Cucumis melo) artificially inoculated with Fusarium pallidoroseum · 3 51 Introduction 52 The melon...

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2 Effect of pulsed light on postharvest disease control-related metabolomic variation in melon

3 (Cucumis melo) artificially inoculated with Fusarium pallidoroseum

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5 Short title: Pulsed light as tool to disease control in melons and its metabolomic changes

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7 Francisco Oiram Filho1; Ebenézer de Oliveira Silva2; Mônica Maria de Almeida Lopes3; Paulo

8 Riceli Vasconselos Ribeiro2; Andréia Hansen Oster2; Jhonyson Arruda Carvalho Guedes4; Patrícia

9 do Nascimento Bordallo2; Guilherme Julião Zocolo2*

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13 1Department of Chemical Engineering, Science Center, Federal University of Ceará, Fortaleza,

14 Ceará, Brazil.

15 2Multiuser Laboratory of Natural Products Chemistry, Embrapa Agroindústria Tropical, Fortaleza,

16 Ceará, Brazil.

17 3 Department of Biochemistry and Molecular Biology, Science Center, Federal University of Ceará,

18 Fortaleza, Ceará, Brazil.

19 4 Department of Analytical and Physical-Chemical Chemistry, Science Center, Federal University

20 of Ceará, Fortaleza, Ceará, Brazil.

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23 *Corresponding author

24 E-mail: [email protected] (GJZ)

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.CC-BY 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted July 10, 2019. . https://doi.org/10.1101/698407doi: bioRxiv preprint

https://doi.org/10.1101/698407

http://creativecommons.org/licenses/by/4.0/

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26 Abstract

27 Pulsed light, as a postharvest technology, is an alternative to traditional fungicides, and can be used

28 on a wide variety of fruit and vegetables for sanitization or pathogen control. In addition to these

29 applications, other effects also are detected in vegetal cells, including changes in metabolism and

30 production of secondary metabolites, which directly affect disease control response mechanisms.

31 This study aimed to evaluate the possible applications of pulsed ultraviolet light in controlling

32 postharvest rot, mainly caused by Fusarium pallidoroseum in yellow melon 'Goldex', in natura, and

33 its implications in the disease control as a function of metabolomic expression to effect fungicidal

34 or fungistatic. The dose of pulsed light (PL) that inhibited F. pallidoroseum growth in melons

35 (Cucumis melo var. Spanish) was 9 KJ m-2. Ultra performance liquid chromatography (UPLC)

36 coupled to a quadrupole time-of-flight (QTOF) mass analyzer identified 12 compounds based on

37 the MS/MS fragmentation patterns. Chemometric analysis by Principal Components Analysis

38 (PCA) and Orthogonal Partial Least Squared Discriminant Analysis (OPLS-DA and S-plot) were

39 used to evaluate the changes in fruit metabolism. PL technology provided protection against

40 postharvest disease in melons, directly inhibiting the growth of F. pallidoroseum through

41 upregulation of specific fruit biomarkers such as pipecolic acid (11), saponarin (7), and orientin (3),

42 which acted as major markers for the defense system against pathogens. PL can thus be proposed as

43 a postharvest technology to avoid chemical fungicides and may be applied to reduce the decay of

44 melon quality during its export and storage.

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51 Introduction

52 The melon (Cucumis melo L.) is a tropical fruit and is an economically crucial part of

53 Brazilian exports. However, the major fragilities in the postharvest chain of melon are incidences of

54 postharvest pathologies, particularly the rot caused by Fusarium pallidoroseum, which is

55 responsible for postharvest losses of melons. The melon is a ground plant whose fruit is in contact

56 with soil, thus facilitating its contamination with F. pallidoroseum [1]. F. pallidoroseum is a

57 widespread and common species in tropical, subtropical, and Mediterranean areas, often isolated

58 from plants with complex diseases; it is also known to be toxigenic [2].

59 Fungal disease from genus Fusarium in fruit is traditionally controlled by application of

60 synthetic fungicides that leave chemical residues, which could be harmful to the consumer, and also

61 encourage the development of fungicide-resistant strains of fungal pathogens. To overcome these

62 challenges, several alternatives or integrative approaches, including physical methods, are

63 imperative to develop fruit with increased natural defense responses through the resistance

64 mechanisms induced by abiotic stress [3].

65 Among these strategies, application of technologies using light as an abiotic factor aiming

66 to promote a regulatory and signaling role in the developmental and metabolic processes of plants is

67 encouraged [4]. Ultraviolet light (UV-C) is a continuous radiation that is widely reported in the

68 induction of resistance mechanisms and control of postharvest diseases, thus extending the shelf-life

69 of fruit and vegetables [5, 6]. However, studies involving pulsed light (PL) as radiation from the

70 perspective of inducing resistance mechanisms are still discreet [7].

71 PL is a new, non-thermal technology, where a lamp containing an inert gas such as xenon

72 emits high-frequency radiation pulses between wavelengths of 180 to 1100 nm [8]. This technology

73 is used to sanitize fruit and vegetable surfaces by acting on microorganism cells and breaking and

74 altering their DNA sequences, thus inhibiting the pathogen reproductive capacity [9]. Moreover, the

75 use of PL as a physical method can stimulate the production of phytochemicals in plant tissues to

76 minimize the possible deleterious effects caused by radiation [10].


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77 Metabolomics has been emphasized within the “omics” sciences, and efficiently evaluates

78 a large part of the metabolites of an organism both quantitatively and qualitatively, at a given time

79 and in a specific situation [11]. Metabolomics can identify a change in the concentration of

80 compounds involved in primary metabolism or production of secondary metabolites. These

81 metabolic alterations may arise from cellular lesions, metabolic adjustments to restore cellular

82 homeostasis, or synthesis and/or accumulation of metabolites resulting from cellular pathways [12].

83 The major objective of this study was to investigate the fungicidal or fungistatic effect to

84 Fusarium pallidoroseum in the melon cv. Spanish by PL treatment using ultra-performance liquid

85 chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-QTOF-MSE) and

86 chemometric tools to identify possible biomarkers associated with metabolic changes in the defense

87 mechanism.

88

89 Material and methods

90 Chemical compounds

91 Acetonitrile (PubChem CID: 6432), formic acid (PubChem CID: 284), methanol

92 (PubChem CID: 887), sodium hypochlorite (PubChem CID: 23665760), MilliQ water (PubChem

93 CID: 962), liquid nitrogen (PubChem CID: 947).

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95 Plant material

96 Melons (C. melo var. Spanish) were obtained at maturity stage (10–12 ºBrix) from a

97 commercial growing field of Norfruit Northeast Fruit, located at Mossoro-RN, Brazil (04”54’9,4”S

98 and 37”21’59,9”W). Mature melons were surface disinfected with 200 μg L-1 sodium hypochlorite

99 solution for 2 min, rinsed, and allowed to drain. The melons were then artificially inoculated.

100

101 Fruit inoculation


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102 F. pallidoroseum spore suspension was produced from pure colonies of the fungus at a

103 concentration of 106 spores mL-1. Melons were inoculated near the peduncles and adjacent regions

104 with 100 µL inoculums of F. pallidoroseum (n = 5 inoculants). After inoculation, the melons were

105 subjected to PL treatment.

106

107 Disease control by PL treatment

108 After 12 h there is already pathogen effect [13] on fruits inoculated with F. pallidoroseum.

109 Then, the fruits were irradiated in a PL chamber (XeMaticA-2LXL model, SteriBeam® GmbH,

110 Germany) equipped with two xenon flash lamps and Teflon® transparent supports that allowed the

111 melons to be uniformly exposed over 360º by both lamps with uniformity at distance of 0.07 m

112 between fruit and lamps. The lamps produced short-time pulses of 0.3 µs, delivering broad-

113 spectrum white light (200-1100 nm) with approximately 15–20 % of UV-C, according to the system

114 built-in photodiode readings. Evaluation of disease control against F. pallidoroseum was carried out

115 by dose screening, as follows: 0 (non-treated with PL), 6, 9, and 12 KJ m-2, according to the

116 standard limits proposed [14]. After PL-screening, the inoculated and PL-treated melons were

117 incubated in a box protected from light for 48 h at 28 ºC ± 1 ºC with relative humidity of 92 % to

118 ensure optimal conditions of mycelium growth, and then stored at room temperature for 21 d. After

119 this time, the occurrence of a severe degree of fungal disease was analyzed by measuring the radial

120 lesion diameter in the inoculum region using a digital pachymeter (Digimess®, São Paulo, Brazil),

121 and expressing it in meters (m) and percentage of disease incidence (%). These analyses were

122 performed using randomized design, each treatment comprised 8 replicates and data were subjected

123 to analysis of variance (ANOVA) followed by Tukey’s test at 5% probability.

124

125 Disease control promoted by suitable PL-dose against F.

126 pallidoroseum


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127 After PL-screening, an additional experiment was conducted using the suitable PL-dose

128 capable of controlling fungal disease in the fruit. To evaluate disease control, melons were

129 inoculated with F. pallidoroseum under the same inoculation conditions described in Fruit

130 inoculation section. Inoculated melons were then irradiated with a PL-dose of 9 KJ m-2 in the same

131 instrumental conditions described in Disease control by PL treatment section. The control group

132 included inoculated and non-inoculated melons. After 96 h, biological triplicates of each treatment

133 were subjected to extraction processes for injection into a UPLC system.

134

135 Extract preparation

136 Extracts were obtained Moore, Farrant (15] with modifications. The pellets were extracted

137 from the different adjacent regions (0.005 m) to the inoculum with thickness approximately of rind

138 melon. Peel melon powder (1.00 g) was resuspended in 4 mL of MeOH/H2O (7:3 v/v). The

139 homogenate was then subjected to sonication (Ultracelaner 1450, Unique®, Brazil) for 30 min,

140 followed by centrifugation at 6,000 × g, for 5 min. The pellet was re-extracted twice using 3 mL of

141 MeOH/H2O (7:3 v/v) using the same conditions of ultrasound and centrifugation. The supernatant

142 was filtered through a 0.22 μm polytetrafluoroethylene membrane (PTFE) (Biotechla®, Bulgaria)

143 and injected directly into a UPLC system.

144

145 Chromatographic analysis by UPLC-QTOF-MSE

146 The analyses were accomplished on an Acquity UPLC (Waters, USA) system coupled to a

147 Xevo Quadrupole and Time-of-Flight mass spectrometer (Q-TOF, Waters). Separations were

148 performed on a C18 column (Waters Acquity® UPLC C18 - 150 mm × 2.1 mm, 1.7 μm). For

149 metabolic fingerprinting, a 2-μL aliquot of phenolic extract was subjected to UPLC using an

150 exploratory gradient with the mobile phase composed of deionized water (A) and acetonitrile (B),

151 both containing formic acid (0.1% v/v). The phenolic extracts from melons were subjected to


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152 exploratory gradient as follows: 2–95% for 15 min, with a flow rate of 500 µL min-1. Ionization was

153 performed with an electrospray ionization source in negative mode ESI, acquired in the range of

154 110–1200 Da. The optimized instrumental parameters were as follows: capillary voltage at -2800 V,

155 cone voltage at -40 V, source temperature at 120ºC, desolvation temperature at 330ºC, flow cone

156 gas at 20 L h-1, desolvation gas flow at 600 L h-1, and the microchannel voltage of the plate (MCP) -

157 detector at -1900 V. The mode of acquisition was MSE and system was controlled using MassLynx

158 4.1 software (Waters Corporation). The extracts were injected in triplicate.

159

160 Statistical analysis

161 The UPLC-MS data were processed using MassLynx® software (Waters Co., Milford,

162 MA, USA), under the following conditions: retention time deviation tolerance ± 0.05 min, mass

163 range 110–1200 Da (accurate mass tolerance of ± 0.05 Da) and noise elimination level 5. For

164 structural identification of metabolites, molecular formulas were considered and the values for m/z

165 were obtained from high-resolution spectra observed in the chromatogram at higher intensity. The

166 relative error is given in ppm for each formula. Margins of error less than 10 ppm are considered for

167 studies in MS/MS.

168 The structural proposals of molecules were performed using MS/MS data through the

169 establishment of rational fragmentation patterns reported in literature [16-19]. A list of identities of

170 peaks was created using the retention time (rt) and error (m/z). For unidentified peaks, all possible

171 molecular formulas were derived (elements C, H, N, and O, with the tolerance of 10 ppm, at least 2

172 carbon atoms) using the elemental composition tools available in MassLynx® software.

173 The UPLC-MS data analyzed by chemometrics were processed using Markerlynx®

174 software for Principal Components Analysis (PCA) and Orthogonal Partial Least Squared

175 Discriminant Analysis (OPLS-DA and S-plot). S-plots were obtained via OPLS-DA analysis to

176 determine potential biomarkers that significantly contributed to the difference among groups [20-

177 23].


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178

179 Results and discussion

180

181 The growth of F. pallidoroseum under PL treatment

182 The lesion diameter of fungal infection (Fig 1) and percentage of disease incidence (data

183 not shown) were analyzed to determine the suitable PL-dose of treatment for the control or

184 inhibition of F. pallidoroseum in melon fruit. The growth of the pathogen on melon fruit inoculated

185 with P. pallidoroseum was strongly inhibited by PL treatment (Fig 1). This is corroborated as

186 shown in Fig 1, by a mean lesion diameter of 0.013 m, with a 100% incidence of fungal disease in

187 melons without PL radiation.

188

189 Fig 1. Screening of PL doses to evaluate the lesion diameter (m) in melon inoculated with F.

190 pallidoroseum. Mean values followed by the same small letter did not differ significantly between

191 PL-treatments, by Tukey’s test at 5% probability.

192

193 Disease progression in the PL-treated group that received 6 KJ m-2 was significantly lower

194 than that in the untreated group, with a significant reduction in lesion diameter (0.007 m) (Fig 1)

195 and 62.5% incidence of fungal disease. Moreover, a dose of 9 KJ m-2 was more effective than all

196 the doses employed and was associated with a low mean lesion diameter of 0.004 m, (Fig 1), and

197 33.3% disease incidence by F. pallidoroseum. The hormesis concept was used to explain the

198 behavior with a dose radiation of 9 KJ m-2.

199 Hormesis is a phenomenon in which low levels of potentially damaging radiation elicit

200 beneficial responses, i.e. physiological stimulation of beneficial responses in plants by low levels of

201 stressors that otherwise elicit harmful responses. Hormetic doses of ultraviolet light (UV-C)

202 radiation are involved in plant susceptibility towards diseases, and are capable of elicit plant


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203 resistance mechanisms including production of anti-fungal compounds such as specific phenolics

204 including phytoalexins that act both as light quenchers that absorb damaging wavelengths of light,

205 and antioxidants that prevent reactive oxygen species (ROS) mediated cellular damage [7, 24, 25].

206 The UV-C radiation might also have fungistatic effect promoted by phenolic compounds which

207 plays a barrier role physically against pathogens attacks and the same barrier reduce water and

208 nutrients diffusion important to the growth pathogen [26].

209 However, a recent study compared the application of low-intensity UV-C and high-

210 intensity PL sources as elicitors of hormesis in tomato fruit (Solanum lycopersicum cv. Mecano)

211 [7]. Curiously, these authors showed that postharvest hormetic treatment with 16 pulses of PL (7.4

212 KJ m-2) with a spectral range (240 – 1050 nm) significantly delayed ripening along with inducing

213 disease resistance to Botrytis cinerea in tomato fruit with 41.7% reduction in disease progression

214 compared to 38.1% reduction with the conventional low-intensity UV-C (254 nm) at 0.37 KJ m-2.

215 Thus, according to the authors, PL treatment, although rich in UV-C (broader spectral output),

216 elicited the same pathways or responses as hormesis induced by conventional low UV sources

217 (narrower spectral range), making the PL-treatment more commercially attractive, as this treatment

218 allows a substantial reduction in treatment time from seconds to microseconds.

219 The last dose applied, 12 KJ m-2, corresponded to a disease incidence statistically equal to

220 the treatment with 9 KJ m-2, but showed a mean lesion diameter twice large (0.008 m) compared to

221 that in treatment with 9 KJ m-2 (Fig 1). These results showed a possible damaging effect on melon,

222 where excess of PL can inhibit the fruit defenses against fungal disease, and that the dose of 12 KJ

223 m-2 stipulated by the [14] was limiting to the conditions assessed.

224 Based on these results, we can hypothesize that the PL-treatment with 9 KJ m-2 applied

225 here acted as a fungistatic factor inhibiting the mycelial growth of F. pallidoroseum and that this

226 behavior is linked to a probable hormetic effect associated with the induction of metabolites

227 synthesis, which possibly activated defense responses against this fungal disease in melon cv.

228 Spanish. For this reason, 9 KJ m-2 was chosen for identification of the metabolites produced using a


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229 UPLC system and chemometric tools to associate the identification of specific metabolites with

230 disease control mechanism in melon infected with F. pallidoroseum.

231

232 Putative metabolite identification by UPLC-QTOF-MS

233 Initially, infected and uninfected melon samples were evaluated in the positive and

234 negative ionization modes. Finally, the negative ionization mode was chosen because it presents a

235 metabolomic profile with a higher number of compounds at higher intensities, and is more selective

236 and sensitive.

237 The chemical profile of melon samples was established by analyzing the negative mode

238 (ESI-) chromatograms, Fig 2, together with mass spectra. The peaks were numbered according to

239 their elution order, the compounds were tentatively identified by interpretation of their MS and

240 MS/MS spectra determined by QTOF-MS, along with data from literature and open-access mass-

241 spectra databases as MassLynx®. Table 1 lists the MS data of tentatively identified compounds,

242 including experimental and calculated m/z for the molecular formula, error, and the fragments

243 obtained by MS/MS, as well as the proposed compound for each peak. In general, 12 metabolites of

244 distinct chemical classes, organic acids, non-protein amino acids, and phenolics, have been

245 tentatively identified.

246

247 Fig 2. Base-peak chromatogram of melon inoculated with F. pallidoroseum and subjected to PL-

248 treatment with 9 KJ m-2.

249


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250 Table 1. Secondary metabolites tentatively identified by UPLC-QTOF-ESI-MSE in melons treated with PL.Positive ion mode Negative ion mode

Peaktr

(min) [M+H]+

Observed

[M+H]+

Calculated

Error

(ppm)

[M-H]-

Observed

[M-H]-

CalculatedMS/MS

Error

(ppm)

Molecular

formulaCompounds

1 3.16 295.1405 295.1393 4.1 293.1232 293.1236 131.0708 -1.4 C12H22O8Hydroxybutanoic acid ethyl

ester-hexoside

2 3.58 611.1619 611.1612 1.1 609.1454 609.1456

489.1016

429.0793

309.0857-0.3 C27H30O16 Isoorientin-2”- O-glucoside

3 3.78 449.1066 449.1084 -4.0 447.0936 447.0927

357.0532

327.0522

285.01972.0 C21H20O11 Orientin

4 3.96 595.1645 595.1663 -3.0 593.1486 593.1506413.0811

293.0408 -3.4 C27H30O15 4′′ -O-glucosylvitexin

5 4.11 625.1752 625.1769 -2.7 623.1591 623.1612443.1021

323.0592 3.4 C28H32O16 Isoscoparin 2″-O-glucoside

6 4.23 433.1118 433.1135 -3.9 431.0968 431.0978341.0652

311.0542 -2.3 C21H20O10 Vitexin

7 4.28 595.1641 595.1663 -3.7 593.1498 593.1506

503.1073

473.9605

341.0679-1.3 C27H30O15 Isovitexin-7”- O-glucoside (Saponarin)

8 4.40 463.1236 463.1240 -0.9 461.1052 461.1084

371.0816

341.0660

299.0436-2.8 C22H22O11 Diosmetin-6-C-glucoside

9 4.49 801.2205 801.2242 -4.6 799.2114 799.2086461.1159

341.0681 3.5 C38H40O19 Isoscoparin 7-O-[6′′-feruloyl]-glucoside


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10 4.59 771.2106 771.2136 -3.9 769.1970 769.1980

623.1699

443.0993

413.0831-1.3 C37H38O18

Isoscoparin-2”-O-(6’’’-ρ-coumaroyl)-

glucoside

11 4.76 130.0866 130.0868 -1.5 128.0710 128.0712 84.0796 -1.6 C6H11NO2 Pipecolic acid*

12 6.62 273.0753 273.0763 -3.7 271.0605 271.0606

177.0180

151.0026

119.0489-0.4 C15H12O5 Naringerin

251 * Compound tentatively identified in negative and positive ionization mode, with the MS/MS ion in positive mode.252


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253 The peak 1 (tr = 3.16 min) was tentatively identified as hydroxybutanoic acid

254 ethyl ester-hexoside. This compound showed the ion m/z 293.1232 [M-H]- in MS and

255 the fragment ion m/z 131.0708 [M-H-162]- in MS/MS, indicating a pattern loss of the

256 hexoside moiety [27].

257 The mass spectrum of peak 2 (tr = 3.58 min) showed the precursor ion m/z

258 609.1454 [M-H]-. The MS/MS spectrum showed fragment ions m/z 489.1016 [M-H-

259 120]-, 429.0793 [M-H-180]-, and 309.0857 [M-H-180-120]-. The losses of 180 and 120

260 u are significant for diglucosides like sophoroside (1-2 linkages of two glucose

261 molecules). Through the correlation of ions observed in MS and MS/MS, the compound

262 was tentatively identified as luteolin-6-C-glucosyl-2"-O-glucoside, also known as

263 isoorientin-2"-O-glucoside [28].

264 The mass spectrum of peak 3 (tr = 3.78 min) presents the precursor ion m/z

265 447.0936 [M-H]- that exhibited fragments ions m/z 357.0532 [M-H-90]-, 327.0522 [M-

266 H-120]-, and 285.0197 [M-H-162]-. The fragment ion m/z 285.0197 [M-H-162]- is the

267 aglycone formed from the loss of the glycosidic group (Table 1 and Fig. 2.) [29]. Thus,

268 the compound was tentatively identified as 8-C-glucosyl luteolin, also known as

269 orientin [28].

270 The compound 4 (tr = 3.96 min) showed in the mass spectrum as the precursor

271 ion m/z 593.1486 [M-H]-, also exhibiting the fragment ions m/z 413.0811 [M-H-180]-

272 and 293.0408 [(aglycone+41)-18)]-, which are characteristic of flavone O-glucosyl-C-

273 glucoside, indicating the presence of sophoroside, and apigenin as aglycone. This

274 compound was characterized as apigenin-6-C-glucosyl-2”-O-glucoside, also known as

275 4’’-O-glucosylvitexin or isovitexin-2”-O-glucoside [28, 30].

276 The peak 5 (tr = 4.11 min) showed in the mass spectrum a precursor ion m/z

277 623.1591 [M-H]- and its respective fragment ion m/z 443.1021 [M-H-162+18]-, which


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278 suggested loss of a glucose moiety and one unit of water and the fragment ion m/z

279 323.0592 [M-H-120]-. These patterns of fragmentation indicate the a presence of a

280 diglucoside linkage and thus, compound 5 was tentatively identified as isoscoparin 2”-

281 O-glucoside [31].

282 The peak 6 (tr = 4.23 min), presented in the mass spectrum the precursor ion

283 m/z 431.0968 [M-H]-. In the MS/MS spectrum, it showed the fragments ions m/z

284 341.0652 [M-H-90]- and 311.0542 [M-H-120]-, indicating the presence of hexose as the

285 monosaccharide and apigenin as aglycone. Therefore, the compound was tentatively

286 identified as 8-C-glucosyl apigenin, also known as vitexin [28].

287 The mass spectrum of peak 7 (tr = 4.28 min) showed the ion m/z 593.1498 [M-

288 H]- and the fragment ions m/z 503.1073 [M-H-90]- and 473.9605 [M-H-120]-. This

289 pattern of loss indicates the presence of a diglucoside similar to that in compound 5 and

290 m/z 341.0679 [M-H-120-132]-, the loss of 132 Da represents a pentoside. Thus, the

291 compound was tentatively identified as isovitexin-7”-O-glucoside, also known as

292 saponarin. [32].

293 Peak 8 (tr = 4.40 min) in the mass spectrum showed the precursor ion m/z

294 461.1052 [M-H]- and its fragments m/z 371.0816 [M-H-90]-, 341.0660 [M-H-120]-, and

295 m/z 299.0436 [M-H-162]-. The fragment ion 299.0436 [M-H-162]- represents the

296 aglycone, formed by the loss of a glucoside moiety. Thus, the compound was

297 characterized as diosmetin-6-C-glucoside [33]

298 The peak 9 (tr = 4.49 min) represents compound 9 with a mass spectrum

299 showing the ion m/z 799.2114 [M-H]- and its fragment m/z 461.1159 [M-H-338]-

300 representing a loss of feruloyl plus a glucoside moiety and m/z 341.0681 [M-H-feruloyl-

301 Glucoside-120]-.Thus, based on fragmentation, the metabolite was tentatively identified

302 as isoscoparin 7-O-[6′′-feruloyl]-glucoside [31]


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303 The peak 10 (tr = 4.59 min) presents in the mass spectrum, the ion m/z

304 769.1970 [M-H]- and its fragments m/z 623.1699 [M-H-146]- showing a loss of the

305 coumaroyl moiety; the fragment ion m/z 443.0993 [M-H-coumaroyl-162-18]- is the

306 result of successive losses of coumaroyl, glucoside, and a unit of water. The correlation

307 of the ions observed indicates that the metabolite in question is isoscoparin-2”-O-(6′′′-p-

308 coumaroyl)-glucoside [4]

309 Negative and positive ionization modes were used to identify pipecolic acid, a

310 non-protein amino acid (homolog of proline). Therefore, the peak 11 (tr = 4.76 min)

311 showed the ions m/z 128.0710 [M-H]- and m/z 130.0866 [M+H]+ in the MS in the

312 negative and positive ionization modes, respectively. Corroborating with chemical

313 identification, the fragment ion m/z 84.0796 [M+H-COOH]+ was observed in the

314 positive mode, referring to an aromatic core obtained from the cleavage of the carboxyl

315 group (Table 1 and Fig 2) [34].

316 In peak 12 (tr = 6.62 min) the ion m/z 271.0605 [M-H]- was observed with a

317 fragmentation pattern in MS/MS showing the loss of ring B at m/z 177.0180 and by

318 Retro Diels-Alder at m/z 151.0026 and m/z 119.0489. Thus, it was tentatively identified

319 as flavanone naringenin [35, 36].

320

321 Chemometric analysis

322 Principal component analysis (PCA) is a multivariate data analysis method that

323 can synthesize data from an original matrix with many variables in the set of smaller

324 orthogonal variables [37, 38]. We confirm that the chemometric analyses were centered

325 in PL-treatment with a dose of 9 KJ m-2, considered here as treatment for disease control

326 in melon inoculated with F. pallidoserum (Fig 1).


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327 Therefore, PCA-2D was performed to discriminate different treatments groups

328 according to their metabolic profiles represented by retention time and the mass-to-

329 charge ratio (rt-m/z) from the UPLC-QTOF-MSE analysis (Fig 3). The PCA-2D showed

330 perfect separation of all groups evaluated with 89% of the total cumulative variance in

331 the diaxial axes PC1 and PC2 (R2X[1] = 0.7401 and R2X[2] = 0.1571), with a data noise

332 level of 6%, showing a robust model regarding data certainty. The formation of groups

333 was related to the similarity between biological triplicates.

334

335 Fig. 3. Discrimination of treatment groups by PCA-2D.

336

337 The PCA-2D in the PC1 showed the inoculated group (negative scores) and

338 non-inoculated group (positive scores); whereas in the PC2 (positive scores),

339 discrimination of the inoculated control and non-PL-treated group was observed. PC2

340 (negative scores) also showed the separation of inoculated PL-treated and non-

341 inoculated groups (Fig 3). The separation of negative and positive groups in PC1 and

342 PC2 is clearly linked to the differences of each metabolomic profile [39]. For this

343 reason, OPLS-DA chemometric analysis was applied to the UPLC-QTOF-MSE data to

344 compare samples according to metabolites that influenced on disease control against F.

345 pallidoroseum in melons treated with PL. OPLS-DA is an analysis method used to

346 study ions that contribute to experimental sample classification, and the classification

347 between two groups in the OPLS-DA model can be visualized in the form of a score

348 chart and scatter plot (S-plot).

349 Fig 4A summarizes the separation between non-inoculated and non-treated

350 fruit (control) and non-inoculated fruit treated with PL (9 KJ m-2), with respect to

351 metabolic responses in the function of PL as abiotic stress, through OPLS-DA


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352 (R2X(cum) = 0.9232). The S-Plot generated from OPLS-DA, with VIP > 1.0 and p <

353 0.05 showed potential biomarkers between the treatments evaluated.

354

355 Fig 4 OPLS-DA, S-Plot graphs, and intensity of biomarkers between non-inoculated

356 control and non-inoculated and PL-treated melons (9 KJ m-2) (A). OPLS-DA, S-Plot

357 graphs, and intensity of markers between inoculated control and inoculated and PL-

358 treated melons (9 KJ m-2) (B).

359

360 The control group showed synthesis of principal secondary metabolites such

361 as naringenin (peak 12), hydroxybutanoic acid ethyl ester-hexoside (1), isoorientin-2”-

362 O-glucoside (2), orientin (3), and 4”-O-glucosylvitexin (4). However, an upregulation

363 of naringenin (12) and hydroxybutanoic acid ethyl ester-hexoside (1) was highlighted

364 (Fig 4A). Naringenin is a flavonoid that serves as a primer for more advanced flavonoid

365 structures and as a substrate for glycosylation reactions. Hydroxybutanoic acid ethyl

366 ester-hexoside is a compound has been previously described in other varieties of melon

367 such as Piel de Sapo, Galia, and Cantaloupe [27]. Interestingly, naringenin (12) and

368 hydroxybutanoic acid ethyl ester-hexoside (1) were downregulated in melons treated

369 with hormetic PL-radiation, whereas specific flavonoid compounds such as orientin (3),

370 4”-O-glucosylvitexin (4), and isoorientin-2”-O-glucoside (2) were up-regulated (Fig

371 4A), thus these compound which were up-regulated might be considered as biomarkers

372 caused by PL-treatment. Accumulation of flavonoid precursors such as phenylalanine

373 ammonia lyase into vacuoles present in the epidermal and subepidermal mesophyll

374 tissues of fruit stimulate plant defense mechanisms under specific PL-radiation

375 conditions [24]. Many phenylpropanoids have been associated with induced disease

376 resistance and disease control. According to Jung et al. (2013) the flavonoids orientin,


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377 isoorientin-2”-O-glucoside, and 4”-O-glucosylvitexin are secondary metabolites

378 involved in antioxidant activities against abiotic stress in rice leaves (Oryza sativa cv.

379 Ilmi) exposed to different conditions of LED light radiation.

380 The differences in inoculated (control) and inoculated plus PL-treated (9 KJ m-

381 2) melons are shown in Fig 4B. Separation of the two treatments groups by the OPLS-

382 DA graph (R2X(cum) = 0.8882) indicated the differences among groups according to

383 their chemical profiles. The S-Plot obtained from the OPLS-DA graph, demonstrated

384 different potential biomarkers between groups for VIP > 1.0. Inoculation of F.

385 pallidoroseum mainly induced the synthesis of glycosylated flavonoids such as

386 diosmetin-6-C-glucoside (8), isoorientin-2"-O-glucoside (2), and 4’’-O-glucosylvitexin

387 (4) (Fig 4B), and this behavior is due to the presence of flavonoids at the infection site

388 that are responsible for defense mechanisms against pathogens [40]. However, these

389 metabolites which are a natural response of fruit against de pathogen were not sufficient

390 to control the growth pathogen as shown by control treatment at Figure 1.

391 Biotic and abiotic stress might regulate systemically the defense mechanism by

392 induce systemic resistance (ISR) or systemic acquired resistance (SAR) [41, 42]. PL-

393 treatment and inoculation with F. pallidoroseum led by SAR to the upregulation of two

394 major biomarkers in melon, pipecolic acid (11) and orientin (3) (Fig 4B), indicating a

395 change in the metabolic pathway, where the fruit preferentially used these specific

396 compounds as phytochemical markers in response to the treatment [43]. The presence of

397 pipecolic acid was verified in both groups that were inoculated with F. pallidoroseum.

398 The presence of pipecolic acid in pathogen inoculation sites is associated with plant

399 defense responses and acts as a regulator of inducible plant immunity [44]. Thus, when

400 the PL-treatment is applied the pipecolic acid together with orientin is up-regulated

401 significantly achieving disease control (fungistatic effect) which also seen at Figure 1 on


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402 treatment 9 KJ.m2. Curiously, pipecolic acid is the supposed precursor of betaines that

403 are accumulated in the cytoplasm as a result of physiological plant responses to stress

404 phenomena induced in a large part by adverse environmental conditions. These

405 compounds that are biochemically inert in the cell are synthesized from some specific

406 amino acids such as serine, alanine, methionine, the non-protein amino acid γ-

407 aminobutyric acid, and some cyclic amino acids such as proline and pipecolic acid.

408 Biosynthesis of betaines in the cytoplasm under abiotic stress conditions is mainly due

409 to the action of methyltransferases, which utilize S-adenosylmethionine as a methyl

410 group donor [43].

411 Fig 5A shows the results of the OPLS-DA graph (R2X(cum) = 0.9684) between

412 the inoculated and non-inoculated groups in the function of F. pallidoroseum as a biotic

413 stress. The S-Plot originating from OPLS-DA data demonstrated potential biomarkers in

414 each group, with values for VIP > 1.0 and p < 0.05.

415

416 Fig 5. OPLS-DA, S-Plot graphs, and intensity of biomarkers between non-inoculated

417 and inoculated melons (A). OPLS-DA, S-Plot graphs, and intensity of markers between

418 treated non-inoculated (9 KJ m-2) and PL-treated inoculated melons (9 KJ m-2) (B).

419

420 The number of compounds up-regulated in non-inoculated melons is much

421 higher when that compared to inoculated melons, highlighting naringenin (12),

422 hydroxybutanoic acid ethyl ester-hexoside (1) and orientin (3) (Fig 5A). However,

423 saponarin (7) was a biomarker present in group inoculated control melons and

424 isoorientin-2"-O-glucoside (2) shows a little up-regulation on the same group (Fig 5A).

425 Hydroxybutanoic acid ethyl ester-hexoside is a compound linked to amino acid groups

426 and findings have indicated significant interconnections between different branches of


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427 amino acid metabolism and plant resistance to pathogens [45, 46]. Naringenin is a

428 flavonoid showing metabolic activity associated with barley resistance to Fusarium

429 graminearum [47].

430 The results observed in Fig. 5B show the separation of distinct groups, where

431 the OPLS-DA plot (R2X(cum) = 0.9798) presents differences between the non-

432 inoculated fruit and inoculated plus PL-treatment. The S-Plot obtained by the OPLS-DA

433 graph points out the potential markers for each group. Upregulation of flavonoids such

434 as naringenin (12), orientin (3) and 4”-O-glucosylvitexin (4) in non-inoculated PL-

435 treated fruit (9 KJ m-2) is much higher when compared to the inoculated and PL-treated

436 (9 KJ m-2) group (Fig. 5B). The synthesis of flavonoids in detriment to abiotic stress

437 observed in PL treatment occurs due to a change in the metabolic pathway of fruit in

438 response to the medium [48]. On the other hand, inoculated and PL-treated melons

439 accumulated a glycosylated flavonoid identified as saponarin (7), and pipecolic acid

440 (11) as biomarkers [49]. Nevertheless, the presence of pipecolic acid on non-inoculated

441 and PL-treated (9 KJ m-2) group (Fig 5B) shows that this metabolite might be

442 synthetized by abiotic stress also. Saponarin is an antioxidant belonging to the flavones

443 and is to inhibiting malonaldehyde formation in barley. In a normal reaction,

444 malonaldehyde is formed from oxidized lipids on the skin surface in barley leaves by

445 UV irradiation [50]. The effect of PL treatment in melons inoculated with fungi resulted

446 in a response mediated by the synthesis of pipecolic acid in the cellular medium, which

447 resulted in increased levels of betaines in the cell, that induce the fruit immunity system.

448

449 Conclusions450


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451 In the present study, a PL-dose of 9 KJ m-2 in melon inoculated with Fusarium

452 pallidoroseum controlled the disease promoted by this pathogen (fungistatic effect), and

453 induced metabolic variation in the fruit defense system. Pipecolic acid (11) and orientin

454 (3) were the two major biomarkers associated with postharvest disease control against

455 F. pallidoroseum in infected melons treated with a pulsed radiation. This study also

456 showed that fruit subjected to biotic and abiotic stresses, separately, demonstrated

457 different metabolic responses, with a chemical profile in response to each stress. In

458 addiction the compounds orientin (3), 4”-O-glucosylvitexin (4), and isoorientin-2”-O-

459 glucoside (2) are the metabolites in response only PL-treatment. Our findings highlight

460 that the application of PL technology provided control against the postharvest disease of

461 melon cv. Spanish by directly controlling the growth of F. pallidoroseum through the

462 synthesis/up-regulation of specific compounds that acted as principal biomarkers of the

463 defense system against pathogen. Thus, PL can be readily proposed as a new

464 postharvest technology alternative to chemical fungicides and may be applied to reduce

465 the delay caused by F. pallidoroseum in melons var. Spanish during their export and

466 storage.

467

468 Acknowledgments

469 The authors are gratefully acknowledged for support from the Brazilian

470 Company of Agricultural Research (EMBRAPA), National Council for Scientific and

471 Technological Development (CNPq), National Institute of Science and Technology

472 (INCT BioNat, Brazil) and commercial growing field of Norfruit Northeast Fruit.

473

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Cucumis melo) artificially inoculated with Fusarium pallidoroseum · 3 51 Introduction 52 The melon...

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