CN116948829A - Protozoon tail trichomonas NJAU-ZG8 for promoting tomato growth and preventing and controlling tomato bacterial wilt and application thereof - Google Patents

Protozoon tail trichomonas NJAU-ZG8 for promoting tomato growth and preventing and controlling tomato bacterial wilt and application thereof Download PDF

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Publication number
CN116948829A
CN116948829A CN202310631922.1A CN202310631922A CN116948829A CN 116948829 A CN116948829 A CN 116948829A CN 202310631922 A CN202310631922 A CN 202310631922A CN 116948829 A CN116948829 A CN 116948829A
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tomato
njau
bacterial wilt
growth
trichomonas
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李�荣
阎致广
沈标
郭赛
刘红军
沈其荣
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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
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    • 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

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Abstract

The invention provides a strain of protozoan tail trichomonas NJAU-ZG8 capable of promoting tomato growth. The strain is preserved in China center for type culture collection (China center for type culture collection), the preservation date is 2023, 5 and 22 days, the preservation number is CCTCC NO: M2023805, and the strain can effectively promote the growth of tomato plants and effectively reduce the incidence of bacterial wilt of tomatoes. The strain is separated from greenhouse soil of a greenhouse of a vegetable science institute of Nanjing, jiangsu province, has good effect of promoting growth of tomato plants, can inhibit bacterial wilt of the tomatoes, has simple culture conditions, is easy to store, is easy to produce and apply, and has good development prospect.

Description

Protozoon tail trichomonas NJAU-ZG8 for promoting tomato growth and preventing and controlling tomato bacterial wilt and application thereof
Technical Field
The invention belongs to the technical field of microbial fertilizers, and particularly relates to a tail trichomonas NJAU-ZG8 for promoting tomato growth, preventing and controlling tomato bacterial wilt and application thereof.
Background
Tomato (Lycopersicon esculentum mill.) is a widely cultivated vegetable crop, the most consumed vegetable worldwide, and one of the most widely cultivated crops worldwide. Due to the high economic value and the characteristics of being popular among people, the planting area and the yield of tomatoes show a steadily increasing trend. Tomato bacterial wilt (bacterial wilt disease of tomato) is a vascular bundle systemic disease caused by a physiological race of the l-type of lactobacillus (Ralstonia solanacearum) of the solanaceae family. The strain Lawsonia of Solanaceae is widely distributed throughout the world and is a gram-negative bacterium that can be transmitted through soil and water. The strain of the family solanaceae survives in soil for a long time, enters plants through infecting the roots of tomatoes, then enters a root vascular bundle system and blocks a catheter, so that the plants cannot normally absorb and transport moisture and nutrients, plant wilting is caused, then toxins are released to destroy the catheter of the plants, thin-wall tissues are infected, the plants are gradually rotted, and yield reduction and death are caused.
Crop disease control is an important part of ensuring agricultural safety, and the control measures include chemical control and biological control. Chemical control: the bacterial wilt can be slowed down in a short time by using chemical pesticides and the like, but the method can kill indigenous microorganisms in soil and can cause huge damage to the environment of soil and water; biological control: the prevention and control measures have no pollution and no residue on the environment, so the prevention and control measures are a key direction of sustainable agricultural development, wherein soil-borne diseases caused by the Lawsonia solanaceae can be effectively prevented and controlled by utilizing the disease inhibiting function of beneficial microorganisms.
Disclosure of Invention
The invention aims at overcoming the defects of the prior art and providing a strain of protozoan tail trichomonas NJAU-ZG8 for promoting the growth of tomatoes and preventing and controlling tomato bacterial wilt.
The invention aims to provide application of the protozoan tail trichomonas NJAU-ZG8.
The aim of the invention can be achieved by the following technical scheme:
a protozoon tail trichomonas (Cercomonassp.) for promoting tomato plant growth and inhibiting bacterial wilt is named as NJAU-ZG8, and the strain is preserved in China center for type culture Collection with a preservation date of 2023, 5 months and 22 days and a preservation number of CCTCC NO: M2023805.
The strain NJAU-ZG8 was identified as Trichomonas pigmentosa (Cercomonassp.) in combination with developmental tree alignment analysis and morphological features constructed from the 18SrDNA sequence.
The strain NJAU-ZG8 has a promoting effect on the growth of tomatoes and an inhibiting effect on tomato bacterial wilt; the field test proves that the strain can effectively promote the growth of tomato plants and effectively inhibit the incidence of bacterial wilt of tomatoes, and shows that the inoculation of functional bacteria can inhibit bacterial wilt of tomatoes and promote the growth of plants.
The application of the trichomonas (Cercomonassp.) NJAU-ZG8 in promoting the growth of tomatoes and/or preventing and treating tomato bacterial wilt.
A culture of the said trichomonas (Cercomonasp.) protozoa NJAU-ZG8.
The application of the culture in promoting the growth of tomatoes and/or preventing and treating tomato bacterial wilt.
The application of the trichomonas (Cercomonassp.) protozoa NJAU-ZG8 in preparing the reagent for promoting the growth of tomatoes and/or preventing and treating the bacterial wilt of the tomatoes.
Advantageous effects
The invention provides a protozoan tail trichomonas strain for promoting tomato growth and resisting bacterial wilt. The culture solution of the trichomonas (Cercomonasp.) protozoon NJAU-ZG8 can effectively promote the growth of tomato plants and effectively inhibit the incidence of tomato bacterial wilt, and after 2 months, compared with a non-inoculated control, the growth state of the tomato plants treated by the culture solution of the trichomonas (Cercomonasp.) protozoon NJAU-ZG8 is obviously better, the incidence of tomato bacterial wilt is obviously lower, the trichomonas (Cercomonasp.) protozoon NJAU-ZG8 has a promotion effect on the tomato plants, and the trichomonas (Cercomonasp.) protozoon NJAU-ZG8 also has an inhibition effect on the tomato bacterial wilt.
Drawings
FIG. 1 shows the form of functional bacterium NJAU-ZG 8;
FIG. 2 is a phylogenetic tree constructed by a functional bacterium NJAU-ZG818sRNA sequence;
FIG. 3 shows the field effect of functional bacterium NJAU-ZG8 on tomato growth promotion.
FIG. 4 shows the field effect of the functional bacteria NJAU-ZG8 on the control of tomato bacterial wilt.
Preservation of biological Material
NJAU-ZG8, classified and named as Cercomonassp, is preserved in China center for type culture Collection, with a preservation address of the university of Wuhan, a preservation date of 2023, 5 months and 22 days, and a preservation number of CCTCC NO: M2023805.
Detailed Description
EXAMPLE 1 isolation and screening of protozoa
Test procedure
1. Test material:
coli: mode E.coli Escherichia coli DH 5. Alpha.
Test soil: the soil is collected from greenhouse (tomato) of greenhouse vegetables (31 DEG 43'N,118 DEG 46' E) of Nanjing, jiangsu, and the local climate condition is subtropical monsoon climate, and the annual average temperature of the area is 15.4 ℃ and the average rainfall is 1106mm. The greenhouse is used for continuously planting tomatoes in 3 months in 2013, the soil to be tested is yellow loam, the soil used in the test is treated by chemical fertilizers collected in 9 months in 2020, and the basic physicochemical properties of the soil are as follows: pH6.33, total nitrogen content 1.890g/kg, available phosphorus content 0.1386g/kg, quick-acting potassium content 0.4648g/kg.
PAS (Page' samoebasaline) buffer: first, solution1 and solution2 were prepared, wherein the components of solution1 were 0.142g disodium phosphate monobasic, 0.136g potassium phosphate dibasic, and water was added to a volume of 500mL. The composition of solution2 was 0.12g sodium chloride, 4mg magnesium sulfate heptahydrate, 4mg dihydrate and calcium chloride, and water was added to a volume of 500mL. The 1LPAS buffer solution comprises 500mLSolution1 and 500mLSolution2, and is sterilized by adding water to a volume of 1L, and sterilizing at 121deg.C for 20 min.
LB liquid medium: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride, adding deionized water, fixing the volume to 1000mL, and sterilizing at 115 ℃ for 30min.
Major reagents and instrumentation: TIANGENDP304 blood genome DNA extraction kit, inverted microscope, gene amplification instrument, vertical shaking incubator, constant temperature incubator, ultraviolet visible spectrophotometer, etc.
2. Isolation of strains:
soil mass of healthy tomato field blocks of greenhouse vegetable was collected from Nanjing vegetable science institute (31 deg. 43'N,118 deg. 46' E). The concrete method is that the tomato plant is pulled up with root system, the large soil is gently shaken, and the shaken-out soil is taken as soil body soil. Mixing the obtained soil sample, adding 10g of the mixed soil into a tissue culture bottle filled with 90mLPAS buffer solution, and placing the tissue culture bottle into 220 r.m -1 Shaking in a shaker at 28 ℃ for 30min to allow the protist in the soil to be fully eluted in PAS buffer. After shaking, the tissue culture flask was allowed to stand for 10min. Inoculating Escherichia coli DH5 alpha into LB liquid medium, and culturing at 220 r.m -1 Shaking culture at 37deg.C for 1 day, centrifuging to remove culture medium, eluting with PAS buffer solution for three times to obtain Escherichia coli suspension, sterilizing the suspension at 115deg.C for 30min, diluting with PAS buffer solution at a ratio of 1:50 to obtain sterilized Escherichia coli suspension, and culturing protozoon. Under aseptic condition, adding 100 μl of sterilized Escherichia coli suspension into each well of 96-well plate, absorbing upper liquid of 100 μl of soil suspension, adding into each well of 96-well plate, sealing 96-well plate with sealing film, and placing in a low temperature incubator at 20deg.C for blackeningDark culture.
The growth of protozoa in 96-well microplates was examined daily with an inverted microscope on days 7 to 14 of the dark culture. The specific test method is as follows: the 96-well microplate is placed on an inverted microscope stage, the light source is adjusted to appropriate brightness, and the microscope magnification and focal length are adjusted until a clear field of view appears. The growth status of the protist per well of each 96-well microplate was examined in turn. When a clear view appears, a clear picture of the movement of the protist such as ciliates, reniform worms, amoebas and the like appears, the aperture is marked, and the protist is further sucked and separated, and the specific operation is as follows: soil protist medium was first prepared, added to a 96-well microplate in an amount of 100. Mu.L per well in an ultra clean bench, the liquid in the above-mentioned labeled well diameter was added to the above-mentioned culture medium-added 96-well microplate in an amount of 1. Mu.L per well, and the culture was allowed to stand in a low-temperature incubator at 20℃for 2 to 7 days in darkness, and the growth of protist in the 96-well microplate was examined daily with an inverted microscope during the culture. If the protozoa are observed to be in an cyst state, the liquid in the pore size is required to be sucked, a soil protozoa culture medium is added into a new 96-well microplate, and the culture is observed after 2 to 3 days of culture. Repeating the above steps, continuously diluting and separating soil protozoa until only 1 to 2 types of protozoa can be observed in the aperture, sucking the liquid in the aperture by using a glass micropipette, placing the micropipette on an inverted microscope slide, observing by using an inverted microscope, transferring the liquid in the glass micropipette to a new 96-well microplate added with soil protozoa culture medium if only 1 type of protozoa can be observed, and checking whether the protozoa in the aperture multiply and the morphology is single after culturing for 2 to 3 days. If the shape is single, then a small amount of the protozoon culture solution is sucked from the protozoon aperture seed with the single shape and is added into a new 96-well micro-pore plate, and the above operation is repeated for 2 to 3 times. This is to isolate and purify the resulting protozoa, which are mostly cleaned of bacteria and fungi in some of the soil. So as to avoid the impurity of the protist during the subsequent DNA extraction and comparison sequence identification.
EXAMPLE 2 morphological observation and identification of functional bacterium NJAU-ZG8
Adding the separated and purified soil protist into a soil protist culture solution, and placing the soil protist culture solution into a low-temperature incubator for light-shielding culture at 20 ℃ for 24 hours. The cells were removed and placed under an inverted microscope and photographs of the protozoon nutrition and cysts were taken with an electronic CCD (FIG. 1).
The 18SrDNA sequences of the 4 isolated protozoa were aligned with the Genbank database and phylogenetic tree was drawn by MEGA10.0 software using the adjacency method (FIG. 2). The results showed that the sequence coverage (Query coverage) of 18SrRNA of NJAU-ZG8 and Cercomonassp.strainaSF20 was 100% and the sequence Identity (Identity) was 99.88%.
Example 3 tomato field effect test of functional bacterium NJAU-ZG8
Tomato field test 2 treatments were set up:
(1) Blank Control (CK);
(2) Inoculating the protozoon NJAU-ZG8 for treatment (NJAU-ZG 8).
The nutrient settings of each treatment in the field test are the same according to the total nitrogen of 225 kg-ha -1 Total phosphorus (P) 2 O 5 )150kg·ha -1 Total potassium (K) 2 O)225kg·ha -1 And (5) fertilizing, and supplementing other nutrients with chemical fertilizers. The different treatments set up three repetitions, each repetition having a cell area of 10m 2 And 40 tomatoes are planted in each district, and the planting time is 3 months after the tomatoes are transplanted into a field.
Pre-culturing the protist: 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, a soil protist medium was prepared, and then the protist NJAU-ZG8 was inoculated thereto. And (3) placing the tissue culture bottle at a low temperature of 20 ℃ for 7 days in dark culture, sucking the crude organism culture solution in the tissue culture bottle in an ultra-clean workbench to count the number of the crude organisms, and diluting the crude organism culture solution into the concentration required by the inoculation field test by using PAS buffer solution for later use.
During field tomato plant planting, adding cultured primordial organisms NJAU-ZG8, and adding primordial organisms with the number of 7.5X10 according to each tomato plant 6 And root irrigationThe protist NJAU-ZG8 was inoculated into the rhizosphere of field tomatoes while the Control (CK) was added with an equal amount of PAS buffer.
At the end of the tomato field test, the incidence of tomato bacterial wilt is counted. The incidence rate of the field tomato bacterial wilt is the percentage of the total plants of the number of plants with tomato bacterial wilt symptoms in each district.
The growth traits of tomato plants (tomato plant height, stem thickness and leaf SPAD value content of each treatment cell) were measured on day 60 of field planting of tomatoes.
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.
Stem thickness: natural stem thickness of field tomato plants was measured using vernier calipers and plant stem thickness was measured 1 cm below the 1 st true leaf of tomato plants.
Leaf chlorophyll determination: SPAD values of tomato inverted two leaves were measured with SPAD-520.
Aboveground biomass determination: the plants are packed into envelopes and the weight of fresh plants on the upper part of the tomato land is accurately weighed by a one-thousandth balance.
Rhizosphere soil sample collection: 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 10000rpm for 10min, discarding supernatant, collecting precipitate as rhizosphere soil sample, and storing in a-80deg.C ultra-low temperature refrigerator for use.
Taking 0.1g of the collected soil sample in sterile water, shaking and uniformly mixing by using a vortex meter to obtain tomato rhizosphere soil suspension, diluting and coating the suspension on a bacterial wilt Selective Medium (SMSA) plate, and placing the plate in dark culture at 30 ℃. After 3 days, the plates were removed for counting.
Results and analysis
As shown in panels a and B of fig. 3, the inoculated protist NJAU-ZG8 treatment was able to significantly increase the plant height (a) and stem thickness (B) of tomato plants (p < 0.05), by 23.2% and 11.0%, respectively, compared to Control (CK).
As shown in panel C of FIG. 3, the inoculation of the protoplast NJAU-ZG8 treatment significantly increased the upper fresh weight of the tomato plants (p < 0.05) by 98.3% compared to the Control (CK).
As shown in panel A of FIG. 4, the inoculated protist NJAU-ZG8 treatment significantly reduced the number of bacterial wilt in rhizosphere soil (p < 0.05), by 19.3% compared to Control (CK).
As shown in panel B of FIG. 4, the inoculation of NJAU-ZG8 significantly reduced the incidence of tomato bacterial wilt (p < 0.05) by 43.0% compared to the control treatment.

Claims (6)

1. A trichomonas (cercomonassp.) protozoa NJAU-ZG8 for promoting tomato growth and controlling tomato bacterial wilt, said strain having been deposited in the chinese collection of typical cultures at the following addresses: the preservation date of the university of Wuhan is 2023, 5 months and 22 days, and the preservation number is CCTCC NO: M2023805.
2. Use of the trichomonas (cercomonassp.) protozoa NJAU-ZG8 of claim 1 for promoting tomato growth.
3. Use of the trichomonas (cercomonassp.) protozoa NJAU-ZG8 of claim 1 for controlling tomato bacterial wilt.
4. A culture of the trichomonas (cercomonassp.) protozoa NJAU-ZG8 of claim 1.
5. Use of the culture according to claim 4 for promoting tomato growth and/or for controlling tomato bacterial wilt.
6. Use of the trichomonas (cercomonassp.) protozoa NJAU-ZG8 of claim 1 for the preparation of an agent for promoting tomato growth and/or controlling tomato bacterial wilt.
CN202310631922.1A 2023-05-31 2023-05-31 Protozoon tail trichomonas NJAU-ZG8 for promoting tomato growth and preventing and controlling tomato bacterial wilt and application thereof Pending CN116948829A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117721022A (en) * 2024-02-07 2024-03-19 南京农业大学三亚研究院 Protozoan amoeba for promoting yield of synthetic flora biomembrane and abundance of bacillus bailii and application of protozoan amoeba
CN117887580A (en) * 2024-03-15 2024-04-16 南京农业大学三亚研究院 Protozoan tail trichomonas for promoting banana growth and preventing and controlling fusarium wilt and application thereof

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117721022A (en) * 2024-02-07 2024-03-19 南京农业大学三亚研究院 Protozoan amoeba for promoting yield of synthetic flora biomembrane and abundance of bacillus bailii and application of protozoan amoeba
CN117721022B (en) * 2024-02-07 2024-04-12 南京农业大学三亚研究院 Protozoan amoeba for promoting yield of synthetic flora biomembrane and abundance of bacillus bailii and application of protozoan amoeba
CN117887580A (en) * 2024-03-15 2024-04-16 南京农业大学三亚研究院 Protozoan tail trichomonas for promoting banana growth and preventing and controlling fusarium wilt and application thereof
CN117887580B (en) * 2024-03-15 2024-05-10 南京农业大学三亚研究院 Protozoan tail trichomonas for promoting banana growth and preventing and controlling fusarium wilt and application thereof

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