CN111607535A - Application of cycloserine and beneficial bacteria in cooperation for preventing and controlling soil-borne bacterial wilt of tomatoes - Google Patents
Application of cycloserine and beneficial bacteria in cooperation for preventing and controlling soil-borne bacterial wilt of tomatoes Download PDFInfo
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- CN111607535A CN111607535A CN202010403070.7A CN202010403070A CN111607535A CN 111607535 A CN111607535 A CN 111607535A CN 202010403070 A CN202010403070 A CN 202010403070A CN 111607535 A CN111607535 A CN 111607535A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- 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
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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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- 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
Abstract
The invention discloses an application of cycloserine and beneficial bacteria in cooperation for preventing and controlling tomato soil-borne bacterial wilt, which adopts a root irrigation method to apply the cycloserine and the beneficial bacteria to soil; wherein, the beneficial bacterium is strain NJQL-A6, is classified and named as Ralstonia pilettii, is preserved in China general microbiological culture Collection center in 9.26.2012, and has the preservation unit address: the preservation number of the strain is CGMCC No. 6628. According to the invention, the capability of beneficial bacteria for inhibiting the soil-borne bacterial wilt can be enhanced in a resource regulation and control mode, the beneficial bacteria and the cycloserine are applied at the same time, and the cycloserine is taken as a resource substance, so that the growth of the beneficial bacteria can be promoted, the inhibiting capability of the beneficial bacteria on the bacterial wilt can be remarkably improved, the morbidity of the tomato bacterial wilt can be reduced, and the tomato soil-borne diseases can be prevented and controlled.
Description
Technical Field
The invention belongs to the technical field of microorganisms, relates to application of resources matched with beneficial bacteria to prevention and control of tomato bacterial wilt, and particularly relates to application of cycloserine and beneficial bacteria NJQL-A6 matched with prevention and control of tomato soil-borne bacterial wilt.
Background
Tomato bacterial wilt is a soil-borne disease caused by Laurella sp (Ralstonia solanacearum for short), is frequently generated in the eastern and southern areas of China, can cause irreversible wilting and death of tomatoes, causes serious yield reduction of the tomatoes, and restricts the development of agricultural economy. By applying pesticides or fumigating and other traditional modes, the soil-borne ralstonia solanacearum can be killed in a short time, and the occurrence of diseases is effectively slowed down. However, the method can kill indigenous microorganisms in the soil and reduce the diversity or abundance of soil species.
At present, the beneficial bacteria are relatively green and sustainable measures for preventing and controlling the tomato bacterial wilt, can inhibit the growth of soil-borne ralstonia solanacearum, and reduce the occurrence of diseases to a certain extent. In the process of screening beneficial bacteria and verifying the bacteriostatic effect of the beneficial bacteria, people often use a culture medium with rich nutrition, and the beneficial bacteria can be massively proliferated or show good bacteriostatic ability. The variety and the quantity of resources in the soil are relatively limited, and after beneficial bacteria are applied to the soil, enough and proper resources cannot be obtained, so that the growth is slowed down, even the survival in the soil environment cannot be realized, and the biocontrol efficiency is inevitably reduced.
Therefore, a resource capable of promoting the growth of beneficial bacteria, providing a resource environment suitable for the growth of beneficial bacteria in soil and enhancing the capability of inhibiting the soil-borne ralstonia solanacearum needs to be found.
Disclosure of Invention
The invention aims to solve the problems that nutrients in soil are limited, and beneficial bacteria are difficult to multiply in a large amount in the soil and exert control effect when applied to control tomato soil-borne diseases. The inventor finds that: in an indoor microporous plate system, cycloserine can promote the growth of beneficial bacteria NJQL-A6 and can also obviously improve the inhibition capacity of the beneficial bacteria on ralstonia solanacearum; in a greenhouse pot experiment, cycloserine and beneficial bacteria NJQL-A6 are applied to soil by a root irrigation method at the same time, so that the disease index of tomato soil-borne bacterial wilt can be reduced. The beneficial bacteria and the cycloserine are used as resource substances, so that the growth of the beneficial bacteria can be promoted, the inhibiting capability of the beneficial bacteria on the ralstonia solanacearum can be obviously improved, the morbidity of the tomato ralstonia solanacearum is reduced, and the tomato soil-borne diseases are prevented and controlled.
The purpose of the invention is realized by the following technical scheme:
the application of cycloserine and beneficial bacteria in cooperation for preventing and controlling soil-borne bacterial wilt of tomatoes; wherein, the beneficial bacterium is strain NJQL-A6, is classified and named as Ralstonia pilettii, is preserved in China general microbiological culture Collection center in 9.26.2012, and has the preservation unit address: the preservation number of the strain is CGMCC No. 6628.
The strain NJQL-A6 is specifically disclosed in an invention patent application filed by the applicant on 12/19/2012, and has the name of: a microbial plant vaccine for controlling tomato bacterial wilt, which has the following application numbers: 201210466659.7.
as a preferred technical scheme for the application of the cycloserine and the beneficial bacteria in cooperation for preventing and controlling the tomato soil-borne bacterial wilt, the invention adopts a root irrigation method, and simultaneously applies the cycloserine and the beneficial bacteria into soil, wherein the final concentration of the cycloserine is 0.1-1 mmol/kg of soil, and the final concentration of the beneficial bacteria is 1 × 107～1×109CFU/g soil.
The invention also aims to provide a method for preventing and controlling the soil-borne bacterial wilt of tomatoes by matching cycloserine and beneficial bacteria, which comprises the following steps: transplanting 3-5 true-leaf tomato seedlings for 7-10 days, and irrigatingAdding cycloserine and beneficial bacterium suspension into soil by root method, wherein the final concentration of cycloserine is 0.1-1 mmol/kg soil, and the final concentration of beneficial bacterium NJQL-A6 is 107～109CFU/g soil.
The concentration of the beneficial bacterium suspension is 1 × 107～1×109CFU/mL。
The beneficial bacterium suspension is prepared by activating strain NJQL-A6 with NA solid culture medium, culturing NJQL-A6 single colony in NA liquid culture medium at 30 deg.C and 170rpm overnight, centrifuging fresh bacterium liquid at 4500rpm for 5min at normal temperature, collecting bacterium, washing bacterium with sterile normal saline, removing culture medium, and regulating bacterium concentration with sterile normal saline to 1 × 107～1×109CFU/mL to obtain NJQL-A6 bacterial suspension.
Drawings
FIG. 1 is a graph showing the effect of cycloserine on the growth of beneficial bacterium NJQL-A6; wherein, FIG. 1A shows the effect of cycloserine on the biomass of beneficial bacterium NJQL-A648 h; FIG. 1B shows the effect of cycloserine on the maximum growth rate of the beneficial bacterium NJQL-A6.
FIG. 2 shows the effect of cycloserine on the growth inhibition of the soil-borne ralstonia solanacearum by the beneficial bacterium NJQL-A6.
FIG. 3 shows the effect of cycloserine on the disease index of tomato soil-borne bacterial wilt.
Biological material preservation information
The strain NJQL-A6 is classified and named as Ralstonia pickettii, is deposited in China general microbiological culture Collection center in 9.2012 and 26.s.i.: the preservation number of the strain is CGMCC No. 6628.
Detailed Description
The technical solution of the present invention will be further explained with reference to the specific embodiments.
Example 1
Indoor effect of cycloserine in promoting growth of beneficial bacteria
NA liquid medium: 10g of glucose, 5g of peptone, 3g of beef extract, 0.5g of yeast extract and 1000mL of deionized water, adjusting the pH value to 7.2-7.4, and carrying out autoclaving at 115 ℃ for 30 min.
The NA solid culture medium is NA liquid culture medium added with 2-3% (w/w) agar.
The strain NJQL-A6 is classified as Ralstonia pickettii with the preservation number of CGMCC No. 6628.
Streaking NJQL-A6 preserved in glycerol on NA solid culture medium, culturing at 30 deg.C for 2 days, selecting single colony on the plate, transferring to NA liquid culture medium, culturing at 30 deg.C and 170rpm overnight, centrifuging at 4500rpm for 5min at room temperature, collecting thallus, washing thallus with sterile physiological saline (0.85% NaCl) for 3 times, removing culture medium, and regulating the concentration of the thallus to 1 × 10 with sterile physiological saline8CFU/mL to obtain bacterial suspension for later use.
The test is carried out by setting 2 treatments, 1) and a cyclic serine group, wherein cyclic serine and beneficial bacteria NJQL-A6 are added, 2) and a control group, only the beneficial bacteria NJQL-A6 are added, cyclic serine is replaced by equal volume of water, the culture medium is a 10% NA culture medium (the NA liquid culture medium is diluted by 10 times by sterile water), the final concentration of the cyclic serine is 0.1mM, and the inoculation amount of the beneficial bacteria NJQL-A6 is 1% (the final concentration is 1 × 10)6CFU/mL), shake-cultured at 30 ℃ and 170rpm for 48h, and OD is measured on a microplate reader600And observing the growth condition of the beneficial bacterium NJQL-A6.
The effect of cycloserine on the growth of beneficial bacteria is shown in fig. 1 and table 1, and the results show that cycloserine can significantly promote the increase of biomass and growth rate of beneficial bacteria NJQL-A6 (P <0.05) compared with the control group.
TABLE 1 influence of cycloserine on biomass and growth rate of beneficial bacterium NJQL-A6
Note: different letters in the same column in the table indicate that the difference reaches a significance level of 0.05, as follows.
Example 2
Cyclic serine enhanced indoor effect of beneficial bacteria on inhibiting soil-borne ralstonia solanacearum
Materials: ralstonia solanacearum QL-Rs1115 isolated from the kylin town of Nanjing[1]Its red fluorescent proteinThe marked strain RS-RFP is a model invasive pathogen of the invention, the strain is obtained by transferring a red fluorescent protein gene mCherry into ralstonia solanacearum QL-Rs1115 through a plasmid pYC12, and the strain can be plated on a NA culture medium of 30 mu g/mL gentamicin[2]Growing upwards and is named as RS-RFP. The beneficial bacterium NJQL-A6 is selected from the Town of kylin of Nanjing, and can inhibit the growth of Ralstonia solanacearum QL-Rs 1115.
Marking beneficial bacterium NJQL-A6 preserved in glycerol on NA solid culture medium, culturing the plate in 30 deg.C incubator for 2d until single colony appears, selecting single colony on the plate, transferring to NA liquid culture medium, culturing overnight at 30 deg.C and 170rpm, taking fresh bacterium liquid 4500rpm, centrifuging at room temperature for 5min, collecting thallus, washing thallus with sterile normal saline (0.85% NaCl) for 3 times, removing culture medium, and adjusting bacterium liquid concentration to 1 × 10 with sterile normal saline8CFU/mL to obtain beneficial bacteria suspension for later use.
Resuscitating Ralstonia solanacearum RS-RFP on an NA solid culture medium containing 30 mu g/mL gentamicin, placing a plate in an incubator at 30 ℃ for 2 days until a single colony appears, selecting the single colony on the plate, transferring the single colony to an NA liquid culture medium, culturing at 30 ℃ and 170rpm overnight, taking fresh bacterial liquid 4500rpm, centrifuging at normal temperature for 5min, collecting the bacterial, washing the bacterial for 3 times by using sterile normal saline (0.85 percent NaCl), removing the culture medium, and finally adjusting the bacterial concentration to 1 × 10 by using the sterile normal saline8And (5) CFU/mL to obtain a pseudomonas solanacearum suspension for later use.
The experiment set up 2 treatments: 1) cycloserine group: adding cycloserine, beneficial bacteria and ralstonia solanacearum; 2) control group: beneficial bacteria and ralstonia solanacearum, and cyclic serine is replaced by equal volume of water. The culture medium is 10% NA culture medium (NA liquid culture medium is diluted by 10 times by sterile water), the final concentration of cycloserine is 0.1mM, the inoculation amounts of beneficial bacterium NJQL-A6 and ralstonia solanacearum RS-RFP are both 1%, shaking culture is carried out for 48h at 30 ℃ and 170rpm, and OD is measured on an enzyme-linked immunosorbent assay600And red fluorescence intensity mCherry (emission: 587nm, absorption: 610nm), the relative growth of Ralstonia solanacearum was calculated: log10 (mCherry/OD)600)。
The results are shown in fig. 2 and table 2, and compared with the control group, cycloserine can remarkably enhance the ability of the beneficial bacterium NJQL-A6 to inhibit ralstonia solanacearum (P < 0.05).
TABLE 2 influence of cycloserine on the inhibition of ralstonia solanacearum by the beneficial bacterium NJQL-A6
Example 3
Pot culture effect of cycloserine for enhancing beneficial bacteria to inhibit soil-borne ralstonia solanacearum
Inoculating beneficial bacteria NJQL-A6 into NA liquid culture medium, shake culturing at 30 deg.C and 170rpm for 48h, taking fresh bacterial liquid 4500rpm, centrifuging at room temperature for 5min, collecting thallus, washing thallus with sterile normal saline (0.85% NaCl) for 3 times, removing culture medium, and adjusting bacterial liquid concentration to 1 × 10 with sterile normal saline8And (5) about CFU/mL to obtain beneficial bacterium suspension for later use.
Inoculating Ralstonia solanacearum QL-Rs1115 to NA liquid culture medium, shake culturing at 30 deg.C and 170rpm for 48h, centrifuging fresh bacterial liquid at 4500rpm for 5min at normal temperature, collecting thallus, washing thallus with sterile physiological saline (0.85% NaCl) for 3 times, removing culture medium, and adjusting bacterial liquid concentration to 1 × 10 with sterile physiological saline8And (5) about CFU/mL to obtain a ralstonia solanacearum suspension for later use.
Tomato variety: red dwarf tomatoes.
The tomato seeds are disinfected on the surface, placed on a sterile flat plate padded with sterile deionized water soaked filter paper and subjected to germination acceleration for 2d at the temperature of 30 ℃. Seeds with uniform germination are planted into a seedling raising plate with 50 holes and a sterilization substrate. And after 3 true leaves of the tomatoes grow, transplanting the seedlings with the same growth vigor into a 6-hole seedling raising tray.
The experiment set up 2 treatments: 1) and cycloserine group: after the seedlings are transplanted for 7 days, the suspension of cycloserine and beneficial bacteria NJQL-A6 is uniformly poured near the roots of the tomatoes by adopting a root irrigation method, the final concentration of the cycloserine is 0.1mmol/kg of soil, and the final concentration of the beneficial bacteria NJQL-A6 is 107CFU/g soil. After 7 days, the bacterial suspension of Ralstonia solanacearum is inoculated by the root irrigation method, and the final concentration is 106CFU/g soil. 2) And a control group: after the seedlings are transplanted for 7 days, the suspension of beneficial bacteria NJQL-A6 is inoculated by a root irrigation method, and cycloserine is replaced by equal volume of water, so that beneficial effects are achievedThe final concentration of the strain NJQL-A6 is 107CFU/g soil. After 7 days, the bacterial suspension of Ralstonia solanacearum is inoculated by the root irrigation method, and the final concentration is 106CFU/g soil. Once tomato has disease symptoms after the ralstonia solanacearum is inoculated, the disease condition is recorded.
The disease grade of the plants is 5 grades[3]0-no disease symptoms, 1-25% of plant parts show disease symptoms, 2-26-50% of plant parts show disease symptoms, 3-51-75% of plant parts show disease symptoms, 4-76-100% of plant parts show disease symptoms or the plants are dead, the disease index is used for representing the disease condition, and the calculation formula is that the plant disease index is (∑ (the number of disease plants in each stage is × corresponding stage)/(the total number of plants investigated is × highest level value)]。
The results are shown in fig. 3 and table 3, and the results show that the disease index of the tomato soil-borne bacterial wilt can be remarkably reduced (P is less than 0.05) after the cycloserine is added, and the disease degree of the bacterial wilt can be reduced.
TABLE 3 Effect of cycloserine on beneficial bacterium NJQL-A6 for inhibiting tomato soil-borne bacterial wilt
Reference documents:
[1]、Wei,Z.,Yang,X.M.,Yin,S.X.,Shen,Q.R.,Ran,W.&Xu,Y.C..Efficacy ofBacillus-fortified organic fertiliser in controlling bacterial wilt of tomatoin the field.Appl.Soil Ecol.,2011,48,152-159.
[2]、Tan,S.Y.,Gu,Y.,Yang,C.L.,Dong,Y.,Mei,X.L.,Shen,Q.R.etal..Bacillusamyloliquefaciens T-5may prevent Ralstonia solanacearum infectionthrough competitive exclusion.Biol.Fertil.Soils,2015,52,341-351.
[3]、Kempe,J.,and L.Sequeira.Biogical control of bacterial wilt ofpotatoes:attempts to induce resistance by treating tubers with bacteria.Plantdisease,1983,67:499–503.
Claims (7)
1. the application of cycloserine and beneficial bacteria in cooperation for preventing and controlling soil-borne bacterial wilt of tomatoes; wherein, the beneficial bacterium is strain NJQL-A6, is classified and named as Ralstonia pilettii, is preserved in China general microbiological culture Collection center in 9.26.2012, and has the preservation unit address: the preservation number of the strain is CGMCC No. 6628.
2. The use of claim 1, wherein the cycloserine and the beneficial bacteria are applied to the soil by root irrigation.
3. Use according to claim 1, characterized in that the final concentration of cycloserine is 0.1-1 mmol/kg of soil.
4. The use according to claim 1, wherein the final concentration of beneficial bacteria is 1 × 107～1×109CFU/g soil.
5. A method for preventing and controlling tomato soil-borne bacterial wilt by combining cycloserine and beneficial bacteria is characterized by comprising the following steps: after the tomato seedlings are transplanted for 7-10 days, adding cycloserine and beneficial bacterium suspension into soil by a root irrigation method, wherein the final concentration of the cycloserine is 0.1-1 mmol/kg of soil, and the final concentration of beneficial bacterium NJQL-A6 is 107～109CFU/g soil.
6. The method of claim 5, wherein the concentration of the suspension of beneficial bacteria is 1 × 107～1×109CFU/mL。
7. The method of claim 5, wherein the beneficial bacteria suspension is prepared by activating NJQL-A6 strain with NA solid medium, selecting NJQL-A6 single colony in NA liquid medium, culturing overnight at 30 deg.C and 170rpm, centrifuging at 4500rpm for 5min, collecting thallus, washing thallus with sterile physiological saline, removing medium, and adjusting the concentration of the thallus to 1 × 10 with sterile physiological saline7～1×109CFU/mL to obtain NJQL-A6 bacterial suspension.
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