CN111269838A - Method for inducing and separating antagonistic bacteria in soil by barley grains infected with gibberellic disease - Google Patents
Method for inducing and separating antagonistic bacteria in soil by barley grains infected with gibberellic disease Download PDFInfo
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Abstract
The invention discloses a method for inducing and separating antagonistic bacteria in soil by barley kernels infected with gibberellic disease, which comprises the steps of burying the barley kernels full of the gibberellic disease in the soil, inducing the antagonistic bacteria in the soil to be attached to the infected barley kernels, and then selecting the antagonistic bacteria which can be fixedly planted on the barley kernels through a bacteriostatic antagonistic experiment. Compared with the traditional method for directly screening the antagonistic bacteria from the soil, the method has the advantages of pertinence, strong practicability, simplicity, effectiveness, small workload, high screening efficiency, high accuracy and easier growth of the screened antagonistic bacteria on the barley grains.
Description
Technical Field
The invention relates to the technical field of microbiology, in particular to a method for inducing and separating antagonistic bacteria in soil by barley grains infected with gibberellic disease.
Background
Wheat scab is also called wheat head withering, wheat head rotting and red wheat head, and is one of the main diseases of wheat. Wheat scab is common worldwide and is mainly distributed in humid and semi-humid areas, especially in temperate areas with humid and rainy climate. The seedling can be damaged from seedling to ear emergence, and the seedling is mainly withered, stalk-base rot, stalk rot and ear rot, wherein the most serious damage is ear rot. Scab is caused by a variety of fusarium, which can parasitize in soil to fly into the air under suitable climatic conditions to infect barley and wheat, causing scab to occur. For example, in winter wheat, such as rainy winter, winter rain provides proper conditions for the growth of gibberellic disease in soil, and provides a large amount of initial bacterial source for the occurrence of gibberellic disease in the next spring, resulting in severe occurrence of gibberellic disease in wheat in the next spring.
The existing control methods of wheat scab comprise physical control, chemical fungicide control, disease-resistant wheat variety breeding and biological control. The biological control is a new method for effectively controlling crop diseases by utilizing beneficial microorganisms and microbial metabolites, and the microbial control can solve the problems of drug resistance, phytotoxicity and the like of chemical control. Although gibberellic fungi can propagate and grow in soil, because the soil contains a large amount of antagonistic bacteria for inhibiting the mass propagation of the gibberellic fungi, spores or sporangia of the gibberellic fungi are rarely observed on the surface of the soil. The microorganisms used for biological control are antagonistic bacteria screened from soil, and comprise bacteria, fungi, a small amount of actinomycetes and the like. How to separate the antagonistic bacteria which can inhibit the growth of the gibberellic disease from the soil is an important link of biological control. The method for separating antagonistic bacteria from soil generally comprises the steps of separating all floras in the soil, and then screening beneficial bacteria with antagonistic action by using an oxford cup method as an inhibition effect experiment. However, the soil is the major nutrient for the life of microorganisms, and 1g of the soil is presentUp to 106～109If soil microorganism separation is carried out firstly and then an antibacterial test is carried out on each microorganism individual, the large soil separation workload is easily caused, and effective microorganisms with inhibiting effects are easily leaked.
Disclosure of Invention
The invention aims to provide a method for inducing and separating antagonistic bacteria in soil by utilizing barley grains infected with gibberellic disease.
The technical scheme for realizing the purpose of the invention is as follows: a method for inducing and separating antagonistic bacteria in soil by using barley grains infected with gibberellic disease comprises the following steps:
1) preparation of barley kernels carrying gibberellic disease: filling the steamed or boiled barley grains into a culture bottle, sterilizing at high temperature, cooling to room temperature, inoculating the gibberellic disease into the bottle filled with the barley grains, culturing at 25-26 ℃ for 5-6 days, and shaking the culture bottle for 2-3 times during the period to ensure that the barley grains are fully full of the gibberellic disease;
2) wrapping barley grains containing gibberellin with gauze, wrapping several barley grains full of gibberellin respectively with several sterile gauzes, tying and tying labels one by one;
3) burying barley grains: burying the barley grains wrapped by each sterile gauze in the soil at a depth of 10-20 cm, and taking out after 25-30 days;
4) bottling: respectively filling barley grains wrapped in each sterile gauze taken out of the soil into a sterilized glass bottle, and marking for later use;
5) preparing a culture dish: taking out the barley grains in each glass bottle, cleaning, sterilizing, cutting the barley grains on sterile filter paper, and then subpackaging the barley grains on flat culture mediums in a plurality of culture dishes for culture;
6) observing the development condition of the barley grain hyphae infected with the bacteria on the culture dish after the culture is carried out for 3-5 days in the step 5), and selecting microbial colonies which are not obviously expanded by the red gibberellic disease and can inhibit the growth of the barley grain hyphae in the corresponding culture dish for carrying out separation culture;
7) screening of antagonistic bacteria: selecting microbial colonies which are selected in the step 6) and subjected to separation culture, respectively coating the microbial colonies on a plate culture medium of a plurality of new culture dishes according to the shape of the Chinese character 'ji', placing one barley grain which is prepared in the step 1) and carries the gibberella zeae in the middle of each culture dish, sealing the edge of the plate culture medium by using a sealing film, culturing for 7-8 days at the temperature of 25-26 ℃, and selecting the microbial colonies in the corresponding culture dishes which do not obviously expand the gibberella zeae.
Further, picking the microbial colonies screened in the step 7), repeating the screening for a plurality of times according to the operation of the step 7), and purifying to obtain single colonies. Compared with microbial colonies, the single bacterial colony has the advantages of high stability and difficult variation.
Further, the method also comprises the step 8) of secondary screening of antagonistic bacteria: in particular, the amount of the solvent to be used,
8.1) selecting a single colony with an antibacterial effect screened in the step 7) and inoculating the single colony into a sterilized barley grain culture bottle, simultaneously inoculating two barley grains which are cultured in the step 1 and carry the fusarium graminearum, wherein a gap for enabling hyphae to grow upwards is formed between the barley grains in the barley grain culture bottle and a bottle opening, then injecting 1mL of sterile water into the barley grain culture bottle, sealing the opening, culturing at 25-26 ℃ for 5-7 d, shaking the glass bottle every day in the culture period, and finally standing for 1-2 d to observe whether the hyphae are inhibited;
8.2) observing the hypha bacteriostasis effect in the barley grain culture bottle in the step 8.1), and selecting the microbial bacteria in the corresponding culture bottle without obvious expansion of the red gibberellic disease as the target antagonistic bacteria.
And step 8) carrying out secondary screening on the single colony with the bacteriostatic action obtained in the step 7) to obtain antagonistic bacteria with better effect of inhibiting the gibberellic disease when the microorganisms with antagonistic action on the gibberellic disease and harmful gibberellic disease simultaneously infect organisms.
Furthermore, fields planted with overlarge wheat and the fields planted with the barley grains in the step 3) are selected, and especially the fields with annual scab are easy to generate antagonistic bacteria.
Further, the high-temperature sterilization process in the step 1) comprises the following steps: bottling the steamed or boiled barley grains, and sterilizing at 120-125 ℃ for 20-25 min;
the inventors have found that antagonistic bacteria exist in soil, and barley scab mainly occurs at ears, so the present invention screens antagonistic bacteria having a certain ability to colonize barley plants by burying barley grains full of gibberellic disease in soil, inducing the antagonistic bacteria in the soil to adhere to the barley grains, and separating the antagonistic bacteria colonized on the barley grains. Compared with the traditional method for directly screening antagonistic bacteria from soil, the method has the advantages of pertinence, strong practicability, simplicity, effectiveness, small workload, high screening efficiency and high accuracy.
Drawings
FIG. 1 is a diagram showing the development of hyphae of the infected barley grain Fusarium in the culture dish of the example having a good inhibitory effect in step 6);
FIG. 2 is a diagram showing the development of hyphae of the infected barley grain Fusarium in the culture dish in the bacteriostatic antagonistic experiment in step 7), wherein the two left culture dishes are blank controls;
FIG. 3 is a graph showing the development of mycelia of barley kernels carrying Fusarium graminearum in the culture flask inoculated with the antagonistic bacteria obtained in step 7) in step 8.1).
Detailed Description
The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
A method for inducing and separating antagonistic bacteria in soil by using barley grains infected with gibberellic disease comprises the following steps:
1) preparation of barley kernels carrying gibberellic disease: sterilizing a steamed or boiled barley grain bottling culture bottle at 120-125 ℃ for 20-25 min, cooling to room temperature, inoculating gibberellic disease into the barley grain bottling culture bottle, culturing for 5-6 d at 25-26 ℃, and shaking the culture bottle for 2-3 times during the period to ensure that the barley grain is fully full of the gibberellic disease;
2) wrapping barley grains containing gibberellin with gauze, wrapping barley grains of Fusarium full of gibberellin with several sterile gauzes, tying labels; each piece of gauze can wrap 100 barley grains;
3) burying barley grains: burying the barley grains wrapped by each sterile gauze in a place which is 10-20 cm below the soil, and taking out after 25-30 days; selecting field blocks planted with overlarge wheat and the like from soil buried by barley grains;
4) bottling: filling the barley grains wrapped in the sterile gauze taken out of each soil into a sterilized glass bottle, and marking for later use;
5) preparing a culture dish: taking out the barley grains in each glass bottle, washing the barley grains with tap water, treating the barley grains with 75% ethanol for 10s respectively, then disinfecting the barley grains with 2% NaClO for 15min, then washing the barley grains with sterile water for 3 times, cutting the barley grains on sterile filter paper, and then culturing the barley grains on a PDA culture medium for 3-5 d; placing 4-5 barley grains in each culture dish, making three repeated culture dishes for each small bottle, and placing the barley grains with gibberellin which are not buried in one culture dish for comparison;
6) observing the development condition of the barley grain hyphae infected with the bacteria on the culture dish after being cultured for 3-5 days in the step 5), and selecting microbial colonies capable of inhibiting the growth of the barley grain hyphae for isolated culture; wherein, fig. 1 is a culture dish with better inhibition effect, the growth of the red gibberellic disease in the culture dish is inhibited, and the culture dish with poor inhibition effect shows that the red gibberellic disease grows out of the whole culture dish;
7) primary screening of antagonistic bacteria: selecting microbial colonies with antagonism in the step 6), respectively coating the selected microbial colonies with a zigzag shape on plate culture mediums of twenty-four new culture dishes, placing one barley grain carrying the fusarium graminearum prepared in the step 1) in the middle of each culture dish for an antibacterial antagonism experiment, setting a culture dish which does not have antagonistic bacteria and only has the fusarium graminearum as a blank control, sealing the edge of the plate culture medium by using a sealing film, culturing for 7-8 days at 25-26 ℃, screening out microbial colonies with good fusarium graminearum inhibition effect, repeating the step 7) for four times, and purifying and selecting single colonies which are not easy to mutate after propagation; as shown in fig. 2, two left culture dishes are blank controls, and the other 24 culture dishes have the bacteriostatic effect of the selected antagonistic bacteria with bacteriostatic effect. The bacteriostatic condition is observed through the back of the culture medium, so that the whole culture dish of a blank control group is full of the hypha of the fusarium graminearum, and the hypha of the barley grains infected with bacteria in the middle of the other culture dishes is obviously inhibited and cannot be expanded;
8) secondary screening of antagonistic bacteria:
8.1) selecting a single colony with the bacteriostatic action in the step 7) to inoculate in a sterilized barley grain culture bottle, simultaneously inoculating two barley grains which are cultured in the step 1 and carry the fusarium graminearum, wherein the distance between the barley grains in the barley grain culture bottle and a bottle opening is 2cm for enabling hyphae to grow upwards, injecting 1mL of sterile water into the barley grain culture bottle, sealing the opening, culturing for 7 days at the temperature of 25-26 ℃, shaking the glass bottle every day for the first 5 days, and stopping shaking and standing for 2 days after 5 days to observe whether the hyphae are inhibited or not;
8.2) observing the hypha bacteriostasis effect in the barley grain culture bottle in the step 8.1), as shown in figure 3, the antagonistic bacteria in the left 4 culture bottles can be rapidly propagated in the barley grains and can effectively inhibit the growth of the fusarium graminearum so that the fusarium graminearum can not grow, and the corresponding microbial bacteria in the culture bottles are the target antagonistic bacteria of the invention.
Wherein, the bacteriostasis condition of each culture dish in the step 7) is shown in the following table 1:
TABLE 1
Hypha growth inhibition ratio (%) in table 1 = (hyphal growth diameter of CK group-colony diameter of example group)/hyphal growth diameter of CK group [ ] 100%
As can be seen from Table 1, the bacteriostatic effects of the compositions 5, 10, 15, 18, 20, 21 and 22 are better, and the bacteriostatic rates respectively reach 84.83%, 92.42%, 83.89%, 86.26%, 70.14%, 84.36% and 84.36%.
The bacteriostasis of the antagonistic bacteria in each culture flask in the step 8) is shown in the following table 2:
table 2:
in Table 2, the bacteriostatic rate for hypha growth (%) = [ control hypha growth height-hypha growth height)/control hypha growth height ]. 100%
As is apparent from Table 2, the numbers 5, 10, 18 and 22 showed good bacteriostatic effects, and the bacteriostatic rates were all 100%, i.e., the Fusarium graminearum was inhibited and completely prevented from growing in the selected microorganism barley grain bottles of 5, 10, 18 and 22.
According to experimental data, the barley kernels full of the fusarium graminearum are buried in the soil, and the antagonistic bacteria in the soil can be induced to be attached to the barley kernels. The invention realizes the rapid screening of the target antagonistic bacteria, and because the antagonistic bacteria are separated from the barley grains, the screened antagonistic bacteria have certain adhesive capacity on the barley grain plants, thereby ensuring the biological control efficiency of the antagonistic bacteria on the living ears. And coating the screened antagonistic bacteria on a plate culture medium, sealing the edge of the plate culture medium by using a sealing film, and culturing at 25-26 ℃ to be used for field experiments.
The steamed or cooked barley grains can be boiled or steamed to half-raw unripe barley grains after being soaked in water, or can be boiled or steamed to unripe barley grains; the number of barley grains of gibberella wrapped by gauze in the step 2) is preferably about 100, sufficient barley grains can be fully buried in soil to be fully contacted with the soil, the requirement of an experiment can be met, too many barley grains are not easy to contact the soil comprehensively, the barley grains are not easy to store and occupy space, too few barley grains are not enough for the experiment, and 100 barley grains are just provided with a 30ML bottle; the method for selecting the monad is a conventional technical means in the field of microorganisms such as a plate-scribing method and the like; the sterilization mode of the barley grains in the step 5) is a conventional laboratory sterilization means, and specifically, the barley grains can be cleaned by tap water, then are respectively sterilized by 2% NaClO for 15min after being treated for 10s by 75% ethanol, and then are washed by sterile water for 3 times; the number of barley grains placed in each culture dish and the number of repeated culture dishes which can be made in each small bottle in the step 5) are selected according to the experimental design; the culture medium can be a conventional culture medium suitable for culturing the fusarium graminearum such as a PDA culture medium.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent flow transformations made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (5)
1. A method for inducing and separating antagonistic bacteria in soil by barley grains infected with gibberellic disease is characterized in that: which comprises the following steps:
1) preparation of barley kernels carrying gibberellic disease: filling the steamed or boiled barley grains into a culture bottle, sterilizing at high temperature, cooling to room temperature, inoculating the gibberellic disease into the bottle filled with the barley grains, culturing at 25-26 ℃ for 5-6 days, and shaking the culture bottle for 2-3 times during the period to ensure that the barley grains are fully full of the gibberellic disease;
2) wrapping barley grains containing gibberellin with gauze, wrapping several barley grains full of gibberellin respectively with several sterile gauzes, tying and tying labels one by one;
3) burying barley grains: burying the barley grains wrapped by each sterile gauze in the soil at a depth of 10-20 cm, and taking out after 25-30 days;
4) bottling: respectively filling barley grains wrapped in each sterile gauze taken out of the soil into a sterilized glass bottle, and marking for later use;
5) preparing a culture dish: taking out the barley grains in each glass bottle, cleaning and sterilizing, cutting the barley grains on sterile filter paper, and then subpackaging the barley grains on flat culture media in a plurality of culture dishes for culture;
6) observing the development condition of the barley grain hyphae infected with the bacteria on the culture dish after the culture is carried out for 3-5 days in the step 5), and selecting microbial colonies which are not obviously expanded by the red gibberellic disease and can inhibit the growth of the barley grain hyphae in the corresponding culture dish for carrying out separation culture;
7) screening of antagonistic bacteria: selecting microbial colonies which are selected in the step 6) and subjected to separation culture, respectively coating the microbial colonies on a plate culture medium of a plurality of new culture dishes according to the shape of the Chinese character 'ji', placing one barley grain which is prepared in the step 1) and carries the gibberella zeae in the middle of each culture dish, sealing the edge of the plate culture medium by using a sealing film, culturing for 7-8 days at the temperature of 25-26 ℃, and selecting the microbial colonies in the corresponding culture dishes which do not obviously expand the gibberella zeae.
2. The method for inducing the isolation of antagonistic bacteria in soil using barley kernels infected with fusarium graminearum according to claim 1, wherein: selecting the microbial colonies screened in the step 7), repeatedly screening for a plurality of times according to the operation of the step 7), and purifying to obtain single colonies.
3. The method for inducing the isolation of antagonistic bacteria in soil using barley kernels infected with fusarium graminearum according to claim 2, wherein: it also comprises the step 8) of secondary screening of antagonistic bacteria: in particular, the amount of the solvent to be used,
8.1) selecting a single colony with an antibacterial effect screened in the step 7) and inoculating the single colony into a sterilized barley grain culture bottle, simultaneously inoculating two barley grains which are cultured in the step 1 and carry the fusarium graminearum, wherein a gap for enabling hyphae to grow upwards is formed between the barley grains in the barley grain culture bottle and a bottle opening, then injecting 1mL of sterile water into the barley grain culture bottle, sealing the opening, culturing at 25-26 ℃ for 5-7 d, shaking the glass bottle every day in the culture period, and finally standing for 1-2 d to observe whether the hyphae are inhibited;
8.2) observing the hypha bacteriostasis effect in the barley grain culture bottle in the step 8.1), and selecting the microbial bacteria in the corresponding culture bottle without obvious expansion of the red gibberellic disease as the target antagonistic bacteria.
4. The method for inducing the isolation of antagonistic bacteria in soil using barley kernels infected with fusarium graminearum according to claim 1, wherein: and 3) selecting field blocks planted with overlarge wheat and the like from the soil embedded with the barley grains in the step 3).
5. The method for inducing the isolation of antagonistic bacteria in soil using barley kernels infected with fusarium graminearum according to claim 1, wherein: the high-temperature sterilization process in the step 1) comprises the following steps: bottling the steamed or boiled barley grains, and sterilizing at 120-125 deg.C for 20-25 min.
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| 周清等: "一株小麦赤霉病拮抗菌的筛选鉴定及防治效果分析", 《生态科学》, vol. 38, no. 3 * |
| 徐丽梅等: "禾谷镰刀菌拮抗菌Bacillus amyloliquefaciens AX-3的分离鉴定及拮抗物质的鉴定", 《中国生物防治学报》, vol. 36, no. 1 * |
| 马誉澄: "抗菌素", 人民卫生出版社, pages: 76 * |
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