1 PosFruit as an alternative to sodium hypochlorite for post-harvest disinfection of

2 citrus fruit

4 Zamuner, C. F. C.1; Dilarri, G.1; Cavalca, L. B.1; Behlau, F.2; da Silva, T. G.2; Bacci, Jr.

5 M.3; Ferreira, H. 1*

7 1 Departamento de Bioquímica e Microbiologia, Instituto de Biociências, Universidade

8 Estadual Paulista, Av. 24A, 1515, Rio Claro, SP, 13506-900, Brazil

9 2 Department of Research & Development, Fundo de Defesa da Citricultura

10 (Fundecitrus), Araraquara, Sao Paulo 14.807-040, Brazil

11 3 Centro de Estudos de Inseto Sociais (CEIS), Instituto de Biociências, Universidade

12 Estadual Paulista, Av. 24A, 1515, Rio Claro, SP, 13506-900, Brazil

13

14 Keywords: Citrus canker, fresh fruit, sanitization

15 Running title: Sanitization of Tahiti limes against X. citri

16

17

18 * corresponding author

19 Tel. +55 19 3526 4187; e-mail address: henrique.ferreira@linacre.oxon.org

20
21

22 Abstract

23

24 1. Introduction

25 Brazil is considered the largest producer of orange juice and second largest producer of

26 citrus fruit in the world, being surpassed only by China. Citriculture is one of the major

27 economic activities in Brazil, generating a revenue of more than US$ 2 billion/year in

28 international trades. Moreover, the citrus industry generates thousands of direct and

29 indirect job positions, thus playing an important social role in the country (Neves, Trombin

30 et al., 2010). The most profitable market within citriculture is the fresh fruit production, in

31 which the product has a high aggregated value and comprised about 20% of the citrus

32 production in Brazil. Within the most cultivated varieties, limes and lemons occupy 5% of

33 the market (Moretti, 2007). However, fruit production and quality are constantly

34 threatened by crop plagues and diseases. Amongst them, a great focus has been given

35 to citrus canker, a disease caused by the Gram-negative bacteria Xanthomonas citri

36 subsp. citri (X. citri). Citrus canker affects all commercial citrus cultivars at different levels

37 and the only viable recuperation is through management (Ference, Gochez et al., 2018,

38 Gottwald, Graham et al., 2002). This bacterial disease has a great dispersion potential

39 through the combined action of wind and rain, and can be introduced in new areas due

40 to contaminated vegetal parts and fruit transport. In this context, the post-harvest

41 sanitization of fruit before their distribution is extremely important.

42 In Brazil, the Normative Instruction MAPA 21 (IN21) published by the ministry of

43 agriculture on April 25, 2018 (Brazil, 2018) began to divide citrus areas in different groups,
44 accordingly with citrus canker incidence and regulates the integrated management

45 strategy in spite of the eradication of plants. In the article 49, sodium hypochlorite is

46 recommended as a sanitizing agent to be used in fruit processing. Upon arriving in the

47 packing house, all fresh fruit harvested from orchards where citrus canker is endemic,

48 should be immersed in a 200 ppm (0,2% v/v) sodium hypochlorite solution at pH 7 for 2

49 minutes. A number of sanitizing products are known and allowed to be used in other

50 countries. For instance, in the United States, the Food and Drug Administration (FDA)

51 approve the use of chlorine dioxide, hydrogen peroxide, peracetic acid and ozone,

52 beyond sodium hypochlorite. In spite of other products, sodium hypochlorite is the only

53 product allowed in Brazil due to a number of researches in relation to its efficacy for

54 sanitizing several cultivars in post-harvest processing and low cost (Moretti, 2007).

55 Furthermore, sodium hypochlorite is considered generally safe, being used for nearly a

56 century as a disinfectant for water supplies (Mishra, Abrol et al., 2018). Nevertheless,

57 some concerns are associated with the used of hypochlorite solutions as they may result

58 in the formation of trihalomethanes that are considered carcinogens, as well as other

59 noxious compounds due to the reaction with organic matter (Richardson, Thruston et al.,

60 2000). In addition, chlorine is a volatile substance that loses its effectiveness as it binds

61 to the organic material (soil, organic matter and other debris from the harvest) becoming

62 necessary to be constantly adjusted in solution (topping off) during the sanitization

63 process, which may mask off the cost associated with its use. Moreover, it has a corrosive

64 effect on industrial equipment and may also damage fruit skin. The use of chlorine-based

65 products may cause environmental damage and risks to packing-house workers as it can

66 cause several damages to eyes and skin (Gil, Selma et al., 2009, Mishra et al., 2018,
67 Moretti, 2007) . Finally, the potential generation of harmful compounds due to the

68 interaction with the organic matter from harvest, may cause fruit refusal in the

69 international market (European Union Commission of Health and Food Safety).

70 In this scenario, although the IN21 implies the use of sodium hypochlorite in Brazil, it

71 also allows the use of other substances that are officially recognized as alternative fruit

72 disinfectants, provided that their efficacy and safety are scientifically demonstrated

73 (article 49, II paragraph). Recently a product named PosFruit was developed based on

74 vegetable oils and organic acids, which has a great potential to be used as a sanitizing

75 agent, once a number of research shows antifungal and antibacterial activity associated

76 with this substance and their derivatives (Chang, Chen et al., 2001, Friedman, Henika et

77 al., 2002, Lee & Ahn, 1998, Shreaz, Wani et al., 2016).

78 In this work, we evaluated the PosFruit action as a sanitizer for citric fruit (Tahiti limes)

79 as an alternative to sodium hypochlorite. We show here that PosFruit has bactericidal

80 action against X. citri. Moreover, we could not detect natural resistance to PosFruit, and

81 longer exposures to the product did not induce resistance in X. citri either. We also

82 show that PosFruit was as effective as hypochlorite to sanitize Tahiti limes artificially

83 contaminated with X. citri. Finally, we tested the sanitization efficacy of PosFruit in an

84 automated sanitization line and observed a reduction of 4 log10 in CFU/mL recovery of

85 X. citri.

86

87 2. Material and methods

88 2.1 Sanitizing agents

 89 The agricultural adjuvant PosFruit (Arvensis; Zaragoza, Spain) and Pluron 444A

90 (positive control) were used as sanitizing agents. PosFruit is a mix of organic acids and

91 vegetable oils, and was obtained from Aurora Agroespecialidades LTDA (Paulínia, São

92 Paulo – Brazil) - Lot number 3690006, 5L pack, manufactured in August 2017 (expire

93 date: August, 2020). Pluron 444A (containing sodium hypochlorite as the principal

94 agent) was produced by the company Mustangpluron (Catanduva, São Paulo, Brazil) -

95 Lot 2138, 5L pack. All experiments were performed from February until August 2018.

96

97 2.2 Bacterial strain and growth conditions

98 The Xanthomonas citri subp. citri strain used was the isolate 306 (IBSBF 1594)

99 (Schaad, Postnikova et al., 2006). Before being exposed to the sanitizing agents,

100 bacteria were cultivated for 16h in NYG/NYG-agar medium (nitrogen-yeast-glycerol: 5

101 g/L of peptone, 3 g/L of yeast extract, 2% glycerol; for solid medium bacterial agar was

102 added to 15 g/L) at 29 oC.

103

104 2.3 Sensitivity assays

105 Liquid cultures had their Optical Densities (O.D. 600 nm) adjusted to 0.3 using fresh

106 NYG medium. Subsequently, cell suspensions were diluted 100x with fresh medium to

107 make test cultures of 5 mL, each containing 106 cells.mL-1. The product PosFruit was

108 added to the test cultures in order to give the final concentrations of 0.0625%, 0.125%,

109 0.25%, 0.5%, and 1% (v/v). As positive control, cells were exposed to Kanamycin at 20

110 µg.mL-1. All treatments were kept at 29 oC for 4 hours and 200 rpm and after incubation,

111 samples of 100 µL of each treatment were spread onto NYG-agar plates for CFU
112 counting. The absence of growth after 96 h incubation indicated bactericidal activity.

113 The minimal inhibitory concentration (MIC) was defined as the lowest concentration of

114 compound where no colonies were detected.

115

116 2.4 Sanitizing tests

117 2.4.1 Preparation of the bacterial cell suspension and fruit

118 Bacteria were inoculated from plate in 250 mL Erlenmeyer flasks containing 100 mL of

119 NYG and incubated at 29 oC under shaking (200 rpm) until cultures reached the O.D.

120 600 nm of 0.3. After incubation, the cultures were centrifuged at 6000 xg for 7 minutes

121 and the supernatant dispensed. Cells were resuspended in phosphate buffered saline

122 (1X PBS; 137 mM NaCl, 2,7 mM KCl, 10 mM Na2HPO4, 1,8 mM KH2PO4; pH 7,4) and

123 the O.D. 600 nm was again adjusted to 0,3 (108 cells/mL). Tahiti limes were selected to

124 have similar sizes of approximately 5 cm in diameter measured in the cross-section

125 perpendicular to the longer axis of the fruit.

126

127 2.4.2 Manual sanitization process

128 Before the tests, fruit were washed with filter-sterilized tap water to remove impurities

129 from the harvest and dried at room temperature overnight. A total of 15 Tahiti limes,

130 divided in 5 groups (five replicates) were spray inoculated until the run-off point using

131 the X. citri cell suspension mentioned in 2.4.1, and let to dry at room temperature (~21

132 oC) for approximately 6 h. Fruit contaminated with X. citri were sprayed with PosFruit at

133 the concentrations of 1%, 2% and 5% until the run-off point. The fruit were exposed to

134 the products for 2 minutes, followed by the removal of the excess product using an air
135 drier (without heat) for 30 seconds. Sodium hypochlorite at 0.2% and 1X PBS were

136 used as positive and negative controls, respectively.

137

138 2.4.3 Automated Sanitization

139 Fruit utilized for the tests in the packing-house line were not washed before processing,

140 since they receive sprays of tap water during the automated sanitization. The whole

141 experiment was composed of two distinct experiments: (1) control and (2) test

142 experiment. For the control experiment, 15 limes, divided in 5 groups of 3 limes each

143 (five replicates), were labeled using a permanent marker for easy identification, and

144 then mixed with other limes to act as carriers throughout the sanitizing process. This

145 was necessary since small quantities of limes cannot be pushed throughout the line

146 efficiently. Each of the five groups was processed through the sanitizing line

147 independently (separate runs). Fruit in the control experiment were sprayed with water

148 only during the whole process. For the test experiment, 15 limes were again divided in 5

149 groups, labeled with a permanent marker, contaminated with X. citri as described in

150 section 2.4.2, and mixed with other limes (not contaminated) to act as carriers during

151 the sanitizing process. The 5 groups were processed independently through the line

152 with the difference that now they were sprayed with 2% of PosFruit in water (v/v) during

153 the run. Marked fruits were collected at the end of the line and allowed to dry at room

154 temperature.

155

156 2.4.4 Colony counting

157 After the treatments, fruit were placed into plastic bags and washed manually using 100

158 mL of 1X PBS during 5 minutes. The washes were collected in 50 mL polypropylene

159 tubes and centrifuged at 6000 xg for 7 minutes and the supernatant discarded. Cells

160 were then resuspended in 3 mL of 1X PBS and 100 µL were spread onto semi-selective

161 MGY-KCC medium (16 µg.mL-1 Kasugamycin, 16 µg.mL-1 Cephalexin, and 50 µg.mL-1

162 Cycloheximide) (Behlau, Jones et al., 2012). Colony growth was evaluated after 72 h of

163 incubation at 29 oC. To confirm the identity of X. citri colonies we performed diagnostic

164 PCR following the procedure described by Coletta-Filho, Takita et al. (2006).

165

166 2.5 Bacterial resistance induction

167 The probability that X. citri developed resistance against PosFruit was evaluated

168 according to (Oz, Guvenek et al., 2014), with modifications. Starting cultures of 5 mL

169 each containing 106 cells.mL-1 were prepared using PosFruit at 1/8x of the MIC and

170 incubated for 24 h at 29 oC and 200 rpm. After the incubation period, 100 µL of the initial

171 cultures (1/8x the MIC) were inoculated on NYG-agar and incubated for 48 h at 29 oC

172 for CFU counting. In addition, 500 µL of the same cultures were used as inocula and

173 transferred to fresh 5 mL cultures (identical to the starting cultures), but now containing

174 PosFruit at 1/4x of the MIC. The transfer scheme was repeated until the maximum

175 concentration of PosFruit reached 4x the MIC. Experiments were conducted in

176 triplicates, and repeated three times.

177

178 2.6 Bacterial persistence

179 Bacteria were inoculated into 5 mL of NYG to a final concentration of 106 cells.mL-1, and

180 PosFruit was added at 0,9x the MIC (starting culture). The bacterial suspension was

181 exposed to the product for 4 h at 29 oC under shaking (200 rpm). After incubation, 100

182 µL of the liquid culture were spread onto NYG-agar, without PosFruit, for the recovery of

183 X. citri cells. Colonies that were able to grow were inoculated into fresh liquid NYG

184 medium, now containing PosFruit at 1x the MIC, and incubated as before. Aliquots of

185 100 µL were spread onto solid media following product exposures of 4 h, 4.5 h and 24

186 h, and incubated for up to 48 h in order to assess for persistent colonies of X. citri.

187

188 3. Results

189 3.1 PosFruit has bactericidal action against X. citri

190 The efficacy of PosFruit to inhibit growth of X. citri was evaluated exposing bacterial

191 cultures to various dilutions of the product prepared directly into the culture medium.

192 PosFruit was able to inhibit X. citri growth in liquid medium at the concentration of

193 0.125% after 4 h of exposure. In addition, no colonies were recovered after plating

194 samples of the cultures on NYG-agar (Fig. 1, C). These results were comparable to our

195 positive control kanamycin, which has bactericidal action against X. citri at 20 g/mL

196 (Fig. 1, B). By decreasing the concentration of the product to 0.06%, there was still a

197 significant inhibition of bacterial growth reflected as a reduced cell turbidity in the culture

198 (not shown). However, upon plating samples of this culture on solid medium, a

199 considerable amount of X. citri colonies were able to grow (Fig. 1, compare D with our

200 negative and positive controls, A and B, respectively). Therefore, 0.125% was defined

201 as the minimal inhibitory concentration (MIC) of PosFruit against X. citri.

202

203 3.2 Bacterial resistance and persistence

204 X. citri cells were exposed to PosFruit at the MIC (0.125%), and following exposure

205 periods of 4 h, 4.5 h (half an hour beyond the incubation period used to define the MIC),

206 and 24 h, culture samples were spread onto NYG-agar (without any product) in order to

207 allow growth of persistent colonies. After several attempts, no colonies could be

208 recovered, which suggests that X. citri could not persist the treatment with PosFruit.

209 Additionally, to check if and how likely natural resistant cells could be found, we

210 prepared serial dilutions of X. citri cultures and plated them on NYG-agar containing

211 PosFruit at the MIC. Plates were incubated for up to a week under standard cultivation

212 conditions, and again, no bacterial colonies could be detected.

213 A final approach to detect resistance to the product was to expose X. citri to

214 increasing concentrations of PosFruit, starting from a sub-lethal concentration and

215 increasing up to as high as fourfold the MIC (Table 1). The rationale here was to test if

216 the product can induce any genetic alteration in the course of the increasing exposures,

217 and that could lead to PosFruit resistance. The experiment was initiated exposing X.

218 citri to 1/8 of the MIC (day 1). After 24 h incubation, culture samples were spread onto

219 NYG-agar, and as a result, we detected colonies in 3 out of 3 trials after 48 h of plate

220 incubation (Table 1, day 1). Fractions of the starting cultures (day 1, after 24 h

221 incubation) were used as inocula to prepare the subsequent cultures in which the

222 concentration of PosFruit was increased twofold (to 1/4 of the MIC). Upon plating

223 samples of cultures collected on the second day, we detected growth in 2 out of 3

224 plates. However, when the concentration of product reached half the MIC, bacterial
225 growth was halted (Table 1, day 3). The passages were continued until the

226 concentration of PosFruit reached four times the MIC (13th day), but neither the

227 increase of cell turbidity in liquid cultures nor the growth of colonies on plate were

228 detected from the third day on. Altogether, results show that the PosFruit action did not

229 induce resistance to itself in X. citri.

230

231 3.3 PosFruit was efficient to sanitize Tahiti limes

232 The efficacy of PosFruit to sanitize Tahiti limes was evaluated by artificially

233 contaminating fruit with X. citri, following their exposure to three concentrations of the

234 product (Fig. 2; 1, 2 and 5%). The time-period of exposure was fixed to 2 min, since this

235 is the time used for the positive control hypochlorite. Treatments with PosFruit at the

236 concentration of 1% (v/v) showed a reduction of approximately 2 log10 in the number of

237 CFU/mL recovered of X. citri (Fig. 2; compare panels A-1 and A-3; B). By further

238 increasing the concentration of the product to 2% we detected a reduction of 3 log10 in

239 the number of CFU/mL recovered, where the efficacy of PosFruit could be compared to

240 the positive control hypochlorite at 0.2% (Fig. 2; compare panels A-1, A-2, and A-4; B).

241 Although some X. citri colonies could still be recovered after treatments with 2%

242 PosFruit, other yellowish epiphytic/contaminants were detected (Fig. 2, A-4). These

243 contaminants were disregarded as false positives based on PCR analysis for X. citri.

244 Finally, exposure of Tahiti limes to PosFruit at 5% completely eliminated all the bacteria,

245 as no colonies could be recovered on NYG-agar plates (Fig. 2, A-5). Altogether,

246 PosFruit was as effective as hypochlorite to sanitize Tahiti limes against X. citri when
247 used at 2% and a minimum exposure time of 2 minutes, leading to a reduction of 3 log10

248 of CFU/mL of X. citri recovered.

249

250 3.4 PosFruit performed better in an automated lime sanitizing system

251 The efficiency of PosFruit to sanitize Tahiti limes against X. citri was subsequently

252 evaluated using a commercial automated sanitization line (Fig. 3, A). Limes were

253 artificially contaminated with X. citri and mixed with X. citri-free fruit in XXX L plastic

254 boxes. Fruit were dropped into the line (Fig. 3, A-1) and let to roll through

255 uninterruptedly for ~2 minutes until the collection point (Fig. 3, A-5). During this process,

256 fruit were spray-washed with tap water (Fig. 3, A-3), followed by PosFruit at 2% for ~30

257 seconds each (Fig. 3, A-4). The rolls of the machine were set to a speed of 1730

258 rpm.min-1, which guaranteed that fruit were thoroughly covered with the product until the

259 run-off point by just passing through the line (Fig. 3, A-2). At the end, fruit were placed

260 in plastic boxes and let to dry at room temperature. Fruit washed with water only, led to

261 recovery of 4 log10 CFU/mL (Fig. 3, B, and C-1, red circles). The identity of X. citri

262 isolates was checked by PCR, and the enormous number of non-X. citri bacterial

263 colonies recovered was probably due to the fact that limes were washed with tap water

264 only before PosFruit application. This procedure was intended to mimic exactly the

265 sanitization process carried out in a commercial packing-house. Astonishingly,

266 treatment with 2% PosFruit in an automated system led to a complete elimination of X.

267 citri, where no colonies could be recovered, and only epiphytic/contaminants were

268 detected on plate (Fig. 3, B, and C-2). The CFU/mL reduction considering our target X.

269 citri was therefore of 4 log10 when compared with the control treatment. We also
270 attribute the better effectiveness of the product to sanitize the limes when applied in an

271 automated system to an extra mechanical cleansing process probably exerted when

272 fruits roll in contact with the cylinders.

273

274 4. Discussion

275 Legislation allows only hypochlorite – however, it is toxic, damages the skin of the fruit

276 and is not allowed for the organic market…cinnamaldehyde is ok.

277 Natural resistance can be found in a population as a result of genetic mutations, for

278 instance, where one or more individuals present a selective advantage when are

279 exposed to a compound. In the case of bacterial persistence, individuals in a population

280 are able to reduce its metabolism at the point that it cannot be affected by the action of

281 a compound during its growth phase (Martins, Merfa et al., 2018). Nevertheless, the

282 persistent colony its not resistant to the selective pressure in which it was exposed.

283 Once the persistent colony its identified and enters in contact with the same compound

284 in a new media, its growth will be influenced by the effects of the compound, which is

285 indicated for a decrease or even complete restriction in development. On the other

286 hand, resistant colonies are able to completely develop in higher concentrations of the

287 compound.

288 The manual treatment adapted here to simulate the sanitizing conditions used in

289 packing-houses showed that sodium hypochlorite was not effective to sanitize limes

290 against X. citri- perhaps this happened because the inoculum was too high!!!
291 This decrease of 1 log in X. citri growth, in comparison to the previous manual test, may

292 be caused due to the carrying process of fruits which are in contact with each other and

293 with the soft surface of the machine rolls. These surfaces can act scrapping off some

294 cells from fruits and this effect can be observed as a minor reduction in CFU.

295

296 5. Conclusion

297

298 Acknowledgments

299 CZ received a MSc Scholarship from Fundação de Amparo à Pesquisa do Estado de

300 São Paulo, FAPESP (201?/???). This work was funded by FAPESP (grant 2015/50162-

301 2) and INCT Citros (FAPESP 2014/50880-0 and CNPq 465440/2014-2). Aline???

302

303 Figure legends

304 Fig. 1. Determination of the Minimal inhibitory concentration (MIC) of PosFruit

305 against X. citri. Bacteria were exposed to different concentrations of PosFruit (from 1%

306 to 0.0625%) for a period of 4 h, and after serially diluted (from 10-1 to 10-6) for CFU

307 counting on plates. A) negative control; B) 20 g.mL-1 kanamycin; C) 0.06% PosFruit,

308 and D) 0.125% PosFruit. Experiments were repeated three times and in triplicates.

309 Here, we show only the plates derived from the cell counting in which PosFruit had

310 bactericidal action (C), and one dilution below (D).

311 Fig. 2. Sanitization of Tahiti limes. Tahiti limes were spray contaminated with X. citri

312 and then manually exposed to PosFruit as indicated. Following treatment, limes were

313 washed and the numbers of viable cells verified on plate growth (A) Cell viability check;

314 a representative experiment. Plates: 1, 1X PBS (negative control); 2, 0.2% sodium

315 hypochlorite/2 min (positive control); 3, 1% PosFruit/2 min; 4, 2% PosFruit/2 min; 5, 5%

316 PosFruit/2 min. (B) colony forming units, CFU, determined for each of the treatments

317 (logarithmic scale). Experiments were repeated three times in triplicates; standard

318 errors are indicated by the vertical lines above the bars.

319

320 Fig. 3. Automated sanitization of Tahiti limes. Previously labeled fruit were

321 contaminated with X. citri, mixed with carrier limes, and loaded on the sanitizing

322 machine (A): 1, fruit drop point; 2, rollers; 3, water spray lines; 4, product spray line, and

323 5, fruit collection point. B) Colony forming units, CFU, derived from the treatments

324 (logarithmic scale). Water, negative control, and PosFruit at 2%. Experiments were

325 repeated three times in triplicates; standard errors are indicated by the vertical lines

326 above the bars. C) Plates from a representative CFU determination experiment: 1) plate

327 inoculated with washes from limes washed with water only (X. citri colonies are labeled

328 using red circles), and 2) plate inoculated with washes from limes exposed to PosFruit

329 at 2%.

330
 Table 1

Bacterial resistance induction

Day 1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th 11th 12th 13th

MIC a 1/8x 1/4x 1/2x 1x 1x 2x 2x 2x 3x 3x 3x 3x 4x

Bacterial growth b 3/3 2/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3

331 a Proportion of product regarding the Minimal Inhibitory Concentration (MIC= 0,125% of PosFruit suspension in water)

332 b Plating was done in triplicate; the values on the left of the slash sign show the number of plates in which any growth

333 could be observed

334

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