Paper Towel Method: In Vitro Inoculation Technique for Rapid and Robust Assessment of Clusterbean and Cowpea Genotypes Against Macrophomina phaseolina

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The document displays deficiencies in composition, orthography, and stylistic elements, with some sections failing to address all necessary elements. Additionally, the methodology lacks clarity, and the writing style hinders replication. I will now enumerate several of the aspects mentioned earlier.

Page 1, line 36: R. bataticola?

Page 2, line 64: indicate the name scientific from cowpeas and clusterbean.

Page 2, line 68: “The research is needed to improve the identification and characterisation of the genetic potential of host defence”. Why is this the relation of the item with the manuscript?

In materials and method: Data on grain samplings, such as the time of year and coordinates, are missing. Acronyms, such as PDA, also lack definitions.

Section 2.3: Mass or mycelia?

Page 3 line 107: gm is unit of measure?

And was the relative humidity not considered?

Line 139: gain?

Was seed germination realized?

In the section of Materials and Methods and Results, there are complete sentences similar to those written by other authors.

Author Response

Comments: The document displays deficiencies in composition, orthography, and stylistic elements, with some sections failing to address all necessary elements. Additionally, the methodology lacks clarity, and the writing style hinders replication. I will now enumerate several of the aspects mentioned earlier.

Authors Response:

Thank you very much for your constructive suggestions. We have made modifications to the abstract, introduction, and discussion. Further, we have made the necessary changes to create more clarity in the methodology.

Comments: Page 1, line 36: R. bataticola?

Authors Response: Thank you, sir, the correction is included

Comments: Page 2, line 64: indicate the name scientific from cowpeas and clusterbean.

Authors Response: Cluster bean (Cyamopsis tetragonoloba) and cowpea (Vigna unguiculata) is added in the text.

Comments: Page 2, line 68: “The research is needed to improve the identification and characterisation of the genetic potential of host defence”. Why is this the relation of the item with the manuscript?

Authors Response:

The robust screening is prerequisite for the identification of durable resistant genotypes; thus, the development of suitable inoculation techniques must be required to define the genetic potential of host defence of different genotypes.

Comments: In materials and method: Data on grain samplings, such as the time of year and coordinates, are missing. Acronyms, such as PDA, also lack definitions.

Authors Response: The disease samples were collected during Kharif 2021 from   Pulses Research Station, S.D. Agricultural University, Gujarat.

Comments: Section 2.3: Mass or mycelia?

Authors Response: It's fungal mycelia, not mass.

Comments: Page 3 line 107: gm is unit of measure? G is And was the relative humidity not considered?

Authors Response: Here, g is used for gram (unit of measurement). Thanking you sir for pointing out this. The relative humidity was not measured. Only moistened sorghum grains were used for mass culturing of pathogen/fungus.

Comments: Line 139: gain?

Authors Response: It's grain. Thanking you sir for your observation.

Comments: Was seed germination realized?

Authors Response:

Seed germination data was taken to realize the seed quality; Almost 100% of seed germination ensured healthy seeds that were used for testing.

Comments: In the section of Materials and Methods and Results, there are complete sentences similar to those written by other authors.

Authors Response:

We have made necessary changes to avoid plagiarism or the structure of the sentences similar to other publications.

Reviewer 2 Report

Comments and Suggestions for Authors

In this study, the Authors investigated the pathogenicity of Macrophomina phaseolina and the resistance to the pathogen of clusterbean and cowpea genotypes through different inoculation techniques.
The investigation is relevant but the potential pathogenic mechanisms of Macrophomina phaseolina against clusterbean and cowpea varieties, as well as defense and resistance processes of these leguminous crops could be discussed more broadly by the Authors, considering the different genotypes and inoculation methods used in the study.

Author Response

In this study, the Authors investigated the pathogenicity of Macrophomina phaseolina and the resistance to the pathogen of clusterbean and cowpea genotypes through different inoculation techniques.
The investigation is relevant but the potential pathogenic mechanisms of Macrophomina phaseolina against clusterbean and cowpea varieties, as well as defense and resistance processes of these leguminous crops could be discussed more broadly by the Authors, considering the different genotypes and inoculation methods used in the study.

Authors Response:

First of all, we would like to thank reviewer #2 for his remark on the research topic in which we are involved and for the manner in which he/she sees the importance of the work.  As per suggestion of the reviewers we have added one separate paragraph in the discussion section citing the defense response of the different released varieties of clusterbean and cowpea under the artificially inoculated condition to justify the importance of the paper towel method.

Reviewer 3 Report

Comments and Suggestions for Authors

Paper towel method: In vitro inoculation technique for rapid 2 and robust assessment of clusterbean and cowpea genotypes 3 against Macrophomina phaseolina This study presents an innovative method for in vitro inoculation of clusterbean and cowpea genotypes with the fungus Macrophomina phaseolina, a known pathogen that causes root diseases in various legumes, impacting the safety of food production. The proposed approach aims to enhance the understanding of the resistance mechanisms in these legumes, contributing to the development of more resilient crop varieties.

The article is useful, informative, and well-constructed.

Comments for author File: Comments.pdf

Author Response

Paper towel method: In vitro inoculation technique for rapid  and robust assessment of clusterbean and cowpea genotypes  against Macrophomina phaseolina This study presents an innovative method for in vitro inoculation of clusterbean and cowpea genotypes with the fungus Macrophomina phaseolina, a known pathogen that causes root diseases in various legumes, impacting the safety of food production. The proposed approach aims to enhance the understanding of the resistance mechanisms in these legumes, contributing to the development of more resilient crop varieties. The article is useful, informative, and well-constructed.

Authors Response:

Thank you very much sir for your positive comments

Comments: The abstract is presented coherently and informatively.

Authors Response: Thank you very much for your observation

Comments: The introduction addresses the central theme of crop loss associated with plant mortality or dehydration as a common issue. Is there any data on the annual loss of crops in terms of hectares or monetary value? Similarly, among the various factors that limit productivity, how severe is the issue of dry root rot compared to moisture, heat, and soil fertility, for example?

Authors Response:

Usually, the yield loss due to the dry root rot varies with the level of infection in field conditions. So far, very little information is available on this aspect. 25-48% yield loss is reported in arid legume crops due to dry root rot as reported by Singh et al., 2024. Further, 30-44% yield losses are reported in mungbean (Bashir & Malik, 1988; Sharma & Singh, 2001) and 29% yield loss in urdbean (Kulkarni et al., 2019) due to dry root rot. But, no infection is available in the monetary count.

The dry root rot is highly severe in many pulses including arid legumes. As we know, arid legume crops like clusterbean and cowpea are well-adapted under dry conditions; thus, the issue of biotic stress due to dry root rot is more profound compared to abiotic stress like moisture stress and heat stress.

Singh Mahabeer, Singh Jitendra, Maurya Shivam, Kumar Sunil, Meena A.K., Sharma Pinki, Lakhran Lalita (2024). Root Rot Disease Incited by Macrophomina phaseolina in Arid Legumes and their Management: A Review . Legume Research. 47(1): 01-07. doi: 10.18805/LR-4714.

Bashir, M. & Malik, B.A. (1988) Diseases of major pulse crops in Pakistan—a review. Tropical Pest Management, 34, 309–314.

Sharma, K. & Singh, T. (2001) Seed and seedling infection of Rhizoctonia bataticola in Vigna radiata. Journal of Mycology and Plant Pathology, 30, 15–18.

Kulkarni, S., Shobharani, M. & Raja. (2019) Integrated management of dry root rot of blackgram caused by Rhizoctonia bataticola. International Journal of Current Microbiology and Applied Science, 8, 853–858.

Comments: It is understood that the interaction between the pathogen and legumes compromises crop productivity in vulnerable areas. However, it is not clearly established that a method for evaluating in vitro tolerance is proposed. That is to say, the introduction is highly
focused on the problem but does not hint at the intentions of assessing which method
may employ fewer resources or be more effective for studying inoculation.

Authors Response:

Thanking very much sir; few points have been incorporated in the introduction to state the different inoculation method and their significance to display the intention of our experimentation.

Comments: In the results section, the characteristics of plumule length, radical length, and total length are compromised by inoculation but also differ among cultivation methods. What benefits or drawbacks are presented in the analysis of resistance to fungal infections for
agroindustry?

Authors Response:

In  our  experiment, the of plumule length, radical length, and total length are compromised by different inoculation methods, rather than cultivation methods.

The analysis of resistance to fungal infection is helpful in selecting robust resistant genotypes having good yield potential.

Comments: What characteristics make the genotypes Cowpea GC-3 and Clusterbean GG3 resistant to developing root rot?

Authors Response:

Some histochemical phenomenon might be associated with root rot resistance in cowpea and clusterbean genotypes; further research is necessary in this aspects.

Reviewer 4 Report

Comments and Suggestions for Authors

Macrophomina phaseolina poses a significant threat to global crop production. This manuscript evaluates the resistance of clusterbean and cowpea to Macrophomina phaseolina under three inoculation techniques. The study offers a comprehensive overview, with well-structured tables and thorough analysis and discussion, leading to sound conclusions. The manuscript identifies the paper towel method as the optimal inoculation technique for studying the pathogenicity of Macrophomina phaseolina in vitro. This research establishes a foundation for developing disease-resistant genotypes.

1. The abstract requires revision; currently, it only reflects the results related to Table 1, and additional findings need to be articulated.

2. The introduction should include relevant advancements in resistance evaluation methods, as well as studies of the three methods discussed in the manuscript in other species.

3. The first occurrence of abbreviations should include full terms, such as PDA, BOD, and DRR.

4. Improve the labeling of Figure 1 by specifying ABCDEF, including magnification levels.

5. Clarify the growth duration of crops in Figures 2-4.

6. Tables 1-4 should include standard deviation in the data and eliminate Inference. Annotations are necessary for the superscript and parenthetical data in Table 1.

7. Why does section 3.2 omit analysis of the in vitro agar inoculation method?

8. Add more recent references from recent years to the manuscript.

Author Response

Macrophomina phaseolina poses a significant threat to global crop production. This manuscript evaluates the resistance of clusterbean and cowpea to Macrophomina phaseolina under three inoculation techniques. The study offers a comprehensive overview, with well-structured tables and thorough analysis and discussion, leading to sound conclusions. The manuscript identifies the paper towel method as the optimal inoculation technique for studying the pathogenicity of Macrophomina phaseolina in vitro. This research establishes a foundation for developing disease-resistant genotypes.

Authors Response:

Thank you very much for your comments and recognition of this study.

Comments: The abstract requires revision; currently, it only reflects the results related to Table 1, and additional findings need to be articulated.

Authors Response: Thank you very much for your suggestions. We have fixed this error in the abstract

2 Comments:. The introduction should include relevant advancements in resistance evaluation methods, as well as studies of the three methods discussed in the manuscript in other species.

Authors Response: Thank you very much for your proposal, we have rephrased some parts of the introduction.

3 Comments:. The first occurrence of abbreviations should include full terms, such as PDA, BOD, and DRR.

Authors Response: Thank you very much for your question. We have fixed this error

4 Comments:. Improve the labeling of Figure 1 by specifying ABCDEF, including magnification levels.

Authors Response: Thank you very much for your observation; improvisation in figure legend was done.

5 Comments:. Clarify the growth duration of crops in Figures 2-4.

Authors Response: The specific growth stages of cowpea and clusterbean were mentioned in Figures 2 to 4.

6 Comments: Tables 1 to 4 should include standard deviation in the data and eliminate Inference. Annotations are necessary for the superscript and parenthetical data in Table 1.

Authors Response: Thank you, sir, for suggestion. Annotations is included the superscript and parenthetical data in Table 1; and standard deviation in the data is added in table 2 and 3 only. This is not possible for disease incidence data in table no 1 and 4, where total no of killed plants were counted.

7 Comments: Why does section 3.2 omit analysis of the in vitro agar inoculation method?

Authors Response: In our pathogenicity test, we observed that in vitro paper towel method was more suitable than in vitro agar inoculation in terms of its rapidity and effectiveness. Further, to confirm its robustness for screening a large number of genotypes in vitro, the paper towel method was compared with soil inoculation method in pot condition; so that a rapid and robust screening techniques can be established for screening the arid legumes crops against DRR pathogen. Thus, in vitro agar inoculation method.

8 Comments: Add more recent references from recent years to the manuscript.

Authors Response: Thank you sir, some new references related to this manuscript were added

Reviewer 5 Report

Comments and Suggestions for Authors

Dear Authors,

 

The manuscript is orgin and novelty. However some revisions should be done.

 

The needed corrections and suggestions were given as follows:

 

1- The abstract should be rewritten by following the background, materials and methods, results and conclusion part. 

2- Please delete "(1999)" in line 38. 

3- The latitude and longitude of the studied area should be added in the materials and methods part.

4- The full name of the "PDA" should be given in the line 89.

5- Why were the clusterbean genotype (RGC-1066) and cowpea genotype (GC 2108) selected?

6- What is the meaning of the "BOD"?

7- When was the study condcuted?

8- The used sterilize soil properties should be given? 

9- The needed references should be given for the subtitle "2.4.2. Agar inoculation method", "2.4.3. Soil inoculation method", "2.5. Reproducibility of the paper towel method in comparison to the soil-inoculation method" and "2.6. Statistical analysis".

10- The experimental design and growing conditions of the stuyd should be given in the materials and methods part by giving subtitle. 

11- The values of "S.Em., CD and C.V. should be given for Disease occurrence (days) in Table 1.

12- The letters for the results in Tables 2, 3, 4 should be given for the 5% significant level.

13- The deeply results should be given for the subtitle "3.2. Reproducibility of the paper towel method in comparison with soil inoculation method".

14- The discussion part should be divided according to results subtitles. 

General suggestions:

 

1. The language of manuscript should be rechecked.

2. The manuscript contains many spelling mistakes and errors. So, the manuscript should be controlled again for the errors.

Best regards,

Comments on the Quality of English Language

The language of manuscript should be revised for the quality of English.

Author Response

Comments: 1- The abstract should be rewritten by following the background, materials and methods, results and conclusion part. 

Authors Response: Thank you sir for your positive remarks and your suggestion of abstract modification was incorporated.

Comments: 2- Please delete "(1999)" in line 38. 

Authors Response:  Thank you sir for pointing out this correction

Comments: 3- The latitude and longitude of the studied area should be added in the materials and methods part.

Authors Response: The GPS of the experimental site was incorporated.

Comments: 4- The full name of the "PDA" should be given in the line 89.

Authors Response: full name of the "PDA" is added

Comments: 5- Why were the clusterbean genotype (RGC-1066) and cowpea genotype (GC 2108) selected?

Authors Response: Both these genotypes were sowing susceptibility against root rot in field conditions; thus chosen for experimentation.

Comments: 6- What is the meaning of the "BOD"?

Authors Response: Biological Oxygen demand (BOD).

Comments: 7- When was the study conducted?

Authors Response: The study was initiated during Kharif 2021 and extended upto Kharif 2022

Comments: 8- The used sterilised soil properties should be given? 

Authors Response: Sorry sir, the property of the sterilized soil was not analyzed before the study.

Comments: 9- The needed references should be given for the subtitle "2.4.2. Agar inoculation method", "2.4.3. Soil inoculation method", "2.5. Reproducibility of the paper towel method in comparison to the soil-inoculation method" and "2.6. Statistical analysis".

Authors Response: Some references related to different inoculation techniques were added in the introductory part.

Comments: 10- The experimental design and growing conditions of the study should be given in the materials and methods part by giving subtitles. 

Authors Response: The details of the experimental design along with their data analysis protocol were described under 2.6 statistical analysis section.

Comments: 11- The values of "S.Em., CD and C.V. should be given for Disease occurrence (days) in Table 1.

Authors Response: Your suggestion was incorporated

Comments: 12- The letters for the results in Tables 2, 3, 4 should be given for the 5% significant level.

Authors Response: Sir, letters are provided for the data present in table 1, where DNMRT was done.

Comments: 13- The deeply results should be given for the subtitle "3.2. Reproducibility of the paper towel method in comparison with soil inoculation method".

Authors Response: Thank you sir, we have made necessary changes.

Comments: 14- The discussion part should be divided according to the results subtitles. 

Authors Response: Thank you sir, we have tried our best to justify our result with separate sections of discussion

Comments: 15. The language of manuscript should be rechecked.

Authors Response:  Thanking you sir, we have corrected most possible errors.

Comments: 16. The manuscript contains many spelling mistakes and errors. So, the manuscript should be controlled again for the errors

Authors Response: Correction made sir

Comments: 17 Comments on the Quality of English Language. The language of the manuscript should be revised for the quality of English.

Author Response: The quality of English was improved extensively.

Reviewer 6 Report

Comments and Suggestions for Authors

This manuscript provides a comparison between three inoculation methods for judging infection in two types of beans for dry rot caused by Macrophomina phaseolina. The highlighted method, using a paper towel packet for the inoculation has been previously reported for the same pathogen. The authors need to better justify why their work is different from that previously reported. Also need to include better justification for why this work needed to be done in the introduction. It is better explained in the discussion, but must be in the introduction. Also need to mention in the introduction that that the method has been used before for this pathogen and for beans. Minor comments are below.

Line 36 - Need to mention that R. bataticola is the old name for Macrophomina phaseolina

Line 60 - need new paragraph here.

Line 89 - Need to write out potato dextrose agar (PDA) at first use and delete 'solidified' since agar will be solid

Line 117 - Need to give Genus and species for both clusterbean and cowpea

Line 124 - Need to define Biological Oxygen Demand (BOD) incubator and explain why it was needed. Also need to give manufacturer and location for the incubator used.

Line 128 - Define/write out DRR here

Line 147 - please give properties of the soil

Line 149 - need to include temperature and lighting

Line 225 - write out Disease Index (DI) here

 

Comments on the Quality of English Language

There are minor English problems throughout the manuscript primarily with omitting words.

Author Response

Comments: This manuscript provides a comparison between three inoculation methods for judging infection in two types of beans for dry rot caused by Macrophomina phaseolina. The highlighted method, using a paper towel packet for the inoculation has been previously reported for the same pathogen. The authors need to better justify why their work is different from that previously reported. Also need to include a better justification for why this work needed to be done in the introduction. It is better explained in the discussion but must be in the introduction. Also need to mention in the introduction that that the method has been used before for this pathogen and for beans.

Response: Thanking sir for your suggestion. We have made the necessary changes.

Comments: Line 36 - Need to mention that R. bataticola is the old name for Macrophomina phaseolina

Response: Yes sir, correction made

Comments: Line 60 - need new paragraph here.

Response: Thank you sir for your comments and it was implemented

Comments: Line 89 - Need to write out potato dextrose agar (PDA) at first use and delete 'solidified' since agar will be solid

Response: PDA is used as an acronyms for potato dextrose agar; this PDA has different states, i.e., molten and solidified; thus, mentioned here.

Comments: Line 117 - Need to give Genus and species for both clusterbean and cowpea

Response: Already included in the introductory part (Line 66).

Comments: Line 124 - Need to define Biological Oxygen Demand (BOD) incubator and explain why it was needed. Also need to give manufacturer and location for the incubator used.

Response: It needs to control the incubation temperature, water quality in relation to oxygen, pollution levels, and improvement of biology studies on plants. Our BOD incubator is manufactured by Remi, India. It is used in our Pulses Research Station

Comments: Line 128 - Define/write out DRR here

Response: DRR is the acronym of dry root rot

Comments: Line 147 - please give properties of the soil

Response: Sorry sir, we did not analyse the property of the sterilized soil before using it for experimentation.

Comments: Line 149 - need to include temperature and lighting     

Response: Sir, temperature and lighting condition was already written

Comments: Line 225 - write out Disease Index (DI) here

Response: Sorry sir, we have counted the disease incidence, rather than the disease index.

Comments: Comments on the Quality of English Language. There are minor English problems throughout the manuscript primarily with omitting words

 

Response: Sir, we have considered your suggestions and made the necessary changes.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Suggestions were addressed, revise the final document without WORD change control.

Author Response

   

Comments: Suggestions were addressed, revise the final document without WORD change control.

Authors’ Response: Thank you very much for your comments and recognition of this study. We have made the necessary changes as per your suggestion.

Reviewer 2 Report

Comments and Suggestions for Authors

The adjustments improved the manuscript.

Author Response

 

Comments: The adjustments improved the manuscript.

Authors’ Response: Thank you very much for your comments and recognition of this study.

Reviewer 4 Report

Comments and Suggestions for Authors

The author has responded to my comments and has meticulously revised the manuscript. The current version of the manuscript is acceptable. It would be preferable if the author could provide a clean version without tracked changes for reviews.

Author Response

 

Comments: The author has responded to my comments and has meticulously revised the manuscript. The current version of the manuscript is acceptable. It would be preferable if the author could provide a clean version without tracked changes for reviews.

Authors’ Response: Thank you very much for your comments and recognition of this study. We have made necessary changes as per your suggestion.

Reviewer 5 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for your revised manuscript. I think it is better than old version. 

Congratulations and success!!!

Best regards,

Author Response

 

Comments: Dear Authors, Thank you for your revised manuscript. I think it is better than the old version. Congratulations and success.

Author’s Response: Thank you very much for your comments and recognition of this study.

Reviewer 6 Report

Comments and Suggestions for Authors

The justification and editing improved the manuscript.

Comments on the Quality of English Language

Please have an editor read through the manuscript one additional time. There are a variety of small changes needed throughout, including the abstract.

Author Response

 

Comments: Please have an editor read through the manuscript one additional time. There are a variety of small changes needed throughout, including the abstract.

Author’s Response: Thank you very much for your comments and recognition of this study. We have made necessary improvement in English grammatical and typo errors as per your suggestion.