无机磷溶解菌RW8的筛选、鉴定及对白三叶促生效果研究

doi:10.11686/cyxb2016282

摘要/Abstract

摘要： 从白三叶根际分离筛选9株溶磷微生物,并对挑选菌株进行形态、细胞以及种属鉴定,并分析其对白三叶的促生效果。通过分析溶磷圈与溶磷菌落直径的大小与比值获得溶磷量与持续溶磷能力较强的RW8菌株。通过形态与扫描电镜观察发现RW8是具有鞭毛的短杆菌,其菌落形态表面光滑,不透明;将RW8 16S rDNA序列比对NCBI数据库发现其与阴沟肠杆菌属(Enterobacter sp.)的同源性高达99.5%,Biolog鉴定其与阴沟肠杆菌(Enterobacter cloacae ss dissolvens)的相似性为0.610。RW8溶磷量达424.85 μg/mL,溶磷能力可能与RW8的产酸性能(12.30 μg/mL)密切相关。RW8对白三叶根系与幼苗的生物产量的分配有显著影响,接种RW8后能抑制白三叶根系伸长,根系鲜重与干重有下降趋势,但差异不显著;而幼苗的生物产量(干重与鲜重)均显著高于对照(P<0.05),并且该现象与植物生长激素IAA无关,其具体的作用机制还有待于进一步研究。

Abstract: Nine strains of inorganic phosphorus-solubilizing bacteria were isolated from the rhizosphere of white clover (Trifolium repens), and each strain was identified to the genus and species level based on its phenotypic and cytological characteristics. The growth-promoting effect of each strain on white clover was also analyzed. Strain RW8, which showed the strongest phosphorus-solubilizing activity, was selected based on analyses of the size and ratio of its plaques and colonies. Scanning electron microscopy observations revealed that RW8 was Bacillus brevis, a flagellated bacterium. Colonies of RW8 were opaque and had a smooth surface. In NCBI blast searches, the 16S rDNA sequence of RW8 showed 99.5% homology with that of Enterobacter sp. A Biolog analysis suggested that RW8 had high similarity (0.610) with Enterobacter cloacae. In a phosphorous-dissolving assay, RW8 produced 424.85 μg/mL dissolved phosphorus, which was possibly related to its strong organic acid production (12.30 μg/mL). RW8 strongly affected the biomass distribution to roots in white clover seedlings. Root elongation and the fresh weight and dry weight of roots were lower in RW8-colonized plants than in control plants, although the differences were not statistically significant. However, the seedling biomass (dry weight and fresh weight) of RW8-colonized white clover was significantly greater than that of control plants (P<0.05), and this phenomenon was not a result of indole acetic acid production by the bacterium. Further research is required to determine the precise mechanism of its growth-promoting effect.

中图分类号:

lixiaodongzl@163.com

蔡璐, 王小利, 陈莹, 王子苑, 李小冬. 无机磷溶解菌RW8的筛选、鉴定及对白三叶促生效果研究[J]. 草业学报, 2017, 26(5): 181-188.

CAI Lu, WANG Xiao-Li, CHEN Yin, WANG Zi-Yuan, LI Xiao-Dong. Isolation and identification of an inorganic phosphorus-solubilizing bacterium RW8 and its growth-promoting effect on white clover (Trifolium repens)[J]. Acta Prataculturae Sinica, 2017, 26(5): 181-188.

参考文献

[1] Sashidhar B, Podile A R. Mineral phosphate solubilization by rhizosphere bacteria and scope for manipulation of the direct oxidation pathway involving glucose dehydrogenase. Journal of Applied Microbiology, 2010, 109(1): 1-12.
[2] Zou X, Dan B, Doxtader K G. A new method for estimating gross phosphorus mineralization and immobilization rates in soils. Plant & Soil, 1992, 147: 243-250.
[3] Podile A R, Kishore G K. Plant growth-promoting rhizobacteria[M]//Gnanamanickam S S. Plant-Associated Bacteria. Netherlands: Springer, 2005: 195-230.
[4] Alloush G A, Boyer D G, Belesky D P, et al . Phosphorus mobility in a karst landscape under pasture grazing system. Agronomie, 2003, 23(7): 593-600.
[5] Zhang Q H. Study on Variation Features of Phosphorus of Karst Rocky Desertification Soil and Ecological Restore in Guizhou[D]. Guizhou: Guizhou University, 2007.
张清海. 贵州喀斯特石漠化地区土壤磷素变异特征及生态恢复研究[D]. 贵州: 贵州大学, 2007.
[6] Tripura C, Sashidhar B, Podile A R. Ethyl methanesulfonate mutagenesis-enhanced mineral phosphate solubilization by groundnut-associated serratia marcescens GPS -5. Current Microbiology, 2007, 54(2): 79-84.
[7] Pérez E, Sulbarán M, Ball M M, et al . Isolation and characterization of mineral phosphate-solubilizing bacteria naturally colonizing a limonitic crust in the south-eastern Venezuelan region. Soil Biology & Biochemistry, 2007, 39(11): 2905-2914.
[8] Reyes I, Valery A, Valduz Z. Phosphate-solubilizing microorganisms isolated from rhizospheric and bulk soils of colonizer plants at an abandoned rock phosphate mine. Plant & Soil, 2006, 287(1/2): 69-75.
[9] Gulati A, Rahi P, Vyas P. Characterization of phosphate-solubilizing fluorescent pseudomonads from the rhizosphere of seabuckthorn growing in the cold deserts of Himalayas. Current Microbiology, 2008, 56(1): 73-79.
[10] Jorquera M A, Hernández M T, Rengel Z, et al . Isolation of culturable phosphobacteria with both phytate-mineralization and phosphate-solubilization activity from the rhizosphere of plants grown in a volcanic soil. Biology & Fertility of Soils, 2008, 44(8): 1025-1034.
[11] Badar R, Qureshi S A. Comparative effect of Trichoderma hamatum and host-specific rhizobium species on growth of Vigna mungo . Journal of Applied Pharmaceutical Science, 2012, (2): 128-132.
[12] Pankaj K, Mishra, Shekhar C, et al . Bioassociative effect of cold tolerant Pseudomonas spp. and Rhizobium leguminosarum -PR1 on iron acquisition, nutrient uptake and growth of lentil ( Lens culinaris L.). European Journal of Soil Biology, 2011, (47): 35-43.
[13] Rong L Y, Yao T, Ma W B, et al . The inoculant potential of plant growth promoting rhizobacteria strains to improve the yield and quality of Trifolium pratense cv.Minshan. Acta Prataculturae Sinica, 2014, 23(5): 231-240.
荣良燕, 姚拓, 马文彬, 等. 岷山红三叶根际优良促生菌对其宿主生长和品质的影响. 草业学报, 2014, 23(5): 231-240.
[14] Ma W B, Yao T, Wang G J, et al . Assessment of rhizobacteria strains for Vicia sativa . Acta Prataculturae Sinica, 2014, 23(5): 241-248.
马文彬, 姚拓, 王国基, 等. 根际促生菌筛选及其接种剂对箭筈豌豆生长影响的研究. 草业学报, 2014, 23(5): 241-248.
[15] Guan P. The Effect of PGPR Fertilizers on the Growth and Quality of Alfalfa under the Different Soil Conditions[D]. Harbin: Harbin Normal University, 2014.
管鹏. 施用 PGPR 菌肥对不同土壤条件下的苜蓿生长及品质的影响[D]. 哈尔滨: 哈尔滨师范大学, 2014.
[16] Wu Y J, Zhang Y, Chi Y K, et al . The leaf morphological diversity of wild white clover in Guizhou karst area. Jiangsu Agriculture Science, 2016, 44(2): 291-296.
吴永洁, 张俞, 池永宽, 等. 贵州省岩溶地区野生白三叶形态多样性. 江苏农业科学, 2016, 44(2): 291-296.
[17] Bai S J, Yang Y S. Effects of white clover on sail fertility and following crop. Pratacultural Science, 1991, 8(3): 31-32.
白淑娟, 杨运生. 白三叶草地的土壤肥力及对后作的影响. 草业科学, 1991, 8(3): 31-32.
[18] Lin Q M, Zhao X R, Sun Y X, et al . Community characters of soil phosphobacteria in four ecosystems. Soil and Environmental Science, 2000, 9(1): 34-37.
林启美, 赵小蓉, 孙焱鑫, 等. 四种不同生态系统的土壤解磷细菌数量及种群分布. 土壤与环境, 2000, 9(1): 34-37.
[19] Li W H, Shi J Y. Isolation, purification, and phosphate-solubilizing capability of phosphorous bacteria in West Lake sediment. Chinese Journal of Applied Ecology, 2006, 17(11): 2112-2116.
李文红, 施积炎. 西湖沉积物中解磷菌的分离纯化及其解磷能力. 应用生态学报, 2006, 17(11): 2112-2116.
[20] Wan L, Kang L H, Liao B W, et al . The relationship between organic acid secreted from phosphorus-solubilizing bacteria. Forest Research, 2004, 17(1): 89-94.
万璐, 康丽华, 廖宝文, 等. 红树林根际解磷菌分离、培养及解磷能力的研究. 林业科学研究, 2004, 17(1): 89-94.
[21] Hafeez F Y, Malik K. Manual on Biofertilizer Technology[M]. Pakistan: National Institute for Biotechnology & Genetic Engineering (NIBGE), 2000.
[22] Xi L Q, Yao T, Yang J J, et al . Property of associative nitrogen-fixing bacteria producing IAA and its promoting growth of oat. Grassland and Turf, 2005, (4): 25-29.
席琳乔, 姚拓, 杨俊基, 等. 联合固氮菌株分泌能力及其对燕麦的促生效应测定. 草原与草坪, 2005, (4): 25-29.
[23] Lin Q M, Wang H, Zhao X R, et al . The phosphorus ability and mechanism analysis in some bacterials and funguses. Microbiology, 2001, 28(2): 26-30.
林启美, 王华, 赵小蓉, 等. 一些细菌和真菌的解磷能力及其机理初探. 微生物学通报, 2001, 28(2): 26-30.
[24] Cheng C, Yang M, Li J X, et al . Biolog microbial ideritificatron system study on the operating regulation of Bacteria identifi cation. Food and Fermentation Industry, 2006, 32(5): 50-54.
程池, 杨梅, 李金霞, 等. Biolog微生物自动分析系统细菌鉴定操作规程的研究. 食品与发酵工业, 2006, 32(5): 50-54.
[25] Wang G H, Zhao Y, Zhou D R, et al . Review of phosphate-solubilizing microorganisms. Ecology and Environment, 2003, 12(1): 96-101.
王光华, 赵英, 周德瑞, 等. 解磷菌的研究现状与展望. 生态环境, 2003, 12(1): 96-101.
[26] Rodrí Guez H, Fraga R. Phosphate solubilizing bacteria and their role in plant growth promotion. Biotechnology Advances, 1999, 17(4/5): 319-339.
[27] Ogut M, Er F, Kandemir N. Phosphate solubilization potentials of soil Acinetobacter strains. Biology & Fertility of Soils, 2010, 46(7): 707-715.
[28] Walpola B C, Yoon M H. Isolation and characterization of phosphate solubilizing bacteria and their co-inoculation efficiency on tomato plant growth and phosphorous uptake. African Journal of Microbiology Research, 2013, 7(3): 7266-7275.
[29] Kumar A, Bhargava P, Rai L C, et al . Isolation and molecular characterization of phosphate solubilizing Enterobacter and Exiguobacterium species from paddy fields of Eastern Uttar Pradesh, India. African Journal of Microbiology Research, 2010, 4(4): 820-829.
[30] Li X J, Wu F G, Wang B X, et al . The identification and application of a phosphorus-solubilizing bacteria in tabacco. Henan Agriculture Science, 2011, 40(6): 66-70.
李晓举, 吴风光, 王豹祥, 等. 一株烤烟根际解磷细菌的鉴定及其在烤烟生产中的应用. 河南农业科学, 2011, 40(6): 66-70.
[31] Lin K M, Guan X F, Ma L N, et al . Organophosphorus pesticide degrading bacteria-the biological characteristics of Enterobacter claocae . Chinese Agricultural Science Bulletin, 2008, 24(9): 382-386.
林抗美, 官雪芳, 马丽娜, 等. 有机磷农药降解菌——阴沟肠杆菌的生物学特性. 中国农学通报, 2008, 24(9): 382-386.
[32] Liu F P, Liu H Q, Zhou H L, et al . Isolation and characterization of phosphate-solubilizing bacteria from betel nut ( Areca catechu ) and their effects on plant growth and phosphorus mobilization in tropical soils. Biology & Fertility of Soils, 2014, 50(6): 927-937.
[33] Banik S, Dey B K. Phytohormone producing of phosphate solubilizing bacteria. Indian Agriculturist, 1978, 22: 93-97.
[34] Vyas P, Gulati A. Organic acid production in vitro and plant growth promotion in maize under controlled environment by phosphate-solubilizing fluorescent Pseudomonas . BMC Microbiology, 2009, 9(1): 174.
[35] Luo S Q, Ling Y, Huang J G. In vitro effects of artemisinin on inorganic phosphate-solubilizing bacteria. African Journal of Microbiology Research, 2013, 7(6): 525-532.
[36] Qiu Y S, Zhou S P, Mo X Z, et al . Study of nitrogen fixation bacteria associated with rice root II. The characteristics of nitrogen fixation by Alcaligenes faecalis strain A-15 and Enterobacter claocae strain E-26. Acta Microbiologica Sinica, 1981, 21(4): 473-476.
丘元盛, 周淑萍, 莫小真, 等. 稻根联合固氮细菌的研究II. 粪产碱菌A-15和阴沟肠杆菌E-26的固氮特性. 微生物学报, 1981, 21(4): 473-476.
[37] Ova E A, Kutman U B, Ozturk L, et al . High phosphorus supply reduced zinc concentration of wheat in native soil but not in autoclaved soil or nutrient solution. Plant and Soil, 2015, 393(1/2): 147-162.
[38] Pace P, Cralle H T, El-Halawany S H, et al . Drought-induced changes in shoot and root growth of young cotton plants. Journal of Cotton Science, 1999, 4(3): 183-187.