{'fraction_non_alphanumeric': 0.043713215623327986, 'fraction_numerical': 0.0222043873729267, 'mean_word_length': 5.1241808650065535, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Secondary Metabolic Profiles of Two Cultivars of Piper nigrum (Black Pepper) Resulting from Infection by Fusarium solani f. sp. piperis

Bragantina and Cingapura are the main black pepper (Piper nigrum L.) cultivars and the Pará state is the largest producer in Brazil with about 90% of national production, representing the third largest production in the world. The infection of Fusarium solani f. sp. piperis, the causal agent of Fusarium disease in black pepper, was monitored on the cultivars Bragantina (susceptible) and Cingapura (tolerant), during 45 days’ post infection (dpi). Gas Chromatography-Mass spectrometry (GC-MS) analysis of the volatile concentrates of both cultivars showed that the Bragantina responded with the production of higher contents of α-bisabolol at 21 dpi and a decrease of elemol, mostly at 30 dpi; while Cingapura displayed an decrease of δ-elemene production, except at 15 dpi. The phenolic content determined by the Folin Ciocalteu method showed an increase in the leaves of plants inoculated at 7 dpi (Bragantina) and 7–15 dpi (Cingapura); in the roots, the infection caused a phenolic content decrease in Bragantina cultivar at 45 dpi and an increase in the Cingapura cultivar at 15, 30 and 45 dpi. High Performance Liquid Chromatography-Mass spectrometry (HPLC-MS) analysis of the root extracts showed a qualitative variation of alkamides during infection. The results indicated that there is a possible relationship between secondary metabolites and tolerance against phytopathogens.


Introduction
Black pepper (Piper nigrum L.) is by far one of the most important spices and has been used for centuries in food, to treat many ailments, and as cosmeceutical products. It is cultivated in Asian countries such as Malaysia, India, Indonesia, Thailand, Vietnam, China and Sri Lanka, in addition to Madagascar and Brazil [1,2]. In Brazil, the cultivation of P. nigrum started in the 17th century and since 1933, it has been economically explored by Japanese immigrants [3]. Pará state is the largest producer

Profiles of Volatile Compounds of the Black Pepper Cultivars
The P. nigrum leaves, from the Bragantina and Cingapura cultivars, after Fusarium infection, were submitted to simultaneous distillation and extraction to obtain their volatile concentrates. Individual components were identified by comparison of both mass spectrum and gas chromatography (GC) retention data with authentic compounds, previously analyzed and stored in the data system. Commercial libraries, which contained retention indices and mass spectra of volatile compounds commonly found in essential oils were used [16,17]. The GC and GC-MS analysis of the volatile concentrates from inoculated plants (IP) and control plants (CP) allowed the identification of 30 constituents in Bragantina cultivar and 54 components in Cingapura cultivar (Tables 1 and 2). In the volatile concentrates of Bragantina cultivar, the identified constituents presented an average of 91.6%, with the predominance of oxygenated sesquiterpenes (IP, 77.4%; CP, 82.0%), where αbisabolol (IP, 56.3%; CP, 51.5%), elemol (IP, 12.6%; CP, 24.0%) were the main compounds, followed

Variation in Volatile Profiles of the Black Pepper Cultivars, after Fusarium Infection
The volatile concentrates of Bragantina cultivar were dominated by oxygenated sesquiterpenes (IP, 77.4%; CP, 82.0%), followed by sesquiterpene hydrocarbons (IP, 10.7%; CP, 10.2%) as minor compounds (Table 1, Figure 2). The production of oxygenated sesquiterpenes showed a drastic decrease (71.5%) in the inoculated plants, just at 30 dpi, which can be associated with the progress of Fusarium disease during that cultivation period. In contrast, the Cingapura cultivar showed sesquiterpene hydrocarbons as main compounds (IP, 76.2%; CP, 72.6%) and oxygenated sesquiterpenes in minor proportions (IP, 17.1%; CP, 20.7%) (Table 2, Figure 2). An increase in production of sesquiterpene hydrocarbons was observed with inoculated plants at 15

Variation in Total Phenolic Compounds Profiles of the Black Pepper Cultivars, after the Fusarium Infection
The variation in total phenolic compounds was evaluated in the leaves and roots of Bragantina and Cingapura cultivars, after the inoculation, and expressed as milligrams of gallic acid equivalents per gram of gram of extract (mg GAE·g −1 ). Bragantina cultivar leaves showed no significant variation, while the Cingapura cultivar presented a significant production in the inoculated plants at 7 dpi, which can be associated as a response to the infection in the early days ( Figure 3). Root total phenolic compounds showed different behavior in comparison to the leaves. In the Bragantina cultivar (susceptible to Fusarium) the only significant change was observed at 45 dpi, which was a dramatic decrease of total phenolic compounds in the inoculated plants, when compared to control plants (IP, 39.9 mg GAE·g −1 ; CP, 91.9 mg GAE·g −1 ). On the other hand, Cingapura cultivar (tolerant to Fusarium) produced higher content of total phenolic compounds in the inoculated plants at 15

Variation in Total Phenolic Compounds Profiles of the Black Pepper Cultivars, after the Fusarium Infection
The variation in total phenolic compounds was evaluated in the leaves and roots of Bragantina and Cingapura cultivars, after the inoculation, and expressed as milligrams of gallic acid equivalents per gram of gram of extract (mg GAE·g −1 ). Bragantina cultivar leaves showed no significant variation, while the Cingapura cultivar presented a significant production in the inoculated plants at 7 dpi, which can be associated as a response to the infection in the early days (Figure 3). Root total phenolic compounds showed different behavior in comparison to the leaves. In the Bragantina cultivar (susceptible to Fusarium) the only significant change was observed at 45 dpi, which was a dramatic decrease of total phenolic compounds in the inoculated plants, when compared to control plants (IP, 39.9 mg GAE·g −1 ; CP, 91.9 mg GAE·g −1 ). On the other hand, Cingapura cultivar (tolerant to Fusarium) produced higher content of total phenolic compounds in the inoculated plants at 15

Discussion
For populations of an agricultural crop and its associated pathogens, it has been seen that the genetic variation for its resistance and infectivity is ubiquitous [18,19]. Although P. nigrum cultivars are not completely resistant to Fusarium, no contrary evidence has been reported yet [20].
The response to infections of Bragantina and Cingapura cultivars promotes the production of 3E-hexenol, which is considered a volatile produced in green leaves in response to a herbivore attack. Cingapura inoculated plants produced 3E-hexenol (7 and 15 dpi), which indicated a response in the early stages. However, in the Bragantina cultivar their emission was observed from 21 to 45 dpi only in inoculated plants. In the oxidation of linoleic acid by the action of lipoxygenase enzymes (LOX), 3Z-hexenal is the first product formed followed by isomerization reactions resulting in the formation of 2E-hexenal. These aldehydes are converted to the corresponding alcohols by dehydrogenase action [29,30].
Among minor compounds, E-β-ocimene was frequent and showed higher concentrations in Bragantina inoculated plants at 7, 21 and 45 dpi and Cingapura plants at 7 dpi. This compound has been reported as important in plant-plant interactions, pathogen resistance and changes in abiotic factors [31]. The emission of E-β-ocimene was correlated during interaction of tobacco and peanut, inoculated by Pseudomonas syringae and Sclerotium rolfsii, respectively [32][33][34][35][36]. The plants present direct and indirect defense responses when they are damaged by herbivores, or inoculated by fungal and bacterial pathogens [37]. The significant change in the production of α-bisabolol observed in Bragantina cultivar at 21 dpi and δ-elemene in Cingapura cultivar at 15 dpi may be a response to plant-pathogen interactions, since some sesquiterpenes are produced as phytoalexins by the plant defense system [38]. PCA analysis of volatile compounds from each cultivar discriminated the cultivar in two groups. For the Bragantina cultivar, the negative loadings in the PC1 were observed for inoculated samples after 21 dpi, such as I30, I45 and I21, except the sample C45. However, the groups in the Cingapura samples did not display a response after the infection.
Oxygenated sesquiterpenes were dominant in Bragantina cultivar (susceptible) and sesquiterpene hydrocarbons in Cingapura cultivar (tolerant). The Bragantina cultivar did not display a direct influence of oxygenated sesquiterpenes during the infection. In general, sesquiterpene levels decreased in inoculated plants as initial response to disease (from 7 to 15 dpi) and after 21 dpi, this difference decreased. However, in Cingapura cultivar, sesquiterpene hydrocarbons showed a direct correlation with infection, because a high production was observed in early stages (7-15 dpi). From 30 dpi, a decrease in this difference was observed ( Figure 2). The increase in concentration of sesquiterpene hydrocarbons was determined as a marker of infection by Fusarium in corn. Inoculated grains produced high concentrations of β-selinene, α-selinene, β-macrocarpene, β-bisabolene and trichodiene during infection by F. graminearum, F. verticillioides and F. subglutinans, while uninoculated grains showed high levels of cycloisosativene and α-ylangene [39]. These results are in contrast with the earlier reports on response of two lines of P. nigrum inoculated by Phytophthora capsici, which the higher accumulation of phenolic compounds in the roots and stem were observed with the susceptible line (Sreekara) in comparison to the tolerant (04-P24) [40].
Plants submitted to stress displace the energy invested in primary metabolism to secondary metabolism and consequently there is a reduction in the growth rate, reproduction and competitiveness. Subsequently, the synthesis of secondary metabolites is altered for survival and results in regulation of carbon flow between primary and secondary metabolism [41]. Resistance to pests or diseases of certain species could be related to the concentration and variety of phenolic compounds [42,43]. Due to their toxicity, compounds such as flavonoids and hydroxycinnamic acids act as passive or inducing barriers against microorganisms and herbivores [43,44].
The infection by Colletotrichum nymphaea, the causal agent of anthracnose in strawberry (Fragaria sp.) in two cultivars "Elsanta" (susceptible) and "Honeoye" (resistant) promoted an increase in the concentration of phenolic compounds as ellagic acid derivatives, flavonols and flavan-3-ols [45]. Furthermore, multivariate analysis of phenolic compounds of different mango cultivars (Mangifera indica) allowed the identification of biomarkers in the groups as tolerant and susceptible to Fusarium infection. The larger levels of phenolic compounds were observed in more tolerant cultivars "Nam docmai" and "Roza" [46]. These results support the hypothesis that phenolic compounds play an important role in host-resistance in inoculated tissue during plant-pathogen interactions [43]. Despite the variation in total phenolic contents in the roots of both cultivars, they displayed a wide diversity of alkamides. Additionally, it is important to emphasize the low selectivity of phenolic compounds by Folin-Ciocalteu assay, once it displays false positives to non-phenolic reducing species, such as vitamin C, amino acids and thiol derivatives [47]. In addition, many nitrogen-containing compounds showed considerable reactivity toward Folin Ciocalteu reagent. These classes include hydrazines, hydroxylamines, guanidines, tertiary amines, aromatic amines, pyrroles, and indoles [48].
The plant with the highest levels of alkamides (AAs) are found in species of Asteraceae, Piperaceae, Solanaceae and Rutaceae [49,50]. There are numerous studies on antimicrobial activities of AAs by different test methods in vitro, e.g., dilution, disk diffusion and bioautography [51]. The antifungal activity of AAs has been reported against Cladosporium species [52,53]. In plants, these compounds play an important role in seedling development in physiological or stress conditions [54]. Kim et al. (2010) [55] described two hypothetical mechanisms of their protective role: (1) NAE (N-acylethanolamines) accumulation could modulate the level of other lipids (e.g., phosphatidic acid) in response to pathogens or (2) NAEs might interfere with the quorum sensing mechanism (i.e., inter-pathogen communication to coordinate their activities) of pathogen.

Plant Materials
Plants of black pepper, from Brangatina and Cingapura cultivars, were provided by Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA)-Campus of Amazônia Oriental, city of Belém, state of Pará, Brazil. Twenty-eight plants of each cultivar, with three months of cultivation, were used in the experiment.

Fungal Strains and Inoculation on Black Pepper
The origin and identification of the fungal strain used to infection experiments are not clear, nor reference given. F. solani f. sp. piperis isolates were collected from black pepper commercial planting located in Baião city (Pará State). The fungal identification was performed based on morphological characteristics of conidia and microconidia in the inoculant solution, which present an oval shape and fusoid, respectively. The fungus was cultured on potato dextrose agar (PDA) for 10 days, at 27 • C, before starting the experiment. The fungi spores were collected in sterile water and the inoculant solution was prepared at a concentration of 1.43 × 10 5 spores/mL. The roots of each plant of black pepper cultivars were inoculated with the spore solution according to Meireles, while plants of the control group were treated with distilled water [7]. The Fusarium infection and the production of secondary metabolites were monitored at 7, 15, 21, 30 and 45 days post inoculation (dpi). After the experimentation, the phytopathogen was again isolated from the roots of the black pepper cultivars under study.

Volatile Concentrates and Extracts Preparation
The volatile constituents were extracted from the leaves (3 g) of each treated plant by simultaneous distillation-extraction process using a Likens-Nickerson apparatus and n-pentane (3 mL) as solvent. After 2 h, the organic fraction was removed. Fresh leaves and roots (5 g) were extracted by percolation (96 h) with ethyl acetate and the residual solvent was removed under reduced pressure.

Volatile Concentrates Analysis
An aliquot (1 µL) of organic fraction of each sample was submitted for analysis of volatile components. The analyses were carried on a GC-MS Thermo Focus DSQ II (Thermo Fisher Scientific, Austin, TX, USA), under the following conditions: DB-5ms (30 m × 0.25 mm; 0.25 mm film thickness, Agilent, Santa Clara, CA, USA) fused-silica capillary column; programmed temperature, 60-240 • C (3 • C/min); injector temperature, 250 • C; carrier gas, helium, adjusted to a linear velocity of 32 cm/s (measured at 100 • C); injection type, split (2 µL); split flow was adjusted to yield 20:1 ratio; septum sweep was a constant 10 mL/min; EIMS (Electron Impact Mass Spectrometry), electron energy, 70 eV; temperature of the ion source and connection parts, 200 • C. The quantitative data regarding the volatile constituents were obtained by peak area normalization using a FOCUS GC/FID (Thermo Fisher Scientific, Austin, TX, USA) operated under similar conditions of the GC-MS, except the carrier gas, which was nitrogen. The retention indices were calculated for all the volatile constituents using a homologous series of n-alkanes (C8-C32, Sigma-Aldrich, St. Louis, MO, USA) [56].

Total Phenolics Content
The amount of total phenolics (TP) of the ethyl acetate extract was determined using the Folin-Ciocalteu procedure (Sigma-Aldrich, St. Louis, MO, USA) [57,58]. The extracts were dissolved in methanol at an initial concentration of 40 mg/mL and then diluted in water. Aliquots (500 µL) of the aqueous solution was mixed with 250 µL of Folin-Ciocalteu reagent (1.0 N, Sigma-Aldrich, St. Louis, MO, USA) and 1250 µL of sodium carbonate (75 g/L). The absorbance was measured after 30 min, at 760 nm and 25 • C (UV-Vis spectrophotometer, Biosystems RA2708, Costa Brava, Barcelona, Spain). The experimental calibration curve was prepared using gallic acid at concentrations of zero, 0.5, 1, 2, 4, 6, 8 and 10 mg/L, which were submitted to the same procedure. The total phenolics content are expressed as gallic acid equivalents (GAE) in milligrams per gram of extract (mg GAE/g).

Extract Samples Analysis
The ethyl acetate extracts were analyzed by a liquid phase chromatograph coupled to a mass spectrometer detector (HPLC-ESI-MS, Shimadzu, Tokyo, Japan), which was equipped with analytical pumps LC-20AD (HPLC-ESI-MS, Shimadzu, Tokyo, Japan) (2), sampler SIL-20AHT (HPLC-ESI-MS, Shimadzu, Tokyo, Japan), detector UV/Vis SPD-20A (HPLC-ESI-MS, Shimadzu, Tokyo, Japan), oven CTO-20A (HPLC-ESI-MS, Shimadzu, Tokyo, Japan) and controller CBM-20A (HPLC-ESI-MS, Shimadzu, Tokyo, Japan). The mobile phase was methanol (MeOH) (plus 0.1% formic acid) and water (plus 0.1% formic acid) in a gradient of 30% of MeOH maintained for 2 min, then, from 2 to 25 min the percentage of MeOH was increased to 100% and kept for an additional 5 min. The column was Luna 5u PFP (2) 100 A, 150 × 2 mm (Phenomenex, Torrance, CA, USA). The UV detector was kept in 254 and 280 nm, the oven was in 40 • C. The flow was 200 µL/min and it was injected directly in the mass spectrometer. The mass spectrometer Bruker micrOTOF-QII (Bruker, Billerica, MA, USA) was in positive mode with nebulizer and dry gas (N 2 , 8 L/min), at 4 Bar. The dry temperature was 200 • C and the collision and quadrupole energy was 12 and 6 eV, respectively. The funnels RF1 and RF2 were set to 200 Vpp.

Statistical Analysis
Samples were assayed in triplicate in phenolic content assay and the results are shown as means ± standard deviation. Analysis of variance was conducted and the differences between variables were tested for significance by one-way analysis of variance (ANOVA) with Tukey's post hoc test using GraphPad version 5.0. (GraphPad Software, San Diego, CA, USA) Differences at p < 0.05 were considered statistically significant. The identified volatile compounds were used as variables in the Principal Component Analysis (PCA) to classify and group the inoculated and control plants. These data were analyzed using the Minitab software (free version, Minitab software, State College, PA, USA).