Cladosporium cladosporium HKLJ-2 and application thereof in prevention and control of tetranychus urticae

Abstract

The invention provides cladosporium cladosporioides HKLJ-2 and application of the cladosporium cladosporioides HKLJ-2 in prevention and control of tetranychus urticae, and relates to the technical field of biocontrol bacteria. The cladosporium cladosporioides HKLJ-2 strain is separated from a tetranychus urticae body dead on an angelica sinensis plant, is identified as cladosporium cladosporioides, is named as cladosporium cladosporioides HKLJ-2, is preserved in the China Center for Type Culture Collection (Wuhan University) on September 10, 2024, has the preservation number of CCTCC NO: M 20241952, and has the preservation number of CCTCC NO: M 20241952. The spore liquid has strong pathogenicity to tetranychus urticae adults in a normal temperature environment, is expected to be developed into biocontrol bacteria of tetranychus urticae, and provides more choices for biological control of tetranychus urticae.

Description

Technical Field

[0001] This invention relates to the field of biocontrol technology, specifically to a strain of Cladosporium HKLJ-2 and its application in controlling two-spotted spider mites. Background Technology

[0002] Two-spotted spider mite ( Tetranychus urticae Koch The two-spotted spider mite (Tetranychus spp.), also known as the red spider mite, belongs to the family Tetranychidae in the order Acari of the class Arachnidae. It is a polyphagous pest widely distributed worldwide, causing severe economic losses, particularly to vegetables, flowers, and medicinal plants in my country. Adults, larvae, and nymphs of the two-spotted spider mite congregate on the underside of leaves to feed. Initially, chlorotic spots appear on the leaves, gradually spreading to the entire leaf, causing chlorosis and, in severe cases, leaf drop, thus affecting plant growth. In recent years, the two-spotted spider mite has been observed in Yunnan province on Angelica sinensis (Angelica sinensis). Angelica sinensis (Oliv.)Diels), Yunnan Polygonatum ( Polygonatum kingianum This disease occurs extensively on medicinal plants such as Coll., causing premature withering and collapse of the plants, death of the above-ground parts, and resulting in serious economic losses.

[0003] Currently, the main method for controlling two-spotted spider mites relies on chemical control. However, due to the long-term use of chemical pesticides, the two-spotted spider mite has developed strong resistance, resulting in limited control effectiveness. Furthermore, the frequent use of chemical pesticides causes pesticide residues and environmental pollution. Therefore, finding a green, efficient, and safe biological control measure for two-spotted spider mites is crucial for agricultural production. The use of biological control measures to control two-spotted spider mites has gradually attracted attention. Among these, the use of biocontrol bacteria has the advantages of being naturally sourced, environmentally friendly, and capable of causing stable disease outbreaks within the population without easily inducing resistance. Currently reported pathogenic fungi of two-spotted spider mites include Beauveria bassiana and Cordyceps sinensis. Therefore, developing more biocontrol bacteria resources for two-spotted spider mites is particularly important for effective control. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a strain of Cladosporium HKLJ-2 and its application in the control of two-spotted spider mites. This strain has strong pathogenicity against two-spotted spider mites and has the potential to be developed into a biocontrol bacterium, which will provide more options for the biological control of two-spotted spider mites.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A species of Cladosporium ( Cladosporium cladosporioides The preservation name of *Cladosporium* HKLJ-2 is *Cladosporium* HKLJ-2. Cladosporium cladosporioides HKLJ-2 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20241952.

[0006] The above-mentioned strain was isolated from two-spotted spider mites infected with fungi, collected from Angelica sinensis plants at a medicinal herb planting base in Gucheng District, Lijiang City, Yunnan Province. It was identified as... Cladosporium (Cladosporium) Cladosporium cladosporioides (Cladosporium).

[0007] The present invention also provides the application of the above-mentioned Cladosporium HKLJ-2 strain in the control of two-spotted spider mites.

[0008] Preferably, the spore concentration of Cladosporium HKLJ-2 used by the biocontrol bacteria to control two-spotted spider mites is 1.0 × 10⁻⁶. 3 -1.0×10 8 per mL.

[0009] This invention provides a strain of Cladosporium HKLJ-2 and its application in controlling the two-spotted spider mite. Compared with existing technologies, its advantages are: This invention isolated a strain HKLJ-2 from the cadavers of two-spotted spider mites that died naturally from infection on Angelica sinensis plants. This strain was identified as Cladosporium (Cladosporium cladosporium). Cladosporium cladosporioides The strain, named Cladosporium HKLJ-2, exhibits strong pathogenicity against adult female Tetranychus tinctoria under normal temperature and humidity conditions and is expected to be developed into a biocontrol agent for Tetranychus tinctoria. Attached Figure Description

[0010] Figure 1 This is a schematic diagram of the morphological characteristics of the Cladosporium HKLJ-2 strain in an embodiment of the present invention; Figure 2 This is a phylogenetic tree of Cladosporium HKLJ-2 strain based on EF1α-rDNA sequence analysis in this embodiment of the invention; Figure 3 This is a schematic diagram of the symptoms of two-spotted spider mite infection in an embodiment of the present invention; Figure 4 This is a slide specimen of adult two-spotted spider mite infected with Cladosporium HKLJ-2 strain in an embodiment of the present invention; Figure 5 The leaves of *Polygonatum yunnanense* used in the pathogenicity test in this embodiment of the invention; Figure 6 The mortality rate of the spore suspension of Cladosporium strain HKLJ2 in this embodiment of the invention against female adult Tetranychus tinctoria. Detailed Implementation

[0011] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0012] The Cladosporium strain HKLJ-2 described in this invention is classified and named as: Cladosporium strain HKLJ-2 Cladosporium cladosporioides HKLJ-2, with accession number CCTCC NO: M 20241952, was received and registered by the China Center for Type Culture Collection (CCTCC, address: Wuhan University Collection Center, Luojia Mountain, Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China) on September 10, 2024. The biological material was tested by the Collection Center on September 17, 2024, and the result showed it to be viable. Example 1:

[0013] Obtaining and identifying Cladosporium HKLJ-2 strain: (1) Obtained: Fungi were isolated from the dead two-spotted spider mites that died naturally from infection on severely damaged Angelica sinensis leaves in Gucheng District, Lijiang City, Yunnan Province. Infected and killed two-spotted spider mites were picked into centrifuge tubes containing 1 mL of sterile water. The tubes were shaken to detach the parasitic fungal spores, yielding a stock solution of spores. The fungus was isolated using a plate spread method. The stock solution of spores was diluted 10-fold, 100-fold, 1000-fold, and 10000-fold, and 200 μL of each dilution was evenly spread onto the surface of PDA solid medium. The medium was incubated at 25°C and continuously observed. After colony formation, edge hyphae were picked using an inoculation needle for transfer and purification. The purified strain was then stored on PDA slant at 4°C.

[0014] The PDA solid culture medium composition, in g / L, is as follows: potato (200 g diced + 1 L distilled water, boiled for 30 min / L), take the filtrate, add glucose (20 g) and agar (17 g), and make up to 1 L.

[0015] (2) Identification: The isolated strains were subjected to morphological and molecular biological identification, and the experimental results are recorded below: A. Morphological characteristics: The colonies of this strain are light olive-green, with a flat or raised surface, often with irregular wrinkles in the center. They are powdery to felt-like, producing light brown conidia. Under a microscope, the hyphae are branched with transverse septa and branches. The conidia are ovoid or obovoid, approximately 4 μm in diameter. Figure 1 .

[0016] B. EF1α gene sequence analysis: The EF1α sequence of the strain was determined (the nucleotide sequence is shown in SEQ ID NO:1).

[0017] EF1α universal primers: EF1αF(5'-CATCGAGAAGTTCGAGAAGG-3'); PCR amplification was performed using EF1αR(5'-TACTTGAAGGAACCCTTACC-3'), and the reaction system is shown in Table 1. Table 1

[0018] The PCR reaction conditions were as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 40 s, 56℃ annealing for 40 s, 72℃ extension for 1 min, for a total of 35 cycles; 72℃ extension for 10 min; 1.8% agarose gel electrophoresis detection; storage at 4℃.

[0019] PCR products were sent to Sangon Biotech for sequencing. The sequencing results were then compiled and submitted to NCBI for BLAST comparison (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) to preliminarily determine the taxonomic position of the target strain. Based on relevant literature on bacterial classification, a phylogenetic tree (Nearest Neighbor (NJ)) was constructed using MEGA7 software for molecular identification, such as... Figure 2 As shown.

[0020] The strain was identified as Cladosporium dendriticum through morphological and molecular identification. Cladosporium cladosporioides It was named Cladosporium HKLJ-2. Example 2:

[0021] Verification of infection of Tetranychus pubescens HKLJ-2 by the two-spotted spider mite: Healthy adult female two-spotted spider mites bred in the laboratory were used for a reintroduction experiment. Cladosporium HKLJ-2 strain was inoculated onto PDA plates and cultured for 5 days. Mycelia and spores were scraped using a sterile inoculation needle and inoculated into sterile water containing 0.1% Tween-80. The inoculated water was transferred to 50 mL centrifuge tubes and then vortexed at 2000 rpm for 5 min to disperse the spores, obtaining a stock spore suspension. The spores were counted using a hemocytometer, and the concentration of the spore suspension was adjusted. The suspension was then diluted to obtain a 1×10⁻⁶ spore suspension. 8 A spore suspension of 1 spore per mL; 1×10 8 Two-spotted spider mite female adult spore suspension was used to infect the plant. A single *Polygonatum yunnanense* plant containing two-spotted spider mite female adult mites was selected for the experiment. The prepared spore suspension was sprayed onto the upper and lower surfaces of the leaves using a handheld sprayer. Seven days later, the leaves were cut off and observed and photographed under a stereomicroscope to capture the symptoms of two-spotted spider mite infection. Figure 3 ), and observed and photographed slides of Cladosporium HKLJ-2 infecting Tetranychus tinctoria under an inverted microscope ( Figure 4 This means that Cladosporium HKLJ-2 can infect Tetranychus tinctoria. Example 3:

[0022] Validation of the lethal intermediate concentration of Cladosporium HKLJ-2 strain against adult female Tetranychus two-spotted spider mites: Cladosporium HKLJ-2 strain was inoculated onto PDA plates and cultured for 5 days. Mycelia and spores were scraped using a sterile inoculation needle and inoculated into sterile water containing 0.1% Tween-80. The inoculated solution was transferred to 50 mL centrifuge tubes and then vortexed at 2000 rpm for 5 minutes to disperse the spores, yielding a stock spore suspension. The spores were counted using a hemocytometer, and the concentration of the spore suspension was adjusted to obtain a concentration of 1.0 × 10⁻⁶ spores. 8 1.0×10 7 1.0×10 6 1.0×10 5 1.0×10 4 1.0×10 3 Prepare a spore suspension at 1 spore / mL for later use.

[0023] The pathogenicity of Cladosporium HKLJ-2 strain against the two-spotted spider mite was determined using a spray method. Healthy, mite-free leaves of similar size from *Polygonatum yunnanense* were selected, washed with clean water, and placed with the underside of the leaves facing upwards in a 0.6% water agar medium to fix the leaves in place. A ring of moistened cotton strips was placed around the leaves to prevent the inoculated two-spotted spider mites from escaping. Figure 5 ).

[0024] Female adult two-spotted spider mites were transferred to the aforementioned leaves using a paintbrush, 35 mites per leaf. One hour later, the activity of the mites on the leaves was checked, and mites with low activity due to mechanical damage were removed, ensuring that each leaf contained at least 30 mites. Different concentrations of prepared spore suspensions were sprayed onto the leaf culture dishes containing female adult mites using a handheld sprayer, with a spray volume of 1 mL per dish. A sterile water treatment containing 0.1% Tween-80 was used as a control. Each treatment was repeated four times. The number of dead mites was counted daily until day 7 after spraying. Mites that did not move when touched by the brush were considered dead. The mite carcasses were then cultured in a moist environment; mites showing growth of the test bacteria were considered effectively dead. The cumulative mortality rate was calculated as: (daily cumulative number of dead mites / total number of tested mites) × 100%. Analysis was performed using Excel to determine the regression equation and calculate the median lethal concentration (LC50).

[0025] Experimental results: With increasing spore concentration and treatment time of Cladosporium HKLJ-2 strain, the cumulative mortality rate of female adult Tetranychus two-spotted spider mites continuously increased. Seven days after inoculation, female adult mites died in all treatments at the concentration of 1.0 × 10⁻⁶. 8 1.0×10 7 The cumulative mortality rate of female adult mites reached its highest value at concentrations of 1.0 × 10⁶ / mL, with the highest concentration being 1.0 × 10⁶ / mL. 8 The highest cumulative mortality rate among adult female mites was observed after treatment with mites / mL, reaching 96.67%; at a concentration of 1.0×10⁶ mite / mL... 7 After treatment with [number] mites / mL, the female adult mites were the second most numerous, reaching 88.33%. Figure 6 The pathogenicity of Cladosporium HKLJ-2 strain against adult female Tetranychus two-spotted spider mites on day 7 was determined using the Probit model, with the regression equation y = -0.2165 + 0.1475x. The LC50 at day 7 was 6.9183 × 10⁻⁶. 4 per mL.

[0026] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Cladosporium ( Cladosporium cladosporioides HKLJ-2, characterized in that: The *Cladosporium* HKLJ-2 species is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20241952.

2. The application of the Cladosporium HKLJ-2 strain as described in claim 1 in the control of two-spotted spider mites.

3. A biocontrol bacterium for controlling two-spotted spider mites, characterized in that: The biocontrol bacteria is the spore of Cladosporium HKLJ-2 as described in claim 1.

4. The biocontrol bacteria according to claim 3, characterized in that: When the biocontrol bacteria were used to control two-spotted spider mites, the spore concentration of Cladosporium HKLJ-2 was 1.0 × 10⁻⁶. 3 -1.0×10 8 per mL.

Images