1 Involvement of abscisic acid in the resistance of citrus fruit 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 hormone 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 fruits 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 ethylene 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 plant hormone 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 hormones, like jasmonic acid (JA), salicylic acid (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 Leptosphaeria maculans (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 carotenoid biosynthetic pathway that results
79 in a yellow-pigmented fruit and a marked reduction in ABA levels following the biosynthesis
80 of carotenoids (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 enzyme in the biosynthesis of ABA in citrus fruit; 3)
164 Norflurazon (NFZ) (PubChem CID: 33775), which inhibits phytoene 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 genes 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 gene 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 mevalonic acid 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).