CN112322589A - Penicillium chrysogenum double-stranded RNA fungal virus for improving growth speed of beauveria bassiana hyphae - Google Patents
Penicillium chrysogenum double-stranded RNA fungal virus for improving growth speed of beauveria bassiana hyphae Download PDFInfo
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Abstract
The invention provides a penicillium chrysogenum double-stranded RNA fungal virus for increasing the growth rate of beauveria bassiana hyphae, which comprises SEQ ID NO 1 or a nucleotide sequence with at least 95%, 96%, 97%, 98% or 99% identity with the SEQ ID NO, SEQ ID NO 2 or a nucleotide sequence with at least 95%, 96%, 97%, 98% or 99% identity with the SEQ ID NO, SEQ ID NO 3 or a nucleotide sequence with at least 95%, 96%, 97%, 98% or 99% identity with the SEQ ID NO and SEQ ID NO 4 or a nucleotide sequence with at least 95%, 96%, 97%, 98% or 99% identity with the SEQ ID NO; and the use of the double-stranded RNA fungal virus of Penicillium chrysogenum for increasing the hyphal growth rate of Beauveria bassiana. The invention also provides a beauveria bassiana strain containing the double-stranded RNA fungal virus of penicillaceae.
Description
Technical Field
The invention relates to a novel double-stranded RNA fungal virus of penicillium chrysogenum, which can improve the hypha growth speed of Beauveria bassiana (Beauveria bassiana), a Beauveria bassiana strain containing the virus and application thereof.
Background
The beauveria bassiana has wide host range and is a biological control agent for controlling crop pests. Beauveria bassiana is widely applied to biological control of crop diseases and insect pests, and the conidia is mainly used for realizing the endogenous property and directly acting on target pests. Studies show that the toxicity of beauveria bassiana is positively correlated with the hypha growth speed, the hypha growth speed is positively correlated with the cell formation process, and the hypha growth speed is closely correlated with the metabolic pathways of the strain, such as lipid metabolism, transport and catabolism, amino acid metabolism and carbohydrate metabolism.
Studies have shown that nearly one fifth or more of the isolates of Beauveria bassiana from around the world contain double stranded RNA viruses. Mainly comprises Totiviviridae, Partitiviridae and Polymycoviridae, and unclassified Narnaviridae containing minimal genomes. At present, it has been reported that the double-stranded RNA virus in Beauveria bassiana belongs to Totiveria dae in a small amount, and to Partiveria dae containing 2 to 4 double-stranded RNAs in a part, and Polymycoviridae is composed of 4 or more double-stranded RNAs. To date, no double-stranded RNA virus of the Penicillium chrysogenum family (Chrysoviridae) has been found in the Beauveria bassiana double-stranded RNA virus classification.
In the aspect of research on the influence of the double-stranded RNA virus on the beauveria bassiana as a host, research shows that the colony growth speed and biomass of the beauveria bassiana strain containing the viruses of Polymycoviridae are lower than those of the strain without the viruses; in the research of insecticidal activity of endogenous viruses on beauveria bassiana, it is reported that the partiiviridae viruses reduce the insecticidal activity of the beauveria bassiana, and the viruses of the family Polymycoviridae improve the toxicity of the beauveria bassiana strains on the greater wax moth. The effect of double-stranded RNA viruses of Penicillium chrysogenum (Chrysoviridae) on the growth rate of beauveria bassiana hyphae has not been reported.
Disclosure of Invention
The invention aims to provide a fungus endogenous double-stranded RNA virus which is positively correlated with the toxicity of beauveria bassiana on pests and the growth speed of bacterial colonies, negatively correlated with the spore yield of the beauveria bassiana and irrelevant with the germination rate of beauveria bassiana spores, and is expected to be used for the purpose of improving the toxicity of the beauveria bassiana in the process of preventing and treating the pests.
In one aspect, the present invention provides a double-stranded RNA virus of Penicillium chrysogenum (Chrysoviridae) capable of increasing the growth rate of beauveria bassiana hyphae.
The virus is endogenous in Beauveria bassiana strain BbOFZK152 isolated from larva of Ostrinia furnacalis Guenee in corn field in pear county of Jilin province. The strain is preserved in China general microbiological culture Collection center (CGMCC) at 3 months and 18 days in 2020, and the preservation number of the strain is CGMCC 19371.
Thus, in one embodiment, the present invention relates to a double stranded RNA fungal virus of the family Penicilliaceae carried by Beauveria bassiana (Beauveria basssaana) with a accession number of CGMCC 19371.
In one embodiment, the invention provides a double stranded RNA fungal virus of the family penicillium chrysogenum comprising SEQ ID No. 1 or a nucleotide sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto, SEQ ID No. 2 or a nucleotide sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto, SEQ ID No. 3 or a nucleotide sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto and SEQ ID No. 4 or a nucleotide sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto.
In one embodiment, the invention provides a double stranded RNA fungal virus of the family penicillium chrysogenum consisting of SEQ ID No. 1 or a nucleotide sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto, SEQ ID No. 2 or a nucleotide sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto, SEQ ID No. 3 or a nucleotide sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto and SEQ ID No. 4 or a nucleotide sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto.
In one embodiment, the invention provides a double stranded RNA fungal virus of the family Penicilliaceae comprising the nucleotide sequences set forth in SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4.
In one embodiment, the invention provides a double-stranded RNA fungal virus of the family Penicilliaceae consisting of the nucleotide sequences shown in SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4.
In one embodiment, the present invention provides a method for increasing the rate of hyphal growth of beauveria bassiana using a double-stranded RNA fungal virus of the family penicillaceae.
In one embodiment, the present invention provides a method for controlling plant pests comprising the step of infecting said penicillaceae double stranded RNA fungal virus with beauveria bassiana.
The inventor of the application unexpectedly finds that by transferring the virus into the beauveria bassiana, a new strain with high toxicity is successfully created, so that the toxicity of the beauveria bassiana on pests is improved, and the production and control cost is reduced.
In another aspect, the present invention provides a strain of beauveria bassiana comprising the double-stranded RNA fungal virus of the penicillaceae family.
In one embodiment, the strain has a accession number of CGMCC 19371.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present specification will control. Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
Drawings
FIG. 1 is an agarose gel electrophoresis image of the result of dsRNA extraction.
FIG. 2 is a diagram showing the alignment of dsRNA 1.
FIG. 3 is a diagram showing the alignment of dsRNA 2.
FIG. 4 is a diagram showing the alignment of dsRNA 3.
FIG. 5 is a diagram showing the alignment of dsRNA 4.
FIG. 6 shows the relationship between the growth rate of double-stranded RNA virus and Beauveria bassiana.
FIG. 7 shows the relationship between double-stranded RNA viruses and the spore yields of Beauveria bassiana.
FIG. 8 shows the relationship between double-stranded RNA viruses and the germination rates of Beauveria bassiana.
FIG. 9 shows the superposition of hyphae growth 15 days after the strain was grown on the counter.
FIG. 10 shows the dsRNA extraction and verification electrophoretogram of the screened strain, wherein M is DNA marker, lanes 1-5 are BbOFZK152, and lanes 6-10 are recipient strain BbOFDH 1-5.
FIG. 11 shows the PCR detection electrophoresis of the screened strains, where M is DNA marker, lanes 1-5 are BbOFZK152, and lanes 6-10 are recipient strain BbOFDH 1-5.
FIG. 12 shows the effect of Beauveria bassiana virus on the growth rate of host hyphae.
Detailed Description
Example 1 isolation and identification of Beauveria bassiana double-stranded RNA Gene
1.1 isolation of double-stranded RNA of Beauveria bassiana Strain
Approximately 0.5g of the mycelia were ground to a powder under liquid nitrogen and transferred to a 2ml centrifuge tube. Mu.l of 2XSTE buffer (release, preservation of nucleic acids), 400. mu.l of phenol-chloroform isoamyl alcohol (25: 24: 1), 183. mu.l of 10% SDS, shaking at room temperature for 10min, and centrifuging for 10min (4 ℃ C., 12000r) were added in this order. Taking 800 μ l of supernatant, adding 0.05g of cellulose powder and 152 μ l of anhydrous ethanol (constant volume is about 15% -16%), filling with 17% ethanol STE solution, performing ice bath for 10min, and centrifuging for 3-5min (4 ℃ C. 12000 r). The supernatant was discarded, and 1ml of 17% ethanol 1XSTE washing buffer was added thereto, followed by mixing, shaking and centrifugation for 1min (4 ℃ C., 12000 r). Repeatedly cleaning for 3-4 times. Discard the supernatant, suck the residual liquid as much as possible with a 200. mu.l pipette, add 640. mu.l 1XSTE, mix well, shake for 5min, stand, centrifuge for 3min (4 ℃ C. 12000 r). Add 600. mu.l to 1.5ml EP tube (sterilized or DEPC treated), add 0.1 volume of 3M NaAC and equal volume of isopropanol, mix well, precipitate at-20 deg.C (over 30 min), centrifuge for 30min (4 deg.C, 12000 r). The supernatant was discarded, washed with 1ml of pre-cooled 75% ethanol, precipitated and centrifuged for 2min (4 ℃, 12000 r). The wash solution was aspirated off with a 200. mu.l pipette and dried at 37 ℃ for 1min (30s staged drying to prevent over-drying). Add 45. mu.l DEPC water and store at-20 ℃.
1.2 viral genome amplification
Viral ds RNA genomic sequence full length cloning strategy: random primer cloning, specific primer mid-gap cloning and end sequence cloning were mainly performed to obtain the full-length genomic sequence of viral ds RNA.
Viral genome reverse transcription process:
using purified dsRNA as a template and a random primer RACE3RT as a reverse transcription primer, adding:
after mixing, reacting for 10-15min at 95-98 ℃ in a PCR instrument, rapidly cooling on ice for 5min, and after completion, sequentially adding a reverse transcription reagent into the reaction solution:
after mixing, the reaction was carried out in PCR. The reaction conditions are as follows: reacting at 25 deg.C for 5min, incubating at 42 deg.C for 1h, heating at 85 deg.C for 5min, storing at 4 deg.C, and terminating the reaction procedure.
RNA degradation and cDNA renaturation in viral dsRNA:
adding 25ul of the reaction solution after reverse transcription into 2.5ul of 1M NaOH solution, reacting for 1h in a 65 ℃ water bath pot or a PCR instrument, cooling at room temperature after the reaction is finished, sequentially adding 2.5ul of 1M Tris-HCl solution, mixing uniformly, adding 2.5ul of 1M HCl solution, mixing uniformly, and placing in a 65 ℃ water bath pot for renaturation for 1-16 h.
Performing PCR amplification of unknown fragments on the cDNA products synthesized by reverse transcription:
the reaction procedure is as follows: pre-denaturation at 95 deg.C for 3min, denaturation at 95 deg.C for 15s, annealing at 56 deg.C for 20s, extension at 72 deg.C for 1min, circulation for 36 times, extension at 72 deg.C for 5min, and storage at 4 deg.C.
After the PCR process is finished, loading the PCR product into agarose gel with the concentration of 1% for electrophoresis detection to obtain a diffuse band, cutting the diffuse band with the length of more than 500bp for purification and recovery, and finally dissolving in 25-30ul ddH2And (4) in O.
T-A cloning, transformation, colony PCR, detection of recovered product:
connecting the purified and recovered fragment with a PMD18-T vector, transferring the fragment into DH5 alpha escherichia coli competent cells, culturing at the constant temperature of 37 ℃ for 12 hours, selecting a single colony, and identifying positive clones, wherein the process is as follows: firstly, selecting a single colony to be repeatedly blown and sucked into a PCR tube with 10ul of sterilized water to be uniformly mixed; adding 5.5ul of bacterial liquid into 300ul of LB + Amp + liquid culture medium, and performing thallus culture at 37 ℃; ③ 4.5ul of bacterial liquid is used for colony PCR, and the reaction system and the reaction conditions are as follows:
and (3) PCR reaction conditions: pre-denaturation at 95 deg.C for 3min, denaturation at 95 deg.C for 15s, annealing at 54 deg.C for 20s, extension at 72 deg.C for 1min, circulation for 36 times, extension at 72 deg.C for 5min, and storage at 4 deg.C.
Fourthly, after 1 percent agarose gel electrophoresis, selecting positive clones and sending the positive clones to a company Limited Biotechnology (Shanghai) for sequencing.
Splicing of viral genome sequences: after sequencing the nucleic acid sequence obtained by cloning the random primer, searching and comparing the nucleic acid sequence through a BLAST online program in an NCBI database, determining the possible source of the extracted ds RNA according to the fed-back final result, splicing the sequence obtained after cloning by using DNASTAR software, and assembling nucleic acid sequence fragments with different sizes into contigs to obtain the size of an unknown sequence. And designing a virus specific primer and a random primer pair to obtain a full-length genome sequence, and verifying again to determine the correctness of the nucleic acid sequence.
1.3. Viral genome sequence alignment identification
The "Sequence and" Protein "functional regions in the DNAMAN software and the NCBI online program ORF-Finder were used to predict the open reading frames and their sizes that the viral dsRNA genome might encode.
The NCBI online program CDD-search was used to analyze the amino acid sequences that the new viral dsRNA genome might encode and the conserved domains that they encode.
1.4. Results
The electropherogram of the dsRNA extraction results is shown in fig. 1. After the sequencing, the full-length genome sequence of the virus is found to be composed of the following four parts: the nucleotide sequences of the dsRNA1, the dsRNA2, the dsRNA3 and the dsRNA4 are shown as SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4 respectively. These sequences are described in more detail below.
The nucleotide sequence of dsRNA1 is shown in SEQ ID NO. 1. The dsRNA1 alignment is schematically shown in figure 2.
Blast homology alignment indicates a maximum homology of 70.71% with known fungal virus nucleic acid sequences, and analysis of the ORF and conserved domains shows that the viral dsRNA1 encodes its RNA-dependent RNA polymerase (RdRp). Therefore, the virus is a new strain of the fungal double-stranded RNA virus and is named Beauveria basssiana chrysovirus 1(BbCV 1).
dsRNA 2: the sequence is shown in SEQ ID NO. 2. The dsRNA2 alignment is schematically shown in figure 3.
Blast homology comparison shows that the homology with the nucleic acid sequence of known fungal virus is up to 66.77%, and the homology with the amino acid sequence of known virus coat protein is up to 64.54%.
dsRNA 3: the sequence is shown in SEQ ID NO. 3. The dsRNA3 alignment is schematically shown in figure 4.
Blast homology alignment shows that the homology with the known fungal virus nucleic acid sequence is 68.83% at the highest, and the homology with the known virus amino acid sequence is 56.36% at the highest.
dsRNA 4: the sequence is shown in SEQ ID NO. 4. The dsRNA4 alignment is schematically shown in figure 5.
Blast homology alignment shows a homology of up to 67.88% with the nucleic acid sequence of known fungal viruses and 63.79% with the amino acid sequence of the coat protein of known viruses.
The result of the alignment of dsRNA1 shows that the first dsRNA chain of the virus encodes the RdRp gene from top to bottom corresponding to the result of an electrophoretogram. The second dsRNA strand encodes its coat protein. The proteins encoding the third and fourth strands, dsRNA3 and dsRNA4, are unknown. According to the comparison result of the RdRp gene, the virus is Beauveria basssiana chrysovirus 1m (BbCV1) of Penicillium chrysogenum.
Example 2 determination of the Effect of double-stranded RNA viruses on host biological Activity during subculture
2.1 Beauveria bassiana Strain subculture
Inoculating beauveria bassiana strain BbOFZK152 identified by morphology and molecular biology on a PDA culture medium, culturing in a constant temperature incubator at 26 ℃ for 10d, scraping off conidia after conidia are produced, putting into a centrifuge tube containing 20mL of sterile 0.1% (v/v) Tween-80 aqueous solution, performing vortex oscillation for 2min, counting by using a blood counting cell counting plate, and preparing into 1 × 107Taking 100 microliter of spore suspension liquid, uniformly coating the spore suspension liquid on a PDA culture medium, placing the PDA culture medium in a constant-temperature incubator at the temperature of 26 ℃ for subculture for 14 days, and continuously culturing for 5 generations. The culture of the 1 st, 3 rd and 5 th generations is taken to carry out the fluorescent quantitative PCR determination of the biological characteristics and the virus content.
2.2 spore yield determination
Preparing conidia of different generations of beauveria bassiana culture into 1 × 10 with sterile 0.1% (v/v) Tween-80 aqueous solution71mL of spore suspension is evenly coated on PDA culture medium and is placed in a constant temperature incubator at the temperature of 26 ℃ for culture for 14 d. Using a 3.5mm diameter punch from the center to the edge 1 of the plateThe colonies were removed at position 2, placed in a centrifuge tube containing 20mL of sterile 0.1% (v/v) Tween-80 aqueous solution, vortexed for 2min, and the amount of spores produced was measured and calculated using a hemocytometer. 3 colonies were randomly selected per plate, and 3 plates were set for each beauveria bassiana isolate as replicates.
2.3 determination of Germination Rate
After spores grow out of the Beauveria bassiana culture subjected to subculture, selecting a proper amount of spores by using an inoculating loop, inoculating the spores into an SDY liquid culture medium, performing shake culture at the temperature of 26 ℃ at 200r/min for 24 hours, performing microscopic examination, respectively observing the spore germination conditions under different visual fields, and calculating the germination rate. Each beauveria bassiana isolate was repeated 3 times.
2.4 determination of colony growth Rate
In the subculture of beauveria bassiana of each generation, 3 single spores of beauveria bassiana isolates were randomly picked and inoculated into a PDA culture medium. Observing once a day, measuring the diameter of the bacterial colony by using a vernier caliper, and observing for 10d. The colony growth rate (cm/d) is colony diameter/10.
2.5 fluorescent quantitative PCR determination of Beauveria bassiana Virus content
0.1g of beauveria bassiana culture of different generations is collected respectively, and Total RNA is extracted by using a Trizol method. First strand cDNA synthesis was performed using AVM synthesis Kit II (Omega). Designing a specific primer according to a virus RdRp (RNA-depedent RNA polymerase) gene, adopting a 2xSG Fast qPCR Master Mix (Shanghai biological engineering Co., Ltd.) kit, using a beauveria bassiana cal (calmodulin) gene as an internal reference gene in a real-time fluorescent quantitative PCR system Quanstudio 6Flex, carrying out expression difference analysis on the beauveria bassiana virus RdRP gene, and respectively detecting the expression quantity of the beauveria bassiana virus RdRP gene of the 1 st generation, the 2 nd generation and the 5 th generation after the beauveria bassiana subculture by a relative quantitative method. Each experimental sample is subjected to 3 independent biological repetitions, the relative expression level is calculated by a 2-delta CT method, and the change of beauveria bassiana virus in subculture is observed.
Real-time PCR reaction system and reaction program
(1) The reaction system is as follows:
(2) the qPCR cycle program was run as follows:
2.6 statistical analysis
Statistical analysis is carried out by adopting software SPSS 26.0, homogeneity of variance (homogeneity of variance) detection method is adopted for homogeneity of variance detection, One-way ANOVA is used for difference significance analysis, and multiple comparison is carried out by adopting LSD method. The sporulation yield, spore germination rate, colony growth speed and the relativity of the toxicity of the beauveria bassiana on the larvae of the ostrinia nubilalis to the virus content are subjected to unitary linear regression analysis by adopting software SPSS 26.0, a regression model is established, and the influence degree of each variable on dependent variables is evaluated. The software Sigmaplot 12.5 was used for plotting.
2.7 results
The biological activity of the double-stranded RNA virus was determined by subculture, and FIG. 6 shows the relationship between the double-stranded RNA virus and the growth rate of Beauveria bassiana; FIG. 7 shows the relationship between double-stranded RNA virus and the spore yield of Beauveria bassiana; FIG. 8 shows the relationship between double-stranded RNA viruses and the germination rates of Beauveria bassiana.
Specifically, the host strain is subjected to 1-5 generations of subculture, the spore yield, the spore germination rate and the colony growth speed of each generation of beauveria bassiana culture are measured, and the result is combined with the fluorescent quantitative PCR measurement of the virus content of each generation in the subculture process, so that the virus content is continuously reduced along with the beauveria bassiana subculture, is positively correlated with the colony growth speed of the beauveria bassiana (figure 6), is negatively correlated with the spore yield of the beauveria bassiana (figure 7), is not correlated with the spore germination rate of the beauveria bassiana (figure 8), and is positively correlated with the toxicity of the beauveria bassiana to the host.
Example 3 Interspecies spread of Beauveria bassiana Virus and Effect on the biological Activity of Beauveria bassiana
3.1 Virus transfer between Beauveria bassiana Virus strains
Respectively activating virus-containing strain BbOFZK152 and laboratory engineering strain BbOFDH1-5 (expressing bar gene), performing dark culture at 26 deg.C for 3d, and respectively taking primary hypha edges of the two strains with a perforator with diameter of 5 mm. And (3) carrying out confrontation culture on a new PDA culture medium, repeating for three times, and carrying out 26 ℃ till hyphae of the two strains grow and coincide. And (3) taking a mycelium block on one side containing the resistance gene at the mycelium boundary to a newly-screened resistance PDA culture medium (with the glufosinate concentration of 200ng \ mul) by using a 5mm puncher, culturing for 15d, collecting conidia, carrying out monospore separation, randomly separating 5 monospores, inoculating to the sterilized PDA culture medium, carrying out dark culture for 15 days, and collecting the mycelium for virus detection.
3.2 Beauveria bassiana Virus detection
And (4) carrying out dsRNA extraction verification on the hypha of the strain after extraction and screening, carrying out primary screening, and removing the strain without dsRNA. And (3) extracting total RNA of the primary screening strain, carrying out reverse transcription on the total RNA, taking the obtained cDNA as a template, and carrying out reverse transcription on the total RNA by using a primer BbCV-F: CCGCCACTTGACGCCGTATAAC, respectively; BbCV-R: CTTCGTGTTCGGGCATGGGATG, PCR amplification was performed for virus-specific sequences. The PCR procedure was as follows:
3.3 Effect of Beauveria bassiana Virus on host hyphal growth Rate
The test method is the same as 2.4, and 3 single spores of beauveria bassiana isolates are randomly picked from the beauveria bassiana strain culture to be tested.
3.4 statistical analysis
The method is the same as 2.6.
3.5 results
The interpenetration of beauveria bassiana virus strains and the effect on the growth rate of beauveria bassiana hyphae are shown in fig. 9, which shows that the hyphae grow coincidently 15 days after the strains are cultured in opposition.
After the virus-containing strain BbOFZK152 and the receptor strain BbOFDH1-5 are subjected to opposite culture (figure 9), single spore separation and propagation expansion are carried out on conidia obtained by one side of the receptor strain through glufosinate-ammonium selection pressure, and double-stranded RNA extraction is carried out, so that the result shows that all the isolates contain double-stranded RNA (figure 10), and RT-PCR verification is carried out by using virus specific primers on the basis, so that the result shows that all the isolates are positive, and the virus has been horizontally transmitted to the receptor strain BbOFDH1-5 by the donor strain BbOFZK 152.
The effect of beauveria bassiana virus on the growth rate of host hyphae is shown in fig. 12. The growth rate of the corresponding group of bacteria was obtained by measuring the hypha diameter of five control strains and five treatment strains (containing BbCV1) for 10d continuously. Through the analysis of the difference significance of the growth rates of the two groups, the result shows that the growth rate of the hyphae of the strain containing the virus is higher than that of the strain without the virus, and the difference is extremely significant. BbCV1 was shown to increase the rate of hyphal growth.
While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.
Reference to the literature
[1]Mascarin GM,Jaronski ST(2016)The production and uses of Beauveria bassiana as a microbial insecticide.World J Microb Biot 32:177.https://doi.org/101007/s11274-016-2131-3
[2]Patricia R.DALZOTOa,Chirlei GLIENKE-BLANCOb,Vanessa KAVA-CORDEIRO,et al.,(2006)Horizontal transfer and hypovirulence associated with double-stranded RNA in Beauveria bassiana.mycological research IIO 1475–1481
[3]Se Won Yie,Mahmoud E.Khalifa,Torque Hahn,et al.,(2014)Molecular characterization of a novel victorivirus from the entomopathogenic fungus Beauveria bassiana,Arch Virol 159:1321–1327
[4]Ioly Kotta-Loizou,Jana Sipkova,Robert H.A.Coutts.(2015)Identification and sequence determination of a novel double-stranded RNA mycovirus from the entomopathogenic fungus Beauveria bassiana.Arch Virol160:873–875
[5]Igor Koloniuk,Lenka Hraba′kova′,Karel Petrzik.(2015)Molecular characterization of a novel amalgavirusfrom the entomopathogenic fungus Beauveria bassiana.Arch Virol 160:1585–1588
[6]Ioly Kotta-Loizou1,Robert H.A.Coutts.(2017)Studies on the Virome of the Entomopathogenic Fungus Beauveria bassiana Reveal Novel dsRNA Elements and Mild Hypervirulence.PLOS Pathogens|DOI:10.1371/journal.ppat.1006183
[7]Najie Shi1,Guogen Yang,Ping Wang,et al.,(2019)Complete genome sequence of a novel partitivirus from the entomogenous fungus Beauveria bassiana in China.Archives of Virology https://doi.org/10.1007/s00705-019-04428-1
[8]Zhengkun Zhang,Yang Lu,Wenjing Xu,Li Sui,Qian Du,Yangzhou Wang,Yu Zhao and Qiyun Li*,(2020)Influence of genetic diversity of seventeen Beauveria bassiana isolates from different hosts on virulence by comparative genomics..BMC genomics,https://doi.org/10.1186/s12864-020-06791-9
[9]Zhengkun Zhang,Yang Lu,Wenjing Xu,Qian Du,Li Sui,Yu Zhao,Qiyun Li*,(2019)RNA sequencing analysis of Beauveria bassiana isolated from Ostrinia furnacalis identifies the pathogenic genes.Microbial Pathogenesis,https://doi.org/10.1016/
Sequence listing
<110> Jilin province academy of agricultural sciences
<120> a double-stranded RNA fungal virus of Penicillium chrysogenum family for increasing growth rate of beauveria bassiana hypha
<130> case number
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3441
<212> DNA
<213> Beauveria bassiana chrysovirus 1(BbCV1)
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<212> DNA
<213> Beauveria bassiana chrysovirus 1(BbCV1)
<400> 2
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<213> Beauveria bassiana chrysovirus 1(BbCV1)
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<212> DNA
<213> Beauveria bassiana chrysovirus 1(BbCV1)
<400> 4
Claims (7)
1. A Penicillium chrysogenum (Chrysoviridae) double stranded RNA fungal virus comprising SEQ ID NO 1 or a nucleotide sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto, SEQ ID NO 2 or a nucleotide sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto, SEQ ID NO 3 or a nucleotide sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto and SEQ ID NO 4 or a nucleotide sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto.
2. A double stranded RNA fungal virus of the family Penicilliaceae consisting of SEQ ID NO 1 or a nucleotide sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto, SEQ ID NO 2 or a nucleotide sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto, SEQ ID NO 3 or a nucleotide sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto and SEQ ID NO 4 or a nucleotide sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto.
3. A double-stranded RNA fungal virus of Penicillium chrysogenum family carried by Beauveria bassiana (Beauveria bassiana) with a collection number of CGMCC 19371.
4. Use of the double-stranded RNA fungal virus of Penicilliaceae family produced according to any one of claims 1 to 3 for increasing the hyphal growth rate of Beauveria bassiana.
5. A method for controlling plant pests comprising the step of infecting beauveria bassiana with the double-stranded RNA fungal virus of penicillaceae family chrysogenum according to claim 1 or 2.
6. A strain of Beauveria bassiana (Beauveria bassiana) comprising the double-stranded RNA fungal virus of penicillaceae family according to claim 1 or 2.
7. Beauveria bassiana (Beauveria bassiana) with the preservation number of CGMCC 19371.
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