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Dynamics of belowground volatile diffusion and degradation
Author’s Accepted Manuscript Dynamics of belowground volatile diffusion and degradation Salina Som, Denis S. Willett, Hans T. Alborn
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S2452-2198(17)30148-9 http://dx.doi.org/10.1016/j.rhisph.2017.07.004 RHISPH76
To appear in: Rhizosphere Received date: 17 July 2017 Accepted date: 17 July 2017 Cite this article as: Salina Som, Denis S. Willett and Hans T. Alborn, Dynamics of belowground volatile diffusion and degradation, Rhizosphere, http://dx.doi.org/10.1016/j.rhisph.2017.07.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Dynamics of belowground volatile diffusion and degradation Salina Som, Denis S. Willett, Hans T. Alborn* Chemistry Research Unit, Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, Gainesville, FL, USA: *[email protected]
Abstract Above ground herbivory can induce release of plant volatiles that attract natural enemies of the herbivores. Similarly, roots can release herbivore induced volatiles that attract beneficial organisms such as entomopathogenic nematodes belowground. Unlike their aboveground counterparts, belowground volatile signals interact with solids, liquids, and gases as they move through soil pore spaces. These interactions influence belowground signaling, can create non-linear diffusion profiles, and result in surface adsorbtion and degradation of volatiles in space and time. By examining diffusion and degradation in sand-filled microcosms, we found that the diffusion profiles of E-β-caryophyllene, d-limonene, pregeijerene, α-pinene, germacrene-d, and linalool were affected by moisture and pH. Furthermore, the common plant volatile linalool was non-diffusive below ground. In addition, we discovered a novel pathway for the degradation of linalool into rapidly diffusing belowground signals. These findings suggest areas for future exploration and highlight the importance of abiotic factors when studying belowground semiochemically-based interactions such as attraction of beneficial entomopathogenic nematodes to plant roots infested by host insects. Keywords: herbivore induced plant volatiles, HIPV, entomopathogenic nematode
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1. Introduction Invertebrates both above and belowground rely upon volatile signaling to communicate within species, identify resources, and navigate their environment (Holopainen and Blande, 2012; Farmer, 2001). Our understanding of aboveground volatile communication has benefited from extensive work on not only the nature of aboveground signals, but also on how those signals diffuse in space and time (Elkinton and Card´e, 1984). Pheromone plumes, for example, are transported considerably in space and time, but in a manner that can be predicted based on previous experimentation, observation, and modeling (Murlis et al., 1992). Understanding of volatile communication belowground, however, is in its nascency. Some modeling of herbicide diffusion has been conducted (Lindstrom et al., 1967). Additionally,through recent studies with entomopathogenic nematodes, we have realized the importance of terpene volatiles in communicating presence of food sources in both cultivated (Turlings et al., 2012; Rasmann et al., 2005; Ali et al., 2010, 2011, 2012) and wild ecosystems (Rasmann et al., 2009). Additionally, these roundworms can be selected for enhanced responsiveness to terpenoids and have a demonstrated caPreprint submitted to Rhizosphere
pability to learn in response to exposure to terpenoids (Willett et al., 2015; Hiltpold et al., 2010). While some pioneering work has been done exploring the diffusion of various belowground potential and known volatile signals, diffusion of volatile signals belowground is complicated by the existence of a third component: the medium itself (Hiltpold and Turlings, 2008; Chiriboga et al., 2017). In contrast to volatile signals aboveground, where a chemical will diffuse primarily in the gas phase with little contact with other media besides the air, volatiles belowground not only diffuse through the gaseous medium, but also, depending on size and polarity, interact to varying degrees with substrate particles (organic matter, sand, silt, clay, etc.) and sometimes high levels of moisture in the substrate pore spaces (Ruiz et al., 1998; Insam and Seewald, 2010). This complex dynamic will affect not only diffusion of volatile signals through the belowground medium, but potentially also lead to degradation of those signals over space and time. In the air, degradation can occur after interacting with reactive oxygen species. Belowground, this can occur as well, but may be magnified by interactions with other soil components. The exact nature of this diffusion and degradation July 20, 2017
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has potentially large implications for the utility of these volatiles for invertebrate signaling belowground. Timing of diffusion and rapidity of degradation may result in certain volatiles being more predictable and useful signals than others in specific situations. To examine the nature of belowground volatile diffusion, we monitored volatile blends moving through sand-filled microcosms at different moisture levels. To further investigate volatile degradation, we manipulated acidity and moisture levels while monitoring volatile conversion.
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2. Materials and Methods
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To examine the effect of moisture on the diffusion rate of belowground volatile signals, volatile blends were injected into sand-filled glass chambers and diffusion monitored through volatile collection onto thermal desorption Tenax filters. Glass diffusion chambers consisted of a series of six glass cylindrical L-tube assays with a straight glass joint adapter attachment (Figure 1A). These chambers were filled with washed Candler sand originally collected from the Florida ridge and adjusted to either 10% or 20% moisture by volume with deionized water. Capped soil probes were permanently inserted into the sand of the central chamber through a teflon plate covering the surface of the sand to avoid difusion into the air. Soil probes consisted of 6ml internal volume 8cm lengths of 0.5cm ID stainless steel tubing perforated with 28 2mm holes along its length (Figure 1B). Volatile blends (5 μL) were injected 2.5cm deep into the arm of the diffusion assay using a 10 μL syringe. Diffused volatiles were collected from the central chamber using Tenax filters attached to soil probes making the total diffusion distance 13cm. At least six replications of each assay were conducted.
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Figure 1: Volatile diffusion assay chambers. (A) Empty glass chamber. (B) Filled with sand and with addition of Tenax filter mounted to belowground probe in center. A Teflon plate was affixed below nut at top of probe to prevent loss of humidity over the course of the experiment. Volatiles diffused through sand following trajectory of blue arrow.
2.3. Volatile Collection Diffused volatiles were collected on Tenax by coupling collection filters to the probes. Air tight connections between the filter and a teflon vacuum tubing was accomplished by utilizing Swagelock straight 1 4 inch (0.635cm) connectors with teflon ferules and viton rubber o-rings as back ferules. The teflon tubing was connected to vacuum through a Aalborg flow meter modified for vaccuum flow and with a 0 to 20 ml/min glass liner. Air from the sand-filled chamber was pulled through the Tenax filter at a rate of 10ml per minute for 1 minute (approximately 2x the probe volume for each sampling). Collections were made at 0 (prior to volatile injection), 1, 10, 60, 180, 1080, 1440 (24 hours), 2880, 4320, and 5720 minutes. Trials with just pregeijerene and just linalool (not the entire volatile blend) were conducted as above, but collected at hourly intervals. After collection, Tenax filters were stored in sealed glass vials at −20o C until analysis on GC-MS. 2.4. Volatile Analysis Samples were introduced from Tenax filters onto the column via a low degradation thermal desorption injector consisting of a regular splitless injector, used as desorption oven, modified with an attached cryofocusing unit allowing for direct on column focusing of desorbed volatiles and no additional flash heating (Alborn and Willms, 2015). The HP 6890 gas chromatograph was equipped with 30-m x 0.25-mm-ID, 0.25-μm film thickness HP-5 capillary column, interfaced to a 5973 Mass Selective Detector (Hewlett Packard, Palo Alto, California) in electron impact mode. The splitless mode injector was set for 2 min desorption at 150o C and the
For blend assays, a partial-neat volatile blend was prepared. The volatile blend comprised 500 μls of each neat E-(β)-caryophyllene (Fluka, 99%), trans-2hexenal (Sigma Aldrich), d-limonene (Sigma Aldrich, 97%), (±)-linalool (Sigma Aldrich, 97%), (R)-(-)-2hexanol (Sigma Aldrich, 99%), 500 μl of 100 ng/μl of pregeijerene (extracted from Common Rue roots, Ruta graveolens, in pentane), and 125 μl of germacrene D (extracted from Goldenrod plants, Solidago spp., in dichloromethane). 2
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were both detectable at low amounts 13.5cm away from the injection site one minute after injection, peak amounts were not detected until hours later (Figure 2A,B). Limonene, the smaller molecule, diffused faster (reached peak amounts) than caryophyllene, at both moisture levels (Figure 2C,D).
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2.5. Conversion and Degradation Despite the lack of α-pinene in the original blend, this compound was consistently detected. To explore the possibility of conversion of linalool to α-pinene, 100 μl of (±)-linalool was added to 3ml of Millipore water (18 MΩ) in a separatory funnel then left for 30 minutes. The solution was then extracted with 3 ml of pentane. Once the layers separated, the organic layer was collected and blown down with N2 gas to 150 μl. This sequence of steps was repeated with addition of 40 μl of formic acid (Fluka) to adjust the water to pH 2, and addition of sodium hydroxide to adjust the water to pH 12. All trials were conducted in conjunction with blank controls in which an identical trial was run in the absence of linalool. To further examine conversion in the presence of sand, 3 ml of Millipore water and 100 μl linalool were added to a glass vial and saturated with washed and sterilized Florida (silica) sand. This was left for 30 minutes whereafter 5 ml pentane was added, filtered to remove the sand, separated, and blown down with N2 gas as above. The procedure was repeated without linalool as a negative control. Resultant solutions were analyzed as above with GC-MS with direct injection instead of thermal desorption. To examine and isolate diffusion and degradation of linalool, diffusion assays were performed an analyzed as above with the exception that 100ng (10 μl of 10 ng/ μl solution) of linalool was injected, not the full blend. Five replicates were conducted and, following volatile collections over the course of 24 hours, solvent extraction was used to confirm the presence of linalool at the injection site. To do so, a 1cm diameter cork borer was used to extract 1ml of sand at the linalool injection site. The sand sample was then washed in pentane, separated, concentrated, and analyzed on a GC-MS as described above.
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Amount (ng)
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oven ramped from 30o C to 260o C at 10 degrees/min after a three minute initial hold. Flow rate was doubled for the first two minutes, then average velocity maintained at 30 cm/min for analysis. Compounds were identified through comparison to standards and quantified by comparison to serial dilution of standards (1 to 100ng in steps of 10ng) utilizing using the same GC/MS system in regular splitless mode.
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Figure 2: Diffusion profiles of d-limonene and E β -caryophyllene at different moisture levels. (A) E - β caryophyllene. (B) d-limonene. (C) 20% moisture. (D) 10% moisture. Lines and shaded regions denote mean and standard error respectively.
Moisture also affected diffusion of other volatiles examined. For α-pinene (degradation product), 2hexanone, and germacrene-d, the increase in moisture from 10% to 20% decreased the time to first detection of the compound (T-Test: P = 0.01, 0.002, 0.08 respectively; Figure 3). For pregeijerene, the opposite was observed; increasing moisture delayed time to first detection (P = 0.001; Figure 3d). 3.2. Volatile Degradation While a small degree of degradation was observed from pregeijerene to geijerene throughout the experiment, the dynamics of geijerene diffusion did not seem to mirror the dynamics of pregeijerene diffusion. Despite limited detection (one replication), pregeijerene at 10% moisture had a clear peak and tail while geijerene maintained a relatively flat diffusion profile (Figure 4A). At 20% moisture, pregeijerene and geijerene were not detected until 24 hours (1440 minutes) after injection (Figure 4B). Importantly, we discovered degradation of linalool into α and β-pinene (Figure 5). This can occur under acidic conditions where acidity is induced by addition
3. Results 3.1. Volatile Diffusion Moisture influenced volatile diffusion profiles (Figure 2). While both E-β -caryophyllene and d-limonene 3
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4. Discussion
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Figure 4: Diffusion and degradation of pregeijerene at 10% (A) and 20% (B). Lines and shaded area denote means and standard error respectively.
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of formic acid, through the presence of sand, or in water alone. No α or β-pinene were detected under basic conditions. Linalool was not recovered in Tenax volatile collections from linalool-only diffusion assays. Additionally, linalool did not completely diffuse away from the injection site; large amounts of the compound were recovered from solvent extraction (Figure 6A). When observing degradation of linalool in isolation (i.e. not in conjunction with a blend) over time, β-pinene seems to diffuse faster and peaks sooner than α-pinene (Figure 6B).
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Figure 3: Time to first detection of compounds introduced into volatile diffusion chambers. Points and error bars denote mean and standard error respectively.
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Figure 5: Production of α and β pinene from linalool under varying pH. Acidic conditions (pH 2) were achieved through addition of formic acid and basic conditions (pH 12) through addition of sodium hydroxide. The sand environment contained saturated sand at pH 5. The water environment had a pH of 5.5.
affect the diffusion rate in soils. Thus, if blends are as important for signaling belowground as they are aboveground, the message may arrive garbled. Predicting signal integrity belowground becomes even more complicated when degradation is taken into account. While we highlight here the recognized degradation of the herbivore-induced plant volatile pregeijerene, we also describe for the first time the degradation of linalool into α-pinene a rapidly diffusing plant signal. α-pinene is often recovered from plant volatile collections belowground but, because of its almost ubiquitous nature, was presumed to be environmental background - perhaps arising from pine needles. Here, we show that α-pinene can arise from the degradation of linalool in slightly acidic sand environments then rapidly diffuse through the system. Linalool, a commonly released plant volatile, seems to not diffuse well likely due to in-
The effect of abiotic factors such as moisture on diffusion of volatile signals belowground holds important implications for invertebrate communication belowground. It is clear that plant roots can release herbivore induced volatiles, such as E-β-caryophyllene and pregeijerene used in our experiments, that have been shown to recruit entomopathogenic nematodes to sites of herbivory (Rasmann et al., 2005; Ali et al., 2010). Additionally, these same signals can be used by belowground herbivores to select suitable host plants (Robert et al., 2012). However, we have shown that entomopathogenic nematodes can learn to respond to other volatiles including α -pinene and limonene (Willett et al., 2015; Filgueiras et al., 2016; Willett et al., 2017). The efficacy of those signals, however, likely varies with abiotic factors such as moisture since, depending on the compound, this will to a varying degree 4
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2012. Subterranean, herbivore-induced plant volatile increases biological control activity of multiple beneficial nematode species in distinct habitats. PLoS One 7 (6), e38146. Ali, J. G., Alborn, H. T., Stelinski, L. L., 2010. Subterranean herbivore-induced volatiles released by citrus roots upon feeding by diaprepes abbreviatus recruit entomopathogenic nematodes. Journal of chemical ecology 36 (4), 361–368. Ali, J. G., Alborn, H. T., Stelinski, L. L., 2011. Constitutive and induced subterranean plant volatiles attract both entomopathogenic and plant parasitic nematodes. Journal of Ecology 99 (1), 26–35. Chiriboga, M. X., Campos-Herrera, R., Jaffuel, G., R¨oder, G., Turlings, T. C., 2017. Diffusion of the maize root signal (e)β-caryophyllene in soils of different textures and the effects on the migration of the entomopathogenic nematode heterorhabditis megidis. Rhizosphere. Elkinton, J. S., Card´e, R. T., 1984. Odor dispersion. In: Chemical ecology of insects. Springer, pp. 73–91. Farmer, E. E., 2001. Surface-to-air signals. Nature 411 (6839), 854– 856. Filgueiras, C. C., Willett, D. S., Junior, A. M., Pareja, M., El Borai, F., Dickson, D. W., Stelinski, L. L., Duncan, L. W., 2016. Stimulation of the salicylic acid pathway aboveground recruits entomopathogenic nematodes belowground. PloS one 11 (5), e0154712. Hiltpold, I., Baroni, M., Toepfer, S., Kuhlmann, U., Turlings, T. C., 2010. Selection of entomopathogenic nematodes for enhanced responsiveness to a volatile root signal helps to control a major root pest. Journal of Experimental Biology 213 (14), 2417–2423. Hiltpold, I., Turlings, T. C., 2008. Belowground chemical signaling in maize: when simplicity rhymes with efficiency. Journal of chemical ecology 34 (5), 628–635. Holopainen, J. K., Blande, J. D., 2012. Molecular plant volatile communication. In: Sensing in nature. Springer, pp. 17–31. Insam, H., Seewald, M. S., 2010. Volatile organic compounds (vocs) in soils. Biology and fertility of soils 46 (3), 199–213. Lindstrom, F. T., Haque, R., Freed, V. H., Boersma, L., 1967. The movement of some herbicides in soils. linear diffusion and convection of chemicals in soils. Environmental science & technology 1 (7), 561–565. Murlis, J., Elkinton, J. S., Carde, R. T., 1992. Odor plumes and how insects use them. Annual review of entomology 37 (1), 505–532. Rasmann, S., Johnson, M. D., Agrawal, A. A., 2009. Induced responses to herbivory and jasmonate in three milkweed species. Journal of chemical ecology 35 (11), 1326. Rasmann, S., K¨ollner, T. G., Degenhardt, J., Hiltpold, I., Toepfer, S., Kuhlmann, U., Gershenzon, J., Turlings, T. C., 2005. Recruitment of entomopathogenic nematodes by insect-damaged maize roots. Nature 434 (7034), 732–737. Robert, C. A., Erb, M., Duployer, M., Zwahlen, C., Doyen, G. R., Turlings, T. C., 2012. Herbivore-induced plant volatiles mediate host selection by a root herbivore. New Phytologist 194 (4), 1061– 1069. Ruiz, J., Bilbao, R., Murillo, M. B., 1998. Adsorption of different voc onto soil minerals from gas phase: Influence of mineral, type of voc, and air humidity. Environmental Science & Technology 32 (8), 1079–1084. Turlings, T. C., Hiltpold, I., Rasmann, S., 2012. The importance of root-produced volatiles as foraging cues for entomopathogenic nematodes. Plant and Soil 358 (1-2), 51–60. Willett, D. S., Alborn, H. T., Duncan, L. W., Stelinski, L. L., 2015. Social networks of educated nematodes. Scientific reports 5. Willett, D. S., Alborn, H. T., Stelinski, L. L., 2017. Multitrophic effects of belowground parasitoid learning. Scientific Reports 7 (1), 2067.
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Figure 6: (A) Compounds recovered from solvent extraction of linalool injection site. (B) Diffusion profiles of α and β pinene produced from linalool. Lines and shaded region denote means and standard error respectively.
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teractions with the sand media and thus might not be detected at all in collection of root volatiles. Rapidly diffusing plant volatiles like α-pinene, pregeijerene, limonene, and E-β-caryophyllene do not present linear diffusion profiles. Instead of a flat release, diffusion of these compounds resembles a front characterized by an initial peak which is followed by a long tail and decline. These profiles raise questions for how belowground organisms interpret and follow these signals. Is the initial peak important for interpretation? How do organisms like entomopathogenic nematodes recruit to sources of volatile release? While these questions beg for further research, a number of practical takeaways is naturally engendered by our findings. First, when introducing entomopathogenic nematodes into an area for biological control, it is important to consider abiotic conditions. Wet conditions may delay diffusion of compounds like pregeijerene, for example. Second, degradation is an important component of belowground signaling and may play a role in how signals are interpreted by various organisms belowground.
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Alborn, H. T., Willms, S. D., Sep. 29 2015. Technique for thermal desorption analyses of thermo labile volatile compounds. US Patent App. 14/869,984. Ali, J. G., Alborn, H. T., Campos-Herrera, R., Kaplan, F., Duncan, L. W., Rodriguez-Saona, C., Koppenh¨ofer, A. M., Stelinski, L. L.,
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References
root-produced volatiles as foraging cues for entomopathogenic nematodes. Plant and Soil 358 (1-2), 51–60. Willett, D. S., Alborn, H. T., Duncan, L. W., Stelinski, L. L., 2015. Social networks of educated nematodes. Scientific reports 5. Willett, D. S., Alborn, H. T., Stelinski, L. L., 2017. Multitrophic effects of belowground parasitoid learning. Scientific Reports 7 (1), 2067.
Alborn, H. T., Willms, S. D., Sep. 29 2015. Technique for thermal desorption analyses of thermo labile volatile compounds. US Patent App. 14/869,984. Ali, J. G., Alborn, H. T., Campos-Herrera, R., Kaplan, F., Duncan, L. W., Rodriguez-Saona, C., Koppenh¨ofer, A. M., Stelinski, L. L., 2012. Subterranean, herbivore-induced plant volatile increases biological control activity of multiple beneficial nematode species in distinct habitats. PLoS One 7 (6), e38146. Ali, J. G., Alborn, H. T., Stelinski, L. L., 2010. Subterranean herbivore-induced volatiles released by citrus roots upon feeding by diaprepes abbreviatus recruit entomopathogenic nematodes. Journal of chemical ecology 36 (4), 361–368. Ali, J. G., Alborn, H. T., Stelinski, L. L., 2011. Constitutive and induced subterranean plant volatiles attract both entomopathogenic and plant parasitic nematodes. Journal of Ecology 99 (1), 26–35. Chiriboga, M. X., Campos-Herrera, R., Jaffuel, G., R¨oder, G., Turlings, T. C., 2017. Diffusion of the maize root signal (e)β-caryophyllene in soils of different textures and the effects on the migration of the entomopathogenic nematode heterorhabditis megidis. Rhizosphere. Elkinton, J. S., Card´e, R. T., 1984. Odor dispersion. In: Chemical ecology of insects. Springer, pp. 73–91. Farmer, E. E., 2001. Surface-to-air signals. Nature 411 (6839), 854– 856. Filgueiras, C. C., Willett, D. S., Junior, A. M., Pareja, M., El Borai, F., Dickson, D. W., Stelinski, L. L., Duncan, L. W., 2016. Stimulation of the salicylic acid pathway aboveground recruits entomopathogenic nematodes belowground. PloS one 11 (5), e0154712. Hiltpold, I., Baroni, M., Toepfer, S., Kuhlmann, U., Turlings, T. C., 2010. Selection of entomopathogenic nematodes for enhanced responsiveness to a volatile root signal helps to control a major root pest. Journal of Experimental Biology 213 (14), 2417–2423. Hiltpold, I., Turlings, T. C., 2008. Belowground chemical signaling in maize: when simplicity rhymes with efficiency. Journal of chemical ecology 34 (5), 628–635. Holopainen, J. K., Blande, J. D., 2012. Molecular plant volatile communication. In: Sensing in nature. Springer, pp. 17–31. Insam, H., Seewald, M. S., 2010. Volatile organic compounds (vocs) in soils. Biology and fertility of soils 46 (3), 199–213. Lindstrom, F. T., Haque, R., Freed, V. H., Boersma, L., 1967. The movement of some herbicides in soils. linear diffusion and convection of chemicals in soils. Environmental science & technology 1 (7), 561–565. Murlis, J., Elkinton, J. S., Carde, R. T., 1992. Odor plumes and how insects use them. Annual review of entomology 37 (1), 505–532. Rasmann, S., Johnson, M. D., Agrawal, A. A., 2009. Induced responses to herbivory and jasmonate in three milkweed species. Journal of chemical ecology 35 (11), 1326. Rasmann, S., K¨ollner, T. G., Degenhardt, J., Hiltpold, I., Toepfer, S., Kuhlmann, U., Gershenzon, J., Turlings, T. C., 2005. Recruitment of entomopathogenic nematodes by insect-damaged maize roots. Nature 434 (7034), 732–737. Robert, C. A., Erb, M., Duployer, M., Zwahlen, C., Doyen, G. R., Turlings, T. C., 2012. Herbivore-induced plant volatiles mediate host selection by a root herbivore. New Phytologist 194 (4), 1061– 1069. Ruiz, J., Bilbao, R., Murillo, M. B., 1998. Adsorption of different voc onto soil minerals from gas phase: Influence of mineral, type of voc, and air humidity. Environmental Science & Technology 32 (8), 1079–1084. Turlings, T. C., Hiltpold, I., Rasmann, S., 2012. The importance of
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PII: DOI: Reference:
S2452-2198(17)30148-9 http://dx.doi.org/10.1016/j.rhisph.2017.07.004 RHISPH76
To appear in: Rhizosphere Received date: 17 July 2017 Accepted date: 17 July 2017 Cite this article as: Salina Som, Denis S. Willett and Hans T. Alborn, Dynamics of belowground volatile diffusion and degradation, Rhizosphere, http://dx.doi.org/10.1016/j.rhisph.2017.07.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Dynamics of belowground volatile diffusion and degradation Salina Som, Denis S. Willett, Hans T. Alborn* Chemistry Research Unit, Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, Gainesville, FL, USA: *[email protected]
Abstract Above ground herbivory can induce release of plant volatiles that attract natural enemies of the herbivores. Similarly, roots can release herbivore induced volatiles that attract beneficial organisms such as entomopathogenic nematodes belowground. Unlike their aboveground counterparts, belowground volatile signals interact with solids, liquids, and gases as they move through soil pore spaces. These interactions influence belowground signaling, can create non-linear diffusion profiles, and result in surface adsorbtion and degradation of volatiles in space and time. By examining diffusion and degradation in sand-filled microcosms, we found that the diffusion profiles of E-β-caryophyllene, d-limonene, pregeijerene, α-pinene, germacrene-d, and linalool were affected by moisture and pH. Furthermore, the common plant volatile linalool was non-diffusive below ground. In addition, we discovered a novel pathway for the degradation of linalool into rapidly diffusing belowground signals. These findings suggest areas for future exploration and highlight the importance of abiotic factors when studying belowground semiochemically-based interactions such as attraction of beneficial entomopathogenic nematodes to plant roots infested by host insects. Keywords: herbivore induced plant volatiles, HIPV, entomopathogenic nematode
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1. Introduction Invertebrates both above and belowground rely upon volatile signaling to communicate within species, identify resources, and navigate their environment (Holopainen and Blande, 2012; Farmer, 2001). Our understanding of aboveground volatile communication has benefited from extensive work on not only the nature of aboveground signals, but also on how those signals diffuse in space and time (Elkinton and Card´e, 1984). Pheromone plumes, for example, are transported considerably in space and time, but in a manner that can be predicted based on previous experimentation, observation, and modeling (Murlis et al., 1992). Understanding of volatile communication belowground, however, is in its nascency. Some modeling of herbicide diffusion has been conducted (Lindstrom et al., 1967). Additionally,through recent studies with entomopathogenic nematodes, we have realized the importance of terpene volatiles in communicating presence of food sources in both cultivated (Turlings et al., 2012; Rasmann et al., 2005; Ali et al., 2010, 2011, 2012) and wild ecosystems (Rasmann et al., 2009). Additionally, these roundworms can be selected for enhanced responsiveness to terpenoids and have a demonstrated caPreprint submitted to Rhizosphere
pability to learn in response to exposure to terpenoids (Willett et al., 2015; Hiltpold et al., 2010). While some pioneering work has been done exploring the diffusion of various belowground potential and known volatile signals, diffusion of volatile signals belowground is complicated by the existence of a third component: the medium itself (Hiltpold and Turlings, 2008; Chiriboga et al., 2017). In contrast to volatile signals aboveground, where a chemical will diffuse primarily in the gas phase with little contact with other media besides the air, volatiles belowground not only diffuse through the gaseous medium, but also, depending on size and polarity, interact to varying degrees with substrate particles (organic matter, sand, silt, clay, etc.) and sometimes high levels of moisture in the substrate pore spaces (Ruiz et al., 1998; Insam and Seewald, 2010). This complex dynamic will affect not only diffusion of volatile signals through the belowground medium, but potentially also lead to degradation of those signals over space and time. In the air, degradation can occur after interacting with reactive oxygen species. Belowground, this can occur as well, but may be magnified by interactions with other soil components. The exact nature of this diffusion and degradation July 20, 2017
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has potentially large implications for the utility of these volatiles for invertebrate signaling belowground. Timing of diffusion and rapidity of degradation may result in certain volatiles being more predictable and useful signals than others in specific situations. To examine the nature of belowground volatile diffusion, we monitored volatile blends moving through sand-filled microcosms at different moisture levels. To further investigate volatile degradation, we manipulated acidity and moisture levels while monitoring volatile conversion.
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2. Materials and Methods
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2.1. Diffusion Assay
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To examine the effect of moisture on the diffusion rate of belowground volatile signals, volatile blends were injected into sand-filled glass chambers and diffusion monitored through volatile collection onto thermal desorption Tenax filters. Glass diffusion chambers consisted of a series of six glass cylindrical L-tube assays with a straight glass joint adapter attachment (Figure 1A). These chambers were filled with washed Candler sand originally collected from the Florida ridge and adjusted to either 10% or 20% moisture by volume with deionized water. Capped soil probes were permanently inserted into the sand of the central chamber through a teflon plate covering the surface of the sand to avoid difusion into the air. Soil probes consisted of 6ml internal volume 8cm lengths of 0.5cm ID stainless steel tubing perforated with 28 2mm holes along its length (Figure 1B). Volatile blends (5 μL) were injected 2.5cm deep into the arm of the diffusion assay using a 10 μL syringe. Diffused volatiles were collected from the central chamber using Tenax filters attached to soil probes making the total diffusion distance 13cm. At least six replications of each assay were conducted.
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2.2. Volatile Blend
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A
B
6.05 cm
12.0 cm 10.69 cm
2.0 cm
2.0 cm
5.85 cm
13.0 cm
Figure 1: Volatile diffusion assay chambers. (A) Empty glass chamber. (B) Filled with sand and with addition of Tenax filter mounted to belowground probe in center. A Teflon plate was affixed below nut at top of probe to prevent loss of humidity over the course of the experiment. Volatiles diffused through sand following trajectory of blue arrow.
2.3. Volatile Collection Diffused volatiles were collected on Tenax by coupling collection filters to the probes. Air tight connections between the filter and a teflon vacuum tubing was accomplished by utilizing Swagelock straight 1 4 inch (0.635cm) connectors with teflon ferules and viton rubber o-rings as back ferules. The teflon tubing was connected to vacuum through a Aalborg flow meter modified for vaccuum flow and with a 0 to 20 ml/min glass liner. Air from the sand-filled chamber was pulled through the Tenax filter at a rate of 10ml per minute for 1 minute (approximately 2x the probe volume for each sampling). Collections were made at 0 (prior to volatile injection), 1, 10, 60, 180, 1080, 1440 (24 hours), 2880, 4320, and 5720 minutes. Trials with just pregeijerene and just linalool (not the entire volatile blend) were conducted as above, but collected at hourly intervals. After collection, Tenax filters were stored in sealed glass vials at −20o C until analysis on GC-MS. 2.4. Volatile Analysis Samples were introduced from Tenax filters onto the column via a low degradation thermal desorption injector consisting of a regular splitless injector, used as desorption oven, modified with an attached cryofocusing unit allowing for direct on column focusing of desorbed volatiles and no additional flash heating (Alborn and Willms, 2015). The HP 6890 gas chromatograph was equipped with 30-m x 0.25-mm-ID, 0.25-μm film thickness HP-5 capillary column, interfaced to a 5973 Mass Selective Detector (Hewlett Packard, Palo Alto, California) in electron impact mode. The splitless mode injector was set for 2 min desorption at 150o C and the
For blend assays, a partial-neat volatile blend was prepared. The volatile blend comprised 500 μls of each neat E-(β)-caryophyllene (Fluka, 99%), trans-2hexenal (Sigma Aldrich), d-limonene (Sigma Aldrich, 97%), (±)-linalool (Sigma Aldrich, 97%), (R)-(-)-2hexanol (Sigma Aldrich, 99%), 500 μl of 100 ng/μl of pregeijerene (extracted from Common Rue roots, Ruta graveolens, in pentane), and 125 μl of germacrene D (extracted from Goldenrod plants, Solidago spp., in dichloromethane). 2
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were both detectable at low amounts 13.5cm away from the injection site one minute after injection, peak amounts were not detected until hours later (Figure 2A,B). Limonene, the smaller molecule, diffused faster (reached peak amounts) than caryophyllene, at both moisture levels (Figure 2C,D).
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A
2.5. Conversion and Degradation Despite the lack of α-pinene in the original blend, this compound was consistently detected. To explore the possibility of conversion of linalool to α-pinene, 100 μl of (±)-linalool was added to 3ml of Millipore water (18 MΩ) in a separatory funnel then left for 30 minutes. The solution was then extracted with 3 ml of pentane. Once the layers separated, the organic layer was collected and blown down with N2 gas to 150 μl. This sequence of steps was repeated with addition of 40 μl of formic acid (Fluka) to adjust the water to pH 2, and addition of sodium hydroxide to adjust the water to pH 12. All trials were conducted in conjunction with blank controls in which an identical trial was run in the absence of linalool. To further examine conversion in the presence of sand, 3 ml of Millipore water and 100 μl linalool were added to a glass vial and saturated with washed and sterilized Florida (silica) sand. This was left for 30 minutes whereafter 5 ml pentane was added, filtered to remove the sand, separated, and blown down with N2 gas as above. The procedure was repeated without linalool as a negative control. Resultant solutions were analyzed as above with GC-MS with direct injection instead of thermal desorption. To examine and isolate diffusion and degradation of linalool, diffusion assays were performed an analyzed as above with the exception that 100ng (10 μl of 10 ng/ μl solution) of linalool was injected, not the full blend. Five replicates were conducted and, following volatile collections over the course of 24 hours, solvent extraction was used to confirm the presence of linalool at the injection site. To do so, a 1cm diameter cork borer was used to extract 1ml of sand at the linalool injection site. The sand sample was then washed in pentane, separated, concentrated, and analyzed on a GC-MS as described above.
10.0
Amount (ng)
129
oven ramped from 30o C to 260o C at 10 degrees/min after a three minute initial hold. Flow rate was doubled for the first two minutes, then average velocity maintained at 30 cm/min for analysis. Compounds were identified through comparison to standards and quantified by comparison to serial dilution of standards (1 to 100ng in steps of 10ng) utilizing using the same GC/MS system in regular splitless mode.
E-β-caryophyllene 0.1
20% Moisture 10% Moisture 1
10
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Time (minutes)
d-limonene
B
20% Moisture 210% Moisture
10.0
Amount (ng)
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0.1
1
10
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1080
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Time (minutes)
Figure 2: Diffusion profiles of d-limonene and E β -caryophyllene at different moisture levels. (A) E - β caryophyllene. (B) d-limonene. (C) 20% moisture. (D) 10% moisture. Lines and shaded regions denote mean and standard error respectively.
Moisture also affected diffusion of other volatiles examined. For α-pinene (degradation product), 2hexanone, and germacrene-d, the increase in moisture from 10% to 20% decreased the time to first detection of the compound (T-Test: P = 0.01, 0.002, 0.08 respectively; Figure 3). For pregeijerene, the opposite was observed; increasing moisture delayed time to first detection (P = 0.001; Figure 3d). 3.2. Volatile Degradation While a small degree of degradation was observed from pregeijerene to geijerene throughout the experiment, the dynamics of geijerene diffusion did not seem to mirror the dynamics of pregeijerene diffusion. Despite limited detection (one replication), pregeijerene at 10% moisture had a clear peak and tail while geijerene maintained a relatively flat diffusion profile (Figure 4A). At 20% moisture, pregeijerene and geijerene were not detected until 24 hours (1440 minutes) after injection (Figure 4B). Importantly, we discovered degradation of linalool into α and β-pinene (Figure 5). This can occur under acidic conditions where acidity is induced by addition
3. Results 3.1. Volatile Diffusion Moisture influenced volatile diffusion profiles (Figure 2). While both E-β -caryophyllene and d-limonene 3
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A
α-Pinene
B
2-Hexanone 60
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Time (min)
Time (min)
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5.0
2.5
600
A
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Moisture 0.0 10%
C
Amount (ng)
0 20%
Germacrene-D 2500
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Pregeijerene
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Sand Moisture
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4. Discussion
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Figure 4: Diffusion and degradation of pregeijerene at 10% (A) and 20% (B). Lines and shaded area denote means and standard error respectively.
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Amount (ng)
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of formic acid, through the presence of sand, or in water alone. No α or β-pinene were detected under basic conditions. Linalool was not recovered in Tenax volatile collections from linalool-only diffusion assays. Additionally, linalool did not completely diffuse away from the injection site; large amounts of the compound were recovered from solvent extraction (Figure 6A). When observing degradation of linalool in isolation (i.e. not in conjunction with a blend) over time, β-pinene seems to diffuse faster and peaks sooner than α-pinene (Figure 6B).
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Time (min)
Figure 3: Time to first detection of compounds introduced into volatile diffusion chambers. Points and error bars denote mean and standard error respectively.
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Sand Moisture
Compound α−Pinene
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β−Pinene
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Acidic
Basic
Sand
Water
Figure 5: Production of α and β pinene from linalool under varying pH. Acidic conditions (pH 2) were achieved through addition of formic acid and basic conditions (pH 12) through addition of sodium hydroxide. The sand environment contained saturated sand at pH 5. The water environment had a pH of 5.5.
affect the diffusion rate in soils. Thus, if blends are as important for signaling belowground as they are aboveground, the message may arrive garbled. Predicting signal integrity belowground becomes even more complicated when degradation is taken into account. While we highlight here the recognized degradation of the herbivore-induced plant volatile pregeijerene, we also describe for the first time the degradation of linalool into α-pinene a rapidly diffusing plant signal. α-pinene is often recovered from plant volatile collections belowground but, because of its almost ubiquitous nature, was presumed to be environmental background - perhaps arising from pine needles. Here, we show that α-pinene can arise from the degradation of linalool in slightly acidic sand environments then rapidly diffuse through the system. Linalool, a commonly released plant volatile, seems to not diffuse well likely due to in-
The effect of abiotic factors such as moisture on diffusion of volatile signals belowground holds important implications for invertebrate communication belowground. It is clear that plant roots can release herbivore induced volatiles, such as E-β-caryophyllene and pregeijerene used in our experiments, that have been shown to recruit entomopathogenic nematodes to sites of herbivory (Rasmann et al., 2005; Ali et al., 2010). Additionally, these same signals can be used by belowground herbivores to select suitable host plants (Robert et al., 2012). However, we have shown that entomopathogenic nematodes can learn to respond to other volatiles including α -pinene and limonene (Willett et al., 2015; Filgueiras et al., 2016; Willett et al., 2017). The efficacy of those signals, however, likely varies with abiotic factors such as moisture since, depending on the compound, this will to a varying degree 4
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A
10.0
Amount (ng)
Amount (ng)
30
20
2012. Subterranean, herbivore-induced plant volatile increases biological control activity of multiple beneficial nematode species in distinct habitats. PLoS One 7 (6), e38146. Ali, J. G., Alborn, H. T., Stelinski, L. L., 2010. Subterranean herbivore-induced volatiles released by citrus roots upon feeding by diaprepes abbreviatus recruit entomopathogenic nematodes. Journal of chemical ecology 36 (4), 361–368. Ali, J. G., Alborn, H. T., Stelinski, L. L., 2011. Constitutive and induced subterranean plant volatiles attract both entomopathogenic and plant parasitic nematodes. Journal of Ecology 99 (1), 26–35. Chiriboga, M. X., Campos-Herrera, R., Jaffuel, G., R¨oder, G., Turlings, T. C., 2017. Diffusion of the maize root signal (e)β-caryophyllene in soils of different textures and the effects on the migration of the entomopathogenic nematode heterorhabditis megidis. Rhizosphere. Elkinton, J. S., Card´e, R. T., 1984. Odor dispersion. In: Chemical ecology of insects. Springer, pp. 73–91. Farmer, E. E., 2001. Surface-to-air signals. Nature 411 (6839), 854– 856. Filgueiras, C. C., Willett, D. S., Junior, A. M., Pareja, M., El Borai, F., Dickson, D. W., Stelinski, L. L., Duncan, L. W., 2016. Stimulation of the salicylic acid pathway aboveground recruits entomopathogenic nematodes belowground. PloS one 11 (5), e0154712. Hiltpold, I., Baroni, M., Toepfer, S., Kuhlmann, U., Turlings, T. C., 2010. Selection of entomopathogenic nematodes for enhanced responsiveness to a volatile root signal helps to control a major root pest. Journal of Experimental Biology 213 (14), 2417–2423. Hiltpold, I., Turlings, T. C., 2008. Belowground chemical signaling in maize: when simplicity rhymes with efficiency. Journal of chemical ecology 34 (5), 628–635. Holopainen, J. K., Blande, J. D., 2012. Molecular plant volatile communication. In: Sensing in nature. Springer, pp. 17–31. Insam, H., Seewald, M. S., 2010. Volatile organic compounds (vocs) in soils. Biology and fertility of soils 46 (3), 199–213. Lindstrom, F. T., Haque, R., Freed, V. H., Boersma, L., 1967. The movement of some herbicides in soils. linear diffusion and convection of chemicals in soils. Environmental science & technology 1 (7), 561–565. Murlis, J., Elkinton, J. S., Carde, R. T., 1992. Odor plumes and how insects use them. Annual review of entomology 37 (1), 505–532. Rasmann, S., Johnson, M. D., Agrawal, A. A., 2009. Induced responses to herbivory and jasmonate in three milkweed species. Journal of chemical ecology 35 (11), 1326. Rasmann, S., K¨ollner, T. G., Degenhardt, J., Hiltpold, I., Toepfer, S., Kuhlmann, U., Gershenzon, J., Turlings, T. C., 2005. Recruitment of entomopathogenic nematodes by insect-damaged maize roots. Nature 434 (7034), 732–737. Robert, C. A., Erb, M., Duployer, M., Zwahlen, C., Doyen, G. R., Turlings, T. C., 2012. Herbivore-induced plant volatiles mediate host selection by a root herbivore. New Phytologist 194 (4), 1061– 1069. Ruiz, J., Bilbao, R., Murillo, M. B., 1998. Adsorption of different voc onto soil minerals from gas phase: Influence of mineral, type of voc, and air humidity. Environmental Science & Technology 32 (8), 1079–1084. Turlings, T. C., Hiltpold, I., Rasmann, S., 2012. The importance of root-produced volatiles as foraging cues for entomopathogenic nematodes. Plant and Soil 358 (1-2), 51–60. Willett, D. S., Alborn, H. T., Duncan, L. W., Stelinski, L. L., 2015. Social networks of educated nematodes. Scientific reports 5. Willett, D. S., Alborn, H. T., Stelinski, L. L., 2017. Multitrophic effects of belowground parasitoid learning. Scientific Reports 7 (1), 2067.
B
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Compound
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α−Pinene β−Pinene
0.1
0 Dihydro linalool
Linalool
Linalool oxide
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1000
1500
Time (minutes)
Figure 6: (A) Compounds recovered from solvent extraction of linalool injection site. (B) Diffusion profiles of α and β pinene produced from linalool. Lines and shaded region denote means and standard error respectively.
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teractions with the sand media and thus might not be detected at all in collection of root volatiles. Rapidly diffusing plant volatiles like α-pinene, pregeijerene, limonene, and E-β-caryophyllene do not present linear diffusion profiles. Instead of a flat release, diffusion of these compounds resembles a front characterized by an initial peak which is followed by a long tail and decline. These profiles raise questions for how belowground organisms interpret and follow these signals. Is the initial peak important for interpretation? How do organisms like entomopathogenic nematodes recruit to sources of volatile release? While these questions beg for further research, a number of practical takeaways is naturally engendered by our findings. First, when introducing entomopathogenic nematodes into an area for biological control, it is important to consider abiotic conditions. Wet conditions may delay diffusion of compounds like pregeijerene, for example. Second, degradation is an important component of belowground signaling and may play a role in how signals are interpreted by various organisms belowground.
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5. References
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References
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Alborn, H. T., Willms, S. D., Sep. 29 2015. Technique for thermal desorption analyses of thermo labile volatile compounds. US Patent App. 14/869,984. Ali, J. G., Alborn, H. T., Campos-Herrera, R., Kaplan, F., Duncan, L. W., Rodriguez-Saona, C., Koppenh¨ofer, A. M., Stelinski, L. L.,
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References
root-produced volatiles as foraging cues for entomopathogenic nematodes. Plant and Soil 358 (1-2), 51–60. Willett, D. S., Alborn, H. T., Duncan, L. W., Stelinski, L. L., 2015. Social networks of educated nematodes. Scientific reports 5. Willett, D. S., Alborn, H. T., Stelinski, L. L., 2017. Multitrophic effects of belowground parasitoid learning. Scientific Reports 7 (1), 2067.
Alborn, H. T., Willms, S. D., Sep. 29 2015. Technique for thermal desorption analyses of thermo labile volatile compounds. US Patent App. 14/869,984. Ali, J. G., Alborn, H. T., Campos-Herrera, R., Kaplan, F., Duncan, L. W., Rodriguez-Saona, C., Koppenh¨ofer, A. M., Stelinski, L. L., 2012. Subterranean, herbivore-induced plant volatile increases biological control activity of multiple beneficial nematode species in distinct habitats. PLoS One 7 (6), e38146. Ali, J. G., Alborn, H. T., Stelinski, L. L., 2010. Subterranean herbivore-induced volatiles released by citrus roots upon feeding by diaprepes abbreviatus recruit entomopathogenic nematodes. Journal of chemical ecology 36 (4), 361–368. Ali, J. G., Alborn, H. T., Stelinski, L. L., 2011. Constitutive and induced subterranean plant volatiles attract both entomopathogenic and plant parasitic nematodes. Journal of Ecology 99 (1), 26–35. Chiriboga, M. X., Campos-Herrera, R., Jaffuel, G., R¨oder, G., Turlings, T. C., 2017. Diffusion of the maize root signal (e)β-caryophyllene in soils of different textures and the effects on the migration of the entomopathogenic nematode heterorhabditis megidis. Rhizosphere. Elkinton, J. S., Card´e, R. T., 1984. Odor dispersion. In: Chemical ecology of insects. Springer, pp. 73–91. Farmer, E. E., 2001. Surface-to-air signals. Nature 411 (6839), 854– 856. Filgueiras, C. C., Willett, D. S., Junior, A. M., Pareja, M., El Borai, F., Dickson, D. W., Stelinski, L. L., Duncan, L. W., 2016. Stimulation of the salicylic acid pathway aboveground recruits entomopathogenic nematodes belowground. PloS one 11 (5), e0154712. Hiltpold, I., Baroni, M., Toepfer, S., Kuhlmann, U., Turlings, T. C., 2010. Selection of entomopathogenic nematodes for enhanced responsiveness to a volatile root signal helps to control a major root pest. Journal of Experimental Biology 213 (14), 2417–2423. Hiltpold, I., Turlings, T. C., 2008. Belowground chemical signaling in maize: when simplicity rhymes with efficiency. Journal of chemical ecology 34 (5), 628–635. Holopainen, J. K., Blande, J. D., 2012. Molecular plant volatile communication. In: Sensing in nature. Springer, pp. 17–31. Insam, H., Seewald, M. S., 2010. Volatile organic compounds (vocs) in soils. Biology and fertility of soils 46 (3), 199–213. Lindstrom, F. T., Haque, R., Freed, V. H., Boersma, L., 1967. The movement of some herbicides in soils. linear diffusion and convection of chemicals in soils. Environmental science & technology 1 (7), 561–565. Murlis, J., Elkinton, J. S., Carde, R. T., 1992. Odor plumes and how insects use them. Annual review of entomology 37 (1), 505–532. Rasmann, S., Johnson, M. D., Agrawal, A. A., 2009. Induced responses to herbivory and jasmonate in three milkweed species. Journal of chemical ecology 35 (11), 1326. Rasmann, S., K¨ollner, T. G., Degenhardt, J., Hiltpold, I., Toepfer, S., Kuhlmann, U., Gershenzon, J., Turlings, T. C., 2005. Recruitment of entomopathogenic nematodes by insect-damaged maize roots. Nature 434 (7034), 732–737. Robert, C. A., Erb, M., Duployer, M., Zwahlen, C., Doyen, G. R., Turlings, T. C., 2012. Herbivore-induced plant volatiles mediate host selection by a root herbivore. New Phytologist 194 (4), 1061– 1069. Ruiz, J., Bilbao, R., Murillo, M. B., 1998. Adsorption of different voc onto soil minerals from gas phase: Influence of mineral, type of voc, and air humidity. Environmental Science & Technology 32 (8), 1079–1084. Turlings, T. C., Hiltpold, I., Rasmann, S., 2012. The importance of
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