CN112553086B - Violet purpurea bacterial strain and application thereof in preventing and treating phyllotreta striolata - Google Patents
Violet purpurea bacterial strain and application thereof in preventing and treating phyllotreta striolata Download PDFInfo
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Abstract
The invention relates to a lilac violet spore bacillus strain and application thereof in the aspect of controlling phyllotreta striolata, belonging to the technical field of biological pesticides. The purpleleaf violet (Purpureocillium lilacinum) PlSC59A3 strain is preserved in Guangdong province microorganism strain preservation center with the preservation date of 2020, 10 months and 16 days, and the preservation number is GDMCC No: 61232. the lilac violet spore bacterium PlSC59A3 strain provided by the invention is highly effective in preventing and treating phyllotreta striolata, is an entomopathogenic fungus, is used as a biological pesticide, has the characteristics of no pollution to the environment, difficulty in generating drug resistance of pests and no toxicity to people and livestock, and is suitable for the production requirement of green healthy agricultural products.
Description
Technical Field
The invention relates to a lilac violet spore bacillus strain and application thereof in the aspect of controlling phyllotreta striolata, belonging to the technical field of biological pesticides.
Background
Phyllotreta striolata belongs to Coleoptera and phylloplasmaceae pests, commonly known as dog lice, vegetable fleas, ground fleas, yellow fleas and the like, and is called flea beetle for short, which is an important pest of cruciferous crops in China. The larva and imago of the flea beetle can damage crucifer crops, the larva lives in soil, gnaws seed roots and buds, and plant roots in adult plant period, so that seedling shortage, ridge breaking, plant wilting and even withering are caused; the adult gnaws the leaves and the tender tips to enable the leaves to form dense oval perforations, and the tender stems of the sprouts are bitten off to cause the sprouts to be dry and even dead. The imagoes have strong jumping capability and are convenient to migrate, so that the damage range is expanded, and the imagoes can cause destructive damage to the vegetables in the seedling stage when serious. The phyllotreta striolata occurs 3-5 generations in the north of China in one year and 7-8 generations in the south of China in one year, the generations are seriously overlapped, the phyllotreta striolata can be propagated in the south of China all the year round, the propagation quantity is large, and the harm is the most serious in spring and autumn.
Spraying a chemical insecticide is a main method for controlling the damage of flea beetles at present, only spraying foliage to control adults is generally adopted in actual production, the control of underground larvae is neglected, and the adults can fly and jump well and easily avoid sprayed chemical agents, and the coleoptera covers the body to ensure that liquid medicine is not easy to attach and permeate, so that the control effect is poor. In order to control the harm, the pesticide is mixed blindly, the dosage is increased, and the dosage frequency is improved, so that the formation and development of the drug resistance of the flea beetle are accelerated, and the vicious circle of pesticide residue of agricultural products exceeding the standard, destroying ecology and the like is caused. The development of the biological control technology of flea beetles is not slow.
Entomogenous fungi are important pest biocontrol resources, soil is an important living place of the entomogenous fungi, and the entomogenous fungi can be used for preventing and controlling flea beetle states in the soil and also can be used for preventing and controlling adult flea beetles on the ground, can supplement and improve the distribution of microorganisms in the soil, and is beneficial to continuously controlling the harm brought by the flea beetles. Related researches on biological control of flea beetles are carried out at home and abroad, and comprise screening and utilizing parasitic natural enemies, predatory natural enemies, insect pathogenic bacteria, nematodes, fungi and the like, wherein a plurality of insect pathogenic bacteria can infect the phyllotreta striolata beetles, such as Metarhizium flaviviridum, Metarhizium anisopliae, Beauveria bassiana and Beauveria bassiana, and bacteria such as Serratia marcescens and Bacillus firmus, and a plurality of related patent publications are disclosed.
Patent application No. CN200510033568.4 discloses a beauveria bassiana strain and application thereof, wherein the beauveria bassiana strain is obtained by separating from a naturally infected petiolatus apetalus body and is effective to the petiolatus beetle and phyllotreta striolata; the patent application number CN201510084020.6 discloses an application of metarhizium anisopliae CQMa421 strain in the preparation of a medicament for preventing and treating phyllotreta striolata or diamondback moth, the strain can infect the phyllotreta striolata and the diamondback moth, the fatality rates can respectively reach 60.9% and 70.8%, and the metarhizium anisopliae CQMa421 strain prepared by the method for enhancing the toxicity has the fatality rate of 100% and has very outstanding prevention and treatment effects; the patent application number CN201610276234.8 discloses a microbial pesticide containing bacillus subtilis, which has good killing effect on phyllotreta striolata; patent application No. CN2013104943. X discloses a serratia marcescens PS-1 strain separated from phyllotreta striolata and application thereof, which have strong pathogenicity on phyllotreta striolata larvae and adults and asparagus caterpillar larvae and adults; patent application No. CN201611000946.3 discloses a Bacillus thuringiensis HAN055 preparation with poisoning activity on phyllotreta striolata and application thereof, and the preparation prepared from the strain has remarkable prevention and treatment effects on phyllotreta striolata. Therefore, at present, researches on prevention and treatment of flea beetles by utilizing microbial pesticides are rare, and the number of strains and strains which can be selected is small.
The purple lilac fungus Purpureocillium lilacinum is a common soil inhabitant, and belongs to Fungi of the kingdom Fungi, Ascomycota of Ascomycota, Sordariomycetes of Ascomycota, Hypocrea of Hypocreales, Purpureocillium of Ophiopedia of nematodiaceae. The bacterium is commonly used for controlling crop root-knot nematodes and has control effect on some insects, but no research report for controlling flea beetles is found.
Disclosure of Invention
The invention aims to solve the technical problem of providing a material for developing a new fungal pesticide and providing a high-efficiency lilac violet spore bacterial strain for preventing and treating striped flea beetle.
The invention also aims to provide application of the purple lilac spore strain in controlling phyllotreta striolata.
In order to achieve the purpose, the invention adopts the technical scheme that: a purple lilac (Purpureocillium lilacinum) strain PLSC59A3, which is preserved in Guangdong province collection of microorganisms with the preservation date of 2020, 10 months and 16 days, and the preservation number is GDMCC No: 61232.
the strain is obtained by separating and purifying soil samples collected from the city of Nangac, Sichuan province and is obtained by biological activityScreening, and finding that the strain is the strain with the highest activity on flea beetles adults in all the tested strains, wherein the activity is 1 multiplied by 108Treatment at spores/mL achieved a corrected mortality rate of 96.30% for the flea beetle adults (Table 1). semi-Lethal Concentration (LC) of 5d, 7d, 9d and 11d after treatment on flea beetle adults50) Are respectively 4.11 multiplied by 106spores/mL、1.03×106spores/mL、0.75×106spores/mL and 0.39X 106spores/mL; at 5X 106spores/mL、1×106spores/mL and 0.5X 106Half-lethal time LT at spores/mL treatment concentration50For 5.9, 9.1 and 13.3 days, respectively (table 2).
The morphological characteristics of said rhodosporidium lilacinum strain PlSC59a 3: as shown in FIG. 1, in the PDA culture medium, the colony was initially white and villous on the surface; the colony is circular when cultured on day 7, the center of the colony is slightly raised, the middle part of the surface is white, the edge is light purple, the hair is villous, the back surface is light purple, and the diameter is (42.30 +/-0.07) mm.
The microstructure of the purple lilac strain PlSC59a 3: as shown in FIG. 2, the hyphae had septa, branches and were transparent under an optical microscope, and had a width of (2.87. + -. 0.11) μm; conidia are single cells, are elliptical to circular, are chain-shaped, smooth, colorless to light yellow, and have the width of 2-4 mu m, irregular branches or recurrent branches with bottle necks are arranged on the sporophores, and the base parts of the bottle stalks are wider. The recurrent branches consist of cylindrical parts, sometimes the branches curve away from the main axis.
The strain is identified in molecular biology, r DNA-ITS gene sequences of the PlSC59A3 strain are amplified by adopting universal primers ITS1 and ITS4, amplification products are separated by electrophoresis (figure 3), and sequencing results show that the amplification products are 559bp in length and the base sequence of the amplification products is shown as SEQ ID NO. 1. The sequence is obtained by Blast in NCBI database, and the comparison result shows that the sequence of the PlSC59A3 strain is highly similar to the sequence of a plurality of lilac purple spore strains; the r DNA-ITS gene sequences of the PlSC59A3 strain, the lilac purple spore strain, the green muscardine fungus and the white muscardine fungus are constructed through MEGA software, and the result shows that the PlSC59A3 strain and all the lilac purple spore strains are on the same branch, and the genetic relationship is high homology (figure 4).
Combining the above morphology and the identification of the ITS1-5.8S-ITS4rDNA sequence phylogenetic tree, the strain PlSC59A3 is classified as belonging to the species Purpureocillium lilacinum of Ascomycota (Ascomycota), Hypocreales (Hypocrea), nemataceae (Ophiococcus, Hypoccipitaceae), and Purpureocillium (Purpureocillium).
When the PlSC59A3 strain infects phyllotreta striolata adults, the phyllotreta striolata appears to have the phenomena of slow trend and cachexia after 2-3 days of treatment, and then die in succession. Under an optical microscope, it can be observed that white hyphae firstly extend out of the body from the dense bristle parts such as the antenna, the foot joints, the chest and the feet and the internode membrane of the phyllotreta striolata corpse, and the whole polypide is wrapped by the white hyphae with the passage of time, and then a large number of conidia are generated (fig. 5).
The invention also provides application of the purple lilac spore bacterium PlSC59A3 strain in preventing and treating phyllotreta striolata. The application is that the PlSC59A3 strain is used for producing conidia, then the conidia are prepared into a spore suspension by using a 0.05% Tween-80 solution, and the spore suspension is applied to the field by adopting a foliar spray method in the period of flea beetle occurrence. The application can also process conidia into a preparation for field flea beetle control.
Compared with the prior art, the invention has the beneficial effects that:
(1) the lilac purple spore strain PlSC59A3 has high efficiency on preventing and treating phyllotreta striolata;
(2) the strain is an entomopathogenic fungus, is used as a biological pesticide, has the characteristics of no pollution to the environment, difficult generation of drug resistance of pests and no toxicity to people and livestock, and is suitable for the production requirement of green healthy agricultural products.
Drawings
FIG. 1 shows the colony morphology of the strain PlSC59A3 cultured on potato dextrose medium (PDA) at 25. + -. 1 ℃ for 12 days; wherein, the left picture is the front of the bacterial colony, and the right picture is the back of the bacterial colony.
FIG. 2 shows the spore-forming structure and conidia of the purple lilac strain PlSC59A 3.
FIG. 3 is an ITS1-5.8S-ITS4rDNA gene specific amplification electrophoresis picture of the purple lilac fungus PlSC59A3 strain.
FIG. 4 shows phylogenetic trees of different strains constructed with ITS1-5.8S-ITS4rDNA gene sequences.
FIG. 5 shows the change of symptoms of Phyllotreta striolata infected with the Strain Lawsonia lilacinus PlSC59A 3; wherein, FIG. 5A shows Phyllotreta striolata adults 4 days after treatment; FIG. 5B shows Phyllotreta striolata adults 7 days after treatment; FIG. 5C shows Phyllotreta striolata adults 12 days after treatment.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the following detailed drawings and examples.
Example 1: isolation and screening of the Strain of purple lilac fungus PlSC59A3
Inoculating slant strain of test strain on PDA plate, culturing for 2-3 weeks, collecting conidia, suspending conidia with 0.05% Tween-80 solution, measuring spore concentration by hemacytometer method, and adjusting suspension to 108spores/mL, soaking the flea beetle imagoes in conidium suspension to be tested for 10s by adopting a dipping method, taking out the imagoes, airing the imagoes, putting the imagoes into a culture dish with wet filter paper at the bottom for feeding, changing the cabbage leaves every day, observing the change of the imagoes, and determining the imagoes as disease-dead imagoes when the imagoes grow mildews, such as picking out dead imagoes, performing moisture-preserving culture and the like. Each treatment was repeated 3 times for 10 adults. Mortality and corrected mortality were calculated formally:
mortality (%). mortality vs. number of dead insects/number of insects before treatment X100%
Corrected mortality (%) (treatment mortality-control mortality)/(1-control mortality) × 100%
The results showed that of the 32 strains tested, the activity of PlSC59a3 strain was highest, with a corrected mortality of 96.30% for phyllotreta striolata adults 11 days after treatment (table 1).
TABLE 1 Activity of different strains of A. lilacinus on A. flavus adults
Example 2: identification of purple lilac fungus PlSC59A3 strain
Example 1 the slant culture of the selected purple lilac strain PlSC59a3 was inoculated on a PDA plate, cultured at 27 ± 1 ℃, and the change in colony growth was observed. As shown in FIG. 1, the colony was initially white and villous on the surface; the colony is circular when cultured on day 7, the center of the colony is slightly raised, the middle part of the surface is white, the edge is light purple, the hair is villous, the back surface is light purple, and the diameter is (42.30 +/-0.07) mm.
The purple lilac spore strain PlSC59A3 screened in example 1 has a hypha with septa, branches and is transparent and has a width of (2.87 +/-0.11) mu m under an optical microscope as shown in FIG. 2; conidia are single cells, are elliptical to circular, are chain-shaped, smooth, colorless to light yellow, and have the width of 2-4 mu m, irregular branches or recurrent branches with bottle necks are arranged on the sporophores, and the base parts of the bottle stalks are wider. The recurrent branches consist of cylindrical parts, sometimes the branches curve away from the main axis.
The porphyridium lilacinum strain PlSC59A3 screened in example 1 is identified in molecular biology, r DNA-ITS gene sequence of the PlSC59A3 strain is amplified by universal primers ITS1 and ITS4, the length of the sequence is 559bp, and the sequence is shown as SEQ ID NO:1 (FIG. 3 is an ITS1-5.8S-ITS4rDNA gene specific amplification electrophoresis picture of the porphyridium lilacinum PlSC59A3 strain). When the blast inputs an ITS1-5.8S-ITS4rDNA gene sequence, the DNA sequence computer comparison result shows that the sequence of the PlSC59A3 strain is highly similar to the sequences of a plurality of violet spore strains; by means of MEGA software, a phylogenetic evolutionary tree (shown in figure 4) is constructed by using ITS1-5.8S-ITS4rDNA gene sequences of the PlSC59A3 strain, the standard lilac purple spore strain and the green muscardine and white muscardine strains, and the result shows that the PlSC59A3 strain and all the lilac purple spore strains are on the same branch and have high homologous relationship. Thus, the strain isolated by comprehensive morphological and ITS1-5.8S-ITS4rDNA sequence phylogenetic tree identification is a purple lilac (Purpureocillium lilacinum) strain classified as belonging to the phylum Ascomycota, Hypocrea, nematoda (Ophiococcus), and Porphyromyces (Purpureocillium). The strain is preserved in Guangdong province microorganism strain preservation center with the preservation date of 2020, 10 and 16 days, and the preservation number is GDMCC No: 61232.
example 3: production of conidia of the L.lilacinus strain PlSC59A3
Plate culture: taking the preserved slant strain, scraping a small amount of conidia, inoculating to a PDA plate, culturing in an incubator at 27 +/-1 ℃ for 7-14 days, and scraping the conidia on the surface layer of the colony for later use.
Production of conidium pure powder: suspending the conidia with 0.05% Tween-80 solution, inoculating to PD seed culture solution, shake-culturing in 27 + -1 deg.C incubator for 2 days to form seed solution, inoculating the seed solution to rice culture medium (plastic bag or solid fermentation tank), fermenting at 27 + -1 deg.C for 2 weeks with an inoculum size of about 10% (v/m), collecting conidia powder with a spore separator, packaging, and refrigerating.
Example 4: toxicity determination of lilac violet spore bacterium PlSC59A3 strain on phyllotreta striolata
The conidium pure powder produced in the above example 3 is applied, the phyllotreta striolata is dipped in spore suspension liquid with different concentrations under the laboratory condition, the mortality of the phyllotreta striolata is continuously investigated every 24 hours after the treatment, the corrected mortality is calculated, the toxicity regression equation is constructed by adopting SPSS, and the semi-Lethal Concentration (LC)50) And time to semi-Lethality (LT)50)。
Mortality (%). mortality vs. number of dead insects/number of insects before treatment X100%
Corrected mortality (%) - (treatment mortality-control mortality)/(1-control mortality) × 100%
The results showed that the strain PlSC59a3 was semilethal to flea beetle adults at 7d, 9d, 11d and 14d after treatment (LC)50) Are respectively 4.11 multiplied by 106spores/mL、1.03×106spores/mL、0.75×106spores/mL and 0.39X 106spores/mL; at 5X 106spores/mL、1×106spores/mL and 0.5X 106Half-lethal time LT at spores/mL treatment concentration50For 5.9, 9.1 and 13.3 days, respectively (table 2).
TABLE 2 results of virulence determination of phyllotreta striolata by different concentrations of spore suspension of PlSC59A3 strain
The purple lilac spore bacterium PlSC59A3 strain is used for bioassay of phyllotreta striolata adults and observation of disease symptoms of the phyllotreta striolata adults, the result is shown in figure 5, and the phyllotreta striolata is slow in dynamic change and low in cachexia and then dies after 2-3d after treatment. Under an optical microscope, it can be observed that white hyphae firstly extend out of the body from the dense bristle parts such as the antenna, the foot joints, the chest and the feet and the like of the phyllotreta striolata corpse and the internode membrane, and the whole polypide is wrapped by the white hyphae along with the passage of time, and then a large number of conidia are generated.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
SEQUENCE LISTING
<110> southern China university of agriculture
<120> purple lilac spore bacterial strain and application thereof in aspect of controlling phyllotreta striolata
<130> 2020
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 559
<212> DNA
<213> rDNA-ITS Gene sequence
<400> 1
aaaaactcca aacccactgt gaaccttacc tcagttgcct cggcgggaac gccccggccg 60
ccggcccccg cgccggcgcc ggacccaggc gcccgccgca gggaccccaa actctcttgc 120
attacgccca gcgggcggaa tttcttctct gagttgcaca agcaaaaaca aatgaatcaa 180
aactttcaac aacggatctc ttggttctgg catcgatgaa gaacgcagcg aaatgcgata 240
agtaatgtga attgcagaat tcagtgaatc atcgaatctt tgaacgcaca ttgcgcccgc 300
cagcattctg gcgggcatgc ctgttcgagc gtcatttcaa ccctcgagcc cccccggggg 360
cctcggtgtt gggggacggc acaccagccg cccccgaaat gcagtggcga ccccgccgca 420
gcctcccctg cgtagtagca cacacctcgc accggagcgc ggaggcggtc acgccgtaaa 480
acgcccaact ttcttagagt tgacctcgga tcaggtagga atacccgctg aacttaagca 540
tatcaataag gcggagaaa 559
Claims (3)
1. The purple lilac spore (purple lilacinum) strain PLSC59A3 is deposited in Guangdong province culture collection center at 2020, 10-16 days and with the deposition number of GDMCC No: 61232.
2. the use of the PlSC59a3 strain of claim 1 for controlling phyllotreta striolata.
3. The use as claimed in claim 2, characterized in that conidia of the PlSC59a3 strain are used as active ingredients directly or processed into preparations for controlling phyllotreta striolata.
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| CN110628649A (en) * | 2019-08-14 | 2019-12-31 | 华南农业大学 | Paecilomyces lilacinus strain, application thereof and method for extracting toxin from paecilomyces lilacinus strain |
| WO2020058196A1 (en) * | 2018-09-19 | 2020-03-26 | Bayer Cropscience Biologics Gmbh | Method for increasing storage stability of fungal spores |
| CN111961598A (en) * | 2020-09-18 | 2020-11-20 | 华南农业大学 | Isaria javanicus strain and application thereof in aspect of preventing and treating phyllotreta striolata |
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| WO2020058196A1 (en) * | 2018-09-19 | 2020-03-26 | Bayer Cropscience Biologics Gmbh | Method for increasing storage stability of fungal spores |
| CN110628649A (en) * | 2019-08-14 | 2019-12-31 | 华南农业大学 | Paecilomyces lilacinus strain, application thereof and method for extracting toxin from paecilomyces lilacinus strain |
| CN111961598A (en) * | 2020-09-18 | 2020-11-20 | 华南农业大学 | Isaria javanicus strain and application thereof in aspect of preventing and treating phyllotreta striolata |
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