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Substrate-associated mycorrhizal fungi promote changes in terpene composition,

antioxidant activity, and enzymes in Curcuma longa L. acclimatized plants

Meire Pereira de Souza Ferrari, Mayara dos Santos Queiroz, Matheus Marquezin de
Andrade, Odair Aberton, José Eduardo Gonçalves, Zilda Cristiani Gazim, Hélida Mara
Magalhães
PII: S2452-2198(19)30223-X
DOI: https://doi.org/10.1016/j.rhisph.2020.100191
Reference: RHISPH 100191

To appear in: Rhizosphere

Received Date: 29 December 2019

Revised Date: 1 February 2020
Accepted Date: 3 February 2020

Please cite this article as: de Souza Ferrari, M.P., Queiroz, M.d.S., Andrade, M.M.d., Aberton, O.,
Gonçalves, José.Eduardo., Gazim, Z.C., Magalhães, Hé.Mara., Substrate-associated mycorrhizal
fungi promote changes in terpene composition, antioxidant activity, and enzymes in Curcuma longa L.
acclimatized plants, Rhizosphere (2020), doi: https://doi.org/10.1016/j.rhisph.2020.100191.

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 Substrate-associated mycorrhizal fungi promote changes in terpene composition,
antioxidant activity, and enzymes in Curcuma longa L. acclimatized plants

Meire Pereira de Souza Ferraria, Mayara dos Santos Queiroza, Matheus Marquezin de Andradea, Odair
Abertona, José Eduardo Gonçalvesb, Zilda Cristiani Gazima, Hélida Mara Magalhãesa
a
Universidade Paranaense, Mascarenhas de Moraes Square, 4282, 87502-210, Umuarama, Paraná,
Brazil
b
Cesumar Institute of Science, Technology and Innovation - ICETI, Av. Guerdner, 1610, Jd. Aclimação,
Maringá, Paraná, Brazil
 1 Substrate-associated mycorrhizal fungi promote changes in terpene composition, antioxidant
2 activity, and enzymes in Curcuma longa L. acclimatized plants

3 Abstract

4 The aim of this study was to evaluate the chemical composition of rhizomes and the biochemical
5 activity of Curcuma longa L. acclimatized plants associated with arbuscular mycorrhizal fungi (AMF)
6 and different substrates. The seedlings were produced in the laboratory using the tissue culture
7 technique, in Murashige and Skoog culture medium. After 120 days, the seedlings were transplanted
8 to pots in a greenhouse. Two types of substrates were used vermiculite and soil and vermiculite,
9 commercial substrate, and vermicompost, with or without the addition of Rhizophagus clarus,
10 Claroideoglomus etunicatum, or a combination of the two fungi. At the end of 240 days, the chemical
11 composition of the essential oil of the rhizomes was evaluated and biochemical activity analysis was
12 performed on the leaves. Oxygenated sesquiterpenes were the most prevalent, with the principal
13 compounds being ar-turmerone and β-turmerone. AMFs in the soil and vermiculite substrate resulted
14 in an increased production of compounds with emphasis on caryophyllene, α-curcumene, β-
15 bisabolene, β-curcumene, cyclohexene, cubenol, β-santalol, α-acorenol, 7-epi-cis-sesquisabinene
16 hydrate, 2,5-Octadecadiynoic acid, methyl ester, and cis-5,8,11,14,17-eicosapentaenoic acid. The
17 opposite was true for the chemical composition of plants grown in substrates with more organic matter
18 (vermiculite, commercial substrate, and vermicompost). AMFs also modified the antioxidant activity
19 and phenolic compounds of the leaves. The free radical scavenging enzymes showed higher activity in
20 plants exposed to AMFs in the substrate with soil and vermiculite. This was mainly true for catalase,
21 the activity of which indicated stress; in this case, C. longa plants were smaller, less vigorous, and
22 produced fewer rhizomes.

23 Keywords: acclimatization; catalase; essential oil; metabolic changes; Zingiberaceae

24 1. Introduction
25 Turmeric (Curcuma longa L. (Zingiberaceae)) has attracted commercial interest because of its
26 pharmacological and medicinal properties (Abdel-Lateef et al., 2016). The most important chemical
27 components of this species belong to a group of compounds called curcuminoids; curcumin is the
28 principal curcuminoid in the essential oil (Nawal, 2018). Curcuminoids are yellow natural terpenes
29 that have anti-oxidant, anti-tumor, anti-inflammatory, and anti-microbial properties (Omar et al.,
30 2013).
31 Curcuma longa is mainly propagated through rhizomes (Berni et al. 2014), which are used
32 commercially (Ferrari et al., 2016) and are the main sources of chemical compounds for the
33 pharmaceutical and cosmetic industries (Kochhar and Kochhar, 2008). The conventional propagation
34 technique has several limitations such as low seedling multiplication, pathogen contamination, and
35 dormancy during the cold season, which interrupts its plantation in new areas during this period
1
36 (Faridah et al., 2011; Ahmadian et al., 2013). Micropropagation is an alternative technique through
37 which C. longa seedlings are produced in the laboratory in a specific culture medium (Singh et al.,
38 2012). This aids high multiplication rates, as a single rhizome can yield up to 300 seedlings; moreover,
39 micropropagation is less time consuming and seedlings are free of pathogens and can be produced at
40 any time of the year, thus nullifying the effect of rhizome dormancy (Oliveira et al., 2015; Ferrari et
41 al., 2016; Silva et al., 2017).
42 The in vitro multiplication protocols of C. longa are well established (Antoniazzi et al., 2016,
43 Ferrari et al., 2016). The final phase of the acclimatization process however, requires more research, as
44 existing studies are inconclusive and do not reveal important plant metabolism aspects of C. longa. In
45 micropropagation, the acclimatization stage is the most delicate, as the plant leaves a low stress
46 environment for a less favorable situation; on this occasion, the plant will need to transition from
47 being heterotrophic to being autotrophic (Silva et al., 2017). This causes the passage of these seedlings
48 to the external environment, which could happen if, for example, vegetation causes high seedling
49 mortality (Assis et al., 2009). This problem could be overcome by the use of special cultivation
50 conditions. Thus, arbuscular mycorrhizal fungi (AMFs) and the type of substrate employed during this
51 process are fundamental factors that guarantee the success of this stage. This is because these fungi
52 form symbiotic associations with plants and increase their survival chances by guaranteeing them
53 greater nutrient and water absorption (Smith and Smith, 2011a).
54 As C. longa is exploited by the pharmaceutical and medical industries and studies have shown
55 changes in its chemical compounds due to its association with AMFs (Urcoviche et al., 2015), it is
56 important to elucidate how this interaction influences the chemical composition of the plant. This
57 information is very relevant for the industry that mainly uses rhizomes as raw material. The use of new
58 technologies that promote metabolic changes in plants with the help of sustainable production
59 techniques has been prominent in the academic field. The results obtained from greenhouse tests can
60 be extrapolated to conventional cultivation conditions, such as the agricultural use of AMFs, in order
61 to optimize nutrient absorption by decreasing chemical fertilizer use (Smith and Smith, 2011a; Smith
62 and Smith, 2011b). In the case of medicinal plants, especially C. longa, understanding how these fungi
63 alter the chemical composition of the plant will also help direct the plant metabolism to the production
64 of compounds of interest (Urcoviche et al., 2015).
65 Some studies have also revealed that mycorrhizal fungi promote drastic changes in the
66 metabolism of primary and secondary plant products (Ceccarelli et al., 2010; Nell et al., 2010). Under
67 stressful conditions, vegetables tend to increase the production of antioxidant compounds and enzymes
68 such as free radical scavengers, superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase
69 (APX), glutathione reductase (GR), peroxidases (POD), and polyphenoloxidase (PPO) (Gutteridge and
70 Halliwell, 2010). This is an attempt to combat and minimize oxidative damage such as lipid

2
 71 peroxidation and protein and nucleic acid damage (Gill and Tujeta, 2010). In the case of C. longa,
72 such studies are scarce; they are however essential as they could effectively contribute to the
73 improvement of the production processes of seedlings, as well as the primary rhizome supply chain of
74 the pharmaceutical and cosmetic industry.
75 Thus, it is expected that fungi will not only positively interfere with plant growth, but also will
76 alter plants’ metabolic activities. The elucidation of these processes becomes paramount to
77 understanding which compounds are favored by each fungus, whether new compounds are stimulated,
78 and the most favored chemical groups. Additionally, it is important to verify plant biochemical
79 processes such as phenolic compound production, antioxidant activity, and antioxidant enzyme
80 activity (Riter-Netto, 2014; Pedone-Bonfim et al., 2012; Pedone-Bonfim et al., 2015).
81 In view of the above, the objective of this study was to evaluate the influence exerted by
82 AMFs on the production of chemical compounds and the biochemical activity of C. longa
83 acclimatized in different substrates and inoculated with two AMFs, Rhizophagus clarus and
84 Claroideoglomus etunicatum.

85 2. Materials and methods

86 2.1 Obtaining plant material

87 Curcuma longa rhizomes were collected from the medicinal garden of the University of
88 Paraná - UNIPAR, Umuarama, PR, Brazil. They were washed in water and dried on sheets of paper in
89 a laboratory environment. Cracked, wilted, and disease-prone rhizomes were eliminated and only
90 those with explants (sprouts) were kept. Explants were removed with the help of a scalpel and
91 standardized to a length of 1.5 cm ± 0.3.

92 2.2 In vitro inoculation of C. longa

93 Shoots were disinfected in a laminal flow chamber by using sodium hypochlorite (2%) for 20
94 minutes; then, they were washed three times in autoclaved water. The shoots were inoculated into 350
95 mL glass vials containing an culture medium Murashige and Skoog - MS (Murashige and Skoog,
96 1962) at full strength. The medium was supplemented with 30 g L-1 sucrose and 6.0 g L-1 agar. Three
97 growth regulators were added to the culture medium: 6-benzylaminopurine (BAP), α-
98 naphthaleneacetic acid (NAA), and kinetin (KIN) in concentrations of 8.88, 2.16, and 0.92 µM,
99 respectively. The pH was adjusted to 5.8 (Antoniazzi et al., 2016).
100 The flasks were autoclaved at 121 °C for 20 minutes. The inoculation was performed in an
101 aseptic chamber and shoots were placed individually in vials containing 50 mL of the culture medium,
102 closed with clear plastic lids and sealed with PVC film. The explants were kept in a growth chamber
103 for 120 days (Fig. 1a-b), under a 24-h light photoperiod achieved with the use of Blumenau®,
104 Blumenal, Brazil - light emitter diodes (LEDS) lamps [LED 10 W 6.000 K, 100-240 V-50/60 Hz,

3
105 power factor: ≥0.92 (High FP)], at a temperature of 25 °C ± 2 °C and a light intensity of 72.02 µmol
106 m−2·s−1.

107 2.3 Acclimatization of C. longa

108 After this step, the plants were removed from the vials, washed in water to remove the
109 residues of the culture medium attached to the roots, and placed in plastic trays with water-dampened
110 papers. Later, they were transferred to PVC vessels with a 3 L capacity. The substrates used in the
111 assay were previously autoclaved at 121 °C for 1 hour. The soil used in the experiment was collected
112 from the UNIPAR experimental area in a 0–20 cm layer and was classified as dystroferric red–yellow
113 latosol with sandy texture and low base saturation (Santos et al., 2013).
114 In the assay, we used two types of substrates: two mycorrhizal fungi used alone or in
115 combination and plots without fungi. The experiment was performed with eight treatments: T1)
116 vermiculite (Brasil Minérios®, Goiânia, Brazil) and soil (1:1 v/v) without mycorrhizal fungi; T2)
117 vermiculite and soil (1:1 v/v) inoculated with R. clarus; T3) vermiculite and soil (1:1 v/v) inoculated
118 with C. etunicatum; T4) vermiculite and soil (1:1 v/v) with a combination of R. clarus and C.
119 etunicatum; T5) vermiculite, commercial substrate (Carolina Soil®, Santa Cruz do Sul, Brazil), and
120 vermicompost (Humus Fertil®, Toledo, Brazil) (composition: nitrogen 5%, cationic capacity 200
121 mmol, organic carbon 10%, minimum CTC/C ratio 10%) (1:1:1 v/v) without mycorrhizal fungi; T6)
122 vermiculite, commercial substrate, and vermicompost inoculated with R. clarus; T7) vermiculite,
123 commercial substrate, and vermicompost inoculated with C. etunicatum; T8) vermiculite, commercial
124 substrate, and vermicompost with a combination of R. clarus and C. etunicatum (Fig. 1c).
125 Rhizophagus clarus and C. etunicatum were sourced from the UNIPAR Glomales bank and
126 were applied to the upper third of each pot in 200 g of inoculate soil (150 spores). One C. longa
127 seedling was planted per pot and was kept in a greenhouse, at a controlled temperature of 25 ± 4 °C,
128 without humidity control for 240 days. At this time, the plants were fertilized with 150 mL Hoagland
129 solution (Hoagland and Arnon, 1950) without phosphorus and were irrigated manually, daily for 30
130 days. The experiment was set up in a completely randomized design with eight replications for each
131 treatment, totaling 64 plots.
132 2.4 Determination of the essential oil composition of the different treatments
133 2.4.1 Essential oil isolation

134 After 240 days, the rhizomes of the plants were collected, sliced, and dried at laboratory room
135 temperature. The rhizome mass was obtained from the eight plants of each treatment. T1 did not
136 produce rhizomes, while T2, T3, T4, T5, T6, T7, and T8 produced 2.244, 1.453, 1.735, 21.311,
137 25.774, 17.294, and 16.953 g, respectively. The rhizomes were then submitted to the hydrodistillation
138 process in a modified Clevenger apparatus. The distillation time was 2 hours and the oil was removed

4
139 with n-hexane, filtered with anhydrous Na2SO4, and refrigerated at 4 °C in amber vials (Santos et al.,
140 2009).

141 2.4.2 Chemical characterization of essential oils

142 The identification of the chemical constituents of the C. longa essential oil was performed by a
143 gas chromatograph (Agilent 7890 B®, Santa Clara, USA), coupled to a mass spectrometer (Agilent
144 5977 A-MSD ®, Santa Clara, USA) equipped with an Agilent HP-Capillary column 5MS UI®, Santa
145 Clara, USA (30 m × 0.250 mm × 0.25 µm). For analysis, a 2.0 µL injection of a dichloromethane
146 solution was used. The conditions of the analysis were: injector temperature 280 °C operating in split
147 mode (20:1), transfer line 280 °C, carrier gas He at a flow rate of 1 mL/min, and initial column
148 temperature of 60 °C (2 min), with a ramp of 1,5 °C/min until a temperature of 185 °C was reached.
149 The temperature remained at this level for 1 min, followed by an increase of 9 °C/min until a
150 temperature of 275 °C was reached. The temperature remained at this level for 2 min, followed by an
151 increase of 25 °C/min until a temperature of 300 °C was reached; the temperature remained at this
152 level for 1 min. The EM detection system was used in 70 eV electron impact ionization scan mode at a
153 mass/charge (m/z) ratio in the range of 40 - 550 (m/z). The temperatures of the ionization source and
154 the quadrupole were 230 and 150 °C, respectively. The compounds were identified by comparing their
155 mass spectra with those found in the NIST 11.0 library as well as by comparing the retention rates
156 (RR) obtained by the homologous n-alkane (C7–C28) series (Adams, 2017).

157 2.4.3 Multivariate statistical analysis

158 The chemical composition of the essential oil - EO was determined by multivariate
159 exploratory analyses. To measure the similarity among the isolate centroids we used the Euclidean
160 distance (dissimilarity measure) for the set of chemical compounds obtained in essential oils of each
161 treatment. For the grouping strategy, we adopted the method of the main components. The principal
162 components analysis - PCA allows for the concentration of the highest amount of original information
163 contained in p variables. The most relevant chemical compounds identified and their area quantity in
164 % (Table 1) were plotted in Excel spreadsheets.
165 From this analysis, we removed the compounds that were not identified (ni). Additionally, the
166 analysis was performed based on the chemical group the total compounds identified in each treatment
167 belonged to. These data were transformed into orthogonal latent variables called main components,
168 which are linear combinations of the original variables created with the eigenvalues of the covariance
169 matrix of the data (Hair, 2005). The Kaiser criterion was used to elect the main components. An
170 eigenvalue preserves relevant information when it is higher than the unit. Multivariate analyses were
171 performed using the program Statistica 13.3 (Statsoft, 2017).

172 2.5 Biochemical assessments

5
173 2.5.1 Obtaining the extract
174 This procedure was adapted from Larrauri et al. (1997). We measured 1 g samples from fresh
175 C. longa leaves and kept them in a freezer at -20 °C.

176 2.5.2 Measurement of antioxidant activity by the DPPH method

177 From the extract, we determined the antioxidant activity based on the absorption extinction of
178 the radical 2,2-diphenyl-1-picryl hydrazyl (DPPH 60 µM), according to the method proposed by
179 Rufino et al., (2009). For the determination of antioxidant activity, a 0.1 mL aliquot of the obtained
180 extract was transferred to a test tube in a dark environment and 3.9 mL of the DPPH radical (0.06
181 mM) was added and homogenized in a tube shaker; three technical and three biological repetitions
182 were performed for all analyses. The readings taken on the (Beckman 640B®, California, USA)
183 spectrophotometer (515 nm) were monitored by a computer system every 30 minutes for a total of four
184 readings, where the absorbance reduction was observed until stabilization. The results were expressed
185 as a percentage of free radical scavenger (% FRS), according to the equation:
186 aa = [(Ac-Am) / Ac] .100
187
188 Where: aa = Antioxidant activity (%)
189 Ca = control absorbance
190 As = absorbance of the sample
191 The value was calculated by creating a graph of the percentage inhibition of DPPH as a
192 function of antioxidant concentration, using three technical and three biological replicates.

193 2.5.3 Measurement of phenolic compounds

194 To determine the total phenol content of C. longa leaves, the ethanolic extract described above
195 was used. The method employed was visible-area spectroscopy using the Folin-Ciocalteu method
196 (Kuskoski et al., 2005). The total phenol content (TP) was determined by the interpolation of the
197 absorbance of the samples against a calibration curve, constructed with gallic acid standards (5 to 40
198 µg/mL), and expressed as mg GAE (gallic acid equivalents) per g of extract. The equation for the
199 gallic acid calibration curve was C = 809.0200A +5.0827, where C is the concentration of gallic acid,
200 A is the absorbance at 750 nm, and R²= (0.999) is the correlation coefficient. Three technical and three
201 biological replicates were used.

202 2.5.4 Statistical analysis

203 The phenolic compound, antioxidant activity and enzymes values were first subjected to the
204 Shapiro-Wilk normality test and then to an analysis of variance test (ANOVA) (p ≤ 0.05). The means
205 were compared by Tukey’s test (p ≤ 0.05) using the software SISVAR 5.6 (Ferreira, 2011).
206 2.5.6 Determination of enzyme activity: SOD, APX, and CAT

6
207 The enzyme extract used in all analyses was obtained according to Bonacina et al. (2017).
208 Enzymatic activities were expressed in enzyme units.
209 SOD (EC 1.15.1.1): The activity of SOD was measured by its ability to inhibit the
210 photoreduction of nitroblue tetrazolium (NBT) as described by Giannopolitis and Ries (1977). The
211 reading was taken at 560 nm, in which one unit of SOD (U) activity was defined as the amount of
212 enzymes required to inhibit 50% reduction in NBT. SOD activity was expressed as U SOD mg-1 MF
213 min-1.
214 CAT (EC 1.11.1.6): CAT activity was measured according to the methodology proposed by
215 Havir and McHale (1987). This activity was determined by the degradation of H2O2 within 1 minute at
216 260 nm. Enzyme activity was quantified using the molar extinction coefficient 36 M-1 cm-1 (Anderson
217 et al. 1995), expressed in mmol H2O2 g-1 MF min-1.
218 APX (EC 1.11.1.11): APX activity was measured according to the methodology proposed by
219 Nakano and Asada (1981). The activity was determined by the degradation of H2O2 within 1 minute at
220 290 nm. Enzyme activity was quantified using the molar extinction coefficient 2.8 m M-1 cm-1
221 (Nakano and Asada, 1981). APX activity was expressed as mmol ascorbic acid g-1 MF min-1.
222 All enzymes were evaluated using 96-well flat-bottom Elisa plates. In all enzymatic assays
223 three technical repetitions and three biological repetitions were performed. The equipment used was
224 the UV-VIS spectrophotometer, (Spectra Max Plus®, Massachusetts, EUA), and the SoftMax
225 (SoftMax Pro 6.5.1®, California, USA) program.

226 3. Results
227 3.1 Chemical characterization of essential oils and cluster analysis
228 The chemical composition of the essential oil of C. longa is presented in Table 1. A total of 48
229 compounds were disclosed, of which 44 were identified. There was a variation in the essential oil
230 composition of C. longa, as a function of mycorrhization and/or substrate type (Table 1). T1
231 containing only soil and vermiculite and not containing AMFs, did not develop rhizomes, so it was not
232 possible to extract the essential oil for evaluation from this treatment.
233 The major EO compounds, regardless of treatment type, were ar-turmerone and β-turmerone
234 (curlone); both compounds were quantified above 50% in all treatments. In T4, ar-turmerone was
235 higher (61.7%) compared to other compounds (Table 1). The substrate consisting of vermiculite/soil
236 (1:1 v/v) and inoculated with R. clarus (T2) produced 34 compounds. Among them was germacrone
237 that was not detected in any other treatment (Table 1). T3 had the highest number of compounds (37 in
238 total); among these were o-cymene, cis-α-bisabolene, caryophyllene oxide, and cis-α-santalol, while
239 ergosta-5,22-dien-3-ol, acetate, and (3β, 22E) were identified only in this treatment (Table 1). In T4,
240 28 compounds were identified; α-phellandrene, which was identified in all other treatments, was not
241 observed in T4.

7
242 Regarding T5, only eight compounds were found (Table 1) and a substantial increase in the
243 concentration of the compound α-phellandrene (4.31%) was observed. In T6, 11 compounds were
244 identified (table 1). The principal compound was trans-Nuciferol, detected in a concentration of
245 3.54%. In T7, β-cymene (0.81%) and the highest amount of 1.8-cineole (3.14%) were detected (Table
246 1). Eight compounds were identified in T8; among these, α-phellandrene had the highest percentage
247 (8.70%), while terpinolene and camphor (1.88%) were also identified (Table 1).
248 In the essential oil of C. longa rhizomes, compounds belonging to the class of oxygenated
249 sesquiterpenes constituted the majority of compounds found in T4 (87.99%), followed by T5 and T6
250 (Fig. 2C). The second most significant class was the hydrocarbon sesquiterpenes, with the highest
251 concentration observed at T2 (10.32%) and the lowest at T4 (6.67%); this class was represented
252 mainly by α-zingiberene (4.60%). In T6, the most abundant sesquiterpene was β-sesquiphellandrene
253 (3.18%) (Fig. 2C, Table 1).
254 Principal component analysis showed a total variation of 99.81%, with principal component 1
255 representing the largest part (99.31%), followed by component 2 (0.50%). It was possible to verify the
256 formation of four groups, the first with ar-turmerone, the second with β-turmerone, and a third with
257 the 1.8-cineole, a-zingiberene, and β-sesquiphellandrene compounds. The remaining compounds were
258 all found in the fourth group (Fig. 2A).
259 The T5 vector was closer to the most abundant compound ar-turmerone, which was away
260 from the center and remained close to vector 1. The T2 vector was closer to the compound β-
261 turmerone. 1.8-cineole, α-zingiberene, and β- sesquiphellandrene were closer to the treatment vectors
262 6, 7, and 8. The other compounds were more influenced by T2, T3, and T4 (Fig. 2A). In general,
263 oxygenated sesquiterpenes and monoterpenes were under the influence of the T4 and T5 treatments
264 (Fig. 2B). The hydrocarbon sesquiterpenes were under the influence of T2. PCA a low relationship of
265 hydrocarbon monoterpenes and other compounds with the treatments used (Fig. 2B).

266 3.2 Antioxidant activity, phenolic compounds, and enzymes

267 All C. longa plants that were grown in a combination of vermiculite, commercial substrate,
268 and vermicompost (T5 to T8), showed an increase in antioxidant activity (Fig. 3A). In T7 and T8
269 (84% and 74.90%, respectively) we found a more significant response of leaf activity. In the other
270 treatments, the antioxidant activity was reduced (Fig. 3A). It was not possible to measure the
271 antioxidant activity in T1, owing to the low production of leaves. T7 resulted in 31.51% more activity
272 compared to T3. T8 resulted in a 49.39% increase in antioxidant activity compared to T4 (Fig. 3A).
273 The C. longa plants that were exposed to soil and vermiculite (T2 to T4) had the lowest antioxidant
274 activity.
275 In the analysis of total phenolic compounds, the values were similar among the seven
276 treatments (between 50 and 80 mg/EAG). Plants that grew in T4 stood out, as this treatment resulted

8
277 in an increase of 71.89%, 72.27%, and 74.18% when compared to the T1, T3, and T5 treatments,
278 respectively (Fig. 3B).
279 In the analysis of antioxidant enzymes, T3 resulted in the increase of the ascorbate peroxidase
280 enzyme, with a substantial increase in its activity (0.46 mM), followed by T2 that resulted in 0.2 mM
281 (Fig. 4A). In the other treatments, C. longa plants had an activity below 0.1 mM (Fig. 3A). T3
282 promoted an increase of 96.06%, 86.25%, and 85.94% in the activity of this enzyme compared to T8,
283 T7, and T1, respectively. CAT had the lowest activity compared to the other enzymes. However, in the
284 C. longa plants that were submitted to the T1, T2, and T3 treatments, this enzyme had higher activity
285 (Fig. 4A). The activity of this enzyme was higher in T3 (0.03 mM), followed by T1 and T2 (0.028 and
286 0.026 mM, respectively). In T5 and T4, this enzyme’s activity increased by 82.33% and 68.41%,
287 respectively (Fig. 4B). Of the three enzymes evaluated, SOD showed more activity; its activity
288 exceeded 40 U in T4, T5, T6, and T7. In T8, the activity of this enzyme was the lowest (5.76 U) (Fig.
289 4C). In this case, T8 was less than 91.25% compared to T7 (Fig. 4C).
290 4. Discussion
291 4.1 Arbuscular mycorrhizal fungi and substrate types changed the chemical composition of the C.
292 longa essential oil

293 AMFs have an established ability to live symbiotically with numerous plants of economic
294 interest, mainly cereals and fruit trees. For these crops, significant increases in the uptake of nutrients
295 such as phosphorus, calcium, potassium, magnesium, and water were highlighted in this study. This
296 nutrient uptake increase results in more vigorous plants, with faster growth, and decreased
297 susceptibility to environmental stresses (Smith and Smith, 2011b). However, the effect of fungi on the
298 growth of medicinal plants remains poorly understood. This situation is aggravated by the biochemical
299 aspects of these plants, especially their chemical composition with emphasis on terpenes (Kapoor et al.
300 2017) and enzyme activities (Folli-Pereira et al., 2012).
301 Medicinal plants have an enormous ability to produce compounds, which have been used for a
302 variety of purposes (Abdel-Lateef et al., 2016). There is a tendency for such compounds to replace
303 chemicals that are toxic to human health and the environment. Curcuma longa has rhizomes rich in
304 secondary metabolism products, especially curcuminoids (Nawal, 2018). It is possible that
305 mycorrhizal fungi could affect plant metabolism in the same manner they affect plant growth. In the
306 present study, this action was dependent on the type of substrate used. This is because these fungi
307 require ideal conditions for the establishment of symbiosis and its development (Smith and Smith,
308 2011a).
309 Interestingly, it was observed that in the treatments where both vermiculite and soil were used
310 (T2, T3, and T4), there was an increase in the amount of compounds produced, with emphasis on
311 caryophyllene, α-curcumene, β-bisabolene, β-curcumene, cyclohexene, cubenol, β-santalol, α-

9
312 acorenol, 7-epi-cis-sesquisabinene hydrate, 2,5-Octadecadiynoic acid, methyl ester, and cis-
313 5,8,11,14,17-eicosapentaenoic acid; however, in these treatments the plants developed less (Fig. 1d).
314 On the other hand, the seedlings acclimatized in vermiculite, commercial substrate, and vermicompost
315 (T6, T7, and T8) showed a decrease in the production of compounds. In these treatments, the plants
316 were probably under less stress and directed their metabolic pathways to the biosynthesis of primary
317 compounds, which are useful for their development (Taarit et al., 2011; Savoi et al., 2016, Bonacina et
318 al., 2017; Kapoor et al., 2017). In addition, this substrate favored the symbiosis of fungi with the root
319 systems of the plants as these turned out to be more vigorous, while an increase of the root system and
320 leaf areas was observed (Fig. 1e).
321 Several factors have been mentioned as influencing the ability of fungi to associate with the
322 root system of plants, such as the presence of phosphorus in the soil (Smith and Read 2008), soil
323 moisture (Wright 2018), presence of organic matter, humic acids, and physical aspects of the soil
324 (Smith and Smith, 2011b). On the other hand, the plant can also influence this association; the
325 mycelium of the fungus that grows into the plant is controlled by the homeostasis of the latter, while
326 the mycelium of the fungus that grows in the soil is controlled by environmental conditions (Smith and
327 Smith 2011a). However, owing to the vermicompost and commercial substrate was richer in organic
328 matter. Our study provides evidence that this composition affected genetic and physiological aspects
329 of C. longa and resulted in more vigorous plants and lower yield of compounds. On the other hand,
330 plants exposed to substrate with less organic matter were noticeably smaller, had low rhizome
331 production, and a significant increase in the yield of compounds (Fig. 1d).
332 One of the first biochemical signs observed in plants under stress is to direct valuable energy
333 resources to other metabolic pathways, while probably decreasing the production of primary
334 metabolites (Kapoor et al., 2017). This hypothesis of equilibrium of growth differentiation, terpenoid
335 synthesis, and AMFs was suggested by Kapoor et al. (2017), as both symbiosis and terpenoid
336 synthesis require resources and are dependent on nutrient availability.
337 The mechanism behind this signaling and the genes that are associated with this type of
338 response remain unknown for most cultivated plants. In the case of terpenoid synthesis, Kapoor et al.
339 (2017) reported that mycorrhizal tomato plants showed induction of genes from the terpene synthase
340 family (TPS) (TPS31, TPS32, and TPS33). Folli- Pereira et al. (2012) suggested that an exchange of
341 biochemical signals and responses occurs in the establishment of symbiosis, involving the expression
342 of so-called “symbiosis genes” in the plant and the emission of symbiotic signals by the fungus
343 (Parniske, 2004). This enables the regulation of plant defense gene expression and the synchronization
344 of morphological and physiological changes in the plant host. Therefore, the compounds observed in
345 plants that received the T2, T3, and T4 treatments could produce signals for the expression of other

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346 genes that control important plant activities, such as production of primary compounds and
347 developmental genes as well as attenuate the stress effect in plants.
348 There was no significant variation in the major compounds ar-turmerone and β-turmerone in
349 any of the treatments. Similar results were obtained for Mentha crispa L. grown in substrate
350 inoculated with C. etunicatum or R. clarus in soil with low and high phosphorus levels. The carvone
351 compound was present in eight different treatments and constituted over 70% of their EOs (Urcoviche
352 et al., 2015). It is interesting that these compounds are produced consistently even when they influence
353 the physiology and biochemistry of the plant. It is likely that because of high relevance of these
354 compounds to the plant, certain mechanisms in the plant aid the function of these compounds more
355 constantly in plant metabolism.
356 Regarding the principal curcuminoids in C. longa, these have been reported to possess anti-
357 inflammatory, anti-viral, anti-bacterial, anti-oxidant, anti-fungal, anti-carcinogenic, and other
358 properties (Gounder and Lingamallu, 2012; Wang et al., 2014; Sueth-Santiago et al., 2015). The ar-
359 turmerone compound has been reported to have anti-microbial activity against Staphylococcus aureus,
360 Pseudomonas aeruginosa, Candida albicans, and Aspergillus niger (Singh et al. 2011). According to
361 Hu et al. (2017), the effects of these compounds appear to disrupt the fungal endomembrane system,
362 the plasma membrane, and mitochondria, thus inhibiting ergosterol synthesis and mitochondrial-
363 related activities such as ATpase, dehydrogenase, and more.
364 Several authors describe ar-turmerone and β-turmerone as the principal essential oil
365 compounds of C. longa rhizomes (Singh et al., 2011). However, studies by Raina and Srivastava
366 (2005) showed that the essential oil of C. longa rhizomes produced in the Himalayas in northern India,
367 consists mainly of α-turmerone (44.1%), β-turmerone (18.5%), and ar-turmerone (5.4%). In agreement
368 with this study. In Orissa, India, essential oil extracted from the rhizomes of C. longa, had α-
369 turmerone (49.1%) as its main constituent, followed by β-turmerone (16.8%), and α-phellandrene
370 (5.3%) (Singh et al., 2011).
371 In the present study, there was a significant variation in α-phellandrene when plants were
372 acclimatized in vermiculite containing substrate, commercial substrate, and vermicompost (1:1:1 v/v)
373 (Table 1). The presence of this compound in rhizomes was probably optimized by the presence of
374 AMFs and nutrient availability (Smith and Read, 2008). The isolated compounds in group 3 were
375 influenced by treatments T6, T7, and T8. The increase in the production of these monoterpenes
376 probably occurred due to the availability of nutrients in the substrate, which may have been increased
377 by the presence of AMFs; adequate amounts of nutrients may increase the production of some
378 compounds (Chrysargyris et al., 2016) and influence enzymes that are important in terpene
379 biosynthesis (Koeduka et al., 2006).

11
380 Oxygenated sesquiterpenes are reported in the literature as the most abundant compounds in
381 C. longa oil (Raina and Srivastava, 2005; Singh et al., 2011), corroborating the results of this study.
382 The T4 and T5 treatments were the ones that resulted in a higher production of these compounds
383 (87.99% and 86.67%, respectively).

384 4.2 Antioxidant activity and total phenolic compounds of C. longa altered under different substrate
385 and mycorrhizal fungi treatments

386 Phenolic production and the antioxidant activity of compounds are characteristics that can be
387 altered by the action of mycorrhizal fungi. Plants of the Zingiberaceae family can produce antioxidant
388 compounds that have the ability to neutralize reactive molecules or radicals that may be deleterious to
389 the plant cell (Omar et al., 2013). Among the compounds that have this capacity are curcumene,
390 santalene, sitosterol (Li et al., 2019), 8,9-dehydro-9-formyl-cycloisolongifolene, germacrone (Xiang et
391 al. 2017), curcumin, desmethoxycurcumin, and bisdemetoxycurcumin (Auger et al., 2018).
392 The seedlings of C. longa that showed significant increases in antioxidant activity (above
393 50%) were those grown under the T7, T8, T5, and T3 treatments, respectively. T4 and T6 plants had
394 lower averages. The antioxidant activity of a plant can be promoted by an isolated compound or by the
395 synergistic effect of other compounds. Among the compounds identified in the EO all have the ability
396 to induce antioxidant activity. However, another compound could have produced the observed results,
397 as suggested by (Awasthi and Dixit, 2009). C. longa leaves tend to have a varied composition when
398 compared to the rhizome.
399 Regarding oxidizing activity, it was expected that it would appear in plants that are under
400 stress conditions and unfit for development; however, in this study this was only demonstrated in T3.
401 Some hypotheses may explain these results; the first is that plants have various biochemical
402 mechanisms to mitigate oxidative damage. When a plant develops in inappropriate conditions, this
403 results in free radical production, which has the ability to destroy mainly lipids, but also proteins,
404 nucleic acids, and other primary metabolites (Gill and Tujeta, 2010; Barbosa et al., 2014). In their
405 evolutionary process, plants have developed a set of substances to counteract the effects of radicals.
406 There are three main groups: antioxidant enzymes (Gill and Tujeta, 2010; Barbosa et al. 2014),
407 phenolic compounds (Matkowski, 2008), and vitamins, especially E and C (Sies and Stahl, 1995).
408 In this study, we evaluated the antioxidant activity and the enzymes that showed significant
409 activity under the fungi, soil, and vermiculite substrate treatments, with a core focus on the catalase
410 enzyme. The biochemical activity of a plant is dynamic, as the plant performs several metabolic
411 processes at the same time; therefore, it is difficult to predict in which situation it will prefer a radical-
412 fighting mechanism and make changes if one of these mechanisms is saturated (Ferrari et al., 2019).
413 Therefore, these modulations were the result of signal exchanges and biochemical responses
414 established by the plant/fungal interaction that regulates the expression of defense genes, thus leading

12
415 to morphological and physiological changes that interfere with both the action and the availability of
416 bioactive compounds (Folli-Pereira et al., 2012).
417 On the other hand, the production of phenolic compounds was similar in most treatments; only
418 the plants developed under the T4 treatment had an increased phenolic compound production. The
419 antioxidant activity of plants was lower in the same treatment, which allowed us to conclude that
420 although T4 resulted in the highest amount of phenols, these did not have antioxidant activity. We
421 suggest that compounds other than phenolics may have been responsible for the antioxidant activity
422 resulting from these treatments (Daglia et al., 2000). The method of investigating antioxidant activity
423 employed in this assay was based on the elimination of the stable free radical 1,1-diphenyl-2-
424 picrylhydrazyl (DPPH) and had a greater affinity for compounds that neutralize the DPPH radical.
425 Another issue to be considered is the complex interaction among antioxidant compounds that cannot
426 be considered individually due to the synergism and antagonism among them (Roginsky and Lissi,
427 2005).
428 However, the mechanisms of action of AMFs in potentiating the amount of secondary
429 compounds remain unclear. The mycorrhizal efficiency in increasing these compounds may be
430 associated with the symbiotic ability to increase the number of plastids, mitochondria, nutrient
431 absorption, and mycorrhizal defense responses; all of these factors may favor the increase of bioactive
432 compounds (Freitas et al., 2004; Ceccarelli et al., 2010). Other studies, however, have not shown a
433 positive effect on the availability of phenolic compounds in mycorrhizal plants (Perner et al., 2008;
434 Nell et al., 2010; Geneva et al., 2010).

435 4.3 Antioxidant enzymes mitigated oxidative damage in C. longa plants

436 Cellular metabolic reactions such as photosynthesis, respiration, photorespiration, and lipid
437 oxidation generate reactive oxygen species (ROS) (Gill and Tujeta, 2010). Several sensors in plants
438 are triggered to monitor the levels of these radicals in cells; when elevated, the levels of these radicals
439 activate a signal translation network that promotes inactivation mechanisms such as the enzymes SOD,
440 APX, and CAT (Hossain and Dietz, 2016). This network modulates metabolic reactions by
441 suppressing or activating pathways that result in the controlled maintenance of ROS levels in cells
442 (Wu et al., 2006; Zhang et al., 2013).
443 Few studies have focused on the presence of mycorrhizal fungi and the response of enzyme
444 activity. The literature shows two initial hypotheses in plants that are subjected to the presence of
445 mycorrhizal fungi. The first hypothesis is that the plant is benefited by the fungi and, therefore, tends
446 to have decreased activity of enzymes. The second hypothesis, as suggested by Folli Pereira et al.
447 (2012), is that plants with a higher antioxidant enzyme activity are more tolerant to different stressors,
448 precisely because mycorrhizae induce increased activity of antioxidant enzymes such as peroxidase,
449 catalase, and superoxide dismutase. Antagonistic results obtained in trials have shown that both

13
450 situations can occur (Wu et al., 2006) in the enzymatic systems of plants that grow symbiotically with
451 mycorrhizal fungi.
452 There was a considerable increase of the APX enzyme (0,5mM) in plants inoculated with C.
453 etunicatum (T3). Wu et al. (2006) reported that APX enzyme activity was 59% higher in mycorrhized
454 Citrus tangerine roots with fungi of the same genus, than in non-mycorrhizal seedlings. The same
455 authors observed an increase of all enzymes studied (SOD, APX, POD, and CAT) in mycorrhizal
456 plants subjected or not to stress (Wu et al., 2006). In other treatments, although enzymatic activity
457 existed and contributed to the removal of radicals, it was lower, which indicates that the plant used
458 other enzymes or mechanisms to promote cell cleansing.
459 On the other hand, CAT showed higher activity under the T1, T2, and T3 treatments where the
460 plants were smaller and less vigorous. Foli Pereira et al. (2012) suggest that increased CAT activity
461 signals a stress condition by the plant. Among the mechanisms of inactivation, the enzyme SOD is
462 considered the first enzyme of the defense line against ROS, dismuting O2• - radicals and generating
463 H2O2 and O2 (Barbosa et al., 2014). In this study, SOD had the highest recorded activity (T7).
464 Although an increase in APX and CAT enzymes that will convert the H2O2 radicals formed by
465 SOD into H2O and O2 (Gill and Tujeta, 2010) is expected, in this study the increase of SOD activity
466 did not cause APX and CAT elevation, as the activities of these enzymes remained relatively stable in
467 the treatments (T6 and T7). Other antioxidant enzymes not investigated in this study may have acted
468 on such radicals; such enzymes are POD, PPO, GR, and glutathione peroxidase (GPX) (Wu et al.,
469 2006; Gill and Tujeta, 2010; Kundu et al., 2018).
470 Studies showing how these associations interfere with the enzyme gene signal network are
471 scarce; however, Borde et al. (2011) suggest that in mycorrhizal associations, ROS act as secondary
472 messengers and alterations in the expression of antioxidant enzymes, such as PODs, are related to the
473 development of symbiosis (Borde et al., 2011). Thus, AMFs can increase the plant’s ability to
474 withstand biotic and abiotic stresses by allowing greater tolerance to ROS (Folli-Pereira et al., 2012).
475 The results of the present study demonstrated that mycorrhizal fungi C. etunicatum or the
476 combination of C. etunicatum and R. clarus associated with the soil + commercial substrate and
477 vermicompost, were efficient in promoting the acclimatization of C. longa after cultivation in vitro, as
478 the plants had 100% survival rate. The chemical composition of the essential oil of C. longa rhizomes
479 was strongly influenced by the aforementioned association. Plants acclimatized in vermiculite and soil
480 showed an increase in the number of chemical compounds, while plants acclimatized in vermiculite,
481 commercial substrate, and vermicompost had a reduced number of compounds. Oxygenated
482 sesquiterpenes were the main compound class in all the treatments, with the principal compounds
483 being ar-turmerone and β-turmerone.

14
484 The afore mentioned associations resulted in biochemical changes in plants, such as increased
485 antioxidant activity and free radical scavenging enzymes. Plants inoculated with C. etunicatum (T7),
486 had higher antioxidant activity. Plants inoculated with both fungi (T4) had the highest percentage of
487 phenolic compounds. The SOD enzyme was more active in plants inoculated with fungi, under the T6
488 and T7 treatments. Both APX and CAT had an increased activity in plants grown under the soil-based
489 substrate and vermiculite treatments (T1, T2, and T3).
490 Although this study was conducted in a greenhouse in order to favor the acclimatization
491 process of plants, the results obtained could serve as the basis for the use of AMFs in cultivations, as
492 they favor the absorption of nutrients and water by the plant, thus reducing the use of chemical
493 fertilizers. The results of this study revealed that the fungi associated with the substrate type resulted
494 in changes in the chemical composition of the plant. This finding creates opportunities for the use of
495 these microorganisms in altering metabolic pathways and directing plant metabolism for the
496 production of compounds of industrial interest by using more sustainable technologies.
497 Acknowledgments
498 We would like to thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
499 n° 001 and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for funding this
500 work. And Universidade Paranaense (UNIPAR) for financial support.
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19
Table 1. Chemical composition of the essential oil of C. longa obtained from rhizomes produced as a function of inoculation with Rizophagos clarus and Claroideoglomus etunicatum and
different substrates.

Relative area (%)

a
Peak Composition RI calc T2 T3 T4 T5 T6 T7 T8 IM
1 α-phellandrene 1005 0.10 0.17 - 4.31 6.70 6.90 8.70 a. b. c
2 o-cymene 1026 - 0.16 - - - - - a. b. c
3 β-cymene 1027 - - - - - 0.81 - a. b.c
4 Cineole 1.8 1032 1.23 1.84 1.07 2.32 2.94 3.14 2.13 a. b. c
5 Terpinolene 1099 0.49 0.79 0.37 - 1.34 1.61 1.88 a. b. c
6 Camphor 1142 0.49 0.79 0.37 - 1.34 1.61 1.88 a. b. c
7 Terpinen-4-ol 1176 0.26 - 0.20 - - - - a. b.c
9 p-cymen-8-ol 1189 0.17 0.56 - - - - - a. b.c
10 α-terpineol 1209 0.47 - - - - - - a. b.c
11 Caryophyllene 1479 0.32 0.41 0.38 - - - - a. b. c
12 α-Curcumene 1492 0.46 0.56 0.45 - - - - a. b. c
13 α-zingiberene 1500 3.10 3.00 2.77 4.03 4.6 4.26 4.41 a. b. c
14 β-bisabolene 1508 2.10 0.37 0.32 - - - - a. b.c
15 cis-α-Bisabolene 1623 - 0.15 - - - - a. b.c
16 β-curcumene 1685 3.54 0.43 0.22 - - - - a. b.c
17 Ni 1747 0.16 - - - - a. b.c
18 β-sesquiphellandrene 1563 0.30 2.46 2.05 2.67 3.1 2.84 2.97 a. b. c
19 Dihydrocurcumene 1567 - 0.38 0.22 - - - - a. b. c
20 Ni 1574 - 0.16 - - - - a. b. c
21 Cyclohexene 1576 0.34 0.44 0.26 - - - - a. b.c
22 Ni 1577 0.20 - - - - a. b.c
23 trans-sesquisabinene hydrate 1577 0.78 0.74 - - - - a. b.c
24 trans-nuciferol 1579 0.34 0.25 4.18 3.54 - - a. b. c
25 Cubedol 1586 0.49 0.39 0.52 - - a. b. c
26 Caryophyllene oxide 1586 - 3.74 - - - - - a. b. c
27 cis-lanceol 1604 - 0.81 - - - - - a. b.c
28 β-santalol 1604 0.83 0.72 0.86 - - - - a. b.c
29 cis-α-santalol 1614 - 0.50 - - - - - a. b.c
30 Cubenol 1631 0.96 2.30 0.96 - - - - a. b. c
31 α-acorenol 1632 0.42 1.02 0.32 - - - - a. b. c
32 7-epi-cis-sesquisabinene hydrate 1631 2.13 0.64 2.32 - - - - a. b. c
 33 ar-tumerone 1634 54.46 50.70 54.43 61.7 54.00 56.33 57.1 a. b.c
34 α-bisabolol 1635 0.72 0.75 0.74 - - - - a. b.c
35 8-cedren-13-ol. 1646 0.40 0.60 - - - - - a. b.c
36 β- tumerone 1691 20.82 19.50 21.6 22.01 20 20.85 20.93 a. b. c
37 4-epi-cubedol 1697 0.35 0.57 - - - - - a. b. c
38 Germacrone 1698 0.24 - - - - - - a. b. c
39 9-cedranone 1739 - - - 1.7 1.04 - a. b.c
40 Longifolenaldehyde 1748 - 1.25 1.32 1.26 - a. b.c
41 Oleic Acid 2105 0.95 0.91 0.86 - 1.4 1.65 - a. b.c
42 2.5-Octadecadiynoic acid. methyl ester 2106 1.51 1.57 1.73 - - - - a. b. c
43 cis-5,8,11,14,17-Eicosapentaenoic acid 2173 0.41 0.38 0.38 - - - - a. b. c
44 Androst-2-en-4-one, 17-(tetrahydropyran-3-yl)oxy- 2265 - 0.32 0.18 - - - - a. b. c
45 Ethyl isso-allocholate 2361 0.3 0.18 - - - - a. b.c
46 Ergosta-5,22-dien-3-ol, acetate, (3β22E)- 2367 - 0.38 - - - - - a. b.c
47 Ni 2621 - 0.03 - - - - - a. b.c
48 Ni 2832 0.20 - - a. b. c
-
Total identified 99.64 99.81 100 100 100 100 100
Hydrocarbon monoterpenes 0.59 1.12 0.37 4.31 8.04 9.32 10.58
Oxygenated monoterpenes 2.62 3.19 1.64 2.32 4.28 4.75 4.01
Hydrocarbon sesquiterpenes 10.32 8.20 6.67 6.70 7.70 7.10 7.38
Oxygenated sesquiterpenes 82.94 83.74 87.99 86.67 78.58 77.18 78.03
Others 3.17 3.56 3.33 - 1.4 1.65 -
a
Compounds listed according to HP-5MS column elution order; bRetention Index (RI) calculated using a homologous series of n-alkanes on a capillary column (HP-5MS); cIdentification based
on comparison of Wiley 275 spectroteca mass spectra; Relative area (%): It is the percentage of the area occupied by the compound within the chromatogram. (-): missing compound in the
sample; n.i = not identified; (-): missing.
T1) did not form rhizomes. so it was not possible to extract essential oil; T2) vermiculite and soil inoculated with R. clarus; T3) vermiculite and soil with C. etunicatum; T4) vermiculite and soil
with R. clarus and C. etunicatum; T5) vermiculite. commercial substrate and vermicompost without mycorrhizal fungi; T6) vermiculite. substrate and vermicompost inoculated with R. clarus;
T7) vermiculite. commercial substrate and vermicompost inoculated with C. etunicatum and T8) vermiculite. commercial substrate and vermicompost with R. clarus and C. etunicatum.
Fig.1. Stages carried through during the acclimatization process of Curcuma longa and chemical structure of
some compounds obtained from the essential oil of rhizomes (a) plants of C. longa cultivated in vitro (b)
seedlings of C. Longa after 120 days of cultivation (c) general vision of seedlings in acclimatization process
(d) treatments T1 the T4 in order C. longa plants inoculated with fungi and cultivated in soil and vermiculita
(e) T5 the T8 plants cultivated inoculated with fungi and cultivated in commercial substrate, vermicompost
and vermiculite.
Fig.2. Principal components - PCA obtained from the chemical composition (A), chemical groups (B) and total
identified (%) (C) of the essential oil of Curcuma longa as a function of inoculation with Rizophagos clarus and
Claroideoglomus etunicatum and different substrates.
T1) did not form rhizomes, so it was not possible to extract essential oil; T2) vermiculite and soil inoculated
with R. clarus; T3) vermiculite and soil with C. etunicatum; T4) vermiculite and soil with R. clarus and C.
etunicatum; T5) vermiculite, commercial substrate and vermicompost without mycorrhizal fungi; T6)
vermiculite, substrate and vermicompost inoculated with R. clarus; T7) vermiculite, commercial substrate and
vermicompost inoculated with C. etunicatum and T8) vermiculite, commercial substrate and vermicompost
with R. clarus and C. etunicatum.
Fig. 3. Antioxidant activity (A) and total phenolic compounds (B) obtained from fresh leaves of Curcuma longa as a function
of inoculation with Rizophagos clarus and Claroideoglomus etunicatum and different substrates.
* Means followed by the same letter in the column do not differ by Tukey test (p≤0.05), n =9
T1) did not form rhizomes, so it was not possible to extract essential oil; T2) vermiculite and soil inoculated with R. clarus;
T3) vermiculite and soil with C. etunicatum; T4) vermiculite and soil with R. clarus and C. etunicatum; T5) vermiculite,
commercial substrate and vermicompost without mycorrhizal fungi; T6) vermiculite, substrate and vermicompost inoculated
with R. clarus; T7) vermiculite, commercial substrate and vermicompost inoculated with C. etunicatum and T8) vermiculite,
commercial substrate and vermicompost with R. clarus and C. etunicatum.
Fig.4. Enzyme activity ascorbate peroxidase – APX (A), catalase – CAT (B) and superoxide
dismutase – SOD (C) obtained from fresh leaves of Curcuma longa as function to inoculation with
Rizophagos clarus and Claroideoglomus etunicatum and different substrates.
* Means followed by the same letter in the column do not differ by Tukey test (p≤0.05), n =9
T1) did not form rhizomes, so it was not possible to extract essential oil; T2) vermiculite and soil
inoculated with R. clarus; T3) vermiculite and soil with C. etunicatum; T4) vermiculite and soil
with R. clarus and C. etunicatum; T5) vermiculite, commercial substrate and vermicompost without
mycorrhizal fungi; T6) vermiculite, substrate and vermicompost inoculated with R. clarus; T7)
vermiculite, commercial substrate and vermicompost inoculated with C. etunicatum and T8)
vermiculite, commercial substrate and vermicompost with R. clarus and C. etunicatum.
Highlights

• Mycorrhizal fungi associated with substrates mainly altered compounds from the
terpene class.

• The most prominent compounds were ar-turmerone, and β- turmerone.

• Oxygenated sesquiterpenes were the main class of compounds in all the

treatments.

• The enzyme catalase had greater activity in the treatments with vermiculite and
soil substrates.
Declaration of interests

The authors declare that they have no known competing financial interestsor personal relationships
that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: