Method for controlling pear tree diseases and insect pests by combining predatory mites and Bacillus amyloliquefaciens

Abstract

The present invention discloses a method for controlling pear tree pests and diseases using a combination of predatory mites and Bacillus amyloliquefaciens. The predatory mite is Neoseiulus barkeri. The method comprises the following steps: 1) preparing biocontrol bacteria: preparing a Bacillus amyloliquefaciens powder; 2) preparing predatory mites: preparing adult Neoseiulus barkeri mites in a matrix material; 3) preparing a mixed biocontrol product: mixing the Bacillus amyloliquefaciens powder with the matrix material containing Neoseiulus barkeri mites before use; the mixed biocontrol product is sealed in a package and stored at 13-17°C; 4) applying the agent: applying the mixed biocontrol product during the second physiological fruit drop period of the pear tree, opening the package and securing it to the trunk of the pear tree; the mixed biocontrol product used per pear tree contains 160-190 million CFU of Bacillus amyloliquefaciens and 2,900-3,100 Neoseiulus barkeri mites. The method can simultaneously control pear mite pests and pear fire blight, achieving a fire blight control efficacy of over 80%.

Description

Technical Field

[0001] The invention relates to the technical field of fruit tree disease and insect pest control, and in particular to a method for controlling pear tree diseases and insect pests by combining predatory mites and Bacillus amyloliquefaciens. Background Art

[0002] The Korla fragrant pear is an important specialty cash crop in Xinjiang and a national geographical indication product in China. To date, its cultivated area has exceeded one million mu (approximately 1,000,000 mu), with a brand value of 15 billion yuan. Korla fragrant pear cultivation is subject to a variety of pests and diseases. Among them, mites and pear fireblight are the most serious constraints to the safe production of Korla fragrant pears, significantly impacting both yield and quality. Pear fireblight is a pear disease caused by the bacterium Erwinia amylovora (Burr) Winslow et al. Currently, mites and pear fireblight are primarily controlled with chemical pesticides. The long-term, monopolistic use of chemical pesticides can lead to a series of serious social and ecological problems. Excessive use not only results in excessive pesticide residues in fruit, damaging the ecological environment, but also contributes to the resurgence of pests and diseases. Therefore, it is necessary to find safe and effective measures to control these major pear pests and diseases.

[0003] Predatory mites, predatory natural enemies of pest mites, belong to the subclass Acari, order Parasitoides. There are numerous species, including the common Neoseiulus, Chilean Phytoseiulus, Decker's Amblyseiulus, and Western Typhlosei. They primarily prey on small pests such as spider mites, gall mites, tarsonema mites, aphids, and scale insects. The development and promotion of biological control products is an ideal approach for pest and disease control. Currently, microbial pesticides and natural enemies of pests are widely used in production, such as predatory mites for pest control and microbial pesticides for controlling major pests and diseases. The promotion and application of biological control products not only reduces the use of chemical pesticides but also plays a significant role in sustainable pest and disease control. Even more significant is the recent development of methods that combine two biological control products for synergistic control of major pests. This expands the scope of pest control, improves pest control effectiveness, and significantly reduces labor, achieving twice the result with half the effort. However, there are currently no reports of the application of products that control both pests and diseases for biological control of fruit tree pests and diseases in Xinjiang. Summary of the Invention

[0004] The object of the present invention is to address the above problems and provide a method for simultaneously preventing pear tree pest mites and amylopectin disease by using mixed biocontrol products.

[0005] In order to achieve its purpose, the present invention adopts the following technical solutions:

[0006] A method for controlling pear tree diseases and insect pests using a combination of predatory mites and Bacillus amyloliquefaciens, characterized in that the predatory mite is Neoseiulus barkeri, comprising the following steps:

[0007] 1) Prepare biocontrol bacteria: Prepare Bacillus amyloliquefaciens powder;

[0008] 2) Preparing predatory mites: preparing adult Neoseiulus barkeri mites in a matrix material;

[0009] 3) Preparing a mixed biocontrol product: Before use, mix the Bacillus amyloliquefaciens powder and the matrix material containing Neoseiulus pasteurii. The mixed biocontrol product is sealed in a package and stored at 13-17°C.

[0010] 4) Application of pesticides: Apply during the second physiological fruit drop period of the pear trees. Open the package of the mixed biocontrol product and fix it on the trunk of the pear tree. The mixed biocontrol product used for each pear tree contains 160-190 million CFU of Bacillus amyloliquefaciens and 2,900-3,100 Neoseiulus pasteurii mites.

[0011] The insect pest is spider mite pest, and the disease is fire blight.

[0012] The Bacillus amyloliquefaciens powder is a wettable powder.

[0013] The packaging is a packaging bag, a packaging box or a packaging bottle.

[0014] The mixed biocontrol product used on each fruiting pear tree contains 160-180 million CFU of Bacillus amyloliquefaciens or 165-175 million CFU of Bacillus amyloliquefaciens and 2930-3070 Neoseiulus pasteurii mites.

[0015] The pear tree is 7 years old or older. Generally, Korla fragrant pear trees 7 years old or older are fruit-bearing trees, and young trees under 7 years old are basically not infected with pear fire blight.

[0016] The matrix material in step 2) is wheat bran.

[0017] In the above technical solution, the mass ratio of Bacillus amyloliquefaciens powder to substrate containing Neoseiius pasteurii in the mixed biocontrol product applied to each pear tree is 1:25-35, 1:27-33, or 1:29-31. The Bacillus amyloliquefaciens powder contains 400-600 million CFU / gram of Bacillus amyloliquefaciens, and each gram of substrate containing Neoseiius pasteurii contains 250-350, 270-330, or 290-310 Neoseiius pasteurii mites. Preferably, 8-12 grams or 9-11 grams of the mixed biocontrol product is applied to each pear tree.

[0018] In the above technical solution, the pear tree is Korla fragrant pear.

[0019] The present invention provides a novel method for synergistically controlling important pear pests and diseases using two biological control products. A mixed product of Neoseiulus pasteurii and Bacillus amyloliquefaciens can simultaneously control pear mites and fire blight. Research into the optimal application period revealed that application during the second physiological fruit drop period of pear trees not only achieved excellent control of the mites, but also achieved over 80% control of fire blight, significantly outperforming other application periods. The present method utilizes only biological control products, eliminating the use of chemical agents. It is environmentally friendly, pollution-free, and offers significant pest and disease control effectiveness. It addresses a series of serious social and ecological issues associated with the long-term use of chemical pesticides, including excessive pesticide residues in fruit and the resurgence of pests and diseases due to chemical pesticide resistance. DETAILED DESCRIPTION

[0020] The present invention will be further described below with reference to the embodiments, but the present invention is not limited thereto.

[0021] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0022] Example 1

[0023] 1. Method for selecting efficient predatory mites by evaluating their predatory ability

[0024] Following the method of McMurtry and Scriven (1964), fresh, round, 0.7 cm diameter Korla fragrant pear leaves were placed in each groove. Three, six, nine, 12, 15, and 18 eggs or adult females of the Turkestani spider mite (Tetranychus turkestani (Ugarov et Nikolski)) were then placed in the grooves. A single 24-hour-starved Neoseiulus barkeri, Neoseiulus cucumeris, or Neoseiulus californicus mite was then placed in the grooves. The leaves were then placed in an artificial climate incubator at 25°C for 24 hours to observe the predation of the three predatory mites on the Turkestani spider mites. Each mite density was considered a treatment, each groove a replicate, and each treatment was replicated 10 times. The experimental data were fitted using a Holling type II functional response (Holling, 1959), using the formula N to calculate the effect. ɑ =ɑTN / (1+ɑT h N), where N ɑ is the number of prey captured; ɑ is the instantaneous attack rate; T is the total time of the experiment; N is the prey density, that is, the initial density of pest mites; T h The time it takes for the predatory mite to process one prey. Take the inverse of the formula and transform it into 1 / N ɑ =1 / ɑTN+T h / T, use the least squares method to obtain the parameters ɑ and T h , use ɑ / T h The values ​​were used to evaluate the predatory ability of the three predatory mites.

[0025] 2. Safety evaluation of Bacillus amyloliquefaciens against Neoseiulus pasteurii

[0026] A 500 million CFU / g wettable powder of Bacillus amyloliquefaciens was diluted 10-fold, 20-fold, and 30-fold with water to treat Neoseiulus barkeri. After the solution dried completely on the mites, nymphs and adults were selected and placed in grooved glass plates to be reared with individual Turkestan spider mites. The temperature was 25±1°C and the relative humidity was 75±5%. Each treatment was repeated four times, with 30 mites treated. Survival of the mites was examined 24, 48, and 72 hours after treatment.

[0027] 3. Mixed production method of predatory mites and Bacillus amyloliquefaciens

[0028] One day before using the two biological control products, 500 million CFU / g of Bacillus amyloliquefaciens wettable powder and wheat bran matrix with predatory mites (Neoseius pasteurii) (300 heads / g matrix) were mixed at a weight ratio of 1:30 and packaged using a packaging machine. Each bag contained 10 grams and was stored at 15°C after packaging.

[0029] 4. The actual control effect of the combination of predatory mites and Bacillus amyloliquefaciens on pear tree pest mites and apricot fire blight

[0030] 4.1 Overview of the test site

[0031] The experimental site was located in Group 1, Bagjigedai Village, Halayugong Township, Korla City, Xinjiang. The test variety was Korla Fragrant Pear, 8-10 years old, with a plant spacing of 5m x 6m, an average plant height of 4.5m, and a canopy width of 5m. The experimental site was a flat plot with good light conditions, uniform growth, and consistent fertilization and water management. The plants grew vigorously and had dense branches and foliage, but weeds such as gray pigweed and field bindweed were present in the field. No pesticides were applied in the orchard 15 days before or after the release of Neoseiulus barkeri. Based on the concept of a "self-sustaining" predatory mite population, predatory mites were released using an inoculation-based hanging method at the end of flowering, the first physiological fruit drop, and the second physiological fruit drop of the Korla Fragrant Pear. During this period, eggs, nymphs, and adults of the Turkestan spider mite were present.

[0032] 4.2 Experimental design and arrangement

[0033] 4.2.1 Processing and numbering

[0034] Table 1 Treatment of pear orchard test with a mixture of Bacillus amyloliquefaciens wettable powder and predatory mites

[0035] Processing number Treatment method 1 Hybrid product late bloom release 2 The mixed product is released during the first physiological fruit drop period 3 The mixed product is released during the second physiological fruit drop period 4 No release of mixed biological control products

[0036] 4.2.2 Community Arrangement

[0037] The plots were arranged in random blocks, with protection strains left between the plots.

[0038] Table 2 Schematic diagram of experimental plots

[0039] 3-1 2-1 1-1 4-1 2-2 4-2 3-2 1-2 4-3 3-3 1-3 2-3 1-4 4-4 2-4 3-4

[0040] 4.2.3 Plot area and repetition

[0041] Plot size: 4 treatments, totaling 10 mu (approximately 10 mu), with 4 trees surveyed per plot, for a total of 16 plots and 64 trees. Replication: 4 replicates (plots) per treatment.

[0042] 4.3 Application method

[0043] The mixed biological control product was released at three different times: the last flowering period (April 26, 2021), the first physiological fruit drop period (May 10, 2021), and the second physiological fruit drop period (June 20, 2021). Plastic bags containing the mixed biological control product were cut and nailed to the bifurcation of the pear tree trunk with the bag opening facing upwards. One bag of biological control product was hung on each tree. Neoseiulus barkeri mites were released after 5:00 PM on the day of release.

[0044] 4.4 Investigation and control effectiveness calculation method

[0045] 4.4.1 Survey of Turkestan spider mites and calculation of control efficacy

[0046] Each tree was marked with a mite-infested spring shoot from the current year at the east, south, west, north, and center of the canopy. Mite populations were surveyed at the time of the last biocontrol application and 7, 14, 21, 28, 38, and 48 days after application. Active mites were counted on two to three leaves from each marked branch, for a total of 25 leaves from two trees in each plot. The total number of insects in each treatment was counted, and the number of Turkestan spider mites on each leaf was calculated. The changes in the Turkestan spider mite population were analyzed, and the insect population reduction rate and corrected control efficacy were calculated for each period. The insect population reduction rate (%) = [(insect population before application - insect population after application) / insect population before application] × 100. The corrected control efficacy (%) = [(insect population reduction rate in the treatment area - insect population reduction rate in the control area) / (100 - insect population reduction rate in the control area)] × 100.

[0047] 4.4.2 Pear Fire Blight Investigation Methods and Control Efficacy Calculation Methods

[0048] Investigation method: For each tree, 10 branches were fixed in each of the four directions of east, south, west and north. The percentage of diseased branches (branches with branch dieback (shepherd's whip) symptoms) in each direction was recorded as a percentage of the total number of branches, and the disease index and prevention and control effect were calculated.

[0049] Grading standards for dead branches (shepherd's whip):

[0050] Level 0: No occurrence;

[0051] Level 1: The number of diseased branches accounts for 1%-10% of the total number of branches surveyed;

[0052] Level 3: The number of diseased branches accounts for 11%-20% of the total number of branches surveyed;

[0053] Level 5: The number of diseased branches accounts for 21%-35% of the total number of branches surveyed;

[0054] Level 7: The number of diseased branches accounts for 36%-50% of the total number of branches surveyed;

[0055] Level 9: The number of diseased branches accounts for more than 51% of the total number of branches surveyed.

[0056] Survey time and frequency: Survey once 7 days and 14 days after the last release of biological control products, for a total of 2 surveys.

[0057] Efficacy calculation method: The data were calculated according to the relevant pesticide field efficacy test guidelines. The data obtained from the test were statistically analyzed using data processing software. The difference significance was analyzed using the new multiple range method. The test results were analyzed and evaluated.

[0058]

[0059]

[0060] 5 Results and Analysis

[0061] 5.1 Comparison of the predatory abilities of different predatory mites against Turkestan spider mites

[0062] The predatory responses of Neoseiulus barkeri, Neoseiulus cucumeri, and Neoseiulus californicus to Turkestan spider mites are shown in Table 3. Among the three predatory mites, Neoseiulus barkeri has the strongest predatory ability against both eggs and adults of Turkestan spider mites, making it the most ideal agent for controlling Turkestan spider mites. Therefore, we selected a mixture of Neoseiulus barkeri and Bacillus amyloliquefaciens for controlling Turkestan spider mites in pear orchards.

[0063] Table 3 Functional responses of different predatory mites to the Turkestan spider mite

[0064]

[0065] 5.2 Safety Analysis of Bacillus amyloliquefaciens and Neoseiulus pasteurii

[0066] The toxicity of Bacillus amyloliquefaciens to Neoseiius pasteurianus is shown in Table 4. After the nymphs and adults of Neoseiius pasteurianus were treated with 10-fold, 20-fold and 30-fold dilutions of 500 million CFU / g of Bacillus amyloliquefaciens wettable powder for 24 hours, 48 ​​hours and 72 hours respectively, there was no significant difference in the number of surviving nymphs and adults of Neoseiius pasteurianus and the blank control (treated with clean water), indicating that Bacillus amyloliquefaciens has no toxic effect on Neoseiius pasteurianus and that Bacillus amyloliquefaciens and Neoseiius pasteurianus can be safely mixed.

[0067] Table 4 Toxicity of Bacillus amyloliquefaciens to Neoseiulus pasteurii

[0068]

[0069] 5.3 Control effect of mixed biological control products of Neoseiulus pasteurii nymphs and Bacillus amyloliquefaciens on Turkestan spider mites in the field

[0070] Seven days after the last release, the mite population reduction rate in the area where the mixed biological control product was released during the second physiological fruit drop period was only 3.94%, and the corrected control efficacy (26.30%) was significantly (p < 0.05) lower than the control efficacy (35.38%) in the area where the biological control product was released during the last flowering period. However, from 14 to 48 days after the release, the corrected control efficacy of the three release treatments gradually increased. By 48 days, the corrected control efficacy of the mite population in the area where the mixed biological control product was released during the second physiological fruit drop period reached 100%, the highest control efficacy. No significant differences in control efficacy were observed during the same period (see Table 5).

[0071] Table 5 Control effect of mixed biological control products of predatory mites and Bacillus amyloliquefaciens on Turkestan spider mites in the field (%)

[0072]

[0073] *Note: The efficacy (%) in the table above is the average of each replicate. Different lowercase and uppercase letters after the data in the same column indicate differences at the 0.05 and 0.01 levels, respectively. The significance was determined using the Duncan's new multiple range (DMRT) method.

[0074] 5.4 Effect of a mixed biological control product of Neoseiulus pasteurii nymphs and Bacillus amyloliquefaciens on fire blight in the field

[0075] The efficacy of the mixed biocontrol product containing predatory mites and Bacillus amyloliquefaciens against fire blight is shown in Table 6. Seven and 14 days after application, the fire blight control rates in the area released during the second physiological fruit drop period were 82.11% and 80.94%, respectively, both significantly higher than those in the area released during the final flowering period and the first physiological fruit drop period.

[0076] Table 6 Control effect of predatory mites and Bacillus amyloliquefaciens mixture on fire blight

[0077]

[0078] *Note: The efficacy (%) in the table above is the average of each replicate. Different lowercase and uppercase letters after the data in the same column indicate differences at the 0.05 and 0.01 levels, respectively. The significance was determined using the Duncan's new multiple range (DMRT) method.

[0079] 5.5 Conclusion

[0080] Systematic research has revealed that Neoseiulus pasteurii is an ideal natural enemy for controlling pear mites. Bacillus amyloliquefaciens has no toxic effects on Neoseiulus pasteurii, and therefore, Neoseiulus pasteurii and Bacillus amyloliquefaciens can be used together. Field results have shown that a 1:30 weight ratio of 500 million CFU / g of Bacillus amyloliquefaciens wettable powder mixed with a substrate containing 300 predatory mites per gram, applied during the second physiological fruit drop period of Korla fragrant pears, achieves optimal control of both Turkestan spider mites and fire blight. This represents a new, highly effective and safe method for controlling major pear pests and diseases.

Claims

1. A method for controlling pear tree diseases and insect pests by using a predatory mite and Bacillus amyloliquefaciens in combination, characterized in that: The predatory mite is Neoseiulus barkeri, the insect pest is spider mite pest, and the disease is fire blight of amylovora, comprising the following steps: Prepare biocontrol bacteria: prepare Bacillus amyloliquefaciens powder; Prepare predatory mites: prepare adult Neoseiulus barkeri mites in a matrix material, the matrix material being wheat bran; Preparation of mixed biocontrol products: Before use, mix Bacillus amyloliquefaciens powder and The mixed biocontrol product comprises a matrix material containing mites, wherein the Bacillus amyloliquefaciens powder contains 400-600 million CFU / gram of Bacillus amyloliquefaciens, and each gram of the matrix containing Neoseiulus pasteurianus contains 250-350 Neoseiulus pasteurianus mites. The mass ratio of the Bacillus amyloliquefaciens powder to the matrix containing Neoseiulus pasteurianus in the mixed biocontrol product is 1:25-35. The mixed biocontrol product is sealed in a package and stored at 13-17°C. Application of pesticide: Apply during the second physiological fruit drop period of the pear tree. Open the package of the mixed biocontrol product. Fixed on the trunk of the pear tree; the mixed biocontrol product used for each pear tree contains 160-190 million CFU of Bacillus amyloliquefaciens and 2,900-3,100 Neoseiulus pasteurii mites.

2. The method according to claim 1, wherein: The Bacillus amyloliquefaciens powder is a wettable powder.

3. The method according to claim 1, wherein: The packaging is a packaging bag, a packaging box or a packaging bottle.

4. The method according to claim 1, wherein: The mixed biocontrol product used on each fruiting pear tree contains 160-180 million CFU of Bacillus amyloliquefaciens and 2,930-3,070 Neoseiulus pasteurii mites.

5. The method according to claim 4, characterized in that: The pear trees are 7 years old or older.

6. The method according to claim 1, wherein: Apply 8-12 grams of mixed biocontrol products per pear tree.

7. The method according to claim 1, wherein: The pear tree is Korla fragrant pear.

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