South African Journal of Botany 76 (2010) 701–709 www.elsevier.com/locate/sajb Volatiles associated with different flower stages and leaves of Acacia cyclops and their potential role as host attractants for Dasineura dielsi (Diptera: Cecidomyiidae) ⁎ M.J. Kotze a, , A. Jürgens b, S.D. Johnson b, J.H. Hoffmann a a Zoology Department, University of Cape Town, Rondebosch 7701, South Africa b School of Biological and Conservation Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa Received 19 June 2010; received in revised form 26 July 2010; accepted 27 July 2010 Abstract Acacia cyclops (Fabaceae) is an Australian species which was introduced into South Africa in the nineteenth century. Because of its invasive status in South Africa, a gall midge, Dasineura dielsi (Diptera: Cecidomyiidae), was released in 2001 in order to impact its reproduction by inducing galls on the flowers and thereby preventing seed set. Nothing is known about the cues used by D. dielsi for locating its host flowers. As part of an initial investigation into whether or not chemical cues might play a role in host finding, we analysed headspace samples of Acacia cyclops volatiles from leaves and reproductive parts at different stages (early bud, late bud, early flowering, and senescing flowering stages) using gas chromatography–mass spectrometry (GC–MS). In total, 72 different compounds were detected of which 62 were identified. The analyses showed that open flowers, the stage used by D. dielsi for oviposition, and yellow buds had similar odour compositions with (Z)-3-hexen-1-ol acetate, 4-oxoisophorone, (Z)-β-ocimene, an unknown aliphatic compound, heptadecane, and nonadecane dominating in open flowers. Leaf volatiles were distinct from those in the reproductive plant parts by their high relative amount of (Z)-β-ocimene. (Z)-3-Hexen-1-ol acetate had its maximum relative amount in the green bud samples and was much lower in the later floral stages. In contrast, 4-oxoisophorone peaked in yellow buds and open flowers with little or none of it found in younger or older stages. The volatile compounds of the different flower stages and leaves are discussed in relation to their potential role as attractants used by the biocontrol agent D. dielsi to locate its host plant. © 2010 SAAB. Published by Elsevier B.V. All rights reserved. Keywords: Acacia cyclops; Biological control; Dasineura dielsi; Headspace analysis; Spatial odour patterns; 4-Oxoisophorone 1. Introduction biocontrol agent can maintain its populations in only one, or a limited number of closely related species of host plants Biological control of weeds is an important discipline of (Zimmermann et al., 2004). Biologically relevant testing methods classical biological control and is practiced in many countries are based on investigations of the physiological, morphological, (Palmer et al., 2010; Timmons, 2005; Zimmermann et al., 2004)to phenological, entomological, and chemical bases of host restric- protect agricultural crops and conservation of biodiversity tion (McEvoy, 1996). Host chemistry however has not often been (Zimmermann et al., 2004). It is a practice in which the alien used in determining the host range of a biocontrol agent, and invasive plants' natural enemies e.g. host-specific plant-feeding therefore the suitability of an insect as a biocontrol agent (Jordon- insects, mites and pathogens are transferred from their country of Thaden and Louda, 2003). After an investigation into the origin and after rigorous host-specificity tests are released into the chemistry of thistles to determine why a biocontrol agent expanded country where the plants have become a problem (Zimmermann its host range from exotic thistles to native thistles, Jordon-Thaden et al., 2004). Host-specificity tests ensure that the introduced and Louda (2003) suggested that consideration of contemporary chemical profiles to supplement host-specificity tests based on ⁎ Corresponding author. phylogenetic relationships will contribute to a clearer prediction of E-mail address: [email protected] (M.J. Kotze). potential host plant use and ecological impacts. 0254-6299/$ - see front matter © 2010 SAAB. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.sajb.2010.07.024 702 M.J. Kotze et al. / South African Journal of Botany 76 (2010) 701–709 Acacia cyclops A. Cunn. ex G. Don (Fabaceae), originating At the same time, little is known about the volatile chemistry of in south-western Australia, became invasive after it was most species of Acacia (Seigler, 2003) whose comprehensive introduced into South Africa in the early 1800s for dune report on the phytochemistry of Acacia, only mentions plant stabilisation (Adair, 2005; Impson et al., 2004; Moseley et al., volatiles from A. farnesiana. Floral volatiles have been described 2009). Reproduction is entirely sexual and relies on the from A. farnesiana, A. berlandieri,andA. rigidula (all three production of copious quantities of seeds which accumulate native to North America) (Flath et al., 1983), A. praecox, A. caven, as large seed banks and which remain viable for many years A. aroma (all three from Argentina) (Lamarque et al., 1998; (Richardson and Kluge, 2008). Today it has invader status Zygadlo et al., 1996), and from A. karroo, an African species (category 2) in South Africa as it impacts negatively on the (Kaiser, 1997). biodiversity of the environment (Henderson, 2001). It also Many earlier studies of plant volatiles, such as the one by grows in and reduces water production in catchment areas, Flath et al. (1983), used a vacuum steam distillation, hydro- amongst other impacts (Richardson and Kluge, 2008). Contrary distillation or solvent extraction method to extract the volatiles. to the many negative ecological aspects, it also has economic All these techniques however remove substances from the plant benefits as it is an excellent source of firewood, and as such it tissue itself, including compounds that may not be emitted by generates considerable income for lower income communities the plant into the surrounding air. Furthermore, such methods in South Africa (Dennill and Donnelly, 1991; Impson et al., may dilute, or entirely miss, emitted volatiles, especially those 2004). For this reason, a compromise was reached by releasing that are synthesized continuously. More reliable information on biological control agents that reduce seed set without affecting the occurrence of naturally emitted blends of volatiles can be vegetative growth and, to date, two complementary biocontrol obtained using dynamic headspace sampling, whereby volatiles agents have been released in South Africa: a weevil, are trapped on a polymer and then extracted with solvent or Melanterius servulus (Coleoptera: Curculionidae) which feeds thermally desorbed. The latter is a highly sensitive method that directly on seeds (Dennill and Donnelly, 1991; Impson et al., was used in the present investigation to characterize volatile 2004) and a midge, Dasineura dielsi Rübsaamen (Diptera: emissions from leaves, green buds, yellow buds, open flowers Cecidomyiidae), which induces galls on the flowers and thereby and senescing flowers of A. cyclops in order to identify potential prevents seed set (Adair, 2005). chemical cues that could be used by D. dielsi to locate flowers. Dasineura dielsi, which also originated from Australia (Kolesik et al., 2005) is short lived as an adult (approximately 2. Methods 2 days) with its total life cycle, from egg stage through three larval stages and a pupal stage, taking place in the gall (Adair, 2.1. Collection of volatile samples from different flowering 2003, 2005; Kolesik et al., 2005). Soon after emergence from stages and leaves the gall, mating takes place and the female starts searching for oviposition sites, depositing her eggs on the perianth tube of the From the perspective of the objectives of this study, in-situ ovaries of open A. cyclops flower heads (Adair, 2003, 2005). headspace collection of A. cyclops flower volatiles was not Gall formation commences with larval feeding. Inflorescences feasible due to the way the inflorescences are borne on the of A. cyclops consist of a number of flowers. Each flower in stems. The globose flower heads occur sporadically, and are which eggs have been laid develops into a multi-chambered individually interspersed among the leaves. Preliminary head- gall and the entire inflorescence forms a spherical cluster of space samples inadvertently included scent from leaves and a protruding, convoluted and elongated galls (Adair, 2003, whole range of flower stages with the flower scent concentra- 2005). tion after GC–MS analysis of the samples too weak to Acacia cyclops is the primary host of D. dielsi (Adair, 2003, confidently compare the scent compositions to those of the 2005). The extended flowering period of A. cyclops sustains the other flower stages. Therefore, despite the risk of obtaining high multivoltine life strategy of D. dielsi which has up to five levels of green leaf volatiles characteristic of wounded foliage generations per annum (Adair, 2003, 2005; Henderson, 2001). (Arimura et al., 2001; Grison et al., 1999), it was decided to pick Other Australian Acacia species invasive in South Africa, such the flowers, buds and leaves separately for headspace volatile as A. melanoxylon, A. longifolia and A. saligna, are occasionally collection. used as hosts by D. dielsi (Post et al., 2010). Considering that Plant cuttings containing leaves, and buds and flowers