CN104988096A - Bio-control bacterium Kg2A capable of efficiently inhibiting fusarium and bacillus anthraci and applications thereof - Google Patents

Bio-control bacterium Kg2A capable of efficiently inhibiting fusarium and bacillus anthraci and applications thereof Download PDF

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CN104988096A
CN104988096A CN201510430348.9A CN201510430348A CN104988096A CN 104988096 A CN104988096 A CN 104988096A CN 201510430348 A CN201510430348 A CN 201510430348A CN 104988096 A CN104988096 A CN 104988096A
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kg2a
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CN104988096B (en
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郑肖兰
李锐
吴伟怀
习金根
贺春萍
易克贤
梁艳琼
郑金龙
秦周
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CATAS Environment and Plant Protection Institute
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Abstract

The invention belongs to the field of microbial pesticide, and specifically relates to a bio-control bacterium Kg2A and applications thereof on controlling pepper wilt and pepper anthracnose. The bio-control bacterium is Bacillus amyloliquefaciens Kg2A, and the preservation number is CGMCC No.10918. The invention also discloses the molecular biological characteristics, physiological-biochemical characteristics, and culture method of the provided bacterium strain, and proves that the provided bio-control bacterium Kg2A has an activity on inhibiting fusarium that may cause pepper wilt and bacillus anthraci that can cause pepper anthracnose.

Description

One plant height effect suppresses biocontrol microorganisms Kg2A and the application thereof of sickle-like bacteria and anthrax-bacilus
Technical field
The invention belongs to microbial pesticide field, be specifically related to a bacillus amyloliquefaciens Kg2A and in control pepper blight and the application of anthrax.
Background technology
Pepper is the main tropical agricultural-food of China, is the important farm crop of Hainan vast farmers extra earning, and a large pillar of Ye Shi Hainan Province Economic development, it has more and more important effect in the evolution of national economy.This is because on the one hand, its seed contains volatile oil, piperine, crude fat, crude protein etc., is the seasonings that people like; On the other hand, medically pepper can be used as peptic, adjusts thermit powder, diuretic(s) and tunica mucosa bronchiorum stimulant, having the effect in loose cold, stomach invigorating, temperature, maldigestion can be treated, having indigestion does not disappear, the various diseases such as stomach cold pain, trusted subordinate's crymodynia, cold-dampness gastric disorder causing nausea, large intestine cold sliding, empty swollen cold long-pending, bronchitis, cold dysentery, syphilis, flu, treating rheumatic ostealgia and lung plug coughing with a lot of sputum; In addition, pepper industry has become the mainstay of Hainan Island agro based economic development and the Main Economic source of peasant programme.On the other hand, in production, pepper is the fragrant pungent plant in a kind of important torrid zone, and originate in the tropical rain forest of the western Ghats of India, (district) is economized in current China Hainan, Guangdong, Guangxi, Yunnan, Fujian etc. all has plantation.Hainan Island is as the main pepper production area of China and pepper breeding key area, plant that green pepper area accounts for that the green pepper total area is planted by China more than 90%, the region of suitable planting pepper is limited, Hainan Province's plantation weather of pepper and land occupation condition richly endowed by nature, the quality of the pepper products comparatively periphery traditional mode of production country such as Vietnam is good.But, Hainan pepper generally every mu can produce peppercorn 200-300 jin every year, 500-600 jin is produced in well-regulated every mu, pepper garden per year, the per mu yield of small area high yield can reach 1200 jin, a traditional unit of weight in 1 year, unit area yield is not high on the whole, trace it to its cause probably I guess many-sided, but having not deniable is having a strong impact on of being brought by pepper disease.For a long time, the generation of the soil-borne diseases such as Hainan Island pepper kind simplification, and long-term continuous cropping is all conducive to repeatedly infecting of various pathogenic bacteria, particularly pepper seasonal febrile diseases is produced to pepper and is brought serious harm.
Biocontrol bacteria occupies very consequence in the control of Plant diseases, and its main advantage has: the complicated and diversified Biocontrol Mechanism of (1) biocontrol bacteria makes pathogenic bacteria not easily develop immunity to drugs.The antifungal mechanism of biocontrol bacteria has multiple, and normally two or more antimicrobial mechanism synergies plays the effect of controlling disease.In control of plant disease practice, simultaneously the method generally adopted uses viable bacteria body and its antagonistic metabolites, and it is short complementary mutually that this is conducive to various antimicrobial mechanism, thus the Biocontrol Potential of biocontrol bacteria is played to greatest extent.In addition, bacteriogenic Substance (as bacteriocin) can affect the multiple pathways metabolism of sensitive organism simultaneously, so, just easy unlike microbiotic to the possibility of bacteriocin generation resistance because pathogenic bacteria is undergone mutation.(2) vivo applications of biocontrol bacteria makes its preventive effect be because biocontrol bacteria is separated by farmland ecosystem mostly more lastingly than other microorganism, generally there is the ecological adaptation identical or close with pathogenic bacteria and the affinity to host plant, surely grow relatively easy, add its breeding rapidly, make application bacterium live body controlling disease become possibility.And for other microbe groups of biological and ecological methods to prevent plant disease, pests, and erosion, majority is the meta-bolites controlling disease utilizing them.By contrast, biocontrol bacteria can amount reproduction set up " live in factory " producing antibacterial substance preventive effect can be more lasting after surely growing.(3) metabolism is vigorous, the generation antagonistic substance cycle is short, and biocontrol bacteria reproduction speed is surprisingly fast compared with biocontrol fungi or actinomycetes, and under appropriate conditions, dozens of minutes with regard to fissiparity once, just produces tens million of offsprings in one day later.Under liquid culture condi, the peak period of producing antagonistic substance within 44 hours, can be reached.This is conducive to saving time, and shortens the production cycle, reduces production cost.(4) be conducive to maintaining the beneficial microorganism eubiosis, the antibacterial substance produced due to biocontrol bacteria is usually direct to work for corresponding pathogenic bacteria, high specificity, so can not have a negative impact to other beneficial microorganism of agroecosystem, contribute to maintaining the beneficial microorganism eubiosis.
From pepper, separation screening obtains biocontrol strain pepper disease blight and anthrax to antagonistic action, the control of pepper disease is had great importance, but correlation technique is still very weak at present, so far the antagonistic bacterium effectively preventing and treating pepper disease has no formal report, does not does not also prevent and treat the report of the biological pesticide of pepper disease.
Summary of the invention
Object of the present invention provides a strain biocontrol microorganisms Kg2A and the application in control pepper withers thereof for the blank of prior art.
Object of the present invention is achieved by following technical proposals.
The bacterium that the present invention adopts is bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and preserving number is CGMCC.No.10918.Address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
Production bacterial strain Kg2A of the present invention is separated the pepper blade from great Po town, Hainan Province, through the microscopic examination of bacterium colony and form, staining reaction, the test of customary physiological biochemical characteristic and 16s rDNA sequential analysis, this identification of strains is a new strains of bacillus amyloliquefaciens (Bacillus amyloliquefaciens), there is following characteristics: (1) bacterial strain is direct rod shape, normal with paired or catenation, tool nose circle or square end; Gemma ellipse, oval, column, circle, without pod membrane; Gram-positive; (2) by physiological and biochemical indexs such as indoles experiment, catalase experiment, oxydase, Citrate trianion growth experiments, and combine " the outstanding Bacteria Identification handbook of uncle ", qualification result Kg2A belongs to bacillus subtilis Pseudomonas, and result is consistent with Molecular Identification.
The minimum medium of biocontrol microorganisms Kg2A is LB substratum or YEP substratum.
The utilized carbon source of biocontrol microorganisms Kg2A has: sorbyl alcohol, rhamnosyl, vitamins C, Threonine, semi-lactosi, proline(Pro), N.F,USP MANNITOL, maltose, Gelucystine, Zulkovsky starch, glucose, tyrosine, fructose.
The adaptive faculty of biocontrol microorganisms Kg2A to environment is stronger, can grow in the temperature range of 4 DEG C-90 DEG C, is that 0-10% all can grow in salt concn.
The present invention is investigated biocontrol microorganisms Kg2A to the antibiotic susceptibility of difference, for chemical prevention lays the first stone.
Bacterial strain Kg2A of the present invention is inhibited to sickle-like bacteria, sickle spore anthrax-bacilus and colletotrichum gloeosporioides Penz.
Bacterial strain Kg2A of the present invention or its fermented liquid can use as biological control agent, for preventing and treating fungal diseases of plants.
The present invention also provides the application of a kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Kg2A, and this bacterial strain may be used for preventing and treating anthracnose of rubber trees, red rot of sugar cane and pepper blight.
The present invention also provides a kind of biological control method of fungal diseases of plants, bacterial strain Kg2A or its fermented liquid is used to the plant with fungal disease, also can use the biological control agent containing above-mentioned bacterial strains or its fermented liquid, also can prevent and treat agents with other biological and use.
Bacterial strain Kg2A of the present invention has broad spectrum antibacterial, and cultivate simple, strong adaptability, antibacterial ability is stablized, and can be widely used in the control of various plants fungal disease.
Accompanying drawing explanation
Fig. 1 is the 16S rDNA PCR primer electrophorogram of Kg2A and other biocontrol microorganisms, and swimming lane M is DNA marker, and 1 is Kg1, and 2 is Kg2, and 3 is Kg2A, and 4 is Kg4.
Fig. 2 is the gramstaining result of biocontrol microorganisms Kg2A.
Fig. 3 is the antagonistic effect figure of biocontrol microorganisms Kg2A to sickle spore anthrax-bacilus.
Fig. 4 is the antagonistic effect figure of biocontrol microorganisms Kg2A to colletotrichum gloeosporioides Penz.
Fig. 5 Kg2A is to the antagonistic effect figure of colletotrichum gloeosporioides Penz.
Fig. 6 Kg2A is to the antagonistic effect figure of sickle-like bacteria.
Embodiment
Embodiment 1
The separation and Culture of bacterial strain Kg2A and screening purifying
1. sample
Sample is the pepper blade in great Po town, Hainan Province, the blade be collected is put into plastics bag and record.
1.2 material surface sterilizations are separated with antagonistic bacterium
1.2.1 clear water rinses the sick leaf with clip
Wash away the dust gravel on blade with clear water, dry.Being good for intersection clip size with scissors in disease is that the vanelets of 5 × 5mm is for subsequent use.
1.2.2 sample sterilization
The minor illness leaf of clip is put into beaker, soaks 30 seconds with mercuric chloride, should note constantly stirring minor illness leaf gently in the process of soaking, then use aseptic water washing three times, dry, for subsequent use.
1.2.3 bacteria distribution is cultivated
Be placed with on PDA solid medium with sterilized tweezers by the vanelets of clip, every ware evenly puts 4 pieces, then is sealed by culture dish preservative film.Be inverted in constant incubator, 28 DEG C of cultivations.After 12h carries out observing 48h, the thalline grown is forwarded on identical substratum, after 28 DEG C of cultivation 48h, carries out purifying; Meanwhile, adopted by the bacterium of gained after purifying in gradient dilution coating LB plate culture medium, after 28 DEG C of cultivation 48h, picking list bacterium colony is connected in corresponding medium slant.
The screening of 1.3 antagonistic bacteriums
1.3.1 the purifying of bacterium
The slant tube be positioned in 8 ~ 10 DEG C of incubators is taken out, in aseptic operating platform, LB liquid medium is dispensed in Erlenmeyer flask, then chooses a small amount of bacterium in Erlenmeyer flask with inoculating needle, sealing.On 28 DEG C of shaking tables, concussion is cultivated about 12 hours, takes out.Then draw 200 μ L on LB solid medium with pipettor, paint daubs is smeared evenly, cultivates in 28 DEG C of incubators.And then obtain single bacterial strain by the process of infinite dilution method.
Embodiment 2
The qualification of bacterial strain Kg2A
1, Microbiological Characteristics:
To ruling in 37 DEG C of incubators, the biocontrol strain cultivated carries out morphological specificity observation, finds that biocontrol strain Kg2A is direct rod shape, less chaining, tool nose circle or square end; Gemma ellipse, oval, column, circle, without pod membrane, are bacillus.After gramstaining, thalline all presents bluish voilet, is gram-positive microorganism.
2, molecular biology identification:
(1) DNA extraction: adopt the general test kit in this area to carry out the extraction of genomic dna;
(2) PCR reaction: bacterial universal primers 8F and 1492R, by the English Weihe River, prompt base (Shanghai) trading company synthesizes, and structure is as follows: 8F 5'-AGAGTTTGATCCTGGCTCAG-3'
1492R 5'-GGTTACCTTGTTACGACTT-3'，
As shown in Figure 1, amplified band length is about 1500bp to the 16S rDNA gene amplification result of biocontrol strain Kg2A, in the same size with expection;
(3) PCR is identified that correct recombinant bacterium send prompt base (Shanghai) trading company in English Weihe River gene sequencing.
By comparing on the net at NCBI, result display is 99% with the homology of bacillus subtilis Pseudomonas, therefore infers that Kg2A bacterial strain belongs to bacillus subtilis Pseudomonas.
3, the Physiology and biochemistry qualification of biocontrol microorganisms
(1) indole test
1. substratum preparation: get the 1% Tryptones aqueous solution, adjust pH 7.2-7.6, being sub-packed in test tube, is highly the 1/3-1/4 of test tube, 121 DEG C of sterilizing 20min;
2., after substratum cooling, get 10 μ L strains tested fresh seeds liquid and be inoculated in Tryptones liquid nutrient medium;
3. 28 DEG C of cultivations, respectively at observations after 2d, 4d;
When 4. observing at every turn, slowly add the high reagent of 3-5mm in nutrient solution surface along tube wall, occur at liquid layer interface red, be positive reaction.If color is not obvious, ether can be added in nutrient solution and be about 1mL, fully shake, leave standstill a moment, after ether is floating to liquid level, adds indole reagent again, occur red at ether and reagent solution bed interface, be positive reaction.
The equal redfree of result produces, and result is as shown in table 1, therefore analyzes biocontrol microorganisms under indoles is physiological and biochemical index situation for negative.
Table 1 indole test result
Note: "+" is positive, "-" is negative.
(2) Citrate trianion growth test
1. substratum preparation: get NaCl 1.0g respectively, MgSO 47H 2o 0.2g, NH 4h 2pO 40.5g, Trisodium Citrate 2.0g, agar 18.0g, 0.04% phenol red aqueous solution 10mL; Distilled water is supplemented to 1000.0mL, adjusts pH to be 7.0, then adds indicator, packing test tube;
2. 121 DEG C of sterilizing 20min, pendulum inclined-plane;
3. strains tested list bacterium colony streak inoculation on inclined-plane of picking fresh culture, cultivates 3-7d for 28 DEG C;
4. observation experiment result: substratum becomes blue or pink into positive, otherwise is negative.
Except fungi-proofing Kg1 nondiscoloration of delivering a child, connect other biocontrol microorganisms and all become pink, all the other biocontrol strains are positive as shown in table 2;
Table 2 Citrate trianion growth test result
Note: "+" is positive, "-" is negative.
(3) the suitableeest utilization of carbon source measures
1. choose Zulkovsky starch, Gelucystine, xitix, proline(Pro), arginine, maltose, tyrosine, N.F,USP MANNITOL, glucose, sorbyl alcohol, glycine, rhamnosyl, fructose, oxalic acid, Threonine, semi-lactosi, etc. utilization of carbon source measure;
2. minimum medium: (NH 4) 2sO 42.0g, NaH 2pO 4h 2o 0.5g, K 2hPO 40.5g, MgSO 47H 2o 0.2g, CaCl 22H 2o 0.1g, distilled water is supplemented to 1000.0mL, pH 6.5, is sub-packed in test tube and (often props up test tube 4mL), 121 DEG C of sterilizing 20min;
3. gained carbon source is mixed with the aqueous solution of 5%, adds after sterilizing in test tube, make its final concentration be 1%;
4. get strains tested seed liquor 10 μ L to be added in different carbon source respectively, and blank is set, then at 28 DEG C, under 200r/min condition, shake training 40h;
If 5. occur obviously muddy, illustrate that this carbon source can be utilized; If nothing, then need therefrom to take out 10 μ L, be transferred in carbon source test tube new accordingly, under similarity condition, cultured continuously 3 times; If still without muddy, then prove that this carbon source is not suitable for being utilized by strains tested.
Experimental result is as follows:
Carbon source can be utilized to have: sorbyl alcohol, rhamnosyl, micro-C, Threonine, semi-lactosi, proline(Pro), N.F,USP MANNITOL, maltose, Gelucystine, Zulkovsky starch, glucose, tyrosine, fructose;
Carbon source can not be utilized to have: arginine, oxalic acid, glycine.
(4) mensuration of optimum growth temperature
1. LB substratum configuration: Tryptones 10.0g, yeast extract 5.0g, sodium-chlor 5.0g, distilled water is supplemented to 1000.0mL, is sub-packed in test tube, cools after 121 DEG C of sterilizing 20min;
2. bacterial strain is accessed in test tube respectively, under being placed in 4 DEG C, 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C, 99 DEG C degrees Celsius respectively, cultivate 10min (40 DEG C of temperature cultivate 12h);
3. streak culture: respectively the bacterial strain under above-mentioned differing temps to be rule, cultivate 24 hours, observe its growing state respectively for 37 DEG C.
Measure the growth temperature of biocontrol microorganisms, result is as follows:
The measurement result of table 3 optimum growth temperature
Note: "+" can grow, and "-" be not for growing.
(6) Antagonistic Fungi Salt resistant test
After cultivating 24h, take out the upgrowth situation observing bacterial strain in each test tube, result is as follows:
Table 4 biocontrol microorganisms Salt resistant test result
Note: "+" is strain growth, and "-" be not for grow.
As can be seen from table 3-2, except bacterial strain Kg1 can only grow when salt concn is 0-7%, all the other 4 kinds of Antagonistic Fungis are that 0-10% all can grow in salt concn, and be that in the environment of 0, upgrowth situation is best in salt concn, in scope of experiment, its growth tendency of increase with salt concn weakens gradually, until salt concn be 12% and after, biocontrol microorganisms not regrowth.
(7) catalase test
1. cultivation strain 16h is shaken with liquid nutrient medium;
2. transfering loop dips the fresh bacterium liquid cultivating 16h, is coated in and is dripping on the slide having 3% aqueous hydrogen peroxide solution;
3. observations: have bubble to be produced as the positive, bubble-free is negative.
Found that the fresh bacterium liquid of Kg2A is coated in when dripping on the slide having 3% aqueous hydrogen peroxide solution, all having bubble to produce; Display catalase experimental result is positive.
Table 5 catalase test result
Note: "+" is positive, "-" is negative
(8) oxidase test
1. cultivation strain 16h is shaken with LB liquid medium;
2. in clean culture dish, put a filter paper, drip 1% dimethyl to the penylene diamine hydrochloride aqueous solution, only make filter paper moistening, can not be excessively wet;
3. get the fresh bacterium liquid cultivating 16h with transfering loop, spread upon on moistening filter paper;
4. observations: the existing red person of the lawn smeared in 10s is for positive, and the existing red person of 10-60s is time-delay reaction, and the existing red person of more than 60s disregards, by negative process.
Result shows: all not colorific change in the 60S after smearing lawn; Whole display is negative.
Table 6 oxidase test result
Note: "+" is positive, "-" is negative
(9) to antibiotic Study of Sensitivity
Measure bacterial strain Kg2A to need not antibiotic susceptibility with filter paper enzyme, prepare the filter paper of diameter 7 millimeters with punch tool, high-temperature sterilization.Be cooled to after dissolving in the LB solid medium of 50 degrees Celsius ~ 55 degrees Celsius and add 1 milliliter of strains tested seed liquor, plate is down flat after shaking up, filter paper after sterilizing is placed in flat board, then on filter paper, antibiotic solution is added, addition is equivalent to penbritin Amp 50 micrograms per millilitre, kantlex Kana 50 micrograms per millilitre, paraxin Chl 50 micrograms per millilitre, Streptomycin sulphate Str 50 micrograms per millilitre, tsiklomitsin Tet 50 micrograms per millilitre, Erythromycin E ry 50 micrograms per millilitre, Rifampin Rif 50 micrograms per millilitre, cephamycin C ef 50 micrograms per millilitre.Be positioned in 28 degrees Celsius of constant incubators and cultivate, respectively at 24 hours, 48 hours observationss.Result is as follows:
Table 7 antibiotics resistance experimental result
Note: "+", for there is inhibition zone, "-" be not for occur inhibition zone.
Embodiment 3
Biocontrol microorganisms Kg2A is to the bacteriostatic test of pathogenic bacteria
In the dull and stereotyped central authorities of the PDA that getting ready in advance, inoculation diameter be 6mm for examination pathogenic bacteria bacterium block, simultaneously in symmetry 2 the place inoculation tested bacteria apart from PDA plate center 25mm, every strain bacterium repeats 3 times, and 28 DEG C of constant temperature are inverted and are cultivated, and observe antibacterial situation after 6d.
Embodiment 4
Form dual test
Substratum: Tryptones 10.0g, yeast extract 5.0g, sodium-chlor 5.0g, agar powder 20g, distilled water is supplemented to 1000.0mL, 121 DEG C of sterilizing 20min.
By carrying out opposite culture test to biocontrol microorganisms Kg2A, in the 1-10 generation of result display Kg2A, stablizes antagonism sickle spore anthrax-bacilus and colletotrichum gloeosporioides Penz.
Disclosedly above be only preferred embodiment of the present invention, interest field of the present invention can not be limited with this, therefore according to the equivalent variations that the claims in the present invention are done, still belong to the scope that the present invention is contained.

Claims (8)

1. a strain biocontrol microorganisms Kg2A, this bacterial strain is bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and preserving number is: CGMCC No.10918.
2. a substratum for biocontrol microorganisms described in claim 1, is characterized in that, described substratum is LB substratum or YEP substratum.
3. the purposes of biocontrol microorganisms Kg2A according to claim 1 in control fungal diseases of plants.
4. purposes as claimed in claim 3, it is characterized in that, described fungal diseases of plants is caused by sickle-like bacteria, sickle spore anthrax-bacilus and/or colletotrichum gloeosporioides Penz.
5. a biological control agent, containing bacterial strain according to claim 1 or its fermented liquid.
6. the purposes of biological control agent according to claim 5 in control fungal diseases of plants.
7. purposes as claimed in claim 6, it is characterized in that, described fungal diseases of plants is caused by sickle-like bacteria, sickle spore anthrax-bacilus and/or colletotrichum gloeosporioides Penz.
8. a biological control method for fungal diseases of plants, is characterized in that, uses the bacterial strain of claim 1 or the biological control agent of claim 5 to the plant with fungal disease.
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