Application of ethephon and chitin in enhancing wheat scab resistance

Abstract

The invention discloses an application of ethephon and chitin in enhancing wheat scab resistance. A method for enhancing wheat scab resistance by using ethephon and chitin comprises the step of spraying a mixed solution of ethephon and chitin to wheat ears before a wheat flowering period. The wheat is treated by using the mixture of chitin and ethephon, so that the strength and durability of wheat defense reaction can be improved, the resistance of wheat to gibberellic disease is further enhanced, and the effect of preventing and controlling diseases is achieved.

Description

Technical Field

[0001] The invention relates to the field of plant disease prevention and control, and in particular to an application of ethephon and chitin in enhancing resistance to wheat fusarium head blight. Background Art

[0002] Wheat fusarium head blight is a fungal disease that often occurs in warm and humid climates. It is one of the major diseases that endangers wheat production worldwide. When the wheat ears are in the flowering period and there is rainy weather with suitable temperatures, the ears are easily infected with fusarium head blight. In the early stage of infection, water-soaked spots will appear on the ears. In the later stage, the ears will dry up and turn white, causing a decrease in wheat yield. The main pathogen of wheat fusarium head blight is Gibberella maydis ( Gibberella zeae ), whose asexual stage is Fusarium graminearum ( Fusarium graminearum ) complex species, which is a semi-biotrophic fungus (Ma Hongxiang et al., 2019). After the spores of the pathogen germinate, hyphae are produced to infect wheat. In the early stage, it absorbs nutrients from the living cells of wheat in a living form, and then secretes toxins to kill the host cells and obtain nutrients from the dead cells of wheat in a dead form. Fusarium graminearum can secrete fungal toxins including deoxynivalenol (DON). These toxins remaining in the grains are resistant to high temperatures and difficult to degrade. After eating wheat grains contaminated by toxins, people and animals will experience vomiting, diarrhea, and refusal to eat (Chen et al. 2019). Wheat fusarium head blight was originally only a regional disease, often occurring in the wheat-growing areas of the middle and lower reaches of the Yangtze River in my country. In recent years, due to factors such as climate warming, increased rainfall, and crop rotation, Fusarium fusarium has frequently broken out and gradually spread to other wheat-growing areas such as the Huanghuai wheat-growing area, becoming one of the main epidemic diseases endangering my country's wheat production (Bai et al. 2018; Ma et al. 2019).

[0003] The prevention and control of wheat fusarium scaly adopts comprehensive prevention and control measures, including spraying pesticides, cultivation management, and planting disease-resistant varieties. Among them, pesticide control is an important part of comprehensive prevention and control. Spraying pesticides before the flowering period of wheat can greatly reduce the harm of fusarium scaly. In actual production, if the prevention and control measures lack the step of pesticide control, even if disease-resistant varieties are planted, fusarium scaly will still occur and cause harm in an environment with sufficient rainfall and suitable temperature. However, in addition to causing environmental pollution, the excessive use of pesticides also creates selection pressure on pathogens, prompting pathogens to change rapidly and develop drug resistance, which in turn leads to the failure of the pesticides. Therefore, the development of new pesticides or prevention and control methods that are environmentally friendly, low-cost, and easy to promote on a large scale is of great significance to reducing the harm of fusarium scaly and protecting the environment.

[0004] Plant disease resistance can be divided into two categories from the perspective of molecular mechanism: PTI and ETI. PTI has a relatively weak resistance level but a broad spectrum, while ETI has a strong resistance level but a narrow spectrum. PTI and ETI can enhance each other and synergistically regulate plant disease resistance (Ngou et al. 2021, Yuan et al. 2021). Wheat resistance to Fusarium head blight belongs to quantitative resistance in the genetic category, corresponding to PTI resistance at the molecular level. Since no immune genes for wheat Fusarium head blight have been found in wheat and its related species, that is, ETI resistance does not exist in the resistance mechanism of wheat Fusarium head blight, activating and enhancing the PTI pathway in wheat Fusarium head blight resistance is an effective means to improve disease resistance. The activation of plant PTI depends on the plant's recognition of conserved substances of pathogens, such as flagellin of pathogenic bacteria and chitin of pathogenic fungi. Exogenous application of these conserved substances of pathogens can significantly enhance the plant's disease resistance to certain diseases (Shibuya and Minami et al. 2001). However, in wheat, chitin-mediated disease resistance is relatively weak (Wang et al. 2024), so increasing the intensity and persistence of this signal is an important strategy to enhance wheat disease resistance. Summary of the invention

[0005] In view of the problem that the above-mentioned commonly used drugs for preventing and controlling wheat scab are easy to cause environmental pollution and at the same time cause selection pressure on pathogens, prompting pathogens to develop drug resistance, thereby causing the loss of drug efficacy, the present invention provides an application of ethephon and chitin in enhancing wheat scab resistance, and spraying wheat with a mixture of ethephon and chitin can significantly enhance wheat scab resistance. The method is environmentally friendly and easy to scale up.

[0006] In order to achieve the above-mentioned object, the present invention provides, on one hand, the use of ethephon and chitin in enhancing the resistance of wheat to fusarium head blight.

[0007] Chitin can activate the PTI resistance of plants, but the effect is weak. The expression of plant disease resistance-related genes can positively feedback regulate the chitin signaling pathway, and ethephon can induce the expression of plant disease resistance-related genes, although the expression level is low but relatively long-lasting. Therefore, the combined application of ethephon and chitin can amplify chitin signal transduction, and the latter further promotes the expression of disease resistance-related genes, thereby increasing the intensity and persistence of disease resistance gene expression and giving plants disease resistance.

[0008] The second aspect of the present invention provides a method for enhancing wheat resistance to fusarium head blight by using ethephon and chitin, which comprises the step of spraying a mixed solution of ethephon and chitin on wheat ears before the flowering period of wheat.

[0009] Specifically, the period before the wheat flowering period is the day before the wheat flowering period.

[0010] Specifically, the mixed solution of ethephon and chitin contains ethephon at a concentration of 0.15‰ to 0.18‰, chitin at a concentration of 20 to 30 μg / mL, and a non-ionic surfactant at a concentration of 0.04% to 0.06% (v / v). The non-ionic surfactant is selected from one or more of Tween-20, Triton X-100, and NP-40.

[0011] Specifically, the spraying is to spray 40-50 mL of the solution per 60 wheat ears until dense mist-like water droplets appear on the ears (avoiding the formation of large water droplets rolling down).

[0012] Preferably, the spraying is twice a day.

[0013] Further preferably, the spraying time is 8 am and 2 pm.

[0014] Through the above technical solution, the present invention achieves the following beneficial effects: 1. The present invention utilizes a mixture of chitin and ethephon to treat wheat, which can improve the intensity and durability of the wheat defense response, thereby enhancing the resistance of wheat to fusarium head blight.

[0015] 2. The present invention has the characteristics of simple and easy large-scale operation, low-cost reagents and environmental friendliness, and bacteria are not easy to develop drug resistance. It can eliminate the public's concerns about chemical agents polluting the environment. It is a perfect alternative to traditional pesticide control methods and has extremely broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a flow chart of improving wheat scab resistance using an aqueous solution of chitin and ethephon in an embodiment of the present invention; Figure 2 The relative expression levels of defense-related genes in wheat spikes after different treatments are detected, where the gene expression levels are relative to the expression levels at 0 h after the first treatment, and each data is the mean (3 replicates) ± standard deviation; Figure 3 The statistical results of different wheat varieties inoculated with Fusarium head blight after different treatments, where each data is the mean (30 data points) ± standard deviation, and double asterisks (**) indicate that in the same wheat variety, the diseased spikelet rate of wheat after treatment is extremely significant compared with that of untreated wheat ( P <0.01) decreased. DETAILED DESCRIPTION

[0017] The specific implementation of the present invention is described in detail below in conjunction with the examples. It should be understood that the specific implementation described herein is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0018] Use Figure 1 The process described verifies the effect of using ethephon and chitin to enhance wheat resistance to fusarium head blight, see Examples 1 to 3 for details.

[0019] Example 1: Preparation of different treatment liquids 1. Preparation of Chitin Solution Commercial shrimp shells or crab shells (purchased from Shenggong Biotechnology (Shanghai) Co., Ltd.) from which minerals and proteins have been removed were cut into long strips or sheets with scissors, ground into fine powder in a commercially available pharmaceutical grinder, and coarse particles were removed with a 200-mesh powder sieve to obtain chitin powder, which was stored at room temperature for a long time.

[0020] When preparing the solution, dissolve chitin powder in sterile deionized water, and use an ultrasonic lysator at a power of 200 W for 4 h to obtain a chitin solution, which is then stored at -20°C for a long term.

[0021] 2. Preparation of Ethephon Aqueous Solution Dilute the commercially available 40% ethephon aqueous solution with tap water and add Tween-20 to obtain the ethephon working solution.

[0022] 3. Preparation of mixed solution Add chitin solution to the above-mentioned ethephon working solution to make the final concentration of chitin 25 μg / mL, the final concentration of ethephon 0.16‰, and the final concentration of Tween-20 0.05% (v / v), and mix thoroughly to obtain an aqueous solution containing chitin and ethephon. The solution should be prepared and used immediately, and the storage time at room temperature after preparation should not exceed 6 hours.

[0023] Example 2: Detection of defense gene expression after pre-treatment of wheat ears with different treatment solutions 1. Pretreatment of wheat ears with different treatment solutions One day before the flowering period of wheat variety Fielder (highly susceptible to ergot) (1-2 florets were blooming on the extracted wheat ears), the wheat ears were sprayed with different solutions in Example 1 until dense mist-like water droplets appeared on the ears (pay attention to control the spraying amount, 50 mL of solution was sprayed on 60 wheat ears to avoid the formation of large water droplets rolling down). The spraying time was once at 8 o'clock in the morning and once again at 2 o'clock in the afternoon.

[0024] 2. Preparation of total cDNA from wheat spikes after different treatments The time point just after the treatment was marked as 0 h, and three spikelets in the middle of the treated wheat spikelets were taken as samples at different time points. Total RNA from the spikelets was extracted using the TransZol conventional RNA extraction kit (Beijing Quanshijin Biotechnology Co., Ltd.) according to the method provided in the instructions attached to the kit. Then 5 μg of total RNA was taken, DNase I (Invitrogen, USA) was added to remove genomic DNA, and then used as a template for reverse transcription using oligo(dT)18 oligo primer and M-MLV reverse transcriptase (Invitrogen, USA) to obtain total cDNA.

[0025] 3. Detection of the expression of defense genes in wheat ears after different treatments Using the total cDNA obtained by reverse transcription as a template, qRT-PCR technology was used to detect the expression changes of four representative defense genes. The detection primers were: TaPR1 The primers were TaPR1-RT-F (5′-CGTCTTCATCACCTGCAACTA-3′) and TaPR1-RT-R (5′-CAAACATAAACACACGCACGTA -3′). TaPAL The primers were TaPAL-RT-F (5′-TTGATGAAGCCGAAGCAGGACC-3′) and TaPAL-RT-R (5′-ATGGGGGTGCCTTGGAAGTTGC-3′). TaWRKY23 The primers were TaWRKY23-RT-F (5′-ACCCTTACCTAGAGCGGCTTC-3′) and TaWRKY23-RT-R (5′-CACGGATGCTAATGGCCACC-3′). TaCERK1 The primers used were TaCERK1-RT-F (5′-TTACCCCATCGACGCCATTC-3′) and TaCERK1-RT-R (5′-TCTGCCGGACATGAGGTTCA -3′). Figure 2 As shown, single treatment with chitin solution and ethephon solution can induce the expression of the four defense genes, but the gene expression induced by chitin occurs in the early stage of treatment, while the gene expression induced by ethephon occurs in the late stage of treatment; in addition, after treatment with the mixed solution of chitin and ethephon, the induced expression intensity and persistence of the four defense genes were significantly improved compared with single treatment, suggesting that the mixed solution treatment of the two can improve the intensity and persistence of the defense response.

[0026] Example 3: Detection of disease resistance of wheat after pre-treating wheat ears with different treatment solutions 1. Preparation of Gibberella spore suspension Take out the spore liquid of Fusarium graminearum (named PH-1, international reference strain number NRRL31084) frozen at -70℃, spread it on potato dextrose agar solid medium, and place it in a constant temperature incubator at 25℃ to culture until the mycelium covers the surface of the medium. Pick the bacterial block from the surface of the medium and inoculate it into the mung bean soup medium, place it in a shaker at 25℃ and shake it at 180r / min for 3 days, take 10 μL of bacterial liquid and examine it under a microscope, count the number of conidia, and dilute all the spore liquid to a concentration of 1×10 with mung bean soup. 5 / mL, stored at 4℃ for future use.

[0027] The culture medium formula for bacterial culture is as follows: Potato glucose agar medium: 200 g of potatoes were cut into small pieces, boiled in distilled water for 20 min, filtered through gauze, and then 20 g of glucose and 15 g of agar were added. The volume was adjusted to 1 L with distilled water, and sterilized at high temperature and high pressure for 20 min.

[0028] Mung bean soup culture medium: Weigh 40 g of mung beans, place them in 1 L of distilled water and boil them for 20 min, keeping the mung bean skin intact, filter the supernatant with fine gauze, and sterilize them at 121℃ and high temperature and high pressure for 15 min.

[0029] 2. Inoculate wheat ears with Fusarium graminearum spores and count the inoculation results The wheat ear at the flowering stage was treated with the treatment method in Example 2, and the treated wheat ear was inoculated by the single flower drip method one day later. The present invention selects representative wheat varieties showing different levels of resistance to fusarium head blight for the test, including the highly susceptible variety Fielder, the medium-resistant varieties Yangmai 158 and Ningmai No. 9. These varieties are commonly used control varieties in wheat fusarium head blight research, and the resistance levels are stable and have been repeatedly verified in actual production.

[0030] The specific operation steps of the single flower drip inoculation method are as follows: use a sterile syringe to absorb the spore suspension, inject 10 μL of spore liquid into a small flower in the middle of the wheat ear, cover the inoculated wheat ear with a plastic bag and tie the bag tightly, and remove the plastic bag after moisturizing for 3 days. 21 days after inoculation, count the number of diseased spikelets and the total number of spikelets on the wheat ear, and calculate the diseased spikelet rate (the percentage of diseased spikelets to the total number of spikelets). The results are shown in Tables 1 and Figure 3 shown.

[0031] Table 1 Diseased spikelet rate of wheat 21 days after inoculation (%)

[0032] Note: 30 wheat spikelets were inoculated in each treatment, and each data is the mean (30 data points) ± standard deviation. Double asterisks (**) indicate that in the same wheat variety, the diseased spikelet rate of wheat after treatment is extremely significant compared with that of untreated wheat (P <0.01) decreased.

[0033] The results showed that compared with the untreated group, all three treatments could significantly reduce the diseased spikelet rate of the highly susceptible variety Fielder, and the mixed treatment had a more significant effect than the single treatment (chitin or ethephon treatment) ( Figure 3 However, single treatment could not improve the disease resistance of the medium-resistant varieties Yangmai 158 and Ningmai 9, while mixed treatment could further improve the disease resistance of these two varieties ( Figure 3 ). The above results show that the mixed treatment of chitin and ethephon can improve the intensity and durability of wheat defense response, thereby enhancing wheat resistance to fusarium head blight.

[0034] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0035] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0036] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. An application of ethephon and chitin in enhancing resistance to wheat fusarium head blight.

2. A method for enhancing wheat fusarium head blight resistance using ethephon and chitin, characterized in that: The method comprises the steps of spraying a mixed solution of ethephon and chitin on wheat ears before the wheat blooming period.

3. The method according to claim 2, characterized in that The wheat before flowering period refers to the day before wheat flowering period.

4. The method according to claim 2, characterized in that The mixed solution of ethephon and chitin contains ethephon with a concentration of 0.15‰ to 0.18‰, chitin with a concentration of 20 to 30 μg / mL, and a non-ionic surfactant with a concentration of 0.04% to 0.06% (v / v).

5. The method according to claim 4, characterized in that The nonionic surfactant is selected from one or more of Tween-20, Triton X-100 and NP-40.

6. The method according to claim 2, characterized in that The spraying step is to spray 40-50 mL of the solution on every 60 wheat ears until dense mist-like water droplets appear on the ears.

7. The method according to claim 2, characterized in that The spraying was performed twice a day.

8. The method according to claim 7, characterized in that The spraying time is 8 am and 2 pm.

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