World Journal of Microbiology and Biotechnology

, Volume 30, Issue 9, pp 2427–2436 | Cite as

Succession patterns of fungi associated to wound-induced agarwood in wild Aquilaria malaccensis revealed from quantitative PCR assay

  • Rozi MohamedEmail author
  • Phai Lee Jong
  • Ismail Nurul Irdayu
Original Paper


Aquilaria malaccensis produces agarwood in response to wounding and fungal attack. However, information is limited regarding Aquilaria‘s interaction with its diverse fungal community. In this study, time-related changes of three natural fungal colonizers in two wounded wild A. malaccensis were tracked, beginning a few hours after wounding up to 12 months. Using species-specific primers derived from their nrITS sequences in quantitative real-time PCR (qPCR), we quantified the amount of Cunninghamella bainieri, Fusarium solani and Lasiodiplodia theobromae. Because time is a major factor affecting agarwood quantity and quality, 14 wood samples were collected at different time points, i.e., 0–18 h, 2–13 days, 2–18 weeks, and 6–12 months after wounding. qPCR data revealed that the abundance of the three species decreased over time. The fungi were detected in high numbers during the first few hours and days after wounding (40- to 25,000-fold higher levels compared with initial counts) and in low numbers (<1- to 3,200-fold higher than initially) many months later. Consistent with its role in defense response, the accumulation of secondary metabolites at the wounding site could have caused the decline in fungal abundance. Succession patterns of the two trees were not identical, indicating that fungal populations may have been affected by tree environment and wound microclimate. Our results are important for understanding the diversity of microbial community in wild Aquilaria species and their association to wound-induced agarwood formation. Fungi could be secondary triggers to agarwood production in situations where trees are wounded in attempt to induce agarwood.


Fungal community Gaharu nrITS Thymelaeaceae Wounding 



This work was supported by the Universiti Putra Malaysia Research University Grant Scheme (Project No. 03-03-11-1438RU). We thank the Department of Forestry, Malacca, Malaysia, for allowing us to carry out this study at the Sungai Udang Recreational Forest in Malacca.


  1. Applied Biosystems (2011) Real-time PCR: understanding Ct. Accessed 26 May 2012
  2. Barden A, Anak NA, Mulliken T, Song M (2000) Heart of the matter: agarwood use and trade and CITES implementation for Aquilaria malaccencis. TRAFFIC International, CambridgeGoogle Scholar
  3. Budge GE, Shaw MW, Colyer A, Pietravalle S, Boonham N (2009) Molecular tools to investigate Rhizoctonia solani distribution in soil. Plant Pathol 58:1071–1080CrossRefGoogle Scholar
  4. CITES (2011) Appendix II of convention on international trade in endangered species of wild fauna and flora. Accessed 26 March 2012
  5. Cui J, Wang C, Guo S, Yang L, Xiao P, Wang M (2013) Evaluation of fungus-induced agilawood from Aquilaria sinensis in China. Symbiosis 60:37–44CrossRefGoogle Scholar
  6. Espy MJ, Uhl JR, Mitchell S, Thorvilson JN, Svien KA, Wold AD, Smith TF (2000) Diagnosis of herpes simplex virus infections in the clinical laboratory by Lightcycler PCR. J Clin Microbiol 38:795–799Google Scholar
  7. Gao ZH, Wei JH, Yang Y, Zhang Z, Xiong HY, Zhao WT (2012) Identification of conserved and novel microRNAs in Aquilaria sinensis based on small RNA sequencing and transcriptome sequence data. Gene 505:167–175CrossRefGoogle Scholar
  8. Haugland RA, Siefring SC, Wymer LJ, Brenner KP, Dufour AP (2005) Comparison of Enterococcus density measurements by quantitative polymerase chain reaction and membrane filter culture analysis at two freshwater recreational beaches. Water Res 39:559–568CrossRefGoogle Scholar
  9. Henson JM, French R (1993) The polymerase chain reaction and plant disease diagnosis. Annu Rev Phytopathol 31:81–109CrossRefGoogle Scholar
  10. Jong PL, Tsan P, Mohamed R (2014) Gas chromatography-mass spectrometry analysis of agarwood extracts from mature and juvenile Aquilaria malaccensis. Int J Agric Biol 16:644–648Google Scholar
  11. Ke D, Ménard C, Picard FJ, Boissinot M, Ouellette M, Roy PH, Bergeron MG (2000) Development of conventional and real-time PCR assays for the rapid detection of group B Streptococci. Clin Chem 46:324–331Google Scholar
  12. Kodsueb R, McKenzie EHC, Lumyong S, Hyde KD (2008) Fungal succession on woody litter of Magnolia liliifera (Magnoliaceae). Fungal Divers 30:55–72Google Scholar
  13. Lin MH, Chen TC, Kuo TT, Tseng CC, Tseng CP (2000) Real-time PCR for quantitative detection of Toxoplasma gondii. J Clin Microbiol 38:4121–4125Google Scholar
  14. Liu YY, Chen HQ, Yang Y, Zhang Z, Wei JH, Meng H, Chen WP, Feng JD, Gan BC, Chen XY, Gao ZH, Huang JQ, Chen B, Chen HJ (2013) Whole-tree agarwood-inducing technique: an efficient novel technique for producing high-quality agarwood in cultivated Aquilaria sinensis trees. Molecules 18:3086–3106CrossRefGoogle Scholar
  15. Liu X, Gong J (2012) Revealing the diversity and quantity of peritrich ciliates in environmental samples using specific primer-based PCR and quantitative PCR. Microbes Environ 27:497–503CrossRefGoogle Scholar
  16. Loeffler J, Henke N, Hebart H, Schmidt D, Hagmeyer L, Schumacher U, Einsele H (2000) Quantification of fungal DNA by using fluorescence resonance energy transfer and the lightcycler system. J Clin Microbiol 38:586–590Google Scholar
  17. Mohamed R, Jong PL, Zali MS (2010) Fungal diversity in wounded stems of Aquilaria malaccensis. Fungal Divers 43:67–74CrossRefGoogle Scholar
  18. Mohamed R, Jong PL, Kamziah AK (2014) Fungi inoculation induced agarwood formation in young Aquilaria malaccensis trees in the nursery. J For Res 21:201–204CrossRefGoogle Scholar
  19. Morrison TB, Weis JJ, Wittwer CT (1998) Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques 24:954–962Google Scholar
  20. Naef R (2011) The volatile and semi-volatile constituents of agarwood, the infected heartwood of Aquilaria species: a review. Flavour Fragr J 26:73–89CrossRefGoogle Scholar
  21. Ng LT, Chang YS, Azizol AK (1997) A review on agar (gaharu) producing Aquilaria species. J Trop For Prod 2:272–285Google Scholar
  22. Nitsche A, Steuer N, Schmidt CA, Landt O, Siegert W (1999) Different real-time PCR formats compared for the quantitative detection of human cytomegalovirus DNA. Clin Chem 45:1932–1937Google Scholar
  23. Nobuchi T, Siripatanadilok SA (2008) The formation of wood in tropical forest trees. In: Nobuchi T, Mohd Hamami S (eds) Cytological observations of Aquilaria crassna wood associated with the formation of aloeswood. UPM Press, Serdang, pp 147–160Google Scholar
  24. Oldfield S, Lusty C, MacKinven A (1998) The world list of threatened trees. World Conservation Press, CambridgeGoogle Scholar
  25. Pojanagaroon S, Kaewrak C (2005) Mechanical methods to stimulate aloes wood formation in Aquilaria crassna Pierre Ex H. LEC. (Kristsana) trees. In: Jatisatienr A, Paratasilpin T, Elliott S, Anusarnsunthorn V, Wedge D, Craker LE, Gardner ZE (eds) III WOCMAP congress on medicinal and aromatic plants—volume 2: conservation, cultivation and sustainable use of medicinal and aromatic plants. ISHS Acta Horticulturae, Chiang MaiGoogle Scholar
  26. Premalatha K, Kalra A (2013) Molecular phylogenetic identification of endophytic fungi isolated from resinous and healthy wood of Aquilaria malaccensis, a red listed and highly exploited medicinal tree. Fungal Ecol 6:205–211CrossRefGoogle Scholar
  27. Promputtha I, Lumyong S, Lumyong P, McKenzie EHC, Hyde KD (2002) Fungal succession on senescent leaves of Manglietia garrettii in Doi Suthep-Pui National Park, northern Thailand. Fungal Divers 10:89–100Google Scholar
  28. Sivichai S, Jones EBG, Hywel-Jones N (2002) Fungal colonisation of wood in a freshwater stream at Tad Ta Phu, Khao Yai National Park, Thailand. In: Hyde KD, Jones EBG (eds) Fungal succession. Fungal Divers, vol 10, pp 113–129Google Scholar
  29. Soehartono T, Mardiastuti A (1997) The current trade in gaharu in West Kalimatan. Biodivers Indones 1:1–10Google Scholar
  30. Somrithipol S, Chatmala I, Jones EBG (2002) Cirrenalia nigrospora sp nov and C tropicalis from Thailand. Nova Hedwigia 75:477–485CrossRefGoogle Scholar
  31. Tamuli P, Boruah P, Nath SC, Samanta R (2000) Fungi from diseased agarwood tree (Aquilaria agallocha Roxb.): two new records. Adv For Res India 22:182–187Google Scholar
  32. Tamuli P, Boruah P, Nath SC, Leclercq P (2005) Essential oil of eaglewood tree: a product of pathogenesis. J Essent Oil Res 17:601–604CrossRefGoogle Scholar
  33. Taniguchi A, Onishi H, Eguchi M (2011) Quantitative PCR assay for the detection of the parasitic ciliate Cryptocaryon irritans. Fish Sci 77:607–613CrossRefGoogle Scholar
  34. Wittwer CT, Herrmann MG, Moss AA, Rasmussen RP (1997) Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques 22:130–134Google Scholar
  35. Wong MT, Siah CH, Faridah QZ, Mohamed R (2013) Characterization of wound-responsive genes in Aquilaria malaccensis. J Plant Biochem Biotechnol 22(2):168–175CrossRefGoogle Scholar
  36. Woodhall JW, Laurenson L, Peters JC (2012) First report of Rhizoctonia solani anastomosis group 5 (AG5) in wheat in the UK. New Dis Rep 26:9CrossRefGoogle Scholar
  37. Zhu F, Massana R, Not F, Marie D, Vaulot D (2005) Mapping of picoeucaryotes in marine ecosystems with quantitative PCR of the 18S rRNA gene. FEMS Microbiol Ecol 52:79–92CrossRefGoogle Scholar
  38. Zulak KG, Bohlmann J (2010) Terpenoid biosynthesis and specialized vascular cells of conifer defense. J Integr Plant Biol 52:86–97CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Rozi Mohamed
    • 1
    Email author
  • Phai Lee Jong
    • 1
  • Ismail Nurul Irdayu
    • 1
  1. 1.Forest Biotech Laboratory, Department of Forest Management, Faculty of ForestryUniversiti Putra Malaysia (UPM)SerdangMalaysia

Personalised recommendations