Phytostimulation and Synergistic Antipathogenic Effect of Tagetes erecta Extract in Presence of Rhizobacteria

Round 1

Reviewer 1 Report

This research describes phytostimulation activity of T.erecta (marigold) extract applied with two Bacillus spp., which has antifungal activity against selected phytopathogens. The paper need to be carefully revised for casual mistakes. The experiments need be described in such manner that the results and methods are in agreement to each other. Some of the concerns are given below:

 

Line 24: .. a to increase the activity against phy.. what activity? antifungal?

Line 25-27: “The hydroalcoholic extract from T. erecta flowers was characterized by …. UHPLC-HESI” rephrase

Line 28: delete ‘seed model’

Why does A. niger has also been selected for this study? Is it a phytopathogen of given crops? If yes then include justifications.

Line 41 and 44 are contradictory. In first line, use of chemicals is said to be harmful, while in next line, use of chemical and phytostimulants has been encouraged. Is this okay?

Line 56-57: Rephrase - Among the main plant compounds with biostimulating effect could be mentioned: polysaccharides, polyphenols, compounds with hormonal activity, protein hydrolysates, amino acids.

Formula in line 88 – use ‘with’ the extract, instead of ‘in’ the….! Also, is it root elongation or root length?

Line 213: correct ‘Patri’

Correct the Fig 1 for the overlapping legends on axis.

Table 1 may be shifted to supplementary file

Fig 2, its apparent that the 12.5% of extract is highly lethal for all three bacteria, compared to 40% alcohol despite to the fact that these are endospore formers. In fact, alcohol is used as disinfectant. It is supposed to reduce viability. Hence, it’s not wise to conclude that concentrations above 12.5 increase cell viability by comparing it with alcohol. Instead, may be sterilized water as control could give better idea. Ethanol could remain as positive control.

Fig 3: Its ‘relative’ seed germination, and ‘relative’ root elongation. Correct the legends. Otherwise, the seed germination can’t be more than 100%.

Fig 4, shift to supplementary file

Line 433: delete – ‘This approach leads to healthier foods….’. This paper is not about the food quality.

Line 465 onwards, fig 7: cell viability and MIC for fungal growth has been described in results. In methods, section 4.2, this has been described differently. Revise. Also, MIC data for bacteria as explained under 4.2, method section, has not been given in results. If this is given as effect of extract on bacterial growth, then it should be described in similar way in methods.

Fig 8: correct ‘fungicid’ activity with appropriate term

Table 7 – rephrase the table title. Correct the spelling mistakes – resistent, complet

Author Response

Dear reviewer,

First of all, we would like to thank you for the efforts you made to improve this manuscript. We made all the proposed corrections and the justification of the statistical analysis carried out in this way, solvent control vs extract. Please check our responses to the observations.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments for author File: Comments.pdf

Author Response

Dear reviewer,

First of all, we would like to thank you for the effort you made to improve this manuscript. We made all the proposed corrections and the justification of the statistical analysis carried out in this way, solvent control vs extract. Please check our responses to the observation.

Author Response File: Author Response.docx

Reviewer 3 Report

The paper responds to current global challenges faced by agriculture – need to reduce synthetic fertilisers and pesticides, growing prices of chemicals, instability of agricultural market and in other hand, expectation to transform into sustainable agriculture. 

It focuses on phytostimulation and antipathogenic effect of Tagetes erecta extract in presence of rhizobacteria. Therefore, the research presented in this paper might play an important role in the application of modern, safe and nature-based methods in agriculture.

I agree that use of microorganisms, especially from the PGPR group, can significantly help to reduce yield losses and maintain agricultural productivity at the current level, when reducing chemicals in the production process. Inoculation of the plant rhizosphere with microorganisms is a potentially attractive method of supporting plant development and resistance. Plant growth-promoting bacteria play a significant role in enhancing plant development under both stress-free and stress-free conditions through a number of direct and indirect mechanisms.

In this paper research has been undertaken on the physicochemical characterization of  T. erecta extract, the antioxidant activity by chemical methods, the influence on rhizobacteria viability, the selection of the extract variant that stimulates bacterial cell proliferation and the evaluation of antifungal effect against three phytopathogenic strains were evaluated for extract and mixture of extract with PGPR. This type of knowledge is worth learning to understand the capacity such a combined approach, involving natural extracts and bacteria.  Even if the paper presents laboratory data, without field testing, it brings very attractive information.

The authors provided interesting results that PGPR strains used  they can be used as biocontrol agents against plant pathogens  stimulating the growth of vegetable seedlings. The authors proved that PGPR strains combined with T. erecta extract can be used as biocontrol agents due to their antagonistic activity against plant pathogenic fungi, in particular F. graminearum. These findings can also be applied to agricultural biotechnology to develop biopesticides to combat plant pathogens.

The article presents the material and methods used in a comprehensive and transparent manner.  The methodology is reliable and accurately described, which positively influences the assessment of this work. The division into individual methodological sections gives the reader a clear picture and proves good flow in the work plan. However it would be good to add 1-2 sentences at the beginning of 2.3 section to inform on the goal of seed germination bioassay.

The photos attached in the manuscript show the effect of the action and are a positive addition to this work. The reader may realize how important the antifungal activity is for the different concentrations of T. erecta extract.

The introductory section is extensive and written concisely and clearly to the reader. The literature is well-chosen and the conclusions clearly refer to the research carried out. The discussion was conducted properly.

Minor issues to be considered:

Line 193. Please explain what was behind selecting these 3 bacterial species.

Line 352, the sentence shall be corrected – seems that existing lost a part of thought.

There is scarce discussion relating this study to other studies on use of bacterial strains as antipathogenic measure. The literature database on the use of PGPR strains is extensive, it is worth mentioning a few literature reports to enrich the outputs.

Please add 1-2 sentences in conclusion section on how the authors imagine potential application of the combination of rhizobacteria and the marigold extract in practice.

Taking into account the importance of the work and well written text, it is worth of publishing.

Author Response

Dear reviewer,

First of all, we would like to thank you for the effort you made to improve this manuscript. We made all the proposed corrections and the justification of the statistical analysis carried out in this way, solvent control vs extract. Please check our responses to the observation.

Author Response File: Author Response.docx

Reviewer 4 Report

The manuscript contains very interesting research results that could be of great importance for organic farming. The manuscript requires minor corrections. Detailed comments below.

Why is there a blank space on the first page?

Line 61, correct [7, 8].

Line 66, correct [11, 12, 13]

Line 77, correct Bacillus sp.

Line 90, please state where the research was carried out, and in which year?

Line 98, correct -20 °C

Line 104, correct 28 °C (for 72h) [17].

Line 111, 118, correct µg/mL

Line 111, correct (R² = 0.9994).

Line 114, correct [19] which

Line 118, correct (R² = 0.9968).

Line 121, correct et al. [20] with

Line 123, correct 90 °C

Line 126, correct (R²= 0.9981).

Line 142, correct [21].

Line 161, 162, correct 37 °C

Line 179, correct and Popa [26]

Line 184, correct 1 °C.

Line 197, correct 37 °C;

Line 203 , correct 37 °C.

Line 211, correct 40-45 °C.

Line 231, correct at 28 ºC,

Line 258, please provide full details of the manufacturer of the statistical software

Line 261 etc., correct p<0.05

Line 265, correct g/mL

Line 285, correct [37] and … [38].

Line 293, correct D-glucose/mL).

Line 300, 301, correct (383.74 mg/L), syringic acid (30.71 mg/L), ellagic acid (26.80 mg/L) and 3,4-dihydroxybenzoic acid (20.50 mg/L).

Line 322, correct Table 4. Antioxidant

Line 344, correct [15, 46, 47, 48]

Line 347, correct Bacillus spp.

Line 349, correct [50] synthesis

Line 351, correct [53], effect

Line 376, correct Table 5. Antibiofilm

Page 17?

Line 501, correct [68, 69].

Line 514, correct marketplace [16].

References, please remove publications before 2010 (9, 10, 18, 24, 25, 33, 35, 37, 38, 42-45, 61, 67-69). 

Author Response

Dear reviewer,

First of all, we would like to thank you for the effort you made to improve this manuscript. We made all the proposed corrections and the justification of the statistical analysis carried out in this way, solvent control vs extract. Please check our responses to the observation

Author Response File: Author Response.docx

Reviewer 5 Report

Title: Phytostimulation and synergistic antipathogenic effect of Tagetes erecta extract in presence of rhizobacteria.

The use of phytostimulants for increasing crop resistance to biotic stress including disease is an interesting topic. However, there are major concerns about the data presented, the as well as the interpretation of the results.

 Below are my comments and suggestions:

Lines 26-27: not clear please revise.

Lines 48: “helps to manage climate change in agriculture” what do you mean by this statement?

Please combine the second and the third paragraphs in the introduction.

Lines 61-64: This section not clear. Does the roots of Marigold (Tagetes) has a suppression effect on nematode? Also, what part of the Tagetes had been used in “Natarajan et al [10]” study?

Line 79: “Increasing reports of fungicide resistance to pathogens” do you mean that the pathogen become more resistance to fungicide? Please revise.    

Lines 99-104: This section not clear at all. What is ATR-FTIR? Are the extract phase is liquid or solid? In previous section you mentioned that extract is semi-solid not dry?  

Line 107 “Over 100 μL of sample” did you dilute it with water or solvent? You extract is semi-solid.

Line 107 add “to” between “added” and “900”.

Line 108: “Folin Ciocalteu reagent” the Folin is powder. Please add one more sentence about how did you prepare the reagent.

Line 113-114: revise, not clear

Lines 114-115: how did you prepare the 2.5% ALCL3 and 10% sodium acetate? Did you dilute them with water or what type of solvent?

Line 146: “Over 100 μl of sample / standard was added 100 μl of 0.3 mM DPPH radical solution.” Not clear

Line 147: what does “λ” mean? Did you use spectrophotometer to measure them?

Line 185: add the equation for germination rate

Lines 195: “disk diffusion technique” how? add more detail about this technique.     

Line 226: what does “RPMI 1640” stand for? It seem to me an institute “location”

Lines 235-239: long sentence please split or revise

Figure 1: what do you mean by plant material? Is it the extract or fresh samples?

Line 275: “4000−650 cm” the smaller number should be first “650-4000” please revise all ranges across the manuscript.

Lines 288-293: all plants extract have carbohydrate. Please compare your carbohydrate value with other previous studies.

Lines 293-294: “In this context, the extract has the ability to function as a nutrient for both plant and microbial cells.”  Is this good or bad? I understand that the extract will promote microbes in the microenvironment and consequently, increase pathogens activity.

Lines 303-310: is there any study that shows the “thresholds” I mean at what concentration the phenolic compound become unfavorable to plant growth.     

 

Lines 323-337: these two paragraphs are not related to your objectives. I suggest to remove them from your manuscript.

Line 352: “we aimed to highlight the concentrations which improved cell proliferation was observed” not clear

Figures 2, 7, 8, 9. In x-axis, the small concentration should be first, left side. Also, across the figures, your statistical analysis was within the each concentration (i.e. ethanol vs. plant extract). In your figures, no statistical analysis was carried out to show the significant between extract levels. I mean you did not analyze the difference between the level 50 and 25 for example. Therefore, all sentences mentioned that the concentration A is better than B should be removed from your manuscript. Or, you should conduct the statistical analysis across the extract levels.  

Lines 424-430: No solid data in your study support this statement. Please remove.

Line 434: “According to Janakiev et al. [64], phytopathogenic fungi reduce up to 80% of crop yield, as well as the quality” not clear. Please revise.

Where is figure 5a and 5b?

Figure 6: please add the extract levels to the figure

Figure 7 not correct. Figure 7a and 7b not exist. I think there is something went wrong when they upload the figures.

 Line 537: “26.67” not significant.

Lines 551-553: Table 7 shows that most treatments are not significant. M. laxa shows complete inhibition for all treatment. A. niger has similar results too.

Table 7: “complete inhibition” not “Complet inhibiton”

In general the result and discussion section should be potentially revised. The authors should compare their results with other previous studies. In fact, the interpretation of the results is very poor, no in-depth discussion was carried out.

Overall, considering the novelty, integrity of the results as well as the interpretation of those results, this paper cannot meet the requirements of publication in this journal at this stage. A major revision is required.

 

Author Response

Dear reviewer,

First of all, we would like to thank you for the effort you made to improve this manuscript. We made all the proposed corrections and the justification of the statistical analysis carried out in this way, solvent control vs extract. Please check our responses to the observation.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors have addressed my concerns. The paper may be accepted

Reviewer 5 Report

The manuscript has been potentially improved. The authors responded properly to my comments. Now, I think the manuscript is ready and I have no further comments.