CN117586931B - Achromobacter xylosoxidans IVF-WK240 and application thereof - Google Patents

Achromobacter xylosoxidans IVF-WK240 and application thereof Download PDF

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CN117586931B
CN117586931B CN202410082698.XA CN202410082698A CN117586931B CN 117586931 B CN117586931 B CN 117586931B CN 202410082698 A CN202410082698 A CN 202410082698A CN 117586931 B CN117586931 B CN 117586931B
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ivf
achromobacter xylosoxidans
cucumber
increasing
cucumber plant
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CN117586931A (en
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杨学勇
秦宇轩
王昆
朱雪莹
张菡
郑盈盈
包翔宇
万丽
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Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, 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
    • C12N1/20Bacteria; Culture media therefor
    • C12N1/205Bacterial isolates
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01NPRESERVATION 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
    • A01N63/00Biocides, 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
    • A01N63/20Bacteria; Substances produced thereby or obtained therefrom
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01PBIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P21/00Plant growth regulators
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, 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
    • C12N1/20Bacteria; Culture media therefor
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12RINDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00Microorganisms ; Processes using microorganisms
    • C12R2001/01Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/025Achromobacter
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02WCLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W30/00Technologies for solid waste management
    • Y02W30/40Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse

Abstract

The invention relates to the technical field of microorganisms, in particular to Achromobacter xylosoxidans IVF-WK240 and application thereof. The invention provides a Achromobacter xylosoxidansAchromobacter xylosoxidans) IVF-WK240, the preservation number is CGMCC No.28311, the achromobacter xylosoxidans IVF-WK240 or a microbial preparation thereof has the effect of promoting plant growth, and the crop yield is increased, including increasing the plant height of plants, increasing the stem thickness of plants, increasing the fruit bearing number of plants, increasing the female flower rate of plants, increasing the leaf area of plants, increasing the dry weight of plants, increasing the fresh weight of plants, increasing the root length of plants and the like.

Description

Achromobacter xylosoxidans IVF-WK240 and application thereof
Technical Field
The invention relates to the technical field of microorganisms, in particular to Achromobacter xylosoxidans IVF-WK240 and application thereof.
Background
CucumberCucumis sativusLThe plant is a common vegetable and fruit, also called cucumber and green melon, and is widely planted in temperate and tropical areas. The cucumber is warm and not cold-resistant, and has high nutritional value, so that the cucumber becomes one of the essential vegetables in modern families, and the demand is large. However, the yield of cucumber is not as good as expected, due to the planting environment, fertilizers and human factors.
It is widely recognized in the art that biological measures not only alleviate stress in agriculture, but also are of great importance in promoting benign development of sustainable agriculture. For example, rhizosphere growth-promoting bacteria not only colonize plant rhizosphere, promote plant growth, promote mineral nutrient absorption and utilization, but also inhibit harmful organisms, but the effect of different rhizosphere growth-promoting bacteria is not the same due to the influence of the source thereof. However, there is no report on the fact that Achromobacter xylosoxidans can significantly promote plant growth, especially increase in cucumber yield.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide the achromobacter xylosoxidans IVF-WK240 and the application thereof, wherein the achromobacter xylosoxidans IVF-WK240 has the advantages of promoting plant growth and remarkably improving plant yield, and especially can improve the stress resistance of cucumbers which are intolerant to low temperature and/or weak light and promote the cucumbers to grow under the low temperature and weak light condition.
For this purpose, the invention provides the following technical scheme:
achromobacter xylosoxidans @Achromobacter xylosoxidans) IVF-WK240 is preserved in China general microbiological culture Collection center (CGMCC) with a preservation address of 1 Xidelu No. 3 of North Chen West Lu of the Korean area of Beijing, 3 of China academy of sciences microbiological study, and with a preservation date of 2023, 8 months and 31 days and a preservation number of 28311.
The Achromobacter xylosoxidans IVF-WK240 is separated from greenhouse cucumber rhizosphere soil of a south oral pilot experiment base of China academy of agricultural sciences; the colony characteristics of the Achromobacter xylosoxidans IVF-WK240 on the LB solid medium are as follows: smooth surface, neat edge, non-transparency and wet adhesion; the thalli are in a blunt round shape, are arranged singly and have no spores under a microscope.
A microbial preparation comprising said Achromobacter xylosoxidans IVF-WK240, a culture thereof, a fermentation product thereof and/or at least one metabolite thereof.
Optionally, in the microbial preparation, the viable count of the Achromobacter xylosoxidans IVF-WK240 is more than or equal to 10 7 cfu/mL。
A method for preparing the microbial preparation, comprising the following steps: the Achromobacter xylosoxidans IVF-WK240 is subjected to expansion culture.
Optionally, in the step of expanding culture, the inoculum size of the Achromobacter xylosoxidans IVF-WK240 accounts for 1% -10% of the total volume of the expanding culture medium, and more preferably 1%; the time of the expansion culture is preferably 6-8 hours, more preferably 8 hours; the rotation speed of the expansion culture is preferably 200-220 rpm, more preferably 220rpm; the temperature of the expansion culture is preferably 28-37 ℃, more preferably 37 ℃; the culture medium for the expansion culture is preferably a TSB liquid culture medium; the components and the dosage of the TSB liquid culture medium are not particularly required, the technology well known in the art is adopted, and the OD is measured at the wavelength of 600nm by an ultraviolet spectrophotometer after the expansion culture 600 Bacterial liquid with value of 1.
After the expansion culture, the invention preferably aims at OD 600 Performing first centrifugation, washing and second centrifugation on bacterial liquid with a value of 1; the speed of the first centrifugation is preferably 3000-6000 g, more preferably 5000g; the time of the first centrifugation is preferably 5-15 min, more preferably 10min; the solvent for the rinsing is preferably sterile water; the rinsed object is preferably a first centrifuged sediment; the dosage of the sterile water and the flushing mode have no special requirements, and the technology well known in the art is adopted; the speed and time of the second centrifugation are the same as those of the first centrifugation, and are not described in detail herein; after the above procedure, a precipitate containing the strain IVF-WK240 was obtained.
After the second centrifugation, the sediment after the second centrifugation is preferably resuspended; the resuspended reagent is preferablyIs sterile water; the sterile water is used in an amount to obtain an OD at a wavelength of 600nm using an ultraviolet spectrophotometer 600 Achromobacter xylosoxidans IVF-WK240 with a value of 0.02; thus, the effective viable count is more than or equal to 10 7 cfu/mL of Achromobacter xylosoxidans IVF-WK240 bacterial liquid.
Obtaining OD 600 After the Achromobacter xylosoxidans IVF-WK240 bacterial liquid with the value of 0.02, plants can be directly applied on one hand, and the Achromobacter xylosoxidans IVF-WK240 fermentation liquid can be prepared on the other hand. The preparation method of the achromobacter xylosoxidans IVF-WK240 fermentation liquid preferably comprises the following steps: for OD 600 Aerobic fermentation is carried out on the bacterial solution of the Achromobacter xylosoxidans IVF-WK240 with the value of 0.02, and fermentation liquor of the Achromobacter xylosoxidans IVF-WK240 is obtained.
The culture medium for aerobic fermentation of the invention takes water as a solvent and also preferably comprises the following components (weight percent): corn flour 0.5-2.0%, glucose 0.1-1.0%, bean cake powder 0.5-3%, fish meal 0.1-1.5%, calcium carbonate 0.31-1.69%, ammonium sulfate 0.05-0.15%, dipotassium hydrogen phosphate 0.01-0.06%, magnesium sulfate 0.01-0.06% and manganese sulfate 0.01-0.06%; during aerobic fermentation, the volume ratio of the inoculum size of the Achromobacter xylosoxidans IVF-WK240 bacterial liquid to the aerobic fermentation culture medium is preferably 1-2: 10 to 30, more preferably 1:10; the aerobic fermentation time is preferably 10-16 h, more preferably 12h; the rotating speed of the aerobic fermentation is preferably 200-220 rpm, more preferably 220rpm; the aerobic fermentation temperature is preferably 28-37 ℃, more preferably 37 ℃, so that fermentation liquor of the Achromobacter xylosoxidans IVF-WK240 can be obtained.
After the fermentation broth is obtained, the fermentation broth is preferably diluted; the diluted solvent is preferably sterile water; the volume ratio of the sterile water to the aerobic fermentation broth is (10-30): (1-2), more preferably 10:1, whereby the diluted fermentation broth can be directly applied to plants.
The Achromobacter xylosoxidans IVF-WK240, the microbial preparation or the microbial preparation prepared by the preparation method has the application of any one of the following:
(1) Use in promoting plant growth;
(2) Use in the preparation of a plant growth regulator;
(3) Use in the preparation of a fertilizer;
optionally, the promoting plant growth comprises at least one of increasing plant height, increasing stem thickness, increasing fruit bearing number, increasing female flower rate, increasing leaf area, increasing dry weight, increasing fresh weight, and increasing root length;
optionally, the promoting plant growth comprises enhancing stress resistance of the plant under low temperature and/or low light conditions; optionally, the weak light includes natural light or artificial light;
optionally, the low temperature range is 10 ℃ to 20 ℃; optionally, the temperature range in the daytime is more than 14 ℃ and less than or equal to 20 ℃, and the temperature range in the night is more than or equal to 10 ℃ and less than or equal to 14 ℃; the time length of the day is 10-12 hours, and the time length of the night is 14-12 hours; the day and night temperatures can be simulated in a climatic chamber.
Optionally, the condition range of weak light is 3000 lx-10000 lx, and the illumination time range is 6 h-8 h.
Optionally, in the low-temperature low-light condition, the temperature is kept to be more than 14 ℃ and less than or equal to 20 ℃ within 10-12 hours, the illumination time is 6-8 hours during the period, the illumination intensity is 3000 lx-10000 lx, the rest time is in a dark state, and then the dark state is kept within 14-12 hours, wherein the temperature is not less than 10 ℃ and not more than 14 ℃ alternately.
Optionally, the plant comprises cucumberCucumis sativusL.);
Optionally, the cucumber is a cucumber which is not resistant to low temperature and/or weak light;
optionally, the cucumber is de Ruite 89, jinchun No. four or Zhongnong Dada No. 11.
A plant growth regulator or fertilizer comprises the Achromobacter xylosoxidans IVF-WK240, the microbial preparation or the microbial preparation prepared by the preparation method of the microbial preparation.
A method for promoting plant growth, comprising applying the Achromobacter xylosoxidans IVF-WK240, the microbial preparation prepared by the preparation method of the microbial preparation or the plant growth regulator or fertilizer; the application mode comprises root irrigation;
when the bacterial liquid of the Achromobacter xylosoxidans IVF-WK240 is applied, the application amount of the bacterial liquid is 10-100 mL per strain each time, and the application frequency is 1-2 times per week; alternatively, the application amount of the bacterial liquid is 50 mL/strain each time, and the application frequency is 1 week; optionally, the effective viable count of the Achromobacter xylosoxidans IVF-WK240 in the bacterial liquid is more than or equal to 10 7 cfu/mL;
And/or when the fermentation diluent of the Achromobacter xylosoxidans IVF-WK240 is applied, the application amount of the fermentation diluent is 70-120 mL per strain each time, and the application frequency is 1-2 times per week; alternatively, the fermentation diluent is applied at a rate of 100 mL/strain each time, with a frequency of 1 week;
and/or, the period of application comprises the entire growth phase of the plant, preferred periods of application comprise the seedling, flowering and/or fruiting phase;
and/or, the plant comprises cucumber.
The technical scheme of the invention has the following advantages:
1. the invention provides a Achromobacter xylosoxidansAchromobacter xylosoxidans) IVF-WK240, the preservation number is CGMCC No.28311, the achromobacter xylosoxidans IVF-WK240 or a microbial preparation thereof has the effect of promoting plant growth, including increasing plant height, increasing stem thickness, increasing fruit bearing number, increasing female flower rate, increasing leaf area, increasing dry weight, increasing fresh weight and/or increasing root length of plants, and also has the effect of promoting plant growth under low temperature and/or low light conditions, enhancing stress resistance of plants to low temperature and/or low light, and especially can promote growth of cucumbers which are intolerant of low temperature and/or low light. Therefore, the Achromobacter xylosoxidans IVF-WK240 or the microbial preparation thereof can promote the growth of plants and increaseAfter the crop yield is applied to the cucumber, compared with a control group with other fertilizers, the yield per mu of the cucumber treated by applying the strain is increased by 8.7%, and the fruit setting per mu is increased by 11.7%.
Furthermore, as the achromobacter xylosoxidans IVF-WK240 or the microbial preparation thereof has the effects of promoting plant growth and improving crop yield, the achromobacter xylosoxidans IVF-WK240 or the microbial preparation thereof can be used for preparing plant growth regulators, fertilizers and other products.
2. According to the method for promoting plant growth, the yield of crops can be remarkably improved by applying the achromobacter xylosoxidans IVF-WK240, the microbial preparation thereof or the plant growth regulator or fertilizer prepared by the achromobacter xylosoxidans IVF-WK240, and compared with a control group only applying other fertilizers, the yield of the treated group cucumber per mu by applying the strain is increased by 8.7% and the fruit setting number per mu is increased by 11.7% after the method is applied to the cucumber.
Biological preservation information
Achromobacter xylosoxidans provided by the inventionAchromobacter xylosoxidans) IVF-WK240, china general microbiological culture Collection center (CGMCC) is preserved in 8 months and 31 days of 2023, the preservation address is North Celu No.1, 3 of the Korean area of Beijing, and the post code is 100101 and the preservation number is CGMCC No.28311.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a colony morphology of Achromobacter xylosoxidans IVF-WK240 in the culture medium in example 1;
FIG. 2 is a colony morphology of Achromobacter xylosoxidans IVF-WK240 in example 1 under a microscope;
FIG. 3 is a phylogenetic analysis of Achromobacter xylosoxidans IVF-WK240 in example 1;
FIG. 4 shows the effect of cucumber growth under different treatments in Experimental example 1;
FIG. 5 is a bar graph of different measurement indexes of cucumber under different treatments in Experimental example 1; in the figures, in contrast to the sterility control,represents p < 0.05, < -> Represents p < 0.01, < ->Represents p < 0.001, < ->Represents p < 0.0001;
FIG. 6 shows the effect of the treatments of control and IVF-240 of Experimental example 2 on cucumber fruit vigor;
FIG. 7 shows the effect of different treatments of the fertilizer IVF-240, the fertilizer control, the fertilizer Y1 and the fertilizer Y2 in experimental example 2 on the yield of cucumber per mu;
FIG. 8 shows the effect of different treatments of the fertilizer IVF-240, the fertilizer control, the fertilizer Y1 and the fertilizer Y2 in experimental example 2 on the cucumber fruit setting number;
FIG. 9 shows the effect of different treatments of IVF-240, control, Y1 and Y2 on cucumber acre yield in Experimental example 2;
FIG. 10 shows the effect of different treatments of IVF-240, control, Y1 and Y2 on cucumber fruit set number in Experimental example 2;
FIG. 11 shows the effect of different treatments of IVF-240, control for fattening, Y1 and Y2 in Experimental example 2 on cucumber acre yield;
FIG. 12 shows the effect of different treatments of IVF-240, control for fattening, Y1 and Y2 in Experimental example 2 on cucumber fruit set.
Detailed Description
The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.
The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.
EXAMPLE 1 isolation and characterization of Achromobacter xylosoxidans IVF-WK240
1. Achromobacter xylosoxidans IVF-WK240 source
And (3) separating and culturing strains in rhizosphere samples of different cucumbers on an LB solid medium by a streaking method, wherein the cucumbers are collected in a southern mouth pilot-scale experiment base greenhouse of China academy of agricultural sciences.
2. Morphological identification
The inoculating loop was used to pick a small amount of target strain, streaked on LB solid medium, cultured at 28℃for 20 hours, then observed the morphology, shape, size, edge, surface, shape of ridges, transparency, color of colony and medium, etc. (see FIG. 1 for results), cultured for 24 hours, and then subjected to microscopic examination, and the morphology of the cells was recorded (see FIG. 2 for results).
As can be seen from FIGS. 1 and 2, after 24. 24h culture in LB medium, the colony form of the target strain is small, round, smooth, flat or convex, clean in edge, opaque, wet-sticky, and gray. Gram staining is negative, and under an optical microscope, the thalli are blunt and round, are arranged singly and have no spores.
3. Molecular biological identification
(1) DNA extraction
Extracting genomic DNA of the target strain IVF-WK240 by adopting a TIANamp Bacteria DNA kit kit; the kit was purchased from Tiangen Biochemical technologies (Beijing) Inc.
(2) PCR amplification and sequencing
Using the genome DNA extracted in the step (1) as a template, and adopting a universal primer to carry out PCR amplification:
an upstream primer: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 1);
a downstream primer: 5'-AAGGAGGTGATCCAGCCGCA-3' (SEQ ID NO. 2);
the PCR reaction system comprises template DNA of 1 mu L, upstream primer 1 mu L of 10 mu mol/L, downstream primer 1 mu L of 10 mu mol/L, nouzan 2X Taq PCR Master Mix 12.5.5 mu L and ddH (polymerase chain reaction) calculated by 25 mu L 2 O is complemented to 25 mu L;
the PCR reaction procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, renaturation at 55℃for 1min; extending at 72 ℃ for 2min for 30 cycles; extending at 72 ℃ for 10min;
the PCR products were detected by agarose gel electrophoresis under standard conditions, and then sent to the Beijing Rui Boxing family biological Co., ltd for sequencing, and the sequencing results were processed and aligned to GenBank.
(3) Construction of phylogenetic tree
The sequencing results were aligned in NCBI to preliminarily determine species, standard sequences of each known Achromobacter genus were obtained from GenBank, aligned using Readseqn software, sequence similarity was calculated using MEGA5.01 and subjected to phylogenetic analysis (see FIG. 3 for the results).
As can be seen from FIG. 3, the target strain Achromobacter xylosoxidans IVF-WK240 and Achromobacter xylosoxidans are at the same development level, and have the closest relationship with the physiological and biochemical identification result, so that the target strain is Achromobacter xylosoxidans, named Achromobacter xylosoxidans IVF-WK240, and then preserved in China general microbiological culture Collection center (CGMCC) at 8/31 of 2023 with the preservation address of Beijing Kogyo North Xielu No.1, national institute of microbiology, post code 100101 and the preservation number of CGMCC No.28311.
EXAMPLE 2 Achromobacter xylosoxidans IVF-WK240 bacterial liquid
The embodiment provides a preparation method of an achromobacter xylosoxidans IVF-WK240 bacterial liquid, which comprises the following steps:
performing expansion culture on Achromobacter xylosoxidans IVF-WK240 in Tryptic Soy Broth (TSB) liquid culture medium, wherein the inoculation amount is 1% v/v; the rotation speed is 220rpm; the temperature is 37 ℃; the OD is measured at a wavelength of 600nm by an ultraviolet spectrophotometer for about 6 hours 600 1, centrifuging the bacterial liquid at 5000g for 10min, cleaning the bacterial liquid with 25mL of sterile water, centrifuging at 5000g for 10min, pouring out the waste liquid, re-suspending the bacterial liquid with sterile water, and measuring OD at 600nm by ultraviolet spectrophotometer 600 A bacterial liquid of 0.02, and the effective viable count is more than or equal to 10 7 cfu/mL of Achromobacter xylosoxidans IVF-WK240 bacterial liquid.
EXAMPLE 3 Achromobacter xylosoxidans IVF-WK240 fermentation dilution
The embodiment provides a preparation method of a fermentation diluent of Achromobacter xylosoxidans IVF-WK240, which comprises the following steps:
(1) Preparing a fermentation medium, which comprises the following components in percentage by weight: corn flour 1%, glucose 0.5%, bean cake powder 1.5%, fish meal 0.8%, calcium carbonate 1%, ammonium sulfate 0.1%, dipotassium hydrogen phosphate 0.03%, magnesium sulfate 0.03%, manganese sulfate 0.03%, and the balance water.
(2) Fermentation: achromobacter xylosoxidans IVF-WK240 strain (OD 600 0.02) 20mL, 200mL of the fermentation medium (500 mL Erlenmeyer flask as a culture vessel) added to the step (1) was cultured overnight (12 h) at 37℃and 220 rpm. Diluting the cultured fermentation broth with 2L of sterile water (the dilution ratio is the volume ratio of the fermentation broth to the sterile water is 1:10), and the effective viable count is not less than 10 7 cfu/mL。
Example 4A method for promoting cucumber growth
The embodiment provides a method for promoting cucumber growth in a plug, which comprises the following steps:
(1) Pre-treatment of seeds: soaking cucumber seeds (low-temperature-intolerant weak light variety DeRuite 89) in warm water at 50 ℃ for 30min, then soaking the cucumber seeds in 75% alcohol for 15s, washing the cucumber seeds with sterile water for 1 time, soaking the cucumber seeds in 6% sodium hypochlorite for 15min, shaking the cucumber seeds at random, pouring out the sodium hypochlorite, and washing the cucumber seeds with sterile water for 6-8 times. Then placing the seeds into a culture dish with the thickness of 100 multiplied by 100mm, adding 2 layers of filter paper at the bottom, adding a proper amount of sterile water, and then placing the seeds into a dark incubator with the temperature of 30 ℃ for germination; selecting seeds with consistent growth vigor for standby after germination;
(2) Sowing the germinated seeds with consistent growth vigor selected in the step (1) in 8-hole trays (the substrate is sterilized Flora gard turfy soil seedling substrate), sowing 1 seed per hole, culturing in a climatic chamber (the temperature is 20 ℃ and 10 ℃ each day, the illumination is carried out for 8 hours each day, the soil humidity is 80%), applying the achromobacter xylosojae IVF-WK240 bacterial solution in the example 2 by a root irrigation method after the cucumber is in a seedling stage, wherein the application amount is 50mL per seed, and the application frequency is 1 week until the fruit setting stage is finished.
Example 5
This example differs from example 4 in that the amount applied was 100 mL/strain per application, with a frequency of 2 weeks.
Example 6
This example differs from example 4 in that the amount of each application was 10 mL/strain, and the frequency of application was 1 week.
Example 7
The embodiment provides a method for promoting cucumber growth in a greenhouse, which comprises the following steps:
(1) And planting cucumber (low-temperature weak light intolerant variety DeRuite 89) by adopting a local planting method and combining the area of an experimental plot.
(2) And when the cucumber enters a seedling stage, applying the achromobacter xylosoxidans IVF-WK240 fermentation diluent in the example 3 by a root irrigation method, wherein the application amount of each time is 100 mL/plant, and the application frequency is once every 1 week until the fruit setting stage is finished.
Example 8
This example differs from example 7 in that the amount of each application was 70 mL/strain, and the frequency of application was 1 week.
Example 9
This example differs from example 7 in that the amount applied was 120 mL/strain and the frequency of application was 2 weeks.
Experimental example 1 growth promoting effect of Achromobacter xylosoxidans IVF-WK240 bacterial liquid in plug tray on cucumber seedling stage
1. Experimental materials: the commercial cucumber variety cultivated in the north is Deruite 89 which is a low-temperature-resistant weak light variety and is used as a test material.
2. Experimental methods and groupings:
(1) Pre-treatment of seeds: soaking cucumber seeds (low-temperature-intolerant weak light variety DeRuite 89) in warm water at 50 ℃ for 30min, then soaking the cucumber seeds in 75% alcohol for 15s, washing the cucumber seeds with sterile water for 1 time, soaking the cucumber seeds in 6% sodium hypochlorite for 15min, shaking the cucumber seeds at random, pouring out the sodium hypochlorite, and washing the cucumber seeds with sterile water for 6-8 times. Then placing the seeds into a culture dish with the thickness of 100 multiplied by 100mm, adding 2 layers of filter paper at the bottom, adding a proper amount of sterile water, and then placing the seeds into a dark incubator with the temperature of 30 ℃ for germination; selecting seeds with consistent growth vigor for standby after germination;
(2) And (3) fertilizing: sowing the seeds which are selected to sprout and grow consistently in the step (1) in 8-hole trays (the substrate is sterilized Flora gard grass peat seedling substrate), sowing 1 seed per hole, performing culture in a climatic chamber (the day and night temperature is respectively 20 ℃ (10 hours) and 10 ℃ (14 hours), alternately performing the culture, the daily illumination time is 6 hours, the illumination intensity is 3000 lx-10000 lx, the rest time is in a dark state, the soil humidity is 80%), applying the leucobacter xylosoxidans IVF-WK240 bacterial liquid of the example 2 by a root irrigation method after cucumber is in a seedling stage, the application rate is 50mL per seed each time, the application frequency is 1 week, simultaneously setting a blank control group, and applying an equal volume of sterile water to the blank control group. Under the condition of not additionally applying other fertilizers, normal management is carried out according to planting requirements, wherein culture conditions (day and night temperature is respectively 20 ℃ (kept for 10 hours) and 10 ℃ (kept for 14 hours) are alternately carried out, the daily white light illumination time is 6 hours, the illumination intensity is 3000 lx-10000 lx, the rest time is in a dark state, and the soil humidity is 80%).
3. Results and analysis:
when the cucumber grows to three leaves at the seedling age and one heart (when the standard is obvious difference), the indexes of plant height, stem thickness, leaf area, dry weight, fresh weight, root length and the like of cucumber seedlings in the experimental group and the blank control group are measured, and the seedling growth promotion effect of the tested strain is evaluated. The data statistical method is T-test. The results are shown in Table 1, FIG. 4 are graphs of cucumber growth under different treatments, and FIG. 5 is a bar graph of different measurement indexes of cucumber under different treatments. The specific measurement criteria are as follows:
plant height: the height from the surface of the plug substrate to the growth point is taken as the reference;
stem thickness: the lower part of the cotyledon node is tightly abutted, and the vernier caliper is used for measuring;
leaf area: taking a picture after the maximum true She Pingzhan, and carrying out statistics by using image J software;
root length: after cleaning the root, measuring the length of the main root by a vernier caliper;
fresh weight: washing the whole plant, spin-drying the excessive moisture, and weighing by an electronic balance;
dry weight: deactivating enzyme for 15min at 105 ℃ and drying at 70 ℃ for more than 48h until the weight is constant; wherein, the plant height and stem thickness are analyzed and counted for 8 repeated data, and 3 of the leaf area, root length, whole plant fresh weight and whole plant dry weight are randomly selected from 8 repeated data for counting and analyzing.
TABLE 1 Effect of different treatments on cucumber growth
As can be seen from table 1, fig. 4 and fig. 5 (the blank control and the aseptic control can be used interchangeably, the IVF-WK240 and the bacteria-adding treatment can be used interchangeably), the average plant height of the blank control group is 7.63±0.62, the average stem thickness is 3.89±0.25, the average root length is 10.40±1.04, the average fresh weight of the whole plant is 6.86±1.69, the average leaf area is 111.58 ±11.81, the average dry weight of the whole plant is 0.60±0.19, cucumber seedlings added with the IVF-WK240 strain are 9.55±0.69, and the growth promoting efficiency of the plant height is increased by 25% compared with that of the blank control group; the average value of the stem thickness is 4.95+/-0.42, and compared with a blank control group, the stem thickness growth promoting efficiency is increased by 27%; the root length average value is 12.33+/-0.76, and compared with a blank control group, the root length growth promotion efficiency is increased by 18%; the average value of the fresh weight of the whole plant is 8.94+/-0.79, and compared with a blank control group, the growth promoting efficiency of the whole plant fresh weight is increased by 30%; the average value of the leaf area is 161.10 +/-21.20, and compared with a blank control group, the growth promoting efficiency of the leaf area is increased by 44.4%; the average dry weight of the whole plant is 0.76+/-0.09, and the growth promoting efficiency of the whole plant is increased by 27 percent relative to that of a blank control group. See table 2 below. Therefore, under the low-temperature weak light condition, the IVF-WK240 strain can improve the stress resistance of the cucumber which is not resistant to the low-temperature weak light and promote the growth of the cucumber seedlings which are not resistant to the low-temperature weak light.
TABLE 2 mean and growth rate of the effect of different treatments on cucumber growth
Experimental example 2 growth promoting effect of Achromobacter xylosoxidans IVF-WK240 on cucumber in greenhouse
1. Experimental materials: the commercial cucumber variety cultivated in the north is Deruite 89 which is a low-temperature-resistant weak light variety and is used as a test material.
2. Experimental methods and groupings:
2.1 grouping
Treatment group 1 (fatliquoring IVF-240): achromobacter xylosoxidans IVF-WK240 fermentation dilutions of example 3 were applied with the additional application of a nitrogen-phosphorus-potassium mass ratio of 20:10:20 (available from smini corporation) and the application amount was determined according to the instructions of the water-soluble compound fertilizer.
Treatment group 2 (fatliquoring control): only the mass ratio of nitrogen, phosphorus and potassium is 20:10:20 (available from smini corporation) and the application amount was determined according to the instructions of the water-soluble compound fertilizer.
Treatment group 3 (fattening Y1): and (5) applying the commercial bacterial liquid Y1 to irrigate seedlings of the cucumbers in the cell 3. Wherein the commercial bacterial liquid Y1 is a bacterial cell purchased from Mu En (Guangzhou) biotechnology Co. Simultaneously, the mass ratio of additionally applied nitrogen, phosphorus and potassium is 20:10:20 (available from smini corporation) and the application amount was determined according to the instructions of the water-soluble compound fertilizer.
Treatment group 4 (fattened Y2): and (5) applying commercial bacterial liquid Y2 to irrigate seedlings of the cucumbers in the cell 4. Wherein the commercial bacterial liquid Y2 is 360 bacterial housekeeper purchased from Zhengzhou chemical industry products Co. Simultaneously, the mass ratio of additionally applied nitrogen, phosphorus and potassium is 20:10:20 (available from smini corporation) and the application amount was determined according to the instructions of the water-soluble compound fertilizer.
Treatment group 5 (IVF-240): achromobacter xylosoxidans IVF-WK240 fermentation dilutions in example 3 were applied without additional application of nitrogen-phosphorus-potassium in a mass ratio of 20:10: 20.
Treatment group 6 (control): no substance is applied.
Treatment group 7 (Y1): and (5) applying the commercial bacterial liquid Y1 to irrigate seedlings of the cucumbers in the cell 7. Wherein, the commercial bacterial liquid Y1 is a bacterial nutrient purchased from Mu En (Guangzhou) biotechnology limited company, and simultaneously the mass ratio of nitrogen, phosphorus and potassium is 20:10: 20.
Treatment group 8 (Y2): and (5) applying commercial bacterial liquid Y2 to irrigate seedlings of the cucumbers in the cell 8. Wherein the commercial bacterial liquid Y2 is 360 bacterial housekeeper purchased from Zhengzhou chemical industry products Co. Meanwhile, the mass ratio of nitrogen, phosphorus and potassium is 20:10: 20.
2.2 Experimental methods
2.2.1 species planting: planting cucumbers in a greenhouse by adopting a local planting method and combining area planting arrangement of an experimental plot;
fertilizing method 1 (adding fertilizer): seedling irrigation is carried out on the cucumbers in the corresponding cells in the treatment groups 1, 3 and 4 from the seedling stage, 100mL of corresponding IVF-WK240 fermentation diluent or bacterial liquid is irrigated to each seedling in each treatment group, 100 mL/plant is applied once per week in the whole growth period of the cucumbers, and meanwhile, the mass ratio of nitrogen to phosphorus to potassium is 20 according to the application instruction: 10:20, and then carrying out normal management according to local planting requirements. Treatment group 2, applied with only a mass ratio of nitrogen to phosphorus to potassium of 20:10: 20.
Fertilizing method 2 (no addition of fertilizer): seedling irrigation is carried out on cucumbers in corresponding communities of treatment groups 5, 7 and 8 from a seedling stage, 100mL of corresponding IVF-WK240 fermentation diluent or bacterial liquid is irrigated to each seedling of each treatment group, 100 mL/plant is applied once per week in the whole growth period of the cucumbers, and meanwhile, the mass ratio of nitrogen, phosphorus and potassium is not additionally applied and is 20:10:20, and carrying out normal management according to the local planting requirements under the condition of the water-soluble compound fertilizer. Treatment group 6, no substance was applied.
Experimental cell description: the experimental cells are located on the same land block (day and night temperatures are respectively 20 ℃ (10 hours) and 10 ℃ (14 hours) of a test base plastic greenhouse in the south of China national academy of agricultural science, the illumination intensity is 6000-10000lx, the illumination time is 8 hours, the rest time is in a dark state, the soil humidity is 80%), namely one land block is divided into 8 cells, wherein the cell 1 is used for treating the group 1, the cell 2 is used for treating the group 2, the cell 3 is used for treating the group 3, the cell 4 is used for treating the group 4, the cell 5 is used for treating the group 5, the cell 6 is used for treating the group 6, the cell 7 is used for treating the group 7, and the cell 8 is used for treating the group 8. The cucumber 9930 cultivated in adjacent cells is used as a protection line; the area of each cell is 7m 2 20 cucumbers are planted in each district, the cucumber plant spacing is 35cm, and the mass ratio of nitrogen, phosphorus and potassium is 20 except that the types of bacterial agents in each treatment are different: 10:20, and other management is the same.
3. Results and analysis
And (3) carrying out statistical analysis on the cucumber planted in each treatment group, namely respectively counting the single plant yield and the mu yield (20 plants in one furrow and about 3500 plants in one mu of land) in the seedling stage, the flowering stage and the fruit setting stage of the cucumber. The data processing method is T-test. The results are shown in Table 3, FIG. 6 to FIG. 12. It can be seen that the mass ratio of nitrogen, phosphorus and potassium is 20:10: under the condition of 20 water-soluble compound fertilizer, the per mu yield of the fermentation dilution liquid of the Achromobacter xylosoxidans IVF-WK240 is increased by 8.7 percent compared with that of a fertilizer adding control, the per mu fruit setting number is increased by 11.7 percent, the per mu yield is increased by 24.5 percent compared with that of a fertilizer adding Y1, the per mu fruit setting number is increased by 25.4 percent, the per mu yield is increased by 45.5 percent compared with that of a fertilizer adding Y2, and the per mu fruit setting number is increased by 6.3 percent, which indicates that the fermentation dilution liquid of the Achromobacter xylosoxidans IVF-WK240 has more remarkable effect of promoting cucumber growth compared with that of the fertilizer adding control, the fertilizer adding Y1 (the existing bacterial liquid) and the fertilizer adding Y2 (the existing bacterial liquid) (see fig. 7 and 8). The yield per mu of the fermentation dilutions of the Achromobacter xylosoxidans IVF-WK240 was increased by 28.1%, 11.8% and 49.4%, and the fruit set per mu was increased by 12.6%,9.8%,0% as compared with the control, Y1 and Y2, respectively, without additional application of other fertilizers, and the fermentation dilutions of the Achromobacter xylosoxidans IVF-WK240 also had a more remarkable effect of promoting cucumber growth (see FIGS. 6, 9 and 10). Under the condition that other fertilizers are not additionally applied, as shown in fig. 11 and 12, compared with the fertilizer adding control, the yield per mu and the fruit setting number per mu of the IVF-240 are equivalent, which indicates that the fermentation diluent of the Achromobacter xylosoxidans IVF-WK240 can replace chemical fertilizers, the environment is more friendly, and compared with the fertilizer adding control, the yield per mu and the fruit setting number per mu of Y1 are obviously lower than those of the fertilizer adding control.
TABLE 3 influence of different treatments on average acre yield, average individual fruit weight and average fruit setting of cucumber
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Claims (10)

1. Achromobacter xylosoxidans @Achromobacter xylosoxidans) IVF-WK240 with preservation number of CGMCC No.28311.
2. A microbial preparation comprising Achromobacter xylosoxidans IVF-WK240 according to claim 1.
3. The microbial preparation according to claim 2, wherein the viable count of Achromobacter xylosoxidans IVF-WK240 in the microbial preparation is not less than 10 7 cfu/mL。
4. A method of preparing a microbial preparation according to claim 2 or 3, comprising: the Achromobacter xylosoxidans IVF-WK240 is subjected to expansion culture.
5. The use of the microorganism preparation of a. Xylosoxidans IVF-WK240 of claim 1, 2 or 3, having any one of the following:
(1) In promoting cucumber productionCucumis sativusLUse in plant growth;
(2) Preparing cucumberCucumis sativusLUse in plant growth regulators;
(3) And the application in preparing fertilizer.
6. The use according to claim 5, wherein the promotion of cucumber plant growth comprises at least one of increasing the plant height of the cucumber plant, increasing the stem thickness of the cucumber plant, increasing the fruit set number of the cucumber plant, increasing the leaf area of the cucumber plant, increasing the dry weight of the cucumber plant, increasing the fresh weight of the cucumber plant and increasing the root length of the cucumber plant;
and/or, the promoting cucumber plant growth comprises enhancing stress resistance of the cucumber plant under low temperature and/or low light conditions; the low temperature is 10-20 ℃, the daytime temperature is more than 14 ℃ and less than or equal to 20 ℃, and the night temperature is more than or equal to 10 ℃ and less than or equal to 14 ℃; the weak light condition range is 3000 lx-10000 lx, and the illumination time range is 6 h-8 h.
7. Use according to claim 5 or 6, characterized in that the cucumber plant is a low temperature and/or low light intolerant cucumber plant.
8. A cucumber plant growth regulator or fertilizer comprising the achromobacter xylosoxidans IVF-WK240 of claim 1, the microbial preparation of claim 2 or 3.
9. A method of promoting cucumber plant growth comprising applying the achromobacter xylosoxidans IVF-WK240 of claim 1, the microbial preparation of claim 2 or 3, or the cucumber plant growth regulator or fertilizer of claim 8; the application mode comprises root irrigation;
when the bacterial liquid of the Achromobacter xylosoxidans IVF-WK240 is applied, the application amount of the bacterial liquid is 10-100 mL per strain each time, and the application frequency is 1-2 times per week;
and/or when the fermentation diluent of the Achromobacter xylosoxidans IVF-WK240 is applied, the application amount of the fermentation diluent is 70-120 mL per strain each time, and the application frequency is 1-2 times per week;
and/or the period of application comprises the whole growth period of the cucumber plant.
10. Method for promoting cucumber plant growth according to claim 9, characterized in that when a bacterial liquid of achromobacter xylosoxidans IVF-WK240 is applied, the effective viable count of achromobacter xylosoxidans IVF-WK240 in the bacterial liquid is not less than 10 7 cfu/mL;
And/or the application amount of the bacterial liquid is 50 mL/strain each time, and the application frequency is once every 1 week;
and/or when a fermentation dilution of Achromobacter xylosoxidans IVF-WK240 is applied, the effective viable count of the Achromobacter xylosoxidans IVF-WK240 in the fermentation dilution is more than or equal to 10 7 cfu/mL;
And/or the application amount of the fermentation diluent is 100 mL/strain each time, and the application frequency is once in 1 week;
and/or the period of application comprises the seedling, flowering and/or fruit setting period of cucumber.
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