A Rhizogenic Biostimulant Effect on Soil Fertility and Roots Growth of Turfgrass

Round 1

Reviewer 1 Report

12 suggest rephrasing sentence to “chemical fertilizers can lead to severe environmental damages” as not all use of chemical fertilizers will lead to groundwater contamination and soil toxicity.

16 change “Fluvic” to “Fulvic acids” check entire document.

20 check spelling errors

38  be specific about which fertilizers (e.g. iron) introduce trace metals. Are heavy or trace metals a concern for the soil type and region examined in this study?

40 and 47 true that some fertilizers are associated with acidification, but this is not at all a concern for this study with soil pH 8.89 and 8.14. It would be more relevant to discuss the barriers to nutrient availability and plant growth posed by high pH soil and how some amendments demonstrate potential to reduce pH and ameliorate these issues.

65-66 Provide a reference for this statement demonstrating biostimulant linked reductions in fertilizer use and reduction in “damage”.

80-81 describe the specific enzyme activity potentials investigated and the specific relationship to biochemical soil properties and function and plant response.

Introduction section: include some information or background on the specific characteristics (e.g. fulvic acids, prebiotics, calcium lignosulfate) of the biostimulant product tested. General assumptions about the soil biochemical and plant growth response to biostimulant products are too broad.

87-90 this paragraph does not seem necessary.

94-98, 103-105, 110, 112, 114, 121-123, 164, 216, 244, 287, 330, check spelling, sentence structure and grammar.

145-146 Were the biostimulant product rates identical for both soil types? Was only a single soil core taken or was it a composite of several cores within each replicate? When were the soil samples collected in relationship to the application of the biostimulants?

148 Clarify if measured nutrients represent the soil-water or exchangeable cations determined with an ammonium acetate procedure.

162-164 no specific protocol referenced for manual root length measurement. Was a consistent volume of soil roots extracted for each treatment rep?

166-174 There appears to be a mistake in the letter designations for loamy sand pH, EC and sandy soil Ca+2? Please clarify the factors in the different soil types were analyzed separately. Treatment plots dimensions and grass species are different between the two soil types and possibly confound the data. They should be analyzed separately.  

258 use of the word “latter” is to distinguish between two different things. Only soil organic matter is mentioned in the previous sentence.

267-268 multi-year data necessary to validate conclusion that biostimulants have had any lasting impact of soil microbial community size and activity and soil fertility.

283 Baseline sodium values for control and biostimulant treatment plots are needed to draw the conclusion increased calcium levels displaced sodium. The low control EC levels do not indicate a sodium issue exists in these soils

285 there is no foliar or root nutrition analysis to validate this statement.

337 laboratory enzyme activity measurements only reflect the potential for these reactions, they are not an absolute measurement of activity.

339 I am skeptical of any relationship between root growth and lower EC measurement, the values are extremely low in either case indicating low nutrient availability.

353-354 This statement regarding potential elemental toxicities for different soil types with different biostimulant application rates is extremely vague. It does not seem likely a recommendation could be made based on the soil characterization described here.

Author Response

Dear reviewer,

Thank you for your comments and suggestions to improve the manuscript. We have take into account all your comments, and we hope that this new improved version completes all the requirements requested by the reviewers. The answers to your comments are below. Thanks again for the time dedicated to the report.

Dr. Pedro V. Mauri

 

Reviewer 1

Comment 1

12. Suggest rephrasing sentence to “chemical fertilizers can lead to severe environmental damages” as not all use of chemical fertilizers will lead to groundwater contamination and soil toxicity.

 

• Answer 1

Sentence is changed in the new manuscript as reviewer suggested.

Comment 2

16. Change “Fluvic” to “Fulvic acids” check entire document.

 

• Answer 2

Corrected in all the text.

 

Comment 3

20. Check spelling errors

 

• Answer 3

Sentences changed in this line.

 

Comment 4

38. Be specific about which fertilizers (e.g. iron) introduce trace metals. Are heavy or trace metals a concern for the soil type and region examined in this study?

 

• Answer 4

Reviewer is right, heavy or trace metals are not problem for the soil types and regions examined in this study. We delete the l 38 sentence.

 

Comment 5

40 and 47 true that some fertilizers are associated with acidification, but this is not at all a concern for this study with soil pH 8.89 and 8.14. It would be more relevant to discuss the barriers to nutrient availability and plant growth posed by high pH soil and how some amendments demonstrate potential to reduce pH and ameliorate these issues.

 

• Answer 5

It is right; this part in the introduction is omitted, since in this study this theme is not a problem (pH 8.89 and 8.14).  

Comment 6

65-66 Provide a reference for this statement demonstrating biostimulant linked reductions in fertilizer use and reduction in “damage”.

 

• Answer 6

Sentence was improved in the new manuscript, and reference was added.

 

Comment 7

80-81 describe the specific enzyme activity potentials investigated and the specific relationship to biochemical soil properties and function and plant response.

 

• Answer 7

Soil enzymes activities and their implication in soil characteristics and activity are added in the new manuscript. Below is the text added in the new manuscript:

 

“Soil agronomical treatments affect strongly the enzymatic activity compared to other soil biochemical parameters [28]. Soil enzymes activities is defined as potential indicators of soil quality [29, 30] due to their relationship to soil biology and the cycles of nutrients in the soil [31]. Catalase and dehydrogenase are among the most known enzymes related to soil biological characteristics and fertility. Dehydrogenases activity is reflected as good indicator of the oxidative metabolism in soils and microbiological activity [32]. Moreover, catalase enzyme is defined an important indicator of soil fertility and aerobic microorganisms [33]. Additionally, the phosphatase soil enzyme activity catalyze the hydrolysis of organic phosphorus compounds and transform them into an inorganic form of phosphorus, which is assimilated by plants and microorganisms [34]. Furthermore, the calcium lignosulfonate is another element added to biostimulant and which can also improve the soil. The calcium lignosulfonate could be used as soil nutrient sources and as a solution additive to resolve the problems of soil degradation and caking [35], it can also improve soil structure and used to stabilize cohesive to non-cohesive soils [36].”

 

 

Comment 8

Introduction section: include some information or background on the specific characteristics (e.g. fulvic acids, prebiotics, calcium lignosulfate) of the biostimulant product tested. General assumptions about the soil biochemical and plant growth response to biostimulant products are too broad.

 

• Answer 8

Introduction section was improved in the submitted manuscript. We have added all specifics characteristics and suggestions proposed by the reviewers, with more references. The following has been added to the new manuscript:

 

“In this context, the authors of [20, 21] informed that prebiotics are natural products, including sewage sludge, compost, humus, animal manure, and chitin-bearing wastes, which improve soil biochemical activity and microbial population. Likewise, the applications of prebiotics in soils can improve crop growth by producing bioactive substances such as hormones and enzymes, controlling soil diseases, accelerating decomposition of soil lignin materials and delevering inorganic nutrients for plant uptake [22]. Moreover, [23] informed that probiotics are accepted as advantageous microorganisms, which once introduced into soil, should develop an impotant biomass level with several plant beneficial traits. Biostimulant composed with fulvic acids also improved soil properties and plant growth. Similary, the addition of fulvic acids substances to soils can have important implications on the soil physical, chemical, and biological characteristics [24, 25]. Humic and fulvic acids contain nutrient cations, such as potassium, calcium and magnesium, in easily assimilable form for plants [26]. Concerning biological effcet of humic substances [27] reported that addition of these sustances enhance soil fertility through its effects on the composition of microbial populations.”

 

Comment 9

87-90 this paragraph does not seem necessary.

 

• Answer 9

This paragraph is delated in the resubmitted manuscript

 

Comment 10

94-98, 103-105, 110, 112, 114, 121-123, 164, 216, 244, 287, 330, check spelling, sentence structure and grammar.

 

• Answer 10

All these sentences were corrected and improved in the new manuscript.

 

Comment 11

145-146 Were the biostimulant product rates identical for both soil types? Was only a single soil core taken or was it a composite of several cores within each replicate? When were the soil samples collected in relationship to the application of the biostimulants?

 

• Answer 11

The dose of biostimulant applied was identical for both types of soils. About the soil sampling, we collected for each replicate various cores and we make a pool (mixture of all core) for each replicate. With this manner, each replicate can take a representative sampling. Collection of soil samples was achieved at the end of the experiment. All this explication as reviewer suggested are added in the material and methods of the new manuscript. The text in blue has been added to the new version of the manuscript.

 

“Soil samples were collected at the final of the experiments, in 0–20 cm of soil depth and in each replicate. Soil Auger with 10.7cm of diameter and 17 cm depth was used for soil sampling. In each replicate, sampling was achieved in various core, to take a representative soil sample, and we a pooling samples of the same replicate was analyzed. “

 

 

Comment 12

148 Clarify if measured nutrients represent the soil-water or exchangeable cations determined with an ammonium acetate procedure.

 

• Answer 12

The nutrients analyze represent exchangeable cations determined with an ammonium acetate procedure. The exchangeable cations of the soil are displaced by successive extractions of a 1N solution of ammonium acetate at pH = 7 and it is determined in this extract. This explication is added in the material and method of the new manuscript. The following text is added in the new manuscript

 

“The exchangeable cations of the soil are displaced by successive extractions of a 1N solution of ammonium acetate at pH = 7 and it is determined in this extract.”

 

Comment 13

162-164 no specific protocol referenced for manual root length measurement. Was a consistent volume of soil roots extracted for each treatment rep?

 

• Answer 13

This part was improved and explained in the mew manuscript. The text in blue has been added to the new version of the manuscript.

 

“Three replicates of roots were sampled for each treatment (at the end of the experiment) using Auger instrument with 10 cm of diameter and 22 cm depth.”

 

Comment 14

166-174 There appears to be a mistake in the letter designations for loamy sand pH, EC and sandy soil Ca+2? Please clarify the factors in the different soil types were analyzed separately. Treatment plots dimensions and grass species are different between the two soil types and possibly confound the data. They should be analyzed separately.

 

• Answer 14

Data of the experiment were analysed separately for each type of soil and the results are organized in two sections, sandy and sandy loam soil. Reviewer is right; the paragraph 166-174 of the statistical analyses should be better explained. This part was clarify and explained in the new manuscript in the section 3.6.

 

Comment 15

258 use of the word “latter” is to distinguish between two different things. Only soil organic matter is mentioned in the previous sentence.

 

• Answer 15

Sentence corrected

 

Comment 16

 

267-268 multi-year data necessary to validate conclusion that biostimulants have had any lasting impact of soil microbial community size and activity and soil fertility.

 

 

• Answer 16

It is right, our conclusion are only from the first experiment realized. For this reason, we will continue with the study for several years and with other parameters. In future work presented in the final of the manuscript, we have cited this point

 

“In future work, we would like to study the effect of biostimulant on the crop and in several years, focusing on the expected enhanced health.”

 

Comment 17

 

283 Baseline sodium values for control and biostimulant treatment plots are needed to draw the conclusion increased calcium levels displaced sodium. The low control EC levels do not indicate a sodium issue exists in these soils.

 

• Answer 17

The reviewer is right; the low control EC levels do not indicate a sodium issue exists in these soils. Soils studied here do not have sodium problems (0.03- 0.38 dS/m), thus this justification has been removed from the new manuscript. Furthermore, the canonical analysis added in the new manuscript confirmed that EC don’t have relation in both soils control and treated with the other parameters as root length or nutritive elements.

 

Comment 18

285 there is no foliar or root nutrition analysis to validate this statement.

 

• Answer 18

We referred to soil nutritive element and this sentence is corrected in the new manuscript.

 

Comment 19

337 laboratory enzyme activity measurements only reflect the potential for these reactions; they are not an absolute measurement of activity.

 

• Answer 19

These sentences in L 337 has been deleted from the present manuscript, since the PCA analysis has been replaced with a canonical analysis by suggestion of a reviewer.

 

Comment 20

339 I am skeptical of any relationship between root growth and lower EC measurement; the values are extremely low in either case indicating low nutrient availability.

 

• Answer 20

Reviewer is right. These sentences concerning the relationship of roots growth with EC were omitted in the new manuscript and replaced by the new results obtained by the canonical data, where we observed clearly that root growth is not closely related to pH and EC.

 

Comment 21

353-354 This statement regarding potential elemental toxicities for different soil types with different biostimulant application rates is extremely vague. It does not seem likely a recommendation could be made based on the soil characterization described here.

 

• Answer 21

Reviewer in right and this affirmation is delated in the new manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

This manuscript reports on the effect of a commercial biostimulant on soil properties and turfgrass root length grown in two soil types. The authors reported positive effects on several soil properties, including organic matter soil microbial respiration, enzyme activities and inorganic nutrients. These data are of interest as an approach to improve soil fertility and plant growth, while potentially decreasing chemical fertilizer use. The elemental composition of the biostimulant was not reported. It would be important to include this in order to better understand its effects on soil fertility. Plant growth data were limited to measurements of root length. This is only one component of root growth. Information on root architecture and biomass would add to the impact of this manuscript, as well as information on growth of above ground plant matter. 

There were significant issues with English language and style and I have made suggestions for improvement as well as other specific comments in the attached file. 

 

Comments for author File: Comments.pdf

Author Response

Dear reviewer,

Thank you for your comments and suggestions to improve the manuscript. We have take into account all your comments, and we hope that this new improved version completes all the requirements requested by the reviewers. The answers to your comments are below. Thanks again for the time dedicated to the report.

Dr. Pedro V. Mauri

 

Reviewer 2

Comment 1

The elemental composition of the biostimulant was not reported. It would be important to include this in order to better understand its effects on soil fertility.

 

• Answer 1

We have added more explication of the composition of the biostimulant in the new text.

 

“The product applied is a rhizogenic biostimulant composed of fulvic acids, prebiotics molecules that are generated from the fermentation of humic acids, probiotics molecules formed by a bacterial strain of Bacillus sublitis (with high capacities of soil and plant promoter) with concentration in the product at least 5E+ 6CFU/ml, and the calcium lignosulfonate.”

 

Comment 2

Plant growth data were limited to measurements of root length. This is only one component of root growth. Information on root architecture and biomass would add to the impact of this manuscript, as well as information on growth of above ground plant matter. 

 

• Answer 2

 

Reviver is right, root analysis is a complex study with multiple variables like root biomass, root architecture, roots weigh, among others. In this first study, we have analysed the most common roots variable (root length). In addition, several studies cited this root length as representative trait of root change under water deficit. The following two papers have reported this fact: Root traits are related to plant water‐use among rangeland Mediterranean species (Fuctionnal ecology 2017, 31, pp1700-1709) and Morphological and biochemical responses of turf grasses to water deficit conditions (Article in Pakistan Journal of Botany October 2010).

Moreover, in this first study we have focused on the effect of biostimulant on the soil chemical and biological properties. However, as the reviewer suggested in our future work (cited al final of the manuscript) we have included these root parameters.

 

“To evaluate the changes in the plant vigour, root architecture and biomass, we will employ different technologies and methodologies such as the ones described in [78].

 

 

Specific comments

- All the specifics comments suggested by the reviewer were corrected in the resubmitted manuscript                                                                                                                                              

1. 31 corrects……improves

- Corrected

 

1. 43 Omit ‘moreover”. It is used too often in these first 2 paragraphs of the introduction and throughout the manuscript.

- Corrected

 

1. 54-55 affects the nitrogen cycle……… and reduces soil pH

- Corrected

 

1. 71 products can

- Corrected

 

1. 78 biostimulant on soil

- Corrected

 

1. 79 plant root growth

- Corrected

 

1. 80 enzyme activities ….. (sandy loam

- Corrected

 

1. 81 root length ……. was also examined

- Corrected

 

1. 83 plant root growth

- Corrected

 

1. 92 years, biostimulants have become

- Corrected

 

1. 93-94 have tested the effects of biostimulants on soil composition and

- Corrected

 

1. 94-95 In this section we present some references of works focused on how biostimulants

- Corrected

 

1. 95-96 and allow greater plant growth.

- Corrected

 

1. 97 Awkward wording. I suggest: The use of biostimulants for soil restoration has been evaluated (25).

- The sentence in L97 is replaced by the new sentence proposed by the reviewer.

 

1. hese authors reported

- Corrected

 

1. 99 augmented soil enzymatic

- Corrected

 

1. 100 supported soil protection ….. contributed

- Corrected

 

1. 103 Biostimulants also improve ……… soil nutrient elements

- Corrected

 

104. 103-106 Eliminate lines 103 and 104. Begin the sentence on line 105: Biostimulants can stimulate …… permitting the improvement of soil nitrogen availability

- Sentences changed

 

1. 107-109 has several studies on biostimulants and defined biostimulants as containing organisms whose function, after application to plants or the rhizosphere, is to stimulate ……

- Sentences changed

 

1. 110 -112 Reword and shorten: Biostimulants help plants tolerate biotic and abiotic stresses, increase their nutrient efficiency and show beneficial effects on soil properties such as pH, E.C. and soil nitrogen (30).

- Sentences changed.

 

1. 114-115 in root growth. Biostimulants improved several vegetative growth characteristics of shoots and roots including length and dry weight (31).

- Sentences changed.

 

1. 116 biostimulants on root growth also indicated in their results that ….

- Sentences changed.

 

 

1. 117 plant roots and biostimulants induce

- Corrected

 

1. 118-120 Replace existing sentence with: Biostimulant products can be introduced to crop leaves, seeds or soil as a means of stimulating root growth (34).

- Sentences replaced

 

1. 121 Delete this sentence.

- Sentences delated

 

1. 122-125 A review (35) of biostimulants highlighted their advantages, suggesting their usefulness …… yield and summarized the various mechanisms proposed to explain soil and plant improvement observed with the use of biostimulants.

- Sentences changed

 

1. 129 sandy loam, not loam sandy – correct throughout

- Corrected in all the manuscript and figures.

 

1. 133 …. Turfgrass Agrostis stolonifera Tee One was used.

- Corrected

 

1. 138 composed of

- Corrected

 

139 prebiotic molecules What prebiotic molecules were added to the biostimulant? I understand that the formulation may be confidential, but it would be useful to have information on the elemental content of the biostimulant.  

 

• More information and details concerning all biostimulant composition(as prebiotic, probiotics) was added to the new manuscript“The product applied is a rhizogenic biostimulant composed of fulvic acids, prebiotics molecules that are generated from the fermentation of humic acids, probiotics molecules formed by a bacterial strain of Bacillus sublitis (with high capacities of soil and plant promoter) with concentration in the product at least 5E+ 6CFU/ml, and the calcium lignosulfonate.”

•  
•  

 

1. 161 Root growth

- Corrected

 

1. 163 root samples

- Corrected

 

1. 164 ruler

- Corrected

 

1. 167 root growth

- Corrected

1. 185 letters within a column and within a soil type

- Sentences added

 

1. 186 Are these significance levels correct? Should the p value for the first one be ***p<0.001?

- Reviewer is right, the significance level ***p<0.000 is misspelled and it is corrected and replaced with ***p<0.001 in the new manuscript.

 

 

Fig. 1A is missing X axis titles l. 216 Fig 3 Y axis misspelled - Root length

- Corrected

 

1. l. 225 and 226 correlation in sandy soil ……..only in sandy soil

- The correlation coefficient of sandy loam soil has been added

 

1. 231 Relationships between root growth

- Corrected

 

1. 233 root growth

- Corrected

 

1. 237 soil strong correlations

- Corrected

 

1. 242 root growth

- Corrected

 

1. 244 soils was conducted

- Corrected

 

1. 250 that higher root length

- Corrected

 

1. 254 root length; whereas pH ……related to root growth

- Corrected

 

1. 257 This study demonstrated

- Corrected

 

1. 258 Indeed, biostimulant application …. Does this statement apply to all biostimulants?

- This sentence is improved, authors of this study inform about organic biostimulant.

 

1. 259 Similar to our results

- Replaced

 

1. 265 Omit Microbial activity

- Omitted

 

1. 268 What prebiotic elements are present in your biostimulant formulation? Re word: the prebiotic elements present in the biostimulant.

 

- The sentence is improved in the new manuscript. We have added more detail of prebiotics and probiotic components of the biostimulant

 

1. 285 plant nutrient elements What nutrient elements are the authors referring to in this sentence?

 

• This sentence has been modified as follows:

 

“According to our results, the decrease in soil pH permitted the assimilation and availability of Ca⁺², Mg⁺², K⁺ and P plants nutrients elements”

 

 

287 fulvic

- Corrected

 

1. 316 [53]; excretion

- Corrected

 

1. 323 root length In this context, there is more than just root length that is important and may be affected by biostimulants – root biomass, root architecture, for example. Why did the authors choose just to measure root length?

 

• Reviewer is right. This comment has been replied in answer 2.

 

1. 329 root performance

- Corrected

 

1. 334 root length

- Corrected

 

1. 338 root growth

- Corrected

 

1. 339 root development. Is it appropriate to say that root growth is related to pH and EC? You showed a correlation, bot a direct relationship.

 

- These sentences is delated in the new manuscript; and according to other reviewer with the same comments.

 

1. 342 soil texture

- Corrected

 

1. 344 improved

- Corrected

 

1. 345 soils, but to a different degree

- Corrected

 

1. 348 a better or more appropriate decision

- Replaced

 

1. 349 number of applications

- Corrected

 

1. 354 elements that could contribute to soil sustainability

- Corrected

 

1. 362 applications

- Corrected

 

1. 399 with different compositions

- Reviewer referred to l.379 and it is corrected in the new manuscript

 

 

 

Author Response File: Author Response.docx

Reviewer 3 Report

Manuscript "A Rhizogenic Biostimulant Effect on Soil Fertility and Roots Growth of Turfgrass" is interesting and important but needs some corrections.

Table 2: "F-prob" not "Sig".
Figures 1, 2, 3: Please, add HSD values.
Tables 2, 3, 4: add standard deviations for each mean value.
Figure 4: A principal component analysis used in the manuscript are not correct statistical method for data with replications presented in this paper. Authors should used canonical varaite analysis or AMMI model.

Paper needs major revision.

 

Author Response

Dear reviewer,

Thank you for your comments and suggestions to improve the manuscript. We have take into account all your comments, and we hope that this new improved version completes all the requirements requested by the reviewers. The answers to your comments are below. Thanks again for the time dedicated to the report.

Dr. Pedro V. Mauri

 

Reviewer 3

 

Comment 1

Table 2: "F-prob" not "Sig".

 

• Answer 1

Sig was replaced by F-prob in the manuscript

Comment 2

Figures 1, 2, 3: Please, add HSD values.

 

• Answer 2

Values are added in the figures 1, 2 and 3 of the new manuscript.

 

Comment 3

Tables 2, 3, 4: add standard deviations for each mean value.

 

• Answer 3

Standard deviation was added to the table 2. No deviations were added to table 3 and 4. Data of these tables are the correlations coefficients of the relationships between root and all parameters measured in the two type soils.

Comment 4

Figure 4: A principal component analysis used in the manuscript are not correct statistical method for data with replications presented in this paper. Authors should used canonical varaite analysis or AMMI model.

 

• Answer 4

PCA analysis was delated, and we have achieved a canonical analysis as suggested the reviewer.

 

Introduction, results and discussion of the new manuscript were improved as suggested reviewer.

 

 

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Second review:

21 “were” to “was”

24 revise sentence to “an 77% increase in calcium…” and “38% increase in potassium”

61 delete “to”

69 spelling error “products”

82 spelling “delivering”

101-105 move calcium lingnosulfonate information to previous paragraph. Insert at line 91.

119 the “related work” section is redundant with the previous section. Consider combining with introduction or deleting.

184 change “at the final” to “at the end”

185 change to “A soil auger” do not capitalize auger

218 spelling error “canonical”

236 table 2, all SE values for EC should be rounded the same number of decimal places as other parameters

240-248, 253-262 report the values and SE shown in figure 1 and 2. For example: “soil organic matter of the treated sandy soil was four times greater (0.40 ± 0.XX) than the control (0.13 ± 0.XX),…”; ”Similarly, dehydrogenase activity (4.30 ± 0.XX) also increased in response to the biostimulant and was greater than control (3.18 ± 0.XX) by 35% in the sandy soil”.

358 superscript K⁺

416 “soils”

444-452 Add a sentence a detailing how future research should also examine if the positive trends in soil nutrition and enzymatic activities found in the first year after application continue after subsequent applications of the biostimulants.  Determine the persistence of the observed changes in soil properties. 

446 specify what "different technologies and methodologies" you will use to evaluate plant vigor. It is easy to say you will use NDVI rather than reference a paper the reader needs to look up.  The reference provided does not employ methods to evaluate root architecture and biomass. 

Author Response

Dear reviewer,

We really appreciated the second review of the article. All suggestions have been of great contribution to improve the manuscript. We have taken into account all the changes suggested. The answers to your comments are below. Thanks again for the time dedicated to the report.

Dr. Pedro V. Mauri

 

Second review: All the comments suggested by the reviewer were corrected in the resubmitted manuscript

 

Comment 1.

21 “were” to “was”

 

Answer 1.

• Corrected

 

Comment 2.

24 revise sentence to “an 77% increase in calcium…” and “38% increase in potassium”

 

Answer 2.

• Sentence revised

 

Comment 3.

61 delete “to”

 

Answer 3.

• Delated

 

Comment 4.

69 spelling error “products”

 

Answer 4.

• Corrected

 

Comment 5.

82 spelling “delivering”

 

Answer 5.

• Corrected

 

Comment 6.

101-105 move calcium lingnosulfonate information to previous paragraph. Insert at line 91.

 

Answer 6.

• These sentences are inserted at line 91 in the new version of the manuscript. In addition, all references have been rearranged.

 

Comment 7.

119 the “related work” section is redundant with the previous section. Consider combining with introduction or deleting.

 

 

Answer 7.

• This part of the text (Related work) was combined with the introduction section in the new version of the manuscript: Lines 88-103 and lines 114-124 (text in blue color). Section Related work was deleted.

 

Comment 8.

184 change “at the final” to “at the end”

 

Answer 8.

• This Word was changed in the new manuscript

 

Comment 9.

185 change to “A soil auger” do not capitalize auger

 

Answer 9.

• Changed

 

Comment 10.

218 spelling error “canonical”

 

Answer 10.

• Corrected

 

Comment 11.

236 table 2, all SE values for EC should be rounded the same number of decimal places as other parameters.

 

Answer 11.

• The SE values with three decimals have been rounded to two decimals (similar to all other parameters), in the Table 2

 

Comment 12.

240-248, 253-262 report the values and SE shown in figure 1 and 2. For example: “soil organic matter of the treated sandy soil was four times greater (0.40 ± 0.XX) than the control (0.13 ± 0.XX),…”; ”Similarly, dehydrogenase activity (4.30 ± 0.XX) also increased in response to the biostimulant and was greater than control (3.18 ± 0.XX) by 35% in the sandy soil”.

 

Answer 12.

• Means values and their SE are reported in the text in the section 4.2 and 4.3 as the reviewer recommended.

 

Comment 13.

358 superscript K⁺

 

Answer 13.

• Corrected

 

 

 

Comment 14.

416 “soils”

 

Answer 14.

• Corrected

 

Comment 15.

444-452 Add a sentence a detailing how future research should also examine if the positive trends in soil nutrition and enzymatic activities found in the first year after application continue after subsequent applications of the biostimulants.  Determine the persistence of the observed changes in soil properties. 

 

Answer 15.

• This sentence was added in the new manuscript. Line 443-445: Additionally, all parameters evaluated in this study (enzyme activity, soil nutrient and microbial activity) will be evaluated in future work, with the aim of determining the persistence of the observed changes in soil properties.

 

Comment 16.

446 specify what "different technologies and methodologies" you will use to evaluate plant vigor. It is easy to say you will use NDVI rather than reference a paper the reader needs to look up.  The reference provided does not employ methods to evaluate root architecture and biomass. 

 

Answer 16.

• This part of the text was improved in the new version of the manuscript as the reviewer suggested and we have deleted the reference provided. The text in blue has been added to the new version of the manuscript.

 

In future work, we would like to study the effect of biostimulant on the crop over several years, focusing on the expected enhanced health. To evaluate the changes in the plant vigour, plant quality and vegetation status, we will use spectral reflectance approach like the Greenseeker device to measure the NDVI (Normalized Difference Vegetation Index) and drone imagery (true color and thermal imagery). Root architecture and biomass will be evaluated with the aim of assessing the effect of biostimulant in the plant radicular system.

Reviewer 2 Report

The authors have addressed many of my concerns. While I appreciate the added information about the constituents of the biostimulant, it still lacks an elemental analysis. Given the frequency of its application it may have added substantially to the concentrations of Ca, Mg, P and K as well as increasing the accessibility of these elements already present in the soil. There was no mention of N content of the biostimulant or the soil prior to biostimulant application. This would also be useful to know.

There are many errors in English usage remaining in the revised manuscript, particularly in the sentences that have been added. I detail the specific changes recommended below.

l. 21 root lengths

l. 32 corrects

l. 51 affects the nitrogen cycle

l. 61 -62 can allow a reduction ... fertilizers ... application cause ...

l. 77 authors of [20,21] state that

l. 82-83 Should this be delivering rather than delevering?   Moreover, [23] stated that

l. 84 develop a significant biomass with several

l. 85 Biostimulants composed of fulvic acids

l. 86 fulvic acid substances - what is meant by substances??

l. 87 implications for soil

l. 90 substances ... through their effects

l. 93 soil enzyme activities are defined

l. 95 among the best known

l. 96 activity is considered as a good

l. 97 of oxidative metabolism in soils and of microbiological

l. 99 Additionally, phosphate ... catalyzes 

l. 100 transforms

l. 101 Omit furthermore   Calcium

l. 102 added to biostimulants

l. 103 Calcium lignosulfonate .... as a soil nutrient source

l. 104 [35]; ....... and is used

l. 108 enzyme activities

l. 110 examined not exanimated

l. 111 plant root growth

l. 120 biostimulants have

l. 121 effect of biostimulants on soil composition

l. 122 In this section

l. 123 studies focused on how biostimulants improve

l. 124 greater plant growth

l. 135 the improvement

l. 136 biostimulants

l. 158 biostimulants

l. 174 probiotic molecules

l. 175 (with high capacity for soil and plant growth promotion)

l. 176 product of at least ... express the concentration as Y x10⁵ CFU/ml  and calcium ...

l. 184 at the end of the

l. 185 A soil auger of 10.7 diameter

l.186-188 In each replicate several core samples were taken and pooled prior to analysis.

l. 201 Johnson and Temple [52]  It appears that the references prior to this one are also incorrect according to the numbering system in the reference list - please check carefully.

l. 206 using an auger of 10 cm diameter and 22 cm depth

l. 213 (HSD) for each type

l. 216 type of soil

l. 218 canonical

l. 220 type of soil

l. 261 control of about

l. 274 Fig 3 Y axis title Root length

l. 307 types

l. 308 treatments .... The two axes

l. 310 associated with

l. 311 related to ....   K⁺, whereas ....  associated with root growth

l. 316 enzyme activity

l. 319 were separated from the two soils

l. 320 enzyme activities and nutrient elements

l. 334 Should this be small molecules rather than small microbes?

l. 335 prebiotic molecules

l. 336 probiotic molecules

l. 355 plant nutrient elements

l. 379 was comparable

l. 387 enzymes is stimulated

l. 394 root length

l. 406 soil enzyme activities

l. 408 biostimulant is related to

l. 409 nutrient content

l. 419 decisions

l. 436 Biostimulants offer

l. 443 on the crop over several years

 

Author Response

Dear reviewer,

We really appreciated the second review of the article. All suggestions have been of great contribution to improve the manuscript. We have taken into account all the changes suggested. The answers to your comments are below. Thanks again for the time dedicated to the report.

Dr. Pedro V. Mauri

 

Comment.

The authors have addressed many of my concerns. While I appreciate the added information about the constituents of the biostimulant, it still lacks an elemental analysis. Given the frequency of its application it may have added substantially to the concentrations of Ca, Mg, P and K as well as increasing the accessibility of these elements already present in the soil. There was no mention of N content of the biostimulant or the soil prior to biostimulant application. This would also be useful to know.

 

Answer.

 

• The product used is enriched with Calcium (10% complex Calcium) and contain minimum traces (insignificant) of nitrogen, or other nutrient elements. For this reason, we reported in the discussion that the improvement of soil nutrient is due to calcium lignosulfonate and prebiotic and probiotic product composition. The Content of calcium and soil nutrient were added in the new version of the manuscript following the reviewer suggestions. The text in blue has been added to the new version of the manuscript. Moreover, soil nitrogen contain was also added to Table 1.

 

 

The product applied is a rhizogenic biostimulant composed of fulvic acids, calcium nutrient (10 % of complex Calcium), calcium lignosulfonate and a minimum trace of nitrogen. Moreover, the biostimulant also contains prebiotic molecules that are generated from the fermentation of humic acids, probiotic molecules formed by a bacterial strain of Bacillus sublitis (with high capacity for soil and plant growth promotion) with concentration in the product of at least 6 x 10⁵ CFU/ml

 

 

Specific changes recommended by the reviewer:

 

• All the specifics comments suggested by the reviewer were corrected in the resubmitted manuscript

 

1. 21 root lengths

• Corrected

 

1. 32 corrects

• Corrected

 

1. 51 affects the nitrogen cycle

• Corrected

 

1. 61 -62 can allow a reduction ... fertilizers ... application cause ...

• Corrected

 

1. 77 authors of [20,21] state that

• Corrected

 

1. 82-83 Should this be delivering rather than delevering?   Moreover, [23] stated that

- The two words were changed in the new manuscript. It is right, delevering is replaced by delivering, and informed by stated.

 

1. 84 develop a significant biomass with several

                - Corrected

 

1. 85 Biostimulants composed of fulvic acids

                - Corrected

 

1. 86 fulvic acid substances - what is meant by substances??

- Substance word was deleted, we are referred only to fulvic acids

 

1. 87 implications for soil

                - Corrected

 

1. 90 substances ... through their effects

- Corrected

 

 

1. 93 soil enzyme activities are defined

- Corrected

 

1. 95 among the best known

- Corrected

 

1. 96 activity is considered as a good

- Corrected

 

1. 97 of oxidative metabolism in soils and of microbiological

- Corrected

 

1. 99 Additionally, phosphate ... catalyzes 

- Corrected

 

1. 100 transforms

- Corrected

 

1. 101 Omit furthermore   Calcium

- Omitted

 

1. 102 added to biostimulants

- Corrected

 

1. 103 Calcium lignosulfonate .... as a soil nutrient source

- Corrected

 

1. 104 [35]; ....... and is used

- Corrected

 

1. 108 enzyme activities

- Corrected

 

1. 110 examined not exanimated

- Corrected

 

1. 111 plant root growth

- Corrected

 

1. 120 biostimulants have

- Corrected

 

1. 121 effect of biostimulants on soil composition

- Corrected

 

1. 122 In this section

- This phrase is deleted in the new manuscript as suggested the reviewer 1 and this section is combined with introduction in the new manuscript following suggestions of reviewer.

 

1. 123 studies focused on how biostimulants improve

- This phrase is deleted in the new manuscript as suggested the reviewer 1 and this section is combined with introduction in the new manuscript following suggestions of reviewer.

 

1. 124 greater plant growth

- This phrase is deleted in the new manuscript as suggested the reviewer 1 and this section is combined with introduction in the new manuscript following suggestions of reviewer.

 

1. 135 the improvement

- Corrected

 

1. 136 biostimulants

- Corrected

 

1. 158 biostimulants

- Corrected

 

1. 174 probiotic molecules

- Corrected

 

1. 175 (with high capacity for soil and plant growth promotion)

- Corrected

 

1. 176 product of at least ... express the concentration as Y x10⁵CFU/ml  and calcium ...

- Corrected

 

1. 184 at the end of the

- Corrected

 

10. 185 A soil auger of 10.7 diameter

- Corrected

 

l.186-188 In each replicate several core samples were taken and pooled prior to analysis.

- Sentence replaced as the reviewer suggests in the new manuscript.

1. 201 Johnson and Temple [52]  It appears that the references prior to this one are also incorrect according to the numbering system in the reference list - please check carefully.

 

- Reviewer is right. All references were rearranged in the new version of the manuscript and in the References section

 

1. 206 using an auger of 10 cm diameter and 22 cm depth

- Corrected

 

1. 213 (HSD) for each type

- Corrected

 

1. 216 type of soil

- Corrected

 

1. 218 canonical

- Corrected

 

1. 220 type of soil

- Corrected

 

1. 261 control of about

- Corrected

 

1. 274 Fig 3 Y axis title Root length

- Corrected

 

1. 307 types

- Corrected

 

1. 308 treatments .... The two axes

- Corrected

 

1. 310 associated with

- Corrected

 

1. 311 related to ....   K⁺, whereas ....  associated with root growth

- Corrected

 

1. 316 enzyme activity

- Corrected

 

1. 319 were separated from the two soils

- Corrected

 

1. 320 enzyme activities and nutrient elements

- Corrected

 

1. 334 Should this be small molecules rather than small microbes?

- Authors of this work refereed to small microbes in their investigation.

 

1. 335 prebiotic molecules

- Corrected

 

1. 336 probiotic molecules

- Corrected

 

1. 355 plant nutrient elements

- Corrected

 

1. 379 was comparable

- Corrected

 

 

1. 387 enzymes is stimulated

- Corrected

 

1. 394 root length

- Corrected

 

1. 406 soil enzyme activities

- Corrected

 

1. 408 biostimulant is related to

- Corrected

 

1. 409 nutrient content

- Corrected

 

1. 419 decisions

- Corrected

 

 

1. 436 Biostimulants offer

- Corrected

 

1. 443 on the crop over several years

- Corrected

 

 

 

Reviewer 3 Report

Now paper is good.

Author Response

Dear reviewer,

We really appreciated the second review of the article. Thanks again for the time dedicated to the report.

Dr. Pedro V. Mauri