Review Reports - Low-Dose Micronized Biochar Enhances Vegetative and Physiological Traits of <i>Vitis vinifera</i> and Improves Soil Quality in Organic Cultivation: A Two Year-Experiment in Tuscany

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

I attach a review of the article „Low-dose micronized biochar promotes Vitis vinifera perfor mances and improves soil quality in organic cultivation: a two year-experiment in Tuscany”.

The studies are very interesting especially from the point of view of application perspective, and circular economy strategies. However, before publication, the manuscript requires corrections. Major revision.

Main notes:

There is a lack of consistency in the presented statistical methods. The information provided in the methodological section is completely different from the information provided in the figures descriptions – see below. This is unacceptable.
The incorrect assumption that there are two treatments – there is a control and one treatment.
As indicated in the methodology (see line 143), soil pH was measured. The results were not presented in this paper. Presenting pH changes is particularly important in the context of decreasing Ca and Mg content in both soil and plants. This issue should be addressed in the discussion.
The chemical composition of the biochar should be given.
Abbreviation B (biochar) is unfortunate because the text gives the impression that boron (B) is being discussed in relation to other elements. I suggest changing the abbreviation.
In the discussion, the authors should have referred to weather conditions. This aspect was not discussed.

Other comments:

Line 100-101: …We hypothesized that (i) micronized biochar enhances soil health parameters…

Rev: soil health parameters? Unfortunate term.

Line 114: …Climatic conditions during the experimental period…

Rev: Rather “weather conditions”.

Line 122-124: …Thanks to its aqueous characteristic and the microscopic (> Ø 40 μm) dimension of its particles, the micronized biochar can be utilized in fertigation

Rev: ? maybe: the microscopic (< Ø 40 μm)?

Line 142-147: …For the other analyses, the same procedure was adopted for both soil characterization and leaf samples. Soil chemical parameters such as pH, TOC, Dissolved Organic Carbon (DOC), total nitrogen (Ntot), nitrates (NO3--N), available phosphorous (available P) and exchangeable K, Ca and Mg (Kex, Caex, Mgex) contents, as well as enzymatic activities (Dehydrogenase; DHA and Alkaline phosphatase; APA – detailed below) were performed in B and CTR samples. Three or five replicates were performed for each assay…

Rev: Where pH data is provided?, this parameter should be presented. The decrease in Ca and Mg content in soil presented by the authors may be correlated with soil acidification – a decrease in pH and consequently the bioavailability of elements.

Line 126-128: …A randomized plot experiment, with two treatments, was setup in 2021 and 2022. The treatments were: (i) fertigation with only water (5 L plant–1; CTR) and (ii) fertigation with micronized biochar (0.5% v/v; 5 L plant–1; B)….

Rev: … two treatments…? Is one treatment. Control this is a group that does not receive the study factor. This serves as a reference point to see if the changes actually occurred due to the treatment.

Must be corrected in all manuscript.

Line 203-205: ... For leaf K+, Ca2+ and Mg2+ content, about 0.2 g of dry powdered leaf samples were put in Teflon tubes with 2 mL of H2O2 (30%, w/v) and 8 mL of HNO3 (70 %, v/v) and incubated overnight at room temperature…

Rev: Cations after incubation in H₂O₂ (30%, w/v) and HNO₃ (70 %)? Rather total forms.

Line 213-218: …Results were compared with a two-tailed Student’s t-test using a significance level of 0.05, considering the treatment with the micronized biochar as variability factor. Data not normally distributed (i.e., gs in July 2021, Ci in September 2021; Pn in July 2022 and Ci in July 2022) were subjected to Welch's t-test prior further analysis Chl and Flav data were analyzed using the non-parametric Mann-Whitney test….

Rev: Which tests are correct? See information in the titles of the figures.

Line 229-231: …Means were subjected to a one-way ANOVA with the use of micronized biochar as variability factor. The lack of asterisks indicates no significant differences among means at p ≤ 0.05 using the post-hoc LSD test…

Rev: Which tests are correct?

Line 240-241: …Means were subjected to a one-way ANOVA with the use of micronized biochar as variability factor. The presence of asterisks indicates significant differences among means at p ≤ 0.05 using the post-hoc LSD test, whilst the lack of asterisks indicates no significant differences among means at p ≤ 0.05 using the same post-hoc test. **: p ≤ 0.01; ***: p ≤ 0.001…

Rev: Which tests are correct? See line: 251-253, 262-266, 274-278, 291-292, 293-295, 312-316, 326-329, 344-347.

Line 292: …LA: leaf area.his is a table…

Rev: ?

Table 2: 17.0 ± 2.0

Rev: Remove spaces.

Line: 451, 492: …status..

Rev: Remove italics.

Line 461-462: …Rosa tomentosa and Cattleya edithiae. while Guo et al. [38] further confirmed the same pattern in Schefflera arboricola…

Rev: edithiae. while – dot.

Rev: The full Latin names of plant species mentioned in the text for the first time should be written consistently.

Line 468-469: …leaf expansion…

Rev: expansion? – too more.

Author Response

Reviewer #1

There is a lack of consistency in the presented statistical methods. The information provided in the methodological section is completely different from the information provided in the figures descriptions – see below. This is unacceptable.

Response: We sincerely apologize for the inconsistency between the statistical methods reported in the Materials and Methods Section and those described in the figure captions. The discrepancy was due to a typographical error in the captions of the figures. All figure captions have now been carefully revised and corrected to ensure full consistency with the Statistical Analysis paragraph, which already contained the correct methodological information. We thank the Reviewer for pointing out this issue and have thoroughly checked the revised manuscript to avoid similar inconsistencies.

The incorrect assumption that there are two treatments – there is a control and one treatment.

Response: We thank the Reviewer for this comment. We agree that the experimental design included one untreated control group and one biochar treatment. Accordingly, we revised the manuscript throughout to clearly distinguish the untreated control condition from the micronized biochar-treated group.

As indicated in the methodology (see line 143), soil pH was measured. The results were not presented in this paper. Presenting pH changes is particularly important in the context of decreasing Ca and Mg content in both soil and plants. This issue should be addressed in the discussion.

Response: We thank the Reviewer for this valuable comment. The reference to soil pH in the Methodology section (line 143) was included to describe the procedure used for the initial soil characterization. However, soil pH was also monitored throughout the experiment, although these data were not reported because no significant variations were observed over time. In addition, the absence of substantial changes in soil organic matter and the limited variations detected in soil enzyme activities further support the stability of soil conditions during the experimental period, including potential pH-related dynamics. Therefore, the explication of the decrease in Ca and Mg content in soil induced by the pH decrease cannot be utilized for this experiment.

The chemical composition of the biochar should be given.

Response: The chemical composition of the micronized biochar was added in paragraph 2.2.

Abbreviation B (biochar) is unfortunate because the text gives the impression that boron (B) is being discussed in relation to other elements. I suggest changing the abbreviation.

Response: We thank the Reviewer for this valuable suggestion. To avoid confusion between biochar and boron (B), we replaced the abbreviation “B” with “MB” (micronized biochar) consistently throughout the manuscript.

In the discussion, the authors should have referred to weather conditions. This aspect was not discussed.

Response: We thank the Reviewer for this valuable suggestion. Since only minor differences in temperature were observed between the two experimental years, the Discussion was revised by referring to the higher precipitation levels recorded during the study period. In particular, increased rainfall was considered as a possible factor explaining the higher DHA activity and MPI values (lines 539-544) as well as the greater leaf area, which may have promoted increased plant biomass production (lines 617-618).

Line 100-101: …We hypothesized that (i) micronized biochar enhances soil health parameters… Rev: soil health parameters? Unfortunate term.

Response: We removed “soil health parameters” and we added “soil fertility”.

Line 114: …Climatic conditions during the experimental period…Rev: Rather “weather conditions”.

Response: due

Line 122-124: …Thanks to its aqueous characteristic and the microscopic (> Ø 40 μm) dimension of its particles, the micronized biochar can be utilized in fertigation àRev: ? maybe: the microscopic (< Ø 40 μm)?

Response: due

Line 142-147: …For the other analyses, the same procedure was adopted for both soil characterization and leaf samples. Soil chemical parameters such as pH, TOC, Dissolved Organic Carbon (DOC), total nitrogen (Ntot), nitrates (NO₃^--N), available phosphorous (available P) and exchangeable K, Ca and Mg (Kex, Caex, Mgex) contents, as well as enzymatic activities (Dehydrogenase; DHA and Alkaline phosphatase; APA – detailed below) were performed in B and CTR samples. Three or five replicates were performed for each assay

Response: We thank the Reviewer for this comment. The reference to soil pH in the Methodology section (line 143) was included to describe the procedure used for the initial soil characterization. However, soil pH was also monitored throughout the experiment, although these data were not reported because no significant variations were observed over time. In addition, the absence of substantial changes in soil organic matter and the limited variations detected in soil enzyme activities further support the stability of soil conditions during the experimental period, including potential pH-related dynamics. Therefore, the explication of the decrease in Ca and Mg content in soil induced by the pH decrease cannot be utilized for this experiment.

Line 126-128: …A randomized plot experiment, with two treatments, was setup in 2021 and 2022. The treatments were: (i) fertigation with only water (5 L plant^–1; CTR) and (ii) fertigation with micronized biochar (0.5% v/v; 5 L plant–1; B)….

Must be corrected in all manuscript.

Response: We thank the Reviewer for this important clarification. We agree that the experimental design included one untreated control group and one micronized biochar treatment, rather than two treatments. Accordingly, the manuscript has been revised throughout to consistently distinguish the control group from the treated group.

Line 203-205: ... For leaf K⁺, Ca²⁺ and Mg²⁺ content, about 0.2 g of dry powdered leaf samples were put in Teflon tubes with 2 mL of H₂O₂ (30%, w/v) and 8 mL of HNO3 (70 %, v/v) and incubated overnight at room temperature…

Rev: Cations after incubation in H₂O₂ (30%, w/v) and HNO₃ (70 %)? Rather total forms.

Response: due

Rev: Which tests are correct? See information in the titles of the figures.

Rev: Which tests are correct?

Rev: Which tests are correct? See line: 251-253, 262-266, 274-278, 291-292, 293-295, 312-316, 326-329, 344-347.

Line 292: …LA: leaf area. This is a table…

Rev: ?

Response: due

Table 2: 17.0 ± 2.0

Rev: Remove spaces.

Response: The unnecessary spaces in Table 2 were removed

Line: 451, 492: …status..

Rev: Remove italics.

Response: due.

Line 461-462: …Rosa tomentosa and Cattleya edithiae. while Guo et al. [38] further confirmed the same pattern in Schefflera arboricola…

Rev: edithiae. while – dot.

Rev: The full Latin names of plant species mentioned in the text for the first time should be written consistently.

Response: We thank the Reviewer for these observations. The punctuation was corrected by replacing the full stop before “while” with a comma. In addition, the full Latin names of plant species were revised throughout the manuscript for consistency, and the corresponding taxonomic authorities were added at their first occurrence.

Line 468-469: …leaf expansion…

Rev: expansion? – too more.

Response: We thank the Reviewer for this suggestion. The term “leaf expansion” was replaced with “leaf development”.

Reviewer 2 Report

Comments and Suggestions for Authors

This paper addresses soil fertility decline in organic vineyards by testing very low doses of micronized biochar applied via fertigation. The topic is relevant and has practical value. The experimental design is generally sound, the data are rich, and the results are credible. The overall quality is good. However, the following issues should be considered by the authors.

(1) The authors did not provide any data on grape yield or quality. However, the title and abstract use "*Vitis vinifera* performances", which could mislead readers. I suggest the authors clarify in the title that the study focuses on vegetative growth and physiological responses. In the discussion, they should explain that the vines were only 2–3 years old (still young), so representative fruit quality data could not be obtained. In the conclusions, they should mention that long‑term trials are needed to assess effects on fruit quality.

(2) The static data only show one or two snapshots per growing season (leaf area, dry weight, leaf number). There are no time‑series data on leaf emergence rate, expansion dynamics, or senescence. The B treatment significantly increased net photosynthesis and final leaf area, but without growth dynamics we cannot tell whether this was due to faster growth, a longer growth period, or both. This is important for understanding the mechanism of biochar action.

(3) The authors measured leaf area and leaf dry weight but did not calculate specific leaf area (SLA). SLA is one of the most basic leaf functional traits. It can show whether leaves are oriented toward light capture or resource conservation. Having the raw data but not calculating or discussing SLA is an omission. I recommend that they calculate SLA, compare treatments statistically, and discuss how B treatment affected SLA and how that relates to the increase in photosynthesis.

(4) Table 2 shows that leaf area increased significantly under B treatment, but leaf number did not change. This means individual leaf area became larger. The authors did not analyze this further: which leaf positions had larger leaves? Did leaf length increase, or width, or both? These data can be obtained by re‑measuring already collected leaf samples, without additional field work.

(5) I suggest at least the following additional analyses or data: calculate SLA and perform statistical comparisons; provide raw data on leaf length and width (or the L/W ratio) to clarify the morphological basis for the increased leaf area; if possible, measure leaf area and chlorophyll content at different leaf positions (e.g., basal, middle, top) to see whether biochar changed the spatial pattern of leaf development.

(6) Provide basic physicochemical properties of the micronized biochar, such as specific surface area, pore structure, and functional groups.

(7) About the biochar particle size: the text says “> 0.40 µm”. Is this a typo? Usually micronized means smaller than a certain size. Please clarify.

(8) In section 2.2, the micronized biochar is described as “suspended into water at a dose of 300 mL L⁻¹”, but the treatment is described as “0.5% v/v” (about 5 mL L⁻¹). These two values differ by an order of magnitude, which is confusing. Is 300 mL L⁻¹ the concentration of the commercial stock solution, and 0.5% the concentration after dilution for field application? Please explain so that other researchers do not get confused when repeating the experiment.

(9) Leaf magnesium content decreased, but chlorophyll content did not change. This needs a clearer explanation or additional physiological indicators (e.g., chlorophyll a/b ratio or photosystem II parameters).

(10) In the bar charts, the meaning of the different treatments (open bars vs. closed bars) should be labeled directly on the graph, rather than only written in the caption as “content in soil irrigated with only water (open bars) or with micronized biochar (closed bars)”.

(11) The formatting of figures and tables is not consistent. Please unify the formatting according to the journal’s requirements.

Author Response

Reviewer #2

Response: We thank the Reviewer for this valuable comment. The title has been modified as suggested in order to better reflect the focus of the study on vegetative growth and physiological responses. In the Discussion section, a clarification regarding the young age of the vines (2–3 years old) and the consequent absence of representative grape production and quality data in the present experiment has been added (lines 629-633). Furthermore, in the final part of the Conclusions section, reference has been made to the need for longer-term experiments to properly assess the effects of micronized biochar application on grape yield and fruit quality (lines 711-712).

Response: We thank the Reviewer for this valuable suggestion. The present study was mainly focused on evaluating the effects of micronized biochar on soil fertility and, consequently, on soil nutrient availability to the plant. Therefore, biometric parameters were assessed only at the end of the first and second experimental years. We agree that time-series measurements of leaf emergence, expansion dynamics, and senescence would provide additional insight into the mechanisms underlying the observed increases in net photosynthesis and leaf area under biochar treatment. We consider the Reviewer’s suggestion highly valuable and will take it into account in future studies aimed at better characterizing vine growth dynamics over time.

Response: We thank the Reviewer for this valuable suggestion. Specific leaf area (SLA) values were calculated and included in Table 2. The calculation method has been described in the Materials and Methods section, while the results have been presented and discussed in the biometric data subsection of the Results section. Furthermore, in the Discussion section, the higher net photosynthesis (P_n) observed in MB-treated plants during 2021 was associated with the increased light interception capacity of the leaves, suggesting that treated plants invested more resources in leaf expansion, as indicated by the higher SLA values (lines 627-629). Conversely, this interpretation could not be extended to 2022, since no significant differences in SLA values were observed between treated and control plants during that year.

Response: We thank the Reviewer for this insightful comment. Leaf length and leaf width have now been included in the biometric results section and in Table 2. Unfortunately, it is not possible to relate leaf size variation to leaf position within the canopy, as this information was not recorded during sampling. We acknowledge that this represents a limitation of the present study. This aspect will be considered in future experiments to better characterize within-canopy variability in leaf morphological traits.

Response: We thank the Reviewer for these valuable and constructive suggestions. In response, we have now included SLA values as well as leaf length and leaf width data, and we have further improved the interpretation of these morphological traits by relating them to the observed photosynthetic responses (see responses to the above reviewer comments). Unfortunately, it was not possible to include measurements of chlorophyll content or to analyze leaf traits according to leaf position (e.g., basal, middle, apical), as leaf sampling was conducted randomly and leaf position was not recorded for individual samples. We acknowledge this as a limitation of the present study. These aspects will be carefully considered in future experiments to better characterize the spatial variability of leaf development and its relationship with physiological responses to biochar application.

(6) Provide basic physicochemical properties of the micronized biochar, such as specific surface area, pore structure, and functional groups.

Response: We thank the Reviewer for this suggestion. The requested information has been added in Section 2.2 (Materials and Methods), where the basic physicochemical properties of the micronized biochar, including specific surface area, pore structure, and functional groups, are now reported. Specifically, we introduced these details: “Indeed, the resulting micronized biochar was suspended into water at a dose of 300 ml L^–1, with a pH of 6.8 and Electric Conductivity of 1.10 mS cm^–1, total organic carbon (TOC) 65%, total N <0.5%, total P 0.034%. This material was characterized by a high specific surface area (ranging from tens to several hundreds of m² g⁻¹) and a well-developed hierarchical pore structure, consisting of both micro- (< 2 Ø nm) and mesopores (Ø 2-50 nm). Its surface was also enriched with oxygen-containing functional groups (e.g.; carboxylic, phenolic, and carbonyl groups), contributing to its chemical reactivity and overall ability to interact with soil nutrients and organic matter.”

(7) About the biochar particle size: the text says “> 0.40 µm”. Is this a typo? Usually micronized means smaller than a certain size. Please clarify.

Response: due

Response: We thank the Reviewer for this important clarification. Indeed, 300 mL L⁻¹ refers to the concentration of the commercial stock suspension of micronized biochar, while the 0.5% v/v corresponds to the final concentration applied as treatment in the field after dilution. This aspect has now been clarified in Section 2.2 (Materials and Methods) to avoid any possible misunderstanding and to ensure reproducibility of the experimental procedure.

Response: We thank the Reviewer for this insightful comment. A possible explanation for the observed decrease in leaf Mg content without a corresponding change in chlorophyll content has been added in the Discussion (lines 685-692). However, chlorophyll measurements were obtained using non-destructive methods; therefore, it was not possible to determine chlorophyll a/b ratio or other photosystem II parameters. Likewise, additional PSII physiological traits were not monitored during the two-year experimental period. We acknowledge that these measurements would have provided a more mechanistic understanding of the observed responses, and they will be considered in future studies to better elucidate the physiological effects of micronized biochar application.

Response: We thank the Reviewer for this helpful suggestion. The figure legends have now been directly incorporated into all bar charts, clearly indicating the meaning of open and closed bars. We agree that this modification improves the clarity and readability of the figures.

(11) The formatting of figures and tables is not consistent. Please unify the formatting according to the journal’s requirements.