A strain of Metarhizium anisopliae (YEC) and its application in controlling teak moth

Abstract

The present invention discloses a strain of Metarhizium flavoviride (YEC) and its use in controlling teak moths. The present strain of Metarhizium flavoviride (YEC) was isolated from soil in Guangdong Province and can infect the mangrove pest teak moth, achieving a high mortality rate and inactivation rate against the moth within a short period of time. The present strain of Metarhizium flavoviride (YEC) is a biological control strain with potential development and application value in controlling mangrove pests.

Description

Technical Field

[0001] The invention belongs to the technical field of microbiology, and particularly relates to a yellow-green Metarhizium anisopliae strain YEC and application thereof in preventing and controlling teak camel moth. Background Art

[0002] In recent years, mangrove pests have become frequent and widespread in various regions of my country, causing widespread damage. This not only poses a serious threat to the overall health of mangrove ecosystems but also has profound impacts on the ecological security of coastal areas. Major pests in mangrove ecosystems include the teak moth, the Guangzhou moth, the tung tree hairy-jawed moth, and the small bag moth. Among them, the teak moth (Hyblaea puera), a member of the Hyblaeidae family (Lepidoptera), is a newly emerging leaf-feeding mangrove pest that poses a serious threat to the ecological health of mangrove communities. The teak moth is an oligophagous insect that feeds on 45 host plant species, primarily from the Lamiaceae, Verbenaceae, and Rhizophoraceae families. Reports indicate that this insect primarily harms teak and Avicennia marina in my country.

[0003] The larvae of the teak moth go through five developmental stages and prefer to feed on young leaves. When an infestation occurs, the leaves, fruits, and young branches of mangrove plants are severely damaged within a short period of time. This prevents the plants from photosynthesizing for an extended period, leading to poor growth. This not only affects flowering and fruiting that year, but also negatively impacts the following year's reproductive cycle. Furthermore, damaged plants are more likely to attract pests such as bark beetles and water lice, increasing the risk of plant death.

[0004] Currently, mangrove pest control methods primarily include chemical, physical, and biological control. Chemical control, as a traditional pest control method, remains important in modern agriculture due to its rapid effectiveness and high efficacy. However, it also poses challenges such as pest resistance, harm to non-target organisms, pesticide residues in agricultural and forestry products, and environmental pollution. Due to the fragility of mangrove ecosystems, biological control, a naturally occurring method with greater safety, is the primary method for mangrove pest control.

[0005] Patent Publication No. CN105493966A discloses a method for controlling leaf-feeding pests in mangroves, including the use of a saturated saline solution for biological control of the teak moth, the Guangzhou moth borer, the hairy-jawed tortoise, and various bagworms. Patent Publication No. CN116210499A discloses a method for controlling mangrove pests using liquid plant glue, insect pheromone reagents, and biological agents. Patent Publication No. CN103283540A discloses a method for biological control of the mangrove pest, the hairy-jawed tortoise, using the natural enemy, the trichogrammatid wasp. These control methods have potential application value in the biological control of mangrove pests.

[0006] Currently, there is no report on the technology of using Metarhizium flavoviride to control mangrove pests. Summary of the Invention

[0007] In view of the deficiencies in the prior art, the present invention aims to provide a strain of Metarhizium anisopliae (YEC) and its application in preventing and controlling teak camel moth.

[0008] The first object of the present invention is to provide a Metarhizium flavoviride strain YEC, with a deposit number of GDMCC No.65143.

[0009] The second object of the present invention is to provide the use of the yellow green Metarhizium anisopliae strain YEC in preventing and controlling mangrove pests.

[0010] Preferably, the application is the application of the yellow-green Metarhizium anisopliae strain YEC in preventing and controlling teak camel moth.

[0011] Preferably, the teak moth is a 2nd to 3rd instar larvae of the teak moth.

[0012] Preferably, the application includes the step of contacting the living cells of the Metarhizium anisopliae strain YEC or a culture containing the living cells of the Metarhizium anisopliae strain YEC with mangrove pests.

[0013] Preferably, the culture containing the live cells of the Metarhizium anisopliae strain YEC is a conidia suspension of the Metarhizium anisopliae strain YEC.

[0014] Preferably, the concentration of the conidia suspension of Metarhizium anisopliae strain YEC is 5.83×10 5 cfu / mL or above.

[0015] Preferably, the concentration of the conidia suspension of Metarhizium anisopliae strain YEC is 3.08×10 8 cfu / mL or above.

[0016] Preferably, the application method is spraying or dipping.

[0017] The third object of the present invention is to provide a biocontrol preparation for controlling mangrove pests, which contains the live bacteria of the yellow-green Metarhizium strain YEC and / or a culture containing the live bacteria of the yellow-green Metarhizium strain YEC.

[0018] Beneficial effects of the present invention:

[0019] The yellow-green Metarhizium anisopliae strain YEC of the present invention has a highly effective insecticidal function, is simple to cultivate, grows rapidly, and produces a large amount of spores. It has a high mortality rate against the mangrove pest teak moth, is non-toxic and pollution-free, and has high biosafety, making it widely used in the prevention and control of teak moth pests in agriculture and forestry.

[0020] 2. The yellow-green Metarhizium anisopliae strain YEC of the present invention is a biological control strain with potential application value in the prevention and control of mangrove pests. It can be developed into a microbial insecticide, providing technical support for the green prevention and control of mangrove pests and has good application prospects.

[0021] Preservation Instructions

[0022] The Metarhizium flavoviride YEC (Metarhizium anisopliae strain YEC) of the present invention was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on September 14, 2024, with the deposit number GDMCC No. 65143, and the deposit address is the 5th floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the colony morphology of the yellow-green Metarhizium anisopliae strain YEC on the 7th day of culture on PPDA medium; among them, A is the front view of the colony of strain YEC, and B is the back view of the colony of strain YEC.

[0024] Figure 2 The morphology of conidia, conidiophores and hyphae of the yellow-green Metarhizium anisopliae strain YEC; among them, A is the conidia of strain YEC, B is the conidiophore of strain YEC, and C is the hyphae of strain YEC.

[0025] Figure 3 This is the phylogenetic tree constructed for strain YEC based on ITS site sequences.

[0026] Figure 4 This is a test of the toxicity of strain YEC to teak moth; among them, A is the healthy teak moth in the control group, B is the early stage of teak moth dead insect after being infected by strain YEC, and C is the late stage of teak moth dead insect infected by strain YEC. DETAILED DESCRIPTION

[0027] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0028] Example 1: Isolation and identification of Metarhizium anisopliae strain YEC

[0029] 1. Isolation of Metarhizium flavoviride strain YEC

[0030] The Metarhizium flavoviride strain YEC was isolated from the soil of Guangdong Province.

[0031] 2. Identification of strains

[0032] 2.1 Morphological identification

[0033] The strain YEC was cultured on PPDA plates, and the morphology of the colonies was as follows: Figure 1 As shown, Figure 1 A in the figure is the front side of the YEC colony. Figure 1 Panel B is the back of the YEC colony.

[0034] from Figure 1 It can be seen that on the PPDA plate, the colonies of strain YEC are round with light yellow back. After 5-7 days of culture, conidia begin to form in the center of the colony, aggregate in green, and are surrounded by scattered white hyphae.

[0035] Observed under a microscope, the hyphae are separated and transparent ( Figure 2 C), molecular spore stalks are solitary or aggregated, branched at the top, and end in a bottle-shaped stalk ( Figure 2 B in the figure); conidia are oblong and transparent ( Figure 2 A), the size is (5.7-)6.4-8.8(-9.0)×(3.0-)3.6-5.5(-5.7)μm (average size 7.7×4.4μm, n=50, n is the statistical number), L / W=1.75, L / W is the aspect ratio.

[0036] 2.2 Molecular Biological Identification

[0037] The strain YEC was transferred to PPDA medium and cultured for 7 days before scraping conidia and hyphae.

[0038] DNA was extracted according to the instructions of the M5 HiPer Fungal Genomic DNA Kit (purchased from Polymer, product number MF070-plus) to obtain genomic DNA of strain YEC.

[0039] The rDNA-ITS sequence was amplified using the universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3', SEQ ID NO. 1) / ITS4 (5'-TCCTCCGCTTATTGATATGC-3', SEQ ID NO. 2) of the fungal ribosomal rDNA region to obtain a PCR amplification product.

[0040] The PCR reaction program for amplifying rDNA-ITS sequences was as follows: pre-denaturation at 95°C for 3 min; 34 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 45 s; and extension at 72°C for 10 min.

[0041] The amplified products were electrophoresed in an electrophoresis tank containing 1% (W / V) agarose gel to confirm the successful amplification of the target DNA fragment, and then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0042] The sequencing results were corrected and spliced ​​using Bioedit and DNAman software to obtain the ITS nucleotide sequence shown in SEQ ID NO. 3. Blast alignment was performed in the GenBank database, and it was found that the ITS nucleotide sequence of the target strain had the highest homology with that of the strain Metarhizium flavoviride. The relevant sequences were then downloaded and aligned using ClustalW in MEGA-X software. The phylogenetic tree of strain YEC was constructed using the Neigbor-joining method. The bootstrap test was performed during the tree construction, and the sampling was repeated 1000 times. The phylogenetic tree is shown in Figure 3 .

[0043] from Figure 3 It can be seen that strain YEC and Metarhizium flavoviride clustered in the same branch. Combining the results of morphological identification and ITS sequence analysis, strain YEC was ultimately determined to be Metarhizium flavoviride. Therefore, strain YEC was named Metarhizium flavoviride YEC.

[0044] Example 2: Pathogenicity determination of Metarhizium anisopliae strain YEC against the mangrove pest, Cercidiphyllum truncatum

[0045] 1. Preparation of conidia suspension

[0046] Add 2 mL of 0.5 g / L Tween-80 aqueous solution to the yellow green Metarhizium anisopliae strain YEC cultured for 7-15 days, fully moisten it and transfer it into a centrifuge tube with a pipette, shake and filter, shake well to obtain a conidia suspension, and continue to dilute it with 0.5 g / L Tween-80 aqueous solution to a concentration of 5.83 × 10 5 cfu / mL, 4.67×10 6 cfu / mL, 1.30×10 7 cfu / mL, 3.08×10 8 cfu / mL, set aside.

[0047] 2. Determination of the pathogenicity of Metarhizium anisopliae strain YEC against the mangrove pest, Cercidiphyllum truncatum

[0048] Immersion method: Place mangrove pests in beakers containing 15 mL of conidia suspension at different concentrations and immerse for 60 seconds. Then transfer them to insect rearing boxes lined with filter paper.

[0049] The concentration of 5.83×10 5 cfu / mL (treatment group Ⅰ), 4.67×10 6 cfu / mL (treatment group II), 1.30×10 7 cfu / mL (treatment group III), 3.08×10 8 cfu / mL (treatment group IV) was treated with a conidia suspension of the yellow-green Metarhizium anisopliae strain YEC, and treated with a 0.5g / L Tween-80 aqueous solution as a control. All test insects were cultured at room temperature of 25°C, and an appropriate amount of fresh teak leaves were added every day to feed the teak camel moth larvae. Observation was conducted once a day for 15 consecutive days, and the number of deaths of the test insects was recorded. The dead insects were placed in an incubator at 25°C and a humidity of 70%-80% to observe the growth of hyphae and spore production. When hyphae and conidia visible to the naked eye grew on the insect corpses, photos were taken and the number of dead insects was recorded. Each group of test insects had 30 heads, and the experiment was repeated 3 times. The mortality rate, dead insect rate and corrected mortality rate were calculated according to the following formula:

[0050] Mortality rate (%) = number of deaths in treatment / mortality rate in control * 100;

[0051] Dead insect rate (%) = number of dead insects / number of dead insects treated*100;

[0052] Corrected mortality (%) = (treatment mortality - control mortality) / (1 - control mortality)*100.

[0053] Table 1 Pathogenicity of Metarhizium anisopliae strain YEC to the teak moth

[0054]

[0055] As can be seen in Table 1, the data reached their highest point by the 7th day of observation. The corrected mortality rates for treatment groups I and II reached over 50% at 4 dpi, for treatment group III at 3 dpi, and for treatment group IV at 2 dpi. All treatment groups began to form dead insects by 3 dpi, with treatment group IV having the highest dead insect rate, exceeding 50%.

[0056] At the same time after inoculation, the adjusted mortality rate and dead insect rate of teak camel moth increased with the increase of spore concentration; within 6 dpi, the adjusted mortality rate of teak camel moth in each treatment group increased with time, that is, all treatment groups reached the highest mortality rate at 6 dpi.

[0057] Table 2 The median lethality of Metarhizium anisopliae strain YEC to teak moth

[0058]

[0059] As can be seen from Table 2, the lethal time of the yellow green Metarhizium anisopliae strain YEC to the 2nd and 3rd instar larvae of the teak camel moth shortened with the increase of the concentration of the spore suspension. 50 The LT of treatment group Ⅲ and treatment group Ⅳ were 2.61d and 2.34d respectively. 50 In the linear regression equation, the correlation coefficients of the four concentration gradients were all close to 1, indicating that the cumulative number of adult deaths was highly positively correlated with the number of days of infection.

[0060] from Figure 4 It can be seen that after the YEC strain infects the mangrove pest teak moth, hyphae grow on the infected teak moth body and produce conidia. Specifically, in the early stage of the teak moth dead insect after being infected by the strain YEC, hyphae expand and colonize in large quantities, but no conidia are formed ( Figure 4 B); In the late stage of the teak camel moth infected by strain YEC, a large number of conidia will form on the insect body ( Figure 4 C).

[0061] In conclusion, the yellow green Metarhizium strain YEC can effectively infect the deadly mangrove pest, the teak camel moth, with a high insect killing rate, and is a biological control strain with potential value in the prevention and control of mangrove pests. Specifically, the conidia of the yellow green Metarhizium strain YEC was 5.83×10 5 -3.08×10 8 cfu / mL can effectively control teak camel moth, when the concentration reaches 3.08×10 8 cfu / mL and above, the teak moth can be killed quickly 4 days after inoculation.

Claims

1. A strain of Metarhizium anisopliae ( Metarhizium flavoviride ) strain YEC, characterized in that The deposit number is GDMCC No.65143.

2. Use of the yellow-green Metarhizium anisopliae strain YEC according to claim 1 in preventing and controlling teak camel moth.

3. The use according to claim 2, characterized in that The teak camel moth is the 2nd to 3rd instar larvae of the teak camel moth.

4. The use according to claim 2, characterized in that The method comprises the step of bringing the living bacteria of the yellow-green Metarhizium anisopliae strain YEC or the culture containing the living bacteria of the yellow-green Metarhizium anisopliae strain YEC into contact with the bodies of the teak moth.

5. The use according to claim 4, characterized in that The culture containing the living bacteria of the yellow-green Metarhizium strain YEC is a conidia suspension of the yellow-green Metarhizium strain YEC.

6. The use according to claim 5, characterized in that The concentration of the conidia suspension of the yellow green Metarhizium strain YEC is 5.83×10 5 cfu / mL or above.

7. The use according to claim 6, characterized in that The concentration of the conidia suspension of the yellow green Metarhizium strain YEC is 3.08×10 8 cfu / mL or above.

8. The use according to claim 4, characterized in that The application method is spraying or dipping.

9. A biocontrol agent for controlling teak camel moth, characterized in that: Containing the living cells of the Metarhizium anisopliae strain YEC according to claim 1 and / or a culture containing the living cells of the Metarhizium anisopliae strain YEC according to claim 1.

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