CN115960752A - Burkholderia JSCBY002 and application thereof - Google Patents

Burkholderia JSCBY002 and application thereof Download PDF

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Publication number
CN115960752A
CN115960752A CN202211083616.0A CN202211083616A CN115960752A CN 115960752 A CN115960752 A CN 115960752A CN 202211083616 A CN202211083616 A CN 202211083616A CN 115960752 A CN115960752 A CN 115960752A
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burkholderia
jscby002
anthracnose
tea
microbial inoculum
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Inventor
徐超
汤茶琴
张红岩
王润贤
刘敏
陶程
黄小忠
曹淼
洪文龙
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Jiangsu Polytechnic College of Agriculture and Forestry
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Jiangsu Polytechnic College of Agriculture and Forestry
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    • 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
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Abstract

The invention discloses Burkholderia (Burkholderia cepacia) JSCBA 002 and application thereof, wherein the Burkholderia JSCBA 002 belongs to the genus Pseudomonas originally and is preserved in China general microbiological culture Collection center (CGMCC) on 8-15 th of 2022, and the preservation number is CGMCC No.:25538. the invention also discloses application of the Burkholderia JSCBY002 in a bactericide for preventing and treating anthracnose of tea trees. The Burkholderia JSCBY002 has obvious control effect on anthracnose of tea trees, can be artificially cultured, has high propagation speed, is convenient for large-scale production, and has good development and application prospects.

Description

Burkholderia JSCBY002 and application thereof
Technical Field
The invention relates to Burkholderia JSBBY 002 and application thereof as a bactericide for preventing and controlling tea tree anthracnose, belonging to the technical field of microorganisms.
Background
Tea is a traditional dominant industry in China, and in recent years, problems such as reduction of biodiversity of tea gardens, unbalance of ecological systems in the tea gardens and the like are caused, so that the problems such as growth and development of tea trees, soil acidification of the tea gardens, occurrence of plant diseases and insect pests and the like are increasingly serious, and sustainable development of the tea gardens is seriously threatened.
With the improvement of the national agricultural importance degree, in the tea tree cultivation process, active strategies need to be adopted, effective measures for optimizing the ecological environment of the tea garden are explored, measures such as biological control and the like are adopted, and the realization of efficient green control of tea garden plant diseases and insect pests becomes a research hotspot for promoting the sustainable development of the tea industry.
Disclosure of Invention
The purpose of the invention is as follows: the first purpose of the invention is to provide Burkholderia JSCBY002; the second purpose of the invention is to provide a strain fermentation liquor containing the Burkholderia JSCBY002; the third purpose of the invention is to provide a microbial preparation containing the Burkholderia JSCBY002 or the fermentation liquor of the strain thereof; the fourth purpose of the invention is to provide an application of Burkholderia JSCBY002 or fermentation liquor or microbial preparation thereof in the preparation of bactericide for preventing and treating anthracnose of tea trees.
The technical scheme is as follows: the Burkholderia JSCBY002 is preserved in China general microbiological culture Collection center (CGMCC) in 2022 at 8 months and 15 days, and the preservation number is CGMCC No.:25538, the collection address is the microbiological research institute of the institute of sciences of China, no. 3, xilu No. 1, beijing, chaoyang district, classified and named as: burkholderia JSCBY002.
Further, burkholderia JSCBY002 is a bacterium isolated from tea garden soil.
Further, morphological feature observation of Burkholderia JSCBY002 shows that: when the bacterial colony is cultured on a PDA plate, the bacterial colony is irregular in shape, raised, milky yellow, neat in edge, dull, rod-shaped and negative in gram stain. And amplifying the complete sequence of the ITS zone, sequencing, and obtaining the ITS sequence obtained by PCR amplification as shown in SEQ ID No:1, the original genus is pseudomonas.
The strain fermentation liquid provided by the invention comprises Burkholderia JSCBY002.
The microbial preparation comprises Burkholderia JSCBY002 or strain fermentation liquor.
The invention also comprises the application of the Burkholderia JSCBY002 or the strain fermentation liquor or the microbial preparation as a bacterial agent for preventing and controlling anthracnose of tea trees.
The application of the invention comprises the following steps: diluting the cultured Burkholderia JSCBY002 liquid microbial inoculum with water, mixing well to obtain a diluent of the liquid microbial inoculum, and spraying the diluent on the surface of the tea plant.
Further, the volume ratio of the Burkholderia JSCBY002 liquid microbial inoculum to water is 1.
Further, the Burkholderia JSCBY002 liquid microbial inoculum comprises a PD culture medium and the Burkholderia JSCBY002.
Further, 10-20L of diluent of liquid microbial inoculum is sprayed on each mu of tea row.
Has the beneficial effects that: compared with the prior art, the invention has the following remarkable advantages:
(1) The Burkholderia cepacia JSBBY 002 obtained by the invention has obvious control effect on the anthracnose of tea trees, can obviously reduce the infection rate of the anthracnose of the tea trees, has a certain effect on reducing the use of chemical pesticides, and is an important means for ensuring the drinking safety of tea leaves.
(2) The Burkholderia cepacia JSBBY 002 is simple in culture condition, easy to store, easy for industrial production and good in development and application prospect.
Drawings
FIG. 1 is a diagram showing the effect of Burkholderia JSCBY002 confronted with tea tree anthrax pathogen flat plate;
FIG. 2 is a diagram of the control susceptibility of Burkholderia JSCBY002 to anthracnose of tea trees in example 2;
FIG. 3 is a disease index chart of Burkholderia JSCBY002 for controlling anthracnose of tea trees in example 2;
FIG. 4 is a graph showing the control susceptibility of Burkholderia JSCBY002 on anthracnose of tea trees in example 3;
FIG. 5 is a disease index chart of Burkholderia JSCBY002 for controlling anthracnose of tea trees in example 3;
FIG. 6 is a diagram of the control susceptibility of Burkholderia JSCBY002 to anthracnose of tea trees in example 4;
FIG. 7 is a disease control index chart of Burkholderia JSCBY002 on anthracnose of tea trees in example 4.
Detailed Description
The technical scheme of the invention is further explained by combining the attached drawings
Example 1
Burkholderia strain JSCBY002 was isolated from tea garden soil with good growth conditions of Nanjing Yarun tea science and technology Limited, nanjing, jiangsu, in 2018 by the applicant.
When cultured on a PDA plate, the colony is observed to have irregular shape, raised, milky yellow, neat edges, no luster, rod shape and negative gram stain.
And amplifying the complete sequence of the ITS region, sequencing, and performing PCR amplification to obtain the complete sequence of the ITS region as shown in SEQ ID No:1, the original genus is pseudomonas.
SEQ ID No：1
AAAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGACCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCATGCACTCGAGTTGCAGAGTGCAATCCGGACTACGATCGGTTTTCTGGGATTAGCTCCCCCTCGCGGGTTGGCAACCCTCTGTTCCGACCATTGTATGACGTGTGAAGCCCTACCCATAAGGGCCATGAGGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCCTTAGAGTGCTCTTGCGTAGCAACTAAGGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTGCGCCGGTTCTCTTTCGAGCACTCCCACCTCTCAGCAGGATTCCGACCATGTCAAGGGTAGGTAAGGTTTTTCGCGTTGCATCGAATTAATCCACATCATCCACCGCTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTTAATCTTGCGACCGTACTCCCCAGGCGGTCAACTTCACGCGTTAGCTACGTTACTAAGGAAATGAATCCCCAACAACTAGTTGACATCGTTTAGGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGTGCATGAGCGTCAGTATTGGCCCAGGGGGCTGCCTTCGCCATCGGTATTCCTCCACATCTCTACGCATTTCACTGCTACACGTGGAATTCTACCCCCCTCTGCCATACTCTAGCCTGCCAGTCACCAATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCGGTCTTAGCAAACCGCCTGCGCACGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTATTCTTCCGGTACCGTCATCCCCCGACTGTATTAGAGCCAAGGATTTCTTTCCGGACAAAAGTGCTTTACAACCCGAAGGCCTTCTTCACACACGCGGCATTGCTGGATCAGGCTTTCGCCCATTGTCCAAAATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCTGGTCGTCCTCTCAGACCAGCTACTGATCGTCGCCTTGGTAGGCCTTTACCCCACCAACTAGCTAATCAGCCATCGGCCAACCCTATAGCGCGAGGCCCGAAGGTCCCCCGCTTTCATCCGTGGATCGTATGCGGTATTAATCCGGCTTTCGCCGGGCTATCCCCCACTACAGGACATGTTCCGATGTATTACTCACCCGTTCGCCACTCGCCACCAGGTGCAAGCACCCGTGCTG。
Plate confrontation test:
(1) Inoculating the pathogen of anthracnose to one side of a PDA culture medium plate by using an inoculating needle on a clean bench, simultaneously inoculating Burkholderia JSCBY002 to the symmetrical side of the PDA culture medium, inversely culturing in a constant temperature incubator at 25 ℃ (light illumination is 8h, dark culture is 16 h), and observing and recording the inhibition effect.
(2) After the plate is artificially cultured for 10 days, the pathogenic bacteria colony radius is measured by a cross method, namely the longitudinal longest part and the transverse widest part of the colony are measured, and the average value of the two values is taken. The inhibitory effect of Burkholderia JSCBY002 on anthrax pathogenic bacteria was observed, and plates inoculated with only the pathogenic bacteria were used as controls. The specific inhibitory effect is shown in fig. 1.
FIG. 1 is a diagram showing the effect of Burkholderia JSCBY002 confronted with tea tree anthrax pathogen flat plate; wherein, the graph a is a colony culture effect graph of the anthrax pathogenic bacteria of a blank control group, and the radius is 4cm; the plot b is the effect of the experimental groups on the stand-off, and the mean radius of the anthrax pathogens is only 1.9cm, as measured by the cross method. As can be seen from FIG. 1, the effect of the Burkholderia JSCBY002 on the inhibition of the anthrax pathogenic bacteria is significant.
Example 2: analysis of field control effect of Burkholderia cepacia JSCBY002 on anthracnose
(1) In the season of high occurrence of anthracnose (7 months), liquid culture (PD culture medium, constant temperature shaking table at 25 deg.C, light culture for 8h, dark culture for 16 h) of Burkholderia JSCBY002 liquid microbial inoculum (liquid microbial inoculum comprises PD culture medium, burkholderia JSCBY002, and bacterial suspension concentration is 10% 5 CFU/ml-10 6 CFU/ml) is added with water according to the volume ratio of 1. And uniformly spraying the liquid microbial inoculum diluent on the surfaces of the tea canopies in the evening, wherein the spraying amount is 15L/mu. The treatment group sprayed with the same amount of clear water was used as a control. Each treatment was 1 mu tea garden and 3 sets were repeated.
(2) And (5) monitoring the disease degree of the anthracnose every day after inoculation, and counting the infection rate and infection index of the anthracnose.
(3) On the 5 th day, the 10 th day, the 15 th day and the 20 th day after the liquid microbial inoculum is applied, the degrees of the anthracnose of the tea garden which is processed by contrast treatment and the tea garden which is processed by the liquid microbial inoculum sprayed by the microbial inoculum are respectively counted, the incidence rate and the incidence index of the anthracnose are counted, and the control effect of the burkholderia on the anthracnose is analyzed, and the results are shown in a figure 2 and a figure 3.
Grading standard of anthracnose disease plants of tea trees: the classification of the anthracnose disease condition mainly comprises the following steps of according to the number and the diameter of scabs on the front surface and the back surface of an inoculated leaf, and the primary classification standard is as follows:
level 0: normal;
stage I: a small amount of disease spots are formed on the front surface and the back surface;
and II, stage: the front and back sides have disease spots, and the disease area accounts for less than 1/4 of the total area of the leaves;
grade III: the front and back sides have disease spots, and the disease area accounts for 1/4-1/2 of the total area of the leaves;
IV stage: the front and back sides have disease spots, and the disease area accounts for 1/2-3/4 of the total area of the leaves;
IV stage: the front and back sides have disease spots, and the disease area accounts for more than 3/4 of the total area of the leaves.
The susceptibility index of anthracnose is calculated according to the following formula:
the infection index = (number of diseased plants at each stage × representative value at the stage) × 100/(number of total plants × representative value at the highest stage);
the susceptibility to anthracnose is calculated according to the following formula:
disease susceptibility = (susceptible plant tree/inoculated plant tree) × 100%.
FIG. 2 is a graph of the disease susceptibility of Burkholderia JSCBY002 on anthracnose of tea trees in example 2; as can be seen from FIG. 2, the infection rates of the treatment groups inoculated with the Burkholderia JSCBY002 are obviously lower than those of the control treatment groups, particularly at 20d after inoculation, the infection rates of the inoculation treatment groups are 45%, and the infection rates of the control treatment groups are up to 72%. The inoculation of Burkholderia JSCBY002 can reduce the incidence of anthracnose of tea trees.
FIG. 3 is a disease-preventing index chart of Burkholderia JSCBY002 on anthracnose of tea trees in example 2; as can be seen from FIG. 3, the susceptibility index of the treatment group inoculated with the Burkholderia JSCBY002 is significantly lower than that of the control treatment group, particularly at the 20 th day after inoculation, the susceptibility index of the inoculation treatment group is 15.6, and the susceptibility index of the control treatment group is as high as 23.6. The inoculation of Burkholderia JSCBY002 can reduce the harm degree of anthracnose to tea trees.
Example 3: field control effect analysis of Burkholderia cepacia JSCBY002 on anthracnose
The experimental procedure was as in example 2: (1) In the season of high occurrence of anthracnose (7 months), liquid culture medium (PD medium, constant temperature shaking table at 25 deg.C, light culture for 8h, dark culture for 16 h) of Burkholderia JSCBY002 liquid bacteria (liquid bacteria component package)Comprises PD culture medium, burkholderia JSCBY002, and the concentration of the bacterial suspension is 10 5 CFU/ml-10 6 CFU/ml) is added with water according to the volume ratio of 1. And uniformly spraying the liquid microbial inoculum diluent on the surfaces of the tea canopies in the evening, wherein the spraying amount is 15L/mu. The control was treated with an equal amount of water spray. Each treatment was 1 mu tea garden and was repeated in 3 groups.
(2) And (5) monitoring the disease degree of the anthracnose every day after inoculation, and counting the infection rate and infection index of the anthracnose.
(3) On the 5 th day, the 10 th day, the 15 th day and the 20 th day after the liquid microbial inoculum is applied, the degrees of the anthracnose of the tea garden which is processed by contrast treatment and the tea garden which is processed by the liquid microbial inoculum sprayed by the microbial inoculum are respectively counted, the incidence rate and the incidence index of the anthracnose are counted, and the control effect of the burkholderia on the anthracnose is analyzed, and the results are shown in a figure 4 and a figure 5.
Grading standard of anthracnose disease plants of tea trees: the disease classification of the anthrax is mainly based on the number and diameter of the scabs on the front and back surfaces of the inoculated leaf, and the initial classification standard, the infection rate and the infection index calculation formula are the same as those in example 2.
FIG. 4 is a graph showing the control susceptibility of Burkholderia JSCBY002 on anthracnose of tea trees in example 3; as can be seen from FIG. 4, the treatment groups inoculated with the B.berghei JSCBY002 all had significantly lower susceptibility than the control treatment group. At 20d, the infection rate of the inoculation group was 40%, and the infection rate of the control group was 72%. The inoculation of Burkholderia JSCBY002 can reduce the incidence rate of anthracnose of tea trees. In this embodiment, the burkholderia liquid microbial inoculum is diluted and mixed by adding water according to the volume ratio of 1 to 9, and compared with the burkholderia liquid microbial inoculum in embodiment 2 which is diluted and mixed by adding water according to the volume ratio of 1 to 14, in this embodiment, the susceptibility is slightly lower, but the difference is not large.
FIG. 5 is a disease-preventing index chart of Burkholderia JSCBY002 on anthracnose of tea trees in example 3; as can be seen from FIG. 5, the treatment groups inoculated with the B.burkholderia JSCBY002 all had significantly lower infection indexes than the control treatment group. At 20d, the infection index of the inoculation group is 14.2, and the infection rate of the control group is 23.6. The inoculation of Burkholderia JSCBY002 can reduce the harm degree of anthracnose to tea trees. In this embodiment, the burkholderia liquid microbial inoculum is diluted and mixed with water according to the volume ratio of 1 to 9, and compared with the burkholderia liquid microbial inoculum in embodiment 2 which is diluted and mixed with water according to the volume ratio of 1 to 14, in this embodiment, the susceptibility index is slightly lower, but the difference is not large.
Example 4: analysis of field control effect of Burkholderia cepacia JSCBY002 on anthracnose
The experimental procedure was as in example 2:
(1) In the season of high occurrence of anthracnose (7 months), liquid culture (PD culture medium, constant temperature shaking table at 25 deg.C, light culture for 8h, dark culture for 16 h) of Burkholderia JSCBY002 liquid microbial inoculum (liquid microbial inoculum comprises PD culture medium, burkholderia JSCBY002, and bacterial suspension concentration is 10% 5 CFU/ml-10 6 CFU/ml) is added with water according to the volume ratio of 1. And uniformly spraying the liquid microbial inoculum diluent on the surface of the tea row tent at evening, wherein the spraying amount is 15L/mu. The control was treated with an equal amount of water spray. Each treatment was 1 mu tea garden and was repeated in 3 groups. (2) And (5) monitoring the disease degree of the anthracnose every day after inoculation, and counting the infection rate and infection index of the anthracnose.
(3) On the 5 th day, the 10 th day, the 15 th day and the 20 th day after the liquid microbial inoculum is applied, the degrees of the anthracnose of the tea garden which is processed by contrast treatment and the tea garden which is processed by the liquid microbial inoculum sprayed by the microbial inoculum are respectively counted, the incidence rate and the incidence index of the anthracnose are counted, and the control effect of the burkholderia on the anthracnose is analyzed, and the results are shown in fig. 6 and fig. 7.
Grading standard of anthracnose disease plants of tea trees: the disease classification of anthrax is mainly based on the number and diameter of the scabs on the front and back surfaces of the inoculated leaf, and the initial classification standard, the infection rate and the infection index calculation formula are the same as those in example 2.
FIG. 6 is a graph showing the control susceptibility of Burkholderia JSCBY002 on anthracnose of tea trees in example 4; as can be seen from FIG. 6, the treatment groups inoculated with Burkholderia jsCBY002 were all less susceptible than the control treatment group. At 20d, the infection rate of the inoculation group is 48 percent, and the infection rate of the control group is 72 percent. The inoculation of Burkholderia JSCBY002 can reduce the incidence rate of anthracnose of tea trees. In this embodiment, the burkholderia liquid microbial inoculum is diluted and mixed with water according to the volume ratio of 1 to 19, and compared with the burkholderia liquid microbial inoculum in embodiment 2 which is diluted and mixed with water according to the volume ratio of 1 to 14, in this embodiment, the susceptibility is slightly increased, but the difference is not obvious.
FIG. 7 is a disease-controlling index chart of Burkholderia JSCBY002 on anthracnose of tea trees in example 3; as can be seen from FIG. 7, the treatment groups inoculated with Burkholderia jsCBY002 all had lower susceptibility indices than the control treatment group. At 20d, the infection index of the inoculation group is 16.3, and the infection rate of the control group is 23.6. The inoculation of Burkholderia JSCBY002 can reduce the harm degree of anthracnose to tea trees. In this embodiment, the burkholderia liquid microbial inoculum is diluted and mixed with water according to the volume ratio of 1 to 19, and compared with the burkholderia liquid microbial inoculum in embodiment 2 which is diluted and mixed with water according to the volume ratio of 1 to 14, in this embodiment, the susceptibility index is slightly increased, but the difference is not obvious.

Claims (10)

1. The Burkholderia JSCBY002 is characterized in that the Burkholderia JSCBY002 is preserved in China general microbiological culture Collection center (CGMCC) in 2022 at 8 months and 15 days, and the preservation number is CGMCC No.:25538.
2. the Burkholderia JSCBY002 according to claim 1, is a bacterium isolated from soil in tea plantation.
3. The burkholderia JSCBY002 of claim 1, wherein the ITS sequence of burkholderia JSCBY002 is as set forth in SEQ ID No:1 is shown.
4. A strain fermentation liquid is characterized by comprising Burkholderia JSCBY002.
5. A microbial preparation comprising a fermentation broth of the Burkholderia JSCBY002 strain according to any one of claims 1 to 3 or the strain according to claim 4.
6. Use of burkholderia JSCBY002 according to any one of claims 1 to 3 or of a fermentation broth of the strain according to claim 4 or of a microbial preparation according to claim 5 as a fungicide for the control of anthracnose of tea trees.
7. Use according to claim 6, characterized in that it comprises the following steps:
diluting the cultured Burkholderia JSCBY002 liquid microbial inoculum with water, mixing uniformly to obtain liquid microbial inoculum diluent, and spraying uniformly on the surface of the tea plant.
8. The use according to claim 7, wherein the ratio of the JSCBKY 002 liquid microbial inoculum to water in volume ratio of 1.
9. The use according to claim 7, wherein the liquid microbial inoculum of Burkholderia JSCBY002 comprises PD medium, burkholderia JSCBY002.
10. The use of claim 7, wherein 10-20L of the diluted liquid microbial inoculum is sprayed per acre of tea row.
CN202211083616.0A 2022-09-06 2022-09-06 Burkholderia JSCBY002 and application thereof Pending CN115960752A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117660267A (en) * 2024-01-31 2024-03-08 广东省科学院微生物研究所(广东省微生物分析检测中心) Burkholderia for preventing and controlling tea tree pathogenic fungi and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117660267A (en) * 2024-01-31 2024-03-08 广东省科学院微生物研究所(广东省微生物分析检测中心) Burkholderia for preventing and controlling tea tree pathogenic fungi and application thereof
CN117660267B (en) * 2024-01-31 2024-04-12 广东省科学院微生物研究所(广东省微生物分析检测中心) Burkholderia for preventing and controlling tea tree pathogenic fungi and application thereof

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