CN113046249A - Verticillium lecanii LL-01 and biocontrol application thereof - Google Patents
Verticillium lecanii LL-01 and biocontrol application thereof Download PDFInfo
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Abstract
The invention discloses verticillium lecanii LL-01 and biocontrol application thereof, belonging to the technical field of microorganisms. The preservation number of the strain is as follows: CGMCC No. 21940; the optimal temperature for growth and sporulation is 26 ℃, and the photoperiod is 6L:18D, the carbon source is fructose, the nitrogen source is casein, the growth speed is high under the culture condition, the spore yield is high, the cumulative fatality rates of the Nanyang gluteus wenshuensis scale and the Lycoris radiata Leoni in different insect ages can respectively reach 84.09% -97.62% and 89.89% -98.85%, the biological control potential is good, the composition can replace part of chemical pesticides for preventing and controlling Leonia Leoni, the dosage of the chemical pesticides is reduced, and the ecological balance is maintained.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to verticillium lecanii LL-01 and biocontrol application thereof.
Background
The mealybugs Planococcus lilacinus and lycoris radiata phenacaccus solani are 2 worldwide fig pests, the mealybugs nanyana can damage more than 100 fruits, woods and ornamental plants of 35 families, such as cocoa, custard litchi, guava, bauhinia, metaphylla, rhododendron and the like, and are listed as entry plant quarantine pests in China in 2007; the lycoris radiata mealybugs can be 131 kinds of ornamental plants and commercial crops in 101 genera of 40 families, such as lycoris radiata, crassulaceae, compositae, leguminosae, solanaceae and the like, and are pests which are mainly concerned by imported succulent plants by the national quality supervision inspection and quarantine bureau in 2014. Although 2 quarantine mealybugs are not widely distributed in China at present, the 2 quarantine mealybugs are seriously harmed in sweetsop orchards and succulent plant planting bases in Fujian province and are obviously overlapped in generations in early-stage investigation, nymphs and adults often gather and stab into harmful fruits, leaves, buds, tender stems and other parts to cause flower falling, leaf falling, fruit falling and growth failure, and honeydew is also secreted to induce sooty smoke, so that the quality and economic value of the sweetsop fruits and the succulent plants are directly or indirectly influenced. Because the two mealybugs are covered with a large amount of waxiness on the body surface and are difficult to prevent and treat, the prevention and control measures adopted in the prior production are also more focused on using chemical pesticides.
However, the long-term use of chemical pesticides easily causes negative problems such as '3R', environmental pollution, biological diversity reduction and the like, and conflicts with the leading guidance of green plant protection in China continuously, and more environment-friendly measures are needed to be found for synergetic pest control.
The entomopathogenic fungi are one of the main factors for controlling the population quantity of insects in nature, the infection lethal proportion accounts for about 60 percent of the natural total number of the insect death caused by diseases, and the excavation of the entomopathogenic fungi is an important measure for green prevention and control of pests.
At present, about 1000 kinds of entomopathogenic fungi are recorded all over the world, but only F061, FM-03 and the like in Metarhizium anisopliae are excavated for preventing and controlling mealybugs and lycoris radiata, and the available strain resources are relatively less.
Therefore, the search for highly effective strains of entomopathogenic fungi to mitigate their harm is a problem that needs to be addressed by those skilled in the art.
Disclosure of Invention
In view of the above, the invention provides verticillium lecanii LL-01 and biocontrol application thereof.
The invention firstly separates and purifies entomopathogenic fungi, then determines the classification status by morphological observation and molecular identification measures, and then determines the extracellular enzyme activity and the pathogenicity of the entomopathogenic fungi to the Nanyang gluteus wenshuni and the lycoris radiata mealybugs, so as to enrich the biocontrol bacteria resources of the 2 kinds of quarantine mealybugs and provide support for the prevention and control of the quarantine mealybugs by utilizing the entomopathogenic fungi.
Preservation information: verticillium lecanii LL-01 was deposited at the general microbiological center of China Committee for culture Collection of microorganisms, the address of No. 3 Homex No. 1, North Kyoho, No. 1, having the preservation number of CGMCC No.21940, in 3.31.2021.
In order to achieve the purpose, the invention adopts the following technical scheme:
a verticillium lecanii strain LL-01 with the preservation number of CGMCC No. 21940.
The invention also provides a medicament containing the verticillium lecanii LL-01.
Preferably: the pesticide is an insecticide, and the target pests are mealybugs of Nanyang gluteus wenshuni and lycoris radiata
The invention also provides application of verticillium lecanii LL-01 in plant biocontrol
Preferably: the plant is Annona squamosa and succulent plant, HONGXINLIAN.
According to the technical scheme, compared with the prior art, the invention discloses and provides a verticillium lecanii strain LL-01 and the biocontrol application thereof, compared with the prior art, the invention separates 1 entomopathogenic fungus LL-01 from the pathogenic body of the Nanyang gluteus went scale, and the verticillium lecanii strain is determined through morphological observation and molecular identification; the optimal temperature for growth and sporulation is 26 ℃, the photoperiod is 6L:18D, the carbon source is fructose, the nitrogen source is casein, the diameter of 10D cultured colony under the condition and the sporulation amount can respectively reach 4.66cm and 3.16 multiplied by 108Spore/cm2(ii) a LC after it is infected by Nanyang gluteus wenshuni and Lycoris radiata Leng of different insect ages for 10 days50Are respectively 0.98 multiplied by 105～9.71×105spores/mL and 0.50X 105～5.30×105Spore/mL，1.00×108The cumulative lethality of the spore/mL treatment was 84.09% -97.62% and 89.89% -98.85%, respectively, LT503.70-5.84 d and 3.48-5.14 d respectively; the activity of the protease and the chitinase reaches peak values of 19.44U/mL and 15.01U/mL respectively at the 5 th d, and the activity of the lipase reaches peak values of 7.68U/m at the 6 th d. In conclusion, the verticillium lecanii LL-01 has high growth speed and high spore yield, has good biological control potential on the 2 quarantine mealybugs, can replace part of chemical pesticides for preventing and controlling the mealybugs, reduces the dosage of the chemical pesticides, and is beneficial to maintaining ecological balance.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a drawing showing colony characteristics (A) and microscopic characteristics (B, C) of LL-01 according to the present invention.
FIG. 2 is a schematic diagram showing the colony diameter and spore yield of Verticillium lecanii LL-01 cultured for 10 days under different temperatures (A), illumination (B), carbon sources (C) and nitrogen sources (D).
FIG. 3 is a drawing showing that Verticillium lecanii LL-01 provided by the present invention infects Ericerus pela (A) and Eremiella lycopersici (B).
FIG. 4 is a schematic diagram showing the mortality of Verticillium lecanii LL-01 after infection of Ericerus pela and Ericerus lycoris for 10 days.
FIG. 5 is a dynamic diagram of the extracellular enzyme activity of Verticillium lecanii LL-01 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. 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.
The embodiment of the invention discloses verticillium lecanii LL-01 and biocontrol application thereof.
Experimental materials and data analysis methods related to the examples:
the test strain is collected from Nanyang buttock pink scale killed by infection of entomopathogenic fungi in Jinxing Xiangfanna litchi orchard (23 degree 76 '22' in northern latitude and 117 degree 26 '42' in east longitude in Zhangzhou city, Fujian province), and Chaochou culture medium (NaNO) is adopted33g、KH2PO41g、MgSO4·7H2O 0.5g、KCl 0.5g、FeSO40.01g, 30g of sucrose, 20g of agar powder and 1000mL of distilled water) and the serial number of LL-01 for later use.
For testing the annona squamosa mealybugs and lycoris radiata mealybugs which are respectively collected from annona squamosa orchards in jinxing county of zhao ampere in Fujian province and succulent planting bases in Jiu lake town of Longhai city, the method comprises the following steps of at room temperature of 25-28 ℃, relative humidity of 50-60%, and photoperiod of 12L: feeding Annona squamosa fruit and succulent plant, such as Rhododendron pulchrum, under 12D, and substituting for nymphs and female adults of first age, second age and third age which hatch or exuviate for 1D for use.
Data analysis
In MEGA 7 software, a phylogenetic tree of strain LL-01 was constructed using the Neighbor join method. In DPS 7 software, performing multiple comparison on the colony diameter and the spore yield of the strain LL-01 under different temperatures, illumination, carbon sources and nitrogen sources by using a Tukey test method; calculating lethal middle concentration LC of strain LL-01 on 2 kinds of Nanyang gluteus went gecko and lycoris radiata powdered scale by using a probability value analysis method50And lethal mid-time LT50And taking the calculated value of the treatment with the actual cumulative mortality rate of more than 50 percent as an effective result.
Example 1
Identification of entomopathogenic fungi
And (3) morphology observation: the strain LL-01 is selected to be inoculated in the center of a Czochralski culture medium plate by single spore inoculation, and is placed at the temperature of 25 +/-1 ℃, the relative humidity of 90 +/-5 percent and the light cycle of 12L: culturing in a GZX-250 BSH-III type illumination incubator of 12D for 10 days, observing and shooting morphological characteristics of bacterial colonies by using a DSLR-A450 type digital single-lens reflex camera, and measuring the diameters of the bacterial colonies; and observing in an E200MV type biological microscope, shooting morphological characteristics of hypha, conidiophores, spore-forming cells and conidia, and preliminarily identifying the classification status of entomogenous fungi.
The results show that:
the strain LL-01 grows well on a Czochralski medium plate; the colony is nearly circular, white in the initial stage, radially grows outwards in an expanding manner, has a diameter of 3.82-4.24 cm after 10 days, and slightly shows a light pink color in the middle area (fig. 1-A). The hypha is slender, has branching and separation and is 1.99-2.37 mu m wide; the conidiophores are arranged on hyphae singly or in an aggregation manner, the width of the conidiophores is 1.57-1.78 mu m, and phialides sporulating cells are arranged at the top ends of the conidiophores; spore-forming cells are single-born, clustered or recurrent, and have a size of (8.33-10.70) μm x (1.81-2.22) μm; conidia are produced singly or in clusters on the top of spore-forming cells, and have an oval or oblong shape with a size of (2.32-5.41) μm x (1.53-1.80) μm (FIG. 1-B, C). The strain LL-01 belongs to the genus Verticillium, Lecanicillium, was preliminarily identified.
And (3) molecular identification: culturing the purified entomogenous fungi strain for 10 days, scraping about 0.2g of hypha and spores on a culture medium by using a sterile scalpel, adding liquid nitrogen, grinding, and extracting the total DNA of the separated strain by using a fungus genome DNA extraction kit; taking total DNA as a template, respectively adopting rDNA-ITS gene universal primers (ITS 1: 5'-TCCGTAG GTGAACCTGCGG-3' and ITS 4: 5'-TCCTCCGCTTATTGATATGC-3'), 18S rDNA gene universal primers (NS 1: 5'-GTAGTCATATGCTTCTCTC-3' and NS 2: 5'-GGCTGCTGGCACCACACTTGC-3') and Nad1 gene universal primers (nad 1A: 5 '-ATGGCSAGTATGCAAAGAAGA-3' and nad 1B: 5 '-GCATGTTCTGTCATAAASCCACTAAC-3') to carry out PCR amplification: PCR reaction (25. mu.l): taq PCR Master Mix 12.5. mu.L, template DNA 2. mu.L (100 ng/. mu.L), 10. mu. mol/L primers 1. mu.L each, add ddH2O to the total volume of 25 mu L; ddH for control2O replaces the template DNA. PCR reaction procedure: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 40s, annealing at 54 ℃ for 40s, and extension at 72 ℃ for 90s for 30 cycles; final extension at 72 ℃ for 10 min); the amplification product was verified by electrophoresis on a 1.5% agarose gel containing GelRed dye and thenAnd purifying and recovering the Gel Extraction Kit Gel recovery Kit, and then determining the sequence.
BLAST comparison is carried out on 3 gene sequences obtained by sequencing, related species sequences are sequentially introduced into DNAMAN 6, phyllosylate 1 and MEGA 7 software, alignment, tandem connection and tree building of multi-gene sequences are respectively carried out, and the types of separated strains are finally judged according to the homology among the sequences.
The results show that:
ITS, 18S and Nad1 amplification is carried out on the total DNA of the strain LL-01, sequence fragments with the lengths of 560bp, 979bp and 528bp are respectively obtained, homology comparison is carried out on the sequence fragments and related species in NCBI/BLAST, and 3 segments of gene sequences respectively achieve 99.82%, 100.00% and 99.80% of similarity with the sequences of verticillium lecanii with the accession numbers of EU284718, AF049155 and EF 512938 and are clustered on a phylogenetic tree. Finally determining the strain verticillium lecanii by combining morphological characteristics.
Preservation information: verticillium lecanii LL-01 was deposited at the general microbiological center of China Committee for culture Collection of microorganisms, the address of No. 3 Homex No. 1, North Kyoho, No. 1, having the preservation number of CGMCC No.21940, in 3.31.2021.
Example 2
Optimizing the culture condition of the entomopathogenic fungi:
temperature selection: selecting single spore of Verticillium lecanii LL-01, inoculating to the center of a Chaudhuri medium plate, and culturing in 3 incubators at 22, 26 and 30 deg.C in the dark, wherein each treatment is repeated for 3 times; after culturing for 10 days, measuring the colony diameter by a cross method; 10mL of sterile water containing 0.05% Tween-80 was added to each treatment petri dish, conidia were scraped off with a sterile slide to prepare a spore suspension, filtered through 3 layers of gauze, and observed with a Nepal blood count plate under an E200 biomicroscope to measure the spore yield of each treatment.
Illumination selection: selecting Verticillium lecanii LL-01 single spore to inoculate in the center of the Czochralski culture medium plate, and then placing in 6L:18D, 12L: 12D and 18L: culturing in an illumination incubator with 3 photoperiods and temperature (26 + -1) deg.C) such as 6D, and repeating each treatment for 3 times; after 10d of culture, the colony diameter and spore yield were determined.
Carbon source selection: on the basis of a Czochralski culture medium, 3 materials such as sucrose, fructose and starch are respectively used as test carbon sources to prepare 3 test culture media, and each treatment is repeated for 3 times; selecting single spore of Verticillium lecanii strain LL-01, inoculating to the center of a Chaudhuri medium plate, and placing at the temperature of 26 +/-1 ℃ and the photoperiod of 6L: after culturing for 10 days in an 18D light incubator, the colony diameter and spore yield were measured.
And (3) selecting a nitrogen source: on the basis of selection of a Chao's medium and a carbon source, 3 materials such as sodium nitrate, yeast extract, beef extract and the like are respectively used as nitrogen sources for testing to prepare 3 test media, and each treatment is repeated for 3 times; selecting single spore of Verticillium lecanii LL-01, inoculating to the center of a Czochralski culture medium plate, and placing at 26 + -1 deg.C with a photoperiod of 6L: after culturing for 10 days in an 18D light incubator, the colony diameter and spore yield were measured.
The results show that:
temperature:
the growth and sporulation of Verticillium lecanii LL-01 are greatly affected by temperature. The growth speed is fastest at 26 ℃ and 30 ℃, the diameters of colonies after 10d of culture reach 4.06cm and 3.93cm respectively, the difference between the two is not obvious, but both are obviously larger than 22 ℃; the spore yield is up to 1.95 × 10 at 26 deg.C8Spore/cm2Significantly higher than the other 2 treatments (fig. 2-a).
Illumination:
the growth and spore production of verticillium lecanii LL-01 are obviously affected differently by illumination. The growth rate was measured at 18L: 6D is fastest, and the colony diameter reaches 4.22cm after 10D culture, although the colony diameter is equal to 12L: 12D did not differ significantly, but significantly greater than 6L: 18D; sporulation yield was measured as 6L:18D is highest and reaches 2.19 multiplied by 108Spore/cm2Significantly higher than the other 2 treatments (fig. 2-B).
Carbon source:
the growth speed and sporulation quantity of verticillium lecanii LL-01 under different carbon sources are obviously different. The growth speed is fastest with fructose, the diameter of a bacterial colony after 10d of culture reaches 4.58cm, and is obviously larger than that of other 2 treatments; the sporulation yield is the highest with fructose and sucrose, and respectively reaches 2.38 multiplied by 108Spore/cm2And 2.24X 108Spore/cm2The difference was not significant, but significantly greater than starch (fig. 2-C).
Nitrogen source:
the growth speed and spore yield of verticillium lecanii LL-01 under different nitrogen sources are obviously different. The growth speed is the fastest with casein and glycine, the colony diameters reach 4.66cm and 4.63cm after 10d of culture, the difference between the two is not obvious, but both are obviously larger than sodium nitrate; the spore yield is the highest by casein, and reaches 3.16 multiplied by 108Spore/cm2Significantly higher than the other 2 treatments (fig. 2-D).
Example 3
Evaluation of biocontrol potential of entomopathogenic fungi:
and (3) determining pathogenicity: on the basis of optimizing culture conditions, taking Verticillium lecanii LL-01 cultured for 10 days, adding 10mL of sterile water containing 0.05% Tween-80 into a culture dish, scraping off conidia by using a sterile slide, filtering by using 3 layers of gauze to obtain a spore stock solution, and calculating the concentration of the spores after fully and uniformly mixing; diluting spore stock solution to 1 × 10 with sterile water8、1×107、1×106、1×105And 1X 104spores/mL 5 treatment concentrations, with 0.05% tween-80 sterile water as control, were replicated 3 times per treatment. Taking plastic preservation boxes with the diameter of 12cm and the height of 10cm for the mealybugs in the south ocean hip, and placing 1 annona squamosa fruit growing for 90d in each preservation box; digging air holes with the diameter of 6cm on the cover of each preservation box, and sticking 1 insect-proof net (80 meshes) with the diameter of 8cm on the air holes by using glue; picking up 30 healthy Nanyang gluteus wenshuensis sources respectively by using a writing brush, soaking the healthy Nanyang gluteus wenshuensis sources respectively in bacterial liquid with various concentrations for 10s, picking the Nanyang gluteus wenshuensis sources onto filter paper, airing the filter paper for 2min, picking the Annona squamosa fruits respectively, and covering the Annona squamosa fruits with a fresh-keeping cover. For lycoris radiata mealybugs, 1 piece of flower mud and 1 piece of filter paper with the same diameter are sequentially laid in a culture dish with the diameter of 9cm, the thickness of the flower mud is 0.5cm, 6mL of water is absorbed, 1 piece of succulent plant red heart lotus leaf is placed on the filter paper, and the petiole is wrapped by wet cotton and is tightly attached to the filter paper; then picking 30 heads of lycoris radiata mealybugs in each age source by using a soft writing brush, soaking the lycoris radiata mealybugs in the bacterial liquid with each concentration for 10s, picking the lycoris radiata mealybugs on filter paper, airing the filter paper for 2min, and picking the leaves of the lycoris radiata mealybugs respectivelyAnd (4) covering the culture dish. Placing the preservation box and the culture dish at the temperature of 26 +/-1 ℃ and the relative humidity of 90 +/-5 percent, wherein the light cycle is 6L: and (3) observing and removing dead insect bodies regularly every day in an MGC-350HP-2 type artificial climate box of 18D, confirming that hyphae growing from the insect bodies are effective infection, and observing until the death number of the test insects in all treatments stops changing. Counting the death rate of 2 quarantine mealybugs, and calculating the lethal medium concentration LC of verticillium lecanii LL-0150And lethal mesogenic LT in different concentrations of spore suspensions50。
And (3) measuring the activity of extracellular enzyme:
preparing extracellular enzyme solution: take 1X 108spore/mL of Verticillium lecanii LL-01 spore solution 1mL was added to 99mL of protease induction medium (gelatin 10g, K)2HPO4NaCl 0.3g and MgSO4·7H2O0.3 g each, dissolved in 1L phosphate buffer solution at a concentration of 0.02mol/L, pH ═ 7), chitinase induction medium (peptone 5g, K2HPO4KCl and MgSO 47·H2O0.5 g each, ZnSO4·7H2O1 g, colloidal chitin 10g, dissolved in 1L distilled water) or lipase induction medium (glucose 2g, peptone 5g, MgSO4·7H2O 0.1g，K2HPO41g, 20ml of olive oil dissolved in 980ml of distilled water), and culturing for 1-10 days on an QYC-2102C type full-temperature culture shaker at 25 ℃ and 180 r/min; respectively taking 2mL of 3 culture solutions every day, centrifuging for 30min by a KDC-140HR high-speed refrigerated centrifuge of 12000r/min, and taking supernate for measuring enzyme activity; each treatment was performed on 3 flasks, i.e., 3 replicates. Protease activity assay: casein is taken as a substrate; taking the inactivated protease solution as a blank control, and measuring the absorbance value of each group of reaction mixed liquor at 680nm by using a UV-1800PC type ultraviolet-visible spectrophotometer; and calculating the protease activity of each group of reaction mixed liquor according to the L-tyrosine standard curve, wherein the enzyme amount for catalytically decomposing casein to generate 1 mu mol of tyrosine per minute is 1 enzyme activity unit. Chitinase activity assay: taking colloidal chitin as a substrate; using inactivated chitinase solution as blank control, measuring absorbance value of each group of reaction mixed solution at 585nm, and catalytically decomposing chitin at every minuteThe enzyme amount for generating 1 mu mol of N-acetylglucosamine is 1 enzyme activity unit; and calculating the chitinase activity of each group of reaction mixed liquor according to the N-acetylglucosamine standard curve. Lipase activity: taking p-nitrophenylpalmitate as a substrate; taking the inactivated lipase solution as a blank control, and determining the absorbance value of each group of reaction mixed liquor at 410 nm; and (3) calculating the lipase activity of each group of reaction mixed liquor according to a p-nitrophenol standard curve, wherein the enzyme amount for catalyzing and decomposing fat into 1 mu mol of p-nitrophenol per minute is 1 enzyme activity unit.
The results show that:
pathogenicity:
verticillium lecanii LL-01 can effectively infect Nanyang gluteus wenchoe and Lycoris radiata (fig. 3-A, B), inoculate LC for one-year, two-year and three-year nymphs and female adults of 2 kinds of mealybugs after 10 days50Are respectively 0.98 multiplied by 105～9.71×105spores/mL and 0.50X 105～5.30×105spores/mL (table 1); the 2 kinds of mealybugs have various insect states of 1 × 104And 1X 105The death rate of spore/mL treatment is not 50%, and LT calculated by a probability value analysis method50The relative sizes of the insects are larger than the death stopping time of the test insects; mortality was significantly improved for all 3 other treatments, LT50All shorten with the increase of the concentration of the bacteria liquid, especially 1X 108Mortality rates at spore/mL treatment concentrations were 84.09% -97.62% and 89.89% -98.85%, respectively (FIG. 4), LT503.70-5.84 d and 3.48-5.14 d respectively (Table 2).
TABLE 1 LC of Verticillium lecanii LL-01 against Ericerus pela and Ericerus lycoris after 10d infection50
TABLE 2 LT of Verticillium lecanii LL-01 against Ericerus pela and Ericerus lycoris Leonii Leoni after 10d infection50
Extracellular enzyme Activity
Among the 3 kinds of extracellular enzymes of Verticillium lecanii LL-01, the change trends of the protease and the lipase activities are similar, the change trends of the protease and the lipase activities are basically similar, and the peak values are respectively reached to 19.44U/mL and 15.01U/mL at the 5 th d; the increase and decrease of lipase activity was relatively smooth and peaked at 6d at 7.68U/mL (FIG. 5).
In conclusion, the growth characteristics of the strain, such as growth speed, sporulation quantity and the like, are important indexes for judging whether the entomopathogenic fungi are excellent or not. The optimal temperature for the growth and spore production of verticillium lecanii LL-01 provided by the invention is 26 ℃, and the photoperiod is 6L:18D, fructose as carbon source and casein as nitrogen source, and the colony diameter and spore yield under the culture condition can reach 4.66cm and 3.16 × 10 respectively8Spore/cm2。
The pathogenicity is an important index for measuring the biocontrol potential and the application prospect of the entomopathogenic fungi. Cumulative fatality rate and LC of verticillium lecanii LL-01 to different insect ages of Nanyang gluteus went50And LT50Finally 84.09% -97.62% and 0.98 × 10 respectively5～9.71×105spores/mL and 3.70-5.84 d; cumulative fatality rate and LC for lycoris radiata mealybugs50And LT50Finally respectively reach 89.89 percent to 98.85 percent and 0.50 multiplied by 105～5.30×105spores/mL and 3.48-5.14 days. The activity values of extracellular enzymes such as protease, chitin, lipase and the like are considered to be one of main factors for determining pathogenicity of entomopathogenic fungi, the activity of the strain LL-01, namely the protease and the chitinase, has large change amplitude, and the time for reaching the peak value is short, so that the activity is supposed to be the main reason that the verticillium lecanii LL-01 can effectively break through infection death in the body walls of 2 quarantine mealybugs; the lipase activity reaches the peak for a long time, but the change amplitude is relatively stable, and the lipase activity is supposed to be another important reason that verticillium lecanii LL-01 can continuously break through and disrupt the body walls of 2 quarantine mealybugs.
Therefore, the verticillium lecanii LL-01 has high growth speed and high spore yield, has strong infection pathogenic activity on the Nanyang gluteus wenshuni and the lycoris radiata mealybugs, can be used for biological control of the 2 mealybugs, has the potential of replacing partial chemical prevention and control agents of the mealybugs, enriches the biological prevention microbial resources of the 2 quarantine mealybugs, promotes the application of entomopathogenic fungi such as verticillium lecanii and the like in the biological control of the mealybugs, can reduce the dosage of chemical pesticides and is beneficial to maintaining ecological balance.
Example 4
A pharmaceutical preparation comprises Verticillium lecanii LL-01.
The preparation method can adopt a conventional process, and the preparation formulation is an insecticide.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (5)
1. A Verticillium lecanii strain (Lecanicillium lecanii) LL-01, which is characterized in that: the preservation number is CGMCC No. 21940.
2. A pharmaceutical preparation comprising Verticillium lecanii LL-01 as defined in claim 1.
3. The agent of claim 2, wherein the agent is an insecticide and the target pests are mealybugs nanyanthes and mealybugs lycoris.
4. Application of verticillium lecanii LL-01 in plant biocontrol.
5. The use as claimed in claim 4, wherein the plant is annona squamosa and succulent "pistachio".
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