Microbial agent combination method for preventing and treating panax notoginseng root rot

Abstract

The invention relates to the field of microbial technology application, in particular to a bacillus methylotrophicus 9912 microbial inoculum and natamycin which are combined in a certain amount. The method is used for field planting of panax notoginseng, has an obvious prevention and treatment effect on panax notoginseng root rot, the prevention and treatment effect can reach 85% or above, growth of panax notoginseng can be promoted, and the fresh weight of underground roots of panax notoginseng plants and the content of notoginsenoside are obviously increased. The method is simple to operate, low in cost, easy to popularize, green and environment-friendly, and lays a good foundation for pollution-free planting of pseudo-ginseng.

Description

Technical Field

[0001] The invention belongs to the field of microbial technology application, and specifically relates to a method for combining microbial agents for preventing and treating Panax notoginseng root rot. Background Art

[0002] Panax notoginseng (Burkill) FH Chen. is a plant of the genus Panax in the family Araliaceae. It has many functions such as reducing swelling and pain, stopping bleeding and removing blood stasis. It is the main ingredient of many traditional Chinese medicine compound preparations and has a wide range of medicinal value. As people pay more attention to health care issues, the market demand for Panax notoginseng is also increasing. The authentic production area of ​​Panax notoginseng is Wenshan, Yunnan. The cultivation area, total output and quality of this area rank first in the country. Therefore, Panax notoginseng cultivation has become a pillar industry for the economic development of Yunnan Province.

[0003] Panax notoginseng is a perennial plant that likes warm and humid climates. It is usually cultivated in a no-tillage and shaded manner. Due to its unique growth environment, it is easily infected by various diseases, among which root rot is the most harmful disease. Panax notoginseng root rot, commonly known as rotten head, is a typical soil-borne disease that occurs throughout the year and occurs throughout the entire growth cycle of Panax notoginseng. Diseases of the aboveground parts of plants can be prevented or controlled through chemical or physical prevention and control measures, but diseases of the underground parts cannot be discovered in advance and are difficult to control. They can only be discovered when the leaves change color. Since the large-scale cultivation of Panax notoginseng, root rot has always been a difficult problem to solve. According to statistics, root rot can cause a 5-20% reduction in the yield of Panax notoginseng, and in severe cases it can reach more than 70%, accounting for 70-85% of the various diseases of Panax notoginseng, and it also seriously affects the quality of Panax notoginseng, greatly reducing the economic benefits of planting. In particular, continuous planting is more likely to lead to the occurrence and spread of diseases such as root rot. In some areas, the "seven gardens" are even destroyed and the harvest is completely lost, seriously affecting the planting income.

[0004] According to research, the main cause of Panax notoginseng root rot is the result of changes in the population of anaerobic bacteria, yeasts, fungi and actinomycetes in the soil. Nematodes can also cause the disease. The main rot pathogens are Fusarium solani, Cy1 indrocarpon sp., Phytophthora cactorum, Pratylenchus Filipev, Pythium Pringsheim, Saprolegnia Nees, etc. In addition, the occurrence and development of Panax notoginseng root rot is also related to changes in the microecology of the rhizosphere soil, improper cultivation and management measures, plant self-toxicity, continuous cropping and many other aspects.

[0005] At present, the methods for preventing and treating Panax notoginseng root rot mainly include soil fumigation and chemical pesticides. Both methods have serious side effects, not only causing damage to the environment and resources, but also causing excessive pesticide residues to seriously affect the quality of Panax notoginseng. In addition, the drug resistance of Panax notoginseng itself causes the prevention and treatment effect to decline year by year, eventually forming a vicious cycle in which the frequency and amount of application of pesticides continue to increase, while the quality of Panax notoginseng continues to decline. In recent years, there have also been studies on the use of microbial agents or Chinese medicines for the prevention and treatment of Panax notoginseng root rot, and some results have been achieved. CN110699301A discloses that the control rate of root rot caused by Panax notoginseng Fusarium solani by using Serratia puerariae PBLJ042 strain is close to 50%, and the bacteriostatic effect of the bacterium on Panax notoginseng Fusarium solani is only strong, and the control rate is limited. The metabolites of the Vietnamese Sophora japonica endophytic fungus SDTE-P used in CN105462850B have an inhibitory effect (100%) on the growth of Panax notoginseng root rot fungus F.solani. This genus of fungi is not yet commercially available and has not been used in field planting. In CN108823105B, the microsporum head group fungus was used to prevent and control Panax notoginseng root rot, with an efficiency of 70.59%, and the effect needs to be improved. CN105594711A mentions that a Chinese herbal medicine formula such as Scutellaria baicalensis, Coptis chinensis, Sophora flavescens, Cnidium monnieri, Stemona japonica, Lycium bark, Licorice, Platycladus orientalis leaves and Artemisia argyi is boiled in water and sprayed on the soil surface or after digging the soil layer. The prevention and control effect can reach 80%, but this method is cumbersome and inconvenient to apply.

[0006] Therefore, finding green, environmentally friendly, safe, side-effect-free and easy-to-implement microbial pesticides has become an inevitable trend in the development of Panax notoginseng cultivation. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a method for combining a microbial agent including a methylotrophic Bacillus 9912 agent and natamycin. This method is used in the process of Panax notoginseng cultivation, which can not only effectively prevent and control Panax notoginseng root rot, but also significantly promote the growth of Panax notoginseng, increase the fresh weight of the underground roots of Panax notoginseng plants and the content of Panax notoginseng saponins.

[0008] In order to solve the above technical problems, the present invention is implemented through the following technical solutions:

[0009] During the growth of Panax notoginseng, methylotrophic Bacillus 9912 and natamycin are used in combination. The specific method is: during the growth period of Panax notoginseng, methylotrophic Bacillus 9912 and natamycin are used together every 1 to 1.5 months. It is applied 3 times in the first year after transplanting Panax notoginseng seedlings, and 1 to 2 times in the second year.

[0010] Preferably, from May to August, methylotrophic Bacillus 9912 is used together with natamycin every 1 to 1.5 months, and is applied 3 times in the first year after transplanting Panax notoginseng seedlings, and 1 to 2 times in the second year.

[0011] Preferably, the methylotrophic Bacillus 9912 bacterial agent is a liquid agent, and the effective viable bacteria count is ≥ 300 million / ml.

[0012] A further improvement of the technical solution of the present invention is that when the preparation is used in the process of Panax notoginseng planting, the dosage of methylotrophic Bacillus 9912 liquid agent is 1 to 10 kg / mu·time; and the dosage of natamycin is 4 to 16 g / mu·time.

[0013] Preferably, the preparation is used in the process of planting Panax notoginseng, and the dosage of methylotrophic Bacillus 9912 liquid agent is 1 to 5 kg / mu / time.

[0014] A further improvement of the technical solution of the present invention is that the preparation is applied in the process of planting Panax notoginseng, and the application method is root irrigation and spraying, and the spraying treatment parts include the Panax notoginseng stems, leaves and the root soil surface.

[0015] A further improvement of the technical solution of the present invention is that: the preparation is used in the process of Panax notoginseng cultivation, and the application concentration of the microbial agent is that the liquid agent of methylotrophic Bacillus 9912 is diluted 50 to 100 times with water, and natamycin and an equal mass of lactose are added and mixed until each liter of solution contains 40 to 160 mg of natamycin.

[0016] In the present invention, the preparation method of the methylotrophic Bacillus 9912 liquid inoculant can refer to patent CN108559719A, and can be prepared according to the following steps:

[0017] a. Preparation of seed solution of methylotrophic Bacillus strain 9912:

[0018] Inoculating a plate culture of the strain methylotrophic Bacillus 9912 into a seed medium to obtain a seed solution;

[0019] b. Preparation of fermentation broth of methylotrophic Bacillus strain 9912:

[0020] Inoculating the cultured seed liquid into a fermentation medium, fermenting and culturing to obtain a fermentation liquid;

[0021] c. Preparation method of liquid microbial agent: After fermentation, add additives to the fermentation liquid, including 0-1% fulvic acid, 0-0.2% amino acid, 0-0.1% preservative and 0-0.1% inhibitor. Add each additive to the fermentation liquid and stir evenly to obtain liquid microbial fertilizer.

[0022] In the present invention, the deposit number of methylotrophic Bacillus 9912 is CCTCC No: M2010343.

[0023] Due to the adoption of the above technical solution, the technical progress achieved by the present invention is:

[0024] The present invention provides a method for combining a microbial agent comprising a methylotrophic Bacillus 9912 agent and natamycin. The method can not only reduce the incidence of root rot of Panax notoginseng plants, but also promote the growth of Panax notoginseng roots, increase the fresh weight of the roots, and the total content of Panax notoginseng saponins in the roots is significantly higher than that in the control group. This lays a good foundation for the pollution-free cultivation of Panax notoginseng, and also opens up new ways for the research and development of microbial source fertilizers and pesticides. DETAILED DESCRIPTION

[0025] Example 1

[0026] Experiment on the antibacterial efficacy of combined application of methylotrophic Bacillus and natamycin against the pathogen of Panax notoginseng root rot:

[0027] The synergistic antifungal activity of a liquid preparation of methylotrophic Bacillus and natamycin against Fusarium oxysporum (the pathogen of Panax notoginseng) was demonstrated in an in vitro test using a 96-well microplate. The method is as follows:

[0028] Natamycin was from North China Pharmaceutical Huasheng Co., Ltd., the methylotrophic Bacillus liquid preparation was homemade, and lactose was purchased from Zhengzhou Dewang Chemical Products Co., Ltd.

[0029] Prepare a spore suspension of Fusarium oxysporum, the concentration of which is 1.0*10 7 pcs / ml, for future use.

[0030] Assay culture medium: Sabouraud's medium (1L): glucose: 40 g; peptone: 5 g, yeast powder 5 g, autoclave at 121°C for 20 min and set aside.

[0031] Preparation of methylotrophic Bacillus liquid preparation group: 20 ml of Sabouraud medium (containing 105 Fusarium oxysporum spores / ml) were added with 2000 μl, 1000 μl, 500 μl, 250 μl, 125 μl, and 62.5 μl of methylotrophic Bacillus 9912 liquid preparation, respectively, to prepare methylotrophic Bacillus 9912 liquid preparation groups of different concentrations.

[0032] Preparation of natamycin dilution: Add an equal amount of lactose to natamycin, dilute with sterile water to a final concentration of 500 μg / ml, and then dilute with sterile purified water to 7 concentrations by two-fold gradient dilution.

[0033] Experimental process:

[0034] 1) Preparation group:

[0035] Mixed preparation group: Add different dilution multiples of methylotrophic Bacillus 9912 liquid preparation group (containing 10 5 / ml) sample 200μl, and then add 10μl of natamycin dilution of each concentration to make the final concentration of natamycin 25μg / ml, 12.5μg / ml, 6.25μg / ml, 3.13μg / ml, 1.56μg / ml, 0.78μg / ml, and 0.39μg / ml.

[0036] Natamycin group: Fusarium oxysporum spore suspension (containing 10 5 Each batch of natamycin was diluted with 200 μl of 1 μg / ml, and then 10 μl of natamycin dilution solution of each concentration was added to make the final concentration of natamycin be 25 μg / ml, 12.5 μg / ml, 6.25 μg / ml, 3.13 μg / ml, 1.56 μg / ml, 0.78 μg / ml, and 0.39 μg / ml.

[0037] 2) Test bacteria control group: Add 10 5 The test culture medium for the spore detection bacterial suspension is 200μl / well.

[0038] 3) Culture medium blank control group: 200µl / well of assay culture medium.

[0039] After culturing in a humidified box at 26°C for 5 days, the growth of Fusarium oxysporum was visually observed. The results are shown in Table 1.

[0040] Table 1 Synergistic effect of natamycin and methylotrophic Bacillus 9912 liquid preparation against Fusarium oxysporum

[0041] “+++”: A large amount of Fusarium oxysporum growth is visible

[0042] “++”: A moderate amount of Fusarium oxysporum growth is visible

[0043] “+”: A small amount of Fusarium oxysporum growth can be seen

[0044] “+-”: Very small amount of Fusarium oxysporum growth can be seen

[0045] “-”: No visible growth of Fusarium oxysporum

[0046] The above results show that natamycin and methylotrophic Bacillus liquid preparation have a synergistic effect. When natamycin is used alone, the inhibitory concentration of Fusarium oxysporum is 3.13μg / ml (natamycin); when methylotrophic Bacillus liquid preparation is used alone, a small amount of bacteria grows when it is diluted 10 to 100 times, and the dilution multiple has little effect on it. When the original solution is used, there is still a very small amount of Fusarium oxysporum growth; and when natamycin and methylotrophic Bacillus liquid preparation are used together, the concentration of natamycin is 0.39μg / ml and the methylotrophic Bacillus liquid preparation is diluted to 100 times, which can kill all Fusarium oxysporum.

[0047] Field trial method to verify the effect of drug use

[0048] A field comparison test was carried out in the same shade shed. The first year is the seedling raising period. After the seedling raising is completed, the plants are transplanted to the field and harvested after two years of continuous cultivation. Microbial agents were applied in the first and second years after transplanting, and the diseases of Panax notoginseng plants were counted. The study was divided into 7 groups: 1 control group (CK-1), 4 groups of embodiments of the present invention, and 2 comparative groups, with 360 Panax notoginseng seedlings in each group. Among them, the control group, embodiment group, and comparative group used different types of microbial agent schemes, and the rest of the field management such as water, fertilizer, disinfection, and pest control were treated the same.

[0049] Experimental design:

[0050] CK-1: Normal field management. Use the conventional commercial fertilizer Qiyoufeng without adding any microbial preparations.

[0051] Comparative Example 1: From May to August, apply methylotrophic Bacillus 9912 liquid agent every 1.5 months, the dosage is 10 kg / mu / time, diluted 50-100 times with water, root irrigation + spraying. Apply 3 times in the first year after seedling transplanting, and apply 2 times in the second year. The rest of the treatment is the same as CK-1.

[0052] Comparative Example 2: From May to August, natamycin was applied every 1.5 months at a dosage of 32 g / mu / time. Natamycin was added with an equal amount of lactose and diluted with water to a solution containing 40-160 mg of natamycin per liter. Root irrigation + spraying was performed. It was applied 3 times in the first year after seedling transplanting and 2 times in the second year. The rest of the treatments were the same as CK-1.

[0053] Example 2: From May to August, apply methylotrophic Bacillus 9912 liquid and natamycin at intervals of 1.5 months. The specific method is: the dosage of methylotrophic Bacillus 9912 liquid is 5 kg / mu·time, and the dosage of natamycin is 4 g / mu·time. Dilute the methylotrophic Bacillus 9912 liquid with water 50-100 times, add natamycin and lactose of equal mass and mix until each liter of solution contains 40-160 mg of natamycin. Apply 3 times in the first year after seedling transplanting, and apply 2 times in the second year. The rest of the treatment is the same as CK-1.

[0054] Example 3: The dosage of methylotrophic Bacillus 9912 liquid agent is 1 kg / mu·time, and the dosage of natamycin is 16 g / mu·time. The rest of the treatment is the same as in Example 2.

[0055] Example 4: The dosage of the liquid agent of methylotrophic Bacillus 9912 is 2.5 kg / mu·time, the dosage of natamycin is 8 g / mu·time, and the other treatments are the same as in Example 2.

[0056] Example 5: The dosage of methylotrophic Bacillus 9912 liquid agent is 2.5 kg / mu·time, and the dosage of natamycin is 8 g / mu·time. It is applied 3 times in the first year after seedling transplanting and once in the second year. The rest of the treatment is the same as CK-1.

[0057] In the first and second years after seedling transplanting, the root rot of Panax notoginseng was investigated and counted, and the control effect was calculated. The classification of Panax notoginseng root rot refers to the description in "Plant Disease Research Methods", namely: Level 0, healthy and asymptomatic; Level 1, lesions occupy less than 5% of the surface area; Level 2, lesions occupy 6% to 10% of the surface area; Level 3, lesions occupy 11% to 25% of the surface area; Level 4, lesions occupy 26% to 50% of the surface area; Level 5, lesions occupy 51% to 75% of the surface area; Level 6, lesions occupy more than 75% of the surface area or completely rotten.

[0058] According to the survey data, the disease index (%) = [Σ(number of disease-grade plants × representative value of this value) / (total number of surveyed plants × highest representative value)] × 100

[0059] Control effect (%) = [(control disease index - experimental disease index) / control disease index] × 100

[0060] The results are shown in Table 2.

[0061] Table 2 The control effect of different drug regimens on Panax notoginseng root rot

[0062] As shown in Table 2, the incidence of root rot was extremely high in the control plots without microbial agents, while the experimental groups using microbial agents had different degrees of control effects on root rot. The combined application of the two agents significantly improved the control effect compared to the separate application.

[0063] Two years after the seedlings were transplanted, Panax notoginseng was harvested. Disease-free plants were selected and the soil on the roots, stems and leaves was washed clean with clean water, and the surface water was dried with absorbent paper. The fresh weight of the aboveground part, the fresh weight of the underground part, and the weight of the main root after cutting off the fibrous roots and lateral roots were measured separately, and the percentage of the main root in the fresh weight of the underground part was calculated (each treatment was repeated 6 times and the average value was taken). The comparison results are shown in Table 3.

[0064] Table 3 Effects of different medication regimens on the growth of Panax notoginseng

[0065] As shown in Table 3, the aboveground fresh weight, underground fresh weight, and taproot percentage of mature Panax notoginseng in the embodiment group were increased to varying degrees compared with the control group. The underground fresh weight and taproot percentage of Panax notoginseng in the embodiment group were significantly increased compared with the control group and the comparative group, indicating that the combination of methylotrophic Bacillus 9912 liquid inoculant and natamycin has a good effect and has a significant growth-promoting effect on Panax notoginseng.

[0066] After the main root part was dried, saponins were extracted according to the 2020 edition of the Pharmacopoeia, and the saponin content was tested by HPLC. The results are shown in Table 4.

[0067] Table 4 Comparison of notoginseng saponin content in different dosing regimens

[0068] As shown in Table 4, the content of notoginsenoside R1 in Examples 2-5 is comparable to that in the control group, while the content of ginsenoside Rg1, ginsenoside Rb1 and the sum of the three saponins are significantly higher than that in the control group and the comparative example group.

[0069] In summary, the combined application of the methylotrophic Bacillus 9912 agent provided by the present invention and natamycin has a good control effect on Panax notoginseng root rot, with a control efficiency of more than 85%, and obviously has the effect of promoting root growth.

[0070] It should be noted that the above-mentioned embodiments are intended to illustrate rather than limit the present invention, and any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention are included in the protection scope of the present invention.

Claims

1. A method for combining microbial agents to prevent and treat Panax notoginseng root rot, characterized in that: During the growth period of Panax notoginseng, methylotrophic Bacillus 9912 and natamycin are used in combination. The specific method is: during the growth period of Panax notoginseng, methylotrophic Bacillus 9912 and natamycin are used together every 1 to 1.5 months, 3 times in the first year after seedling transplanting, and 1 to 2 times in the second year.

2. The method for combining microbial agents for preventing and treating Panax notoginseng root rot according to claim 1, characterized in that: During the growth period of Panax notoginseng, from May to August, methylotrophic Bacillus 9912 and natamycin are used together every 1 to 1.5 months, 3 times in the first year after seedling transplanting, and 1 to 2 times in the second year.

3. The method according to claim 1, characterized in that The methylotrophic Bacillus 9912 bacterial agent is a liquid agent, and the effective live bacteria count is ≥ 300 million / ml.

4. The method for combining microbial agents for preventing and treating Panax notoginseng root rot according to claim 3, characterized in that: The dosage of liquid agent of methylotrophic Bacillus 9912 is 1 to 10 kg / mu·time; the dosage of natamycin is 4 to 16 g / mu·time.

5. The method for combining microbial agents for preventing and treating Panax notoginseng root rot according to claim 4, characterized in that: The dosage of methylotrophic Bacillus 9912 liquid agent is 1 to 5 kg / mu / time.

6. A method for combining microbial agents for preventing and treating Panax notoginseng root rot according to claim 5, characterized in that: The application method is root irrigation and spraying, and the spraying treatment parts include: Panax notoginseng stems, leaves and root soil surface.

7. A method for combining microbial agents for preventing and treating Panax notoginseng root rot according to claim 6, characterized in that: The application concentration of the microbial agent is: the methylotrophic Bacillus 9912 liquid agent is diluted 50 to 100 times with water, and natamycin and lactose of equal mass are added and mixed until each liter of the solution contains 40 to 160 mg of natamycin.

8. The method for combining microbial agents for preventing and treating Panax notoginseng root rot according to claim 1, wherein the methylotrophic Bacillus 9912 has a preservation number of CCTCC No: M2010343.

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