阅读说明：本技术 一种花生腐烂病病原真菌的致病性鉴定方法 (Method for identifying pathogenicity of peanut rot pathogenic fungi ) 是由 王冕 张朝昕 陈明娜 王通 潘丽娟 许静 禹山林 迟晓元 陈娜 于 2019-11-08 设计创作，主要内容包括：本发明公开了一种花生腐烂病病原真菌的致病性鉴定方法,属于病原真菌致病性鉴定技术领域。本发明致病性鉴定方法是将感病花生品种的种子和不同组织分别经消毒、冲洗、微创处理后,接入病原真菌菌丝体块,使病原真菌与花生种子和花生不同组织实现共培养,5天后观察侵染致病情况。本发明方法能够随时对腐烂病病原真菌的致病性进行检测,快速简便、稳定性高、重复性好,有利于快速鉴定大量花生抗病种质资源,对花生抗病品种选育及腐烂病防控研究具有重要意义。(The invention discloses a pathogenicity identification method of peanut rot pathogenic fungi, belonging to the technical field of pathogenic fungi pathogenicity identification. The pathogenicity identification method comprises the steps of respectively sterilizing, washing and carrying out minimally invasive treatment on seeds and different tissues of a susceptible peanut variety, inoculating a pathogenic fungus mycelium block to realize co-culture of pathogenic fungi, the peanut seeds and the different tissues of the peanut, and observing infection pathogenicity after 5 days. The method can detect the pathogenicity of the rot pathogenic fungi at any time, is quick, simple and convenient, has high stability and good repeatability, is favorable for quickly identifying a large number of peanut disease-resistant germplasm resources, and has important significance for the breeding of peanut disease-resistant varieties and the prevention and control research of rot.)

1. A method for identifying pathogenicity of peanut rot pathogenic fungi is characterized in that seeds and different tissues of susceptible peanut varieties are respectively disinfected, washed and subjected to minimally invasive treatment, and then a pathogenic fungi mycelium block is inoculated, so that the pathogenic fungi, the peanut seeds and the different tissues of peanuts are cultured together, and the infection pathogenic condition is observed after 5 days.

2. The method for identifying the pathogenicity of a pathogenic fungus of peanut rot disease according to claim 1, wherein the pathogenic fungus of peanut rot disease is fusarium oxysporum.

3. The method for identifying the pathogenicity of pathogenic fungi of peanut rot disease according to claim 2, wherein the fusarium oxysporum is fusarium oxysporum Hs-f, which is deposited in 29 th month 08 m 2019: the China general microbiological culture Collection center (CGMCC) has a collection number of CGMCC NO:18130 and an address of: west road No.1, north zhou yang ward, beijing, the requested depository is peanut institute of shandong province.

4. The method for identifying the pathogenicity of pathogenic fungi of peanut rot disease according to claim 3, wherein the Fusarium oxysporum Hs-f is cultured by the following method:

(1) activating strains: inoculating the frozen strains into 5ml of PDA liquid culture medium, and performing shake culture at 25 ℃ and 180rpm for 8 h;

(2) enrichment culture: transferring the activated 5ml culture solution into 50ml PDA liquid culture medium, and performing shake culture at 25 ℃ and 180rpm overnight;

(3) and (3) amplification culture: and (3) coating 1ml of the enrichment culture solution on a PDA culture medium plate, airing, repeatedly coating twice, airing, and performing dark culture at 25 ℃ overnight.

5. The method for identifying the pathogenicity of pathogenic fungi of peanut rot disease according to claim 4, wherein the cryopreserved strain is a strain with stable pathogenicity, which is preserved by a single colony which is separated and purified and is rapidly passaged to 50 generations;

the rapid passage is as follows:

(1) selecting a single colony, inoculating the single colony in 5ml of PDA liquid culture medium, and performing shake culture at 25 ℃ and 180rpm for 8 hours;

(2) taking 5ml of the culture solution in the step (1), transferring the culture solution into 50ml of PDA liquid culture medium, and performing shake culture at 25 ℃ and 180rpm overnight;

(3) finally, coating the culture solution in the step (2) on a PDA culture medium plate, drying in the air, repeatedly coating twice, and performing dark culture at 25 ℃ overnight after drying in the air;

(4) repeating the steps (1) to (3) to passage to 50 generations.

6. The method for identifying the pathogenicity of pathogenic fungi causing peanut rot disease according to claim 1, wherein the susceptible peanut variety is Huayu No. 20.

7. The method for identifying the pathogenicity of pathogenic fungi causing peanut rot disease according to claim 1, wherein the different tissues of the peanut are peanut seedling root system, peanut seedling stem part, peanut seedling cotyledon and peanut leaf.

8. The method for identifying the pathogenicity of pathogenic fungi of peanut rot disease according to claim 1, wherein the conditions for co-culturing the pathogenic fungi with peanut seeds and different tissues of peanuts are as follows: culturing at 25 deg.C under 16h/8h for 7 days in a constant temperature incubator.

9. The method for identifying the pathogenicity of pathogenic fungi of peanut rot according to claim 1, wherein the inoculation amount of the mycelium block of the pathogenic fungi is a cylindrical block with a diameter of 0.3 cm.

10. The method for identifying the pathogenicity of pathogenic fungi of peanut rot disease according to claim 1, wherein the judgment standard of the pathogenicity is from yellowing and blackening of an inoculation point to spreading of rot.

Technical Field

The invention belongs to the technical field of pathogenic fungus pathogenicity identification, and particularly relates to a method for identifying pathogenicity of peanut rot pathogenic fungi.

Background

Peanuts are a temperature-loving leguminous crop which blooms on the ground and then shoots into the ground to fruit, and are oil and economic crops widely planted worldwide. But is susceptible to soil-borne diseases due to the nature of underground results. A plurality of peanut soil infectious diseases are reported, including fungal diseases, bacterial diseases, nematode diseases and the like. The fungal diseases mainly comprise stem rot, root rot, fruit rot and the like. These fungal diseases often result in peanut seedling death or underground root rot. The large-area rotting and yield reduction and even the top harvest of peanuts caused by fungal diseases are mostly reported, and the situation that the rotting and yield reduction of peanuts is aggravated in the northern production area is in the next year, so that the screening, the culture and the pathogenicity research of pathogenic fungi of the rotting and disease-causing peanut become more important, the method is a precondition and a basis for researching the mechanism of resisting the fungal rotting and disease-preventing peanut and breeding disease-resistant varieties, and is also a key for preventing and controlling the rotting and disease of the peanuts.

At present, a field test and a potted plant tieback method are mostly adopted in a peanut pathogenic fungus pathogenicity identification method; the detection method has long period and is greatly restricted by the external environment; the method for rapidly identifying the pathogenicity of the peanut pathogenic fungi has important significance for screening disease-resistant seeds of peanuts.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide the method for identifying the pathogenicity of the peanut rot pathogenic fungi, which is quick, simple, convenient, high in stability and good in repeatability, and overcomes the defects that field and potted plant tieback detection is limited by the planting period and the detection period is long.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a method for identifying the pathogenicity of pathogenic fungus of peanut rot includes such steps as respectively disinfecting, washing and minimally invasive treating the seeds and different tissues of peanut, inoculating the mycelial block of pathogenic fungus, co-culturing the pathogenic fungus with the seeds and different tissues of peanut, and observing the infection pathogenicity after 5 days.

On the basis of the scheme, the pathogenic fungus of the peanut rot disease is fusarium oxysporum.

On the basis of the scheme, the fusarium oxysporum is fusarium oxysporum Hs-f, and is preserved in the following steps in 29 months in 2019 and 08 months: the China general microbiological culture Collection center (CGMCC) has a collection number of CGMCC NO:18130 and an address of: west road No.1, north zhou yang ward, beijing, the requested depository is peanut institute of shandong province.

On the basis of the scheme, the fusarium oxysporum Hs-f is cultured by the following method:

(1) activating strains: inoculating the frozen strains into 5ml of PDA liquid culture medium, and performing shake culture at 25 ℃ and 180rpm for 8 h;

(2) enrichment culture: transferring the activated 5ml culture solution into 50ml PDA liquid culture medium, and performing shake culture at 25 ℃ and 180rpm overnight;

(3) and (3) amplification culture: and (3) coating 1ml of the enrichment culture solution on a PDA culture medium plate, airing, repeatedly coating twice, airing, and performing dark culture at 25 ℃ overnight.

On the basis of the scheme, the frozen strain is a pathogenicity-stable strain which is preserved by a single colony which is separated and purified and is quickly passaged to 50 generations.

The rapid passage is as follows:

(1) selecting a single colony, inoculating the single colony in 5ml of PDA liquid culture medium, and performing shake culture at 25 ℃ and 180rpm for 8 hours;

(2) taking 5ml of the culture solution in the step (1), transferring the culture solution into 50ml of PDA liquid culture medium, and performing shake culture at 25 ℃ and 180rpm overnight;

(3) finally, coating the culture solution in the step (2) on a PDA culture medium plate, drying in the air, repeatedly coating twice, and performing dark culture at 25 ℃ overnight after drying in the air;

(4) repeating the steps (1) to (3) to passage to 50 generations.

On the basis of the scheme, the susceptible peanut variety is No. 20 Huayu.

On the basis of the scheme, the different tissues of the peanut comprise a peanut seedling root system, a peanut seedling stem part, a peanut seedling cotyledon and a peanut leaf.

On the basis of the scheme, the co-culture conditions of the pathogenic fungi, the peanut seeds and different tissues of the peanuts are as follows: culturing at 25 deg.C under 16h/8h for 7 days in a constant temperature incubator.

On the basis of the scheme, the inoculation amount of the mycelium blocks of the pathogenic fungi is a cylindrical block with the diameter of 0.3cm (the mycelium blocks are cylindrical blocks with the diameter and the height of 0.3 cm).

On the basis of the scheme, the pathogenicity judgment standard is as follows: the inoculated point becomes yellow and black until the rot spreads.

The technical scheme of the invention has the advantages

The method can detect the pathogenicity of the rot pathogenic fungi at any time, is quick, simple, convenient, high in stability and good in repeatability, overcomes the defects that field and potted plant tieback detection is limited by the planting period and the detection period is long, can simultaneously screen the pathogenicity of the root, stem, leaf, cotyledon and seed to the peanut rot pathogenic fungi, establishes a peanut anti-infection model, is beneficial to quickly identifying a large number of peanut disease-resistant germplasm resources, and has important significance on the research of peanut disease-resistant variety breeding and rot disease prevention and control.

The specific implementation mode is as follows:

terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified.

The present invention will be described in further detail with reference to the following data in conjunction with specific examples. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.