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a Dep. of Horticultural Science, Univ. of Minnesota, 305 Alderman Hall, 1970 Folwell Ave., St. Paul, MN 55108
b Dep. of Plant Biology and Pathology, Rutgers Univ., 59 Dudley Rd., New Brunswick, NJ 08901
c Enanta Pharmaceuticals Inc., 500 Arsenal St., Watertown, MA 02472
* Corresponding author (ewatkins{at}umn.edu)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: JA, jasmonic acid • GLV, green-leaf volatiles • MeJA, Methyl jasmonate
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Plants are always producing volatiles, the most common of which are the typical green-leaf volatiles (GLVs), which include several saturated and unsaturated six-carbon alcohols, aldehydes, and esters. These GLVs are typically released in higher amounts after mechanical damage (Pare and Tumlinson, 1999). Plants can deter or attract insects through volatile production. Airborne volatiles collected from cowpea [Vigna unguiculata (L.) Walp.] included a number of chemicals that are known to play a role in affecting the response of phytophagous insects: hexanal, 
-pinene, ß-pinene, limonene, hexyl acetate, and (Z)-3-hexen-1-ol acetate (Lwande et al., 1989).
Plants also have the ability to produce volatiles after being induced by insect feeding. Induction often results in a greater quantity of total volatiles emitted, as well as a greater diversity of volatile compounds. Typically, the most obvious result of induction is a marked increase in the amount of terpenes produced by the plant, especially 1,3,6-octatriene, 3,7-dimethyl (ß-ocimene), and 1,6-octadien-3-ol, 3,7-dimethyl (linalool) (Pare and Tumlinson, 1999).
Induced volatile production can lead to attraction of natural insect enemies of the herbivorous insect (Turlings et al., 1993). There is typically a delay between the onset of insect feeding and emission of induced volatile release, therefore, a series of biochemical reactions must be involved (Pare and Tumlinson, 1999). The induced volatile production is likely caused by a factor in the regurgitate of the herbivore (Turlings et al., 1993). Not only are induced volatiles produced and released from these damaged leaves, but the plant can produce these volatiles in other parts of the plant (Alborn et al., 1996). Other factors also play a role in this complex relationship: insect damage history (Alborn et al., 1996), larval age (Takabayashi et al., 1995), leaf stage (Takabayashi et al., 1994), light (Loughrin et al., 1994; Maeda et al., 2000), plant species (Geervliet et al., 1997), and cultivar (Loughrin et al., 1995; Rapusas et al., 1996; Gouinguene et al., 2001).
Some of the effects of insect feeding on plants, primarily terpenoid production, can be mimicked by the application of jasmonic acid (Gols et al., 1999). Jasmonic acid is a hormone found in plants formed from -linolenic acid and it can have both inducing and inhibiting effects on a plant (Sembdner and Parthier, 1993). The compound is involved in the expression of several genes that play a role in plant development and defense. When jasmonates are applied to plants, several genes are up-regulated including those involved in jasmonate biosynthesis, secondary metabolism, cell-wall formation, and defense proteins (Cheong and Choi, 2003). Methyl jasmonate (MeJA), the volatile form of the compound, may be able to alert neighboring plants of attack (Farmer and Ryan, 1990).
Jasmonates have also been shown to be an integral part of insect defense in plants (McConn et al., 1997; Stotz et al., 2002). Jasmonate induction can occur quite rapidly after insect feeding begins (Halitschke et al., 2000; Schittko et al., 2000).
Research on turfgrass biochemistry is limited. Johnson et al. (2002) studied the green leaf chemistry profiles of six turfgrass species and showed that differences between species could be used as identification tools; however, the study did not examine volatile production. Yue et al. (2001) reported the volatile compounds from turf-type tall fescue (Festuca arundinacea Schreb.) at two temperatures. At 25°C, the most abundant volatile found was (Z)-3-hexen-1-ol acetate, and at 32°C, the most abundant volatile was nonanal. Several other compounds were detected at both temperatures including several aldehydes, ketones, alcohols, and monoterpenes.
Turfgrass breeders have had success developing germplasm with improved resistance to billbugs in other species (Kindler et al., 1982; Johnson-Cicalese et al., 1989; Funk et al., 1993). The role of induced volatile production in turfgrasses has not been studied extensively. This study was initiated to evaluate the volatile profile of various tufted hairgrass germplasm lines. In addition to evaluating intact plants with no previous damage history, the effect of JA on the volatile profile of the various collections was studied. Turfgrass undergoes constant mechanical damage, especially from mowing; therefore, the effect of mechanical damage on volatile production was also investigated. Identifying and quantifying volatiles from tufted hairgrass plants may aid in the development of germplasm with improved insect resistance.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Seedlings were established from the original collection seed in the greenhouse (approx. 25°C/20°C day/night, 12 h photoperiod). After approximately 70 d, seedlings were transferred to 10.2 cm pots and maintained in the greenhouse for an additional 5 wk. All plants were grown in Pro-Mix ‘BX’ peat-based growing media (Premier Horticulture, Dorval, QC, Canada). Plants were fertilized and watered as needed and rust disease was controlled with two applications of triadimefon [1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl) butanone]. Fungicide applications were early in establishment and therefore did not affect results. The plants were not clipped at any time before the experiment.
Volatile Collection
Using excised leaves has been shown to affect the quantity of volatiles released (Schmelz et al., 2001); therefore, whole plant volatiles were collected by dynamic headspace analysis using a modification of the methods described by Yue et al. (2001) Plants were placed individually inside a 3000 mL reaction kettle, which served as the collection chamber. The collection apparatus (3–1 glass chamber with air inlet, flowmeter and Tenax trap tube) was placed under fluorescent lights at 25°C and a 12-h-photoperiod. The temperature inside the chamber was monitored to ensure a consistent temperature of 25°C. Ambient air was allowed to flow through the chamber after being filtered through a Tenax tube (the same type of tube used for final collection). Air was passed over the plant at a rate of 50 mL min–1 and pumped into the chamber with a small air pump. Volatiles were collected for approximately 24 h for each sample on Tenax absorbent. The volatile collection period began approximately 3 h after the beginning of the photoperiod and lasted 24 h. Volatile collections were made periodically from both an empty chamber and a chamber containing only a pot of the moistened growing media to account for contaminants in the system. Plant material consisted of germplasm lines 14350, 14469, 14541, and 14393, all originating from Norway.
Tufted hairgrass plants were treated with JA and compared to untreated checks for total volatile production following the procedure used by Yue et al. (2001). From a stock solution of JA (200 µmol mL–1) (Sigma-Aldrich, St. Louis, MO) 50 µL was diluted into 20 mL of dH2O and sprayed onto tufted hairgrass plants (20 mL of solution used to spray four plants). Volatiles were collected as previously described on the same plant material starting 24 h after JA was applied.
The effect of simulated mowing on volatile production of tufted hairgrass was also tested. Plants were hand-clipped to a height of 7.5 cm at 3 h after the beginning of the 12-h photoperiod (control plants representing each line were not clipped). Plants were immediately placed in chambers and volatiles were collected every 3 h for the remainder of the photoperiod. A final collection consisted of volatiles produced during the 12-h dark period and the first 3 h of the photoperiod. Plant material consisted of 14350 and 14469 (Norway), 12837 (Pyrenees) and 12806 (Finland).
For each experiment, three plants from each germplasm line were analyzed per treatment (an untreated control including the same number of plants per line was included in each experiment). All data were subjected to analysis of variance according to the general linear model procedure of SAS (SAS Institute, Inc., Cary, N.C.). Significant differences between treatments or cultivars were evaluated using Tukey's mean separation, P 
0.05.
The Short Path Thermal Desorption Model TD-4 (SIS, Ringoes, NJ) (250°C for 4 min.) was used to desorb collected volatiles from the Tenax tube into the GC injector. An HP 6890 GC (Agilent Technologies, Palo Alto, CA) was fitted with a DB-I capillary column (30 m, I.D. = 0.23 mm, film thickness = 0.15 µm) (J&W Scientific, Folson, CA). The injector temperature was 250°C. Initial oven temperature was 29°C and it was then raised to 170°C at a rate of 2°C/min. Split ratio was 2:1. An HP 5973 Selective Detector (Agilent Technologies, Palo Alto, CA) was connected to the GC with an interface tube at 280°C. The electron ionization voltage was 70 eV and the mass range scanned was 35–350. Mass spectral comparisons were made with authentic standards when available; otherwise, tentative identifications were made using a NIST (National Institute of Standards and Technology) mass spectral library.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Hexenyl acetate was found in the headspace of plants in both treatments (Table 1); however, none was collected from collection line 14541 in either treatment (Table 2). This was the only compound that showed a significant intraspecific difference in this study.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, the volatile collection system may not have been sufficient to detect small amounts of certain volatiles. Terpenes, such as ocimene and linalool, are often detected in small amounts from non-induced plants (Buttery and Ling, 1984; Lwande et al., 1989).
The results of this study concur with results from other studies on the effect of JA application to plants. The major volatiles produced in response to JA application were ocimene and linalool. After treatment with JA, plants typically produce several terpenoids including ocimene and linalool (Gols et al., 1999; Pare and Tumlinson, 1999; Halitschke et al., 2000; Yue et al., 2001). Ocimene has been shown to attract parasitic wasps (Rose et al., 1998), aphid parasitoids (Du et al., 1998), and predatory mites (Takabayashi et al., 1994; Gols et al., 1999). In addition to serving as cues for predators of insects, some terpenoids have been shown to have an adverse effect on larval development (Mabry and Gill, 1979). In a native population of Nicotiana attenuata Torr. ex Wats, linalool was shown to decrease oviposition rates of an insect herbivore and increase egg predation of insect herbivores by a generalist predator (Kessler and Baldwin, 2001). The ultimate effect of the volatile response that has been demonstrated in tufted hairgrass is not known; however, parasitoids of billbug may exist (Vittum et al., 1999) and future research on this species should examine this area further.
The effect of a volatile blend with an increased diversity of compounds may enhance attractiveness to certain insects (Loughrin et al., 1998). In the present study, when the defense response was induced, a more complex blend of volatile compounds was released from tufted hairgrass plants. A more thorough screening of current tufted hairgrass germplasm for induced volatile emissions may find variability for the characteristic.
Although jasmonate-induced responses appear to be an advantage for a plant, they may cause long-term disadvantages. Induced defense can allow a species to forgo costs when defense is unnecessary and fitness costs can exist when a plant is induced but escapes attack (Baldwin, 1998). Terpene emission from maize takes longer than cotton (Gossypium hirsutum L.); it has been proposed that perennial plants, such as cotton, produce constitutive defenses that aid in long-term success and survival, while annual plants, such as maize, may benefit from only using defenses when under attack, due to their short life cycle (Loughrin et al., 1994). Plants that naturally exhibit anti-herbivore defenses generally have slower growth rates (Coley et al., 1985). If, in fact, increased volatile production would lead to decreased growth, breeding for this trait may not be advantageous in many crop species; however, reduced growth could be an advantage in a turfgrass species. Potential negative effects of induced responses are important for a plant breeder to consider when developing insect-resistant germplasm.
This study found intraspecific variation for the production of hexenyl acetate. Hexenyl acetate has been shown to be a potential attractant to natural enemies of herbivores (Du et al., 1998) and has been shown to be systemically released by herbivore-damaged cotton (Rose et al., 1996). Although the current study did not find any hexenyl acetate released from germplasm line 14541, small amounts of the compound may have been emitted below detection levels. Hexenyl acetate was, however, produced in much higher quantities in mechanically damaged plants, suggesting a possible role in wound response. Variation for production of this compound may be useful to a germplasm improvement program.
Because our program is developing tufted hairgrass for use as a turfgrass, the effect that mowing has on volatile production is also important to consider. If these plants are sending out chemical cues in a turf situation, the effect of mowing should be considered in any volatile research that is done with the species. The increase in GLVs after mechanical damage was expected. Mechanical damage did not lead to the production of additional terpene. The most abundant GLV released by the damaged plants was hexenyl acetate.
The ultimate usefulness of intraspecific variation for volatile production is the development of cultivars with enhanced volatile production. Utilizing germplasm altered for quantity and quality of volatile production and release could be an effective insect resistance strategy. Before making plant selections based on volatile production, a breeding population must be evaluated for the type of volatiles that are produced. If significant variation is found for the trait, the plant breeder can develop populations with increased production of certain volatiles. The entire germplasm collection that forms the basis of our tufted hairgrass breeding program is made up of plant material from other parts of the world. Attempts should be made to integrate germplasm lines collected from North America—naturalized and native ecotypes may provide natural resistance to insect pests.
Plant breeders may be able to develop cultivars that make use of induced chemical production as parasitoid attraction can depend on plant genotype. Rapusas et al. (1996) studied the effect of brown planthopper (Nilaparvata lugens Stål) infection on fifteen rice genotypes. They found that a parasitoid (Cyrtorhinus lividipennis Reuter) of the planthopper was more strongly oriented to certain rice genotypes than others. On certain genotypes, this response occurred even in the absence of the insect herbivore. Halitschke et al. (2000) found genotype differences in Nicotiana attenuata for volatile production following treatment with MeJA, caterpillar feeding (Manduca Sexta L.), and the treatment of mechanical wounds with larval oral secretion. Loughrin et al. (1995) found that an insect-damaged naturalized cotton cultivar (from south Florida) produced greater amounts of volatiles than insect-damaged commercial cotton cultivars. Additionally, they found that beet armyworm larvae (Spodoptera exigua Huebner) preferred to feed on the leaves of the commercial cultivars compared to the naturalized cultivar. This variation suggests that it may be possible to use classical breeding techniques to alter plant populations so that natural production of chemical deterrents is increased.
In order for increased volatile production to be an effective strategy for developing insect-resistant turfgrass cultivars, further research should study the attractiveness of insect predators to terpene-producing tufted hairgrass germplasm. This approach might also be effective in other important turfgrass species.
Received for publication February 13, 2006.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
