Rapid detection of Ganoderma-infected oil palms by microwave ergosterol extraction with HPLC and TLC M.S. Muniroh a , M. Sariah a, ⁎, M.A. Zainal Abidin a , N. Lima b , R.R.M. Paterson b a Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia b IBB — Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, 4710-057 Braga, Portugal abstract article info Article history: Received 17 July 2013 Received in revised form 13 March 2014 Accepted 16 March 2014 Available online 26 March 2014 Keywords: Microwave assisted extraction Ganoderma Ergosterol Oil palm TLC HPLC Detection of basal stem rot (BSR) by Ganoderma of oil palms was based on foliar symptoms and production of basidiomata. Enzyme-Linked Immunosorbent Assays-Polyclonal Antibody (ELISA-PAB) and PCR have been pro- posed as early detection methods for the disease. These techniques are complex, time consuming and have accu- racy limitations. An ergosterol method was developed which correlated well with the degree of infection in oil palms, including samples growing in plantations. However, the method was capable of being optimised. This cur- rent study was designed to develop a simpler, more rapid and efficient ergosterol method with utility in the field that involved the use of microwave extraction. The optimised procedure involved extracting a small amount of Ganoderma, or Ganoderma-infected oil palm suspended in low volumes of solvent followed by irradiation in a conventional microwave oven at 70 °C and medium high power for 30 s, resulting in simultaneous extraction and saponification. Ergosterol was detected by thin layer chromatography (TLC) and quantified using high per- formance liquid chromatography with diode array detection. The TLC method was novel and provided a simple, inexpensive method with utility in the field. The new method was particularly effective at extracting high yields of ergosterol from infected oil palm and enables rapid analysis of field samples on site, allowing infected oil palms to be treated or culled very rapidly. Some limitations of the method are discussed herein. The procedures lend themselves to controlling the disease more effectively and allowing more effective use of land currently employed to grow oil palms, thereby reducing pressure to develop new plantations. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Palm oil is a major commodity used in ca. 30% of foods and cos- metics. Increasingly the oil is used as a biofuel and contributes consider- ably to the economies of many nations and particularly Malaysia and Indonesia (Paterson et al., 2013). Oil palms suffer from the major dis- ease, basal stem rot (BSR), caused by the white rot fungus Ganoderma. According to Idris et al. (2011), 632 out of 1061 estates (59.57%) that responded to a survey, reported incidences of BSR disease. The average incidence of BSR in Malaysia was 3.71% with an affected area of 59,148 ha. The economic losses are between $68 and $455 million a year in Malaysia alone (Chong, 2012). The disease is currently detected based on development of foliar symptoms and production of basidiomata in mature fields. However, visible symptoms indicate that the (a) palms are already at a serious stage of infection and (b) fungus has killed approximately half of the basal tissue. More effective methods to detect the disease early are required urgently so that remedial action (e.g. culling) can be taken quickly to prevent spread to healthy oil palms causing even more dis- ease. Better control of the disease may create ecological benefits from reduced pressure to create new plantations as the yields from existing fields increase. The Enzyme-Linked Immunosorbent Assay-Polyclonal Antibody (ELISA-PAB) and PCR have been proposed (Mohd Aswad et al., 2011), but these methods are not applicable for large scale field monitoring be- cause they are complex, time consuming and have accuracy limitations. For example, PCR can be subjected to inhibition (Paterson, 2007a) and ELISA-PAB suffers from cross reactivity (Idris and Rafidah, 2008). Ganoderma boninense, the causal fungal pathogen of BSR in oil palm, col- onises the roots and leads to an incremental increase in fungal biomass during disease development. Also, the fungus appears to colonise from one oil palm to another by producing vast numbers of spores (Sanderson, 2005). One of the common cell membrane components, er- gosterol, is sufficiently specific(Mille-Lindblom et al., 2006) that it is used to quantify fungi in soil (Grant and West, 1986; Frostegard and Baath, 1996), roots (Bindler et al., 1988), cereal grains (Seitz et al., 1977), and decaying plant material (Newell et al., 1988). The compound is used to quantify ectomycorrhizae (Salmanowicz and Nylund, 1988; Journal of Microbiological Methods 100 (2014) 143–147 ⁎ Corresponding author. Tel.: +60 3 894 8947; fax: +60 3 89381014. E-mail address: muniroh_mdsaad@yahoo.com (M. Sariah). http://dx.doi.org/10.1016/j.mimet.2014.03.005 0167-7012/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth