CN111642520A

CN111642520A - Paecilomyces lilacinus wettable powder and application thereof

Info

Publication number: CN111642520A
Application number: CN202010683602.7A
Authority: CN
Inventors: 杜丹超; 鹿连明; 胡秀荣; 黄振东; 蒲占湑; 陈国庆; 刘顺民; 吕佳
Original assignee: Zhejiang Citrus Research Institute
Current assignee: Zhejiang Citrus Research Institute
Priority date: 2020-07-16
Filing date: 2020-07-16
Publication date: 2020-09-11
Anticipated expiration: 2040-07-16
Also published as: CN111642520B

Abstract

The invention provides paecilomyces lilacinus wettable powder and application thereof, belonging to the technical field of biological pest control and comprising the following components in percentage by mass: 10% of paecilomyces lilacinus conidium pure spore powder, 3% of wetting agent, 5% of dispersing agent and 0.25% of ultraviolet protective agent; 0.1% of spore germination accelerant; 0.05% of adhesive; and 81.6% of carrier. The paecilomyces lilacinus wettable powder overcomes the problems of environmental pollution, pesticide residue, pest drug resistance and the like caused by chemical pesticides, is a high-efficiency, safe and environment-friendly biocontrol bacterium preparation, and provides a new material for biological control of diaphorina citri, panonychus citri and cotton aphid.

Description

Paecilomyces lilacinus wettable powder and application thereof

Technical Field

The invention belongs to the technical field of biological pest control, and particularly relates to paecilomyces lilacinus wettable powder and application thereof.

Background

Diaphorina citri, panonychus citri and cotton aphid are important pests on citrus. These pests often accumulate on the tips, leaves or fruits of citrus fruit and cause sap feeding damage, resulting in dead citrus tips, fallen leaves and fruit, and may produce secretions that induce soot disease. The diaphorina citri is the only natural transmission medium of citrus greening disease which is the most difficult and most threatening destructive disease in the current citrus production, and at present, an ideal prevention and control medicament is still lacked for the citrus greening disease, and the 'insect prevention and disease prevention' is usually a key technical measure for preventing and controlling the disease. At present, chemical pesticides are mainly used for controlling the pests, but the problems of drug resistance, environmental pollution, pesticide residue and the like caused by frequent use of the chemical pesticides are becoming serious. With the concern of people on the problems of ecological environment, food safety and the like, the development of safe and efficient prevention and control agents and the establishment of new pest prevention and control measures become an urgent task in the current agricultural production, and are also important subjects related to the sustainable development of the citrus industry. The biological pesticide has the characteristics of no residue, strong specificity, difficult generation of drug resistance, good environmental compatibility, simple production process and the like, and people pay more and more attention to the biological pesticide.

In the research aspect of using biocontrol bacteria to prevent and control citrus pests, scholars at home and abroad successively develop some beneficial explorations. Among the fungi reported to be pathogenic to diaphorina citri, there are paecilomyces fumosoroseus, soot citrus bacteria, etc., fungi pathogenic to panonychus citri, such as polymannua thosporum, etc., and fungi pathogenic to citrus aphids, such as Aphis citricola, etc., such as Fusarium cubense, etc. However, the existing research mostly focuses on the laboratory exploration stage, and the commercialized microbial pesticide product in the market can be applied to the prevention and control of citrus pests.

Paecilomyces lilacinus (A)Paecilomyces lilacinus) Also known as purple lilac spore bacterium (Purpureocillium lilacinum) Is an important biocontrol fungus. The existing research shows that the bacterium has strong parasitism and pathogenicity on various plant nematodes, has certain antagonism on various plant pathogenic bacteria such as rice sheath blight, corn microsporum, citrus green mold and the like, can promote seed germination and plant root system and plant growth by metabolites, and has certain pesticide degradation effect on various produced functional enzymes. Paecilomyces lilacinus ZJPL08 is a fungus separated from diaphorina citri in Taizhou Zhejiang by the laboratory. The pathogenicity analysis determines that the strain has strong pathogenicity to the diaphorina citri; biological characteristic analysis proves that the strain has stronger stress resistance to high and low temperature, ultraviolet rays and the like, and has the excellent characteristics of high growth speed, large spore yield, high spore germination rate and the like, so the strain has good development and application prospects. At present, no report on the control of pests such as diaphorina citri by using the paecilomyces lilacinus wettable powder is available at home and abroad. Therefore, the paecilomyces lilacinus wettable powder with stable quality and convenient application is prepared by comparing dosage forms, screening auxiliary materials and the like, and a new choice is provided for preventing and controlling pests such as diaphorina citri, panonychus citri and cotton aphid.

Disclosure of Invention

The invention aims to solve the technical problem of making up the defect that a fungus biological agent is lacked in the current pest control of diaphorina citri, panonychus citri and cotton aphid and provides paecilomyces lilacinus wettable powder.

The second purpose of the invention is to provide a preparation method of the wettable powder.

The third purpose of the invention is to provide the application of the wettable powder in pest control, and specifically, the application of the wettable powder in pest control of diaphorina citri, panonychus citri and cotton aphid.

The purpose of the invention is realized by the following technical scheme:

the paecilomyces lilacinus wettable powder comprises the following components in percentage by mass: 10% of paecilomyces lilacinus conidium pure spore powder, 3% of wetting agent, 5% of dispersing agent and 0.25% of ultraviolet protective agent; 0.1% of spore germination accelerant; 0.05% of adhesive; and 81.6% of carrier.

The paecilomyces lilacinus is paecilomyces lilacinus (A)Paecilomyces lilacinus) The strain ZJPL08 (published in 201310117537.1) is registered and preserved in China general microbiological culture Collection center (CGMCC) at 3/15 in 2013, and the preservation number is CGMCC number 7318.

Preferably, the spore content of the pure spore powder of the paecilomyces lilacinus conidium is 2.96-3.74 × 10¹¹The number per gram, the viable spore rate is 98.31-99.43%, and the water content is 6.06-6.44%.

Preferably, the wetting agent is sodium dodecyl sulfate, the dispersing agent is sodium lignosulfonate, the ultraviolet protective agent is humic acid, the spore germination promoter is calcium chloride, the adhesive is xanthan gum, and the carrier is diatomite.

The preparation method of the wettable powder comprises the following steps: mixing the carrier, the wetting agent, the dispersing agent, the ultraviolet protective agent, the spore germination promoter, the adhesive and the like according to a certain proportion, adding the paecilomyces lilacinus conidium powder, stirring uniformly, sieving by a 325-mesh standard sieve, and fully stirring and uniformly mixing to obtain the paecilomyces lilacinus wettable powder.

The paecilomyces lilacinus wettable powder is applied to control diaphorina citri, panonychus citri and cotton aphid.

Compared with the prior art, the invention has the following beneficial effects:

the paecilomyces lilacinus wettable powder has excellent product performance index and spore content of 2.98 × 10¹⁰The spore count per gram is 92.34%, the water content is 3.59%, the wetting time is 85.32 s, the spore suspension rate is 84.76%, the fineness is 96.52%, and the spore passes through a 325-mesh sieve, and the pH value is 6.85.

The paecilomyces lilacinus wettable powder can be used for preventing and treating diaphorina citri, panonychus citri and cotton aphid. LC against Pediculus citri 7 d after treatment under room conditions₅₀Is 1.39 × 10⁴Each mL, concentration of 1.0 × 10⁸LT of wettable powder per mL for diaphorina citri₅₀2.64 d, at a concentration of 1.99 × 10⁷The corrected mortality rates of panonychus citri and cotton aphid are respectively 66.67 percent and 78.26 percent 3 d after being treated by the wettable powder per mL and 95.65 percent and 100.00 percent 7 d after being treated, and the concentration is 1.99 × 10 under the field condition⁷The corrected mortality rate of diaphorina citri is 42.83% 3 d after treatment with wettable powder per mL, and the corrected mortality rate of diaphorina citri is 100% 14 d after treatment.

The paecilomyces lilacinus wettable powder overcomes the problems of environmental pollution, pesticide residue, pest drug resistance and the like caused by chemical pesticides, is a high-efficiency, safe and environment-friendly biocontrol bacterium preparation, and provides a new material for biological control of diaphorina citri, panonychus citri and cotton aphid.

Detailed Description

The present invention will be described in further detail below by way of examples. The following experimental methods are conventional in the art unless otherwise specified. All the ingredients, reagents or materials used, unless otherwise specified, are commercially available ingredients, reagents or materials. The invention should not be limited to the scope of the examples.

Example 1: preparation and quality determination of paecilomyces lilacinus ZJPL08 conidium pure spore powder

1. Strain activation

Transferring the strain stored in a potato glucose agar culture medium (PDA, formula is: potato 200 g, glucose 20 g, agar 20 g, distilled water 1000 mL, natural pH) slant at 4 deg.C to PDA plate culture medium, and culturing at constant temperature in an incubator at 28 deg.C for 7 d.

2. Seed liquid preparation

The activated strain ZJPL08 spores are picked by a sterile inoculating loop to a seed culture solution potato sucrose liquid culture medium (PSB, the formula is that potato 200 g, sucrose 20 g, distilled water 1000 mL, natural pH), and the mixture is placed in a constant temperature shaking table (28 ℃, 120 rpm/min) to be cultured for 3 d, and the seed solution is used for inoculating a solid culture medium for amplification culture.

3. Solid fermentation culture

Putting rice into a 1000 mL beaker, soaking overnight in water, draining, sterilizing at 121 ℃ for 21 min in an autoclave, cooling the sterilized rice to room temperature, inoculating the cultured seed solution into the solid rice according to the inoculation amount of 10% in a clean bench, stirring uniformly, and transferring into culture dishes with about 100 g per dish for 3 times. Placing in an incubator at 28 ℃ for constant-temperature culture for 7 d.

4. Harvesting of pure sporopouenin

And (3) replacing the dish cover of the culture dish with sterilized newspaper after culturing for 7 d to rice culture medium to grow dark purple spore powder, placing the dish cover in a 35 ℃ blast drying oven for drying for 36 h, taking out the culture dish, and screening the culture dish in an ultraclean workbench by using a 325-target standard sample sieve to obtain the pure spore powder of the strain ZJPL 08.

5. Quality index determination of pure sporopollen

Measuring spore content by weighing 0.01 g spore powder, adding into 1mL sterile water tube containing 0.5% OP-10, vortex oscillating for 5 min to disperse the spores into uniform suspension, diluting the spore suspension by 100 times, counting under microscope with blood counting plate, repeating for 5 times, calculating average value, wherein the specification of the blood counting plate is 25 grids × grids, and the total number of spores in four grids and one grid at the center in a double-line counting area is calculated³× dilution multiple × 100 the results are shown in Table 1.

And (3) determining the viable spore rate: 0.01 g of conidium powder is weighed and added into 1mL of potato glucose liquid culture medium (PDB, the formula is that 200 g of potatoes, 20 g of glucose and 1000 mL of distilled water are contained, and the mixture is naturally mixed to prepare spore suspension. 0.1mL of spore suspension is absorbed by a liquid transfer machine and dropped in the center of a sterile glass slide, then the glass slide is placed on a U-shaped glass rod in a culture dish, sterile filter paper is paved at the bottom of the culture dish, 2 mL of sterile water is dropped on the filter paper for moisture preservation, then the culture dish is placed in a microorganism incubator for constant temperature culture at 28 ℃, and 3 times of treatment are carried out. After culturing for a period of time, taking out the slide, placing the slide under a microscope, counting 200-300 spores randomly, and taking the half of the length of the bud tube in the visual field, which is larger than the width of the spore, as the standard of spore germination. Calculating the spore germination rate according to the following formula: spore germination rate (%) = number of spores germinated in count region/total number of spores in count region × 100. The results are shown in Table 1.

And (3) water content determination: accurately weighing 0.1 g of spore powder in a culture dish of 9 cm with constant weight, drying in a digital display air drying oven at 100 ℃ to constant weight, weighing the spore powder and the dish together, and calculating the water content of the spore. The experiment was performed in 5 replicates and the average was taken. The water content calculation method is shown in the formula: water content (%) = (total mass of schoenoprass before constant weight-total mass of schoenoprass after constant weight)/total mass of spore x 100. The results are shown in Table 1.

TABLE 1 quality index of pure spore powder of Paecilomyces lilacinus ZJPL08

Example 2: screening of paecilomyces lilacinus ZJPL08 wettable powder carrier and auxiliary agent

1. Screening of vectors

Vectors used for screening were: calcium carbonate, kaolin, talc, diatomaceous earth, and attapulgite, available from Shanghai Shize Biotech, Inc.; flour, purchased in local supermarkets.

(1) Effect of the vector on spore germination: mixing the above carrier powder with melted PDA culture medium at a ratio of 5% (w/v), sterilizing, and making into plate. 0.1mL of spore with a concentration of about 10³Individual/mL of a conidia suspension of strain ZJPL08 was spread on each plate containing different carriers. PDA plates without vector were used as controls. 3 replicates were set per treatment. All treated plates were incubated at 28 ℃ for 2-3 d. The number of colonies forming units of Paecilomyces lilacinus growing from each plate was observed and the spore germination rate was calculated.

(2) Effect of vector on hyphal growth: plates containing various carriers were prepared as described above. Taking a hypha block which grows vigorously on the edge of 7 d Paecilomyces lilacinus cultured by a puncher with the diameter of 5 mm, inoculating the hypha block to each culture medium, setting 3 times of treatment, culturing at the constant temperature of 28 ℃, and measuring the diameter of each colony when the culture is different, wherein the diameter is recorded as d 1. The daily increase in colony diameter was calculated according to the following formula: the daily increase in colony diameter (mm/d) = d 1/day of growth.

The results of the biocompatibility determination of strain ZJPL08 with each vector are shown in table 2. The results show that other carriers except kaolin and talcum powder have no adverse effect on spore germination of the strain ZJPL08, and the attapulgite and flour even promote the spore germination; CaCO₃The diatomite and the flour have a promoting effect on the growth of hyphae, and the attapulgite has small influence on the growth of the hyphae. Comprehensively considering the performance and cost of each carrier, diatomite is selected as the carrier for preparing the wettable powder of the paecilomyces lilacinus wettable powder ZJPL 08.

TABLE 2 biocompatibility of different vectors with Paecilomyces lilacinus

Note: the numbers in the same column followed by different lower case letters indicate significant differences at a level of P <0.05

2. Wetting agent screening

Wetting agents used for screening were: sodium Dodecyl Sulfate (SDS), a product of Amersham corporation; sodium dodecyl benzene sulfonate SDBS is a product of Tianjin Bodi chemical corporation.

(1) And (3) measuring the wetting force: the wetting agents were added to diatomaceous earth in the proportions of 1%, 3% and 5% (w/v) and 10% spore powder, and the mixture was mixed well to prepare a sample. Accurately weighing 0.1 g of sample, uniformly pouring the sample onto a 150 mL beaker page containing 100 mL of standard water from the position flush with the opening of the beaker in one step, immediately recording the time by using a stopwatch when the sample is added until the sample is completely wetted, recording the wetting time, and repeating the treatment for 3 times by using the sample without the wetting agent as a control.

(2) And (3) measuring the biocompatibility: mixing various wetting agents with PDB liquid culture medium at final mass concentrations of 100, 200, 400, 800, 1500 and 3000 mu g/mL respectively, weighing 0.01 g of conidium powder, adding into 1mL of prepared solution, and uniformly mixing to obtain molecular spore suspension. Then 0.1mL of the suspension was pipetted into the center of a sterile slide and incubated at 28 ℃ in an incubator with moisture, 3 replicates per treatment set-up, using spore suspension without wetting agent as a control. After culturing for a period of time, placing the slide under an optical microscope, observing the spore germination condition and calculating the spore germination rate by the same method, and analyzing the influence of each wetting agent on the germination of the conidia of the strain ZJPL 08. Mixing various wetting agents and PDA culture medium to prepare 100, 200, 400, 800, 1500 and 3000 mug/mL mass concentration, and autoclaving to prepare the plate. In the same manner as above, a paecilomyces lilacinus block with a diameter of 5 mm was inoculated, then cultured at a constant temperature of 28 ℃ for 3 replicates per treatment, and the diameter of each colony was measured and the daily growth of the hyphae was calculated using the hyphae block inoculated on a PDA plate containing no wetting agent as a control at different culture times.

The wetting effect of each wetting agent on conidia of strain ZJPL08 is shown in table 3, and the biocompatibility with strain ZJPL08 is shown in tables 4 and 5. As can be seen from table 3, the wetting power of each wetting agent varies with the type and concentration, the difference between sodium dodecyl sulfate and sodium dodecyl benzene sulfonate is significant, and the wetting power of each wetting agent at different concentrations is significantly different. The wetting force of sodium lauryl sulfate increases with concentration and decreases with a minimum wetting time of 11.24 s at 5%. The wetting power of sodium dodecyl benzene sulfonate is poor, and at a concentration of 5%, the wetting power is low and is 180.5 s. As can be seen from Table 4, different wetting agents have different influences on the spore germination rate of the strain, sodium dodecyl sulfate has obvious influence on the spore germination rate, the sodium dodecyl sulfate has obvious inhibition effect on the spore germination of the strain ZJPL08 when the concentration is higher, the sodium dodecyl sulfate hardly has influence on the spore germination rate of the strain ZJPL08 when the concentration is 100 mu g/mL, and the sodium dodecyl benzene sulfonate has little influence on the spore germination rate of the strain, and even when the concentration reaches 3000 mu g/mL, the spore germination rate can still reach 78.70%. As can be seen from Table 5, sodium dodecyl sulfate had a significant effect on the hyphal growth of strain ZJPL08, and at higher concentrations (> 1500. mu.g/mL), the hyphal growth was completely inhibited, while at low concentrations of 100. mu.g/mL, there was almost no effect on the hyphal growth. In contrast, sodium dodecylbenzenesulfonate has little influence on the growth of hyphae, and the daily growth of hyphae can still reach 2.78mm at a high concentration of 3000 mug/mL. Comprehensively considering the wetting power and the biocompatibility of each wetting agent, the low-concentration sodium dodecyl sulfate is selected as the wetting agent for preparing the wettable powder of the paecilomyces lilacinus ZJPL 08.

TABLE 3 wetting Effect of various wetting agents on conidia of Strain ZJPL08 at different concentrations

TABLE 4 Effect of different concentrations of different wetting agents on spore germination Rate of Strain ZJPL08

Note: the numbers in the same row are marked with different capital letters to indicate that the difference is remarkable at the level P <0.05, and the numbers in the same column are marked with different lower-case letters to indicate that the difference is remarkable at the level P <0.05

TABLE 5 Effect of different concentrations of wetting agent on hyphal growth of Strain ZJPL08

3. Screening of dispersants

Dispersants used for screening were: sodium lignosulfonate, calcium lignosulfonate and sucrose fatty acid ester were all available from Shanghai Shize Biotech, Inc.

(1) And (3) measuring the dispersing force: coarse screening of a dispersing agent: after mixing the carrier, the wetting agent and the spore powder, 5% of the dispersant to be selected is added to be processed into a sample, then 0.05 g of the sample containing 1% of the wetting agent is weighed and poured into an 80 mL small beaker containing 50 mL of standard hard water, and the dispersion condition of the sample is visually observed. The coarse screen dispersion was graded into three grades: and (3) excellent: it is automatically dispersed in water in the form of cloud, and has no visible particles sinking, and is indicated by "+". Good: the particles are automatically dispersed in water, and sink, and the sinking particles can be slowly dispersed or dispersed after being slightly shaken, and are indicated by "+ -". Poor: it can not be automatically dispersed in water, and can be dispersed after being strongly shaken, and it is indicated by "-". Fine screening of a dispersing agent: the fine screening takes the suspension rate of the active ingredients of the preparation as an index, and the operation steps are carried out according to GB/T14825-2006. The operation is repeated for 3 times, and the spore suspension rate calculation method is shown in a formula: spore suspension (%) = (spore content in mother liquor-spore content in lower suspension)/spore content in mother liquor x 10/9 x 100.

(2) And (3) measuring the biocompatibility: mixing various dispersants with PDB liquid culture medium at final mass concentrations of 100, 200, 400, 800 and 1500 mu g/mL respectively, weighing 0.01 g of conidium powder, adding into 1mL of prepared solution, and uniformly mixing to obtain conidium suspension. Then 0.1mL of the suspension is pipetted into the center of a sterile glass slide and placed in an incubator at 28 ℃ for moisturizing culture, 3 replicates per treatment set, and spore suspension without dispersant is taken as a control. After culturing for a period of time, placing the slide glass under an optical microscope, observing the spore germination condition and calculating the spore germination rate by the same method, and analyzing the influence of each dispersant on the germination of the conidia of the strain ZJPL 08. Mixing various dispersants with PDA culture medium to obtain mass concentrations of 200, 400, 800, 1500, 3000, 5000 μ g/mL, autoclaving, and making into plate. In the same manner as above, a paecilomyces lilacinus block with a diameter of 5 mm was inoculated, then cultured at a constant temperature of 28 ℃ for 3 replicates per treatment, and the diameter of each colony was measured and the daily growth of the mycelia was calculated at different culture times with the hypha block inoculated on a PDA plate without a dispersant as a control.

The dispersing effect of each dispersant in the case of coarse screening is shown in table 6, the dispersing effect in the case of fine screening is shown in table 7, and the biocompatibility of each dispersant with strain ZJPL08 is shown in table 8. As can be seen from Table 6, the sodium lignosulfonate and the sucrose fatty acid ester have good dispersibility in Paecilomyces lilacinus spores and are relatively stable, and the sample is automatically dispersed in the form of mist in water without visible particle sinking. As can be seen from Table 7, the conidium suspension rate of the strain ZJPL08 can be remarkably improved after the dispersant is added, and the effects of sodium lignin sulfonate and sucrose fatty acid ester are not remarkably different. From tables 8 and 9, it can be seen that calcium lignosulfonate and sodium lignosulfonate have no influence on hypha growth and spore germination of the strain ZJPL08, even sodium lignosulfonate with low concentration has a promoting effect on hypha growth, and sucrose fatty acid ester has a significant difference compared with a control and has an obvious inhibiting effect on hypha growth and spore germination of the strain. Comprehensively considering the dispersing effect of each dispersing agent and the biocompatibility of each dispersing agent and the strain ZJPL08, sodium lignosulfonate is selected as the dispersing agent to be used for preparing paecilomyces lilacinus ZJPL08 wettable powder.

TABLE 6 dispersant effect on coarse screening of dispersant

TABLE 7 conidia suspension percentage after addition of each dispersant

TABLE 8 Effect of different concentrations of different dispersants on spore germination Rate of Strain ZJPL08

TABLE 9 Effect of different concentrations of different dispersants on hyphal growth of Strain ZJPL08

4. Screening of UV protectant

Examples of UV protection agents for screening are: ascorbic acid, a product of Sigma company; humic acid and sodium fluorescein, available from Shanghai Zeze Biotech, Inc.

(1) Determination of protective efficacy: mixing the ultraviolet protective agents to be tested with the PDB liquid culture medium at a final mass concentration of 0.25%, weighing 0.01 g of conidium powder, adding the conidium powder into 1mL of prepared solution, and uniformly mixing to obtain conidium suspension. Then 0.1mL of the suspension was pipetted into the center of a sterile glass slide, placed under an ultraviolet lamp (30 w, 120 Lux) at 20 cm for 10 min, and then the slide was placed flat on a U-shaped glass rod in a petri dish, which was humidified with a moist sterile filter paper. Then, the culture was carried out at 28 ℃ and the number of germinated spores was counted by sampling under a microscope to calculate the germination rate. Each treatment was set up in 3 replicates and UV irradiation was performed on a suspension of Paecilomyces lilacinus spores without UV protectant as a control.

(2) And (3) measuring the biocompatibility: mixing the ultraviolet protective agents to be tested with the PDB liquid culture medium at a final mass concentration of 0.25%, weighing 0.01 g of conidium powder, adding the conidium powder into 1mL of prepared solution, and uniformly mixing to obtain conidium suspension. Then 0.1mL of the suspension was pipetted into the center of a sterile slide and the slide was placed flat on a U-shaped glass rod in a petri dish, which was humidified with moist sterile filter paper. Thereafter, the cells were cultured at 28 ℃ and the number of germinated spores was counted under a microscope to calculate the germination rate. Each treatment was set up in 3 replicates and a suspension of Paecilomyces lilacinus spores without UV protectant was used as a control.

The protective efficacy and biocompatibility of each uv protectant against strain ZJPL08 are shown in table 10. As can be seen from Table 10, the most effective protective agents for conidia of strain ZJPL08 are humic acid and fluorescein sodium, and when the two protective agents are added respectively, the germination rates of the conidia of strain ZJPL08 after ultraviolet irradiation for 10 min can still reach 93.73% and 77.16%. As can be seen from the results of the spore germination rates measured without ultraviolet irradiation after the ultraviolet protective agents are added, each ultraviolet protective agent does not have obvious influence on the spore germination of the strain ZJPL08, and has good biocompatibility. Therefore, the protection efficacy of each ultraviolet protective agent and the biocompatibility of the ultraviolet protective agent and the strain ZJPL08 are comprehensively considered, and humic acid is used as the ultraviolet protective agent for preparing the wettable powder of the paecilomyces lilacinus ZJPL 08.

TABLE 10 protective efficacy and biocompatibility of each UV protectant against strain ZJPL08

5. Screening of spore germination promoters

Spore germination promoters for screening were: zinc sulfate, calcium chloride and potassium chloride, all of which are products of Amersham company. The spore germination promoters to be tested were mixed with the PDB liquid medium at final mass concentrations of 0.1%, 0.2% and 0.5%, respectively, 0.01 g of conidium powder was weighed into 1mL of the prepared solution, and conidium suspension was prepared by mixing well. Then 0.1mL of the suspension was pipetted into the center of a sterile slide and the slide was placed flat on a U-shaped glass rod in a petri dish, which was humidified with moist sterile filter paper. Then, the culture was carried out at 28 ℃ and the number of germinated spores was counted by sampling under a microscope to calculate the germination rate. Each treatment was repeated 3 times, and the treatment without accelerator was used as a control. The effect of different germination promoters on conidium germination is shown in table 11. As can be seen from Table 11, calcium chloride and potassium chloride both significantly increased the conidium germination rate of strain ZJPL08, and at 0.1% concentration, the conidium germination rates reached 90.42% and 85.34%, respectively, which were significantly higher than the germination rate of the control without promoter by 76.38%. Therefore, calcium chloride is selected as a spore germination promoter to be used for preparing wettable powder of paecilomyces lilacinus ZJPL 08.

TABLE 11 Effect of spore germination promoters on conidium germination of Strain ZJPL08

6. Adhesive screening

The adhesives used for screening were: xanthan gum, available from Qingdao Youso chemical science and technology, Inc.; sucrose, product of Amersham; soluble starch, a product of Sigma company.

(1) And (3) measuring the adhesive force: the adhesive to be tested is added into the mixture with the concentration of 10 according to the proportion that the final mass concentration is 0.05 percent⁵pieces/mL of spore suspension were sprayed with a watering can onto citrus leaves until dripping. After 4 h, taking leaves with the same height (punched into small disks by a puncher) and grinding the leaves in 30 mL of sterile water, uniformly coating 0.1mL of liquid on a PDA culture medium, culturing the PDA culture medium at 28 ℃ for 2-3 d, observing the colony forming unit number grown on each plate and calculating the spore germination rate. Let the treatment without adhesive be a control, repeated 4 times per treatment.

(2) And (3) measuring the biocompatibility:

mixing each tested adhesive with a PDB liquid culture medium at a final mass concentration of 0.05%, weighing 0.01 g of conidium powder, adding into 1mL of prepared solution, and uniformly mixing to obtain a conidium suspension. Then 0.1mL of the suspension was pipetted into the center of a sterile slide and the slide was placed flat on a U-shaped glass rod in a petri dish, which was humidified with moist sterile filter paper. All treated plates were then incubated at 28 ℃ and the number of germinated spores was counted and the germination rate was calculated by sampling under a microscope. Each treatment was set up in 3 replicates and spore suspension without agglutinating was used as a control.

The adhesion of each adhesive and its biocompatibility with strain ZJPL08 are shown in table 12. As can be seen from Table 12, the number of colonies generated on PDA medium after the conidia suspension of strain ZJPL08 added with xanthan gum was sprayed on citrus leaves was 96.67, which is significantly higher than the control without adhesive, indicating that xanthan gum can improve the adhesion of conidia of strain ZJPL08 on citrus leaves. None of the 3 adhesives had a significant effect on conidiophorous germination of strain ZJPL 08. Therefore, xanthan gum is selected as a sticking agent for preparing wettable powder of Paecilomyces lilacinus ZJPL 08.

TABLE 12 adhesion and biocompatibility of each adhesive with Strain ZJPL08

Example 3: preparation of paecilomyces lilacinus ZJPL08 wettable powder and quality index determination

1. Preparation of paecilomyces lilacinus ZJPL08 wettable powder: the carriers, the wetting agent, the dispersant, the ultraviolet protective agent, the spore germination accelerant, the adhesive and the like screened by the tests are mixed according to a proportion and stirred uniformly, then the paecilomyces lilacinus conidium powder is added, and the mixture is sieved by a 325-mesh standard sieve and stirred uniformly to obtain the paecilomyces lilacinus conidium wettable powder.

The formula of the conidium wettable powder of the paecilomyces lilacinus ZJPL08 is as follows: the active ingredient is paecilomyces lilacinus conidium powder with the content of 10 percent; the wetting agent is sodium dodecyl sulfate, the content is 3%; the dispersant is sodium lignosulfonate with the content of 5 percent; the ultraviolet protective agent is humic acid, the content is 0.25 percent; the spore germination accelerant is calcium chloride, the content is 0.1%; the adhesive is xanthan gum with the content of 0.05%; the carrier is diatomite with a content of 81.6 percent.

2. Quality index determination of paecilomyces lilacinus ZJPL08 wettable powder:

(1) and (3) measuring the spore content: the procedure is as in example 1.

(2) And (3) determining the viable spore rate: the procedure is as in example 1.

(3) And (3) water content determination: the procedure is as in example 1.

(4) And (3) measuring the wetting time: the method is carried out according to GB/T5451-2001.

(5) And (3) suspension rate determination: according to GB/T14825-2006.

(6) Measurement of fineness: according to the "wet sieve method" of GB/T16150-.

(7) And (3) pH value measurement: the procedure was as described in GB/T1601 + 1993.

The results of measuring the quality index of the paecilomyces lilacinus ZJPL08 wettable powder are shown in Table 13, and it can be seen from Table 13 that the conidium wettable powder has a spore content of 2.98 × 10¹⁰The spore count per gram is 92.34%, the water content is 3.59%, the wetting time is 85.32 s, the spore suspension rate is 84.76%, the fineness is 96.52%, and the spore is sieved by a 325-mesh sieve. The performance index of the prepared wettable powder meets the requirement of pesticide preparation products.

TABLE 13 quality index of Paecilomyces lilacinus ZJPL08 wettable powder

Example 4: prevention effect determination of paecilomyces lilacinus ZJPL08 wettable powder

1. Prevention effect of strain ZJPL08 wettable powder on diaphorina citri

(1) Indoor toxicity determination, 1 g of wettable powder of the strain ZJPL08 is weighed and added into 298 mL of sterile water to be mixed evenly, and the spore concentration is 1.0 × 10⁸Conidium suspension of seed/mL, then diluted with sterile water gradient to concentration of 1.0 × 10⁷、1.0 × 10⁶、1.0 × 10⁵And 1.0 × 10⁴And (3) filling the spore suspension into a small sprayer, and then uniformly spraying the adult diaphorina citri. Picking the treated diaphorina citri to the in-vitro murraya paniculata leaves in a glass culture dish by using a writing brush, covering the leaves with a 60-mesh insect-proof net, putting the glass dish into an artificial climate box, and illuminating at the temperature of 28 ℃, the illumination L: D =14:10 and the relative humidity>Culturing under 90% of conditions. Each treatment was set to 3 replicates, each replicate 30 diaphorina citri, with sterile water treatment as a negative control. After culturing for different time, counting the number of diaphorina citri dead insects, and calculating the death rate according to the following formula: corrected mortality (%) = (point)Help mortality-control mortality)/(1-control mortality) × 100 Linear regression equation was generated using software SAS v9.2 to calculate the lethal middle concentration (LC)₅₀) And median time to death (LT)₅₀）。

The pathogenic effects of the wettable powder of strain ZJPL08 on diaphorina citri under indoor conditions are shown in tables 14 and 15. As can be seen, the wettable powder of the strain ZJPL08 has strong pathogenicity on the diaphorina citri, and the higher the spore concentration is, the better the pathogenicity effect on the diaphorina citri is. The longer the culture time after inoculation is, the higher the mortality rate of diaphorina citri is under the same concentration condition. 3 d after treatment, LC thereof₅₀Is 9.67 × 10⁷one/mL, and 7 d after treatment, LC thereof₅₀Is only 1.39 × 10⁴one/mL. The higher the conidia concentration, the more LT against Pediculus citri₅₀The shorter the concentration is, the 1.0 × 10⁴LT of wettable powder per mL for diaphorina citri₅₀Is 6.17 d and the concentration is 1.0 × 10⁸LT of wettable powder per mL for diaphorina citri₅₀Only 2.64 d.

TABLE 14 lethal middle concentration (LC) of paecilomyces lilacinus ZJPL08 wettable powder against diaphorina citri₅₀）

TABLE 15 median lethal time of wettable powder of Paecilomyces lilacinus ZJPL08 on diaphorina citri (LT)₅₀）

(2) The field control effect determination is that a proper amount of wettable powder of the strain ZJPL08 is weighed and added into clear water according to the proportion of 1:1500 to prepare the spore with the concentration of 1.99 × 10⁷Adding a proper amount of pure spore powder of strain ZJPL08 into clear water to prepare into liquid medicine with concentration of 1.99 × 10⁷Bacterial strain ZJPL08 pure spore suspension per ml, adding 10% imidacloprid wettable powder into clear water at a ratio of 1:2000 to prepare liquid medicine, and adding 30% nitenpyram soluble liquid at a ratio of 1:4500For example, clear water is added to prepare a medicinal solution. The liquid medicine is put into a sprayer to uniformly spray the diaphorina citri on the murraya paniculata plants raised in the insect rearing cages respectively, only clear water is sprayed as a control, 5 times of treatment are set for each treatment, 60 times of diaphorina citri imagoes are repeated for each treatment, and 3 times of experiments are repeated. And (3) feeding the treated diaphorina citri under natural conditions (average temperature is 25 ℃ and relative humidity is 71%), observing and recording the number of diaphorina citri dead insects at different times, and calculating the death rate according to the formula. And (3) carrying out difference significance test on the processed data by using a Duncan's new repolarization method of SAS v9.2 software.

The results of the field control effect measurement are shown in Table 16. The result shows that the strain ZJPL08 wettable powder has good control effect on diaphorina citri under natural conditions. 3 d after treatment, the fatality rate of the strain ZJPL08 wettable powder to the diaphorina citri is 42.83%, the control effect of the strain ZJPL08 wettable powder to the diaphorina citri is lower than that of 10% imidacloprid wettable powder, but the control effect of the strain ZJPL08 wettable powder to the diaphorina citri is equivalent to that of 30% nitenpyram soluble solution, and the strain ZJPL08 pure spore suspension is obviously higher than that of the strain. The diaphorina citri mortality rate of each treatment increased with time. The mortality rate of the diaphorina citri treated by the strain ZJPL08 wettable powder is remarkably increased until 14 days after treatment, the mortality rate of the diaphorina citri treated by the strain ZJPL08 wettable powder to the diaphorina citri reaches 100 percent, the rate is remarkably higher than that of two chemical insecticides such as 10 percent imidacloprid wettable powder and 30 percent nitenpyram soluble solution, and the rate is higher than that of a pure spore suspension of the strain ZJPL 08. Therefore, the formulation of the paecilomyces lilacinus ZJPL08 is beneficial to the full exertion of the efficacy of conidium and improves the control effect of the bacterial strain on the diaphorina citri. In terms of quick-acting property, the wettable powder of the strain ZJPL08 is lower than that of 10% imidacloprid wettable powder, but the effect is stronger, and new conidia are generated on diaphorina citri bodies infected with the strain ZJPL08 to form a new infection cycle. Therefore, from the above results, it can be seen that the wettable powder of strain ZJPL08 can be used alone or in combination with other insecticides for the control of diaphorina citri.

TABLE 16 control of diaphorina citri by wettable powder of strain ZJPL08 under natural conditions

Note: the numbers in the same column followed by different lower case letters indicate significant differences at a level of P < 0.05.

2. Wettable powder of strain ZJPL08 for preventing panonychus citri and cotton aphid

Collecting citrus branches with panonychus citri or cotton aphid in field orange garden, adding clear water into wettable powder of strain ZJPL08 at a ratio of 1:1500 to obtain spore with concentration of 1.99 × 10⁷Respectively and uniformly spraying panonychus citri and cotton aphid after each/mL suspension, inserting the treated citrus branches into a water-containing plastic pipe with the caliber of about 1 cm, plugging the pipe orifice with cotton, putting the citrus branches into a large plastic bottle with an insect-proof net on the top, then placing the plastic bottle into a climatic chamber, and irradiating at the temperature of 28 ℃, the illumination L =14:10 and the relative humidity>Culturing under 90% of conditions. Each treatment was set to 6 replicates and sterile water treatment was used as a negative control. After culturing for different time, observing and counting the dead number of panonychus citri and cotton aphid under a stereoscopic microscope, and calculating and correcting the death rate.

The control effect of the strain ZJPL08 wettable powder on panonychus citri and cotton aphid is shown in Table 17. As can be seen, the wettable powder of the strain ZJPL08 has better control effect on panonychus citri and cotton aphid. At 3 d post-treatment, the corrected mortality rate for panonychus citri was 66.67%, whereas that for Aphis gossypii was higher, 78.26%. At 7 days after treatment, the corrected mortality rates of panonychus citri and cotton aphid can reach 95.65% and 100.00% respectively. The wettable powder of the strain ZJPL08 can be used for preventing and controlling panonychus citri and cotton aphid.

TABLE 17 wettable powder of ZJPL08 strain for controlling panonychus citri and Aphis gossypii

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

Claims

1. The paecilomyces lilacinus wettable powder is characterized by comprising the following components in percentage by mass: 10% of paecilomyces lilacinus conidium pure spore powder, 3% of wetting agent, 5% of dispersing agent and 0.25% of ultraviolet protective agent; 0.1% of spore germination accelerant; 0.05% of adhesive; and 81.6% of carrier.

2. The wettable powder of claim 1, wherein the paecilomyces lilacinus is paecilomyces lilacinus (Paecilomyces lilacinus)) (Paecilomyces lilacinus) Strain ZJPL 08.

3. The paecilomyces lilacinus wettable powder according to claim 1, wherein the spore content of the pure spore powder of the conidia of the paecilomyces lilacinus is 2.96-3.69 × 10¹¹The number per gram, the viable spore rate is 98.31-99.43%, and the water content is 6.06-6.44%.

4. The paecilomyces lilacinus wettable powder according to claim 1, wherein the wetting agent is sodium dodecyl sulfate, the dispersing agent is sodium lignosulfonate, the ultraviolet protective agent is humic acid, the spore germination promoter is calcium chloride, the adhesive is xanthan gum, and the carrier is diatomite.

5. Use of a wettable powder of paecilomyces lilacinus as claimed in any one of claims 1 to 4 for the control of diaphorina citri, panonychus citri and aphis gossypii.