CN113303340A - Composition of biocontrol bacteria and fish protein and application of composition in cucumber cultivation - Google Patents
Composition of biocontrol bacteria and fish protein and application of composition in cucumber cultivation Download PDFInfo
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Abstract
A biocontrol bacteria and fish protein composition comprises a bacillus composite bacteria culture solution and a fish protein diluent, wherein the bacillus composite bacteria culture solution comprises XY-1, XY-13 and XY-53; XY-1 and XY-13 are Bacillus amyloliquefaciens, and XY-53 is Bacillus subtilis; wherein XY-1, XY-13 and XY-53 are all preserved in China Center for Type Culture Collection (CCTCC) at 24 months and 3 months in 2021, and Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) XY-1 has a preservation number: CCTCC NO: M2021269; bacillus amyloliquefaciens (Bacillus amyloliquefaciens) XY-13 preservation number: CCTCC NO: M2021270; bacillus subtilis XY-53 with the deposit number: CCTCC NO: M2021271. Through field tests, the biocontrol bacteria and fish protein composition disclosed by the invention has a remarkable effect of preventing and treating root rot, and also has a remarkable effect of promoting the growth of root systems, stems and leaves of cucumbers. In addition, the biocontrol bacteria and fish protein composition also has a certain effect on soil remediation.
Description
Technical Field
The invention relates to the technical field of cultivation of vegetable crops, in particular to a composition of biocontrol bacteria and fish protein and application thereof in cucumber cultivation.
Background
The cucumber is crisp and tasty, is rich in various nutrient elements such as vitamin A, vitamin C and the like, can clear away heat and toxic materials, beautify the face, reduce summer heat and the like, and is a good vegetable product which is popular in the life of people. Cucumber is a main cultivation variety of facility vegetables, is a vegetable crop with better economic benefit in agricultural planting, and is one of the most important edible vegetables in daily diet of people. With the gradual deepening of the modern agricultural operation idea, more and more farmers, family farms and modern agricultural parks are engaged in the scale production of vegetables. At present, with the improvement of living standard of people, cucumbers become conventional vegetables, the market demand is increased day by day, and the cucumber vegetable has wide market prospect in annual demand.
Due to successive cropping and excessive fertilization, the continuous cropping obstacle of soil is caused, which is mainly characterized in that soil-borne diseases are serious, and diseases of cucumber roots caused by various fungi are gradually increased, wherein the cucumber root rot caused by fusarium solani is serious, and the production of cucumbers is seriously threatened. How to improve the continuous cropping obstacle soil and dig the cucumber production potential is an important way for ensuring the cucumber production in China, improving the comprehensive production capacity of the cucumber and promoting the sustainable development of the cucumber industry.
Disclosure of Invention
In view of the above, there is a need for a composition of biocontrol bacteria and fish protein for preventing and treating root rot of cucumber and promoting cucumber growth.
A composition of biocontrol bacteria and fish protein comprises a bacillus composite bacteria culture solution and a fish protein diluent, wherein the components of the bacillus composite bacteria culture solution comprise XY-1, XY-13 and XY-53; XY-1 and XY-13 are Bacillus amyloliquefaciens, and XY-53 is Bacillus subtilis;
wherein XY-1, XY-13, XY-53 are preserved in China Center for Type Culture Collection (CCTCC) at 24/3/2021,
bacillus amyloliquefaciens (A)Bacillus amyloliquefaciens) XY-1, accession number: CCTCC NO: M2021269;
bacillus amyloliquefaciens (A)Bacillus amyloliquefaciens) XY-13 accession number: CCTCC NO: M2021270;
bacillus subtilis (A), (B) and (C)Bacillus subtilis) XY-53, accession number: CCTCC NO: M2021271.
Preferably, the concentration of the bacillus composite bacteria culture solution is 108cfu/mL, wherein the fish protein diluent is 200 times of the fish protein diluent, the volume ratio of the fish protein diluent to the bacillus composite bacteria culture solution is 1:1, and the concentration ratio of XY-1, XY-13 and XY-53 is 1:1: 1.
preferably, the bacillus subtilis and the bacillus amyloliquefaciens are prepared by placing a screened strain in an LB liquid culture medium at 30 ℃ and shaking culture at 180rmp/min for 24 hours, centrifuging at 10000r/min for 5 minutes, taking supernatant, and adjusting the concentration to 1 x 108cfu/mL.
Preferably, the LB liquid medium comprises 10.0g of tryptone, 5.0g of yeast extract powder, 10.0g of sodium chloride and 1000mL of distilled water, and the pH of the LB liquid medium is 7.0 +/-0.2.
The invention also provides application of the biocontrol bacterium and fish protein composition in preventing and treating cucumber root rot.
The invention also provides application of the biocontrol bacterium and fish protein composition in promoting cucumber growth.
Has the advantages that: passing field tests:
compared with single bacillus subtilis, bacillus amyloliquefaciens or fish protein, the biocontrol bacteria and fish protein composition has obvious effect of preventing and treating cucumber root rot, has obvious effect of preventing and treating root rot compared with single bacteria and fish protein, and has obvious effect of preventing and treating root rot compared with the mixing of two bacteria liquids of XY-1, XY-13 and XY-53 and fish protein.
Compared with single bacillus subtilis, bacillus amyloliquefaciens or fish protein, the biocontrol bacterium and fish protein composition has the obvious effect of promoting the growth of root systems and stem leaves of cucumbers, has the obvious effect of promoting the growth of the root systems and the stem leaves of the cucumbers compared with single bacteria, and has the obvious effect of promoting the growth of the root systems and the stem leaves of the cucumbers compared with the mixing of two bacteria solutions of XY-1, XY-13 and XY-53 and the fish protein.
In addition, the biocontrol bacteria and fish protein composition also has a certain effect on soil improvement, can increase the content of organic matters and quick-acting nutrients in soil, promotes plants to absorb the nutrients in the soil, and improves continuous cropping obstacles.
Drawings
FIG. 1 shows the 16SrDNA amplification sequence electrophoresis chart of XY-1, XY-13 and XY-53 strains according to the present invention.
FIG. 2 shows a phylogenetic tree of the strains XY-1, XY-13 and XY-53 of the invention.
FIG. 3 is a graph showing the growth of strain XY-1 under the fish protein of the present invention.
FIG. 4 is a graph showing the growth of strain XY-13 under the fish protein of the present invention.
FIG. 5 is a graph showing the growth of strain XY-53 under the fish protein of the present invention.
Detailed Description
The present invention will be described below with reference to specific examples, but the present invention is not limited thereto.
The test methods used in the following examples are all conventional methods unless otherwise specified; the fish proteins used in the following examples were purchased commercially, and the Bacillus subtilis and Bacillus amyloliquefaciens culture medium was screened for plant rhizosphere disease-resistant strains and cultured in shake flasks under laboratory conditions.
The fish protein is prepared from deep-sea wild fish as a raw material by low-temperature high-pressure crushing and then carrying out biological enzymolysis, and generally contains 2-20 amino acids, 35-42% of fish protein, more than 50% of protein polymeric organic matter, more than 35% of small peptide, 35-45% of 18 amino acids, more than 2.1% of organic matter, more than 1.1% of organic phosphorus and 5-6.5 of pH. Can be purchased in the market, such as the Zhoushanfengyutsu ocean biological fertilizer company Limited.
The bacillus subtilis XY-53 and the bacillus amyloliquefaciens XY-1 and XY-13 are prepared by placing a screened strain in LB liquid culture medium at 30 ℃ and shaking culture at 180rmp/min for 24h, centrifuging at 10000r/min for 5min, taking supernatant, mixing the three supernatants according to the ratio of 1:1:1, and adjusting the concentration to 1 × 108cfu/mL. The LB liquid culture medium comprises 10.0g of tryptone, 5.0g of yeast extract powder, 10.0g of sodium chloride and 1000mL of distilled water, and the pH value of the LB liquid culture medium is 7.0 +/-0.2. The selected strains were obtained from soil.
After the selection strain was PCR-amplified with 16SrDNA, a band of about 1,500bp was observed in agarose gel, as shown in FIG. 1. A phylogenetic tree was constructed based on the gene sequences of biocontrol bacteria XY-1, XY-13 and XY-53 as shown in FIG. 2. Please refer to the gene sequence table, the gene sequences of XY-1 and XY-13 and MN365047.1:92-1383Bacillus siamensis strain、MN149347.1:79-1370 Bacillus vallismortis strain、MN365058.1:59-1350 Bacillus subtilis strain、MN559948.1:60-1351 Bacillus amyloliquefaciens strain、MN700268.1:46-1337 Bacillus velezensis strain、MN756640.1:82-1373 Bacillus amyloliquefaciensThe gene sequences of strain have 99% similarity, are in the same branch, and are analyzed by combining morphological characteristics, culture characteristics and physiological and biochemical characteristics, XY-1 and XY-13 are identified as bacillus amyloliquefaciens; the gene sequence of XY-53 is related to MN750777.1:64-1354Bacillus tequilensis strain、MN750756.1:65-1355 Bacillus subtilisStrain and MN704467.1:87-1377Bacillus velezensis strain、MN365236.1:65-1355 Bacillus siamensisThe gene sequence of strain has 99% similarity, is in the same branch, and is identified as bacillus subtilis by combining morphological characteristics, culture characteristics and physiological and biochemical characteristic analysis XY-53.
Wherein XY-1, XY-13, XY-53 are preserved in China Center for Type Culture Collection (CCTCC) at 24/3/2021,
bacillus amyloliquefaciens (A)Bacillus amyloliquefaciens) XY-1, accession number: CCTCC NO: M2021269;
bacillus amyloliquefaciens (A)Bacillus amyloliquefaciens) XY-13, accession number: CCTCC NO: M2021270;
bacillus subtilis (A), (B) and (C)Bacillus subtilis) XY-53, accession number: CCTCC NO: M2021271.
The following examples 1 to 3 are tests of the pharmaceutical composition of the present invention for controlling cucumber root rot.
Example 1: single bacterium disease resistance test.
Cucumber seeds (Dell 99) purchased from Tianjin Derui Special industries, Inc., are sterilized, soaked, cultured, grown to two leaves and one heart (15 days), and subjected to evaluation of prevention and control effects of the strains XY-1, XY-13 and XY-53 potted plants, which are respectively 10mL104The cfu/mL fusarium solani suspension is treated by root irrigation and is completely inoculated with 15mL10 mL after 24 hours8And (3) resuspending cfu/mL strain resuspension, treating 15 strains each, repeating for 3 times, investigating the disease condition 7 days after inoculation, and calculating the disease index and the prevention effect.
Treatment 1: only irrigate 10mL104cfu/mL fusarium solani suspension
And (3) treatment 2: 10mL104cfu/mL eggplant sickleBacterial suspension +15 mL108cfu/mL XY-1 Strain resuspension
And (3) treatment: 10mL104cfu/mL Fusarium solani suspension +15 mL108cfu/mL XY-13 Strain resuspension
And (4) treatment: 10mL104cfu/mL Fusarium solani suspension +15 mL108cfu/mL XY-53 Strain resuspension
The potting effect shows that after the inoculation of the bacterial suspensions XY-1, XY-13 and XY-537 days, the disease indexes are 33.33, 26.67 and 13.33, and the control effects are 65.12%, 72.09% and 82.86% respectively.
Table one: single bacterium field test effect
Example 2: and (4) performing compound bacteria disease resistance test.
Cucumber seeds (Dell 99) purchased from Tianjin Derui Special industries, Inc., are sterilized, soaked, cultivated, grown to have two leaves and one heart (15 days), and potted control effect evaluation is carried out on the compound strains XY1-13, XY1-53, XY-13 and XY-53, respectively using 10mL104The cfu/mL fusarium solani suspension is treated by root irrigation and is inoculated with 15mL10 after 24 hours8cfu/mL composite strain resuspension (total concentration of composite strain is 10)8cfu/mL), 15 strains are treated, the treatment is repeated for 3 times, the disease condition is investigated 7 days after inoculation, and the disease index and the control effect are calculated.
Treatment 1: only irrigate 10mL104cfu/mL fusarium solani suspension
And (3) treatment 2: 10mL104cfu/mL Fusarium solani suspension +15 mL108cfu/mL XY-1, XY-13 Strain resuspension
And (3) treatment: 10mL104cfu/mL Fusarium solani suspension +15 mL108cfu/mL XY-1, XY-53 Strain resuspension
And (4) treatment: 10mL104cfu/mL Fusarium solani suspension +15 mL108cfu/mL XY-13, XY-53 Strain resuspension
And (4) treatment 5: 10mL104cfu/mL Fusarium solani suspension +15 mL108cfu/mL XY-1, XY-13, XY-53 Strain resuspension
The potting effect shows that after the inoculation of the bacterial suspensions XY1-13, XY1-53, XY13-53 and XY 1-13-537, the control effect is respectively 60.00%, 80.00%, 73.33% and 86.67%.
Table two: field test effect of compound bacteria
Example 3: disease resistance test of the complex bacteria and fish protein composition.
Cucumber seeds (Dell 99) purchased from Tianjin Derui Special industries, Inc., are disinfected, soaked, cultured, grown to two leaves and one heart (15 days), and then are subjected to fish protein diluent, compound strains XY-1, XY-13 and XY-53 potted plant control effect evaluation, 10mL10 is respectively used4The root of the cfu/mL fusarium solani suspension is treated by root irrigation, and after 24 hours, 15mL of 200-fold fish protein diluent (FPH) is inoculated200Double dilution), 15mL108cfu/mL composite strain resuspension (total concentration of composite strain is 10)8cfu/mL), 15 strains are treated, the treatment is repeated for 3 times, the disease condition is investigated 7 days after inoculation, and the disease index and the control effect are calculated.
Process 1 (CK process): only irrigate 10mL104cfu/mL fusarium solani suspension
And (3) treatment 2: 10mL104cfu/mL Fusarium solani suspension +15 mL108cfu/mL XY-1, XY-13 Strain resuspension +15mL FPH200Double dilution liquid
And (3) treatment: 10mL104cfu/mL Fusarium solani suspension +15 mL108cfu/mL XY-1, XY-53 Strain resuspension +15mL FPH200Double dilution liquid
And (4) treatment: 10mL104cfu/mL Fusarium solani suspension +15 mL108cfu/mL XY-13, XY-53 Strain resuspension +15mL FPH200Double dilution liquid
And (4) treatment 5: 10mL104cfu/mL Fusarium solani suspension +15 mL108cfu/mL XY-1, XY-13 and XY-53 Strain resuspension +15mL FPH200Double dilution liquid
The addition of the compound bacteria and the fish protein can effectively prevent and treat cucumber root rot caused by fusarium solani. After inoculation of XY1-13, XY1-53, XY13-53 and XY1-13-53 of compound bacterial suspension and 200 times of fish protein liquid for 7 days, the control effects on fusarium solani are 84.93%, 86.30%, 89.04% and 90.41% respectively.
Table three: control effect of medicine for controlling cucumber root rot
As can be seen from the first table, the second table and the third table, the combination of the XY-1, XY-13 and XY-53 compound bacteria and the fish protein has a remarkable effect of preventing and treating cucumber root rot compared with the combination of a single bacterium or any two of the XY-1, XY-13 and XY-53.
The following examples 4 to 8 are growth promotion tests of the pharmaceutical composition of the present invention.
Example 4: fish protein seed soaking test.
The cucumber seeds and the culture dish are sterilized by 3 percent sodium hypochlorite solution and 75 percent alcohol for standby. Soaking the seeds in the same amount of fish protein diluent and distilled water for 8 hr, soaking 30 seeds in each treatment, repeating for three times, culturing in dark box at 25 deg.C, and supplementing fish protein diluent or distilled water with appropriate concentration according to evaporation amount every day.
Treatment 1: blank control
And (3) treatment 2: 600-fold dilution of fish protein
And (3) treatment: 800-fold dilution liquid of fish protein
And (4) treatment: 1000-fold dilution liquid of fish protein
And (4) treatment 5: 1500-fold dilution liquid of fish protein
The germination vigor, the germination index and the vitality index of the fish protein processed by 1000 times of the solution are the highest and are respectively and obviously higher than 13.07 percent, 36.28 percent and 122.96 percent of the fish protein processed by CK.
Table four: growth promoting effect of fish protein on cucumber seed germination
Example 5: root irrigation test of fish protein.
The method comprises the steps of sterilizing and accelerating germination of cucumber seeds, sowing the cucumber seeds in a 50-hole seedling raising plug tray, performing a fish protein root irrigation test after the cotyledons of the cucumbers are flattened, respectively performing root irrigation on the sowed 10 th day and 20 th day, wherein each plant is 15mL, and sowing for 30 days to measure the growth vigor of the seedlings.
Treatment 1: blank control
And (3) treatment 2: 50-fold dilution liquid of fish protein
And (3) treatment: 100-fold dilution of fish protein
And (4) treatment: 200-fold dilution of fish protein
And (4) treatment 5: fish protein 400-fold diluent
Compared with treatment 1, the treatment of the fish protein diluent significantly increases the plant height, stem thickness and chlorophyll content. The stem diameter and chlorophyll content of the fish protein treated by 100 times of the diluent are the highest, and are obviously higher than 62.65% and 89.71% of that of the fish protein treated by CK. The height of the fish protein diluted liquid is 200 times of the highest height, and is obviously higher than 82.81% of CK treatment.
Table five: growth promoting effect of fish protein on cucumber seedlings
Compared with treatment 1, the root-irrigation treatment of the fish protein obviously increases the root volume, the root surface area and the total root length of the cucumber plants. The volume of the roots treated by the fish protein 200 times of the diluent is the largest, and is obviously different from that treated by other fish proteins, and is obviously higher than that treated by CK (CK) by 153.73%. The surface area of the root treated by the fish protein 200 times of diluent is the largest, and has no obvious difference with the fish protein 400 times of diluent, and is obviously higher than that treated by a blank control, the fish protein 50 times of diluent and the fish protein 100 times of diluent. The total root length of the fish protein 200-fold diluent is longest, and has no obvious difference with the fish protein 400-fold diluent, and is obviously higher than that of a blank control, the fish protein 50-fold diluent and the fish protein 100-fold diluent.
Table six: growth promoting effect of fish protein on root system of cucumber seedling
Example 6: and (3) growth promotion test of the single bacterium on cucumber seedlings.
Cucumber seeds (Dell 99) purchased from Tianjin Derui Special industries, Inc., are sterilized, soaked, cultured, grown to two leaves and one heart (15 days), and subjected to evaluation of prevention and control effects of the strains XY-1, XY-13 and XY-53 potted plants, which are respectively treated with 10mL104The cfu/mL fusarium solani suspension is treated by root irrigation and is completely inoculated with 15mL10 mL after 24 hours8cfu/mL bacterial strain re-suspension, each 15 strains, repeat 3 times, inoculated 7 days after statistics of seedling growth index.
Treatment 1: only irrigate 10mL104cfu/mL fusarium solani suspension
And (3) treatment 2: 10mL104cfu/mL Fusarium solani suspension +15 mL108cfu/mL XY-1 Strain resuspension
And (3) treatment: 10mL104cfu/mL Fusarium solani suspension +15 mL108cfu/mL XY-13 Strain resuspension
And (4) treatment: 10mL104cfu/mL Fusarium solani suspension +15 mL108cfu/mL XY-53 Strain resuspension
The potting effect shows that the plant height, stem thickness and chlorophyll of the cucumber seedlings are obviously higher than those of the cucumber seedlings treated by the treatment 1 after the inoculation of the bacterial suspensions XY-1, XY-13 and XY-537 days.
TABLE VII: growth promoting effect of single bacterium on cucumber seedlings
Example 7: and (4) compound bacteria growth promotion test.
Cucumber seeds (Dell 99) purchased from Tianjin Derui Special industries, Inc., are sterilized, soaked, cultivated, grown to have two leaves and one heart (15 days), and are subjected to evaluation of prevention and control effects of pot culture by composite strains XY1-13, XY1-53 and XY13-53, respectively using 10mL104The cfu/mL fusarium solani suspension is treated by root irrigation and is inoculated with 15mL10 after 24 hours8cfu/mL bacterial strain re-suspension, each 15 strains, repeat 3 times, inoculated 7 days after statistics of seedling growth index.
Treatment 1: only irrigate 10mL104cfu/mL fusarium solani suspension
And (3) treatment 2: 10mL104cfu/mL Fusarium solani suspension +15 mL108cfu/mL XY1-13 Strain resuspension
And (3) treatment: 10mL104cfu/mL Fusarium solani suspension +15 mL108cfu/mL XY1-53 Strain resuspension
And (4) treatment: 10mL104cfu/mL Fusarium solani suspension +15 mL108cfu/mL XY13-53 Strain resuspension
And (4) treatment 5: 10mL104cfu/mL Fusarium solani suspension +15 mL108cfu/mL XY1-13-53 Strain resuspension
The potting effect shows that after 10 days of inoculation of the strain suspensions XY1-13, XY1-53, XY13-53 and XY1-13-53, the differences of the plant height, the stem thickness and the chlorophyll are obvious from the treatment 1, the XY1-13-53 has the most obvious effects on the stem thickness and the chlorophyll growth promotion of cucumber plants, and the plant height, the stem thickness and the chlorophyll growth promotion rate are 29.84%, 34.72% and 31.28% respectively relative to the treatment 1.
Table eight: growth promoting effect of composite bacteria on cucumber seedlings
Example 8: growth promotion test of the composite bacteria and fish protein composition.
And (4) opening a pot culture test in the practical training base of the university of Ningxia, Yinchuan in China in 2019, 9 months and 22 days. The pot experiment consisted of 6 treatments, 10 each, 3 replicates, randomized block arrangement. The substrate was sterilized continuously (121 ℃, 20 min) three times and then cooled to room temperature for use. Sowing the cucumber seeds after germination acceleration in 50-hole trays with 1 grain in each hole, and respectively using 10mL10 when cucumber seedlings grow for 20 days4The cfu/mL fusarium solani suspension is treated by root irrigation and inoculated with 15mL10 after 24h8cfu/mL re-suspension of complex strain and 15mL 200-fold fish protein diluent (FPH)200Multiple dilutions), and counting the seedling growth index on day 7 after inoculation and sterilization, respectively.
Treatment 1: only irrigate 10mL104Fusarium solani cfu/mLBacterial suspension
And (3) treatment 2: 10mL104cfu/mL Fusarium solani suspension +15 mL108cfu/mL XY1-13+15mL FPH200Double dilution liquid
And (3) treatment: 10mL104cfu/mL Fusarium solani suspension +15 mL108cfu/mL XY1-53+15mL FPH200Double dilution liquid
And (4) treatment: 10mL104cfu/mL Fusarium solani suspension +15 mL108cfu/mL XY13-53+15mL FPH200Double dilution liquid
And (4) treatment 5: 10mL104cfu/mL Fusarium solani suspension +15 mL108cfu/mL XY1-13-53+15mL FPH200Double dilution liquid
The plant height, stem thickness and chlorophyll content of the inoculated composite bacteria and the fish protein treated by the method are obviously different from those of a control. The stem thickness and chlorophyll content were highest in treatment 5, and compared with treatment 1, the stem thickness was increased by 37.09%, and the chlorophyll content was increased by 40.78%. Compared with treatment 1, the plant heights under the combined bacteria and fish protein inoculation are respectively increased by 25.00%, 18.55%, 29.84% and 27.42%.
Table nine: growth promoting effect of composition on cucumber seedlings
TABLE Ten: effect of the composition on cucumber matrix soil nutrients
It can be seen from examples 4 to 8 that the composition of biocontrol bacteria and fish protein of the present invention has a significant growth promoting effect on the growth of cucumber seedlings, and the effect is significantly higher than that of other control groups.
Example 9: and (3) testing the activity of the fish protein on the spore strain.
Inoculating a single bacillus colony into a 50mL triangular flask containing 10mL LB broth, culturing at 180r/min at 30 ℃ for 24h to obtain a seed solution; taking 1mL of seed solution in a 250mL triangular flask containing 50mL of LB broth, adding 200-fold fish protein diluent according to 0.5%, 1%, 2%, 2.5%, 3% and 4% of the inoculum size (50 mL), culturing at 30 deg.C at 180r/min, and determining OD at 0, 4, 8, 12, 16, 20, 24, 48 and 72h respectively600。
Treatment 1: bacterial strain +0.25ml 200 times fish protein diluent
And (3) treatment 2: bacterial strain +0.5ml 200 times fish protein diluent
And (3) treatment: bacterial strain +1ml 200 times fish protein diluent
And (4) treatment: bacterial strain +1.25ml 200 times fish protein diluent
And (4) treatment 5: bacterial strain +1.5ml 200 times fish protein diluent
And (6) treatment: bacterial strain +2ml 200 times fish protein diluent
And (7) treatment: inoculation of the Strain alone
Referring to fig. 3 to 5, the activity of the added fish protein on the bacterial strain shows that the 200 times of liquid treatment of the fish protein has the most obvious effect of increasing the activities of XY-1, XY-13 and XY-53 within 72 hours, and the activity decline of the bacteria is delayed. Wherein 1.0 percent of the addition amount of the culture medium is cultured for 24-72h, and the effects of promoting the activity of the spore strain and delaying the decay period of the strain are comprehensively shown.
Sequence listing
<110> Ningxia university
<120> composition of biocontrol bacteria and fish protein and application thereof in cucumber cultivation
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cttcggcggc tggctcctaa aaggttacct caccgacttc gggtgttaca aactctcgtg 60
gtgtgacggg cggtgtgtac aaggcccggg aacgtattca ccgcggcatg ctgatccgcg 120
attactagcg attccagctt cacgcagtcg agttgcagac tgcgatccga actgagaaca 180
gatttgtggg attggcttaa cctcgcggtt tcgctgccct ttgttctgtc cattgtagca 240
cgtgtgtagc ccaggtcata aggggcatga tgatttgacg tcatccccac cttcctccgg 300
tttgtcaccg gcagtcacct tagagtgccc aactgaatgc tggcaactaa gatcaagggt 360
tgcgctcgtt gcgggactta acccaacatc tcacgacacg agctgacgac aaccatgcac 420
cacctgtcac tctgcccccg aaggggacgt cctatctcta ggattgtcag aggatgtcaa 480
gacctggtaa ggttcttcgc gttgcttcga attaaaccac atgctccacc gcttgtgcgg 540
gcccccgtca atttcctttg agtttcagtc ttgcgacccg tactcccccc agggcggagt 600
gcttaatgcg ttagctgcag cactaagggg cggaaacccc ctaacacttt agcactccat 660
cgtttacggc gtggactacc agggtatcta atcctgttcg ctccccacgc tttcgctcct 720
cagcgtcagt tacagaccag agagtcgcct tcgccactgg tgttcctcca catctctacg 780
catttcaccg ctacacgtgg aattccactc tcctcttctg cactcaagtt ccccagtttc 840
caatgaccct ccccggttga gccgggggct ttcacatcag acttaagaaa ccgcctgcga 900
gccctttacg cccaataatt ccggacaacg cttgccacct acgtattacc gcggctgctg 960
gcacgtagtt agccgtggct ttctggttag gtaccgtcaa ggtgccgccc tatttgaacg 1020
gcacttgttc ttccctaaca acagagcttt acgatccgaa aaccttcatc actcacgcgg 1080
cgttgctccg tcagactttc gtccattgcg gaagattccc tactgctgcc tcccgtagga 1140
gtctgggccg tgtctcagtc ccagtgtggc cgatcaccct ctcaggtcgg ctacgcatcg 1200
tcgccttggt gagccgttac ctcaccaact agctaatgcg ccgcgggtcc atctgtaagt 1260
ggtagccgaa gccacctttt atgtctgaac catgcggttc aaacaaccat ccggtattag 1320
ccccggtttc ccggagttat cccagtctta caggcaggtt acccacgtgt tactcacccg 1380
tccgccgcta acatcaggga gcaagctccc atctgtccgc tcgacttgca gtat 1434
<210> 2
<211> 1427
<212> DNA
<213> Bacillus amyloliquefaciens XY-13(Bacillus amyloliquefaciens XY-13)
<400> 2
ttcggcggct ggctcctaaa aggttacctc accgacttcg ggtgttacaa actctcgtgg 60
tgtgacgggc ggtgtgtaca aggcccggga acgtattcac cgcggcatgc tgatccgcga 120
ttactagcga ttccagcttc acgcagtcga gttgcagact gcgatccgaa ctgagaacag 180
atttgtggga ttggcttaac ctcgcggttt cgctgccctt tgttctgtcc attgtagcac 240
gtgtgtagcc caggtcataa ggggcatgat gatttgacgt catccccacc ttcctccggt 300
ttgtcaccgg cagtcacctt agagtgccca actgaatgct ggcaactaag atcaagggtt 360
gcgctcgttg cgggacttaa cccaacatct cacgacacga gctgacgaca accatgcacc 420
acctgtcact ctgcccccga aggggacgtc ctatctctag gattgtcaga ggatgtcaag 480
acctggtaag gttcttcgcg ttgcttcgaa ttaaaccaca tgctccaccg cttgtgcggg 540
cccccgtcaa ttcctttgag tttcagtctt gcgaccgtac tccccaggcc ggagtgctta 600
atgcgttagc tgcagcacta agggggcgga aaccccccta acccattagc actcatcgtt 660
tacggcgtgg actaccaggg gtatctaatc ctgttcgctc cccacgcttt cgctcctcag 720
cgtcagttac agaccagaag agtcgccttc gccaactggt gttcctccac attctctacg 780
catttcaccg ctacaccgtg gaattccact ctccctcttc tgcactcaag ttccccagtt 840
tccaatgacc ctcccccggt tgagccgggg gctttcacat cagacttaag aaaccgcctg 900
cgagcccttt acgcccaata attccggaca acgcttgcca cctacgtatt accgcggctg 960
ctggcacgta gttagccgtg gctttctggt taggtaccgt caaggtgccg ccctatttga 1020
acggcacttg ttcttcccta acaacagagc tttacgatcc gaaaaccttc atcactcacg 1080
cggcgttgct ccgtcagact ttcgtccatt gcggaagatt ccctactgct gcctcccgta 1140
ggagtctggg ccgtgtctca gtcccagtgt ggccgatcac cctctcaggt cggctacgca 1200
tcgtcgcctt ggtgagccgt tacctcacca actagctaat gcgccgcggg tccatctgta 1260
agtggtagcc gaagccacct tttatgtctg aaccatgcgg ttcagacaac catccggtat 1320
tagccccggt ttcccggagt tatcccagtc ttacaggcag gttacccacg tgttactcac 1380
ccgtccgccg ctaacatcag ggagcaagct cccatctgtc cgctcga 1427
<210> 3
<211> 1428
<212> DNA
<213> Bacillus subtilis XY-53(Bacillus subtilis XY-53)
<400> 3
cttcggcggc tggctcctaa aaggttacct caccgacttc gggtgttaca aactctcgtg 60
gtgtgacggg cggtgtgtac aaggcccggg aacgtattca ccgcggcatg ctgatccgcg 120
attactagcg attccagctt cacgcagtcg agttgcagac tgcgatccga actgagaaca 180
gatttgtggg attggcttaa cctcgcggtt tcgctgccct ttgttctgtc cattgtagca 240
cgtgtgtagc ccaggtcata aggggcatga tgatttgacg tcatccccac cttcctccgg 300
tttgtcaccg gcagtcacct tagagtgccc aactgaatgc tggcaactaa gatcaagggt 360
tgcgctcgtt gcgggactta acccaacatc tcacgacacg agctgacgac aaccatgcac 420
cacctgtcac tctgcccccg aaggggacgt cctatctcta ggattgtcag aggatgtcaa 480
gacctggtaa ggttcttcgc gttgcttcga attaaaccac atgctccacc gcttgtgcgg 540
gcccccgtca attcctttga gtttcagtct tgcgaccgta ctccccaggc ggagtgctta 600
atgcgttagc tgcagcacta aggggcggaa accccctaac acttagcact catcgtttac 660
ggcgtggact accagggtat ctaatcctgt tcgctcccca cgctttcgct cctcagcgtc 720
agttacagac cagagagtcg ccttcgccac tggtgttcct ccacatctct acgcatttca 780
ccgctacacg tggaattcca ctctcctctt ctgcactcaa gttccccagt ttccaatgac 840
cctccccggt tgagccgggg gctttcacat cagacttaag aaaccgcctg cgagcccttt 900
acgcccaata attccggaca acgcttgcca cctacgtatt accgcggctg ctggcacgta 960
gttagccgtg gctttctggt taggtaccgt caaggtaccg ccctattcga acggtacttg 1020
ttcttcccta acaacagagc tttacgatcc gaaaaccttc atcactcacg cggcgttgct 1080
ccgtcagact ttcgtccatt gcggaagatt ccctactgct gcctcccgta ggagtctggg 1140
ccgtgtctca gtcccagtgt ggccgatcac cctctcaggt cggctacgca tcgttgcctt 1200
ggtgagccgt tacctcacca actagctaat gcgccgcggg tccatctgta agtggtagcc 1260
gaagccacct tttatgtttg aaccatgcgg ttcaaacaac catccggtat tagccccggt 1320
ttcccggagt tatcccagtc ttacaggcag gttacccacg tgttactcac ccgtccgccg 1380
ctaacatcag ggagcaagct cccatctgtc cgctcgactt gcatgtat 1428
Sequence listing
<110> Ningxia university
<120> composition of biocontrol bacteria and fish protein and application thereof in cucumber cultivation
<141> 2021-05-20
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1434
<212> DNA
<213> Bacillus amyloliquefaciens XY-1(Bacillus amyloliquefaciens XY-1)
<400> 1
cttcggcggc tggctcctaa aaggttacct caccgacttc gggtgttaca aactctcgtg 60
gtgtgacggg cggtgtgtac aaggcccggg aacgtattca ccgcggcatg ctgatccgcg 120
attactagcg attccagctt cacgcagtcg agttgcagac tgcgatccga actgagaaca 180
gatttgtggg attggcttaa cctcgcggtt tcgctgccct ttgttctgtc cattgtagca 240
cgtgtgtagc ccaggtcata aggggcatga tgatttgacg tcatccccac cttcctccgg 300
tttgtcaccg gcagtcacct tagagtgccc aactgaatgc tggcaactaa gatcaagggt 360
tgcgctcgtt gcgggactta acccaacatc tcacgacacg agctgacgac aaccatgcac 420
cacctgtcac tctgcccccg aaggggacgt cctatctcta ggattgtcag aggatgtcaa 480
gacctggtaa ggttcttcgc gttgcttcga attaaaccac atgctccacc gcttgtgcgg 540
gcccccgtca atttcctttg agtttcagtc ttgcgacccg tactcccccc agggcggagt 600
gcttaatgcg ttagctgcag cactaagggg cggaaacccc ctaacacttt agcactccat 660
cgtttacggc gtggactacc agggtatcta atcctgttcg ctccccacgc tttcgctcct 720
cagcgtcagt tacagaccag agagtcgcct tcgccactgg tgttcctcca catctctacg 780
catttcaccg ctacacgtgg aattccactc tcctcttctg cactcaagtt ccccagtttc 840
caatgaccct ccccggttga gccgggggct ttcacatcag acttaagaaa ccgcctgcga 900
gccctttacg cccaataatt ccggacaacg cttgccacct acgtattacc gcggctgctg 960
gcacgtagtt agccgtggct ttctggttag gtaccgtcaa ggtgccgccc tatttgaacg 1020
gcacttgttc ttccctaaca acagagcttt acgatccgaa aaccttcatc actcacgcgg 1080
cgttgctccg tcagactttc gtccattgcg gaagattccc tactgctgcc tcccgtagga 1140
gtctgggccg tgtctcagtc ccagtgtggc cgatcaccct ctcaggtcgg ctacgcatcg 1200
tcgccttggt gagccgttac ctcaccaact agctaatgcg ccgcgggtcc atctgtaagt 1260
ggtagccgaa gccacctttt atgtctgaac catgcggttc aaacaaccat ccggtattag 1320
ccccggtttc ccggagttat cccagtctta caggcaggtt acccacgtgt tactcacccg 1380
tccgccgcta acatcaggga gcaagctccc atctgtccgc tcgacttgca gtat 1434
<210> 2
<211> 1427
<212> DNA
<213> Bacillus amyloliquefaciens XY-13(Bacillus amyloliquefaciens XY-13)
<400> 2
ttcggcggct ggctcctaaa aggttacctc accgacttcg ggtgttacaa actctcgtgg 60
tgtgacgggc ggtgtgtaca aggcccggga acgtattcac cgcggcatgc tgatccgcga 120
ttactagcga ttccagcttc acgcagtcga gttgcagact gcgatccgaa ctgagaacag 180
atttgtggga ttggcttaac ctcgcggttt cgctgccctt tgttctgtcc attgtagcac 240
gtgtgtagcc caggtcataa ggggcatgat gatttgacgt catccccacc ttcctccggt 300
ttgtcaccgg cagtcacctt agagtgccca actgaatgct ggcaactaag atcaagggtt 360
gcgctcgttg cgggacttaa cccaacatct cacgacacga gctgacgaca accatgcacc 420
acctgtcact ctgcccccga aggggacgtc ctatctctag gattgtcaga ggatgtcaag 480
acctggtaag gttcttcgcg ttgcttcgaa ttaaaccaca tgctccaccg cttgtgcggg 540
cccccgtcaa ttcctttgag tttcagtctt gcgaccgtac tccccaggcc ggagtgctta 600
atgcgttagc tgcagcacta agggggcgga aaccccccta acccattagc actcatcgtt 660
tacggcgtgg actaccaggg gtatctaatc ctgttcgctc cccacgcttt cgctcctcag 720
cgtcagttac agaccagaag agtcgccttc gccaactggt gttcctccac attctctacg 780
catttcaccg ctacaccgtg gaattccact ctccctcttc tgcactcaag ttccccagtt 840
tccaatgacc ctcccccggt tgagccgggg gctttcacat cagacttaag aaaccgcctg 900
cgagcccttt acgcccaata attccggaca acgcttgcca cctacgtatt accgcggctg 960
ctggcacgta gttagccgtg gctttctggt taggtaccgt caaggtgccg ccctatttga 1020
acggcacttg ttcttcccta acaacagagc tttacgatcc gaaaaccttc atcactcacg 1080
cggcgttgct ccgtcagact ttcgtccatt gcggaagatt ccctactgct gcctcccgta 1140
ggagtctggg ccgtgtctca gtcccagtgt ggccgatcac cctctcaggt cggctacgca 1200
tcgtcgcctt ggtgagccgt tacctcacca actagctaat gcgccgcggg tccatctgta 1260
agtggtagcc gaagccacct tttatgtctg aaccatgcgg ttcagacaac catccggtat 1320
tagccccggt ttcccggagt tatcccagtc ttacaggcag gttacccacg tgttactcac 1380
ccgtccgccg ctaacatcag ggagcaagct cccatctgtc cgctcga 1427
<210> 3
<211> 1428
<212> DNA
<213> Bacillus subtilis XY-53(Bacillus subtilis XY-53)
<400> 3
cttcggcggc tggctcctaa aaggttacct caccgacttc gggtgttaca aactctcgtg 60
gtgtgacggg cggtgtgtac aaggcccggg aacgtattca ccgcggcatg ctgatccgcg 120
attactagcg attccagctt cacgcagtcg agttgcagac tgcgatccga actgagaaca 180
gatttgtggg attggcttaa cctcgcggtt tcgctgccct ttgttctgtc cattgtagca 240
cgtgtgtagc ccaggtcata aggggcatga tgatttgacg tcatccccac cttcctccgg 300
tttgtcaccg gcagtcacct tagagtgccc aactgaatgc tggcaactaa gatcaagggt 360
tgcgctcgtt gcgggactta acccaacatc tcacgacacg agctgacgac aaccatgcac 420
cacctgtcac tctgcccccg aaggggacgt cctatctcta ggattgtcag aggatgtcaa 480
gacctggtaa ggttcttcgc gttgcttcga attaaaccac atgctccacc gcttgtgcgg 540
gcccccgtca attcctttga gtttcagtct tgcgaccgta ctccccaggc ggagtgctta 600
atgcgttagc tgcagcacta aggggcggaa accccctaac acttagcact catcgtttac 660
ggcgtggact accagggtat ctaatcctgt tcgctcccca cgctttcgct cctcagcgtc 720
agttacagac cagagagtcg ccttcgccac tggtgttcct ccacatctct acgcatttca 780
ccgctacacg tggaattcca ctctcctctt ctgcactcaa gttccccagt ttccaatgac 840
cctccccggt tgagccgggg gctttcacat cagacttaag aaaccgcctg cgagcccttt 900
acgcccaata attccggaca acgcttgcca cctacgtatt accgcggctg ctggcacgta 960
gttagccgtg gctttctggt taggtaccgt caaggtaccg ccctattcga acggtacttg 1020
ttcttcccta acaacagagc tttacgatcc gaaaaccttc atcactcacg cggcgttgct 1080
ccgtcagact ttcgtccatt gcggaagatt ccctactgct gcctcccgta ggagtctggg 1140
ccgtgtctca gtcccagtgt ggccgatcac cctctcaggt cggctacgca tcgttgcctt 1200
ggtgagccgt tacctcacca actagctaat gcgccgcggg tccatctgta agtggtagcc 1260
gaagccacct tttatgtttg aaccatgcgg ttcaaacaac catccggtat tagccccggt 1320
ttcccggagt tatcccagtc ttacaggcag gttacccacg tgttactcac ccgtccgccg 1380
ctaacatcag ggagcaagct cccatctgtc cgctcgactt gcatgtat 1428
Claims (6)
1. A composition of biocontrol bacteria and fish protein, characterized by: the fish protein composite culture solution comprises a bacillus composite culture solution and a fish protein diluent, wherein the bacillus composite culture solution comprises XY-1, XY-13 and XY-53; XY-1 and XY-13 are Bacillus amyloliquefaciens, and XY-53 is Bacillus subtilis;
bacillus amyloliquefaciens (Bacillus amyloliquefaciens) XY-1 with a preservation number: CCTCC NO: M2021269;
bacillus amyloliquefaciens (Bacillus amyloliquefaciens) XY-13 with a preservation number: CCTCC NO: M2021270;
bacillus subtilis XY-53 with the deposit number: CCTCC NO: M2021271.
2. The biocontrol bacteria and fish protein composition of claim 1, wherein: the concentration of the bacillus composite bacteria culture solution is 108cfu/mL, wherein the fish protein diluent is 200 times of the fish protein diluent, the volume ratio of the fish protein diluent to the bacillus composite bacteria culture solution is 1:1, and the concentration ratio of XY-1, XY-13 and XY-53 is 1: 1.
3. As claimed in claim2 the biocontrol bacteria and fish protein composition is characterized in that: the bacillus subtilis and bacillus amyloliquefaciens are prepared by screening strains, placing LB liquid culture medium at 30 ℃, shaking and culturing for 24h at 180rmp/min, centrifuging for 5min at 10000r/min, taking supernatant, mixing the three supernatants according to the proportion of 1:1:1, and adjusting the concentration to 1 × 108cfu/mL.
4. The biocontrol bacteria and fish protein composition of claim 3, wherein: the LB liquid culture medium comprises 10.0g of tryptone, 5.0g of yeast extract powder, 10.0g of sodium chloride and 1000mL of distilled water, and the pH value of the LB liquid culture medium is 7.0 +/-0.2.
5. Use of a composition of biocontrol bacteria and fish protein as defined in any one of claims 1 to 4 for the control of cucumber root rot.
6. Use of a composition of biocontrol bacteria and fish protein as defined in any one of claims 1 to 4 for promoting cucumber growth.
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| CN115812719A (en) * | 2022-12-08 | 2023-03-21 | 宁夏大学 | Compound composition of chitosan oligosaccharide, bacillus amyloliquefaciens and lentinan and application of compound composition in promoting growth of cucumber seedlings |
| CN115843806A (en) * | 2022-12-08 | 2023-03-28 | 宁夏大学 | Compound composition of seaweed oligosaccharide, bacillus subtilis and lentinan and application of compound composition in promoting growth of cucumber seedlings |
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| CN113303340B (en) | 2022-05-31 |
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