Ascophyllum nodosum and Silicon-Based Biostimulants Differentially Affect the Physiology and Growth of Watermelon Transplants under Abiotic Stress Factors: The Case of Drought

Round 1

Reviewer 1 Report

The study is very interesting and its objective was to test the efficacy of two biostimulants to mitigate the water deficiency conditions that watermelon transplants endure until flowering. The manuscript was found to be very well written, I don't see any problem in how clearly it was written. It has an adequate experimental design, the methods used as well as the way of analyzing them are correct. The results are adequately presented and discussed. I congratulate the work team. It is an excellent work.

Author Response

The authors would like to express their gratitude to the reviewer for the time invested to assess our manuscript, as well as for the kind words.

Reviewer 2 Report

The submitted manuscript entitled “Ascophyllum nodosum and silicon-based biostimulants differentially affect the physiology and growth of watermelon transplants under abiotic stress factors: the case of drought” is a useful manuscript suggested a useful method for enhancement of drought tolerance on watermelon. This research has been meticulously planned. However, I have a few questions and comments to improve the impact of the manuscript on the readers. Questions and comments to the author are the following:

Abstract and Introduction: Well written

Materials and Methods:

Lines 87-88: 75% and 50% of field capacity. When is this field water condition? Is this soil moisture condition immediately after irrigation or before irrigation? From Fig A & C, leaf number and plant area is larger in 50% Asc than 50% Si treatments. I think that the amount of transpiration is greater in 50% Asc than 50% Si, but is it possible to accurately adjust the soil moisture like 50% field capacity?

Lines 92-94: Should be explain how to prepare Silicon-based and seaweed biostimulants. If authors got a company, should be write company name. And please explain pH of these two solutions.  If possible, explain nutrients such as nitrogen and potassium and amino acid concentration.

Lines 92-94: Why a difference in the amount of foliar application between the two biostimulants? Si (30kg/ha), Asc (4L/ha).

Line 110: Explain the organ (leaf or shoot) from which RWC was measured. If author used a leaf, please explain which number leaf from the top used, and time that author sampled leaf, and how long keep under water for turgid treatment.

Line 125: In Result section, author explain on stem diameter. But in Materials and Methods author not explain the method of stem diameter measurement.

Results and Discussion:

1) Table 1 indicated RCC. But author did not explain the result of RCC in Results and Discussion section. Explain how to calculate Relative Chlorophyll Content.

2) Table 1: Should be explain unit all parameters. (likes Stem diameter (cm)?)

3) From Table 1, 50% field capacity, number of female flowers is zero (0). Author indicated, in abstract, “On the other hand, A. nodosum alleviated the negative effects of water deficit especially in the harshest conditions.”, however I don't think the fruit can be harvested for no flowers. I think author need to spend a long time researching to see that Asc effect.

4) Lines 273-282, author explain watermelon withstand extreme water deficit without considerably damaging their photosynthetic apparatus. I don’t understand this sentence. Author should explain more by citing other papers. On the other hand, Nitrogen concentration of leaf affect chlorophyll content. Authors should be present nitrogen content of leaf, and more discuss.

Author Response

The submitted manuscript entitled “Ascophyllum nodosum and silicon-based biostimulants differentially affect the physiology and growth of watermelon transplants under abiotic stress factors: the case of drought” is a useful manuscript suggested a useful method for enhancement of drought tolerance on watermelon. This research has been meticulously planned. However, I have a few questions and comments to improve the impact of the manuscript on the readers.

Response: The authors would like to express their gratitude to the reviewers for the time they invested for assessing our manuscript and their useful comments and suggestions. The comments were responded one-by-one below.

Questions and comments to the author are the following:

Abstract and Introduction: Well written

Materials and Methods:

Lines 87-88: 75% and 50% of field capacity. When is this field water condition? Is this soil moisture condition immediately after irrigation or before irrigation? From Fig A & C, leaf number and plant area is larger in 50% Asc than 50% Si treatments. I think that the amount of transpiration is greater in 50% Asc than 50% Si, but is it possible to accurately adjust the soil moisture like 50% field capacity?

Response, L91-94: The field water capacity percentages refer to the time period immediately after irrigation. It is now specified in the manuscript. Indeed, 50% Asc had greater leaf number compared to 50% Si, but plant area was not significantly different between these treatments. Therefore, we believe that water dynamics regarding field water capacity were not considerably different among the water treatments.

Lines 92-94: Should be explain how to prepare Silicon-based and seaweed biostimulants. If authors got a company, should be write company name. And please explain pH of these two solutions.  If possible, explain nutrients such as nitrogen and potassium and amino acid concentration.

Response, L102-104: Asc is comprised of seaweed extract of Ascophyllum 19,5% (w/v), P2O5 (9,8%), K2O (13,7%). Si biostimulant is comprised of >85% SiO2 (w/v). It is now mentioned in the manuscript.

Lines 92-94: Why a difference in the amount of foliar application between the two biostimulants? Si (30kg/ha), Asc (4L/ha).

Response, L104-105: The amounts of foliar application for each biostimulant were in accordance to the instructions on the label for watermelon crops.

Line 110: Explain the organ (leaf or shoot) from which RWC was measured. If author used a leaf, please explain which number leaf from the top used, and time that author sampled leaf, and how long keep under water for turgid treatment.

Response, L122-125: The plants were grown until blooming of the first few flowers at DAT 20. Afterwards, RWC was measured on three fully developed leaves (4th, 5th, and 6th leaf from the lateral bud) per sample. Turgor weight was measured after the leaves were moisturized in plastic bags filled with water for 24 h. It is now mentioned in the manuscript.

Line 125: In Result section, author explain on stem diameter. But in Materials and Methods author not explain the method of stem diameter measurement.

Response, L109-110: Stem diameter was measured with a digital caliper. It is now mentioned in the manuscript.

Results and Discussion:

1) Table 1 indicated RCC. But author did not explain the result of RCC in Results and Discussion section. Explain how to calculate Relative Chlorophyll Content.

Response: The results of relative chlorophyll content were explained and discussed in L273-288. Relative chlorophyll content was measured with a SPAD-type chlorophyll meter (CCM-200 plus, Opti-Sciences, Hudson, NH, USA) which provides dimensionless values, as mentioned in L110-112.

2) Table 1: Should be explain unit all parameters. (likes Stem diameter (cm)?)

Response: The parameters’ units were included in Table 1, as suggested.

3) From Table 1, 50% field capacity, number of female flowers is zero (0). Author indicated, in abstract, “On the other hand, A. nodosum alleviated the negative effects of water deficit especially in the harshest conditions.”, however I don't think the fruit can be harvested for no flowers. I think author need to spend a long time researching to see that Asc effect.

Response: As stated in the introduction, the objective of our study was to test the efficiency of two biostimulants to mitigate the water deficiency conditions endured by watermelon transplants until flower blooming. Our intention was to evaluate the plant responses during the period of transplanting shock in the first few weeks after transplantation (now mentioned in L61-66). We did not aim to evaluate overall flowering and yield production. That would require further investigation in the field and would include the risk of precipitations. However, that is an excellent subject for research.

4) Lines 273-282, author explain watermelon withstand extreme water deficit without considerably damaging their photosynthetic apparatus. I don’t understand this sentence. Author should explain more by citing other papers. On the other hand, Nitrogen concentration of leaf affect chlorophyll content. Authors should be present nitrogen content of leaf, and more discuss.

Response: Regarding the photosynthetic mechanism, Fv/Fm (φP0) has been reported to reach values of 0.78-0.86 in non-stressed plants, as mentioned in L247-248. In our case, all treatments had values above 0.78 showing that the plants were not considerably stressed regardless of the water deficit. Moreover, the correlation between chlorophyll and nitrogen contents was discussed as suggested, in L284-288.

Reviewer 3 Report

This research investigated the effect of foliar application of two biostimulants on three rates of drought stress of watermelon seedlings until flower blooming.  The author did a good job in data collection, and literature review in the whole manuscript. However, the experiment design and decipherment were not well managed. Here are some comments the author should carefully think about for this research project and any future experience designs:

1. The research was conducted for only 20 days, which was too short to observe any detectable response by biostimulantces.

2. The drought stress and foliar application were operated in DAT 2, which was too early. The right way is to culture the seedlings until 2-3 new leaves established or at least after root settled. Giving stress and application at so early stage will not eliminate transplanting stress.

3. The foliar application was given only one time before the drought, which was totally not enough. The author should consider apply once per week or follow the instruction of biostimulant products.

4. The author stated that watermelon showed more stress performance (lower chlorophyll fluorescence parameters) in A. nodosum treatment indicating an alleviation of stress effect. This statement is totally wrong. A stress alleviation should be reflected by higher chlorophyll fluorescence performance and better growth parameters, which were not observed or consistent in this experiment, which was another evidence of bad experience design.

Based on the review of the whole manuscript, the reviewer strongly suggests the authors self-review the dataset and redo the analysis. A follow-up experience with reasonable stress management and application design are needed before publishing these data.

Author Response

This research investigated the effect of foliar application of two biostimulants on three rates of drought stress of watermelon seedlings until flower blooming.  The author did a good job in data collection, and literature review in the whole manuscript. However, the experiment design and decipherment were not well managed.

Response: The authors would like to express their gratitude to the reviewers for the time they invested for assessing our manuscript and their useful comments and suggestions. The comments were responded one-by-one below.

Here are some comments the author should carefully think about for this research project and any future experience designs:

1. The research was conducted for only 20 days, which was too short to observe any detectable response by biostimulantces.

Response: As stated in the introduction, the objective of our study was to test the efficiency of two biostimulants to mitigate the water deficiency conditions endured by watermelon transplants until flower blooming. Our intention was to evaluate the plant responses during the period of transplanting shock in the first few weeks after transplantation (now mentioned in L61-66). Longer experimentation would require further investigation in the field and would include the risk of precipitations. However, that is an excellent subject for research.

2. The drought stress and foliar application were operated in DAT 2, which was too early. The right way is to culture the seedlings until 2-3 new leaves established or at least after root settled. Giving stress and application at so early stage will not eliminate transplanting stress.

Response: Since our objective was to evaluate the effect of biostimulants during the transplanting shock phase, it only made sense to apply the biostimulants as early as possible in order to achieve the maximum efficiency. In real life conditions, it is highly possible that plants may be transplanted in regions with low water availability from day one. In any case, watermelon requires about 3-4 days to form 2-3 new leaves after transplantation.

3. The foliar application was given only one time before the drought, which was totally not enough. The author should consider apply once per week or follow the instruction of biostimulant products.

Response: The amounts and frequency of foliar applications for each biostimulant were in accordance to the instructions on the label for watermelon crops. It is now mentioned in L104-105.

4. The author stated that watermelon showed more stress performance (lower chlorophyll fluorescence parameters) in A. nodosum treatment indicating an alleviation of stress effect. This statement is totally wrong. A stress alleviation should be reflected by higher chlorophyll fluorescence performance and better growth parameters, which were not observed or consistent in this experiment, which was another evidence of bad experience design.

Response: Regarding the photosynthetic mechanism, Fv/Fm (φP0) has been reported to reach values of 0.78-0.86 in non-stressed plants, as mentioned in L247-248. Indeed, lower fluorescence values indicated a bit more stressed plants. However, in our case all treatments had values above 0.78 showing that the plants were not considerably stressed regardless of the water deficit. As stated in L262-270, diminished photosynthetic activity was exhibited with foliar application of A. nodosum, ultimately leading to a positive response (eustress) displayed by greater biomass, plant area, and root system development.

Based on the review of the whole manuscript, the reviewer strongly suggests the authors self-review the dataset and redo the analysis. A follow-up experience with reasonable stress management and application design are needed before publishing these data.

Response: A preliminary experiment was already conducted in order to determine the proper field water capacity treatments for the case of watermelon. It is now mentioned in L96-98.

Reviewer 4 Report

Inserted some revisions for the ms in the attached pdf file. Maybe a longer time of the experiment coould have helped in havig more significant differences among the treatments in the case of the photosynthetic efficiency (i.e., Fv/Fm). However, some positive effects of Si and seaweed are clear.

Comments for author File: Comments.pdf

Author Response

Inserted some revisions for the ms in the attached pdf file. Maybe a longer time of the experiment coould have helped in havig more significant differences among the treatments in the case of the photosynthetic efficiency (i.e., Fv/Fm). However, some positive effects of Si and seaweed are clear.

Response: The authors would like to express their gratitude to the reviewers for the time they invested for assessing our manuscript and their useful comments and suggestions. The comments from the pdf file were responded one-by-one below.

Responses to the pdf comments:

L58-60: more recent data were included.

L66-68: OJIP was explained as suggested.

L94-96: In order to determine 100% field water capacity we used a number of pots (including the watermelon plants) which were not involved in the experiment. On the first day, we watered the pots until a runoff of 15-20% water which is a typical practice of irrigation in potted plants. We measured the total water used for irrigation which was 200 mL (100% field water capacity). In the 75% (150 mL) and 50% (100 mL) treatments there was no runoff water from the pots.

L90-94: For each water treatment we used 18 pots. Six pots for non-biostimulant, six pots for Ascophyllum nodosum, and six pots for silicon biostimulant. Accordingly, for each biostimulant treatment we used 18 pots. Six pots for 100% water, six pots for 75% water, and six pots for 50% water.

L108: Our aim was to grow the plants for the first few weeks after transplantation in order to evaluate the potential biostimulant effect on the transplanting shock, especially under the influence of water deficit. This period coincides with blooming in watermelon, at about 20 days after transplantation.

L124-125: TW was measured after the leaves were moisturized in plastic bags filled with water for 24 h.

L142-148: Watermelon is a monoecious plant where male flowers start to bloom a few days earlier than the female ones. As mentioned in the manuscript, the experiment lasted until blooming of the first flowers, thus, female flowers did not manage to exhibit potential significant differences among the different treatments, but this was not our objective. In addition, stem diameter was also similar in all treatments which can be attributed to the transplanting shock which ensues the first few weeks after transplantation. The above are now mentioned in the manuscript.

L156-159: Indeed, plants irrigated with greater amounts of nutrient solution responded better compared to the water deficient ones, an effect which was visible even after a rather short period of 20 days after transplantation. It is now mentioned in the manuscript.

L170: Table 1. We deleted the “a” letters in the cases where no significant differences were detected. Also, 100% Asc had 0.17 male flowers.

L187-188: An explanation was included as suggested.

L214-217: An explanation was included as suggested.

L225: Regarding the length of the experiment, our intention was to evaluate the plant responses during the period of transplanting shock in the first few weeks after transplantation. We did not aim to evaluate overall flowering and yield production. That would require further investigation in the field and would include the risk of precipitations. However, that is an excellent subject for research.

L254: Figure 1. Indeed, the letters of statistical analysis are correct.

The rest of the comments in the pdf file were answered in L31, L48, L135, L136-137, L156, L229.

Also, suggested citations were included in the manuscript (L199-201, L222-224).

Round 2

Reviewer 2 Report

The paper is written accurately by my comments, and I think the data and reference used in the treatise is also no problem.

Reviewer 3 Report

After an edition, the author did not make much progress or add a follow-up research. Their response cannot answer my questions.

1. The author replied that “The amounts and frequency of foliar applications for each biostimulant were in accordance to the instructions on the label for watermelon crops.” But no product information about these two biostimulants were provided. The reviewers would like to check the instructions.

2. The author stated that watermelon showed more stress performance (lower chlorophyll fluorescence parameters) in A. nodosum treatment indicating an alleviation of stress effect. This statement is totally wrong. A stress alleviation should be reflected by higher chlorophyll fluorescence performance and better growth parameters, which were not observed or consistent in this experiment, which was another evidence of bad experience design.

The author replied that their Fv/Fm readings were above 0.78 meaning no stress. Then it means no plants were stressed by the experiment design, which again means bad experience design as the objective of this research was investigating the biostimulant effect on draught stress alleviation.

Based on the review of the whole manuscript, a follow-up experience with reasonable stress management and application design are needed before publishing these data.