Arbuscular mycorrhizal fungus inoculant for improving yield of medicago truncatula in low-phosphorus soil as well as preparation method and application of arbuscular mycorrhizal fungus inoculant

Abstract

The invention discloses an arbuscular mycorrhizal fungus inoculant for improving the yield of medicago truncatula in low-phosphorus soil as well as a preparation method and application of the arbuscular mycorrhizal fungus inoculant. The arbuscular mycorrhizal fungus inoculant comprises a culture medium, spores and ectogenic hyphae of arbuscular mycorrhizal fungi mixed in the culture medium, and a host plant root system infected by the arbuscular mycorrhizal fungi, the culture medium is river sand, and the arbuscular mycorrhizal fungi comprise glomus terrestris, Aureobasidium microconcave, glomus intraradices, glomus mosseae, glomus twistula and glomus macrosporium; the root system of the host plant infected by the arbuscular mycorrhizal fungi is the root system of the corn and alfalfa infected by the arbuscular mycorrhizal fungi. According to the method, different arbuscular mycorrhizal fungi strains such as glomus terrestris, aureoconcave aureoconcave, glomus mosseae, glomus intraradices, glomus twistula and pearl macrosporium are mixed according to a certain proportion, so that the phosphorus obtaining capacity of the host plant in a low-phosphorus soil environment can be effectively enhanced, and the yield of the host plant is further remarkably increased; meanwhile, the arbuscular mycorrhizal fungus inoculant can partially or completely replace phosphate fertilizer according to the soil phosphorus level of a specific pasture planting environment, so that related environmental pollution is reduced, and sustainable agricultural development of environmental ecology is facilitated.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural microbial engineering and plant yield increase, and in particular to an arbuscular mycorrhizal fungal agent for increasing the yield of forage Medicago truncatula in low-phosphorus soil, and a preparation method and application thereof. Background Art

[0002] Due to the uneven distribution and low mobility of inorganic phosphate ions in the soil, phosphorus is an important factor limiting plant growth in many ecosystems, seriously affecting the species richness and productivity of the ecosystem. In order to cope with the problem of phosphorus deficiency in agricultural production, a large amount of phosphorus fertilizer is used, which has caused environmental problems such as eutrophication of water bodies, seriously affecting the natural ecological balance and sustainable development. Therefore, it is urgent to study environmentally friendly means to solve the problem of phosphorus deficiency.

[0003] Plants have two main strategies to cope with low-phosphorus stress environments. One is to evolve root traits for efficient resource acquisition (such as root branching, root hairs, and root rows); the other is to form a symbiosis with mycorrhizal fungi, especially arbuscular mycorrhizal fungi, to obtain phosphorus resources that were previously difficult to utilize.

[0004] Arbuscular mycorrhizal fungi (AMF) are a type of ancient fungi that originated more than 400 million years ago. They can form mycorrhizal symbioses with about 80% of terrestrial plant species. Arbuscular mycorrhizal fungi can absorb water and nutrients (such as nitrogen and phosphorus) that are difficult for the host plant roots to reach through abundant extraradical hyphae, and exchange nutrients with the host plant through the arbuscular structures formed in the cortical cells of the host plant roots, thus forming a mutually beneficial symbiosis with the host plant. Arbuscular mycorrhizal fungi can enhance the phosphorus acquisition capacity of the host plant by expanding the absorption volume of the plant root system, dissolving inorganic phosphorus, and mineralizing organic phosphorus. At the same time, the infection of arbuscular mycorrhizal fungi can open an effective phosphorus acquisition pathway that does not involve direct phosphorus absorption pathways such as the root epidermis and root hairs. In addition, the symbiosis of arbuscular mycorrhizal fungi can activate the expression of a series of phosphorus stress-induced genes, including phosphorus-hydrogen ion-ATP ion pumps, arbuscular mycorrhizal-induced inorganic phosphate ion transporters, and acid phosphatases, which together enhance the phosphorus acquisition capacity of the host plant roots.

[0005] However, the current single-species arbuscular mycorrhizal fungal agents have little effect on improving the phosphorus acquisition ability of host plants in low-phosphorus environments, and cannot significantly increase the yield of host plants in low-phosphorus environments. Summary of the invention

[0006] The invention provides an arbuscular mycorrhizal fungal agent for improving the yield of forage Medicago truncatula in low-phosphorus soil, and a preparation method and application thereof, so as to solve the above problems.

[0007] In order to achieve the above object, the technical solution of the present invention is:

[0008] The present invention provides an arbuscular mycorrhizal fungal agent for increasing the yield of forage Medicago truncatula in low-phosphorus soil, comprising a culture matrix, spores and exophytic hyphae of arbuscular mycorrhizal fungi mixed in the culture matrix, and a host plant root system infected by the arbuscular mycorrhizal fungi;

[0009] Wherein, the culture medium is river sand, and the arbuscular mycorrhizal fungi include Geospora, Sessile Spores, Glomus in the roots, Glomus mosseae, Glomus twisted and Giant Sporangium pearlis;

[0010] The host plant root systems infected by the arbuscular mycorrhizal fungi are the root systems of corn and alfalfa infected by the arbuscular mycorrhizal fungi.

[0011] Furthermore, the spore count ratio of Geospora spp., Sessile Spores, Glomus radicifolia, Glomus mosseae, Glomus twisted, and Giant Sporangium pearlii in the arbuscular mycorrhizal fungal agent is 5:1:1:1:1:1:1;

[0012] The ratio of the number of arbuscular mycorrhizal fungi spores in the arbuscular mycorrhizal fungi agent to the target soil mass is: 200 arbuscular mycorrhizal fungi spores / kg target soil;

[0013] The amount of fungi contained in the arbuscular mycorrhizal fungal agent is: 988 spores / 100g of agent.

[0014] Another aspect of the present invention provides a method for preparing the arbuscular mycorrhizal fungal agent for increasing the yield of Medicago truncatula in low-phosphorus soil, comprising the following steps:

[0015] The single pure cultured arbuscular mycorrhizal fungi species is uniformly mixed with river sand, a culture medium, to obtain a mixture, which is placed in a turnover box, and corn seeds and alfalfa seeds whose surfaces have been disinfected are planted in the mixture in the turnover box as host plants, and the mixture is cultured in a greenhouse under light, watered daily, and supplemented with nutrient solution every 7 days. After culturing in the greenhouse for 5 months, watering and nutrient solution are stopped, and after the host plants die naturally, the above-ground parts of the host plants alfalfa and corn are cut off, and the underground parts of the host plants are shredded, which are fully and uniformly mixed with river sand to obtain an arbuscular mycorrhizal fungal agent for increasing the yield of forage Medicago truncatula in low-phosphorus soil.

[0016] Furthermore, the mass ratio of the single pure cultured arbuscular mycorrhizal fungus species to the river sand is 1:19.

[0017] Furthermore, the quantity ratio of the corn seeds to the alfalfa seeds is 1:6.

[0018] Furthermore, the nutrient solution is Hoagland's nutrient solution, and the volume-mass ratio of the nutrient solution added each time to the mixed material is 1L:20kg.

[0019] Furthermore, the light intensity of the culture in the warm net room is 10000-15000Lx, and the light cycle is 13h light / 11h dark; the environmental temperature of the culture is 17-30°C; and the environmental humidity of the culture is 50%-70%.

[0020] In another aspect, the present invention provides the arbuscular mycorrhizal fungal agent for increasing the yield of forage Medicago truncatula in low-phosphorus soil or the use of the arbuscular mycorrhizal fungal agent prepared by the preparation method in increasing the yield of forage Medicago truncatula in low-phosphorus soil.

[0021] Furthermore, the spore number ratio of Geospora, Sessile Spore, Glomus intraradica, Glomus mosseae, Glomus twisting, and Giant Sporangium pearlis in the arbuscular mycorrhizal fungal agent is 5:1:1:1:1:1:1; and the ratio of the dosage of the applied arbuscular mycorrhizal fungi and the target soil mass is: 200 arbuscular mycorrhizal fungal spores / kg target soil.

[0022] The beneficial effects of the present invention are:

[0023] The invention discloses an arbuscular mycorrhizal fungal agent for improving the yield of forage Medicago truncatula in low-phosphorus soil. Different species of arbuscular mycorrhizal fungal species, such as Geospora, Sessile Mycorrhiza, Glomus radicifolia, Glomus mosesi, Glomus twisted and Giant Sporangium pearlii, are mixed to improve the promoting effect of the arbuscular mycorrhizal fungal agent on the phosphorus acquisition ability of the host plant in the low-phosphorus environment. Meanwhile, different arbuscular mycorrhizal fungal species are mixed in a certain proportion to further effectively enhance the phosphorus acquisition ability of the host plant in the low-phosphorus soil environment, thereby significantly improving the yield of the host plant. Meanwhile, the arbuscular mycorrhizal fungal agent can also partially or completely replace phosphorus fertilizer according to the soil phosphorus level of the specific forage planting environment, thereby reducing related environmental pollution and contributing to the environmental ecological sustainable agricultural development. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 The results of the underground biomass (Root), aboveground biomass (Shoot) and fruit biomass (Fruit) of the host plant under three different soil treatments in Example 2 of the present invention are shown in FIG.

[0026] Figure 2This is a graph showing the results of biomass phosphorus of host plants under three different soil treatments in Example 2 of the present invention. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Embodiment 1:

[0029] An arbuscular mycorrhizal fungal agent for improving the yield of forage Medicago truncatula in low-phosphorus soil, comprising a culture matrix, spores and exophytic hyphae of arbuscular mycorrhizal fungi mixed in the culture matrix, and a host plant root system infected by the arbuscular mycorrhizal fungi;

[0030] Wherein, the culture medium is river sand, and the arbuscular mycorrhizal fungi include Funneliformis geosporum (Fg, numbered BGC GZ01), Acaulospora scrobiculata (As, numbered BGC HK02A), Rhizophagus intraradices (Ri, numbered BGC BJ09), Funneliformis mosseae (Fmo, numbered BGC XJ01), Glomus tortuosum (Gt, numbered BGC NM03A) ​​and Gigaspora margarita (Gma, numbered BGC ZJ03);

[0031] The host plant root system infected by the arbuscular mycorrhizal fungi is the root system of corn and alfalfa infected by the arbuscular mycorrhizal fungi;

[0032] The spore number ratio of each arbuscular mycorrhizal fungus species in the arbuscular mycorrhizal fungus agent is Fg:As:Ri:Fmo:Gt:Gma=5:1:1:1:1:1;

[0033] When the arbuscular mycorrhizal fungi agent is applied, the ratio of the number of arbuscular mycorrhizal fungi spores in the arbuscular mycorrhizal fungi agent to the mass of the target soil is: 200 arbuscular mycorrhizal fungi spores / kg target soil;

[0034] The amount of fungi contained in the arbuscular mycorrhizal fungal agent is: 988 spores / 100g of agent.

[0035] 2) Source of strains:

[0036] The above-mentioned arbuscular mycorrhizal fungi species all come from the "Chinese Arbuscular Mycorrhizal Fungi Resource Bank" (Institute of Plant Nutrition and Resources, Beijing Academy of Agriculture and Forestry Sciences), and are then obtained through standard strain propagation and cultivation.

[0037] 3) Strain propagation process:

[0038] First, 1kg of a single pure culture of arbuscular mycorrhizal fungi was evenly mixed with 19kg of sterilized river sand and placed in a 40L turnover box. Then, 5 corn seeds and 30 alfalfa seeds with surface disinfection were planted in each turnover box as host plants. The culture conditions in the warm net room were as follows: the light intensity was 10000-15000Lx, the light cycle was 13h light / 11h dark; the ambient temperature was 17-30℃; the ambient humidity was 50%-70%, and watering was carried out appropriately every day. At the same time, 1L Hogland's nutrient solution was added to each turnover box every 7 days. After 5 months of cultivation in the warm net room, watering and nutrient solution were stopped. After the host plants died naturally, the above-ground parts of the host plants alfalfa and corn were cut off, and the underground parts of the host plants were obtained and cut into pieces. After being fully mixed with the river sand, the arbuscular mycorrhizal fungi agent was formed.

[0039] Embodiment 2:

[0040] By artificial controlled experiments, the yield of Medicago truncatula and the phosphorus content of the plants were measured in a low-phosphorus soil to verify the effect of the arbuscular mycorrhizal fungal agent of the present invention on increasing the yield of Medicago truncatula in low-phosphorus soil (conducted in a hot spring room of Zhejiang University (120°19′E, 30°26′N)):

[0041] Materials: The original low-phosphorus soil was obtained from the low-phosphorus paddy field soil of the nutrient long-term positioning test site of the Changxing Agricultural Experiment Station of Zhejiang University (119°91′E, 31°02′N). The basic physical and chemical properties of the soil are as follows: Total soil phosphorus 236.75 mg kg -1 , total nitrogen 1323.75 mg kg -1 , soil available phosphorus (Olsen-P) 5.7 mg kg -1 , organic matter 12.4 g kg -1 , pH value 6.9 (soil:water=1:5).

[0042] The specific steps are:

[0043] (1) The seeds of Medicago truncatula were immersed in 98% concentrated sulfuric acid for 7 minutes, then rinsed with sterile water for 3 times, immersed in 5% sodium hypochlorite solution for 30 seconds, and then rinsed with sterile water for 3 times; finally, the treated seeds were placed in a culture dish containing a double layer of filter paper moistened with sterile water, placed in a dark environment, maintained at an ambient temperature of 20°C, and germinated for 18 hours.

[0044] (2) The arbuscular mycorrhizal fungal agent prepared in Example 1 was inoculated into a 3L pot containing 2kg of original low-phosphorus soil that had been sterilized twice with 121°C high-pressure steam for 30 min at an amount of 400 spores per pot, and 50ml of the original low-phosphorus soil filtrate (sieve size 20μm) was added. The pot was then left to stand at room temperature for 2 weeks to allow the microbial community in the soil other than arbuscular mycorrhizal fungi to recover on its own. This was the arbuscular mycorrhizal fungi mixed inoculation treatment (Mix), with a total of 5 pots.

[0045] At the same time, 3L pots each containing 2kg of original low-phosphorus soil sterilized twice with 121℃ high-pressure steam for 30min were used. These pots were not inoculated with arbuscular mycorrhizal fungi, but 50ml of mixed spore filtrate (sieve size 20μm) was added to maintain the initial soil nutrient state consistent with the arbuscular mycorrhizal fungi mixed inoculation treatment (Mix). At the same time, 50ml of original low-phosphorus soil filtrate (sieve size 20μm) was added, and then left to stand at room temperature for 2 weeks to restore the microbial community in the soil except arbuscular mycorrhizal fungi. This is the blank control treatment without arbuscular mycorrhizal fungi (NAMF), with a total of 5 pots.

[0046] At the same time, 3 L pots, each containing 2 kg of natural low-phosphorus paddy field soil and not inoculated with arbuscular mycorrhizal fungi (no treatment), were used as the original low-phosphorus soil control treatment (OHS), for a total of 5 pots.

[0047] (3) In each of the above treatments, 5 Medicago truncatula seeds were evenly sown 2 cm from the soil surface after germination for 18 hours. The pots were then placed in a warm net room with a light intensity of 10,000 to 15,000 Lx, a photoperiod of 13 h light / 11 h dark, an ambient temperature of 17 to 30°C, and an ambient humidity of 50% to 70%. At the same time, the soil was watered appropriately according to the plant's water requirement every day to keep the soil moist.

[0048] (4) Four weeks after sowing, the Medicago truncatula seeds were thinned to one plant in each test pot. By the time of sample harvest, all Medicago truncatula survived.

[0049] (5) After about 4 months of cultivation, the aboveground parts of the host plants under the three different soil treatments were cut off from the soil surface, weighed, and sterilized at 105°C for 30 minutes, then dried at 65°C until constant weight, and weighed again. The fruits were air-dried and weighed. The roots of the host plants were washed, air-dried, and weighed. The results of the underground biomass (Root), aboveground biomass (Shoot), and fruit biomass (Fruit) of the host plants under the three different soil treatments are shown in Figure 2. Figure 1The values ​​in the figure are the means of each treatment (±SE, n=5). In each biomass (ordinate) index, the same letters in the figure represent no significant difference in the multiple comparison test (P>0.05); NAMF: blank control without AMF; Mix: mixed inoculation with AMF; OHS: original low-phosphorus soil control; Abbreviations: AMF: arbuscular mycorrhizal fungi; SE: standard error; the ordinate Biomass is biomass (g·pot -1 );

[0050] Depend on Figure 1 It can be seen that compared with the treatment without arbuscular mycorrhizal fungi and the original low-phosphorus soil, the inoculation of arbuscular mycorrhizal fungi can significantly increase the aboveground biomass (forage yield) and underground biomass of Medicago truncatula in low-phosphorus soil. The aboveground biomass (forage yield) of the inoculated arbuscular mycorrhizal fungi was 3.18 g·pot -1 , the underground biomass is 0.836 g·pot -1 The aboveground biomass of the treatment without inoculation of arbuscular mycorrhizal fungi increased by about 108 times, and the aboveground biomass of the treatment with original low-phosphorus soil increased by about 0.66 times; the underground biomass of the treatment without inoculation of arbuscular mycorrhizal fungi increased by about 34.88 times, and the underground biomass of the treatment with original low-phosphorus soil increased by about 0.96 times.

[0051] The total phosphorus of the host plants under the three different soil treatments was extracted by concentrated sulfuric acid-hydrogen peroxide method, and then the biomass phosphorus of the host plants under the three different soil treatments was determined by flow analyzer (ICP-OES; Optima 3300DV, PerkinElmer). The results are shown in Figure 2. Figure 2 The values ​​in the figure are the means of each treatment (±SE, n=5); the same letters in the figure represent no significant difference in the multiple comparison test (P>0.05); NAMF: blank control without AMF; Mix: mixed inoculation with AMF; OHS: original low-phosphorus soil control; abbreviations: AMF: arbuscular mycorrhizal fungi; SE: standard error; the ordinate Biomass P is biomass phosphorus (mg·pot -1 ).

[0052] Depend on Figure 2 It can be seen that the phosphorus content of Medicago truncatula biomass treated with this arbuscular mycorrhizal fungus agent was 5.64 mg·pot -1 It was about 183 times higher than the treatment without inoculation of arbuscular mycorrhizal fungi, and about 0.92 times higher than the treatment of original low-phosphorus soil, indicating that inoculation of this arbuscular mycorrhizal fungi can significantly increase the phosphorus content of Medicago truncatula plants in low-phosphorus soil.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An arbuscular mycorrhizal fungal agent for increasing the yield of Medicago truncatula in low-phosphorus soil, characterized in that: The method comprises a culture matrix, spores and exophytic hyphae of arbuscular mycorrhizal fungi mixed in the culture matrix, and a host plant root system infected by the arbuscular mycorrhizal fungi; Wherein, the culture medium is river sand, and the arbuscular mycorrhizal fungi include Geospora, Sessile Spores, Glomus in the roots, Glomus mosseae, Glomus twisted and Giant Sporangium pearlis; The host plant root systems infected by the arbuscular mycorrhizal fungi are the root systems of corn and alfalfa infected by the arbuscular mycorrhizal fungi.

2. The arbuscular mycorrhizal fungal agent for increasing the yield of Medicago truncatula in low-phosphorus soil according to claim 1, characterized in that: The spore count ratio of Geospora spp., Sessile Spores, Glomus radicifolia, Glomus mosseae, Glomus twisting, and Giant Sporangium pearlii in the arbuscular mycorrhizal fungal agent is 5:1:1:1:1:1:1; The ratio of the number of arbuscular mycorrhizal fungi spores in the arbuscular mycorrhizal fungi agent to the target soil mass is: 200 arbuscular mycorrhizal fungi spores / kg target soil; The amount of fungi contained in the arbuscular mycorrhizal fungal agent is: 988 spores / 100g of agent.

3. A method for preparing the arbuscular mycorrhizal fungal agent for increasing the yield of Medicago truncatula in low-phosphorus soil according to claim 1, characterized in that: The following steps are involved: The single pure cultured arbuscular mycorrhizal fungi species is uniformly mixed with river sand, a culture medium, to obtain a mixture, which is placed in a turnover box, and corn seeds and alfalfa seeds whose surfaces have been disinfected are planted in the mixture in the turnover box as host plants, and the mixture is cultured in a greenhouse under light, watered daily, and supplemented with nutrient solution every 7 days. After culturing in the greenhouse for 5 months, watering and nutrient solution are stopped, and after the host plants die naturally, the above-ground parts of the host plants alfalfa and corn are cut off, and the underground parts of the host plants are shredded, which are fully and uniformly mixed with river sand to obtain an arbuscular mycorrhizal fungal agent for increasing the yield of forage Medicago truncatula in low-phosphorus soil.

4. The preparation method according to claim 3, characterized in that: The mass ratio of the single pure cultured arbuscular mycorrhizal fungus species to the river sand is 1:

19.

5. The preparation method according to claim 3, characterized in that: The quantity ratio of the corn seeds to the alfalfa seeds is 1:

6.

6. The preparation method according to claim 3, characterized in that: The nutrient solution is Hoagland's nutrient solution, and the volume mass ratio of the nutrient solution added each time to the mixed material is 1L:20kg.

7. The preparation method according to claim 3, characterized in that: The light intensity of the culture in the warm net room is 10000-15000Lx, and the light cycle is 13h light / 11h dark; the environmental temperature of the culture is 17-30°C; and the environmental humidity of the culture is 50%-70%.

8. Use of the arbuscular mycorrhizal fungal agent for increasing the yield of Medicago truncatula in low-phosphorus soil as claimed in any one of claims 1 to 2 or the arbuscular mycorrhizal fungal agent prepared by the preparation method as claimed in any one of claims 3 to 7 in increasing the yield of Medicago truncatula in low-phosphorus soil.

9. The use according to claim 8, characterized in that: The spore number ratio of Geospora, Sessile Spore, Glomus intraradica, Glomus mosesi, Glomus twisting, and Giant Sporangium pearlis in the arbuscular mycorrhizal fungal agent is 5:1:1:1:1:1:1; and the ratio of the dosage of the applied arbuscular mycorrhizal fungi and the target soil mass is: 200 arbuscular mycorrhizal fungal spores / kg target soil.

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