min, washing with sterile water for 5 times, placing into 75% ethanol for disinfection for 30s, washing with sterile water for 5 times, and sucking water for later use;
(4) Preparation of PDA medium: dissolving 200g potato, 20g glucose and 20g agar in 1000mL distilled water, and spreading on a flat plate to obtain PDA culture medium;
(3) Culturing, separating and purifying: placing the product B in a PDA culture medium, placing in a constant temperature incubator at 28 ℃ for culturing for 4d, and selecting fungi with typical growth morphology difference in the PDA culture medium by using a fungus colony edge selection method, transferring the fungi into a new PDA culture medium, culturing and purifying in the constant temperature incubator at 28 ℃ for 4 times, thus obtaining the phosphorus-dissolving blueberry endophytic trichoderma.
Example 2: tissue separation of phosphorus-dissolving blueberry endophytic trichoderma
(1) Cleaning: selecting a blueberry root with healthy surface and no disease spots, cleaning with clear water, and airing;
(2) Sterilizing: immersing the cleaned and dried blueberry root in 75% ethanol solution for 2s in an ultra-clean workbench, washing with sterile water for 4 times, disinfecting in 3% (V/V) NaClO solution for 4min, washing with sterile water for 4 times, placing in 75% ethanol for disinfecting for 20s, washing with sterile water for 6 times, and sucking out the water for later use;
(4) Preparation of PDA medium: dissolving 150 g potato, 15g glucose and 15g agar in 1000mL distilled water, and spreading in a flat plate to obtain PDA culture medium;
(3) Culturing, separating and purifying: placing the product B in a PDA culture medium, placing in a constant temperature incubator at 28 ℃ for culturing for 5 days, and selecting fungi with typical growth morphology differences in the PDA culture medium by using a fungus colony edge selection method, transferring the fungi into a new PDA culture medium, culturing and purifying in the constant temperature incubator at 28 ℃ for 5 times, thus obtaining the phosphorus-dissolving blueberry endophytic trichoderma.
Example 3: tissue separation of phosphorus-dissolving blueberry endophytic trichoderma
(1) Cleaning: selecting a blueberry root with healthy surface and no disease spots, cleaning with clear water, and airing;
(2) Sterilizing: immersing the cleaned and dried blueberry root in 75% ethanol solution for 3s in an ultra-clean workbench, washing with sterile water for 6 times, transferring into 3% (V/V) NaClO solution for disinfection for 3min, washing with sterile water for 4 times, placing into 75% ethanol for disinfection for 35s, washing with sterile water for 6 times, and sucking water for later use;
(4) Preparation of PDA medium: dissolving 250g of potato, 25g of g glucose and 25g of agar in 1000mL of distilled water, and spreading the mixture on a flat plate to prepare a PDA culture medium;
(3) Culturing, separating and purifying: placing the product B in a PDA culture medium, placing in a constant temperature incubator at 28 ℃ for culturing for 4d, selecting fungi with typical growth morphology difference in the PDA culture medium, transferring the fungi to a new PDA culture medium, culturing and purifying in the constant temperature incubator at 28 ℃ for 5 times, and obtaining the phosphorus-dissolving blueberry endophytic trichoderma.
Example 4: tissue separation of phosphorus-dissolving blueberry endophytic trichoderma
(1) Cleaning: selecting a blueberry root with healthy surface and no disease spots, cleaning with clear water, and airing;
(2) Sterilizing: immersing the cleaned and dried blueberry root in 75% ethanol solution for 3s in an ultra-clean workbench, washing with sterile water for 5 times, transferring into 3% (V/V) NaClO solution for disinfection for 3min, washing with sterile water for 5 times, placing into 75% ethanol for disinfection for 30s, washing with sterile water for 5 times, and sucking water for later use;
(4) Preparation of PDA medium: dissolving 220 g potato, 15g glucose and 20g agar in 1000mL distilled water, and spreading in a flat plate to obtain PDA culture medium;
(3) Culturing, separating and purifying: placing the product B in a PDA culture medium, placing in a constant temperature incubator at 28 ℃ for culturing for 4d, and selecting fungi with typical growth morphology difference in the PDA culture medium by using a fungus colony edge selection method, transferring the fungi into a new PDA culture medium, culturing and purifying in the constant temperature incubator at 28 ℃ for 4 times, thus obtaining the phosphorus-dissolving blueberry endophytic trichoderma.
A large number of analysis and verification experiments are carried out, and the following experimental study results are shown in the invention:
1. separation and identification of blueberry endophytic trichoderma
1.1 separation and identification method for blueberry endophytic trichoderma
The method comprises the steps of utilizing a tissue separation method to separate and culture endophytic fungi at blueberry roots collected from a high-slope ecological blueberry garden (106 DEG 50 '14' in east longitude and 26 DEG 16 '46' in north latitude) of Guiyang, guizhou, selecting blueberry roots with healthy surfaces and no disease spots, washing with clear water, and airing. Immersing blueberry roots with healthy surfaces and no disease spots in a 75% ethanol solution for 3s times in an ultra-clean workbench, washing with sterile water for 5 times, transferring into a 3% (V/V) NaClO solution for 3min for sterilization, washing with sterile water for 5 times, placing 75% ethanol for sterilization 30s, washing with sterile water for 5 times and sucking water, placing 5 sections of blueberry root sections in each PDA (potato 200g, 20g glucose, 20g agar and 1000mL distilled water) culture medium, repeating 20 PDA culture mediums, and placing in a 28 ℃ constant temperature incubator for culturing for 3-5d; meanwhile, a plant tissue marking method is adopted, blueberry roots with the surfaces disinfected are directly smeared on the surfaces of the culture mediums, and 5 PDA culture mediums are cultivated under the same conditions to serve as a control. And (3) selecting fungi with typical growth morphology differences in the PDA plate culture medium by using a fungus colony edge selection method, transferring the fungi to a new PDA culture medium, culturing in a constant temperature incubator at 28 ℃, and purifying for 3-5 times. Separating and purifying the obtained strain, inoculating the strain into PDA culture medium, and culturing at constant temperature of 28 ℃.
Colony features (colony color, colony shape, and edge features) were observed and photographed with a camera. And observing the mycelium morphology and spores of the strain by using a microscope, and completing morphological identification. The colony features (colony color, colony shape, and edge features) were photographed with a camera. The DNA of the strain was extracted using Fungal DNA Midi Kit. The sequences of the strains were obtained using the universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') and the DNA was amplified by Polymerase Chain Reaction (PCR). And directly sending the PCR amplification product to Chongqing department biological testing company for gel cutting, purification and sequencing to finish molecular biology identification.
Separation and identification result
On PDA medium, hyphae flourished, and 3d colonies had grown over the entire PDA dish. The colonies showed no apparent ring, were initially white, and produced green conidia after 6d (FIG. 1A, FIG. 1B) A. The invention relates to a method for producing a fibre-reinforced plastic composite Conidiophores monospore stand up, and conidiophores peduncles directly produce bottle peduncles or produce secondary branches (fig. 1C, fig. 1D). Multiple sequence comparison analysis is carried out on the ITS sequence and sequences of the same genus and different species in the Blastn comparison result, and simultaneously the ITS sequence and the Blastn comparison result are introducedNectriaberolinensisThe exogenous genus uses Clustal W in MEGA7.0 software for multiple sequence alignment, and a systematic IST developmental tree is constructed using the Neighbor-Joining method (MEGA 7.0). Strain GL41 was found to be identical toTrichodermakoningiopsisOne branch of phylogenetic tree with 100% support is located, and the branches are gathered into one group (figure 2); identification of endophytic fungi by combining morphological characteristics and microscopic morphological characteristics of colonies into Trichoderma pseudokoningii GL41Trichodermakoningiopsis。
2. Detection test
The detection test is carried out on the phosphorus-dissolving blueberry endophytic trichoderma obtained after separation and identification, and the test process is as follows:
2.1 determination of phosphorus-dissolving Capacity of blueberry endophytic Trichoderma
Quantitative determination: beating activated fungus blocks into fungus cakes with the diameter of 5mm by using a puncher, and inoculating PKO inorganic phosphorus culture medium (Zn) into an ultra-clean bench 3 （PO 4 ) 25.0g of 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, 1000mL of distilled water, 20.0g of agar and natural pH), and after the stable growth, observing whether a transparent phosphorus dissolving ring appears on the periphery.
Qualitative determination: and (3) taking PKO culture medium without adding Agar as a liquid culture medium, adding 150 mL to each conical flask, under aseptic condition, taking 5 bacterial cakes with the diameter of 8.0 mm, respectively putting the bacterial cakes into conical flasks with fermentation liquid, transferring the bacterial cakes to a shaking table, culturing the bacterial cakes at the temperature of 28 ℃ and the rotating speed of 150 r/min for 7 d, sampling and measuring the effective phosphorus content in the fermentation liquid every day after culturing, and measuring the pH value by using a pH meter.
(1) The method for measuring the content of the available phosphorus comprises the following steps: taking 5mL supernatant, centrifuging at 12000 r/min for 5 min, taking 3ml supernatant as a liquid to be detected, placing the liquid to be detected in 50ml conical flasks, adding 1 drop of 2, 4-dinitrophenol indicator into each flask, gradually developing the color of the liquid, adding 4 mol/L NaOH solution into each flask to make the fermentation liquid of the conical flask yellow, and adding 1mol/L sulfuric acid solution into the flask to adjust the pH value to make the liquid just slightly yellow. Adding 5ml of molybdenum-antimony anti-chromogenic agent, adding distilled water to a volume of 50ml, properly shaking to make the liquid uniform, standing on a horizontal table surface for 30 min, performing color comparison by using an ultraviolet-visible spectrophotometer with a wavelength of 700 nm, taking PKO liquid fermentation broth without bacteria as reference liquid, setting an absorption value to be zero, respectively measuring the color values of different fermentation broths, and calculating the phosphorus concentration on a phosphorus standard curve.
(2) Drawing a phosphorus standard curve: diluting 100 mg/L of phosphorus standard solution to 5 mg/L, taking 0, 2,4, 6, 8 and 10 mL of phosphorus standard solution of 5 mg/L into a 50mL conical flask, adding distilled water to 30 mL, then respectively adding 2 drops of 2, 4-dinitrophenol indicator, then gradually adding 4 mol/L of NaOH into the conical flask to show yellowish color, adding 1mol/L of H2SO4 to fade out, respectively adding 5mL of molybdenum-antimony anti-color developing agent, rapidly shaking uniformly, adding distilled water to 50mL of marked line position, developing 2H at normal temperature, and measuring absorbance value by using a spectrophotometer 700 nm wavelength. And drawing a phosphorus standard curve, wherein the abscissa is the phosphorus concentration, and the ordinate is the absorbance value.
(3) The calculation formula is as follows: effective phosphorus content P (μg/mL) = (ρ×v×ts)/Vo
The mass concentration (mug/mL) of the available phosphorus (p) searched from the working curve; v: volume to volume (mL) of the color developing solution (here 50 mL); ts, division multiple (here, 17 times); vo, the volume (mL) of the fermentation broth (here 3 mL) was determined.
Detection result:
the transparent ring was observed on day 4 of inoculation using PKO solid medium as basal medium (FIG. 3), and as can be seen from FIG. 4, GL41TrichodermakoningiopsisThe total appearance is that the trend of descending is followed by ascending and then descending, the effective phosphorus content of the 1 st d to the 5 th d gradually rises, the 5 th d reaches the maximum value of 273.90 mu g/ml, and the seventh day is reduced to 128.19 mu g/ml.
Tieback test of blueberry endophytic trichoderma in blueberry tissue culture seedlings
Endophytic fungi are inoculated on aseptic blueberry seedlings, cultured for 1 month, and the colonization condition of the strain is observed under a microscope. The method comprises the following steps: and (3) filling 1/3 moss matrix into the tissue culture bottles for sterilization, cleaning root agar, and transplanting the blueberry tissue culture seedlings which have rooted into the tissue culture bottles, wherein 2 blueberry seedlings are planted in each bottle. After one week of seeding, 3ml of MEA broth with the strain blocks was added to the above described tissue culture flask containing sterile seedlings. Strain colonisation was observed after 30 d.
Observation of colonisation rate: the aseptic roots are washed by distilled water, transferred into a flat plate containing lactic acid for neutralization for 10min, transferred into acid fuchsin containing 0.5% for dyeing for 30s, transferred into lactic acid glycerol liquid (lactic acid: glycerol: distilled water=1:1:1) for decolorization, finally placed on a glass slide for tabletting, and the infection condition of trichoderma is observed under a microscope.
Test results:
GL41 colonized in root tissue of blueberry tissue culture seedlings, there was no significant abnormality in root tissue cells compared to control treatment (fig. 5). GL41 was observed in GL 41-inoculated tissue culture seedling tissueTrichodermakoningiopsisHyphae (fig. 6). Indicating that GL41 can successfully colonize blueberry roots.
Inoculation test
Under aseptic conditions, 3 activated endophytic fungi cakes (diameter=5 mm) were taken with an inoculating loop, inoculated into a 250 mL conical flask containing 100 mL PDB medium, autoclaved at 121 ℃ for 30 minutes, and bacterial culture was prepared at 150 rpm for 7 days. Then, the number of spores in each bacterial culture was measured by a hemocytometer, and the spore concentration was adjusted to 1X 10 5 CFU/mL was ready for use.
The inoculation test uses potting test, 2 treatments, namely control group (CK), and GL41 spore suspension is inoculated, and each pot is 1 strain, and 18 experimental pots are used.
Potting experiments were performed in a greenhouse. Healthy, disease-free and consistently growing one year old blueberry seedlings are selected, potted with sterilized humus soil, and inoculated with root injury irrigation: the disinfected scalpel was inserted into the soil near the plants to cause root wounds, and each blueberry was infused with 100 mL spore suspension, with seedlings inoculated with equal amounts of sterile distilled water as a control group. No fertilizer is applied in the growing period, water is poured once every three days, and the plants are harvested after unified cultivation and management for 90 days.
Growth medium: organic humus, pine needle crushed, leaves, high-quality sawdust and imported coconut coir mixed fermentation humus, which is purchased from Lv Yuan Mei Jia agricultural technology Co., ltd, and autoclaved for 2 hours at 121 ℃ before use. Removing microorganisms on the substrate.
Influence of blueberry endophytic trichoderma on blueberry growth promotion
Observing the colonisation condition as 3; the root system structure is scanned by using a desktop scanner; biomass was weighed (dry and fresh weight): drying in a drying oven at 45 ℃ for 48 hours; measuring the plant height and the ground diameter of each basin by a ruler and a vernier caliper respectively; chlorophyll, soluble protein, soluble sugar, SOD enzyme, CAT enzyme, total nitrogen, total phosphorus and total potassium content are measured by acetone extraction method, coomassie brilliant blue G-250, visible spectrophotometry, ultraviolet spectrophotometry, alkaline hydrolysis diffusion method, molybdenum-antimony colorimetry and flame method.
Test results:
the potted seedling setting result is the same as that of the tissue culture seedling (figure 7), and compared with the control, the GL41 inoculation has obvious effect on the promotion of the blueberry potted seedling (figures 8-20).
Sequencing results
Trichoderma pseudokoningii GL 41%Trichodermakoningiopsis) GenBank accession No.: MW750424
TGTGACCATACCAAACTGTTGCCTCGGCGGGGTCACGCCCCGGGTGCGTCGCAGCCCCGGAACCAGGCGC
CCGCCGGAGGGACCAACCAAACTCTTTCTGTAGTCCCCTCGCGGACGTTATTTCTTACAGCTCTGAGCAA
AAATTCAAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAA
ATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCA
GTATTCTGGCGGGCATGCCTGTCCGAGCGTCATTTCAACCCTCGAACCCCTCCGGGGGGTCGGCGTTGGG
GATCGGGAACCCCTAAGACGGGATCCCGGCCCCGAAATACAGTGGCGGTCTCGCCGCAGCCTCTCCTGCG
CAGTAGTTTGCACAACTCGCACCGGGAGCGCGGCGCGTCCACGTCCGTAAAACACCCAACTTCTGAAATG
TTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAT。
Claims (6)
1. The phosphorus-dissolving blueberry endophytic trichoderma is characterized in that: the phosphorus-dissolving blueberry endophytic trichoderma is trichoderma pseudokoningii @ andTrichoderma koningiopsis) GL41 is preserved in China general microbiological culture collection center (CGMCC) with a preservation number of CGMCC No.40241 in 2022 and 7 and 11 days.
2. The use of the phosphorus-dissolving blueberry endophytic trichoderma according to claim 1, wherein: the application of the phosphorus-dissolving blueberry endophytic trichoderma in preparing the preparation for dissolving inorganic phosphorus in a culture medium.
3. The use of the phosphorus-dissolving blueberry endophytic trichoderma according to claim 1, wherein: the application of the phosphorus-dissolving blueberry endophytic trichoderma in preparing a preparation for promoting the growth of blueberry rhizosphere.
4. A blueberry growth promoting bacterial product, which is characterized in that: the active ingredient of the product comprises the phosphorus-dissolving blueberry endophytic trichoderma reesei of claim 1.
5. A blueberry growth promoting bacterial product, which is characterized in that: the active ingredient of the product is the phosphorus-dissolving blueberry endophytic trichoderma reesei of claim 1.
6. A preparation method of a blueberry growth promoting bacterial product is characterized by comprising the following steps: use of the phosphorus-solubilizing blueberry endophytic trichoderma reesei of claim 1 as an active ingredient or one of the active ingredients for the preparation of said product.
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