Effect of Beauveria bassiana-Seed Treatment on Zea mays L. Response against Spodoptera frugiperda

Round 1

Reviewer 1 Report

1. The first field trial experiment was performed in mid-February in which S. frugiperda larvae were detected in both negative control and B. bassiana treated plants. However, the second field trial experiment was done in mid-April, in which S. frugiperda larvae were not detected in both the negative control and B. bassiana treated plants. The different periods may impact to S. frugiperda outbreak leading to affect the results. The experiment should be performed in the same period that S. frugiperda usually outbreak in that area.
2. In Fig. S2, the second field trial's dimension design was different from the first The second field trial has only five furrows in each treatment, much lower than the first trial with 25 furrows in the negative control and 33 furrows in B. bassiana treated plants. Why did you decrease furrows in the second field trial and grow near the B. bassiana treated plants of the first field?
3. Table 1 showed that the weight of corn cobs in the MCGHA treatment from the second field trial were lower than the negative control to me even if you mentioned that it has no significant differences. I wonder whether the corn cobs were damaged?
4. In line 296, you mentioned that it has more than two corn cobs in B. bassiana treated plants of the second field trial compared to the negative control. How about the first field trial? Is it possible that B. bassiana is a growth promoter?
5. In Table 2, MCPTG4 treated plants affected S. frugiperda development in the laboratory conditions. The percentage of pupa (63.33%) was lower than that of MCGHA treated plants (100%). MCPTG4 should be used in the field rather than MCGHA.
6. In Fig. 3, the weight of S. frugiperda 6th instar larval from MCPTG4 treated plants was lower than the negative control and MCGHA treatment. In contrast, after they were transferred to artificial diet, the weight of S. frugiperda pupal from the MCPTG4 treated plants was the same as the negative control and became higher than that of MCGHA treatment (Fig.4). Is it affected by the artificial diet?
7. Since we know that B. bassiana can survive in the soil, did you observe B. bassiana colonization in the soil, plant root, and stem, and infect insects after B. bassiana treated seeds were planted?
8. In bioassays under laboratory conditions, one larva/plant is too little. I would recommend using 5-10 larvae per plant. 

Author Response

Reviewer 1

Open Review

(x) I would not like to sign my review report

( ) I would like to sign my review report

English language and style

( ) Extensive editing of English language and style required

( ) Moderate English changes required

(x) English language and style are fine/minor spell check required

( ) I don't feel qualified to judge about the English language and style

Yes      Can be improved        Must be improved      Not applicable

Does the introduction provide sufficient background and include all relevant references?

(x)        ( )         ( )         ( )

Is the research design appropriate?

( )         (x)        ( )         ( )

Are the methods adequately described?

( )         (x)        ( )         ( )

Are the results clearly presented?

( )         (x)        ( )         ( )

Are the conclusions supported by the results?

( )         (x)        ( )         ( )

Comments and Suggestions for Authors

The first field trial experiment was performed in mid-February in which S. frugiperda larvae were detected in both negative control and B. bassiana treated plants. However, the second field trial experiment was done in mid-April, in which S. frugiperda larvae were not detected in both the negative control and B. bassiana treated plants. The different periods may impact to S. frugiperda outbreak leading to affect the results. The experiment should be performed in the same period that S. frugiperda usually outbreak in that area.

Response: Thanks for your comment. In the region where the field trails were done, there are three corn cultivation periods. Early (planting time in February), Intermediate (planting time in April) and Late season (planting time in August). Also, there are all year long monitoring programs to detect economically important pests. For Fall armyworm there are three different expected peaks for its presence: March, May and September. So, for 2019, this insect was detected in our region and the commonly followed control programs were applied. We included in the revised manuscript a brief description of the phytosanitary status of this insect in our region and how it is detected and controlled, with the citation to the website where you can download the description of the monitoring programs in our region. It is in Spanish, but it can be easily translated.

In Fig. S2, the second field trial's dimension design was different from the first The second field trial has only five furrows in each treatment, much lower than the first trial with 25 furrows in the negative control and 33 furrows in B. bassiana treated plants. Why did you decrease furrows in the second field trial and grow near the B. bassiana treated plants of the first field?

Response: Thanks for your comment. We decrease the number of furrows because there was not more cultivation space available. Because our control plants were not treated with GHA, we could use this space to plant a new set of controls and GHA-treated plants with the rationale that the fungus was not present in the soil and that we could see an effect, if we increase the concentration of GHA when compared to the control treatment.

Table 1 showed that the weight of corn cobs in the MCGHA treatment from the second field trial were lower than the negative control to me even if you mentioned that it has no significant differences. I wonder whether the corn cobs were damaged?

Response: Thanks for your comment. No, the corn cobs obtained from the GHA-treated plants were not damaged. The field owner was very kind and give us some corn cobs for our family’s consumption. However, the corn cobs harvested from the negative controls had some larvae of Helicoverpa zea. They were a few and unfortunately, we did not monitor how many of the control corn cobs were damaged, if any. It is a good point, and we hope to continue our field trials to do a more exhaustive study of each stage during the cultivation cycle.

In line 296, you mentioned that it has more than two corn cobs in B. bassiana treated plants of the second field trial compared to the negative control. How about the first field trial? Is it possible that B. bassiana is a growth promoter?.

Response: Thanks for your comment: We think that yes, B. bassiana is a growth promoter, although under field conditions it is not very obvious. it was not possible to harvest the control treatment from the first trial because we discarded these plants to plant the second trial. However, in a previous article, we reported that the B. bassiana treatment induced early flowering in corn and the formation of more corn cobs that the control-treated plants (we have included this reference in the revised manuscript). This study was done under greenhouse conditions; therefore, it is possible that in the field, GHA-treatment had effect on flowering and therefore, on more corn cobs production, although the weight and size of these corn cobs were not significantly different than the obtained from the controls.

In Table 2, MCPTG4 treated plants affected S. frugiperda development in the laboratory conditions. The percentage of pupa (63.33%) was lower than that of MCGHA treated plants (100%). MCPTG4 should be used in the field rather than MCGHA.

Response: Thanks for your comment. Yes, we agree, and we are planning on doing more field trials with MCPTGA. We were very glad that the field owner was willing to try to use this technology. However, he wanted to use a well-known product. When we explained what we wanted to do, he tried to convince us to use Bacillus thuringiensis. Fortunately, when we mentioned Beauveria bassiana strain GHA he agreed. Now, based on the data presented in this manuscript, he wants to use the MCPTG4 treatment.

In Fig. 3, the weight of S. frugiperda 6th instar larval from MCPTG4 treated plants was lower than the negative control and MCGHA treatment. In contrast, after they were transferred to artificial diet, the weight of S. frugiperda pupal from the MCPTG4 treated plants was the same as the negative control and became higher than that of MCGHA treatment (Fig.4). Is it affected by the artificial diet?

Response: Thanks for your comment. Yes, apparently the artificial diet could be compensating the effect of the MCPTG4 treatment. For this manuscript we can only report and discuss the results based on the use of the artificial diet and the comparison among treatments, but it is worth to consider a procedure where we can keep the larvae feeding on the plants until pupae formation, if any.

Since we know that B. bassiana can survive in the soil, did you observe B. bassiana colonization in the soil, plant root, and stem, and infect insects after B. bassiana treated seeds were planted?

Response: Thanks for your comment. No, for this manuscript we did not do these analyses in the field trials. The main purpose was to try to apply this technology under “close to commercial” conditions. We had access to a commercial field. The owner and his employees did the best to help us, but we indeed had a lot of limitations regarding on what we could do. In our laboratory experiments, we analyzed if B. bassiana was colonizing death larvae. It was not. We mentioned this in the discussion of the submitted manuscript. Also, we made suggestions about analyzing the gut microbiota of feeding insects. Regarding colonization, in a previous manuscript, under laboratory and greenhouse conditions, we reported a high endophytic colonization percentage in corn roots and stems (both GHA and PTG4 strains, in addition to other strain: PTG6). We have not analyzed the soil and we agree that it is also important to do.

In bioassays under laboratory conditions, one larva/plant is too little. I would recommend using 5-10 larvae per plant.

Response: Thanks for your suggestion. We would take this in count in our future work and also, we think that the age of the larvae is important. Probably it is better to use neonates and to keep them feeding on treated plants until reaching pupa stage. Sounds challenging but it is worth to try.

Submission Date

17 December 2020

Date of this review

24 Dec 2020 06:13:51

Reviewer 2 Report

The authors could benefit from editing the manuscript to improve English style to improve clarity and grammar (ie, in vs on, extra spaces or no spaces in places). I think the whole manuscript could be better organized overall. The second field trial, unfortunately, adds nothing to the paper since they did not detect fall armyworms in either the treatment or negative control (the population could have crashed locally for the moment for some reason). You ideally would have 3 separate field trials to draw conclusions from the experiments but really need at least two consistent ones. Thus, I would suggest emphasizing the lab work first, then present the first field trial and then frame the second field trial as not as a field trial for measuring insect management but as collection of yield parameters for the corn.

The end of the introduction could include predictions for the experiments to help fill out the end of the section. It also be beneficial to differentiate your study more and go just a bit more into detail on the other entomopathogenic fungal studies you mention in the previous paragraph. Your study is more about inoculating the plants to provide systemic resistance against pests but the fugus can also be sprayed in formulation on the undersides of leaves in larger amounts to encourage and increase the chance that it will infect insect pests and it's not clear from your description of these studies if they are like yours or different like the later. 

Line 170: Insects were monitored how? Visually scouted? Beat sheet? Net? Please elaborate.

Second Field Trial: Why did you increase the inoculation rate 2 orders of magnitude. It's hard to compare the two different field trials because of this...

Also, did you harvest the plants from this trial. It seems you only harvested plants from the first trial, but then describe parameters for corn yield in the second trial which is confusing.  

Line 190: Five ears of corn per treatment seems low to asses yield. From reading it that make only 10 ears total.

Section 2.4 should be moved earlier before the field experiments.

Figure 1. The analysis is inappropriate. The way you describe these sections almost sound like blocks. Even then though edge effects on field plots are a problem when sampling insects. Regardless, you need to incorporate random effects into your ANOVA to account for the different sections/blocks, or switch to a generalized linear mixed effects model.

Table 2. IT be more transparent to gain the actual number of individuals used in the experiments if the data was also displayed as fractions (ie. 100%; 15/15)

Line 351/Figure 7: That's not parthenogenesis. If you leave virgin females isolated or prevent them from matting they eventually lay unfertile eggs. This is common for MANY insects. Further, the wing deformations are pretty common if you don't remove them from their container fast enough - you can tell they were left in the container/cage and tried to fly/escape because a lot of their scales are gone too. This should be removed from the paper and is not evidence of the fungal pathogen inoculation effects.    

Supporting Information: Figure legends could use much more detail. S5: Although interesting, this doesn't really add anything to the paper, especially since you did not provide identifications for the insects!

Author Response

Reviewer 2

Open Review

(x) I would not like to sign my review report

( ) I would like to sign my review report

English language and style

(x) Extensive editing of English language and style required

( ) Moderate English changes required

( ) English language and style are fine/minor spell check required

( ) I don't feel qualified to judge about the English language and style

Yes      Can be improved        Must be improved      Not applicable

Does the introduction provide sufficient background and include all relevant references?

( )         ( )         (x)        ( )

Is the research design appropriate?

( )         ( )         (x)        ( )

Are the methods adequately described?

( )         ( )         (x)        ( )

Are the results clearly presented?

( )         ( )         (x)        ( )

Are the conclusions supported by the results?

( )         ( )         (x)        ( )

Comments and Suggestions for Authors

The authors could benefit from editing the manuscript to improve English style to improve clarity and grammar (ie, in vs on, extra spaces or no spaces in places). I think the whole manuscript could be better organized overall. The second field trial, unfortunately, adds nothing to the paper since they did not detect fall armyworms in either the treatment or negative control (the population could have crashed locally for the moment for some reason). You ideally would have 3 separate field trials to draw conclusions from the experiments but really need at least two consistent ones. Thus, I would suggest emphasizing the lab work first, then present the first field trial and then frame the second field trial as not as a field trial for measuring insect management but as collection of yield parameters for the corn.

Response: Thanks for your comments and suggestions. We have checked the manuscript and made the changes on grammar and tried to improve the clarity. We hope to have now a very good revised version. Also, we re-organized the manuscript. We moved the laboratory work first. Regarding the field work, there are three corn cultivation periods: Early (planting time in February), Intermediate (planting time in April) and Late season (planting time in August). So, we could consider a new cultivation cycle starting in April as the second field trial mentioned in the submitted manuscript. Also, there are all year long monitoring programs to detect economically important pests. For Fall armyworm there are three different expected peaks for its presence: March, May and September. For 2019, this insect was detected in our region and the commonly followed control programs were applied. We included in the revised manuscript a brief description of the phytosanitary status of this insect in our region and how it is detected and controlled, with the citation to the website where you can download the description of the monitoring programs in our region. It is in Spanish, but it can be easily translated.

The end of the introduction could include predictions for the experiments to help fill out the end of the section. It also be beneficial to differentiate your study more and go just a bit more into detail on the other entomopathogenic fungal studies you mention in the previous paragraph. Your study is more about inoculating the plants to provide systemic resistance against pests but the fugus can also be sprayed in formulation on the undersides of leaves in larger amounts to encourage and increase the chance that it will infect insect pests and it's not clear from your description of these studies if they are like yours or different like the later.

Response: Thanks for your comments and suggestions. We added sentences in Lines 144-152 addressing these points.

Line 170: Insects were monitored how? Visually scouted? Beat sheet? Net? Please elaborate.

Response: They were visually scouted. We added a sentence in the revised manuscript. Line 276.

Second Field Trial: Why did you increase the inoculation rate 2 orders of magnitude. It's hard to compare the two different field trials because of this...

Also, did you harvest the plants from this trial. It seems you only harvested plants from the first trial, but then describe parameters for corn yield in the second trial which is confusing.

Response: We increased the inoculation due to the interest of the field owner. He wanted to repeat the field trial as soon as possible due to the effects on Spodoptera frugiperda population. For the intermediate corn cultivation cycle then we decided to use more inoculum and then harvest the first and second trial. We did not have enough space for new planting, then we discarded the controls from the first trial, thus we only reported the harvest data from the second trial (we have control and treatment data). The main purpose was to try to apply this technology under “close to commercial” conditions. We had access to a commercial farm. The owner and his employees did the best to help us, but we indeed had a lot of limitations regarding on what we could do.

Line 190: Five ears of corn per treatment seems low to asses yield. From reading it that make only 10 ears total.

Response: Yes, we agree that It is indeed a very low number, but we want to mention that it is an estimate. It was following what was recommended by the field owner and his employees, particularly that it has to be random.

Section 2.4 should be moved earlier before the field experiments.

Response: Change was done.

Figure 1. The analysis is inappropriate. The way you describe these sections almost sound like blocks. Even then though edge effects on field plots are a problem when sampling insects. Regardless, you need to incorporate random effects into your ANOVA to account for the different sections/blocks, or switch to a generalized linear mixed effects model.

Response: Thanks for your suggestion. We have opted to show the frequency of larvae per furrow therefore we now present a new figure 7 (with the changes in the section’s order, Figure 1 is now Figure 7)

Table 2. IT be more transparent to gain the actual number of individuals used in the experiments if the data was also displayed as fractions (ie. 100%; 15/15)

Response: Thanks for your comment. We have added the total number of individuals used in the experiments (45 larvae per treatment).

Line 351/Figure 7: That's not parthenogenesis. If you leave virgin females isolated or prevent them from matting they eventually lay unfertile eggs. This is common for MANY insects. Further, the wing deformations are pretty common if you don't remove them from their container fast enough - you can tell they were left in the container/cage and tried to fly/escape because a lot of their scales are gone too. This should be removed from the paper and is not evidence of the fungal pathogen inoculation effects.

Response: Thanks for your comments. We have removed the part of the damaged wings from the manuscript. We have added a sentence indicating that neonates were observed hatching from these eggs, so it is indeed an apparent parthenogenesis.

Supporting Information: Figure legends could use much more detail. S5: Although interesting, this doesn't really add anything to the paper, especially since you did not provide identifications for the insects!

Response: Thanks for your comments. We consider that how it is supplementary information it is worth to show the pictures of the insects observed during the field trials, even we did not do the taxonomic ID.

Submission Date

17 December 2020

Date of this review

29 Dec 2020 01:29:02

Reviewer 3 Report

Title: The title is not informative as it does not reflect the scope of the research carried out. They concerned the effect not only on S. frugiperda but also on maize plants.

Introduction: the Introduction chapter, is too extensive and contains too much information on the spread and harmfulness of S. frugiperda, while information on B. bassiana is contained in only a few lines (lines 107-119). Information on the pathogen's endophytic development capacity, supported by numerous published information, is definitely lacking.

Material and Methods: Lack of information on the origin of the tested PTG4 strain and confirmation of its taxonomic status by genetic methods (were they performed?). The basic methodological error of the reviewed work is the lack of an experiment confirming the endophytic development of the tested B. bassiana strains in maize plants after seed inoculation. This applies to both experiments carried out in laboratory and field conditions as well. . In the Discussion (lines 533-536) the authors wrote: “It was not possible to determine the presence of B. bassiana  in the plant tissues that were used to fed the larvae, but it is routine in our lab to find B. bassiana as endophyte in Zea mays plants that were  grown after B. bassiana-methyl cellulose seed treatments.” What does it mean? Whether such tests could not be performed, or was their result negative? By the way, referring to routine is not scientific evidence.

            In my opinion, for an article to be published, the authors must supplement the data with tests (or information from previously published works) that both isolates showed endophytic development in plants.Without this, the entire interpretation of the results and the conclusions drawn from it are only half-baked.

The following information is also missing in the Material and Methods section: Was the germination capacity of the blastospores tested prior to inoculation of the maize seeds? If so, how it was done. How long (time) did the seed inoculation process take? Line 215-216 - how many replications was this experiment performed? How many larvae, pupae and adults were used in the tests in total? There is also no information on how the observations on the occurrence of beneficial insects, including bees, were carried out on plants in field experiments. Since authors cite this information in their results and even in their conclusions (line 590), it must be scientifically documented. Photos in supplementary material are certainly not enough!

Discussion: it is rather poorly organized and largely repeats the statements already presented in the results chapter. These repetitions should be omitted e.g. line 452-462; 467-474 etc.

Author Response

Reviewer 3

Open Review

(x) I would not like to sign my review report

( ) I would like to sign my review report

English language and style

( ) Extensive editing of English language and style required

(x) Moderate English changes required

( ) English language and style are fine/minor spell check required

( ) I don't feel qualified to judge about the English language and style

Yes      Can be improved        Must be improved      Not applicable

Does the introduction provide sufficient background and include all relevant references?

( )         ( )         (x)        ( )

Is the research design appropriate?

( )         (x)        ( )         ( )

Are the methods adequately described?

( )         ( )         (x)        ( )

Are the results clearly presented?

( )         (x)        ( )         ( )

Are the conclusions supported by the results?

( )         ( )         (x)        ( )

Comments and Suggestions for Authors

Title: The title is not informative as it does not reflect the scope of the research carried out. They concerned the effect not only on S. frugiperda but also on maize plants.

Response: Thanks for your comment. We have changed the title. We hope that is clearer now.

Introduction: the Introduction chapter, is too extensive and contains too much information on the spread and harmfulness of S. frugiperda, while information on B. bassiana is contained in only a few lines (lines 107-119). Information on the pathogen's endophytic development capacity, supported by numerous published information, is definitely lacking.

Response: Thanks for your comment. We have changed the title. We hope that is clearer now and we have added more information about B. bassiana effects depending on treatment and what it is known about B. bassiana as an endophyte.

Material and Methods: Lack of information on the origin of the tested PTG4 strain and confirmation of its taxonomic status by genetic methods (were they performed?).

Response: Yes, we have included the origin of the tested PTG4 strain and its GenBank number.

The basic methodological error of the reviewed work is the lack of an experiment confirming the endophytic development of the tested B. bassiana strains in maize plants after seed inoculation. This applies to both experiments carried out in laboratory and field conditions as well. . In the Discussion (lines 533-536) the authors wrote: “It was not possible to determine the presence of B. bassiana  in the plant tissues that were used to fed the larvae, but it is routine in our lab to find B. bassiana as endophyte in Zea mays plants that were  grown after B. bassiana-methyl cellulose seed treatments.” What does it mean? Whether such tests could not be performed, or was their result negative? By the way, referring to routine is not scientific evidence.

Response: Thanks for your comments. The aim of this manuscript is to analyze the effects of seed treatments in laboratory bioassays and in the field, particularly against S. frugiperda. Specifically, for these plants (both from bioassays and from the field) we did not confirm that they were colonized by GHA and PTG4 as endophytes. It was not our goal for this work as we had previously published an article reporting that GHA, PTG4 and another strain PTG6 colonize corn plants as endophytes under laboratory and greenhouse conditions. We agree that more information was needed to clarify this, therefore we have included the citation to our previous work and rephrased the sentences the reviewer highlighted.

In my opinion, for an article to be published, the authors must supplement the data with tests (or information from previously published works) that both isolates showed endophytic development in plants.Without this, the entire interpretation of the results and the conclusions drawn from it are only half-baked.

Response. We have added the citation to our previous work with these strains and rephrased the sentences the reviewer highlighted.

The following information is also missing in the Material and Methods section: Was the germination capacity of the blastospores tested prior to inoculation of the maize seeds? If so, how it was done. How long (time) did the seed inoculation process take? Line 215-216 - how many replications was this experiment performed? How many larvae, pupae and adults were used in the tests in total? There is also no information on how the observations on the occurrence of beneficial insects, including bees, were carried out on plants in field experiments. Since authors cite this information in their results and even in their conclusions (line 590), it must be scientifically documented. Photos in supplementary material are certainly not enough!

Response. We have added the citation to our previous work where we explained the seed inoculation procedure. Also, we have done our best to include as much information as possible in the material and methods and results section regarding our laboratory experiments. About the observation of occurrence of insects shown in the supplementary material we think it is important to show what we observed. As a matter of fact, we want to emphasize that the main purpose was to try to apply this technology under “close to commercial” conditions. We had access to a commercial farm. The owner and his employees did the best to help us, but we indeed had a lot of limitations regarding on what we could do. As an extra and unexpected outcome, we can now say that other farmers what to try our seed treatments. We hope to continue with this field work and then to try to response the many questions we still have, including the ones you have mentioned.

Discussion: it is rather poorly organized and largely repeats the statements already presented in the results chapter. These repetitions should be omitted e.g. line 452-462; 467-474 etc.

Response. Thanks for your comments and suggestions. We have reorganized the Discussion and we hope is clearer now.

Submission Date

17 December 2020

Date of this review

28 Dec 2020 13:38:20

Round 2

Reviewer 2 Report

Looking back over the paper I see the authors have made improvements to the manuscript but there are still areas that can be enhanced. Although the Figure 7 associated with the first field experiment is better and helps visualize the fall armyworm distribution in the plots there now seems to be no statistical analysis to support the authors claims for the first field experiment – thinking about that further, given the nature of the data being counts of fall armyworms, a GLM fitted with a quasipoisson or negative binomial distribution would be appropriate between the treatments. I don’t think the sections would actually constitute a block factor to be incorporated as a random effect anymore since given that there are only 2 treatments thinking about them on the scale of the whole plot might be better. Still, it’s near impossible to draw conclusions from one field trial. 2 more successful field trials, with trial as a random effect, would be sufficient to interpret the field data. As it is now though this data is preliminary and does not seem publishable yet.

I thank the authors for clarifying that the females in their laboratory experiments were unmated – the fact that they laid fertile eggs that hatched is interesting! For Figure 6 the caption could be clearer, and perhaps should say “Representative adult virgin female having laid fertile eggs demonstrating apparent parthenogenesis.”. For lines 421 – 425 consider saying: We also observed apparent parthenogenesis, with fertile eggs that hatched viable neonates from virgin female moths that had feeding on plants treated with B. bassiana (Fig. 6), indicating important changes to S. frugiperda physiology caused by this entompathogen. It also be nice to say this was not ever observed in the control treatment and/if parthenogenesis occurred more frequently or not at all on specific fugal treatment strains. This arguably is your most interesting finding from the laboratory experiment and should actually be tested to see what percent of females on the different entomopathogenic strains exhibited parthenogenesis.

616-618: “An explanation to this could be that because there were lesser number of males, they were urged to do parthenogenesis.” This is by no means an adequate explanation and needs to be developed further. It doesn’t make much sense – if the moths for all treatments were kept isolated in individual cups, thus all individuals were virgins, no female moths would not have access to male, so even the control/negative control moths would also not know the sex ratio in the population.

682 – 704: This whole section about volatile mediated repellents given off by the entomopathogen really does not make sense since you found insects in your first field trial. Further, the untreated plants in the negative control also had no fall armyworms in the second field experiment. It’s complete conjecture and should be removed from the discussion. Volatiles are extremely important, and entomopathogens might be able to disrupt/alter plant volatile synthesis influencing insect host plant preference, but your results do not lend to that interpretation. Again, the field data in reference to differential fall armyworm abundance is too preliminary to draw any conclusions from.

Figure S5 still is rather irrelevant given the context of the study and could be removed. The two stripped beetles might actually be the same species as well (Diabrotica virgifera or, but less likely, Acalymma trivittatum/Acalymma vittatum) but its hard to tell just from the pictures.

On line 393 P = 0.000 should be P < 0.001

Author Response

We really appreciate your comments and suggestions. Thanks for the time you have invested helping us present a very much improved manuscript.

Looking back over the paper I see the authors have made improvements to the manuscript but there are still areas that can be enhanced. Although the Figure 7 associated with the first field experiment is better and helps visualize the fall armyworm distribution in the plots there now seems to be no statistical analysis to support the authors claims for the first field experiment – thinking about that further, given the nature of the data being counts of fall armyworms, a GLM fitted with a quasipoisson or negative binomial distribution would be appropriate between the treatments. I don’t think the sections would actually constitute a block factor to be incorporated as a random effect anymore since given that there are only 2 treatments thinking about them on the scale of the whole plot might be better.

Response: We have done the GLM analysis, fitted with negative binomial distribution using the fall armyworm counts in the CC and GHA furrows. We now present the results and discussion of this analysis on the text and on the legend of Figure 7.

Still, it’s near impossible to draw conclusions from one field trial. 2 more successful field trials, with trial as a random effect, would be sufficient to interpret the field data. As it is now though this data is preliminary and does not seem publishable yet.

Response: Yes, we agree that more field trials are needed therefore we have modified the Discussion section addressing this and highlighting the importance of our findings as foundations for future research. Field trials take time and even preliminary, they are worth to be published. In our research area there are plenty of solid and complete studies under laboratory conditions. Due to the urgent need for alternatives to control biotic and abiotic stressors, it is very important to test them under “real life” conditions. The more studies are published, the more people would be interested on doing field trials and hopefully, the agriculture industry would try to use this technology.

I thank the authors for clarifying that the females in their laboratory experiments were unmated – the fact that they laid fertile eggs that hatched is interesting! For Figure 6 the caption could be clearer, and perhaps should say “Representative adult virgin female having laid fertile eggs demonstrating apparent parthenogenesis.”. For lines 421 – 425 consider saying: We also observed apparent parthenogenesis, with fertile eggs that hatched viable neonates from virgin female moths that had feeding on plants treated with B. bassiana (Fig. 6), indicating important changes to S. frugiperda physiology caused by this entompathogen. It also be nice to say this was not ever observed in the control treatment and/if parthenogenesis occurred more frequently or not at all on specific fugal treatment strains. This arguably is your most interesting finding from the laboratory experiment and should actually be tested to see what percent of females on the different entomopathogenic strains exhibited parthenogenesis.

Response: Yes, we thank your enthusiastic suggestions to improve the way we explain this part of the results and their discussion. We hope that now is clearer. Indeed, we did not find this in the control treatments, but do not have complete data regarding the frequency of this “apparent parthenogenesis” in the treatments, as it was totally unexpected. We plan to analyze the effect of the treatments on several generations of FAW in future studies under laboratory conditions.

616-618: “An explanation to this could be that because there were lesser number of males, they were urged to do parthenogenesis.” This is by no means an adequate explanation and needs to be developed further. It doesn’t make much sense – if the moths for all treatments were kept isolated in individual cups, thus all individuals were virgins, no female moths would not have access to male, so even the control/negative control moths would also not know the sex ratio in the population.

Response: We have deleted this part.

682 – 704: This whole section about volatile mediated repellents given off by the entomopathogen really does not make sense since you found insects in your first field trial. Further, the untreated plants in the negative control also had no fall armyworms in the second field experiment. It’s complete conjecture and should be removed from the discussion. Volatiles are extremely important, and entomopathogens might be able to disrupt/alter plant volatile synthesis influencing insect host plant preference, but your results do not lend to that interpretation. Again, the field data in reference to differential fall armyworm abundance is too preliminary to draw any conclusions from.

Response: Yes, we agree with you comment. As we have changed the way we presented the discussion of the field trials, we have rephrased the volatiles part as a perspective for future studies, taking into account recent evidences published that indicate the importance of some volatiles released by fungi (Beauveria bassiana) or plants, that can function as insect repellents or attractants.

Figure S5 still is rather irrelevant given the context of the study and could be removed. The two stripped beetles might actually be the same species as well (Diabrotica virgifera or, but less likely, Acalymma trivittatum/Acalymma vittatum) but its hard to tell just from the pictures.

Response: We have deleted this part.

On line 393 P = 0.000 should be P < 0.001

Response: We have corrected this part.

Reviewer 3 Report

Since in the revised version the authors have applied the vast majority of my comments made in the previous review, I conclude that the paper can be published in the current version.

Author Response

We really appreciate your comments and suggestions. They have helped us to present a very much improved manuscript. 

Round 3

Reviewer 2 Report

The phrasing of the GLM with the negative binomial in the manuscript is incorrect, "Negative binomial distribution analysis" is said. Also that analysis seems not to be conducted correctly. Normally an F value of 1 would mean your p value is not significant?  

I understand field trials take time, but doing experiments multiple times in the field is to help with the lack of reproducibility we see across science as a whole. Research studies are also great and can be useful for teasing apart confounding effects you might not be able to control for in the field. Given how this paper is set up it could easily be two, one for the field (once you have more data) and another with the lab results if you are adamant about publishing it. I also agree due to the urgent need for alternatives to control insect pests it is important to test these methods in the real world, but one field trial is still not adequate. It really needs to be three given this scale, bare minimum two trials with the same trends. Publishing unfinished and incomplete work, that even you had trouble replicating, is not acceptable, especially if you want it to be compelling for adoption by producers and industry. 

The rephrased section on the volatiles in the discussion can still be better stated. You still use the findings (lack there of from field trial 2) to justify the importance of volatiles produced by B. bassiana as an insect repellent. You need to clearly differentiate it. Something like: Others studies have shown that volatiles produced by B. bassiana may act as an insect repellent. Although our field experiment can not corroborate this finding, with . Including the insect abundance field work at this time is rather presumptuous.

Author Response

We want to thank the reviewer's comments and, more importantly, his/her suggestions. We have taken a more detailed view of our results, based on the GLM analysis suggested and on what it is presented in Table 1. Also we have changed several parts of the discussion with the hope to address his/her concerns.

The phrasing of the GLM with the negative binomial in the manuscript is incorrect, "Negative binomial distribution analysis" is said. Also that analysis seems not to be conducted correctly. Normally an F value of 1 would mean your p value is not significant?

We apologize for no explaining better the analysis done in the previous version. That part was not edited properly before submission. In the current version, we have explained with more detail the analyses done. They are in materials and methods, results and the legend of Fig. 7. In Materials and methods: "To interpret the putative changes in the frequency of S. frugiperda larva found in the first field trial, analysis with a Generalized Linear Model (GLM), fitted with negative binomial distribution, was used. Pearson chi-square value > 0.05 indicated goodness of fit of the data. Omnibus test with P < 0.05 indicated significant differences therefore the results of the Test of Model effects Type III were reported" . In Results and in the legend of Fig. 7: "The analysis GLM-fitted with negative binomial distribution showed Pearson chi-square value of 0.818 indicating goodness of fit of the data. The Omnibus test value of 0.0001 indicated a statistically significant model. The Type III Test of Model effects showed P=0.013 for Treatments (CC/GHA) and P=0.0001 for Furrows indicating significant differences on larvae distribution". We thank the reviewer for the suggestion to use this method.

I understand field trials take time, but doing experiments multiple times in the field is to help with the lack of reproducibility we see across science as a whole. Research studies are also great and can be useful for teasing apart confounding effects you might not be able to control for in the field. Given how this paper is set up it could easily be two, one for the field (once you have more data) and another with the lab results if you are adamant about publishing it. I also agree due to the urgent need for alternatives to control insect pests it is important to test these methods in the real world, but one field trial is still not adequate. It really needs to be three given this scale, bare minimum two trials with the same trends. Publishing unfinished and incomplete work, that even you had trouble replicating, is not acceptable, especially if you want it to be compelling for adoption by producers and industry.

We hope to have taken into account the reviewer's comments. We have specified that these are preliminary results in the field. We also have added several paragraphs indicating that these preliminary studies are a follow up of what we observed in the laboratory experiments. Mainly on the putative avoidance observed in the larvae fed on B. bassiana GHA strain-treated plants (MCGHA in Table 1). We respectfully ask the reviewer to consider these preliminary results to be publishable based on the reasoning we presented. We have also emphasized in the discussion that more repetitions of these two field trials are needed. 

The rephrased section on the volatiles in the discussion can still be better stated. You still use the findings (lack there of from field trial 2) to justify the importance of volatiles produced by B. bassiana as an insect repellent. You need to clearly differentiate it. Something like: Others studies have shown that volatiles produced by B. bassiana may act as an insect repellent. Although our field experiment can not corroborate this finding, with . Including the insect abundance field work at this time is rather presumptuous.

Yes, we agree. We modified the section on the volatiles in the discussion to only be presented as topics of analyses for future studies.

Round 4

Reviewer 2 Report

Thank you for addressing my comments. I have looked at the GLM for the Spodoptera abundance field data provided and still have concerns. It seems in the process it was still analyzed wrong. From Figure 7 and the supplemental data I reproduced this data frame to try and figure out where the discrepancy was. Furrow appears to be placed as a covariate linearly over the whole of the two treatments (CC starts at 1 with GHA ending at the last furrow 58 – the 5 furrow space in-between the treatments does not seem to be taken into account either), rather than within treatments (CC is between 1 and 25 and GHA is 1 to 33). The former way would not reproduce a sound model or experimental design since treatments are not randomized by furrow (low numbered furrows are only represented in CC while high numbered furrows are only in GHA; more CC and GHA plots would be needed ideally in a matrix to do this). I conducted some analyses of this data frame in R and SPSS as well, and the analysis which I believe you are presenting in the manuscript while having significant results also estimates the mean/coefficient of the GHA treatment significantly higher than the CC treatment (the model significantly predicts more caterpillars on GHA than CC).

From a methodological perspective too you say that this is indicative of a difference of distribution for the worms in the treatments, but have collapsed the analysis of space into only one dimension rather than keep 2 dimensions, and while the latter is harder to analyze it be better since the way the experiment was conducted the wild moths had access over the width and length of the treatment plots.    

Given all of that, furrow should not really be incorporated in the analysis. The data was not really collected to incorporate differences in spatial distribution within/between treatment plots and not analyzed to interpret it that way either.

As an aside, going back through the data I was able to reproduce and reanalyzing it the best I could get for treatment was a nonsignificant or trending effect on caterpillar abundance.  

Author Response

We thank the reviewer for his/her generously given feedback. Your comments help us to prepare better our next field trials, when we finally can go back after COVID. For the data presented in this manuscript, we did the GLM in SPSS again, comparing only the treatments (CC/GHA). And yes, the results indicated that there is a non-significant difference thus we modified the text accordingly and hope to have improved it.