Impact of Beauveria bassiana and Metarhizium anisopliae on the Metabolic Interactions between Cucumber (Cucumis sativus L.) and Cucumber Mosaic Virus (CMV)
Gaëtan Guignard
Round 1
Reviewer 1 Report
Dear colleagues,
This review is concerning a research work entitled “Impact of Beauveria bassiana and Metarhizium anisopliae on the metabolic interactions between Cucumis sativus L. and Cucumber mosaic virus (CMV)”, by Roshan Shaalan, Ludmilla Ibrahim, Falah Assadi and Walid El Kayal. As detailed data and interpretation, I recommend it for an international audience in this journal, however several points have to be precised and a major revision is requested.
Please notice that in order to bring a broad audience to this article and to this journal, for specialists and non-specialists, the eight major points of my comments (at the beginning) are very important (mandatory…) for a suitable value of the article. Minor points are also enhanced at the end of this review.
I deeply hope to see this good article published soon,
The eight major points are:
11- Although I am not English-native speaker, this text is fully understandable, however some parts are not very fluent and a precise re-writing would make it much more english-standardized and more attractive for this international journal.
22- In the introduction (and abstract, and conclusion?), in order to have a general view of this research, the authors should clearly write that this topics concerns insect (vector?)-plant(s) (host(s))-fungi-virus (parasite(s)) interaction(s), this case being quite complex with several organisms involved and though of great research interest; moreover insert a clear scheme of this interaction plant-insect-fungi-virus system and precise the (only) points of your present study in this whole interaction(s) cycle(s).
33- As I am involved in plant taxonomy I am very sensible to correct taxa names, which should be inserted at least the first time they appear in the text. So from the introduction, insert latin names in italics (and author(s)) of all plants cited (species and variety, check them one by one all through the text, even cited only once as some are apparently missing like tomato, cucumber (in addition to the title, appearing in the text)…). Use international Plant Names Index (IPNI) https://www.ipni.org/) for plants, or equivalent; for viruses an international list should be also consulted (ictv https://talk.ictvonline.org) or equivalent; for fungi fungi list see https://www.mycobank.org/ or equivalent.
44- In order to be more attractive especially for non-specialists, a figure including detailed photos of the cultivar(s) (infected or not) is necessary (with so called "clear mosaic and blistering symptoms on").
55- The authors have to be more cautious about the precision of each word in their sentence "This is the first metabolomics study of entomopathogenic endophytes treated virus diseased plants..." as metabolomics-plants-virus papers are quite numerous only in 2022.
66- For "fig. 10" (which apparently does not appear in the text?), this Venn diagram has to be used and detailed in the discussion: enhance the incidence(s) of the (very) various values (from 8 to 67) of this diagram for the knowledge of the organisms involved (in terms of biology, genetics, metabolomics or etc...).
77- As many molecules are involved in this study, the already detailed discussion has to propose a clear table ((s) with groups of molecules?) summarizing (even with "?" if it is "only" putative) the effects (positive or negative) of all these induced (increase or decrease) molecules.
88- References already taken in account by the authors are of real interest, however checking briefly in the word of science WOS and scilit (from mdpi) with the key-words of the abstract, other references (research article(s), editorial(s), especially very recent ones…) appear and references should be once more selected and used (if relevant…) in order to provide a larger view of this interesting research. Among these are the followings:
[1-19]
1. Balogun, M.; Maroya, N.; Augusto, J.; Ajayi, A.; Kumar, L.; Aighewi, B.; Asiedu, R. Relative efficiency of positive selection and tissue culture for generating pathogen-free planting materials of yam (Dioscorea spp.). Czech Journal of Genetics and Plant Breeding 2017, 53, 9-16.
2. Farahat, A.S.; El-Morsi, A.A.; Soweha, H.E.; Sofy, A.R.; Refaey, E.E. METABOLIC CHANGES OF CUCUMBER PLANTS DUE TO TWO CMV EGYPTIAN ISOLATES. Arab Universities Journal of Agricultural Sciences 2018, 26, 2019-2028.
3. Weng, Y.; Garcia-Mas, J.; Levi, A.; Luan, F. Editorial: Translational Research for Cucurbit Molecular Breeding: Traits, Markers, and Genes. Frontiers in Plant Science 2020, 11.
4. Abdelkhalek, A.; Király, L.; Al-Mansori, A.-N.A.; Younes, H.A.; Zeid, A.; Elsharkawy, M.M.; Behiry, S.I. Defense Responses and Metabolic Changes Involving Phenylpropanoid Pathway and PR Genes in Squash (Cucurbita pepo L.) following Cucumber mosaic virus Infection. Plants 2022, 11.
5. Ali, A.K.; Ahmed, I.A.; Al-Kuawiti, N. First report of Cucumber mosaic virus subgroup IB isolates infecting cucumber and cowpea in Iraq; 2022.
6. Broufas, G.; Ortego, F.; Suzuki, T.; Smagghe, G.; Broekgaarden, C.; Diaz, I. Editorial: Plant-Pest Interactions Volume I: Acari and Thrips. Frontiers in Plant Science 2022, 12.
7. Chan, Y.-L.; Saidov, N.; Lee, L.-M.; Kuo, F.-H.; Shih, S.-L.; Kenyon, L. Survey of Viruses Infecting Tomato, Cucumber and Mung Bean in Tajikistan. Horticulturae 2022, 8.
8. Frąckowiak, P.; Wrzesińska, B.; Wieczorek, P.; Sanchez-Bel, P.; Kunz, L.; Dittmann, A.; Obrępalska-Stęplowska, A. Deciphering of benzothiadiazole (BTH)-induced response of tomato (Solanum lycopersicum L.) and its effect on early response to virus infection through the multi-omics approach. Plant and Soil 2022.
9. Vinodhini J, Rajendran L, Jeya Sundara Sharmila D. Karthikeyan G Comparative coat protein annotation of two biologically distinct strains of Cucumber mosaic virus in chilli; 2022.
10. Lu, X.; Zhang, L.; Huang, W.; Zhang, S.; Zhang, S.; Li, F.; Zhang, H.; Sun, R.; Zhao, J.; Li, G. Integrated Volatile Metabolomics and Transcriptomics Analyses Reveal the Influence of Infection TuMV to Volatile Organic Compounds in Brassica rapa. Horticulturae 2022, 8.
11. Maravi, D.K.; Kumar, S.; Sahoo, L. NMR-Based Metabolomic Profiling of Mungbean Infected with Mungbean Yellow Mosaic India Virus. Applied Biochemistry and Biotechnology 2022.
12. Mesquita, P.; Barbosa, N.; Santos, F.d.; Rodrigues, F.; Abreu, E.; Leite, K.; Jesus, O.; Tumelero, A.; Schnadelbach, A.; Barbosa, C. Metabolomic and Histological Response of Passiflora cincinnata Infected with Cowpea aphid-borne mosaic virus (CABMV) Reveals Changes in Asymptomatic and Symptomatic Phases. Journal of the Brazilian Chemical Society 2022.
13. Mo, Q.; Lv, B.; Sun, Y.; Wu, X.; Song, L.; Cai, R.; Tang, X. Screening and production of dsRNA molecules for protecting Cucumis sativus against Cucumber mosaic virus through foliar application. Plant Biotechnology Reports 2022.
14. Patryk, F.; Barbara, W.; Przemysław, W.; Paloma, S.-B.; Laura, K.; Antje, D.; Aleksandra, O.-S. Deciphering of BTH-induced response of tomato (Solanum lycopersicum L.) and its effect on plant virus infection through the multi-omics approach; 2022.
15. Radeva-Ivanova, V.P.; Pasev, G.; Lyall, R.; Angelov, M.; Nankar, A.N.; Kostova-Protochristova, D. First Report of Cucurbit Aphid-Borne Yellows Virus Causing Yellowing Disease on Pumpkin (Cucurbita pepo) and Cucumber (Cucumis sativus) in Bulgaria. Plant Dis 2022, 106.
16. Routhu, G.K.; Borah, M.; Siddappa, S.; Nath, P.D. Exogenous application of coat protein-specific dsRNA inhibits cognate cucumber mosaic virus (CMV) of ghost pepper. J Plant Dis Protect 2022.
17. Scortichini, M.; Fiallo-Olivé, E. Editorial: Insights in Microbe and Virus Interactions With Plants: 2021. Frontiers in Microbiology 2022, 13.
18. Shahzad, G.-I.-R.; Passera, A.; Maldera, G.; Casati, P.; Marcello, I.; Bianco, P.A. Biocontrol Potential of Endophytic Plant-Growth-Promoting Bacteria against Phytopathogenic Viruses: Molecular Interaction with the Host Plant and Comparison with Chitosan. Int J Mol Sci 2022, 23.
19. Vial, T.; Phakeovilay, C.; Watanabe, S.; Chan, K.W.K.; Peng, M.; Deharo, E.; Chassagne, F.; Vasudevan, S.G.; Marti, G. Antiviral potential of medicinal plants: a case study with guava tree against dengue virus using a metabolomic approach; 2022.
Minor points are:
1 the interesting abstract is far too long, looking like an introduction; please restrict it to the key words of the present study and move most of the sentences in the introduction;
2 in the introduction, in the paragraph "according to our recent studies..." line 93, write "fungi" instead of "fungus";
3 in the introduction, the last sentence "It directly demonstrates..." should move in the conclusion?
4 in material and methods, in 2.1 explain in a few words the meaning of "negative control... positive control...";
5 in material and methods, indicate (in 2.1-2.4…) the number of cucumber plants cultivated, the number of seedlings, leaf samples as in 2.3, "freeze dried samples" as in 2.4;
6 in 2.4, line 176 write "mins" instead of "mints" (see with the editor’s recommendations);
7 in 3.3 line 306, write "the comparisons showing significant differences" instead of "...that show...";
8 for figures 2-9, explain in a few words in each caption the meaning of the “z score” and colours (yellow, orange; white and red being almost absent (do you use this in the discussion as the range of “colours” (= values) of the present study is overall quite limited?) using one detailed example, very useful especially for non-specialists of this kind of representation. Explain more clearly the meaning of BT and MT, use bracket characters below the labels to gather two BT or MT, to make them more rapidly understandable. The text-paragraph "the columns in..." should be inserted (or in shorter sentences) in the caption of each figure? Explain also (and use in the discussion?) the histogram on each left part of each figure. I also wonder if all original values table(s) have to be inserted as supplementary material (see with the editor);
9 for figure 10, explain more clearly in the caption the values 11 and 32, 25 and 51, as apparently for a non-specialist they do not seem to be comparisons between two-three-four entities according to the title of this figure ("common differentially”...) but seem to be a single-entity value (e.g. 32 represents the value for the only 6B ?).
Author Response
1- Although I am not English-native speaker, this text is fully understandable, however some parts are not very fluent and a precise re-writing would make it much more english-standardized and more attractive for this international journal.
Thank you for your input; the material has now been proofread by a native English speaker. If necessary, we may also employ the journal's editorial office as a second round.
2- In the introduction (and abstract, and conclusion?), in order to have a general view of this research, the authors should clearly write that this topics concerns insect (vector?)-plant(s) (host(s))-fungi-virus (parasite(s)) interaction(s), this case being quite complex with several organisms involved and though of great research interest; moreover insert a clear scheme of this interaction plant-insect-fungi-virus system and precise the (only) points of your present study in this whole interaction(s) cycle(s).
Thank you for this suggestion, in order to clarify the full interaction between the vector-plant-fungi-virus, the authors contributed an illustrated schema (Figure S1) to the text in response to this suggestion.
3- As I am involved in plant taxonomy I am very sensible to correct taxa names, which should be inserted at least the first time they appear in the text. So from the introduction, insert latin names in italics (and author(s)) of all plants cited (species and variety, check them one by one all through the text, even cited only once as some are apparently missing like tomato, cucumber (in addition to the title, appearing in the text)…). Use international Plant Names Index (IPNI) https://www.ipni.org/) for plants, or equivalent; for viruses an international list should be also consulted (ictv https://talk.ictvonline.org) or equivalent; for fungi fungi list see https://www.mycobank.org/ or equivalent.
This essential point was taken into account; all latin names are in italics, and plant species are identified by genus, species, and variety (when possible). This was double checked throughout the document.
4- In order to be more attractive especially for non-specialists, a figure including detailed photos of the cultivar(s) (infected or not) is necessary (with so called "clear mosaic and blistering symptoms on").
Figure 2 is added in Line 727 to illustrate the infected and non-infected cultivars. Symptoms are clearly shown. Please see the paragraph “Assessment of CMV infection”.
5- The authors have to be more cautious about the precision of each word in their sentence "This is the first metabolomics study of entomopathogenic endophytes treated virus diseased plants..." as metabolomics-plants-virus papers are quite numerous only in 2022.
This statement is now rephrased in lines 817-818.
6- For "fig. 10" (which apparently does not appear in the text?), this Venn diagram has to be used and detailed in the discussion: enhance the incidence(s) of the (very) various values (from 8 to 67) of this diagram for the knowledge of the organisms involved (in terms of biology, genetics, metabolomics or etc...).
The Venn diagram represents the numbers of overlapping differentially expressed metabolites among comparisons (Dpi3-Control vs. Dpi6-Metarhizium, Dpi3-Control vs. Dpi6-Beauveria, Dpi3-Control vs. Dpi10-Metarhizium, Dpi3-Control vs. Dpi10-Beauveria). Also, it shows the numbers of differentially expressed metabolites which are specific for each of these comparisons. For example, the numbers of differentially expressed metabolites which are not overlapped with other studied comparisons and specific for comparisons 3C vs. 6M and 3C vs. 6B are 11 and 32 metabolites, respectively. The specific metabolites for 3C vs. 6M comparison includes; 2-Oxoglutaramic acid, Valyl-Hydroxyproline, Phenylpyruvic acid and many others. However, some of the 32 metabolites specific for 3C vs. 6B comparison are Glyceric acid, 3-O-p-Coumaroylquinic acid, Pyroglutamic acid, Homoserine, Methionine Sulfoxide, Vitexin and D-Lysopine. The other overlapped and non-overlapped metabolites among comparisons in the Venn diagram are present in Supplementary Table S2.
7- As many molecules are involved in this study, the already detailed discussion has to propose a clear table ((s) with groups of molecules?) summarizing (even with "?" if it is "only" putative) the effects (positive or negative) of all these induced (increase or decrease) molecules.
Metabolites are present in supplementary table 2.
8- References already taken in account by the authors are of real interest, however checking briefly in the word of science WOS and scilit (from mdpi) with the key-words of the abstract, other references (research article(s), editorial(s), especially very recent ones…) appear and references should be once more selected and used (if relevant…) in order to provide a larger view of this interesting research. Among these are the followings:
The most suitable references were chosen from the list the reviewer kindly proposed and were added in lines 544, 550, 552, 558.
Minor points are:
- the interesting abstract is far too long, looking like an introduction; please restrict it to the key words of the present study and move most of the sentences in the introduction;
The abstract is now more concise and all key words were moved to the introduction part.
- in the introduction, in the paragraph "according to our recent studies..." line 93, write "fungi" instead of "fungus";
The amendment is applied.
- in the introduction, the last sentence "It directly demonstrates..." should move in the conclusion?
Yes this sentence has moved to the conclusion line 561.
- in material and methods, in 2.1 explain in a few words the meaning of "negative control... positive control...";
A brief description of the negative and positive control was added to the materials and methods (lines 107-108).
- in material and methods, indicate (in 2.1-2.4…) the number of cucumber plants cultivated, the number of seedlings, leaf samples as in 2.3, "freeze dried samples" as in 2.4;
Numbers of plants, replicates and processed samples were added in the material and methods section.
- in 2.4, line 176 write "mins" instead of "mints" (see with the editor’s recommendations);
Amendment is done.
- in 3.3 line 306, write "the comparisons showing significant differences" instead of "...that show...";
Amendment is done.
8 for figures 2-9, explain in a few words in each caption the meaning of the “z score” and colours (yellow, orange; white and red being almost absent (do you use this in the discussion as the range of “colours” (= values) of the present study is overall quite limited?) using one detailed example, very useful especially for non-specialists of this kind of representation. Explain more clearly the meaning of BT and MT, use bracket characters below the labels to gather two BT or MT, to make them more rapidly understandable. The text-paragraph "the columns in..." should be inserted (or in shorter sentences) in the caption of each figure? Explain also (and use in the discussion?) the histogram on each left part of each figure. I also wonder if all original values table(s) have to be inserted as supplementary material (see with the editor);
The z score is a measure of distance, in standard deviations, from the plate mean. A well with a Z score of 0 has the same raw value as the plate mean. A well with a Z score of 1.0 is exactly one standard deviation above the plate mean and a Z score of -0.5 is half a standard deviation below the plate mean. The histogram contains all the values we have obtained in our matrix m (value vs frequency) and how they correspond to the specified heatmap colour range. Using the scale argument, each value was transformed in m to a row Z-score, or the number of standard deviations above or below the mean of its row. This gives a distribution centred around the midpoint of the colour scale, so the heatmap has more contrast and is easier to interpret.
- for figure 10, explain more clearly in the caption the values 11 and 32, 25 and 51, as apparently for a non-specialist they do not seem to be comparisons between two-three-four entities according to the title of this figure ("common differentially”...) but seem to be a single-entity value (e.g. 32 represents the value for the only 6B ?):
We have improved the caption of this figure as follow: The Venn diagram represents the numbers of overlapping differentially expressed metabolites among comparisons (Dpi3-Control vs. Dpi6-Metarhizium, Dpi3-Control vs. Dpi6-Beauveria, Dpi3-Control vs. Dpi10-Metarhizium, Dpi3-Control vs. Dpi10-Beauveria). Also, it shows the numbers of differentially expressed metabolites which are specific for each of these comparisons. For example, the numbers of differentially expressed metabolites which are not overlapped with other studied comparisons and specific for comparisons 3C vs. 6M and 3C vs. 6B are 11 and 32 metabolites, respectively. The specific metabolites for 3C vs. 6M comparison includes; 2-Oxoglutaramic acid, Valyl-Hydroxyproline, Phenylpyruvic acid and many others. However, some of the 32 metabolites specific for 3C vs. 6B comparison are Glyceric acid, 3-O-p-Coumaroylquinic acid, Pyroglutamic acid, Homoserine, Methionine Sulfoxide, Vitexin and D-Lysopine. The other overlapped and non-overlapped metabolites among comparisons in the Venn diagram are present in Supplementary figure S2. The Pathway for each expressed metabolite is determined by P number beside each metabolite. The correlated pathway for each determined number is present in Supplementary table 1
Reviewer 2 Report
Attached file
Comments for author File: 
Comments.pdf
Author Response
- line 11: delete “:” in front of *
Done
- Line 18: Cucumber Mosaic Virus to Cucumber mosaic virus (CMV) OR Cucumber mosaic virus without initials
Done
- Lines 63 and 75: The citations here are lacking “,”
Done
- The authors explained that specific primers was used to test for CMV from symptomatic cucumber plants from Lebanon. The genetic population of CMV is with several strains due to recombinant event. These strains differ in host range, symptom expression, mode of transmission and characteristic. A better inside into the reaction and result from the metabolomics data will be to further characterize the CMV strain used in their assay. This will then provide a complete story about which strain of CMV is in action. Please address this issue.
CMV Lebanese isolate was detected using PCR CMV kit (BIOREBA-Qualiplante) which includes the primers. For this kit, the primers were designed on the capsid protein gene. They are based on CMV-Q RNA 3 and amplify an 870 bp fragment for all CMV isolates.
Round 2
Reviewer 1 Report
Dear colleagues
I was glad to read all these corrections, however I still maintain the two points of my first review:
Point 6. Although explanations now appear in the text, this Venn diagram is apparently not used in the discussion where it has to be, as just kept as a result it has no meaning.
Point 8. All the interesting explanations in the answer from the authors to the editor have to appear (even in shorter sentences) in the caption of each figure or at least once in the text (results or discussion), especially for non-specialists of this kind of representation.
Moreover, two points have to be considered for this new version:
1 the new welcome figure 2 (photo on the right) has apparently a problem as it looks compressed vertically (? Format problem?)
2 I cannot see “A scheme illustrating this interaction between the host, fungi, and virus is shown in the supplemental figure 1 (Fig. S1)”, as written in the new text. Moreover, this scheme should be in the main text, not as supplementary data.
Author Response
Point 6. Although explanations now appear in the text, this Venn diagram is apparently not used in the discussion where it has to be, as just kept as a result it has no meaning.
The Venn diagram has now been discussed in a full paragraph section Lines 607-621.
Point 8. All the interesting explanations in the answer from the authors to the editor have to appear (even in shorter sentences) in the caption of each figure or at least once in the text (results or discussion), especially for non-specialists of this kind of representation.
Each figure legend now contains a brief description of the preceding analysis.
Moreover, two points have to be considered for this new version:
1 the new welcome figure 2 (photo on the right) has apparently a problem as it looks compressed vertically (? Format problem?)
Indeed, the original version of this image has been uploaded.
2 I cannot see “A scheme illustrating this interaction between the host, fungi, and virus is shown in the supplemental figure 1 (Fig. S1)”, as written in the new text. Moreover, this scheme should be in the main text, not as supplementary data.
This diagram of plant-fungal endophyte-virus metabolomic interaction has now been moved from the supplemental figures section to the main text (Figure 12).
Author Response File: 
Author Response.docx
