CN117025398B - Protozoan flagellate NJAU-W1 for promoting tomato growth and preventing and controlling bacterial wilt and application thereof - Google Patents

Protozoan flagellate NJAU-W1 for promoting tomato growth and preventing and controlling bacterial wilt and application thereof Download PDF

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CN117025398B
CN117025398B CN202311301829.0A CN202311301829A CN117025398B CN 117025398 B CN117025398 B CN 117025398B CN 202311301829 A CN202311301829 A CN 202311301829A CN 117025398 B CN117025398 B CN 117025398B
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njau
tomato
bacterial wilt
protozoan
flagellate
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CN117025398A (en
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熊武
吕启汇
王艺锦
沈其荣
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Sanya Research Institute Of Nanjing Agricultural University
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Sanya Research Institute Of Nanjing Agricultural University
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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
    • C12N1/10—Protozoa; Culture media therefor
    • C12N1/105—Protozoal isolates
    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P21/00—Plant growth regulators
    • 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
    • C12N1/10—Protozoa; Culture media therefor
    • 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
    • C12R2001/90—Protozoa ; Processes using protozoa

Abstract

The invention provides a protozoan flagellate NJAU-W1 for promoting crop growth and preventing and controlling tomato bacterial wilt and application thereof. Protozoan flagella NJAU-W1 is preserved in China Center for Type Culture Collection (CCTCC) NO: C2023264, and the flagella can effectively promote the growth of tomato plants and effectively reduce the incidence of tomato bacterial wilt. The flagella is separated from the experimental base tomato greenhouse soil (118 DEG 57'E,32 DEG 03' N) in the area Jiang Ning of Nanjing city in Jiangsu province, has good effect of promoting the growth of tomato plants, has the function of potential inhibition of tomato bacterial wilt, has simple culture conditions, is easy to store, is easy to produce and apply, and has good development prospect.

Description

Protozoan flagellate NJAU-W1 for promoting tomato growth and preventing and controlling bacterial wilt and application thereof
Technical Field
The invention belongs to the technical field of microbial fertilizers, and particularly relates to a protozoan flagellate NJAU-W1 with the functions of promoting tomato growth and biomass accumulation and preventing and controlling bacterial wilt.
Background
Tomato (Lycopersicon esculentum mill.) is an important vegetable, fruit, widely cultivated around the world, one of the most consumed vegetables worldwide. Bacterial wilt of tomato is caused by Laurencia solanaceaeRalstonia solanacearum) The diseases caused by the method are widely distributed in tomato producing areas worldwide, and have serious influence on the yield and quality of tomatoes. Pathogenic bacteria of tomato bacterial wilt mainly exist in soil, and can invade from the wound of the root or stem base of a plant, propagate in vascular bundles after invasion, spread to the duct tissue upwards, propagate in the plant and cause pathology, destroy the vascular bundles of the plant, so that the upper stem and leaf cannot obtain normal water supply to wilt, and yield reduction and death are caused.
How to improve the crop yield and effectively prevent and control crop diseases has become an important link in agricultural production. In China, chemical prevention and control measures are mainly relied on, pesticides or other chemical reagents are used for reducing the content of pathogenic bacteria in soil, but at the same time, ecological balance and sustainable productivity of the soil are also affected. With the deep research, biological control methods are also gradually possible, and the microbial agent plays a role in inhibiting pathogenic bacteria by regulating and controlling soil microbial communities. Meanwhile, the biological control method can regulate and control soil microbial communities by adding exogenous microbial agents, improve the quantity of beneficial bacteria, better exert the functions of the beneficial bacteria and better promote plant growth and inhibit soil-borne diseases. The mode can reduce the harm of chemical control measures to the sustainable development of soil to the greatest extent.
Disclosure of Invention
The invention aims to provide a protozoan flagellate NJAU-W1 which has the functions of promoting the growth of tomato plants and improving the biomass of tomatoes, and has a certain potential biological control effect on tomato bacterial wilt.
It is a further object of the present invention to provide the use of the protozoan (flagellate) NJAU-W1.
The aim of the invention can be achieved by the following technical scheme:
protozoan flagellate capable of promoting growth of tomato plants and inhibiting bacterial wiltDimastigella trypaniformis) Classified and named as flagellate NJAU-W1%Dimastigella trypaniformis NJAU-W1), the strain is preserved in China center for type culture Collection, the preservation date is 2023, 8 months and 28 days, and the preservation number is CCTCC NO: C2023264.
The strain NJAU-W1 is identified as the flagellate by combining the developmental tree comparison analysis result and morphological characteristics constructed by the 18S rDNA sequenceDimastigella trypaniformis）。
The protozoan flagella isDimastigella trypaniformis) Use of NJAU-W1 for promoting tomato growth.
The protozoan flagella isDimastigella trypaniformis) Application of NJAU-W1 in preventing and treating tomato bacterial wilt is provided.
The protozoan flagella isDimastigella trypaniformis) Application of NJAU-W1 in preparing products for promoting tomato growth and/or preventing and treating tomato bacterial wilt.
The protozoan flagella isDimastigella trypaniformis) Cultures of NJAU-W1.
The application of the culture in promoting the growth of tomatoes and/or preventing and treating tomato bacterial wilt.
Advantageous effects
The invention provides a protozoan flagella strain for promoting tomato growth and inhibiting bacterial wiltDimastigella trypaniformis) NJAU-W1. Using protozoan flagellateDimastigella trypaniformis) The NJAU-W1 culture solution irrigation can effectively promote the growth of tomato plants, the height and biomass of the tomato plants are obviously improved, and protozoan flagella are @Dimastigella trypaniformis) After being irrigated, the NJAU-W1 can obviously reduce the content of the bacterial wilt in the soil, which proves that the tomato bacterial wilt inhibitor has potential inhibition effect on tomato bacterial wilt and can reduce the possibility of infection of tomatoes by the bacterial wilt.
Drawings
FIG. 1 shows the form of functional bacteria NJAU-W1 in vivo and cysts.
FIG. 2 is a phylogenetic tree constructed by a functional bacterium NJAU-W1 sRNA sequence.
FIG. 3 is a graph showing the effect of functional bacterium NJAU-W1 on tomato growth promotion.
FIG. 4 shows the function of the functional bacterium NJAU-W1 in promoting tomato plant height.
FIG. 5 shows the accelerating function of functional bacteria NJAU-W1 on tomato dry weight.
FIG. 6 shows the inhibitory effect of functional bacterium NJAU-W1 on bacterial wilt in tomato rhizosphere soil.
Preservation of biological Material
NJAU-W1 classified and named as flagellate NJAU-W1%Dimastigella trypaniformis NJAU-W1), the preservation address is the university of Wuhan preservation center, the preservation date is 2023, 8 months and 28 days, and the preservation number is CCTCC NO: C2023264.
Detailed Description
Example 1 isolation and identification of protozoa
1. Test material:
test soil: the post-town village (118°57'e,32°03' n) in kylin area Jiang Ning in south kyo city was tested as a base tomato greenhouse, which was continuously grown for many years. The soil is yellow brown soil, and the basic physicochemical properties of the soil are as follows: the organic matter content is 32.88 and g/kg, the total nitrogen content is 1.27 and g/kg, the effective phosphorus content is 145.63 mg/kg, the quick-acting potassium content is 220.15 mg/kg, and the soil pH is 6.23.
Coli: mode escherichia coli Escherichia coli DH5 alpha.
LB liquid medium: tryptone 10 g, yeast extract 5g, sodium chloride 10 g, deionized water was added to a volume of 1000 mL,115℃and sterilized for 30min.
PAS (Page's amoeba sample) buffer: firstly, preparing a solution 1 and a solution 2, wherein the component of the solution 1 is 0.142 g disodium phosphate monobasic, 0.136 g potassium phosphate di-cyanide, and adding water to a constant volume of 500 mL. The ingredients of solution 2 were 0.12 g sodium chloride, 4mg magnesium sulfate heptahydrate, 4mg calcium chloride dihydrate, and water was added to a volume of 500 mL. The components of 1L PAS buffer solution are 500 mL solution 1 and 500 mL solution 2, and water is added to fix the volume to 1L, 121 ℃ and sterilization is carried out for 20 min.
Major reagents and instrumentation: QIAGEN soil DNA extraction kit (DNeasy PowerSoil Kit, QIAGEN), inverted microscope, gene amplification apparatus, vertical shaking incubator, ultraviolet-visible spectrophotometer, etc.
2. Isolation of strains:
soil of a tomato greenhouse of an experimental base was collected in a kylin post town village (118°57'e,32°03' n) in Jiang Ning region of south kyo city. 5g of tomato rhizosphere soil is placed into a tissue culture bottle, 200mL of sterile water is added, the tissue culture bottle is placed into a shaking table, and the shaking table is fully vibrated under the condition of 180rpm/min until protozoa are uniformly dispersed in a soil suspension system, and the tissue culture bottle is kept stand for a moment until soil particles are precipitated. 10uL of supernatant was pipetted into a 96-well plate, followed by 90uL PAS buffer and 10uL of inactivated E.coli solution prepared with sterile water as the sole food source in each well. The 96-well plates were placed in an incubator for shading culture, maintained at 20℃and taken out every 24 hours, the protozoa growth of each well was observed at 400-fold with an inverted microscope, and inactivated E.coli liquid was periodically replenished in each well.
Protozoa growth in 96-well plates were observed daily using an inverted microscope on days 7 to 14 of dark culture. After continuing the culture for a while, 10uL of the stock solution was aspirated from the stock well and added to a new buffer-E.coli culture system, and the previous culture operation was repeated until only a single protozoa could be observed in a well, and the well protozoa were aspirated alone and then added with the buffer and E.coli solution for the expansion culture. If only 1 protozoa were observed, the liquid in the glass micropipettes was transferred to a new 96 well microplate with soil protozoa medium and after 2 to 3 days of incubation examined if the protozoa in the wells had propagated and if the morphology was single. If the form is single, then 10uL of the culture system of the protozoa with the single form is sucked into a new 96-well micro-pore plate, and the protozoa culture solution is added, and the above operation is repeated for 2 to 3 times. This is to isolate and purify the protozoa, which are mostly cleaned of bacteria and fungi in some of the soil. So as to avoid the impurity of protozoa during the subsequent extraction of DNA comparison sequence identification.
EXAMPLE 2 morphological observation and identification of functional bacterium NJAU-W1
Adding the separated and purified protozoa into a protozoa culture solution, and placing the protozoa into a low-temperature incubator for light-shielding culture at 20 ℃ for 24 hours. The protozoan trophosomes and cysts were taken under an inverted microscope and photographed with an electronic CCD (FIG. 1). As shown in FIG. 1, the left side shows the living form of the protozoa NJAU-W1, and the right side shows the cyst form under stress conditions. In living form it takes on a spindle shape and has a flagellum at the head; the cyst forms a sphere, the volume is about 1/3 of that of a living body, the diameter is about 4um, and the morphological difference among individuals is not large.
The isolated 18S rDNA sequences of the protozoa were aligned with the Genbank database and phylogenetic tree was drawn by MEGA 10.0 software using the adjacency method (FIG. 2). The results show that NJAU-W1 andDimastigella trypaniformisthe 18SrRNA sequence coverage (Query coverage) of SH II was 100% and the sequence Identity (Identity) was 98.87%.
EXAMPLE 3 potted plant effect of functional bacterium NJAU-W1 tomato
1. Tomato potting experiments set 2 treatments:
(1) Blank Control (CK);
(2) Inoculation of protozoa NJAU-W1 treatment (NJAU-W1).
Each treatment was performed in 12 replicates
2. Soil basin loading:
soil from a greenhouse potting test is collected from a long-term locating point of the subject group in Hainan city of south China, jiangsu province, and the soil of the locating point is subjected to chemical fertilizer treatment for a long time and has a disease history of tomato bacterial wilt. The collected soil passes through a 2 mm screen, and after plant residues in the soil are removed, the soil is uniformly mixed again. Uniformly mixing the soil after sieving and uniformly mixing with quartz sand and vermiculite subjected to high-temperature sterilization, wherein the volume ratio of the three is that of the soil: quartz sand: vermiculite = 3:1:2, the evenly mixed soil is filled into the basin according to the amount of each basin 500 and g for standby.
3. Pre-culturing protozoa: in the early stage, a large number of 1L tissue culture bottles are used, 700mL of PAS buffer solution is added into each bottle, and 2X 10 is added 11 Mode E.coli was prepared as a soil protozoan medium into which protozoan NJAU-W1 was inoculated. The tissue culture bottle is placed at a low temperature of 20 ℃ for 5 days of dark culture, the protozoa culture solution in the tissue culture bottle is sucked in an ultra-clean workbench to count the number of protozoa, and PAS buffer solution is used for dilution to the concentration required by the inoculation field test for standby.
4. Addition of protozoa: selecting tomato seedlings with consistent growth vigor in a period of two leaves and one heart, transplanting the tomato seedlings into a flowerpot filled with 500g of soil, and planting one tomato in each pot. Adding counted protozoan culture solution into soil 3 days after transplanting, wherein the concentration is 1.0X10 per gram of dry soil 3 And (5) watering the protozoan cells regularly, and performing potting management.
5. Tomato plant height: the natural plant height of the tomato plant, i.e. the vertical distance from the highest point of the tomato plant when all the leaves are naturally stretched, to the base of the plant, was measured using a tape measure.
As shown in FIG. 3, treated tomato plants inoculated with the protozoa NJAU-W1 grew significantly better than the Control (CK). As shown in fig. 4, the treatment with the protozoan NJAU-W1 was able to significantly increase the plant height of tomato (p < 0.05), by 31.5% compared to the Control (CK).
6. Tomato plant aerial biomass assay: accurately weighing the weight of the dried plants on the upper part of the tomato land by using a one-thousandth balance. As shown in fig. 5, the treatment with the protozoan NJAU-W1 significantly increased the dry weight of tomato (p < 0.05), by 36.8% compared to the Control (CK).
7. Rhizosphere soil sample collection and bacterial wilt quantification: 6 tomato plants are randomly selected in each treatment, soil on the surface of a tomato root system is buffed, and then the root system of the tomato plants is filled into a sterile self-sealing bag. During treatment, the collected tomato root system tissue is sheared into small sections with the length of 2cm by using sterile scissors, and then the small sections are put into a tissue culture bottle containing 200mL of sterile water, and the tissue culture bottle is placed into a constant temperature shaking table and is vibrated for 30min under the conditions of 30 ℃ and 220 rpm. Taking out the root system with sterile forceps, centrifuging the soil suspension at 4deg.C and 8000rpm for 10min, discarding supernatant, collecting precipitate as rhizosphere soil sample, and storing in a-80deg.C ultra-low temperature refrigerator. DNA of tomato rhizosphere soil is extracted, and bacterial wilt is quantified. PCR amplification was performed with the bacterial wilt-specific primer peh A#3/6 (5'-CAGCAGAACCCGCGCCTGATCCAG-3'/5'-ATCGGACTTG ATGCGCAGGCCGTT-3'). The PCR reaction system (25. Mu.L) was 2X Easy Taq PCR Super Mix12.5. Mu.L, 1. Mu.L each of the peh A#3/6 primer (10. Mu. Mol/L), 1. Mu.L of the DNA template, and 9.5. Mu.L of dd H2O. The PCR reaction conditions are 96 ℃ for 1 min, 96 ℃ for 30 s,70 ℃ for 30 s,72 ℃ for 1 min, and 2 cycles; 94℃for 30 s,70℃for 30 s,72℃for 1 min,33 cycles; and at 72℃for 5 min.
As shown in FIG. 6, the treatment with the protozoan NJAU-W1 significantly reduced the number of bacterial wilt in the rhizosphere soil of tomatoes (p < 0.05) by 12.7% compared to the Control (CK).
In conclusion, the protozoan NJAU-W1 has good promotion effect on the growth condition and biomass accumulation of tomato plants, has the capability of inhibiting bacterial wilt, and has the potential of being developed into biological bacterial agents and biological organic fertilizers for promoting the growth of tomatoes.

Claims (4)

1. Flagellate insectDimastigella trypaniformis Application of NJAU-W1 in promoting tomato plant growth; the said flagellaDimastigella trypaniformis NJAU-W1 has been deposited in China center for type culture Collection with the following deposit address: the preservation date of the university of Wuhan is 2023, 8 months and 28 days, and the preservation number is CCTCC NO: C2023264.
2. Flagellate insectDimastigella trypaniformisApplication of NJAU-W1 in preventing and treating tomato bacterial wilt; the said flagellaDimastigella trypaniformis NJAU-W1 has been deposited in China center for type culture Collection with the following deposit address: the preservation date of the university of Wuhan is 2023, 8 months and 28 days, and the preservation number is CCTCC NO: C2023264.
3. Flagellate insectDimastigella trypaniformisApplication of NJAU-W1 in preparing products for promoting growth of tomato plants and/or preventing and treating tomato bacterial wilt; the said flagellaDimastigella trypaniformis NJAU-W1 has been deposited in China center for type culture Collection with the following deposit address: the preservation date of the university of Wuhan is 2023, 8 months and 28 days, and the preservation number is CCTCC NO: C2023264.
4. Flagellate insectDimastigella trypaniformisUse of a culture of NJAU-W1 for promoting the growth of tomato plants and/or for controlling tomato bacterial wilt; the said flagellaDimastigella trypaniformis NJAU-W1 has been deposited in China center for type culture Collection with the following deposit address: the preservation date of the university of Wuhan is 2023, 8 months and 28 days, and the preservation number is CCTCC NO: C2023264.
CN202311301829.0A 2023-10-10 2023-10-10 Protozoan flagellate NJAU-W1 for promoting tomato growth and preventing and controlling bacterial wilt and application thereof Active CN117025398B (en)

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