CN114231442B - Parlerian Long Nishi pseudomonas and application thereof in prevention and treatment of oomycete pathogenic bacteria soil-borne diseases - Google Patents
Parlerian Long Nishi pseudomonas and application thereof in prevention and treatment of oomycete pathogenic bacteria soil-borne diseases Download PDFInfo
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Abstract
The invention belongs to the technical field of crop control, and discloses a P.parr Long Nishi pseudomonas and application thereof in control of oomycetes pathogenic bacteria soil-borne diseases, wherein the preservation number of the strain is CCTCC NO: M2020821. The strain is reported for the first time to be used for preventing and controlling oomycete soil-borne diseases such as damping-off in melon seedling stage, tobacco black shank, pepper phytophthora blight and the like, and has no cross drug resistance with the existing bactericide and insecticide. Compared with the existing chemical synthetic pesticide, the pesticide has the advantages of high efficiency, low toxicity, no environmental pollution, difficult generation of drug resistance and the like due to the natural environment, and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of crop control, and particularly relates to a P.parr Long Nishi pseudomonas and application thereof in control of oomycete pathogenic bacteria soil-borne diseases.
Background
The oomycete pathogenic bacteria are one of important plant pathogenic bacteria, and the host range of the oomycete pathogenic bacteria is wide, such as part of grain crops and economic crops, vegetables, fruit trees, cotton, hemp and the like. The diseases caused by the bacillus subtilis mainly comprise pathogenic bacteria such as Pythium spp, phytophthora spp, peronospora spp and the like, and the pathogenic bacteria can cause diseases of crops such as melons, fruits, vegetables and the like, such as pepper blight, vegetable seedling damping-off, tobacco black shank and the like. Oomycete disease causes devastating damage to many crops and flower plants, causing billions of dollars of losses each year around the world. According to statistics, the current oomycete bactericide accounts for 21 percent of the market share of the bactericide, and with the continuous improvement of the commercial production level of agricultural products, the chemical prevention and control demand of oomycete diseases can be increased. With the continuous improvement of the requirements on safety, high efficiency and low toxicity of the bactericide, the bactericide with better using effect and lower toxicity is a selective bactericide, and the problem is that the bactericide is easy to generate drug resistance after being continuously used, which is one of the important difficulties in preventing and treating oomycete diseases in China. In addition, the use of a large amount of chemical bactericides can cause pesticide residue and environmental pollution, and a brand new action mechanism and a safer and more effective prevention and control agent are urgently needed in production. In recent years, the microbial agent is widely applied to prevention and control of crop diseases due to the fact that the microbial agent is safer to human bodies and more optimized to the environment.
Pseudomonas (Pseudomonas) is one of the ordinary habitat dominant bacteria, widely activates around plants (including Plant rhizosphere and leaf surfaces), and many strains in the Pseudomonas are Plant Growth-Promoting Rhizobacteria (PGPR), and have the functions of resisting Plant diseases and Promoting Plant Growth. Currently, as for the use of biocontrol pseudomonads in the control of oomycete diseases, pseudomonas fluorescens (Pesudomonas fluorescens) is mainly reported, and Pseudomonas chlororaphis (Pseudomonas chlororaphis) and Pseudomonas putida (Pseudomonas putida) are also reported.
It is believed that antagonistic bacteria isolated from the root system or rhizosphere of a particular crop provide better biocontrol effects because these strains have a more consistent environment with the crop and pathogenic bacteria and thus are more likely to function. In addition, the colonization effect of the biocontrol bacteria on the root system is important for the biocontrol effect, and the biocontrol bacteria effectively inhibit the occurrence of diseases by competing with pathogenic bacteria for nutrition and infecting space sites.
The P.paler Long Nishi pseudomonad HG258 is a biocontrol bacterium separated from the rhizosphere of cucumber seedling culture matrix soil. Currently, reports on the plant disease control of Pseudomonas paler Long Nishi are few, pseudomonas palleroniana SAW15 reports that the Pseudomonas palenoniana SAW15 has bacteriostatic activity on Rhizoctonia solani (inhibition rate of 69.3%), fusarium solani (inhibition rate of 81.8%) and Pythium arrhenomans (inhibition rate of 66.2%), and SAW15 has good control effect on kidney beans inoculated with the three pathogenic bacteria under potting conditions.
The invention discloses a Pahler Long Nishi pseudomonad HG258 which is a pseudomonad separated from rhizosphere soil of cucumber and has good control effect on crop oomycete soil-borne diseases, and discloses application of the Pahler Long Nishi pseudomonad HG258 in controlling seedling damping-off caused by Pythium aphanidermatum, tobacco black shank caused by Phytophthora nicotianae and Phytophthora capsici caused by Phytophthora capsici on melons. The application carries out the prevention and control effects of HG258 on the cucumber damping-off, the tobacco black shank and the pepper phytophthora blight and the evaluation of the colonization ability of the root system of the cucumber, and aims to lay a foundation for the application of the HG258 in the later period.
Disclosure of Invention
The invention aims to provide an isolated pseudomonad, which is a P.parr Long Nishi pseudomonad HG258 with the preservation number of CCTCC NO: M2020821. The pseudomonas can be used for preventing and treating plant diseases such as damping-off of melons, tobacco black shank, pepper phytophthora blight and the like.
Another object of the present invention is to provide a method for preparing a liquid fermentation broth of Pseudomonas aeruginosa HG258 in Parler Long Nishi.
The last purpose of the invention is to provide the application of the Parlerian Long Nishi pseudomonad HG258 in preparing the plant pathogenic bacteria inhibitor, in particular to preparing the drug for preventing and controlling the soil-borne diseases of oomycetes of crops.
In order to achieve the purpose, the invention adopts the following technical scheme to realize
An isolated pseudomonad, isolated from rhizosphere soil at cucumber seedling stage, identified as Pseudomonas paler Long Nishi (Pseudomonas pallroniana), which has been sent to the chinese type culture collection for storage at 12 months 02 of 2020, under taxonomic nomenclature: pseudomonas pallroniana HG258; the preservation number is CCTCC NO of M2020821; a place: wuhan university in Wuhan, china.
The P.parler Long Nishi pseudomonad HG258 grew well in LB medium colonies, with the colonies being yellow-green (FIG. 1). The preparation method of the pseudomonas paler Long Nishi comprises the following steps:
inoculating the secondary seed liquid of the pseudomonas HG258 of the Parler Long Nishi into an HG258 fermentation culture medium according to the inoculation amount of 0.1-0.2%, controlling the tank pressure to be 0.02-0.05 Mpa, the stirring speed to be 120-150rpm, the aeration ratio to be 1, and culturing for 32-38h at the temperature of 28-35 ℃;
the HG258 fermentation medium is as follows: 0.5-1.5% of cane sugar, 0.5-1.5% of glycerol, 2-4% of yeast extract, 0.5-1.5% of soybean meal, 0.01-0.04% of magnesium sulfate, 0.001-0.005% of potassium dihydrogen phosphate and pH6.5-7.5 of culture medium;
the application of Parlerian Long Nishi pseudomonad HG258 in preparing plant pathogenic bacteria inhibitors, in particular to preparing medicaments for preventing and treating soil-borne diseases of oomycetes of crops;
in the above applications, preferably, the pathogenic bacteria are Pythium aphanidermatum (Pythium aphanidermatum), phytophthora nicotianae (Phytophthora nicotiana), phytophthora capsici (Phytophthora capsici), sclerotinia sclerotiorum (sclerotiorum), rhizoctonia solani (Rhizoctonia solani), colletotrichum gloeosporioides (Colletotrichum gloeosporioides) and/or Botrytis cinerea (Botrytis cinerea).
Compared with the prior art, the invention has the following advantages:
(1) The invention provides a Pahler Long Nishi pseudomonas, the strain is reported for the first time to be used for preventing and controlling damping-off of melon seedling stage, tobacco black shank and pepper phytophthora blight, compared with the prior art, the Pahler Long Nishi pseudomonas provided by the invention has wider antibacterial spectrum and has no cross drug resistance with the existing bactericide and insecticide.
(2) Compared with the existing chemical synthetic pesticide, the pesticide has the advantages of high efficiency, low toxicity, no environmental pollution, difficult generation of drug resistance and the like because of being from natural environment.
(3) The screened pseudomonas palettii Long Nishi has cucumber rhizosphere colonization characteristics, and is particularly suitable for preventing and treating cucumber damping-off.
Drawings
FIG. 1HG258 strain colony morphology.
FIG. 2 shows the bacteriostatic activity of HG258
FIG. 3 is a schematic diagram of HG258 for preventing and controlling cucumber damping-off
Detailed Description
In order to better explain the invention, the following further illustrate the main content of the invention in connection with specific examples, but the content of the invention is not limited to the following examples. The technical solutions of the present invention are, unless otherwise specified, conventional in the art, and the reagents or materials, unless otherwise specified, are commercially available.
Example 1:
obtaining and identifying Pseudomonas paler Long Nishi
An isolated bacterium, which is separated from rhizosphere soil of cucumber, belongs to Pseudomonas paler Long Nishi (Pseudomonas pallroniana) after being identified by 16s rDNA combination physiology and biochemistry (figure 1, table 1 and table 2), and is delivered to China center for type culture Collection for preservation in 12 months and 02 months in 2020 year, and is classified and named as follows: pseudomonas pallroniana HG258; the preservation number is CCTCC NO of M2020821; a place: china, wuhan university.
In the present invention, parler Long Nishi is Pseudomonas HG258 or simply HG258.
Morphological characteristics: the colony is round, gray white at the initial stage, and turns yellow green after 5 days.
Physiological and biochemical characteristics of the strain:
TABLE 1 physiological and biochemical characteristics of the strain HG 258-enzyme activity, carbon source assimilation
+: positive reaction; -: negative reaction; w-weakly positive reaction
TABLE 2 physiological and biochemical characteristics of Strain HG 258-carbon source assimilation
+: positive reaction; -: negative reaction; w-weak positive reaction
Example 2:
fermentation of pseudomonas HG258 at parler Long Nishi (the percentages in the following medium formula are mass fractions):
1) First-stage seed culture: placing 100ml of first-stage seed culture medium in 500ml triangular flask, performing moist heat sterilization at 121 deg.C for 15-30min, inoculating HG258 freeze-dried tube strain in the first-stage seed culture medium, and performing shake culture at 30 deg.C and 180rpm for 8 hr; the raw materials and the dosage of the culture medium used for the first-stage seed culture are as follows: 2% of glucose, 1% of beef extract, 1% of peptone, 0.3% of sodium chloride and 7% of culture medium pH;
2) Secondary seed culture: 250L of secondary seed culture medium is filled in a 500L fermentation tank, moist heat sterilization is carried out for 15-30min at 121 ℃, and 200ml of primary seeds are inoculated in the secondary seed culture medium; culturing at 30 deg.C for 10 hr; controlling the tank pressure to be 0.05Mpa, the stirring speed to be 200rpm, the aeration ratio to be 1.5, and the raw materials and the dosage of the culture medium used for the second-stage seed culture as follows: 0.5 percent of glucose, 0.2 percent of yeast extract, 1 percent of peptone, 0.002 percent of monopotassium phosphate, 0.01 percent of magnesium sulfate and pH6.5-7.5 percent of culture medium;
3) Fermentation: 6 tons of HG258 fermentation medium is filled in a 10 ton fermentation tank, moist heat sterilization is carried out for 30min at 121 ℃, 350L of secondary seed liquid is transferred into the fermentation medium, the tank pressure is controlled to be 0.05Mpa, the stirring speed is 150rpm, the aeration ratio is 1; the raw materials and the dosage of the culture medium used for fermentation are as follows: 1.5% of sucrose, 0.5% of glycerol, 2% -4% of yeast extract, 1.5% of soybean meal, 0.01% of magnesium sulfate, 0.002% of potassium dihydrogen phosphate and pH7 of a culture medium;
the effective bacteria content after canning is 300 hundred million cfu/mL.
Example 3: bacteriostatic activity of Parler Long Nishi pseudomonad HG258 and cucumber damping-off prevention and control effect
The tested cucumber damping-off pathogenic bacteria are separated from cucumber damping-off plants, tobacco black shank pathogenic bacteria are separated from tobacco black shank plants, pepper phytophthora blight pathogenic bacteria are separated from pepper phytophthora blight plants and are preserved on a V8 inclined plane at 10 ℃, and other pathogenic fungi are preserved on a PDA inclined plane at 4 ℃. The slant strain is transferred to PDA plate for activation and cultured at 30 deg.c for 6 days. The sterilized filter paper sheets were placed on both ends of the PDA plate, and then 7. Mu.L of HG258 fermentation broth was dropped. And after 24h, inoculating the pathogenic bacteria bacterial dishes by using a 4mm puncher, taking the inoculated sterile water as a blank control, taking the pseudomonas fluorescens separated from the soil as a control, and setting the distance between the pathogenic bacteria and the filter paper to be 25mm. After the control full plate is provided, the diameters of the treated and control bacterial colonies are counted, and the bacteriostasis rate is calculated.
Bacteriostasis rate = [ (control colony diameter-treated colony diameter)/(control colony diameter-cake diameter) ] × 100%
In vitro bacteriostasis tests show that HG258 has good bacteriostatic activity on three kinds of oomycetes (figure 2), and HG258 has better bacteriostatic effect on pythium aphanidermatum compared with pseudomonas fluorescens. (Table 3).
TABLE 3 bacteriostatic activity of Pseudomonas fluorescens HG258 on pathogenic bacteria
The cucumber seeds are soaked in 1% sodium hypochlorite for 2min for disinfection, then washed with sterile water for 3 times, placed in a moistened gauze for germination acceleration at 30 ℃, and then soaked in HG258 fermentation broth stock solution (prepared in example 2) for 30min, wherein the control medicament is pyraclostrobin with 1000 mu g/mL of active ingredient, and the blank control is sterile water. Culturing pythium aphanidermatum on a PDA (personal digital assistant) plate in a dark environment at 30 ℃ for 3d, cutting a triangular hypha block with the side length of 15mm by using a sterile scalpel as an inoculated pathogen, and placing the hypha face upwards on the surface of a seedling substrate (turf: vermiculite: perlite (volume ratio) = 1). The sample loading amount of the seedling substrate is 2cm away from the surface of the plug tray. The treated seeds were placed on a plug of hyphae, followed by attachment of a seedling substrate, 3 replicates per treatment, 72 seeds per replicate. And (5) carrying out illumination culture at 28 ℃ for 10 days, counting the number of seedlings and calculating the morbidity and the control effect.
Morbidity rate = (number of sown seeds-number of normal seedlings)/number of sown seeds × 100%
Control effect = (control incidence-treatment incidence)/control incidence × 100%.
The test result shows that (figure 3), the HG258 fermentation liquid stock solution has the cucumber damping-off prevention effect of 98.51 percent and has good prevention and control effects.
TABLE 4 prevention and control effect of HG258 fermentation broth on cucumber damping-off
Example 4: colonization effect of Parler Long Nishi pseudomonad HG258 on cucumber root
Stable rifampicin (100. Mu.L. ML) was obtained by stepwise increasing the induction concentration -1 ) Pseudomonas fluorescens (the strain in example 3) and HG258 strain, inoculating a single colony to NA liquid culture medium, shaking and culturing for 16h by using a shaking table at 200r/min, and then adjusting the cell concentration to 10 by using sterile water 9 cfu/mL, irrigating root 5 mL/plant after the cotyledon of cucumber is spread, taking out plant after 10 days, cleaning root with sterile water, weighing 1g, grinding, diluting, and smearing with rifampicin (100 μ g. ML) -1 ) The TSA medium plate is placed in a constant temperature box at 28 ℃ for culturing for 48 hours, and when a colony is formed, the number of the colony is detected and the colonization density is converted.
Research results show that the HG258 strain separated from the rhizosphere of the cucumber has better colonization ability in the cucumber than pseudomonas fluorescens.
TABLE 5 Pseudomonas fluorescens and HG258 ability to colonize cucumber root system
Claims (4)
1. An isolated Pseudomonas paler Long Nishi (Pseudomonas sp. (R))Pseudomonas palleroniana) HG258, which is 12 months in 2020Delivering the culture to China center for type culture collection for preservation in 02 days, wherein the preservation number is CCTCC NO: M2020821, and the preservation address is university in Wuhan, china.
2. The method of claim 1, wherein the steps of culturing the pseudomonas palustris Long Nishi HG258 comprise:
the Parler Long Nishi pseudomonad HG258 secondary seed liquid as claimed in claim 1 is inoculated into HG258 fermentation medium according to the inoculum size of 0.1% to 0.2%, the pressure of the tank is controlled to be 0.02MPa to 0.05MPa, the stirring speed is controlled to be 120 rpm to 150rpm, the aeration ratio is controlled to be 0.5 to 1.0, and the pseudomonad HG258 secondary seed liquid is cultured for 32 h to 38h at the temperature of 28 ℃ to 35 ℃;
the HG258 fermentation medium is as follows: 0.5 to 1.5 percent of sucrose, 0.5 to 1.5 percent of glycerol, 2 to 4 percent of yeast extract, 0.5 to 1.5 percent of soybean meal, 0.01 to 0.04 percent of magnesium sulfate, 0.001 to 0.005 percent of potassium dihydrogen phosphate and the pH value of a culture medium of 6.5 to 7.5.
3. Use of the strain of Perilla Long Nishi Pseudomonas HG258 of claim 1 for preparing a plant pathogen inhibitor, wherein the pathogen is Pythium aphanidermatum (Pythium aphanidermatum), phytophthora nicotianae (Phytophthora nicotiana), phytophthora capsici (Phytophthora capsicii), sclerotium sclerotiorum (Sclerotinia sclerotiorum), rhizoctonia solani (Rhizoctonia solani), colletotrichum gloeosporioides (Colpolytrichoides), botrytis cinerea (Botrytis cinerea).
4. The use of the strain of Parlerian Long Nishi Pseudomonas HG258 of claim 1 in the preparation of a medicament for controlling a soil-borne disease of a crop of Oomycetes, wherein the pathogenic bacteria of the soil-borne disease of the crop of Oomycetes are Pythium aphanidermatum (Pythium aphanidermatum), phytophthora nicotianae (Phytophthora nicotiana), phytophthora capsici (Phytophthora capsici), sclerotinia sclerotiorum (Sclerotinia sclerotiorum), rhizoctonia solani (Rhizoctonia solani), colletotrichum gloeosporioides (Colletotrichytrium cinerea), and Vitis glaucophyllum (Botrytis cinerea).
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| CN1291078A (en) * | 1997-11-20 | 2001-04-11 | 美国政府农业部 | Biocontrol agent for control of root diseases |
| CN104254611A (en) * | 2012-02-28 | 2014-12-31 | 马罗内生物创新公司 | Control of phytopathogenic microorganisms with pseudomonas sp. and substances and compositions derived therefrom |
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| CN1291078A (en) * | 1997-11-20 | 2001-04-11 | 美国政府农业部 | Biocontrol agent for control of root diseases |
| CN104254611A (en) * | 2012-02-28 | 2014-12-31 | 马罗内生物创新公司 | Control of phytopathogenic microorganisms with pseudomonas sp. and substances and compositions derived therefrom |
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