CN110591928A - Beauveria bassiana and application thereof - Google Patents
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Abstract
The invention discloses Beauveria bassiana, and relates to the technical field of biological control of pests, wherein the strain is Beauveria bassiana (Beauveria bassiana) Bb2032, the preservation number is CGMCC No.18118, and the preservation date is 2019, 8 months and 12 days. The invention also provides a method for promoting germination of beauveria bassiana Bb2032 spores, a tea aphid prevention and control microbial inoculum, a finger preparation method of the tea aphid prevention and control microbial inoculum, and application of the beauveria bassiana in prevention and control of tea aphids. The invention has the beneficial effects that: the mortality rate of beauveria bassiana Bb2032 to adult tea aphids and nymphs 8d is more than 90%, and the 8d cumulative mortality rates of the adult tea aphids and the nymphs are respectively 100% and 96.23% at the highest.
Description
Technical Field
The invention relates to the technical field of biological control of pests, and particularly relates to beauveria bassiana and application thereof.
Background
The tea aphids are also called binary citrus aphids, are widely distributed in each tea production area in China, and also harm plants such as oil tea, citrus and the like. The insect is damaged in a piercing and sucking mode, has two forms of winged and wingless, has strong reproductive capacity and high growth speed, and is one of important pests in tea gardens. The tea aphids in China occur for 20-30 generations in the year, about 20 generations in Zhejiang, more than 25 generations in Anhui, about 30 generations in Fujian, 15-20 days in spring tea and 6-8 days in summer. Saliva secreted by tea aphids into tea tree bodies in the process of sucking host juice can destroy normal physiological metabolism of the tea trees, so that plant leaves curl and shrink, and growth of serious people is stopped; the excrement honeydew is easy to cause tea coal disease, so that the leaves of plants become black, the photosynthesis activity is reduced, the tree vigor is weakened, and the tea yield is reduced.
Patent CN201910029859.8 discloses a method for preventing and treating chrysanthemum aphids by using nitrogen fertilizer, which comprises the following steps: in the process of cultivating chrysanthemum in a greenhouse, when aphids occur, uniformly spraying chrysanthemum leaves by using a mixed solution of a urea 0.3% solution, an ammonium bicarbonate 0.5% solution and an ammonia water 0.3% solution, spraying once every 5 days, and continuously spraying for 3 times. The nitrogen fertilizer solution is used for treatment, so that the effect of preventing and treating the aphid damage of the whole chrysanthemum plant reaches more than 98 percent. The invention provides an effective method for preventing and treating aphid damage of chrysanthemum by using nitrogen fertilizer, and plants without aphid damage of chrysanthemum are obtained.
At present, the prevention and control technology for tea aphids mainly depends on chemical prevention, such as 3000 times liquid of 2000 times pirimicarb wettable powder, 1500 times liquid of 50% pirimiphos-methyl, 3000 times liquid of 40% dimethoate missible oil or 3000 times liquid of 10% beta-cypermethrin missible oil. The use of a large amount of pesticides can not only cause the tea aphids to generate drug resistance, but also pollute the environment and influence the quality of tea leaves, so that the exploration and development of safe and effective green prevention and control are urgently needed.
Disclosure of Invention
One of the technical problems to be solved by the invention is to provide beauveria bassiana with good prevention and treatment effect on tea aphids.
The invention solves the technical problems through the following technical means:
the strain is Beauveria bassiana Bb2032, the preservation unit is China general microbiological culture Collection center (CGMCC), the preservation number is CGMCC No.18118, and the preservation date is 2019, 8 months and 12 days.
Has the advantages that: the lethality of beauveria bassiana Bb2032 to adult tea aphids and 8d to nymph aphids reaches over 90 percent, and the 8d cumulative mortality of the adult tea aphids and the 8d to the nymph aphids is respectively 100 percent and 96.23 percent at the highest.
The second technical problem to be solved by the invention is to provide a method for promoting the germination of beauveria bassiana Bb2032 spores.
The invention solves the technical problems through the following technical means:
a method for promoting germination of beauveria bassiana Bb2032 spores comprises the following steps:
(1) preparing a beauveria bassiana Bb2032 spore suspension;
(2) adding nutrient substances into the suspension of Beauveria bassiana Bb2032 spores, and culturing at 22-28 deg.C for 12-24 h.
Has the advantages that: the nutrient substances can promote the germination of the spores, the germination rate of the spores is reduced along with the temperature rise at 28-35 ℃, the germination rate of the spores reaches 88.64% in 24h at 25 ℃, the optimal growth temperature of the beauveria bassiana Bb2032 is 22-28 ℃, and the main germination time of the spores is within 12h at the temperature.
Preferably, the nutrients comprise sugars, amino acids or insect powder.
Preferably, the saccharide is one of glucose, fructose, maltose, sucrose, trehalose and lactose.
Preferably, the amino acid is one of methionine, glycine, lysine, valine, leucine, isoleucine and histidine.
Preferably, the insect powder is galleria mellonella or yellow mealworm live insect freeze-dried powder.
Preferably, the preparation method of the beauveria bassiana Bb2032 spore suspension comprises the following steps:
(1) inoculating beauveria bassiana Bb2032 into a culture medium, culturing for 10d under the condition of 22-28 ℃ by illumination, carrying out sporulation, and shoveling the produced spore powder to obtain beauveria bassiana Bb2032 spores;
(2) and (2) taking a sterilized 0.05 wt% Tween 80 solution, putting the beauveria bassiana Bb2032 spores obtained in the step (1), and dispersing to obtain a beauveria bassiana Bb2032 spore suspension.
The invention aims to solve the technical problem of providing the tea aphid preventing and controlling microbial inoculum with good effect on preventing and controlling the tea aphids.
The invention solves the technical problems through the following technical means:
a tea aphid control agent comprising beauveria bassiana Bb2032 according to claim 1.
Has the advantages that: the mortality rate of beauveria bassiana Bb2032 to adult tea aphids and nymphs 8d is more than 90%, and the 8d cumulative mortality rates of the adult tea aphids and the nymphs are respectively 100% and 96.23% at the highest.
Preferably, the beauveria bassiana Bb2032 is beauveria bassiana Bb2032 spore suspension.
Preferably, the preparation method of the beauveria bassiana Bb2032 spore suspension comprises the following steps:
(1) inoculating beauveria bassiana Bb2032 into a culture medium, culturing for 10d under the condition of 22-28 ℃ by illumination, carrying out sporulation, and shoveling the produced spore powder to obtain beauveria bassiana Bb2032 spores;
(2) and (2) taking a sterilized 0.05 wt% Tween 80 solution, putting the beauveria bassiana Bb2032 spores obtained in the step (1), and dispersing to obtain a beauveria bassiana Bb2032 spore suspension.
Preferably, the culture medium is a SMAY culture medium, a PPDA culture medium or a PDA culture medium.
Preferably, the beauveria bassianaThe concentration of spore suspension of the bacterium Bb2032 is 1 × 106-1×108Spores/ml.
Preferably, the prepared beauveria bassiana Bb2032 spore suspension is subjected to pregermination, and the pregermination comprises the following steps: and (3) carrying out shaking culture on the prepared beauveria bassiana Bb2032 spore suspension for 4-12h on a shaking table.
Has the advantages that: the Beauveria bassiana Bb2032 spore pre-germination can improve the pathogenicity to the Aphis pyricularis, the 8d cumulative mortality rate of the pre-germination 8h reaches 78.57%, and the 8d cumulative mortality rate of the control group without the pre-germination (0h) is only 57.14%.
Preferably, the nutrient substance is added during the shaking culture.
Preferably, the light culture condition is 14L:10D photoperiod.
Preferably, the nutrients comprise sugars, amino acids or insect powder.
Preferably, the saccharide is one of glucose, fructose, maltose, sucrose, trehalose and lactose.
Preferably, the amino acid is one of methionine, glycine, lysine, valine, leucine, isoleucine and histidine.
Preferably, the insect powder is galleria mellonella or yellow mealworm live insect freeze-dried powder.
Has the advantages that: the addition of the tenebrio molitor powder reduces the pathogenic effect of the beauveria bassiana Bb2032 on the adult tea aphids, but the pathogenicity of the tea aphids is improved, and both methionine and glucose have a synergistic effect on the pathogenicity of the beauveria bassiana Bb 2032; the addition of six sugars under the concentration of 1% has a promoting effect on the germination of beauveria bassiana Bb2032 spores, the promoting effect of trehalose and glucose with the germination rate of 24h under the concentration of 0.1% is obvious, and seven amino acids basically have a promoting effect on the germination of Bb2032 spores; the two insect powder of yellow mealworm and greater wax moth have better promotion effect on the germination of Bb2032 spores.
Preferably, the pesticide also comprises plant source pesticide.
Preferably, the botanical pesticide is a matrine aqueous solution, a veratrine solvent solution or a rotenone microemulsion.
Preferably, the volume ratio of the plant-derived pesticide to the beauveria bassiana Bb2032 spore suspension is 1: 1.
Has the advantages that: after the plant-derived pesticide and the beauveria bassiana Bb2032 spore suspension are compounded, the toxicity to the tea aphids is higher than that of a single pesticide, the mixing effect of the rotenone and the beauveria bassiana Bb2032 is the best, and the lethality is relatively short.
The fourth technical problem to be solved by the invention is to provide a preparation method of the tea aphid preventing and treating microbial inoculum.
The invention solves the technical problems through the following technical means:
a preparation method of a tea aphid control microbial inoculum comprises the following steps:
(1) inoculating beauveria bassiana Bb2032 into a culture medium, culturing for 10d under the condition of 22-28 ℃ by illumination, carrying out sporulation, and shoveling the produced spore powder to obtain beauveria bassiana Bb2032 spores;
(2) and (2) taking a sterilized 0.05 wt% Tween 80 solution, adding the beauveria bassiana Bb2032 spores obtained in the step (1), and dispersing to obtain a beauveria bassiana Bb2032 spore suspension, namely the tea aphid control microbial inoculum.
Has the advantages that: the fatality rate of the tea aphid fungicide prepared by the method to the tea aphid imagoes and nymphs is more than 90%, and the 8d cumulative mortality rate of the imagoes and nymphs is 100% and 96.23% respectively.
Preferably, the culture medium is a SMAY culture medium, a PPDA culture medium or a PDA culture medium.
Preferably, the concentration of the beauveria bassiana Bb2032 spore suspension is 1 × 106-1×108Spores/ml.
Preferably, the method also comprises the following steps of pre-germinating the prepared beauveria bassiana Bb2032 spore suspension, wherein the pre-germination comprises the following steps: adding nutrient substances into the suspension of Beauveria bassiana Bb2032 spores, and culturing at 25 deg.C for 0-12h under illumination.
Preferably, the light culture condition is 14L:10D photoperiod.
Preferably, the nutrients comprise sugars, amino acids or insect powder.
Preferably, the saccharide is one of glucose, fructose, maltose, sucrose, trehalose and lactose.
Preferably, the amino acid is one of methionine, glycine, lysine, valine, leucine, isoleucine and histidine.
Preferably, the insect powder is galleria mellonella or yellow mealworm live insect freeze-dried powder.
Preferably, the pesticide also comprises a plant source pesticide, and the plant source pesticide is mixed with the beauveria bassiana Bb2032 spore suspension.
Preferably, the botanical pesticide is a matrine aqueous solution, a veratrine solvent solution or a rotenone microemulsion.
Preferably, the volume ratio of the plant-derived pesticide to the beauveria bassiana Bb2032 spore suspension is 1: 1.
The fifth technical problem to be solved by the invention is to provide the application of beauveria bassiana in preventing and treating tea aphids.
The invention solves the technical problems through the following technical means:
the application of beauveria bassiana in preventing and treating tea aphids comprises the following steps: the tea aphid prevention and control microbial inoculum prepared by the preparation method is sprayed on tea aphids.
Has the advantages that: the mortality rate of beauveria bassiana Bb2032 to adult tea aphids and nymphs 8d is more than 90%, and the 8d cumulative mortality rates of the adult tea aphids and the nymphs are respectively 100% and 96.23% at the highest.
The invention has the advantages that:
(1) the lethality rate of beauveria bassiana Bb2032 to the adult tea aphids and nymphs for 8d is more than 90%, and the 8d cumulative mortality rates of the adult tea aphids and the nymphs for 100% and 96.23% respectively are the highest;
(2) the pathogenic force of beauveria bassiana Bb2032 spores can be improved by pre-germination, the 8d cumulative mortality of 8h of pre-germination reaches 78.57%, and the 8d cumulative mortality of a control group without pre-germination (0h) is only 57.14%;
(3) the addition of the nutrient substances has a synergistic effect on the pathogenicity of beauveria bassiana Bb 2032;
(4) after the plant source pesticide and the beauveria bassiana Bb2032 spore suspension are compounded, the toxicity to the tea aphids is higher than that of a single pesticide, and meanwhile, the mixed preparation of the beauveria bassiana Bb2032 and the rotenone sub-lethal concentration (multiplied by 10) is used for preventing and treating the tea aphids, so that the using amount of the pesticide is reduced.
Drawings
FIG. 1 is a graph showing the results of measuring the pathogenicity of adult aphids by Beauveria bassiana Bb2032 with different concentrations in example 1 of the present invention;
FIG. 2 is a graph showing the results of measuring the pathogenicity of Mycoplasma bassiana Bb2032 on Aphis pyricularis in different concentrations in example 1 of the present invention;
FIG. 3 is a morphogram of the early stage of Aphis citricola infection in example 1 of the present invention;
FIG. 4 is a morphogram of the middle stage of the infection of Aphis theophylla in example 1 of the present invention;
FIG. 5 is a morphogram of the late stage of Physalis aurantiaca infection in example 1 of the present invention;
FIG. 6 is a graph of the dynamic results of the pre-germination time of beauveria bassiana Bb2032 spores versus the cumulative mortality of aphids in example 4 of the present invention;
FIG. 7 is a graph showing the effect of different saccharides on spore germination of Beauveria bassiana Bb2032 in example 4 of the present invention;
FIG. 8 is a graph showing the effect of different amino acids on the germination of Beauveria bassiana Bb2032 spores in example 4 of the present invention;
FIG. 9 is a graph showing the effect of different insect powders on the germination of beauveria bassiana Bb2032 spores in example 4 of the present invention;
FIG. 10 is a graph of the cumulative mortality dynamic results of aphid infested by spores germinated for 4 hours with different nutrients added in example 4 of the present invention;
FIG. 11 is a graph of the cumulative mortality dynamic results of 4h of aphid infestation by spore germination with different nutrients added in example 4 of the present invention;
FIG. 12 is a graph of the cumulative mortality dynamic results of 12h infested aphids after spore germination with different nutrients added in example 4 of the present invention;
beauveria bassiana (Beauveria bassiana) Bb2032, the preservation unit is China general microbiological culture Collection center, the preservation number is CGMCC No.18118, and the preservation date is 2019, 8 months and 12 days.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Test materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The specific techniques or conditions not specified in the examples can be performed according to the techniques or conditions described in the literature in the field or according to the product specification.
0.5% veratrine soluble solution is purchased from Jianhua plant pesticide factory in Hebei Handan city;
6% rotenone microemulsion was purchased from Beijing Sanpu Baicao Green plant preparations, Inc.;
0.3% matrine aqua is purchased from Shanxi Dewei Biochemical Limited liability company;
beauveria bassiana (Beauveria bassiana) Bb2032 with the preservation number of CGMCC No.18118, the preservation date of 2019, 8 months and 12 days, and the preservation place is the common microorganism center of China Committee for culture Collection of microorganisms.
The tested tea aphids are collected from tea trees without pesticide application in a combined fertilizer high and new technology agricultural garden, the tea aphids are cut off with branches and are taken back to be identified, then the tea aphids are raised on indoor (25 +/-1 ℃) fresh tea branches subjected to water culture, the wingless aphids are selected every two days after being raised for several generations and transferred to the fresh insect-free tea branches under the same culture conditions, and the tea aphids are continuously raised for bioassay.
Example 1
Screening of pathogenic Strain
(1) Selecting Beauveria bassiana separated from aphids and hemiptera insects and provided by the research center of microbial epiphyte of agricultural university of Anhui for toxicity screening; the basic information of the strains is shown in a table 1;
table 1 shows the source and collection area of Beauveria bassiana
(2) Preparation of beauveria bassiana spore suspension: inoculating the strain on the prepared SDAY culture medium, putting the strain into an artificial intelligent illumination incubator (photoperiod 14L: 10D) at 25 ℃ for culturing for 10D, and waiting for full spore production. Preparing a 50ml conical flask, containing 20ml of TW-80 solution with the concentration of 0.05%, sterilizing, cooling, scraping spore powder on a culture medium into the conical flask by using a spore shovel, shaking for 15min on a vortex magnetic oscillator at the frequency of 150r/min to uniformly disperse the spores, filtering by using a sterilized cotton needle tube filled with degreased cotton to obtain spore suspension, counting the number of the spores by using a blood cell counting plate under a microscope, calculating the concentration of the spore suspension, and continuously using the 0.05% TW-80 (Tween 80) solution to dilute according to proportion to obtain the spore suspensions with different concentrations according to requirements.
(3) Primary screening: inoculating the bred robust wingless adult aphid on a sterile culture dish with a wet soft capillary pen, and uniformly spraying 2ml of 1 × 10 concentration on the culture dish with a Potter spray tower under 6-7psi7Conidium suspension of different species of beauveria bassiana of spore/ml, taking out the culture dish after spraying, transferring the tea aphids to dry filter paper until the insect body is sprayed and dried, transferring the treated tea aphids to an aseptic raising box with a fresh tea tip (the tea tip takes a part with buds and 3-4 tender leaves at the front end of a tea branch, the lower part is wrapped by absorbent cotton soaked in sterile water for moisture preservation), placing the raising box in an artificial intelligent climate box with the relative humidity of 70% at 25 ℃ after marking, adhering the wall with the soaked sterile filter paper in the front of the raising box for 24h to ensure that the humidity is more than 95%, and taking out the filter paper in the later period to ensure that the humidity in the box is consistent with that in the climate box. The tea tips are replaced once every 2d, newly born aphids on the tea tips are removed every 24h, the disease condition of the newly born aphids is observed, death is judged according to the non-reflective behaviors of stiff bodies, light touch of the lower abdomen of the bodies, leg parts and the like of the insects by a writing brush, and the time of 7d is continuously recorded. The spore suspension of different strains with the same concentration is respectively one treatment, each treatment is provided with 3 repetitions, and each repetitionMore than 20 test insects are reused, and sterile water sprayed by a spraying tower is used as a control group. And (4) placing the dead test insects in a culture dish padded with wet filter paper for continuous culture, and confirming that the insects covered by the hyphae are infected and dead by the beauveria bassiana through microscopic examination. Counting the death number and the stiff worm number of each group of beauveria bassiana after treatment, and performing further experiments;
(4) re-screening: preparation of 1X 106、1×107、1×108The pathogenicity of tea aphids is measured by each strain of beauveria bassiana spore suspension with three concentration gradients, a second-instar nymph experiment group is added on the basis of a wingless adult experiment, adults and nymphs are treated respectively, each treatment is repeated for 3 times, more than 20 test insects are used repeatedly, sterile water is sprayed by a spray tower as a control group, and inoculation, infection and statistics are carried out according to the operation of preliminary screening. Counting the death number and the stiff worm number of each group of beauveria bassiana after treatment, and calculating the death rate, the infection rate and the LC50、LT50(in case of death), the most pathogenic strains were selected by comprehensive evaluation.
(II) results of the experiment
(1) Screening a strain Beauveria bassiana (Beauveria bassiana) Bb2032 with high pathogenicity to tea aphids, wherein the lethal result is shown in Table 2;
table 2 shows the lethal effect of Beauveria bassiana Bb2032 on tea aphids (7d)
(2) The results of determining the virulence of tea aphids by Beauveria bassiana Bb2032 (Beauveria bassiana) at different concentrations are shown in FIG. 1, and the virulence of adult aphids is 1X 106At spore/ml concentration, the difference between treatments in the inoculated strain 3d is not obvious, and the Beauveria bassiana Bb2032 treatment is started at the 4 th dRapid increase in group mortality, 1X 108The death peak appears at the 4 th d under the concentration of spores/ml, wherein the cumulative mortality of the beauveria bassiana Bb2032 at the 8 th d can reach 100 percent;
as shown in FIG. 2, the number of aphids is 1X 106At spore/ml concentration, the 8d cumulative mortality was 66%, at 1X 107Under the concentration of spores/ml, the cumulative mortality rate after 4d rapidly increases, and the death peak appears at 6d, wherein the cumulative mortality rate of Bb2032 at 8d reaches 91%; 1X 108The cumulative mortality rate of each group treated under the concentration of spores/ml is increased and accelerated within 4-7 days.
(3) LC of beauveria bassiana Bb2032 on tea aphids50The measurement results are shown in table 3;
table 3 shows LC of different concentrations of beauveria bassiana Bb2032 for treating tea aphids50
As can be seen from table 3, the concentration of spore suspension required for green tea aphids to reach half-death decreases gradually as the number of days of treatment increases, if the aphids are lethal to a higher concentration than the adults. The lethal middle concentration of 8d adult aphids is 2.102X 104Spore/ml, aphid 5.813X 105Spores/ml.
(4) As shown in fig. 3, 4 and 5, fig. 3 is a morphological diagram of the early stage of tea aphid infection, fig. 4 is a morphological diagram of the middle stage of tea aphid infection, and fig. 5 is a morphological diagram of the later stage of tea aphid infection, and it can be seen that the tea aphid is gradually covered by hypha.
Example 2
Influence of the Medium on the spore production of Beauveria bassiana Bb2032
(1) Sucking 100ul of liquid with a 0-100ul adjustable pipette with a concentration of 1X 106Inoculating spore suspension of highly pathogenic strain with spore/ml onto each dish, uniformly coating spore suspension with a coating rod, standing for 2min, sealing with a sealing film, culturing in an artificial intelligence illumination incubator (light period 14L: 10D) at 25 deg.C for 10D, repeating five groups for each culture medium, and drilling five holes in each dish with a 4mm diameter hole puncher, with the punching positions being intermediate and intermediate to the dishPlacing the taken-down bacteria and culture medium into a conical flask containing 30ml of 0.05% TW-80 solution at 1/2 positions of edge connecting lines in four directions, oscillating for 15min on a vortex magnetic oscillator at the frequency of 150 r.min, filtering by using a sterilized cotton needle tube filled with degreased cotton to obtain spore suspension, and counting the number of spores by using a blood cell counting plate under a microscope; the different media types and compositions are shown in table 4;
table 4 shows the types and the main components of the culture media
(II) experimental results: the results of the experiment are shown in table 5;
table 5 shows the sporulation yield of Beauveria bassiana Bb2032 on different media
Note: upper and lower case letters in the table indicate significant differences at the 0.01 and 0.05 levels for each group, respectively.
As can be seen from Table 5, the strain produced spores on PDA best, reaching 2.36X 107/cm2The strains grown on SDA produced the least spores.
Example 3
(I) influence of culture temperature on spore germination of Beauveria bassiana Bb2032
(1) Preparing Water Agar (WA) culture medium (20g agar is dissolved in 1000ml water, sterilized at 120 deg.C for 20min, and cooled to about 50 deg.C, and adding 2ml agar into disposable sterile culture dish (D ═ 40mm) for completely cooling);
(2) sucking 1ul of the liquid with a concentration of 1 × 10 with an adjustable pipette of 0.2-2ul6Inoculating spore suspension of highly pathogenic strain with spore/ml into culture dish, standing for 2min, sealing with parafilm, and respectively placing at 18 deg.C, 22 deg.C, 25 deg.C, 28 deg.C,In an artificial intelligent constant-temperature incubator at the temperature of 30 ℃ and the temperature of 35 ℃, each temperature is one treatment, and each treatment is repeated by four groups;
(3) at the time of culturing for 12h and 24h, the spore germination rate of 2 replicates was counted by microscopic examination for each treatment. When the inverted microscope is used for microscopic examination, the sealing film on the culture dish is taken down, the culture dish is cut to be even along the edge of agar, a small amount of trypan blue light dots are dipped in the middle of the culture dish by using a rubber-tipped dropper, the culture dish is turned over on an objective table after being slightly air-dried, the position is adjusted, and the spore germination condition is observed. Taking 1/2 with the length of the visible germ tube of the spore being larger than the diameter of the spore as a germination standard, randomly counting all spores under 3 visual fields in each repeating group, wherein the number of the spores in each visual field is not less than 80, and calculating the germination rate;
(II) experimental results: the results of the experiment are shown in table 6;
table 6 shows the effect of different temperatures on the germination of Beauveria bassiana Bb2032 spores
Upper and lower case letters in the table indicate significant differences at the 0.01 and 0.05 levels for each group, respectively.
As can be seen from Table 6, the germination rate of the spores increases with the increase of the temperature between 18 ℃ and 25 ℃, the germination rate of the spores decreases with the increase of the temperature between 28 ℃ and 35 ℃, the germination rate of the spores reaches 88.64% after 24h at 25 ℃, the spores still partially germinate at 30 ℃, but the germination rate is only 34.60% after 24 h. Comparing the germination rates of 12h and 24h, the germination amount of the spores can reach more than 70% of that of the spores in 24h at 12h, the germination rate of the spores is still greatly improved after 12h at 18 ℃, and the germination rate of the spores is increased within 10% after 12h at 22-35 ℃. Thus, the optimum growth temperature of beauveria bassiana Bb2032 is 22-28 ℃, and the main germination time of spores at the temperature is within 12 h.
Example 4
Influence of pregermination of Beauveria bassiana Bb2032 spores on pathogenicity of Aphis theophylla
(1) Diluting Bb2032 strain 5 × 10 with 0.1% yellow mealworm powder filtrate8Spore/ml spore suspension to 1X 107Spore/ml, 1X 10 of the prepared7Putting the pure spore suspension of spore/ml into a constant temperature shaking incubator at 25 ℃ for shaking culture, and recording the time as 0 h;
(2) spraying 2ml of diluted spore suspension to the wingless adult aphids and the second-instar nymphs respectively by using a Potter spray tower, and continuously culturing the residual spore suspension; after 4h (4h), spraying for the second time, and spraying for the third time and the fourth time again after 6h and 8 h;
(3) taking the statistical data of 0h as a control group for each spore suspension, performing treatment for the rest of the spore suspensions by one time, setting 3 groups of repetition for each treatment of adult aphids and young aphids, and culturing a bioassay box in an artificial intelligent climate box at 25 ℃ after each spraying, wherein the culture conditions are the same as those in example 1;
(4) observing spore suspensions of 0h, 4h, 6h and 8h by using a blood cell counting plate, counting the spore germination rate at the spraying time, and determining the method in the same way as the example 3;
(II) influence of nutrient substances on spore germination rate
(1) Respectively preparing 6 kinds of saccharides (glucose, fructose, maltose, sucrose, trehalose and lactose), 7 kinds of amino acids (methionine, glycine, lysine, valine, leucine, isoleucine and histidine) and 2 kinds of insect powder (prepared by freeze-drying and grinding galleria mellonella and tenebrio molitor) into 0.1% and 1% aqueous solutions, and filtering and sterilizing by using a filter membrane for later use;
(2) prepared at a concentration of 5X 108Spore/ml of highly pathogenic beauveria bassiana spore suspension, diluting the spore suspension to 1 × 10 with 0.1% and 1% of the above nutrient solutions6Spores/ml for use. Sucking 1ul of diluted spore suspension with 0.2-2ul of adjustable pipette, dibbling into the middle of Water Agar (WA) culture dish, standing for 2min, marking with parafilm seal, culturing in 25 deg.C artificial intelligent illumination incubator under the same conditions as in example 1, treating each solution for one treatment, repeating each treatment for four times, and determining spore germination rate3；
Influence of nutrient substances added in pregermination of beauveria bassiana Bb2032 spores on pathogenicity of tea aphids
(1) Selecting three nutrient substances of 0.1 percent of yellow meal worm, 1 percent of glucose and 1 percent of methionine from the determination result in the step (II) to carry out a pathogenicity experiment of spore germination gain on the tea aphids;
(2) dilution 5X 108Spore/ml spore suspension to 1X 107And (3) putting the spores/ml into a constant-temperature shaking incubator at 25 ℃ for shaking culture, and prolonging the pre-germination time of the spores to 12 h. Taking out a proper amount of spore suspension after culturing for 4h (4h), respectively spraying 2ml of wingless adult aphids and second-instar nymphs by using a Potter spray tower, continuously culturing the residual spore suspension, respectively spraying for the second time and the third time after 8h and 12h, and putting a bioassay box into an artificial intelligent climate box at 25 ℃ after each spraying;
(3) each spore suspension was treated one at a time, 3 replicates were set for each adult and young aphid, and the control group was sprayed with a spore suspension diluted with a 0.05% strength TW-80 solution, under time conditions consistent with the other groups.
(IV) results of the experiment
(1) The pathogenicity of each treatment to adult and several aphids is shown in table 7;
table 7 shows the germination rates of spores at different pregermination times
As shown in FIG. 6, for aphid virulence, the cumulative mortality within 8d increased with the prolongation of the pre-germination time of the spores, and the cumulative mortality of 8d increased upon inoculation, wherein the cumulative mortality of 8d for the pre-germination 8h reached 78.57%, while the cumulative mortality of 8d for the control group without pre-germination (0h) was only 57.14%.
(2) Effect of nutrients on spore germination Rate
The effect of sugars, amino acids, and 3-type nutrients on spore germination of beauveria bassiana Bb2032 is shown in FIG. 7, FIG. 8, and FIG. 9. Wherein, the addition of six saccharides under the concentration of 1% has the promotion effect on the germination of beauveria bassiana Bb2032 spores, seven amino acids basically have the promotion effect on the germination of the beauveria bassiana Bb2032 spores, the promotion effect of methionine and isoleucine is obvious under the addition concentration of 1%, and the germination rate of the spores for 12 hours is higher than 95%; under the condition of 0.1 percent of addition concentration, the spore germination rate of methionine, glycine, lysine and leucine for 24 hours can reach more than 95 percent, which is obviously higher than 87.72 percent of that of a control group (CK). The two insect powder of yellow mealworm and greater wax moth have better promotion effect on the germination of Bb2032 spores, the germination rates of the spores in 24 hours of the treatment group with 1% addition concentration are respectively 99.36% and 97.90, and the influence is obvious.
(3) Effect of different pregermination Nutrients on the pathogenicity of Bb2032
As shown in fig. 10, 11 and 12, the addition of the 0.1% tenebrio molitor powder increased the virulence to aphid, increasing from 57.14% to 72.73% of the control treatment. The pathogenicity of beauveria bassiana Bb2032 is synergized by adding 1% of methionine and 1% of glucose, the increase of the cumulative mortality of the test insects between 3d and 6d of the inoculated bacteria is accelerated, the cumulative mortality of the adult aphids and the 8d of the adult aphids are higher than that of the control treatment, and the difference of the experimental results of the pre-germination of the spores for 4h, 8h and 12h is not large.
Example 5
Pathogenic force of plant pesticide on tea aphid
(1) Toxicity of single plant source pesticide on tea aphids
Three plant-derived pesticides selected in the experiment are prepared into conventional use concentrations by using sterile water, then are respectively diluted by 5 times (sub-lethal concentration) and 10 times (sub-sublethal concentration), and are filtered and sterilized for later use, wherein the specific pesticide preparation conditions are shown in table 8. Healthy wingless adults and second-instar nymphs are selected as test insects, 2ml of prepared plant source pesticides with various dilution concentrations are sprayed on all the groups of insects respectively through a Potter spray tower, the sprayed test insects are transferred to a feeding box, the death condition is observed and recorded, and the specific spraying, feeding and statistical methods are the same as the screening operation in the example 1. One treatment is carried out on the liquid medicine with each concentration, each adult and nymph treatment is carried out repeatedly, and 2ml of sterile water is sprayed on a spraying tower to serve as a control.
Table 8 shows dilution factor of test botanical pesticide
(2) Compatibility of beauveria bassiana Bb2032 and three botanical pesticides
Nine kinds of liquid medicines, including three kinds of plant-derived pesticides shown in Table 8, including common concentration, sub-lethal concentration (x 5), and sub-lethal concentration (x 10), were used at a volume ratio of 1:1 to 1 of beauveria bassiana (balsamo) Vuillemin with high pathogenicity7Uniformly mixing spore suspension with spore/ml concentration, accurately sucking 5ul of mixed solution with 0-10ul of pipette, spot-planting on WA culture medium (D is 40mm), uniformly coating with sterilized triangular rod, sealing, culturing in 25 deg.C artificial intelligent constant temperature incubator, and performing microscopic examination for 12h and 24h to count germination rate. The mixed preparation with different concentrations of each pesticide is one treatment, each treatment is repeated in four groups, and the mixed preparation added with sterile water according to the volume ratio of 1:1 is used as a control group.
(3) Beauveria bassiana Bb2032 and plant pesticide are mixed to match toxicity of tea aphids
Will be 1 × 107Uniformly mixing spore/ml high-pathogenicity beauveria bassiana spore suspension and prepared plant source pesticides with various dilution concentrations according to the volume ratio of 1:1 to obtain a test liquid medicine, selecting healthy wingless adults and second-instar nymphs as test insect bodies, carrying out other treatment methods in the same step (1), and observing and recording the death condition.
(II) results of the experiment
(1) Toxicity of single plant source pesticide on tea aphids
The toxicity results of different botanical pesticides on tea aphids at three concentrations are shown in table 9. From table 9, with the increase of the dilution factor, the toxicity of the three botanical pesticides to the green aphids is reduced, wherein the toxicity of veratrine and rotenone to the green aphids is higher than that of the adult aphids.
Table 9 shows the toxicity of three botanical pesticides on tea aphids
(2) Influence of three plant-derived pesticides on Bb2032 spore germination
The effect of three plant-derived pesticides on spore germination of beauveria bassiana Bb2032 is shown in table 10. As can be seen from Table 10, under the conventional use concentration, matrine and veratrine have certain promotion effects on the germination of beauveria bassiana Bb2032 spores, wherein the matrine promotion effect is most obvious, the spore germination inhibition rate in 24h is-6.1%, and the veratrine with better compatibility with the germination of the beauveria bassiana Bb2032 spores is veratrine.
TABLE 10 Effect of three botanical pesticides on spore germination of Beauveria bassiana Bb2032
(3) Plant source pesticide and beauveria bassiana Bb2032 are mixed to match toxicity of tea aphids
The toxicity results of the three plant-derived pesticides and the beauveria bassiana Bb2032 spore suspension mixture on the tea aphids are shown in Table 11.
Table 11 shows the toxicity of the plant pesticide and Bb2032 mixture on tea aphids
The different lower case letters in the table indicate significant differences at the 0.05 level for each group;
as can be seen from table 11, in the compounding of the bacterial agents, almost all the mixed agents have higher toxicity to tea aphids than the single pesticide, and the sub-lethal concentration (x 10) of rotenone and beauveria bassiana Bb2032 have the best mixing effect, and the mixing effect is relatively short in killing, so that the mixed agents of the beauveria bassiana Bb2032 and the sub-lethal concentration (x 10) of rotenone can be used for controlling the tea aphids, and the usage amount of the pesticide is reduced.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. A strain of beauveria bassiana is characterized in that: the strain is named as Beauveria bassiana (Beauveria bassiana) Bb2032, the preservation number is CGMCC No.18118, and the preservation date is 2019, 8 months and 12 days.
2. A method for promoting germination of beauveria bassiana Bb2032 spores is characterized in that: the method comprises the following steps:
(1) preparing a spore suspension of beauveria bassiana Bb2032 of claim 1;
(2) adding nutrient substances into the suspension of Beauveria bassiana Bb2032 spores, and culturing at 22-28 deg.C for 12-24 h.
3. The method for promoting germination of beauveria bassiana Bb2032 spores as claimed in claim 2, wherein: the nutrient substances comprise saccharides, amino acids or insect powder.
4. The tea aphid preventing and controlling microbial inoculum is characterized in that: comprises beauveria bassiana Bb2032 as defined in claim 1.
5. The agent for controlling tea aphids according to claim 4, which is characterized in that: the beauveria bassiana Bb2032 is beauveria bassiana Bb2032 spore suspension.
6. The agent for controlling tea aphids according to claim 4, which is characterized in that: also comprises botanical pesticide.
7. The agent for controlling tea aphids according to claim 5, which is characterized in that: the preparation method of the beauveria bassiana Bb2032 spore suspension comprises the following steps:
(1) inoculating beauveria bassiana Bb2032 into a culture medium, culturing for 10d under the condition of 22-28 ℃ by illumination, carrying out sporulation, and shoveling the produced spore powder to obtain beauveria bassiana Bb2032 spores;
(2) and (2) taking a sterilized 0.05 wt% Tween 80 solution, putting the beauveria bassiana Bb2032 spores obtained in the step (1), and dispersing to obtain a beauveria bassiana Bb2032 spore suspension.
8. A preparation method of a tea aphid control microbial inoculum is characterized by comprising the following steps: the method comprises the following steps:
(1) inoculating the beauveria bassiana Bb2032 of claim 1 into a culture medium, culturing for 10 days at 22-28 ℃ under illumination, carrying out sporulation, and shoveling the produced spore powder to obtain beauveria bassiana Bb2032 spores;
(2) and (2) taking a sterilized 0.05 wt% Tween 80 solution, adding the beauveria bassiana Bb2032 spores obtained in the step (1), and dispersing to obtain a beauveria bassiana Bb2032 spore suspension, namely the tea aphid control microbial inoculum.
9. The preparation method of the agent for controlling tea aphids according to claim 8, which is characterized by comprising the following steps: further comprising the steps of: the preparation method comprises the following steps of (1) carrying out pregermination on the prepared beauveria bassiana Bb2032 spore suspension, wherein the pregermination comprises the following steps: adding nutrient substances into the suspension of Beauveria bassiana Bb2032 spores, and culturing at 25 deg.C for 0-12h under illumination.
10. Use of beauveria bassiana according to claim 1 for controlling tea aphids.
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