Optimization of Culture Medium of Serendipita indica Based on Response Surface Methodology

doi:10.11924/j.issn.1000-6850.casb2021-0229

Abstract

Abstract:

The root endophytic fungus Serendipita indica can be cultured on artificial medium. The present study aims at optimizing the medium to enhance the growth rate of S. indica. In this experiment, the single-factor experiment was selected for evaluating the effect of six parameters on the growth rate of S. indica, and the optimization design of the medium was performed by the response surface methodology (RSM). The results showed that the optimum temperature was 31±2℃, the optimum pH was 6.9±0.2, the best carbon source was sucrose, the best nitrogen source was NaNO₃, and the best inorganic salt was MgSO₄·7H₂O. The optimal medium formulation was determined by RSM as following: MgSO₄·7H₂O 0.824 g/L, KH₂PO₄ 1.118 g/L, NaNO₃ 1.055 g/L, NaCl 0.03 g/L, CaCl₂ 0.05 g/L and sucrose 10 g/L. In the optimal medium, the average growth rate of S. indica reached 1.8083 cm/d, which was greatly faster than that in the original MMN medium. In this study, medium optimization based on RSM improved the growth rate of S. indica, which laid a foundation for the large-scale production of S. indica and its application in field trials.

Key words: Serendipita indica, medium, response surface method, single factor optimization, growth rate

CLC Number:

S5-33

LI Jiahuan, WANG Xiaohui, JIANG Mingguo, SONG Fuqiang, CHANG Wei. Optimization of Culture Medium of Serendipita indica Based on Response Surface Methodology[J]. Chinese Agricultural Science Bulletin, 2022, 38(3): 102-109.

Figures/Tables 10

References 26

[1]	VERMA S, VARMA A, REXER K, et al. Piriformospora indica, gen. et sp. Nov., a new root-colonizing fungus[J]. Mycologia, 1998, 90(5):896-903. doi: 10.2307/3761331 URL
[2]	宋凤鸣, 毛克克, 吴铖铖, 等. 印度梨形孢的生物学效应及其作用机制[J]. 浙江大学学报:农业与生命科学版, 2011, 37(1):1-6.
[3]	张玲秀, 白建华, 郝瑞林, 等. 印度梨形孢发酵条件优化及玉米田间应用试验[J]. 江苏农业科学, 2018, 46(24):334-337.
[4]	舒珊, 高中男, 袁听, 等. 印度梨形孢最适培养基的筛选及其对水稻的促生作用研究[J]. 福建农业学报, 2019, 34(2):155-161.
[5]	SWETHA S, VARMA A, PADMAVATHI T. Statistical evaluation of the medium components for the production of high biomass, α-amylase and protease enzymes by Piriformospora indica using Plackett-Burman experimental design[J]. Biotech, 2014(4):439-445.
[6]	石小保, 郑振泽, 伍俊为, 等. 印度梨形孢对金柑种子萌发及幼苗生长的影响[J]. 园艺与种苗, 2020, 40(12):1-5.
[7]	LI Q, KUO Y W, LIN K H, et al. Piriformospora indica colonization increases the growth, development, and herbivory resistance of sweet potato (Ipomoea batatas L.)[J]. Plant cell reports, 2020, 40(2):339-350. doi: 10.1007/s00299-020-02636-7 URL
[8]	吴楚, 税蓉, 韦巧, 等. 根内生真菌印度梨形孢侵染对黑麦草气体交换和生长发育的影响[J]. 草地学报, 2018, 26(3):786-790.
[9]	黄化刚, 吕立新, 张艳茗, 等. 微生物帮助烟草抗旱的机理及其应用[J]. 应用生态学报, 2017, 28(9):3099-3110.
[10]	潘锐, 邓晶, 胡爱兵, 等. 印度梨形孢促进棉花幼苗生长及诱导提高苗期抗旱性研究[J]. 干旱地区农业研究, 2019, 37(5):249-256.
[11]	FATEMEH H, MOHAMMAD REZA M, ANTHONY ROGER D, et al. Maize water status and physiological traits as affected by root endophytic fungus Piriformospora indica under combined drought and mechanical stresses[J]. Planta, 2018, 247(5):1229-1245. doi: 10.1007/s00425-018-2861-6 URL
[12]	LI L, LI L, WANG X Y, et al. Plant growth-promoting endophyte Piriformospora indica alleviates salinity stress in Medicago truncatula[J]. Plant physiology and biochemistry, 2017, 119:211-223. doi: 10.1016/j.plaphy.2017.08.029 URL
[13]	李亮, 陈希, 王奋, 等. 印度梨形孢通过激活抗氧化物酶活性及诱导P5CS基因表达提高紫花苜蓿耐盐性[J]. 河北工业大学学报, 2016, 45(4):29-36.
[14]	高娅, 梁玉, 董智, 等. 盐胁迫下印度梨形孢对核桃幼苗生长的影响[J]. 干旱资源与环境, 2019, 33(8):194-198.
[15]	LIN H F, XIONG J, ZHOU H M, CHEN C M, et al. Growth promotion and disease resistance induced in Anthurium colonized by the beneficial root endophyte Piriformospora indica[J]. BMC plant biology, 2019, 19(1):40. doi: 10.1186/s12870-019-1649-6 URL
[16]	彭兵, 刘剑, 惠非琼, 等. 印度梨形孢诱导烟草对黑胫病的抗性及其机理的初步研究[J]. 农业生物技术学报, 2015, 23(4):432-440.
[17]	BAKHSHANDEH E, PIRDASHTI H, LENDEH K S, et al. Effects of plant growth promoting microorganisms inoculums on mineral nutrition, growth and productivity of rice (Oryza sativa L.)[J]. Journal of plant nutrition, 2020, 43(11):1643-1660. doi: 10.1080/01904167.2020.1739297 URL
[18]	WU M Y, WEI Q, XU LE, et al. Piriformospora indica enhances phosphorus absorption by stimulating acid phosphatase activities and organic acid accumulation in Brassica napus[J]. Plant and soil, 2018, 432(1):333-344. doi: 10.1007/s11104-018-3795-2 URL
[19]	韦巧, 武美燕, 张文英. 不同磷水平下印度梨形孢对生菜生长及磷素吸收利用的影响[J]. 河南农业科学, 2017, 46(1):100-104.
[20]	DABRAL S, VARMA A, NATH M, et al. Biopriming with Piriformospora indica ameliorates cadmium stress in rice by lowering oxidative stress and cell death in root cells[J]. Ecotoxicology and environmental safety, 2019, 186:109741. doi: 10.1016/j.ecoenv.2019.109741 URL
[21]	SABRA M, AMAL A, FRANKEN P, et al. Beneficial Root Endophytic Fungi Increase Growth and Quality Parameters of Sweet Basil in Heavy Metal Contaminated Soil[J]. Frontiers in plant science, 2018, 9:1726. doi: 10.3389/fpls.2018.01726 URL
[22]	SEPEHRI M, KHATABI B. Combination of siderophore-producing bacteria and Piriformospora indica provides an efficient approach to improve cadmium tolerance in alfalfa[J]. Microbial ecology, 2020:1-14.
[23]	刘慧春, 朱开元, 张加强, 等. 印度梨形孢液体发酵产物的制备及其在日本青葱上的应用[J]. 浙江农业科学, 2019, 60(5):779-782.
[24]	刘远开. 红松外生菌根真菌与凋落物分解相关性研究[D]. 哈尔滨:黑龙江大学, 2010.
[25]	YANG L, CAO J L, ZOU Y N, et al. Piriformospora indica: a root endophytic fungus and its roles in plants[J]. Notulae botanicae horti agrobotanici cluj-napoca, 2020, 48(1):1-13. doi: 10.15835/nbha48111761 URL
[26]	谢勇武, 谭属琼. 响应面法优化南境金线莲组培培养基工艺研究[J]. 贵州师范大学学报:自然科学版, 2019, 37(5):29-36.

培养基	配方
PDMA	葡萄糖20 g;20%马铃薯汁500 mL;麦芽浸粉（波美3度）500 mL;琼脂15 g
综合PDA	葡萄糖20 g;20%马铃薯汁1000 mL;VB1(0.05 g);MgSO₄·7H₂O 1.5 g;KH₂PO₄ 3 g;琼脂15 g
PDA	葡萄糖20 g;土豆200 g;水1000 mL;琼脂15 g
MMN	葡萄糖10 g;麦芽汁（波美3度）100 mL;VB1 0.1 g;MgSO₄·7H₂O 0.15 g;KH₂PO₄ 0.5 g;NaCl 0.025 g; CaCl₂ 0.05 g;(NH₄)₂HPO₄ 0.25 g; FeCl₃ (1%) 1.2 mL;柠檬酸0.2 g;水900 mL;琼脂15 g
改良MMN	葡萄糖10 g;麦芽汁3 g;VB1(0.05 g);MgSO₄·7H₂O 0.15 g;KH₂PO₄ 0.5 g;NaCl 0.025 g;CaCl₂ 0.05 g; (NH₄)₂HPO₄ 0.25 g;FeCl₃(1%) 1.2 mL;牛肉膏15 g;蛋白胨15 g;水1000 mL;琼脂15 g
KM	葡萄糖20 g;水解酪蛋白1 g;酵母提取物1 g;蛋白胨2 g;20×salt stock solution 50 mL;100×Microelement stock solution 10 mL;Fe-EDTA10 mL;1000×Vitamin stock solution 1 mL;水930 mL;琼脂15 g;20×salt stock solution：NaNO₃ 12 g;KCl 10.4 g;MgSO₄·7H₂O 10.4 g;KH₂PO₄ 30.4 g;水1000 mL;100×Microelement stock solution：ZnSO₄·7H₂O 2.2 g;H₃BO₄ 1 g;MnSO₄·H₂O 0.5 g;CoCl·5H₂O 0.16 g;CuSO₄·5H2O 0.16 g;(NH₄)₆MO₇O₂₄ 0.11 g;水1 000 mL;Fe-EDTA：FeSO₄ 0.13 g;EDTA 0.18 g;水100 mL;1000×Vitamin stock solution：VB1 (0.05 g);VB3 (0.01 g);VB6 (0.01 g);甘氨酸0.04 g;1000 mL

培养基	配方
PDMA	葡萄糖20 g;20%马铃薯汁500 mL;麦芽浸粉（波美3度）500 mL;琼脂15 g
综合PDA	葡萄糖20 g;20%马铃薯汁1000 mL;VB1(0.05 g);MgSO₄·7H₂O 1.5 g;KH₂PO₄ 3 g;琼脂15 g
PDA	葡萄糖20 g;土豆200 g;水1000 mL;琼脂15 g
MMN	葡萄糖10 g;麦芽汁（波美3度）100 mL;VB1 0.1 g;MgSO₄·7H₂O 0.15 g;KH₂PO₄ 0.5 g;NaCl 0.025 g; CaCl₂ 0.05 g;(NH₄)₂HPO₄ 0.25 g; FeCl₃ (1%) 1.2 mL;柠檬酸0.2 g;水900 mL;琼脂15 g
改良MMN	葡萄糖10 g;麦芽汁3 g;VB1(0.05 g);MgSO₄·7H₂O 0.15 g;KH₂PO₄ 0.5 g;NaCl 0.025 g;CaCl₂ 0.05 g; (NH₄)₂HPO₄ 0.25 g;FeCl₃(1%) 1.2 mL;牛肉膏15 g;蛋白胨15 g;水1000 mL;琼脂15 g
KM	葡萄糖20 g;水解酪蛋白1 g;酵母提取物1 g;蛋白胨2 g;20×salt stock solution 50 mL;100×Microelement stock solution 10 mL;Fe-EDTA10 mL;1000×Vitamin stock solution 1 mL;水930 mL;琼脂15 g;20×salt stock solution：NaNO₃ 12 g;KCl 10.4 g;MgSO₄·7H₂O 10.4 g;KH₂PO₄ 30.4 g;水1000 mL;100×Microelement stock solution：ZnSO₄·7H₂O 2.2 g;H₃BO₄ 1 g;MnSO₄·H₂O 0.5 g;CoCl·5H₂O 0.16 g;CuSO₄·5H2O 0.16 g;(NH₄)₆MO₇O₂₄ 0.11 g;水1 000 mL;Fe-EDTA：FeSO₄ 0.13 g;EDTA 0.18 g;水100 mL;1000×Vitamin stock solution：VB1 (0.05 g);VB3 (0.01 g);VB6 (0.01 g);甘氨酸0.04 g;1000 mL

培养条件	A	B	C	D	E	F
温度	22℃	25℃	28℃	31℃	34℃	37℃
pH	6.3	6.5	6.7	6.9	7.1	7.3
碳源	无	牛肉膏	麦芽糖	蔗糖	葡萄糖	秸秆
氮源	无	尿素	硝酸钠	蛋白胨	磷酸氢二铵	酵母浸粉
无机盐	无	硫酸镁	磷酸二氢钾	氯化钠	氯化钙	氯化铁

培养条件	A	B	C	D	E	F
温度	22℃	25℃	28℃	31℃	34℃	37℃
pH	6.3	6.5	6.7	6.9	7.1	7.3
碳源	无	牛肉膏	麦芽糖	蔗糖	葡萄糖	秸秆
氮源	无	尿素	硝酸钠	蛋白胨	磷酸氢二铵	酵母浸粉
无机盐	无	硫酸镁	磷酸二氢钾	氯化钠	氯化钙	氯化铁

变量	水平
变量	(-1)	(0)	(1)
硫酸镁/(g/L)	0.15	0.65	1.15
磷酸二氢钾/(g/L)	0.4	1.4	2.4
硝酸钠/(g/L)	0.4	1.4	2.4