阅读说明：本技术 一种月季灰霉病菌生理小种的鉴定方法 (Identification method of physiological races of gray mold of Chinese rose ) 是由 邱显钦 张颢 晏慧君 蹇洪英 陈敏 唐开学 王其刚 张婷 于 2020-11-28 设计创作，主要内容包括：一种月季灰霉病菌生理小种的鉴定方法,该方法包括灰霉病菌的分离纯化和扩繁,月季灰霉病菌孢子悬浮液制备,6份鉴定寄主材料,离体花瓣鉴定,以及花瓣病情等级划分。该方法仅用6份鉴定寄主材料,即可高效准确地在我国各地鉴定出月季灰霉病菌生理小种,节约成本、省工省时。(A method for identifying physiological races of gray mold of China rose comprises the steps of separation, purification and propagation of gray mold, preparation of gray mold spore suspension of China rose, 6 portions of identification host materials, in-vitro petal identification and petal disease grading. The method can efficiently and accurately identify the physiological races of gray mold of China in each place by only using 6 parts of identification host materials, thereby saving cost, labor and time.)

1. A method for identifying physiological races of gray mold of Chinese rose is characterized in that: the method comprises the following steps of respectively separating, purifying and propagating botrytis cinerea on roseleaves infected with botrytis cinerea in different collection places, wherein the botrytis cinerea material in one collection place is taken as a sample, and the samples are respectively prepared into a rose botrytis cinerea spore suspension; collecting petals without diseases and insect pests on an identified host material, firstly, carrying out anatomy microscopic examination on fungus-free bacterial colonies or hyphae, then, cleaning with 70% v/v ethanol and sterile water, sucking water on the surfaces of the petals with sterile filter paper, then, paving the petals in culture dishes containing nutrient substrates, and placing each petal of the identified host material in one culture dish; inoculating each China rose botrytis cinerea spore suspension liquid on petals in each culture dish for culture, identifying the China rose botrytis cinerea physiological races according to the inoculated petal disease grades, and dividing the inoculated petal disease grades into 2 grades: level 0: disease resistance, no disease spots on petals or disease attack in the area of less than 20% of petals; level 1: the disease is infected by more than or equal to 20 percent of petal area.

2. The method for identifying physiological races of Botrytis cinerea according to claim 1, characterized in that: the separation, purification and propagation process of the botrytis cinerea is as follows:

firstly, on a super-clean workbench, respectively dipping botrytis cinerea from roses infected with botrytis cinerea collected from different places to transfer to petals of a rose tissue culture seedling in a tissue culture bottle by using a sterilized dissecting needle, wherein a new sterilized dissecting needle needs to be replaced when each dip of botrytis cinerea material is used, so that the botrytis cinerea from different collection places is not mixed, the botrytis cinerea from wild species of the roses is transferred to the petals of a costus root (Rosa pseudo-dangsae) tissue culture seedling, and the botrytis cinerea from a Chinese rose culture variety is transferred to the petals of a rose (Rosa 'Yunmei') tissue culture seedling; inoculating a botrytis cinerea material sample on petals of a Chinese rose tissue culture seedling in a tissue culture bottle, and then placing the tissue culture bottle inoculated with the botrytis cinerea in a tissue culture room under the culture conditions that: the illumination intensity is 1800-3200lx, the light cycle is 10-16h light period and 8-14h dark period, the relative humidity of indoor air is 80-95%, and the cultivation is carried out for 3-7 d at the room temperature of 22-28 ℃;

secondly, transferring the single botrytis cinerea colonies on the tissue culture seedlings to the corresponding petals of the new Chinese rose tissue culture seedlings by using the same method in the step I, and culturing for 3-7 days under the same culture conditions in the step I;

thirdly, repeating the step two for 3 times;

fourthly, repeating the step 1 time, wherein the culture time is 10-15 days;

during the culture period from the first step to the fourth step, the culture substrates in the tissue culture bottle are MS +6-BA0.2mg/L + NAA0.02mg/L + sucrose 35g/L + agar 7.0g/L, and the mouth of the tissue culture bottle is sealed by an air-permeable plastic film.

3. The method for identifying physiological races of Botrytis cinerea according to claim 1, characterized in that: the process for preparing the botrytis cinerea spore suspension comprises the following steps:

brushing the botrytis cinerea subjected to separation, purification and propagation in sterile distilled water containing 0.03% v/v of Tween 20 by using a sterilized brush, wherein the sterilized brush needs to be replaced when each part of the botrytis cinerea is brushed, and the botrytis cinerea at different collection places are brushed into the sterile distilled water containing 0.03% v/v of Tween 20 in different containers to ensure that the botrytis cinerea at different collection places are not mixed; the concentration of Botrytis cinerea spores in each container was adjusted to 1X 10 with sterile distilled water containing 0.03% v/v Tween 20⁴～5×10⁴And (5) obtaining the gray mold spore suspension of the Chinese rose.

4. The method for identifying physiological races of Botrytis cinerea according to claim 1, characterized in that: the identification host material is as follows: ' the peach's day ' (Rosa ' Taozhiyaoyao '), ' the month powder ' (Rosa chinensis ' Pallida '), ' the meadow lily ' (Rosa ' Jinfenlian '), ' the star volunteers ' (Rosa ' xinfeixingyuan '), ' the reflection flower of Japanese ' (Rosa ' yingrihua ') and ' the spring ' (Rosa ' Sichun ').

5. The method for identifying physiological races of Botrytis cinerea according to claim 1, characterized in that: the petals without plant diseases and insect pests on the collection and identification host material are petals without plant diseases and insect pests on the 1 st to 2 nd layers of flowers from inside to outside on the collection and identification host material.

6. The method for identifying physiological races of Botrytis cinerea according to claim 1, characterized in that: the washing process with 70% v/v ethanol and sterile water was as follows: treating the petals with 70% v/v ethanol for 1min, and then cleaning with sterile water for 3-5 times; the petals are laid in the culture dish containing the nutrient medium in a way that the right side of the petals faces upwards and the petals are laid in the culture dish containing the nutrient medium, and the nutrient medium formula is as follows: MS + benzimidazole 0.4g/L + agar 8.0 g/L.

7. The method for identifying physiological races of Botrytis cinerea according to claim 1, characterized in that: the method is characterized in that each China rose botrytis cinerea spore suspension is respectively inoculated on petals in each culture dish for culture: inoculating the gray mold germ spore suspension of the Chinese rose to each petal in the culture dish by adopting a spraying method or a dropping method, and ensuring that each petal is covered by the gray mold germ spore suspension by naked eyes; after inoculation, the culture dish is cultured for 5-7 days under the conditions of indoor light intensity of 1800-3200lx, light period of 10-16h, dark period of 8-14h, indoor air relative humidity of 85-95% and room temperature of 22-28 ℃.

8. The method for identifying physiological races of Botrytis cinerea according to claim 7, characterized in that: 3-5 replicates were set for each sample.

9. The method for identifying physiological races of Botrytis cinerea according to claim 1, characterized in that: adopting SAS V9.0 software to carry out ANOVA analysis on the inoculated petal disease grade division data; the final data of the area of the petal lesion is as follows: total lesion area/total petal area × 100% + -standard error.

10. The method for identifying physiological races of gray mold rose as claimed in any one of claims 1 to 9, characterized in that: the identification of the physiological races of the gray mold of Chinese rose is to use 0 to represent the level of the inoculated petal disease and 1 to represent the level of the inoculated petal disease, correspondingly list the resistance/sensitivity results between different identification host materials and the gray mold of Chinese rose from different collection places one by one, and compare the results with the comparison table of the physiological races of gray mold of Chinese rose of the following table 1 to identify the types of the physiological races of gray mold of Chinese rose;

TABLE 1 comparison table of physiological races of Botrytis cinerea

0 in Table 1: disease resistance; 1: infection;

physiological races of botrytis cinerea type a are represented: the anti-infection/infection identification results between the 6 identification host materials and the physiological races of the botrytis cinerea A are disease-resistant;

physiological races of botrytis cinerea type B: in the 6 parts of identification host materials, only 1 part of 'Sichun' identification host material and the physiological race of the botrytis cinerea type B are susceptible, and the other 5 parts of identification host materials and the physiological race of the botrytis cinerea type B are all resistant;

physiological races of gray mold of type C: the 6 identification host materials comprise 2 identification host materials of spring and 2 identification host materials of anemone asteroides and 2 identification host materials of the ashbya once C type physiological races, and the rest 4 identification host materials and the physiological races of the ashbya once C type physiological races are all susceptible;

physiological races of gray mold of type D: the 6 parts of identification host materials comprise 3 parts of 'Sichun', 'Yingri lotus' and 'xing Yuan' and the anti-susceptible identification results between the identification host materials and the physiological races of the D-type gray mold fungus are all susceptible, and the anti-susceptible identification results between the other 3 parts of identification host materials and the physiological races of the D-type gray mold fungus are all resistant;

physiological races of gray mold of type E: the 6 parts of identification host materials comprise 4 parts of 'Sichun', 'Yingrihua', 'Xinyuxing' and 'radix clematidis', and the anti-susceptible identification results between the identification host materials and the physiological races of the gray mold bacteria of the type E are all susceptible, and the anti-susceptible identification results between the other 2 parts of identification host materials and the physiological races of the gray mold bacteria of the type E are all disease-resistant;

physiological races of botrytis cinerea type F: the 6 identification host materials only have the premature graying of peach and the anti-susceptible identification results between the physiological races of the botrytis cinerea grisea F are disease-resistant, and the rest 5 identification host materials and the physiological races of the botrytis cinerea grisea F are susceptible.

Technical Field

The invention belongs to the technical field of woody flower disease-resistant breeding, and particularly relates to an identification method of physiological races of gray mold of Chinese rose.

Background

China rose (Rosa hybrida) is a plant of the Rosa genus (Rosa L.) of the Rosaceae family (Rosaceae), and is the most abundant fresh cut flower species in the world at present. The main production areas of the cut flower Chinese rose of China are concentrated in Kunming city and Dian Zhongwan, Yunnan province.

The gray mold of Chinese rose is a fungal disease caused by Botrytis cinerea (Botrytis cinerea) and is the most serious fungal disease in the transportation process of cut-flower Chinese roses. It not only infects stems and leaves, but also is the most harmful to flowers. At present, the occurrence of gray mold of Chinese rose is mainly prevented and controlled by repeatedly applying chemical agents in production, so that the production cost is increased, the pollution and the damage to the environment are caused, and the ornamental value of the Chinese rose is also seriously reduced.

In order to reduce economic losses of Chinese rose manufacturers and distributors and reduce environmental pollution caused by pesticide application, researchers at home and abroad are performing breeding scientific research works on Chinese rose gray mold resistant varieties. However, as the physiological species of the gray mold fungus of the Chinese rose are more and the conditions of variation and the like exist, the modern Chinese rose variety with long-acting and broad-spectrum gray mold fungus resistance does not appear at home and abroad at present.

The precondition for breeding the gray mold resistant Chinese rose variety is to know the physiological race condition of the local gray mold pathogen. So far, no research report on physiological races of gray mold of China rose exists at home and abroad. At present, the traditional identification method for other physiological races of botrytis cinerea at home and abroad mainly comprises the following steps: the monospore germs of different geographical sources are respectively inoculated on the differential host plants, and different physiological races are identified by observing and evaluating the morbidity condition of the host petals after a certain morbidity period.

On one hand, because the division of the physiological races of the gray mold fungus of Chinese rose has obvious regionality and specificity, the identification host plant material preferably selects the material (same genus but different species/variety) of the local identification area; on the other hand, if foreign differentiation host plant materials are adopted, the steps and processes of patent expense, introduction, domestication, propagation and the like of host plants are involved, and the procedures not only waste time and labor and cost for physiological race identification work, but also cause that foreign patent varieties are not necessarily suitable for domestic identification work because different geographic environments distribute different physiological races, thereby possibly influencing the accuracy of subsequent identification work. At present, no related report on identification of physiological races of gray mold of China rose exists at home and abroad, so a differential host system and a method suitable for carrying out identification of physiological races of gray mold of China rose are not determined.

The method can accurately predict the species of the botrytis cinerea of the Chinese rose, timely detect the differentiation succession of the physiological races of the Chinese rose, and has important significance for preventing and treating the botrytis cinerea of the Chinese rose, introducing disease-resistant materials and breeding new disease-resistant varieties. In view of the fact that a set of differential host system suitable for identifying the physiological races of the gray mold fungus in China is not available in China, and the technical problems of time and labor consumption, long identification period and the like exist in the host morbidity statistical method adopted in the traditional resistance identification method, a set of differential host system and method which are accurate in identification and rapid in screening and suitable for identifying the physiological races of the gray mold fungus in China is urgently explored.

Disclosure of Invention

The invention aims to provide the identification method of the physiological races of the gray mold of China rose, which has the characteristics of simple operation, accurate identification and quick screening.

The purpose of the invention is realized by the following technical scheme:

a method for identifying physiological races of gray mold of Chinese rose is characterized in that: the method comprises the following steps of respectively separating, purifying and propagating botrytis cinerea on roseleaves infected with botrytis cinerea in different collection places, wherein the botrytis cinerea material in one collection place is taken as a sample, and the samples are respectively prepared into a rose botrytis cinerea spore suspension; collecting petals without diseases and insect pests on an identified host material, firstly, carrying out anatomy microscopic examination on fungus-free bacterial colonies or hyphae, then, cleaning with 70% v/v ethanol and sterile water, sucking water on the surfaces of the petals with sterile filter paper, then, paving the petals in culture dishes containing nutrient substrates, and placing each petal of the identified host material in one culture dish; inoculating each China rose botrytis cinerea spore suspension liquid on petals in each culture dish for culture, identifying the China rose botrytis cinerea physiological races according to the inoculated petal disease grades, and dividing the inoculated petal disease grades into 2 grades: level 0: disease resistance, no disease spots on petals or disease attack in the area of less than 20% of petals; level 1: the disease is infected by more than or equal to 20 percent of petal area.

Further, the separation, purification and propagation process of the botrytis cinerea is as follows:

firstly, on a super-clean workbench, respectively dipping botrytis cinerea from roses infected with botrytis cinerea collected from different places to transfer to petals of a rose tissue culture seedling in a tissue culture bottle by using a sterilized dissecting needle, wherein a new sterilized dissecting needle needs to be replaced when each dip of botrytis cinerea material is used, so that the botrytis cinerea from different collection places is not mixed, the botrytis cinerea from wild species of the roses is transferred to the petals of a costus root (Rosa pseudo-dangsae) tissue culture seedling, and the botrytis cinerea from a Chinese rose culture variety is transferred to the petals of a rose (Rosa 'Yunmei') tissue culture seedling; inoculating a botrytis cinerea material sample on petals of a Chinese rose tissue culture seedling in a tissue culture bottle, and then placing the tissue culture bottle inoculated with the botrytis cinerea in a tissue culture room under the culture conditions that: the illumination intensity is 1800-3200lx, the light cycle is 10-16h light period and 8-14h dark period, the relative humidity of indoor air is 80-95%, and the cultivation is carried out for 3-7 d at the room temperature of 22-28 ℃;

secondly, transferring the single botrytis cinerea colonies on the tissue culture seedlings to the corresponding petals of the new Chinese rose tissue culture seedlings by using the same method in the step I, and culturing for 3-7 days under the same culture conditions in the step I;

thirdly, repeating the step two for 3 times;

fourthly, repeating the step 1 time, wherein the culture time is 10-15 days;

during the culture period from the first step to the fourth step, the culture substrates in the tissue culture bottle are MS +6-BA0.2mg/L + NAA0.02mg/L + sucrose 35g/L + agar 7.0g/L, and the mouth of the tissue culture bottle is sealed by an air-permeable plastic film.

Further, the process for preparing the gray mold rose spore suspension is as follows:

brushing the botrytis cinerea subjected to separation, purification and propagation into sterile distilled water containing 0.03% v/v Tween 20 by using a sterilized brush, wherein each brush is used for brushing the botrytis cinerea, a new sterilized brush needs to be replaced, the botrytis cinerea at different collection places are brushed into the sterile distilled water containing 0.03% v/v Tween 20 in different containers, and the months at different collection places are ensuredThe botrytis cinerea is not mixed; the concentration of Botrytis cinerea spores in each container was adjusted to 1X 10 with sterile distilled water containing 0.03% v/v Tween 20⁴～5×10⁴And (5) obtaining the gray mold spore suspension of the Chinese rose.

Further, the identifying host material is: ' the peach's day ' (Rosa ' Taozhiyaoyao '), ' the month powder ' (Rosa chinensis ' Pallida '), ' the meadow lily ' (Rosa ' Jinfenlian '), ' the star volunteers ' (Rosa ' xinfeixingyuan '), ' the reflection flower of Japanese ' (Rosa ' yingrihua ') and ' the spring ' (Rosa ' Sichun ').

Furthermore, the petals without plant diseases and insect pests on the collection and identification host material are petals without plant diseases and insect pests on the 1 st to 2 nd layers of flowers from inside to outside on the collection and identification host material.

Further, the washing process with 70% v/v ethanol and sterile water was as follows: treating the petals with 70% v/v ethanol for 1min, and then cleaning with sterile water for 3-5 times; the petals are laid in the culture dish containing the nutrient medium in a way that the right side of the petals faces upwards and the petals are laid in the culture dish containing the nutrient medium, and the nutrient medium formula is as follows: MS + benzimidazole 0.4g/L + agar 8.0 g/L.

Further, the inoculation of each of the botrytis cinerea spore suspensions on petals in each culture dish respectively comprises the following steps: inoculating the gray mold germ spore suspension of the Chinese rose to each petal in the culture dish by adopting a spraying method or a dropping method, and ensuring that each petal is covered by the gray mold germ spore suspension by naked eyes; after inoculation, the culture dish is cultured for 5-7 days under the conditions of indoor light intensity of 1800-3200lx, light period of 10-16h, dark period of 8-14h, indoor air relative humidity of 85-95% and room temperature of 22-28 ℃.

Further, 3-5 replicates were set for each sample.

Further, adopting SAS V9.0 software to carry out ANOVA analysis on the inoculated petal disease grade division data; the final data of the area of the petal lesion is as follows: total lesion area/total petal area × 100% + -standard error.

Further, the identification of the physiological races of gray mold of China rose is to use 0 to represent the level of the inoculated petal disease, 1 to represent the level of the inoculated petal disease, correspondingly list the anti/inductive results between different identification host materials and gray mold of China rose from different collection sites one by one, and compare the results with the comparison table of the physiological races of gray mold of China rose as shown in the following table 1 to identify the types of the physiological races of gray mold of China rose;

TABLE 1 comparison table of physiological races of Botrytis cinerea

0 in Table 1: disease resistance; 1: infection;

physiological races of botrytis cinerea type a are represented: the anti-infection/infection identification results between the 6 identification host materials and the physiological races of the botrytis cinerea A are disease-resistant; i.e. the statistical result is 0.

Physiological races of botrytis cinerea type B: only 1 part of 'Sichun' of the 6 parts of identification host materials has an anti-susceptible/susceptible identification result between the identification host materials and the physiological races of the botrytis cinerea B, namely a statistical result is 1; the resistance/susceptibility identification results between the other 5 parts of identification host materials and the physiological race of the botrytis cinerea B are all disease-resistant, namely the statistical result is 0.

Physiological races of gray mold of type C: the 6 identification host materials comprise 2 identification host materials of 'spring of spring' and 'lotus florida', and the anti-susceptible identification results between the identification host materials and the physiological races of the botrytis cinerea type C are all susceptible, namely the statistical result is 1; the resistance/susceptibility identification results between the other 4 identification host materials and the physiological races of the botrytis cinerea type C are all disease-resistant, namely the statistical result is 0.

Physiological races of gray mold of type D: the 6 parts of identification host materials comprise 3 parts of identification host materials of spring, lotus and star volunteer, and the anti-susceptible/susceptible identification results between the identification host materials and the physiological races of the D-type botrytis cinerea are all susceptible, namely the statistical result is 1; the resistance/susceptibility identification results between the rest 3 identification host materials and the D type gray mold fungus physiological microspecies are all disease-resistant, namely the statistical result is 0.

Physiological races of gray mold of type E: the 6 parts of identification host materials comprise 4 parts of 'Sichun', 'Yingri', 'Star wish' and 'radix clematidis', and the anti-infection/infection identification results between the identification host materials and the physiological races of the gray mold bacteria of the type E are all susceptible, namely the statistical result is 1; the resistance/susceptibility identification results between the rest 2 identification host materials and the physiological race of the gray mold of the type E rose are all disease-resistant, namely the statistical result is 0.

Physiological races of botrytis cinerea type F: the resistance/susceptibility identification result between the 'premature peach' host material and the F-type botrytis cinerea physiological microspecies in the 6 parts of identification host materials is disease resistance, namely the statistical result is 0; the anti-susceptible/susceptible identification results between the other 5 parts of identification host materials and the physiological races of the botrytis cinerea of type F are all susceptible, namely the statistical result is 1.

Compared with the prior art, the invention has the main innovation points and beneficial effects that:

1. through a large number of experiments, 6 parts of host materials are screened from 873 parts of collected rosa plants to serve as identification host materials of the invention, and only the 6 parts of identification host materials can be used for efficiently and accurately identifying the physiological races of botrytis cinerea in China, so that the cost is saved, the labor and the time are saved, and the identification host materials and the identification method are suitable for identifying the physiological races of botrytis cinerea in China.

2. The identification host materials are all from China, not only have independent intellectual property rights, but also have adaptability to cultivation and propagation in the whole country, and simultaneously have strong identification capability. The method provides operable host materials for local identification of physiological races of gray mold of Chinese rose by researchers in China, and provides guidance for selection of breeding materials for resisting gray mold of Chinese rose.

3. The method for identifying the gray mold of the Chinese rose by adopting the in-vitro petal inoculation method is characterized in that the gray mold of the Chinese rose is inoculated on the in-vitro petals of the identification host material. And determining that the petal has no disease spots or the incidence of the petal area of less than 20 percent is set as 0 grade, which is represented by 0, and the incidence of the petal area of more than or equal to 20 percent of the petal disease spots is set as 1 grade, which is represented by 1; inputting 0 and 1 into comparison table of physiological races of Botrytis cinerea to compare, and determining the types of physiological races. If the disease area of the petals is not 20% of the disease area of the petals, the grades of the gray mold of the isolated petals of the Chinese rose are classified, and different physiological races cannot be distinguished.

4. The identification time is short, only 5-7 days are needed from inoculation of the botrytis cinerea suspension to identification of the petals of the host material to completion of physiological race identification, and more than 14 days are needed in the traditional method (the traditional method is to inoculate and identify the whole plant or leaves, and a large amount of time is used for plant propagation and other works in the early stage).

5. The identification host plant materials adopted by the invention are all cultivars flowering in four seasons, so that the identification work of physiological races of gray mold of Chinese rose can be carried out in each season; furthermore, the in-vitro petals of the host material are identified, because the petals are host plant tissues with the largest gray mold, and the traditional method is a cutting seedling, the method not only occupies less space, but also is simple and convenient to operate, and the result is accurate and reliable; the invention carries out the experiment of infecting host with botrytis cinerea in a relatively closed culture dish, can eliminate the possibility that different physiological race bacteria types are mixed through air, thereby ensuring the accuracy of the experimental result.

The above-mentioned biomaterials to which the present invention relates may all be purchased from commercial sources. Such as available from Yunnan Lidu flower development, Inc., the company address: four towns in the city of Tonghai, Yuxi, Yunnan province, zip code: 652702.

meanwhile, the above-mentioned biomaterials of the present invention are disclosed in the non-patent documents listed in Table 8, and the applicants have living bodies to store the biomaterials in their bases, and they are guaranteed to be distributed to the public within 20 years from the date of the present patent application. The contact address of the applicant is Beijing Lou 2238 of Panlongdistrict, Kunming, Yunnan province, and the institute of flowers and plants, postcode, of agricultural academy of sciences, of Yunnan province: 650205.

Detailed Description

The following examples are given without specific indication of conventional methods. Materials such as reagents are commercially available.

Example 1 identification of physiological races of Botrytis cinerea in our country

As Yunnan province is the distribution center of China rose wild resources and the cultivation center of China rose varieties, and is also the main occurrence place of China rose gray mold. Therefore, the implementation case mainly takes Yunnan province as the main part, and covers other 6 provinces in China, and the total of 12 different areas are the collection places.

(1) Separation, purification and propagation of botrytis cinerea

Firstly, on a super clean workbench, using sterilized dissecting needles (sterilization method: sterilizing at 120 ℃ for 20 minutes) to respectively dip botrytis cinerea from 12 roses infected with botrytis cinerea at different places in China and transfer the botrytis cinerea to petals of a rose tissue culture seedling in a tissue culture bottle cultured according to a conventional plant tissue culture method, wherein each time one part of botrytis cinerea material is dipped, a new sterilized dissecting needle is required to be replaced, so that the botrytis cinerea at different collection places are not mixed, the botrytis cinerea from wild species of the roses is transferred to the petals of a costus root (Rosa pseudo-baumanniae) tissue culture seedling, and the botrytis cinerea from a rose cultivar is transferred to the petals of a rose (Rosa 'Yunmei') tissue culture seedling; the method comprises the following steps of taking a botrytis cinerea material in a collection place as a sample, inoculating the sample to petals of a Chinese rose tissue culture seedling in a tissue culture bottle, and then placing the tissue culture bottle inoculated with the botrytis cinerea in a tissue culture room, wherein the culture conditions are as follows: the illumination intensity is 2800lx, the light cycle is 12h light period and 12h dark period, the relative humidity of indoor air is 85%, and the cultivation is carried out for 3-7 d at the room temperature of 24-25 ℃. Botrytis cinerea roses infected with Botrytis cinerea can be diagnosed by a diagnostic method in the literature (Noack R.Breeding and selection strategies for disease and pest resistance. in: Encyclopedia of Rose science AV, Debener T, Guidin S eds., Oxford: electronic Academic Press,2003,1: 144-.

Secondly, transferring the single botrytis cinerea colonies on the tissue culture seedlings to the corresponding petals of the new Chinese rose tissue culture seedlings by using the same method of the step (1), and culturing for 3-7 days under the same culture conditions of the step (1);

thirdly, repeating the step (1) and the step (3);

fourthly, repeating the step (1) and the step (1) for 1 time, wherein the culture condition time is changed to 10-15 days;

during the culture period from the step (1) to the step (1), the culture substrates in the tissue culture bottle are MS +6-BA0.2mg/L + NAA0.02mg/L + sucrose 35g/L + agar 7.0g/L, and the mouth of the tissue culture bottle is sealed by an air-permeable plastic film.

12 different locations within the country: 6 counties and cities in Yunnan province: daizhou Dali City (abbreviation: DL), Pu' er City (abbreviation: PE), Honghe Mongolian City (abbreviation: MZ), Yuxi City (abbreviation: YX), Kunming City (abbreviation: KM), Qujing City (abbreviation: QJ); 1 city in Sichuan province: metropolis (abbreviated: CD); 1 city, Guizhou province: guiyang city (abbreviated as GY); beijing, Inc. (abbreviated as: BJ); 1 city in Henan province: nanyang City (abbreviation: NY); 1 city in Guangdong province: shenzhen city (abbreviation: SZ); 1 city in Jiangsu province: tin-free city (abbreviated: WX).

(2) Preparation of a suspension of gray mold spores of the rose

Brushing botrytis cinerea on the petals of the tissue culture seedlings in the step (1) to sterile distilled water containing 0.03% (volume fraction) of Tween 20 by using a sterilized brush (a sterilization method: sterilizing at high temperature of 120 ℃ for 20 minutes), wherein each brush is used for brushing botrytis cinerea material, a new sterilized brush needs to be replaced, and the botrytis cinerea in different collection places are brushed into the sterile distilled water containing 0.03% (volume fraction) of Tween 20 in different containers, so that the botrytis cinerea in different collection places are not mixed; the concentration of Botrytis cinerea spores in each container was adjusted to 5X 10 with sterile distilled water containing 0.03% (volume fraction) Tween 20⁴And (5) obtaining the gray mold spore suspension of the Chinese rose for inoculation and standby.

(3) Preparation of Chinese rose host material

Collecting identification host materials, collecting petals without plant diseases and insect pests on the 1 st to 2 nd layers of flowers of the identification host materials from inside to outside, firstly, carrying out microscope examination on the collected petals by using a dissecting mirror, processing the petals for 1min by using ethanol with the volume fraction of 70% after ensuring that the collected petals do not contain any fungal colony or hypha, then, cleaning the petals for 3-5 times by using sterile water, sucking water on the surfaces of the petals by using sterile filter paper, and spreading the petals in culture dishes containing nutrient substrates with the front faces upward (the back faces of the petals are smooth, fungi are not easy to successfully inoculate, the front faces are favorable for successful inoculation, and 3 petals in each culture dish), wherein the petals of each identification host material are placed in one culture dish; the nutrient medium formula comprises the following components: MS + benzimidazole 0.4g/L + agar 8.0 g/L;

the identification host material is as follows: ' the peach's day ' (Rosa ' Taozhiyaoyao '), ' the month powder ' (Rosa chinensis ' Pallida '), ' the meadow lily ' (Rosa ' Jinfenlian '), ' the star volunteers ' (Rosa ' xinfeixingyuan '), ' the reflection flower of Japanese ' (Rosa ' yingrihua ') and ' the spring ' (Rosa ' Sichun ').

(4) Inoculation and culture of gray mold of Chinese rose

Respectively inoculating the botrytis cinerea spore suspension of 12 collection places in the step (2) onto each petal in each culture dish in the step (3) by adopting a spraying method or a dropping method, ensuring that the front of each petal is covered by the botrytis cinerea spore suspension, wherein the botrytis cinerea spores of one collection place are one sample, each sample is provided with 3 repetitions, and the botrytis cinerea spore suspension of one collection place is respectively inoculated onto each petal of each culture dish in the step (3); after inoculation is finished, culturing the culture dish for 5-7 days under the conditions of indoor light intensity 2800lx, light period of 12h, dark period of 12h, air relative humidity of 85% and room temperature of 24-25 ℃;

(5) observation and statistics of disease conditions of Chinese rose host material

The inoculated petal disease was divided into 2 grades: level 0: disease resistance, no disease spots on petals or disease attack in the area of less than 20% of petals; level 1: the disease is infected by more than or equal to 20 percent of petal area.

Performing ANOVA analysis on the inoculated petal disease division data by adopting SAS V9.0 statistical analysis software (STATISTICAL ANALYSIS SYSTEM, SAS for short), wherein the final data of the infected petal lesion area after the physiological race of botrytis cinerea is inoculated to petals at each different collection site is as follows: total lesion area/total petal area × 100% + -standard error.

(6) Identification of physiological races

And (3) correspondingly listing the anti-sensitivity/sensibility results between different identification host materials and the botrytis cinerea from different collection sites into a table (shown in the following tables 2 and 3) according to the statistical results of the step (5), wherein the level 0 is represented by level 0, the level 1 is represented by level 1, the 0/1 combination matrix type which is visually reflected in the table is the type of the physiological race of the botrytis cinerea, the 0/1 combination matrix type is compared with the comparison table of the types of the physiological races of the botrytis cinerea in the table 1, and the type of the physiological race of the botrytis cinerea is judged and identified (from type A to type F, tables 2 and 3).

And (3) test results:

according to the following tables 2 and 3, the types of collected physiological races of gray mold of rose in 12 county and city areas nationwide, that is, 6 physiological races (types a, B, C, D, E, and F) in total from types a to F, can be visually counted. From tables 2 and 3, the type of the physiological race of a certain region can be searched and judged, for example, the physiological race of botrytis cinerea C type collected from beijing region, which is susceptible to the disease of 2 of the 6 identified host materials, i.e., the statistical result is 1, and the rest 4 identified host materials are all disease-resistant, i.e., the statistical result is 0. The method not only can identify the type of the physiological race of the gray mold fungus of Chinese rose, but also can guide the breeding of the Chinese rose in the area and select breeding materials with resistance to the physiological race of the gray mold fungus of the Chinese rose in the area.

TABLE 2 example 1 anti-susceptible results between different identified host materials and 6 different geographical sources of Botrytis cinerea

TABLE 3 results of resistance/sensitization between different identified host materials and 6 different geographical sources of Botrytis cinerea of example 1

Note: 0 in tables 2 and 3: disease resistance; 1: is susceptible to diseases.

Comparative experiment 1: inoculating the gray mold spore suspension of China rose to the isolated leaf of the identified host material

Comparative experiment 1 the same procedure as in example 1 was used to compare the difference between the isolated petals of botrytis cinerea spores suspension inoculated into the identifying host material and the isolated leaves of the identifying host material inoculated with the botrytis cinerea spores suspension described in example 1, except that the petals of the identifying host material were changed to the leaves of the identifying host material. The statistical results of comparative experiment 1 are: there are only two cases of inoculated leaves, one is no lesion, the other is < 20% of the leaf area, the disease condition of inoculated leaves can be classified into 1 grade, i.e. 0 grade: finally, the identification work of the physiological races of the gray mold of the Chinese rose in different collection places cannot be finished.

Comparative experiment 2 inoculated petal disease grading comparison (petal disease area 30% as benchmark)

Comparative experiment 2 the procedure of example 1 was otherwise the same as in example 2 except that the disease level of petals was rated based on the area of petals affected at 30% (see tables 4 and 5 below): the original 6 physiological races of botrytis cinerea (types A to F) (tables 2 and 3) will be identified as 4 physiological races (only types A, B, C and D), the results not only confuse the physiological races, but also the inaccuracy of the identification results will delay the development of scientific research work such as breeding for disease resistance and the like.

TABLE 4 comparison of the resistance/susceptibility results between the different identified host materials of experiment 2 and Botrytis cinerea of 6 different geographical origins

Note: 0 in Table 4: disease resistance; 1: is susceptible to diseases.

TABLE 5 comparison of the resistance/susceptibility results between the different identified host materials of experiment 2 and Botrytis cinerea of 6 different geographical origins

Note: 0 in Table 5: disease resistance; 1: is susceptible to diseases.

Comparative experiment 3: grading comparison of inoculated petal disease conditions (petal disease area 10% as benchmark)

Comparative experiment 3 the procedure of example 1 was otherwise the same as in example 3 except that the disease level of petals was rated based on the area of 10% of the petals, and the results were as follows (see tables 6 and 7): the original 6 physiological races of botrytis cinerea (types A to F) (tables 2 and 3) will be identified as 4 physiological races (only B, C, E and F), the results not only confuse the physiological races, but also the inaccuracy of the identification results will delay the development of scientific research work such as breeding for disease resistance and the like.

TABLE 6 comparison of the resistance/susceptibility results between the different identified host materials and 6 Botrytis cinerea of different geographical origin in experiment 3

Note: 0 in Table 6: disease resistance; 1: is susceptible to diseases.

Table 7 comparison of the resistance/susceptibility results between the different identified host materials of experiment 3 and 6 Botrytis cinerea of different geographical origin

Note: 0 in Table 7: disease resistance; 1: is susceptible to diseases.

The comparison experiment 2 and the comparison experiment 3 fully show that if the disease area of the petals is not 20 percent, the disease area is used as the grade of the gray mold of the isolated petals of the Chinese rose, different physiological races cannot be distinguished.

TABLE 8 publication of biological materials related to the present invention in non-patent literature