Bacillus subtilis PA8 and application thereof
A technology of Bacillus subtilis and inoculum, applied in the field of microorganisms, can solve problems such as few studies, and achieve the effects of improving seedling quantity, excellent siderophore-producing ability, and excellent growth-promoting effect.
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Embodiment 1
[0021] Embodiment 1: Separation and purification of bacterial strains
[0022] 1. Medium
[0023] LB medium: tryptone 10g, yeast extract 5g, NaCl 10g, agar powder 20g, distilled water 1L, sterilized at 120°C for 20 minutes.
[0024] 2 test steps
[0025] 2.1 Culture medium preparation
[0026] Prepare LB medium as described in 1, and prepare liquid medium and slant medium.
[0027] 2.2 Preparation of soil dilution
[0028] Soil samples were collected from paddy fields in Ping’an Township, Fengjie County, Chongqing City. Weighed 5g of soil samples, put them into a triangular flask filled with 45mL of sterile water, oscillated on a shaker at 180rpm for 30min, and placed them in a water bath at 80°C for 20min. to kill other bacteria. Make soil suspension, adopt 10-fold dilution method to prepare concentration gradient to be 10 -2 , 10 -3 、10 -4 soil suspension samples.
[0030] take 10 -2 、10 -3 、10 -4 100 μL of the soil dilution solutio...
Embodiment 2
[0031] Example 2: Determination of auxin and screening and identification of bacterial strain PA8
[0032] 1 medium
[0033] King's medium (King): protein building 20g, MgSO 4 ·7H 2 O 1.5g, K 2 HPO 4 1.15g, glycerin 15mL, distilled water 1L, pH 6.8 for the determination of IAA production capacity.
[0034] S2 colorimetric solution: FeCl 3 4.5g, 10.8mol / L H 2 SO 4 The measuring range of 1L is 5-200ug / mL, generally more than 10ug / mL needs to dilute the colorimetric solution.
[0035] 2 test steps
[0036] The auxin was determined by the Salkowski colorimetric method, and the specific steps were as follows:
[0037] (1) Qualitative determination of plant auxin IAA: inoculate the isolated and purified bacterial suspension into the above-mentioned King's medium (King) according to 1% of the medium volume, and shake and cultivate it on a constant temperature shaker at 30°C and 180r / min for 7d , centrifuge at 12000r / min for 10min, take 0.1mL of the supernatant and drop i...
Embodiment 3
[0042] Embodiment 3: Determination of siderophore ability
[0043] 1 medium
[0044] CAS medium: 1 mL of 20% sucrose solution per 100 mL, 3 mL of 10% acid hydrolyzed casein, 1 mmol / L CaCl 2 100μL, 1mmol / L MgSO 42mL, 1.8g agar, slowly add phosphate buffer saline and 5mL of CAS staining solution at about 60°C to obtain CAS blue medium.
[0045] Table 1: 0.1mol / L phosphate buffer (pH6.8), diluted 10 times before use.
[0046] NaH 2 PO 4 2H 2 o
0.5905g K H 2 PO 4
0.075g NH 4 Cl
0.250g NaCl 0.125g Deionized water Dilute to 100mL
[0047] Siderophore detection CAS dye:
[0048] Solution A: Dissolve 0.079g CAS (chrome azure) in 50mL deionized water, then add 10mL 1mmol / L FeCl 3 Solution (containing 12mmol / LHCl);
[0049] Solution B: 0.069g hexadecyltrimethylammonium bromide (HDTMA) was dissolved in 40mL of deionized water;
[0050] CAS blue detection solution (100mL): Slowly add solution A into solution B along the beak...
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