Biocontrol bacteria for preventing and controlling rice planthopper and application thereof

Abstract

This invention provides a biocontrol agent for controlling rice planthoppers and its application. The biocontrol agent is classified and named *Penicillium oxalate* (…). Penicillium oxalicum FS-PNC, a biocontrol agent of this invention, was deposited at the China General Microbiological Culture Collection Center on December 26, 2022, with accession number CGMCC NO.40461. The biocontrol agent is *Penicillium oxalate* (FS-PNC). Penicillium oxalicum When spore suspensions of FS-PNC are sprayed onto rice, the field control efficacy against rice planthoppers can reach 66%, indicating that this biocontrol fungus has good application prospects in the control of rice planthoppers.

Description

Technical Field

[0001] This invention belongs to the field of biological pesticides, specifically relating to a biocontrol fungus for controlling rice planthoppers and its application. Background Technology

[0002] Rice planthoppers are a major pest of rice, often causing outbreaks due to their migratory habits. Due to the continuous and inappropriate use of chemical pesticides, rice planthoppers have developed resistance to various pesticides. Currently, rice planthoppers have developed resistance levels hundreds to thousands of times higher to commonly used pesticides such as imidacloprid, chlorpyrifos, thiamethoxam, pymetrozine, imidacloprid, and thiamethoxam, leading to decreased pesticide effectiveness and a vicious cycle of increasing dosage and frequency of application. Using fungi to control pests is an effective biological control technique. Entomopathogenic fungi can cause widespread epidemics among crop pests, and are highly selective, mostly safe for humans and animals, harmless to natural enemies of pests, and do not pollute the environment, playing a significant role in promoting green agricultural development. Currently, only one entomopathogenic fungus, *Metarhizium anisopliae* CQMa421, is registered on the China Pesticide Information Network for controlling rice planthoppers, representing a single species. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a biocontrol agent for controlling rice planthoppers and its application.

[0004] The objective of this invention is achieved through the following technical solution: A biocontrol agent for controlling rice planthoppers, named Penicillium oxalicum FS-PNC, was deposited on December 26, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNO.40461, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0005] The application of the above-mentioned biocontrol fungus in the control of rice planthoppers.

[0006] A biocontrol agent comprising conidial powder of the aforementioned biocontrol agent, Penicillium oxalicum FS-PNC.

[0007] The application of the above-mentioned biocontrol agent in the control of rice planthoppers.

[0008] One method for controlling rice planthoppers involves spraying the aforementioned biocontrol agent onto the rice.

[0009] The significant advantages of this invention are: The biocontrol agent Penicillium oxalicum FS-PNC of this invention can effectively infect rice planthoppers. Field trials have shown that the spore suspension of Penicillium oxalicum FS-PNC has a significant control effect on rice planthoppers, with a control efficacy of up to 66%. Attached Figure Description

[0010] Figure 1 Colony characteristics of *Penicillium oxalicum* FS-PNC on CYA medium. Left: Front view of colony; Right: Back view of colony.

[0011] Figure 2 Microstructural features of *Penicillium oxalicum* FS-PNC. Left: conidiophore; Right: conidia.

[0012] Figure 3 Infection of brown planthoppers by Penicillium oxalicum FS-PNC. Left: Symptoms of infection in brown planthopper nymphs; Right: Symptoms of infection in brown planthopper adults. Detailed Implementation

[0013] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0014] Example 1: Separation of FS-PNC The infected brown planthoppers were glued onto aluminum foil and placed in a petri dish lined with filter paper. The dish was then incubated at 28°C with humidity. Once the planthoppers were fully colonized with conidia, the foil containing PDA culture medium was used to seal the Erlenmeyer flask, with the planthopper side facing inwards. After 12 hours, the foil was removed, and the flask was sealed with a silicone stopper. After another 3 days of incubation, colonies were picked and inoculated onto PDA plates for further culture. After 7 days, spores were collected from the plates and prepared into a spore suspension using 0.05% Tween 80. This suspension was transferred to an Erlenmeyer flask containing glass beads and shaken at 120 rpm for 2–3 hours to ensure complete conidial dispersion. The suspension was then diluted to different concentrations for single-spore isolation. The resulting strains were sprayed onto live brown planthoppers, and the strain with the strongest pathogenicity against brown planthoppers was selected and named FS-PNC.

[0015] Example 2: Identification of FS-PNC 1. Identification of strain morphology The morphological characteristics of strain FS-PNC on different culture media are shown below: When cultured on Czapek's yeast extract agar (CYA) at 25°C for 7 days, the colonies were 53-55 mm in diameter, relatively thick, with a few radial raised stripes on the surface and neat edges; the texture was velvety; the conidial structure was grayish-green; the colony edges were white; there was colorless exudate; there was no soluble pigment; there was no special odor, and the back was bright ivory (ral1015). After being cultured on Czapek's yeast extract agar (CYA) at 30°C for 7 days, the colonies were 62-65 mm in diameter, flat, with gray central conidial structures; and no exudate was observed. After being cultured at 37°C for 7 days on Czapek's yeast extract agar (CYA), the colony diameter was 35-41 mm, with irregular radial grooves on the surface and no exudate. No growth was observed after culturing at 5°C for 7 days on Czapek's yeast extract agar (CYA) medium.

[0016] When cultured on malt extract agar (MEA) at 25°C for 7 days, the colonies were 47–52 mm in diameter, relatively thick, and velvety in texture. The colonies had indistinct concentric rings, with granular white mycelium in the center and white mycelium at the edge. The conidia were grayish-green, the exudate was colorless and contained no soluble pigment, and the reverse side was pale yellow (ral1001).

[0017] Incubated on YES plates at 25°C for 7 days, the colonies were 51–54 mm in diameter, relatively thick, with regular radial grooves (18–22 grooves) on the surface and neat edges; the texture was velvety; the conidia were grayish-green; the center of the colony contained cottony white mycelium, the colony edges were neat and white; there was a colorless exudate; there was no soluble pigment; the reverse side was pale yellow (ral1001).

[0018] When cultured on DG18 plates at 25°C for 7 days, the colonies were 23–24 mm in diameter, relatively thick, with neat edges; the texture was velvety; the conidia were grayish-green; the colony edges were neat and white; there was no exudate or soluble pigment; the reverse side was bright ivory (ral1015).

[0019] When cultured on CREA plates at 25°C for 7 days, the colonies were 18–19 mm in diameter, thin, and had poor acid production capacity.

[0020] No sclerotia were observed to form after culturing on oat agar (OA) medium at 25°C for 30 days.

[0021] The conidiophores are arranged in two whorls, occasionally in a single whorl; the conidia are ellipsoidal, arranged in chains, and have smooth walls.

[0022] 2. ITS sequence determination of the strain The ITS sequence data (SEQ ID NO.1) amplified from strain FS-PNC was submitted to GenBank. Homology comparison was performed using the online software Blast from the NCBI website. The FS-PNC strain showed the highest homology (over 99%) with *Penicillium oxalicum* A28, leading to the preliminary identification of FS-PNC as *Penicillium oxalicum*. On December 26, 2022, strain FS-PNC was deposited at the China General Microbiological Culture Collection Center (CGMCC), classified and named *Penicillium oxalicum* FS-PNC, with accession number CGMCC NO.40461, and the deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0023] Example 3: Application of FS-PNC FS-PNC sporulation medium formulation and culture method: Sporulation plate culture medium formula: 20g wheat bran, 20g rice bran, 20g agar, 1000ml water; sterilize at 121℃ for 20min.

[0024] Solid-phase fermentation medium formula: wheat bran: rice bran: corn flour: rice husk = 1:1:1:1 by weight, add water to a moisture content of 65%; sterilize at 121℃ for 45 minutes.

[0025] Preparation of spore seed solution: FS-PNC was cultured on a sporulation plate at 28°C for 7 days to produce a large number of conidia. Conidia were collected from the plate and prepared into a suspension with 0.1% Tween-80. The suspension was shaken to ensure full dispersion of conidia, and then the conidia concentration was adjusted to 10. 8 Spores / ml, to obtain spore seed solution, for later use.

[0026] Inoculation and culture method: Spread the solid-phase fermentation medium evenly in a stainless steel tray to a thickness of 100 mg / cm². 2 Then, spray the obtained spore seed solution at a rate of 100 ml / m³. 2 After inoculation, the rice plants were covered with plastic wrap to keep them moist and cultured in the dark at 28°C for 5 days. After that, the plastic wrap was removed, and the plants were covered with newspaper and cultured for another 2 days. After the culture was completed, the plants were transferred to a ventilated and dried place at 34±1°C for 24 hours. The rice plants were then sieved through a 150-mesh sieve to obtain FS-PNC conidial powder. Water was added to prepare a uniform suspension, which was then sprayed in the field to control rice planthoppers.

[0027] The rice variety is Yiyou 673, with a planting area of ​​66.7m² per plot. 2 Five treatments and one water control were set up, with three replicates per treatment, arranged in a randomized block design. Application was performed after 8 PM. The pesticide dosage and amount per acre are as follows: deal with medicine Dosage (ml) T1 FS-PNC 7 billion spores / ml 90 T2 FS-PNC 8 billion spores / ml 90 T3 FS-PNC 9 billion spores / ml 90 T4 Metarhizium anisopliae 8 billion spores / ml 90 T5 0.3% matrine 150 CK Clear water 150 Calculation formula: Insect population reduction rate = (Number of live insects before treatment - Number of live insects after treatment) / Number of live insects before treatment × 100%; Corrected control efficacy = [1 - (Insect population base before treatment in the control area × Number of insects after treatment in the treated area) / (Insect population after treatment in the control area × Number of insects before treatment in the treated area)] × 100.

[0028] The results are shown in the table below. FS-PNC has a significant effect on the control of rice planthoppers.

[0029] Note: The data in the table are the average of three replicates. Lowercase letters in the same column indicate 5% significance, and ** indicates that each treatment is highly significant compared to the control.

[0030] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A biocontrol agent for controlling rice planthoppers, characterized by: The biocontrol bacteria is classified and named Penicillium oxalate ( Penicillium oxalicum FS-PNC was deposited at the China General Microbiological Culture Collection Center on December 26, 2022, with accession number CGMCC NO.40461, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The application of the biocontrol bacteria as described in claim 1 in the control of rice planthoppers.

3. A biocontrol agent, characterized in that: The biocontrol agent comprises the conidial powder of the biocontrol bacteria as described in claim 1.

4. The application of the biocontrol agent as described in claim 3 in the control of rice planthoppers.

5. A method for controlling rice planthoppers, characterized in that: The biocontrol agent described in claim 3 is sprayed onto rice.

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