阅读说明：本技术 一种荞麦灰斑病抗性鉴定方法 (Resistance identification method for gray leaf spot of buckwheat ) 是由 卢文洁 王莉花 王艳青 尹桂芳 孙道旺 于 2021-07-21 设计创作，主要内容包括：本发明涉及一种荞麦灰斑病抗性鉴定方法,包括以下步骤：1)选取健康的活体荞麦植株,用灭菌针头刺伤叶片表面,无菌水喷雾湿润针刺点,将荞麦灰斑病病原菌圆形菌饼接种于叶片针刺点,得接种植株；2)将接种植株在温室内进行培养,培养完成后观察记录接种植株的叶片病斑侵染情况；3)根据病斑扩展情况进行分级,根据所属的级别计算叶片的病情指数,进行抗性评价。本发明所提供的鉴定方法操作便捷、高效、准确、稳定,可应用于大批量荞麦种质资源材料的抗病性鉴定。(The invention relates to a resistance identification method for gray leaf spot of buckwheat, which comprises the following steps: 1) selecting healthy living buckwheat plants, stabbing the surfaces of leaves by using a sterilizing needle, spraying sterile water to wet the stabbed points, and inoculating the circular cakes of the pathogenic bacteria of the gray leaf spot of the buckwheat to the stabbed points of the leaves to obtain inoculated plants; 2) culturing the inoculated plant in a greenhouse, and observing and recording the infected condition of the leaf spots of the inoculated plant after the culture is finished; 3) grading according to the extension condition of the lesion spots, calculating the disease index of the leaves according to the grade of the lesion spots, and evaluating the resistance. The identification method provided by the invention is convenient, efficient, accurate and stable to operate, and can be applied to the disease resistance identification of large-batch buckwheat germplasm resource materials.)

1. The identification method for the resistance to gray leaf spot of buckwheat is characterized by comprising the following steps:

selecting healthy buckwheat plants growing for 15-18 days, stabbing the surfaces of buckwheat leaves, inoculating the pathogenic bacteria of gray leaf spot to the stabbed parts of the leaves, inoculating and culturing for 10-14 days, and observing the disease spot infection condition of the buckwheat leaves.

2. The method of claim 1, wherein the puncture comprises, avoiding veins, puncturing the surface of the leaf, without piercing the leaf; the buckwheat plants after the leaves are inoculated with pathogenic bacteria still normally survive in the soil.

3. The identification method according to claim 2, wherein the pathogen used for inoculation is a cake of a pathogen round of gray leaf spot, which is activated and cultured in PDA medium at a constant temperature of 25 ℃ in the dark;

preferably, the fungus cake is a circular fungus cake with the diameter of 5 mm.

4. The method of claim 3, wherein the pathogen cake is removed 36 hours after inoculation.

5. The method of claim 4, wherein the inoculation culture is: culturing the buckwheat plant inoculated with pathogenic bacteria at 25-28 deg.C and relative humidity of 75-90%.

6. The identification method according to any one of claims 1 to 5, further comprising grading the disease according to the lesion infection, calculating an index of disease of buckwheat according to the grading, and evaluating the resistance according to the index of disease.

7. The method according to claim 6, wherein the disease is classified according to the size of lesion:

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 0.51-2.00 mm;

and 5, stage: the diameter of the lesion is 2.01-4.00 mm;

and 7, stage: the diameter of the lesion is 4.01-6.00 mm;

and 9, stage: the diameter of the lesion is more than or equal to 6.01 mm.

8. The method of claim 7, wherein the determination of the level of resistance comprises:

(1) calculating the disease index according to the disease grade,

(2) the resistance grade is divided according to the disease index,

high resistance: the disease index is less than or equal to 25;

resisting: 25< disease index less than or equal to 40;

sensing: disease index is less than or equal to 55 after 40;

high feeling: disease index > 55.

9. Use of the method of any one of claims 1 to 8 for buckwheat germplasm resource innovation.

10. Use of the method of any one of claims 1 to 8 for increasing buckwheat production.

Technical Field

The invention relates to a method for identifying crop disease resistance, in particular to a method for identifying resistance to gray leaf spot of buckwheat.

Background

The gray leaf spot of buckwheat is a fungal disease caused by Pestalotiopsis microspora (Pestalotiopsis microspora). The gray leaf spot of buckwheat mainly infects and harms leaves, and at the early stage of disease occurrence, the scab on the leaves is round or nearly round, the center of the scab is sunken downwards, the edge of the scab is slightly raised, and the boundary of the scab and the scab has obvious yellow halo. With the progress of disease, the disease spots gradually expand to the periphery and become elliptical or oblong disease spots, and when the disease spots are serious, the disease spots are mutually connected to form irregular large disease spots, so that the leaves are dry and fall off. At present, the gray leaf spot of buckwheat is one of the main diseases which seriously affect the production of buckwheat. Therefore, effective prevention and control of the gray leaf spot of buckwheat becomes the key for the continuous and stable development of the buckwheat industry.

The disease-resistant variety is the most economic and effective prevention and control measure for the gray leaf spot of the buckwheat. At present, no identification method for the resistance of the gray spot disease of the buckwheat and a buckwheat germplasm resource with disease resistance exist, and the breeding of disease-resistant varieties of the gray spot disease of the buckwheat is severely restricted. Therefore, the establishment of a resistance identification method of the gray leaf spot of the buckwheat becomes a key for screening disease-resistant germplasm resources and breeding disease-resistant varieties. The method for identifying the resistance of the grey speck disease of the buckwheat is convenient, efficient, accurate and stable to operate, can provide an important technical support for screening disease-resistant buckwheat germplasm resources and disease-resistant breeding research, and provides an important research foundation for effectively preventing and controlling the grey speck disease of the buckwheat.

Disclosure of Invention

In order to rapidly judge the gray leaf spot resistance carried by unknown buckwheat varieties, a pathogenic bacterium resistance identification method which has strong universality and high accuracy and does not migrate along with the buckwheat varieties and a matched resistance evaluation system are urgently needed. The pseudoplectania pilosula, a pathogen of the gray leaf spot of the buckwheat, is mainly used for infecting leaves of a plant to cause diseases by infecting the leaves of the plant, the disease of the plant leaves is connected when the disease is serious, the leaves are rotten or withered and shed under the rainy or drought condition, the plant consolidation rate is influenced, and the yield loss of the buckwheat is serious.

The invention aims to provide an identification method of resistance to gray leaf spot of buckwheat, which comprises the following steps:

selecting healthy buckwheat plants growing for 15-18 days, stabbing the surfaces of buckwheat leaves, inoculating pathogenic bacteria of gray leaf spot to the stabbed parts of the leaves, inoculating and culturing for 10-14 days, and observing the disease spot infection condition of the buckwheat leaves.

In the identification method provided by the invention, the puncture comprises the steps of avoiding veins by using a needle, puncturing the surface of the leaves and not puncturing the leaves. The puncture method provided by the invention is beneficial to infecting the leaves from the punctured wound by the spores of pathogenic bacteria and accelerating the morbidity of inoculated leaves.

In the identification method provided by the invention, the pathogenic bacteria used for inoculation are gray spot pathogenic bacteria circular fungus cakes which are subjected to constant-temperature dark activation culture on a PDA culture medium at 25 ℃;

preferably, the fungus cake is a circular fungus cake with the diameter of 5 mm.

According to the identification method provided by the invention, the pathogenic bacteria cake is removed after inoculation for 36 hours.

In the identification method provided by the invention, the inoculation culture comprises the following steps: culturing the buckwheat plant inoculated with the pathogenic bacteria at the temperature of 25-28 ℃ and the relative humidity of 75-90%.

In the identification method provided by the invention, classification is carried out according to the lesion infection condition, the disease index of buckwheat is calculated according to the classification condition, and resistance evaluation is carried out according to the disease index.

The different growth stages of the buckwheat and the infection time of pathogenic bacteria lead the buckwheat to show great size difference of disease spots after being infected by the pathogenic bacteria. According to the invention, long-term experimental investigation is carried out, the infection condition of buckwheat leaves after being inoculated with pathogenic bacteria for 10-14 days in 15-18 days is determined, the whole resistance capability of buckwheat plants can be stably and accurately reflected, and the disease grade of gray leaf spot of the buckwheat leaves is formulated according to the size of stable disease spots.

In the identification method provided by the present invention, the classification of disease grade according to the size of lesion comprises:

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 0.51-2.00 mm;

and 5, stage: the diameter of the lesion is 2.01-4.00 mm;

and 7, stage: the diameter of the lesion is 4.01-6.00 mm;

and 9, stage: the diameter of the lesion is more than or equal to 6.01 mm.

In the identification method provided by the present invention, the determination of the resistance rating comprises:

(1) calculating the disease index according to the disease grade,

(2) the resistance grade is divided according to the disease index,

high resistance: the disease index is less than or equal to 25;

resisting: 25< disease index less than or equal to 40;

sensing: disease index is less than or equal to 55 after 40;

high feeling: the disease index is greater than 55.

According to the understanding of the technical personnel in the field, the invention also claims the application of the identification method in buckwheat germplasm resource innovation and buckwheat yield improvement.

The invention has the beneficial effects that:

(1) according to the method, the buckwheat plants in 15-18 days are selected for the inoculation and identification of the leaf pathogenic bacteria, so that the identification time is saved, and the most accurate resistance result of the buckwheat plants is obtained.

(2) According to the invention, after 10-14 days of inoculation, the scab infection condition of the leaves is observed and recorded, and at the moment, the obtained infection result can reflect the resistance level of the buckwheat plant.

(3) The method of the invention directly inoculates the fungus cake of the pathogenic bacteria to the needle-pricked part of the buckwheat leaves, and has the advantages of simple operation, fast disease onset, low cost and high efficiency.

(4) According to the growth characteristics of the gray leaf spot pathogenic bacteria on different buckwheat variety leaves, 9 levels of 6 different levels with relative grade values of 0, 1, 3, 5, 7 and 9 are adopted to grade the diameters of the disease spots of inoculated leaves, the disease index is calculated, and the disease resistance is judged according to the severity; the disease grading provided by the invention is to sort under an Excel system according to the diameter data of the investigated lesion spots, the part severity index of a sample corresponding to an asymptomatic condition is set as 0 grade, 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 6 different levels of 0, 1, 3, 5, 7 and 9 are graded; the disease index is calculated according to the infection degrees of the leaves at different levels, the data is subjected to significance analysis by adopting SPSS19.0 statistical software, and the division of the disease resistance and infection resistance disease index is determined according to the significance. The results confirm that the resistance of the buckwheat can be accurately evaluated by the evaluation method determined by the invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit and substance of the invention.

Unless otherwise specified, test materials, reagents, instruments and the like used in the examples of the present invention are commercially available; all technical measures in the examples of the present invention are conventional measures well known to those skilled in the art, unless otherwise specified.

Example 1

This example is a specific evaluation example of resistance to gray leaf spot of buckwheat using the resistance identification method provided by the present invention.

The test uses high-resistance buckwheat material No. 10, medium-resistance buckwheat material No. 5, medium-sense buckwheat material ZY81 and high-sense buckwheat material ZY57 as test materials in the field. The buckwheat material seeds are provided by biotechnology of agricultural science institute of Yunnan province and a germplasm resource storage center of crop germplasm resource research institute.

The pathogenic bacterial strain and source of the gray spot disease are as follows: the strongly pathogenic strain HM-12 of grey spot disease pathogenic bacteria is provided by the buckwheat disease topic group of the institute of biotechnology and germplasm resources of agricultural academy of sciences in Yunnan province.

In this example, the steps of the resistance identification method are as follows:

(1) selecting relatively consistent healthy leaves of the buckwheat plants when the buckwheat plants grow for 15-18 days, pricking the leaf surfaces with a sterilizing needle slightly to avoid the leaf veins but not pierce the leaves, and wetting the punctured parts with sterile water spray.

(2) Transplanting the pathogenic bacteria of the gray leaf spot of the buckwheat to a PDA culture medium flat plate, carrying out constant-temperature dark activation culture for 5 days, and punching holes on the edges of bacterial colonies by using a puncher with the diameter of 5mm to obtain circular bacterial cakes for later use.

(3) And (4) picking the bacterial circular cakes by using a sterilization inoculating needle to inoculate the leaf puncture points, and taking off the bacterial cakes after inoculating for 36 hours.

(4) And culturing the inoculated plant in a greenhouse with the temperature of 25-28 ℃ and the relative humidity of 75-90%, and observing and recording the disease spot infection condition of the buckwheat leaves after culturing for 10-14 days.

(5) Grading according to the disease spread diameter, calculating the disease index of the leaves according to the grades to which the disease indexes belong, and evaluating the resistance (see table 1), wherein the grading standard of the disease degree and the grading standard of the resistance of the gray leaf spot of the buckwheat are as follows:

disease grade classification standard:

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 0.51-2.00 mm;

and 5, stage: the diameter of the lesion is 2.01-4.00 mm;

and 7, stage: the diameter of the lesion is 4.01-6.00 mm;

and 9, stage: the diameter of the lesion is more than or equal to 6.01 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 25;

anti-Medium (MR): 25< disease index less than or equal to 40;

(iii) feeling (MS): disease index is less than or equal to 55 after 40;

high feeling (HS): the disease index is greater than 55.

TABLE 1 identification result of resistance to artificial inoculation of pathogenic bacteria of gray leaf spot of tested buckwheat material

*: lesion diameter and disease index are averages of 5 replicates.

From the above results, it can be seen that the resistance identification results of buckwheat materials exhibiting resistance in the field are consistent with known practical conditions through resistance identification tests.

Example 2

In this example, the same method as in example 1 was used to identify gray spot resistance in ZY57, ZY26, yunqao No. 2, ZY81, beige No. 5, mihei No. 10 and quassian No. 9.

The resistance identification procedure of this example is as follows:

after healthy plants of 7 different buckwheat materials were numbered by one experimenter using arabic numerals, the plants with only the numbers were handed over to another experimenter, and the experiment was performed in the same manner as in example 1. After the experiment is finished, the name of the material is checked by comparing the serial numbers. The results showed that the resistance of 7 different buckwheat materials to gray spot was significantly different, and the resistance results of 7 different buckwheat materials, ZY57, ZY26, yun qiao No. 2, ZY81, beige No. 5, beige No. 10, gray bitter No. 9, were consistent with the results actually expressed in the field (see table 2). 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 gray leaf spot of the buckwheat.

TABLE 2 identification results of resistance to artificial inoculation of grey speck disease pathogenic bacteria of different buckwheat materials

Symptoms and signs	Material numbering	Lesion spot diameter (mm)	Index of disease	Type of resistance	Name of breed
						With disease spots	1	6.25	64.07	HS	ZY26
With disease spots	2	2.33	32.51	MR	Mi Zong No. 5
						With disease spots	3	3.75	48.06	MS	Yunqiao No. 2
With disease spots	4	1.85	23.45	HR	Rice black 10
						With disease spots	5	4.95	50.49	MS	ZY81
With disease spots	6	3.92	29.18	MR	Ash bitter No. 9
						With disease spots	7	5.74	62.37	HS	ZY57

*: lesion diameter and disease index are averages of 5 replicates.

Therefore, the resistance identification method has the advantages of simple, accurate, efficient and stable operation, short detection period and the like, is suitable for quickly identifying the disease resistance of large-batch materials, and has wide application prospect.

Comparative example 1 buckwheat plants of different growth periods

High-resistance buckwheat material No. 10, medium-resistance buckwheat material No. 5, medium-resistance buckwheat material No. ZY81 and high-resistance buckwheat material ZY57 are respectively used as test materials in the field. And selecting Mihei No. 10, Mizuo No. 5, ZY81 and ZY57 in different growth periods to inoculate grey speck pathogenic bacteria, and observing the spread condition of the specks after inoculating and culturing for 12 days (see table 3).

Experiments show that the buckwheat plant growing for 5 days only has 2 cotyledons, does not grow true leaves, and is not suitable for inoculation. 2 true leaves of buckwheat plants growing for 10 days grow, but the leaves are small and tender, cannot bear pathogenic bacteria cakes, and the petioles are easy to break. The buckwheat plants growing for 15 days and 18 days have 2-3 true leaves, the size of the leaves is moderate, pathogenic bacteria fungus cakes can be inoculated, after inoculation, pathogenic bacteria infect quickly, the leaves are quick in disease occurrence, and disease spots are obvious. The buckwheat plant growing for 20 days has large buckwheat leaves, pathogenic bacteria infection after inoculation has slow morbidity, and the disease spot is slow to expand. The buckwheat plants growing for 25 days have wide buckwheat leaves, pathogenic bacteria infection after inoculation is slow in morbidity, and disease spots are slow in expansion. Therefore, the experimental results show that the optimum inoculation of buckwheat plant leaves growing for 15 days and 18 days can obtain more accurate resistance level on the basis of saving identification time.

TABLE 3 plaque diameter of test materials artificially inoculated with gray leaf spot pathogenic bacteria for 12 days at different growth stages

*: lesion diameter is the average of 5 replicates.

Comparative example 2 different observation periods

High-resistance buckwheat material No. 10, medium-resistance buckwheat material No. 5, medium-resistance buckwheat material No. ZY81 and high-resistance buckwheat material ZY57 are respectively used as test materials in the field. After the test material grows for 16 days, the leaves of the plant are inoculated with the buckwheat scab pathogenic bacteria, and the inoculation symptoms are observed respectively in inoculation culture for 2 days, 5 days, 10 days, 12 days, 14 days, 16 days, 20 days and 25 days.

The results in Table 4 show that the inoculum sites of the four test materials showed no symptoms after 2 days of inoculum culture. After 5 days of inoculation, the high-resistance buckwheat material rice black No. 10 and the medium-resistance buckwheat material rice brown No. 5 inoculation points show fading symptoms, and the medium-sense buckwheat material ZY81 and the high-sense buckwheat material ZY57 inoculation points only show tiny scabs. Investigation on inoculation for 10 days, 12 days and 14 days shows that the scab of the four test materials is gradually enlarged, and the scab difference among different materials is obvious. After 16 days, 20 days and 25 days of inoculation, the scab diameters of the high-resistance buckwheat material Mihei No. 10 and the medium-resistance material Mizuo No. 5 are basically unchanged, and the scab diameters of the sensed buckwheat material ZY81 and the high-sensitivity buckwheat material ZY57 are only slightly increased. Therefore, after the inoculation of pathogenic bacteria, the optimal observation time is 10 days, 12 days and 14 days of inoculation culture, during the observation period, the lesion of the test material is basically stable and has no obvious change, and not only can the effect be well distinguished from the symptoms, but also the more accurate resistance level can be obtained.

TABLE 4 lesion diameters at different observation times after test materials were artificially inoculated with gray leaf spot pathogens

*: lesion diameter is the average of 5 replicates.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.