CN114365749A - Composition for preventing and treating black rot and black shank of tobacco root and improving character and quality - Google Patents
Composition for preventing and treating black rot and black shank of tobacco root and improving character and quality Download PDFInfo
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- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
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Abstract
The invention provides a composition for preventing and treating tobacco root black rot and black shank and improving characters and quality, which comprises bacillus amyloliquefaciens cd5bc, streptomyces oxysporum WS-29246 and methyloxanil-manganese zinc; the preservation number of the bacillus amyloliquefaciens cd5bc is CCTCC NO: M2016232; the preservation number of the streptomyces oxysporum WS-29246 is CCTCC NO: M2012413. The invention can achieve the control effect of more than 90% aiming at the tobacco black shank and the tobacco root black rot, and overcomes the technical problem of low control effect of the conventional biological control method; the dosage of the first frost, manganese and zinc can be very low, and the harm to soil and environment is small; the invention can effectively reduce the cadmium accumulation in the tobacco, effectively reduce the lysine in the tobacco, improve the quality of the tobacco and reduce the harm to human bodies, and has good application prospect.
Description
Technical Field
The invention belongs to the technical field of agriculture, relates to a tobacco planting technology, and particularly relates to a technology for preventing and treating tobacco root black rot and black shank and improving tobacco characters and tobacco related substance content.
Background
Root rot in tobacco production[1]And black shank[2]Is a serious soil-borne disease and has been highly concerned by researchers. The root black rot and the black shank mainly occur in Yunnan, Guizhou, Hubei, Hunan, Sichuan and other places in China, the incidence rate of serious disease fields can reach more than 30 percent, and if the root black rot and the black shank are not controlled, the harm of the serious disease fields can be increased year by year.
Root black rot is caused by rhizomatoid moniliforme: (Thielaviopsis basicola) The hyphae of the tobacco are colorless in the initial stage and brown after the hyphae are branched, the tobacco can generate black root rot from the seedling stage to the adult stage, and the black root rot has great influence on the yield and the quality of the tobacco[3]. The black shank is caused by phytophthora nicotianae, can be attacked in seedling stage and field stage, and is easier to attack under the conditions of long continuous cropping annual limit and excessive concentration of varieties of tobaccos[4]。
At present, the control methods for epidemic diseases including root black rot and black shank can be divided into chemical control methods, biological control methods, soil control methods, botanical drug control methods, and comprehensive control methods combining various methods.
The chemical prevention method is to use chemical agents to block pathogenic bacteria infection or inhibit and kill pathogenic bacteria. Common chemical agents for root rot include thiram, thiophanate methyl, and nail cream
Manganese, zinc, carbendazim, etc[5]The common chemical agents for the black shank comprise fluocinolone acetonide, thiophanate methyl, dimethomorph, azoxystrobin, methoprene, manganese zinc and the like[6]. However, although the chemical control method has good and quick effect, the long-term use of high dose is not beneficial to agricultural development, and has important influence on soil, environment, animals and human beings.
The soil control method is to improve soil or to plant crops using special soil. Generally, researchers believe that the incidence of root black rot is lower when the pH of the soil is lower[7]This may be followed bypH influences the distribution and activity of antagonistic microorganisms and pathogens in soil[8]. In addition to adjusting physical properties such as pH of soil, researchers mainly use drugs[9]Or a microorganism[10]The soil is treated to a level suitable for reducing morbidity.
The botanical drug control method is more and more favored by people because of better environmental protection property, although it still uses drugs for control than the generally recognized chemical control method. Chinese patent 'a plant source medicinal fertilizer and a preparation method and application thereof' (publication No. CN 106804632A) obtains the plant source medicinal fertilizer capable of preventing and treating tobacco black shank by fermenting a plurality of Chinese medicinal materials.
Among all control methods, currently, the biological control method is generally considered to have a better prospect, and the method mainly utilizes exogenous microorganisms as fertilizers, can avoid the problem of '3R' caused by high-dose application of chemical agents, and is a green and environment-friendly method. Luoyanyan, a current state of biological control of black shank and black root rot[11]Henhaiying[12]All of which are discussed in greater detail in the Master thesis. At present, the biological control microorganisms which are often selected mainly include bacillus, pseudomonas and azotobacter. For example, Liujianjin et al[13]The bacillus amyloliquefaciens YN48 strain is adopted as a biocontrol bacterium to obtain good effect on preventing and treating the tobacco black shank; yilong et al[14]The method has the advantages that a spore strain R27 is separated, the growth and pathogenicity of hypha of the black rot of tobacco roots can be well inhibited, and the control effect of carbendazim and thiophanate methyl is approximate to that of the control effect of the carbendazim and thiophanate methyl in the aspects of the morbidity and the disease index of tobacco seedlings; zhang Meng et al[15]Stenotrophomonas maltophilia YCYM-04 and Bacillus belgii YCYM-09 are separated, and the control effect of the two strains on tobacco black shank is more than 50%; royuying et al[16]Two strains of bacillus amyloliquefaciens and one strain of bacillus subtilis are screened, and the control effect on the tobacco black shank reaches 60.2-70.1%; further, as in CN108135178A, the agricultural biocenosis of the United states has screened a pathogen against a number of pathogensThe bacillus subtilis AIP61892 has prevention and treatment effects.
In the above-exemplified biological control method, a strain having a control efficacy is mainly screened in a large amount or discovered by chance, which causes difficulties in finding the corresponding strain, thereby making the research of the biological control method slow. In fact, although the existing strains with control effects on black rot and black shank are mainly concentrated in a few genera such as bacillus, pseudomonas and azotobacter, the strains with control effects are found to be no different from canhai millet compared with the total amount of the strains in the genera, and the difficulty is great when seeking new strains with control effects from the known strains in the genera. In the prior art, the discovery of new strains is generally to discover soil with a low incidence rate, then to perform a large amount of strain screening work aiming at the soil, and to perform a large amount of detailed microorganism experiments, and finally to determine whether the strains obtained by corresponding separation have the control efficacy.
In addition to the prevention and treatment of epidemic diseases by using a single strain alone, in the field of biological prevention and treatment, a method of combining a plurality of strains is usually adopted, such as Liuchang and the like[17]The method comprises the steps of enabling pseudomonas to be matched with trichoderma viride, enabling bacillus to be matched with trichoderma harzianum, and enabling the bacillus to be matched with the trichoderma harzianum
The control of the fusarium root rot of the tobacco and the improvement of the agronomic traits achieve better effects, but the control may be in the reason of confidentiality, and the report does not disclose specific information of related strains; plum seedling, etc[18]In order to solve the problem of unstable control effect of a single strain, a large number of tests are carried out to screen out Bacillus belgii GY1, Bacillus amyloliquefaciens GY10 and Bacillus subtilis GY12, and the three are mixed, so that the potted plant control effect reaches 74.53%.
In addition to the combination of multiple strains, some researchers have also studied the control effect of combining specific microorganisms and chemicals in the field of tobacco blight. Such as Liujianjin[19]The combination of bacillus amyloliquefaciens YN48 and flumioaluminum, refined methyl frost and manganese zinc is considered to have better control effect. However, the microorganism is combined with a drugThere has been a decrease in control, probably due to the tendency of chemical agents to inhibit microbial growth or to antagonize microbial metabolites, such as Xuyuru et al[20]The control effect is greatly reduced after the cream A and the manganese zinc are added on the basis of the trichoderma.
However, while biological control methods have advantages over existing methods, the control obtained by biological control methods is generally low relative to chemical control. Such as Yan Qing Yan[21]The control effect of 5 common chemical agents on the black shank can reach more than 70 percent through investigation, while the control effect of more than 70 percent is difficult to obtain through a biological control method, and the control effect of only a few biological control bacteria approaches or slightly exceeds 70 percent. Meanwhile, as mentioned above, the biocontrol bacteria are not easily obtained, and a large amount of screening work is required.
In addition, the control of tobacco quality, such as reducing heavy metal absorption, controlling the content of corresponding amino acid, etc., often depends on tedious cultivation system or chemical agent[22]The microorganisms are extremely easy to be inhibited by chemical agents, so that the obvious defect exists in the tobacco epidemic disease prevention and control by utilizing the microorganisms, namely the occurrence of the epidemic disease can be usually prevented, but the quality of the tobacco is difficult to further improve.
In summary, there is a need in the art to provide a biological control technology with high control efficiency, which can improve the growth traits of tobacco and improve the quality of tobacco, aiming at the disadvantages faced by the existing biological control methods.
References in this section:
[1] molecular identification and pathogenicity analysis of Nicotiana tabacum of Henan province [ J ] tobacco science and technology, 2017, 50(3):7.
[2] Occurrence and comprehensive control of tobacco black shank by Lemna minor [ J ] southern agriculture, 2018, 12(35):2.
[3] Zhao Wen Jun, Yang Jiu Wen, Von Yu, and so on, the research on the prevention and treatment of black rot of tobacco root [ J ] Hunan agricultural science 2015(1):3.
[4] Zhaohui, Wangxi English, Liu national right, Tan Zhiyong, tobacco black shank occurrence factor and comprehensive prevention and treatment research progress [ J ] Hunan agricultural science 2020(11):5.
[5] Juan Song Yu, Trichoderma asperellum T-6 has growth promoting and disease resisting effects on tobacco [ D ] Shandong agricultural university, 2019.
[6] Yanqingyan, the control effect of different medicaments on tobacco black shank is compared and analyzed, wherein the Yanqingyan, Henan agriculture, 2019(11) and 3.
[7]Doran W L．Relation of the adjustment of soil reaction to black root rot of tobacco.Science,1927,66：661~662.
[8]Kaufmann D DWilliams L E∙Effect of mineral fertilization and soil reaction on soil fungi.Phytopathology,1964,54：134~139.
[9] Huangshiwang, Wangchun, Wenyun, etc. research on the prevention and control effect of soil fumigation on tobacco soil-borne diseases [ J ] Hunan agricultural science 2021(11) 59-61.
[10] Study on the improvement of soil biological characteristics and the prevention and treatment of tobacco black shank by using a compound microbial preparation [ J ] China soil and fertilizer, 2021(1).
[11] Research on screening, identification and biological control of rhizosphere antagonistic bacteria of Royun Yan tobacco root black rot [ D ]. northwest university of agriculture and forestry science and technology, 2021.
[12] Penhaiying, biological control technology research on tobacco black shank and root black rot [ D ] Shandong agricultural university 2020.
[13] Liu Jian jin, Liu Zi apparatus, Luoshan, etc. adopts biocontrol bacterium YN48 to prevent and control tobacco black shank [ J ] in the early stage, Jiangxi agricultural science, 2019, 31(4):4.
[14] Screening of antagonistic spore strains for preventing and treating tobacco root black rot [ J ] plant pathology report 2011, 41(3):4.
[15] Zhang Meng, Liang Junyang, Wang Qing Fu, Dou ya Fei, Zhang Jie, Liu Qiang, Pabo, Peng Jianfei tobacco Black shank biocontrol bacteria identification and prevention efficacy determination [ J/OL ]. Henan university of agriculture bulletin 1-13[2022-01-05].
[16] Screening of tobacco black shank biocontrol bacillus and field control effect thereof [ J ] Guizhou agricultural science, 2019, 47(1):6.
[17] Liu Chang, Yaoheng 34387, Li Xiaojie, and the like.
[18] The prevention and treatment effect of plum seedling, king phoenix dragon, king xiaoqiang, etc. on tobacco black shank by biocontrol bacteria recombination [ J ] Chinese tobacco science 2020, 41(2):7.
[19] The study on the prevention and treatment of tobacco black shank by matching of the bacillus amyloliquefaciens YN48 and a bactericide [ J ]. the school newspaper of Yunnan agricultural university: nature science edition 2020,35 (2):6.
[20] Xuyuru, Yangshaoxing, Quzhengyuan, etc. the prevention and cure effect of different medicines on tobacco black shank [ J ] Liaoning agricultural science, 2021(5):5.
[21] Yanqingyan, the control effect of different medicaments on tobacco black shank is compared and analyzed, wherein the Yanqingyan, Henan agriculture, 2019(11) and 3.
[22] Huang Zhen Rui, Lin Aten, Li Ji Du, Mazhu Wen, Li Shu Ling-Nai' an and humic acid promote the growth of flue-cured tobacco and reduce the tobacco leaf cadmium pollution synergistic effect [ J ]. southern agricultural science, 2021,52(02): 429-.
Disclosure of Invention
In view of the drawbacks and practical needs of the prior art, the object of the present invention is to provide a composition that solves the following disadvantages of the prior art: (1) the problem that the control effect of the existing biological control method is too low for tobacco black shank and root black rot is solved; (2) the problem that the growth character of the tobacco is difficult to improve under the condition of low control effect is solved; (3) solves the problem that the content of heavy metal and related amino acid in tobacco can not be effectively improved by the existing biological control method.
On the basis, the invention specifically aims to provide a composition which has the control effects on tobacco black shank and tobacco root black rot of more than 90 percent, effectively improves the growth characteristics of tobacco, obviously reduces the content of tobacco septa and the content of lysine in tobacco and a preparation method thereof.
In order to achieve the above purpose, the invention provides the following technical scheme:
a composition for preventing and treating tobacco root black rot and black shank and improving characters comprises bacillus amyloliquefaciens cd5bc, streptomyces oxysporum WS-29246 and bloom-forming manganese zinc.
Bacillus amyloliquefaciens cd5bc is disclosed in Chinese patent "A Bacillus amyloliquefaciens cd5bc and its application" (publication No. CN 105907683A), with the preservation number of CCTCC NO: M2016232, and is used for preventing and treating pathogenic bacteria of capsicum sclerotinia, bitter melon fusarium wilt, cucumber fusarium wilt, yellow black spot, rice blast, rice sheath blight, broad bean red spot, broad bean brown spot, broad bean red spot, and broad bean brown spot. Streptomyces oxysporus WS-29246 is disclosed in Chinese patent "Streptomyces oxysporus and its application in preventing and treating clubroot of Cruciferae", with the preservation number of CCTCC NO: M2012413, and is used for preventing and treating clubroot of Cruciferae.
As shown in one test example of the invention, the invention unexpectedly discovers that the combination of the bacillus amyloliquefaciens cd5bc, the streptomyces oxysporum WS-29246 and the cream-methyl manganese zinc has remarkable control effect on tobacco black shank and tobacco black root rot, and the dosage of the cream-methyl manganese zinc can be extremely low. This unexpected result was discovered by the inventors in their work to try to utilize whether Streptomyces oxysporum has a preventive effect against related blight of tobacco. To the best of the inventor, no report exists in the prior art for preventing and treating related epidemic diseases of tobacco by using streptomyces oxysporum.
Encouraged by the unexpected discovery, the inventor researches the combination of Streptomyces oxysporum WS-29246 and other reported strains with control efficacy on tobacco black shank and black root rot and the cream-A-Mn-Zn, and examines the control efficacy of the combination on the tobacco black shank and the black root rot. Unfortunately, however, although selected strains have been reported to have control efficacy, the control efficacy after combination is very limited. The inventors speculate that this may be due to antagonism between strains or inhibition of the drug against the selected strains, and confirmed this by the present invention through test examples 3 and 4.
As shown in the embodiment of the invention, in the research process, the composition not only has good control effect on tobacco black shank and black root rot, but also can well improve the agronomic characters of tobacco. Particularly unexpectedly, when the inventor detects the related content in the tobacco, the composition of the invention is also found to effectively reduce the accumulation of heavy metal cadmium and reduce the content of lysine.
The research of screening special strains to reduce heavy metal accumulation is still in the preliminary stage at present, and related reports are few. Many studies have been made mainly on the basis of the regulation of the pH in the soil by specific strains to modify the uptake of heavy metals by crops, such as Wangxin[23]Screening out an alcaligenes Bacillus XT-4 which can obviously increase the pH value of rhizosphere soil and lead CaCl in the soil2The content of the extracted cadmium is reduced, so that the content of heavy metal Cd on the overground part of the pakchoi is reduced; there are reports of the Xuhong Master thesis of the university of Hunan industry based on the same principle[24]. There have been studies on the regulation of heavy metal content in crops by screening heavy metal-fixing bacteria and reducing the effectiveness and migratory ability of heavy metals in soil, such as Sunleni[25]A technology of cadmium tolerance immobilized bacteria Pseudomonas sp.YM 4 in inhibiting wheat cadmium absorption is reported.
However, through experiments, the composition of the present invention cannot significantly increase the pH of soil, and whether the composition of the present invention can produce effects similar to those of heavy metal-immobilized bacteria remains to be further studied.
The content of amino acids in tobacco is influenced by variety, region, different parts and cultivation measures, and the content is shown in Zhao Tian Master thesis "influence of different cultivation and modulation measures on amino acid content of burley tobacco and flue-cured tobacco[26]Is described in detail in (1). At present, the reports of using microorganisms to regulate lysine in tobacco are less, and the regulation is mainly carried out by changing the cultivation and baking modes and modifying genes, such as Chinese patent 'cultivation and baking method for reducing the lysine content of flue-cured tobacco' (CN 109804879A) and Wang Yi Tung's paper' increase of lysine content in transgenic tobacco[27]. As shown in the embodiment of the invention, the lysine in the tobacco can be effectively reduced, so that the ammonia in the tobacco can be reduced, and the damage of the tobacco to human bodies can be reduced. Since the research of regulating and controlling specific amino acids in tobacco by using microorganisms has not been carried out on a large scale, the theoretical mechanism in the aspect needs to be further investigated.
As an alternative embodiment of the invention, the Bacillus amyloliquefaciens cd5bc and Streptomyces oxysporum WS-29246 are added in the form of fermentation broth; the cream-a-methyl and manganese-zinc wettable powder is 72 percent of cream-a-methyl and manganese-zinc wettable powder.
The 72% wettable powder of the first frost and the manganese zinc is also called 72% of the first frost and the manganese zinc (wettable powder) and is a product under the HG/T4946-.
Preferably, the concentration of the bacillus amyloliquefaciens cd5bc fermentation liquor is 1 x 106cfu/mL~1×107cfu/mL; in the Streptomyces oxysporus WS-29246 fermentation liquor, the bacterial concentration is 1 × 106cfu/mL~1×107cfu/mL。
Preferably, the cream-methyl-manganese-zinc is 1000-5000 times of a diluent of 72% cream-methyl-manganese-zinc wettable powder.
Preferably, the cream-methyl manganese-zinc is 5000 times of diluent of 72% cream-methyl manganese-zinc wettable powder.
According to the preferable technical scheme, the composition comprises bacillus amyloliquefaciens cd5bc, streptomyces oxysporum WS-29246 and methyloxanil/manganese zinc according to the volume ratio of (10-30) to (10-20) to 1. More preferably, the composition consists of bacillus amyloliquefaciens cd5bc, streptomyces oxysporum WS-29246 and zinc manganese methyl acetate in a ratio of 15:10:1 by volume.
As one alternative embodiment of the invention, the Streptomyces oxysporum WS-29246 fermentation broth is obtained by inoculating Streptomyces oxysporum WS-29246 and fermenting, wherein the used culture medium comprises the following components: 100g of cane sugar, 10g of glucose, 0.1g of casamino acid, 5g of yeast extract powder, 21g of propionic acid and K2SO4 0.25g，MgCl2·6H2O10 g, trace element 1mL, water 1000mL, pH = 7.0; wherein the microelement formula is MnSO4·7H2O 100mg，CuSO4·7H2O 50mg，ZnSO4·7H2O 100mg，CoCl2100mg and 100mL of purified water; the fermentation liquor of the bacillus amyloliquefaciens cd5bc is obtained by inoculating bacillus amyloliquefaciens cd5bc and fermenting, and the used culture medium is LB culture medium.
The preparation method of the composition provided by the invention comprises the following steps: mixing the above components uniformly.
The invention provides application of the composition in preventing and treating tobacco root black rot and black shank.
The invention has the beneficial effects that:
1. the invention can achieve the control effect of more than 90% aiming at the tobacco black shank and the tobacco root black rot, and overcomes the technical problem of low control effect of the conventional biological control method; the dosage of the first frost, manganese and zinc can be very low, and the harm to soil and environment is small.
2. The method can effectively reduce the cadmium accumulation in the tobacco, improve the quality of the tobacco and enlarge the planting range of the tobacco.
3. The invention can effectively reduce lysine in tobacco, reduce ammonia in tobacco and reduce the damage of tobacco to human body.
References in this section:
[23] wangxin, the research on the screening, identification and prevention and control of vegetable cadmium accumulation effect of Alcaligenes Bacillus XT-4 [ D ]. Nanjing agriculture university, 2019.
[24] Xuhong, influence and mechanism of the alkaline-producing microbial compound inoculant on cadmium accumulation of rice are explored to [ D ]. Hunan university of industry, 2021.
[25] Sunleni, Guo Ying Xue Ting, Hou Jie, Yang Zhang, Chen Meng, Tianwei, cadmium tolerance fixed bacteria screening and control effect on cadmium absorption of different varieties of wheat [ J ] agricultural environmental science reports 2020,39(09): 1878-.
[26] Effect of different cultivation and modulation measures on amino acid content of burley tobacco and flue-cured tobacco [ D ]. university of agriculture in south of hewn, 2012.
[27] Wang Ying, Zheng jin Gui, Xie Gui Zi increase content of lysine in transgenic tobacco [ J ]. application and environmental biology report, 2005(01): 32-35.
Detailed Description
The present invention is described in detail below by way of examples, and it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention.
In each of the examples and test examples of the present invention, the strains used were purchased commercially or given away, and the present invention does not relate to screening of new strains.
Example 1
1. Raw materials
The bacillus amyloliquefaciens cd5bc with the preservation number of CCTCC NO of M2016232 is sold in the market;
streptomyces oxysporum WS-29246 with preservation number of CCTCC NO: M2012413, available on the market;
72 percent of a wettable powder of methyl cream and manganese zinc, Fuda agriculture and chemical company of Jiangsu province, Jiangyin city;
LB culture medium: self-preparation in a laboratory;
streptomyces oxysporum WS-29246 medium, laboratory self-prepared: 100g of cane sugar, 10g of glucose, 0.1g of casamino acid, 5g of yeast extract powder, 21g of propionic acid and K2SO4 0.25g，MgCl2·6H2O10 g, trace element 1mL, water 1000mL, pH = 7.0; wherein the microelement formula is MnSO4·7H2O 100mg，CuSO4·7H2O 50mg，ZnSO4·7H2O 100mg，CoCl2100mg and 100mL of purified water;
test soil: disease-free soil in tobacco planting field
Tobacco seedling: yunyan 87, owned by the laboratory.
2. Potted plant control effect experiment
Fermenting the fermentation liquor (bacterial concentration 1 × 10) of the bacillus amyloliquefaciens cd5bc6cfu/mL), Streptomyces oxysporus WS-29246 fermentation broth (concentration 1X 10)6cfu/mL) and 72% of methyloxathite-manganin wettable powder (diluted by 5000 times of water) are mixed according to the volume ratio of 1:1:0.1 to obtain a mixed medicament; after 40 days of tobacco seedling transplantation, the tobacco seedlings are divided into two groups, each plant in the two groups is irrigated with 20mL of mixed medicament, one group is inoculated with phytophthora nicotianae and the other group is inoculated with the tobacco root rot pathogen after 5 days, and the disease incidence condition is investigated according to the standard GB/T23222-2008.
Through detection, the prevention effect of the experiment on the tobacco black shank reaches 91.3%, and the prevention effect on the tobacco root black rot reaches 90.7%. The prevention effect of the invention exceeds the medicament composition (CN 108633913B) provided by Chinese patent depending on chemical medicaments applied in 2018, and the breakthrough of preventing and controlling tobacco black shank and tobacco black root rot by using microorganisms is realized.
On the basis, the fermentation liquor (bacterial concentration is 1 multiplied by 10) of the bacillus amyloliquefaciens cd5bc6cfu/mL), Streptomyces oxysporus WS-29246 fermentation broth (concentration 1X 10)6cfu/mL) and 72% wettable powder of methyl cream and manganese zinc (diluted with 5000 times of water) were adjusted to 3:1:0.1, 2:1:0.1 and 1:2:0.1 by volume ratio, and then the same experiment was performed, and the results are shown in Table 1, wherein the experiment groups are respectively designated as experiment group 1, experiment group 2 and experiment group 3.
TABLE 1
On the basis of the above, the 72% wettable powders of methyl cream and manganese zinc were adjusted to 1000 times, 2000 times, 3000 times and 4000 times, respectively, and the same experiments were carried out under the same conditions, and the results were respectively set as experiment group 4, experiment group 5, experiment group 6 and experiment group 7, and are shown in table 2.
TABLE 2
3. Agronomic trait experiment of potted plant
After sowing, mixed medicament (Bacillus amyloliquefaciens cd5bc fermentation liquor (bacterial concentration 1X 10) in 1 pot control effect experiment of root irrigation respectively after thinning, first leaf cutting and second leaf cutting6cfu/mL), Streptomyces oxysporus WS-29246 fermentation broth (concentration 1X 10)6cfu/mL) and 72% methyloxanil-manganese-zinc wettable powder (diluted by 5000 times of water) in a volume ratio of 1:1: 0.1), and 20mL of the mixed agent is added into each plant, and the mixed agent is not added to serve as a control group. The potting soil is not inoculated with tobacco root black rot and tobacco phytophthora. The stem height, stem circumference and seedling uniformity were investigated at the heart stage of 5 leaves and 1 in tobacco seedlings, and the results are shown in Table 3.
TABLE 3
As shown in Table 3, the present invention can effectively improve the agronomic characteristics of tobacco.
4. Lysine content investigation experiment
While the experiment of the agricultural character of the potted plant is carried out, the lysine content in the experimental group and the control group is also considered. The method for measuring the lysine content comprises the following steps: 10g of leaf was taken from each plant and analyzed for amino acid determination, the percentage of lysine = the lysine content of the sample ÷ the total amino acid content of the sample. The results are shown in FIG. 4.
TABLE 4
As can be seen from Table 4, the lysine content in tobacco can be remarkably increased.
5. Cadmium reduction experiment
Cadmium is added into the tested soil until the content of cadmium in an effective state in the soil is measured to be 3.8 mg/kg by the method in GB/T17141-1997, the cadmium content in the lower leaves, the middle leaves and the upper leaves is examined according to the experiment of pot agronomic characters, and the result is shown in Table 5.
TABLE 5
As can be seen from Table 5, the present invention significantly reduces the cadmium content in tobacco.
The following test examples are provided to illustrate the procedure for obtaining the compositions according to the invention.
Test example 1
Raw materials:
the fermentation liquor of the bacillus amyloliquefaciens cd5bc and the streptomyces oxysporum WS-29246 are obtained by liquid culture fermentation of bacillus amyloliquefaciens cd5bc and streptomyces oxysporum WS-29246 strains;
the 72% wettable powder of the cream-methyl and the manganese zinc is obtained by the market;
tobacco seedling: yunyan 87, owned by the laboratory;
the tobacco root black rot and tobacco phytophthora are owned by the laboratory.
Potted plant control effect test: fermenting the fermentation liquor (bacterial concentration 1 × 10) of the bacillus amyloliquefaciens cd5bc6cfu/mL), Streptomyces oxysporus WS-29246 fermentation broth (concentration 1X 10)6cfu/mL) and 72% of methyloxathite-manganin wettable powder (diluted by 5000 times of water) are mixed according to the volume ratio of 1:1:0.1 to obtain a mixed medicament; after 40 days of tobacco seedling transplantation, the tobacco seedlings are divided into two groups, each plant in the two groups is irrigated with 20mL of mixed medicament, one group is inoculated with phytophthora nicotianae and the other group is inoculated with the tobacco root rot pathogen after 5 days, and the disease incidence condition is investigated according to the standard GB/T23222-2008.
Through detection, the control effect of the test on the tobacco black shank reaches 91.3%, and the control effect on the tobacco root black rot reaches 90.7%.
Test example 2
The test 1 is one of test schemes randomly carried out by the inventor in the process of determining whether the streptomyces aculeatus has the control effect on the tobacco blight. Encouraged by the results of the test 1, the inventor researches the combination of the streptomyces oxysporum WS-29246 and other bacillus amyloliquefaciens reported to have good control effect, and researches the effects of different combinations on the control of tobacco black shank and tobacco root black rot according to the method of the test 1.
The strain source is as follows:
bacillus amyloliquefaciens YN48 purchased from the research center of microbial fermentation engineering of Yunnan province, Co., Ltd;
bacillus amyloliquefaciens X60 with the preservation number of CCTCC M2013375, which is sold in the market;
bacillus amyloliquefaciens GUMT319 with the preservation number of CCTCC NO: m2018872, commercially available.
Referring to test example 1, fermentation broths (OD) of the above three strains600= 0.3) and Streptomyces oxysporum WS-29246 fermentation liquor (OD) according to the volume ratio of 1:1:1600= 0.3) and 72% wettable powder of methyl cream and manganese zinc: (1000-fold dilution) to obtain mixed medicaments which are respectively named as a bacillus amyloliquefaciens YN48 scheme (scheme one), a bacillus amyloliquefaciens X60 scheme (scheme two) and a bacillus amyloliquefaciens GUMT319 scheme (scheme three). After 40 days of tobacco seedling transplantation, the tobacco seedlings are divided into two groups, each plant in the two groups is irrigated with 20mL of mixed medicament in each scheme, one group is inoculated with phytophthora nicotianae after 5 days, the other group is inoculated with phytophthora nicotianae, the disease incidence condition is investigated according to the standard GB/T23222-2008, and the results are shown in Table 6:
TABLE 6
As shown in Table 6, the control effect of the bacillus amyloliquefaciens YN48 scheme and the bacillus amyloliquefaciens GUMT319 scheme on tobacco black shank and tobacco root black rot is not more than 30%, and the control effect of the bacillus amyloliquefaciens X60 scheme on tobacco black shank and tobacco root black rot is not more than 40%, so that the control effect is very limited.
From the test results, the bacillus amyloliquefaciens which has good control effect on the black shank or the black root rot at present has poor control effect on the black shank and the black root rot of the tobacco after being combined with the streptomyces oxysporum WS-29246 and the frost-methyl manganese zinc.
Test example 3
After obtaining the results of test example 2, the inventors hypothesized that antagonism between strains may lead to a decrease in control efficacy. Based on this guess, the inventors conducted an inter-strain antagonism test. In the test, antagonism among strains is explored through a flat plate cross scribing method, and the growth condition of the strains at the cross points is observed. Antagonism between microorganisms refers to the growth and metabolic activity of one microorganism that exerts inhibitory, interfering or deleterious effects on other microorganisms, resulting in the growth being affected.
In addition to the strains used in experiments 1 and 2, this experiment further examined Bacillus subtilis (accession number CGMCC NO: 10248), Pseudomonas glanadensis PF3402 (accession number CCTCC NO: M2018545), Bacillus amyloliquefaciens GUMT319 (accession number CCTCC NO: M2018872), Bacillus amyloliquefaciens F11 (obtained by gift), and Bacillus amyloliquefaciens YN48 (obtained from microbial fermentation engineering research center, Inc. of Yunnan province).
In the test, the adopted preservation number of the bacillus subtilis is CGMCC No. 10248, and the bacillus subtilis is disclosed in Chinese patent of microbial agent for resisting both tobacco root rot and black shank and a preparation method and application thereof (publication No. CN 110511891A); the adopted preservation number of the pseudomonas glaradida PF3402 is CCTCC NO: m2018545, published in Chinese patent "a strain of Pseudomonas glanadensis capable of preventing and treating tobacco black shank and root black rot simultaneously and having growth promoting effect" and its application "(CN 109136154A); the adopted collection number of the bacillus amyloliquefaciens GUMT319 is CCTCC NO: m2018872, published in Chinese patent "Bacillus amyloliquefaciens GUMT319 and its application" (CN 109504639A); the adopted bacillus amyloliquefaciens F11 is a strain in an article published by von Rong (von Rong, Liuli, Chenhai reciting, Yangyoshu, Cynanchum lechleri and Dingxifeng. research on antifungal activity of the bacillus amyloliquefaciens F11 [ J ]. journal of agricultural resources and environment, 2021,38(05): 849-; the bacillus amyloliquefaciens YN48 is purchased from a limited company of the research center of microbial fermentation engineering in Yunnan province, and related reports are published in articles published by Liujian gold (Liujian gold, Liuzi instrument, Wangchao, Zhanyan, Xuyanpeng, Lujie, Luoshan and Haoyouqiu. the bacillus amyloliquefaciens YN48 is matched with a bactericide to prevent and treat tobacco black shank research [ J ]. the university of Yunnan university of agriculture (Nature science), 2020,35(02):361 + 365+ 370.). The bacterial strains are all bacterial strains with good control effect on black shank and/or black root rot.
As shown in Table 7, except for Bacillus amyloliquefaciens cd5bc, Streptomyces oxysporum WS-29246 was antagonistic to other strains.
TABLE 7
Note: in the table, "-" indicates no antagonism, and "+" indicates antagonism
As is clear from Table 7, it is found that the antagonistic action of Bacillus amyloliquefaciens F11, Bacillus amyloliquefaciens YN48 and Streptomyces oxysporum WS-29246 is not likely to be the reason why it is difficult to obtain a good control effect in the manner of test example 1 by mixing the Streptomyces oxysporum WS-29246 with the above-mentioned both bacteria. The mechanism is that the antagonism inhibits the growth of the bacteria or prevents the generation of some important metabolites, thereby reducing the control effect of the corresponding strains. Due to the fact that Streptomyces oxysporum WS-29246 and CGMCC NO: 10248. CCTCC NO: m2018545, CCTCC NO: m2018872 has antagonism, and the control effect is necessarily reduced after being mixed respectively, so that the inventor does not perform a specific control effect test.
Test example 4
The inventors further examined the case where the 72% wettable powder of methyl cream and manganese zinc was replaced with another drug in addition to test example 1.
As shown in table 8, when the 72% wettable pulvis Fumi Carbonisatus-Mandarin was replaced by 1500-fold diluted Fluorubitril-Pericarb suspension (comparative drug group 1) or 1500-fold diluted wettable thiophanate-methyl powder (comparative drug group 2), the control effect was significantly reduced.
TABLE 8
| Scheme(s) | Tobacco black shank control effect (%) | Tobacco root rot control (%) |
| Comparative drug group 1 | 53.4 | 43.1 |
| Comparative drug group 2 | 59.5 | 53.2 |
The inventors examined the inhibition of 72% methyloxanil-mancozeb wettable powder, flurobacter-propamocarb suspending agent and thiophanate-methyl wettable powder on the strain in test example 3, and used MIC (minimum inhibitory concentration) as an examination standard. As shown in Table 9, the MIC of the 72% wettable powder of methyl frost and manganese zinc to Bacillus amyloliquefaciens cd5bc and Streptomyces oxysporum WS-29246 is more than 5000 mug/mL, but the MIC of the fluorine bacterium and propamocarb suspending agent and the wettable powder of thiophanate-methyl to Bacillus amyloliquefaciens cd5bc and Streptomyces oxysporum WS-29246 are both small, and the bactericidal activity is strong. Therefore, it can be seen that after 72% of the wettable pulvis methyloxanil and manganese-zinc is replaced on the basis of the test example 1, the bacillus amyloliquefaciens cd5bc and the streptomyces oxysporum WS-29246 are greatly inhibited, which may be partially responsible for the lower control effect of the comparative drug group 1 and the comparative drug group 2.
TABLE 9
Note: in the table, "> 100 and < 500" indicates that the MIC is between 100 and 500. mu.g/mL, and no further experiment is performed to determine the specific MIC, since it is not necessary to determine the exact value.
Test example 5
On the basis of the experimental example 2, the inventor inspects the influence of the first scheme to the third scheme on the cadmium accumulation and the lysine content of the tobacco, and finds that the first scheme, the second scheme and the third scheme have no influence on the cadmium accumulation and the lysine, and the related data have no statistical difference.
Claims (10)
1. A composition for preventing and treating tobacco root black rot and black shank and improving characters and quality, which is characterized by comprising bacillus amyloliquefaciens cd5bc, streptomyces oxysporum WS-29246 and methyloxanil-manganese zinc;
the preservation number of the bacillus amyloliquefaciens cd5bc is CCTCC NO: M2016232; the preservation number of the streptomyces oxysporum WS-29246 is CCTCC NO: M2012413.
2. The composition according to claim 1, characterized in that the bacillus amyloliquefaciens cd5bc and the streptomyces oxysporum WS-29246 are added in the form of a fermentation broth; the cream-a-methyl and manganese-zinc wettable powder is 72 percent of cream-a-methyl and manganese-zinc wettable powder.
3. The composition as claimed in claim 2, wherein the bacillus amyloliquefaciens cd5bc fermentation broth has a concentration of 1 x 106cfu/mL~1×107cfu/mL; in the Streptomyces oxysporus WS-29246 fermentation liquor, the bacterial concentration is 1 × 106cfu/mL~1×107cfu/mL。
4. The composition as claimed in claim 3, wherein the cream-methyl-manganese-zinc is 1000-5000 times of a 1000-5000 times dilution of 72% cream-methyl-manganese-zinc wettable powder.
5. The composition of claim 4, wherein the methyloxanil-manganese zinc is a 5000-fold dilution of 72% methyloxanil-manganese zinc wettable powder.
6. The composition according to claim 5, which consists of bacillus amyloliquefaciens cd5bc, streptomyces oxysporum WS-29246 and zinc manganese methyl frost in a ratio of (10-30): 10-20): 1 by volume.
7. The composition according to claim 6, characterized in that it consists, by volume, of bacillus amyloliquefaciens cd5bc, streptomyces oxysporum WS-29246 and zinc manganese formate in a ratio of 15:10: 1.
8. The composition as claimed in any one of claims 2 to 7, wherein the fermentation broth of Streptomyces oxysporum WS-29246 is obtained by inoculating Streptomyces oxysporum WS-29246 and fermenting, and the medium used comprises the following components: 100g of cane sugar and grape10g of sugar, 0.1g of casamino acid, 5g of yeast extract powder, 21g of propanesulfonic acid and K2SO4 0.25g，MgCl2·6H2O10 g, trace element 1mL, water 1000mL, pH = 7.0; wherein the microelement formula is MnSO4·7H2O 100mg，CuSO4·7H2O 50mg，ZnSO4·7H2O 100mg，CoCl2100mg and 100mL of purified water;
the fermentation liquor of the bacillus amyloliquefaciens cd5bc is obtained by inoculating bacillus amyloliquefaciens cd5bc and fermenting, and the used culture medium is LB culture medium.
9. A method for preparing a composition for preventing and treating black rot and black shank of tobacco roots and improving properties, which is characterized in that the composition is the composition as claimed in any one of claims 1 to 8, and the method for preparing the composition is to uniformly mix the components in the composition.
10. Use of a composition according to any one of claims 1 to 8 for controlling tobacco root rot and black shank.
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