CN117887634B - A bacteria with growth-promoting effect and its application - Google Patents

Abstract

The invention discloses a bacterium with growth promoting effect and application thereof, and relates to the technical field of plant growth, wherein the bacterium is Bacillus sp, and the strain is registered and preserved in the university of Guangdong academy of sciences microbiological analysis and detection center of Guangdong province, and the preservation number is GDMCC.No. 64088. After the strain provided by the invention is inoculated to plants, the strain can be planted in the plants and can influence the growth of the plants, and after the strain provided by the invention is inoculated to wheat, the growth of the wheat can be promoted, so that a research basis is provided for the academic world to explore the flora structure and action mechanism of endophytes of the plants.

Description

A bacteria with growth-promoting effect and its application

Technical Field

The invention relates to the technical field of plant growth, and in particular to a bacterium with growth-promoting effect and application thereof.

Background technique

Endophytic bacteria are microorganisms that can colonize in healthy plant tissues and establish a harmonious relationship with plants. They can directly promote the growth of host plants. They have two main functions: first, plant endophytic bacteria can be used as biological control agents; second, the growth-promoting effect of plant endophytic bacteria. The growth-promoting effect of plant endophytic bacteria can promote plant growth by improving nutrient forms to promote absorption; enhancing tolerance to stresses such as cold, heat, and drought; enhancing plant disease resistance by antagonizing, competing, inducing, or activating plant systemic resistance; and producing (or synergizing) regulating plant hormones.

Since tissue preference and regional differences are the main factors affecting the distribution of endophytic bacteria in plants, the development and utilization of endophytic bacteria in plants has been hindered. In addition, there is no complete and suitable technical system for the utilization of endophytic bacteria in plants, which can effectively study endophytic bacteria in plants. Therefore, the understanding of the flora structure and mechanism of action of endophytic bacteria in plants has become a hot topic in academic research.

Summary of the invention

The purpose of the present invention is to provide a strain of bacteria with a growth-promoting effect and its application. The bacterium is an endophytic bacterium extracted from the aril of Paris polyphylla seeds, and belongs to the genus Bacillus sp. The strain has been deposited in the Guangdong Provincial Microbiological Collection Center (Guangdong Provincial Microbiological Analysis and Testing Center) on November 30, 2023, with a collection number of GDMCC.No64088, and the collection address is Building 59, No. 100 Xianlie Middle Road, Guangzhou.

The objective of the present invention is achieved through the following technical solutions:

The isolation and purification of the bacteria include:

A. Sampling: Collect the seeds of Yunnan Paris polyphylla, soak them in 75% ethanol for 1 min, rinse them with sterile water 3 times, soak them in sodium hypochlorite solution with 5% effective chlorine for 3 min, and rinse them with sterile water 4 times to disinfect the surface of Paris polyphylla seeds. The disinfected Paris polyphylla seeds are stored for later use. Use a 1000 μL pipette to take 120 μL of the sterile water used for the last wash, inoculate it on a beef extract peptone solid culture medium plate, and then spread it evenly with a sterile stainless steel coating rod. After completion, place it in a water-tight incubator for culture at 28 °C. After 3 days of culture, observe that no bacteria grow on the culture dish, indicating that the disinfection is successful.

B. Preparation of culture medium: Preparation of beef extract peptone culture medium: base beef extract: 3.0 g, peptone 10.0 g, sodium chloride 5.0 g, water 1000 ml, pH 7.4-7.6, agar 20 g;

1/2 PDA: 100 g potato, 10 g glucose, 20 g agar, 1000 ml water, natural pH.

C. Isolation and purification: Under sterile conditions, peel off the aril of the sterilized Polygonum yunnanensis seeds, put the aril of the Polygonum yunnanensis seeds in a sterile mortar, grind them thoroughly, and dilute them appropriately. Use a 1000 μL pipette to take 200 μL of the grinding liquid and inoculate it on a beef extract peptone medium plate, and then spread it evenly with a stainless steel coating rod. After completion, place it in a water-proof incubator at a temperature of 28 ℃ for 7 days. In this process, after the bacterial colonies grow, pick the grown colonies and inoculate them into new culture media for culture. Repeat several times and purify them by streaking to obtain a single clone.

D. Strain Identification:

1) Morphological identification: Refer to the results of the eighth edition of Microbiology Experimental Course edited by Zhou Deqing and Xu Deqiang, the eighth edition of Bergey's Manual of Bacterial Identification, and the bacterial classification table for routine identification of bacteria:

a. Colony characteristics of bacteria on solid culture medium: The colonies are yellow, round, highly transparent, with a smooth and sticky surface, and neat and moist edges.

b. Colony characteristics of bacteria in liquid culture medium: Select a small amount of bacterial moss, inoculate it into liquid culture medium, culture it in a shaking incubator at 25°C and 170 rpm for 24 h, and then let it stand for 24 h to observe the bacterial liquid.

The bacterial solution is uniformly turbid, the precipitate is granular, and there is no sterile film.

c. Gram staining: Prepare a clean slide, take a loop of water on the slide, use an inoculation loop to pick up a small amount of bacterial moss, spread it in the water, make it thin and even, and complete the smear. Fire the bacterial surface upward 2 to 3 times to complete the fixation. Add ammonium oxalate crystal solution to the fixed smear for 1 to 2 minutes, wash with water, and complete the staining. Add iodine solution to stain for 1 to 2 minutes, wash with water, and complete the mordant. Absorb the residual water, add 95% ethanol to the bacterial surface and shake it back and forth to decolorize, 30 seconds each time, repeat three times and immediately wash with water to complete the decolorization. Add safranin to stain for 2 to 3 minutes, wash with water, and complete the re-staining. After staining, observe the shape and color of the bacteria under a microscope.

Under the microscope, the bacteria are red, rod-shaped, and are Gram-negative bacteria.

2) Molecular identification: The purified bacteria were sent to Tianyi Huiyuan Biotechnology Co., Ltd. for sequencing. The 16S rDNA sequences of endophytic bacteria were amplified using 27F and 1492R primers. After sequencing, the 16S rDNA sequences of endophytic bacteria were compared and analyzed using BLAST in Geneious.

3) Physical and chemical property experiments: The bacteria were subjected to contact enzyme test. If bubbles were produced during the experiment, it meant that the bacteria was positive. The bacteria were subjected to oxidase test. If the filter paper turned red within 60 seconds during the experiment, it meant that the bacteria was positive. The bacteria were subjected to sugar fermentation test. If bubbles were produced during the experiment, it meant that the bacteria produced gas through fermentation.

Combining the morphological characteristics, molecular identification results and physicochemical properties experimental results, the strain was finally identified as Bacillus sp.

The beneficial effects of the present invention are:

After inoculating plants with Bacillus GDMCC 64088, it can colonize in the plant body and affect the growth of the plant. After inoculating wheat with the strain provided by the present invention, the growth of wheat can be promoted, providing a research basis for the academic community to explore the flora structure and action mechanism of plant endophytes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the morphology of the colonies of Bacillus GDMCC 64088 on a culture medium;

FIG2 is a microscopic image of Bacillus GDMCC 64088 after Gram staining;

Figure 3 shows the growth of wheat seedlings in the experimental group after 7 days of co-cultivation;

Figure 4 shows the growth of wheat seedlings in the control group after 7 days of co-cultivation.

Detailed ways

The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

Example 1: Isolation, screening and identification of Bacillus GDMCC 64088

A. Sampling: Collect the seeds of Yunnan Paris polyphylla, soak them in 75% ethanol for 1 min, rinse them with sterile water 3 times, soak them in sodium hypochlorite solution with 5% effective chlorine for 3 min, and rinse them with sterile water 4 times to disinfect the surface of Paris polyphylla seeds. The disinfected Paris polyphylla seeds are stored for later use. Use a 1000 μL pipette to take 120 μL of the sterile water used for the last wash, inoculate it on a beef extract peptone solid culture medium plate, and then spread it evenly with a sterile stainless steel coating rod. After completion, place it in a water-tight incubator for culture at 28 °C. After 3 days of culture, observe that no bacteria grow on the culture dish, indicating that the disinfection is successful.

B. Preparation of culture medium: Preparation of beef extract peptone culture medium: base beef extract: 3.0 g, peptone 10.0 g, sodium chloride 5.0 g, water 1000 ml, pH 7.4-7.6, agar 20 g;

1/2 PDA: 100 g potato, 10 g glucose, 20 g agar, 1000 ml water, natural pH.

C. Isolation and purification: Under sterile conditions, peel off the aril of the sterilized Polygonum yunnanensis seeds, put the aril of the Polygonum yunnanensis seeds in a sterile mortar, grind them thoroughly, and dilute them appropriately. Use a 1000 μL pipette to take 200 μL of the grinding liquid and inoculate it on a beef extract peptone medium plate, and then spread it evenly with a stainless steel coating rod. After completion, place it in a water-proof incubator at a temperature of 28 ℃ for 7 days. In this process, after the bacterial colonies grow, pick the grown colonies and inoculate them into new culture media for culture. Repeat several times and purify them by streaking to obtain a single clone.

D. Strain Identification:

1) Morphological identification: Refer to the results of the eighth edition of Microbiology Experimental Course edited by Zhou Deqing and Xu Deqiang, the eighth edition of Bergey's Manual of Bacterial Identification, and the bacterial classification table for routine identification of bacteria:

a. Colony characteristics of bacteria on solid culture medium: The colonies are yellow, round, highly transparent, with a smooth and sticky surface, and neat and moist edges.

b. Colony characteristics of bacteria in liquid culture medium: Select a small amount of bacterial moss, inoculate it into liquid culture medium, culture it in a shaking incubator at 25°C and 170 rpm for 24 h, and then let it stand for 24 h to observe the bacterial liquid.

The bacterial solution is uniformly turbid, the precipitate is granular, and there is no sterile film.

c. Gram staining: Prepare a clean slide, take a loop of water on the slide, use an inoculation loop to pick up a small amount of bacterial moss, spread it in the water, make it thin and even, and complete the smear. Fire the bacterial surface upward 2 to 3 times to complete the fixation. Add ammonium oxalate crystal solution to the fixed smear for 1 to 2 minutes, wash with water, and complete the staining. Add iodine solution to stain for 1 to 2 minutes, wash with water, and complete the mordant. Absorb the residual water, add 95% ethanol to the bacterial surface and shake it back and forth to decolorize, 30 seconds each time, repeat three times and immediately wash with water to complete the decolorization. Add safranin to stain for 2 to 3 minutes, wash with water, and complete the re-staining. After staining, observe the shape and color of the bacteria under a microscope.

Under the microscope, the bacteria are red, rod-shaped, and are Gram-negative bacteria.

2) Molecular identification: The purified bacteria were sent to Tianyi Huiyuan Biotechnology Co., Ltd. for sequencing. The 16S rDNA sequences of endophytic bacteria were amplified using 27F and 1492R primers. After sequencing, the 16S rDNA sequences of endophytic bacteria were compared and analyzed using BLAST in Geneious.

3) Physical and chemical property experiments: The bacteria were subjected to contact enzyme test. If bubbles were produced during the experiment, it meant that the bacteria was positive. The bacteria were subjected to oxidase test. If the filter paper turned red within 60 seconds during the experiment, it meant that the bacteria was positive. The bacteria were subjected to sugar fermentation test. If bubbles were produced during the experiment, it meant that the bacteria produced gas through fermentation.

Combining the morphological characteristics, molecular identification results and physical and chemical properties experimental results, the strain was finally identified as Bacillus sp.

Example 2: Effect of Bacillus GDMCC 64088 on wheat growth

A. Prepare bacterial liquid: Use an inoculation loop to pick up a small amount of bacterial moss and inoculate it into beef extract peptone liquid culture medium (10 ml/tube). Incubate in a shaking incubator at 25°C and 170 rpm for 24 h. Set aside.

B. Wheat seed treatment: Disinfect the surface of wheat seeds with 75% ethanol for 3 minutes, then soak with sterile water for 2 minutes, repeat 5 times, and finally use sterile filter paper to absorb the moisture, let it dry naturally for 12 hours, and then set aside.

C. Growth promotion culture: Take a 350 mL tissue culture bottle, pour 50 mL of 1/2 PDA medium into it, sterilize, and set aside after solidification. Take 10 μL of bacterial solution and inoculate it into the tissue culture bottle, and then evenly spread the bacterial solution on the 1/2 PDA medium with a stainless steel coating rod. Finally, use sterile tweezers to inoculate the surface-sterilized wheat seeds into the tissue culture bottle. Inoculate 12 wheat seeds in each tissue culture bottle as the experimental group, and set up three replicates. Directly inoculate the surface-sterilized wheat seeds in the tissue culture bottle without bacterial solution as the control group. Similarly, 12 wheat seeds per bottle, a total of three replicates. After completion, place it in an artificial climate incubator for 7 days. The conditions of the artificial climate incubator are set to 28 ℃, 39% humidity, and the light and dark time are 14 h and 10 h, respectively. After 7 days, the survival of wheat seedlings was observed, and their root length, plant height, fresh weight and root number were measured. The plant height was measured from the base of the plant stem to the petiole of the first true leaf. The root length was measured from the three longest roots on the left, middle and right, and the average was taken as the final root length. The root number was based on the roots grown on the wheat embryo.

The statistical data on the effect of Bacillus GDMCC 64088 strain on the growth of white wheat seedlings are shown in Table 1; the growth of wheat seedlings in the experimental and control groups co-cultivated for 7 days are shown in Figures 3-4.

Table 1 Determination of morphological indexes of co-cultured wheat seedlings

Group Plant height/cm Plant height growth rate/% Root length/cm Root length growth rate/% Number of roots Root number growth rate/% Fresh weight/g Fresh weight growth rate/% Control group 3.15±2.8 - 2.29±1.3 - 6 - 0.098 - test group 7.96±.4.4 152.7 3.12±1.4 36.2 7 16.6 0.185 88.7

Summarize:

Bacillus GDMCC 64088 strain significantly promoted the growth of wheat seedlings. The plant height, root length, root number and fresh weight of wheat seedlings co-cultured with Bacillus GDMCC 64088 strain increased compared with those of the control. The growth rate of plant height was 152.7%, the growth rate of root length was 36.2%, the growth rate of root number was 16.6%, and the growth rate of fresh weight was 88.7%.

The above is only a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art do not deviate from the spirit and scope of the present invention, and should be within the scope of protection of the claims attached to the present invention.

Claims (2)

1.A bacterium having a growth promoting effect, characterized by: the bacteria are of the genus Bacillus (Bacillus sp.) and the strains have been deposited under accession number gdmcc.no 64088 at the institute of microbiology (microbiological analysis and detection center, cantonese province).

2. Use of the bacterium according to claim 1 in wheat planting.