阅读说明：本技术 一种用于防控马铃薯根腐病的哈茨木霉高产菌株的选育方法 (Breeding method of trichoderma harzianum high-yield strain for preventing and controlling potato root rot ) 是由 王喜刚 郭成瑾 沈瑞清 焦杨 张丽荣 杨波 于 2019-07-21 设计创作，主要内容包括：本发明公开了一种用于防控马铃薯根腐病的哈茨木霉高产菌株的选育方法,该哈茨木霉高产菌株通过出发菌株进行紫外诱变、接种发酵瓶、发酵液过滤、平板培养基的准备、抗性筛选的步骤选育得到,根据菌种选育确定的数量,优先选择对马铃薯根腐病致病菌镰刀菌抑菌圈大的菌株进行分离纯化传代,完成哈茨木霉高产菌株的快速筛选。本发明提供的哈茨木霉高产菌株选育方法,可有效提供菌种选育效率,从而缩短菌种选育周期,以前菌种选育要经过6～8代选育,现在仅需3～4代即可完成,时间缩短一半；本发明提高菌种筛选效率和选育优良菌种的准确性,从而为哈茨木霉发酵提供优良的菌种,在防控马铃薯镰刀菌根腐病方面提供了可靠保证。(The invention discloses a breeding method of a trichoderma harzianum high-yield strain for preventing and controlling potato root rot, the trichoderma harzianum high-yield strain is obtained by breeding through the steps of ultraviolet mutagenesis of an initial strain, inoculation of a fermentation bottle, filtration of fermentation liquor, preparation of a plate culture medium and resistance screening, and according to the number determined by strain breeding, strains with large inhibition zones of fusarium, pathogenic bacteria of potato root rot are preferentially selected for separation, purification and passage so as to complete the rapid screening of the trichoderma harzianum high-yield strain. The method for breeding the trichoderma harzianum high-yield strain can effectively improve the strain breeding efficiency, so that the strain breeding period is shortened, the strain breeding is carried out for 6-8 generations before, and can be completed for only 3-4 generations at present, and the time is shortened by half; the invention improves the strain screening efficiency and the accuracy of breeding excellent strains, thereby providing excellent strains for the fermentation of trichoderma harzianum and providing reliable guarantee in the aspect of preventing and controlling the fusarium root rot of potatoes.)

1. A breeding method of Trichoderma harzianum high-yield strains for preventing and controlling potato root rot is characterized by comprising the following steps:

step one, spore suspension preparation: inoculating Trichoderma harzianum original strain M-17 strain to PDA culture medium slant for culture, washing the slant with physiological saline, scattering with glass beads, and removing mycelium to obtain suspension containing spore;

step two, ultraviolet mutagenesis: placing the suspension containing the spores obtained in the step one in a flat-bottom culture dish, and carrying out ultraviolet mutagenesis;

step three, plate separation and screening: then diluting the suspension liquid obtained after the mutagenesis in the step two, coating the suspension liquid on a PDA culture medium plate, culturing at 25-28 ℃ under a dark condition, after bacterial colonies grow out, selecting bacterial strains with changed colony morphologies, carrying out single bacterial colony streaking separation, and carrying out secondary separation and purification to obtain relatively pure mutagenic bacterial strains;

step four, inoculating a fermentation bottle: connecting the mutagenic strain obtained in the third step to a fermentation bottle in an aseptic chamber by adopting a digging and inoculating method, putting the fermentation bottle on a shaking table for shaking, filtering the fermentation bottle fermented to the end point after the culture is finished, and taking the filtrate;

step five, preparing a culture medium of the plate: preheating and melting a PDA culture medium, pouring the PDA culture medium into a flat plate in a sterile room, and pouring a detection culture medium containing sensitive bacteria after the culture medium is solidified;

step six, resistance screening: directly connecting the filtrate of the fermentation liquor with a sample injector on a detection culture medium containing sensitive bacteria, wherein the distance between every two sample applications is more than 20mm, putting the plate culture medium into an incubator for culture after all the sample applications are finished, and culturing for 8-10 days at 25-28 ℃ under the dark condition;

and step seven, taking the plate cultured in the step six for 8-10 days out of the incubator, measuring each inhibition zone, preferentially selecting the strains with large inhibition zones for confirmation according to the number determined by strain breeding, and completing the rapid screening of the high-yield strains of trichoderma harzianum.

2. The breeding method of the trichoderma harzianum high-yield strain for preventing and controlling potato root rot according to claim 1, wherein the PDA culture medium slant and plate making method in the first step and the third step comprises the following steps:

1) firstly, weighing the components of the culture medium according to the following mixture ratio: 200g of peeled potatoes, 20g of glucose, 15-20 g of agar and 1000ml of distilled water;

2) cutting potatoes into small pieces, putting the small pieces into a pot, adding 1000ml of water, heating the small pieces on a heater until the water is boiled, maintaining the boiling for 20-30 min, filtering the small pieces on a measuring cup by using 2 layers of gauze while the small pieces are hot, removing filter residues, and supplementing distilled water into filtrate to 1000 ml;

3) putting the filtrate into a pot, adding 20g of glucose and 15-20 g of agar, then putting the mixture on an asbestos net, heating the mixture with soft fire, continuously stirring the mixture by using a glass rod, subpackaging the mixture after the agar is completely dissolved, sterilizing the mixture at 121 ℃ for 25-30 min, naturally cooling the mixture, pouring the mixture into a slope and a flat plate, and solidifying the mixture for later use.

3. The breeding method of the Trichoderma harzianum high-yield strain for preventing and controlling potato root rot according to claim 1, wherein the slant culture condition in the first step is a dark condition, and the strain is cultured at 25-28 ℃ for 5-7 days.

4. The breeding method of the trichoderma harzianum high-yield strain for preventing and controlling the potato root rot according to claim 1, wherein in the second step, ultraviolet mutagenesis conditions are that spores are placed 30cm under a vertical distance of a 15W ultraviolet lamp, and magnetic stirring irradiation is carried out for 30-180 s.

5. The breeding method of trichoderma harzianum high-yield strain for preventing and controlling potato root rot according to claim 1, characterized in that in the fourth step, the formula of the fermentation bottle is one or more of wheat flour, corn flour, whole potato flour, potato starch, soluble starch and bran flour with 2% of carbon source addition; the nitrogen source is added with 2 percent of one or more of soybean meal, grape wine peel residue powder, ammonium nitrate, urea, ammonium sulfate and sodium nitrate; the growth promoter is one or more of leachate of Glycyrrhrizae radix and radix Isatidis, and the concentration is 30%, 50%, and 70%, respectively.

6. The breeding method of the trichoderma harzianum high-yield strain for preventing and controlling potato root rot according to claim 1, wherein the culture conditions of the fermentation bottle in the fourth step are that the rotating speed of a shaking table is 220rpm, the culture temperature is 25-28 ℃, and the culture time is 120-168 h.

7. The breeding method of the trichoderma harzianum high-yield strain for preventing and controlling potato root rot according to claim 1, wherein in the fifth step, the thickness of the PDA culture medium is controlled to be 2-3 mm when the PDA culture medium is preheated and melted.

8. The breeding method of the trichoderma harzianum high-yield strain for preventing and controlling potato root rot according to claim 1, wherein in the fifth step, the thickness of the detection culture medium containing sensitive bacteria is controlled to be 2-3 mm.

9. The breeding method of the trichoderma harzianum high-yield strain for preventing and controlling potato root rot according to claim 1, wherein the sensitive bacteria in the detection medium in the step five are one or more of fusarium oxysporum, fusarium equiseti, fusarium sambucinum, fusarium solani and fusarium acuminatum.

10. The breeding method of the trichoderma harzianum high-producing strain for preventing and controlling potato root rot according to claim 5, wherein the fermentation bottle formula is one of corn flour and bran flour with 2% of carbon source addition; the nitrogen source is added into 2 percent of one of soybean meal and wine peel residue powder; the growth promoter is one or more of leachate of Glycyrrhrizae radix and radix Isatidis, and the concentration is 30%, 50%, and 70%, respectively.

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to a breeding method of a trichoderma harzianum high-yield strain for preventing and controlling potato root rot.

Background

Trichoderma is a microorganism widely existing in nature, Trichoderma belongs to Deuteromycota, Hyphomycetales, Trichoderma, and common Trichoderma includes Trichoderma viride, Trichoderma koningii, Trichoderma asperellum, Trichoderma atroviride, Trichoderma harzianum, Trichoderma longibrachiatum, etc. The hyphae of trichoderma harzianum are fine and colorless, have separation and multiple branches, conidiophores grow from lateral branches of the hyphae, grow oppositely or generate alternately, generally have 2-3 branches, are bottle-shaped or cone-shaped, are mostly spherical, and have verruca verrucosa on the sporoderm and are blue-green.

Trichoderma harzianum, a biocontrol bacterium, can be used to prevent plant diseases caused by pathogenic bacteria such as Pythium, Rhizoctonia solani, Fusarium, Rhizopus nigricans, Staphylotrichum, Sclerotinia sclerotiorum, etc. The fusarium root rot, the dry rot and the blight caused by infecting the potatoes by fusarium are world soil-borne fungal diseases, the root rot of the potatoes can be attacked in seedling culture periods and field growth periods, the fusarium root rot of the potatoes caused by various fusarium can seriously damage the production of the potatoes in recent years, the yield of commonly attacked areas is reduced by 13-35%, and the potatoes die in serious areas and even die in top of the areas. The control of the potato root rot is generally treated by adopting a chemical bactericide, the residue problem often exists when the chemical bactericide is used for treatment, simultaneously fusarium in a production field can not remove roots and is easy to relapse in the coming year, and the like, so that the trichoderma harzianum is used for biologically controlling the root rot, the control is environment-friendly, safe and effective, and the control can be carried out for a long time.

However, the naturally bred trichoderma harzianum strain has limited inhibition effect on fusarium, which is a pathogenic bacterium of potato root rot, and can be inhibited only by a large dose, and the inhibition effect is limited, so that a high-yield strain capable of improving the metabolic product of trichoderma harzianum needs to be developed and bred by a technical means, the prevention and control effect on the potato root rot is improved, and the production cost can be reduced.

Disclosure of Invention

Based on the prior art, the invention aims to provide a breeding method of a trichoderma harzianum high-yield strain for preventing and controlling potato root rot, the breeding method of the trichoderma harzianum high-yield strain provided by the invention can effectively improve strain breeding efficiency, so that the strain breeding period is shortened, the previous strain breeding needs 6-8 generations of breeding, only 3-4 generations of breeding are needed at present, and the time is shortened by half; the invention improves the strain screening efficiency and the accuracy of breeding excellent strains, thereby providing excellent strains for the fermentation of trichoderma harzianum and providing reliable guarantee in the aspect of preventing and controlling the fusarium root rot of potatoes.

The starting strain Trichoderma harzianum M-17 used in the invention has a preservation number: CCTCC NO: m2018538, the Latin article name is Trichoderma harzianum M-17, the preservation unit is China center for type culture Collection, the preservation date is 8 months and 13 days in 2018, and the preservation address is Wuhan university in Wuhan, China.

The technical scheme adopted by the invention is as follows: a breeding method of Trichoderma harzianum high-yield strains for preventing and controlling potato root rot comprises the following steps:

step one, spore suspension preparation: inoculating Trichoderma harzianum original strain M-17 strain to PDA culture medium slant for culture, washing the slant with physiological saline, scattering with glass beads, and removing mycelium to obtain suspension containing spore;

step two, ultraviolet mutagenesis: placing the suspension containing the spores obtained in the step one in a flat-bottom culture dish, and carrying out ultraviolet mutagenesis;

step three, plate separation and screening: then diluting the suspension liquid obtained after the mutagenesis in the step two, coating the suspension liquid on a PDA culture medium plate, culturing at 25-28 ℃ under a dark condition, after bacterial colonies grow out, selecting bacterial strains with changed colony morphologies, carrying out single bacterial colony streaking separation, and carrying out secondary separation and purification to obtain relatively pure mutagenic bacterial strains;

step four, inoculating a fermentation bottle: connecting the mutagenic strain obtained in the third step to a fermentation bottle in an aseptic chamber by adopting a digging and inoculating method, putting the fermentation bottle on a shaking table for shaking, filtering the fermentation bottle fermented to the end point after the culture is finished, and taking the filtrate;

step five, preparing a culture medium of the plate: preheating and melting a PDA culture medium, pouring the PDA culture medium into a flat plate in a sterile room, and pouring a detection culture medium containing sensitive bacteria after the culture medium is solidified;

step six, resistance screening: directly connecting the filtrate of the fermentation liquor with a sample injector on a detection culture medium containing sensitive bacteria, wherein the distance between every two sample applications is more than 20mm, putting the plate culture medium into an incubator for culture after all the sample applications are finished, and culturing for 8-10 days at 25-28 ℃ under the dark condition;

and step seven, taking the plate cultured in the step six for 8-10 days out of the incubator, measuring each inhibition zone, preferentially selecting the strains with large inhibition zones for confirmation according to the number determined by strain breeding, and completing the rapid screening of the high-yield strains of trichoderma harzianum.

In order to better implement the invention, further, the manufacturing method of the PDA culture medium inclined plane and the plate in the first step and the third step comprises the following steps:

1) firstly, weighing the components of the culture medium according to the following mixture ratio: 200g of peeled potatoes, 20g of glucose, 15-20 g of agar and 1000ml of distilled water;

2) cutting potatoes into small pieces, putting the small pieces into a pot, adding 1000ml of water, heating the small pieces on a heater until the water is boiled, maintaining the boiling for 20-30 min, filtering the small pieces on a measuring cup by using 2 layers of gauze while the small pieces are hot, removing filter residues, and supplementing distilled water into filtrate to 1000 ml;

3) putting the filtrate into a pot, adding 20g of glucose and 15-20 g of agar, then putting the mixture on an asbestos net, heating the mixture with soft fire, continuously stirring the mixture by using a glass rod, subpackaging the mixture after the agar is completely dissolved, sterilizing the mixture at 121 ℃ for 25-30 min, naturally cooling the mixture, pouring the mixture into a slope and a flat plate, and solidifying the mixture for later use.

In order to better realize the method, in the step one, the slant culture condition is a dark condition, and the slant culture is carried out for 5-7 days at 25-28 ℃.

In order to better realize the method, further, in the second step, the ultraviolet mutagenesis condition is that spores are placed at a vertical distance of 30cm under a 15W ultraviolet lamp, and the spores are irradiated for 30-180 s under magnetic stirring.

In order to better realize the invention, the formula of the fermentation bottle in the fourth step is one or more of wheat flour, corn flour, potato starch, soluble starch and bran powder with the carbon source addition amount of 2%; the nitrogen source is added with 2 percent of one or more of soybean meal, grape wine peel residue powder, ammonium nitrate, urea, ammonium sulfate and sodium nitrate; the growth promoter is one or more of leachate of Glycyrrhrizae radix and radix Isatidis, and the concentration is 30%, 50%, and 70%, respectively.

In order to better realize the method, the culture conditions of the fermentation bottle in the fourth step are that the rotating speed of a shaking table is 220rpm, the culture temperature is 25-28 ℃, and the culture time is 120-168 hours.

In order to better realize the method, further, the thickness of the culture medium is controlled to be 2-3 mm when the PDA culture medium is preheated and melted in the fifth step.

In order to better realize the method, the thickness of the detection culture medium containing the sensitive bacteria is controlled to be 2-3 mm in the fifth step.

In order to better realize the invention, the sensitive bacteria in the detection culture medium in the step five are one or more of fusarium oxysporum, fusarium equiseti, fusarium sambuci, fusarium solani and fusarium acuminatum.

In order to better realize the invention, further, the formula of the fermentation bottle is one of corn flour and bran powder with the carbon source addition amount of 2 percent; the nitrogen source is added into 2 percent of one of soybean meal and wine peel residue powder; the growth promoter is one or more of leachate of Glycyrrhrizae radix and radix Isatidis, and the concentration is 30%, 50%, and 70%, respectively.

Advantageous effects

The invention has the following beneficial effects and innovation points:

1. ultraviolet mutagenesis and resistance screening are generally breeding methods for actinomycetes, and are independently and separately combined with traditional natural breeding for use;

2. the resistance screening is generally carried out by using specific antibiotics, such as streptomycin and penicillin resistance screening, and few viable bacteria are used for screening, and the invention creatively uses sensitive bacteria, namely functional fusarium to carry out resistance screening, thereby improving the breeding efficiency;

3. the judgment of excellent strains, the conventional breeding needs to be carried out by culturing the strains again, and the verification is carried out according to the shaking result to obtain high-yield strains;

4. the traditional Chinese medicine extracting solution is added into the fermentation culture medium during strain screening, which is a big characteristic that general culture media do not have, and the traditional Chinese medicine extracting solution can improve the yield of the product of the bred strain and increase the resistance to pathogenic fusarium, thereby further obtaining high-quality and high-efficiency strains.

Detailed Description

The present invention will be described in further detail with reference to specific examples.

The manufacturing method of the PDA culture medium inclined plane and the plate comprises the following steps:

1) firstly, weighing the components of the culture medium according to the following mixture ratio: 200g of peeled potatoes, 20g of glucose, 15-20 g of agar and 1000ml of distilled water;

2) cutting potatoes into small pieces, putting the small pieces into a pot, adding 1000ml of water, heating the small pieces on a heater until the water is boiled, maintaining the boiling for 20-30 min, filtering the small pieces on a measuring cup by using 2 layers of gauze while the small pieces are hot, removing filter residues, and supplementing distilled water into filtrate to 1000 ml;

3) putting the filtrate into a pot, adding 20g of glucose and 15-20 g of agar, then putting the mixture on an asbestos net, heating the mixture with soft fire, continuously stirring the mixture by using a glass rod, subpackaging the mixture after the agar is completely dissolved, sterilizing the mixture at 121 ℃ for 25-30 min, naturally cooling the mixture, pouring the mixture into a slope and a flat plate, and solidifying the mixture for later use.