Application of a strain of *Porphyromonas hygroscopicus* and its microparticles in the control of red imported fire ants

Abstract

This invention discloses the application of *Porphyromonas hygrostis* strain and its microtablets in the control of red imported fire ants. *Porphyromonas hygrostis* PTG35 was isolated from diseased and dead litchi stink bugs in the field (accession number GDMCC No. 65103). This fungus exhibits high pathogenicity against red imported fire ants of different grades and developmental stages. Microtablets prepared using *Porphyromonas hygrostis* PTG35 conidia as the active ingredient are applied to the control of red imported fire ants, effectively controlling their population. *Porphyromonas hygrostis* PTG35 and its microtablets possess advantages such as natural composition, strong resistance to adverse conditions, and environmental friendliness, showing good application potential in the biological control of red imported fire ants.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to the application of a strain of *Porphyromonas aeruginosa* and its microparticles in the control of red imported fire ants. Background Technology

[0002] Red imported fire ants (Solenopsis invicta) are among the world's 100 most harmful invasive alien species, posing a significant threat to human safety, the ecological environment, and biodiversity in the invaded areas. Due to their strong adaptability and the inability of conventional chemical control measures to achieve long-term effective eradication, the safety and sustainability of control processes have become paramount in recent years. Fungi, an important group of insecticidal microorganisms, possess complex pathogenic mechanisms and are less likely to induce pest resistance. Considered a "nature-based solution," they represent one of the most promising biological control methods for red imported fire ant management.

[0003] The production level of fungal insecticides in my country remains low, and the pace of formulation updates is slow. Existing fungal formulations are mostly suited to the life habits of fruit, vegetable, and forest pests, and are still limited to powders, wettable powders, suspensions, and oils—types that rely on adhesion to plant leaf surfaces and require high-performance equipment. These formulations often contain large amounts of organic solvents, easily causing environmental pollution and poor storage stability. There are no widely successful cases of using these formulations to control red imported fire ants, a social insect with soil-dwelling and gregarious habits.

[0004] As eusocial insects, red imported fire ants have very different living habits from conventional pests. Individuals of specific castes take on defense tasks in an organized and orderly manner, evolving unique social behaviors to protect the population from pathogenic microorganisms. Commonly used fungal insecticides are contact insecticides, which infect pathogenic insects by having their conidia in contact with the host's epidermis. For example, Chinese patent applications CN107418899A (A strain of Beauveria bassiana BbL25 for controlling red imported fire ants and its application), CN115927006A (A strain of Beauveria bassiana and its application in controlling red imported fire ants), and CN115505538A (Metarhizium anisopliae strain CIPPMa0941 for controlling red imported fire ants and its application and fungal agent) disclose the application of some highly pathogenic fungal strains and their spore suspensions. However, these technical solutions have the following problems in control: By contacting the red imported fire ants through the body surface, the fungal conidia are used for contact killing. Red imported fire ants that have not come into contact with the fungal agent can detect the characteristic volatile odor of pathogenic fungi and sense the potential threat. They maintain nest safety and self-cleaning through avoidance, mutual cleaning, and other defensive behaviors. Furthermore, contact fungicides can only temporarily control worker ants that are out foraging, reducing the size of the nest in a short period of time. Since the reproductive queen has a strong reproductive capacity, unless the active ingredient conidia are transmitted to the queen through interbreeding by worker ants and are eaten, simply reducing the number of worker ants will not result in enough worker ants to transmit the spores to the reproductive queen. The surviving individuals can also migrate and rebuild new nests, leading to the failure of control.

[0005] Another recognized and effective bait control method involves transmitting the bait between individual ants through the digestive tract via feeding behavior. The bait can kill the queen and destroy the nest. Its active ingredient is a low-toxicity chemical agent, which reduces the dosage to some extent. However, its effect is relatively slow compared to contact pesticides. Due to differences in bait dosage and carrier granulation size among manufacturers, there are significant differences in safety, stability, and control costs. On the other hand, fungal infective units, conidia, can be produced in large quantities through fermentation, which is low-cost and easily meets actual production needs. This is a green technology that ensures ecological security and aligns with the principles of ecological civilization and sustainable development. However, to widely apply fungal products, it is necessary to address the "empty target effect" or "off-target effect" caused by the red imported fire ant's defensive behavior, and to overcome the short shelf life of conidia due to their poor resistance and storage tolerance. Therefore, it is necessary to explore fungal formulations that address these unfavorable factors. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention isolates, purifies, and identifies the highly pathogenic fungus *Porphyromonas aquatilis* PTG35, and, based on the life habits of red imported fire ants, develops a microtablet formulation containing conidia, the effective active ingredient of this highly pathogenic fungus. This formulation has natural ingredients and a good control effect on red imported fire ants, exhibiting significant innovation and unique advantages, especially in places with high safety standards for pesticide use, such as fishponds, schools, and residential areas.

[0007] The first objective of this invention is to provide a strain of *Purpureocillium takamizusanense* PTG35, with accession number GDMCC No. 65103.

[0008] A second objective of this invention is to provide a microtablet containing *Porphyromonas high water content* PTG35.

[0009] Preferably, by weight, the *Porphyromonas high water content* PTG35 microtablets consist of 1 part of *Porphyromonas high water content* PTG35 mycelial powder, 98 parts of excipients and 1 part of magnesium stearate, wherein the excipients consist of 4-4.5 parts of starch, 2-4.5 parts of sucrose and 1 part of dextrin.

[0010] More preferably, the excipients consist of 4.5 parts starch, 4.5 parts sucrose and 1 part dextrin by weight.

[0011] The third objective of this invention is to provide a method for preparing high-water-content PTG35 microtablets, comprising the following steps:

[0012] S1. By weight, take 4.5 parts starch, 4.5 parts sucrose and 1 part dextrin; add starch to water and mix evenly at a final concentration of 0.25 g / mL, heat at 100℃ for 10-15 min to make starch slurry, grind sucrose into powder, then mix sucrose and dextrin with starch slurry and stir until uniform and firm granules are formed.

[0013] S2. The granules are sieved at 24 rpm through an electric vibrating screen with an upper 12 mesh and a lower 40 mesh for 30 minutes, and then dried in a drying oven at 55°C. The moisture content after drying is controlled at 1.5%-2.0%. The granules are then sieved through a 24-mesh granulation screen to remove clumps and obtain the excipients for tableting.

[0014] S3. By weight, add 1 part of the above-mentioned PTG35 high water mold powder to 98 parts of excipients and stir evenly, then add 1 part of magnesium stearate and mix evenly. The mixed material is then processed into PTG35 high water mold micro tablets with a particle size of 2-5 mm using a rotary tableting machine.

[0015] Preferably, the mycelial powder of *Porphyromonas highwaterii* PTG35 is a freeze-dried powder of conidia of *Porphyromonas highwaterii* PTG35.

[0016] Preferably, the content of *Porphyromonas purpurea* PTG35 conidia, the active ingredient in the microtablets, is greater than 5 × 10⁻⁶. 9 1 spore / plate.

[0017] The fourth objective of this invention is to provide the application of the aforementioned *Porphyromonas hygroscopicus* PTG35 and the aforementioned *Porphyromonas hygroscopicus* PTG35 microparticles in the control of red imported fire ants.

[0018] Preferably, the red imported fire ants are winged female red imported fire ants, winged male red imported fire ants, worker ants, larvae or pupae.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. The microbial agent microtablets prepared from the conidial powder of PTG35 high water purpureus are isolated from naturally diseased and dead litchi stink bugs. When applied to control red imported fire ants worker ants, the worker ant reduction rate and ant nest reduction rate reach more than 80%.

[0021] 2. The high-water-content PTG35 microtablets of the present invention have highly efficient infection characteristics, especially effective against red imported fire ant larvae, pupae and winged ants.

[0022] 3. Because the sucrose in the high-water-content PTG35 microtablet formulation has three properties—preservative, fungal-promoting, and insect-attracting—it not only enhances the control efficacy but also extends the shelf life of the formulation, thus improving its applicability in the control of red imported fire ants.

[0023] 4. Since the formulation of PTG35 microtablets contains no chemical pesticides, and fungi are organisms that naturally exist in nature, originating from nature and returning to nature, there is no pollution or residue during application, making it a biologically environmentally friendly fungal strain with good application prospects in the field of microbial control of red imported fire ants.

[0024] Preservation instructions:

[0025] Purpureocillium takamizusanense PTG35 of the present invention was deposited on September 5, 2024 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCC No. 65103, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences Institute of Microbiology. Attached Figure Description

[0026] Figure 1Morphological characteristics of strain PTG35 on PDA medium; where A is the colony morphology of strain PTG35 after 7 days of growth on PDA medium, B is the colony morphology of strain PTG35 after 12 days of growth on PDA medium, C is the morphology of conidiophores and flask morphologies of strain PTG35, and D is the morphology of conidia of strain PTG35.

[0027] Figure 2 Electrophoresis diagram of the amplification products of strain PTG35 based on ITS universal primers.

[0028] Figure 3 Phylogenetic tree of strain PTG35 based on rDNA-ITS sequence.

[0029] Figure 4 Symptoms of different grades and life stages of red imported fire ants infected with strain PTG35 (4 days) are shown in the image. Among them, A is infected worker ant, B is infected larva, C is infected pupa, D is infected winged female ant, and E is infected winged male ant.

[0030] Figure 5 The cumulative mortality rate over time was observed for different grades and life stages of red imported fire ants treated with spore suspensions of strain PTG35 at different concentrations. Here, AE represents the cumulative mortality rate over time for larvae, pupae, worker ants, winged female ants, and winged male ants, respectively.

[0031] Figure 6 The selection rate of red imported fire ant workers on sand treated with strain PTG35 is shown in Figure A. A represents the selection rate of red imported fire ant workers on sand treated with strain PTG35, and B represents the apparatus used to test the selection preference of red imported fire ants.

[0032] Figure 7 The image shows the microstructure of PTG35 microtablets containing *Porphyromonas aquatilis*; where A is the surface view of the microtablet and B is the cross-sectional view of the microtablet.

[0033] Figure 8 The effect of different dosages of PTG35 microtablets on worker ants was studied.

[0034] Figure 9 The effect of different dosages of PTG35 microtablets on ant nest control. Detailed Implementation

[0035] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0036] Example 1: Identification of fungal strain PTG35

[0037] A highly pathogenic fungus, designated PTG35, was isolated from the litchi stink bug. The strain was identified by morphological and molecular methods under laboratory conditions.

[0038] I. Experimental Design

[0039] 1. Strains Isolation and Culture

[0040] The infected insects were placed in sterile petri dishes, and a small amount of spores were picked up with an inoculation needle and inoculated onto potato dextrose agar (PDA) medium (200 g / L potato, 20 g / L glucose, 20 g / L agar, with the remainder being distilled water) using the streak method. The dishes were then incubated in a constant temperature incubator at 27 ± 1 °C, a photoperiod L:D = 12 h:12 h, and a relative humidity of 80% ± 5%. After 7 days of incubation, relatively intact mycelial blocks were selected, and a small amount of spores were transferred to a new medium for further purification and culture for 15 days before use.

[0041] 2. Morphological identification

[0042] Based on the "Handbook of Fungal Identification" edited by Wei Jingchao, the morphological characteristics of the strain were identified. The strain was then identified using the methods described in "Microbiology Experimental Handbook" edited by Zhou Deqing et al. (Shanghai: Science and Technology Press).

[0043] 3. Molecular identification

[0044] A small number of single colonies were selected, and genomic DNA was extracted from the strains using a fungal reagent kit. Using the genomic DNA as a template, rDNA-ITS sequence PCR amplification was performed using universal fungal primers: ITS1: 5'-TCCGTAGGTGAACCTGCGG-3', ITS4: 5'-TCCTCCGCTTATTGATATGC-3'. The PCR reaction volume was 50 μL, containing 25 μL of I5 Mix, 1 μL of DNA template, 1 μL each of forward and reverse primers, and 22 μL of ddH2O. The PCR amplification conditions for ITS were: 98℃ pre-denaturation for 2 min; 35 cycles of 98℃ for 10 s, 55℃ for 15 s, and 72℃ for 15 s; extension at 72℃ for 5 min. The PCR products were detected by 1% agarose gel electrophoresis (150V, 100mA, 20 min) and sent to Beijing Qingke Xinyue Biotechnology Co., Ltd. for sequencing. The sequences were then corrected using Chromas sequence assembly software, and homology was compared in the NCBI gene bank (http: / / blast.ncbi.nlm.nih.gov / ). A phylogenetic tree was constructed using the neighbor-joining method with MEGA 7.0 software for phylogenetic analysis.

[0045] II. Experimental Results

[0046] 1. Morphological characteristics of the strain

[0047] like Figure 1 As shown, where Figure 1 A and Figure 1 In the figure, B represents the colony morphology of strain PTG35 after 7 days and 12 days of growth on PDA medium at 27°C. Figure 1 In the text, C represents the morphology of the conidiophores and sporophores of strain PTG35. Figure 1 In the diagram, D represents the conidial morphology of strain PTG35. On day 7 of cultivation on PDA medium, strain PTG35 produces pale purple powdery mature spores. The colony center is raised, with a low concentric ring extending from the center to the outer third, and a radial, milky-white hyphae ring approximately 12 mm wide extending from the center to the outer two-thirds of the colony. Figure 1 (A) On day 12 of culture, the strain produced more sporulation, and the colonies turned purple from the center outwards, with a low, flat powdery layer. The colony edges were dark green or purple, and the edges were relatively regular. Figure 1 (B) Conidiophores are 1.0–1.9 μm thick and vary in length. After multiple branching, the conidiophores produce asymmetrical sterigmata. Bottleneck-shaped whorled branches develop on the conidiophores. The sterigmata are wider at the base, becoming slender and bottle-shaped upwards, solitary or broom-like, deviating from the main axis, and measuring (5.0–9.0 μm) × (1.0–2.8 μm). Figure 1 (C) Conidia are oval or fusiform, arranged in chains, transparent and smooth, measuring (1.1–3.0 μm) × (1.2–2.5 μm). Figure 1 (D in the middle).

[0048] 2. rDNA-ITS sequence analysis of the strain

[0049] The rDNA-ITS of the target strain was amplified by PCR, and the electrophoresis image of the amplification product is shown below. Figure 2 As shown, the sequencing results indicated an amplified fragment of 569 bp, with its nucleotide sequence as shown in SEQ ID NO.1. A BLAST comparison of this sequence with the NCBI gene bank revealed that the rDNA-ITS sequence of the target strain showed over 99% similarity to the corresponding sequences of several previously reported *Porphyromonas aquatilis* strains. Related sequences were selected, and a phylogenetic tree was constructed using MEGA 7.0. Figure 3 Based on the morphological characteristics and rDNA-ITS sequence analysis of the strain, strain PTG35 was identified as *Purpureocillium takamizusanense*, and therefore named *Purpureocillium takamizusanense* PTG35.

[0050] Example 2: Indoor bioassay of the pathogenicity of PTG35 of *Porphyromonas aquatilis* against red imported fire ants

[0051] I. Experimental Design

[0052] 1. Treatment of the test strains

[0053] Conidia of *Porphyromonas aquatilis* PTG35 strain were preserved in 20% glycerol at -80°C. After activation, the strain was inoculated onto PDA plates and cultured in a constant temperature incubator at 27±1°C, a photoperiod L:D = 12h:12h, and a light intensity of 3000 lx. After 7 days of culture, once colonies had sporulated, mycelia and spores were scraped and placed in sterile 0.05% Tween-80 solution. After shaking and filtration, spores were collected and sampled into a hemocytometer to prepare 1×10⁻⁶ spores. 4 1×10 5 1×10 6 1×10 7 1×10 8 Five concentration gradients of spore suspensions were prepared, with each concentration constituting one treatment. A sterile 0.05% Tween-80 solution was used as a control.

[0054] 2. Test Red Imported Fire Ants and Pathogenicity Bioassay

[0055] The red imported fire ant nests used in the experiment were collected from the Baiyun Base of the Guangdong Academy of Agricultural Sciences in Zhongluotan Town, Baiyun District, Guangzhou. The ant colony was separated from the soil using the drip method and then reared in shallow plastic dishes (40cm×30cm×10cm). They were fed honey water, frozen crickets, and mealworms to establish an indoor experimental population. Healthy winged ants, adult worker ants, larvae, and pupae were placed in a spore suspension of the test concentration, immersed for 10 seconds, then removed and placed on filter paper to absorb excess water. They were then transferred to plastic culture dishes (bottom diameter = 15cm, height 1.75cm) with a gypsum-lined bottom for moisture retention. Each dish contained approximately 100 ants. A small amount of talcum powder was applied to the inner wall of the dish to prevent escape. Cotton balls soaked in honey water were placed inside the dish for feeding. The dish opening was sealed with plastic wrap, and several small holes were pricked in the wrap with a needle for ventilation. The treated red imported fire ants were reared in an artificial climate chamber (26±1℃, relative humidity 80%±5%, 14L:10D). Each treatment contained 20–30 ants of each caste, repeated three times, and observed for 6 days. The number of worker ant deaths was recorded daily. Dead ants were kept moist and observed to determine if death was caused by fungal infection. The cumulative mortality rate for each treatment was calculated, and the cumulative and corrected mortality rates of worker ants for each concentration were statistically analyzed. The lethal median time (LT) was calculated using the Probit method. 50 Find the regression equation and calculate the median lethal concentration (LC). 50 ).

[0056] II. Experimental Results

[0057] All castes of live red imported fire ants can be infected with strain PTG35. In the early stages of infection, the behavior and external morphology of all castes of red imported fire ants are indistinguishable from healthy controls. Two days after inoculation, the pupae show reduced tactile response, and worker ants and winged ants significantly reduce or cease feeding; some worker ants die. On the fourth day after inoculation, individuals of all castes died due to infection with strain PTG35, their bodies covered with hyphae and conidia; the conidia are initially milky white (…). Figure 4 ).

[0058] The spore suspension of *Porphyromonas high-water-content* PTG35 exhibits strong pathogenicity against red imported fire ant larvae, pupae, adult worker ants, winged male ants, and winged female ants. With increasing spore concentration and over time, the cumulative mortality rate across all stigmas gradually increases. Figure 5 When the spore concentration reaches 1×10⁻⁶ 8 At a concentration of 1 spore / mL, the cumulative corrected mortality rate of worker ants, winged female ants, and winged male ants all reached 100%, while that of larvae and pupae reached 93.17% and 90.00%, respectively.

[0059] The Probit model was used to calculate the median lethal concentration (LC50) of red imported fire ants treated with PTG35 of *Porphyromonas aquatilis*. 50 (Table 1) LC50 of larvae, pupae, winged females, and winged males on day 7 post-inoculation. 50 Both are approximately 10 3 spores / mL, LC50 of worker ants 50 The highest is about 10. 5 spores / mL. Red imported fire ant larvae, pupae, and winged ants are more sensitive to PTG35 of *Porphyromonas aquatilis*, while worker ants are the least sensitive.

[0060] Table 1. Regression equations for the pathogenicity of *Porphyromonas purpureus* PTG35 against different strains of red imported fire ants.

[0061]

[0062]

[0063] Table 2 shows the lethal intermediate time (LT) of *Pseudomonas aeruginosa* PTG35 against various grades of red imported fire ants. As the concentration of the spore suspension increased, the LT of each grade of red imported fire ants decreased. 50 The value decreases, in 1×10 4 ~1×10 8 Within the range of spores / mL, the LT of larvae 50 The LT of the pupa decreased from 5.23 days to 2.51 days. 50 The worker ant's LT decreased from 6.26 days to 4.08 days. 50 The LT of winged female ants decreased from 6.55 days to 3.60 days. 50 The LT (Long-Term Duration) of winged male ants decreased from 6.19 days to 3.62 days. 50It decreased from 6.49 days to 3.75 days.

[0064] Table 2. Lethal median time of *Porphyromonas aurea* PTG35 against various red imported fire ant strains.

[0065]

[0066]

[0067] Indoor bioassay results showed that *Porphyromonas high-water-content mold* PTG35 has a strong pathogenicity to red imported fire ants, as evidenced by its lethal concentration and lethal time in five castes of red imported fire ants. Seven days after inoculation with *Porphyromonas high-water-content mold* PTG35, the LC50 of red imported fire ant larvae, pupae, winged females, and winged males... 50 Both are approximately 10 3 spores / mL, LC50 of larvae 50 The lowest, only 0.22×10 3 1 spore / mL. The spore suspension concentration reached 1×10⁻⁶. 8 At 1 spore / mL, the LT of red imported fire ant larvae, pupae, worker ants, winged females, and winged males... 50 The pathogenicity of *P. hydrophila* PTG35 to different castes of red imported fire ants, from strongest to weakest, was as follows: larvae > workers > winged females > winged males > pupae, with time limits of 2.51 days, 4.08 days, 3.60 days, 3.62 days, and 3.75 days respectively. *P. hydrophila* PTG35 was lethal to multiple castes of red imported fire ants, indicating that *P. hydrophila* PTG35 has significantly high pathogenicity to red imported fire ants.

[0068] Example 3: Attraction effect of PTG35 of *Porphyromonas hygroscopicus* on red imported fire ants

[0069] Red imported fire ants, being social insects, can typically recognize the characteristic volatile odors of pathogenic fungi in their environment. They avoid contact with pathogens through defensive behaviors typical of social insects, such as escaping, refusing food, or rapidly migrating, thus affecting the effectiveness of fungicides. However, the presence and frequent occurrence of fungal parasitism in ants in nature indicates that not all pathogenic microorganisms can be identified; some may exhibit repulsive, attractive, or non-taxis. This example employs a selective experiment to investigate the tropism of red imported fire ants towards *Porphyromonas aquatilis* PTG35, considering the practical application potential of this strain.

[0070] I. Experimental Design

[0071] The apparatus used in this experiment (such as...) Figure 6Figure B in the diagram shows a designed and improved indoor bioassay method that utilizes the burrowing habits of red imported fire ants (RAPs) to assess their preference for test samples. Four 2mL polystyrene sample tubes were glued to the bottom of a 18cm diameter × 6cm high plastic petri dish using hot melt adhesive, with each sample tube 6cm from the center of the dish. A 4mm diameter access hole was drilled above the top of two opposite sample tubes, passing through the bottom of the dish and the cap of the sample tube, allowing worker RAPs to pass through and dig out the sand. The other two sample tubes were left undrilled and used only for support. Polytetrafluoroethylene (PTFE) was applied to the inner wall of the petri dish to prevent the RAPs from escaping.

[0072] The experiment used sand mixed with *Porphyromonas highwaterii* PTG35 as a treatment, and pure sand as a control. 5 mL of the *Porphyromonas highwaterii* PTG35 spore suspension (concentration 1×10⁻⁶) was used as the test. 8 (Spores / mL) were poured into a beaker containing 20g of sand; for the control, pure sand was poured into an equal volume (5mL) of pure water. After thorough mixing, the mixture was filled into sample tubes until completely full, and then combined with the sand-digging device. Red imported fire ant worker ants were introduced into the sand-digging device, and the device was placed in an artificial climate chamber (26±1℃, relative humidity 80%±5%, complete darkness) for 24 hours. The mass of sand dug out by the red imported fire ants from each sample bottle was then weighed. The experiment was repeated 6 times, with 100 test worker ants used each time. To eliminate the influence of factors such as direction and odor, two additional sample tubes containing pure sand were set up as blank controls. The device (excluding the sample tubes) was recycled and cleaned after each use, and the inner wall was wiped with 75% alcohol before reuse. The sample tubes were for single use only, and new tubes were used for each test.

[0073] II. Experimental Results

[0074] The selection experiment showed that red imported fire ants did not avoid or reject sample tubes containing *P. high-water-content purpureus* PTG35; on the contrary, they exhibited attracted and oriented behavioral responses. In the pure sand vs. pure sand treatment, there was no significant difference in the amount of sand dug by red imported fire ants in the two sample tubes (t = 0.706, df = 5, P = 0.519). In the fungicide-laden sand vs. pure sand treatment, red imported fire ants exhibited digging behavior in both sample tubes. Analysis of variance showed that sand containing *P. high-water-content purpureus* PTG35 had an attractive effect on red imported fire ants, and the attraction effect was highly significant (t = 5.917, df = 5, P = 0.004). Figure 6 A) indicates that PTG35 does not cause avoidance behavior in red imported fire ants, but rather has a strong attraction effect on them.

[0075] Example 4: Preparation and shelf-life quality testing of PTG35 microtablets containing *Porphyromonas aurea*.

[0076] Fungal conidia exposed to the natural environment are susceptible to the effects of temperature, humidity, and ultraviolet radiation. To further explore the potential of the strain, formulation of *Porphyromonas highwaterii* PTG35 helps enhance the conidia's resistance to stress in nature, enabling it to fully exert its pathogenicity in practical applications. Simultaneously, the efficacy of live biological preparations is easily affected by storage environment and duration; the stability of the viability of the active ingredient, the conidia, during its shelf life is also a key factor in practical application. This embodiment describes the formulation of the active ingredient, conidia, of *Porphyromonas highwaterii* PTG35, into a microtablet dosage form, and a comprehensive analysis and evaluation of the quality and shelf life of this dosage form are conducted.

[0077] I. Experimental Design

[0078] 1. Strains culture and pretreatment

[0079] PTG35 of *Porphyromonas aquatilis* was inoculated onto PDA medium. Large-sized culture dishes (d = 15 cm) were selected for large-scale culture. The inoculated culture dishes were placed in a constant temperature incubator with a temperature of 27±1℃, a photoperiod L:D = 12h:12h, a relative humidity of 80%±5%, and a light intensity of 3000 lx for 10-14 days. The conidial powder was harvested and placed in a vacuum freeze dryer and dried for 48 hours under the conditions of a vacuum pump of 20 Pa and a cold hydrazine temperature of -10℃ to -20℃ to obtain *Porphyromonas aquatilis* PTG35 mycelial powder.

[0080] 2. Microtablet preparation and formulation evaluation

[0081] High-water-content PTG35 microtablets were prepared using magnesium stearate as a lubricant and wet granulation and tableting. Common tablet ingredients, starch, sucrose, and dextrin, were used as excipients. Using sucrose as the experimental factor, different mass ratios (g / mL) of starch, sucrose, and dextrin were set for excipient premixing: ① 4∶2∶1; ② 4.5∶4.5∶1; ③ 4∶5∶1. First, starch was added to pure water at a final concentration of 0.25 g / mL and mixed thoroughly. The mixture was heated at 100℃ for 10-15 min to form a starch slurry, which was then stored in a clean container. Sucrose was ground into a flour-like consistency. The sucrose and dextrin were then mixed with the starch slurry and stirred electrically for 30 min (stirring speed 24 rpm) to dry-mix the materials until uniform, firm granules were formed, increasing the material's flowability and compressibility. The prepared wet granules were sieved at 24 rpm through an electric vibrating screen (12 mesh upper layer, 40 mesh lower layer) for 30 minutes, then placed in a drying oven and dried at 55℃ for 70-120 minutes. Depending on the dryness of the granules (moisture content controlled at 1.5%-2.0% after drying), they were sieved a second time through a 24-mesh granulation screen to remove clumps of granules that were stuck together, thus obtaining the excipients for tableting, which were then bagged for later use. At 10℃, 1% of *Porphyromonas highwaterii* PTG35 mycelial powder was added to the excipients and stirred evenly. Then, 1% of magnesium stearate was added and mixed evenly to prepare compressible granules. *Porphyromonas highwaterii* PTG35 microtablets with a diameter of 2-5 mm were then produced using a rotary tableting machine.

[0082] 3. Sensory evaluation of microtablets

[0083] The appearance of the compressed tablets was evaluated in accordance with the standards of the Chinese Pharmacopoeia. The evaluation criteria included the sieving of soft material, adhesion and impaction, surface smoothness, hardness, disintegration and friability. The impact of each index factor (Table 3) on the product was evaluated.

[0084] Table 3 Comprehensive Evaluation Table of Various Indicators

[0085] index Judgment criteria Soft material properties Excellent; Good; Slightly sticky; Average; Loose Soft material sieving condition Excellent, easy; Good, relatively easy; Average, not easy. Adhesion and punching conditions Pass / Fail; Fail / Yes Angle of repose / ° The angle should be between 30° and 50°, the smaller the better. Appearance Excellent: smooth and clean; Good: relatively smooth and clean; Average: not smooth and clean. Hardness / N Between 40-100N Disintegration rate / min Between 5 and 10 minutes, the faster the better. Friability / % Less than 1%

[0086] 4. Shelf-life quality testing of microtablets

[0087] Microtablets and bacterial powder were stored at 4°C, and the following biological characteristics were examined at 3 and 6 months:

[0088] (1) Conidia germination rate determination: i.e., germination rate. Appropriate amounts of PTG35 microtablets and mycelial powder of *Porphyromonas high-water-content* were dissolved in sterile 0.05% Tween-80 solution and prepared into formulations with the same concentration of 1×10⁻⁶. 8A spore suspension of 1 spore / mL was inoculated into a 50mL Erlenmeyer flask containing 9mL of sterile germination medium (0.5% peptone, 1% glucose, 1% OP). The flask was then placed in a shaker at 26±1℃ and 110r / min for 24h. The number of germinating and non-germinating spores was counted under a microscope using a hand counter. The count was repeated 3 times, and the spore germination rate (%) was calculated.

[0089] (2) Conidia viability assay: i.e., viable cell count. Weigh 1.0 g of microparticles and 0.5 g of bacterial powder into sterile 2 mL centrifuge tubes, add 1 mL of sterile 0.05% Tween-80 solution to each tube, shake to dissolve, and prepare mixed bacterial solutions. Then dilute the two mixed bacterial solutions with sterile water to the same concentration (10). 6 CFU·mL -1 Take 100 μL and spread it on a PDA plate (200 g potato, 20 g glucose, 20 g agar, add H2O to make up to 1000 mL, pH = 7.0). Incubate at 26 ± 1℃ and L:D = 14:10 for 3 days until colonies grow on the plate. Repeat 3 times and count the total number of colonies (×10). 6 CFU·mL -1 ).

[0090] (3) Colony growth rate determination: Weigh 1.0 g of micro-tablets and 0.5 g of bacterial powder into 10 mL of sterile 0.05% Tween-80 solution, mix thoroughly, and then inoculate onto PDA plates. Repeat 4 times. Incubate at 26±1℃ and L∶D=14∶10 for 20 days. Measure the colony diameter every 5 days using the cross-cross method, for a total of 4 measurements. Calculate the growth rate (mm·d). -1 ).

[0091] II. Experimental Results

[0092] 1. Quality inspection results after microtablet preparation

[0093] The comprehensive evaluation of the microtablets prepared with different sucrose ratios is shown in Table 4. When the mass ratio of starch, sucrose and dextrin is 4:2:1 and 4.5:4.5:1 (the sucrose ratios are 28.6% and 45.0% respectively), all the indexes are judged to be qualified. The microtablets prepared under the two ratios have uniform color, showing light purple, with an average diameter of 3.5 mm, a tablet thickness of 1.5 mm, and a tablet weight of 0.125 g (the floating range of the single tablet weight compared with the average tablet weight is within ±2%). After testing, the microtablets prepared under the two ratios have smooth and complete surfaces, no defects, easy screening of excipients, no sticking to punches, small angle of repose, moderate hardness, good disintegration performance, and can be completely disintegrated within 10 minutes without adding disintegrants. Since sucrose is the key factor to attract worker ants to forage, the high ratio (45%) of sucrose content (4.5:4.5:1) is selected as the first choice. Pressing tablets with this ratio has good forming quality, simple and feasible formula and process, and meets the requirements of large-scale production. Figure 7 )

[0094] Table 4 Preparation of microtablets with different sucrose ratios

[0095]

[0096]

[0097] 2. Experimental results of the quality detection of the microtablets during the shelf life

[0098] Table 5 shows that when the PTG35 microtablets of Hypomyces chrysospermus are stored for 6 months without adding preservatives, there is no moisture absorption and mildew, and the quality indexes of the active ingredients are still relatively high. The germination rate of conidia reaches more than 80%, the viable bacteria count and the colony growth rate are relatively stable, showing no significant difference from the measurement results at 3 months, and all indexes are significantly higher than the detection results of the PTG35 mycelium powder of Hypomyces chrysospermus. It can be seen that the compatibility between the excipient components of the microtablet dosage form and the conidia of Hypomyces chrysospermus PTG35 is good, which does not affect the mycelial growth and the spore germination rate. Moreover, the high content of sucrose in the formula can also take into account the advantages of anti-corrosion and bacteria cultivation (sucrose is also contained in the common culture medium for fungi), which better solves the problems of poor stress resistance and poor storage resistance of entomopathogenic fungal conidia in the natural environment, and is an ideal dosage form of the microbial agent for extending the shelf life.

[0099] Table 5 Quality detection results of the active ingredients of the microtablets

[0100]

[0101] Note: Different lowercase letters in the same row indicate significant differences between the two dosage forms (P < 0.05), and different capital letters in the same row indicate significant differences between the same dosage form at different storage periods (P < 0.05).

[0102] Example 5: High-water purpureus PTG35 microparticles and their field control efficacy against red imported fire ants

[0103] Previous experiments have shown that *P. tumefaciens* PTG35 has an attractant effect on red imported fire ants. Microtablets were prepared by combining *P. tumefaciens* PTG35 conidial powder with excipients that do not contain chemical insecticides. To further enhance the role of *P. tumefaciens* PTG35 in production practice, this embodiment conducted a field efficacy trial to determine the field efficacy of *P. tumefaciens* PTG35 microtablets on red imported fire ants, and to consider the potential of this formulation for practical application in controlling red imported fire ants.

[0104] I. Experimental Design

[0105] 1. Test site and test materials

[0106] The experimental materials were *Porphyromonas aquatilis* PTG35 microtablets prepared in Example 4 (in which the mass ratio of starch, sucrose and dextrin in the excipients was 4.5:4.5:1), and *Porphyromonas aquatilis* PTG35 mycelial powder obtained by the method in step 1 of Example 4 above. No treatment was used as a blank control. The experiment was conducted at the Baiyun Experimental Base of Guangdong Academy of Agricultural Sciences. The experimental site had little human interference, red imported fire ants appeared in clusters, and the ant nests were clearly raised and relatively evenly distributed.

[0107] 2. Field trial design

[0108] Rectangular test plots were set up according to the actual conditions of the test area. The minimum area of ​​each test plot was 10m². 2 Each plot should contain at least four live ant nests. When the density of live ant nests is low, the plot area should be appropriately increased to ensure that each plot contains at least four live ant nests. Live ant nests should be marked with colored tags before treatment. Experimental and control plots were arranged in a randomized block design. No fungicides or insecticides were applied to the ant nests in the control plots. The experiment included five treatments: four different dosages of microparticles and a fungicide powder treatment, with each treatment replicated three times; the control plot was replicated three times, for a total of 18 plots.

[0109] 3. Application method and test weather conditions

[0110] Microparticles were applied to individual ant nests using a placement method. 5g, 10g, 15g, and 20g microparticles were placed on filter paper (11cm x 11cm) within a 50cm radius of the nest, avoiding damage to the ant mound. When ant nests were densely distributed or inconspicuous, the microparticles were evenly distributed throughout the entire area, ensuring the nests were not disturbed during application and not mixed with other fertilizers or granules. For powder treatment, 20g of powder was applied to each nest. First, a ring of powder (10g) was sprinkled around the nest's perimeter. Then, the nest was stirred with a stick until a large number of red imported fire ants emerged. The remaining 10g of powder was then quickly and evenly sprinkled onto the ants until the powder covered the entire ground area of ​​the nest. Nests treated with different methods were marked. During application, the temperature ranged from 10℃ to 32℃, with cloudy or overcast skies, a relative humidity of 85%, and dry ground. Application was conducted between 8:00 and 10:00 AM, with no rainfall for 3-5 days before and 5-7 days after application.

[0111] 4. Survey and Calculation

[0112] Before application of the pesticide, the number of live nests and worker ants attracted in each experimental plot were investigated. A ham sausage bait method was used to investigate the population size of red imported fire ants. Ham sausage was placed in centrifuge tubes (10 mL), placed flat on the ground approximately 50 cm away from the ant nest. Three bait tubes were set up in each plot. After approximately 30 minutes, the tubes were collected and the ants were counted. Surveys were conducted at 5, 10, and 15 days after application, recording the number of worker ants attracted three times. During the final survey, the nests were excavated to observe the survival of the ant colony, and the number of live red imported fire ant nests was recorded.

[0113] The effectiveness of worker ant control is calculated based on the reduction rate of worker ant numbers before and after each treatment, using the following formula:

[0114] Worker ant control effect (%) = (1 - (average number of worker ants in the control area before treatment × average number of worker ants in the treated area after treatment) / (average number of worker ants in the control area after treatment × average number of worker ants in the treated area before treatment)) × 100.

[0115] The effectiveness of ant control is calculated based on the ant nest reduction rate before and after microparticle treatment, using the following formula:

[0116] Ant nest control effect (%) = (1 - (number of live ant nests in the control area before treatment × number of live ant nests in the treated area after treatment) / (number of live ant nests in the control area after treatment × number of live ant nests in the treated area before treatment)) × 100.

[0117] II. Experimental Results

[0118] The worker ant control efficacy of each treatment is as follows: Figure 8As shown, the worker ant reduction rate was the lowest among all treatments when using fungal powder. The highest worker ant control efficacy (11.46%) was achieved 15 days after application, significantly lower than the efficacy of 5g / nest of micro-tablets applied at the same time point (F = 3.081, df = 8, P < 0.001). The worker ant control efficacy varied with different micro-tablet application rates. At micro-tablet application rates of 5g / nest and 10g / nest, the worker ant reduction rates after 15 days were 40.74% and 60.53%, respectively. At application rates of 15g / nest and 20g / nest, the highest worker ant control efficacy (84.35% and 88.55%) was achieved 10 days after application, and the efficacy did not increase further after 15 days. The difference between the two periods after application was not significant (F = 1.065, df = 8, P = 0.726). In terms of worker ant reduction rate, treatments with an application rate of 15g / nest and 20g / nest showed the best control effect on red imported fire ant workers, reaching over 80%.

[0119] After 15 days of treatment, the ant nest control efficacy of each treatment was as follows: Figure 9 As shown, the ant nest reduction rate in the blank control group was 6.67%, while the ant nest reduction rate after applying fungal powder was 5.45%, with no significant difference between the two (F=2.429, df=8, P=0.563). When the dosage of micro-tablets was 5g / nest, the ant nest reduction rate was 20%, which was significantly different from the fungal powder treatment (F=6.861, df=8, P=0.028). When the dosage was 10g / nest and 15g / nest, the ant nest reduction rates reached 62.5% and 78.57%, respectively, with a significant difference in control efficacy between the two dosages (F=8.975, df=8, P=P=0). 035); When the dosage was 20g / nest, the ant nest reduction rate reached its highest value of 83.33%, which was not significantly different from the control efficacy of 15g / nest (F=1.362, df=8, P=0.739), but the control efficacy was extremely significant compared with 10g / nest and 5g / nest (F=1.899, df=8, P=0.008 and F=4.724, df=8, P<0.001), and the differences between the fungal powder and the blank control were extremely significant (F=6.434, df=8, P<0.001 and F=6.795, df=8, P<0.001).

[0120] In summary, when the dosage of micro-tablets is 15g / nest and 20g / nest, the reduction rate of worker ants and nests on days 10-15 is significantly better than that of fungal powder in the field. PTG35 micro-tablets of *Porphyromonas aurea* can achieve ideal control effect with the above dosage, and have the characteristics of high efficiency and long-lasting effect. They can be mass-produced for field application.

Claims

1. A strain of *Porphyromonas aeruginosa* ( Purpureocillium takamizusanense PTG35, characterized in that, The accession number is GDMCC No. 65103.

2. A high-water-content PTG35 microtablet, characterized in that, Contains the high-water purpureus PTG35 as described in claim 1.

3. The high-water-content PTG35 microtablets according to claim 2, characterized in that, By weight, the *Porphyromonas high water content* PTG35 microtablets consist of 1 part of *Porphyromonas high water content* PTG35 mycelial powder, 98 parts of excipients and 1 part of magnesium stearate. The excipients consist of 4-4.5 parts of starch, 2-4.5 parts of sucrose and 1 part of dextrin.

4. The high-water-content PTG35 microtablets according to claim 3, characterized in that, The excipients, by weight, consist of 4.5 parts starch, 4.5 parts sucrose, and 1 part dextrin.

5. A method for preparing high-water-content PTG35 microtablets, characterized in that, Includes the following steps: S1. By weight, take 4.5 parts starch, 4.5 parts sucrose and 1 part dextrin; add starch to water and mix evenly at a final concentration of 0.25 g / mL, heat at 100℃ for 10-15 min to make starch slurry, grind sucrose into powder, then mix sucrose and dextrin with starch slurry and stir until uniform and firm granules are formed. S2. The granules are sieved at 24 rpm through an electric vibrating screen with an upper 12 mesh and a lower 40 mesh for 30 minutes, and then dried in a drying oven at 55°C. The moisture content after drying is controlled at 1.5%-2.0%. The granules are then sieved through a 24-mesh granulation screen to remove clumps and obtain the excipients for tableting. S3. By weight, 1 part of the PTG35 mycelium powder of claim 1 is added to 98 parts of excipients and stirred evenly, then 1 part of magnesium stearate is added and mixed evenly. The mixed material is then processed into PTG35 micro tablets with a particle size of 2-5 mm using a rotary tableting machine.

6. The preparation method according to claim 5, characterized in that, The aforementioned *Porphyromonas high water content* PTG35 mycelial powder is freeze-dried powder of conidia of *Porphyromonas high water content* PTG35.

7. The preparation method according to claim 5, characterized in that, The active ingredient in the *Porphyromonas purpureus* PTG35 microtablets, *Porphyromonas purpureus* PTG35 conidia, contains more than 5 × 10⁻⁶ mol / L. 9 1 spore / piece.

8. The application of the PTG35 microparticles of *Porphyromonas high water content* according to claim 1 and the PTG35 microparticles of *Porphyromonas high water content* according to claim 2 in the control of red imported fire ants.

9. The application according to claim 8, characterized in that, The red imported fire ants mentioned refer to winged female red imported fire ants, winged male red imported fire ants, worker ants, larvae, or pupae.

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