CN112980744B - Pseudomonas and application thereof in prevention and treatment of potato late blight - Google Patents
Pseudomonas and application thereof in prevention and treatment of potato late blight Download PDFInfo
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- 241000589774 Pseudomonas sp. Species 0.000 claims description 6
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- 244000061456 Solanum tuberosum Species 0.000 abstract description 21
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- 230000000694 effects Effects 0.000 abstract description 9
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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
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- 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
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Abstract
The invention discloses pseudomonas and application thereof in preventing and treating potato late blight. The strain is preserved in China Center for Type Culture Collection (CCTCC) in 12 months and 7 days in 2020, and the preservation address is as follows: china Wuhan university CCTCC NO: m2020868. The fermentation liquor of the strain has biological control effect on late blight bacteria, late blight infection in the storage process of potatoes after harvesting can be prevented, and tuber control effect of four different potato varieties can reach more than 90%. The strain can be used for preparing a biological agent for preventing and treating late blight bacteria in the storage process of potato tubers. Meanwhile, the strain has activity of generating IAA, the yield of IAA reaches 50mg/L after fermentation for 48 hours, and the strain has potential activity of promoting plant growth.
Description
Technical Field
The invention belongs to the field of microbial control, and particularly relates to pseudomonas and application thereof in controlling potato late blight.
Background
The potato is the fourth crop of the world after rice, wheat and corn and is prepared from late blight bacterium: (Phytophthora infestans) The potato late blight caused by the disease occurs in potato planting areas all over the world, and is an oomycete disease causing the most serious potato damage. The late blight is a multicycle disease, can infect plants for many times in the potato planting period, can invade potato tubers, and becomes a primary infection source in the next year. The potato tubers lose up to 50% of their decay during storage due to late blight.
The main methods for preventing and treating late blight bacteria at present are disease-resistant varieties and bactericide. Because the late blight bacteria have high evolution speed, the resistance of the disease-resistant variety to the late blight is reduced or lost after the disease-resistant variety is cultivated in the field for 3 to 5 years. At present, the prevention and treatment of the main late blight mainly depends on chemical bactericides. After long-term use of the chemical bactericide, pathogenic bacteria easily generate drug resistance and cause environmental pollution. Due to the problems of food safety and the like, no suitable chemical bactericide for preventing and treating late blight from infecting potato tubers exists in the tuber preservation process at present. The use of microbial agents has the advantage that pathogenic bacteria are not prone to develop resistance to drugs and are environmentally friendly. The metabolite auxin Indole Acetic Acid (IAA) of the microorganism has the functions of promoting the germination of seeds, the development of lateral roots and cell division of plants and the cell elongation.
Disclosure of Invention
The invention aims to provide pseudomonas and application thereof in preventing and treating potato late blight, and solves the defects that the existing prevention and treatment of the potato late blight mainly depends on chemical bactericides and the problem that no bactericides are available in the storage process of potato tubers.
In order to achieve the purpose, the invention adopts the following technical scheme:
a strain of pseudomonas which is pseudomonas (pseudomonas:)Pseudomonas sp.) X49, which has been deposited with the China center for type culture Collection on 12/7/2020 with the deposition number: CCTCC NO: m2020868. The address is Wuhan university.
Biological preparation containing the pseudomonas.
The pseudomonas is applied to preventing and treating potato late blight.
The invention has the advantages that:
the invention provides a strain of pseudomonas (Pseudomonas sp.) X49, the strain has IAA production ability and can promote the growth of potato seedlings. The inhibition rate of the plate confronting against late blight bacteria is 64%, the activity of the plate confronting against late blight bacteria in the early storage period of potato tubers reaches 90%, and the bacteria have the activity of inducing potato tubers to resist the late blight bacteria. Has good potential application value for storing potato tubers.
Drawings
FIG. 1 shows a schematic view of aPseudomonas sp.X49In LB solid MediumColony morphology and gram stain.
FIG. 2Pseudomonas sp.X49Protecting potato blocks.
FIG. 3Pseudomonas sp.X49And (4) analyzing the prevention effect of late blight bacteria.
Detailed Description
Example 1
LB liquid medium (10 g peptone, 10g sodium chloride, 5 g yeast extract, 1L distilled water, pH7.0), 121 deg.C, high pressure 20 min.
LB solid medium (10 g peptone, 10g sodium chloride, 5 g yeast extract, 12g agar powder, 1L distilled water, pH 7.0), 121 deg.C, high pressure 20 min.
Rye culture medium: 60g of rye, soaking overnight at room temperature, grinding, carrying out water bath at 65 ℃ for 2h, filtering with four layers of gauze, and metering the volume to 1L by using distilled water. 121 deg.C, high pressure for 20 min.
Strain isolation and identification: pseudomonas bacteria (Pseudomonas sp.) X49 was isolated from the rhizosphere soil of vetch (Vicia villosa Roth var). The separation method comprises the following steps: placing 10g rhizosphere soil of smooth vetch in 90 mL sterile water, oscillating at 120r/min for 30min, standing for 20min to obtain soil bacterial suspension, diluting step by step, selecting 105Multiple sum of 106Adding 100 mu L of soil suspension diluent to the surface of an LB culture medium respectively, uniformly coating, and then placing in a constant-temperature incubator at 27 ℃ for culture; when bacterial colonies grow out, selecting single bacterial colonies with different forms for pure culture, and storing at 4 ℃ for later use. Screening the biocontrol strain by a plate confrontation method.
The plate method is used for measuring the activity of late blight bacteria: taking late blight bacteria, culturing and recovering the late blight bacteria for 15 days by adopting a rye culture medium, taking late blight bacteria cakes with the diameter of 3mm, inoculating the late blight bacteria cakes in the center of a flat plate which is about 2 cm away from the rye culture medium, inoculating pseudomonas X49 strain by using an inoculating loop, contrasting with an inactivated strain, then placing the flat plate in an 18 ℃ incubator for culturing for 7 days, recording the diameter of the late blight bacteria, and calculating the inhibition rate of the late blight bacteria to be 64%.
The colonies of X49 on LB solid medium were characterized as pale yellow, opaque colonies, smooth-edged, rod-shaped, gram-negative. (FIG. 1).
And (3) molecular identification: inoculating Pseudomonas sp X49 in LB solid culture medium by flat plate drawing method, culturing at 27 deg.C for 48 hr, selecting single colony, inoculating in LB liquid culture medium, culturing at 27 deg.C for 170 r.min－1Culturing for 48h, extracting strain DNA with kit TransZol UP, and performing PCR amplification of strain 16S rRNA gene using bacterial universal primers 16SPF (5 ' -AGAGTTTGATCCTGGCTCAGAACGAACGCT) and 16SPR (5'-TACGGCTACCTTGTTACGACTTCACCCC-3'). And recovering the purified target fragment, sending the target fragment to a gene company for sequencing, carrying out BLAST similarity comparison on a sequencing result in a GenBank database, downloading related strain sequences with higher homology, and constructing a phylogenetic tree by using MEGA-X software and adopting a Maximum Likelihood method (Maximum-Likelihood). Determination of the X49 Strain as Pseudomonas (M.sub.) (Pseudomonas.sp）。
Pseudomonas.spThe nucleic acid sequence of the 16s rRNA of X49 is as follows:
TGCAGTCGAGCGGTAGAGAGAAGCTTGCTTCTCTTGAGAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGATAACGTTCGGAAACGGACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTTGCCTAATACGTAACTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTTGTTAAGTTGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCAAAACTGACTGACTAGAGTATGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGAAGCCTTGAGCTTTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATCCAATGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACATTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCACCAGAAGTAGCTAGTCTAACCTTCGGAGGACGGTAC。
x49 has IAA-producing activity: inoculating X49 fermentation liquid into LB liquid medium containing tryptophan (200 mg/L) at 1% inoculation amount, at 30 deg.C for 170 r.min－1And (5) culturing. Supernatants were taken at 24h and 48h and IAA production was determined using a standard curve. The IAA can be produced by 33.12mg/L in 24 hours, and the IAA can be produced by 50.08mg/L in 48 hours.
Preparation of late blight bacterium sporangium suspension: inoculating late blight bacteria to rye culture medium, culturing at 18 deg.C for 15 days, adding sterile water into culture dish, filtering mycelium with four layers of gauze, centrifuging the filtrate at 3000rrpm, collecting sporangium, and adjusting sporangium concentration to 1 × 10 with sterile water3One per ml.
Preparation of LB broth X49: an X49 single colony on LB solid medium was picked, inoculated in 3mLLB liquid medium, cultured at 27 ℃, 170rpm to OD600=1, and re-inoculated at 1: 1000 volume ratio, inoculating LB fermentation liquor of X49 into LB liquid culture medium with new configuration, culturing at 27 deg.C and 170rpm by shaking to OD600=1。
Potato tuber protection test 2 holes 1.0cm in height and 5mm in diameter were drilled on the surface of different varieties of potato tubers with a 5mm diameter punch, approximately 3cm apart. Adding 200uL LB fermentation liquid of X49 into the hole, standing for 30min, and adding late blight bacteria spore suspension (1 × 10)3Pieces/ml) 200uL was added to the wells. A control (CK X49) to which only the fermentation broth of Pseudomonas sp.X 49 was added and a control (CK Pi) to which only the spore suspension of Phytophthora infestans was inoculated were set at the same time. The potato pieces were placed in an incubator at 18 ℃. And cutting the potato blocks equally along the punched positions for 10-12 days to determine the disease area of the potato blocks. The prevention effect of the X49 fermentation liquor on the late blight bacteria is calculated and measured on four varieties respectively, and the results show that the simulation reaches more than 90 percent (figure 3).
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> Fujian agriculture and forestry university
<120> pseudomonas and application thereof in prevention and treatment of potato late blight
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tgcaagcgtt aatcggaatt actgggcgta aagcgcgcgt aggtggtttg ttaagttgga 540
tgtgaaatcc ccgggctcaa cctgggaact gcattcaaaa ctgactgact agagtatggt 600
agagggtggt ggaatttcct gtgtagcggt gaaatgcgta gatataggaa ggaacaccag 660
tggcgaaggc gaccacctgg actgatactg acactgaggt gcgaaagcgt ggggagcaaa 720
caggattaga taccctggta gtccacgccg taaacgatgt caactagccg ttggaagcct 780
tgagctttta gtggcgcagc taacgcatta agttgaccgc ctggggagta cggccgcaag 840
gttaaaactc aaatgaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc 900
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ggtgccttcg ggaacattga gacaggtgct gcatggctgt cgtcagctcg tgtcgtgaga 1020
tgttgggtta agtcccgtaa cgagcgcaac ccttgtcctt agttaccagc acgttatggt 1080
gggcactcta aggagactgc cggtgacaaa ccggaggaag gtggggatga cgtcaagtca 1140
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Claims (3)
1. A strain of pseudomonas, which is characterized in that: the pseudomonas is pseudomonas (Pseudomonas sp.) X49, which has been deposited with the China center for type culture Collection on 12/7/2020 with the deposition number: CCTCC NO: m2020868.
2. A biological agent comprising the Pseudomonas bacterium according to claim 1.
3. Use of the pseudomonads of claim 1 for controlling potato late blight.
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