Common gas phase molecules from fungi affect seed germination and plant health in Arabidopsis thaliana

Volatiles organic compounds (VOCs) are low molecular mass compounds with high vapor pressure and low to medium water solubility that exist in the gaseous state at room temperature (Herrmann [2010]). Approximately 250 VOCs have been identified from fungi (Chiron and Michelot [2005]) as the products of both primary and secondary metabolism (Turner and Aldridge [1983]; Korpi et al. [2009]). These gas phase molecules are emitted in complex mixtures that vary quantitatively and qualitatively depending not only on the age and genetic profiles of the producing species but also on extrinsic variables such as substrate, temperature, moisture level and pH (Sunesson et al. [1995]; Claeson et al. [2002]; Matysik et al. [2008]). Fungal volatiles have distinctive odorant properties and they have been studied extensively for their positive and negative sensory properties. They impart unique aromas and flavors to mold-ripened cheeses, Japanese koji and other mold-fermented food products (Steinkraus [1983]; Kinderlerer [1989]), and are responsible for the bouquet of gourmet mushrooms such as boletes, chanterelles and truffles (Cho et al. [2008]; Fraatz and Zorn [2010]). On the negative side, when foods are contaminated by molds, they produce off flavors. VOCs have been used as an indirect indicator of fungal spoilage in agricultural products (Borjesson et al. [1992]; Jelen and Wasowicz [1998]; Schnürer et al. [1999]) and of mold contamination in water-damaged buildings (Kuske et al. [2005]; Sahlberg et al. [2013]). Finally, because VOCs can diffuse through the atmosphere and the soil, they are well adapted for signaling between species that share a common ecological niche. Both bacterial and fungal VOCs play competitive roles in chemical interactions between microorganisms (Beattie and Torrey [1986]; Morath et al. [2012]). Many plant and microbial volatile molecules function as semiochemicals, otherwise known as “infochemicals”, and there is a large literature on the ability of fungal VOCs to mediate arthropod behavior, where they have properties as synomones, allomones, and kairomones (Rohlfs et al. [2005]; Mburu et al. [2011]; Davis et al. [2013]). The fungal VOC commonly called “mushroom alcohol” (1-octen-3-ol) is responsible for much of the musty odor associated with mold contamination and is an important insect semiochemical. It attracts many insect species, including the malaria mosquito (Takken and Knols [1999]; Thakeow et al. [2008]).

In contrast, the interactions between fungal VOCs and plants have not received much scientific attention (Bitas et al. [2013]). Based on the observation that there is very little vegetation in areas known to have truffles (subterranean gourmet fungi), it has been hypothesized that these fungi may have the ability to suppress plant growth through their volatiles (Splivallo et al. [2007]). Kishimoto et al. ([2007]) have shown that 1-octen-3-ol enhances resistance of mature plants of Arabidopsis thaliana to Botrytis cinerea and activates some of the same defense genes turned on by ethylene and jasmonic acid signaling, important plant hormones involved in plant defense.

We hypothesized that we could distinguish between bioactive and inactive fungal vapors by using chemical standards of individual VOCs and then exposing plants to controlled concentrations in a model habitat. We selected A. thaliana as our test species due to the many benefits associated with the use of a well recognized model system including but not limited to: small size, short life cycle, genetic tractability, and comprehensively researched background. Preliminary studies have also shown that tomato plants exposed to VOCs are affected in a similar fashion to A. thaliana exposed to the same VOCs indicating that A. thaliana is a good model organism for study. Similarly, the effects of fungal VOCs on plant development have been demonstrated in several plants in Brassicaceae family including radish, cabbage, rape, and broccoli (Ogura et al. [2000]). The aim of our study was to evaluate the effect of individual fungal VOCs on seed germination, vegetative plant growth and chlorophyll concentration in a controlled environment. In this report, our specific objectives have been to create standardized model exposure habitats in order to compare the possible stimulatory and inhibitory effects of fungal VOCs from different chemical classes (e.g., alcohols, aldehydes, ketones, and so forth) and to conduct exposure studies using A. thaliana seeds and two-week-old vegetative plants.

In order to study the influence of individual fungal VOCs on plant health, we used A. thaliana as our test organism and developed standardized protocols for exposing seeds and young vegetative plants. We investigated a representative sample of common fungal VOCs encompassing seven alcohols, two alkenes, five aldehydes, four ketones, and a single representative isothiocyanate, carboxylic acid, and furan. In addition, we tested both isomers of the terpene 1-methyl-4-(1-methyetenyl)-cyclohexene, commonly known as limonene. We used a 72 hour exposure period, and exposed either seeds or young vegetative plants to 1 ppm of chemical standards of the 23 fungal VOCs in a contained chamber.

Seed germination assays with lettuce, cucumber and other economically important species have been widely employed as low-cost, ethically acceptable toxicity tests to screen for dangerous levels of industrial contamination in water and soils (Banks and Schultz [2005]; Wang et al. [2001]). Almost all of these assay studies have involved aqueous phase compounds, although there have been a few scattered reports describing inhibitory effect of plant volatiles on the germination of seeds from crop and weed species (Holm [1972]; Bradow and Connick [1990]). The physiological basis for this inhibitory action has not been elucidated. A. thaliana, though not agriculturally important, offers a number of experimental advantages for studying basic aspects of plant biology, including seed germination, because of its many genetic resources (Koornneef et al. [2002]). Our development of an A. thaliana exposure system for studying VOC effects under controlled conditions offers the promise of being able to use this species to dissect the germination inhibition response at the molecular level.

In contrast to seed germination studies, which all report inhibition of germination by VOCs (Holm [1972]; French et al. [1975]; Bradow and Connick [1990]). To date, published studies of the effects of VOCs on vegetative plants report VOC--associated growth stimulation by mixtures of VOCs emitted by growing bacteria or fungi (Ryu et al. [2003]; Minerdi et al. [2011]; Hung et al. [2013]; Paul and Park [2013]). Many soil dwelling microbes emit VOCs that mediate various chemical “conversations” between the rhizosphere and plants (Wenke et al. [2010], [2012]). For example, plant growth promoting rhizobacteria (PGPR) produce mixtures of VOCs that enhance growth in a wide variety of species (Farag et al. [2006]; Vespermann et al. [2007]; Lugtenberg and Kamilova [2009]). In some cases, microbial VOCs induce systemic resistance (Van Loon et al. [1998]; Ryu et al. [2003]) or inhibit the growth of plant pathogens (Minerdi et al. [2009], [2011]). Volatiles of Cladosporium cladosporioides enhance growth of tobacco plants (Paul and Park [2013]) and our laboratory has shown that vegetative plants of A. thaliana seedlings grown in a shared atmosphere with volatiles emitted by living cultures of the biocontrol fungus Trichoderma viride, displayed increased size and vigor (Hung et al. [2013]). Fungal VOCs may contribute to the ability of certain species to outcompete neighboring plants. For example, the VOCs produced by Muscodor yucantanensis were toxic to the roots, and inhibited seed germination, of amaranth, tomato and barnyard grass (Macias-Rubalcava et al.[2010]). In all of these cases, the growth-enhancing effects were mediated by mixtures of naturally emitted VOCs that change with both growth phase and extrinsic environmental parameters.

In our studies, we used a controlled system and exposed plants to low concentrations of individual VOCs. In this controlled habitat, the VOCs tested either had neutral or negative effects on vegetative plant growth, suggesting that the known growth enhancing effects of bacterial and fungal VOCs maybe by synergistic mixtures working in concert. A parallel example is provided by the antibiotic effects of volatiles produced by Muscodor albus. This species produces a mixture of VOCs that inhibit and kill a wide range of plant pathogenic fungi and bacteria (Strobel et al. [2001]). Nevertheless, when Muscodor VOCs were tested individually, the inhibitory effects were not observed, suggesting that the antifungal activity required a suite of VOCs working in concert (Strobel et al. [2001]).

It should be recognized that studies on transkingdom signalling mediated by fungal VOCs are technically difficult to conduct. Biogenic VOCs exhibit enormous heterogeneity chemically, spatially, and temporally; are found in low concentrations; and by definition have innate evaporative properties that make it difficult to investigate their impact on plant growth and development in natural settings. Our exploratory research shows that by using controlled concentrations of pure synthetic compounds in model habitats, individual VOCs can be studied one by one, thereby isolating their distinct growth-promoting, growth-inhibiting and other physiological effects. The genetic and genomic resources available for A. thaliana make this organism well suited for future research on the mechanistic basis of VOC mediated interactions and for analysis of the consequent biological responses. In summary, the study of gas phase fungal metabolites offers many interesting prospects for enlarging our understanding of the way in which fungi interact with plants in nature and may have potential for commercial application of VOCs in greenhouse agriculture.