African Journal of Microbiology Research Vol. 7(7), pp. 577-585, 12 February, 2013 Available online at http://www.academicjournals.org/AJMR DOI: 10.5897/AJMR12.1727 ISSN 1996-0808 ©2013 Academic Journals Full Length Research Paper Acaricidal activity of Hypocrella raciborskii Zimm. (Hypocreales: Clavicipitaceae) crude extract and some pure compounds on Tetranychus urticae Koch (Acari: Tetranychidae) Suradet Buttachon1, Angsumarn Chandrapatya1*, Winanda Himaman2 and Anake Kijjoa3 1Departmant of Entomology, Faculty of Agriculture, Kasetsart University, Chatuchak, Bangkok 10900, Thailand. 2Forest and Plant Conservation Research Office, Department of National Parks, Wildlife and Plant Conservation, Bangkok 10900, Thailand. 3ICBAS - Instituto de Ciências Biomédicas de Abel Salazar - CIIMAR, Universidade do Porto, 4099-003, Porto, Portugal. Accepted 7 February, 2013 The acaricidal activity of secondary metabolites from an entomopathogenic fungus, Hypocrella raciborskii Zimm. in controlling two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae) is explored in this study as a new agent for pest control. The crude extract was prepared in ethyl acetate. Secondary metabolites were elucidated using Nuclear Magnetic Resonance and High- Resolution Mass Spectrometry analysis. The major secondary metabolites of the crude extract were ergosterol, dustanin (15α, 22-dihydroxyhopane) and 3β-acetoxy-15α,22-dihydroxyhopane. The crude extract and pure compounds were used to determine their toxicity, repellency and oviposition deterrence against T. urticae, under the laboratory conditions. Crude extract of 3% w/v showed a strong contact toxicity (97% mortality, one day after treatment) and residual toxicity (80%, three days after treatment). Ergosterol induced 75% mite mortality in a residual toxicity bioassay. Females topically sprayed with 3% crude extract had their egg production reduced by 97%. Direct spraying of 3β-acetoxy- 15α, 22-dihydroxyhopane and dustanin reduced egg production of T. urticae by 71 and 70%, respectively. Both crude extract and pure compounds had relatively slight repellency effect on T. urticae. Key words: Fungal metabolites, bioassay, two-spotted spider mite. INTRODUCTION Fungi from genus Hypocrella are pathogens which tropical region, especially in the moist old-growth forests belong to Division Ascomycota, Family Clavicipitaceae. where the epizootics normally occur on their hosts They are known as entomopathogens infesting scale (Hywel-Jones and Evans, 1993; Chaverri et al., 2008). insects (Coccidae, Homoptera) and whiteflies Several entomopathogenic fungi produce secondary (Aleyrodidae, Homoptera) and are quite common in metabolites, many of which are reported to exhibit insecticidal, phytotoxic, cytotoxic, antiviral, antifungal antitumor and antibacterial activities (Krasnoff et al., 1996; Pelaez, 2004; Jin-Ming, 2006). However, there is a *Corresponding author. E-mail: [email protected]. Tel: 662-942- paucity of information regarding secondary metabolites or 8350. Fax: 662-561-4882. toxins produced by Hypocrella due to the lack of available 578 Afr. J. Microbiol. Res. cultures for studies (Isaka et al., 2003). Zhenjun and Extraction of the fungal metabolites Lown (1990) isolated hypocrellins A and B which are H. raciborskii was cultured on potato dextrose agar (PDA) plates pigments from Hypocrella bambusae (Berk. & Broome) and incubated at 27 to 28°C for 7 days. Ten fungal mats (1 cm in Petch found in People's Republic of China. These sub- diameter) were taken from each plate and placed in an Erlenmeyer stances showed potential to be used as photodynamic flask containing 100 ml of malt extract broth (MEB) before being therapy of tumors. Consequently, hypocrellins A, B and C incubated at 27 to 28°C in the rotary shaker set at 180 rpm for four were identified as secondary metabolites from Hypocrella days. These fungal biomasses were used as inoculums throughout sp. collected in China where hypocrellins A exhibited the study. A plastic bag containing 200 g of cooking rice that was autoclaved at 121°C for 15 min was inoculated with 3 µl broth photosensitive nematicidal activity and hypocrellins B and culture and incubated at 28°C for 30 days (total of 120 bags). Ten C showed only photodynamic activity (Jin-Ming, 2006). moldy rice bags were then emptied into 5 L Erlenmeyer flasks and Watts et al. (2003) demonstrated that extracts of 2500 ml of ethyl acetate were added to each flask. All flasks were Hypocrella raciborski Zimm., Hypocrella discoidea (Berk. kept for three days before the fungal materials and solvents were & Broome) Sacc. and Hypocrella tamurai (Henn.) had cy- filtered through a Bucher funnel using #2 Whatman filter paper. The totoxic activity against sf9 insect cell lines with ID50 value two aqueous layers were then separated using a separating funnel and the ethyl acetate solution was concentrated at reduced ≤ 10 µg/ml. Isaka et al. (2009) investigated the secondary pressure yielding 52.34 g of crude extract. About 15 g of crude metabolites of Hypocrella and their Ashersonia were set aside to be tested in an acaricidal activity bioassay. Then anamorphs collected in Thailand and reported 3 hopane 37.34 g was dissolved in 500 ml of a 4:1 mixture of chloroform and triterpenes which are zeorin (6α,22-dihydroxyhopane), ethyl acetate and then washed with water (3 × 500 ml). The organic dustanin (15α,22- dihydroxyhopane) and 3β-acetoxy-15α, layer was dried with anhydrous Na2SO4, filtered and evaporated 22- dihydroxyhopane) and postulated that hopane triter- under reduced pressure to give 36.38 g of crude chloroform extract, which was applied on a column chromatography of silica gel (380 g) penes may be suitable to be used as chemotaxonomic and the fractions were collected as follows: fractions 1-77 (CHCl3- markers for Hypocrella and their Ashersonia anamorphs. petrol, 1:1), fractions 78 to 151 (CHCl3-petrol, 7:3), fractions 152- Recently, Isaka et al. (2011) reported seven new 194 (CHCl3-petrol, 9:1), fractions 195 to 209 (CHCl3), fractions 210- lanostane-type triterpenes hypocrellols A-G and six new 245 (CHCl3-Me2CO, 9:1), fractions 246 to 269 (CHCl3-Me2CO, 7:3), hopane-type triterpenes from Hypocrella sp. BCC14524 fractions 270-294 (CHCl3-Me2CO, 3:7) and fraction 295 (MeOH). collected in Thailand where some biological activities Fractions 42-52 (2.2 g) were combined and precipitated from a mixture of CHCl3 and petrol to give a white solid of ergosterol (0.8 were also mentioned such as growth inhibition against g). Fractions 109-118 (0.79 g) and fractions 119 to 153 (4.7 g) were Mycobacterium tuberculosis and cytotoxic activity against combined and precipitated from a mixture of CHCl3 and petrol to human cancer cell lines. However, acaricidal activity of give white solid of dustanin (15α, 22- dihydroxyhopane) (5.5 g). these triterpenes is not yet investigated. Fractions 154-159 (1.12 g) and fractions 160-182 (2.78 g) were Mortality of insects induced by ascomycetous fungi is combined and precipitated from a mixture of MeOH and petrol to give white solid of 3β-acetoxy-15α, 22-dihydroxyhopane (2.78 g). thought to be caused by toxic substances released by the fungus (Roberts, 1981; Evans, 1988). Hence, fungal se- condary metabolites may play an important role in fungi- Stock colony of Tetranychus urticae insect interactions and such compounds may have insec- A colony of T. urticae was obtained from Department of Agriculture, ticidal properties (Mongkolsamrit et al., 2010). In this Ministry of Agriculture and Cooperatives, Bangkok, Thailand. All study, the secondary metabolites of H. raciborskii Zimm. mites were cultured on mulberry leaves (Morus alba L.) placed on were elucidated and both crude extract and pure com- moistened cotton pads resting on sponges in plastic boxes. The pounds were investigated for the acaricidal activity on the mulberry leaves were examined every few days and replaced with two-spotted spider mite, Tetranychus urticae Koch (Acari: fresh ones when over-crowding of mites and yellow leaves were observed. The colonies were maintained at room temperature (27 Tetranychidae), which is one of the important phytopha- to 28°C, 60 to 65% RH). Adult females were placed on new leaves gous mite infesting several economic plants in Thailand. for 10 days and newly emerged adults were used for each The information gained from this study will be useful for experiment. All bioassays were conducted and carried out under further development of alternative pesticides for mite control. the same environmental conditions as the culture. MATERIALS AND METHODS Bioassays Fungal extract The test materials included: the crude extract that was prepared at 0.1, 0.5, 1, 2 and 3% (w/v) using 95% ethanol as the solvent, 3 pure Fungal material compounds (ergosterol, dustanin (15α,22-dihydroxyhopane) and 3β-acetoxy-15α,22-dihydroxyhopane) prepared at 0.5% (w/v), The fungus used in this study was isolated from a scale insect dicofol and neem extract (0.1%) were used as a positive and a collected in Kui Buri National Park, Prachuap Khiri Khan Province, standard, respectively. Thailand in January 2009 and identified by Miss Winanda Himaman. Fungal sample with accession number IF 01781 has Residual toxicity been deposited in the Forest Entomology and Microbiology Group, Forest and Wild Plant Conservation Research Office, Department of An aliquot of 50 μl of each tested material was painted on the upper National Parks, Wildlife and Plant Conservation, Bangkok, surface of each mulberry leaf disc (2 cm in diameter). Mulberry leaf Thailand. discs painted with ethanol 95% were used as the control while Buttachon et al. 579 those painted with dicofol and neem extract served as a positive control. At the end of this experiment, only 10 to 34.5% of and a standard, respectively. Dried leaf discs were then placed on mites treated with 0.5 to 3% crude extracts remained moistened cotton pads in glass Petri dishes (9 cm in diameter) and alive which were significantly different from 98 to 99% of 20 T. urticae females (two to three days-old) were transferred from the culture with a small artist’s brush into the center of each leaf survival mites in the controls.