CN108192829B - Streptomyces luteorusand biological control preparation and application thereof - Google Patents
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Abstract
The invention discloses streptomyces luteorusus, a biological control preparation and application thereof, and belongs to the technical field of biological control of plant diseases.A streptomyces luteorusus strain HY61 is separated from seabed sediments in the sea area of a tobacco terrace and is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 15060. The streptomyces luteorusus strain HY61 has the advantages of high sporulation speed, wide action spectrum for preventing and treating various plant diseases, strong genetic stability, good environmental compatibility, long disease prevention lasting period and the like, thereby having good application prospect.
Description
Technical Field
The invention relates to the technical field of biological control of plant diseases, and particularly relates to streptomyces luteorubidus, a biological control preparation and application thereof.
Background
Plant diseases are antagonistic symbioses of the host plant and the pathogen, and their occurrence and prevalence are the result of the interaction of the host plant and the pathogen. Diseases of crops and trees occur, so that national economy and people's life are seriously lost. In recent years, with the general public concern about the safety of chemical pesticides in foods and environments and the banning of some dangerous chemical pesticides, people are prompted to seek other safe and effective new ways for controlling plant diseases, and the prevention and treatment of plant diseases by using beneficial microorganisms are increasingly paid attention. It is characterized by that it introduces exogenous antagonistic bacteria beneficial to plant into the broken natural ecological system of plant or utilizes the regulation of environment to promote growth of natural beneficial microbial population and express biocontrol activity so as to attain the goal of preventing diseases by means of interaction between microbes.
The actinomycetes are widely existed in nature, most of the actinomycetes are moderate in temperature and saprophytic, and few of the actinomycetes are high in temperature, halophilic, acidophilic, alkalophilic, anaerobic and parasitic, mainly grow in the land and fresh water, have low water content and exist in the atmosphere of neutral or slightly alkaline soil with good ventilation. There are many reports on biocontrol actinomycetes separated and screened from soil and used for preventing and controlling plant diseases, and Yan Jianfang and the like separate a plurality of actinomycetes from soil of Xinjiang and Liaoning and screen actinomycetes strains with antagonistic action on melon wilt pathogens. The Suqiang et al screen and separate one actinomycete strain of streptomyces rimosus subspecies fissuring from soil of vegetable protected land in northeast region, and the test result shows that the fermentation liquid has antifungal activity.
The marine environment is special, has the characteristics of high salinity, high pressure, low temperature, lack of nutrition and the like, and the marine actinomycetes living in the special environment has a unique metabolic pathway different from terrestrial actinomycetes. The search for new species of microorganisms and physiologically active substances with special functions from the ocean becomes a research hotspot at home and abroad, and the marine microorganisms become new resources of agricultural disease-resistant active substances. In recent years, more than 50% of newly discovered marine microbial active substances are produced by marine actinomycetes, and secondary metabolites of a plurality of actinomycetes have the application in the aspects of medicine and plant protection, and have been widely used as antibacterial, anti-plant pathogenic fungi and antitumor drugs.
Disclosure of Invention
The invention provides streptomyces luteorubii, a biological control preparation and application thereof.
The technical scheme of the invention is as follows:
the Streptomyces luteoflavus strain HY61 provided by the invention is separated from seabed sediments in the sea area of a tobacco station, is preserved in the China general microbiological culture preservation management center, has the preservation date of 2017, 12 months and 13 days, is classified and named as Streptomyces luteoflavus (Streptomyces luteoviticus), and has the preservation number of: CGMCC No. 15060.
Further, the 16S rRNA gene sequence of the strain is shown in SEQ. NO. 1.
The invention provides application of Streptomyces luteovirticus strain HY61 in biological control of plant diseases.
The invention provides a biological control preparation, which comprises Streptomyces luteoviticus strain HY61 and a metabolite thereof.
The invention provides a method for preparing a biological control preparation, which comprises the following steps:
(1) strain activation: picking strains, streaking and inoculating the strains to a solid slant culture medium, culturing for 3-7 d in a constant-temperature incubator at 25-32 ℃, and transferring liquid seeds after a large number of spores grow on the slant;
(2) preparing liquid seeds: selecting an activated strain, inoculating the activated strain into a container filled with a seed culture medium, sealing the container with gauze, carrying out shaking culture on the container at a temperature of between 25 and 32 ℃ and at a speed of between 150 and 200r/min for 24 to 48 hours, and growing a large number of mycelium pellets or mycelia to obtain liquid seeds;
(3) liquid fermentation: inoculating the liquid seeds prepared in the step (2) into a fermentation medium according to the inoculation amount of 1-15% of the volume ratio, rotating at the speed of 200-700 r/min, controlling the volume ratio of dissolved oxygen at 10-40%, controlling the pH at 6.0-7.5, and culturing at the temperature of 25-32 ℃ for 48-72 h in a ventilating way, so that the fermentation liquid can be used as a biological control preparation.
Further, the components and final concentrations of the solid slant culture medium are as follows: 4g/L yeast extract powder, 10g/L malt extract powder, 4g/L glucose, 18g/L agar, pH7.0, and high pressure steam sterilizing at 121 deg.C for 20 min;
further, the components and final concentrations of the liquid seed culture medium are as follows: 4g/L yeast extract powder, 10g/L malt extract powder, 4g/L glucose, pH7.0, and sterilizing with high pressure steam at 121 deg.C for 20 min;
further, the components and final concentrations of the fermentation medium are as follows: 15g/L of starch, 5g/L of glycerol, 11g/L of peptone, 4.4g/L of yeast extract powder, 0.4g/L of potassium nitrate, 4g/L of magnesium sulfate and KH2PO4 0.2g/L，KCl 0.2g/L，CaCl2 10mg/L，VB10.5mg/L, riboflavin 0.5mg/L, VH0.5mg/L, nicotinic acid 1mg/L, FeSO4 0.1mg/L，VB120.5mg/L, pH7.0, autoclaving at 121 deg.C for 20 min.
The invention provides application of the biological control preparation in a medicament for controlling plant diseases. The plant diseases comprise Colletotrichum gloeosporioides, Botrytis cinerea, Fusarium oxysporum, Fusarium graminearum Schw, Curvularia zeae and the like.
Compared with the prior art, the invention has the beneficial effects that: the streptomyces luteorusus strain HY61 has the advantages of high sporulation speed, wide action spectrum for preventing and treating various plant diseases, strong genetic stability, good environmental compatibility, long disease prevention lasting period and the like, thereby having good application prospect.
Drawings
FIG. 1 colony morphology of Streptomyces luteus strain HY61
FIG. 2 phylogenetic Tree based on 16S rRNA sequence
The Streptomyces luteoflavus strain HY61 is separated from seabed sediments in the sea area of a tobacco station, is preserved in the China general microbiological culture preservation management center, has the preservation date of 2017, 12 and 13 days, is classified and named as Streptomyces luteovirticus (Streptomyces luteovirticus), and has the preservation number of: CGMCC No. 15060.
Detailed Description
The invention will now be further illustrated by the following non-limiting examples in order to clarify the characteristics of the invention, without thereby limiting the scope of the invention.
Example 1: strain HY61 separation and screening
1) Separation: the strain HY61 is obtained by separation by a dilution coating method, and the specific separation method comprises the following steps: weighing 10g of a submarine sediment sample, dissolving the sample in 90mL of sterile water, shaking the sample evenly to mix the sample with the water fully, and coating the sample in a Gao's first culture medium plate after gradient dilution. Culturing the plate in 28 deg.C incubator for 5d, streaking colonies of different forms on the culture medium in a blank Gao's No. one culture medium plate, purifying the obtained strains, numbering and storing respectively. Wherein the culture medium of the Hirschmannin I comprises the following components: soluble starch 20g/L, KNO3 1g/L，K2HPO4 0.5g/L，MgSO4 0.5g/L，NaCl 0.5g/L，FeSO40.01g/L, agar 20g/L, pH7.4, and autoclave sterilization at 121 deg.C for 20 min.
2) Screening: inoculating Curvularia lunata (L.) pers to the midpoint of PDA culture medium by using a puncher with diameter of 5mm, inoculating 10 μ L of antagonistic bacteria liquid to two symmetrical sides of the bacteria block at a distance of 2cm from the center, inverting, and culturing at 28 deg.C for 5d, wherein a culture dish without inoculated antagonistic bacteria is used as a control. The test results were checked when the control dish had hyphae growing over the dish. Wherein the PDA culture medium comprises the following components: 200g/L of potato, 20g/L of glucose, 20g/L of agar, natural pH and high-pressure steam sterilization at 121 ℃ for 20 min.
A bacterial strain HY61 capable of efficiently preventing and treating various plant pathogenic bacteria is obtained through a large amount of screening work, and experiments prove that the biological control preparation prepared from the bacterial strain is a biological control preparation with a great application prospect.
Example 2: strain identification of HY61
1) Characteristics of the bacterial species
Culturing strain HY61 with Gao's I culture medium for 5d, wherein the colony has circular shape, shrivelled surface (shown in figure 1), powdery shape, and nearly grey white color, and has fishy smell; the aerial fungi are white, the hyphae in the substrate are light brown yellow, and the soluble brown pigment is secreted. On a Gao's first culture medium flat plate observed by the insert, aerial hyphae form longer axial filaments, branches grow at intervals, secondary rotation occurs, the spore filaments are broken into spores after being mature, the spores are in an ellipsoidal shape, and the branches of the intrabasal hyphae are bent.
Strain HY61 can decompose glucose, fructose, glycerol and starch, and cannot utilize sucrose, lactose, cellulose and chitosan. The test results of milk coagulation, pigment production, gelatin liquefaction and the like are positive, and the test results of methyl red, casein hydrolysis and the like are negative. The growth was good in 3% NaCl medium and the test results are detailed in Table 1.
TABLE 1 physiological and biochemical characteristics of Strain HY61
2) Identification of strains
The length of a 16S rRNA gene sequence obtained by DNA extraction, PCR amplification and sequencing is 1430bp, BLAST comparison is carried out on GenBank, the base similarity of the strain and Streptomyces luteoviticus (NR118282) is up to 99 percent, and HY61 strain is identified as Streptomyces luteoviticus (see figure 2) by combining morphological and physiological and biochemical indexes. The 16S rRNA sequence information is as follows:
caacgggcagtcaccttcgacggctcctccacaagggttgggccaccggcttcgggtgttaccgactttcgtgacgtgacgggcggtgtgtacaaggcccgggaacgtattcaccgcagcaatgctgatctgcgattactagcaactccaacttcatggggtcgagttgcagaccccaatccgaactgagaccggctttttgagattcgctccacctcacggcatcgcagctcattgtaccggccattgtagcacgtgtgcagcccaagacataaggggcatgatgacttgacgtcgtccccaccttcctccgagttgaccccggcagtctcctgtgagtccccatcaccccgaagggcatgctggcaacacaggacaagggttgcgctcgttgcgggacttaacccaacatctcacgacacgagctgacgacagccatgcaccacctgtacaccgaccacaagggggcgaccatctctggccgtttccggtgtatgtcaagccttggtaaggttcttcgcgttgcgtcgaattaagccacatgctccgctgcttgtgcgggcccccgtcaattcctttgagttttagccttgcggccgtactccccaggcggggaacttaatgcgttagctgcggcacggacgacgtggaatgtcgcccacacctagttcccaacgtttacggcgtggactaccagggtatctaatcctgttcgctccccacgctttcgctcctcagcgtcagtatcggcccagagatccgccttcgccaccggtgttcctcctgatatctgcgcatttcaccgctacaccaggaattccgatctcccctaccgaactctagcctgcccgtatcgaatgcagacccggggttaagccccgggctttcacatccgacgcgacaagccgcctacgagctctttacgcccaataattccggacaacgcttgcgccctacgtattaccgcggctgctggcacgtagttagccggcgcttcttctgcaggtaccgtcactttcgcttcttccctgctgaaagaggtttacaacccgaaggccgtcatccctcacgcggcgtcgctgcatcaggctttcgcccattgtgcaatattccccactgctgcctcccgtaggagtctgggccgtgtctcagtcccagtgtggccggtcgccctctcaggccggctacccgtcgtcgccttggtaggccatcaccccaccaacaagctgataggccgcgggctcatcctgcaccgccggagctttccaccaccaaccatgcggtcagtagtcatatccggtattagaccccgtttccagggcttgtcccagagtgcagggcagattgcccacgtgttactcacccgttcgccactaatccaccccgaagggcttcatcgttcgactgcatggtagccccccgcacggggc
3) genetic stability
The streptomyces luteorubii HY61 is continuously passed on a solid slant culture medium for 50 times by adopting a scribing method, and the thallus morphology, the growth speed and the capability of resisting corn curvularia are measured, so that the method has no obvious difference from primary strains and has good genetic stability.
4) Strain preservation
The Streptomyces luteoviteris HY61 of the invention has been deposited in China general microbiological culture Collection center at 12/13 th 2017 with the preservation number: CGMCC No.15060, preservation Unit Address: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing.
Example 3 Streptomyces lutescens HY61 fermentation Process
(1) Strain activation: picking strains, streaking and inoculating to a solid slant culture medium: 4g/L of yeast extract powder, 10g/L of malt extract powder, 4g/L of glucose, 18g/L of agar, 7.0 of pH, high-pressure steam sterilization at 121 ℃ for 20min, culturing for 7d at 28 ℃ in a constant-temperature incubator until a large amount of spores are grown on the inclined plane for transferring liquid seeds;
(2) preparing liquid seeds: selecting an activated strain, inoculating the activated strain into a seed culture medium (yeast extract powder 4g/L, malt extract powder 10g/L, glucose 4g/L, pH7.0, and autoclaving at 121 ℃ for 20min), sealing with gauze, and performing shake culture at 30 ℃ and 200r/min for 48h to grow a large amount of mycelial balls or mycelia to obtain liquid seeds;
(3) liquid fermentation: inoculating the fermentation medium (15 g/L starch, 5g/L glycerol, 11g/L peptone, 4.4g/L yeast extract, 0.4g/L potassium nitrate, 4g/L magnesium sulfate, KH) according to the volume ratio of 1-15%2PO4 0.2g/L，KCl 0.2g/L，CaCl210mg/L，VB10.5mg/L, riboflavin 0.5mg/L, VH0.5mg/L, nicotinic acid 1mg/L, FeSO4 0.1mg/L，VB120.5mg/L, pH7.0, high pressure steam sterilization at 121 ℃ for 20min) to the liquid seeds prepared in the step 2, rotating speed 500r/min, controlling dissolved oxygen by 20 percent, pH7.0, temperature 30 ℃, and ventilating and culturing for 72h, thus obtaining the biological control preparation.
Example 4 Streptomyces lutescens HY61 fermentation broth disease resistance test
The test of the resistance of Streptomyces luteus HY61 fermentation broth to phytopathogens was carried out by the tube-disc method, and the results are shown in Table 2. The Streptomyces luteus HY61 fermentation liquor has significant antagonistic effect on the growth of plant pathogenic bacteria such as Colletotrichum gloeosporioides, Botrytis cinerea, Fusarium oxysporum, Gibberella tritici, Curvularia zeae Schw, Curvularia lunata, etc. The inhibition effect on the Curvularia lunata is most obvious, and the average inhibition diameter of 10 mu L fermentation liquor reaches 4 cm; has good inhibitory effect on other plant pathogenic bacteria, has average inhibitory diameter of more than 2.0cm, and has development and utilization potential.
TABLE 2 diameter and area of inhibition zone for strain HY61 fermented liquid to inhibit plant pathogenic bacteria
Example 5 field Effect verification of Streptomyces lutescens HY61 fermentation broth on leaf spot of Curvularia lunata
Puncturing 6 needles on the middle leaves of the corn by using a No. 5 syringe needle, then inoculating corn curvularia leaf spot at the puncturing position, repeating for 3 times when 50 strains are treated, spraying 500 times of diluent of Streptomyces luteus HY61 fermentation liquor, 500 times of 50% carbendazim and clear water without application of medicine after 2d for 3 treatments, investigating disease indexes 7 days after application of medicine, and calculating the prevention and treatment effect. The test results (table 3) show that the control effect of the 500-fold diluent of the streptomyces luteoflavus HY61 fermentation liquid is 73.2 percent and is obviously higher than that of the 500-fold diluent of 50 percent carbendazim 7 days after the application.
TABLE 3 prevention and treatment effects of Streptomyces luteus HY61 fermentation broth on leaf spot of Curvularia lunata
Sequence listing
<110> university of Ludong
<120> Streptomyces luteorusis, biological control preparation and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1430
<212> DNA
<213> Streptomyces luteovirticus (Streptomyces luteoviterilliatus)
<400> 1
caacgggcag tcaccttcga cggctcctcc acaagggttg ggccaccggc ttcgggtgtt 60
accgactttc gtgacgtgac gggcggtgtg tacaaggccc gggaacgtat tcaccgcagc 120
aatgctgatc tgcgattact agcaactcca acttcatggg gtcgagttgc agaccccaat 180
ccgaactgag accggctttt tgagattcgc tccacctcac ggcatcgcag ctcattgtac 240
cggccattgt agcacgtgtg cagcccaaga cataaggggc atgatgactt gacgtcgtcc 300
ccaccttcct ccgagttgac cccggcagtc tcctgtgagt ccccatcacc ccgaagggca 360
tgctggcaac acaggacaag ggttgcgctc gttgcgggac ttaacccaac atctcacgac 420
acgagctgac gacagccatg caccacctgt acaccgacca caagggggcg accatctctg 480
gccgtttccg gtgtatgtca agccttggta aggttcttcg cgttgcgtcg aattaagcca 540
catgctccgc tgcttgtgcg ggcccccgtc aattcctttg agttttagcc ttgcggccgt 600
actccccagg cggggaactt aatgcgttag ctgcggcacg gacgacgtgg aatgtcgccc 660
acacctagtt cccaacgttt acggcgtgga ctaccagggt atctaatcct gttcgctccc 720
cacgctttcg ctcctcagcg tcagtatcgg cccagagatc cgccttcgcc accggtgttc 780
ctcctgatat ctgcgcattt caccgctaca ccaggaattc cgatctcccc taccgaactc 840
tagcctgccc gtatcgaatg cagacccggg gttaagcccc gggctttcac atccgacgcg 900
acaagccgcc tacgagctct ttacgcccaa taattccgga caacgcttgc gccctacgta 960
ttaccgcggc tgctggcacg tagttagccg gcgcttcttc tgcaggtacc gtcactttcg 1020
cttcttccct gctgaaagag gtttacaacc cgaaggccgt catccctcac gcggcgtcgc 1080
tgcatcaggc tttcgcccat tgtgcaatat tccccactgc tgcctcccgt aggagtctgg 1140
gccgtgtctc agtcccagtg tggccggtcg ccctctcagg ccggctaccc gtcgtcgcct 1200
tggtaggcca tcaccccacc aacaagctga taggccgcgg gctcatcctg caccgccgga 1260
gctttccacc accaaccatg cggtcagtag tcatatccgg tattagaccc cgtttccagg 1320
gcttgtccca gagtgcaggg cagattgccc acgtgttact cacccgttcg ccactaatcc 1380
accccgaagg gcttcatcgt tcgactgcat ggtagccccc cgcacggggc 1430
Claims (8)
1. The streptomyces luteoflavus strain HY61 is characterized in that the strain HY61 is preserved in China general microbiological culture collection center with the preservation date of 2017, 12 months and 13 days and is classified and named as streptomyces luteoflavus (Streptomyces luteoflavus)Streptomyces luteoverticillatus) The preservation number is: CGMCC number 15060.
2. A biocontrol agent comprising the Streptomyces lutescens strain HY61 of claim 1.
3. A process for the preparation of a biocontrol agent as described in claim 2, characterized in that it comprises the steps of:
(1) strain activation: selecting the Streptomyces luteus strain HY61 as claimed in claim 1, streaking and inoculating to a solid slant culture medium, culturing at 25-32 deg.C in a constant temperature incubator for 3-7 days, and inoculating liquid seeds after a large amount of spores grow on the slant;
(2) preparing liquid seeds: inoculating the activated streptomyces lutescens strain HY61 in the step (1) into a container filled with a seed culture medium, sealing with gauze, performing shake culture at 25-32 ℃ and 150-200 r/min for 24-48 h by using a shaking table, and growing a large amount of mycelium pellets or mycelia to obtain liquid seeds;
(3) liquid fermentation: inoculating the liquid seeds prepared in the step (2) into a fermentation medium according to the inoculation amount of 1-15% of the volume ratio, rotating at the speed of 200-700 r/min, controlling the volume ratio of dissolved oxygen at 10-40%, controlling the pH at 6.0-7.5, controlling the temperature at 25-32 ℃, and performing aeration culture for 48-72 hours to obtain a fermentation liquid, namely the biological control preparation.
4. The process for preparing a biocontrol agent as claimed in claim 3, characterized in that the composition and final concentration of said solid slant medium: 4g/L yeast extract powder, 10g/L malt extract powder, 4g/L glucose, 18g/L agar, pH7.0, and autoclaving at 121 deg.C for 20 min.
5. The method of claim 3, wherein the liquid seed culture medium comprises the following components in final concentrations: 4g/L yeast extract powder, 10g/L malt extract powder, 4g/L glucose, pH7.0, and high-pressure steam sterilization at 121 deg.C for 20 min.
6. The process for the preparation of a biocontrol agent as claimed in claim 3, characterized in that the components and final concentrations of the fermentation medium are: 15g/L of starch, 5g/L of glycerol, 11g/L of peptone, 4.4g/L of yeast extract powder, 0.4g/L of potassium nitrate, 4g/L of magnesium sulfate and KH2PO4 0.2 g/L，KCl 0.2 g/L，CaCl2 10 mg/L，VB1 0.5mg/L, riboflavin 0.5mg/L, VH0.5mg/L, nicotinic acid 1mg/L, FeSO4 0.1mg/L，VB120.5mg/L, pH7.0, autoclaving at 121 deg.C for 20 min.
7. Use of the biocontrol agent of claim 2 in the manufacture of a medicament for controlling plant diseases.
8. The plant diseases according to claim 7 include colletotrichum gloeosporioides, botrytis cinerea, fusarium oxysporum, gibberella cerealis and curvularia zeae.
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