阅读说明：本技术 一种蝴蝶兰软腐病抗性鉴定方法 (Identification method for resistance of soft rot of phalaenopsis ) 是由 杨祁云 林壁润 沈会芳 张景欣 孙大元 蒲小明 于 2020-06-12 设计创作，主要内容包括：本发明公开了提供一种蝴蝶兰软腐病抗性鉴定方法,剪取蝴蝶兰品种中下部叶片,用酒精将叶片及叶柄伤口擦拭一遍,自然晾干。将病原菌株接种于NA培养液,用无菌水配制成细菌悬浮液。用无菌大头针在叶片中央密集轻刺,在针刺处滴细菌悬浮液,放入磁盘中保湿密封,黑暗培养。当叶片出现水渍状病斑时,观察并记录叶片发病情况。以平均防效进行分级,评价品种的抗感特性。(The invention discloses a method for identifying the soft rot resistance of phalaenopsis, which comprises the steps of shearing the middle and lower leaves of phalaenopsis, wiping the leaves and the petiole wounds with alcohol once, and naturally drying. The pathogenic strains are inoculated in NA culture solution, and bacteria suspension is prepared by using sterile water. Puncturing the leaf with sterile pin, dripping bacterial suspension, placing in magnetic disk, and culturing in dark. When water stain-like spots appear on the leaves, the disease condition of the leaves is observed and recorded. The average control effect is graded to evaluate the anti-influenza characteristics of the variety.)

1. The identification method for the soft rot resistance of phalaenopsis comprises the following steps:

s1 pathogenic bacteria separation and preparation, obtaining a butterfly orchid soft rot sample, sterilizing according to the conventional method, cutting tissues at the joint of disease and health into thin slices by using a sterile knife, transferring the thin slices onto an NA culture medium for culture, separating and purifying, and verifying and determining by the Koehz' S rule; culturing on NA plate at 32 deg.C to obtain pure culture of pathogenic bacteria; inoculating pathogenic strain to NA culture solution, culturing at 32 deg.C and 150r/min for 36h, and preparing into 10 with sterile water⁶-10⁷CFU/mL of bacterial suspension;

s2, placing the sterilized filter paper in a culture dish, and spraying sterile water until the filter paper is wetted for later use;

s3 variety preparation, namely selecting 28 butterfly orchid varieties, preparing 5 leaves of each variety, wherein the diameter of each variety is 50-70 mm, cleaning and airing the leaves, disinfecting the leaves with 75% alcohol by mass concentration, and contrasting with high-susceptibility soft rot GD 1; respectively placing the sterilized butterfly orchid leaves in a sterilized flat plate with good moisture retention;

s4 inoculation identification, using sterile pin to prick 5 times densely at the center of the leaf, dripping 0.2mL of bacterial suspension obtained in the step S1 at the pricked position, repeating for 4 times and repeating for 5 leaves;

s5 investigation and evaluation, placing in a magnetic disk for moisture-proof sealing, and culturing at 30 ℃ in the dark. Observing and recording the disease condition of the leaves every 24h when the leaves have water stain-shaped disease spots;

s6, grading according to the grading standard and calculating the disease index, and evaluating the anti-influenza characteristics of the variety according to the disease index.

2. The method for identifying the soft rot resistance of phalaenopsis according to claim 1, wherein the soft rot resistance level of phalaenopsis varieties is determined according to the following criteria:

high resistance: the disease index is less than or equal to 20;

disease resistance: the disease index is more than 20 and less than or equal to 40;

resisting: the disease index is more than 40 and less than or equal to 60;

the infection: the disease index is more than 60 and less than or equal to 80;

high feeling: the disease index is less than 80.

3. The method for identifying resistance to soft rot of butterfly orchid according to claim 1, characterized in that the grading criteria are:

grade 0, no water stain-like disease spots exist at the acupuncture position; level 1, water stain-like disease spots appear at the acupuncture position, and the disease spots do not spread out of the acupuncture range by 0.2 cm;

the 2-grade scab spreads out of the 0.2cm distance outside the acupuncture range, and the area of the scab is less than or equal to 25 percent of the area of the leaf;

3, the area of 25 percent of the leaves is less than the area of the scab and less than or equal to 50 percent of the area of the leaves;

4, the area of 50 percent of the leaves is less than the area of the scab and less than or equal to 75 percent of the area of the leaves;

grade 5, 75% of the leaf area is less than the lesion area.

4. The method for identifying the resistance to soft rot of butterfly orchid according to claim 1, wherein the disease index is calculated by the formula:

Technical Field

The invention relates to a method for identifying crop pest resistance, in particular to a method for identifying butterfly orchid soft rot resistance, and belongs to the field of agricultural plant protection.

Background

The soft rot disease of phalaenopsis (phalaenopsis soft rot disease) is caused by dicke's bacterium dickeya dieffenfenbachiae, is one of the main diseases of phalaenopsis, is widely distributed and occurs in all phalaenopsis producing areas in China. After the disease occurs, the pathogenic bacteria secrete pectinase, and the pectic substance in the cell wall is dissolved, so that the tissue is soft and rotten, and spreads rapidly, and serious warp extrusion loss is caused. The breeding of disease-resistant varieties is the main means for controlling the diseases. However, at present, no method for identifying the approved resistance of the phalaenopsis does not exist at home and abroad, so that the method for accurately evaluating the resistance of the phalaenopsis appears to be particularly important.

Disclosure of Invention

In view of the defects of the prior art, the invention aims to provide a method for identifying the resistance of the soft rot of the butterfly orchid.

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

a method for identifying resistance to soft rot of phalaenopsis, which comprises the following steps:

s1 pathogenic bacteria separation and preparation, obtaining a butterfly orchid soft rot sample, sterilizing according to the conventional method, cutting tissues at the joint of disease and health into thin slices by using a sterile knife, transferring the thin slices onto an NA culture medium for culture, separating and purifying, and verifying and determining by the Koehz' S rule; culturing on NA plate at 32 deg.C to obtain pure culture of pathogenic bacteria; inoculating pathogenic strain to NA culture solution, culturing at 32 deg.C and 150r/min for 36h, and preparing into 10 with sterile water⁶-10⁷CFU/mL of bacterial suspension;

s2, placing the sterilized filter paper in a culture dish, and spraying sterile water until the filter paper is wetted for later use;

s3 variety preparation, namely selecting 28 butterfly orchid varieties, preparing 5 leaves of each variety, wherein the diameter of each variety is 50-70 mm, cleaning and airing the leaves, disinfecting the leaves with 75% alcohol by mass concentration, and contrasting with high-susceptibility soft rot GD 1; respectively placing the sterilized butterfly orchid leaves in a sterilized flat plate with good moisture retention;

s4 inoculation identification, using sterile pin to prick 5 times densely at the center of the leaf, dripping 0.2mL of bacterial suspension obtained in the step S1 at the pricked position, repeating for 4 times and repeating for 5 leaves;

s5 investigation and evaluation, placing in a magnetic disk for moisture-proof sealing, and culturing at 30 ℃ in the dark. Observing and recording the disease condition of the leaves every 24h when the leaves have water stain-shaped disease spots;

s6, grading according to the grading standard and calculating the disease index, and evaluating the anti-influenza characteristics of the variety according to the disease index.

Preferably, the level of soft rot resistance of a butterfly orchid variety is determined according to the following criteria:

high resistance: the disease index is less than or equal to 20;

disease resistance: the disease index is more than 20 and less than or equal to 40;

resisting: the disease index is more than 40 and less than or equal to 60;

the infection: the disease index is more than 60 and less than or equal to 80;

high feeling: disease index is more than 80;

preferably, the ranking criteria are:

grade 0, no water stain-like disease spots exist at the acupuncture position; level 1, water stain-like disease spots appear at the acupuncture position, and the disease spots do not spread out of the acupuncture range by 0.2 cm;

stage 2, spreading the scab to a distance of 0.2cm outside the acupuncture range, and simultaneously, the area of the scab is less than or equal to 25 percent of the area of the leaf;

3, the area of 25 percent of the leaves is less than the area of the scab and less than or equal to 50 percent of the area of the leaves;

4, the area of 50 percent of the leaves is less than the area of the scab and less than or equal to 75 percent of the area of the leaves;

grade 5, 75% of the leaf area is less than the lesion area.

Preferably, the disease index is calculated by the formula:

it should be noted that the formulation of the NA medium used in the present invention is: beef extract 0.3%, yeast extract 0.1%, peptone 0.5%, glucose 1.0%, agar 1.8%, water 1000ml, pH6.8-7.0;

the method has the advantages of scientific identification method, time and labor saving, simple process and easy operation, and is most effective for resistance evaluation according to lesion classification and disease index calculation.

Detailed Description

The present invention will be further described with reference to the following examples, which should be construed as being exemplary in nature and not limiting the scope of the present invention.

The invention relates to a method for identifying the resistance of soft rot of phalaenopsis, which comprises the following steps:

s1 pathogenic bacteria separation and preparation, obtaining a butterfly orchid soft rot sample, sterilizing according to the conventional method, cutting tissues at the joint of disease and health into thin slices by using a sterile knife, transferring the thin slices onto an NA culture medium for culture, separating and purifying, and verifying and determining by the Koehz' S rule; culturing on NA plate at 32 deg.C to obtain pure culture of pathogenic bacteria; inoculating pathogenic strain to NA culture solution, culturing at 32 deg.C and 150r/min for 36h, and preparing into 10 with sterile water⁶-10⁷CFU/mL of bacterial suspension;

s2, placing the sterilized filter paper in a culture dish, and spraying sterile water until the filter paper is wetted for later use;

s3 variety preparation, namely selecting 28 butterfly orchid varieties, preparing 5 leaves of each variety, wherein the diameter of each variety is 50-70 mm, cleaning and airing the leaves, disinfecting the leaves with 75% alcohol by mass concentration, and contrasting with high-susceptibility soft rot GD 1; respectively placing the sterilized butterfly orchid leaves in a sterilized flat plate with good moisture retention;

s4 inoculation identification, using sterile pin to prick 5 times densely at the center of the leaf, dripping 0.2mL of bacterial suspension obtained in the step S1 at the pricked position, repeating for 4 times and repeating for 5 leaves;

s5 investigation and evaluation, placing in a magnetic disk for moisture-proof sealing, and culturing at 30 ℃ in the dark. Observing and recording the disease condition of the leaves every 24h when the leaves have water stain-shaped disease spots;

s6, grading according to the grading standard and calculating the disease index, and evaluating the anti-influenza characteristics of the variety according to the disease index.

Preferably, the level of soft rot resistance of a butterfly orchid variety is determined according to the following criteria:

high resistance: the disease index is less than or equal to 20;

disease resistance: the disease index is more than 20 and less than or equal to 40;

resisting: the disease index is more than 40 and less than or equal to 60;

the infection: the disease index is more than 60 and less than or equal to 80;

high feeling: disease index is more than 80;

preferably, the ranking criteria are:

grade 0, no water stain-like disease spots exist at the acupuncture position; level 1, water stain-like disease spots appear at the acupuncture position, and the disease spots do not spread out of the acupuncture range by 0.2 cm;

stage 2, spreading the scab to a distance of 0.2cm outside the acupuncture range, and simultaneously, the area of the scab is less than or equal to 25 percent of the area of the leaf;

3, the area of 25 percent of the leaves is less than the area of the scab and less than or equal to 50 percent of the area of the leaves;

4, the area of 50 percent of the leaves is less than the area of the scab and less than or equal to 75 percent of the area of the leaves;

grade 5, 75% of the leaf area is less than the lesion area.

Preferably, the disease index is calculated by the formula: