CN110157638A - A kind of gram lining institute series bacillus YY-1 and its application - Google Patents

A kind of gram lining institute series bacillus YY-1 and its application Download PDF

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CN110157638A
CN110157638A CN201910380414.4A CN201910380414A CN110157638A CN 110157638 A CN110157638 A CN 110157638A CN 201910380414 A CN201910380414 A CN 201910380414A CN 110157638 A CN110157638 A CN 110157638A
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series bacillus
lining institute
rot
bacterial strain
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CN110157638B (en
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翟立峰
王殿东
张美鑫
赵行
朱菲
伍维兰
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Yangtze Normal University
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    • A—HUMAN NECESSITIES
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    • 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
    • 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
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Abstract

The invention discloses a kind of gram lining institute's series bacillus and its application, bacterial strain for a gram lining institute series bacillus (Paenibacillus kribbensis) YY-1, be preserved in China typical culture collection center, depositary institution address: the Chinese Wuhan Wuhan University, the deposit date is on April 8th, 2019, deposit number was CCTCC NO.M2019242.The bacterial strain has significant inhibiting effect to the disease fungus black rot of pear bacterium of trunks of fruit trees, can effectively preventing black rot of pear, and the bacterial strain is harmless to ecological environment, will not cause the drug resistance of cause of disease, and condition of culture is simple, is easy to save；Biocontrol agent preparation method provided by the invention is simple, easy to industrialized production, with good development and application prospects.

Description

A kind of gram lining institute series bacillus YY-1 and its application
Technical field
The invention belongs to technical field of biological control, in particular to one kind gram lining institute series bacillus YY-1 and its answer With.
Background technique
Black rot of pear, also known as scurf, it is widely distributed in each pears producing region in China, it is one of important disease of China pear tree. Disease main harm pear tree limb, fruit and the blade, limb is aggrieved to promote branch early ageing, and blade is aggrieved to cause blade early It falls, fruit is aggrieved to cause a large amount of decayed fruits, seriously affect yield and quality.By black rot of pear bacterium (Botryosphaeria dothidea) caused by ring spot be that pears producing region occurs and one of the serious disease that causes harm throughout the year, especially with rotten caused by storage phase Fruit loss is most heavy, and while infecting pear tree limb is difficult to clean off, and provides germ source to infect again.China is every year because of pears wheel Line disease causes the operatic circle underproduction about 25%, and decayed fruit rate is more than 80% when serious.The germ not only infects pear tree, apple, grape, peach, It is also harmful on the fruit trees such as Kiwi berry, blueberry.
Black rot of pear bacterium be Ascomycota grape seat chamber Cordycepps (Botryosphaeriaceae) Botryosphaeria (Botryosphaeria) type sepecies, can be saprophytic or parasitize in plant, or within raw fungal morphology exist.The bacterium is alive Boundary various regions are widely distributed, can infect a variety of fruit trees and forest, cause citrus fruit rot, kiwi fruit maize ear rot, ring rot of apple, Black rot of pear, pears dry rot, Kiwi berry branch-rot, grape limb canker, blueberry branch-rot, peach bleeding disease, cherry canker, The multiple diseases such as Walnut Canker, Dothiorella Gregaria Sacc, dothiorella gregaria and Canker.Grape seat chamber bacterium mainly passes through bud, hole skin, stomata Host's vascular tissue is infected with wound etc., initial stage causes host's limb ulcer or young sprout withered, and later period scab causes in flakes Whole strain is dead, causes destructive consequence to ecological forest and economic fruit production.
Prevention and treatment for black rot of pear, relies primarily on cultivation management measure at present and chemical pesticide is prevented and treated.But due to Black rot of pear has the characteristics that longer incubation period, longer disease cycle and multiple spot endanger more, and a life is needed in prevention and treatment Long period repeatedly handles, multiple applications chemical pesticide, could preferably control its harm.There are control cost height, time-consuming, dirty The problems such as contaminating environment.Therefore, seek new economical and efficient, environmentally protective Prevention Technique and approach and have become current planting fruit trees Industry need to be solved the problems, such as urgently.
Bacillus genus (Paenibacillus) bacterium be a kind of amphimicrobian, endospore formed bacterial species, should Class bacterium initially belong to bacillus (Bacillus), it is then re-classified as one and individually belongs to.Bacillus genus Bacterial strain it is widely distributed, have detection from a variety of environment such as soil, water, rhizosphere, vegetables, feed and insect larvae and material. Paenibacilli growth is fast, nutrition is simple, is capable of forming the gemma with stronger anti-adversity ability, and living bacteria count compared with Non- bacillus is high, performance is stablized, therefore is widely used in product development.This kind of bacterium can generate various cells Exoenzyme, such as polysaccharide degrading enzyme and protease, the various synthetic reactions of catalysis biological field of fuel production, also can produce antibacterial Metabolite, inhibits the growth of pathogen, and induces plant generation system resistance.In addition, some series bacillus are as plant Rhizosphere growth-promoting endophytic bacteria (plant growth-promoting rhizobacteria, PGPR), can competitively be colonized and plant Object root system is not only used as bio-feritlizer, but also can play inhibiting effect to root system of plant pathogenetic bacteria, fungi and nematode.In addition, gram in Cloth institute series bacillus (Paenibacillus kribbensis) also have the characteristics that antagonistic phytopathogen and potassium decomposing, phosphorus decomposing With a variety of effects such as fixed nitrogen, crop yield can be improved in agricultural production, improve quality of agricultural product.Therefore gram lining institute class bud Spore bacillus is agriculturally having a good application prospect.
Currently, using gram lining institute series bacillus controlling plant diseases at home and abroad it has been reported that how bright equal from Thailand Gram lining institute series bacillus 4-531 being separated in mountain soil earth to cigarette brown spot pathogen (Alternaria alternate), it is yellow Cucurbit wilt bacterium (Fusarium oxysporum) and rhizoctonia rot of cotton bacterium (Rhizoctonia solani) show it is stronger Bacteriostatic activity；The phosphoric acid ester substance that gram lining institute series bacillus POC115 is generated to hungmao mentagrophyte (Trichophyton rubrum) there is antibacterial activity (identification of 1 plant of Antagonistic Bacteria Inhibiting Against Plant Pathogenic Fungi, Journal of Northwest Sci Tech University of Agriculture and Forestry, 2007)； The efficient phosphate-solubilizing potassium decomposing gram lining institute series bacillus strain CX-7 that Wang Wen etc. is screened from soil to verticillium dahliae, Cotton-wilt fusarium etc. has bacteriostatic activity (screening of antagonism gram lining institute series bacillus CX-7 bacterial strain and point of antibacterial protein From Agricultural University Of Hebei, 2012)；Gram lining institute series bacillus T-9 separated from soil can seriously inhibit botrytis cinerea (Botrytis cinerea), anthrax-bacilus (Colletotricum acutatum), Fusarium oxysporum (Fusarium oxysporum), Pyricularia oryzae (Magnaporthe oryzae), phytophthora blight of pepper (Phytophthora capsici), stand it is withered Rhizoctonia (Rhizoctonia solani) and white rot pyrenomycetes (Sclerotium cepivorum) bacterium colony growth, and can prevent Harm caused by these diseases are controlled on host plant；Gram lining institute series bacillus PS04 that Liao Meide etc. is separated from soil To Rhizoctonia solani Kuhn (Rhizoctonia solani) there is stronger bacteriostatic activity, which, which mainly passes through, inhibits pathogen Growth and induction host plant generate resistance play the role of diseases prevention (antifungal activity of PS04 bacterial strain, Changjiang University's journal, 2008).It can be seen that gram lining institute's series bacillus is because strain difference controlling plant diseases are also different, without foresight with And corresponding enlightenment.And also about gram lining institute's series bacillus not there is the related of prevention and treatment fruit tree ring spot to report at present Road.
Summary of the invention.
In view of the defects existing in the prior art, the purpose of the present invention is to provide one kind gram lining institute series bacillus YY- 1, which can prevent and treat trunks of fruit trees disease fungus black rot of pear bacterium, inhibit growth, breeding and the field planting of black rot of pear bacterium, rich Rich biocontrol microorganisms libraries, provide more choices for the genetic modification of plant.
To achieve the above object, the present invention adopts the following technical scheme: the present invention provides a kind of gram lining institute class gemma Bacillus strain, the bacterial strain for a gram lining institute series bacillus bacterial strain (Paenibacillus kribbensis) YY-1, it is preserved in China typical culture collection center, depositary institution address: the Chinese Wuhan Wuhan University, the deposit date is April 8 in 2019 Day, deposit number is CCTCC NO.M2019242.
The present invention also provides above-mentioned gram of lining institute series bacillus YY-1 answering in prevention and treatment trunks of fruit trees disease fungus With.
Further, the disease fungus is black rot of pear bacterium.
A kind of biocontrol agent, effective component are above-mentioned gram of lining institute series bacillus YY-1.
Further, described gram of lining institute series bacillus YY-1 is the culture bacteria suspension, fermentation liquid or fermentation point of the bacterial strain From supernatant.
Further, the viable count of the fermentation liquid and gram lining institute series bacillus YY-1 in culture bacteria suspension is not less than 1 ×105cfu/mL。
Further, the separation of fermentative broth supernatant, which is adopted, is prepared by the following steps: by gram lining institute series bacillus YY-1 It is resuspended to obtain bacteria suspension with sterile PD fluid nutrient medium after activation, then the bacteria suspension is inoculated in PD liquid with 5% inoculum concentration In culture medium, shaken cultivation 48h~72h at 120~200r/min, 25~30 DEG C, then by obtained fermentation liquid in 4 DEG C 6000 ~ 8000rpm centrifuging and taking supernatant is finally filtered supernatant up on the separation of fermentative broth using 0.22 nm sterilised membrane filter Clear liquid.
The present invention also provides above-mentioned biocontrol agents in prevention and treatment ring rot of apple, black rot of pear, pears dry rot, macaque peach branch Application in blight, grape limb canker or blueberry branch-rot.
Compared with prior art, the invention has the following beneficial effects:
1, present invention screening on the cockscomb blade of health obtains a gram lining institute series bacillus YY-1, which passes through generation Antibacterial substance come inhibit black rot of pear bacterium (Botryosphaeria dothidea) growth and infect, therefore gram lining institute class Bacillus YY-1 can not only directly inhibit the growth and breeding of pathogen, and can with field planting of the Antagonistic Fungus on host, To inhibit the occurrence degree of plant disease.
2, after the present invention is handled pear tree branch using gram lining institute series bacillus YY-1 fermentation liquid, it is inoculated with black rot of pear Disease incidence is decreased obviously after bacterium, and lesion diameter significantly reduces, it was demonstrated that a gram lining institute series bacillus YY-1 can efficiently inhibit pears Target spot pathogen bacterial strain has preferable biological control effect, has good effect, and the bacterial strain pair to prevention and treatment black rot of pear evil Ecological environment is harmless, will not cause the drug resistance of cause of disease, and condition of culture is simple, is easy to save.
3, biocontrol agent provided by the invention has preparation simple, and at low cost, easy to industrialized production, control efficiency is good It is good, and not will cause environmental pollution, pesticide can be replaced to prevent and treat black rot of pear, with good development and application prospects.
Detailed description of the invention
Fig. 1 is a gram colonial morphology figure of the lining institute series bacillus YY-1 in PDA culture medium；
Fig. 2 is a gram colonial morphology figure of the lining institute series bacillus YY-1 on LB culture medium；
Fig. 3 is a gram electron-microscope scanning figure of lining institute series bacillus YY-1；
Fig. 4 for gram lining institute series bacillus YY-1 16S rDNA sequential evolution analysis figure；
Fig. 5 is a gram influence of the lining institute series bacillus YY-1 fermentation liquid to black rot of pear bacterium JNT1111 hypha form；
Figure A is control group；Figure B is experimental group；
Fig. 6 grows black rot of pear bacterium mycelia for gram various concentration separation of fermentative broth supernatant of lining institute series bacillus It influences；
Influence of the Fig. 7 for inoculation gram lining institute series bacillus YY-1 bacteria suspension to black rot of pear bacteria pathogenic.
Specific embodiment
Invention is further described in detail with attached drawing combined with specific embodiments below, but the scope of the present invention not office It is limited to the content.Raw material described in embodiment is ordinary commercial products unless otherwise specified.Experiment as described in the examples Method is without special instruction, i.e., routinely experimental methods of molecular biology operates.
The separation screening of 1 bacterial strain gram lining institute series bacillus YY-1 of embodiment and identification
Separation method: taking the cockscomb blade of collection health from Chongqing City Fuling District, and the pear tree shoot tissue cleaning of health is taken to disappear Poison cuts the tissue that diameter is 5 mm, successively 45 s, 75% ethanol postincubation, 1 min is handled with sodium hypochlorite, at sterile water 1 min, sterile 1 min of water process are managed, is placed in after aseptic filter paper dries and is inoculated in 28 DEG C of PDA solid medium cultures.The plate 28 DEG C stationary culture 2-3 days.It is picked out according to the form, color, size of the bacterial clump screened on plate different thin Bacterium is simultaneously transferred to PD fluid nutrient medium.
Screening technique: antagonistic bacterium is screened using opposite culture method, disinfection inoculation ring dips a small amount of bacterium solution, in PDA plate Center scribing line, and the black rot of pear bacterium JNT1111 mycelia block for being 5 mm in two sides inoculation diameter, are placed in 28 DEG C of 5 d of culture, observe Diameter between bacterium colony, every group repeats three times, only will be set as compareing in the plate of two sides inoculation JNT1111 mycelia block.To plate On the bacterium with bacteriostatic activity that screens be transferred to PD fluid nutrient medium, purified, cultivated to get to the suppression of purifying Active bacterial strain processed is named as YY-1.
Identification method: bacterial strain YY-1 is smooth, regular edges in PDA culture medium, intermediate in spherical milky outstanding Bacterium colony (Fig. 1) is then smooth, edge sawtooth shape, flat white colony (Fig. 2) on LB solid medium.Bacterial strain YY-1's Thallus is (Fig. 3) in the shape of a rod under a scanning electron microscope, can be changed bacterium for Gram's staining.
The DNA that bacterial strain YY-1 of the present invention is extracted with the bacteria total DNA of raw work biology Co., Ltd, then uses bacterium 16s RDNA universal primer 16sF/R carries out PCR amplification, obtains amplified fragments, amplified fragments sequencing, particular sequence such as SEQ ID Shown in NO.1.Sequence is compared through BLAST in GenBank, with a gram lining institute series bacillus (Paenibacillus kribbensis) affiliation is nearest.By to the 16s rDNA sequence construct in other representatives of YY-1 and bacillus genus Phyletic evolution development tree, the results showed that YY-1 andPaenibacillus kribbensisRepresentative strain is gathered in same branch (figure 4) and be novel bacterial, bacterial strain of the present invention for a gram lining institute series bacillus (Paenibacillus kribbensis), it is named as Gram lining institute series bacillus YY-1.
The application of 2 bacterial strain gram lining institute series bacillus YY-1 of embodiment
The preparation of separation of fermentative broth supernatant: with sterile PD fluid nutrient medium after gram lining institute series bacillus YY-1 is activated Resuspension obtains bacteria suspension, then the bacteria suspension is inoculated in PD fluid nutrient medium with 5% inoculum concentration, in 180r/min, 28 DEG C Supernatant is finally used 0.22 nm then by obtained fermentation liquid in 4 DEG C of 6000rpm centrifuging and taking supernatants by lower shaken cultivation 72h Sterilised membrane filter filters up to the separation of fermentative broth supernatant, spare.
1, influence of the YY-1 to spore germination.
With aseptic water washing black rot of pear bacteria strain JNT1111 cirrus, spore liquid is adjusted to suitable in 40 power microscopes Concentration.It takes isometric separation of fermentative broth supernatant and spore liquid to mix, draws 20 uL mixed liquors and glass slide center, be placed in nothing In bacterium plate, 28 DEG C of 2.5 h of sprouting, as experimental group；Control group replaces separation of fermentative broth supernatant, other steps with sterile water It is identical.It observes and records, calculates germination rate, as a result as shown in Figure 5.
It is obtained from microscopically observation: compared with the control group, bacterial strain can be caused after fermented liquid separation supernatant processing The mycelia of JNT1111 grows deformity, mycelia shortened internodes, and has the phenomenon that expanding, and the separation of fermentative broth supernatant of 50% concentration Liquid is 100% to the inhibiting rate of bacterial strain JNT1111 spore germination.
2, the antibacterial control effect testing of bacterial strain YY-1
PDA culture medium fusing is cooled to 40 ~ 50 DEG C, by separation of fermentative broth supernatant respectively with 0%, 2%, 3%, 4%, 5% ratio The PDA plate that the supernatant of separation of fermentative broth containing various concentration is prepared in culture medium, the pears wheel for being then 5 mm by diameter is added Line germ bacterial strain JNT1111 inoculated by hypha block observes and records hypha growth condition in above-mentioned PDA plate center, 28 DEG C of cultures.Knot Fruit is as shown in Figure 6.
It can be seen from the figure that in the medium be added various concentration fermentation liquid after bacterial strain JNT1111 growth by Different degrees of inhibition, and the higher bacterial strain JNT1111 growth inhibition of concentration of separation of fermentative broth supernatant is stronger, and bacterial strain The growth of JNT1111 bacterium colony is irregular.And the strain growth of control group is rapid, bacterium colony growth is vigorous, gas natural disposition is strong.The result shows that bacterium Antibacterial substance concentration in the separation of fermentative broth supernatant of strain YY-1 is higher, and Antibacterial Activity is stronger.
3, the living body control effect testing of bacterial strain YY-1
Intercepting pear tree branch fresh, healthy, that size and form is almost the same is material, and clear water is cleaned, and 75% alcohol disinfecting dries, Paraffin sealing moisturizing punches its surface with the punch of disinfection, and experimental group is outstanding in the bacterium that wound is inoculated with 10 uL bacterial strain YY-1 Liquid (viable count about 1 × 105Cfu/mL after) drying, the black rot of pear bacteria strain JNT1111 mycelia block that inoculation diameter is 5 mm, bacterium Silk faces wound, preservative film moisturizing；Control group replaces bacterial strain YY-1 bacteria suspension, other same experimental groups of step, experiment with sterile water 3 repetitions are set, and 28 DEG C of moisturizing culture 3d record wound incidence, as a result as shown in Figure 7.
From figure 7 it can be seen that after culture 3d, the black rot of pear when lesion diameter of experimental group (YY-1+JNT1111) and inoculation The diameter change of bacteria strain JNT1111 mycelia block is unobvious, illustrates that experimental group is not fallen ill；And control group (PD+JNT1111) Lesion diameter variation is more obvious, is about inoculated with 7 times of fungus block diameter, shows that bacterial strain YY-1 can be with Antagonistic Fungus gram lining institute class Field planting of the bacillus on host.
To sum up, of the invention gram lining institute series bacillus (Paenibacillus kribbensis) YY-1 can efficiently press down Black rot of pear bacteria strain processed has preferable biological control effect, and not will cause environmental pollution, pesticide can be replaced to pears wheel Line disease is prevented and treated.
Finally, it is stated that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although referring to compared with Good embodiment describes the invention in detail, those skilled in the art should understand that, it can be to skill of the invention Art scheme is modified or replaced equivalently, and without departing from the objective and range of technical solution of the present invention, should all be covered at this In the scope of the claims of invention.
SEQUENCE LISTING
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caccaagggt atcgaaaccc ctaacaccta gcattcatcg tttacggcgt ggactaccag 720
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agtcgccttc gccactggtg ttcctccaca tctctacgca tttcaccgct acacgtggaa 840
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ttctcccatg caggaaaagg atgtatcggg tattagctac cgtttccggt agttatccct 1380
gtcttgtggg caggttgcct acgtgttact cacccgtccg ccgc 1424

Claims (8)

1. one kind gram lining institute series bacillus (Paenibacillus kribbensis) YY-1, during the bacterial strain is preserved in State's Type Tissue Collection, deposit number are CCTCC NO. M2019242.
2. application of the as described in claim 1 gram of lining institute series bacillus YY-1 in prevention and treatment trunks of fruit trees disease fungus.
3. applying according to claim 2, which is characterized in that the disease fungus is black rot of pear bacterium.
4. a kind of biocontrol agent, it is characterised in that: effective component is described in claim 1 grams of lining institute series bacillus YY-1.
5. biocontrol agent according to claim 4, which is characterized in that described gram of lining institute series bacillus YY-1 is the bacterial strain Culture bacteria suspension, fermentation liquid or fermentation separation supernatant.
6. biocontrol agent according to claim 4, which is characterized in that gram lining institute class in the fermentation liquid and culture bacteria suspension The viable count of bacillus YY-1 is not less than 1 × 105cfu/mL。
7. biocontrol agent according to claim 4, which is characterized in that the separation of fermentative broth supernatant uses following steps system : it will be resuspended to obtain bacteria suspension with sterile PD fluid nutrient medium after gram lining institute series bacillus YY-1 activation, then by the bacterium Suspension is inoculated in PD fluid nutrient medium with 5% inoculum concentration, at 120~200r/min, 25~30 DEG C shaken cultivation 48h~ 72h, it is finally that supernatant is sterile using 0.22 nm then by obtained fermentation liquid in 4 DEG C of 6000 ~ 8000rpm centrifuging and taking supernatants Membrane filtration is up to the separation of fermentative broth supernatant.
8. biocontrol agent as claimed in claim 4 prevention and treatment ring rot of apple, black rot of pear, pears dry rot, Kiwi berry branch-rot, Application in grape limb canker or blueberry branch-rot.
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