Application of Biocat G, Selenium, and Chitosan to Counteract the Negative Effects of Cd in Broccoli Plants Grown in Soilless Culture

Round 1

Reviewer 1 Report

The manuscript can be accepted after the revisions carried out by the authors.

Author Response

Reviewer 1. Accepted

Thank very much.

Reviewer 2 Report

The authors have significantly improved article. However, article is not acceptable for publication in its present form. The authors need to follow the following instructions to improve this article.

Line 96. Please check “16”

Line 101-102. Please delete this sentence - this sentence is repeated

Line 143. The length of the day was 10- 12 hours? The study was conducted in winter!

Line 149. Whether the agrochemicals used could contain cadmium?

Line 161. The authors vaguely describe the application of Biocat G. How 4 grams were applied? In solution form? Rock wool was mixed with Biocat G?

Line 241. Please check “...under an excess of Cd…” All seven treatments?

Line 263. Please check “stem diameter” – “stem day”? (Table 1)

Line 272. Table 2. Could you explain RWC and RWC AP?

Line 350. Table 4. Could you explain n.q.?

Line 376, 382. Please correct “Chitosan” with “chitosan”

Line 392. Table 5. In the caption is –Cd/SP. In the table are the results for 6 treatments. Please check

Line 428. Value of 43% is unclear, how it was calculated, what does the rest mean?

Line 439. Please add the reference at the end of sentence.

Line 456. Please check “It seem”

Line 488. “…it seems that…” The conclusion should be rewrite. You cannot speculate.

Author Response

Reviewer 2 (R2)

R2. Line 96. Please check “16”.

Authors. Done

R2. Line 101-102. Please delete this sentence - this sentence is repeated.

Authors. Done

R2. Line 143. The length of the day was 10-12 hours? The study was conducted in winter!

Authors. In Spain, in the Murcia geographical area, during the period in which the experiment was carried out (October to January) there are usually between 10 and 12 hours of light: in October 11.5 hours and in January 9.9 hours.

R2. Line 149. Whether the agrochemicals used could contain cadmium?

Authors. Not likely. The products are periodically tested for quality to ensure that there is no presence of heavy metals in their content. This product used in agriculture are Cd freee.

R2. Line 161. The authors vaguely describe the application of Biocat G. How 4 grams were applied? In solution form? Rock wool was mixed with Biocat G?

Authors. Biocat G was used to the dose of 4 g plant^-1 applied manually in solid on the substrate, around the stem. This has been introduced in the ms.

R2. Line 241. Please check “...under an excess of Cd…” All seven treatments?

Authors. These sentence have been changed to “. were assayed on broccoli plants one control treatment (T1) grown under conditions without Cd and six different treatments (T2, T3, T4, T5, T6 and T7) grown under an excess of Cd.

R2. Line 263. Please check “stem diameter” – “stem day”? (Table 1)

Authors. Done

R2. Line 272. Table 2. Could you explain RWC and RWC AP?

Authors. RWC is the relative water content of the shoot, and it’s the same of RWC AP.

R2. Line 350. Table 4. Could you explain n.q.?

Authors: n.q. sample no presence in analysis results.

R2. Line 376, 382. Please correct “Chitosan” with “chitosan”

Authors. Done

R2. Line 392. Table 5. In the caption is –Cd/SP. In the table are the results for 6 treatments. Please check.

Authors. Done: The next sentence has been added. Values of EDI (mg (kg BW day)^-1), THQ and ORD indexes, according to the Cd concentration in the inflorescence for plants grown in the presence of Cd with different treatments (+Cd/SP, +Cd/BioG, +Cd/SeF, +Cd/SeR, +Cd/ChiF, +Cd/ChiR).

R2. Line 428. Value of 43% is unclear, how it was calculated, what does the rest mean?

Authors. The Cd content in the edible part for Biocat G is 57% compared to plants grown with Cd without treatment (100% Cd). This means that it reduces the Cd content by 43% with respect to that treatment, in addition to reducing it with respect to the rest of the treatments by a minimum of 38%.

R2. Line 439. Please add the reference at the end of sentence.

Authors. Done

R2. Line 456. Please check “It seem”

Authors. The sentence has been corrected: “Therefore, Se can increase the concentration of glutathione….”.

R2. Line 488. “…it seems that…” The conclusion should be rewrite. You cannot speculate.

Authors. Conclusion has been rewritten avoiding specalations.

Reviewer 3 Report

The experiment conducted by the authors broadens the knowledge on the ‘Application of Biocat G, selenium, and chitosan to counteract the negative effects of Cd in broccoli plants grown in soilless culture’. Indeed, evaluation of the effects of Biocat G, selenium, and chitosan under Cd stressful condition  is of special interest. There are a lot of comments that should be taken into account by authors, which I believe are significant and important aspects that need to be thoroughly addressed in authors revision.

The main concern is:

Abstract:

Abstract section should be re-written. Language used in this section is very bad.   

(1) Lines 16-17. This sentence should be rephrased.

(2) Lines 18-19. Replace them by

The current study, was aimed to evaluate the effect of the exogenous applications of Biocat G (fulvic/humic acids), selenium (Se), and chitosan to roots and leaves of broccoli plants exposed to Cd stress.

(3) Lines 20-24. Replace them by  

The applied treatments were: i) T1: Hoagland nutrient solution (NS), ii) T2: NS + Cd at 3 mg L^-1 (NS + Cd), iii) T3: NS + Cd + root application of Biocat G (NS + Cd + BioG), iv) T4: NS + Cd + foliar application of Se (NS + Cd + Se1), v) T5: NS + Cd + root application of Se (NS + Cd + Se2), vi) T6: NS + Cd + foliar application of chitosan (NS + Cd + chitosan1), and vii) T7: NS + Cd + root application of chitosan (NS + Cd + chitosan2).

(4) Lines 25-26. Remove this sentence.

Throughout the     ................................ and inflorescence.

(5) Lines 26-30. Replace them by  

The results showed that the exogenous application of Biocat G and Se (T3 and T5) ameliorated the adverse effects caused by Cd toxicity and significantly improved plant growth rate by increasing ………………...........  These treatments (T3 and T5) yielded the best results.

(6) The presentation of the results should be carefully and completely revised.

(7) Lines 30-31. Remove this sentence.

This is greatly …………………………. low as possible.

(8) At the end of the Abstract section, please add conclusion.

Introduction:

The introduction missing details of some important issues.

(9) Please illustrate by more details the benefit impacts of using Biocat G, selenium, and chitosan on plants under Cd stressful condition.

(10) Lines 103-118. This paragraph should be rewrite.

 Material and methods:

(11) Please refer to your seven treatments as I mention in the Abstract section.

1. i) T1: Hoagland nutrient solution (NS), ii) T2: NS + Cd at 3 mg L^-1 (NS + Cd), iii) T3: NS + Cd + root application of Biocat G (NS + Cd + BioG), iv) T4: NS + Cd + foliar application of Se (NS + Cd + Se1), v) T5: NS + Cd + root application of Se (NS + Cd + Se2), vi) T6: NS + Cd + foliar application of chitosan (NS + Cd + chitosan1), and vii) T7: NS + Cd + root application of chitosan (NS + Cd + chitosan2).

(12) Please also correct all of them in the Figures and Tables.

Discussion:

This section missing many important issues.

(13) The mechanism of action of Cd toxicity as well as different treatments (Biocat G, selenium, and chitosan) is missing in all parameters. Why and how Cd toxicity increase or decrease a parameter and why and how the different treatments (Biocat G, selenium, and chitosan) increases or decreases a parameter……what are the possible mechanisms, should be mentioned in the discussion part.

(14) Authors should discuss (give explanations and avoid repeating the results) the effect of each one of the different treatments (Biocat G, selenium, and chitosan) on each one of the measured parameters under both non-stressed and stressed conditions.

(15) Authors did not mention many literature on this topic and do not compare their results with those of previously published papers. Some papers should be added to enhance this part.

(16) At the end of this section, please show how your results fill the gap of previous studies.

Conclusion:

This section must be rewritten.

(17) Authors should include specific results of their research, which extend the current state of knowledge. 

Linguistic quality:

(18) The English language should be revised.

Comments for author File: Comments.pdf

Author Response

Reviewer 3 (R3)

Abstract:

1. R. Abstract section should be re-written. Language used in this section is very bad.   

• Lines 16-17. This sentence should be rephrased.

Authors. Done. The accumulation of cadmium in plants produces phytotoxic damage and a decrease in crop yield. To avoid this effect, it is necessary to prevent its absorption by roots and reduce its toxicity in plant tissues.

The English has been revised.

• Lines 18-19. Replace them by: The current study, was aimed to evaluate the effect of the exogenous applications of Biocat G (fulvic/humic acids), selenium (Se), and chitosan to roots and leaves of broccoli plants exposed to Cd stress.

Authors. Done

• Lines 20-24. Replace them by: The applied treatments were: i) T1: Hoagland nutrient solution (NS), ii) T2: NS + Cd at 3 mg L^-1 (NS + Cd), iii) T3: NS + Cd + root application of Biocat G (NS + Cd + BioG), iv) T4: NS + Cd + foliar application of Se (NS + Cd + Se1), v) T5: NS + Cd + root application of Se (NS + Cd + Se2), vi) T6: NS + Cd + foliar application of chitosan (NS + Cd + chitosan1), and vii) T7: NS + Cd + root application of chitosan (NS + Cd + chitosan2).

Authors. Done

• Lines 25-26. Remove this sentence: Throughout the     ................................ and inflorescence.

Authors. Done

• Lines 26-30. Replace them by: The results showed that the exogenous application of Biocat G and Se (T3 and T5) ameliorated the adverse effects caused by Cd toxicity and significantly improved plant growth rate by increasing ………………...........  These treatments (T3 and T5) yielded the best results.

Authors. This sentence has been corrected. The results showed that the exogenous application of Biocat G and Se (T3 and T5) ameliorated the adverse effects caused by Cd toxicity and significantly improved plant growth rate by decreasing Cd toxicity, besides, Biocat G was able to limit the transport of Cd from the leaves to the inflo-rescences, reducing the content of Cd in the edible part. These treatments (T3 and T5) yielded the best results.

• The presentation of the results should be carefully and completely revised.

Authors. This has been checked in the whole ms.

• Lines 30-31. Remove this sentence. This is greatly …………………………. low as possible.

Authors. Done

• At the end of the Abstract section, please add conclusion.

Authors. This conclusion has been added: These treatments (T3 and T5) yielded the best results, act on the plants by deactivating Cd toxicity, but they did not affect its accumulation in the plant tissue. In addition, Biocat G limits the transport of Cd from the non-edible to the edible part.

Introduction:

The introduction missing details of some important issues.

• Please illustrate by more details the benefit impacts of using Biocat G, selenium, and chitosan on plants under Cd stressful condition.

Authors. Done

• Lines 103-118. This paragraph should be rewrite.

Authors. Done

Material and methods:

• Please refer to your seven treatments as I mention in the Abstract section.

T1: Hoagland nutrient solution (NS), ii) T2: NS + Cd at 3 mg L^-1 (NS + Cd), iii) T3: NS + Cd + root application of Biocat G (NS + Cd + BioG), iv) T4: NS + Cd + foliar application of Se (NS + Cd + Se1), v) T5: NS + Cd + root application of Se (NS + Cd + Se2), vi) T6: NS + Cd + foliar application of chitosan (NS + Cd + chitosan1), and vii) T7: NS + Cd + root application of chitosan (NS + Cd + chitosan2).

Authors. Done

• Please also correct all of them in the Figures and Tables.

Authors. Done

Discussion:

This section missing many important issues.

• The mechanism of action of Cd toxicity as well as different treatments (Biocat G, selenium, and chitosan) is missing in all parameters. Why and how Cd toxicity increase or decrease a parameter and why and how the different treatments (Biocat G, selenium, and chitosan) increases or decreases a parameter……what are the possible mechanisms, should be mentioned in the discussion part.

Authors. Some sentences has been introduced to solve this comment. Overall with respect to Cd treatment without product application. We would like to say that in this experiment have been measured growth parameters, leaf chlorophyll, and leaf Cd and Se concentration. And these parameters has been related among them. But, to reach possible mechanism we are doing new experiments with a wide high range of measurements.

• Authors should discuss (give explanations and avoid repeating the results) the effect of each one of the different treatments (Biocat G, selenium, and chitosan) on each one of the measured parameters under both non-stressed and stressed conditions.

Authors. To avoid repetitions, we have delated this sentence: “Thus, the plants treated with Se through the roots, obtained concentrations of Se and Cd of 3.30 and 5.40 mg kg-1, respectively, while for those that received Se via the leaves, the concentrations were (mg kg-1) 1.62 for Se and 6.19 for Cd (Table 4). Other sentences (few) have been maintained to easy the reader the discussion about specific aspect.

One paragraph has been introduced about the chitosan. So, now the discussion contains, one paragraph about Cd toxicity, one paragraph about Se, one paragraph about biocat G, one paragraph about Chitosan.

• Authors did not mention many literature on this topic and do not compare their results with those of previously published papers. Some papers should be added to enhance this part.

Authors. New references have been introduced in the new paragraphs.

• At the end of this section, please show how your results fill the gap of previous studies.

Authors. This have been introduced in every paragraph of the discussion. It is not necessary to repeat at the end of the discussion.

Conclusion:

This section must be rewritten.

• Authors should include specific results of their research, which extend the current state of knowledge.
• Linguistic quality:

The English language should be revised.

Authors. English language will revise by the English editing service of the journal if needed in the final version of the ms.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Manuscript Title: Foliar and root application of biostimulant active materials to 2 counteract the negative effects of Cd in broccoli plants

Recommendation

Title: In vivo would be in italics and this is not meaningful

Abstract:

Q.1 Line no. 22, What is SP, NS? not mentioned the full form in the start of the manuscript

Q.2 Why fulvic/humic acids are used?

Inroduction:

Q.1 How cadmium Affect inflorescence part of Brocolli not mentioned, should mentioned.

Q.2 Tolerance level of Cd, should mention

Materials and method:

Q.1 Bioaccumulation factors should be there

Q.2 No references in the different parameters, should mention the references in 2.3.1, 2.3.2, 2.3.3 and 2.3.4

Results:

Q.1 Line no 258, “very tolerant”, on the basis of what?

Discussion: How fulvic acid and humic acid reduce the Cd stress,  that is not explained in the discussion.

Conclusion: Rewrite it

Comments for author File: Comments.doc

Reviewer 2 Report

This paper needs major revisions. Find the attached.

Comments for author File: Comments.pdf

Reviewer 3 Report

The study evaluated the effectiveness of reducing stress due to excess cadmium in broccoli plants grown in a glasshouse using different strategies. Selenium, chitosan and the commercial preparation Biocat were tested in the experiment. Selecting such different substances at only one concentration in one treatment is confusing. The plants grew in rockwool. This fact is very questionable. The authors refer to growing crops in soil in many places [lines 54,56,61,85,89, 337] in the paper, and they should be referring to soilless crops. The authors state that Biocat is applied to the soil [lines 135- 136]. And what is known about the performance of this product in soilless systems? 12 plants in one treatment is too few, no information if data had normal distribution, I'm afraid not.

The title is false because the commercial formulation was not applied foliar. Whether selenium can be called a material? Materials are something else. Simply suggests: Application of Biocat, selenium and chitosan to counteract the negative effects of Cd in broccoli plants grown in soilless culture.

The introduction makes virtually no reference to Biocat, selenium and chitosan. No data are available on the effects of cadmium on Brassica vegetables or plants grown in soilless culture.

No data on seedlings, how and where they were grown.

Temperature and humidity must be shown how they have changed.

The exact dates of planting and measurement, the number of days after sowing must be given.

What does 8 hours photoperiod mean? What was the source of light?

Cadmium was used in the form of sulphate. EC and pH results of the solutions must be available. Whether the sulphate has lowered the pH?

What the authors mean "with dose of 4 g/plant (Line 134)” How exactly was it delivered to the roots? In wool? 4 grams of preparation?

What kind of chitosan was used, what was its molecular weight and deacetylation degree? - this should be given

On what basis the concentrations of chitosan (in my opinion too high) and selenium were used?

The authors do not state how many times selenium and chitosan were applied, how many total?

The description of the morphological measurements is unclear, you should specifically state how many days after sowing they were taken.

Description of mineral analysis too general, missing literature, number and size of samples, how results were standardized.

Results from a laboratory where there is little variability can be evaluated using the Duncan's test, but in these studies, conducted over only one season, a severe test such as Tukey's should be used.

Results in tables must be with standard deviation

Spad is not chlorophyll, it's called relative chlorophyll content. Is the value of 134.6 SPAD (Table 3) correct?

When exactly were the photos taken? How toxicity was assessed?

In the discussion, the authors form false theses. the studies they cite are about soil [31,32]. Mineral wool has no sorption complex.

The authors in the selenium discussion only compare the results and do not make an attempt to explain the mechanisms.

The conclusion is tiresome, containing an excess of abbreviations; should be rewritten.

There are many meaningless sentences in the Author Contributions section. It says that all the authors have read the paper. It is strange that so many authors did not notice the working sentences in this part of the paper.

The bibliographic record in many places does not comply with MDPI requirements.

Reviewer 4 Report

The manuscript must carry out an in-depth review of the material and methods and perform a better presentation of the results for a consistent discussion. More suggestions are in the text.

Comments for author File: Comments.pdf