Nitrogen fixing Paenibacillus 1-43 and application thereof
A Bacillus and nitrogen-fixing technology, applied in the application, bacteria, fungicides and other directions, can solve the problems of limiting the application of Paenibacillus species and the late start of research on autogenous nitrogen-fixing bacteria, and achieves germination growth promotion, reduction of usage, plant growth The effect of promotion
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Embodiment 1
[0024] The cultivation and identification of embodiment 1 Paenibacillus nitrogen-fixing 1-43 bacterial strains
[0025] (1) Cultivation of strains
[0026] Production of medium: liquid medium (1L): 20g sucrose; 12.06g K 2 HPO 4 ;3.4gKH 2 PO 4 ;0.2g MgSO 4 ·7H 2 O; 0.01g NaCl; 0.01g FeCl 3 ;0.002gNaMoO 4 2H 2 O; H 2 O 1L. Sterilize at 120°C for 25 minutes. The production of solid medium is to add 14g agar per liter of liquid medium. Inoculate Paenibacillus nitrogen-fixing bacteria into solid or liquid medium, and cultivate them in a 30°C incubator for 3 days.
[0027] (2) Extraction of bacterial total DNA The extraction method refers to the method in the literature [Ausubel et al. Current Protocols in Molecular Biology. New York: Wiley.1987].
[0028] (3) Identification of strains and amplification of nitrogenase structural gene nifH and 16S rDNA
[0029] Extract genomic DNA from the pure culture of bacterial strain Paenibacillus sp.1-43 of the present invention, ...
Embodiment 2
[0042] The determination of embodiment 2 nitrogenase activity
[0043] 1. Inoculate 1-43 strains in 5mL medium for measuring enzyme activity, culture overnight at 30°C, transfer to a 50mL Erlenmeyer flask at 1% inoculum size, culture overnight at 30°C, and then collect the bacteria. Suspend the specific medium with an appropriate amount of medium for measuring enzyme activity, and adjust the OD 600 to 1. Inoculate and culture 1.5mL of bacterial liquid for 2h, take 100μL of gas and measure the ethylene content by gas chromatography.
[0044] The enzyme activity medium is: 26.3g Na 2 HPO 4 12H 2 O; 3.4 g KH 2 PO 4 ; 10 μg biotin; 26 mg CaCl 2 2H 2 O; 30mg MgSO 4 ; 0.33mg MnSO 4 ·H 2 O; 36 mg ferric citrate; 7.6 mg Na 2 MoO 4 2H 2 O; 10 μg p-aminobenzoic acid; 4 g glucose. Add glucose separately after sterilizing at 115°C for 30 minutes, and other reagents can be sterilized at 121°C for 30 minutes after preparation.
[0045] 2. Determination of protein content (Br...
Embodiment 3
[0061] Antibacterial activity experiment of embodiment 3 Paenibacillus nitrogen-fixing 1-43 bacterial strains
[0062] PDA medium (potato 200g, glucose 20g, agar powder 16g, add distilled water to 1L). Plant pathogenic bacteria and Paenibacillus 1-43 provided by the present invention were inoculated respectively at two points at 2 cm from the center of the PDA plate, and then the plate was placed in a 30° C. Inhibition, the results are shown in Figure 3. The plant pathogens used in this example are: Fusarium oxysporum, Rhizoctonia cerealis, Sclerotinia sclerotiorum, and Botrytis cinerea.
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