Application of Gibberellic Acid-Deficient Mutants in Promoting Spore Production of Arbuscular Mycorrhizal Fungi

Abstract

The present invention discloses the use of a gibberellin-deficient mutant in promoting spore production in arbuscular mycorrhizal fungi. The present invention utilizes the hairy roots of the gibberellin-deficient mutant as a host plant to inoculate the arbuscular mycorrhizal fungi, significantly increasing the spore production of the arbuscular mycorrhizal fungi. The hairy roots of the gibberellin-deficient mutant can significantly increase the spore production of the arbuscular mycorrhizal fungi under both normal pH conditions and acidic conditions. The gibberellin-deficient mutant can be widely used to promote spore production in arbuscular mycorrhizal fungi. The present invention also provides a preparation and method for promoting spore production in arbuscular mycorrhizal fungi. The preparation and method can propagate the arbuscular mycorrhizal fungi under sterile conditions with high propagation efficiency, significantly increase the spore production of the arbuscular mycorrhizal fungi, are simple to operate, low in cost, and have a short cycle time, thus having broad prospects for promotion and application.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and more specifically relates to the application of a gibberellin-deficient mutant in promoting spore production of arbuscular mycorrhizal fungi. Background Art

[0002] AM fungi (arbuscular mycorrhiza, AM) are ubiquitous symbiotic fungi in the soil, belonging to the class Glomeromycetes. AM fungi can establish symbiotic relationships with more than 80% of terrestrial plants. AM fungi absorb nutrients from the soil through their exophytic hyphae and transfer them to the plants, obtaining carbon sources from the host plants to support their own growth, development, and reproduction. This mutually beneficial symbiotic relationship can increase the tolerance of host crops to many biotic and abiotic stress conditions and improve the growth status of host crops. This in turn increases crop yield and improves crop quality. Therefore, arbuscular mycorrhizal fungi have great application potential in agricultural production.

[0003] There are two main methods for propagating spores of arbuscular mycorrhizal fungi. One is to use a combination of sorghum and clover (or other plant combinations) as host plants, propagating them in a matrix containing a certain ratio of soil, river sand, and diatomaceous earth. This method produces an inoculum with soil as a carrier. The other method involves establishing a dual culture system of arbuscular mycorrhizal fungi and hairy roots (such as carrots and tomatoes) in a petri dish, propagating spores under sterile conditions. Because the spores obtained from the dual culture system are sterile, they are widely used in scientific research and production. Spores are the reproductive bodies of arbuscular mycorrhizal fungi, and the number of spores is an important indicator of inoculum quality. Therefore, improving spore production is a key factor in arbuscular mycorrhizal fungi propagation technology and an important way to effectively improve inoculum quality.

[0004] AM fungi and host plants need to exchange signals continuously to achieve mutualistic symbiosis. Plant hormones, as important signaling molecules, have been shown to play an important role in regulating the symbiosis between plants and AM, which in turn affects spore formation. Exogenous abscisic acid can promote the infection of AM fungi on the root system and the development of arbuscules. The patent CN111073842A previously applied for by the inventors shows that exogenous addition of ABA can promote the production of spores. Arbuscules are the core structure of AM fungi that promote growth. The formation of arbuscules is severely inhibited or promoted, which is related to the significant reduction or increase in lipids provided to AM fungi by the host plant. Spores are fungal structures that store lipids. Plant hormones may play a role in spore formation by affecting the development of arbuscules.

[0005] Therefore, it is of great significance to use plant hormone mutants to study a spore propagation method for arbuscular mycorrhizal fungi that can be propagated under sterile conditions, improve propagation efficiency, be simple to operate, and significantly increase spore production. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an application of a gibberellin-deficient mutant in promoting spore production of arbuscular mycorrhizal fungi.

[0007] The purpose of the present invention is to provide application of a gibberellin-deficient mutant in promoting spore production of arbuscular mycorrhizal fungi.

[0008] Another object of the present invention is to provide the use of a gibberellin-deficient mutant in the preparation of a preparation for promoting spore production of arbuscular mycorrhizal fungi.

[0009] Another object of the present invention is to provide a preparation for promoting spore production of arbuscular mycorrhizal fungi.

[0010] Another object of the present invention is to provide a method for promoting spore production of arbuscular mycorrhizal fungi.

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0012] Through creative exploratory experiments, the present invention significantly increases the production of arbuscular mycorrhizal fungi spores by co-culturing the hairy roots of a tomato gibberellin-deficient mutant with arbuscular mycorrhizal fungi spores. Whether under normal pH environmental conditions (pH 6.5) or under acidic stress conditions of pH 4.5, the spore production of the hairy roots of the tomato gibberellin-deficient mutant after symbiosis with the fungus DAOM197198 was significantly higher than that of the wild type. Furthermore, compared with the wild type, the arbuscular mycorrhizal fungi spore production of the hairy roots of the tomato gibberellin-deficient mutant under acidic stress conditions was 3.4 times that of the wild type under normal pH conditions. The hairy roots of the tomato gibberellin-deficient mutant can significantly increase the spore production of arbuscular mycorrhizal fungi in a dual culture system. Therefore, the following applications should all be within the scope of protection of the present invention:

[0013] Application of gibberellin-deficient mutants in promoting spore production in arbuscular mycorrhizal fungi.

[0014] Application of gibberellin-deficient mutants in the preparation of preparations for promoting spore production of arbuscular mycorrhizal fungi.

[0015] Preferably, the arbuscular mycorrhizal fungus is Rhizophagus irregularis.

[0016] More preferably, the Rhizospora heteromorpha is Rhizospora heteromorpha DAOM197198.

[0017] Based on the above applications, the present invention provides a preparation for promoting spore production of arbuscular mycorrhizal fungi, wherein the preparation comprises hairy roots of a gibberellin-deficient mutant.

[0018] The invention also provides a method for promoting spore production of arbuscular mycorrhizal fungi, comprising mixing the hairy roots of a gibberellin-deficient mutant with the arbuscular mycorrhizal fungi.

[0019] Preferably, the method comprises: using the hairy roots of a gibberellin-deficient mutant as a host plant, inoculating arbuscular mycorrhizal fungi, and culturing in a dual culture system.

[0020] Preferably, the arbuscular mycorrhizal fungus is Heterosporangium.

[0021] More preferably, the Rhizospora heteromorpha is Rhizospora heteromorpha DAOM197198.

[0022] Preferably, the gibberellin-deficient mutant is a tomato gibberellin-deficient mutant.

[0023] More preferably, the tomato gibberellin-deficient mutant is the tomato gibberellin-deficient mutant gib3.

[0024] The present invention has the following beneficial effects:

[0025] The present invention provides the use of a gibberellin-deficient mutant for promoting spore production in arbuscular mycorrhizal fungi. The present invention utilizes a dual culture of hairy roots of the gibberellin-deficient mutant with spores of the arbuscular mycorrhizal fungi, significantly increasing spore production. Furthermore, the hairy roots of the gibberellin-deficient mutant significantly promote spore production in the arbuscular mycorrhizal fungi compared to the wild type, both under acidic stress (pH 4.5) and normal pH conditions (pH 6.5).

[0026] The present invention also provides a preparation and method for promoting the spore production of arbuscular mycorrhizal fungi, which can propagate arbuscular mycorrhizal fungi under sterile conditions with high propagation efficiency, and can significantly increase the spore production of arbuscular mycorrhizal fungi. The operation is simple, the cost is low, and the cycle is short. Therefore, the gibberellin-deficient mutant has broad application prospects in promoting the spore production of arbuscular mycorrhizal fungi or preparing preparations that promote the spore production of arbuscular mycorrhizal fungi. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The figures are dual culture systems of the hairy roots of the gibberellin-deficient tomato mutant gib3 and the hairy roots of the wild-type tomato 'Moneymaker' with arbuscular mycorrhizal fungi in Examples 1 and 2; wherein, Figure (A) shows a dual culture system of the hairy roots of the gibberellin-deficient tomato mutant gib3 and the heteromorphic rhizocystis fungus DAOM197198; and Figure (B) shows a dual culture system of the hairy roots of the wild-type tomato 'Moneymaker' and the heteromorphic rhizocystis fungus DAOM197198.

[0028] Figure 2This is a graph showing the number of spores in each culture dish determined using the grid drawing method in Examples 1 and 2.

[0029] Figure 3 Figures 1 and 2 show the spore production in the dual culture system of tomato hairy roots and arbuscular mycorrhizal fungi; Figure (A) shows the spore production in the dual culture system of hairy roots of wild-type tomato 'Moneymaker' and heteromorphic rhizocystis DAOM197198 under normal pH conditions; Figure (B) shows the spore production in the dual culture system of hairy roots of tomato gibberellin-deficient mutant gib3 and heteromorphic rhizocystis DAOM197198 under acidic stress conditions. DETAILED DESCRIPTION

[0030] The present invention is further described below with reference to specific examples, which, however, are not intended to limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.

[0031] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0032] Example 1 Hairy roots of tomato gibberellin-deficient mutants promote spore production of arbuscular mycorrhizal fungi under normal pH conditions

[0033] This example uses the commonly used arbuscular mycorrhizal fungus species Heterosporangium heterorhizogenes DAOM197198 to study the effect of the hairy roots of a tomato gibberellin-deficient mutant on the spore production of arbuscular mycorrhizal fungi under normal pH conditions (pH 6.5). The specific experimental methods and results are as follows:

[0034] 1. Experimental methods

[0035] In this example, Agrobacterium rhizogenes ACCC10060 was used to transform the hairy roots of the gibberellin-deficient tomato mutant gib3 and the hairy roots of the wild-type tomato 'Moneymaker'.

[0036] The culture dishes used were sterile plastic culture dishes with a diameter of 15 cm.

[0037] The culture medium used was MSR culture medium, and the formula was (addition amount in 1 L): MgSO4·7H2O: 739 mg; KNO3: 76 mg; KCl: 65 mg; Ca(NO3)2·4H2O: 359 mg; KH2PO4: 4.1 mg; MnSO4·4H2O: 2.45 mg; ZnSO4·7H2O: 0.28 mg; H3BO3: 1.85 mg; CuSO4·5H2O: 0.22 mg; (NH4)6Mo7O 244H2O: 0.034mg; NaMoO4·2H2O: 0.0024mg; Thiamine hydrochloride (VB1): 1mg; Pyridoxine hydrochloride (VB6): 0.9mg; Niacin: 1mg; Calcium pantothenate: 0.9mg; Vitamin B 12 : 0.4mg; Biotin: 0.0009mg; NaFeEDTA: 8mg.

[0038] (1) Prepare MSR medium according to the above recipe, add 10 g / L sucrose, adjust the pH of the MSR medium to 7.0 with 1 M NaOH solution, and dispense into 1 L conical flasks, with 500 mL in each flask. Add 2.5 g / L plant gel to each conical flask, seal the flask, and sterilize it at 121°C for 15 min. Pour the mixture into a 15 cm culture dish on a clean bench and allow to cool and solidify before use.

[0039] (2) Half of the culture of Heteromorpha rhizocystis DAOM197198 on the spore culture medium, which had been propagated for 3 months in the hairy root-arbuscular mycorrhizal fungus dual culture system, was transferred to a 50 mL centrifuge tube, and 3 volumes of 10 mM sodium citrate solution (pH 6.0) were added. The tube was shaken on a shaker (shaking speed of 120 rpm, temperature of 25°C) for 1 h, and the solution was filtered with a 25 μm nylon membrane to obtain the reserved spores.

[0040] (3) Use a dissecting needle to disperse the spores under a stereomicroscope and inoculate them into MSR culture medium, with about 100 spores inoculated into each culture medium.

[0041] (4) After 5 days of culture, five 3 cm long hairy roots of the gibberellin-deficient mutant gib3 of tomato (gib3 group) were inoculated at a distance of 0.5 cm from the spores; hairy roots of the wild-type tomato 'Moneymaker' (MM group) were inoculated as a control treatment, with 7 replicates for each treatment.

[0042] (5) Seal the dish with sealing film and place it in a constant temperature incubator for dark culture (culture temperature is 25℃) for 12 weeks. Then take out the dish and draw a grid on the bottom of the dish (such as Figure 2 ). Place the culture dish upside down under a stereomicroscope and count the number of spores in each grid on each culture dish and add them up to calculate the number of spores in each culture dish.

[0043] 2. Experimental results

[0044] In Example 1, the dual culture system of hairy roots of the gibberellin-deficient tomato mutant gib3 and the hairy roots of the wild-type tomato 'Moneymaker' and arbuscular mycorrhizal fungi was as follows: Figure 1As shown in the figure, the hairy roots of wild-type tomato 'Moneymaker' produce spores in the dual culture system with Heteromorpha rhizosporium DAOM197198 under normal pH conditions. Figure 3 As shown in Figure (A), the effects of the hairy roots of a tomato gibberellin-deficient mutant on the spore production of arbuscular mycorrhizal fungi are shown in Table 1. It can be seen that under normal pH conditions, the spore production of the tomato gibberellin-deficient mutant group was significantly higher than that of the wild-type group (P=0.025). This indicates that the hairy roots of the tomato gibberellin-deficient mutant can significantly increase the spore production of arbuscular mycorrhizal fungi in the dual culture system.

[0045] Table 1 Effects of hairy roots of tomato gibberellin-deficient mutants on spore production of arbuscular mycorrhizal fungi under normal pH conditions

[0046] Treatment group Spore count gib3 6769±655* MM 4221±501

[0047] Note: “gib3” and “MM” represent the tomato gibberellin-deficient mutant and its wild type ‘Moneymaker’, respectively. “*” indicates significant differences at the P ≤ 0.05 level in T-test.

[0048] Example 2 Hairy roots of tomato gibberellin-deficient mutants promote spore production of arbuscular mycorrhizal fungi under acidic pH conditions

[0049] 1. Experimental methods

[0050] The hairy roots (gib3 group, MM group), culture dishes, and culture medium used in this example were the same as those in Example 1.

[0051] (1) Prepare MSR medium according to the above formula, add 10 g / L sucrose, adjust the pH of the solution to 4.5, and dispense into 1 L conical flasks, 500 mL per bottle; add 3.0 g / L plant gel to each conical flask, seal the flask, and sterilize it at 121 °C for 15 min; pour it into a 15 cm culture dish on a clean bench, cool and solidify it, and then set aside.

[0052] (2) Half of the culture of Heteromorpha rhizocystis DAOM197198 on the spore culture medium, which had been propagated for 3 months in the hairy root-arbuscular mycorrhizal fungus dual culture system, was transferred to a 50 mL centrifuge tube, and 3 volumes of 10 mM sodium citrate solution (pH 6.0) were added. The tube was shaken on a shaker (shaking speed of 120 rpm, temperature of 25°C) for 1 h, and the solution was filtered with a 25 μm nylon membrane to obtain the reserved spores.

[0053] (3) Use a dissecting needle to disperse the spores under a stereomicroscope and inoculate them into MSR culture medium, with about 100 spores inoculated into each culture medium.

[0054] (4) After 5 days of culture, five 3 cm long hairy roots of the gibberellin-deficient tomato mutant gib3 (gib3 group) were inoculated at a distance of 0.5 cm from the spores. Hairy roots of the wild-type tomato 'Moneymaker' (MM group) were inoculated as a control treatment. Each treatment was replicated 7 times.

[0055] (5) Seal the dish with sealing film and place it in a constant temperature incubator for dark culture (culture temperature is 25℃). After 8 weeks, take out the dish and draw a grid on the bottom of the dish (such as Figure 2 ). Place the culture dish upside down under a stereomicroscope and count the number of spores in each grid on each culture dish and add them up to calculate the number of spores in each culture dish.

[0056] 2. Experimental results

[0057] In Example 2, the dual culture system of hairy roots of the gibberellin-deficient tomato mutant gib3 and the hairy roots of the wild-type tomato 'Moneymaker' and arbuscular mycorrhizal fungi was as follows: Figure 1 As shown in Table 2, the effect of the hairy roots of the tomato gibberellin-deficient mutant on the spore production of arbuscular mycorrhizal fungi under acidic stress conditions (pH 4.5) is shown. It can be seen that under acidic stress conditions, the spore production of the tomato gibberellin-deficient mutant treatment group was significantly higher than that of the wild-type treatment group (P=0.014). The average number of spores in each culture dish of the gib3 group was as high as 14,334, more than twice that of the wild type and 3.4 times the number of spores in the wild-type treatment group in Example 1.

[0058] In Example 2, the hairy roots of the gibberellin-deficient tomato mutant gib3 were cultured in a dual culture system with Rhizospora heteromorpha DAOM197198 under acidic stress conditions. Figure 3 As shown in Figure (B), compared with the wild type, the spore production of Heterosporium heterorhizogenes DAOM197198 in the hairy root-arbuscular mycorrhizal fungus dual culture system of the tomato gibberellin-deficient mutant was greatly increased, and a large number of new spores were produced.

[0059] Table 2 Effects of hairy roots of tomato gibberellin-deficient mutants on spore production of arbuscular mycorrhizal fungi under acidic stress conditions

[0060] Treatment group Spore production gib3 14334±950* MM 6331±2217

[0061] Note: “gib3” and “MM” represent the tomato gibberellin-deficient mutant and its wild type ‘Moneymaker’, respectively. “*” indicates significant differences at the P ≤ 0.05 level in T-test.

[0062] The above embodiments are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Application of gibberellin-deficient mutants in promoting spore production in arbuscular mycorrhizal fungi; The gibberellin-deficient mutant is a tomato gibberellin-deficient mutant gib3; The arbuscular mycorrhizal fungus is Heterosporangium heterorhizogenes.

2. Application of gibberellin-deficient mutants in the preparation of preparations for promoting spore production of arbuscular mycorrhizal fungi; The gibberellin-deficient mutant is a tomato gibberellin-deficient mutant gib3; The arbuscular mycorrhizal fungus is Heterosporangium heterorhizogenes.

3. The use according to claim 1 or 2, characterized in that The heteromorphic Rhizospora is heteromorphic Rhizospora DAOM197198.

4. A preparation for promoting spore production of arbuscular mycorrhizal fungi, characterized in that The preparation includes hairy roots of a gibberellin-deficient mutant; The gibberellin-deficient mutant is a tomato gibberellin-deficient mutant gib3; The arbuscular mycorrhizal fungus is Heterosporangium heterorhizogenes.

5. A method for promoting spore production of arbuscular mycorrhizal fungi, characterized in that: Hairy roots of gibberellin-deficient mutants were mixed with arbuscular mycorrhizal fungi; The gibberellin-deficient mutant is a tomato gibberellin-deficient mutant gib3; The arbuscular mycorrhizal fungus is Heterosporangium heterorhizogenes.

6. The method according to claim 5, characterized in that The hairy roots of a gibberellin-deficient mutant were used as host plants and inoculated with arbuscular mycorrhizal fungi and cultured in a dual culture system.

7. The method according to claim 6, characterized in that The heteromorphic Rhizospora is heteromorphic Rhizospora DAOM197198.

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