CN104877943A - Antagonistic bacterium for controlling radix rehmannia root rot and application of antagonistic bacterium - Google Patents
Antagonistic bacterium for controlling radix rehmannia root rot and application of antagonistic bacterium Download PDFInfo
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Abstract
The invention provides an antagonistic bacterium for controlling radix rehmannia root rot. The antagonistic bacterium is named as pseudomonas aeruginosa LK-12 (the Preservation No.: CGMCC 10966) after authentication. The pseudomonas aeruginosa LK-12 disclosed by the invention is obtained by screening from continuous cropping rhizosphere soil of radix rehmannia root through an agar disk diffusion method. Fusarium oxysporum and aspergillus flavus pathogenic bacteria separated from lesion parts of the radix rehmannia root have a high antagonistic effect; in the meanwhile, fusarium oxysporum, fusarium moniliforme, talaromyces sp. and other pathogenic bacteria separated from lesion parts of radix pseudostellariae also have a remarkable restriction effect and can be used for effectively controlling root rot diseases of medicinal plants comprising the radix rehmannia root to provide a strain for overcoming or relieving replant diseases of the medicinal plants. Therefore, the antagonistic bacterium is a potential biological control strain which is good in biological control effect, free of pollution as well as safe and ecological, and is wide in development and application prospects.
Description
Technical field
The present invention relates to a strain bacterial isolates, particularly the antagonistic bacterium of a strain control Radix rehmanniae root rot, can be used for overcoming or alleviate continuous cropping glutinous rehmannia soil-borne disease problem, belongs to microorganism and technical field of biological control.
Background technology
Understanding for Soil-sickness Problem is long-standing, and a lot of food crop (as wheat, potato), cash crop (as tobacco, cotton), oil crops (as soybean, peanut), vegetables (cucumber, tomato), garden crop (as watermelon, strawberry), medicinal plant (as ginseng, glutinous rehmannia, pseudo-ginseng) and artificial forest (as willow, China fir) all exist Soil-sickness Problem in various degree.But, the Soil-sickness Problem of long-standing problem China agriculture production, show particularly serious in Chinese medicinal materials cultivation and production, all there is Soil-sickness Problem in various degree in the block medicinal plants of about 70%, as the medicinal plants such as glutinous rehmannia, pseudo-ginseng, Radix Angelicae Sinensis, ginseng all exist serious Soil-sickness Problem.
Glutinous rehmannia (
rehmannia glutinosal.) being scrophulariaceae per nnial herb, being used as medicine, its medication is with a long history with block root, clinical efficacy is remarkable, is the famous famous-region drug of China.But the Soil-sickness Problem of glutinous rehmannia is particularly serious, and under continuous cropping, glutinous rehmannia plant often shows the symptom of retarded growth: the short and small reduction of overground part plant, leaf photosynthesis weaken, easily withered lodging; Underground part block root cannot normally expand, root/shoot ratio imbalance; Reduction in the life period, effective component cannot effectively accumulate, and medicinal quality declines; Disease and pest occurs rampant, and block root is perishable, the withered phenomenon of normal occurrence of large-area (as shown in Figure 1).Usually, must within 8 ~ 10 years, again can plant on same plot at interval after every stub land Huang results.Glutinous rehmannia continuous cropping not only cause output, quality sharply to decline and soil-borne disease serious, more alarmingly be, when the continuous cropping obstacle origin cause of formation and the mechanism of action are still not clear, major part peasant household attempts to maintain output by enriching the measure such as fertilizer, pesticide abuse, effect is often not good, not only increase Productive statistics, also cause that environmental pollution, Chinese medicinal materials agriculture are residual to exceed standard and the series of problems such as farmland ecosystem functional deterioration, make the cultivation and production of natural resources of Chinese medicinal materials be absorbed in vicious cycle.Therefore, a kind of reasonable effective measures are built for overcoming or alleviating the important content that continuous cropping obstacle and soil-borne disease problem become resources of medicinal plant ecological study.Seminar's early-stage Study finds, Fusarium oxysporum (as shown in Figure 2) is the important factor causing glutinous rehmannia continuous cropping obstacle, soil-borne disease wildness, and its content is done time limit increase with Herba Munroniae henryi and constantly increases.And the biological control utilizing Antagonistic Fungi to carry out Plant diseases is considered to a kind of science, reasonable, efficient, environmentally friendly measure.The present invention is directed to glutinous rehmannia specialized form Fusarium oxysporum, separation screening, to the antagonistic bacterium of this pathogenic fungi of plant height effect antagonism, is accredited as Pseudomonas aeruginosa, called after
pseudomonas aeruginosalK-12.
Summary of the invention
The object of this invention is to provide the antagonistic bacterium of a strain control Radix rehmanniae root rot, for root rot problem common in the continuous planting process of biological control glutinous rehmannia, provide biocontrol strain for overcoming or alleviating glutinous rehmannia Soil-sickness Problem.
Technical scheme of the present invention is as follows:
Antagonistic bacterium provided by the present invention be Pseudomonas aeruginosa (
pseudomonas aeruginosalK-12), this bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center of the Institute of Microorganism, Academia Sinica of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on June 9th, 2015, deposit number is CGMCC 10966.
Pseudomonas aeruginosa of the present invention (
pseudomonas aeruginosalK-12) to glutinous rehmannia specialized pathogen bacterium-Fusarium oxysporum, there is very strong antagonistic action.
Useful Antagonistic Fungi of the present invention has following advantage:
(1) Antagonistic Fungi-Pseudomonas aeruginosa of the present invention (
pseudomonas aeruginosalK-12) be screened by a large amount of screening operations, screen from 2946 strain soil bacterias and obtain, to glutinous rehmannia specialized form Fusarium oxysporum, there is very strong antagonistic effect.
(2) Antagonistic Fungi-Pseudomonas aeruginosa of the present invention (
pseudomonas aeruginosalK-12) to screen in glutinous rehmannia pathology root flavus and screen, in medicinal plant Root of Heterophylly Faalsestarwort continuous cropping soil or the Fusarium oxysporum of diseased region, naked joint mould, beading mould, also there is very strong antagonistic action.
(3) Antagonistic Fungi-Pseudomonas aeruginosa of the present invention (
pseudomonas aeruginosalK-12) derive from continuous cropping glutinous rehmannia rhizosphere soil, be not derived from other habitats, rhizosphere colonization effect is better and safe and reliable, often plants crop (as wheat, corn, paddy rice, soybean, peanut etc.) all without pathogenic infection ability to glutinous rehmannia and other.
(4) Antagonistic Fungi-Pseudomonas aeruginosa of the present invention (
pseudomonas aeruginosalK-12) it is 5 ~ 9.5 that culture temperature ranges preferably from 25 ° of C ~ 45 ° C, pH scope, and the temperature and the pH scope that are applicable to growth are comparatively wide, strong adaptability.
Accompanying drawing explanation
Fig. 1 is glutinous rehmannia overground part and the underground part growing state of the different continuous cropping time limit.
Fig. 2 is the colonial morphology of pathogenic bacteria-Fusarium oxysporum that glutinous rehmannia plant diseases root is separated to. A: represent bacterium colony front; B: represent bacterium colony reverse side.
Fig. 3 be Antagonistic Fungi Pseudomonas aeruginosa (
pseudomonas aeruginosalK-12) gramstaining and microscopic morphology are observed.
Fig. 4 be Pseudomonas aeruginosa (
pseudomonas aeruginosalK-12) with the opposite culture photo of pathogenic fungi. plate center inoculates all kinds of pathogenic fungi, wherein A: Fusarium oxysporum (glutinous rehmannia of originating); B: flavus (source glutinous rehmannia); C: Fusarium oxysporum (source Root of Heterophylly Faalsestarwort); D: ankle joint mould (source Root of Heterophylly Faalsestarwort); E: fusarium moniliforme (source Root of Heterophylly Faalsestarwort). B is that potato sucrose substratum (PSA) is dull and stereotyped, and all the other are potato glucose (PDA) substratum.
Fig. 5 be Pseudomonas aeruginosa (
pseudomonas aeruginosalK-12) meta-bolites suppresses Fusarium oxysporum growth. A: add LK-12 fermented liquid; B: do not add LK-12 fermented liquid.
Fig. 6 be Pseudomonas aeruginosa (
pseudomonas aeruginosalK-12) prevention and control Fusarium oxysporum infects glutinous rehmannia tissue cultured seedling. A: represent the tissue cultured seedling growing state vertical view only inoculating Fusarium oxysporum (left side) and inoculation Fusarium oxysporum and pseudomonas (right side); B: represent the tissue cultured seedling growing state side elevational view only inoculating Fusarium oxysporum (left side) and inoculation Fusarium oxysporum and pseudomonas (right side).
Fig. 7 be Pseudomonas aeruginosa (
pseudomonas aeruginosalK-12) prevention and control Fusarium oxysporum infects glutinous rehmannia detoxification transplanted seedling. and left three basins represent only inoculates Fusarium oxysporum; Right three basins represent inoculation pseudomonas (apart from root 2 cm inoculation one circle) and Fusarium oxysporum (apart from root 4 cm inoculation one circle).
Embodiment
Below in conjunction with specific embodiment, technical scheme of the present invention is described in detail.Following examples only for instruction and explanation of the present invention, and do not form the restriction to technical solution of the present invention.
Embodiment 1 Antagonistic Fungi Pseudomonas aeruginosa (
pseudomonas aeruginosalK-12) screening and qualification thereof
1, glutinous rehmannia specialized form Fusarium oxysporum Antagonistic Fungi
pseudomonas aeruginosathe screening of LK-12
Antagonistic Fungi Pseudomonas aeruginosa of the present invention (
pseudomonas aeruginosalK-12) screen from continuous cropping glutinous rehmannia rhizosphere soil and obtain.
1.1 pseudomonas selective medium is prepared
Pseudomonas selective medium (pseudomonas selective isolation agar, PSIA) compound method is as follows: take 20 g Soybean-casein digest nutrient agar (soybean casein digest agar, SCD) (BD, USA), add 495.5 mL distilled water heated and stirred, after abundant dissolving, add 1 mL 0.1%(wt/vol again) Viola crystallina storing solution, 121 ° of C autoclave sterilization 15 min, when being cooled to about 50 ° of C, 3.5 mL 5%(wt/vol are added again in substratum) Nitrofurantoin storing solution, after abundant mixing, rapid a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices, after cooled and solidified, namely obtained pseudomonas selective medium is dull and stereotyped.
Wherein, 0.1%(wt/vol) compound method of Viola crystallina storing solution is: take 0.1 g Viola crystallina (Sangon, Shanghai), be dissolved in 100 mL distilled waters, room temperature preservation is for subsequent use; The compound method of Nitrofurantoin storing solution is: take 5 g furadantins (Sangon, Shanghai), and join in 90 mL DMFs (Sangon, Shanghai) and fully dissolve, then be settled to 100 mL, room temperature keeps in Dark Place for subsequent use.
Pseudomonas separation screening in 1.2 rhizosphere soils
Take the fresh rhizosphere soil of 5 g, be dissolved in the sterilized water of 45 mL coolings, fully vibration shakes up, and obtains 10
-1diluent, gets 5 mL 10
-1in the sterilized water that dilution Soil Slurry cools to another bottle 45 mL, fully vibration shakes up, and obtains 10
-2diluent, again dilute 10
-3diluent, draws the soil dilution liquid of 60 μ L, and even spread is on the pseudomonas selective medium flat board made, and each extent of dilution is all coated with 3 flat boards, is placed in 32 ° of C constant incubator lucifuges and cultivates 30 h.Picking individual colonies is clear and legible and growth is uniform dull and stereotyped, is forwarded to the dull and stereotyped upper 32 ° of C of new selective medium and continues to cultivate, and when bacterium colony is high-visible, is placed in that 4 ° of C refrigerators are of short duration to be saved backup.
The Antagonistic Fungi screening of 1.3 glutinous rehmannia specialized form Fusarium oxysporums
Preparation potato sucrose substratum (PSA) or potato dextrose medium (PDA), be formulated as follows: take potato 200 g cleaning peeling, be cut into small pieces, add suitable quantity of water and boil rear timing 25 min, by four layers of filtered through gauze, add in filtrate sucrose 30 g(or glucose 30 g), agar 15 g, continue heated and stirred mixing, add water again and be settled to 1000 mL, packing after cooling a little, a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices or save backup after autoclaving.
Through PSA or the PDA substratum of 121 ° of C autoclave sterilizations, rapid a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices, obtained dull and stereotyped after cooled and solidified, bottom culture dish, cross the center of circle to cross frame, the pseudomonas of the activated cultivation of distance cm place, the center of circle 2.5 inoculation, be placed in after 28 ° of C constant incubator lucifuges cultivate 48 h, inoculate glutinous rehmannia specialized form Fusarium oxysporum in the central position of culture dish, be placed in 28 ° of C constant incubator lucifuge opposite culture a couple of days, the size that has that it's too late of Real Time Observation inhibition zone.Opposite culture is after 5 days, and filter out by observing inhibition zone size a strain to have strong antagonistic effect pseudomonas to glutinous rehmannia specialized form Fusarium oxysporum, carried out preservation to this bacterial strain, deposit number is CGMCC 10966.The strong antagonistic strain screened is carried out purifying cultivation, and inclined-plane saves backup.
2, the Antagonistic Fungi qualification of glutinous rehmannia specialized form Fusarium oxysporum
Through microscopic examination, 2.1 Antagonistic Fungis of the present invention (deposit number is: CGMCC 10966) can see that this bacterium is bacillus, tool list flagellum, Gram-negative bacteria (as shown in Figure 3).LB solid medium is flat, moistening bacterium colony, bacterium colony metal luster.Fluorochrome can be produced.Meanwhile, the physio-biochemical characteristics of this Antagonistic Fungi are: oxidase positive, can oxygenolysis glucose, produce acid not aerogenesis, can decompose fructose, glycerol, N.F,USP MANNITOL, proline(Pro), arginine, L-Ala simultaneously, but not decomposing sucrose and inositol.Can not hydrolyzed starch, can liquefy gelatin, decomposable asymmetric choice net urea, ammonium sulfate, ammonium chloride, ammonium nitrate, saltpetre.
2.2 16s-23s rRNA gene interval PCR identify
The strong antagonistic strain filtered out (deposit number is: CGMCC 10966) is forwarded to LB liquid nutrient medium and carries out enlarged culturing, extract genomic dna, pcr amplification 16s-23s rRNA gene interval, identifies for bacteria molecule.Bacterial genomes DNA extraction method is as follows: get 1 mL and shake pseudomonas bacterium liquid (the 28 ° of C spent the night, 180 rpm), centrifugal 5 min of 10000 rpm, remove supernatant, 950 μ L TE damping fluids suspension precipitations are added in precipitation thalline, and add 50 μ L 10% SDS solution and 5 μ L Proteinase Ks (20mg/mL), mixing, 37 ° of C water-bath 1 h, add 150 μ L 5 mol/mL NaCl solution and 150 μ L CTAB/NaCl solution (10% CTAB again, 4.1% NaCl), mixing, 65 ° of C water-bath 20 min, add isopyknic Fen ︰ Lv Fang ︰ primary isoamyl alcohol (volume ratio 25 ︰ 24 ︰ 1) solution and carry out extracting, centrifugal 10 min of 12000 rpm, by supernatant liquor, with isopyknic chloroform/primary isoamyl alcohol (24/1), extracting is once again, supernatant liquor equal-volume Virahol precipitation at room temperature 30 min, centrifugal 10 min of 12000 rpm, abandon supernatant, DNA precipitation is cleaned with 70% ethanol, dissolve with 50 μ L sterilized waters after natural air drying.
Adopt 16s-23s rRNA gene interval pcr amplification technology to carry out Molecular Identification to the pseudomonas of separation screening, primers designed sequence is: 1405f(5'-TGYACACACCGCCCGT-3') and 456r(5'-CCTTT CCCTCACGGTACTG-3').PCR amplification system (25 μ L) is: 12.5 μ L Taq PCR Master Mix (2 ×) (Sangon, Shanghai), 1.0 μ L upstream and downstream primers (10 μMs), 20 ng DNA profilings.Pcr amplification program is: 94 ° of C denaturation 5 min, and 94 ° of C sex change 1 min, 61 ° of C anneal 1 min, and 72 ° of C extend 90 sec, 35 circulations, continue 72 ° of C and extend 10 min.16s-23s rRNA gene fragment 1% agarose gel electrophoresis of amplification detects, and with Universal DNA Purification Kit glue recovery test kit (TIANGEN, Beijing) purifying recovery object band, and serves Hai Shenggong order-checking portion and check order.Sequencing sequence adopts BLAST instrument and ncbi database (Nucleotide collection(nr/nt)) compare.This Antagonistic Fungi of molecular biology identification is Pseudomonas aeruginosa, called after
pseudomonas aeruginosalK-12.
The fungistatic effect of embodiment 2 Antagonistic Fungi and antagonistic substance thereof detects
(1) Antagonistic Fungi opposite culture
Preparation PSA or PDA substratum, rapid a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices after 121 ° of C autoclave sterilizations, after cooled and solidified, obtained PSA or PDA is dull and stereotyped, bottom culture dish, cross the center of circle to cross frame, the pseudomonas of the activated cultivation of distance cm place, the center of circle 2.5 inoculation, be placed in after 28 ° of C constant incubator lucifuges cultivate 48 h, inoculate pathogenic fungi in the central position of culture dish, be placed in 28 ° of C constant incubator lucifuge opposite culture a couple of days, the formation of Real Time Observation inhibition zone.Cultivate discovery in 5 days, Pseudomonas aeruginosa (
pseudomonas aeruginosalK-12) can efficiently antagonism glutinous rehmannia specialized form Fusarium oxysporum, flavus and Root of Heterophylly Faalsestarwort specialized form Fusarium oxysporum, fusarium moniliforme, ankle joint mould, suppress its mycelial growth (as shown in Figure 4).
(2) fungistatic effect of antagonistic substance detects
The Antagonistic Fungi of preservation is inoculated in shaking culture 48h in LB liquid nutrient medium, and after high speed centrifugation, supernatant is degerming with 0.22 μm of filtering with microporous membrane, filtrate is adopted the dull and stereotyped additive process of metabolite to carry out fungistatic effect evaluation.Result shows, and with the addition of the test group of meta-bolites, pathogenic bacteria Fusarium oxysporum mycelial growth is suppressed, and does not add the control group of meta-bolites, and pathogenic bacteria mycelial growth is normal.By measuring little 1 cm(of the mycelia radius ratio control group of test group Fusarium oxysporum that finds to add meta-bolites as shown in Figure 5).
Wherein the experimental procedure of the dull and stereotyped additive process of metabolite is: be heated to by sterilized PDA substratum and melt completely, be cooled to about the 45 DEG C Antagonistic Fungi fermented liquids (2.5%) adding heat sterilization, pour in culture dish, treat that it solidifies after mixing.Access the glutinous rehmannia specialized form Fusarium oxysporum activated afterwards, be placed in 28 ° of C constant incubator lucifuges and cultivate a couple of days, Real Time Observation Fusarium oxysporum grows.
Embodiment 3 Antagonistic Fungi prevention and control Fusarium oxysporum infringement glutinous rehmannia tissue cultured seedling effect assessment
Preparation glutinous rehmannia tissue cultured seedling MS substratum (MS+0.2 mg/L 6-BA+0.2 mg/L IBA+30 g/L sucrose+7 g/L agar), each tissue culture bottle adds 30 mL substratum, through autoclave sterilization, to be cooled solidify after, a ditch is burnt in media surface with tweezers, in the strain of ditch side inoculation glutinous rehmannia seedling 2, being placed in 25 ° of C constant temperature group training rooms cultivates after 45 days, the pseudomonas LK-12 bacterium liquid 300 μ L of overnight incubation is added in ditch, control group adds equivalent LB substratum and replaces, glutinous rehmannia specialized form Fusarium oxysporum is inoculated at the opposite side (namely away from glutinous rehmannia tissue cultured seedling side) of ditch, period continues in ditch, add pseudomonas LK-12 bacterium liquid, dynamic observation Fusarium oxysporum growth and situation is infected to glutinous rehmannia tissue cultured seedling.Result shows, be added with between pathogenic bacteria and tissue cultured seedling in the test group of Antagonistic Fungi, Fusarium oxysporum growth is suppressed, only grow in ditch distal extent, and add in the control group of equivalent LB substratum, Fusarium oxysporum growth fast, ditch can be crossed, infect glutinous rehmannia tissue cultured seedling plant, cause tissue cultured seedling stem rot rotten, final dead (as shown in Figure 6).
Embodiment 4 Antagonistic Fungi prevention and control Fusarium oxysporum infringement glutinous rehmannia detoxification transplanted seedling effect assessment
Transplant the cultivation glutinous rehmannia tissue cultured seedling of 45 days in the culture medium of autoclave sterilization process, 1 strain planted by every basin, and domestication cultivation two weeks, the glutinous rehmannia transplanted seedling selecting growing way consistent carries out follow-up test.Around glutinous rehmannia transplanted seedling root, the pseudomonas LK-12 bacterium liquid of overnight incubation is enclosed in 2 cm place inoculations one, after 2 days, around root, 4 cm places (i.e. pseudomonas LK-12 outer ring) inoculates glutinous rehmannia specialized form Fusarium oxysporum, continue to cultivate in 25 ° of C constant temperature group training rooms, every 3 days of period added a pseudomonas LK-12 bacterium liquid, control group replaces to add equivalent LB substratum, dynamically observes Fusarium oxysporum and infects situation to glutinous rehmannia transplanted seedling.Result shows: only add in the control group of pathogenic bacteria, there is disease states very soon in glutinous rehmannia transplanted seedling, started to occur withered phenomenon the 10th day time, to perish substantially when 15 days, choose bacterium again sequence verification find that control group soil surface and dead plant have a large amount of Fusarium oxysporum mycelial growth, and the glutinous rehmannia detoxic seedling well-grown of test group, whole duration of test does not all have pathology phenomenon (as shown in Figure 7).
As fully visible, the strain Antagonistic Fungi (preserving number is CGMCC 10966) that the present invention filters out has very strong antagonistic action to glutinous rehmannia specialized pathogen bacterium, can effectively to the infringement of glutinous rehmannia, have broad application prospects in this field by prevention and control Fusarium oxysporum pathogenic bacteria.
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
SEQUENCE LISTING
<110> University Of Agriculture and Forestry In Fujian
The Antagonistic Fungi of <120> mono-strain control Radix rehmanniae root rot and application thereof
<130> 2
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 16
<212> DNA
<213> artificial sequence
<400> 1
tgyacacacc gcccgt 16
<210> 2
<211> 19
<212> DNA
<213> artificial sequence
<400> 2
cctttccctc acggtactg 19
Claims (2)
1. one strain control Radix rehmanniae root rot Antagonistic Fungi, it is characterized in that: described Antagonistic Fungi be Pseudomonas aeruginosa (
pseudomonas aeruginosa) LK-12, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on June 9th, 2015, culture presevation is numbered CGMCC 10966.
2. prevent and treat the application of Antagonistic Fungi in control glutinous rehmannia, Root of Heterophylly Faalsestarwort medicinal plant cultivating disease and soil-borne disease of Radix rehmanniae root rot as claimed in claim 1 for one kind.
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| CN105462894A (en) * | 2015-12-23 | 2016-04-06 | 广西大学 | Application of sophora tonkinensis endophytic bacterium B29 in preventing and controlling panax notoginseng anthracnose |
| CN105779342A (en) * | 2016-03-28 | 2016-07-20 | 福建农林大学 | Antagonistic bacterial strain of special-form fusarium moniliforme of radix pseudostellariae and application of antagonistic bacterial strain |
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| CN113717890A (en) * | 2021-09-02 | 2021-11-30 | 华南农业大学 | Bacillus salinus strain and application thereof |
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| CN114703101A (en) * | 2022-04-11 | 2022-07-05 | 河南省农业科学院烟草研究所 | Pseudomonas aeruginosa YZ66 and application thereof in preventing and treating fusarium root rot of tobacco and promoting growth |
| CN115039794A (en) * | 2022-06-30 | 2022-09-13 | 中国烟草总公司辽宁省公司 | Biocontrol microbial inoculum for preventing and treating tobacco target leaf spot disease as well as preparation method and application thereof |
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