CN112358994B - Pseudomonas and application thereof - Google Patents
Pseudomonas and application thereof Download PDFInfo
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- CN112358994B CN112358994B CN202011285453.5A CN202011285453A CN112358994B CN 112358994 B CN112358994 B CN 112358994B CN 202011285453 A CN202011285453 A CN 202011285453A CN 112358994 B CN112358994 B CN 112358994B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/38—Pseudomonas
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/27—Pseudomonas
Abstract
The invention discloses pseudomonas and application thereof, belonging to the technical field of biology. The invention separates and screens pseudomonas strain 33-1 with high-efficiency biocontrol antagonistic fusarium wilt from rhizosphere microorganisms of greenhouse cucumber in Gansu through a plate confrontation experiment and a bacteriostatic activity experiment of fermentation liquor of new-born control strain. The strain can better inhibit the growth of pathogenic bacteria, has a better inhibiting effect on fusarium oxysporum cucumber specialized strains of cucumber fusarium wilt pathogenic bacteria, has an inhibition rate of 76.4 percent, can also well inhibit the growth of fusarium oxysporum hyphae of fermentation products, and has an inhibition rate of 80.65 percent. The 33-1 bacterium is pseudomonas and is classified and named as fredricksburg pseudomonas, and is preserved in China general microbiological culture Collection center (CGMCC) at 27.09.2020, with the preservation number of CGMCC No. 20833.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to pseudomonas and application thereof.
Background
Cucumber Fusarium wilt is infected by Fusarium oxysporum cucumber specialized (Fusarium oxysporum f.sp. cucumerinum) fungi, which can occur in open field and protected field cultivation of cucumbers, and is a devastating soil-borne disease affecting cucumber production in the world. The morbidity can reach 20% -30%, the cucumber yield is reduced, and can reach 80% -90% in serious cases, so that the cucumber yield is seriously reduced. At present, the disease becomes one of important factors for restricting the yield and quality of cucumbers in production, the cucumbers are main vegetable types in out-of-season cultivation in a sunlight greenhouse, and the cultivation area accounts for more than 50% of the production area of the sunlight greenhouse, so the disease also becomes an important factor for restricting the agricultural development.
At present, chemical pesticides are used for preventing and treating greenhouse cucumber fusarium wilt, the chemical pesticides can effectively control the greenhouse cucumber fusarium wilt, pesticide residues generated by long-term use of the chemical pesticides pollute soil and underground water and harm human and livestock health, pathogenic bacteria are easy to generate drug resistance along with the use of a large amount of systemic bactericides, and the prevention effect is gradually reduced. Therefore, it is highly desirable to screen out strains with high-efficiency biocontrol antagonistic fusarium wilt.
Disclosure of Invention
Aiming at the defects and shortcomings of the prior art, the invention provides pseudomonas and application thereof.
The invention aims to provide a Pseudomonas frederiksbergensis (Pseudomonas frederiksbergensis)33-1 separated from rhizosphere soil of cucumber, which is deposited in China general microbiological culture Collection center, and has the address: the preservation time is 2020, 09 months and 27 days.
The second purpose of the invention is to provide the application of a strain of Pseudomonas frederiksbergensis 33-1, the first purpose is that the strain is used for preventing and treating cucumber fusarium wilt, and the second purpose is that the growth of cucumber seedling stage is promoted.
Meanwhile, the invention also provides application of a microbial inoculum containing the pseudomonas, wherein the first application is the prevention and the treatment of cucumber fusarium wilt by using the microbial inoculum, and the second application is the promotion of cucumber seedling growth.
The pseudomonas is the most abundant bacterial group of plant roots and rhizosphere, has various biological activities, has a promoting effect on the growth of plants, has favorable conditions as biocontrol bacteria, and can well play a biocontrol role. The invention separates antagonistic bacteria from rhizosphere microorganisms of cucumber in Gansu greenhouse, and screens out pseudomonas strain 33-1 with high-efficiency biocontrol antagonistic fusarium wilt. The bacterium is finally determined to be Pseudomonas fredrickbergii (Pseudomonas frederiksbergensis) through morphological observation, physiological and biochemical identification and 16S rDNA molecular identification.
The strain 33-1 can better inhibit the growth of pathogenic bacteria, has better inhibiting effect on fusarium oxysporum cucumber specialized strain of cucumber fusarium wilt pathogenic bacteria, has the bacteriostasis rate of 76.4 percent, can also better inhibit the growth of fusarium oxysporum hyphae of fermentation products, and has the inhibition rate of 80.65 percent. The test result of the disease prevention effect of the indoor pot culture shows that the strain 33-1 can better inhibit the fusarium wilt of cucumber in a greenhouse and has a certain promotion effect on the growth of the cucumber in the seedling stage indoors.
Compared with the prior art, the invention has the following beneficial effects: the 33-1 screened by the invention belongs to the Pseudomonas fredrickbergii in Pseudomonas, has antagonistic bactericidal activity, has obvious effects on preventing diseases and promoting growth of cucumber in seedling stage, and has great potential in preventing and controlling soil-borne plant diseases. The bacterial liquid of the strain 33-1 or a microbial inoculum product taking the strain 33-1 as a core strain can be applied to the prevention and treatment of cucumber fusarium wilt and the growth promotion of cucumber seedling stage.
Preservation description:
the preservation number is as follows: CGMCC No.20833
Taxonomic nomenclature: pseudomonas fredrickbergii
Latin name: pseudomonas frederiksbergensis
The strain number is as follows: 33-1
The preservation organization: china general microbiological culture Collection center
The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)
Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district
The preservation date is as follows: 09/27 days 2020.
Drawings
FIG. 1 is a graph showing the inhibitory effect of the antagonistic strain in example 2 on Fusarium oxysporum.
FIG. 2 is a graph showing the inhibitory effect of the antagonistic bacteria fermentation broth on the growth of Fusarium oxysporum hyphae in example 2.
Detailed Description
The present invention will be described in detail with reference to specific embodiments. The following examples are presented to assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any manner.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Fusarium oxysporum cucumber specialization type (Fusarium oxysporum f.sp. cuumberin) used in the following examples was purchased from the chinese collection of microorganisms. The strain 33-1 is separated and screened in rhizosphere soil of cucumber in a solar greenhouse in the Liangzhou district of Wuwei city, Gansu province, and is preserved in the China general microbiological culture Collection center. The test agent was 50% carbendazim wettable powder.
Other materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
Isolation and identification of highly antagonistic Strain 33-1
Placing 5g of soil sample (collected from cucumber greenhouse soil in Wuwei City of Gansu province) into a triangular flask, adding 50ml of sterile water, sealing, shaking for 20min in a shaking table to prepare suspension, absorbing 5ml of suspension, adding the suspension into 100ml of beef extract peptone culture medium, culturing at 28 ℃, absorbing 5ml of bacterial solution after 72h, adding 100ml of beef extract peptone culture medium, continuing culturing, and performing single colony separation after repeating the culture for three times. The separation adopts a plate marking method and a gradient dilution method: the plate-scribing method is to use an inoculating loop to dip the bacterial liquid and perform scribing separation on a beef extract peptone solid culture medium; the gradient dilution method comprises culturing the bacterial liquid according to the ratio of 10-2、10-4、10-6、10-8The dilution was made into a suspension with sterile water, and 0.1ml of the suspension was pipetted and spread evenly on a beef extract peptone solid medium. And (3) performing static culture on the coated plate at 28 ℃, selecting a single colony after 5-7d, and performing strain purification by using a plate marking method. One of the isolated and purified strains was named 33-1.
The strain 33-1 is subjected to morphological microscopic examination and physiological and biochemical reaction detection. The classification and identification of 33-1 are carried out according to the handbook of identification of common bacteria systems. The results of the physiological and biochemical reaction are shown in Table 1.
TABLE 1 part of the physio-biochemical characteristics of Strain 33-1
| Test items | Characteristics of | Test items | Characteristics of |
| Gram of | - | Hydrolysis of gelatin | + |
| Methyl Red | - | Growth at 4 deg.C | + |
| Starch hydrolysis | - | Produce H2S | - |
| Catalase enzyme | + | Oxidase enzyme | + |
| Nitrate reduction | + | 5％Nacl | - |
| V.p. test | + | 7％Nacl | - |
Note: "+", "-" represent positive and negative reactions, respectively.
The observation result of the flat bacterial colony is visible, and the bacterial colony of the bacterial strain 33-1 is round and raised, and the edge is neat and moist; through microscopic examination, the strain 33-1 is gram-negative bacteria, rod-shaped, moving and does not produce spores; the result of physiological and biochemical reaction of the strain is combined, and the strain 33-1 is preliminarily confirmed to be pseudomonas.
Molecular biological identification of the strain 33-116S rDNA. The 16S rDNA of the strain 33-1 was determined by Shanghai Kangkang Biotechnology Ltd, the determination result is shown in SEQ ID NO.1, and after performing blast alignment of the sequence obtained by sequencing with NCBI database, 33-1 was identified as Pseudomonas fredrickbergensis (Pseudomonas frederiksbergensis).
Example 2
Bacteriostatic action test of Strain 33-1
(1) Inoculating 33-1 into a beef extract peptone slant culture medium, activating at a constant temperature of 28 ℃ for 48h, inoculating the activated strain into a beef extract peptone plane culture medium, and culturing at a constant temperature of 28 ℃ for 24 h. Fusarium oxysporum is inoculated in a PDA (potato dextrose agar) plane culture medium and activated for 96h at a constant temperature of 26 ℃. The method comprises the steps of uniformly coating fusarium oxysporum in a 9cm culture dish, placing an oxford cup with the inner diameter of 6mm in the center of the culture dish, adding 33-1 bacterial suspension into the oxford cup, culturing in a constant-temperature incubator at 26 ℃, setting blank control CK (sterile water treatment), repeating test bacteria for three times, measuring the diameter of an antibacterial colony when the colony of a control plate grows full, and calculating the antibacterial rate according to a formula 2-1, wherein the result is shown in figure 1 (the plate with the code of 1 represents 33-1 bacterial strain, and 2 represents CK).
Bacteriostasis rate is treated growth diameter/contrast growth diameter x 100% (2-1)
TABLE 2 inhibitory Effect of antagonistic strains on Fusarium oxysporum
As can be seen from Table 2, the strain 33-1 has a good inhibition effect on Fusarium oxysporum, and the highest bacteriostasis rate can reach 76.4%.
(2) The antibacterial activity of the fermentation product of the strain is measured by adopting a hypha growth rate method, namely the hypha growth inhibition effect of the fermentation liquid on pathogenic bacteria. Inoculating 33-1 into a beef extract peptone liquid culture medium, culturing at a constant temperature of 28 ℃ for 48h to prepare a fermentation liquid, inoculating fusarium oxysporum in a PDA planar culture medium, activating at a constant temperature of 26 ℃ for 96h, uniformly coating 200 mul of 33-1 fermentation liquid on a PDA plate, placing a pathogenic bacteria cake of 5mm in the center of the plate, culturing at 26 ℃ for 7d by taking a sterile culture solution as a control CK, repeating for three times, and measuring the colony diameter of pathogenic fungi by a cross method, wherein the plate coded as 1 represents 33-1 strain and 2 represents CK, and the result is shown in figure 2. The hyphal growth inhibition rate was calculated according to the formula 2-2, and the results are shown in Table 3.
TABLE 3 inhibitory Effect of antagonistic bacteria fermentation broth on the growth of Fusarium oxysporum hyphae
As can be seen from Table 3, 33-1 has an obvious inhibition effect on the growth of pathogenic bacteria hyphae, and the inhibition rate of 33-1 on the growth of pathogenic bacteria hyphae can reach 80.65%.
Example 3
Test of prevention and treatment effect and growth promotion effect of strain 33-1 on cucumber fusarium wilt
Inoculating the strain 33-1 into a liquid beef extract peptone culture medium, culturing at 28 deg.C for 2d for later use, inoculating pathogenic bacteria Fusarium oxysporum in a PDA liquid culture medium, culturing at 26 deg.C for 7d, and making into Fusarium oxysporum suspension for later use. Soaking uncoated cucumber seed in 45 deg.C water for 1 hr, sterilizing with 70% ethanol for 1min, and sterilizing with HgCl2Sterilizing for 1min, washing with sterile water for several times, germinating at 25 deg.C for 9 days, scratching the root of the bud, soaking the scratched root in the previously cultured suspension of Fusarium oxysporum for 5s, inoculating into 70-hole seedling tray, filling 6ml of the suspension of Fusarium oxysporum in each hole at a distance of 1cm from the stem, filling 6ml of the suspension of antagonistic bacteria 33-1 after 24h, and treating with bacterial liquid to obtain B (stock solution) and A (10)-1)、C(10-2)、D(10-3)、E(10-4) Five concentrations. Each treatment was repeated 30 times, and CK blank control (no pathogen), CK (pathogen + clear water), CK culture control (pathogen + medium), and CK drug control (pathogen + 50% carbendazim wettable powder diluted 1000 times) were provided. The temperature is 24-28 ℃, and the relative humidity is 50-70%. And (5) investigating the disease condition after 30d, counting the results according to the cucumber fusarium wilt grading standard shown in the table 4, calculating the control effect, and measuring the plant height, the root length and the total weight of the plants.
TABLE 4 cucumber blast classification criteria
The disease degree of the plant and the disease grading standard are calculated according to the formula l and 2.
Equation 1: disease index (%). sigma (disease level value x number of strains in disease level)/(highest disease level value x total number of strains) × 100%
Equation 2: relative prevention and treatment effect (%) (control disease index-treatment disease index)/control disease index is multiplied by 100 percent
The experimental data were analyzed for variance and significance using Excel 2007 and SPSS16.0 statistical analysis software, and the results are shown in table 5.
TABLE 5 control Effect test results of indoor potting of Strain 33-1
Note: the same lower case after the same row data indicates no significant difference at the 0.05 level.
The same capital letters after the same row of data indicate no significant difference at the 0.01 level.
As can be seen from Table 5, the disease indexes of the five treatments are reduced compared with CK under indoor conditions, wherein the disease indexes of C and D treatment are the lowest, namely 5.36 percent and 12.5 percent respectively, D has equivalent effect with CK medicament, the disease degree of C treated plants is obviously smaller than 14.28 percent of CK medicament, the effects of C and D are especially prominent in prevention effect, namely 86.95 percent and 69.56 percent respectively, and C is obviously higher than 65.23 percent of prevention effect of CK medicament. The experimental data show that the strain 33-1 has good prevention and treatment effects on cucumber fusarium wilt.
In the aspect of growth promoting effect, as can be seen from the above table, A, C, D strain is higher than CK, wherein the height of C is significantly higher than CK, and C is not significantly different from CK medicament, which indicates that the concentration of C can promote the growth of plants, and the concentration is not significantly different from that of the commercial pesticide in the growth of plants. A, B, C, D, E has no significant difference from CK and CK medicaments and has very significant difference from CK blank on the influence on the root length of the plant, which shows that although the effect of pathogenic bacteria on the growth of the root length of the plant is not improved by microbial inoculum with various concentrations, the inhibition effect on the growth of the root length is not increased. From the influence of each concentration of medicament on the total weight of plants, C is very different from CK, CK blank and CK medicament, D is not different from CK medicament, A, B, E is very different from CK and CK blank, which shows that five concentrations of medicament have certain weight gain effect on plants, and the C, D effect is particularly obvious. The strain 33-1 has obvious growth promoting effect on cucumber seedling stage.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Sequence listing
<110> institute of biological research of science institute of Gansu province
<120> Pseudomonas and use thereof
<130> 2020.10.13
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1414
<212> DNA
<213> Pseudomonas fredrickbergii ()
<400> 1
taccatgcag tcgagcggca gcacgggtac ttgtacctgg tggcgagcgg cggacgggtg 60
agtaatgcct aggaatctgc ctggtagtgg gggataacgc tcggaaacgg acgctaatac 120
cgcatacatc ctacgggaga aagcagggga ccttcgggcc ttgcgctatc agatgagcct 180
aggtcggatt agctagttgg tgaggtaatg gctcaccaag gcgacgatcc gtaactggtc 240
tgagaggatg atcagtcaca ctggaactga gacacggtcc agactcctac gggaggcagc 300
agtggggaat attggacaat gggcgaaagc ctgatccagc catgccgcgt gtgtgaagaa 360
ggtcttcgga ttgtaaagca ctttaagttg ggaggaaggg catttaccta atacgtaagt 420
gttttgacgt taccgacaga ataagcaccg gctaactctg tgccagcagc cgcggtaata 480
cagagggtgc aagcgttaat cggaattact gggcgtaaag cgcgcgtagg tggttcgtta 540
agttggatgt gaaatccccg ggctcaacct gggaactgca ttcaaaactg tcgagctaga 600
gtatggtaga gggtggtgga atttcctgtg tagcggtgaa atgcgtagat ataggaagga 660
acaccagtgg cgaaggcgac cacctggact gatactgaca ctgaggtgcg aaagcgtggg 720
gagcaaacag gattagatac cctggtagtc cacgccgtaa acgatgtcaa ctagccgttg 780
ggagccttga gctcttagtg gcgcagctaa cgcattaagt tgaccgcctg gggagtacgg 840
ccgcaaggtt aaaactcaaa tgaattgacg ggggcccgca caagcggtgg agcatgtggt 900
ttaattcgaa gcaacgcgaa gaaccttacc aggccttgac atccaatgaa ctttccagag 960
atggattggt gccttcggga acattgagac aggtgctgca tggctgtcgt cagctcgtgt 1020
cgtgagatgt tgggttaagt cccgtaacga gcgcaaccct tgtccttagt taccagcacg 1080
ttaaggtggg cactctaagg agactgccgg tgacaaaccg gaggaaggtg gggatgacgt 1140
caagtcatca tggcccttac ggcctgggct acacacgtgc tacaatggtc ggtacagagg 1200
gttgccaagc cgcgaggtgg agctaatccc agaaaaccga tcgtagtccg gatcgcagtg 1260
tgcaactcgt ctgcgtgaag tcggaatcgc tagtaatcgc gaatcagaat gtcgcggtga 1320
atacgttccc gggccttgta cacaccgccc gtcacaccat gggagtgggt tgcaccagaa 1380
gtagctagtc taacctttgg gaggacggta ccta 1414
Claims (6)
1. The Pseudomonas strain (Pseudomonas frederiksbergensis)33-1 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of No.1 Xilu-Chen in the area of the south of the morning of Beijing, the preservation date is 09 and 27 days in 2020 and the preservation number is CGMCC No. 20833.
2. Use of the pseudomonads (Pseudomonas frederiksbergensis)33-1 according to claim 1 for the control of cucumber fusarium wilt.
3. Use of Pseudomonas frederiksbergensis (Pseudomonas frederi) 33-1 according to claim 1 for promoting cucumber seedling growth.
4. A microbial inoculum comprising Pseudomonas frederiksbergensis 33-1 as defined in claim 1.
5. The use of the microbial agent of claim 4 for controlling cucumber fusarium wilt.
6. Use of the microbial agent of claim 4 for promoting cucumber seedling growth.
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