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Method for separating metabolite capable of inducing cucumber resisting powdery mildew from molecule modifying trichoderma harzianum

A technology against powdery mildew and Trichoderma, applied in the direction of microorganism-based methods, botany equipment and methods, biochemical equipment and methods, etc., can solve problems such as increased difficulty and unstable effect of induced resistance, and achieve efficient screening, The control effect is stable and the control effect is obvious

Inactive Publication Date: 2008-05-28
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although many previous studies have proved that Trichoderma can obviously induce the expression of potential resistance-related genes in host plants under living conditions, they have encountered some difficulties in practical application: (1) Although the inducer mainly based on a single living Trichoderma can continuously Propagate in the species enclosure and rhizosphere to produce resistance-inducing metabolites, but in practical applications, the population and activity of living microorganisms are easily affected by various environmental factors in the soil, resulting in unstable induction of resistance
(2) Due to the increasing difficulty of screening excellent strains from wild Trichoderma or isolating new antagonistic chemical substances from antagonistic Trichoderma, few studies have reported isolation of new compounds with the function of inducing resistance from Trichoderma

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] 1. Preparation of Trichoderma Protoplasts

[0021] Cultivate Trichoderma on potato dextrose agar medium, wash the spores with sterile water, then add them to potato dextrose solution, cultivate them at 28°C and 120 rpm for 18-20 hours, filter mycelia, and wash bacteria with 0.7 molar NaCl Silk.

[0022] Preparation concentration is that the cell wall lyase solution of 13 mg / ml is used for processing Trichoderma hyphae, and the ratio of mycelium in the cell wall lyase solution is 30%; Shake culture 3 hours under 130 rev / min and 28 ℃ of conditions, obtain Protoplasts; then filter, wash with 0.7M NaCl solution, centrifuge at 4°C and 4000 rpm for 15 minutes, collect the protoplasts, add 5 ml of sorbitol buffer, centrifuge at 4°C and 4000 rpm for 15 minutes, re- Suspend protoplasts in sorbitol buffer and adjust the concentration of protoplasts to 10 7 individual / ml.

[0023] 2. Plasmid Extraction

[0024] Take 1.5 ml of bacterial culture solution cultivated to the logari...

Embodiment 2

[0038] 1. Preparation of Protoplasts from Trichoderma Strains

[0039] Cultivate Trichoderma on potato dextrose agar medium, wash the spores with sterile water, then add them to potato dextrose solution, cultivate them at 28°C and 120 rpm for 19-22 hours, filter mycelia, and wash bacteria with 0.7 molar NaCl Silk.

[0040] Preparation concentration is that the cell wall lyase solution of 11 mg / ml is used for processing Trichoderma hyphae, and the ratio of mycelia in the cell wall lyase solution is 40%; Vibrate and cultivate 4 hours under 130 rev / min and 26 ℃ of conditions, obtain Protoplasts; then filter, wash with 0.7M NaCl solution, centrifuge at 4°C and 4000 rpm for 15 minutes, collect the protoplasts, add 5 ml of sorbitol buffer, centrifuge at 4°C and 4000 rpm for 15 minutes, re- Suspend protoplasts in sorbitol buffer and adjust the concentration of protoplasts to 1.5×10 7 individual / ml.

[0041] 2. Plasmid Extraction

[0042] Take 2 ml of the bacterial culture solutio...

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PUM

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Abstract

The invention relates to a process which is capable of inducing cucumbers to resist powdery mildew metabolites separated from trichoderma harzianum with lecular modification, which takes advantages of gene integration technique of restriction endonuclease mediated, constructs transformant of trichoderma harzianum by preparing strain protoplast of trichoderma harzianum and extracting plasmid, then the transformant is cultivated and fermented, extracellular metabolites of trichoderma harzianum transformant capable of systematic performance to induce cucumbers to resist powdery mildew are selected from the fermentation liquid. The extracellular metabolites of trichoderma harzianum transformant of the invention can overcome the weakness that living trichoderma harzianum is not stable in induced resistance effect. The invention not only has remarkable control effect to powdery mildew, but also facilitates growth of cucumber seedlings, and provides an efficient process for rapidly and highly efficiently selecting novel microbial pesticide guide compound from trichoderma harzianum for preparation of green molecular pesticide.

Description

technical field [0001] The invention relates to a method for isolating inducible cucumber powdery mildew resistance metabolites from molecularly improved Trichoderma, and the obtained metabolites are used to promote the growth of cucumber seedlings and improve the systemic resistance of cucumber seedlings to powdery mildew (Sphaerotheca fuliginea). Background technique [0002] Trichoderma is currently the most widely used biological control bacteria in the world. Its disease control mechanism not only has hyperparasitic and antibacterial effects on pathogenic bacteria, but also has the function of obviously inducing the expression of potential resistance-related genes in host plants. It has been proved that Trichoderma can induce crops such as cucumber, cotton, tobacco, lettuce and pepper to produce various diseases such as gray mold (Botrytis cinerea), anthracnose (Colletotrichum indemuthianum, C.orbicular, C.graminiicolar), Phytophthora ( Phytophthoracapsci), early blight...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15A01N63/02C12R1/885
Inventor 陈捷屈中华刘力行
Owner SHANGHAI JIAO TONG UNIV
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