Trichoderma asperellum ACCC30536 inoculation improves soil nutrition and leaf artemisinin production in Artemisia annua

  • Tongtong Zhai
  • Yuefeng Wang
  • Changli Liu
  • Zhaoying Liu
  • Min Zhao
  • Yuan Chang
  • Rongshu ZhangEmail author
Original Article


Artemisia annua L. is the main source of artemisinin, currently the most effective treatment for malaria. However, an affordable and abundant supply of artemisinin remains elusive. Trichoderma is a biocontrol agent that stimulates plant growth and defense responses, and improves soil quality. To date, relatively few studies have focused on improving leaf biomass and artemisinin production in A. annua using Trichoderma. To investigate the role of T. asperellum ACCC30536 in improving the artemisinin yield of A. annua, field-grown A. annua was inoculated with T. asperellum conidia and grown for 60 days. The results showed that leaf artemisinin concentration and dry weight were increased significantly after inoculation. The optimal inoculation dose was 200 mL of conidia suspension at 1 × 107 colony-forming units (cfu)/mL, the highest artemisinin concentration was 8.83 mg/g, and the highest artemisinin production was 70.6 g on day 50. The results of qRT-PCR revealed that expression of genes encoding key enzymes for artemisinin biosynthesis, namely HMGR1, FPS, ADS, CYP71AV1, CPR, DBR, DXS1, and DXR1, was generally upregulated during days 20–50 following induction by Trichoderma. In addition, the moisture, pH stability, organic matter content, and availability of nitrogen, phosphorus, and kalium in inoculated soil were significantly improved. Thus, application of T. asperellum ACCC30536 may offer a novel approach for improving artemisinin production by upregulating the expression of key enzymes for artemisinin biosynthesis, increasing leaf yield, and improving soil fertility.


Trichoderma asperellum Artemisia annua Artemisinin Soil fertility Leaf yield 



This work was supported by the National Natural Science Foundation of China (NSFC: 31370642), the State Key Laboratory of Tree Genetics and Breeding (Northeast Forestry University; 201202), and the Natural Science Foundation of Heilongjiang Province of China (C201216).

Supplementary material

11738_2019_2836_MOESM1_ESM.doc (306 kb)
Supplementary material 1 (DOC 306 KB)
11738_2019_2836_MOESM2_ESM.doc (178 kb)
Supplementary material 2 (DOC 177 KB)
11738_2019_2836_MOESM3_ESM.docx (89 kb)
Supplementary material 3 (DOCX 88 KB)
11738_2019_2836_MOESM4_ESM.xlsx (15 kb)
Supplementary material 4 (XLSX 15 KB)


  1. Arsenault PR, Vail DR, Wobbe KK, Weathers PJ (2010) Effect of sugars on artemisinin production in Artemisia annua L.: transcription and metabolite measurements. Molecules 15(4):2302–2318CrossRefGoogle Scholar
  2. Boughalleb-M’Hamdi N, Salem IB, M’Hamdi M (2018) Evaluation of the efficiency of Trichoderma, Penicillium, and Aspergillus species as biological control agents against four soil-borne fungi of melon and watermelon. Egypt J Biol Pest Control 28:25. CrossRefGoogle Scholar
  3. Bryant L, Flatley B, Patole C, Brown GD, Cramer R (2015) Proteomic analysis of Artemisia annua-towards elucidating the biosynthetic pathways of the antimalarial pro-drug artemisinin. BMC Plant Biol 15:175–188. CrossRefPubMedPubMedCentralGoogle Scholar
  4. Chen LX (2005) Soil experiment and practice course. Northeast Forestry University Press, Harbin (in Chinese) Google Scholar
  5. Cui YQ, Ma JY, Sun W, Sun JH, Duan ZH (2015) A preliminary study of water use strategy of desert plants in Dunhuang, China. J Arid Land 7(1):73–81. CrossRefGoogle Scholar
  6. Czechowski T, Larson TR, Catania TM, Harvey D, Brown GD, Graham IA (2016) Artemisia annua mutant impaired in artemisinin synthesis demonstrates importance of nonenzymatic conversion in terpenoid metabolism. Proc Natl Acad Sci 113(52):15150–15155CrossRefGoogle Scholar
  7. El-Katatny MH (2010) Enzyme production and nitrogen fixation by free, immobilized and coimmobilized inoculants of Trichoderma harzianum and Azospirillum brasilense and their possible role in growth promotion of tomato. Food Technol Biotechnol 48(2):161–174Google Scholar
  8. Ferreira JFS, Simon JE, Janick J (1997) Artemisia annua: botany, horticulture, pharmacology. Hort Res 19:319–371Google Scholar
  9. Francesco V, Krishnapillai S, Emilio LG, Sheridan LW, Marco N, Roberta M, Nadia L, Alberto P, Michelina R, Stefania L, Gelsomina M, Matteo L (2014) Trichoderma secondary metabolites active on plants and fungal pathogens. Open Mycol J 8(5):127–139Google Scholar
  10. Guo RT, Wang ZY, Huang Y, Fan HJ, Liu ZH (2018) Biocontrol potential of saline- or alkaline-tolerant Trichoderma asperellum mutants against three pathogenic fungi under saline or alkaline stress conditions. Braz J Microbiol 388:1–10Google Scholar
  11. Jha P, Ram M, Khan MA, Kiran U, Abdin MZ (2011) Impact of organic manure and chemical fertilizers on artemisinin content and yield in Artemisia annua L. Ind Crops Prod 33(2):296–301CrossRefGoogle Scholar
  12. Jimenez J, Lei H, Steyer J-P, Houot S, Patureau D (2017) Methane production and fertilizing value of organic waste: organic matter characterization for a better prediction of valorization pathways. Biores Technol 241:1012–1021CrossRefGoogle Scholar
  13. Kazaz B, Webster S, Yadav P (2016) Interventions for an artemisinin-based malaria medicine supply chain. Prod Oper Manag 25(9):1576–1600. CrossRefGoogle Scholar
  14. Kong P, Hong CX (2017) Biocontrol of boxwood blight by Trichoderma koningiopsis Mb2. Crop Prot 98:124–127CrossRefGoogle Scholar
  15. Li C, Li J, Wang G, Li X (2016) Heterologous biosynthesis of artemisinic acid in Saccharomyces cerevisiae. J Appl Microbiol 120(6):1466–1478. CrossRefPubMedGoogle Scholar
  16. Li YT, Hwang SG, Huang YM, Huang CH (2018) Effects of Trichoderma asperellum on nutrient uptake and Fusarium wilt of tomato. Crop Prot 110:275–282CrossRefGoogle Scholar
  17. Luo SQ, Huang JG, Yuan L (2014) Nutrients and microorganisms in soils with wild Artemisia annua L. Acta Pedol Sin 51(4):868–879 (in Chinese) Google Scholar
  18. Lv MM, Liu ZH, Wang H, Zhu GD, Yang XT, Zhang RS (2015) Effects of Trichoderma asperellum on the physical and chemical properties and nutrient components of the pot soil culturing tissue-cultured Populus davidiana × P. bolleana seedlings. Bull Bot Res 35(2):289–296 (in Chinese) Google Scholar
  19. Mercke P, Bengtsson M, Bouwmeester HJ, Posthumus MA, Brodelius PE (2000) Molecular cloning, expression, and characterization of amorpha-4, 11-diene synthase, a key enzyme of artemisinin biosynthesis in Artemisia annua L. Arch Biochem Biophys 381:173–180. CrossRefPubMedGoogle Scholar
  20. Ndoungue M, Petchayo S, Techou Z, Nana WG, Nembot C, Fontem D, Ten Hoopen GM (2018) The impact of soil treatments on black pod rot (caused by Phytophthora megakarya) of cacao in Cameroon. Biol Control 123:9–17CrossRefGoogle Scholar
  21. Patel S, Saraf M (2017) Biocontrol efficacy of Trichoderma asperellum MSST against tomato wilting by Fusarium oxysporum f. sp. lycopersici. Arch Phytopathol Plant Prot 50:223–230CrossRefGoogle Scholar
  22. Peng M, Chen M, Chen R, Lan X, Hsieh MH, Liao Z (2011) The last gene involved in the MEP pathway of Artemisia annua: cloning and characterization and functional identification. J Med Plants Res 5(2):223–230Google Scholar
  23. Qiao Y, Miao S, Han X, Yue S, Tang C (2017) Improving soil nutrient availability increases carbon rhizodeposition under maize and soybean in Mollisols. Sci Total Environ 603:416–424CrossRefGoogle Scholar
  24. Shen Q, Yan TX, Fu XQ, Tang KX (2016) Transcriptional regulation of artemisinin biosynthesis in Artemisia annua L. Sci Bull 61(1):18–25. CrossRefGoogle Scholar
  25. Shin K, Diepen G, Blok W, Bruggen AHC (2017) Variability of effective microorganisms (EM) in bokashi and soil and effects on soil-borne plant pathogens. Crop Prot 99:168–176CrossRefGoogle Scholar
  26. Tchameni SN, Sameza ML, O’donovan A, Fokom R, Mangaptche Ngonkeu EL, Wakam Nana L, ETOA F-X, NWAGA D (2017) Antagonism of Trichoderma asperellum against Phytophthora megakarya and its potential to promote cacao growth and induce biochemical defence. Mycology 8(2):84–92. CrossRefGoogle Scholar
  27. Wang XR, Su SM, Zeng XB, Bai LY, Li LF, Duan R, Wang YN, Wu CX (2015) Inoculation with chlamydospores of Trichoderma asperellum SM-12F1 accelerated arsenic volatilization and influenced arsenic availability in soils. J Integr Agric 14(2):389–397CrossRefGoogle Scholar
  28. Wei SG, Ma XJ, Feng SX, Huang RS, Dong QS, Yan ZG, Huang QG (2008) Evaluation on germplasm resources of main production area of Artemisia annua in China. China J Chin Mater Med 33(3):241–244 (in Chinese) Google Scholar
  29. Wu T, Wang YJ, Guo DJ (2012) Investigation of glandular trichome proteins in Artemisia annua L. using comparative proteomics. PloS One. CrossRefPubMedPubMedCentralGoogle Scholar
  30. Wu Q, Sun R, Ni M, Yu J, Li Y, Yu C, Dou K, Ren J, Chen J (2017) Identification of a novel fungus, Trichoderma asperellum GDFS1009, and comprehensive evaluation of its biocontrol efficacy. PloS One. CrossRefPubMedPubMedCentralGoogle Scholar
  31. Xiang L, Zhu S, Zhao T, Zhang M, Liu W, Chen M, Lan X, Liao Z (2015) Enhancement of artemisinin content and relative expression of genes of artemisinin biosynthesis in Artemisia annua by exogenous MeJA treatment. Plant Growth Regul 75(2):435–441. CrossRefGoogle Scholar
  32. Xiao L, Tan HX, Zhang L (2016) Artemisia annua glandular secretory trichomes: the biofactory of antimalarial agent artemisinin. Sci Bull 61(1):26–36. CrossRefGoogle Scholar
  33. Xue AG, Guo W, Chen YH (2017) Effect of seed treatment with novel strains of Trichoderma spp. on establishment and yield of spring wheat. Crop Prot 96:97–102CrossRefGoogle Scholar
  34. Yao ZH, Baloch AM, Liu ZH, Zhai TT, Jiang CY, Liu ZY, Zhang RS (2018) Cloning and characterization of an AUX/IAA gene in Populus davidiana x P. alba var. pyramidalis and the correlation between its time course expression and the levels of indole-3-acetic in saplings inoculated with trichoderma. Pak J Bot 50(1):169–177Google Scholar
  35. Zhang RS, Zhao M, Zhou YD, Han S (2009) Artemisinin content and biomass yield of introduced Artemisia annua. Sci Silvae Sinicae 45(4):151–155 (in Chinese) Google Scholar
  36. Zhang RS, Baloch AM, Li SH, Li SH, Liu ZH, Jiang CY, Wang H, Baloch AW, Diao GP (2018) Improvement in biomass, IAA levels and auxin signaling related gene expression in shanxin poplar seedlings (Populus davidiana x p. alba var. pyramidalis) induced by Trichoderma asperellum. Pak J Bot 50(4):1629–1636Google Scholar
  37. Zhou LY, Yang G, Sun HF, Tang JF, Yang J, Wang YZ, Garran TA, Guo LP (2017) Effects of different doses of cadmium on secondary metabolites and gene expression in Artemisia annua L.. Front Med 11(1):137–146CrossRefGoogle Scholar
  38. Zhu MM, Zhang FY, Lv ZY, Shen Q, Zhang L, Lu X, Jiang WM, Fu XQ, Yan TX, Chen LX, Wang GF, Tang KX (2014) Characterization of the promoter of Artemisia annua Amorpha-4,11-diene synthase (ADS) gene using homologous and heterologous expression as well as deletion analysis. Plant Mol Biol Rep 32(2):406–418. CrossRefGoogle Scholar

Copyright information

© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2019

Authors and Affiliations

  1. 1.College of Landscape ArchitectureNortheast Forestry UniversityHarbinChina
  2. 2.Key Laboratory of Photobiology, Photosynthesis Research Center, Institute of BotanyChinese Academy of SciencesBeijingChina
  3. 3.College of Life ScienceNortheast Forestry UniversityHarbinChina

Personalised recommendations