Isaria javanica strain JAM-1 and application of Isaria javanica strain JAM-1 in prevention and control of baccarpus pseudocerasus
By using the Javan spore strain JAM-1 as a microbial insecticide, the problems of pesticide resistance and environmental pollution caused by chemical control of cherry fruit bees were solved, achieving a highly efficient and safe biological control effect.
Patent Information
- Application Number
- CN202511217793.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-28
- Publication Date
- 2025-10-28
Smart Images
Figure CN120843299A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbiology technology, specifically relating to a strain of *Cladosporium javanicum* JAM-1 and its application in controlling cherry fruit bees. Background Technology
[0002] The cherry fruit wasp belongs to the order Hymenoptera, family Tenthredinidae, and genus Analcellicampa. Adult females are 6-8 mm long with a wingspan of 12-15 mm, entirely black and glossy, with filiform antennae of 9 segments. Males are slightly smaller, 5-7 mm long, with 9-segmented antennae bearing two rows of long ring hairs. The cherry fruit wasp is mainly distributed in cherry-producing areas of Beijing, Hebei, Henan, Shaanxi, Hubei, and Sichuan, and its damage is more severe than that of the cherry fruit fly. The cherry fruit wasp primarily damages flowers and fruits as larvae. After hatching, the larvae feed on the calyx, petals, and stigma. As they grow larger, they bore into the fruit, feeding on the pulp and seeds. Generally, one larva can damage 1-2 fruits. The larval stage lasts 20-30 days, after which they detach from the fruit and burrow into the soil to overwinter in a cocoon. This pest begins to damage cherry fruit from the young fruit stage, causing fruit deformities, rot, and fruit drop. In severe cases, the rate of infested fruit can reach over 90%, causing enormous harm to the cherry industry in various regions.
[0003] Currently, the control of cherry fruit wasps relies primarily on chemical control, supplemented by physical and agricultural methods. However, due to the cherry fruit wasp's remarkable resilience and strong resistance to pesticides, coupled with the lack of highly effective targeted pesticides and single, effective control measures, field control results remain unsatisfactory. To curb outbreaks, producers are often forced to increase the use of chemical pesticides, creating a vicious cycle of "increased resistance - further increases in pesticide use," which in turn triggers a chain reaction leading to a sharp decline in natural enemies, environmental pollution, and excessive pesticide residues in agricultural products.
[0004] Biological control is a relatively ideal and environmentally friendly method for controlling cherry fruit bees. Entomopathogenic fungi, as an important class of pathogens of pests, play a crucial role in the biological control of pests. For example, *Metarhizium anisopliae*, *Beauveria bassiana*, *Isaria fumosorosea*, and *Bacillus thuringiensis* are widely used in the control of agricultural pests. However, there are currently no reports on the use of *Isaria javanica* to control cherry fruit bees.
[0005] The information disclosed in this background section is only intended to enhance understanding of the overall background of the invention and should not be considered as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the Invention
[0006] The purpose of this invention is to provide a strain of *Cladosporium javanicum* JAM-1 and its application in controlling cherry fruit bee, so as to solve the problems existing in the use of chemical methods to control cherry fruit bee in the prior art.
[0007] To achieve the above objectives, the present invention provides the following technical solution:
[0008] The first objective of this invention is to provide a strain of Isariajavanica JAM-1, which was isolated and purified from infected pine caterpillars collected in Shiyan City, Hubei Province. Morphological and molecular biological identification confirmed it as Isariajavanica. It was deposited on June 25, 2025, at the China Center for Type Culture Collection (CCTCC, Wuhan University Collection Center, Postcode: 430072), with accession number CCTCC M 20251476.
[0009] The present invention also provides the application of the Javan spore strain JAM-1 in the control of cherry fruit bees.
[0010] The present invention also provides a microbial insecticide comprising the Javan spore strain JAM-1.
[0011] Compared with the prior art, the present invention has the following beneficial effects:
[0012] (1) The Javan spore strain JAM-1 of the present invention is an insect pathogenic fungus. It has been found that it has high pathogenicity against cherry fruit bee larvae, can effectively kill cherry fruit bee larvae, and has a high stunted larvae rate.
[0013] (2) The present invention contains the strain JAM-1 of *Bryum ovale*, which is environmentally friendly, safe for humans and animals, suitable for the needs of organic agricultural production, and has the potential to be developed into a microbial insecticide.
[0014] Preservation Information
[0015] Isariajavanica JAM-1, accession number: CCTCC M 20251476, accession date: June 25, 2025, depositary institution: China Center for Type Culture Collection (CCTCC), depositary address: Wuhan University Collection Center. Attached Figure Description
[0016] Figure 1Front view of colony morphology of Java sp. strain JAM-1;
[0017] Figure 2 The reverse side view of the colony morphology of *Cladosporium javanicum* strain JAM-1;
[0018] Figure 3 Sporulation structure and conidia of the Javan Fibrosporium strain JAM-1;
[0019] Figure 4 Phylogenetic tree of the Javan spore strain JAM-1;
[0020] Figure 5 The infection morphology of the Javan spore strain JAM-1 on 4th instar cherry fruit bee larvae 3 days later. Detailed Implementation
[0021] The technical solution of this invention patent will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.
[0022] 1. Isolation and purification of *Cladosporium javanicum* strain JAM-1
[0023] The bodies of pine caterpillars infected with the disease were collected from Saiwudang, Shiyan City, Hubei Province. The caterpillars were placed on a clean bench and first disinfected with 75% alcohol for 30 seconds, then soaked in 1% sodium hypochlorite for 2 minutes, and then rinsed three times with sterile water. Finally, the caterpillars were placed in a sterile petri dish lined with sterile filter paper and dissected into small pieces with sterile forceps and scalpel.
[0024] On a clean bench, the tissue blocks were aseptically inoculated onto PDA (potato dextrose agar) plates, three blocks per plate. The plates were then incubated at 25°C in a biochemical incubator. Once mycelia appeared, the transformed strain was picked and placed on a PDA plate for purification. This purification process was repeated three times. The purified fungus was designated JAM-1, and the purified mycelia were inoculated into PDA slant tubes. After the strain had fully colonized the slant, it was stored at 4°C.
[0025] 2. Morphological identification of *Cladosporium javanicum* strain JAM-1
[0026] 2.1 Cultivation and Observation
[0027] The strain was inoculated onto PDA medium and cultured for observation. Colony morphology characteristics are shown in [see attached image]. Figure 1 and Figure 2 .
[0028] Depend on Figure 1 and Figure 2 It can be seen that on the PDA plate, the colonies are white, thick and fluffy, with a pale yellow back and a light purple-gray spore layer.
[0029] 2.2 Observation and photography under an optical microscope
[0030] See results Figure 3 .
[0031] Depend on Figure 3 Under an optical microscope, the hyphae are colorless and transparent, with a width of 2.35±0.31μm. The conidiophores are similar to the hyphae, with 2-4 phialides forming whorls on each conidiophore. The base of the phialides is elliptical and swollen, gradually tapering upwards. Conidia grow on the phialides to form conidial chains. The conidia are transparent, smooth, and oblong, with pointed ends, measuring 5.23±0.45μm in length and 2.06±0.13μm in width.
[0032] 3. Molecular biological identification of *Cladosporium javanicum* strain JAM-1
[0033] Fungal DNA was extracted according to the instructions of the bioengineering fungal genomic DNA rapid extraction kit to obtain the genomic DNA of *Cladosporium javanicum* JAM-1. Primers were designed and synthesized to amplify the ribosomal transcription spacer sequence (ITS), elongation factor-α (EF1-α), and β-tubulin gene, respectively, yielding PCR amplification products. The primer sequences are shown in Table 1 below, and the PCR reaction system is shown in Table 2 below.
[0034] Table 1. Relevant primer sequences
[0035]
[0036] Table 2 PCR reaction system
[0037]
[0038] PCR reaction program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 35 cycles; 72℃ extension for 10 min.
[0039] The amplified products were recovered, purified, ligated, transformed, and sequenced. The nucleotide sequences of ITS, EF1-α, and β-tubulin were obtained and are shown in SEQ ID No. 1 to SEQ ID No. 3, respectively. A phylogenetic tree was constructed using MEGA 11 based on the ITS, EF1-α, and β-tubulin sequences using the neighbor-joining method. The results are shown in […]. Figure 4 .
[0040] Depend on Figure 4 It is known that the *Isaria javanica* strain JAM-1 belongs to the same species as *Isaria javanica*. Based on morphological characteristics and phylogenetic analysis, the strain is clearly classified as *Isaria javanica*, belonging to the phylum Ascomycota, class Sordariomycetes, order Hypocreales, family Cordycipitaceae, and genus *Isaria*. Therefore, it is named *Isariajavanica* JAM-1.
[0041] 4. Virulence determination of *Cladosporium javanicum* strain JAM-1 against 4th instar larvae of the cherry fruit wasp.
[0042] 4.1 Preparation of conidial suspension
[0043] Take the preserved slant culture of *Cladosporium javanicum* strain JAM-1, scrape off a small amount of conidia and inoculate them onto a PDA plate. Incubate in an incubator at 25-28℃ for 1-2 weeks. After conidia are produced, scrape off the conidia and place them in a centrifuge tube. Add 0.05% Tween-80 solution, shake well, and measure the conidia concentration using a hemocytometer. Prepare a conidia suspension of the required concentration for later use.
[0044] 4.2 Impregnation Test
[0045] Under laboratory conditions, 4th instar cherry fruit wasp larvae were treated with a conidial suspension of *Cladosporium javanicum* strain JAM-1 by immersion method. The number of larvae was 30, and the treatment was repeated three times. A 0.05% Tween-80 aqueous solution was used as a control for the larvae. Observations and records were recorded at 3, 5, and 7 days after treatment.
[0046] The results showed that after treating cherry fruit wasp larvae with a suspension of conidial spores of *Cladosporium javanicum* strain JAM-1 for 3 days, the 4th instar larvae began to die, exhibiting stiffness. Hyphae of *Cladosporium javanicum* strain JAM-1 were observed extending from the larval surface and producing numerous conidia (see...). Figure 5 ).
[0047] The number of larvae was recorded, and the corrected mortality rate of 4th instar cherry fruit wasp larvae treated with different concentrations of Java sp. strain JAM-1 conidial suspensions was calculated, as well as the larval stunting rate of cherry fruit wasp after treatment with different concentrations of Java sp. strain JAM-1 conidial suspensions. The results are shown in Table 3.
[0048] Table 3. Pathogenicity of *Cladosporium javanicum* strain JAM-1 against 4th instar larvae of the cherry fruit bee.
[0049]
[0050] Note: Corrected mortality rate (%) = (treatment mortality rate - control mortality rate) / (1 - control mortality rate) × 100%.
[0051] Table 3 shows that, under different concentrations of conidial suspension treatment, the corrected mortality rate and stunted rate of 4th instar larvae of the cherry fruit wasp increased with increasing conidial concentration. Furthermore, with prolonged treatment time, the corrected mortality rate of 4th instar larvae of the cherry fruit wasp increased in all treatment groups. (20×10) 6 The corrected mortality rate for the spore / mL treatment reached 98.42%.
[0052] In addition, the toxicity of the Javan spore strain JAM-1 to the 4th instar larvae of the cherry fruit bee was calculated by virulence regression analysis, and the results are shown in Table 4.
[0053] Table 4. Virulence of *Cladosporium javanicum* strain JAM-1 against 4th instar larvae of the cherry fruit bee.
[0054]
[0055] Table 4 shows that after 7 days of treatment, the LC50 of *Cladosporium javanicum* strain JAM-1 against 4th instar larvae of the cherry fruit bee was... 50 1.04×10 6 Conidia / mL.
[0056] In summary, the Java sp. JAM-1 strain described in this invention has high pathogenicity against cherry fruit wasp, can effectively kill cherry fruit wasp larvae, and has a high larval stunting rate. It is a biological control strain with potential significance in the control of cherry fruit wasp.
[0057] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.
Claims
1. A strain of *Cladosporium javanicum* JAM-1, characterized in that, Its taxonomic name is Isariajavanica, and it was deposited at the China Center for Type Culture Collection on June 25, 2025, with accession number CCTCC M 20251476.
2. The application of the Javan Species strain JAM-1 according to claim 1 in the control of cherry fruit bees.
3. A microbial insecticide, characterized in that, Includes the Javan spore strain JAM-1 as described in claim 1.