Cues from a specialist herbivore increase tolerance to defoliation in tomato

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1 Functional Ecology 2014, 28, doi: / Cues from a specialist herbivore increase tolerance to defoliation in tomato Timothy Korpita 1, Sara Gomez* 1,2 and Colin M. Orians 1 1 Department of Biology, Tufts University, Medford, Massachusetts 02155, USA; and 2 Department of Biological Sciences, University of Rhode Island, Kingston, Rhode Island 02881, USA Summary 1. Recent studies have shown that herbivore cues trigger rapid shifts in primary metabolism and resource allocation within a plant. This has been suggested to be an induced mechanism of tolerance to defoliation but there is little evidence supporting this. Here, we investigated, using a cultivar of tomato (Solanum lycopersicum), whether prior herbivore damage confers plants an advantage in terms of increased growth and initial flower production following defoliation. 2. Plants were mechanically wounded and treated with either regurgitant of the specialist Manduca sexta, deionized water or received no damage for five consecutive days. Chlorophyll content, plant growth and biomass allocation were measured during the 5-day damage period. After the damage treatments, all leaves were removed to simulate defoliation in the three treatment groups, and regrowth capacity was followed until flowering. The number of regrown leaves, the initial number of flowers and biomass production and allocation were quantified at harvest. 3. During the 5-day damage period, plants treated with M. sexta regurgitant grew less in height than undamaged plants (but had thicker stems). Chlorophyll content also decreased sharply in these plants. However, regurgitant-treated plants recovered more quickly from the defoliation treatment, producing more new leaves compared with undamaged and mechanically damaged treatments. 4. Our results support the hypothesis that induced changes in resource allocation in response to specialist herbivores results in increased regrowth ability and may be adaptive in the event of severe defoliation. Future studies on wild plants could help inform the evolutionary significance of this response. Key-words: induced sequestration, induction, Manduca sexta, regrowth, resource reallocation, Solanum lycopersicum, tolerance Introduction Plants have evolved adaptations to minimize negative effects and to maximize recovery in response to a variety of biotic and abiotic stresses (Rosenthal & Janzen 1979). While most plant species respond to herbivore damage by producing defence compounds that directly and negatively affect the performance of herbivores, many species have also evolved tolerance traits that increase regrowth capacity and/or reproduction (reviewed by Karban & Baldwin 1997; Stowe et al. 2000; Tiffin 2000; Fornoni 2011; Orians, Thorn & Gomez 2011). Tolerance may be a particularly important strategy against specialist herbivores that have evolved to either tolerate or detoxify the plant s direct chemical defences (Karban & Baldwin 1997; Strauss & *Correspondence author. sara.gomez@tufts.edu Agrawal 1999), as is the case for Manduca sexta, a specialist on Solanaceae (Wink & Theile 2002). In general, a plant s capacity to tolerate damage largely depends upon its inherent growth rate and extent of stored resources prior to the damage event (van der Meijden, Wijn & Verkaar 1988; Huhta, Tuomi & Rautio 2000b; Huhta et al. 2000a; Stowe et al. 2000; Tiffin 2000; Wise & Abrahamson 2005; Schwachtje & Baldwin 2008; Fornoni 2011; Hochwender et al. 2012; Zhao & Chen 2012). These are considered constitutive traits. Additionally, there are herbivore-inducible mechanisms that can increase tolerance, such as increase in photosynthetic rates, activation of dormant meristems and compensatory growth (reviewed in Tiffin 2000). More recently, it has been hypothesized that plants can also increase their tolerance to herbivores by reallocating resources away from tissues under attack and using those resources for future regrowth after the 2013 The Authors. Functional Ecology 2013 British Ecological Society

2 396 T. Korpita et al. herbivory event. Evidence of herbivore-induced resource reallocation includes increased transport of recently fixed carbon to roots and/or stems in response to both herbivores and jasmonates (Holland, Cheng & Crossley 1996; Babst et al. 2005, 2008; Schwachtje et al. 2006; Henkes et al. 2008; Kaplan et al. 2008; Gomez et al. 2012; Moreira, Zas & Sampedro 2012; Poveda et al. 2012; Ferrieri et al. 2013). Similarly, jasmonic acid treatment of roots modifies carbon partitioning in Nicotiana (Henkes et al. 2008). Remobilization of nitrogen in response to (simulated) herbivory has also been shown (Newingham, Callaway & BassiriRad 2007; Gomez et al. 2010). For example, Newingham, Callaway & BassiriRad (2007) observed a shift in allocation of nitrogen towards the leaves after root herbivory in Centaurea maculosa. Some evidence suggests that these herbivore-induced resource shifts might support storage in inaccessible tissues. In Populus, for example, jasmonates increase protein concentration in stem tissue (Beardmore, Wetzel & Kalous 2000). This storage role of reallocated resources might support increased regrowth after the attack, thereby increasing tolerance to herbivory. Additionally, resource shifts can also support defence production in local and systemic tissues (Arnold et al. 2004; Ferrieri et al. 2013). In the tomato system specifically, we have previously shown herbivory-induced resource allocation away from treated leaves and into storage organs (Gomez et al. 2010, 2012) along with rapid changes in the concentrations of primary metabolites locally and systemically (Steinbrenner et al. 2011; Gomez et al. 2012). Interestingly, cues of the specialist herbivore, Manduca sexta, largely induced metabolites associated with carbon and nitrogen transport while cues of a generalist herbivore, Helicoverpa zea, induced many more metabolites that are precursors of defence induction (Steinbrenner et al. 2011). This suggests that plants might cope with some specialist herbivores by enhancing regrowth capacity instead of direct chemical defence; defences that tend to have less impact on specialist herbivores. Perhaps when cues signal the potential for extensive defoliation, not uncommon among specialist herbivores, plants undergo a rapid and specific response that minimizes the loss of resources to the herbivore and maximizes the amount of resources available for later regrowth. Despite the increasing evidence that invertebrate herbivory triggers resource reallocation, whether such changes result in improved tolerance expressed as increase regrowth and flowering production is underexamined (but see Schwachtje et al. 2006). Here, we examined whether cues of M. sexta increase the regrowth capacity and initial flower production of a cultivated tomato variety. First, we hypothesized that during a five-day damage treatment period, plants in the simulated herbivory treatment (damage + M. sexta regurgitant) would exhibit a decrease in growth compared with mechanically damaged plants (damage + water) or undamaged control plants. This would be consistent with our previous work demonstrating shifts in resource allocation from leaves to stems and roots in tomato. Secondly, we hypothesized that, if induced sequestration of resources increases tolerance, subsequent defoliation would result in faster and more regrowth and greater initial flower production in response to herbivore cues as compared to mechanically damaged or undamaged control plants. Materials and methods PLANTS Tomato (Solanum lycopersicum, First Lady II Cultivar) plants were grown in potting mix (Metromix, Sun Gro Horticulture) in 10 cm 9 10 cm 9 20 cm pots. The plants were grown in a greenhouse under natural light during June and July. Plants were fertilized three times a week with 50 ml of ½ strength Hoagland solution for the duration of the experiment. Treatments began approximately 5 weeks after germination, when the plants were 247 (03) cm tall on average (seven leaf stage). INDUCTION TREATMENTS A total of 45 plants were divided equally among three experimental groups: an undamaged control group and two damage treatments. In the two damage treatments (hereafter referred to as induction treatments ), three leaves (leaves 2, 3 and 4, counting down from the apex) were damaged for five consecutive days (Fig. 1; Induction phase). This time frame was chosen to mimic herbivores with limited movement feeding on the same plant for several days. Previous experiments have shown that this amount of time elicits quantifiable changes in tomato s primary metabolism (T. Korpita, unpublished data). Leaf 1 was defined as the newest leaf that was over 50% expanded. A fabric trace wheel was used to make puncture wounds along the edge of every leaflet of each of the three leaves. After damage, 80 ll of either Manduca sexta regurgitant or deionized water were applied with a paintbrush ( Manduca and Water treatment, respectively). Caterpillar regurgitant is well known to elicit a specific response compared with mechanical damage (Kessler & Baldwin 2002; Halitschke et al. 2003). Caterpillar regurgitant was collected from fifth instar Induction phase (leaves 2, 3, 4) Defoliation (all leaves) Time (days) Post-defoliation phase Harvest Fig. 1. Experimental set-up. Tomato plants were either undamaged ( Control ) or received damage with a fabric trace wheel on leaves 2, 3 and 4. Deionized water ( Water treatment ) or Manduca sexta regurgitant ( Manduca treatment ) was applied on the wounds on damaged plants. Damage was repeated daily on five consecutive days (day 1 to day 5; Induction phase). Chlorophyll content was non-destructively assessed daily on leaves 0, 2 and 5 during the induction phase. On day 6, all plants was completely defoliated, and leaf development was monitored until day 29 (Post-defoliation phase), when all plants were harvested.

3 Herbivore-induced increases in regrowth and flowering 397 M. sexta larvae fed on tomato plants for at least 48 h and stored at 80 C until use. The water treatment was designed to test for the effects of mechanical damage alone. DEFOLIATION Plants in the three treatments were defoliated one day after the induction phase (day 6) by removing all leaves and the apex of the plant with a razor blade (Fig. 1) and were allow to regrow until flowering. A subset of five plants in each treatment were harvested at the end of the induction phase to determine whether herbivore cues had an effect on biomass allocation patterns (Table 1; day 6). The harvested plants were divided into different tissues (apex, new leaves, old leaves, damaged leaves, stem and roots) and weighed. We note that a complete defoliation of a tomato plant is common once M. sexta larvae reach the later instars (personal observation). All leaves removed in the defoliated plants were weighed, and plants were monitored for regrowth. PLANT RESPONSES TO INDUCTION TREATMENTS Chlorophyll was measured as an indirect measure of photosynthetic potential. Starting on day 0 (the day before damage began) and continuing through day 6 (immediately prior to the defoliation treatment), chlorophyll measurements were made on the undamaged terminal leaflet of leaf 0 (<50% expanded), 2 (young fully expanded, subjected to damage treatment) and 5 (mature fully expanded, untreated) using a chlorophyll meter (Opti- Sciences, CCM 200). Three measurements per leaf were made and averaged. The relative change of chlorophyll content was measured as the ratio between values measured on day 6 divided over the values quantified on day 1 prior to the beginning of the induction treatment. Over the same period, we measured changes in apical growth measured as changes in stem height (from the cotyledons to the base of the newest visibly separated leaf) and changes in stem width (at the centre point of the stem between leaf 2 and 3, marked at day 0) between day 1 and day 6. Changes in these two variables were quantified simultaneously as a ratio (hereafter termed stem height: width growth ratio) to provide information on putative trade-offs between plant growth (changes in height) and stem storage (estimated as changes in stem basal diameter). Table 1. Mean (SE) fresh biomass (g) of the different vegetative tissues immediately before defoliation (day 6; n = 5-15) and at harvest (day 29; n = 10) Tissue Control Water Manduca Day 6 (at defoliation) Apex 22 (02) 21 (02) 23 (02) New leaves 30 (02) 30 (03) 27 (02) Old leaves 24 (02) 23 (03) 25 (02) Damaged leaves 56 (02) 54 (02) 60 (01) Stem 209 (05) 199 (11) 181 (01) Roots 60 (03) 66 (13) 79 (13) Total 408 (14) 395 (26) 386 (20) Day 29 (at harvest) Apex 55 (01) 66 (06) 69 (20) Leaves 24 (04) 20 (04) 26 (22) Stem 194 (11) 196 (09) 205 (16) Roots 50 (07) 45 (04) 50 (13) Total 323 (26) 327 (16) 349 (25) PLANT RESPONSES TO DEFOLIATION We quantified plant regrowth ability after defoliation as a proxy of vegetative tolerance by measuring the production of new leaves every 3 4 days to be able to detect effects of the induction treatment at different time points during the post-defoliation phase. At day 23 post-defoliation, plants were harvested, divided into four tissues (roots, stem, regrown leaves and regrown apex), weighed and flash frozen in liquid nitrogen to estimate total biomass and tissue specific biomass. Initial flower production at harvest (number of flower buds) was measured to capture how quickly plants began flowering. STATISTICAL ANALYSES Statistical tests were performed using JMP 9.0. A one-way MANOVA was performed to test the effect of the induction treatment on the relative change in chlorophyll from day 1 to day 6 on three different age leaves (Leaves 0, 2 and 5; dependent variables). Chlorophyll relative change in leaf 2 was log-transformed. A oneway MANOVA was also used to test the effect of the induction treatment on biomass allocated to different tissues (dependent variables; see table 1) at defoliation and at harvest, respectively. Individual one-way ANOVAS were applied on the dependent variables if the overall induction treatment effect was significant. To test for the effect of the induction treatment over time on the number of regrown leaves, repeated measures ANOVA was used, using time as the repeated factor and induction as the betweensubjects factor. The stem height: width growth ratio during the induction treatment and the number of flowers at the final harvest were analysed using one-way ANOVAs. Tukey s honest significant difference (HSD) test was used to assess differences between treatment means. Results Leaves in all treatments experienced a decline in chlorophyll content during the induction phase. However, the magnitude of the decline differed among treatments as indicated by the overall significant induction treatment effect (one-way MANOVA; Wilks k = 0662; F 6,80 = 306; P = 001; Fig. 2a). This was driven by a significant induction treatment effect on leaf 2 (young fully expanded leaf F 2,42 = 415; P = 002) and a marginally significant effect (F 2,42 = 301; P = 006) on leaf 0, the youngest expanding leaf. The chlorophyll content in leaf 2 of plants treated with M. sexta regurgitant declined significantly more over time than in control plants. Chlorophyll in regurgitant-treated plants declined 36% by day 6, whereas that in undamaged control plants it declined 23%. Chlorophyll content in mechanically damaged plants treated with water was not significantly different to either control or regurgitanttreated plants (Fig. 2a). In leaf 0, this decline was 28% in regurgitant-treated plants compared with 16% in control plants (Fig. 2a). However, induction did not affect chlorophyll content on leaf 5 (mature, fully expanded, undamaged source leaf (F 2,42 = 227; P = 012)). There was a strong effect of the induction treatment on plant stem height: width growth ratio after 5 days of damage (F 2,32 = 897; P < 0001). Control, undamaged plants had a higher ratio, driven by greater apical growth and

4 398 T. Korpita et al. reduced stem diameter (Fig. 2b). On average, by the end of the induction treatment, plants treated with M. sexta regurgitant were about 33% shorter but had a 222% thicker stem than undamaged control plants. Mechanically damaged plants treated with water did not significantly differ from control or regurgitant-treated plants in apical growth (height) or stem width growth. Damage prior to complete defoliation (induction treatment) had a significant and positive effect on regrowth capacity and initial flower production. Plants previously challenged with Manduca regurgitant showed the greatest (a) Relative change in chlorophyll (b) Stem height growth (cm) P = 0 06 P = 0 02 P = 0 12 a ab Leaf 0 Leaf 2 Leaf 5 a ab Stem width growth (mm) Fig. 2. Induction phase. (a) Mean (SE) relative change in chlorophyll content from days 1 to 6, prior to defoliation, on leaf 0 (youngest leaf; undamaged), leaf 2 (young leaf; damaged) and leaf 5 (old leaf; undamaged) in undamaged Control (white), Water (light grey) and Manduca (dark grey) treatments. Statistical differences among treatments (Tukey s HSD P < 005), indicated by different letters above individual bars, are only reported for tissues that had a significant overall induction treatment effect. (b) Mean (SE) stem height growth (Y axis) and width growth (X axis) during the induction phase for plants in the undamaged Control (white), Water (light grey) and Manduca (dark grey) treatments. Statistical differences among treatments (Tukey s HSD P < 005) are indicated by different letters. b b regrowth capacity, producing overall more leaves than control, undamaged plants (Fig. 3; Induction treatment effect F 2,27 = 517; P = 001). This effect was visible soon after the defoliation treatment was applied. Specifically, 1-day post-defoliation, plants in the two damage treatments had more new leaf buds than undamaged plants (Fig. 3). Moreover, the damage effects on the amount of leaves produced post-defoliation did not vary over time (no time by treatment interaction; Fig. 3). At harvest, day 23 post-defoliation, plants in the Manduca treatment had an average of regrown leaves compared with and regrown leaves in the Water and Control treatments, respectively (Fig. 3). There was no significant effect of the induction treatment on total plant biomass. Interestingly, while the biomass M. sexta regurgitant-treated plants tended to be lower than controls at the end of the induction treatment (day 6), they were higher at the final harvest (23 days postdefoliation; Table 1). In addition, there was no significant induction treatment effect on the relative allocation to vegetative tissues. The induction treatment significantly affected initial flower production at harvest (Fig. 4; F 2,27 = 393; P = 003). At the time of harvest, plants treated with Manduca regurgitant had produced an average of 36 flowers per plant, while undamaged controls produced 20 flowers per plant; initial flower production on plants mechanically damaged (treated with water) showed intermediate values (28 flowers per plant, Fig. 4). Discussion Our results show that simulated herbivory by the specialist M. sexta elicited rapid changes in a tomato cultivar resulting in greater regrowth capacity and initial flower production after a severe defoliation event. The effect of mechanical damage alone was similar but weaker than Regrown leaves (no.) Days after defoliation Time *** Induction * Time x Induction n.s. Fig. 3. Post-defoliation phase. Mean (SE) number of new leaves regrown after complete defoliation at day 6 in Control (solid), Water (dashed) and Manduca (dotted) treatments at different time points.

5 Herbivore-induced increases in regrowth and flowering 399 Fig. 4. Harvest. Mean (SE) number of flowers at harvest (23 days post-defoliation). Statistical differences among treatments (Tukey s HSD P < 005) are indicated by different letters. those elicited by herbivore cues. To our knowledge, this is one of the first studies to show that herbivore cues can elicit a change in resource allocation that may ultimately increase a plant s tolerance to herbivory. There are several implications of our results. First, herbivore cues caused a shift in the pattern of growth that could be linked to the increase in tolerance. Although both leaf chlorophyll and apical growth declined during the induction phase, stem width growth did not. The observed decline in chlorophyll content in younger leaves [the main pool of leaf nitrogen (Evans 1989)] and the tendency for less biomass accumulation following treatment with M. sexta regurgitant support a shift in allocation patterns away from immediate growth (Gomez et al. 2010, 2012). This pattern is consistent with the general observation that younger leaves are more inducible (Arnold et al. 2004; Gutbrodt et al. 2011). Additionally, plants in the Manduca treatment exhibited a larger increase in basal stem diameter than did mechanically damaged or control plants. Perhaps, storage of carbon and proteins in the basal part of the stem saved those resources from M. sexta and made them available for subsequent regrowth after defoliation. Importantly, we note that the effects can be subtle; even though there was no significant change in total biomass or relative biomass allocation among the treatments, the small changes in basal stem diameter were associated with significant differences in the timing of regrowth (see discussion below). To our knowledge, no previous work has examined this issue. Historically, decreases in apical growth following herbivory have been assumed to be related to allocation costs of plant defence traits (Green & Ryan 1972; Herms & Mattson 1992; Karban & Baldwin 1997; Zavala & Baldwin 2006). While this may be a component of the reduced apical growth during the induction phase, our results suggest that greater allocation to stem tissue may have contributed to this pattern. Importantly, given the increasing evidence for short-term resource sequestration induced by herbivores (reviewed by Orians, Thorn & Gomez 2011), more attention should focus on how leaf-feeding herbivores alter patterns of resource allocation to growth, storage and defence. Secondly, we showed that changes in resource allocation caused by herbivory alter a plant s regrowth capacity following defoliation, indicating that improving short-term tolerance is a part of the integrated plant responses induced by herbivory. Plants that had been exposed to regurgitant by the specialist M. sexta were clearly able to recover from the loss of all their leaf tissue better than previously undamaged plants. We note that this effect was observed even 1-day post-damage, suggesting that herbivore cues were altering patterns of apical dominance. Not only did they produce new leaves at a faster rate, but also had higher initial flower production. Plant tolerance to leaf damage by herbivores is well studied (Paige 1999; Strauss & Agrawal 1999; Tiffin 2000). Traditionally, it has been viewed as a function of stored reserves and release from apical dominance through increased photosynthesis and growth of new tissues following the loss of the apical meristem (Tschaplinski & Blake 1989a,b; Huhta, Tuomi & Rautio 2000b; Tiffin 2000). A rapid herbivore-induced shift in growth during or just after the actual herbivory event may be another important mechanism of tolerance. This would not require an investment of resources in the absence of herbivory, as would other tolerance characteristics, such as constitutive storage. Based on work in tomato and other systems (Holland, Cheng & Crossley 1996; Schwachtje et al. 2006; Babst et al. 2008; Kaplan et al. 2008; reviewed by Erb et al. 2009; Anten & Pierik 2010), we conclude that the mechanisms of increased tolerance in response to M. sexta are likely driven by changes in carbon and nitrogen allocation to sites of short-term storage (especially sites near sites of new shoot growth) and by hormonal changes that affect patterns of apical dominance. Finally, we suggest a well-tailored response to the type of herbivory simulated in this study is relevant in natural settings provided that plants have time to regrow and produce seeds before the season ends. Some herbivores, like M. sexta, can defoliate entire plants once they reach later instars. Similarly, gregarious feeders can also cause considerable damage during later developmental stages. We predict that these herbivores will trigger changes in growth that will increase regrowth capacity once the threat is no longer present (Orians, Thorn & Gomez 2011). Perhaps the observed induced mechanisms to increase tolerance are a common response to specialist herbivores that are well adapted to their host plant s defence compounds. Not only are they capable of detoxifying or tolerating the defence chemicals of their host plants (as is the case with M. sexta on Solanaceae) (Wink & Theile 2002), but also they are likely to continue feeding on the same plants (Wittstock et al. 2004) and thus provide a reliable cue to the plant. In general, induced secondary defences are expected to be more important against generalist herbivores (Orians, Thorn & Gomez 2011). In addition, an induced tolerance

6 400 T. Korpita et al. response may be important in plants subject to periodic insect outbreaks if plants have the appropriate anatomical structures for storage of resources. Specifically, we predict that plant species that experience periodic outbreaks (Barbosa, Letourneau & Agrawal 2012; Zvereva, Zverev & Kozlov 2012) will, in response to cues of those herbivores (perhaps above a critical threshold), undergo a metabolic reorganization that increases resource allocation to inaccessible tissues and failure to respond will compromise regrowth and other fitness measures when outbreaks do occur. In summary, these results provide a new perspective on the induced responses of plants, and to our knowledge, provide the first evidence of specialist herbivore cues increasing regrowth after a defoliation event. While this induced mechanism of tolerance may potentially be ecologically relevant, it is important to note that this study was conducted on a domesticated cultivar grown in the greenhouse with regular fertilization. Future studies should focus on determining whether this is a common response to specialist herbivory across systems, in both wild and cultivated plant species across a range of ecologically relevant environments. They should also elucidate the details of the underlying mechanisms that contribute to the documented increase in tolerance. A study that simultaneously documented post-herbivory changes in resource allocation to growth, defence and storage would go a long way towards determining the relative significance of this induced response. Acknowledgements The authors thank B. Trimmer for providing Manduca sexta larvae and L. Sampedro and two anonymous reviewers for their insightful comments on previous versions of the manuscript. 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