阅读说明：本技术 一种硒砂瓜生物防治菌株的筛选方法 (Screening method of selenium-containing biological control strain of sand melon ) 是由 杨国平 张琇 张慧兄 刘艳 李壮 吴凯华 谢引弟 尚洁 张殊 高洪渤 陈澎生 于 2020-07-21 设计创作，主要内容包括：本发明涉及生物防治技术领域,具体涉及一种硒砂瓜生物防治菌株的筛选方法。包括如下步骤：S1：用PDA培养基平板培养致病菌,备用；S2：利用种植硒砂瓜的土壤制作土壤浸出培养基,灭菌,放入培养皿中,并制备成斜面培养基；S3：将硒砂瓜种子消毒,然后用无菌水漂洗；再将种子用无菌湿纱布包裹,恒温催芽；S4：将发芽后的硒砂瓜种子放置于土壤浸出培养基表面,将待测生防菌株滴加到发芽种子根部,恒温培养；S5：将致病菌培养物打孔,放置于硒砂瓜种子根部附近,恒温培养；S6：检查硒砂瓜种子,筛选对致病菌具有防病作用的生物防治菌株。本发明的筛选方法操作简单,以活硒砂瓜植株作为平台,直接观察硒砂瓜是否发病,获得生防菌株。(The invention relates to the technical field of biological control, in particular to a screening method of selenium sand melon biological control strains. The method comprises the following steps: s1: using a PDA culture medium to plate and culture pathogenic bacteria for later use; s2: preparing a soil leaching culture medium by utilizing soil for planting selenium sand melons, sterilizing, putting the soil leaching culture medium into a culture dish, and preparing a slant culture medium; s3: sterilizing selenium watermelon seeds, and rinsing with sterile water; wrapping the seeds with sterile wet gauze, and accelerating germination at constant temperature; s4: placing the germinated selenium sand melon seeds on the surface of a soil leaching culture medium, dripping the biocontrol strain to be detected to the roots of the germinated seeds, and culturing at constant temperature; s5: perforating a pathogenic bacteria culture, placing the pathogenic bacteria culture near the root of the selenium sand melon seed, and culturing at constant temperature; s6: and (3) inspecting the selenium sand melon seeds, and screening biological control strains with disease prevention effects on pathogenic bacteria. The screening method is simple to operate, and by taking the live selenium sand melon plants as a platform, whether the selenium sand melons are attacked is directly observed, so that the biocontrol strain is obtained.)

1. A screening method of selenium sand melon biological control strains is characterized by comprising the following steps: the method comprises the following steps:

s1: using a PDA culture medium to plate and culture pathogenic bacteria to obtain a pathogenic bacteria culture for later use;

s2: preparing a soil leaching culture medium by utilizing soil for planting selenium sand melons, then sterilizing the soil leaching culture medium, putting the soil leaching culture medium into a culture dish, and placing the culture dish into an inclined plane to enable the soil leaching culture medium to be solidified to form an inclined plane culture medium;

s3: sterilizing selenium sand melon seeds, and rinsing the seeds with sterile water; wrapping the disinfected selenium sand melon seeds with sterile wet gauze, and accelerating germination at constant temperature;

s4: placing the selenium-containing watermelon seeds germinated in the step S3 on the surface of the soil leaching culture medium in the step S2, then dropwise adding the biocontrol strain to be detected to the roots of the germinated seeds, and culturing at constant temperature;

s5: punching the pathogenic bacteria culture prepared in the step S1, placing the pathogenic bacteria culture in the vicinity of the root of the selenium-enriched watermelon seed obtained in the step S4, setting a soil leaching culture medium in which a biocontrol strain to be detected is not placed as a positive control group, setting a soil leaching culture medium in which a biocontrol strain to be detected is not placed and a pathogenic bacteria culture is not inoculated as a negative control group, shading the root of the soil leaching culture medium, and culturing at constant temperature;

s6: after the culture is finished, the selenium watermelon seeds are checked after the positive control group has pathogenic symptoms, and if the selenium watermelon seeds are healthy, the selenium watermelon seeds have a disease prevention effect on pathogenic bacteria.

2. The screening method of the selenium-containing citrullus lanatus biocontrol strain according to claim 1, characterized in that: in step S2, the preparation of the soil leaching culture medium includes the following steps:

taking 180-220g selenium-enriched muskmelon planting soil, adding 500-1000ml purified water, stirring for 15-30min, standing for 50-70min, pouring the supernatant into a clean beaker, adding 0.3-0.8g of cane sugar and 0.05-0.15g of yeast extract powder, uniformly stirring, fixing the volume to 1000ml, finally adding 8g/L agar, and uniformly stirring to obtain the soil extraction culture medium.

3. The screening method of the selenium-containing citrullus lanatus biocontrol strain according to claim 1, characterized in that: in the step S2, the soil leaching culture medium is sterilized by steam.

4. The screening method of the selenium-containing citrullus lanatus biocontrol strain according to claim 3, characterized in that: the temperature of the steam sterilization is 116-125 ℃, and the time is 12-18 min.

5. The screening method of the selenium-containing citrullus lanatus biocontrol strain according to claim 1, characterized in that: in the step S3, the disinfection treatment of the selenium sand melon seeds adopts mercuric chloride with the mass fraction of 0.05-0.2% to disinfect for 1-3 minutes, and then the seeds are rinsed 6-8 times by using sterile water.

6. The screening method of the selenium-containing citrullus lanatus biocontrol strain according to claim 1, characterized in that: in the step S3, the temperature condition for constant-temperature germination acceleration is 28-32 ℃.

7. The screening method of the selenium-containing citrullus lanatus biocontrol strain according to claim 1, characterized in that: in the step S4, the constant temperature culture temperature is 25-30 ℃, and the culture time is 20-28 h.

8. The screening method of the selenium-containing citrullus lanatus biocontrol strain according to claim 1, characterized in that: in the step S5, the conditions of the constant temperature culture are as follows: 14-18h of illumination treatment, wherein the illumination intensity is 4500-; then dark processing is carried out for 6-10h, and the temperature is 15-20 ℃.

9. The screening method of the selenium-containing citrullus lanatus biocontrol strain according to claim 1, characterized in that: after the step S6, the method further includes a step S7: the strain with disease prevention effect is subjected to pot biological control test and field biological control test, and the strain passing the tests is used for producing biological control products.

Technical Field

The invention relates to the technical field of biological control, in particular to a screening method of selenium sand melon biological control strains.

Background

The selenium-containing watermelon is a large amount of fruits, the sand pressing land of nearly 100 ten thousand mu exists in Ningxia, the selenium-containing watermelon is a main economic crop in Ningxia Zhongwei city, various diseases commonly occur due to long-term continuous cropping, the most serious harm is selenium-containing watermelon Fusarium wilt, pathogenic bacteria are Fusarium oxysporum and invade into the selenium-containing watermelon body from the root, the selenium-containing watermelon is propagated in vascular bundle tissues and spread to branches and tendrils, the pathogenic bacteria can be separated from the whole plant body, hypha blocks vascular bundles and generates mycotoxin to wither the whole selenium-containing watermelon plant. Due to the adsorption effect of soil on chemical agents, the general chemical prevention and treatment effect is not ideal, fusarium oxysporum separated from dead plants of Ningxia selenium sand melon has very strong pathogenicity, hypha can invade roots of selenium sand melon and cause blight as long as the hypha touches selenium sand melon roots, and the prevention and treatment of the continuous cropping obstacle of sand melon in Ningxia Gansu and other places are well-known worldwide problems and mainly expressed in the following two aspects.

Firstly, practices prove that the chemical pesticide can not prevent and treat the selenium watermelon fusarium wilt. The commercial bactericide which can be bought in agricultural material market is used for melon agriculture, but the problem of seedling death due to fusarium wilt of selenium sand melons cannot be solved.

Secondly, the traditional biological control microbial inoculum is difficult to effectively control the selenium watermelon fusarium wilt. The traditional method is to adopt a counter-acting method to screen strains with antagonistic action, and the specific method is to inoculate pathogenic microorganisms on a microorganism culture medium and inoculate strains to be detected at the same time, wherein the distance between the pathogenic microorganism colonies and the strains to be detected is 1-3 cm, after several days of culture, if the pathogenic bacteria colonies and the strains to be detected can not be contacted all the time, and the growth radius of the pathogenic bacteria colonies in other directions exceeds the distance between the pathogenic bacteria and the strains to be detected, the strains to be detected are shown to have the capability of antagonizing (inhibiting) the pathogenic bacteria. In general, a strain having a strong antagonistic activity is selected for biological control.

In theory, antagonistic strains are used as biocontrol strains, but in practical application, microorganisms with antagonistic ability can kill or inhibit pathogenic bacteria, and the antagonistic effect occurs on the microorganism culture medium, which can antagonize pathogenic bacteria, but on or in plants, it is difficult to antagonize or inhibit pathogenic bacteria, and the main reasons include: 1. the difference between the nutrient conditions provided by the plants and the nutrient conditions provided by the culture medium is very large, and the growth speeds of the antagonistic bacteria and the pathogenic bacteria are different from those of the culture medium; 2. the interaction of plants with microorganisms is extremely complex and microorganisms and their metabolites may strengthen or weaken the immune competence of plants. Although some microorganisms cannot antagonize pathogenic bacteria, the microorganisms can stimulate plants to immunize against some pathogenic bacteria so as to achieve the effect of preventing diseases. It follows that there is no necessary link between antagonism and biocontrol effects occurring on the medium.

Disclosure of Invention

In order to overcome the defects and shortcomings in the prior art, the invention aims to provide the screening method of the selenium sand melon biocontrol strain, the screening method is simple to operate, does not depend on the traditional antagonistic index as a screening basis, takes a live selenium sand melon plant as a platform, directly observes whether the selenium sand melon is attacked through the interaction among the biocontrol strain, the selenium sand melon plant and blight bacteria, obtains the biocontrol strain capable of protecting the selenium sand melon from being infected by the blight and has good disease prevention effect.

The purpose of the invention is realized by the following technical scheme: a screening method of selenium sand melon biological control strains comprises the following steps:

s1: using a PDA culture medium to plate and culture pathogenic bacteria to obtain a pathogenic bacteria culture for later use;

s2: preparing a soil leaching culture medium by utilizing soil for planting selenium sand melons, then sterilizing the soil leaching culture medium, putting the soil leaching culture medium into a culture dish, and placing the culture dish into an inclined plane to enable the soil leaching culture medium to be solidified to form an inclined plane culture medium;

s3: sterilizing selenium sand melon seeds, and rinsing the seeds with sterile water; wrapping the disinfected selenium sand melon seeds with sterile wet gauze, and accelerating germination at constant temperature;

s4: placing the selenium-containing watermelon seeds germinated in the step S3 on the surface of the soil leaching culture medium in the step S2, then dropwise adding the biocontrol strain to be detected to the roots of the germinated seeds, and culturing at constant temperature;

s5: punching the pathogenic bacteria culture prepared in the step S1, placing the pathogenic bacteria culture in the vicinity of the root of the selenium-enriched watermelon seed obtained in the step S4, setting a soil leaching culture medium in which a biocontrol strain to be detected is not placed as a positive control group, setting a soil leaching culture medium in which a biocontrol strain to be detected is not placed and a pathogenic bacteria culture is not inoculated as a negative control group, shading the root of the soil leaching culture medium, and culturing at constant temperature;

s6: after the culture is finished, the selenium watermelon seeds are checked after the positive control group has pathogenic symptoms, and if the selenium watermelon seeds are healthy, the selenium watermelon seeds have a disease prevention effect on the pathogenic bacteria.

The screening method of the selenium sand melon biocontrol strain is simple to operate, does not rely on the traditional antagonistic index as a screening basis, takes the live selenium sand melon plant as a platform, and directly observes whether the selenium sand melon is attacked through the interaction among the biocontrol strain-selenium sand melon plant-blight bacteria, so that the biocontrol strain capable of protecting the selenium sand melon from being infected by blight is obtained, and the disease prevention effect is good.

Wherein, the plants are used as a key screening platform, which is different from the traditional flat plate confronting method that a microorganism culture medium is used as a platform; in addition, the complete living plant is used as a key material and cultured in a plant incubator, different from in vitro tissues such as cut leaves or cut roots, the immune response of the complete plant which continuously grows and the in vitro plant tissues is different, so that the invention adopts the living plant seeds as a screening platform, can reflect the real biocontrol effect better and can accurately screen out the strains which can be applied to crop production.

Wherein the selenium Cucumis sativus seeds can be selected from Cucumis sativus seeds No. 5 of Jincheng.

Wherein, in the step S4, the selenium fructus cucurbitae seeds germinate until the radicle is about 1.5-2cm long, then the germinated selenium fructus cucurbitae seeds are placed on the surface of the soil leaching culture medium, and 5-6 plants are placed on each culture medium surface; in step S5, the pathogenic bacteria culture is made into a small round disc containing hyphae by using a sterile puncher, and the disc containing hyphae is placed at a position 5mm away from the root of the selenium-containing watermelon seed by using sterile forceps, as shown in figure 1.

In step S5, the positive control group is a soil leaching culture medium without a biocontrol strain to be tested placed, and only a pathogenic bacteria culture is inoculated; the negative control group is a soil leaching culture medium which is not provided with a biocontrol strain to be detected and inoculated with a pathogenic bacteria culture; the experimental group of the invention is a soil leaching culture medium for placing the biocontrol strain to be tested and inoculating the pathogenic bacteria culture.

More preferably, the screening method of the biological control strain can be suitable for screening various plant disease biological control strains, and is not limited to watermelon fusarium wilt.

Preferably, in step S2, the preparation of the soil leaching culture medium includes the following steps:

taking 180-220g selenium-enriched muskmelon planting soil, adding 500-1000ml purified water, stirring for 15-30min, standing for 50-70min, pouring the supernatant into a clean beaker, adding 0.3-0.8g of cane sugar and 0.05-0.15g of yeast extract powder, uniformly stirring, fixing the volume to 1000ml, finally adding 8g/L agar, and uniformly stirring to obtain the soil extraction culture medium.

According to the invention, the soil leaching culture medium is prepared through the steps, so that the nutrient components are similar to those of the soil environment, the microorganism is cultured, and the screened biocontrol strain can play a biocontrol role in the field soil seeds. However, the strains traditionally obtained on microbial culture media are not necessarily able to grow under soil conditions and therefore are difficult to exert biocontrol effect, which is one of the reasons why strains screened by traditional methods are rarely used for production.

The surface of the culture medium prepared by 8g/L of agar is soft, so that selenium-containing watermelon seeds can slightly sink into the culture medium, and the selenium-containing watermelon seeds are fixed stably; the surface of the culture medium made of 15g/L agar is too hard, so that selenium sand melon seeds are easy to slide off and are not fixed stably.

Preferably, in step S2, the soil leaching medium is sterilized by steam sterilization.

Preferably, the temperature of the steam sterilization is 116-125 ℃, and the time is 12-18 min.

According to the method, the steam sterilization mode is adopted, and the temperature and time of the steam sterilization are strictly controlled, so that the microorganisms in the soil leaching culture medium can be killed, and the prevention and control effects of the microorganisms on the biocontrol strain to be detected in the subsequent biocontrol strain screening processes of the step S4 and the step S5 are avoided.

Preferably, in the step S3, the disinfection treatment of the selenium-containing watermelon seeds is carried out by disinfecting the seeds for 1 to 3 minutes by using 0.05 to 0.2 mass percent of mercuric chloride, and then rinsing the seeds for 6 to 8 times by using sterile water; the method can disinfect and kill microorganisms on the surfaces of selenium sand melon seeds, and avoids the influence of the microorganisms on the control effect of the to-be-detected biocontrol strains in the subsequent biocontrol strain screening process.

Preferably, in the step S3, the temperature condition for constant-temperature germination is 28-32 ℃, which is suitable for the germination and growth of selenium-containing watermelon seeds and promotes germination.

Preferably, in the step S4, the constant temperature culture is carried out at 25-30 ℃ for 20-28 h; can promote the growth and the propagation of the biocontrol strain to be detected at the root of the germinated seed, and realize the protection, the prevention and the treatment of the root of the germinated seed.

Preferably, in step S5, the conditions for the isothermal culture are: 14-18h of illumination treatment, wherein the illumination intensity is 4500-; then dark processing is carried out for 6-10h, and the temperature is 15-20 ℃. The conditions of the constant-temperature culture are simulated day and night environments, and the screening influence on the biocontrol strains to be detected, caused by conditions such as illumination, temperature and the like, is eliminated.

Preferably, after the step S6, the method further includes the step S7: the strain with disease prevention effect is subjected to pot biological control test and field biological control test, and the strain passing the tests is used for producing biological control products.

Through pot biological control experiments and field biological control experiments, the biological control strain can be verified to play a better biological control role in farming production.

Wherein, the potted plant biocontrol test specifically operates as follows: preparing biocontrol strain into 1x10⁶CFU/ml bacterial suspension, and 100ml bacterial liquid is applied to every 500g soil. Culturing at 28 deg.C for 24 hr to allow the biocontrol strain to colonize in soil. After 24h, inoculate 1x10⁶The root of the selenium-containing muskmelon seed with the root length of about 1.5 cm is planted in the soil containing bacteria, and the root of the selenium-containing muskmelon seed treated by the method is placed in a plant incubator to be cultured.

The culture conditions are as follows: 16h of illumination, 28 ℃ and 5000 Lx; 8h dark, 18 ℃. The onset was observed after 14 days.

The invention has the beneficial effects that: the screening method of the selenium sand melon biocontrol strain is simple to operate, does not rely on the traditional antagonistic index as a screening basis, takes the live selenium sand melon plant as a platform, and directly observes whether the selenium sand melon is attacked through the interaction among the biocontrol strain-selenium sand melon plant-blight bacteria, so that the biocontrol strain capable of protecting the selenium sand melon from being infected by blight is obtained, and the disease prevention effect is good.

Wherein, the plants are used as a key screening platform, which is different from the traditional flat plate confronting method that a microorganism culture medium is used as a platform; in addition, the complete living plant is used as a key material and cultured in a plant incubator, different from in vitro tissues such as cut leaves or cut roots, the immune response of the complete plant which continuously grows and the in vitro plant tissues is different, so that the invention adopts the living plant seeds as a screening platform, can reflect the real biocontrol effect better and can accurately screen out the strains which can be applied to crop production.

Drawings

FIG. 1 is a picture of the soil leaching medium of the experimental group inoculated with a pathogenic bacteria culture at step S5 in the screening method of the present invention.

Detailed Description

For the understanding of those skilled in the art, the present invention will be further described with reference to the following examples and the accompanying fig. 1, and the description of the embodiments is not intended to limit the present invention.