Method of Optical Diagnostics of Grain Seeds Infected with Fusarium

Round 1

Reviewer 1 Report

I think that the manuscript entitled “Method of optical diagnostics of grain seeds infected with Fusarium" deserves publication in Applied Sciences after major revision. The manuscript deals with current problems, but needs improvement

 

Line 29: please change “Fusarium” into „Fusarium”

Line 56: please change “Puccinia striiformis” into „Puccinia striiformis” and other Latin names

Line 57: please change “in vivo” into „in vivo”

Introduction: no information on the global scale of the problem (the values) and why are toxins so dangerous

Line 131: please describe statystical methods and program/s

Figure 1 and 2; please complain on the Figures for wheat and barley seeds

Toxin content (T-2 taxine)?

Discussion: no, reference of the obtained results to the literaturę data. Needed to be completed.

Line 223: the humidity was not measured there is no reference

Author Response

We, the authors, appreciate all the time spent by the reviewers and all the ideas, suggestions, and comments presented. We addressed all the ideas, suggestions, and comments below in our revised manuscripts.

 

Comments and Suggestions for Authors:

 

1. Line 29: please change “Fusarium” into „Fusarium”

Response to reviewer: The authors thank the Reviewer for the comment. The replacement has been made.

 

2. Line 56: please change “Puccinia striiformis” into „Puccinia striiformis” and other Latin names

Response to reviewer: The authors thank the Reviewer for the comment. The replacement has been made.

 

3. Line 57: please change “in vivo” into „in vivo”.

Response to reviewer: The authors thank the Reviewer for the comment. The replacement has been made.

 

4. Introduction: no information on the global scale of the problem (the values) and why are toxins so dangerous.

Response to reviewer: Information about the global fusarium problem has been added to the Introduction section.

 

5. Line 131: please describe statystical methods and program/s.

Response to reviewer: Statistical methods and programs are described in more detail in the source [24], a link to which is added to the section Materials and Methods.

 

6. Figure 1 and 2; please complain on the Figures for wheat and barley seeds.

Response to reviewer: The excitation and photoluminescence characteristics of oat, wheat and barley seeds are similar to each other and when presented in the same figure, the seed graphs of all three crops will be located very close to each other and visually difficult to distinguish. The presentation of graphs for oats, barley and wheat on separate graphs will lead to a sharp increase in the volume of the article due to the appearance of four more similar drawings. Therefore, the authors used a compromise option with the publication of Figures 1 and 2 for oats as an example and a description of the type of similar graphs in the text part of the article. For wheat, the excitation and photoluminescence graphs are given and described in more detail in the article [26], a reference to which is introduced in this article.

 

7. Toxin content (T-2 taxine)?

Response to reviewer: The content of the T-2 toxin in the grain is over 100 ng.

 

8. Discussion: no, reference of the obtained results to the literaturę data. Needed to be completed.

Response to reviewer: In the Discussion section, when analyzing the results obtained, there is a link to the article [27]. In general, there are very few articles devoted to the use of luminescent spectroscopy for the diagnosis of fusarium seeds of grain plants. There is an article by Lens, E.C., Belles que, J. & Marcus, L.G. Detection of citrus cancer in citrus plants using laser induced fluorescence spectroscopy. Precision Agric 10, 319–330 (2009). https://doi.org/10.1007/s11119-009-9124-2 , but it is dedicated to the diagnosis of citrus fruits.

 

9. Line 223: the humidity was not measured there is no reference.

Response to reviewer: Measurements were carried out for seeds of conditioned humidity.

Author Response File: Author Response.docx

Reviewer 2 Report

Please see the attachment file.

Comments for author File: Comments.pdf

Author Response

We, the authors, appreciate all the time spent by the reviewers and all the ideas, suggestions, and comments presented. We addressed all the ideas, suggestions, and comments below in our revised manuscripts.

 

Comments and Suggestions for Authors:

 

1. Some abbreviations which are used in the manuscript text do not introduce anywhere. One example is in the first line of introduction Fusarium ssp. or HSI in line 39.

Response to reviewer: The authors thank the Reviewer for the careful reading of the manuscript. Comments corrected. The transcripts of all abbreviations used are given.

 

2. In the introduction you do a good literature review but none of them is about detection of fusarium infection by optical method. It is good to add some references about it.

Response to reviewer: Detection of fusarium infection by the HSI method refers to optical, including in the infrared region. There are links to articles about this [2-10]. There is very little information about the method of luminescent spectroscopy for the diagnosis of fusarium. Added a link to the authors' article [26].

 

3. It is good also to compare your results (for example your sensitivity) with some reported results in measuring the degree of fusarium infection and write about the preferences of your work than others.

Response to reviewer: Many thanks to the Reviewer for this recommendation. Unfortunately, calculations of the sensitivity of fusarium detection methods are not presented in any of the articles we found. Other authors do not pay due attention to the definition of sensitivity. The advantages of the methodology developed by us are added to the Conclusion section.

 

4. In the second paragraph of section 2 (Materials and methods) line #2 you mentioned the mass of the sample is 2 kg and after that you tell the weight is not less that 25g. How it is possible? Furthermore, it is good to change the word weight here with mass because the unit for measuring the weight is not g it is N.

Response to reviewer: The authors thank the Reviewer for the careful reading of the manuscript. In the manuscript, we are talking about the mass of a 2 kg sample from which 25 g "weights" are selected for a specific analysis. Corrections have been made to the manuscript.

 

5. The citation to reference #24 in text (third paragraph of section 2 line#2) is different with other citation inside the text.

Response to reviewer: The link has been corrected. The authors thank the reviewer for pointing out the inaccuracy.

 

6. 1 in the result section is plotted just for oat seed and the results for wheat and barley just mentioned in the text without bringing the curves. I think you should add the wheat and barley curves. The same situation is for Fig.2.

Response to reviewer: The excitation and photoluminescence characteristics of oat, wheat and barley seeds are similar to each other and when presented in the same figure, the seed graphs of all three crops will be located very close to each other and visually difficult to distinguish. The presentation of graphs for oats, barley and wheat on separate graphs will lead to a sharp increase in the volume of the article due to the appearance of four more similar drawings. Therefore, the authors used a compromise option with the publication of Figures 1 and 2 for oats as an example and a description of the type of similar graphs in the text part of the article. For wheat, the excitation and photoluminescence graphs are given and described in more detail in the article [26], a reference to which is introduced in this article.

 

7. In Result section (section 3) under the figure 1 line #4 you mentioned there is a clear shift to the left where is this shift. I can not see this shift in fig.1.

Response to reviewer: In Figure 1, the curve shift to the left is small for oat seeds. It is much more noticeable for wheat and barley, which is what is written in the text. More detailed information is available in another article by the authors [26], a link to which is added to the text of the manuscript.

 

8. In Fig.1 and 2 you compare the uninfected and infected oat seed but the degree of infection in not mentioned anywhere.

Response to reviewer: The degree of infection of oat seeds in Figures 1 and 2 is 82.4%.

 

9. Same as comment number 6, in Fig.3 you bring the result just for barley. In all of your inspection you should bring all your result for all seeds. But I cannot understand why you plot the curves for just one of your seeds and why this seed is changed in different figures.

Response to reviewer: Qualitatively, the characteristics of oat, barley and wheat seeds vary the same way. The representation of seed plots of all three crops in one figure leads to an overload of the figure with dependency curves. The presentation of graphs for oats, barley and wheat on separate graphs will lead to a sharp increase in the volume of the article due to the appearance of several similar drawings. Therefore, the authors used a compromise option with the publication of drawings for oats as an example and a description of the type of similar graphs in the text part of the article. Formulas are given in full for all types of seeds. For wheat, the excitation and photoluminescence graphs are given and described in more detail in the article [26], a reference to which is introduced in this article.

 

10. The result in Table 1 shows that you analyze the oat seed just for two degrees of infection. Therefore, your result can not be reliable for oats. Whys you did not increase the number of samples with different degree of infection in oat like other seeds?

Response to reviewer: The authors thank the Reviewer for the comment.

Table 1 contains additional data for other degrees of infection of oat seeds.

 

11. The final block diagram which you proposed which is very good diagram is based on the result of barley and wheat and can not be applied for oat. I think it is good to change some part of the paper so that some ambiguity is removed.

Response to reviewer: The authors thank the Reviewer for the comment.

The article has been amended and supplemented related to the diagnosis of oats.

Author Response File: Author Response.docx

Reviewer 3 Report

Please find in the following my comments about the review of a manuscript under the title (Method of optical diagnostics of grain seeds infected with Fusarium):

In this study, the main purpose is to take up the spectral luminescent properties of healthy and infected seeds and to develop a technique of optical diagnostics of fusariosis of grain plants.

The main question addressed by the research

This study focused on the spectral characteristics of excitation and luminescence of wheat, oat and barley seeds which were measured on a diffraction spectrofluorimeter in the range of 180-700nm. Based on the methodology of determining the degree of infection of agricultural plant seeds, an optoelectronic device can be designed for remote monitoring of infection of their seeds. The proposed device can be assembled in the form of a nozzle on a UAV or a separate device.

Originality and relevance

• The study is interesting for reading and relevant in the field as it discusses the taking up the spectral luminescent properties of healthy and infected seeds and developing a technique of optical diagnostics of fusariosis of grain plants
• The study has moderate scientific quality.
• The study is relevant to the scope of this journal.
• The manuscript is clear, relevant for the field and its presentation need fine modifications.

Comments:

Methods:

• Add all the references to the methods used in the study.

Results:

• The title of the table should be informative and self-explanatory? Revise.
• The legends of the figures should be informative and self-explanatory? Revise.
• The interpretation and description of the results are need more description.

 

Discussion:

• The findings need more interpretations as the authors did not explain their findings in sufficient way and go to the conclusion.
• It is better to add a section under the title (limitations of study) and add the limitations of your study

General:

• Add a list of abbreviations.

 

Author Response

We, the authors, appreciate all the time spent by the reviewers and all the ideas, suggestions, and comments presented. We addressed all the ideas, suggestions, and comments below in our revised manuscripts.

 

Comments and Suggestions for Authors:

 

1. Add all the references to the methods used in the study.

Response to reviewer: The authors thank the Reviewer for the careful reading of the manuscript. References to the methods used in the study are found in the sources [25-27].

 

2. The title of the table should be informative and self-explanatory? Revise.

Response to reviewer: The table name has been corrected.

 

3. The legends of the figures should be informative and self-explanatory?

Response to reviewer: Changes have been made to the legends of the figures.

 

4. The interpretation and description of the results are need more description.

Response to reviewer: Additions have been made with the interpretation of the results.

 

5. The findings need more interpretations as the authors did not explain their findings in sufficient way and go to the conclusion.

Response to reviewer: Additions have been made with the interpretation of the results.

 

6. It is better to add a section under the title (limitations of study) and add the limitations of your study.

Response to reviewer: The method developed by the authors for diagnosing the infection of seeds with fusarium has limitations: seeds must be of conditioned humidity and have low contamination (no more than 5%). Additions have been made to the text of the manuscript.

 

7. Add a list of abbreviations.

Response to reviewer: Only three abbreviations are used in the text of the article, one of them (PCR) refers to common ones that do not require definition in the text (Table 2 https://www.mdpi.com/authors /layout#_bookmark14).All abbreviations are deciphered in the text.

 

Author Response File: Author Response.docx

Reviewer 4 Report

See the attachment.

Comments for author File: Comments.pdf

Author Response

We, the authors, appreciate all the time spent by the reviewers and all the ideas, suggestions, and comments presented. We addressed all the ideas, suggestions, and comments below in our revised manuscripts.

 

Comments and Suggestions for Authors:

 

1. The authors present the absorption spectroscopy diagnosis, but there is no sufficient data and no valuable analysis.
2. The measurement on oat seeds was only done on sample without infection and sample with 82.4% degree of infection. But the measurement on other degrees of infection is also needed to develop an accurate relation between absorption and degree of infection.
3. The description of absorption spectroscopic measurement on wheat and barley samples is just one sentence—“There is a clear shift to the left and the parameter H increases by 65.8% and 62.1% for the spectra of wheat and barley, respectively.” First, it is necessary to show the spectra to illustrate the shift of absorption peaks using a figure. Second, the degree of infection of wheat and barley samples should be given.
4. The only analysis regarding the absorption spectroscopy diagnosis is 1 st paragraph of Section 4–Discussion, which says “the excitation spectrum is closer to that of immature seeds.” But it is just a superficial comment. A deeper quantitative and qualitative analysis is needed.
5. It seems like the authors have never intends to emphasize the absorption spectroscopy measurement, because it is not mentioned at all in Section 5–Conclusions. If this is the case, it would be better to get rid of any description of the measurement.

Response to reviewer: The authors thank the Reviewer for the careful reading of the manuscript and the valuable comments and recommendations for improvement.

1. The results for oats of various degrees of infection have been added to Table 1.
2. The presentation of graphs for oats, barley and wheat on separate graphs will lead to a sharp increase in the volume of the article due to the appearance of several similar drawings. Therefore, the authors used a compromise option with the publication of drawings for oats as an example and a description of the type of similar graphs in the text part of the article. The degree of infection of wheat and barley is 98%. This information is added to the text of the manuscript.
3. A more detailed analysis of the absorption spectra is presented in another article by these authors [26], a reference to which is added to the manuscript.
4. Absorption spectroscopy in this article is not an independent method of seed diagnostics, but is the first stage in the development of a method of luminescent diagnostics. First, the informative absorption ranges (wavelengths) are determined from the absorption spectra, and then, in the obtained ranges (wavelengths), the seeds are excited and the radiation spectrum (photoluminescence) is recorded, followed by the determination of integral fluxes. For more information, see [25-27].

 

2. The introduction is overly detailed. It gives too many details about the use of spectroscopy to diagnose different agriculture samples.

Response to reviewer: The introduction contains brief information (no more than one sentence per information source) from articles published in leading journals in the period 2003-2021.

It would be possible to shorten something, but Reviewer 1 and Reviewer 2, on the contrary, insist on increasing the volume of the Introduction.

 

3. The photoluminescence spectra on wheat and barley samples were measured on several different degrees of infection. But I do not think the way the authors prepared the samples is correct. In my opinion, different degrees of infection mean different stages of infection, e.g. early and late stages, and can possess different photoluminescence wavelength peaks. However, what the authors did was mixing the sample without infection with the sample with 98% degree of infection in a specific proportion. This way does change the luminescence intensity, but it does not change the wavelength peaks at all and cannot truly represent different degrees (or stages) of infection.

Response to reviewer: The authors thank the Reviewer for his very valuable advice. The study of different stages of infection of seeds with fusarium will necessarily be implemented in the further stages of our scientific work.

 

4. The authors should use a figure to show the good linear fitting between photoluminescence flux ratio and degree of infection, rather than just claiming “The determination coefficients for equations (14) and (15) are 0.92 and 0.98, respectively.”

Response to reviewer: The authors thank the Reviewer for the recommendation. The drawing has been added to the text of the manuscript.

 

5. For the photoluminescence measurement on the barley samples with 424 nm excitation source, why is the linear fitting between the flux and degree of infection so bad?

Response to reviewer: The authors associate a relatively small value of the determination coefficient for the dependence on the excitation of 424nm for barley seeds with a large spread of photoluminescence flux values.

 

6. The acronym NIR is not used properly. First, the definition does not comply with the common standard. The authors use NIR to stand for “near infrared range”, but NIR is already a common acronym in science topics and stands for near infrared. Second, after defining NIR, the authors still use “near infrared range” in the manuscript.

Response to reviewer: The authors agree with the Reviewer's comment. Corrections have been made to the text of the Manuscript.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

I think that the re-submitted the manuscript entitled “Method of optical diagnostics of grain seeds infected with Fusarium" deserves publication in Applied Sciences after major revision.

Unfortunately, the authors did not include all pertinent comments

Introduction: no information on the global scale of the problem (the values) and why are toxins so dangerous

Figure 1 and 2; please complain on the Figures for wheat and barley seeds. The answer that was not added because it increased the volume of the manuscript is evasive. Referring to another article to see the results of this work all the more. The results of the experiments can always be added to the supplementary material, it will not increase the volume of the manuscript.

 

Discussion: no, reference of the obtained results to the literature data. Needed to be completed.

“Response to reviewer: In the Discussion section, when analyzing the results obtained, there is a link to the article [27]. In general, there are very few articles devoted to the use of luminescent spectroscopy for the diagnosis of fusarium seeds of grain plants.” Please refer to the available literature.

Author Response

The authors thank the Reviewer for re-reviewing the manuscript and his comments and recommendations.

Comments and Suggestions for Authors:

 

1. Introduction: no information on the global scale of the problem (the values) and why are toxins so dangerous

Response to reviewer: Information about the global fusarium problem has been added to the Introduction section (Lines 31-33).

 

2. Figure 1 and 2; please complain on the Figures for wheat and barley seeds. The answer that was not added because it increased the volume of the manuscript is evasive. Referring to another article to see the results of this work all the more. The results of the experiments can always be added to the supplementary material, it will not increase the volume of the manuscript.

Response to reviewer: On the recommendation of the Reviewer, the spectral characteristics of excitation and photoluminescence of wheat and barley seeds are published in the supplementary material to this article.

 

3. Discussion: no, reference of the obtained results to the literature data. Needed to be completed. “Response to reviewer: In the Discussion section, when analyzing the results obtained, there is a link to the article [27]. In general, there are very few articles devoted to the use of luminescent spectroscopy for the diagnosis of fusarium seeds of grain plants.” Please refer to the available literature.

Response to reviewer: A link to the article [24] has been added to the text of the manuscript (lines 98-100).

The authors will be sincerely grateful to the Reviewer if he points out other more modern publications on the luminescent diagnosis of diseases of grain plants.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments for author File: Comments.pdf

Author Response

The authors thank the Reviewer for re-reviewing the manuscript and his comments and recommendations.

 

Comments and Suggestions for Authors:

 

1. In answering to the comment #6 you emphasized that the curves for barley and wheat is the same as oat and you believe that there is no need to bring them. How it is possible that the curve be the same but the H parameter calculation be different for them, and the shift to the left be different in them. Furthermore, the degree of infection for barley and wheat is also different than oat. According to your answers, the result for wheat is not a new result and it was brought in ref [26]. So, the paper should change so that the emphasize would be on oat and barley. And the title may be changed. At the end, I think the paper in not write neat and tidy and the reader may be confused when they read the paper. In my opinion the paper should change in some parts and explain more about some ambiguity in the text about the curves and the reason why you bring some curves for oat seeds and some other for barley or wheat. Please add more explanation to your manuscript.

 

Response to reviewer: The authors thank the Reviewer for his comments and suggestions.

Spectral characteristics of excitation and photoluminescence of wheat seeds (Figures 1-6) and barley (Figures 7-11) have been added to the additional materials of the manuscript.

In general, this article is the second part of the development of a method and device for optical diagnostics of fusarium seeds. The first part is devoted to the study of optical spectral luminescent properties of healthy and infected seeds. Some results for wheat seeds are presented in the article [27].

The second part of the research is devoted to the development of a method for controlling the degree of fusarium infection of seeds of grain plants (including wheat, barley, oats). These results are presented in this article. It generalizes the spectral properties of all seeds and develops a control method.

At the third stage of research, it is planned to develop an optical photoluminescent control device for the implementation of this method.

Author Response File: Author Response.docx

Reviewer 4 Report

Comments for author File: Comments.pdf

Author Response

The authors thank the Reviewer for re-reviewing the manuscript and his comments and recommendations.

 

Comments and Suggestions for Authors:

 

1. I just do not see the point of putting absorption spectroscopy content in the manuscript. Like I mentioned last time, there is no sufficient data and no valuable analysis. If the authors really would like to add this content, there should be more measurement and deeper analyses, such as fitting a quantitative relation between the degree of infection and absorption. A simple comparison of the absorption under two degrees of infection, like what the authors are doing, is just preliminary data and cannot reach the level of journal publication.                                                                         A.The description of absorption spectroscopic measurement on wheat and barley samples is just one sentence—“There is a clear shift to the left and the parameter H increases by 65.8% and 62.1% for the spectra of wheat and barley, respectively.” It is necessary to show the spectra to illustrate the shift of absorption peaks using a figure.                            B.The only analysis regarding the absorption spectroscopy diagnosis is 1st paragraph of Section 4—Discussion, which says “the excitation spectrum is closer to that of immature seeds.” But it is just a superficial comment. A deeper quantitative and qualitative analysis is needed.                               C. I do not think the authors have ever intends to emphasize the absorption spectroscopy measurement, because it is not mentioned at all in Section 5—Conclusions. If this is the case, the authors should get rid of any description of the measurement.

 

Response to reviewer: The method of absorption spectroscopy in this study is not a separate independent method, but is part of the method of optical photoluminescence diagnostics. In this method, at the first stage, the excitation (absorption) spectrum of the seeds of η_e (λ) is measured by synchronous scanning of spectrofluorimeter monochromators. Analysis of the obtained spectra of η_e (λ) makes it possible to determine the wavelengths (or spectral regions) with which to excite photoluminescence. At the second stage, the seeds are excited by the radiation determined at the previous stage, and the photoluminescence spectra φ_l(λ) are measured using a spectrofluorometer or an integral flux Φ using, for example, a photodiode. This description has been added to the text of the manuscript (lines 127-132)

A.  Spectral characteristics of excitation (absorption) of wheat and barley seeds are added to the section of additional materials (Figures 1 and 7, respectively).

B.  Added discussion of changes in excitation (absorption) spectra from the standpoint of biochemistry (lines 190-196).

C. Measurement and analysis of the excitation (absorption) spectra of healthy and infected seeds allow us to draw two extremely important conclusions:

1. When seeds are infected with fusarium, their optical properties change – the excitation spectrum (qualitatively and quantitatively), and therefore the photoluminescence spectra change.
2. To further study the properties of photoluminescence of seeds during infection, it is necessary to excite seeds with radiation of wavelengths 232nm, 362nm, 424nm, 485nm.

If you delete the description of the measurement of the excitation (absorption) spectrum, then readers of the journal will immediately have questions: how are the wavelengths of seed excitation determined?

Author Response File: Author Response.docx