The Effects of Surface-AppliedJasmonic and Salicylic Acids on Caterpillar Growth and Damage to Tomato Plants1

AARONL. IVERSON,LOUIS R. IWRSON,AND STEVEESHITA, Buckeye Valley High Schools, Delaware, OH 43015 and USDA Forest Service, Delaware, OH 43015

ABSTRACT.We tested the role of (SA) and jasmonic acid (JA)in altering the tomato plant's defense against herbivory by tobacco hornworm. Treatments of SA or JA were topically applied to tomato plants, hornworm consumption was allowed to proceed for 12 days, and harvest analyses were performed Measurements taken included a subjective plant rating (1-10 score), plant dry mass, caterpillar mass, and the number of times the caterpillars fell off the plant. Results showed significant effects of exogenously applied SA and JA on the defense of tomato plants against insect herbivory. Plants treated with SA had little resistance to the feeding caterpillars and the plant lost more biomass to them. JA, in contrast, apparently increased the defensive mechanisms of the plant, resulting in lower caterpillar growth and increased caterpillar detachment from plants. The data are consistent with a model where JA, endogenous or exogenously applied, is necessary for defense against insect herbivory and SA disrupts JA biosynthesis and/or pool accumulation.

OHIO J SCI 101 (5):S)O-94, 2001

INTRODUCTION Linolenic Acid Jasmonic acid UA) is an endogenous plant growth 4 13-Iipoxgenase regulator widely distributed in higher plants (Meyer and Hydroperoxy-octadecatrienoicAcid others 1984; Tizio 1996). In response to injury, a plant SA block 4 4 4 allene oxide synthare (A08 may produce JA; which induces the expression of de- AIlene Oxide fensive compounds such as insect proteinase inhibitors. 4 alkoxid cylcae (AOC) JA may also be systemically distributed throughout the 12-0x0-phytodienoic Acid (PDA) plant and create volatile gases, which in turn may induce 4 12-0~0-PDAreductuse, oxidation neighboring plants to increase their defense allocations 7-epi-Jasmonic Acid (JA) as well as attract parasitic wasps to attack the infesting 4 herbivores (Creelman and Mullet 1995; McConn and Gene Expression others 1997; Thaler and others 1996; Turlings and others 1995). The synthesis of jasmonic acid takes place via the FIGLIRE1. 1';ithway of jasmonic acid systhesis in plants, also showing the polnt uf inhibition by salicylic acid in the pathway (after Pan and octadecanoid pathway (Fig. 1). The precursor of jas- others 1998). monic acid is linolenic acid. Linolenic acid is converted to hydroperoxylenolenic acid by lipoxygenase. After reactions catalyzed by allene oxide synthase (AOS) and allene oxide cyclase, phytodienoic acid is formed and others have shown cross protection for both insect and through oxidation, jasmonic acid is formed (Creelman pathogen resistance (for example, Inbar and others 1998; ancl Mullet 1997; Pan and others 1998). The jasmonic acid Thaler and others 1999). The plant response apparently then facilitates the induction of plant defensive genes. depends on the plant-challenger system and the type Salicylates, when synthesized or applied to plants, in- and strength of the elicitor. hibit AOS activity, which in turn inhibits the production Several investigations have centered on the defense of jasmonic acid and proteinase inhibitors (Fig. 1) (Ras- of the tomato (Lycopersicon esculentum) and their insect kin 1992; Doares and others 1995; Pan and others 1998). pests. Howe and others (1996) found that a tomato Consequently, a plant given salicylates will become less mutant that was deficient in the capacity to induce de- able to defend itself against insect attack. In contrast, fense genes, via the octadecanoid pathway, was much elevated SA in plants has often been associatecl with more susceptible to damage by the tobacco hornworm increased pathogen resistance (Yang and others 1997). (Manducu sexta). Thaler and others (1996) found that However, the relationship between pathogen and in- exogenously applied JA increased defense against beet sect resistance is still under debate (Apriyanto and armyworm (Spodopteru exigzta), and showed that field- Potter 1990; Hatcher 1995). Some studies show mutual applied JA enhances the production of chemical de- antagonism of JA vs. SA pathways, with consequent in- fenses in tomato. Thaler (1999a) also showed that JA, crease in pathogen resistance but decrease in insect re- when exogenously applied to tomato, not only induced sistance with the exogenous application of SA, while additional plant resistance to beet armyworm damage, but also doubled the incidence of of the en- doparasitic wasp Hyposoter exigz~aon the armyworm. 'Manuscript received 23 October 2000 :~ndin revised form 1 March These results may indicate a potential use of JA in 2001 (#OO-18). inhibiting agricultural pests. OHIO JOURNAL OF SCIENCE A L IVERSON, L R. IVERSON, AND S. ESHITA 9 1

In this experiment, we investigate the tomato-hornworm Caterpillar Treatment system, a well-studied and economically important Tobacco hornworm (~Ma~zd~~casexta) eggs were ob- crop-insect pest system. We tcst the effect of surface- tained from the North Carolina State University Insectary. applied salicylic acid (SA) and jasmonic acid UA) on Eggs were placed in a 25 x 25 cm plastic container with tomato plants, in conjunction with the damage induced artificial diet at room temperature and in the natural light by herbivory by the tobacco hornworm. regime for January in Ohio. The artificial diet was ob- tained also from the NCSU Insectary and consisted of METHODS a mixture of wheat germ, casein, sucrose, tol-ula yeast, Plant Growth Wesson salt mixture, sorbic acid, cholesterol, methyl Heirloom tomato seecilings (Lycopersicon escc~rlentuna paraben, streptomycin sulphate, agar, vitamin mixture (L.) Mill., cv. Moskovich) were germinated under kluo- (USB no. 234301, ascorbic acid, and formalin. Eggs hatched rescent lights (1711:7h, L:D, 21" C) in seed starters and in 4-6 days. Four 1- to 2-day old larvae were placed on transplanted as 16-day old seedlings into 10 crn pots each caterpillar-treat~llent plant and allowed to con- and placed in a greenhouse (sodium lights; 17h:7: L:D; sume foliage for 12 clays. Caterpillars were counted daily 24-29" C photophase and 16-18" C scotophase). Plants on each treatment plant; frequently some caterpillars hacl were allowed to grow for 12 days following transplan- detached from the plant to the soil surface of the pot tation before chemical or caterpillar treatments were or table just below the plant. TIicse caterpillars were applied. Plant5 were randomly placed in rows according tallied as 'cletached' and replaced. to each treatment. Each row of six replicate plants was separated from other rows (treatments) by at least 60 Analysis Of Treatment Effects cm. This spacing was necessary to reduce possible The plants were subjectively scored beginning the clay effects of spreading volatile gases or caterpillars among after the treatments were applied ancl continuing every the various treatments. A total of 24 plants were given two clays until the clay of harvest. The levels of rating caterpillar treatments, with six replicates each of four were as follows: surface-applied treatments (SA, JA, both, or water). An 1 = No damage, perfectly healthy additional 18 plants, to serve as controls for caterpillars, 2 = Slight leaf discoloi-ation or chlorosis were not given caterpillars but three surface-applied 3 = Slight leaf wilting or cc~rling treatments (SA, JA, or water). Pots within rows and rows 4 = More leaf curling or wilting themselves were shuffled every 2-3 days to account for 5 = Some leaves curled or wiltetl over half any unequal illumination of light among the plants. The 6 = Some leaves curled or wilted over three-foourths plants were evaluated daily and watered as needed 7 = All leaves curled or wilted over half throughout the experiment. 8 = All leaves curled or wilted full 9 = Dead leaves Phytohormone Application 10 = Plant dead Solutions of 0.01% JA (methyl , IUPAC name: (-)-1a,2P-3-0~0-2-(cis-2-pentenyl)-cyclo- After the 12 clays, the 6-week old plants were cut at pentaneacetic acid methyl ester, obtained as a gift from the base, placed in a paper bag, dried for 48 hours in a R.A. Creelman) and 0.05% SA (methyl salicylate, IUPAC drying oven at 70" C, ancl weighed (in bags) with a Fisher name: 2-hydroxybenzoic acid methyl ester; obtained top-loading balance accurate to 0.01 g. Because the 18 from Sigma Chemical Co., St. Louis, MO) were prepared caterpillar-control plants also served as controls for for the chemical treatments. Concentration levels were another experiment testing the effects of UV-C light in chosen based on previous work by Browse (Browse, conjunction with SA and JA (not reported here but personal communication). First, stock solutions of SA which experiment was terminated 4 days earlier), these (5%) and JA (1%) were prepared with ethanol, as was control plants were harvested 4 days earlier than the rest a control with neither SA nor JA. Ten ml of each stock of the experiment Therefore, one would expect those solution was then added to 990 ml of deionized water plants to have slightly less dry weight than they would to obtain the desired concentrations of 0.05% for SA, be if alloweci to grow as long as the caterpillar-applied 0.01% for JA, and control. In each of the three solutions, plants. Those data are included in some of the analyses six drops of Tween 20 (detergent, obtained from R.A. because of the adclitional information obtained. Data of Creelman) were added to allow equal distribution of score, plant weight, caterpillar weight, and number of liquid on the plant leaf surface and to allow the plant to caterpillar detachnlents were statist~callyanalyzed in S- absorb the solution more readily (Creelman, personal Plus software (Statistical Sciences 1993) using an analysis communication; Browse, personal communication). of variance (ANOVA) w~thmultiple comparisons tested The plants were sprayed with hand spray bottles with the Tukey method. The P value used for declaring two times---one day prior and one hour prior-to the significant effects was 0.05. application of the caterpillars. Each plant was sprayed with a fine mist of 2-2.5 ml of SA, JA, control, or both RESULTS SA and JA solutions. The solutions had dried prior to Plant Scores initial application of the caterpillars. Plants were then Plants with no caterpillars, regardless of chemical sprayed every two days over a 12-day period until the treatment were vigorous and healthy throughout the ex- experiment was terminated. periment (score of 1; Table I). Thus, the JA and SA did JA AND SA EFFECTS ON TOLMATO HERBIVORY

Q"ects ofs~l@ce-nppliecl./A arzd SA on toma!opla?z! vigor and weizht.

Caterpillars added No C;lterpillars aclclecl (Control)' V;tri;~hleTreatment Water S A J A SA+JA Water S A J A

Mean plant v~gor.score 3 70 f 4.03 f 3.20 f 3.57 f .28 .27 .20 .15

Mean plant weight, g' 0.63 f 0.41 i 0.82 f 0.69 + .09 b .14 a .I I c 08 b

' Recauw the planrs with no c:~rerpillars were hanured 4 clays earlier, only general comparisons should be made to the caterpillar-treated plants. Different letters ~ntl~catesignificantly clifferenr (P ~0.05)results using Tukey's lnultiple comparison test afier the nnalys~sof variance. not impact plant vigor. With caterpillar treatment, all exogenous JA overcoming the SA block of JA bio- plants sufferecl damage, with the SA-treated plants having synthesis. greater damage as compared to controls, both, and especially the JA treatment. Caterpillar Detachment This metric is a measure of the total number of times Plant Weight caterpillars had dropped from the plant to the pot or Plants treated with caterpillars had generally lower tablc, cumulative over the 12 days of caterpillar con- hionlass compared to plants with no caterpillars (Table 1). sumption. Though no statistical analysis was possible This effect of a reduction in plant biomass via caterpillar on these data, the JA-treated plants had nearly twice ingestion is apparent even though the control plants that the number of detachments compared to water-treated had no caterpillars applied were harvested four days plants, and three times the detachments of SA-treated earlier than the caterpillar-treated plants. plants (Table 2). Those plants treated with both JA and Among the plants treated with caterpillars, aboveground SA had nearly the same number of detachments as dry matter data showed that the SA-treated plants had compared to the JA-treated plants. These data suggest that significantly lower total yield compared to water-treated caterpillars were making choices on preferred food, and plants, while JA-treated plants had higher yields compared the JA-treated plants were not preferred. Because the to water-treated plants and especially when compared to plants were widely spaced within the greenhouse, no evi- SA-treated plants (Table 1).JA-treated plants had twice the dence of crossover of caterpillars between treatments biomass of the SA-treated plants. When SA and JA were was detected. applied together, there was a slight, but not statistically different, increase in biomass conlpared to controls (0.69 DISCUSSION f .08 vs. 0.63 f .09 g per plant). In general, the effects of Evidence from plant weight, caterpillar weight, and JA \is. SA negated each other. Conceivably. exogenous JA the cumulative count of caterpillar detachments shows should be able to bypass and eventually overcome the SA that the surface application of SA made tomato plants block on endogenous JA production, if enough exogenous more susceptible to caterpillar damage and that JA made JA was applied and absorbed. Perhaps this effect is tomato plants less susceptible to caterpillar damage. beginning to be apparent in this experiment. Anlong the controls that had no caterpillars added, SA- treated plants had a significantly higher biomass relative to the JA- or water-treated plants (Table 1). Further, the JA- treated plants had a slightly, but statistically insignificant, brects of s~~~jfhce-applzed.JAand SA on lower mean compared to the water-treated plants. These catetpillnr weights and derachn~tr?ts trends are opposite to those observed when caterpillars were added to the plants. Trearment Water S A JA JAcSA Caterphr Weight Caterpillars on SA-treated plants grew 55% larger than Mean caterpillar 87 * 33 b 135 f 36 c 42 f 8 a 53 f 16 ab those on controls, and 221% larger than those grown on weight, mgl JA-treated plants (Table 2). In contrast, caterpillars grown on JA-treated plants showed a 52% reduction in growth Total Detachments 17 11 31 30 compared to controls, while those grown- on plants treated both SA and JA had a 43?'6 reduction in growth I Different Inters indicate significantly different (P <0.05:) results using 'l'ukry's compared to controls. This trend is consistent with multipie comparison test after the analysis OF variance. OHIO JOURVAL OF SCIENCE A. L. IVERSON, L R. IVERSON, AND S. ESHITA 93

SA-treated plants had proportionately more plant tissue by the community, but some recent work cloes not sup- converted to caterpillar larvae tissue than did JA-treated port this model (for example, Agrawal and others 1999). plants. In both plant and caterpillar weight, SA and JA- A second speculation could be that the additional SA treated plants were significantly different from the could lead to an increased systemic resistance to a control treatment, but in opposite directions. pathogen that might have been present in the plants, Plants treated with salicylic acid (SA) apparently have thereby allowing better growth. a diminished resistance to caterpillar damage. This trend is consistent with a model where SA blocks the produc- General Interpretations tion of allene oxide synthase (AOS), which is a necessary This study provides further evidence of the elaborate enzyme to produce jasmonic acid (JA) through the octa- chemical communication and defense systems of plants. decanoid pathway (Fig. l).JA is one of the signals for Surface application of JA adds resistance to herbivory, a the plant to produce a defensive reaction against insect trait which holds great potential application in agri- attack. With its defense impaired, a SA-influenced plant culture to aid in pest management. Altliough not assayed is unable to produce the necessaly defensive compounds in this experiment, the surface-applied JA apparently such as polyphenol oxidase (as well as peroxidase and enhances the production of secondary metabolites via lipoxygenase) or proteinase inhibitors (digestibility the octadecanoid pathway, so that additional defensive reducers) and becomes more susceptible to the herbi- compo~~ndscould be produced. vore (Stout and others 1998a, 1998b; Ficlantsef and others 1999). As a result, the plant is consumed at a ACKX~WLEDGME~~~.The authors are obliged to many people who facilirated this experiment for a high school science fair project of faster rate and consequently has a smaller final mass the senior author. Dr. Kal~)hBackhaus, Arizona State University. than control plants. Thus, caterpillars feeding on SA provided the primary idea for the caterpillal- experiment. Dr. Robert plants have a larger mass as they are not as inhibited Creelman, Texas AWM University, provided guidance and donated by the proteinase inhibitors or other defense responses the methyl jasrnonate UA). Ms. Beverley Pagura, North Carolina State IJni\:ersiry Insecta~y,provided the tobacco hornwonn eggs. Dr. John that can affect the growth and reproduction of the insect. Browse, Washington St;lte University, also provided insights and Plants treated with JA, on the other hand, exhihit the research papers for the experiment. Dr. Joanne Rebheck and Dave opposite effect. JA is the end product of the octadecanoid Carey, USDA Forest Service. assisted with greenhouse space, while Margzrret Iverson provided technical assistance. Dr. Keith Davis, defense pathway that leads to the activation of defense George Keeney, Mark Lagrimini, and David Denlinger, The Ohio genes and production of defensive proteins. In our State IJniversity, provided technical guidance. Thanks also to Ms. moclel, exogenous JA is absorbed and stimulates a Rose Blanchard, Buckeye Valley High School, for ideas and greater production of these defensive compounds and encourdgenlent. makes the plant more resistant than a control plant with fewer activated defensive compounds. Caterpillars LITERATURE CITED feecling on JA-treated plants will, therefore, have a Agrawal .4A, Gorski PM,. Tallamnv DN'. 1999. Polymorphism in plant defense against herbivory: constitutive and induced resistance in smaller mass than caterpillars feeding on control plants. Czic~rmissatir/rts.J Chem Ecol 252285-304. As such, the JA-treated plant is also consumed at a Apriyanto D, Potter DA. 1990. Pathogen activated induced resistance slonrer rate and has a greater Inass than the controls. of cucumber: response of arthropod herbivores to systematically A large effort is underway to find and test suitable protected leaves. Oecologia 8525.31. Creelrnan RA, Mullet JE. 1995. 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