CN114009307A - Functional cucumber seedling-raising biological matrix containing Shewanella and preparation method thereof - Google Patents
Functional cucumber seedling-raising biological matrix containing Shewanella and preparation method thereof Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/20—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/05—Fruit crops, e.g. strawberries, tomatoes or cucumbers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention relates to the technical field of seedling culture substrates, in particular to a functional cucumber seedling culture biological substrate containing Shewanella and a preparation method thereof. The preparation method comprises the step of adding Shewanella A0B14 into a conventional substrate, wherein the Shewanella A0B14 is preserved in the common microorganism center of China general microbiological culture Collection center in 2018, 11 and 30 months, is classified and named as Shewanella sp, and has the preservation address of No. 3 Hokko No.1 Samura of the sunward region in Beijing, and the preservation number is CGMCC No. 16843. The Shewanella A0B14 used in the invention is suitable for various conventional matrixes, can survive in the matrixes for a long time, can stably fix the cucumber in the rhizosphere, and has remarkable growth promoting effect.
Description
Technical Field
The invention relates to the technical field of seedling culture substrates, in particular to a functional cucumber seedling culture biological substrate containing Shewanella and a preparation method thereof.
Background
With the development of facility agriculture, the problem of continuous cropping obstacle of facility soil becomes more serious, and the continuous development of the facility vegetable industry is restricted. Meanwhile, the demand of protected agriculture for healthy and disease-resistant seedlings is increasing continuously. The rapid development of industrial seedling culture is promoted, the industrial seedling culture becomes a main mode of seedling production in recent years, the substrate used for the industrial seedling culture is light in texture and good in water retention, fertilizer retention and air permeability, the problem of poor disease resistance of seedlings is still not solved, and the quality and survival of the seedlings are ensured mainly by using a large amount of bactericides and fertilizers. In recent years, researchers try to add biocontrol microbial agents into substrates to improve the disease resistance of seedlings, but in the practical application process, the microorganisms are difficult to colonize, short in lasting period and unstable in effect, so that the popularization and application of the technology are limited.
Related studies of crop rhizosphere microorganisms have revealed important ecological functions of rhizosphere microorganisms on plants, especially growth-promoting functions of rhizosphere bacteria. The existing scholars screen some bacteria from the rhizosphere of crops and add the bacteria into a seedling or cultivation substrate to obtain better effect, and the used bacterial strains comprise acinetobacter, belief bacillus, enterobacter cloacae, bacillus amyloliquefaciens, acinetobacter calcoaceticus, bacillus, paenibacillus, bacillus vallismortis, trichoderma harzianum and the like. The method provides a new direction for developing a novel microbial seedling and culture medium.
At present, the types of substrate products containing plant growth-promoting bacteria are quite limited, and new plant growth-promoting bacteria resources are continuously searched for developing novel microorganism seedling culture substrates. In addition, the product containing the plant growth-promoting microorganism matrix has the problem of ensuring the long-term survival of the growth-promoting microbial inoculum in the matrix, but the shelf life of the current general matrix product is 2-6 months, and no related product can ensure the survival of the plant growth-promoting microbial inoculum.
Disclosure of Invention
Aiming at the technical problems that the variety of matrix products containing plant growth-promoting bacteria is quite limited and microorganisms in the matrix products are difficult to survive for a long time, the invention provides a functional cucumber seedling-raising biological matrix containing Shewanella and a preparation method thereof, and the Shewanella A0B14 used by the invention is suitable for various conventional matrixes (commercially available matrixes and/or self-made matrixes), can survive for a long time in the matrix and can stably fix the cucumber rhizosphere, thereby having remarkable growth-promoting effect.
In a first aspect, the invention provides a preparation method of a functional cucumber seedling biological matrix containing Shewanella, Shewanella A0B14 is added into a conventional matrix, Shewanella A0B14 is disclosed in Chinese patent application CN201910049412.7, and the specific preservation information is as follows:
the collection date is 11 months and 30 days in 2018, the collection unit is China general microbiological culture Collection center, the collection address is No. 3 of Xilu No.1 of Beijing Korean district, and the collection number is CGMCC No. 16843.
Further, the method comprises the following steps:
(1) preparing Shewanella A0B14 fermentation liquor;
(2) according to 5% of the dry weight of the substrate, Shewanella A0B14 fermentation liquor is added into the conventional substrate, and the mixture is uniformly mixed and bagged.
Further, the preparation method of the Shewanella A0B14 fermentation liquor comprises the following steps:
shewanella A0B14 is inoculated on a TSB culture medium and is used as a first-level seed after overnight culture at 28 ℃ and 180 rpm; the first-class seeds are inoculated in a soybean meal culture medium according to the inoculation amount of 5 percent for fermentation production, the pH is natural, the fermentation temperature is 25-35 ℃, the fermentation rotation speed is 150-.
Further, the preparation method of the soybean meal culture medium comprises the following steps:
adding defatted soybean flour 5% into tap water, pH natural, and sterilizing with high pressure steam at 121 deg.C for 15 min.
In a second aspect, the invention provides a functional cucumber seedling raising biological matrix containing Shewanella, which is prepared by the preparation method.
Furthermore, the number of viable bacteria in the functional cucumber seedling biological matrix is more than or equal to 1.0 multiplied by 107CFU/g substrate dry weight, substrate organic matter is more than or equal to 15%, total nutrient is 1.5% -5%, water is less than or equal to 35%, pH value is 6.5-7.5, Ec is less than or equal to 2.5ms/cm, wherein the total nutrient refers to N content and P2O5Content and K2Sum of O content.
Furthermore, after the functional cucumber seedling raising biological matrix is placed at normal temperature for 6 months, the number of viable bacteria is more than or equal to 9 multiplied by 108CFU/g dry weight of matrix.
The cucumber seedling substrate product containing Shewanella A0B14 and the preparation method thereof have the advantages that Shewanella respiratory systems are various, more than 20 organic and inorganic compounds can be utilized, the Shewanella respiratory systems comprise two-carbon and three-carbon compounds, amino acids and various sugars generated by fermentation, the Shewanella can survive in a conventional substrate for a long time after being inoculated with Shewanella A0B14, and the number of beneficial viable bacteria is more than or equal to 9 multiplied by 10 after being stacked for 6 months8CFU/g dry weight of substrate; the Shewanella A0B14 can stably fix the cucumber rhizosphere and has obvious growth promoting effect.
Drawings
In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.
FIG. 1 is a graph of total viable count versus time.
FIG. 2 is a graph showing the change in viable count of Shewanella over time.
FIG. 3 is a photograph comparing growth promoting effects of a conventional substrate and a biological substrate.
FIGS. 1 and 2 represent the significant difference between the control matrix and the biological matrix at each sampling time (independent sample t-test).
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution in the embodiment of the present invention will be clearly and completely described below with reference to the drawings in the embodiment of the present invention, and it is obvious that the described embodiment is only a part of the embodiment of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The TSB medium used in the embodiment of the invention is a commercial product;
the soybean meal culture medium is prepared by the following preparation method:
adding defatted soybean flour 5% into tap water, pH natural, and sterilizing with high pressure steam at 121 deg.C for 15 min.
Example 1
A functional cucumber seedling-raising biological matrix containing Shewanella is prepared by the following preparation method:
(1) preparing Shewanella A0B14 fermentation liquor:
shewanella A0B14 is inoculated on a TSB culture medium and is used as a first-level seed after overnight culture at 28 ℃ and 180 rpm; inoculating the first-class seed in soybean powder culture medium according to an inoculation amount of 5% for fermentation production, wherein the pH is natural, the fermentation temperature is 25 ℃, the fermentation speed is 300rpm, the fermentation time is 48 hours, and the colony count after fermentation is about 2 × 108CFU/mL；
(2) Adding Shewanella A0B14 fermentation liquid into sterilized conventional matrix (obtained from Shandong commercial Biotechnology Co., Ltd., component composed of 25% starch resin foam particles, 35% cow dung straw fermentation product and 40% peat) according to 5% of dry weight of the matrix, mixing well and bagging to obtain functional cucumber seedling-raising biological matrix (hereinafter referred to as Shewanella)Biological matrix) with viable count not less than 1.0 × 107CFU/g substrate dry weight, substrate organic matter is more than or equal to 15 percent, total nutrient (N + P)2O5+K2O) 1.5-5 percent, water content less than or equal to 35 percent, pH value 6.5-7.5 and Ec less than or equal to 2.5 ms/cm.
Example 2
Example 2 is different from example 1 in that the fermentation conditions of Shewanella A0B14 in step (1) are different, and the fermentation conditions of example 2 are 35 ℃ of fermentation temperature, 150rpm of fermentation speed and 36 hours of fermentation time.
Comparative example 1
Replacing Shewanella A0B14 fermentation broth with soybean meal culture medium of the same volume, adding into sterilized conventional matrix, mixing well, and bagging to obtain control matrix.
Verification example 1
The biological matrix of example 1 and the control matrix of comparative example 1 were tested for viable cell count trend over time by counting viable cells in 1g of sample every 30 days and repeating 4 times. Putting each gram of sample into a 50ml centrifuge tube, adding 10ml of sterile water, shaking, and then diluting to 10 degrees by using sterile water in a gradient manner8Coating, counting the total viable count and the viable count of Shewanella in the matrix during plate counting, identifying Shewanella according to colony morphology, identifying the strain by using 16S rRNA gene to determine the accuracy of Shewanella morphology identification, and identifying and finding that Shewanella in the plate can be basically determined by morphology.
The counting results are shown in fig. 1 and fig. 2, compared with the control matrix, the total viable count in the biological matrix is obviously increased, and the viable count of Shewanella in the biological matrix is more than or equal to 9 multiplied by 10 after the biological matrix is placed for half a year8CFU/g dry weight of substrate, demonstrating that the shelf life of the biomatrix prepared according to the invention is significantly longer than 6 months.
Verification example 2
Cucumber seedling raising tests were conducted using 6 months of the bio-substrate of example 1 and a conventional substrate, and the growth promoting effects of the two were compared. The test was carried out in an artificial greenhouse (temperature 25 ℃, photoperiod L: D16: 8, light intensity 20000lx) at institute of ecology, academy of sciences, Shandong province, in 5-month-middle-ten of 2021. The cucumber is Qiangsheng 719 (Shandong Lushou agriculture group).
The test method comprises the following steps: filling two substrates in a disposable paper cup, then dibbling a cucumber seed in each paper cup, covering the surface with the substrates, culturing in an artificial greenhouse, watering 10mL every other day after seedling emergence, and measuring the fresh weight of the plant height, the stem thickness, the overground part and the underground part 7 days after seedling emergence. Measuring the length from the root to the growing point by using a ruler; measuring the thickness of the stem at a position 2cm below the cotyledon in a direction parallel to the cotyledon by using a vernier caliper; weighing the fresh weight of the ground and the fresh weight of the lower part by using one ten-thousandth electronic balance; the dry weight of the upper part and the lower part is that fresh samples of the seedlings are put into an oven for deactivation of enzymes at 105 ℃ for 15min, then dried to constant weight at 80 ℃, and then weighed by a ten-thousandth electronic balance; strong seedling index (stem thickness/plant height + dry underground/dry above ground) x dry single plant weight. The experiment was repeated 6 times.
As shown in FIG. 3, compared with the conventional substrate, the dry weight of the roots of the cucumber seedlings of the biological substrate is increased by about 87.2%, the weight of the stems is increased by about 66.5%, the root-stem ratio is increased by about 10.1%, the dry weight of the plants is increased by about 70.1%, and the seedling strengthening index is increased by 109.7%. In conclusion, the seedling raising substrate has a remarkable growth promoting effect, and the shelf life of the biological substrate prepared by the invention is proved to be obviously longer than 6 months.
Verification example 3
Cucumber seedlings were planted using the biological substrate of example 1, cucumber plants were harvested on days 7, 14, 21, 35, 49, 63 after planting, the overground parts of the plants were cut off after harvesting, parts of the main root, lateral root and adventitious root were cut off after shaking off the soil attached to the roots to approximately 10g into 50mL sterilized centrifuge tubes, and after each harvest of one plant, the scissors were sterilized with 75% ethanol and rinsed three times with sterilized water before use. After the collection, the centrifuge tube with the root sample is placed in an ice box and transported to a laboratory. Subsequently, 35mL of sterilized 1 XPBS buffer was added to each sample tube, the tube was washed in a shaker at 180rpm for 20min, then the surface of the root was dried by blotting water using a sterile filter paper, and the washing was repeated 3 times, 0.2g of the above sample was thoroughly ground with sterile water, and the ground sample was diluted 10-fold in a gradient manner to 10-fold8Post-plating on TSB plates for enumeration, identification of Shewanella according to colony morphology, and strain identification using 16S rRNA geneAnd identifying and determining the accuracy of Shewanella morphotype recognition. The determination shows that the planting amount of the Shewanella in the rhizosphere after 7, 14, 21, 35, 49 and 63 days after cucumber planting is 1.34 multiplied by 104、1.28×103、1.69×104、1.71×104、1.52×104、1.48×103CFU/g root, which shows that Shewanella A0B14 can be planted on cucumber rhizosphere for a long time in the biological matrix.
Verification example 4 colonisation survival of different strains in a matrix
The same procedure was followed using the bio-matrix of example 1 to prepare a bio-matrix of 4 additional commercially available strains, four strains: shewanella ATCC700550, Shewanella ATCC8071, Bacillus belgii BMZ141112 and Paenibacillus ATCC 9995. After standing for half a year, 1g of each of the five matrixes is taken and put into a 50ml centrifuge tube, 10ml of sterile water is added for shaking, and then the mixture is diluted to 10 degrees by using sterile water in a gradient manner8Coating was counted and repeated 4 times. The viable bacteria count result is: the number of viable bacteria in the biological matrix containing A0B14, ATCC700550, ATCC8071, BMZ141112 and ATCC9995 is 9.1 × 108、2.7×105、3.6×104、3.2×104、1.7×103CFU/g dry weight of matrix.
Verification example 5 Shewanella A0B14 colonization on different formula substrates
A, B, C, D four matrixes are prepared according to the following weight ratio, Shewanella A0B14 fermentation liquor is added into the four matrixes respectively according to 5% of the dry weight of the matrixes (the preparation method is the same as that in example 1), the four matrixes are uniformly mixed and bagged, and the four matrixes all meet the condition that the viable count is more than or equal to 1.0 multiplied by 107CFU/g dry weight of matrix.
A. Peat soil: perlite: vermiculite: 4, thoroughly decomposed straw: 1: 0.5: 4.5;
B. peat soil: perlite: vermiculite: and (3) thoroughly decomposed cow dung is 4: 1: 0.5: 4.5;
C. peat soil: perlite: vermiculite: decomposed shredded coconut 4: 1: 0.5: 4.5;
D. peat soil: perlite: vermiculite: and (3) thoroughly decomposing oyster mushroom planting mushroom dregs which are 4: 1: 0.5: 4.5.
placing the halfAfter the year, 1g of each of the four matrixes is taken and put into a 50ml centrifuge tube, 10ml of sterile water is added, the mixture is shaken and then is diluted to 10 degrees by using the sterile water in a gradient way8Coating was counted and repeated 4 times. The viable bacteria count result is: A. b, C, D Shewanella lively A0B14 in four matrixes are 9.7 × 10 respectively8、9.2×108、9.1×108、1.1×109CFU/g dry weight of matrix.
Although the present invention has been described in detail by referring to the drawings in connection with the preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made on the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and these modifications or substitutions are within the scope of the present invention/any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention.
Claims (8)
1. A preparation method of a functional cucumber seedling biological matrix containing Shewanella is characterized in that Shewanella A0B14 is added into a conventional matrix, the Shewanella A0B14 is preserved in China general microbiological culture Collection center in 11 and 30 months in 2018, is classified and named as Shewanella sp, and is preserved in the No. 3 Hosth West Lu No.1 of the sunward area in Beijing, and the preservation number is CGMCC No. 16843.
2. The method of claim 1, comprising the steps of:
(1) preparing Shewanella A0B14 fermentation liquor;
(2) according to 5% of the dry weight of the substrate, Shewanella A0B14 fermentation liquor is added into the conventional substrate, and the mixture is uniformly mixed and bagged.
3. The method according to claim 2, wherein the Shewanella A0B14 fermentation broth is prepared by:
shewanella A0B14 is inoculated on a TSB culture medium and is used as a first-level seed after overnight culture at 28 ℃ and 180 rpm; the first-class seeds are inoculated in a soybean meal culture medium according to the inoculation amount of 5 percent for fermentation production, the pH is natural, the fermentation temperature is 25-35 ℃, the fermentation rotation speed is 150-.
4. The method of claim 3, wherein the soy flour medium is prepared by:
adding defatted soybean flour 5% into tap water, pH natural, and sterilizing with high pressure steam at 121 deg.C for 15 min.
5. A functional cucumber seedling-raising biological matrix containing Shewanella provided by the preparation method of any one of claims 1-4.
6. The functional cucumber seedling biological matrix as claimed in claim 5, wherein the viable count in the functional cucumber seedling biological matrix is more than or equal to 1.0 x 107CFU/g substrate dry weight, substrate organic matter is more than or equal to 15%, total nutrient is 1.5% -5%, water is less than or equal to 35%, pH value is 6.5-7.5, and Ec is less than or equal to 2.5 ms/cm.
7. The functional cucumber seedling-raising biological matrix as claimed in claim 6, wherein the total nutrients refer to N content and P content2O5Content and K2Sum of O content.
8. The functional cucumber seedling biological matrix as claimed in claim 5, wherein the viable count of the functional cucumber seedling biological matrix is not less than 9 x 10 after the functional cucumber seedling biological matrix is placed at normal temperature for 6 months8CFU/g dry weight of matrix.
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