CN103173394A - Bacillus subtilis for controlling citrus canker - Google Patents

Abstract

The invention relates to a bacillus subtilis CCCQ080 with stronger activity prevention for citrus canker, belonging to the field of agriculture disease biological control. The bacillus subtilis is obtained by being separated from natural environment host leaves and is identified according to the morphological characteristics and molecular biology. The bacillus subtilis has very strong inhibition effect on the citrus canker, and also has good control effect on the citrus canker. Furthermore, the bacillus subtilis also has broad-spectrum antagonistic activity, has stronger inhibition effect on pathogenic bacteria such as citrus canker pathogen and tobacco angular leaf spot pathogen, and also has good inhibition effect on pathogenic fungus such as tobacco botrytis cinerea, apple canker pathogenic bacteria and thielaviopsis basicola. The secondary metabolite of the bacillus subtilis contains cellulase, protease, siderophore and the like. The biological film of the canker pathogenic bacteria can be destroyed by the fermentation filtrate of the bacillus subtilis, and the environment-friendly biopesticide can be produced by bacterial fermentation or secondary metabolite extraction technology, so that the bacillus subtilis has important business development and application value.

Description

One strain is used for the subtilis of control citrus ulcer

Technical field

The present invention relates to Plant diseases biological control technical field, particularly relate to the subtilis CCCQ080 bacterial strain that a strain is used for the control citrus ulcer.

Background technology

Citrus is the large fruit of the first in the world, in 138 countries (area) such as Brazil, China, the U.S., plantation is arranged, and the total area is 8,000,000 hm approximately ², more than 1.2 hundred million tons of ultimate productions.China is the most important original center of citrus, and present citrus culture area is 1,900,000 hm approximately ², approximately 2,000 ten thousand tons of annual production rank first in the world respectively and second.Citrus ulcer (citrus canker) be by carpetweed Xanthomonas campestris citrus cause a disease mutation ( Xanthomonas axonopodispv. Cirri) the citrus destructive disease that causes, be domestic and international great Quarantine Objects, mainly infect Rutaceae both citrus, Poncirus and Fortunella plant.This disease mainly is distributed in more than 30 countries in Asia, Africa and America, accounts for to produce 1/3rd of citrus countries and regions, wherein occurs the most general with the Asia.

Citrus ulcer has also produced great effect to China's citrus industry, this disease in Guangdong, the coastlands such as Guangxi, Fujian, Zhejiang, Hainan generally occur always.Jiangxi Province's cultivated area is 150,000 hm approximately ², citrus ulcer generation area is 2.5 ten thousand hm approximately ²Citrus In Hunan cultivated area more than 280,000 hm ², citrus ulcer generation area more than 10,000 hm ²Citrus ulcer generation area in Guizhou Province's surpasses 3,000 hm ²

At present to the control of citrus ulcer still take chemical agent as main, thereby cause the serious consequences such as enhancings of pathogenic bacteria resistance, environmental pollution, finally occur prevention effect bad, endanger the problems such as ecological safety and human health.

Along with further carrying out in a deep going way of scientific research, discovery overcome the resistance of Plant diseases, reduce chemical bactericide to the pollution of environment, ecological destruction and agricultural byproducts in aspect chemical pesticide residual, Biocontrol microorganism has unique advantage, utilize the biological pesticide of the secondary metabolite preparation of biocontrol microorganisms, have pollution-free, noresidue, be difficult for making harmful organism develop immunity to drugs, high with Environmental compatibility, to characteristics such as person poultry safeties, become the main body of nuisanceless green agricultural chemicals and the developing direction of pesticide in future.Therefore, Efforts To Develop biological control research is the important channel of agricultural sustainable development, has important production practical significance.

Summary of the invention

The purpose of this invention is to provide the subtilis that a strain is used for the control citrus ulcer, this bacterial strain has stronger control effect to citrus ulcer.Cultivate proterties, morphological specificity and Physiological-biochemical Characters and the analysis of 16S rDNA sequence alignment according to the bacterial strain bacterium colony, preliminary definite bacterial strain CCCQ080 is subtilis, and it has been carried out antimicrobial spectrum mensuration, show that it has good bacteriostatic action, has a broad antifungal spectrum to multiple pathogenic bacteria.

The screening of CCCQ080 bacterial strain: from Chongqing City's soil and healthy plant blade, separate to obtain a plurality of bacterial strains, through primary dcreening operation and multiple sieve, finally by crossing the sick preventive effect test of greenhouse control, determine to be the CCCQ080 bacterial strain to what citrus ulcer had a better preventive and therapeutic effect.

The identification of morphology of CCCQ080 bacterial strain: gram-positive microorganism (Fig. 2), peritrichous (Fig. 1) produces gemma (Fig. 3).

The biochemical reactions of CCCQ080 bacterial strain: grow in 7% NaCl, the pH=10 growth, L-arabinose, D-wood sugar, PEARLITOL 25C, D-Glucose all produce acid, V.P is positive, the casein hydrolysis, and indole reaction is positive, reduction nitrate becomes nitrite, and is aerobic, produces hydrogen sulfide etc.According to grouped data, this pathogenic bacteria is initially identified as subtilis.

Molecular biology identification: by extracting CCCQ080 genomic dna and 16S rDNA pcr amplification, the 16S rDNA fragment length that CCCQ080 amplifies is 1470bp(Fig. 4), CCCQ080 and bacillus ( Bacillussp .) approach in heredity, in conjunction with cultural characters, morphological specificity, gramstaining and physiological and biochemical test result, with bacterial strain CCCQ080 be defined as subtilis ( Bacillus subtilis).

The live body pure culture of this bacterial classification has been preserved in ' China Committee for Culture Collection of Microorganisms's common micro-organisms center ' (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on 06 15th, 2012, Institute of Microorganism, Academia Sinica, postcode 100101, phone: 010-64807355), preserving number: CGMCC No.6224.

The CCCQ080 Metabolite is analyzed: CCCQ080 can not decompose and contain chitin composition substratum, shows in product without chitinase (Fig. 5); CCCQ080 has produced the yellow transparent circle on the Mierocrystalline cellulose flat board, illustrate all to contain cellulase in its meta-bolites energy decomposition of cellulose (Fig. 6); CCCQ080 produces obvious transparent circle on the cow's milk protein substratum, show to contain proteolytic enzyme (Fig. 7) in product; The detected result of having a liking for the iron element shows, has produced the yellow transparent circle on flat board, shows that CCCQ080 has chelating Fe ³⁺The ability of ion (Fig. 8).

CCCQ080 bacterial strain antimicrobial spectrum is measured: the CCCQ080 bacterial strain shows stronger bacteriostatic action to pathogenetic bacteria citrus ulcer bacteria (Fig. 9), tobacco ralstonia solanacearum and Tobacco Angular Leaf Spot Disease bacterium, and inhibition zone is between 5.6 ~ 15.6mm.The 21 kind of plant pathogenic fungies such as tobacco ash arrhizus bacteria, Valsa mali and black root of tobacco bacterium are all had certain fungistatic effect, and antibacterial bandwidth is between 5 ~ 22mm.

CCCQ080 bacterial strain Controlling effect: CCCQ080 has the sick effect of good control to citrus ulcer, and excised leaf control disease in greenhouse shows, the bacterial strain fermentation liquor prevention sprays after 24h spray inoculation ulcer bacteria again, can suppress disease fully (Figure 10) occurs.Plant control disease in greenhouse shows, the bacterial strain fermentation liquor prevention sprays after 24h stab inoculation ulcer bacteria again, and preventive effect can reach 43.26-61.24%, shows to control preferably effect.

The CCCQ080 bacterial strain is to citrus ulcer bacteria biomembrane damage effect: CCCQ080 bacterial strain fermentation liquor supernatant liquor is processed germ, its bacterium liquid specific conductivity and reducing sugar content are increased, show that namely its fermentation liquor treatment can make the ulcer bacteria cell damage, the protoplasma seepage.

Advantage of the present invention

Subtilis CCCQ080 bacterial strain from plant leaf surface separation acquisition, it is a new bacterial strain of finding first and identifying, itself has very strong restraining effect to various plants pathogenic bacterias such as citrus ulcers on flat board, its fermented liquid has good control effect to citrus ulcer.Because this bacterial strain is to obtain from plant surface separation, has natural harmonious blending with natural, ecological, compare with chemical pesticide that soil ecology has no side effect, noresidue, therefore, have potential business development and using value in the biological control practice of disease.

Description of drawings

Fig. 1 is bacterial strain CCCQ080 peritrichous

Fig. 2 is bacterial strain CCCQ080 gramstaining

Fig. 3 is bacterial strain CCCQ080 spore staining

Fig. 4 is the 16S rDNA base fragment sequence of CCCQ080 bacterial strain

Fig. 5 is the active detection of bacterial strain CCCQ080 chitinase

Fig. 6 is that bacterial strain CCCQ080 cellulase activity detects

Fig. 7 is the active detection of bacterial strain CCCQ080 protease

Fig. 8 is that bacterial strain CCCQ080 has a liking for the active detection of iron element

Fig. 9 is that bacterial strain CCCQ080 is to the restraining effect of citrus ulcer bacteria

Figure 10 is that bacterial strain CCCQ080 is to the excised leaf control action kou of citrus ulcer

Embodiment

The invention will be further described below in conjunction with embodiment

The evaluation of embodiment 1. subtilis CCCQ080 bacterial strains

(1) morphological observation and evaluation: bacterial strain CCCQ080 is being cultivated 1-3d on the NA culture plate under 28 ℃, observing its colonial morphology (shape, color gloss, surface be bending, edge shape etc. whether);

(2) biochemical reactions is observed: bacterial strain CCCQ080 is inoculated in the test media such as the macromolecular cpds decomposition such as temperature, potential of hydrogen, salt tolerance, sugar alcohol fermentation, Citrate trianion utilization, utilizations of nitrogen compound, starch and product enzyme the observing response situation;

(3) molecular biology identification: the genomic dna that extracts bacterial strain, with bacterium universal primer fD1:5'-AGAGTTTGATCCTGGCTC AG-3', rP1:5'-ACGGTTACCTTGTTA CGACTT-3', take the genomic dna of bacterial strain CCCQ080 as template, the PCR amplification is checked order by electrophoresis detection; Utilize Blast software and BioEdit software on the NCBI website to carry out sequence homology analysis, (Neighbor-Joining) constructing system is grown evolutionary tree to the contiguous method of employing Mega 4.0 softwares, proves conclusively thus the kind level taxonomic category title of CCCQ080.Determine the classification position of this bacterium.

Embodiment 2. subtilis CCCQ080 bacterial strain Controlling effects

2.1 CCCQ080 bacterial strain fungistatic effect: the bacterial strain point is seeded in the NA flat board, and 28 ℃ of constant temperature culture are after 2 days, then use the hedgehog hydnum atomizer with 3 * 10 ⁸The citrus ulcer bacteria suspension of cfu/mL is sprayed on dull and stereotyped upper (approximately 100 μ L/ wares), repeats 3 times.Cultivate under 28 ℃ of constant temperatures after 3 days, measure the diameter of inhibition zone.Determine that the CCCQ080 bacterial strain is to the inhibition of citrus ulcer.

2.2 CCCQ080 bacterial strain Controlling effect

Leaf method: get healthy sweet orange class Citrus leaf some, with distilled water, its surface is cleaned, dry.Then 75% alcohol solution dipping 30 s clean with sterilized water, dry.Blade evenly is divided into A, B, C and 4 processing of D at random.A processes the blade face and only sprays aqua sterilisa, and B only sprays the fermented liquid of Antagonistic Fungi, and C sprays c itrus canker germ bacteria suspension after processing the fermented liquid that first sprays Antagonistic Fungi in the blade face, and D processes the bacteria suspension that ulcer bacteria is only sprayed on the blade face.Pack into after spraying is processed in the pallet of sealing of sterilization, 26 ℃ of moisturizings are cultivated.Every processing repeats for 3 times.Observed afterwards in 7 days.Determine to control effect on the excised leaf of CCCQ080 bacterial strain to citrus ulcer.

Plants method: choose suitable sweet orange plant, spray Antagonistic Fungi stab inoculation c itrus canker germ after 1 day, replace Antagonistic Fungi process and only inoculate ulcer bacteria as contrast take sterilized water.5 leaves of every processing repeat for 3 times, and every blade is at 6 points of zygomorphy stab inoculation of master pulse.Again sample is placed in 26 ℃ ~ 28 ℃, cultivates in the intelligent growth cabinet of relative humidity 80% and observed afterwards in 25 days.Determine to control effect on the greenhouse plant of CCCQ080 bacterial strain to citrus ulcer.

Embodiment 3. CCCQ080 Metabolites are analyzed

Detect 3.1 chitinase is active

Sole carbon source substratum (Chi-Ayers): primary ammonium phosphate 1.0g, sal epsom 0.2g, Repone K 0.2g, agar 20g, 1% colloidal chitin are settled to l000mL, pH:7.0.

The chitinous preparation of colloidal: the 20g chitin is dissolved in the 350mL concentrated hydrochloric acid, places 24h at 4 ℃, filters through glass wool, filtrate adds-20 ℃ of ice dehydrated alcohol 2L that spend the night, and the 10000 centrifugal 20min of r/min, tap water rinse and are precipitated to neutral pH, lyophilize ,-20 ℃ of sealings are preserved.When using the colloidal chitin, grind more than 5 times with manual homogenizer.

Antagonistic Fungi is connected on the flat board that above-mentioned substratum makes, cultivated 7-10 days for 28 ℃, observe the size that transparent circle has that it's too late and measure transparent circle, show that if any transparent circle bacterial strain has produced chitinase.Determine that with this CCCQ080 produces the chitinase situation.

3.2 cellulase detects

The Congo red substratum of Mierocrystalline cellulose: potassium primary phosphate 0.5g/L, sal epsom 0.25g/L, Xylo-Mucine 1.88 g/L, gelatin 2.0g/L, Congo red 0.2g/L, agar 20g/L, pH:7.0

Bacterium to be measured is received on above-mentioned substratum, cultivated after 7-10 days for 28 ℃, with Congo red (lg/mL) dyeing lh, after outwelling Congo red dye liquor, then use sodium-chlor (1M) to soak lh, observe having or not of transparent circle, show that if any transparent circle bacterial strain has produced cellulase.Determine CCCQ080 cellulase-producing situation with this.

3.3 the detection of proteolytic enzyme

Protein culture medium: skim-milk 40g, agar 16g is settled to 400mL.

Bacterium to be measured is inoculated on above-mentioned protein culture medium, cultivated 3 ~ 5 days for 25 ℃, observe periphery of bacterial colonies and have or not transparent circle.Show that if any transparent circle bacterial strain has produced proteolytic enzyme.Determine that with this CCCQ080 produces the chitinase situation.

3.4 have a liking for the detection of iron element

Have a liking for iron element substratum: A:a. 60.5mg chrome azurol S is dissolved in the 50mL deionized water; B. 10mL ferric iron solution (1mM ferric chloride (FeCl36H2O), 10mM hydrochloric acid are solvent); C. the 72.9mg hexadecyl trimethyl ammonium bromide is dissolved in the 40mL deionized water.Above-mentioned a, b, three kinds of solution of c are settled to 100mL after mixing, and transfer pH to neutral, 121 ℃ of sterilization 20min.B:1000mL NA nutrient solution transfers to 6.8,121 ℃ of sterilization 20min with NaOH solution with nutrient solution pH.Mix A after sterilization finishes, make flat board after B liquid, inoculate bacterium to be measured, cultivate for 28 ℃ and observed colour-change in 7-10 days, show if any haloing that bacterial strain has produced and have a liking for the iron element.Determine with this that CCCQ080 produces and have a liking for iron element situation.

Embodiment 4. CCCQ080 bacterial strains are to citrus ulcer bacteria biomembrane damage effect

4.1 bacterial strain CCCQ080 is carried out fermentation culture, then centrifugal and filter, get supernatant liquor dilution 0 times (stoste), 2 times, 5 times, 10 times and 20 times, get and respectively process 1mL and join respectively in 20mL NB nutrient solution, then to add concentration be 3 * 10 ⁸Cfu/mL c itrus canker germ bacteria suspension 400uL.Separately arrange one group and do not add the citrus ulcer bacteria processing as parallel control, add the 1mL aseptic deionized water as blank with NB liquid.Every processing repeats for 3 times.Then measure electrical conductivity of solution at 0h, 12h, 24h and 36h respectively.

4.2 bacterial strain CCCQ080 is carried out respectively fermentation culture 48h, then centrifugal and filter, get supernatant liquor dilution 0 times (stoste), 2 times, 5 times, 10 times and 20 times, get and respectively process 1mL and join respectively in 20mL NB nutrient solution, adding the 1mL sterilized water as blank, then to add concentration be 3 * 10 ⁸Cfu/mL c itrus canker germ bacteria suspension 400uL.Centrifugal and filtration after fermentation culture 48h is got supernatant liquor and is measured reducing sugar content.Every processing repeats for 3 times.

Measure the CCCQ080 bacterial strain to the effect of citrus ulcer bacteria biomembrane damage with this.

16S rDNA fragment sequence:

1 CCTCTGTCAC TTCAGCGGCT GGCTCCATAA AGGTTACCTC ACCGACTTCG GGTGTTACAA

61 ACTCTCGTGG TGTGACGGGC GGTGTGTACA AGGCCCGGGA ACGTATTCAC CGCGGCATGC

121 TGATCCGCGA TTACTAGCGA TTCCAGCTTC ACGCAGTCGA GTTGCAGACT GCGATCCGAA

181 CTGAGAACAG ATTTGTGGGA TTGGCTTCAA CTCTCGGCGG TTTCGCGTGC CCTTTGTTCT

241 GTCCATTGTA GCACGTGTGT TAGCCCAGGT CCATAAGGGG CATGAGTGAT TTGACGTCAA

301 TCCCCCACAC TCTCCTCCGG TTTGTCACCG GCAGTCACCT TAGAGGTGCC CAACTGAATG

361 CTGGCAACTA AGATCAAGGG TTGCGCTCGT TGCGGGACTT AACCCAACAT CTCACGACAC

421 GAGCTGACGA CAACCATGCA CCACCTGTCA CTCTGCCCCC GAAGGGGACG TCCTATCTCT

481 AGGATTGTCA GAGGATGTCA AGACCTGGTA AGGTTCTTCG CGTTGCTTCG AATTAAACCA

541 CATGCTCCAC CGCTTGTGCG GGCCCCCGTC AATTCCTTTG AGTTTCAGTC TTGCGACCGT

601 ACTCCCCAGG CGGAGTGCTT AATGCGTTAG CTGCAGCACT AAGGGGCGGA AACCCCCTAA

661 CACTTAGCAC TCATCGTTTA CGGCGTGGAC TACCAGGGTA TCTAATCCTG TTCGCTCCCC

721 ACGCTTTCGC TCCTCAGCGT CAGTTACAGA CCAGAGAGTC GCCTTCGCCA CTGGTGTTCC

781 TCCACATCTC TACGCATTTC ACCGCTACAC GTGGAATTCC ACTCTCCTCT TCTGCACTCA

841 AGTTCCCCAG TTTCCAATGA CCCTCCCCGG TTGAGCCGGG GGCTTTCACA TCAGACTTAA

901 GAAACCGCCT GCGAGCCCTT TACGCCCAAT AATTCCCGAC AACGCTTGCC ACCTACGTAT

961 TACCGCGGCT GCTGGCACGT AGTTAGCCGT GGCTTTCTGG TTAGGTACCG TCAAGGTGCC

1021 GCCCTATTTG AACGGCACTT GTTCTTCCCT AACAACAGAG CTTTACGATC CGAAAACCTT

1081 CATCACTCAC GCGGGCGTTG CTCCGTCAGA CTTTCGTCCA TTGCGGAAGA TTCCCTACTG

1141 CTGCCTCCCC TAAGGAGTCT GGGCGGTGTC TCAATCCCAA GTGTGGCCGA TCACCCTCTC

1201 AGGTCGGCTA CGCCATCGTC TGCCTTGGTG AGCCGTTACC TCACCAACTA GCTAATGCGC

1261 CGCGGGTCCA TCTGTAAGTG GTGAGCCGAA GCCACCTTTT ATGTCTGAAC CATGCGGTTC

1321 AGACAACCAT CCGGTATTAG CCCCGGTTTC CCGGAGTTAT CCCAGTCTTA CAGGCAGGTT

1381 ACCCACGTGT TACTCACCCG TCCGCCGCTA ACATCAGGGA GCAAGCTCCC ATCTGTCCGC

1441 TCGACTGCAT GTATAGCACG CCGCCCCGTT

Claims (1)

1. One strain be used for the control citrus ulcer subtilis ( Bacillus subtilis) CCCQ080, its preserving number is CGMCC NO.6224.

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