A rapeseed endogenous Trichoderma dark green retv2 strain and its preparation method and application
A technology of Trichoderma viridans and strains, applied in biochemical equipment and methods, botany equipment and methods, and methods based on microorganisms, which can solve problems that have not been seen before
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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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