CN117467585B - Isolated geobacillus for promoting generation of morchella primordium and ascocarp and application thereof - Google Patents
Isolated geobacillus for promoting generation of morchella primordium and ascocarp and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- 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
- A01G18/00—Cultivation of mushrooms
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P21/00—Plant growth regulators
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Abstract
The invention relates to the technical field of microbial agents. The invention provides a isolated geobacillus for promoting generation of morchella primordium and ascocarp and application thereof, wherein the isolated geobacillus has the preservation name ofPedobacter insulaeZP30, latinPedobacter insulaePreservation date: 2023, 8, 31, deposit unit: china center for type culture Collection, accession number: cctccc NO: m20231568. The isolated geobacillus can be used as an auxiliary added beneficial microbial agent in Morchella cultivation production to promote absorption and utilization of Morchella to external nutrition or improve soil flora structure, and finally improve Morchella cultivation stability. The strain can promote the generation of morchella primordia and increase the yield of morchella.
Description
Technical Field
The invention relates to the technical field of microbial agents, in particular to a geobacillus isolated for promoting generation of morchella primordia and ascocarp and application thereof.
Background
Morchella (L.) pers(Morchellaspp.) belong to Ascomycota (Ascomycota), pantoea (Pezizomycetes), pantoea (Pezizales) in classification, are rare edible fungi distributed worldwide, and have certain edible and medicinal values; however, morchella cultivation has the problems of low primordium generation amount, low primordium survival rate, unsatisfactory yield, continuous cropping and the like, and limits the healthy development of Morchella industry.
Researches in Pion and 2013 show that morchella crassipesM. crassipes) And Pseudomonas putida [ ]Pseudomonas putida) The morchella crassipes has obvious reciprocal symbiotic relation, the morchella crassipes can actively transmit the pseudomonas putida, the growth and the reproduction of the pseudomonas putida are promoted to a certain extent, and finally the morchella crassipes is used as nutrient substances for digestion, absorption and utilization; morchella crassipes is integrally like a conscious peasant, sows bacteria, and after the bacteria grow for a certain time, the bacteria are harvested and converted into self food. In such a symbiotic environment, bacteria grow and spread over a long distance through the hyphal network of fungi, which reach a reciprocal situation by taking certain bacteria as food. Observation of Morchella mycelium cultivated in a field shows that a large amount of bacteria are attached to the Morchella mycelium, and histology data also show that the growth and development of Morchella are accompanied by significant change of bacterial flora structure, and obvious interaction relationship exists between Morchella and bacteria.
Therefore, the development of the microbial agent suitable for Morchella esculenta has broad prospects in increasing the occurrence of Morchella esculenta primordium, improving the yield of Morchella esculenta and reducing the continuous cropping problem of Morchella esculenta cultivation.
Disclosure of Invention
The invention aims to provide isolated geobacillus for promoting generation of morchella primordia and ascocarp and application thereof, which can increase the occurrence rate of the morchella primordia and increase the yield of the morchella.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a isolated geobacillus for promoting morchella primordium and ascocarp to occur, the isolated geobacillus has the preservation name ofPedobacter insulaeZP30, latinPedobacter insulaePreservation date: 2023, 8, 31, deposit unit: china center for type culture Collection, accession number: cctccc NO: m20231568.
The invention also provides application of the isolated geobacillus in Morchella cultivation production, wherein the cultivation comprises potting, shelf cultivation and field cultivation.
The invention also provides application of the isolated geobacillus in promoting generation of morchella primordia and ascus fruits.
The invention also provides a method for promoting the generation of morchella primordium and ascus by using the isolated geobacillus, which comprises the following steps: spraying isolated geobacillus bacteria liquid into soil, ploughing the soil after spraying, and then cultivating Morchella.
Preferably, the humidity of soil in the field before spraying is 22% -28%.
Preferably, the ploughing depth is 20-25 cm during ploughing.
Preferably, the concentration of the bacteria in the isolated geobacillus bacteria solution is 10 4 ~10 5 And the spraying amount of the isolated geobacillus bacteria liquid is 10-15L/mu.
The invention provides a isolated geobacillus for promoting generation of morchella primordium and ascocarp and application thereof, wherein the isolated geobacillus has the preservation name ofPedobacter insulaeZP30, latinPedobacter insulaePreservation date: 2023, 8, 31, deposit unit: china center for type culture Collection, accession number: cctccc NO: m20231568. The isolated geobacillus can be used as a beneficial microbial agent added in an auxiliary way in Morchella cultivation production to promote sheepThe absorption and utilization of the morchella to external nutrition or the improvement of the soil flora structure, and finally the cultivation stability of the morchella. The strain can promote the generation of morchella primordia, increase the generation rate of the morchella primordia and improve the yield of the morchella.
Preservation description
Isolated geobacillus deposit namePedobacter insulae ZP30, latinPedobacter insulaePreservation date: 2023, 8, 31, deposit unit: china center for type culture collection, preservation address: chinese, university of armed chinese, accession number: cctccc NO: m20231568.
Drawings
FIG. 1 is a 16s rDNA phylogenetic tree of isolated Geobacillus ZP30 strain;
FIG. 2 is a graph showing that the effect of isolated Geobacillus ZP30 strain on the occurrence of morchella primordia and the number of ascos, with significant differences between treatments with different lower case letters (P < 0.05);
FIG. 3 is a graph of a non-harmful evaluation test of isolated Agrobacterium ZP30 strain, the upper graph showing the first and seventh days of the CK control group, and the lower graph showing the first and seventh days of the ZP30 puncture test group.
Detailed Description
The invention provides a isolated geobacillus for promoting morchella primordium and ascocarp to occur, the isolated geobacillus has the preservation name ofPedobacter insulaeZP30, latinPedobacter insulaePreservation date: 2023, 8, 31, deposit unit: china center for type culture Collection, accession number: cctccc NO: m20231568.
The invention also provides application of the isolated geobacillus in Morchella cultivation production, wherein the cultivation comprises potting, shelf cultivation and field cultivation.
The invention also provides application of the isolated geobacillus in promoting generation of morchella primordia and ascus fruits.
The invention also provides a method for promoting the generation of morchella primordium and ascus by using the isolated geobacillus, which comprises the following steps: spraying isolated geobacillus bacteria liquid into soil, ploughing the soil after spraying, and then artificially cultivating Morchella.
In the present invention, the humidity of the soil in the field before spraying is preferably 22% to 28%, more preferably 25%.
In the invention, the ploughing depth is preferably 20-25 cm, more preferably 22-23 cm.
In the present invention, the concentration of the bacteria in the isolated geobacillus bacteria solution is preferably 10 4 ~10 5 Each ml, more preferably 5X 10 4 The spraying amount of the isolated geobacillus bacteria liquid is preferably 10-15L/mu, and more preferably 12-13L/mu.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1Pedobacter insulae ZP30 bacterial strain separation, screening and identification
1. Isolation and screening of ZP30 bacterial Strain
Isolation of strains: soil samples were collected at 28 days of 2022, 6 months, at the stage of morchella primordium development in arillus araba, si. During sampling, from the original base generation position of Morchella, taking the soil from the ground surface to the depth of 10cm, taking 3 sampling points in total, storing at a low temperature of 4 ℃, taking the materials back to a laboratory, mixing the materials in a sterile container, and then using the materials for subsequent separation experiments. Weighing 10g of fresh soil sample, mixing with 90ml of sterile water under shaking, standing for 5min, collecting supernatant with sterile syringe, and filtering with sterile filter with 3 μm aperture to obtain 10 -1 Sample diluent is subjected to gradient dilution by adopting sterile water, the dilution is 10 times each time, and finally 200 mu m concentration is 10 -4 、10 -5 、10 -6 The sample dilutions of (2) are spread and inoculated on LB, R2A, TSA, PCA and PCA+Morchella polysaccharide 5 culture medium plates, each gradient is repeated for 3 times, the culture is inverted at 21 ℃ after sealing, single colony prokaryotic microorganisms are selected after 3 days, the components of the culture medium and the number of the obtained single colonies are shown in table 1, 563 bacterial colonies are selected in total, and the selected single colonies are placed in LB liquid culture mediumCulturing, growing at 21 ℃ for 3 days, and storing at 4 ℃ for subsequent experiments.
Screening of candidate beneficial bacteria: culturing 563 strain single colony microorganism and Morchella mycelium in CYM liquid culture medium (Yeast extract 2 g/L, peptone 2 g/L, K) 2 HPO 4 1 g/L，MgSO 4 0.5 g/L，KH 2 PO 4 0.46 g/L and glucose 20 g/L), taking the normal growth of Morchella mycelium after 5 days of culture as a screening condition, screening 14 candidate bacterial strains which are harmless and symbiotic with Morchella for the first time, re-screening the 14 candidate bacterial strains, observing the growth state of Morchella mycelium every day, taking the bacterial content reduction after 5 days of culture as a screening condition, and finally obtaining 1 bacterial strain ZP30 by a screening test, wherein the growth vigor of Morchella is best taken as a candidate beneficial bacterium when the Morchella mycelium is co-cultured.
Table 1 Medium and number of single colonies
2. Identification of ZP30 bacterial Strain
The bacterial strain is identified through 16S rDNA gene and genome homology analysis, a colony PCR method is adopted for 16S acquisition, and the following primers are synthesized by Beijing qingke biotechnology Co., ltd:
BSF5'-AGAGTTTGATCCTGGCTCAG-3'（SEQ ID NO.1）；
BSR5'-AAGGAGGTGATCCAGCCGCA-3'（SEQ ID NO.2）。
PCR reaction system 1 XPCR buffer Mg 2+ plus, rTaq 0.5U, primer 10nM, bacterial suspension 1. Mu.L; the PCR reaction conditions were 95℃for 3min,95℃for 15s,58℃for 1min,72℃for 25s, and 72℃for 3min, and amplification was performed; the amplified products were detected by agarose gel electrophoresis and sent to Beijing qingke biosciences, inc. for two-way sequencing.
The result showed that 16S rDNA fragment length of the ZP30 strain was 1455bp. Submitting it to NCBI public database for sequence alignment, comparing it with the sequence number and NR_044083 has a high degree of similarity of 99.10%. Downloading a sequence similar to the sequence, constructing a phylogenetic tree, firstly comparing the sequences by using MAFFT v7.407, converting the compared file into a phy format, then using IQtree2 software, automatically detecting an optimal sequence substitution model, constructing the phylogenetic tree by using a maximum likelihood method ML with boottrap set as 1000, and the result is shown in figure 1, and shows thatPedobacterThe genus is obviously distinguished from the adjacent genus, and the ZP30 strain obtained by separation is separated from the strain with the sequence number of NR_044083 under the support rate of 98 percentPedobacter insulaeStrains clustered together, with distinct differentiation from other candidate strains, consistent with NCBI results.
The colony is circular, convex, moist, and the cell is in a rod shape, 1.8-2.5 μm (length) ×0.6-0.8 μm (width), no spore is formed, and gram staining is negative. The phylogenetic data of the combined 16r rDNA can basically confirm that the ZP30 strain separated in the experiment isPedobacter insulae. The 16s rDNA sequence of ZP30 strain was submitted to NCBI public database to obtain the sequence No. OR486031.
Example 2 ZP30 Strain promotes the development of Morchella primordia and ascocarpus
In the middle ten days of 11 months 2022-4 months in Kunming city plant research institute test field of Yunnan province and happy and persuaded Morchella cultivation base of Kunming city of Yunnan province, two places are cultivated and tested, 2 treatments are set up by adopting random block design, 3 times of each treatment are repeated, and the strain is not added as a control, wherein 6 pots are added in the Kunming city plant test field, and the area of each pot is 0.144m 2 The method comprises the steps of carrying out a first treatment on the surface of the The cultivation area of each cell of the Min-an-le base is not less than 2m 2 A total of 3 replicates. Firstly, performing LB enrichment culture on ZP30, centrifuging the cultured thalli, cleaning with sterile physiological saline, adjusting the concentration, and finally obtaining the culture medium according to 10 4 Spraying 10L of the water for each mu of land per ml of use concentration, spraying the same volume of physiological saline for a control group, and ploughing the land after spraying, wherein the ploughing depth is 25cm; the soil humidity before spraying is 25%; sowing, earthing, adding exogenous nutrition bags and managing production according to conventional Morchella cultivation in field after spraying, and using amount of strain175kg of exogenous nutrient bags are used in each mu of land, and 1800 bags are used in each mu of land, namely about 750kg. In the production process, the primordium (40 d after sowing, the primordium size is 1-2mm in diameter) and the number of the ascus fruits (65 d after sowing, and the ascus fruits height is more than 5 cm) are counted in detail.
As shown in fig. 2, the ZP30 strain showed a remarkable promoting effect on the growth of the morchella, the primordium number and the ascus number of the morchella were remarkably increased after the ZP30 strain was added, the primordium number was increased by 96.87% and the ascus number was increased by 49.62% compared with the control treatment, and the ZP30 strain was considered to have a promoting effect on the growth of the morchella in a relatively complex production environment by the potting test and the data of the increase of the primordium and ascus number in the field test.
Example 3 innocuous detection of ZP30 Strain
Activating ZP30 strain indoors, culturing with 100ml LB culture medium at 18deg.C 160rmp for 48 hr, centrifuging at 5000rmp, cleaning with sterile physiological saline for 2 times, and regulating bacterial suspension concentration to 10 with sterile physiological saline 9 The secondary is per milliliter. Healthy ascus fruits are selected in a Morchella cultivation field, ZP30 bacterial suspension is dipped by sterile toothpicks, the tissue of Morchella stipe is punctured and infected, each ascus fruit is punctured and inoculated with 5 holes, sterile physiological saline is adopted for puncture in comparison treatment, and each treatment is not less than 6 ascus fruits. After one week, the puncture mushrooms are observed, the growth state of the morchella ascus is checked, and if the ascus can still grow normally after being punctured by the ZP30 strain, the strain is harmless to the ascus. As shown in FIG. 3, the Morchella esculenta can still grow normally after the ZP30 strain punctures for one week, which indicates that the ZP30 strain is harmless to the Morchella esculenta.
As can be seen from the above examples, the present invention provides a isolated Geobacillus for promoting the generation of morchella primordia and ascocarp, and its applicationPedobacter insulaeThe preservation name of the bacteria isPedobacter insulaeZP30, latinPedobacter insulaePreservation date: 2023, 8, 31, deposit unit: china center for type culture Collection, accession number: cctccc NO: m20231568. The invention is characterized in thatPedobacter insulaeThe strain can be used as an auxiliary added beneficial bacteria agent in Morchella cultivation production to promote the absorption and utilization of Morchella to external nutrition or improve the soil flora structure, and finally improve the cultivation stability of Morchella. The strain can promote the generation of morchella primordia, increase the generation rate of the morchella primordia and increase the yield of ascocarp.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (7)
1. A strain of isolated geobacillus for promoting generation of morchella primordia and ascocarp is characterized in that the isolated geobacillus has the preservation name ofPedobacter insulaeZP30, latinPedobacter insulaePreservation date: 2023, 8, 31, deposit unit: china center for type culture Collection, accession number: cctccc NO: m20231568.
2. Use of isolated geobacillus in Morchella culture production according to claim 1, wherein said cultivation comprises potting, shelf cultivation and field cultivation.
3. Use of isolated geobacillus in accordance with claim 1 for promoting the development of morchella primordia and ascocarp.
4. A method for promoting the generation of morchella primordia and asco by using the isolated geobacillus of claim 1, comprising the steps of: spraying isolated geobacillus bacteria liquid into soil, ploughing the soil after spraying, and then cultivating Morchella.
5. The method of claim 4, wherein the humidity of the soil in the field prior to spraying is 22% -28%.
6. The method according to claim 5, wherein the ploughing depth is 20-25 cm.
7. The method according to claim 6, wherein the concentration of the bacteria in the isolated geobacillus liquid is 10 4 ~10 5 And the spraying amount of the isolated geobacillus bacteria liquid is 10-15L/mu.
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| BR112016030493B1 (en) * | 2014-06-26 | 2022-10-04 | Indigo Ag, Inc | SYNTHETIC COMBINATION, PLURALITY OF SYNTHETIC COMBINATION, AND AGRICULTURAL PRODUCT |
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