Interactive Effects of Drought and Saline Aerosol Stress on Morphological and Physiological Characteristics of Two Ornamental Shrub Species
Round 1
Reviewer 1 Report
Journal: Horticulture
Manuscript Title: Interactive effects of drought and saline aerosol stress on morphological and physiological characteristics of two ornamental shrub species
Comment to authors
Line 13. three replications replaced with triplicates
Line 32. stresses (drought and salt), which have a cumulative negative
Line 33. You are saying many references and you have quoted one reference. Please add more references or modify the statement.
Line 37. Statement needs reference.
Line 40. Acreage means?
Line 41. due to the importance to have amenity??? This whole sentence is not clear, please rewrite it.
Line 47. individuate plants that are able to tolerate environmental stresses.
Line 79-81. Very short paragraph. Please merge it.
Line 83. role in European
Line 90. In this view, it appeares quite relevant
Line 109. Saline solution sentence is not clear.
Line 116. following the protocol of Tribulato et al. [31]
Line 126. For each species triplicates of 12 plants
Line 129. relative humidity levels were 66%
Line 136. Please merge with previous sentence.
Line 142. Above you have used time as A.m. and P.m. Now you are using in another format.
Line 182. reported in Table 2 and Table 3.
Line 183. In Callistemon, the total dry biomass
Line 188. In Callistemon, the epigeous
Line 189. by ~10, 24, and 33% in WC 30%, WC 20% and WC 10% respectively as compared with the control
Line 198. Also, for what regards leaf number. What does it mean?
In table 2: As compared to So, there is a decrease in S1 and then increase (increase and then decrease). This is almost every column as well as every WC%. Why it is like that? Please elaborate.
Line 214. Biomass, which showed
Line 215. By is not a suitable preposition here.
Line 218. ble 3) while the highest decrease
Line 272. Why it is in a single line? Merge with previous line.
Other comments:
I think P values should be added where you will use the word “significant”
Please avoid short paragraphs in the whole paper. Try to merge with next or previous paragraphs.
Author Response
Response to Reviewer 1 Comments
The authors would like thank for your comments and evaluation. Corrections and suggestions have been implemented in the current version of the manuscript. The modification are highlighted in yellow in the manuscript. We hereby provide a point-by-point answer. The manuscript has been accordingly revised also in English language.
The authors
Point 1: Line 13. three replications replaced with triplicates
Response 1: Done
Point 2: Line 32. stresses (drought and salt), which have a cumulative negative
Response 2: The sentence was modified
Point 3: Line 33. You are saying many references and you have quoted one reference. Please add more references or modify the statement.
Response 3: The sentence was modified
Point 4: Line 37. Statement needs reference.
Response 4: Done, the reference was added.
Point 5: Line 40. Acreage means?
Response 5: The sentence was modified
Point 6: Line 41. due to the importance to have amenity??? This whole sentence is not clear, please rewrite it.
Response 6: The sentence was rewritten
Point 7: Line 47. individuate plants that are able to tolerate environmental stresses.
Response 7: Done
Point 8: Line 79-81. Very short paragraph. Please merge it.
Response 8: The paragraph was merged
Point 9: Line 83. role in European
Response 9: Done
Point 10: Line 90. In this view, it appeares quite relevant
Response 10: Done
Point 11: Saline solution sentence is not clear.
Response 11: The sentence was rewritten
Point 12: Line 116. following the protocol of Tribulato et al. [31]
Response 12: Done
Point 13: Line 126. For each species triplicates of 12 plants
Response 13: Done
Point 14: Line 129. relative humidity levels were 66%
Response 14: Done
Point 15: Line 136. Please merge with previous sentence.
Response 15: The two sentences have been merged.
Point 16: Line 142. Above you have used time as A.m. and P.m. Now you are using in another format.
Response 16: The format has been standardized
Point 17: Line 182. reported in Table 2 and Table 3.
Response 17: Done
Point 18: Line 183. In Callistemon, the total dry biomass
Response 18: Done
Point 19: Line 188. In Callistemon, the epigeous
Response 19: Done
Point 20: Line 189. by ~10, 24, and 33% in WC 30%, WC 20% and WC 10% respectively as compared with the control
Response 20: The sentence was modified
Point 21: Line 198. Also, for what regards leaf number. What does it mean?
Response 21: The sentence was rewrite.
Point 22: In table 2: As compared to So, there is a decrease in S1 and then increase (increase and then decrease). This is almost every column as well as every WC%. Why it is like that? Please elaborate.
Response 22: Actually, the S1 versus So, almost all parameters declined. As it can be observed from the means of the parameters analysed. It can be observed an opposite trend in the WC20% and WC10% but the differences were not significant.
Point 23: Line 214. Biomass, which showed
Response 23: Done
Point 24: Line 215. By is not a suitable preposition here.
Response 24: Done
Point 25: Line 218. ble 3) while the highest decrease
Response 25: Done
Point 26: Line 272. Why it is in a single line? Merge with previous line.
Response 26: The two sentence was merged.
Other comments:
Point 27: I think P values should be added where you will use the word “significant”
Response 27: The statistical analysis was performed for all physiological parameters. In the text P values have been reported where the data were significant.
Point 28: Please avoid short paragraphs in the whole paper. Try to merge with next or previous paragraphs.
Response 28: Thanks for your suggestion. The short paragraphs were merged in whole paper.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
All comments are written in manuscript PDF
Comments for author File: Comments.pdf
Author Response
Response to Reviewer 2 Comments
The authors would like thank for your comments and evaluation. Corrections and suggestions have been implemented in the current version of the manuscript. The modification are highlighted in yellow in the manuscript. We hereby provide a point-by-point answer.
The authors
Point 1: Line 130: Please explain if these temperature and humidity conditions were the same throughout the research?
Response 1: Figure S1 shows the trend of temperature and humidity during the course of the trial. The mean data has been reported in the manuscript.
Point 2: Line 143: During measurement of leaf gas exchanges, net photosynthetic rate, chlorophyll a fluorescence and stomatal conductance, It should be write values of temperature, humidity and PAR for each day of measurement separately
Response 2: the values of temperature, humidity and PAR for each day of measurement separately were added
Point 3: Line 147: please briefly explain the chlorophyll a fluorescence and the meaning of the studied parameter Fv / Fm?
Response 3: The methodology was modified.
Point 4: Line 224: this statement is not shown in Figure 2a!
Response 4: Sorry for the mistake; for this parameter there was no interaction effect but only main effect.
Point 5: Figure 2: , Wc10% severe drought stress
Response 5: Done
Point 6: Line 245: in S0, significant decrease was not observed, according to Fig 3a
Response 6: sorry for the mistake; the sentence was rewritten
Point 7: Line 245: in viburnum plants.
Response 7: The sentence was modified
Point 8: Figure 3: ; WC10% severe drought stress
Response 8: Done
Point 9: Line 254: This part should be better explained. It is unclear. Since An and Gs were monitored over a period of 60 days, the results should be presented chronologically.
Response 9: The sentence has been revised and improved: “Gas exchange measurements in Callistemon plants were severely affected under water deficit. The AN was reduced by water deficit and plants exposed to severe water stress (WC20% and WC10%) for 30 days showed a reduction by 30 and 70% compared to control. At the end of the trial, 60 days, with a reduction by 45 and 53% in WC20% and WC10% plants. The addition of saline aerosol stress, after 30 days, differences were amplified comparing treatments with control plants (Figure 4a)”.
Point 10: Figure 4: S0 is not shown in Fig 4a and b
Response 10: The values of S0 are underneath with others, therefore it seems that are not present.
Point 11: Figure 4: ; WC10% severe drought stress
Response 11: The correction was done
Point 12: Figure 4 and 5: the results in Fig 4 and 5 should be supplemented by a Tukey s test to see a significant difference and significant reduction of these parameters. Due to overlapping lines it is difficult to follow the results. Authors should consider a different presentation of the results
Response 12: Thank you for the comment and suggestions. The data have been subjected to different statistical analysis depending on the factors involved. The water and salinity stresses were applied at different time points during the experimental period. It means that during the first 30 days there were two factors (water deficit and time), while after at 45 and 60 days a salinity stress was added reaching three factors (water deficit, salinity, and time). Therefore, for the first 30 days a two-way ANOVA. Results have been reported in tables. Difference among means were determined with Tukey’s post-test was performed, while for the 45 and 60 days a Multivariate analysis was performed.
Point 13: Line 272: Figure 5 b represents gs only in viburnum plants!
Response 13: Sorry for the mistake; the sentence was rewritten according your suggestion.
Point 14: Figure 5: ; WC10% severe drought stress
Response 14: Done
Point 15: Line 284: This sentence is unclear ... between WC 30% and?
Response 15: The sentence was modified
Point 16: Figure 6: ; WC10% severe drought stress
Response 16: Done
Point 17: Figure 6: Authors commented in the text significant differences but this was not shown in the graf.
Response 17: The statistical analysis has been improved and additional information has been reported.
Point 18: Figure 7: WC10% severe drought stress
Response 18: Done
Point 19: Line 308: it should be emphasized that the decrease was more pronounced in Calistemon plants as seen in the figure 8. Please explain, this sentence is unclear. Line 309. According to Fig 8. values of leaf number were more reduced in viburnum under stress treatment, and authors wrote the opposite. According to Fig 8 only FV/Fm was more reduced in WC10%S2 treatment, in Calistemon plants. While RWC, An and Gs were more reduced in Viburnum. Autors wrote that these parameters were more reduced in Calistemon. Please explaine. Line 312: SPAD values are not commented.
Response 19: The reduction of leaf number was more evident in Callistemon (28%) than in Viburnum (15%). In the heat map (figure 8) the normalization of the data, due to a different leaf number of two species (over 1000 leaves in Callistemon and 140 in Viburnum), determines the difficulty to appreciate this differences (but the values are clearly indicated in tables 2 and 3 and figures 1 and 2. About FV/FM, RWC, An, and Gs, we modified the sentence according your suggestion in “Irrigation and saline aerosol treatments noticeably affected the leaf relative water content (RWC), chlorophyll a fluorescence (Fv/Fm) in the most stressed Callistemon plants; the differences in net photosynthetic activity (AN) and stomatal conductance (gs) were more prominently in Viburnum plants”. The SPAD values were now commented.
Point 20: Figure 8:, WC10% severe drought stress
Response 20: Done
Point 21: Line 319: maximum quantum efficiency of PSII
Response 21: Done
Point 22: Line 321: 9
Response 22: Done
Point 23: Line 365: fresh mass was not shown in results
Response 23: the sentence was modified according your suggestion
Point 25: Line 366: please clearify ... was it salt-only treatment or combined salt treatment with drought treatment?
Response 25: the sentence was rewritten
Point 26: Line 379: Please commented and discussed yours results regarding leaf area!
Response 26: thanks for the suggestion: In our trial the total leaf area of Callistemon showed interaction effects with differences due to saline aerosol in the control and light drought stress, while in Viburnum plants no interaction effects were observed
Point 27: Line 420: The reduction of chlorophyll a fluorescence and derived parameters such as Fv/Fm can help to assess the tolerance of different species to the different stresses. Line 420: the values of parameter
Response 27: The sentence was modified
Point 28: Line 422: What significant changes? significant decrease or increase?
Response 28: decrease; the sentence was modified
Point 29: Line 427: Instead, in the severe drought stress treatments and in combination of the two stresses, the lower Fv/Fm values indicated that PSII had been damaged, and the heat dissipation capacity was reduced in the leaves of both species Authors should commented results and compared them with other similar research
Response 29: The sentence was modified according your suggestion
Point 30: Line 438: this statement (RWC indicates the water status of plant tissues during drought stress. It decreases with the water deficit increase, although this reduction is genotype specific [62]. In particular, this reduction was more evident in Callistemon than in Viburnum) is not supported with statistic, and it is not shown in grafs
Response 30: In particular, in Callistemon the reduction for effect of drought stress was of about 37%; in Viburnum differences were observed only in correspondence of saline aerosol. In our study, the lowest RWC was obtained at 10%WC treatment in Callistemon and at 10%WC, S1 and S2 in Viburnum.
Point 31: Line 472: Missing conclusion ... which one had better tolerance to investigated conditions?
Response 31: The conclusions have been improved.
Point 32: References: must review references carefully because some of them are not written according to instructions
Response 32: The references were modified according to author guidelines.
Author Response File: Author Response.pdf
Reviewer 3 Report
This manuscript deals with the combined effects of drought and salt stress on two Mediterranean ornamental shrubs. This is a study of great interest and the topic is within the scope of the journal. The manuscript is very well structured and written, and the bibliography is very up-to-date, but the description of the methodology and the presentation of results need to be improved. Also, some modifications are necessary in the other sections of the manuscript.
I recommend a major revision before being accepted for publication.
See attached file for particular and minor comments. It contains paragraphs highlighted in yellow and comments.
Comments for author File: Comments.pdf
Author Response
Response to Reviewer 3 Comments
The authors would like thank for your comments and evaluation. Corrections and suggestions have been implemented in the current version of the manuscript. The modification are highlighted in yellow in the manuscript. We hereby provide a point-by-point answer.
The authors
Point 1: Line 17: Some numerical values of the results should be shown in the abstract. Especially highlight significant differences between treatments or variation with respect to the non-stressed treatment.
Response 1: The information was added in the abstract section
Point 2: Line 26: This study focuses on two shrub cultivars used in gardening. Apart from line 62, there are no numerical values in the entire Introduction section that gives the reader an idea of the magnitude of the problem: affected area (in a country or in the Mediterranean basin), affected species, differences between species, percentage of defoliation, mortality, loss of growth, economic losses, intensity of drought, amount of salt deposited on the leaves by aerosols, amount of Na and Cl absorbed by a plant, etc.
Not all of the aforementioned aspects should be addressed, but it would be advisable to include values for some of them.
Response 2: The information was added in the introduction section
Point 3: Line 104: What was the size of the plants at the beginning of the experiment?
Response 3: At the beginning of the experiment, the dry biomass of the plants was on average 27.8±2.09 g and 33.6±3.28 g for Callistemon and Viburnum, respectively
Point 4: Line 106: Please replace "(14/13/13, N, P, K + microelements)" by "(14-13-13 + microelements)", since for all fertilizers, the standard notation "N-P-K" denotes the ratio N:P2O5:K2O. Therefore, it is not necessary to add "N, P, K" in the text.
Response 4: Done
Point 4: Line 114: As the water consumption of the plants, within the same treatment, varies depending on the plant size and only two sensors were used per treatment, how was the homogeneity of the substrate moisture controlled within each treatment? Were the plants similar in size? please clarify.
Response 5: Two sensors were used for each treatment and for each replicate; moreover the plants used for the trial were homogeneous with each other.
Point 6: Line 118: Has the substrate water potential for each volumetric moisture percentage been determined? The water available to plants is better related to the substrate water potential than to the volumetric water, since the relationship is not usually linear. So, it is essential to know the relationship between substrate water potential and volumetric moisture content in order to better interpret the differences between irrigation treatments.
Response 6: Sensors were calibrated following the protocol of Starr and Paltineanu [2002] and Tribulato et al. [2019]. Sensors were calibrated where a series of each measurements is taken in connection with samples of volumetric soil to quantify the relationship between the substrate (measured with the 5TM) and the volumetric water content (WC). The sensors were installed 10 cm below the substrate surface at the center of each sample pot. The use of the probes was preceded by their calibration to determine the real content of water in the substrate samples, which were placed in a thermo-ventilated oven at 70 °C until the constant weight (W cal.) reached (R2 = 0.9742).
Point 7: Later in the text, WC30%, WC20% and WC10% are defined as light, moderate and severe drought stress, respectively. Why that name? if the relationship between volumetric water and substrate water potential is not shown. Please, clarify.
Response 7: The methodology was modified
Point 8: Line 119: Does the control treatment (WC40%) correspond to the field capacity? Which WC% corresponds to the field capacity? Please clarify.
Response 8: Yes, the WC40% correspond to field capacity.
Point 9: Line 119: Please clarify when irrigation was interrupted.
Response 9: Same threshold values were adopted to determine irrigation interruption.
Point 10: Was irrigated from the minimum value established in each treatment, to field capacity? to the threshold of the next treatment? up to exceeding WC40%?
Response 10: The irrigation was activated when the water content dropped below the pre-set threshold values of WC10%, WC20%, WC30% and WC40% (control) of the volume of the substrate.
Point 11: Line 120: During the first 4 weeks the water availability treatments were applied. During the second 4 weeks, were WC and S treatments applied together or only S treatments? Please clarify.
Response 11: After 4 weeks, at the same time as the drought stress, the plants were also subjected to the three aerosol treatments mentioned above for another 4 weeks.
Point 12: Line 126: ?
Response 12: the sentence was modify to be more comprehensible in “triplicates of four plants for 12 treatments (144 plants)”.
Point 13: Line 139: Replace "RWC" by "Relative water content" since previously these acronyms with the complete words have not been shown (it appears on line 148).
Response 13: Done
Point 14: Line 142: solar time? Please, clarify.
Response 14: Done
Point 15: What were the measurement conditions? (Temperature, relative hunidity and solar radiation). Did the gas analyzer have a temperature and/or light control modules? If that is not the case, please, include the range of variation of Temperature, Relative Humidity and solar radiation during the measurements of the same day, as well as the differences between measurement dates.
Response 15: the requested information was added
Point 16: Line 149: At what time were they collected? What was the hourly range of collection? As mentioned above, the water status of the plants can vary between the beginning of the sampling and the end.
Response 16: The Relative Water Content (RWC) was measured between 12:00 a.m. and 2:00 p.m. under conditions of maximum stress for the plant.
Point 17: Line 150: In dark conditions? Clarify.
Response 17: Yes, in dark conditions. The sentence was modified
Point 18: Line 151: Why did the authors use 70 ºC for moisture content (line 134) and 75 ºC for RWC? The results may vary slightly due to the emission of temperature-dependent volatile substances? Please clarify.
Response 18: The moisture content and RWC were determined using different oven, since, the dry biomass required an oven with large capability, while leaf discs for RWC could be performed in smaller oven. However, there is no difference between 70 or 75 °C because both lead to complete dehydration. The drying was completed in both cases when the dry weight was constant.
Point 19: Line 158: Since in Table 1 the Salt treatment has been called "Aerosol", the same should be done here. Then, I suggest to replace "drought, salt, and species" by "drought (D), saline aerosol (A), and species (S)" Therefore, D, A and S have been defined and can be typed on line 171.
Response 19: The correction was done
Point 20: Line 171: D, A and S effects have been mentioned on line 158.
Response 20: The correction was done
Point 21: Line 176: italics, please.
Response 21: The correction was done in all manuscript
Point 22: Table 1: Please, describe in a footnote what F is.
Response 22: Done
Point 23: Table 1 and footnotes: In some cases the level of significance appears with a lower case (p) and in other cases with a capital letter (P). Please be consistent.
Response 23: The table was modified.
Point 24: F-test from ANOVA
Response 24: Done
Point 25: Line 202: The letters denoting significant differences between saline and irrigation treatments have the same format (capital letters), and this can lead to confusion. I suggest changing the format of some of them, for example, change the letters of the saline treatments to lower case.
Response 25: Thank you for your suggestion. The letter format of the saline treatments has been changed to lowercase.
Point 26: Line 233: Same comment as in table 2.
Response 26: Thank you for your suggestion. The letter format of the saline treatments has been changed to lowercase.
Point 27: Line 255: Was the reduction significant from date to date?
Response 27: thank you for the comment and suggestions. The data have been subjected to statistical analysis. See the following response.
Point 28: If it was measured on different dates on the same plants, then the data may be autocorrelated. In that case, data could be analyzed with a repeated measures ANOVA to know the significance of the differences between treatments and between measurement dates. If that was not the case, then a three-factor ANOVA (irrigation, salinity, date) can be performed.
Response 28: thank you for the comment and suggestions. The data have been subjected to statistical analysis. The water and salinity stresses were applied at different time points during the experimental period. It means that during the first 30 days there were two factors (water deficit and time), while after at 45 and 60 days a salinity stress was added reaching three factors (water deficit, salinity, and time). Therefore, for the first 30 days a two-way ANOVA. Results have been reported in tables. Difference among means were determined with Tukey’s post-test was performed, while for the 45 and 60 days a Multivariate analysis was performed.
Point 29: Line 256: Was it significant?
Response 29: The P value was reported
Point 30: Line 265: Significance of the differences? Different letters for different homogeneous groups?
Response 30: additional information on the statistical analyses have been reported in tables.
Point 31: Line 267: Same comment as for Callistemon in line 256.
Response 31: the text has been revised and improved.
Point 32: Line 278: Same comment as for Callistemon in Fig. 4.
Response 32: The p-values were reported
Point 33: Line 282: p-value?
Response 33: The p-values were reported
Point 34: Line 290: Same comments as in Fig. 4 and 5.
Response 34: we tried to add the significant letters considering the statistical results, but the figure appeared more confused. Therefore, we added statistical tables were it is possible to identify the differences.
Point 35: Line 300: Please, indicate the p-values and the different letters indicating statistical differences.
Response 35: p-values of the statistical analyses have been directly reported in the text.
Point 36: Line 303: Figure 8 summarizes the relative value of the responses to drought and salt treatments. It is the relationship between variables and treatments, which are shown as a heat map. However, is it a correlation-based map? If the columns are the variables, what do the rows represent in this correlation? Please, clarify.
Response 36: Heat map analysis summarizing the morphological and physiological changes of potted Callistemon and Viburnum responses to drought (WC40%: control; WC30%: light drought stress; WC20%: moderate drought stress; WC10% severe drought stress) and saline aerosol treatment
Point 37: Line 321: Replace "8" by "9"
Response 37: Sorry for the mistake. Done
Point 38: Line 332: These two sentences are more typical of the Discussion section than of the results. Please move forward.
Response 38: The two sentences have been moved in the discussion section.
Point 39: Line 343: These first two paragraphs are more appropriate for the Discussion section, since they do not analyze the results obtained. It is suggested to reduce this content to 3-4 lines, in order to present the general approach, and take the rest to the Introduction section.
Response 39: The text was modified according your suggestion.
Point 40: Line 388: This statement does not fully conform to the results obtained and what is stated in this paragraph. In Callistemon, the water stress treatment (WC10%) did not alter the R/S ratio. This ratio was ONLY altered when Water and Salt stresses were combined, and ONLY for Callistemon, not for Viburnum. Please modify the wording for further clarification.
Response 40: The sentence was modified according to your comment.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Great work.
Author Response
The authors would like thank for your comment.
Reviewer 3 Report
This is a revised and improved version of an earlier version. The authors have taken into account the comments of the reviewers. Therefore, I have no problem to recommend this manuscript for publication but after a few minor details.
See attached file for particular and minor comments. It contains paragraphs highlighted in green and comments. They are mainly about sentence checking and text editing.
Comments for author File: Comments.pdf
Author Response
Response to Reviewer 3 Comments
Point 1: This is a revised and improved version of an earlier version. The authors have taken into account the comments of the reviewers. Therefore, I have no problem to recommend this manuscript for publication but after a few minor details.
See attached file for particular and minor comments. It contains paragraphs highlighted in green and comments. They are mainly about sentence checking and text editing. This is a revised and improved version of an earlier version. The authors have taken into account the comments of the reviewers. Therefore, I have no problem to recommend this manuscript for publication but after a few minor details.
See attached file for particular and minor comments. It contains paragraphs highlighted in green and comments. They are mainly about sentence checking and text editing.
Response 1: The authors would like thank for your comments and evaluation. Corrections and suggestions have implemented in the current version of the manuscript. The modification are highlighted with “Track Changes” function in the manuscript. We hereby provide a point-by-point answer.
Point 2: Line 17: Do the authors mean "increased drought" instead of "drought increased? Please check this sentence.
Response 2: Thanks for the suggestion; the sentence was rewritten.
Point 3: Line 27: A parenthesis is missing. "... 10%), by..."
Response 3:. Done
Point 4: Line 54: is a word missing? ... the garden what determines....
Response 4: Thanks for the suggestion; the sentence was rewritten.
Point 5: Line 190: italics.
Response 5: Done
Point 6: Line 201: Although it has dried to constant weight, it is not the same to dry at 70 ºC, than at 75 ºC. In plants there are other substances apart from water that volatilize depending on the drying temperature, in such a way that the higher the temperature, the greater the amount of volatilized substances. That is why I made a comment in the first round about why two different temperatures were used.
Anyway, from my previous experiences, 70 and 75 ºC are two temperatures so close that the difference in drying is insignificant, so the procedure followed can be considered valid. However, for future studies, it is advisable to use the same drying temperature in all cases.
Response 6: Thanks for the suggestion. In our future studies we will use the same drying temperature in all cases.
Author Response File: Author Response.pdf
