CN104962497B - The dinitrogen of a kind of canker resistant pathogenic bacteria supports cholerae strain - Google Patents

The dinitrogen of a kind of canker resistant pathogenic bacteria supports cholerae strain Download PDF

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CN104962497B
CN104962497B CN201510410134.5A CN201510410134A CN104962497B CN 104962497 B CN104962497 B CN 104962497B CN 201510410134 A CN201510410134 A CN 201510410134A CN 104962497 B CN104962497 B CN 104962497B
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bacterial strain
dinitrogen
citrus
vibrio
present
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CN104962497A (en
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唐前君
周志成
肖启明
戴良英
李迅
刘双清
李魏
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Hunan Agricultural University
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    • C—CHEMISTRY; METALLURGY
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    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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Abstract

Foster vibrio (Vibrio diazotrophicus) the T24 bacterial strain of dinitrogen that a kind of canker resistant is sick, is deposited in China typical culture collection center on April 10th, 2015, and deposit number is CCTCC NO:M2015221.The fermentation liquid of bacterial strain T24 of the present invention is inhibited to citrus processing, utilizes needle point method to record it, without fermented liquid, citrus processing extracorporeal inhibiting rate is reached more than 90%.

Description

The dinitrogen of a kind of canker resistant pathogenic bacteria supports cholerae strain
Technical field
The invention belongs to biological technical field, the dinitrogen relating to a kind of canker resistant pathogenic bacteria supports cholerae strain, it is specifically related to a kind of dinitrogen and supports vibrio (Vibriodiazotrophicus) T24 bacterial strain, also relate to this bacterial strain T24 application in preventing and treating citrus processing.
Background technology
Citrus are that a kind of subtropical evergreen fruit trees kind, its yield and cultivated area rank first in the middle of the fruit in the whole world, are distributed in the countries and regions of some benign climates of Perenniporia martius.Orange yield, the Northern Hemisphere almost occupies the quantum of output of half, and the Southern Hemisphere then occupies the supply of about 32%, and remaining part comes from Mediterranean.Perenniporia martius region, the yield of developing country accounts for the 65% of total yield.2007, under the leading of some countries such as Brazil of Orange Producing big country and the U.S., orange yield and the cultivated area in the whole world respectively reach 1.2 hundred million tons and 8,320,000 hectares, total amount accounts for the 20% of whole world fruit total output, its volume of trade and yield are only second to Semen Maydis and Semen Tritici aestivi, become the big trade agricultural product in third place in the world.
Citriculture has had the history of several thousand in China, and not only cultivated area is wide, and the kind planted is very many.Mostly all large-area in some Shelter in South China Cities such as the Fujian of China, Guangdong, Guangxi, Yunnan, Hunan plant the kinds such as orange class (such as Niu Heer, friend's Na etc.), loose-skinned oranges, Fructus Citri grandis class (such as shatian pomelo, grapefruit etc.).Especially in recent years the Citrus Industry of China welcome the development of great-leap-forward, has become as orange yield and occupies world-class big producing country.
While enjoying the achievement of Citrus Industry splendidness development, we also should pay attention to the factor of some the biological aspects affecting orange yield and quality and the abiotic aspect run in Orange Producing process, and to take the measure of corresponding active defense.Insect pest and disease are the maximum problems run in plant growing process, also it is the either large or small threatening factors affecting harvest, a kind of important disease being listed in quarantine harmful organisms register by the Ministry of Agriculture is had in planting such as Citrus, also it is one of the three big diseases that Citrus Industry is had devastating impact of quarantine object in the whole world, the citrus bacterial canker disease (Citrusbacterialcankerdisease) namely caused by Xanthomonasaxonopodispv.Citri.This disease not only affects the quality of Citrus, greatly affects the yield of Citrus especially, becomes a global problem in Citrus plantation.
Along with society progress, the expansion gradually of the market demand, and competitive pressure be continuously increased, Citrus Industry is able to fast development.But owing to the cultivation of some susceptible variety is lacked effective management, the seriousness of citrus disease is incremented by gradually and also becomes a kind of inevitable trend therewith, how to take reasonable effective measures to be managed and preventing and treating in time, to advancing Citrus to plant this mainstay industry, there is very great and profound significance.Preventing and treating citrus bacterial canker disease mostly adopted chemical method to be main in the past, in recent years, Biological control receives everybody concern because of the advantage of its each side, and the scholar of research Biological control gets more and more, and utilizes the method for Biological control to have very big potentiality to tackle citrus bacterial canker disease.
Summary of the invention
It is an object of the invention to provide a kind of dinitrogen and support vibrio (Vibriodiazotrophicus) T24 bacterial strain, for the microbial resources that the Biological control offer of citrus processing is new.
Dinitrogen of the present invention supports vibrio T24 (Vibriodiazotrophicus) T24 bacterial strain, it is deposited in China typical culture collection center on April 10th, 2015, depositary institution address: Wuhan, China Wuhan University, deposit number is CCTCCNO:M2015221.
The characteristic of bacterial strain T24 of the present invention:
1, the characteristic of aspect is cultivated
The bacterial strain T24 of the present invention is in 30 DEG C of opaque colonies that are creamy white on LB flat board, and matt, edge is irregular, and bacterium colony looks relatively dry smooth, and front does not have projection, sees Fig. 1.
The characteristic of 2, morphology aspect
Thalline is Gram-negative, amphitrichous (Fig. 2), and without pod membrane, without spore, thalline is elongated shaft-like.
3, physiological and biochemical property
Starch Hydrolysis: negative；Cellulose decomposition: negative；C.I. 13020. (M.R.): positive；Acetyl methyl carbinol (V.P.): positive；Catalase: negative；Gelatin liquefaction: positive；Produce hydrogen sulfide: negative；Nitrate reduction: positive；Indole: negative.
The qualification of bacterial strain T24 of the present invention:
1, the Biolog of bacterial strain T24 of the present invention identifies
What adopt is the Biolog microbial identification system of Rapid identification microorganism, and concrete steps are with reference to the Guide Book of the MicroLogTMSystem of Biolog company.
1), flat board selects: select BiologGN2 plate for bacterial strain T24
Sample preparation: will be inoculated in Biolog inoculation liquid (GN/GP-IF) by cultured bacterial strain T24 on solid medium, controls suitable concentration well, and making inoculation turbidity is 52%T；
2), application of sample: take the bacterium solution (150ul/ hole) of certain volume, in the parallel addition each hole of BiologGN2 plate (totally 96 hole)；
3), cultivating and reading: be positioned in 30 DEG C of calorstats constant temperature and cultivate 4-6h, can carry out first time reading, 16-24h can carry out second time reading, and for the result read at 4-6 hour, SIM value is required >=0.75 it is only acceptable result.When reading result after 16-24 hour cultivates, SIM value is required >=0.5 it is only acceptable result.The qualification result of this bacterial strain T24 such as table 1 below:
The Biolog qualification result of table 1 bacterial strain T24
Species Probability Similarity The distance of positions Type
=> 1) the foster vibrio of dinitrogen 84 0.548 5.39 GN-NENT OXI+
2) vibrio harveyi 13 0.077 6.04 GN-NENT OXI+
3) Vibrio cincinnatiensis 3 0.015 6.57 GN-NENT OXI+
4) Aeromonas group 10 0 0.000 7.75 GN-NENT OXI+
5) Vibrio metschnikovii 0 0.000 7.84 GN-NENT OXI-
6) Vibrio furnissii 0 0.000 8.09 GN-NENT OXI+
7) river orphan bacterium 0 0.000 8.68 GN-NENT OXI+
8) Aeromonas media group 5B 0 0.000 8.70 GN-NENT OXI+
9) Aeromonas caviae group 4 0 0.000 9.00 GN-NENT OXI+
10) Listonella anguillarum 0 0.000 9.14 GN-NENT OXI+
Bacterial genomes DNA, the 16SrDNA fragment extracting bacterial strain of the present invention adopts universal primer 27F/1492R to carry out pcr amplification, and amplified fragments is by the order-checking of Bo Shang biotechnology (Shanghai) Co., Ltd., and the sequence recorded carries out tetraploid rice on NCBI.Result shows that the foster vibrio homology of its 16SrDNA sequence and dinitrogen is the highest, reaches more than 99%.Detecting through Biolog, this bacterial strain T24 and Vibriodiazotrophicus similarity is 0.548, much larger than other bacterial strain similarity, meets qualification condition.By morphologic observation and ITS sequence Molecular Identification, it is determined that bacterial strain of the present invention is the novel species that dinitrogen supports vibrio (Vibriodiazotrophicus), and called after dinitrogen supports vibrio (Vibriodiazotrophicus) T24 bacterial strain.
Bacterial strain T24 of the present invention is inoculated fermentation culture in weight access LB fluid medium with 4-5% and can prepare fermentation liquid, fermentation culture conditions is: temperature 28-37 DEG C, pH6.5-7.5, time 24-36h and rotating speed 180-200r/min, and best fermentation culture conditions is: temperature 30 DEG C, pH7.0, time 24h and rotating speed 180r/min.This fermentation liquid is inhibited to citrus processing, utilizes local scab method to record it, without fermented liquid, citrus processing extracorporeal inhibiting rate is reached more than 90%.
Therefore, bacterial strain T24 of the present invention can effectively suppress citrus processing, for the microbial resources that the Biological control offer of citrus processing is new；And by Physiology and biochemistry detection, Biolog microbial identification system and this bacterium 16SrDNA sequence analysis, the present invention all shows that this bacterium is the member that dinitrogen supports in vibrio, accurately the T24 of the present invention can be categorized as dinitrogen and support vibrio, thus establish a kind of quickly (the utilizing Biolog microbial identification system, 4-6h or 16-24h can obtain qualification result) of taxonomic identification microorganism, reliably, approach accurately.
Accompanying drawing explanation
Fig. 1 is single bacterium colony figure of bacterial strain T24 of the present invention.
Fig. 2 is the flagellum figure of bacterial strain T24 of the present invention.
Fig. 3 is the antibacterial figure of bacterial strain T24 of the present invention.
Fig. 4 is the fermentation liquid antagonistic effect figure to citrus processing of bacterial strain T24 of the present invention.
Wherein, A: be sprayed with the blade of bacterial strain fermentation liquor of the present invention；B: the blade (comparison blade) that clear water processes.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in detail further.
Below in conjunction with specific embodiment, such scheme is described further.These embodiments are an illustration for the present invention and are not limited to restriction the scope of the present invention.The implementation condition adopted in embodiment can do further adjustment according to the condition of producer, and not marked implementation condition is generally the condition in normal experiment.
The preparation of embodiment 1 bacterial strain T24 of the present invention
Gather soil sample from Xiangxi Autonomous Prefecture Longshan County, every kind of soil sample takes 10g, is added separately in the triangular flask equipped with 90ml sterilized water (having bead), shakes 1h, crosses elimination precipitation, bacteria suspension is diluted to 10 successively-2、10-3、 10-4、10-5、10-6.Antibacterial adopts mode of averaging, fungus and actinomycetes employing to smear method and be easily separated.At 28 DEG C, 48h cultivated by LB fluid medium, chooses the bacterium colony that form, size, color and luster are different, is purified, and slant medium preserves.
Pour in sterilizing culture dish after citrus ulcer bacteria is made the LB solid medium mixing of bacterium solution and thawing, the gel flat of solidification punches, it is respectively prepared certain density bacteria suspension by separating the bacterial strain obtained, take in the circular hole that 100ul bacterium solution is added to corresponding flat board with liquid-transfering gun, if 3 parallel.The culture dish that labelling is good is placed in the calorstat of 28 DEG C, observes the formational situation of an inhibition zone every 24h.Select have the bacterial strain Odontothrips loti of antagonistic effect to carry out multiple sieve.
The embodiment 2 bacterial strain of the present invention T24 indoor inhibitory action to citrus processing
Pour in sterilizing culture dish after citrus ulcer bacteria is made the LB solid medium mixing of bacterium solution and thawing, the gel flat of solidification punches, it is respectively prepared certain density bacteria suspension by separating the T24 bacterial strain obtained, take in the circular hole that 100ul bacterium solution is added to corresponding flat board with liquid-transfering gun, if 3 parallel.The culture dish that labelling is good is placed in the calorstat of 30 DEG C, observes the formational situation of an inhibition zone every 24h.The meansigma methods of the antibacterial circle diameter that result is formed is at about 45mm, and inhibition zone edge slightly swells, and sees Fig. 3.
Embodiment 3 bacterial strain T24 of the present invention prevents and treats citrus processing on Citrus live body
Picking bacterial strain T24 of the present invention is inoculated in 25mlLB fluid medium, 37 DEG C, and 180r/min cultivates 24h, obtains fermentation liquid.Choose the similar mandarin tree of upgrowth situation 3, each tree is chosen the fully deployed young leaves four that growth promoter degree is similar, with sterilized Inoculating needle, blade upper epidermis is punctured (every leaf 20 hole), first by nebulization, citrus processing is sprayed on blade upper surface and lower surface, after 1d, the fermentation liquid of prepared bacterial strain T24 of the present invention is sprayed on 3 Citrus young leaves in each process group that citrus processing processed as process, remaining 1 leaf is comparison, comparison blade does not spray the fermentation liquid of bacterial strain of the present invention and sprays clear water, spray this bacterial strain T24 fermentation liquid and after one month, observe incidence.Each tree is a process group, and result is shown in table 2 below, in conjunction with referring to Fig. 4.
Result shows, compares (CK) and is respectively formed citrus bacterial canker disease typical case's scab, and the scab suppression ratio spraying the blade of bacterial strain T24 fermentation liquid of the present invention reaches more than 90% in 3 process groups.
The table 2 bacterial strain T24 fermentation liquid of the present invention inhibitory action to citrus bacterial canker disease

Claims (8)

1. the dinitrogen that canker resistant is sick supports vibrio (Vibriodiazotrophicus) T24 bacterial strain, is deposited in China typical culture collection center on April 10th, 2015, and preserving number is CCTCCNO:M2015221.
2. the bacterial strain as claimed in claim 1 application in suppressing citrus processing.
3. the bacterial strain as claimed in claim 1 application in preventing and treating citrus processing.
4. the method preparing canker resistant pathogenic bacteria fermentation liquid, it is characterised in that the method is that with 4-5% inoculum concentration, the bacterial strain described in claim 1 is accessed fermentation culture in LB fluid medium.
5. method as claimed in claim 4, it is characterised in that described fermentation culture conditions is: temperature 28-37 DEG C, pH6.5-7.5, time 24-36h and rotating speed 180-200r/min.
6. method as claimed in claim 5, it is characterised in that described fermentation culture conditions is: temperature 30 DEG C, pH7.0, time 24h and rotating speed 180r/min.
7. the fermentation liquid that prepared by method as according to any one of claim 4-6 application in suppressing citrus processing.
8. the fermentation liquid that prepared by method as according to any one of claim 4-6 application in preventing and treating citrus processing.
CN201510410134.5A 2015-07-14 2015-07-14 The dinitrogen of a kind of canker resistant pathogenic bacteria supports cholerae strain Expired - Fee Related CN104962497B (en)

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