Received: February 09, 2016 Accepted: February 11, 2016 Published: February 25, 2016
Citation: Erisléia-Meireles N, Luciana-Xavier P, Alessandra-Ramos R, José- Guilherme MS, William-Setzer N, et al. (2016) Phenylpropanoids Produced by Piper divaricatum, A Resistant Species to Infection by Fusarium solani f. sp. piperis, the Pathogenic Agent of Fusariosis in Black Pepper. J Plant Pathol Microbiol 7:333. doi:10.4172/2157-7471.1000333
Copyright: © 2016 Erisléia-Meireles N, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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In a previous study, the essential oil (EO) of Piper divaricatum showed strong antifungal activity in vitro against Fusarium solani F. sp. piperis. For this reason, the seedlings of P. divaricatum were inoculated with the pathogen to evaluate the in-vivo resistance. Evaluation of symptoms and analysis of secondary metabolite production from infected and non-infected plants occurred at 7, 21, 30 and 45 days post inoculation (dpi). Throughout the experiment, the infected seedlings did not display symptoms of infection or significant variation of total phenolic compounds. However, lipoxygenase (LOX) enzymatic activity, which is involved in the defense mechanism, had increased in the 21 and 45 dpi. In addition, the EOs were analyzed by GC-MS. The EOs from infected plants showed a substantial increase in the concentrations of phenylpropanoid compounds, particularly on days 21 and 45 dpi. The main constituent was methyl eugenol, which reached 95.3% in the infected sample at 45 dpi, followed by eugenol acetate with a variation of 6.1% to 10.7%, between 21 and 30 dpi. These results suggest an incompatible plant-pathogen interaction and involvement of various volatile compounds from P. divaricatum in the resistance mechanism.
Piper nigrum; Plant-pathogen interaction; Volatile compounds; Methyl eugenol; Lipoxygenase activity; Phenolic content
Piper divaricatum G. Mey. [syn: Arthanthe adenophylla Miq., A. colubrina (Link ex Kunth) Miq., Peltobryon attenuatum Klotzsch, Piper apiculatum C. DC, P. belemense C. DC, P. colubrinum Kunth, P. crassifolium (Miq.) C. DC, P. crassum Vell, P. glabellum (Miq.) C. DC, P. glabrilimbum C. DC, P. insipiens Trel. and Yunck., P. nitidum Vahl, Schilleria colubrina Link ex Kunth, among others] is distributed in northeastern and southeastern South America [1-3]. It is a shrublike aromatic plant growing up to 3 m and is known in the Brazilian Amazon as “piper-pau-de-angola”.
There have been several previous studies on the phytochemistry of P. divaricatum. The ethanolic extract of a specimen from Antioquia, Colombia, used locally as a medicinal and insecticide, showed 4,5-dioxyaporphine alkaloids, γ-asarone and 4-hydroxy-2,5- dimethoxy-allylbenzene, as the main constituents . The seasonal and circadian studies of the essential oil of a specimen collected in Breves, Pará state, Brazil, showed methyl eugenol and eugenol as the principal volatile compounds . Hierarchical cluster analysis of the leaf oils of eleven samples of P. divaricatum, collected in the Brazilian Amazon, resulted in the discovery of two different chemical types: a methyl eugenol + eugenol chemotype and an elemicin-rich chemotype . The antioxidant and antifungal activities of the methyl eugenol + eugenol chemotype of P. divaricatum has also been reported . Another specimen of P. divaricatum, harvested in Itabuna, Bahia state, Brazil, has provided oil from the leaves indicating a safrole-rich chemotype . More recently, it was reported that the EO of the methyl eugenol + eugenol chemotype showed high in-vitro fungicidal potential against the phytopathogen Fusarium solani f. sp. piperis, which causes fusariosis in black pepper (Piper nigrum L.) .
Black pepper (Piper nigrum L., Piperaceae) is one of the most famous and oldest spices in the world, with culinary and food preservative uses. The largest producers of black pepper are Brazil and India. The harvest in Brazil has been about 50 thousand tons per year, with about 90% produced in the Pará State . The cultivation of black pepper has been threatened by infestations of Phytophthora capsici (in India) and Fusarium solani f. sp. piperis (in Brazil), two phytopathogenic fungi that cause severe diseases to the plant. Fusariosis leads to obstruction of the conducting vessels of the plant, root rot and death . Control measures for the disease caused by Fusarium have been taken. However, variability and low homogeneity in the cultivation of P. nigrum has contributed to the spread of the fungus. Thus, strategies to increase black pepper resistance have been performed, including micrografting, micropropagation and in-vitro plant regeneration, development of molecular markers and identification of genes related to the infection process .
The resistance of different plants to pathogens may be related to the production of primary and secondary metabolites that act as natural antimicrobials . In plant-pathogen interactions, several reactions occur simultaneously, and often the pathogen can overcome the structural and biochemical defenses of the plant in order to colonize its tissue . Studies have suggested flavonoid and phenylpropanoid compounds to be responsible for the induction of fungal infection resistance, as observed in citrus fruits infected by Penicillium digitatum . Some studies with native Piper species in the Amazon, such as P. aduncum L., P. colubrinum Kunth (syn: P. divaricatum), P. tuberculatum Jacq., P. hispidinervum C. DC. and P. hispidum Sw. were carried out in search of specimens resistant to infection by Fusarium, aiming at development of interspecific hybrids . Piper colubrinum showed strong resistance to infection in the soil by F. solani f. sp. piperis and, when used as a rootstock in P. nigrum, showed high tissue compatibility and fixation (98%). Unfortunately, the grafted seedlings have shown delayed compatibility issues, which decreased the life cycle in the field .
In this perspective, the aim of this study was to evaluate the resistance of P. divaricatum to Fusarium disease and analyze the production of metabolites after inoculation of the pathogen. In this context, it should be realized that metabolomics studies have assisted in understanding the biological system and have shown success in many plants, including, for example, Arabidopsis thaliana (L.) Heynh. and tobacco (Nicotiana tabacum L.) [18,19].
Plant and fungus material
Piper divaricatum was collected in the municipality of Breves, Pará state, Brazil, in July 2012. The cuttings containing two nodes were propagated in propylene bags with commercial fertilizer. The seedling growth was monitored for six months until root development. As a control group susceptible to Fusarium disease, some ten-month-old seedlings of Piper nigrum, which were obtained from the Bragantina cultivar, were used. Fusarium solani f. sp. piperis was isolated from an individual of P. nigrum with the symptoms of Fusarium disease, after twenty days of infection. Small pieces were cut near the site of infection, cleaned with ethanol, sterile water, and 10% sodium hypochlorite, for 1 min. After 10 days of contact with the medium (PDA, potato dextrose agar), at temperature of 27ºC, the fungus was isolated and cultured in Petri dishes for use in the tests, which took place after 15 days of growth.
To prepare the solution of inoculum, 10 mL of sterile water was added to the Petri dishes and the conidia were removed with a spatula. The concentration of spores in the suspension was 2.1 × 106 mL-1, measured in a Neubauer chamber. For inoculation, 18 seedlings of P. divaricatum and 18 seedlings of P. nigrum were removed from the plastic bags and their roots washed under running water to remove excess soil. Micro wounds were made in the roots of the seedlings to facilitate the penetration of spores, followed by immersion for 5 min in the conidial suspension. For the control group, only distilled water was used. The seedlings were replanted and maintained in a greenhouse for 45 days with water irrigation twice per day. The monitoring of secondary metabolites in the plant and observation of disease symptoms were made at intervals of 7, 21, 30 and 45 days post-inoculation (dpi). After 65 days, the pathogen was again isolated from the seedlings of P. divaricatum. Figure 1 illustrates the experimental design.
Folin-Ciocalteu total phenolics determination
The leaves of infected and uninfected plants were collected at random in steps 7, 21, 30 and 45 dpi. Fresh plant material (1 g) was cut and extracted with 20 ml of ethanol/water (7: 3), at 60°C for 30 min. The extracts were obtained in triplicate and stored in amber vials under refrigeration at 4°C. The total phenolics content was determined by the Folin-Ciocalteu method . An aliquot of 500 μL was used to react with 250 μL of reagent (1N) and 1250 μL of sodium carbonate (75 g L-1). After 30 min incubation in the dark, the absorbance of the mixture was read at 760 nm using a UV-Visible spectrophotometer. The experimental calibration curve was prepared using gallic acid at concentrations of 0.5 and 10.0 mg L-1 and the content of total phenolics was expressed as gallic acid equivalents (GAE) in milligrams per gram of dry leaves (mg GAE g-1).
Lipoxygenase (LOX) activity
To prepare the substrate, 78 μL of linoleic acid (Sigma-Aldrich, USA) and 90 μL Tween 20 (Sigma-Aldrich) were mixed with 10 mL of boiling water and a few drops of sodium hydroxide (0.5 N). The final volume was adjusted to 25 mL, resulting in a sodium linoleate solution (10 mM), which was stored at -20°C. The LOX activity determination was carried out with 5 μL of crude leaf extract and 50 μL of sodium linoleate (10 mM), mixed with 1950 μL of sodium phosphate buffer (50 mM) at pH 6.5. The absorbance at 234 nm for the reaction was monitored for 150 s, at continuous intervals of 30 s, using a UV-Visible spectrophotometer. The increase in absorbance at 234 nm indicated the presence of a conjugated double bonds system in the formed hydroperoxide .
The volatile concentrate of leaves (3 g) of P. divaricatum was obtained by hydrodistillation with continuous extraction with n-pentane using a Likens-Nickerson apparatus for 2 h. After extraction, an aliquot of 1 μL was analyzed by gas chromatography. Qualitative analysis was carried out on a THERMO DSQ II GC–MS instrument, under the following conditions: DB-5 ms (30 m 0.25 mm; 0.25 mm film thickness) fused-silica capillary column; programmed temperature: 60-240°C (3ºC min-1); injector temperature: 250ºC; carrier gas: helium, adjusted to a linear velocity of 32 cm s-1 (measured at 100°C); injection type: 1 μL of solution, splitless mode, split flow was adjusted to yield a 20:1 ratio; septum sweep was a constant 10 mL min-1; EIMS: electron energy, 70 eV; temperature of ion source and connection parts: 200°C. Quantitative data regarding the volatile constituents were obtained by peak-area normalization using a FOCUS GC/FID operated under GCMS similar conditions, except for the carrier gas, which was nitrogen. The retention indices were calculated for all volatile constituents using an n-alkane (C8–C40, Sigma–Aldrich) homologous series. Individual components were identified by comparison of both mass spectrum and GC retention data with authentic compounds, which were previously analyzed and stored in private library, as well as with the aid of commercial libraries containing retention indices and mass spectra of volatile compounds commonly found in essential oils .
The extracts and essential oils were obtained of leafs collected from five individuals. The chemical composition of volatile compounds is represented as mean. In the Total phenolic content and LOX activity assays, the samples were assayed in triplicate, and the results are shown as means ± standard deviation. Analysis of variance was conducted, and the differences between variables were tested for significance by Tukey test, using GraphPad Prism 5.0 software. Differences at p < 0.05 were considered statistically significant.
The resistance of P. divaricatum seedlings were compared to the resistance of P. nigrum seedlings to the infection and symptoms of Fusarium disease for 45 days (Figure 2). Throughout the duration of the experiment, no wilting symptoms or drying of the branches of P. divaricatum were observed. The plants were healthy and some of the individuals started their normal reproductive period with flowering and fruiting. In the experiment with P. nigrum, however, the first signs of yellowing leaves began on 21 dpi. By 30 dpi there was obvious necrosis of some leaves, with the advancement of these effects until the 45th day. The progression of infection in P. nigrum, with the total drying of the branches and death of most plants, occurred by 65 dpi (Figure 2).
During the experiment, the total phenolic content ranged from 10 to 18 mg GAE g-1 in the fresh leaf matter. No statistically significant differences were observed between infected and non-infected plants. On the other hand, the lipoxygenase activity was higher at days 21 and 45 dpi. At day 21, the lipoxygenase activity in infected plants was 2.2 ± 0.4 × 10-7 M s-1, which corresponded to an increase of about 50% over the uninfected plants (1.5 ± 0.3 × 10-7 M s-1). On 45 dpi, the lipoxygenase activity of the infected plants was 1.6 ± 0.2 × 10-7 M s-1, which corresponded to twice the rate observed for the uninfected plants (0.8 ± 0.1 × 10-7 M s-1) (Figure 3).
The essential oils of P. divaricatum were analyzed by GC and GCMS. These concentrates were obtained from infected and non-infected seedlings, between 7 and 45 dpi. Eighteen constituents were identified in the essential oils and phenylpropanoids were the main components, comprising about 94% of the total composition. Additionally, there was a substantial increase in phenylpropanoid concentrations in the leaf oils of infected plants compared to non-infected plants, particularly in the samples collected at 21 and 45 dpi (Table 1).
RIa = Calculated retention index (DB-5ms capillary column, C8-C30 n-alkanes); RIb = Literature retention index (Adams, 2007); *Constituent emission only in infected plants; +Constituents with increased content in infected plants; -Constituents with reduced content in infected plants; bold letters/numbers = phenylpropanoids
Table 1: Variation in the essential oil compositions of Piper divaricatum infected and non-infected by Fusarium solani f. sp. piperis, over a period of 45 days.
At 7 dpi was possible to visualize qualitative and quantitative differences in the essential oils of P. divaricatum, depending on the infection. α-Pinene, myrcene and (Z)-β-ocimene were identified only in infected seedlings, but there was an increase in production of methyl eugenol (88.6 to 91.0%) and (E)-β-ocimene (0.3 to 1.1%), and a decrease in the production of β-caryophyllene and germacrene D. At 21 dpi, in addition to the presence of linalool, there was an increase in the production of methyl eugenol (81.5 to 88.3%) and (E)-β-ocimene (1.3 to 2.0%), a decrease in eugenol acetate, β-elemene and β-caryophyllene, and the absence of β-copaene, α-humulene, bicyclogermacrene and germacrene A. At 30 dpi, in addition to production of eugenol, there was a decrease in the production of methyl eugenol (93.3 to 85.7%), increased production of eugenol acetate (2.3 to 10.7%) and an absence of germacrene D, bicyclogermacrene and germacrene A. Finally at 45 dpi, there was a significant increase in methyl eugenol content (80.6 to 95.3%), decreased production of eugenol acetate (9.1 to 1.0%), and lack of α-pinene, myrcene and limonene.
Incompatibility can explain the absence of symptoms of Fusarium infection in P. divaricatum in plant-pathogen response. In the compatible reactions, if the pathogen is virulent and the host susceptible, the disease occurs. In incompatible reactions if the pathogen is virulent and the host resistant, the disease is not installed . For Piper nigrum, the positive control group of Fusarium disease, the wilting of aerial parts after 21 dpi was observed, and after that, necrosis of the root tissue.
Pathogens that cause wilting in plants, such as F. solani f. sp. piperis, have a hemi-biotrophic life cycle. Initially, it is installed in living tissue and then in the dead tissue . After colonization of the root tissue, distribution in the vascular system occurs and, with the death of the plant, the fungus in the infected tissue survives as a saprophyte . During the development stage, toxins are released in the host tissue, which in turn induces the production of defense molecules in an attempt to inhibit invasion. This process occurs within the vascular system, causing the obstruction of vessels that carry nutrients and water .
The plant response to pathogen attack is a complex mechanism, involving several defense strategies and different biochemical pathways . The chain of events begins with the recognition of elicitors (molecules responsible for activating the integrated signaling network) by specific receptors of the host, and ends with the activation of resistance genes . This induced-defense mechanism is represented by two steps: a hypersensitivity reaction that induces the rapid elimination of cells located at the site of infection and the systemic response acquired, more lasting and effective, which spreads in all plant tissues .
The interaction with pathogens directly affects the metabolism of the host plant, which reorganizes its primary metabolism to create defense mechanisms . The production of total phenolic compounds in the leaves of P. divaricatum did not vary as a result of the infection (Figure 2). However, in the infected seedlings, the lipoxygenase activity had increased in 21 and 45 dpi, as well as the concentration of the phenylpropanoids in the leaf essential oils (Table 1).
Lipoxygenases catalyze the stereospecific addition of molecular oxygen in the cis,cis-1,4-pentadiene system of polyunsaturated fatty acids to form hydroperoxides, which are metabolized enzymatically to produce jasmonic acid and methyl jasmonate . The stress caused by pathogen attack raises the levels of jasmonic acid and its methyl derivative which, in turn, upregulate the biosynthesis of phenolic and terpene compounds through the accumulation of phenylalanine ammonia-lyase and terpene-synthase enzymes, respectively [32-34].
The variation of volatile constituents in P. divaricatum was highly significant, when caused by Fusarium infection, given that some compounds were produced or changed, presumably for self-protection of the plant, in response to damage caused by the pathogen. In this regard, it is to highlight the increase in the content of phenylpropanoid compounds and the emission of monoterpene compounds, throughout the experiment [35,36]. Phenylpropanoids have numerous functions in plants, ranging from structural constituents, growth, and reproductive biochemistry and physiology to chemoecological interactions with microbes, animals (particularly insects), and neighboring plants . Except for the 30th day, the emission (increase) of methyl eugenol was significant in the infected seedlings. Several studies of the inhibitory properties of methyl eugenol against plant pathogens have been described . Both eugenol and eugenol acetate have shown biological activity against various pathogens.
In summary, Piper divaricatum showed no Fusarium disease symptoms during inoculation with F. solani f. sp. piperis. Also, there was increased lipoxygenase activity and variation in the production of volatile compounds such as monoterpene and sesquiterpene hydrocarbons. The disease resistance is likely related to the presence and increased production of phenylpropanoids.
We are grateful for CNPQ, CAPES and FAPESPA/PA for their financial support.