CN103614312A - Pseudomonas chloroaphis with effects of effective zea mays sheath blight control and zea mays growth promotion - Google Patents
Pseudomonas chloroaphis with effects of effective zea mays sheath blight control and zea mays growth promotion Download PDFInfo
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- CN103614312A CN103614312A CN201310432792.5A CN201310432792A CN103614312A CN 103614312 A CN103614312 A CN 103614312A CN 201310432792 A CN201310432792 A CN 201310432792A CN 103614312 A CN103614312 A CN 103614312A
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Abstract
The invention relates to a strain of Pseudomonas chloroaphis with effects of effective zea mays sheath blight control and zea mays growth promotion, and belongs to the technical field of microorganisms. The strain is Pseudomonas chloroaphis PSJ1, is isolated from wheat rhizosphere, and has the preservation number of CGMCC NO.7932. The Pseudomonas chloroaphis PSJ1 has effects of zea mays growth promotion, Rhizoctonia solani growth inhibition, and sheath blight occurrence reduction.
Description
Technical field
The invention belongs to microorganism field, relate to a kind of Pseudomonas chlororaphis and application, particularly relate to a kind of effective inhibition dry thread Pyrenomycetes (Rhizoctonia solani) and also can promote the Pseudomonas chlororaphis (Pseudomonas chlororaphis) of corn growth.
Background technology
Corn (Zea mays) is China's main food, fodder crop and important industrial raw material, in Chinese national economy and agriculture production, occupies critical role, and Maize Production direct relation China's grain strategic security.The disease such as corn banded sclerotial blight, helminthosporium maydis and the mould leaf spot of curved spore occurs more and more generally in recent years, and harm increases the weight of day by day, has seriously restricted the development of Maize Production.
In the integrated control of agricultural pest, chemical prevention occupies critical role, but long-term unreasonable a large amount of uses of chemical pesticide contaminate environment is not only destroyed the eubiosis, people and animals' health is brought to disadvantageous effect simultaneously.Along with social progress and growth in the living standard, people are more and more higher to the requirement of environmental quality and green non-pollution food.
In soil, exist various microorganisms, comprising bacterium, fungi, unwrapping wire mattress, protozoon and algae etc.Wherein some microorganism has good restraining effect to phytopathogen, has important using value in control of plant disease.Many bacterial strains in Rhodopseudomonas, particularly fluorescent Pseudomonads distribute extensively, quantity is many, and nutritional needs is simple, breeding is fast, competition colonization power is strong, can produce Multiple Classes of Antibiotics and active substance, can induce certain plants produce systemic and receive much concern.
The present invention is separated to a strain Pseudomonas chlororaphis (Pseudomonas choloeaphis), called after PSJ1 from wheat rhizosphere.This bacterial strain can effectively suppress the growth of dry thread Pyrenomycetes, reduces the generation of corn banded sclerotial blight, and can promote milpa growth.
Summary of the invention
The object of the invention is to provide Pseudomonas chlororaphis (Pseudomonas chloroaphis) the PSJ1 bacterial strain of a strain antagonism corn sheath blight fungus, its biological control in corn banded sclerotial blight etc., promotes the aspects such as crop yield to have extraordinary effect.
The present invention adopts following technical scheme to realize: bacterial strain PSJ1 separation is from Taian Shandong wheatland.By indoor flat plate stand facing each other experiment and greenhouse preventive effect measure, filter out the antagonistic strain with good prophylaxis effect, called after PSJ1.By this bacterium 16SrRNA sequencing and phylogenetic analysis are accredited as to Pseudomonas chlororaphis (Pseudomonas choloeaphis).
The bacterial strain the present invention relates to has antagonistic action to multiple corn disease fungal pathogens.Indoor antagonistic experiment shows that this bacterial strain all has good inhibition to corn sheath blight fungus (Rhizoctonia solani), southern corn leaf blight (Bipolaris maydis), character studies on curvularia lunata (Curvularia lunata Boedijn) etc.
During sowing, by concentration, be 10
8the PSJ1 bacteria suspension of CFU/ml waters, and treats that corn grows to the 2-3 leaf phase, then with above-mentioned bacteria suspension, plant is carried out to blade spraying, measures the growth-promoting effect of PSJ1 to corn after 14d.With contrast, compare, with PSJ1, process and can significantly promote milpa growth, plant ground, underground fresh weight increase respectively 54% and 43%.
Greenhouse preventive effect experiment shows, the milpa of processing with biocontrol microorganisms PSJ1 is compared with contrast after the former bacterium of inoculation corn banded sclerotial blight, and scab expansion is suppressed, and reaches conspicuous level (p<0.05), and protection effect is obvious.
Preservation information
The preservation time: on July 16th, 2013
Depositary institution's title: the common micro-organisms center C GMCC of China Committee for Culture Collection of Microorganisms
Deposit number: CGMCC NO.7932
Depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Classification And Nomenclature: Pseudomonas chlororaphis (pseudomonas chloroaphis)
accompanying drawing explanation
The antagonistic action of Fig. 1 Pseudomonas chlororaphis PSJ1 to pathogenic fungi
A is corn sheath blight fungus, and B is fusarium graminearum, and C is southern corn leaf blight.Left side is for being moistened with the filter paper of PSJ1 bacterium liquid, and right side is clear water.
The amplified production of Fig. 2 bacterial strain PSJ116S rDNA gene
Fig. 3 uses NJ method according to the systematic evolution tree of 16S rDNA sequence construct
Plant incidence after 7 days after Fig. 4 corn leaf sheath inoculation sheath blight fungus
A is Nongda108, and B is Zheng Dan 958
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should understand these embodiment is only not used in and limits the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, carries out according to normal condition conventionally.
Embodiment 1: the separation of plant growth-promoting rhizobacteria and primary dcreening operation
The separation of plant growth-promoting rhizobacteria adopts the method for Fang Zhongda (1979).Get the soil sample 10g collecting, join in the triangular flask that fills 90mL sterilized water, triangular flask is put on shaking table, the 180r/min 20min that vibrates, makes soil supension.By the standing 5min of the soil supension preparing, get supernatant liquor, adopt 10 times of serial dilution methods, be diluted to successively 10
-2, 10
-3, 10
-4with 10
-5, each concentration is drawn 200 μ L on NA flat board, and evenly, each concentration is in triplicate in coating.Then the NA flat board coating is cultivated to 24h in 28 ℃ of constant incubators.
While there is bacterial colony on NA flat board, by geotrichum candidum (Geotrichum candidum) spore suspension (10
5individual spore/mL) evenly spray in growing on the NA flat board of bacterium colony.Spraying is wanted evenly, the about 200 μ L of every ware.The amount spraying is not too much, to prevent that forming current breaks up the bacterium colony of having grown.
Choose the bacterium colony that occurs obvious inhibition zone around, with transfering loop picking thalline, at the flat lining out of NA, flat board is put into 28 ℃ of incubators and cultivate 12h.After growing single bacterium colony, picking colony line again, in triplicate, obtains pure bacterium.With the bacterium colony that the screening of toothpick picking obtains, put into the test tube that fills 5mL liquid LB substratum, 28 ℃ of shaking culture 14-16h.Get 1mL bacterium liquid, join in the centrifuge tube of 2mL, and then add 15% glycerine to mix, in-80 ℃ of refrigerators, preserve.
Embodiment 2: the mensuration of plant growth-promoting rhizobacteria antimicrobial spectrum
Adopt face-off culture method (Sijam etc., 2005).The dull and stereotyped central authorities of the PDA that is 90mm at diameter connect corn sheath blight fungus Rhizoctonia solani, fusarium graminearum Gibberella zeae that diameter is 9mm and the bacterium cake of southern corn leaf blight Bipolaris maydis, then with sealed membrane, seal flat board, put into 28 ℃ of incubators and cultivate 2 days.The sterilizing filter paper that is 6mm by diameter dips ready Bacteria liquid (10
8cfu/mL), move on to apart from 15mm place, pathogenic bacteria edge, symmetrical placement, contrast only connects pathogenic bacteria bacterium cake, and sealing, finally puts into 28 ℃ of incubators and cultivates.Each is processed and repeats 3 times.After 3d, measure and connect bacterium place pathogenic bacteria colony diameter and contrast pathogenic bacteria colony diameter, calculate inhibiting rate.
Result shows, PSJ1 is to having extraordinary inhibition (Fig. 1) for examination pathogenic bacteria.PSJ1 is respectively 71.1%, 82.4% and 85.5% to the inhibiting rate of Rhizoctonia solani, Gibberella zeae, Bipolaris maydis.
Embodiment 3: the acquisition of bacterial strain 16S rRNA sequence
Pseudomonas chlororaphis (Pseudomonas choloaphis) genome of take is template, by its 16S rDNA of pcr amplification.Upstream and downstream primer used is respectively 16S-F:5 '-AGAGTTTGATYMTGGCTCAG-3 ', 16S-R:5 '-AGGGYTACCTTGTTACGACTT-3 '.PCR response procedures is 94 ℃ of 3min; 94 ℃ of 30s, 54 ℃ of 45s, 72 ℃ of 2min, 30 circulations; 72 ℃ are extended 10min; 4 ℃ of preservations.After reaction finishes, PCR product is placed in to 1.0% sepharose and carries out electrophoretic analysis.With reference to the U.S. PCR of AXYGEN company purifying, reclaim test kit and reclaim PCR product, in 16 ℃ of water-baths, be connected and spend the night with pMD18-T.Ligation system is: 10 * T4DNA Ligase buffer1 μ L, pMD18-T(50ng/ μ L) 0.3 μ L, target DNA 7.7 μ L, T4DNA Ligase(350U/ μ L) 1 μ L.
Utilize heat shock method will connect product transformed competence colibacillus cell.Concrete grammar is: 100 μ L competent escherichia coli cells are placed on ice and are melted, add 10 μ L to connect product, stir gently to mix, mixture is placed to 20min on ice, then 42 ℃ of water-bath heat shock 90S, place rapidly 5min on ice, then add 800 μ L LB liquid nutrient mediums (not containing microbiotic), mix, 37 ℃, the 220r/min 45min that vibrates.The centrifugal 5min of 6000rpm, takes out supernatant to remaining 200 μ L, and resuspended thalline, is evenly coated in thalline on the microbiotic flat board that contains penbritin, then dull and stereotyped forward is placed to 30min, is inverted for 37 ℃ and cultivates 16-20h, to growing single bacterium colony.
Alkaline lysis method of extracting plasmid DNA (Han Zhiyong etc., 2000).The plasmid of extraction is placed in to 1.0% sepharose and carries out electrophoresis, with empty pUC18 plasmid in contrast.If electrophoresis swimming rate is identical with pUC19, illustrates without external source fragment and insert; If the electrophoresis swimming speed than pUC19 is slow, illustrates and in pMD18-T, inserted external source fragment.Select the slow plasmid of swimming speed to carry out PCR evaluation.PCR program used: 94 ℃ of 3min; 94 ℃ of 30s, 54 ℃ of 45s, 72 ℃ of 2min, 30 circulations; 72 ℃ are extended 10min.After reaction finishes, PCR product is placed in to 1.0% sepharose and carries out electrophoretic analysis.The bacterial strain that contains recombinant plasmid of having identified, serves the order-checking of Hai Boshang company with bacterium liquid form.2 clones of each bacterium liquid sample.Sequencing result is compared to the 16S rDNA sequence of relevant genus kind in GenBank with BLAST software, utilize Mega software building evolutionary tree.
Electrophoresis detection shows, from PSJ1 genome, increases to the fragment of about 1.3kb (Fig. 2).Through measuring, PSJ1 bacterial strain 16SrDNA contains 1287bp, and concrete sequence is as shown in SEQ.NO.1.
According in the genealogical tree of 16SrDNA sequence construct, PSJ1 and pin pseudomonas cluster to together with, illustrate that PSJ1 belongs to Pseudomonas chlororaphis (Fig. 3).
The indoor growth-promoting test of embodiment 4:PSJ1 bacterial strain to corn
Choose Zheng Dan 958 corn seeds of the same size and carry out surface sterilization, the sterilized water Steeping and budding 10h for corn seed completely that will sterilize, changed one time water every 2-3 hour.Seed after vernalization carries out greenhouse pot culture experiment, and in the flowerpot (190mm * 160mm) of sterilizing soil is housed, every basin program request is 4, and each processes 6 basins, repeats for three times.
With sterilized water, adjusting PSJ1 bacteria concentration is 10
8cFU/ml, every basin pouring 40ml; Treat that corn grows to the 2-3 leaf phase, it is 10 that blade face evenly sprays bacteria concentration
8the PSJ1 bacteria suspension of CFU/ml, control group sprays equivalent clear water.Process that to measure plant height (native face is above to plant highest point), radical, root after 14d long, and the fresh weight of ground and underground part.The results are shown in Table 1.
The impact of table 1PSJ1 on corn growth proterties
As can be seen from Table 1, with PSJ1 bacteria suspension, process and can significantly promote the growth of milpa and the growth of root.Meanwhile, PSJ1 processes the increase promoted plant fresh weight, with contrast, compares on the ground and underground fresh weight increases respectively 54% and 43%.
The preventive effect effect of embodiment 5:PSJ1 bacterial strain to corn banded sclerotial blight
Choose corn seed of the same size and carry out surface sterilization, the sterilized water Steeping and budding 10h for corn seed completely that will sterilize, changed one time water every 2-3 hour.Seed after vernalization carries out greenhouse pot culture experiment, and in the flowerpot (190mm * 160mm) of sterilizing soil is housed, every basin program request is 4, and each processes 6 basins, repeats for three times.With sterilized water, adjusting PSJ1 bacteria concentration is 10
8cFU/ml, every basin 40ml waters.Treat that corn grew to for two one heart stages of leaf, carried out root irrigation with bacteria suspension, every dish 40ml(10
8cFU/ml) fill with every three days root once, continuous three times, with clear water, be treated to contrast.After filling with for the third time root, within one week, inoculate respectively strong virulence corn sheath blight fungus (YWK-100) in rhizosphere, suitably moisturizing, observes corn morbidity with scab spread scenarios and adds up.The results are shown in Figure 4 and table 2.
Table 2 rhizosphere inoculation YWK-100 plant scab extension length and plant sickness rate
As can be seen from the table, processing on plant scab extension length will be significantly lower than contrast, and PSJ1 processes Zheng Dan 958 and Nongda108 can significantly improve disease resistance of plant.
The above, only indivedual embodiment of the present invention, not the present invention is done to any pro forma restriction, any simple modification, equivalent variations and modification that every foundation technical spirit of the present invention is done above embodiment, all still belong in the scope of technical solution of the present invention.
Claims (2)
1. a strain Pseudomonas chlororaphis, is characterized in that called after Pseudomonas chlororaphis PSJ1, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, depositary institution's abbreviation: CGMCC; Preserving number: CGMCC NO.7932; Its 16SrRNA nucleotide sequence is as shown in SEQ.NO.1.
2. the application of strain Pseudomonas chlororaphis PSJ1 in control of maize banded sclerotial blight as claimed in claim 1, described pathogenic bacteria is Rhizoctonia solani.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2020006555A1 (en) * | 2018-06-29 | 2020-01-02 | AgBiome, Inc. | Compositions comprising bacteria and methods for controlling plant pests and improving plant health |
| WO2020092381A1 (en) * | 2018-10-30 | 2020-05-07 | AgBiome, Inc. | Bacterial compositions and methods for controlling plant pests and improving plant health |
| CN112812988A (en) * | 2020-12-28 | 2021-05-18 | 南京绿正生物科技有限公司 | Pseudomonas chlororaphis strain, biocontrol microbial inoculum and preparation method and application thereof |
| CN112899205A (en) * | 2021-03-31 | 2021-06-04 | 慕恩（广州）生物科技有限公司 | Pseudomonas chlororaphis MN225969 and application thereof |
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| CN1148791A (en) * | 1994-04-18 | 1997-04-30 | 瑞典农场主全国经济协会 | Composition and method for controlling plant diseases |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2020006555A1 (en) * | 2018-06-29 | 2020-01-02 | AgBiome, Inc. | Compositions comprising bacteria and methods for controlling plant pests and improving plant health |
| WO2020092381A1 (en) * | 2018-10-30 | 2020-05-07 | AgBiome, Inc. | Bacterial compositions and methods for controlling plant pests and improving plant health |
| CN113631040A (en) * | 2018-10-30 | 2021-11-09 | 农业生物群落股份有限公司 | Compositions and methods for controlling plant pests and improving plant health |
| CN113631040B (en) * | 2018-10-30 | 2023-07-07 | 农业生物群落股份有限公司 | Compositions and methods for controlling plant pests and improving plant health |
| CN112812988A (en) * | 2020-12-28 | 2021-05-18 | 南京绿正生物科技有限公司 | Pseudomonas chlororaphis strain, biocontrol microbial inoculum and preparation method and application thereof |
| CN112812988B (en) * | 2020-12-28 | 2022-05-06 | 南京绿正生物科技有限公司 | Pseudomonas chlororaphis strain, biocontrol microbial inoculum and preparation method and application thereof |
| CN112899205A (en) * | 2021-03-31 | 2021-06-04 | 慕恩（广州）生物科技有限公司 | Pseudomonas chlororaphis MN225969 and application thereof |
| CN112899205B (en) * | 2021-03-31 | 2022-05-31 | 慕恩（广州）生物科技有限公司 | Pseudomonas chlororaphis MN225969 and application thereof |
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