An arbuscular mycorrhizal fungal inoculant for promoting the survival rate of tea grafts and its preparation method

Abstract

This invention discloses an arbuscular mycorrhizal fungal agent for promoting the grafting survival rate of tea trees and its preparation method, belonging to the fields of microbial preparations and plant cultivation technology. The arbuscular mycorrhizal fungal agent includes *Funneliformis mosseae* and *Claroideoglomus etunicatum*, with a mass ratio of *Funneliformis mosseae* to *Claroideoglomus etunicatum* of 2:1. Experimental results show that after applying the arbuscular mycorrhizal fungal agent of this invention, the grafting survival rate of tea trees increased from 60% in the control group to 80%. This invention overcomes the limitations of relying on physical methods for tea tree grafting, achieving for the first time the efficient colonization of arbuscular mycorrhizal fungi at the grafting interface, and has significant agricultural application value.

Description

Technical Field

[0001] This invention relates to the fields of microbial preparations and plant cultivation technology, and in particular to an arbuscular mycorrhizal fungal agent that promotes the survival rate of grafted tea trees and its preparation method. Background Technology

[0002] Tea grafting is a core technology for variety improvement, resistance enhancement, and rapid tea garden renewal. Its core challenge lies in the efficient connection of vascular bundles between the scion and rootstock and the coordinated development of callus tissue. Traditional grafting techniques optimize cutting angles, interface wrapping materials, and temperature and humidity control; that is, existing improvement techniques mostly focus on physical optimization. However, these methods fail to address the root cause problem at the level of cellular metabolic regulation.

[0003] Arbuscular mycorrhizal fungi (AMFs), as key mediators of plant-microbe interactions, have been shown to improve soil aggregate structure by secreting glomerulimycin proteins and mediate the transroot transport of nutrients such as phosphorus and zinc through hyphal networks. In non-grafting systems, AMFs can induce host plants to synthesize signaling molecules such as jasmonic acid (JA) and salicylic acid (SA), coordinating the expression of stress-resistance genes. However, existing AMF application research is mostly limited to root inoculation of single crops, and has not yet addressed the colonization needs of microbial communities in the unique microenvironment of the grafting interface. Tea trees, as typical mycorrhizally independent plants, exhibit significantly lower symbiotic efficiency with AMFs compared to legumes or grasses, further limiting the direct application of AMFs in tea cultivation. These limitations have resulted in a long-standing research gap in AMF research in tea grafting, necessitating the development of suitable inoculants and preparation methods to overcome these technical bottlenecks. Summary of the Invention

[0004] The purpose of this invention is to provide an arbuscular mycorrhizal fungal inoculant for promoting the survival rate of tea tree grafts and its preparation method, thereby solving the problems existing in the prior art. The arbuscular mycorrhizal fungal inoculant provided by this invention can promote the survival rate of tea tree grafts, expanding the direct application of AMF in tea tree grafting cultivation.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] Technical Solution 1: An arbuscular mycorrhizal fungal agent for promoting the survival rate of tea grafts, obtained by inoculation and culture of Funneliformis mosseae and Claroideoglomus etunicatum, wherein the mass ratio of Funneliformis mosseae to Claroideoglomus etunicatum is 2:1.

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

[0008] Technical Solution 2: A method for preparing the aforementioned arbuscular mycorrhizal fungal inoculant includes the following steps: mixing *Hymenopterus mossicae* and *Hymenopterus juvenileus* at a mass ratio of 2:1 and inoculating them onto the roots of maize seedlings; culturing them in a culture medium composed of fine river sand, perlite, and biochar; separating the culture medium and maize roots; chopping the roots and mixing them with the culture medium to obtain the original inoculant; inoculating the original inoculant into a propagation substrate for cultivation; collecting the mycelium and spores; and mixing them with a carrier to obtain the aforementioned arbuscular mycorrhizal fungal inoculant.

[0009] Furthermore, the fine river sand is obtained through sieving, washing, sterilization, and air drying.

[0010] Furthermore, the culture conditions for inoculating the original bacterial agent into the propagation substrate include 12,000 lux of light per day, cultured at 28°C for 13 hours, and cultured at 18°C ​​in the dark for 11 hours.

[0011] Furthermore, the Hoagland nutrient solution is applied every two weeks during the cultivation process.

[0012] Furthermore, the carrier is sterilized vermiculite.

[0013] Technical Solution 3: A method for promoting the survival rate of grafted tea trees, comprising the step of applying the aforementioned arbuscular mycorrhizal fungal agent to the roots of the tea tree rootstock.

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

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

[0016] This invention's inoculant, through mycorrhizal symbiosis and metabolite regulation, achieves a synergistic improvement in the grafting survival rate and stress resistance of tea trees. This invention elucidates the effect of arbuscular mycorrhizal fungi inoculants on improving the grafting survival rate of tea trees, providing a theoretical basis and technical support for the large-scale promotion of this inoculant. The arbuscular mycorrhizal fungi inoculant provided by this invention is prepared through propagation on a specific culture medium (fine river sand, perlite, and biochar in a volume ratio of 3:2:1), exhibiting high preparation efficiency. Optimized culture medium ratios and propagation conditions (light / temperature cycle) ensure a spore yield ≥500 spores / g. Experimental results of this invention show that after applying this inoculant, the grafting survival rate of tea trees increased from 60% in the control group to 80%. In summary, this invention overcomes the limitations of relying on physical methods for tea tree grafting, achieving for the first time the efficient colonization of arbuscular mycorrhizal fungi at the grafting interface, and has significant agricultural application value. Detailed Implementation

[0017] 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0019] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0020] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0021] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

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

[0023] The samples of *M. mossiosporium* and *M. jussiosporium* were provided by the Institute of Plant Nutrition and Resources, Beijing Academy of Agricultural and Forestry Sciences.

[0024] Example 1

[0025] Preparation of arbuscular mycorrhizal fungal inoculants:

[0026] (1) Mix Moses tuberculosis and young glomerulosa at a mass ratio of 2:1 (40g of Moses tuberculosis and 20g of young glomerulosa) and inoculate them onto the roots of corn seedlings that have been sown for 2 weeks. The inoculated corn seedlings will grow for 3 months on a culture medium (the volume ratio of fine river sand, perlite and biochar is 3:2:1 and the thickness of the culture medium is 10cm). During the growth process, the seedlings will be watered with Hoagland nutrient solution once every 2 weeks.

[0027] (2) Remove the above-ground parts of the corn, separate the culture medium and the corn roots, cut the corn roots into pieces, and then mix them with the culture medium again to obtain the arbuscular mycorrhizal fungi original inoculum. The original inoculum contains arbuscular mycorrhizal fungi spores, hyphae and culture medium of infected root segments.

[0028] (3) The prepared arbuscular mycorrhizal fungi inoculum was inoculated into the propagation substrate fine river sand at a ratio of 20wt%, and mixed well to obtain a propagation substrate containing the arbuscular mycorrhizal fungi inoculum. Then, the propagation substrate containing the arbuscular mycorrhizal fungi inoculum was evenly spread in a rectangular culture box with a thickness of 20cm, and distilled water was poured to 50% of the field capacity. Corn seeds were sown, and after sowing, the surface was covered with a 1cm layer of fine river sand containing the arbuscular mycorrhizal fungi inoculum. The corn was cultured for 10 weeks, including 13 hours of culture under 12000 lux light and 28℃ temperature, and 11 hours of culture under 18℃ darkness. During the growth process, watering was carried out according to the dryness and wetness conditions, and the substrate moisture content was controlled to 30% of the field capacity. Watering was carried out every 2 days. Hoagland nutrient solution was applied once every 2 weeks until the substrate moisture content reached 30% of the field capacity.

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

[0030] Example 2

[0031] Three-year-old tea seedlings (Yuncha No. 1) were used as rootstocks; Yunkang No. 22 tea cultivar was used as scions. 10g of the prepared microbial agent (prepared in Example 1) was applied to the roots of each rootstock as the experimental group; the control group received no microbial agent. Each group had 30 replicates, randomly arranged in a greenhouse (temperature 25±2℃, humidity 70%). Cleft grafting was used, and the graft union was wrapped with plastic film. Post-grafting management was routine, maintaining soil moisture content at 65%.

[0032] The following indicators were measured respectively:

[0033] (1) Survival rate of the experimental group and the control group: The proportion of plants with complete healing of the graft interface and new shoot growth was counted 60 days after grafting.

[0034] (2) Determine the callus formation rate of the experimental group and the control group: Samples were taken on the 10th, 20th and 30th day after grafting, and the thickness of the callus at the interface was measured (microscopic sectioning method).

[0035] (3) Root activity of the experimental and control groups was measured: TTC method was used to measure root dehydrogenase activity of grafted seedlings;

[0036] (4) chlorophyll content of the experimental and control groups was determined: SPAD value of leaves was determined by acetone extraction method;

[0037] The statistical results of the above measurement indicators are detailed in Table 1.

[0038] Table 1

[0039] Measurement indicators experimental group control group Grafting survival rate 80％ 60％ Callus thickness (30 days) 1.72±0.25mm 1.02±0.22mm Root activity (μgTTF / 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, all other data are expressed as mean ± standard deviation.

[0041] In addition, the mycorrhizal infection rate of tea tree roots in the experimental group and the control group was observed by electron microscopy. It was found that, compared with the control group, the mycorrhizal infection rate of tea tree roots in the experimental group was over 85%, which significantly promoted nutrient absorption. The lignin deposition rate at the grafting interface of the experimental group was accelerated, and the vascular bundle connection time of the rootstock and scion was shortened by 25% compared with the control group.

[0042] Comparative Example 1

[0043] The only difference from Example 1 is the type of microbial strain used in this comparative example. This comparative example uses a single microbial strain treatment, and the effects of each treatment on the survival rate and root vigor of the tea grafts are shown in Table 2.

[0044] Table 2

[0045] Handling method Grafting survival rate Root activity (μgTTF / g / h) Moses tubulosa mold only (60g) 73％ 31.2±2.8 Only juvenile *Glomus hygroscopicus* (60g) 70％ 29.7±3.0 Example 1 Finished Microbial Agent 80％ 35.4±3.4

[0046] Conclusion: The synergistic effect of mixed bacterial agents is significantly better than that of single bacterial strain treatment.

[0047] Comparative Example 2

[0048] The only difference from Example 1 is the different mixing ratio of the microbial strains used in this comparative example. The effects of different mixing ratios on the survival rate and root vigor of the tea grafts are shown in Table 3.

[0049] Table 3

[0050] Microbial strain ratio (Moses: young strain) Grafting survival rate Root activity (μgTTF / 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: A mass ratio of 2:1 is the optimal ratio.

[0052] In summary, the fungal agent of this invention improves the grafting survival rate of tea trees through mycorrhizal symbiosis and metabolite regulation. This invention elucidates the effect of arbuscular mycorrhizal fungal agents in improving the grafting survival rate of tea trees, providing a theoretical basis and technical support for the large-scale promotion of this fungal agent.

[0053] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of an arbuscular mycorrhizal fungal inoculant in improving the grafting survival rate of tea trees, characterized in that, The arbuscular mycorrhizal fungal inoculum was obtained by inoculating and culturing Funneliformis mosseae and Claroideoglomus etunicatum, with a mass ratio of Funneliformis mosseae to Claroideoglomus etunicatum of 2:

1.

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

3. The application according to claim 1, characterized in that, The preparation method of the arbuscular mycorrhizal fungal inoculant includes the following steps: Mixing *M. mossiocarpa* and *M. juba* at a mass ratio of 2:1, and inoculating them onto the roots of maize seedlings; culturing them in a culture medium composed of fine river sand, perlite, and biochar; separating the culture medium from the maize roots; chopping the roots and mixing them with the culture medium to obtain the original inoculum; inoculating the original inoculum into a propagation substrate; collecting the mycelium and spores; and mixing them with a carrier to obtain the arbuscular mycorrhizal fungal inoculum.

4. The application according to claim 3, characterized in that, The fine river sand is obtained through sieving, washing, sterilization and air drying.

5. The application according to claim 3, characterized in that, The culture conditions for inoculating the original bacterial agent into the propagation substrate include 12,000 lux of light per day, cultured at 28°C for 13 hours, and cultured at 18°C ​​in the dark for 11 hours.

6. The application according to claim 5, characterized in that, During the cultivation process, Hoagland nutrient solution is applied once every two weeks.

7. The application according to claim 3, characterized in that, The carrier is sterilized vermiculite.

8. A method for promoting the survival rate of grafted tea trees, characterized in that, This includes the step of applying arbuscular mycorrhizal fungi to the roots of tea tree rootstocks; The arbuscular mycorrhizal fungal inoculum was obtained by inoculating and culturing Funneliformis mosseae and Claroideoglomus etunicatum, with a mass ratio of Funneliformis mosseae to Claroideoglomus etunicatum of 2:

1.

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