CN116649372A - Microbial composition and application thereof in prevention and control of coleopteran pests - Google Patents

Microbial composition and application thereof in prevention and control of coleopteran pests Download PDF

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CN116649372A
CN116649372A CN202310921042.8A CN202310921042A CN116649372A CN 116649372 A CN116649372 A CN 116649372A CN 202310921042 A CN202310921042 A CN 202310921042A CN 116649372 A CN116649372 A CN 116649372A
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ippbiote33
cfu
holotrichia parallela
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bacillus thuringiensis
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CN116649372B (en
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耿丽丽
张�杰
束长龙
米良
王泽宇
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Institute of Plant Protection of Chinese Academy of Agricultural Sciences
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/22—Bacillus
    • A01N63/23—B. thuringiensis
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P7/00—Arthropodicides
    • A01P7/04—Insecticides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/075—Bacillus thuringiensis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
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  • Chemical & Material Sciences (AREA)
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  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Virology (AREA)
  • Microbiology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Pest Control & Pesticides (AREA)
  • Plant Pathology (AREA)
  • Environmental Sciences (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
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  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Agronomy & Crop Science (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Insects & Arthropods (AREA)
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Abstract

The invention relates to a microbial composition and application thereof in preventing and controlling coleopteran pests. The composition comprises bacillus thuringiensis and enterobacteria.

Description

Microbial composition and application thereof in prevention and control of coleopteran pests
Technical Field
The invention relates to the field of biological control, in particular to a microbial composition for controlling coleopteran pests.
Background
Coleoptera (Coleoptera) is the most abundant and widely distributed insect species, and causes no harm in the agroforestry area other than Lepidoptera (Lepidoptera), with scarab beetles being the most serious of the most harmful species in Coleoptera.
At present, the prevention and treatment of coleopteran pests mainly depend on chemical prevention and treatment, but the abuse of chemical pesticides can cause the problems of environmental pollution, food safety and the like. Biological control is currently rapidly developing as a sustainable, safe and environment-friendly control mode. The advent of bacterial biopesticides represented by bacillus thuringiensis (Bacillus thuringiensis, bt) has provided the potential for rational replacement and reduced application of chemical pesticides. In order to achieve continuous and efficient control of coleopteran pests, better and effective biological control means are continually sought.
Disclosure of Invention
One aspect of the invention provides a composition comprising bacillus thuringiensis and enterobacteria.
In a specific embodiment, the bacillus thuringiensis is a Bt185 strain and the enterobacteria is an IPPBiotE33 strain.
In a specific embodiment, the cfu ratio of the bacillus thuringiensis to the enterobacteria is from 1:10 to 10:1.
In a specific embodiment, the cfu ratio of the bacillus thuringiensis to the enterobacteria is 1:1.
The second invention provides the use of a composition according to any of the inventions for controlling gill-gold tortoise (Melonoshidae).
In a specific embodiment, the gill-gold turtle is Holotrichia parallela (Holotrichia parallela).
In a specific embodiment, the gill-gold turtle is a larval stage Holotrichia parallela (Holotrichia parallela).
The invention has the beneficial effects that: the invention discovers that the combined use of bacillus thuringiensis and enterobacter has a synergistic effect on the insecticidal activity of Holotrichia parallela for the first time.
Detailed Description
The above-described aspects of the invention are described in further detail below in the form of preferred embodiments, which are not to be construed as limiting the invention.
The microorganism IPPBiotE33 strain disclosed by the invention is disclosed in an authorized patent ZL 202010979164.9 for the first time, and is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of 20449 and the preservation date of 2020, 07 and 27 days with the preservation address of: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing. Its system is classified as Enterobacter sp.
The microbial Bt185 strain of the invention is disclosed in an authorized patent ZL 200410009808.2 for the first time, and is preserved in the China general microbiological culture Collection center (CGMCC No. 1242), the preservation date is 11 months and 5 days in 2004, and the preservation address is: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing. The system is classified as bacillus thuringiensis (Bacillus thuringiensis).
Reagents for use in the examples of the invention are commercially available unless otherwise specified.
Example 1
Preparation of IPPBiotE33
IPPBiotE33 was inoculated into a test tube containing 5 mL liquid LB medium (tryptone 10.0 g/L, yeast extract 5.0 g/L, naCl 10.0 g/L,121 ℃ C. Sterilized for 20 min), cultured at 30 ℃ C. For 12 hours for activation, then the activated bacterial liquid was transferred to a solid LB medium (LB liquid medium plus agar 15 g/L, dish diameter 15 cm), cultured at 30 ℃ C. For 12 hours, the bacterial cells were collected with a sterile spatula, uniformly suspended with PBS buffer to obtain an IPPBiotE33 suspension, and the IPPBiotE33 suspension was subjected to plate counting.
Example 2
Determination of insecticidal Activity of IPPBiotE33 against Holotrichia parallela (Holotrichia parallela)
And carrying out gradient dilution on the IPPBiotE33 suspension to obtain IPPBiotE33 dilutions with different concentrations.
The method comprises the steps of preparing carrot filaments, washing the carrot filaments with clear water, and airing until the surface of the carrot filaments has no water. Soaking radix Dauci Sativae filaments in each IPPBiotE33 diluent for 20 min, taking out, placing in 6-hole biological test plate, mixing with about 4-5 per hole, and stirring with 60 g soil to obtain final biological test concentration of 1×10 5 、1×10 6 、1×10 7 、1×10 8 And 1X 10 9 cfu/g. And (3) uniformly subpackaging the mixed soil into 6-hole raw measurement plates with corresponding concentration, inoculating 1 head of larvae of Holotrichia parallela which is initially hatched for 2 days into each hole, and placing the larvae into an incubator with the temperature of 25 ℃ and the illumination of L:D=16:8 for feeding. Each repeat 20 heads, 5 replicates. PBS buffer was used as a negative control. After 7 days, the numbers of dead and live insects were investigated, the average mortality was calculated, the mortality was corrected, and LC was calculated using SPSS software 50 。
Wherein corrected mortality = (average mortality of bacterial treatment-average mortality of negative controls)/(average mortality of 1-negative controls) ×100%.
Results IPPBiotE33 exhibits insecticidal Activity against Holotrichia parallela (H. Parallela) initially hatched larvae, LC 50 8.81X 10 7 cfu/g,95% letter interval of 1.53×10 7 -22.40×10 7 cfu/g。
Example 3
Preparation of Bt185
The strain Bt185 was inoculated into a test tube containing 5 mL liquid LB medium (tryptone 10.0 g/L, yeast extract 5.0 g/L, naCl 10.0 g/L,121℃for 20 min) and cultured at 30℃for 12 hours for activation, and then the bacterial liquid obtained by the activation was transferred to solid 1/2 LB medium (tryptone 5.0 g/L, yeast extract 2.5 g/L, naCl5.0 g/L plus agar 15 g/L,121℃for 20 min, dish diameter 15 cm) and cultured at 30℃for 24 hours, and about 80% was lysed by microscopic observation. The cells were collected with a sterile spatula, uniformly suspended with PBS buffer to obtain Bt185 suspension, and the obtained Bt185 suspension was plate-counted.
Example 4
Determination of insecticidal Activity of Bt185 on Holotrichia parallela (Holotrichia parallela)
And carrying out gradient dilution on the Bt185 suspension bacteria liquid to obtain Bt185 dilution liquid with different concentrations.
The method comprises the steps of preparing carrot filaments, washing the carrot filaments with clear water, and airing until the surface of the carrot filaments has no water. Soaking radix Dauci Sativae filaments in each Bt185 diluent for 20 min, taking out, placing in 6-hole biological test plate, 4-5 strips per hole, and mixing the rest bacterial liquid with 6In 0 g soil, the final measured concentration is 1×10 5 、1×10 6 、1×10 7 、1×10 8 And 1X 10 9 cfu/g. And (3) uniformly subpackaging the mixed soil into 6-hole raw measurement plates with corresponding concentration, inoculating 1 head of larvae of Holotrichia parallela which is initially hatched for 2 days into each hole, and placing the larvae into an incubator with the temperature of 25 ℃ and the illumination of L:D=16:8 for feeding. Each repeat 20 heads, 5 replicates. PBS buffer was used as a negative control. After 7 days, the numbers of dead and live insects were investigated, the average mortality was calculated, the mortality was corrected, and LC was calculated using SPSS software 50 。
Results Bt185 exhibits insecticidal Activity against Holotrichia parallela larvae, LC 50 6.59X10 7 cfu/g,95% letter interval of 1.44X10 7 -14.14×10 7 cfu/g。
Example 5
Mixing the IPPBiotE33 suspension and the Bt185 suspension according to the ratio of cfu of 1:1, and carrying out gradient dilution to obtain mixed dilutions with different concentrations.
The method comprises the steps of preparing carrot filaments, washing the carrot filaments with clear water, and airing until the surface of the carrot filaments has no water. Soaking radix Dauci Sativae filaments in the mixed diluents for about 20 min, taking out, placing in 6-hole biological test plate, with about 4-5 strips per hole, mixing the rest diluents in 60 g soil to obtain final biological test total concentration of IPPBiotE33 and Bt185 of 1×10 5 、1×10 6 、1×10 7 、1×10 8 And 1X 10 9 cfu/g. And (3) uniformly subpackaging the mixed soil into 6-hole raw measurement plates with corresponding concentration, inoculating 1 head of larvae of Holotrichia parallela which is initially hatched for 2 days into each hole, and placing the larvae into an incubator with the temperature of 25 ℃ and the illumination of L:D=16:8 for feeding. Each repeat 20 heads, 5 replicates. PBS buffer was used as a negative control. After 7 days, the numbers of dead and live insects were investigated, the average mortality was calculated, the mortality was corrected, and LC was calculated using SPSS software 50 。
Results the mixed bacterial solution of IPPBiotE33 and Bt185 shows insecticidal activity on Holotrichia parallela initially hatched larvae, LC 50 Is 2.06X10 7 cfu/g,95% letter interval of 0.38X10 7 -4.63×10 7 cfu/g。
Example 6
Analysis of biological test results
The synergy coefficient (SR) was calculated using the Wadley method as follows:
wherein LC is 50exp Is a composition LC 50 Predicted value of LC (L) 50obs Is a composition LC 50 Is measured by the above method. P (P) A And P B Is the ratio of A to B in the composition. SR.gtoreq.1.5 means having a synergistic effect of 1.5>SR>0.5 indicates that there is an additive effect, and SR.ltoreq.0.5 indicates that there is an antagonistic effect.
LC of IPPBiotE33 on Holotrichia parallela larvae 50 8.93X 10 7 cfu/g; LC of Bt185 on Holotrichia parallela larvae 50 6.29×10 7 cfu/g; results of the biological assay LC on Holotrichia parallela larvae in combination of IPPBiotE33 and Bt185 at a cfu ratio of 1:1 50 Is 2.07×10 7 cfu/g。
The synergistic effect of IPPBiotE33 and Bt185 was analyzed by the Wadley method: the synergy coefficient (SR) of IPPBiotE33 and Bt185 was 3.66 > 1.5, indicating that these two strains have a synergy at a cfu ratio of 1:1.
Example 7
Potting experiment
Mixing nutrient soil and vermiculite according to a ratio of 2:1 to obtain planting soil, filling the planting soil into a flowerpot, and then respectively mixing PBS suspension bacteria liquid of IPPBiotE33, PBS suspension bacteria liquid of Bt185 and PBS suspension bacteria liquid of IPPBiotE33 and Bt185 (cfu ratio is 1:1) into the planting soil to make the final concentration of the combination of IPPBiotE33, bt185, IPPBiotE33 and Bt185 in the planting soil be 1 multiplied by 10 9 cfu/g, where IPPBiotE33 is combined with Bt185, 1X 10 9 cfu/g is the total concentration of IPPBiotE33 and Bt185. PBS buffer was used as a negative control. And (3) planting peanuts, growing the peanuts under the natural illumination condition at room temperature, after the peanuts sprout for 7 days, inoculating 2 Holotrichia parallela larvae (test insects) with the age of 2 days near the root of each seedling, and repeating each treatment for 3 times for 30 peanut seedlings.After 7 days of larva inoculation, physiological indexes such as plant height, fresh weight, weight change rate of test insects and the like of the seedlings are counted, and the results are shown in table 1.
Wherein, the rate of change in body weight of the test insects= (average body weight of the negative control group-average body weight of the treated group)/average body weight of the negative control group.
From the results of table 1, it can be seen that, although the physiological index of peanut seedlings after applying the IPPBiotE33, bt185 and the combination of both was significantly higher than that of the negative control, the treatment groups using the combination of IPPBiotE33 and Bt185 were significantly higher than that of the individual treatment groups of IPPBiotE33 and Bt185, and the rate of change of the test body weight of the treatment groups using the combination of IPPBiotE33 and Bt185 was significantly higher than that of the individual treatment groups of IPPBiotE33 and Bt185. This indicates that IPPBiotE33 and Bt185 show a synergistic effect against holotrichia parallela larvae when applied in admixture to peanut seedlings, thereby facilitating peanut growth.
TABLE 1
Different lowercase letters indicate significant differences (P < 0.05) passing Dunn multiple comparison test (n=20).

Claims (7)

1. A composition comprising bacillus thuringiensis and enterobacteria.
2. The composition of claim 1, wherein the bacillus thuringiensis is a Bt185 strain and the enterobacteria is an IPPBiotE33 strain.
3. The composition of claim 1, wherein the cfu ratio of bacillus thuringiensis to enterobacteria is from 1:10 to 10:1.
4. The composition of claim 1, wherein the cfu ratio of bacillus thuringiensis to enterobacteria is 1:1.
5. Use of a composition according to any one of claims 1 to 4 for controlling gill scara (Melolonthidae).
6. The use according to claim 5, characterized in that the gill-mossback is Holotrichia parallela (Holotrichia parallela).
7. The use according to claim 5, characterized in that the gill-mossback is a larval-stage Holotrichia parallela (Holotrichia parallela).
CN202310921042.8A 2023-07-26 2023-07-26 Microbial composition and application thereof in prevention and control of coleopteran pests Active CN116649372B (en)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6063605A (en) * 1989-05-09 2000-05-16 Zeneca Limited Bacillus thuringiensis endotoxin genes and methods of use
CN1327048A (en) * 2001-06-15 2001-12-19 中国科学院武汉病毒研究所 A strain of recombined broad spectrum bacillus thuringiensis
CN1401773A (en) * 2001-08-20 2003-03-12 中国农业科学院植物保护研究所 Bt gene with high toxicity to Lepidoptera and Coleoptera insects, expression vector and engineering bacteria
CN1609191A (en) * 2004-11-16 2005-04-27 中国农业科学院植物保护研究所 Bacillus thringiensis strain and gene with high effect on coleoptera pests
CN101130762A (en) * 2007-08-07 2008-02-27 中国农业科学院植物保护研究所 Efficient bacillus thuringiensis cry8H gene, protein for vaginata destructive insect and uses of the same
CN111996152A (en) * 2020-09-17 2020-11-27 中国农业科学院植物保护研究所 Enterobacter strain with insecticidal activity on Holotrichia parallela
CN113302199A (en) * 2019-01-10 2021-08-24 先正达农作物保护股份公司 Compositions and methods for controlling insect pests

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6063605A (en) * 1989-05-09 2000-05-16 Zeneca Limited Bacillus thuringiensis endotoxin genes and methods of use
CN1327048A (en) * 2001-06-15 2001-12-19 中国科学院武汉病毒研究所 A strain of recombined broad spectrum bacillus thuringiensis
CN1401773A (en) * 2001-08-20 2003-03-12 中国农业科学院植物保护研究所 Bt gene with high toxicity to Lepidoptera and Coleoptera insects, expression vector and engineering bacteria
CN1609191A (en) * 2004-11-16 2005-04-27 中国农业科学院植物保护研究所 Bacillus thringiensis strain and gene with high effect on coleoptera pests
CN101130762A (en) * 2007-08-07 2008-02-27 中国农业科学院植物保护研究所 Efficient bacillus thuringiensis cry8H gene, protein for vaginata destructive insect and uses of the same
CN113302199A (en) * 2019-01-10 2021-08-24 先正达农作物保护股份公司 Compositions and methods for controlling insect pests
CN111996152A (en) * 2020-09-17 2020-11-27 中国农业科学院植物保护研究所 Enterobacter strain with insecticidal activity on Holotrichia parallela

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