Metarhizium lepidatum xbm-z and application thereof in preventing and treating mangrove pests

Abstract

The application discloses a Metarhizium rileyi (XBM-z) and application thereof in prevention and treatment of mangrove forest pests. Metarhizium rileyi The Metarhizium rileyi XBM-z provided by the application has a preservation number of GDMCC NO: 65954, is separated from a Rhynchophorus phoenicis Hope sclerotia collected from a mangrove nature reserve in Guangdong, has strong pathogenicity on Rhynchophorus phoenicis Hope and Xylotrupes jucundus Hope, which are outbreak pests of the mangrove forest, and has strong halophilicity, promotes mycelium growth and improves spore production under the condition that the salt concentration is less than 3%, is suitable for application in the high-salinity environment of the mangrove forest, is a biocontrol bacterium with good application potential in the prevention and treatment of the mangrove forest pests, and has important significance for the prevention and treatment of the mangrove forest pests.

Description

Technical Field

[0001] This invention belongs to the field of microbial control technology, specifically relating to a strain of Metarhizium anisopliae XBM-z and its application in controlling mangrove pests. Background Technology

[0002] Mangroves are a unique type of evergreen plant community that grows in the intertidal zone of tropical and subtropical coasts. They play a vital role in purifying seawater, preventing wind and waves, sequestering and storing carbon, and maintaining biodiversity. In my country, mangroves are mainly distributed along the coasts of five regions: Guangdong, Guangxi, Hainan, Fujian, and Zhejiang. Guangdong Province accounts for approximately 40% of the country's mangrove area. Among these, *Gnaphalium affine*, *Gnaphalium affine*, and *Kandelia candel* cover a relatively large area. Due to their relatively simple community structure and harsh living environment, they are prone to large-scale pest and disease outbreaks. *Gnaphalium affine* (also known as the sea olive borer) is a significant pest. Ptyomaxia syntaractis The Guangzhou small spotted moth (Pyralidae), also known as the mangrove moth, belongs to the order Lepidoptera and the family Pyralidae. It is the most common, major, and voracious leaf-eating pest of the mangrove plant *Rhizophora stylosa*. Teak camel moth ( Hyblaea puera The teak leafhopper, also known as the teak leafhopper or teak leafroller, belongs to the family Hyblaeidae in the order Lepidoptera. It is a newly emerging leaf-eating mangrove pest that has seriously threatened the ecological health of mangrove communities. Outbreaks of mangrove pests cause widespread damage, affecting the growth and function of mangrove plants. Furthermore, damaged mangrove plants are more susceptible to attack by harmful organisms such as bark beetles and leafhoppers, increasing the risk of death.

[0003] There are several methods for controlling mangrove pests, including chemical control, physical control, and biological control. Chemical control has advantages such as rapid effectiveness and high efficacy, but its improper use can easily cause environmental pollution and lead to pesticide resistance in pests. Biological control is a nature-based method, in which entomopathogenic fungi are widely distributed and abundant in nature, playing an important role in regulating insect populations in the ecosystem. Furthermore, biological control offers higher safety and longer-lasting effects, and is expected to become an effective alternative to chemical pesticides.

[0004] Currently, there is limited research on Metarhizium anisopliae, which can survive and infect pests in the high-salinity environment of mangroves. Furthermore, there are no reports on techniques for isolating Metarhizium anisopliae from the mummified insects of mangrove pests to control them. Developing new strains is of great significance for controlling mangrove pests. Summary of the Invention

[0005] Based on existing defects and shortcomings, the present invention aims to provide a strain of Metarhizium anisopliae (… Metarhizium rileyiXBM-z (the preservation number of this fungus is GDMCC NO: 65954, the preservation date is February 28, 2025, the preservation unit is GDMCC-Guangdong Provincial Microbial Culture Collection Center) and its application in the control of mangrove pests.

[0006] The first objective of this invention is to provide a strain of Metarhizium anisopliae (… Metarhizium rileyi XBM-z, with accession number GDMCC NO: 65954.

[0007] A second objective of this invention is to provide a biological agent containing live cells of Metarhizium anisopliae XBM-z or a culture of live cells of Metarhizium anisopliae XBM-z as the active ingredient.

[0008] Preferably, the culture of live cells of Metarhizium anisopliae XBM-z in the preparation is a suspension of conidia of Metarhizium anisopliae XBM-z.

[0009] Preferably, the concentration of the Metarhizium anisopliae XBM-z conidial suspension in the formulation is 1×10⁻⁶. 4 cfu / mL or higher.

[0010] Preferably, the concentration of the Metarhizium anisopliae XBM-z conidial suspension in the formulation is 1×10⁻⁶. 4 ~1×10 8 cfu / mL.

[0011] A third objective of this invention is to provide the application of the described Metarhizium anisopliae XBM-z or the described preparation in the control of mangrove pests.

[0012] Preferably, the application includes the step of applying live cells of Metarhizium anisopliae XBM-z or the preparation to mangrove pests.

[0013] Preferably, the mangrove pests are the sea olive female twig borer or the teak camel moth.

[0014] Preferably, the *Hylocereus laniceps* or *Camelus hainanensis* is a third-instar larva of the *Hylocereus laniceps* or *Camelus hainanensis*.

[0015] Preferably, the method of application is impregnation.

[0016] The beneficial effects of this invention are:

[0017] The Metarhizium anisopliae XBM-z provided by this invention exhibits strong pathogenicity against the explosive lepidopteran pests of mangroves, namely the sea olive female twig borer and the teak camel moth. It also has strong halophilicity, promoting mycelial growth and increasing sporulation at salt concentrations below 3%, making it suitable for application in the high-salinity environment of mangroves. It is a biocontrol agent with good application potential in the control of mangrove pests and is of great significance for the control of mangrove pests.

[0018] Preservation Instructions

[0019] The present invention Metarhizium rileyi XBM-z (Metarhizium anisopliae XBM-z) was deposited on February 28, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCC NO: 65954. The depository address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Institute of Microbiology. Attached Figure Description

[0020] Figure 1 The images show the colony morphology of Metarhizium anisopliae XBM-z on SMAY medium on day 15. In the images, A is the front view of a Metarhizium anisopliae XBM-z colony, and B is the back view of a Metarhizium anisopliae XBM-z colony.

[0021] Figure 2 The images show the morphology of conidia, conidiophores, and hyphae of Metarhizium reesei XBM-z; where A is a conidia of Metarhizium reesei XBM-z, B is a conidiophore of Metarhizium reesei XBM-z, and C is a hyphae of Metarhizium reesei XBM-z.

[0022] Figure 3 This is a phylogenetic tree constructed for Metarhizium anisopliae XBM-z based on ITS site sequences.

[0023] Figure 4 This study aimed to determine the toxicity of *Metarhizium anisopliae* XBM-z to *Metarhizium anisopliae* and *Camelus hainanensis*. A represents healthy *Metarhizium anisopliae* larvae from the control group; B represents *Metarhizium anisopliae* larvae that died 2 days after infection with *Metarhizium anisopliae* XBM-z; C represents *Camelus hainanensis* larvae that died 7 days after infection with *Metarhizium anisopliae* XBM-z; D represents healthy *Camelus hainanensis* larvae from the control group; E represents *Camelus hainanensis* larvae that died 3 days after infection with *Metarhizium anisopliae* XBM-z; and F represents *Camelus hainanensis* larvae that died 7 days after infection with *Metarhizium anisopliae* XBM-z. Detailed Implementation

[0024] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0025] Example 1

[0026] 1. Isolation and identification of strain XBM-z

[0027] (1) Strains Isolation

[0028] In May 2023, *Bombyx mori* larvae were collected from female leaves of *Bombyx mori* in a mangrove nature reserve in Guangdong Province. The collected insects were placed in 5 mL sterile EP tubes and brought back to the laboratory. A small number of conidia were picked from the insects in a clean bench and inoculated onto SMAY medium (40 g maltose, 10 g yeast powder, 10 g peptone, and 15 g agar powder, diluted to 1 L with water) using the spot inoculation method. After culturing for 5–7 days, mycelia from the edge of the colonies were picked and isolated on fresh SMAY medium. The above operation was repeated to obtain pure strain XBM-z.

[0029] (2) Morphological identification

[0030] The isolated and purified strain XBM-z was cultured at 25°C on SMAY medium plates (9 cm in diameter), and the morphological characteristics of the colonies were observed and recorded daily. After 7 to 10 days of culture, mycelia were picked and the morphological characteristics of the mycelia and conidiophores of the strain were observed under an optical microscope. After 12 to 15 days, mature conidia were picked and the morphological characteristics of the conidia were observed.

[0031] After obtaining pure culture strains, their morphological characteristics were identified. On SMAY medium, strain XBM-z initially showed white, raised, umbilicated colonies with a small number of green spores in the center; by day 15, the colony edges were pale white, with numerous dark green spores produced on the inner edge, and the colony back was brownish-yellow. Figure 1 ).

[0032] Microscopic observation revealed that the hyphae of strain XBM-z were smooth and septate; the conidiophores were spike-like, arising from the vegetative hyphae, with several conidiophores on each branch, short and cylindrical, slightly swollen near the base and slightly pointed at the apex; the conidia were colorless and transparent, oblong, with a smooth surface, and measured (1.01~1.85) μm × (0.52~1.19) μm. Figure 2 ).

[0033] (3) Molecular identification

[0034] Using the purified genomic DNA of strain XBM-z as a template, the rDNA-ITS sequence of strain XBM-z was amplified by PCR using universal fungal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3', SEQ ID NO.2) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3', SEQ ID NO.3). The PCR reaction mixture (50 μL) consisted of 25 μL of 2×Es Taq MasterMix (Dye), 1 μL of DNA template, 0.2 μL each of 10 μmol / L forward and reverse primers, and ddH2O to a final volume of 50 μL. The amplification program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; and a final extension at 72℃ for 5 min. The PCR products were analyzed by 1.0% agarose gel electrophoresis and then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were BLAST-aligned in the NCBI database, and sequences with high similarity to the target sequence were downloaded. A phylogenetic tree was constructed using the Neighbour-joining method, and a repeatability test was performed 1000 times using the bootstrap method.

[0035] Based on the ITS sequencing results, poor-quality sequences at both ends were removed, resulting in the nucleotide sequence shown in SEQ ID NO.1. A BLAST comparison with the NCBI database revealed that this strain was consistent with the sequence in the NCBI database. Metarhizium rileyi isolate The similarity of strain BUM901 was 99.65%.

[0036] Further phylogenetic analysis ( Figure 3 The results showed that strain XBM-z clustered with other Metarhizium repens strains in the database, indicating a high degree of similarity between this strain and the Metarhizium repens strains in the database. Based on comprehensive morphological identification and ITS sequence similarity analysis, the taxonomic position of this strain was determined to be Metarhizium repens, and it was named Metarhizium repens (…). Metarhizium rileyi XBM-z.

[0037] 2. Halophilicity determination of Metarhizium anisopliae XBM-z

[0038] First, SMAY agar plates containing 0.5%, 1%, 2%, and 3% NaCl were prepared, with the untreated plate serving as a control. XBM-z strain mycelial discs (0.5 mm in diameter) were inoculated into the center of each SMAY agar plate at different salt concentrations. After incubation at 25°C for 15 days, the size of the mycelial zones under different salt concentrations was measured. Conidia were washed with an aqueous solution containing Tween-80, diluted, and the number of conidia was counted using a hemocytometer to calculate the sporulation yield of the strain under different salt concentrations. This was repeated three times.

[0039] Table 1. Effects of different salt concentrations on the growth and sporulation of Metarhizium anisopliae XBM-z (15 days)

[0040]

[0041] Note: Different letters in the same column indicate significant differences in the average diameter or average sporulation rate of the mycelial rings under different salt concentrations (Duncan's test). P <0.05).

[0042] Table 1 shows that adding a certain concentration of NaCl can promote the growth of *Metarhizium anisopliae* XBM-z and increase sporulation. By day 15 of cultivation, compared with the control, treatment with 0.5%–3% NaCl significantly promoted the growth of *Metarhizium anisopliae* XBM-z, and treatment with 0.5%–2% NaCl significantly increased the sporulation of *Metarhizium anisopliae* XBM-z. These results indicate that *Metarhizium anisopliae* XBM-z has a certain degree of halophilicity, and a certain salt concentration can promote its mycelial growth and sporulation.

[0043] 3. Pathogenicity determination of Metarhizium anisopliae XBM-z against mangrove pests *Metarhizium anisopliae* and *Teak moth*.

[0044] The pathogenicity of Metarhizium anisopliae XBM-z to the larvae of the sea olive female worm and the teak camel moth was determined by immersion method.

[0045] First, the conidia of *Metarhizium anisopliae* XBM-z were prepared into 1×10⁻⁶ concentrations using a 0.5% (v / v) Tween-80 aqueous solution. 8 1×10 7 1×10 6 1×10 5 and 1×10 4Third-instar larvae of uniform growth were selected and immersed in a spore suspension of cfu / mL for 10 s, then immediately transferred to disposable plastic containers for rearing (rearing conditions: temperature 25±1℃, relative humidity 70±5%, photoperiod 14 L:10 D). Third-instar larvae treated with a 0.5% (v / v) Tween-80 aqueous solution served as a control. Each treatment was repeated three times, with 10 larvae per replicate. Fresh female leaves of *Rhizophora stylosa* were provided daily, and dead larvae were removed and transferred to new plastic containers for humidified culture. Mycelial growth and sporulation were observed.

[0046] At the same time, five other strains of Metarhizium anisopliae (Metarhizium anisopliae Ma3297) were also tested. M. anisopliae Ma3297), Metarhizium anisopliae Mf985 ( M. acridum Mf985), Metarhizium anisopliae Mf1245 ( M. flavoviride Mf1245), Metarhizium anisopliae Ma30 ( M. lepidiotade Ma30), Metarhizium anisopliae Shuang ( M. biformisporae Shuang) underwent the same treatment as described above, and the pathogenicity of these 5 strains of Metarhizium anisopliae to the teak moth was determined.

[0047] The number of larval deaths is counted daily, and the mortality rate and corrected mortality rate are calculated using the following formulas:

[0048] Mortality rate (%) = Number of deaths in the treatment / Control mortality rate × 100%;

[0049] Corrected mortality rate (%) = (treatment mortality rate) (Control mortality rate) / (100) (Comparison mortality rate) × 100%.

[0050] Table 2. Pathogenicity of Metarhizium anisopliae XBM-z against 3rd instar larvae of the sea moth and the teak camel moth.

[0051]

[0052] Table 2 shows that the pathogenicity of the conidial suspension of *Metarhizium anisopliae* XBM-z to the third instar larvae of *Tamarix chinensis* and *Tamarix chinensis* was positively correlated with conidial concentration and treatment time. The optimal conidial concentration was 1 × 10⁻⁶. 6 At a concentration of cfu / mL, it exhibited strong pathogenicity against both pests, with a mortality rate of approximately 70% after 7 days of treatment, while the conidial concentration was 1×10⁻⁶. 8 At cfu / mL, the cumulative corrected mortality rate of third-instar larvae of *Spodoptera litura* reached 100% on day 7, and the cumulative corrected mortality rate of third-instar larvae of *Pterocarya stenoptera* also reached over 88%. The median lethal concentration (LC50) of XBM-z conidial suspension against third-instar larvae of *Spodoptera litura* was determined. 50The value is 7.55 × 10 4 LC50 of cfu / mL against 3rd instar larvae of the teak moth 50 It is 1.76 × 10 5 cfu / mL (Table 3).

[0053] Table 3. Median lethal concentration (LC50) of Metarhizium anisopliae XBM-z against 3rd instar larvae of the sea moth and the teak moth at 7 days. 50 )

[0054]

[0055] Table 4. Lethal median time (LT) of Metarhizium anisopliae XBM-z against 3rd instar larvae of the sea moth and the teak moth. 50 )

[0056]

[0057] Metarhizium anisopliae XBM-z showed LT levels in 3rd instar larvae of the sea olive leafminer and the teak camel moth. 50 The timeframe shortened with increasing treatment concentration, showing a strong positive correlation. At the same concentration, *Metarhizium anisopliae* XBM-z resulted in a faster lethality against *Tamarix chinensis* than against *Teak moth*, especially at a treatment concentration of 1×10⁻⁶. 8 The difference between the two is most pronounced at cfu / mL, LT 50 The d and d were 2.96 days and 3.99 days, respectively (Table 4).

[0058] like Figure 4 As shown, in 1×10 8 At a concentration of CFU / mL, *Metarhizium anisopliae* XBM-z exhibited significant toxicity against the larvae of *Metarhizium anisopliae* and *Metarhizium spp.* var. * ... Figure 4 In case A), after inoculation with Metarhizium anisopliae XBM-z, larvae showed reduced or no feeding on day 1, and some larvae died on day 2, with the peak mortality period on day 3; two days after larval death, the body surface of the larvae was covered with white mycelium ( Figure 4 (B in the text), but little or no spore production was observed; by the 7th day after death, a large number of powdery green spores had formed ( Figure 4 The C in the sample formed typical symptoms of Metarhizium anisopliae infection in larvae. Compared with the control healthy teak moth larvae (C), Figure 4 (D in the text) After inoculation with Metarhizium anisopliae XBM-z for 2 days, the larvae showed symptoms of infection such as lethargy, and the mortality peaked 4 days later; 3 days after the larvae died, a large number of mycelia were visible, and a small number of green spores had been produced. Figure 4 (E in the text); by the 7th day after death, a large number of green spores formed on the surface of the mummified worm ( Figure 4 (F in the middle).

[0059] Table 5. Pathogenicity of different Metarhizium anisopliae to 3rd instar larvae of the teak moth

[0060]

[0061] Further comparison was made of the pathogenicity of Metarhizium anisopliae XBM-z with five other Metarhizium anisopliae strains (Metarhizium anisopliae Ma3297, Metarhizium anisopliae Mf985, Metarhizium chrysogenum Mf1245, Metarhizium scabra Ma30, and Metarhizium bifidum Shuang) against the third instar larvae of the teak camel moth.

[0062] All tested Metarhizium anisopliae could infect the third instar larvae of the teak camel moth. The infected larvae became sluggish and lost their appetite. They began to die gradually 2 to 3 days after inoculation, and a large number of them died 5 to 7 days after inoculation. The dead insects were dark yellowish-brown or dark black, and white mycelium gradually grew on their bodies, eventually producing green spores.

[0063] However, the pathogenicity varies among different strains. Table 5 shows that, at the same concentration, the teak moth treated with *Metarhizium anisopliae* XBM-z exhibited a higher mortality rate in a shorter time. Specifically, at a conidial concentration of 1×10⁻⁶... 5 cfu / mL~1×10 7 At a concentration of cfu / mL, the mortality rate of third-instar larvae of the teak moth treated with *Metarhizium anisopliae* XBM-z was the highest from 2 to 7 days after treatment; at a conidial concentration of 1×10⁻⁶, the mortality rate was also the highest. 8 At cfu / mL, the mortality rate of third-instar larvae of the teak moth treated with Metarhizium anisopliae XBM-z for 2 days was 20%, significantly higher than the mortality rates of other Metarhizium anisopliae treatments (highest 16.67%, lowest only 6.67%). This indicates that Metarhizium anisopliae XBM-z has a faster onset of action than other Metarhizium anisopliae and can provide better control of the teak moth in a shorter time.

[0064] In addition, when the conidial concentration is 1×10 8 At that time, after 7 days of treatment, Metarhizium anisopliae XBM-z had the highest mortality rate of 88.89% against the third instar larvae of the teak camel moth, which was superior to the pathogenicity of other Metarhizium anisopliae (the mortality rate of the third instar larvae of the teak camel moth was 80%~88%).

[0065] Table 6. Lethal median concentration (LC50) of different Metarhizium anisopliae against 3rd instar larvae of the teak moth. 50 )

[0066]

[0067] Table 7. Lethal median time (LT) of different Metarhizium anisopliae pathogens on 3rd instar larvae of the teak moth. 50 )

[0068]

[0069] Table 6 shows the LC50 of Metarhizium anisopliae XBM-z at 5 days. 50 It is 9.17×10 6 The cfu / mL concentration was significantly lower than that of other Metarhizium anisopliae. 50 (All in 10) 7 (cfu / mL or higher). From LT 50 The results (Table 7) show that at 1×10 6 cfu / mL~1×10 8 At a concentration of CFU / mL, the LT of Metarhizium anisopliae XBM-z 50 All were lower than the LT values ​​of the other five Metarhizium anisopliae strains. 50 The durations were 5.74 days, 4.73 days, and 3.99 days, respectively.

[0070] In conclusion, Metarhizium anisopliae XBM-z exhibits significant pathogenicity against the sea olive female twig borer and the teak camel moth, and also demonstrates good halophilicity, making it suitable for application in the high-salinity environment of mangroves. It is a biocontrol bacterium with good application potential in the control of mangrove pests.

Claims

1. A strain of Metarhizium anisopliae ( Metarhizium rileyi XBM-z, characterized in that, Its accession number is GDMCCNO:65954.

2. A biological agent, characterized in that, The active ingredient is a live cell of Metarhizium anisopliae XBM-z as described in claim 1 or a culture of live cells of Metarhizium anisopliae XBM-z as described in claim 1.

3. The formulation according to claim 2, characterized in that, The culture of live cells of Metarhizium anisopliae XBM-z in the preparation is a suspension of conidia of Metarhizium anisopliae XBM-z.

4. The formulation according to claim 3, characterized in that, The concentration of the *Metarhizium anisopliae* XBM-z conidial suspension in the formulation is 1 × 10⁻⁶. 4 cfu / mL or higher.

5. The formulation according to claim 4, characterized in that, The concentration of the *Metarhizium anisopliae* XBM-z conidial suspension in the formulation is 1 × 10⁻⁶. 4 ~1×10 8 cfu / mL.

6. The application of Metarhizium anisopliae XBM-z as described in claim 1 or the preparation as described in claim 2 in the control of mangrove pests, wherein the mangrove pests are the third instar larvae of the sea moth or the teak moth.

7. The application according to claim 6, characterized in that, The procedure includes applying live cells of Metarhizium anisopliae XBM-z or the preparation thereof to mangrove pests.

8. The application according to claim 7, characterized in that, The method of application is impregnation.

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