A Cryptobiotic Trichoderma WGJ7 and Its Bacterial Agent and Application

Abstract

The invention relates to the field of microbial technology, and in particular to a cryptophytic trichoderma WGJ7 and a bacterial agent and application thereof. The invention has the following beneficial effects: the strain WGJ7 of the invention is separated from the rhizosphere soil of pears by the applicant, has good IAA production, phosphorus dissolving, peroxidase production and protease production capabilities, and through potted plant experiments, it is found that the strain WGJ7 can significantly promote the growth of pear seedlings, improve the root development morphology of pear seedlings, increase the biomass of seedlings, and has a good growth-promoting effect on pears; the strain and discarded mushroom residues are fermented to prepare a biological bacterial agent, and field experiments are carried out, and then it is found that the biological bacterial agent can significantly improve the quality of pear fruit, wherein the soluble solids, single fruit weight, longitudinal diameter and transverse diameter of the fruit are significantly improved; it is explained that the strain of the present application has a good field quality improvement and yield-increasing effect on pear trees, and the raw material of the bacterial agent is simple, and can be promoted and utilized as a biological organic fertilizer material.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a cryptobiotic Trichoderma WGJ7, a bacterial agent and an application thereof. Background Art

[0002] The development of the pear industry has reached a critical point in the transition from unilateral production increase to quality improvement and efficiency increase. Improving fruit quality has become an important task for industrial development. Improving soil fertility, especially biological fertility, is the key to ensuring the healthy growth, high and stable yield of fruit trees, and improving and improving the quality and taste of fruits. Trichoderma bio-organic fertilizer has played a positive role in increasing soil organic matter, improving soil physical and chemical properties, and optimizing the soil rhizosphere environment, thereby improving the yield and quality of fruit trees. However, few of the existing bacterial fertilizers on the market are targeted at pear crops. Those skilled in the art know that microorganisms have selectivity and preference for hosts, and most strains are not able to promote the growth of all plants, and often have high specificity. Moreover, the existing technology focuses more on increasing yield, and the fruit quality is relatively neglected. The bacterial fertilizer that can promote the growth of pear trees may not necessarily play a role in improving the quality of the fruit.

[0003] Based on this, it is necessary for us to screen and verify microorganisms for pear crops, to obtain biological fertilizers suitable for the growth of pear trees and improve the quality of pear fruits, so as to effectively promote the development of the pear industry. Summary of the invention

[0004] In view of the above, it is necessary to screen and verify microorganisms for pear crops, to obtain biological fertilizers suitable for the growth of pear trees and improve the quality of pear fruits, so as to effectively promote the development of the pear industry.

[0005] In order to achieve the above purpose, the present invention screened out a new strain: Cryptophytic Trichoderma Trichoderma afarasin Strain WGJ7, whose taxonomic designation is: Trichoderma afarasin WGJ7, Chinese classification name: Cryptobiotic Trichoderma WGJ7, preservation number is CCTCC NO: M 20242472; the strain is deposited in China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the preservation date is November 7, 2024.

[0006] The present invention also includes the cryptobiotic Trichoderma Trichoderma afarasin Inoculum of strain WGJ7.

[0007] The present invention also includes a method for preparing the bacterial agent, the method comprising: Trichoderma afarasin The strain WGJ7 was inoculated into PDB medium and cultured on a shaking table to obtain Trichoderma fermentation broth, and then the Trichoderma fermentation broth was inoculated into mushroom residues for solid fermentation to obtain the product.

[0008] The present invention also includes the cryptobiotic Trichoderma Trichoderma afarasin Application of strain WGJ7 or the bacterial agent in the preparation of biological organic fertilizer and / or foliar fertilizer.

[0009] The present invention also includes the cryptobiotic Trichoderma Trichoderma afarasin Application of strain WGJ7 or the bacterial agent in promoting the growth of pear seedlings and / or improving the quality of pear fruits.

[0010] Furthermore, the specific indicators of the pear fruit quality are: soluble solid content of the fruit, fruit weight, fruit transverse diameter and / or fruit longitudinal diameter.

[0011] The present invention also includes the cryptobiotic Trichoderma Trichoderma afarasin Application of strain WGJ7 or the bacterial agent in producing IAA, solubilizing phosphate, producing peroxidase and / or producing protease.

[0012] The present invention also includes the use of the cryptobiotic Trichoderma Trichoderma afarasin A method for planting plants with strain WGJ7 or the bacterial agent, the method comprising: adding a bacterial suspension containing strain WGJ7, biological organic fertilizer and / or foliar fertilizer during the seedling stage of the plant.

[0013] Furthermore, the plant is pear.

[0014] The invention has the following beneficial effects: the strain WGJ7 of the invention is separated from the rhizosphere soil of pear by the applicant, has good IAA production, phosphorus solubilization, peroxidase production and protease production capabilities, and through potted plant experiments, it is found that the strain WGJ7 can significantly promote the growth of pear seedlings, improve the root development morphology of pear seedlings, increase the biomass of seedlings, and has a good growth-promoting effect on pear; the strain and discarded mushroom residues are fermented to prepare a biological microbial agent, and field experiments are carried out, and then it is found that the biological microbial agent can significantly improve the quality of pear fruit, wherein the soluble solids, single fruit weight, longitudinal diameter and transverse diameter of the fruit are significantly improved; it is explained that the strain of the present application has a good field quality improvement and yield-increasing effect on pear trees, and the raw materials of the microbial agent are simple, and can be promoted and utilized as a biological organic fertilizer material. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the colony morphology of strain WGJ7.

[0016] Figure 2 This is the phylogenetic tree of strain WGJ7 constructed based on the rpb2 sequence.

[0017] Figure 3It is a result diagram of the enzyme production capacity of strain WGJ7; in the figure, A is the PDA+aniline blue medium without inoculation, and B is the PDA+aniline blue medium after inoculation; C is the culture condition of the Trichoderma strain WGJ5 screened in the same batch in the skim milk powder medium, and D is the culture condition of the strain WGJ7 of the present application in the skim milk powder medium.

[0018] Figure 4 The phenotype of pear seedlings in potted experiments; the upper group of plants in the figure are the experimental group inoculated with strain WGJ7, and the lower group of plants are the control CK group that is not inoculated with the strain.

[0019] Figure 5 The effect of strain WGJ7 on the fresh and dry weight of pear seedlings, among which, Figure 5 Figure A shows the experimental results of the fresh weight of the aboveground part of the pear seedlings, Figure B shows the experimental results of the fresh weight of the underground part of the pear seedlings, Figure C shows the experimental results of the dry weight of the aboveground part of the pear seedlings, and Figure D shows the experimental results of the dry weight of the underground part of the pear seedlings.

[0020] Figure 6 The effects of strain WGJ7 on plant height, leaf number and leaf area of ​​pear seedlings are shown in Figure 2. Figure 6 Figure A shows the experimental results of pear seedling height, Figure B shows the experimental results of leaf number, and Figure C shows the experimental results of leaf area.

[0021] Figure 7 The effect of strain WGJ7 on the root system of pear seedlings, among which, Figure 7 Figure A shows the experimental results of root length of pear seedlings, Figure B shows the experimental results of root surface area of ​​pear seedlings, and Figure C shows the experimental results of root tip number.

[0022] Figure 8 Schematic diagram of solid fermentation of strain WGJ7.

[0023] Fig. 9 These are the results of the effect of strain WGJ7 on the quality of pear fruit; Figure A is the experimental result of fruit soluble solids, Figure B is the experimental result of single fruit weight, Figure C is the experimental result of fruit transverse diameter, and Figure D is the experimental result of fruit longitudinal diameter.

[0024] Biomaterial Deposit Information

[0025] The strain information of this application is: Cryptophytic Trichoderma Trichoderma afarasin Strain WGJ7, whose taxonomic designation is: Trichoderma afarasin WGJ7, Chinese classification name: Cryptobiotic Trichoderma WGJ7, preservation number is CCTCCNO: M 20242472; the strain is deposited in China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the preservation date is November 7, 2024. DETAILED DESCRIPTION

[0026] All features disclosed in this specification, or steps in all methods or processes disclosed, except mutually exclusive features and / or steps, can be combined in any manner.

[0027] Any feature disclosed in this specification (including any appended claims and abstract), unless otherwise stated, is merely an example of a series of equivalent or similar features. Example 1

[0028] This example is the isolation and identification of Cryptozoic Trichoderma WGJ7.

[0029] 1. Isolation of strain WGJ7: The strain was isolated from the rhizosphere of pear trees in a high-quality early-maturing pear orchard in Fengxian County, Xuzhou City, Jiangsu Province.

[0030] 2. Identification of strain WGJ7: ① Morphological identification The specific operation is: inoculate WGJ7 spore liquid on PDA medium and place it in a constant temperature incubator for cultivation. After 2 days of cultivation, the diameter of the colony on the plate is about 6 cm, spider-like, dark green, and the spore-producing surface is green under scattered light. The spores are distributed in concentric circles, and mature spores can be produced without light. It meets the morphological characteristics of Cryptozoic Trichoderma WGJ7 (see Figure 1 ); ② After extracting the strain DNA and sequencing it, molecular identification was performed, and the ITS sequence of the strain was amplified. Based on the comparison of rpb2 sequence and phylogenetic tree analysis and identification, it was determined that the strain was Trichoderma afarasin Closely related, see the evolutionary tree Figure 2 Combined with morphological identification, the strain was classified and named Trichoderma afarasin . Example 2

[0031] This example is a growth-promoting function assay of strain WGJ7.

[0032] 1. Quantitative determination of IAA function of strains: IAA production was detected by the Salkowski colorimetric method (Glickmann and Dessaux, 1995). The spore solution of Cryptozoic Trichoderma WGJ7 was added to a 1% inoculum containing 200 mg·L -1 Tryptophan in PDB medium, 28°C in the dark, 170 r·min -1 The bacterial suspension was shaken for 5 days and then rotated at 8000 r·min -1 Centrifuge at 4°C for 10 min. Take 100 μL of the fermentation supernatant and add Salkowski colorimetric solution at a ratio of 1:1. React in the dark for 30 min and measure OD with an enzyme reader. 530The value was calculated using the corresponding standard curve to calculate the amount of IAA produced by the strain. The standard curve was prepared using IAA standard (Sigma). The concentrations of the prepared IAA solutions were 0, 10, 20, 30, 40, and 50 mg / L, and 100 μL of the above gradient solution was taken, and 100 μL of the colorimetric solution was added. The OD was measured after reacting in the dark for 30 min. 530 The TSB blank medium with colorimetric solution added was used as the control, and three replicates were set up for each, and a standard curve was drawn.

[0033] 2. Determination of the phosphorus solubilizing ability of the strain: The molybdenum antimony colorimetric method was used to determine the effective phosphorus content in the culture medium. The bacterial cakes were cut with a hole puncher (d = 5 mm) and inoculated into the Montana organic phosphorus liquid culture medium. Each treatment group was set up for 3 replicates and placed in a constant temperature shaker at 28 °C and 170 r·min. -1 After shaking culture for 5 days, the culture medium was rotated at 10000 r·min -1 Centrifuge for 15 min, use a UV spectrophotometer to compare colors at 700 nm and measure the OD value to determine the strain's ability to dissolve organic phosphorus, draw a standard curve, and use the standard curve to calculate the strain's effective phosphorus content. The formula for calculating the effective phosphorus content is as follows: , where: P is the phosphorus concentration found in the standard curve; V is the fixed volume during color development; Ts is the fractionation multiple; V0 is the volume of the fermentation liquid to be measured; AP is the effective phosphorus content; the results are shown in Table 1.

[0034]

[0035] As shown in Table 1, strain WGJ7 has strong IAA production and phosphate solubilization capabilities. We speculate that this strain has a certain growth-promoting function on plants. Example 3

[0036] This example is a test for the enzyme production ability of strain WGJ7.

[0037] Method for determining the ability of strains to produce peroxidase and protease: Inoculate the activated strain to be tested on milk agar medium and place it in a constant temperature incubator at 28 °C for 5 days. If a transparent circle is produced, it means that it has the ability to produce protease. Inoculate the activated strain to be tested on PDA+aniline blue medium. If the blue color fades, it means that peroxidase is produced. The specific results are as follows: Figure 3 As shown: Figure 3 The result diagram of the enzyme production capacity of strain WGJ7; in the figure, A is the PDA+aniline blue medium without inoculation, and B is the PDA+aniline blue medium after inoculation; C is the culture of the Trichoderma strain WGJ5 screened in the same batch in the skim milk powder medium, and D is the culture of the strain WGJ7 of the present application in the skim milk powder medium, Figure 3 A in the figure is the uninoculated PDA+aniline blue medium. After inoculation with WGJ7, the blue color faded ( Figure 3 B in the figure indicates that strain WGJ7 produces peroxidase, which means it has the potential to efficiently degrade lignin; Figure 3 C and Figure 3 As shown in D, the transparent zone produced by WGJ7 on skimmed milk powder is significantly larger than that of Trichoderma WGJ5, indicating that it has a stronger ability to produce protease. We speculate that this strain has good biocontrol potential. Example 4

[0038] This example is a potted experiment of strain WGJ7.

[0039] 1. Inoculate the cryptobiotic Trichoderma WGJ7 strain on PDA medium and culture at 28°C for 7 days until complete sporulation. Scrape the spores and adjust the concentration of the spore suspension to 10 with sterile water. 8 cfu / ml.

[0040] 2. Pear seeds were soaked in sterile deionized water for one day in advance, soaked in 75% alcohol for 1 min, rinsed with sterile water 3-5 times, soaked in 2% sodium hypochlorite solution for 20 min, and rinsed with sterile water 5 times. The sterilized Pear seeds were evenly spread on sterilized filter paper for germination for 3-5 days, and the germinated seeds were selected and sown in the sterilized plug tray matrix. After the pear seedlings grew three true leaves, pear seedlings with consistent growth were selected and transplanted into pots filled with matrix (100g). After 10 days of seedling acclimatization, the roots were irrigated with Trichoderma spore suspension. The roots of each pear seedling were inoculated with Trichoderma WGJ7 spore suspension (final concentration was 10 6 CFU / g soil), and the blank control CK was set as the non-inoculated treatment. A second inoculation was performed 10 days after inoculation, and the operation was the same as the first inoculation. The harvest was carried out after 2 months of cultivation.

[0041] 3. Harvesting seedlings: After 2 months of cultivation, the growth of the pear seedlings was measured, including plant height, dry and fresh weight of the aboveground part and root system, and leaf area. The root system of the pear seedlings was scanned and analyzed for total root length and root surface area.

[0042] The results of the pot experiment are as follows Figure 4-Figure 7 As shown: Figure 4 The pear seedling phenotype of the potted experiment shows that the growth of the pear seedlings inoculated with WGJ7 spores is significantly better than that of the CK control treatment. Inoculation with Trichoderma WGJ7 significantly promotes the growth of the aboveground part and root system of the pear seedlings.

[0043] Figure 5 The effect of strain WGJ7 on the fresh and dry weight of pear seedlings, among which, Figure 5Figure A shows the experimental results of the fresh weight of the aboveground part of the pear seedlings, Figure B shows the experimental results of the fresh weight of the underground part of the pear seedlings, Figure C shows the experimental results of the dry weight of the aboveground part of the pear seedlings, and Figure D shows the experimental results of the dry weight of the underground part of the pear seedlings; Figure 5 It can be seen that compared with CK, the fresh weight and dry weight of the aboveground pear seedlings inoculated with Cryptotrophic Trichoderma WGJ7 increased significantly by 23.9% and 29.22%, respectively ( Figure 5 A in Figure 5 C in the aboveground parts); the fresh weight and dry weight of the underground parts increased significantly by 69.1% and 13.4%, respectively ( Figure 5 B in Figure 5 D in.

[0044] Figure 6 The effects of strain WGJ7 on plant height, leaf number and leaf area of ​​pear seedlings are shown in Figure 2. Figure 6 Figure A shows the experimental results of the height of pear seedlings, Figure B shows the experimental results of the number of leaves, and Figure C shows the experimental results of the leaf area. Figure 6 It can be seen that compared with CK, the plant height, leaf number and leaf area of ​​pear seedlings inoculated with Cryptotrophic Trichoderma WGJ7 were significantly increased by 26.0%, 18.0% and 52.2%, respectively.

[0045] Figure 7 The effect of strain WGJ7 on the root system of pear seedlings, among which, Figure 7 Figure A shows the experimental results of root length of pear seedlings, Figure B shows the experimental results of root surface area of ​​pear seedlings, and Figure C shows the experimental results of root tip number. Figure 7 It can be seen that the number of root tips, root length and root surface area of ​​pear seedlings inoculated with Cryptochloroplast WGJ7 were significantly better than those of CK treatment. The experimental group inoculated with strain WGJ7 increased by 37.7%, 15.7% and 10.9% respectively compared with the CK group without strain inoculation.

[0046] In summary, in the potted experiment of pear seedlings, the spore liquid of strain WGJ7 can significantly increase the biomass of pear seedlings and promote the aboveground growth and root development of pear seedlings. Example 5

[0047] This example is a field experiment of strain WGJ7.

[0048] Solid fermentation of Trichoderma strains: The selected Trichoderma strain WGJ7 was inoculated into PDB medium and incubated in a shaker at 28°C and 170 r·min -1, and carry out liquid fermentation for 3-4 days to obtain Trichoderma fermentation liquid. Dilute the hydrolyzed amino acids 20 times and add them to the turnover box containing mushroom residue (collected from Ningliang Bioengineering Co., Ltd.) and soak for 24 hours. Wring the soaked mushroom residue dry and sterilize at 115℃ for 1 hour. After cooling, inoculate the prepared Trichoderma fermentation liquid in a shallow dish, adjust the water content to about 65%, and culture at a constant temperature of 28℃ for 7 days. During the fermentation, keep the water content at 60% to obtain Trichoderma solid strains, which are stored in a refrigerator at 4℃. After microscopic examination, the number of Trichoderma spores per g (dry weight) of Trichoderma solid inoculant was 1.3×10 9 The specific solid fermentation diagram of the strain is as follows Figure 8 As shown in the figure, it can be seen that the mushroom residue after solid fermentation is covered with dark green Trichoderma hyphae.

[0049] Evaluation of the effect of Trichoderma inoculant in the field: The solid Trichoderma inoculant was diluted with water at an addition rate of 1.5% (kg / L). Field experiments were carried out in an early-maturing pear orchard in Fengxian County, Xuzhou City, Jiangsu Province. 10L of the liquid fertilizer was applied to each tree hole during the small fruit stage. A total of 12 trees were applied, and a blank control was made for 12 trees in a row. Pear fruits were collected during the mature stage of the pear trees, and their single fruit weight, soluble solids and other indicators were measured.

[0050] The results of the field experiment are as follows Fig. 9 As shown, Fig. 9 The results of the effect of strain WGJ7 on the quality of pear fruit are shown in Figure A. Figure B shows the experimental results of the soluble solids in the fruit, Figure C shows the experimental results of the horizontal diameter of the fruit, and Figure D shows the experimental results of the vertical diameter of the fruit. Fig. 9 As shown in the figure, the application of the fungicide can significantly increase the soluble solids, single fruit weight and size of early-maturing pear fruits. The soluble solids increased significantly by 4.2%, the single fruit weight increased significantly by 17.2g, or 5.6%, and the longitudinal and transverse diameters increased significantly by 2.6mm and 2.7mm, respectively.

[0051] In summary, the cryptobiotic Trichoderma WGJ7 screened by the applicant of the present invention has good abilities of producing IAA, solubilizing phosphate, producing peroxidase and protease. Through potted plant experiments, it was found that the cryptobiotic Trichoderma WGJ7 can significantly promote the growth and root development of Pyrus betula seedlings. After the strain was solid-fermented to prepare a bacterial agent, it was applied to the field planting of pear trees, which can significantly improve the fruit quality of pears. This shows that the strain of the present application has a good growth-promoting ability for plants, Trichoderma WGJ7 is easy to use, has a good effect of improving quality and increasing yield in the field, and can be promoted and utilized as a biological organic fertilizer material.

[0052] The above-described embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. Cryptobiotic Trichoderma Trichoderma afarasin Strain WGJ7, its deposit number is CCTCC NO: M 20242472.

2. Containing the cryptobiotic Trichoderma described in claim 1 Trichoderma afarasin Inoculum of strain WGJ7.

3. A method for preparing the bacterial agent as claimed in claim 2, characterized in that: The method comprises: Trichoderma afarasin The strain WGJ7 was inoculated into PDB medium and cultured on a shaking table to obtain Trichoderma fermentation broth, and then the Trichoderma fermentation broth was inoculated into mushroom residues for solid fermentation to obtain the product.

4. Cryptobiotic Trichoderma as claimed in claim 1 Trichoderma afarasin Use of strain WGJ7 or the bacterial agent as claimed in claim 2 in the preparation of biological organic fertilizer and / or foliar fertilizer.

5. Cryptobiotic Trichoderma as claimed in claim 1 Trichoderma afarasin Use of strain WGJ7 or the bacterial agent as described in claim 2 in promoting the growth of pear seedlings and / or improving the quality of pear fruits.

6. The use according to claim 5, characterized in that: The specific indicators of the pear fruit quality are: the soluble solid content of the fruit, the fruit weight, the fruit transverse diameter and / or the fruit longitudinal diameter.

7. Cryptobiotic Trichoderma as claimed in claim 1 Trichoderma afarasin Use of strain WGJ7 or the bacterial agent as claimed in claim 2 in producing IAA, solubilizing phosphate, producing peroxidase and / or producing protease.

8. Use of the cryptobiotic Trichoderma as claimed in claim 1 Trichoderma afarasin The method for planting plants using strain WGJ7 or the bacterial agent as claimed in claim 2, characterized in that: The method comprises the following steps: adding a bacterial suspension containing strain WGJ7, biological organic fertilizer and / or foliar fertilizer to the plants during the seedling stage.

9. The method according to claim 8, characterized in that The plant is pear.

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