CN103060241B - Acinetobacter calcoaceticus for promoting rhizosphere growth of walnuts and application thereof - Google Patents
Acinetobacter calcoaceticus for promoting rhizosphere growth of walnuts and application thereof Download PDFInfo
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- CN103060241B CN103060241B CN2013100165315A CN201310016531A CN103060241B CN 103060241 B CN103060241 B CN 103060241B CN 2013100165315 A CN2013100165315 A CN 2013100165315A CN 201310016531 A CN201310016531 A CN 201310016531A CN 103060241 B CN103060241 B CN 103060241B
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Abstract
The invention discloses acinetobacter calcoaceticus for promoting rhizosphere growth of walnuts. The acinetobacter calcoaceticus is named acinetobacter calcoaceticus (Acinetobacter calcoaceticus) X128 in class, and preserved at the general microbiological center of the China Committee for Culture Collection of Microorganisms on December 31, 2012; and the preservation number is CGMCC No.7071. The invention also provides application of the acinetobacter calcoaceticus for promoting rhizosphere growth of the walnuts. The acinetobacter calcoaceticus for promoting rhizosphere growth of the walnuts is evenly fed to the periphery of the walnut root in a pouring manner; root growth can be obviously facilitated; the quantity of lateral roots is improved; and growth of the bottom diameter of the walnut and the height of seedling is facilitated. In contrast, the difference reaches a significant level. Therefore, excellent strain resources are provided for developing a special microbial fertilizer for the walnut in the future.
Description
Technical field
The present invention relates to strain walnut rhizosphere growth-promoting acinetobacter calcoaceticus and an application thereof.
Background technology
Walnut (Juglans regia L) is walnut section, Juglans plants, is listed as first of the large nut in the world four, have the title of " king of woody oleiferous plants ", and its kind of benevolence has high nutritive value and nourishing function.Walnut is again important in material and ecological afforestation seeds, is widely cultivated in mountain area, hills, rural area and town and country, has economy, ecology and social multiple benefits.China is maximum in the world walnut producing country and country of consumption, and walnut cultivated area and nut ultimate production all occupy first place in the world, and the whole nation has more than 20 province (autonomous region, municipality directly under the Central Government) to have walnut to distribute.According to statistics, within 2010, Chinese walnut total cultivated area surpasses 2,000,000 hectares, and a year nut ultimate production breaks through 900,000 tons.
In recent years, along with the rapid raising of walnut planting benefit, the walnut fertilising drops into constantly and increases, and fertilising has become important control measures of Walnut cultivation.Present stage the fertilizer used of walnut, be mainly that compound manure, farm manure that chemical fertilizer is combined into are main organic fertilizer etc.Be implanted in Hills because walnut is multiple, the soil water-reataining fertilizer-keeping ability, the fertilizer nutrient utilization ratio of composite fertilizer is low, has caused the eutrophication of surface water and the pollution of underground water; Though the farm manure of take is main organic fertilizer and can improves soil physico-chemical property, the germ that organic character wherein is very different, contain and sick worm's ovum etc. easily cause soil pollution.Microbial fertilizer can not only be improved soil, improves ecological environment of soil, can also effectively improve plant absorbing soil nutrient.Therefore, seek the new-type fertilizer that is applicable to walnut, especially microbial fertilizer, receive much attention with the research that reduces fertilizer amount, lifting fertilizer effect.
The domestic report of the research about the walnut microbial fertilizer is less.Yang Chunhua etc. (1998) from forest leaf isolation and selection 6 bacterial isolates preparations, the walnut seedling is all had to the growth-promoting effect; Gu Bin etc. (2002) select take the commodity bio-fertilizers such as biological potassium fertilizer that the biologic phosphorus fertilizer that genus bacillus is major ingredient and the silicate bacteria of take be major ingredient, excellent kind of walnut children, has carried out application test in orchard age; Tang Zhoubin etc. (2003) utilize the PGPR microbial inoculum of having applied on multiple kinds of crops, and Juglans is sprayed, and have produced growth-promoting functions preferably.
Plant growth-promoting rhizobacteria (plant growth-promoting rhizobacteria, PGPR) is the important sources of microbial fertilizer effective strain.PGPR refers to and survives in plant rhizosphere, root table plant-growth is had to the beneficial bacteria general designation that promotes or pathogenic bacteria is had to antagonistic action.Studies confirm that in a large number, PGPR has certain tailored version, indigenous, and therefore by some, microorganism commonly used is directly applied in walnut is produced, and is difficult to continual and steady generation and is similar to the experiment effect in farm crop.The walnut growth cycle is long, with respect to general farm crop, more easily forms some metastable biological community structures.Therefore, be necessary to screen the PGPR with growth-promoting functions for walnut.At present, the screening for the special-purpose PGPR of walnut has no report.
Summary of the invention
The objective of the invention is for overcoming above-mentioned the deficiencies in the prior art, strain walnut rhizosphere growth-promoting acinetobacter calcoaceticus and an application thereof is provided.
The present invention is achieved by the following technical solutions:
One strain walnut rhizosphere growth-promoting acinetobacter calcoaceticus, its Classification And Nomenclature is acinetobacter calcoaceticus (Acinetobacter calcoaceticus) X128, on December 31st, 2012, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is: CGMCC No.7071.
Colony characteristics and the thalli morphology of described walnut rhizosphere growth-promoting acinetobacter calcoaceticus:
After this bacterium being cultivated on the LB flat board to 24h, the formation bacterium colony is glossy, and circle is translucent.Smooth surface, flat, neat in edge; The somatic cells size is the μ m of (1-1.2) μ m * (0.5-0.6); Produce gemma, G-, cell is rod-short.
The physiological and biochemical property of described walnut rhizosphere growth-promoting acinetobacter calcoaceticus:
The gelatin hydrolysis negative, the citrate test positive, it is positive that glucose produces the acid test, the L-glutamic acid transfer enzyme test positive, growth temperature test: 37 ℃ of positives (can grow), 41 ℃ of feminine genders, 44 ℃ of feminine genders.
By the 16S rDNA sequence of described walnut rhizosphere growth-promoting acinetobacter calcoaceticus and the sequence alignment in the GenBank database, result shows: X128 and Acinetobacter calcoaceticus are in together in a minimum branch, and the similarity of its 16S rDNA sequence and Acinetobacter calcoaceticus (AJ888983) reaches 99%.In conjunction with colonial morphology, physiological and biochemical property and 16S rDNA sequential analysis, be accredited as acinetobacter calcoaceticus (Acinetobacter calcoaceticu).
Described walnut rhizosphere growth-promoting acinetobacter calcoaceticus can produce indolylacetic acid (IAA), and coordinate plant growth and growth are had to vital role.
Beneficial effect of the present invention:
Walnut rhizosphere growth-promoting acinetobacter calcoaceticus of the present invention is made microbial inoculum and is inoculated into walnut, can obviously promote root growth, improves the lateral root number, increases the growth of walnut leading thread and height of seedling, and compared with the control, difference reaches conspicuous level.Therefore, the present invention provides good strain resource for the microbial fertilizer of developing in the future the walnut special use.
Embodiment
Below in conjunction with embodiment, the present invention is further described.
One strain walnut rhizosphere growth-promoting acinetobacter calcoaceticus, its Classification And Nomenclature is: acinetobacter calcoaceticus (Acinetobacter calcoaceticus) X128, on December 31st, 2012, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is: CGMCC No.7071.
The screening of embodiment 1 walnut rhizosphere growth-promoting acinetobacter calcoaceticus
(1) screening process:
By the gradient dilution method, from the walnut Rhizosphere sampling of model town, business section, Zhangqiu, Shandong Province, isolate 342 strain isolates.By three zoning collimation methods, the bacterium be separated to is carried out to purifying, and judge whether purifying of bacterial strain by microscopy, the bacterium after purifying is numbered, picking list bacterium colony, be transferred on the LB inclined-plane and save backup.Filter out 21 strains and protect green effect bacterial strain preferably by protecting green method, in conjunction with radish cotyledon weightening finish method, finishing screen is selected the bacterium that 1 strain has good growth-promoting effect, is designated as X128.
(2) colony characteristics and thalli morphology:
After X128 being cultivated on the LB flat board to 24h, the formation bacterium colony is glossy, and circle is translucent.Smooth surface, flat, neat in edge; The somatic cells size is the μ m of (1-1.2) μ m * (0.5-0.6); Produce gemma, G-, cell is rod-short.
(3) physiological and biochemical property:
The gelatin hydrolysis negative, the citrate test positive, it is positive that glucose produces the acid test, the L-glutamic acid transfer enzyme test positive, growth temperature test: 37 ℃ of positives (can grow), 41 ℃ of feminine genders, 44 ℃ of feminine genders.
(4) producing the IAA ability measures
X128 is inoculated in to the LB substratum, and 28 ℃, the 180rpm shaking table is cultivated 4d.By the OD600 value of spectrophotometry bacteria suspension, then by the centrifugal 10min of bacteria suspension 10000rpm, get supernatant liquor and add equal-volume Salkowski (50mL35%HClO
4+ 1mL0.5M FeCl
3) color solution, the standing 30min of lucifuge, measure its OD530 value.Calculating bacteria concentration OD600 value is 1 o'clock, the content of IAA in the unit volume fermented liquid, the content of the typical curve calculating IAA by the IAA gradient dilution.By measuring, it is 26.44 μ g/ (mLOD that acinetobacter calcoaceticus X128 produces IAA content
600)
-1.
(5) 16S rDNA sequential analysis
16S rDNA gene order, be shown in shown in the nucleotides sequence list.By the sequence alignment in surveyed 16S rDNA sequence and GenBank database, result shows: X128 and Acinetobacter calcoaceticus are in together in a minimum branch, and the similarity of its 16S rDNA sequence and Acinetobacter calcoaceticus (AJ888983) reaches 99%.In conjunction with colonial morphology, physiological and biochemical property and 16S rDNA sequential analysis, be accredited as acinetobacter calcoaceticus (Acinetobacter calcoaceticu).
Embodiment 2 pot experiments
The walnut rhizosphere growth-promoting acinetobacter calcoaceticus X128 of preservation is activated on the LB inclined-plane, picking one articulating enters beef-protein medium (extractum carnis 0.3%, peptone 1%, sodium-chlor 0.5%, agar 2%, pH7.0-7.2, above equal quality per-cent) in, in 37 ℃, under the 180rpm condition, shaking culture is 2 days, centrifugal 5min under fermented liquid 6000rpm, with after stroke-physiological saline solution rinse thalline 3 times, it is 2.5 * 10 that stroke-physiological saline solution is regulated the bacteria suspension viable count
8Cfu/m(suspension A), standby.Field planting after Juglans is stained with to root in suspension A.By stroke-physiological saline solution, the bacterial concentration of suspension A is adjusted to 1 * 10
8Then get 50ml suspension B and evenly water around the Juglans root cfu/ml(suspension B).After Walnut Roots is stained with to root in stroke-physiological saline solution, pouring equivalent stroke-physiological saline solution is (CK) in contrast, and normal management is measured main root length, lateral root number, height of seedling and the leading thread of Walnut Roots after two months.It is as shown in the table for result.
Main root length, lateral root number, height of seedling and the leading thread of the walnut that as can be seen from the table, walnut rhizosphere growth-promoting acinetobacter calcoaceticus suspension A processes have improved 19.65%, 18.68%, 23.07% and 20.27% than contrast respectively.Difference all reaches conspicuous level.More than show: the growth of 128 pairs of walnuts of strain X has obvious promoter action.
In same row, same letter means that difference is not remarkable, otherwise means significant difference (p<0.05).
Claims (2)
1. a strain walnut rhizosphere growth-promoting acinetobacter calcoaceticus, it is characterized in that, its Classification And Nomenclature is acinetobacter calcoaceticus (Acinetobacter calcoaceticus) X128, on December 31st, 2012, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is: CGMCC No.7071.
2. walnut rhizosphere growth-promoting acinetobacter calcoaceticus claimed in claim 1 application in promoting the walnut growth.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20080054047A (en) * | 2006-12-12 | 2008-06-17 | (주)친농바이오 | Phosphate solubilizing bacteria acinetobacter calcoaceticus cn7 of bioremediation for the recovery of salt injury soil |
| KR100908412B1 (en) * | 2009-01-07 | 2009-07-20 | 바이프롬 주식회사 | Acinetobacter calcoaceticus strains and microbial agent for the treatment of soil having the same |
| CN101914470A (en) * | 2010-07-22 | 2010-12-15 | 山东省科学院生物研究所 | Acinetobacter calcoaceticus and culture method and application thereof |
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2013
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20080054047A (en) * | 2006-12-12 | 2008-06-17 | (주)친농바이오 | Phosphate solubilizing bacteria acinetobacter calcoaceticus cn7 of bioremediation for the recovery of salt injury soil |
| KR100908412B1 (en) * | 2009-01-07 | 2009-07-20 | 바이프롬 주식회사 | Acinetobacter calcoaceticus strains and microbial agent for the treatment of soil having the same |
| CN101914470A (en) * | 2010-07-22 | 2010-12-15 | 山东省科学院生物研究所 | Acinetobacter calcoaceticus and culture method and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| 《四川核桃生产区根际解磷细菌研究》;余旋;《中国博士学位论文全文数据库》;20120228(第2期);摘要 * |
| 《耐盐碱解磷菌的筛选及其对羊草生长的促进作用研究》;胡纯国;《中国优秀硕士学位论文全》;20090228(第2期);摘要，第42页 * |
| Dijkshoorn,L.,等.《Acinetobacter calcoaceticus partial 》.《GenBank登录号:AJ888983》.2005,全文. * |
| 余旋.《四川核桃生产区根际解磷细菌研究》.《中国博士学位论文全文数据库》.2012,(第2期),摘要. |
| 胡纯国.《耐盐碱解磷菌的筛选及其对羊草生长的促进作用研究》.《中国优秀硕士学位论文全》.2009,(第2期),摘要，第42页. |
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Inventor after: Ma Hailin Inventor after: Xing Shangjun Inventor after: Liu Fangchun Inventor after: Du Zhenyu Inventor after: Ma Bingyao Inventor after: Du Binghai Inventor after: Ding Yanqin Inventor after: Yao Liangtong Inventor after: Chen Bo Inventor before: Ma Hailin Inventor before: Xing Shangjun Inventor before: Liu Fangchun Inventor before: Du Zhenyu Inventor before: Ma Bingyao Inventor before: Du Binghai Inventor before: Ding Yanqin Inventor before: Yao Liangtong Inventor before: Chen Bo |
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