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CN-122012280-A - Method for efficiently screening rhizobia with functions of promoting growth and inhibiting aspergillus flavus and application thereof

CN122012280ACN 122012280 ACN122012280 ACN 122012280ACN-122012280-A

Abstract

The invention discloses a method for efficiently screening rhizobia with functions of promoting growth and inhibiting aspergillus flavus and application thereof, according to the invention, the field ecological pre-enrichment effect of the ARC functional microbial agent is introduced into a rhizobia screening process for the first time, and the target flora abundance is directionally increased from the source through the toxicity control and enrichment effect. The method comprises the steps of synchronously collecting three samples of rhizosphere soil, root system and rhizobium, comprehensively separating rhizobium, constructing a high-flux primary screening system with parallel functions of growth promotion and bacteriostasis, and synchronously evaluating the nitrogen fixation growth promotion capability and the bacteriostasis effect of volatile substances of candidate strains in early stage through a potting test and a double-dish buckling method. And further carrying out functional quantification, polygenic phylogenetic identification and histology mechanism analysis on the positive strain. The invention overcomes the defects of single function and low efficiency of the traditional method, and provides an effective way for rapidly, directionally and systematically screening the multifunctional rhizobia resource from the optimized rhizosphere ecological system.

Inventors

  • ZHANG QI
  • ZHOU YANG
  • LI PEIWU

Assignees

  • 中国农业科学院油料作物研究所

Dates

Publication Date
20260512
Application Date
20260212

Claims (10)

  1. 1. A method for efficiently screening rhizobia with functions of promoting growth and inhibiting aspergillus flavus is characterized by comprising the following steps: (1) Ecological pre-enrichment treatment is carried out on leguminous crop rhizosphere soil by utilizing an ARC functional microbial agent; (2) Synchronously collecting three types of samples of rhizosphere soil, root systems and root nodules from the crops treated in the step (1); (3) Respectively preprocessing and gradient diluting three types of samples, coating the samples on a rhizobium selective culture medium, separating and purifying to obtain rhizobium single colonies, and establishing a candidate strain library; (4) Performing difunctional parallel primary screening on strains in the candidate strain library, and selecting strains with both types of primary screening results reaching standards as primary screening positive strains; (41) Primary screening of growth promoting function, namely evaluating the promoting effect of the primary screening on the growth and nodulation of host plants through a potting test; (42) Primary screening of antibacterial function, namely evaluating the inhibition effect of volatile substances on the growth of Aspergillus flavus hypha by a double-dish buckling method; (5) And re-screening the primary screened positive strain to quantify the growth promotion and bacteriostasis efficacy of the primary screened positive strain, and carrying out species identification and functional mechanism analysis on the optimized strain.
  2. 2. The method of claim 1, wherein the ARC functional microbial agent in step (1) comprises at least three of Bacillus amyloliquefaciens, brevibaciens laterosporus, bacillus mucilaginosus and Enterobacter ludwigii, wherein the effective viable count of the Bacillus amyloliquefaciens is greater than or equal to 22x10 9 cfu/gram, the effective viable count of the Brevibaciens laterosporus is greater than or equal to 22x10 9 cfu/gram, the effective viable count of the Bacillus mucilaginosus is greater than or equal to 1x10 10 cfu/gram, and the effective viable count of the Enterobacter ludwigii is greater than or equal to 1x10 10 cfu/gram.
  3. 3. The method according to claim 1, wherein in the step (41), the standard of standard for the primary screening of the growth promoting function is that the plant height of the host plant is not less than 25% relative to the blank control after 30 days of inoculation of the candidate strain, and the number of individual root nodules is not less than 150% of the blank control.
  4. 4. The method according to claim 1, wherein in the step (42), the standard of the primary screening of the antibacterial function is that the relative inhibition rate of the candidate strain to the colony diameter of the aspergillus flavus is more than or equal to 50%.
  5. 5. The method according to claim 1, wherein the re-screening step in the step (5) is specifically characterized in that the criterion of promoting the effective energy is that the plant height and the root nodule number of a host plant are remarkably improved, and the criterion of inhibiting the bacterial efficacy is that the inhibition rate of a candidate strain on the colony diameter of aspergillus flavus is more than or equal to 60%, the inhibition rate of the conidium yield is more than or equal to 90% and the inhibition rate of the aflatoxin B1 yield is more than or equal to 90%.
  6. 6. The method of claim 1, wherein the identifying of the species in step (5) is based on phylogenetic identification of multiple housekeeping genes, comprising sequence analysis of the strain' S16S rRNA gene and at least two housekeeping genes selected from atpD, recA, dnaK, glnII genes and construction of a phylogenetic tree.
  7. 7. The method according to claim 1, wherein the parsing of the functional mechanism in the step (5) includes: (51) Identifying characteristic volatile antibacterial substances generated by the strain by adopting a headspace solid-phase microextraction-gas chromatography mass spectrometry combined technology; (52) The strains were analyzed for upregulation of host plant symbiotic related gene expression and/or downregulation of aflatoxin synthesis and spore development related gene expression by transcriptome sequencing techniques.
  8. 8. The method of claim 7, wherein the characteristic volatile bacteriostatic substance in step (51) is dimethyl disulfide or 2-methylbutyric acid.
  9. 9. The rhizobia with the functions of promoting growth and inhibiting aspergillus flavus obtained by screening by adopting the method as claimed in any one of claims 1-8 is characterized by comprising soybean slow-growing rhizobia APFJPT-23-4 or Guangzhou slow-growing rhizobia APHBXY-23-1, wherein the classification of soybean slow-growing rhizobia APFJPT-23-4 is named Bradyrhizobium japonicum, and is preserved in China center for type culture collection with the preservation number of CCTCC M20253050, and the classification of Guangzhou slow-growing rhizobia APHBXY-23-1 is named Bradyrhizobium guangzhouense, and is preserved in China center for type culture collection with the preservation number of CCTCC M20253048.
  10. 10. Use of a rhizobia with both growth promoting and aspergillus flavus inhibiting functions according to claim 9 for the preparation of a microbial agent for promoting growth of leguminous crops, nodulation and nitrogen fixation and/or inhibiting aflatoxin contamination.

Description

Method for efficiently screening rhizobia with functions of promoting growth and inhibiting aspergillus flavus and application thereof Technical Field The invention belongs to the technical field of agricultural microbial resource screening and evaluation, and particularly relates to a method for efficiently screening rhizobia with functions of promoting growth and inhibiting aspergillus flavus and application thereof. Background Leguminous crops such as peanuts, soybeans and the like are globally important grain and oil sources, and sustainable production of the leguminous crops faces two major core challenges, namely, dependence on nitrogen fixation by symbiotic with rhizobia, low nodulation efficiency under natural conditions and restriction of yield improvement, and easiness in pollution of fruits by aflatoxin and serious threat to food safety. The traditional solution is to apply growth promoting bacteria such as rhizobia and biocontrol bacteria respectively, and has the problems of single function, increased cost, unstable effect and the like. In recent years, a breakthrough has been made in the technology of ARC (Aflatoxin control, rhizobium-nodulation induction, coupling) functional microbial agents developed by the group Li Peiwu academy of agricultural sciences in china. The technology realizes the synergistic effect that the abundance of field aspergillus flavus toxigenic bacteria is reduced by more than 60%, the enrichment and nodulation quantity of peanut rhizobia is obviously increased, and the yield is averagely improved by 19.67% under the condition that exogenous rhizobia is not applied by treating soil through a specific compound microbial agent. The achievement proves that the dual purposes of poison control and nitrogen fixation are completely and possibly realized synchronously by regulating and controlling the rhizosphere microorganism system. The ARC technology creates a microenvironment in the field that facilitates the colonization and expression of multifunctional beneficial microorganisms (especially rhizobia that may have both growth promoting and bacteriostatic properties). However, there is still a lack of systematic way to efficiently and accurately isolate and identify rhizobia strains that are responsible for the core coupling function from this optimized ecosystem and develop them into directly inoculated microbial products. The existing rhizobia screening method focuses on single nitrogen fixation and growth promotion capability, the separation source is usually limited to rhizobia, and the important biocontrol property of inhibiting aspergillus flavus is not brought into a core screening process, so that the screened strain has single function and cannot meet the urgent requirement of green production on one-bacterium multiple-effect microbial preparation. Therefore, the ecological advantage created by the ARC technology can be effectively utilized, the ecological advantage is systematically separated from the rhizosphere multidimensional habitat, and the efficient screening method with dual functions of growth promotion and bacteriostasis is evaluated in parallel, so that the novel multifunctional rhizobia resource is excavated, the ARC technology mechanism is deepened, and the novel microbial inoculant has important theoretical and application values. Disclosure of Invention In order to solve the defects in the prior art, the invention provides a method for efficiently screening rhizobia with functions of promoting growth and inhibiting aspergillus flavus and application thereof. In order to solve the technical problems, the invention provides the following technical scheme: the first object of the invention is to provide a method for efficiently screening rhizobia with functions of promoting growth and inhibiting aspergillus flavus, which comprises the following steps: (1) Ecological pre-enrichment treatment is carried out on leguminous crop rhizosphere soil by utilizing an ARC functional microbial agent; (2) Synchronously collecting three types of samples of rhizosphere soil, root systems and root nodules from the crops treated in the step (1); (3) Respectively preprocessing and gradient diluting three types of samples, coating the samples on a rhizobium selective culture medium, separating and purifying to obtain rhizobium single colonies, and establishing a candidate strain library; (4) Performing difunctional parallel primary screening on strains in the candidate strain library, and selecting strains with both types of primary screening results reaching standards as primary screening positive strains; (41) Primary screening of growth promoting function, namely evaluating the promoting effect of the primary screening on the growth and nodulation of host plants through a potting test; (42) Primary screening of antibacterial function, namely evaluating the inhibition effect of volatile substances on the growth of Aspergillus flavus hypha by a double-dish buckling meth