CN107090487B - Method for identifying resistance of buckwheat black spot - Google Patents
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Abstract
The invention relates to a method for identifying resistance to black spot disease of buckwheat, which comprises the following steps: 1) after the surfaces of healthy buckwheat leaves are treated by sterile water, pricking the front surfaces of the leaves by using a needle, and inoculating a spore suspension of pathogenic bacteria of black spot to the pricking points to obtain inoculated leaves; 2) culturing the inoculated leaves under a constant temperature condition, and measuring the size of the lesion spots after the culture is finished; 3) grading the size of the lesion, calculating the disease index of the leaves according to the grade of the lesion, and evaluating the resistance. The method provided by the invention is simple to operate, short in period, accurate and reliable in evaluation result, and suitable for large-scale popularization and application.
Description
Technical Field
The invention relates to the field of crop pest resistance identification, in particular to a resistance identification method for black spot disease of buckwheat.
Background
The black spot disease of buckwheat is an important fungal disease caused by Alternaria alternata (Alternaria alternata), is widely distributed in buckwheat planting areas in China, and has a disease incidence rate of 65-80% when the disease is serious. The disease mainly infects buckwheat leaves, causes withered and yellow leaves, premature senility and slow plant growth, causes serious reduction of yield and quality, and causes serious economic loss for buckwheat production. The occurrence of black spot of buckwheat is one of the main factors which seriously affect the high yield and high quality of buckwheat in China at present. Therefore, scientific and effective prevention and control of the black spot of the buckwheat becomes the key for quality improvement, efficiency improvement and continuous and stable development of buckwheat production at the present stage.
The disease-resistant variety is the most economic and effective important measure for preventing and treating the black spot of the buckwheat, and the screening of the disease-resistant buckwheat germplasm resource is the basis for breeding the disease-resistant buckwheat variety. At present, no method for identifying the resistance of the black spot disease of the buckwheat and disease-resistant buckwheat germplasm resources exist in China, and the breeding of disease-resistant varieties of the black spot disease of the buckwheat is severely restricted, so that the establishment of the method for identifying the resistance of the black spot disease of the buckwheat is urgently researched. The accurate and stable resistance identification method for the black spot disease of the buckwheat can provide an important technical guarantee for screening of disease-resistant buckwheat germplasm resources and research of disease-resistant breeding in the future, and lays an important research foundation for effectively preventing and controlling the black spot disease of the buckwheat.
Disclosure of Invention
The invention aims to provide a method for identifying resistance to black spot disease of buckwheat, which is characterized by comprising the following steps:
1) after the surfaces of healthy buckwheat leaves are treated by sterile water, pricking the front surfaces of the leaves by using a needle, and inoculating a spore suspension of pathogenic bacteria of black spot to the pricking points to obtain inoculated leaves;
2) culturing the inoculated leaves under a constant temperature condition, and measuring the size of the lesion spots after the culture is finished;
3) and (4) carrying out resistance identification according to the size of the lesion.
Preferably, the buckwheat leaves are leaves of buckwheat plants in the 4-5 leaf stage. The growth period of the buckwheat is short (about 90 days), the size of the buckwheat leaves is proper in the 4-5 leaf period, and the late-stage observation of the inoculation disease symptoms is more suitable.
Preferably, the needle-pricked part needs to avoid the veins of the leaves. The disease mainly has symptoms on leaves, and disease spots are mainly on mesophyll areas, so that veins are avoided during inoculation, and the disease is more consistent with the symptom characteristics of the disease.
Preferably, the specific operation of pricking the leaves is to prick the mesophyll portion of the front face of the leaves with a needle. During the process of acupuncture, only the puncture wound is noticed and no puncture is made.
Preferably, the concentration of the spore suspension of the pathogenic bacterium of black spot is
1×106CFU/ml。
Preferably, the spore suspension is inoculated in an amount of 5. mu.L. The concentration of the bacterial liquid enables the quantity of the germ spores to reach a certain degree, and the spore germination and the infection of the host are facilitated during inoculation; such an amount of inoculation may be sufficient to cover the needle-prick site.
Preferably, the inoculated leaves are cultured in an illumination incubator, the culture temperature is 25 +/-1 ℃, the illumination intensity is 8000-10000 lux, illumination and darkness are alternated once every 12 hours, and the size of the scab of the inoculated leaves is measured after 4 days. The conditions are closer to the natural growth conditions of plants, and the natural environment conditions can be simulated to the maximum extent.
Preferably, the step 3) is specifically:
A. dividing the disease grade according to the size of the disease spots
Level 0: no disease spots and no disease;
level 1: the diameter of the lesion is 0.01-0.50 mm;
and 3, level: the diameter of the lesion is 0.51-2.50 mm;
and 5, stage: the diameter of the lesion is 2.51-4.50 mm;
and 7, stage: the diameter of the lesion is 4.51-6.50 mm;
and 9, stage: the diameter of the lesion is more than or equal to 6.51 mm;
B. and substituting the disease grade into a disease index calculation formula to obtain a disease index:
wherein, the "representative value of each stage" in the formula is the disease level of the leaf, such as 0, 1, 3, 5, 7, 9, and the highest disease level is 9.
C. Identifying the resistance of the buckwheat according to the resistance grade divided by the disease index;
the dividing standard of the resistance grade is as follows:
high Resistance (HR): the disease index is less than or equal to 35;
the Medium Resistance (MR) is 35< the disease index is less than or equal to 45;
(iii) feeling (MS): disease index is less than or equal to 55 when the disease index is more than 45;
high feeling (HS): disease index > 55.
The method of the present invention preferably comprises the following steps:
1) washing healthy leaves of buckwheat plants in the 4-5 leaf stage with sterile water, drying the surface water, stabbing the front surfaces of the leaves with a needle, and then adding 5uL of water with the concentration of 1 × 106Inoculating the CFU/ml pathogen spore suspension to the acupuncture point to obtain an inoculated leaf;
2) culturing the inoculated leaves in an illumination incubator at the culture temperature of 25 +/-1 ℃ and the illumination intensity of 8000-10000 lux, alternately illuminating and darkness every 12h, and measuring the size of lesion spots of the inoculated leaves after 4 d;
3) a, classifying the disease grade according to the size of the disease spots
Level 0: no disease spots and no disease;
level 1: the diameter of the lesion is 0.01-0.50 mm;
and 3, level: the diameter of the lesion is 0.51-2.50 mm;
and 5, stage: the diameter of the lesion is 2.51-4.50 mm;
and 7, stage: the diameter of the lesion is 4.51-6.50 mm;
and 9, stage: the diameter of the lesion is more than or equal to 6.51 mm;
B. and substituting the disease grade into a disease index calculation formula to obtain a disease index:
C. identifying the resistance of the buckwheat according to the resistance grade divided by the disease index;
the dividing standard of the resistance grade is as follows:
high Resistance (HR): the disease index is less than or equal to 35;
the Medium Resistance (MR) is 35< the disease index is less than or equal to 45;
(iii) feeling (MS): disease index is less than or equal to 55 when the disease index is more than 45;
high feeling (HS): disease index > 55.
The method has the following beneficial effects:
1) the method of the invention can directly inoculate the spore suspension liquid on the surface of the punctured leaf, and has the advantages of simple operation, quick disease onset, low cost and short detection period.
2) The evaluation method of the invention applies 9 grades of 6 different levels with relative grade values of 0, 1, 3, 5, 7 and 9 to grade the disease spots of inoculated leaves, calculates the disease index and judges the disease resistance according to the severity. Disease grading is to sort under an Excel system according to the size data of the detected lesion, a part severity index of a sample corresponding to no symptom is set as 0, the continuous reading numerical value of the rest data from low to high is divided into 5 sections, and each section respectively represents one disease severity grade, namely 9 grades of 0, 1, 3, 5, 7 and 9 and 6 different levels are graded. Calculating disease indexes according to different grades of leaves, performing significance analysis on the data by adopting JMPVER.5.0.1(SAS Institute Inc 2000) statistical software, and determining division of disease resistance and infection disease indexes according to significance. The evaluation method determined by the method can accurately evaluate the resistance of the buckwheat.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
This example relates to a specific evaluation example of resistance to black spot disease of buckwheat:
the test uses a variety Mihei No. 3, a medium-resistant variety Mihei No. 2, a medium-sensitive variety YNK7 and a high-sensitive variety YNK43 which respectively show high resistance in the field as test materials.
The buckwheat seeds are provided by germplasm resource preservation center of agricultural science institute of Yunnan province.
The pathogenic bacterial strain and source of the black spot disease are as follows: the highly pathogenic bacterial strain HN-3 of the black spot pathogen was isolated from a sample of Yunnan anning by the buckwheat pathology research laboratory in this laboratory.
The method comprises the following specific steps:
(1) transplanting the strain of the pathogenic bacteria of black spot of buckwheat to PDA culture medium, activating and culturing for 7 days, scraping the culture, and preparing with sterile water to obtain a culture with a concentration of 1 × 106CFU/mL spore suspension, ready for use.
(2) In the 4-5 leaf period of the buckwheat plant, taking fresh and healthy buckwheat leaves to be identified, cleaning the buckwheat leaves with sterile water, placing the buckwheat leaves on sterilized absorbent paper in a stainless steel disc with the specification of 16cm multiplied by 40cm (the surface of the stainless steel disc is sterilized, and two layers of sterilized filter paper are laid on the bottom of the box), and airing the buckwheat leaves.
(3) After the front surface of the leaves was lightly pricked with a sterilized pin (to avoid the veins), 5. mu.L of spore suspension was applied to the pricked spots of the buckwheat leaves by a pipette for inoculation.
(4) After the inoculation of the leaves is finished, the inoculated leaves are placed in an illumination incubator for culture, the illumination intensity is 8000-10000 lux, illumination and darkness are alternated once every 12h, the morbidity of the inoculated leaves is observed and recorded every day, and the size of disease spots of the inoculated leaves is measured after 4 d.
(5) Grading according to the disease grading standard, calculating the disease index, and evaluating the resistance level of the black spot of the buckwheat according to the disease index, wherein the disease grading standard and the resistance grading standard of the black spot of the buckwheat are as follows:
disease grade classification standard:
level 0: no disease spots and no disease;
level 1: the diameter of the lesion is 0.01-0.50 mm;
and 3, level: the diameter of the lesion is 0.51-2.50 mm;
and 5, stage: the diameter of the lesion is 2.51-4.50 mm;
and 7, stage: the diameter of the lesion is 4.51-6.50 mm;
and 9, stage: the diameter of the lesion is more than or equal to 6.51 mm.
The disease index calculation formula is as follows:
resistance rating criteria:
high Resistance (HR): the disease index is less than or equal to 35;
the Medium Resistance (MR) is 35< the disease index is less than or equal to 45;
(iii) feeling (MS): disease index is less than or equal to 55 when the disease index is more than 45;
high feeling (HS): disease index > 55.
TABLE 1 identification result of resistance to artificial inoculation of black spot spore suspension of test variety
| Symptoms and signs | Name of breed | Lesion spot diameter (mm) | Index of disease | Type of resistance |
| With disease spots | No. 3 rice black | 1.22 | 30.37 | HR |
| With disease spots | Brown and bitter taste No. 2 | 2.00 | 42.71 | MR |
| With disease spots | YNK7 | 2.56 | 51.65 | MS |
| With disease spots | YNK34 | 3.48 | 78.56 | HS |
Lesion diameter and disease index are mean values of 3 replicates.
From the above results, it can be seen that the resistance identification results of buckwheat varieties exhibiting resistance in the field match known practical conditions through resistance identification tests.
Example 2
The black spot resistance identification is carried out on the rice black No. 3, the Ningqiao No. 1, the brown bitter No. 2, the Mongolian 0353 and YNK43, the cloud buckwheat No. 1 and the cloud buckwheat No. YNK7 by the method 1. Healthy leaves of the 7 different buckwheat varieties are collected by one experimenter, the 7 different varieties are numbered by Arabic numerals, and the leaves only with the numbers are handed to another experimenter to carry out experiments according to the steps of the method 1. After the experiment is finished, looking up the name of the variety by contrasting the serial numbers. The results show that the resistance of 7 different buckwheat varieties to black spot is obviously different, wherein the resistance results of the rice black No. 3, the brown bitter No. 2, YNK7 and YNK43 are consistent with the results actually expressed in the field. The resistance identification verification test proves that the resistance identification result is matched with the known actual situation again. Therefore, the method can be used for rapidly identifying the resistance of the black spot disease of the buckwheat.
TABLE 2 identification results of resistance to artificial inoculation of black spot spore suspension of different buckwheat varieties
Lesion diameter and disease index are mean values of 3 replicates.
Therefore, the resistance identification method has the advantages of easiness in operation, high efficiency, stability, short detection period and the like, is suitable for quickly identifying the disease resistance of large-batch materials, and has wide application prospect.
Claims (1)
1. The method for identifying the resistance of the black spot disease of the buckwheat is characterized by comprising the following steps:
1) after the surfaces of the leaves in the 4-5 leaf stage of the healthy buckwheat plants are treated by sterile water, stabbing the front mesophyll parts of the leaves by using a needle head, and setting the concentration of 5 mu L to be 1 × 106Inoculating a spore suspension of CFU/ml black spot pathogenic bacteria to a needle point to obtain an inoculated leaf;
2) placing the inoculated leaves in an illumination incubator, wherein the incubation temperature is 25 +/-1 ℃, the illumination intensity is 8000-10000 lux, illumination and darkness are alternated once every 12h, and the size of the lesion is measured after 4 d;
3) carrying out resistance identification according to the size of the lesion;
wherein, the step 3) is specifically as follows:
A. dividing the disease grade according to the size of the disease spots
Level 0: no disease spots and no disease;
level 1: the diameter of the lesion is 0.01-0.50 mm;
and 3, level: the diameter of the lesion is 0.51-2.50 mm;
and 5, stage: the diameter of the lesion is 2.51-4.50 mm;
and 7, stage: the diameter of the lesion is 4.51-6.50 mm;
and 9, stage: the diameter of the lesion is more than or equal to 6.51 mm;
B. and substituting the disease grade into a disease index calculation formula to obtain a disease index:
C. identifying the resistance of the buckwheat according to the resistance grade divided by the disease index;
the dividing standard of the resistance grade is as follows:
high Resistance (HR): the disease index is less than or equal to 35;
the Medium Resistance (MR) is 35< the disease index is less than or equal to 45;
(iii) feeling (MS): disease index is less than or equal to 55 when the disease index is more than 45;
high feeling (HS): disease index > 55.
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| CN108486033A (en) * | 2018-02-23 | 2018-09-04 | 云南省德宏热带农业科学研究所 | A kind of in-vitro verification method of coffee germplasm rust resistance |
| CN108486214B (en) * | 2018-03-07 | 2022-03-11 | 云南省农业科学院生物技术与种质资源研究所 | Buckwheat stem blight resistance identification method |
| CN108893520B (en) * | 2018-05-23 | 2022-02-01 | 云南省农业科学院花卉研究所 | Method for identifying resistance of Chinese rose downy mildew |
| CN111593088A (en) * | 2020-06-12 | 2020-08-28 | 广东省农业科学院植物保护研究所 | Identification method for resistance of soft rot of phalaenopsis |
| CN113403365A (en) * | 2021-07-21 | 2021-09-17 | 云南省农业科学院生物技术与种质资源研究所 | Identification method for resistance to damping-off of buckwheat |
| CN113462740B (en) * | 2021-07-21 | 2023-04-14 | 云南省农业科学院生物技术与种质资源研究所 | Resistance identification method for gray leaf spot of buckwheat |
| CN115044643A (en) * | 2022-07-12 | 2022-09-13 | 中国林业科学研究院亚热带林业研究所 | Evaluation method for camellia disease resistance |
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| CA2903704A1 (en) * | 2013-03-14 | 2014-10-02 | Georgia State University Research Foundation, Inc. | Inhibiting or reducing fungal growth |
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| CN102640671A (en) * | 2012-05-02 | 2012-08-22 | 湖北省农业科学院果树茶叶研究所 | Method for identifying Alternaria altemata (Fr.) Keissler resistance of pear germplasm |
| CA2903704A1 (en) * | 2013-03-14 | 2014-10-02 | Georgia State University Research Foundation, Inc. | Inhibiting or reducing fungal growth |
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| 新疆枣果黑斑病病原菌鉴定及生物学特性;刘晓琳等;《西北林学院学报》;20150323;132-138 * |
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