CN117987281B - Beauveria bassiana Bbzy230628 strain and application thereof - Google Patents
Beauveria bassiana Bbzy230628 strain and application thereof Download PDFInfo
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Abstract
The invention discloses a beauveria bassiana Bbzy230628 strain and application thereof, wherein the strain is beauveria bassiana, and the preservation name is beauveria bassiana Beauveria bassiana; the microbial strain is preserved in China general microbiological culture Collection center (China Committee for culture Collection), and the preservation address is North Star Xiyu No.1, 3 in the Korean area of Beijing; preservation date: 2023, 11, 17; preservation number: CGMCC No.40927. The beauveria bassiana Bbzy230628 provided by the invention is a biocontrol fungus capable of being used for controlling the major crop pests, namely the cratoxyla schinifolium Holotrichia scrobiculata Brenske, has the characteristics of strong pathogenicity to adult cratoxyla, safety to environment and difficulty in causing the pests to generate drug resistance, and can be used for biologically controlling the cratoxyla.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a beauveria bassiana Bbzy230628 strain and application thereof.
Background
The larch Holotrichia scrobiculataBrenske belongs to Coleoptera (Coleoptera), the gill tortoise (Melolonthidae), the adult of the larch takes on leaves which harm the overground parts of crops and fruit trees, and the larva takes on the root systems of the crops and the fruit trees underground, so the larch is an important pest in agriculture of China and is widely distributed in China. In recent years, the insect is harmful to the planting areas such as gingko, blueberry and the like, the occurrence of the coarse fineleaf holotrichia is serious along with the adjustment of the planting structure and the change of the global environment climate, the insect feeding of the coarse fineleaf holotrichia damages the aerial part leaves of the gingko, the insect feeding of the larvae damages the underground root systems of crops and plants, the gingko plants are dry and dead, serious economic loss is caused to agricultural production, and especially the damage is aggravated along with the global climate change and the adjustment of the planting structure in recent years. At present, the prevention and treatment measures for the cratoxylem are mainly prevention and treatment measures mainly based on pesticide. Then, no specific agent for preventing and controlling the crassostrea gigas exists at present, and meanwhile, although the chemical pesticide has quick effect, the application of the chemical pesticide can not only cause pesticide residues of agricultural products and serious environmental pollution, but also easily kill natural enemies of the crassostrea gigas and cause target pests to generate drug resistance, so that the requirements of sustainability and ecological control of pest control are not met. The biological control measures have the characteristics of ecology and continuous control, so that the biological control is an important way for ecologically and continuously controlling the holotrichia crassifolia.
The entomopathogenic fungi have the characteristics of strong natural popularity, no pollution to the environment, safety to people, livestock and plants, little or no resistance to pests and contribution to environmental protection, and become an important measure for biological pest control. Therefore, the research on biocontrol fungi of the holotrichia crassipes provides a basis for biocontrol of the holotrichia crassipes and also provides a guarantee for reducing drug resistance and environmental pollution caused by chemical control.
Beauveria bassiana (Beauveria bassiana) belongs to the genus Beauveria (Beauveria) belonging to the phylum ascomycota (Ascomycota), the phylum Paniculata (Pezizomycotina), the genus Sphaerotheca (Sordariomycetes), the order of Sarcodactylis (Hypocreales), the family Cordyceps (Cordycipitaceae), and the genus Beauveria (Beauveria). As an excellent biocontrol bacterium, beauveria bassiana has the characteristics of strong pathogenicity, wide host range, low production cost, no environmental pollution and the like, and is widely used for preventing and controlling agriculture and forestry diseases and insect pests of lepidoptera, coleoptera, homoptera and the like. Unfortunately, however, no report of naturally infecting adults of the holotrichia crassipes has been found, nor has the original strain of beauveria bassiana isolated from naturally infested holotrichia crassipes been found. In addition, because beauveria bassiana has rich genetic diversity and a certain host specificity, the screening of the strain with high efficiency on the target high toxicity prevention and treatment is the basis of application.
Disclosure of Invention
The invention aims to provide a beauveria bassiana Bbzy2308 strain and application thereof, and aims to overcome the defect that the existing beauveria bassiana cannot be infected with lethal crassipes angustifolia Holotrichia scrobiculata Brenske under natural conditions, prevent and treat armyworms by a biotechnology means, and avoid or reduce the problem of drug resistance caused by unreasonable use of chemical pesticides.
In order to solve the problems in the prior art, the invention provides the following technical scheme: the beauveria bassiana Bbzy230628 strain provided by the invention is beauveria bassiana Bbzy230628 strain, and the preservation name is beauveria bassiana Beauveria bassiana; the microbial strain is preserved in China general microbiological culture Collection center (China Committee for culture Collection), and the preservation unit address is North Star Xiyu No.1, no. 3 in the Korean area of Beijing; preservation date: 2023, 11, 17; preservation number: CGMCC No.40927.
Further, the ITS gene sequence of the beauveria bassiana Bbzy230628 strain is a nucleotide sequence shown as SEQ ID No. 1.
Further, when the strain is cultured on an SDAY culture medium at 25 ℃ until the strain reaches 10d, the diameter of a bacterial colony is 9.25-10.34 mm, the front surface of the bacterial colony is white, fluff is provided, the center of the bacterial colony is slightly convex, the color of the back surface of the bacterial colony changes from light to dark from edge to center, and the position from yellow brown to 1/2 of the center changes into black brown; the mycelium of the fungus is smooth, the mycelium is separated, and the diameter of the mycelium is 2-3 mu m; conidiophores stand upright to be in grape spike shape; the conidium is elliptic, has smooth surface and one end pointed, and one end rounded, and has a size of 4.65 μm×2.15 μm.
The invention separates beauveria bassiana Bbzy and 2308 wild strains from adult disease-carrying crude beauveria bassiana, and the wild strains are inoculated back to the adult crude beauveria bassiana to rejuvenate the strain to obtain beauveria bassiana Bbzy and 2308 strains, and the strains are subjected to monospore separation and culture according to a conventional method to obtain pure beauveria bassiana with strong pathogenicity.
On an SDAY culture medium, colony growth of the beauveria bassiana Bbzy230628 strain is irregularly round, slightly bulges at the center and is mostly white at the initial stage. After sporulation, the colony surface is light gray, and later becomes light yellow, and the back is colorless or light yellow to pink. The conidiophore stands upright, is on the top end of the nutritional mycelium or on the branches of the nutritional mycelium, is nearly spherical or oval, is colorless and transparent, and has the size of (2.0-5.8) mu m multiplied by (1.9-6.0) mu m.
The beauveria bassiana Bbzy230628 microbial inoculum prepared from beauveria bassiana Bbzy230628 strain.
Further, the active ingredients are at least one of the following (a), (b) and (c):
(a) Conidium suspension of beauveria bassiana Bbzy230628 strain;
(b) The primary extract of the fermentation broth of the beauveria bassiana Bbzy230628 strain culture is obtained;
(c) The obtained beauveria bassiana Bbzy230628 strain cells were subjected to ultrasonic lysis to obtain supernatants.
The preparation method of the beauveria bassiana Bbzy230628 microbial inoculum provided by the invention comprises the following steps:
(1) Separating beauveria bassiana Bbzy230628 from collected morbid crude holotrichia Holotrichia scrobiculata Brenske, culturing on an SDAY medium, transferring conidium to a newly prepared SDAY medium, and purifying;
(2) Then separating the single spore to obtain purified beauveria bassiana Bbzy230628;
(3) Taking conidium of beauveria bassiana Bbzy230628, putting the conidium into sterile water containing 0.05% Tween-80 to prepare a beauveria bassiana Bbzy230628 spore suspension with the concentration of 1.0X10- 8 spores/mL, and obtaining the beauveria bassiana Bbzy230628 microbial inoculum.
(4) And (3) taking the prepared beauveria bassiana Bbzy230628 microbial inoculum, carrying out spray inoculation on the crude holotrichia angustifolia, and spraying 2-3mL microbial inoculum on each larva.
The invention discloses application of beauveria bassiana Bbzy230628 strain or beauveria bassiana Bbzy230628 microbial inoculum in preparation of a crude stenocardia base Holotrichia scrobiculata Brenske control preparation.
The beneficial effects are that: the beauveria bassiana Bbzy230628 provided by the invention is a biocontrol fungus capable of being used for controlling the major crop pests, namely the cratoxyla schinifolium Holotrichia scrobiculataBrenske, has the characteristics of strong pathogenicity to adult cratoxyla, safety to environment and difficulty in causing the pests to generate drug resistance, and can be used for biologically controlling the cratoxyla.
Compared with the prior art, the invention has the following advantages: (1) The invention is a strain separated from the morbid adult of the crude holotrichia crassifolia for the first time, and has the advantages of simple culture, high growth speed and high spore yield.
(2) The conidium of the invention has strong pathogenicity to the adult of the holotrichia crassifolia, is environment-friendly and pollution-free, is not easy to generate drug resistance, can be widely used for preventing and controlling the cratoxylem. The beauveria bassiana Bbzy strain Bbzy and 2308 obtained by separating the infected scarab beetle from the infected scarab beetle in the team has high death speed and high death capacity on the scarab beetles, and the cumulative correction mortality rate reaches 100%, 100% and 100% at 10 days after 1.65X10 5、1.65×106、1.65×107 and 1.65X10 8 spores/mL inoculation treatment. LT 50 was 4.8, 4.2, 3.8, 3.5, 2.9, 2.5 days at 10 4、105、106、107 and 10 8 spores/mL treatments, respectively.
(3) The inventor discovers that beauveria bassiana is popular in a large number of adult scarab beetles in the scarab beetle population during investigation of gingko forests in the dipping region of the triangulariella volvacea in the city of triangularis of Yunnan, in 2023, 8 months, and causes a large number of adult scarab beetles to be infected and die. Therefore, the strain is parasitic entomogenous fungi with strong popularity which are first discovered in China at present, and has great development and application prospects in preventing and controlling the entomogenous fungi.
Drawings
FIG. 1 is a chart showing the morphology of conidia of beauveria bassiana Bbzy2308 of the present invention.
FIG. 2 is a colony morphology of beauveria bassiana Bbzy2308 of the present invention.
FIG. 3 is a diagram of the dorsal and ventral aspects of an adult, fatigus angustifolia, which is lethal to a natural infection of beauveria bassiana Bbzy2308 of the present invention. Fig. 3A is a back view of a beauveria bassiana Bbzy2308 adult of a natural infection-lethal adult of a crude fineleaf holly, and fig. 3B is a ventral view of a natural infection-lethal adult of a crude fineleaf holly of beauveria bassiana Bbzy 2308.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Examples
The beauveria bassiana Bbzy230628 strain provided by the invention is beauveria bassiana Bbzy230628 strain, and the preservation name is beauveria bassiana Beauveria bassiana; the microbial strain is preserved in China general microbiological culture Collection center (China Committee for culture Collection), and the preservation unit address is North Star Xiyu No. 1, no. 3 in the Korean area of Beijing; preservation date: 2023, 11, 17; preservation number: CGMCC No.40927.
The beauveria bassiana Bbzy230628 strain is applied to the prevention and treatment of the tortoise Holotrichia scrobiculataBrenske with holotrichia crassifolia.
The ITS gene sequence of the beauveria bassiana Bbzy230628 strain is the nucleotide sequence shown as SEQ ID No. 1.
As shown in figures 1 and 2, when the strain is cultured on an SDAY culture medium at 25 ℃ for 10d, the diameter of a bacterial colony is 9.25-10.34 mm, the front surface of the bacterial colony is white and has villus, the center of the bacterial colony is slightly convex, the color of the back surface of the bacterial colony changes from light to deep from edge to center, and the color of the back surface of the bacterial colony changes from yellow brown to black at 1/2 of the center; the mycelium of the fungus is smooth, the mycelium is separated, and the diameter of the mycelium is 2-3 mu m; conidiophores stand upright to be in grape spike shape; the conidium is elliptic, has smooth surface and one end pointed, and one end rounded, and has a size of 4.65 μm×2.15 μm.
As shown in figure 3, the invention separates beauveria bassiana Bbzy and 2308 wild strains from adult disease-causing crassostrea angustifolia, and the wild strains are inoculated back to adult crassostrea angustifolia and rejuvenated to obtain beauveria bassiana Bbzy and 2308 strains, and the strains are subjected to monospore separation and culture by a conventional method to obtain pure beauveria bassiana with strong pathogenicity.
On the SDAY culture medium, the colony of the beauveria bassiana Bbzy230628 strain is irregularly round in the early growth stage, slightly raised in the center and mostly white in the early stage. After sporulation, the colony surface is light gray, and later becomes light yellow, and the back is colorless or light yellow to pink. The conidiophore stands upright, is on the top end of the nutritional mycelium or on the branches of the nutritional mycelium, is nearly spherical or oval, is colorless and transparent, and has the size of (2.0-5.8) mu m multiplied by (1.9-6.0) mu m.
The beauveria bassiana Bbzy230628 microbial inoculum prepared from beauveria bassiana Bbzy230628 strain. The active ingredients are at least one of the following (a), (b) and (c):
(a) Conidium suspension of beauveria bassiana Bbzy230628 strain;
(b) The primary extract of the fermentation broth of the beauveria bassiana Bbzy230628 strain culture is obtained;
(c) The obtained beauveria bassiana Bbzy230628 strain cells were subjected to ultrasonic lysis to obtain supernatants.
The preparation method of the beauveria bassiana Bbzy230628 microbial inoculum provided by the invention comprises the following steps:
(1) Separating beauveria bassiana Bbzy230628 from collected morbid crude holotrichia Holotrichia scrobiculata Brenske, culturing on an SDAY medium, transferring conidium to a newly prepared SDAY medium, and purifying;
(2) Then separating the single spore to obtain purified beauveria bassiana Bbzy230628;
(3) Taking conidium of beauveria bassiana Bbzy230628, putting the conidium into sterile water containing 0.05% Tween-80 to prepare a beauveria bassiana Bbzy230628 spore suspension with the concentration of 1.0X10- 8 spores/mL, and obtaining the beauveria bassiana Bbzy230628 microbial inoculum.
(4) And (3) taking the prepared beauveria bassiana Bbzy230628 microbial inoculum, carrying out spray inoculation on the crude holotrichia angustifolia, and spraying 2-3mL microbial inoculum on each larva.
The invention discloses application of beauveria bassiana Bbzy230628 strain or beauveria bassiana Bbzy230628 microbial inoculum in preparation of a crude stenocardia base Holotrichia scrobiculata Brenske control preparation.
Test example 1
Isolation and identification of pathogenic bacteria
1.1 Materials and methods
1.1.1 Material
An adult coarse finfish Holotrichia scrobiculataBrenske, which is infested and killed by a fungus, is collected in Yi county, triajing, yunnan province.
Sajo medium (SDAY): 1% peptone, 1% yeast powder, 4% glucose, 1.5-2% agar powder and 1000 ml water.
Aseptic operating conditions: all vessels and appliances were subjected to high temperature sterilization (121 ℃,30 min) and inoculation etc. in an ultra clean bench.
Culture conditions: culturing in a 28 deg.C illumination (12L: 12D) incubator, transferring to a test tube SDAY slant after colony formation, culturing for 2-3 days, and storing in a 4 deg.C refrigerator.
1.1.2 Isolation and purification of pathogenic bacteria
Separating: the pathogenic bacteria are isolated from adult carcasses of morbid macrobrachium rosenbergii. The armyworm larva is brought back to a laboratory, the adult corpse of the armyworm is soaked in 70% alcohol for 30 seconds, then soaked in 0.1% mercuric chloride solution for 3 minutes (thorough disinfection), finally washed three times with sterile water, conidium or mycelium growing on the adult armyworm of the armyworm is taken out by an inoculating needle, placed on an SDAY culture medium, cultivated at 28 ℃, subjected to monospore separation and cultivation according to a conventional method after a large number of mycelium grows, to obtain pure strain beauveria bassiana with strong spore production capability Beauveria bassianaBbzy230628, transferred to an SDAY inclined plane to grow for 3-5 days, and placed in a refrigerator at 4 ℃ for storage after the growth.
Rejuvenation: culturing the separated strain on SDAY for 2-3 days to generate a large amount of conidium, then picking the conidium into sterile water containing 1% Tween-80, uniformly stirring with a glass rod to obtain spore suspension with proper concentration (10 8 spores/mL), uniformly spraying on the surface of adult scarab with a small sprayer (the surface of the insect is moist), and keeping the relative humidity above 80%. Collecting dead bodies, preserving moisture, and separating the obtained dead bodies according to the method to obtain the strain with stronger pathogenicity.
Purifying: the conidium powder on the culture medium is picked up and inoculated on a new culture medium again. The strain is purified after 2 to 3 generations of culture.
1.1.3 Identification of pathogenic bacteria
Morphological identification: and (5) identifying according to the culture property of pathogenic bacteria and the shapes of hyphae and conidium. And (3) microscopic examination of the forms of bacterial colony hyphae, conidia and conidiophores by using a 40×10-fold microscopic optical microscope, photographing and recording, and classifying and identifying according to the strain forms.
Strain rDNA-ITS sequence analysis. The strain DNA was obtained using a fungal genomic DNA extraction kit (supplied by Dingguo Biotechnology Co., guangzhou). PCR amplification of the rDNA-ITS sequence of the strain was performed using the fungal universal primers ITS1 (5'-TCCGTAGGTCCGTAGGTGAACCTGCGG-3')/ITS 4 (5'-TCCTCCGCTTATTGATATGC-3'). 50 mu.l of PCR reaction system, 25. Mu.l of I-5 TM.2 Xhigh-FIELDLITY MASTER Mix, 1. Mu.l of DNA template, 2. Mu.l (10. Mu. Mol/L) of each of the upstream and downstream primers, and 20. Mu.l of ddH 2 O. Amplification procedure: pre-denaturation at 94℃for 5 min; 94℃for 30s,56℃for 30s,72℃for 90s, 35 cycles; extending at 72℃for 10min. The PCR product was detected by 1% agarose gel electrophoresis and sent to Guangzhou Rui Biotech Co.Ltd for sequencing. And comparing the obtained DNA fragment sequences in NCBI database to determine the species relationship of the strain.
1.2 Results
1.2.1 Morphological identification results
Separating and obtaining a wild strain from an adult coarse holothurian gill-like turtle naturally infected by entomogenous fungi, culturing on an SDAY (Sa agar medium), inoculating back to a mythic larva for rejuvenation to obtain a strain, and separating single hypha of the strain to obtain a purified strain, namely beauveria bassiana Bbzy230628.
On the SDAY culture medium, the colony of the beauveria bassiana Bbzy230628 strain is irregularly round in the early growth stage, slightly raised in the center and mostly white in the early stage. After sporulation, the colony surface is light gray, and later becomes light yellow, and the back is colorless or light yellow to pink. The conidiophore stands upright and is arranged on the top end of the nutritional mycelium or on branches of the nutritional mycelium, and the conidiophore is nearly spherical or oval, colorless and transparent, and has the size of (2.0-5.8) mu m multiplied by (1.9-6.0) mu m.
1.2.2 Molecular characterization results
The sequence of PCR amplified product fragment of Bbzy230628 strain rDNA-ITS sequence analysis is determined, ITS gene sequence of beauveria bassiana Bbzy230628 strain is nucleotide sequence shown in SEQ ID No.1, and the result is as follows:
TTCCTCCGCTTATTGATATGCTTAAGTTCAGCGGGTAGTCCTACCATGATTCGAGGTCAACGTTCAGAAGTTGGGTGTTTTACGGCGTGGCCGCGTCGGGGTTCCGGTGCGAGCTGTATTACTACGCAGAGGTCGCCGCGGACGGGCCGCCACTCCATTTCAGGGCCGGCGGTGGTGCTGCCGGTCCCCAACGCCGACCTCCCCAAGGGGAGGTCGAGGGTTGAAATGACGCTCGAACAGGCATGCCCGCCAGAATGCTGGCGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGGATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCAGAGCCAAGAGATCCGTTGTTGAAAGTTTTGATTCATTTGTTTTGCCTTGCGGCGTATTCAGAAGATGCTGGAATACAAGAGTTTGAGGTCCCCGGCGGGCCGCTGGTCCAGTCCGCGTCCGGGCTGGGGCGAGTCCGCCGAAGCAACGATAGGTAGGTTCACAGAAGGGTTAGGCAGTATGAAAACTCGGTAATGATCCCTCCGCAGGTCCCCCCTACGGAA
And submitting the sequence to a GenBank database for Blast comparison, selecting related sequences, and constructing a phylogenetic tree by adopting a Neighbor-joining method. The comparison result shows that Bbzy2308 strain is beauveria bassiana.
The strain is determined to be beauveria bassiana Beauveria bassiana according to field infection symptoms, collection and indoor separation observation of the morbid insect bodies, identification according to morphological characteristics of beauveria bassiana infection symptoms, conidium and hypha and rDNA-ITS sequence analysis results.
Test example 2
Biological characteristics of purified strain of beauveria bassiana Bbzy230628
2.1 Materials and methods
2.1.1 Test strains
And selecting a dish which grows vigorously and uniformly after purification as a test strain. The mycelium was inoculated again onto SDAY and cultured in a constant temperature light (12L: 12D) incubator at 28 ℃.
2.1.2 Determination of colony growth Rate and sporulation
One dish of beauveria bassiana Bbzy2308 which is cultivated in advance is perforated by a puncher with the diameter of 8mm, inoculated on another culture medium, repeated for 3 times, cultivated in a constant-temperature illumination (12L: 12D) incubator with the temperature of 28 ℃, and the diameter is measured and recorded at regular time every day until the colony grows up to the culture medium. The bacterial cake was taken at the same position of the culture medium by using a puncher with the diameter of 8mm, and then inoculated to the surface of the SDAY culture medium, and cultured in an incubator for 7 days, and the colony diameter was measured and recorded once a day to determine the colony growth condition.
2.2 Results
The results in Table 1 show that beauveria bassiana Bbzy230628 grows faster on the culture medium when cultured for 1-2d, the average diameter of the colony increases at a lower speed, the colony grows faster when cultured for 3d, the average diameter of the colony increases at a speed of 0.33cm/d, and the average diameter of the colony increases at a speed of 0.89cm/d when cultured for 6 d. The colony growth rate measurement results are shown in Table 1:
TABLE 1
As can be seen from Table 2, the beauveria bassiana Bbzy230628 strain was cultured on SDAY medium until the production of spores was started on day 6, and the spore production amount was 1.12X10: 10 7 spores/unit colony on day 6. As the culture time increases, the spore production speed is high, the spore production amount increases, and the spore production amount at the 8 th time is 1.30X10 8 spores/unit colony. The spore yield at 10 was 1.51X10 10 spores/unit colony. The biological characteristics of the strain are better, and the growth condition is better. The sporulation measurement results are shown in table 2:
TABLE 2
Test example 3
Indoor toxicity determination of beauveria bassiana Bbzy230628 on crassipes
3.1, Materials and methods
3.1.1 Test insect source
Larvae of adult Testudinis (Holotrichia scrobiculata Brenske) with gill thickness, which are fed with folium Ginkgo in a light incubator with photoperiod of 12 L:12D at 25+ -1deg.C and humidity 70+ -5% in the laboratory. Healthy and uniform 3-day-old adults are obtained as test insects.
3.1.2 Preparation of spore suspension
The conidia of the beauveria bassiana Bbzy230628 with good growth vigor are washed out by sterile water and filtered to prepare 10 8 spores/ml conidia suspension, the filtrate is dripped on a blood cell counting plate by a sterile capillary dropper, spores are counted under a microscope and data are recorded, the conidia suspension is diluted into 10 8、107、106、105 and 10 4 spore suspension with a concentration gradient of 10.5% Tween 80 in sequence by sterile water, and the sterile water with 0.5% Tween 80 is used as a blank control for toxicity determination.
3.1.3 Dipping method
The adult Testudinis is treated by soaking method. The test insects are respectively soaked in beauveria bassiana Bbzy2308 spore suspension with the concentration of 1.0X10 8 spores/mL, the mixture is gently shaken, the test insects are taken out and dried after 30 s, and the test insects with high activity are selected for testing. The test insects are placed in an octagonal bottle (4.5 cm multiplied by 19.5 cm), sterilized soil with the thickness of 1cm is paved at the bottom of the octagonal bottle, ginkgo leaves are used as food, and the ginkgo leaves are fed in a constant temperature incubator with the temperature of (28+/-1). The adult crude holotrichia angustifolia treated with sterile water at 0.05% tween-80 was used as a control, 3 replicates were set for each treatment, 30 replicates each were tested, and 10d was observed continuously. The death number of the adult test of the holotrichia crassipes and the number of stiff worms with beauveria bassiana spores on the surfaces of the adults are recorded every day, and the average death rate and the stiff worm rate are calculated. The data were subjected to linear regression statistical analysis using DPS 14.0 software.
3.2 Results
Toxicity measurement is carried out on adult fineleaf crassostrea, and the conidium of beauveria bassiana Bbzy230628 infects the adult fineleaf crassostrea. The experimental results show that: the beauveria bassiana Bbzy230628 strain has higher toxicity to the holotrichia crassipes, and the cumulative corrected mortality rate reaches 93.33%, 100% and 100% at 10 days after 1.65X10 4、1.65×105、1.65×106、1.65×107 and 1.65X10 8 spores/mL inoculation treatment.
From the lethal time effect, LT 50 was 4.8, 4.2, 3.8, 3.5, 2.9, 2.5 days, respectively, in the lethal of the crude fineleaf holotrichia under 10 4、105、106、107 and 10 8 spores/mL treatments. The strain has high toxicity to the holotrichia crassifolia and high infection pathogenic speed.
The strain is a wild strain obtained by separating and purifying adult crude holotrichia angustifolia which die from natural infection, namely beauveria bassiana Bbzy230628 and CGMCC No. 40927, and can grow rapidly on an SDAY culture medium at the temperature of 25 ℃ and the relative humidity of more than 70%, and has large spore yield and high spore germination rate. The spore suspension has good pathogenicity to the adult of the holotrichia crassifolia, and can be widely used for preventing and controlling the adult of the holotrichia crassifolia. Meanwhile, the strain has wide sources of culture raw materials, low price and simple culture method, is easy to produce in large quantity, has great development and application potential, is used for preventing and controlling the cratoxylem with the beauveria bassiana Bbzy230628 strain, namely typical biological prevention and control, can avoid the problems of drug resistance, environmental pollution and the like caused by chemical pesticides, and provides a basis for green prevention and control of the cratoxylem.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the foregoing embodiments, which have been described in the foregoing embodiments and description merely illustrates the principles of the invention, and various changes and modifications may be made therein without departing from the spirit and scope of the invention, the scope of which is defined in the appended claims, specification and their equivalents.
Claims (5)
1. A beauveria bassiana (Beauveria bassiana) Bbzy230628 strain, characterized in that: the beauveria bassiana Bbzy230628 strain is beauveria bassiana, and the preservation name is beauveria bassiana Beauveria bassiana; the microbial strain is preserved in China general microbiological culture Collection center (China Committee for culture Collection), and the preservation unit address is North Star Xiyu No.1, no. 3 in the Korean area of Beijing; preservation date: 2023, 11, 17; preservation number: CGMCC No.40927.
2. The beauveria bassiana Bbzy230628 microbial agent prepared from beauveria bassiana Bbzy230628 strain as claimed in claim 1.
3. The beauveria bassiana Bbzy230628 preparation according to claim 2, whose active ingredient is a conidium suspension of beauveria bassiana Bbzy230628 strain according to claim 1.
4. The preparation method of the beauveria bassiana Bbzy230628 microbial inoculum as claimed in claim 2, which is characterized by comprising the following steps:
(1) Culturing beauveria bassiana Bbzy230628 of claim 1 on an SDAY medium, transferring the conidia to a newly prepared SDAY medium, and purifying;
(2) Then separating the single spore to obtain purified beauveria bassiana Bbzy230628;
(3) Taking conidium of beauveria bassiana Bbzy230628, putting the conidium into sterile water containing 0.05% Tween-80 to prepare a beauveria bassiana Bbzy230628 spore suspension with the concentration of 1.0X10- 8 spores/mL, and obtaining the beauveria bassiana Bbzy230628 microbial inoculum.
5. Use of a beauveria bassiana Bbzy230628 strain of claim 1 or a beauveria bassiana Bbzy230628 inoculant of claim 2 in the preparation of a crassipes (Holotrichia scrobiculata Brenske) control formulation.
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