Introduction

“Oxylipin” is the group name of compounds derived from the oxidation of unsaturated fatty acids and includes fatty acid hydroperoxides, hydroxyl fatty acids, epoxy fatty acids, keto fatty acids, volatile aldehydes, and cyclic compounds. They are often found as autocatalytic breakdown products of unsaturated fatty acids [9]; however, they also may be produced by enzymatically controlled pathways and frequently function as cellular messengers in eukaryotic systems. Important animal oxylipins include prostaglandins and leukotrienes [22]; important plant oxylipins include jasmonic acid [47]. Further, there is increasing evidence that oxylipins play essential roles in fungal morphogenesis and pathogenesis [1, 7, 10, 59].

Those oxylipins that easily vaporize are often classified as volatile organic compounds (VOCs), i.e., low molecular mass compounds with high vapor pressure that exist in the gaseous state at room temperature [27]. Many compounds emitted by fungi such as ammonia, 1-butanol, farnesol, and non-anoic acid are important chemical languages that allow fungi to communicate with one another and to respond to environmental signals [40]. Volatile oxylipins are important components of aromas. Autoxidation products of unsaturated fatty acids have been widely studied because they cause rancid off-odors in food [28, 44]. Several six-carbon oxylipins are the dominant compounds released by plant material after tissue damage and are responsible for the “green notes” of fruits and vegetables [8, 63]. The various aldehydes associated with the distinctive odor of cut grass, collectively called “green leaf volatiles,” are formed from linolenic or linoleic acids [21, 43]. Many of these volatile metabolites, as well as aromatic oxylipins found in fruit and vegetables, and in essential oils of spices and herbs, are biologically active against fungal pathogens [58]. Because of their anti-fungal activity, (E)-2-hexenal (also called (E)-hex-2-enal) and 1-hexanol, have been tested for controlling postharvest fungal pathogens [15, 16, 18, 23, 50].

In contrast to the abundant six-carbon oxylipins from plants, eight-carbon compounds are the most common oxylipins from mushrooms and molds. Mushroom alcohol, or 1-octen-3-ol, is often the single most abundant aroma compound produced by mushrooms [20, 57] and is a well-known flavoring agent and insect attractant [45]. It is formed through enzymatic oxidation and cleavage of linoleic acid by a lipoxygenase and a hydroperoxide lyase, as well as by autoxidation of linoleic acid [14]. 1-Octen-3-ol is also one of the major fungal VOCs emitted by molds such as those commonly found in water-damaged buildings [34, 38, 48]. Because mushroom alcohol has a chiral carbon, it can exist as two optical isomers or enantiomers: (R)-(−)-1-octen-3-ol and (S)-(+)-1-octen-3-ol [46]. (R)-(−)-1-octen-3-ol exhibits the typical aroma of fresh mushrooms, while (S)-(+)-1-octen-3-ol smells more moldy [14]. Chiral discrimination plays a central role in the activity of many biosystems [25], however, most laboratory studies on 1-octen-3-ol have used the racemic form.

Several eight-carbon oxylipins serve hormonal functions in fungi. For example, under conditions of high spore density, 1-octen-3-ol functions as a self-inhibitor in Penicillium paneum and Agaricus bisporus [11, 12, 52]. Self-inhibitors inhibit spore germination reversibly, possibly through effects on the plasma membrane [12]. At certain concentrations, the eight-carbon oxylipins 1-octen-3-ol, 3-octanone, and 3-octanol stimulate aerial conidiation in Aspergillus nidulans [26]. In Agaricus bisporus, 1-octen-3-ol inhibits primordium formation [52]. 1-Octen-3-ol has also been used to minimize dry bubble disease caused by Lecanicillium fungicola in the cultivation of the white button mushrooms [3].

It is well known that olfaction plays important roles in insect behavior serving as attractants, repellants, and sexual pheromones. Less well known is that some of these insect pheromones and other “semiochemicals” (signaling compounds) are common plant and fungal volatiles. Many six- and eight-carbon oxylipins serve as semiochemicals for arthropods. For example, 1-octen-3-ol is an attractant for midges [6], mosquitoes [37], and tsetse flies [56]; 1-hexanol attracts German cockroaches [35]; and (E)-2-hexenal is a component of the bedbug pheromone [41]. See Davis et al. for an excellent review of insect chemoreception of microbial VOCs [17].

The majority of the research on the physiological effects of six-carbon and eight-carbon oxylipins has focused on single system assays. Therefore, in order to compare the activities of three common volatile oxylipins in a controlled environment, we studied them in both fungal spore germination assays and an insect bioassay. The aims of this study were to: (1) test the effects of different concentrations of chemical standards of mushroom alcohol and two green leaf aldehydes on colony formation of Aspergillus niger and Penicillium chrysogenum; (2) observe if the enantiomers of 1-octen-3-ol have differential effects on spore germination; and (3) determine if these common volatile oxylipins had developmental effects on the morphogenesis (metamorphosis) of Drosophila melanogaster. A. niger is found in many habitats, is a common postharvest plant pathogen, and infects a variety of vegetables, fruits, and crops including onions, grapes, maize, grains, and other major crops [55]. P. chrysogenum is best known for its production of the antibiotic penicillin, but is also a common spoilage mold [54]. Drosophila is a well-known model organism for the study of interkingdom genetics. However, these insects are globally destructive pests of pre- and postharvest fruits [19, 39]. Here, we employ the fruit fly as a model insect pest that may be controllable by the use of fungal volatiles. Our results will help inform applied biologists that volatile oxylipins have multiple, complex, and multi-kingdom functional roles in ecosystems and have potential uses as biofumigants.

Materials and Methods

Fungal Samples and Culture Conditions

Aspergillus niger and Penicillium chrysogenum isolates were obtained from Dr. Marshall Bergen, Rutgers University, and had been identified by standardized taxonomic protocols [36, 53]. Cultures were grown on potato dextrose agar (PDA) (Difco) and incubated at 26 °C. Conidial suspensions were harvested from 3-day-old confluent plates by flooding the surface with a sterile solution of 10 mL 10 % glycerol and 1 % Tween 80, and gently stirring. The resulting spore-glycerol suspensions were transferred to sterile centrifuge tubes and stored at 4 °C. Viable spore counts were determined by serial dilutions in sterile water. Dilutions of 1 × 10−3–10−7 spores were plated on PDA and incubated at 26 °C for 3–5 days before conducting viable counts.

Chemicals

Chemical standards of the three fungal oxylipins, (E)-2-hexenal (≥95 %), 1-hexanol (≥99 %), and racemic 1-octen-3-ol (>99 %), were purchased from Sigma (Missouri, USA). In some observational experiments, fungi were exposed to vapors of the enantiomers of 1-octen-3-ol. Both (R)-(−)-1-octen-3-ol (99.6 %) and (S)-(+)-1-octen-3-ol (99.9 %) were obtained as gifts from Bedoukian Research Inc. (Connecticut, USA). The industrial solvents used as positive controls were acetone, benzene, formaldehyde (37 %), toluene, and m-xylene. All except acetone were purchased from Sigma. Acetone was obtained from Alfa Aesar (Massachusetts, USA). The names (including synonyms), the chemical structures, odor, and a summary of some of the functional properties of the fungal oxylipins are given in Table 1.

Table 1 The chemical names and structures, odor properties, and some examples of functionality of the compounds used in this study
Full size table

Exposure Conditions for Fungal Spore Germination Studies

Plastic Petri plates (100 mm × 15 mm) divided in half (sometimes called “I” plates) were used for spore germination studies. One half of the plate was filled with 10 mL of PDA and inoculated with 10 µL of a spore suspension of A. niger or P. chrysogenum that had been serially diluted in order to obtain estimated plate counts of 50–100 colony-forming units (CFUs). A sterile glass cover slip (22 mm × 22 mm) was placed into the second, empty side of the two-compartment Petri dish, and then aliquots of liquid 1-hexanol, (E)-2-hexenal, or racemic 1-octen-3-ol were pipetted onto the cover slip in order to deliver concentrations of 10, 50, and 100 ppm. The Petri dishes were sealed immediately using two layers of Parafilm and placed into 4 L glass containers with tightly fitting polypropylene lids. The added agar was also considered when we calculated the concentrations, however, we recognize that the final concentrations are estimates because there was some leakage of VOCs from the plates into the containers. For controls, the empty segment of the divided Petri dish was untreated, i.e., germinating spores were not exposed to any VOCs. Three independent experiments of three replicates (n = 9) were conducted for each fungal VOC. For positive controls, acetone, benzene, toluene, m-xylene, and formaldehyde were tested at 100 and 1000 ppm against A. niger in the same divided Petri plate exposure set up as was done for the chemical standards of the fungal VOCs.

All experiments were incubated at 26 °C. For A. niger, the number of CFUs was counted after 2 days; for P. chrysogenum, the CFUs were counted after 3 days. For both species, the diameter of individual mold colonies was measured after 3 days.

Effects of Enantiomers of 1-Octen-3-ol on Colony Morphology of A. niger and P. chrysogenum

Aspergillus niger was cultured for 3 days and P. chrysogenum for 4 days with and without treatment with 50 ppm racemic 1-octen-3-ol and its two enantiomers, (R)-(−)-1-octen-3-ol and (S)-(+)-1-octen-3-ol. Exposure and culture conditions were the same as described above.

Exposure Conditions for Drosophila melanogaster Third Instar Larvae

The conditions for exposure of D. melanogaster (y1, w1118) larvae to VOCs were adapted from Inamdar et al. [31]. However, flies were exposed to the volatile oxylipins within a 500-mL glass dish. Briefly, all fly stocks were maintained on Ward’s Instant Drosophila medium (blue) and all experiments were performed at 25 °C. Fruit juice agar plates were used for egg collection. For 500 mL of fruit juice medium, the following ingredients were combined: 126 mL of Strawberry Banana 100 % Juice (V8 brand), 15 g agar, 6 g sucrose, and 376 mL ddH2O. After autoclaving, when the medium had cooled to 60–65 °C, 10 mL of ethanol and 5 mL of acetic acid were added, mixed well, and then, using a 10-mL pipetting device, 10 mL of medium was pipetted into the bottom of a small (35 mm × 100 mm) Petri dish. Thirty mature third instar larvae (i.e., larvae collected at the 6th day after eggs had been laid) were collected from the fruit juice agar and then transferred onto 1 % agar medium in the deep Petri dish (500 mL, Pyrex, 100 × 80, No. 3250) containing the vaporized oxylipins (3 ppm). Then the deep Petri culture dish containing the vaporized oxylipin and Drosophila larvae, was sealed with two layers of Parafilm and placed on an orbital shaker at 50 rpm to maintain even distribution of the volatile compounds. The number of larvae, pupae, and/or adult stages was counted daily for 10 days. The percent of fly larvae progressing to each stage was calculated for three replicates and repeated three times (30 larvae/replicate, for 270 larvae in total for each treatment). Repeated analysis of variance (ANOVA) analysis and Duncan’s multiple-range tests were performed with SAS 8.2 software (SAS Institute, Cary, NC, USA).

Results

Positive Controls

The industrial solvents used as positive controls were acetone, benzene, formaldehyde, toluene, and m-xylene. A. niger conidia were exposed at 100 and 1000 ppm, and CFUs were counted at 48 and 72 h. Vapors of formaldehyde prevented germination of A. niger at 100 ppm, however, vapors of acetone, benzene, toluene, and m-xylene had no effect on the number of CFUs observed, even at 1000 ppm (data not shown).

The Effects of 1-Octen-3-ol, 1-Hexanol, and (E)-2-hexenal on the Germination Rate and Colony Diameters of A. niger and P. chrysogenum

The effect of vapors of 1-octen-3-ol, 1-hexanol, and (E)-2-hexenal on the germination efficiency (×103 CFU/mL) and colony diameters of A. niger are shown in Fig. 1. At 10 and 50 ppm, exposure to all three VOCs yielded a slight increase in the number of CFUs. At 100 ppm, 1-octen-3-ol completely inhibited colony formation, while (E)-2-hexenal and 1-hexanol had smaller effects compared to control without any volatiles (Fig. 1a). At concentrations where colonies did form, after the second day of inoculation, radial growth and spore formation were inhibited for all three volatiles at all concentrations (Fig. 1b). The strongest inhibition of radial growth was in the presence of (E)-2-hexenal.

Fig. 1
figure 1

Effects of vapors of 1-octen-3-ol, 1-hexanol, and (E)-2-hexenal oxylipins on Aspergillus niger a the number of colony-forming units and b the colony diameters (2 days)

Full size image

Similar results were obtained for P. chrysogenum (Fig. 2). A slight increase in the number of CFUs was observed in the presence of 10 ppm of 1-octen-3-ol and (E)-2-hexenal; at higher concentrations fewer CFUs were observed for all three volatile oxylipins (Fig. 2a). (E)-2-hexenal had the strongest inhibitory effect on the CFUs of P. chrysogenum, then 1-octen-3-ol, followed by 1-hexanol. P. chrysogenum did not grow in the presence of 100 ppm of vapors of 1-octen-3-ol and (E)-2-hexenal. Conidial formation was delayed and colony diameters were smaller in the presence of all three volatile oxylipins at each concentration tested (Fig. 2b).

Fig. 2
figure 2

Effects of vapors of 1-octen-3-ol, 1-hexanol, and (E)-2-hexenal oxylipins on Penicillium chrysogenum. a The number of colony-forming units and b the colony diameters (3 days)

Full size image

Effects of Enantiomers of 1-Octen-3-ol on Colony Morphology of A. niger and P. chrysogenum

When spores were germinated in the presence of 50 ppm of volatilized racemic 1-octen-3-ol and its two enantiomers, differences in colony morphology were observed (Fig. 3). After 4 days of growth, control colonies of A. niger had heavily sporulating black colonies and grew with an average colony diameter of 15 ± 0.1 mm. Cultures grown in the presence of (S)-(+)-1-octen-3-ol and racemic 1-octen-3-ol had smaller colony diameters (5.6 ± 0.5 mm), with sporulation limited to the center of the colony surrounded by halos of non-sporulating mycelia. Those cultures grown in the presence of (R)-(−)-1-octen-3-ol showed the greatest growth inhibition effect with average colony diameters of 1.5 ± 0.4 mm and only a small pinpoint of black conidial formation in the center of the colony (Fig. 3a).

Fig. 3
figure 3

a Aspergillus niger and b Penicillium chrysogenum cultivated for 3 days with/without treatment with racemic 1-octen-3-ol and its enantiomers, (R)-(−)-1-octen-3-ol and (S)-(+)-1-octen-3-ol (50 ppm). Aspergillus niger was cultured for 3 days and P. chrysogenum was cultured for 4 days

Full size image

Similar results were obtained when P. chrysogenum was grown in the presence of 50 ppm of racemic 1-octen-3-ol and its two enantiomers. At 3 days, control cultures of P. chrysogenum had a heavily sporulating center with bluish-green areas of sporulation surrounded by a halo of non-sporulating white mycelium (Fig. 3b). P. chrysogenum grew very sparingly when exposed to 50 ppm of (R)-(−)-1-octen-3-ol, with a few tiny colonies. Compared to controls, colonies grown in the presence of racemic 1-octen-3-ol and (S)-(+)-1-octen-3-ol formed smaller whitish blue–green colonies with a colony diameter of 1.3 ± 0.5 mm.

Effects of Three Vaporized Oxylipins on Metamorphosis and Viability of Third Instar D. melanogaster Larvae

We also tested the effects of these three VOCs on D. melanogaster. Third instar larvae of Drosophila were exposed to 3 ppm of each compound and then the subsequent development of larvae, pupae, and adults was monitored daily for 10 days of continuous exposure. Survival of exposed larvae was similar to controls (Fig. 4a) but pupae exposed to 1-octen-3-ol exhibited significantly delayed development (Fig. 4b). The adults firstly experienced a small increase in emergence and then loss of viability (Fig. 4c). Total death rates increased significantly with 10 days of continuous exposure. In Fig. 4d, the dead larvae, pupae, and adults were counted together and after 10 days. The mortality rates for 1-octen-3-ol, (E)-2-hexenal, and 1-hexanol were 100, 58, and 40 %, respectively. Although about 80 % of larvae pupated after treatment with 1-octen-3-ol, only a small number of exposed pupae (7 %) emerged into adults (Fig. 4c). We also observed that these three oxylipins caused morphological abnormalities in Drosophila larval, pupal, and adult stages. As compared to controls (Fig. 5a–c) body color of larvae and pupae became darker in the presence of 1-octen-3-ol (Fig. 5d, e); for adults, wings were abnormally developed (Fig. 5 f).

Fig. 4
figure 4

Metamorphosis of mature third instar Drosophila larvae over 10 days of continuous exposure to 3 ppm of vapors of (E)-2-hexenal, 1-hexanol, and racemic 1-octen-3-ol. a Percent living larvae, b percent pupae, c percent living adults, d percent mortality over 10 days. The number of larvae, pupae, and adults was counted daily for 10 days, and 30 larvae were exposed to each oxylipin in each experiment and the experiment was repeated three times. (N = 270) **P < 0.01, ***P < 0.001

Full size image
Fig. 5
figure 5

Effects of 1-octen-3-ol on the development of Drosophila. Examples of a control larvae, b control pupae, c control adults. Examples of 1-octen-3-ol treated d larvae, e pupae, and f adults

Full size image

Discussion

Like plants and animals, fungi use diverse signal molecules, some of which are volatile, to control processes critical to their own growth and reproduction such as nutrient acquisition, spore germination, and sexual development. We are only beginning to understand the role of these reactive volatile molecules within complex and multi-organismal ecological systems. In our study, “mushroom alcohol” (1-octen-3-ol) and the “green leaf volatiles” (E)-2-hexenal and 1-hexanol inhibited the germination and growth of A. niger and P. chrysogenum. A concentration of 100 ppm of any of the oxylipins almost completely inhibited spore germination and growth. However, even high concentrations (1000 ppm) of well-known toxic industrial solvents such as acetone, benzene, toluene, and xylene did not have any detectable inhibitory effect on spore germination and colony growth. These findings support the hypothesis that the volatile phase oxylipins may have specifically evolved as signaling agents in the fungal life cycle.

Our data support the findings described for other fungal species. Radial growth of Aspergillus parasiticus is completely inhibited by 100 μL of both hexanal and octanol [61]. Similarly, (E)-2-hexenol inhibited the growth of Rhizoctonia solani and Sclerotium rolfsii [60] and both 1-hexanol and (E)-2-hexanol inhibited Fusarium avenaceum and Fusarium graminearum [16]. (E)-2-hexenal vapors inhibit both A. flavus growth and aflatoxin production in corn [18] and give good control of Penicillium expansum, the causal agent of blue mold disease in pears [50]. Additionally, 1-hexanol can be used as a postharvest fumigant to control gray mold (Botrytis cinerea) during postharvest storage of strawberry, blackberry, and grape [2]. Although it has not been used for postharvest control of pathogens, 1-octen-3-ol inhibits spore germination and induces microconidiation in Penicillium paneum [11, 12]. The ability of eight-carbon volatiles to affect germination of conidia was also reported with Aspergillus nidulans [26] and Lecanicillium fungicola [3]. Our data supplement previous studies that demonstrate that at appropriate concentrations these volatile compounds can be used as possible postharvest fumigants to control mold pathogens in fruits, vegetables, and crops. More quantitative work needs to be done to simulate natural conditions to better use these volatile compounds for diseases and spoilage control.

On the other hand, both A. niger and P. chrysogenum formed more CFUs in the presence of low concentrations of 1-octen-3-ol. In a study on Trichoderma species [49], dark grown cultures also produced more conidiation when exposed to low concentrations of vapors of 1-octen-3-ol, 3-octanol, and 3-octanone between 1 and 100 μM. At 500 µM of 1-octen-3-ol, both conidiation and growth were greatly inhibited. However at 500 μM, 3-octanone increased condition. Similar hermetic effects of oxylipins whereby lower concentrations promote, while higher concentrations impede both growth and conidiation [14, 26].

Most of the published literature on 1-octen-3-ol focuses on its properties as a mushroom flavor compound [62] or its importance in attracting biting insects [4, 13, 42]. Across systems, most researchers have tested the racemic form of the compound. However, 1-octen-3-ol is registered in two forms as a pesticide: (a) the racemic mixture that includes both enantiomers and (b) (R)-(−)-1-octen-3-ol. In cases where it has been studied, the R enantiomer is frequently the more active component. For example, the R form is somewhat more effective in retarding Arabidopsis seed germination, seedling, and vegetative growth [29]. Conversely, in human embryonic stem cells, (S)-(+)-1-octen-3-ol showed stronger toxic activity than (R)-(−)-1-octen-3-ol or the racemic form [30]. Recently, an odorant receptor (AaegOR8) was demonstrated to be selectively sensitive to (R)-(−)-1-octen-3-ol [24]. In our studies, (R)-(−)-1-octen-3-ol imposed stronger inhibitory activities than (S)-(+)-1-octen-3-ol on the growth of A. niger and P. chrysogenum. Previous reports show that (R)-(−)-1-octen-3-ol is the main component of natural mushroom flavor and it is largely synthesized in the cap and gills of the mushroom [14]. Analysis of several cultivated mushrooms for the enantiomeric ratio of 1-octen-3-ol found the optical purity of (R)-(−)-1-octen-3-ol to range in amounts over 82.1 % in Xerocomus badius to over 98.5 % in Agaricus bisporus [62]. This suggests preferential biosynthesis of the R enantiomer. The underlying mechanisms behind selective response and production of 1-octen-3-ol enantiomers need further study, and we recommend that future molecular studies that focus on the possible hormonal action of 1-octen-3-ol should include an examination of both enantiomers.

In the previous work from our laboratory, we developed a Drosophila bioassay system using third instar larvae for testing the toxicity of volatile phase compounds and showed that aerial exposure to vapors of both industrial solvents and fungal VOCs could cause delays in metamorphosis and death of larvae, pupae, and adults [31] and that 1-octen-3-ol disrupts dopamine packaging and causes neurodegeneration in flies [32]. Here we confirm the high toxicity of 1-octen-3-ol to Drosophila. In addition, our data demonstrated that like 1-octen-3-ol, both 1-hexanol and (E)-2-hexenal disrupt Drosophila metamorphosis. With the additional knowledge that many fungal volatiles are more toxic to Drosophila than benzene, formaldehyde, toluene, and xylene [31], it is possible that we may be able to develop biofumigants with fungal volatiles at concentrations well below those toxic to humans.

Deciphering the chemical language of VOCs will be a multidisciplinary challenge [5, 33]. As biologists become more aware of the interkingdom fumigant properties of fungal VOCs, we predict that interdisciplinary collaborations will become more common. In conclusion, our study supports the hypothesis that fungal volatiles have inhibitory effects on fungal pathogens and insects. We plan to use this information to develop biofumigants for postharvest pathogens.