Biogenic Silver and Copper Nanoparticles: Potential Antifungal Agents in Rice and Wheat Crops

Round 1

Reviewer 1 Report

The authors needs to focus the following comments to improve the quality of the manuscript

#1.    In page, 1 abstract, lines 18-20, “ High Resolution- … of the nanoparticles” are not required. Delete the sentence.

#2.    Line 24, “showed antifungal activity at low concentrations”- include the results (value)

#3.    The abstract can be improved with the proper findings of this manuscript

#4.    What is the role of seed germination in anti-fungal activity? How is this experiment significant in the aspect of anti-phytopathogens?

#5.    Once the silver nanoparticles are abbreviated as AgNPs, then no need to mention the silver in the next mentions, either use AgNPs or Silver nanoparticles, do not mix both terms

#6.    Line 104, “Growth was carried out at 28 °C,” rewrites the growth as fungi growth

#7.    Line 113, “Based in the same protocol” checks grammatical issue

#8.    How the plug of 5 mm can be placed in the center of the PDA plate?

#9.    Seed germination should be tested by immersing/ treating seed with fungal strain to claim their relevancy on the anti-fungal activity

#10. Lines 195-197 “The biomass from …. of 250–800 nm ” are already mentioned in the methodology section, they are not suitable to be placed in the results section

#11. What is the correlation of the highest band in pH-based synthesis of nanoparticle study? Explain

#12. Why was the pH variation tested only for CuNPs and not for AgNPs?

#13. Similar way, why precursor concentrations are varied only for AgNPs, not for CuNPs? Justify

#14. In the results section, do not discuss or compare with citations, it should be done in the discussion section

#15. The figure caption should start with the main heading, not with the sub legends like a) and (b), etc.

#16. The SEM image of CuNPs is not clear, why is it so? Explain the reasons

#17. Why high percentage of C observed in the TEM-EDS analysis for both NPs? Clarify the reason

#18. MIC results of AgNPs mentioned it as  0,034, what this value means? Is it correct

#19. Why CuCl2 not showed the MIC, discuss it in the discussion section with a proper comparison of the literature

#20. Why the MIC concentrations of  AgNps and CuNPs are not measured against R. oryzae-sativae and S. oryzae?

#21. Provide the antifungal plate results, which are mandatory to be included in the manuscript. The table with only readings is not valid proof to claim the results

#22. Seed germination results need to be properly treated with a fungal solution in order to justify its inclusion in this manuscript or else remove this section.

#23. The manuscript elaborately deals with the characterization but the antifungal results are very limited.

#24. The discussion section can be improved with an appropriate comparison with suitable literature

#25. The conclusion section can be improved

#26. Language and grammatical issues are there in the manuscript, which need further attention

 

 

Moderate revision required to improve English language

Author Response

RESPONSES TO REVIEWER 1

#1.    In page, 1 abstract, lines 18-20, “ High Resolution- … of the nanoparticles” are not required. Delete the sentence.

R: Thank you for the suggestion. This sentence was deleted.

#2.    Line 24, “showed antifungal activity at low concentrations”- include the results (value)

R: According to the suggestion the results were included in the revised version.

#3.    The abstract can be improved with the proper findings of this manuscript

R: Thank you. The abstract was improved and completed with the main findings of the work.

#4.    What is the role of seed germination in anti-fungal activity? How is this experiment significant in the aspect of anti-phytopathogens?

R: Thank you for the questions. The purpose of the seed germination test was as an indicator of toxicity, demonstrating that the treatment with nanoparticles does not have a negative effect on germination. In order to clarify it we have written a paragraph in the discussion section, including a citation, in the revised version.

Although seeds are the most basic means of production in agriculture, information on the effects of nanoparticle treatments on seed germination/growth is still limited. In order to take advantage of the positive effects of the inclusion of nanotechnology in agronomic practices, the possible toxicity that these metallic nanoparticles could cause in plants must be thoroughly analyzed. In this sense, the study of the impact of nanoparticles on seed germination is relevant as an indicator of toxicity (Spagnoletti et al., 2019). In this work, the germination percentage of rice and wheat seeds treated with nanoparticles was evaluated to verify that the nanoparticles did not negatively affect germination.

 

#5.    Once the silver nanoparticles are abbreviated as AgNPs, then no need to mention the silver in the next mentions, either use AgNPs or Silver nanoparticles, do not mix both terms

R: Thank you for the comment. It was corrected in the revised version of the manuscript.

#6.    Line 104, “Growth was carried out at 28 °C,” rewrites the growth as fungi growth

R: Thanks, it was rewritten.

#7.    Line 113, “Based in the same protocol” checks grammatical issue

R: Thanks for the correction, this sentence was rewritten.

#8.    How the plug of 5 mm can be placed in the center of the PDA plate?

R: Thanks for the question. Agar blocks (plugs) of fresh mycelium were punched out from the edge of the colony using a 5 mm diameter sterile punch. Plates were inoculated in the center with one plug per plate. This explanation was included in the Methods section in the revised manuscript, and this method was previously reported in reference 24.

#9.    Seed germination should be tested by immersing/ treating seed with fungal strain to claim their relevancy on the anti-fungal activity

R: Thanks for the comment. As explained in #4 the purpose in this work was to demonstrate that the treatment with nanoparticles does not have a negative effect on germination (toxicity). Future work should include the evaluation of treated seeds in pot trials, other toxicity tests, as well as challenge trials with phytopathogens to assess plant symptoms with and without nanoparticle treatment.

#10. Lines 195-197 “The biomass from …. of 250–800 nm ” are already mentioned in the methodology section, they are not suitable to be placed in the results section

R: Thank you for the suggestion, this paragraph was rewritten and details of the methodology were deleted.

#11. What is the correlation of the highest band in pH-based synthesis of nanoparticle study? Explain

R: Thank you for the question. The appearance and increase over time of a characteristic band corresponding to the SPR of the metallic nanoparticles during the monitoring of the reaction is a first indicator of the synthesis. In the same reaction time where only one condition is changed (in this case pH) a band with lower absorbance suggests lower production of nanoparticles. For this reason we concluded that at pH=7 the synthesis was favored, showing the highest band at 390 nm, followed by pH=9 and pH=5.

#12. Why was the pH variation tested only for CuNPs and not for AgNPs?

R: Thanks for the questions. It has been widely reported that the biological synthesis of silver nanoparticles can be monitored through the appearance of bands near 400-450 nm in the UV-vis spectrum (Sanguiñedo et al., 2018, Fouda et al., 2020). However, for the biogenic synthesis of copper nanoparticles, monitoring is more difficult since previous studies have reported bands at different wavelengths, attributing them to copper or copper oxide nanoparticles. Likewise, it was reported that the pH could produce shifts in the bands obtained in the UV-vis spectrum of copper nanoparticles (Cuevas et al, 2015). Taking this into account, we decided to evaluate and report the results obtained for the synthesis of copper nanoparticles at different pHs. In this work, although at pH 5 no shift was observed in the spectrum, a decrease in the band at 390 nm was observed. In the case of the AgNPs, the synthesis was carried out at pH 6, observing, as previously reported under the same synthesis conditions (Sanguiñedo et al., 2018), the band at 440 nm corresponding to the SPR of the AgNPs.

 

#13. Similar way, why precursor concentrations are varied only for AgNPs, not for CuNPs? Justify

Thanks for the question. As explained in the Discussion section the biological synthesis of copper nanoparticles has been less studied than that of other metallic nanoparticles such as gold and silver. As reported in the literature [Cuevas et al., 2015; Awwad & Amer, 2020], copper NPs show more variable composition and properties. Furthermore, the approaches in extracellular biosynthesis by fungi show that the bioactive metabolites produced would be responsible for the reduction of the metal and it is expected that the same mechanism could be involved in both Ag+ or Cu²⁺ reduction for the nanoparticle synthesis (Ovais et al., 2018). Taking all this into account, in this work we decided to evaluate the synthesis of copper nanoparticles based on what was previously reported by Cuevas et al. (2015), using 5mM CuCl₂ for the synthesis and evaluating different pH as described in #12. Future studies may include the evaluation of the synthesis of nanoparticles in other conditions, for example in other concentrations of CuCl₂.

 

#14. In the results section, do not discuss or compare with citations, it should be done in the discussion section

R: Thanks for the comment. The results section was revised and comparisons with other works and citations were deleted.

#15. The figure caption should start with the main heading, not with the sub legends like a) and (b), etc.

R: Thank you. Figure captions (Figure 3 and Figure 7) were corrected according to the suggestion.

#16. The SEM image of CuNPs is not clear, why is it so? Explain the reasons

R: Although a lower resolution is achieved, the SEM analysis allowed us to demonstrate the synthesis of the nanoparticles through a not so expensive analysis. With these auspicious results we decided to carry out the analyzes by HR-TEM. Then, the main purpose of the study by HR-TEM and EDS was to improve the resolution of the images obtained by SEM to determine the size of the nanoparticles, complementing it with the DLS measurements, as well as to perform the elemental analysis of the nanoparticle.

#17. Why high percentage of C observed in the TEM-EDS analysis for both NPs? Clarify the reason

R: Thanks for the question. This explanation was included in the Discussion in the revised version.

EDS has been reported as a useful technique to obtain the elementary mapping of the biogenic nanoparticles synthesized by microorganisms (Fouda et al., 2022). The presence of C can be attributed to the scattering of capping agents such as carbohydrates or proteins.

 

#18. MIC results of AgNPs mentioned it as  0,034, what this value means? Is it correct

R: Thanks for the question. Yes, MIC was 0.034 ηM.

The determination of Minimum Inhibitory Concentration in Broth is a standardized method for quantification of antimicrobial activity. In particular, to determine antifungal activity of filamentous fungi by this method it is necessary to obtain a spore suspension which is used as inoculum in the assay. Then, the MIC is defined as the minimal concentration that causes visible inhibition of fungal growth; at concentrations below the MIC as well as in the growth controls (without antifungal) the spores germinate and mycelium growth is observed. According to our results 0.034 ηM was the minimal concentration that caused visible inhibition of fungal growth in the microdilution test.

#19. Why CuCl2 not showed the MIC, discuss it in the discussion section with a proper comparison of the literature

R: Thank you for the question. MIC could not be established for CuCl₂ at the tested concentrations (no inhibition was observed), which is indicative of a much higher MIC than values obtained for nanoparticles. This coincides with findings by Shende et al (2015) who found that 100 mM copper salt solutions did not show any significant activity against three strains of Fusarium spp.  In other recent work, although 60 mM copper solution showed antifungal activity against phytopathogens, copper nanoparticles showed higher activity at lower concentrations (Ahmad et al., 2020). This result was discussed and compared with previous works in the discussion section.

#20. Why the MIC concentrations of  AgNps and CuNPs are not measured against R. oryzae-sativae and S. oryzae?

R: Thank you for the question. Both methods described in this work are widely used in Microbiology for the evaluation of antifungal activity of any chemical compound or nanomaterial. The purpose of these in vitro assays was to evaluate if the nanoparticles are able to inhibit the fungal growth. The determination of Minimum Inhibitory Concentration in Broth is a standardized method for quantification of antimicrobial activity. In particular, to determine antifungal activity of filamentous fungi by this method it is necessary to obtain a spore suspension which is used as inoculum in the assay, as described for Fusarium graminearum and Pyricularia oryzae. Then, the MIC is defined as the minimal concentration that causes visible inhibition of fungal growth; at concentrations below the MIC as well as in the growth controls (without antifungal) the spores germinate and mycelium growth is observed. However, some fungi do not frequently produce spores, such as Sclerotium oryzae and Rhizoctonia oryzae sativa. Taking it into account for these fungi we determined the inhibition of mycelium growth in solid medium, using vegetative inoculum (a plug of fresh mycelium) as described, for example, in the reference cited in the manuscript (Mohammed et al., 2019). Both methods showed nanoparticles were able to inhibit the fungal growth, under the evaluated in vitro conditions.

 

#21. Provide the antifungal plate results, which are mandatory to be included in the manuscript. The table with only readings is not valid proof to claim the results

R: Thank you for the suggestion. The antifungal methods are standardized ones and the results are generally reported as MIC or % inhibition values in order to compare with other results.In response to the reviewer´s suggestion we include figures as supplementary material showing these trials (Figures S1 and S2).

 

 

Figure S1. Determination of Minimum Inhibitory Concentration (MIC) against Pyricularia.oryzae. SC: sterile control. GC: growth control

 

Figure S2.  Antifungal activity of AgNPs against  R. oryzae sativae (% inhibition growth). GC: growth control (PDA). AgNPs: PDA supplemented with AgNPs

 

 

#22. Seed germination results need to be properly treated with a fungal solution in order to justify its inclusion in this manuscript or else remove this section.

R: Thank you for the comments. As explained in #4 the purpose of the seed germination test was as an indicator of toxicity, demonstrating that the treatment with nanoparticles does not have a negative effect on germination. In order to clarify it we have written a paragraph in the discussion section, including a citation, in the revised version. With this promissory result future works should include the evaluation of treated seeds in pot trials, other toxicity tests, as well as challenge trials with phytopathogens to assess plant symptoms with and without nanoparticle treatment.

#23. The manuscript elaborately deals with the characterization but the antifungal results are very limited.

R: Thank you for the comment. Indeed, in this work we elaborately deal with the biological synthesis and characterization of the metallic nanoparticles what we consider essential for being published in a journal in the chemistry area, particularly in the Nanoscale Chemistry research area. Likewise, our interdisciplinary work demonstrated that the nanomaterials developed had the potential to be used in a specific application, in the control of phytopathogens of rice and wheat. As mentioned before, based on the promising results of this work, plant and field trials will be carried out.

#24. The discussion section can be improved with an appropriate comparison with suitable literature

R: Thanks for the suggestion, we have extended the discussion and included new references.

#25. The conclusion section can be improved

R: Thanks for the suggestion. The conclusion section was revised and improved in the revised version.

#26. Language and grammatical issues are there in the manuscript, which need further attention

R: Thank you, according to the suggestion we revised all the manuscript.

 

References

 

Ahmad, H., Venugopal, K., Bhat, A.H., Kavitha, K., Ramanan, A., Rajagopal, K., Srinivasan, R., Manikandan, E.  Enhanced Biosynthesis Synthesis of Copper Oxide Nanoparticles (CuO-NPs) for their Antifungal Activity Toxicity against Major Phyto-Pathogens of Apple Orchards. Pharm Res 2020, 37(246). https://doi.org/10.1007/s11095-020-02966-x

Awwad A, Amer M. Biosynthesis of copper oxide nanoparticles using Ailanthus altissima leaf extract and antibacterial activity. Chem Int 2020, 6(4), 210-217. https://doi: 10.5281/zenodo.3670918

Cuevas R, Durán N, Diez M, Tortella G, Rubilar O. Extracellular biosynthesis of copper and copper oxide nanoparticles by stereum hirsutum, a native white-rot fungus from chilean forests. J Nanomater 2015, 1-7. https://doi.org/10.1155/2015/789089

Fouda A, Hassan SE, Abdo AM, El-Gamal MS. Antimicrobial, antioxidant and larvicidal activities of spherical silver nanoparticles synthesized by endophytic streptomyces spp. Biol Trace Elem Res 2020, 195(2), 707-724. https://doi.org/10.1007/s12011-019-01883-4

Fouda A, Hassan S, Eid A, Awad M, Althumayri K, Badr N, Hamza M. Endophytic bacterial strain, Brevibacillus brev-is-mediated green synthesis of copper oxide nanoparticles, characterization, antifungal, in vitro cytotoxicity, and larvi-cidal activity. Green Process Synth 2022, 11(1), 931-950 31.       

Lamichhane, J.R., Osdaghi, E., Behlau, F., Köhl, J., Jones, J.B., Aubertot, J-N. Thirteen decades of antimicrobial copper compounds applied in agriculture. A review. Agron Sustain Dev 2018, 38(28). https://doi.org/10.1007/s13593-018-0503-9https://doi.org/10.1515/gps-2022-0080

Ovais M, Khalil AT, Ayaz M, Ahmad I, Nethi SK, Mukherjee S. Biosynthesis of metal nanoparticles via microbial enzymes: a mechanistic approach. Int J Mol Sci Technol 2018, 19 (12), 4100. https://doi.org/10.3390/ijms19124100

Sanguiñedo P, Fratila R, Estevez MB, Martínez de la Fuente J, Grazú V, Alborés S. Extracellular biosynthesis of silver nanoparticles using fungi and their antibacterial activity. Nano Biomed Eng 2018, 10(2) https://doi.org/10.5101/nbe.v10i2.p165-173

Shende, S., Ingle, A.P., Gade, A., Raid, M. Green synthesis of copper nanoparticles by Citrus medica Linn. (Idilimbu) juice and its antimicrobial activity. World J Microbiol Biotechnol 2015, 31, 865–873. https://doi.org/10.1007/s11274-015-1840-3

Spagnoletti FN, Spedalieri C, Kronberg F, Giacometti R. Extracellular biosynthesis of bactericidal Ag/AgCl nanoparticles for crop protection using the fungus Macrophomina phaseolina. J Environ Manag 2019, 231, 457-466. https://doi.org/10.1016/j.jenvman.2018.10.081

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

The paper focuses on a very relevant issue which is the biological synthesis of silver and copper oxide nanoparticles using Trichoderma harzianum TA2 and their use in crop protection. This type of knowledge is worth learning to understand potential of biogenic metallic nanoparticles from T. harzianum TA2, and whether they could be considered as candidates for the control of phytopathogens affecting important crops. This is a very promising approach to replace toxic chemical pesticides.

The paper deals with important issues: on the size, distribution, morphology, surface  charge and functional groups of the nanoparticles and in vitro antifungal activity of the nanoparticles  against pathogens of rice and wheat, as well as their effect on seed germination were evaluated.

The authors provided interesting results that showed that silver and copper biogenic nanoparticles are produced by Trichoderma harzianum TA2. The authors were able to purify and measure the biogenic nanoparticles. The biotests in use of the nanoparticles against fungal diseases gave promising results, since they were effective at low concentrations.  Therefore, they could be a biotechnological alternative contributing to the integrated disease management in wheat and rice crops. No negative effects on crops were also found.

 

The article presents in a legible and transparent manner the material and methods used in a given research work. The methodology is clear and described concisely. Introduction section is comprehensive and is also written in an concise and clear manner. The literature is well-chosen and the conclusions clearly refer to the conducted research.

There are, however some minor weaknesses in discussion, therefore I recommend minor revision of the paper.

 

Minor issues to be corrected:

·         Introduction

It would be good to add a citation to the statement (line 36-38).

·         Discussion

Reference to organisms (line 437) - please add the info on the organisms biosynthesizing the nanoparticles: species, where they were isolated from, whether they are rhizosphere or endophytic organisms.

I suggest to extend the literature citation on biogenic metal nanoparticles as a new tool for more sustainable management of agricultural diseases, especially comparing own result to others.

·         Results

3.1. Biological synthesis of metallic nanoparticles (line 195-198), it seems that this description should be moved to the material and methods chapter.

3.3. Antifungal activity (table 1),  was the analysis performed in technical repetitions? if yes, it is worth adding a statistical analysis when comparing the results, possibly a standard deviation.

·         Conclusions

Conclusions could be a bit more developed – especially extracted from the own results, not only the broader but not detailed statements

 

In summary, the paper is worth publishing in the Journal.

Author Response

RESPONSES TO REVIEWER 2

The paper focuses on a very relevant issue which is the biological synthesis of silver and copper oxide nanoparticles using Trichoderma harzianum TA2 and their use in crop protection. This type of knowledge is worth learning to understand potential of biogenic metallic nanoparticles from T. harzianum TA2, and whether they could be considered as candidates for the control of phytopathogens affecting important crops. This is a very promising approach to replace toxic chemical pesticides.

The paper deals with important issues: on the size, distribution, morphology, surface  charge and functional groups of the nanoparticles and in vitro antifungal activity of the nanoparticles  against pathogens of rice and wheat, as well as their effect on seed germination were evaluated.

The authors provided interesting results that showed that silver and copper biogenic nanoparticles are produced by Trichoderma harzianum TA2. The authors were able to purify and measure the biogenic nanoparticles. The biotests in use of the nanoparticles against fungal diseases gave promising results, since they were effective at low concentrations.  Therefore, they could be a biotechnological alternative contributing to the integrated disease management in wheat and rice crops. No negative effects on crops were also found.

 The article presents in a legible and transparent manner the material and methods used in a given research work. The methodology is clear and described concisely. Introduction section is comprehensive and is also written in an concise and clear manner. The literature is well-chosen and the conclusions clearly refer to the conducted research.

There are, however some minor weaknesses in discussion, therefore I recommend minor revision of the paper.

In summary, the paper is worth publishing in the Journal. 

 

Minor issues to be corrected:

 

• Introduction

It would be good to add a citation to the statement (line 36-38).

R: Thanks for the suggestion. A citation was added in the revised version.

• Discussion

Reference to organisms (line 437) - please add the info on the organisms biosynthesizing the nanoparticles: species, where they were isolated from, whether they are rhizosphere or endophytic organisms.

R: Thank you. We have completed this information in the revised manuscript.

I suggest to extend the literature citation on biogenic metal nanoparticles as a new tool for more sustainable management of agricultural diseases, especially comparing own result to others.

R: Thanks for the suggestion, we have extended the discussion and included new references.

• Results

3.1. Biological synthesis of metallic nanoparticles (line 195-198), it seems that this description should be moved to the material and methods chapter.

R: Thank you. According to the suggestion this paragraph was removed from the results.

3.3. Antifungal activity (table 1) was the analysis performed in technical repetitions? if yes, it is worth adding a statistical analysis when comparing the results, possibly a standard deviation.

R: Thank you for the question. All experiments were carried out in duplicates, or triplicates, and results were indicated as the modal value when distinct values were found, as reported in Melhem et al. (2022).

• Conclusions

Conclusions could be a bit more developed – especially extracted from the own results, not only the broader but not detailed statements

R: Thanks for the suggestion. The conclusion section was more developed in the revised version. 

References

Ahmad, H., Venugopal, K., Bhat, A.H., Kavitha, K., Ramanan, A., Rajagopal, K., Srinivasan, R., Manikandan, E.  Enhanced Biosynthesis Synthesis of Copper Oxide Nanoparticles (CuO-NPs) for their Antifungal Activity Toxicity against Major Phyto-Pathogens of Apple Orchards. Pharm Res 2020, 37(246). https://doi.org/10.1007/s11095-020-02966-x

Hermosa, R., Viterbo, A., Chet, I., Monte, E. Plant-beneficial effects of Trichoderma and of its genes. Microbiology 2012, 158, 17-25.  https://doi.org/10.1099/mic.0.052274-0

Lamichhane, J.R., Osdaghi, E., Behlau, F., Köhl, J., Jones, J.B., Aubertot, J-N. Thirteen decades of antimicrobial copper compounds applied in agriculture. A review. Agron Sustain Dev 2018, 38(28). https://doi.org/10.1007/s13593-018-0503-9

Melhem, M. S. C., Coelho, V. C., Fonseca, C. A., Oliveira, L., Bonfietti, L. X., Szeszs, M. W., Magri, M. M. C., Dorneles, F. S., Taguchi, H., Moreira, D. V. S., Motta, A. L., Batista, M. V., Kamei, K., Shikanai-Yasuda, M. A. Evaluation of the Sensititre YeastOne and Etest in Comparison with CLSI M38-A2 for Antifungal Susceptibility Testing of Three Azoles, Amphotericin B, Caspofungin, and Anidulafungin, against Aspergillusfumigatus and Other Species, Using New Clinical Breakpoints and Epidemiological Cutoff Values. Pharmaceutics 2022, 14(10), 2161. https://doi.org/10.3390/pharmaceutics14102161

Shende, S., Ingle, A.P., Gade, A., Raid, M. Green synthesis of copper nanoparticles by Citrus medica Linn. (Idilimbu) juice and its antimicrobial activity. World J Microbiol Biotechnol 2015, 31, 865–873. https://doi.org/10.1007/s11274-015-1840-3

Usman, M., Farooq, M., Wakeel, A., Nawaz, A., Cheema, S. A., Rehman, H. ur, Ashraf, I., Sanaullah, M. Nanotechnology in agriculture: Current status, challenges and future opportunities. Sci Total Environ 2020, 721, 137778. https://doi.org/10.1016/j.scitotenv.2020.13777

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

 

#1.  The title says “Biogenic metallic nanoparticles” in general, but the work dealt with only Cu and Ag NPs. So this title will not suit to this manuscript. Mention the specific NPs in the title itself

 

 #2.  Author response to my question “Why was the pH variation tested only for CuNPs and not for AgNPs?” is not convincing. If one NPs production tested for pH variation its obvious that other NPs also to be tested, we can’t leave as it mentioned or reported in literature. Because here the sample used for the synthesis of NPs is different, hence the result also might be differed

 

#3.  Author response to my question “Why the MIC concentrations of AgNps and CuNPs are not measured against R. oryzae-sativae and S. oryzae?” is not convincing.  Authors just giving a explanation of what is MIC assay for fungi. If authors claiming the synthesized NPs are antifungal, then the detailed evidence is required. Due to the variation of Fungi species the same MIC concentration cannot be used for other Fungi. So, the MIC study must be extended for R. oryzae-sativae and S. oryzae.  Also, the MIC should be compared with previously reported anti-fungal results of various biogenic synthesized Cu and Ag NPs in a table format.

 

 #4.  The title highlighted the major application is Antifungal activity, but the results for this justification is too minimum as per my view.

 

#5.  As per the author claim if the seed germination is performed to know the toxicity of the NPs, then it can be renamed as phytotoxicity assay or in other suitable section title.

 

 

Paragraph formation and sentence alignment needs a minor attention 

Author Response

RESPONSES TO REVIEWER 1

1. The title says “Biogenic metallic nanoparticles” in general, but the work dealt with only Cu and Ag NPs. So this title will not suit to this manuscript. Mention the specific NPs in the title itself.

R: Thanks to the referee for the response. According to the suggestion the title was changed in the revised version, specifically mentioning silver and copper nanoparticles.

2. Author response to my question “Why was the pH variation tested only for CuNPs and not for AgNPs?” is not convincing. If one NPs production tested for pH variation its obvious that other NPs also to be tested, we can’t leave as it mentioned or reported in literature. Because here the sample used for the synthesis of NPs is different, hence the result also might be differed

R: Thanks for the comment. We agree that synthesis can change with modifications in the reaction conditions. In fact, our group has evaluated, in other previous works, different conditions for the biogenic synthesis of silver nanoparticles using other fungi (Estevez et al., 2021; Sanguiñedo et al., 2019; Estevez et al., 2019).

In this work, the objectives were not to evaluate many nanoparticle synthesis conditions, but rather to evaluate whether the fungal strain was capable of producing nanoparticles under particular conditions. As the results were promising under the selected conditions, future trials will include the evaluation of changes in various reaction conditions (including pH) in the production of silver nanoparticles. In order to clarify, this sentence was included in the discussion section in the revised version of the manuscript.

3. Author response to my question “Why the MIC concentrations of AgNps and CuNPs are not measured against  oryzae-sativae and S. oryzae?” is not convincing.  Authors just giving a explanation of what is MIC assay for fungi. If authors claiming the synthesized NPs are antifungal, then the detailed evidence is required. Due to the variation of Fungi species the same MIC concentration cannot be used for other Fungi. So, the MIC study must be extended for R. oryzae-sativae and S. oryzae.  Also, the MIC should be compared with previously reported anti-fungal results of various biogenic synthesized Cu and Ag NPs in a table format.

R: Thank you for the comment. For R. oryzae-sativae and S. oryzae it is not possible to determine the MIC by the microdilution method in both because these fungi do not produce spores (therefore no spore suspension is available for assay). However, the method to determine the antifungal activity for fungi that do not produce spores is the one we used on solid medium (PDA, with agar). In our work the results showed that the nanoparticles had antifungal activity against both fungi because they partially inhibited the mycelial growth. The evidence of this antifungal activity is clearly observed by the smaller growth diameter of each fungus with nanoparticles than without nanoparticles (control plate, GC), as shown in Figure S2.

Figure S2.  Antifungal activity of AgNPs against  R. oryzae sativae (% inhibition growth). GC: growth control (PDA). AgNPs: PDA supplemented with AgNPs. Arrows show mycelial growth.

4. The title highlighted the major application is Antifungal activity, but the results for this justification is too minimum as per my view.

R: Thanks for the opinion. The title is specific, it highlighted biogenic nanoparticles against rice and wheat phytopathogens fungi, so in this work the antifungal activity was demonstrated against four important fungi isolated from these crops. Results evidenced that at very low concentrations AgNPs inhibited all fungi and CuONPs inhibited three fungi. Furthermore, as previously explained, in this work we elaborately deal with the biological synthesis and characterization of silver and copper nanoparticles, what we consider essential for being published in a journal in the chemistry area, particularly in the Nanoscale Chemistry research area.

5. As per the author claim if the seed germination is performed to know the toxicity of the NPs, then it can be renamed as phytotoxicity assay or in other suitable section title.

R: Thank you for the comment. Both in Methods and in Results sections ¨Antifungal activity¨ and ¨Seed germination¨ are in different section titles. According to the suggestion the title was changed as ¨Seed germination toxicity test¨

References

Estevez, M.B., Raffaelli, S., Mitchell, S.G., Faccio, R., Alborés, S. Biofilm eradication using biogenic silver nanoparticles. Molecules 2021, 25(9). https://doi: 10.3 390/molecules25092023}

Estevez, M.B.,  Mitchell, S.G., Faccio, R., Alborés, S. Biogenic silver nanoparticles: understanding the antimicrobial mechanism using Confocal Raman Microscopy. Mater Res Express 2020, 6(12). https://doi.org/10.1088/2053-1591/ab6636

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