CN106676033B - One Bacillus species and its application - Google Patents
One Bacillus species and its application Download PDFInfo
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- CN106676033B CN106676033B CN201611027470.2A CN201611027470A CN106676033B CN 106676033 B CN106676033 B CN 106676033B CN 201611027470 A CN201611027470 A CN 201611027470A CN 106676033 B CN106676033 B CN 106676033B
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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- A—HUMAN NECESSITIES
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- A01G9/00—Cultivation in receptacles, forcing-frames or greenhouses; Edging for beds, lawn or the like
- A01G9/02—Receptacles, e.g. flower-pots or boxes; Glasses for cultivating flowers
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Abstract
The invention discloses a Bacillus species, its classification naming is series bacillus (Paenibacillus sp.), China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on July 06th, 2016, deposit number is CGMCC No.12738.Series bacillus of the invention can significantly improve the growth conditions of Populus and Salix xylophyta, such as increase root long, radical, height of seedling and biomass, survival rate be improved, during allowing to apply the growth and breeding in Populus and Salix xylophyta.
Description
Technical field
The present invention relates to microorganisms technical fields, particularly relate to a Bacillus species and its application.
Background technique
Populus and Salix are woody due to tree-like beautiful, adaptable, easy the advantages that surviving in Salicaceae, become common and prevent
It protects a forest and green tree species, can also make commerical tree species.With the continuous mature and widespread adoption of tissue rapid propagation technology, these tree species
The forming process that can accelerate sapling by group culturation rapid propagating technology improves the efficiency of afforestation.But it is woody in Populus and Salix
In plant fast propagation and growth course, due to the influence of external environment, it be easy to cause the phenomenon that Reducing sugar is poor, such as root long
It is short, radical is few, height of seedling is low, survival rate is low, biomass is low etc., so limit Populus and Salix woody plant tissure be fast
Numerous efficiency.
Endophytic bacterium (endophytic bacteria) is different from the bacterium for being grown in plant surface, is distributed in strong
In health plant, have population diversity abundant, host plant will not be made to show apparent infection symptoms.As the micro- life of plant
The component part of state system, they may promote the adaptability of host plant.As people realize conservation of natural environment
Gradually increase, their research and application are for reducing pest and disease damage or adverse circumstances to the harm of plant and promoting plant
Growth and nitrogen fixation etc. are of great significance and application value, and new think of is provided using endophytic bacterium for people
Direction is examined, vast potential for future development is presented.
Summary of the invention
The purpose of the present invention is to provide a Bacillus species, and the series bacillus is enabled to significantly improve Populus
With the growth conditions of Salix xylophyta, such as increase root long, radical, height of seedling and biomass, improves survival rate, allow to answer
During the growth and breeding of Populus and Salix xylophyta.
In order to solve the above technical problems, the present invention adopts the following technical scheme:
One Bacillus species, classification naming is series bacillus (Paenibacillus sp.), in July, 2016
It is preserved within 06th China Committee for Culture Collection of Microorganisms's common micro-organisms center, depositary institution address is Beijing's southern exposure
The institute 3 of area North Star West Road 1, deposit number are CGMCC No.12738.
Wherein series bacillus (Paenibacillus sp.) Gram-negative has gemma, and bacterium colony is in circle
Shape, surface is smooth mellow and full, and neat in edge is creamy white.
Series bacillus of the present invention is isolated from the poplar rhizosphere soil of Hebei province Baoding, is named as P26.Through being sequenced
The 16S rDNA nucleotide sequence size of bacterial strain P26 is about 1501bp.Through compared with the similar sequences in Genebank, bacterial strain
(registration number is by the nucleotide sequence of P26 and Paenibacillus La1 (registration number AM906090), P.sp.Ma-2
) and the 16S rDNA nucleotide sequence homology of P.humicus (registration number is respectively AM411529, EU867348) AM906089
Highest similitude is up to 99%；(registration number is with P.sepulcri (registration number DQ291142), P.castaneae
EU099594), P.mendelii (registration number AF537343), P.favisporus (registration number NR_029071),
The 16SrDNA nucleotide sequence homology similitude of P.borealis (registration number AJ011327) reaches 96%.To bacterial strain P26
Further form and Physiology and biochemistry identification, the results show that P26 Gram-negative, there is gemma, bacterium colony is rounded, surface light
Cunning is mellow and full, and neat in edge is creamy white.Physio-biochemical characteristics are shown in Table 1:
Table 1
According to the morphological feature of bacterial strain P26 and 16S rDNA nucleotide alignments as a result, choosing bacillus genus
The 16S rDNA nucleotide sequence phylogenetic tree construction of several bacterial strains (see Fig. 1), the results showed that, bacterial strain P26 and bacterial strain
P.sp.La1 (AM906090) and P.sp.Ma-2 (AM906089) is located at same group, and affiliation is nearest.
According to the morphological feature of bacterial strain P26, physio-biochemical characteristics and 16SrDNA nucleotide sequence analysis and systematic growth
Analysis, referring to " Bergey's Manual of Systematic Bacteriology " and " common bacteria system identification handbook "
Bacterial strain P26 is accredited as series bacillus Paenibacillus..
The present invention connects bacterium on low nitrogen culture medium to Triploid of Populus Tomentosa tissue-cultured seedling using bacterial strain P26 and cultivates, and to willow
Bacterium processing is connect with the outdoor cuttage cuttings of European-American Poplar 107, detection bacterial strain P26 is to the energy for promoting sibling species plant establishment and growth
Power.Test result shows: bacterial strain P26 is significant to Triploid of Populus Tomentosa tissue culture seedling rooting and growth-promoting effect；To European-American Poplar 107
It takes root and grows also and have significant facilitation with dry land willow hardwood cuttings.
Based on above-mentioned technical effect, the invention proposes the series bacillus to promote Populus and Salix xylophyta raw
In length, Populus and Salix woody plant tissure it is fast it is numerous in, and in preparation Populus and Salix xylophyta bio-stimulant
In application.Wherein, the Populus xylophyta is preferably white Mao Yang or European-American Poplar；The Salix xylophyta is preferably drought
Willow.
Meanwhile the present invention also provides a kind of tissue rapid propagation methods of Populus and Salix xylophyta, comprising:
A, the series bacillus that deposit number is CGMCC No.12738 is incubated overnight work in TSA broth bouillon
Change；
B, by dipping 10s-10min in the culture in step of the cutting of Populus or Salix xylophyta or tissue-cultured seedling,
Then it is bred in culture substrate.
It is common to be also possible to the tissue rapid propagations such as vermiculite, perlite, river sand either field soil for the culture substrate
Matrix.
Preferably, the culture substrate includes the low nitrogen BEMB200 culture medium containing 2.5mM ammonium nitrate, further
The culture medium includes IBA, and IBA concentration can be 0.15-0.3mg/L.
Preferably, the culture substrate includes IBA, it is further preferable that the IBA concentration is 0.15-0.3mg/L.
Biomaterial preservation information explanation:
P26, classification naming: series bacillus (Paenibacillus sp.) is preserved in China on July 06th, 2016
Microbiological Culture Collection administration committee common micro-organisms center, address are city, BeiJing, China, North Star West Road 1, Chaoyang District institute 3
Number, Institute of Microorganism, Academia Sinica, deposit number is CGMCC No.12738.
The utility model has the advantages that series bacillus of the invention, can increase Populus and Salix xylophyta root long, radical, seedling
High and biomass improves survival rate, has significant growth-promoting functions, can be applicable to and promotes Populus and the growth of Salix xylophyta
In, Populus and Salix woody plant tissure it is fast it is numerous in, and in preparation Populus and Salix xylophyta bio-stimulant.
Detailed description of the invention
Fig. 1 show the 16S rDNA sequential system development tree of bacterial strain P26.
Specific embodiment
The embodiment of the invention discloses a Bacillus species and its application, those skilled in the art can be used for reference in this paper
Hold, is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are to those skilled in the art
For be it will be apparent that they are considered as being included in the present invention.Bacterial strain of the present invention and application have passed through preferably real
Example is applied to be described, related personnel obviously can not depart from the content of present invention, in spirit and scope to product as described herein,
Methods and applications are modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
Just a Bacillus species provided by the present invention are described further below.
One, the 16S rDNA sequencing and Phylogenetic analysis of bacterial strain P26
The 16S rDNA of PCR amplification bacterial strain P26, is then surveyed by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd
Sequence identification.The sequence of obtained strains is submitted in Genbank and carries out similar sequences comparison using Blast program, is therefrom selected
The higher sequence of similitude carries out sequence comparative analysis using Claustal X, then uses MEGA software component systematic evolution tree,
And Bootstrap analysis is carried out, number of repetition is 1000 times.
16S rDNA nucleotide sequence size through bacterial strain P26 is sequenced is about 1501bp.Through to the similar sequence in Genbank
Column compare, the nucleotide sequence and Paenibacillus La1 (registration number AM906090) and P.sp.Ma-2 of bacterial strain P26
The 16S rDNA nucleotide sequence homology highest similitude of (registration number AM906089) is up to 99%.
According to the morphological feature of bacterial strain P26 and 16S rDNA nucleotide alignments as a result, choosing bacillus genus
The 16S rDNA nucleotide sequence phylogenetic tree construction of several bacterial strains (see Fig. 1), the results showed that, bacterial strain P26 and bacterial strain
P.sp.La1 (AM906090) and P.sp.Ma-2 (AM906089) is located at same group, and affiliation is nearest.
According to the morphological feature of bacterial strain P26, physio-biochemical characteristics and 16SrDNA nucleotide sequence analysis and systematic growth
Analysis, referring to " Bergey's Manual of Systematic Bacteriology " and " common bacteria system identification handbook "
Bacterial strain P26 is accredited as series bacillus Paenibacillus sp..
Two, triploid white poplar connects bacterium culture experiment
1, test strain
Series bacillus P26 of the present invention；It is isolated through reflecting in same time, environment and soil with P26
Fixed is equally the P27 (such as P.humicus) of series bacillus；
2, culture medium
Used medium is low nitrogen BEMB200 culture medium (containing 2.5mM ammonium nitrate) in test.By configured culture medium
(culture solution is to be impregnated with perlite to be advisable) is distributed into the triangular flask for filling the 100ml of 40ml perlite, and packing is completed to be placed on
Sterilize 20min in 120 DEG C of high-pressure sterilizing pots.
3, bacterium test method and result are connect
Cultured Triploid of Populus Tomentosa tissue-cultured seedling is placed on superclean bench, by single stem cutting, every section is with 2
The stem section of the 1.5cm long of leaflet.3 processing of test setting, CK (not connecing bacterium), P26 (meeting bacterium P26), P27 (meeting bacterium P27).It will cut
Good stem section impregnates 10s in the TSA broth bouillon respectively containing P26 and P27 overnight incubation, and control (CK) does not connect bacterium then.
The stem section handled well is respectively connected to cultivate in the triangular flask for filling low nitrogen perlite culture medium.Each 24 bottles of processing, every bottle connects 2
A stem section.Culture starts rear every 10d and randomly selects 8 tissue-cultured seedling from each processing to be investigated, the master of measurement record tissue-cultured seedling
Radical amount, root long and height of seedling, and observe its upgrowth situation.When cultivating 30d, tissue-cultured seedling is taken out, in 35 DEG C of baking ovens after constant,
Measure the dry mass of seedling.It the results are shown in Table 2.
Influence of 2 bacterial strain of table to Triploid of Populus Tomentosa tissue culture seedling rooting and growth
Note: the same incubation time same letter of same row indicates no significant difference (p < 0.05)
As can be seen from Table 2, on the low nitrogen perlite culture medium of inoculation P26 bacterial strain, Triploid of Populus Tomentosa stem section root growth,
Seedling height growth and biomass are significantly improved trend, the indices of each incubation time bacterial strain processing with control strain P27
Reach significant difference with CK, and there were significant differences for P27 group and CK group.
When cultivating 10d, the mean elements 3.24 of P26 processing group, average root long 1.96cm, average height of seedling 2.31cm are right
According to group mean elements 1.21, average root long 0.93cm, average height of seedling 1.92cm；The mean elements of P26 processing group, average root long
With average height of seedling respectively than not meeting the CK high 167.77%, 110.75%, 20.31% of bacterium, than P27 high 126.57%, 56.8%,
5.96%.With the extension of incubation time, the effect of P26 bacterial strain hestening rooting and growth is still obvious: when arriving 30d, P26 processing
The mean elements 5.71 of group, average root long 4.14cm, average height of seedling 5.26cm；The average root long of P26 processing group, mean elements and
Average height of seedling improves 191.32%, 80.0% and 20.92% than not meeting the CK of bacterium respectively, than P27 high 65.02%, 34.41% and
29.56%.In test, it is also observed that when being inoculated with 10d, the CK group part tissue-cultured seedling for not connecing bacterium processing is not taken root, and in life
With slight vitrification phenomenon in growth process, and the tissue-cultured seedling of P26 group then grows vigorous, and nursery stock is more healthy and stronger.
4, bacterial strain and IBA Combined Treatment are tested
After cultured Triploid of Populus Tomentosa group training material is dipped P26 bacterium solution by method in 3, be respectively connected to containing
0.15mg/L, 0.30mg/L IBA concentration BEMB200 perlite culture medium triangular flask in.Each 16 bottles of processing, every bottle connects 2
A stem section.Culture starts to carry out survey when rear 30d, other investigation methods are the same as 3.It the results are shown in Table 3.
3 difference IBA concentration of table and different strains handle the influence grown to Triploid of Populus Tomentosa tissue culture seedling rooting
| Processing | Mean elements | Average root long (cm) | Average height of seedling (cm) |
| P26 | 5.65±2.68ab | 4.09±2.65bc | 5.20±1.37ab |
| P26+0.15mg/L IBA | 4.74±2.42b | 4.91±2.59ab | 4.87±1.50b |
| P26+0.30mg/L IBA | 6.35±3.05a | 5.43±3.02a | 5.81±2.47a |
| CK | 2.11±1.72c | 2.43±0.49d | 4.55±1.35c |
Note: same row same letter indicates no significant difference (p < 0.05)
Table 3 the result shows that, the processing of P26 bacterial strain and bacterial strain and IBA Combined Treatment are taken root to Triploid of Populus Tomentosa and grows and is equal
There is apparent facilitation, is significantly better than control treatment.Bacterial strain has compared with only being handled with bacterial strain with IBA Combined Treatment effect
Certain improvement effect is especially combined with the IBA under 0.3mg/L concentration.
Embodiment described above is only that preferred embodiment of the invention is described, and is not carried out to the scope of the present invention
It limits, without departing from the spirit of the design of the present invention, those of ordinary skill in the art make technical solution of the present invention
Various changes and improvements, should all fall into claims of the present invention determine protection scope in.
SEQUENCE LISTING
<110>Agricultural University Of Hebei
<120>one Bacillus species and its application
<130>2016
<160>1
<170>PatentIn version 3.3
<210>1
<211>1258
<212>DNA
<213>Paenibacillus
<400>1
gacgaacgct ggcgtcgtgc ctaatacatg caagtcgagc ggatcttgtc cttcgggaca 60
aggttagcgg cggacgggtg actaacacgt aggcaagctg ccctcacgac tgggataacc 120
tccgtaaacg gatgctaata ccggatatgc ggtctctcct cctggaggga tcgggaaaga 180
cggagcaatc tgtcgcttgg ggatgggcct gcggcgcatt agctagttgg tgrggtaacg 240
gctcaccaag gcgacgatgc gtagccgacc tgagagggtg atcggccaca ctgggactga 300
gagacggccc agactcctac tggaggcagc agtagggcat cttccgcaat ggacgcgagt 360
ctgactgagc aacgccgcgt gagtgaggaa ggccttctgg tcgtaaagct ctgttgccag 420
ggaagaacgg gtggaagagt aactgcttcc gccatgacgg tacctgagaa gaaagccccg 480
gctaactacg tgccagcagc cgcggtaata cgtagggcgc aagcgttgtc cggaattatt 540
gggcgtacag cgcgcgcagg cggctttgta agtccggtgt ttaatcttgg ggctcaaccc 600
caagtcgcac gggaaactgc aaggcttgag gaagaacggg tgcaagagta actgcttccg 660
ccatgacggt acctgagaag aaagccccgg ctaactacgt gccagcagcc gcggtaatac 720
gtagggggca agcgttgtcc ggaattattg ggcgtaaagc gcgcgcagcg gctttgtaag 780
tccggtgttt aatcttgggg ctcaacccca agtcgcacgg gaaactgcaa ggcttgagtg 840
cagaagagga aagtggaatt ccacgtgtag cggtgaaatg cgtagagatg tggaggaaca 900
ccagtggcga aggcgacttt ctgggctgta actgacgctg aggcgcgaaa gcgtggggag 960
caaacaggat tagataccct ggtagtccac gccgtaaacg atgaatgcta ggtgttaggg 1020
gtttcgatac ccttggtgcc gaagttaaca caataagcat tccgcctggg gagtacgctc 1080
gcaagagtga aactcaaagg aattgacggg gacccgcaca agcagtggag tatgtggttt 1140
aattcgaagc aacgcgaaga accttaccag gtcttgacat ccccctgaat cacctagaga 1200
taggtgcggc cttcgggaca ggggagacag gtggtgcatg gttgtcgtca gctcgtgt 1258
Claims (6)
1. a kind of tissue rapid propagation method of Populus and Salix xylophyta, which comprises the steps of:
A, the series bacillus that deposit number is CGMCC No.12738 is incubated overnight activation in TSA broth bouillon；
B, by dipping 10s-10min in the culture in step of the cutting of Populus or Salix xylophyta or tissue-cultured seedling, then exist
It is bred in culture substrate.
2. the tissue rapid propagation method of Populus according to claim 1 and Salix xylophyta, which is characterized in that the Populus
Xylophyta is white Mao Yang or European-American Poplar.
3. the tissue rapid propagation method of Populus according to claim 1 and Salix xylophyta, which is characterized in that the Salix
Xylophyta is dry land willow.
4. the tissue rapid propagation method of Populus according to claim 1 and Salix xylophyta, it is characterised in that: the culture
Matrix includes the low nitrogen BEMB200 culture medium containing 2.5mM ammonium nitrate.
5. the tissue rapid propagation method of Populus according to claim 4 and Salix xylophyta, it is characterised in that: the culture
Matrix includes IBA.
6. the tissue rapid propagation method of Populus according to claim 5 and Salix xylophyta, it is characterised in that: the IBA
Concentration is 0.15-0.3mg/L.
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