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Interaction between Piriformospora indica and Azotobacter chroococcum governs better plant physiological and biochemical parameters in Artemisia annua L. plants grown under in vitro conditions

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Abstract

Artemisinin and its derivatives have been used in artemisinin combination therapies (ACTs) for treatment of malaria. The drug artemisinin is produced in the leaves of Artemisia annua. The escalating demands of ACTs together with minor concentration of artemisinin in leaves (0.01–1.1%) of A. annua L. has necessitated advent of viable methods to meet this growing demand. In the present study, symbiotic microbes; Piriformospora indica (Pi) and Azotobacter chroococcum (Az) were used singly and in combination (Pi +Az) to investigate their effect on in vitro grown A. annua L. plants. For co-cultivation of A. annua L. plantlets with symbiotic microbes, four weeks old rooted plantlets were used. Results showed that interaction of in vitro raised plantlets with dual inoculants significantly enhanced the plant growth, photosynthetic pigments, total soluble sugar, soluble protein, flavonoids content compared to control plants. The plant growth and rest of the parameters were enhanced in the treatments with single inoculants and were higher in plants inoculated with Pi than Az. A significant enhancement of 2-fold in artemisinin content was also observed in treatments receiving both inoculants. The physiological and biochemical parameters in these tested treatments improved significantly in the order (Pi +Az) > (Pi) > (Az).

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References

  • Abdel-Lateif K, Bogusz D, Hocher V (2012) The role of flavonoids in the establishment of plant roots endosymbioses with arbuscular mycorrhiza fungi, rhizobia and Frankia bacteria. Plant Signal Behav 7:636–641

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Abdin MZ, Israr M, Rehman RU, Jain SK (2003) Artemisinin, a novel antimalarial drug: biochemical and molecular approaches for enhanced production. Planta Med 69:289–299

    Article  CAS  PubMed  Google Scholar 

  • Ahmad F, Ahmad I, Khan MS (2008) Screening of free-living rhizospheric bacteria for their multiple plant growth promoting activities. Microbiol Res 163:173–181

    Article  CAS  PubMed  Google Scholar 

  • Antunes PM, Rajcan I, Goss MJ (2006) Specific flavonoids as interconnecting signals in the tripartite symbiosis formed by arbuscular mycorrhizal fungi, Bradyrhizobium japonicum (Kirchner) Jordan and soybean (Glycine max (L.) Merr.) Soil Biol Biochem 38:533–543

    Article  CAS  Google Scholar 

  • Arnon DI (1949) Copper enzyme in isolated chloroplast polyphenoloxidase in Beta vulgaris L. Plant Physiol 24:1–15

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Arora M, Saxena P, Choudhary DK, Abdin MZ, Varma A (2016) Dual symbiosis between Piriformospora indica and Azotobacter chroococcum enhances the artemisinin content in Artemisia annua L. World J Microbiol Biotechnol 32:19

    Article  PubMed  Google Scholar 

  • Awasthi A, Bharti N, Nair P et al (2011) Synergistic effect of Glomus mosseae and nitrogen fixing Bacillus subtilis strain Daz26 on artemisinin content in Artemisia annua L. Appl Soil Ecol 49:125–130

    Article  Google Scholar 

  • Bagayoko M, George E, Romheld V, Buerkert A (2000) Effects of mycorrhizae and phosphorus on growth and nutrient uptake of millet, cowpea and sorghum on a west African soil. J Agric Sci 135:399–407

    Article  Google Scholar 

  • Bagyaraj and Menge (1978) Interaction between a VAM and Azotobacter and their effects on rhizosphere microflora and plant growth. New Phytol 80:567–573

    Article  Google Scholar 

  • Baishya D, Deka P, Kalita MC (2015) In vitro co-cultivation of Piriformospora indica filtrate for improve biomass productivity in Artemisia annua (L.) Symbiosis 66:37–46

    Article  Google Scholar 

  • Baslam M, Goicoechea N (2012) Water deficit improved the capacity of arbuscular mycorrhizal fungi (AMF) for inducing the accumulation of antioxidant compounds in lettuce leaves. Mycorrhiza 22:347–359

    Article  CAS  PubMed  Google Scholar 

  • Bhattacharjee S, Sharma GD (2012) Effect of dual inoculation of arbuscular mycorrhiza and rhizobium on the chlorophyll, nitrogen and phosphorus contents of Pigeon Pea (Cajanus cajan L.) Adv Microbiol 2:561–564

    Article  Google Scholar 

  • Bhuyan SK, Bandyopadhyay P, Kumar P et al (2015) Interaction of Piriformospora indica with Azotobacter chroococcum. Sci Rep 5:13911

    Article  Google Scholar 

  • Boby VU, Bagyaraj DJ (2003) Biological control of root-rot of Coleus forskohlii Briq. using microbial inoculants. World J Microbiol Biotechnol 19:175–180

    Article  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Chang CC, Yang MH, Wen HM, Chern JC (2002) Estimation of total flavonoid content in propolis by two complementary colorimetric methods. J Food Drug Anal 10:178–182

    CAS  Google Scholar 

  • Clark RB, Zeto SK, Zobel RW (1999) Arbuscular mycorrhizal fungal isolate effectiveness on growth and root colonization of Panicum virgatum in acidic soil. Soil Biol Biochem 31:1757–1763

    Article  CAS  Google Scholar 

  • Dangash A, Ram M, Niranjan R et al (2015) In vitro selection and hormonal regulation in cell culture of Artemisia annua L. plant. JSM Cell Dev Biol 3:1013

    Google Scholar 

  • Das A, Kamal S, Shakil NA et al (2012) The root endophyte fungus Piriformospora indica leads to early flowering, higher biomass and altered secondary metabolites of the medicinal plant, Coleus forskohlii. Plant Signal Behav 7:103–112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Das J, Ramesh KV, Maithri D, Mutangana D, Suresh CK (2014a) Response of aerobic rice to Piriformospora indica. Indian J Exp Biol 52:237–251

    PubMed  Google Scholar 

  • Das A, Tripathi S, Varma A (2014b) In vitro plant development and root colonization of Coleus forskohlii by Piriformospora indica. World J Microbial Biotechnol 30:1075–1084

    Article  CAS  Google Scholar 

  • Davies MJ, Atkinson CJ, Burns C et al (2009) Enhancement of artemisinin concentration and yield in response to optimization of nitrogen and potassium supply to Artemisia annua. Ann Bot 104:315–323

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dhingra V, Rao KV, Narasu ML (1999) Artemisinin: present status and perspectives. Biochem Mol Biol Educ 27:105–109

    CAS  Google Scholar 

  • Evelin H, Kapoor R (2014) Arbuscular mycorrhizal symbiosis modulates antioxidant response in salt-stressed Trigonella foenum-graecum plants. Mycorrhiza 24:197–208

    Article  CAS  PubMed  Google Scholar 

  • Ferreira JF, Luthria DL, Sasaki T, Heyerick A (2010) Flavonoids from Artemisia annua L. as antioxidants and their potential synergism with artemisinin against malaria and cancer. Molecules 15:3135–3170

    Article  CAS  PubMed  Google Scholar 

  • Fuentes P, Zhou F, Erban A, Karcher D, Kopka J, Bock R (2016) A new synthetic biology approach allows transfer of an entire metabolic pathway from a medicinal plant to a biomass crop. elife 5:e13664

    Article  PubMed  PubMed Central  Google Scholar 

  • Gengmao Z, Quanmei S, Yu H, Shihui L, Changhai W (2014) The physiological and biochemical responses of a medicinal plant (Salvia miltiorrhiza L.) to stress caused by various concentrations of NaCl. PLoS One 9:e89624. https://doi.org/10.1371/journal.pone.0089624

    Article  PubMed  PubMed Central  Google Scholar 

  • Glick BR (2012) Plant growth-promoting bacteria: mechanisms and applications. Scientifica (Cairo) 2012:1–15. https://doi.org/10.6064/2012/963401

  • González R, Laudat T, Arzola M et al (2011) Effect of Azotobacter chroococcum on in vitro pineapple plants growth during acclimatization. In Vitro Cell Dev Biol Plant 47:387–390

    Article  Google Scholar 

  • Gosal SK, Karlupia A, Gosal SS, Chhibba IM, Varma A (2010) Biotization with Piriformospora indica and Pseudomonas fluorescens improves survival rate, nutrient acquisition, field performance and saponin content of micropropagated Chlorophytum sp. Ind J Biotechnol 9:289–297

    CAS  Google Scholar 

  • Gueye M (1990) Effect of inoculation with rhizobium and rhizobium plus Glomus mosseae on N2 fixation by Bambara Grundnut. 4th AABNF Conference held at Ibadan, Nigeria, 24–29 September 1990

  • Guo YA, Ni Y, Huang J (2010) Effects of rhizobium, arbuscular mycorrhiza and lime on nodulation, growth and nutrient uptake of Lucerne in acid purplish soil in China. Trop Grassl 44:109–114

    Google Scholar 

  • Gupta R, Singh A, Gupta MM, Pandey R (2016) Cumulative role of bioinoculants on growth, antioxidant potential and artemisinin content in Artemisia annua L. under organic field conditions. World J Microbiol Biotechnol 32:167

    Article  PubMed  Google Scholar 

  • Harper SH, Lynch JM (1979) Effect of Azotobacter chroococcum on barley seed germination and seedling development. Microbiology 112:45–51

    Google Scholar 

  • Hiscox JT, Israelstam GF (1979) A method for the extraction of chlorophyll from leaf tissue without maceration. Can J Bot 57:1332–1334

    Article  CAS  Google Scholar 

  • Huang Y, Hui L, Peng D, Wang Y, Ren Q, Guo Y (2016) The production and exportation of artemisinin- derived drugs in China: current status and existing challenges. Malar J 15:365

    Article  PubMed  PubMed Central  Google Scholar 

  • Jensen HL (1942) Nitrogen fixation in leguminous plants. II. Is symbiotic nitrogen fixation influenced by Azotobacter? Proc Linnean Soc NSW 67:205–212

    CAS  Google Scholar 

  • Jogawat A, Saha S, Bakshi M et al (2013) Piriformospora indica rescues growth diminution of rice seedlings during high salt stress. Plant Signal Behav 8:e26891

    Article  PubMed Central  Google Scholar 

  • Kapoor R, Chaudhary V, Bhatnagar AK (2007) Effects of arbuscular mycorrhizal and phosphorus application on artemisinin concentration in Artemisia annua L. Mycorrhiza 17:581–587

    Article  CAS  PubMed  Google Scholar 

  • Karthikeyan A, Sakthivel KM (2011) Efficacy of Azotobacter chroococcum in rooting and growth of Eucalyptus camaldulensis stem cuttings. Res J Microbiol 6:618–624

    Article  Google Scholar 

  • Kazi RP, Lakshman HC (2008) Combined inoculation of arbuscular mycorrhizal fungi and Azotobacter beneficial to Proralea coryliflia L. Asian J Bio Sci 3:11–14

    Article  Google Scholar 

  • Khaosaad T, Krenn L, Medjakovic S et al (2008) Effect of mycorrhization on the isoflavone content and the phytoestrogen activity of red clover. J Plant Physiol 165:1161–1167

    Article  CAS  PubMed  Google Scholar 

  • Khatabi B, Molitor A, Lindermayr C et al (2012) Ethylene supports colonization of plant roots by the mutualistic fungus Piriformospora indica. PLoSOne 7:e35502

    Article  CAS  Google Scholar 

  • Kilam D, Saifi M, Abdin MZ, Agnihotri A, Varma A (2015) Combined effects of Piriformospora indica and Azotobacter chroococcum enhance plant growth, antioxidant potential and steviol glycoside content in Stevia rebaudiana. Symbiosis 66:149–156

    Article  CAS  Google Scholar 

  • Kumar A, Sharma S, Mishra S (2009) Effect of alkanity on growth performance of Jatropha curcus inoculated with PGPR and AM fungi. J Phytol 1:177–184

    Google Scholar 

  • Kumar A, Sharma S, Mishra S (2015) Evaluating effect of arbuscular mycorrhizal fungal consortia and Azotobacter chroococcum in improving biomass yield of Jatropha curcas. Plant Biosyst-An International Journal Dealing with all Aspects of Plant Biology 150:1056–1064

    Article  Google Scholar 

  • Larraburu EE, Carletti SM, Caceres EA, Llorente BE (2007) Micropropagation of photonia employing rhizobacteria to promote root development. Plant Cell Rep 26:711–717

    Article  CAS  PubMed  Google Scholar 

  • Mandal S, Evelin H, Giri B, Singh VP, Kapoor R (2013) Arbuscular mycorrhizal enhances the production of stevioside and rebaudioside-A in Stevia rebaudiana via nutritional and non-nutritional mechanisms. Appl Soil Ecol 72:187–194

    Article  Google Scholar 

  • Mortimer PE, Le Roux MR, Perez-Fernandez MA et al (2013) The dual symbiosis between arbuscular mycorrhizal and nitrogen fixing bacteria benefits the growth and nutrition of the woody invasive legume Acacia Cyclops under nutrient limiting conditions. Plant Soil 366:229–241

    Article  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Ovando-medina I, Chavez-Auguilar A-AL et al (2007) Ex vitro survival and early growth of Alpinia purpurata plantlets inoculated with Azotobacter and Azospirillium. Pak J Biol Sci 10:3454–3457

    Article  CAS  PubMed  Google Scholar 

  • Phillips JM, Hayman DS (1970) Improved procedures for clearing roots and staining parasitic and VAM fungi for rapid assessment of infection. Trans Br Mycol Soc 55:158–161

    Article  Google Scholar 

  • Prajapati K, Yami KD, Singh A (2008) Plant growth promotional effect of Azotobacter chroococcum, Piriformospora indica and Vermicompost on Rice Plant. Nepal J Sci Technol 9:85–90

    Google Scholar 

  • Salmeron V, Martinez-Toledo MV, Gonzalez-Lopez J (1990) Nitrogen fixation and production of auxins, gibberellins, and cytokinins by an Azotobacter chroococcum strain isolated from the root of Zea mays in the presence of insoluble phosphate. Chemosphere 20:417–422

    Article  CAS  Google Scholar 

  • Satheesan J, Narayanan AK, Sakunthala M (2012) Induction of root colonization by Piriformospora indica leads to enhanced asiaticoside production in Centella asiatica. Mycorrhiza 22:195–202

    Article  CAS  PubMed  Google Scholar 

  • Saxena P, Ahlawal S, Ali A, Khan S, Abdin MZ (2016) Gene expression analysis of the withanolide biosynthetic pathway in hairy root cultures of Withania somnifera elicited with methyl jasmonate and the fungus Piriformospora indica. Symbiosis 71:143–154

    Article  Google Scholar 

  • Schäfer P, Pfiffi S, Voll LM et al (2009) Phytohormones in plant root-Piriformospora indica mutualism. Plant Signal Behav 4:669–671

    Article  PubMed  PubMed Central  Google Scholar 

  • Selvarathi P, Ramasubramanian V, Jeyaprakash R (2010) Bioremedial effect of Azotobacter and Phosphobacterium on the growth and biochemical characteristics of paper mill effluent treated with Lycopersicum esculentum Mill. J. Biosci Res 1:58–64

    Google Scholar 

  • Sharma G, Agrawal V (2013) Marked enhancement in the artemisinin content and biomass productivity in Artemisia annua L. shoots co-cultivated with Piriformospora indica. World J Microbiol Biotechnol 29:1133–1138

    Article  CAS  PubMed  Google Scholar 

  • Sharma P, Kharkwal AC, Abdin MZ, Varma A (2014) Piriformospora indica improves micropropagation, growth and phytochemical content of Aloe vera L. plants. Symbiosis 64:11–23

    Article  CAS  Google Scholar 

  • Shi SM, Chen K, Gao Y et al (2016) Arbuscular mycorrhizal fungus species dependency governs better plant physiological characteristics and leaf quality of mulberry (Morus alba L.) seedlings. Front Microbiol 7:1030. https://doi.org/10.3389/fmicb.2016.01030

    PubMed  PubMed Central  Google Scholar 

  • Singh A, Gupta R, Srivastava M, Gupta MM, Pandey R (2016) Microbial secondary metabolites ameliorate growth, in planta contents and lignification in Withania somnifera (L.) Dunal. Physiol Mol Biol Plants 22:253–260

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sirrenberg A, Gobel C, Grond S et al (2007) Piriformospora indica affects plant growth by auxin production. Physiol Plant 131:581–589

    Article  CAS  PubMed  Google Scholar 

  • Sreeramulu KR, Hanumanthappa M, Andani G, Murthy KN, Jayasheela N (2000) Dual inoculation of Azotobacter chroococcum and Glomus fasciculatum improves growth and yield of sunflower under field conditions and saves N and P fertilizer application. Environ Ecol 18:380–384

    Google Scholar 

  • Suthar RK, Purohit SD (2012) Biopriming of micropropagated Boswellia serrata Roxb. Plantlets-role of endophytic root fungus Piriformospora indica. Ind J Biotechnol 11:304–308

    CAS  Google Scholar 

  • Vadassery J, Ritter C, Venus Y et al (2008) The role of Auxins and Cytokinins in the mutualistic interaction between Arabidopsis and Piriformospora indica. Mol Plant-Microbe Interact 21:1371–1383

    Article  CAS  PubMed  Google Scholar 

  • Vafadar F, Amooaghaie R, Otroshy M (2014) Effects of plant-growth-promoting rhizobacteria and arbuscular mycorrhizal fungus on plant growth, stevioside, NPK, and chlorophyll content of Stevia rebaudiana. J Plant Interact 9:128–136

    Article  CAS  Google Scholar 

  • Varma A, Verma S, Sudah SN, Franken P (1999) Piriformospora indica, a cultivable plant growth-promoting root endophyte. Appl Environ Microbiol 65:2741–2744

    CAS  PubMed  PubMed Central  Google Scholar 

  • Varma A, Singh A, Sahay NS et al (2001) Piriformospora indica: An axenically culturable mycorrhiza-like endosymbiotic fungus. In: Hock B (ed) Mycota IX. Springer, Berlin, Heidelberg, New York, pp 123–150

    Google Scholar 

  • Vyas S, Nagori R, Purohit SD (2008) Root colonization and growth enhancement of micro-propagated Feronia Limonia (L) Swingle by Piriformospora indica- A cultivable root endophyte. Int J Plant Dev Biol 2:128–132

    Google Scholar 

  • Walker V, Couillerot O, Von Felten A et al (2012) Variation of secondary metabolite levels in maize seedling roots induced by inoculation with Azospirillum, Pseudomonas and Glomus consortium under field conditions. Plant Soil 356:151–163

    Article  CAS  Google Scholar 

  • White NJ (2008) Qinghaosu (artemisinin): the price of success. Science 320:330–334

    Article  CAS  PubMed  Google Scholar 

  • WHO (2015) World malaria report September 2015. World Health Organization, Geneva

    Google Scholar 

  • WHO (2016) Artemisinin and artemisinin- based combination therapy resistance. World Health Organization, WHO/HTM/GMP/201611

  • Wu QS, Zou YN, He XH, Luo P (2011) Arbuscular mycorrhizal fungi can alter some root characters and physiological status in trifoliate orange (Poncirus trifoliate L. Raf.) seedlings. Plant Growth Regul 65:273–278

    Article  CAS  Google Scholar 

  • Yang Y-Z, Zhang J-M, Zhang Y, Zhu J-Q, Dong S-Q (2013) Effects of Pirifomospora indica on Chinese cabbage production and resistance to waterlogged stress. J Chem Pharm Res 5:520–524

    Google Scholar 

  • Zhang Z, Zhang J, Huang Y (2014) Effects of arbuscular mycorrhizal fungi on the drought tolerance of Cyclobalanopsis glauca seedlings under greenhouse conditions. New For 45:545–556

    Article  CAS  Google Scholar 

  • Zhao SS, Zeng MY (1986) Determination of Qinghaosu in Artemisia annua L. by high performance liquid chromatography. Chin J Pharm Anal 6:3–5

    Google Scholar 

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Acknowledgements

The research was supported by a grant from Department of Biotechnology (DBT), Ministry of Science and Technology, Govt. of India under the DBT’s Twinning Programme for North East. Monika Arora gratefully acknowledges the DBT for providing Research fellowship. The authors are thankful to Prof. M. Z. Abdin from Jamia Hamdard for providing instrumentation facility and expertise to carry out the present investigation.

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  1. Amity Institute of Microbial Technology (AIMT), Amity University Campus, Noida, India

    Monika Arora & Ajit Varma

  2. Centre for Transgenic Plant Development, Faculty of Science, Hamdard University, New Delhi, India

    Parul Saxena & M. Z. Abdin

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  1. Monika Arora
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  2. Parul Saxena
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  3. M. Z. Abdin
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  4. Ajit Varma
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Correspondence to Ajit Varma.

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Arora, M., Saxena, P., Abdin, M.Z. et al. Interaction between Piriformospora indica and Azotobacter chroococcum governs better plant physiological and biochemical parameters in Artemisia annua L. plants grown under in vitro conditions. Symbiosis 75, 103–112 (2018). https://doi.org/10.1007/s13199-017-0519-y

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