CN111575199B - Pseudomonas aeruginosa JT86 and application thereof in preventing and treating sclerotinia rot - Google Patents
Pseudomonas aeruginosa JT86 and application thereof in preventing and treating sclerotinia rot Download PDFInfo
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Abstract
本发明公开了一株铜绿假单胞菌JT86及其在防治菌核病中的应用。本发明从土壤中筛选得到了一株铜绿假单胞菌JT86，该菌株已于2019年9月30日保藏于广东省微生物菌种保藏中心，保藏号为GDMCC NO：60799。该菌株及其挥发物能够完全抑制水稻纹枯病菌、花生白绢病菌和油菜菌核病菌的菌核萌发，抑制其菌核的菌丝的生长，使得菌核的菌丝出现片状、皱缩，且该菌株对水稻纹枯病菌、花生白绢病菌、油菜菌核病菌、稻瘟病菌、香蕉枯萎病菌、菜心炭疽病菌、茶炭疽病菌、辣椒炭疽病菌也具有很好的抑制效果；因此，该菌株能够针对性处理土壤中顽拗的土传病原菌，有效抑制菌核病的发生，在制备防治土传真菌病害的土壤处理剂中具有良好的应用前景。
The invention discloses a strain of Pseudomonas aeruginosa JT86 and its application in preventing and treating sclerotinia. In the present invention, a strain of Pseudomonas aeruginosa JT86 was screened from the soil, and the strain was deposited in the Guangdong Provincial Microbial Culture Collection Center on September 30, 2019, and the preservation number is GDMCC NO: 60799. The strain and its volatiles can completely inhibit the sclerotia germination of Rhizoctonia oryzae, Peanut Sclerotinia and Sclerotinia sclerotiorum, inhibit the growth of their sclerotium hyphae, and make the sclerotium hyphae appear flaky and wrinkled. It also has good inhibitory effect on rice sheath blight, peanut white silk, rape sclerotiorum, rice blast, banana fusarium wilt, cabbage anthracnose, tea anthracnose, and pepper anthracnose; Therefore, the strain can target the recalcitrant soil-borne pathogenic bacteria in the soil, effectively inhibit the occurrence of sclerotinia, and has a good application prospect in the preparation of a soil treatment agent for preventing and controlling soil-borne fungal diseases.
Description
Technical Field
The present invention belongs to the field of biological plant disease preventing and controlling technology. More particularly, relates to pseudomonas aeruginosa JT86 and application thereof in preventing and treating sclerotinia rot.
Background
Soil-borne diseases caused by plant pathogenic fungi are important bottlenecks which restrict sustainable development of agricultural production in China. The soil physicochemical environment is generally not conducive to the growth of plant pathogenic fungi compared to plant pathogens such as bacteria, nematodes and the like, and thus plant pathogenic fungi are usually present in the soil as some propagules (e.g., sclerotia, conidia scleotiorum) or saprophytic to plant roots and crop residues. As a highly specialized, mutually selective and coevolution system is formed in the interaction process of the plant and the pathogenic fungi, a variety of soil-borne pathogenic fungi have different specialization types; for example, fusarium fungi can cause disease in cotton, cucumber, watermelon, and other crops. Some soil-borne diseases are not formed by single pathogenic bacteria, but are the result of complex infection of multiple pathogenic bacteria, which further complicates the pathogenic mechanism of the soil-borne diseases.
The rice sheath blight disease is one of three fungal diseases of rice caused by Rhizoctonia solani (Rhizoctonia solani), is distributed in all rice producing countries in the world, causes the most serious damage to Asian rice regions, obviously reduces the quality of the infected rice, and causes failure harvest in severe cases. In China, the disease area of the disease per year reaches more than 2 hundred million mu, and besides rice, the bacterium can infect more than 200 other crops to cause important damping-off and the like. The rhizoctonia solani overwinter or cross bad environment by producing sclerotium in nature, and plant diseases caused by the rhizoctonia solani are important sclerotium-producing soil-borne diseases. Chinese is a big rape planting country, and Sclerotinia sclerotiorum (Sclerotinia sclerotiorum) caused by Sclerotinia sclerotiorum is a main disease affecting the yield of rape in China. The disease is in a gradually serious trend at present, the yield is reduced by 10 to 20 percent in light years, and the yield is reduced by more than 50 percent in heavy years. Sclerotinia has a wide host range, and can damage 400 plants in Cruciferae, Leguminosae, Solanaceae, Rutaceae, etc., to cause sclerotinia. Southern blight caused by Sclerotium rolfsii (Sclerotium rolfsii) and Verticillium wilt caused by Verticillium dahliae (Verticillium dahliae), which can also harm up to 200 crops and cause huge economic loss to agricultural production in China. These fungi all produce sclerotia or microsclera. The sclerotium is a special dormant structure formed by some filamentous fungi, and is formed by high aggregation of hyphae of pathogenic bacteria, winding to form an initial entanglement body, then high differentiation, cell wall thickening and melanin secretion, and finally spherical deep black sclerotium is formed. The sclerotium can be preserved in soil for more than ten years and still has activity without being infected by host plants. The pathogenic bacteria mainly live through the winter by sclerotium, and the number of the overwintering sclerotium is a key factor for the occurrence of the sclerotinia sclerotiorum. As the sclerotinia sclerotiorum has the characteristics of wide host range, easy propagation, persistent vitality and the like, no particularly effective control method exists at present, and the sclerotinia sclerotiorum is mainly controlled by chemical pesticides. However, the use of chemical pesticides throughout the year causes great damage to humans and the natural environment, making disease control more difficult. Therefore, the search for beneficial microbes which are friendly to the environment and the biological control of the sclerotinia has become a research hotspot and receives more and more attention.
Many biocontrol bacteria can metabolize substances for inhibiting the growth of pathogenic bacteria in the metabolic process, such as special protein bacterial toxins, antibiotics, chitins and other enzymes, and the like, so that the growth and metabolic activity of the pathogenic bacteria are directly inhibited, and even the pathogenic bacteria can be inactivated under the action of antibacterial substances with extremely strong effects. The biocontrol bacteria have the characteristics of quick growth and reproduction, large quantity, multiple varieties, simple acquisition way, various action modes and the like, and have remarkable effect in the research and practice of biological control of plant diseases. Therefore, the method further screens out high-efficiency biocontrol bacteria with broad-spectrum antibacterial activity, and has important significance for the biological control of soil-borne diseases and the development of green biological agents.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects and shortcomings of the existing soil-borne fungal disease control technology and provides a pseudomonas aeruginosa JT86 and application thereof in the control of sclerotinia rot. The invention aims at preventing and treating sclerotinia, screens out biocontrol bacteria (pseudomonas aeruginosa JT86) which can effectively inhibit sclerotium germination and development from the environment, can be used for preparing soil treatment agents and antifungal preparations, and has wide application space in the green prevention and control of soil-borne fungal diseases and plants.
The invention aims to provide a Pseudomonas aeruginosa (Pseudomonas aeruginosa) JT 86.
The invention also aims to provide application of the pseudomonas aeruginosa JT86 in controlling soil-borne fungal diseases or preparing a soil treatment agent for controlling soil-borne fungal diseases.
The invention also aims to provide application of the pseudomonas aeruginosa JT86 in preventing and treating fungal diseases or preparing a fungicide.
It is still another object of the present invention to provide a soil treatment agent for controlling soil-borne fungal diseases.
It is a further object of the present invention to provide a fungicidal preparation.
The above purpose of the invention is realized by the following technical scheme:
the invention provides a Pseudomonas aeruginosa (Pseudomonas aeruginosa) JT86, which is preserved in Guangdong province microorganism strain collection center in 2019, 9 and 30 months, and the preservation number is GDMCC NO: 60799 and the preservation address is No. 59 building 5 of No. 100 Dazhong Jie-Lu-100 Guangzhou city.
The pseudomonas aeruginosa JT86 is obtained by screening from soil, the strain and volatile matters thereof can completely inhibit sclerotium germination of rhizoctonia solani, southern blight bacteria and sclerotinia sclerotiorum of rice, inhibit the growth of hyphae of sclerotium thereof, enable the hyphae of the sclerotium to be flaky and shrivelled, and can be used as a soil treatment agent to prevent and treat soil-borne diseases caused by sclerotinia sclerotiorum; the strain also has good inhibition effect on rice sheath blight bacteria, peanut sclerotium rolfsii, sclerotinia sclerotiorum, rice blast, banana wilt bacteria, colletotrichum brassicae, tea colletotrichum and pepper colletotrichum; therefore, the following applications should be within the scope of the present invention:
the pseudomonas aeruginosa JT86 is applied to the prevention and treatment of soil-borne fungal diseases or the preparation of soil treatment agents for the prevention and treatment of the soil-borne fungal diseases.
Preferably, the soil-borne fungal disease is sclerotinia.
Preferably, the sclerotinia sclerotiorum is sclerotinia sclerotiorum caused by rhizoctonia solani, southern blight or sclerotinia sclerotiorum.
The pseudomonas aeruginosa JT86 is applied to the prevention and treatment of fungal diseases or the preparation of fungicide preparations.
Preferably, the fungus is Rhizoctonia solani, southern blight of peanut, sclerotinia sclerotiorum, rice blast, banana wilt, colletotrichum brassicae, tea colletotrichum or pepper colletotrichum.
The invention also provides a soil treatment agent for preventing and treating soil-borne fungal diseases, which comprises the pseudomonas aeruginosa JT86 or fermentation liquor thereof.
The invention also provides a fungicide preparation which comprises the pseudomonas aeruginosa JT86 or fermentation liquor thereof.
The invention has the following beneficial effects:
the pseudomonas aeruginosa JT86 and volatile matters thereof obtained by screening from soil can completely inhibit sclerotium germination of rhizoctonia solani, peanut sclerotium rolfsii and rape sclerotinia sclerotiorum and inhibit the growth of hyphae of the sclerotium, so that the hyphae of the sclerotium are flaky and shriveled, and can be used as a soil treatment agent to prevent and treat soil-borne diseases caused by sclerotinia sclerotiorum; in addition, the strain has good inhibition effect on rice sheath blight bacteria, peanut sclerotium rolfsii, sclerotinia sclerotiorum, rice blast, banana wilt bacteria, colletotrichum brassicae, tea colletotrichum and pepper colletotrichum capsici; therefore, the application of the strain in preventing and treating soil-borne fungal diseases or the preparation of soil treatment agents for preventing and treating soil-borne fungal diseases can reduce the use of chemical pesticides and reduce pesticide residues, provides a new way for gradually replacing chemical prevention and treatment with biological prevention and treatment, and has wide development and application prospects.
Drawings
FIG. 1 is a map of the morphological characteristics of JT86 strain; wherein, the graph (A) is a gram stain graph of a colony of JT86 strain; (B) the image is an electron microscope scanning image of JT86 strain.
FIG. 2 is a 16S rDNA gene clade of JT86 strain.
FIG. 3 is a gyrB gene clade of JT86 strain.
FIG. 4 is a graph showing the inhibitory effect of Pseudomonas aeruginosa JT86 on sclerotia of southern blight bacteria; wherein (A1) is a control group 10 μm electron micrograph, (A2) is a control group 20 μm electron micrograph, (B1) is a control group 10 μm electron micrograph, and (B2) is a control group 20 μm electron micrograph.
FIG. 5 is a graph showing the inhibitory effect of Pseudomonas aeruginosa JT86 on Rhizoctonia solani; wherein, the graph (A) is a control group; (B) the figure is a treatment group.
FIG. 6 is a graph showing the inhibitory effect of Pseudomonas aeruginosa JT86 on southern blight; wherein, the graph (A) is a control group; (B) the figure is a treatment group.
FIG. 7 is a graph showing the inhibitory effect of Pseudomonas aeruginosa JT86 on Pyricularia oryzae; wherein, the graph (A) is a control group; (B) the figure is a treatment group.
FIG. 8 is a graph showing the inhibitory effect of Pseudomonas aeruginosa JT86 on Fusarium oxysporum; wherein, the graph (A) is a control group; (B) the figure is a treatment group.
FIG. 9 is a graph showing the inhibitory effect of Pseudomonas aeruginosa JT86 on colletotrichum gloeosporioides; wherein, the graph (A) is a control group; (B) the figure is a treatment group.
FIG. 10 is a graph showing the inhibitory effect of Pseudomonas aeruginosa JT86 on Colletotrichum theacrinum; wherein, the graph (A) is a control group; (B) the figure is a treatment group.
FIG. 11 is a graph showing the inhibitory effect of Pseudomonas aeruginosa JT86 on Colletotrichum capsici; wherein, the graph (A) is a control group; (B) the figure is a treatment group.
FIG. 12 is a graph showing the inhibitory effect of Pseudomonas aeruginosa JT86 on Sclerotinia sclerotiorum; wherein, the graph (A) is a control group; (B) the figure is a treatment group.
FIG. 13 is a graph showing the effect of volatiles of Pseudomonas aeruginosa JT86 on the inhibition of buckled culture of southern sclerotium; wherein, the graph (A) is a control group; (B) the figure is a treatment group.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 isolation and screening of Pseudomonas aeruginosa (Pseudomonas aeruginosa) JT86
1. Collecting samples:
soil samples were collected from rice fields jumping into northern farm at the southern agricultural university of the Tianhewan, Guangzhou, Guangdong province, and placed in sterile centrifuge tubes to be brought back to the laboratory for strain isolation.
2. And (3) separation of biocontrol bacteria:
uniformly mixing soil samples, putting 5g of soil samples into a 50mL sterile centrifuge tube, adding sterile water to 50mL, violently shaking the suspended soil samples, and marking the suspended soil samples as 1 multiplied by 10-1. Then, 1mL of 1X 10 was aspirated by a pipette-1The soil sample suspension is put into a new centrifugal tube filled with 9mL of sterile water and sequentially diluted into 1 × 10-2、1×10-3The series of concentration soil solutions of (1). 0.1mL of 1X 10 was aspirated-2、1×10-3And (3) putting the soil solution on a solid LB plate (yeast extract 5g, tryptone 10g, NaCl 5g, water 1000mL, pH7.2, agar powder 15g, sterilizing at 121 ℃ for 15min), uniformly coating by using a coating rod, airing, and then putting in an incubator at 30 ℃ for culturing for 1-2 d. And picking a single colony for primary screening.
3. Primary screening of biocontrol bacteria:
the pellet was punched out with a 6mm punch and inoculated into the center of a PDA plate. Inoculating the bacteria (single colony) obtained in the step 2 at a position 2.5cm away from the pathogenic bacteria block, culturing 4 different candidate bacteria in each dish at 28 ℃ by taking the non-inoculated bacteria as a control until the pathogenic bacteria colony of the control group grows over the culture dish, and observing whether an inhibition zone appears around the bacteria. And (5) preserving and numbering strains capable of generating bacteriostatic zones. Then, a 6mm puncher is used for punching a bacterium block, the bacterium block is inoculated on the right side of a PDA (personal digital assistant) plate, and then the strain capable of generating the inhibition zone is inoculated at a position 3cm away from the pathogenic bacterium block, 1 bacterium in each dish, and the non-inoculated strain is used as a control. And (3) culturing at 28 ℃ until the control group pathogenic bacteria colony grows to fill the culture dish, and observing whether an obvious inhibition zone appears around the bacteria. And (4) storing the strains which can generate obvious inhibition zones separately, and recording the serial numbers of the strains.
4. Rescreening biocontrol bacteria:
pathogenic bacteria: rhizoctonia solani, sclerotinia sclerotiorum, verticillium dahliae, pyricularia oryzae, fusarium oxysporum, colletotrichum brassicae, colletotrichum lecanii, colletotrichum tea, colletotrichum capsici, tobacco target spot bacteria and botrytis cinerea;
the pathogen inoculation method is the same as that in the step 3, the candidate biocontrol bacteria are inoculated with 4 strains of bacteria with the same number in each dish, the bacteria are cultured at the temperature of 28 ℃ by taking the non-inoculated biocontrol bacteria as a reference until the pathogen colony of the control group grows over the culture dish, the diameter of the pathogen colony of the control group and the diameter of the pathogen colony of the control group are measured and recorded, and the bacteriostasis rate is calculated according to the following formula:
the bacteriostatic rate (%) is (diameter of control hypha-diameter of treated hypha)/diameter of control hypha x 100%;
the bacterium with the strain number of JT86 obtained by the method has high control effect on various pathogenic fungi.
Example 2 identification of Pseudomonas aeruginosa (Pseudomonas aeruginosa) JT86
1. Morphological identification
(1) Experimental methods
The JT86 strain screened in example 1 was cultured in LB medium, and morphological characteristics were observed and physiological and biochemical analyses were performed.
(2) Results of the experiment
The morphological characteristic diagram of JT86 strain is shown in FIG. 1, and it can be seen that the colony of the strain is round, light yellow, flat in edge, convex in center, smooth in surface, matt, translucent, gram-negative, and shows that the thallus is rod-shaped with short oval shape and unit cell under electron microscope, and the thallus size is 1.8 × 3.0 μm.
Further, as a result of physiological and biochemical tests, it was found that the JT86 strain was positive in physiological and biochemical tests such as catalase reaction, oxidase reaction, motility measurement, V-P test, gelatin liquefaction, and nitrate reduction, and negative in starch hydrolysis, methyl red reaction, hydrogen sulfide reaction, and the like, and was aerobic, and glucose, fructose, lactose, mannitol, sucrose, and maltose were not available.
The result of combining the morphological characteristics and physiological and biochemical characteristics of the above JT86 strain was preliminarily identified as Pseudomonas (Pseudomonas).
2. Molecular identification
(1) Experimental methods
Genomic DNA extraction procedure of JT86 Strain DNA concentration was determined using a Nanodrop 2000 micro ultraviolet Spectrophotometer (Thermo Scientific, Wilmington, DE) with reference to the product (Bacterial DNA Kit D3350, OMEGA) instructions.
PCR amplification was carried out using the extracted genomic DNA of JT86 strain as a template, and bacterial 16S rDNA universal primers (forward primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', reverse primer 1492R: 5'-ACGGCTACCTTGTTACGACTT-3') and gyrB gene universal primers (forward primer 5'-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3', reverse primer 5'-AGCAGGATACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3', Y represents C or T, N represents A, T, C or G R represents A or G); the 16S rDNA universal primer and the gyrB gene universal primer of the bacteria are synthesized by Beijing Optimalaceae New Biotechnology Limited.
The reaction system for PCR amplification was (25. mu.L): 2 XPCR Mix 12.5. mu.L, forward primer (10. mu. mol/L) 1.0. mu.L, reverse primer (10. mu. mol/L)1. mu.L, DNA template 1. mu.L, deionized water to make up to 25. mu.L. And (3) packaging the reaction solution into PCR tubes, and performing PCR reaction after uniformly mixing. The reaction procedure for PCR amplification was: pre-denaturation at 98 deg.C for 2min, annealing at 98 deg.C for 10s, annealing at 56 deg.C for 1min, extension at 72 deg.C for 1min, 30 cycles, and final extension at 72 deg.C for 10 min.
After the fragments obtained by PCR amplification reaction are recovered and purified, sequencing is carried out by Beijing Optimalaceae New Biotechnology Limited, and the obtained sequence is submitted to NCBI for sequence comparison. A phylogenetic tree is constructed by comparing the 16S rDNA sequence and the gyrB gene sequence of the JT86 strain with the existing sequence of Pseudomonas aeruginosa (Pseudomonas aeruginosa) in a database.
(2) Results of the experiment
The 16S rDNA gene evolutionary tree of JT86 strain is shown in FIG. 2, and the gyrB gene evolutionary tree of JT86 strain is shown in FIG. 3, and the results show that JT86 strain and Pseudomonas aeruginosa are gathered into one branch.
In combination with morphological and molecular characterization, the JT86 strain was identified as Pseudomonas aeruginosa (Pseudomonas aeruginosa) JT86 and deposited at 2019, 30 months, 9 at the guangdong collection of microorganisms with accession number GDMCC NO: 60799 and the preservation address is No. 59 building 5 of No. 100 Dazhong Jie-Lu-100 Guangzhou city.
Example 3 inhibition of Sclerotinia sclerotiorum Germinosa JT86 by Pseudomonas aeruginosa
1. Experimental methods
Activating Pseudomonas aeruginosa JT86 preserved by glycerol at-80 ℃ on LB solid culture medium at 30 ℃ for 48h, dipping activated single colony of Pseudomonas aeruginosa JT86 into 100mL NA liquid culture medium, and culturing at 28 ℃ with shaking at 200rpm for 48 h. And performing secondary culture according to the inoculation amount of 10%, namely taking 10% bacterial liquid to culture in 100mL NA liquid culture medium at 28 ℃ for 48h with bacteria shaking at 200rpm to obtain the pseudomonas aeruginosa JT86 fermentation liquid.
Picking 120 sclerotium of rice sheath blight bacteria, peanut sclerotium rolfsii and rape sclerotinia sclerotiorum with the same size, setting a control group and a treatment group to repeat each 3 times, carrying out surface disinfection on the sclerotium, namely disinfecting for 5 minutes by using 10% sodium hypochlorite (effective chlorine content), then washing the sclerotium by using sterile water, fully airing the sclerotium on sterile filter paper, and putting the sclerotium into 100mL of pseudomonas aeruginosa JT86 fermentation liquor after airing, wherein the control group is an NA liquid culture medium. After culturing the sclerotium for 24 hours by shaking on a shaking table at 28 ℃ and 200rpm, taking out the sclerotium, washing the sclerotium by using sterile water, fully drying the sclerotium on sterile filter paper, placing the sclerotium on a water agar plate, recording the germination number of the sclerotium, and calculating the germination inhibition rate of the sclerotium according to the following formula:
inhibition/% ([ (control germination number-treatment germination number)/control germination number ] × 100.
After shaking out the mycelium pellet by shaking on a shaking table at 28 ℃ and 200rpm for 24h (the sclerotinia sclerotiorum sclerotium germinates slowly, the shaking time needs to be prolonged), the experimental group picks up the mycelium pellet by using sterilized tweezers and puts the mycelium pellet into pseudomonas aeruginosa JT86 fermentation liquor, then the culture is carried out for 24h, and the control group is shaken in an NA liquid culture medium. Set 3 sets of replicates. Respectively selecting a sclerotium, subpackaging in a 1.5mL centrifuge tube, and then adding glutaraldehyde with the volume being three times that of the mycelium for fixing for two or three days. And (4) observing by a scanning electron microscope, firstly fixing the sample, then dehydrating the sample by using alcohol with a series of concentrations, and drying by using a special instrument. After the sample is dried, a vacuum spraying instrument is used for spraying carbon, then spraying is carried out, the spraying is uniform, and after all the steps are finished, the shape of the thallus is observed by using an electric mirror (XL-30-ESEM) and FeI in the Netherlands.
2. Results of the experiment
The inhibition rates of pseudomonas aeruginosa JT86 on the sclerotium germination of the three sclerotium-producing fungi are shown in Table 1, and it can be seen that the sclerotium of the three sclerotium-producing fungi (rhizoctonia solani, southern sclerotium blight and sclerotinia sclerotiorum colza) in the control group completely germinates, while the sclerotium of the three sclerotium-producing fungi treated by the pseudomonas aeruginosa JT86 fermentation liquor can not germinate, and the inhibition rates of the sclerotium germination are all 100%; the pseudomonas aeruginosa JT86 can completely inhibit the sclerotium germination of rhizoctonia solani, southern blight and sclerotinia sclerotiorum.
TABLE 1 inhibition of Sclerotinia germination by Pseudomonas aeruginosa JT86 on three Sclerotinia-producing fungi
FIG. 4 shows the inhibitory effect of Pseudomonas aeruginosa JT86 on sclerotia of southern blight bacteria, wherein (A1) is a control 10 μm electron micrograph, (A2) is a control 20 μm electron micrograph, and (B1) is a control 10 μm electron micrograph, and (B2) is a control 20 μm electron micrograph; as can be seen, the hyphae of the sclerotium of the southern blight fungus in the control group can normally grow, and the hyphae have smooth surface and full shape; the hypha of the sclerotium treated by the fermentation liquor of pseudomonas aeruginosa JT86 appears flaky and shrivelled; the result shows that the pseudomonas aeruginosa JT86 can completely inhibit the growth of the hypha of the sclerotium of the peanut sclerotium.
Example 4 challenge test of Pseudomonas aeruginosa (Pseudomonas aeruginosa) JT86 to pathogenic fungi
1. Experimental methods
The bacteriostasis rate of pseudomonas aeruginosa JT86 on pathogenic fungi (rhizoctonia solani, southern blight, sclerotinia sclerotiorum, rice blast, banana wilt, colletotrichum brassicae, tea colletotrichum and pepper colletotrichum capsici) is determined by adopting a confronting method in a dish.
Inoculating a pathogenic fungus cake with the diameter of 5mm to the center of a PDA (personal digital assistant) plate, placing sterile filter paper sheets with the diameter of 5mm at four positions 25mm away from pathogenic fungi, respectively dripping 10 mu L of sterile water/pseudomonas aeruginosa JT86 fermentation liquor (the control group is sterile water, and the treatment group is pseudomonas aeruginosa JT86 fermentation liquor) on the filter paper sheets by using a liquid transfer gun, and airing on a sterile ventilation table. Observing after constant temperature culture at 28 ℃ for 1-2d, measuring the diameter of a bacteriostatic zone and calculating the bacteriostatic rate according to the following formula:
the bacteriostasis rate is (the growth diameter of the pathogenic bacteria of the control group-the growth diameter of the pathogenic bacteria of the treatment group)/the growth diameter of the pathogenic bacteria of the control group multiplied by 100 percent.
2. Results of the experiment
The results of the bacteriostasis rate of the pseudomonas aeruginosa JT86 on pathogenic fungi are shown in Table 2, the bacteriostasis effects of the pseudomonas aeruginosa JT86 on rhizoctonia solani, southern blight bacteria, sclerotinia sclerotiorum, rice blast, fusarium oxysporum f.sp.cubense, colletotrichum cochlioides, tea anthracnose and pepper anthracnose are shown in the figures 5-12 in sequence, and the results of the tables 2 and the figures 5-12 show that the pseudomonas aeruginosa JT86 has higher bacteriostasis rate on the above 8 pathogenic fungi, and the bacteriostasis rate is 59.48-87.02%.
TABLE 2 results of the bacteriostatic rate of Pseudomonas aeruginosa JT86 on pathogenic fungi
Example 5 bacteriostatic Effect of volatiles of Pseudomonas aeruginosa JT86 on pathogenic fungi
1. Experimental methods
Volatile make-up experiment: dropwise adding 100 mu L of pseudomonas aeruginosa JT86 fermentation liquor on an LB flat plate, uniformly coating by using a coating rod, and culturing for 24h in a 30 ℃ culture box; covering a layer of sterilized cellophane on a PDA flat plate, removing air bubbles to make the PDA flat plate smooth, processing and assembling three kinds of produced sclerotinia (Rhizoctonia solani, southern blight and sclerotinia sclerotiorum) in the center of the PDA flat plate, buckling the two, sealing, and placing in a 30 ℃ incubator; the control group was coated with sterile water. During observation, the cellophane is torn off, and a small amount of the hyphae at the outermost edge is cut out and observed under a microscope. By comparing with the form of normal hypha, judging whether the pseudomonas aeruginosa JT86 has influence on the growth of the hypha of the rice sheath blight germ, the peanut sclerotinia sclerotiorum and the like.
VOCs of the pseudomonas aeruginosa JT86 are identified by a SPME-GC-MS (gas chromatography-mass spectrometry). Inoculating 1mL of pseudomonas aeruginosa JT86 fermentation liquor into a 50mL triangular flask containing 20mL of PDA liquid culture medium, sealing the triangular flask by using tin foil paper, carrying out shaking culture in a shaking table at 28 ℃ and 200rpm for 2d, then extracting generated VOCs by using an SPME extraction head, and carrying out GC-MS (gas chromatography-mass spectrometry) analysis.
GC-MS (gas chromatography-mass spectrometry) conditions: 7890A-5975C GC/MSD GC/MS; the column was an HP-5 quartz capillary column (30 m.times.0.25 μm, Agilent). The injection port temperature is 280 ℃, and the transmission line temperature is 250 ℃. The temperature programming was as follows: the initial column temperature is 100 ℃ and stays for 1 min; raising the temperature to 200 ℃ in 5 ℃ min; then raising the temperature to 300 ℃ at a speed of 10 ℃/min; finally, the temperature is increased to 250 ℃ at the speed of 8 ℃/min and the temperature is kept for 5 min. The carrier gas is high-purity helium (99.999%); the column flow rate was 1 mL/min. The sample size was 1.0. mu.L (no split).
GC-MS analysis: the GC conditions were as described above. The MS conditions were as follows: the ion source temperature is 280 ℃; ionization mode EI, ionization energy 70 eV; full scan acquisition, mass detector (MSD) detection. The components to be tested were determined by NIST (2011) spectral library search.
2. Results of the experiment
The bacteriostatic effect of the volatile matter of the pseudomonas aeruginosa JT86 on pathogenic fungi is shown in Table 3, and the bacteriostatic rate of the volatile matter of the pseudomonas aeruginosa JT86 on three sclerotium-producing fungi is 100 percent.
The buckling culture inhibition effect diagram of the volatile matter of pseudomonas aeruginosa JT86 on southern sclerotium rolfsii is shown in FIG. 13, and it can be seen that the southern sclerotium rolfsii of the control group germinates and hyphae normally grow and extend on the PDA plate, while the southern sclerotium rolfsii treated by the volatile matter of pseudomonas aeruginosa JT86 of the treatment group cannot germinate on the PDA plate and hyphae cannot grow and extend very much; the volatile matter of the pseudomonas aeruginosa JT86 has obvious effect of inhibiting the growth of hypha of rhizoctonia solani, southern blight bacteria and sclerotinia sclerotiorum.
In addition, volatile matters produced by pseudomonas aeruginosa JT86 are analyzed by SPME-GC-MS, and VOCs experimental results show that: the volatile substances are mostly alkenes, alkanes, bromoforms, hydrocarbons and other compounds.
TABLE 3 bacteriostatic effect of Pseudomonas aeruginosa JT86 on pathogenic fungi
Example 6 inhibitory Effect of Pseudomonas aeruginosa (Pseudomonas aeruginosa) JT86 on sclerotia in soil
1. Experimental methods
2kg of sterile mixed soil (nutrient soil and sandy soil) is filled in a sterilized plastic pot with a height of 20cm in advance, and the soil is up to 10cm of the pot. Leveling soil surface, placing 3 bags of sclerotium with surface sterilized in sterile tea bag at 10cm position of soil layer, each bag containing 10 sclerotium, and each pot containing 30 sclerotium. One pathogen was treated with one pot each. The sclerotium to be tested includes those produced by Rhizoctonia solani, southern blight and Sclerotinia sclerotiorum. Then cover the sterile mixed soil (nutrient soil and sandy soil) to full basin. I.e. the total depth of the soil is 20cm, and the sclerotium sample is placed in the middle of the soil sample. 400mL of pseudomonas aeruginosa JT86 fermentation liquor is poured on each pot of soil to ensure that the soil layer is completely wet. A control group was set up and 400mL of NA medium was poured onto the control group. And (4) after the soil sample is treated, placing the soil sample on a laboratory workbench for storage at room temperature. Then 20mL of sterile water is poured into each pot of soil sample every day to keep the soil sample moist. Taking out 1 pack of sclerotium from one pot every 3 days, washing with sterile water for 3 times, culturing on water agar, observing germination condition of sclerotium, and calculating germination inhibition rate according to the following formula:
inhibition/% ([ (control germination number-treatment germination number)/control germination number ] × 100.
2. Results of the experiment
The inhibition rates of the pseudomonas aeruginosa JT86 soil treatment on the sclerotium germination of the three sclerotium-producing fungi are shown in Table 4, and it can be seen that after the sclerotium in the soil treated by the pseudomonas aeruginosa JT86 fermentation liquor is 3d, the germination of the sclerotium in the soil is completely inhibited, and the inhibition rates of the germination are all 100%; the pseudomonas aeruginosa JT86 fermentation liquid can be used for soil treatment to prevent and treat sclerotinia rot caused by rhizoctonia solani, southern blight bacteria and sclerotinia sclerotiorum.
TABLE 4 inhibition of Sclerotinia germination by Pseudomonas aeruginosa JT86 soil treatment on three Sclerotinia-producing fungi
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (5)
1. Pseudomonas aeruginosaPseudomonas aeruginosa) JT86, wherein the strain was deposited at 30.9.2019 with the microbial cultures Collection of Guangdong province under the accession number GDMCC NO: 60799.
2. use of pseudomonas aeruginosa JT86 according to claim 1 for the control of soil-borne fungal diseases, which are sclerotinia sclerotiorum caused by rhizoctonia solani, sclerotinia rot of peanut or sclerotinia sclerotiorum, or for the preparation of a soil treatment for the control of soil-borne fungal diseases.
3. Use of Pseudomonas aeruginosa JT86 according to claim 1 for controlling a fungal disease or for preparing a fungicide for controlling a fungal disease, wherein the fungus is Rhizoctonia solani, southern blight, Sclerotium napellus, Pyricularia oryzae, Fusarium oxysporum, Colletotrichum brassicae, Colletotrichum theae, or Colletotrichum capsici.
4. A soil treatment agent for controlling soil-borne fungal diseases, comprising Pseudomonas aeruginosa JT86 or a fermentation broth thereof according to claim 1.
5. A fungicidal formulation comprising pseudomonas aeruginosa JT86 or its fermentation broth according to claim 1.
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