Lack of an Induced Response Following Fire and Herbivory of Two Chemotypes of Melaleuca Quinquenervia and Its Effect on Two Biol

Biological Control 39 (2006) 154–161 www.elsevier.com/locate/ybcon

Lack of an induced response following ﬁre and herbivory of two chemotypes of Melaleuca quinquenervia and its eﬀect on two biological control agents

G.S. Wheeler a,*, K.M. Ordung b

a USDA/ARS, Invasive Plant Research Lab and University of Florida, 3225 College Ave., Ft. Lauderdale, FL 33314, USA b SCA/Americorps, 3225 College Ave., Ft. Lauderdale, FL 33314, USA

Received 4 January 2006; accepted 31 May 2006 Available online 9 June 2006

Abstract

Induced responses following damage from herbivory or fire may reduce the nutritional quality of plants for subsequent herbivores. If compatible, the combination of fire and biological control has the potential of effectively controlling invasive weeds. Potentially plants subjected to previous herbivory from biological control agents or damage from fire, either natural or deliberate, will be less susceptible to biological control because of decreases in the nutritional quality of the plant from changes in physical defenses and/or accumulated secondary compounds. In the fire-adapted species, Melaleuca quinquenervia, the impact of previous herbivory by the weevil biological control agent Oxyops vitiosa and burning by a propane torch was studied on the first replacement leaves produced following damage. Two chemical variants or chemotypes of M. quinquenervia responded similarly with decreased leaf toughness in leaves produced following burning; little change was found in the levels of foliar nitrogen or volatile constituents in response to treatments. Moreover, survival decreased when the burn replacement leaves were fed to O. vitiosa larvae and Boreioglycaspis melaleucae psyllid nymphs; growth and development time were not affected. These results indicate that although survival will decrease in both species fed the first replacement leaves following burning, previous herbivory by weevils is compatible with subsequent damage by both biological control agents. 2006 Elsevier Inc. All rights reserved.

Keywords: Fire-adapted species; Induction of secondary metabolites; Induced responses; Insect nutrition; Integrated control; Plant quality; Secondary metabolites; Terpenoids; Leaf toughness

1. Introduction Gorchov, 2000). The induced chemical (e.g., secondary metabolites) and physical (e.g., toughness) responses to New foliage produced in plants following severe herbi- damage are well-documented in many plant systems (e.g., vore or fire damage can have dramatically different nutri- Tallamy and Raupp, 1991; Karban and Baldwin, 1997; tional suitability for subsequent herbivores. Immediately Agrawal et al., 1999). Generally, induced responses in following severe herbivory or fire damage, insect presence plants result in reduced nutritional value (Schultz and may decline markedly because of the nature of the damage Baldwin, 1982). This reduced nutritional value of the foli- and the lack of suitable food. However, the replacement age can be a result of several factors including increased foliage produced by surviving plants after severe damage accumulation of secondary metabolites (reviewed by Kar- may be highly attractive to specialized herbivores (Carroll ban and Baldwin, 1997) or increased physical resistance and Hoffman, 1980; Vieira et al., 1996; Negroń-Ortiz and such as leaf toughness (Schultz and Baldwin, 1982; but see Vieira et al., 1996). Fire damage can also change the * Corresponding author. Fax: +1 954 476 9169. nutritional quality of the surviving plants due to increased E-mail address: [email protected] (G.S. Wheeler). availability of soil nutrients and sunlight (Briese, 1996;

Rieske, 2002). However, not all responses to damage have observed exploiting the foliage produced following natural been shown to protect the plant against herbivory. Feeding M. quinquenervia fires and following severe herbivore dam- on previously damaged plants may increase, decrease, or age by conspecifics (Wheeler personal observation). have no effect on the subsequent herbivore performance In many aromatic plant species, as in the family Myrta- (Coleman and Jones, 1991; Karban and Baldwin, 1997; ceae (Brophy and Doran, 1996) different chemical variants Nyka¨nen and Koricheva, 2004). are well known and have been identified as distinct chemo- Different weed control techniques have evolved indepen- types. Examples of chemotype or secondary compound dently and although integration may significantly improve variability in invasive weeds targeted for biological control weed management, little information is available describing include Euphorbia esula L. Euphorbiaceae (Holden and their compatibility (Hatcher and Melander, 2003). While Mahlberg, 1992), Hypericum perforatum L. Clusiaceae little work has been conducted integrating natural ecologi- (Sirvent et al., 2002), Lantana camara L. Verbenaceae cal processes such as fire into biological control implemen- (Randrianalijaona et al., 2005), and Senecio jacobaea tation (Briese, 1996; Paynter and Flanagan, 2004), L. Asteraceae (Macel et al., 2002). The variation in constit- biological control has been studied in combination with uents studied from the leaves of M. quinquenervia in its pesticides (Paynter and Flanagan, 2004), plant growth reg- native range (Ireland et al., 2002) and in Florida (Wheeler, ulators (Van and Center, 1994), mowing (Tipping, 1991), 2006) indicates that at least two chemotypes exist within and mechanical harvesting (Sheldon and O’Bryan, 1996). the species. One chemotype is referred to here by its pri- However, the plant response to damage may mitigate bio- mary sesquiterpene E-nerolidol (chemotype I) and another logical control when these responses decrease the nutrition- is referred to here by a primary sesquiterpene viridiflorol al quality or quantity of the food to be eaten by biological (chemotype II). These variable levels of secondary metabo- control agents. Leaves produced by long-lived trees follow- lites have a significant impact on the performance and ing these damage events may not have the same physical fecundity of these biological control agents (Wheeler and and chemical qualities as the undamaged leaves. Changes Ordung, 2005; Wheeler, 2006). Moreover, previous may be immediate and ephemeral (Schultz and Baldwin, research indicated that the weevil O. vitiosa had reduced 1982) or they may require months and persist for one or larval survival, performance, and fecundity when fed more years (Haukioja, 1980; Neuvonen and Haukioja, M. quinquenervia leaves of relatively high toughness 1984). These changes in plant quality can potentially influ- (Wheeler, 2001), and low nitrogen (Wheeler, 2001, 2003). ence biological control agent populations and their effects The purpose of this study was to determine the influence on the target weeds (Awmack and Leather, 2002; Schweit- of burning and herbivory on the plant quality factors leaf zer et al., 2004). toughness, nitrogen, and the quantitative and qualitative The environmental weed Melaleuca quinquenervia (Cav.) changes in secondary metabolites of replacement foliage S.T. Blake (Myrtaceae) is a fire-adapted species that threat- of both chemotypes of M. quinquenervia. The impact of ens the biodiversity of the south Florida ecosystem includ- these nutritional changes was assessed on the survival, ing the Everglades National Park and adjacent natural growth, and development of immature stages of the biolog- areas (FLEPPC Plant List Committee, 2003). This species ical control agents O. vitiosa and B. melaleucae. These was introduced as early as 1900 from Australia and was results are relevant to the compatibility of fire as a natural planted widely in Florida as an ornamental and for erosion phenomenon or as a deliberate weed management tool and control (Turner et al., 1998). Currently, M. quinquenervia herbivory by biological control agents. Additionally, they occupies more than 200,000 ha in South Florida and infests examine the compatibility of two biological control agents a variety of habitats (Bodle et al., 1994). In its native Aus- introduced to control M. quinquenervia. tralian range and in its Florida adventive range, this species experiences periodic fires that burn the bark and plant can- 2. Methods opy (Turner et al., 1998; Flowers, 1991). Following fire, large trees will produce new leaves that may be highly 2.1. Plants attractive to adapted herbivore species. Herbivore species that feed on fire-adapted hosts may have specialized physi- Melaleuca quinquenervia experimental plants were ologies or behaviors to exploit the replacement foliage pro- grown from cuttings taken from plants of known chemo- duced following fire (Everaerts et al., 2000; Suckling et al., type grown in a screenhouse located at the Invasive Plant 2001). Research Laboratory (IPRL) USDA/ARS in Fort Lauder- Biological control activities against M. quinquenervia dale, Florida, USA. The chemotype of the plants was have resulted in the release of three insect species the weevil determined by gas chromatography (GC) and gas chroma- Oxyops vitiosa Pascoe (Center et al., 2000), a psyllid Bore- tography/mass spectroscopy (GC/MS; see below). Plants ioglycaspis melaleucae Moore (Wineriter et al., 2003), and a (N = 16) of each chemotype were fertilized at 90 g/11.4 l gall fly Fergusonina turneri Taylor (Rayamajhi et al., 2002). pot in a slow-release ‘Southern’ formulation (Florikan All but the gall fly are widely established in Florida and 13-13-13, N-P-K; Sarasota, FL). This fertilizer level was impacting the host (Pratt et al., 2005; Pratt unpublished known from previous research (Wheeler, 2003) to produce data). Population increases of O. vitiosa have been M. quinquenervia plants with foliar nitrogen levels that 156 G.S. Wheeler, K.M. Ordung / Biological Control 39 (2006) 154–161 were representative of field conditions. As these plants mixed by vortexing for 30 s. The mixture was extracted apparently have high water requirements, growing fre- with an equal volume of CHCl3 and vortexed for 60 s. quently in flooded wetlands and freshwater marshes (Turn- To separate the different solvent layers, each sample was er et al., 1998), all plants received constant drip irrigation. centrifuged at 10,000 rpm for 10 min. An aliquot (200 ll) Once the plants were about 1 m tall they were subjected to of the CHCl3 layer was removed and dried over Na2SO4. the treatments described below. An internal standard n-tridecane (5 ng/ll) was added and Plants were damaged by either weevil larval feeding or each sample was analyzed by gas chromatography (GC) burning (N = 4). The damage treatments began in Decem- and compound identities were confirmed by GC/mass spec- ber, during the start of the south Florida winter/dry sea- troscopy (GC/MS). son. This schedule corresponded with the beginning of the period when plant tip growth is greatest (Van et al., 2.3. Gas chromatography 2002), field populations of weevil larvae are most abundant (Pratt et al., 2004), and M. quinquenervia fires are locally To quantify the foliar constituents, extracts were ana- common (Platt and Schwartz, 1990). Additionally, two lyzed with an Agilent (Hewlett-Packard) model 6890 GC. control treatments included undamaged plants grown Data collection, storage, and analysis were conducted with either inside and outside the same cage described below. the Agilent ChemStation (Wilmington, DE) data system. For the herbivory treatment, 50 3rd and 4th instar O. viti- Helium was used as a carrier gas at a linear flow rate of osa larvae were released on each plant and allowed to feed 37 cm/s. All samples were splitless, where the entire sample on all available tips for 1 week. More than 75% of the (1 ll) was injected on a fused silica capillary column (DB- available tips were damaged by the larvae. The plants sub- 17MS Agilent; 30 m · 0.32 mm i.d., 0.25 lm thick film). jected to the herbivory and cage control treatments were Injector temperature was 250 C and FID temperature caged for only the duration of the insect exposure (1 week). was 250 C. The oven temperature was held at 50 C for For the burn treatment, the methods follow those of Stein 2 min then increased at 8 C/min to 250 C where it was et al. (1992) where all leaves and branches were uniformly held for 10 min. ignited with a propane torch in an effort to simulate an To confirm these compound identities GC/MS was per- intense fire that might occur in the field. Tissues burned formed with an Agilent 6890 instrument fitted with either a until all fuels were consumed and the fire extinguished HP-5MS (Agilent, 30 m · 0.25 mm, 0.25 lm film thickness) itself. Following damage new plant growth was stimulated or a DB-17MS (J&W Scientific, 30 m · 0.32 mm, 0.25 lm in all plants by applying liquid fertilizer (Peter’s 20:20:20; thick film) FSOT column with helium at 36 or 42 cm/s N:P:K; 3 g/pot). New leaves were produced on all damaged (HP-5MS and DB-17MS, respectively) as a carrier gas, plants during the subsequent 3 months. injector port (split 1:20) at 250 C, mass selective detector (HP 5973) at 250 C (source) and 150 C (quad) with trans- 2.2. Chemical analysis fer line 280 C and ion source filament voltage of 70 eV. Component identification was made on the basis of mass Percent nitrogen was determined from the pooled sec- spectral fragmentation, retention index with n-paraffins, ond and third position leaves produced 3 months after comparison with authentic constituents when available, the treatments from four plants of each chemotype by and mass spectral and retention matching with commercial treatment combination. The leaves were dried for 2 d at libraries (NIST, Wiley, and Adams). 60 C, ground in a Wiley mill (60 mesh), and the nitrogen content was determined using a CHNS/O analyzer (Per- 2.4. Insects kin-Elmer 2400 series II). The organic analytical standard, acetanilide was analyzed before and after each sample. Weevil adults were collected from M. quinquenervia tip Leaf toughness was measured using a modified gram gauge leaves of undetermined chemotype located at IPRL, (Wheeler, 2001) that estimates the pressure required to USDA/ARS, Ft. Lauderdale, FL, USA. Eggs were puncture leaf tissues. Toughness estimates were collected obtained from weevil male and female pairs that were on leaves 1–10 counting from the leaf tip toward the base. maintained in Plexiglas cylindrical cages (30 · 15 cm) and Sub-samples (N = 4) were collected within each leaf that fed M. quinquenervia tip leaves of undetermined chemotype were pooled for analysis from four trees per chemotype as described previously (Wheeler, 2006). Larval perfor- and treatment combination. mance was determined beginning with neonates when fed The M. quinquenervia foliar organic constituents were leaves of the different chemotype · treatment combinations identified by standard methods (Wheeler et al., 2003). To (N = 20). All weevil larvae were reared individually in plas- obtain an estimate of the levels of volatile constituents, tic Petri dishes (15 · 2 cm) lined with moistened filter paper one tip (100 ± 5 mg) from each plant was collected and fro- and sealed with Parafilm to retain moisture. Experimental zen. Leaf components were extracted by a modified micro- leaves were replaced every 3 d. The use of excised leaves wave digestion where leaf samples were immersed in EtOH for insect bioassays has been shown to be a good replace- (1 ml) and irradiated (750 W) for 60 s. A 500-ll aliquot was ment for intact leaves (Janssen, 1993, 1994; Schmelz combined with an equal volume of water (d.i.) and then et al., 2001). The results from laboratory experiments have G.S. Wheeler, K.M. Ordung / Biological Control 39 (2006) 154–161 157 been shown to represent those collected from field experi- 3. Results ments when determining the influence of plant quality on immature insect performance (Koricheva et al., 1998). Pre- 3.1. Plant quality pupae were transferred to individual 30 ml plastic cups for pupation containing ground floral blocks (Smithers-Oasis Nitrogen levels (mean ± se) of the replacement leaves Co., Kent, OH, USA) (Wheeler and Zahniser, 2001). All produced after treatments were not influenced significantly rearing was conducted at 28 C 90% RH (measured inside by chemotypes (P > 0.8), treatments (P > 0.2), or their the Petri dish) and under a 14:10 h photoperiod. Data were interaction (P > 0.3). collected on survival, adult biomass (±0.1 mg fresh mass), Leaf toughness was influenced by chemotype, treat- and development time (±1.0 d) to reach pupation. ments, and the covariate leaf position and the interaction Psyllid adults were collected from M. quinquenervia tip between treatment and chemotype (Fig. 1). Subsequent leaves of undetermined chemotype located at IPRL, USDA/ARS, Ft. Lauderdale, FL as described previously (Wheeler and Ordung, 2005). Eggs were obtained from psyllid male and female pairs that were maintained in Plex- iglas cylindrical cages (30 · 15 cm) and fed M. quinquenervia tip leaves of undetermined chemotype. The recently (within 8 h) hatched neonates (N = 40) were carefully transferred to a single experimental M. quinquenervia leaf (positions 4–12) with a small paintbrush. The leaves were placed in Petri dishes (9.5 · 1 cm) lined with moistened filter paper and sealed with Parafilm to retain moisture (90% RH). The nymphs were observed daily and the filter paper moistened as needed until adulthood. Psyllid survival, growth, and development were similar when fed leaves excised or attached to the plant (Pratt, unpublished data). Each adult was sexed and weighed on a microbalance (±0.1 lg; Perkin-Elmer AD6). Data were collected on nymphal percent survival, development time (d) from eclo- sion to the adult stage, and final biomass (fresh mass).

2.5. Data analysis

All analyses were conducted with SAS/PC (PROC GLM) unless otherwise noted (SAS Institute, Inc., 1990). Select comparisons were performed with a contrast proce- dure where the burn treatment results were compared with those of the uncaged control and the herbivory results were compared with those of the caged control. To determine if foliar nitrogen levels were influenced by chemotypes or treatments the results were analyzed by two-way ANOVA (chemotype and treatment). The leaf toughness results were Fig. 1. Mean (± SE) leaf toughness (g/mm2)ofM. quinquenervia from analyzed on leaves 1–10 counting from the branch tip and two chemotypes from the branch tip toward the base. Collections included comparisons of regression coefficients of the different replacement leaves following burning or herbivory, leaves from a cage control, and a non-caged control. Leaf toughness was influenced by chemotypes and treatments were performed by two-way chemotype (F1,239 = 7.97; P = 0.0051), treatments (F3,239 = 23.49; (chemotype and treatment) ANCOVA where leaf position P < 0.0001), and the covariate leaf position (F1,239 = 17.43; P < 0.0001) served as the covariate. To determine if constituent levels and the interaction between treatment and chemotype (F3,239 = 4.95; were influenced by chemotypes or treatments, a two-way P = 0.0024). When analyzed by chemotype (A or B) both treatments (E- ANOVA (chemotype and treatments) was conducted with nerolidol: F3,138 = 21.78; P < 0.0001; viridiflorol: F3,98 = 6.34; P = 0.0006) and the covariate leaf position (E-nerolidol: F = 6.02; P = 0.0154; interaction. To determine if diets influenced insect survival, 1,138 viridiflorol: F1,98 = 8.53; P = 0.0043) influenced leaf toughness significant- two-way (chemotype and treatment) logistic regressions ly, while their interaction was not significant (>0.2). Select comparisons (PROC LOGISTIC) were performed with interaction with the appropriate controls indicated that the toughness elevations for (Neter et al., 1989). To determine if plant chemotype, dam- the burn leaves were significantly lower for leaves from both chemotypes age treatments, and insect gender influenced biomass and compared with the control leaves (E-nerolidol: t1,141 = 14.27; P < 0.0001; viridiflorol: t1,101 = 10.04; P < 0.0001). Moreover, the toughness elevation development time of weevils and psyllids, three-way ANO- for the leaves from only the E-nerolidol plants (B) produced following the

VAs with two-way interactions were conducted on each herbivory treatment was lower (t1,141 = 4.53; P < 0.0001) than the leaves insect species. from the cage control treatment. 158 G.S. Wheeler, K.M. Ordung / Biological Control 39 (2006) 154–161 analysis for each chemotype separately indicated that both treatment and the covariate leaf position influenced leaf toughness significantly; however their interaction was not significant. The leaves produced following the burn treatment had the lowest toughness values from both chemotypes and these were significantly less than their respective control (uncaged control) leaves (Figs. 1A and B). Additionally, the replacement leaves produced following the herbivory treatment from the E-nerolidol plants only had lower toughness compared with the caged control leaves (Fig. 1B). The change in leaf toughness with different leaf positions was similar among treatments as their slope values did not differ significantly (all P > 0.2). The total foliar terpenoid content was influenced only by chemotypes; leaves from the viridiflorol chemotype contained significantly greater levels compared with those of the E-nerolidol chemotype. The effect of treatments did not significantly influence the total levels of constituents in the replacement foliage nor were the two-way interactions among these terms significant (P > 0.3). The levels of individual terpenoids were also significantly greater from the leaves of the viridiflorol chemotype, as has been demonstrated previously (Wheeler and Ordung, 2005; Wheeler, 2006). As the chemotype · damage treatment interactions were significant for a-pinene, the treatment factor was analyzed separately by chemotype. An increase in a-pinene levels (F1,12 = 8.79; P = 0.0118) occurred only in viridiflorol leaves from the herbivory treatment (8.2 ± 0.9 lg/mg) compared with those of the Fig. 2. Survival of O. vitiosa weevil larvae (A) and B. melaleucae psyllid cage control (4.7 ± 1.0 lg/mg). nymphs (B) when fed M. quinquenervia leaves from two chemotypes subjected to different damage treatments. Leaves fed to larvae included 3.2. Insects replacement leaves following burning or herbivory, leaves from a cage control, and a non-caged control. Weevil survival (A) was significantly 2 influenced by chemotype ðX 1 ¼ 9:8698; P = 0.0017) and treatment 2 Weevil survival was reduced when fed the burn replace- (X 3 = 14.1233; P = 0.0027) where survival was significantly reduced when ment leaves compared with those from the control treat- fed the burn replacement leaves compared with those of the control 2 ment, whereas weevil survival was not different between (X 1 = 12.1886; P = 0.0005). Psyllid nymphal survival (B) was significantly 2 influenced by chemotype (X 1 = 2.8071; P = 0.09) and treatment those fed the herbivory and the cage control leaves 2 (X 3 = 8.3144; P = 0.0399) where survival was significantly reduced in (Fig. 2A). Additionally, survival was significantly reduced nymphs fed the burn replacement leaves compared with those of the 2 when the larvae were fed the viridiflorol leaves compared control (X 1 = 6.6385; P = 0.0100). with those fed the E-nerolidol leaves (Fig. 2A). The interaction between these two effects (treatment · chemotype) was not significant (P > 0.5). Weevil pupal biomass was affected by sex and the treatment · sex interaction. Weevil pupal Similarly, psyllid nymphal survival was reduced when biomass, regardless of treatment, averaged 51.4 (±1.1 mg) individuals were fed the burn replacement leaves compared and 47.9 (±0.9 mg) for females and males, respectively. with leaves of the control treatment (Fig. 2B). Additional- However, pupal mass was not affected by treatment when ly, psyllid survival was reduced when fed the E-nerolidol analyzed separately by sex (females F3,27 = 2.15; leaves (P = 0.09) compared with those fed the viridiflorol P = 0.1167; males F3,32 = 2.31; P = 0.0947). Additionally, chemotype (Fig. 2B). The interaction between these two regardless of treatment, pupal biomass was affected by effects was not significant (P > 0.5). Psyllid development chemotype; those individuals fed the E-nerolidol leaves time was significantly affected only by the gender of the (F1,58 = 5.93; P = 0.0179) had greater biomass individuals; females (16.8 ± 0.5 d) required more time to (50.5 ± 0.9 mg) than those fed the viridiflorol leaves complete development than males (15.2 ± 0.5 d). Similarly, (46.9 ± 1.1 mg). Weevil development time to the pupal psyllid final biomass was affected significantly only by the stage (overall mean: 20.8 ± 0.4 d) was not influenced by gender of the individuals; females (0.268 ± 0.01 mg) had chemotype (P > 0.6), treatments (P > 0.5), sex (P > 0.1), greater biomass (2.7-fold) compared with males or their two-way interactions (P > 0.2). (0.158 ± 0.01 mg). G.S. Wheeler, K.M. Ordung / Biological Control 39 (2006) 154–161 159

4. Discussion responses to damage, thresholds, or these responses may be affected by environmental factors (Coleman and Jones, Leaves that are replaced on surviving trees following 1991). Our results included two M. quinquenervia chemical severe defoliation by either fire or herbivory may be a dif- varieties or chemotypes, one (viridiflorol) more resistant to ferent nutritional resource than leaves from undamaged O. vitiosa herbivory (Wheeler, 2006). The only change trees. Our results indicate that the newly flushed M. quinqu- detected following herbivory was in the viridiflorol chemo- enervia leaves from the burn treatment had the lowest type leaves; greater concentrations of a-pinene (1.75-fold) toughness values and the O. vitiosa weevil larvae and occurred following herbivory. Regardless of this apparent B. melaleucae psyllid nymphs fed these leaves had reduced induced response to herbivory, the subsequent bioassays survival (means of 68% and 58% reductions, respectively). of each species indicated no impact on insect survival or Of the plant quality factors examined, only leaf toughness performance. Possibly this lack of an effect on these oli- was consistently associated with these differences where the gophagous species may be the result of the herbivores’ high leaves produced following the burn treatment had the low- degree of specificity to the host allowing them to better est toughness values (viridiflorol 272–397 g/mm2 and cope with the nutritional changes that occur in response E-nerolidol 358–544 g/mm2). This association between to damage compared with a generalist herbivore. For low toughness leaves produced after burning and low larval example, specialist species like those used for biological survival is contrary to previous results (Wheeler, 2001) that control, may be better able to detoxify, avoid, or tolerate indicated just the opposite, that larvae fed M. quinquener- these induced responses compared with generalist species via leaves with similar low leaf toughness values (200– (Cohen et al., 1992). Although induced responses have 350 g/mm2) had greater survival. Thus, these reductions been found in diverse plant families (Karban and Baldwin, in both weevil and psyllid survival when fed the low tough- 1997), possibly M. quinquenervia lacks the ability to ness burn replacement leaves are difficult to interpret. respond to damage as it is already well defended by high Other nutritional changes occur associated with concentrations of diverse terpenoids. Several studies that decreased foliar toughness including increased moisture examined species most closely related to Melaleuca, from (Feeny, 1970; Coley, 1983; Wheeler, 2001) nitrogen (Feeny, the genus Eucalyptus (family Myrtaceae), found induced 1970; Coley, 1983; Wheeler, 2001), and fiber and cellulose susceptibility or that the induced trees were preferred over content (Coley, 1983; Choong et al., 1992). Preliminary undamaged trees (Wallace, 1970; Landsberg, 1990). Final- results, not included here (Wheeler unpublished data), ly, induction of diverse classes of phytochemicals has been examined water content in M. quinquenervia viridiflorol reported in plants damaged by herbivores, including terpe- (only) leaves produced following these treatments. Con- noids like those described in our analyses (Constabel, trast analysis of these results indicated that the burn 1999). Despite the impression that induced responses are replacement leaves had greater moisture levels universal, the lack of chemical changes in herbivore-dam- (81.0 ± 0.6%) compared with the control (76.7 ± 1.4%; aged plants as seen here is not uncommon (Karban and F1,26 = 6.65; P = 0.0159); no difference was found between Baldwin, 1997). the herbivory (70.0 ± 1.2%), and cage-control The integration of fire and herbivory by biological con- (70.5 ± 1.3%) treatments. A similar increase in moisture trol agents may be an effective control of weeds in natural content was found in Chestnut oak leaves following burn- areas (Briese, 1996). The results presented here suggest that ing (Rieske, 2002). Generally, relatively high foliar water induced responses will have little effect that will compro- content is required by insects, especially insects feeding mise the combination of these control methods. Unpub- on trees (Mattson and Scriber, 1987). However, excessive lished field observations (Rayamajhi unpublished data) levels can dilute essential nutrients and in an attempt to indicate that in response to herbivory by defoliating O. viti- acquire sufficient amounts herbivores may compensate by osa larvae, M. quinquenervia plants increase branching with increasing consumption (Slansky, 1993). Increased leaf an additional number (1.3-fold) of tips and corresponding consumption simultaneously will increase the dose of sec- leaves. Not only will more leaves be produced after weevil ondary compounds that occur in the leaves possibly over- damage but these leaves will apparently be well suited whelming the detoxifying enzymes that metabolize nutritionally for the flush-feeding O. vitiosa larvae (Wheel- potentially toxic dietary constituents (Slansky and Wheel- er, 2001). The O. vitiosa weevil population increases er, 1992). This response in combination with induction of observed in the field following defoliation from either fire additional plant quality factors not detected here could or previous herbivory (Wheeler unpublished data) were explain this decreased survival. probably a result of the abundant replacement leaves pro- In contrast to the burn treatment, few changes were duced. However, our results indicate that the first leaves detected following herbivory in the quality of replacement produced on plants damaged by fire may not be compatible leaves or in the performance of the insects fed the foliage. with these biological control agents. Possibly the induced This apparent lack of an induced response could have been response that causes increased mortality in these species a result of several factors. Research on plant genetic varie- decreases with time and this should be determined in future ties may account for erroneous conclusions regarding research. Finally, the second biological control agent induced responses as different genotypes may have different released against M. quinquenervia, the psyllid is apparently 160 G.S. Wheeler, K.M. Ordung / Biological Control 39 (2006) 154–161 compatible with damage caused by the first, as we could Everaerts, C., Cusson, M., McNeil, J.N., 2000. The influence of smoke find no induced defenses in the leaves damaged by weevils volatiles on sexual maturation and juvenile hormone biosynthesis in nor was their an indication of decreased survival or perfor- the black army cutworm, Actebia fennica (Lepidoptera: Noctuidae). Insect Biochem. Molec. Biol. 30, 855–862. mance by the immature psyllids. Feeny, P.P., 1970. Seasonal changes in oak leaf tannins and nutrients as a cause of spring feeding by winter moth caterpillars. Ecology 51, Acknowledgments 565–581. FLEPPC Plant List Committee. 2003. Florida Exotic Pest Plant Council’s 2003 list of invasive species. Wild. Weeds 6, supplement. We are indebted to the technical assistance of Luke Flowers, J.D.I., 1991. Subtropical fire suppression in Melaleuca quinqu- Kasarjian, Rosa Leidi-Ferrer, USDA/ARS, Ft. Lauder- enervia. In: Center, T.D., Doren, R.F., Hofstetter, R.L., Myers, R.L., dale, FL, to I.A. Southwell (New South Wales Agriculture, Whiteaker, L.D. (Eds.), Proceedings of the Symposium on Exotic Pest Wollongbar Agricultural Institute, Wollongbar, Australia) Plants, Miami, FL, pp. 151–158. Hatcher, P.E., Melander, B., 2003. Combining physical, cultural and who generously provided assistance in terpenoid identifica- biological methods: prospects for integrated non-chemical weed tion and generous donations of viridiflorol and 2,4-dihy- management strategies. Weed Res. 43, 303–322. droxy-6-methoxytoluene standards. Mention of trade Haukioja, E., 1980. On the role of plant defenses in the fluctuation of names or commercial products in this publication is solely herbivore populations. 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