阅读说明：本技术 一种甘蔗细菌性病害抗性的鉴定方法 (Identification method of sugarcane bacterial disease resistance ) 是由 孔春艳 陆鑫 毛钧 刘新龙 林秀琴 田春艳 李纯佳 徐超华 李旭娟 刘洪博 于 2021-06-18 设计创作，主要内容包括：本发明涉及植物抗性检测领域,特别是涉及一种甘蔗细菌性病害抗性的鉴定方法。本发明提供了一种甘蔗细菌性病害抗性的鉴定方法,包括以下步骤：将甘蔗细菌性病害的病原菌液接种到离体的甘蔗叶片后,对接种所得甘蔗叶片进行培养和抗性鉴定,根据经所述培养后甘蔗叶片上条纹病斑长度,判断甘蔗对细菌的抗性和发病等级；发病等级和抗性水平划分标准如下：病斑长度≤5.0cm,发病等级为1级,高抗；5.0cm＜病斑长度≤10.0cm,发病等级为2级,抗病；10.0cm＜病斑长度＜15.0cm,发病等级为3级,感病；病斑长度≥15.0cm,发病等级为4级,高感。本发明所述方法具有操作简单、不受季节和外界环境的限制、接种鉴定稳定、效率高和周期短的优势。(The invention relates to the field of plant resistance detection, in particular to an identification method of sugarcane bacterial disease resistance. The invention provides a method for identifying sugarcane bacterial disease resistance, which comprises the following steps: inoculating a pathogenic bacteria liquid of sugarcane bacterial diseases to in-vitro sugarcane leaves, culturing and identifying resistance of the sugarcane leaves obtained by inoculation, and judging the resistance and disease level of the sugarcane to bacteria according to the length of the stripe disease spots on the sugarcane leaves after culture; the criteria for grading the grade of onset and the level of resistance are as follows: the length of the disease spot is less than or equal to 5.0cm, the disease grade is 1 grade, and the disease resistance is high; the length of the disease spot is more than 5.0cm and less than or equal to 10.0cm, the disease grade is 2, and the disease is resisted; the length of the disease spot is more than 10.0cm and less than 15.0cm, the disease grade is 3 grade, and the disease is infected; the length of the disease spot is more than or equal to 15.0cm, the disease grade is 4 grade, and the feeling is high. The method has the advantages of simple operation, no limitation by seasons and external environments, stable inoculation identification, high efficiency and short period.)

1. The method for identifying the resistance of sugarcane to bacterial diseases is characterized by comprising the following steps:

inoculating a pathogenic bacteria liquid of sugarcane bacterial diseases to in-vitro sugarcane leaves, culturing and identifying resistance of the sugarcane leaves obtained by inoculation, and judging the resistance and disease level of the sugarcane to bacteria according to the length of the stripe disease spots on the sugarcane leaves after culture;

the criteria for grading the grade of onset and the level of resistance are as follows:

the length of the disease spot is less than or equal to 5.0cm, the disease grade is 1 grade, and the disease resistance is high;

the length of the disease spot is more than 5.0cm and less than or equal to 10.0cm, the disease grade is 2, and the disease is resisted;

the length of the disease spot is more than 10.0cm and less than 15.0cm, the disease grade is 3 grade, and the disease is infected;

the length of the disease spot is more than or equal to 15.0cm, the disease grade is 4 grade, and the feeling is high.

2. The method of claim 1, wherein the time of inoculation is 24 hours and the temperature is 28 ℃; the temperature of the culture is 28 ℃ and the time is preferably 10 d.

3. The identification method according to claim 1, wherein the method for preparing the pathogenic bacteria solution comprises the following steps:

(1) putting pathogenic bacteria of sugarcane bacterial diseases into an XAL liquid culture medium for activated culture to obtain activated bacteria liquid;

(2) placing the activated bacterium liquid in an XAS solid culture medium for culturing to obtain a pathogenic bacterium single colony of sugarcane bacterial diseases;

(3) selecting a single pathogenic bacterium colony, inoculating the single pathogenic bacterium colony into an XAS solid culture medium, and culturing to obtain purified bacteria;

(4) and (3) placing the single colony of the purified bacteria in an XAL liquid culture medium for culture, and diluting the single colony of the purified bacteria by using the XAL liquid culture medium to obtain the pathogenic bacteria liquid.

4. The identification method according to claim 3, wherein the XAL liquid medium comprises the following components at the following concentrations: 10g/L sucrose, 5g/L bacto-grade peptone, 5g/L yeast extract, 0.5g/L monopotassium phosphate, 0.25g/L magnesium sulfate heptahydrate, 0.05g/L sodium sulfite, 5g/L potassium bromide, 0.01g/L benomyl, 25mg/L cephalexin, 30mg/L novobiocin, 50mg/L vernalin and 100mg/L cycloheximide.

5. The identification method according to claim 3, wherein the XAS solid medium comprises the following components in concentrations: 10g/L of sucrose, 5g/L of bacto-grade peptone, 5g/L of yeast extract, 0.5g/L of monopotassium phosphate, 0.25g/L of magnesium sulfate heptahydrate, 0.05g/L of sodium sulfite, 5g/L of potassium bromide, 0.01g/L of benomyl, 15g/L of agar powder, 25mg/L of cephalexin, 30mg/L of novobiocin, 50mg/L of vernalicin and 100mg/L of cycloheximide.

6. The identification method according to claim 3, wherein the temperature of the activation culture in step (1) is 28-32 ℃, the rotation speed is 200rpm, and the time is 18-24 h.

7. The identification method according to claim 3, wherein the temperature of the culture in step (2) and the culture in step (3) are both 28-32 ℃ and the time is 4-5 days.

8. The identification method according to claim 3, wherein the temperature of the culture in the step (4) is 28 to 32 ℃, the rotation speed is 200rpm, and the time is 36 to 48 hours.

9. The identification method according to any one of claims 1 to 8, wherein the concentration of the pathogenic bacterium solution is 1X 10⁸cfu/mL。

10. The identification method according to claim 1, wherein the sugarcane leaf is 20cm in length.

Technical Field

The invention relates to the field of plant resistance detection, in particular to an identification method of sugarcane bacterial disease resistance.

Background

Sugarcane canebelt disease is caused by Xanthomonas albilineans (Xanthomonas albilineans), has destructive bacterial diseases on sugarcane, and is one of the three major bacterial diseases of sugarcane. The Xanthomonas albus is listed in the entry plant quarantine harmful organism directory of the people's republic of China in 2007, and is one of the main objects of the import quarantine of sugarcane germplasm in China.

After the Xanthomonas albilineans infects sugarcane plants, the sugarcane grows slowly, and plant necrosis is caused when the sugarcane is serious, so that serious loss is brought to sugarcane production. In recent years, the disease has been reported in sugarcane areas such as Guangxi, Guangdong, Yunnan and Hainan provinces in China. Onset symptoms of sugarcane stripe disease: the leaf surface is seen to appear as a white to yellow, dull pencil-line stripe parallel to the main vein, with the color of the stripe often turning reddish over time, with the edges widening, with many small red dots in the stripe.

The sugarcane canebellosis is a bacterial seed-borne disease with strong infectivity, and is mainly propagated by long-distance propagation through transportation of diseased seedlings, propagation of harvesting tools such as cutters and the like, and propagation of air flow and rainwater, so that the disease is easily spread by open type living inoculation. The disease usually does not show symptoms or symptoms are not obvious in a long latent period, and the disease suddenly breaks out once meeting a proper environment, and particularly when a sugarcane variety is planted to be a disease-resistant variety, large-area infection can cause serious yield loss. The latent and sudden characteristics bring great difficulty to the prevention and the quarantine of the sugarcane canker. In order to reduce the harm of the canker to the production of the sugarcane, the breeding, popularization and application of disease-resistant varieties are the most economic and effective measures for preventing and controlling the green canker of the sugarcane.

The traditional inoculation identification method for the sugarcane leaf-streak disease mainly comprises a leaf-cutting method and a head-cutting method, and the leaf-cutting method is mainly characterized in that: dipping a sterile scalpel into the xanthomonas albilineans suspension in the sugarcane seedling stage, cutting off 1/3 parts of plant leaf tips, and dipping the scalpel into a bacterial solution again after 1 plant is inoculated; the leaf cutting method is mainly characterized in that: cutting off the head of the sugarcane +1 leaf at the leaf ear, and smearing 100 mu L of prepared bacterial suspension on the cut position by using a liquid transfer gun; the identification and evaluation of the sugarcane variety canker resistance are carried out by adopting a leaf cutting method and a cutting method, although the operation is simple, the influence of the limitation of sugarcane growing seasons and the change of external environment is larger, the inoculation identification is unstable, the efficiency is low, and the period is longer.

Disclosure of Invention

In order to solve the above problems, the present invention provides a method for identifying resistance to sugarcane bacterial diseases. The method provided by the invention is simple to operate, is not limited by seasons and external environments, and has the advantages of stable inoculation identification, high efficiency and short period.

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

the invention provides a method for identifying sugarcane bacterial disease resistance, which comprises the following steps:

inoculating a pathogenic bacteria liquid of sugarcane bacterial diseases to in-vitro sugarcane leaves, culturing and identifying resistance of the sugarcane leaves obtained by inoculation, and judging the resistance and disease level of the sugarcane to bacteria according to the length of the stripe disease spots on the sugarcane leaves after culture;

the criteria for grading the grade of onset and the level of resistance are as follows:

the length of the disease spot is less than or equal to 5.0cm, the disease grade is 1 grade, and the disease resistance is high;

the length of the disease spot is less than or equal to 10.0cm when the disease spot is less than 5.0cm, the disease grade is 2 grade, and the disease is resisted;

the disease is infected when the length of the disease spot is less than 10.0cm and the disease grade is 3, and the length of the disease spot is less than 15.0 cm;

the length of the disease spot is more than or equal to 15.0cm, the disease grade is 4 grade, and the feeling is high.

Preferably, the inoculation time is 24 hours, and the temperature is 28 ℃; the temperature of the culture is 28 ℃ and the time is preferably 10 d.

Preferably, the preparation method of the pathogenic bacteria liquid comprises the following steps:

(1) putting pathogenic bacteria of sugarcane bacterial diseases into an XAL liquid culture medium for activated culture to obtain activated bacteria liquid;

(2) placing the activated bacterium liquid in an XAS solid culture medium for culturing to obtain a pathogenic bacterium single colony of sugarcane bacterial diseases;

(3) selecting a single pathogenic bacterium colony, inoculating the single pathogenic bacterium colony into an XAS solid culture medium, and culturing to obtain purified bacteria;

(4) and (3) placing the single colony of the purified bacteria in an XAL liquid culture medium for culture, and diluting the single colony of the purified bacteria by using the XAL liquid culture medium to obtain the pathogenic bacteria liquid.

Preferably, the XAL liquid medium comprises the following components at the following concentrations: 10g/L sucrose, 5g/L bacto-grade peptone, 5g/L yeast extract, 0.5g/L monopotassium phosphate, 0.25g/L magnesium sulfate heptahydrate, 0.05g/L sodium sulfite, 5g/L potassium bromide, 0.01g/L benomyl, 25mg/L cephalexin, 30mg/L novobiocin, 50mg/L vernalin and 100mg/L cycloheximide.

Preferably, the XAS solid medium comprises the following components in the following concentrations: 10g/L of sucrose, 5g/L of bacto-grade peptone, 5g/L of yeast extract, 0.5g/L of monopotassium phosphate, 0.25g/L of magnesium sulfate heptahydrate, 0.05g/L of sodium sulfite, 5g/L of potassium bromide, 0.01g/L of benomyl, 15g/L of agar powder, 25mg/L of cephalexin, 30mg/L of novobiocin, 50mg/L of vernalicin and 100mg/L of cycloheximide.

Preferably, the temperature of the activation culture in the step (1) is 28-32 ℃, the rotating speed is 200rpm, and the time is 18-24 h.

Preferably, the temperature of the culture in the step (2) and the culture in the step (3) are both 28-32 days, and the time is 4-5 days.

Preferably, the temperature of the culture in the step (4) is 28-32 ℃, the rotating speed is 200rpm, and the time is 36-48 h.

Preferably, the concentration of the pathogenic bacteria liquid is 1 × 10⁸cfu/mL。

Preferably, the length of the sugarcane leaf is 20 cm.

Has the advantages that: the invention provides a method for identifying sugarcane bacterial disease resistance, which comprises the following steps: inoculating a pathogenic bacteria liquid of sugarcane bacterial diseases to in-vitro sugarcane leaves, culturing and identifying resistance of the sugarcane leaves obtained by inoculation, and judging the resistance and disease level of the sugarcane to bacteria according to the length of the stripe disease spots on the sugarcane leaves after culture; wherein the disease grade and resistance level are divided into the following criteria: the length of the disease spot is less than or equal to 5.0cm, the grade 1 is high, and the disease resistance is high; disease-resistant, 2-grade, disease-resistant, with the length of the disease spot being less than or equal to 10.0cm and less than 5.0 cm; disease infection is caused when the length of lesion is 10.0cm and the length of lesion is less than 15.0cm and grade 3; the length of the lesion is more than or equal to 15.0cm, 4 grades and high feeling. The method can rapidly and accurately identify the disease resistance of the material by utilizing the sugarcane in-vitro leaves, has the characteristics of simple operation, good controllability, high efficiency and the like, can ensure the continuity of pathogen inoculation and infection and the consistency and controllability of the pathogenic environment, and has symptoms when the pathogen is infected for 1d, and is a simple, convenient and efficient inoculation identification method. The method is carried out under a controllable environment condition, so that the continuity, stability and uniformity of pathogen inoculation and infection conditions are ensured, errors caused by the environment condition are reduced, and the method is higher in accuracy compared with the traditional method; the inoculation identification result is stable, and the repeatability is good; the method is simple and convenient, has short period (diseases are caused by infection for 1 d), has high inoculation efficiency, can repeatedly identify a single plant, and is suitable for large-scale inoculation identification of materials. The invention can perform multiple inoculation identification on 8 sugarcane varieties, and has stable performance.

Compared with the traditional method, the method has the advantages of short identification period, simple and convenient operation, good repeatability and the like, can save a large amount of resources and resources, realizes the purpose of large-scale quick identification, and has important significance for the identification of the resistance of sugarcane bacterial diseases and the breeding for disease resistance, especially the identification of the resistance of sugarcane bacterial diseases and the research work of the breeding for disease resistance.

Drawings

FIG. 1 is a standard schematic view of blade classification (stages 1-4);

FIG. 2 is a level 1 standard schematic of FIG. 1;

FIG. 3 is a standard level 2 schematic of FIG. 1;

FIG. 4 is a standard level 3 schematic of FIG. 1;

fig. 5 is a standard 4-level diagram of fig. 1.

Detailed Description

The components used in the present invention are all those conventionally purchased by those skilled in the art, unless otherwise specified.

The invention provides a method for identifying sugarcane bacterial disease resistance, which comprises the following steps:

inoculating a pathogenic bacteria liquid of sugarcane bacterial diseases to in-vitro sugarcane leaves, culturing and identifying resistance of the sugarcane leaves obtained by inoculation, and judging the resistance and disease level of the sugarcane to bacteria according to the length of the stripe disease spots on the sugarcane leaves after culture;

the criteria for grading the grade of onset and the level of resistance are as follows:

the length of the disease spot is less than or equal to 5.0cm, the disease grade is 1 grade, and the disease resistance is high;

the length of the disease spot is less than or equal to 10.0cm when the disease spot is less than 5.0cm, the disease grade is 2 grade, and the disease is resisted;

the disease is infected when the length of the disease spot is less than 10.0cm and the disease grade is 3, and the length of the disease spot is less than 15.0 cm;

the length of the disease spot is more than or equal to 15.0cm, the disease grade is 4 grade, and the feeling is high.

The method comprises the steps of inoculating pathogenic bacteria liquid of sugarcane bacterial diseases to in-vitro sugarcane leaves, culturing the sugarcane leaves obtained by inoculation and identifying the resistance, and judging the resistance and disease level of the sugarcane to bacteria according to the length of the stripe disease spots on the sugarcane leaves after culture. In the present invention, the sugar cane leaves are preferably fresh sugar cane leaves; the growth period of the sugarcane leaves is not limited at all, and the sugarcane leaves are preferably an elongation period or a mature period, and more preferably an elongation period; the concentration of the pathogenic bacteria liquid is preferably 1 × 10⁸cfu/mL; the color of the disease spots of the stripe diseases at the initial stage is preferably white or cream yellow, and the color of the disease spots at the later stage is preferably red; the lesions are preferably selected fromThe veins of the sugarcane leaves are parallel; the species of the bacteria in the specific embodiment of the invention is not particularly required, preferably the sugarcane smut, more preferably the sugarcane smut pathogen strain LB-1 (abbreviated as strain LB-1), and the sugarcane smut pathogen strain LB-1 has been disclosed in isolation and identification of the sugarcane smut pathogen in Guangxi (Weichun, Wajinju, Zhang autumn, Zhang Bao, Song Xiong, Lidewei, Suzheng Qiang, Liyan Rui. phytosanitary, 2019, 33 (01): 19-23).

The inoculation mode of the invention is preferably that the base part of the sugarcane leaf is placed in the pathogenic bacteria liquid, the tip of the leaf which is not immersed in the pathogenic bacteria liquid is sprayed with sterile water, and the preservative film is sealed; the time for inoculation is preferably 24 hours; the temperature of the inoculation is preferably 28 ℃. According to the invention, the bacterial liquid is preferably poured off after the inoculation is finished.

After the inoculation, the sugarcane leaves obtained by the inoculation are preferably fully wetted and cultured after being sealed by a preservative film. The invention preferably adopts sterile water for wetting; the standard of sufficient wetting is that the water on the leaf surface is covered by water, but water drops cannot flow down; the temperature of the culture is preferably 28 ℃; the time for the cultivation is preferably 10 days.

The length of the sugarcane leaf is preferably 20 cm; the sugarcane leaves are preferably cut from healthy, disease-and-pest-free and disinfected sugarcane leaves; the cut is required to be neat.

In the present invention, the disinfection is preferably performed by sequentially using alcohol disinfection and sodium hypochlorite aqueous solution disinfection; the volume percentage content of the alcohol is 75 percent; the time for alcohol sterilization is preferably 3 min.

After the alcohol disinfection, the method preferably further comprises cleaning the leaves obtained after the alcohol disinfection; the cleaning is preferably performed by using sterile distilled water; the number of washing is preferably 3.

In the invention, the mass percentage of the available chlorine in the sodium hypochlorite aqueous solution is 0.1%; the time for the sterilization is preferably 10 min.

After the sodium hypochlorite aqueous solution is disinfected, preferably, cleaning and airing the blades obtained by the disinfection of the sodium hypochlorite aqueous solution; the cleaning is preferably performed by using sterile distilled water; the number of washing is preferably 3; the air drying mode is not limited at all, and the mode known by the person skilled in the art can be adopted; preferably, the airing temperature is 20-28 ℃, and most preferably 25 ℃; the criterion for drying is that the leaves show no water droplets.

The preparation method of the pathogenic bacteria liquid preferably comprises the following steps:

(1) putting pathogenic bacteria of sugarcane bacterial diseases into an XAL liquid culture medium for activated culture to obtain activated bacteria liquid;

(2) placing the activated bacterium liquid in an XAS solid culture medium for culturing to obtain a pathogenic bacterium single colony of sugarcane bacterial diseases;

(3) selecting a single pathogenic bacterium colony, inoculating the single pathogenic bacterium colony into an XAS solid culture medium, and culturing to obtain purified bacteria;

(4) and (3) placing the single colony of the purified bacteria in an XAL liquid culture medium for culture, and diluting the single colony of the purified bacteria by using the XAL liquid culture medium to obtain the pathogenic bacteria liquid.

The volume ratio of pathogenic bacteria of sugarcane bacterial diseases to the XAL liquid culture medium is preferably (100-1000) muL to (1-10) mL, and more preferably 100 muL to 1 mL; the temperature of the culture is preferably 28 ℃; the rotation speed of the culture is preferably 200 rpm; the culture time is preferably 18-24 hours, and more preferably 19-23 hours.

The XAL liquid medium of the invention preferably comprises the following components in concentration: 10g/L of sucrose, 5g/L of bacto-grade peptone, 5g/L of yeast extract, 0.5g/L of monopotassium phosphate, 0.25g/L of magnesium sulfate heptahydrate, 0.05g/L of sodium sulfite, 5g/L of potassium bromide, 0.01g/L of benomyl, 25mg/L of cephalexin, 30mg/L of novobiocin, 50mg/L of vernalicin and 100mg/L of cycloheximide; the components of the XAL liquid medium can be referred to in Evaluation of selective media and immunoassays for detection of Xanthomonas albumins, the clinical agent of biochemical leaf area (Davis M J, Rott P, Baudin P, Dean J L. plant area, 1994, 78: 78-82).

The preparation method of the XAL liquid culture medium preferably comprises the following steps: dissolving sucrose, bacterial grade peptone, yeast extract, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, sodium sulfite, potassium bromide and benomyl in 900mL of pure water, adjusting pH to 7.0, and fixing volume to 1L to obtain a culture medium; subpackaging and autoclaving the culture medium, and adding an antibiotic mother liquor storage solution when the temperature of the culture medium obtained after autoclaving is not higher than 55 ℃ to enable the final use concentrations to be 25mg/L of cefalexin, 30mg/L of novobiocin, 50mg/L of vernacin and 100mg/L of cycloheximide to obtain the XAL liquid culture medium.

In the invention, the antibiotic mother liquor storage liquid preferably comprises a cefalexin mother liquor storage liquid, a neomycin mother liquor storage liquid, a kasugamycin mother liquor storage liquid and a cycloheximide mother liquor storage liquid which are independently subpackaged.

The preparation method of the cefalexin mother liquor storage liquid preferably comprises the following steps: when the prepared concentration of cefalexin is 25mg/L, weighing 125mg of cefalexin, adding the cefalexin into 10mL of sterilized distilled water, and filtering by using a bacterial filter to obtain cefalexin mother liquor storage liquid; the concentration of the cefalexin mother liquor is preferably 12.5mg/mL, and the cefalexin mother liquor is subpackaged into small parts and stored at-20 ℃. When the cefalexin mother liquor storage liquid is used, preferably 2 mu L of cefalexin mother liquor storage liquid is taken out of each milliliter of culture medium and added into the culture medium, so that the use concentration of cefalexin is 25 mg/L. The present invention is not limited in any way to the manner of filtration, and may be performed in a manner known to those skilled in the art.

The preparation method of the neomycin mother liquor stock solution preferably comprises the following steps: when the concentration of the prepared neomycin is 30mg/L, weighing 150mg of neomycin, adding the neomycin into 10mL of sterilized water, and filtering by using a bacterial filter to obtain a neomycin mother liquor stock solution; the concentration of the stock solution of the neomycin mother liquor is preferably 150mg/mL, and the stock solution is divided into small parts to be stored at the temperature of minus 20 ℃. When the neomycin mother liquor stock solution is used, 2 mu L of neomycin mother liquor stock solution is preferably taken in per ml of culture medium and added into the culture medium, so that the use concentration of neomycin is ensured to be 30 mg/L. The present invention is not limited in any way to the manner of filtration, and may be performed in a manner known to those skilled in the art.

The preparation method of the kasugamycin mother liquor stock solution preferably comprises the following steps: when the prepared kasugamycin concentration is 50mg/L, 250mg of kasugamycin is weighed and added into 10mL of sterilized water, and a mother liquor storage solution of the kasugamycin is obtained after the filtration by a bacterial filter; the concentration of the kasugamycin mother solution is preferably 250mg/mL, and the kasugamycin mother solution is subpackaged into small parts and stored at the temperature of-20 ℃. When the kasugamycin mother solution storage solution is used, 2 mu L of the kasugamycin mother solution storage solution is preferably taken out of each milliliter of culture medium and added into the culture medium, so that the use concentration of the kasugamycin is ensured to be 50 mg/L. The present invention is not limited in any way to the manner of filtration, and may be performed in a manner known to those skilled in the art.

The preparation method of the cycloheximide mother liquor storage solution preferably comprises the following steps: when the configured concentration of the cycloheximide is 100mg/L, weighing 500mg of cycloheximide, adding the cycloheximide into 10mL of sterilized water, and filtering by using a bacterial filter to obtain a cycloheximide mother liquor storage solution; the concentration of the cycloheximide mother liquor is preferably 500mg/mL, and the cycloheximide mother liquor is subpackaged into small parts and stored at the temperature of minus 20 ℃. When the cycloheximide mother liquor storage solution is used, preferably 2 mu L of cycloheximide mother liquor storage solution is taken out of every milliliter of culture medium and added into the culture medium, so that the use concentration of cycloheximide is ensured to be 100 mg/L. The present invention is not limited in any way to the manner of filtration, and may be performed in a manner known to those skilled in the art.

The preparation method of the antibiotic mother liquor storage liquid can save time and can prepare the storage liquid with proper concentration.

In the present invention, the temperature of the autoclaving is preferably 121 ℃; the time for the autoclaving is preferably 21 min.

The volume-mass ratio of the activated bacterial liquid to the XAS solid culture medium is preferably (10-200) muL to (10-20) mL, and more preferably 100 muL to 15 mL; the temperature of the activation culture is preferably 28-32 ℃, and the most preferably 28 ℃; the activation culture time is preferably 4-5 d, and more preferably 5 d; during the activation culture, the diameter of a culture dish is preferably 90 mm; the height of the culture dish is preferably 20 mm.

The XAS solid culture medium comprises the following components in concentration: 10g/L of sucrose, 5g/L of bacto-grade peptone, 5g/L of yeast extract, 0.5g/L of monopotassium phosphate, 0.25g/L of magnesium sulfate heptahydrate, 0.05g/L of sodium sulfite, 5g/L of potassium bromide, 0.01g/L of benomyl, 15g/L of agar powder, 25mg/L of cephalexin, 30mg/L of novobiocin, 50mg/L of vernalicin and 100mg/L of cycloheximide; the components of the XAS solid medium can be referred to in Evaluation of selective media and immunoassays for detection of Xanthomonas albumins, the clinical agent of biochemical leaf area (Davis M J, Rott P, Baudin P, Dean J L. plant area, 1994, 78: 78-82).

The preparation method of the XAS solid culture medium preferably comprises the following steps: dissolving sucrose, bacterial grade peptone, yeast extract, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, sodium sulfite, potassium bromide and benomyl in 900mL of pure water, adjusting the pH to 7.0, fixing the volume to 1L, and adding 15g of agar powder to obtain a culture medium; subpackaging and autoclaving the culture medium, and adding an antibiotic mother solution storage solution when the temperature of the culture medium obtained after autoclaving is 50-55 ℃, more preferably 50 ℃ to obtain the XAS solid culture medium. The autoclaving method of the present invention is described above, and is not described herein.

The single colony of pathogenic bacteria is preferably selected by using an inoculating loop.

The method for obtaining the single bacterial colony of the purified bacteria is preferably to pick the single bacterial colony by using a sterile sucker; the volume of the sterile tip is preferably 10 μ L; the dosage of the XAL liquid culture medium is preferably 10-20 mL, and more preferably 15 mL; the culture temperature is preferably 28-23 ℃, and more preferably 28 ℃; the rotation speed of the culture is preferably 200 rpm; the culture time is preferably 36-48 h, and more preferably 38-46 h; during the culture, the diameter of the culture dish is preferably 90 mm; the height of the culture dish is preferably 20 mm.

The disease grade and resistance level division standard of the invention is as follows:

the length of the disease spot is less than or equal to 5.0cm, the disease grade is 1 grade, and the disease resistance is high;

the length of the disease spot is less than or equal to 10.0cm when the disease spot is less than 5.0cm, the disease grade is 2 grade, and the disease is resisted;

the disease is infected when the length of the disease spot is less than 10.0cm and the disease grade is 3, and the length of the disease spot is less than 15.0 cm;

the length of the lesion is more than or equal to 15.0cm, 4 grades and high feeling.

For further illustration of the present invention, the following detailed description will be made of a method for identifying sugarcane bacterial disease resistance provided by the present invention with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.

Example 1

1. For identifying sugarcane varieties

In the embodiment, the tested materials are Co1146, cassia sugar 97-69, LCP85-384, Yunzan 08-1609, Yunzan 05-51, ROC20, cassia sugar 03-1462 and YueGan No. 50 8 varieties.

2. LB-1 culture of pathogenic strain of sugarcane white stripe disease

Unfreezing a frozen stock solution of the Xanthomonas albus strain LB-1 stored at-80 ℃, putting 100 mu L of the stock solution into 1mL of XAL liquid culture medium, and carrying out shake culture at 28 ℃ and 200rpm for 18-24 h; and then putting 100 mu L of the shake-cultured bacterial liquid into a sterile XAS solid culture medium, uniformly coating, putting into a constant temperature box at 28 ℃ for culturing for 5d, then using an inoculating loop to select a single bacterial colony of the Xanthomonas albus on the XAS solid culture medium in a sterile operation mode, inoculating into a new XAS solid culture medium, and culturing for 5d in the constant temperature box at 28 ℃ to obtain the purified Xanthomonas albus. Picking out single colony of purified Xanthomonas albilineans with 10 μ L sterile suction head, placing into 20mLXAL liquid culture medium, performing shaking culture at 28 deg.C and 200rpm for 36-48 h, and preparing into 1 × 10 bacteria concentration with sterilized XAL liquid culture medium⁸cfu/mL pathogen suspension was used immediately for inoculation.

3. Preparation and preparation of XAL liquid Medium

Pouring prepared and autoclaved XAL liquid culture medium into a container for standby, wherein the XAL liquid culture medium comprises the following components in parts by weight: dissolving 10g of sucrose, 5g of bacterial grade peptone, 5g of yeast extract, 0.5g of dipotassium phosphate, 0.25g of magnesium sulfate heptahydrate, 0.05g of sodium sulfite, 5g of potassium bromide and 0.01g of benomyl in 900mL of pure water, adjusting the pH value to 7.0 and then fixing the volume to 1L; subpackaging the culture medium, and sterilizing at 121 deg.C for 21 min; when the prepared concentration of cefalexin is 25mg/L, weighing 125mg of cefalexin, adding the cefalexin into 10mL of sterilized distilled water, filtering by using a bacterial filter to obtain cefalexin mother liquor storage liquid, wherein the concentration of the cefalexin mother liquor storage liquid is 12.5mg/mL, subpackaging into small parts, storing at-20 ℃, taking 2 mu L of cefalexin mother liquor storage liquid out of every milliliter of culture medium during use, and adding the cefalexin into the culture medium to ensure that the use concentration of the cefalexin is 25 mg/L; when the concentration of the prepared neomycin is 30mg/L, weighing 150mg of neomycin, adding the neomycin into 10mL of sterilized water, filtering by using a bacterial filter to obtain a neomycin mother liquor stock solution, wherein the concentration of the neomycin mother liquor stock solution is 150mg/mL, subpackaging into small parts, storing at-20 ℃, taking 2 mu L of the neomycin mother liquor stock solution from each milliliter of culture medium during use, adding the fresh neomycin mother liquor stock solution into the culture medium, and ensuring that the use concentration of the neomycin is 30 mg/L; when the configured concentration of the kasugamycin is 50mg/L, 250mg of the kasugamycin is weighed and added into 10mL of sterilized water, and the sterilized water is filtered by a bacterial filter to obtain a kasugamycin mother liquor stock solution, wherein the concentration of the kasugamycin mother liquor stock solution is 250mg/mL, the kasugamycin mother liquor stock solution is subpackaged into small parts and stored at the temperature of minus 20 ℃, 2 mu L of the kasugamycin mother liquor stock solution is taken out of every milliliter of a culture medium during use and added into the culture medium, and the use concentration of the kasugamycin is ensured to be 50 mg/L; when the configured concentration of the cycloheximide is 100mg/L, weighing 500mg of cycloheximide, adding the cycloheximide into 10mL of sterilized water, filtering by using a bacterial filter to obtain a cycloheximide mother liquor storage solution, wherein the concentration of the cycloheximide mother liquor storage solution is 500mg/mL, subpackaging into small parts, storing at-20 ℃, taking 2 mu L of cycloheximide mother liquor storage solution from every milliliter of culture medium during use, adding the cycloheximide mother liquor storage solution into the culture medium, and ensuring that the use concentration of the cycloheximide is 100mg/L to obtain an antibiotic mother liquor storage solution; when the temperature of the culture solution after autoclaving is not higher than 55 ℃, adding an antibiotic mother solution storage solution to make the final concentration of the antibiotic mother solution be 25mg/L of cefalexin, 30mg/L of novobiocin, 50mg/L of kasugamycin and 100mg/L of cycloheximide.

Preparation and preparation of XAS solid Medium

The XAS solid culture medium comprises the following components in parts by weight: dissolving 10g of sucrose, 5g of bacterial grade peptone, 5g of yeast extract, 0.5g of dipotassium phosphate, 0.25g of magnesium sulfate heptahydrate, 0.05g of sodium sulfite, 5g of potassium bromide and 0.01g of benomyl in 900mL of pure water, adjusting the pH to 7.0, fixing the volume to 1L, adding 15g of agar powder, and uniformly mixing; subpackaging the culture medium, and sterilizing at 121 deg.C for 21 min; when the prepared concentration of cefalexin is 25mg/L, weighing 125mg of cefalexin, adding the cefalexin into 10mL of sterilized distilled water, filtering by using a bacterial filter to obtain cefalexin mother liquor storage liquid, wherein the concentration of the cefalexin mother liquor storage liquid is 12.5mg/mL, subpackaging into small parts, storing at-20 ℃, taking 2 mu L of cefalexin mother liquor storage liquid out of every milliliter of culture medium during use, and adding the cefalexin into the culture medium to ensure that the use concentration of the cefalexin is 25 mg/L; when the concentration of the prepared neomycin is 30mg/L, weighing 150mg of neomycin, adding the neomycin into 10mL of sterilized water, filtering by using a bacterial filter to obtain a neomycin mother liquor stock solution, wherein the concentration of the neomycin mother liquor stock solution is 150mg/mL, subpackaging into small parts, storing at-20 ℃, taking 2 mu L of the neomycin mother liquor stock solution from each milliliter of culture medium during use, adding the fresh neomycin mother liquor stock solution into the culture medium, and ensuring that the use concentration of the neomycin is 30 mg/L; when the configured concentration of the kasugamycin is 50mg/L, 250mg of the kasugamycin is weighed and added into 10mL of sterilized water, and the sterilized water is filtered by a bacterial filter to obtain a kasugamycin mother liquor stock solution, wherein the concentration of the kasugamycin mother liquor stock solution is 250mg/mL and is subpackaged into small parts for storage at the temperature of-20 ℃, 2 mu L of the kasugamycin mother liquor stock solution is taken out of each milliliter of culture medium during use and added into the culture medium, and the use concentration of the kasugamycin is ensured to be 50 mg/L; when the configured concentration of the cycloheximide is 100mg/L, weighing 500mg of cycloheximide, adding the cycloheximide into 10mL of sterilized water, filtering by using a bacterial filter, and obtaining a cycloheximide mother liquor storage solution, wherein the concentration of the cycloheximide mother liquor storage solution is configured to be 500mg/mL, subpackaging into small parts and storing at-20 ℃, and adding 2 mu L of cycloheximide mother liquor storage solution into a culture medium in each milliliter of the culture medium during use so as to ensure that the use concentration of the cycloheximide is 100 mg/L; when the temperature of the culture solution after autoclaving is 50 ℃, adding an antibiotic mother solution storage solution to make the final concentration of the antibiotic mother solution be 25mg/L of cefalexin, 30mg/L of novobiocin, 50mg/L of kasugamycin and 100mg/L of cycloheximide.

4. Sugarcane in vitro leaf inoculation

Collecting 3 healthy fresh sugarcane leaves without diseases and insect pests and in an elongation period, taking 1 leaf from each sugarcane leaf, washing with tap water, sterilizing with 75% alcohol for 3min, washing with sterile water for 3 times, sterilizing with sodium hypochlorite solution with effective chlorine of 0.1% for 10min, washing with sterile water for 3 times, and air drying. Cutting the leaves into 20 cm-long segments with high-pressure sterilized scissors, wherein the cut ends are required to be neat; then inserting one end of the base part of the leaf into a 2L plastic measuring cup filled with 50mL of pathogenic bacteria liquid, spraying sterile water on the tip of the leaf which is not immersed in the bacterial suspension, and sealing by using a preservative film; placing the measuring cup in an incubator at 28 ℃, culturing for 24h, and then pouring out the bacterial liquid (sterilizing the poured bacterial liquid); then, fully wetting the detached leaves with sterilized water, putting the detached leaves back into the measuring cup, and sealing the measuring cup with a preservative film; continuously culturing in an incubator at 28 ℃ for 10 days to complete the inoculation of the isolated leaves; mock inoculation with sterile XAL broth uninoculated with the pathogen served as a control.

5. Investigating leaf morbidity and statistical analysis

After the in vitro leaf inoculation is completed, measuring the length of a stripe disease spot (the color of the stripe disease spot at the initial stage of the disease is white or cream yellow, and the color of the disease spot at the later stage of the disease is red) parallel to the veins by using a ruler, and calculating the average value; and calculating the disease grade of each sample according to the length average value of the disease spots and counting the disease resistance of each sample. The disease grade and the level of resistance are shown in Table 1.

TABLE 1 in vitro leaf inoculation incidence grade division standard for sugarcane leaf streak disease

Grade of disease onset	Description of the symptoms	Level of resistance
			Level 1	The length of the stripe disease spot along the vein direction is less than or equal to 5.0cm	Gao Kang
Stage 2	The length of the stripe disease spot along the vein direction is 5.1-10.0 cm	Disease resistance
			Grade 3	The length of the stripe disease spot along the vein direction is 10.1-15.0 cm	Infection of the disease
4 stage	The length of the stripe disease spot along the vein direction is more than or equal to 15.1cm	Feeling of height

6. Analysis of results

After 11 days of inoculation treatment, 8 varieties of sugarcane were investigated, and the results of the investigation are shown in Table 2.

TABLE 2 identification results of resistance levels of different sugarcane varieties against Rhizopus graminis

Name of Material	Length/cm of lesion	Level of resistance
			Osmanthus sugar 03-1462	18.63	Feeling of height
Yunzhan 05-51	14.7	Infection of the disease
			ROC20	13.03	Infection of the disease
Yuncao 08-1609	10.97	Infection of the disease
			LCP85-384	7.60	Disease resistance
Sweet osmanthus 97-69	6.27	Disease resistance
			Co1146	3.53	Gao Kang
Yue gan No. 50	3.37	Gao Kang

As shown in Table 2, the leaf spots of Osmanthus 03-1462 among the 8 sugarcane varieties were the largest, the leaf spots of Yunzan 05-51, Yunzan 08-1609 and ROC20 were large, the leaf spots of Osmanthus 97-69, LCP85-384 and Co1146 were small, and the leaf spot of Yuugan 50 was the smallest. The lowest resistance level of the cinnamic sugar 03-1462 in the 8 tested identification materials is seen, and the materials are high-sensitivity materials; the sugarcane 08-1609 and ROC20 are disease-sensitive materials; 97-69 parts of cassia sugar and 85-384 parts of LCP as disease-resistant materials; co1146 and Yue gan No. 50 are high-resistance materials. The in vitro leaf resistance identification result is basically consistent with the performance in production, and the in vitro leaf identification method is proved to be capable of distinguishing the resistance of different sugarcane varieties to the sugarcane canebullosis.

According to the method disclosed by the embodiment, the disease resistance of the material can be rapidly and accurately identified by utilizing the sugarcane in-vitro leaves, the method has the characteristics of simplicity in operation, good controllability, high efficiency and the like, the persistence of pathogen inoculation and infection and the consistency and controllability of the pathogenic environment can be ensured, and the pathogen infection 1d has symptoms, so that the method is a simple, convenient and efficient inoculation identification method. The method is carried out under a controllable environment condition, so that the continuity, stability and uniformity of pathogen inoculation and infection conditions are ensured, errors caused by the environment condition are reduced, and the method is higher in accuracy compared with the traditional method; the inoculation identification result is stable, and the repeatability is good; the method is simple and convenient, has short period and high inoculation efficiency, can repeatedly identify a single plant, and is suitable for large-scale inoculation identification of materials.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.