Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

After carefully reading this manuscript, I present my comments with the aim of contributing to its improvement and enhancing clarity and readability for the audience. I have conducted a point-by-point analysis to facilitate corrections, if necessary.

Regarding the abstract, it adequately summarizes the objectives, methodology, and main findings of the study; however, the research gap could be stated more explicitly, with a direct sentence such as: “However, the physiological mechanisms underlying its effect on S. furcifera remain unclear.” Additionally, some keywords repeat terms already presented in the title, and replacing them with broader indexing terms could improve the visibility of the work (endophytic fungi; biological control; rice planthopper; insect physiology; detoxification enzymes; plant–microbe–insect interaction).

The title reflects the scope of the study but could be strengthened to more clearly highlight the main biological and applied findings, such as reproductive reduction, multi-enzymatic interference, and potential as a biocontrol agent. The current title only suggests a “physiological effect,” whereas the study goes beyond that.

The introduction contextualizes the importance of S. furcifera as a pest, the impact of SRBSDV, and the use of endophytic fungi. However, the mechanistic gap is not strongly articulated. It remains implicit and could be formulated more directly, for example: “However, the physiological mechanisms by which O. guangxiensis X22 influences S. furcifera remain unclear.” In addition, the state of the art regarding physiological and metabolic mechanisms in insects exposed to endophytes could be discussed in greater depth. To further strengthen the introduction, I suggest clarifying the central scientific question and clearly highlighting the study's novel contribution.

The methodological description presents insufficient detail on certain experimental conditions. Please clarify the environmental conditions (temperature, humidity); the exposure time to the treatment should be described more clearly; it is not entirely clear whether controls were included to account for possible indirect effects of plant colonization; additional detail on the confirmation of fungal colonization would be beneficial; although the study measures TOR and Vg expression, it does not explore the metabolic pathway in depth; the mechanistic inference remains more correlational than causal. Although statistical tests are mentioned, there is no complete specification of the exact number of biological versus technical replicates, nor of any sample size or power analysis.

It is important to verify that all methodological aspects are reflected in the results. While this appears to be the case, if the methodology describes symbiotic colonization of the plant, the results should include quantitative or visual confirmation (e.g., colonization rate, PCR confirmation, re-isolation). If this is not clearly demonstrated, an indirect gap emerges, since interpretation depends on effective colonization. Temperature and humidity are described in the methodology, yet the results do not contextualize these conditions when presenting biological data; although this is not an error, it reduces interpretative robustness. Furthermore, what is the functional connection between TOR, Vg, and antioxidant enzymes? Another aspect is that the results present stability of the reference genes (RPL9, RPL10), but the methodology does not fully describe how this stability was statistically validated (e.g., geNorm, NormFinder).

The Discussion section addresses all major findings of the study; however, some results, particularly the non-significant changes in TOR and Vg gene expression, are not critically explored. A more integrative interpretation linking enzymatic alterations, feeding suppression, and reproductive decline would strengthen the scientific rigor of the manuscript.

Conclusion. The study evaluated specific metabolic pathways (TOR, Vg) and antioxidant enzymes; however, the conclusion emphasizes practical application (biocontrol) and does not fully integrate the molecular findings into a robust mechanistic interpretation.

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

L 47-100: The authors clearly introduce the rationale for the research by describing the relevance of rice to human nutrition and the environmental costs and human safety concerns that arise from the overuse of insecticides to control Sogatella furcifera. One of the alternatives presented by the authors to reduce the use of insecticides is the use of resistant cultivars, however, there are limitations.

The use of endophytic entomopathogenic fungi is a promising alternative in pest management practices, as it has shown stimulating results against insects, in addition to acting, in many cases, as a plant growth promoter. One of the relevant novelties of this work is linked to the fact that they worked with the fungus Ochroconis guangxiensis. The vast majority of published articles work with Beauveria bassiana or Metarhizium anisopliae. In this sense, a new species of entomopathogenic fungus is very promising.

L 101-213: Material and methods - The methodology described by the authors is detailed and widely used in the literature. In order to contribute to the manuscript, I would like the authors to evaluate the comments below:
L103-106: For how long are these insects kept in the laboratory?
L107: There is no need to state the beginning and end of the experiment.
L109-111: If you did not isolate O. guangxiensis, could you please cite the reference of who did isolate it?
L123: Dear authors, regarding the tradition of keeping the seeds in the mycelial suspension for 30 minutes, please consider citing the source. Also, isn't a suspension with 5×10⁵ CFU/mL a low concentration? In general, we see concentrations of 107 or 108.
L157: A reference 21 (Huang, S.; Huang, F.; Wu, B.; Long, L.; Ling, Y. Studies on biotypes of the brown and white-backed leafhopper in China and Vietnam. Chin. J. Appl. Entomol. 2014, 51, 525-533) cannot be found in search engines. Please consider replacing it. Thank you.
L174: Explain what CK is?
L 215-320: Results - The results are robust and adequate to the methodology employed by the authors. The figures and tables presented are relevant and assist the reader in interpreting the results. In order to contribute to this topic, please consider the observation below.
L 217-218: Dear authors, endophytic fungi present as growth promoters are a highly random characteristic. However, I recommend removing the comment, as to make this statement, there is a need to present more data to support it. There is a lot of work in the literature on this. In this context, I recommend a specific manuscript on the growth-promoting effect of O. guangxiensis on rice.
L 322-389: The discussion topic is appropriate to the results, where the authors adequately discuss the results found, basing themselves on the current literature. In order to contribute to the manuscript, I ask the authors to observe the comments below.
L 324: “Rubber drills” is an appropriate term. Check the more appropriate terms.
L355: Aspergillus oryzae – italics
L356: Alternaria sp. – Italics
L 390-400: The implications are important and fundamental in robust results.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Please see the attached file. 

Comments for author File: Comments.pdf

Author Response

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Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

This study offers an alternative biocontrol method to the earlier reported Phialocephala fortinii J2PC4 endophytic fungus against the white back planthopper Sogatella furcifera, a key pest and virus vector of rice. This alternative method is based on another endophytic fungus, Ochroconis guangxiensis strain X22 which, as claimed by the Authors of the recent submission, is more effective, than the previously reported one. The paper is well written, tests were carried out with great accurracy, and carefully evaluated. Although the overall reduction may not be drastically that big, rather modest, it is very carefully documented by experimental results. Also this is a valuable submission, which fits well to the high standard of ACRICULTURE. My only general remark is that the power of X22 would have been more comparable to the literature data on J2PC4, if that latter strain had also been included into the tests. However, this does not lower the scientifc merit of the present results on X22.

Minor suggestions:

Lines 2-3: „Effects of the Ochroconis guangxiensis X22 Strain on the Physiological and Metabolic State of the Sogatella furcifera” – I suggest to extend a little bit the title, to be more explanatory to Readers, like this (just a suggestion, feel free to develop another wording): Entomopathogenic Effects of the Plant-associated Fungus, Ochroconis guangxiensis X22 Strain on the Physiological and Metabolic State of the Rice-pest planthopper, Sogatella furcifera

Line 23: „efficient transmission of” – instead: „efficiently transmit”

Lines 31-32: „TOR and Vg genes” – Full names of the gene and that of vitallogenin should be given, as these are the first mentionings.

Line 32: „were downregulated” – instead: „were modestly downregulated” – If the difference to control was not statistically significant you should be cautious.

Line 58: „forproduction” – for production (space)

Line 103: Please state whether TN1 rice cultivar, used for the tests, was free from any symbiotic fungus. Please briefly describe how they were kept / grown to ensure that it was free from any infections. Pls see also the next comment (Line 119)

Line 119: Pls affirm whether the inner parts of TN1 rice seed might incidentally contain some endophytic fungi, or can this possibility be firmly excluded.

Line 135: „exemined” - for what? Surely, it is evident for you, but not neccesserily for the Readers.

Line 274 / Fig 5: Check „A” on the graphs. One of them should be „B”, and on that one, the label on the y-axis should be AChE.

Line 274 / Fig 5: Altough the statistical evaluation was performed pairwise between checks and treated insects (which is correct), it is conspucious that the enzyme activity even in checks declined by time. Why? The same holds also for Figs 6 and 7. These should be treated in the Discussion.

Line 294 / Fig 6: On charts „A” and „C” the activities of superoxide dismutase and catalyse show sharp V-shaped trends. It looks strange why they were upregulated at 72 and 120 h, but downregulated at 96h. The same holds for similar odd tendencies with the respective enzyme activites at Fig 7.

Line 324: „rubber borers” – please indicate at least family name.

Line 359: „insignificantly” – Your results proved significant reduction! Maybe the extent of reduction was not that drastical, however, the difference was statistically significant according to your calculation. 

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors carefully considered my comments and suggestions, and eventually produced a clearer, more direct and more scientifically rigorous text, which greatly contributed to the improvement of this manuscript.