A method and application for promoting arbuscular mycorrhizal fungal infection

Abstract

The invention belongs to the field of biotechnology, and relates to a method and application for promoting arbuscular mycorrhizal fungi infection. The invention significantly improves the infection of arbuscular mycorrhizal fungi to the root system by adding potassium myristate to the culture matrix. The addition of potassium myristate can also significantly increase the infection of different arbuscular mycorrhizal fungi to the root system and the number of spores in the roots in the soil potting system. The method can be widely used in promoting arbuscular mycorrhizal fungi infection and improving the propagation efficiency of arbuscular mycorrhizal fungi, is simple to operate, low in cost, short in cycle, and has a wide prospect of promotion and application.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and more specifically, relates to a method and application for promoting infection of arbuscular mycorrhizal fungi. Background Art

[0002] Arbuscular mycorrhizal fungi are ubiquitous fungi of the subphylum Glomeromycetes in the soil, and can establish a symbiotic relationship with the roots of more than 80% of terrestrial plants. Arbuscular mycorrhizal fungi can greatly promote the absorption of water and mineral nutrients, such as phosphorus and nitrogen, by the roots of host crops, and improve the ability of crops to resist biotic and abiotic stresses, such as improving the tolerance of crops to nutrient deficiency, drought, heavy metal poisoning, pH stress, pests and diseases, and improving the growth potential of crops, thereby increasing the yield and quality of crops. They are known as "biofertilizers" and "biopesticides." Therefore, arbuscular mycorrhizal fungi have great application potential in sustainable agricultural development.

[0003] In the process of establishing a symbiotic relationship between arbuscular mycorrhizal fungi and plants, spores germinate under suitable conditions such as moisture and temperature, and the germination tube continues to elongate and branch. Plant roots growing near arbuscular mycorrhizal fungi release substances such as strigolactones to guide the germination tubes of arbuscular mycorrhizal fungi to grow toward the plant root branches. At the same time, arbuscular mycorrhizal fungi also release mycorrhizal factors such as lipochitosan oligosaccharides to communicate signals with plants. After the germination tubes of arbuscular mycorrhizal fungi reach the root system, they will form mycopods in the epidermis, invade the plant cortical cells from the mycopods, form arbuscles, spores and vesicles in the root cells, and then continue to grow outside the roots to form extraradical hyphae and spores.

[0004] Arbuscular mycorrhizal fungi inoculants generally contain spores, hyphae and infected root segments, which are all propagules of arbuscular mycorrhizal fungi. Promoting the propagules is an important method to effectively improve the quality of inoculants. In the existing technology, when studying to improve the quality of inoculants, most of the focus is on spore production, while infected root segments, as important propagules of arbuscular mycorrhizal fungi, have been less studied in improving the quality of inoculants. Therefore, studying a propagation method that can significantly promote the root infection rate of arbuscular mycorrhizal fungi is of great significance for improving the quality of inoculants and agricultural production. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the above-mentioned existing arbuscular mycorrhizal fungi propagation technology, and provide a method and application for promoting arbuscular mycorrhizal fungi infection. The present invention can significantly improve the infection of arbuscular mycorrhizal fungi to the root system, and has simple operation, low cost and short cycle, and has broad application prospects in improving the quality of arbuscular mycorrhizal fungi inoculants or preparing preparations for promoting arbuscular mycorrhizal fungi infection.

[0006] The technical solution of the present invention is:

[0007] A culture matrix for promoting the infection of arbuscular mycorrhizal fungi, the culture matrix comprising river sand or soil, and potassium myristic acid; the potassium myristic acid addition concentration is 0.5 mmol / kg to 1 mmol / kg.

[0008] The present invention also provides a method for promoting arbuscular mycorrhizal fungal infection, comprising the following steps:

[0009] S1 used white clover and sorghum as host plants, and used a mixture of river sand and diatomaceous earth as a propagation medium after high temperature and high pressure sterilization (121°C, 60 min). The volume ratio of river sand to diatomaceous earth in the mixture was 1:2-3. The arbuscular mycorrhizal fungi were propagated with inoculants. After 4 months of growth under conventional management, the arbuscular mycorrhizal fungi inoculants were harvested and set aside.

[0010] S2: The test plant seeds were first surface-sterilized with a 5% sodium hypochlorite solution for 8-12 minutes, and then cultured in a dark incubator at 25-30°C for germination;

[0011] After S3 seeds germinated, they were sown in perlite sterilized by high temperature, high pressure and wet heat (121°C, 60 min), watered with deionized water every day, and Hoagland nutrient solution was applied twice a week, 5 ml each time;

[0012] S4 is cultivated in sand culture system or soil potting system.

[0013] Furthermore, the arbuscular mycorrhizal fungi in step S1 are Rhizophagus irregularis, Rhizophagus intraradices or Funneliformis osseae.

[0014] Furthermore, the specific steps of culturing in the sand culture system in step S4 are:

[0015] When two true leaves are fully expanded, select seedlings with similar growth potential and transplant them into pots. Add the above-mentioned culture medium that promotes arbuscular mycorrhizal fungi infection for cultivation. The specific operation is as follows:

[0016] (1) The river sand was passed through a 2 mm sieve, sterilized by high temperature and high pressure heat (121°C, 60 min), and then air-dried for later use;

[0017] (2) Add sterilized river sand and the arbuscular mycorrhizal fungi agent obtained in step S1 in a "sandwich" manner, transplant one tomato seedling into each pot, water it with deionized water every day, and add 15 ml of Hoagland nutrient solution with reduced P (1 / 10 concentration) to each pot every week;

[0018] (3) Dissolving potassium myristic acid in deionized water to prepare a potassium myristic acid solution, and applying 10 ml of the potassium myristic acid solution once every 7 days in the first 4 weeks after inoculation;

[0019] (4) All pots were placed in a growth chamber at 28°C for 6 weeks with a photoperiod of 16 h light / 8 h dark.

[0020] Furthermore, the specific operation of adding the sterilized river sand and the arbuscular mycorrhizal fungi agent obtained in step S1 in a "sandwich" manner in step (2) is: 245g sterilized river sand in the bottom layer + 35g arbuscular mycorrhizal fungi agent obtained in step S1 in the middle layer + 75g sterilized river sand in the upper layer.

[0021] Furthermore, the specific steps of culturing in the soil potting system in step S4 are: adding the above-mentioned culture matrix that promotes arbuscular mycorrhizal fungi infection for culturing, and the specific operations are:

[0022] (a) orchard soil is air-dried and crushed, passed through a 2 mm sieve to mix evenly, and then sterilized at high temperature and high pressure (121° C., 60 min) and dried to obtain treated soil;

[0023] (b) weighing 400 g of the treated soil obtained in step (a), adding potassium myristic acid powder and mixing thoroughly to obtain treated soil;

[0024] (c) adding the treated soil obtained in step (b) and the arbuscular mycorrhizal fungal agent obtained in step S1 in a "sandwich" manner;

[0025] (d) When the tomato seedlings have two fully expanded true leaves, select those with similar growth potential and transplant them into pots;

[0026] (e) Each pot was treated with CH 4 N 2 O.KH 2 PO 4 , K 2 SO 4 100 mg / kg N, 30 mg / kg P and 60 mg / kg K were added in the form of

[0027] (f) Carefully manage tomato seedlings and cultivate them for 6 weeks.

[0028] Furthermore, the specific operation of adding the treated soil obtained in step (b) and the arbuscular mycorrhizal fungal agent obtained in step S1 in a "sandwich" manner in step (c) is: 300 g of the treated soil obtained in step (b) in the bottom layer + 50 g of the arbuscular mycorrhizal fungal agent obtained in step S1 in the middle layer + 100 g of the treated soil obtained in step (b) in the upper layer.

[0029] Another object of the present invention is to provide an application of the method for promoting arbuscular mycorrhizal fungal infection in the preparation of a preparation for promoting arbuscular mycorrhizal fungal infection.

[0030] The present invention has found that adding potassium myristate to arbuscular mycorrhizal fungi culture substrates such as sand culture systems and soil potted culture systems can significantly increase the infection of arbuscular mycorrhizal fungi on the root system.

[0031] The number of spores or vesicles in the roots treated with potassium myristate in the method of the present invention is about 3 times that of the roots without potassium myristate. The exogenous addition of potassium myristate in the present invention significantly increases the mycorrhizal infection rate in different culture systems, wherein F% (mycorrhizal infection frequency in the root system) increases by 29.69% to 159.32%, M% (mycorrhizal infection intensity in the root system) increases by 93.31% to 372.89%, and A% (arbuscular abundance in the root system) increases by 222.35% to 653.94%. In the method of the present invention, the exogenous addition of potassium myristate significantly increases the infection of arbuscular mycorrhizal fungi.

[0032] Compared with the prior art, the present invention has the following advantages:

[0033] (1) The present invention can significantly increase the mycorrhizal infection rate of arbuscular mycorrhizal fungi.

[0034] (2) The present invention is simple to operate, low in cost, and short in cycle, and has broad application prospects in improving the quality of arbuscular mycorrhizal fungal agents or preparing preparations that promote arbuscular mycorrhizal fungal mycorrhizal infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The experiment is described in detail below with reference to the accompanying drawings and specific implementation methods:

[0036] Figure 1 It is the infection situation of Rhizospora heteromorpha and Glomus mosseae. DETAILED DESCRIPTION

[0037] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0038] Unless otherwise specified, the reagents and materials used in the following examples are commercially available. The strains used in the present invention are common strains, such as Rhizophagus irregularis, DAOM197198, purchased from Premier Tech, Canada); Rhizophagus intraradices (BGCJX04B) purchased from the Institute of Plant Nutrition and Resources, Beijing Academy of Agricultural and Forestry Sciences; Funneliformis mosseae is a commercially available product of Guangdong Libainong Biotechnology Co., Ltd.

[0039] Example 1: A method for promoting infection of arbuscular mycorrhizal fungi

[0040] S1 used white clover and sorghum as host plants, and sterilized the mixture of river sand and diatomaceous earth at high temperature and high pressure (121℃, 60min) as the propagation matrix. The volume ratio of river sand and diatomaceous earth in the mixture was 1:2.5. The inoculum was propagated on Heteromorpha rhizocystis (inoculation amount was 10%). After 4 months of growth under conventional management, the arbuscular mycorrhizal fungi inoculum was harvested and set aside.

[0041] S2: The tomato seeds of the test plant were first surface-sterilized with a 5% sodium hypochlorite solution for 10 min, and then cultured in a dark incubator at 28°C for germination;

[0042] After S3 seeds germinated, they were sown in perlite sterilized by high temperature and high pressure heat (121°C, 60 min), watered with deionized water every day, and Hoagland nutrient solution was applied twice a week, 5 ml each time;

[0043] S4 is cultured in a sand culture system. The specific steps of the culture in the sand culture system are: when the two true leaves are fully expanded, seedlings with similar growth potential are selected and transplanted into pots, and a culture matrix that promotes the infection of arbuscular mycorrhizal fungi is added for culture. The specific operation is:

[0044] (1) The river sand was passed through a 2 mm sieve, sterilized by high temperature and high pressure heat (121°C, 60 min), and then air-dried for later use;

[0045] (2) Sterilized river sand and the arbuscular mycorrhizal fungi agent obtained in step S1 were added in a "sandwich" manner, i.e., 245 g sterilized river sand at the bottom layer + 35 g arbuscular mycorrhizal fungi agent obtained in step S1 at the middle layer + 75 g sterilized river sand at the top layer. One tomato seedling was transplanted into each pot, and the pot was irrigated with deionized water every day. 15 ml of Hoagland nutrient solution with reduced P (1 / 10 concentration) was added to each pot every week.

[0046] (3) Dissolve potassium myristic acid in deionized water to prepare a 5 mM potassium myristic acid solution. Apply 10 ml of potassium myristic acid solution every 7 days in the first 4 weeks after inoculation. Finally, the total amount of potassium myristic acid added is 0.56 mmol / kg.

[0047] (4) All pots were placed in a growth chamber at 28°C for 6 weeks with a photoperiod of 16 h light / 8 h dark.

[0048] Example 2: A method for promoting infection of arbuscular mycorrhizal fungi

[0049] S1 used white clover and sorghum as host plants, and used a mixture of river sand and diatomaceous earth as a propagation medium after high temperature and high pressure sterilization (121℃, 60min). The volume ratio of river sand and diatomaceous earth in the mixture was 1:2.5. The inoculum was propagated on Heteromorpha rhizocystis (inoculation amount was 10%). After 4 months of growth under conventional management, the arbuscular mycorrhizal fungi inoculum was harvested and set aside.

[0050] S2: The tomato seeds of the test plant were first surface-sterilized with a 5% sodium hypochlorite solution for 10 min, and then cultured in a dark incubator at 28°C for germination;

[0051] After S3 seeds germinated, they were sown in perlite sterilized by high temperature and high pressure heat (121°C, 60 min), watered with deionized water every day, and Hoagland nutrient solution was applied twice a week, 5 ml each time;

[0052] S4 is cultured in a soil potting system. The specific steps of the culture in the soil potting system are: adding a culture matrix that promotes the infection of arbuscular mycorrhizal fungi for culture. The specific operations are:

[0053] (a) orchard soil is air-dried and crushed, passed through a 2 mm sieve to mix evenly, and then sterilized at high temperature and high pressure (121° C., 60 min) and dried to obtain treated soil;

[0054] (b) weighing 400 g of the treated soil obtained in step (a), adding 120 mg of potassium myristic acid powder and mixing thoroughly to obtain treated soil;

[0055] (c) adding the treated soil obtained in step (b) and the arbuscular mycorrhizal fungi agent obtained in step S1 in a "sandwich" manner, i.e., 300 g of the treated soil obtained in step (b) in the bottom layer + 50 g of the arbuscular mycorrhizal fungi agent obtained in step S1 in the middle layer + 100 g of the treated soil obtained in step (b) in the upper layer, and the final concentration of potassium myristic acid is 1 mmol / kg;

[0056] (d) When the tomato seedlings have two fully expanded true leaves, select those with similar growth potential and transplant them into pots;

[0057] (e) Each pot was treated with CH 4 N 2 O.KH 2 PO 4 , K 2 SO 4 100 mg / kg N, 30 mg / kg P and 60 mg / kg K were added in the form of

[0058] (f) Carefully manage tomato seedlings and cultivate them for 6 weeks.

[0059] Example 3: A method for promoting infection of arbuscular mycorrhizal fungi

[0060] The difference from Example 1 is that Rhizosporon heteromorpha is replaced by Glomus intraradices.

[0061] Example 4: A method for promoting infection of arbuscular mycorrhizal fungi

[0062] The difference from Example 2 is that Rhizospora heteromorpha is replaced by Glomus mosseae.

[0063] Comparative Example 1: A method for promoting infection of arbuscular mycorrhizal fungi

[0064] The difference from Example 1 is that potassium myristate solution was not applied.

[0065] Comparative Example 2: A method for promoting infection of arbuscular mycorrhizal fungi

[0066] The difference from Example 3 is that potassium myristate solution was not applied.

[0067] Comparative Example 3: A method for promoting infection of arbuscular mycorrhizal fungi

[0068] The difference from Example 2 is that potassium myristic acid powder was not added.

[0069] Comparative Example 4: A method for promoting infection of arbuscular mycorrhizal fungi

[0070] The difference from Example 4 is that potassium myristic acid powder is not added.

[0071] Experimental Example 1: Determination of the infection effect of arbuscular mycorrhizal fungi in sand culture system

[0072] After culturing for six weeks according to the methods of Example 1, Example 3, Comparative Example 1, and Comparative Example 2, samples were taken, and the tomato roots were washed and cut into small sections of about 1 cm, and the mycorrhizal infection rate was determined by trypan blue staining. The results of the determination of the infection effect of Heteromorpha rhizocystis in the sand culture system are shown in Table 1. The results of the determination of the infection effect of Glomus intraradici in the sand culture system are shown in Table 2.

[0073] Table 1 Determination results of the infection effect of heteromorphic rhizosporium in sand culture system

[0074]

[0075]

[0076] Note: "*" indicates that there is a significant difference at the 5% level in the T test. F%: frequency of mycorrhizal infection in the root system, M%: intensity of mycorrhizal infection in the root system, A%: abundance of arbuscules in the root system. "-myr" means that the comparative example 1 was not treated with potassium myristic acid, and "+myr" means that the potassium myristic acid was added to the example 1.

[0077] Table 2 Results of the determination of the infection effect of Glomus in the root in the sand culture system

[0078] Mycorrhizal infection index Comparative Example 2 (-myr) Example 3 (+myr) F％ 16.25±5.15 42.14±4.68* M％ 4.94±1.57 19.97±2.79* A％ 1.94±0.81 7.89±1.64* Number of spores or vesicles in roots 0.39±0.17 1.27±0.26*

[0079] "*" indicates that there is a significant difference at the 5% level in the T test. F%: frequency of mycorrhizal infection in the root system, M%: intensity of mycorrhizal infection in the root system, A%: abundance of arbuscules in the root system. "-myr" means that the comparative example 2 was not treated with potassium myristic acid, and "+myr" means that the potassium myristic acid was added to the example 3.

[0080] As can be seen from Table 1 and Table 2, the method of the present invention significantly promotes three infection parameters of two arbuscular mycorrhizal fungi, F% (mycorrhizal infection frequency in the root system), M% (mycorrhizal infection intensity in the root system), and A% (arbuscular abundance in the root system). Among them, the F%, M% and A% of the heteromorphic rhizosporon in Example 1 of the present invention in the sand culture system increased by 29.69%, 93.31% and 222.35% respectively compared with the comparative example 1, and the F%, M% and A% of the root glomus in Example 3 of the present invention in the sand culture system increased by 159.32%, 304.25% and 306.70% respectively compared with the comparative example 2. In addition, the number of spores or vesicles in the roots of the heteromorphic rhizosporon in Example 1 of the present invention is 2.94 times that of the comparative example 1, and the number of spores or vesicles in the roots of the root glomus in Example 3 of the present invention is 3.26 times that of the comparative example 2. It can be seen that the method of the present invention can significantly promote the formation of intraradical spores or vesicles of Rhizosporium heteromorphum and Glomus intraradicalum.

[0081] Experimental Example 2: Determination of the infection effect of arbuscular mycorrhizal fungi in soil potting system

[0082] After 6 weeks of cultivation according to the methods of Example 2, Example 4, Comparative Example 3, and Comparative Example 4, samples were taken, the tomato roots were washed and cut into small sections of about 1 cm, and the mycorrhizal infection rate was determined using trypan blue staining. The results of the determination of the infection effect of Heteromorpha rhizocystis in the soil potting system are shown in Table 3. The results of the determination of the infection effect of Glomus mosseae in the soil potting system are shown in Table 4. The infection of Heteromorpha rhizocystis and Glomus mosseae are shown in Table Figure 1 As shown, "Ri" represents Rhizospora heteromorpha and "Fm" represents Glomus mosseae.

[0083] Table 3 Determination results of the infection effect of Heteromorpha rhizosporangiophora in soil potting system

[0084] Mycorrhizal infection index Comparative Example 3 (-myr) Example 2 (+myr) F％ 28.89±4.44 48.89±4.01* M％ 4.50±1.33 21.28±6.65* A％ 1.65±0.58 12.44±4.97*

[0085] Note: "*" indicates that there is a significant difference at the 5% level in the T test. F%: frequency of mycorrhizal infection in the root system, M%: intensity of mycorrhizal infection in the root system, A%: abundance of arbuscules in the root system. "-myr" means that the comparative example 3 was not treated with potassium myristic acid, and "+myr" means that the potassium myristic acid was added to the example 2.

[0086] Table 4 Determination results of infection effect of Glomus mosseae in soil potting system

[0087] Mycorrhizal infection index Comparative Example 4 (-myr) Example 4 (+myr) F％ 28.89±2.94 52.22±4.84* M％ 7.78±1.39 18.94±3.38* A％ 1.54±0.19 6.07±1.52**

[0088] "*" indicates that there is a significant difference at the 5% level in the T test. F%: frequency of mycorrhizal infection in the root system, M%: intensity of mycorrhizal infection in the root system, A%: abundance of arbuscules in the root system. "-myr" means that the comparative example 4 was not treated with potassium myristic acid, and "+myr" means that the potassium myristic acid was added to the example 4.

[0089] It can be seen from Table 3 and Table 4 that the F%, M% and A% of Rhizosporium heteromorphum in Example 2 of the present invention in the soil potting system increased by 69.23%, 372.89% and 653.94% respectively compared with the comparative example 3, and the F%, M% and A% of Glomus mosseae in Example 4 of the present invention in the soil potting system increased by 80.75%, 143.44% and 294.16% respectively compared with the comparative example 4. Figure 1 It can be seen that the method of the present invention can significantly increase the mycorrhizal infection rate of two arbuscular mycorrhizal fungi, Sporangiophora heteromorpha and Glomus mosseae.

[0090] The above embodiments are preferred implementation modes of the present invention, but the implementation modes 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 equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for promoting infection of arbuscular mycorrhizal fungi, characterized in that: The following steps are involved: S1 used white clover and sorghum as host plants, and used a mixture of river sand and diatomaceous earth as a propagation medium after being sterilized at 121℃ and high temperature and high pressure for 60 minutes. The volume ratio of river sand to diatomaceous earth in the mixture was 1:2-3. The arbuscular mycorrhizal fungi were propagated with inoculants. After 4 months of growth under conventional management, the arbuscular mycorrhizal fungi inoculants were harvested and set aside. S2: The test plant seeds were first surface-sterilized with a 5% sodium hypochlorite solution for 8-12 min, and then germinated in a dark incubator at 25-30°C; After S3 seeds germinated, they were sown in perlite sterilized at 121°C for 60 min, watered with deionized water every day, and Hoagland nutrient solution was applied twice a week, 5 ml each time; S4 was cultivated in a soil potting system; The specific steps of culturing in the soil potting system in step S4 are: adding a culture matrix that promotes the infection of arbuscular mycorrhizal fungi for culturing, and the specific operations are: (a) Orchard soil was air-dried and crushed, passed through a 2 mm sieve to mix evenly, and then sterilized at 121°C and dried at high temperature and high pressure for 60 min to obtain the treated soil; (b) weighing 400 g of the treated soil obtained in step (a), adding potassium myristic acid powder and mixing thoroughly to obtain treated soil; the potassium myristic acid addition concentration is 0.5 mmol / kg to 1 mmol / kg; (c) adding the treated soil obtained in step (b) and the arbuscular mycorrhizal fungal agent obtained in step S1 in a "sandwich" manner; (d) When the tomato seedlings have two fully expanded true leaves, select those with similar growth potential and transplant them into pots; (e) 100 mg / kg N, 30 mg / kg P, and 60 mg / kg K were added to each pot in the form of CH4N2O, KH2PO4, and K2SO4, respectively; (f) Carefully manage tomato seedlings and cultivate them for 6 weeks; The specific operation of adding the treated soil obtained in step (b) and the arbuscular mycorrhizal fungi agent obtained in step S1 in a "sandwich" manner in step (c) is: 300 g of the treated soil obtained in step (b) in the bottom layer + 50 g of the arbuscular mycorrhizal fungi agent obtained in step S1 in the middle layer + 100 g of the treated soil obtained in step (b) in the upper layer.

2. Use of the method for promoting arbuscular mycorrhizal fungal infection according to claim 1 in preparing a preparation for promoting arbuscular mycorrhizal fungal mycorrhizal infection.

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