Application of chaetomium globosum in prevention and treatment of wheat stem rot
A technology for Chaetomium globosa, stem rot, applied in the direction of application, microbe-based methods, fungicides, etc.
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Embodiment 1
[0018] Isolation of Chaetomium globosa Strain 12XP1-2-3
[0019] At the wheat grain filling stage, the unaffected plants in the wheat take-all field were collected in Xiping County, Henan Province and put into paper bags. The samples were stored in a refrigerator at 4°C. Rinse the root of the sample, and then take 2-3cm of wheat root tissue, disinfect the surface with 75% absolute ethanol for 10-30s, disinfect with 1% NaCIO for 1.5min, rinse with sterile water for 3 times, and blot dry with sterile filter paper After hydration, cut into small sections of about 0.5 cm, and place them on a potato dextrose agar medium (PDA, raw material composition: 300 g of peeled potatoes, 20 g of glucose, 20 g of agar and 1000 ml of distilled water) with sterile tweezers. After 5-7 days of cultivation under the same conditions, pick off the edge mycelia and grow on a new PDA plate. After 5-7 days under the same conditions, pick off the mycelium pieces with uniform colonies and place them on a...
Embodiment 2
[0021] Extraction of DNA from Chaetomium globosa Strain 12XP1-2-3
[0022] Pick the chaetomium strain 12XP1-2-3 onto the PDA medium, and after culturing at 20°C for 5-7 days, pick 5 edge mycelium pieces and place them evenly on the PDA plate covered with sterilized cellophane. After 3-5 days, scrape the mycelium with a sterilized small shovel, freeze it with liquid nitrogen, and store it in a refrigerator at -20°C. When extracting DNA, take out 20-25 mg mycelium and put it into a pre-cooled 1.5ml EP tube; add a little liquid nitrogen and grind the mycelium to powder in the EP tube with a pre-cooled iron nail; add 500 μl Extraction Buffer (50mM Tris -Cl (pH 8.0), 150mM NaCl, 100mM EDTA (8.0)), shake the suspended pellet on a vortex shaker and mix well; , mixed upside down, placed in a water bath at 37°C for 1-3 hours; then added 75 μl of 5M NaCl, mixed upside down; added 65 μl of CTAB / NaCl (10% CTAB, 0.7M NaCl) solution, bathed in water at 65°C for 30 minutes ; Add an equal v...
Embodiment 3
[0024] Molecular identification of Chaetomium globosa strain 12XP1-2-3
[0025] In this test, primers ITS1 (5'-TCC GTA GGT GAA CCT GCG G-3') and ITS4 (5'-TCC TCCGCT TAT TGA TAT GC-3') were used to amplify the strains containing ITS1, 5.8S rDNA and ITS2 of DNA fragments. The system refers to Daval et al. (2010). Amplification reaction conditions: The PCR reaction program is: 95°C for 3min; 95°C for 45s, 50°C for 30s, 72°C for 1min, 35 cycles; 72°C for 10min. After the PCR products were sequenced, BLAST sequence alignment analysis was carried out in NCBI. The result of the sequence alignment shows that the biocontrol bacterium 12XP1-2-3 is a globular hair shell.
[0026] Table 1. PCR reaction system
[0027] Element concentration Dosage (μl) 10×PCR buffer 10mM 5.0 dNTP mix 10mM 1.0 TaqDNase 5u 0.5 Primer 1 20μM 1.0 Primer 2 20μM 1.0 DNA template 10-50ng / μl 1.0 double distilled water pure water 40.5 Tot...
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