A Highly Effective Insecticidal Fungiformis and Its Application

Abstract

This invention provides a highly effective insecticidal strain of *Ceratophyllum demersum* and its applications. A strain of *Ceratophyllum demersum* 3166 with a high growth rate and large spore production was isolated from soil. Strain 3166 can effectively control rice stem borers, whiteflies, leafminers, and tomato nematodes. By analyzing the genome and transcriptome of strain 3166, highly expressed gene clusters related to insecticidal activity were identified. Optimizing the fermentation conditions of strain 3166 in a 5-liter fermenter resulted in a spore concentration of 3 × 10⁻⁶. 9 100 spores / mL or higher. Pilot-scale fermentation at the same level was finally completed in 42-liter and 500-liter fermenters. This invention further enriches the means of pest control in agricultural production.

Description

Technical Field

[0001] This invention relates to the fields of microbiology and plant protection technology, and more specifically, to a highly effective insecticidal strain of *Cercospora aegypti* and its applications. Background Technology

[0002] While chemical pesticides have indeed played a crucial role in the control of plant diseases and pests, they have also brought about many problems that cannot be ignored. Statistics show that the global effective utilization rate of pesticides is less than 30%. Due to the extensive use of chemical pesticides, pests are developing increasing resistance, pesticide residues are becoming increasingly serious, and environmental pollution is extremely severe. These problems have already posed a threat to the ecological environment and human health. Although biological control technology does not have these drawbacks, the low efficacy and instability of existing microbial strains severely restrict its development and application in the field of plant pest control in my country.

[0003] Rice planthoppers are insects belonging to the family Plantiphaga in the order Hemiptera. They are herbivorous, with the brown planthopper and the white-backed planthopper being exclusively feeding on rice. Adult rice planthoppers begin laying eggs 3-5 days after emergence, reaching peak egg production within 7-10 days, laying 200-600 eggs in their lifetime. Both adult and nymph rice planthoppers suck the sap from rice plants, and the toxic substances secreted from their salivary glands can clog vascular tissues or cause rice plants to wither. Furthermore, their excrement can promote fungal growth, thus affecting rice photosynthesis and respiration, and transmitting plant viral diseases. The brown planthopper, white-backed planthopper, and gray planthopper can transmit rice dwarf virus, rice black-streaked dwarf virus, and rice stripe leaf blight, respectively.

[0004] Western flower thrips, belonging to the genus *Thrips* in the family Thripidae of the order Thysanoptera, are extremely harmful to crops. These insects feed on the stems, leaves, flowers, and fruits of plants using their rasping-sucking mouthparts, causing petals to fade, leaves to wrinkle, and scarring of stems and fruits, ultimately leading to plant wilting. Furthermore, western flower thrips can transmit various viruses, including tomato spotted wilt virus. Therefore, preventing the spread of western flower thrips is of paramount importance. Strict quarantine measures should be implemented, employing both physical and chemical control methods.

[0005] *Acer glabripennis* ( Lecanicillium *Attenuatum* is an important entomopathogenic fungus belonging to the class Ascomycetes, order Hypocreales, and genus *Attenuatum*, with a wide host distribution. *Attenuatum* typically kills pests or pathogens by growing inside the host and decomposing host tissues. They produce specific enzymes and toxins that can damage the host's cell walls and cell membranes, leading to host death. Because *Attenuatum* is a natural biocontrol agent with no negative impacts on the environment and human health, it offers a sustainable solution for pest control in agricultural production. Summary of the Invention

[0006] The purpose of this invention is to provide a highly effective insecticidal strain of *Ceratophyllum demersum* and its applications.

[0007] To achieve the objectives of this invention, in a first aspect, this invention provides a strain 3166 isolated from soil, characterized by high growth rate and high sporulation yield. This strain is classified and named *Ceratophyllum demersum*. Lecanicillium attenuatum The sample is now deposited at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, accession number CGMCC NO.40860, deposited on October 25, 2023.

[0008] Secondly, the present invention provides a bacterial agent containing *Cercospora acervulata* 3166.

[0009] Thirdly, the present invention provides any of the following applications of *Cladosporium alginate* 3166 or its inoculum:

[0010] (1) Used to control plant pests;

[0011] (2) Used to prepare products for the prevention and control of plant pests.

[0012] In this invention, the pests include, but are not limited to, rice stem borer, whitefly, leaf miner, and tomato nematode, with leaf miner and tomato nematode being preferred.

[0013] The plant is a dicotyledonous plant or a monocotyledonous plant.

[0014] Fourthly, the present invention provides a product for controlling plant pests, which contains *Cladosporium acutum* 3166 or its fermentation broth.

[0015] Furthermore, the product includes pesticides or fertilizers.

[0016] The preparation method of fermentation broth of *Ceratophyllum demersum* includes: fermenting and culturing *Ceratophyllum demersum* to obtain the fermentation broth of the bacteria.

[0017] Furthermore, the fermentation temperature is 15–30℃, and the fermentation time is 20–96 h;

[0018] Furthermore, the fermentation medium used consisted of 10–60 g / L glucose, 5–25 g / L peptone, 5–20 g / L yeast extract, 0.5–5 g / L dipotassium hydrogen phosphate, 1–10 g / L calcium carbonate, 0.02–0.5 g / L manganese sulfate, and 0.02–0.5 g / L magnesium sulfate.

[0019] Fifthly, the present invention provides a method for controlling plant pests by applying *Clerodendrum thunbergii* 3166 or its fermentation liquid or a product containing the above-mentioned components to the plant.

[0020] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0021] Strain 3166 was found to be highly effective in controlling rice planthoppers and western flower thrips. Analysis of the genome and transcriptome of strain 3166 identified highly expressed gene clusters associated with insecticidal activity. Optimization of fermentation conditions in a 5-liter fermenter resulted in a spore concentration of 3 × 10⁻⁶. 9 100 spores / mL or higher. Pilot-scale fermentation at the same level was finally completed in 42-liter and 500-liter fermenters. This invention further enriches the means of pest control in agricultural production. Attached Figure Description

[0022] Figure 1 The results of indoor toxicity tests of *Aureobasidium acutum* against rice stem borer (A), whitefly (B), leafminer (C), and tomato nematode (D) are presented in a preferred embodiment of the present invention.

[0023] Figure 2 The growth curve of *Cercospora acervulata* is shown in a preferred embodiment of the present invention.

[0024] Figure 3 The effect of culture temperature on the growth curve and sporulation of *Cercospora acervulata* in a preferred embodiment of the present invention.

[0025] Figure 4 The inoculum amount of *Ceratophyllum demersum* in the fermenter is optimized in a preferred embodiment of the present invention.

[0026] Figure 5 The dissolved oxygen in the fermenter of *Ceratophyllum demersum* is optimized in a preferred embodiment of the present invention.

[0027] Figure 6 The KOG classification results of the genome genes of *Ceratophyllum demersum* in a preferred embodiment of the present invention are shown. Detailed Implementation

[0028] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0029] Example 1: Isolation, purification, and identification of *Streptococcus acutissima* 3166

[0030] This invention isolates a strain of *Strombus aegyptium* with a high growth rate and large spore production from soil. Lecanicillium attenuatum3166, Microscopic characteristics of hyphae and spores: No specialized conidiophores; conidiophores are lanceolate, straight or curved, 2-4 whorled at the tips of hyphae or their branches, or solitary on the sides of hyphae, (12-30) μm × (0.5-1.5) μm. Conidia are oblong, colorless, with smooth walls, (2.5-6.5) μm × (0.8-1.5) μm.

[0031] Example 2: Indoor toxicity determination of *Streptococcus acutissima* 3166 against *Chilodonella esculenta*.

[0032] 1. Preparation of gradient concentration conidial solutions of *Streptococcus faecium* 3166

[0033] *Ceratophyllum demersum* 3166 was inoculated into liquid culture medium and cultured with shaking at 25°C and 200 rpm for 3 days to obtain a suspension of *Ceratophyllum demersum* 3166 spores. Using the culture medium, 10... 9 A suspension of *Ceratophyllum demersum* spores / mL was serially diluted to 10... 8 10 7 and 10 6 1 spore / mL, with culture medium as a control.

[0034] 2. Indoor toxicity testing methods

[0035] The toxicity to rice stem borer was determined using the immersion method. Third-instar larvae of the rice stem borer were immersed in a spore suspension for 15 seconds, then transferred to a rearing box (13.5 cm long, 9 cm wide, and 7 cm high) containing water chestnuts (approximately 5 cm long, 3 cm wide, and 0.5 cm thick) with 9 small ventilation holes in 3 rows at the top. Three replicates were set up for each concentration, with 10 larvae per replicate. The boxes were placed in a light incubator at 27 ℃, 80% relative humidity, and a photoperiod of 14 L:10 D. The number of larval deaths was recorded 3–7 days after treatment.

[0036] 3. Indoor toxicity test results

[0037] The experimental results are shown in Table 1. 10 9 10 spores / mL and 10 8 After treatment with 1 spore / mL for 7 days, the corrected mortality rates of the rice stem borer reached 65.56% and 55.19%, respectively. LC50 50 The lethal concentration was 3.77 × 10⁻⁶. 7 Spores / mL. *Cercospora acutangiella* is effective in inhibiting the rice stem borer, a common pest on rice.

[0038] In addition to the rice stem borer, this invention also determined the toxicity of *Agrostis stenoptera* to whiteflies, leafminers, and tomato nematodes, with corrected mortality rates of 84.45%, 90.22%, and 90.43%, respectively. Figure 1 ).

[0039] Table 1 Corrected mortality rates of rice stem borer, whitefly, leafminer fly, and tomato nematode after treatment with *Ceratophyllum demersum*.

[0040]

[0041] Note: Different lowercase letters indicate significant differences.

[0042] Example 3: Fermentation optimization of *Streptococcus acutissima* 3166

[0043] 1. Preparation of seed liquid

[0044] Strain 3166 of *Streptococcus acutissima* was inoculated onto PDA medium and cultured at 25 °C for 7 days in a biochemical incubator. The spores were then washed away with sterile physiological saline containing 0.01% Tween 80 to prepare 5 × 10⁻⁶ spores. 7 A spore suspension of 1 spore / mL was prepared and inoculated at 1-10% onto modified YPD medium (formula: 40 g / L glucose, 10 g / L peptone, 10 g / L yeast extract, 2 g / L dipotassium hydrogen phosphate, 5 g / L calcium carbonate, 0.1 g / L manganese sulfate, 0.1 g / L magnesium sulfate), and cultured for 72 h until OD500 reached. 600 The concentration should be 40-60. Then, the culture solution is mixed evenly with 50% sterile glycerol in an equal volume ratio to prepare a seed solution, which is then frozen and stored at -80°C for later use.

[0045] 2. Growth curve of *Streptococcus aureus* 3166 in shake flasks

[0046] 2% of the seed culture of *Streptococcus acutissima* strain 3166 was inoculated into Erlenmeyer flasks containing 100 ml of modified YPD medium. The shaker was set at 25°C and 200 rpm. During the culture process, samples were taken to measure the OD (octane rating) of the bacterial culture. 600 .

[0047] like Figure 2 As shown, the growth curve of *Streptococcus acutissima* strain 3166 reached the plateau phase after 40 hours of fermentation in a shake flask, at which point the OD... 600 It can reach over 70.

[0048] Optimization of fermentation conditions in a 3.5L fermenter

[0049] 3.1 Screening of culture temperature

[0050] To optimize the fermentation temperature in the fermenters, four treatment groups were established: 20, 25, 30, and 35 °C. 2% of the seed culture of *Streptococcus acutissima* strain 3166 was inoculated into 5L fermenters containing 2L of modified YPD medium, and the incubation temperatures were set at 20, 25, 30, and 35 °C. Dissolved oxygen was controlled at 10%. Oxygen saturation (OD) of the bacterial culture was measured during the incubation process.600 The value was determined by counting the spore concentration using a hemocytometer.

[0051] By optimizing the growth and sporulation curves of *Strombus aegyptium* at temperature, it was determined that the growth rate and sporulation number of *Strombus aegyptium* were superior to those at other temperature conditions. Figure 3 ).

[0052] 3.2 Optimization of vaccination volume

[0053] To optimize the inoculum size for fermentation in the fermenter, three inoculum size treatments were set up: 2%, 4%, and 6%. Seed culture of *Streptococcus acutissima* strain 3166 was inoculated at 2%, 4%, and 6% in 5L fermenters containing 2L of modified YPD medium, respectively. The culture temperature was set at 25℃, and dissolved oxygen was controlled at 10%. Oxygen saturation (OD) of the bacterial culture was measured during the culture process. 600 The value was determined by counting the spore concentration using a hemocytometer.

[0054] The effect of inoculum size on the growth curve and sporulation yield shows that the larger the inoculum size of *Strombus aegyptium*, the faster the growth rate. Figure 4 When the inoculum size is 6%, sporulation is relatively rapid, reaching its maximum after approximately 28 hours of culture, with a maximum sporulation yield of about 2.43 × 10⁻⁶. 9 The number of spores / mL was 1, but there was no significant difference in biomass and sporulation in the later stages of fermentation. Subsequently, a 2% inoculum was selected for further experiments.

[0055] 3.3 Dissolved oxygen optimization

[0056] To optimize dissolved oxygen levels during fermentation in the fermenter, four dissolved oxygen gradient treatments were established: 4%, 8%, 10%, and 20%. 2% of *Streptococcus acutissima* strain 3166 seed culture was inoculated into a 5L fermenter containing 2L of modified YPD medium, and the culture temperature was set at 25℃. Dissolved oxygen levels were controlled at 4%, 8%, 10%, and 20%. Oxygen oxidative stress (OD) of the bacterial culture was measured during the culture process. 600 The value was determined by counting the spore concentration using a hemocytometer.

[0057] The higher the dissolved oxygen in the fermenter of *Ceratophyllum demersum*, the more favorable it is for the growth of the bacteria and sporulation. Figure 5 However, under dissolved oxygen control conditions of 10% and 20%, there was no significant difference in cell growth and sporulation. From the perspectives of environmental protection, energy conservation, emission reduction, and cost savings, the relatively lower dissolved oxygen control should be prioritized. The maximum sporulation reached 3.76 × 10⁻⁶ after approximately 40 hours of fermentation. 9 spores / mL.

[0058] 3.4 Fermentation in 42L and 500L fermenters

[0059] Based on the above results, fermentation conditions of 25℃, 2% inoculum, and 10% dissolved oxygen were selected, and fermentation was carried out for 40 hours. A pilot-scale 500L fermenter fermentation of *Streptococcus acutissima* was completed, achieving the same level as a 5L fermenter, with a sporulation yield of 3.6 × 10⁻⁶. 9 spores / mL.

[0060] Example 4: Genome analysis of *Ceratophyllum demersum* 3166

[0061] Whole-genome DNA was extracted from strain 3166, and the samples were tested. A library was constructed using the qualified samples. First, the large DNA fragments were broken into fragments of about 500 bp. Then, the sticky ends formed by the fragments were repaired into blunt ends. Then, an "A" base was added to the 3' end so that the DNA fragments could be ligated to adapters with "T" bases at the 3' end. The ligation products of the target fragments were recovered by electrophoresis. Then, the DNA fragments with adapters at both ends were amplified by PCR. Finally, cluster preparation and sequencing were performed using the qualified libraries.

[0062] Based on the sequencing results, the genomes of 3166 strains were assembled, and coding genes and non-coding RNAs were predicted, resulting in a total of 10524 predicted genes. The genome length was 35.45 Mb, and the GC content was 57.79%. Gene functions were annotated based on these predictions. Figure 6 As shown, the KOG database classification results indicate that strain 3166 involves 378 genes related to the biosynthesis, transport, and catabolism of secondary metabolites. Among these, 175 genes are involved in the transmission of mycelium through the insect epidermis, including proteases (83 genes), cutinases (15 genes), glycosidases (33 genes), chitinases (22 genes), and lipases (22 genes); and 49 genes are involved in insecticidal activities such as endotoxins / insecticides.

[0063] The gene sequences of 5.8S rRNA, 18S rRNA and 28S rRNA of strain 3166 are shown in SEQ ID No:1-3.

[0064] Example 5: Transcriptome analysis of *Ceratophyllum demersum* 3166

[0065] The transcriptomic results are shown in Table 2. After 72 h and 144 h of culture of *Streptococcus acutissima*, the expression of 9916 (94.22%) and 10104 (96.01%) genes were detected, respectively. Among them, 2287 (23.06%) and 2393 (23.68%) were highly expressed genes (FPKM>60).

[0066] The highly expressed genes include 37 proteases, 2 keratinases, 6 glycosidases, 6 chitinases, 2 lipase-related genes, and 2 endotoxin / insecticide genes.

[0067] Table 2 Relative gene expression levels

[0068]

[0069] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Lecanicillium attenuatum 3166, accession number CGMCC NO.40860.

2. A bacterial agent containing the bacteria described in claim 1.

3. Any of the following applications of the bacteria of claim 1 or the bacterial agent of claim 2: (1) Used to control plant pests; (2) Used to prepare products for the prevention and control of plant pests; The pests mentioned are rice stem borer, whitefly, leaf miner, and tomato nematode.

4. A product for controlling plant pests, characterized in that, Contains the bacteria of claim 1 or its fermentation broth; The pests mentioned are rice stem borer, whitefly, leaf miner, and tomato nematode.

5. The product according to claim 4, characterized in that, The products include pesticides or fertilizers.

6. The product according to claim 4 or 5, characterized in that, The method for preparing the fermentation broth of *Ceratophyllum demersum* according to claim 1 includes: fermenting and culturing *Ceratophyllum demersum* to obtain the fermentation broth of the bacteria; The fermentation temperature is 15–30℃, and the fermentation time is 20–96 h. The fermentation medium used consisted of 10–60 g / L glucose, 5–25 g / L peptone, 5–20 g / L yeast extract, 0.5–5 g / L dipotassium hydrogen phosphate, 1–10 g / L calcium carbonate, 0.02–0.5 g / L manganese sulfate, and 0.02–0.5 g / L magnesium sulfate.

7. A method for controlling plant pests, characterized in that, Applying the bacteria of claim 1 or its fermentation broth or the product of any one of claims 4-6 to plants; The pests mentioned are rice stem borer, whitefly, leaf miner, and tomato nematode.

Images