CN116396889B - Iron-producing carrier golden fungus for preventing and controlling soil-borne bacterial wilt and promoting tomato growth and application thereof - Google Patents
Iron-producing carrier golden fungus for preventing and controlling soil-borne bacterial wilt and promoting tomato growth and application thereof Download PDFInfo
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- CN116396889B CN116396889B CN202310058200.1A CN202310058200A CN116396889B CN 116396889 B CN116396889 B CN 116396889B CN 202310058200 A CN202310058200 A CN 202310058200A CN 116396889 B CN116396889 B CN 116396889B
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Classifications
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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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
-
- 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
-
- 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
- A01P3/00—Fungicides
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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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
-
- 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
-
- 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
Abstract
The invention discloses a golden yellow bacillus CHR6, which is classified under Chryseobacterium rhizoplanae, and is preserved in China general microbiological culture Collection center (CGMCC) with a strain preservation number of 25522 in 8-10-year 2022. The strain CHR6 has the functions of promoting the growth of tomato plants and preventing and treating tomato soil-borne bacterial wilt. The invention also discloses application of the golden yellow bacillus CHR6 in promoting the growth of tomato plants or preventing and controlling tomato bacterial wilt. The invention also discloses a siderophore chryseochelin A with a structure shown in the formula I. The chryseochelin A has the functions of promoting the growth of tomato plants and preventing and treating tomato soil-borne bacterial wilt. The invention also discloses an application of chryseochelin A in preventing and controlling tomato bacterial wilt or promoting tomato plant growth.
Description
Technical Field
The invention belongs to the field of agricultural resource environment, and relates to a golden fungus CHR6 with disease resistance and growth promotion functions, application of golden fungus CHR6 in biological control of soil-borne tomato bacterial wilt and promotion of ferrite nutrient absorption of tomato plants, siderophores chryseochelin A separated from golden fungus CHR6 secretion, and application of chryseochelin A in biological control of soil-borne tomato bacterial wilt and promotion of ferrite nutrient absorption of tomato plants.
Background
Bacterial wilt of tomato is a bacterial vascular bundle soil-borne disease caused by Lawsonia solanaceae (Ralstonia solanacearum), and is frequently found in the south of China, mainly in warm and moist south areas. Bacterial wilt can cause wilting and even death of the solanaceous crops, cause a large amount of yield reduction of the solanaceous crops and cause great economic loss. The disease control method by utilizing rhizosphere microorganisms is a disease control method which accords with modern agricultural health and food safety standards, for example, beneficial bacteria with the function of antagonizing pathogenic bacteria are applied, so that plant growth can be promoted, and disease occurrence can be reduced.
Beneficial bacteria protect plants from pathogenic bacteria and promote plant growth in processes where secreted metabolites typically participate and function, including siderophores. Iron is a trace element essential for the growth of most living organisms, but its biological effectiveness in the environment is low, which results in very vigorous competition for iron in the rhizosphere area by bacteria. Under such iron-limiting conditions, bacteria secrete siderophores into the environment to obtain the ferrite nutrients required for growth. The beneficial bacteria can limit pathogenic bacteria to obtain iron by secreting the specially identifiable siderophores, so that plants are protected from being affected by the pathogenic bacteria, but in the aspect of soil-borne disease prevention and control, the antibacterial siderophores are not put into practical production and application. Because of the great difficulty in identifying siderophores, their chemical nature remains unclear, which prevents the study of their precise mechanism of action and the synthesis of commercial compounds for use.
Disclosure of Invention
Aiming at the current situation of preventing and controlling tomato bacterial wilt, the invention provides a novel method for biologically preventing and controlling and promoting plant ferrite nutrient absorption, and provides a theoretical basis for developing novel efficient biological bacterial wilt-resistant and plant ferrite nutrient absorption-promoting microbial agents by utilizing beneficial microbial siderophore effect.
The inventor screens out a strain of high-yield siderophore golden yellow bacillus CHR6 in tomato fields, detects basic biological characteristics (colony morphology and bacterial strain 16S rDNA gene homology comparison) of the strain, the golden yellow bacillus CHR6 has the siderophore secretion function, inoculates the golden yellow bacillus CHR6 on a CAS blue qualitative detection plate, and after standing culture for 3-4 days in a 30 ℃ incubator, yellow halos appear around the bacterial strain; the bacterial culture broth of Flavobacterium aureobacterium CHR6 reacted with the CAS detection solution to make it bluish and orange red, indicating that the fermentation broth of Flavobacterium CHR6 has siderophores.
The effect of the strain CHR6 on preventing and treating tomato soil-borne bacterial wilt and promoting ferrite nutrient absorption of tomato plants is detected through an indoor experiment and a greenhouse potting experiment, and the strain CHR6 is found to have the functions of promoting growth of tomato plants and preventing and treating tomato soil-borne bacterial wilt, can be used for preparing biological organic fertilizer or pathogenic bacteria inhibitor, and has wide application prospect.
The inventor carries out separation and purification on the siderophore secreted by the chrysobacterium CHR6, and finally obtains the novel siderophore chryseochelin A.
The effect of siderophore chryseochelin A on preventing and treating tomato soil-borne bacterial wilt and promoting the ferrite nutrient absorption of tomato plants is detected through an indoor experiment and a greenhouse potting experiment, and the siderophore chryseochelin A is found to have the growth promoting effect of the tomato plants; and chryseochelin A can be strongly combined with residual iron in a growth medium, so that iron deficiency and growth stagnation of bacterial wilt are induced, and the tomato soil-borne bacterial wilt prevention and treatment effect is achieved. The chryseochelin A can be used for preparing biological organic fertilizer or pathogenic bacteria inhibitor, and has wide application prospect.
The aim of the invention can be achieved by the following technical scheme:
the golden yellow bacillus CHR6 is classified and named Chryseobacterium rhizoplanae, and is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on the 8 th month of 2022, and the address of a preservation unit: the collection number of the strain is CGMCC No.25522, and the collection number of the strain is 1 rd hospital No. 3 of North Chen West road in the Chaoyang area of Beijing city.
Another object of the present invention is to provide a method for preparing a bacterium chrysogenum CHR6, comprising: inoculating single colony of Flavobacterium aureum CHR6 into 10mL TSB medium, placing in shaking table, culturing at 30deg.C and 170rpm for 24 hr, regulating OD of seed solution with sterile water 600 Transferring to 0.5, transferring to 5 mL-300 mL TSB culture medium, culturing at 30deg.C and 170rmp for 48 hr in shaking table, washing the obtained bacterial suspension with sterile water twice, and regulating OD of the bacterial solution with sterile water 600 To 0.5, obtaining the golden yellow bacillus CHR6 bacterial liquid.
The sterile water washing twice comprises the following steps: the bacterial suspension is centrifuged for 5 minutes at 5000rmp, the supernatant is removed, sterile water is added for shaking up, the centrifugation at 5000rmp is continued for 5 minutes, the supernatant is removed, sterile water is added for shaking up, and the centrifugation at 5000rmp is continued for 5 minutes, so that the supernatant is removed.
Another object of the invention is to provide the use of said golden fungus CHR6 for promoting tomato growth.
Preferably, the application is the application of the golden fungus CHR6 in preparing a bio-organic fertilizer for promoting the growth of tomatoes.
A method of promoting tomato growth comprising: inoculating the golden yellow bacillus CHR6 into soil by adopting a root irrigation method.
Preferably, when tomato seedlings grow to three true leaf stage, the golden fungus CHR6 is inoculated into soil by a root irrigation method.
More preferably, when the tomato seedlings grow to the three true leaf stage, a root irrigation method is adopted, and each tomato seedling is inoculated with 10 mL-20 mL OD 600 0.5 of golden yellow bacillus CHR6 bacterial liquid.
The invention also aims to provide the application of the golden fungus CHR6 in preventing and controlling tomato bacterial wilt.
Preferably, the application is the application of the golden fungus CHR6 in preparing a pathogenic bacteria inhibitor for preventing and controlling tomato bacterial wilt.
A method for preventing and controlling tomato bacterial wilt, comprising: inoculating the golden yellow bacillus CHR6 into soil by adopting a root irrigation method.
Preferably, when tomato seedlings grow to three true leaf stage, the golden fungus CHR6 is inoculated into soil by a root irrigation method.
More preferably, when the tomato seedlings grow to the three true leaf stage, a root irrigation method is adopted, and each tomato seedling is inoculated with 10 mL-20 mL OD 600 0.5 of golden yellow bacillus CHR6 bacterial liquid.
Another object of the present invention is to provide a citrate-based siderophore chryseochelin a having a structure as shown in formula i, having a double fumaric acid structure:
another object of the invention is to provide the use of chryseochelin a for controlling bacterial wilt of tomatoes.
A method for preventing and controlling tomato bacterial wilt by using chryseochelin a, comprising the following steps: and adding chryseochelin A into soil by adopting a root irrigation method.
Preferably, when tomato seedlings grow to a three-leaf stage, chryseochelin A is added into soil by a root irrigation method.
More preferably, when the tomato seedlings grow to the three true leaf stage, a root irrigation method is adopted, and 50-100 mu g chryseochelin A of each tomato seedling is applied.
Further preferably, when tomato seedlings grow to a three-leaf stage, a root irrigation method is adopted, and 5-10 mL of chryseochelin A aqueous solution with the concentration of 10 mug/mL is applied to each tomato seedling.
It is another object of the present invention to provide the use of chryseochelin a for promoting tomato growth.
A method of promoting tomato growth using chryseochelin a comprising: and adding chryseochelin A into soil by adopting a root irrigation method.
Preferably, when tomato seedlings grow to a three-leaf stage, chryseochelin A is added into soil by a root irrigation method.
More preferably, when the tomato seedlings grow to the three true leaf stage, a root irrigation method is adopted, and 50-100 mu g chryseochelin A of each tomato seedling is applied.
Further preferably, when tomato seedlings grow to a three-leaf stage, a root irrigation method is adopted, and 5-10 mL of chryseochelin A aqueous solution with the concentration of 10 mug/mL is applied to each tomato seedling.
Drawings
FIG. 1 shows colony morphology of Flavobacterium aureofaciens CHR 6.
FIG. 2 is a phylogenetic tree established by the adjacency method based on the gene sequence of 16S rDNA of Flavobacterium aureum CHR 6.
FIG. 3 is a graph comparing greenhouse potted plant pro-active fruits; left, inoculating CHR6; adding aseptic fermentation liquor containing chryseochelin A; right, blank.
FIG. 4 is a graph showing comparison of bacteriostatic effects of potted plants in a greenhouse; left, blank control; inoculating CHR6; right, a sterile fermentation broth containing chryseochelin a was added.
FIG. 5 is a CHR6 siderophore capacity CAS plate assay.
FIG. 6 shows the Base Peak Chromatograms (BPC) of CHR6 supernatant without iron (upper panel) and with iron (lower panel) (m/z 50-2000).
FIG. 7 shows fragmentation behavior of chryseochelin A (m/z 621) at a collision energy of 35eV (square labeled precursor).
FIG. 8 is a chryseochelin A dose response curve of ralstonia solanacearum under iron-rich and iron-limited conditions.
Biological preservation information:
the golden yellow bacillus CHR6 is classified and named Chryseobacterium rhizoplanae, and is preserved in China general microbiological culture Collection center, and preserving unit address: the collection number of the strain is CGMCC No.25522, and the collection number of the strain is 1 rd hospital No. 3 of North Chen West road in the Chaoyang area of Beijing city.
Detailed Description
The technical scheme of the invention is further modified through the specific embodiments.
TSB medium: deionized water (1000 mL), tryptone (15.0 g), soy peptone (5.0 g), sodium chloride (5.0 g).
TSA medium: deionized water (1000 mL), tryptone (15.0 g), soy peptone (5.0 g), sodium chloride (5.0 g), agar (20 g).
CAS test solution formulation:
the following four solutions were prepared:
2mM CAS stock: 0.2421g CAS (chrome azure Chrome azurol sulphonate) was dissolved in 200mL deionized water to give a CAS stock solution;
1mM FeCl 3 stock solution: 0.2703g of ferric chloride hexahydrate was dissolved in 1L of 10mM hydrochloric acid to give FeCl 3 A stock solution;
0.0219g of cetyltrimethylammonium bromide (HTDMA) was weighed and dissolved in 50mL of water to obtain cetyltrimethylammonium bromide solution;
4.3079g of anhydrous piperazine is weighed and dissolved in 30mL of water, and the pH value is adjusted to 5.6 by hydrochloric acid to obtain piperazine solution;
1.5mL CAS stock solution was taken and 7.5mL FeCl was added 3 The stock solution was mixed well, 50mL of cetyltrimethylammonium bromide solution was added with stirring, then 30mL of piperazine solution was added, and finally deionized water was added to prepare 100mL of LCAS test solution.
CAS blue detection plate, chrome Azure (CAS) 60.5mg, cetyl trimethylammonium bromide (HDTMA) 72.9mg,1 mmol.L -1 FeCl 3 ·6H 2 O(10mmol·L -1 HCl formulation) 10mL,0.1 mol.L -1 Phosphate buffer 50mL, distilled water 940mL, agar 0.9%.
Phosphate buffer (g/L): naH (NaH) 2 PO 4 ·2H 2 O 5.905，Na 2 HPO 4 ·12H 2 O 24.270，NH 4 Cl 2.500，KH 2 PO 4 0.750,NaCl 1.250,pH 6.8, diluted 10-fold in use.
MKB limited-iron medium: 5.0g of casein amino acid, 15mL of glycerol (glycerol) and 785mL of deionized water; 2.5g of dipotassium hydrogen phosphate is dissolved in 100mL of deionized water; 2.5g of magnesium sulfate heptahydrate is dissolved in 100mL of deionized water; after the preparation is completed, the three solutions are independently sterilized respectively, sterilized at 115 ℃ for 30min and pH 7.2, and when in use, the three solutions are mixed to obtain the MKB iron-limiting culture medium.
MKB iron-rich medium: adding 1mM FeCl into MKB limited-iron culture medium 3 Stock solution, feCl 3 The final concentration is 50 mu mol.L -1 。
CAA medium: deionized water 1000mL, acid hydrolyzed casein (10.0 g), K 2 HPO 4 ·3H 2 O(1.18g)、MgSO4·7H 2 O(0.25g)。
Example 1
Isolation and identification of Flavobacterium aureum CHR6
1. Isolation of Flavobacterium aureofaciens CHR6
Rhizosphere soil samples were collected from tomato planting greenhouse (115°51'E,28°41' N) in Nanchang, jiangxi province and used as 906 tomato rhizosphere soil. 1g of rhizosphere soil was mixed with 9ml of SM buffer in a sterilized 50ml Erlenmeyer flask, and the mixture was transferred in a shaker at a rotation speed of 170rpm and a temperature of 30℃for 30min to obtain a soil suspension.
Dilution of soil suspension concentration to 10 Using sterile Water -5 -10 -6 The specific method comprises the following steps: taking 100 mu L of soil suspension, adding into a centrifuge tube containing 900 mu L of sterile water, and swirling to obtain a dilution gradient of 10 -1 Repeating the step for gradient dilution until a diluted concentration of 10 is obtained -5 -10 -6 Is a soil suspension of (a).
100. Mu.L of the diluted soil suspension was pipetted into a solid plate containing 1/10 of the diluted TSA medium and spread evenly.
Placing the coated flat plate into a constant temperature incubator, culturing for 48 hours at 30 ℃ in a dark place, picking single bacterial colonies, scribing on a TSA solid flat plate, and continuing culturing for 48 hours at 30 ℃ in a dark place; picking single colony to be connected into a 96-well plate containing 100 mu L of TSB culture medium in each well, putting the well plate into a shaking table, and culturing overnight at the temperature of 30 ℃ and the rotating speed of 170 rpm; and taking out the bacterial liquid, fully mixing the bacterial liquid with 30% glycerol according to the volume ratio of 1:1, and storing in a refrigerator at the temperature of minus 80 ℃.
2. Morphology observations of Flavobacterium aureofaciens CHR6
Taking out glycerol strain stored in-80deg.C refrigerator, streaking and inoculating on TSA plate, and culturing at 30deg.C in dark place. The strain CHR6 grows on the surface of a TSA solid flat plate for 48 hours, the shape of the strain CHR6 is shown in figure 1, and the colony is yellow and round, opaque, glossy at the edge and moist and wrinkle-free on the surface.
3. Genome information of Flavobacterium aureofaciens CHR6
Amplification was performed using the universal primer for 16S rDNA:
27F：5’-AGAGTTTGATCCTGGCTCAG-3’；
1492R：5’-GGTTACCTTGTTACGACT T-3’。
PCR reaction System (25. Mu.L): 1. Mu.L of DNA template, 12.5. Mu.L of reaction mixture, 1. Mu.L of front and rear primers, ddH, respectively 2 O 10.5μL。
PCR reaction conditions: after 5min of pre-denaturation at 95 ℃, entering into thermal cycle: denaturation at 94℃for 30s, annealing at 58℃for 30s, elongation at 72℃for 1min for 30s, for 30 cycles. Finally, the extension is carried out for 10min at 72 ℃.
The PCR amplified products were purified and sequenced by Shanghai Bioengineering Co. BLAST alignment analysis was performed on the NCBI website based on the sequencing result of the gene sequence of 16S rDNA.
The strain 16S rDNA sequence was aligned with that in the Genbank database and phylogenetic tree was drawn by MEGA 7.1 software using the adjacency method (FIG. 2). The results showed that the CHR6 strain was Flavobacterium aureobacteria (Chryseobacterium).
The golden yellow bacillus CHR6 is classified and named Chryseobacterium rhizoplanae, and is preserved in China general microbiological culture Collection center, and preserving unit address: the collection number of the strain is CGMCC No.25522, and the collection number of the strain is 1 rd hospital No. 3 of North Chen West road in the Chaoyang area of Beijing city.
Example 2
Preparation of golden yellow bacillus CHR6 bacterial liquid
Activating strain from stored glycerol tube, inoculating single colony into 50mL triangular flask containing 10mL TSB culture medium, culturing at 30deg.C with 170rpm for 24 hr, and regulating OD of seed solution with sterile water 600 Transferring 5mL to a 1L triangular flask containing 300mL TSB culture medium, culturing at 30deg.C and 170rmp for 48 hr in a shaker, centrifuging the obtained bacterial suspension 5000rmp for 5min, removing supernatant, adding sterile water, shaking, centrifuging for 5min, removing supernatant, and regulating OD of the bacterial solution with sterile water 600 To 0.5, obtaining the golden yellow bacillus CHR6 bacterial liquid.
Potted plant experiment for promoting tomato plant growth by chrysobacterium CHR6
Soaking small red dwarf cherry tomato seeds in 70% alcohol for 1min, washing with sterile water for 3 times to remove residual alcohol, soaking in 3% sodium hypochlorite solution for 5min for surface sterilization, and rinsing with sterile water for 6 times. Placing the sterilized tomato seeds into a sterile flat plate filled with sterile water to soak filter paper, uniformly spreading, placing the flat plate in a constant temperature incubator at 30 ℃ to accelerate germination for 48 hours, then selecting good-germination sprouts, sowing the good-germination sprouts into a seedling tray, raising the seedlings to a trefoil stage, and selecting seedlings with consistent growth vigor for transplanting.
The red dwarf cherry small tomato seedlings with consistent growth vigor are transplanted into 6-hole trays (each hole in the 6-hole trays is independently separated) filled with the sterilized soil, and one tomato plant is transplanted in each hole. 2 treatments were set: strain treated and control groups (CK, without inoculation of any microorganisms), each treated 5 replicates, one tray, i.e. 6 tomato plants, for one replicate, for a total of 60 tomato plants. When the tomato seedlings reach the three true leaf stage, a root irrigation method is adopted, and each tomato plant is inoculated with 10mL OD 600 0.5 of golden yellow bacillus CHR6 bacterial liquid, all plantsGrowing in a greenhouse with natural temperature of 30-35 ℃ and humidity of 60-80% RH, measuring the growth index of tomato plants after 30 days, harvesting the plants, and measuring the dry weight of the plants and the iron content in roots, stems and leaves.
And detecting growth promotion related indexes such as plant height, chlorophyll content, dry fresh weight of overground parts, dry fresh weight of underground parts, thick stem, iron content of plants and the like of tomato plants in the inoculated strain treatment group and the control group.
The results are shown in Table 1 and FIG. 3, and the golden fungus CHR6 significantly increases the chlorophyll content, iron content, overground and underground dry weight and other growth-promoting related indexes of tomato plants.
TABLE 1 growth promoting index of strain CHR6
Potting experiment for inhibiting tomato bacterial wilt by chrysobacterium CHR6
Pattern pathogenic bacteria: rhizoctonia solani QL-Rs1115 (Wei et al 2011), abbreviated as RS, with strong pathogenicity is separated from kylin town disease tomato plants in Nanjing.
Soaking small red dwarf cherry tomato seeds in 70% alcohol for 1min, washing with sterile water for 3 times to remove residual alcohol, soaking in 3% sodium hypochlorite solution for 5min for surface sterilization, and rinsing with sterile water for 6 times. Placing the sterilized tomato seeds into a sterile flat plate filled with sterile water to soak filter paper, uniformly spreading, placing the flat plate in a constant temperature incubator at 30 ℃ to accelerate germination for 48 hours, selecting good-germination sprouts, sowing the good-germination sprouts into a seedling tray, raising the seedlings to a trefoil stage, and selecting seedlings with consistent growth vigor for transplanting.
Seedlings of consistent vigor were transplanted into 6-well trays (each well in one tray being independently separated) containing sterilized soil, and one tomato plant was transplanted per well. Setting 3 treatments: strain treated group, positive control group (bacterial wilt only) and negative control group (no microorganism). When tomato seedlings grow to three true leaf periods, a root irrigation method is adopted, and each strain of tomato in the strain treatment group is inoculated with 10mL OD 600 0.5 of Flavobacterium aurumCHR6 bacterial liquid, and OD is inoculated to each tomato after one week 600 5mL of bacterial wilt RS bacterial liquid with the concentration of 0.5. Each treatment was repeated 5 times, 6 tomato plants each, and a total of 90 tomato plants were used. All plants were grown in a greenhouse with natural temperature 30-35 ℃ and humidity 60-80% RH, potting locations were changed twice a week and watered periodically. Plants were monitored daily for incidence until the end of the experiment.
Plant incidence (%) = number of affected plants/total number of plants x 100%
As shown in FIG. 4, the control effect of the golden yellow bacillus CHR6 is as high as 92% + -14% of the positive control group, and the incidence of the CHR6 treatment is 17% + -15%, which is obviously lower than that of the positive control group, thus showing that the inoculated CHR6 shows good control effect.
Iron-producing carrier ability of chrysobacterium CHR6
The stored CHR6 glycerol tube was removed from the-80℃refrigerator, 5. Mu.L to 96 well plates were removed, each containing 195. Mu.L of TSB medium, and incubated overnight at a temperature of 30℃and a rotation speed of 170 rpm.
mu.L of CHR6 bacteria cultured overnight was taken out into a 96-well plate containing 190. Mu.L of MKB iron-limited medium, and cultured at a temperature of 30℃and a rotation speed of 170rpm for 48 hours.
Centrifuging CHR6 bacterial liquid passing through MKB limited-iron culture medium with an ELISA plate centrifuge, respectively, at 4000 r.min -1 Centrifuging for 5min, and filtering the supernatant with 96-well plate filter membrane (0.22 μm) to obtain sterile supernatant. Mixing the sterile supernatant with CAS detection solution according to the volume ratio of 1:1, standing for 2h, and measuring OD by using an enzyme-labeling instrument 630 The mark is A; deionized water is used as a reference, the deionized water is mixed with CAS detection solution according to the volume ratio of 1:1, and the mixture is stood for reaction for 2 hours, and an enzyme-labeled instrument is used for measuring OD 630 And is denoted as Ar.
The relative siderophore content (SU, calculated formula: su=1-a/Ar) was calculated to be 0.95, indicating that chrysobacterium aurum CHR6 has a strong siderophore-producing ability.
After inoculating Flavobacterium aureum CHR6 on a CAS blue qualitative detection plate and standing and culturing for 4d in a 30 ℃ incubator, the result is shown in figure 5, and yellow halos appear around the strain, thereby further confirming that the CHR6 has the capacity of secreting siderophores.
Example 3
Preparation, separation and purification of 1.chryseochelin A
Liquid culture of Flavobacterium aureofaciens CHR 6: mu.L of glycerol stock containing the strain Flavobacterium aureum (stored at-80 ℃) was inoculated into 195. Mu.L of TSB medium in 96-well plates and incubated at a temperature of 28℃for 22 hours at 170 rpm; 10. Mu.L of overnight culture was transferred to a new 96-well plate containing 190. Mu.L of CAA medium and incubated at 28℃for 48 hours at 170 rpm; the liquid culture was centrifuged at 10733rcf for 10min at room temperature, and the supernatant was filtered through a 0.22 μm filter and stored at-20 ℃.
Freezing the supernatant with liquid nitrogen, lyophilizing overnight to obtain dry solid powder, dissolving in 80mL of methanol, filtering with filter paper to remove insoluble residue, evaporating organic solvent under mild nitrogen flow to obtain dry extract, dissolving the extract in 5mL of deionized water again, centrifuging at 14000rpm for 10min at 4deg.C, collecting supernatant as solution containing siderophore, and transferring into new centrifuge tube for preservation. From a centrifuge tube, two 60. Mu.L solutions, one of which was supernatant without iron and the other with 3. Mu.L FeCl 3 Aqueous solution (100 mM) to obtain iron complex of siderophore, i.e. supernatant plus Fe (III), was used for mass spectrometry.
The crude siderophores obtained were purified on an Shimadzu HPLC system and chromatographed at room temperature using a YMC-act Triat C18 column (150X 30mm,5 μm), mobile phase A: water, mobile phase: b methanol, flow rate 40mL/min, gradient elution:
(i)0-2min 5％B；
(ii) Linearly increasing to 30% b until 15 minutes;
(iii) Linearly increasing to 100% b until 30 minutes;
(iv) The column was rinsed with 100% solution for up to 50 minutes;
all peaks having an absorption wavelength of 270nm were collected with a fraction collector.
All collected fractions were simply measured by mass spectrometry to confirm the presence or absence of the desired chryseochelin molecular mass. The chryseochelin a fraction eluted for about 14 minutes. Packing the collected chryseochelin A components into a plurality of 15mL centrifuge tubes, and concentrating the components at 25 ℃ by using a vacuum concentrator to obtain off-white powder; the off-white powder was readily soluble in water, and the off-white powder was dissolved in 25. Mu.L deionized water and subjected to a second purification step on a Thermo Fisher analytical liquid phase RP-HPLC. Chromatographic separation was carried out at 40℃using a Waters CORTECS C18 column (150X 4.6mm,2.7 μm), mobile phase A: water +0.1% formic acid, mobile phase B: acetonitrile +0.1% formic acid, flow rate: gradient elution was performed at 1.0 mL/min:
(i) 4%B isocratic, from 0.0-0.5min;
(ii) Linearly increasing to 11% b until 5.25 minutes;
(iii) The linear increase was 95% until 7.0 minutes;
(iv) Hold 95% B for up to 10.0 min
(vi) Returning to the starting condition of 4%B for up to 10.5 minutes;
(vii) Equilibrate for 4.5 minutes until the next run;
the absorption wavelength of 270nm was again monitored. The fractions were collected manually and the collected fractions were lyophilized to give a white powder which was stored at-20 ℃.
Structural identification of 2.chryseochelin A
The exact molecular mass of chryseochelin A was determined by UHPLC-HR-MS and its structural formula was predicted. FIG. 6 shows two chromatographic separations of supernatant with Fe (III) and without iron. In the iron-free supernatant, the peak at 3.45 min was chryseochelin A and the corresponding m/z values were 621 ([ m+H)] + ). After addition of iron, the peak shifted forward (chryseochelin A-iron complex 1.74 min) and M/z value 647, which corresponds to [ M-2H+Fe (III)] + Is the case in (a). The presence of iron atoms is confirmed by their characteristic isotopic pattern distribution. According to seven golden rules of structural prediction, the structural formula of chryseochelin A is predicted as C 24 H 36 N 4 O 15 . No reasonable match was found in the Natural Products Atlas and PubChem et al 450 more known siderophores or universal databases, and therefore detected chryseochelin a was a novel compound.
UHPLC-HR-MS shows [ m+H ]] + The ion has an accurate molecular mass of 621.22385 and accords with the recommended chemical formula C 24 H 36 N4O 15 (1.8 ppm), this high carbon to oxygen ratio is typically found only in citrate-based siderophores. At a collision energy of 35eV (FIG. 7), a characteristic fragmentation fragment H from the parent ion is obtained 2 O (-m/z 18) and CO 2 H 2 (-m/z 46), loss of both ion fragments and other major fragments indicates the presence of hydroxyl and citrate moieties. By cleavage of the amide bond in the citrate moiety, the main fragment has a molecular weight of 233.11318 (C 9 H 17 N 2 O 5 + 0.1 ppm) and 389.1921 (C) 15 H 21 N 2 O 10 + -0.4 ppm) reveals the connectivity of the molecular substructure. The fumaric acid fragment appeared in a molecular weight of 99.00787 (C 4 H 3 O 3 + 2.0 ppm) at molecular weight 523.22621 (C) 20 H 35 N 4 O 12 + -3.1 ppm) of the signal whose opposite fragment is lower. 3-hydroxy cadaverine (3-hydroxy cadaverine) moiety is distributed in a molecular weight 100.07560 (C 5 H 10 NO + 0.9 ppm) and 117.10203 (C 5 H 13 N 2 O + 1.8 ppm).
At D 2 Chrysochelin A was measured in O 1 H and 13 c NMR spectra (. Gtoreq.99.8% at.D), chemical shifts are shown in Table 2. At the position of 1 In H, the presence of citrate moieties can be seen by the characteristic bimodal at δH2.73 and 2.58ppm, and the coupling constant of 14.7Hz, which belongs to the chemically unequal proton at the C3/C3' position, due to the stereocenter at C2. In the range of 1.5-3.8ppm, CH with unequal protons can be seen 2 Multiple peaks, C8/C8 'and C9/C9' of the group. This complexity stems from the chiral center at C7/C7', where the hydroxyl group is attached. The presence of hydroxyl groups is typical of δH23.64 and δC66.5ppm 1 H and 13 the C shift was confirmed. For both sides of the molecule, two different chiralities of the hydroxyl group relative to the citrate center are assumed, because of the two carbon atomsThe chemical shift signals at 66.50 and 66.53ppm are very close, similar in intensity, and a broad proton multiplex is obtained at the C7/C7' position. Above 6ppm, a signal of two protons at the double bond at positions C11/C11 'and C12/C12' appears. The high chemical shift results from conjugated carboxylic acid and hydroxamic acid systems. The cleavage of the proton signal at positions C9/C9', C10/C10' and C11/C11' can be explained by the different chemical shifts caused by the isomers of hydroxamic acid. This is also demonstrated by the occurrence of inverse cross peaks in the NOESY spectrum of exchangeable protons. Will be measured by HSQC and DEPT 90 and 135 13 The C signal being assigned to the corresponding 1 H atoms confirm the number of protons attached to the carbon. Carbonyl compounds (C1, C4/C4', C10' and C13, C13 ') are distinguished by their high chemical shift (. Delta.C)>160 ppm) and is easy to dispense. 1 H– 1 HCOSY is used to distribute the connectivity of the carbon chain (C5/C5 ' -C9/C9 '), confirming the hydroxylation position at carbon C7/C7 '. TOCSY shows an isolated CH at the C3/C3' position 2 The protons at the radicals and double bonds (C10/C10 'and C11/C11' positions) are the separate spin systems and then pass 1 H– 13 C HMBC linkages revealing the correlation of protons at the C9/C9' position with carbon C10/C10' and the correlation of protons at the C5/C5' and C3/C3' positions with carbon C4/C4 '. Finally, NOESY was measured to demonstrate the correlation of protons at positions C9/C9 'and C11/C11', supporting the positional assignment of double bond protons previously based on chemical shift.
Data 1H and 13C of Table 2.chryseochelin A. Multiple signals due to hydroxamic acid isomerization
Example 4
Effect of chryseochelin a in inhibiting growth of ralstonia solanacearum
1. Indoor well plate experiment
Inoculating the ralstonia solanacearum RS into 4mL of LB culture medium, and growing at 30 ℃ and 220 rpm; the culture product was washed twice with 0.8% NaCl solution, and the OD of the bacterial wilt RS solution was adjusted with 0.8% NaCl solution 600 To 0.1.
For dose response assays, chryseochelin A was first diluted to 25mg/mL with ultra-pure sterile water and incubated in CAA (iron limited) medium (i.e., pure CAA medium without iron) and 50. Mu.M FeCl, respectively 3 Further diluted to a final concentration of 100. Mu.g/mL in CAA (iron-rich) medium. The chryseochelin a solution obtained above was filter sterilized and serially diluted with ultra-pure sterile water in nine steps (2-fold dilution). Then 198. Mu.L of each dilution was transferred to a 96-well plate and repeated and 2. Mu.L of OD was added 600 A bacterial suspension of the genus Rhizoctonia at 0.1 was prepared by using a pure CAA medium (200. Mu.L) containing no chryseochelin A as a control. In a microplate reader, 96-well plates were incubated statically at 30℃and OD was measured every 20 minutes 600 For a total of 71 hours, shake for 1 minute before each measurement.
Under iron limiting conditions, bacterial growth steadily decreased with increasing chryseochelin a concentration (fig. 8), at a maximum concentration of 100 μg/mL, bacterial growth was reduced by 88.45% compared to control. In contrast, the inventors did not observe growth inhibition in iron-rich conditions except for the highest concentration of chryseochelin a. It was shown that chryseochelin a itself does not have antibiotic properties, but is able to bind strongly to residual iron in the growth medium, thereby inducing iron deficiency and growth arrest in ralstonia solanacearum.
2. Greenhouse potting experiment
Pattern pathogenic bacteria: rhizoctonia solani QL-Rs1115 (Wei et al 2011), abbreviated as RS, with strong pathogenicity is separated from kylin town disease tomato plants in Nanjing.
Soaking small red dwarf cherry tomato seeds in 70% alcohol for 1min, washing with sterile water for 3 times to remove residual alcohol, soaking in 3% sodium hypochlorite solution for 5min for surface sterilization, and rinsing with sterile water for 6 times. Placing the sterilized tomato seeds into a sterile flat plate filled with sterile water to soak filter paper, uniformly spreading, placing the flat plate in a constant temperature incubator at 30 ℃ to accelerate germination for 48 hours, then selecting good-germination sprouts, sowing the good-germination sprouts into a seedling tray, raising the seedlings to a trefoil stage, and selecting seedlings with consistent growth vigor for transplanting.
Seedlings with consistent growth vigor are transplanted into 6-hole trays filled with sterilized soil, and one tomato plant is transplanted in each hole. Setting 3 treatments: chryseochelin a treatment group, positive control group, negative control group; each treatment was repeated 5 times, 6 tomato plants each, and a total of 90 tomato plants were used. When tomato seedlings grow to a three-leaf period, each tomato plant of the chryseochelin A treatment group is simultaneously inoculated with 5mL of chryseochelin A sterile water solution with the concentration of 10 mug/mL and 5mL of OD by a root irrigation method 600 0.5 of bacterial wilt RS bacterial liquid, and 5mL of OD is connected into each tomato plant of the positive control group by a root irrigation method 600 The bacterial wilt RS bacterial liquid is 0.5, and the negative control group is not inoculated with any fermentation liquid and microorganism. All plants were grown in a greenhouse with natural temperature of 30-35 ℃ and humidity of 60-80% RH, potting locations were changed twice a week and watered periodically. Plants were monitored daily for incidence until the end of the experiment.
Plant incidence (%) = number of affected plants/total number of plants x 100%
The control effect of chryseochelin a is shown in fig. 4. The inoculation of chryseochelin A shows good prevention and control effects. The incidence of the positive control group is as high as 92% ± 14%, while the incidence of CHR6 treatment is 25% ± 14%, significantly lower than the positive control group.
Potted plant experiment of chryseochelin A for promoting tomato plant growth
Soaking small red dwarf cherry tomato seeds in 70% alcohol for 1min, washing with sterile water for 3 times to remove residual alcohol, soaking in 3% sodium hypochlorite solution for 5min for surface sterilization, and rinsing with sterile water for 6 times. Placing the sterilized tomato seeds into a sterile flat plate filled with sterile water to soak filter paper, uniformly spreading, placing the flat plate in a constant temperature incubator at 30 ℃ to accelerate germination for 48 hours, and then selecting and sowing the well-germinated buds into seedling trays. And (5) raising seedlings to a trefoil stage, and selecting and transplanting seedlings with consistent growth vigor.
The red dwarf cherry tomato seedlings with consistent growth vigor are transplanted into 6-hole trays (each hole in one tray is independently separated) filled with the sterilized soil, and one tomato plant is transplanted in each hole. 2 treatments were set: chryseochelin a treatment group, control group (CK), each treated 5 replicates, one tray, i.e. 6 tomato plants, for one replicate, for a total of 60 tomato plants. When tomato seedlings reach the three true leaf stage, each tomato plant is inoculated with 5mL of chryseochelin A sterile water solution with the concentration of 10 mug/mL by a root irrigation method, the tomato plants of a control group are not inoculated with any fermentation liquor and microorganisms, all plants grow in a greenhouse with the natural temperature of 30-35 ℃ and the humidity of 60-80% RH, the growth index of the tomato plants is measured after 30 days, and the dry weight of the plants and the iron content in roots, stems and leaves are measured by harvesting the plants.
And detecting growth-promoting related indexes such as plant height, chlorophyll content, dry fresh weight of the overground part, dry fresh weight of the underground part, thick stem, iron content of the plants and the like of the tomato plants in the chryseochelin A treatment group and the control group.
The results are shown in Table 3 and FIG. 3, wherein chryseochelin A significantly increases the chlorophyll content, iron content, dry weight of aerial parts and other relevant growth promoting indexes of tomato plants.
TABLE 3 growth promoting index of siderophores chryseochelin A
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Claims (9)
1. The golden yellow bacillus CHR6 is classified and named Chryseobacterium rhizoplanae, and is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 25522 in the 8 th month and 10 th day of 2022.
2. Use of chrysobacterium CHR6 according to claim 1 for promoting the growth of tomato plants, characterized in that: inoculating 10 mL-20 mL OD to each tomato seedling by root irrigation 600 0.5 of golden yellow bacillus CHR6 bacterial liquid.
3. Use of chrysobacterium aurum CHR6 according to claim 1 for the preparation of a bio-organic fertilizer for promoting tomato growthThe application is characterized in that: inoculating 10 mL-20 mL OD to each tomato seedling by root irrigation 600 0.5 of golden yellow bacillus CHR6 bacterial liquid.
4. Use of chrysobacterium aurum CHR6 according to claim 1 for controlling tomato bacterial wilt, characterized in that: inoculating 10 mL-20 mL OD to each tomato seedling by root irrigation 600 0.5 of golden yellow bacillus CHR6 bacterial liquid.
5. Use of chrysobacterium aurum CHR6 according to claim 1 for the preparation of a pathogen inhibitor for controlling tomato bacterial wilt, characterized in that: inoculating 10 mL-20 mL OD to each tomato seedling by root irrigation 600 0.5 of golden yellow bacillus CHR6 bacterial liquid.
6. The structure secreted by chrysobacterium aurum CHR6 according to claim 1 is shown in formula i as citrate-based siderophore chrysoechelin a:
7. use of chryseochelin a according to claim 6 for promoting the growth of tomato plants, characterized in that: the root irrigation method is adopted, and 50 mug to 100 mug g chryseochelin A of each tomato seedling is applied.
8. Use of chryseochelin a according to claim 6 for controlling tomato bacterial wilt, characterized in that: the root irrigation method is adopted, and 50 mug to 100 mug g chryseochelin A of each tomato seedling is applied.
9. Use of chryseochelin a according to claim 6 for the preparation of a pathogen inhibitor for controlling tomato bacterial wilt, characterized in that: the root irrigation method is adopted, and 50 mug to 100 mug g chryseochelin A of each tomato seedling is applied.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101402930A (en) * | 2008-11-15 | 2009-04-08 | 山西师范大学 | A plant growth-promotiong rhizosphere |
| CN104726383A (en) * | 2015-04-15 | 2015-06-24 | 华南农业大学 | Bacillus amyloliquefaciens JK6 and biological fertilizer and application |
| CN114196585A (en) * | 2021-12-16 | 2022-03-18 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Burkholderia for preventing and treating tomato bacterial wilt and application thereof |
| CN115369062A (en) * | 2022-09-02 | 2022-11-22 | 上海市农业科学院 | Tomato bacterial wilt antagonistic bacterium WJB0802 and application thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101402930A (en) * | 2008-11-15 | 2009-04-08 | 山西师范大学 | A plant growth-promotiong rhizosphere |
| CN104726383A (en) * | 2015-04-15 | 2015-06-24 | 华南农业大学 | Bacillus amyloliquefaciens JK6 and biological fertilizer and application |
| CN114196585A (en) * | 2021-12-16 | 2022-03-18 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Burkholderia for preventing and treating tomato bacterial wilt and application thereof |
| CN115369062A (en) * | 2022-09-02 | 2022-11-22 | 上海市农业科学院 | Tomato bacterial wilt antagonistic bacterium WJB0802 and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| Chryseobacterium rhizoplanae sp. nov., isolated from the rhizoplane environment;Peter Kämpfer等;Antonie van Leeuwenhoek;第107卷(第2期);第533-538页，参见全文 * |
| Competition for iron drives phytopathogen control by natural rhizosphere microbiomes;Shaohua Gu等;Nature microbiology;第5卷(第8期);第1002-1010页，参见全文 * |
| 复合微生物菌剂对番茄青枯病的生防效应王文丽;王文丽等;南京农业大学学报;第45卷(第6期);第1174-1182页，参见全文 * |
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