Spodoptera litura infested rice leaves released much higher amounts of volatiles than those in mechanical damaged and healthy plants. The contents of several green leaf volatiles ( (E)-2-hexenal, (Z)-3-hexen-1-ol, (E)-2-hexen-1-ol), methyl salicylate (MeSA) and terpenoids were increased dramatically in S. litura infested plants. Green leaf volatiles, MeSA and methyl jasmonate (MeJA) significantly inhibited the seedling growth of tomato seedlings in a dose- dependent manner. Linalool and β-caryophyllene had a slightly inhibitory effect on the seedling growth of tomato at low concentrations. The expression of LoxD and AOS, two important genes in the biosynthesis of jasmonates, were induced in tomato leaves as consequence of herbivore-induced rice green leaf volatiles treatment. However, no AOS transcripts were detected in either linalool or β-caryophyllene treated tomato leaves. The effects of the exogenous MeJA on transcript accumulation of LoxD and AOS in tomato leaves were also investigated. The results showed that MeJA treatment induced strong expression of LoxD and AOS exactly as green leaf volatiles treatment did. Further studies showed that herbivore-induced green leaf volatiles had similar effects on the expression of tomato herbivore defense-related genes PI-¢ò.
Herbivore-induced plant volatiles can inhibit the growth of other plant nearby, which may benefit the plant in natural ecosystem and evolutionary course.
Allelopathic activity, herbivore-induced volatiles, methyl jasmonate, green leaf volatiles, rice
It has been well documented that a variety of plants can emit volatile compounds when damaged by herbivorous arthropods. These induced volatiles provide natural enemies of the herbivores with important information, which make plant indirectly control of herbivore populations by herbivore’s predators and parasitoids (Dicke 2000). However, when herbivore-induced plant volatiles are released into the environment, any organism in the environment, including other plants, microorganisms and herbivores, are potential receptor of them. Some studies showed that herbivore- induced plant volatiles also have repellent effects on conspecific or intraspecific herbivores (Kessler and Baldwin 2001; Xu et al. 2002). Moreover, some evidence suggests that the same volatiles can indeed act against microorganisms (Cardoza et al. 2002). Herbivore- induced plant volatiles can influence not only animals and microorganisms but also other plants in the vicinity. Plant-plant communication though herbivore-induced plant volatiles has been a focus point of chemical ecology in recent years (Arimura et al. 2000; Dicke and Bruin 2001).
Allelopathy is defined as a direct or indirect interaction, whereby chemicals released by one plant influence the physiological processes of a neighboring plant (Rice 1984). One of the classical works in allelopathy has focused on the volatiles of sagebrush (Artemisia L.) and their ability to deter growth of other species under and near its canopy. Among the volatiles credited with the allelopathic effects are the major constituents in sagebrush emissions: camphor and cinelole. Seed germination and seedlings growth of several weeds were inhibited by application of camphor and cineole. Interestingly, the contents of these volatiles were increased under mechanical and herbivory damage. Some of the volatiles produced by plants under herbivore attack such as caryphyllene have phytotoxic effects (Kong et al. 1999). It seems possible that some volatiles produced by plants under herbivory may serve as allelopathic agents. This will be an additional benefit to the plant and should be added to the positive side of the equation when calculating the costs and benefits of herbivore-induced volatiles emissions.
Rice volatiles can attract the natural enemies of the brown planthopper (BPH). This attraction effect of rice volatiles was significantly increased when rice stems were infested by BPH. Spodoptera litura infested rice plants released much higher amounts of volatiles than those infested by BPH, and the contents of several green leaf volatiles, methyl salicylate (MeSA) and terpenoids increased dramatically (Xu et al. 2002). In dual-choice flight tunnel experiments, rice plants damaged by S. litura had a clearly repellent effect on BPH adult females compared to healthy undamaged plants, mechanical damaged plants or the plants infested by BPH (Xu et al. 2002). However, little is known about their bioactivities on other plants. In the present study, we tested the effects of synthetic forms of the commercially available compounds identified from the volatile profile emitted by S. litura infested rice plants on the seedling growth of tomato and the expression of the defense related genes.
Plants and insects
Rice variety. Rice variety oryza sativa L. JX 89 was used in this study. Rice seeds were germinated in laboratory and planted in an insect-free net room after germination. Twenty days later, rice seedlings were transplanted to plastic pots (φ10 × 15 cm) in the same net room with 50 seedlings per pot. The seedlings watered and fertilized with Hoagland nutrient solution at regular intervals. The plants were used for experiments 30-35 d after germination. Spodoptera litura was reared with artificial diets in laboratory.
Plant treatment for collecting volatiles
We transferred a plastic pots with 50 seedlings into a screen case (35 × 35 × 35 cm) for following treatment: 1) mechanical damaged plants: each uninfested plants leaves were inflicted with a hole-puncher (3 holes /leaf, ~0.5 cm2) 24 hours before collecting the volatiles and another hole did so immediately before the experiment, 2) S. litura infested plants was obtained by placing two third instar caterpillars on each seedling and the larvae was allowed to feed on the plants for 24 h, and removed with their by-products immediately before experiment, 3) undamaged healthy plants: rice seedlings did not receive any treatment, but were of the same age and size as treated plants.
Chemical analysis of volatiles from different treated rice seedlings.
The headspace volatiles were collected and analyzed according to the method described by Xu (2002)
Allelopathic effects of herbivore-induced rice volatile compounds.
Methyl jasmonate and six herbivore-induced rice plant volatile compounds: (E)-2-hexenal, (Z)-3-hexen-1-ol, (E)-2-hexen-1-ol, linalool, methyl salicylate, β-caryophyllene (Sigama-Aldrich Chemical company, St. Louis, Missouri) were used in the experiment. These compounds were selected because they are present in the blend of compounds emitted by the herbivore-induced plants and because they were commercially available. Ten uniform, germinated seedlings (with radicle lengths approx. 2 mm) of the receptor plants were placed into a beaker (250 ml, two layers of moistened filter paper on the beaker bottom) as a circle. A cap of centrifuge tube (1.5 ml) was placed at the center of the circle. Three levels of the volatile compounds, 0.5, 1 μl, and 5μl, were diluted in 100 μl hexane and added to the cap. The beaker was sealed with the parafilm. This method prevented the direct contact of the solution with the seedling and the seedling was exposed only to the vapors of the volatile compounds emitted from the cap for the duration of the experiment. A similar set-up but without induced rice plant volatiles served as control. For each treatment, six replicates were maintained. The entire set-up was kept in an environmentally controlled growth chamber at 25 ± 2°C with a photoperiod of 14/10 h day/night. After 5 days, length of shoot and root of the seedlings was measured.
Data Analyses. Impact effects were evaluated by the method of Williamson and Richardson (1988). ANOVA with Duncan’s multiple-range test was used to analyze the data.
Analysis of gene expression
Expression patterns of defense-related genes in different treated tomato leaves were analyzed using reverse transcription- polymerase chain reaction (RT-PCR) according to the method described by Xu (2003).
The headspace volatiles of rice seedlings with different treatment were collected with Tenax-TA trap and analyzed with GC and GC-MS. Sixteen components were isolated and identified from different treated rice seedlings, including 7 terpenoids, 3 alcohols, 2 ketones, 1 aldehyde, 1 acetate, 1 hydrocarbon and 1 indole (Figure 1). Significant differences in volatile emissions were observed for rice plants with different treatments. S. litura infested plants released much higher amounts of volatiles than those in all other treatments and the contents of several green leaf volatiles ((E)-2-hexenal, (Z)-3-hexen-1-ol, (E)-2-hexen-1-ol), methyl salicylate and terpenoids (limonene, ocimene, β-caryophyllene) were increased dramatically in S. litura infested plants. Linalool; (3E)-4,8-dimethyl-1,3,7-nonatriene, indole (E)-α-Bergamotene and an unidentified sesquiterpene were only detected in S. litura damaged plants (Figure 1).
Figure 1. Typical chromatographic profiles of headspace volatiles of different treated rice.
A, Healthy rice seedlings; B, Mechanical damaged rice seedlings; C, herbivore Spodopera litura infested rice seedlings. 1. (E)-2-hexenal; 2. (Z)-3-hexen-1-ol; 3. (E)-2-hexen-1-ol; 4. 2-heptanone; 5. 2-heptanol; 6. limonene; 7. ocimene; 8.; 10. methyl salicylate; 11. indole; 12. β-caryophyllene; 13. (E)-α-bergamotene; 14. n-octadecane; 15. (E)-nerolidol; 16. 6,10-dimethyl-2-undecanone; unk. unkown sesquiterpene; imp. impurity.
The allelopathic effects of herbivore induced rice volatiles on the seedling growth of tomato were investigated. The results showed that green leaf volatiles, (E)-2-hexenal, (Z)-3-hexen-1-ol, (E)-2-hexen-1-ol, MeSA and MeJA significantly inhibited the seedling growth of tomato seedlings in a dose-dependent manner. Linalool and β-caryophyllene had a slightly inhibitory effect on the seedling growth of tomato in low concentrations (Figure 2).
Figure 2. Allelopathic effects of herbivore-induced plant volatiles and methyl jasmonate on the seedling growth of tomato. A, B, C means 1, 2, 5 μl volatile compounds dissolved in 100 μl n-hexane separately.
Lipoxygenase (LOX) and Allene oxide synthase (AOS) are two important enzymes in the biosynthesis of jasmonates. The expression patterns of these two genes were analyzed in herbivore-induced volatiles and MeJA treated tomato leaves using RT-PCR (Figure3). The expression of LoxD and AOS gene were induced in tomato leaves as consequence of herbivore-induced rice green leaf volatiles treatment. However, no AOS transcripts were detected in either linalool or β-caryophyllene treated tomato leaves. The effects of the exogenous MeJA on transcript accumulation of LoxD and AOS in tomato leaves were also investigated. The results showed that MeJA treatment induced strong expression of LoxD and AOS exactly as green leaf volatiles treatment did. Further studies showed that herbivore-induced green leaf volatiles had similar effects on the expression of tomato herbivore defense-related genes PI-¢ò..
Figure 3. Expression patterns of defense-related genes in methyl jasmonate and herbivore-induced plant volatiles treated tomato seedlings LoxD, Lipoxygenase; AOS, Allene oxide synthase; PI-¢ò., Proteinase inhibitor ; elF4A, Internal standard of RT-PCR. 1, healthy plants; 2, (E)-2-hexenal; 3, (Z)-3-hexen-1-ol; 4, (E)-2-hexen-1-ol; 5, linalool; 6, β-caryophyllene; 7, methyl salicylate; 8, methyl jasmoante. bp, base pairs.
The main or primary function of herbivore-induced plant volatiles will be very hard to determine, but clearly any benefit that the plant derives from emitting the volatiles will contribute to selective pressures that have shaped their production and release. To understand the costs and benefits of inducible indirect defense through herbivore-induced plant volatiles, one needs to study all interactions that are mediated by these infochemicals. This related to interactions between plants and carnivores, plants and herbivores, and plants and other plants. Based on the present study, it seems possible that some volatiles produced by plants under herbivory may serve as allelopathic agents. This will be an additional benefit or cost to the plant and should be added to the positive or negative side of the equation when calculating the costs and benefits of herbivore-induced volatiles emissions. It was suggested that the reason of the allelochemicals inhibiting the growth of receptor plants was these chemicals elicited plant jasmonate-signaling pathway, which induced the plant investing the resources and energies to synthesize the defenses, and decreased the resources and energies allocated to the growth and development of the plants consequently.
This work was supported by the National Natural Science Foundation of China (Grant No. 30270270 , 30470335) and Natural Science Foundation of Guangdong Province in China (Grant No. 039254, 04020569)
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