**3. Biotechnological approach: genomics-proteomics-metabolomics**

Plants in nature are constantly challenged by several harmful phytopathogens including bacteria, fungi, nematodes, or virus, producing a high and negative impact on crop productivity worldwide [51]. An uncontrolled amount of synthetic and chemical pesticides used during past decades makes necessary to adopt new strategies allowing a sustainable plant protection in crops and forest systems. The use of natural compounds as plant biostimulators of growth or biotic and abiotic stress responses in plants is getting importance in the last decade because of legal restrictions on the use of phytosanitary products on crops [52, 53]. European Union policy works upon a significant reduction in pesticide use in the short future [54]. One alternative are natural origin compounds with priming capacities, such as the essential oils (EOs) [55]. This section describes examples of recent molecular approaches studying EOs and discusses the use of EOs as an alternative of nonpollutant primers to induce plant resistance for environmental-friendly plant protection.

#### **3.1. The "priming" process**

climate shift to milder winters and wetter summers. Dutch elm disease (DED) is frequently mentioned in forest pathology reviews as the best example of a destructive disease of alien origin since it almost destroyed the elm populations of Europe, North America, and parts of Asia. DED reemerged in the 1970s in Europe as a devastating disease, which killed also elm genotypes that had been resistant in the first epidemic (at the beginning of the twentieth century). This new epidemic was caused by the emergence of the separate and highly virulent species *Ophiostoma novo-ulmi* [32] consisting of the subspecies *novo-ulmi* and *Americana* [33]. Also, [34] provided strong evidence that *Mycosphaerella populorum*, the Septoria canker of poplars, has adapted to infect, colonize, and cause mortality on poplar woody stems as a result of horizontal transfer of the necessary gene battery from ascomycete fungi associated with wood

In fungal pathogens of woody plants, emergence of new interspecific hybrids was described in *Melampsora* [35], *Phytophthora* [36], *Ophiostoma* [37], *Cronartium* [38], and *Heterobasidion* [39]. An up-to-date case of a worrisome forest pathogen that may have a latency period in asymptomatic infected plants is *H. fraxineus*, the agent of European ash dieback, which penetrates into wood tissues from infected leaves and may not produce external necroses until the next growing season [40]. The *Botryosphaeriaceae* are a classical example of a very diverse group of fungi, which comprises well-studied endophytes and latent pathogens of woody plants that typically cause disease associated with some types of stress [41]. A key factor in the spread of *Diplodia sapinea* and *D. scrobiculata* is the latency period within host plant tissues. These fungi are able to live within the host without causing any visible symptoms but rapidly shift to a pathogenic interaction when an environmental stress factor primes the host (e.g., local or

An example of new association between vectors and pathogens is the spread of *C. parasitica* on chestnuts by *Dryocosmus kuriphilus*, the oriental chestnut gall wasp, in Europe [42]. *D. kuriphilus* is an invasive insect of Asian origin. Also, a new association was recently reported between *D. sapinea* and *Leptoglossus occidentalis* [43], the so-called western conifer seed bug (WCSB), an invasive coreid, accidentally introduced to Italy from the US in 1999 [44], and nowadays present in several parts of Europe [45]. This association might be beneficial for both partners: the insect enables the fungus to reach a higher number and variety of host trees, either pines or other conifers, while the fungus stimulates the tree's production of monoterpenes, signaling the status of weakness of the tree and attracting more insects [43]. Regarding the new silvicultural practices, commercial plantations of poplars may be severely damaged by emerging plant pathogens worldwide [46]. In northeastern and north-central North America, one of the most harmful poplar diseases is *Mycosphaerella populorum* (Peck). Also, the epidemics of *Phytophthora ramorum* on *Larix kaempferi* (Lamb.) Carr.) in UK might have been driven by the intrinsic fragility of clonal monocultures on great areas due to ecosystem simplification, extreme mechanization, and reduced genetic diversity [47, 23]. Looking ahead, authors of [48] propose an evolutionary ecology perspective that could provide new directions for forest research or disease management: (1) fungal evolutionary diversity (species diversity of forest pathogens and their ecological niches), (2) pathogen evolution (how forest pathogens become adapted to their hosts), (3) forest resistance to disease, especially in relation to tree breeding (trade-offs, tolerance, emerging properties in populations), and (4) the role of hyperparasites

decay and from prokaryotes.

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large-scale climate change) [31].

Priming is "the physiological state that enables cells to respond to very low levels of a stimulus in a more rapid and robust manner than non-primed cells. In plants, priming plays a role in defense and development" [56, 57]. A classical priming defense strategy consists in the use of very well-conserved molecules into the phytopathogen structure called damage/pathogen/ microbe-associated molecular patterns (DAPMPs/PAMPs/MAMPs), such as the lipopolysaccharides (LPS, peptidoglycan (PGN), bacterial flagellin, fungal chitin, bacterial Ax21, or elongation factor Tu (EF-Tu). MAMPs are recognized by plasma-membrane receptors in plants called pattern recognition receptors (PRRs). PAMPs recognition activates a pattern-triggered immunity (PTI) associated with the increase in intracellular calcium, phosphorylation processes mediated by MAPKinase cascades, production of reactive oxygen species (ROS), plant protective compounds, induction of defense-related transcription factors, and corresponding plant pathogenesis-related proteins (PRs) such as glucanases and chitinases, as well as proteins and compounds involved in plant cell wall fortification, such as callose or lignin. PTI might be suppressed by host-adapted phytopathogens, producing an effector-triggered susceptibility (ETS), and adapted plants might block those effectors, activating a robust effector-triggered immunity (ETI) [53, 58–60]. In parallel to the PAMP response, each pathogen specifically triggers a cascade of signaling pathways mediated by phytohormone receptor and recognition of salicylic acid (SA), jasmonate acid (JA), or ethylene (ET). Commonly, it is well accepted that SA is induced by biotrophic and hemibiotrophic phytopathogens, while ET and JA are activated by necrotrophic ones and also by some hemibiotrophs. Those pathways are also interconnected, in order that the activation of one of them currently down-regulate the other one or vice versa [56]. A new mechanism called EMPIS (ETI-Mediating and PTI-inhibited sector) inhibits unnecessary immune responses in plants, limiting the fitness cost of the robust ETI, when PTI is enough effective [61]. Additionally to MAMPs, hormone-mimic-related compounds have been used as classical biostimulators of priming on plants; some examples are the synthetic chemical compounds such as: benzo (1,2,3)-thiadiazole-7carbothiolic acid (BTH), a SA analog which activates systemic acquired resistance (SAR) in crops [62], and the β-aminobutyric acid (BABA), a nonprotein amino acid priming compound with a direct fungitoxic effect [63] or the nonprotein amino acid pipecolic acid [64]. The recent advances in metabolic profiling have led to the discovering of certain new plant secondary metabolites that play significant roles as priming molecules at nature, during biotic and abiotic plant stress responses and in the plant-to-plant communication; at this point, EOs might play an important role in future biotechnological approaches [65, 66].

are essential for plasma membrane structure. A similar mechanism of action was observed on

Antifungal Effect of Essential Oils

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http://dx.doi.org/10.5772/intechopen.78008

Emerging molecular studies try to elucidate the molecular effects of EOs produced by plants to the surrounding ones [52]. This old natural process is currently known as "allelopathy" or the ability of a plant to produce biomolecules, especially secondary metabolites, to affect another plant beneficially or vice versa [73]. In 1997, [74] demonstrated that methyl salicylate (MeSA), the volatile benzenoid and secondary metabolite, which is easily metabolized on the plant to SA, activates disease resistance and the expression of defense-related genes in neighboring plants and in the healthy tissues of the infected plants. Later on, other research works have shown that MeSA mediates plant-plant communications during immune responses. MeSA, which is an important insect-attracting pollinators [75], is not induced by wounding but is induced by tobacco mosaic virus and *Pseudomonas syringae* pv. *maculicola ES*4326 and *Pst DC*3000 pv. *tomato*, where both are SA inducers [76]. The plant molecular response to MeSA has been studied into essential oil extracts from Gaultheria procumbent (GEO), whose metabolic profile has been characterized recently [77]. GEO induced defense response against the hemibiotrophic fungus *Colletotrichum higginsianum* and was very effective in inducing SA plant defense-related genes similarly to the synthetic MeSA and also induced some marker genes of JA pathway [78]. A recent study investigated the role of volatile organic compounds inducing systemic acquired resistance (SAR). The headspace exposure of arabidopsis to a mixture of the bicyclic monoterpenes, α-pinene and β-pinene, induced the accumulation of ROS and the expression of SA- and SAR-related genes, including AZELAIC ACID INDUCED1 (AZI1) and three of its paralogs. Pinene-induced resistance was dependent on SA biosynthesis and signaling and on AZI1. Arabidopsis geranylgeranyl reductase1 mutants with reduced monoterpene biosynthesis were SAR-defective, but showed normal local resistance and MeSAinduced defense responses, suggesting that monoterpenes act independently of SA-mediated pathway. The volatile emissions composed by α-pinene, β-pinene, and camphene induced plant defense in neighboring plants, activating SAR responses on them. The impaired SAR immunity lines eds1-2 and *ggr-*1*-*1 showed reduced emissions of α-pinene, β-pinene, and camphene [79]. *Pseudomonas syringae* pv. *maculicola ES*4326 also induced terpenoid production of (E,E)-4,8,12 trimethyl-1,3,7,11-tridecatetraene (TMTT), β-ionone, and α-farnesene, depending on JA signaling and independently on SA pathway in *Medicago truncatula* [80]. Copper sulfate, which activates JA biosynthesis in plant by camalexin biosynthesis, induced VOs in arabidopsis wild-type plants but not in *tps*4 mutant showing that TMTT is induced by JA pathway [80]. TMTT and other VOs were also induced in lima beans by herbivory [81].

However, the significance on the *Pst* induction of TMTT in plants is still unknown.

**4. EOs in the control of phytopathogenic fungi in agricultural crops**

In agriculture, the losses caused by plant diseases reach an average of 12% per year. Among the pathogens, fungi are considered the most important. There are around 8.000 species of fungi that attack plants, distributed in more than 64 genera of fungi [82]. Added to the importance of plant diseases caused by phytopathogenic fungi, we have two other factors that must be considered.

carvacrol and thymol acting against vineyard and wine spoilage yeast [72].

**3.4. Plant signaling pathways and EOs**

#### **3.2. Metabolic engineering improving EO yield**

A line of research on EO biotechnology consists in improving EO yield in plants using metabolic engineering. One of the plant species in which biotechnology approaches has been applied because its commercial interest is peppermint, and [67] transformed peppermint with various gene constructs by overexpressing genes involved in the supply of precursors through the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. The overexpression of the MEP pathway gene 1-deoxy-D-xylulose 5-phosphate reductoisomerase increased up to 78% of the oil yield over wild-type controls in a multiyear field trials. Current genetic manipulation on EO synthesis pathway was also useful improving the EO production in the same species [68]. The inhibition of the mevalonate pathway also enhanced the carvacrol biosynthesis and DXR gene expression in shoot cultures of *Satureja khuzistanica Jamzad*. *S. khuzistanica* shoots were treated with fosmidomycin (an inhibitor of the nonmevalonate pathway) and mevinolin (an inhibitor of the mevalonate pathway). The last one induced the gene expression of DXR, measured by heterologous QRT-PCR, increasing the DXR enzyme activity and allowing higher levels in carvacrol biosynthesis on plants compared to controls [69].

#### **3.3. Molecular mechanism of EOs in fungi**

Recent studies have been made in order to elucidate the molecular mechanisms underlying the phytotoxic effect for some of those compounds on phytopathogenic fungi, but still are limited. The lipophilic or hydrophobic nature of many EO components allows them to interact directly with the fungal membrane, resulting in the alteration of membrane properties including the fluidity. An active transport via trans-membrane pumps has not been yet demonstrated [55]. A recent study based on RNA-Seq-transcriptomic analysis of the fungus *Fusarium oxysporum* f. sp. *niveum*, responding to thymol, shows that most of glycosphingolipid and sphingolipid metabolism-related fungal genes were downregulated upon this treatment, while genes involved in an antioxidant activity, chitin biosynthesis, and cell wall modification were up-regulated. The authors propose that the thymol acts by disrupting fungal cell wall and cell membranes through increasing the production of ROS on the fungal cell surface as well as by blocking the fungal molecular genes necessary for cell wall fortification and cell membrane synthesis [70]. Those molecular data are in line with the results obtained by [71], showing that thymol strongly inhibited conidial production and hyphal growth on *Fusarium graminacearum* via inducing lipid peroxidation and disrupting ergosterol biosynthesis, which are essential for plasma membrane structure. A similar mechanism of action was observed on carvacrol and thymol acting against vineyard and wine spoilage yeast [72].

#### **3.4. Plant signaling pathways and EOs**

sector) inhibits unnecessary immune responses in plants, limiting the fitness cost of the robust ETI, when PTI is enough effective [61]. Additionally to MAMPs, hormone-mimic-related compounds have been used as classical biostimulators of priming on plants; some examples are the synthetic chemical compounds such as: benzo (1,2,3)-thiadiazole-7carbothiolic acid (BTH), a SA analog which activates systemic acquired resistance (SAR) in crops [62], and the β-aminobutyric acid (BABA), a nonprotein amino acid priming compound with a direct fungitoxic effect [63] or the nonprotein amino acid pipecolic acid [64]. The recent advances in metabolic profiling have led to the discovering of certain new plant secondary metabolites that play significant roles as priming molecules at nature, during biotic and abiotic plant stress responses and in the plant-to-plant communication; at this point, EOs might play an

A line of research on EO biotechnology consists in improving EO yield in plants using metabolic engineering. One of the plant species in which biotechnology approaches has been applied because its commercial interest is peppermint, and [67] transformed peppermint with various gene constructs by overexpressing genes involved in the supply of precursors through the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. The overexpression of the MEP pathway gene 1-deoxy-D-xylulose 5-phosphate reductoisomerase increased up to 78% of the oil yield over wild-type controls in a multiyear field trials. Current genetic manipulation on EO synthesis pathway was also useful improving the EO production in the same species [68]. The inhibition of the mevalonate pathway also enhanced the carvacrol biosynthesis and DXR gene expression in shoot cultures of *Satureja khuzistanica Jamzad*. *S. khuzistanica* shoots were treated with fosmidomycin (an inhibitor of the nonmevalonate pathway) and mevinolin (an inhibitor of the mevalonate pathway). The last one induced the gene expression of DXR, measured by heterologous QRT-PCR, increasing the DXR enzyme activity and

allowing higher levels in carvacrol biosynthesis on plants compared to controls [69].

Recent studies have been made in order to elucidate the molecular mechanisms underlying the phytotoxic effect for some of those compounds on phytopathogenic fungi, but still are limited. The lipophilic or hydrophobic nature of many EO components allows them to interact directly with the fungal membrane, resulting in the alteration of membrane properties including the fluidity. An active transport via trans-membrane pumps has not been yet demonstrated [55]. A recent study based on RNA-Seq-transcriptomic analysis of the fungus *Fusarium oxysporum* f. sp. *niveum*, responding to thymol, shows that most of glycosphingolipid and sphingolipid metabolism-related fungal genes were downregulated upon this treatment, while genes involved in an antioxidant activity, chitin biosynthesis, and cell wall modification were up-regulated. The authors propose that the thymol acts by disrupting fungal cell wall and cell membranes through increasing the production of ROS on the fungal cell surface as well as by blocking the fungal molecular genes necessary for cell wall fortification and cell membrane synthesis [70]. Those molecular data are in line with the results obtained by [71], showing that thymol strongly inhibited conidial production and hyphal growth on *Fusarium graminacearum* via inducing lipid peroxidation and disrupting ergosterol biosynthesis, which

important role in future biotechnological approaches [65, 66].

**3.2. Metabolic engineering improving EO yield**

150 Potential of Essential Oils

**3.3. Molecular mechanism of EOs in fungi**

Emerging molecular studies try to elucidate the molecular effects of EOs produced by plants to the surrounding ones [52]. This old natural process is currently known as "allelopathy" or the ability of a plant to produce biomolecules, especially secondary metabolites, to affect another plant beneficially or vice versa [73]. In 1997, [74] demonstrated that methyl salicylate (MeSA), the volatile benzenoid and secondary metabolite, which is easily metabolized on the plant to SA, activates disease resistance and the expression of defense-related genes in neighboring plants and in the healthy tissues of the infected plants. Later on, other research works have shown that MeSA mediates plant-plant communications during immune responses. MeSA, which is an important insect-attracting pollinators [75], is not induced by wounding but is induced by tobacco mosaic virus and *Pseudomonas syringae* pv. *maculicola ES*4326 and *Pst DC*3000 pv. *tomato*, where both are SA inducers [76]. The plant molecular response to MeSA has been studied into essential oil extracts from Gaultheria procumbent (GEO), whose metabolic profile has been characterized recently [77]. GEO induced defense response against the hemibiotrophic fungus *Colletotrichum higginsianum* and was very effective in inducing SA plant defense-related genes similarly to the synthetic MeSA and also induced some marker genes of JA pathway [78]. A recent study investigated the role of volatile organic compounds inducing systemic acquired resistance (SAR). The headspace exposure of arabidopsis to a mixture of the bicyclic monoterpenes, α-pinene and β-pinene, induced the accumulation of ROS and the expression of SA- and SAR-related genes, including AZELAIC ACID INDUCED1 (AZI1) and three of its paralogs. Pinene-induced resistance was dependent on SA biosynthesis and signaling and on AZI1. Arabidopsis geranylgeranyl reductase1 mutants with reduced monoterpene biosynthesis were SAR-defective, but showed normal local resistance and MeSAinduced defense responses, suggesting that monoterpenes act independently of SA-mediated pathway. The volatile emissions composed by α-pinene, β-pinene, and camphene induced plant defense in neighboring plants, activating SAR responses on them. The impaired SAR immunity lines eds1-2 and *ggr-*1*-*1 showed reduced emissions of α-pinene, β-pinene, and camphene [79]. *Pseudomonas syringae* pv. *maculicola ES*4326 also induced terpenoid production of (E,E)-4,8,12 trimethyl-1,3,7,11-tridecatetraene (TMTT), β-ionone, and α-farnesene, depending on JA signaling and independently on SA pathway in *Medicago truncatula* [80]. Copper sulfate, which activates JA biosynthesis in plant by camalexin biosynthesis, induced VOs in arabidopsis wild-type plants but not in *tps*4 mutant showing that TMTT is induced by JA pathway [80]. TMTT and other VOs were also induced in lima beans by herbivory [81]. However, the significance on the *Pst* induction of TMTT in plants is still unknown.
