CN103548594B - Method for controlling Codonopsis pilosula yield and metabolite accumulation by utilizing rhizosphere beneficial bacteria - Google Patents
Method for controlling Codonopsis pilosula yield and metabolite accumulation by utilizing rhizosphere beneficial bacteria Download PDFInfo
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Abstract
The invention relates to a method for controlling Codonopsis pilosula yield and metabolite accumulation by utilizing bacteriabeneficial beneficial bacteria. The method is characterized by comprising the following steps of seedling cultivation: germinating Codonopsis pilosula seeds, transplanting the germinated Codonopsis pilosula seeds to hole trays filled with vermiculite, watering the germinated Codonopsis pilosula seeds by 1/2 Hoagland nutrient solution and cultivating the germinated Codonopsis pilosula seeds in a greenhouse, wherein in the greenhouse, the diurnal temperature is 26-30 DEG C, illumination is performed for 12-14 hours per day with light intensity of 800-1000mumol/m<2>*s, and the relative humidity is 60-80%. The plant bacteriabeneficial beneficial microorganism promotes plants to absorb nutrient substances and relieve plant diseases, therefore, growth conditions of the plants are improved, root development and growth of the Codonopsis pilosula are promoted, and then contents of some metabolic substances in the Codonopsis pilosula can be changed. Bacillus subtilis has the advantages of being free of toxicity and harm to people and livestock, free of pollution to environment, non-pathogenic and strong in stress resistance, and can produce a variety of growth hormone-like chemicals and rich antimicrobial substances, promote plant growth and control accumulation of plant metabolite.
Description
Technical field
The invention belongs to microbiology and plant physiology, relate to and utilize beneficial rhizosphere bacteria B. subtilis GB03 to regulate and control the growth of Radix Codonopsis root system development, regulation activity composition and Metabolite Accumulation, thus make Radix Codonopsis output increased and the improved method of quality.
Background technology
Bacillus subtilis (Bacillus subtilis) has nontoxic to people and animals, free from environmental pollution, the feature such as non-pathogenic and strong stress resistance, it can produce multiple growth hormone material and abundant antibacterial material, Promoting plant growth, improve vine growth and development, increase plant to the defensive ability/resistance ability of the adverse circumstances such as salt stress, thus improve the disease resistance of plant.
In recent years, Radix Codonopsis achieves new progress in faces such as treatment coronary heart disease, hyperlipemia, hypopiesis, its consumption speed is made to increase (Ma Xuemei and Wu are towards peak 2009), but because Radix Codonopsis seed is little, life-span is short, and during direct sowing and seedling, germination percentage is low, and during cultivation, application rate is large, cost is high, does not reach production requirement.According to investigations, the demand of domestic annual Radix Codonopsis is considerably beyond production, and since commercially concluding the business from Radix Codonopsis as commodity, the phenomenon of big rise and big fall just often appears in the production and selling of Radix Codonopsis, and price fluctuation is also very large.At present, national Radix Codonopsis wild resource reserves are about 1.58 ten thousand tons, but resource is disperseed, output presents downward trend year by year, and the commodity amount that can provide is extremely limited, Radix Codonopsis market supply and demand contradiction very outstanding (Liu Dejun etc. 2001).Show from Chinese medicine Radix Codonopsis resource investigation result, Radix Codonopsis main product in the market in Shanxi, Gansu, Shaanxi, the province such as Sichuan and Hubei, be mainly cultivation product, a small amount of is wild, Gansu is except original producton location, the plantation of nearly large area development decades Radix Codonopsis, expands the production scale of element flower Radix Codonopsis simultaneously, becomes the maximum producing region of Chinese medicine Radix Codonopsis (Xiangdong Zhang etc. 2013).
At present, along with the enforcement of the modernization of Chinese medicine and the status of China in World Economics more and more higher, the production of Chinese medicine and all steady growth of outlet, the outlet of China's Radix Codonopsis in 2007 has exceeded 0.3 ten thousand tons according to estimates, as herbal pharmaceutical raw material, the consumption of Radix Codonopsis also significantly increases.In recent years, the market price repeatedly lifting of Radix Codonopsis, and obviously decline due to national Radix Codonopsis total output, its price is significantly raised.Codonopsis pilosula supply falls short of demand become Chinese medicine produce in a great problem, therefore, improve Radix Codonopsis and produce soil quality, regulation activity composition and Metabolite Accumulation, improving the first-selection that Radix Codonopsis quality has become Chinese medicine plant husbandry, is also problem demanding prompt solution during Radix Codonopsis is produced.
Summary of the invention
The object of the invention be to provide a kind of regulate and control the method for Radix Codonopsis output and Metabolite Accumulation.Utilize beneficial rhizosphere bacteria B. subtilis bacterial strain GB03 to regulate and control the accumulation of Radix Codonopsis metabolite, the method is simple to operate, and cost is low, efficiently, nontoxic and environmentally friendly harmless.
The present invention seeks to be achieved through the following technical solutions: a kind of regulate and control the method for Radix Codonopsis output and Metabolite Accumulation, its main feature is to include following steps:
(1) seedling culture: by Radix Codonopsis seed, germinate, after germination, transplant in the cave dish that vermiculite is housed, pouring 1/2Hoagland nutrient solution, cultivates in greenhouse; The diurnal temperature in greenhouse is 26-30 DEG C, illumination 12-14h/d, and luminous intensity is 800-1000 μm of ol/m2s, relative humidity 60-80%.
Described beneficial rhizosphere bacterial regulatory Radix Codonopsis output and the method for Metabolite Accumulation, also include following steps:
(2) seedling replanting: treat growth of seedling 28-32 days, transplanting to the diameter 18-22cm degree of depth is in the plastic flowerpot of 28-30cm, the matrix of flowerpot is that turfy soil and farmers' soil 1:1 mix, dry 3d at 158-162 degree high temperature oven, fill basin afterwards, weigh, control that irrigation amount is maxmun field capacity 60%;
(3) bacterium solution preparation: preparation LB fluid nutrient medium: according to the proportions of sodium chloride 5g/L, tryptone 10g/L and dusty yeast 5g/L, adjust pH to 6.8-7.2, temperature be 121-126 DEG C of sterilizing after cooling, the LB fluid nutrient medium of every 100mL adds the GB03 bacterium liquid of 50-100 μ L, under rotating speed is 180r/min, temperature is on the constant-temperature shaking incubator of 25-28 DEG C after overnight incubation, nucleic acid-protein detector (NanoDrop1000, Germany) mensuration bacterial concentration is used to take out for subsequent use when being OD600=0.8-1.0.
Described beneficial rhizosphere bacterial regulatory Radix Codonopsis output and the method for Metabolite Accumulation, also include following steps:
(4) seedling process: add bacterium liquid after transplanting two weeks, adds bacterium once later every other month, draws the bacillus subtilis GB03 of 0.5-1mL, be applied to uniformly on the soil around every strain seedling basal part of stem with liquid-transfering gun.
(5) index determining a: Growing season terminates rear mensuration biomass, middle amino acid content and lobetyolin.
Beneficial effect of the present invention:
Plant rhizosphere beneficial microbe by promoting the absorption of Plant To Nutrient material and alleviating plant disease, thus improves the condition of plant growth, then changes the content of some metabolites in plant.Simultaneously, bacillus subtilis (Bacillus subtilis) has nontoxic to people and animals, free from environmental pollution, the feature such as non-pathogenic and strong stress resistance, multiple growth hormone material and abundant antibacterial material can be produced, Promoting plant growth, the simultaneously accumulation of controllable plant metabolites.Radix Codonopsis contains the compositions such as abundant terpene, glycoside, amino acid and polysaccharide.Lobetyolin's rich content, has the effects such as anticancer, antibacterial, anti-inflammatory, calmness, step-down, anti-gastric acid secretion.In addition, Radix Codonopsis contains necessary 7 seed amino acids of human body, makes it have more purposes.The present invention, by the growth of regulation and control Radix Codonopsis, improves output, strengthens the accumulation of metabolite, beneficiating ingredient in its body is accumulated in a large number, has more use value.Processing method of the present invention is simple, can extensive use, has important society, economy and Eco-economic Value.
Accompanying drawing explanation
Fig. 1: bacillus subtilis GB03 impact on Radix Codonopsis root dry weight (output); In figure, four pillars represent the Radix Codonopsis root dry weight (output) after bacillus subtilis strain GB03 bacterium liquid, bacillus coli DH 5 alpha bacterium liquid, LB fluid nutrient medium and water treatment Radix Codonopsis seedling respectively.
Fig. 2: bacillus subtilis GB03 is on the impact of Radix Codonopsis root total amino acid; In figure, four pillars represent the Radix Codonopsis root total amino acid content after bacillus subtilis strain GB03 bacterium liquid, bacillus coli DH 5 alpha bacterium liquid, LB fluid nutrient medium and water treatment Radix Codonopsis seedling respectively.
Fig. 3: bacillus subtilis GB03 is on the impact of Radix Codonopsis root 7 kinds of essential amino acid total contents; In figure, four pillars represent the Radix Codonopsis root 7 kinds of essential amino acid total contents after bacillus subtilis strain GB03 bacterium liquid, bacillus coli DH 5 alpha bacterium liquid, LB fluid nutrient medium and water treatment Radix Codonopsis seedling respectively.
Fig. 4: lobetyolin measures collection of illustrative plates; In figure, four pillars represent the Radix Codonopsis root alkynes glycosides content high-performance liquid chromatogram determination collection of illustrative plates after bacillus subtilis strain GB03 bacterium liquid, bacillus coli DH 5 alpha bacterium liquid, LB fluid nutrient medium and water treatment Radix Codonopsis seedling respectively.
Fig. 5: bacillus subtilis GB03 impact on Radix Codonopsis root alkynes glycosides; In figure, four pillars represent the Radix Codonopsis root alkynes glycosides content after bacillus subtilis strain GB03 bacterium liquid, bacillus coli DH 5 alpha bacterium liquid, LB fluid nutrient medium and water treatment Radix Codonopsis seedling respectively.
Detailed description of the invention
Be described principle of the present invention and feature below, example, only for explaining the present invention, is not intended to limit scope of the present invention.The those of ordinary skill of described technical field, according to above content disclosed by the invention and scope that each parameter is got, all can realize object of the present invention.
Embodiment 1: seedling culture
By Radix Codonopsis seed, germinate, after germination, transplant in the cave dish that vermiculite is housed, pouring 1/2Hoagland nutrient solution, cultivates in greenhouse; The diurnal temperature in greenhouse is 26-30 DEG C, illumination 12-14h/d, and luminous intensity is 800-1000 μm of ol/m2s, relative humidity 60-80%.
Embodiment 2: seedling replanting
Treat growth of seedling 28-32 days, transplanting to the diameter 18-22cm degree of depth is in the plastic flowerpot of 28-30cm, the matrix of flowerpot is that turfy soil and farmers' soil 1:1 mixs, at 158-162 degree high temperature oven baking 3d, fill basin afterwards, weigh, control that irrigation amount is maxmun field capacity 60%;
Embodiment 3: bacterium solution preparation
Preparation LB fluid nutrient medium: according to the proportions of sodium chloride 5g/L, tryptone 10g/L and dusty yeast 5g/L, adjust pH to 6.8-7.2, temperature be 121-126 DEG C of sterilizing after cooling, the LB fluid nutrient medium of every 100mL adds the GB03 bacterium liquid of 50-100 μ L, under rotating speed is 180r/min, temperature is on the constant-temperature shaking incubator of 25-28 DEG C after overnight incubation, and using detection of nucleic acids instrument to measure bacterial concentration is OD
600take out for subsequent use during=0.8-1.0;
Embodiment 4: seedling process
Add bacterium liquid after transplanting two weeks, add bacterium every other month once later, draw the bacillus subtilis GB03 of 0.5-1mL with liquid-transfering gun, be applied to uniformly on the soil around every strain seedling basal part of stem.
Embodiment 5: index determining
A Growing season terminates rear mensuration biomass, root and plant above ground portion amino acid content and lobetyolin.
Experimental example 1: a kind of method of beneficial rhizosphere bacterial regulatory Radix Codonopsis output and Metabolite Accumulation
1 materials and methods
1.1 materials are cultivated
After germination, transplant in the cave dish that vermiculite is housed, pouring 1/2Hoagland nutrient solution also carries out vegetable material cultivation in greenhouse; The diurnal temperature in greenhouse is (28 ± 2) DEG C/(23 ± 2) DEG C, illumination 14h/d, luminous intensity about 1200 μm of ol/m
2s, relative humidity 60%-80%.
1.2 processing method
After 3 week age of growth of seedling, process, be divided into bacillus subtilis strain GB03, coli strain DH5 α, LB fluid nutrient medium and the process of 4, water, processing method is consistent with previous experiments, when growth of seedling one month, transplanting to the diameter 20cm degree of depth is in the plastic flowerpot of 30cm, during transplanting, points for attention are: the related vermiculite of seedling in being coiled in cave digs out together, all put into flowerpot, the matrix of flowerpot is that turfy soil and farmers' soil 1:1 mix, dry 3d at 160 degree of high temperature ovens, fill basin afterwards, weigh, to control irrigation amount consistent.Add bacterium liquid after transplanting two weeks, add bacterium every other month once later, sample after Radix Codonopsis seedling grows 1 year, measure index of correlation, often process 6 repetitions.
1.3 index determining
Main root measuring diameter: use slide measure to measure main root diameter, unit is cm.
Root stereometry: dug out by Radix Codonopsis root after cleaning, wipe surface moisture, fill 200mL distilled water in 500mL volumetric flask, be all immersed in water by Radix Codonopsis root system with filter paper, reads liquid level rising number, and record.
Overground part and root dry weight and fresh weight of plant seedlings measure: use ten thousand/balance to take each process fresh weight after sampling, and record data, are contained in Envelope bag by having claimed the sample of fresh weight, finish writing label, be placed on constant temperature oven, set temperature 105 DEG C, dry and take dry weight after 48 hours, record data.
Determined amino acid: take levigate Radix Codonopsis plant dry sample 0.5g, add the sulfosalicylic acid of 5mL deionized water and 5mL8%, abundant vibration 15min, centrifugal 20min under 10000rpm, get supernatant and use Hitachi 83-50 type automatic amino acid analyzer, with reference to GB/T182462000 method determined amino acid and analysis are carried out to sample, result is repeat the measured values of biased samples 3 times.
Lobetyolin measures: adopt the method for HPLC to measure lobetyolin's content, key instrument is Agilent Agilent1100 type high performance liquid chromatograph, and chromatographic column is Agilent TC-C
18post, using acetonitrile-water (30:70) as mobile phase, flow velocity is 1mL/min, and determined wavelength is 267nm, and the temperature of chromatographic column is 30 DEG C.The method adopted is the hot dipping that Chinese Pharmacopoeia is included for 2005, and the ethanol with 45%, as solvent, measures the extract (%) of Radix Codonopsis.Get lobetyolin's reference substance appropriate then accurately weighed, add methyl alcohol and make every milliliter containing the solution of 96 μ g, keep for subsequent use.Respectively each codonopsis pilosula processed is pulverized (crossing a number sieve), accurately weighed sample is about 2g, be placed in the conical flask of the band stopper of 150mL, add methyl alcohol 50mL accurately, after weighed quality, use ultrasonic extraction 30min, supply the quality of less loss afterwards, shake up and filter, accurate absorption filtrate 25mL, is placed in solvent evaporated in water-bath, is dissolved by remaining residue methyl alcohol and transfers in the volumetric flask of 10mL in batches, constant volume also shakes up, and keeps for subsequent use with the miillpore filter filtration of 0.45 μm.
2 results and analysis
2.1GB03 on the impact of Radix Codonopsis biomass
As can be seen from Figure 1, bacillus subtilis GB03 significantly (P<0.05) improves the dry weight of Radix Codonopsis root, and comparatively DH5 α, LB and water improve 49.8%, 55.4% and 37.7% respectively.
2.2GB03 on the amino acid whose impact of Radix Codonopsis
As can be seen from Table 1, GB03 is DH5 α, LB and water comparatively, raising in various degree is all had to asparatate, threonine, serine, glutamic acid, glycine, valine, methionine, isoleucine, leucine, lysine, histidine, proline, cystine and the tryptophan in Radix Codonopsis root 18 seed amino acid, wherein comparatively large to the raising degree of methionine, tryptophan, threonine and proline, improve 53.6%, 31.4%, 21.5% and 19.0% respectively.
As can be seen from Figure 2, comparatively DH5 α, LB and moisture you can well imagine high Radix Codonopsis root total amino acid 10.6%, 6.2% and 5.6% to bacillus subtilis GB03.As can be seen from Figure 3, bacillus subtilis GB03 is DH5 α, LB and water comparatively, improves Radix Codonopsis root 7 kinds of essential amino acids (threonine, valine, phenylalanine, methionine, isoleucine, leucine and lysine) total content 11.8%, 6.7% and 7.7% respectively.
Table 1GB03 is on the impact of Radix Codonopsis root 18 seed amino acid content
Table 2 different disposal is on the impact of Radix Codonopsis overground part 18 seed amino acid
As can be seen from Table 2, GB03 all has raising in various degree compared with DH5 α, LB and water to threonine, serine, glycine, alanine, valine, isoleucine, leucine, phenylalanine, lysine and the tryptophan in Radix Codonopsis overground part 18 seed amino acid, wherein the raising effect of tryptophan, lysine, leucine and glycine is comparatively obvious, improves 30.6%, 17.3%, 16.3% and 16.1% respectively.
2.3GB03 is on the impact of ammonia alkynes glycosides content in Radix Codonopsis root
Measure collection of illustrative plates from Fig. 4 lobetyolin, the peak area of GB03, far away higher than contrast DH5 α, LB and water, reflects that from figure the alkynes glycosides content in the Radix Codonopsis seedling body of GB03 is high compared with other process intuitively.Through calculating the content (Fig. 5) of alkynes glycosides in Radix Codonopsis root, bacillus subtilis GB03 comparatively DH5 α, LB and water significantly improves the alkynes glycosides content of Radix Codonopsis (P<0.05), and increase rate is respectively 75.2%, 71.5% and 51.9%.
3 discuss
The growth indexes of this research to growth 1 year Radix Codonopsis measures, find GB03 comparatively DH5 α, LB and water, improve Radix Codonopsis plant height 18.1%, 30.1% and 26.4% respectively, increase main root diameter 45.9%, 42.7% and 36.6% respectively, improve root volume 6.5%, 37.3% and 25.2% respectively, significantly improve Radix Codonopsis root dry weight 49.8%, 55.4% and 37.7%.Radix Codonopsis is mainly used as medicine with root, and many functions and drug effect are also played by root, and therefore, the raising of Radix Codonopsis root volume and main root diameter, has great significance for Radix Codonopsis volume increase.In addition, this research also finds, GB03 process is DH5 α, LB and water comparatively, significantly (P<0.05) improves Radix Codonopsis overground part fresh weight 14.9%, 57.1% and 45.3% respectively, significantly (P<0.05) improves Radix Codonopsis root fresh weight 45.2%, 91.2% and 51.4%, and significantly (P<0.05) improves Radix Codonopsis overground part dry weight 36.5%, 47.2% and 47.6% respectively.
Amino acid is the material that human life activity is indispensable, and high molecular protein can not directly be absorbed by the body in food, but just can be utilized after being broken down into amino acid Small molecular.Research shows, the Jiu Zhaigou amino acid contained kind of each growth year Radix Codonopsis is more complete, containing 7 kinds of essential amino acid (threonines, valine, phenylalanine, methionine, isoleucine, leucine and lysine), total amino acid content is higher, and increase year by year with growth year, research shows, total amino acid contained by Radix Codonopsis and essential amino acids content are all higher than Vegetables and fruit (Zou Yuanfeng etc. 2010), as can be seen here, Radix Codonopsis has higher edibility, this measuring 18 seed amino acids of Radix Codonopsis overground part and root, the several amino acids of the Radix Codonopsis of GB03 process all has raising in various degree compared with LB, and the total amino and 7 kinds of necessary amino acid of human body of Radix Codonopsis root comparatively LB improves 10.6% respectively, 6.2%, 5.6% and 11.8%, 6.7% and 7.7%, the total amino acid of overground part and 7 kinds of essential amino acids improve 10.8% respectively, 0.5%, 4.3% and 13.8%, 2.5% and 5.0%, in addition, this research finds, the amino acid content of GB03 process all in various degree higher than contrast, but do not reach remarkable.This may be mainly because Radix Codonopsis is generally perennial medicinal plant, its root system is as the main composition that is used as medicine, generally plant in In Longxi Area of Gansu and just gather in the crops for 2 years, and our experiment is by the restriction of various condition, the Radix Codonopsis cultivated is 1 year, incubation time is shorter, and the multiple metabolin of synthesis is not also transported to root system, and experimental result also can prove this point.No matter we is 18 seed amino acids if finding, or total amino acid, 7 kinds of needed by human amino acid, be the 2-3 of root system doubly at Radix Codonopsis overground part.
Research finds, bacillus subtilis Tu-100 has obvious chemotaxis (Hu little Jia etc. 2010) to the amino acid contained by rape root secretion.Separately there are some researches show, bacillus subtilis GB03 can produce abundant volatile chemical to promote that pale reddish brown Herba Lysimachiae foenumgraeci grows, and these volatility signals can induce the accumulation of essential oil in the pale reddish brown Herba Lysimachiae foenumgraeci body of different growing stage and release: GB03 to improve terpinol and Eugenol content about 2 times and 10 times (Banchio et a12009) respectively.In addition, GB03 bacterial strain can significantly improve the content (Zhang et al2010c) of choline in pattern plant Arabidopsis thaliana body under osmotic stress.The experimental result of Song Dan etc. (2008) shows, lobetyolin has anticancer, antibacterial, anti-inflammatory, calmness, step-down and regulates the effects such as prostaglandin metabolism, and in addition, it can also resist gastric acid secretion significantly, thus protection stomach lining.This experiment finds, the accumulation of GB03 to Radix Codonopsis metabolite has great facilitation, significantly improves the content of root alkynes glycosides, and comparatively DH5 α, LB and water significantly improve respectively and is respectively 75.2%, 71.5% and 51.9%.This shows, GB03 bacterial strain can improve the accumulation of plant interior metabolism product to a great extent.Studies have found that, find in the cauline leaf of Radix Codonopsis to enrich volatile oil, trace element, alkaloid and starch (Wang Kangcai etc. 2002), therefore, study useful soil bacteria and Radix Codonopsis is done mutually, will play an important role for the accumulation improving Radix Codonopsis interior metabolism product.
4 brief summaries
(1) comparatively DH5 α, LB and water remarkable (P<0.05) improve the biomass of Radix Codonopsis to bacillus subtilis GB03, significantly (P<0.05) improves root fresh weight 45.2%, 91.2% and 51.4% respectively, improves Radix Codonopsis root dry weight 49.8%, 55.4% and 37.7%.
(2) bacillus subtilis GB03 all has raising in various degree compared with DH5 α, LB and water to asparatate, threonine, serine, glutamic acid, glycine, valine, methionine, isoleucine, leucine, lysine, histidine, proline, cystine and the tryptophan in amino acid in 18 of Radix Codonopsis root, wherein comparatively large to the raising degree of methionine, tryptophan, threonine and proline, improve 53.6%, 31.4%, 21.5% and 19.0% respectively.Comparatively DH5 α, LB and water, improves Radix Codonopsis root total amino acid 10.6%, 6.2% and 5.6% respectively, improves root 7 kinds of essential amino acids 11.8%, 6.7% and 7.7%.Raising in various degree is all had to threonine, serine, glycine, alanine, valine, isoleucine, leucine, phenylalanine, lysine and the tryptophan in overground part 18 seed amino acid, wherein the raising effect of tryptophan, lysine, leucine and glycine is comparatively obvious, improve 30.6%, 17.3%, 16.3% and 16.1% respectively, comparatively DH5 α, LB and water, improve Radix Codonopsis overground part total amino acid 10.8%, 0.5% and 4.3%, improve overground part 7 kinds of essential amino acids 13.8%, 2.5% and 5.0%.
(3) bacillus subtilis GB03 comparatively DH5 α, LB and water, significantly improve alkynes glycosides content in Radix Codonopsis root, increase rate is respectively 75.2%, 71.5% and 51.9% (P<0.05).
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (1)
1. a method for beneficial rhizosphere bacterial regulatory Radix Codonopsis output and Metabolite Accumulation, is characterized in that including following steps:
(1) seedling culture: by Radix Codonopsis seed, germinate, after germination, transplants in the cave dish that vermiculite is housed, waters 1/2 Hoagland nutrient solution, cultivate in greenhouse; The diurnal temperature in greenhouse is 26-30 ° of C, illumination 12-14h/d, and luminous intensity is 800-1000 μm of ol/m2s, relative humidity 60-80 %;
(2) seedling replanting: treat growth of seedling 28-32 days, transplanting to the diameter 18-22cm degree of depth is in the plastic flowerpot of 28-30cm, the matrix of flowerpot is that turfy soil and farmers' soil 1:1 mix, dry 3 d at 158-162 degree high temperature oven, fill basin afterwards, weigh, control that irrigation amount is maxmun field capacity 60%;
(3) bacterium solution preparation: preparation LB fluid nutrient medium: according to the proportions of sodium chloride 5 g/L, tryptone 10 g/L and dusty yeast 5 g/L, adjust pH to 6.8-7.2, temperature be 121-126 DEG C of sterilizing after cooling, the LB fluid nutrient medium of every 100 mL adds the GB03 bacterium liquid of 50-100 μ L, under rotating speed is 180 r/min, temperature be 25-28 ° of C constant-temperature shaking incubator on after overnight incubation, use detection of nucleic acids instrument to measure bacterial concentration for subsequent use for taking out during OD600=0.9;
(4) seedling process: add bacterium liquid after transplanting two weeks, adds bacterium once later every other month, draws the bacillus subtilis GB03 of 0.5-1 mL, be applied to uniformly on the soil around every strain seedling basal part of stem with liquid-transfering gun.
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