Biofertilizer Application Enhances Drought Stress Tolerance and Alters the Antioxidant Enzymes in Medicinal Pumpkin (Cucurbita pepo convar. pepo var. Styriaca)

Round 1

Reviewer 1 Report

The article “Biofertilizer application enhances drought stress tolerance and alters the antioxidant enzymes in medicinal pumpkin (Cucurbita pepo convar. pepo var. Styriaca)” discusses the effects of different fertilizers on drought amelioration in pumpkin. Some of observations on this paper are

• The paper should be thoroughly checked for grammatical mistakes and language errors.
• Provide details for stress application? How different field capacity levels were achieved? At which stage drought was applied and for how many days? How horizontal moisture transfer was prevented among control and drought treated plots? At what stage, sampling was done?
• Give details for β-. sitosterol assay
• Give formula for protein determination
• Though drought stress was applied but no data has been provided for soil moisture or plant water content which could have confirmed drought in plants
• The discussion should be further strengthened by adding some more details/explanations on the obtained results.

 

 

Author Response

Reviewer 1 Report

Comments and Suggestions for Authors

The article “Biofertilizer application enhances drought stress tolerance and alters the antioxidant enzymes in medicinal pumpkin (Cucurbita pepo convar. pepo var. Styriaca)” discusses the effects of different fertilizers on drought amelioration in pumpkin. Some of observations on this paper are

• The paper should be thoroughly checked for grammatical mistakes and language errors.

Authors’ response: The grammatical mistakes and language errors have been rectified in the paper.

 

• Provide details for stress application? How different field capacity levels were achieved? At which stage drought was applied and for how many days? How horizontal moisture transfer was prevented among control and drought treated plots? At what stage, sampling was done?

Authors’ response: In order to obtain the different drought stress levels in soil as well as application of drought stress treatments, we tried not irrigated plots (Krizek, 1985). For this purpose, once the experimental plots irrigated and the soil moisture was reached to field capacity level, the irrigation was stopped. For measurement of soil moisture level and determination of irrigation time we used of TDR device (Noborio et al., 1999). The available water content was determined based on soil moisture curves, so, the soil water content was calculated for different treatments. It should be mentioned that we used also the weight method for confirmation of calculated soil moisture. All the experimental plots were irrigated similarly up to 1 week before flowering followed by application of drought stress for 1 month up to starting fruits coloring. In order to get of 85% (low stress), 70% (moderate stress) and 55% (severe stress) levels of field capacity, it was allowed that soil was reached to its equal moisture level and then irrigated. For better irrigation management, we used of drip irrigation method. Lines 117-127.

• Give details for β-. sitosterol assay

Authors’ response: For this purpose, the samples oil was extracted, and the β-sitosterol content was measured using the protocol proposed by Smith et al. [25] at 231.5nm with a spectrophotometer. Lines 190-195.

• Give formula for protein determination

Authors’ response: Completely described. Lines 152-158.

• Though drought stress was applied but no data has been provided for soil moisture or plant water content which could have confirmed drought in plants

Authors’ response: Since the drip irrigation method was used and there are distance among stress treatments plots and control plot, so, the stress treatments plots were not affected by control plots moisture. Sampling was done after fruits coloring.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript titled "Biofertilizer application enhances drought stress tolerance and alters the antioxidant enzymes in medicinal pumpkin (Cucurbita pepo convar. pepo var. Styriaca)” by Najafi et al investigated the effect of bio-fertilizers (Nitroxin, mycorrhiza, and Thiobacillus) on the medicinal pumpkin against the impact of drought stress and to increase drought tolerance. Overall the work is technically sound and well written, however, the significance of this research needs to be more emphasized. Here are several points that can improve the manuscript.

• In the first sentence (line 24); please mention the name of biofertilizers in place of ‘some biofertilizers’
• As the paper is on drought stress, I did not see any relevant content in the Introduction section. Please add previous studies carried out on pumpkin on drought tolerance using biofertilizer.
• Line 89-90; In the sentence ‘which results in higher absorption of some useable nutrients’ what are those nutrients? Please specify.
• Lines 91-92; authors have talked about the effect of Nitroxin on plants' root and shoot growth but in this study, but they did not measure it. They should add the effect of biofertilizers on plant biomass under drought.
• It would be better if the author could add the results of physiological parameters under control and drought conditions with and without biofertilizers applications in pumpkin plants. Please show the effect of biofertilizer on RWC, Ion leakage, water loss rate, MDA, and proline content. Do at least a few if not all.
• Lines 224-226 and Fig. 1d, e; under the severe stress conditions, only the mycorrhiza treatment had a positive effect on the GR and GPX enzymes. Please justify why others do not?
• Line 238-239: please replace ‘Nitorxin’ with ‘Nitroxin’.
• Authors could also show the accumulation of ROS components using NBT and DAB staining in the leaves of pumpkin as they talked about ROS in the manuscript.
• What will be the effect if consortia of Nitroxin, mycorrhiza, and Thiobacillus are applied to the pumpkin plant under drought? Will it provide more tolerance compared to the individual one?
• It would be great if authors could add plant images showing tolerance to drought stress after biofertilizer application.

 

Author Response

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript titled "Biofertilizer application enhances drought stress tolerance and alters the antioxidant enzymes in medicinal pumpkin (Cucurbita pepo convar. pepo var. Styriaca)” by Najafi et al investigated the effect of bio-fertilizers (Nitroxin, mycorrhiza, and Thiobacillus) on the medicinal pumpkin against the impact of drought stress and to increase drought tolerance. Overall the work is technically sound and well written; however, the significance of this research needs to be more emphasized. Here are several points that can improve the manuscript.

 

• In the first sentence (line 24); please mention the name of biofertilizers in place of ‘some biofertilizers’

Authors response: Agreed and Mentiuoned. Line 27.

• As the paper is on drought stress, I did not see any relevant content in the Introduction section. Please add previous studies carried out on pumpkin on drought tolerance using biofertilizer.

Authors’ response: Agreed and described. Lines 76-84.

• Line 89-90; In the sentence ‘which results in higher absorption of some useable nutrients’ what are those nutrients? Please specify.

Authors’ response:  There are Sulfur and Phosphate. Line 96.

• Lines 91-92; authors have talked about the effect of Nitroxin on plants' root and shoot growth but in this study, but they did not measure it. They should add the effect of biofertilizers on plant biomass under drought.

Authors’ response: This sentence is revised. Line No.98. The present study was aimed to evaluate the effect of these biofertilizers in improving enzyme activities.

• It would be better if the author could add the results of physiological parameters under control and drought conditions with and without biofertilizers applications in pumpkin plants. Please show the effect of biofertilizer on RWC, Ion leakage, water loss rate, MDA, and proline content. Do at least a few if not all.

Authors’ response: These parameters were not measured in our study.

• Lines 224-226 and Fig. 1d, e; under the severe stress conditions, only the mycorrhiza treatment had a positive effect on the GR and GPX enzymes. Please justify why others do not?

Authors’ response: Mentioned. Line 237-238.

• Line 238-239: please replace ‘Nitorxin’ with ‘Nitroxin’.

Authors’ response: Corrected.

• Authors could also show the accumulation of ROS components using NBT and DAB staining in the leaves of pumpkin as they talked about ROS in the manuscript.

Authors’ response: The above mentioned parameter was not measured in our study.

• What will be the effect if consortia of Nitroxin, mycorrhiza, and Thiobacillus are applied to the pumpkin plant under drought? Will it provide more tolerance compared to the individual one?

Authors’ response: Although the individual applications of mycorrhiza and Thiobacillus and nitrogen fixing bacteria has good effects in providing drought tolerance in pumpkin, their consortia is expected to have improved tolerance compared to the application of individual biofertilizers.

• It would be great if authors could add plant images showing tolerance to drought stress after biofertilizer application.

Authors’ response: Unfortunately, there is no image in this manner in hand.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

In this manuscript, the authors describe the effects of biofertilizers on antioxidant enzyme activities under drought stress conditions of medicinal pumpkin. This is an interesting topic with many potential applications.

• The main concern is the clarity of manuscript, in particular table 1 and the figures and legends. Please, make them clearer. For example, in Figures 1 and 2 please explain the meaning of the letters written above the histograms
• Please, provide more details about material and methods, e.g. extraction of soluble proteins
• Line 126: AM stands for “arbuscular mycorrhiza” as far as I know; please clarify
• Line 276: SE stands for “standard error” as far as I known; please clarify
• There are a few typos and grammar mistakes in the text, e.g. Lines 38,39 are not clear. Thus, careful proofreading of the final manuscript is required
• Please, specify the Thiobacillus species used in the study

In my opinion, this manuscript should be rewritten in order to improve its quality and clarity

Author Response

Reviewer 3 Report

Comments and Suggestions for Authors

In this manuscript, the authors describe the effects of biofertilizers on antioxidant enzyme activities under drought stress conditions of medicinal pumpkin. This is an interesting topic with many potential applications.

 

• The main concern is the clarity of manuscript, in particular table 1 and the figures and legends. Please, make them clearer. For example, in Figures 1 and 2 please explain the meaning of the letters written above the histograms

Authors response: Mentioned at the footnote of Table 1 (Line 209-218), Fig 1 (Line 288-292), Fig 2 (Line 324-326)

• Please, provide more details about material and methods, e.g. extraction of soluble proteins

Authors’ response: Added. Line 152-158

• Line 126: AM stands for “arbuscular mycorrhiza” as far as I know; please clarify

Authors’ response: Extend form provided. Line 45 and 345

• Line 276: SE stands for “standard error” as far as I known; please clarify

Authors’ response: Extend form provided. Line 47

• There are a few typos and grammar mistakes in the text, e.g. Lines 38,39 are not clear. Thus, careful proofreading of the final manuscript is required

Authors’ response: The text was revised and the typos and grammar mistakes were corrected. Also, the sentences were edited.

• Please, specify the Thiobacillus species used in the study

Authors’ response: As per the manufacturer it was Thiobacillus sp.

• In my opinion, this manuscript should be rewritten in order to improve its quality and clarity

Authors’ response: The Manuscript is significantly revised to improve its quality.

 

Reviewer 4 Report

Comments and suggestions for the authors:

The manuscript by Najafi et al. describes a technically sound piece of scientific research. Experiments have been conducted rigorously, with appropriate controls, replication, and sample sizes. While the study seems interesting, this article needs a thorough revision with language and structure before potential publication in "horticulturae". The paper must be edited by someone expert in academic writing. I could see a lot of grammatical mistakes in the MS. Authors should go through the manuscript and correct grammatical error. The quality of the figures should be increased to meet the requirements of the journal. Both introduction and discussion need to be revised and supplemented with the recent literature available on biofertilizer role in stress tolerance and antioxidant enzyme. Lines 65-74 needs to be restructured. It would be better if the authors provide map, geographical coordinates, altitude and soil type of the study area.

Author Response

Reviewer 4 Report

Comments and suggestions for the authors:

• The manuscript by Najafi et al. describes a technically sound piece of scientific research. Experiments have been conducted rigorously, with appropriate controls, replication, and sample sizes. While the study seems interesting, this article needs a thorough revision with language and structure before potential publication in "horticulturae". The paper must be edited by someone expert in academic writing. I could see a lot of grammatical mistakes in the MS. Authors should go through the manuscript and correct grammatical error.

Authors’ response: The authors are thankful to the reviewer for providing excellent comments. The manuscript was thoroughly revised. Language and grammar was corrected with Licensed software; Grammalry

• The quality of the figures should be increased to meet the requirements of the journal.

Authors’ response: It was tried to put the other figure with better quality.

• Both introduction and discussion need to be revised and supplemented with the recent literature available on biofertilizer role in stress tolerance and antioxidant enzyme.

Authors’ response: The new references were added. Line 76-84.

• Lines 65-74 needs to be restructured. It would be better if the authors provide map, geographical coordinates, altitude and soil type of the study area.

Authors’ response: The characteristics were added to manuscript. Line 130-133.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The authors have made substantial changes in the manuscript and they also improved the manuscript. It may be accepted in its present form.