Salinity Tolerance of Novel and Established Olive (Olea europaea L.) Cultivars for Super-High-Density Systems

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript addresses a pressing challenge for modern olive production: identifying cultivars that can maintain productivity in super high density systems under increasing salinity. By coupling precise ionomic profiling with detailed physiological measurements, the study delivers valuable new data that can inform breeding programmes and agronomic management strategies in regions facing soil and irrigation water salinisation. This integrated approach underscores the work’s significance and positions it as a potentially influential contribution for researchers and growers seeking sustainable solutions amid climate driven water constraints.
To maximise the scientific and practical impact of these findings, the following comments focus on enhancing statistical robustness, improving the extrapolation of results, and clarifying key methodological details. The suggestions are offered in a constructive spirit and with full respect for the substantial effort already invested, aiming to consolidate the validity of the conclusions and broaden their applicability under real world field conditions.

1. The abstract correctly anchors the study in the context of salt induced yield loss in super high density (SHD) olives and outlines a factorial NaCl assay coupled with ionomics and gas exchange metrics. However, at ~230 words it exceeds the 200 word cap and dilutes impact with second order numbers (e.g., exact shoot length, individual ion values). Compress the background to a single clause linking SHD expansion to soil salinisation, report cultivar trends qualitatively (“Arbosana showed the smallest biomass reduction; Lecciana sustained the highest K⁺/Na⁺ ratio”), and finish with a single outcome oriented sentence (“These data identify Arbosana as a robust candidate for saline SHD orchards and highlight Lecciana’s ion regulation traits for breeding”).
   I recommend replacing the original keyword list with a set that more accurately reflects the experimental scope and enhances database retrieval. Substitute the broad term “abiotic stress” with “salinity tolerance”, which precisely describes the stress factor under investigation. Retain “ion homeostasis” and “photosynthetic efficiency”, as they capture the core physiological processes analysed. The descriptor “genetic variability” is misaligned with the study—no genetic diversity metrics were collected—so replace it with “super high density (SHD)” or, alternatively, “ionomics”, both of which better represent the agronomic framework and analytical approach. Finally, keep the cultivar names Coriana, Lecciana and Siquitita, correctly spelled, to facilitate cultivar specific searches. The revised set—salinity tolerance; ion homeostasis; photosynthetic efficiency; super high density; Coriana; Lecciana; Siquitita —will improve the article’s thematic visibility and indexing accuracy in specialised databases.
2. The introduction charts the rise of SHD systems and salinity risk, citing classical and recent agronomic sources, but lacks a quantitative statement of the problem and omits the latest molecular literature. Insert one sentence quantifying global SHD acreage and projected saline hectare increases, then integrate 2022–2025 studies on SOS1/HKT1/NHX transporters and heat–salinity co‑stress. Close with a precise gap (“no comparative ionomic screen of newest SHD cultivars under moderate–high salinity exists”) and two testable hypotheses: (i) Arbosana will outperform Arbequina at 75 mM NaCl; (ii) Lecciana will maintain superior K⁺/Na⁺ ratios but incur a growth penalty. Briefly define “ion homeostasis” for cross‑disciplinary readers and ensure numerical citations are sequential. These focussed edits will align both sections with journal limits while sharpening novelty and mechanistic relevance.
3. Experimental Design and Internal Validity
   • Plant material. Comparing three emerging cultivars (Lecciana, Coriana, Siquitita) with the commercial standards Arbequina and Arbosana is appropriate, yet adding a clearly salt sensitive genotype (e.g., Picual) would have broadened the tolerance gradient.
   • Salinity gradient. The range 0–75 mM NaCl (≈0.8–9.2 dS m⁻¹) spans moderate to high field conditions, but an upper tier ≥100 mM would help define lethal thresholds.
   • Nutrient control. Although the 0 mM treatment uses identical Hoagland solution, the manuscript does not state whether osmolarity was balanced with non toxic salts; this could confound osmotic versus ionic effects.
   • Replication. Four plants per cultivar × treatment (n = 4) are insufficient to detect higher order interactions in a perennial with high biological variance.
   • Environment and duration. A five month greenhouse study ensures tight control but cannot capture field level co stresses (heat, VPD) or reproductive performance.
4. Analytical Methods and Statistics
   •    Ionic quantification. The pXRF + AAS approach is innovative; reported R² > 0.95 is convincing, yet root mean square error and bias should be disclosed.
   •    Statistical framework. The authors employ robust factorial ANOVA with post hoc tests after checking assumptions; however, given the small n, a permutation ANOVA or mixed effects model would yield more reliable inference.
   •    Growth modelling. Cubic splines capture non linear trajectories but risk over fitting; segmented or logistic models would be more parsimonious and biologically interpretable.
   •    Multiple testing. No adjustment for family wise error is documented despite extensive genotype × organ × salinity contrasts—raising the likelihood of Type I errors.

5. Interpretation of Results
•    Arbosana plasticity. The cultivar exhibits the smallest biomass loss ( 5.4 % at 75 mM) and sustained photosynthesis, yet the absolute advantage over Arbequina ( 11 %) is modest; its agronomic relevance should be statistically confirmed.
•    Lecciana paradox. High K⁺/Na⁺ ratios coexist with strong growth inhibition, implying an energetic penalty for ion homeostasis that the discussion should address.
•    Ion compartmentalisation. Root sequestration of Na⁺/Cl⁻ is well documented, but mechanistic links to transporter genes (SOS1, NHX, HKT1) are absent, limiting causal insight.

6. Limitations and Biases

• Greenhouse conditions may under represent combined heat–salinity stress typical of Mediterranean summers.
• Juvenile plants (five months) do not inform on flowering, fruit set or oil quality—critical metrics for cultivar recommendation.
• Low replication restricts statistical power and generalisability.

7. Contribution and Applications
•    Provides the first comparative ionomic physiological dataset for Lecciana, Coriana and Siquitita under super high density (SHD) salinity.
•    Highlights Arbosana’s relative vegetative resilience and Lecciana’s efficient K⁺/Na⁺ regulation—valuable traits for pre breeding.
•    Practical recommendations remain provisional until validated under field conditions and reproductive stages.

 

8. Recommendations for Authors
1.    Include a salt susceptible cultivar and/or extend the NaCl range to ≥100 mM.
2.    Increase replication (≥6–10 plants) or adopt split plot designs with subsampling.
3.    Employ permutation tests or mixed models and report effect sizes (η², 95 % CI).
4.    Apply false discovery rate or Bonferroni corrections for multiple comparisons.
5.    Provide RMSE/bias for pXRF validation and describe osmotic balancing in controls.
6.    Incorporate RT qPCR data for key transporters and record reproductive/quality traits in extended trials.

9. Overall Conclusion
The manuscript combines modern ion profiling techniques with a detailed factorial design and delivers promising insights into cultivar specific salt responses in SHD olive systems. Nevertheless, limited replication, potential osmotic confounds, and absence of field level validation temper the strength of its conclusions. Addressing the statistical and mechanistic gaps outlined above would substantially enhance the manuscript’s scientific rigour and agronomic impact.

Comments on the Quality of English Language

Assessment of English‐language quality
Overall, the manuscript is written in sound, intelligible scientific English; terminology is accurate and the syntax poses no barrier to comprehension. However, the prose can be tightened and smoothed to achieve the level of clarity and concision expected by an international readership. Four recurrent issues merit attention:
1.    Over long, multi clause sentences. For example, the first sentence of the Abstract runs 45 words and packs three ideas—industry transition, labour shortages and salinity—into a single structure . Breaking such sentences into two will improve readability.
2.    Redundant or imprecise wording. Phrases such as “degrading irrigation water quality” (better: “deteriorating irrigation water quality”) and “remarkable photosynthetic resilience” could be replaced with more direct alternatives to avoid subjective tone .
3.    Inconsistent tense and register between sections. The Introduction properly employs the present tense for established knowledge, but the Results occasionally slip into present perfect or present continuous (“has become increasingly critical… is becoming inevitable”) where simple present would suffice.
4.    Minor mechanical issues. These include sporadic missing hyphens in compound modifiers (“five month study” → “five month study”), uneven abbreviation usage (e.g., first use of “EC” without expansion) and occasional extra spaces around parentheses.

Recommendation
The English is serviceable and does not obscure the scientific content; nevertheless, a focused language edit—aimed chiefly at shortening sentences, pruning redundancy and harmonising tense and style—would sharpen the manuscript and ensure seamless communication to the broad plant science community.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors  

Dear Authors,

first of all I would like to let you know that I liked your manuscript, even if you should revise it thoroughly.

General:
This manuscript addresses a highly relevant and practically important topic: the effects of salt stress on the growth and physiological responses of five olive cultivars in high-density (SHD) planting systems.

Introduction:
Line 41 - Add references
Add more context regarding the economic importance of SHD olive plantations
Add more context regarding global salinisation

Materials and Methods:
Lines 118-122: Is salinity 0 a control? Please explain this in the text.
Lines 208-231: Statistical Analysis: please indicate where you explain the statistical method used for the chlorophyll parameter / Fluorimetry
Missing replicates per treatment

Results
Lines 462-468: 3.3 Changes in Mineral Content: consider rephrasing and move it to Statistical Analysis

Discussion
Should be rewritten.
The Discussion section is too detailed and too long. You should consider shortening it.
Too many results in the discussion section: you describe the results for each cultivar separately and discuss them insufficiently.
You have written the best example of well-discussed results in 4.3.2. Potassium Accumulation Patterns.
Please do not repeat the results in the discussion.
You should also address the general differences and relationship between parents and progeny between olive cultivars, especially in terms of plant growth and other phenological parameters.
Line 638: „some olive cultivars'“ name cultivars
In section 4.6 Study Limitations and Future Research you do not address this but draw several conclusions. However, I believe that some parts could be used to discuss your results.

Conclusions
Should be rewritten.
Most of this section could be useful for a discussion.

Comments on the Quality of English Language

No additional comments on English.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for addressing my previous comments so thoroughly. The science is now sound; only minor editorial refinements remain. Please shorten the Abstract to ≤ 200 words by deleting a few superfluous adverbs and end it with the sentence: “These data identify Coriana and Sikitita as promising candidates for salinized SHD orchards.” Arrange the keywords in strict alphabetical order, avoiding any duplication of title terms. Ensure that all figures are supplied as TIFF files at ≥ 300 dpi, include an appropriate scale bar, and that all statistical symbols follow MDPI style. With these small adjustments, the manuscript will be ready for publication.

Author Response

Reviewer comment: Thank you for addressing my previous comments so thoroughly. The science is now sound; only minor editorial refinements remain. Please shorten the Abstract to ≤ 200 words by deleting a few superfluous adverbs and end it with the sentence: “These data identify Coriana and Sikitita as promising candidates for salinized SHD orchards.” Arrange the keywords in strict alphabetical order, avoiding any duplication of title terms. Ensure that all figures are supplied as TIFF files at ≥ 300 dpi, include an appropriate scale bar, and that all statistical symbols follow MDPI style. With these small adjustments, the manuscript will be ready for publication.

Authors' response:
We sincerely thank the Reviewer for their time and constructive feedback. We are pleased that they find the science of our manuscript to be sound. We have addressed all the remaining editorial refinements as requested:

• The abstract has been revised. It is now 184 words and concludes with a strong statement identifying the two most tolerant cultivars, suggested by the Reviewer.
• The keywords have been arranged in strict alphabetical order, and no terms from the title are now duplicated ("salinity tolerance" has been replaced with "abiotic stress").
• We confirm that all figures have been uploaded as separate high-resolution files. Furthermore, all statistical symbols throughout the manuscript have been revised to conform to MDPI style guidelines.