Streptomyces cinnamoneus and application thereof in preventing and treating bemisia tabaci

Abstract

The present application belongs to the technical field of pest control, and particularly relates to a Streptomyces cinnamocastaneus and application thereof in the prevention and treatment of Bemisia tabaci. The present application discloses a Streptomyces cinnamocastaneus XJ33, which is preserved in China Typical Culture Collection Center on March 10, 2025, and the preservation number is CCTCC M 2025414. After the original liquid of the Streptomyces cinnamocastaneus, the bacterial liquid of the Streptomyces cinnamocastaneus diluted by 10 times, the bacterial liquid of the Streptomyces cinnamocastaneus diluted by 100 times and the clear water are treated, the average mortality of Bemisia tabaci in 72 hours is 96.32%, 83.93%, 75.11% and 5.40%, which indicates that the Streptomyces cinnamocastaneus has a significant technical effect in the prevention and treatment of Bemisia tabaci.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pest control, and particularly relates to a Streptomyces cinnamocastaneus and application thereof in prevention and treatment of Bemisia tabaci. BACKGROUND

[0002] Bemisia tabaci belongs to Hemiptera Aleyrodidae and is an important pest of vegetables and economic crops, which is widely distributed in the world and mainly harms Solanaceae, melons and legumes and flowers. Bemisia tabaci causes plants to wilt and dry up by sucking plant sap and secreting honeydew as nymphs and adults, and the adults can also transmit plant viruses to cause crop yield reduction or even absolute yield reduction. At present, chemical pesticides are the main measure for field prevention and control of Bemisia tabaci, and chemical control is the most effective emergency method during the outbreak period of Bemisia tabaci. However, Bemisia tabaci has serious overlapping generations, fast reproduction speed and wide transmission range, and the field population has developed resistance to various insecticides to varying degrees, which increases the difficulty of prevention and control. It has been found that Bemisia tabaci has developed resistance to organophosphorus, neonicotinoid, pyrethroid and antibiotic insecticides. Some insecticides with high toxicity, high residual and high resistance are not suitable for the current green agricultural development, and it is urgent to seek new insecticidal products with high efficiency, low toxicity and low residual.

[0003] Microbial control is a method of using microorganisms and their metabolites to control crop diseases and pests. It includes using pathogenic microorganisms (such as bacteria, fungi, viruses, etc.) to directly infect and kill pests, and using beneficial microorganisms (such as actinomycetes, yeasts, etc.) to produce antibiotics or other bacteriostatic substances to inhibit or kill harmful microorganisms. Microbial control has the characteristics of high efficiency, strong specificity, environmental friendliness and the like, and is an important part of the sustainable development of modern agriculture. SUMMARY

[0004] In order to solve the above technical problems, the application provides the following technical solutions:

[0005] The application discloses a Streptomyces cinnamocastaneus XJ33, which is preserved in the China Center for Type Culture Collection on March 10, 2025, and the preservation number is CCTCC M2025414.

[0006] The application further discloses a microbial agent containing the Streptomyces cinnamocastaneus XJ33.

[0007] The present invention also discloses a microbial fermentation broth, which is obtained by fermentation of Streptomyces cinnamocastaneus XJ33.

[0008] Preferably, the method for preparing the microbial fermentation broth is as follows:

[0009] (1) Streptomyces cinnamocastaneus XJ33 was inoculated into TSBY medium and shaken to activate it, thus obtaining activated Streptomyces cinnamocastaneus XJ33.

[0010] (2) The activated Streptomyces cinnamocastaneus XJ33 was inoculated into YEME medium for fermentation to obtain the microbial fermentation broth.

[0011] Preferably, the preparation method of the TSBY culture medium in step (1) is as follows: weigh 30 g / L of trypsin soybean broth and 5 g / L of yeast extract, and sterilize by steam at 121°C for 15 min.

[0012] Preferably, the conditions for activating the bacteria by shaking in step (1) are: the shaker is set to 28°C, 220 r / min, and shaken for 48 h.

[0013] Preferably, the preparation method of the YEME culture medium in step (2) is as follows: weigh 3 g / L yeast extract, 3 g / L malt extract, 5 g / L protein, 340 g / L sucrose and 10 g / L glucose, and sterilize at 113-114℃ for 15 min; add MgCl2·6H2O before use to make Mg 2+ The final concentration was 5 mM.

[0014] Preferably, the fermentation conditions in step (2) are: the shaker is set to 28°C, 220 r / min, and shaken for 72 h. The volume ratio of the activated Streptomyces cinnamocastaneus XJ33 to YEME medium is 1:5.

[0015] Preferably, the concentration of Streptomyces cinnamocastaneus XJ33 in the microbial fermentation broth is 1.3-1.4 billion CFU / ml.

[0016] The application of Streptomyces cinnamocastaneus XJ33 or microbial agents containing it or microbial fermentation broth fermented from it in the control of whiteflies.

[0017] The application of Streptomyces cinnamocastaneus XJ33 or microbial agents containing it or microbial fermentation broth fermented from it in the preparation of products for the prevention and control of whiteflies.

[0018] The beneficial effects of this invention are:

[0019] This invention discloses a *Streptomyces cinnamocastaneus* XJ33 strain, which was deposited at the China Center for Type Culture Collection (CCTCC) on March 10, 2025, with accession number CCTCC M2025414. Treatment with the stock solution of *Streptomyces cinnamocastaneus*, a 10-fold diluted bacterial suspension, a 100-fold diluted bacterial suspension, and water resulted in average mortality rates of 96.32%, 83.93%, 75.11%, and 5.40% for whiteflies after 72 hours, respectively, indicating that *Streptomyces cinnamocastaneus* has significant technical effects in controlling whiteflies. Attached Figure Description

[0020] Figure 1 Colony diagram of Streptomyces cinnamocastaneus XJ33;

[0021] Figure 2 Phylogenetic analysis diagram of Streptomyces cinnamocastaneus;

[0022] Figure 3 The efficacy of the stock solution of Streptomyces cinnamocastaneus XJ33 in killing whiteflies;

[0023] Figure 4 The effect of a 10-fold dilution of the stock solution of Streptomyces cinnamocastaneus XJ33 on killing whiteflies;

[0024] Figure 5 The effect of a 100-fold dilution of Streptomyces cinnamocastaneus XJ33 on killing whiteflies;

[0025] Figure 6This is a diagram showing the results of water treatment. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0027] Example 1: Classification and Identification of Bacteria

[0028] The species of strain XJ33 was identified using 16S rDNA identification technology. 16S rRNA gene amplification was performed using universal bacterial primers. After PCR amplification, the PCR products were detected by electrophoresis on a 1% agarose gel. The gene product obtained from PCR was sequenced, and the 16S rRNA gene sequence of strain XJ33 is shown in SEQ ID NO.1.

[0029] SEQ ID NO.1

[0030]

[0031] The 16S rRNA gene sequence of strain XJ33 was entered into the NCBI database and analyzed using BLAST software. Strains with high homology to strain XJ33 were selected, and multiple sequence alignment and phylogenetic tree construction were performed using MEGA 7.0 software. (See below) Figure 2 As shown. The 16S rRNA gene sequence of strain XJ33 was obtained by PCR amplification. The alignment results showed that strain XJ33 was most similar to Streptomyces cinnamocastaneus. Therefore, strain XJ33 was identified as Streptomyces cinnamocastaneus. This strain was named Streptomyces cinnamocastaneus XJ33, deposited at the China Center for Type Culture Collection, address: Wuhan, China (Wuhan University), with accession number CCTCC M2025414, and deposit date: March 10, 2025.

[0032] Example 2: Preparation of Microbial Inoculants

[0033] Experimental methods

[0034] (1) Take the Streptomyces strain XJ33 screened from the activated bacterial library and purify it for no more than 3 generations to ensure the activity of the bacteria;

[0035] (2) In a 50ml Erlenmeyer flask, fill it with 20ml of TSBY medium. In a clean bench, use a sterile inoculation stick to pick a single Streptomyces strain XJ33 and inoculate it into the Erlenmeyer flask containing TSBY medium that has been prepared, sterilized at 121℃ for 20min and cooled. Add 7mm glass beads to the flask. Set the shaker to 28℃ and 220r / min and shake for 48h to obtain TSBY bacterial solution. Then transfer 20ml of TSBY bacterial solution that has been shaken for 48h to a 250ml Erlenmeyer flask containing 100ml YEME medium and continue to culture. Set the shaker to 28℃ and 220r / min and shake for 72h. Add 7mm glass beads to the shaker.

[0036] In the laminar flow hood, using sterile forceps and a pipette, 10 μL of bacterial culture was dropped onto a hemocytometer, covered with a coverslip, and the viable bacteria were counted under an electron microscope. The viable bacteria count for XJ33 was 1.3-1.4 billion CFU / ml.

[0037] TSBY medium

[0038] Weigh 30g of tryptone soya broth and 5g of yeast extract, bring the volume to 1L with dd water, and sterilize by steaming at 121℃ for 15min.

[0039] YEME medium

[0040] Weigh out 3g of yeast extract, 3g of malt extract, 5g of egg white, 340g of sucrose, and 10g of glucose, and dilute to 1L with dd water. Sterilize at 113-114℃ for 15 minutes. Before use, add MgCl2·6H2O to Mg... 2+ The final concentration was 5 mM.

[0041] Example 3: Application of *Streptomyces cinnamonis* XJ33 microbial inoculant

[0042] The bioassay method for adult whiteflies uses the leaf immersion agar moistening method, and the specific steps are as follows:

[0043] (1) Prepare a flat-bottomed glass finger tube with a diameter of about 2.2 cm and a length of about 8 cm. Use a dropper to add 2-3 mL of 2% agar solution to the bottom of the glass finger tube. During this process, avoid spilling agar solution onto the glass tube wall and avoid generating air bubbles in the added agar solution. Seal the tube and let it cool. After the agar solidifies and all water vapor on the flat-bottomed glass tube wall has completely evaporated, the bioassay can be performed.

[0044] (2) Prepare the insecticide concentration for the test according to the field recommended concentration and the results of the preliminary test. Dilute the tested Streptomyces cinnamonis XJ33 microbial agent in a gradient to the concentration to be tested and use it for later use. Use water treatment as a control.

[0045] (3) Take the cultivated cotton leaves and use a punch to make leaf discs with a diameter of 22mm on the flat part of the leaf, avoiding the large veins. Immerse the leaf discs in the prepared microbial agent of Streptomyces cinnamonis XJ33 for 20 seconds and then take them out. Let the back of the leaves air dry naturally.

[0046] (4) Use blunt-tipped tweezers to pick up the dried cotton leaf dish, place it face down into the flat-bottomed glass finger tube where the agar has solidified, and make sure the cotton leaf dish and agar are tightly fitted together to remove air bubbles. After plugging with cotton, mark the bottom of the flat-bottomed glass finger tube for later use.

[0047] (5) If the whiteflies are densely covered on the leaves, they can be collected by patting the leaves. Otherwise, use a suction tool to drive the whiteflies into a flat-bottomed glass finger tube that has been treated and dried. Each tube should contain a certain number of whiteflies. Plug the cotton into the tube to halfway down so that the whiteflies can crawl on the leaf discs inside the flat-bottomed glass tube.

[0048] (6) Place the flat-bottomed glass finger tube containing whiteflies upside down in the prepared light incubator. Set the temperature to 26℃±2℃, the humidity to 70%±10%, and the light cycle to 16h:8h (L:D). After 48 hours, check the survival of the whiteflies in the flat-bottomed glass finger tube. If the whiteflies do not move or cannot move normally, they are considered dead.

[0049] Experimental results are as follows Figures 3-6 As shown in Table 1:

[0050] Table 1 Experimental Results

[0051]

[0052] Note: In Table 1, the number before the slash represents the number of whiteflies that died, and the number after the slash represents the total number of whiteflies.

[0053] Results analysis: The average mortality rates of whiteflies after 72 hours were 96.32%, 83.93%, 75.11%, and 5.40% for the original solution of *Streptomyces cinnamonis*, the bacterial suspension diluted 10 times, the bacterial suspension diluted 100 times, and water, respectively. This indicates that *Streptomyces cinnamonis* has a significant technical effect in the control of whiteflies.

Claims

1. A strain of *Streptomyces cinnamon* ( Streptomyces cinnamocastaneus XJ33, characterized in that, It was deposited at the China Center for Type Culture Collection on March 10, 2025, with accession number CCTCC M 2025414.

2. A microbial inoculant, characterized in that, The microbial agent contains *Streptomyces cinnamonis* as described in claim 1. Streptomyces cinnamocastaneus )XJ33.

3. A microbial fermentation broth, characterized in that, The microbial fermentation broth is composed of *Streptomyces cinnamonis* as described in claim 1. Streptomyces cinnamocastaneus XJ33 was obtained through fermentation.

4. The microbial fermentation broth according to claim 3, characterized in that, The preparation method of the microbial fermentation broth is as follows: (1) The *Streptomyces cinnamonis* as described in claim 1 ( Streptomyces cinnamocastaneus XJ33 was inoculated into TSBY medium and activated by shaking to obtain activated Streptomyces cinnamonis. Streptomyces cinnamocastaneus XJ33; (2) The activated Streptomyces cinnamonis ( Streptomyces cinnamocastaneus XJ33 was inoculated into YEME medium for fermentation to obtain the microbial fermentation broth.

5. The microbial fermentation broth according to claim 4, characterized in that, The preparation method of TSBY culture medium in step (1) is as follows: weigh 30 g / L of trypsin soybean broth and 5 g / L of yeast extract, and sterilize by steaming at 121℃ for 15 min.

6. The microbial fermentation broth according to claim 5, characterized in that, The conditions for activating the bacteria by shaking in step (1) are: the shaker is set to 28°C, 220 r / min, and shaken for 48 h.

7. The microbial fermentation broth according to claim 6, characterized in that, The fermentation conditions described in step (2) are as follows: the shaker is set to 28°C, 220 r / min, and shaken for 72 h; the activated Streptomyces cinnamonis ( Streptomyces cinnamocastaneus The volume ratio of XJ33 to YEME culture medium is 1:

5.

8. The microbial fermentation broth according to claim 4, characterized in that, The cinnamon chestnut streptomyces in the microbial fermentation broth ( Streptomyces cinnamocastaneus The concentration of XJ33 is 1.3-1.4 × 10⁻⁶. 9 cfu / ml.

9. The *Streptomyces cinnamonis* as described in claim 1 ( Streptomyces cinnamocastaneus The application of the microbial agent of claim XJ33 or claim 2 or the microbial fermentation liquid of any one of claims 3-8 in the prevention and control of whiteflies.

10. The *Streptomyces cinnamonis* as described in claim 1 ( Streptomyces cinnamocastaneus The application of the microbial agent of claim XJ33 or claim 2 or the microbial fermentation broth of any one of claims 3-8 in the preparation of products for the prevention and control of whiteflies.

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