CN109022305A - Polygonatum cyrtonema endogenetic bacteria bacterial strain ZJU-C612-1 and its application - Google Patents
Polygonatum cyrtonema endogenetic bacteria bacterial strain ZJU-C612-1 and its application Download PDFInfo
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Abstract
The invention discloses a kind of polygonatum cyrtonema endogenetic bacteria bacterial strain ZJU-C612-1, are Bei Laisi bacillus ZJU-C612-1 Bacillus velezensis ZJU-C612-1, deposit number: CCTCC NO:M 2018311.The invention also discloses its purposes: inhibiting the growth of dendrobium candidum anthrax bacteria, polygonatum cyrtonema leaf blight germ, soybean anthracnose, fusarium graminearum, Fusarinm solani, cotton standing dead silk core germ, withered germ of water-melon；And promote polygonatum kingianurn tissue-cultured seedling, more yellow rhizoma polygonati tuber growths.
Description
Technical field
The invention belongs to microorganism field, antibacterial, growth-promoting functions the polygonatum cyrtonema endogenetic bacteria ZJU- of specially one plant tool
C612-1 and its application.
Background technique
Endophyte of plant (Endophyte) refers to that a certain stage in the full history of life or the history of life parasitizes plant tissue
In, and the microorganism for not causing clearly visible disease and function to change to plant itself.Studies have shown that many endophyte of plant tools
There are biological and ecological methods to prevent plant disease, pests, and erosion and growth-promoting functions.Endophyte Biocontrol Effect mechanism mainly include nutrition and space site competition, generate antibacterial substance and
Induce disease resistance of plant.And, mainly there are two aspects in the reason of endophyte generation growth-promoting functions, on the one hand, many endophytes can produce
Auxins, these hormones such as raw auxin, gibberellin and cytokinin can effectively facilitate the growth and development of plant；
On the other hand, endophyte can enhance the nitrogen fixing capacity of plant and the absorbability to nutrients such as potassium, calcium, phosphorus, iron.
Polygonatum cyrtonema is Liliaceae HUANGJING ZANYU CAPSULE, is one of 3 kinds of selected " Chinese Pharmacopoeia ".Medicinal as collection,
The crude drug in whole of the Chinese medicine rhizoma polygonati of edible, ornamental and health care, plump underground rhizome are that polygonatum cyrtonema is main medicinal
Position, dry rhizome have boosting qi and nourishing yin, invigorating the spleen, moistening lung, kidney-nourishing and other effects.Polygonatum cyrtonema main product in Hunan, Anhui, Jiangxi,
Zhejiang and other places are produced and are preferred with Propagation of Rhizomes, because the function values such as its medical value and nutrition and health care are constantly excavated by people,
Present rhizoma polygonati raw material are in the situation that supply falls short of demand.In recent years, with the classification of medicinal plant, chemical pharmacology and cultivation side
After the further investigation of formula etc., the diversity of endophyte has become the hot spot studied now.However, only a small amount of at present
The research of document report rhizoma polygonati endophyte, the research for carrying out more yellow rhizoma polygonati endophytes thus are of great significance.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of Bei Laisi bacillus with biological and ecological methods to prevent plant disease, pests, and erosion and growth-promoting functions
(Bacillus velezensis) new strains ZJU-C612-1 and application thereof.
In order to solve the above technical problem, the present invention provides a kind of polygonatum cyrtonema endogenetic bacteria bacterial strain ZJU-C612-1 (shellfishes
Lay this bacillus velezensis ZJU-C612-1), preservation title: Bei Laisi bacillus ZJU-C612-
1Bacillus velezensis ZJU-C612-1, depositary institution: China typical culture collection center, preservation address: China
Wuhan Wuhan University, deposit number: CCTCC NO:M 2018311, preservation date: on May 28th, 2018.
Polygonatum cyrtonema endogenetic bacteria bacterial strain ZJU-C612-1 is able to suppress colletotrichum (dendrobium candidum anthrax bacteria
(Colletotrichum gloeosporioides), soybean anthracnose (C.truncatum)), Fusarium ((polygonatum cyrtonema
Leaf blight germ (Fusarium oxysporum), fusarium graminearum (F.graminearum), Fusarinm solani
(F.solami), withered germ of water-melon (F.oxyporum f.sp.Niveum)), Rhizoctonia (cotton standing dead silk core germ
(Rhizoctonia.solani)) growth of 3 categories, 7 kinds of plant pathogen mycelia.
Polygonatum cyrtonema endogenetic bacteria bacterial strain ZJU-C612-1 can promote polygonatum kingianurn tissue-cultured seedling and more yellow rhizoma polygonati stem tubers raw
It is long.
Present invention discover that one plant of bacterial strain ZJU-C612-1 with biological and ecological methods to prevent plant disease, pests, and erosion, growth-promoting functions, which is isolated from more yellow rhizoma polygonatis, is
Polygonatum cyrtonema endogenetic bacteria；That is, the present invention has found that this plant has biological and ecological methods to prevent plant disease, pests, and erosion and growth-promoting functions in polygonatum cyrtonema endogenetic bacteria
Polygonatum cyrtonema endogenetic bacteria bacterial strain ZJU-C612-1, the identified bacterium are Bei Laisi bacillus.It can generate a variety of antibacterial
Property active material, including protease, 1,4 beta-glucanase, cellulase, thermophilic iron element and lipopeptide antibiotic, to dendrobium candidum anthrax
Germ, polygonatum cyrtonema leaf blight germ, soybean anthracnose, fusarium graminearum, Fusarinm solani, cotton standing dead silk core germ and
The growth of 7 kinds of plant pathogen mycelia of withered germ of water-melon 3 categories has good inhibiting effect.In addition, the bacterium can also produce
Raw gibberellin (GA), zeatin (ZT), kinetin (KT), 4 Plant Hormone of auxin (IAA), to polygonatum kingianurn tissue-cultured seedling and more
The growth of rhizoma polygonati stem tuber has facilitation.The development and utilization for being found to be polygonatum cyrtonema endogenetic bacteria of the bacterium provides good
Good basis.
Although informing Bei Laisi bacillus in patent CN 102703342 and CN 102286412 can use respectively
In prevention and treatment Hybrid Bamboo top dry and tomato wilt, but the pathogenic bacteria of both diseases and 7 kinds of plant pathogens of the invention are not
Together.In addition, there is document report Bei Laisi bacillus to can be used in preventing and treating wheat powdery mildew (Cai et al., 2017), wild cabbage
Diseases, these diseases and 7 kinds of diseases of the invention such as black spot (Shuna et al., 2011) are not also identical.
The fermentation liquid of bacterial strain ZJU-C612-1 can promote the growth of polygonatum kingianurn tissue-cultured seedling and more yellow rhizoma polygonati stem tubers.Meng Q
It can promote to some extent beet Deng (2016) discovery Bei Laisi bacillus BAC03, carrot, cucumber, pepper, potato, trailing plants
Foretell, pumpkin, the growth of totally 9 kinds of plants such as tomato and radish, but promotes polygonatum kingianurn tissue-cultured seedling and more about Bei Laisi bacillus
The document of the growth of rhizoma polygonati field seedling has not been reported.
Detailed description of the invention
Specific embodiments of the present invention will be described in further detail with reference to the accompanying drawing.
Fig. 1 bacterial strain is ZJU-C612-1 screening experiment figure, polygonatum cyrtonema leaf blight germ.
Fig. 2 is bacterial strain ZJU-C612-1 morphological feature figure；A: the bacterial strain ZJU-C612-1 colonial morphology on LB culture medium；
B: the thalli morphology of bacterial strain ZJU-C612-1 electricity microscopic observation；C is the aspect graph in the sporulation later period, and D is that the part of C is put
Big schematic diagram；
Fig. 3 is that bacterial strain ZJU-C612-1 produces protease, cellulase, 1,4 beta-glucanase, thermophilic iron element, the survey of chitinase ability
Determine result figure；A: protease；B: cellulase；C: 1,4 beta-glucanase；D: thermophilic iron element；E: chitinase.
Fig. 4 is that bacterial strain ZJU-C612-1 produces lipopeptide antibiotic mass spectrogram；Intens.[a.u.]；
The mass spectrogram of A:m/z 800~3000；B: the partial enlargement of m/z 1000~1100 in mass spectrogram A；C: mass spectrogram A
The partial enlargement of middle m/z 1400~1500.
The chromatogram of GA, KT, ZT, IAA in Fig. 5 bacterial strain ZJU-C612-1 fermentation extracting solution；
A1, A2, A3, A4 figure are respectively the chromatogram of GA, KT, ZT, IAA standard specimen；
B1, B2, B3, B4 figure are respectively the chromatogram of GA, KT, ZT, IAA in fermentation liquid.
Fig. 6 is antagonistic effect figure of the bacterial strain ZJU-C612-1 to 6 kinds of plant pathogens；A: B: fusarium graminearum is stood withered
Rhizoctonia, C: soybean anthracnose, D: colletotrichum gloeosporioides Penz, E: withered germ of water-melon, F: Fusarinm solani.
Fig. 7 is growth-promoting functions figure of the bacterial strain ZJU-C612-1 to polygonatum kingianurn group bottle seedling；A:MS；B:MS+ hormone；C: cell-free
Filtrate and MS ratio are 1:100；D: cell-free filtrate is 3:100 with MS ratio；E: cell-free filtrate is 5:100 with MS ratio.
Fig. 8 is growth-promoting functions figure of the bacterial strain ZJU-C612-1 to polygonatum cyrtonema stem tuber；A: sterile LB control；B:OD600=
0.5；C:OD600=0.05.
Specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in
This:
The screening of embodiment 1, bacterial strain ZJU-C612-1
Polygonatum cyrtonema be collected in Zhejiang Province Jiangshan City (north latitude 28 " 22 ' 26.97, east longitude 118 " 30 ' 36.14), by Zhejiang Province
Jiangshan City security personnel township technical advice station provides, and the separation of endogenetic bacteria (can refer to using conventional organization partition method
CN103122331A " a kind of separation method of endophyte of plant "), it isolates and obtains 11 plants of endogenetic bacterias.
The screening of antagonistic strain uses tablet face-off method, comprises the concrete steps that:
(1) endogenetic bacteria is activated on LB solid plate, chooses the LB liquid medium that a ring single colonie is inoculated into 1mL
In, 37 DEG C are placed in, culture 12h is inoculated with interior raw thin as strain in 200r/min shaken cultivation case with 1% (volume %) inoculum concentration
Bacterium is into new LB liquid medium (4mL/10mLEP pipe), and 37 DEG C, 200r/min shaken cultivation for 24 hours, obtains endogenetic bacteria hair
Zymotic fluid is spare.
(2) using polygonatum cyrtonema leaf blight pathogen as indicator bacteria, the screening of Antagonistic Fungi is carried out.Take polygonatum cyrtonema leaf blight sick
In opportunistic pathogen inoculated by hypha block to new PDA culture medium, the activation culture 7d at 28 DEG C takes diameter with punch along colony edge
The bacteria cake of 5mm is put into plate center, sets 2ul endogenetic bacteria fermentation liquid away from selecting at the 2.0cm of bacterium colony two sides, observe each bacterial strain to finger
Show the inhibitory effect of bacterium, measures the antibacterial bandwidth in two sides after 4d, and average.
Obtaining one plant by the screening experiment of Antagonistic Fungi has preferable fungistatic effect to polygonatum cyrtonema leaf blight pathogen
Bacterial strain ZJU-C612-1, as shown in Figure 1.
Antibacterial data: colony radius 1.52cm, percentage mycelial inhibition 62.1%.
The bacterial strain ZJU-C612-1 preservation is subjected to, preservation title: Bei Laisi bacillus ZJU-C612-
1Bacillusvelezensis ZJU-C612-1, depositary institution: China typical culture collection center, preservation address: China
Wuhan Wuhan University, deposit number: CCTCC NO:M 2018311, preservation date: on May 28th, 2018.
The identification of embodiment 2, bacterial strain ZJU-C612-1
Amplification sequencing is carried out with 16S rDNA gene order of the universal primer 27F and 1492R to bacterial strain ZJU-C612-1, is obtained
The sequence for being 1425bp to size, sequence is uploaded to and obtains gene accession number in GenBank: MH 298776.The sequence is existed
Blast is carried out on the website NCBI and compares analysis, as the result is shown itself and Bacillus velezensis strain GQJK49
(CP021495.1) similarity is 99%.
Bacterial strain ZJU-C612-1 is cultivated for 24 hours on LB culture medium at 28 DEG C, and single colonie is similar round, and edge is irregular, wrinkle
Pleated protrusion, rough surface is opaque, dry, and bacterium colony is in light yellow (Fig. 2A).Scanning electron microscopic observation is the results show that thallus is in bar
Shape, size are (0.5~0.7) μ m (1~3) μm (Fig. 2 B), and gemma is oval, middle life or end life, a length of (0.6~1) μm (figure
2C).The bacterial strain can 20~40 DEG C at a temperature of grow, optimum growth temperature be 37 DEG C；It is cultivated in the LB of 1%~7%NaCl
It can be grown on base；Growing pH range is 5.0~9.0.Its feature is consistent with bacillus (Bacillus sp.).
Referring to the physio-biochemical characteristics of the culture based assays bacterial strain ZJU-C612-1 recommended in " common bacteria identification handbook "
It was found that oxidizing ferment, catalase, V-P measurement, Starch Hydrolysis, lecithinase, indoles experiment are the positive, methyl red is feminine gender
(table 1).Referring to " common bacteria system identification handbook ", lecithin cannot be generated according to bacillus subtilis and bacillus amyloliquefaciens
Esterase determines that bacterial strain ZJU-C612-1 is Bei Laisi bacillus.
The physio-biochemical characteristics of table 1, bacterial strain ZJU-C612-1
Embodiment 3, bacterial strain ZJU-C612-1 produce the measurement of antibacterial substance
1, the measurement of protease ability is produced
By bacterial strain ZJU-C612-1 point be connected to protease detection plate (A: skimmed milk power 8g is dissolved in 300mL distilled water, 115
DEG C sterilizing 10min, B: agar 8g adds distilled water to be settled to 300mL, and 121 DEG C of sterilizing 20min, A are mixed after being sterilized separately with B) on,
30 DEG C of dark culture 2d, the discovery edge bacterial strain ZJU-C612-1 generate apparent transparent circle (Fig. 3 A), illustrate bacterial strain ZJU-C612-1
Protease can be generated.
2, the measurement of chitinase ability is produced
Bacterial strain ZJU-C612-1 point is connected to the culture medium (ammonium dihydrogen phosphate using colloidal chitin as sole carbon source
1.0g, potassium chloride 0.2g, Magnesium sulfate heptahydrate 0.2g, w=1% colloidal chitin 100mL, agar 20g, distilled water 1000mL,
PH7.0), 30 DEG C of culture 3d, discovery bacterial strain ZJU-C612-1 do not have transparent circle (Fig. 3 E), illustrate that bacterial strain ZJU-C612-1 can not
Generate chitinase.
3, the measurement of 1,4 beta-glucanase ability is produced
Bacterial strain ZJU-C612-1 point is connected to aniline blue glucan agar medium plate (glucose 1.0g, Poria cocos powder
4.0g, dipotassium hydrogen phosphate 1.0g, disodium hydrogen phosphate 3.0g, aniline blue 0.06g, green vitriol 0.5g, agar powder 12g,
Distilled water 1000mL, pH 7.0), 30 DEG C of 3~5d of culture, discovery bacterial strain ZJU-C612-1 has transparent circle (Fig. 3 C), illustrates bacterial strain
ZJU-C612-1 can generate 1,4 beta-glucanase.
4, thermophilic iron element ability measurement is produced
Bacterial strain ZJU-C612-1 point, which is connected to chromium Austria alcohol (CAS) culture medium, (takes 60.5mg chromium Austria alcohol to be dissolved in 50mL Fe3+
(1mmol/L FeCl3·6H2O, 10mmol/L HCl) solution be uniformly mixed, while agitating, be slowly added 16 amino
After baked base ammonium bromide (HDTMA) solution (72.9mg is dissolved in 40mL water), pH to 7.0 is adjusted, 100mL is then settled to.It finally will be upper
State after solution sterilization with 900mL WA culture medium (agar containing 20g, peptone 4.5g, glucose 9g, beef extract 2.7g, NaCl
4.5g, pH 7.0) mixing), 30 DEG C of 3~5d of culture, discovery bacterial strain ZJU-C612-1 has crocus haloing (Fig. 3 D), illustrates bacterial strain
ZJU-C612-1 can generate thermophilic iron element.
5, cellulase-producing ability measures
Bacterial strain ZJU-C612-1 point is connected to cellulase activity detection plate ((NH4) of pH 7.02SO42g, MgSO4`
7H2O 0.5g, KH2PO41g, NaCl 0.5g, CMC-Na 2g, Congo red 0.4g, agar 20g, distilled water 1L) on, 37 DEG C of perseverances
Temperature culture 4d；The Congo red solution of appropriate l mg/mL is added into culture dish, dyes l h；Dye liquor is discarded, appropriate lmol/ is added
The NaCI solution of L washs l h, then pours into the hydrochloric acid of l mol/L, and discovery bacterial strain ZJU-C612-1 has clearly transparent circle (figure
3B), illustrate that bacterial strain ZJU-C612-1 can generate cellulase.
6, the measurement of lipopeptide antibiotic ability is produced
Bacterial strain ZJU-C612-1 is lined 37 DEG C of constant temperature incubations on LB solid medium, and for 24 hours, each bacterial strain after activation is pressed
1% volume ratio is inoculated in LB liquid medium, and 37 DEG C, 200rpm, shaken cultivation 12h.Then it is coated with after being diluted with sterile water
In 37 DEG C of constant temperature incubation 48h of LB solid medium.2 single bacteriums of picking are fallen on the target of Target Board hole, and 1 μ L auxiliary matrix is added to mix,
MALDI-TOF-MS is detected after natural air drying.Instrument parameter are as follows: reflex mode of operation, positive ion detection, detection range 100~2
000Da, the every map 50 of laser hits, laser frequency 30.0Hz, ion source acceleration voltage 20kV, reflected voltage 23.5kV pulse
Ion.MALDI-TOF-MS testing result shows that bacterial strain ZJU-C612-1 can generate withered grass element (Iturin), Feng Yuansu
(Fengycin) and Surfactin (Surfactin) three classes lipopeptide antibiotic (table 2, Fig. 4).
Her withered grass element, Feng Yuansu, Surfactin testing result of table 2, bacterial strain ZJU-C612-1
Embodiment 4, bacterial strain ZJU-C612-1 produce the measurement of plant exogenous hormone
Strain ZJU-C612-1 is seeded to activation on solid LB media (after 28 DEG C of cultures for 24 hours), then takes a ring list
Colony inoculation transfer 1mL LB liquid medium in, 37 DEG C of 200rpmmin-1Shaken cultivation 12h, is made seed liquor.Take kind
Sub- liquid is seeded in the LB liquid medium of 100mL according to 1% (volume %) inoculum concentration, 37 DEG C, 200rpmmin-1Oscillation training
72h is supported, fermentation liquid is obtained.Fermentation liquid is extracted with the ethyl acetate of 2 volumes times later, extract liquor of the fetch bit in upper layer carries out
Rotary evaporation concentration is saved by 0.22 μm of membrane filtration in 4 DEG C, is surveyed with high performance liquid chromatography-mass spectrometry instrument (LC-MS)
Determine IAA, KT, GA3With the content of ZT, measurement result shows its GA in fermentation liquid3Content be 0.07 μ g/L, KT content be
The content of 21.34 μ g/L, ZT is 0.02ng/L, and the content of IAA is 8.29 μ g/L (Fig. 5).Mass Spectrometry Conditions are as follows: using electron spray
Positive negative ion mode, capillary voltage 3.0KV, nebulizer pressure 45Psi, dry gas (N2) flow velocity 5L/min, dry gas
325 DEG C of temperature, 350 DEG C of temperature of protective gas (N2), protective gas flow velocity 11L/min.
Embodiment 5, bacterial strain ZJU-C612-1 are to the bacteriostatic experiments of 6 kinds of plant pathogens
Using agar diffusion method, the dendrobium candidum anthrax bacteria, soybean anthracnose, fusarium graminearum, eggplant of diameter 5mm are taken
6 kinds of sick sickle-like bacteria, cotton standing dead silk core germ and withered germ of water-melon plant pathogen bacteria cakes be put in PDA plate center, away from
(d=5mm) is punched from the 2.5cm of center, 2ul ZJU-C612-1 fermentation liquid (preparation of embodiment 1 gained) is added in hole, with
LB measures mycelial growth inhibition rate after control group mycelia covers with entire culture dish as control, 28 DEG C of culture dark culturings.
Each processing is repeated 3 times.Measurement result as shown in fig. 6, show bacterial strain ZJU-C612-1 be able to suppress dendrobium candidum anthrax bacteria,
Soybean anthracnose, fusarium graminearum, Fusarinm solani, cotton standing dead silk core germ and withered germ of water-melon, mycelia growth
Inhibiting rate is followed successively by 71.8%, 79%, 55%, 58.4%, 75.1%, 66.4%.
Mycelial growth inhibition rate %=((control colony radius-processing colony radius)/control colony radius) × 100
Table 3, bacterial strain ZJU-C612-1 are to the fungistatic effects of 6 kinds of plant bacterium opportunistic pathogens
Note: the English alphabet in row after number is the significance difference analysis in the level of p < 0.0 5.
The Biological control of embodiment 6, bacterial strain ZJU-C612-1 yellow rhizoma polygonati stem tuber to polygonatum kingianurn tissue-cultured seedling and mostly
Endogenetic bacteria is activated on LB solid plate, a ring single colonie is chosen and is inoculated into the LB liquid medium of 1mL,
37 DEG C are placed in, culture 12h is inoculated with endogenetic bacteria as strain with 1% (volume %) inoculum concentration in 200r/min shaken cultivation case
Into new LB liquid medium (100mL/500mL conical flask), 37 DEG C, 200r/min shaken cultivation 48h, endogenetic bacteria is obtained
After fermentation liquid, carries out 5000rpm and be centrifuged 10min, obtain supernatant, it is rear with being 0.22 μm of sterilizing filter filtering with aperture, it obtains
Obtain cell free fermentation liquid.
Take growing way identical (plant height 2cm), the polygonatum kingianurn bottle seedling of fresh weight identical (0.15g) is respectively placed in following culture medium,
1), MS culture medium；2), MS+6-BA 1mg/mL+NAA 0.1mg/mL+ agar 7.5g+ sucrose 30g, pH=5.8；3), cell-free
Filtrate mixes well with MS volume ratio for 1:100；4) cell-free filtrate is mixed well with MS volume ratio for 3:100；5) cell-free
Filtrate mixes well with MS volume ratio for 5:100, and each tissue culture bottle connects 3, and when inoculation cuts off root, and each processing is inoculated with tissue culture
Seedling 21.Condition of culture are as follows: 16h illumination, intensity of illumination are 30~40 μm of olm-2·s-1, temperature is (27 ± 1) DEG C；8h is dark
Culture, temperature are (21 ± 1) DEG C；Above-mentioned illumination and dark alternately, are observed after 30 days and are recorded without born of the same parents' filtrate to polygonatum kingianurn
Growth effect.As a result, it has been found that: the processing group without born of the same parents' fermentation liquid is added and the experimental result indifference of HORMONE TREATMENT group, card is added
Bright no born of the same parents' fermentation liquid is consistent with functions of hormones for the growth-promoting functions of tissue-cultured seedling (table 4, Fig. 7).
Identical 3 years raw polygonatum cyrtonema stem tubers of growing way are taken to carry out surface sterilization 15min, sterile water with 0.5% sodium hypochlorite
Rinsing 3~4 times, is seeded in matrix (turf: vermiculite: perlite=2:1:1).Carry out following 3 kinds of processing: A:LB Liquid Culture
Base carries out pouring root；B: antagonism bacterium solution (OD is used600=0.5) (1 × 108CFU/mL pouring root) is carried out；C: antagonism bacterium solution (OD is used600=
0.05)(1×107CFU/mL pouring root) is carried out.3 basins of every processing, 3 repetitions, condition of culture (25 DEG C, 16h illumination, the training of 8h dark
Support), the growing state of polygonatum cyrtonema is observed, records the variation of each biomass after two months.The result shows that after bacterium solution processing,
The long difference of polygonatum cyrtonema bud is unobvious, but root long, single plant radical and bud number difference are obvious, as shown in fig. 7, root after bacterium solution processing
Long, radical all obviously increases, OD600The polygonatum cyrtonema part root long of=0.5 processing group, single plant radical increase compared to control group
44.6%, 102.4%, OD600The polygonatum cyrtonema part root long of=0.05 processing group, single plant radical increase compared to control group
20.7%, 64.5%, and the bud length on processing group rhizome is significantly less than control group (table 5, Fig. 8).
Antagonism bacterium solution (OD600=0.5) the preparation method comprises the following steps: endogenetic bacteria of the invention is activated on LB solid plate,
It chooses a ring single colonie to be inoculated into the LB liquid medium of 1mL, is placed in 37 DEG C, cultivates 12h in 200r/min shaken cultivation case
As strain, endogenetic bacteria is inoculated with to new LB liquid medium (100mL/500mL conical flask) with 1% (volume %) inoculum concentration
In, 37 DEG C, 200r/min shaken cultivation 48h, acquisition endogenetic bacteria fermentation liquid is spare, is diluted to OD with LB solution later600=
0.5 (spectrophotometer measurement), OD600=0.05.
Table 4, bacterial strain ZJU-C612-1 are to the growth-promoting effect of polygonatum kingianurn group bottle seedling
| Plant height (cm) | Fresh weight (g) | Proliferation times | Chlorophyll content | |
| ms | 5.4±2.87a | 0.31±0.87b | 1.52±0.67b | 22.88±4.60a |
| Ms+ hormone | 5.64±1.35a | 0.57±0.25a | 2.43±1.29a | 25.15±5.76a |
| 1% | 5.19±1.37a | 0.48±0.19a | 2.14±1.15a | 21.81±6.56a |
| 3% | 5.94±1.56a | 0.42±0.12a | 2.24±1.14a | 25.44±3.93a |
| 5% | 5.95±1.94a | 0.41±0.13a | 1.86±0.73a | 25.85±5.31a |
Note: the English alphabet in row after number is the significance difference analysis in the level of p < 0.0 5
Table 5, bacterial strain ZJU-C612-1 are to the growth-promoting effect of polygonatum cyrtonema stem tuber
| Radical | Root long (cm) | Lateral bud number | Bud is long (cm) | |
| LB | 2.06±0.56b | 5.85±0.77b | 3.00±1.00b | 0.21±0.18a |
| OD600=0.05 | 3.39±0.75a | 7.06±0.45a | 3.67±1.33b | 0.16±0.06a |
| OD600=0.5 | 4.17±0.48a | 8.46±0.33a | 5.33±0.67a | 0.10±0.08a |
Note: the English alphabet in row after number is the significance difference analysis in the level of p < 0.0 5.
Existing Bei Laisi bacillus is carried out bacteriostatic experiment, acquired results by comparative experiments 1 as described in embodiment 5
Comparison with invention bacterial strain ZJU-C612-1 is described in table 6 below.
Table 6, mycelial growth inhibition rate
Comparative experiments 2 carries out existing Bei Laisi bacillus to polygonatum kingianurn tissue-cultured seedling (only as described in embodiment 6
It is arranged 3%) and (OD is only arranged in the Biological control of more yellow rhizoma polygonati stem tubers600=0.5), acquired results and invention bacterial strain ZJU-C612-
1 comparison is described in table 7 below.
Table 7
The above list is only a few specific embodiments of the present invention for finally, it should also be noted that.Obviously, this hair
Bright to be not limited to above embodiments, acceptable there are many deformations.Those skilled in the art can be from present disclosure
All deformations for directly exporting or associating, are considered as protection scope of the present invention.
Claims (3)
1. polygonatum cyrtonema endogenetic bacteria bacterial strain ZJU-C612-1, it is characterized in that: being Bei Laisi bacillus ZJU-C612-
1Bacillus velezensis ZJU-C612-1, deposit number: CCTCC NO:M 2018311.
2. the purposes of polygonatum cyrtonema endogenetic bacteria bacterial strain ZJU-C612-1 as described in claim 1, it is characterized in that: inhibiting iron sheet
Dendrobium nobile anthrax bacteria (Colletotrichum gloeosporioides), polygonatum cyrtonema leaf blight germ (Fusarium
Oxysporum), soybean anthracnose (C.truncatum), fusarium graminearum (F.graminearum), Fusarinm solani
(F.solami), cotton standing dead silk core germ (Rhizoctonia.solani), withered germ of water-melon (F.oxyporum
F.sp.Niveum growth).
3. the purposes of polygonatum cyrtonema endogenetic bacteria bacterial strain ZJU-C612-1 as described in claim 1, it is characterized in that: promoting Yunnan yellow
Smart tissue-cultured seedling, more yellow rhizoma polygonati tuber growths.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109554317A (en) * | 2018-12-29 | 2019-04-02 | 陕西省微生物研究所 | Bei Laisi bacillus and its application in wheat sharp eyespot disease prevention growth-promoting |
| CN110117557A (en) * | 2019-03-27 | 2019-08-13 | 海南大学 | A kind of biocontrol microorganisms HN-2 and extract inhibit the application on disease fungus preparation in preparation |
| CN110301335A (en) * | 2019-07-10 | 2019-10-08 | 浙江大学 | The cultivation of the lateral bud breeding bulb of west safflower |
| CN112029691A (en) * | 2020-09-28 | 2020-12-04 | 广西壮族自治区农业科学院植物保护研究所 | Application of Bacillus beleisi YM-11-C in prevention and treatment of mango anthracnose |
| CN112920966A (en) * | 2020-12-31 | 2021-06-08 | 浙江省农业科学院 | Bacillus belgii, microbial agent thereof, preparation method of microbial agent and application of microbial agent |
| CN115707782A (en) * | 2022-08-10 | 2023-02-21 | 贵州省植物园（贵州省园林科学研究所、贵州省植物研究所） | Method for screening polygonatum cyrtonema pathogenic fungi |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20100055625A (en) * | 2008-11-18 | 2010-05-27 | 한국화학연구원 | Bacillus velezensis s3-5 strain and method for the biological control of plant diseases using same |
| KR20120010017A (en) * | 2010-07-23 | 2012-02-02 | 대한민국(농촌진흥청장) | A novel Bacillus velezensis B-42 for multi function |
| CN108004185A (en) * | 2018-01-09 | 2018-05-08 | 中国农业科学院植物保护研究所 | One plant of tool diseases prevention, growth-promoting, drought resisting function plant endogenesis Bei Laisi bacillus and its application |
| CN108265012A (en) * | 2016-12-30 | 2018-07-10 | 北京绿色农华作物科技有限公司 | A kind of Bei Laisi Bacillus strains and its microbial inoculum and application |
-
2018
- 2018-07-18 CN CN201810791036.4A patent/CN109022305B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20100055625A (en) * | 2008-11-18 | 2010-05-27 | 한국화학연구원 | Bacillus velezensis s3-5 strain and method for the biological control of plant diseases using same |
| KR20120010017A (en) * | 2010-07-23 | 2012-02-02 | 대한민국(농촌진흥청장) | A novel Bacillus velezensis B-42 for multi function |
| CN108265012A (en) * | 2016-12-30 | 2018-07-10 | 北京绿色农华作物科技有限公司 | A kind of Bei Laisi Bacillus strains and its microbial inoculum and application |
| CN108004185A (en) * | 2018-01-09 | 2018-05-08 | 中国农业科学院植物保护研究所 | One plant of tool diseases prevention, growth-promoting, drought resisting function plant endogenesis Bei Laisi bacillus and its application |
Non-Patent Citations (1)
| Title |
|---|
| 宗英，等: "一株贝莱斯芽孢杆菌抑制禾谷镰刀菌的研究", 《核农学报》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109554317A (en) * | 2018-12-29 | 2019-04-02 | 陕西省微生物研究所 | Bei Laisi bacillus and its application in wheat sharp eyespot disease prevention growth-promoting |
| CN109554317B (en) * | 2018-12-29 | 2022-04-01 | 陕西省微生物研究所 | Bacillus belgii and application thereof in preventing diseases and promoting growth of wheat sheath blight |
| CN110117557A (en) * | 2019-03-27 | 2019-08-13 | 海南大学 | A kind of biocontrol microorganisms HN-2 and extract inhibit the application on disease fungus preparation in preparation |
| CN110301335A (en) * | 2019-07-10 | 2019-10-08 | 浙江大学 | The cultivation of the lateral bud breeding bulb of west safflower |
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