CN104031863B - One strain is for the bacillus subtilis of antagonism Radix Pseudostellariae specialized form Fusarium oxysporum - Google Patents
One strain is for the bacillus subtilis of antagonism Radix Pseudostellariae specialized form Fusarium oxysporum Download PDFInfo
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Abstract
The present invention relates to the strain bacillus subtilis for antagonism Radix Pseudostellariae specialized form Fusarium oxysporum.This bacterial strain be bacillus subtilis (<i>bacillus subtilis</i>) BS-C, register preservation on May 12nd, 2014 at China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCCNo.9145.The separation of this Antagonistic Fungi can provide new biocontrol agent for the Biological control of Radix Pseudostellariae root rot.The bacillus subtilis of the present invention (<i>bacillus subtilis</i>) BS-C, can efficiently suppress the specialized form Fusarium oxysporum of Radix Pseudostellariae rhizosphere soil, having a extensive future in this field.
Description
Technical field
The present invention relates to the strain bacillus subtilis for antagonism Radix Pseudostellariae specialized form Fusarium oxysporum.
Background technology
Radix Pseudostellariae (Pseudstellariaeheterophylla) is the Chinese crude drug that a kind of comparison is famous and precious, containing aminoacid, saccharide, glycoside, phospholipid, cyclic peptide and trace element etc..Have invigorating qi and benefiting blood, the effect of the tonifying the spleen and stomach that promotes the production of body fluid, be suitable to children's's summer heat for a long time and do not move back, cough, diet is depressed, the disease of the weaknesses such as cardiopalmus and children's weak unable, night sweat after being ill, mouth equality disease.Day by day deep along with the research of Radix Pseudostellariae pharmacological effect, Radix Pseudostellariae, as tonification medicine, is increasingly becoming one of conventional bulk medicinal materials of China.
Radix Pseudostellariae is of many uses, and the market demand is very big.But, its continuous cropping obstacle is extremely serious, and when namely planting Radix Pseudostellariae continuously in same piece of land, even if when enhancing field management, its upgrowth situation is deteriorated, and even has no harvest, and its quality deteriorates, and polysaccharide and total content of ginsenoside are remarkably decreased.Applicant confirms through research: after Radix Pseudostellariae continuous cropping, in rhizosphere soil, Radix Pseudostellariae specialized form Fusarium oxysporum (Fusariumoxysporumf.sp.heterophylla) quantity pole dramatically increases, and continuous cropping Radix Pseudostellariae specialized form Fusarium oxysporum (Fusariumoxysporumf.sp.heterophylla) quantity is nearly 5 times of comparison；Being inoculated in by the Radix Pseudostellariae specialized form Fusarium oxysporum being separated in the healthy soil of plantation Radix Pseudostellariae, Radix Pseudostellariae specialized form Fusarium oxysporum breeds in a large number in healthy soil, and infects Radix Pseudostellariae root, causes Radix Pseudostellariae disease.
In recent years, along with the research of particular mechanism of action of green agriculture, organic agriculture and microbial control is just flourish, obtains efficient Antagonistic Fungi and make it act on to perform to maximum, be the basis of Biological control.Biological control research in other crops (such as Fructus Musae, Citrullus vulgaris, Fructus Cucumidis sativi, Fructus Fragariae Ananssae etc.) of Fusarium oxysporum is relatively more, and the Antagonistic Fungi kind separating report is also more comprehensive.Such as Trichoderma spp., AMF (AM), Nonpathogenic Fusarium oxysporum, Paecilomyces lilacinus, false single-cell bacteria, bacillus cereus, actinomycetes etc., Bacillus subtilis BRF-2 was once used for Fructus Cucumidis sativi and tomato wilt by the beautiful grade of such as old snow, but the bacterial strain that this research separates compares source difference with them, for object also different, owing to Fusarium oxysporum also exists the reason of genetic polymorphism.As a kind of universal soil-borne disease fungal pathogens, the pathogenic strain in Fusarium oxysporum kind has the host speciality of height, Radix Pseudostellariae specialized form Fusarium spp. might not be had good effect from the Bacillus subtilis BRF-2 separated elsewhere.At present the separated Antagonistic Fungi kind for Radix Pseudostellariae specialized form Fusarium oxysporum only has " Paenibacillus polymyxa for antagonism Radix Pseudostellariae rhizosphere soil Fusarium oxysporum " (patent No.: the CN102676435B) of this seminar, yet there are no the relevant report of bacillus amyloliquefaciens for Radix Pseudostellariae specialized form Fusarium oxysporum.This seminar is in early-stage Study, the bacillus subtilis (Bacillussubtilis) being separated to a strain capable of high-efficiency suppression Radix Pseudostellariae specialized form Fusarium oxysporum continuous cropping Radix Pseudostellariae Rhizosphere sampling by diluting painting flat band method and flat board dual test to gather from experimental plot, Radix Pseudostellariae GAP base, Xi Yuan village, Ningde City Zherong County, Fujian Province, called after BS-C.The separation of this Antagonistic Fungi can provide new biocontrol agent for the Biological control of Radix Pseudostellariae root rot.
Summary of the invention
For this, the present invention screens the strain bacillus subtilis for antagonism Radix Pseudostellariae specialized form Fusarium oxysporum from Radix Pseudostellariae continuous cropping rhizosphere soil.
A strain provided by the invention is for the bacillus subtilis of antagonism Radix Pseudostellariae rhizosphere soil Radix Pseudostellariae specialized form Fusarium spp., for bacillus subtilis (Bacillussubtilis) BS-C, registering preservation on May 12nd, 2014 at China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCCNo.9145.
This bacterium can in bacillus cereus collective media cultivation and fermentation.Medium component: tryptone (Tryptone) 10g/L, yeast extract (Yeastextract) 5g/L, sodium chloride (NaCl) 5g/L, pH7.0.
Observing this bacterium Gram’s staining result under (eyepiece 10X, object lens 100X) under oil mirror, this bacterium is bacillus, and Gram’s staining is negative.This bacterial strain is through morphology and 16sRNA molecular biology identification, it is determined that for bacillus subtilis (Bacillussubtilis).
This bacterial strain wide can utilize carbon source, including: L-rhamnose, sucrose, D-Glucose and PEARLITOL 25C, but part carbon source is not utilized, including: D-Fructose, D-xylose, L-arabinose, L-inositol, maltose and cellobiose, also wide can utilize nitrogenous source.
Bacillus subtilis (Bacillussubtilis) BS-C of the present invention, can efficiently suppress the specialized form Fusarium oxysporum of Radix Pseudostellariae rhizosphere soil, having a extensive future in this field.This bacterial strain is finish China Committee for Culture Collection of Microorganisms's common micro-organisms center registration guarantor on May 12nd, 2014, and it is called for short CGMCC, and address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and deposit number is CGMCCN0.9145.
Accompanying drawing explanation
Fig. 1 is that bacillus subtilis (Bacillussubtilis) BS-C is at NA culture medium growth figure.
Fig. 2 is bacillus subtilis (Bacillussubtilis) BS-C fungistatic effect to Radix Pseudostellariae specialized form Fusarium oxysporum.Left figure is the comparison of Radix Pseudostellariae specialized form Fusarium oxysporum；Right figure is the fungistatic effect of bacillus subtilis BS-C antagonism Radix Pseudostellariae specialized form Fusarium oxysporum.
Fig. 3 is that bacillus subtilis (Bacillussubtilis) BS-C is to Radix Pseudostellariae toxicity test.In picture, first row is the comparison Radix Pseudostellariae Potted orchard not adding bacillus subtilis BS-C, and second row is the Radix Pseudostellariae Potted orchard after the process of addition bacillus subtilis BS-C.
Fig. 4 is that bacillus subtilis (Bacillussubtilis) BS-C, Radix Pseudostellariae specialized form Fusarium oxysporum Radix Pseudostellariae tissue cultured seedling train test altogether.
Detailed description of the invention
1, the isolation and purification of bacillus subtilis BS-C
The collection of soil sample 2012 gathers continuous cropping Radix Pseudostellariae Rhizosphere sampling from experimental plot, Radix Pseudostellariae GAP base, Xi Yuan village, Ningde City Zherong County, Fujian Province.Adopting 5 point sampling methods, mix homogeneously is as sample.
Adopting isolation by dilution method to soil sample microorganism separation purification, weigh in the triangular flask that 10g soil is equipped with 90mL sterilized water, 120r/min, vibrate 30min, and gained soil supension is for the separation about microorganism.The soil supension shaken is made 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7Etc. the different dilution soil liquid.The suspension of above-mentioned dilution is applied in equipped with LB culture medium (formula is as follows: tryptone (Tryptone) 10g/L, yeast extract (Yeastextract) 5g/L, sodium chloride (NaCl) 10g/L, the pH of this culture medium is regulated with NaOH, to 7.0 ~ 7.4, every liter adds 10g ~ 15g agar powder) flat band method on, each concentration repeats 3 times, it is placed in overnight incubation in the incubator of 30 DEG C, choose the flat board that clump count is suitable, picking single bacterium colony therein, adopt the method for parallel scribing to be purified, continuous 3 switchings, confirm as single antibacterial.The soil microorganism taken after separating purification is inoculated in NA culture medium respectively, 30 DEG C activate 2 days, then receive in PDA culture medium, beat with card punch and take Radix Pseudostellariae Fusarium oxysporum bacterium cake and receive PDA (Rhizoma Solani tuber osi 200g/L, glucose 20g/L, every liter of agar adds 15 ~ 20 grams, natural PH) flat board carries out flat board face-off experiment, choose after the bacterial strain of obvious antagonism is purified preserve, standby.
2, the screening of bacillus subtilis BS-C and fungistatic effect measure
Opposite culture method is adopted to carry out screening further with Radix Pseudostellariae Fusarium oxysporum each bacterial strain having certain antagonism of above-mentioned purification: the bacterium solution 100 μ L taking out above-mentioned purification pours on LB plating medium, it is coated with rod with the glass of sterilizing to be coated with uniformly by diluent, cultivates 7 days for 30 DEG C.On good PDA plate with diameter 5mm card punch at the plating medium that diameter is 9cm while punching, access Antagonistic Fungi truffle, and by an equal amount of Fusarium oxysporum truffle access the other end, carry out flat board opposite culture.It is identified by the bacterial strain that screening antagonistic effect is good, through morphology and 16srRNA molecular biology identification, it is determined that for bacillus subtilis (Bacillussubtilis), called after BS-C.
Bacillus subtilis BS-C bacterial strain ferments through seed tank, and after amplifying fermentation further to pilot scale fermentation tank, after Radix Pseudostellariae is planted, spraying immediately is applied to soil, or after bacterium solution fermentation, mixes base manure and be applied to the soil being about to plantation Radix Pseudostellariae.Bacterium solution spraying is used or is mixed base manure and uses, and its application concentration is 106-108CFUs/mL.Radix Pseudostellariae yield and quality will be significantly improved after using.
3, the fungistatic effect of bacillus subtilis BS-C measures
In the lab, with card punch, Fusarium oxysporum and bacillus subtilis BS-C respectively broken into small bacteria block that diameter is 5mm and move it into the two ends of PDA plate culture medium respectively.The truffle of the PDA plate culture medium center access formed objects not inoculating this bacterium shines for sky.28 DEG C of constant temperature culture 5 days, carry out colony diameter size measurement and suppression ratio calculates, it is determined that the antagonism performance of bacterial strain.
Bacteriostasis rate computing formula: Fusarium oxysporum colony diameter × 100% of bacteriostasis rate (%)=(the Fusarium oxysporum colony diameter of this bacterium of the Fusarium oxysporum colony diameter of comparison-inoculate)/comparison.
As a result, 5 days bacteriostasis rates of Fusarium oxysporum are reached 92.1% by this bacterial strain, as shown in Figure 2.
4, bacillus subtilis BS-C bacterial strain toxicity test
This bacillus subtilis is accessed in 100mLLB culture medium, each 3 bottles, after cultivating 3 days, take this bacterium bacterium solution 100mL sterilized water of cultivation dilute apply after 5 times Radix Pseudostellariae potted plant in, observe the growing state of Radix Pseudostellariae every day, observe this bacillus subtilis to the whether toxic effect of Radix Pseudostellariae.As it is shown on figure 3, compared with the control, Radix Pseudostellariae group Potted orchard well-grown, it was shown that Radix Pseudostellariae growth itself is not poisoned by this bacillus subtilis BS-C, will not make Radix Pseudostellariae cause a disease.
5, bacillus subtilis BS-C, Radix Pseudostellariae Fusarium oxysporum and Radix Pseudostellariae tissue cultured seedling train test altogether.
This Antagonistic Fungi bacterium solution of 200 μ L is joined around Radix Pseudostellariae tissue cultured seedling rhizosphere, in tissue culture bottle edge access Fusarium spp. after cultivating 2 days, observes and make effect mutually between pathogen, Antagonistic Fungi, Radix Pseudostellariae tissue cultured seedling.
As shown in Figure 4, Radix Pseudostellariae tissue cultured seedling well-grown, only access the Radix Pseudostellariae tissue cultured seedling of Radix Pseudostellariae Fusarium oxysporum and then entered Radix Pseudostellariae Fusarium oxysporum and infect and withered gradually and finally dead.And in the Radix Pseudostellariae tissue culture bottle being connected to Radix Pseudostellariae Fusarium oxysporum and bacillus subtilis BS-C, bacillus subtilis BS-C can suppress Radix Pseudostellariae Fusarium oxysporum grow to Radix Pseudostellariae tissue cultured seedling and form inhibition zone, cause Radix Pseudostellariae Fusarium oxysporum cannot cross inhibition zone and Radix Pseudostellariae is caused a disease, and crown prince's seedling upgrowth situation is the same with blank grows fine.
6, the application prospect of bacillus subtilis BS-C
Plant disease 80% is above by fungus-caused, controls fungal disease of crucial importance for improving crop yield.Fusarium oxysporum is the soil-borne disease fungus of a quasi-representative, in soil and organic matter, quantity is many and active, it lives away from home scope widely, the 100 various plants generation droops such as melon, Solanaceae, Fructus Musae, cotton, pulse family and flowers can be caused, agricultural production causes serious crop failure, reduces quality, impact supply and export trade, even cause person poultry poisoning, cause serious economic loss.Applicant confirms through research: after plantation Radix Pseudostellariae, in rhizosphere soil, Radix Pseudostellariae specialized form Fusarium oxysporum quantity pole dramatically increases；Being inoculated in by the Fusarium oxysporum being separated in the healthy soil of plantation Radix Pseudostellariae, Fusarium oxysporum breeds in a large number in healthy soil, and infects Radix Pseudostellariae root, causes Radix Pseudostellariae disease.The annual total cultivated area of Radix Pseudostellariae about more than 50,000 mu in the whole nation, by existing market price budget, the whole nation output value about more than 5,000,000,000 yuan, but in Radix Pseudostellariae rhizosphere soil, the pathogenic bacterium such as specialized form Fusarium oxysporum and pathogen are increased after continuous cropping, yield is made sharply to drop to less than the 1/3 of the first planting (comparison), produce serious economic loss, be a major issue being badly in need of solving.Bacillus subtilis BS-C can efficiently suppress Radix Pseudostellariae specialized form Fusarium oxysporum, having a extensive future in this field.
Claims (1)
1. a strain is for bacillus subtilis (Bacillussubtilis) BS-C of antagonism Radix Pseudostellariae specialized form Fusarium oxysporum, registering preservation on May 12nd, 2014 at China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCCNo.9145.
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| CN102676435B (en) * | 2012-05-24 | 2013-06-12 | 福建师范大学福清分校 | Paenibacillus polymyxa for antagonizing Fusarium oxysporum in rhizosphere soil of Radix Pseudostellariae |
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