A strain of java clonostachys rosea dmc01 and its application in preventing and treating caloptilia theivorella elliott

Abstract

The present application belongs to the technical field of microbiology, and particularly relates to a strain of Isaria javanica DMC01 and application thereof in prevention and treatment of Chrysoritis basalis. The strain of Isaria javanica DMC01 is taxonomically named as Isaria javanica, and was preserved in the Guangdong Microbial Culture Collection Center on April 28, 2021, with a preservation number of GDMCC No. 61632. The Isaria javanica DMC01 of the present application has high pathogenicity to Chrysoritis basalis, can effectively kill Chrysoritis basalis, and has a high rate of cadaver. The Isaria javanica DMC01 of the present application is a biological control strain with potential significance in the prevention and treatment of Chrysoritis basalis.

Description

Technical Field

[0001] This invention belongs to the field of microbiology technology, specifically relating to a strain of Java sp. DMC01 and its application in controlling the yellow-winged silkworm moth. Background Technology

[0002] The yellow-winged silkworm moth (Pyralidae) is a pest belonging to the genus Pyralidae in the family Pyralidae of the order Lepidoptera. It is a significant pest of tropical fruit trees such as jackfruit, durian, and breadfruit. Adult females lay eggs on the surface of tender shoots, fruit stalks, or flower buds of the host plant. After hatching, the larvae feed on the surrounding tissues. Around the second instar, they bore into tender branches, flower buds, and fruits to feed. Mature larvae can pupate inside the tunnels or emerge and spin silk to pupate within leaves. The accumulation of frass inside and outside the tunnels attracts fruit fly larvae, causing the affected parts of the fruit to turn brown and rot, leading to poor fruit development or fruit drop and reduced yield.

[0003] Chemical pesticides are currently the primary method for controlling the yellow-winged silkworm moth. However, the extensive use of chemical pesticides easily leads to environmental pollution and pesticide resistance in the moth. Biological control is a more ideal and environmentally friendly method for controlling the yellow-winged silkworm moth. Entomogenic fungi, as an important type of pathogenic fungi of pests, play an important role in the biological control of pests. Currently, there are no reports on the use of *Isaria javanica* for the control of the yellow-winged silkworm moth.

[0004] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Java sp. DMC01 and its application in the control of the yellow-winged silkworm moth.

[0006] A strain of Isaria javanica, DMC01.

[0007] The strain DMC01 of Isaria javanica provided by this invention was obtained by collecting naturally infected insects, isolating and purifying it, and identified as Isaria javanica by morphology and molecular biology. It was deposited on April 28, 2021, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, postcode: 510070), with accession number GDMCC No. 61632.

[0008] This invention also provides the application of the Javan spore strain DMC01 in the control of the yellow-winged silkworm moth. Those skilled in the art can formulate the Javan spore strain DMC01 into a microbial insecticide for the control of the yellow-winged silkworm moth.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] (1) The present invention isolates and purifies the strain, and through morphological observation and molecular identification, the obtained strain DCM01 is identified as Isaria javanica; secondly, the applicant also determined its toxicity to the yellow-winged silkworm moth. The test results showed that the Isaria javanica DMC01 of the present invention has high pathogenicity, can effectively kill the yellow-winged silkworm moth, and has a high rate of stunted insects.

[0011] (2) The Java sporophyllum DMC01 of the present invention is a biological control strain with potential significance in the control of yellow-winged silkworm moth. It can be developed into a microbial insecticide, providing technical support for the green control of yellow-winged silkworm moth.

[0012] Preservation Information

[0013] Isaria javanica DMC01, accession number GDMCC No.61632, accession date April 28, 2021, deposited at Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No.100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0014] Figure 1 This is a frontal view of the colony morphology of strain DMC01;

[0015] Figure 2 This is a reverse image of the colony morphology of strain DMC01;

[0016] Figure 3 This is a morphological image of *Cladosporium javanica* under a microscope.

[0017] Figure 4 Phylogenetic diagram of strain DMC01;

[0018] Figure 5 and Figure 6 The infection morphology of strain DMC01 on the yellow-winged silkworm moth. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to specific embodiments. These descriptions are for explanation purposes only and are not intended to limit the scope of the invention.

[0020] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Unless otherwise specified, all experimental methods used in the following examples are conventional methods.

[0021] Example

[0022] I. Isolation and purification of *Cladosporium javanicum* strain DMC01

[0023] The bodies of eucalyptus caterpillars were collected from Qinlian Forest Farm, Qinzhou City, Guangxi Zhuang Autonomous Region. On a clean bench, the caterpillars were immersed in 75% alcohol for 30 seconds for surface disinfection, and then soaked in 0.1% mercuric chloride for 3 minutes for disinfection. They were then transferred to three bottles of sterile water for rinsing, and then placed on sterilized filter paper to dissect the caterpillars into small pieces.

[0024] Five tissue blocks were placed on PDA medium supplemented with 50 μg / ml streptomycin per plate and incubated at 25°C. After mycelium growth, the transformed strain was picked and transferred to PDA plates for purification. The purification process was repeated three times. One selected fungus was designated DMC01, and the purified mycelium was inoculated into PDA slant tubes. After the strain had fully colonized the slant tubes, they were stored at 4°C as a culture medium.

[0025] II. Identification of *Cladosporium javanicum* strain DMC01

[0026] The bacterial strain was cultured at 25℃, and the colonies were observed during the culture process. The morphology of the colonies was as follows: Figure 1 and Figure 2 As shown.

[0027] from Figure 1 and Figure 2 As can be seen, on the PDA plate, the colonies are round, the hyphae are white, the texture is thick and fluffy, the back of the colony is pale yellow, and the spore layer is light purple-gray.

[0028] Under a microscope, the hyphae are colorless and transparent, with whorls of 2-4 phialides growing on each sporophore. The base of the phialides is elliptical and swollen, gradually tapering upwards. Conidia grow on the phialides to form chains of conidia. The conidia are transparent, smooth, and elongated elliptical. Figure 3 .

[0029] Based on the above characteristics such as colony morphology and spore morphology, the strain was preliminarily identified as Isariajavanica.

[0030] III. Molecular Identification

[0031] 1. DNA was extracted according to the instructions of the NuClean Plant Genomic DNA Kit from the Kangwei Century strain DMC01 to obtain the genomic DNA of the Javan Fibrosium strain.

[0032] 2. The target gene ribosomal transcription spacer sequence (ITS), elongation factor-α (EF1-α), and β-tubulin gene were selected for amplification to obtain PCR amplification products. The relevant primer sequences are shown in Table 1 below, and the PCR reaction system is shown in Table 2 below.

[0033] Table 1 Sequences of relevant primers

[0034]

[0035] Table 2 PCR reaction system

[0036]

[0037] PCR reaction procedure for ITS and β-tubulin: denaturation at 95℃ for 3 min; denaturation at 95℃ for 30 s, annealing at 52℃ for 30 s, extension at 72℃ for 1 min, for a total of 35 cycles; extension at 72℃ for 10 min.

[0038] PCR reaction program for EF1-α: denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 55℃ for 45 s, extension at 72℃ for 90 s, for a total of 35 cycles; extension at 72℃ for 7 min.

[0039] 3. The amplified products were recovered, purified, ligated, transformed, and sequenced. The nucleotide sequences of ITS, EF1-α, and β-tubulin were obtained by amplification and are shown in SEQ ID No. 7 to SEQ ID No. 9, respectively.

[0040] 4. The sequencing results were processed using Bioedit and DNAman software, and BLAST alignment was performed in the GenBank database. High-reliability related sequences were downloaded and aligned using MEGA 7.0. A phylogenetic tree of strain DMC01 was constructed using the neighbor-joining method. A bootstrap test was performed during tree construction, and the sampling was repeated 1000 times. The results are shown in […]. Figure 4 .

[0041] from Figure 4It can be seen that strain DMC01 belongs to the same clade as Isaria javanica CHE-CNRCB 305 and Isariajavanica CBS134.22, and its morphological characteristics are consistent with those of Isaria javanica. Therefore, strain DMC01 is identified as Isaria javanica. Thus, it is named Isaria javanicaDMC01.

[0042] IV. Virulence determination of *Cladosporium javanicum* strain DMC01 against *Hemiberlesia lataniae*.

[0043] 1. Using an inoculation loop, scrape the conidia of the isolated and purified *Cladosporium javanicum* strain into a 50 mL Erlenmeyer flask, add 20 mL of an aqueous solution containing 0.05% Tween-80, shake thoroughly, and count the spores under a microscope using a hemocytometer to obtain the initial concentration of the spore stock solution. Prepare a 1×10⁻⁶ spore solution. 5 1×10 6 1×10 7 1×10 8 Four concentration gradients of spores / mL were established, with each concentration constituting one treatment. A 0.05% Tween-80 aqueous solution was used as a control. Each treatment was replicated in three groups, with 20 3-year-old yellow-winged silkworm moths in each replicate.

[0044] 2. Evenly spray the prepared spore solutions of various concentrations, as well as the 0.05% Tween-80 aqueous solution for the control treatment, onto the *Hemiberlesia lataniae* until the spore solution no longer drips. Then, place each *Hemiberlesia lataniae* individually into a rearing dish and incubate at 25℃ for 12 hours in both light and dark. Feed them daily with fresh jackfruit leaves, and observe and record the mortality rate of each treatment daily for 8 consecutive days. If any insects die, remove them from the rearing dish and place them in a sterilized glass petri dish lined with filter paper for humidified cultivation. Observe whether mycelium grows. Results are shown in […]. Figure 5 and Figure 6 .

[0045] from Figure 5 and Figure 6 It can be seen that after the DMC01 strain infects the yellow-winged silkworm moth, mycelium will grow on the body of the yellow-winged silkworm moth.

[0046] The number of mummified insects was recorded, and the corrected mortality rate of *Gnaphalium affine* under different concentrations of *Gnaphalium affine* DMC01 and the mummified insect rate of *Gnaphalium affine* after treatment with different concentrations of *Gnaphalium affine* DMC01 were calculated. The results are shown in Table 3.

[0047] Table 3. Pathogenicity of different concentrations of *Cladosporium javanicum* DMC01 against *Hemiberlesia lataniae*.

[0048]

[0049] Table 3 shows that, for the same culture time, the corrected mortality rate of *Gnaphalium affineum* increased with increasing spore concentration. After inoculation, 1×10-1 5 spores / mL and 1×10 6 The corrected mortality rate and stunted worm rate were both low at spore / mL treatment, 1×10 7 After 8 days, the corrected mortality rate and scab rate were both 66.10% for spore / mL treatment, while for 1×10 8 After 8 days of treatment with spores / mL, the corrected mortality rate reached 100%, and the rate of stunted insects also reached 100%. The *Cladosporium javanicum* DMC01 of this invention exhibits high pathogenicity against the yellow-winged silkworm moth. Specifically, 1×10 8 A spore solution with spores / mL can quickly and effectively kill the yellow-winged silkworm moth, and has a high rate of insect stunting.

[0050] In summary, the Javan-based strain DMC01 exhibits high pathogenicity against the yellow-winged silkworm moth, effectively killing it and achieving a high rate of insect stunting. It is a potentially significant biological control strain for the yellow-winged silkworm moth.

[0051] The foregoing description of specific exemplary embodiments of the present invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed. The exemplary embodiments were chosen and described 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 modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. The application of a strain of *Cladosporium javanicum* DMC01 in the control of the yellow-winged silkworm moth, characterized in that, The taxonomic name of the Javan Fibrosporium strain DMC01 is... Isaria javanica It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on April 28, 2021, with accession number GDMCC No. 61632.

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