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.
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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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