1 Involvement of abscisic acid in the resistance of citrus to Penicillium 2 digitatum infection

3 4 María T. Lafuente*, Ana-Rosa Ballester, Luis González-Candelas 5 6 Instituto de Agroquímica y Tecnología de Alimentos, IATA-CSIC, Calle Agustín Escardino 7 7, 46980 Paterna, Valencia, Spain 8 9 Ana-Rosa Ballester, [email protected] 10 Luis González-Candelas, [email protected] 11 * Corresponding author. Tel.: +34 900 022; fax: +34 963 636 301 12 E-mail address: [email protected]

13

1 14 ABSTRACT

15 The involvement of abscisic acid (ABA) in citrus fruit resistance to Penicillium

16 digitatum infection was investigated. To this end, the effect of both exogenous ABA and

17 inhibitors of ABA biosynthesis in plants on the resistance of sweet oranges to be infected by

18 this major postharvest pathogen, and also on in vitro fungal growth, was examined. The

19 ability of both P. digitatum to infect wild-type Navelate orange fruit (Citrus sinesis (L.)

20 Osbeck) and its spontaneous ABA-deficient mutant Pinalate was compared. The results

21 showed that the Pinalate orange was more susceptible than its parental to infection, and

22 exogenous ABA reduced the percentage of infected and also the maceration zone of

23 this mutant without affecting in vitro fungal growth. The global results indicated that the

24 ABA of the fruit plays a defensive role in citrus fruit against P. digitatum infection. This

25 work demonstrates, for the first time, the ability of P. digitatum to produce ABA. The

26 hormone decreased in fruit in early stages as a consequence of infection. Thereafter when

27 tissue maceration became evident, ABA increased, and this rise seemed related to the P.

28 digitatum-induced rise in production. This was suggested by the fact that ABA

29 increased in Navelate orange, but not in its ABA-deficient mutant Pinalate in this infection

30 stage, while exogenous ethylene barely increased ABA in the mutant. Finally, the sharp

31 marked rise in free ABA in later stages was likely related to free ABA being released from

32 conjugated ABA, and also to fungal ABA. However, fungal ABA was not a primary

33 virulence factor in citrus fruit infection.

34

35 KEYWORDS

36 Abscisic acid-deficient orange mutant; free and conjugated abscisic acid; fungal ABA;

37 postharvest; resistance; stress.

2 38 1. INTRODUCTION

39

40 Abscisic acid (ABA) is a that plays an important role in plant stress

41 responses, but can be also synthesised by some fungal species (Chanclud and Morel, 2016;

42 Ding et al., 2016). Upon pathogen attack, infected plant cells induce signalling molecules to

43 initiate mechanisms in the surrounding cells to reduce pathogen spread. Plenty of information

44 is available about the role of plant , like (JA), (SA) and

45 ethylene, in the interaction between pathogens and plants (Robert-Seilaniantz et al., 2011;

46 Romanazzi et al., 2016; Zhou et al., 2018). Fewer studies are available about the involvement

47 of ABA in the susceptibility of plants, and especially of fruit crops, to be infected by

48 phytopathogenic fungi. However, it is known that ABA may affect the outcome of plant-

49 microbe interactions either positively or negatively (Robert-Seilaniantz et al., 2011;

50 Chanclud and Morel, 2016). The results obtained by using the ABA-deficient mutants of

51 tomato plants (Audenaert et al., 2002) and strawberry fruit (Li et al., 2013) have shown that

52 ABA promotes the infection caused by the necrotrophic fungus Botrytis cinerea. Similarly,

53 ABA may lead to enhanced pathogen susceptibility in different fruit crops like pepper

54 (Hwang et al., 2008), banana (Li et al., 2017) and tomato (Gong et al., 2017; Wilkinson et

55 al., 2018). In contrast, a positive correlation between ABA and disease resistance has been

56 found in grapes infected by Glomerella cingulata (Wang et al., 2015), and also in Brassica

57 napus and Arabidopsis infected by (Kaliff et al., 2007). Such

58 differences in the effect of ABA on the resistance to pathogens may depend on the ABA-

59 induced mechanisms in a specific plant-pathogen interaction or crop.

60 Penicillium digitatum (Pers.:Fr.) Sacc. is the major pathogen that causes postharvest

61 disease in the citrus fruit grown under Mediterranean conditions. Previous research has

3 62 demonstrated that ethylene (Porat et al., 1999; Marcos et al., 2005) and JA (Droby et al.,

63 1999) are involved in the defence of citrus fruit against this pathogen. The role played by

64 ABA in the response of this important agronomic fruit crop to P. digitatum infection remains

65 unknown, but endogenous ABA levels are known to increase in response to ethylene in citrus

66 peel (Lafuente et al., 1997).

67 This work aimed to determine variations in ABA levels during the fruit infection

68 process and to elucidate whether the hormone is involved in the defence response of citrus

69 fruit to infection by P. digitatum. To this end, the spatio-temporal changes in the ABA levels

70 in citrus fruit infected with this pathogen were examined. We also determined the effect of

71 both exogenous ABA and inhibitors of the hormone on the resistance of sweet oranges to P.

72 digitatum infection. We compared the ability of the fungus to infect wild-type Navelate

73 orange fruit (Citrus sinesis (L.) Osbeck) and its ABA-deficient spontaneous mutant Pinalate.

74 The availability of artificially generated mutants is not very common in woody plants.

75 Therefore by considering the limitations of pharmacological experiments, this ABA-deficient

76 yellow sweet orange mutant characterised by Rodrigo et al. (2003) has been a valuable tool

77 to investigate the involvement of ABA in the response of citrus fruit to infection. This mutant

78 has a fruit-specific biochemical blockage in the biosynthetic pathway that results

79 in a yellow-pigmented fruit and a marked reduction in ABA levels following the biosynthesis

80 of (Rodrigo et al., 2003). The putative involvement of conjugated-ABA, ethylene

81 and fungal ABA in the pathogen-induced changes in ABA are also discussed.

82

83 2. MA TERIAL AND METHODS

84 2.1. Fruit material

4 85

86 Full mature Navelina and Lane Late sweet oranges (Citrus sinensis (L.) Osbeck) were

87 harvested from the trees grown in commercial orchards in Lliria (Valencia, Spain). The fruit

88 of the Navelate (Citrus sinensis (L.) Osbeck) sweet orange and its spontaneous ABA-

89 deficient yellow mutant (Pinalate) were randomly harvested from the adult trees grown in

90 experimental orchards according to normal cultural practices in the ‘The Spanish Citrus

91 Germoplasm Bank’ at the Instituto Valenciano de Investigaciones Agrarias (IVIA, Moncada,

92 Valencia) in Spain.

93 Oranges from all the cultivars were immediately delivered to the laboratory. The fruit

94 with no damage or visual defects were surface-sterilised with commercial bleach and then

95 rinsed with abundant tap water as previously described (Ballester et al., 2010). After drying

96 at room temperature, fruit were divided into groups to be immediately inoculated with P.

97 digitatum conidia or treated with chemicals (see Section 2.3) before being inoculated with

98 the fungus.

99 Two groups of oranges of each cultivar were used to compare changes in disease

100 incidence, and in free and conjugated ABA and ethylene production. The fruit in the first

101 group were inoculated with 10 µL of the conidial suspension, while those of the second group

102 were inoculated with the same volume of water (control fruit). The oranges in each group

103 were divided into two subgroups. Three replicates of five fruit were included in the first

104 subgroup to determine disease evolution. The second subgroup included three replicates of

105 at least five fruit per incubation period. These fruit were used to periodically determine ABA

106 and ethylene levels in peel or flavedo (outer coloured part of peel) discs (7 mm in diameter)

107 taken around the inoculation site. The ethylene production of discs was determined

108 immediately as described below (Section 2.7), while the discs used for the free and

5 109 conjugated ABA analyses were immediately frozen, homogenised in liquid nitrogen and kept

110 at -80ºC for later determinations.

111 To test whether the application of ABA, and of the inhibitors of its synthesis (Section

112 2.3), modified the response of oranges to P. digitatum, fruit were divided into four groups.

113 Two groups were treated with either ABA or the solutions of the ABA inhibitors (Groups 1

114 and 2). The other two groups (3 and 4) were treated only with water containing the same

115 volume of ethanol used to dissolve the hormone, or the inhibitors, and were used as the

116 controls of the chemically treated fruit. After drying at room temperature, the oranges in

117 Groups 1 and 3 were inoculated with the fungus, whereas those in Groups 2 and 4 were

118 inoculated only with water. The oranges in each group were divided into two subgroups, as

119 described above to evaluate disease incidence and for the ABA analysis.

120 To examine the spatial changes in the free and bound ABA, the flavedo samples were

121 taken from several well-defined concentric zones of flavedo, which included tissue taken: 1)

122 around the inoculation point with abundant spores; 2) in the contiguous area with abundant

123 mycelium; 3) from the macerated zone; 4) from the healthy non-infected area. In order to

124 obtain these samples on the same day, fruit were infected and kept in plastic boxes at 20ºC

125 and 90-95% relative humidity (RH) for 6 d. To obtain enough tissue from the different zones

126 for the ABA analysis, 15 fruit were included in each replicate and three biological replicates

127 were used in the experiment.

128 In order to understand whether the differences observed in the changes in ABA during

129 the infection of Pinalate and Navelate fruit could be related to the rises in the hormone

130 ethylene, two additional groups of fruit of each cultivar were used. The fruit from the first

131 group were treated with ethylene (2 or 40 µL L-1, see Section 2.3) and those of the second

132 group with air (control samples). During the treatments, all the fruit were maintained at 20ºC

6 133 and 90-95% RH to avoid dehydration. In this experiment, the flavedo samples were taken

134 from the total fruit surface, and were frozen and ground as described above for the ABA

135 analysis. Three biological replicates of five fruit per replicate and cultivar were collected

136 during each sampling period.

137

138 2.2. Fungal material, and disease incidence and severity

139

140 To infect fruit, P. digitatum conidia suspensions were prepared in sterile distilled

141 water from the 7-day-old cultures grown on potato dextrose agar (PDA) at 24ºC. Experiments

142 were performed using the Penicillium digitatum (Pers.:Fr.) Sacc. isolate Pd1 (CECT 20795)

143 (Marcet-Houben et al., 2012). The conidia concentration was measured with a

144 haemocytometer. The conidial suspensions were adjusted to concentrations ranging between

145 104 and 106 conidia mL-1. Fruit were inoculated by wounding peel tissue with a flame

146 sterilized needle (4 mm depth) to reach the albedo (inner white part of peel), or at a depth of

147 2 mm to inoculate fruit in the limit between flavedo and albedo tissues. Ten µL of conidial

148 suspensions were used to inoculate each wound. The wounds in the control samples were

149 inoculated with the same volume of water.

150 To determine disease incidence and severity, the percentage of infected wounds and

151 lesion diameter (cm) of the fruit macerated zone were periodically determined in two

152 perpendicular directions during fruit incubation in plastic boxes, kept in the dark at 90-95%

153 RH and 20ºC. For each treatment, determinations were made in three replicate samples of

154 five fruit and with four equidistant wounds in the equatorial zone per fruit.

155

156 2.3. Chemical treatments.

7 157

158 To test whether ABA was involved in the resistance of citrus fruit to P. digitatum infection,

159 fruit were treated with either ABA or the inhibitors of ABA biosynthesis in plants. The

160 selected inhibitors were: 1) Tungstate (Tungs) (PubChem CID: 24465), which inhibits the

161 last ABA biosynthesis step by inhibiting ABA aldehyde oxidase; 2) Nordihydroguaiaretic

162 acid (NDGA) (PubChem CID: 453483489), an inhibitor of 9-cis epoxycarotenoid

163 dioxygenase (NCED), which is a key in the biosynthesis of ABA in citrus fruit; 3)

164 Norflurazon (NFZ) (PubChem CID: 33775), which inhibits desaturase at the

165 beginning of carotenoid biosynthesis (Supplementary Fig. S1). All the compounds were

166 obtained from Sigma-Aldrich (St. Louis, MO, USA). Fruit were treated by dipping them for

167 2 min in aqueous solutions containing concentrations of each compound (indicated in the

168 Results section) and 0.5% ethanol to dissolve them. The control fruit were simply treated

169 with water containing the same proportion of ethanol according to the same procedure.

170 Treatments were performed before wounding the fruit. These solutions were also used to test

171 whether either ABA or the inhibitors may affect in vitro fungal growth.

172 Ethylene treatments were performed in Navelate and Pinalate fruit as previously

173 described (Lafuente et al., 2014). The fruit of both cultivars were treated for 7 d with 2 µL

174L -1 ethylene at 90-95% RH and 20ºC. Thereafter, the effect of ethylene on the ABA levels of

175 the mutant and parental fruit was further examined by treating the fruit for 3 d with a higher

176 ethylene concentration (40 µL L-1). The control fruit were air-treated under the same

177 experimental conditions used for the ethylene treatments.

178

179 2.4. In vitro experiments

180

8 181 The in vitro experiments were performed as previously described (Marcet-Houben et

182 al., 2012; Ballester et al., 2015) in PDB (potato dextrose broth) and/or PDA to test whether

183 ABA and inhibitors Tungs, NDGA and NFZ affected P. digitatum growth. Briefly, the

184 experiments in PDB were performed in 96-well microtiter plates. Wells contained 104 conidia

185 mL-1 in the PDB medium supplemented with either ABA or the inhibitors of ABA

186 biosynthesis in plants. These chemicals were assayed at final concentrations ranging between

187 0.1 and 100 mM. Three replicate wells per compound and concentration were used. Plates

188 were incubated at 24ºC and fungal growth was measured as A492 for up to 6 d. The PDA

189 experiments were also conducted by centrally inoculating 9-cm PDA plates containing the

190 desired concentration of the tested chemical, with a 5 µL drop of conidia (106 conidia mL-1),

191 and by incubating plates at 24ºC in the dark. Chemicals were added after autoclaving PDA

192 when the medium was still warm. Colony diameter (cm) was recorded. The percentage of in

193 vitro growth inhibition was also calculated as previously described (Ballester and Lafuente,

194 2017) according to this formula:

195 Percentage of growth inhibition = 100 x (GC-GSL)/GC

196 GC is the diameter of the fungal colony of the control plates and GSL is that of the plates

197 treated with the ABA-inhibitors.

198

199 2.5. Analysis of the free ABA in both fruit and fungus

200

201 ABA was determined in three biological replicate samples of previously frozen peel

202 tissues (whole peel or flavedo, as indicated in each experiment) taken from different fruit

203 lots, and also in P. digitatum spores. To determine the temporal changes in ABA that

204 occurred in response to infection, the free ABA was determined in the peel or flavedo discs

9 205 (7 mm) taken around the inoculation zone. A minimum of eight discs per fruit and of five

206 independent fruit (40 discs) were used in each replicate. ABA was analysed as previously

207 reported (Lafuente et al., 1997; Romero et al., 2012) in the representative homogenised

208 samples taken from the infected fruit and their respective control samples inoculated only

209 with water. ABA was extracted from 1 g of fresh weight tissue samples with 80% acetone

210 containing 0.1 g L−1 butylated hydroxytoluene and 0.5 g L−1 citric acid. After 5 min of

211 centrifugation at 3000 x g, the supernatants of each sample were diluted with cold TBS (6.05

-1 -1 -1 212 g L Tris, 0.2 mg L MgCl2 and 8.8 g L NaCl) at pH 7.8 to reach the final ABA

213 concentrations, which fell within the linear range of the ABA standard curve. The extracted

214 samples were analysed in duplicate by an indirect ELISA method using the ABA-4’-BSA

215 conjugate. This compound was synthesised as described by Weiler (1980) with some

216 modifications (Norman et al., 1988). To determine the effect of ethylene on the ABA levels

217 of the Navelate and Pinalate fruit, the free ABA was analysed in the flavedo of the fruit of

218 both cultivars treated with either ethylene or air (control). In this experiment, three replicate

219 samples containing five fruit each were used and the flavedo samples were taken from the

220 whole fruit. ABA was expressed per kg peel or flavedo, as indicated in the figure captions,

221 and on a fresh weight basis.

222 To know whether P. digitatum was able to produce ABA, the conidial suspensions

223 from the cultures grown at 24ºC on PDA during different periods were adjusted to 108 conidia

224 mL-1.After 5 min of centrifugation at 1,3000 x g, ABA was extracted from the precipitated

225 conidia with 1 mL of the same solvent used to extract the hormone from fruit. Extracts were

226 concentrated with a SpeedVac and the resulting residues were diluted with 400 µL of the

227 TBS solution used to determine the free ABA in duplicate following the same indirect ELISA

228 method described above. Three biological replicate samples of spores were used per fungal

10 229 growth period and the results were expressed as pg ABA x 108 per spore unit. To this end,

230 the number of spores was determined before extracting the hormone. This method was

231 validated by also analysing ABA by reverse UHPLC-MS in the same samples taken from the

232 7-day-old cultures, as previously reported (Seo et al., 2011), at the phytohormones

233 quantification service of the IBMCP/CSIC (Valencia, Spain).

234

235 2.6. Conjugated ABA analysis

236

237 The conjugated ABA was analysed, as previously described (Lafuente et al., 1997),

238 by determining the total ABA and calculating the conjugated ABA concentration by the

239 difference between the total and free ABA. Briefly, 970 µL of the TBS solution (pH 7.8)

240 were added to 30 µL of the diluted extracts used to determine the free ABA in glass tubes.

241 Thereafter, pH was adjusted to pH 11 with 0.5 N NaOH and hydrolysis was performed by

242 heating tubes at 60ºC for 1 h. The extract was neutralised with 2 N HCl, diluted twice with

243 TBS and the total ABA was analysed as described above for the free ABA.

244

245 2.7. Determination of ethylene production

246

247 Ethylene production was determined in three replicate samples of the flavedo discs

248 taken around the inoculation site, as previously described (Lafuente et al., 2001). Discs were

249 taken from the same fruit used to determine ABA. Six 7-mm diameter discs taken from three

250 independent fruit (two discs per fruit) were included in each replicate and ethylene was

251 measured during P. digitatum progression in the discs taken from the fruit inoculated with

11 252 either the fungus or water (control samples). Each replicate sample was incubated in sealed

253 15-mL glass tubes at 20 C, and 1 mL of gas was drawn from each tube with a hypodermic

254 syringe and injected into a Gas Chromatograph (GC) (Perkin Elmer Autosampler), equipped

255 with an activated alumina column. The chromatograph was equipped with a flame ionisation

256 detector and the oven temperature was maintained at 140ºC.

257

258 2.8. Statistical analysis

259 All the values are provided as the mean of three replicate samples±standard error. A

260 mean comparison using the Tukey’s test was made to determine whether the mean values

261 significantly differed (P ≤ 0.05). The analyses were performed with the Statgraphics Plus 4.0

262 Software (Manugistics, Inc.).

263

264 3. RESULTS

265

266 3.1. Free ABA decreases in sweet oranges in early P. digitatum infection stages

267

268 In the first experiment run to know whether citrus fruit showed changes in ABA in

269 response to P. digitatum infection, the Navelina oranges harvested in November were

270 infected with the Pd1 strain. A high conidia concentration (106 conidia mL-1) was applied to

271 induce a rapid marked response to infection. Fruit were also inoculated at a depth of 4 mm.

272 In this way, infection started in albedo, which is more susceptible than flavedo (Ballester et

273 al., 2006). In this experiment, ABA was analysed in the frozen samples of peel discs, which

274 included both albedo and flavedo tissues. Under these experimental conditions, the ABA

12 275 levels in the control samples barely changed, but an early decrease in the free ABA was

276 observed in the infected fruit, which lasted until day 2 (Fig. 1A). An approximate 2-fold

277 increase in ABA occurred when peel maceration started (Fig. 1B), and 10 % of the wounds

278 inoculated with the pathogen showed disease symptoms. This pattern of changes in ABA was

279 also observed in the fruit of the same cultivar harvested in January which, in this cultivar,

280 corresponded to the latest commercial maturity stage during the citrus season

281 (Supplementary Fig. S2).

282 In a subsequent experiment, performed in Lane Late sweet oranges (Citrus sinensis

283 L. Osbeck), the changes that occurred only in flavedo at the inoculation depth of 2 mm, at

284 the limit between flavedo and albedo tissues, were examined. This approach was performed

285 because of: 1) the poor penetration of exogenous compounds in citrus fruit peel, which is

286 very hydrophobic as the aim was to evaluate the effect of both exogenous ABA and inhibitors

287 of its synthesis in plants; 2) the ABA deficiency in the Pinalate mutant was evident mainly

288 in flavedo; 3) the ABA levels were much higher in flavedo than in albedo (Lafuente et al.,

289 1997). Under these experimental conditions, infection progression (Fig. 2B) was much

290 slower than in the Navelina fruit infected at a depth of 4 mm (Fig. 1B). By day 3, the

291 percentage of wounds showing disease symptoms was only 22% (Fig. 2B). As shown in Fig.

292 2A, the ABA levels in the flavedo of the infected fruit were lower than the ABA levels of the

293 wounded control fruit by day 3, which was 2 d later than in the above-mentioned experiments.

294 Therefore, by considering that the susceptibility of flavedo to be infected was lower than that

295 of albedo, the ABA sampling period was extended in subsequent experiments when ABA

296 was determined in this outer part of peel and/or when inoculation was performed between

297 albedo and flavedo at a depth of 2 mm.

298

13 299 3.2. ABA is involved in the resistance of citrus fruits to P. digitatum infection

300 The above results suggested that ABA is involved in the susceptibility of citrus fruit

301 to P. digitatum infection. Therefore, the effect of both exogenous ABA and the inhibitors of

302 ABA synthesis in plants was examined on the incidence of P. digitatum infection in citrus

303 fruit. The effect of Tungs was first evaluated because it inhibits the last ABA biosynthesis

304 step in plants and does, therefore, not affect other precursors of the hormone in the

305 biosynthetic pathway (Supplementary Fig. S1). A high (10 mM) Tungs concentration was

306 applied to fruit because, as mentioned above, penetration of exogenous compounds in the

307 peel of citrus fruit is poor. Tungs reduced disease in the mature Lane Late sweet oranges

308 (Supplementary Fig. S3A) but, according to the in vitro experiments, Tungs also had a strong

309 effect as it reduced fungal growth in a dose-dependent manner (Supplementary Figs S3B and

310 S3C). Therefore, the effect of NDGA, an inhibitor of NCED, was assayed. NCED is a key

311 enzyme in ABA biosynthesis in citrus fruit (Supplementary Fig. S1). Like Tungs, NDGA

312 affected both in vitro and in vivo growth (data not shown). In view of these results, the in

313 vitro effect of NFZ, which inhibits phytoene desaturase, was evaluated at the beginning of

314 carotenoid biosynthesis (Supplementary Fig. S1). This inhibitor did not modify fungal

315 growth (data not shown). Similarly, the application of 1 mM ABA did not alter the growth

316 of the fungus (Supplementary Fig. S4).

317 After considering these results, and as the application of 1 mM ABA significantly

318 increased ABA levels in citrus fruit (Romero et al., 2012), the effect of both 1 mM ABA and

319 1 mM NFZ was examined on the resistance of Navelate and its ABA-deficient mutant

320 (Pinalate) to P. digitatum infection. In this experiment, a low conidia concentration was used

321 and inoculation was performed at a depth of 2 mm to make the putative differences in P.

322 digitatum infection after the ABA or NFZ application more evident, as well as the differences

14 323 between cultivars in their resistance to the fungus. Under these experimental conditions, the

324 ABA-deficient mutant (Pinalate) was more susceptible to infection than the parental fruit

325 (Figs 3A and 3B). The percentage of wounds showing disease by the end of the experiment

326 was about 3-fold higher in the ABA-deficient mutant (Fig. 3C). The results also showed that

327 ABA and NFZ had a mild effect on infection in Navelate fruit (Fig. 3A), which already had

328 high ABA levels in flavedo (Fig. 3D). However, in the fruit of the ABA-deficient mutant,

329 which presented about one fourth of the ABA levels compared to the parental (Fig. 3D), the

330 exogenous ABA had a clear effect by reducing infection (Fig. 3B). It was noteworthy to find

331 that NFZ favoured infection in the ABA-deficient mutant (Fig. 3B), but barely affected

332 disease severity in the parental fruit (Fig. 3A). However, this compound also favoured peel

333 damage in the Pinalate fruit, which was manifested as tissue darkening while fruit were kept

334 in the dark (Supplementary Fig. S5). The NFZ-related tissue darkening was not observed in

335 the Navelate fruit. Therefore, this inhibitor was also ruled out and research focused on

336 comparing the behaviour of both the mutant and the parental fruit rather than using

337 pharmacological approaches in subsequent experiments.

338

339 3.3. The P.digitatum-induced rise in ethylene production precedes the rise in free ABA in

340 the infected citrus fruits

341

342 It has long since been known that ethylene increases in citrus fruit in response to P.

343 digitatum infection (Chalutz et al., 1978), and that this hormone increases ABA in citrus fruit

344 flavedo (Lafuente et al., 1997). To know whether a link exists between ABA and the

345 infection-induced rise in ethylene, changes in ABA and ethylene production were measured

346 in parallel in the flavedo of both the Navelate and the Pinalate fruit. Ethylene production of

15 347 the flavedo discs taken from the infected fruit of both cultivars transiently increased after 2

348 d (Fig. 4A and 4B). ABA initially decreased in response to infection (depth of 4 mm) in the

349 Navelate fruit flavedo (Fig. 4C). Then it increased after a rise in ethylene. In the Pinalate

350 fruit, the ABA levels rapidly increased in both the wounded and infected fruit (Fig. 4D). Then

351 the ABA levels slightly dropped in the control and infected samples, despite the rise in

352 ethylene observed in the infected fruit (Fig. 4B). Thus the effect of exogenous ethylene on

353 ABA content in the flavedo of the fruit of both cultivars was determined in a subsequent

354 experiment. Ethylene (2 µL L-1) induced a sharp transient increase in ABA in the Navelate

355 fruit (Fig. 5A). This increase occurred by day 3, but the ABA levels were not maintained

356 after subsequently exposing fruit to ethylene (Fig. 5A). A slight increase in ABA was

357 observed in the ethylene-treated Pinalate fruit by day 3, but ABA levels were negligible

358 compared to those found in the parental flavedo (Figs 5A and 5B). It was also confirmed that

359 applying a 20-fold higher ethylene level (40 µL L-1) did not induce higher ABA

360 concentrations (Supplementary Fig. S6).

361

362 3.4. The release of ABA from its conjugate might be responsible for the marked rise in the

363 free ABA in a later P. digitatum infection stage.

364

365 The preliminary experiments revealed that the free ABA levels were very high in

366 fruit, and displayed abundant sporulation. Such high free ABA levels could be related to a

367 new hormone synthesis in later fruit infection stages, but also to the release of the free ABA

368 from its conjugated form, or even to putative fungal ABA. The experiment shown above in

369 Fig. 3 ended by day 7, when flavedo tissue was taken to determine ethylene, and ABA still

370 showed poor sporulation (Supplementary Fig. S7A). Therefore, in order to test these

16 371 hypotheses, the experimental period was extended and determined both the free and

372 conjugated ABA at later time points after fungal inoculation.

373 The assay was performed in the Navelate and Pinalate fruits, which were inoculated

374 at the limit between flavedo and albedo (2 mm). Disease symptoms were evident by day 4

375 (Fig. 6A). During this period, the percentage of wounds showing disease was about 3-fold

376 higher in the ABA-deficient mutant (62% in Pinalate, 21% in Navelate), tissue maceration

377 was barely observed in the parental fruit and the macerated zone diameter was about 8-fold

378 bigger in the mutant (Fig. 6A). The differences between both cultivars decreased thereafter,

379 but the Pinalate fruit showed higher disease incidence and severity for up to 7 d. Sporulation

380 was evident only by day 6 in Pinalate and 1 d later in Navelate (Fig. 6B). By day 12, the fruit

381 of both cultivars were completely rotten and spores were abundant in flavedo, but not in

382 albedo (Supplementary Fig. S7B). By day 18, albedo was almost disintegrated and the peel

383 tissue structure was already disorganised (Supplementary Fig. S7C).

384 Marked increases in the free ABA were observed at later times during infection in

385 both cultivars despite the mutant’s ABA deficiency (Figs. 6C and 6D). Such increases were

386 evident only by day 12, when sporulation was obvious in flavedo, but not in albedo.

387 Thereafter, the free ABA levels continued to increase with sporulation. To test whether the

388 marked rise in the free ABA was a consequence of releasing the hormone from the conjugated

389 ABA, which is very abundant in citrus fruit flavedo (Lafuente et al., 1997), the conjugated

390 ABA was also determined in these samples. As expected, the conjugated ABA (Figs. 6E and

391 6F) was higher in the parental fruit. The ABA conjugated form levels were more abundant

392 than the free ABA (Figs. 6C and 6D) in the freshly harvested fruit of both the Navelate and

393 Pinalte fruits. Conjugated ABA lowered from 12 d to 18 d in the flavedo of both the parental

394 and mutant fruits, and these decreases were concomitant with the rise in the free ABA (Figs

17 395 6C-F). Given this result, it was not possible to rule out that the rise in the free ABA that took

396 place during this later period was due, at least in part, to the conjugated ABA. By day 12,

397 when the rise in free ABA was evident, no difference in the conjugated ABA levels were

398 observed between the flavedo of the control and infected Navelate fruit. Therefore, it is

399 interesting to consider that fungal ABA might contribute to the rise in the free ABA observed

400 after 12 d.

401 The results found when examining the spatial changes in the free and conjugated

402 ABA by taking samples of different flavedo tissue zones (healthy, macerated, with mycelium,

403 and sporulated) from the same fruit also revealed that the free ABA was higher in the

404 sporulated zone (Fig. 7A). However, differences in the conjugated ABA among the several

405 zones were negligible (Fig. 7B). Thus experiments were performed to determine whether P.

406 digitatum was indeed able to produce ABA.

407

408 3.5. P. digitatum is able to produce ABA

409

410 ABA levels were determined in the spores obtained from the cultures grown on PDA

411 medium during different periods. The results showed that the necrotrophic fungus P.

412 digitatum is able to produce ABA, and that the ability of the fungus to produce the hormone

413 varied with its age (Fig. 8). The maximum ABA levels were found when spores were taken

414 from the 7-day-old cultures. Thereafter, ABA lowered with fungal age.

415

416 4. DISCUSSION

417

18 418 Few studies are available about the role of ABA in the response of fruit crops to

419 phytopathogenic fungi infection. However, it has been demonstrated that this hormone may

420 favour, but also reduce the infection caused by different pathogens (Kaliff et al., 2007; Wang

421 et al., 2015; Wilkinson et al., 2018). As far as we know, there is only one report about citrus

422 fruit which shows that the hormone may participate in reducing Alternaria brown spot disease

423 (Llorens et al., 2013). Some data about evaluations made of natural disease (non-inoculated

424 fruit) incidence in fruit stored under conditions that favour peel damage may also suggest a

425 protective role of ABA against P. digitatum in citrus fruit (Alférez et al., 2005). Deng et al.

426 (2018) found that the expression of some involved in ABA biosynthesis and signalling

427 may be up- and down-regulated in response to P. digitatum infection. Yet whether ABA is

428 indeed involved in the disease resistance of citrus fruit against this pathogen remains

429 unknown. In this work, we investigated the possible involvement of ABA in the response of

430 orange fruit to the major postharvest pathogen P. digitatum.

431 The results showed that the drop in ABA in peel constitutes a rapid early response of

432 citrus fruit related to P. digitatum infection (Fig. 1A and Supplementary Fig. S2) and,

433 therefore, suggest that ABA is indeed involved in the susceptibility of citrus fruit to infection

434 by this necrotrophic fungus. A faster drop in ABA and quicker infection progression were

435 observed when profound inoculation, which affected peel tissue that is more susceptible to

436 infection (albedo) (Ballester et al., 2006), was performed (Figs. 1 and 2; a depth of 4 mm

437 versus 2 mm). The fact that ABA decreased before tissue maceration development became

438 relevant, and given that the faster infection progression was, the earlier ABA lowered (Fig.

439 1 and 2), both suggest that the ability of P. digitatum to infect citrus fruit is related to its

440 capacity to lower ABA levels. So these results suggest that ABA would play a defensive role

441 in citrus fruit against P. digitatum infection, which would agree with findings in grapes

19 442 infected with Glomerella cingulata (Wang et al., 2015), and in Brassica napus and

443 Arabidopsis infected with Leptosphaeria maculans (Kaliff et al., 2007). Unfortunately, very

444 little information about the role of ABA has been obtained by pharmacological experiments

445 when applying inhibitors of ABA biosynthesis. They have been widely used to determine the

446 role of ABA in different physiological processes and stress cues in plants. Many authors

447 mention them as specific ABA inhibitors, but they may have additional effects related mostly

448 to oxidative stress (Xiong et al., 2011). Under our experimental conditions, Tungs and NDGA

449 had a clear effect by reducing the in vitro growth of P. digitatum. According to this result,

450 the lesser disease incidence found in the fruit treated with these inhibitors could be related,

451 at least in part, to their direct effect on fungal viability. Consequently, we cannot fairly

452 conclude from this pharmacological approach that ABA plays a role in the defence response.

453 So our subsequent experiment concentrated on comparing the behaviour of the Pinalate

454 (ABA-deficient mutant) and the Navelate (parental) fruit, and examined the effect of NFZ

455 and exogenous ABA, which did not affect in vitro fungal growth. The fact that the ABA-

456 deficient mutant was more susceptible to infection reinforces the idea that ABA plays a

457 defensive role in citrus fruit against P. digitatum infection. In this context, it is worth

458 mentioning that although ABA may have direct antifungal activities (Khedr et al., 2018), the

459 hormone does not alter P. digitatum growth (Supplementary Fig. S4). Therefore, the

460 differences in the susceptibility to infection between Navelate and the ABA-deficient oranges

461 (Pinalate) are not related to a direct effect of ABA on the fungus. It was surprising to find

462 that NFZ favoured infection in the ABA-deficient mutant, but not in the parental fruit.

463 However, it caused dark areas to develop in Pinalate fruit peel, which indicated that NFZ

464 induces cell damage in this cultivar. Moreover, the NFZ concentration used in this

465 experiment did not modify the ABA levels compared to the control fruit, and a higher

20 466 concentration would further enhance peel damage. Therefore, by also considering that NFZ

467 might inhibit not only ABA, but also other metabolites (Supplementary Fig. S1), this

468 pharmacological approach was ruled out. Another finding which confirmed the hypothesis

469 that ABA may play a defensive role against P. digitatum infection in citrus fruit arose from

470 the fact that exogenous ABA had a clear effect by reducing infection in the ABA-deficient

471 mutant (Fig. 3B), and the Pinalate ABA-treated fruit showed similar ABA levels (Fig. 3D)

472 and a similar susceptibility to infection (Fig. 3B) as the untreated Navelate fruit (Fig. 3A).

473 Adding exogenous ABA to the parental fruit barely affected disease progression. However,

474 it should be taken into account that the parental fruit flavedo already has very high ABA

475 levels, and endogenous phytohormone levels might suffice to trigger cellular processes to

476 cope with pathogen attack. This agrees with previous findings which have shown that

477 exogenous ABA has very little effect on dehydration in Navelate, but triggers changes in the

478 expression profile of the ABA-perception system and reduces water loss in ABA-deficient

479 mutant Pinalate (Romero et al., 2012; 2014). Hence the above results indicate that ABA plays

480 a defensive role against P. digitatum in citrus fruit, but we ought to consider: 1) Pinalate fruit

481 is a spontaneous ABA-deficient mutant with a blockage in the carotenoid biosynthetic

482 pathway rather than a knockout mutant, in which the responsible for the last ABA

483 biosynthesis step is inoperative; 2) the partial effect of exogenous ABA on disease reduction

484 in Pinalate fruit. Therefore, we should contemplate that ABA could contribute to reduce

485 disease, but additional responses should participate in the complex network of the

486 mechanisms participating in plant disease resistance.

487 The results of the present work further revealed different factors that influence

488 variations in ABA content during the P. digitatum infection process (Fig. 9). The early ABA

489 decrease appears indicative of a fruit infection perception response. This decrease was

21 490 followed by a sharp increase in hormone levels in the parental fruit, which might be mediated

491 by ethylene. Previous findings have shown that exogenous ethylene induces changes in ABA

492 metabolism (Establés-Ortiz et al., 2016) and increases hormone levels in citrus fruit

493 (Lafuente et al., 1997). As expected (Chalutz et al., 1978), ethylene increased in both the

494 Navelate and Pinalate fruit in response to infection (Fig. 4A and 4B), but the rise in ABA

495 following the rise in ethylene occurred only in the parental fruit (Fig. 4C and 4D). The peel

496 of the fruit from both cultivars presented maceration, and even sporulation, by day 7

497 (Supplementary Fig. S7A). These results, together with those indicating that the ABA levels

498 were very low in the ethylene-treated Pinalate fruit (Fig. 5B), suggest that the rise in ABA

499 following the early infection-induced reduction in ABA in the infected Navelate fruit (Fig.

500 4) is more likely related to the infection-induced rise in ethylene than to fruit reaction to the

501 tissue maceration caused by P. digitatum.

502 Finally, at the later fruit infection times (Fig. 6) when sporulation was evident

503 (Supplementary Figs. S7B and S7C), a marked rise in ABA occurred in both the parental and

504 ABA-deficient fruit. This result, together with the results obtained from examining the

505 spatio-temporal (Fig. 7) changes in the free and conjugated ABA, suggest that such a rise in

506 ABA is the consequence of releasing the free ABA from the conjugated ABA, which is very

507 abundant in citrus fruit flavedo (Lafuente et al., 1997). Although plant β-glucosidases have

508 been involved in this release (Lee et al., 2006), advanced tissue degradation suggests that

509 fungal β-glucosidases are more likely to be responsible for conjugated ABA hydrolysis than

510 fruit β-glucosidases, a hypothesis that deserves future research. Whether the rise in the free

511 ABA is merely a consequence of the release from its conjugate without affecting fungal

512 colonisation, or whether it plays a role in the resistance of citrus fruits against the fungus,

513 remains elusive. Nevertheless, such a release occurs too late, when the fungus has already

22 514 colonised the fruit. Another interesting idea is that P. digitatum is able to produce the

515 hormone. This was confirmed by analysing ABA in the spores obtained from PDA cultures

516 (Fig. 8), although the ABA levels produced by the fungus were much lower than those

517 detected in rotten fruit. As far as we know, this is the first report to show the ability of P.

518 digitatum to produce ABA and, therefore, the putative function of this ability is unknown.

519 Other reports have demonstrated that ABA may be produced by other necrotrophic fungi

520 (Nambara and Marion-Poll, 2005) through the direct pathway, while ABA

521 production in plants is derived from the indirect pathway (Lievens et al., 2017). There is

522 converging evidence for ABA used as a virulence factor (Chanclud and Morel, 2016) and

523 indicating that ABA may act as a pathogen effector and an immune regulator (Lievens et al.,

524 2017). The fact that the ABA derived from P. digitatum mainly increased when sporulation

525 was evident indicates that fungal ABA is not a primary virulence factor of this fungus, but

526 might help infection progression. In line with the present work, it is worth mentioning the

527 proposed role of ABA in altering cell wall properties that may influence necrotrophic

528 infection outcome (Nafisi et al., 2015). Therefore, the massive rise in ABA in later P.

529 digitatum infection stages might contribute to favour the pathogen entering in citrus fruit,

530 and hence its colonisation. The aim of this paper was to understand how the fruit ABA

531 influences the susceptibility of oranges to infection by this pathogen. Our experiments

532 demonstrate, for the first time, that P. digitatum is able to produce ABA and, therefore, open

533 up new research lines on the role of fungal ABA in infection progression.

534

535 5. CONCLUSIONS

536

23 537 In short, both, citrus fruit and P. digitatum, the main postharvest pathogen that causes

538 rots in this important agronomic crop, produce ABA. In fruit, ABA seems to play a defensive

539 role against infection caused by the pathogen. Fungal ABA appears not to be a primary

540 virulence factor, but it cannot be ruled out that the fungal hormone may help fruit

541 colonisation. The results also show, for the first time, that the trend in ABA changes varies

542 during fruit infection because of different factors (Fig. 9). ABA decreases in early stages as

543 a consequence of P.digitatum infection. Thereafter, it increases and this increase is likely

544 related to the P. digitatum-induced rise in ethylene. This is demonstrated by the fact that

545 ABA does not increase in Pinalate in this infection stage, and exogenous ethylene barely

546 increases hormone levels in this ABA-deficient cultivar. Finally, fungal ABA, but also the

547 release of ABA from the conjugated ABA, could be responsible for the marked rise in the

548 free ABA in the later infection stage.

549

550 APPENDIX A: SUPPLEMENTARY DATA

551

552 The Supplementary Material related to this article can be found in the online version.

553

554 ACKNOWLEDGEMENTS

555 The technical assistance of J. Cerveró and M. Ferragut is gratefully acknowledged.

556 We also thank Dr. G. Ancillo (IVIA) for allowing us to use the Spanish Citrus Germoplasm

557 Bank, and Dr. I. Lopez-Diaz and Dr. E. Carrera for the ABA quantification by UPHLC

558 carried out at the Plant Hormone Quantification Service, IBMCP, Valencia, Spain. This work

559 was supported by the Spanish Ministry of Economy and Competitiveness (Research Grants

24 560 AGL2014-55802-R and AGL2017-88120-R) and the Generalitat Valenciana, Spain (Grant

561 PROMETEOII/2014/027).

562

563 FUNDING

564 This work was supported by the Spanish Ministry of Economy and Competitiveness

565 (Research Grants AGL2014-55802-R and AGL2017-88120-R) and the Generalitat

566 Valenciana, Spain (Grant PROMETEOII/2014/027).

567

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680

681

28 682 Figure Legends

683 Figure 1. Changes in the free ABA in the peel discs (A) and the lesion diameter of the

684 macerated area (B) of the Navelina fruit harvested in November and inoculated at a depth of

685 4 mm () with 10 µL of P. digitatum (106 conidia mL-1). The free ABA is expressed per kg

686 of peel. The control samples () were mock-inoculated with 10 µL of water. After infection,

687 fruit were maintained in the dark at 20ºC. The error interval indicates the standard error of

688 the estimated mean value. Different letters mean significant differences (p ≤ 0.05) between

689 the infected and control fruit of the same cultivar for the same storage period.

690

691 Figure 2. Changes in the free ABA in the flavedo discs (A) and the lesion diameter of the

692 macerated area (B) of the Lane Late fruit inoculated at a depth of 2 mm () with 10 µL of P.

693 digitatum (106 conidia mL-1). The free ABA is expressed per kg of flavedo. The control

694 samples () were mock-inoculated with 10 µL of water. After infection, fruit were

695 maintained in the dark at 20ºC. The error interval indicates the standard error of the estimated

696 mean value. Different letters mean significant differences (p ≤ 0.05) between the infected

697 and control fruit of the same cultivar for the same storage period.

698

699 Figure 3. Effect of ABA and NFZ on the changes in the lesion diameter of the macerated

700 area of the Navelate (A) and Pinalate (yellow ABA-deficient mutant) (B) fruit infected at a

701 depth of 2 mm with 104 conidia mL-1 of P. digitatum (10 µL). The percentage of decay in the

702 Navelate () and Pinalate (□) fruit is shown in Fig. 3C. The effect of exogenous ABA on the

703 hormone levels (measured immediately after applying ABA) on the flavedo of the fruit of

704 both cultivars is found in Fig. 3D. The free ABA is expressed per kg of flavedo. To visualise

705 fruit colour in the photographs in Figs. 3A and 3B, readers are referred to the web version of

29 706 this article. The error interval indicates the standard error of the estimated mean value.

707 Different letters mean significant differences (p ≤ 0.05) among the various conditions

708 evaluated for the same fruit cultivar and storage period (Fig. 3B and 3C). No significant

709 differences in the lesion diameter were found for the same day between the control Navelate

710 fruit and Navelate fruit treated with ABA or NFZ. Therefore, no letters are included in Fig.

711 3A. The significant differences (p ≤ 0.05) between the Navelate and Pinalate fruit, treated or

712 not with ABA, are indicated by distinct letters in Fig. 3D.

713

714 Figure 4. Changes in ethylene production (A and B) and in fthe ree ABA levels (C and D)

715 in the flavedo of the Navelate (A and C) and Pinalate (B and D) fruit inoculated at a depth of

716 4 mm with 104 conidia mL-1 of P. digitatum (10 µL) () and their respective control samples

717(). Ethylene production and the free ABA are expressed per kg of flavedo. Fruit were

718 maintained in the dark at 20ºC. The error interval indicates the standard error of the estimated

719 mean value. Distinct letters mean significant differences (p ≤ 0.05) between the control and

720 infected fruit for the same cultivar and storage period.

721

722 Figure 5. Effect of exogenous ethylene on the free ABA levels in the flavedo of the Navelate

723 (A) and Pinalate (B) fruit. The free ABA is expressed per kg of lavedo. Fruit were treated for

724 up to 7 d with 2 µL L-1 ethylene (grey bars) or maintained in air (control, black bars) at 20ºC.

725 The error interval indicates the standard error of the estimated mean value. Distinct letters

726 mean significant differences (p ≤ 0.05) between the fruit treated with air and ethylene for the

727 same cultivar and storage period.

728

30 729 Figure 6. Changes in the lesion diameter of the macerated (A) and sporulated (B) zones of

730 the Pinalate (□) and Navelate () fruit inoculated at a depth of 2 mm with 10 µL of P.

731 digitatum (104 conidia mL-1). The changes in the free (C and D) and conjugated (E and F)

732 ABA levels in the flavedo of the infected () and control () Navelate (C and E) and Pinalate

733 (D and F) fruit are also shown. The free and conjugated ABA are expressed per kg of flavedo.

734 After infection, fruit were maintained in the dark at 20ºC. The error interval indicates the

735 standard error of the estimated mean value. Distinct letters mean significant differences (p ≤

736 0.05) between the infected and the control fruit for the same citrus cultivar and storage period.

737

738 Figure 7. The spatial changes in the free (A) and conjugated (B) ABA levels in the flavedo

739 of the Lane Late oranges inoculated at a depth of 4 mm with 105 conidia mL-1 of P. digitatum

740 (10 µL). The free and conjugated ABA are expressed per kg of flavedo. Samples were taken

741 at 6 dpi. The error interval indicates the standard error of the estimated mean value. Distinct

742 letters mean significant differences (p ≤ 0.05) among the different zones.

743

744 Figure 8. The free ABA levels of the P. digitatum spores obtained from the cultures grown

745 during different periods at 24ºC. The error interval indicates the standard error of the

746 estimated mean value.

747

748 Figure 9. Schematic diagram of the factors influencing the free ABA evolution during citrus

749 fruit infection. To visualise the figure colour, readers are referred to the web version of this

750 article.

31 Fig. 1

Peel 4 mm depth Navelina A 1.0 a a A 0.8 a B A

1 0.6 - g

k b 0.4 g b m 0.2 b 0.0 wounding B a ) 2.0 infection m

c 100 % (

infection r

e 1.5 t e m

a 1.0 i d

n o

i 0.5 s

e b L 0.0

0 1 2 3 Days at 20 ºC Fig. 2

Flavedo 2 mm depth Lane Late A 2.0 A

B 1.5 a A a 1 a - g k

1.0 a g m 0.5 b b

0.0 Wounding B Infection ) 2.0 m c (

r 1.5 e t e

m 1.0 a 22 % i

d infection

n

o 0.5 i

s a e L 0.0 b 0 1 2 3 Days at 20 ºC Fig. 3

A B Navelate Pinalate Control 8 NFZ 1 mM ABA 1 mM a ) m c

( 6

r e t e

m 4 a b a i d

n a o i 2 b s e L b c 0 b c 0 2 4 6 8 10 0 2 4 6 8 10 C Days at 20 ºC Days at 20 ºC D Navelate Navelate Pinalate Pinalate aControl 8 NFZ 1 mM 80 ABA 1 mM a ) ab 3 m c

a ( 6

r A e

60 a t B y e b A a

m 4 a b c a 2 1 i e - d

g D

40 n k a

o i b % 2 g s e

a m L b c 20 0 1 b b c a b c 0 2 4 6 8 10 0 2 4 6 8 10 0 Days at 20 ºC Days at 20 ºC 0 0 2 4 6 8 10 IN A V A P B A B A N +A Days at 20 ºC + V IN A P N Fig. 4

Navelate Pinalate

a A Wounding B Infection

300 300 n n

a o i o i t

) t

) a 1 c - 1 c

- a u s u

s d

1 d - 1 o - o

200 200 r g r g k p

p

k l

l e o e o n

n a m

a e l m e p l y p ( y

( 100 100

a h h a t t a E E a a b a a a a b b a 0 b b a a a 0 C D 2.4 a 2.4 a 2.0 a a a 2.0 A A a B B 1.6 b a 1.6 A A

a 1 1 - - g g 1.2 a 1.2 k k b

g g b m m 0.8 0.8

0.4 0.4

0.0 0.0 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 Days at 20 ºC Days at 20 ºC Fig. 5

Navelate Pinalate Air A B 0.8 6 Ethylene a 0.6 A A B B A A

4 a 1 1 - - g g 0.4 k k

g g a a a a m

m b 2 a a a b a 0.2

0 0.0 0d 3d 7d 0d 3d 7d Fig. 6

Macerated Sporulated ) Pinalate

m 8 A a B

c Navelate (

3

r a Navelate Pinalate

e 6 a t e 2 m 4 a a a i d 1 n 2 a a a

o b i s

e 0 b b b 0 L 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 Days at 20 ºC

Navelate Pinalate 5 C a D wounding ) infection A 4

B a A a A B

A

1 3

- e g e k a

r 2 g F

m 1 a ( b b b 0 a b

a

) E F A 40 10 A B a B A

a

A 8 d 30 a a e 1 t -

a 6 g g k 20 a

u j g 4

n a

m a o b ( 10 a b

C 2 b 0 0 0 3 6 9 12 15 18 0 3 6 9 12 15 18 Days at 20 ºC Days at 20 ºC Fig. 7

A ) 2.0 a A B A

1 - 1.5 g b k

g b

m b (

1.0 A B A

e 0.5 e r F

) 0.0

A B

B a a A

16 1 -

g a a k

g 12 m (

A B

A 8

d e t a

g 4 u j n o

C 0 y e d th t m e l ra liu t a e e la e c c ru H a y o M M p S Fig. 8

Pd1

20

8 - 0

1 15 * ) A B A 1

- 10 e r o p s 5 g p (

0 0 5 10 15 20 Days at 24 ºC Fig. 9 Supplemental Fig. S1. Schematic diagram of ABA biosynthesis in plants and the effect of NFZ, NDGA and Tungstate inhibiting its synthesis.

Supplemental Fig. S2. Changes in free ABA in peel discs of Navelina fruit harvested in January and inoculated at 4 mm depth () with 10 µL of P. digitatum (106 conidia mL-1). Control samples () were mock-inoculated with 10 µL water after wounding the tissue at 4 mm depth. After infection, fruis were maintained under darkness at 20 ºC. The error interval indicates the standard error of the estimated mean value. Different letters mean significant differences (p ≤ 0.05) between the infected and the control fruit for the same storage period.

Janauary 1.0

0.8

A a

B a A

1 0.6

- a g

k a

g 0.4 m 0.2 b wounding infection 0.0 b 0 1 2 3 Days at 20 ºC Supplemental Fig. S3. Effect of Tungs (10 mM) on lesion diameter (cm) of macerated circular zone (A) of Lane Late oranges inoculated with 10 µL of P. digitatum (106 conidia mL-1); and of different Tungs concentrations, ranging from 0.1 to 100 mM, on the in vitro fungal growth in PDA (B), expressed as % inhibition fungal growth (section 2.4) after determining the fungal growth diameter, and in PDB (D), expressed as optical density at A492. The error interval indicates the standard error of the estimated mean value.

Fruit PDA PDB 4 Control A 100 mM a a a a B 10 mM C 10 mM 1.2 Tungs a 100 1 mM 1 mM 0 mM a n 0.1 mM a b b b o 1.0 i )

t 0 mM

i 80

m 3 b i c h (

0.8 n r i

e b b

t 60 h 2 t e a 9 4 w 0.6 m o

2 A a r

i b g d

40

l n a c 0.4 o g

i a n s a b

u c e f 20 bc c

1 L a 0.2 b d c % c d d b 0 e e e de 0.0 0 4d 6d 0 2 4 6 0 2 4 6 Days at 24 ºC Days at 24 ºC

Supplemental Fig. S4. Effect of 1 mM ABA on the in vitro fungal growth in PDA, expressed as fungal growth diameter (cm), and in PDB, expressed as optical density at A492. The error interval indicates the standard error of the estimated mean value. No significant differences between control and ABA-treated samples were found at any time of the experiment.

PDA PDB ABA 1mM 6 ABA 1 mM Control Control 1.2 ) m

c 5 ( 1.0

r e t e 4 0.8 m a i 2 d 9

3 0.6 4 h t A w o r 0.4 g 2

l a g

n 1 0.2 u F 0 0.0

0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 Days at 24 ºC Days at 24 ºC Supplemental Fig. S5. Pinalate fruit showing NFZ-induced tissue darkening.

Supplemental Fig. S6. Effect of treating Navelate and Pinalate fruit for 3 d with 40 µL L-1 ethylene or maintaining them in air at 20 ºC on free ABA levels in the flavedo. The error interval indicates the standard error of the estimated mean value. Different letters mean significant differences (p ≤ 0.05) among different samples for the same cultivar. FH: freshly harvested fruit.

Pinalate Navelate a 3 A B

A 2 b 1 - g

k b

g m 1

a b b 0 ir e ir e FH n FH n A e A e yl yl th th E E Supplemental Fig. S7. Navelate and Pinalate flavedo disks showing poor sporulation, taken 7 d after fruit inoculation with 10 µL of P. digitatum (4 mm depth, 104 conidia mL-1) (A). Fruit were completely rotten by 12 d post-inoculation (dpi) (B); and the albedo was almost disintegrated and the peel tissue structure was disorganized by 18 dpi (C).