CN114231588A - Wheat stem basal rot resistance identification method - Google Patents
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Abstract
The invention provides a method for identifying resistance to 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 the optimal inoculation concentration and injection time point. The identification method of the invention inoculates FCR by directly injecting spore suspension into the stem of wheat seedling for the first time, and can complete disease evaluation within 22 days according to inoculation concentration and injection time point. The identification method provided by the invention can obviously improve the 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
Basal stem rot (FCR) is a serious soil-borne fungal disease in many wheat growing areas of the world. Typical symptoms of FCR include turning of the infected plant stem to tan and formation of white ears at the mature stage due to stalk necrosis affecting the filling. FCR can be caused by a variety of fusarium fungi, with fusarium pseudograminearum being the most common pathogenic bacterium. Studies have shown that most wheat varieties worldwide are poorly resistant to FCR, and only a few genotypes show moderate resistance to the disease. FCR can result in significant yield loss in wheat producing areas.
Reliable FCR assessment methods are crucial for identifying resistant germplasm and for host resistance studies. According to the experimental environment, two types are common at present: field-based resistance identification and greenhouse-based resistance identification. The field test is often influenced by various factors, such as resident pathogen population in soil, soil type, weather conditions and the like, which can significantly influence the FCR resistance, and the field test can be evaluated only once in one growing season, so that the period is long.
A conventional greenhouse-based identification method includes placing a spore suspension on a seedling stem (shoot base liquid drop), soaking 1 to 4-day-old seedlings in the spore suspension (seedling soaking), planting wheat seeds in soil covered with soil, and the like, and evaluating seedling resistance after onset. Compared with field tests, the greenhouse test has higher repeatability, environmental factors influencing the development of diseases are easier to control, and the method is widely applied to screening of wheat stem basal rot resistance sources and genetic research. However, these greenhouse identification methods take 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 have important significance for the research of the wheat stem basal rot.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for identifying resistance to wheat stem basal rot, which has high efficiency and good repeatability, and can eliminate germplasm with poor sensitivity to FCR at an early stage.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for identifying resistance to wheat stem basal rot, which comprises the following steps: an identification method for detecting resistance of wheat stem basal rot comprises the following steps: injecting a spore suspension of fusarium at a single leaf and single heart stage, wherein the concentration of the spore suspension is 107one/mL.
Preferably, the wheat comprises hexaploid wheat.
Preferably, the injection comprises injecting the spore suspension into the stem of a greenhouse-grown wheat seedling, and stopping the injection after the spore suspension overflows the top of the seedling.
Preferably, the height of the injected stem is 0.4-0.7 cm from the soil surface.
Preferably, the evaluation of the resistance to stalk-base rot is carried out 15d after said injection.
Preferably, the preparation method of the spore suspension comprises the following steps: culturing the spores of the fusarium by using a carboxymethyl cellulose culture medium, and mixing the spores with tween 20 to obtain the spore suspension;
the volume of tween 20 was 0.5% of the volume of the spore suspension.
Preferably, the wheat is susceptible or resistant according to the disease index.
Preferably, the method of assessment comprises calculating a disease index based on the severity of basal stem rot;
the severity of the stem base rot is expressed by 0-6, wherein: 0 indicates no symptoms or small brown spots around the injection site; 1 indicates 1/4 where the sheath of leaf 1 has significant browning, but less than the total leaf length; 1/2 showing browning area exceeding first sheath length; 3 indicates that the 1 st leaf sheath is completely browned and necrotic; 4, significant browning and necrosis of the second leaf sheath; 5 indicates that the 3 rd leaf sheath has obvious browning and necrosis; 6 indicates severe to complete necrosis of the whole plant;
the disease index DI ═ (Σ nX/6N) × 100, formula I; where X is the severity scale, N is the number of a certain severity, and N is the total number of tests performed on the same batch.
The invention also provides application of the identification method in resistance breeding.
The invention also provides application of the identification method in screening of wheat germplasm with stem rot resistance.
Has the advantages that: the invention provides a method for identifying the resistance of wheat stem basal rot, wherein wheat seedlings growing in a greenhouse are inoculated with FCR (fast cell response) by injecting a spore suspension into seedling stems, and the phenotype identification can be carried out 15d after injection (or 22d after planting) by determining the inoculation concentration and the injection time point. In the embodiment of the invention, 36(04z36 for short) in the disease-resistant material 04 and Xinmai 26(XM for short) as the susceptible material are used for verifying the effectiveness of the identification method; meanwhile, the repeatability of the identification method is verified by utilizing a plurality of wheat germplasms.
The identification method of the invention inoculates FCR disease by directly injecting spore suspension into the stem of wheat seedling for the first time, and can complete disease evaluation within 22 days according to inoculation concentration and injection time point. The identification method provided by the invention can obviously improve the 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 FCRs;
figure 2 is a graph of the effect of inoculation concentration and injection time on the severity of stem base rot in 04z36 and XM seedlings, where a: injection at one leaf stage; b: injecting in one-leaf one-heart stage; c: injecting in the two-leaf one-heart stage; each group of data in each histogram is 04z36 and XM from left to right in sequence;
fig. 3 is a sample paraffin section at day 15 after one-leaf one-heart injection of 04z36 and XM seedling stems, where a: an uninoculated 04z36 plant; b: uninoculated XM plants; c: 04z36 plant CMC; d: CMC is injected into XM plants; e: 04z36 plant injection 107A spore suspension; f: XM plant injection 107A spore suspension; scale bar, 400 μm;
fig. 4 is a correlation study of the degree of root rot of two wheat varieties, with a very significant correlation coefficient (r ═ 0.95) (p < 0.01).
Detailed Description
The invention provides a method for identifying resistance to wheat stem basal rot, which comprises the following steps: injecting a spore suspension of fusarium at a single leaf and single heart stage, wherein the concentration of the spore suspension is 107one/mL.
The wheat of the present invention preferably includes hexaploid wheat, and the source of the germplasm resource of the wheat of the present invention is not particularly limited, and may be a conventional commercially available variety or a newly bred variety (line). In the embodiment of the invention, the effectiveness of the identification method is verified by utilizing 36 of a commercially available disease-resistant material 04 and a disease-sensitive material Xinmai 26; meanwhile, 92 hexaploid wheat introduction lines derived from 21 wild emmer, 04z36, Csccr 6, Handan 87-1 and XM are used for verifying the effectiveness and repeatability of the identification method.
The construction method of 92 wild biddy wheat-derived hexaploid wheat provided by the embodiment of the invention comprises the following steps: 21 parts of wild two-grain wheat collected from Israel are used as female parents and respectively hybridized with a Chinese hexaploid wheat variety Handan 87-1, backcrossed for 2-3 generations by Handan 87-1, and then progeny 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 has overflowed the spore suspension. The height of the stem injected by the invention is preferably 0.4-0.7 cm, more preferably 0.5cm from the soil surface. In the present invention, histological observations can be made 15d after the injection and an assessment of basal stem rot can be made to determine whether wheat is susceptible or resistant.
The preparation method of the spore suspension preferably comprises the following steps: culturing the spores of the fusarium by using a carboxymethyl cellulose (CMC) culture medium, and mixing the spores with tween 20 to obtain a spore suspension; the volume of tween 20 was 0.5% of the volume of the spore suspension.
The species of the Fusarium is not particularly limited in the present invention, but is not to be considered as the full scope of the present invention, as exemplified by Fusarium graminearum isolate WZ-8A (Jin, identification of a novel genetic region associated with a resistance to Fusarium crown in wheat straw. 2020) used in the examples. In the present example, it is preferable that the WZ-8A is cultured on 1/2 potato dextrose agar plates for 5 to 7 days at room temperature, and when white hyphae grow on the plates and show red pigmentation, 10 sheets of 6mm by 4mm medium with the hyphae are taken out of the plates and then transferred to a conical flask containing 400mL of carboxymethyl cellulose medium; the Erlenmeyer flask was placed in a shaker at a speed of 160rpm and a temperature of 25 ℃ for about 72 hours, the spores were collected and the concentration of the spore suspension was adjusted. Tween 20 was added to the spore suspension at a final concentration of 0.5% v/v before use for inoculation.
The invention carries out histological observation 15 days after the spore suspension is injected, carries out evaluation on the stem basal rot and determines that the wheat variety is susceptible or resistant. The method of assessment according to the present invention preferably comprises calculating a disease index based on the severity of basal stem rot;
the severity of the stem base rot is expressed by 0-6 (as shown in figure 1), wherein: 0 indicates no symptoms or small brown spots around the injection site; 1 indicates 1/4 where the sheath of leaf 1 has significant browning, but less than the total leaf length; 1/2 showing browning area exceeding first sheath length; 3 indicates that the 1 st leaf sheath is completely browned and necrotic; 4, significant browning and necrosis of the second leaf sheath; 5 indicates that the 3 rd leaf sheath has obvious browning and necrosis; 6 indicates severe to complete necrosis of the whole plant;
the disease index DI ═ (Σ nX/6N) × 100, formula I; where X is the severity scale, N is the number of a certain severity, and N is the total number of tests performed on the same batch. The method for histological observation is not particularly limited, and in the embodiment, the observation is performed by a paraffin tissue section method, tissue staining is performed by using 1% safranine and 0.5% fast green, xylem cell walls are stained red, cellulose is stained green, and different influences of fusarium pseudograminearum on the influenza materials are observed at a cell level.
In the invention, when the disease index is less than or equal to 20, the disease is resistant; the medium resistance is more than 20 and less than or equal to 30; more than 30, the feeling is less than or equal to 40; if > 40, the disease is infected.
The invention also provides application of the identification method in resistance breeding.
In the present invention, a single germplasm resistance may interact with the strains, and therefore, mixed strains from different geographical sources may be selected for inoculation when performed, helping to determine the potential value of the target germplasm in a local FCR improved breeding program.
By utilizing the identification method, the resistance evaluation time can be greatly shortened from 45 days to 22 days (about 7 days from sowing to one leaf and one heart), the screening efficiency is obviously improved, the large-scale germplasm screening and gene function research are facilitated, and 92 parts of hexaploid wheat derived from wild emmer can screen the germplasm for FCR infection at the early development stage.
The invention also provides application of the identification method in screening of wheat germplasm with stem rot resistance.
The application of the present invention is preferably the same as described above and will not be described further herein.
The method for identifying the resistance to wheat stem basal rot and the application thereof provided by the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Plant material
The best identification method is determined by taking 36(04z36) and new wheat 26(XM) in the anti-FCR and high-sensitivity FCR wheat variety 04 as materials. 92 hexaploid wheat introduction lines derived from wild emmer are taken as materials to verify the effectiveness and repeatability of the optimization identification method.
2. Preparation of inoculated bacterial liquid
The fusarium graminearum isolate WZ-8A of dominant pathogenic species in Huang-Huai-Mai area in China, which is provided by the Li-Hon-Li-Honglian professor of the plant protection institute of Henan university of agriculture, is used for FCR inoculation. The isolate was cultured 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 hyphae were removed from the plate and then transferred to Erlenmeyer flasks containing 400mL of carboxymethyl Cellulose Medium (CMC). The Erlenmeyer flasks were incubated for about 72 hours on a shaker at a speed of 160rpm and a temperature of 25 ℃. The spores were collected and the concentration of the spore suspension was adjusted to meet the experimental requirements. Tween 20 was added to the spore suspension at a final concentration of 0.5% v/v before use for inoculation.
3. Improved vaccination methods
Four inoculation concentrations and three seedling ages are designed, and the total number of the treatments is 12, wherein the inoculation concentrations are respectively as follows: 104、105、106And 107spores/mL inoculated with 04z36 and XM seedlings, respectively, at one leaf stage, one heart stage (first leaf fully expanded, second leaf just emerged, leaf still curled) and two heart stages (first and second leaf fully expanded, third leaf just emerged, leaf curled), each treatment contained four replicates, each of ten seedlings of each variety were reused.
04z36 and XM seeds were first surface sterilized with 75% ethanol, rinsed twice with sterile water, and then germinated in a petri dish at 25 ℃ for about three days. After germination, seedlings were planted in 4cm square seedling trays containing autoclaved soil. The seedling culture hole trays are arranged in a greenhouse of China agriculture university according to random block groups, the illumination period is 16h, the dark period is 8h, and the day and night temperature is 25 +/-2 ℃/20 +/-2 ℃ respectively. For inoculation, the spore suspension was injected into the seedling stem using a 10mL needle syringe (0.7 mm outer diameter). The stem is injected at a distance of about 0.5cm from the soil surface. The injection was stopped when the suspension flowed out of the top of the seedling. Control plants were inoculated with CMC solution only.
When susceptible genotype "XM" dies severely, the severity of FCR is assessed. Evaluation was carried out 15 days after inoculation (22 days after planting) at the earliest, depending on the development of seedling disease symptoms. The severity of FCR was assessed using the phenotypic grade index established in the present invention, divided into 6 grades, where 0 indicates no symptoms or small brown spots around the injection site; 1. significant browning of sheath No. 1 (but less than 1/4 for total leaf length); 2. 1/2 where the browning area exceeds the length of the primary sheath; 3. 1, complete browning and necrosis of leaf sheath; 4. the second leaf sheath has obvious necrotic browning focus; 5. the 3 rd leaf sheath has obvious necrotic browning focus; 6. the whole plant was severe to complete necrosis. Then grading the disease index DI (Sigma nX/6N) x 100 according to the disease index, wherein the formula I is shown in the specification; where X is the severity scale, N is the number of a certain severity, and N is the total number of tests performed on the same batch. 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 stem was harvested and fixed with formalin-acetic acid-ethanol solution (FAA) overnight at 4 ℃. After dehydration in ethanol and xylene, the tissues were embedded in paraffin. The tissue sections were cut into 5mm slices and the samples were observed with 1% safranine and 0.5% fast green (G1031, https:// www.servicebio.cn /). the samples were viewed using a microscope imaging system (DS-U3, Nikon, Japan).
Analysis of variance showed that both inoculation concentration and injection time had a significant effect on FCR severity (table 1). 04z36 and XM disease severity increased linearly with increasing concentration of molecular spore suspension and delay in injection time (figure 2). In the injection of highly concentrated suspensions (e.g. 10)6Or 107spores/mL) a small brown spot was observed around the injection site of the FCR sensitive variety XM seedling stem 2-3 days later.
TABLE 104 z36 ANOVA of severity of Stem rot in 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 |
2 | 105.2 | ＜0.001 |
| Inoculation concentration |
6 | 312.7 | ＜0.001 |
| Material x inoculum concentration |
6 | 25.1 | ＜0.001 |
| Residual error | 72 | 3.8 | |
| Total of | 96 |
Of the 12 treatments, 04z36 and XM seedlings were injected with 10 injections7spore/mL, and injection concentration is 10 in one heart period with two leaves7Individual spores/mL of spore suspension, produced the most severe FCR symptoms: 04z36 and XM disease indices reached 45.2 and 93.5, respectively, 12 days after injection (about 20 days after planting); the disease index of 04z36 and XM is 27.1 and 68.8 respectively for one-leaf one-heart injection.
When the injection concentration is 104At spore/mL, 04z36 and XM seedlings when evaluated for FCR resistance 10 days post-injection, the suspension at one leaf stage produced the lowest disease index among the 12 treatments (2.3 for 04z36 and 5.4 for XM). Control seedlings injected with CMC solution showed no FCR symptoms (table 2).
TABLE 2 disease indices with different treatments
One-leaf one-heart phase injection 107spore/mL suspensions were identified as the best vaccination methods due to their relatively high FCR severity and better ability to distinguish between disease and infection genotypes.
To verify theEffectiveness of the method, stem tissue of 04z36 and XM seedlings was injected 10 in one leaf and one heart stage7spore/mL suspension was used for histological analysis. No significant difference was observed between 04z36 and XM seedlings injected with no or only CMC solution (a, b, c, d in figure 3). In contrast, during FCR infection, impaired seedling stem structure was observed for both XM and 04z36 infused plants. Severe degradation of xylem cell wall (red staining) and cellulose (green staining) was observed in XM seedlings, while slight degradation was also observed in 04z36 seedlings (e, f in fig. 3).
4. Optimizing effectiveness of vaccination methods
The effectiveness of the optimized vaccination method was evaluated based on the determined reproducibility using a set of 92 wild emmer-derived hexaploid wheat introgression 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 for these two experiments were calculated using Microsoft Excel. In addition, the genotype that showed better FCR resistance in the first trial was used in the second trial to confirm that it had a better level of resistance.
There was a high degree of significance (p <0.01) and strong correlation (r ═ 0.95) between the FCR severity of the two trials (fig. 4). For hexaploid wheat introgression lines constructed with wild emmer, the average severity of FCR was between 28.1 and 88.5, with an overall average of 59.0 (table 3). The average disease indices of the resistant control 04z36 and Cscr6 were 27.8 and 23.4, respectively, and the average disease indices of the backcross parent Handan 87-1 and the sensitive control XM were 59.8 and 68.4, respectively.
TABLE 3 resistance validation test of wheat genotypes to FCR
TABLE 42 disease indices for wheat genotypes exhibiting consistent resistance to FCR
It was verified that two resistant germplasm, including two hexaploid wheat (LR-43 and LR-61) derived based on wild emmer, were co-screened to show similar levels of disease resistance as disease resistance controls 04z36 and Cscr 6. The mean disease indices for these two resistant germplasms were 28.8, 28.1, similar to those for Cscr6(23.4) and 04z36(27.8) in two rounds of disease assessment.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method for identifying resistance to basal rot of wheat stems comprises the following steps: injecting a spore suspension of fusarium at a single leaf and single heart stage, wherein the concentration of the spore suspension is 107one/mL.
2. The method of identifying of claim 1, wherein said wheat comprises hexaploid wheat.
3. The method of claim 1, wherein the injecting comprises injecting the spore suspension into the stem of a greenhouse-grown wheat seedling and stopping the injecting after the spore suspension overflows the top of the seedling.
4. The method of claim 1 or 3, wherein the injected stem is located 0.4 to 0.7cm high from the soil surface.
5. The identification method according to claim 1, characterized in that the evaluation of the resistance to stalk-base rot is carried out 15d after said injection.
6. The method of claim 1 or 3, wherein the spore suspension is prepared by a method comprising: culturing the spores of the fusarium by using a carboxymethyl cellulose culture medium, and mixing the spores with tween 20 to obtain the spore suspension;
the volume of tween 20 was 0.5% of the volume of the spore suspension.
7. The method of claim 5, wherein the wheat is susceptible or resistant according to the disease index.
8. The method of claim 8, wherein the method of assessing comprises calculating a disease index based on the severity of the stalk break;
the severity of the stem base rot is expressed by 0-6, wherein: 0 indicates no symptoms or small brown spots around the injection site; 1 indicates 1/4 where the sheath of leaf 1 has significant browning, but less than the total leaf length; 1/2 showing browning area exceeding first sheath length; 3 indicates that the 1 st leaf sheath is completely browned and necrotic; 4, significant browning and necrosis of the second leaf sheath; 5 indicates that the 3 rd leaf sheath has obvious browning and necrosis; 6 indicates severe to complete necrosis of the whole plant;
the disease index DI ═ (Σ nX/6N) × 100, formula I; where X is the severity scale, N is the number of a certain severity, and N is the total number of tests performed on the same batch.
9. Use of the identification method of any one of claims 1 to 8 in resistance breeding.
10. Use of the identification method of any one of claims 1 to 8 for screening wheat germplasm for resistance to stalk rot.
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