CN114045240A

CN114045240A - Rhizobium, microbial inoculum prepared from same and application of rhizobium

Info

Publication number: CN114045240A
Application number: CN202111371804.9A
Authority: CN
Inventors: 李永; 薛寒; 姜宁; 朴春根; 汪来发
Original assignee: Research Institute of Forest Ecology Environment and Protection of Chinese Academy of Forestry
Current assignee: Research Institute of Forest Ecology Environment and Protection of Chinese Academy of Forestry
Priority date: 2021-11-18
Filing date: 2021-11-18
Publication date: 2022-02-15
Anticipated expiration: 2041-11-18
Also published as: CN114045240B

Abstract

The invention discloses Rhizobium sp, a microbial inoculum prepared from the Rhizobium sp and application of the Rhizobium sp. The microbial inoculum prepared by the rhizobium strain 11760 can effectively promote the rapid healing of poplar wounds and has a good healing promoting effect on plant wounds, and field treatment experiments show that the microbial inoculum can obviously promote the rapid healing of the wounds when being applied at the early stage of the formation of the poplar wounds, and is suitable for popularization and application in plant wound management practice.

Description

Rhizobium, microbial inoculum prepared from same and application of rhizobium

Technical Field

The application relates to the field of healing of plant wounds, in particular to a rhizobium strain, a microbial inoculum prepared from the rhizobium strain and application of the rhizobium strain in healing of the plant wounds.

Background

A large number of wounds are caused in the processes of plant trimming, plant transplanting and field operation, and if the wounds are not properly treated, nutrient components in plants can be lost, or pathogenic bacteria such as bacteria and fungi invade the plants along with the wounds, so that the trees are weakened and even die, and how to promote the plant wounds to heal quickly is an important work.

At present, the plant wound healing agent used in production and application contains a large amount of chemical agents including bactericides, nutritional agents and protective agents, the chemical components are easy to cause environmental pollution, most of the high molecular film forming agents contained in the commercially available wound dressing agents are polyacrylamide, acrylate, waterborne polyurethane, modified epoxy resin and the like, and the film forming agents have certain toxicity to plant forming layer cells and influence the healthy growth of plants.

Disclosure of Invention

The invention provides a Rhizobium strain which is named 11760 and is classified and named as Rhizobium Rhizobium sp, and is preserved in the China general microbiological culture Collection center of the culture Collection of microorganisms; the address of the depository: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North; the preservation time is 2018, 02 and 08 days; the preservation number is: CGMCC No. 15351.

The invention also provides a bacterial agent which is prepared from the rhizobium strain 11760.

Preferably, the bacterial agent contains the active bacterial number of the strain 11760 more than or equal to 10⁸CFU/mL。

The invention also provides application of the rhizobium bacteria or the microbial inoculum in repairing plant wounds. The plant is preferably a poplar.

Specifically, the application is to apply the microbial inoculum prepared by the strain 11760 to the wound;

the application method is spraying or smearing;

the application concentration is that the number of active bacteria of the strain 11760 is more than or equal to 10⁸CFU/mL；

After being applied, the wound part can be wrapped by a film material such as a preservative film and the like for moisture preservation;

the period of application is preferably within 1-2 hours after the wound has occurred.

The beneficial effects of the invention include:

in poplar endophytic bacteria separated from poplar barks, the Rhizobium sp strain 11760 is found to be capable of promoting the healing of poplar wounds. The microbial inoculum prepared by the rhizobium 11760 strain can effectively promote the rapid healing of poplar wounds and has a good healing promoting effect on plant wounds, and field wound healing treatment experiments show that the microbial inoculum can obviously promote the rapid healing of the wounds when being applied in the early stage of the formation of the plant wounds, can heal for more than 10 days earlier than a control, and is suitable for popularization and application in plant wound management practice.

Drawings

FIG. 1 is a graph of bark healing at day 3 of the inoculation test in example 2 of the present invention, wherein FIG. 1A is a negative control; fig. 1B applies bacterial wound healing agent 11760.

Detailed Description

The present invention will be further illustrated and described with reference to the following examples, but the examples described are only a part of the examples of the present invention, and not all of the examples. All other inventions and embodiments based on the present invention and obtained by a person of ordinary skill in the art without any creative effort belong to the protection scope of the present invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 screening and identification of strains

(1) Obtaining and identifying wound healing strains

Taking poplar bark, grinding the poplar bark with a mortar, adding sterile water for dilution, coating a plate, putting the plate in an incubator at 30 ℃ for inverted culture for 24 hours, picking out a single colony, and carrying out streak culture on a new culture dish.

In the field inoculation experiment of poplar pathogenic bacteria, a colony cultured independently needs to be inoculated to a wound of a tree body to measure the pathogenicity of the colony, and in the experimental process, when the bacterial strain 11760 is inoculated to the wound of a plant, the wound healing speed of the plant at the inoculated part is higher than that of other bacterial strains and a control experiment.

(2) Bacterial identification

DNA of the strain is extracted by a CTAB method, PCR amplification is carried out by using a general primer 27f/1492r (5 '-AGAGAGnTGATCCTGGCTCAG-3'/5'-GGTTACCTTGTTACGACTT-3') of bacteria, and a PCR product is sent to Beijing Bomaide gene technology company Limited for sequencing. Then, the strain is identified by a network identification tool (https:// eztaxon-e. ezbiocloud. net/identification), the 16S rRNA gene sequence information is input into a database for comparison, and the strain 11760 is compared with a model strain Rhizobium alkalisoli CCBAU 01393^TThe homology was 98.5%.

According to the molecular identification result, the strain 11760 is identified as Rhizobium sp and is preserved in the China general microbiological culture Collection center of the culture Collection of microorganisms; the address of the depository: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North; the preservation time is 2018, 02 and 08 days; the preservation number is: CGMCC No. 15351.

Example 2 field wound healing test

(1) Preparation of microbial inoculum

The backup of the preserved strain is streaked, and then a single colony is picked and continuously transferred and cultured for 3 generations. Single colonies are picked and transferred into 150mL TSB liquid culture medium for propagation culture, and shaking culture is carried out for 24h at 220rpm under the culture condition of 30 ℃. Centrifuging at 10000rpm for 5min, precipitating the cultured strain, and diluting the precipitate with sterile water to 10⁸CFU/mL or so.

(2) Experiment in the field

The sterile absorbent cotton is soaked in sterile water for inoculation.

The field experiment is carried out in Puyang city Lin academy of sciences nursery and China forestry science research institute greenhouse, the test tree species is poplar, and 5 seedlings are randomly selected in the nursery to carry out the wound healing experiment.

Using a sterile scalpel to make a wound with the same size on the bark as an inoculation point, wherein the wound is deep to the surface layer of the xylem; covering absorbent cotton soaked with sterile water after inoculation, and wrapping and moisturizing by using a preservative film; each tree was inoculated with 2 points, from top to bottom:

inoculation point 1: inoculation with sterile water (negative control);

inoculation point 2: inoculating 11760 microbial inoculum;

the specific inoculation method comprises the following steps: the negative control group is prepared by inoculating 100ul sterile water on artificial wound, covering soaked absorbent cotton for moisturizing, and wrapping with preservative film; the 11760 inoculum test group was 100ul of 10 inoculum inoculated to an artificial wound⁸Covering 11760 microbial inoculum prepared in the step (1) of CFU/mL with soaked absorbent cotton for moisturizing, and then wrapping a preservative film.

On days 3, 8 and 13 after inoculation, the recorded wound healing was observed, as shown in fig. 1, at day 3 after inoculation, the wound applied with the inoculant of the present invention healed, while the negative control wound edge began to darken and showed no healing signs, and the negative control gradually developed callus by day 13. The microbial inoculum has good effect of promoting the healing wound of the poplar, and can be widely applied to the repair of a large number of wounds caused in the transplanting, trimming and other field operation processes of the poplar.

Claims

1. A Rhizobium sp (Rhizobium sp.) characterized in that the Rhizobium sp is named as Rhizobium 11760 and is deposited at the China general microbiological culture Collection center; the address of the depository: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North; the preservation time is 2018, 02 and 08 days; the preservation number is: CGMCC No. 15351.

2. A bacterial preparation prepared from Rhizobium strain 11760 of claim 1.

3. The bacterial agent of claim 2, wherein the bacterial agent contains 11760-containing active strains with the number not less than 10⁸CFU/mL。

4. Use of a rhizobia as defined in claim 1 or a bacterial agent as defined in any one of claims 2 to 3 in the repair of wounds in plants.

5. The use of claim 4, wherein the use is the application of a bacterial agent prepared from strain 11760 to a wound.

6. Use according to claim 5, characterized in that the application method is spraying or painting.

7. The use as claimed in claim 5, wherein the microbial inoculum concentration is that the number of active bacteria of the strain 11760 is more than or equal to 10⁸CFU/mL。

8. Use according to any one of claims 5 to 7, wherein the wound site is wrapped for moisturization after application.

9. Use according to any one of claims 5 to 7, wherein the period of administration is within 1 to 2 hours after the wound has occurred.