Arbuscular mycorrhizal fungus inoculant for promoting grafting survival rate of tea trees and preparation method of arbuscular mycorrhizal fungus inoculant

Abstract

The invention discloses an arbuscular mycorrhizal fungus inoculant for promoting the grafting survival rate of tea trees and a preparation method of the arbuscular mycorrhizal fungus inoculant, and belongs to the technical field of microbial preparations and plant cultivation. The invention relates to an arbuscular mycorrhizal fungus fungicide, which is characterized in that the arbuscular mycorrhizal fungus fungicide is prepared from fungiporus mosseae and clostridium etunicatum, and the mass ratio of the fungiporus mosseae to the clostridium etunicatum is 2 to 1. Experimental results show that after the arbuscular mycorrhizal fungus inoculant is applied, the survival rate of tea tree grafting is increased to 80% from 60% of a control group. The method breaks through the limitation that tea tree grafting depends on physical means, efficient colonization of arbuscular mycorrhizal fungi on a grafting interface is achieved for the first time, and the method has great agricultural application value.

Description

Technical Field

[0001] The present invention relates to the technical fields of microbial agents and plant cultivation technologies, and particularly relates to an arbuscular mycorrhizal fungal agent for promoting the survival rate of tea tree grafting and a preparation method thereof. Background Art

[0002] Tea tree grafting is a core technology for variety improvement, resistance enhancement, and rapid renewal of tea gardens. The core challenge lies in the efficient connection of the vascular bundles of the scion and the rootstock and the coordinated development of callus. Traditional grafting techniques optimize the cutting angle, the interface wrapping material, and the temperature and humidity control. That is, existing improvement techniques mostly focus on optimizing physical means, but these methods fail to solve the root problem from the level of cell metabolism regulation.

[0003] Arbuscular mycorrhizal fungi (AMF), as a key medium for plant-microbe interaction, have been proven to improve soil aggregate structure by secreting glomalin proteins and mediate the cross-root transport of nutrients such as phosphorus and zinc through the hyphal network. In non-grafted systems, AMF can induce host plants to synthesize signal molecules such as jasmonic acid (JA) and salicylic acid (SA) and coordinate the expression of stress-resistant genes. However, existing AMF application research is mostly limited to the inoculation of single-crop roots and has not yet involved the colonization requirements of the microbial community in this special microenvironment of the grafting interface. As a typical mycorrhizal non-dependent plant, the symbiotic efficiency of tea trees with AMF is significantly lower than that of leguminous or gramineous crops, which further limits the direct application of AMF in tea tree cultivation. The above limitations have left AMF in the long-term research blank in the field of tea tree grafting, and there is an urgent need to develop adaptable agents and preparation methods to break through the technical bottleneck. Summary of the Invention

[0004] The purpose of the present invention is to provide an arbuscular mycorrhizal fungal agent for promoting the survival rate of tea tree grafting and a preparation method thereof to solve the problems existing in the above-mentioned prior art. The arbuscular mycorrhizal fungal agent provided by the present invention can promote the survival rate of tea tree grafting and expands the direct application of AMF in tea tree grafting cultivation.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] Technical Solution 1: An arbuscular mycorrhizal fungal agent for promoting the survival rate of tea tree grafting, which is obtained by inoculating and culturing Funneliformis mosseae and Claroideoglomus etunicatum, and the mass ratio of Funneliformis mosseae to Claroideoglomus etunicatum is 2:1.

[0007] Furthermore, the fungal content in the arbuscular mycorrhizal fungal agent is ≥500 spores / g.

[0008] Technical solution 2: A preparation method of the arbuscular mycorrhizal fungal agent includes the following steps: Mix Funneliformis mosseae and Claroideoglomus etunicatum in a mass ratio of 2:1, and inoculate them onto the roots of corn seedlings; Cultivate in a culture medium composed of fine river sand, perlite, and biochar, separate the culture medium and the corn roots, cut the roots into pieces and mix them with the culture medium to obtain the original strain agent; Inoculate the original strain agent into the propagation substrate for cultivation, collect the mycelium and spores, and mix them with the carrier to obtain the arbuscular mycorrhizal fungal agent.

[0009] Further, the fine river sand is obtained through sieving, rinsing, sterilization, and air-drying treatments.

[0010] Further, the cultivation conditions for inoculating the original strain agent into the propagation substrate include 12000 lux of light per day, culturing at 28°C for 13 hours, and culturing at 18°C for 11 hours under dark conditions.

[0011] Further, Hoagland nutrient solution is irrigated once every 2 weeks during the cultivation process.

[0012] Further, the carrier is sterilized vermiculite.

[0013] Technical solution 3: A method for promoting the survival rate of tea tree grafting includes the step of applying the above-mentioned arbuscular mycorrhizal fungal agent to the roots of the tea tree rootstock.

[0014] Further, the grafting method is the cleft grafting method.

[0015] The present invention discloses the following technical effects:

[0016] The agent of the present invention realizes the synergistic improvement of the survival rate and stress resistance of tea tree grafting through mycorrhizal symbiotic effects and metabolite regulation. The present invention clarifies that the arbuscular mycorrhizal fungal agent has the effect of improving the survival rate of tea tree grafting, providing a theoretical basis and technical support for the large-scale promotion of this agent. The arbuscular mycorrhizal fungal agent provided by the present invention is prepared by propagation in a specific culture medium (the volume ratio of fine river sand, perlite, and biochar is 3:2:1), with high preparation efficiency, and the culture medium ratio and propagation conditions are optimized (light / temperature cycle) to ensure that the spore yield is ≥500 spores / g. The experimental results of the present invention show that after applying this agent, the survival rate of tea tree grafting is increased from 60% in the control group to 80%. In summary, the present invention breaks through the limitation of tea tree grafting relying on physical means, and for the first time realizes the efficient colonization of arbuscular mycorrhizal fungi at the grafting interface, having great agricultural application value. Specific embodiments

[0017] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0018] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0019] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0020] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are merely exemplary.

[0021] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0022] In the examples, the fine river sand was pretreated as follows: the fine river sand was passed through a 2 mm sieve, washed with water until the water was clear, then soaked in distilled water for 3 days, and rinsed with distilled water 3 more times; then, under the condition of 121 °C, sterilized for 2 h, cooled, spread into a thin layer less than 1 cm in a sterile environment, and air-dried naturally for 1 week.

[0023] Funneliformis mosseae and Claroideoglomus etunicatum were provided by the Institute of Plant Nutrition and Resources, Beijing Academy of Agriculture and Forestry Sciences.

[0024] Example 1

[0025] Preparation of arbuscular mycorrhizal fungal inoculum:

[0026] (1) Mixing the mossea spores and the young glomerulone in a mass ratio of 2:1 (40 g of mossea spores and 20 g of the young glomerulone) was inoculated onto the root system of corn seedlings sown for 2 weeks. The inoculated corn seedlings were grown on a culture medium (the volume ratio of fine river sand, perlite and biochar was 3:2:1, and the thickness of the culture medium was 10 cm) for 3 months, and the Hoagland nutrient solution was irrigated once every 2 weeks during the growth process;

[0027] (2) removing the above-ground part of the corn, separating the culture matrix from the corn root system, chopping the corn root system, and then mixing it with the culture matrix again to obtain an arbuscular mycorrhizal fungus stock inoculant, which contains arbuscular mycorrhizal fungus spores, hyphae, and a culture matrix of infected root segments;

[0028] (3) The prepared arbuscular mycorrhizal fungus original seed inoculant was inoculated into the fine river sand of the propagation matrix at an inoculation rate of 20wt%, and mixed to obtain the propagation matrix containing the arbuscular mycorrhizal fungus original seed inoculant, and then the propagation matrix containing the arbuscular mycorrhizal fungus original seed inoculant was evenly spread in a rectangular culture box with a thickness of 20cm, and watered with distilled water to 50% of the field water holding capacity; corn seeds were sown, and after sowing, the surface was covered with 1cm of fine river sand containing the arbuscular mycorrhizal fungus original seed inoculant, and the corn was cultured for 10 weeks, wherein the corn was cultured for 13h under the conditions of 12000lux and 28℃, and for 11h in the dark at 18℃; during the growth process, watering was carried out according to the dry and wet conditions, and the moisture content of the matrix was controlled to be 30% of the field water holding capacity, and watering was carried out once every 2 days; Hoagland nutrient solution was irrigated once every 2 weeks, and the amount used was based on the amount of the matrix moisture content to reach 30% of the field water holding capacity;

[0029] (4) After the corn has grown for 10 weeks, cut off the above-ground part of the corn plant, separate the propagation matrix from the corn root system, cut the corn root system into pieces less than 0.5 cm, and then mix it with the propagation matrix again to obtain the cultured matrix. The cultured matrix is suspended in water and passed through sieves of different apertures (such as 200 mesh and 400 mesh) to collect spores and mycelium. Use sterilized vermiculite (inert and good water retention) as a carrier and mix it with the mycelium at a volume ratio of 1:3 to make a finished bacterial agent (bacterial content ≥ 500 spores / g).

[0030] Example 2

[0031] Take 3-year-old tea tree seedlings (Yuncha No. 1) as rootstocks; Yunkang No. 22 tea tree varieties as scions. Apply 10g of the finished bacterial agent prepared in Example 1 to the root of each rootstock as the test group; the control group is not applied with bacterial agent. Each group is set up with 30 replicates, randomly arranged in a greenhouse (temperature 25±2℃, humidity 70%). Among them, the grafting method adopts cleft grafting, the interface is wrapped with plastic film, and routine management is performed after grafting to maintain the soil moisture content at 65%.

[0032] Measure the following indicators respectively:

[0033] (1) Survival rates of the experimental group and the control group: Count the proportion of plants with completely healed interfaces and new shoot growth 60 days after grafting;

[0034] (2) Measure the callus formation rates of the experimental group and the control group: Take samples on the 10th, 20th, and 30th days after grafting and measure the thickness of the callus at the interface (microscopic section method);

[0035] (3) Measure the root activities of the experimental group and the control group: Measure the dehydrogenase activity of the roots of grafted seedlings by the TTC method;

[0036] (4) Measure the chlorophyll contents of the experimental group and the control group: Measure the SPAD value of leaves by acetone extraction method;

[0037] For the statistical results of the above measured indicators, please refer to Table 1.

[0038] Table 1

[0039] Measurement indicators Experimental group Control group Grafting survival rate 80％ 60％ Callus thickness (30 days) 1.72 ± 0.25 mm 1.02 ± 0.22 mm Root activity (μg TTF / g / h) 35.4±3.4 24.3±3.2 Chlorophyll SPAD value 44.2±1.8 32.5±2.3

[0040] Note: Except for the grafting survival rate, the other data are expressed as mean ± standard deviation.

[0041] In addition, the mycorrhizal infection rates of the tea tree roots in the experimental group and the control group were observed by electron microscopy, and it was found that compared with the control group, the mycorrhizal infection rate of the tea tree roots in the experimental group reached more than 85%, significantly promoting nutrient absorption; the lignin deposition rate at the grafting interface of the experimental group was accelerated, and the connection time of the vascular bundles between the rootstock and the scion was shortened by 25% compared with the control group.

[0042] Comparative Example 1

[0043] The difference from Example 1 is only that the types of strains in this comparative example are different. Among them, this comparative example is a single-strain treatment, and the effects of each treatment method on the grafting survival rate and root activity of tea trees are shown in Table 2.

[0044] Table 2

[0045] Treatment method Grafting survival rate Root activity (μg TTF / g / h) Only Funneliformis mosseae (60 g) 73％ 31.2±2.8 Only Claroideoglomus etunicatum (60 g) 70％ 29.7±3.0 Finished product microbial inoculum of Example 1 80％ 35.4±3.4

[0046] Conclusion: The synergistic effect of the mixed microbial inoculum is significantly better than that of the single-strain treatment.

[0047] Comparative Example 2

[0048] The difference from Example 1 is only that the mixing ratio of the strains in this comparative example is different. Among them, the effects of different mixing ratios on the grafting survival rate and root activity of tea trees are shown in Table 3.

[0049] Table 3

[0050] Strain ratio (Funneliformis mosseae: Claroideoglomus etunicatum) Grafting survival rate Root activity (μg TTF / g / h) 1:1 76.6％ 31.2±2.8 2:1 (Example 1) 80％ 35.4±3.4 1:2 73.3％ 38.6±2.4

[0051] Conclusion: The mass ratio of 2:1 is the optimal ratio.

[0052] In summary, the bacterial agent of the present invention improves the survival rate of tea tree grafting through the mycorrhizal symbiotic effect and metabolite regulation. The present invention clarifies the effect of the arbuscular mycorrhizal fungal agent in improving the survival rate of tea tree grafting, providing a theoretical basis and technical support for the large-scale promotion of this bacterial agent.

[0053] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. An arbuscular mycorrhizal fungal inoculant for promoting the survival rate of tea tree grafting, characterized in that, Obtained by inoculating and culturing with Funneliformis mosseae and Claroideoglomus etunicatum, and the mass ratio of Funneliformis mosseae to Claroideoglomus etunicatum is 2:

1.

2. The arbuscular mycorrhizal fungal inoculant according to claim 1, characterized in that The spore content of the arbuscular mycorrhizal fungal inoculant is ≥500 spores / g.

3. A method for preparing the arbuscular mycorrhizal fungal inoculant according to claim 1, characterized in that, It includes the following steps: Mix Funneliformis mosseae and Claroideoglomus etunicatum according to a mass ratio of 2:1, and inoculate them onto the roots of corn seedlings; culture them in a culture medium composed of fine river sand, perlite and biochar, separate the culture medium and the corn roots, cut the roots into pieces and mix them with the culture medium to obtain the original inoculant; inoculate the original inoculant into the propagation substrate for culture, collect the mycelium and spores, and mix them with the carrier to obtain the arbuscular mycorrhizal fungal inoculant.

4. The preparation method according to claim 3, characterized in that, The fine river sand is obtained through sieving, rinsing, sterilization and air-drying treatments.

5. The preparation method according to claim 3, wherein, The culture conditions for inoculating the original inoculant into the propagation substrate include 12000 lux of light per day, culturing at 28 °C for 13 hours, and culturing at 18 °C for 11 hours under dark conditions.

6. The preparation method according to claim 5, wherein During the culturing process, Hoagland nutrient solution is irrigated once every 2 weeks.

7. The preparation method according to claim 3, wherein The carrier is sterilized vermiculite.

8. A method for promoting the survival rate of tea tree grafting, characterized in that, It includes the step of applying the arbuscular mycorrhizal fungal inoculant described in claim 1 to the roots of the tea tree rootstock.

9. The method according to claim 8, characterized in that The grafting method is the cleft grafting method.