CN114231588B - Wheat stem-based rot resistance identification method - Google Patents
Wheat stem-based rot resistance identification method Download PDFInfo
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Abstract
The invention provides a method for identifying the resistance of wheat stem basal rot, and relates to the technical field of disease resistance detection. The identification method of the invention can perform phenotype identification 15 days after injection (or 22 days after planting) by injecting spore suspension into wheat seedling stems growing in a greenhouse for FCR inoculation and determining optimal inoculation concentration and injection time point. The identification method of the invention is to inoculate FCR by directly injecting spore suspension into wheat seedling stem, and disease evaluation can be completed within 22 days according to inoculation concentration and injection time point. The identification method can obviously improve screening efficiency and is beneficial to large-scale germplasm screening and gene function research.
Description
Technical Field
The invention belongs to the technical field of resistance detection, and particularly relates to a method for identifying resistance of wheat stem basal rot.
Background
Stem rot (FCR) is a serious soil borne fungal disease in many wheat growing areas of the world. Typical symptoms of FCR include the stem of the infected plant becoming tan and the formation of white ears at the maturity stage due to stalk necrosis affecting the grouting. FCR can be caused by a variety of fusarium fungi, with fusarium pseudograminearum being the most common pathogen. Studies have shown that most wheat varieties worldwide are poorly resistant to FCR, with only a few genotypes exhibiting moderate resistance to the disease. FCR can result in significant yield loss in the wheat producing area.
Reliable FCR assessment methods are critical for the identification of resistant germplasm and host resistance studies. There are two types of tests that are currently common depending on the test environment: field-based resistance identification and greenhouse-based resistance identification. Field trials are often affected by a variety of factors, such as the population of resident pathogens in the soil, the type of soil, and weather conditions, etc., which can significantly affect FCR resistance, and field trials are typically only assessed once in one growing season, for long periods.
Conventional greenhouse-based identification methods include placing a spore suspension on the stems of seedlings (stem-based droplets), soaking seedlings of 1 to 4 days old in the spore suspension (seedling soaking), and planting wheat seeds in soil with a fungus soil covered on the surface layer for waiting for the disease to develop, and evaluating seedling resistance. Compared with field tests, the greenhouse test has higher repeatability, environmental factors influencing disease development are easier to control, and the greenhouse test has wider application in screening of wheat stem-based rot resistance sources and genetic research. However, these greenhouse identification methods require about 45 days from seed germination to complete resistance identification. Therefore, the improvement of the existing FCR inoculation identification method and the shortening of the identification period are of great significance to the research of wheat stem basal rot.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for identifying the resistance of wheat stem rot, which has high effectiveness and good repeatability, and can eliminate germplasm with poor sensitivity to FCR in early stages.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for identifying the resistance of wheat stem basal rot, which comprises the following steps: an identification method for detecting the resistance of wheat stem basal rot, which comprises the following steps: the spore suspension of Fusarium was injected in one-leaf one-heart period at a concentration of 10 7 And each mL.
Preferably, the wheat comprises hexaploid wheat.
Preferably, the injecting comprises injecting the spore suspension into the stems of greenhouse-cultured wheat seedlings, and stopping the injection after the tops of the seedlings overflow the spore suspension.
Preferably, the injected stems are located 0.4 to 0.7cm high from the soil surface.
Preferably, the evaluation of the resistance to stalk rot is performed 15d after the injection.
Preferably, the preparation method of the spore suspension comprises the following steps: culturing spores of fusarium in a carboxymethyl cellulose culture medium, and mixing with tween 20 to obtain a spore suspension;
the volume of tween 20 was 0.5% of the volume of the spore suspension.
Preferably, the wheat is judged to be susceptible or resistant according to the disease index.
Preferably, the method of assessing comprises calculating a disease index based on the severity of stem basal rot;
the severity of the stem basal rot is expressed as 0 to 6, wherein: 0 represents asymptomatic or small brown spots around the injection site; 1 represents that the leaf sheath of 1 has obvious browning, but is less than 1/4 of the total leaf length; 2 represents a browning area exceeding 1/2 of the length of the first leaf sheath; 3 represents complete browning necrosis of leaf sheath 1; 4 indicates that the second leaf sheath has obvious browning necrosis; 5 indicates that the 3 rd leaf sheath has obvious browning necrosis; 6 represents severe to complete necrosis of the whole plant;
the disease index di= (Σnx/6N) ×100, formula I; where X is a scale of severity, N is the number of severity levels, and N is the total number of tests performed on the same lot.
The invention also provides application of the identification method in resistance breeding.
The invention also provides application of the identification method in screening of the wheat germplasm resisting the stem basal rot.
The beneficial effects are that: the invention provides a wheat stem rot resistance identification method, wheat seedlings growing in a greenhouse are inoculated with FCR by injecting spore suspension into seedling stems, and phenotype identification can be carried out at 15d after injection (or 22d after planting) by determining inoculation concentration and injection time point. In the embodiment of the invention, 36 in the disease-resistant material 04 (04 z36 for short) and new wheat 26 of the disease-resistant material (XM for short) are utilized to verify the effectiveness of the identification method; the reproducibility of the identification method was also verified using multiple wheat germplasm.
The identification method of the invention is to inoculate FCR disease by directly injecting spore suspension into wheat seedling stem, and disease evaluation can be completed within 22 days according to inoculation concentration and injection time point. The identification method can obviously improve screening efficiency and is beneficial to large-scale germplasm screening and gene function research.
Drawings
FIG. 1 is a schematic diagram of stages 0-6 of FCR;
FIG. 2 is the effect of inoculation concentration and injection time on the severity of stem basal rot in 04z36 and XM seedlings, where a: injection in the period of one leaf; b: injecting in a leaf-heart period; c: injecting in a period of two leaves and one heart; each group of data in each bar graph is 04z36 and XM in sequence from left to right;
FIG. 3 is a sample paraffin section taken on day 15 after injection of 04z36 and XM shoot at the leaf-by-core stage, where a: unvaccinated 04z36 plants; b: non-inoculated XM plants; c:04z36 plants were injected with CMC; d: injecting CMC into XM plants; e:04z36 plant injection 10 7 Spore suspension; f: XM plant injection 10 7 Spore suspension; scale bar, 400 μm;
fig. 4 is a correlation study of the degree of root rot of two wheat varieties, the correlation coefficient (r=0.95), and is extremely remarkable (p < 0.01).
Detailed Description
The invention provides a method for identifying the resistance of wheat stem basal rot, which comprises the following steps: the spore suspension of Fusarium was injected in one-leaf one-heart period at a concentration of 10 7 And each mL.
The wheat of the present invention preferably includes hexaploid wheat, and the source of the germplasm resources of the wheat is not particularly limited, and may be a conventional commercial variety or a newly cultivated variety (line). In the embodiment of the invention, the effectiveness of the identification method is verified by using 36 in the commercial disease-resistant material 04 and the new wheat 26 of the disease-resistant material; the validity and reproducibility of the identification method were also verified using 92 hexaploid wheat introduction lines derived from 21 parts of wild two-grain wheat, 04z36, cscr6, handan 87-1 and XM.
The construction method of the hexaploid wheat derived from 92 wild two-grain wheat comprises the following steps: 21 parts of wild two-grain wheat collected from israel is used as a female parent, hybridized with Chinese hexaploid wheat variety Handan-1 respectively, backcrossed for 2-3 generations with Handan-87-1, and then the offspring is selfed for 6 generations until most of agronomic characters are fixed.
The injection according to the invention preferably comprises injecting the spore suspension into the stem of the seedling, and stopping the injection after the top of the seedling overflows the spore suspension. The location of the injected stems according to the invention is preferably 0.4 to 0.7cm high, more preferably 0.5cm high, from the soil surface. In the invention, histological observation can be carried out 15d after injection, and evaluation of stem basal rot can be carried out, so as to determine that wheat is susceptible or resistant.
The preparation method of the spore suspension of the invention preferably comprises the following steps: culturing spores of fusarium with carboxymethyl cellulose (CMC), and mixing with tween 20 to obtain spore suspension; the volume of tween 20 was 0.5% of the volume of the spore suspension.
The type of the fusarium is not particularly limited in the present invention, and the fusarium pseudograminearum isolate WZ-8A (Jin, identifcation of a novel genomic region associated with resistance to Fusarium crown rot in heat.2020) used in the examples is not limited to the whole scope of the present invention. In the examples of the present invention, it is preferable that the WZ-8A is cultured on a 1/2 potato dextrose agar plate at room temperature for 5 to 7 days, and when white hyphae grow on the plate and show red pigmentation, 10 pieces of the 6mm X4 mm medium with the hyphae are taken out from the plate, and then transferred to a conical flask containing 400mL of carboxymethyl cellulose medium; the conical flask was placed in a shaker at 160rpm and 25℃for about 72 hours, spores were collected and the concentration of the spore suspension was adjusted. Tween 20 was added to the spore suspension prior to use for inoculation, at a final concentration of 0.5% v/v.
The invention carries out histological observation 15d after the spore suspension is injected, and carries out evaluation of stem basal rot, and the wheat variety is determined to be susceptible or resistant. The method of assessment of the present invention preferably comprises calculating a disease index based on the severity of stem basal rot;
the severity of the stem basal rot is represented at 0 to 6 (as in fig. 1), wherein: 0 represents asymptomatic or small brown spots around the injection site; 1 represents that the leaf sheath of 1 has obvious browning, but is less than 1/4 of the total leaf length; 2 represents a browning area exceeding 1/2 of the length of the first leaf sheath; 3 represents complete browning necrosis of leaf sheath 1; 4 indicates that the second leaf sheath has obvious browning necrosis; 5 indicates that the 3 rd leaf sheath has obvious browning necrosis; 6 represents severe to complete necrosis of the whole plant;
the disease index di= (Σnx/6N) ×100, formula I; where X is a scale of severity, N is the number of severity levels, and N is the total number of tests performed on the same lot. The method for histological observation is not particularly limited, in the embodiment, the observation is performed by a paraffin tissue section method, tissue staining is performed by 1% safranine and 0.5% fast green, the xylem cell wall is stained red, cellulose is stained green, and different influences of fusarium pseudograminearum on the anti-influenza material are observed at the cell level.
In the invention, when the disease index is less than or equal to 20, the disease resistance is realized; the neutralization resistance is more than 20 and less than or equal to 30; the neutral feeling is more than 30 and less than or equal to 40; > 40 is susceptible to disease.
The invention also provides application of the identification method in resistance breeding.
In the present invention, a single germplasm resistance may interact with strains, so that when vaccinated, a selection of mixed strains from different geographical sources may be vaccinated, helping to determine the potential value of the target germplasm in a local FCR improved breeding program.
The identification method can greatly shorten the time for resistance evaluation from 45 days to 22 days (about 7 days from sowing to one leaf and one heart period), remarkably improve screening efficiency, is beneficial to large-scale germplasm screening and gene function research, and can screen germplasm for FCR (human immunodeficiency virus) infection in early development by verifying 92 parts of hexaploid wheat derived from wild two-grain wheat.
The invention also provides application of the identification method in screening of the wheat germplasm resisting the stem basal rot.
The application of the present invention is preferably the same as that described above, and will not be described again here.
The following describes in detail a method for identifying resistance to wheat stem rot and application thereof with reference to examples, but they should not be construed as limiting the scope of the invention.
Example 1
1. Plant material
The best identification method is determined by taking 36 (04 z 36) and new wheat 26 (XM) in medium-resistance FCR and high-sensitivity FCR wheat variety 04 as materials. 92 hexaploid wheat introduction lines derived from wild two-grain wheat are used as materials, and the effectiveness and the repeatability of the optimized identification method are verified.
2. Preparation of inoculant liquid
Fusarium pseudograminearum isolate WZ-8A of dominant pathogenic species in Huang-Huai wheat regions of China provided by Li Hongli Hongyuan professor of Henan agricultural university is utilized for FCR inoculation. The isolate was incubated on 1/2 strength potato dextrose agar plates for 5-7 days at room temperature. When white hyphae grew on the plate and showed red pigmentation at the bottom, 10 pieces of 6mm×4mm medium with mycelium were removed from the plate and transferred to a conical flask containing 400mL of carboxymethyl Cellulose Medium (CMC). The flask was incubated in a shaker at a speed of 160rpm and a temperature of 25℃for about 72 hours. Spores were collected and the concentration of the spore suspension was adjusted to meet experimental requirements. Tween 20 was added to the spore suspension prior to use for inoculation, at a final concentration of 0.5% v/v.
3. Improved inoculation method
Four inoculation concentrations and three seedling ages were designed for a total of 12 treatments, the inoculation concentrations were: 10 4 、10 5 、10 6 And 10 7 spores/mL, one leaf stage, one leaf one core stage (first leaf fully expanded and second leaf just appeared with leaves still curled) and two leaf one core stage (first and second leaf fully expanded and third leaf just appeared with leaves curled) of 04z36 and XM seedlings, respectively, each treatment contained four replicates, each replicate using ten seedlings per variety.
Seeds of 04z36 and XM were first surface sterilized with 75% ethanol, rinsed twice with sterile water, and germinated in petri dishes at 25 ℃ for about three days. After germination, seedlings were planted in 4cm square seedling trays containing autoclaved soil. The seedling culture trays are arranged in a greenhouse of Chinese agricultural university according to random group, the illumination period is 16 hours, the darkness period is 8 hours, and the day and night temperatures are 25+/-2 ℃/20+/-2 ℃ respectively. At the time of inoculation, the spore suspension was injected into the seedling stems using a 10mL needle syringe (0.7 mm outer diameter). The injection site of the stems was about 0.5cm from the soil surface. Injection is stopped when the suspension flows out from the top of the seedling. Control plants were inoculated with CMC solution only.
The severity of FCR was assessed when the susceptibility genotype "XM" was severely necrotic. The evaluation can be made at the earliest 15 days after inoculation (22 days after planting) according to the development of disease symptoms of seedlings. The severity of FCR was assessed using the phenotypic grade indicators formulated in the present invention and divided into 6 grades, where 0 indicates no symptoms or small brown spots around the injection site; 1. the 1 st leaf sheath is significantly browned (but less than 1/4 of the total leaf length); 2. the browning area exceeds 1/2 of the length of the first leaf sheath; 3. complete browning and necrosis of leaf sheath 1; 4. the second leaf sheath has obvious necrotic brown lesions; 5. the 3 rd leaf sheath has obvious necrotic brown lesions; 6. the whole plant was severely to completely necrotized. Then, grading the disease index DI= (ΣnX/6N) multiplied by 100 according to the disease index, wherein the formula I is shown in the specification; where X is a scale of severity, N is the number of severity levels, and N is the total number of tests performed on the same lot. Analysis of variance (ANOVA) was performed using IBM SPSS 22.0 software.
At 15 days post injection, histological observations were performed: the middle of the infected area on the seedling stems was harvested and fixed overnight with formalin-acetic acid-ethanol solution (FAA) at 4 ℃. After dehydration in ethanol and xylene, the tissues were embedded in paraffin. Tissue sections were cut into 5mm slices and samples were visualized using a microscopic imaging system (DS-U3, nikon, japan) with 1% saffron and 0.5% fast green (G1031, https:// www.servicebio.cn /).
Analysis of variance showed that both the inoculation concentration and the injection time had a significant effect on FCR severity (table 1). The disease severity of 04z36 and XM increased linearly with increasing molecular spore suspension concentration and with delay in injection time (fig. 2). After injection of a high concentration suspension (e.g. 10 6 Or 10 7 spores/mL) 2-3 days after injection of the FCR sensitive cultivar XM seedling stemsSmall browning points were observed.
TABLE 1 analysis of variance of severity of stem basal rot in 04z36 and XM seedlings
| Classification | Degree of freedom | Mean square | |
| Material | |||
| 1 | 10092.1 | ＜0.001 | |
| |
3 | 9887.1 | ＜0.001 |
| Period of |
2 | 2342.1 | ＜0.001 |
| Material |
3 | 1734.4 | ＜0.001 |
| Material x period of |
2 | 105.2 | ＜0.001 |
| Inoculation concentration |
6 | 312.7 | ＜0.001 |
| Material x inoculation concentration |
6 | 25.1 | ＜0.001 |
| Residual error | 72 | 3.8 | |
| Totals to | 96 |
Of the 12 treatments, 04z36 and XM seedlings were injected 10 7 The concentration of the spores per mL is 10 in the two-leaf one-heart injection 7 Individual spores/mL of spore suspension produced the most severe FCR symptoms: the disease index of 04z36 and XM reached 45.2 and 93.5 at 12 days post injection (about 20 days post-implantation), respectively; the disease indices of 04z36 and XM were 27.1 and 68.8, respectively, by one-leaf one-heart injection.
When the injection concentration is 10 4 spores/mL 04z36 and XM seedlings when assessed for FCR resistance 10 days after injection, suspensions of one leaf phase produced the lowest disease index (04 z36 of 2.3 and XM of 5.4) of the 12 treatments.Control seedlings injected with CMC solution did not show FCR symptoms (table 2).
TABLE 2 disease index under different treatments
One-leaf one-heart injection 10 7 spore/mL suspensions are considered the best vaccination method because of their relatively high FCR severity and better ability to distinguish between disease resistant and disease susceptible genotypes.
To verify the effectiveness of this method, stem tissue of 04z36 and XM seedlings was injected 10 a leaf-by-leaf heart time period 7 The spore/mL suspension was used for histocytology analysis. No significant difference was observed between 04z36 and XM seedlings without or with CMC solution injected only (a, b, c, d in fig. 3). In contrast, during FCR infection, damaged seedling stem structures infused with XM and 04z36 plants were observed. Xylem cell walls (stained red) and cellulose (stained green) were observed to be severely degraded in XM seedlings, while slight degradation was also observed in 04z36 seedlings (e, f in fig. 3).
4. Optimizing the effectiveness of vaccination methods
The effectiveness of the optimized vaccination procedure was evaluated based on the determined reproducibility using a collection of 92 wild-type, two-grain wheat-derived hexaploid wheat lines and 4 wheat genotypes of known FCR resistance levels (including Cscr6 and 04z 36). Two trials were performed, 10 seedlings per genotype in each trial.
The correlation coefficients of these two experiments were calculated using Microsoft Excel. In addition, genotypes exhibiting better FCR resistance in the first trial were used in the second trial to confirm that they had better resistance levels.
There was a high significance (p < 0.01) and strong correlation (r=0.95) between the FCR severity of the two trials (fig. 4). For hexaploid wheat importation lines constructed using wild-type two-grain wheat, the average severity of the FCR was between 28.1 and 88.5, with an overall average of 59.0 (table 3). The average disease index of the resistant controls 04z36 and Cscr6 was 27.8 and 23.4, respectively, and the average disease index of the back-crossed parent Handan 87-1 and the sensitive control XM was 59.8 and 68.4, respectively.
TABLE 3 wheat genotype resistance verification test against FCR
Table 4 2 disease index of wheat genotypes exhibiting consistent resistance to FCR
It was verified that co-screening to two resistant germplasm, including two hexaploid wheat derived based on wild two-grain wheat (LR-43 and LR-61), exhibited similar levels of disease resistance as disease resistant controls 04z36 and Cscr 6. In two rounds of disease assessment, the average disease index for the two resistant germplasm was 28.8, 28.1, similar to that of Cscr6 (23.4) and 04z36 (27.8).
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (7)
1. A method for identifying the resistance of wheat stem basal rot, which comprises the following steps: the spore suspension of Fusarium was injected in one-leaf one-heart period at a concentration of 10 7 individual/mL;
the preparation method of the spore suspension comprises the following steps: culturing spores of fusarium in a carboxymethyl cellulose culture medium, and mixing with tween 20 to obtain a spore suspension;
the volume of tween 20 was 0.5% of the volume of the spore suspension;
the injection comprises injecting the spore suspension into stems of wheat seedlings cultured in a greenhouse, and stopping the injection after the tops of the seedlings overflow the spore suspension;
identification of resistance to stalk rot was performed 15 days after the injection.
2. The identification method of claim 1, wherein the wheat comprises hexaploid wheat.
3. The method of identification of claim 1, wherein the injected stems are located 0.4 to 0.7cm high from the soil surface.
4. The method of claim 1, wherein the wheat is determined to be susceptible or resistant based on the index of the disease.
5. The method of claim 4, wherein the method of identifying comprises calculating an index of disease based on the severity of stem basal rot;
the severity of the stem basal rot is expressed as 0 to 6, wherein: 0 represents asymptomatic or small brown spots around the injection site; 1 represents that the leaf sheath of 1 has obvious browning, but is less than 1/4 of the total leaf length; 2 represents a browning area exceeding 1/2 of the length of the first leaf sheath; 3 represents complete browning necrosis of leaf sheath 1; 4 indicates that the second leaf sheath has obvious browning necrosis; 5 indicates that the 3 rd leaf sheath has obvious browning necrosis; 6 represents severe to complete necrosis of the whole plant;
the disease index di= (Σnx/6N) ×100, formula I; where X is a scale of severity, N is the number of severity levels, and N is the total number of tests performed on the same lot.
6. Use of the identification method according to any one of claims 1 to 5 in resistance breeding.
7. Use of the identification method according to any one of claims 1 to 5 for screening wheat germplasm against stalk rot.
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