CN105296396A - Cotton endophytic bacterium HB3S-13 and application of same in prevention and treatment of cotton verticillium wilt - Google Patents
Cotton endophytic bacterium HB3S-13 and application of same in prevention and treatment of cotton verticillium wilt Download PDFInfo
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Abstract
The invention relates to phytopathology, in particular to a cotton endophytic bacterium HB3S-13 and application of same in prevention and treatment of cotton verticillium wilt. The cotton endophytic bacterium HB3S-13 is obtained through isolation and screening of the stem and roots of a healthy cotton plant, classified and named as (Agrobacterium sp.) HB3S-13 and preserved in China Center for Type Culture Collection, the preservation number is CCTCC No. M2015604, and the preservation date is the thirteenth, October, 2015. The cotton endophytic bacterium HB3S-13 can be cultured on an artificial culture medium and is easy to obtain and culture so that factory production and application and popularization can be facilitated; the cotton endophytic bacterium HB3S-13 is good in prevention effect on cotton verticillium wilt, the prevention and treatment effect is 64.87-75.21%, the prevention effect is stable, and compared with bacillus subtilis which is applied much to production, wherein the prevention effect of bacillus subtilis is 46.0-52.9%, the prevention and treatment effect is improved by approximately 50%. In the prior art, no report about the biocontrol effect of the bacterium on cotton verticillium wilt is found, and the cotton endophytic bacterium HB3S-13 has great potential for being developed into a commercial biocontrol agent.
Description
Technical field
The present invention relates to plant pathology, specifically, the present invention relates to Endophytic Bacteria in Cotton HB3S-13 and the application in cotton verticillium wilt control thereof.
Background technology
Cotton verticillium wilt (Verticilliumwiltofcotton) is the important disease of one on cotton, cotton verticillium wilt is the silborne fungal diseases caused by verticillium dahliae (VerticilliumdahliaeKleb.), and it can cause cotton Severe Reduction, and even plant is dead.The conventional control method of cotton verticillium wilt has cultivates disease-resistant variety, agricultural measures and chemical prevention etc.Due to the variability of verticillium dahliae, the cultivation of disease-resistant variety and popularization are restricted.Rice field-upland field rotation is proved to be one comparatively effective means, but it is higher to the requirement at cotton field structure and water source.Chemical prevention and control method can cause the pollution to environment, and this method not only kills pathogenic bacteria, also kills the beneficial microorganism in soil, and the maintenance of the latter to nature or agricultural soil system all has important effect.Given this, people are devoted to find effective alternative method.Biological control is a kind of method having application prospect, utilizes biological control to prevent and treat cotton verticillium wilt, can guarantee the safety of physical environment, have the strategic importance of Sustainable development while improving plant growth and output.
The occurrence injury utilizing beneficial microorganism to control Plant diseases has economy, eco-friendly feature.About the research preventing and treating cotton verticillium wilt with endophyte as biocontrol microorganisms, domestic report is not rarely seen yet, but accomplish scale production at present and can be used in the but also few of production practice, a wherein subtilis comparatively successful example at last, the mechanism of action of its biological control is mainly produce the multiple antimicrobial substances such as bacitracin to suppress pathogenic bacteria, to the control of verticillium, there is certain effect, but in production application, there is many problems, wherein topmost problem is that control in field effect is lower, the subtilis formulation of domestic registration to the prevention effect of cotton verticillium wilt about 45%, and it is unstable in different year effect.Therefore, be necessary to excavate out the more effective microorganism resource of the control of cotton verticillium wilt, for future, the exploitation of biogenic pesticide provides corresponding basis.
As the advantage of biocontrol microorganisms, endogenetic bacteria is that bacterium is unicellular, growth and breeding is fast, is convenient to large-scale fermentative production and preservation.The mechanism of bacterium controlling plant diseases mainly contains antibiosis, the Competition in nutrition and site, hyperparasitism and inducing plant resistance effect.
Summary of the invention
Goal of the invention: the present inventor, for the deficiencies in the prior art, the object of this invention is to provide a kind of Endophytic Bacteria in Cotton HB3S-13, to realize carrying out biological control with it to cotton verticillium wilt.
Another object of the present invention is to provide the application of Endophytic Bacteria in Cotton HB3S-13 described above in cotton verticillium wilt control.
Technical scheme: in order to realize foregoing invention object of the present invention, the technical solution used in the present invention is: a kind of Endophytic Bacteria in Cotton, its Classification And Nomenclature is: (Agrobacteriumsp.) HB3S-13, be preserved in China typical culture collection center (CCTCC), address: Wuhan, China Wuhan University, its deposit number is: CCTCCNO:M2015604, preservation date: on October 13rd, 2015.
Described Endophytic Bacteria in Cotton HB3S-13, be obtain from healthy cotton plant stem and root separation screening to cotton verticillium wilt, there is better biocontrol effect and the endogenetic bacteria of preventive effect effect stability, this endogenetic bacteria well surely can be grown in cotton body, to people and animals, insect, plant safety non-toxic, also there is following character:
1, morphological specificity
The colonial morphology of bacterial strain HB3S-13 on NA substratum is faint yellow, and bacterium colony is circular, has projection; Gramstaining is carried out to bacterial strain HB3S-13, judges that HB3S-13 is Gram-negative rod-shaped bacterium.
2,16SrRNA analyzes
With the corresponding primer amplification of 16SrRNAITS sequence, amplified production carries out purifying and order-checking, sequence on sequencing result and NCBI finds after comparing, the homology of the 16SrRNAITS sequence of HB3S-13 endogenetic bacteria and rhizobium reaches 100%, therefore, HB3S-13 is attributed to rhizobium (Agrobacteriumsp.).
In order to realize another object of the present invention, the invention provides the application of Endophytic Bacteria in Cotton HB3S-13 described above in cotton verticillium wilt control.
Beneficial effect of the present invention:
1, HB3S-13 can cultivate on artificial medium, and easily obtain and cultivate, the bacterial strain for biological control must be able to be cultivated on artificial medium, is beneficial to factorial praluction and applies.
2, Endophytic Bacteria in Cotton HB3S-13 of the present invention has the bacterium of favorable effect to cotton verticillium wilt, its prevention effect is 64.87 ~ 75.21%, and preventive effect is stablized, to plant growth without toxic side effect, to people and animals, insect and crop safety, environmental friendliness, compared with above applying more subtilis (preventive effect is 46.0% ~ 52.9%) with production, prevention effect improves about 50%; In addition, Endophytic Bacteria in Cotton HB3S-13 of the present invention is through identifying that this bacterium is Gram-negative rod-shaped bacterium, for rhizobium (Agrobacteriumsp.), still the biocontrol effect of this bacterium on cotton verticillium wilt does not report to have the biological prevention and control agent that larger potential development becomes business.
Accompanying drawing explanation
Fig. 1 is the form of HB3S-13 on NA substratum;
Fig. 2 a, Fig. 2 b are respectively the aspect graph after gramstaining HB3S-13;
Fig. 3 is the comparison result figure of 16SrRNA sequence on NCBI of HB3S-13;
Fig. 4 is the antagonistic action result figure of HB3S-13 to verticillium dahliae;
Fig. 5 is the prevention effect figure of 30d after the mixed solution 1 obtained after verticillium dahliae spore liquid mixes with HB3S-13 Bacteria liquid equal-volume is inoculated cotton seedling;
Fig. 6 is the prevention effect figure of 30d after the mixed solution 2 obtained after verticillium dahliae spore liquid mixes with HB3S-13 ferment product equal-volume is inoculated cotton seedling;
Fig. 7 is the cotton seedling growth control figure (positive control picture group) only inoculating 30d after verticillium dahliae spore liquid;
Fig. 8 be HB3S-13 bacterium cultivate with verticillium dahliae make-up contrast speed of growth comparison diagram, wherein (a) is the colony growth situation of cultivating verticillium dahliae after 14d with HB3S-13 bacterium make-up, and (b) is the colony growth situation of cultivating verticillium dahliae after 14d with blank ware make-up.
Endogenetic bacteria HB3S-13, is preserved in China typical culture collection center (CCTCC), address on October 13rd, 2015: Wuhan, China Wuhan University, deposit number: CCTCCNO:M2015604.
Embodiment
Be below specific embodiments of the invention, technical scheme of the present invention is done to describing further, but protection scope of the present invention be not limited to these embodiments.Every do not deviate from the present invention's design change or equivalent substituting include within protection scope of the present invention.
The culture medium prescription that following examples use is:
Cha Shi culture medium prescription: saltpetre 2g, dipotassium hydrogen phosphate 1g, magnesium sulfate 0.5g, Repone K 0.5g, ferrous sulfate 0.01g, sucrose 30g, agar 15g, distilled water 1L, pH7.3 ± 0.2;
PDA slat chain conveyor based formulas: potato 200g, sucrose 20g, agar 20g, distilled water 1L;
NA culture medium prescription: extractum carnis 3g, peptone 10g, sodium-chlor 5g, agar 20g, pH7.3,
Distilled water 1L.
Embodiment 1
The separation method of cotton endophyte HB3S-13.
The sepn process of cotton endophyte is all carried out in super worktable, and the stem stalk mainly for cotton is separated with main root.The stem stalk of cotton and main root are cut into the plant fragments sample that 3cm is long, then the plant sample be cut into is carried out surface sterilization according to following program: the alcohol disinfecting 1min utilizing 70% (v/v), then the hypochlorite disinfectant 5min of 5% (v/v) is used, again use the alcohol disinfecting 30s of 70% (v/v), finally use rinsed with sterile water 3 ~ 5 times, each 3min.The plant sample of having sterilized is placed on aseptic dry filter paper, blots the moisture on surface under aseptic condition.With the scalpel of sterilization, sterilizing agent is immersed at the sample two ends of stem stalk and root, rest part sterile surgical cutter section, about 2mm is thick for every sheet.The sample sheet cut is placed on ready PDA flat board or NA substratum at once, and be placed in 25 DEG C of cultivations, the fungi of acquisition or bacterial colony are further purified by monospore or single mycelia.
Embodiment 2 endogenetic bacteria HB3S-13 carries out identification of morphology.
The colonial morphology of bacterial strain HB3S-13 on NA substratum is faint yellow, and bacterium colony is circular, has projection; Thalline is shaft-like, sees Fig. 1.Carry out gramstaining to bacterial strain HB3S-13, judge that it is Gram-negative rod-shaped bacterium, its form is as shown in Fig. 2 a, Fig. 2 b.
Embodiment 316SrRNA Molecular Identification.
With the corresponding primer amplification of 16SrRNA sequence, the length of 16SrRNA is 1333bp, specifically as shown in SEQIDNo.1.
SEQIDNo.1
The sequence sequence surveyed on result and NCBI finds after comparing, and the homology of the 16SrRNAITS sequence of itself and Agrobacteriumsp. reaches 100%, HB3S-13 is attributed to rhizobium (Agrobacteriumsp.).Comprehensive HB3S-13 morphological specificity, physiological characteristic and Molecular Identification result, be attributed to Agrobacteriumsp. by HB3S-13.
Embodiment 4 endogenetic bacteria HB3S-13 is to the antagonistic action (indoor) of verticillium dahliae.
By the central authorities of fresh verticillium dahliae V225 pure culture biscuits involvng inoculation at PDA flat board, after 25 DEG C of cultivation 3d, absorption 50ulHB3S-13 bacterium liquid is placed in four angles from colony edge 2cm place respectively, observes two bacterium and whether there is antagonistic action.As shown in Figure 4, result shows opposite culture result, there is not antagonistic action, equal well-grown between two bacterium.
Embodiment 5 endogenetic bacteria HB3S-13 is to the restraining effect of cotton verticillium wilt.
Verticillium dahliae V225 (defoliation bacterial strain) bacterium block is inoculated in Cha Shi substratum, 25 DEG C, 150rpm shake bacterium cultivate 7 ~ 10d, then obtain verticillium wilt pathogen spore liquid with after sterilizing 4 layers of filtered through gauze, described spore liquid concentration is 5 ╳ 10
6individual spore/ml, for subsequent use;
The bacterium liquid of HB3S-13 is inoculated in NA substratum, 28 DEG C, 150rpm shakes bacterium and cultivates after 3d, is 1 ╳ 10 with blood counting chamber adjustment bacterial concentration
8individual/ml, then moves in 50ml centrifuge tube by bacterium liquid, centrifugal 10 minutes of 6000r/min under normal temperature, and after centrifugal, supernatant liquor is the meta-bolites of HB3S-13 bacterial strain, i.e. HB3S-13 ferment product; The lower sediment sterilized water obtained after centrifugal is suspended, obtains HB3S-13 Bacteria liquid;
Mixed bacteria liquid 1 is obtained after above-mentioned verticillium dahliae spore liquid being mixed with HB3S-13 Bacteria liquid equal-volume, mixed bacteria liquid 2 is obtained after verticillium dahliae spore liquid and HB3S-13 ferment product being carried out equal-volume Homogeneous phase mixing, in addition also respectively using verticillium dahliae spore liquid 3 and sterilized water 4 as positive control and negative control group, the process of leaching root is carried out to follow-up cotton seedling.
With susceptible variety Ji cotton 11 for experimental cultivar, after the cotton-seed ginning of susceptible variety Ji cotton 11, soak 1h, plant in cotton matrix, when cotton seedling grows to 1 ~ 2 true leaf, cotton seedling is shifted out from matrix, use to be seeded; The method inoculated immerses in mixed bacteria liquid 1 described above, mixed bacteria liquid 2, verticillium dahliae spore liquid 3, sterilized water 4 by the root of cotton seedling respectively, plant respectively in disposal plastic cup by matrix again after 20min, the cotton seedling of 15 strains is managed in every component other places, be divided in 5 plastic cups, 3 young plant in each cup.
After inoculation, 15d institutes an inquiry disease index, and 25d after investigation disease index to inoculation, calculates disease index, diseased plant rate and prevention effect every three days.Experiment repetition 3 times, test-results is as shown in Fig. 5, Fig. 6, Fig. 7, and Disease investigation the results are shown in Table one and table two, and result shows, the disease index of positive control refers to higher than the disease after HB3S-13 process far away, and sterilized water negative control is not fallen ill.Repeated result shows that the average prevention effect of HB3S-13 Bacteria liquid to cotton verticillium wilt be the average prevention effect of 71.15%, HB3S-13 ferment product to cotton verticillium wilt is 60.67%.
Table one: greenhouse state of an illness exponential case questionnaire
Table two: repeatedly repeat experiment preventive effect result table
The volatile matter of embodiment 6 endogenetic bacteria HB3S-13 is to the restraining effect of verticillium dahliae.
Adopt make-up culture method, cut-off footpath is two, the culture dish of 90mm, the PDA substratum of 20ml is poured respectively at the bottom of ware (b) at the bottom of (a), ware, then by the PDA substratum in (b) at the bottom of verticillium dahliae pure culture biscuits involvng inoculation to ware, endogenetic bacteria HB3S-13 bacterium liquid 100ul is uniformly coated in the PDA substratum in (a) at the bottom of ware, by two ware make-ups, with ParafilmTM, be placed in 25 DEG C of constant incubators to cultivate, not connect bacterium in contrast in (a) at the bottom of ware.Experiment in triplicate.Observe the upgrowth situation of verticillium dahliae, 8d starts to adopt right-angled intersection method to calculate colony diameter and calculates its speed of growth, surveys once, until 14d every 1d.With the inhibiting rate that the numerical evaluation HB3S-13 endogenetic bacteria Volatile Metabolites measured by 14d grows verticillium dahliae, after 14 days, the upgrowth situation of verticillium dahliae is shown in Fig. 8 (b).In addition, design one group of control experiment, prepare the culture dish of above-mentioned formed objects, carry out make-up cultivation by above-mentioned same step to verticillium dahliae, difference is only not to be coated with endogenetic bacteria HB3S-13 bacterium liquid in (a) at the bottom of ware.
Inhibiting rate=(contrast mean diameter-process mean diameter)/contrast mean diameter × 100%.The results are shown in Table three.
Result shows that volatile matter that HB3S-13 bacterium liquid produces can suppress the growth of verticillium wilt pathogen, and inhibiting rate is 52%.
Verticillium dahliae growing state comparison sheet in table three: HB3S-13 and verticillium dahliae make-up cultivation group and control group
Embodiment 7 endogenetic bacteria HB3S-13 is on the impact of cotton seeds seedling rate.
With susceptible variety Ji cotton 11 for experimental cultivar, after the seed lint of susceptible variety Ji cotton 11, be immersed in after soaking 12h in HB3S-13 bacterium liquid, plant in cotton matrix; With the seed of Ji cotton 11 water-treated clearly as a control group, its seedling rate is calculated after 3d.Result shows, the seeding ratio after the process of HB3S-13 bacterium liquid is 68.67%, and seed (control group) seedling rate after clear water process is 75.5%, and illustrate, the impact of HB3S-13 endogenetic bacteria on seeding ratio is little.
Claims (3)
1. Endophytic Bacteria in Cotton HB3S-13, its Classification And Nomenclature be (
agrobacteriumsp.) HB3S-13, be preserved in China typical culture collection center (CCTCC), its deposit number is: CCTCCNO:M2015604, preservation date: on October 13rd, 2015.
2. Endophytic Bacteria in Cotton HB3S-13 according to claim 1 is for controlling the application of cotton verticillium wilt.
3. Endophytic Bacteria in Cotton HB3S-13 according to claim 1 is for suppressing the application of verticillium dahliae.
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| CN109988727A (en) * | 2018-12-25 | 2019-07-09 | 南阳师范学院 | Verticillium dahliae endophyte and its application |
| CN113151043A (en) * | 2021-02-04 | 2021-07-23 | 塔里木大学 | Rhizobium DG3-1 for resisting cotton verticillium wilt and application thereof |
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| CN113151043A (en) * | 2021-02-04 | 2021-07-23 | 塔里木大学 | Rhizobium DG3-1 for resisting cotton verticillium wilt and application thereof |
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