CN107460151B - Bacillus sphaericus and application thereof in preventing and treating meloidogyne incognita - Google Patents
Bacillus sphaericus and application thereof in preventing and treating meloidogyne incognita Download PDFInfo
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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
- C12R2001/07—Bacillus
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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
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- C—CHEMISTRY; METALLURGY
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- 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 strain is identified as the spherical lysine bacillus (L ysinibacillus sphaericus) with the strain name of RQ11, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, has the preservation number of CGMCC No.14201, has the preservation date of 2017, 05 and 27 days, has good lethal activity on the southern root-knot nematode, and can obviously promote the growth of tomatoes.
Description
Technical Field
The invention belongs to the technical field of microbial biology, and particularly relates to bacillus sphaericus and application thereof in preventing and treating meloidogyne incognita.
Background
Plant parasitic nematodes are one of the important pathogens of plant infectious diseases, are common and serious in the world. Root-knot nematodes are plant parasitic nematodes which seriously harm agricultural production, are mainly parasitic on more than 2000 host species such as vegetables, grain crops, economic crops, fruit trees, ornamental plants, weeds and the like, and are particularly serious in plant damage of tropical, subtropical and temperate zones, and the yield of the plant is generally reduced by about 10% after the disease occurs, and the serious disease is more than 75%. Especially, the incidence area of the meloidogyne incognita is wide, and almost all cultivated plants can be infected. Root-knot nematodes mainly damage the roots of hosts, lateral roots and fibrous roots are the most seriously damaged, and aerial parts sometimes have diseases. The root-knot nematode damages the host root by destroying the direct mechanical damage of the epidermal cells of the root and by using the needle to injure the host and then secreting saliva, so that the normal function of the root cells is destroyed, and finally the root is deformed. After the root system is damaged, nodular root knots with different sizes and shapes can be formed, the root system is white in color and soft in texture, and then the root system is deepened into light brown and has cracks on the surface. The capacity of absorbing and transporting nutrients and water of the root system is reduced after the root system is diseased, so that the diseased yield of the plants is reduced. The overground part of the seriously ill plant is more obvious in performance, the plant is seriously malnourished, thin, weak, wilted, slow in growth, yellow and small in leaf, deformed and small in fruit or shed in flower and fruit, and the whole plant is seriously withered.
Due to the lack of resistant varieties, high multiple breeding indexes and the like, the prevention and treatment of plant parasitic nematodes are mainly based on chemical agents. The chemical nematocide has the disadvantages of high toxicity, environmental pollution, threat to the health of people and livestock and the like, a plurality of effective nematocides such as Shennongdan, carbofuran and the like are forbidden, and people urgently need safe and environment-friendly prevention and treatment measures along with the advocation of green agriculture and sustainable development strategy. The biological control has the advantages of environmental compatibility, environmental protection and the like, and has wide application background. Avermectin is a nematicidal substance separated from a fermentation broth of Streptomyces avermitilis (Streptomyces avermitilis), and is considered to be the best biological agent at present, but some nematodes have already developed resistance to avermectin. In conclusion, stable and efficient root-knot nematode biocontrol agents are still lacking at present.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a tomato endophytic bacterium Lysinibacillus sphaericus strain RQ11 and application thereof as tomato root-knot nematode biocontrol bacteria. Experiments prove that the Lysinibacillus sphaericus strain RQ11 has a good effect of preventing and treating tomato root-knot nematodes. The method has important significance for further reducing the pesticide consumption and promoting the development of the vegetable industry of China towards the green pollution-free direction.
The invention provides a bacillus sphaericus lysine which is identified as bacillus sphaericus (L ysin bacillus sphaericus), the strain name is RQ11, the bacillus sphaericus is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of No.1 North road of West Chen of the sunward district of Beijing City, China institute of microbiology, the preservation number is CGMCC No.14201, and the preservation date is 27 months in 2017 and 05 months.
The lysine bacillus sphaericus is obtained by separating and screening from the inside of tomato roots.
The lysine bacillus sphaericus RQ11 strain provided by the invention is a spore-producing bacterium, and can be preserved by any technical method for preserving bacillus provided by the published documents in the field.
The strain Lysinibacillus sphaericus RQ11 strain has a control effect on Meloidogyne incognita. In order to realize the effects of storage, transportation and field application of the strain, the use dosage form of the strain is further researched.
Therefore, the invention also provides a microbial inoculum containing the lysine bacillus sphaericus.
The preparation method of the microbial inoculum comprises the following steps:
(1) culturing the Lysinibacillus sphaericus strain RQ11 to logarithmic phase as culture solution by a conventional method;
(2) the microbial inoculum is prepared from a culture solution by a conventional freeze drying method, and the microbial inoculum is prepared by the specific preparation method that the culture solution is adsorbed by skimmed milk powder, 50-80 parts of the culture solution is adsorbed by 5-10 parts of skimmed milk powder, 1-2 parts of NaCl and 1-3 parts of peptone together in volume proportion, and the microbial inoculum is obtained by freeze drying.
The thallus content in the microbial inoculum is 1.00 × 108～1.00×109CFU/g, and the microbial inoculum is stored in a dry state.
The invention also provides application of the spherical lysine bacillus in preventing and treating meloidogyne incognita, the spherical lysine bacillus strain is fermented to obtain a culture solution, and then the culture solution is centrifuged to obtain a fermentation supernatant, or a bacterial suspension prepared from the strain, so that the strain can be used for preventing and treating meloidogyne incognita.
The invention also provides application of the microbial inoculum for preventing and treating meloidogyne incognita, which comprises the steps of activating and drying the microbial inoculum by warm water at the temperature of 30-50 ℃ before use, adding warm water 50-100 parts by volume into 1-2 parts of the microbial inoculum, uniformly stirring, standing for 5-8 hours, and applying a root-damaging hole application method (namely digging a pit with the depth of 3-6 cm from the root of a tomato seedling to apply uniformly-vibrated bacterial suspension) with the use amount of 10-30 m L for each seedling.
The invention finally provides the spherical lysine bacillus and application of the microbial inoculum prepared by the spherical lysine bacillus to tomato growth promotion.
The endophytic bacteria exist in a host body, the living environment is stable, the endophytic bacteria are not easily interfered by the external environment, the endophytic bacteria existing in roots and the root-knot nematode have the same ecological niche, and the unique ecological advantages make the endophytic bacteria become a research hotspot of biological control resources. Therefore, the endophytic bacteria bred in tomato roots can effectively prevent and control meloidogyne incognita and can also obviously promote the growth of tomatoes.
The technical effects are as follows: compared with the prior art, the obtained spherical lysine bacillus RQ11 strain is an endophytic bacterium bred in tomato roots, is not easily influenced by external environment, and overcomes the influence of biocontrol bacteria on the environment changed in soil. Because the RQ11 has higher application value in the aspect of preventing and controlling the harm of the meloidogyne incognita, especially the bacterium is tomato endophytic bacterium, the bacterium can continuously act on the meloidogyne incognita, and the defect of low control effect or instability caused by the fact that general biocontrol bacteria cannot be bred in roots is overcome.
Detailed description of the invention
The present invention is illustrated by way of example, but is not limited to the following examples.
The plant endophytic bacteria related to the invention are separated from various plant bodies of suburban farmland of Nanjing and stored in a microbiological laboratory of the institute of Life sciences of Nanjing university, the tomatoes are purchased from the market, the variety is Shanghai tomato institute cooperative 903 red tomato (L ycopencicleson) and Meloidogyne incognita, which is offered by professor Lihongmei Mei of Nanjing university of agriculture, and the tomatoes are extended to propagate.
The materials, reagents, instruments and the like used in the present invention are well known in the art, and other experimental devices well known in the art can be applied to the following embodiments of the present invention.
Isolation and screening of the B.lysimachiae RQ11 strain:
the method comprises the steps of collecting tomato roots, sterilizing the surfaces of underground parts of normally growing tomatoes by cutting the tomato roots into blocks, utilizing a conventional method as long as bacteria on the surfaces are killed by the conventional method, placing the sterilized root tissue blocks into a tissue homogenizer sterilized at high temperature for grinding, pouring the ground and crushed tomato tissue blocks into a conventional bacterial culture medium for dilution, placing the ground and crushed tomato tissue blocks into a 28-DEG C incubator for culture, selecting all separated bacterial strains, respectively co-culturing all separated bacterial strains and second-instar larvae of the southern root-knot nematode through bacterial suspension and fermentation supernatant to obtain bacterial strains with nematicidal activity, observing the bacterial morphology of each bacterial strain with primary activity under a microscope, numbering the rod-shaped bacterial strains are RG1, RG2 and RG3 … …, numbering the rod-shaped bacterial strains are RQ1, RQ2 and RQ3 … …, further artificially inoculating the root-knot nematode strains of the tomato with the active strains with higher control effect through a method of artificially inoculating the root-knot nematode and inoculating bacterial suspensions of the strains of the tomato with higher control effect, wherein the tomato root-knot-nematode-promoting bacterial strains are obtained by a method of artificially inoculating tomato root-inoculating strain with a mixed strain after inoculating soil and soil, inoculating tomato root-nematode-1, and inoculating tomato root-10-growing-nematode-growing-root-nematode-root-nematode-root-.
Example 1: in vitro screening of biocontrol bacteria of meloidogyne incognita-initial screening of hydrolytic gelatin activity
The 59 plant endophytic bacteria obtained by separation are streaked and activated on an NA plate, the culture is carried out for 48h at 28 ℃, a single colony is selected and inoculated in a 5m L NB culture medium (beef extract 0.3% (w/v), peptone 1% (w/v), NaCl 0.5% (w/v), pH 7.0-7.2, sterilization is carried out for 20min at 121 ℃), seed liquid is prepared by culturing for 12h at 28 ℃ at 180rpm, 5m L seed liquid is sucked and inoculated in a 50m L NB culture medium, the seed liquid is cultured for 24h at 180rpm and 28 ℃, the bacterial culture liquid is centrifuged for 20min at 5000rpm to remove supernatant, the thalli is washed twice by using sterile distilled water and resuspended, the concentration of the bacterial suspension is diluted to 1.34 × 10 by using a dilution coating method8CFU/m L gradient dilution of the bacterial suspension, spreading on gelatin medium (gelatin 0.5% (w/v), K)2HPO40.05％ (w/v)、MgSO4·7H2O0.02% (w/v), agar 1.5% (w/v)) and incubated at 28 ℃ for 48 h. Strains producing a transparent circle around the colony, i.e., a hydrolysis circle, were selected, the hydrolysis circle diameter and the colony diameter were measured and the ratio of the hydrolysis circle diameter to the colony diameter (HC value) was calculated, and each treatment was repeated three times. And (3) test results: among the 59 endogenous bacteria, 19 bacterial colonies surrounded a clear circle, in which the H value of the B.sphaericus RQ11 strain was 2.07.
Example 2: in vitro screening-nematocide activity screening of biocontrol bacteria of meloidogyne incognita
The 19 bacterial strains with hydrolyzed gelatin obtained in example 1 were cultured on NB medium for 60h, after centrifugation, the supernatants were collected and sterilized again by filtration through a 0.22 μm microporous membrane twice, the supernatants were diluted 5-fold with sterile distilled water, 200 μ L supernatants were pipetted into 24-well plates, 10 μ L nematode suspension (containing about 50 juveniles of two-instar larvae of meloidogyne incognita) was added to each well, cultured at 25 ℃ for 12h, 10 μ L% NaCl was added to each well to stimulate nematodes, survival of nematodes was observed under a microscope (40X), the live nematodes were bent flexibly, the bodies were stiff after stimulation of the dead nematodes were counted, the number of live and dead nematodes were counted and the corrected mortality of the fermentation supernatant to the larvae of two-instar nematodes was calculated 3 times per treatment, NB culture broth diluted 5-fold was used as a control, the nematode suspension was pipetted, 200 μ L strain suspension of the strain was pipetted into 24-well plates, 10 μ L% were added to each well, cultured 24 h.nacl was used for 3 times per treatment, the total number of the nematode was observed under a microscope, the corrected mortality of the strain was found to be equal to the corrected mortality of the strain, and the strain was found to be the corrected mortality of the strain, wherein the corrected mortality was found to be equal to 100% in 5% of.
Example 3: potted plant control effect determination of biocontrol bacteria of meloidogyne incognita
The method comprises the steps of selecting tomato seeds with consistent seed filling, sowing the tomato seeds into a seedling pot filled with sterilized organic matters, culturing in a greenhouse with the temperature of 28 ℃/20 ℃, the illumination of 14 h/darkness of 10h (day/night) and the humidity of 70%, transplanting the tomato seeds into a flowerpot filled with 1000g of nutrient soil after 2 true leaves are grown, mixing the nutrient soil with the sterilized healthy loam and the organic matters 1: 1(v: v), cutting 3-5 cm deep ditches at a position 1cm away from roots on one side of the seedling by a knife, injecting bacterial suspension to the side of the cut roots, inoculating 10m L sterile distilled water into negative Control (CK), diluting 10m L by one hundred times to be positive control, pricking 2-3 cm small holes at positions 2cm away from the roots on two sides of the tomato after one week, adding 1m L into each hole by a pipette to each hole to obtain negative Control (CK), adding 1m L sterile distilled water into each hole to obtain positive control, performing positive control on each hole, performing negative control on each hole punching, and performing check on each hole, wherein the number of the Root, the Root.
Example 4: growth promotion of tomato by Lysinibacillus sphaericus RQ11 strain
The tomato seeds are surface-sterilized by 0.5 percent NaClO for 2min, washed twice by sterile distilled water and respectively soaked in 4 strains of biocontrol bacteria suspension for 8 h. The seeds are sowed in flowerpots after being dried in the shade, 3 seeds are sowed in each flowerpot, and 5 pots are treated in each flowerpot. After emergence, the roots were drenched with the bacterial suspension and re-inoculated with sterile distilled water as Control (CK), and each treatment was repeated three times. After the culture is carried out for 60 days conventionally in a greenhouse, the fresh weight, the plant height, the fresh weight and the root length of the overground part are investigated, and the dry weight of the overground part are weighed after the overground part is dried to constant weight at 60 ℃. And (3) test results: the lysinibacillus sphaericus RQ11 strain can remarkably promote the fresh weight and stem length of the overground part of the tomato, and compared with a control, the fresh weight and stem length of the overground part of the tomato are respectively increased by 68.91% and 22.70%.
Example 5: colonization dynamics of Lysinibacillus sphaericus RQ11 strain on tomato root
The strain of Lysinibacillus sphaericus RQ11 is subjected to resistance marking by streptomycin sulfate so that the strain can normally grow in NA culture medium containing 300 mu g/m L streptomycin sulfate, and the strain is inoculated with 10m L1.34 × 10 after the tomato is transplanted for one week8CFU/m L bacterial suspension, investigating every 5d after inoculation, randomly selecting 3 tomato seedlings for each treatment, weighing fresh weight 0.2g for each root, sterilizing the surface with 0.5% NaClO and 70% ethanol for 5min, washing with sterile distilled water for 5 times, adding 1m L sterile distilled water into a sterilized homogenizer, fully grinding the tomato roots, sucking 1m L homogenate, and performing 0, 10, 100 and 1000 times of homogenizationDiluting, coating the homogenate obtained after 200 mu L dilution on NA plates, coating 3 plates on each dilution gradient, counting the number of bacterial colonies after culturing for 48h at 28 ℃, calculating the number of 4 bacteria in tomato roots according to the number, collecting rhizosphere soil of each tomato, naturally drying for 24h at room temperature, sieving the dried soil with a sieve of 1mm, weighing 10g of the soil and shaking and uniformly mixing with 90m L sterile distilled water, standing for 30min, sucking 1m L supernatant for 10, 100, 1000 and 10000 times of dilution, coating the supernatant obtained after 200 mu L dilution on the NA plates, coating 3 plates on each dilution gradient of the supernatant, counting the number of bacterial colonies after culturing for 48h at 28 ℃, calculating the number of RQ11 strains in the rhizosphere soil according to the number, and keeping the colonization of the Lysinium sphaericum RQ11 strain in the tomato roots at 4.29 × 104CFU/m L, stable maintenance in rhizosphere soil of 4.66 × 106CFU/mL。
Example 6: disease nursery control effect determination of spherical lysine bacillus RQ11 microbial inoculum
The method comprises the following steps of fully and uniformly mixing a 50m L globular lysine bacillus RQ11 strain culture solution, 8g skimmed milk powder, 1g NaCl and 1g peptone, putting the mixture into a-80 ℃ refrigerator for 2h to completely freeze the liquid, then putting the mixture into a freeze dryer to process the mixture for 48h, taking the mixture out, crushing a massive microbial inoculum, and measuring the amount of live bacteria in the microbial inoculum by using a gradient dilution coating method, transplanting tomato seedlings with two true leaves into a disease nursery (3m × m), inoculating 10m 567 g/m L to injured roots of each tomato seedling in an experimental group after one week, inoculating 10m L g/m L empty Carriers (CK) to injured roots of each tomato seedling in a negative control group, inoculating 10m L to abamectin diluted by one hundred times, inoculating 1000 southern Root knot-extracted eggs to roots of each tomato seedling after one week, separating the treated 20 tomato seedlings by using a ClO Root-Root nematode internal Root-egg separation method, and reducing the soil knot of rhizoid nematodes by using a Root-wheat Root knot nematode centrifugal test method, wherein the soil knot of the soil inoculation method is that the soil knot of the soil is 23% of the soil.
Example 7: identification of Lysinibacillus sphaericus RQ11 Strain
Molecular identification selects gene sequence of 16S rRNA, an Ezup column type genome DNA extraction kit (Shanghai ' S) is used for extracting genome DNA of bacteria, amplification of 16S rRNA gene sequence adopts common primers 27F and 1492R of bacteria, a forward primer is 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID No: 1), a reverse primer is 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID No: 2), a reaction system (25 mu L) is 20 × Taq enzyme mix (Takara)12.5 mu ×, a forward primer is 1 mu ×, a reverse primer is 1 mu L template 1 mu L, double distilled water is 9.5 mu L, reaction conditions are pre-denatured at 94 ℃, denatured at 94 ℃ for 0.5min, annealed at 52 ℃ for 0.5min, extended at 72 ℃ for 1min, 30 cycles, sequencing at 72 ℃ for 1min, heat treatment of 16S gene obtained by using a heat treatment method, the strain obtained by using a Huaqinbie bacteria strain heat treatment, the strain is subjected to a colony morphology observation, gram staining and spore staining, molecular identification selects gene sequence of 16S rRNA, amplification of 16S rRNA gene sequence, amplification adopts common primers 27F and 1492R, a forward primer 5'-AGAGTTTGATCCTGGCTCAG-3' mu × (SEQ ID No.1, reverse primer No. 3 template 3. mu. 80. template, double distilled water 9.5 mu. L. and a reaction system, reaction conditions are 94 ℃ for pre-denatured for obtaining a strain, the sequence of a strain, the strain is subjected to a strain, the strain obtained by using a strain by a strain of a strain of a strain of yellow strain of a strain of 8 RQ 7 strain of 8.
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Claims (8)
1. The bacillus sphaericus bred in tomato roots is characterized by being identified as bacillus sphaericus (L ysinibacillus sphaericus), the strain name is RQ11, the bacillus sphaericus is preserved in China general microbiological culture Collection center, the preservation number is CGMCC No.14201, and the preservation date is 2017, 05 and 27 days.
2. The bacillus sphaericus lysine according to claim 1, wherein the bacillus sphaericus lysine is obtained by separating and screening from the inside of tomato roots.
3. A microbial agent comprising the lysine bacillus sphaericus of claim 1 or 2.
4. Use of the lysinibacillus sphaericus strain of claim 1 or 2 for the control of meloidogyne incognita.
5. The use of claim 4, wherein the L.sphaericus strain is fermented to obtain a culture solution, and the culture solution is centrifuged to obtain a fermentation supernatant, or a bacterial suspension prepared from the strain, and can be used for controlling Meloidogyne incognita.
6. Use of the inoculant according to claim 3 for the control of meloidogyne incognita.
7. Use of the lysinibacillus sphaericus according to claim 1 or 2 to promote the growth of tomatoes.
8. Use of the microbial inoculant of claim 3 for promoting tomato growth.
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| CN108676741B (en) * | 2018-04-26 | 2022-04-22 | 南京农业大学 | Functional composite microorganism seedling culture substrate and preparation method and application thereof |
| CN108676762B (en) * | 2018-07-11 | 2021-08-27 | 中国农业大学 | Lysine bacillus for preventing and controlling plant nematodes and application thereof |
| CN110055188A (en) * | 2019-03-07 | 2019-07-26 | 南京师范大学 | One plant of Paenibacillus polymyxa XW4 for producing bacteriostatic peptide and its separation screening and application |
| CN111763646B (en) * | 2020-07-24 | 2021-11-30 | 湖南省植物保护研究所 | Long lysine bacillus, microbial agent, biocontrol agent, and preparation method and application thereof |
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