CN106967633A - One plant of Kluyvera ascorbata and its application - Google Patents
One plant of Kluyvera ascorbata and its application Download PDFInfo
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- CN106967633A CN106967633A CN201710185236.0A CN201710185236A CN106967633A CN 106967633 A CN106967633 A CN 106967633A CN 201710185236 A CN201710185236 A CN 201710185236A CN 106967633 A CN106967633 A CN 106967633A
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Abstract
One plant of Kluyvera ascorbata and its application, microorganism belonging to genus technical field.The bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC), address on March 7th, 2017：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, deposit number：CGMCC No.13726, Classification And Nomenclature：Kluyvera ascorbata Kluyvera ascorbata.Bacterial strain of the present invention is to screen out to come from Yunnan field Eupatorium adenophorum parasitic insect Herba Lycopi trypetid (Procecidochares utilis Stone) alimentary canal, quickly eupatorium adenophorum rhizome can be infected under natural environment, cause rhizome lesion, tender stem, root corruption, until the whole strain of plant is withered, for the prevention and control of Eupatorium adenophorum, the good cost of effect is low.
Description
Technical field
The present invention is microbial technology field, specially one plant Kluyvera ascorbata (Kluyvera
Ascorbata) bacterial strain and its application.
Background technology
Eupatorium adenophorum (Eupatorium adenophorum), composite family Eupatorium, perennial herb or undershrub, can be quick
Single dominantcommunity is formed, and other plant growths can be suppressed in many ways, is a kind of global malignant weed, is also China
A kind of maximum adventitious plant of existing harm.Because Eupatorium adenophorum contains a variety of poisonous components and malodor components, only Herba Lycopi etc.
Only a few species have feeding phenomenon to it.Although improvement of the people to Eupatorium adenophorum in recent years takes the obligate natural enemy of dispensing, removed
The means such as careless agent, plant prevention and control, but produce little effect.
Microorganism has as the modern of a kind of efficient, safety, noresidue in modern agricultural production and scientific research
Very important status.Correspondingly, the microbial pesticide research and development for Eupatorium adenophorum also turn into Eupatorium adenophorum control in recent years
One Main way in field, but because Eupatorium adenophorum contains a variety of antibacterial substances in itself, cause R＆D work also to be produced effects so far
It is little.
The content of the invention
It is an object of the invention to provide one plant of Kluyvera ascorbata and its application, with ascorbic acid Ke Lvwo
You are used for the prevention and control of Eupatorium adenophorum by bacterium for the microbial inoculum of active component, and the good cost of effect is low.
Kluyvera ascorbata (Kluyvera ascorbata) provided by the present invention, the bacterial strain of patent applicant
Numbering is ZLSY22；The bacterial strain is preserved in the common micro- life of China Committee for Culture Collection of Microorganisms on March 7th, 2017
Thing center (abbreviation CGMCC), address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, protects
Hide numbering：CGMCC No.13726, Classification And Nomenclature：Kluyvera ascorbata Kluyvera ascorbata.
Bacterial strain ZLSY22 CGMCC No.13726 of the present invention biological characteristics is：
LB culture mediums (LB Medium) culture bacterium colony be milky, rounded protuberance, neat in edge, the smooth moistening in surface,
There is stink；
Thalline is small rhabdocyte, and 0.5~0.7 2~3 μm of μ m, Gram-negative is moved with dilute peritricha.
Starch Hydrolysis experiment is negative, and grease hydrolysis experiment is negative, and litmus milk experiment is positive, and urea experiment is negative；Fermentation
Glucose can produce acid, but not aerogenesis；Indoles experiment is positive, and citrate experiment is positive, and hydrogen sulfide experiment is negative, and V-P is negative, M-
R is positive.
Bacterial strain of the present invention is from Yunnan field Eupatorium adenophorum parasitic insect Herba Lycopi trypetid (Procecidochares utilis
Stone) alimentary canal screens out what is come.Bacterial strain of the present invention quickly can infect eupatorium adenophorum rhizome under natural environment, cause rhizome
Lesion, tender stem, root are quickly corrupt, until the whole strain of plant is withered, are a kind of micro- with good result and developmental research prospect
It is biological.
Bacterial strain ZLSY22 CGMCC No.13726 of the present invention are used for prevention and control Eupatorium adenophorum.
Bacterial strain ZLSY22 CGMCC No.13726 of the present invention can be isolated and purified by following methods to be obtained：
Herba Lycopi trypetid is picked up from the Eupatorium adenophorum near the chemical building of Yunnan Prov Agriculture University, and the Eupatorium adenophorum for having insect gall is adopted back
Laboratory.To solution cuts larva in superclean bench after the insect gall overall disinfection adopted back, larva is rinsed three times with 75% alcohol,
Two minutes every time, dissect after rinsed with sterile water three times, obtain Herba Lycopi's trypetid alimentary canal.
1. sterilizing
Culture dish used, centrifuge tube, test tube, the experiment equipment such as pipette tips and sterilized water use autoclaving, 0.1MPa,
121 DEG C, sterilize 30 minutes.
2. grinding
Herba Lycopi's trypetid digestive tract is put into sterile centrifugation tube, 1ml sterilized waters is added, is ground with grinding rod.
3. gradient dilution
The juice 1ml after grinding is taken, is put into the test tube for filling 9ml sterilized waters, as 10-1The bacterium solution of dilution factor, then from
10-11ml is taken to be put into the test tube for filling 9ml sterilized waters in the bacterium solution of dilution factor, as 10-2The bacterium solution of dilution factor, by that analogy
It is made 10-3、10-4、10-5、10-6、10-7The bacterium solution of dilution factor.
4. the configuration of culture medium
The culture medium that the separation of bacterium is conventional is beef-protein medium (NA culture medium), the conventional LB cultures of purifying
Base.
NA culture mediums：Beef extract 3g, peptone 10g, NaCl 5g, agar 18g, water 1000ml, pH 7.0-7.2,121 DEG C
Sterilize 20min.
LB culture mediums：Peptone 10g；Yeast extract 5g；Sodium chloride 10g；Agar 15g；Distilled water 1L；PH 7.0,121
DEG C sterilizing 20min.
Plate is down flat, each culture dish about 15ml culture medium keeps flat standing, to be cooled standby.
5. coating
Bacterium solution 10 will be diluted-4、10-5、10-6、10-7It is respectively coated on flat board, each gradient applies three wares, and do ware is low
Upper mark.
6. culture
Plate is inverted to cultivate 3-5 days in 28 DEG C of incubators.
7. the purifying of bacterium
Aseptically with choose pin by the single bacterium colony picking grown on NA culture mediums on LB culture mediums rule cultivate.It is pure
Change the single bacterium colony that can be obtained after purification for 3-4 times.
Kluyvera ascorbata condition of culture is studied
1. temperature
Kluyvera ascorbata is inoculated in pH 7.0 LB culture mediums, respectively 15 DEG C, 25 DEG C, 30 DEG C, 35 DEG C,
Under 40 DEG C of temperature conditionss, rotating speed is 220r/min shaking table culture, and the light at wavelength 600nm is measured by sampling when having cultivated 24h
Absorption value (OD600).The OD of Kluyvera ascorbata nutrient solution under the table 1 seen below, relatively more each temperature conditionss600Value can
Know, the most suitable cultivation temperature of Kluyvera ascorbata is 25 DEG C, and convenient growth scope is between 20 DEG C -30 DEG C.
The influence of the temperature Ascorbic Acid gram Lv Wal bacteria growing of table 1
2.pH values
Kluyvera ascorbata is inoculated in the LB culture mediums that pH is 5.5,6,6.5,7,7.5,8 and 8.5 respectively, is shaking
Bed (rotating speed is 220r/min, and temperature is 25 DEG C) culture, the absorbance value at wavelength 600nm is measured by sampling in 24h
(OD600).2 are shown in Table, compares the OD of Kluyvera ascorbata nutrient solution under the conditions of each pH600Value is understood, in 5.5-8.5pH
Kluyvera ascorbata can preferably grow between scope, and wherein pH grows best when being 7.5.
The influence of the pH Ascorbic Acid gram Lv Wal bacteria growing of table 2
3. culture medium
Kluyvera ascorbata is inoculated in pH 7.0 LB culture mediums, TSB culture mediums and NA culture mediums respectively, is shaking
Bed (rotating speed is 220r/min, and temperature is 25 DEG C) culture, the absorbance value at wavelength 600nm is measured by sampling in 24h
(OD600).As shown in Table 3, in three kinds of culture mediums, LB culture mediums are best suitable for the growth of Kluyvera ascorbata.
The influence of the culture medium Ascorbic Acid gram Lv Wal bacteria growing of table 3
Note：NA culture mediums：Beef extract 3g, peptone 10g, NaCl 5g, agar 18g, water 1000ml, 121 DEG C of sterilizings
20min。
LB culture mediums：Peptone 10g；Yeast extract 5g；Sodium chloride 10g；Agar 15g；Distilled water 1L；121 DEG C of sterilizings
20min。
TSB culture mediums：Tryptone 15g；Soy peptone 5g；Sodium chloride 5g；Agar 15g；Distilled water 1L；121 DEG C go out
Bacterium 20min.
Microbial inoculum prevention and control Eupatorium adenophorum is made with bacterial strain ZLSY22 CGMCC No.13726 of the present invention can be by the following method：
A ZLSY22 stage expands numerous culture：With standard solid LB culture mediums, the purple stem pool of 0.2% ratio addition by volume
Blue water extract, adjusts pH value 5.5~8.5, is down flat after the cooling of plate culture dish, is inoculated with ZLSY22 bacterial strains, 15 DEG C of -40 DEG C of cultures of temperature
1-3 days, see that bacterium colony covers with culture medium.
ZLSY22 two-stage expands breeding culture medium：Normal fluid LB culture mediums, the purple stem pool of 0.5% ratio addition by volume
Blue water extract, pH value 5.5-8.5.Access second step expand it is numerous after cover with the solid medium of bacterial strain, in 15 DEG C~40 DEG C of temperature
Culture 1~3 day, is once stirred, inoculative proportion is for every 12 hours：1cm2Two-stage solid medium is inoculated with 2L Liquid Cultures
The ratio of base is inoculated with.
ZLSY22 use liquid, is cultivated the nutrient solution completed the two-stage, and 8 DEG C refrigerate 8 hours, are stood after taking-up to normal temperature
By 1:5~1:After 10 ratio is diluted with water, directly it is sprayed to Eupatorium adenophorum stem or is poured by irrigation method to plant root
Portion.
Described Eupatorium adenophorum water extract is：Eupatorium adenophorum fresh goods plant (contains blade), after crushing at 40 DEG C ± 5 DEG C,
According to mass ratio 1:9~1:12 ratio is extracted 2 hours, is taken after supernatant, 0.22 μm of membrane filtration and be can be used after removal solid.
Different administration mode, fatal rate are obtained with applying the relation of rear time by experiment, Fig. 1 is seen.Fig. 1 ordinate
For the fatal rate of eupatorium adenophorum, abscissa is time of application.Fatal rate and time of application observation statistics be the 4th, 6,10,12,
14th, 18,20 days.Lines 1 in Fig. 1 is pour, and lines 2 are foliage spray, and lines 3 are blank control.
As can be seen from Figure 1, after using the bacterium solution containing bacterium of the present invention, eupatorium adenophorum can quickly fall ill and dead, its peak period
The 8th to 10 day is appeared in, to 20 days.Not yet dead plant, shows not to be infected or produce resistance, concrete reason and mechanism are still
Treat further research.
Beneficial effects of the present invention：The Kluyvera ascorbata that the present invention is provided is used for the prevention and control of Eupatorium adenophorum, effect
Really good cost is low.
Brief description of the drawings
Fig. 1 is different administration mode, using rear time and the graph of a relation of Eupatorium adenophorum fatal rate.
Embodiment：
Embodiment 1.
The first step：Purified ZLSY22 bacterial strains will be sieved, after standard solid LB medium sterilizations, will be added before not solidifying
Plus 0.2% purple stem boneset extract, and adjust pH value and be down flat plate to 5.5., it is to be condensed after ZLSY22 is seeded on culture medium,
It is after being cultivated 1 day on 25 DEG C of culture mediums, the fritter for being divided into top surface area to be 1 square centimeter with bacterium culture medium is standby.
Second step：Configuration standard LB liquid medium.Added by volume after sterilizing 0.5% purple stem boneset extract,
PH value is adjusted to 5.5,25 DEG C are cooled to, the 1cm that previous step is ready to carry disease germs2Fritter culture medium is inoculated with (addition) in proportion
Into fluid nutrient medium, cultivate 3 days, once stirred at 25 DEG C within every 12 hours.Taking-up is cooled to 8 DEG C and refrigerated 8 hours.
3rd step：The fluid nutrient medium that carries disease germs refrigerated is taken out, the ratio for the 10 parts of volumes that added water by the culture medium of 1 part of volume
After example dilution, Eupatorium adenophorum stem or bastem portion are sprayed to.
Embodiment 2.
The first step：First choice will sieve purified ZLSY22 bacterial strains, after standard solid LB medium sterilizations, not solidify
0.2% purple stem boneset extract of preceding addition, and adjust pH value and be down flat plate to 7.0, it is to be condensed after ZLSY22 is seeded to culture medium
On, it is after being cultivated 3 days on 29 DEG C of culture mediums, the fritter for being divided into top surface area to be 1 square centimeter with bacterium culture medium is standby.
Second step：Configuration standard LB liquid medium, added by volume after sterilizing 0.5% purple stem boneset extract,
PH value is adjusted to 6.5,29 DEG C are cooled to, the 1cm that previous step is ready to carry disease germs2Fritter culture medium is inoculated with (addition) in proportion
Into fluid nutrient medium, cultivate 5 days, once stirred at 29 DEG C within every 12 hours.Taking-up is cooled to 8 DEG C and refrigerated 8 hours.
3rd step：The fluid nutrient medium that carries disease germs refrigerated is taken out, is by volume culture medium：Water=1:10 ratio adds
After water dilution, pour to plant base portion.
Embodiment 3：
The first step：First choice will sieve purified ZLSY22 bacterial strains, after standard solid LB medium sterilizations, not solidify
0.2% purple stem boneset extract of preceding addition, and adjust pH value and be down flat plate to 7.5, it is to be condensed after ZLSY22 is seeded to culture medium
On, it is after being cultivated 5 days on 25 DEG C of culture mediums, the fritter for being divided into top surface area to be 1 square centimeter with bacterium culture medium is standby.
Second step：Configuration standard LB liquid medium, added by volume after sterilizing 0.5% purple stem boneset extract,
PH value is adjusted to 7.5,25 DEG C are cooled to, the 1cm that previous step is ready to carry disease germs2Fritter culture medium is inoculated with (addition) in proportion
Into fluid nutrient medium, cultivate 3 days, once stirred at 25 DEG C within every 12 hours, taking-up is cooled to 8 DEG C and refrigerated 8 hours.
3rd step：The fluid nutrient medium that carries disease germs refrigerated is taken out, is by volume culture medium：Water=1:5 ratio is dilute
After releasing, Eupatorium adenophorum stem or bastem portion are sprayed to.
Embodiment 4.
100 plants each to the Eupatorium adenophorum of artificial growth, carry out the experiment of Contrast on effect.Blank control is Eupatorium adenophorum fresh goods
Plant (contains blade), after crushing at 45 DEG C, according to mass ratio 1:10 ratio is extracted 2 hours, and supernatant is taken after removing solid,
10 times of water are added to be diluted to blank control liquid after 0.22 μm of membrane filtration, remaining uses the bacterium solution of embodiment 2.Usage amount：Spray and be
100 plants of 2L, Irrigation is 100 plants of 5L, and 20 days cumulative datas of Continuous Observation are as follows after use once：
Data above can be seen that multinomial using can significantly induce Eupatorium adenophorum generation after ZLSY22 bacterial strains in 20 days
Lesion, and it is dead to quickly result in Eupatorium adenophorum.
Claims (3)
- Kluyvera ascorbata 1. (Kluyvera ascorbata) ZLSY22CGMCC No.13726.
- 2. Kluyvera ascorbata (Kluyvera ascorbata) ZLSY22 CGMCC as claimed in claim 1 No.13726, its application is prevention and control Eupatorium adenophorum.
- 3. Kluyvera ascorbata (Kluyvera ascorbata) ZLSY22 CGMCC as claimed in claim 2 No.13726 application, it is characterised in that according to the following steps：A ZLSY22 stage expands numerous culture：With standard solid LB culture mediums, the Eupatorium adenophorum water of 0.2% ratio addition by volume Extract, adjusts pH value 5.5~8.5, is down flat after the cooling of plate culture dish, is inoculated with ZLSY22 bacterial strains, 15 DEG C of -40 DEG C of culture 1-3 of temperature My god, see that bacterium colony covers with culture medium；ZLSY22 two-stage expands breeding culture medium：Normal fluid LB culture mediums, the Eupatorium adenophorum water of 0.5% ratio addition by volume Extract, pH value 5.5-8.5 covers with the solid medium of bacterial strain after access second step expansion is numerous, in 15 DEG C~40 DEG C cultures 1 of temperature ~3 days, once stirred within every 12 hours, inoculative proportion is：1cm2Two-stage solid medium inoculation 2L fluid nutrient mediums Ratio is inoculated with；ZLSY22 use liquid, is cultivated the nutrient solution completed the two-stage, and 8 DEG C refrigerate 8 hours, are stood after taking-up to normal temperature by 1: 5~1:After 10 ratio is diluted with water, directly it is sprayed to Eupatorium adenophorum stem or is poured by irrigation method to plant root；Described Eupatorium adenophorum water extract is：Eupatorium adenophorum fresh goods plant (contains blade), after crushing at 40 DEG C ± 5 DEG C, according to Mass ratio 1:9~1:12 ratio is extracted 2 hours, is taken after supernatant, 0.22 μm of membrane filtration and be can be used after removal solid.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113215048A (en) * | 2021-05-21 | 2021-08-06 | 中国农业科学院农业资源与农业区划研究所 | Kluyveromyces AZ981 for improving nitrogen fixation capacity of nitrogen-fixing bacteria and application thereof |
Citations (1)
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|---|---|---|---|---|
| US20070154459A1 (en) * | 2003-04-07 | 2007-07-05 | Hargis Billy M | Method for bacteriophage delivery and amplification |
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2017
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070154459A1 (en) * | 2003-04-07 | 2007-07-05 | Hargis Billy M | Method for bacteriophage delivery and amplification |
Non-Patent Citations (2)
| Title |
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| BHOJ RAJ SINGH等: "Multiple-herbal-antimicrobial-resistance (MHAR) in microbes of animals,birds, fish, food, lizard and water origin", 《INTERNATIONAL CONFERENCE AND 28TH ANNUAL CONVENTION OF IAVMI-2014 ON "CHALLENGES AND OPPOTUNITIES IN ANIMAL HEALTH AT THE FACE OF GLOBALIZATION AND CLIMATE CHANGE"》 * |
| 兰明先等: "泽兰实蝇幼虫内生细菌的分离鉴定及除草活性研究", 《江西农业学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113215048A (en) * | 2021-05-21 | 2021-08-06 | 中国农业科学院农业资源与农业区划研究所 | Kluyveromyces AZ981 for improving nitrogen fixation capacity of nitrogen-fixing bacteria and application thereof |
| CN113215048B (en) * | 2021-05-21 | 2022-03-18 | 中国农业科学院农业资源与农业区划研究所 | Kluyveromyces AZ981 for improving nitrogen fixation capacity of nitrogen-fixing bacteria and application thereof |
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