Compatibility of Plant Defense Elicitors with Aphid- and Nematode-Resistant Tomato Varieties in Integrated Pest Management

Final Report for GS03-026

Project Type: Graduate Student
Funds awarded in 2003: $10,034.00
Projected End Date: 12/31/2004
Region: Southern
State: Arkansas
Graduate Student:
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Project Information

Summary:

The goals of this project were to evaluate the effects of several forms of host-plant resistance in tomato (Lycopersicon esculentum) on potato aphids (Macrosiphum euphorbiae) and root-knot nematodes (Meloidogyne javanica), and to determine if these forms of resistance can be used simultaneously for enhanced protection against these pests. The first form of resistance we evaluated was Mi-mediated resistance. Mi-1.2 is a single dominant resistance gene (R-gene) that confers resistance against potato aphids, root-knot nematodes, and sweet-potato whiteflies. The goal of this study was to determine whether Mi-mediated resistance is compatible with induction of two different forms of acquired resistance. One form of acquired resistance we evaluated was systemic acquired resistance (SAR), a form of acquired resistance that is dependent upon salicylic acid (SA), and is primarily associated with pathogen protection. Foliar application of an SA analog, benzothiadiazole (BTH), was used to induce SAR in a susceptible tomato cultivar that lacks Mi-1.2, and a resistant cultivar that carries the gene. BTH treatment reduced the population growth of two different aphid isolates on both the susceptible and the resistant tomato cultivars. These results indicate that SAR is an effective defense against aphids, and can be combined with R-gene mediated resistance for enhanced aphid control.

This study also examined another form of acquired resistance in tomato, jasmonate-dependent induced resistance (IR). IR is known to deter feeding by chewing insects, but its effects on piercing-sucking insects such as aphids have not previously been extensively investigated. A foliar application of synthetic jasmonic acid (JA) was used to induce IR in both susceptible (Mi-) and resistant (Mi+) tomato cultivars. Induction of IR triggered expression of a JA-inducible proteinase inhibitor in both the susceptible and the resistant tomato cultivars, although the effects of JA treatment on aphid performance differed between these cultivars. JA-treatment significantly reduced aphid population growth on the susceptible tomato cultivar, but it did not influence aphid numbers on a near-isogenic resistant tomato cultivar. Thus, although JA-dependent IR is an effective defense against aphids, it does not appear to interact synergistically with Mi-mediated aphid resistance.

The mechanisms of different forms of resistance may influence whether they have a synergistic effect against attackers. In order to characterize the mode of action of IR, this study also examined the effects of JA application on the life parameters of individual potato aphids and their progeny on resistant (Mi+) and susceptible (Mi-) tomato plants with and without JA treatment. JA-treatment did not influence the survivorship or fecundity of aphids on resistant plants, which confirms the results of our previous population growth studies. In contrast, on the susceptible tomato cultivar, JA-dependent defenses significantly reduced the longevity and net reproduction of adult aphids, and reduced the number of juveniles to survive to maturity. These results indicate that JA application induces systemic defenses in susceptible tomato that have a direct negative effect on aphid survivorship. In an additional experiment, aphid excretion rates were used as an indirect measure of aphid feeding to determine if IR and/or Mi-mediated resistance had antixenotic effects. The average honeydew excretion per aphid was lower on resistant plants carrying Mi-1.2, confirming previous findings that Mi-mediated resistance deters aphid feeding. However, honeydew excretion was comparable on plants with and without JA treatment, indicating that JA-dependent defenses did not deter feeding. This suggests that the effect of JA on aphid performance was due to anti-digestive and/or toxic factors. These results could explain why activation of JA-dependent defenses didn’t enhance resistance conferred by Mi-1.2. Potentially, the effects that Mi-mediated resistance has on aphid feeding could inhibit aphids from coming into contact with JA-induced resistance factors.

We also evaluated whether artificial induction of acquired resistance would enhance Mi-mediated resistance against root-knot nematodes. Mi-1.2 is the only known source of resistance against root-knot nematodes in cultivated tomato, and this resistance has remained stable throughout decades of use. Despite the importance of resistant cultivars, the effectiveness of Mi-mediated resistance is limited in certain growing regions. Mi-mediated resistance is temperature-sensitive, and becomes unstable at soil temperatures above 30°C. This could limit the use of resistant cultivars in tropical and subtropical tomato growing regions. Major goals of this study were to determine the effects of JA-dependent defenses in tomato on root-knot nematode performance, and to determine if JA treatment can enhance nematode protection offered by Mi-1.2. This study investigated whether artificial induction of JA could provide tomato plants with protection against nematode infection at high soil temperatures. The results from this study indicate that JA application induces a systemic defense response in the roots of susceptible tomato, and has a negative impact on root-knot nematodes. Furthermore, we found that JA-dependent defenses are heat-stable, and protect tomato at temperatures that reduce the effectiveness of Mi-mediated resistance. At 25°C, JA application did not significantly enhance or inhibit resistance conferred by Mi-1.2; however, JA treatment may potentially enhance nematode control on resistant plants at 30°C. These results are the first to indicate that JA-dependent defenses are effective against a parasitic nematode, and to suggest that artificial induction of JA could be used to control root-knot nematodes on tomato.

Introduction

Tomato production is an important component of American agriculture. On a list of the top 34 vegetable crops in the U.S., fresh market tomatoes rank sixth in total acreage and second in total value grossing over one billion dollars in 2001 (United States Department of Agriculture, 2002). Tomato production is particularly important to the economy of the Southern United States. Of the thirteen states that represent the Southern growing region, as defined by the Sustainable Agriculture Research and Education (SARE) program, commercial tomato production is found in nine of these states (AL, AR, FL, GA, NC, SC, TN, TX, and VA). In fact, the South comprises 49% of the country’s fresh market tomato acreage, and produces 57% of the national commodity (United States Department of Agriculture, 2002). Tomato is also important because it acts as an alternative crop for small Southern tobacco farms affected by declined profitability in tobacco production (SARE, 2002).

Despite its economic importance, the fresh market tomato industry is currently endangered due to its dependence on pesticides that are disappearing from the market. Southern tomato growers are particularly threatened by the phase-out of methyl bromide and the proposed ban on organophosphate and carbamate pesticides. Methyl bromide is an effective soil fumigant that is widely used to control root-knot nematodes on tomato (Bloem et at. 2001). The Environmental Protection Agency (EPA) mandated the phase-out of methyl bromide fumigation by the year 2005. The loss of this pesticide could cause a substantial reduction in the nation’s total tomato acreage; for example, it is predicted that Florida could experience a 69% reduction in tomato acreage (Spreen et al., 1995). To date, no suitable alternative offers the same broad-spectrum control (Bloem et al., 2001). Tomato producers also rely heavily on organophosphates and carbamates to control insects and other pests, including the potato aphid and root knot nematodes. Many insecticides belonging to these two pesticide families have already been banned or heavily restricted. Legislation is pending that will mandate the ban of both pesticide families entirely from the market. The loss of these insecticides is predicted to result in a 13% increase in production costs for tomato growers, 15% decreased yield, and increased competition from foreign markets (FFBF, 1999). The state most vulnerable to reduced yields and increased production costs is Florida, with a predicted 21% loss in yield production and 20% cost increase (FFBF, 1999). In addition to reduced yields and increased production cost, the ban of these pesticides will result in more food imports, higher food prices for American consumers, and less consumption of nutritionally important fruits and vegetables, according to a study compiled by Texas A & M University’s Agricultural and Food Policy Center (Knutson, et al., 1999).

The continuing loss of widely used pesticides from the market clearly demonstrates the need to develop effective alternative pest control strategies for tomato. The primary goal of this research was to identify effective alternative management strategies for two important pests of tomato: root-knot nematodes (Meloidogyne spp.), and potato aphids (Macrosiphum euphorbiae). Root-knot nematodes are soil-borne roundworms that parasitize the root systems of various vegetable crops including tomato. Symptoms include production of root galls, increased susceptibility to drought stress and pathogen attack, and premature death. Nematode infestations are known to cause substantial tomato yield reduction, making root-knot nematodes among the most significant economical pests worldwide (Williamson et. al. 1996). Potato aphids are an emerging pest of tomato that cause leaf curling, chlorosis, production of sooty mold, and increased feeding by hemipterous insects resulting in yield reduction (Walgenbach, 1997). This insect is becoming an increasingly significant problem for tomato growers because of the continuing loss of broad-spectrum insecticides from the market (Walgenbach, 1997). The combined use of resistant tomato varieties and plant defense elicitors could offer effective control against aphids and nematodes in the absence of the disappearing pesticides.

Many commercial tomato varieties are resistant to aphids and certain species of nematodes, due to a single dominant gene (Mi-1.2) that was introduced through traditional breeding practices. Mi-1.2 is used as the only source of nematode resistance in cultivated tomato and varieties possessing this gene have been widely used for decades. These resistant varieties provide highly effective nematode control at moderate soil temperatures, but have reduced effectiveness at high temperatures. Nematode resistance begins to diminish at soil temperatures above 28°C, and is nearly 100% susceptible at 32°C (Dropkin, 1969). This suggests the use of resistant varieties could be limited to cooler spring temperatures in some southern states (Noling, 2002). Mi-mediated resistance is also effective against potato aphids with some limitations. Unlike nematode resistance that is expressed throughout the entire life of the plant, resistance against aphids is not expressed until about the plant’s reproductive age, or about six weeks after germination (Kaloshian, et al., 1995). This leaves juvenile resistant varieties susceptible to aphid infestation. Another limitation to aphid resistance is that Mi-1.2 is not effective against all biotypes of potato aphid (Goggin et al., 2000). These limitations suggest the need for additional alternative control methods.

The use of plant defense elicitors to “immunize” plants could offer promising additional control strategies against nematodes and aphids in tomato. Plant defense elicitors are molecules that trigger induced defensive responses in plants, and thereby render plants more resistant to pests. Defense elicitors have been used to develop many new reduced risk pesticides and plant activators; for example, defense elicitors are used as active ingredients in Actigard (Syngenta Crop Protection), Messenger (Eden Bioscience), ReZist, and CaB’y (Stoller Enterprises). Of the plant elicitors that have been studied most extensively, most act by inducing one of two distinct defensive pathways in the plant: salicylic acid mediated resistance and jasmonic acid mediated resistance.

Salicylic acid (SA) is a plant-signaling compound that plays a critical role in the induction of systemic acquired resistance to certain pests, such as many bacterial, viral, and fungal pathogens. Elicitation of SA-mediated defenses is associated with induction of pathogenesis-related proteins in the plant, and localized cell death surrounding the invading pest. Elicitors of SA-dependent defenses have been shown to reduce nematode performance on susceptible tomato varieties (K. Sitaramaiah et al., 1979; Nandi, B. et al., 1999). The effects of SA-mediated defenses on aphid performance on tomato have not yet been determined; however, SA appears to be involved in aphid resistance in winter wheat (Mohase, 2002). The first objective of this project was to determine if an SA elicitor protects susceptible tomato varieties against aphid infestation. Another objective of this work was to determine if an SA elicitor is appropriate for use on resistant varieties. Induction of SA is required for the function of Mi-1.2 (Branch et al., 2004). Potentially, prior induction of SA may enhance aphid and/or nematode resistance in tomato varieties that carry Mi-1.2. Alternatively, an SA elicitor could interfere with the function of Mi-1.2 (Vasyukova et al., 1999); therefore, it is important to determine if SA elicitors are compatible with aphid and nematode resistant varieties.

We also tested the effects of another plant signaling compound, jasmonic acid (JA), on aphids and nematodes. JA is involved in induced resistance to many insects, and triggers increased polyphenol oxidase activity and enhanced expression of proteinase inhibitors in plants. In tomato, JA is known to play a role in induced defenses against the tobacco hornworm, corn earworm, and other chewing insects. The first goal of this study was to determine if JA application reduces aphid and/or nematode performance on a tomato cultivar lacking Mi-1.2. Another goal was to investigate whether or not JA elicitors can be used on resistant tomato varieties. Signaling conflicts are known to exist between SA and JA; for example, induction of JA-mediated insect resistance in tomato renders plants more susceptible to bacterial speck (Thaler et al., 1999). Therefore, it is important to determine whether JA elicitors will enhance or inhibit the effectiveness of aphid- and nematode-resistant tomato varieties.

References

Bloem, S., Mizell, R.E. 2001. Tomato IPM in Florida. Department of Entomology and Nematology, Florida Coop. Ext. Serv., Institute of Food and Agricultural Sciences, University of Florida 1-22.

Branch C, Hwang CF, Navarre DA and Williamson VM. 2004. Salicylic acid is part of the Mi-1-mediated defense response to root-knot nematode in tomato. Mol. Plant Microbe Interact. 17, 351-356

Dropkin, V.H. 1969. The necrotic reaction of tomatoes and other hosts resistant to Meloidogyne: Reversal by temperature. Phytopathol. 59, 1632-1637.

FFBF (Floridia Farm Bureau Federation). 1999. All Pain, No Gain: FFBF News Release. 1-6.

Goggin, F.L., Williamson, V.M., and Ullman, D.E. 2001. Variability in response of Macrosiphum
euphorbiae and Myzus persicae (Hemiptera: Aphididae) to the tomato resistance gene Mi.
Environ. Entomol. 30(1), 101-106.

Knutson, R.D., Smith, E.G. 1999. Economic impacts of the elimination of organophosphates and carbamates onTexas Agriculture. AFPC Policy Working Paper 99-3. Agricultural and Food Policy Center, Department of Agriculture Experiment Station. Texas Agriculture Extension Service pp. 11.

Kaloshian, I., Lange, W.H., Williamson, V.M. 1995. An aphid-resistance locus is tightly linked to the nematode-resistance gene, Mi, in tomato. Proc. Natl. Ac. Sci. 92, 662-625

Mohase, L. Westhuizen A.J. 2002. Salicylic acid is involved in resistance responses in the Russian wheat aphid–wheat interaction. J. Plant Physiol. 159(6), 585-590.

Nandi, B., Sukul, N.C. and Sinha Babu, S.P. 2000. Exogenous salicylic acid reduces Meloidogyne incognita infestation of tomato. Allelopathy J. 7, 285-288

Noling, J.W. Nematodes and their management. University of Florida Coop Extension. http://edis.ifas.ufl.edu/BODY_CV112.

Sitaramaiah, K., Pathak, K.N. 1979. Effect of phenolics and an aromatic acid on Meloidogyne javanica infecting tomato. Nematologica 25, 281-287.

Spreen, T.H., J.J. VanSickle, A.E. Moseley, M.S. Deepak and L. Mathers. 1995. Use of methyl bromide and the economic impact of it’s proposed ban on the Florida fresh fruit and vegetable industry. IFAS Bulletin 898 (tech), University of Florida, Gainsville, FL.

Sustainable Agriculture Research and Education Program (SARE). 2002. Meeting the diverse needs of limited-resource producers. www.sare.org/bulletin /limited-resource/profile8.htm.

Thaler, J.S., Stout, M.J., Karban, R., and Duffey, S.S. 2001. Jasmonate-mediated induced plant resistance affects a community of herbivores. Ecol. Entomol. 26, 213-324.

Thaler, J.S., Fidantsef, A.L., Duffey, S.S. Bostock, R.M. 1999. Trade-offs in plant defense against pathogens and herbivores: A field demonstration of chemical elicitors of induced resistance. J. Chem. Ecol. 25, 1597-1609.

USDA-NASS (United States Department of Agriculture-National Agricultural Statistics Service). 2002. Vegetables 2001 Summary. 4-43.

Vasyukova, N.I., Zinov’eva, Il’inskaya, Gerasimova, Udalova. 1999. Salicylic acid and interaction between tomato plants and root-knot nematode, Meloidoagyne incognita. Doklady, Biol. Sci. 364, 82-84.

Walgenbach, J.F. 1997. Effect of potato aphid (Homoptera: Aphididae) on yield, quality, and economics of staked-tomato production. Hort. Entomol. 90(4), 996-1004.

Williamson, V.M., Hussey, R.S. 1996. Nematode pathogenesis and resistance in plants. Plant Cell8, 1735-1745.

Zinov’eva, Vasyukova, Ozeretskovskaya, Sonin. 1998. Resistance of tomato plants to root-knot nematodeMeloidogyne incognita induced by arachidonic acid and methyl jasmonate. Doklady, Biol.Sci. 363, 587-589.

Project Objectives:

A major objective of this study was to determine if elicitors of SA- and JA-mediated plant defenses could protect tomato plants against aphid and root-knot nematode infestation. A second objective was to determine if the use of these elicitors is compatible with the use of aphid- and nematode-resistant tomato varieties. Specific objectives included:

1. Explore the impact of SA-dependent defenses against the potato aphid on tomato cultivars with and without the resistance gene Mi-1.2

2. Explore the impact of JA-dependent defenses against the potato aphids on tomato cultivars with and without the resistance gene Mi-1.2.

3. Explore the impact of JA-dependent defenses against root-knot nematodes on tomato cultivars with and without the resistance gene Mi-1.2, and determine whether JA-dependent defenses protect resistant tomato at higher temperatures.

Research

Materials and methods:

1) Effects of SA-Dependent Defenses on Aphid Performance.

Plant and insect materials. Two near-isogenic tomato cultivars with and without Mi-1.2 were used for our studies: Moneymaker (Mi-), and Motelle (Mi+). All plants were grown in 3.8 liter pots of LC1 Sunshine potting mix (Sungro Horticulture, Belevue, WA) under stable greenhouse conditions (~24-27°C; 16:8 L:D photoperiod). Plants were watered daily with a dilute nutrient solution containing 1000 mg/L CaNO3 (Hydro Agri North America, Tampa, FL), 500 mg/L MgSO4 (Giles Chemical Corp, Waynesville, NC), and 500 mg/L Hydroponic 4-18-38 Growmore fertilizer (Growmore, Gardena, CA).

Two potato aphid isolates, which we designated as WU11 and WU12, were utilized for this study. Isolate WU11 originated from the laboratory colony of Dr. Yvan Rahbe, and aphid isolate WU12 was obtained from the laboratory colony of Dr. Stuart Seah. Aphids were maintained in Conviron growth chambers (Controlled Environments, Inc., Winnipeg, Canada) under optimal conditions for aphid development (20°C, 16:8 L:D photoperiod).

Benzothiadiazole application. SA-dependent defenses were induced in tomato by applying a foliar treatment of benzothiadiazole (BTH), a synthetic analog of SA. Exogenous treatments of SA and BTH have both been shown to induce acquired resistance to pathogens and induction of associated pathogenesis-related proteins (Friedrich et al., 1996; Gorlach et al., 1996; Lawton et al., 1996). We chose to use BTH rather than SA as a defense elicitor because it lacks the phytotoxic effects associated with SA, and has stronger systemic effects (Friedrich et al., 1996). BTH (Syngenta Crop Protection, Greenboro, N.C.) was dissolved in acetone at a rate of 28 g/l and dispersed in water to achieve a 1.2 mM BTH solution (Friedrich et al., 1996). For the control treatments, an equal quantity of acetone (without BTH) was dispersed in water. Approximately six weeks after germination, at which stage Mi-mediated resistance is active in the foliage, tomato plants were sprayed with BTH solution or control solution applied at a rate of 1 ml per leaf using an atomizer (~12 ml/plant). The eighth leaf from the cotyledon of each plant was protected from treatment using a plastic bag, which was removed when all other leaves were dry. Aphid bioassays (described below) were performed using this untreated leaf, so that we could measure the effects of acquired resistance on aphids independent of any effects that BTH residue might have on the insects.

Effects of SA-dependent defenses on aphid population growth. The effects of BTH on population growth of two aphid isolates (designated WU11 and WU12) were measured using two independent bioassays. In each assay, aphid performance was measured on Moneymaker (Mi-) or Motelle (Mi+) sprayed with either BTH or control solution. Forty-eight hours after treatment application, the terminal leaflet of the eighth leaf from the cotyledon was inoculated with 15 age-synchronized aphids, which were confined to single leaflets using a sleeve cage (12 plants/treatment for the WU11 assay; 8 plants/treatment for the WU12 assay). Six days after inoculation, aphid performance was evaluated by counting the total number of aphids/cage. Bioassays were performed in a Conviron growth chamber (20°C; 16:8 L:D photoperiod).

For each assay, foliar application and genotype were compared as independent fixed factors by full factorial two-way ANOVA using JMP version 5.01 (SAS, Cary, North Carolina). Treatment combinations were analyzed by Tukey’s HSD statistics and paired t-tests using JMP version 5.01.

2) Effects of JA-Dependent Defenses on Aphid Performance

Plant and insect materials. We used the plant cultivars, insect isolates, and growing conditions described above for the BTH assays.

Jasmonic acid application. JA-dependent defenses were induced in tomato by applying a foliar treatment of exogenous JA, as previously described by Thaler et al. (1999). Jasmonic acid (Sigma Chemicals, St. Louis, MO) was dissolved in acetone at a rate of 1 g/ml and dispersed in water to achieve a 1.5 mM JA solution (Thaler et al., 1999). An equal quantity of acetone was dispersed in water (without JA) for control treatment. JA and control treatments were applied six weeks after germination as described for BTH. One leaf per plant was protected from treatment for use in aphid bioassays.

Aphid population growth assays. The effects of JA on aphid isolates WU11 and WU12 were tested in the same manner as the effects of BTH described above (13 plants/treatment for the WU11 assay; 9 plants/treatment for the WU12 assay). Plants sprayed with JA versus control treatments were maintained in separate growth chambers to insure that volatiles from plants treated with JA would not affect control plants.

Adult longevity and fecundity. Individual adult aphid performance was compared on susceptible Moneymaker (Mi-) with and without JA treatment. Forty-eight hours after JA application, WU11 aphids were caged singly on the penultimate leaflets of the study leaf (16 replicate plants/treatment; 2 cages/plant). All aphids used for this study were apterous adults that had emerged to adulthood within 24 hours prior to the experiment. Aphids were confined to single leaflets using Tape Shape traps (Converters Inc, Huntington, PA) with organza covers to allow airflow and easy access to the aphid while minimizing damage to the leaflets. Aphid status (dead or alive) and fecundity were recorded daily until all aphids died. Juvenile aphids were removed from the cages on a daily basis. A complete adult life table was constructed to calculate life expectancy (e), survivorship (lx), net reproduction (number of offspring/female), and instantaneous rate of increase (number of offspring/female/unit time over which progeny were produced) (Carey 1993). This study was performed under controlled greenhouse conditions (~18-22°C; 16:8 L:D photoperiod). Plants were separated according to foliar treatment to prevent volatiles induced by JA from eliciting defenses in control plants. This study was repeated on resistant Motelle tomato (Mi+).

Adult longevity was analyzed using log-rank Chi Square test of equality over strata (PROC LIFETEST) (Allison 1995), using SAS version 7.0 (SAS, Cary, North Carolina). Average lifetime fecundity/female and average daily fecundity/female was analyzed by two-way analysis of variance (ANOVA) using JMP version 5.01 (SAS, Cary, North Carolina).

Juvenile development and mortality. The effects of JA-dependent defenses on aphid juveniles were evaluated on susceptible Moneymaker (Mi-) tomato (10 replications/treatment; 2 sub-replications/plant). To produce age-synchronized first instar aphids, apterous adult aphids (isolate WU11) were caged singly on study leaflets in Tape Shape traps, and then removed twenty-four hours later after producing at least one juvenile. All but one juvenile were removed from each cage so that development of individual aphids could be monitored. The status (dead or alive) was recorded daily until all aphids died or reached maturity. Aphid development was studied by monitoring the production of exuvia. We also evaluated the effect of IR on juvenile survivorship and development time of the second generation of aphids born on JA induced plants. Adults that reached maturity were removed and JA reapplied to all plants as described above. After all leaves were dried, aphids were placed back on respective plants. Adults were allowed to produce juveniles as previously described and juvenile performance evaluated and analyzed in the same manner as the previous generation. This study was repeated on resistant Motelle (Mi+) tomato.

Juvenile mortality was analyzed by assigning “death” with a value of 1, and alive (reached adult status) with a value of 0, and analyzed by ChiSquare test of likelihood ratio using JMP version 5.01. Development time (number of days to reach adulthood) was analyzed by two-way ANOVA using JMP version 5.01.

Aphid feeding rates. Aphid feeding was compared on susceptible Moneymaker (Mi-) and resistant Motelle (Mi+) with and without JA treatment in a full factorial design. Feeding rates were evaluated by assessing honeydew excretion, which has been shown to correlate with rates of sap ingestion (Banks et al. 1964). Third instar juvenile aphids (isolate WU11), rather than adults, were used for this study so that the honeydew production per aphid could be measured without any honeydew contribution from offspring. Forty-eight hours after JA application, juveniles were placed in pairs on the terminal leaflet of each study leaf. Aphids were confined to single leaflets using an organza sleeve cage. Sheets of VWR 415 qualitative filter paper (VWR, West Chester, PA) were cut to the size of the sleeve cages and placed on the underside of each leaflet. Status (dead or alive) and location of aphids were checked daily. All aphids remained on the underside of the leaflets throughout the duration of the study. After 3 days of feeding, filter papers were removed, stained with 1% ninhydrin/ethanol solution, and developed at 60°C for 10 minutes (Minks et al. 1987). Feeding rates were evaluated by calculating the number of honeydew droplets/aphid/day. Data were analyzed as independent factors in a full factorial analysis using JMP 5.01, and mean separation was achieved using LSD statistics.

3) Effects of JA-dependent Defenses Against Root Knot Nematodes

Plant material. Two near-isogenic cultivars of tomato, Moneymaker (Mi-), and Motelle (Mi+), were used for our bioassays. All plants were grown in 0.95 liter styrofoam cups (Dart Container Company, Mason, MI) of autoclaved sand (Quikrete, Atlanta, GA) under stable greenhouse conditions (~24-27°C; 16:8 L:D photoperiod). Plants were watered daily with the modified Gromore solution described above.

JA application. Entire plants were sprayed with JA or control solutions described above at the 4-leaf stage at a rate of 1 ml per leaf.
Effects of JA application on nematode performance. The effects of JA application on M. javanica were measured on tomato cultivars, Moneymaker (Mi-) and Motelle (Mi+), sprayed with either JA or carrier solution. Forty-eight hours after chemical treatment, the sand surrounding the roots of each plant were injected with ~3,000 second-stage juvenile (J2) avirulent VW4 nematodes. Nematode performance was compared between treatment groups by measuring the reproduction of the emerging adults 7 weeks after inoculation. Nematode egg masses were stained by immersing washed root systems for 10 minutes in a solution of eurioglaucine (0.1g/liter water) (Sigma Chemicals, St. Louis, MO) (Yaghoobi et al. 1995). Total egg mass numbers per plant were counted and the root systems were oven-dried and weighed.

In order to compensate for variation in root weights, we evaluated nematode performance using the number of egg masses/dry root weight/plant. Unequal variances were stabilized by log transformation using the equation log(Y+1) where Y is egg masses/dry root weight/plant (Gomez et al. 1984). Statistical comparisons were performed using JMP version 5.01 (SAS, Cary, NC). For each assay, foliar treatment and genotype were compared as independent fixed factors by full factorial two-way ANOVA. Comparisons between treatment combinations were analyzed by paired t-test.

The effects of temperature on JA-Dependent Defenses. The effects of temperature on JA-dependent defenses were measured on tomato cultivars Moneymaker (Mi-) and Motelle (Mi+) at 32°C (7 reps/treatment group). Twenty-four hours prior to chemical treatment, plants were placed in a Conviron growth chamber to achieve the desired sand temperature. All plants were treated with a foliar application of JA or carrier solution and inoculated with VW4 nematodes as described above. Plants were maintained in growth chambers for seven days after nematode inoculation to allow enough time for establishment, and were then moved back to greenhouse benchtops. Plants were watered twice daily by drip irrigation with the modified Gromore solution described above. Nematode performance was measured 7 weeks after inoculation as described above.

This experiment was repeated with the addition of a second set of plants kept at 25°C. In order to control root temperatures more accurately, plants were watered by hand twice a day with nutrient solution acclimated to the growth chambers’ temperature. Nematode performance was measured 7 weeks after inoculation as described above. Data were analyzed as described above for nematode bioassays.

A third study was performed at 30°C (20 reps/treatment group) using experimental procedures described above.

References

Allison PD. 1995. Survival analysis using the SAS system; A practical guide. SAS Institude Inc., Cary, NC.

Banks CJ and MacCaulay EDM. 1964. The feeding, growth and reproduction of Aphis fabae on Vicia faba under experimental conditions. Ann. Appl. Biol. 53: 229-242

Carey JR. 1993. Applied Demography for Biologists with Special Emphasis on Insects. Oxford University Press, New York.

Friedrich L, Lawton K, Wilhelm R, Masner P, Specker N, Rella MG, Meier B, Dincher S, Staub T, Uknes S, Metraux JP, Kessmann H and Ryals J. 1996. A benzothiadiazole derivative induces systemic acquired resistance in tobacco. Plant J. 10: 61-70

Gomez KA and Gomez AA. 1984. Statistical procedures for agricultural research. Wiley-Interscience, New York, NY.

Gorlach J, Volrath S, Knauf-Beiter G, Hengy G, Beckhove U, Kogel KH, Oostendorp M, Staub T, Ward E, Kessmann H and Ryals J. 1996. Benzothiadiazole, a novel class of inducers of systemic acquired resistance, activates gene expression and disease resistance in wheat. Plant Cell B: 629-643

Lawton K, Friedrich L, Hunt M, Weymann K, Delaney TP, Kessmann H, Staub T and Ryals J. 1996. Benzothiadiazole induces disease resistance in Arabidopsis by activation of the systemic acquired resistance signal transduction pathway. Plant J. 10: 71-82

Minks AK and Harrewijn P. 1987. Aphids: their biology, natural enemies, and control. Elsevier,
Amsterdam, NY.

Thaler JS, Fidantsef AL, Duffey SS and Bostock RM. 1999. Trade-offs in plant defense against pathogens and herbivores: A field demonstration of chemical elicitors of induced resistance. J. Chem. Ecol. 25: 1597-1609

Yaghoobi J, Kaloshian I, Wen Y and Williamson VM. 1995. Mapping a new nematode resistance locus in Lycoperiscon peruvianum. Theoret. Appl. Genet. 91: 457-464

Research results and discussion:

1) Effects of SA-Dependent Defenses on Aphid Performance.

Effects of BTH application on aphid population growth. WU11 aphid numbers after 6 days of feeding differed significantly among treatment groups (P=<0.0001). There was no significant interaction between plant genotype and foliar treatment (P=0.265). Aphid numbers were significantly lower on Motelle (Mi+) plants compared to Moneymaker (Mi-) (P=<0.0001), which confirms previous findings that WU11 is impacted by Mi-1.2 (Goggin et al. 2001). Aphid numbers were also reduced by BTH application (P=0.0131). Although BTH treatment reduced aphid numbers on the susceptible cultivar Moneymaker (P=0.0127), it did not significantly enhance aphid control on the resistant cultivar Motelle (P=0.2783). WU12 aphid numbers after six days of feeding differed significantly among treatments
(P=<0.0001). There was no significant
interaction between Mi-mediated resistance and BTH application (P=0.161). Aphid numbers were significantly lower on Motelle versus Moneymaker plants (P=0.0076), which confirm previous findings that Mi-1.2 is active against aphid isolate WU12 (Goggin et al. 2004). Aphid numbers were also significantly lower on plants treated with BTH compared to control plants (P=<0.0001). BTH treatment significantly reduced aphid numbers on both Moneymaker and Motelle (P=<0.001 and P=0.022 respectively). Discussion. The results from this population growth study reveals that artificial induction of SA-dependent defenses using BTH application has a negative impact on potato aphid populations, and does not interact antagonistically with resistance conferred by Mi-1.2. Furthermore, BTH treatment can enhance Mi-mediated resistance against certain potato aphid isolates. These results suggest that this elicitor could potentially be a useful pest management strategy against potato aphids on resistant and susceptible tomato cultivars. Experiments are underway to also evaluate the effects of BTH on nematode performance and Mi-mediated nematode resistance.
2) Effects of JA-Dependent Defenses on Aphid and Nematode Performance

Effects of JA application on aphid population growth. WU11 aphid numbers differed significantly among treatments after 6 days of feeding (P=<0.0001). There was a significant interaction between plant genotype and foliar application (P=0.0083). Comparisons between Moneymaker and Motelle plants sprayed with the control treatment revealed that Mi-1.2 had a strong negative effect on aphid numbers (P=<0.0001). JA treatment significantly reduced aphid numbers on the susceptible cultivar Moneymaker (P=0.0013), but did not influence aphid numbers on the resistant cultivar Motelle (P=0.6322). WU12 aphid numbers after 6 days of feeding differed significantly among treatments (P=0.049). There was no significant interaction between genotype and JA application (P=0.1579). The overall effect of the plant genotype was not statistically significant at a=0.05 confidence interval (P=0.0815), although aphid numbers on control plants were significantly lower on Motelle versus Moneymaker control plants (P=0.0022). Aphid numbers were significantly reduced on plants sprayed with JA compared to control plants (P=0.0022), although JA did not significantly enhance aphid control on the resistant cultivar Motelle (P=0.1846). Adult longevity and fecundity. Aphid longevity was significantly reduced on susceptible tomato (Mi-) sprayed with JA compared to tomato sprayed with carrier solution (P=0.0001). Aphids on plants treated with JA reached 50% (lx=0.5) and 90% (lx=0.1) mortality in half the time required for aphids on control plants. Compared to aphids on plants sprayed with carrier solution, the life expectancy (e) of aphids on induced plants at days zero, ten and twenty are 2.5, 3.8, and 1.7 times lower, respectively. Average lifetime fecundity/female was significantly lower on susceptible (Mi-) plants treated with JA compared to susceptible plants treated with carrier solution (P=0.0007). In contrast, average daily fecundity/living female was not significantly reduced by JA treatment (P=0.9261). JA treatment on resistant (Mi+) tomato did not impact adult longevity (P=0.7495), daily fecundity (P=0.0662), or total lifetime fecundity (P=0.2451). Juvenile development and mortality. The nutritional uptake of the parent aphid can have an impact on the mortality rates and development time of its offspring (Kennedy et al. 1959). It is therefore important to evaluate the effect of IR on multiple generations of aphids. In this experiment, juvenile mortality was significantly higher on susceptible plants sprayed with JA compared to control for both generations studied. For the first generation of juveniles born on treated plants, mortality was increased by ~25% on JA-induced plants compared to plants treated with carrier solution (P=0.0289). Second-generation mortality was increased by 40% on JA-induced plants (P=0.0065). Juvenile development times were not significantly different on susceptible (Mi-) plants treated with JA or carrier solution for first generation aphids (P=0.9918) or second-generation aphids (P=0.2587). The number of days required to reach adulthood was ~9 days for each treatment group, and all aphids that survived to adulthood passed through a normal number of juvenile instars. Juvenile mortality rates of the first generation of aphids born on resistant tomato were not significantly different between treatments (P=0.1105). Due to the high mortality of first generation aphids on resistant plants, second generation performance and development time could not be measured. Aphid feeding rates. Treatment differences exist for aphid feeding response (P=0.0367). Aphid feeding was significantly reduced on resistant tomato compared to susceptible tomato, confirming previous findings that Mi impacts aphid feeding (P=0.0177) (Kaloshian et al. 1997). In contrast, JA application did not inhibit aphid feeding (P=0.1046). There was no significant interaction between genotype and foliar treatment (P=0.4872). Discussion. These results reveal that JA- dependent defenses have a negative impact on potato aphid populations, and do not interact antagonistically with resistance conferred by Mi-1.2. In order to characterize the impact of JA-dependent defenses on aphid population growth, we also tested the effects of JA treatment on individual aphid life parameters. Our results indicate that JA treatment reduces adult longevity and lifetime fecundity, but not daily fecundity. Since JA treatment did not have an impact on daily fecundity, the reduced lifetime fecundity is probably a result of reduced longevity. Our results also indicate that JA treatment has a negative impact on the number of juveniles to reach maturity, but does not impact development time. These results indicate that JA-dependent defenses primarily impact aphid population growth by reducing the survivorship of juvenile and adult aphids. We also tested the effects of JA treatment on aphid feeding rates. The results from our feeding assays confirm that Mi-mediated resistance, but not JA-dependent defenses, deters aphid feeding. This suggests that the impact JA-dependent defenses have on aphids is due to a toxic and/or anti-digestive rather than an anti-feeding factor. This could explain why JA-dependent defenses are not effective on resistant tomato. Potentially, reduced aphid feeding by Mi-1.2 could inhibit contact with JA induced toxic/anti-digestive compounds. 3) Effects of JA-dependent Defenses Against Root-Knot Nematodes Effects of JA application on nematode performance. Nematode egg mass production by nematode differed significantly among treatment groups (P=<0.0001). There was a significant interaction between tomato cultivars and chemical treatment (P=0.0.0137), and so the effects of plant genotype and JA treatment were analyzed separately. Nematode performance was dramatically reduced on the resistant cultivar Motelle (Mi+) compared to the susceptible cultivar Moneymaker (Mi-) (P=<0.0001 for plants sprayed with control solution; P=<0.0001 for plants sprayed with JA), which confirms previous findings that that Mi-mediated resistance is effective against nematode isolate VW4 (Milligan et al. 1998; Lambert et al. 1999). On Moneymaker, egg mass production was significantly lower on plants treated with JA than on plants sprayed with control solution (P=0.0002). In contrast, JA application did not impact nematode performance on Motelle tomato (P=0.5594). Effects of temperature on JA-dependent defenses. When the effects of JA treatment on Moneymaker and Motelle were tested at 32°C, nematode egg mass production differed significantly among treatment groups (P=<0.0001). There was no significant interaction between plant genotype and chemical treatment (P=0.3665). Although nematode egg mass production was greater than typically observed at lower temperatures, the overall effect of plant genotype was significant, indicating that Mi-1.2 was not completely inhibited by high soil temperature (P=<0.0001). The overall effect of JA treatment was statistically significant, which demonstrates that JA-dependent defenses are effective against nematodes at 32°C (P=0.0500). Nematode reproduction on the susceptible cultivar Moneymaker was significantly reduced by JA application (P=0.0454). In contrast, JA application did not dramatically reduce nematode performance on the resistant cultivar Motelle (P=0.4266). An additional experiment was performed to compare the effects of JA treatment and Mi-mediated resistance at 25°C versus 32°C. Nematode egg mass production differed significantly among treatment groups (P=<0.0001), and there were no significant interactions among plant genotype, chemical treatment, and temperature (P=0.2288). Nematode reproduction was significantly lower on Motelle (Mi+) than on Moneymaker (Mi-) at both soil temperatures (P=<0.0001 for plants at 25°C; P=0.0043 for plants at 32°C), although egg mass numbers on Motelle increased dramatically with temperature (P=<0.0001). JA treatment reduced nematode performance on Moneymaker at both soil temperatures (P=0.0081 for plants at 25°C; P=0.0496 for plants at 32°C). On Motelle, JA treatment had no effect at 25°C (P=0.9328). At 32°C, nematode performance was lower on Motelle plants treated with JA than on Motelle sprayed with control solution, but this difference was not significant at the a=0.05 confidence interval (P=0.0808). A third experiment was performed to compare the effects of JA treatment and Mi-mediated resistance at 30°C. Nematode egg mass production differed significantly among treatment groups (P=<0.0001), and there were significant interactions between plant genotype and chemical treatment (P=0.3619). Nematode reproduction was significantly lower on Motelle (Mi+) than on Moneymaker (Mi-) (P=0.0001). The overall effect of JA treatment was significant, which confirms that JA-dependent defenses are effective at 30°C. JA application reduced nematode performance on both tomato cultivars tested (P=0102 on susceptible Moneymaker; P=0343 for resistant Motelle). Discussion. The results from our studies indicate that JA-dependent defenses reduce root-knot nematode performance on susceptible tomato and do not impact Mi-mediated resistance. Our results also indicate that JA-dependent defenses are also heat-stable, and may offer protection on resistant tomato against root-knot nematodes at high temperatures. These findings are the first to report that JA-dependent defenses are effective against a parasitic nematode. References Goggin FL, Williamson VM and Ullman DE. 2001. Variability in the response of Macrosiphum euphorbiae and Myzus persicae (Hemiptera: aphididae) to the tomato resistance gene Mi. Environ. Entomol. 30: 101-106 Goggin FL, Gowri S, Williamson VM and Ullman DE. 2004. Developmental regulation of Mi-mediated aphid resistance is independent of Mi-1.2 transcript levels. Mol. Plant Microbe In. 17: 532-536 Kennedy JS and Stroyan HLG. 1959. Biology of Aphids. Annu. Rev. Entomol. 4: 139-160 Lambert KN, Ferrie BJ, Nombela G, Brenner ED and Williamson VM. 1999. Indentification of genes whose transcripts accumulate rapidly in tomato after root-knot nematode infection. Physiol. Plant Pathol. 55: 341-348 Milligan SB, Bodeau J, Yaghoobi J, Kaloshian I, Zabel P and Williamson VM. 1998. The root knot nematode resistance gene Mi from tomato is a member of the leucine zipper, nucleotide binding, leucine rich repeat family of plant genes. Plant Cell 10: 1307-1319

Participation Summary

Educational & Outreach Activities

Participation Summary

Education/outreach description:

Presentations
Cooper, W. R., and Goggin, F. L. 2003. Compatibility of Induced Resistance and Mi-
mediated Aphid Resistance in Tomato. National Entomological Society Meeting
in Cincinnati OH. Poster Session.

Cooper, W. R. and Goggin F. L. 2004. Effects of jasmonate-dependent defenses in tomato on the potato aphid, Macrosiphum euphorbiae. To be presented in a Poster Session at the 2004 National Entomological Society Meeting in Salt Lake City UT.

Cooper, W. R., and Goggin, F. L. 2004. Jasmonate Dependent Defenses in Tomato Against the Potato Aphid. Ohio Valley Entomological Society Meeting in Hanover Indiana. Student Presentation.

Publications
Cooper, W. R., Jia, L., and Goggin, F. L. 2004. Acquired and R-gene mediated resistance against the potato aphid in tomato. Journal of Chemical Ecology. In
Press.

Cooper, W. R. and Goggin, F. L. 2004. Effects of jasmonate-dependent defenses in tomato on the potato aphid, Macrosiphum euphorbiae. Submitted to Entomologia Experimentalis et Applicata.

Cooper, W. R. and Goggin, F. L. 2004. Effects of jasmonate-dependent defenses in tomato on root-knot nematodes, Meloidogyne javinica. To be submitted to Journal of Nematology.

Project Outcomes

Project outcomes:

The use of plants’ natural defenses is becoming increasing important to tomato production due to the continuing loss of broad-spectrum pesticides from the market. Commercially available products that induce JA and SA defenses are available for crop protection against certain insects and pathogens. The results from this study show that artificial induction of JA and SA significantly reduces both aphids and root-knot nematodes. This work is of practical value because susceptible tomato varieties play an important role in commercial tomato production. Although many tomato varieties carry the Mi resistance gene, susceptible tomato lines in some cases have desirable agronomic traits that are lacking in resistant varieties. Another benefit of susceptible tomato varieties is that, when grown in rotation with resistant lines, they may delay the emergence of resistance-breaking aphid and nematode strains. Therefore, it is important to identify effective means of controlling aphids and root-knot nematodes on susceptible varieties.

It is also important to evaluate the interactions between acquired resistance and Mi-mediated resistance. The results from this study suggest that BTH can enhance aphid control on resistant varieties and JA can enhance nematode resistance at high soil temperatures. These results also suggest that these defense elicitors could potentially be used for caterpillar control or some other purpose without compromising the function of Mi. This information not only will help optimize aphid and nematode control, but also has practical value to growers who wish to use SA or JA elicitors to control other pests. For example, Actigard, a commercial preparation of BTH, is currently recommended for the control of bacterial speck on tomato (2002 product label, Syngenta Crop Protection). The results from these studies indicate that products such as Actigard are can be used simultaneously with resistant tomato cultivars.

Economic Analysis

N/A

Farmer Adoption

N/A

Recommendations:

Areas needing additional study

This work funded by USDA-SARE has laid the foundation for future studies involving induced resistance against phloem-feeding insects and interactions between acquired resistance and other R-Genes.

1) Role of JA-induced volatiles on aphid host selection. One area of potential research involves the role JA-induced volatiles have on host selection of potato aphids. Cis-jasmone, a volatile catabolite of JA, has been shown to repel the damson-hop aphid (Phorodon humuli), lettuce aphid (Nasonovia ribis-nigri), and the grain aphid (Sitobion avenae) (Birkett et al. 2000; Bruce et al. 2003). Very little is known, however, about the effects of JA induced volatiles on host-selection behavior of the potato aphid. If JA-induced volatiles repel potato aphids, the use of a JA elicitor on field tomato could have additional pest management advantages not revealed by our studies.

3) Role of JA-induced volatiles on natural enemies of potato aphids. While volatiles released by induction of JA may repel herbivores, they also attract many natural enemies. Induction of JA-dependent defenses is known to attract parasitoids and predators of caterpillars and leafminers (Turlings et al. 1990; Thaler et al. 2001; Thaler et al. 2002). Cis-jasmone has been shown to attract the seven-spotted ladybird (Coccinella septempunctata) and the aphid parasitoid Aphidius ervi (Bruce et al. 2003). If JA-induced volatiles attract natural enemies, the use of a JA elicitor on field tomato could have additional pest management advantages not revealed by our studies.

4) Interactions between acquired resistance and other R-genes. Many other R-genes that confer resistance to pathogens have been identified in tomato and other crops. Many of these R-genes also require SA synthesis for function. It is important to evaluate the compatibility of other R-genes besides Mi-1.2 in order to utilize commercial defense elicitors effectively.

References

Birkett MA, Campbell CA, Chamberlain K, Guerrieri E, Hick AJ, Martin JL, Matthes M, Napier JA, Pettersson J, Pickett JA, Poppy GM, Pow EM, Pye BJ, Smart LE, Wadhams GH, Wadhams LJ and Woodcock CM. 2000. New roles for cis-jasmone as an insect semiochemical and in plant defense. P. Natl. Acad. Sci. USA 97: 9329-9334

Bruce TJ, Martin JL, Pickett JA, Pye BJ, Smart LE and Wadhams LJ. 2003. Cis-Jasmone treatment induces resistance in wheat plants against the grain aphid, Sitobion avenae (Fabricius) (Homoptera: Aphididae). Pest Manag. Sci. 59: 1031-1036

Thaler JS, Stout MJ, Karban R and Duffey SS. 2001. Jasmonate-mediated induced plant resistance affects a community of herbivores. Ecol. Entomol. 26: 213-324

Thaler JS, Farag MA, Pare PW and Dicke M. 2002. Jasmonate-deficient plants have reduced direct and indirect defences against herbivores. Ecol. Lett. 5: 764-774

Turlings TCJ, Tumlinson JH and Lewis WJ. 1990. Exploitation of herbvore-induced plant odors by host-seeking parasitic wasps. Science 250: 1251-1253

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.