Exploiting Plant Volatile Organic Compounds (VOCs) in Agriculture to Improve Sustainable Defense Strategies and Productivity of Crops

1Reports analyzing the status of agriculture worldwide forewarn that a significant increase of the present agricultural production would be necessary to meet the future demand for food, as the world population is expected to rise from 7.3 to 9.7 billion by 2050. As a consequence, FAO recently projected that an increase of food production (70%) may be required and thus a third green revolution needs to be attained. Enhanced crop yields in the past 50 years were made possible by the introduction of mechanization, the progresses in genetics and the use of improved crop varieties, as well as the extensive use of chemicals such as fertilizers and pesticides. However, the yield of grain crops considered the main sources of human and livestock calories (e.g., rice) have already reached a “plateau”. Natural resources providing fertilizers (especially phosphate, ) are also depleting, and the use of chemicals has caused serious problems with food safety and environmental pollution. Moreover, climate change is forecasted to increase the severity and frequency of drought events that will cause both a reduction in plant primary production and a future progressive exposure of agricultural soils to degradation and loss of fertility. Climate change will also favor the spreading of plant pathogens into larger geographical areas where new hosts may be found, leading to more frequent epidemics. In this context, agriculture is called to provide solutions to increase yields while preserving natural resources and the environment.

2This perspective article explores the potential of natural Volatile Organic Compounds (VOCs) emitted by plants as an eco-sustainable strategy to implement future smart agricultural practices and enhance plant protection and productivity. As airborne signals, VOCs allow quick defense signaling between distant plant organs and the communication between plants. In addition, VOCs can “prime” the defense system of plants for an enhanced resistance to an upcoming stress. However, to date, VOCs have been mostly applied in the field to repel herbivores or to attract herbivores’ parasitoids or predators.

3Emission of VOCs can be induced at any time from leaves of all plant species following abiotic or biotic stresses. Results from many studies have demonstrated that emission of isoprenoids, the most abundant group of VOCs, is stimulated by abiotic stresses and improves plant resistance either by direct quenching of reactive oxygen species (ROS), or indirectly by stabilizing cell membranes. However, protection of cell membranes to avoid toxic accumulation of ROS is only one among the many roles of VOCs that may be exploited in agriculture (Figure 1).

4Plants maintain memory of any stress event they have experienced, and this memory is able to influence the response to forthcoming stressful situations. Factors able to shape the plant’s stress memory are referred to as “priming stimuli”, among which plant VOCs play a crucial role because, due to their volatility, they can quickly reach distant plant parts. A “primed” plant shows an earlier, stronger, and faster response upon further stress occurrence, thereby resulting in increased resistance and/or tolerance. VOCs have been extensively demonstrated to prime defenses against herbivorous insects, pathogens, and environmental stresses. Defense priming against pathogens has also been considered as a sort of “green vaccination”. Green leaf volatiles (GLVs) such as Z-3-hexenyl acetate, ubiquitously and rapidly released after mechanical damage of leaf tissues, have been reported to prime resistance of wheat plants to the fungal pathogen F. graminearum and to reduce the damage occurring to maize plants during cold stress. Other VOCs such as methyl salicylate (MeSA) and monoterpenes (i.e., camphene and pinene) have been found to actively participate in the mechanisms leading to systemic acquired resistance (SAR). Low concentrations of methyl jasmonate (MeJA) have been demonstrated to prime plant defenses by modifying the epigenetic status of wound-inducible genes in rice, thereby enhancing responsiveness to wounding. Even methanol, ubiquitously emitted from plant leaves during cell division and cell wall expansion, seems to act as a priming stimulus when released from damaged tobacco leaves by enhancing resistance to the pathogenic bacterium Ralstonia solanacearum. In addition, antibacterial defenses have also been reported to be primed by VOCs such as nonanal in lima bean plants treated with benzothiadiazole (BTH), a synthetic salicylic acid analog. Compared to the direct induction of defenses in plants, priming does not incur in an energetically costly activation of metabolic pathways and therefore represents a sustainable method to develop novel crop protection strategies.

5A number of experimental trials have shown the capacity of various VOCs produced by leaves to inhibit germination and growth of plant pathogens, yet the mechanisms of action remain unknown. Citral, carvacrol, and trans-2-hexenal were reported to be effective in hampering in vitro growth and germination of Monilinia laxa, the agent of brown rot of stone fruit. In particular, trans-2-hexenal provided protection also when tested in vivo on apricot, nectarine, and peach fruits as a postharvest biofumigant. In addition, the growth of Colletotrichum acutatum, causing citrus post-bloom fruit drop, was moderately inhibited in vitro when exposed to linalool. Botrytis cinerea, a necrotrophic fungus with a very broad host range, has been reported to be highly sensitive to the in vitro application of monoterpenes, such as (+)-limonene. However, exposure to (+)-limonene stimulated in vitro growth of the fungal pathogen Penicillium digitatum, whereas this fungus was highly inhibited by the application of citral. In a recent work, performed a screening on the efficacy of 22 different VOCs, known to be emitted from leaves, against the fungal pathogens Colletotrichum lindemuthianum, Fusarium oxysporum, and B. cinerea. These fungi were grown in Petri dishes in which the headspace had been enriched, each time, with a single VOC. Results showed that exposure to nonanal, (+)-carvone, citral, trans-2-decenal, L-linalool, nerolidol, or eugenol significantly inhibited the growth of all these three fungal species, with eugenol demonstrating the strongest activity. Other VOCs such as cuminaldehyde and p-cymene have been also demonstrated to possess antifungal activity against B. cinerea, F. oxysporum, Verticillium dahliae, and Alternaria mali.

6Limited information exists concerning the effects of plant VOCs on crop productivity. Emission of VOCs from leaves can have allelopathic effects and impair the growth of other competitive plant species. Hexenal and isoprenoids (mono- and sesqui-terpenes), for instance, have been demonstrated to inhibit seed germination and root growth. By mediating competition between plant species, VOCs may allow to control weeds and thus enhance crop productivity through a more efficient acquisition of nutrients, water, and light.

7Moreover, it was recently proposed that VOCs (i.e., isoprenoids) may work in synergy with other secondary metabolites (i.e., carotenoids) and hormones (i.e., cytokinins) which are all synthesized by the methyl erythritol phosphate (MEP) pathway to regulate senescence. Aging of plant tissues is controlled by changes in hormone levels and may lead to uncontrolled accumulation of ROS following damage to cellular organs (i.e., membranes) and other macromolecules (i.e., DNA). Therefore, a sustained production of volatile isoprenoids may synergize with the biosynthesis of cytokinins and increase antioxidant activity at the foliar level. This could prevent cell degradation and death, thus prolonging the life span of leaves and flowers with a positive impact on the whole plant production process.

8The trade-off between benefits and costs of VOC emission as stress relief compounds is not clear and difficult to assess. Experiments with transgenic plants suggest that the metabolic cost for emitting isoprene (the most abundant VOC released from leaves) outweighs benefits.

9Despite the demonstrated efficacy of VOCs in enhancing plant fitness and defense against stress in many controlled trials in the laboratory, their effectiveness to enhance plant defenses have been tested in the field only in a few cases. To date, VOCs are applied in agriculture solely for the “push-pull strategy”, where the crop of interest is both intercropped with plant species that emit VOCs able to repel (“push”) herbivores, and surrounded with plants emitting VOCs that simultaneously attract (“pull”) herbivores away from the field (Figure 1).

10Why are VOCs not more intensively used in agriculture for integrated and eco-friendly plant protection? One reason could be that laboratory experiments demonstrating VOC efficacy have been performed with concentrations far higher than those achievable in open field. Trials to test the antimicrobial activity of VOCs are often performed in Petri dishes by applying pure liquid solution, without even quantifying the real VOC concentration present in the headspace over the course of the experiment. Moreover, while the high biodegradability of VOCs may minimize both their impact and the occurrence of long-term non-targeted effects, at the same time this limits VOC persistence and activity. Indeed, VOCs are often very reactive with the environment. Reports have demonstrated that VOCs disappear more rapidly in polluted environments where they can react with NOx, OH− radicals, and ozone. Therefore, high reactivity of VOCs limits the distance they travel across the fields and their overall range of protection on crops. Reduction of VOC concentration in air may thus modulate their effect on insects, microbes, and plants. Finally, the very essence of VOCs (volatility) makes these compounds highly influenced by meteorological factors such as wind speed and direction, humidity and rain, and temperature, among others. It is also important to highlight that VOC emissions from leaves in the field can hardly be faithfully reproduced. In fact, complex blends rather than single VOCs are released by plants, and their biosynthesis is controlled by a network of different biochemical pathways. In addition, the composition of VOC mixtures is subjected to strong plant genotypic and phenotypic plasticity and can vary according to the plant ontogenetic state and environmental conditions. Modern techniques of micro- and nano-encapsulation, which allow a more controlled release of synthetic blends that mimic the natural release of VOCs, are expected to improve VOC efficacy. Further efforts should be also made to decipher the perception mechanism of VOCs within plant tissues, since it is not yet clear how VOCs are perceived by plants. Indeed, molecules acting as VOC receptors have not been identified so far.

11Some studies have reported other possible pitfalls of the application of VOCs in open fields that need to be carefully evaluated. For instance, the release of synthetic GLVs in the field synergistically affected isoprenoid emission from maize plants and unexpectedly increased herbivore damage without significantly attracting beneficial insects. Methanol emitted by wounded tobacco leaves was reported to enhance antibacterial defenses in neighboring non-wounded plants, but also facilitated the spreading of tobacco mosaic virus (TMV). In other cases, VOC phyto-toxicity was observed. For example, demonstrated that the concentration of trans-2-hexenal required to exert the antimicrobial activity was phytotoxic for some fruits (i.e., apricot, peach, and nectarine), whereas plum fruits could be protected without damage to plant tissues. Importantly, application of Z-3-hexenyl acetate enhanced resistance of wheat plants against the pathogenic fungus F. graminearum while boosting the production of deoxynivalenol (DON), a dangerous mycotoxin for human health.

12Technical limits and drawbacks may not have been the only constraints limiting the exploitation of VOCs in agricultural practices. High costs associated with the process of formulation, mass production, registration, and marketing of synthetic VOCs can make their sale price prohibitive, and initial investments unproductive. Depending on the screening procedures, 5–10 years may be required for research and development of effective and specific synthetic blends of VOCs. Elevated production costs and scalability limitations have inevitably slowed down the use of VOCs in agriculture. Besides, the management of VOC-based products in the field may become expensive, and require specific technical skills that usually growers and retailers do not possess. Nevertheless, the current increasing demand for eco-friendly and sustainable solutions to protect crops and enhance their productivity, also due to increasingly stringent limitations on chemical pesticides (see the European legislation on Plant Protection Products), could attract future investments making the use of VOCs in agriculture competitive, especially in conditions where VOC application can be controlled more effectively and problems with synthetic chemicals can manifest (e.g., greenhouses).

13Over the last years, domestication and man-driven crop selection have mainly focused on high yields. However, this has at the same time selected against VOCs and other secondary metabolites that, in unstressed plants, may compete with primary production and reduce yields. Coping with fast climate change and heavier biotic and abiotic stress conditions now requires rediscovery and exploitation of the arsenal of natural defenses that has allowed wild crops to thrive in hostile, unfarmed conditions. This could be attained by selecting plants with different VOC emission blends to be used for novel intercropping schemes, or by co-cultivating modern crops with wild varieties to exploit VOC-driven plant-to-plant signaling and priming. If properly intercropped, stress-sensitive VOC-emitting plant species could act as “sentinel plants” and signal the occurrence of an imminent stressful situation to other neighboring crops of interest, which would be primed and become more resistant. Salt-stressed broad beans, wounded tobacco and pathogen-infected Arabidopsis have shown the ability to enhance resistance of neighboring unstressed plants through emission of VOCs. Alternatively, priming could be elicited by exposing plantlets to synthetic VOCs in confined environments (i.e., growth chamber or greenhouses) before transplanting in the field, thereby exploiting the long-term duration of the enhanced protection. Indeed, the time interval in which plants remain in the state of alertness (primed state) and therefore are more reactive to stress has demonstrated to last for days, weeks, or even be inherited through generations. Modern crops could also be ‘rewildered’ by directing breeding strategies toward the reintroduction of a more efficient capacity to produce and emit VOCs lost during domestication, but of use in plant protection. Recently developed techniques of genetic engineering (i.e., gene editing) could also be employed to restore desired wild/old traits into the plants of agricultural interest. This could allow either ‘de novo’ emission of inducible VOCs (e.g., isoprenoids, ), or modification of the blend already biosynthesized in leaves, such as GLVs. However, difficulties may arise when engineering the biosynthesis of VOCs due to unexpected pleiotropic effects.

14Nowadays, the availability of new analytical technologies such as high-resolution Proton Transfer Reaction “Time-of-Flight’ mass spectrometry (PTR-TOF-MS) makes possible instantaneous and highly sensitive detection of the whole spectra of VOCs with high resolving power. This can provide in vivo a complete and high-throughput measurement of the entire blend of VOCs (the “volatome”) emitted from plant leaves. Phenotyping the volatome could allow non-invasive screening of plant VOC profiles, assisting breeders in the selection of cultivars that successfully perform under changing environmental conditions and associated biotic stressors. PTR-TOF-MS analysis could also enable a real-time diagnosis of the crop health status, by monitoring in air the occurrence of specific VOC emissions (i.e., MeSA, sesquiterpenes) as stress biomarkers triggered by abiotic and biotic constraints. Moreover, variations of VOC emission patterns over time can be used for precision agriculture purposes to monitor plant growth and development in the field. Likewise genomics and high throughput platforms for imaging and remote-sensing, real-time highly resolved VOC detection generate massive amount of data. This production of ‘big data’ requires computational analysis to extract patters and identify features useful for phenotyping. Implementation of machine learning tools to process information on VOC emissions along with environmental parameters collected in the field by multiple sensors will allow exploration of big data in order to measure plant performance and recognize early symptoms of stress.

15FB and IB conceived and wrote the manuscript. FL critically revised the content and contributed to the writing.

16The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.