CN104082344A - Application of bacillus axarquiensis to prevention and treatment of verticillium dahliae - Google Patents
Application of bacillus axarquiensis to prevention and treatment of verticillium dahliae Download PDFInfo
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Abstract
The invention provides a prevention and treatment effect on verticillium dahliae by using antagonistic fungi, namely bacillus axarquiensis. An antagonistic strain TUBP1 which has high antifungal activity on verticillium dahliae is obtained by separating via preliminary screening and secondary screening through an agar plate diffusion method. The strain is determined to be bacillus axarquiensis through morphologic observation, physiological and biochemical experiments and gene sequence analysis of 16S rDNA (ribosome Deoxyribose Nucleic Acid) and gyrB; the antifungal activity of fermentation liquid on verticillium dahliae is determined by using a growth rate method and an oxford cup method respectively; the growth inhibition rate on mycelium of verticillium dahliae is 78.62+/-3.48% and the diameter of an inhibition zone is 21+/-3.86mm; in a room-temperature pot experiment, the bio-control effect of the TUBP1 is 42.56%; in a plot experiment, the bio-control effect of the TUBP1 is 40.16%. An in vitro live test shows that the antagonistic fungi, namely bacillus axarquiensis can be used for preparing a medicine for preventing and treating verticillium dahliae.
Description
Technical field
The present invention relates to the application of the sub-bacillus of Amado Azar base on control cotton verticillium wilt, belong to agricultural technology field.
Background technology
Cotton verticillium wilt (Cotton verticillium wilt) is that the one soil being caused by verticillium dahliae (Verticillium dahliae Kleb) passes vascular bundle fungal disease, also be one of destructive disease in Cotton Production, have a strong impact on output of cotton and quality.Verticillium dahliae can be propagated by seed, soil and the carry disease germs various ways such as diseased plant and approach, and can form Microsclerotia and in soil, survive a lot of years, very difficult in control.Since the nineties in 20th century, cotton verticillium wilt increases the weight of year by year, and causes huge economic loss.According to incompletely statistics, cause the annual underproduction 10%～30%.
Utilize biological control cotton verticillium wilt to enjoy people to pay close attention to.Biological control is pollution-free, noresidue, nontoxic to person poultry safety, and is difficult for making pathogen to produce drug resistance, and production technology is simpler, and some biocontrol microorganisms can also promote crop growth, is subject to many researchers' favor.Antagonistic microbe kind has mould, actinomycetes, bacterium, and wherein that most study is bacillus (Bacillus spp.), because bacillus is widely distributed, reproduction speed is fast, high-output stress-resistance.Cotton Fields in Xinjiang is taking continuous cropping as main, and very limited to the biological bactericide of control cotton in Xinjiang verticillium wilt, is precondition so obtain the efficient Antagonistic Fungi of Verticillium dahliae from Cotton in Xinjiang.The sub-bacillus of Amado Azar base (Bacillus axarquiensis) is a kind for bacillus, has the features such as breeding is fast, strong stress resistance, has the potentiality as advantage biocontrol bacteria.
According to literature survey, at present to Amado Azar Ji Ya bacillus study limitation in its taxonomy aspect, and its biologically active aspect is had no to correlative study, particularly it has no relevant report to cotton verticillium wilt prophylaxis effect.This seminar, by separation, screening, pot experiment and small plot experiment etc. to antagonistic bacterium in 15 years cotton root soils of Southern Xinjiang continuous cropping, filters out the antagonistic strain of a strain to verticillium dahliae.This research is by form, Physiology and biochemistry and clear and definite its taxonomy of molecular biology method, and further determine that by potted plant and small plot experiment it is to cotton verticillium wilt preventive and therapeutic effect, for the good biocontrol fungicide of exploitation Xinjiang continuous cropping cotton field cotton verticillium wilt lays the foundation.
Summary of the invention
Technical problem to be solved by this invention is to provide the purposes of the sub-bacillus of Amado Azar base in control cotton verticillium wilt.In the present invention, confirmed that by vitro, potted plant and small plot experiment the sub-bacillus of Amado Azar base has fine preventive and therapeutic effect to cotton verticillium wilt.
Isolated test: adopt growth rate method and Oxford agar diffusion method to detect the sub-fermentation of bacillus liquid of Amado Azar base in vitro to verticillium dahliae inhibitory action, growth rate method and Oxford agar diffusion method have inhibitory action to verticillium dahliae, adopting growth rate method to survey the sub-fermentation of bacillus liquid of Amado Azar base is 78.62 ± 3.48% to verticillium dahliae inhibiting rate in vitro, and it is 21 ± 3.86mm to verticillium dahliae antibacterial circle diameter in vitro that Oxford agar diffusion method is surveyed the sub-fermentation of bacillus liquid of Amado Azar base.Show that by isolated test result the sub-bacillus antagonism of Amado Azar base verticillium dahliae has inhibitory action.
Pot experiment: from greenhouse pot culture result of the test, the sub-bacillus of Amado Azar base has certain Biocontrol Effect to cotton verticillium wilt.After inoculation, 30～120d control group cotton verticillium wilt disease index is 10.87～39.88, and the sub-bacillus processed group of Amado Azar base disease index is 3.85～21.28.Show that the sub-bacillus of Amado Azar base can reduce the generation of cotton verticillium wilt.30～120d after inoculation, the sub-fermentation of bacillus liquid of Amado Azar base is 36.54%～42.56% to verticillium dahliae relative control effect.
Small plot experiment: the sub-bacillus of Amado Azar base has certain Biocontrol Effect to cotton verticillium wilt.After inoculation, 60～140d control group cotton verticillium wilt disease index is 13.87～49.08, disease index shows a rising trend, it is 49.08 that the rear 140d disease index of inoculation reaches maximum, and the sub-bacillus processed group of Amado Azar base disease index is respectively 3.85～21.28, the sub-bacillus of Amado Azar base has certain biocontrol effect to cotton verticillium wilt, and its relative biological and ecological methods to prevent plant disease, pests, and erosion value is 38.04%～40.16%.130d after inoculation, the sub-fermentation of bacillus liquid of Amado Azar base is 43.07% to the maximum to verticillium dahliae relative control effect.140d after inoculation, relative control effect is 40.16%.
In a word, the sub-bacillus of Amado Azar base of the present invention has antagonism to verticillium dahliae, can be used for cotton verticillium wilt control.The sub-fermentation of bacillus immersion foam flower seed of available Amado Azar base or fill with in cotton root.From another perspective, the invention also discloses a kind of biopesticide of preventing and treating cotton verticillium wilt, wherein contain the sub-bacillus of Amado Azar base or zymotic fluid.So, the invention also discloses the purposes of the sub-bacillus of Amado Azar base on the biopesticide of preparation control cotton verticillium wilt.
Brief description of the drawings
Fig. 1 is the thalli morphology (A: light microscope of Antagonistic Fungi TUBP1 of the present invention; B: ESEM).
Fig. 2 builds TUBP1 phylogenetic tree for the present invention is based on 16S rDNA (A) and gyrB (B) gene order BLAST result.
Fig. 3 is Bacillus axarquiensis zymotic fluid In Vitro Anti verticillium dahliae activity (A: front of the present invention; B: the back side; 1: growth rate method; 2: Oxford agar diffusion method).
Embodiment
Embodiment 1: Antagonistic Fungi primary dcreening operation and multiple sieve
Verticillium dahliae bacterium solution preparation: verticillium dahliae V.dahliae ATCC36211 inoculation, containing on PDA culture medium flat plate, is cultivated 15d for 25 DEG C, and flat board adds sterile water, scraping spore, with blood counting chamber counting, adjusting bacterial concentration is 6 × 10
6spore mL
-1, for subsequent use.
The Isolation and screening of antagonistic bacterium: separate routinely soil bacteria method and carry out.Single colony inoculation of separation and purification, on NA medium, is cultivated to 48h for 180rmin37 DEG C, make bacterium cake with 6mm card punch.On PDA medium, be coated with 200 μ L6 × 10
6mL
-1verticillium dahliae spore suspension, then by the reversal connection of separation and purification bacterial strain bacterium cake in flat board.Cultivate 4d for 25 DEG C, screening produces the antagonistic bacterium of inhibition zone.
This experiment is divided into from obtaining 219 strain bacterial strains from soil sample, wherein 127 strains in healthy soil (accounting for 54.7%), 92 strains (accounting for 42.6%) (table 1) in morbidity soil, obtain 23 strains through primary dcreening operation and produce the bacterial strain that antibacterial circle diameter is large, bacteriostasis is stronger.Primary dcreening operation bacterial strain has been carried out to multiple sieve, and what wherein resistant activity was strong has 16 strain bacterial strains: antibacterial circle diameter is greater than bacterial strain 12 strains (table 2) of 10mm, and wherein the antibacterial circle diameter of TUBP1 bacterial strain is 21 ± 3.86mm.
Table 1 different parts separates antagonistic strain number
Table 2 12 strain antagonisms are carefully to verticillium dahliae antibacterial circle diameter
Note: a: Antagonistic Fungi derives from healthy plant rhizosphere soil; B: Antagonistic Fungi derives from disease plant rhizosphere soil.
Embodiment 2: antagonistic bacterium TUBP1 Morphology and physiology biochemical identification.The antagonistic bacterium TUBP1 inoculation of activation is cultivated to 48h in dull and stereotyped upper 37 DEG C of NA, and carry out Gram’s staining, observe colonial morphology and thalline and gemma and Physiology and biochemistry qualification (methyl red test, Starch Hydrolysis test, glycitols fermentation test, fluorchrome test etc.).
Bacterium colony and the morphological feature of verticillium dahliae antagonistic strain TUBP1, TUBP1 grows on NA medium, the mono-bacterium colony of TUBP1 is rounded, surface shrinkage, dry, matt, light tight, edge is rough, milky (Figure 1A), Gram’s staining is positive (G+), and has gemma, and motility, metachromatic granule experiment are positive.The micro-Microscopic observation of surface sweeping, thalline is shaft-like (Figure 1B)
Verticillium dahliae antagonistic strain TUBP1 Physiology and biochemistry measurement result, verticillium dahliae antagonistic strain TUBP1 Physiology and biochemistry measurement result is in table 3, bacterial strain TUBP1 Morphology and physiology biochemical character is contrasted with corresponding genus and species in " common bacteria system identification handbook ", the characteristic feature of itself and bacillus is basically identical, and Preliminary Identification bacterial strain TUBP1 belongs to bacillus (Bacillus spp.).
Table 3 physiological and biochemical test result
| Project | Experimental phenomena | Phenomenon analysis | Result |
| Produce ammonia test | There is yellowish-brown precipitation | Test organisms can produce ammonia | + |
| Catalase test | Produce a large amount of bubbles | Test organisms has catalase | + |
| Methyl red test | Be orange colour | The further metabolism of acid that test organisms produces | + |
| V-P test | Redden | Can utilize V-P medium | + |
| Starch Hydrolysis test | There is transparent circle | Test organisms can produce amylase | + |
| Fluorchrome test | Without fluorescence | Test organisms does not produce fluorchrome | – |
| Tartrate utilizes | Green, in a large number precipitation | Test organisms does not utilize tartrate | – |
| Nitrate reduction test | It is rose-red that medium turns | Test organisms has nitrate reductase | + |
| Phenylalanine dehydrogenase test | Nondiscolouring | Test organisms does not produce phenylalanine deaminase | – |
| Urease test | Nondiscolouring | Test organisms does not produce urase | – |
| Gelatin liquefaction test | Gelatin liquefaction | Test organisms liquefy gelatin | + |
| The generation test of hydrogen sulphide | Paper slip blackening | Test organisms can produce hydrogen sulphide | + |
| Glycitols fermentation test | Medium flavescence | Test organisms energy fermentation and acid | + |
| Indole test | Medium reddens | Test organisms has tryptophan hydrolase | + |
| Citrate utilization test | Medium becomes peach | Citrate is utilized | + |
Note: (negative Wei –, the positive be+)
Embodiment 3: the phylogenetic analysis of verticillium dahliae Antagonistic Fungi TUBP1 based on 16S rDNA and gyrB gene order BLAST result
Antagonistic bacterium TUBP1 gene order pcr amplification, the pcr amplification of 16S rDNA, extracting antagonistic bacterium TUBP1 genomic DNA is template, adopts universal primer to carry out 16S rDNA sequence pcr amplification.Extension amplification outcome is also served the order-checking of Hai Shenggong biotechnology Services Co., Ltd, and result is as shown in SEQ ID No1.
The pcr amplification of gyrB gene order, extract Antagonistic Fungi TUBP1DNA, and adopt degenerate primer UP-1S (5'GAA GTCATCATGACCGTTCTGCA3', SEQ ID No2) and UP-2Sr (5'AGCAGGGTAC GG ATGTGCG A GCC3', SEQ ID No3) amplification gyrB gene, reaction condition: 95 DEG C of 5min; 95 DEG C of 30s, 58 DEG C of 45s, 72 DEG C of 2min, 30 circulations; 72 DEG C of 10min.Extension amplification outcome is also served the order-checking of Hai Shenggong biotechnology Services Co., Ltd, and result is as shown in SEQ ID No4.
Based on the structure of 16S rDNA and gyrB Phylogenetic Tree, all prokaryotes of having measured in the 16S rDNA of TUBP1 bacterial strain and gyrB sequence and GenBank are carried out to BLAST comparison.According to comparative result, utilize PHYLD program phylogenetic tree construction, recycling Treeview rebuilds.Phyletic evolution distance is according to Kimura model assessment, with the cluster analysis of MEGA5.0 (Molecular Evolutionary Genetics Analysis) software employing adjacent method (Neighbor-Joining), and construct systematic evolution tree.Meanwhile, 100 values of bootstrapping of repeated sampling (Bootstrap value) analysis is carried out the stability of the topological structure of evaluating system chadogram.
All prokaryotes 16srDNA sequences of having measured in the 16S rDNA sequence of Antagonistic Fungi TUBP1 and GenBank are compared, obtain the evolutionary distance of the relevant bacterial strain of antibacterial TUBP1 and built phylogenetic tree (Fig. 2 A).Fig. 2 A shows, the reference culture homology similarity major part of strains tested and bacillus is more than 99%, tentatively this bacterial strain is classified as to bacillus, the autoploidy of TUBP1 and reference culture Bacillus axarquiensis (DQ993670) approaches (similarity is 99.80%) the most simultaneously.With gyrB gene order comparison result phylogenetic tree construction (Fig. 2 B), utilizing gyrB gene can precise Identification bacterial strain TUBP1 be also Bacillus axarquiensis (similarity is 99.00%).Therefore, according to 16S rDNA sequence and gyrB similarity analysis, learn and observe and Physiology and biochemistry test at combining form, determine that Antagonistic Fungi TUBP1 is Bacillus axarquiensis.
Embodiment 4: Antifungal Activity in Vitro test determination, zymotic fluid preparation: by Antagonistic Fungi Bacillus axarquiensis inoculation in NA culture fluid, 37 DEG C, 180rmin
-1under condition, cultivate 48h, centrifugal (6000rmin
-120min), get supernatant, through miillpore filter, (m) filtration sterilization of 0.22 μ, packing are placed in 4 DEG C of Refrigerator stores for subsequent use to supernatant.
Growth rate method antibacterial tests: add 20mL without fermented liquid in 100mL PDA medium, pour in the flat board of sterilizing after shaking up, treating that it is paved solidifies, and accesses in the central verticillium dahliae bacterium cake.Not add the zymotic fluid of Antagonistic Fungi as control group, each processing repeats 6 times; Cultivate 15d for 25 DEG C, measure colony growth diameter.Calculate colony diameter and growth inhibition ratio: colony diameter (cm)=measurement colony diameter mean value-0.09, colony growth inhibiting rate (%)=(control group colony diameter-processed group colony diameter)/control group colony diameter × 100.
Oxford agar diffusion method bacteriostatic test: inoculate verticillium dahliae bacterium cake in PDA flat board, cultivate after 7d for 25 DEG C, periphery of bacterial colonies is put into Oxford cup and is added 200 μ L antagonistic bacterium Bacillus axarquiensis without fermented liquid (0.22 μ m filtering with microporous membrane), 25 DEG C are continued to cultivate after 8d, measure its antibacterial circle diameter with slide measure.
Adopt growth rate method and Oxford agar diffusion method to detect Bacillus axarquiensis zymotic fluid in vitro to verticillium dahliae inhibitory action, as shown in Figure 3, growth rate method (Fig. 3-1A and Fig. 3-1B) and Oxford agar diffusion method (Fig. 3-2A and Fig. 3-2B) have inhibitory action to verticillium dahliae, adopting growth rate method to survey TUBP1 zymotic fluid is 78.62 ± 3.48% to verticillium dahliae inhibiting rate in vitro, and it is 21 ± 3.86mm to verticillium dahliae antibacterial circle diameter in vitro that Oxford agar diffusion method is surveyed Bacillus axarquiensis zymotic fluid.Show that by isolated test result Antagonistic Fungi Bacillus axarquiensis antagonism verticillium dahliae has inhibitory action.
Embodiment 5: pot experiment, Antagonistic Fungi Bacillus axarquiensis to cotton verticillium wilt potted plant control tested in the 2012-2013 4-9 month and carry out in the controlled environment chamber, chamber humidity is 60%～80%.Temperature is (25 ± 5) DEG C, and cotton seeds is in new land 36,1% clorox sterilization 10min for seed, and distilled water flushing, dries.
In antagonistic bacterium Bacillus axarquiensis inoculation NB medium, 37 DEG C of 180rmin
-1shaken cultivation 48h, the centrifugal 10min of 5000g, thalline aseptic 2% sodium carboxymethylcellulose (CMC) that suspends, cotton seeds is soaked in antagonistic bacterium Bacillus axarquiensis suspension 4h (comprising 200 cotton seeds and 1g antagonistic bacterium in 10mL2%CMC), not add antagonistic bacterium as control group, dry, before sowing, adjusting bacterial concentration by dilution plate counting method is 6 × 10
8cFUmL
-1.
V.dahliae ATCC36211 inoculation is 25 DEG C of cultivation 15d in PDA medium, and bacterium cake is made in the punching of 6mm card punch, is inoculated in 25 DEG C of 160rmin in PDB medium
-1shaken cultivation 15d, 8 layers of sterile gauze filter, and collect conidium, and adjusting bacteria concentration with blood counting chamber is 6 × 10
6mL
-1.
The control test of cotton verticillium wilt: (1) control group: V.dahliae ATCC36211; (2) processed group: V.dahliae ATCC36211+ Antagonistic Fungi; It is 6 × 10 that V.dahliae ATCC36211 conidium is inoculated in aseptic soil adjustment bacteria concentration
6g
-1, Antagonistic Fungi Bacillus axarquiensis concentration is 6 × 10
8g
-1, each processing 20 basin cottons, repeat 3 times by (each processing is 60 basins) totally.30d, 60d, 80d, 90d and 120d statistics disease index and relative control efficiency after inoculation.
From greenhouse pot culture result of the test (table 4), Antagonistic Fungi Bacillus axarquiensis has certain Biocontrol Effect to cotton verticillium wilt.After inoculation, 30～120d control group cotton verticillium wilt disease index is 10.87～39.88, and Antagonistic Fungi Bacillus axarquiensis processed group disease index is 3.85～21.28.Show that Antagonistic Fungi Bacillus axarquiensis can reduce the generation of cotton verticillium wilt.30～120d after inoculation, Antagonistic Fungi Bacillus axarquiensis zymotic fluid is 36.54%～42.56% (table 4) to verticillium dahliae relative control effect.
Table 4 Bacillus axarquiensis pot experiment control cotton verticillium wilt biocontrol effect
Embodiment 6: small plot experiment, Antagonistic Fungi Bacillus axarquiensis to the field control effectiveness test of cotton verticillium wilt in the 4-10 month in 2012 and 2013, connect cotton field in 12 28 of Production and Construction Corps of Xinjiang's agriculture one teachers and carry out, select to occur the heavier plot of verticillium wilt throughout the year.Community area is 30m
2, random district group is arranged.Sowing time is on April 15th, 2012 and 2013, and cotton variety is in new land 36, and soil is sandy loam, middle fertility, and irrigation conditions is better.One film 4 row, every row 30 strain cottons, each processing 120 strain cottons are repeated 3 times, and in the time that cotton seedling grows to 5～6 true leaves, the cotton seedling of the every strain of processed group is filled with the antibacterial Bacillus axarquiensis50mL of root inoculation short of money, and (bacteria concentration is 6 × 10
8mL
-1), control group is not inoculated Antagonistic Fungi, 60d, 90d, 110d, 130d and 140d statistics disease index and relative control efficiency, same pot experiment after inoculation.
Table 5 Bacillus axarquiensis field trial control cotton verticillium wilt effect
Small plot experiment result is as known in table 5, and Antagonistic Fungi Bacillus axarquiensis has certain Biocontrol Effect to cotton verticillium wilt.After inoculation, 60～140d control group cotton verticillium wilt disease index is 13.87～49.08, disease index shows a rising trend, it is 49.08 that the rear 140d disease index of inoculation reaches maximum, and Bacillus axarquiensis processed group disease index is respectively 3.85～21.28 (tables 5), Antagonistic Fungi Bacillus axarquiensis has certain biocontrol effect to cotton verticillium wilt, and its relative biological and ecological methods to prevent plant disease, pests, and erosion value is 38.04%～40.16%.130d after inoculation, Antagonistic Fungi Bacillus axarquiensis zymotic fluid is 43.07% to the maximum to verticillium dahliae relative control effect.140d after inoculation, relative control effect is 40.16%.
Claims (5)
1. the purposes of the sub-bacillus of Amado Azar base on control cotton verticillium wilt.
2. the purposes of the sub-bacillus of Amado Azar base on the biopesticide of preparation control cotton verticillium wilt.
3. prevent and treat a biopesticide for cotton verticillium wilt, wherein contain the sub-bacillus of Amado Azar base.
4. the purposes of biopesticide on control cotton verticillium wilt described in claim 3.
5. a method of preventing and treating cotton verticillium wilt, is characterized in that, soaks cotton seeds or fills with in cotton root with the sub-bacillus of Amado Azar base or the biopesticide that contains the sub-bacillus of Amado Azar base.
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