阅读说明：本技术 一种快速筛选抗黄萎病棉花品种的方法 (Method for rapidly screening verticillium wilt-resistant cotton varieties ) 是由 赖成霞 阳妮 玛依拉 石必显 师维军 李春平 郑子漂 于 2021-08-11 设计创作，主要内容包括：本发明公开了一种快速筛选抗黄萎病棉花品种的方法,通过提取大丽轮枝菌V991孢内毒素接种叶圆盘,统计分析叶圆盘荧光参数各指标变化与棉花抗黄萎病品种的关系,确定筛选抗(耐)黄萎病棉花品种(系)的关键性因素为光系统II实际光量子产量Y(II)、非调节性能量耗散的量子产量Y(NO)和非光化学淬灭系数(NPQ)三个特征曲线,利用其筛选出抗黄萎病棉花品种,与采用孢内毒素叶盘和分生孢子浸根水培技术对棉花抗性鉴定的结果高度一致,表明本发明提供的快速筛选棉花抗黄萎病品种的方法简单可靠,解决了利用分生孢子悬浮液浸根水培法存在的因孢子生长一致性差,造成鉴定结果参差不齐的问题,同时为棉花抗黄萎病鉴定方法提供新的毒素浸染源。(The invention discloses a method for rapidly screening verticillium wilt resistant cotton varieties, which comprises the steps of extracting verticillium dahliae V991 spore endotoxin to inoculate a leaf disc, statistically analyzing the relation between the index change of the fluorescence parameter of the leaf disc and the verticillium wilt resistant cotton varieties, determining three characteristic curves of the key factors of screening the verticillium wilt resistant cotton varieties (systems) including the actual light quantum yield Y (II), the non-adjustable energy dissipation quantum yield Y (NO) and the non-photochemical quenching coefficient (NPQ), screening the verticillium wilt resistant cotton varieties by using the characteristic curves, and highly consistent with the result of identifying the resistance of cotton by using spore endotoxin leaf disc and conidium root-soaking water culture technology, thereby showing that the method for rapidly screening the verticillium wilt resistant cotton varieties provided by the invention is simple and reliable, solving the problem that the conidium root-soaking water culture method by using conidium suspension liquid has poor consistency due to spore growth, causes the problem of uneven identification results, and provides a new toxin-infecting source for the identification method of the verticillium wilt resistance of cotton.)

1. A method for rapidly screening a verticillium wilt-resistant cotton variety is characterized by comprising the following steps:

(1) preparing a conidium suspension of verticillium dahliae V991 of cotton verticillium wilt pathogen, and extracting endotoxin of verticillium dahliae V991 of cotton verticillium wilt pathogen;

(2) in the full-bloom stage of cotton, selecting 20 cotton plants from each strain, taking the third last leaf of each strain, punching 20-30 leaf discs with the diameter of 1.0cm on each leaf by using a puncher, then soaking the leaf discs into 50mL of spore endotoxin solution obtained in the step (1) with the concentration of 16 mu g/mL, culturing for 24 hours by using Czapek's culture solution, taking out the leaf discs, sucking dry surface attachment liquid by using absorbent paper, and counting disease index by taking yellowing and patch forming area of the leaf discs as grading basis;

(3) dark adaptation is carried out on the leaf discs processed in the step (2) for 30min, then the leaf discs are placed on a chlorophyll fluorescence imaging system sample stage, an area of interest AOI with the diameter of 0.5-1.0cm is set on each leaf disc, chlorophyll fluorescence imaging pictures of the leaf discs inoculated with spores are collected, and the kinetic change curve of chlorophyll fluorescence parameters of each leaf disc is detected;

(4) counting analysis data, analyzing the relation between each index change of the fluorescence parameters and the verticillium wilt resistant cotton variety, determining the key influence factors for screening the verticillium wilt resistant cotton variety, establishing an evaluation system for screening the verticillium wilt resistant cotton variety, and screening the verticillium wilt resistant cotton variety.

2. The method for rapidly screening the cotton variety with verticillium wilt resistance as claimed in claim 1, wherein the extraction method of spore endotoxin in step (1) is as follows: inoculating cotton verticillium wilt fungus cake with diameter of 0.5cm into Czapek's culture solution sterilized at 121 deg.C for 15min, and shake-culturing in a shaker at 25 deg.C and rotation speed of 130rpm/min for 5 days until conidia have cluster structure on the wall of triangular flask; filtering the liquid obtained by culturing through a steel sieve with 140 meshes and 400 meshes in a double layer manner to obtain a spore suspension; centrifuging the obtained spore suspension at room temperature at 14000rpm/min for 10min, collecting precipitate, washing the collected precipitate with sterilized water, centrifuging at 14000rpm/min for 10min, collecting precipitate, adding into 0.05M phosphate buffer solution with pH of 6.5, suspending the precipitate, and adjusting the spore concentration of the obtained suspension to 10⁷Per mL; repeatedly grinding the spore suspension by a homogenizer at 120Mpa and 10 deg.C for 3-4 times, centrifuging at 4 deg.C and 18000rpm/min for 25min, discarding supernatant, and filtering with 0.45 μm microporous membrane to remove mixed bacteria to obtain filtrate as spore endotoxin.

Technical Field

The invention belongs to the technical field of cotton disease resistance detection, and particularly relates to a method for rapidly screening a cotton variety (line) with verticillium wilt resistance.

Background

Cotton verticillium wilt is a soil-borne and vascular bundle fungal disease called "cancer" of cotton, and causes very serious economic loss to cotton production. Verticillium dahliae (Verticillium dahliae) and Verticillium albo-atrum (Verticillium albo-atrum) are the main pathogenic bacteria. In China, the main pathogenic bacterium of cotton verticillium wilt is verticillium dahliae (V.dahliae). The pathogenic bacteria have the characteristics of wide host range, multiple types, difficult control of chemical pesticides, quick variation, long survival time, strong stress resistance and the like, verticillium dahliae can be infected in the whole cotton growth stage, and systemic infection is formed to cause wilting and yellowing of leaves of cotton plants, browning of vascular bundles, dwarf and small plants, withering of leaves and withering of plants in severe cases, and finally, the plants fall into polished rods, thereby severely restricting the cotton production in China. Aiming at cotton verticillium wilt, no effective prevention and control method exists up to now. The cultivation of resistant varieties is an effective method for preventing and treating cotton verticillium wilt, but for a long time, verticillium wilt resistance breeding has not been greatly developed due to the lack of germplasm resources with immunity or high resistance to verticillium dahliae.

At present, the method for screening the cotton verticillium wilt-resistant varieties mainly comprises an indoor mode and an outdoor mode, wherein outdoor identification adopts the establishment of disease nursery and utilizes natural environment for identification and screening, and because of the change of the geographical environment and the climatic condition of natural climate, the disease occurrence states of the cotton varieties are different, and the cotton verticillium wilt-resistant varieties can be completed in several years, which is time-consuming and labor-consuming; the indoor identification and screening method mainly comprises a conidium root soaking method and a spore exotoxin identification method, wherein the conidium root soaking method is a main mode for identifying indoor verticillium wilt-resistant varieties, but has high requirements on cotton planting, pure water needs to be poured in the whole process, and the root injury part and degree are difficult to control, so that the probability that the resistance of an identification result is false positive is high; the toxin identification method is a new identification mode developed in recent years, researchers generally consider that the pathogenic toxin is of an extracellular secretion type, and have separated out the related toxin from liquid culture mediums of strains such as T9, VD8, SS-43 and the like, the culture mediums are large in quantity and quantity to meet the toxin required by the experiment, the cost is high, the time is consumed, and the method is one of the main reasons causing the slow research at present, but the method has no related research on the pathogenic toxin of other strains, and therefore, the method has a blank in the aspect of researching the intracellular toxin of other strains. The verticillium dahliae V991 is a highly pathogenic and defoliating strain, and can quickly attack crops by using a conidium root soaking method, a spore exotoxin root soaking method and a leaf disc method, but the identification of the verticillium wilt resistant cotton variety by extracting spore endotoxin of the verticillium dahliae as an infection source is not reported in documents and patents.

Disclosure of Invention

Aiming at the problems of the existing identification method of the cotton variety with verticillium wilt resistance, the identification result is uneven due to the poor consistency of spore growth in the conidium root soaking method, and the technical current situation that the cotton variety with verticillium wilt resistance is screened by using the extracted verticillium dahliae V991 spore endotoxin as an infection source is not reported in documents and patents, the invention aims to provide a method for rapidly screening the cotton variety with verticillium wilt resistance, a leaf disc is inoculated by extracting the verticillium dahliae V991 spore endotoxin, the relation between the change of each index of the fluorescence parameter of the leaf disc and the cotton variety with verticillium wilt resistance is statistically analyzed, the key factors influencing the screening of the cotton variety (system) with verticillium wilt resistance are determined to be three characteristic curves of photosystem II actual light quantum yield Y (II), non-adjustable energy dissipation quantum yield Y (NO) and non-photochemical quenching coefficient (NPQ), an evaluation system for screening the cotton variety with verticillium wilt resistance is established, the method for screening the cotton variety resistant to the verticillium wilt can cause the change of chlorophyll fluorescence parameters after infecting the cotton, and the change also has consistency in the cotton variety resistant to (resistant) the verticillium wilt due to the uniformity of the spore endotoxin, so the change of the fluorescence parameters of the cotton variety resistant to the verticillium wilt can be used as the screening basis of the cotton variety resistant to the verticillium wilt.

In order to achieve the technical purpose, the invention is realized by the following technical scheme:

the invention specifically provides a method for rapidly screening a verticillium wilt-resistant cotton variety, which specifically comprises the following steps:

(1) preparing conidium suspension of verticillium dahliae V991 as pathogen of cotton verticillium wilt, and extracting endotoxin from verticillium dahliae V991 as pathogen of cotton verticillium wilt.

(2) In the full-bloom stage of cotton, 20 cotton plants are selected from each strain, the third last leaf of each strain is taken, 20-30 leaf discs with the diameter of 1.0cm are punched on the leaves by a puncher, then the leaf discs are immersed into 50mL of spore endotoxin solution obtained in the step (1) with the concentration of 16 mu g/mL, the spore endotoxin solution is cultured for 24 hours and then taken out, the surface attachment liquid is sucked dry by absorbent paper, and the disease index is counted by taking the yellowing area of the leaf discs as a grading basis.

(3) Dark adaptation is carried out on the leaf discs processed in the step (2) for 30min, then the leaf discs are placed on a chlorophyll fluorescence imaging system sample stage, an area of interest AOI with the diameter of 0.5-1.0cm is set on each leaf disc, chlorophyll fluorescence imaging pictures of the leaf discs inoculated with endotoxin are collected, and the kinetic change curve of chlorophyll fluorescence parameters of each leaf disc is detected.

(4) Counting analysis data, analyzing the relation between each index change of the fluorescence parameters and the verticillium wilt resistant cotton variety, determining the key influence factors for screening the verticillium wilt resistant cotton variety, establishing an evaluation system for screening the verticillium wilt resistant cotton variety, and screening the verticillium wilt resistant cotton variety.

In the method for rapidly screening the verticillium wilt-resistant cotton variety, the conidium suspension in the step (1) is prepared by the following steps: inoculating cotton verticillium wilt fungus cake with diameter of 0.5cm into Czapek's culture solution sterilized at 121 deg.C for 15min, and shake-culturing in a shaker at 25 deg.C and rotation speed of 130rpm/min for 5 days until conidia have cluster structure on the wall of triangular flask; filtering the cultured liquid with steel sieve of 140 meshes and 400 meshes to obtain spore suspension(ii) a Centrifuging the obtained spore suspension at room temperature at 14000rpm/min for 10min, collecting precipitate, washing the collected precipitate with sterilized water, centrifuging at 14000rpm/min for 10min, collecting precipitate, adding into 0.05M phosphate buffer solution with pH of 6.5, suspending the precipitate, and adjusting the spore concentration of the obtained suspension to 10⁷Per mL; repeatedly grinding the spore suspension by a homogenizer at 120Mpa and 10 deg.C for 3-4 times, centrifuging at 4 deg.C and 18000rpm/min for 25min, discarding supernatant, and filtering with 0.45 μm microporous membrane to remove mixed bacteria to obtain filtrate as spore endotoxin.

In the method for rapidly screening the cotton variety with the verticillium wilt resistance, the key influencing factors for screening the cotton variety (line) with the verticillium wilt resistance are determined in the step (4) and are three characteristic curves of the actual light quantum yield Y (II) of a photosystem II, the non-adjustable energy dissipation quantum yield Y (NO) and the non-photochemical quenching coefficient (NPQ).

Meanwhile, the invention discloses application of the method for rapidly screening the verticillium wilt-resistant cotton variety.

By implementing the specific technical scheme provided by the invention, the following beneficial effects can be achieved by implementing the content of the invention:

(1) the invention provides a method for rapidly screening cotton variety with verticillium wilt resistance, which comprises extracting verticillium dahliae V991 spore endotoxin to infect leaf disc, comparing the disease index of cotton with conidium root soaking method, the relation between each index of fluorescence parameter and cotton variety with verticillium wilt resistance, determining the key factors influencing the characteristic curve of cotton variety (system) with verticillium wilt resistance as three characteristic curves of photosystem II actual light quantum yield Y (II), non-adjustable energy dissipation quantum yield Y (NO) and non-photochemical quenching coefficient (NPQ), establishing evaluation system for screening cotton variety with verticillium wilt resistance, adopting spore endotoxin to inoculate leaf disc and conidium root soaking technique to identify the cotton resistance, highly consistent with the identification result of cotton variety resistance obtained by the day change of fluorescence parameter, therefore, the change of the cotton variety fluorescence parameters after inoculation of verticillium wilt endotoxin can be used as the screening basis of the disease-resistant cotton variety.

(2) According to the method for screening the verticillium wilt-resistant cotton varieties by virtue of the change of the chlorophyll fluorescence parameters of cotton leaves caused by inoculating verticillium dahliae to cotton, due to the uniformity of endotoxin of verticillium dahliae V991 spore, the change of the fluorescence parameters of verticillium dahliae is consistent in the verticillium wilt-resistant cotton varieties, and the problem that identification results are uneven due to poor spore growth consistency in the method for screening the verticillium wilt-resistant cotton varieties by utilizing a conidium suspension root soaking method is solved.

Drawings

FIG. 1 is a comparison chart of disease index of different cotton varieties by using a water culture method and a disc method of intracellular toxin soaking.

FIG. 2 shows the appearance characteristics of leaf discs and the chlorophyll fluorescence imaging change chart of different cotton varieties inoculated with verticillium dahliae V991 spore endotoxin. In the figure, A is the appearance characteristic of a leaf disc after different cotton varieties are inoculated with verticillium dahliae V991 spore endotoxin; b is the fluorescence change of FV/FM after different cotton varieties are inoculated with verticillium dahliae V991 spore endotoxin; c is the fluorescence change of endotoxin NO of spore after different cotton varieties are inoculated with verticillium dahliae V991; d is the fluorescence change of Y (II) after different cotton varieties are inoculated with verticillium dahliae V991 spore endotoxin; e is the fluorescence change of NPQ after different cotton varieties are inoculated with verticillium dahliae V991 spore endotoxin; f is the fluorescence intensity value.

FIG. 3 shows the change of day of chlorophyll fluorescence imaging after inoculation of endotoxin from Verticillium dahliae V991 spore with different cotton varieties.

FIG. 4 is a graph showing the change of the fluorescence parameters of cotton variety after inoculation of endotoxin from Verticillium dahliae V991 spore.

FIG. 5 is a statistical chart showing disease index of cotton of different varieties treated by different dyeing sources.

Detailed Description

Strain material:

the cotton verticillium wilt pathogenic bacteria adopted in the invention: verticillium daliae Kleb, a common strain, is publicly available from the plant protection institute of the Chinese academy of agricultural sciences.

Cotton variety: all cotton varieties (lines) of Xiang cotton No. 13, 2890, Zhongmian cotton No. 12, Liao cotton No. 15 and Yu cotton No. 1 used for the test are provided by the institute of economic crops of Xinjiang academy of agricultural sciences, and the cotton variety used by the conidium root soaking method and the toxin disc infection method is Liao cotton No. 15.

The Chachi (Czapek) medium (1L) formulation method employed in this application: weighing 30g of glucose and NaNO₃2 g，K₂HPO₄·3H₂O 1g，KCl 0.5g，MgSO₄·7H₂O 0.5g，FeSO₄·7H₂0.01g of O is poured into 1L of deionized water in sequence, and after the O is fully dissolved, the mixture is sterilized for 15min at high temperature and high pressure.

The preparation method of the 5mmol phosphate buffer solution mother liquor (1L) adopted in the application comprises the following steps: 0.78g NaH was weighed₂PO₄·2H₂O, adding deionized water to constant volume of 1L, and weighing 1.79g of Na₂HPO₄·2H₂And O, and the volume is increased to 1L by deionized water.

The preparation method of 5mmol of phosphate buffer solution pH 7.0(1L) adopted in the application comprises the following steps: respectively measuring 610mLNaH₂PO₄·2H₂O，390mLNa₂HPO₄·2H₂And O, and the volume is increased to 1L by deionized water.

The reagents and materials can be purchased through public channels, and the equipment and instruments adopted in the process are common equipment in the field.

In the test treatment, data analysis and processing are performed by using Microsoft Excel, data analysis and drawing are performed by using Sigmaplot 14.0 software, and pictures are processed by Photoshop 2017.

All materials, reagents and equipment and data processing methods selected for use in the present invention are well known in the art, but do not limit the practice of the present invention, and other reagents and equipment well known in the art may be suitable for use in the practice of the following embodiments of the present invention.

The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

Example 1: method for rapidly screening verticillium wilt-resistant cotton varieties

A method for rapidly screening a verticillium wilt-resistant cotton variety comprises the following steps:

(1) preparing conidium suspension of verticillium dahliae V991 as pathogen of cotton verticillium wilt, and extracting endotoxin from verticillium dahliae V991 as pathogen of cotton verticillium wilt.

(2) In the full-bloom stage of cotton, 20 cotton plants are selected from each strain, the third last leaf of each strain is taken, 20-30 leaf discs with the diameter of 1.0cm are punched on the leaves by a puncher, then the leaf discs are immersed into 50mL of spore endotoxin solution obtained in the step (1) with the concentration of 16 mu g/mL, the spore endotoxin solution is cultured for 24 hours and then taken out, the surface attachment liquid is sucked dry by absorbent paper, and the disease index is counted by taking the yellowing area of the leaf discs as a grading basis.

(3) Dark adaptation is carried out on the leaf discs processed in the step (2) for 30min, then the leaf discs are placed on a chlorophyll fluorescence imaging system sample stage, an area of interest AOI with the diameter of 0.5-1.0cm is set on each leaf disc, chlorophyll fluorescence imaging pictures of the leaf discs inoculated with endotoxin are collected, and the kinetic change curve of chlorophyll fluorescence parameters of each leaf disc is detected.

(4) Counting analysis data, analyzing the relation between each index change of the fluorescence parameters and the verticillium wilt resistant cotton variety, determining the key influence factors for screening the verticillium wilt resistant cotton variety, establishing an evaluation system for screening the verticillium wilt resistant cotton variety, and screening the verticillium wilt resistant cotton variety.

Wherein, the preparation process of the conidium suspension in the step (1) is as follows: inoculating cotton verticillium wilt fungus cake with diameter of 0.5cm into Czapek's culture solution sterilized at 121 deg.C for 15min, and shake-culturing in a shaker at 25 deg.C and rotation speed of 130rpm/min for 5 days until conidia have cluster structure on the wall of triangular flask; filtering the liquid obtained by culturing through a steel sieve with 140 meshes and 400 meshes in a double layer manner to obtain a spore suspension; centrifuging the obtained spore suspension at room temperature at 14000rpm/min for 10min, collecting precipitate, washing the collected precipitate with sterilized water, centrifuging at 14000rpm/min for 10min, collecting precipitate, adding into 0.05M phosphate buffer solution with pH of 6.5, suspending the precipitate, and adjusting the spore concentration of the obtained suspension to 10⁷Per mL;repeatedly grinding the spore suspension by a homogenizer at 120Mpa and 10 deg.C for 3-4 times, centrifuging at 4 deg.C and 18000rpm/min for 25min, discarding supernatant, and filtering with 0.45 μm microporous membrane to remove mixed bacteria to obtain filtrate as spore endotoxin.

Example 2: test for determining key influence factors for screening verticillium wilt-resistant cotton varieties

This example is based on example 1 and examined the key influencing factors for screening cotton varieties (lines) resistant to (resistant to) verticillium wilt.

1. Test method

A conidium root soaking method and a spore endotoxin leaf soaking disc method are adopted to count the disease resistance index of cotton, and a spore endotoxin soaking disc chlorophyll fluorescence imaging picture is collected to detect the dynamics change curve of each leaf disc chlorophyll fluorescence parameter.

2. Detecting the index

(1) Index of disease statistics

Investigating the incidence of verticillium wilt of cotton by a conidium root-dipping water culture method, investigating in 15 days, counting according to 5-level system of incidence of seedling stage of greenhouse cotton, calculating disease index, dividing the incidence type of verticillium wilt, and grading standard: grade 0-healthy plant, no symptoms; 1 grade-1 to 2 pieces of cotyledon; grade 2-1 true leaf onset; more than 3 grades-2 true leaves are attacked or fall off, and only heart leaves are left; grade 4-plant growing point or whole plant withered.

Investigating the disease index of the cotton leaf disc by taking each cotton leaf disc as a unit and adopting a comprehensive investigation method, wherein the disease index is classified as follows: level 0: the leaves are not sick; level 1: the lesion spots account for 1-25% of the whole leaf area, and brown spots appear on the leaves; and 2, stage: the lesion area in the whole leaf area accounts for 26-50%, and the leaves turn yellow or have part of brown lesions; and 3, level: the lesion area in the whole leaf area accounts for 51-74%, and the leaf turns yellow or has most brown lesions; 4, level: the lesion area in the whole leaf area accounts for 75%, and the leaves are basically yellow or brown. The breed resistance was classified according to disease index: disease means 0, immune; disease refers to less than or equal to 10, high resistance; disease resistance means 10.1-20.0; disease refers to 20.1-35.0 resistant disease, and disease refers to more than 35.1 susceptible disease.

(2) Chlorophyll fluorescence imaging parameter statistics

After dark adaptation is carried out on cotton leaf discs for 30min, the cotton leaf discs are placed on a sample stage of a chlorophyll fluorescence imaging system, an area of interest AOI with the diameter of 0.5-1.0cm is set on each leaf disc, and a dynamics change curve of chlorophyll fluorescence parameters of each leaf disc is detected in a Kinetics window of software and can be derived from a Report window. Measuring chlorophyll fluorescence parameters such as the maximum light quantum yield (Fv/Fm) of a light system II, the actual light quantum yield (Y (II)), the non-photochemical quenching coefficient (NPQ), the non-adjustable energy dissipation quantum yield (Y (NO)), and the like by adopting a chlorophyll fluorescence imaging system, and analyzing the fluorescence parameters and pictures.

3. Data analysis

(1) Reliability identification of verticillium wilt-resistant cotton varieties by spore endotoxin soaking leaf disc method

The verticillium wilt resistant variety of cotton is identified by respectively adopting a conidium root soaking method and a spore endotoxin leaf soaking disc method, as shown in attached figure 1, 2890, in the identification of a water culture method and a disc method, the index of the disease of the water culture method is 13.3, the index of the disease of the disc method is 16.6 and is within 20, the variety can be identified as the resistant variety according to the identification standard of the resistance of the cotton variety, Xiang cotton No. 13 is identified in the identification of the water culture method and the disc method, the index of the disease of the water culture method is 28.3, the index of the disease of the disc method is 33.3 and is within 21-35, the variety can be identified as the resistant variety according to the identification standard of the resistance of the cotton variety, Yu cotton No. 1 is identified as the index of the water culture disease of 53.3, the index of the disease of the disc method is 56.25, Liaoling cotton No. 15 is 76.6, the index of the disease of the disc method is 72.91, the index of the disease of the medium cotton No. 12 is 73.3, the index of the disease of the water culture method is 54.16, the index of the disease of the disc method is 54.16, the resistance of the cotton variety can be identified as the sensitive variety, the intracellular toxin extracted by verticillium dahliae V991 is consistent with the resistance of a conidium root soaking method to cotton verticillium wilt, and the method is suitable for identifying the verticillium wilt-resistant cotton variety.

(2) Appearance characteristics of leaf discs and chlorophyll fluorescence imaging changes of different cotton varieties after inoculation of verticillium dahliae V991 intracellular toxins

Observing the appearance characteristics of the leaf discs and the chlorophyll fluorescence imaging changes of different cotton varieties inoculated with verticillium dahliae V991 intracellular toxins, as shown in figure 2, observing that after the different cotton varieties are infected by verticillium dahliae V991 intracellular toxins for 3d, comparing the different cotton varieties with the infected 1d, the disease-resistant varieties 2890 have obvious differences, NO matter the disease-resistant varieties are observed under a scanner or analyzed, the healthy tissue forms are maintained, the disease-resistant varieties Xiang cotton No. 1, the toxin infection 3d, the leaf disc appearance and Fv/Fm, Y (II), NPQ fluorescence parameter images have NO obvious changes, while the fluorescence images Y (NO) have obvious changes in the 3d color, the disease-sensitive varieties Yu cotton No. 1, Zhongmian cotton No. 12, the Liaomian cotton No. 15 Fv/Fm, Y (II), NPQ and NO four fluorescence images have obvious changes in the 3d, wherein the Zhongmian cotton No. 12, as the toxin infection time is prolonged, the Fv/Fm, Y (II), NPQ, Y (NO) fluorescence image response becomes weaker, and after 5d of toxin infection, the Fv/Fm, Y (II), NPQ and even fluorescence response become 0 gradually. Therefore, four fluorescence images of Fv/Fm, Y (II), NPQ, Y (NO) can be used as a mode for identifying the verticillium dahliae resistant varieties by using the verticillium dahliae intracellular toxin method.

(3) Change of chlorophyll fluorescence parameters after inoculation of verticillium dahliae V991 spore endotoxin on different cotton varieties

After the cotton leaf disc is infected by cotton verticillium wilt V991 toxin, as shown in figure 3, the variation range of the maximum light quantum yield (Fv/Fm) of the photosystem II of each cotton variety is 0.50-0.76, wherein the disease-resistant variety and the disease-resistant variety are fluctuated in a microwave wave mode in the range of 0.72, the disease-sensitive variety Liaomian No. 15, Yumian No. 1 and Miao No. 12 are large in amplitude, and are fluctuated in a large wave mode in the range of 0.5-0.76, and the Mimian No. 12 is even decreased in a gradient mode. The actual maximum light quantum quantity Y (II) of each cotton variety has a variation range of 0.10-0.27, wherein the fluctuation of the Liaomian cotton No. 15 is most obvious, like an italic Z shape, the Liaomian cotton No. 3d firstly presents an ascending trend, quickly descends after 5-7d, fluctuates in a wave form of 9-11d, the descending slope of the disease-resistant variety 2890 and the Hunan cotton No. 13 infected for 7 days is smaller than that of the susceptible variety, and the disease-resistant variety can present an obvious spoon shape after 11d infection, the spoon handle formed after 1-7d is gentle, and the susceptible variety has no characteristics.

The variation range of the non-photochemical quenching coefficient (NPQ) of each cotton variety (line) is 0.07-0.26, wherein the disease-resistant variety 2890 and the disease-resistant variety Xiang cotton No. 13 fluctuate above and below 0.13 and present a W shape after being infected for 11 days, a central point in the W shape is formed after being infected for 5 days, the fluctuation range of the disease-sensitive variety is large, and the fluctuation range cannot form a compact W shape.

The quantum yield Y (NO) of the non-regulatory sexual power dissipation of the photosystem II is 0.43-0.81, the disease-resistant variety 2890 and the disease-resistant variety Xiang cotton No. 13 fluctuate from 0.43 to 0.66, an obvious M type is formed after infection for 9 days, a central point of M is formed after infection for 5 days, while the cotton 12 in the disease-sensitive variety can also form an obvious M type after infection for 9 days, but the central point of M can be formed after infection for 7 days and fluctuates in the range of 0.57-0.72, while other disease-sensitive varieties are in oscillation fluctuation in the range of 0.49-0.81.

(4) The change of the fluorescent light of the cotton variety after inoculation of verticillium wilt endotoxin for a plurality of days is used as the basis for screening the disease-resistant cotton variety

In the previous studies, photosystem II actual maximum light quantity Y (II) decreases more gradually during 11 days of toxin infestation of the disease-resistant variety, and forms a scoop shape in the first 7 days. The quantum yield Y (NO) of non-regulatory performance dissipation of photosystem II can form a compact W type, the non-photochemical quenching coefficient (NPQ) can form an M type, the three characteristics can be used as characteristic curves for screening cotton varieties (lines) with resistance (anti) verticillium wilt, 2890 and Xiang cotton No. 13 are used as references of cotton varieties with resistance/anti verticillium wilt, and Zhongmian No. 12 is used as a reference of susceptible varieties, and Y (II), Y (NO) and NPQ fluorescence parameters which change every leaf disc day after cotton leaf discs of 18 varieties (lines) are infected by cotton verticillium wilt V991 toxin are compared as shown in figure 4, Ji cotton No. 8 meets the three basic characteristics, and other cotton varieties (lines) do not have the characteristics, so that Ji cotton No. 8 can be judged as the variety with resistance (anti) verticillium wilt.

The above tests show that: the key influencing factors for screening the cotton variety (line) resisting (resistant) verticillium wilt are three characteristic curves of the actual light quantum yield Y (II) of a photosystem II, the quantum yield Y (NO) of non-adjustable energy dissipation and the non-photochemical quenching coefficient (NPQ).

Example 3: verification of identification of anti-verticillium wilt cotton varieties by different screening methods of anti-verticillium wilt cotton varieties

In order to further verify that the change of the fluorescent light of the cotton varieties after inoculation of verticillium wilt spore endotoxin is used as the basis for screening disease-resistant cotton varieties, the verticillium wilt resistance identification of the 18 cotton varieties is carried out by respectively adopting a conidium root soaking method and a intracellular endotoxin leaf soaking disc method and adopting a conventional disease index procedure, as shown in the attached drawing 5, the disease indexes of No. 8 Ji cotton in the identification of the water culture method and the disc method are 21.6667 respectively, 25 respectively and within 35 respectively, the disease indexes can be identified as disease-resistant varieties according to the identification standards of the resistance of the cotton varieties, and the disease indexes of other cotton varieties (lines) are all above 35 and are all susceptible varieties. According to the disease index identification method, the result of cotton resistance identification by spore endotoxin leaf discs and conidium root soaking technology is highly consistent with the cotton variety resistance identification result obtained by the change of the fluorescence parameters day. Therefore, the change of the cotton variety fluorescence parameters after inoculation of verticillium wilt endotoxin can be used as the screening basis of the disease-resistant cotton variety.

In summary, the method for rapidly screening the cotton variety with verticillium wilt resistance provided by the invention comprises the steps of inoculating a leaf disc with verticillium dahliae V991 spore endotoxin, determining the identification of the cotton variety with verticillium wilt resistance by counting the disease resistance index of the leaf disc, statistically analyzing the relation between a fluorescence imaging picture and the change of fluorescence parameters of the leaf disc and the cotton variety with verticillium wilt resistance, and finally determining the three characteristic curves of the actual light quantum yield Y (II), the non-adjustable energy dissipation quantum yield Y (NO) and the non-photochemical quenching coefficient (NPQ) of a light system II as the basis for screening the cotton variety, and further verifying and finding that the result of identifying the cotton resistance by a spore leaf disc and a conidium root soaking technology is highly consistent with the cotton variety resistance identification result obtained by the change of the fluorescence parameters day, which shows that the method for rapidly screening the cotton variety with verticillium wilt resistance provided by the invention is simple, the method is reliable, solves the problem of uneven identification results caused by poor spore growth consistency in the method for screening the verticillium wilt-resistant cotton varieties by using a conidium suspension root soaking method, and provides a new toxin soaking source for the method for identifying the verticillium wilt resistance of cotton.

The above examples are merely illustrative for clearly illustrating the present invention and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications can be made while remaining within the scope of the present invention.