Method for constructing antagonistic yeast for preventing and controlling tomato gray mold

A technology for biological control of yeast and tomato gray mold, applied in the direction of microorganism-based methods, biochemical equipment and methods, botany equipment and methods, etc. Inconvenience and other issues to achieve sustainable development strategies and reduce usage

Inactive Publication Date: 2011-02-23
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Adopting high-level production management technology is an important measure to prevent and control tomato gray mold, but its effect is limited, and its application is restricted by various conditions, which also makes many agricultural producers feel inconvenient
However, it is very difficult to obtain tomato varieties resistant to Botrytis cinerea through conventional breeding methods. This is mainly due to the long process of breeding new varieties, and there is no genetic resource for tomato Botrytis as a parent for hybrid breeding. lack of parental resources
At present, the control of Botrytis cinerea in production mainly relies on chemical control. As the frequency of application continues to increase, the concentration of use is increasing, the number of resistant bacteria is gradually increasing, and the control effect is declining year by year. Serious problem of pesticide residues and environmental pollution

Method used

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  • Method for constructing antagonistic yeast for preventing and controlling tomato gray mold
  • Method for constructing antagonistic yeast for preventing and controlling tomato gray mold
  • Method for constructing antagonistic yeast for preventing and controlling tomato gray mold

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specific Embodiment approach 1

[0011] Specific embodiment one: the construction method of the biocontrol yeast of present embodiment preventing and treating tomato cinerea is carried out according to the following steps: one, adopt the TRIzol reagent method to extract the total RNA of erythrocytosanus WY-1, according to chitinase For the Crchi1 gene, use the Primer Premier5.0 software to design specific upstream primers Crchi1F and downstream primers Crchi1R, and then reverse-transcribe the total RNA of R. Carry out PCR amplification as the template to obtain the target gene; 2. After the target gene is separated by 1% agarose gel electrophoresis, it is connected to the YEp73-T carrier, and the resulting connection product is transformed into Escherichia coli JM109 and positive clones are screened and identified, and then The plasmid vector YEp73 was extracted by a small amount method, and then the target gene and the plasmid vector YEp73 were subjected to double digestion with Sal I and BamH I respectively,...

specific Embodiment approach 2

[0074] Specific embodiment two: the difference between this embodiment and specific embodiment one is that in step one, the reaction system and cycle parameters for PCR amplification using the first strand of cDNA as a template are as follows:

[0075] (1) Reaction system:

[0076] Ingredients Dosage

[0077] cDNA first strand 1 μl

[0078] 10×PCR Hifi Buffer II (with Mg 2+ ) 5μl

[0079] dNTP (10mM) 1μl

[0080] Crchi1F (10μmol / L) 1μl

[0081] Crchi1R (10μmol / L) 1μl

[0082] Taq Hifi DNA polymerase (5U / μl) 0.5μl

[0083] wxya 2O 39.5 μl

[0084] (2) PCR amplification program: denaturation at 94°C for 2 min, denaturation at 94°C for 1 min, annealing at 56°C for 1 min, extension at 72°C for 1 min, a total of 35 cycles, extension at 72°C for 10 min, and incubation at 4°C. Other steps and parameters are the same as those in Embodiment 1.

specific Embodiment approach 3

[0085] Specific embodiment three: the difference between this embodiment and specific embodiment one is that the system of double enzyme digestion in step two is as follows:

[0086] Ingredients Dosage

[0087] Target gene 6 μL

[0088] Restriction enzyme Sal I 1 μL

[0089] Restriction enzyme BamH I 1 μL

[0090] 10×Buffer BamHI 2μL

[0091] wxya 2 O 10 μL.

[0092] Other steps and parameters are the same as those in Embodiment 1.

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Abstract

The invention discloses a method for constructing antagonistic yeast for preventing and controlling tomato gray mold and relates to a method for constructing the antagonistic yeast. The method comprises the following steps of: 1, extracting total ribonucleic acid (RNA) of bionectria ochroleuca WY-1, performing reverse transcription to synthesize a first cDNA chain and performing polymerase chain reaction (PCR) amplification so as to obtain a target gene; 2, linking the target gene with a YEp73-T vector, converting escherichia coli, extracting a plasmid vector YEp73, performing double digestion on the target gene and the plasmid vector YEp73, recycling a target segment and linking and constructing a recombinant plasmid YEp73-Crchi1; 3, recovering cryptococcus albidus WY-1, performing induced mutation and screening so as to obtain an Leu-auxotrophic mutant strain; and 4, converting the recombinant plasmid YEp73-Crchi1 into the Leu-auxotrophic mutant strain so as to construct the antagonistic yeast. The antagonistic yeast for preventing and controlling the tomato gray mold constructed by the method can effectively prevent and control the gray mold and the bacteriostatic efficacy index is up to 76.7 percent.

Description

technical field [0001] The invention relates to a construction method of biocontrol yeast. Background technique [0002] Tomato gray mold is a worldwide distribution disease. With the continuous expansion of tomato cultivation area, tomato gray mold caused by Botrytis cinerea Pers. is becoming more and more serious, and has become the main disease of vegetable production in my country. one. Especially in protected areas, due to the intensive cultivation of heavy cropping and continuous cropping, coupled with high humidity conditions, the pathogens reproduce in large numbers all year round, creating more favorable conditions for the epidemic of the disease. The annual loss of tomato production is 20% to 30%, and the year of severe disease is about 50%. , In severe cases, it may even cause the tomato to be out of production. [0003] Adopting high-level production management technology is an important measure to control tomato gray mold, but its effect is limited, and its app...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01P3/00C12N15/63A01N63/04C12N15/56C12N1/19C12N15/01C12R1/645
Inventor 王傲雪杨依军李景富陈秀玲张晓烜魏丹张丽莉王迎春
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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