1. A method for identifying the resistance of buckwheat to damping-off is characterized by comprising the following steps:

selecting stem base of buckwheat plant growing for 12-15 days and inoculating rhizoctonia rot pathogenic bacteria; covering the inoculated part with wet absorbent cotton after inoculation, and wrapping and fixing the absorbent cotton with a sealing film; and observing and recording the lesion infection condition of the stem base of the plant after inoculating and culturing for 15-20 days.

2. The method of claim 1, wherein the buckwheat plant is selected to grow vigorously, and the stem base is punctured with a sterilized needle, so that the punctured buckwheat plant can still stand in the soil and normally survive.

3. The method according to claim 2, wherein the stem base is the first stem node after the buckwheat is unearthed.

4. The method of claim 3, wherein the pathogenic bacteria of rhizoctonia solani used for inoculation is hypha of pathogenic bacteria of rhizoctonia solani after being activated and cultured in PDA culture medium at 25 ℃ in the dark at constant temperature.

5. The method of claim 4, wherein pathogenic hyphae are removed 48 hours after inoculation.

6. The method of claim 5, wherein the seed culture is: culturing the buckwheat plant inoculated with the pathogenic bacteria at the temperature of 25-30 ℃ and the relative humidity of 70-90%.

7. The method according to claim 6, further comprising grading the disease according to the lesion infection, calculating the disease index of buckwheat according to the grading, and evaluating the resistance according to the disease index.

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

(1) 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 2.01-3.50 mm;

and 7, stage: the diameter of the lesion is 3.51-5.00 mm;

and 9, stage: the diameter of the lesion is more than or equal to 5.01 mm;

(2) calculating the disease index according to the disease grade:

(3) resistance ratings were assigned according to disease index:

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

resisting: 25< disease index less than or equal to 45;

sensing: 45< disease index less than or equal to 65;

high feeling: disease index > 65.

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.

Background

The buckwheat Rhizoctonia solani is a fungal disease caused by Rhizoctonia solani (Rhizoctonia solani). The incidence of the plants can reach 30 to 45 percent. In the early disease stage of the plant, the stem base part of the plant has irregular dark brown disease spots, the disease spots at the stem base part are spread along the stem of the buckwheat along with the development of the disease, the disease spots are obviously sunken, the stem base part is damaged by the disease in the seedling period and is easy to rot and break, the plant is lodging, serious economic loss is caused to the production of the buckwheat, and the sustainable development of the buckwheat industry is influenced. At present, the buckwheat rhizoctonia solani becomes one of the main diseases which seriously affect the production of buckwheat. Therefore, scientific and effective prevention and control of buckwheat damping-off has become a key to safe production and sustained and stable development of buckwheat.

The cultivation and utilization of disease-resistant varieties are the most economic, effective and safe prevention and control measures for the rhizoctonia solani of the buckwheat. At present, no identification method for the resistance of the buckwheat rhizoctonia rot and a disease-resistant buckwheat germplasm resource exist in China, and the breeding of disease-resistant varieties of the buckwheat rhizoctonia rot is severely restricted. The identification method for the resistance of the buckwheat rhizoctonia rot is simple, accurate, efficient and stable to operate, and can provide an important technical support for screening of disease-resistant buckwheat germplasm resources and research on disease-resistant breeding in future, so that an important research foundation is laid for effective prevention and control of the buckwheat rhizoctonia rot and research on disease-resistant breeding.

Disclosure of Invention

Different microorganisms have different infection capacities on different buckwheat varieties, and in order to quickly judge the resistance of microorganisms carried by unknown buckwheat varieties, a pathogen 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 buckwheat plant is infected by rhizoctonia solani in a different mode from other pathogenic bacteria infecting buckwheat stems, and the pathogenic bacteria cause harm to the plant mainly by infecting the base part (close to the first stem node of soil) of the buckwheat stem, so that the base part of the plant is infected and attacked, and the base part of the plant is rotten or the plant is lodging in severe cases.

The invention aims to provide a method for identifying the resistance of buckwheat rhizoctonia rot, which comprises the following steps: selecting stem base of buckwheat plant growing for 12-15 days and inoculating rhizoctonia rot pathogenic bacteria; covering the inoculated part with wet absorbent cotton after inoculation, and wrapping and fixing the absorbent cotton with a sealing film; and observing and recording the lesion infection condition of the stem base of the plant after inoculating and culturing for 15-20 days.

The stem base part of the buckwheat which grows to 12-15 days is suitable for inoculation and later symptom observation. The sealing film is used for wrapping and fixing the absorbent cotton and the hyphae, so that the spore germination of pathogenic bacteria is facilitated, and the disease attack of the base of the stem is accelerated.

In the identification method provided by the invention, the sterilization needle is used for stabbing the base part of the buckwheat stem, and the stabbed buckwheat plant can still stand in the soil to normally survive; the stem base part is a first stem node after the buckwheat emerges, and the first stem node is selected to be beneficial to infecting plants from the acupuncture wound by pathogenic bacteria spores, so that the buckwheat plants are rapidly attacked.

In the identification method provided by the invention, the rhizoctonia solani pathogenic bacteria used for inoculation are hyphae of the rhizoctonia solani pathogenic bacteria after being activated and cultured in a PDA culture medium at a constant temperature of 25 ℃ in the dark.

In the identification method provided by the invention, pathogenic bacteria hyphae are removed 48 hours after inoculation.

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 25-30 ℃ and the relative humidity of 70-90%.

Specifically, the identification method for the resistance to the rhizoctonia rot of buckwheat comprises the following steps:

(1) cleaning the surfaces of stem bases of healthy buckwheat plants growing for 12-15 days with sterile water, slightly stabbing a first stem node of the buckwheat after the buckwheat is unearthed with a sterilized needle head, inoculating hyphae of damping-off pathogenic bacteria to a needle point, covering the hyphae with sterile wet absorbent cotton, and wrapping and fixing the absorbent cotton and the hyphae with a sealing film;

(2) culturing the inoculated plant at the temperature of 25-30 ℃ and the relative humidity of 70-90%, and observing and recording the lesion expansion condition of the stem base of the plant after the culture is finished;

(3) observing and recording the size of the lesion spots after inoculating and culturing for 15-20 days, grading the size of the lesion spots, calculating the disease index of the stem base according to the grade of the lesion spots, and evaluating the resistance.

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

In the identification method provided by the present invention, the resistance evaluation comprises:

(1) 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 2.01-3.50 mm;

and 7, stage: the diameter of the lesion is 3.51-5.00 mm;

and 9, stage: the diameter of the lesion is more than or equal to 5.01 mm;

(2) calculating the disease index according to the disease grade:

(3) resistance ratings were assigned according to disease index:

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

resisting: the disease index is more than 25 and less than or equal to 45;

sensing: the disease index is more than 45 and less than or equal to 65;

high feeling: disease index > 65.

In the identification method provided by the invention, the buckwheat plant growing robustly is selected, the stem base is punctured by using the sterilizing needle, and the punctured buckwheat plant can still stand in the soil and normally survive.

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) the buckwheat plant growing for 12-15 days is selected for pathogen inoculation and identification, and the most accurate resistance result of the buckwheat plant is obtained.

(2) After the inoculation culture is carried out for 15-20 days, the disease spot infection condition of the stem base of the plant is observed and recorded, and the most accurate resistance evaluation method is obtained.

(3) The method of the invention can directly inoculate the hypha of the pathogenic bacteria to the acupuncture part of the stem base part, and has the advantages of simple operation, fast disease onset, low cost and high efficiency.

(4) According to the growth characteristics of the rhizoctonia rot pathogenic bacteria on the stem base parts of different resistant buckwheat varieties, 9 different levels of 9 levels including relative level numerical values of 0, 1, 3, 5, 7 and 9 are adopted to grade the lesion diameter of the inoculated stem part, 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 stems of different levels, the data is subjected to significance analysis by SPSS19.0 statistical software, and division of disease resistance and infection resistance disease indexes is determined according to 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

In this example, highly resistant buckwheat materials, i.e., milo No. 35, milo No. 27, medium-resistant buckwheat materials, i.e., milo No. 22, medium-sensitive buckwheat materials, i.e., YZ39, and highly sensitive buckwheat materials, i.e., YZ62, were used 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 bacterial strain and the source of the damping-off pathogenic bacteria are as follows: a highly pathogenic strain LK-43 of damping-off pathogenic bacteria is provided by the buckwheat disease subject of the institute of biotechnology and germplasm resources of agricultural academy of sciences in Yunnan province.

The specific steps of resistance identification are as follows:

(1) transplanting the pathogenic bacteria of the buckwheat rhizoctonia rot to a PDA culture medium flat plate, carrying out dark activation culture at a constant temperature of 25 ℃ for 7 days, and taking a small amount of pathogenic bacteria hyphae of the rhizoctonia rot by using an inoculating needle for later use.

(2) Selecting healthy plants with relatively consistent buckwheat plants after the buckwheat plants grow for about 12-15 days, and cleaning stem base parts with sterile water.

(3) Pricking stem base (3 cm higher than ground) with sterilized needle, picking up mycelium with sterilized inoculating needle, inoculating at the pricking point of the stem base, covering the mycelium with sterile wet absorbent cotton, wrapping and fixing absorbent cotton and mycelium with sealing film, and inoculating for 48 hr, and removing mycelium.

(4) And culturing the inoculated plant in a greenhouse with the temperature of 25-30 ℃ and the relative humidity of 70-90%, and observing and recording the lesion infection condition of the stem base of the plant after culturing for 15-20 days.

(5) Grading according to the extended diameter of the disease spots, calculating the disease index of the stem base according to the grade of the disease, and evaluating the resistance (see table 1), wherein the grading standard and the grading standard of the resistance of the buckwheat damping-off 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 spot is 2.01-3.50 mm;

and 7, stage: the diameter of the lesion is 3.51-5.00 mm;

and 9, stage: the diameter of the lesion is more than or equal to 5.01 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): the disease index is more than 25 and less than or equal to 45;

(iii) feeling (MS): the disease index is more than 45 and less than or equal to 65;

high feeling (HS): disease index > 65.

TABLE 1 identification results of resistance to damping-off pathogenic bacteria artificial inoculation of test buckwheat materials

Symptoms and signs	Name of Material	Lesion spot diameter (mm)	Index of disease	Type of resistance
					With disease spots	Mi Zong No. 35	1.07	20.25	HR
With disease spots	Miku No. 27	1.85	36.40	MR
					With disease spots	YZ39	3.36	54.81	MS
With disease spots	YZ62	5.62	69.13	HS

*: 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, damping-off resistance assays were performed on YZ39, YZ5, YZ62, Mizuo No. 35, Mikukou No. 27, Yuqiao No. 3 and Mikui No. 42 by the same resistance assay method as in example 1.

The method for identifying resistance provided in this example comprises the following steps:

after 7 healthy plants of different buckwheat materials were numbered with letters by one experimenter, only the numbered plants were handed to another experimenter, and the experiment was performed according to the procedure of the method for identifying resistance 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 blight was significantly different, and the resistance results of 7 different buckwheat materials, YZ39, YZ5, YZ62, mizuo No. 35, mikuqiao No. 27, yunyao No. 3, and mihei No. 42, were consistent with the results actually expressed in the field (see table 2).

The resistance identification verification test of this example again confirms that the resistance identification result agrees with the known practice. Therefore, the method provided by the invention can be used for rapidly identifying the resistance of the rhizoctonia solani.

TABLE 2 identification results of resistance to rhizoctonia rot pathogenic bacteria of different buckwheat materials by artificial inoculation

*: 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. 35, medium-resistance buckwheat material No. 27, medium-resistance buckwheat material YZ39 and high-resistance buckwheat material YZ62 are respectively used as test materials in the field. The rice palm No. 35, the rice bitter No. 27, the YZ39 and the YZ62 in different growth periods are selected to be inoculated with the rhizoctonia solani pathogenic bacteria, and the disease spot condition is observed after the inoculation culture is carried out for 18 days, and the result is shown in table 3.

Experiments show that buckwheat plants growing for 5 days and 8 days are not suitable for inoculation because the internodes at the stem base part are short and small, are close to soil and are not easy to operate. The length of the internodes of the stem base part of the buckwheat plant with the growth period of 12 days and 15 days is moderate, and pathogenic bacteria are infected quickly, the disease is fast, and the disease spots are obvious. The lignification degree of the stem base tissue of the buckwheat plant with the growth period of 20 days or 25 days is high, the hardness is high, the acupuncture is difficult, the pathogenic bacteria infection is slow, and the disease spot is slow to expand. Therefore, the stem base of the buckwheat plant with the growth period of 12 days and 15 days is inoculated, not only can good inoculation symptoms be obtained, but also a more accurate resistance level can be obtained.

TABLE 3 diameter of lesion 18 days after inoculation of Rhizoctonia solani pathogens on test materials at different growth stages

*: lesion diameter is the average of 5 replicates.

Comparative example 2 different observation periods

High-resistance buckwheat material No. 35, medium-resistance buckwheat material No. 27, medium-resistance buckwheat material YZ39 and high-resistance buckwheat material YZ62 are respectively used as test materials in the field. After the test material grows to 14 days, the stem base of the plant is inoculated with the buckwheat rhizoctonia solani pathogenic bacteria, and the inoculation symptoms are observed when the plant is inoculated and cultured for 2 days, 5 days, 10 days, 15 days, 18 days, 20 days, 25 days and 30 days respectively.

Table 4 results show that the four test materials showed no symptoms at the inoculation site after 2 days of inoculation culture. After 5 days of inoculation culture, no symptoms appear in the high-resistance material Mizuo No. 35 and the medium-resistance material Mikui No. 27, and the inoculation points of the medium-sensitivity material YZ39 and the high-sensitivity material YZ62 show chlorosis but no scab. The investigation after 10 days of inoculation revealed that the four test material inoculated spots had spots, but the spots were tiny. Investigation on 15 days, 18 days and 20 days of inoculation shows that the scab of the four test materials is gradually enlarged, and the scab difference among different materials is obvious. After 25 days and 30 days of inoculation, the scab diameter of the high-sensitivity material YZ62 and the scab diameter of the medium-sensitivity material YZ39 are only slightly increased, and the scab diameters of the high-resistance material Mizuo No. 35 and the medium-resistance material Mikui No. 27 are basically unchanged. Therefore, the experiment shows that the optimal observation time of the material after being inoculated with pathogenic bacteria is 15 days, 18 days and 20 days of inoculation culture, and the lesion of the test material is basically stable and has no obvious change during the observation period, so that the resistance level of the test material can be effectively distinguished.

TABLE 4 lesion diameters at different observation times after inoculation of the test materials with pathogenic bacteria of damping-off

*: 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.