A strain of nematophilic Serratia, insecticide, preparation method and application thereof

Abstract

The present invention discloses a strain of Serratia nematodiphila, named as Serratia nematodiphila FQ268, which was deposited in China Center for Type Culture Collection on September 22, 2023, with a deposit number of CCTCC M 20231765. The present invention also discloses an insecticide containing the Serratia nematodiphila, a preparation method thereof, and an application thereof in preventing and controlling diseases and insect pests. The Serratia nematodiphila FQ268 of the present invention has a control effect on red spider mites of the order Acarina, thrips of the order Thysanoptera, and nematodes of the order Serratia, especially a significant control effect on pepper red spider mites in the field.

Description

Technical Field

[0001] The invention belongs to the technical field of green agricultural production, and in particular relates to a nematophilic Serratia, an insecticide, and a preparation method and application thereof. Background Art

[0002] Vegetables in my country have become the second largest crop after grain crops, and the planting area and total output are both ranked first in the world. During the planting process, the economic losses caused by pests and diseases to vegetables can reach more than 50%, especially red spiders, thrips, nematodes, diamondback moths, leaf beetles, peach aphids, leafminers and other pests. At present, chemical control is the main method for vegetable pest control, but due to the blind use of chemical drugs, problems such as product quality and safety, environmental pollution and increased pest resistance often occur. With the rapid development of facility agriculture, the area of ​​protected cultivation has expanded year by year. In addition, the environmental conditions in protected areas are suitable for pest reproduction, which has gradually aggravated pests. The increase in the number of drug use has led to excessive pesticide residues in vegetables, affecting human health. The use of biocontrol bacteria for biological pest control measures not only meets people's demand for green food, but also reduces the use of chemical pesticides, reduces pesticide residues, ensures production quality, improves yield, increases farmers' benefits, protects the environment, maintains ecological balance, and provides reliable guarantees for the sustainable development of agriculture. Therefore, the development and application of safe microbial pesticides that are not likely to cause pests to develop resistance has become a hot topic in current research.

[0003] At present, the most studied is Bacillus thuringiensis, which can control a variety of lepidopteran vegetable pests, such as diamondback moth, cabbage worm, beet armyworm, etc. There are also biocontrol bacteria such as green muscardine, white bassiana, and Bacillus subtilis, but these biocontrol bacteria have certain requirements for the environment and have not yet been widely used in the field. Therefore, the research and development of microbial pesticides to screen and dig out biocontrol strains that can be promoted on a large scale is of great significance for green control of vegetable pests. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the above background technology and provide a strain of Serratia nematophila, an insecticide and a preparation method and application thereof.

[0005] In order to solve the above technical problems, the technical solution proposed by the present invention is:

[0006] A strain of Serratia nematodiphila, named Serratia nematodiphila FQ268, was deposited in the China Center for Type Culture Collection on September 22, 2023, with the deposit number CCTCC M 20231765.

[0007] As a general inventive concept, the present invention also provides an insecticide fungicide, which comprises the Serratia nematophila according to claim 1, a fermentation broth of the Serratia nematophila or a metabolite of the Serratia nematophila.

[0008] In the above-mentioned insecticide fungicide, preferably, the viable bacteria count of Serratia nematophila FQ268 in the insecticide fungicide is 20-30 billion CFU / mL.

[0009] As a general inventive concept, the present invention also provides a method for preparing the above-mentioned insecticide fungicide, comprising the following steps:

[0010] (1) inoculating Serratia nematophila FQ268 on LB solid culture medium for activation to obtain activated Serratia nematophila FQ268;

[0011] (2) inoculating the activated Serratia nematophila FQ268 into a seed culture medium for primary fermentation to obtain a primary seed solution of Serratia nematophila;

[0012] (3) subjecting the primary seed liquid of Serratia nematophila to secondary fermentation to obtain a secondary seed liquid of Serratia nematophila;

[0013] (4) The secondary seed liquid of Serratia nematophila is subjected to tertiary fermentation. After the fermentation is completed, the culture liquid is directly packaged into a liquid dosage form, that is, a microbial insecticide containing Serratia nematophila FQ268.

[0014] In the above preparation method, preferably, in step (1), the formula of the LB solid culture medium is: 8-10 g / L peptone, 5-6 g / L yeast extract, 8-10 g / L sodium chloride, 15-18 g / L agar, and pH is 7.2-7.3;

[0015] The activation temperature is 28-30° C. and the activation time is 22-24 hours.

[0016] In the above preparation method, preferably, in step (2), the formula of the seed culture medium is: 18-20 g / L peptone, 8-10 g / L sodium chloride, 8-10 g / L yeast extract powder, and the pH is 7.2-7.3;

[0017] The process conditions of the primary fermentation include: liquid volume 60-70%, inoculation amount 7%-10%, culture temperature 28-30°C, and shaking culture at 180-200 rpm for 18-20 hours.

[0018] In the above preparation method, preferably, in step (3), the process conditions of the secondary fermentation include: liquid volume 60-70%, inoculation amount 7%-10%, culture temperature 28-30°C, and shaking culture at 180-200 rpm for 18-20 hours.

[0019] In the above preparation method, preferably, in step (4), the process conditions of the tertiary fermentation include: liquid volume 60-70%, inoculation amount 7%-10%, culture temperature 28-30°C, and shaking culture at 150-180rpm for 22-24h.

[0020] In the above preparation method, preferably, in step (3) and step (4), the formula of the culture medium used in the secondary fermentation and the tertiary fermentation is: 20-30 g / L glucose, 50-60 g / L bran, 10-12 g / L yeast powder, 2-5 g / L magnesium sulfate, 3-5 g / L disodium hydrogen phosphate, 2-5 g / L sodium chloride, and the pH is 7.2-7.3.

[0021] As a general inventive concept, the present invention also provides a use of the above-mentioned nematophilic Serratia or the above-mentioned insecticide fungicide or the insecticide fungicide prepared by the above-mentioned preparation method in preventing and controlling pests and diseases.

[0022] In the above application, preferably, the pests and diseases include red mites of the order Acarina, thrips of the order Thysanoptera, and nematodes of the order Sciaenidae, and further preferably are red mites of the order Acarina, thrips of the order Thysanoptera, and nematodes of the order Sciaenidae on peppers, cabbages, and melons.

[0023] Compared with the prior art, the advantages of the present invention are:

[0024] (1) The nematophilic Serratia marcescens FQ268 of the present invention has a control effect on Acarina red spider mites, Thysanoptera thrips, and Cynocephala nematodes, especially a significant control effect on pepper red spider mites in the field.

[0025] (2) The insecticide fungicide containing the nematophilic Serratia, fermentation broth products or metabolites of the present invention has high activity and a long shelf life.

[0026] (3) The insecticide fungicide of the present invention has enhanced insecticidal efficacy and can significantly prevent and control the occurrence of insect pests on vegetables, thereby increasing vegetable yields. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the colony morphology of Serratia nematophila FQ268 on the front of the plate;

[0028] Figure 2 Phylogenetic tree analysis of the 16S rDNA sequence of strain FQ268 constructed by the neighbor-joining method;

[0029] Figure 3 It is a statistical line graph of the indoor toxicity of Serratia nematophila FQ268 to pepper spider mites;

[0030] Figure 4This is an indoor toxicity test of Serratia nematophila FQ268 against pepper spider mites;

[0031] Figure 5 The toxicity effect of different concentrations of the nematophilic Serratia marcescens FQ268 on cabbage thrips. DETAILED DESCRIPTION

[0032] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings and preferred embodiments of the specification, but the protection scope of the present invention is not limited to the following specific embodiments.

[0033] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0034] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0035] Embodiment 1:

[0036] 1. Isolation and screening of bacteria

[0037] 1. At the tomato base in Haitang District, Sanya City, Hainan Province, use a sampling shovel to scrape off the surface of the tomato soil, take 100g of soil at a depth of 20-100mm and put it into a sterile kraft paper bag, number and record the collection site and time, and bring it back to the laboratory for strain isolation and purification.

[0038] 2. Take 0.1 g of sample with a sterilized spoon and pour it into a 1.5 mL centrifuge tube containing 0.9 mL of LB liquid medium. Incubate at 30°C and 100 rpm for 30 min, let stand for 20 min, and dilute to 10 by 10-fold gradient dilution. -3 Gradient, take 200 μL of the gradient dilution, evenly spread it on LB medium, pick a single colony for bacterial purification, the front colony morphology is as follows Figure 1 shown.

[0039] 2. The morphological, cultural, physiological and biochemical characteristics of Serratia nematophila FQ268 strain are as follows:

[0040] The strains obtained above were subjected to catalase reaction, methyl red test, indole production, citric acid utilization, determination of sugar alcohols (mannitol, glucose, fructose and inositol) utilization and antibiotic determination according to the "Methods for Plant Pathology Research". The statistical results are shown in Table 1. It can be seen from Table 1 that strain FQ268 can utilize mannitol, fructose and glucose, but cannot utilize inositol and citric acid; catalase reaction and methyl red test are negative, indole is produced, and it is insensitive to the antibiotics ampicillin sodium and tetracycline, but sensitive to rifampicin.

[0041] Table 1 Physiological and biochemical results and carbohydrate utilization of strain FQ268

[0042] Measurement items FQ268 Mannitol + fructose + glucose + Inositol － Catalase － Methyl red test － Citric acid utilization － Production of indole + Ampicillin Sodium Insensitive Rifampin 7-10mm tetracycline Insensitive

[0043] “+” means positive reaction, “-” means negative reaction.

[0044] The strain FQ268 was identified by 16S rDNA identification technology. The 16S rRNA gene was amplified using bacterial universal primers (sequences shown in SEQ ID: 1 and SEQ ID: 2). After PCR amplification, the PCR product was detected by electrophoresis using 1% agarose gel; the gene product obtained by PCR was sequenced, and the obtained sequence was entered into the NCBI database and compared and analyzed using BLAST software. Strains with higher homology to the strain were selected, and multiple sequence alignment was performed using MEGA 7.0 software to establish a phylogenetic tree, see Figure 2 As shown. The 16S rRNA gene fragment of strain FQ268 was obtained by PCR amplification, and its sequence length was 1398bp, as shown in SEQ ID: 3. The sequence analysis and comparison showed that the similarity between strain FQ268 and Serratianematodiphila reached 99.93%, and its morphological characteristics and physiological and biochemical characteristics were closest to those of Serratia nematodiphila. Therefore, strain FQ268 was identified as Serratia nematodiphila. The strain was named Serratia nematodiphila FQ268 and was preserved in the China Center for Type Culture Collection, Wuhan (Wuhan University). The strain preservation number is CCTCC NO: M 20231765, and the preservation time is September 22, 2023.

[0045] SEQ ID: 1: 27F 5'-AGAGTTTGATCCTGGCTCAG-3';

[0046] SEQ ID: 2: 1492R 5'-TACGGCTACCTTGTTACGACTT-3';

[0047]

[0048] 3. Indoor toxicity test of Serratia nematophila against pepper red spider

[0049] The strain FQ268 preserved in glycerol was spread on LB solid plate medium, placed in a 30℃ incubator for overnight culture, and a single colony was picked and fermented in a triangular flask containing 50mL LB liquid medium. The fermentation conditions were: temperature 30℃, speed 180rmp, time 24h, and the bacterial solution was obtained for standby use. The formula of LB liquid medium: 10g peptone, 5g yeast powder, 10g sodium chloride, dilute to 1000mL with distilled water, and sterilize at 121℃ for 30min. The leaf immersion method was adopted, three layers of filter paper were placed in the culture dish, and the filter paper was soaked with the bacterial solution using a 10mL pipette. Three pieces of fresh pepper leaves of the same size were cut and placed on the filter paper. The petiole of the leaf was wrapped with a moistened sterilized cotton ball, and then the cotton ball was surrounded with plastic wrap; the red spider was picked up on the leaf with a fine brush, and 20 insects were tested in each group of experiments, and the same experiment was repeated in 2 groups. The control group CK used sterile water to treat the red spider. After treatment, all disposable culture dishes were placed in a biological incubator. The environmental conditions were a temperature of (24+1)°C, a relative humidity of (80+2)%, a light duration of L: dark duration of D = 16:8, and the number of red spider mites in each group was observed and counted every 12 hours to calculate the mortality rate. The mortality rate (%) = (number of dead insects / number of test insects) x 100%. The results are shown in Figure 3 .also, Figure 4 The control effect of FQ268 on red spider mites after 60 hours of treatment is shown in the figure. It can be seen from the figure that in the different time periods recorded (0-60 hours), the mortality rate of FQ268 on red spider mites is very high. After 60 hours of spraying the bacterial solution, the mortality rate of FQ268 on red spider mites reached 100%.

[0050] 4. Preparation method of insecticide

[0051] This embodiment provides a method for preparing an insecticide, and the specific process is as follows:

[0052] (1) Strain activation: Serratia nematodiphila was inoculated on LB solid medium and cultured at 28° C. for 24 h to obtain activated Serratia nematodiphila; wherein the formula of LB solid medium is: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar 15 g / L, pH 7.2;

[0053] (2) Primary fermentation (50 L): The activated Serratia nematophila was inoculated into a seed culture medium for primary fermentation, with a liquid volume of 70%, an inoculum of 10%, a culture temperature of 28° C., and a shaking culture of 190 rpm for 20 h to obtain a primary seed solution of Serratia nematophila; wherein the formula of the seed culture medium is: 18 g / L peptone, 10 g / L sodium chloride, 10 g / L yeast extract powder, and a pH of 7.2;

[0054] (3) Secondary fermentation (500 L): The primary seed liquid was subjected to secondary fermentation, with a liquid volume of 70%, an inoculation amount of 10%, and a culture temperature of 30° C. The culture was shaken at 190 rpm for 20 h to obtain a secondary seed liquid of Serratia nematophila, wherein the formula of the culture medium used in the secondary fermentation was: 20 g / L glucose, 50 g / L bran, 10 g / L yeast powder, 3 g / L magnesium sulfate, 3 g / L disodium hydrogen phosphate, 3 g / L sodium chloride, and a pH of 7.2;

[0055] (4) Tertiary fermentation (50t): The secondary seed liquid was subjected to tertiary fermentation, with a liquid volume of 65%, an inoculation amount of 8%, a culture temperature of 30°C, and a shaking culture at 180rpm for 24h. The formula of the culture medium used in the secondary fermentation was: 25g / L glucose, 55g / L bran, 10g / L yeast powder, 3g / L magnesium sulfate, 4g / L disodium hydrogen phosphate, 3g / L sodium chloride, and a pH of 7.2. The effective viable bacteria count of the nematophilic Serratia marcescens obtained by fermentation was 28 billion CFU / mL. After the fermentation in the production tank was completed, the culture liquid was directly packaged into a liquid dosage form, which was an insecticide containing the nematophilic Serratia marcescens.

[0056] 5. Test on the toxicity of insecticides to pepper red spider mites

[0057] Select existing red spider pepper seedlings of the same size, keep 3 leaves on the left and right of the same plant, cut paper pieces to separate the left and right sides of the plant, use a fine brush to pick red spider mites onto the leaves, and test 50 insects on each left and right leaf. Spray the 100-fold dilution of the insecticide prepared above on the left, and spray sterile water on the right as a control. Spray until the leaf surface is moistened. Repeat 3 times for each group. Put the sprayed plants in a biological incubator under the environmental conditions of temperature (24+1)°C, relative humidity (80+2)%, and light duration L: dark duration D=16:8. After 2 days, count the number of surviving red spider mites in each group, and calculate the mortality rate and relative mortality rate. Mortality rate (%)=(number of dead insects / number of test insects)×100%; relative mortality rate (%)=(mortality rate of treatment group-mortality rate of control group) / (1-mortality rate of control group)×100%.

[0058] Table 2 Toxicity determination of insecticides to pepper red spider mites

[0059]

[0060] From the analysis in Table 2, it can be seen that in the greenhouse potted experiment, compared with the control group, the mortality rate of red spider mites on the plants after spraying the FQ268 fungicide reached 70%, and the relative mortality rate was 68.1%. It has a strong toxic effect on Acarina red spider mites and has a significant control effect.

[0061] VI. Test on the toxicity of insecticides to cabbage thrips

[0062] The nematophilic Serratia marcescens FQ268 insecticide prepared as above was used for the test. The original bacterial solution and 100-fold diluted solution were set as the test group, and sterile water was used as the control group. Each group was repeated 3 times, and each repetition had 20 thrips larvae. The thrips were first sprayed with sterile water for disinfection, and the water was dried after the treatment. Then the thrips were sprayed. The treated thrips and vegetable leaves were placed in a culture dish padded with wet cotton and filter paper moistened with sterile water. After the culture dish was sealed with transparent plastic wrap, 70 small holes were pierced on the transparent film with a needle and marked separately. The control group was numbered CK and placed in an incubator for constant temperature and moisture culture. It was observed every 24 hours, and the data was recorded and the mortality rate was calculated. Mortality rate (%) = (number of dead insects / total number of insects in the test) × 100%. The criterion for death is: lightly touch the abdomen of the thrips with a fine brush. Those that do not move are considered dead. The results are shown in the table below. Figure 5 As shown. Figure 5 It can be seen that compared with the control group, both the original solution and the 100-fold diluted solution have a stronger toxic effect on thrips. The thrips mortality rate reached 100% 48 hours after spraying the original solution, and the thrips mortality rate reached 95.6% 72 hours after spraying the 100-fold diluted solution.

[0063] VII. Test on the toxicity of insecticides to melon nematodes

[0064] The larvae of the second-instar root-knot nematodes in the melon root and soil samples were separated by the shallow plate method for later use. 2 mL of the 100-fold dilution of the insecticide prepared above was taken into a culture dish, and 30 second-instar larvae of the root-knot nematodes were picked up and placed in a constant temperature incubator at 28°C. After 2 days, the life and death of the second-instar larvae were observed under a stereoscope. Distilled water was used as the control group. Each group was treated 3 times, and the relative mortality rate was used to judge the toxicity of the insecticide against the root-knot nematodes. Relative mortality rate = (treatment nematode mortality rate - control mortality rate) / (1-control mortality rate) × 100%.

[0065] Table 3 shows the statistical results of the toxicity test of FQ268 on melon nematodes. According to Table 3, after spraying FQ268, the nematode mortality rate reached 96.7%, and the relative mortality rate was 96.5%. It has a strong toxic effect on melon nematodes and has a significant control effect.

[0066] Table 3 Toxicity determination of spraying different bacterial solutions on melon nematodes

[0067]

[0068] 8. Field application of insecticides

[0069] This embodiment uses the insecticide prepared above, and sets 3 treatments, namely, clear water control; treatment 1: 100-fold dilution of nematophilic Serratia FQ268 insecticide; treatment 2: chemical fungicide 25% nitenpyram, each treatment is repeated 4 times, and the treatment object is pepper. During the occurrence period of pepper red spider mites, a manual sprayer is used to spray evenly once, and the spray is evenly sprayed so that all the leaves are evenly covered with medicine on both sides. No other insecticides are applied during the test. Two plants are surveyed in each plot, and 5 damaged shoots are fixed in the east, south, west, north and middle of each plant. The number of red spiders on the top 5 to 10 leaves of each shoot is investigated. The base number of insects is investigated before applying the pesticide, and the number of live spiders on each treatment mark is investigated 15 days after applying the pesticide. The results are shown in Table 4. The average and control effect are calculated according to the "Guidelines for Field Efficacy Tests of Pesticides". Control effect (%) = 1-(the number of live insects in the clean water control area before the drug application × the number of live insects in the treatment area after the drug application) / (the number of live insects in the clean water control area after the drug application × the number of live insects in the treatment area before the drug application)] × 100%. The peppers were harvested and the yield was measured after they matured.

[0070] Table 4 Field control effect of insecticides on pepper red spider mites

[0071]

[0072] According to the test data in Table 4, 15 days after spraying, the control effect of spraying 100-fold diluted solution of Serratia nematophila FQ268 on pepper red spider mites was better than spraying 25% chemical fungicide nitenpyram, and the field control effect reached 62.8%.

[0073] Table 5 Effects of different pesticide treatments on pepper yield

[0074]

[0075] According to the test data in Table 5, the pepper yield per mu of treatment 1 (100-fold diluted fungicide of Serratia nematophila FQ268) was 2659.7 kg / mu, which was significantly increased by 14.9% and 44.6% compared with treatment 2 (chemical fungicide 25% nitenpyram) and the clear water control, respectively.

[0076] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A strain of Serratia nematophila (Serratia nematodiphila) , characterized in that, The nematophilic Serratia is named Serratia nematophila FQ268, which was deposited in the China Center for Type Culture Collection on September 22, 2023, with the deposit number CCTCC M 20231765.

2. An insecticide, characterized in that: The insecticide fungicide comprises the Serratia nematophila according to claim 1 and a fermentation liquid of the Serratia nematophila.

3. The insecticide fungicide according to claim 2, characterized in that In the insecticide, the viable bacteria count of Serratia nematophila FQ268 is 20-30 billion CFU / mL.

4. A method for preparing the insecticide fungicide according to claim 2 or 3, characterized in that: The following steps are involved: (1) inoculating Serratia nematophila FQ268 on LB solid culture medium for activation to obtain activated Serratia nematophila FQ268; (2) inoculating the activated Serratia nematophila FQ268 into a seed culture medium for primary fermentation to obtain a first-level seed solution of Serratia nematophila; (3) subjecting the primary seed liquid of Serratia nematophila to secondary fermentation to obtain a secondary seed liquid of Serratia nematophila; (4) The secondary seed liquid of Serratia nematophila is subjected to tertiary fermentation. After the fermentation is completed, the culture liquid is directly packaged into a liquid dosage form, which is a microbial insecticide containing Serratia nematophila FQ268.

5. The preparation method according to claim 4, characterized in that: In step (1), the formula of LB solid medium is: peptone 8-10 g / L, yeast extract 5-6 g / L, sodium chloride 8-10 g / L, agar 15-18 g / L, pH 7.2-7.3; The activation temperature is 28-30°C and the activation time is 22-24 h.

6. The preparation method according to claim 4, characterized in that: In step (2), the formula of the seed culture medium is: 18-20 g / L peptone, 8-10 g / L sodium chloride, 8-10 g / L yeast extract powder, and the pH is 7.2-7.3; The process conditions for primary fermentation include: liquid volume 60~70%, inoculation amount 7%~10%, culture temperature 28~30℃, and shaking culture at 180~200rpm for 18~20 hours.

7. The preparation method according to claim 4, characterized in that: In step (3), the process conditions of the secondary fermentation include: liquid volume 60-70%, inoculation amount 7-10%, culture temperature 28-30°C, and shaking culture at 180-200 rpm for 18-20 h.

8. The preparation method according to claim 4, characterized in that: In step (4), the process conditions of the tertiary fermentation include: liquid volume 60-70%, inoculation amount 7-10%, culture temperature 28-30°C, and shaking culture at 150-180 rpm for 22-24 h.

9. The preparation method according to claim 4, characterized in that: In step (3) and step (4), the formula of the culture medium used in the secondary fermentation and the tertiary fermentation is: 20-30 g / L glucose, 50-60 g / L bran, 10-12 g / L yeast powder, 2-5 g / L magnesium sulfate, 3-5 g / L disodium hydrogen phosphate, 2-5 g / L sodium chloride, and pH 7.2-7.

3.

10. Use of the nematophilic Serratia according to claim 1, or the insecticide fungicide according to claim 2 or 3, or the insecticide fungicide prepared by the preparation method of any one of claims 4 to 9 in preventing and controlling pests and diseases, wherein the pests and diseases include red spiders of the order Acarina, thrips of the order Thysanoptera, and nematodes of the order Sphenopterida.

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