Application of Purpureocillium spp. And microtablets thereof in prevention and control of Solenopsis invicta

Abstract

The invention discloses application of Purpureocillium spp. And microtablets thereof in prevention and treatment of Solenopsis invicta. According to the invention, the Purpureocillium aureum PTG35 is separated from the death lychee bugs in the field, and the preservation number of the Purpureocillium aureum PTG35 is GDMCC No.65103; the bacterium has high pathogenicity to solenopsis invicta of different grades and insect states. The micro-tablets prepared by taking the Purpureocillium spp. PTG35 conidia as the active ingredient are applied to prevention and treatment of Solenopsis invicta, and the population quantity of Solenopsis invicta can be effectively controlled. The Purpureocillium aureum PTG35 and the micro-tablet thereof have the advantages of natural components, strong stress resistance, environmental friendliness and the like, and have good application potential in biological control of Solenopsis invicta.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microorganisms, and particularly relates to application of a strain of Purpurosporium truncatum and a micro-tablet thereof in preventing and controlling red imported fire ants. Background Art

[0002] The red fire ant Solenopsis invicta is one of the 100 most harmful invasive alien species in the world, posing a great threat to human safety, ecological environment and species diversity in the invaded areas. Since the red fire ant has strong adaptability and conventional chemical control measures cannot effectively eradicate it in the long term, the safety and sustainability of the control process have been placed at the core in recent years. Fungi are an important group of insecticidal microorganisms. Their pathogenic mechanism is complex and they are not easy to cause pest resistance. They are regarded as "nature-based solutions" and are one of the most promising biological control methods in the current prevention and control of red fire ants.

[0003] The production level of fungal insecticides in my country is still low, and the formulations are updated slowly. The existing fungal formulations are mostly suitable for the living habits of fruit, vegetable and forest pests. They still remain in the categories of powders, wettable powders, suspoemulsions, oils, etc., which rely on the adhesion of plant leaves and have high requirements for equipment performance. The formulas often contain a large amount of organic solvents, which are prone to cause environmental pollution and storage intolerance. There have been no large-scale successful cases of using the above formulations to control the red imported fire ants, a social insect with soil-dwelling and gregarious habits.

[0004] As a eusocial insect, the living habits of red fire ants are very different from those of conventional pests. Individuals of a specific caste undertake defense tasks in an organized and orderly manner, and have evolved unique social behaviors to protect the population from pathogenic microorganisms. Common insecticide fungal formulations are contact killing, infecting pathogenic host insects by contacting the host epidermis with the conidia of its infection structure. For example, Chinese patent applications with publication numbers CN107418899A (a strain of Beauveria bassiana BbL25 for controlling red fire ants and its application), CN115927006A (a strain of Beauveria bassiana and its application in controlling red fire ants) and CN115505538A (a strain of Metarhizium anisopliae CIPPMa0941, and its application and fungal agent for controlling red fire ants) disclose the application of some highly pathogenic fungal strains and their spore suspensions. However, these technical solutions have the following problems in control: through the body surface route, the fungal conidia contact the living red fire ants to kill them by contact, and the red fire ants that have not been exposed to the fungal agent recognize the characteristic volatile odor of the pathogenic fungi, perceive the potential threat, and maintain the safety of the nest and the cleanliness of themselves through defensive behaviors such as avoidance and mutual cleaning. In addition, contact fungicides can only temporarily control worker ants that go out to forage and reduce the size of the nest for a short period of time. Since the reproductive queen ant has a strong reproductive capacity, unless the active ingredient conidia is passed to the queen ant through worker ant trophallaxis and eaten, otherwise, by only reducing the number of worker ants, there will not be enough worker ants to pass the spores to the reproductive queen ant, and the remaining individuals can also rebuild new nests through migration, resulting in failure of prevention and control.

[0005] Another recognized long-lasting bait control method is to transfer the agent between ant colonies through the digestive tract through trophobia. The bait can kill the queen ant and destroy the nest. Its active ingredient is a low-toxic chemical agent, which can reduce the dosage to a certain extent, but the effect is slower than that of contact killing agents. Due to the different bait dosages and carrier granulation sizes of each manufacturer, there are large differences in safety, stability and control costs. In terms of advantages, the conidia, the infectious unit of the fungus, can be obtained in large quantities through fermentation production. It is low-cost and easy to meet actual production needs. It is a green technology that ensures ecological safety and complies with the essence of ecological civilization and sustainable development. However, in order to apply fungal products on a large scale, it is necessary to solve the "empty target effect" or "off-target effect" caused by the defensive behavior of red fire ants, and to overcome the short shelf life of conidia due to poor stress resistance and poor storage resistance. Therefore, it is necessary to explore the formulation of fungal agents for the above-mentioned unfavorable factors. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention separates, purifies and identifies the pathogenic fungus PTG35 which is highly pathogenic to red fire ants, and develops a micro-tablet dosage form containing the conidia of the effective active ingredient of the highly pathogenic fungus according to the living habits of red fire ants. The dosage form has natural ingredients and has a good control effect on red fire ants, especially in places with high drug safety levels such as fish ponds, schools and residential areas, which has great innovation and unique advantages.

[0007] The first object of the present invention is to provide a strain of Purpureocillium takamizusanense PTG35, whose deposit number is GDMCC No.65103.

[0008] The second object of the present invention is to provide a high water purpurogenous spore PTG35 micro-tablet containing the high water purpurogenous spore PTG35.

[0009] Preferably, the microtablets of PTG35 of Psoralea corylifolia are composed of 1 part of powder of PTG35 of Psoralea corylifolia, 98 parts of auxiliary materials and 1 part of magnesium stearate, and the auxiliary materials are composed of 4-4.5 parts of starch, 2-4.5 parts of sucrose and 1 part of dextrin.

[0010] More preferably, the auxiliary material consists of 4.5 parts of starch, 4.5 parts of sucrose and 1 part of dextrin by weight.

[0011] The third object of the present invention is to provide a method for preparing a micro-tablet of PTG35, comprising the following steps:

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

[0013] S2. The granules were sieved through an electric vibrating sieve with an upper 12-mesh and a lower 40-mesh sieve at 24 rpm for 30 min, and then dried in a drying oven at 55°C. The moisture content after drying was controlled at 1.5%-2.0%; the granules were sieved through a 24-mesh sieve to remove the agglomerated particles to obtain the excipients for tableting;

[0014] S3. By mass, 1 part of the powder of the high water purpurogenous mold PTG35 is added to 98 parts of auxiliary materials and stirred evenly, and then 1 part of magnesium stearate is added and mixed evenly. The mixed material is made into high water purpurogenous mold PTG35 micro-tablets with a particle size of 2-5mm by a rotary tablet press.

[0015] Preferably, the fungus powder of Psoralea corylifolia PTG35 is freeze-dried conidia powder of Psoralea corylifolia PTG35.

[0016] Preferably, the content of conidia of the active ingredient of the high water purpurogenous spore PTG35 micro tablet is greater than 5×10 9 Spores / piece.

[0017] The fourth object of the present invention is to provide the use of the high water purpurogenous spore PTG35 and the high water purpurogenous spore PTG35 micro-tablets in preventing and controlling red imported fire ants.

[0018] Preferably, the red fire ant is a winged female ant, a winged male ant, a worker ant, a larva or a pupa of the red fire ant.

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

[0020] 1. The high water purpurogenous spore mold PTG35 disclosed in the present invention is separated from the dead litchi stink bugs that died of natural diseases. The microbial agent microtablet prepared with the conidia powder of high water purpurogenous spore mold PTG35 is used to prevent and control red fire ant worker ants, and the worker ant reduction rate and ant nest reduction rate are respectively more than 80%.

[0021] 2. The PTG35 micro-tablets of the present invention have high-efficiency infection characteristics, especially significant infection effects on red fire ant larvae, pupae and winged ants.

[0022] 3. Since the sucrose in the formula of the PTG35 micro-tablets has the three properties of preservation, bacteria cultivation and insect attraction, it not only promotes the prevention effect but also extends the shelf life of the preparation, thereby improving the applicability of this dosage form in the prevention and control of red fire ants.

[0023] 4. Since the formula of the PTG35 micro-tablets of Pseudomonas aeruginosa does not contain chemical pesticides, and fungi are organisms that exist naturally, originating from nature and returning to nature, there is no pollution, no residue, and biological environmental protection during the application process. It is a fungal strain with good application prospects in the field of microbial control of red fire ants.

[0024] Storage Instructions:

[0025] The Purpureocillium takamizusanense PTG35 (Purpureocillium takamizusanense PTG35) of the present invention was deposited in the Guangdong Microbiological Culture Collection Center (GDMCC) on September 5, 2024, with a deposit number of GDMCC No. 65103, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1are the morphological characteristics of strain PTG35 on PDA medium; wherein, A is the colony morphology of strain PTG35 grown on PDA medium for 7 days, B is the colony morphology of strain PTG35 grown on PDA medium for 12 days, C is the conidiophore and phialopsis morphology of strain PTG35, and D is the conidia morphology of strain PTG35.

[0027] Figure 2 This is the electrophoresis diagram of the amplification product of strain PTG35 based on ITS universal primers.

[0028] Figure 3 This is the phylogenetic tree of strain PTG35 based on rDNA-ITS sequence.

[0029] Figure 4 Symptom diagram of red imported fire ants of different grades and stages infected with strain PTG35 (4 days); A is infected worker ants, B is infected larvae, C is infected pupae, D is infected winged female ants, and E is infected winged male ants.

[0030] Figure 5 The figure shows the trend of the cumulative mortality of red imported fire ants of different grades and stages treated with different concentration gradient spore suspensions of strain PTG35 over time; among them, AE corresponds to the trend of the cumulative mortality of larvae, pupae, worker ants, winged female ants and winged male ants over time.

[0031] Figure 6 It is the selection rate of red fire ants worker ants to sand treated with strain PTG35; wherein, A is the selection rate of red fire ants worker ants to sand treated with strain PTG35, and B is the device for testing the selection preference of red fire ants.

[0032] Figure 7 It is the microstructure diagram of the microtablet of PTG35 of high water purple spore mold; wherein, A is the surface view of the microtablet, and B is the cross-section view of the microtablet.

[0033] Figure 8 This is the control effect of worker ants with different dosages of PTG35 micro-tablets of Polysporus spp.

[0034] Fig. 9 This is the control effect of ant nests with different dosages of PTG35 micro-tablets. DETAILED DESCRIPTION

[0035] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0036] Example 1: Identification of fungal strain PTG35

[0037] A pathogenic fungus with high pathogenicity to red imported fire ants was isolated from the body of the litchi stink bug, numbered PTG35. The species of the strain was identified by morphological and molecular identification methods under laboratory conditions.

[0038] 1. Experimental plan

[0039] 1. Isolation and culture of strains

[0040] Place the infected insects in a sterile culture dish, pick a small amount of spores with an inoculation needle, and inoculate them on a potato dextrose agar (PDA) medium (potato 200g / L, glucose 20g / L, agar 20g / L, and the balance is distilled water) by streaking, and place them in a constant temperature incubator at 27±1°C, a photoperiod of L:D=12h:12h, and a relative humidity of 80%±5%. After 7 days of cultivation, select relatively complete bacterial blocks and pick a small amount of spores to transfer to a new culture medium, and continue to purify and culture for 15 days for use.

[0041] 2. Morphological identification

[0042] According to the "Handbook of Fungal Identification" compiled by Wei Jingchao, the morphological characteristics of the strains were clarified. The strains were identified according to the methods introduced in the "Microbiology Experiment Manual" compiled by Zhou Deqing et al. (Shanghai: Science and Technology Press).

[0043] 3. Molecular Identification

[0044] A small number of single colonies were picked and the genomic DNA of the strain was extracted using a fungal kit. Using genomic DNA as a template, the rDNA-ITS sequence was PCR amplified using the universal fungal primers: ITS1: 5'-TCCGTAGGTGAACCTGCGG-3', ITS4: 5'-TCCTCCGCTTATTGATATGC-3'. The PCR reaction system was 50μL, containing 25μL of I5 Mix, 1μL of DNA template, 1μL of each forward and reverse primer, and 22μL of ddH2O. The PCR amplification conditions of ITS were: 98℃ pre-denaturation for 2min; 98℃ 10s, 55℃ 15s, 72℃ 15s, 35 cycles; 72℃ extension for 5min. The PCR product was detected by 1% agarose gel electrophoresis (150V, 100mA, 20min) and sent to Beijing Qingke Xinye Biotechnology Co., Ltd. for sequencing. The sequence was then corrected by Chromas sequence assembly software, and homology was compared in the NCBI (http: / / blast.ncbi.nlm.nih.gov / ) gene library. The phylogenetic tree was constructed using the neighbor-joining method using MEGA7.0 software for phylogenetic analysis.

[0045] 2. Experimental Results

[0046] 1. Morphological characteristics of strains

[0047] like Figure 1 As shown, Figure 1 A and Figure 1 B in the figure is the colony morphology of strain PTG35 grown on PDA medium at 27°C for 7 and 12 days. Figure 1 C in the figure is the morphology of conidiophores and phialopsis of strain PTG35. Figure 1 D in the figure is the conidia morphology of strain PTG35. On the 7th day of culture on PDA medium, the colony of strain PTG35 produced mature spores in the form of pale purple powder. The center of the colony was bulging, and there were flat concentric rings from the center to the 1 / 3 of the edge. There was a radiating milky white hyphae ring about 12 mm wide from the center to the 2 / 3 of the edge ( Figure 1 A in the figure). On the 12th day of culture, the strain produced more spores, and the colonies showed purple from the center to the outside, with a low and flat powder layer, and the edges of the colonies were dark green or purple, and the edges were relatively regular ( Figure 1 B in the figure). The conidiophores are 1.0 to 1.9 μm thick and vary in length. The conidiophores branch multiple times to produce asymmetrical pedicels, which have bottleneck-shaped whorled branches. The base of the pedicel is relatively wide, and it becomes slender and bottle-shaped upwards. They are solitary or broom-like branches, deviating from the main axis, and are (5.0 to 9.0 μm) × (1.0 to 2.8 μm) ( Figure 1 C in the figure). Conidia are ovoid or spindle-shaped, arranged in chains, transparent and smooth, with a size of (1.1-3.0 μm) × (1.2-2.5 μm) ( Figure 1 D).

[0048] 2. rDNA-ITS sequence analysis of strains

[0049] PCR was used to amplify the rDNA-ITS of the target strain. The electrophoresis of the amplified product was shown in Figure 2 As shown, the sequencing results showed that the amplified fragment was 569 bp, and its nucleotide sequence was shown in SEQ ID NO.1. The sequence was blasted in the NCBI gene library, and it was found that the similarity between the rDNA-ITS sequence of the target strain and the corresponding sequences of multiple reported high-water purpurogenous spore strains reached more than 99%. Relevant sequences were selected and a phylogenetic tree was constructed using MEGA7.0 ( Figure 3 ). Combined with the morphological characteristics of the strain and rDNA-ITS sequence analysis, strain PTG35 was identified as Purpureocillium takamizusanense, and was therefore named Purpureocillium takamizusanense PTG35.

[0050] Example 2: Indoor bioassay of pathogenicity of PTG35 to red imported fire ants

[0051] 1. Experimental plan

[0052] 1. Treatment of test strains

[0053] The conidia of the PTG35 strain of Pseudomonas aeruginosa were preserved in a -80°C ultra-low temperature refrigerator with 20% glycerol. After the strain was activated, it was inoculated on a PDA plate and cultured in a constant temperature incubator at 27±1°C, a photoperiod of L:D=12h:12h, and a light intensity of 3000lx. After 7 days of culture, when the colony produced spores, the mycelium and spores were scraped and placed in a sterilized 0.05% Tween-80 solution. After shaking, the spores were filtered and the samples were applied to a hemacytometer and prepared into 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 The spore suspensions were prepared with 5 concentration gradients of spores / mL, each concentration being a treatment, and the treatment with sterilized 0.05% Tween-80 solution was used as a control.

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

[0055] The red fire ant nests used in the experiment were collected from the Baiyun Base of Guangdong Academy of Agricultural Sciences in Zhongluotan Town, Baiyun District, Guangzhou. The ant colonies were separated from the soil by the drip method and raised in a plastic shallow dish (40cm×30cm×10cm). They were fed with honey water, frozen crickets and mealworms to establish indoor experimental populations. Healthy winged ants, worker ants (referred to as worker ants), larvae and pupae were placed in the spore suspension of the test concentration, soaked for 10s, picked out, placed on filter paper to absorb excess water, and moved to a plastic culture dish (bottom diameter = 15cm, height 1.75cm) with gypsum moisture at the bottom. There were about 100 ants in each box. A small amount of talcum powder was applied to the inner wall of the dish to prevent the red fire ants from escaping. Cotton balls dipped in honey water were placed in the dish for them to eat. The mouth of the dish was sealed with plastic wrap, and several small holes were pierced in the film with a needle for ventilation. The treated red fire ants were placed in an artificial climate chamber for breeding (26±1℃, relative humidity 80%±5%, 14L:10D). The number of each grade in each treatment was 20 to 30, repeated 3 times, and observed for 6 days. The number of worker ants who died was recorded every day, and the dead insects were observed to keep them moist to determine whether they were killed by bacteria. The cumulative mortality of insects in each treatment was calculated, and the cumulative mortality and cumulative corrected mortality of red fire ants in each concentration treatment were counted. The Probit method was used to calculate the lethal median time (LT 50 ), find the regression equation and calculate the median lethal concentration (LC 50 ).

[0056] 2. Experimental Results

[0057] All castes of red fire ants can be infected by strain PTG35. In the early stage of infection, the behavior and external morphology of red fire ants of all castes were no different from those of healthy controls. Two days after inoculation, the touch response of pupae weakened, the feeding of workers and winged ants decreased significantly or stopped, and some workers died. On the fourth day after inoculation, all castes appeared dead individuals infected with strain PTG35, with hyphae and conidia covered on the body surface. The conidia were milky white in the early stage ( Figure 4 ).

[0058] The spore suspension of PTG35 had strong pathogenicity to larvae, pupae, adult worker ants, winged male ants and winged female ants of red fire ants. With the increase of spore concentration and the passage of time, the cumulative mortality rate of each grade gradually increased ( Figure 5 ). When the spore concentration reaches 1×10 8 spores / mL, the cumulative corrected mortality rates of worker ants, winged female ants and winged male ants all reached 100%, and those of larvae and pupae were 93.17% and 90.00% respectively.

[0059] The Probit model was used to calculate the lethal median concentration (LC) of each grade of red imported fire ants treated with PTG35. 50 (Table 1), LC of larvae, pupae, alate female ants and alate male ants on the 7th day after inoculation 50 About 10 3 spores / mL, LC for worker ants 50 The highest is about 10 5 Spores / mL. The larvae, pupae and winged ants of red imported fire ants were more sensitive to PTG35, while worker ants were the least sensitive.

[0060] Table 1 Regression equation of pathogenicity of PTG35 to different grades of red imported fire ants

[0061]

[0062]

[0063] The lethal time of PTG35 to different grades of red fire ants is shown in Table 2. As the concentration of spore suspension increases, the LT of different grades of red fire ants increases. 50 The value decreases, in 1×10 4 ~1×10 8 spores / mL, the LT of larvae 50 From 5.23d to 2.51d, the LT of the pupa 50 From 6.26d to 4.08d, the LT of worker ants 50 From 6.55d to 3.60d, the LT of winged female ants 50 From 6.19d to 3.62d, the LT of winged male ants 50From 6.49d to 3.75d.

[0064] Table 2 The median lethality of PTG35 to different grades of red imported fire ants

[0065]

[0066]

[0067] The results of indoor bioassays showed that PTG35 has a strong pathogenicity to fire ants, which can be seen from its lethal concentration and lethal time for five grades of fire ants. 50 About 10 3 spores / mL, LC of larvae 50 The lowest, only 0.22×10 3 spores / mL. The concentration of spore suspension reaches 1×10 8 spores / mL, the LT of larvae, pupae, workers, winged female ants and winged male ants of red fire ants 50 The pathogenicity of PTG35 to different grades of red fire ants is in the following order: larvae > worker ants > winged female ants > winged male ants > pupa. PTG35 is lethal to multiple grades of red fire ants, indicating that PTG35 has a significant high pathogenicity to red fire ants.

[0068] Example 3: The attracting effect of PTG35 on red imported fire ants

[0069] Red fire ants have a social lifestyle and are usually able to identify the characteristic volatile odors of pathogenic fungi in the environment. They avoid contact with pathogenic microorganisms through defense behaviors unique to social insects, such as escape, refusal to eat, or rapid nest migration, which affects the control effect of the fungicide. However, the phenomenon of fungi parasitizing ants exists and often occurs in nature, which shows that not all pathogenic microorganisms can be identified, and there are many possibilities such as repelling, attracting, or no tropism. This example uses a selective experiment to explore the tropism of red fire ants to high water purpurogenous fungi PTG35 and consider the practical application potential of this strain.

[0070] 1. Experimental plan

[0071] The equipment used in this experiment (such as Figure 6(As shown in Figure B in the figure), it is a designed and improved indoor bioassay method that uses the digging habits of red fire ants in the soil to evaluate the selection preference of red fire ants for test samples. Four 2mL polystyrene plastic sample tubes were glued to the bottom of a plastic culture dish with a diameter of 18cm and a height of 6cm by hot melt glue. The distance between each sample tube and the center of the culture dish was 6cm; a 4mm diameter access hole was drilled above each of the two opposite sample tubes. The access hole passed through the bottom of the culture dish and the cover of the sample tube, so that the red fire ants could pass through smoothly and dig out the sand. The other two sample tubes were not drilled and were only used for support. Polytetrafluoroethylene was applied to the inner wall of the culture dish to prevent the red fire ants from escaping.

[0072] In the experiment, sand mixed with PTG35 was used as treatment, and pure sand was used as control. 5 mL of spore suspension of PTG35 to be tested (concentration 1×10 8 spores / mL) were poured into a beaker containing 20g of sand; for the control, pure sand was poured into the same volume of pure water (5mL); after thorough mixing, the sample tube was filled until it was full without leaving any gaps, and then combined with the sand-digging device. The red fire ant worker ants were introduced into the sand-digging device, and the device was placed in an artificial climate box (26±1℃, relative humidity 80%±5%, complete darkness) for 24h, and then the mass of sand dug out from each sample bottle by the red fire ants in the culture dish was weighed respectively. The experiment was repeated 6 times, and 100 test workers were used each time without reuse. In order to eliminate the influence of factors such as direction and odor, two sample tubes were set up with pure sand as blank controls. The device (except the sample tube) was recovered and cleaned after each use, and the inner wall was wiped with 75% alcohol before reuse. The sample tube was disposable, and a new tube was replaced for each test.

[0073] 2. Experimental Results

[0074] The selection experiment showed that the red fire ants did not avoid or reject the sample tube containing the sand containing PTG35, but instead showed a behavioral response of being attracted and tending to it. In the pure sand vs. pure sand treatment, there was no significant difference in the amount of sand dug by the red fire ants in the two sample tubes (t=0.706, df=5, P=0.519); in the bacterial sand vs. pure sand treatment, the red fire ants dug sand in both sample tubes. The variance analysis showed that the sand containing PTG35 had an attracting effect on the red fire ants, and the attracting effect reached an extremely significant level (t=5.917, df=5, P=0.004) ( Figure 6 A); This indicates that PTG35 does not induce avoidance behavior of red fire ants, but has a strong attractant effect on red fire ants.

[0075] Example 4: Preparation of Microtablets of PTG35 and Quality Inspection of Shelf Life

[0076] Fungal conidia are exposed to the natural environment and are susceptible to the influence of temperature, humidity and ultraviolet rays. In order to further explore the potential of the strain, the formulation of high water purpurogenous spore mold PTG35 helps to enhance the resistance of conidia in nature and help it to give full play to its pathogenicity in practical applications. At the same time, the protective effect of biological living preparations is easily affected by the storage environment and duration, and the stability of the vitality of the active ingredient conidia during its shelf life is also a key point in practical application. In this example, the active ingredient conidia of high water purpurogenous spore mold PTG35 are made into micro-tablet dosage forms, and the quality and shelf life of the dosage form are comprehensively analyzed and evaluated.

[0077] 1. Experimental plan

[0078] 1. Strain culture and pretreatment

[0079] The high water purple spore mold PTG35 was inoculated on the PDA culture medium, and a large culture dish (d=15 cm) was selected for expanded culture. The inoculated culture dish was placed in a constant temperature incubator with a temperature of 27±1°C, a photoperiod of L:D=12h:12h, a relative humidity of 80%±5%, and a light intensity of 3000lx for culture for 10-14 days. The conidia powder was harvested and placed in a vacuum freeze dryer, and dried for 48 hours under the conditions of a vacuum pump of 20pa and a temperature of -10°C to -20°C in a cold hydrazine to obtain the high water purple spore mold PTG35 powder.

[0080] 2. Micro-tablet production and formula evaluation

[0081] Magnesium stearate was used as a lubricant, and the micro-tablets of PTG35 were prepared by wet granulation and tableting. The common tablet ingredients: starch, sucrose and dextrin were used as excipients. Sucrose was used as the experimental factor, and the three components of starch, sucrose and dextrin were set to different mass ratios (g weight ratio) as follows: ①4:2:1; ②4.5:4.5:1; ③4:5:1 for premixing of excipients. First, starch was added to pure water at a final concentration of 0.25g / mL and mixed evenly. The starch was heated at 100℃ for 10-15min to make starch slurry, which was then put into a clean container for standby use. The sucrose was ground into flour, and then the sucrose and dextrin were mixed with the starch slurry. The materials were stirred electrically for 30min (stirring speed 24rpm), and the materials were dry mixed until uniform and firm particles were formed to increase the fluidity and compressibility of the materials. The obtained wet granules were sieved at 24 rpm through an electric vibrating screen (12 meshes on the upper layer and 40 meshes on the lower layer) for 30 min, then placed in a drying oven, set at 55 ℃ for drying for 70-120 min, and according to the granule drying condition (water content after drying was controlled at 1.5%-2.0%), screened twice, passed through a 24-mesh granulation net, removed the particles that were mutually bonded into agglomerates, and obtained the auxiliary materials for tableting, which were packed for later use. In a 10 ℃ environment, the high water purple spore mold PTG35 bacterial powder was added to the auxiliary materials at a ratio of 1% and stirred evenly, and then magnesium stearate was added at a ratio of 1% and mixed evenly to make compressible granules, and the high water purple spore mold PTG35 micro-tablets of 2-5 mm diameter particle size specifications were made with a rotary tableting mechanism.

[0082] 3. Sensory evaluation of micro tablets

[0083] With reference to the standards of the Chinese Pharmacopoeia, the appearance of the compressed tablets was evaluated, and the screening of soft materials, sticking, tablet surface smoothness, hardness, disintegration, friability and other indicators were used as inspection criteria to evaluate the impact of various indicators (Table 3) on the product.

[0084] Table 3 Comprehensive evaluation table of various indicators

[0085] index Criteria Soft material properties Excellent, good; good, slightly sticky; average, loose Soft material screening Excellent, easy; good, relatively easy; average, not easy Sticking situation Pass, no; fail, yes Angle of repose / ° Between 30° and 50°, the smaller the better Appearance Excellent, smooth; Good, relatively smooth; Average, not smooth Hardness / N Between 40-100N Disintegration rate / min 5 to 10 minutes, the sooner the better Friability / % Less than 1%

[0086] 4. Microtablet shelf life quality inspection

[0087] The microtablets and bacterial powder were stored at 4°C and the following biological characteristic parameters were examined at 3 and 6 months:

[0088] (1) Determination of conidia germination rate: that is, germination rate. Take appropriate amounts of PTG35 microtablets and bacterial powder of P. hygrophila, dissolve them in sterilized 0.05% Tween-80 solution, and prepare them into the same concentration of 1×10 81 mL of the spore suspension with spores / mL was pipetted into a 50 mL Erlenmeyer flask filled with 9 mL of sterile germination solution (0.5% peptone, 1% glucose, 1% OP), and the flask was placed in a shaker at 26±1°C and 110 r / min for 24 h. The number of germinated and ungerminated spores was counted with a handheld counter under a microscope. This was repeated 3 times to calculate the spore germination rate (%).

[0089] (2) Conidia activity determination: that is, the number of viable bacteria. Weigh 1.0 g of microtablets and 0.5 g of bacterial powder and place them in sterile 2 mL centrifuge tubes. Add 1 mL of sterilized 0.05% Tween-80 solution to each tube and shake to dissolve. 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 PDA plate (200 g potato, 20 g glucose, 20 g agar, add H2O to 1000 mL, pH = 7.0), place it at 26 ± 1 ° C, L: D = 14: 10, and incubate it 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) Determination of colony growth rate: Weigh 1.0 g of microtablets and 0.5 g of bacterial powder respectively in 10 mL of sterilized 0.05% Tween-80 solution, mix thoroughly, and then inoculate on a PDA plate. Repeat 4 times. Culture at 26±1°C and L:D=14:10 for 20 days. Use the cross method to measure the colony diameter every 5 days for a total of 4 measurements and calculate the growth rate (mm·d -1 ).

[0091] 2. Experimental Results

[0092] 1. Quality test results of micro tablets after preparation

[0093] The comprehensive evaluation of micro-tablets prepared according to different sucrose ratios is shown in Table 4. When the mass ratios of starch, sucrose and dextrin are 4:2:1 and 4.5:4.5:1 (the sucrose ratios are 28.6% and 45.0%, respectively), all indicators are judged to be qualified. The micro-tablets prepared under the two ratios are uniform in color, light purple, with an average diameter of 3.5mm, a tablet thickness of 1.5mm, and a tablet weight of 0.125g (the single tablet weight fluctuates within the range of ±2% compared with the average tablet weight). After testing, the micro-tablets prepared by the two ratios have a smooth and complete surface without defects, the auxiliary materials are easy to sieve, do not stick to the punch, have a small angle of repose, moderate hardness, and good disintegration performance. Even without the addition of a disintegrant, they can be completely disintegrated within 10 minutes. Since sucrose is a key factor in attracting worker ants to forage, a high proportion (45%) of sucrose content (4.5:4.5:1) is the first choice. Tableting with this ratio has good molding quality, simple formula and process, and is suitable for large-scale production requirements ( Figure 7 ).

[0094] Table 4 Preparation of micro tablets with different sucrose ratios

[0095]

[0096]

[0097] 2. Micro-tablet shelf life quality test results

[0098] The results in Table 5 show that when the micro-tablets of PTG35 high water purpurogenous spores were stored for 6 months, without the addition of preservatives, there was no moisture absorption and mold, and the quality indicators of the effective ingredients were still high, the conidia germination rate reached more than 80%, the number of viable bacteria and the colony growth rate were relatively stable, with no significant difference from the test results at 3 months, and all indicators were significantly higher than the test results of PTG35 high water purpurogenous spores powder. It can be seen that the auxiliary ingredients of the micro-tablet dosage form have good compatibility with the conidia of PTG35 high water purpurogenous spores, and do not affect the mycelial growth and spore germination rate. The high content of sucrose in the formula can also take into account the advantages of preservation and fungus cultivation (sucrose is also contained in the commonly used fungal culture medium), which better solves the problems of poor stress resistance and poor storage resistance of conidia of insect pathogenic fungi in natural environments, and is an ideal microbial formulation for extending the shelf life.

[0099] Table 5 Quality test results of active ingredients in micro tablets

[0100]

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

[0102] Example 5: Microtablets of PTG35 and its field efficacy test against red imported fire ants

[0103] Preliminary experiments have shown that PTG35 has an attractant effect on red fire ants. Micro-tablets were made using conidia powder of PTG35 and excipients without chemical pesticides. In order to further play the role of PTG35 in production practice, this example uses field efficacy tests to determine the field efficacy of PTG35 micro-tablets on red fire ants, and considers the potential of this dosage form for practical application in preventing and controlling red fire ants.

[0104] 1. Experimental plan

[0105] 1. Test site and test materials

[0106] The test materials are the PTG35 microtablets of high water purpurogenous spores prepared in Example 4 (wherein the mass ratio of starch, sucrose and dextrin in the auxiliary materials is 4.5:4.5:1), and the PTG35 powder of high water purpurogenous spores obtained by the method of step 1 in the above Example 4. A blank control is used without any treatment. The test site is located in the Baiyun Experimental Base of Guangdong Academy of Agricultural Sciences. There is less human interference in the test site, red fire ants occur in patches, the ant nests are obviously raised, and the distribution is relatively uniform.

[0107] 2. Field trial design

[0108] A rectangular test plot is set up according to the actual situation of the test area. The minimum area of ​​the test plot is 10m 2 , the number of live ant nests in each plot is greater than or equal to 4. When the density of live ant nests is low, the plot area should be appropriately expanded to ensure that the number of live ant nests in each plot is not less than 4. Use colored plugs to mark live ant nests before treatment. The experimental plots and control plots are arranged in random blocks, and no fungicides or pesticides are applied to the ant nests in the blank control plots. The experiment has 5 treatments, including 4 dosages of micro-tablets and bacterial powder treatment, each treatment is repeated 3 times; the blank control is repeated 3 times, with a total of 18 plots.

[0109] 3. Application method and test weather conditions

[0110] Microtablets were placed to treat a single ant nest. Filter paper (11cm×11cm) was used to carry 5g, 10g, 15g, and 20g microtablets, which were placed within 50cm of the ant nest to avoid damaging the anthill. When the ant nests were densely distributed and not obvious, microtablets were evenly placed in the entire area. The ant nests were not disturbed during application, and the microtablets were not mixed with other fertilizers or granules. When the fungus powder was treated, 20g was used for each single ant nest. First, a circle of fungus powder (10g) was sprinkled around the periphery of the ant nest, and then the ant nest was poked with a wooden stick. After a large number of red fire ants emerged, the remaining 10g of fungus powder was quickly and evenly sprinkled on the insect body until the fungus powder covered the entire ground area of ​​the ant nest. The ant nests with different treatments were marked. During the application period of each treatment, the temperature was 10℃~32℃, cloudy or overcast, the relative humidity was 85%, and the ground was dry. The application time was between 8:00-10:00 in a day, and there was no rainfall 3-5d before application and 5-7d after application.

[0111] 4. Investigation and calculation

[0112] Before applying the pesticide, investigate the number of live nests and the number of trapped worker ants in each test plot. Use the ham sausage bait trap method to investigate the population of red fire ants. Put ham sausage in a centrifuge tube (10mL) and place it flat on the ground about 50cm away from the ant nest. Set up 3 trap tubes in each plot. Collect the tubes and count them after about 30 minutes. Survey once each at 5d, 10d and 15d after applying the pesticide, and record the number of trapped worker ants three times in total. During the last survey, dig the nest to observe the survival of the ant colony and record the number of live nests of red fire ants.

[0113] The control effect of worker ants was calculated based on the reduction rate of the number of trapped worker ants before and after each treatment. The formula is as follows:

[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 ant nest control effect is calculated based on the ant nest reduction rate before and after the micro-tablet treatment. The formula is as follows:

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

[0117] 2. Experimental Results

[0118] The control effect of worker ants in each treatment is as follows Figure 8As shown in the results, when the bacterial powder was used for treatment, the worker ant reduction rate was the lowest in all treatments. The worker ant control efficiency reached the highest value of 11.46% 15 days after the application of the bacteria, which was significantly lower than the control efficiency of 5g / nest of micro-tablets in the same period (F=3.081, df=8, P<0.001). There were differences in the worker ant control efficiency of different micro-tablet application amounts. When the micro-tablet application amount was 5g / nest and 10g / nest, the worker ant reduction rate was 40.74% and 60.53% 15 days after the application of the bacteria, respectively; when the application amount was 15g / nest and 20g / nest, the worker ant control efficiency reached the highest value of 84.35% and 88.55% 10 days after the application of the bacteria, respectively, and the control efficiency no longer increased after 15 days. The difference between the two periods after the application of the bacteria was not significant (F=1.065, df=8, P=0.726). Judging from the worker ant reduction rate, the treatments with a dosage of 15g / nest and 20g / nest have the best control effect on red fire ant worker ants, which can reach more than 80%.

[0119] After 15 days of treatment, the control effect of ant nests in each treatment was as follows: Fig. 9 As shown in the results, the ant nest reduction rate of the blank control was 6.67%, and the ant nest reduction rate after the application of the bacterial powder was 5.45%, and the difference between the two was not significant (F = 2.429, df = 8, P = 0.563); when the micro-tablet dosage was 5g / nest, the ant nest reduction rate was 20%, and the difference between the treatment and the bacterial powder reached a significant level (F = 6.861, df = 8, P = 0.028); when the dosage was 10g / nest and 15g / nest, the ant nest reduction rate reached 62.5% and 78.57% respectively, and there was a significant difference in the control effect between the two dosages (F = 8.975, df = 8, P = 0. 035); when the dosage was 20g / nest, the nest reduction rate reached the highest value of 83.33%, which was not significantly different from the control effect of 15g / nest (F=1.362, df=8, P=0.739), and was extremely significantly different from the control effects of 10g / nest and 5g / nest (F=1.899, df=8, P=0.008 and F=4.724, df=8, P<0.001), and was extremely significantly different from the bacterial powder and blank control (F=6.434, df=8, P<0.001 and F=6.795, df=8, P<0.001).

[0120] In summary, when the micro-tablet dosage is 15g / nest and 20g / nest, the worker ant number reduction rate and ant nest reduction rate on the 10th-15th day have good effects, which are significantly higher than the field control effect of bacterial powder. The micro-tablet of PTG35 can achieve the ideal control effect at the above dosage. It has the characteristics of high efficiency and lasting effect, and can be mass-produced for field application.

Claims

1. A strain of Purpureocillium takamizusanense PTG35, characterized in that: The deposit number is GDMCC No.65103.

2. A high water purpurogenous mold PTG35 micro tablet, characterized in that, Contains the high water purple spore mold PTG35 according to claim 1.

3. The micro-tablet of high water purpurogenous mold PTG35 according to claim 2, characterized in that, The microtablets of PTG35 of high water purpurogenous spores are composed of 1 part of powder of PTG35 of high water purpurogenous spores, 98 parts of auxiliary materials and 1 part of magnesium stearate by weight, and the auxiliary materials are composed of 4-4.5 parts of starch, 2-4.5 parts of sucrose and 1 part of dextrin.

4. The high water purpurogenous mold PTG35 micro-tablet according to claim 3, characterized in that, In terms of weight, the auxiliary material consists of 4.5 parts of starch, 4.5 parts of sucrose and 1 part of dextrin.

5. A method for preparing a micro-tablet of PTG35, characterized in that: The following steps are involved: S1. Take 4.5 parts of starch, 4.5 parts of sucrose and 1 part of dextrin by weight; add starch to water at a final concentration of 0.25 g / mL and mix evenly, heat at 100°C for 10-15 min to make starch slurry, grind sucrose into powder, then mix sucrose and dextrin with the starch slurry, and stir until uniform and firm particles are formed; S2. The granules were sieved through an electric vibrating sieve with an upper 12-mesh and a lower 40-mesh sieve at 24 rpm for 30 min, and then dried in a drying oven at 55°C. The moisture content after drying was controlled at 1.5%-2.0%; the granules were sieved through a 24-mesh sieve to remove the agglomerated particles to obtain the excipients for tableting; S3. By mass, add 1 part of the fungus powder of the high water purpurogenous spore PTG35 described in claim 1 to 98 parts of auxiliary materials and stir evenly, then add 1 part of magnesium stearate and mix evenly, and the mixed material is made into high water purpurogenous spore PTG35 micro-tablets with a particle size of 2-5mm using a rotary tablet press.

6. The preparation method according to claim 5, characterized in that: The bacterial powder of PTG35 is freeze-dried powder of conidia of PTG35.

7. The preparation method according to claim 5, characterized in that: The content of conidia of the active ingredient of the high water purpurogenous spore PTG35 micro tablet is greater than 5×10 9 Spores / piece.

8. Use of the high water purpurogenous spore PTG35 described in claim 1 and the high water purpurogenous spore PTG35 micro-tablets described in claim 2 in preventing and controlling red imported fire ants.

9. The use according to claim 8, characterized in that: The red fire ants are winged female ants, winged male ants, worker ants, larvae or pupae of red fire ants.

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