CN114181873B - Streptomyces rohdea and biocontrol agent for preventing and treating soil-borne wilt of greenhouse cucumbers - Google Patents
Streptomyces rohdea and biocontrol agent for preventing and treating soil-borne wilt of greenhouse cucumbers Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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Abstract
The invention discloses a Streptomyces rohdea and biocontrol agent for preventing and treating greenhouse cucumber soil-borne wilt, belonging to the field of biological control of plant diseases, wherein the Streptomyces rohdea (Streptomyces rochei) is named as G6 and is preserved in China general microbiological culture collection center (CGMCC) No.24046. The Streptomyces rohdea of the invention has stable and durable control effect on soil-borne wilt of greenhouse cucumbers, simple fermentation process for mass production, low production cost, safety to human and livestock, no pollution and residue, and is favorable for protecting environment and maintaining ecological balance.
Description
Technical Field
The invention relates to the field of biological control of plant diseases, in particular to Streptomyces rohdea and a biological control preparation for controlling soil-borne wilt of greenhouse cucumbers.
Background
Along with the continuous improvement of the living standard of people and the vegetable planting level, the demands for vegetables are greatly increased. However, greenhouse vegetable soil-borne diseases are increasingly exacerbated by the annual planting of single species of vegetables. In the production process of greenhouse vegetables, chemical pesticides are excessively used, so that soil hardening, soil structure deterioration and pathogen base increase are caused, and meanwhile, pathogen generates drug resistance (resistance). The overuse of chemical pesticides seriously harms human health and pollutes ecological environment, so that an efficient and pollution-free control method is urgently needed to be searched, and the application prospect of replacing chemical bactericides with effective microorganisms for controlling vegetable diseases is huge.
Disclosure of Invention
In view of the above, the invention provides a Streptomyces rohdea for preventing and treating soil-borne wilt of greenhouse cucumbers, which is applied to the prevention and treatment effect of the soil-borne wilt of the greenhouse cucumbers, is safe and pollution-free, and is suitable for popularization and application.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
streptomyces rohdea (Streptomyces rochei) named G6 for preventing and treating greenhouse cucumber soil-borne wilt is preserved in China general microbiological culture Collection center (CGMCC) No.24046 with preservation number: no. 3 of North Chen Xili No. 1, the North Chaoyang area of Beijing is preserved for 2021, 12 months and 06 days.
The biocontrol preparation prepared from the Streptomyces rohdea for preventing and treating greenhouse cucumber soil-borne wilt is prepared by the strain.
The preparation method of the biocontrol agent comprises the following steps:
(1) Activating Streptomyces rohde G6;
(2) Liquid culture:
inoculating the activated strain into a liquid culture medium, and culturing at 28 ℃ for 48 hours at 180r/min to obtain a strain culture solution;
(3) Preparation of biocontrol preparation:
100g of biocontrol agent matrix: the strain culture solution is inoculated onto a biocontrol agent matrix according to the proportion of 5mL strain culture solution, and is cultured for 6-7d at 28 ℃ to ensure that the bacterial content reaches more than 2 hundred million CFU/g, dried and crushed, and stored in a dark place.
Preferably, in step (3), the biocontrol agent matrix is 60 mesh fine ground, corn flour, water at 90:10: mixing at a mass ratio of 25, and sterilizing.
Preferably, in the step (3), the drying process avoids direct sunlight, and the crushed materials pass through a 60-mesh sieve.
The application of the biocontrol preparation in preventing and controlling cucumber fusarium wilt in a greenhouse.
Preferably, the biocontrol agent is applied into a well-watered transplanting hole 1-2 days before transplanting the greenhouse cucumbers, and the application dosage is 10 g/plant.
Preferably, when the biocontrol agent is used for controlling fusarium wilt of cucumber in a greenhouse, the bactericide cannot be used for greenhouse soil.
The Streptomyces rohdea (Streptomyces rochei) G6 has remarkable control effect on soil-borne wilt of greenhouse cucumbers, and researches in examples show that the biocontrol strain preparation can remarkably inhibit the occurrence of the wilt of greenhouse cucumbers for more than 6-7 months; the contrast group without applying the biocontrol agent has obvious symptoms of the wilt disease after 3-4 months after transplanting, and the disease is faster and more serious along with the rising of the temperature, so that the plants die quickly after the symptoms of the wilt disease are displayed, and the vegetable production and the income of vegetable farmers are seriously influenced. And the Streptomyces rohdea (Streptomyces rochei) G6 strain is separated from greenhouse soil, and is harmless to the soil and crops.
In conclusion, the active ingredient Streptomyces rohdea (Streptomyces rochei) G6 of the biocontrol agent provided by the invention has stable and durable control effect on greenhouse cucumber soil-borne wilt, and has the advantages of simple fermentation process for large-scale production, low production cost, safety to people and livestock, no pollution and residue, environmental friendliness and ecological balance maintenance.
Drawings
FIG. 1 shows the colony morphology of Streptomyces rohdea G6;
FIG. 2 shows the mycelium morphology of Streptomyces rohdea G6;
FIG. 3 shows the spore (silk) morphology of Streptomyces rohdea G6;
FIG. 4 shows PCR results of specific primers of Streptomyces rohdea G6;
lanes 1-4 in the figure are Streptomyces G6, and lane 5 is a negative control;
FIG. 5 shows PCR results of Streptomyces rohdea G6 universal primer;
lanes 1-4 in the figure are Streptomyces G6, and lane 5 is a negative control;
FIG. 6 shows the control effect of Streptomyces rohdea G6 biocontrol agent on cucumber fusarium wilt;
FIG. 7 shows cucumber fusarium wilt symptoms without treatment with G6 biocontrol agents.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 identification of strains
The G6 strain isolated from greenhouse soil was taken and identified as follows:
(1) Morphological characteristics:
g6 strain was grown in solid medium of Gao's No. 1 (soluble starch 20G, KNO) 3 1.0g，K 2 HPO 4 0.5g，MgSO 4 ·7H 2 O 0.5g，NaCl 0.5g，FeSO 4 ·7H 2 0.01g of O, 20g of agar, 1000mL of distilled water, pH7.2-7.4 and high-pressure steam sterilization at 121 ℃ for 17 min) and culturing for 7-8d, the colony is compact, the surface and the edge are smooth and flat, and the colony is gray brown as shown in figure 1. Hyphae and spores (silk) of G6 were clearly observed under the microscope; g6 hyphae are not transverse, are not broken and are multi-branched, as shown in figure 2; the aerial hyphae form spore chains, the spore filaments are in a wave curve shape, the spores are elliptical, and the surfaces are smooth, as shown in figure 3.
(2) Physiological and biochemical characteristics:
the strain G6 can decompose cellulose, liquefy gelatin and hydrolyze starch, solidify milk, but cannot produce hydrogen sulfide and melanin by using tyrosine by adopting a culture medium and a test method recommended by Streptomyces appraisal handbook and Streptomyces appraisal of China and Yan Xun for initial editing; can better utilize carbon sources such as glucose, fructose, rhamnose, mannose, lactose and the like, and can not utilize sucrose and sorbitol.
(3) 16S rDNA sequence analysis:
the G6 strain was identified using 16S rDNA universal primers and Streptomyces specific primers, both primers being synthesized from Hua Dagene (Beijing), the specific sequences being shown in Table 1.
TABLE 1 primers
The PCR reaction system and the procedure used were as follows:
(1) PCR system:
(2) PCR procedure:
95℃3min；
95℃30s，59/60℃30s，72℃45s，30cycle；
72℃5min；
4 ℃ infinity/preservation.
The PCR detection results are shown in FIGS. 4-5, and specific bands of about 200bp (FIG. 4) and universal primer bands of about 1500bp (FIG. 5) were obtained.
The 16SrDNA universal primer and the specific primer PCR product are sent to a Hua big gene (Beijing) sequencing part for sequencing, and the sequence is obtained as follows:
(1) the nucleotide sequence obtained by sequencing the streptomycete specific primer is as follows:
GGGGACGTCGGGGTCACTCGGAGGAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTACAATGAGCTGCGATACCGCGAGGTGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTTGCAACTTCGAG，SEQ ID NO.1。
(2) the sequencing result of the 16SrDNA universal primer is finished to obtain the nucleotide sequence as follows:
GGGGGGGTGCTTACCTGCAAGTCGAACGATGAACCACTTCGGTGGGGATTAGTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCTGCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGATACTGATCCTCGCAGGCATCTGCGAGGTTCGAAAGCTCCGGCGGTGCAGGATGAGCCCGCGGCCTATCAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGTTGTGAAAGCCCGGGGCTTAACCCCGGGTCTGCAGTCGATACGGGCAGGCTAGAGTTCGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCGATACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGCACTAGGTGTGGGCAACATTCCACGTTGTCCGTGCCGCAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATACACCGGAAAACCCTGGAGACAGGGTCCCCCTTGTGGTCGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCCGTGTTGCCAGCAGGCCCTTGTGGTGCTGGGGACTCACGGGAGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTACAATGAGCTGCGATACCGCGAGGTGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGCCCAACCCCTTGTGGGAGGGAGCTGTCGAAGGTGGGACTGGCGATGGGACGAAGTCGAACAAGGTGTCAT，SEQ ID NO.2。
the resulting sequences were aligned in the NCBI-BLAST database and the results showed that the Streptomyces test was most likely Streptomyces rohdea (Streptomyces rochei).
The strain G6 was identified as Streptomyces rohdea (Streptomyces rochei) by combining morphological characteristics, physiological and biochemical characteristics and 16S rDNA sequence analysis.
Example 2
The biocontrol preparation based on Streptomyces rohdei G6 is prepared by the following steps:
1) Preparation of the culture medium:
solid medium No. 1 gao: soluble starch 20g, KNO 3 1.0g，K 2 HPO 4 0.5g，MgSO 4 ·7H 2 O 0.5g，NaCl 0.5g，FeSO 4 ·7H 2 0.01g of O, 20g of agar, 1000mL of distilled water, pH7.2-7.4 and sterilizing at 121 ℃ for 17min by high-pressure steam;
liquid medium No. 1 gao: soluble starch 20g, KNO3 1.0g, K 2 HPO 4 0.5g，MgSO 4 ·7H 2 O 0.5g，NaCl 0.5g，FeSO 4 ·7H 2 0.01g of O, 1000mL of distilled water, pH7.2-7.4, and steam sterilization at 121 ℃ for 17min.
2) Activation of strain G6:
inoculating the preserved Streptomyces rohder G6 into solid culture medium of Gaoshi No. 1 according to aseptic operation requirement, and culturing at constant temperature of 28deg.C for 6-8d.
3) Liquid culture of the Streptomyces rohdii strain:
scraping the strain activated in the step 2) by an inoculating shovel according to the aseptic operation requirement, inoculating the strain in a liquid culture medium of Gao's first, and carrying out shake culture at 28 ℃ and 180r/min for 48 hours to obtain a Streptomyces rohdea strain culture solution;
4) Preparation of Streptomyces rohdii G6 biocontrol agent:
100g of biocontrol agent matrix: 5ml of culture solution, inoculating the culture solution prepared in the step 3) into a biocontrol agent matrix, and performing shake culture at the constant temperature of 28 ℃ for 6-7d to ensure that the bacterial content reaches more than 2 hundred million CFU/g. The biocontrol agent matrix is prepared by sieving fine soil, corn flour and tap water through a 60-mesh sieve according to the mass ratio of 90:10:25 is evenly mixed and then sterilized by high-pressure steam at 121 ℃ for 17min.
And after the cultivation is finished, taking out, drying under the environment of avoiding direct sunlight, crushing, sieving with a 60-mesh sieve, and storing at low temperature or normal temperature in a dark place for later use.
Example 3
The Streptomyces rohderi G6 biocontrol preparation prepared by the method of example 2 is used for preventing and controlling fusarium wilt of greenhouse cucumber.
At the beginning of 12 months in the year, on the premise that bactericide is not used in greenhouse soil and all preparation work is completed before transplanting greenhouse cucumbers, the streptomyces rohdea G6 biocontrol agent is applied into transplanting holes 1-2d in advance according to the dosage of 10G/plant, then the cucumbers are transplanted normally, and other water and fertilizer management is normal.
The disease condition of cucumber fusarium wilt is investigated in the field in the next 6 months (figures 6-7), and the results show that the cucumbers treated by the streptomyces rohdea G6 biocontrol agent have no disease, but the cucumbers which are not treated by the biocontrol microbial inoculum have serious disease; the control effect of the streptomyces rohdea G6 biocontrol agent for controlling cucumber fusarium wilt is as long as 6 months, and the effect is 100%.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
<110> Yanbian Korea national academy of agricultural sciences (Yanbian specialty research institute)
<120> Streptomyces rohdea and biocontrol agent for preventing and treating soil-borne wilt of greenhouse cucumber
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 172
<212> DNA
<213> Artificial
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ggggacgtcg gggtcactcg gaggaggtgg ggacgacgtc aagtcatcat gccccttatg 60
tcttgggctg cacacgtgct acaatggccg gtacaatgag ctgcgatacc gcgaggtgga 120
gcgaatctca aaaagccggt ctcagttcgg attggggtct tgcaacttcg ag 172
<210> 2
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<213> Artificial
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gggggggtgc ttacctgcaa gtcgaacgat gaaccacttc ggtggggatt agtggcgaac 60
gggtgagtaa cacgtgggca atctgccctg cactctggga caagccctgg aaacggggtc 120
taataccgga tactgatcct cgcaggcatc tgcgaggttc gaaagctccg gcggtgcagg 180
atgagcccgc ggcctatcag ctagttggtg aggtaacggc tcaccaaggc gacgacgggt 240
agccggcctg agagggcgac cggccacact gggactgaga cacggcccag actcctacgg 300
gaggcagcag tggggaatat tgcacaatgg gcgaaagcct gatgcagcga cgccgcgtga 360
gggatgacgg ccttcgggtt gtaaacctct ttcagcaggg aagaagcgaa agtgacggta 420
cctgcagaag aagcgccggc taactacgtg ccagcagccg cggtaatacg tagggcgcaa 480
gcgttgtccg gaattattgg gcgtaaagag ctcgtaggcg gcttgtcacg tcggttgtga 540
aagcccgggg cttaaccccg ggtctgcagt cgatacgggc aggctagagt tcggtagggg 600
agatcggaat tcctggtgta gcggtgaaat gcgcagatat caggaggaac accggtggcg 660
aaggcggatc tctgggccga tactgacgct gaggagcgaa agcgtgggga gcgaacagga 720
ttagataccc tggtagtcca cgccgtaaac ggtgggcact aggtgtgggc aacattccac 780
gttgtccgtg ccgcagctaa cgcattaagt gccccgcctg gggagtacgg ccgcaaggct 840
aaaactcaaa ggaattgacg ggggcccgca caagcggcgg agcatgtggc ttaattcgac 900
gcaacgcgaa gaaccttacc aaggcttgac atacaccgga aaaccctgga gacagggtcc 960
cccttgtggt cggtgtacag gtggtgcatg gctgtcgtca gctcgtgtcg tgagatgttg 1020
ggttaagtcc cgcaacgagc gcaacccttg tcccgtgttg ccagcaggcc cttgtggtgc 1080
tggggactca cgggagaccg ccggggtcaa ctcggaggaa ggtggggacg acgtcaagtc 1140
atcatgcccc ttatgtcttg ggctgcacac gtgctacaat ggccggtaca atgagctgcg 1200
ataccgcgag gtggagcgaa tctcaaaaag ccggtctcag ttcggattgg ggtctgcaac 1260
tcgaccccat gaagtcggag tcgctagtaa tcgcagatca gcattgctgc ggtgaatacg 1320
ttcccgggcc ttgtacacac cgcccgtcac gtcacgaaag tcggtaacac ccgaagccgg 1380
tggcccaacc ccttgtggga gggagctgtc gaaggtggga ctggcgatgg gacgaagtcg 1440
aacaaggtgt cat 1453
Claims (8)
1. Streptomyces rohdea for preventing and treating fusarium wilt of cucumber in greenhouseStreptomyces rochei) The microbial strain collection agent is characterized by being named as G6 and being preserved in China general microbiological culture Collection center (CGMCC) No.24046.
2. A biocontrol agent prepared from a strain of streptomyces rohdea for controlling fusarium wilt of cucumber in a greenhouse according to claim 1.
3. The method for preparing the biocontrol agent as claimed in claim 2, which comprises the following steps:
(1) Activating Streptomyces rohde G6;
(2) Liquid culture:
inoculating the activated strain into a liquid culture medium, and culturing at 28 ℃ for 48 hours at 180r/min to obtain a strain culture solution;
(3) Preparation of biocontrol preparation:
100g of biocontrol agent matrix: the strain culture solution is inoculated onto a biocontrol agent matrix according to the proportion of 5mL strain culture solution, and is cultured for 6-7d at 28 ℃ to ensure that the bacterial content reaches more than 2 hundred million CFU/g, dried and crushed, and stored in a dark place.
4. The method for preparing a biocontrol agent as claimed in claim 3, wherein,
in the step (3), the biocontrol agent matrix is prepared by sieving fine soil, corn flour and water with a 60-mesh sieve according to a ratio of 90:10: mixing at a mass ratio of 25, and sterilizing.
5. The method for preparing a biocontrol agent as claimed in claim 3, wherein,
in the step (3), direct sunlight is avoided in the drying process, and the crushed materials are sieved by a 60-mesh sieve.
6. The use of the biocontrol agent of claim 2 or the biocontrol agent prepared by any one of claims 3-5 for controlling fusarium wilt of cucumber in a greenhouse.
7. The use according to claim 6, wherein,
the biocontrol preparation is applied into a transplanting hole which is watered thoroughly 1-2 days before transplanting the greenhouse cucumber, and the application dosage is 10 g/plant.
8. The use according to claim 7, wherein,
when the biocontrol agent is used for controlling cucumber fusarium wilt in a greenhouse, a bactericide cannot be used in greenhouse soil.
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