CN116590199B - Paenibacillus piri and application thereof in prevention and treatment of corn ear rot - Google Patents
Paenibacillus piri and application thereof in prevention and treatment of corn ear rot Download PDFInfo
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Abstract
The invention discloses paenibacillus piri which is paenibacillus piri SAAS-3-B4 and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M20221703 and application thereof in corn ear rot. The in vitro antagonistic flat plate counter experiment and in vivo antagonistic leaf in vitro experiments prove that the strain can better prevent and treat corn ear rot caused by Fusarium verticillium. As a biological control material for corn ear rot, the strain has good application prospect.
Description
Technical Field
The invention relates to the field of biological control of plant diseases, in particular to paenibacillus piri and application thereof in control of corn ear rot.
Background
Endophyte is a microorganism that survives within plants but does not affect the host, and that is living in the plant cell environment to perform symbiotic specific functions, such as the synthesis of secondary metabolites or signal molecules as internal and external stimuli in the reciprocal process. Endophytes are a source of novel biomolecules for the biochemical and pharmaceutical industries. They produce bioactive metabolites including immunosuppressive compounds, anticancer agents, plant growth promoters, antibacterial volatiles, pesticides, antioxidants and antibiotics, and have great application potential in medicine, pharmaceutical industry. In addition, the endophyte can promote the growth of plants under severe conditions such as nutritional stress, temperature stress, salinity stress, trace metal stress or drought stress, and the growth of plants in polluted environments is assisted by degrading harmful compounds, so that the endophyte is also an important research object and resource treasury in the agricultural field. In addition, the endophytes are also an important treasury of plant disease biocontrol bacteria resources. In recent years, scientists have also screened a plurality of biocontrol bacteria for dealing with important plant diseases, so that the biocontrol technology is gradually paid attention to.
Corn is one of the main food crops in china and is one of the crops whose global production is currently in the first place. The total amount of planting area of the corn is only lower than that of wheat and rice. The corn ear rot is an infectious disease which occurs in the middle and later stages of the growth and development of corn, and is found in all large corn producing areas in the world, the loss rate in the field is generally 5% -10%, and the serious loss rate can reach more than 50%. These fungal diseases not only directly cause significant production and economic losses of corn and wheat crops, but also the mycotoxin products and many secondary metabolic reaction products caused by the infection of these pathogenic bacteria seriously jeopardize the safety quality of corn grain production, and long-term eating of these corn grains polluted and deteriorated by mycotoxins also seriously jeopardize the reproductive health of human life and domestic fowl and livestock, and even cause death.
The damage caused by corn ear rot to corn yield is increasing, and selecting corn disease-resistant varieties and using chemical microbiocides are common effective means for preventing corn ear rot. However, since selecting disease-resistant varieties consumes huge time and energy and a large amount of chemical agents are adopted to prevent the problems of pollution, ecological imbalance and the like caused by the corn ear rot, an effective and safe corn ear rot control scheme must be searched.
In view of this, the present invention has been made.
Disclosure of Invention
Aiming at the problems, the invention provides paenibacillus piri and application thereof in preventing and controlling corn ear rot.
Research has found that Fusarium verticillium (Fusarium verticillioides) is one of the most well known diseases during the growth and development of corn, which is often affected by a variety of pathogenic bacteria. Fusarium verticillium can infect multiple crops such as corn, sorghum, wheat and the like, causing a plurality of epidemic diseases such as seedling blight, stem rot, ear rot, seed rot and the like of corn.
Wherein, the corn ear rot caused by the fusarium verticillium is changed into: during the corn kernel filling period, fusarium verticillium can infect corn kernels or cobs, causing cobs and kernels to mould and rot, thus severely affecting corn yield. Based on the above, the present invention screens out the above paenibacillus pierce from a plurality of strains and uses it for controlling corn ear rot caused by fusarium verticillium.
In a first aspect, the present invention provides a paenibacillus piri, named paenibacillus piri SAAS-3-B4, deposited in the chinese collection of typical cultures; the preservation unit address is Chinese, wuhan, university of Wuhan; the preservation number is CCTCC NO, M20221703; the preservation time is 2022, 10 and 31 days; the taxonomical name of this strain is Paenibacillus peoriae.
According to the invention, more than one hundred strains of strains are obtained from a corn sample collected from a farm laboratory test station in Shanghai city, 1 antagonistic strain for better preventing and treating corn ear rot is obtained through screening, and through further research, the 16S rDNA sequence of the antagonistic strain is shown as SEQ ID NO:1, belonging to Paenibacillus peoriae strains, which were designated as Paenibacillus piri SAAS-3-B4.
In a second aspect, the invention also provides application of the paenibacillus piri in corn disease control.
In a third aspect, the invention also provides an application of the paenibacillus piri in controlling plant diseases caused by fusarium verticillium.
In a fourth aspect, the present invention also provides a Fusarium verticillium inhibitor comprising the Paenibacillus piri described above; the live bacteria content of the Paenibacillus aurei in the Fusarium verticillium inhibitor is 10 10～1011 CFU/Kg.
In a fifth aspect, the invention also provides a microbial inoculum for preventing and treating corn ear rot, the microbial inoculum comprises the paenibacillus piri, and the living bacterial content of the paenibacillus piri in the microbial inoculum is 10 10～1011 CFU/Kg.
In a sixth aspect, the invention also provides a method for preventing and treating corn ear rot, which comprises applying the bacterial liquid of paenibacillus piri or the bacterial agent to diseased parts of plants.
The invention has the following beneficial effects:
The invention separates and identifies endophytic bacteria of corn plants, screens an antagonistic strain capable of better preventing and controlling corn ear rot caused by fusarium verticillium through an in vitro antagonistic flat plate counter experiment and an in vivo antagonistic leaf in vitro experiment, and further, has good application prospect no matter developing new biopesticide or biocontrol microbial inoculum as a biological prevention and control material for corn ear rot.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the result of the facing culture of Paenibacillus cereus SAAS-3-B4 in example 1;
FIG. 2 is a colony morphology and gram stain of Paenibacillus piri SAAS-3-B4 of example 1;
FIG. 3 is a phylogenetic tree of Paenibacillus piri SAAS-3-B4 in example 1;
FIG. 4 is a graph showing the effect of Paenibacillus piri SAAS-3-B4 in example 2 on controlling maize leaves;
FIG. 5 is a graph showing the leaf area statistics under different experimental conditions in example 2;
FIG. 6 is a comparison of potting results in example 2.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
This example is the isolation, screening and identification of Paenibacillus piri SAAS-3-B4
1. Material acquisition:
(1) Corn samples collected at a laboratory test station of Shanghai national academy of agricultural sciences are placed in sterile bags and stored in a 4 ℃ refrigerator of a timely transportation laboratory.
(2) Classifying and marking according to the sample collecting parts, respectively washing corn samples with sterile water for 5min, washing for 2 times, soaking with 75% alcohol solution for 3min, and washing with sterile water for 3 times; then, the mixture was immersed in a 3% sodium hypochlorite solution for 3min, and finally, the mixture was rinsed with sterile water for 4 times.
2. Isolation of endophyte:
(1) The method for separating the crop endophytes comprises the following steps: according to different sampling positions, the corn samples are divided into 6 types, namely sweet corn leaves, waxy corn leaves, corn ears, corn aerial roots, corn stems and corn roots. The sterilized corn samples were cut into small pieces by a sterile surgical scissors and put into a mortar for grinding to obtain a ground sample suspension, the sample suspension was diluted to 10 -1、10-2、10-3 times of dilution, 200 μl of each of the different dilutions was sequentially extracted and respectively applied to three solid media (NB, PDA and YPD), and each treatment was repeated three times. During the cultivation, colonies and mycelia growing on the plates were observed daily, and the grown colonies and mycelia were transferred to a new plate medium in time for the next step of isolation and purification.
(2) And (3) surface disinfection effect inspection: in the process of sterilizing the corn surface, a set of blank control experiments are required to check whether the sterilization is thorough or not so as to ensure that the separated endophytes are not polluted by microorganisms attached to the corn surface. The specific method comprises the following steps: the wash solution from the last sterile water wash of corn surface was collected and spread on NB planar medium and co-cultured with the experimental group.
If no colony grows in the blank control plate, the corn surface is thoroughly disinfected and sterilized, and the colony grown by the experimental group is corn endophyte. If colonies grow out in the blank control plate, the surface of the corn is proved to be not thoroughly disinfected and sterilized, and non-corn endophytes are mixed in.
3. Purification of endophyte:
Colonies growing in the three media were observed and colonies were selected for further culture on new solid media. Purified maize endophyte was stored in a refrigerator at 4℃and-80℃respectively.
4. Extraction of genomic DNA:
(1) Performing amplification culture on the purified single colony, picking 1-2-ring thalli from an amplification culture plate, uniformly mixing in 200 mu L of sterile water, adding a proper amount of 0.5mm glass beads, adopting a DNA rapid preparation system, extracting DNA by shaking to obtain crude extract of corn endophytic bacteria DNA, centrifuging, and taking 1 mu L of supernatant as template DNA for subsequent PCR amplification.
(2) 16S rDNA Gene amplification: 1 PCR tube (200. Mu.L) was used, and the mixture was sequentially added according to the composition shown in Table 5, and the mixture was divided into 2 PCR tubes (25. Mu.L). The general primers for bacterial 16S rDNA were used, and the primer sequences are shown in Table 1.
TABLE 1 primers for identifying species classifications
(3) Detecting PCR products by gel electrophoresis: ① Agarose gel was prepared at a concentration of 1%. ② Dye (20 ml plus 1 μl) was added, and the mixture was poured into a gel making tank into which a comb was inserted. ③ And (5) gelling and fixing, pulling out the comb, and putting the gel making groove into the electrophoresis groove. ④ The corresponding substances were added 5. Mu.L each to each well. ⑤ And taking out the gel after electrophoresis, and putting the gel into a gel imaging analyzer. ⑥ The electrophoresis results were observed with a gel imaging analyzer.
(4) 16S rDNA sequence determination: and (3) sending the PCR amplified product to Shanghai biological company for sequencing, and completing sequence splicing by using DNAMAN software according to the obtained sequencing result. The spliced sequences were aligned using the NCBI BLAST tool.
Through analysis, 148 bacterial strains were obtained in this example, and these 148 bacterial strains were distributed among 16 families and 26 genera, wherein the genera with higher abundance were Bacillus (Bacillus), escherichia (Enterobacter), and Pantoea (Pantoea), respectively.
5. Screening of strains
(1) Primary screen
Screening antagonistic bacteria of 148 corn endophytes by a plate counter experiment: inoculating Fusarium verticillium serving as a pathogenic bacterium to a PDA culture medium for activation, taking a bacterial cake from a pathogenic bacterium flat plate by using a puncher with the aperture of 5mm, placing the bacterial cake in the center position of a new PDA flat plate, inoculating 4 different corn endophytic bacteria in the cross direction of the bacterial cake, culturing at the constant temperature of 28 ℃ for 5 days, and checking whether a bacteriostasis ring appears.
(2) Double screen
Antagonistic strains obtained in Primary Inspection screens were subjected to a plate counter method to determine the in vitro antagonistic capacity: the bacterial cake is obtained from the pathogenic bacteria flat plate by using a puncher with the aperture of 5mm, the bacterial cake is placed at the center of a new PDA flat plate, antagonistic bacteria are inoculated at the position 5cm away from the bacterial cake in the cross direction of the center of the flat plate, the antagonistic bacteria at four inoculated positions are connected into a square shape, each treatment is repeated three times, the constant temperature culture is carried out at 28 ℃, the diameter of bacterial colony is measured when the pathogenic bacteria hypha of a control group is fully paved in a culture dish, and the bacteriostasis rate of different antagonistic bacteria is calculated.
Inhibition (%) = (control colony diameter-experimental colony diameter)/control colony diameter×100
The experimental results are shown in FIG. 1. According to statistics, the paenibacillus piri SAAS-3-B4 has the best antibacterial effect, and the average antibacterial rate is 64%.
6. Identification of strains
(1) Morphological observation
Endophyte colony morphology: after 24h incubation of the coated medium plates, single colonies were observed and the colony size, color, appearance, boundary, permeability, etc. were recorded. Endophytic bacteria were stained by gram staining and observed with an optical microscope.
Endophytic fungus mycelium morphology: the mycelia of the pathogenic B17-1 strain were observed in morphology after 5d culture on PDA medium. Inoculating endophytic fungus spores, shake culturing at 28deg.C and 200rpm in sterile liquid culture medium for 3d, and observing the characteristics such as spore shape by using an optical microscope.
According to observation, the paenibacillus piri SAAS-3-B4 is in a rod shape, so that spores can be generated, and single bacterial colonies of the paenibacillus piri SAAS-3-B are irregularly round, convex on the surface, fine and opaque, pale yellow in bacterial colony color and complete in boundary. The result of the gram staining is red purple, and the identification of the Paenibacillus piri SAAS-3-B4 as gram negative bacteria is proved. As shown in fig. 2, the left side is a colony morphology; on the right are gram staining charts.
And (3) analyzing the evolutionary tree: 16S rDNA and gyrB gene sequence analysis of Paenibacillus piri SAAS-3-B4 and phylogenetic tree establishment: the PCR amplification of the 16S rDNA gene fragment and the gyrB gene was completed using the antagonistic strain genomic DNA as a template, the amplification system and the procedure for the 16S rDNA gene fragment amplification were as shown in Table 2, and the amplification system and the procedure for the gyrB gene amplification were as shown in Table 3.
TABLE 2 System and procedure for 16S rDNA amplification of Gene
TABLE 3 procedure for gene gyrB amplification
The amplified products were sent to Shanghai Biotechnology Inc. for sequencing, and the resulting 16S rDNA and gyrB sequencing results were aligned in NCBI, from which 8 Paenibacillus species and corresponding genomes were selected as the outer population, scopulibacillus daqui. And (3) comparing by using a ClustalW method in MEGA 7 to construct a phylogenetic tree.
Phylogenetic tree As shown in FIG. 3, paenibacillus piri SAAS-3-B4 (3-B4 for short) is in the same branch as Paenibacillus piri (Paenibacillus peoriae), so 3-B4 is considered Paenibacillus piri (Paenibacillus peoriae).
Example 2
The embodiment is the indoor disease prevention effect verification of paenibacillus piri SAAS-3-B4
1. Cultivation of maize seedlings
Taking Shen Kenuo corn seeds, washing with sterile water for about 5min, washing for 2 times, soaking with 75% alcohol solution for 3min, and washing with sterile water for 3 times; then, the mixture was immersed in a 3% sodium hypochlorite solution for 3min, and finally, the mixture was rinsed with sterile water for 4 times.
60 Corn seeds are selected and soaked in warm water with the water temperature of 35 ℃ for 4 hours, two layers of sterilized filter paper are paved on 4 large flat plates, water is added for soaking, the corn seeds are divided into 4 parts, and every 15 corn seeds are evenly paved in the flat plates. Placing the plant into a plant tissue culture chamber for culture.
2. Determination of biological control effect of antagonistic strain on corn ear rot
In order to further verify the resistance function of Paenibacillus piri SAAS-3-B4 to corn ear rot, corn leaf in vitro verification experiments are carried out, the experiments are divided into 4 groups, and the leaves are inoculated with pathogenic bacteria Fusarium verticillium (B17-1) by a needle-punching inoculation method. The treatment method of the experimental group is shown in table 4:
TABLE 4 in vitro blade test treatments
The experimental treatments were divided into 4 groups according to the above table, and leaf spot sizes were measured after 5d of indoor culture to observe the disease conditions of maize leaves. Wherein the experimental group 3 (the pinhole inoculation pathogenic bacteria B17-1, spraying 3-B4 suspension after 12 hours) is a treatment group; experimental group 4 (spraying 3-B4 suspension, pinhole inoculation of pathogenic bacteria B17-1 after 12 h) was a prophylactic group. The plaque area size of each experimental group was measured and statistically analyzed using ImageJ software, and the experimental results are shown in fig. 4.
As shown in fig. 5, the control treatment of the single inoculated pathogen in the experimental group 2 had an average plaque area of 0.8cm 2, the treatment group had an average plaque area of 0.29cm 2, and the prevention group had an average plaque area of 0.40cm 2, both of which had an average plaque area smaller than that in the experimental group 2.
As shown in the experimental results of the potted plant in FIG. 6, the group A is a control group without SAAS-3-B4 sprayed thereon, the disease spots are in an extended and enlarged shape, the leaves are curled and rotten, the group B is an experimental group, the disease spots have obvious limits, and the area of the disease spots is far smaller than that of the control group, which indicates that the paenibacillus piri SAAS-3-B4 has obvious inhibition effect in the treatment and prevention of the spike rot.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. The paenibacillus piri is characterized in that the paenibacillus piri (Paenibacillus peoriae) is paenibacillus piri SAAS-3-B4, and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M20221703.
2. Use of paenibacillus piri according to claim 1 for the treatment and prevention of ear rot of corn caused by fusarium verticillium.
3. A microbial agent for preventing and treating corn ear rot, which is characterized by comprising the paenibacillus pierce according to claim 1;
the live bacteria content of Paenibacillus piri in the microbial inoculum is 10 10～1011 CFU/Kg.
4. A method for controlling corn ear rot caused by fusarium verticillium, characterized in that the bacterial liquid of paenibacillus piri according to claim 1 or the bacterial agent according to claim 3 is applied to diseased parts of plants.
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