Application of Novel Microorganism-Based Formulations as Alternative to the Use of Iron Chelates in Strawberry Cultivation

Round 1

Reviewer 1 Report

The manuscript authored by Puglisi et al., shows some promising results about the positive effect of a novel microorganism- based formulated (MBF) in mitigating the chlorosis symptoms in strawberry plants compared with a well-known synthetic chelating "sequestrene".  The topic is of great interest for the plant nutrition field since synthetic chelates are widely used by growers nowadays and this treatment entails a high economic cost and some environmental problems. However, based on the results showed, it is not clear for me the benefit for growers in using this MBF, the percentage of soluble Fe is very similar in both (6% in sequestrene and 5% in MBF) and Fe-EDDHA, although it is lower is 2%, It remains considerable.

Manuscript could be improved. I would recommended authors to emphasize the importance of using agronomic alternatives less aggressive with the environment and the possible economic impact that it could have to growers.

1. Introduction. In general, this section is fine but I find it a little bit concise, mainly, the paragraph in which talks about the possible benefits of microorganism to plants nutrition (Line 80-84). I would suggest to authors to dig a little more about the role of microorganisms use in improving iron nutrition in plants and the possible mode of action of them. On my point of view, the manuscript in the current version is a little bit poor in this sense. There are a lot of examples in the literature describing beneficial effects of microorganisms use to improve plant nutrition and it should be interesting to include some examples.
2. Materials and Methods. I would suggest to authors to reorganize the section "2.1. Experimental design" in order to make it easier to follow for readers.

I would divide this section into at least three-four subsections:

• Plant material and growth conditions.
• Soil characterization.
• Treatments/ Inoculum preparation.
• Inoculation method used.

Besides this, some important information in this section is missing.

• Where the plants were grown? In a greenhouse or a shade house? Or outdoors? At room temperature seems ambiguous, at least, I would include a range of temperatures because is not the same to carry out the experiments at room temperature in Córdoba (Spain) in summer where temperatures rise 40º or in winter.
• Authors should clarify the origin and the characterization of the microorganisms strain used in this work.
• Line 138: Trichoderma spores. Which concentration (Spores/ml)?

Author Response

RESPONSE TO REVIEWERS

Reviewer 1

The manuscript authored by Puglisi et al., shows some promising results about the positive effect of a novel microorganism- based formulated (MBF) in mitigating the chlorosis symptoms in strawberry plants compared with a well-known synthetic chelating "sequestrene".  The topic is of great interest for the plant nutrition field since synthetic chelates are widely used by growers nowadays and this treatment entails a high economic cost and some environmental problems. However, based on the results showed, it is not clear for me the benefit for growers in using this MBF, the percentage of soluble Fe is very similar in both (6% in sequestrene and 5% in MBF) and Fe-EDDHA, although it is lower is 2%, It remains considerable.

Authors: authors thank reviewer for its comments. Reviewer is right as regard the MBF formulate, which anyway contained a lower dosage of Fe-EDDHA, thus reducing the impact on the soil and the environmental. However, the aim of this work was really evaluating the effect of the inoculum (indicated in the manuscript as thesis In) without the presence of Fe-EDDHA. The results seems to be very promising also using the inoculum alone. This represents the evident novelty of the paper. In order to underline that both MBF and In were tested and evaluated, authors suggest to change the title as follows: “Application of Novel Microorganism-based Formulates as Alternative to the Use of Iron Chelates in Strawberry Cultivation”. 

Manuscript could be improved. I would recommended authors to emphasize the importance of using agronomic alternatives less aggressive with the environment and the possible economic impact that it could have to growers.

Authors: authors followed the reviewer’s suggestion and underlined the importance of using environmentally sustainable agronomic alternatives as suggested by FAO (pp. 3, lines 108-111).

Introduction. In general, this section is fine but I find it a little bit concise, mainly, the paragraph in which talks about the possible benefits of microorganism to plants nutrition (Line 80-84). I would suggest to authors to dig a little more about the role of microorganisms use in improving iron nutrition in plants and the possible mode of action of them. On my point of view, the manuscript in the current version is a little bit poor in this sense. There are a lot of examples in the literature describing beneficial effects of microorganisms use to improve plant nutrition and it should be interesting to include some examples.

Authors: authors improved the introduction section about the possible benefits of microorganism to plants nutrition, including also other references as suggested by reviewer (pp. 2, lines 80-97).

Materials and Methods. I would suggest to authors to reorganize the section "2.1. Experimental design" in order to make it easier to follow for readers.

I would divide this section into at least three-four subsections:

Plant material and growth conditions.

Soil characterization.

Treatments/ Inoculum preparation.

Inoculation method used.

Besides this, some important information in this section is missing.

Authors: the section "2.1. Experimental design" was divided into 3 sections, as suggested by reviewer (2.1. Plant material and growth conditions; 2.2. Soil characterization; 2.3. Treatments).  The inoculation method was described in treatments, since it consisted in diluting the inoculum provided by manufacturer in water. Anyway, the missing information were provided (pp. 3-4, lines 117-186).

Where the plants were grown? In a greenhouse or a shade house? Or outdoors? At room temperature seems ambiguous, at least, I would include a range of temperatures because is not the same to carry out the experiments at room temperature in Córdoba (Spain) in summer where temperatures rise 40º or in winter.

Authors: plants were grown in laboratory condition in a climatic chamber, and temperature range was provided (pp. 3, lines 118-134).

Authors should clarify the origin and the characterization of the microorganisms strain used in this work.

Authors: microorganism strains used are Trichoderma asperellum e Streptomyces avermitilis (pp.4, lines 171-172).

Line 138: Trichoderma spores. Which concentration (Spores/ml)?

Authors: 10⁸ UFC/g Trichoderma asperellum spores.

Reviewer 2

Utilization of biological materials to improve nutrition control is one of the hot topics recently. Generally, authors showed positive results using their biological materials about supporting iron absorption in strawberry, however, I would like to clear some points before discussing the details as follows,

1. The phenomenon that authors are mentioning in this paper seems not “iron deficiency” but “impairment of iron absorption (under high pH condition)”.

For strawberry, pH seems extremely high as authors mentioned, and EC is also very high through experiment. Based on my experience in my country, pH 5.5-6.5 and EC 0.4 dS/m in summer and 1.0 dS/m in winter is good, approximately and these values may be not so different among various countries. If the pH exceed this value, “impairment of iron absorption” occurs even if iron is not deficient. Also, high EC give damage to strawberry roots. So that, to amend water pH has priority in the field than thinking individual elements such as Fe. What is author’s opinion?

Authors: authors agree with reviewer that the experimental condition are not suitable for strawberries. Moreover, it is clear that the iron deficiency in strawberries (and not in the soil) is due to the pH of the soil in which they were grown: this is the aim of the work (pp. 3, lines 112-115). Unfortunately, in Sicily (authors’ country) the presence of calcareous soil, and high level of pH are very frequent conditions. The aim of this work was indeed to evaluate the effect of microorganism based formulates on calcareous soil in order to increase the Fe solubility in this extreme soil (it also reported in pp.1, lines 34-37 and pp. 2, lines 47-61). The same consideration is linked to the EC values, that in Sicily are very high due to the salt water, which is often used for irrigation use.

2. Through whole manuscript, information is not enough. For example,

Cultivation

There is no description of cropping condition. Did you use a climate chamber? What about seasonal change of management such as water and nutrient?

Authors: these information were provided in the added section 2.1 Plant material and growth conditions (pp. 4, lines 118-135).

Tested materials

There is no information about materials such as “sequestrate”, “MBF and “In”. What is the substrate of “In”? No iron? By the way, do such compounds influence to soil pH and other chemical properties?

Authors: the composition of Sequestrene, MBF and In were provided in section 2.3 Treatments (pp.4, lines 165-171). In the inoculum is not present iron neither EDDHA. Inoculum was added to the soil combined with molasses diluted in water. The soil pH and other chemical properties were not evaluated after the addition of inoculum, since the aim of the work was to evaluate the effect on chlorosis symptoms of the plants.

3. Experimental design is unclear. Authors described 85 plants were used in total. Then, 4 treatments x 5 plants x 4 measurement timing + 5 plants for initial measurement = 85?

Authors: experimental design was rewritten making 3 different subparagraphs as suggested by the previous reviewer. Plants were 85 in total, for each replica. Only 5 plants were used for the T0, and 20 seedlings for each treatment. Experimental design was detailed in the new paragraph 2.3 Treatments. 

4. The test period is too short. Only 20 days of chronological change is shown. Strawberry is usually cultivated for more than half year and it takes more than one month from flowering till harvest at the shortest. The indicated result may not involve the duration of the effect of MBF and In. To evaluate the practicability, yield data should be shown.

Authors: strawberries were cultivated for 42 days in inert substrate, then plants were transplanted in soil, adapted for 7 days, and cultivated for others 20 days after the treatments, in total 69 days (2 months and half). The plants, as shown in Figure 5, showed chlorosis symptoms, and the aim of the work was to evaluate if the treatments could alleviate this problem due to the low iron availability, therefore yield was not evaluated by authors.

4. Small points

Unify units (e.g. uS/cm and dS/m)

Authors: done. In table 1 was reported dS/m as indicated in text (pp.4, table 1).

To values in Figure 1,2,3 and 4 look same among all treatments. I guess these are initial values at starting point. Linear charts are recommendable.

Authors: Figures 1, 2, 3, 4 and 6 were modified according to the reviewer’s suggestion.

Author Response File: Author Response.docx

Reviewer 2 Report

Utilization of biological materials to improve nutrition control is one of the hot topics recently. Generally, authors showed positive results using their biological materials about supporting iron absorption in strawberry, however, I would like to clear some points before discussing the details as follows,

1. The phenomenon that authors are mentioning in this paper seems not “iron deficiency” but “impairment of iron absorption (under high pH condition)”.
For strawberry, pH seems extremely high as authors mentioned, and EC is also very high through experiment. Based on my experience in my country, pH 5.5-6.5 and EC 0.4 dS/m in summer and 1.0 dS/m in winter is good, approximately and these values may be not so different among various countries. If the pH exceed this value, “impairment of iron absorption” occurs even if iron is not deficient. Also, high EC give damage to strawberry roots. So that, to amend water pH has priority in the field than thinking individual elements such as Fe. What is author’s opinion?

2. Through whole manuscript, information is not enough. For example,
Cultivation
There is no description of cropping condition. Did you use a climate chamber? What about seasonal change of management such as water and nutrient?
Tested materials
There is no information about materials such as “sequestrate”, “MBF and “In”. What is the substrate of “In”? No iron? By the way, do such compounds influence to soil pH and other chemical properties?

3. Experimental design is unclear. Authors described 85 plants were used in total. Then, 4 treatments x 5 plants x 4 measurement timing + 5 plants for initial measurement = 85?
There is no n in the figure.

4. The test period is too short. Only 20 days of chronological change is shown. Strawberry is usually cultivated for more than half year and it takes more than one month from flowering till harvest at the shortest. The indicated result may not involve the duration of the effect of MBF and In. To evaluate the practicability, yield data should be shown.

4. Small points
Unify units (e.g. uS/cm and dS/m)
To values in Figure 1,2,3 and 4 look same among all treatments. I guess these are initial values at starting point. Linear charts are recommendable.

Author Response

RESPONSE TO REVIEWERS

Reviewer 1

The manuscript authored by Puglisi et al., shows some promising results about the positive effect of a novel microorganism- based formulated (MBF) in mitigating the chlorosis symptoms in strawberry plants compared with a well-known synthetic chelating "sequestrene".  The topic is of great interest for the plant nutrition field since synthetic chelates are widely used by growers nowadays and this treatment entails a high economic cost and some environmental problems. However, based on the results showed, it is not clear for me the benefit for growers in using this MBF, the percentage of soluble Fe is very similar in both (6% in sequestrene and 5% in MBF) and Fe-EDDHA, although it is lower is 2%, It remains considerable.

Authors: authors thank reviewer for its comments. Reviewer is right as regard the MBF formulate, which anyway contained a lower dosage of Fe-EDDHA, thus reducing the impact on the soil and the environmental. However, the aim of this work was really evaluating the effect of the inoculum (indicated in the manuscript as thesis In) without the presence of Fe-EDDHA. The results seems to be very promising also using the inoculum alone. This represents the evident novelty of the paper. In order to underline that both MBF and In were tested and evaluated, authors suggest to change the title as follows: “Application of Novel Microorganism-based Formulates as Alternative to the Use of Iron Chelates in Strawberry Cultivation”. 

Manuscript could be improved. I would recommended authors to emphasize the importance of using agronomic alternatives less aggressive with the environment and the possible economic impact that it could have to growers.

Authors: authors followed the reviewer’s suggestion and underlined the importance of using environmentally sustainable agronomic alternatives as suggested by FAO (pp. 3, lines 108-111).

Introduction. In general, this section is fine but I find it a little bit concise, mainly, the paragraph in which talks about the possible benefits of microorganism to plants nutrition (Line 80-84). I would suggest to authors to dig a little more about the role of microorganisms use in improving iron nutrition in plants and the possible mode of action of them. On my point of view, the manuscript in the current version is a little bit poor in this sense. There are a lot of examples in the literature describing beneficial effects of microorganisms use to improve plant nutrition and it should be interesting to include some examples.

Authors: authors improved the introduction section about the possible benefits of microorganism to plants nutrition, including also other references as suggested by reviewer (pp. 2, lines 80-97).

Materials and Methods. I would suggest to authors to reorganize the section "2.1. Experimental design" in order to make it easier to follow for readers.

I would divide this section into at least three-four subsections:

Plant material and growth conditions.

Soil characterization.

Treatments/ Inoculum preparation.

Inoculation method used.

Besides this, some important information in this section is missing.

Authors: the section "2.1. Experimental design" was divided into 3 sections, as suggested by reviewer (2.1. Plant material and growth conditions; 2.2. Soil characterization; 2.3. Treatments).  The inoculation method was described in treatments, since it consisted in diluting the inoculum provided by manufacturer in water. Anyway, the missing information were provided (pp. 3-4, lines 117-186).

Where the plants were grown? In a greenhouse or a shade house? Or outdoors? At room temperature seems ambiguous, at least, I would include a range of temperatures because is not the same to carry out the experiments at room temperature in Córdoba (Spain) in summer where temperatures rise 40º or in winter.

Authors: plants were grown in laboratory condition in a climatic chamber, and temperature range was provided (pp. 3, lines 118-134).

Authors should clarify the origin and the characterization of the microorganisms strain used in this work.

Authors: microorganism strains used are Trichoderma asperellum e Streptomyces avermitilis (pp.4, lines 171-172).

Line 138: Trichoderma spores. Which concentration (Spores/ml)?

Authors: 10⁸ UFC/g Trichoderma asperellum spores.

Reviewer 2

Utilization of biological materials to improve nutrition control is one of the hot topics recently. Generally, authors showed positive results using their biological materials about supporting iron absorption in strawberry, however, I would like to clear some points before discussing the details as follows,

1. The phenomenon that authors are mentioning in this paper seems not “iron deficiency” but “impairment of iron absorption (under high pH condition)”.

For strawberry, pH seems extremely high as authors mentioned, and EC is also very high through experiment. Based on my experience in my country, pH 5.5-6.5 and EC 0.4 dS/m in summer and 1.0 dS/m in winter is good, approximately and these values may be not so different among various countries. If the pH exceed this value, “impairment of iron absorption” occurs even if iron is not deficient. Also, high EC give damage to strawberry roots. So that, to amend water pH has priority in the field than thinking individual elements such as Fe. What is author’s opinion?

Authors: authors agree with reviewer that the experimental condition are not suitable for strawberries. Moreover, it is clear that the iron deficiency in strawberries (and not in the soil) is due to the pH of the soil in which they were grown: this is the aim of the work (pp. 3, lines 112-115). Unfortunately, in Sicily (authors’ country) the presence of calcareous soil, and high level of pH are very frequent conditions. The aim of this work was indeed to evaluate the effect of microorganism based formulates on calcareous soil in order to increase the Fe solubility in this extreme soil (it also reported in pp.1, lines 34-37 and pp. 2, lines 47-61). The same consideration is linked to the EC values, that in Sicily are very high due to the salt water, which is often used for irrigation use.

2. Through whole manuscript, information is not enough. For example,

Cultivation

There is no description of cropping condition. Did you use a climate chamber? What about seasonal change of management such as water and nutrient?

Authors: these information were provided in the added section 2.1 Plant material and growth conditions (pp. 4, lines 118-135).

Tested materials

There is no information about materials such as “sequestrate”, “MBF and “In”. What is the substrate of “In”? No iron? By the way, do such compounds influence to soil pH and other chemical properties?

Authors: the composition of Sequestrene, MBF and In were provided in section 2.3 Treatments (pp.4, lines 165-171). In the inoculum is not present iron neither EDDHA. Inoculum was added to the soil combined with molasses diluted in water. The soil pH and other chemical properties were not evaluated after the addition of inoculum, since the aim of the work was to evaluate the effect on chlorosis symptoms of the plants.

3. Experimental design is unclear. Authors described 85 plants were used in total. Then, 4 treatments x 5 plants x 4 measurement timing + 5 plants for initial measurement = 85?

Authors: experimental design was rewritten making 3 different subparagraphs as suggested by the previous reviewer. Plants were 85 in total, for each replica. Only 5 plants were used for the T0, and 20 seedlings for each treatment. Experimental design was detailed in the new paragraph 2.3 Treatments. 

4. The test period is too short. Only 20 days of chronological change is shown. Strawberry is usually cultivated for more than half year and it takes more than one month from flowering till harvest at the shortest. The indicated result may not involve the duration of the effect of MBF and In. To evaluate the practicability, yield data should be shown.

Authors: strawberries were cultivated for 42 days in inert substrate, then plants were transplanted in soil, adapted for 7 days, and cultivated for others 20 days after the treatments, in total 69 days (2 months and half). The plants, as shown in Figure 5, showed chlorosis symptoms, and the aim of the work was to evaluate if the treatments could alleviate this problem due to the low iron availability, therefore yield was not evaluated by authors.

4. Small points

Unify units (e.g. uS/cm and dS/m)

Authors: done. In table 1 was reported dS/m as indicated in text (pp.4, table 1).

To values in Figure 1,2,3 and 4 look same among all treatments. I guess these are initial values at starting point. Linear charts are recommendable.

Authors: Figures 1, 2, 3, 4 and 6 were modified according to the reviewer’s suggestion.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

I think the manuscript was well revised.

In my opinion, the Introduction became a bit too redundant. Some added sentences don’t have direct relationship with this study, e.g., this paper doesn’t treat plant hormones. Defeating world hunger is the subject of prime importance, but I don’t feel any necessity to be written in this paper.

Based on the reply to Reviewer 1, does “Trichoderma spores” means spores of “Trichoderma asperellum”? should be written clearly.

This manuscript is not a review, so subjective expression such as “interestingly” should not be used in an original article. Only readers can decide interesting or not.

Figure 1B in T5, alphabets should be deleted because same letter (a) was given to all treatments.

Author Response

RESPONSE TO REVIEWERS

Reviewer 2

I think the manuscript was well revised.

Authors: authors thank very much the reviewer for the comments, which greatly improved the final version of the manuscript.

In my opinion, the Introduction became a bit too redundant. Some added sentences don’t have direct relationship with this study, e.g., this paper doesn’t treat plant hormones. Defeating world hunger is the subject of prime importance, but I don’t feel any necessity to be written in this paper.

Authors: the consideration about hormones were deleted (pp. 3, lines 88-95). “Defeat the world hunger” was deleted (pp. 3, line 107).

Based on the reply to Reviewer 1, does “Trichoderma spores” means spores of “Trichoderma asperellum”? should be written clearly.

Authors: authors specified in the Material and Methods section “Trichoderma asperellum spores” (pp. 5, line 166).

This manuscript is not a review, so subjective expression such as “interestingly” should not be used in an original article. Only readers can decide interesting or not.

Authors: authors deleted the expression “interestingly” in the discussion section (pp. 13, line 301).

Figure 1B in T5, alphabets should be deleted because same letter (a) was given to all treatments.

Authors: authors thank reviewer for this suggestion, because it was an oversight, therefore “a” letter from the sampling T5 in figure 1 B were deleted.

Author Response File: Author Response.docx