Composite microbial fertilizer and applications of composite microbial fertilizer in Taxus chinensis var.mairei growth promotion and taxol accumulation
A technology of complex microorganisms and southern yew, which is applied in compound fertilizers to promote the growth of southern yew and the accumulation of paclitaxel, can solve the problems that have not been reported in applied research, and achieve the effect of promoting growth and development and combining excellent strains
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Embodiment 1
[0025] Embodiment 1: the preparation of inoculant
[0026] (1) The spores of the AM fungus G.versiforme were treated with 30% H 2 o 2 Disinfect for 10 minutes, then rinse with sterile water for several times, use white clover (Trifolium Nepens L.) as the carrier to enrich and cultivate the spores of the rinsed AM fungi, and the culture medium is a mixture of sterilized soil and sand (2:1), After cultivating for 2 months, remove the above-ground part, and make a mixture containing spores, hyphae and infected root segments as the AM fungal inoculant;
[0027] (2) After activating CLW17 for 2 to 3 times, use an inoculation loop to pick a small amount of bacteria and inoculate it into 100 mL of triangular culture medium containing 50 mL of NB medium (3g of beef extract, 10g of peptone, 5g of sodium chloride, 1000mL of distilled water, pH 7.2 to 7.4). In the bottle, 30°C, 200r / min shaking culture for 48h as inoculant.
Embodiment 2
[0028] Embodiment 2: Bacteria agent inoculation greenhouse pot test
[0029]The inoculants prepared in Example 1 were respectively inoculated on the roots of the 1-year-old seedlings of Taxus chinensis, and the blank NB culture medium and the substrate for the propagation of the AM bacterial agent were used as controls, and a total of 4 treatments were set up. The four treatments were (1) G.versiforme, the inoculation amount was 25 g / plant; (2) CLW17, the inoculation amount was 50 mL / plant; (3) CLW17+G.versiforme, the AM inoculation amount was 25 g / plant, The inoculation amount of phosphate-solubilizing bacteria is 50mL / strain; (4) Blank control CK. Fifteen replicates per treatment were placed in the greenhouse for unified management, with 12 hours of light per day and timely watering.
[0030] 360-day growth situation of southern yew seedlings (see Table 1 ). From Table 1 It can be seen that the inoculation of CLY07 bacterial agent significantly promoted the growth of t...
Embodiment 3
[0034] Embodiment 3: detection test of paclitaxel content in greenhouse potted seedlings
[0035] One year after the inoculation of the southern yew seedlings in Example 2, the roots, stems, and leaves of different treatments were dried and ground at 56° C. to form powders for later use. Weigh 5 grams of powder and place them in a 500ml Erlenmeyer flask with a stopper, measure 250ml of methanol for leaching for 1h, then ultrasonically extract for 20min, and filter. Repeat the above steps 3 times. The filtrates were combined and evaporated to dryness under vacuum at 37°C. The obtained dry matter was dissolved in 10ml of methanol, and passed through a 0.45 μm filter membrane to obtain a crude paclitaxel extract, which was stored at -20°C for use. Paclitaxel (Sigma) was used as a standard substance, and the content of paclitaxel in different treatments was determined by HPLC.
[0036] Depend on Table 2 It can be seen that the single inoculation of the AM fungus G.versiforme a...
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