CN108384734B - Biocontrol microbial inoculum for preventing and treating tobacco root and stem diseases and preparation method thereof - Google Patents
Biocontrol microbial inoculum for preventing and treating tobacco root and stem diseases and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of microorganism application, and particularly relates to a biocontrol microbial inoculum for preventing and treating tobacco root and stem diseases and a preparation method thereof. The biocontrol microbial inoculum for preventing and treating the diseases of tobacco roots and stems is characterized by comprising fermentation liquor obtained by mixed fermentation of two bacteria, namely a strain 16029 and a strain 16098. The biocontrol microbial inoculum can simultaneously have stronger inhibiting effect on pathogenic bacteria of 3 main root diseases of tobacco bacterial wilt, tobacco black shank and tobacco root black rot, and can be used as the biocontrol microbial inoculum for the tobacco root diseases. The microbial inoculum of the invention has good application prospect in preventing and treating tobacco rhizome diseases as a biological control microbial inoculum.
Description
Technical Field
The invention belongs to the technical field of microorganism application, and particularly relates to a biocontrol microbial inoculum for preventing and treating tobacco root and stem diseases and a preparation method thereof.
Background
Tobacco is an important leaf economic crop and is a main economic source for farmers to delight and enrich in tobacco areas, but the growth of tobacco is seriously influenced by rhizomatic diseases such as bacterial wilt, black shank and root black rot, and the tobacco rhizomatic diseases are particularly serious in tropical and subtropical tobacco areas and are destructive diseases threatening the world tobacco production. The 3 main rhizome diseases are frequently mixed to cause diseases, the harm degree is increased, and the phenomena of disease occurrence in the whole field and no yield in dead fields of old diseased fields or dry land tobacco fields in a plurality of serious disease areas occur every year, so that the direct output value loss caused by the reduction of the yield and the quality of the tobacco leaves is very serious. Tobacco bacterial wilt is a bacterial vascular bundle disease caused by Ralstonia solanacearum. The disease is commonly generated in main tobacco areas (autonomous regions) in southern China, such as Guangxi, Guangdong, Fujian, Hunan, Zhejiang and the like, the disease area is large, the disease is serious, in recent years, the disease tends to expand to the tobacco areas in northern China, such as Shandong, Henan, Shaanxi, Liaoning and the like, the distribution and damage of the disease are recorded, and local tobacco areas are also serious. The tobacco black shank is one of the most devastating diseases in tobacco production, also called tobacco epidemic diseases, and various main tobacco producing areas in China occur in different degrees, wherein Anhui, Shandong and Henan provinces are historically serious disease areas, and the disease is often mixed with tobacco bacterial wilt, so that the harm is more serious. Since the area of continuous cropping tobacco fields is enlarged in recent years, the disease is a remarkable increase in the risk. The tobacco phytophthora parasitica tobacco pathotype (phytophthora parasitica var. nicotianae Tucker) is a fungus of the class oomycetes. The disease is mainly caused by stem damage, the base of the stem of the disease-affected plant is initially in water stain-like black spots, the disease-affected plant expands to the upper and lower parts and the medulla part, the plant wilts and dies when the disease-affected stem is wound around the stem for one week, and the medulla part is seen to be in black brown necrosis and is in a laminated shape and full of white flocculent hypha when the disease-affected stem is longitudinally cut. The tobacco root black rot is also a worldwide disease, and is serious in provinces such as Yunnan, Guizhou, Hubei, Guangxi and the like in China, the incidence rate of serious plots can reach more than 30%, and the harm trend is increased in recent years. The pathogenic fungus basidiospora basicola of the tobacco root black rot disease belongs to fungi of deuteromycotina. The disease is harmful in the seedbed stage and field planting, the disease occurs in the seedling stage, pathogenic bacteria firstly invade from the soil surface part of the seedling, disease spots surround the stem part and invade the cotyledon upwards to cause rot and destroy the tobacco seedling; the disease is developed in the field, the overground part of the tobacco plant at the initial stage shows growth retardation and is easy to wither, the root tip can be seen to be browned after root extraction, the black brown is rotted, all the diseased roots are in specific black in severe cases, and fibrous roots are often difficult to extract and remain in the soil.
The main infection sources of the 3 tobacco rootstock diseases are all from soil, particularly the disease field over the years, and great difficulty exists in prevention and treatment. Especially tobacco bacterial wilt, which belongs to bacterial diseases, and plant infection belongs to systemic infection, so that the prevention and the treatment are very difficult; tobacco black shank and root black rot belong to fungal diseases, although some relatively effective agents are used for chemical prevention and control at present, the harm of pathogenic bacteria is still difficult to be radically treated, and once field plants are infected, the disease condition is difficult to control. In addition, the chemical agent has a plurality of defects on the control of tobacco root diseases, pathogenic bacteria are easy to generate drug resistance to chemical pesticides, and the environmental pollution and the increase of the production cost are caused by using a large amount of chemical pesticides. In recent years, biological control and disease resistance induction have been studied more frequently. The biological control has the advantages of no public nuisance, no pollution and the like, and is an economic, effective and sustainable development approach for controlling the diseases of the tobacco roots. The prevention and control of bacterial wilt by using biocontrol bacteria is one of more researches in recent years, for example, antagonistic Pseudomonas spp is used for Roche and the like, and the good prevention effect is achieved in the greenhouse for preventing and controlling the tobacco bacterial wilt, but the biocontrol bacteria is not applied to a field; p.fluoroscens is used for carrying out control effect tests on various crop bacterial wilt by Anuratha and Gnanamantick in India, and good control effects are obtained. Researches of Hayward and the like in Philippines find that mycorrhizal fungi can reduce the occurrence of bacterial wilt, and Suresh and the like find that the arbuscular mycorrhizal extract of the cystolifera has strong bacteriostatic action on the growth and the propagation of ralstonia solanacearum. The existing biological control method can reduce the harm of bacterial wilt to a certain extent, but the existing practical application has the limitations of unstable control effect and the like. Because the phenomenon that various root diseases are mixed and harmful occurs in the field, how to screen the biocontrol microbial inoculum capable of simultaneously inhibiting the 3 big root diseases and the optimal use method thereof are problems to be solved in the aspect of controlling the tobacco root diseases.
Burkholderia cepacia (Burkholderia cepacia) is a gram-negative bacillus widely present in water, soil, plants and human bodies, and is a major member in biocontrol because it can produce various antibacterial components. Recent studies have shown that Burkholderia cepacia is not a species, but a group of phenotypically similar but genotypically different complexes, called the Burkholderia Cepacia Complex (BCC), which has been found to comprise 20 different genotypes, and thus represents different species due to the low DNA hybridization rate between these genotypes. In the beginning of the 80 th of the last century, people find that part of BCC strains can degrade some chemical substances which exist in pesticides and herbicides for a long time and have potential carcinogenic effects on human beings in research, can also purify sewage, and have very good ecological and economic values; meanwhile, some BCC strains have the effects of preventing and treating plant diseases and insect pests and promoting plant growth, are prepared into biological insecticides and biological bactericides and play an important role in agricultural production. However, at the same time, some strains of this bacterium have been found to be pathogenic for human conditions. Some countries in Europe and America abandon or limit the use of BCC biological pesticides because part of strains are conditional pathogenic bacteria, but at present, many countries still produce and use biological pesticides prepared by taking BCC biological pesticides as raw materials, and China also registers the products in 1996.
BCC is composed of 20 species (or genotypes) including b.cepacia, b.multivibratan, b.cenocepacia, b.stabilia, b.vietnamiensis, b.dolosa, b.ambifaria, b.anthina, b.pyrrocinia, b.ubomensis, b.laths, b.diffua, b.arboris, b.semilaris, b.metalllica, b.contidans, b.lata, b.pseudobulbotevorans, b.staralli and b.terrorisi. These species are ubiquitous in nature and are particularly closely related to plants. It has now been found that BCC pathogenic strains are associated with specific virulence genes (BCESM genes). The BCESM gene is considered as a genetic marker linked with human pathogenicity/toxicity in BCC bacteria, and registration is cancelled for registered Burkholderia cepacia strains once the existence of the marker is found. Thus, by detection of the gene of the virulence factor BCESM, pathogenic and non-pathogenic strains of BCC can be distinguished. Of course, it is also necessary to further test strains for pathogenicity using animal models (mice) or plant models in order to ensure the safety of use of BCC biocontrol strains. Alfalfa is generally used as a model plant for the determination of the pathogenicity of nosocomial respiratory infections of bacteria. It was found that alfalfa could be used as a model plant to measure the toxicity of BCC flora to mammals instead of mice, and that the alfalfa model even showed higher sensitivity than the mouse model. Therefore, the alfalfa model can be used for simply and quickly detecting whether the BCC strain has pathogenicity to mammals, and lays a foundation for safely utilizing the BCC strain.
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
Disclosure of Invention
The invention aims to provide a biological control agent and a preparation method thereof, the biological control agent can effectively control 3 diseases of tobacco bacterial wilt, black shank and root black rot, and the biological control agent has a remarkable promoting effect on the growth of tobacco.
The technical scheme provided by the invention is as follows:
the active component of the biocontrol microbial inoculum consists of fermentation liquor obtained by mixed fermentation of two bacteria, namely a strain 16029 and a strain 16098.
Preferably, the strain 16029 is classified and named Burkholderia anthina, and is preserved in the China general microbiological culture Collection center in 2018, month 01 and day 10, with the preservation number of CGMCC NO. 15165.
Preferably, the strain 16098 is classified and named Burkholderia cenocepacia, and is preserved in the China general microbiological culture Collection center in 2018, month 01 and day 10, with the preservation number of CGMCC NO. 15166.
The invention also provides a preparation method of the biocontrol microbial inoculum for preventing and treating the tobacco rhizome diseases, which comprises the following steps:
(1) preparing a seed solution: respectively inoculating the strain 16029 and the strain 16098 on a nutrient gravy culture medium NA for independent culture, performing shake culture at 30 ℃ for 20-24h, and mixing the culture solutions of the two strains in equal amount to obtain a seed solution of the biocontrol microbial inoculum;
(2) obtaining a microbial inoculum fermentation liquid: inoculating the seed solution of the cultured biocontrol microbial inoculum to a fermentation medium, and performing shake culture at 25-28 ℃ for 30-36h to obtain fermentation liquor of the biocontrol microbial inoculum;
(3) the invention relates to a method for obtaining a biocontrol microbial inoculum: adding a microbial inoculum additive into the fermentation liquor of the biological control microbial inoculum, and uniformly stirring to obtain the biological control microbial inoculum.
Preferably, the fermentation medium in step (2) is: 2.0g/L of malt extract powder, 8.0g/L of cane sugar, 2g/L of yeast powder, 0.5g/L of monopotassium phosphate, 0.3g/L of manganese sulfate, 3.0g/L of NaCl and 6.5-7.0 of pH value.
Preferably, the inoculation amount of the seed solution in the step (2) is 3%.
Preferably, the microbial inoculum additive in the step (3) is obtained by mixing, drying and crushing wheat bran and bean cake powder according to the mass ratio of 2: 1.
Preferably, the addition amount of the microbial inoculum additive in step (3) is 2%.
Compared with the prior art, the invention has the following beneficial effects:
(1) the biocontrol microbial inoculum can simultaneously have stronger inhibiting effect on pathogenic bacteria of 3 main root diseases of tobacco bacterial wilt, tobacco black shank and tobacco root black rot, and can be used as the biocontrol microbial inoculum for the tobacco root diseases. The microbial inoculum of the invention has good application prospect in preventing and treating tobacco rhizomatic diseases as a biological control microbial inoculum, and the potted plant control test result shows that the prevention and treatment effect of the inoculated microbial inoculum on tobacco bacterial wilt reaches 84.77 percent; the control effect of the field control test on tobacco bacterial wilt also reaches 80.64%, the control effect on black shank and root black rot reaches more than 85%, the control effect is obviously superior to that of chemical agents, and the harm of tobacco root diseases is well controlled.
(2) The biocontrol microbial inoculum of the invention has the characteristics of dissolving phosphorus and potassium, and has obvious promotion effect on the growth of tobacco when being inoculated.
(3) The biocontrol microbial inoculum is easy to culture, simple in fermentation condition and easy to operate, and as long as a seed solution with good growth is provided, a common tobacco grower can perform enlarged culture by using the simple fermentation condition to obtain microbial inoculum fermentation liquor, and the cultured fermentation liquor can be directly applied to production after being added with the biocontrol auxiliary agent, so that the biocontrol microbial inoculum has good popularization and application prospects.
(4) The biocontrol microbial inoculum has important significance for reducing the use of chemical fertilizers and pesticides and protecting the ecological environment.
Drawings
FIG. 1 shows the bacteriostatic effect of two BCC strains;
FIG. 2 is the growth promoting effect of the biocontrol microbial inoculum of the invention on tobacco;
FIG. 3 shows the pot culture test effect of the biocontrol microbial inoculum of the invention on controlling tobacco bacterial wilt;
FIG. 4 is the result of detecting the pathogenicity of the BCC strain used by the biocontrol microbial inoculum to tobacco;
FIG. 5 shows the result of detecting the pathogenicity of the BCC strain used in the biocontrol microbial inoculum of the invention to alfalfa.
Description of preservation information
Burkholderia anthina, the preservation number is CGMCC NO.15165, the preservation date is 2018, month 01 and day 10, the preservation unit is the China general microbiological culture Collection center (CGMCC for short, address: microorganism research institute of China academy of sciences No. 3, North West Lu No.1 Hotel, Chaozhou, China), and the postal code is 100101);
burkholderia cenocepacia, the preservation number is CGMCC NO.15166, the preservation date is 2018, 01 month and 10 days, and the preservation unit is the China general microbiological culture Collection center (CGMCC for short, address: microorganism research institute of China academy of sciences No. 3, North West Lu No.1 Hotel, Ind. of Beijing City, Kogyo, and postal code: 100101).
Detailed Description
The following examples are given to better understand the present invention and are not intended to limit the present invention. The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified.
In the quantitative experiments in the following examples, three replicates were set up and the results averaged.
Example 1: screening and identification of strains
(1) Screening of strains
2 biocontrol bacteria (the original numbers of the strains are 16029 and 16098 respectively) contained in the compound microbial agent are separated and screened from cassava root soil in a cassava main production area in Hepu county, North sea, autonomous region of Guangxi Zhuang, the strains are stored in China general microbiological culture Collection center (CGMCC) in 2018 for 01 months, and the preservation numbers of the strains 16029 and 16098 are CGMCC No.15165 and CGMCC No.15166 respectively.
(2) Classification and identification of strains
Through analysis of colony morphology, 16S rDNA sequence analysis, a Biolog bacteria identification system, BCC flora specific amplification PCR, BCC different genotype identification primer detection and the like, two strains of bacteria with preservation numbers of CGMCC No.15165 and CGMCC No.15166 are respectively identified as Burkholderia plantarii (Burkholderia antifhina) and Burkholderia cepacia (Burkholderia cenocepacia).
Example 2: preparation of biocontrol microbial inoculum
The fermentation liquor of the biocontrol microbial inoculum is obtained by mixed culture of strain 16029 and strain 16098 seed solutions, and is based on the following experimental screening results:
(1) both strain 16029 and strain 16098 have a potassium phosphate solubilizing effect, but the potassium phosphate solubilizing effect of strain 16029 is better than that of strain 16098.
(2) Both strains can inhibit important plant pathogenic bacteria such as tobacco bacterial wilt and the like, and have biocontrol potential, but the biocontrol effect of the strain 16098 is better than that of 16029. The bacterial suspension and the culture stock solution of the two strains have stronger bacteriostatic action, but the bacteriostatic effect of the sterile filtrate is not obvious. The screening of the bacteriostatic effect of two BCC bacteria is shown in figure 1.
(3) The growth curves of the two strains were substantially identical under identical culture conditions.
(4) Determining the seed culture medium of the microbial inoculum to be a common nutrient meat juice culture medium NA (3.0 g of beef extract, 10.0g of peptone and 5.0g of NaCl5.0g, the volume is increased to 1L, and the pH value is 7.0) by measuring the influence of different carbon sources, nitrogen sources and inorganic salts on the growth of the two strains; the culture medium for the microbial inoculum expansion culture (fermentation) is as follows: 2.0g/L of malt extract powder, 8.0g/L of cane sugar, 2g/L of yeast powder, 0.5g/L of monopotassium phosphate, 0.3g/L of manganese sulfate, 3.0g/L of NaCl and 6.5-7.0 of pH value.
The preparation method of the biocontrol microbial inoculum for preventing and treating the tobacco rhizome diseases comprises the following steps:
(1) the preparation method comprises the following steps: respectively inoculating strain 16029 and strain 16098 on NA culture medium, culturing independently, shaking at 30 deg.C for 20-24h, and mixing the two strain culture solutions to obtain the seed solution of the microbial inoculum.
(2) Obtaining a microbial inoculum fermentation liquid: inoculating the cultured seed liquid to a fermentation medium, wherein the inoculation amount is 3%, and performing shaking culture at 25-28 ℃ for 30-36h to obtain the fermentation liquid of the microbial inoculum.
(3) The preparation of the additive (auxiliary agent) of the microbial inoculum comprises the following steps: mixing testa Tritici and bean cake powder at a weight ratio of 2:1, oven drying, pulverizing, sterilizing, and sealing for storage.
(4) The invention relates to the acquisition and the application of a biocontrol microbial inoculum: 2 percent of the microbial inoculum additive (auxiliary agent) is added into microbial inoculum fermentation liquor to obtain the biological control microbial inoculum of the invention, and the biological control microbial inoculum can be directly used in production after being diluted by corresponding times according to requirements.
The microbial inoculum fermentation medium is easy to prepare, the fermentation conditions are simple, in order to meet the requirement of large field use amount, a common tobacco grower can prepare the fermentation medium and perform simple fermentation culture under the condition of obtaining seed liquid and microbial inoculum assistance, and the cultured fermentation liquid can be directly used after being mixed with the auxiliary agent according to the proportion.
Example 3: the plant growth promoting property of the biocontrol microbial inoculum of the invention
(1) Method for determining phosphorus and potassium dissolving effect of microbial inoculum by soil culture method
Mixing basic cultivation soil laterite and seedling culture medium in equal volume, adding a proper amount of calcium phosphate and potassium feldspar powder, uniformly mixing, bottling, sterilizing, inoculating the microbial inoculum fermentation liquor with the inoculation amount of 3%, inoculating an equal amount of sterile culture solution, and measuring the effective phosphorus and the effective potassium content of each treatment after 20 days. The available phosphorus is measured by molybdenum-antimony colorimetric method, and the available potassium is measured by flame photometry.
The experimental result shows that the content of effective phosphorus and the content of effective potassium of the mixed matrix at the early stage of inoculation treatment are respectively 8.2mg/kg and 117.6 mg/kg; after 20 days, the effective phosphorus and the effective potassium contents of the inoculation agent treatment are 14.2mg/kg and 144.5mg/kg respectively, while the contents of the control treatment are 8.6mg/kg and 119.0mg/kg respectively.
Therefore, the content of available phosphorus and the content of available potassium of the substrate after being inoculated with the biocontrol microbial inoculum for 20 days are greatly improved, and are respectively improved by 65.12 percent and 21.43 percent compared with the control treatment.
(2) The biological control agent of the invention has the growth promoting effect on tobacco seedlings
The tobacco seedling raising pot is subjected to conventional seedling raising treatment, after two true leaves grow on a tobacco seedling, the tobacco seedling is sprayed for 2 times (at an interval of 7d) by using 30 times of diluent of the microbial inoculum, and the same amount of clear water is sprayed in contrast.
As shown in figure 2, the growth vigor of tobacco seedlings treated by the biocontrol microbial inoculum is obviously superior to that of a control, and the leaf area and the chlorophyll content of tobacco seedling leaves subjected to microbial inoculum inoculation treatment are respectively improved by 27.5 percent and 34.8 percent compared with that of the control.
Example 4: control test of biocontrol microbial inoculum of the invention on tobacco root and stem diseases
(1) Pot culture inoculation test for preventing and treating tobacco bacterial wilt by using microbial inoculum
The cultivation soil in the pot experiment is the plough layer soil of the healthy paddy field, the tobacco is cultivated according to the conventional seedling cultivation and transplantation, each pot is planted with a single plant, the diluted fermentation liquor of 30 times of the microbial inoculum is respectively inoculated after 7d, 14d and 21d of transplantation, each single plant is inoculated with 200mL of the diluted fermentation liquor, and the same amount of clear water is inoculated in comparison. Inoculating ralstonia solanacearum, separating and screening 3 ralstonia solanacearum mixed bacterial liquid with strong pathogenicity from ralstonia solanacearum in Guangxi Baiseo main production area by using the research team, and inoculating the bacterial liquid with the concentration of 108cfu/mL, 150mL of root-irrigation inoculation bacterial suspension per basin (strain); the first inoculation time of the ralstonia solanacearum is 10 days after transplantation, then the same amount of pathogen bacterial liquid is inoculated for 1 time every 3 days, and the inoculation is continuously carried out for 5 to 8 times until obvious contrast appearsSymptoms of bacterial wilt. Continuously investigating the disease condition of tobacco after the Control (CK) begins to show the typical bacterial wilt symptoms, recording the number of diseased plants, and calculating the relative control effect of the inoculation of the microbial inoculum according to the following formula:
control effect { (blank control treatment plant rate-control treatment area plant rate)/blank control treatment plant rate } × 100.
Test research results show that after the tobacco is transplanted for 25d (the bacterial wilt is inoculated for 15d), the bacterial wilt symptoms appear in comparison, and the tobacco seedlings inoculated with the biocontrol microbial inoculum of the invention have individual plant disease symptoms for the first time after being transplanted for 30 d; the disease rate of the tobacco inoculated with the microbial inoculum after 50 days of transplantation is 11.5 percent and all are grade 1 diseased plants, while the disease rate of the control is 75.5 percent and 30 percent of diseased plants reach grade 7-9. The prevention and treatment effect of the microbial inoculum on the tobacco bacterial wilt is calculated to reach 84.77 percent according to the disease rate.
(2) The field test of the biocontrol microbial inoculum for preventing and treating 3 main rhizome diseases of tobacco
In the field plot control test, 3 treatments are set, namely the microbial inoculum treatment, the chemical agent control treatment and a blank control (CK, no control treatment is carried out), and the treatment scheme is detailed in Table 1.4 repeated cells are arranged for each treatment, the area of each cell is about 0.08 mu, and the number of tobacco plants is 80, namely the total number of the tobacco plants in each treatment test is 320.
TABLE 1 tobacco rootstock disease plot control test treatment scheme
The investigation and classification standards of tobacco bacterial wilt, tobacco black shank and tobacco root black rot are according to the tobacco disease classification and investigation method (national tobacco industry standard YC/T39-1996).
The disease index and the prevention and treatment effect are calculated according to the following formulas:
disease index { ∑ (number of disease plants at each stage × disease-level value)/(total number of investigated plants × highest-level value) } × 100
Relative prevention and treatment effect (%) { (control disease index-treatment disease index)/control disease index } × 100
The disease index and the prevention and treatment effect of each treatment were calculated according to the last investigation results (see table 2).
TABLE 2 tobacco 3 kinds of rhizome diseases control effect in small area test
As shown in Table 2, the biocontrol microbial inoculum of the invention has the control effect on tobacco bacterial wilt of 80.64 percent, the control effects on black shank and root black rot of 85.13 percent and 87.83 percent respectively, the control effect is obviously better than that of chemical agents, and the biocontrol microbial inoculum can effectively control the harm of 3 diseases of tobacco.
Example 5: safety evaluation of the biocontrol microbial inoculum of the invention
Respectively determining whether the BCC strains used by the biocontrol microbial inoculum have pathogenicity on onions, tobacco leaves and alfalfa; meanwhile, whether the strain contains a BCC flora pathogenic factor (BCESM) is detected through PCR specific amplification, so that whether the microbial inoculum is safe to plants, human beings, livestock and ecological environment is proved.
(1) Pathogenicity detection for onion and tobacco lamina
Activating and culturing different BCC strains, and detecting pathogenicity of Bulbus Allii Cepae by sucking 100 μ l of 10-content solution with 1ml disposable sterilizing syringe7Individual cell test strains were suspended in a bacterial suspension, needle-inoculated with fresh and healthy onion bulbs, and compared with sterile water. Culturing in a climatic chamber at 30 deg.C under 95% relative humidity and 12h light/12 h dark condition, and observing whether the onion bulb is rotten every day. Tobacco was inoculated with the same concentration of bacterial suspension, and 1ml of the bacterial suspension was pressed into the leaf using a disposable inoculation syringe to inject 1ml of sterile water as a control. After 5-7 days, the injection site was observed for the presence of brown allergic necrotic lesions.
As shown in fig. 4, after inoculation of onion bulbs at 7d, there was no evidence of rot in the onion bulbs; after the tobacco leaves are inoculated, the tobacco leaves are also not different from the control, and allergic necrotic spots are not generated. The research shows that all two BCC strains of the microbial inoculum have no pathogenicity on onion and tobacco, and the strains are not phytopathogens and are safe to plants.
(2) Detection of the pathogenicity of test strains on alfalfa
Alfalfa is a model plant for determining the toxicity of BCC to animals instead of mice, and can be used to easily and rapidly detect whether a strain of BCC is pathogenic to mammals.
Respectively inoculating two strains to be tested into LB culture medium, culturing at 30 deg.C with shaking table (180rpm) for 24h, centrifuging to collect thallus, adjusting the concentration of suspension to 10 with sterile water8the/mL was used for inoculation. Soaking alfalfa seeds in 98% concentrated sulfuric acid for 20min, washing with sterile water for 4 times, soaking in sterile water at 30 deg.C for 7h, washing for 2 times, changing water, soaking for 10h, and placing the seeds on water agar plate for inoculation. Seeds on agar plates were pricked with a small needle, 20 μ L of the prepared bacterial suspension was spotted onto the wound, and the needle-pricked seeds inoculated with sterile water were used as controls. And culturing the inoculated seeds in a 28 ℃ illumination incubator, and checking the morbidity of the inoculation after 7 d.
As shown in FIG. 5, the treatment of the inoculated test strains had no disease symptoms, no necrosis at the vicinity of the inoculated site, good growth of both the seed roots and cotyledons, and no co-yellowing phenomenon, as in the case of the sterile water control. The results indicate that the test strains are safe for mammals.
(3) Detection of test strains whether to contain BCC (BCC virulence factor) BCESM (bacillus coli-associated protein)
Detection of the virulence factor of the BCC flora B. cepacia epidemic strain marker (BCESM) sequence primer BCESM1 designed by Mahentihiragangam (1997): 5'-CCACGGACGTGACTAACA-3' and BCESM 2: 5'-CGTCCATCCGAACACGAT-3' PCR amplification was performed and the amplification products were detected by electrophoresis on a 1.5% agarose gel and imaged with a gel image analyzer.
Through PCR detection, about 1400bp BCESM pathogenic factor sequences are not amplified in both test strains.
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Sequence listing
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<120> a biocontrol microbial inoculum for preventing and treating tobacco root and stem diseases and a preparation method thereof
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acatcggaac atgtcctgta gtgggggata gcccggcgaa agccggatta ataccgcata 120
cgatctacgg atgaaagcgg gggaccttcg ggcctcgcgc tatagggttg gccgatggct 180
gattagctag ttggtggggt aaaggcctac caaggcgacg atcagtagct ggtctgagag 240
gacgaccagc cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtggg 300
gaattttgga caatgggcga aagcctgatc cagcaatgcc gcgtgtgtga agaaggcctt 360
cgggttgtaa agcacttttg tccggaaaga aatccttggc tctaatacag ccgggggatg 420
acggtaccgg aagaataagc accggctaac tacgtgccag cagccgcggt aatacgtagg 480
gtgcaagcgt taatcggaat tactgggcgt aaagcgtgcg caggcggttt gctaagaccg 540
atgtgaaatc cccgggctca acctgggaac tgcattggtg actggcaggc tagagtatgg 600
cagagggggg tagaattcca cgtgtagcag tgaaatgcgt agagatgtgg aggaataccg 660
atggcgaagg cagccccctg ggccaatact gacgctcatg cacgaaagcg tggggagcaa 720
acaggattag ataccctggt agtccacgcc ctaaacgatg tcaactagtt gttggggatt 780
catttcctta gtaacgtagc taacgcgtga agttgaccgc ctggggagta cggtcgcaag 840
attaaaactc aaaggaattg acggggaccc gcacaagcgg tggatgatgt ggattaattc 900
gatgcaacgc gaaaaacctt acctaccctt gacatggtcg gaatcctgct gagaggcggg 960
agtgctcgaa agagaaccgg cgcacaggtg ctgcatggct gtcgtcagct cgtgtcgtga 1020
gatgttgggt taagtcccgc aacgagcgca acccttgtcc ttagttgcta cgcaagagca 1080
ctctaaggag actgccggtg acaaaccgga ggaaggtggg gatgacgtca agtcctcatg 1140
gcccttatgg gtagggcttc acacgtcata caatggtcgg aacagagggt tgccaacccg 1200
cgagggggag ctaatcccag aaaaccgatc gtagtccgga ttgcactctg caactcgagt 1260
gcatgaagct ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg 1320
gtcttgtaca caccgcccgt cacaccatgg gagtgggttt taccagaagt ggctagtcta 1380
accgcaagga ggac 1394
<210> 2
<211> 1393
<212> DNA
<213> Burkholderia sp.
<400> 2
gcagtcgaac ggcagcacgg gtgcttgcac ctggtggcga gtggcgaacg ggtgagtaat 60
acatcggaac atgtcctgta gtgggggata gcccggcgaa agccggatta ataccgcata 120
cgatctatgg atgaaagcgg gggaccttcg ggcctcgcgc tatagggttg gccgatggct 180
gattagctag ttggtggggt aaaggcctac caaggcgacg atcagtagct ggtctgagag 240
gacgaccagc cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtggg 300
gaattttgga caatgggcga aagcctgatc cagcaatgcc gcgtgtgtga agaaggcctt 360
cgggttgtaa agcacttttg tccggaaaga aatccttggc tctaatacag tcgggggatg 420
acggtaccgg aagaataagc accggctaac tacgtgccag cagccgcggt aatacgtagg 480
gtgcgagcgt taatcggaat tactgggcgt aaagcgtgcg caggcggttt gctaagaccg 540
atgtgaaatc cccgggctca acctgggaac tgcattggtg actggcaggc tagagtatgg 600
cagagggggg tagaattcca cgtgtagcag tgaaatgcgt agagatgtgg aggaataccg 660
atggcgaagg cagccccctg ggccaatact gacgctcatg cacgaaagcg tggggagcaa 720
acaggattag ataccctggt agtccacgcc ctaaacgatg tcaactagtt gttggggatt 780
catttcctta gtaacgtagc taacgcgtga agttgaccgc ctggggagta cggtcgcaag 840
attaaaactc aaaggaattg acggggaccc gcacaagcgg tggatgatgt ggattaattc 900
gatgcaacgc gaaaaacctt acctaccctt gacatggtcg gaatcctgct gagaggcggg 960
agtgctcgaa agagaaccgg cgcacaggtg ctgcatggct gtcgtcagct cgtgtcgtga 1020
gatgttgggt taagtcccgc aacgagcgca acccttgtcc ttagttgcta cgcaagagca 1080
ctctaaggag actgccggtg acaaaccgga ggaaggtggg gatgacgtca agtcctcatg 1140
gcccttatgg gtagggcttc acacgtcata caatggtcgg aacagagggt tgccaacccg 1200
cgagggggag ctaatcccag aaaaccgatc gtagtccgga ttgcactctg caactcgagt 1260
gcatgaagct ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg 1320
gtcttgtaca caccgcccgt cacaccatgg gagtgggttt taccagaagt ggctagtcta 1380
accgcaagga gga 1393
Claims (5)
1. The biocontrol microbial inoculum for preventing and treating the diseases of tobacco roots and stems is characterized in that the active component of the biocontrol microbial inoculum consists of fermentation liquor obtained by mixed fermentation of two bacteria, namely a bacterial strain 16029 and a bacterial strain 16098; the tobacco root diseases are tobacco bacterial wilt, tobacco black shank or tobacco root black rot;
taxonomical nomenclature of said Strain 16029Burkholderia anthinaThe strain is preserved in the China general microbiological culture Collection center in 2018, month 01 and day 10 with the preservation number of CGMCC NO. 15165;
taxonomical nomenclature of said Strain 16098Burkholderia cenocepaciaAnd is preserved in China general microbiological culture Collection center (CGMCC) at 2018, month 01 and day 10 with the preservation number of CGMCC NO. 15166.
2. The preparation method of the biocontrol microbial inoculum for controlling the tobacco rhizome diseases as claimed in claim 1, which is characterized by comprising the following steps:
(1) preparing a seed solution: respectively inoculating the strain 16029 and the strain 16098 of claim 1 on a nutrient gravy culture medium NA for independent culture, shaking and culturing at 30 ℃ for 20-24h, and mixing the culture solutions of the two strains in equal amount to obtain a seed solution of the biocontrol microbial inoculum;
(2) obtaining a microbial inoculum fermentation liquid: inoculating the seed solution of the cultured biocontrol microbial inoculum to a fermentation medium, and performing shake culture at 25-28 ℃ for 30-36h to obtain fermentation liquor of the biocontrol microbial inoculum;
(3) obtaining the biocontrol microbial inoculum: adding a microbial inoculum additive into the fermentation liquor of the biological control microbial inoculum, and uniformly stirring to obtain the biological control microbial inoculum; the microbial inoculum additive is obtained by mixing wheat bran and bean cake powder according to the mass ratio of 2:1, drying and crushing.
3. The preparation method of the biocontrol microbial inoculum for controlling the tobacco rhizome diseases according to the claim 2, characterized in that the fermentation medium in the step (2) is: 2.0g/L of malt extract powder, 8.0g/L of cane sugar, 2g/L of yeast powder, 0.5g/L of monopotassium phosphate, 0.3g/L of manganese sulfate, 3.0g/L of NaCl and 6.5-7.0 of pH value.
4. The preparation method of the biocontrol microbial inoculum for controlling tobacco rhizome diseases according to claim 2, characterized in that the inoculation amount of the seed liquid in the step (2) is 3%.
5. The preparation method of the biocontrol microbial inoculum for controlling tobacco rhizome diseases according to claim 2, characterized in that the additive amount of the microbial inoculum in the step (3) is 2%.
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| CN109234202A (en) * | 2018-10-29 | 2019-01-18 | 华侨大学 | A kind of fermentation culture method of banana blight Antagonistic Fungi bulkholderia cepasea |
| CN110683875A (en) * | 2019-11-13 | 2020-01-14 | 贵州中烟工业有限责任公司 | Biological bacterial fertilizer for preventing bacterial wilt of flue-cured tobacco, preparation method and application |
| CN114214259B (en) * | 2022-01-17 | 2023-07-18 | 中国农业科学院烟草研究所 | Intelligent biocontrol microbial agent and preparation method and application thereof |
| CN117187077B (en) * | 2023-07-24 | 2024-03-08 | 广东省科学院南繁种业研究所 | Composite flora and application thereof, and control method of bacterial wilt of plants |
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Effective date of registration: 20210618 Address after: 530007, 174 East University Road, the Guangxi Zhuang Autonomous Region, Nanning Patentee after: GUANGXI ACADEMY OF AGRICULTURAL SCIENCES Address before: No. 174, Daxue East Road, XiXiangTang District, Nanning City, Guangxi Zhuang Autonomous Region Patentee before: INSTITUTE OF MICROBIOLOGY, GUANGXI ZHUANG AUTONOMOUS ACADEMY OF AGRICULTURAL SCIENCES |