荧光假单胞菌的溶磷机制及其在杨树菌根际的定殖动态

doi:10.11707/j.1001-7488.20210309

摘要/Abstract

摘要：

目的: 探究荧光假单胞菌菌株JW-JS1的溶磷能力及其在杨树根际和菌根际的定殖动态，揭示菌株的溶磷机制并进一步阐明溶磷细菌与外生菌根真菌的互作机制，为杨树专用复合菌剂的开发与应用提供理论依据。方法: 通过液体培养试验分析荧光假单胞菌菌株JW-JS1培养液可溶性磷含量、pH和可滴定酸含量的变化，采用高效液相色谱（HPLC）法测定有机酸种类和含量，利用抗利福平标记法筛选稳定的标记菌株，运用灌根法研究其在杨树根际和菌根际的定殖动态。结果: 1）JW-JS1菌株培养液可溶性磷含量随接种时间延长逐渐增加，培养液pH与可溶性磷含量呈极显著负相关（r=-0.889^**），可滴定酸含量与可溶性磷含量呈极显著正相关（r=0.958^**）；2）JW-JS1菌株分泌的总有机酸量（434.39 mg·L^-1）明显高于CK（25.94 mg·L^-1）（P < 0.05），共检测出草酸、酒石酸、柠檬酸、顺丁烯二酸和反丁烯二酸5种有机酸，其中，草酸含量（273.69 mg·L^-1）明显高于其他种类有机酸，约占总有机酸量的63.01%；3）筛选出含有300 μg·mL^-1利福平抗性的标记菌株JW-JS1^Rif，与原始菌株相比，其菌落形态和溶磷能力均未发生明显变化（P < 0.05）；4）标记菌株在杨树根际和菌根际均能长期稳定存活并保持一定的定殖数量，随接种时间延长定殖数量呈下降趋势，定殖动态基本一致，其中，接种50天后定殖数量分别为5.2×10⁴和4.5×10⁴ cfu·g^-1。结论: 荧光假单胞菌菌株JW-JS1的溶磷能力与培养液pH、可滴定酸含量和有机酸密切相关，特别是菌株分泌的草酸可能在溶磷过程中发挥重要作用。荧光假单胞菌菌株JW-JS1在杨树根际和菌根际均能长期稳定存活并保持一定的定殖数量。荧光假单胞菌菌株JW-JS1与红绒盖牛肝菌Xc是构建杨树专用功能复合微生物肥料的理想材料。

关键词: 荧光假单胞菌, 有机酸, 草酸, 杨树, 菌根际, 定殖

Abstract:

Objective: This research aimed to explore the changes of phosphate-dissolving ability of Pseudomonas fluorescens at its strain JW-JS1 and the colonization dynamics in rhizosphere and mycorrhizosphere of poplars, to reveal the phosphate-dissolving mechanisms of this strain and further elucidate its interaction mechanisms with ectomycorrhizal fungi, so as to provide a theoretical basis for developing and exploiting the compound microbial fertilizer for poplar. Method: A liquid fermentation experiment was conducted to investigate the changes of soluble phosphorus contents, pH, and titratable acid contents in the broth of P. fluorescens JW-JS1. Kinds and concentrations of organic acids in the broth were also determined by HPLC. The stable marker of antibiotic rifampin was screened by using the rifampin-resistant mutant method, and the colonizing dynamics of mutant strain in rhizosphere and mycorrhizosphere of poplars were investigated by root irrigating method. Result: 1) The results showed that the soluble phosphorus contents in broth of JW-JS1 gradually increased with the time. Correlation analysis showed that the pH was negatively correlated with soluble phosphorus contents (r=-0.889^**), while the titratable acid contents was positively correlated with soluble phosphorus contents (r=0.958^**). 2) The total amounts of organic acid secreted by JW-JS1 (434.39 mg·L^-1) was significantly higher than that of control (25.94 mg·L^-1) (P < 0.05). Five kinds of organic acids were detected in the broth, including oxalic acid, tartaric acid, citric acid, maleic acid and fumaric acid. Among them, the amounts of oxalic acid (273.69 mg·L^-1) was significantly higher than that of other kinds of organic acids, which was about 63.01% of the total organic acid concent. 3) A strain of with 30 μg·mL^-1rifampicin resistance was selected. Compared with wild strain, there was no obvious change in colony morphology and phosphate-dissolving ability of JW-JS1^Rif strain (P < 0.05). 4) The strain of JW-JS1^Rif could effectively survive and maintain a certain colonizing population in the rhizosphere and mycorrhizosphere of poplar, and the colonizing population was a downward trend as the time of inoculation was prolonged, and the colonization dynamics were basically consistent. After 50 days of inoculation, the colonizing population was 5.2×10⁴ cfu·g^-1 and 4.5×10⁴ cfu·g^-1, respectively. Conclusion: The phosphate-dissolving ability of P. fluorescens JW-JS1 is closely related to the pH, titratable acid contents and organic acid in the culture medium, especially oxalic acid secreted by the strains may play an important role in the phosphate-dissolving process. P. fluorescens JW-JS1 can stably survive in the rhizosphere and mycorrhizosphere of poplar. The above results indicate that P. fluorescens JW-JS1 and Xerocomus chrysenteron are ideal materials for constructing functional compound microbial fertilizer for poplar.

Key words: Pseudomonas fluorescens, organic acid, oxalic acid, poplar, mycorrhizosphere, colonization

中图分类号:

S718.7

刘辉,吴小芹,叶建仁,陈丹. 荧光假单胞菌的溶磷机制及其在杨树菌根际的定殖动态[J]. 林业科学, 2021, 57(3): 90-97.

Hui Liu,Xiaoqin Wu,Jianren Ye,Dan Chen. Phosphate-Dissolving Mechanisms of Pseudomonas fluorescens and Its Colonizing Dynamics in the Mycorrhizosphere of Poplars[J]. Scientia Silvae Sinicae, 2021, 57(3): 90-97.

图/表 5

图1

图2

表1

表2

图3

参考文献 0

	陈哲, 吴敏娜, 秦红灵, 等. 土壤微生物溶磷分子机理研究进展. 土壤学报, 2009, 46 (5): 925- 931. doi: 10.3321/j.issn:0564-3929.2009.05.022
	Chen Z , Wu M N , Qin H L , et al. Advances in research on molecular mechanisms of phosphate-solubilizing microorganisms in soil. Acta Pedologica Sinica, 2009, 46 (5): 925- 931. doi: 10.3321/j.issn:0564-3929.2009.05.022
	冯哲叶, 陈莎莎, 王文超, 等. 几株溶磷细菌的筛选和鉴定及其溶磷效果. 南京农业大学学报, 2017, 40 (5): 842- 849.
	Feng Z Y , Chen S S , Wang W C , et al. Screening and identification of several phosphate-solubilizing bacteria and effect of their P-solubility. Journal of Nanjing Agricultural University, 2017, 40 (5): 842- 849.
	焦子伟, 张相锋, 努尔买买提, 等. pqq基因簇在Escherichia coli DH5α中表达及对其溶磷促生的影响. 农业资源与环境学报, 2016, 33 (1): 43- 48.
	Jiao Z W , Zhang X F , Nuer M , et al. Expression pqq gene cluster and its effects on mineral phosphate solubilization and plant promotion in Escherichia coli DH5α. Journal of Agriculutral Resources and Environment, 2016, 33 (1): 43- 48.
	居正英. 2008. 茄子内生枯草芽孢杆菌(Bacillus subtilis)29-12防病促生生理生化研究. 福州: 福建农林大学硕士学位论文.
	Ju Z Y. 2008. The research on biochemistry mechanism of biological control diseases and promotion growth of plants by endophytic bacteria(Baeillus subtilis)29-12. Fuzhou: MS thesis of Fujian Agriculture and Forestry University. [in Chinese]
	李阜棣. 土壤微生物学. 北京: 中国农业出版社, 1996.
	Li F D . Soil microbiology. Beijing: China Agriculture Press, 1996.
	刘辉, 吴小芹, 陈丹. 4种外生菌根真菌对难溶性磷酸盐的溶解能力. 西北植物学报, 2010, 30 (1): 143- 149.
	Liu H , Wu X Q , Chen D . Ability of dissolving insoluble phosphate by four ectomycorrhizal fungi. Acta Botanica Boreali-Occidentalia Sinica, 2010, 30 (1): 143- 149.
	刘辉, 吴小芹, 任嘉红, 等. 荧光假单胞菌与红绒盖牛肝菌共接种对杨树氮代谢和矿质元素含量的影响. 林业科学, 2018, 54 (10): 56- 63. doi: 10.11707/j.1001-7488.20181007
	Liu H , Wu X Q , Ren J H , et al. Effect of co-inoculation Pseudomonas fluorescens and Xerocomus chrysenteron on the nitrogen metabolism and mineral element contents of poplar. Scientia Silvae Sinicae, 2018, 54 (10): 56- 63. doi: 10.11707/j.1001-7488.20181007
	刘辉, 吴小芹, 任嘉红, 等. 荧光假单胞菌与红绒盖牛肝菌共接种对杨树根际土壤酶活性及微生物多样性的影响. 林业科学, 2019, 55 (1): 22- 30.
	Liu H , Wu X Q , Ren J H , et al. Effect of co-inoculation with Pseudomonas fluorescens and Xerocomus chrysenteron on the soil enzyme activity and microbial diversity in Poplar rhizosphere. Scientia Silvae Sinicae, 2019, 55 (1): 22- 30.
	刘辉, 吴小芹, 任嘉红, 等. 一株荧光假单胞菌的溶磷特性及其对杨树的促生效果. 林业科学, 2013, 49 (9): 112- 118.
	Liu H , Wu X Q , Ren J H , et al. Phosphate-dissolving characteristics and growth promoting effect of Pseudomonas fluorescens JW-JS1 on poplar seedlings. Scientia Silvae Sinicae, 2013, 49 (9): 112- 118.
	孙真, 郑亮, 邱浩斌. 植物根际促生细菌定殖研究进展. 生物技术通报, 2017, 33 (2): 8- 15.
	Sun Z , Zheng L , Qiu H B . Research advances on colonization of plant growth-promoting rhizobacteria. Biotechnology Bulletin, 2017, 33 (2): 8- 15.
	王恒煦, 刘泽平, 王志刚, 等. 3株芽孢杆菌在水稻根际定殖促生及其在土壤中的存活. 生态与农村环境学报, 2019, 35 (7): 892- 899.
	Wang H X , Liu Z P , Wang Z G , et al. Colonization and growth promotion of three Bacillus strains in rice rhizosphere and their survival in soil. Journal of Ecology and Rural Environment, 2019, 35 (7): 892- 899.
	王树起, 韩晓增, 严君, 等. 低分子量有机酸对大豆磷积累和土壤无机磷形态转化的影响. 生态学杂志, 2009, 28 (8): 1550- 1554.
	Wang S Q , Han X Z , Yan J , et al. Effects of low molecular weight organic acids on P accumulation in soybean (Glycine max L.) and inorganic P form transformation in soil. Chinese Journal of Ecology, 2009, 28 (8): 1550- 1554.
	王长方, 游泳, 王俊, 等. 利福平标记的青枯菌株在姜块中增殖动态研究初报. 福建农业学报, 2006, 21 (2): 109- 112.
	Wang C F , You Y , Wang J , et al. Proliferation of Ralstonia solanacearum marked with rifampicin on the tuber of ginger. Fujian Journal of Agricultural Sciences, 2006, 21 (2): 109- 112.
	杨顺, 杨婷, 林斌, 等. 两株溶磷真菌的筛选、鉴定及溶磷效果的评价. 微生物学报, 2018, 58 (2): 264- 273.
	Yang S , Yang T , Lin B , et al. Isolation and evaluation of two phosphate-dissolving fungi. Acta Microbiologica Sinica, 2018, 58 (2): 264- 273.
	杨美英, 王春红, 武志海, 等. 不同条件下两株溶磷菌溶磷量及葡萄糖脱氢酶基因表达与酶活分析. 微生物学报, 2016, 56 (4): 651- 663.
	Yang M Y , Wang C H , Wu Z H , et al. Phosphorus dissolving capability, glucose dehydrogenase gene expression and activity of two phosphate solubilizing bacteria. Acta Microbiologica Sinica, 2016, 56 (4): 651- 663.
	姚如斌, 吴小芹. 高效解磷细菌与菌根真菌菌剂交互作用对杨树的促生效应. 南京林业大学学报: 自然科学版, 2012, 36 (5): 170- 173.
	Yao R B , Wu X Q . Interaction between high effective phosphate-solubilizing bacteria and mycorrhizal fungi and its effects on poplar growth. Journal of Nanjing Forestry University: Natural Science Edition, 2012, 36 (5): 170- 173.
	曾丽琼. 2010. 几种优良外生菌根菌的应用和胶丸菌剂的制备. 南京: 南京林业大学硕士学位论文.
	Zeng L Q. 2010. Field application of ectomycorrhizal fungi and study on the process of capsules microbial agent. Nanjing: MS thesis of Nanjing Forestry University. [in Chinese]
	张英, 芦光新, 谢永丽, 等. 溶磷菌分泌有机酸与溶磷能力相关性研究. 草业学报, 2015, 23 (5): 1033- 1038.
	Zhang Y , Lu G X , Xie Y L , et al. The relationship between organic acid secreted from phosphorus-solubilizing bacteria and the phosphate-solubilizing ability. Acta Agrestia Sinica, 2015, 23 (5): 1033- 1038.
	赵小蓉, 林启美, 李保国. 溶磷菌对4种难溶性磷酸盐溶解能力的初步研究. 微生物学报, 2002, 42 (2): 236- 241.
	Zhao X R , Lin Q M , Li B G . The solubilization of four insoluble phosphates by some microorganisms. Acta Microbiologica Sinica, 2002, 42 (2): 236- 241.
	朱培淼, 杨兴明, 徐阳春, 等. 高效解磷细菌的筛选及其对玉米苗期生长的促进作用. 应用生态学报, 2007, 18 (1): 107- 112.
	Zhu P M , Yang X M , Xu Y C , et al. High effective phosphate-solubilizing bacteria: Their isolation and promoting effect on corn seedling growth. Chinese Journal of Applied Ecology, 2007, 18 (1): 107- 112.
	Antoun H . Beneficial microorganisms for the sustainable use of phosphates in agriculture. Procedia Engineering, 2012, 46, 62- 67.
	Babu-Khan S , Yeo T C , Martin W L , et al. Cloning of a mineral phosphate-solubilizing gene from Pseudomonas cepacia. Applied and Environmental Microbiology, 1995, 61 (3): 972- 978.
	Chen Q , Liu S . Identification and characterization of the phosphate-solubilizing bacterium Pantoea sp. S32 in reclamation soil in Shanxi, China. Frontiers in Microbiology, 2019, 10, 2171.
	Chen Y P , Rekha P D , Arun A B , et al. Phosphate solubilizing bacteria from subtropical soil and their tricalcium phosphate solubilizing abilities. Applied Soil Ecology, 2006, 34 (1): 33- 41.
	Collavino M M , Sansberro P A , Mroginski L A , et al. Comparison of in vitro solubilization activity of diverse phosphate-solubilizing bacteria native to acid soil and their ability to promote Phaseolus vulgaris growth. Biology and Fertility of Soils, 2010, 46 (7): 727- 738.
	Gupta M , Bisht S , Singh B , et al. Enhanced biomass and steviol glycosides in Stevia rebaudiana treated with phosphate-solubilizing bacteria and rock phosphate. Plant Growth Regulation, 2011, 65 (3): 449- 457.
	Johansson J F , Paul L R , Finlay R D . Microbial interactions in the mycorrhizosphere and their significance for sustainable agriculture. FEMS Microbiology Ecology, 2004, 48 (1): 1- 13.
	Khan M S , Zaidi A , Wani P A . Role of phosphate-solubilizing microorganisms in sustainable agriculture-a review. Agronomy for Sustainable Development, 2007, 27 (1): 29- 43.
	Kucey R M N . Effect of Penicillium bilaji on the solubility and uptake of P and micronutrients from soil by wheat. Canadian Journal of Soil Science, 1988, 68 (2): 261- 270.
	Kucey R M N , Janzen H H , Leggett M E . Microbially mediated increases in plant-available phosphorus. Advances in Agronomy, 1989, 42, 199- 228.
	Li Z , Bai T S , Dai L T , et al. A study of organic acid production in contrasts between two phosphate solubilizing fungi: Penicillium oxalicum and Aspergillus niger. Scientific Report, 2016, 6, 25313.
	Liu H , Wu X Q , Ren J H , et al. Isolation and identification of phosphobacteria in poplar rhizosphere from different regions of China. Pedosphere, 2011, 21 (1): 90- 97.
	Mamta , Rahi P , Pathania V , et al. Stimulatory effect of phosphate-solubilizing bacteria on plant growth, stevioside and rebaudioside-a contents of Stevia rebaudiana Bertoni. Applied Soil Ecology, 2010, 46 (2): 222- 229.
	Oliveria C A , Alves V M C , Marriel I E , et al. Phosphate solubilizing microorganisms isolated from rhizosphere of maize cultivated in an oxisol of the Brazilian Cerrado Biome. Soil Biology and Biochemistry, 2009, 41 (9): 1782- 1787.
	Park K H , Lee C Y , Son H J . Mechanism of insoluble phosphate solubilization by Pseudomonas fluorescens RAF15 isolated from ginseng rhizosphere and its plant growth-promoting activities. Letters in Applied Microbiology, 2009, 49 (2): 222- 228.
	Seshadri S , Ignacimuthu S , Lakshminarasimhan C . Effect of nitrogen and carbon sources on the inorganic phosphate solubilizing by different Aspergillus niger strains. Chemical Engineering Communications, 2004, 191 (8): 1043- 1052.
	Song O R , Lee S J , Lee Y S , et al. Solubilization of insoluble inorganic phosphate by Burkholderia cepacia DA23 isolated from cultivated soil. Brazilian Journal of Microbiology, 2008, 39 (1): 151- 156.
	Suleman M , Yasmin S , Rasul M , et al. Phosphate solubilizing bacteria with glucose dehydrogenase gene for phosphorus uptake and beneficial effects on wheat. PLoS ONE, 2018, 13 (9): e0204408.
	Theoduloz C , Vega A , Salazar M , et al. Expression of a Bacillus thuringiensis δ-endotoxin cry1Ab gene in Bacillus subtilis and Bacillus licheniformis strains that naturally colonize the phylloplane of tomato plants(Lycopersicon esculentum, Mills). Journal of Applied Microbiology, 2003, 94 (3): 375- 381.
	Tyl C, Sadler G D. 2017. pH and titratable acidity//Nielsen S. (eds) Food Analysis. Food Science Text Series. Springer, Cham: 389-406.
	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, 174.
	Wang Z , Xu G Y , Ma P D , et al. Isolation and characterization of a phosphorus-solubilizing bacterium from rhizosphere soils and its colonization of Chinese Cabbage(Brassica campestris ssp. chinensis). Frontiers in Microbiology, 2017, 8, 1270.

处理 Treatments	有机酸含量 Contents of organic acids/(mg·L^-1)									可溶性磷含量 Contents of soluble P/(mg·L^-1)
处理 Treatments	乙酸 Ethanoic acid	草酸 Oxalic acid	酒石酸 Tartaric acid	柠檬酸 Citric acid	顺丁烯二酸 Maleic acid	反丁烯二酸 Fumaric acid	丁二酸 Succinic acid	乳酸 Lactic acid	总有机酸 Total organic acid	可溶性磷含量 Contents of soluble P/(mg·L^-1)
JW-JS1	—	273.69^*	136.80^*	13.58^*	3.82	6.51	—	—	434.39^*	637.77±24.92^*
CK	—	12.50	2.27	3.80	2.74	4.63	—	—	25.94	49.31±4.21

处理 Treatments	可溶性磷含量 Contents of soluble P/(mg·L^-1)
JW-JS1	584.17±9.45^*
JW-JS1^Rif	579.26±11.03^*
CK	34.96±4.92

[1]	孙伟博,宫新栋,周燕,李红岩. 转玉米PEPC和PPDK基因杨树苗期的光合生理特性[J]. 林业科学, 2020, 56(7): 33-43.
[2]	何经纬,张伊莹,田呈明,熊典广,梁英梅. 区域景观格局对杨树锈病为害流行的影响——以北京延庆地区银白杨为例[J]. 林业科学, 2020, 56(4): 99-108.
[3]	刘文鑫,陈志成,代永欣,万贤崇. 水通道蛋白PIP1基因过表达杨树的光合生理过程对干旱和复水的响应[J]. 林业科学, 2020, 56(2): 69-78.
[4]	沈阔程,陈倩文,齐梅,彭子嘉,樊军锋,余仲东. 杨树叶片结构与抗锈菌侵染的相关性[J]. 林业科学, 2020, 56(12): 75-82.
[5]	孙伟博,魏朝琼,马晓星,魏辉,诸葛强. 3类转基因南林895杨田间试验的安全性评估[J]. 林业科学, 2020, 56(10): 53-62.
[6]	张超, 王进茂, 赵洁, 庞丁玮, 张德健, 杨敏生. 转多基因欧美杨Bt基因表达特征[J]. 林业科学, 2019, 55(9): 61-70.
[7]	辛福梅, 闫小莉, 张长耀, 贾黎明. 西藏拉萨河谷区藏川杨和北京杨树干液流特征及其对环境因子的响应[J]. 林业科学, 2019, 55(2): 22-32.
[8]	刘辉, 吴小芹, 任嘉红, 陈丹. 荧光假单胞菌与红绒盖牛肝菌共接种对杨树根际土壤酶活性及微生物多样性的影响[J]. 林业科学, 2019, 55(1): 22-30.
[9]	张扬, 杜琳, 唐贤丰, 刘唤唤, 周功克, 柴国华. 杨树BAG基因的鉴定及表达模式分析[J]. 林业科学, 2019, 55(1): 138-145.
[10]	李建庆, 梅增霞, 杨忠岐. 不同林分杨树云斑天牛种群空间格局地统计学分析[J]. 林业科学, 2018, 54(3): 83-90.
[11]	刘辉, 吴小芹, 任嘉红, 陈丹. 荧光假单胞菌与红绒盖牛肝菌共接种对杨树氮代谢和矿质元素含量的影响[J]. 林业科学, 2018, 54(10): 56-63.
[12]	金勤, 朱丹雪, 周国英, 李河, 何苑皞, 张茜. 绿色荧光蛋白标记枯草芽孢杆菌Y13^UV在油茶体内的定殖[J]. 林业科学, 2017, 53(7): 111-117.
[13]	黄绢, 陈存, 张伟溪, 丁昌俊, 苏晓华, 黄秦军. 干旱胁迫对转JERF36银中杨苗木叶片解剖结构及光合特性的影响[J]. 林业科学, 2017, 53(5): 8-15.
[14]	尹吴, 孙伟博, 周燕, 诸葛强. 毛果杨Rubisco活化酶基因的克隆与功能分析[J]. 林业科学, 2017, 53(4): 83-95.
[15]	章小铃, 王留强, 孙佩, 吴立栓, 樊玮, 张进, 卢孟柱, 胡建军. 杨树不同种质花粉萌发和致敏蛋白的差异[J]. 林业科学, 2017, 53(2): 54-64.