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Rapeseed endogenous trichoderma atroviride ReTv2 strain and preparation method and application thereof

A technology of Trichoderma dark green, strains, applied in biochemical equipment and methods, botanical equipment and methods, microorganism-based methods, etc., can solve the problems that have not been seen before.

Inactive Publication Date: 2015-01-07
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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  • Rapeseed endogenous trichoderma atroviride ReTv2 strain and preparation method and application thereof
  • Rapeseed endogenous trichoderma atroviride ReTv2 strain and preparation method and application thereof
  • Rapeseed endogenous trichoderma atroviride ReTv2 strain and preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0036] Isolation and identification of strains

[0037] 1. Isolation location and isolation method of the strain

[0038] Healthy rapeseed root tissues from diseased rapeseed fields in Zhijiang area, Hubei Province were collected, cleaned, soaked in 70% ethanol for 1 min, soaked in 5% NaOCl for 5 min, and then washed 3 times with sterile water. Cut the roots into 0.5cm small pieces and put them on the PDA medium, culture them at 25°C for 14 days, dilute the grown fungal spores on the PDA plate, and perform single spore purification; obtain a fungal strain and number it as For ReTv2, its colony, spore peduncle and spore morphology are shown in figure 1 , further combined with ITS DNA sequence for identification.

[0039] 2. 16S rDNA identification of strains

[0040] The above-mentioned strain ReTv2 was inoculated on a PDA plate covered with cellophane, cultured at 28°C for 1 day, and the hyphae were scraped off.

[0041]The DNA of the strain was extracted by the CTAB metho...

Embodiment 2

[0048] The preparation method of Trichoderma dark green ReTv2, specifically as follows:

[0049] The ReTv2 strain was cultured on PDA based on 28°C activation culture for 2-4d, then inoculated on the PDA slope, cultured at 28°C for 4-6d, added sterilized deionized water, and adjusted the concentration of spore liquid to 5×10 5 Spores / mL, add 1 mL of spore liquid to 250 mL of PDB medium, culture at 180 rpm and 25 °C for 6 days, the concentration can reach 1×10 9 Spores / mL, the spores were collected to prepare a spore suspension preparation.

[0050] PDA medium: 200 g of potatoes, 20 g of glucose, 10 g of agar, supplemented with distilled water to 1000 mL, adjusted the pH to 7.0, and sterilized at 121 ° C for 30 min.

[0051] PDB medium: 200 g of potatoes, 20 g of glucose, supplemented with distilled water to 1000 mL, adjusted the pH to 7.0, and sterilized at 121 ° C for 30 min.

Embodiment 3

[0053] Endogenous Verification of Strain ReTv2 in Rapeseed Roots

[0054] The RFP expression vector was constructed with pCAMBIA1301 as the backbone, and the conidia were collected after culturing Trichoderma dark green ReTv2 strain on PDA for 5-7 days, and the transformation method mediated by Agrobacterium was used (Li Moxiao et al., FEMS Microbiol Lett, 2005, 243 (2 ): 323-329) the RFP vector is transformed into Trichoderma dark green ReTv2 bacterial strain, select transformant on the PDA containing 50ug / mL hygromycin, and carry out the second round of selection on the PDA containing 100ug / mL hygromycin, Finally, the strains that can emit red fluorescence and have no change in biological performance are selected ( figure 2 Middle A).

[0055] Inoculate 1×10 cells of the transformed strain capable of emitting red fluorescence in 200 mL of PDB medium. 6 The spores were cultured at 28°C and 200rpm for 15h, centrifuged at 4000rpm for 5min to collect the spores, and washed 3 ...

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Abstract

The invention discloses a rapeseed endogenous trichoderma atroviride ReTv2 strain and a preparation method and application thereof. The trichoderma atroviride ReTv2 strain is separated from a rapeseed root tissue by the applicant, and the number CCTCC NO is M2014407. The ReTv2 strain is activated and cultured in a PDA culture medium at the temperature of 28 DEG C for 2-4d to obtain conidiospores, and the spores are inoculated into a PDB culture medium for enlarged culture for 6d, wherein the content of the conidiospores can achieve 1.0*10<9> / mL. The trichoderma atroviride ReTv2 strain disclosed by the invention is the rapeseed endogenous strain, can be successfully colonized at the root of rapeseed and has an effect on promoting growth of the rapeseed, a fermentation broth has an obvious effect on inhibiting germination of resting spores of pathogenic bacteria of rapeseed clubroot, and the control effect of the strain against the rapeseed clubroot achieves 66.4%; furthermore, the ReTv2 strain can be directly applied during seeding, is simple in an application method, can save labor force and further has the property of being safe to people and animals of biological pesticides.

Description

technical field [0001] The invention belongs to the technical field of plant disease biological control, in particular to a bacterial strain Trichoderma dark green ReTv2 bacterial strain used for the biological control of rape clubroot, and also relates to a preparation method of a biocontrol bacteria Trichoderma dark green ReTv2 bacterial agent, It also relates to the application of a Trichoderma dark viridans ReTv2 strain in the preparation of medicines for treating or preventing clubroot of cruciferous plants such as rapeseed. Background technique [0002] Rapeseed is one of the important oil crops in my country, and it is threatened by various pathogenic bacteria in the production process of rapeseed. Clubroot is an important disease caused by Plasmodiophora brassicae of the protozoan kingdom Plasmodiophora brassicae. Various other cruciferous plants. At present, there is no effective control method for clubroot, and the comprehensive control measures of conventional d...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N3/00C12P1/02A01N63/04A01P3/00A01P21/00C12R1/885
CPCA01N63/30C12N1/14C12N3/00C12P1/02C12N1/145C12R2001/885
Inventor 程家森穆罕默德·哈森姜道宏付艳苹谢甲涛
Owner HUAZHONG AGRI UNIV
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