CN115812739A - Streptomyces wetland bacterial strain 13-3 and application of fermentation liquor thereof in prevention and treatment of corn stalk rot - Google Patents
Streptomyces wetland bacterial strain 13-3 and application of fermentation liquor thereof in prevention and treatment of corn stalk rot Download PDFInfo
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Abstract
The invention relates to an application of streptomyces wetland strain 13-3 in preventing and treating corn stalk rot, wherein the streptomyces wetland strain 13-3 is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms with the preservation number of CGMCC No.22830, and the preservation date is as follows: 7/6/2021. The invention has the advantages that: the streptomyces wetlands strain 13-3 has antagonistic action on 2 pathogenic bacteria (fusarium graminearum and fusarium verticillioides) causing the corn stalk rot, the fermentation liquor can obviously inhibit the hypha growth and spore germination of the 2 pathogenic bacteria (fusarium graminearum and fusarium verticillioides) causing the corn stalk rot, and the fermentation liquor has excellent control effect on the corn stalk rot.
Description
Technical Field
The invention relates to the technical field of crop disease control, in particular to a Streptomyces paludis (Streptomyces paludis) strain 13-3 and application of fermentation liquor thereof in controlling corn stalk rot.
Background
Corn is a main food crop, feed crop and important industrial raw material in China, and plays an important role in national economy and agricultural production. China is a big country for corn production and a big country for corn import. In 2021, the seeding area of the corn in China is increased by 5 percent compared with 2020, the seeding area reaches 4350 hectare, and the yield reaches 2.725 million tons (https:// www.askci.com/news/data/chanxiao/20220119/1145301728429. Shtml). The corn import amount in China is rapidly increased from 2020, the corn import amount reaches 2835 ten thousand tons in 2021, and the corn import amount is increased by 222.9 percent in a same way (https:// www.askci.com/news/data/maoyi/20220124/1000411733214. Shtml). Therefore, the yield and the safe production of the corn directly influence the food safety in China and the healthy development of the extended industry thereof.
Research shows that the occurrence and prevalence of corn diseases and insect pests cause yield loss and quality reduction, and are one of the main factors causing yield reduction of corn. In recent years, due to the popularization of a straw returning system, the corn continuous cropping cultivation and the lack of high-stem-rot-resistance corn varieties in production and the like, the number of field pathogenic bacteria population is increased, the damage of corn stem rot is increased year by year, the annual incidence rate is 10-20%, and the serious annual incidence rate can reach 50-60%. The corn stalk rot pathogen is complex, wherein fusarium graminearum and fusarium verticillioides are main pathogenic bacteria of the corn stalk rot, and the main pathogenic bacteria not only harm corn stalks to cause the stalk rot, but also harm corn ears to cause the mildew and rot of the ears to cause serious yield loss, and in addition, a large amount of mycotoxins generated by the fusarium graminearum also cause influence on the health of people and livestock. At present, the bactericide is still a main means for preventing and treating the stem rot of the corn, but chemical prevention and treatment are easy to cause the crop to generate drug resistance after long-term use, so that the disease prevention and treatment effect is reduced.
Actinomycetes are microbial resources of which metabolites contain a large number of bioactive substances, and the generated bioactive substances have the advantages of good activity, difficulty in generation of resistance and the like, and are widely applied to agricultural production or used as precursor compounds of novel bactericides. In addition, secondary metabolites of actinomycetes are widely applied to aspects such as steroid conversion, petroleum dewaxing, sewage treatment, biological nitrogen fixation and the like. The validamycin produced by streptomyces hygroscopicus validamycin is the most successful biopesticide developed in China, has good control effect on rice sheath blight and long lasting period, can effectively control the occurrence of diseases, and is widely used for preventing and treating the rice sheath blight. Zambrano and the like isolate actinomycetes spp.M2A2 from soil around rice, can inhibit the growth of hypha of rhizoctonia solani, further delay the occurrence of diseases, influence the expansion of pathogenic bacteria in a susceptible variety Fedearroz 68, and have the effect equivalent to the control effect of difenoconazole.
The biological control has the advantages of no pollution to the environment, no toxic or side effect on crops, difficult generation of resistance of pathogenic bacteria and the like, and the corn stalk rot disease control by using biological control measures conforms to the agricultural sustainable development strategy.
Disclosure of Invention
The invention aims to solve the problems that the corn stalk rot is serious, the healthy development of the corn industry is hindered, the environmental pollution is caused by the use of a large amount of bactericides, the drug resistance of pathogenic bacteria is high and the like, and provides the application of the Streptomyces paludis 13-3 strain in the prevention and treatment of the corn stalk rot.
The invention also aims to provide application of Streptomyces wetlands (Streptomyces paludis) strain 13-3 fermentation liquor in preventing and treating corn stalk rot.
The purpose of the invention is realized by the following technical scheme:
the application of the Streptomyces wetlands (Streptomyces paludis) 13-3 in preventing and treating the corn stalk rot is characterized in that the Streptomyces wetlands (Streptomyces paludis) 13-3 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation address is as follows: the preservation number of No. 1 Xilu of Beijing, chaoyang, is CGMCC No.22830, and the preservation date is as follows: 7/6/2021.
An application of Streptomyces paludus (Streptomyces paludus) 13-3 fermentation liquor in preventing and treating corn stalk rot is disclosed, wherein the fermentation liquor is prepared by inoculating Streptomyces paludus (Streptomyces paludus) 13-3 strain in claim 1 into ISP2 solid culture medium, fermenting at 26-28 ℃ for 7-8 days, extracting with ethyl acetate, concentrating the extracting solution under reduced pressure, evaporating to dryness and dissolving with DMSO.
The Streptomyces wetland (Streptomyces paludis) strain 13-3 fermentation liquor is applied to inhibition of hypha growth of Fusarium graminearum (Fusarium graminearum) and Fusarium verticillioides (Fusarium verticillioides).
The Streptomyces wetland (Streptomyces paludis) strain 13-3 fermentation liquor is applied to inhibition of spore germination of Fusarium graminearum (Fusarium graminearum) and Fusarium verticillioides (Fusarium verticillioides).
Compared with the prior art, the invention has the advantages that: the Streptomyces wetlands (Streptomyces paludis) strain 13-3 has antagonism on 2 pathogenic bacteria (Fusarium graminearum and Fusarium verticillioides) causing the corn stalk rot, the fermentation liquor can obviously inhibit the hypha growth and spore germination of the 2 pathogenic bacteria (Fusarium graminearum and Fusarium verticillioides) causing the corn stalk rot, and the fermentation liquor has excellent control effect on the corn stalk rot.
In addition, the Streptomyces paludis (Streptomyces paludis) strain 13-3 is obtained from soil, is harmonious and compatible with the natural environment, and has good application prospect.
Drawings
FIG. 1 shows the characteristics of Streptomyces wetland (Streptomyces paludis) 13-3 cultured in the culture medium of Gao's I.
FIG. 2 shows the inhibitory effect of Streptomyces wetland (Streptomyces paludis) strain 13-3 on Fusarium graminearum (Fusarium graminearum) and Fusarium verticillium (Fusarium verticillioides) under the culture conditions. Note: a is Fusarium graminearum, B is Fusarium verticillium, and CK is control group.
FIG. 3 shows the effect of fermentation broth of Streptomyces wetland (Streptomyces paludis) strain 13-3 on the growth of Fusarium graminearum (Fusarium graminearum) and Fusarium verticillium (Fusarium verticillioides) hyphae. Note: a is Fusarium graminearum, B is Fusarium verticillium, and CK is control group.
FIG. 4 shows the effect of fermentation broth of Streptomyces wetland (Streptomyces paludis) strain 13-3 on the germination of Fusarium graminearum (Fusarium graminearum) and Fusarium verticillium (Fusarium verticillioides) spores. Note: a is Fusarium graminearum, B is Fusarium verticillium, and CK is control group.
Detailed Description
The invention is described in detail below with reference to the drawings and examples of the specification:
the first embodiment is as follows: and (3) separating and identifying the Streptomyces paludis strain 13-3.
1. Collection of soil samples
Collecting 3 parts of soil sample from Jiangxi Jinggang mountain, removing soil on the surface, collecting the soil sample at the depth of 5-20cm, marking, and taking back to a laboratory for natural air drying.
2. Isolation of Actinomycetes
The separation was performed by plate dilution. Grinding the air-dried soil sample with a mortar, weighing 1g of the sample, suspending the sample in 9mL of sterile water, shaking at 40 ℃ and 180rpm for 30min, standing for 5min, sequentially diluting by 10 times, and respectively preparing into 10 -2 、10 -3 、10 -4 The suspension is respectively sucked to 0.1mL of each suspension with different concentrations and added to a modified HVA culture medium (potassium dichromate with the final concentration of 100-200 ppm) plate, the plate is evenly coated and placed upside down at 28 ℃ for culture and observation, different single colonies are picked after 5-7 days and streaked and purified, and the purified strain is stored in a refrigerator at-80 ℃ by adopting a glycerol method.
3. Identification of Streptomyces paludis (Streptomyces paludis) strain 13-3
(1) Morphological feature observation
Streptomyces paludis (Streptomyces paludis) strain 13-3 grows well on most media, and does not produce soluble pigments (Table 1). Under an optical microscope, the strain 13-3 has straight, flexible, hooked, loose and compact spiral spore filaments and elliptic and cylindrical spores.
TABLE 1 culture characteristics of Streptomyces paludis 13-3
(2) Physiological and biochemical characteristics
The characteristics of Streptomyces wetland (Streptomyces paludis) 13-3, such as starch hydrolysis, nitrate reduction, carbon and nitrogen source utilization, were determined by reference to the method described in the Streptomyces identification Manual, and the results are shown in Table 2.
TABLE 2 physiological and biochemical characteristics of Streptomyces paludis 13-3
(3) Sequence analysis
Extracting Streptomyces wetlands (Streptomyces paludis) 13-3 genome DNA by using a bacterial genome extraction kit, respectively carrying out 16S rRNA and rpoB gene amplification to obtain sequences with full lengths of 1410bp and 767bp, submitting the obtained sequences to a GenBank database for BLAST comparison, and analyzing morphological characteristics, physiological and biochemical characteristics, 16S rRNA and rpoB genes to obtain the strain 13-3 which is Streptomyces wetlands (Streptomyces paludis).
Example two: determination of antagonistic action of Streptomyces paludis (Streptomyces paludis) 13-3 strain on 2 pathogenic bacteria causing corn stalk rot
The antagonistic action of the Streptomyces wetlands (Streptomyces paludis) 13-3 strain on fusarium graminearum and fusarium verticillioides is determined by adopting a plate confronting culture method. Firstly, inoculating strains 13-3,3 to both sides of the edge of a PDA culture medium in a streaking manner, inoculating a test pathogenic bacteria cake with the diameter of 5mm in the center of a flat plate, culturing 5d at 28 ℃ to measure the inhibition bandwidth of the Streptomyces wetae (Streptomyces paludis) strain 13-3 to the test pathogenic bacteria (table 3), and taking the test pathogenic bacteria without antagonistic bacteria as a control, the result shows that the Streptomyces wetae (Streptomyces paludis) strain 13-3 has very strong antagonistic action on fusarium graminearum and fusarium verticillioides (fig. 2).
TABLE 3 antagonistic Effect of strains 13-3 on 2 species of Fusarium zeae
Example three: inhibition effect of Streptomyces wetlands (Streptomyces paludis) 13-3 fermentation liquid on 2 pathogenic bacteria causing corn stalk rot
Activating a Streptomyces wetae (Streptomyces paludis) strain 13-3 in a Gao's I culture medium, culturing for 5 days at a constant temperature of 28 ℃, inoculating spores of the Streptomyces wetae (Streptomyces paludis) strain 13-3 into an ISP2 solid culture medium, fermenting for 7-8 days at 26-28 ℃, extracting for 3 times by using ethyl acetate, decompressing, concentrating and evaporating an extracting solution, dissolving by using DMSO, and preparing a 20mg/mL fermentation liquid for later use.
Taking 100 mu L of fermentation liquor of Streptomyces paludis 13-3 strain, adding the fermentation liquor into 100mL of PDA culture medium which is cooled to about 50 ℃ (no scalding hands), uniformly mixing, pouring the mixture into a culture dish, inoculating a corn stalk rot pathogen bacterial cake with the diameter of 5mm into the center of a PDA culture medium plate, culturing at 28 ℃ for 5 days, and measuring the diameter of a bacterial colony. The inhibition rate of the fermentation broth on the growth of hyphae of 2 species of corn stalk rot fungi was calculated by using the same amount of DMSO as a control (Table 4). The results show that the fermentation liquid of the Streptomyces wetlands (Streptomyces paludis) strain 13-3 has stronger inhibition effect on the hypha growth of fusarium graminearum and fusarium verticillioides (figure 3).
TABLE 4 inhibitory Effect of fermentation broth of Strain 13-3 on 2 species of Fusarium zeae
Example four: inhibition effect of Streptomyces wetlands (Streptomyces paludis) strain 13-3 fermentation liquor on spore germination of 2 pathogenic bacteria causing corn stalk rot
Inoculating Fusarium verticillium cake with diameter of 5mm into PDB liquid culture medium, inoculating Fusarium graminearum cake into semen Phaseoli Radiati liquid culture medium, shake culturing at 28 deg.C and 180rpm for 7d, filtering to collect spore suspension, and adjusting its concentration to 1 × 10 5 one/mL.
Taking 1 mu L of fermentation liquid of the Streptomyces wetlands (Streptomyces paludis) strain 13-3 in the third example, adding the fermentation liquid into 1mL of spore suspension, taking DMSO with the same amount as a control, respectively culturing the spore suspension of fusarium graminearum and fusarium verticillioides at 28 ℃ for 10h and 4h, and calculating the inhibition rate of the fermentation liquid of the Streptomyces wetlands (Streptomyces paludis) strain 13-3 on spore germination of 2 corn stalk rot germs (Table 5). The results show that the fermentation liquid of the Streptomyces wetlands (Streptomyces paludis) strain 13-3 obviously inhibits the spore germination of fusarium graminearum and fusarium verticillioides (figure 4).
TABLE 5 inhibitory Effect of fermentation broth of Strain 13-3 on the germination of conidia of 2 species of Fusarium zeae
Example five: application of Streptomyces wetlands (Streptomyces paludis) strain 13-3 fermentation liquor in preventing and treating stem rot of corn in seedling stage.
Culturing fusarium graminearum and fusarium verticillioides to be tested for 5d at 28 ℃, punching fungus cakes on the edges of colonies by using a puncher with the diameter of 5mm, respectively transferring the fungus cakes into sterilized wheat grain culture media, culturing for 10d at 28 ℃ in the dark, respectively inoculating the fusarium graminearum and the fusarium verticillioides into sterile soil, namely respectively and uniformly mixing the cultured wheat grain culture media with the sterile soil according to the mass ratio of 1.
The test site is selected from Cambodisan in the Shangan region of Fuzhou, fujian province, 6 treatments are set in the test, and the treatment of inoculating fusarium graminearum is as follows: a.3% of difenoconazole WG 4 ml/kg of seeds, B, fermentation liquor 10 ml/kg of Streptomyces wetlands (Streptomyces paludis) strain 13-3, and C, clear water control; the treatment for inoculating the fusarium verticillioides comprises the following steps: d.3% difenoconazole WG 4 ml/kg seed, fermentation liquor 10 ml/kg seed of Streptomyces wetland (Streptomyces paludis) strain 13-3, F. clear water control. Weighing each treatment agent according to the weight of the corn seeds, dressing the seeds, spreading out, airing and sowing the seeds in nutrition bowls, wherein 10 corn seeds are treated in each bowl, and the treatment is repeated for 4 times when 5 nutrition bowls are treated.
And (5) investigating the disease condition when the length of the jade reaches the 4-5 leaf stage.
The investigation method comprises the following steps: lifting seedlings, and investigating the disease conditions of corn plants and roots according to the following grading standards:
level 0: the whole plant grows normally without diseases;
level 1: the plant grows normally basically, but a small amount of disease spots can be seen at the root, the area of the disease spots accounts for less than 1/4 of the surface area of the root, and the root group is visually observed to have brown color in white;
and 2, stage: the plant growth is weak, the leaf color is light, the plant height is short, the plant height is only 3/4 of the contrast, the side roots are few and short, fibrous roots are not present, the scab area is 1/4-1/2 of the total area of the root surface, and the root group is observed to have the same color of white and brown visually;
and 3, level: the plant grows extremely abnormally, the overground part can be seen to be withered and yellow withered, the plant height is only 1/2 of the contrast, the lateral roots are few, the lesion area accounts for 1/2-3/4 of the total area of the root surface, and the root group is visually observed to have white color in brown;
4, level: the seeds germinate but do not emerge, almost suffocate and cover the scab, and the roots are dark brown;
disease index = (number of diseased plants at each stage × relative disease value) ÷ (total number of investigated plants × 4) × 100;
control effect (%) = [ (CK disease index-treatment disease index) ÷ CK disease index ] × 100;
TABLE 6 preventive effect of fermentation broth of Strain 13-3 on Fusarium culm rot of maize
The experimental result shows that the control effect of fermentation liquor 10 ml/kg of A.3% difenoconazole WG 4 ml/kg of seeds and B. Streptomyces wetland (Streptomyces paludis) strain 13-3 on stem rot caused by fusarium graminearum is 66.46% and 63.90% respectively, and the strain is safe to corn and has no chemical injury. The control effect of 10 ml/kg of fermentation liquor of Streptomyces wetlands (Streptomyces paludis) strain 13-3 on stem rot caused by fusarium graminearum is not obvious in the control effect difference of 5% significant level and 1% very significant level with 3% difenoconazole WG 4 ml/kg of seeds.
D.3% of difenoconazole WG 4 ml/kg of seeds, 10 ml/kg of fermentation liquor of Streptomyces paludis (Streptomyces paludis) strain 13-3, respectively has 63.57% and 61.23% of control effect on stem rot caused by fusarium verticillioides of corn, is safe to corn and has no phytotoxicity. The control effect of 10 ml/kg of fermentation liquor of Streptomyces wetlands (Streptomyces paludis) strain 13-3 on stem rot caused by fusarium verticillium is not obvious from the control effect difference of 3% difenoconazole WG 4 ml/kg of seeds on 5% obvious level and 1% extremely obvious level.
Therefore, the prevention effect of the fermentation liquid 10 ml/kg of Streptomyces wetland (Streptomyces paludis) strain 13-3 on fusarium zeae stalk rot is equivalent to the prevention effect of 3% difenoconazole WG 4 ml/kg of seeds, and the application prospect is good.
Claims (4)
1. The application of the Streptomyces paludis (Streptomyces paludis) 13-3 strain in preventing and treating the corn stalk rot is characterized in that: the Streptomyces wetland (Streptomyces paludis) strain 13-3 is preserved in the China general microbiological culture Collection center of the culture Collection of microorganisms with the preservation number of CGMCC No.22830 and the preservation date: 7/6/2021.
2. The application of the Streptomyces paludis (Streptomyces paludis) 13-3 fermentation liquor in preventing and treating the corn stalk rot is characterized in that:
the fermentation liquid is prepared by inoculating Streptomyces wetlands (Streptomyces paludis) 13-3 strain in claim 1 to ISP2 solid culture medium, extracting with ethyl acetate at the fermentation temperature of 26-28 ℃ for 7-8 days, concentrating the extracting solution under reduced pressure, evaporating to dryness, and dissolving with DMSO.
3. Use according to claim 2, characterized in that: the Streptomyces wetland (Streptomyces paludis) strain 13-3 fermentation liquor is applied to inhibition of hypha growth of Fusarium graminearum (Fusarium graminearum) and Fusarium verticillioides (Fusarium verticillioides).
4. Use according to claim 2, characterized in that: the Streptomyces wetland (Streptomyces paludis) strain 13-3 fermentation liquor is applied to inhibition of spore germination of Fusarium graminearum (Fusarium graminearum) and Fusarium verticillioides (Fusarium verticillioides).
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