A strain of Trichoderma spinulosa JR701 and its bacterial agent and application

Abstract

The invention relates to the field of microbial technology, and in particular to a strain of Trichoderma spinulosum JR701 and a bacterial agent and application thereof. The invention has the following beneficial effects: the strain JR701 of the invention is isolated by a research group from rhizosphere soil of a pear tree, and has good IAA production, phosphorus solubilization, phosphorus dissolving, laccase production and protease production capabilities. The strain and discarded mushroom residues are fermented to prepare a biological bacterial agent, and field experiments are carried out. It is then found that the biological bacterial agent can significantly improve the quality of early-maturing pear fruits, especially the soluble solids and single fruit weight of the pears. In addition, after applying the bacterial agent, the leaf drop of the tree can be significantly alleviated, and the secondary flowering and new leaf growth phenomena are less; it is explained that the strain of the present application has a positive effect on the health of the pear tree and the quality and yield of the pear fruit in the second year, has a good field effect, 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 strain of Trichoderma spinulosum JR701, a bacterial agent and an application thereof. Background Art

[0002] With global warming and frequent extreme disasters, the early and massive leaf fall of sand pears has occurred to varying degrees in the southern pear producing areas of my country. The sand pear producing areas in the provinces south of the Yangtze River, such as Fujian and Jiangxi, have experienced large-scale and serious damage. Under normal circumstances, the leaf fall of sand pears in the south is after November, while the early and massive leaf fall is mostly concentrated in June to August, which is the critical period for the ripening of pears and the growth of trees after fruit harvesting. After the large-scale early leaf fall, the supply and demand of nutrients in the body are unbalanced, and the combined effects of external climate factors such as high temperature and heavy rain prompt the flower buds that have completed differentiation to sprout and bloom again in August to October, accompanied by the growth of a large number of new leaves, which is called the "secondary flower", "return to flower" and "return to green". The "return to flower" and "return to green" of sand pears will consume a lot of nutrients in the tree, resulting in a low fruit setting rate and a decrease in yield in the following year. "Cui Guan", "Sucui No. 1" and "Cui Yu" are the three early-maturing varieties with the largest cultivation area in the sand pear production area south of the Yangtze River Basin. The disease is very common and serious, which greatly hinders the development of the pear industry in these areas. Preventing and controlling early leaf drop is of great significance to maintaining tree health and improving fruit yield and quality.

[0003] Improving soil fertility, especially soil biological fertility, is an important way to alleviate early leaf fall. At present, the existing technology mostly uses biological organic fertilizers to increase the content of soil organic matter, improve soil physical and chemical properties and optimize the rhizosphere environment of crops to promote crop growth. However, we found in actual work that biological organic fertilizers are mostly fermented and prepared by microorganisms with growth-promoting effects. However, microorganisms have differences in their hosts. Different microorganisms can promote the growth of a certain crop but not another crop. Moreover, the biological organic fertilizers in the existing technology mostly study the effects of biological fertilizers on plant growth, and rarely study the effects on fruit quality. Therefore, it is necessary to target the technical defects of large-scale early leaf fall of pear trees, which affects the growth of fruit trees and thus affects the quality of fruit, and screen out strains for pear crops that can alleviate early leaf fall of pear trees and improve fruit quality and yield, which are of great significance to the development of the early-maturing pear industry. Summary of the invention

[0004] In view of the above, it is necessary to address the technical defects of large-scale early leaf fall of pear trees, which affects the growth of fruit trees and thus affects the quality of fruit, and screen out strains for pear crops that can alleviate the early leaf fall of pear trees and improve fruit quality and yield, which is of great significance to the development of the early-maturing pear industry.

[0005] In order to achieve the above purpose, the present invention screened out a new strain: Trichoderma spinulosumTrichoderma asperelloides Strain JR701, its taxonomic designation is: Trichoderma asperelloides JR701, Chinese classification name: Trichoderma acanthosporum JR701, preservation number is CCTCC NO: M20242473; 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 Trichoderma spinulosa Trichoderma asperelloides Inoculum of strain JR701.

[0007] The present invention also includes a method for preparing the bacterial agent, the method comprising: Trichoderma asperelloides The strain JR701 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 Trichoderma spinulosa Trichoderma asperelloides Application of strain JR701 or the bacterial agent in preparing biological organic fertilizer.

[0009] The present invention also includes the Trichoderma spinulosa Trichoderma asperelloides Application of strain JR701 or the bacterial agent in alleviating premature leaf drop of pear trees and / or improving the quality of pear fruits.

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

[0011] The present invention also includes the Trichoderma spinulosa Trichoderma asperelloides Application of strain JR701 or the bacterial agent in producing IAA, solubilizing phosphate, dissolving phosphate, producing laccase and / or producing protease.

[0012] The present invention also includes the use of the Trichoderma spinulosa Trichoderma asperelloides The invention discloses a method for planting plants with strain JR701 or the microbial agent, which comprises adding the microbial agent containing strain JR701 and / or biological organic fertilizer during the young fruit stage of the plants.

[0013] Furthermore, the plant is pear.

[0014] The present invention has the following beneficial effects: the strain JR701 of the present invention is separated from the rhizosphere soil of pears by the applicant, and has good IAA production, phosphate solubilization, phosphate dissolving, laccase production and protease production capabilities. The strain and discarded mushroom residues are fermented to prepare a biological agent, and field experiments are carried out. It is then found that the biological agent can significantly improve the quality of early-maturing pear fruits, especially the soluble solids and single fruit weight of the pears. In addition, after applying the agent, the leaf drop of the tree can be significantly alleviated, and the secondary flowering and new leaf growth phenomena are less; it is explained that the strain of the present application has a positive effect on the health of the pear tree and the quality and yield of the pear fruit in the second year, has a good field effect, 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 JR701.

[0016] Figure 2 This is the phylogenetic tree of strain JR701 constructed based on the rpb2 sequence.

[0017] Figure 3 The results of the effect of strain JR701 on fruit quality; in the figure, A is the experimental result of fruit soluble solids, and B is the experimental result of single fruit weight.

[0018] Figure 4 It is a result diagram of the enzyme production capacity of strain JR701; in the figure, A is a schematic diagram of the growth of strain JR701 in PDA + guaiacol medium, Figure B is the culture of Trichoderma strain WGJ5 screened in the same batch in skimmed milk powder medium, and Figure C is the culture of strain JR701 of the present application in skimmed milk powder medium.

[0019] Figure 5 This is a picture of the overall planting situation of the pear orchard after the fungicide JR701 was applied.

[0020] Figure 6 This is a picture of the overall planting situation of the pear orchard without the application of the fungicide JR701.

[0021] Biomaterial Deposit Information

[0022] The strain information deposited in this application is: Trichoderma spinulosa Trichoderma asperelloides Strain JR701, its taxonomic designation is: Trichoderma asperelloides JR701, Chinese classification name: Trichoderma acanthosporum JR701, preservation number is CCTCC NO: M20242473; 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

[0023] 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.

[0024] 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

[0025] This example is the separation and identification of Trichoderma acanthosporum JR701.

[0026] 1. Isolation of strain JR701: The strain was isolated from the rhizosphere of pear trees in a high-quality early-maturing pear orchard in Jurong City, Zhenjiang City, Jiangsu Province.

[0027] 2. Identification of strain JR701: ① Morphological identification The specific operation is: inoculate JR701 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, light 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 is consistent with the morphological characteristics of Trichoderma acanthosporum JR701; ② Extract the strain DNA and sequence it for molecular identification, amplify the ITS sequence of the strain, and identify it based on the comparison of the ITS sequence and the phylogenetic tree analysis of the rpb2 gene fragment, and determine that the strain is consistent with Trichoderma asperelloides Closely related, see the evolutionary tree Figure 2 Combined with morphological identification, the strain was classified and named Trichoderma asperelloides . Example 2

[0028] This example is a growth-promoting function assay of strain JR701.

[0029] 1. Determination of the ability of strains to produce iron carriers: 1 mL of fresh Trichoderma spore suspension was added to 100 mL of LNM iron-deficient medium and incubated at 28 °C and 170 r·min -1 Oscillating culture for 5 days. Then centrifuge the bacterial suspension at 8000 r·min-1, 4℃ for 10min. Take 100 μL of the fermentation supernatant, add an equal volume of CAS detection solution, mix thoroughly, let stand in the dark for 0.5~1 h, measure the absorbance (As) at 630 nm with an enzyme marker, and use ultrapure water as a control for zeroing. Take another medium without spores and mix it with an equal volume of CAS detection solution, invert and mix, treat in the dark for the same time, and use its absorbance as the reference value (Ar). Then calculate the relative content of iron carriers according to the formula [(Ar-As) / Ar]×100%.

[0030] 2. Quantitative determination of IAA function of strains: IAA production was detected by the Salkowski colorimetric method (Glickmann and Dessaux, 1995). The spore solution of Trichoderma aculeatus JR701 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 minutes. Measure OD with an enzyme reader 530 The 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.

[0031] 3. Determination of the ability of strains to dissolve and solubilize phosphorus: The molybdenum antimony colorimetric method was used to determine the effective phosphorus content in the culture medium. The bacterial cake was cut with a hole puncher (d = 5 mm) and inoculated into the Montkina organic phosphorus liquid medium and the PVK inorganic phosphorus medium respectively. Each treatment group was set up for 3 replicates and placed in a constant temperature shaker at 28 ℃ 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 and inorganic 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.

[0032]

[0033] As shown in Table 1, strain JR701 has strong IAA production ability, phosphate solubilization ability, phosphate dissolving ability and iron carrier production. We speculate that this strain has a certain growth-promoting function on plants and can promote the absorption of iron by plants. Example 3

[0034] This example is a test for the enzyme production ability of strain JR701.

[0035] Method for determining the ability of strains to produce peroxidase and protease: Inoculate the activated strain to be tested on skimmed milk powder medium and place it in a constant temperature incubator at 28°C for 3 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+guaiacol medium and place it in a constant temperature incubator at 28°C for 3 days. If a red color reaction is produced, it means that laccase is produced. The specific results are as follows: Figure 4 As shown: Figure 4 The result diagram of the enzyme production capacity of strain JR701; in the figure, A is a schematic diagram of the growth of strain JR701 in PDA + guaiacol medium, B is the culture of Trichoderma strain WGJ5 screened in the same batch in skim milk powder medium, and C is the culture of strain JR701 of the present application in skim milk powder medium; Figure 4 As shown in A, strain JR701 can grow in PDA+guaiacol medium and produce red circles, indicating that it can produce laccase and has the potential to efficiently decompose organic matter; Figure 4 B in Figure 4 As shown in C, the transparent zone produced by JR701 on the skimmed milk powder medium is significantly larger than that of Trichoderma WGJ5, indicating that it has a strong ability to produce protease and has the potential for biocontrol. Example 4

[0036] This example is a field experiment of strain JR701.

[0037] Preparation of strain JR701 inoculant: The selected Trichoderma strain JR701 was inoculated into PDB medium and shaken at 28°C and 170 r / min. -1 , and liquid fermentation was carried out for 3-4 days to obtain Trichoderma fermentation liquid. The hydrolyzed amino acids were diluted 20 times and added to the turnover box containing mushroom residue (collected from Ningliang Bioengineering Co., Ltd.) and soaked for 24 hours. The soaked mushroom residue was wrung dry and sterilized at 115 °C for 1 hour. After cooling, the prepared Trichoderma fermentation liquid was added to a shallow dish, and the water content was adjusted to about 65%. It was cultured at a constant temperature of 28 °C for 7 days, and the water content was maintained at 60% during the fermentation period to obtain Trichoderma solid strains, which were stored in a refrigerator at 4 °C. After microscopic examination, the number of Trichoderma spores in the Trichoderma solid inoculant was 2.4×10 9 cfu / g.

[0038] Evaluation of the effect of field application of Trichoderma agent: The above-mentioned Trichoderma solid agent was diluted with water at an addition rate of 1.5% (kg / L), and field trials were carried out in the early-maturing pear orchards of Jiangsu Academy of Agricultural Sciences (Pear Orchard 1) and Yancheng Dafeng (Pear Orchard 2). Among them, Pear Orchard 1 was the Cuiyu and Sucui No. 2 varieties, and Pear Orchard 2 was the Sucui No. 1 variety. 10L of the above-mentioned agent was applied to each tree hole during the young fruit stage, and a total of 12 trees were applied. A blank control was made for 12 trees in a row, and the tree vigor and fruit retention were kept consistent among different treatments. Pear fruits were collected during the fruit maturity period, and their single fruit weight, soluble solids and other indicators were measured. The state of the trees was observed in September, including early leaf fall, secondary flowering, and new leaf growth.

[0039] Effects on fruit quality Figure 3 As shown, Figure 3 Figure A shows the experimental results of fruit soluble solids, and Figure B shows the experimental results of single fruit weight. Figure 3 As shown in the results, the application of this fungus can significantly increase the fruit solidification and single fruit weight of early-maturing pears, among which the solidification of Sucui No. 1, Cuiyu and Sucui No. 2 increased by 4.8%, 8.2% and 9.4% respectively; the single fruit weight increased by 21.7%, 11.4% and 20.0% respectively. This shows that this strain can effectively improve the quality and yield of pear fruits.

[0040] The results of leaf fall are as follows: Figure 5-Figure 6 As shown, Figure 5 This is the overall planting situation of the pear orchard where the fungicide JR701 was applied. Figure 6 This is the overall planting situation of the pear orchard without the application of the fungicide JR701; it can be seen from the figure that Figure 5 The pear trees are densely foliaged with no obvious leaf drop, and they rarely bloom again. Figure 6 The pear trees had obvious leaf drop, especially the red frame part at the bottom of the pear tree, where the leaves were basically all gone and there was a second flowering. This shows that the strain JR701 inoculant of the present application can effectively alleviate the premature leaf drop of pear trees, and has a positive effect on the health of the tree and the quality and yield of pear fruit in the second year.

[0041] In summary, the Trichoderma sp. JR701 screened by the applicant of the present invention has good abilities of producing IAA, solubilizing phosphate, dissolving phosphate, producing siderophore, producing laccase and protease. After the strain is solid-fermented to prepare the bacterial agent, it is applied to the field planting of pear trees, which can significantly improve the fruit quality of pears. At the same time, it can also effectively alleviate the premature leaf fall of pear trees, and have a positive effect on the health of the trees and the quality and yield of pear fruits in the second year. This shows that the strain of the present application has good field quality improvement and yield-increasing ability for pear plants, and can be promoted and utilized as a biological organic fertilizer material.

[0042] 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. Trichoderma acanthosporum Trichoderma asperelloides strain JR701, whose deposit number is CCTCC NO:M20242473.

2. Containing the Trichoderma spinulosa described in claim 1 Trichoderma asperelloides Inoculum of strain JR701.

3. A method for preparing the bacterial agent as claimed in claim 2, characterized in that: The method comprises: Trichoderma asperelloides The strain JR701 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. Trichoderma as claimed in claim 1 Trichoderma asperelloides Use of strain JR701 or the bacterial agent as claimed in claim 2 in the preparation of biological organic fertilizer.

5. The method of claim 1 Trichoderma asperelloides Use of strain JR701 or the bacterial agent as claimed in claim 2 in alleviating premature leaf drop of pear trees 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 and / or the fruit weight.

7. The Trichoderma spinulosa as claimed in claim 1 Trichoderma asperelloides Use of strain JR701 or the bacterial agent as claimed in claim 2 in producing IAA, phosphate solubilization, phosphate dissolving, laccase production and / or protease production.

8. Use of the Trichoderma spinulosa as claimed in claim 1 Trichoderma asperelloides The method for planting plants using strain JR701 or the bacterial agent as claimed in claim 2, characterized in that: The method comprises: adding a bacterial agent containing strain JR701 and / or a biological organic fertilizer during the young fruit stage of the plant; the plant is a pear tree.

Images