CN1325635C - Endogenetic polymexa bacillus of plant for prophyiaxis and promoting growth and application thereof - Google Patents
Endogenetic polymexa bacillus of plant for prophyiaxis and promoting growth and application thereof Download PDFInfo
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Abstract
本发明涉及一种防病促生长植物内生多粘类芽孢杆菌(Paenibacillus polymyxa)及其应用，属于农作物防病控病技术领域。本发明采用的菌株Jaas cd已于2005年3月10日在中国微生物菌种保藏管理委员会普通微生物中心保藏，保藏号为CGMCC No.1325。该菌株可在较低的菌液浓度下进入植物体内，并能在植物体内定殖、繁殖和传导；对植物同时具有促进生长、提高产量和防病的作用，特别是对于土传维管束病害具有良好的防治效果。该菌株抑菌谱广，用于制剂生产成本低廉，操作简单，有利于保护农田生态环境，减少农药残留，适合于我国无公害农产品生产的要求。The invention relates to a plant endogenous Paenibacillus polymyxa for disease prevention and growth promotion and an application thereof, belonging to the technical field of crop disease prevention and control. The bacterial strain Jaas cd used in the present invention has been preserved in the General Microorganism Center of China Microorganism Culture Collection Management Committee on March 10, 2005, and the preservation number is CGMCC No.1325. The strain can enter the plant at a lower bacterial concentration, and can colonize, reproduce and conduct in the plant; it can promote growth, increase yield and prevent disease for plants, especially for soil-borne vascular diseases Has a good control effect. The bacterial strain has a wide antibacterial spectrum, and the production cost of the preparation is low, and the operation is simple, which is beneficial to the protection of the farmland ecological environment and the reduction of pesticide residues, and is suitable for the production requirements of pollution-free agricultural products in my country.
Description
Technical field:
The present invention relates to a kind of endogenetic polymexa bacillus of plant for prophyiaxis and promoting growth (Paenibacillus polymyxa) and, belong to the sick technical field of farm crop diseases prevention control in the application in biological control field.
Background technology:
Endophyte of plant is meant and can lives in the plant materials, but the normal microorganism of not causing manifest symptom.This quasi-microorganism can be grown in vegetable cell gap or cell surely, and sets up the relation of harmonious associating with host plant.At the beginning of eighties of last century, people have just begun the research to endogenetic bacteria, result of study in recent years show endophytic bacterium not only with host plant as its place of perching, and host plant had biological actions such as short lifes, diseases prevention, fixed nitrogen, the while endogenetic bacteria still imports foreign gene the intravital good carrier of plant.Therefore, endophytic bacterium is the natural resource bacterium of plant pest biological control, has very theoretical investigation widely and development and application values.Endophytic bacterium is compared with the antagonistic bacterium that is separated to from soil, influence with the envrionment conditions of not being subject to, in plant materials, has stable living space, can be in plant materials advantage such as fissiparity and transmission independently, screen useful endogenetic bacteria and might become the microbial pesticide of tool potentiality in the biological control, yield increasing fungus or be used as potential biological and ecological methods to prevent plant disease, pests, and erosion carrier bacterium.Utilize endogenetic bacteria more outstanding to the advantage that soil-borne vascular bundle disease carries out biological control, because behind the pathogen infection of soil-borne vascular bundle disease, just in the host plant vascular bundle, grow, breed, cause harm, cause chemical agent control difficulty, the undesirable contaminate environment again of effect, and endogenetic bacteria can with pathogenic bacteria contention living space, produce antagonistic substance and directly the pathogenic bacteria effect be waited the purpose that reaches diseases prevention control disease by growing in that plant materials is decided at the higher level but not officially announced.
Along with the development of modern science and technology, the mechanization of agriculture degree improves, and causes the simplification of farming operation mode, and a large amount of uses of chemical fertilizer, agricultural chemicals cause environmental pollution to cause species diversity decline in the agroecosystem, even ecological balance damage.And endophytic bacterium with the long-term coevolution process of host plant in form close mutual relationship; and the existence microenvironment is stable; be the best rival of chemical fertilizer, agricultural chemicals and other microbial preparations; its reasonable use will reduce the environmental pollution that chemical pesticide causes; improve the species diversity in the agroecosystem, help protecting the eubiosis to realize sustainable development.Growth in the living standard and development of science and technology make people more and more higher to restriction use chemical pesticide and requirement on environmental protection, utilize endogenetic bacteria to carry out biological control and are fit to the requirement that China's non-polluted farm product is produced.Have though have the report of the endophytic bacterium of disease-resistant growth-promoting effect, mostly be subtilis, how red grade is separated to 2 bacillus subtilis BS-1 and BS-2 in the capsicum body.At present, domestic research for Paenibacillus polymyxa focuses mostly on aspect associative nitrogen fixation, does not also have the report of the endogenetic polymexa bacillus of prophyiaxis and promoting growth effect.
Summary of the invention:
The purpose of this invention is to provide and a kind ofly can decided at the higher level but not officially announcedly grow, multiple pathogenic bacteria is had antagonistic action at plant materials, and can promote the endogenetic polymexa bacillus of plant of plant-growth, thereby can develop novel biocontrol fungicide, satisfy the requirement that non-polluted farm product is produced.
Endophytic bacterium provided by the present invention is nineteen ninety-five to separate acquisition from the cotton tissue of NanJing City, Jiangsu Province,China, during 2003-2005, this bacterial strain has been carried out strain identification, and the result is Paenibacillus polymyxa (PaeniibacillussPolymyxa).2004 with this bacterial strain called after Jaas cd, and on March 10th, 2005 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preserving number is CGMCC No.1325.This bacterial strain has following character:
1. colony morphology characteristic
This bacterial strain is well-grown on the NA substratum, and single bacterium colony is half glass pearl, neat in edge, and surface wettability is smooth, the bacterium colony light yellow, tarnish, protuberance is opaque.
2. morphological features
Somatic cells is shaft-like, forms pod membrane, produces gemma, middle life, and sporangiocyst expands.
3. major physiological biochemical character
The Gram-reaction positive; The catalase reaction positive; Utilize pectinose, wood sugar, N.F,USP MANNITOL to produce acid, glucose aerogenesis; The V.P reacting positive, gelatin hydrolysate, casein, starch.
4. genetics feature
With 968f-gc and 1401r is the partial sequence 423bp that primer carries out the 16SrRNA gene of this bacterial strain that pcr amplification goes out, and submits to GenBank, and accession number is AY942618.The partial sequence of the 16SrRNA gene of this bacterial strain is:
1 AGAACCTTAC?CAGGTCTTGA?CATCCCTCTG?ATCGGTCTAG?AGATAGATCT?TTCCTTCGGG
61 ACAGAGGAGA?CAGGTGGTGC?ATGGTTGTCG?TCAGCTCGTG?TCGTGAGATG?TTGGGTTAAG
121 TCCCGCAACG?AGCGCAACCC?TTATGCTTAG?TTGCCAGCAG?GTCAAGCTGG?GCACTCTAAG
181 CAGACTGCCG?GTGACAAACC?GGAGGAAGGT?GGGGATGACG?TCAAATCATC?ATGCCCCTTA
241 TGACCTGGGC?TACACACGTA?CTACAATGGC?CGGTACAACG?GGAAGCGAAA?TCGCGAGGTG
301 GAGCCAATCC?TAGAAAAGCC?GGTCTCAGTT?CGGATTGTAG?GCTGCAACTC?GCCTACATGA
361 AGTCGGAATT?GCTAGTAATC?GCGGATCAGC?ATGCCGCGGT?GAATACGTTC?CCGGGTCTTG
421 TAC
This sequence is carried out similarity relatively with the 16S rRNA gene order of listed part bacterial strain among BLAST instrument and the GenBank on the www.ncbi.nlm.nih.gov website, the result shows and the similarity of Paenibacillus polymyxa (Paenibacilluspolymyxa) reaches 99%.In addition, the qualification result of morphological specificity, Physiology and biochemistry and the Biolog automatic bacteria assessing instrument that this bacterial strain is carried out according to Chinese agriculture microbial strains preservation administrative center, endophytic bacterium of the present invention belongs to (Paenibacilluspolymyxa).
Microbial fermentation processes by routine can be cultivated endophytic bacterium of the present invention, and to make with endophytic bacterium of the present invention be the biocontrol fungicide of effective constituent.The optimal pH of this bacterium is 6.0-8.0, and the suitableeest culture temperature is 25-30 ℃, and the general fermentor tank in the available general fermentation industry is cultivated.When manufacturing is biocontrol fungicide with endophytic bacterium of the present invention, can use the general substratum of known biocontrol fungicide in this technical field equally, be mixed with formulation arbitrarily such as aqua, pulvis.
Described endogenetic polymexa bacillus of plant of the present invention and what make is that the biocontrol fungicide of effective constituent compared with prior art has following advantage with it.
1, can grow conduction in that the plant body is decided at the higher level but not officially announced.
Endogenetic polymexa bacillus of plant of the present invention is carried out interior giving birth to anti-Rifampin and antagonism pathogenic fungi double-tagging measure, in the plant body, reclaim separation after several days.The result shows that this bacterial strain can grow in that the various crop body is decided at the higher level but not officially announced, and can conduction up and down in the plant body.
2, can promote plant strain growth, raising output.
Sprinkle cotton seed and the eggplant seedling water after the vernalization with endogenetic polymexa bacillus of plant bacterium liquid of the present invention, can significantly improve height of seedling and the fresh weight of plant.With endogenetic polymexa bacillus of plant bacterium liquid of the present invention cotton is irritated root, can significantly improve the output of cotton.
3, soil-borne vascular bundle disease had outstanding prevention effect.
With endogenetic polymexa bacillus of plant bacterium liquid of the present invention plant is irritated root soil-borne vascular bundle diseases such as cotton verticillium wilt, eggplant verticillium wilt are had outstanding prevention effect.
4, has the disease-resistant effect of inducing plant.
With endogenetic polymexa bacillus of plant inoculation plant of the present invention, measure the isozyme zymogram of the intravital peroxidase of plant, superoxide-dismutase, esterase after several days, the result shows in various degree appearance enzyme band phenomenons of increasing, widen and deepening of these zymograms; Mensuration to peroxidase, superoxide dismutase activity shows that also the active of these two kinds of enzymes obviously strengthens.Illustrate that this bacterial strain can induce plant disease-resistant by the activity of these and disease resistance relevant enzyme in the inducing plant body.
5, the various crop pathogenic fungi had antagonistic activity.
Indoor antagonistic activity measurement result shows that endogenetic polymexa bacillus of plant of the present invention has wider antimicrobial spectrum, and multiple pathogenic fungies such as verticillium dahliae, eggplant verticillium wilt bacterium, cotton-wilt fusarium, withered germ of water-melon, cucumber fusarium axysporum, wax gourd wilt, Sclerotinia sclerotiorum, rhizoctonia cerealis, fusarium graminearum are all had tangible antagonistic activity.
6, can use with fertilizer.
Endogenetic bacteria of the present invention can be survived in multiple fertilizer such as the green source of xanthohumic acid treasured.
Embodiment:
Be described in further detail the present invention with embodiment below, but content of the present invention is not limited thereto.
Embodiment 1 liquid preparation
To the test tube slant, substratum is NA with endogenetic polymexa bacillus of plant (Paenibacillus polymyxa) Jaas cd inoculation, 25 ℃ constant temperature culture 2-4 days, obtain the test tube kind.
The test tube kind is inoculated in the NB substratum, after 28 ℃ of constant-temperature shaking culture are spent the night, transfers in the NB substratum by 0.1% inoculum size, the nutrient solution of 28 ℃ of constant-temperature shaking culture after 14 hours promptly can be preparation.Also switchable is to carry out fermentative production in the fermentor tank of soybean cake powder substratum to fermention medium.
Prescription one, common bacteria NA substratum (weight %)
Beef extract 0.3%, yeast extract paste 0.1%,
Peptone 0.5%, glucose 1.0%,
Agar 1.5-2.0%, pH 6.8-7.0.
Prescription two, common bacteria NB substratum (weight %)
Beef extract 0.3%, yeast extract paste 0.1%,
Peptone 0.5%, glucose 1.0%,
pH?6.8-7.0。
Prescription three, soybean cake powder substratum (weight %)
Soybean cake powder 2.5%, starch 1.0%
The volume increase test of 2 pairs of cottons of embodiment
Under the identical prerequisite of cotton variety, density, fertilizer amount, cultivation management measure, establish with Jaas cd bacterium liquid irrigating root after the dilution and water and irritate 2 processing of root, randomized block design, each is handled and repeats 4 times, and the sub-district area is not less than 20m
2Irritate the root administration time and be respectively July 13 and July 22.Investigated big bell number, skellat number and blow-of-cottons number on September 10.The result shows the control group volume increase 8.22% of irritating root with the endogenetic polymexa bacillus of plant bacterium liquid irrigating root of the present invention's employing than water.
The growth promoting function of 3 pairs of cotton seedlings of embodiment
The cotton seeds of vernalization is seeded in the greenhouse basin alms bowl, Jaas cd bacterium liquid after will diluting simultaneously sprinkles and waters in cotton seed and basin alms bowl soil, measure height of seedling after 60 days and claim fresh weight, test-results shows that this bacterial strain has tangible growth promoting function to cotton seedling, the plant height comparison is according to increasing by 29.9%, and the fresh weight comparison is according to increasing by 45.2%.
The preventive and therapeutic effect of 4 pairs of cotton verticillium wilts of embodiment
Cotton seedling in the greenhouse basin alms bowl grows to 2 leaf periods, at the cotton stem apart from soil layer 2cm place inoculation endogenetic bacteria of the present invention through the NB liquid culture, after 5 days under distance microbionation point about 1cm place use equally the stab inoculation verticillium dahliae, not inoculate endogenetic bacteria of the present invention is contrast, the state of an illness of continuous observation verticillium.Endogenetic bacteria of the present invention is 67.21% to the biocontrol effect of cotton verticillium wilt.
Under the identical prerequisite of cotton variety, density, fertilizer amount, cultivation management measure, establish with Jaas cd bacterium liquid irrigating root after the dilution and water and irritate 2 processing of root, randomized block design, each is handled and repeats 4 times, and the sub-district area is not less than 20m
2In the medication for the first time of cotton verticillium wilt their early stage on July 13, medication for the second time on July 22.Investigation state of an illness radix before the 1st medication, July 31, investigation on September 10 disease index.After the result showed the endogenetic polymexa bacillus of plant bacterium liquid irrigating root that adopts with the present invention, the prevention effect on July 31 was 51.2%, and September 10, the purpose prevention effect was 49.9%, had the long lasting period.
The effect of 5 pairs of eggplant prophyiaxis and promoting growths of embodiment
When the eggplant seedling in the greenhouse basin alms bowl grew to 4-5 sheet leaf period, with the Jaas cd bacterium liquid irrigating root after the dilution, inoculation eggplant verticillium wilt bacterium after 3 days was used the Jaas cd bacterium liquid irrigating root after diluting again after 7 days.Not inoculate endogenetic bacteria of the present invention is contrast, the state of an illness of continuous observation verticillium, the plant height and the fresh weight of 40 days " Invest, Then Investigate " eggplant seedlings.The result shows that endogenetic bacteria of the present invention has tangible growth promoting function to eggplant, has good prevention effect to eggplant verticillium wilt.
The antagonistic action of 6 pairs of multiple pathogenic fungies of embodiment
Connecing diameter in the dull and stereotyped center of PDA is 5mm pathogenic fungi bacterium cake, 25 ℃, cultivate after 1-3 days, and at the good Jaas cd of taking over a job of colony edge 1.5cm place, measure inhibition zone behind the 6-7d.The result shows that endogenetic polymexa bacillus of plant of the present invention has wider antimicrobial spectrum, and multiple pathogenic fungies such as verticillium dahliae, eggplant verticillium wilt bacterium, cotton-wilt fusarium, withered germ of water-melon, cucumber fusarium axysporum, wax gourd wilt, Sclerotinia sclerotiorum, rhizoctonia cerealis, fusarium graminearum are all had tangible antagonistic activity.
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| CN101235406B (en) * | 2007-01-29 | 2012-02-22 | 上海医药工业研究院 | Method for synthesizing polymyxin E by fermentation method |
| CN101519639B (en) * | 2008-02-27 | 2011-04-13 | 中国科学院生态环境研究中心 | Paenibacillus polymyxa for preventing and treating plant fungal diseases and production method thereof |
| CN102249754A (en) * | 2010-05-17 | 2011-11-23 | 中国科学院生态环境研究中心 | Paenibacillus polymyxa microbial fertilizer produced through sweet potato starch waste water recovery |
| CN102153400B (en) * | 2010-12-13 | 2013-03-20 | 绵阳台沃农业科技有限责任公司 | Multi-effect nutritional agent special for wheat basal application |
| CN102168053B (en) * | 2011-02-13 | 2013-09-25 | 南京农业大学 | Paenibacilluspolymyxa strain for coproduction of LI-F type antibiotic and dibutyl phthalate and application thereof |
| CN102352331B (en) * | 2011-10-21 | 2013-01-16 | 中国农业大学 | Nitrogen-fixing Paenibacillus sp. 1-33 and application thereof |
| CN102352333B (en) * | 2011-10-21 | 2013-01-16 | 中国农业大学 | Nitrogen-fixing Paenibacillus sp. 1-49 and application thereof |
| CN102676435B (en) * | 2012-05-24 | 2013-06-12 | 福建师范大学福清分校 | Paenibacillus polymyxa for antagonizing Fusarium oxysporum in rhizosphere soil of Radix Pseudostellariae |
| CN102851243A (en) * | 2012-09-07 | 2013-01-02 | 北京市农林科学院 | Paenibacillus polymyxa and applications thereof |
| CN103773708A (en) * | 2012-11-16 | 2014-05-07 | 江苏省农业科学院 | A plant endophytic bacterium for disease prevention and growth promotion |
| CN105132324B (en) * | 2015-09-07 | 2018-09-28 | 哈尔滨师范大学 | One plant of wheat rhizosphere Paenibacillus polymyxa WXD 6-4 and its application |
| CN107711890A (en) * | 2017-07-27 | 2018-02-23 | 上海万力华生物科技有限公司 | A kind of Paenibacillus polymyxa sterilization aqua and preparation method thereof |
| CN111876361B (en) * | 2020-08-12 | 2022-05-27 | 浙江农林大学 | Biocontrol Paenibacillus separated from healthy hickory woodland and application thereof |
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