阅读说明：本技术 一种鉴定评价秀珍菇对奈氏西地西菌诱发的黄斑病抗性水平的方法 (Method for identifying and evaluating resistance level of pleurotus geesteranus to physalospora naissacensis induced maculopathy ) 是由 刘增亮 陈雪凤 吴圣进 张雯龙 周双云 于 2021-11-08 设计创作，主要内容包括：本发明公开了一种鉴定评价秀珍菇对奈氏西地西菌诱发的黄斑病抗性水平的方法,该方法包括以下步骤：栽培料带接种秀珍菇菌种后,进行菌丝培养80天,待菌丝生理成熟后,温差刺激菌棒出菇；取奈氏西地西菌菌株,震荡培养,获得病原菌悬浮液；选取子实体多、整齐健康的菌棒,在子实体菌盖大小为0.5～1.0cm时接种；置于密闭保鲜盒内再移至合适温度湿度条件下进行培养；培养2天后,取出菌棒,对秀珍菇黄斑病发病情况进行调查,记录菌棒发病子实体的数量及发病等级,计算病情指数,并据此判定秀珍菇品种黄斑病抗性水平。该方法可以准确的鉴定不同品种秀珍菇的黄斑病抗性水平,结果可靠,对推动秀珍菇黄斑病抗病品种的选育工作具有重要意义。(The invention discloses a method for identifying and evaluating resistance level of pleurotus geesteranus to northern leaf blight induced by western Naringi, which comprises the following steps: inoculating pleurotus geesteranus strains on the cultivation material belt, culturing mycelia for 80 days, and stimulating mushroom sticks to grow mushrooms by temperature difference after the mycelia are physiologically mature; taking a western nernstia bacterial strain, and performing shake culture to obtain a pathogenic bacteria suspension; selecting a neat and healthy fungus stick with a plurality of sporocarps, and inoculating when the size of a pileus of the sporocarps is 0.5-1.0 cm; placing the mixture in a sealed fresh-keeping box, and then transferring the mixture to a proper temperature and humidity condition for culture; after culturing for 2 days, taking out the fungus sticks, investigating the disease incidence of pleurotus geesteranus yellow spot, recording the number and disease incidence grade of diseased fruiting bodies of the fungus sticks, calculating disease indexes, and judging the resistance level of pleurotus geesteranus yellow spot. The method can accurately identify the resistance level of the yellow spot of the pleurotus geesteranus with different varieties, has reliable results, and has important significance for promoting the breeding work of the disease-resistant pleurotus geesteranus yellow spot.)

1. A method for identifying and evaluating the resistance level of pleurotus geesteranus yellow spot by utilizing fruiting body inoculation is characterized by comprising the following steps:

(1) preparation of Pleurotus geesteranus stick

Inoculating pleurotus geesteranus strains on the cultivation material belt, culturing mycelia for 80 days, and stimulating mushroom sticks to grow mushrooms by temperature difference after the mycelia are physiologically mature;

(2) preparation of a suspension of pathogenic bacteria

Taking a bacterial strain of the pathogenic bacteria of the maculopathy, placing the bacterial strain in a liquid NB culture medium, and performing shake culture for 2 days to obtain a pathogenic bacteria suspension;

(3) pleurotus geesteranus fruiting and pathogenic bacteria inoculation

Selecting regular and healthy fungus sticks with more sporocarps, and inoculating when the size of a pileus is 0.5-1.0 cm;

when the pathogenic bacteria are inoculated, a sterile brush is adopted to dip the pathogenic bacteria suspension liquid, and the pathogenic bacteria suspension liquid is uniformly smeared on the pleurotus geesteranus sporocarp;

after inoculating pathogenic bacteria, placing the fungus sticks in a closed preservation box for culturing;

(4) evaluation of disease resistance level

After the culture is finished, the fungus sticks are taken out, the disease occurrence condition of the pleurotus geesteranus yellow spot is investigated, the number and disease level of the diseased fruiting bodies of each fungus stick are recorded, the disease index is calculated, and the resistance level of the pleurotus geesteranus yellow spot is judged according to the disease index.

2. The method for identifying and evaluating the resistance level of pleurotus geesteranus yellow spot by utilizing fruiting body inoculation according to claim 1, wherein the pathogenic strain of macular disease is west neisseria naissance.

3. The method for identifying and evaluating the resistance level of Pleurotus geesteranus yellow spot by using fruiting body inoculation according to claim 1, wherein in step (2), the shake culture condition is 28 ℃ and 180rpm/min for 48 h.

4. The method for identifying and evaluating the level of resistance to yellow spot of Pleurotus geesteranus by inoculating fruiting bodies according to claim 1, wherein in step (2), after the shake culture is finished, the bacterial solution is centrifuged, the precipitate is collected to obtain bacterial cells, and the bacterial cells are diluted with sterile water to obtain the pathogenic bacteria suspension.

5. The method for identifying and evaluating the resistance level of Pleurotus geesteranus yellow spot by utilizing fruiting body inoculation according to claim 4, wherein the centrifugation is 5000rpm/min for 2 min.

6. The method for identifying and evaluating the resistance level of Pleurotus geesteranus yellow spot by inoculating fruiting bodies according to claim 4, wherein the number of pathogenic bacteria in the pathogenic bacteria suspension is 1x10⁸Each strain rod is inoculated with 5ml of pathogenic bacteria liquid.

7. The method for evaluating the level of resistance to yellow spot disease of Pleurotus geesteranus by utilizing fruiting body inoculation identification as claimed in claim 1, wherein in step (3), the number of the fungus sticks inoculated with pathogenic bacteria per variety is not less than 10, and the number of fruiting bodies on each fungus stick is not less than 20.

8. The method for evaluating the level of resistance to yellow spot of Pleurotus geesteranus by inoculating fruiting bodies according to claim 1, wherein in the step (3), the mushroom sticks are placed in the sealed fresh-keeping box at a distance of 2-8cm from the box wall and the fruiting surface is placed at a distance of 10-20cm from the box wall.

9. The method for identifying and evaluating the level of resistance to northern leaf blight of pleurotus geesteranus by using fruiting body inoculation according to claim 1, wherein in the step (3), the culture conditions are a temperature of 25-30 ℃ and a humidity of 85-100%, and the culture is carried out for 48 hours.

Technical Field

The invention relates to the field of crop disease resistance evaluation, in particular to a method for identifying and evaluating the resistance level of pleurotus geesteranus to sigatoka induced by west neisseria naissance.

Background

Pleurotus geesteranus, the famous pleurotus citrinopileatus, is an edible fungus with rich nutrition and delicious taste. The protein content of the vegetable is close to that of meat, is 3-6 times higher than that of common vegetables, and is a delicious and cheap top-grade dish on a dining table of people for a long time.

The yellow spot is the most important disease in pleurotus geesteranus production. The early test results show that the pathogenic bacteria of the maculopathy is West Neisseria. As the environment humidity is high, the temperature is high, the ventilation is not smooth during the fruiting period of the pleurotus geesteranus, and the yellow spot is easy to occur. If the mushroom is not treated in time, the mushroom is easy to spread and explode, which causes that the whole mushroom shed is dead in production and dead in harvest, and causes great economic loss.

Maculopathy occurs in the fruiting body of Pleurotus geesteranus, and only in the fruiting stage. The fruiting period of edible mushrooms such as pleurotus geesteranus is short, if the medicament is applied in the fruiting period, medicament residues are easily caused to cause food poisoning, further serious influence is caused on product circulation and consumption, and sometimes serious chemical injury is caused to the edible mushrooms, so that the medicament is strictly forbidden to be applied in the fruiting period of the pleurotus geesteranus. In addition, the yellow spot is generated rapidly, so that the control of the yellow spot of the pleurotus geesteranus is very difficult.

In view of this, the breeding and popularization of disease-resistant varieties is undoubtedly the most economic, effective and environment-friendly method for preventing and controlling the yellow spot of pleurotus geesteranus. The establishment of a simple and effective resistance identification technical system is the premise and key for developing the research, but no report of an identification evaluation standard method for pleurotus geesteranus yellow spot exists at present, so that the development of the resistance identification method for pleurotus geesteranus yellow spot is of great significance for promoting the research on pleurotus geesteranus yellow spot.

Disclosure of Invention

The invention aims to provide a method for identifying and evaluating the level of resistance of pleurotus geesteranus to the yellow spot disease induced by western Navier nakai, so as to solve the problems in the prior art and lay a technical foundation for cultivating and screening anti-yellow spot pleurotus geesteranus varieties, identifying pathogenic bacteria pathogenic strength and weakness and preventing and treating pleurotus geesteranus yellow spot disease.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides a method for identifying and evaluating the resistance level of pleurotus geesteranus yellow spot by utilizing fruiting body inoculation, which comprises the following steps:

(1) preparation of Pleurotus geesteranus stick

Inoculating pleurotus geesteranus strains into the cultivation material bag, culturing mycelia for 8 days, and stimulating mushroom sticks to grow mushrooms by temperature difference after the mycelia are physiologically mature;

(2) preparation of a suspension of pathogenic bacteria

Taking a bacterial strain of the pathogenic bacteria of the maculopathy, placing the bacterial strain in a liquid NB culture medium, and performing shake culture for 2 days to obtain a pathogenic bacteria suspension;

(3) pleurotus geesteranus fruiting and pathogenic bacteria inoculation

Selecting regular and healthy fungus sticks with more sporocarps, and inoculating when the diameter of the pileus of the sporocarps is 0.5-1.0 cm;

when the pathogenic bacteria are inoculated, a sterile brush is adopted to dip the pathogenic bacteria suspension liquid, and the pathogenic bacteria suspension liquid is uniformly smeared on the pleurotus geesteranus sporocarp;

after inoculating pathogenic bacteria, placing the fungus sticks in a closed preservation box for culturing;

(4) evaluation of disease resistance level

After the culture is finished, the fungus sticks are taken out, the disease occurrence condition of the pleurotus geesteranus yellow spot is investigated, the number and disease level of the diseased fruiting bodies of each fungus stick are recorded, the disease index is calculated, and the resistance level of the pleurotus geesteranus yellow spot is judged according to the disease index.

Further, in the step (2), the shaking culture condition is that the culture is carried out at the temperature of 28 ℃ and the rpm/min of 180 for 48 h.

Further, in the step (2), after the shake culture is finished, the bacterial liquid is centrifuged, the precipitate is collected to obtain the thallus, and the thallus is diluted by sterile water to obtain the pathogenic bacteria suspension.

Further, the pathogenic strain of the macular disease is West Neelicidium.

Further, the centrifugation is 5000rpm/min for 2 min.

Further, the number of pathogenic bacteria in the pathogenic bacteria suspension is 1x10⁸One per ml.

Further, in the step (3), the number of the fungus sticks inoculated with pathogenic bacteria of each variety is not less than 10, and the number of the fruit bodies on each fungus stick is not less than 20.

Further, in the step (3), during cultivation, the fungus sticks are placed in the sealed preservation box and are 2-8cm away from the box wall, and the fruiting surface is 10-20cm away from the box wall.

Further, in the step (3), the culture conditions are that the temperature is 25-30 ℃, the humidity is 85-100%, and the culture is carried out for 48 hours.

The invention discloses the following technical effects:

the selection of the inoculation time is very critical, the diameter of the pileus of the fruiting body is selected to be 0.5-1.0cm for inoculation, and in the actual production, in order to promote the growth of the stipe of the fruiting body, the environment of the mushroom shed at the stage requires high oxidation concentration, but the disease resistance of young mushrooms is weak;

the inoculation mode is simple, no puncture is needed, and the requirements of natural morbidity in production are met;

the environmental conditions (temperature, humidity and closed environment) during the culture after inoculation are very important, and the conditions for normal fruiting of pleurotus geesteranus and suitable disease attack are created.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is the morphology of a pathogen of maculopathy on NA medium;

FIG. 2 shows the morphology of pathogenic bacteria of maculopathy under electron microscope;

FIG. 3 shows the natural onset of yellow spot of Pleurotus geesteranus;

FIG. 4 is a Pleurotus geesteranus stick with fruiting body of suitable size for inoculating pathogenic bacteria;

FIG. 5 shows the disease of Pleurotus geesteranus of different species after inoculation of pathogenic bacteria of yellow spot;

FIG. 6 shows the fruiting body of Pleurotus geesteranus after inoculation with overlarge pileus;

FIG. 7 shows the fruiting body of Pleurotus geesteranus after the cap is too small to be inoculated.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Example 1

1. Preparation of pleurotus geesteranus stick

The method comprises the steps of preparing raw materials by adopting a conventional formula of pleurotus geesteranus, bagging, carrying out thorough sterilization, cooling, and inoculating pleurotus geesteranus of different varieties (strains) according to aseptic operation. After inoculation, the culture medium is placed into a culture room for hypha culture. And (5) stimulating the fungus sticks to produce mushrooms when the hypha reaches physiological maturity.

2. Pathogenic bacteria preparation

The pathogenic bacterium of Yellow spot is West Neeli (Liu Z L, Zhou S, Zhang W, et al. first Report of Cedecea neteri consuming Yellow Rot Disease in Pleurotus pulmonarius in China [ J ] Plant Disease,2020.) which is isolated from the fruiting body of Pleurotus geesteranus infected with Yellow spot in Guangxi and deposited in the institute of microbiology of Guangxi institute of agricultural sciences, the form of the pathogenic bacterium on NA medium is shown in FIG. 1, and the form under electron microscope is shown in FIG. 2. The pathogenic bacteria cause the natural attack of Pleurotus geesteranus as shown in FIG. 3.

Strain activation: placing the macular disease pathogenic bacteria strain in a refrigerator at-80 deg.C for long-term storage, taking out part of the strain stored at-80 deg.C before inoculation, placing in a liquid NB culture medium, performing shake culture at 28 deg.C and 180rpm/min for 48 hr to obtain bacterial liquid, centrifuging the bacterial liquid (5000rpm/min, 2min), collecting precipitate to obtain thallus, diluting with sterile water to 1x10⁸And (4) obtaining an inoculation bacterial suspension for later use.

3. Pleurotus geesteranus fruiting and pathogenic bacteria inoculation method

And selecting bacteria sticks with multiple fruiting bodies, tidiness and health for carrying out subsequent pathogen inoculation tests, wherein the number of the bacteria sticks inoculated to each variety is not less than 10. When the size of pileus of fruiting body is 0.5-1.0cm, inoculation (figure 4) is too big to cause disease (figure 6), and when inoculation is too small, fruiting body is easy to wither and die directly and cannot grow up (figure 7). And (3) dipping the pathogenic bacteria suspension by using a sterile brush, uniformly coating the pathogenic bacteria suspension on pleurotus geesteranus fruiting bodies for inoculation, and coating 5ml of bacterial liquid on each bacterial stick. After inoculation, the fungus sticks are moved to a sealed fresh-keeping box to be horizontally placed in a layered mode (the fungus sticks are preferably 3-8cm away from the box wall in the box, the fruiting surface is preferably 10-20cm away from the box wall, and the distance between the fruiting surface and the box wall is preferably 10-20cmIf the size is too small, fruiting bodies cannot be completely unfolded, and if the size is too large, high CO in the preservation box cannot be kept₂Concentration, resulting in difficult development of fruiting body). The temperature of the preservation box is kept at 25-30 ℃, and the humidity is kept at 85-100%. The fungus sticks are taken out after 48h, the pictures are taken (figure 5), the number of the fruiting bodies of each fungus stick (whether pileus or stipe, if symptoms exist, the disease is calculated), grading is carried out according to the disease incidence, and the disease index is calculated.

4. Disease grade grading standard

And calculating the total quantity of the pathogenic sporocarps and the sporocarps on each bacteria stick, dividing the pathogenic sporocarps and the sporocarps to obtain the incidence of the single bacteria stick, and grading the bacteria sticks respectively according to the incidence of the incidence. The classification criteria are as follows:

5. calculation of disease indexes of different varieties

Disease index (n)₀*0+n₁*1+n₃*3+n₅*5+n₇*7)*100/(N*7)。

N represents the total number of bacteria sticks, N represents the number of bacteria sticks per disease grade, and 0, 1, 3, 5, and 7 represent disease grades.

6. The resistance of the pleurotus geesteranus yellow spot of different varieties is classified according to the disease index, and the standard is as follows:

7. statistics of resistance levels of different varieties of pleurotus geesteranus yellow spot

As shown in table 1.

TABLE 1 statistics of the resistance level to yellow spot of various Pleurotus geesteranus species

As can be seen from the results in the table above, pathogenic bacteria of yellow spot can infect the Pleurotus geesteranus materials of the above varieties and cause plant diseases. The results confirm that Xiuzhen 12 is indeed a high-resistant variety against maculopathy, consistent with the actual planting experience. Therefore, the method for identifying and evaluating the pleurotus geesteranus yellow spot resistance level by utilizing the sporocarp inoculation can accurately identify the yellow spot resistance level of different varieties of pleurotus geesteranus, has reliable results, and has important significance for promoting the breeding work of the disease-resistant pleurotus geesteranus yellow spot varieties.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.