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1 Manuscript type: original paper

2 Investigating the effects of ethphon (2-chloroethylphosphonic acid) on

3 Persian walnut
4 Abstract: Walnut orchards in Iran encounter two significant challenges, namely dark kernels
5 and uneven ripening of the fruit. In order to address these issues, the research here is conducted
6 to investigate the impact of ethephon on walnut ripening. The study followed a randomized
7 complete block design with three replicates and nine treatments. Parameters such as fruit
8 maturity, kernel quality, and dry matter percentage were evaluated. The findings revealed that
9 the highest quality kernel was obtained when harvested on Aug 22nd, which is approximately
10 three to four weeks prior to the normal harvest time. As the regular harvest time approached,
11 the quality of the kernel diminished. To maintain nuts quality and facilitate the peeling of the
12 green hull around Aug 22nd, a concentration of 1920 ppm of ethephon proved effective.
13 Furthermore, for fruits harvested between Aug 31st and Sept 9th, the application of 1920 ppm
14 of ethephon exhibited a positive influence on kernel quality. Consequently, the combination of
15 early harvesting and the utilization of ethephon yielded the whitest walnut kernels.
16 Keywords: Ethephon, Kernel darkness, Ripening, Walnut

17

18 Walnuts, scientifically known as Juglans regia, belong to the Juglandaceae family, which
19 encompasses 20 different species. The Iranian walnut variety is particularly susceptible to
20 various factors that can impact its quality (Akin and Erdem, 2018; Askary et al., 2014),rich in
21 phospholipids, proteins, vitamin E, and non-saturated fatty acids, walnuts offer numerous
22 nutritional benefits (Geng et al., 2021). Furthermore, they are known for their high levels of
23 arginine/lysine, antioxidants, phytosterols, and polyesters (MelkamuA et al., 2008). Studies
24 have demonstrated the potential of walnuts in preventing cardiovascular issues, highlighting
25 their positive impact on health (Ni et al., 2022). Fresh walnut kernels, compared to dried ones,
26 have higher moisture content and are richer in phenolic acids and antioxidants (Chatrabnous et
27 al., 2018). The distinct flavor of fresh walnuts makes them more appealing to consumers than
28 completely dried kernels (Grosso et al., 2020). However, fresh walnuts, whether peeled or
29 unpeeled, are more susceptible to microbial contamination and pathogen infestation during the
30 post-harvest period (Shirani et al., 2020). The quality of walnuts significantly relies on pre-
31 harvest processing practices (Aini et al., 2019; Jha et al., 2012). Delaying the harvest negatively
32 affects fruit quality, while the presence of endogenous ethylene has been found to have positive

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33 effects on crop protection (Aini et al., 2019). Typically, there is a 2-3 weeks gap between kernel
34 ripening and the regular harvest of walnuts. During this period, the walnut kernels tend to
35 darken in color (Ortiz et al., 2019). As the fruit ripens, the quality of the kernel gradually
36 declines until the nut is completely dried. Previous research has indicated that delaying the
37 harvest due to prolonged fruit ripening can adversely impact kernel quality, including factors
38 such as kernel transparency and susceptibility to insect infestation (Hartel et al., 2018; Ochami,
39 2021). Therefore, it is crucial to explore methods and substances that can facilitate ripening
40 acceleration without causing any detrimental effects.
41 Ethephon is a synthetic compound that serves as an exogenous source of ethylene in horticulture
42 (Keshavarzi et al., 2021). Its application has been found to enhance both the quality and quantity
43 of sugar in plants from the Saccharum genus, while also improving their resistance against
44 pathogenic organisms (Liao et al., 2003). Furthermore, appropriate concentrations of ethephon
45 have been shown to increase the levels of active polyphenols, flavonoids, and phenylalanine
46 ammonia-lyase in soybean leaves (Wang et al., 2011). Additionally, treating plants with
47 ethephon and ethylene has been found to augment the vitamin C content and disease resistance
48 (Li et al., 2022).
49 Ethephon has been recognized for its ability to expedite fruit ripening, allowing for a full harvest
50 without an extended drying period (Liao et al., 2003). Studies have shown that the application
51 of ethephon, which activates ethylene, can effectively stimulate the synthesis of defense
52 proteins in tomatoes. This mechanism enhances the plant's natural resistance against pathogens,
53 bolstering its ability to combat diseases and infections (Keshavarzi et al., 2021; Liao et al.,
54 2003). Given these findings, the present study was conducted to examine the impact of ethephon
55 on walnut ripening. The evaluation focused on factors such as kernel color, the ripening of the
56 green shell at the appropriate time, and the harvesting process. By investigating these aspects,
57 the study aimed to address the challenges observed in walnut production and explore the
58 potential of ethephon as a solution.
59

60 METHODS AND MATERIALS

61 For the investigation into the effect of ethephon and harvest time on walnut kernel quality, a
62 selection of walnut trees in the Mazandaran province, was made. The choice of this region was
63 deliberate, as it exhibits a warmer climate and higher relative humidity compared to other
64 walnut-growing regions like Azerbaijan. This specific climate in the selected region allows for

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65 the study of harvest time and fruit quality due to a significant time gap between kernel
66 maturation and mesocarp maturation, which negatively impacts kernel quality. Approximately
67 60-85% of the harvested walnut kernels in this region tend to exhibit black or brown coloration.
68 Therefore, it was feasible to examine the impact of harvest dates and ethephon application on
69 peeling, quality, and kernel density in this area.

70 Experimental design. The test was conducted using a split-plot design with a randomized
71 complete block design, comprising three replicates. The study focused on two factors: harvest
72 date (Factor A) and ethephon application (Factor B). Harvest date was explored at three
73 different levels, while ethephon treatment, which utilized a 48% concentration solution, was
74 examined across nine levels after dilution (Table 1).
75 Table 1. The studied factors
A
A1= 22 Aug A2= 31 Aug A3= 9 Sept
B
B1=0(control) B2=120 B3=240

B4=480 B5=720 B6=960

B7=1200 B8=1920 B9=4800
76 A – harvest date; B – ethephon application

77 Accordingly, a total of 27 treatment combinations were allocated within each block. It is

78 noteworthy that the levels in Factor B were expressed in parts per million (ppm), with the
79 inclusion of a ninth level set at a concentration of 4800 ppm. This specific level aimed to
80 investigate the potential detrimental impacts associated with high ethephon concentrations on
81 the quality of Iranian walnut cultivars.

82 Conducting the experiment. To conduct the test, each group of three trees was treated as a
83 separate block. The fruits from each tree were harvested on one of the three specified dates and
84 subsequently immersed in a solution containing ethephon. It is important to note that ethylene
85 production can be induced by various forms of injury, such as mechanical damage, high winds,
86 birds pecking, pests, and pathogens; In the case of fruit being damaged during harvest or
87 transportation, autocatalytic ethylene production is triggered, which can overlap with the
88 ethylene produced by the ethephon. Therefore, conventional harvesting methods could not be
89 employed as they may damage the mesocarp, leading to inconsistent experimental units. To

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90 ensure the integrity of the experiment, the fruits were carefully collected at different times from
91 each tree, taking precautions to prevent any damage. Following the harvest, the fruits from each
92 individual tree were carefully placed into separate bags to ensure their protection during
93 transportation. These bags were then consolidated into a larger sack. The fruits underwent a
94 thorough washing process to eliminate any dust or debris. Subsequently, the fruit tails were
95 trimmed to the level of the green rind, ensuring that no damage caused by the tails of
96 neighbouring fruits remained. From each tree, only healthy fruits were selected, and they were
97 then divided into nine equal-sized lots for further analysis and evaluation.
98 The fruits from each batch were immersed in the ethephon solution, corresponding to one of
99 the specific concentration levels, for approximately 20 seconds. Subsequently, the treated fruits
100 were carefully placed inside pre-prepared cloth bags made of cotton, measuring approximately
101 22 x 19 cm. After carefully placing the fruits inside bags, the bags were sewn shut and subjected
102 to a controlled environment with a relative humidity ranging between 80% and 85% and a
103 constant temperature of 18 °C. This specific combination of temperature and humidity has been
104 observed to promote the maximum production of ethylene by the ethephon. It is worth noting
105 that the optimal temperature range for ethylene synthesis through the action of ethephon falls
106 within the interval of 16 to 29 °C. Moreover, maintaining a humidity level of 80-85% helps
107 prevent significant moisture loss in the fruits, which could lead to adverse effects on the
108 mesocarp when exposed to ethephon, thereby accelerating the ripening process.After an 8-day
109 period, the bags were opened, and the effectiveness of ethephon in facilitating the peeling of
110 the green rind was assessed and ranked on a scale from 0 to 25. A score of 0 indicated complete
111 attachment of the mesocarp to the endocarp, while a score of 25 indicated complete cracking
112 and detachment of the green skin from the endocarp without the need for a knife. Intermediate
113 scores were assigned for fruits exhibiting varying degrees of detachment.
114 To evaluate color quality, the fruit skin was initially divided into four sections using a knife,
115 and each section was marked individually. Subsequently, the nuts were subjected to a 3-day
116 period of sunlight exposure for drying. After the mesocarp was shelled and dried, the nuts were
117 cracked open, and the kernels were assessed and scored from zero to 20 based on their color
118 quality. Kernels that were completely dark in color received a score of zero, while brown-
119 amber-colored kernels were assigned scores ranging from 2 to 4, and creamy or amber-colored
120 kernels received scores of 4 to 5. Following the marking of the kernels, the dry matter content
121 in the nuts was measured. In this section, only levels one and three of the main factors (harvest
122 date) were examined.

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123 To assess the weight of walnut kernels in each experimental unit, the kernels were initially
124 crushed and weighed individually. The empty containers and the containers with the crushed
125 kernels were subsequently subjected to autoclaving at a temperature range of 110-100°C.
126 Following autoclaving, moisture percentage was obtained using Equation 1:

127 Moisture percentage =

weight of the plate and samples before drying−weight of the plate and samples after drying
128 Eq (1)
weight of the plate and samples before drying−empty plate weight

129 The dry matter percentage of the kernel was determined through the application of Equation 2,
130 which is as follows:

131 𝐾𝑒𝑟𝑛𝑒𝑙′𝑠 𝑑𝑟𝑦 𝑚𝑎𝑡𝑡𝑒𝑟 𝑝𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 = 100 − 𝑚𝑜𝑖𝑠𝑡𝑢𝑟𝑒 𝑝𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 Eq (2)

132 Data analysis. Walnuts were assessed for peelability and kernel quality eight days after being
133 immersed in an ethephon solution. Subsequently, they were stored in specialized bags under
134 controlled conditions of 80-85% humidity and 18°C. The average scores of the samples
135 obtained from each experimental unit were computed. The data were analyzed and visualized
136 using Harvard Graf, Mstatc, and Quatro Pro software. It is important to mention that Duncan's
137 test was employed to evaluate the statistical significance of mean comparisons in this
138 experiment.
139
140 RESULTS

141 Investigating the effect of ethephon on fruit ripening. The mean concentration of ethephon
142 during each harvest date (Table 2).
143
144 Table 2. Ethephon level average in each of the harvesting dates
Ethephon
concentration
(ppm)
B1=0 B2=120 B3=240 B4=480 B5=720 B6=960 B7=1200 B8=1920 B9=4800
Harvest date

A1: 22 Aug 12.000 11.973 12.127 13.500 14.627 15.710 17.043 17.293 16.917
A2:31 Aug 13.167 13.167 16.500 16.400 17.167 18.467 19.540 19.777 19.233

A3:9 Sept 17.567 18.767 19.833 20.100 20.233 21.333 21.267 21.667 22.100
145

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146 Table 2 presents data showing minimal fruit ripening in groups at each harvest moment.

147 Furthermore, a positive correlation is observed between higher concentrations of ethephon and
148 increased fruit ripening, leading to the separation of the mesocarp. Furthermore, the highest
149 values across the various ethephon levels are consistently associated with the harvest date of
150 Sept 9th.
151 The data obtained from this experiment were initially analyzed using a split-plot design.
152 Subsequently, Ea (trial error associated with the main factor) was assessed in conjunction with
153 Eb (experimental error associated with the secondary factor). As no significant differences were
154 observed, a polarization operation was conducted (Table 3).
155
156 Table 3. Analysis of data variance in terms of fruit ripening walnuts
Source of change Degree of freedom Sum of squares
Block 2 510.041 **
Factor A (harvest date) 2 223.418 **
Factor B (Ethephon concentration) 8 35.822 **
A×B 16 1.526 ns
Error 52 4.341
Total 80
157 **: significant at 0.01 level; Ns: insignificant

158 The results indicate statistically significant variations in fruit maturation levels among the
159 experimental blocks, as well as among the different harvest dates and concentrations of
160 ethephon. Moreover, there is no observed correlation between factor A (harvest date) and factor
161 B (ethephon concentration), suggesting that these factors independently influence fruit
162 ripening. The experiment demonstrates a high level of accuracy, as determined by the following
163 Equation (3):

12
164 % × 100 = 𝑚𝑠𝑒 = 𝑐. 𝑣 Eq (3)
01
𝑥000

165 The Effects of Treatments. The comparison of average fruit ripeness among different harvest
166 dates were performed using Duncan's test at a significance level of 0.01 (Table 4).
167

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168
169 Table 4. Comparison of average ripeness across different harvest dates
Harvest date Average ripeness Class
A1 14.577 C
A2 17.135 B
A3 20.319 A
170 Table 4 illustrates significant variations in the average fruit maturity across different harvest
171 dates. The highest average maturity is observed on Sept 9th, while the lowest average is
172 recorded on Aug 22nd. Consequently, during the normal harvest period, mesocarp separation
173 becomes easier (Figure 1). The "class A" concentration is identified as the most effective for
174 promoting walnut ripening. The findings indicate that as the ethephon concentration increases,
175 fruit ripening improves. This trend continues until the eighth level of factor B (1920 ppm
176 ethephon). Notably, there is a significant difference between the eighth level and the control
177 level at the 5% level. However, fruit ripening is reduced at the ninth level of factor B (4800
178 ppm) compared to the eighth level (Figure 2).

30

25
20.319
Average maturity (%)

20
17.135
14.577
15

10

Harvesting dates
179
180 Figure 1. Comparison of maturity levels across different harvesting dates
181

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25

19.283 19.579 19.417

20 18.503
17.342
16.667
Average maturity (%)

16.153
14.902
15 14.244

10

0
0 120 240 460 720 960 1200 1920 4800

Ethephon concentration (ppm)

182
183 Figure 2. Average fruit maturity in different Ethephon concentrations

184 The influence of ethephon on walnut kernel quality. Following the removal of the green skin
185 and sun drying, the nuts were broken and evaluated for kernel quality (Table 5). Additionally,
186 the results of comparing the average fruit maturity at various ethephon concentration levels
187 using Duncan's test at a significance level of 5% are shown in the mentioned table.

188 Table 5. Comparing the average fruit maturity in different levels of ethephon concentration
Ethephon concentration (ppm) Average ripeness Class
B1 (control batch) 14.244 F
B2 (120) 14.902 EF
B3 (240) 16.153 DEF
B4 (480) 16.667 CDF
B5 (720) 17.342 BCD
B6 (960) 18.503 ABC
B7 (1200) 19.283 AB
B8 (1920) 19.579 A
B9 (4800) 19.417 AB

189 The highest quality within each level B corresponds to the harvest on Aug 22nd, whereas the
190 lowest quality is observed on Sept 9th (Table 6). Notably, there is a significant decline in
191 average fruit maturity in the A1 and A2 levels lots as the ethephon concentration reaches Level
192 B9 (4800 ppm). The excessive ethylene present in this concentration of ethephon is likely to

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193 react with the biochemical compounds in the kernel, consequently diminishing its quality
194 (Table 7).
195
196 Table 6. The mean of kernel quality data
Ethephon
concentration
(ppm) B1=0 B2=120 B3=240 B4=480 B5=720 B6=960 B7=1200 B8=1920 B9=4800

Harvest date
A1: 22 Aug 12.720 12.92 14.238 14.334 14.250 14.380 14.960 15.020 9.860
A2:31 Aug 10.070 10.783 10.61 10.813 10.960 11.800 11.600 12.557 9.570
A3:9 Sept 9.820 8.793 7.850 6.710 5.917 7.613 6.693 6.557 7.775
197

198 Table 7. Walnut kernel quality (kernel color) variance analysis

Source of change Degree of freedom Sum of the squares
block 2 317.636 **
Factor A (harvest date) 2 236.583 **
A error 4 18.855
Factor B (Ethephon concentration) 8 4.411 ns
A×B 16 5.362 ns
B error 41* 703.4
Total 75
199 C.V.=20.371%; *: Due to the loss of 5 units, the error rate of the test error was reduced by 5
200 digits; **: significant on 0.05 level; ns: insignificant
201 The results reveal statistically significant differences in the effects of the test blocks, as well as
202 a notable distinction in kernel maturity among different harvest dates (P<0.05). However, there
203 is no significant difference in fruit maturity across various ethephon concentrations.
204 Additionally, the correlation analysis indicates that factor A (harvest date) and factor B
205 (ethephon concentration) exert independent influences on fruit ripening.

206 The average comparison. Table 8 presents a comparative analysis of walnut kernel quality
207 among various harvest dates.

208 Table 8. Comparison of walnut kernel quality across different harvest dates
Harvest date Average quality Class
A1: 22 Aug 13.425 A
A2: 31 Aug 10.981 A
A3: 9 Sept 7.533 B

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209 The results demonstrate significant variations in the average quality of walnuts harvested on
210 Sept 9th compared to the walnuts harvested on the other two dates (P<0.05). There is no
211 significant difference in kernel quality between walnuts harvested on Aug 22nd and Aug 31st,
212 with both exhibiting relatively favorable levels of quality. However, the lowest kernel quality
213 is observed in walnuts harvested on Sept 9th. This suggests that as the typical harvest date
214 approaches, there is a decline in kernel quality (Figure 3).

14
13 13.425
12 10.981
Average quality

11
10
9
7.533
8
7
6
22 August 31 August 9 September

different harvest dates (factor A)

215
216 Figure 3. The comparison of average walnut kernel quality across different harvest dates

217 The comparison of ethephon concentrations. According to the variance table, the different
218 concentration levels of ethephon (factor B) did not yield significant changes. The discrepancy
219 may be attributed to a significant decline in quality observed with an increase in the ethephon
220 level in factor A3 (harvest date: Sept 9th), as well as data overlap between this subfactor and
221 others. To address this issue, the third level of factor A (harvest date: Sept 9th) should be
222 excluded. Table 9 presents the revised analysis after removing subfactor A3 and its associated
223 data, considering only subfactors A1 and A2.
224
225 Table 9. Variance analysis of walnut kernel quality without the A3 sub-factor
Sources of change Degree of freedom Sum of squares
Block 2 227.523 ns
Factor A (harvest date) 1 80.596 ns
A error 2 29.747
Factor B (Ethephon concentration) 8 9.190 **
A ×B 8 1.465 ns
Error 29 606.2
total 50
226 C.V.= 13.23%; **: significant at 0.01 level; NS: Non-significant

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227 The results of comparing kernel qualities, excluding sub-factor A3 from the calculations, are
228 presented in Table 10.

229 Table 10: The average kernel quality comparison across the two harvest dates
Ethephon concentration Average quality Class at 0.05 level
Control batch 11.395 BC
120 ppm 11.852 AB
240 ppm 11.495 BC
480 ppm 12.608 AB
720 ppm 12.605 AB
960 ppm 13.09 AB
1200 ppm 13.28 AB
1920 ppm 13.788 A
4800 ppm 9.715 C

230 Table 10 illustrates that an increased concentration of ethephon corresponds to improved nut
231 quality. However, when the fruit is treated with a concentration of 4800 ppm, the kernel quality
232 is significantly reduced, resulting in the lowest average quality observed at this level (Figure
233 4).

15

13.788
14
13.28
13.09
13 12.608 12.605
Average quality (%)

11.852
12 11.495
11.395

11

10

9.715
9

8
0 120 240 480 720 960 1200 1920 4800

Ethephon concentaration (factor B)

234
235 Figure 4. Comparison of mean walnut kernel quality across different ethephon levels in 2
236 harvest dates

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237 The effects of ethephon on walnut kernels’ dry matter (density). The comparison is made
238 between the dry matter of the first level of factor A (representing the initial harvest time
239 associated with the highest quality) and the third level of factor A (representing the final harvest
240 time where mesocarp separation occurs easily even without the use of ethephon) (Table 11).
241 The comparison of these levels is performed by examining the average values of the
242 aforementioned levels.

243 Table 11. Walnut kernel average dry matter data

Ethephon
concentration
(ppm)
B1=0 B2=120 B3=240 B4=480 B5=720 B6=960 B7=1200 B8=1920 B9=4800

Harvest date

22 Sept 95.412 95.412 95.215 95.289 95.245 95.565 94.785 94.778 94.479

31 Sept 94.833 94.782 93.993 96.187 95.591 94.230 93.841 94.434 94.657

244

245 The outcomes of the kernel density analysis of variance are outlined in Table 12 (data were first
246 analyzed as splits and then polarized because Ea or Eb was not significant).
247
248 Table 12. Variance analysis of walnut kernels’ dry matter
Sources of change Degree of freedom Sum of squares
Block 2 52.541 **
Factor A (harvest date) 1 1.326 ns
Factor B (Ethephon concentration) 8 1.547 *
A× B 8 0.608 ns
Error 30 0.620
Total 49
249 C.V. = 0.852%; **: significant at 0.01 level; *: significant at 0.05 level; ns: non-significant

250 The findings displayed in Table 12 reveal a significant difference for kernel density (P<0.01)
251 and a consistent impact on kernel density across various harvest dates (P<0.01). Additionally,
252 a statistically significant difference was observed in ethephon concentration concerning kernel

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253 density (P<0.05). No significant correlation was detected between factor A (harvest date) and
254 factor B (ethephon concentration).
255 Table 13 presents the mean kernel density values for the experimental groups subjected to
256 varying levels of ethephon treatment.
257
258 Table 13. The mean of kernel density across different ethephon concentrations
Ethephon concentration Average kernel density Class
(B1) 0 ppm 95.123 ABC
(B2) 120 ppm 94.999 ABC
(B3) 240 ppm 94.641 BC
(B4) 480 ppm 95.716 A
(B5) 720 ppm 95.578 AB
(B6) 960 ppm 94.507 C
(B7) 1200 ppm 94.220 C
(B8) 1920 ppm 94.606 BC

259 Table 13 reveals that the highest mean values are associated with the fourth treatment, while
260 the lowest average percentage of dry matter in the kernel is observed in the sixth and seventh
261 treatments. As a result, the fourth treatment exhibits a significant difference from the sixth and
262 seventh treatments, while the remaining treatments do not differ significantly from each other.
263 Comparing Figure 2 with Figure 4 and 5, it becomes evident that there is no conclusive
264 relationship between ethephon concentration and the percentage of dry matter in the kernel,
265 contrary to what was observed for fruit quality and maturation traits.

96 95.716
95.578
95.5
95.123
average kernel density (%)

94.999
95
94.641 94.606
94.507
94.5 94.22

94

93.5

93
0 120 240 480 720 960 1200 1920
266 Ethephon concentration levels

267 Figure 5. Comparison of average walnut kernels’ dry matter (density) across different ethephon
268 levels.

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269
270 DISCUSSION

271 The findings of this study emphasize the significance of employing ethephon for the efficient
272 removal of mesocarp residues and the attainment of uniform nuts. Several noteworthy
273 observations are worth mentioning in this section. A higher ethephon concentration facilitated
274 the easier separation of the mesocarp (Figure 6). Additionally, in certain experimental instances,
275 the green layer exhibited darkening when subjected to a high ethephon concentration. At such
276 concentrations, small and shallow cracks were observed in the mesocarp. Notably, the impact
277 of ethephon on green peel separation was more pronounced in trees with late-maturing fruits.

278 Figure 6. Comparison of fruits treated by different ethephon concentrations

279 Figure 7 displays two nut samples in which the mesocarp is detached from the endocarp. The
280 first sample (a) was treated without the use of ethephon, while the second sample (b) was treated
281 with 960 ppm of ethephon.
282 It is evident that in the control sample, the mesocarp remains firmly attached to the endocarp,
283 necessitating the manual removal of the mesocarp using a knife. Previous investigations have
284 revealed that the closer the harvest date, the more effortless it becomes to separate the mesocarp
285 and obtain clean nuts devoid of any mesocarp remnants.

286

287 Figure 7. A nut harvested on 22 August, (a) not treated with ethephon and (b) treated with 960
288 ppm of ethephon

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289 The experiment demonstrated that as the normal harvest date approaches, the required amount
290 of ethephon for facilitating the separation of the mesocarp from the endocarp decreases. In fact,
291 during the normal harvest period, the ethylene naturally synthesized by the fruit acts
292 synergistically with ethephon, enhancing its effectiveness.

24
22.1
21.667
22 21.333 21.267
20.1 20.233
19.833 19.54 19.777
20 19.233
18.767 18.467
17.567
average maturity (%)

18 17.167 17.043 17.293

16.917
16.5 16.4
15.71
16
14.672
13.5
14 13.167 13.167
12.127
12 11.972
12
A1 A2 A3
10
0 120 240 480 720 960 1200 1920 4800
level of Ethephon concentration (factor B)
293

294 Figure 8. Average fruit ripening at different levels of factor B on 3 harvest dates (scoring
295 based on the ease of separating the mesocarp from the endocarp)

296 As depicted in Figure 8, the control group and the treated fruit with the lowest concentration of
297 ethephon exhibit the lowest scores. However, superior fruit quality in terms of mesocarp
298 maturation is achieved by increasing the ethephon concentration and harvesting the fruits at the
299 A3 factor level. It is worth noting that if the mesocarp separation score exceeds 15, farmers
300 typically do not encounter any issues in this regard. However, for the purpose of highlighting
301 yield differences in this experiment, we have set the scoring range to 25.
302 Therefore, it appears unnecessary to employ ethephon for mesocarp peeling on Sept 9th, which
303 is close to the regular harvest time. During this period, the mesocarp can be easily separated
304 from the endocarp without the use of ethephon. In other words, the fruits have undergone
305 sufficient maturation stages, resulting in effortless separation of the mesocarp from the
306 endocarp. Consequently, employing ethephon for this purpose at one week before the regular
307 harvest time lacks economic value. However, utilizing ethephon 3-4 weeks prior to the regular

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308 harvest leads to the production of uniform nuts without any mesocarp residues compared to the
309 control group.
310 The color analysis of walnut kernels harvested on three different dates reveals distinct
311 variations. Upon reviewing the images (Figure 9-11), it is evident that the highest quality is
312 associated with the samples collected on Aug 22nd, while the lowest quality is observed in the
313 kernels harvested on Sept 9th.

314 Figure 9. Walnut kernels harvested on 22nd Aug

315 (factor A1)
316

317

318 Figure 10. Comparison of kernels harvested on 2 different dates under the influence of the same
319 ethephon concentration (1920 ppm)

320

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321
322 Figure 11. Increasing the number of brown nuts or dark nuts between two harvest dates (22nd
323 Aug – 9th Sept)

324 The findings indicate that the application of ethephon at a dosage of 4800 has a detrimental
325 impact on walnuts. At this dose, ethephon likely interacts with the phenolic compounds present
326 in the walnut seeds, leading to a modification in kernel color.
327 Analyzing the dry matter content of the walnut kernels revealed that the timing of harvest
328 minimally affects the dry matter composition. Consequently, it can be inferred that early fruit
329 harvest not only yields white, high-quality kernels but also has a negligible influence on the
330 amounts of dry kernels obtained.
331 According to the study conducted by Martin (Martin, 1971), the dry matter content of kernel is
332 influenced by the initial moisture level at harvest, while exposure to ethephon does not affect
333 dry matter. However, in the case of treated trees, the green skin of the fruit tends to crack,
334 resulting in loss of nut moisture. Consequently, when measuring dry matter, these samples may
335 exhibit higher values compared to the control group. Although the analysis of the variance table
336 reveals a significant difference in dry matter and ethephon concentration, this distinction is
337 attributed to the cracking of the green skin and the subsequent reduction in the moisture content
338 of nuts. In other words, ethephon does not enhance dry matter, but it does diminish the initial
339 moisture content.
340
341 CONCLUSIONS
342 Although walnuts are classified as climacteric fruits, it is crucial for walnut fruits to reach their
343 maximum maturity. In cases where the mesocarp is immature, the application of ethephon can
344 be employed to expedite fruit ripening.When temperatures exceed 30 °C, conditions conducive
345 to walnut browning arise, resulting in a significant proportion of harvested walnuts displaying

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346 dark brown coloration and inadequate quality. In this experiment conducted under the specified
347 conditions, the optimal harvest time for walnut trees is determined to be Aug 21st. By
348 harvesting the crop at this specific time, the walnut kernels achieve the highest quality. This
349 timing ensures that the mesocarp of the walnuts remains completely immature. Treatment with
350 ethephon at a concentration of 1920 ppm facilitates mesocarp maturation, thereby facilitating
351 the separation of the mesocarp from the endocarp in the most effective manner. The impact of
352 various ethephon concentrations on walnut dry matter content revealed that ethephon
353 augmented carbohydrate production, thereby expediting fruit ripening and consequently
354 increasing the dry matter percentage of walnuts. As the harvest date approaches on Sept 9th,
355 the percentage of dark-colored kernels tends to rise. A comparison of walnut kernels harvested
356 at multiple dates, all treated with the same ethephon concentration, demonstrates a transition in
357 kernel coloration from light cream to light brown and eventually to black between the two
358 harvest dates of Aug 22nd and Sept 9th.
359

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