CN101698828B - Pseudomonas aeruginosa D10, and preparation method and applications thereof - Google Patents
Pseudomonas aeruginosa D10, and preparation method and applications thereof Download PDFInfo
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- CN101698828B CN101698828B CN2009101929211A CN200910192921A CN101698828B CN 101698828 B CN101698828 B CN 101698828B CN 2009101929211 A CN2009101929211 A CN 2009101929211A CN 200910192921 A CN200910192921 A CN 200910192921A CN 101698828 B CN101698828 B CN 101698828B
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Abstract
The invention discloses a pseudomonas aeruginosa D10 which is preserved in China Typical Culture Preservation Center, and the preservation number is CCTCC NO: M 209104. A colony on a beef paste peptone culture medium appears brown, round, glossy and wet, and has trim edges; the diameter of the colony is 0.2-0.5 cm; and the thallus is in a short bar shape and does not have gemma. The invention also discloses a preparation method and applications of the pseudomonas aeruginosa D10. The pseudomonas aeruginosa D10 of the invention can inhibit corn northern and southern leaf blight, has strong antergic functions on corn northern leaf blight, corn southern leaf blight, corn curvalaria leaf spot, cotton verticillium wilt, wheat fusarium graminearum, banana colletotrichum toilette and banana fusarium wilt, has the advantages of wide bacteriostatic range, high prevention efficiency and favorable environmental safety, and has favorable prospects for development and applications.
Description
Technical field
The present invention relates to the plant protection field, be specifically related to a kind of Pseudomonas aeruginosa D10 and preparation method thereof and application.
Background technology
The leaf blight of corn is caused with the leaf portion maize diseases that to produce large-scale scab symptom be the master by the big spot bristle of Ascomycota mycosphaerella ball chamber bacterium [Setosphaeria turcica (Luttrell) Leonard et Suggs]; Its distribution is wider; Endanger heavylier, this disease of main corn producing region all has in various degree generation and harm all over the world.It is reported,, can cause the corn underproduction 50% in the serious popular of leaf blight of corn time and area, in addition more.
Corn southern leaf blight is the important leaf diseases that is belonged to the caused warm moist corn of different cochliobolus [Cochliobolus heterostrophus Drechsler] producing region by the Ascomycota cochliobolus; The main harm blade; Also can endanger leaf sheath, bract, fruit ear and seed, be one of disease that generally takes place on the domestic and international corn.It is about 10%～20% that general according to estimates medium morbidity time susceptible variety is lost, and can reach 30%～80% when serious, even ruin kind of a total crop failure.
At present, the control to corn northern leaf blight and southern leaf blight mainly is cultural control and chemical prevention.Cultural control mainly is through the cultivation disease-resistant variety, but some kinds are easy to lose disease resistance, so realize preventing and treating disease through disease-resistant variety fully certain degree of difficulty are arranged.Though the generation of disease and popular can be effectively controlled in chemical agent control, secular use meeting produces directly murder by poisoning to non-target organism, causes environmental pollution, has had a strong impact on human beings'health and environment.Therefore, seeking new nuisanceless method has become the task of top priority, and biological control method will more and more come into one's own.
The antagonism endogenetic bacteria plays a part very important in biocontrol of plant disease as a kind of important biocontrol microorganisms.Its main advantage is: kind and the One's name is legion of (1) bacterium exist at plant rhizosphere and overground part in a large number; (2) bacterium is wider to the mode of action of pathogenic bacteria, can be through competition, and antagonism and parasitism, modes such as inducing plant generation resistance exert an influence to pathogenic bacteria; (3) has surprising reproduction speed; (4) many bacteriums are present in plant rhizosphere and overground part, and are more suitable to the ecotope of plant; (5) bacterium can pass through artificial culture mostly, is convenient to control, and is easy to operate in practice; (6) some bacterium can not only prevent and treat disease but also can produce and increase income effect.
Therefore, in the sweet corn healthy plant, screen the interior living biocontrol bacteria bacterial strain that obtains corn northern leaf blight and southern leaf blight is had antagonistic action, significant for the control of corn northern leaf blight and southern leaf blight.
Summary of the invention
The objective of the invention is to deficiency to prior art; A kind of Pseudomonas aeruginosa D10 that the corn northern leaf blight and southern leaf blight bacterium is had antagonistic action is provided; This bacterial strain can effectively suppress corn northern leaf blight and southern leaf blight, and is significant to the control of corn northern leaf blight and southern leaf blight.
Another object of the present invention is to provide the preparation method of above-mentioned Pseudomonas aeruginosa D10.
A further object of the invention provides the application of above-mentioned Pseudomonas aeruginosa D10 in the plant disease control.
The present invention is achieved through following technical scheme:
Pseudomonas aeruginosa D10 of the present invention, Pseudomonas aeruginosa D10 is preserved in Chinese typical culture collection center, and the preservation address is a China. Wuhan. Wuhan University, preserving number is CCTCC NO:M 209104.
Said Pseudomonas aeruginosa D10 bacterium colony on beef-protein medium is brown, and circle is glossy, moistening, neat in edge, and colony diameter 0.2～0.5cm, thalline becomes rod-short, and size is about 1.10um * 0.57um, no gemma.
Said Pseudomonas aeruginosa D10 has following biochemical characteristic: Exserohilum turcicum, southern corn leaf blight, the curved spore leaf spot fungi of corn, verticillium dahliae, fusarium graminearum, Glorosprium musarum Cookeet Mass and banana blight bacteria are had antagonistic action; Wherein, bacillus subtilis A 16 also has dull and stereotyped antagonistic action to Exserohilum turcicum and southern corn leaf blight.
Said Pseudomonas aeruginosa D10 also has biological actions such as fixed nitrogen, promotion plant-growth, enhancing host resistance.
The preparation of Pseudomonas aeruginosa D10 of the present invention comprises the steps:
(1) clip is treated isolating corn tissue, and handle with 1%NaClO the sterilization back, uses aseptic water washing again;
(2) corn tissue that will pass through surface sterilization adds sterilized water grinds, and dilutes 1 * 10 successively according to gradient concentration
2, 1 * 10
3, 1 * 10
4Doubly, separate application places 28 ℃ of constant incubators to cultivate on the NA flat board;
(3) treat that flat board grows bacterium colony after, according to the morphological specificity of bacterium colony, the single bacterium colony of picking, through line, obtain Pseudomonas aeruginosa D10 behind the purifying, 4 ℃ of refrigerators are kept on the NA inclined-plane.
Above-mentioned preparation method is preferably following steps:
(1) clip is treated the isolating 2g of corn tissue, behind the 75% alcohol disinfecting 3min, handles 10min with 1%NaClO, uses aseptic water washing again 4 times, gets last washing fluid and stays and do contrast;
(2) corn tissue that will pass through surface sterilization adds mortar that the 10mL sterilized water places sterilization and is ground to tissue and pulverizes, and leaves standstill 3～10min, dilutes 1 * 10 successively according to gradient concentration
2, 1 * 10
3, 1 * 10
4Doubly, draw 100 μ L respectively and coat on the NA flat board, place 28 ℃ of constant incubators to cultivate;
(3) treat that flat board grows bacterium colony after, according to the morphological specificity of bacterium colony, the single bacterium colony of picking through line, repeatedly behind the purifying, obtains bacillus subtilis A 16,4 ℃ of refrigerators are kept on the NA inclined-plane.
When Pseudomonas aeruginosa D10 of the present invention is used to promote plant-growth and controlling plant diseases; After this Pseudomonas aeruginosa D10 is inoculated in maize seedling, cotton seedling, stem and leaf of Wheat or banana seedlings; Can obtain to the curved spore leaf spot of the leaf blight of corn, corn southern leaf blight and corn have disease resistance milpa, to cotton verticillium wilt have disease resistance cotton plants, the wheat mycosis is had the wheat plant of disease resistance or the banana plant that banana anthracnose and banana blight are had disease resistance, and the upgrowth situation of plant significantly improves.
Compared with prior art, the present invention has following beneficial effect:
Pseudomonas aeruginosa D10 of the present invention is the useful endogenetic bacteria of plant; Restraining effect to the corn northern leaf blight and southern leaf blight bacterium is strong; Multiple pathogenic bacteria is had antagonistic action, and antimicrobial spectrum is wide, and environmental safety is good; Can be applicable in the biological control of Plant diseases, especially significant to the control of corn northern leaf blight and southern leaf blight; The culture condition of bacillus subtilis A 16 of the present invention is simple, and easy storing is convenient to suitability for industrialized production, has the excellent development application prospect.
Description of drawings
Fig. 1 is the restraining effect of Pseudomonas aeruginosa D10 to the southern corn leaf blight mycelia;
Fig. 2 is the restraining effect of Pseudomonas aeruginosa D10 to the Exserohilum turcicum mycelia;
Fig. 3 is the restraining effect of Pseudomonas aeruginosa D10 to verticillium dahliae, corn curved spore leaf spot fungi, fusarium graminearum and banana blight bacteria, and wherein A is the restraining effect of Pseudomonas aeruginosa D10 to verticillium dahliae; B is the restraining effect of Pseudomonas aeruginosa D10 to the curved spore leaf spot fungi of corn; C is the restraining effect of Pseudomonas aeruginosa D10 to fusarium graminearum; D is the restraining effect of Pseudomonas aeruginosa D10 to banana blight bacteria (FOC4).
Embodiment
Come further to explain the present invention below in conjunction with embodiment, but embodiment does not do any qualification to the present invention.
Embodiment 1 Pseudomonas aeruginosa D10 is to the dull and stereotyped antagonistic action of southern corn leaf blight and Exserohilum turcicum
Pseudomonas aeruginosa D10 obtains through flat board face-off screening.PDA (wherein adds the 5g/L peptone at the potato sucrose substratum; The pH value is adjusted to 6.5) to insert diameter be the corn northern leaf blight and southern leaf blight bacterium mycelia piece of 5mm in dull and stereotyped central authorities; At every ware symmetry sterilization filter paper that to connect three diameters be 5mm, filter paper width between centers central authorities mycelia piece is 25mm subsequently, on every filter paper, inserts inoculum 10 μ L at last; With the flat board that does not connect the Pseudomonas aeruginosa nutrient solution is blank; Repeat for three times, place 28 ℃ of incubators to cultivate 7d after, measure the inhibition zone radius; It is thus clear that Pseudomonas aeruginosa D10 significantly suppresses the growth of southern corn leaf blight, significantly suppress the growth of Exserohilum turcicum by Fig. 1 by the visible Pseudomonas aeruginosa D10 of Fig. 2.
The indoor preventive effect of embodiment 2 Pseudomonas aeruginosa D10 is measured
Will (bacteria containing amount be 1.2 * 10 with the antagonistic strain nutrient solution through the sweet corn seeds after the 0.1%NaClO sterilization
9Cfu/ml) behind the seed soaking 5h, be sowed at (earth sterilization) in the flowerpot, 10 seeds of every basin sowing are established the sterilized water seed soaking and are contrast.10 basins are sowed in every processing, grow to three leaves one when new (about 21d), and spray corn northern leaf blight and southern leaf blight bacterium spore suspension is placed in the greenhouse after the inoculation and cultivates, 7d investigation incidence behind the inoculation pathogenic bacteria.The result shows that the disease index of the large and small pinta of handling through Pseudomonas aeruginosa D10 bacterial strain of milpa is compared respectively and has shone low 35.1,57.4.
Embodiment 3 Pseudomonas aeruginosa D10 are to the antagonistic action of multiple pathogenic bacteria
Adopt dull and stereotyped face-off method, test Pseudomonas aeruginosa D10 is to the antagonistic action of the curved spore leaf spot fungi of corn, verticillium dahliae, fusarium graminearum, Glorosprium musarum Cookeet Mass, 5 kinds of pathogenic bacterias of banana blight bacteria.Screen with reference to dull and stereotyped face-off method.PDA (wherein adds the 5g/L peptone at the potato sucrose substratum; The pH value is adjusted to 6.5) to insert diameter be the pathogenic bacteria mycelia piece of 5mm in dull and stereotyped central authorities, apart from 25mm place, pathogenic bacteria both sides, with transfering loop streak inoculation endogenetic bacteria; Only to connect pathogenic bacteria is contrast; In 28 ℃ of incubators, cultivate 7d, measure antibacterial bandwidth, every processing repetition 3 times.The result sees table 1 and Fig. 3.From Fig. 3 figure A, can see the restraining effect of Pseudomonas aeruginosa D10 to verticillium dahliae; Can see the restraining effect of Pseudomonas aeruginosa D10 among the B to the curved spore leaf spot fungi of corn; Can be observed the restraining effect of Pseudomonas aeruginosa D10 among the C to fusarium graminearum; Can be observed the restraining effect of Pseudomonas aeruginosa D10 among the D to No. 4 microspecies of banana blight bacteria (FOC4).
Table 1 antagonistic strain is to the restraining effect of various pathogenic bacteria
The antibacterial band of " ++ " expression is 6～8mm; " +++" represent that antibacterial band is 8～10mm;
" ++ ++ " represented antibacterial band＞10mm.
Claims (6)
1. a Pseudomonas aeruginosa D10 is characterized in that said Pseudomonas aeruginosa D10, and Pseudomonas aeruginosa D10 is preserved in Chinese typical culture collection center, and preserving number is CCTCC NO:M 209104.
2. Pseudomonas aeruginosa D10 according to claim 1 is characterized in that said Pseudomonas aeruginosa D10 bacterium colony on beef-protein medium is brown, circle; Glossy, moistening, neat in edge; Colony diameter 0.2～0.5cm, thalline becomes rod-short, no gemma.
3. Pseudomonas aeruginosa D10 according to claim 1 is characterized in that said Pseudomonas aeruginosa D10 has following biochemical characteristic: Exserohilum turcicum, southern corn leaf blight, the curved spore leaf spot fungi of corn, verticillium dahliae, fusarium graminearum, Glorosprium musarum Cookeet Mass and banana blight bacteria are had antagonistic action.
4. Pseudomonas aeruginosa D10 according to claim 3 is characterized in that said Pseudomonas aeruginosa D10 has dull and stereotyped antagonistic action to Exserohilum turcicum and southern corn leaf blight.
5. the said Pseudomonas aeruginosa D10 of claim 1 is used for the purposes of control of maize leaf blight, corn southern leaf blight, the curved spore leaf spot of corn, cotton verticillium wilt, wheat mycosis, banana anthracnose and banana blight.
6. purposes according to claim 5; It is characterized in that utilizing Pseudomonas aeruginosa D10CCTCC NO.M209104 to be inoculated in maize seedling, cotton seedling, stem and leaf of Wheat or banana seedlings, obtain to the curved spore leaf spot of the leaf blight of corn, corn southern leaf blight and corn have disease resistance milpa, to cotton verticillium wilt have disease resistance cotton plants, the wheat mycosis is had the wheat plant of disease resistance or the banana plant that banana anthracnose and banana blight are had disease resistance.
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| CN102533603B (en) * | 2012-01-10 | 2013-04-10 | 南京农业大学 | Pseudomonas sp. 841P-3 for preventing cotton verticillium wilt and use thereof |
| CN102586120B (en) * | 2012-01-31 | 2013-10-09 | 中国农业科学院棉花研究所 | Endophytic fungi CEF08111 of cotton and application thereof in prevention and treatment of cotton verticillium wilt |
| CN103361284B (en) * | 2012-04-09 | 2015-11-25 | 董金皋 | One strain can produce pseudomonas aeruginosa strains and the Raw toxin preparation method thereof of herbicidal active compounds |
| CN102703353B (en) * | 2012-06-07 | 2014-08-27 | 东北农业大学 | Biocontrol strain PA-2 (Pseudomonas Aeruginosa) for preventing and curing tobacco angular leaf spot |
| CN103509740B (en) * | 2013-08-13 | 2015-04-29 | 浙江大学 | Pseudomonas chlororaphis for preventing and treating crop fusarium disease and applications thereof |
| CN103602621B (en) * | 2013-12-02 | 2015-04-15 | 江苏省农业科学院 | Pseudomonas choloeaphtis and application |
| CN105462882B (en) * | 2015-12-14 | 2018-07-31 | 浙江大学 | A kind of pseudomonas aeruginosa and its application for preventing crop verticillium wilt |
| CN105779365B (en) * | 2016-05-17 | 2019-01-25 | 山东省林业科学研究院 | The raw pseudomonas aeruginosa of one plant of red-spotted stonecrop rhizosphere growth-promoting and its application |
| CN106635870B (en) * | 2016-09-27 | 2019-09-13 | 安徽瑞虎肥业有限公司 | Bacillus and its application |
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| .荧光假单胞菌株M18防治甜瓜蔓枯病害.《上海交通大学学报 》.2001,第35卷(第7期),1062-1065. |
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