CN109234206B - Raoultella ornithinolytica HA011 for preventing and treating rice seedling stage damping-off and application thereof - Google Patents

Raoultella ornithinolytica HA011 for preventing and treating rice seedling stage damping-off and application thereof Download PDF

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CN109234206B
CN109234206B CN201811328858.5A CN201811328858A CN109234206B CN 109234206 B CN109234206 B CN 109234206B CN 201811328858 A CN201811328858 A CN 201811328858A CN 109234206 B CN109234206 B CN 109234206B
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王晓莉
王云鹏
袁志香
赵婕如
史雨琪
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Huaiyin Institute of Technology
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Abstract

The invention discloses a Raoultella ornithinolytica HA011 for preventing and treating seedling blight of rice and application thereof. The strain is Raoultella ornithinolytica, is preserved in China general microbiological culture collection center for 19 months in 2017, and has a preservation number of CGMCC No. 14644. The biocontrol microbial inoculum prepared by liquid fermentation culture of the HA011 strain HAs better effect on preventing and treating rice seedling blight. The biocontrol microbial inoculum HA011 is used for controlling the greenhouse test control effect and the plot test control effect of the rhizoctonia solani in the rice seedling stage to reach 79.38 percent and 71.33 percent respectively.

Description

Raoultella ornithinolytica HA011 for preventing and treating rice seedling stage damping-off and application thereof
Technical Field
The invention belongs to the field of agricultural microorganisms, and relates to Raoultella ornithinolytica HA011 for preventing and treating seedling blight of rice and application thereof.
Background
The seedling stage damping-off of rice is a common disease in spring seedlings in various parts of China, the disease rate of a general nursery is between 20% and 50%, and the disease rate can reach more than 80% in severe land. The rice damping-off belongs to soil-borne diseases. The fungi are caused by infection with various pathogenic bacteria, and mainly include Fusarium oxysporum Schelcht (Fusarium oxysporum), Fusarium graminearum Schw (Fusarium graminearum), Fusarium equeiti Sacc (Fusarium equiseti), Fusarium solani (Mart.) App.Et Wr. (Fusarium solani), Fusarium moniliforme Scheld (Fusarium moniliforme), Rhizoctonia solani Kuhn.of Rhizoctonia of Nostolonium, Rhizoctonia solani of Rhizoctonia, Pythium spp. Fusarium is generally harmful to the disease of hypha and posterior chlamydospore overwintering in the diseased residues and soil of various hosts, and conidia generated under proper environmental conditions are spread by airflow and infected. The rhizoctonia is harmful to the hypha and the sclerotium, which overwinter in host disease residual body and soil, spread among seedlings by hypha and infect. The occurrence of seedling stage damping-off of rice causes a large amount of lodging, withering and rotting of rice seedlings, and can also directly cause festering of rice seeds when the rice seedlings are not budded, thereby seriously affecting the growth of the rice seedlings and causing great economic loss to the rice production.
At present, the prevention and treatment of the rice damping-off disease mainly depends on selecting disease-resistant varieties and chemical agents for prevention and treatment. Has no great breakthrough in disease-resistant breeding, and no immune and high-resistance variety is found. Chemical control is the most common control method, and commonly used medicaments such as metalaxyl, diniconazole, oxadixyl, Aimiao, Mansui, and King Feng, etc. Although the chemical prevention and control can control the development of the disease condition in the field to a certain extent, secondary pollution is easily caused to the ecological environment and the wheat production, the dosage is increased and the production cost is increased due to the fact that antibiotics and chemicals which are easy to generate drug resistance are repeatedly used for a long time, the pesticide residue problem exists when the environment is polluted, the barrier is brought to the export of agricultural products, and the threat is brought to the safety of people and livestock. Therefore, environmental friendly biological control technologies are gradually gaining attention. The biological control strategy has the characteristics of wide biological control resource sources, environmental friendliness and the like, so that the application of the biological control strategy in the prevention and control of the seedling blight of rice has a better development prospect.
Disclosure of Invention
The invention aims to provide a Laurus ornithine-degrading HA011 for preventing and treating rice seedling blight and application thereof, aiming at the current situation that no biocontrol agent with higher prevention effect on the rice seedling blight in the prior art exists.
The purpose of the invention can be realized by the following technical scheme:
the ralstonia ornithinolytica HA011 for preventing and treating rice seedling blight is identified to belong to the ralstonia ornithinola, and is preserved in the China general microbiological culture collection center in 9-19 th in 2017, with the strain preservation number of CGMCC No. 14644.
The application of the Laurushibara.ornithine HA011 in preventing and treating rice seedling blight or preparing a biocontrol microbial inoculum for preventing and treating rice seedling blight.
A biocontrol microbial inoculum for preventing and treating seedling blight of rice comprises the Raoultella ornithine HA 011. As a preferred technical scheme, the living bacteria in the biocontrol microbial inoculumThe concentration is not less than 1 × 108 CFU/mL。
The preparation method of the microbial inoculum comprises the following steps: inoculating the seed bacterial liquid of the Raoultella ornithinolytica HA011 into an LB fermentation culture medium in a volume ratio of 1:500 for fermentation culture, carrying out shaking culture at 28 ℃ for 40-48 h at 150-200 rpm, then centrifuging at 5000-6000 rpm for 10-15 min, taking precipitate to obtain the biocontrol bacterial agent HA011, diluting with sterilized water to prepare the bacterial agent, wherein the total viable bacteria concentration in the finished bacterial agent is 1 x 108~1×109 CFU/mL。
Of course, the preparation of the biocontrol microbial inoculum by using the Laurus ornithine HA011 for preventing and treating seedling blight of rice is not limited to the above method. All the methods which can culture HA011 in large quantity and keep the biocontrol activity can be used for preparing the biocontrol microbial inoculum claimed by the invention.
The preparation method of the Raoultella ornithinolytica HA011 seed bacterial liquid with the preservation number of CGMCC No.14644 comprises the following steps: inoculating the Raoultella ornithinolytica HA011 strain with the preservation number of CGMCC No.14644 into LB culture solution, culturing at 28 ℃, shaking at 180rpm, and culturing until the OD value at 600nm is 0.8.
A method for preventing and controlling seedling blight of rice uses the biocontrol microbial inoculum to inoculate the rice.
After the biocontrol microbial inoculum is diluted by 100-fold and 200-fold, the inoculation treatment is respectively carried out on the rice in greenhouse pot culture and plot experiment, and the state of illness begins to be counted when the disease occurs. The greenhouse control effect and the cell test control effect of the Raoultella ornithinolytica HA011 on rice seedling blight are respectively 79.38% and 71.33%.
The invention has the beneficial effects that:
the invention relates to a biocontrol preparation developed specially for rice seedling blight. Because the wheat bran biological agent is a biological agent, a series of problems caused by the use of chemical pesticides are completely avoided, the wheat bran biological agent is beneficial to the pollution-free production of wheat, farmers can not use or reduce the use amount of other chemical pesticides, the expenditure of the farmers can be saved, and the residues of chemical agents are reduced.
Greenhouse experiments show that: the Lauracillus ornithinolyticus HA011 (CGMCC No. 14644) can obviously reduce the occurrence of seedling blight of rice. The biocontrol microbial inoculum prepared by the strain is diluted by 100-200 times for root irrigation treatment, and the greenhouse control effect and the cell test control effect on seedling blight of rice can respectively reach 79.38% and 71.33%.
Detailed Description
Example 1
Biological sample preservation information
The Raoultella ornithinolytica HA011 is classified and named Raoultella ornithinolytica, is preserved in China general microbiological culture Collection center (CGMCC) in 19 th 9 th 2017, and HAs the preservation address of No. 3 of the Xilu-1 of the Hongyang area of Beijing city and the preservation number of CGMCC No.14644 of the institute of microbiology of the Chinese academy of sciences.
The screening method comprises the following steps: the Raoultella ornithinolytica HA011 (CGMCC No. 14644) is separated from rice seedling root soil in the rice paddy field at the side of the firewood river in Huaian city, Jiangsu province. Cutting root system of rice seedling into 1cm pieces, soaking with 1% sodium hypochlorite for 5min, soaking with 70% alcohol for 1-2 min, and washing with sterile water for 3 times (coating 100 μ l of the solution obtained by the last washing on R2A solid culture medium for culture, and considering surface sterilization and sterility if aseptic growth is carried out after 48 h). 3 g of the sterilized sample was placed in a bacterial mortar, 27ml of sterile 0.85% NaCl was added, the tissue was macerated, ground and diluted with sterile 0.85% NaCl in a gradient. The dilution of 10-1, 10-2 and 10-3 is smeared on R2A in 100. mu.l each, and cultured at 28 ℃ for 48 h. The largest single colony is selected and inoculated to a fresh R2A solid culture medium, and the purified strain is obtained through repeated transfer. The purified strain was stored in an ultra-low temperature refrigerator at-70 ℃ with 40% glycerol.
The identification method comprises the following steps: the strain is identified through morphological characteristics, physiological and biochemical experiments and 16S rDNA sequence analysis.
Morphological characteristics: gram-print bacteria, no movement, facultative anaerobism, rod shape with pod membrane, straight rod shape bacteria, positive catalase, indole reaction and methyl red experiment.
16S ITS rRNA gene sequence (1403 bp) of Raoultella ornithinolytica HA 011:
tgcaagtcga gcggtagcac agagagcttg ctctcgggtg acgagcggcg gacgggtgag
taatgtctgg gaaactgcct gatggagggg gataactact ggaaacggta gctaataccg cataacgtcg
caagaccaaa gtgggggacc ttcgggcctc atgccatcag atgtgcccag atgggattag ctagtaggtg
gggtaatggc tcacctaggc gacgatccct agctggtctg agaggatgac cagccacact ggaactgaga
cacggtccag actcctacgg gaggcagcag tggggaatat tgcacaatgg gcgcaagcct gatgcagcca
tgccgcgtgt atgaagaagg ccttcgggtt gtaaagtact ttcagcgagg aggaaggcat taaggttaat
aaccttagtg attgacgtta ctcgcagaag aagcaccggc taactccgtg ccagcagccg cggtaatacg
gagggtgcaa gcgttaatcg gaattactgg gcgtaaagcg cacgcaggcg gtctgttaag tcagatgtga
aatccccggg ctcaacctgg gaactgcatt tgaaactggc aggcttgagt cttgtagagg ggggtagaat
tccaggtgta gcggtgaaat gcgtagagat ctggaggaat accggtggcg aaggcggccc cctggacaaa
gactgacgct caggtgcgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca cgctgtaaac
gatgtcgact tggaggttgt tcccttgagg agtggcttcc ggagctaacg cgttaagtcg accgcctggg
gagtacggcc gcaaggttaa aactcaaatg aattgacggg ggcccgcaca agcggtggag catgtggttt
aattcgatgc aacgcgaaga accttaccta ctcttgacat ccagagaact tagcagagat gctttggtgc
cttcgggaac tctgagacag gtgctgcatg gctgtcgtca gctcgtgttg tgaaatgttg ggttaagtcc
cgcaacgagc gcaaccctta tcctttgttg ccagcgattc ggtcgggaac tcaaaggaga ctgccagtga
taaactggag gaaggtgggg atgacgtcaa gtcatcatgg cccttacgag tagggctaca cacgtgctac
aatggcatat acaaagagaa gcgacctcgc gagagcaagc ggacctcata aagtatgtcg tagtccggat
tggagtctgc aactcgactc catgaagtcg gaatcgctag taatcgtaga tcagaatgct acggtgaata
cgttcccggg ccttgtacac accgcccgtc acaccatggg agtgggttgc aaaagaagta ggtagcttaa
ccttcgggag ggc
16S rRNA gene amplification and sequence analysis, the Raoultella ornithine HA011 is cultured in an LB culture medium at 28 ℃ to logarithmic phase, thalli is collected by centrifugation at 12000r/min for 5min, the genome DNA of bacteria is extracted by adopting a genome DNA rapid extraction kit of Botryobo bioengineering (Dalian) Co., Ltd, and the 16S rRNA gene fragment is amplified from the genome DNA by using the extracted DNA product as a template and using universal primers U8-27(5 '-AGAGTTTGATC(AC) TGGCTCAG-3') and L1494-1514(5 '-CTACGG (AG) TACCTTGTTACGAC-3') for bacterial 16S rRNA amplification. The PCR product is sent to Shanghai biological engineering Co., Ltd for sequencing. Homology comparisons of the determined 16S rRNA gene sequences were performed by BLAST software, and the results were profiled, and therefore, the strain was identified as Raoultella ornithinolytica (Raoultella ornithinolytica).
Table 1 HA011 sequencing alignment results
Bacterial strains Most similar strains and accession numbers Degree of similarity (%)
HA011 Raoultella ornithinolytica CP023888 .1 100
Example 2 preparation of HA011 biocontrol bacterial agent
Inoculating Raoultella ornithinolytica HA011 (CGMCC No. 14644) strain into LB culture solution, performing shaking culture at 28 deg.C and 180rpm for 16h, and culturing every timeSampling in a clean bench at intervals of 2h, measuring the OD value at 600nm, and finishing the culture when the OD value is 0.8, wherein the bacterial liquid is used as seed bacterial liquid. Inoculating the seed bacterial liquid into LB fermentation culture liquid according to the volume ratio of 1:500 for fermentation culture, culturing at 28 ℃ and 180rpm for 48h, then centrifuging at 6000rpm for 10 min, and taking the precipitate to obtain the biocontrol microbial inoculum HA 011. The biological control agent HA011 is diluted by sterilized water, and the total concentration of viable bacteria in the obtained biological control agent is 1 multiplied by 108~1×109 CFU/mL。
Example 3 greenhouse and plot experiments verify the control effect of biocontrol bacteria HA011 on seedling blight of rice
Experiment 1: greenhouse test for detecting the control effect of HA011 on seedling blight of rice (2017.03-2017.08)
A place: huaiyin institute of Industrial and scientific colleges greenhouse
Pathogenic bacteria: fusarium graminearum (laboratory preservation, isolated from rice diseased plants)
And (3) contrast: LB culture medium (formula)
Biocontrol bacteria: raoultella ornithinolytica HA011 (laboratory preservation)
Chemical agents: 30% hymexazol wettable powder
And (3) culturing pathogenic bacteria: fusarium is activated and inoculated in 0.6% mung bean soup liquid culture medium sterilized at 121 deg.C
Culturing at 25 deg.C in a constant temperature oscillator for 5 days, filtering with multiple layers of gauze, and directly storing. Under the microscope, the spore suspension concentration was adjusted to 105 spores/ml.
Greenhouse pot experiment: culturing rice seedlings in a greenhouse, 8 seedlings per pot, and culturing Raoultella ornithinolytica in one leaf stage
HA011 1×108The method comprises the following steps of (1) irrigating roots of CFU/mL bacterial liquid (the using amount of 20mL per pot), 30% hymexazol 600 times liquid (the using amount of 20mL per pot) and a control LB liquid culture medium (the using amount of 20mL per pot), and inoculating 105 spores/mL of fusarium graminearum (the inoculation amount of 10mL per pot) after 7 days; after inoculation, the seeds are kept for 48 hours at 25 ℃ and grow at constant temperature. Each treatment was performed on 24 plants in triplicate. The disease condition of the rice seedlings was investigated every day.
Dividing according to the severity and actual conditions of the damping-off disease:
grade 0, no obvious symptoms at the base of the plant;
grade 1, incidence of leaf blade and leaf sheath disease at the base;
2, the disease of each leaf or leaf sheath below the inverted three leaves;
grade 3, the disease is developed by falling each leaf or leaf sheath below two leaves;
grade 4, the disease at the base of the stem and the leaf is serious, and the whole plant dies.
Disease severity (disease index) = Σ (disease progression × number of diseased plants)/(highest disease progression × total plants)
Number) × 100%
Biocontrol effect = (control group disease severity-treatment group disease severity)/control group disease severity × 100%
Note: calculating disease index and prevention effect of different treatments, and performing difference significance analysis by using DPS data analysis software
And (4) performing multiple comparisons.
The investigation result after the biocontrol microbial inoculum HA011 is treated for 15 days shows that the biocontrol microbial inoculum in the greenhouse pot experiment HAs 79.38 percent of biological control effect (hereinafter referred to as control effect) on the seedling blight of rice (Table 2).
Table 2 biocontrol effect of biocontrol microbial inoculum HA011 on seedling blight of rice in greenhouse pot experiment
Figure 256985DEST_PATH_IMAGE001
Experiment 2: cell test for detecting the control effect of HA011 rice seedling blight at seedling stage (2017.09-2018.02)
The location: huaian chaihei agricultural science and technology development limited seedling culture base
Pathogenic bacteria: fusarium graminearum (extracted from rice diseased plant)
And (3) contrast: LB culture medium (formula)
Biocontrol bacteria: raoultella ornithinolytica HA011 (laboratory preservation)
Chemical agents: 30% hymexazol wettable powder
And (3) culturing pathogenic bacteria: fusarium is activated and inoculated in 0.6% mung bean soup liquid culture medium sterilized by autoclaving at 121 deg.C, cultured in a constant temperature oscillator at 25 deg.C for 5 days, and filtered with multiple layers of gauze for direct storage. Under microscope, the spore suspension concentration was adjusted to 105Spores per ml.
Cell test: the rice seedling raising adopts a seedling raising tray for seeding, and the size of the seedling raising tray is as follows: 54cm multiplied by 28cm multiplied by 4.6cm, about 500 seedlings are randomly placed in each tray, and each tray is treated with 10 trays; the experiment was repeated three times. In one leaf stage, biocontrol Raoultella ornithinolytica HA 0111X 108The root irrigation treatment is carried out on CFU/mL bacterial solution (the dosage is 1000mL per dish), 30% hymexazol 600 times solution (the dosage is 1000mL per basin) and a control group LB (the dosage is 1000mL per basin), each treatment is repeated three times, and after 7 days, the pathogenic bacteria 10 is inoculated5Root irrigation treatment is carried out on spores/ml fusarium graminearum (the inoculation amount is 500ml per pot); after inoculation, the seeds were kept moist for 48h at 25 ℃. The disease condition of the rice seedlings is observed and investigated every day.
Dividing according to the severity and actual conditions of the damping-off disease:
grade 0, no obvious symptoms at the base of the plant;
grade 1, incidence of leaf blade and leaf sheath disease at the base;
2, the disease of each leaf or leaf sheath below the inverted three leaves;
grade 3, the disease is developed by falling each leaf or leaf sheath below two leaves;
grade 4, the disease at the base of the stem and the leaf is serious, and the whole plant dies.
Disease severity (disease index) = Σ (disease progression × number of diseased plants)/(highest disease progression × total plants)
Number) × 100%
Biocontrol effect = (control group disease severity-treatment group disease severity)/control group disease severity × 100%
Note: calculating disease index and prevention effect of different treatments, and performing difference significance analysis by using DPS data analysis software
And (4) performing multiple comparisons.
The investigation result after the biocontrol bacterial agent HA011 is treated for 15 days shows that the biological control effect (hereinafter referred to as control effect) of the biocontrol bacterial agent HA011 on the seedling blight of rice in a plot experiment reaches 71.33 percent (Table 3).
Table 3 biological control effect of biological control fungicide HA011 on seedling blight of rice in plot experiment
Figure 846229DEST_PATH_IMAGE002

Claims (7)

1. The Raoultella ornithinolytica HA011 for preventing and treating rice seedling blight is identified to belong to Raoultella ornithinolytica, and is preserved in China general microbiological culture Collection center in 2017, 9-19 months and 14644 with the strain preservation number of CGMCC No. 14644.
2. The use of the ralstonia ornithinolytica HA011 of claim 1 in preventing and treating seedling blight of rice.
3. The application of the Raoultella ornithine HA011 of claim 1 in preparing a biocontrol microbial inoculum for preventing and treating seedling blight of rice.
4. A biocontrol microbial inoculum for controlling seedling blight of rice, which is characterized by comprising the Raoultella ornithine-degrading HA011 of claim 1.
5. The biocontrol microbial inoculum for controlling seedling blight of rice as claimed in claim 4, wherein the total concentration of viable bacteria in the biocontrol microbial inoculum is not less than 1 x 108 CFU/mL。
6. The use of the biocontrol microbial inoculum of claim 4 or 5 in preventing and controlling seedling blight of rice.
7. A method for controlling seedling blight of rice, characterized in that the biocontrol microbial inoculum according to claim 4 or 5 is used for inoculation treatment of rice.
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Application publication date: 20190118

Assignee: Nanjing native agricultural science and Technology Co.,Ltd.

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Denomination of invention: A strain of ornithine releasing raoulia ha011 for controlling rice seedling blight and its application

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Record date: 20211027