Endophytic bacteria biopesticide and preparation method thereof

Abstract

The invention relates to an endophytic bacterium biopesticide and a preparation method thereof. The endophytic bacterium biopesticide is characterized in that the active ingredient of a viable bacterium is plant endophytic bacterium Calidifetibacillus erzurumensis. According to the preparation method of the biological pesticide, the Calidifetibacillus erzurumensis ZL34 is adopted as a producing strain, and the biological pesticide is produced through a microbial fermentation culture technology. The compound bactericide is an agricultural biological bactericide and has a special effect on dry rot of carya cathayensis.

Description

Technical field:

[0001] The invention relates to a live bacteria biological pesticide and a preparation method thereof, belonging to the technical field of microbial fermentation engineering. Background technology:

[0002] Carya cathayensis belongs to the Juglandaceae family. It is a unique economic tree species in my country. It is mainly distributed in the Tianmu Mountains at the junction of Zhejiang and Anhui provinces, including Lin'an District, Chun'an County, Anji County, Tonglu County in Zhejiang Province and Ningguo County and Jixi County in Anhui Province. It is an important economic source for farmers in mountainous areas to get rich. Dry rot caused by Botryosphaeria dothidea is currently the most important disease in pecan forests. It is common in pecan planting areas, with an incidence rate of 80% to 90% in some areas. In 2018, the total area of ​​pecan forests in Lin'an District was 32,000 hm2. 2 , of which the area of ​​walnut dry rot is 29,400 hm 2 , accounting for more than 90% of the total area of ​​disease; in 2021, the Hangzhou municipal forestry pest monitoring and reporting point randomly surveyed in Lin'an, and the incidence rate of new lesions of pecan dry rot was as high as 95.7%. Dry rot not only affects the fruit yield of pecan, but also weakens the tree vigor and even causes the death of trees, which seriously restricts the sustainable development of the pecan industry. At present, the common prevention and control methods are to scrape off small lesions, break large lesions, spray chemical agents and apply them to the stem, but there are many disadvantages. Pecans generally grow on steep slopes, and scraping and breaking lesions is time-consuming, laborious and difficult to operate; artificially caused new wounds are prone to re-invasion of pathogens, and multiple breaking causes great damage to the tree. The most problematic thing is that the pathogen has the characteristics of latent infection, overwintering in the xylem and deep infection of the trunk, which makes it difficult for conventional pesticides to reach effectively, the pathogen cannot be completely eradicated, and old lesions recur every year and are difficult to cure.

[0003] Purpose of the invention:

[0004] The purpose of the present invention is to overcome the defects of the prior art and provide a new plant endophytic bacteria live bacteria biological pesticide to fundamentally solve the problem of pecan dry rot prevention and control.

[0005] The technical solutions adopted to achieve the above objectives are:

[0006] The live bacteria biological pesticide of the present invention has a live bacteria component, which is an endophytic bacterium Calidifontibacillus erzurumensis ZL34 with strong inhibitory activity against the pecan dry rot pathogen Calidifontibacillus erzurumensis ZL34. Calidifontibacillus erzurumensis ZL34 is a plant endophytic bacterium that can reproduce and spread in the plant body and easily reach various parts of the plant. It can effectively solve the problem that conventional pesticides are difficult to effectively reach the deep part of the plant, control the pathogen from the source, and thus fundamentally solve the problem of pecan dry rot prevention and control.

[0007] The specific content of the present invention:

[0008] 1. Identification of strain ZL34

[0009] ⑴Morphological, physiological and biochemical characteristics

[0010] ZL34 appears as a creamy colony on LB medium, with a smooth surface in the early stage and obvious wrinkles and protrusions in the later stage, and irregular edges. Gram staining is positive, the bacteria are rod-shaped, with blunt ends, oval spores, and mainly mesophytes. It grows aerobically, can utilize glucose, sucrose, lactose, maltose and mannitol, ferment glucose to produce acid, hydrolyze starch and gelatin, can utilize citrate, can decompose tryptophan to form indole, and VP, oxidase, catalase and nitrate reduction reactions are all positive. Based on the above characteristics, ZL34 is preliminarily judged to be Bacillus sp.

[0011] (2) 16S rDNA sequence analysis

[0012] The 16S rDNA sequence of strain ZL34 (SEQ ID NO.1) has been submitted to GenBank for deposit, with NCBI accession number OR394552, and was compared with related genera and species to construct a phylogenetic tree ( Figure 1 ). It can be seen that ZL34 and the model strain P2 of Calidifontibacillus erzurumensis are clustered in the same branch, with a homology of more than 99%. Combining morphological and physiological and biochemical characteristics, strain ZL34 was identified as Calidifontibacillus erzurumensis.

[0013] 2. Antimicrobial spectrum test results of strain ZL34

[0014] As shown in Table 1, strain ZL34 has strong antibacterial activity against a variety of fungi such as Botrytis cinerea, Fusarium oxysporum and a variety of bacteria such as Staphylococcus aureus and Escherichia coli K88, indicating that the strain has a broad-spectrum antibacterial activity.

[0015] Table 1 Antimicrobial spectrum of strain ZL34

[0016]

[0017] Note: ten ten ten ten means the diameter of the inhibition zone is above 30mm, ten ten ten means the diameter of the inhibition zone is between 25-30mm, ten ten means the diameter of the inhibition zone is between 15-20mm, and ten means the diameter of the inhibition zone is between 10-15mm.

[0018] 3. Inhibitory effect of strain ZL34 on Botrytis cinerea

[0019] ⑴ Effect on the growth of mycelium in the grape cavity

[0020] As can be seen from Table 2, there are extremely significant differences in the inhibition rate of different dilution multiples of ZL34 fermentation supernatant on the growth of mycelium of Botrytis cinerea (P < 0.01), and the inhibition rate of 5-fold dilution is the highest, which is 96.48%. As the dilution multiple increases, the inhibition rate gradually decreases. The inhibition rate of 10-fold dilution of ZL34 fermentation supernatant is still greater than 85%. Optical microscopy shows that the mycelium of this concentration treatment group has obvious pigment accumulation, among which the aerial mycelium has constriction and swelling, the mycelium between the mycelial septa at the base is shortened, and a large number of conidia are produced. In contrast, the mycelium of the control group is uniform and smooth, with no pigment accumulation in the mycelium and no conidia produced. Under the electron microscope, it can be seen that in addition to swelling, the mycelium of the treatment group also shows shrinkage and protrusions, and some protrusions break or fall off to form holes, which further confirms the destructive effect of ZL34 fermentation supernatant on mycelium.

[0021] Table 2 Inhibitory effect of ZL34 fermentation supernatant on spore germination and mycelial growth of Botrytis cinerea

[0022]

[0023] ⑵ Effect on spore germination of Botrytis cinerea

[0024] As can be seen from Table 2, the spore germination rate of Botrytis cinerea treated with ZL34 fermentation supernatant at different dilution multiples was not significantly different from that of the control group (Table 2). From the morphological observation, the growth of newly germinated hyphae in the treatment group was significantly inhibited, as shown by fewer hyphae branches and multiple swellings, while the hyphae in the control group were uniform in thickness, strong, with many branches and stretched in a net-like shape ( Figure 3 ). This indicates that ZL34 produced active substances against Botrytis cinerea during fermentation, which could significantly inhibit the growth of new hyphae after spore germination.

[0025] 4. Preparation process:

[0026] ⑴Production strains:

[0027] The microbial strain used in the present invention is Calidifontibacillus erzurumensis ZL34, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit name being Calidifontibacilluserzurumensis ZL34, the deposit number being CGMCC No: 28458, and the deposit date being September 14, 2023.

[0028] ⑵ Culture medium:

[0029] a. Seed slant culture medium:

[0030] Peptone 1%, glucose 0.5%, yeast extract 0.5%, NaCI 0.5%, pH 7.2-7.4.

[0031] b. Fermentation medium:

[0032] The nutrient sources in the culture medium are commonly used carbon sources and nitrogen sources. The carbon source can be corn flour, sweet potato starch, dextrin, sucrose, glucose, etc.; the nitrogen source can be soybean cake powder, corn steep liquor, urea, ammonium sulfate, peptone, yeast powder, etc. On this basis, some other nutrients are added, such as salt, phosphate, potassium salt, calcium salt, etc.

[0033] ⑶Cultivation conditions:

[0034] The culture temperature is 26-38°C, the culture pH is 6-9, and the culture time is 30 to 92 hours.

[0035] ⑷Product post-processing:

[0036] The culture fluid is adjusted to a certain spore content, and after adding appropriate amounts of preservatives and emulsifiers, it is packaged to form an aqueous product; or after adding appropriate amounts of adsorbents for adsorption, drying and crushing, it is packaged to form a powder product.

[0037] For specific process flow, please see the attached manual Figure 4 .

[0038] The significant advantages of the present invention are:

[0039] 1. High efficiency: The pathogen of pecan dry rot has the characteristics of latent infection, overwintering in the wood and deep infection in the trunk, which makes it difficult for conventional pesticides to reach effectively, the pathogen cannot be completely eradicated, and old lesions recur every year and are difficult to cure. Calidifontibacillus erzurumensis ZL34 is an endophytic bacterium that can reproduce in the plant body and easily reach all parts of the plant. It can effectively solve the problem that conventional pesticides are difficult to effectively reach deep into the plant, control the pathogen from the source, and thus effectively solve the problem of pecan dry rot prevention and control.

[0040] 2. Safety: The pecan dry rot control pesticide of the present invention is a live bacteria biological pesticide, and its active ingredient is the plant endophytic bacterium Calidifontibacillus erzurumensis ZL34, which is separated from a healthy pecan tree, and thus has obvious safety compared with conventional pesticides.

[0041] 3. Low price: The pesticidal pesticide for preventing and controlling pecan dry rot of the present invention is prepared by using microbial fermentation technology. The main components of the fermentation medium used are all common agricultural and sideline products, and the fermentation cycle is relatively short, so the production cost is relatively low. Description of the drawings:

[0042] Figure 1 Phylogenetic tree of strain ZL34 based on 16S sequence

[0043] Figure 2 Effects of ZL34 fermentation supernatant on the mycelial morphology of Botrytis cinerea:

[0044] ① Optical microscope (400X): A-aerial hyphae in the treatment group: constriction (arrow), pigment accumulation, and swelling (circle); B-basal hyphae in the treatment group: conidia (arrow), shortened intervals, and pigment accumulation (circle); C-hyphae in the control group;

[0045] ② Cryo-scanning electron microscopy: D-Hyphae in the treatment group: wrinkled (asterisk), swollen (triangle), protruded (circle), and broken (arrow); E-Hyphae in the control group

[0046] Figure 3 Effects of ZL34 fermentation supernatant on newly germinated hyphae of Botrytis cinerea spores: A-treatment group; B-control group

[0047] Figure 4 Process flow chart of biopesticide product preparation Specific implementation method:

[0048] The present invention has the following embodiments:

[0049] Example 1: Preparation of an aqueous solution containing 10 billion spores / ml

[0050] In a 70L fermentation tank, 4% soybean meal, 4% corn meal, 0.5% urea, 0.6% (NH4)2HPO4, and 0.35% CaCO3 are prepared into a 50L culture medium, the pH is adjusted to 7.0, 121°C, cooled to 38°C after 30 minutes, inoculated with 5% production bacteria ZL34, and cultured at 38°C for 36 hours to obtain a fermentation liquid with a spore content of 5.5 billion / mL. After precipitation and concentration, the fermentation liquid is diluted to a suspension with a spore content of 10 billion / ml, and then 0.12% sodium benzoate and 10% saponin are added as preservatives and emulsifiers respectively to obtain the product. The product has a spore content of 10 billion / ml, a suspension rate of 65-90%, a pH of 4.5-7.0, and a fineness of 95% passing through a 100-mesh sieve.

[0051] Example 2: 10 billion / ml aqueous solution product for preventing and controlling pecan dry rot

[0052] 1. Test reagent and concentration:

[0053] The 10 billion / ml aqueous solution product in Example 1 was diluted 100 times with clean water as the test agent, the control agent was a 500-fold dilution of 30% difenoconazole suspension, and the blank control was clean water.

[0054] 2. Test methods:

[0055] In the pecan forest, nine plots with similar disease conditions were randomly divided for control efficacy test, with three plots in each treatment. Spray the entire diseased trunk, spray once every 10 days, and spray twice in succession. Investigate the disease index 10 days after the second spraying and calculate the control efficacy. Refer to Table 3 for the grading standard of pecan dry rot.

[0056]

[0057] Table 3 Classification standards for pecan dry rot

[0058]

[0059] 3. Test results:

[0060] It can be seen from Table 4 that the 100-fold dilution of ZL34 preparation has a good control effect on pecan dry rot, which is 79.6%, slightly inferior to the 500-fold dilution of 30% difenoconazole suspension, and better than the 1000-fold dilution of 30% difenoconazole suspension.

[0061] Table 4 The efficacy of ZL34 product in preventing pecan dry rot

[0062]

[0063]

[0064] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined by the claims.

[0065] Sequence Listing

[0066] SEQ ID NO.1

[0067] 16S rDNA sequence of strain ZL34:

[0068]

Claims

1. A plant endophytic bacterium, which was systematically identified as Calidifontibacillus erzurumensis.

2. The method for preparing the endophytic bacteria of claim 1, which is produced by microbial fermentation technology, is characterized in that The producing bacterium is Calidifontibacillus erzurumensis, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit name being Calidifontibacillus erzurumensis ZL34 and the deposit number being CGMCC No: 28458.

3. The agricultural and forestry disease control pesticide with the plant endophytic bacteria as the active ingredient as claimed in claim 1.

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