CN114317356B - Pseudomonas stutzeri Q1-7 strain in forest land and application thereof in prevention and treatment of bacterial wilt - Google Patents
Pseudomonas stutzeri Q1-7 strain in forest land and application thereof in prevention and treatment of bacterial wilt Download PDFInfo
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Abstract
The invention provides a woodland pseudomonas (Pseudomonas forestsoilum) Q1-7 and application thereof in prevention and treatment of bacterial wilt. The woodland pseudomonas Q1-7 strain is deposited in the microorganism strain collection of Guangdong province on the 11 th month 12 of 2021, and the deposition number is GDMCC No:62061, the preservation address is: guangzhou city first middle road No. 100 college No. 59 building 5. The pseudomonas Q1-7 strain in the woodland can effectively treat and/or prevent the horsetail bacterial wilt, the peanut bacterial wilt or the tomato bacterial wilt caused by the bacterial wilt, so that the pseudomonas Q1-7 strain in the woodland is suitable for preparing a biocontrol preparation for resisting the bacterial wilt.
Description
Technical Field
The invention belongs to the technical field of biological control of plant diseases, and particularly relates to a pseudomonas Q1-7 strain for woodland and application thereof in controlling bacterial wilt.
Background
Bacterial wilt (Ralstonia solanacearum), abbreviated as bacterial wilt, is one of ten plant pathogenic bacteria in the world, and can cause host to cause plant bacterial wilt, such as horsetail bacterial wilt, peanut bacterial wilt, tomato bacterial wilt and the like, so that the agriculture and forestry industry severely reduces the yield and suffers from great economic loss. The bacterial wilt has the advantages of high propagation speed, various propagation paths, obvious differentiation of bacterial strains, high variation speed and complex pathogenesis, so the control difficulty is high. The existing control method mainly comprises the following steps: (1) The disease-resistant variety is planted, which is the most economical and effective method for preventing and treating bacterial wilt at present, however, with the extension of the planting time of the single disease-resistant variety, pathogenic bacteria gradually generate new physiological micro-seeds, the resistance of the original variety is overcome, and the disease-resistant variety is changed into a disease-sensitive variety; (2) The chemical agent is mainly ethylicin and agricultural streptomycin sulfate, but the agricultural streptomycin sulfate is forbidden, the single control effect of ethylicin is insufficient, the chemical agent has certain toxicity to people and livestock, and the problems of pathogen resistance, water resource pollution and the like are caused. Therefore, finding a suitable biological control method becomes a research and development hotspot and an effective way for controlling bacterial diseases.
The prior art discloses pseudomonas strains (2) -29 with antagonism to tomato bacterial wilt, but the mode of directly killing pathogenic bacteria by the strains easily causes mutation of the pathogenic bacteria, and super bacteria are generated, so that the control effect of the bacterial wilt is seriously affected, and therefore, the searching of microorganisms capable of controlling the bacterial wilt through group quenching is very necessary.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide a pseudomonas woodland (Pseudomonas forestsoilum) Q1-7 strain for quenching quorum sensing signals generated by the bacterial wilt so as to treat and/or prevent the bacterial wilt.
The invention aims at providing a Pseudomonas terrestris (Pseudomonas forestsoilum) Q1-7 strain.
The invention also aims to provide the application of the pseudomonas woodland Q1-7 strain in preparing medicines for treating and/or preventing bacterial wilt.
It is still another object of the present invention to provide a medicament for treating and/or preventing bacterial wilt.
The above object of the present invention is achieved by the following technical solutions:
the invention provides a Pseudomonas terrestris (Pseudomonas forestsoilum) Q1-7 strain which is preserved in the Guangdong province microorganism strain collection (GDMCC) on 11-12 days of 2021, wherein the preservation number is GDMCC No:62061 the classification is named Pseudomonas sp.Q1-7, deposit address: guangzhou city first middle road No. 100 college No. 59 building 5.
The invention collects the soil of the root part of the horsetail from the horsetail forest land of Zhanjiang ocean head, separates and screens to obtain the strain Q1-7, determines that the strain Q1-7 belongs to pseudomonas through morphological identification and molecular identification (16S rDNA gene sequence analysis and rpoD gene sequence analysis), estimates that the strain Q1-7 is likely to be a new species of pseudomonas, determines that the strain Q1-7 is a new species of pseudomonas through whole genome sequencing, and determines that the strain Q1-7 is planted as a new species of pseudomonas in woodland (Pseudomonas forestsoilum nov.sp.).
Experiments of the invention find that the forest land pseudomonas Q1-7 strain can inhibit pathogenicity of the bacterial wilt by quenching quorum sensing signal-3-hydroxy methyl palmitate (3-OH PAME) generated by the bacterial wilt, so that the bacterial wilt of ephedra, peanut bacterial wilt or tomato bacterial wilt caused by the bacterial wilt can be effectively treated and/or prevented, and therefore, the application of the forest land pseudomonas Q1-7 strain in preparing medicaments for treating and/or preventing bacterial wilt is in the protection scope of the invention.
Preferably, the bacterial wilt is a plant disease caused by the bacterial wilt.
Further preferably, the plant disease is one or more of horsetail bacterial wilt, peanut bacterial wilt and tomato bacterial wilt.
Preferably, the treatment and/or prevention of bacterial wilt is inhibition of the pathogenicity of the bacterial wilt.
According to the invention, the root injury and fungus filling method is adopted to inoculate the pseudomonas Q1-7 strain in the root soil of the horsetail, the pseudomonas Q1-7 strain in the root soil of the horsetail is adopted to inoculate the stem parts of tomatoes and peanuts, and the growth conditions of the horsetail, tomatoes and peanuts in the experimental group are found to be obviously superior to those of the positive control group, so that the bacterial strain Q1-7 can effectively inhibit the pathogenicity of bacterial wilt and effectively treat and/or prevent the bacterial wilt of the horsetail, the bacterial wilt of the tomatoes and the bacterial wilt of the peanuts caused by the bacterial wilt.
Further preferably, the inhibiting the pathogenicity of the bacterial wilt is quenching quorum sensing signals produced by the bacterial wilt.
Most preferably, the quorum sensing signal is 3-OH PAME.
Methyl 3-hydroxy palmitate (3-OH PAME) plays a critical role in the pathogenic control network of bacterial wilt. Therefore, the pseudomonas solanacearum Q1-7 strain can effectively inhibit the pathogenicity of the bacterial wilt by quenching the quorum sensing signal 3-OH PAME generated by the bacterial wilt, so as to effectively treat and/or prevent the bacterial wilt of the ephedra, the bacterial wilt of the peanut or the bacterial wilt of the tomato caused by the bacterial wilt.
The invention also applies the strain Q1-7 to the bacterial wilt strain GM1000, and discovers that the content of extracellular polysaccharide of the strain GM1000 is obviously reduced, which proves that the strain Q1-7 can effectively weaken the generation of bacterial wilt virulence factors (extracellular polysaccharide) by quenching 3-OH PAME, thereby inhibiting the pathogenicity of the bacterial wilt strain.
The invention also provides a medicine for treating and/or preventing bacterial wilt, which takes the pseudomonas solanacearum Q1-7 strain as an active ingredient. The medicine also comprises pharmaceutically acceptable carriers or excipients, and can be made into different dosage forms, such as injection, liniment or aqua.
Preferably, the concentration of the Pseudomonas terrestris Q1-7 strain in the medicament is OD 600 1.0～1.5。
Preferably, the drug is administered by inoculation.
Further preferably, the inoculation is root-injured bacteria inoculation or injection inoculation.
The invention has the following beneficial effects:
1. the isolated pseudomonas woodland (Pseudomonas forestsoilum) Q1-7 strain is a new species of pseudomonas, and can inhibit the pathogenicity of the pseudomonas through quenching quorum sensing signals 3-OH PAME generated by the pseudomonas, so as to effectively treat and/or prevent the bacterial wilt of the horsetail, the peanut bacterial wilt or the tomato bacterial wilt caused by the pseudomonas woodland, so that the pseudomonas woodland Q1-7 strain is suitable for being prepared into a biological control preparation for resisting the bacterial wilt.
2. The forest land pseudomonas Q1-7 strain does not influence the normal growth of the horsetail ephedra, the peanut and the tomato in a greenhouse potting test, and the prevention and treatment effect on the bacterial wilt are realized by inhibiting the pathogenicity of the bacterial wilt instead of killing the bacterial wilt, so that the forest land pseudomonas Q1-7 strain acts on the bacterial wilt and is not easy to generate drug resistance.
Drawings
FIG. 1 shows the results of the concentration of 3-OH PAME in MSM medium.
FIG. 2 is a statistical result of degradation rate of the bacteria liquid to be tested on 3-OH PAME.
FIG. 3 is a cluster map of strains Q1-7 based on the 16S rDNA gene sequence.
FIG. 4 is a cluster map of strains Q1-7 based on rpoD gene sequence.
FIG. 5 shows the production of extracellular polysaccharide in the medium.
FIG. 6A is the inoculation onset of Muhuang, FIG. 6B is the inoculation onset of tomato, FIG. 6C is the inoculation onset of peanut,
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
MSM medium: (NH) 4 ) 2 SO 4 2.0g，Na 2 HPO 4 ·12H 2 O 1.5g，NaH 2 PO 4 1.5g，MgSO 4 ·7H 2 O 0.2g，CaCl 2 ·2H 2 O 0.01g，FeSO 4 ·7H 2 O 0.001g，1000mL H 2 O，pH 6.5；
LB medium: naCl 10.0g,Tryptone 10.0g,Yeast extract 5.0g,1000mL H 2 O，pH 7.0。
EXAMPLE 1 isolation and screening of Pseudomonas stutzeri Q1-7 Strain
1. Isolation of strains
(1) Sample collection: collecting soil at the root part of the clematis stem in the clematis stem field of Zhanjiang Taiwan, bagging and preserving, and carrying the soil back to a laboratory for strain separation.
(2) Isolation of strains: firstly, fully and uniformly mixing a collected soil sample, weighing 5.0g, soaking and shaking the soil sample in 200mL of MSM culture medium containing 3-OH PAME with the final concentration of 1 mu M/mL, then carrying out shaking culture for 7d at 28 ℃ and 200rpm of shaking table, extracting the soil sample with 10mL of methylene dichloride for three times to leave a lower layer, eluting the soil sample with 1mL of chromatographic methanol after rotary evaporation, detecting the content of 3-OH PAME by using LC-MS, taking +Na as a characteristic ion, the molecular weight is 309, the peak time is 6.0-6.5 min, analyzing the degradation rate of the soil sample to 3-OH PAME, carrying out gradient dilution on the culture solution by using the MSM culture medium, and taking the dilution factor as 10 5 ～10 9 100 mu L of the dilution of (C) is coated on an LB plate, and after drying, the plate is sealed and is inversely cultured in a culture box at 28 ℃ for 24 hours. Single colonies growing on LB plates were picked, numbered, placed in 2mL centrifuge tubes containing 500. Mu.L of liquid LB, and shake cultured at 28℃and 180rpm for 12h for use.
2. Strain screening
100. Mu.L of the bacterial liquid to be tested was added to 10mL of MSM medium containing 3-OH PAME (final concentration 1. Mu. Mol/L) as the sole carbon source as the experimental group (Q1-7 group), and MSM medium containing 3-OH PAME without bacterial liquid to be tested was used as the control group (control CK group). And (3) wrapping the two groups of bacteria with tinfoil paper, placing the bacteria into a shaking table, culturing at 28 ℃ and 200rpm for 7d, measuring the concentration of 3-OH PAME in the two groups of MSM culture media by using LC-MS (figure 1), calculating according to figure 1 to obtain the degradation rate of the bacteria liquid to be detected on the 3-OH PAME (figure 2), picking the bacterial strain with the degradation rate of 3-OH PAME being more than 85%, and naming the bacterial strain as bacterial strain Q1-7, thus obtaining the biocontrol bacterial strain with excellent quenching effect of the 3-OH PAME.
EXAMPLE 2 identification and preservation of Pseudomonas terrestris Q1-7 Strain
1. Identification of Strain Q1-7
(1) Morphological identification:
strains Q1-7 are gram negative; the colony on LB culture medium is white, round, slightly convex in the middle, smooth in surface, round in the earlier stage edge and diffuse in the later stage edge; the LB liquid medium is diffusely turbid, and the gelatin is liquefied.
(2) Molecular identification:
the strain Q1-7 was identified by phylogenetic tree analysis (Phylogenetic analysis) to define the taxonomic status of the strain Q1-7. The method specifically comprises the following steps: firstly, a 16S rDNA gene sequence is analyzed, then a conserved housekeeping gene rpoD sequence is combined, MEGA 5.0 software is used, a phylogenetic tree is constructed by adopting a Neighbor-joining method, and the evolutionary relationship of the strain Q1-7 is comprehensively analyzed.
As shown in FIG. 3, the clustering diagram of the strain Q1-7 based on the 16S rDNA gene sequence shows that the 16S rDNA sequence of the strain Q1-7 has the homology of 97.34% with the strains of the Pseudomonas Pseudomonas aeruginosa 12939 and F23197, the evolution coefficient is most similar to Pseudomonas aeruginosa 12939, the homology with other strains of the Pseudomonas is lower than 97%, and the strain Q1-7 belongs to the Pseudomonas and forms a cluster on the evolution tree. As shown in FIG. 4, the clustering diagram of strains Q1-7 based on rpoD gene sequences revealed that strains Q1-7 were closest to Pseudomonas pharmacofabricae. Thus, the phylogenetic tree analysis result combining the 16S rDNA and rpoD genes can confirm that the strain Q1-7 belongs to Pseudomonas and is likely a new species of Pseudomonas.
The strain Q1-7 was subjected to whole genome sequencing, and by comparative genome analysis, the closest genomic edges of strains Q1-7 and Pseudomonas resinovorans NBRC 106553 were found, but ANI was only 88.565%, which was significantly lower than the threshold (95%) of the species, indicating that strain Q1-7 was indeed a novel species of Pseudomonas, and thus, strain Q1-7 was inoculated as a novel species of Pseudomonas woodland (Pseudomonas forestsoilum nov.sp.).
2. Preservation of Strain Q1-7
Pseudomonas woodland Q1-7 strain was deposited at 11.12.2021 with the microorganism strain collection, cantonese province, accession number GDMCC No:62061, the preservation address is: guangzhou city first middle road No. 100 college No. 59 building 5.
EXAMPLE 3 inhibition of Pseudomonas solanacearum Q1-7 Strain on Rhizoctonia solani GM1000
A fusion plasmid containing the epsA-LacZ promoter (carrying the tetracyclic resistance gene) was transformed into a GM1000 wild-type strain to give a GM1000 reporter strain. Activating GM1000 reporter strain on CPG solid plate containing tetracycline, activating strain Q1-7 on LB solid plate, selecting single colony of GM1000 reporter strain, placing into centrifuge tube containing 10mL CPG liquid culture medium containing tetracycline, selecting single colony of strain Q1-7, placing into centrifuge tube containing 10mL LB, shake culturing two groups of centrifuge tubes at 28deg.C at 200rpm for 12 hr.
OD is set to 600 Strains Q1-7, GM1000 reported strains at 0.4 were as follows: a1000 ratio was added to 24-well plates containing 1mL CPG (320. Mu.g/mL final x-gal) as an experimental group (GM 1000+Q1-7 group) and the individual GM1000 reporter strain as a control group (GM 1000 group), and both groups were shaken in a shaker at 28℃and 200rpm for 8d, respectively, and photographed for recording (FIG. 5).
As can be seen from FIG. 5, the exopolysaccharide content in the experimental group (GM 1000+Q1-7 group) was significantly reduced, while the exopolysaccharide content in the control group (GM 1000 group) was not significantly changed, and it was found that the strain Q1-7 was effective in attenuating the production of bacterial wilt virulence factor (exopolysaccharide) by quenching 3-OH PAME.
EXAMPLE 4 prevention and treatment action of Pseudomonas stutzeri Q1-7 Strain on bacterial wilt
Activating the bacterial wilt NS25 on CPG solid plate, activating the bacterial strain Q1-7 on LB solid plate, selecting bacterial wilt NS25 single colony, placing into a centrifuge tube filled with 10mL PG, selecting bacterial strain Q1-7 single colony, placing into a centrifuge tube filled with 10mL LB, and shake culturing the two groups of centrifuge tubes at 28 ℃ and 200rpm for 12h.
1. Prevention and treatment effect of pseudomonas solanacearum Q1-7 strain on horsetail bacterial wilt
OD is set to 600 Q1-7, NS25 of 1.2 is as follows: 100 are respectively added into 100mL LB and CPG culture medium, put into a shaking table at 28 ℃ and 200rpm to shake to OD 600 1.2. The experiments were divided into three groups: (1) Mixing 30mL of the Q1-7 bacterial liquid with equal amount of the NS25 bacterial liquid to obtain an experimental group (NS 25+Q1-7 group); (2) 30mL of NS25 bacterial liquid was added to the same amount of LB and mixed as a positive control group (NS 25 group); (3) LB was culturedThe base served as the negative control (CK group).
Inoculating casualty ephedra by root injury bacteria irrigation method, specifically comprising: 10mL of the three groups of mixed solutions are respectively poured into the root soil of the cut-off casuarina equisetifolia of Xu Genxu by using a 50mL centrifuge tube, 3 repetitions are carried out, the growth condition of casuarina equisetifolia is observed every day, and records are made.
As shown in FIG. 6A, the growth condition of the casuarina equisetifolia in the experimental group (NS 25+Q1-7 group) is significantly better than that of the positive control group (NS 25 group), which indicates that the strain Q1-7 can effectively inhibit the pathogenicity of bacterial wilt and effectively treat and/or prevent the casuarina equisetifolia bacterial wilt caused by the bacterial wilt.
2. Prevention and treatment effect of pseudomonas solanacearum Q1-7 strain on tomato bacterial wilt and peanut bacterial wilt
OD is set to 600 Q1-7, NS25 of 1.2 is as follows: 100 are respectively added into 100mL LB and CPG culture medium, put into a shaking table at 28 ℃ and 200rpm to shake to OD 600 1.2. The experiments were divided into three groups: (1) 1mL OD 600 1.2 of a Q1-7 bacterial liquid was mixed with an equal amount of an NS25 bacterial liquid to prepare an experimental group (NS25+Q1-7 group); (2) Adding NS25 bacterial liquid into 1mL LB bacterial liquid, and mixing to obtain a positive control group (NS 25 group); (3) LB medium was used as a negative control group (CK group).
200 mu L of the three groups of mixed solutions are respectively taken by a 1mL syringe and injected into stems of tomatoes and peanuts, 3 repetitions are carried out, and growth conditions of the tomatoes and the peanuts are observed every day and recorded.
The growth conditions of tomatoes are shown in fig. 6B, and the growth conditions of peanuts are shown in fig. 6C, so that the growth conditions of tomatoes and peanuts in an experimental group (NS 25+Q1-7 group) are obviously better than those of positive control groups (NS 25 group), and the bacterial strain Q1-7 can effectively inhibit the pathogenicity of bacterial wilt and effectively treat and/or prevent tomato bacterial wilt and peanut bacterial wilt caused by bacterial wilt.
In addition, the disease index evaluation criteria of bacterial wilt are: 0, no withering; 1,1% -25% of leaf withering; 2, 26% -50% of the leaves wither; 3,51% -75% of leaf withers, 4, 76% -99% of leaf withers; 5, death. The average disease index is the arithmetic average of the disease index counted for each group. Calculated, the above positiveThe average disease indexes of plants (casuarina equisetifolia, tomato and peanut) of the control group are respectively as follows: 2.33 of horsetail-weed herb (18 days of inoculation), 3.33 of peanut (11 days of inoculation), and 3 of tomato (9 days of inoculation); the average disease indexes of the plants in the experimental groups are respectively as follows: 0.67 parts of casuarina equisetifolia, 0 parts of peanut and 0 parts of tomato. Controlling effect according to formula
The calculation shows that the prevention and treatment effect of the strain Q1-7 on the bacterial wilt of the horsetail-ephedra is 71.2%, and the prevention and treatment effects of the strain Q1-7 on the bacterial wilt of the peanut and the bacterial wilt of the tomato are 100%, so that the strain Q1-7 can effectively treat and/or prevent the bacterial wilt of the horsetail-ephedra, the bacterial wilt of the peanut and the bacterial wilt of the tomato, and especially has the best prevention and treatment effects on the bacterial wilt of the peanut and the bacterial wilt of the tomato.
In conclusion, the pseudomonas solanacearum (Pseudomonas forestsoilum) Q1-7 strain isolated by the invention is a new species of pseudomonas, and can inhibit the pathogenicity of the pseudomonas solanacearum by quenching quorum sensing signals 3-OH PAME generated by the pseudomonas solanacearum, so that the ephedra bacterial wilt, peanut bacterial wilt or tomato bacterial wilt caused by the pseudomonas solanacearum can be effectively treated and/or prevented, and therefore, the pseudomonas solanacearum Q1-7 strain is suitable for being prepared into a biological control preparation for resisting the bacterial wilt.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (9)
1. Pseudomonas strain for woodlandPseudomonas forestsoilum) The strain Q1-7 is characterized in that the strain Q1-7 of the pseudomonas woodland is deposited with the microorganism strain collection of Guangdong province at the 11 th month 12 of 2021, and the deposition number is GDMCC No:62061.
2. the use of the pseudomonas solanacearum Q1-7 strain in woodland for the preparation of a medicament for treating and/or preventing bacterial wilt, according to claim 1, characterized in that the bacterial wilt is a plant disease caused by the bacterial wilt.
3. The use according to claim 2, wherein the plant disease is one or more of horsetail-rot, peanut-rot, tomato-bacterial-rot.
4. The use according to claim 2, wherein the treatment and/or prevention of bacterial wilt is inhibition of the pathogenicity of bacterial wilt.
5. The use according to claim 4, wherein the inhibition of the pathogenicity of the bacterial wilt is the quenching of quorum sensing signals produced by the bacterial wilt.
6. The use of claim 5, wherein the quorum sensing signal is 3-OH PAME.
7. A medicament for treating and/or preventing bacterial wilt, which is characterized in that the medicament takes the pseudomonas solanacearum Q1-7 strain in woodland as an active ingredient, and the bacterial wilt is a plant disease caused by the bacterial wilt.
8. The medicine according to claim 7, wherein the concentration of the Pseudomonas terrestris Q1-7 strain in the medicine is OD 600 1.0～1.5。
9. The medicament of claim 7, wherein the medicament is administered by inoculation.
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| CN102972220A (en) * | 2012-12-11 | 2013-03-20 | 湛江师范学院 | Simple method for testing disease resistance of pathogenic bacteria quorum-quenching gene prokaryotic expression product |
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| CN112522136A (en) * | 2020-11-19 | 2021-03-19 | 贵州省烟草科学研究院 | Pseudomonas and application thereof in controlling bacterial wilt of solanaceae crops |
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| CN102972220A (en) * | 2012-12-11 | 2013-03-20 | 湛江师范学院 | Simple method for testing disease resistance of pathogenic bacteria quorum-quenching gene prokaryotic expression product |
| CN106978373A (en) * | 2017-05-09 | 2017-07-25 | 福建农林大学 | One plant is used for pseudomonad and the application that bacteriosis is prevented and treated |
| CN112522136A (en) * | 2020-11-19 | 2021-03-19 | 贵州省烟草科学研究院 | Pseudomonas and application thereof in controlling bacterial wilt of solanaceae crops |
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