Potential of Pseudomonas and Trichoderma from the Brazilian Amazon as Biocontrol Agents against the Wheat Blast Disease

Round 1

Reviewer 1 Report

Reviewer 3:

Comments to the Author

General comments: The manuscript by Pereira et al., (submitted) describes the potential of Pseudomonas and Trichoderma from the Brazilian Amazon as biocontrol agents against the wheat blast disease. This is an interesting study which shows a potential biocontrol strategy to minimize yield losses due to wheat blast while providing insights into the mode of action of effective beneficial microbes. In general, the justification of this work is sound, results are clear and the manuscript has been well written. However, for this manuscript to be accepted, kindly address specific comments below.

Specific comments

Line 21: Replace “dropped low” with “reduced”

Line 26: Insert “in ” between both and in vitro

Line 26: Replace “it” with “we”

Line 30: Delete “also”

Lines 36-75: Previous work on controlling rice blast showed the efficacy of Pseudomonads especially (Omoboye et al., 2019). (will be interesting to include a few lines about the efficacy of Pseudomonas spp. In managing Pyricularia oryzae on rice both here in the introduction and also during discussion.

Lines 40-41: rephrase “In certain Northern America and European Union countries, this disease has been designated to be a major quarantine disease”.

Line 43: rephrase “ mainly attacks…”

Line 44: replace “center” with “centered”

Line 44: replace “fungal” with “fungus”

Line 53: remove double space before “regional”

Line 56: rephrase “hindering IDM which is fully dependent…”

Line  57: replace “to the” with “on the”

Line 62: delete “(and perhaps the sole alternative)”; this is too specific

Line 87: replace “is accepted” with “holds true”

Lines 90-94: This sentence is too long; not concise and difficult to understand.

Line 183: replace “dark” with “darkness”

Figures 1, 3 and 7: I propose that the chart type be changed to bars for clarity and better interpretation. Also for Figure 7, it is better to order the treatments such that the cultivar with or without wheat blast follows each other so the difference can be clear. Not well presented as it is now

Line 314: rephrase: “grew aggressively”

Line 317: Rephrase “With respect to general mechanisms of biocontrol, there have been …”

Line 463: Italicize latin names 

Comments for author File: Comments.docx

Author Response

Thank you very much for the several comments made to improve the quality of our manuscript writing and its scientific message. We implemmented all your comments as follows:

Line 21: Replace “dropped low” with “reduced”: replaced as suggested.

Line 26: Insert “in ” between both and in vitro: inserted as suggested.

Line 26: Replace “it” with “we”: replaced as suggested.

Line 30: Delete “also”: deleted as suggested.

Lines 36-75: Previous work on controlling rice blast showed the efficacy of Pseudomonads especially (Omoboye et al., 2019). (will be interesting to include a few lines about the efficacy of Pseudomonas spp. In managing Pyricularia oryzae on rice both here in the introduction and also during discussion: Thanks for the suggestion. We included this interesting citation in the discussion section. 

Lines 40-41: rephrase “In certain Northern America and European Union countries, this disease has been designated to be a major quarantine disease”. Rephrased as suggested.

Line 43: rephrase “ mainly attacks…”. Rephrased as suggested.

Line 44: replace “center” with “centered”: replaced as suggested.

Line 44: replace “fungal” with “fungus”: replaced as suggested.

Line 53: remove double space before “regional”. removed as suggested.

Line 56: rephrase “hindering IDM which is fully dependent…” . Rephrased as suggested..

Line  57: replace “to the” with “on the”: replaced as suggested.

Line 62: delete “(and perhaps the sole alternative)”; this is too specific. Deleted as suggested.

Line 87: replace “is accepted” with “holds true”: replaced as suggested.

Lines 90-94: This sentence is too long; not concise and difficult to understand. Rephased for better understading.

Line 183: replace “dark” with “darkness”. Replaced as suggested.

Figures 1, 3 and 7: I propose that the chart type be changed to bars for clarity and better interpretation. Also for Figure 7, it is better to order the treatments such that the cultivar with or without wheat blast follows each other so the difference can be clear. Not well presented as it is now. We kept the chart type as boxplots to better depict the distribution of the data around the median and mean, as opposed to bars that will not allow for such detailed representation. Following the reviewer´s suggestions, we reordered the treatment so that the effect of treating or not with the biocontrol agents follows each other so the difference can be clear. Thanks for the suggestion.

Line 314: rephrase: “grew aggressively”. Rephrased as suggested.

Line 317: Rephrase “With respect to general mechanisms of biocontrol, there have been …”. Rephrased as suggested.

Line 463: Italicize latin names. Changed as suggested.

Reviewer 2 Report

Wheat blast disease is one of the most important wheat diseases. Organic farming is a very important area. Therefore, it is the important to develop biological control agents for controlling wheat blast. This work is significant. Some comments and suggestions are as follows.

1. Please supplement the experimental results in the abstract.

2. For wheat blast, are there some fungicides reported?

3. Please note some formatting errors in this article, such as Line 104, 167-170: g L^-1, Line 123, 171: oC, Line 263: ..

4. In the methods, was the experiment repeated once? This is unscientific and unreasonable.

5. ‘12 h photoperiod’ and ‘12-hour photoperiod’ should be consistent.

6. The authors conducted a comprehensive study, but conclusions chapter should be expanded in accordance with the results obtained. 

Author Response

Thanks a lot for reinforcing the importance of biocontrol for the management of wheat blast. We will approach your suggestion here and implement the changes in the manuscript.

1. Please supplement the experimental results in the abstract. We expanded the Abstract, including information about what further research in the topic should be focused on, once there is potential for developing formulations of the biocontrol agents found in our study. Thanks for the suggestion.
2. For wheat blast,are there some fungicides reported? Yes, there are many fungicides labled by the Ministry of Agriculture in Brazil for managing wheat blast (Available at:  https://agrofit.agricultura.gov.br/agrofit_cons/principal_agrofit_cons). However, the effectiveness of the major site-specific systemic fungicide classes labeled for wheat diseases management (such as strobilurins, triazoles and succinate dehydrogenase inhibitors) is considered very low due to the widespread distribution of resistance in the country [Check references 8–11]. 
3. Please note some formatting errors in this article, such as Line 104, 167-170: g L^-1, Line 123, 171: oC, Line 263: ... Thanks for indicating these units for correcting. We are sure that Agronomy MDPI will check all units and implement the changes as needed to keep the standards for its papers.
4. In the methods,was the experiment repeated once? This is unscientific and unreasonable. Here there might has been some kind of misunderstanding. Yes, the experiments were repeated once, as recommended by most of high impact scientific journal, which means that they were conducted twice: the experiment and its replicate.This is scientifically sound and reasoble. 
5. ‘12 h photoperiod’and ‘12-hour photoperiod’ should be consistent. Corrected for consistency.
6. The authors conducted a comprehensive study, but the conclusion chapter should be expanded in accordance with the results obtained. Thanks for the suggestions. Conclusion was expanded as suggested.

Reviewer 3 Report

The manuscript entitled: "Potential of Pseudomonas and Trichoderma from the Brazilian Amazon as biocontrol agents against the wheat blast disease", addresses an important topic related to biocontrol agents against phytopathogens to minimize the yield losses. However, there are some important concerns which should be addressed before it’s further processing.  

Author should provide the reason: why they only focused on amazon soil of Brazil?  What are characteristics of amazon soil?  Moreover in introduction, authors also did not discuss about antagonism and their role is disease control.  

Figure 5-6 are cited earlier in results than figure 2. Check the sequence and arrangements of figures again carefully. First cited figure should be numbered first.

Figures 2 has no description in the text.

How authors calculated general inhibitory effect percentage?

Figure 5, hyphae and biofilm are not indicated within the SEM panels. Please use arrows etc. to show their position. When the samples were collected for SEM ultra structural analysis? 

Generally, The results section is weaker is terms of detailed description of figures.

Discussion section also need to be strengthened to justify results rather than repeating results, as few parts are simply showing results again.

In vivo experiments of wheat blast biocontrol analysis, what was the concentration of spores that were used during treatment, and how the severity and incidence of Blast disease were recorded and what was the ratio of empty seeds in each treatment?

Minor comments:

Line 22, Widespread distribution of resistance, reconsider these words.

Line 46,remove in color

Line  78, line 197 check if the use of abbreviations

Line 95-96 should be part of introduction.

Figure 1,legends statistical analysis showing the description is confusing. Please check its grammar

line 219, change 3 to three

Author Response

Thanks a lot for all your comments. We approached all suggestions here and in the manuscript as appropriate.

Author should provide the reason: why they only focused on amazon soil of Brazil?  What are characteristics of amazon soil?   

The justification for why we focused on soils from the Amazon on this study can be derived from a recent contribution from our research group: the fungal and bacterial antagonists under scrutiny as biocontrol agents "were previously obtained from undisturbed and possibly naturally suppressive soils within areas of native rainforest from the Amazon. These organisms with biocontrol abilities would be well adapted to the tropical Brazilian agroecosystems when delivered as biofungicides against the target diseases (Vicentini et al. 2022, Agronomy).

Moreover in introduction, authors also did not discuss about antagonism and their role is disease control.  Following the reviewer´s suggestion we added the followin statement in the manuscript: "Fungal and bacterial antagonists play an important role, as microbial biocontrol agents (BCAs), in managing plant pathogens and diseases and they can be delivered as biofungicides [Check references 13,14 in the manuscript]." 

Figure 5-6 are cited earlier in results than figure 2. Check the sequence and arrangements of figures again carefully. First cited figure should be numbered first. Thanks for detecting that mistake. In fact, in that paragraph we should have called Figures 1 and 2. We fixed the miscalling along the text and now the order is correct.

The paragraph should read now:  3.1. In vitro antagonism of fluorescent Pseudomonas against the wheat blast pathogen

While the mycelial growth of PoTl isolates was significantly reduced (p ≤ 0.05) by the three strains of fluorescent Pseudomonas species tested (Table 2, Figures 1 and 2).

Figures 2 has no description in the text. We fixed this error, as described above.

_________________________________________________________________________________

How authors calculated general inhibitory effect percentage? We calculated and compared the the fungal pathogen mycelial growth (C)  using the methodology of Camporota [34] adapted by Vicentini et al. [30], in which C = DT/DE*100, where DT is the growth radius of the PoTl colony towards the antagonistic Pseudomonas or Trichoderma and DE, the distance separating the two colonies. 

Figure 5, hyphae and biofilm are not indicated within the SEM panels. Please use arrows etc. to show their position. Thanks for the suggestion. We included arrows and legends to indicate fungal hyphae and biofilm formation.

When the samples were collected for SEM ultra structural analysis?  Colony disks from these antagonism experiments were sampled at 7 days after the pairings. We added this information in the manuscritp now.

Generally, The results section is weaker is terms of detailed description of figures. We checked every Table and Figure presented in the manuscript for their appropriate description.

Discussion section also need to be strengthened to justify results rather than repeating results, as few parts are simply showing results again. We followed your suggestion and strenghthened the discussion including and citing more relevant literature.

In vivo experiments of wheat blast biocontrol analysis, what was the concentration of spores that were used during treatment, and how the severity and incidence of Blast disease were recorded and what was the ratio of empty seeds in each treatment?  As indicated in material and methods, "the conidia suspension was prepared in sterile distilled water plus 0.01% tween 20 and adjusted to ≈ 10⁴ conidia mL^-1 using a Neubauer chamber, for subsequent spraying the leaves and ears of wheat plants." This is how we measured disease severity: "The evaluation of the biocontrol treatments effect was done 14 days after inoculation by determining the severity of blast symptoms on wheat ears, which were digitally photographed". Although we have not recorded the ratio of empty grains per ear, the white (bleached) wheat ears (as depitcted in figure 8, M and N) had partial or total sterile spikelets, from the infection point in the rachis, with empty grains. 

Minor comments:

Line 22, Widespread distribution of resistance, reconsider these words. We replaced the word distribution with the word prevalence.

Line 46,remove in color. Removed as suggested.

Line  78, line 197 check if the use of abbreviations. Checked and fixed as suggested.

Line 95-96 should be part of introduction. Moved to introduction as suggested.

Figure 1, legends statistical analysis showing the description is confusing. Please check its grammar. Thanks for noticing that. In fact that statistics is from Table 1 and not from Figure 1. It was simply an artifact of the positioning of the Figure after the Table. We fixed "Significant", which was mispelled. 

line 219, change 3 to three. Fixed as suggested.

Reviewer 4 Report

This manuscript "Potential of Pseudomonas and Trichoderma from the Brazilian Amazon as biocontrol agents against the wheat blast disease'' is about using Pseudomonas and Trichoderma as a biocontrol to control wheat blast disease caused by the ascomycetous fungal pathogen Pyricularia oryzae Triticum lineage. It is well written. This manuscript is recommended for publication after major revision.

1.   In tables 2 and 3, It is not necessary to present this data in the main manuscript because these data just show the statical analysis the authors can present in the supplementary data.

2.    SEM data (Figures 5 and 6) should be present on the same scale size because the author used a different scale it is hard to explain this data.

3.    The SEM data is not enough to explain the mechanism’s interaction. It needs more experiments to explain how Pseudomonas and Trichoderma can inhibit the Pyricularia oryzae Triticum lineage.

4.    More research and discussion into how Pseudomonas and Trichoderma control wheat blast disease is required.

Author Response

Thank you very much for your comments and suggestion. We approached them in here and also in the manuscript, when appropriate.

1.  In tables 2 and 3, It is not necessary to present this data in the main manuscript because these data just show the statical analysis; the authors can present in the supplementary data.  Because the journal does not limit the number of pages, we kept the two tables in the body of the manuscript so the reader do not have to search from them in the supplementary material. 
2. SEM data (Figures 5 and 6) should be present on the same scale size because the author used a different scale it is hard to explain this data. Different scale were necessary to present distinct close-up views of the processes depicted. What we have done to attend the reviewer's suggestions was to redistribute the images as portrait other than on a landscape view. This allowed us to enlarge the hole image composition, without compromising resolution, and the discrimination of the distinct processess depicted. 
3. The SEM data is not enough to explain the mechanism’s interaction. It needs more experiments to explain how Pseudomonas and Trichoderma can inhibit the Pyricularia oryzae Triticum lineage.  The SEM analyses allowed us to reveal the interactions among the distinct bacterial and fungal antagonists and PoTl at the microscopic level, such as extensive colonization and biofilm formation, pathogen's hyphae damage and parasitism. The information presented is robust enough for the message we are conveying in this manuscript.
4. More research and discussion into how Pseudomonas and Trichoderma control wheat blast disease is required. We complemented the discussion incluing more literature support on the ability of Pseudomonas and Trichoderma to control Pyricularia, as recommended.

Reviewer 5 Report

The manuscript describes the antagonistic activities of three Pseudomonas strains and three Trichoderma strains against PoTl, the phytopathogen which causes wheat blast. The in vitro dual culture assay and in vivo greenhouse study were applied to determine the antagonistic activity. Moreover, the antagonistic mechanisms were revealed by SEM. The results were novel and provided new resources for the biocontrol of wheat blast.  

Despite the paper is well prepared, some revisions should be done before it is published:

1.     Line 100 & 122, please indicate the source of the pathogenic PoTl.

2.     Line 201, why did the authors use Figures 5-6 to illustrate the inhibition effect of mycelial growth? The figures seemed not relative to the aspect.

3.     Section 3.4, how is the colonization ability of the antagonistic strains on wheat?

4.     Figure 6, please indicate the meaning of panel F in the figure legend. 

Minor issues;

1.     Line 151, 171&185, the °C symbol is not shown properly.

2.     Line 268, what is the meaning of the asterisk?

Author Response

Thanks a lot for your comments on the novelty of our study, which could indeade provide new resources for the biocontrol of wheat blast.

We will approach your comments and suggestions and introduce the changes in the manuscript.

1. Line 100 & 122, please indicate the source of the pathogenic PoTl :Informed as requested. These isolates belong to our collection of wheat blast. The isolates 12.1.146, 12.1.207, and 12.1.047 were obtained in 2012 from infected wheat plants sampled in Mato Grosso do Sul, Rio Grande do Sul and Brasilia, respectively.
2. Line 201, why did the authors use Figures 5-6 to illustrate the inhibition effect of mycelial growth? The figures seemed not relative to the aspect. These SEM figures were prepared from the in vitro interaction between the pathogen and the biocontrol agents and they truly represent the antagonistic effect at microscopic level. 
3. Section 3.4, how is the colonization ability of the antagonistic strains on wheat? We have not examined the colonization of the wheat ears by the antagonistic bacterial and fungal biocontrol agents. We examined the reduction in disease severity, though, which was very pronounced, depending on the biocontrol agent inoculated. So, we assumed that colonization was successful to garantee disease contrl.
4. Figure 6, please indicate the meaning of panel F in the figure legend. Thanks for calling the attention about this issue. We corrected it in the manuscript. 

   (F) Abundant Trichoderma conidia (Tr.co.) in detailed close-up.

Minor issues;

1. Line 151, 171 & 185, the °C symbol is not shown properly. We checked and fixed as suggested. There was only one case of improper representation. There might have been a problem that occurred after fiel conversion.
2. Line 268, what is the meaning of the asterisk? That should be x (by): treatment x experiment interaction.