CN111424028A - Preparation method of metarhizium strain by utilizing ARTP and ultraviolet composite mutagenesis - Google Patents
Preparation method of metarhizium strain by utilizing ARTP and ultraviolet composite mutagenesis Download PDFInfo
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Abstract
The invention discloses a preparation method of a Metarrhizium anisopliae strain by utilizing ARTP and ultraviolet composite mutagenesis, which relates to the technical field of fungi, in particular to a preparation method of a Metarrhizium anisopliae strain by utilizing ARTP and ultraviolet composite mutagenesis. According to the preparation method of the Metarrhizium anisopliae strain by utilizing ARTP and ultraviolet composite mutagenesis, normal-pressure room-temperature plasma and ultraviolet composite mutagenesis are combined, a good strain AU34 which is full in growth, fast in spore production, strong in ultraviolet resistance and strong in toxicity is successfully screened, the spore production amount of the good strain AU34 is increased by 1.6 times compared with that of an original strain, after the spore production amount of six times of passage is detected, the mutagenized strain has good genetic stability, a novel research method is provided for obtaining a high-performance Metarrhizium anisopliae strain, and the application capability of the Metarrhizium anisopliae is improved.
Description
Technical Field
The invention relates to the technical field of fungi, in particular to a preparation method of a metarhizium strain by utilizing ARTP and ultraviolet composite mutagenesis.
Background
Metarhizium anisopliae (Metarhizium anisopliae) is a broad-spectrum entomopathogenic fungus, has the advantages of no environmental pollution, no residue, no drug resistance of pests, no harm to human and livestock and the like, and is considered to be an environment-friendly pesticide. The pathogenesis of the metarhizium anisopliae is that conidia of the metarhizium anisopliae are adhered to the body surface of an insect, nutrient substances such as amino acid, protein and the like are utilized for germination, protease, chitinase and the like are secreted to penetrate through the epidermis of the insect, and the insect is propagated in a large amount in the insect body to generate toxin, so that the insect is killed. But adverse environmental conditions can affect the exertion of microbial agents, and ultraviolet light, humidity and temperature are all factors affecting the germination and growth of metarhizium anisopliae conidia. In field experiments, the ultraviolet rays and the high temperature reduce the survival rate of spores, greatly reduce the toxicity and block the action of conidia of metarhizium anisopliae. If the spore yield, the ultraviolet resistance and other performances of the metarhizium anisopliae can be improved, the vitality, the germination capacity and the toxicity of conidia can be kept, and the application capacity of the metarhizium anisopliae can be improved.
An atmospheric room temperature plasma mutagenesis system (ARTP), an emerging mutagenesis technology, is capable of generating plasma jets with high active particle concentrations, causing death or mutation of organisms, thereby causing random mutation of genes. The ARTP mutagenesis technology is successfully applied to hundreds of microorganisms such as bacteria, fungi and the like, and has the advantages of simple and convenient operation, safety, no toxicity, low cost, wide mutation spectrum and high mutation rate.
Ultraviolet mutagenesis is a method which is used earliest, has the longest use time, is used most widely and has quite obvious use effect. Ultraviolet rays act on DNA molecules to form pyrimidine dimers between adjacent pyrimidines in DNA strands, thereby preventing normal pairing between bases, and causing mutation or death of microorganisms.
In recent years, in order to obtain excellent strains of Metarrhizium anisopliae, most of the strains are obtained by ultraviolet mutagenesis. For example, ultraviolet ray and nitrous acid are used for compound mutagenesis to breed a storage-resistant mutagenic strain M105-32 with obviously improved spore trehalose content; a mutant strain which grows faster and has stronger ultraviolet tolerance and heat resistance is screened through ultraviolet mutagenesis. However, the report of using the ARTP mutagenesis technology is not available, and the ARTP mutagenesis technology and the ultraviolet mutagenesis technology are not combined to be used with the breeding of the metarhizium anisopliae strain, so that the preparation method of the high-performance metarhizium anisopliae strain is limited, and the development of the pesticide made of the metarhizium anisopliae is also limited.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a preparation method of a metarhizium anisopliae strain by utilizing ARTP and ultraviolet composite mutagenesis, and solves the problems in the background technology.
In order to achieve the purpose, the invention is realized by the following technical scheme: a preparation method of Metarrhizium anisopliae strain by utilizing ARTP and ultraviolet composite mutagenesis comprises the following steps:
s1, preparing spore suspension, namely, pouring a Tween80 solution 5m L on a fresh culture medium plate, washing spores of an original metarhizium anisopliae strain down, putting the washed spores into a 10m L centrifugal tube containing glass beads, uniformly oscillating, filtering by using four layers of sterilized mirror wiping paper, and diluting by corresponding times;
s2, ARTP mutagenesis, namely, putting a slide glass on an alcohol lamp outer flame for burning for 30S, putting the slide glass into a sterilized flat plate, cooling, uniformly coating 10 mu L bacterial liquid on the slide glass, opening an ARTP mutagenesis breeding instrument, starting an ultraviolet lamp for sterilization for 30min, fixing a 1.5m L EP tube filled with 990 mu L sterile water in a clamping groove of the ARTP mutagenesis instrument, transferring a sterilized culture dish filled with the slide glass into an ARTP operating chamber, sequentially putting the slide glass into the corresponding clamping groove by using sterile tweezers, and setting ARTP mutagenesis time;
s3, ARTP mutagenesis culture, after the ARTP mutagenesis of the step S2, shaking and eluting an EP tube with a slide glass, diluting by corresponding times, sucking 100 mu L, coating the tube in a culture medium plate, culturing in a dark place, and calculating the death rate of the ARTP mutagenesis;
s4, ultraviolet mutagenesis, namely, putting a magnetic stirrer into a clean bench, cleaning the clean bench and the magnetic stirrer by alcohol, fixing the distance between the clean bench and the magnetic stirrer to be 30cm, opening an ultraviolet lamp for sterilization for 20min, absorbing 5m of L spore suspension, adding the suspension into a sterilized flat plate with a rotor, opening the magnetic stirrer and a culture dish cover, irradiating by the ultraviolet lamp under dark conditions, setting ultraviolet mutagenesis time, and closing the ultraviolet lamp after irradiation is finished;
s5, ultraviolet mutagenesis culture, namely diluting the bacterial liquid with different irradiation times in the step S4 by a proper multiple, absorbing 100 mu L, coating the bacterial liquid into a culture medium plate, culturing in a dark place, and calculating the ultraviolet mutagenesis lethality;
s6, ARTP combined ultraviolet composite mutagenesis: subjecting the spore suspension to ARTP mutagenesis of step S2 and ultraviolet mutagenesis of S4 in sequence;
s7, ARTP combined ultraviolet composite mutation culture: diluting the spore suspension obtained in the step S6, coating the diluted spore suspension on a culture medium plate, repeating the step for 3 times, culturing in a dark place, and calculating the ARTP combined ultraviolet composite mutagenesis lethality rate;
s8, observing the growth conditions of the mutagenic strain in the S7 on a culture medium plate at different time, counting bacterial colonies on the culture medium plate, carrying out primary screening on the mutagenic strain, dibbling the bacterial colonies growing on the culture medium plate on a new culture medium plate, measuring and recording the growth diameter of the bacterial colonies on the 6 th day, and screening the mutagenic strain which can produce spores, has high growth speed of the bacterial colonies and large growth diameter after mutagenesis;
s9, re-screening: comprises screening the anti-ultraviolet capability and screening the toxicity,
the screening method for the ultraviolet resistance comprises the following steps: irradiating the primarily screened strains for 5min under an ultraviolet lamp, and selecting strains with high germination rate and strong ultraviolet resistance;
the virulence screening method comprises the following steps: carrying out a diamondback moth toxicity test on the original strain and the mutant strain obtained after screening the ultraviolet resistance, and selecting a strain with strong toxicity to the diamondback moth;
s10, determination of mutant strain stability: the obtained excellent mutagenic strain is continuously passed on a culture medium plate for dibbling, a 4mm puncher is used for digging out a strain block, the spore yield of the strain is measured for six continuous generations, and the stability of the strain is detected.
Optionally, the original strain adopts metarhizium anisopliae 421 as a material.
Optionally, the culture medium plate is made of materials including 200g of potatoes, 20g of glucose, 20g of agar and 1000ml of water.
Optionally, the virulence screening comprises the following specific steps:
a. test No. 1 × 10 was prepared by soaking7The metarhizium anisopliae spores per m L are inoculated to the 2-instar larvae of the diamondback moth;
b. adopting Tween80 solution as a control, regularly observing and recording the symptoms of the diamondback moth every day, counting the death number of the diamondback moth, and calculating the corrected death rate;
c. after the larva dies, the dead body is subjected to moisture preservation and culture, and after 5 days, the body is checked whether hypha and conidium grow out, and whether the death is caused by the metarhizium anisopliae infection is confirmed.
Optionally, the ARTP mutagenesis time is set to 0S, 10S, 20S, 30S, 40S, 50S, 60S, and the mutagenesis conditions of the ARTP mutagenesis apparatus are set to 100W of discharge power and 10S L M of air flow.
Optionally, the ultraviolet mutagenesis irradiation time is set to 0s, 30s, 60s, 90s, 120s, 150s, 180s, 210s, 240s, and 270s, and the ultraviolet lamp power is set to 30W.
Optionally, the concentration of the Tween80 solution is 0.05%, and the 0.05% Tween80 solution is prepared by dissolving 50 μ L Tween80 in 100ml of water.
Optionally, the spore suspension is diluted by a factor of 107Left and right.
Optionally, the temperature of the light-shielding culture is 28 ℃, and the time of the light-shielding culture is 3 days.
The invention provides a preparation method of a metarhizium strain by utilizing ARTP and ultraviolet composite mutagenesis, which has the following beneficial effects:
1. according to the preparation method of the metarhizium anisopliae strain by utilizing ARTP and ultraviolet composite mutagenesis, a good strain AU34 which is full in growth, fast in spore production, strong in ultraviolet resistance and strong in toxicity is successfully screened by combining normal-pressure room-temperature plasma and ultraviolet composite mutagenesis, the spore production amount of the good strain AU34 is increased by 1.6 times compared with that of an original strain, and after the spore production amount of six times of passage is detected, the mutagenized strain has good genetic stability.
2. The preparation method of the metarhizium anisopliae strain by utilizing the ARTP and ultraviolet composite mutagenesis has the advantages that the stable mutant strain is easily obtained by utilizing the ARTP mutagenesis technology, and is combined with the traditional ultraviolet mutagenesis, so that the mutation rate of the strain is greatly improved, the mutation diversity of the strain is enriched, and the preparation method is favorable for obtaining the excellent mutant strain with high spore yield, strong ultraviolet resistance and strong toxicity.
3. According to the preparation method of the Metarhizium anisopliae strain by utilizing the ARTP and ultraviolet composite mutagenesis, the high-performance Metarhizium anisopliae strain is obtained by combining the normal-pressure room-temperature plasma with the ultraviolet composite mutagenesis, so that a new material is provided for preparing the environment-friendly pesticide without pollution and residue, the use effect of the existing pesticide is favorably improved, and the requirements of ecological civilization construction and green agriculture development of China are met.
Drawings
FIG. 1 is a comparison chart of spore production of the original strain and the ARTP-ultraviolet complex mutation strain at the same time;
FIG. 2 is a comparison of the colony sizes of the original strain and the ARTP-ultraviolet complex mutation strain according to the present invention;
FIG. 3 is a graph showing the lethality of ARTP mutagenesis in the present invention;
FIG. 4 is a graph of UV mutagenesis lethality in accordance with the present invention;
FIG. 5 is a graph showing the lethality of ARTP-ultraviolet composite mutagenesis in accordance with the present invention;
FIG. 6 shows the survival rate of the original strain according to the present invention after UV irradiation;
FIG. 7 shows the survival rate of AU3 after UV irradiation according to the present invention;
FIG. 8 shows the survival rate of AU34 after UV irradiation according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Referring to fig. 1 to 8, the present invention provides a technical solution: a preparation method of Metarrhizium anisopliae strain by utilizing ARTP and ultraviolet composite mutagenesis comprises the following steps:
s1, preparing spore suspension, namely, pouring 5m L of Tween80 solution on a fresh culture medium plate, wherein the culture medium plate is made of 200g of potatoes, 20g of glucose and 20g of agar, 1000ml of water original strain is made of metarhizium anisopliae 421, spores of the original metarhizium anisopliae strain are washed down and put into a 10m L centrifugal tube containing glass beads, the centrifugal tube is uniformly vibrated, four layers of sterilized mirror wiping paper are used for filtering, the corresponding dilution times are obtained, and the dilution times of the spore suspension are 107On the left and right, i.e.during dilution, the concentration of the spore suspension was adjusted to 10 by counting with a haemocytometer under an optical microscope7Left and right;
s2, ARTP mutagenesis, namely, putting a slide glass on an alcohol lamp outer flame for burning for 30S, putting the slide glass into a sterilized flat plate, cooling, uniformly coating 10 mu L bacterial liquid on the slide glass, opening an ARTP mutagenesis breeding instrument, starting an ultraviolet lamp for sterilization for 30min, fixing a 1.5M L EP tube filled with 990 mu L sterile water in a clamping groove of the ARTP mutagenesis instrument, transferring a sterilized culture dish filled with the slide glass into an ARTP operating chamber, sequentially putting the slide glass into corresponding clamping grooves by using sterile tweezers, and setting the ARTP mutagenesis time which is set to be 0S, 10S, 20S, 30S, 40S, 50S and 60S, wherein the mutagenesis conditions of the ARTP mutagenesis instrument are set to be 100W of discharge power and 10S L M of air flow;
s3, performing ARTP mutagenesis culture, namely after the ARTP mutagenesis in the step S2, oscillating and eluting an EP tube with a slide, diluting by corresponding times, sucking 100 mu L, coating the tube into a culture medium plate, performing light-proof culture at the temperature of 28 ℃ for 3 days, and calculating the ARTP mutagenesis lethality;
s4, ultraviolet mutagenesis, namely, putting a magnetic stirrer into a clean bench, cleaning the clean bench and the magnetic stirrer by alcohol, fixing the distance between the clean bench and the magnetic stirrer to be 30cm, opening an ultraviolet lamp for sterilization for 20min, absorbing 5m L spore suspension into a sterilized flat plate with a rotor, opening the magnetic stirrer and a culture dish cover, irradiating by the ultraviolet lamp under dark conditions, setting ultraviolet mutagenesis time, wherein the ultraviolet mutagenesis irradiation time is set to be 0S, 30S, 60S, 90S, 120S, 150S, 180S, 210S, 240S and 270S, the ultraviolet lamp power is set to be 30W, and after irradiation is finished, closing the ultraviolet lamp;
s5, ultraviolet mutagenesis culture, namely diluting the bacterial liquid with different irradiation times in the step S4 by a proper multiple, absorbing 100 mu L, coating the bacterial liquid into a culture medium plate, performing dark culture at 28 ℃ for 3 days, operating under infrared light, and calculating the ultraviolet mutagenesis lethality rate;
s6, ARTP combined ultraviolet composite mutagenesis: subjecting the spore suspension to ARTP mutagenesis of step S2 and ultraviolet mutagenesis of S4 in sequence;
s7, ARTP combined ultraviolet composite mutation culture: diluting the spore suspension obtained in the step S6, coating the diluted spore suspension on a culture medium plate, repeating the step for 3 times, culturing in a dark place, and calculating the ARTP combined ultraviolet composite mutagenesis lethality rate;
s8, observing the growth conditions of the mutagenic strain in the S7 on a culture medium plate at different time, counting bacterial colonies on the culture medium plate, carrying out primary screening on the mutagenic strain, dibbling the bacterial colonies growing on the culture medium plate on a new culture medium plate, measuring and recording the growth diameter of the bacterial colonies on the 6 th day, and screening the mutagenic strain which can produce spores, has high growth speed of the bacterial colonies and large growth diameter after mutagenesis;
s9, re-screening: comprises screening the anti-ultraviolet capability and screening the toxicity,
the screening method for the ultraviolet resistance comprises the following steps: irradiating the primarily screened strains for 5min under an ultraviolet lamp, and selecting strains with high germination rate and strong ultraviolet resistance;
the virulence screening method comprises the following steps: carrying out toxicity test of diamondback moth on the original strain and the mutant strain obtained after screening the ultraviolet resistance, selecting the strain with strong toxicity to the diamondback moth,
the specific steps of virulence screening are:
a. test No. 1 × 10 was prepared by soaking7The metarhizium anisopliae spores per m L are inoculated to the 2-instar larvae of the diamondback moth;
b. adopting Tween80 solution as control, observing and recording symptoms of diamondback moth every day, counting death number of diamondback moth, and determining corrected death rate, and simultaneously performing correlation analysis on death rate every day to determine L T50Regression equation of virulence, using SPSS software to make numbersAccording to the statistical analysis, the method comprises the following steps of,
c. after the larvae die, carrying out moisture preservation and culture on dead bodies, checking whether mycelia and conidia grow out from the bodies after 5 days, and confirming whether the dead bodies die due to the infection of the metarhizium anisopliae, thereby screening the metarhizium anisopliae strains with high infection capacity, wherein the toxicity screening is divided into an original strain and a stronger mutant strain screened by the ultraviolet resistance capacity, the two strains are respectively repeated three groups of toxicity screening experiments, and each group of experiments is inoculated with 20 plutella xylostella.
S10, determination of mutant strain stability: the obtained excellent mutagenic strain is continuously passed on a culture medium plate for dibbling, a 4mm puncher is used for digging out a strain block, the spore yield of the strain is measured for six continuous generations, and the stability of the strain is detected.
The apparatus adopted in the preparation method comprises a constant temperature and humidity incubator, an ARTP-II type mutation breeding apparatus, an HVE-50 autoclave, an SZ-97 automatic triple pure water distiller, a Mixer 4K micro vortex Mixer, an SJ-CJ-2FD super clean workbench and an 85-2 constant temperature magnetic stirrer, wherein the preparation materials of the culture medium plate used in the preparation method comprise 200g of potatoes, 20g of glucose, 20g of agar and 1000ml of water, the reagent used in the preparation method is a Tween80 solution, the concentration of the Tween80 solution is 0.05 percent, and the preparation method of the 0.05 percent Tween80 solution is that 50 mu L Tween80 is dissolved in 100ml of water.
The results of the three mutagenesis methods through steps S1 to S9 were analyzed as follows:
as shown in figure 3: the death rate is gradually increased along with the increase of ARTP mutagenesis time by taking metarhizium anisopliae 421 spore suspension as an initial strain. The lethality was found to approach 100% at 60s by ARTP mutagenesis. When the irradiation time was 40s, the lethality was 80.06%. In order to obtain a good strain with fast growth and high yield, 80% to 90% of lethality is generally selected as the mutagenesis time, so the ARTP mutagenesis treatment time of the method is selected to be 40 s.
According to the attached FIG. 4: after the irradiation treatment under a 30W ultraviolet lamp for different time, the lethality rate is gradually increased along with the time extension, and the lethality rate reaches 100% when the ultraviolet mutagenesis time is irradiated for 270s, so the ultraviolet mutagenesis time is selected as 270 s.
As shown in fig. 5: the lethality rates of the ARTP combined with the ultraviolet composite mutagenesis were 70.38%, 94.81% and 97.97% at 120s, 150s and 180s, respectively, and 120s was used as the mutagenesis time in consideration of the fact that the method combines the ARTP and the ultraviolet composite mutagenesis to ensure sufficient strains.
The preliminary screening results were as follows:
after the ARTP combined ultraviolet composite mutagenesis, colonies and original strains grown after mutagenesis are spotted on a new culture medium plate, 28 mutagenized strains with the growth diameter larger than that of the original strains are obtained by comparing the growth diameter and the spore production speed of the original strains and the ARTP combined ultraviolet composite mutagenized strains, and the results are shown in table 1.
TABLE 1 growth diameter of original and mutagenized strains
5 mutant strains AU6, AU23, AU34, AU43 and AU48 have faster sporulation, and the sporulation characteristics are shown in figure 1, wherein the 5 mutant strains start to sporulate after 48h, and the original strain starts to sporulate after 60 h. The sporulation behavior of the remaining mutagenized strains was consistent with that of the original strain, but the colony size was larger than that of the original strain, and the results are shown in FIG. 2.
The rescreening results were as follows:
screening the ultraviolet resistance: after the 28 mutant strains obtained by primary screening are irradiated under an ultraviolet lamp for 5min, the original strains AU3 and AU34 are found to have the survival rates of 0.40%, 2.36% and 3.18% respectively after being irradiated for 5min, the mutant strains AU3 and AU34 are more resistant to ultraviolet irradiation than the original strains, wherein the strain AU34 has stronger ultraviolet resistance, and ultraviolet irradiation survival rate graphs of the original strain AU3 and the mutant strain AU34 are respectively shown as the attached figures 6, 7 and 8.
II, virulence screening: and (3) acting spore suspensions of the original strains, AU3 and AU34 on the plutella xylostella, observing the death number of the plutella xylostella at regular time every day, and performing data statistical analysis. The results are shown in Table 2.
TABLE 2 toxicity of Metarhizium anisopliae against Plutella xylostella
From table 2, it can be seen that the corrected mortality rates of the original strain, mutant strain AU3 and mutant strain AU34 are 80.36%, 82.14% and 85.71%, respectively, wherein the mortality rate of strain AU34 is higher, and from half the lethal time, L T50 used by strain AU34 is shortest and 6.12d, so strain AU34 has the best toxicity to diamond back moth.
The results of determining the stability of the mutant strains were as follows:
the original strain had a spore yield of 0.995. + -. 0.316 (10) by measuring the spore yield for 6d growth8Spore/cm2) The spore yield of strain AU34 was 1.6 times higher than that of the original strain. The result of the genetic stability test of the strain AU34 is shown in Table 3, and the strain AU34 has good genetic stability through continuous six-generation spore yield detection.
TABLE 3 genetic stability of the Strain AU34
In summary, the preparation method of the metarhizium anisopliae strain by utilizing the ARTP and ultraviolet composite mutagenesis comprises the steps of utilizing three mutagenesis methods of the ARTP mutagenesis, the ultraviolet mutagenesis and the ARTP combined ultraviolet mutagenesis, selecting proper mutagenesis time of the ARTP combined ultraviolet mutagenesis according to the lethality statistics of strains of the ARTP mutagenesis, the ultraviolet mutagenesis and the ARTP ultraviolet composite mutagenesis, thereby obtaining the strains of the ARTP combined ultraviolet mutagenesis, primarily screening the strains obtained by the ARTP combined ultraviolet mutagenesis, selecting high-performance mutagenesis strains with high spore production, high colony growth speed and large growth diameter, screening the high-performance mutagenesis strains by ultraviolet resistance and toxicity respectively, obtaining the high-performance mutagenesis strains with strong ultraviolet resistance and toxicity, and finally, carrying out strain genetic stability determination on the high-performance mutagenesis strains.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (9)
1. A preparation method of Metarrhizium anisopliae strain by utilizing ARTP and ultraviolet composite mutagenesis is characterized by comprising the following steps: the method comprises the following steps:
s1, preparing spore suspension, namely, pouring a Tween80 solution 5m L on a fresh culture medium plate, washing spores of an original metarhizium anisopliae strain down, putting the washed spores into a 10m L centrifugal tube containing glass beads, uniformly oscillating, filtering by using four layers of sterilized mirror wiping paper, and diluting by corresponding times;
s2, ARTP mutagenesis, namely, putting a slide glass on an alcohol lamp outer flame for burning for 30S, putting the slide glass into a sterilized flat plate, cooling, uniformly coating 10 mu L bacterial liquid on the slide glass, opening an ARTP mutagenesis breeding instrument, starting an ultraviolet lamp for sterilization for 30min, fixing a 1.5m L EP tube filled with 990 mu L sterile water in a clamping groove of the ARTP mutagenesis instrument, transferring a sterilized culture dish filled with the slide glass into an ARTP operating chamber, sequentially putting the slide glass into the corresponding clamping groove by using sterile tweezers, and setting ARTP mutagenesis time;
s3, ARTP mutagenesis culture, after the ARTP mutagenesis of the step S2, shaking and eluting an EP tube with a slide glass, diluting by corresponding times, sucking 100 mu L, coating the tube in a culture medium plate, culturing in a dark place, and calculating the death rate of the ARTP mutagenesis;
s4, ultraviolet mutagenesis, namely, putting a magnetic stirrer into a clean bench, cleaning the clean bench and the magnetic stirrer by alcohol, fixing the distance between the clean bench and the magnetic stirrer to be 30cm, opening an ultraviolet lamp for sterilization for 20min, absorbing 5m of L spore suspension, adding the suspension into a sterilized flat plate with a rotor, opening the magnetic stirrer and a culture dish cover, irradiating by the ultraviolet lamp under dark conditions, setting ultraviolet mutagenesis time, and closing the ultraviolet lamp after irradiation is finished;
s5, ultraviolet mutagenesis culture, namely diluting the bacterial liquid with different irradiation times in the step S4 by a proper multiple, absorbing 100 mu L, coating the bacterial liquid into a culture medium plate, culturing in a dark place, and calculating the ultraviolet mutagenesis lethality;
s6, ARTP combined ultraviolet composite mutagenesis: subjecting the spore suspension to ARTP mutagenesis of step S2 and ultraviolet mutagenesis of S4 in sequence;
s7, ARTP combined ultraviolet composite mutation culture: diluting the spore suspension obtained in the step S6, coating the diluted spore suspension on a culture medium plate, repeating the step for 3 times, culturing in a dark place, and calculating the ARTP combined ultraviolet composite mutagenesis lethality rate;
s8, primary screening: observing the growth conditions of the mutagenic strains in the S7 on culture medium plates at different time, counting bacterial colonies on the culture medium plates, carrying out primary screening on the mutagenic strains, dibbling the bacterial colonies growing on the culture medium plates onto new culture medium plates, measuring and recording the growth diameter of the bacterial colonies at the 6 th day, and screening the mutagenic strains which can produce spores, have high growth speed and large growth diameter after mutagenesis;
s9, re-screening: comprises screening the anti-ultraviolet capability and screening the toxicity,
the screening method for the ultraviolet resistance comprises the following steps: irradiating the primarily screened strains for 5min under an ultraviolet lamp, and selecting strains with high germination rate and strong ultraviolet resistance;
the virulence screening method comprises the following steps: carrying out a diamondback moth toxicity test on the original strain and the mutant strain obtained after screening the ultraviolet resistance, and selecting a strain with strong toxicity to the diamondback moth;
s10, determination of mutant strain stability: the obtained excellent mutagenic strain is continuously passed on a culture medium plate for dibbling, a 4mm puncher is used for digging out a strain block, the spore yield of the strain is measured for six continuous generations, and the stability of the strain is detected.
2. The method for preparing Metarhizium anisopliae strain by using ARTP and ultraviolet composite mutagenesis as claimed in claim 1, wherein: the original strain adopts metarhizium anisopliae 421 as a material.
3. The method for preparing Metarhizium anisopliae strain by using ARTP and ultraviolet composite mutagenesis as claimed in claim 1, wherein: the culture medium plate is made of materials including 200g of potatoes, 20g of glucose, 20g of agar and 1000ml of water.
4. The method for preparing Metarhizium anisopliae strain by using ARTP and ultraviolet composite mutagenesis as claimed in claim 1, wherein: the virulence screening method comprises the following specific steps:
a. test No. 1 × 10 was prepared by soaking7The metarhizium anisopliae spores per m L are inoculated to the 2-instar larvae of the diamondback moth;
b. adopting Tween80 solution as a control, regularly observing and recording the symptoms of the diamondback moth every day, counting the death number of the diamondback moth, and calculating the corrected death rate;
c. after the larva dies, the dead body is subjected to moisture preservation and culture, and after 5 days, the body is checked whether hypha and conidium grow out, and whether the death is caused by the metarhizium anisopliae infection is confirmed.
5. The method for preparing Metarhizium anisopliae strain by ARTP and ultraviolet composite mutagenesis as claimed in claim 1, wherein the ARTP mutagenesis time is set to 0S, 10S, 20S, 30S, 40S, 50S, 60S, the mutagenesis condition of the ARTP mutagenesis apparatus is set to 100W of discharge power and 10S L M of air flow.
6. The method for preparing Metarhizium anisopliae strain by using ARTP and ultraviolet composite mutagenesis as claimed in claim 1, wherein: the ultraviolet mutagenesis irradiation time is set to be 0s, 30s, 60s, 90s, 120s, 150s, 180s, 210s, 240s and 270s, and the ultraviolet lamp power is set to be 30W.
7. The method for preparing Metarhizium anisopliae strain by using ARTP and ultraviolet composite mutagenesis as claimed in claim 1, wherein the concentration of the Tween80 solution is 0.05%, and the 0.05% Tween80 solution is prepared by dissolving 50 μ L Tween80 in 100ml of water.
8. The method for preparing Metarhizium anisopliae strain by using ARTP and ultraviolet composite mutagenesis as claimed in claim 1, wherein: the dilution factor of the spore suspension is 107Left and right.
9. The method for preparing Metarhizium anisopliae strain by using ARTP and ultraviolet composite mutagenesis as claimed in claim 1, wherein: the temperature of the light-proof culture is 28 ℃, and the time of the light-proof culture is 3 days.
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