Analysis of Increasing Glyphosate Resistance and Growth-promoting Effects in Soybean by Desmodesmus subspicatus

doi:10.13560/j.cnki.biotech.bull.1985.2024-0270

Abstract

Abstract:

【Objective】 This study is to characterize the tolerance of Desmodesmus subspicatus, a microalga, to glyphosate as well as effects of adsorbing or degrading glyphosate. Also it is to evaluate the effects of D. subspicatus application on improving glyphosate resistance and promoting growth of soybean（Glycine max）seedlings, and removing glyphosate residues from rhizosphere. These findings may offer new insights into elucidation of the absorption or degradation mechanism of glyphosate and other organophosphorus pesticides by microalgae. 【Method】 Different dosages of glyphosate were added to the liquid medium, respectively, in which D. subspicatus was cultured for 5 d. Then microalgal growth and photosynthesis, together with glyphosate residue in the culture solution were examined. Glyphosate was added to the soybean seedling hydroponic solution for 7 d of cultivation, and then the growth and photosynthesis of soybean seedlings were measured. The algal cultures of D. subspicatus were added to the soybean seedling hydroponic solution for 14 d of cultivation under glyphosate stress. After that, physiological parameters were examined for the soybean seedlings, followed by analyzing levels of glyphosate residues in both the seedlings and hydroponic solution. 【Result】 The D. subspicatus had a certain degree of tolerance to glyphosate and maintained effective growth under 100 mg/L glyphosate stress. The removal rate of glyphosates added to the culture solution was high up to 72.26% after 5-day cultivation of the microalga. Glyphosate stress significantly inhibited soybean seedling growth. D. subspicatus application enhanced the plant height, root length, fresh weight and dry weight of soybean seedlings cultivated in the glyphosate-stressed solution by 15.98%, 49.03%, 49.20% and 25.63%, respectively, compared with the glyphosate-stressed controls. Similarly, values of chlorophyll fluorescence parameters Y（II）, ETR and Fv/Fm increased while NPQ value decreased. Importantly, glyphosate residues in the soybean seedlings and the culture solution significantly reduced by 26.3% and 34.6%, respectively. 【Conclusion】 D. subspicatus cells could absorb or degrade glyphosate in large quantities. The application of D. subspicatus may effectively alleviate the damage of glyphosate stress on the photosynthesis of soybean seedlings, and also significantly reduce the absorption and enrichment of glyphosate in soybean seedlings, thus promoting soybean seedling growth and blocking the migration of glyphosate residues.

Key words: Desmodesmus subspicatus, soybean seedling, glyphosate residue, resistance to stress, growth-promoting effect

LI Jiong-shan, YANG Ze, YAN Xing, LIU Yi-zhen, GUO Yu-shuang, XUE Jin-ai, SUN Xi-ping, JI Chun-li, ZHANG Chun-hui, LI Run-zhi. Analysis of Increasing Glyphosate Resistance and Growth-promoting Effects in Soybean by Desmodesmus subspicatus[J]. Biotechnology Bulletin, 2024, 40(11): 236-247.

Figures/Tables 11

Table 1 Glyphosate treatments on D. subspicatus and soybean seedlings

处理 Treatment	培养液及微藻或大豆 Medium and microalga or soybean	草甘膦浓度Glyphosate concentration/（mg·L^-1）
DS0	BG11+四尾栅藻（D. subspica-tus）	0
DS50		50
DS100		100
DS200		200
BG100	BG11	100
GM0	Hoagland+大豆（G. max）	0
GM5		5
GM10		10
GM20		20
GM50		50
GM100		100
GM-DS0	Hoagland+大豆（G. max）+四尾栅藻（D. subspicatus）	0
GM-DS10		10
GM-DS20		20
GM-DS50		50
HG50	Hoagland	50

Fig. 1 Growth（A）and OD680 values（B）of D. subspi-catus on day 0, 2 and 5 under glyphosate stress at different dosages DS: D. subspicatus. Numbers: Glyphosate dose（mg/L）. The same below

Fig. 2 Growth of soybean seedlings after day 7 of cultivation under glyphosate stresses A: Hydroponic culture growth phenotypes of soybean seedlings in different treatment groups; B: aboveground and belowground growth of soybean seedlings in different treatments. GM: Soybean. Numbers: Glyphosate dose（mg/L）. The same below

Fig. 3 Plant height, root length, fresh weight and dry weight of soybean seedlings under different dosages of glyphosate stresses Different letters indicate significant differences among treatment at 0.05 level（P<0.05）. The same below

Fig. 4 Effects of D. subspicatus on the plant heights, root lengths, fresh weights and dry weights of soybean seedlings under glyphosate stresses GM-DS: Soybean+ D. subspicatus

Fig. 5 Accumulative pigment contents in D. subspicatus and soybean seedlings at the last day of cultivation under different glyphosate stresses A: Pigment levels in D. subspicatus after 5 d of cultivation under different glyphosate stresses. B: Pigment contents in soybean seedlings after 7 d of cultivation under different dosages of glyphosate stresses

Fig. 6 Effects of D. subspicatus on the photosynthetic pigment contents in soybean seedlings under different dosages of glyphosate stresses

Fig. 7 Impacts of D. subspicatus on the chlorophyll fluorescence parameters of soybean seedlings under different dosages of glyphosate stresses

Table 2 Glyphosate content in the culture solution collected from the microalgal cultures at day 5 of culture of D. subspicatus under glyphosate treatment

处理 Treatment	草甘膦施加量 Glyphosate dosage/（mg·L^-1）	藻液草甘膦残留量 Glyphosate residual content in the culture solution/（mg·L^-1）	去除率 Removal rate/%
DS0	0	0^d	0^d
DS50	50	16.339±0.464^c	64.49^b
DS100	100	24.915±2.190^b	72.26^a
BG100	100	97.168±1.306^a	2.83^c

Table 3 Glyphosate residual content in the glyphosate- and microalgal cell-added soybean seedlings culture solutions at day14 of cultivation

处理 Treatment	草甘膦施加量 Glyphosate dosage/（mg·L^-1）	水培液草甘膦残留量 Glyphosate residual content in the culture solution/（mg·L^-1）	去除率 Removal rate/%
GM0	0	0^h	0^f
GM10	10	2.132±0.224^f	76.50^c
GM20	20	4.481±0.473^d	75.42^d
GM50	50	11.182±0.701^b	75.46^d
GM-DS0	0	0^h	0^f
GM-DS10	10	1.395±0.135^g	83.87^a
GM-DS20	20	3.059±0.146^e	82.53^a
GM-DS50	50	9.524±0.531^c	78.77^b
HG50	50	48.912±0.223^a	2.18^e

Table 4 Glyphosate contents in the leaves of soybean seedlings cultivated in the glyphosate and microalgal cell-added culture solutions at day 14 of cultivation

处理 Treatment	草甘膦施加量Glyphosate dosage/（mg·L^-1）	叶片草甘膦残留量Glyphosate residual content/（μg·g^-1）
GM0	0	0
GM10	10	0.379±0.061^d
GM20	20	1.045±0.307^c
GM50	50	5.478±0.589^a
GM-DS0	0	0
GM-DS10	10	0.287±0.061^d
GM-DS20	20	0.912±0.123^c
GM-DS50	50	4.028±0.205^b

References 53

[1]	Modesto KA, Martinez CBR. Roundup causes oxidative stress in liver and inhibits acetylcholinesterase in muscle and brain of the fish Prochilodus lineatus[J]. Chemosphere, 2010, 78(3): 294-299.
[2]	Śliwińska-Wilczewska S, Sylwestrzak Z, Maculewicz J, et al. The effects of allelochemicals and selected anthropogenic substances on the diatom Bacillaria paxillifera[J]. Edukacja Biologiczna I Środowiskowa, 2016, 1: 21-27.
[3]	Woźniak B, Dera J, Ficek D, et al. Dependence of the photosynthesis quantum yield in oceans on environmental factors[J]. Oceanologia, 2002, 44(4): 439-459
[4]	Pieniazek D, Bukowska B, Duda W. Glyphosate--a non-toxic pesticide?[J]. Med Pr, 2003, 54(6): 579-583.
[5]	陈楸健. 微生物与生物炭协同修复草甘膦的效果与修复机理研究[D]. 苏州: 苏州科技大学, 2019.
	Chen QJ. Study on the effects and mechanism of glyphosate remediation by microorganism and biochar[D]. Suzhou: Suzhou University of Science and Technology, 2019.
[6]	林永东, 孙彦龙, 郑彤, 等. MnO₂/Al₂O₃吸附草甘膦及微波紫外耦合降解再生工艺[J]. 环境工程学报, 2015, 9(4): 1815-1822.
	Lin YD, Sun YL, Zheng T, et al. MnO₂ /Al₂O₃ adsorption of glyphosate and microwave UV coupling degradation and regeneration process[J]. Chin J Environ Eng, 2015, 9(4): 1815-1822
[7]	周长印. 改性聚苯乙烯树脂制备及吸附/氧化去除水中草甘膦的研究[D]. 青岛: 青岛科技大学, 2018.
	Zhou CY. Removal of glyphosate from aqueous solution by modified polystyrene resin[D]. Qingdao: Qingdao University of Science & Technology, 2018.
[8]	杨昊博, 接伟光, 林厚泽, 等. 微生物降解大豆农药残留研究现状[J]. 粮食与油脂, 2023, 36(5): 13-18.
	Yang HB, Jie WG, Lin HZ, et al. Research status of microbial degradation of pesticide residues in soybean[J]. Cereals Oils, 2023, 36(5): 13-18.
[9]	Wang X, Balamurugan S, Liu SF, et al. Hydrolysis of organophosphorus by diatom purple acid phosphatase and sequential regulation of cell metabolism[J]. J Exp Bot, 2021, 72(8): 2918-2932.
[10]	胡壮壮, 王路路, 姜雪冰, 等. 我国大豆产业发展现状分析及对策[J]. 大豆科技, 2023(4): 1-11.
	Hu ZZ, Wang LL, Jiang XB, et al. Analysis and countermeasure of soybean industry development status in China[J]. Soybean Sci Technol, 2023(4): 1-11.
[11]	曾学明. 我国大豆产业发展战略规划研究[J]. 中国农业资源与区划, 2017, 38(9): 89-97.
	Zeng XM. The strategic planning of soybean industry development in China[J]. Chin J Agric Resour Reg Plan, 2017, 38(9): 89-97.
[12]	于文波, 李孝忠. 扩种背景下大豆供需矛盾分析及对策建议[J]. 大豆科技, 2023(6): 1-8.
	Yu WB, Li XZ. The contradiction analysis between soybean supply and demand and countermeasures under the background of planting expansion[J]. Soybean Sci Technol, 2023(6): 1-8.
[13]	原向阳, 郭平毅, 张丽光, 等. 不同时期喷施草甘膦对大豆生理指标的影响[J]. 中山大学学报: 自然科学版, 2009, 48(2): 90-94.
	Yuan XY, Guo PY, Zhang LG, et al. Impact of spraying glyphosate on physiologi physioligical index of soybean at different growth stages[J]. Acta Sci Nat Univ Sunyatseni, 2009, 48(2): 90-94.
[14]	Jun SH, Yang JS, Jeon H, et al. Stabilized and immobilized carbonic anhydrase on electrospun nanofibers for enzymatic CO₂ conversion and utilization in expedited microalgal growth[J]. Environ Sci Technol, 2020, 54(2): 1223-1231.
[15]	Nayak M, Karemore A, Sen R. Performance evaluation of microalgae for concomitant wastewater bioremediation, CO₂ biofixation and lipid biosynthesis for biodiesel application[J]. Algal Res, 2016, 16: 216-223.
[16]	Chen GY, Zhao L, Qi Y. Enhancing the productivity of microalgae cultivated in wastewater toward biofuel production: a critical review[J]. Appl Energy, 2015, 137: 282-291.
[17]	Rawat I, Kumar RR, Mutanda T, et al. Biodiesel from microalgae: A critical evaluation from laboratory to large scale production[J]. Appl Energy, 2013, 103: 444-467.
[18]	Salam S, Verma TN. Appending empirical modelling to numerical solution for behaviour characterisation of microalgae biodiesel[J]. Energy Convers Manag, 2019, 180: 496-510.
[19]	Wang HY, Wu B, Jiang N, et al. The effects of influent chemical oxygen demand and strigolactone analog concentration on integral biogas upgrading and pollutants removal from piggery wastewater by different microalgae-based technologies[J]. Bioresour Technol, 2023, 370: 128483.
[20]	申婷, 邢冠润, 杜学振, 等. 不同微藻处理猪场废水的净化效果研究[J]. 畜牧与兽医, 2022, 54(2): 52-56.
	Shen T, Xing GR, Du XZ, et al. Purification effect of different microalgae on treatment of piggery wastewater[J]. Animal Husb Vet Med, 2022, 54(2): 52-56.
[21]	Guo JH, Selby K, Boxall ABA. Effects of antibiotics on the growth and physiology of chlorophytes, cyanobacteria, and a diatom[J]. Arch Environ Contam Toxicol, 2016, 71(4): 589-602.
[22]	Deng ZK, Zhu JY, Yang L, et al. Microalgae fuel cells enhanced biodegradation of imidacloprid by Chlorella sp.[J]. Biochem Eng J, 2022, 179: 108327.
[23]	张晶. 混合营养模式下利用斜生栅藻同步去除养分和重金属[D]. 太原: 太原理工大学, 2022.
	Zhang J. Synchronous removal of nutrients and heavy metals by Scenedesmus obliquus under mixotrophic mode[D]. Taiyuan: Taiyuan University of Technology, 2022.
[24]	程方贝贝, 甘婷婷, 赵南京, 等. 4种淡水微藻对水体重金属铅吸附特性研究[J]. 环境科技, 2021, 34(6): 1-6.
	Cheng F, Gan TT, Zhao NJ, et al. Adsorption characteristics of four freshwater microalgae to heavy metal lead in water[J]. Environ Sci Technol, 2021, 34(6): 1-6.
[25]	Goh PS, Lau WJ, Ismail AF, et al. Microalgae-enabled wastewater treatment: A sustainable strategy for bioremediation of pesticides[J]. Water, 2022, 15(1): 70.
[26]	Hussein MH, Abdullah AM, Badr EI Din NI, et al. Biosorption potential of the microchlorophyte Chlorella vulgaris for some pesticides[J]. J Fertil Pestic, 2017, 8(1): 1000177.
[27]	Rambaldo L, Ávila H, Escolà Casas M, et al. Assessment of a novel microalgae-cork based technology for removing antibiotics, pesticides and nitrates from groundwater[J]. Chemosphere, 2022, 301: 134777.
[28]	Ni Y, Lai JH, Wan JB, et al. Photosynthetic responses and accumulation of mesotrione in two freshwater algae[J]. Environ Sci Process Impacts, 2014, 16(10): 2288-2294.
[29]	张效铭, 刘颖颖, 崔红利, 等. 化学吸收剂单乙醇胺强化小球藻生长代谢及固碳效应的研究[J]. 激光生物学报, 2022, 31(4): 311-320.
	Zhang XM, Liu YY, Cui HL, et al. A study on the enhancement of growth, metabolism and carbon sequestration in Chlorella sp. by chemical absorbent monoethanolamine[J]. Acta Laser Biol Sin, 2022, 31(4): 311-320.
[30]	苏胜. 微藻对草甘膦的响应及提高小麦抗草甘膦残留的效应分析[D]. 太谷: 山西农业大学, 2022.
	Su S. Response of microalgae to glyphosate and effect analysis of improving resistance of wheat to glyphosate residue[D]. Taigu: Shanxi Agricultural University, 2022.
[31]	张其德. 测定叶绿素的几种方法[J]. 植物学通报, 1985, 20(5): 60-64.
	Zhang QD. Several methods for determination of chlorophyll[J]. Chin Bull Bot, 1985, 20(5): 60-64.
[32]	刘颖颖, 朱梅, 崔红利, 等. 不同培养模式及生物膜含水量对栅藻生长和油脂积累的影响[J]. 植物生理学报, 2022, 58(3): 543-553.
	Liu YY, Zhu M, Cui HL, et al. Effects of cultivation mode and biofilm moisture content on the growth and lipid accumulation of Scenedesmus dimorphus[J]. Plant Physiol J, 2022, 58(3): 543-553.
[33]	中华人民共和国生态环境部. 水质草甘膦的测定高效液相色谱法: HJ 1071-2019[S]. 北京: 中国环境出版集团, 2020.
	Ministry of Ecology and Environment of the People's Republic of China. Water quality-determination of glyphosate -high performance liquid chromatography: HJ 1071-2019[J]. Beijing: China Environment Publishing Group, 2020.
[34]	成婧, 王美玲, 龚强, 等. 液相色谱-串联质谱法检测植物源食品中草甘膦及其代谢物的残留量[J]. 食品安全质量检测学报, 2016, 7(1): 138-144.
	Cheng J, Wang ML, Gong Q, et al. Determination of glyphosate and aminomethylphosphonic acid residues in plant-derived foodstuff by high performance liquid chromatography-tandem mass spectrometry[J]. J Food Saf Qual, 2016, 7(1): 138-144.
[35]	乔成奎, 王超, 黄玉南, 等. 桃果实叶片和土壤草甘膦和氨甲基膦酸残留的HPLC-MS/MS检测方法[J]. 园艺学报, 2017, 44(3): 566-574.
	Qiao CK, Wang C, Huang YN, et al. Determination of glyphosate and aminomethylphosphonic acid residues in peach fruit, leaf and soil by high performance liquid chromatography-tandem mass spectrometry[J]. Acta Hortic Sin, 2017, 44(3): 566-574.
[36]	Nanda M, Kumar V, Fatima N, et al. Detoxification mechanism of organophosphorus pesticide via carboxylestrase pathway that triggers de novo TAG biosynthesis in oleaginous microalgae[J]. Aquat Toxicol, 2019, 209: 49-55.
[37]	Tansay S, Issakul K, Ngearnpat N, et al. Impact of environmentally relevant concentrations of glyphosate and 2, 4-D commercial formulations on Nostoc sp. N1 and Oryza sativa L. Rice Seedlings[J]. Front Sustain Food Syst, 2021, 5: 661634.
[38]	张子莲, 陈秋兰, 陈博, 等. 农药对海洋微藻中肋骨条藻的毒性效应及其生物降解[J]. 中国科学: 地球科学, 2023, 53(3): 644-655.
	Zhang ZL, Chen QL, Chen B, et al. Toxic effects of pesticides on the marine microalga Skeletonema costatum and their biological degradation[J]. Sci China Earth Sci, 2023, 53(3): 644-655.
[39]	卢玮. 山仔水库两种水华蓝藻对不同形态磷吸收及响应机制研究[D]. 福州: 福建师范大学, 2015.
	Lu W. Absorption and mechanism response of two waterbloom-forming cyanobacteria at different forms of phosphorus in the Shanzai reservior[D]. Fuzhou: Fujian Normal University, 2015.
[40]	Wang X, He GH, Wang ZY, et al. Purple acid phosphatase promoted hydrolysis of organophosphate pesticides in microalgae[J]. Environ Sci Ecotechnol, 2023, 18: 100318.
[41]	Wong SL, Nakamoto L, Wainwright JF. Identification of toxic metals in affected algal cells in assays of wastewaters[J]. J Appl Phycol, 1994, 6(4): 405-414.
[42]	Fernández C, Asselborn V, Parodi ER. Toxic effects of chlorpyrifos, cypermethrin and glyphosate on the non-target organism Selenastrum capricornutum(Chlorophyta)[J]. An Acad Bras Cienc, 2021, 93(4): e20200233.
[43]	王宇, 张锴, 王艳丽, 等. 冀东野生大豆对草甘膦的耐性鉴定及耐性机制初步研究[J]. 核农学报, 2022, 36(6): 1108-1114.
	Wang Y, Zhang K, Wang YL, et al. Identification of glyphosate-resistance and preliminary research on the resistant mechanism of wild soybean in eastern Hebei province[J]. J Nucl Agric Sci, 2022, 36(6): 1108-1114.
[44]	Schulz A, Münder T, Czytko HH, et al. Glyphosate transport and early effects on shikimate metabolism and its compartmentation in sink leaves of tomato and spinach plants[J]. Z Für Naturforschung C, 1990, 45(5): 529-534.
[45]	King C, Purcell L, Vories E. Plant growth and nitrogenase activity of glyphosate-tolerant soybean in response to foliar glyphosate applications[J]. Agron J, 2001, 93(1): 179-186.
[46]	周垂帆, 李莹, 张晓勇, 等. 草甘膦毒性研究进展[J]. 生态环境学报, 2013, 22(10): 1737-1743.
	Zhou CF, Li Y, Zhang XY, et al. Research advance in ecotoxicity of glyphosate[J]. Ecol Environ Sci, 2013, 22(10): 1737-1743.
[47]	杨国斌, 陈才志, 温欣宇, 等. 草甘膦、草铵膦喷施对槟榔根系形态及生理的影响[J]. 中国南方果树, 2024, 53(1): 74-82.
	Yang GB, Chen CZ, Wen XY, et al. Effects of glyphosate and glufosinate-ammonium application on root morphology and physiology of Areca catechu[J]. South China Fruits, 2024, 53(1): 74-82.
[48]	徐雯. 藻-藻共培养净化牛场废水联产生物肥及其对小麦的促生效应[D]. 太谷: 山西农业大学, 2021.
	Xu W. Algae-algae co-culture to purify cattle-ranch wastewater coupled with bio-fertilizer and its growth-promoting effects on wheat[D]. Taigu: Shanxi Agricultural University, 2021.
[49]	S Sido MY, Tian YP, Wang XG, et al. Application of microalgae Chlamydomonas applanata M9V and Chlorella vulgaris S3 for wheat growth promotion and as urea alternatives[J]. Front Microbiol, 2022, 13: 1035791.
[50]	Xiao R, Zheng Y. Overview of microalgal extracellular polymeric substances(EPS)and their applications[J]. Biotechnol Adv, 2016, 34(7): 1225-1244.
[51]	张华, 沈英, 俞涛. 微藻生物膜去污技术应用研究进展[J]. 可再生能源, 2022, 40(3): 299-306.
	Zhang H, Shen Y, Yu T. Applications and research progress in the wastewater treatment technology of microalgae biofilm[J]. Renew Energy Resour, 2022, 40(3): 299-306.
[52]	Sukačová K, Trtílek M, Rataj T. Phosphorus removal using a microalgal biofilm in a new biofilm photobioreactor for tertiary wastewater treatment[J]. Water Res, 2015, 71: 55-63.
[53]	Pereira A, Castro J, Ribeiro V, et al. Organomineral fertilizers pastilles from microalgae grown in wastewater: Ammonia volatilization and plant growth[J]. Sci Total Environ, 2021, 779: 146205.