Application of gellan gum in preparation of preparation for preventing and treating plant bacterial wilt

Abstract

The invention discloses application of gellan gum to preparation of a preparation for preventing and treating plant bacterial wilt. According to the method, the gellan gum is added into the tomatoes by dissolving and irrigating the roots, the gellan gum can stably resist invasion of ralstonia solanacearum to the roots, the actual prevention and control effect is practically improved, the biocontrol rate of the gellan gum with the concentration of 50 mg / kg is 65.4%, the biocontrol rate of the gellan gum with the concentration of 100 mg / kg is 61.5%, and the method has an obvious prevention and control effect on the ralstonia solanacearum of the tomatoes.

Description

Technical Field

[0001] The present invention relates to the field of bacterial wilt prevention and treatment, and in particular to application of gellan gum in the preparation of a preparation for preventing and treating bacterial wilt of plants. Background Art

[0002] Bacterial wilt is a serious disease in plant production. It develops rapidly, spreads rapidly, and in severe cases can cause widespread plant death. The fungus primarily infects crop roots in the soil. It has a strong survival ability in the soil and can survive for long periods deep in the soil and migrate, becoming a new source of infection the following year. This makes prevention and control extremely difficult, presenting an urgent challenge in agricultural production.

[0003] Due to environmental pollution caused by chemical pesticides and the conflict between the disease resistance and economic benefits of disease-resistant varieties, biological control has gradually become a hot topic and focus of research. Currently, the most commonly used microbial agents are mainly Streptomyces, Bacillus, Pseudomonas, and non-pathogenic Ralstonia solanacearum. However, the complex field environment and environmental conditions significantly affect their colonization and the activity of their secondary metabolites. In practical use, these agents often suffer from unstable control effects and poor late-stage efficacy. Therefore, developing more effective methods for controlling bacterial wilt is an important research direction.

[0004] Gellan gum can be used as a thickener and stabilizer in a wide range of food applications, including puddings, surface smoothing agents, candies, icings, and seasonings. It is also used in non-food applications, such as microbial culture media. Gellan gum is a microbial extracellular polysaccharide produced by microbial fermentation. It is naturally renewable, biocompatible, biodegradable, environmentally friendly, and pollution-free. Currently, there are no reports on the use of gellan gum in plant disease control. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to address the deficiencies in the prior art and provide an application of gellan gum in the preparation of a preparation for preventing and treating plant bacterial wilt.

[0006] Specifically, the CAS number of the gellan gum is 71010-52-1.

[0007] When used, the gellan gum is dissolved in water to prepare a gellan gum solution with a concentration of 50 to 100 mg / kg.

[0008] Wherein, the water is deionized water.

[0009] Preferably, the temperature is 50-70°C during configuration.

[0010] Preferably, when applying, the cold-setting gum solution is added to the roots of the plant by rhizosphere irrigation method. The cold-setting gum solution is added to the roots 7 to 14 days after transplanting the tomato seedlings. The concentration of the cold-setting gum solution is 50 to 100 mg / kg, and the amount added is based on the weight of soil or fertilizer / kg.

[0011] Preferably, the plant is a tomato.

[0012] The present invention also provides a method for preventing and controlling bacterial wilt of plants, which comprises using a cold-setting gum solution to irrigate the roots of the plants.

[0013] Wherein, the concentration of the cold-setting gum solution is 50-100 mg / kg.

[0014] The CAS number of the cold-setting gum is 71010-52-1.

[0015] Preferably, the plant is a crop susceptible to bacterial wilt, preferably tomato.

[0016] Beneficial effect: The present invention dissolves gellan gum and adds it to tomatoes through root irrigation, so that the gellan gum can effectively improve the actual prevention and control effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the antagonism diagram between gellan gum and Ralstonia solanacearum;

[0018] Figure 2 This is a comparison chart of the incidence rates between the gellan gum group and the control group.

[0019] Figure 3 This is a trend chart of the incidence rate after gellan gum and the control group were inoculated with pathogens.

[0020] Figure 4 This is a comparison chart of the incidence of plants in the gellan gum and control groups, from left to right, 50 mg / kg gellan gum treatment group, 50 mg / kg xanthan gum treatment group, CK (blank control), 100 mg / kg gellan gum treatment group, 100 mg / kg xanthan gum treatment group. DETAILED DESCRIPTION

[0021] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0022] In the following examples, the bacterial pathogen tested was the highly pathogenic Ralstonia solanacearum QL-Rs1115 (China General Microbiology Culture Collection Center, accession No. 9487), which was kindly provided by Nanjing Agricultural University. The pathogen was isolated from the roots of diseased tomato plants (Reference: Wei, Z., Yang, XM, Yin, SX, Shen, QR, Ran, W. & Xu, YC. Efficacy of Bacillus-fortified organic fertiliser in controlling bacterial wilt of tomato in the field. Appl. Soil Ecol., 2011, 48, 152-159.).

[0023] The culture medium used in the present invention includes:

[0024] NA solid medium: glucose 10 g / L, peptone 5 g / L, beef extract 3 g / L, yeast powder 0.5 g / L, agar powder 20 g / L, adjust pH to 7.2-7.4, and autoclave at 115°C for 30 min.

[0025] NA liquid culture medium consists of: glucose 10 g / L, peptone 5 g / L, beef extract 3 g / L, yeast powder 0.5 g / L, adjusted to pH 7.2-7.4, and sterilized at 115°C for 30 min.

[0026] The gellan gum used in the following examples is G875406, purchased from Shanghai MacLean Biochemical Technology Co., Ltd., with a CAS number of 71010-52-1. It is high acyl gellan gum with a purity of ≥99%.

[0027] Example 1 Antagonistic effect of gellan gum on Ralstonia solanacearum.

[0028] Activation of strains:

[0029] Take out the Ralstonia solanacearum stored at -80℃ and streak it onto a NA solid plate. Place it in a 37℃ constant temperature incubator for 1-2 days to grow a single colony for use. Pick a single colony of Ralstonia solanacearum and transfer it to 5mL NA liquid culture medium and shake it at 200rpm and 30℃ for 12h. Centrifuge the above culture solution at 8000rpm for 10min, discard the supernatant and retain the bacteria. Resuspend it with sterile water, repeat the above operation 2-3 times to fully wash away the culture medium, and use an enzyme reader to measure the absorbance of the bacterial solution when the absorbance value is 600. Dilute or concentrate it to OD according to the absorbance. 600=0.5 for standby use, the effective viable count of the diluent or concentrate is ≥1×10 7 CFU / mL.

[0030] Inhibition zone test:

[0031] Take 100 μl of Ralstonia solanacearum suspension (OD 600 =0.5) was applied to a NA solid plate and wells were punched using a 6 mm diameter hole punch. Sterilized gellan gum solutions at concentrations of 0.5 g / L, 1 g / L, and 2 g / L were filtered through a 0.22 μm membrane and 100 μL was added to each well. Sterile water was used as a control. The plates were incubated at 30°C for 24-36 hours and observed for the appearance of inhibition zones.

[0032] Figure 1 The results of the antibacterial experiment are as follows. Figure 1 The results of the antibacterial test showed that no inhibition zone appeared on the plate for 0.5 g / L, 1 g / L and 2 g / L gellan gum solutions, indicating that gellan gum had no direct antagonistic effect on Ralstonia solanacearum.

[0033] Example 2: Effect of gellan gum on the prevention and treatment of tomato bacterial wilt.

[0034] Activation of strains: Take out the Ralstonia solanacearum stored at -80℃ and streak it onto a NA solid plate, place it in a 37℃ constant temperature incubator for 1-2 days, and grow a single colony for use. Pick a single colony of Ralstonia solanacearum, transfer it to 5mL NA liquid culture medium and shake it at 200rpm and 30℃ for 12h, then transfer it to 100mL NA medium for expansion culture, and culture it at 200rpm and 30℃ for 36h. Centrifuge the above culture solution at 8000rpm for 15min, discard the supernatant and retain the bacteria, resuspend it with sterile water, repeat the above operation 2 to 3 times to fully wash away the culture medium, and use an enzyme reader to measure the absorbance of the bacterial solution when the absorbance value is 600, and dilute or concentrate it to OD according to the absorbance. 600 =20, the effective viable count of the dilution or concentrate is 1×10 9 CFU / mL.

[0035] Tomato seed germination and sowing, the specific operations are as follows:

[0036] Soak tomato seeds in 75% alcohol for one minute, surface-sterilize with 30% NaClO, and rinse the sterilized seeds four times with sterile water. Place the washed seeds on semi-solid MS medium (containing 0.7% agar and 1% sucrose) and germinate in the dark at 28°C for three days. After germination, remove the infected seeds and transfer the uninfected seeds to a sterile seedling tray. Place four germinated seeds in each well and incubate under white fluorescent light for 16 hours (25±2°C), watering regularly with sterile water.

[0037] When tomato seedlings have three leaves and one heart, carefully remove them from the seedling trays and transplant them into pots. Add approximately 700 g of dry soil to each pot, transplanting two seedlings per pot. Each treatment has five replicates. The experiment included five treatments, each with 10 tomato seedlings. The soil was maintained at 60% of its maximum moisture content by weighing and adding sterile water.

[0038] After the tomato seedlings have grown stably in the soil for 2 weeks, gellan gum and xanthan gum solutions with a concentration of 50-100 mg / kg were added respectively. Each group was set up with 5 replicates, that is, each group required 175-350 mg of gellan gum and xanthan gum. Gellan gum and xanthan gum were added to 50 mL of deionized water and dissolved at 60 ° C. After the solution cooled, the same volume (10 mL) of gellan gum and xanthan gum solution was added to each pot of soil. After 48 hours of treatment, the bacterial solution of Ralstonia solanacearum was added, and 7 mL of OD 600 =20 dilution or concentrate of Ralstonia solanacearum.

[0039] There are five treatments in this experiment, namely A: 50 mg / kg gellan gum solution + Ralstonia solanacearum solution; B: 100 mg / kg gellan gum solution + Ralstonia solanacearum solution; C: 50 mg / kg xanthan gum solution + Ralstonia solanacearum solution; D: 100 mg / kg xanthan gum solution + Ralstonia solanacearum solution; E (control group): only Ralstonia solanacearum solution is added.

[0040] All seedlings were moved to a greenhouse with high temperature and high humidity. After the tomatoes had grown for 4-5 weeks, the wilting degree of the tomato leaves was observed. The severity of the disease for each plant was evaluated and recorded on a 0-4 scale (i.e., disease index) based on the wilting leaf area (0 = no wilting symptoms, 1 = 1-25% of the leaf area wilted, 2 = 26-50% of the leaf area wilted, 3 = 51-75% of the leaf area wilted, 4 = all leaf area wilted or plant death). The incidence rate of each treatment was calculated according to the following formula.

[0041] Plant incidence rate = (number of diseased plants / total number of plants) × 100%

[0042] Disease incidence = ∑ (disease index × number of plants with the disease index) / (total number of plants surveyed × 4) × 100%

[0043] Plant biocontrol rate = [(incidence rate of control group - incidence rate of treatment group) / incidence rate of control group] × 100%

[0044] Depend on Figures 2 to 4It can be seen that gellan gum of different concentrations has a better control effect on tomato bacterial wilt than xanthan gum. The biocontrol rate of 50mg / kg gellan gum is 65.4%, and the biocontrol rate of 100mg / kg gellan gum is 61.5%. Combined with the antibacterial test results in Example 1, it can be seen that 0.5g / L, 1g / L and 2g / L gellan gum solutions did not show inhibition zones on the plate, and had no direct antagonistic effect on bacterial wilt. It is speculated that after gellan gum is added to the soil, it may inducing plant systemic disease resistance or indirectly enriching beneficial microorganisms in the soil to resist bacterial wilt invasion of the root system, thereby effectively improving the actual control effect. In the xanthan gum treatment group with the same concentration gradient, 50mg / kg xanthan gum had no control effect on tomato bacterial wilt, and the biocontrol rate of 100mg / kg xanthan gum was only 42.3%.

[0045] The present invention provides a method and concept for applying gellan gum to control bacterial wilt in plants. There are numerous methods and approaches for implementing this technical solution. The foregoing description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.

Claims

1. Application of gellan gum in the preparation of preparations for preventing and treating plant bacterial wilt.

2. The use according to claim 1, characterized in that The CAS number of the gellan gum is 71010-52-1.

3. The use according to claim 1, characterized in that When used, the gellan gum is dissolved in water to prepare a gellan gum solution with a concentration of 50-100 mg / kg, which is then added to the roots of plants.

4. The use according to claim 3, characterized in that The water is deionized water, and the temperature during preparation is 50-70°C.

5. The use according to claim 1, characterized in that When applying, the cold-setting gum solution is added to the roots of the plant by rhizosphere irrigation; 7 to 14 days after transplanting the tomato seedlings, the cold-setting gum solution with a concentration of 50~100 mg / kg is added to the roots.

6. The use according to claim 1, characterized in that When applied, the amount of gellan gum added is 50~100 mg per 1 kg of soil.

7. The use according to claim 1, characterized in that The plant is a tomato.

8. A method for preventing and controlling plant bacterial wilt, characterized in that: Use cold-setting gum solution to irrigate the roots of the plants.

9. The method according to claim 8, characterized in that The concentration of the gellan gum solution is 50-100 mg / kg, and the amount of gellan gum added per 1 kg of soil is 50-100 mg.

10. The method according to claim 8, characterized in that The CAS number of the cold-setting gum is 71010-52-1.

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