CN117402796B - Degradable microplastic and straw as well as Raoultella planticola with growth promoting and stress resisting functions and application thereof - Google Patents
Degradable microplastic and straw as well as Raoultella planticola with growth promoting and stress resisting functions and application thereof Download PDFInfo
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- CN117402796B CN117402796B CN202311720520.5A CN202311720520A CN117402796B CN 117402796 B CN117402796 B CN 117402796B CN 202311720520 A CN202311720520 A CN 202311720520A CN 117402796 B CN117402796 B CN 117402796B
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Abstract
The invention belongs to the field of microorganisms, and particularly discloses a plant-grown Raoulet bacterium capable of degrading microplastic and straw and having a growth-promoting stress-resistant function and application thereof; the invention discloses a strain which is classified and named as Raoult fungus (Raoultella planticola) TYQ8, and is preserved in China general microbiological culture Collection center (CGMCC) 27928; the invention discovers that the strain has various characteristics and functions, including the characteristic of resisting the broad-spectrum antibiotic tylosin; can degrade the property of PVC microplastic remained in industrial and agricultural production; also has growth promoting property for dissolving inorganic phosphorus and secreting auxin; the plant growth promoting agent has the functions of degrading agricultural waste straws and the capability of promoting plant growth and assisting plant stress resistance; therefore, the strain has great application prospect.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly discloses a plant-grown Raoult fungus with functions of promoting growth and resisting stress, and application thereof.
Background
The plant rhizosphere beneficial microorganism refers to beneficial bacteria which can be freely living in soil or attached to plant root systems, can promote plant growth and the absorption and utilization of mineral nutrition, and can inhibit harmful organisms. According to the existing research reports, a large number of rhizosphere beneficial microorganism strain resources are screened and identified at present, and the resources are applied to agricultural production, so that the plant growth and development can be improved, the plant diseases and insect pests can be prevented, the application of chemical fertilizers can be reduced, and the method has important significance for realizing sustainable development of agriculture.
At present, antibiotics are mainly widely applied to clinical medicine, livestock breeding and other aspects. Tylosin is one type of macrolide antibiotics, and is widely used in livestock and poultry farming for promoting animal growth and preventing or treating animal diseases. With the wide application and mass production of tylosin, most of residues are untreated and enter soil, water and other environments along with various ways, and high-concentration antibiotics accumulated in the soil environment have certain toxicity to soil organisms, so that the application of beneficial microorganisms in agricultural production is restricted. Thus, beneficial strains with antibiotic resistance would be more suitable for the current production environment.
Plastics and straws are common wastes in agricultural production, the degradation period of the plastics and the straws in soil is long, and the degradation incompletely causes pollution to the soil environment. Microplastic is a tiny particle with a size smaller than 5mm formed by the influence of artificial or natural factors. The microplastic cannot be automatically degraded in the environment, and the microplastic in the environment can be thoroughly degraded in about 400 years, so that the pollution degree of the microplastic to the environment is increased. Numerous studies have shown that microorganisms have the potential to convert various compounds, which under certain specific conditions can carry out biochemical reactions using nondegradable compounds as energy sources, and that there are a large number of data showing that microorganisms exist in nature that can decompose plastic articles. Because the main component lignocellulose of the straw has a complex structure and is difficult to decompose under natural conditions, in the process of utilizing the straw, the straw is generally decomposed by means of the secretion of degrading enzymes by microorganisms. At present, strain resources which simultaneously degrade microplastic and straw are freshly reported.
The action mechanism of plant rhizosphere beneficial microorganisms generally comprises two aspects, on one hand, the rhizosphere beneficial microorganisms can show growth promotion effect by synthesizing substances (such as auxins) with direct action on plant growth and development or changing the morphology of effective elements in soil, and on the other hand, the plant rhizosphere beneficial microorganisms can inhibit or relieve the influence of certain adversity stress (including biotic stress and abiotic stress) on plant growth and development and yield. The research shows that the beneficial plant rhizosphere microorganisms have multiple functions, such as plant growth-promoting microorganisms have biological control effects, and the biological control bacteria can also have growth-promoting stress-resistant effects on host plants, but the microbial resources which have been reported at present and have the growth-promoting and stress-resistant functions are fewer.
The agricultural production environment is complex and changeable, and many single-function microbial agents cannot exert normal functions after being applied to the soil environment, so that the development of the microbial strains with various functions has important significance for the application of the microbial agents in actual agricultural production and the promotion of high-quality and high-efficiency agricultural production.
Disclosure of Invention
Aiming at the problems, the invention discloses a plant-grown Raoult fungus with functions of promoting growth and resisting stress, which can degrade microplastic and straw and application thereof.
The invention comprises the following technical scheme:
on one hand, the invention discloses a degradable microplastic, straw and plant-grown Raoult bacteria with growth-promoting stress-resistant function, wherein the strain is separated from cucumber rhizosphere soil in China agricultural university laboratory, and is classified and named as Raoult bacteria (Raoultella planticola) TYQ8, and is preserved in China general microbiological culture collection center (CGMCC 27928) at 7 months and 17 days in 2023.
The microbiological characteristics of the Raoult fungus TYQ8 are as follows: the single colony of the strain is round, milky and opaque, the colony has the shape and size of 2-3mm, the surface is smooth and moist, the edges are regular, the center is micro-convex, the thallus is in a rod shape, gram staining is negative, and the optimal growth temperature is 28-37 ℃. The ability of TYQ8 to utilize or oxidize preselected different carbon sources was examined using Biolog GenIII microwell plates, thereby constructing a metabolic fingerprint of TYQ8 utilizing different carbon sources. TYQ8 was found to utilize 71 carbon sources to varying degrees and to exhibit insensitive properties to 19 chemical reagents.
The similarity of the strain Raoult plantlet (Raoultella planticola) reached 99.85% by amplifying the 16S rDNA fragment of the strain and performing BLAST homology comparison, and therefore the strain was identified as a strain of Raoultella planticola genus, named Raoult plantlet (Raoultella planticola) TYQ8.
In another aspect, the invention provides a microbial agent comprising Raoultella planticola (Raoultella planticola) TYQ8 as described above.
Further, in the microbial inoculum, the plant growth isThe viable count of Raoult bacteria (Raoultella planticola) TYQ8 is 0.5x10 5 -1x10 9 cfu/g。
Further, in the microbial agent, the preparation method of the microbial agent comprises the steps of picking TYQ8 single colonies, activating in LB liquid medium, and shake culturing until the bacterial liquid OD600 is between 0.6 and 0.8.
In another aspect, the invention discloses the use of the strain or agent described above in at least one of the following:
(1) Degrading PVC microplastic;
(2) Degrading the straw;
(3) Dissolving phosphorus;
(4) Secretion of auxin IAA;
(5) Promoting plant growth;
(6) Improving drought resistance of plants;
(7) Improving the resistance of plants to meloidogyne incognita
(8) Ability to resist the broad spectrum antibiotic tylosin.
In some embodiments, TYQ8 was determined to be resistant to tylosin at a concentration of 100mg/L by antibiotic susceptibility testing, and this concentration may inhibit the growth of other bacteria.
In some embodiments, the PVC microplastic is used as the sole carbon source, and the PVC microplastic and TYQ8 are subjected to shake culture in a liquid carbon-deficient culture medium, and the degradation rate of TYQ8 to the PVC microplastic after 40 days of culture is 1.17%.
Further, in the above application, the straw in (2) is derived from one of cucumber, tomato, pepper and eggplant straw.
In some embodiments, cucumber, tomato, capsicum and eggplant straws are respectively taken as unique carbon sources, and are shake-cultured with TYQ8 in a liquid carbon-deficient culture medium, and the degradation rate of TYQ8 on cucumber, tomato, capsicum and eggplant straws after 14 days of culture is 68.26%, 47.16%, 28.24% and 29.32%, respectively.
Further, in the above application, the plant of (5) is cucumber or tomato.
In some embodiments, TYQ8 was identified to secrete auxin at an IAA secretion concentration of 25.99 μg/mL in the presence of tryptophan by qualitative and quantitative analysis; TYQ8 can dissolve indissoluble phosphate in inorganic phosphorus culture medium, and the phosphorus dissolving capability is stronger. TYQ8 has been shown to have the potential to promote plant growth.
In some embodiments, TYQ8 broth is irrigated to the root soil when the young leaves are flattened, 10mL per seedling. The application leads the plant height and stem thickness of the seedlings to be increased, promotes biomass accumulation and is beneficial to the cultivation of strong seedlings.
Further, in the above application, the plant of (6) or (7) is cucumber.
In some embodiments, when the young leaves are flattened, TYQ8 broth is irrigated in drought treated or root knot nematode infested root enclosure soil, 10mL per seedling. The application improves the drought tolerance of cucumber seedlings and effectively reduces the damage of drought stress to plant cell membranes; in addition, the application reduces the number of single plant root knots of cucumber seedlings, and is beneficial to early prevention and control of root knot nematodes.
Preservation information: the strain is classified and named as Raoult plantlet (Raoultella planticola) TYQ8, and is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at the following preservation address: the collection number is CGMCC27928, and the collection center 2023, 7 months and 17 days of the collection are used for detecting the viable strains.
The invention has the following beneficial effects:
the Raoult fungus TYQ8 used in the invention has the characteristic of drug resistance to a broad-spectrum antibiotic tylosin, and can degrade PVC microplastic remained in industry and agricultural production, and the degradation rate of PVC microplastic particles reaches 1.17% within 40 days. Meanwhile, the strain has the function of degrading agricultural waste straws, vegetable straws are used as the only carbon source to carry out shake culture on the strain, and the degradation rates of the strain on cucumber, tomato, capsicum and eggplant straws respectively reach 68.26%, 47.16%, 28.24% and 29.32% within two weeks. In addition, the strain can play a remarkable beneficial role in promoting plant growth and assisting plant stress resistance. In the aspect of growth promotion, the strain has the growth promotion characteristics of dissolving inorganic phosphorus and secreting auxin, can obviously promote the growth of cucumber and tomato seedlings, and the strong seedling indexes of the cucumber and tomato seedlings are obviously increased by 62.34 percent and 37.70 percent respectively compared with the contrast, thereby being beneficial to the cultivation of strong seedlings. Under drought stress, TYQ8 inoculation can promote chlorophyll synthesis of cucumber seedlings, can maintain water content of overground parts of cucumber seedlings, remarkably reduce ion permeability of leaves, and relieve damage of drought stress to plant cell membranes. Under the stress of the root-knot nematodes, the single plant root-knot number of cucumber seedlings is obviously reduced by 38.63% compared with that of a control by inoculating TYQ8, which proves that TYQ8 can effectively assist the cucumber in resisting the infection of the root-knot nematodes and is beneficial to early prevention and control of the root-knot nematodes.
Drawings
FIG. 1 is a fingerprint of TYQ8 versus different carbon source utilization capacities;
FIG. 2 is a phylogenetic tree diagram of TYQ 8;
FIG. 3 is a graph of the effects of TYQ8 on tylosin resistance;
FIG. 4 is a graph of TYQ8 versus degradation rate of four vegetable stalks;
FIG. 5 is a graph of the phosphorus dissolution profile of TYQ 8;
FIG. 6 is a graph showing the effect of TYQ8 on promoting cucumber seedling growth;
FIG. 7 is a graph showing the effect of TYQ8 on promoting tomato seedling growth;
FIG. 8 is a graph showing the effect of TYQ8 on cucumber seedling growth index;
FIG. 9 is a graph showing the effect of TYQ8 on tomato seedling growth index;
FIG. 10 is a graph showing the effect of TYQ8 on assisting drought tolerance of cucumber seedlings;
FIG. 11 is a graph showing the effect of TYQ8 on physiological index of cucumber seedlings under drought stress;
FIG. 12 is a graph showing root knot comparison of cucumber root systems after TYQ8 inoculation;
fig. 13 is a graph showing the effect of TYQ8 on cucumber physiological index under root-knot nematode stress.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. The following embodiments and features of the embodiments may be combined with each other without conflict.
The detailed description of the embodiments of the invention provided below is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Isolation, purification and identification of Raoult (Raoultella planticola) TYQ8.
1. Isolation and purification of Raoult (Raoultella planticola) TYQ8
The Raoult (Raoultella planticola) TYQ8 strain is isolated from cucumber rhizosphere soil in a laboratory of China agricultural university, and the isolation method is a soil dilution method. The method comprises the following specific steps:
uniformly collecting soil samples from rhizosphere of cucumber plants in China agricultural university laboratory, uniformly collecting 4 parts of the soil samples, 1g each part, uniformly mixing four parts of the soil samples, rapidly placing the mixture into a 15mL centrifuge tube filled with 9mL of sterile distilled water, then placing the centrifuge tube into a shaking table, taking out the test tube after 20 minutes, taking 1mL of soil suspension into a new centrifuge tube, adding 9mL of sterile water, diluting the suspension by 10 times, performing serial gradient dilution by analogy, then sucking 100 mu L of the soil suspension with corresponding dilution times onto an LB solid culture medium plate, uniformly smearing the soil suspension, culturing the soil suspension in a culture box at 28 ℃ for 2 days, and observing growth conditions of colonies at regular time. And (3) when a single colony grows on the flat plate, picking the single colony for three continuous streaking separation and purification.
2. Identification of Raoultella planticola (Raoultella planticola) TYQ8 Strain
(1) Microbiological characteristics
The obtained strain is inoculated on an LB culture medium flat plate, and after 2 days of culture at 28 ℃, single bacterial colony of the strain is round, milky and opaque, the bacterial colony is 2-3mm in shape and size, smooth and moist in surface, regular in edge and protruding in the center, the bacterial colony is in a rod shape, gram staining is negative, and the optimal growth temperature is 28-37 ℃. The Biolog GenIII microwell plate is used for detecting the capability of TYQ8 to utilize or oxidize different carbon sources selected in advance, and the metabolic fingerprint of TYQ8 utilizing the different carbon sources is formed by measuring the absorbance value of the reaction solution at OD590 nm. As a result, as shown in FIG. 1, the higher the OD value in the graph, the higher the utilization of the detection substance, and TYQ8 was found to utilize 71 carbon sources to various degrees and to exhibit insensitivity to 19 chemical reagents.
(2) Molecular biological Properties
Single colony was picked up and shake-cultured in a 1.5mL centrifuge tube containing 1mL LB medium at 28℃and 180rpm/min for 24 hours, and 16s rRNA sequence amplification was performed using the bacterial liquid as a template and primers 27F and 1492R. PCR amplification reaction system is 50 mu L, including 25 mu L2 xTaq enzyme, 1 mu L27F primer, 1 mu L1492R primer, 1 mu L fungus liquid and 22 mu L ddH 2 O. The amplification conditions were: pre-denaturation at 95℃for 3min, denaturation at 94℃for 25s, annealing at 55℃for 25s, extension at 72℃for 1min,32 cycles, extension at 72℃for 5min, and amplification product storage at 4 ℃. The amplified products were identified by 1% agarose gel electrophoresis and the PCR products were sent to Beijing Qingke biosciences, inc. for two-way sequencing.
BLAST homology comparison is carried out on the measurement result, and the result shows that the similarity of the strain and Raoultella planticola (Raoultella planticola) reaches 99.64%, so that the strain is identified as a strain of Raoultella planticola, named Raoultella planticola (Raoultella planticola) TYQ8, and the species with relatively close relativity are screened for phylogenetic analysis based on the 16S rRNA gene sequence, and a phylogenetic tree is shown in figure 2. The strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC27928.
Example 2
Antibiotic susceptibility testing.
Antibiotics with high residual amounts in agricultural soil environments, including gentamicin, tylosin, tetracycline, oxytetracycline and sulfadiazine, are selected and the resistance of TYQ8 to these antibiotics is determined by a paper sheet diffusion method.
1. Preparation of drug sensitive paper sheet
Selecting filter paper sheets (quantitative filter paper) with good quality, punching into discs with the diameter of 6mm by using a puncher, placing the discs into a small bottle, sterilizing the discs at the temperature of 121 ℃ for 15min under high pressure, and placing the discs into a 65 ℃ oven for drying for standby. Accurately preparing antibiotic solutions with different concentrations, wherein the concentration of gentamicin is 50mg/L, the concentration of tylosin, tetracycline, terramycin and sulfadiazine is 100mg/L, respectively soaking the dried filter paper discs in the different antibiotic solutions for 10min after the preparation, taking out, and continuously drying in a 65 ℃ oven.
2. Plate assay
Firstly, TYQ8 single bacteria are selected and activated in LB liquid culture medium, shake culture is carried out until bacterial liquid OD600 is about 0.5, 100 microliter bacterial liquid is absorbed and evenly coated in LB solid culture medium, after blow-drying in an ultra-clean workbench, filter paper discs containing antibiotics with different concentrations are placed in the center of an LB flat plate, after the flat plate is sealed by a sealing film, the flat plate is placed in a 28 ℃ incubator for culturing for 24 hours, whether a bacteriostasis ring is formed around the filter paper discs is observed, if the bacteriostasis ring is formed, the bacterial strain is sensitive to the antibiotics, otherwise, the bacterial strain has drug resistance to the antibiotics. In this example, a strain CK existing in a laboratory is used as a control strain, and experimental results are shown in table 1, and TYQ8 is found to grow around a filter paper disc on which tylosin is placed, and a bacteriostasis zone is formed around other antibiotic filter paper discs (figure 3), which shows that TYQ8 has drug resistance to tylosin at a concentration of 100 mg/L.
TABLE 1 TYQ8 resistance to different antibiotics
Note that: "-" means no drug resistance, "+" means drug resistance.
Example 3
Degradation of PVC microplastic particles by TYQ8
In the embodiment, white spherical PVC micro-plastic particles with the diameter of 3mm are taken as a carbon source, and are subjected to shake culture with TYQ8 in a liquid carbon-deficient culture medium to investigate whether TYQ8 has degradation characteristics on PVC micro-plastic, and the specific operation method is as follows:
1. microplastic pretreatment
Placing PVC microplastic particles into a dry aseptic flat plate in an ultra-clean bench, adding absolute ethyl alcohol, soaking for more than 4 hours, washing with aseptic water, placing into an aseptic dry culture dish, and sterilizing by irradiation of an ultraviolet lamp for 15min.
2. Microplastic degradation rate determination
About 0.5g of pretreated microplastic particles was added to a flask containing 100mL of carbon-deficient liquid selective medium formulated to contain the following ingredients per liter of medium: mgSO (MgSO) 4 ·7H 2 O 0 .4g，K 2 HPO 4 1 .3238g，KH 2 PO 4 0 .3266g，CaCl 2 ·2H 2 O 0 .05g，FeSO 4 ·7H 2 O 0 .01g，MnSO 4 ·H 2 O 2.1mg，ZnSO 4 ·7H 2 O0.25 mg, pH 7.0-7.5. 5mL TYQ8 bacterial liquid activated at night is added into a culture medium, 5mL sterile water is added as a control, a triangular flask is placed in a shaking table with the parameter of 28 ℃ and 180rpm/min to shake for 40 days, micro plastic particles in a container are collected, dried and weighed, and the degradation rate of PVC micro plastic is calculated according to the calculation formula:
the original weight of the PVC microplastic particles in the culture medium is 0.5113g, and the weight of the residual PVC microplastic particles after 40 days of degradation is 0.5053g. The weight loss of the PVC microplastic particles without microbiological treatment was 0.22%. After 40 days of biodegradation with TYQ8, the weight loss of the microplastic particles was 1.17%, which indicates that TYQ8 has the property of degrading PVC microplastic.
Example 4
In the embodiment, four common fruit and vegetable straws including cucumber, tomato, pepper and eggplant straws are taken as materials, and are subjected to shake culture with TYQ8 in a liquid carbon-deficient culture medium to explore whether TYQ8 has degradation characteristics on vegetable straws, and the specific method is as follows:
1. vegetable straw pretreatment
Cutting tomato, capsicum, eggplant and cucumber straw into 3-4cm small sections, placing in an oven for drying, placing in a 250mL triangular flask, sealing with a sealing film, placing in an autoclave, and sterilizing at 121 ℃ for 15 minutes.
2. Vegetable straw degradation rate determination
Respectively adding 5g of straw into a triangular flask containing 125mL of carbon-deficient liquid culture medium, wherein the formula of the carbon-deficient liquid culture medium is the same as that of the embodiment 3, then inoculating 5mL of TYQ8 bacterial liquid activated at night into the culture medium, sealing the triangular flask with a sealing film, placing the triangular flask on a shaking table with the temperature of 28 ℃ and the rotating speed of 180rpm/min for shaking 15, recovering the straw, cleaning, drying and weighing, and respectively calculating the degradation rate of the four straws, wherein the calculation formula is as follows:
according to analysis, TYQ8 can degrade four kinds of vegetable straws to different degrees (figure 4), wherein the degradation rate of the straw for cucumber is 68.26%, and the straw explanation rate for pepper and eggplant is lower, probably because the lignification degree of the two kinds of straws is higher, and the straw is not easy to degrade because of containing more lignin.
Table 2 degradation rate of TYQ8 on four kinds of vegetable stalks
Example 5
Growth promoting effect of TYQ8 on plants
Phosphorus dissolution Properties of TYQ8
The ability of TYQ8 to dissolve poorly soluble phosphorus was initially evaluated using inorganic phosphorus solid medium: sucking 10 mu L of TYQ1 bacterial liquid cultured overnight on the surface of an inorganic phosphorus solid culture medium, sealing by a sealing film, placing the culture medium in a 28 ℃ incubator for culturing for 5 days, and observing whether a phosphorus dissolving ring is formed around a colony.
And then further adopting a molybdenum-antimony anti-colorimetry to quantitatively analyze the phosphorus dissolving capacity of TYQ8. The seed solution of TYQ8 is transferred into an inorganic phosphorus liquid culture medium with an inoculum size of 1 percent, a control group without bacteria is arranged at the same time, shake culture is carried out for 3 days under the condition of 28 ℃ and 180rpm/min, and the supernatant is taken as the liquid to be detected. 10mL of the supernatant was aspirated, 5mL of the molybdenum-antimony anti-developer was added, and after 30 mm of standing at room temperature, the absorbance was measured at 700 nm wavelength.
The result shows that TYQ8 can form a circle of obvious transparent phosphorus-dissolving ring around the colony after being cultured in the inorganic phosphorus solid culture medium for 5 days (figure 5), and the further quantitative analysis result shows that TYQ8 is cultured in the inorganic phosphorus liquid culture medium in a shaking way for 3 days, and the content of soluble phosphorus in the bacterial liquid supernatant is 157.54 mg/L.
Characteristics of tyq8 secretion of auxin
Seed solution of TYQ8 is inoculated into LB liquid culture medium containing filtered sterilized tryptophan in an inoculum size of 1%, so that the final concentration of tryptophan is 500 mu g/mL, meanwhile, a control group without bacteria is arranged, and shake culture is carried out for 3 days under the conditions of 28 ℃ and 180 rpm/min. Taking a fermentation broth, centrifuging at 10000rpm/min for 10min, taking 500 mu L of supernatant in a 10mL centrifuge tube, adding 1mLSalkowaki color developing agent, standing at room temperature for 30min for reaction, observing whether the solution changes color, and if the solution changes into pink, indicating that IAA is generated. The preparation method of the Salkowaki color reagent comprises the following steps: 0.811g FeCl was taken 3 Dissolving in 10mL distilled water to obtain FeCl with concentration of 0.5M 3 The solution was taken and 1mL was added to 49mL of 35% HClO 4 And (5) uniformly mixing the solution to obtain the Salkowaki color reagent.
The ability of TYQ8 to secrete auxin was further quantified by the following method: absorbing 500 mu L of centrifugal supernatant of TYQ8 strain, adding 4 mL IAA color-developing agent, and measuring OD after light-shielding for 30min at room temperature 530 Absorbance at nm with blank mediumIn contrast, the IAA-producing content of TYQ1 strain was calculated using pure IAA as a standard curve.
The results show that TYQ8 can produce IAA using tryptophan and that the amount of IAA produced is 25.99 μg/mL.
3. Potting test
The capability of TYQ8 for promoting plant growth is determined by using cucumber and tomato seedlings as test materials and using two existing strains in a laboratory as control strains through a potting experiment. The specific implementation steps are as follows:
(1) Seed disinfection
Selecting full and nondestructive cucumber and tomato seeds, carrying out surface sterilization, soaking in 75% ethanol for 30s, washing with sterile distilled water for 3 times, soaking the seeds in 3% NaClO solution for 10min, washing with sterile distilled water for 3 times until no reagent remains, soaking the seeds in sterile water for 6h, transferring the seeds into a culture dish filled with wet sterile filter paper, and carrying out germination acceleration in a 28 ℃ incubator.
(2) Strain inoculation
When radicle grows out, the seeds are sown in 50-hole trays filled with sterilizing matrixes (turf: vermiculite=3:1), when cotyledons of seedlings are flattened, TYQ8 fermentation liquor is poured, 10mL of each seedling is poured, sterile water is poured into a control group, and the seedlings are sampled after two weeks of culture.
The preparation method of the sterilizing matrix comprises the steps of mixing turf and vermiculite according to a volume ratio of 3:1, sterilizing at 121 ℃ for 1h under high pressure, and sterilizing at high temperature again after 24 h.
The preparation method of TYQ8 fermentation broth comprises the following steps: TYQ8 was inoculated into LB liquid medium, and the bacterial liquid was cultured at 180rpm/min and 28℃until the OD600 became 0.8.
(3) Analysis of results
As can be seen from tables 3 and 4, the inoculation of TYQ8 bacteria significantly promoted the increase in plant height and stem thickness of cucumber and tomato seedlings and promoted the accumulation of dry matter of cucumber and tomato seedlings (FIGS. 8 and 9), wherein the dry weight of the whole plant of cucumber seedlings was increased by 57.57% as compared to the control and the dry weight of the whole plant of tomato seedlings was increased by 47.20% as compared to the control. Meanwhile, the inoculation of TYQ8 is favorable for the cultivation of strong seedlings of cucumbers and tomatoes, the strong seedling index of cucumber seedlings is remarkably increased by 62.34% compared with the contrast, and the strong seedling index of tomato seedlings is remarkably increased by 37.70% compared with the contrast and is higher than that of two contrast strains. Thus, TYQ8 strain has remarkable growth promoting effect on plants, and the effect is shown in FIG. 6 and FIG. 7.
TABLE 3 influence of TYQ8 on cucumber seedling growth index
TABLE 4 Effect of TYQ8 on tomato seedling growth index
Example 6
TYQ8 assists effect of cucumber seedling drought tolerance
The cucumber seedlings are used as test materials, drought treatment is applied to the cucumber seedlings through a potting experiment, and the effect of TYQ8 on improving drought resistance of the cucumber seedlings is analyzed, wherein the specific implementation scheme is as follows:
(1) Seed disinfection
The specific procedure was as in example 5.
(2) Strain inoculation
The specific procedure was as in example 5.
(3) Applying drought
After TYQ8 is inoculated, after the seedlings are in a leaf-by-core period, drought treatment is started to be applied to the seedlings, and the specific implementation method is as follows: stopping watering the seedlings for one week, and after the leaves of the seedlings are obviously wilted, pouring 10mL of sterile water into each seedling to rehydrate the seedlings, and sampling and counting related indexes after rehydrating for two days.
(4) Analysis of results
As can be seen from table 5, under drought treatment, the dry weight of the whole plant of cucumber seedlings under TYQ8 inoculation was significantly increased compared to the control, i.e. TYQ8 was still able to promote accumulation of cucumber seedling dry matter under drought stress (fig. 11). In addition, TYQ8 inoculation can promote cucumber seedling chlorophyll synthesis, and the chlorophyll content of cucumber leaf after TYQ8 handles is showing and is increasing 10.99% than the contrast, simultaneously, TYQ8 inoculation can maintain cucumber seedling upper portion water content to show the ion permeability of reduction leaf, indicate TYQ inoculation has reduced the injury of leaf cell membrane under the arid treatment. Thus, TYQ8 inoculation improved drought tolerance of cucumber seedlings, and the effect is shown in FIG. 10.
TABLE 5 influence of TYQ8 on physiological index of cucumber seedlings under drought treatment
Example 7
TYQ8 assists cucumber seedlings in resisting root-knot nematodes
The cucumber seedlings are used as test materials, the cucumber seedlings are inoculated with the southern root-knot nematodes through potting experiments, the capability of TYQ8 for assisting the cucumber in resisting the southern root-knot nematodes is analyzed, and the specific implementation scheme is as follows:
(1) Seed disinfection
The specific procedure was as in example 5.
(2) Strain inoculation
The specific procedure was as in example 5.
(3) Inoculating meloidogyne incognita
When the cucumber seedlings are in a one-leaf one-heart period, the seedlings are inoculated with second-instar larvae of the meloidogyne incognita. Two small holes with the depth of about 1cm are marked on the root periphery of cucumber seedlings by using a gun head, a root knot nematode suspension is added into the small holes, and each seedling is inoculated with 300 root knot nematodes. 21 days after the nematode inoculation, the root knot number of the cucumber root system is counted.
(4) Analysis of results
As shown in Table 6, after being treated by TYQ8 and the cucumber seedlings are infected by the root-knot nematodes for 21 days, the biomass of the cucumber seedlings is obviously improved compared with that of a control, meanwhile, the single plant root-knot number of the cucumber seedlings is obviously reduced by 38.63% compared with that of the control by inoculating TYQ8 (figure 13), and the control efficiency is higher than that of the two control strains, so that the TYQ8 can effectively assist the cucumber to resist the infection of the root-knot nematodes (figure 12), and is beneficial to early control of the root-knot nematodes.
TABLE 6 influence of TYQ8 on the physiological index of cucumber seedlings under root-knot nematode infestation
As can be seen from the above examples, the Raoult fungus TYQ8 used in the invention has the property of resisting the broad-spectrum antibiotic tylosin, and can degrade PVC microplastic remained in industry and agricultural production, and the degradation rate of PVC microplastic particles reaches 1.17% within 40 days. Meanwhile, the strain has the function of degrading agricultural waste straws, vegetable straws are used as the only carbon source to carry out shake culture on the strain, and the degradation rates of the strain on cucumber, tomato, capsicum and eggplant straws respectively reach 68.26%, 47.16%, 28.24% and 29.32% within two weeks. In addition, the strain can play a remarkable beneficial role in promoting plant growth and assisting plant stress resistance. In the aspect of growth promotion, the strain has the growth promotion characteristics of dissolving inorganic phosphorus and secreting auxin, can obviously promote the growth of cucumber and tomato seedlings, and the strong seedling indexes of the cucumber and tomato seedlings are obviously increased by 62.34 percent and 37.70 percent respectively compared with the contrast, thereby being beneficial to the cultivation of strong seedlings. Under drought stress, TYQ8 inoculation can promote chlorophyll synthesis of cucumber seedlings, can maintain water content of overground parts of cucumber seedlings, remarkably reduce ion permeability of leaves, and relieve damage of drought stress to plant cell membranes. Under the stress of the root-knot nematodes, the single plant root-knot number of cucumber seedlings is obviously reduced by 38.63% compared with that of a control by inoculating TYQ8, which proves that TYQ8 can effectively assist the cucumber in resisting the infection of the root-knot nematodes and is beneficial to early prevention and control of the root-knot nematodes.
Finally, it should be noted that: the scope of the present invention is not limited to the above embodiments, and those skilled in the art will understand that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions are intended to be included within the scope of the present invention.
Claims (7)
1. Degradable microplastic and straw and plant-grown Raoultella strain with growth-promoting stress-resistant functionRaoultella planticola) The strain TYQ8 is characterized by having a preservation number of CGMCC NO.27928.
2. A microbial agent comprising the Raoultella planticola strain TYQ8 of claim 1.
3. The microbial agent according to claim 2, wherein the living bacterial count of the Raoult fungus strain TYQ8 is 0.5x10 5 -1x10 9 cfu/g。
4. A microbial agent according to claim 2 or 3, characterized in that the preparation method of the microbial agent comprises: and (3) selecting TYQ8 single colonies to activate in an LB liquid culture medium, and performing shake culture and fermentation until the OD600 of the bacterial liquid is between 0.6 and 0.8.
5. Use of a strain of ralstonia planticola TYQ8 according to claim 1 or a microbial agent according to claim 2 for at least one of:
(1) Degrading PVC microplastic;
(2) Degrading the straw;
(3) Dissolving phosphorus;
(4) Promoting plant growth;
(5) Improving drought resistance of plants;
(6) Improving the capability of the plant for resisting the meloidogyne incognita;
(7) Ability to resist the broad spectrum antibiotic tylosin;
the straw in the step (2) is derived from one of cucumber, tomato, capsicum or eggplant straw;
the plant in the step (4) is cucumber or tomato;
the plant in (5) or (6) is cucumber.
6. A method for promoting plant growth comprising the steps of: after cotyledons of seedlings are flattened, irrigating TYQ8 fermentation liquor, and irrigating 5-15mL of each seedling; the plant is cucumber or tomato, and the TYQ8 fermentation broth is produced by fermenting the Raoultella planticola strain TYQ8 according to claim 1.
7. A method for improving stress resistance of cucumber plants, comprising the steps of: when the young seedling leaves are flattened, irrigating TYQ8 fermentation liquid into arid or root-knot nematode infected root surrounding soil, and irrigating 5-15mL of each seedling; the stress resistance is drought resistance or resistance to infection of the meloidogyne incognita of the cucumber plant; the TYQ8 fermentation broth is produced by fermentation of the Raoult planticola strain TYQ8 of claim 1.
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