CN112359001B - Bacillus amyloliquefaciens microbial agent and application thereof - Google Patents
Bacillus amyloliquefaciens microbial agent and application thereof Download PDFInfo
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Abstract
The invention discloses a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 microbial agent and application thereof, wherein the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 separated from flowers of fragrant pears in a fragrant pear garden in Xinjiang Korla is used for preparing a medicament for preventing and treating fragrant pear blossom blight, the microbial agent prepared by the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 is used for preparing the medicament for preventing and treating the fragrant pear blossom blight, the diameter of a bacteriostatic circle of a strain xl96-3 to cotton fragrant pear blight is 9.33mm, the prevention effect of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 microbial agent stock solution and the dilution by 5000 times is the highest, the prevention effect on the fragrant pear blossom blight is 5754% and 3252% respectively, the prevention effect on the fragrant pear blossom blight is 3532% and the special prevention effect on the fragrant pear blossom is 3525% and the biological preservative effect on the fragrant pear blossom is further provided for statistics of the biological fragrant pear blossom and the biological preservative yield of the fragrant pear blossom is increased by the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and the biological preservative.
Description
Technical Field
The invention relates to the technical field of microbial strains and application thereof, in particular to a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 microbial strain and application thereof in preparing a medicine for preventing and treating pear blossom diseases.
Background
China is the first big pear producing country in the world, and Chinese pears play a very important role in the development of the world pear industry. The blossom blight is a bacterial disease causing rosa, apple, hawthorn, crabapple, quince and other rosaceous plants, and symptoms of blossom rot, branch withering, big branch and trunk withering, and severe whole tree withering. The disease causes serious harm to industries such as Xinjiang pear and apple, and has huge loss. Especially, the Korla bergamot pears are extremely sensitive to the disease, so that the Korla bergamot pears are popular in 17 years, the safety of the bergamot pear industry is influenced, and the government is investing in huge financial and technical force to actively prevent and control.
The pathogenesis of the flower rot is that sclerotium formed by pathogenic bacteria on diseased fruits, diseased leaves and diseased branches overwinter. In spring of the next year, when the soil temperature is above 2 ℃ and the soil humidity is above 30%, sclerotium generates an ascospore disc, and the ascospore disc is easy to generate ascospore at the temperature of above 5 ℃, is spread along with wind and invades leaves and flowers to cause leaf rot and flower rot. Conidia generated on diseased leaves and flowers invade stigma, reach embryo sac through pollen tube, reach surface through ovary wall to cause fruit rot, and cause branch rot. The pear flower rot overwinter on bark ulcer spots of fruit trees, and is spread to flowers through rainwater and insects in spring to cause flower rot, and then causes branch withering and branch withering. The prevention and control of the flowering phase is the key to the prevention and control of the diseases.
At present, chemical pesticides are mostly adopted to prevent and treat field diseases, chemical bactericides are mostly artificially synthesized substances, and the nature generally has no corresponding microbial degradation way, so that a great amount of pesticide degradation residual toxicity exists in fruit trees and soil, and the pesticide degradation residual toxicity is accumulated in the environment and enters a biological chain through material circulation. After the chemical bactericide is used for a certain time, pathogenic bacteria often generate drug resistance, so that the research and development of the bactericide with stronger drug action are forced, but the drug resistance of the pathogenic bacteria caused by long-term use of pesticides cannot be eliminated. In the blooming period of the bergamot pear, the chemical bactericide also becomes the first killer of the bees and the potential safety hazard of the honey source, and causes serious economic loss. The nation advocates the policy of 'two reductions', and how to develop a new generation of ecologically safe bactericide becomes an urgent problem to be solved. At present, many people begin to search answers from the perspective of biocontrol, and many biological pesticides are applied to production practice to achieve certain economic and ecological benefits in the aspect of controlling apple, hawthorn and loquat flower rot, but research on the aspect of controlling pear flower rot is not reported.
Disclosure of Invention
Aiming at the technical current situation that the application of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 microbial agent in the field of the Bacillus amyloliquefaciens in the aspect of preventing and treating the flower rot of the bergamot pear and the application thereof are not recorded in the prior art, the invention aims to provide the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 microbial agent and the application thereof, the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 microbial agent is obtained by separating the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 from the bergamot pear flowers in a Kurla Xinjiang bergamot pear garden, the microbial agent prepared by the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 is applied to the preparation of the medicine for preventing and treating the flower rot of the bergamot pear, the bacterial strain xl96-3 has the diameter of a zone for preventing and treating the branch rot of the cotton bergamot pear, the stock solution of the Bacillus amyloliquefaciens xl96-3 microbial agent and the stock solution diluted by 5000 times have the highest control effect, the control efficiency on the pear flower rot is 96.32% and 94.97%, the control efficiency on the pear branch blight is 94.88% and 92.79%, the single fruit weight and the yield of the pears are further counted, and the single fruit weight and the yield are obviously increased, so that the Bacillus amyloliquefaciens xl96-3 and the microbial agent thereof have specificity and high efficiency on the pear flower rot, do not have side effects on bees, and have wide application value on biological control of the pear flower rot.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
the invention provides a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 microbial agent which is obtained by fermenting and metabolizing a new strain Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3, wherein the viable count of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 is not less than 10 8 CFU/mL。
In the invention, a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 separation sieve is selected from pear trees in a pear garden in the Kigelle pear garden in Xinjiang, and the molecular level identification of a strain system and the test verification of a physiological and biochemical system of the strain which are well known and accepted in the field prove that the obtained Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 belongs to a typical new strain in the scope of the obtained Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and the strain Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 is preserved in the common microorganism center of the China Committee for culture Collection of microorganisms with the preservation number: CGMCC No:20561, date of deposit: 28/08/2020.
The gene sequence of the xl96-3 strain of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is shown as SEQ ID NO. 1 and SEQ ID NO. 2.
In the invention, an LB culture medium for separating and purifying Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl 96-3: 3g of beef extract, 7g of peptone, 5g of NaCl, 17g of agar, and adding distilled water to 1.0L of pH value of 7.0;
in the present invention, starch is destructurizedEnrichment Landy liquid medium of Bacillus (Bacillus amyloliquefaciens) xl 96-3: glucose 20g, L-glutamic acid 5g, mgSO 4 0.5 g，KCl 0.5g，KH 2 PO 4 1.0g，Fe 2 SO 4 ·6H 2 O 0.15mg，MnSO 4 ·H 2 O 5.0mg，CuSO 4 ·5H 2 O0.16 mg, distilled water was added to 1.0L, and the pH was 7.0.
Meanwhile, the invention provides a preparation method of a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 microbial agent, which comprises the following steps:
(1) Activating the xl96-3 of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) stored at low temperature on an LB plate culture medium, selecting a single colony on the LB slant culture medium, and culturing for 20-26h in an incubator at 28-32 ℃ to obtain the activated xl96-3 strain of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens).
(2) And (2) scraping a ring of the activated Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 strain in the step (1) by using a sterile inoculating ring, inoculating the strain into 100mL of LB liquid culture medium, and performing shake culture for 20-26h at the temperature of 28-32 ℃ and the rotating speed of a shaking table of 120-140r/min to obtain a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 seed solution.
(3) Inoculating the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 seed liquid obtained in the step (2) into a triangular flask of a Landy liquid culture medium according to the inoculation amount of 4-6% by mass volume ratio for shake flask fermentation, and performing shake culture at the temperature of 28-32 ℃ and 180-220r/min for 32-48h to obtain the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 fermentation liquid.
(4) And (3) detecting the quantity of the bacteria and spores in the fermentation liquid obtained in the step (3), stopping fermentation culture when mature spores in the fermentation liquid account for 90% of the total quantity of the spores and the bacteria, and preparing a liquid or solid preparation of the Bacillus amyloliquefaciens xl96-3 microbial agent according to needs.
The invention relates to a preparation method of a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 microbial agent, wherein the inoculation amount of a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 seed solution is 5%, the fermentation temperature is 30 ℃, and the fermentation time is 40h
The invention provides application of a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 microbial agent in preparation of a medicine for preventing and treating pear blossom rot.
By implementing the content of the invention through the implementation of the above specific technical scheme provided by the invention, the following beneficial effects can be obtained:
(1) The invention provides a new strain Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3, which is proved to belong to a typical new strain with the strain number xl96-3 in the category of Bacillus amyloliquefaciens by the well-known and recognized molecular level identification of a strain system and the test verification of a physiological and biochemical system of the strain, and further needs to be preserved according to the legal requirements.
(2) The Bacillus amyloliquefaciens xl96-3 and the microbial agent thereof are applied to preparation of a medicine for preventing and treating the pear blossom blight, the average diameter of the inhibition zones of the Bacillus amyloliquefaciens xl96-3 to the pear branch blight is 9.33mm, the Bacillus amyloliquefaciens xl96-3 is sprayed and released on the leaf surfaces of the pear bees in the full flowering period, the biocontrol effect of the Bacillus amyloliquefaciens xl96-3 microbial agent provided by the invention can reach more than 90%, the yield is improved by 19.6-23.5% through yield statistics, and the application value of the Bacillus amyloliquefaciens in biological prevention and treatment of the pear blossom rot is wide.
Drawings
FIG. 1 is a diagram of a phylogenetic tree constructed by strain xl96-3 based on 16S rDNA sequence.
FIG. 2 is a phylogenetic tree diagram constructed by strain xl96-3 based on gyrB gene sequence.
FIG. 3 is a drawing of the antagonism of strain xl96-3 to the bacterial wilt bacterium plate.
FIG. 4 is a graph showing the effect of strain xl96-3 on the pollen germination of Dangshan pear, wherein A is pure pollen and B is pollen plus biocontrol bacteria.
FIG. 5 is a graph showing the influence of the biocontrol strain spraying during the full-bloom stage of the strain xl96-3 on the incidence rate of the flower rot.
Detailed Description
The present invention will be described below by way of examples, but the present invention is not limited to the following examples. All raw and auxiliary materials selected for use in the present invention, as well as methods for culturing the selected bacterial species, are well known and used in the art, and all percentages referred to herein are by weight unless otherwise indicated.
An LB culture medium for separating and purifying Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl 96-3: 3g of beef extract, 7g of peptone, 5g of NaCl, 17g of agar, and adding distilled water until the pH value is 1.0L and 7.0;
enrichment Landy liquid culture medium of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl 96-3: glucose 20g, L-glutamic acid 5g, mgSO 4 0.5 g，KCl 0.5g，KH 2 PO 4 1 g，Fe 2 SO 4 ·6H 2 O 0.15mg，MnSO 4 ·H 2 O 5.0mg，CuSO 4 ·5H 2 O0.16 mg, distilled water was added thereto to 1.0L, and the pH was 7.0.
The determination method comprises the following steps:
(1) The weight of a single fruit: before the bergamot pears are ripe and harvested, measuring is carried out, five points are selected in a test field, 30 bergamot pears are randomly picked in east, west, south and north directions of a bergamot pear tree at each point, the bergamot pears are respectively weighed, and the average value is the single fruit weight.
(2) Yield: the total number of fruits of each tree is accurately counted, the yield of each plant is calculated according to the product of the total number of the fruits of each tree and the average single fruit quality of the tree, and the total yield which is reduced to hectare after the yield of each plant is measured.
Example 1 isolation, screening and characterization of Bacillus amyloliquefaciens xl96-3
(I) separation and purification
The fragrant pear flowers were collected from the fragrant pear garden of Kuerle city, xinjiang in 2019 years, 10 of the fragrant pear flowers were placed in a soil sample and 20mL of physiological saline, shaken on a shaking table for 30min, and left to stand for clarification. The supernatant is taken and diluted with sterile water 10 respectively -1 、10 -2 、10 -3 、10 -4 、10 -5 Five concentration gradients, water bath at 80 ℃ for 10min. The diluted solution was pipetted at 200. Mu.L and spread on an LB medium plate, and the plate was cultured in an incubator at 30 ℃ for 24 hours. Single colonies were picked from the plates and inoculated into fresh isolatesPurification was performed in plates, and 3-4 times repeated for purification. Inoculating the purified Bacillus amyloliquefaciens xl96-3 on a culture medium, culturing at constant temperature of 30 ℃ for 3-6 days in an incubator, and storing at 4 ℃ for later use.
(II) Classification and identification
Sequencing and analysis of 16SrRNA and gyrB genes of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 (hereinafter, referred to as 'strain xl 96-3') are as follows:
(1) Extraction of PCR template DNA
Inoculating the strain xl96-3 into an LB culture medium, carrying out shake culture at the rotation speed of 180r/min and the temperature of 30 ℃ for 10h, centrifugally collecting thalli, and extracting the genome DNA by adopting a novel plant genome DNA rapid extraction kit.
(2) PCR amplification
PCR amplification primers for 16Sr RNA:
UP1f：5’-GGATCCTAATACATGCAAGTCGAGCGG-3’；
UP2r：5’-GGATCCACGTATTACCGCGGCTGCTGGC-3’。
PCR amplification primers for the gyrB gene:
UP1f：5’-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3’；
UP2r：5’-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3’。
reaction system:
| composition of | Volume/. Mu.L |
| 2XMix | 25 |
| UP2r | 1 |
| UP2r | 1 |
| Fungus sample template | 1 |
| |
22 |
The PCR amplification conditions were: pre-denaturation at 95 deg.C for 4min; denaturation at 94 deg.C for 1min; annealing at 62 deg.C for 1min; extending for 72 ℃ for 2min;34 cycles of treatment; extension at 72 deg.C, 10min, and storage at 4 deg.C.
(3) Sequence determination
The PCR amplification product is subjected to electrophoresis detection and purification, then sequencing is carried out, 16S rDNA and gyrB sequences of the strain xl96-3 are sequenced, DNAstar software is used for splicing to obtain partial ordered sequences of 16SrDNA, the lengths of the partial ordered sequences are 1400bp and 1200bp respectively, the sequences are shown as SEQ ID NO 1 and SEQ ID NO 2, BLAST homologous sequence retrieval is carried out on Gen Bank respectively, MEGA 5.0 software commonly adopted in the field is used for establishing a phylogenetic tree (repeated sampling is carried out for 1000 times) through a Neighbor-Joining method, and the result is shown in attached figures 2-3. Through comparison analysis, the 16Sr DNA gene sequence of the strain xl96-3 has the highest homology with Bacillus amyloliquefaciens Lx-11, the similarity is 99 percent, and the gyrB gene sequence of the strain xl96-3 has the highest homology with Bacillus amyloliquefaciens CC178, the similarity is 96 percent; in a phylogenetic tree constructed by a 16Sr DNA gene sequence, the 16Sr DNA sequence of the strain xl96-3 has the closest genetic relationship with Bacillus amyloliquefaciens (accession number: HQ 179100.1), and the confidence coefficient is 99; in a phylogenetic tree constructed by a gyrB gene sequence, the gyrB sequence of a strain xl96-3 is also closest to the genetic relationship of Bacillus amyloliquefaciens (accession number: JQ 658430.1), the confidence coefficient is 98, the support rate of the strain xl96-3 as a new strain is very high, the strain xl96-3 has excellent stability in the phylogenetic tree, and the strain number xl96-3 in the category of the obtained Bacillus (Bacillus) belongs to a typical new strain through comprehensive judgment of similarity and homology of the strain and molecular level identification of a well-known and accepted strain system in the field.
Based on the biological characteristics, the strain xl96-3 is identified as a new strain in the branch of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens). The strain has been deposited in the Budapest treaty International Collection of microorganisms: china general microbiological culture Collection center (CGMCC), address: west road No. 1, north west of the township, beijing, ministry of sciences, china, institute of microbiology, zip code: 100101, date of deposit: and 28 days 8 and 28 in 2020, the preservation number is CGMCC No:20561.
example 2: bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 CGMCC No:20561 measurement of physiological and biochemical Properties
The physiological and biochemical properties of strain xl96-3 are shown in Table 1.
Strain xl96-3 gram-positive bacterium, rod-shaped, which is able to utilize D-xylose, citrate, hydrolyze starch, but not L-arabinose, D-mannitol, and propionate. The physiological and biochemical tests such as V-P test, gelatin liquefaction, nitrate reduction and the like are positive. The test of salt demand and salt tolerance shows that the pH value is 5.7, and the salt concentration is 7 percent, which is positive. According to morphological characteristics and physiological and biochemical characteristics of the strain xl96-3 and Bergey's Manual of identification of bacteria, the physiological and biochemical characteristics have greater similarity with gram-positive bacillus.
Table 1: measurement of physiochemical Properties
| Physiological and biochemical characteristics | xl96-3 |
| Gram stain | + |
| D-xylose | + |
| L-arabinose | - |
| D-mannitol | - |
| Citrate utilization | + |
| Propionate salts | - |
| Nitrate reduction | + |
| Starch hydrolysis | + |
| Liquefaction of gelatin | + |
| V-P | + |
| Growth at pH5.7 | + |
| 7% NaCl growth | + |
Through the test of the physiological and biochemical properties of the strain xl96-3, the strain xl96-3 is identified as a new species of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) according to the Bacillus and the microbial taxonomy and the general bacteria system identification manual.
By integrating the 16S rDNA gene and gyrB sequence, homology comparison analysis and physicochemical test comparison results, the strain xl96-3 provided by the invention has a distinct difference with the strains in the same genus range of common Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and has the characteristics of new strains in the same genus of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens).
Example 3: preparation of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 microbial agent
In this embodiment, on the basis of embodiment 1-2, the preparation method of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 microbial agent specifically includes the following steps:
(1) Activating the xl96-3 of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) stored at low temperature on an LB plate culture medium, selecting a single colony on the LB slant culture medium, and culturing for 20-26h in an incubator at 28-32 ℃ to obtain the activated xl96-3 strain of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens).
(2) And (2) scraping a ring of the activated Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 strain in the step (1) by using a sterile inoculating ring, inoculating the strain into 100mL of LB liquid culture medium, and performing shake culture for 20-26h at the temperature of 28-32 ℃ and the rotating speed of a shaking table of 120-140r/min to obtain a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 seed solution.
(3) Inoculating the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 seed liquid obtained in the step (2) into a triangular flask of a Landy liquid culture medium according to the inoculation amount of 4-6% by mass volume ratio for shake flask fermentation, and performing shake culture at the temperature of 28-32 ℃ and 180-220r/min for 32-48h to obtain the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 fermentation liquid.
(4) And (3) detecting the quantity of the bacteria and spores in the fermentation liquid obtained in the step (3), stopping fermentation culture when mature spores in the fermentation liquid account for 90% of the total quantity of the spores and the bacteria, and preparing a liquid or solid preparation of the Bacillus amyloliquefaciens xl96-3 microbial agent according to needs.
Example 4: preparation of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 microbial agent
In this embodiment, on the basis of embodiments 1 to 3, a preparation method of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 microbial agent is provided, wherein the inoculation amount of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 seed liquid is 5%, the fermentation temperature is 30 ℃, and the fermentation time is 40h.
Example 5: preparation of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 microbial agent
This example provides a preparation method of Bacillus amyloliquefaciens xl96-3 microbial inoculum based on examples 1-3, wherein the inoculation amount of Bacillus amyloliquefaciens xl96-3 seed liquid is 4%, the fermentation temperature is 28 ℃, and the fermentation time is 32h.
Example 6: preparation of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 microbial agent
This example provides a preparation method of Bacillus amyloliquefaciens xl96-3 microbial inoculum based on examples 1-3, wherein the inoculation amount of Bacillus amyloliquefaciens xl96-3 seed liquid is 6%, the fermentation temperature is 32 ℃, and the fermentation time is 48h.
Example 7: preparation of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 microbial agent
This example provides a preparation method of Bacillus amyloliquefaciens xl96-3 microbial inoculum based on examples 1-3, wherein the inoculation amount of Bacillus amyloliquefaciens xl96-3 seed liquid is 5%, the fermentation temperature is 30 ℃, and the fermentation time is 44 hours.
Example 8: bacteriostatic action of strain xl96-3 on pear blossom blight
And (3) absorbing 5mL of liquid culture 24h of the bacteria liquid of the pear flower rot pathogenic bacteria by adopting a plate bacteriostasis method, uniformly shaking in an LB culture medium cooled to about 40 ℃, and then pouring the mixture into a plate for blow-drying. And (3) sterilizing a filter paper sheet in the flat plate, sucking 5 mu L of bacillus liquid cultured for 24 hours in liquid, dropwise adding the bacillus liquid on the filter paper sheet, and culturing for 20 hours at the constant temperature of 30 ℃. The bacteriostatic effect of the strain xl96-3 on the pear blossom blight bacteria is observed, and the result is shown in figure 3.
As can be seen from the results shown in the attached figure 3, the diameter of the inhibition zone of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 on the Pyrolusitum roseum is 9.33mm, which shows that the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 has obvious inhibition effect on the growth of the Pyrolusitum roseum and has the biological control potential for preventing and controlling the Pyrolusitum roseum.
Example 9: application of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 microbial agent in prevention and control of pyricularia bergamia
(1) Effect test of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 microbial agent on Dangshan pear rot disease
Collecting large buds of Dangshan pear in the flowering period, collecting pollen after the pollen is dried in the shade, mixing the pollen with a biocontrol microbial inoculum 1:1, sowing the pollen on a solid culture medium of 0.1g/L boric acid, 100g/L cane sugar and 10g/L agar, sowing the pollen alone as a control, repeating the steps for 3 times, performing dark culture at the temperature of 20 ℃ for 4 hours, and counting the pollen germination rate. For each treatment 3 fields were observed, with pollen grains not less than 30 per field, and germination was considered as the pollen tube length being greater than the pollen grain diameter, with specific results shown in FIG. 4.
As shown in the result of the attached figure 4, the germination rates of the independently sowed pollen and the mixed germination rates of the biocontrol bacteria and the pollen are all about 83 percent, and no obvious difference exists, which indicates that the Bacillus amyloliquefaciens xl96-3 strain provided by the invention has no bacteriostatic action on the pear flower rot germination.
(2) Effect-preventing test of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 microbial agent for pear blossom rot
In the experiment, the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 microbial agent prepared in the embodiment 4 is diluted by 500 times, 1000 times and 5000 times respectively and then subjected to leaf surface spraying treatment and a bee guide box, the commercially available plant source control agent, the bee guide box, the chemical control agent and the bee guide box are sprayed according to instructions, meanwhile, the embodiments 5 to 7 of the invention are also tested, the morbidity and the final yield of the rosewood flower rot are counted, and the specific results are shown in the table 2 and the attached drawing 5.
Table 2: compared with the control effect of other fragrant pear flower rot control agents
As can be seen from the data in table 2 and attached fig. 5, when bees are sprayed and released on the leaf surfaces of bergamot pears in the full-bloom period, the bio-control effect of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 microbial inoculum is remarkably enhanced compared with that of commercially available plant-source control agents and chemical control agents, and the bio-control effect on the flower rot and branch blight of the bergamot pears can reach over 90%, wherein the bio-control effect of the microbial inoculum stock solution and the microbial inoculum diluted 5000 times provided in example 4 is the best, the weight and the yield of a single fruit are remarkably improved, the weight of the single fruit is improved by 4.3% -7.7% compared with that of a commercially available plant-source control agent and chemical control agent test group, and the yield is improved by 19.6% -23.5%; further statistics is carried out on the number of bees, and the Bacillus amyloliquefaciens xl96-3 microbial inoculum provided by the invention is found to have small influence on the bees in the spraying process, the phenomenon that the number of the bees is rapidly reduced is avoided, the number of the bees in the spraying process of the chemical control agent is obviously reduced, and the phenomenon that the bees die is caused, so that the Bacillus amyloliquefaciens xl96-3 microbial inoculum provided by the invention has small side effect and good control effect on the pear blossom blight.
The tests show that the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) xl96-3 separated and screened from pear flowers in a Kurla fragrant pear orchard in Xinjiang, the strain xl96-3 and a microbial agent thereof have remarkable biological control capacity on the flower rot of fragrant pears, the average diameter of a bacteriostatic circle of the strain xl96-3 on the branch blight of fragrant pears is 9.33mm, the biological control effect of the fragrant pears can reach more than 90 percent by spraying and releasing bees on leaf surfaces in the full-bloom period of fragrant pears, the yield is improved by 19.6 to 23.5 percent through yield statistics, and the biological control method has wide application value on the flower rot of fragrant pears.
The above examples are merely illustrative for clearly illustrating the present invention and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications can be made while remaining within the scope of the present invention.
Sequence listing
<110> Bazhou Calamu agricultural science and technology Co., ltd
<120> bacillus amyloliquefaciens microbial agent and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1451
<212> DNA
<213> Bacillus amyloliquefaciens (Bacillus amyloliquefaciens XL 96-3)
<400> 1
agaggcgggg tgctataatg caagtcgagc ggacagatgg gagcttgctc cctgatgtta 60
gcggcggacg ggtgagtaac acgtgggtaa cctgcctgta agactgggat aactccggga 120
aaccggggct aataccggat ggttgtttga accgcatggt tcagacataa aaggtggctt 180
cggctaccac ttacagatgg acccgcggcg cattagctag ttggtgaggt aacggctcac 240
caaggcgacg atgcgtagcc gacctgagag ggtgatcggc cacactggga ctgagacacg 300
gcccagactc ctacgggagg cagcagtagg gaatcttccg caatggacga aagtctgacg 360
gagcaacgcc gcgtgagtga tgaaggtttt cggatcgtaa agctctgttg ttagggaaga 420
acaagtgccg ttcaaatagg gcggcacctt gacggtacct aaccagaaag ccacggctaa 480
ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttgtccggaa ttattgggcg 540
taaagggctc gcaggcggtt tcttaagtct gatgtgaaag cccccggctc aaccggggag 600
ggtcattgga aactggggaa cttgagtgca gaagaggaga gtggaattcc acgtgtagcg 660
gtgaaatgcg tagagatgtg gaggaacacc agtggcgaag gcgactctct ggtctgtaac 720
tgacgctgag gagcgaaagc gtggggagcg aacaggatta gataccctgg tagtccacgc 780
cgtaaacgat gagtgctaag tgttaggggg tttccgcccc ttagtgctgc agctaacgca 840
ttaagcactc cgcctgggga gtacggtcgc aagactgaaa ctcaaaggaa ttgacggggg 900
cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt 960
cttgacatcc tctgacaatc ctagagatag gacgtcccct tcgggggcag agtgacaggt 1020
ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc 1080
aacccttgat cttagttgcc agcattcagt tgggcactct aaggtgactg ccggtgacaa 1140
accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg ggctacacac 1200
gtgctacaat ggacagaaca aagggcagcg aaaccgcgag gttaagccaa tcccacaaat 1260
ctgttctcag ttcggatcgc agtctgcaac tcgactgcgt gaagctggaa tcgctagtaa 1320
tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca 1380
ccacgagagt ttgtaacacc cgaagtcggt gaggtaacct tttaggagcc agccgccgaa 1440
ggtcacagat t 1451
<210> 2
<211> 1193
<212> DNA
<213> Bacillus amyloliquefaciens (Bacillus amyloliquefaciens XL 96-3)
<400> 2
gggcacatga cgctagatta cctgctggga tacgcgcttt ttcaagatta aaatcttctc 60
cgattcctgt tccgagggcc gtgatcattg atctgacctc attgtttgag agaattttat 120
caagtctggc tttctcaacg ttcagaatct taccgcgcag cggcagaatg gcttggaaat 180
gacggtcccg tccctgtttc gctgatccgc ccgcagagtc accctctacg atatacagct 240
cggaaatgct cggatcttta gaagaacagt ccgccagttt gcccggcaga ttggaaatct 300
caagcgcact tttgcggcgg gtcaattccc gcgctttttt cgctgccatc cgcgctcttg 360
cggccattaa acctttttca acgattttgc gggctgagtc cggattttca agaaggaatg 420
tttccagcgc agaagaaaac agcgtatcag tgatcgttct cgcttcggag ttgccgagct 480
tcgttttcgt ctgcccttcg aattgcggat cagggtgctt aattgaaata atggcagtca 540
gcccttctct cacatcatcc ccgcttaaat tcggatcatt ttctttgaaa atcccttttc 600
ttcttgcata gtcgtttatg acacgggtca gaccggtttt aaatccggct tcgtgcgtcc 660
cgccttcgta tgtgttgatg ttatttgtga aagaataaat gttgcttgta tagctgtcgt 720
tgtattgcaa cgcaacttca accgttatgc cgtctttctc gccttcgata taaatcggct 780
cttcatgaac gacttctttg gaacggttta agtactcaac atagcttttg attccgcctt 840
cgtagtggta ctcgtttttc cgttcttgtc cttcacgttt gtcttcaatc gtgatgttta 900
cgccttttgt caggaaggcc aattcccgga cacggtttga aagcagatca tagtcgtata 960
cgattgtttc tttgaagatt tccggatccg gaacgaagtg cgtaatcgtt ccggtcttat 1020
cagtatcacc gatcacttca agatcagcca caggtacacc gcgctcgtac gcctgatagt 1080
ggatttttcc gtcacgatga accgtaacgt caagagtggt cgacaaggcg tttacgacgg 1140
acgcccctac accgtgaaga ccgccggata catatatccg ctgcagtatc cgc 1193
Claims (4)
1. Bacillus amyloliquefaciens (A)Bacillus amyloliquefaciens) The xl96-3 microbial agent is characterized in that the microbial agent is prepared from bacillus amyloliquefaciens (Bacillus amyloliquefaciens)Bacillus amyloliquefaciens) xl96-3 is obtained by fermentation; the Bacillus amyloliquefaciens (A), (B), (C) and (C)Bacillus amyloliquefaciens) The xl96-3 is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 20561.
2. The Bacillus amyloliquefaciens of claim 1(Bacillus amyloliquefaciens) The preparation method of the xl96-3 microbial agent is characterized by comprising the following steps:
(1) Bacillus amyloliquefaciens to be preserved at low temperature (Bacillus amyloliquefaciens) xl96-3 is activated on an LB plate culture medium, a single colony is selected on an LB slant culture medium and cultured for 20-26h in an incubator at 28-32 ℃ to obtain the activated bacillus amyloliquefaciens (A)Bacillus amyloliquefaciens) xl96-3 strain;
(2) Scraping a ring of the activated Bacillus amyloliquefaciens of step (1) with a sterile inoculating ring (Bacillus amyloliquefaciens) Inoculating xl96-3 CGMCC No. 20561 strain into 100ml LB liquid culture medium, shake culturing at 28-32 deg.C and 120-140-r/min to 20-26-h to obtain Bacillus amyloliquefaciens (CGMCC No. 20561)Bacillus amyloliquefaciens) xl96-3 seed liquid;
(3) The bacillus amyloliquefaciens obtained in the step (2) ((Bacillus amyloliquefaciens) Inoculating xl96-3 seed solution into a triangular flask of Landy liquid culture medium according to the inoculation amount of 4-6% by mass-volume ratio for shake flask fermentation, and performing shake culture at 28-32 ℃ and 180-220r/min for 32-48h to obtain the bacillus amyloliquefaciens (Bacillus amyloliquefaciens)Bacillus amyloliquefaciens) xl96-3 fermentation broth;
(4) Detecting the quantity of the bacteria and spores in the fermentation liquid obtained in the step (3), stopping fermentation culture when mature spores in the fermentation liquid account for 90 percent of the total quantity of the spores and the bacteria, and preparing and obtaining the bacillus amyloliquefaciens according to the requirementBacillus amyloliquefaciens) A liquid or solid formulation of xl96-3 microbial agent.
3. The Bacillus amyloliquefaciens of claim 2Bacillus amyloliquefaciens) A process for producing an xl96-3 microorganism bacterium agent, which comprises hydrolyzingBacillus amyloliquefaciens (A)Bacillus amyloliquefaciens) The inoculation amount of xl96-3 seed liquid is 5%, the fermentation temperature is 30 ℃, and the fermentation time is 40h.
4. The Bacillus amyloliquefaciens of claim 1Bacillus amyloliquefaciens) An application of an xl96-3 microbial agent in preparation of a medicament for preventing and treating pear flower rot.
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