CN110200018B

CN110200018B - Optimal DSE inoculation amount for promoting plant rooting

Info

Publication number: CN110200018B
Application number: CN201910505631.1A
Authority: CN
Inventors: 毕银丽; 薛子可; 全文智
Original assignee: Beijing Heshengyuan Ecological Environment Engineering Technology Co ltd; China University of Mining and Technology Beijing CUMTB
Current assignee: Beijing Heshengyuan Ecological Environment Engineering Technology Co ltd; China University of Mining and Technology Beijing CUMTB
Priority date: 2019-06-12
Filing date: 2019-06-12
Publication date: 2020-10-16
Anticipated expiration: 2039-06-12
Also published as: CN110200018A

Abstract

The invention discloses an optimal DSE inoculation amount for promoting plant rooting. The optimal DSE inoculation amount for promoting plant rooting disclosed by the invention is that the dry weight of hyphae is (0.468-1.404) mg/mL, the plant tissue or organ is soaked by the liquid containing the dark septate endophytic fungi with the dry weight of the hyphae being (0.468-1.404) mg/mL, the plant rooting is promoted, the root length, the root number and the root diameter of the plant organ treated by the culture solution of the dark septate endophytic fungi are obviously increased, and the optimal concentration dilution of the dark septate endophytic fungi with the concentration of 20% (namely the dry weight of the hyphae is 0.468mg/mL) is realized. The invention utilizes the optimal DSE inoculation quantity of the plant rooting to process the plant tissues or organs, can assist the plant rooting and promote the plant growth, has positive ecological significance for improving the land reclamation efficiency, and also provides a microbial technical support for the land reclamation and ecological reconstruction of the mining area.

Description

Optimal DSE inoculation amount for promoting plant rooting

Technical Field

The invention relates to the field of biotechnology, and discloses an optimal DSE inoculation amount for promoting plant rooting.

Background

Dark Septate Endophytes (DSE) have biological functions similar to mycorrhizal fungi, and mainly colonize the epidermis, cortex and even the intracellular space of vascular bundle tissues of healthy plant roots to form symbionts without causing plant diseases. DSEs are ecologically widespread, and colonization among different habitat plants suggests that they have little or no host specificity and can establish a reciprocal mutual benefit and a mutually regulated physiological whole with the plant, but each has its morphological characteristics. Researches show that DSE plays a more important role in the stress environment, the absorption area of the plant root system is enlarged through the growth of hypha, and the absorption capacity of nutrient elements outside the original root system absorption range is improved.

Disclosure of Invention

The invention aims to solve the technical problem of how to promote plant rooting.

In order to solve the technical problems, the invention firstly provides a method for promoting plant rooting, which comprises the following steps: the plant tissue or organ is treated by the dark-color septate endophytic fungi to realize the promotion of the plant rooting.

In the above method, treating the plant tissue or organ with a dark-colored septal endophyte may comprise: soaking the plant tissue or organ with a liquid containing the dark-colored septate endophytic fungi.

In the above method, the dry weight of hyphae in the liquid containing the dark septate endophyte may be (0.468-1.404) mg/mL. The dry weight of hyphae in the liquid containing the dark septal endophyte can be specifically 0.468mg/mL or 1.404 mg/mL. The dry weight of the hyphae is obtained by filtering liquid containing the dark septate endophytic fungi by using filter paper, collecting the hyphae and drying the hyphae to constant weight.

In the above method, the liquid containing the dark septate endophyte may be a culture solution of the dark septate endophyte or a dilution of the culture solution.

The culture solution can be obtained by culturing the dark septal endophytic fungi by using an MMN culture medium.

The diluent can be obtained by diluting the culture solution with water.

The MMN medium can be obtained by adding CaCl₂0.05g，MgSO₄0.15g，NaCl 0.025g，FeCl₃0.01g，KH₂PO₄0.5g, 0.0001g of Vitamin B1 (thiamine), (NH)₄)₂HPO₄0.25g, Glucose (Glucose) 10g, Citric acid (Citric acid) 0.2g and Maltextract (Malt extract) 10g were mixed, 1000ml of distilled water was added thereto, and sterilized.

In the above method, when the plant tissue or organ is soaked with the liquid containing the dark-colored septate endophytic fungi, half of the plant tissue or organ may be in the air.

During treatment, half of the plant tissue or organ can be left in the air by adding the MMN medium.

In the above method, the treatment time is 15 days.

The conditions of the treatment may be: the first 3 days are carried out in the dark, the temperature is 22 ℃, and the relative humidity of air is 70 percent; after three days of treatment, the culture is carried out under illumination, the illumination intensity is 1500-.

In the above method, the promotion of plant rooting may be embodied in promotion of root elongation, root number increase and/or root thickening of the plant.

In the above method, the plant organ may be a plant seed.

In the method, the dark color septate endophytic fungus can be deposited in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 17464.

In the above method, the plant may be a1) or a2) or a 3):

a1) a monocot or dicot;

a2) a gramineous plant;

a3) corn.

The plant tissue or organ may be a sterile tissue or organ.

The treatment may be performed in a sterile environment.

Any one of the following applications of the method for promoting plant rooting also belongs to the protection scope of the invention:

x1) plant production;

x2) plant growing;

x3) promoting plant growth;

x4) promoting land reclamation.

The plant may be a1) or a2) or a 3):

a1) a monocot or dicot;

a2) a gramineous plant;

a3) corn.

In one embodiment of the invention, the corn is medium waxy No. one.

In the method for promoting the plant to take root, the root length, the root number and the root diameter of the plant organ treated by the culture solution of the dark-color septal endophytic fungi are obviously higher than those of a control group which is not treated by the dark-color septal endophytic fungi, and the concentration of the dark-color septal endophytic fungi is the best 20 percent when the plant organ is treated by the dark-color septal endophytic fungi. The method for promoting the plant to take root can assist the plant to take root and promote the plant growth, has positive ecological significance for improving the land reclamation efficiency, and also provides a microbial technical support for the land reclamation and ecological reconstruction of the mining area.

Biological material preservation instructions

Classification nomenclature of biological materials: darksidea zeta

Strain number of biological material: needle A1-3

Deposit name of biological material: china general microbiological culture Collection center

The preservation unit of the biological material is abbreviated as: CGMCC (China general microbiological culture Collection center)

Deposit unit address of biological material: west road No.1, north west of the township, beijing, ministry of sciences, china, institute of microbiology, zip code: 100101

Preservation date of biological material: 4 and 8 months in 2019

Accession number to the collection of biological materials: CGMCC No.17464

Drawings

FIG. 1 shows the effect of different concentrations of DSE medium seed soaking on corn roots. CK denotes seed soaking with sterile water, MMNCK denotes seed soaking with sterile MMN liquid medium, 20%, 40% and 60% denote seed soaking with 20% DSE medium, 40% DSE medium and 60% DSE medium, respectively; in the same figure, data marked with different lower case letters have significant differences, and data marked with the same lower case letters have no significant differences.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

In the following examples, the experimental inoculation method was carried out in a sterile operating table unless otherwise specified. DSE was co-cultured with corn seeds in a climatic chamber (RXZ Intelligent climatic chamber).

Example 1 Effect of different concentrations of DSE Medium on corn rooting

Dark Septal Endophyte (DSE) (Darksidea zeta) (named needle a 1-3): the obtained bacterial strain is separated and purified from the root system of the pinocembria arundinacea in the northern electric power winning mining area of the union of the autonomous region of inner Mongolia, and is identified as the dark-color septate endophytic fungi according to the following method: and detecting the ITS sequence of the strain, wherein the sequence is the sequence 1 in the sequence table.

The strain is preserved in China general microbiological culture Collection center (CGMCC) at 2019, 4 and 8 months, and the preservation number is CGMCC No. 17464.

The corn variety is Zhongnuo No. one (authentication No. 0103006-.

First, culture of DSE

PDA culture medium: mixing potato extract powder 3.0g, glucose 20.0g and agar 14.0g, adding distilled water 1000ml, boiling to dissolve, packaging, and autoclaving at 121 deg.C for 15 min.

The DSE was inoculated into PDA medium and cultured in dark for 15 days in an inverted state at 28 ℃.

Preparation of DSE culture solution

MMN liquid medium: adding CaCl₂0.05g，MgSO₄0.15g，NaCl 0.025g，FeCl₃0.01g，KH₂PO₄0.5g, 0.0001g of Vitamin B1 (thiamine), (NH)₄)₂HPO₄0.25g, 10g Glucose, 0.2g Citric acid and 10g Malt extract, adding 1000ml distilled water, packaging, and autoclaving at 121 deg.C for 15 min.

And (2) after the culture in the first step is finished, inoculating a fungus cake with the diameter of 6mm into an MMN liquid culture medium, carrying out shake culture at 25 ℃, wherein the rotation speed of a shaking table is 180r/min in the first day and 160r/min in the second day, obtaining a DSE culture solution after the culture is carried out for 15 days, and the dry weight of DSE hyphae in 1mL of the DSE culture solution (namely the dry weight of the hyphae after the DSE culture solution is filtered by filter paper and the hyphae is collected and dried to constant weight) is 2.34mg for later use.

Influence of DSE culture solution with different concentrations on plant rooting effect

Diluting the DSE culture solution obtained in the second step with sterile water to obtain three concentrations of culture solutions, namely 20% DSE culture solution (20% DSE culture solution: 20ml of the DSE culture solution obtained in the second step is taken and diluted to 100ml with sterile water), 40% DSE culture solution (40% DSE culture solution: 40ml of the DSE culture solution obtained in the second step is taken and diluted to 100ml with sterile water) and 60% DSE culture solution (60% DSE culture solution: 60ml of the DSE culture solution obtained in the second step is taken and diluted to 100ml with sterile water) for later use.

A sterile triangular flask (100ml) is divided into five groups of 3, and 5 waxy corn seeds are placed in each triangular flask. The three concentrations of DSE culture solution obtained above were added to three sets of triangular flasks, one concentration of DSE culture solution per set, ensuring 5ml of culture solution per flask (i.e. the solution just submerged half of the seeds). The remaining group was controlled with sterile water and the other group with sterile MMN broth. All triangle bottles were sealed with sterile PVA film. And adding a sterile MMN nutrient solution every 5 days for 15 days of co-culture, and carrying out statistical analysis on the growth condition of the corn root system by using CI600 software. Culturing in dark 3 days before, at 22 deg.C and air relative humidity of 70%; after three days of culture, the culture is carried out by illumination, the illumination intensity is 1800Lux (in the incubator, the illumination is in the range of 1500-.

The influence results of the DSE culture solution with different concentrations on the seed soaking and root promoting are shown in figure 1 and table 1, the average single-plant total root length and the average root diameter of the corn added with 20 percent of the DSE culture solution are obviously higher than the corresponding indexes of the root systems of other groups of plants, and the average single-plant root number of the corn added with 20 percent of the DSE culture solution and the average single-plant root number of the corn added with 60 percent of the DSE culture solution are obviously higher than those of other groups of plants. The 20% DSE culture solution is helpful for promoting corn seed rooting.

TABLE 1 influence of different concentrations of DSE medium seed-soaking on corn roots

In table 1, the total root length represents the average total root length per plant, and the number of roots represents the average number of roots per plant; in the data in the same column, the data marked with different lower case letters have significant difference, and the data marked with the same lower case letters have no significant difference.

<110> university of mineral industry (Beijing), Beijing Synbiotic ecological environmental engineering technology Limited

<120> an optimum amount of DSE inoculum for promoting plant rooting

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Claims

1. The method for promoting the rooting of the Zhongnuo No. one rice comprises the following steps: the tissue or organ of the medium glutinous rice I is treated by the dark-color endophytic fungi to promote the rooting of the medium glutinous rice I; the dark color septate endophytic fungi is a strain with the preservation number of CGMCC No.17464 in the China general microbiological culture Collection center.

2. The method of claim 1, wherein: waxy tissue or organ treated with a dark septal endophytic fungus comprises: and soaking the tissue or organ of the medium glutinous rice No. I with the liquid containing the dark septate endophytic fungi.

3. The method of claim 2, wherein: the liquid containing the dark septate endophyte has hypha dry weight of 0.468-1.404 mg/mL.

4. The method of claim 2, wherein: the liquid containing the dark septate endophyte is a culture solution of the dark septate endophyte or a dilution of the culture solution.

5. The method of claim 2, wherein: when the medium glutinous rice tissue or organ is soaked in the liquid containing the dark septal endophytic fungi, half of the medium glutinous rice tissue or organ is in the air.

6. The method of claim 1, wherein: the treatment time is 15 days;

and/or the treatment conditions are as follows: the first 3 days are carried out in the dark, the temperature is 22 ℃, and the relative humidity of air is 70 percent; after three days of treatment, the culture is carried out under illumination, the illumination intensity is 1500-.

7. The method according to any one of claims 1-6, wherein: the root promotion of the medium glutinous rice I is embodied in the aspects of promoting the elongation, the increase of the number of the roots and/or the thickening of the roots of the medium glutinous rice I;

and/or the medium glutinous rice first organ is medium glutinous rice first seed.

8. Use of any of the following methods of claims 1-7:

x1) production of glutinous rice one;

x2) planting glutinous rice I;

x3) promoting growth of medium waxy rice No. one;

x4) promoting land reclamation.