Metarhizium aureoviride strain YEC and application thereof in prevention and treatment of teak camel moths

Abstract

The invention discloses a metarhizium aureoviride strain YEC and application of the metarhizium aureoviride strain YEC in prevention and treatment of teak camel moths. The Metarhizium anisoviride YEC provided by the invention is separated from soil in Guangdong province, can infect mangrove forest pests, namely teak camel moths, and can have relatively high fatality rate and stiff insect rate on the teak camel moths within a short time. The metarhizium aureoviride strain YEC is a biological control strain with potential development and application value in control of mangrove forest pests.

Description

Technical Field

[0001] The invention belongs to the technical field of microbiology, and specifically 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 in different regions of my country have been frequent and widespread, causing large-scale damage, which not only poses a serious threat to the overall health of the mangrove ecosystem, but also has a profound impact on the ecological security of the coastal areas. In the mangrove ecosystem, the main harmful insects 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) belongs to the Hyblaeidae family of the Lepidoptera order. It is a newly emerged leaf-feeding mangrove pest in recent years and has seriously threatened the ecological health of the mangrove community. The teak moth is an oligophagous insect that can feed on 45 host plants, mainly plants from the Lamiaceae, Verbenaceae, and Rhizophoraceae families. It has been reported that the insect mainly harms plants such as teak and Avicennia marina in my country.

[0003] The larvae of the teak camel moth go through five developmental stages and prefer to feed on young leaves. When an insect pest breaks out, the leaves, fruits and young branches of mangrove plants will be severely damaged in a short period of time, which will cause the plants to be unable to photosynthesize for a long time, leading to poor growth, which will not only affect the flowering and fruiting of the current year, but also have a negative effect on the reproduction cycle of the next year. In addition, damaged plants are more likely to attract attacks from pests such as bark beetles and water lice, increasing the risk of plant death.

[0004] At present, the main methods of controlling mangrove pests include chemical control, physical control, biological control and other means. Among them, chemical control, as a traditional means of pest control, still occupies an important position in modern agriculture due to its advantages such as rapid effect and good prevention effect. However, it also has problems such as pests easily developing drug resistance, harming non-target organisms, pesticide residues in agricultural and forestry products and polluting the environment. Due to the fragility of the mangrove ecosystem, biological control is a natural control method with higher safety and is the primary method of mangrove pest control.

[0005] Patent publication number CN105493966A discloses a method for controlling leaf-feeding pests in mangroves, which discloses the use of saturated saline solution to biologically control teak camel moth, Guangzhou moth borer, hairy jawed moth and various bag moths. Patent publication number CN116210499A discloses a report on the use of liquid plant glue, insect pheromone reagents and biological agents to control mangrove pests. Patent publication number CN103283540A discloses a method for biologically controlling the mangrove pest Trichogramma tungii using the natural enemy yellow-eyed bee. These reports on control methods have potential application value for the biological control of mangrove pests.

[0006] At present, 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 strain of Metarhizium flavoviride YEC, with the deposit number being 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 camel moth is a 2nd-3rd instar larvae of the teak camel moth.

[0012] Preferably, the application comprises the step of contacting the live cells of the Metarhizium anisopliae strain YEC or a culture containing the live 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 the yellow green Metarhizium 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 biological control preparation for controlling mangrove pests, containing 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] 1. The yellow green Metarhizium anisopliae strain YEC of the present invention has a highly effective insecticidal function, a simple cultivation method, rapid growth, and a large spore production. It has a high lethality rate against teak camel moth among mangrove pests, is non-toxic and pollution-free, has high biological safety, and can be widely used in the prevention and control of teak camel 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] Collection Instructions

[0022] The Metarhizium flavoviride YEC (yellow-green Metarhizium anisopliae strain YEC) of the present invention was deposited in the Guangdong Microbiological Culture Collection Center (GDMCC) on September 14, 2024, with a deposit number of 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 a colony morphology image of the yellow-green Metarhizium anisopliae strain YEC cultured on the PPDA medium on the 7th day; wherein A is a front view of the colony of the strain YEC, and B is a back view of the colony of the strain YEC.

[0024] Figure 2 The morphological diagram of conidia, conidiophores and hyphae of the yellow-green Metarhizium anisopliae strain YEC; wherein A is the conidia of strain YEC, B is the conidiophores of strain YEC, and C is the hyphae of strain YEC.

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

[0026] Figure 4 The toxicity of strain YEC to teak moth is determined; wherein 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, rather than to limit the present invention.

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

[0029] 1. Isolation of Metarhizium flavoviride strain YEC (Metarhizium flavoviride)

[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. The colony morphology was as follows Figure 1 As shown, Figure 1 A in the figure is the front side of the YEC colony. Figure 1 B in the figure is the back of the colony of strain YEC.

[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, aggregated in green, 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 terminated 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 after 7 days of culture, conidia and hyphae were scraped off.

[0038] DNA was extracted according to the instructions of M5 HiPer Fungal Genomic DNA Kit (purchased from Polymer, catalog 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 sequence was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 45 s, for 34 cycles; 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, and Blast comparison was performed in the GenBank database. It was found that the ITS nucleotide sequence of the target strain had the highest homology with the strain Metarhizium flavoviride. Subsequently, the relevant sequences were downloaded, and the sequences were aligned using ClustalW of MEGA-X software. The phylogenetic tree of strain YEC was constructed using the Neigbor-joining method. The bootstrap test was performed when constructing the phylogenetic tree, 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 are clustered in the same branch. Combining the results of morphological identification and ITS sequence analysis, strain YEC was finally determined to be Metarhizium flavoviride. Therefore, strain YEC was named Metarhizium flavoviride YEC.

[0044] Example 2: Pathogenicity determination of Metarhizium anisopliae strain YEC to mangrove pests

[0045] 1. Preparation of conidia suspension

[0046] Add 2 mL of 0.5 g / L Tween-80 aqueous solution to the yellow green Metarhizium strain YEC cultured for 7-15 days, transfer it into a centrifuge tube with a pipette after fully moistening, 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, for future use.

[0047] 2. Determination of pathogenicity of Metarhizium anisopliae strain YEC to the mangrove pest, Leptothrix edulis

[0048] Immersion method: Place mangrove pests in beakers with 15 mL of conidia suspension of different concentrations and immerse them for 60 seconds, then transfer them to insect 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 a 0.5g / L Tween-80 aqueous solution was used 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. Observe once a day for 15 consecutive days and record the number of deaths of the test insects. The dead insects were placed in an incubator at 25°C and a humidity of 70%-80% to observe the growth of mycelium and spore production. When mycelium and conidia visible to the naked eye grow on the insect corpses, take photos and record the number of dead insects. There were 30 test insects in each group, 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 (%) = dead insect number / treatment death number * 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 from Table 1, the data reached the highest level on the 7th day of observation, with the corrected mortality rates of treatment groups I and II reaching more than 50% at 4 dpi, the corrected mortality rate of treatment group III reaching more than 50% at 3 dpi, and the corrected mortality rate of treatment group IV reaching more than 50% at 2 dpi. All treatment groups basically began to form dead insects at 3 dpi, and the dead insect rate of treatment group IV reached more than 50%.

[0056] At the same time after inoculation, the corrected mortality rate and dead insect rate of teak camel moth increased with the increase of spore concentration; within 6 dpi, the corrected mortality rate of teak camel moth in each treatment group increased with the delay of 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 The linear regression equation showed that the correlation coefficients of the four concentration gradients were close to 1, indicating that the cumulative death of adults 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 will grow on the infected teak moth body and produce conidia. Specifically, in the early stage of the teak moth dead insect infected by the strain YEC, the 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 summary, the yellow green Metarhizium strain YEC can effectively infect the deadly mangrove pest teak camel moth, and the dead insect rate is high. It 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 camel moth can be killed quickly 4 days after inoculation.

Claims

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

2. Use of the yellow-green Metarhizium anisopliae strain YEC described in claim 1 in preventing and controlling mangrove pests.

3. The use according to claim 2, characterized in that: The invention relates to the application of the yellow-green Metarhizium anisopliae strain YEC in preventing and controlling teak camel moth.

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

5. The use according to claim 2, characterized in that: The method comprises the step of contacting the live bacteria of the yellow green Metarhizium anisopliae strain YEC or the culture containing the live bacteria of the yellow green Metarhizium anisopliae strain YEC with the bodies of mangrove pests.

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

7. The use according to claim 6, 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.

8. The use according to claim 7, 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.

9. The use according to claim 5, characterized in that: The application method is spraying or dipping.

10. A biological control agent for controlling mangrove pests, characterized in that: A living cell containing the yellow green Metarhizium anisopliae strain YEC as claimed in claim 1 and / or a culture containing the living cell containing the yellow green Metarhizium anisopliae strain YEC as claimed in claim 1.

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