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CN-121975714-A - Method for simply and efficiently promoting growth of AM fungal hyphae and non-co-production of spores and application

CN121975714ACN 121975714 ACN121975714 ACN 121975714ACN-121975714-A

Abstract

The invention discloses a simple and efficient method for promoting the growth of AM fungal hyphae and non-co-production of spores and application thereof. The method can preserve AM fungus agent in double culture in long-term cold storage for 3-38 months. Spores obtained after long-term preservation are inoculated on a culture medium under a non-symbiotic condition, and C14 fatty acid is added into the culture medium. After exogenous addition of C14 fatty acid, the long-term refrigerated spores can obviously increase the quantity of hyphae and spore production of non-symbiotic formation of AM fungi, the hyphae density is 7.3 times that of the non-symbiotic formation spores without adding fatty acid, and each mother spore can form about 54 sporozoites which are about 10 times that of the non-symbiotic formation spores treated by adding C14 fatty acid and an exogenous stimulator. The method can propagate the AM fungus microbial inoculum under the non-symbiotic condition, has high propagation efficiency, can obviously increase the non-symbiotic spore yield of the AM fungus, is simple and convenient to operate, has low cost and short period, and has wide popularization and application prospects.

Inventors

  • LIU XIAODI
  • ZHU HONGHUI
  • DENG MINGRONG
  • YAO QING
  • LI YANXUAN

Assignees

  • 广东省科学院微生物研究所(广东省微生物分析检测中心)

Dates

Publication Date
20260505
Application Date
20260326

Claims (10)

  1. 1. A simple and efficient method for promoting the growth of AM fungal hyphae and/or non-co-producing spores is characterized by comprising the following steps of carrying out long-term refrigeration on AM fungal spores combined with exogenous fatty acid treatment, wherein the long-term refrigeration is carried out for 3-40 months at 4-10 ℃.
  2. 2. The method of claim 1, wherein the temperature of the long-term refrigeration is 4-8 ℃.
  3. 3. The method of claim 1, wherein the long-term refrigeration time is 3-38 months.
  4. 4. The method of claim 1, wherein the fatty acid is a C14 fatty acid.
  5. 5. The method of claim 4, wherein the C14 fatty acid concentration is from 0.5 to 1 mM.
  6. 6. The method according to claim 1, wherein the exogenous fatty acid is a medium containing exogenous fatty acid, the medium contains MgSO 4 ·7H 2 O 739 mg;KNO 3 76 mg;KCl 65 mg;Ca(NO 3 ) 2 ·4H 2 O 359 mg;KH 2 PO 4 4.1 mg;MnSO 4 ·4H 2 O 2.45 mg;ZnSO 4 ·7H 2 O 0.28 mg;H 3 BO 3 1.85 mg;CuSO 4 ·5H 2 O 0.22 mg;(NH 4 ) 6 Mo 7 O 24 ·4H 2 O 0.034 mg;NaMoO 4 ·2H 2 O 0.0024 mg; thiamine hydrochloride (VB 1 ) 1 mg, pyridoxine hydrochloride (VB 6 ) 0.9 mg, niacin 1 mg, calcium pantothenate 0.9 mg, vitamin B 12 0.4.4 mg, biotin 0.0009 mg;NaFeEDTA 8 mg, sucrose 10 g, and potassium myristate 0.5 mmol per liter.
  7. 7. The method of claim 1, wherein the AM fungal spores are spores obtained by propagation using a tomato hairy root-AM fungal dual culture system.
  8. 8. The method of claim 7, wherein the propagation is for 3 months.
  9. 9. The method of claim 1 or 7, wherein the AM fungus is rhizopus deleteri DAOM197198.
  10. 10. Use of the method according to any one of claims 1-9 for promoting the growth of hyphae and/or non-co-production of spores of AM fungus, said AM fungus being a rhizopus morphous DAOM197198.

Description

Method for simply and efficiently promoting growth of AM fungal hyphae and non-co-production of spores and application Technical Field The invention belongs to the field of biotechnology. More particularly, relates to a simple and efficient method for promoting the growth of AM fungal hyphae and non-co-production of spores and application thereof. Background Arbuscular mycorrhizal (arbuscular mycorrhizal, AM) fungi are saccharum mycotina fungi, can establish symbiotic relation with more than 80% of root systems of land plants, greatly promote the root systems of crops to absorb water and mineral nutrients such as phosphorus and nitrogen, improve the stress resistance of crops, such as improving the tolerance of crops to nutrient impoverishment, drought, heavy metal poisoning, pH value stress, plant diseases and insect pests and the like, improve the growth vigor of the crops, further improve the yield of the crops and improve the quality of the crops. Therefore, AM fungi have great application potential in agricultural production. Since AM fungi have not been truly pure cultivated so far, the expansion of AM fungal spores is premised on establishing symbiotic relationships. At present, the propagation of AM fungal spores mainly comprises two modes, namely, using a sorghum and clover combination (or other plant combinations) as host plants, propagating in a certain proportion of soil, river sand, diatomite and other matrixes, wherein the obtained microbial inoculum takes the soil as a carrier, and establishing a double culture system of AM fungi and hairy roots (such as carrots and tomatoes) under aseptic conditions of a culture dish and the like, and propagating spores and the like under aseptic conditions. As spores obtained by the hairy root double culture system are sterile, the spores are widely applied in the fields of scientific research, production and the like. The prior art (application number 201911320670.0) discloses application of abscisic acid in promoting spore production of AM fungi, and the abscisic acid can directly act on the spores of the AM fungi by adding the abscisic acid into the spores of the AM fungi, so that the number of the spores of the AM fungi is remarkably increased. The application of gibberellin-deficient mutant in promoting the spore production of AM fungi (patent number: ZL 202010718350.7) remarkably improves the spore yield of AM fungi by inoculating AM fungi with hairy roots of tomato gibberellin-deficient mutant, and remarkably improves the spore number of AM fungi under normal pH conditions (pH 6.5) and acidic conditions (pH 4.5). The spores propagated by the method are sterile, and are favorable for intensive research on AM fungi in scientific research. However, the method needs to take root systems as host plants, the AM fungi can generate spores after establishing symbiotic relation with the AM fungi, the steps are complicated, and the period of spore production is long. The prior art (patent number: ZL 202110518816.3) discloses a method for promoting AM fungi to produce spores in a non-co-mode and application thereof, and potassium myristate, strigolactone, methyl jasmonate and peptone are added into AM fungi spores to remarkably improve the production quantity of AM fungi spores, and 5.5 sporozoites can be formed by AM fungi single parent spores in the addition treatment. The addition of potassium myristate, strigolactone, methyl jasmonate and peptone can significantly increase AM fungal spore production under non-symbiotic conditions. The symbiotic relation with the host plant is not needed, but more compounds are needed to be exogenously added, so that the operation is complex. And the added strigolactone and methyl jasmonate are very expensive in price and high in production cost, and are not beneficial to the expansion production of AM fungal inoculants and the application of AM fungal inoculants in agricultural industry. Therefore, the research on the AM fungus propagation method which has the advantages of simple propagation operation, low cost, short period, high propagation efficiency, less dependence on the external environment and capability of remarkably increasing the spore yield under the non-symbiotic condition has important significance, and further spans a step forward in the aspect of realizing the pure culture of the AM fungus. Disclosure of Invention The invention aims to overcome the defects and the shortcomings of the conventional AM fungus propagation technology, and provides a method for promoting the non-symbiotic spore production of AM fungi, which provides technical support for the development of biological agriculture and related industries. A first object of the present invention is to provide a method for promoting non-symbiotic cultivation of AM fungi comprising the step of inoculating AM fungal spores to a medium containing 0.1-5 mM C14 fatty acid after cooling at 4-10℃for 3-38 months. Preferably, the refrigeration is at 4-6deg.C fo