Field Efficacy of Proteolytic Entomopathogenic Fungi against Ceratovacuna lanigera Zehntner
Round 1
Reviewer 1 Report
The research reported in this manuscript is very interesting and it was presented very clearly. I appreciated the work of the authors.
Few question observation follows:
Figure 1: picture A. I suppose that the white spots in the plates are due to skim milk powder not completely suspended. If you had a best picture, you could replace the one reported in the manuscript.
Paragraph 3.4. Why did you use Rhizoctonia solani as outgroup? Why did you not consider B. bassiana or another Ascomycetes?
Figures from 5 to 10. The name of abscissa axis is "duration of the spray" but it is not correct because I understand that the duration of the observation is represented. Please, rephrase.
Discussion. About increase of leaf size, brix and plant weight, did you consider a putative biostimulant effect? I suggest to add one comment about that in the discussion.
Author Response
Reviewer comments
The research reported in this manuscript is very interesting and it was presented very clearly. I appreciated the work of the authors.
Response: Thank you very much for your insightful observation, valuable suggestions and appreciated our works.
Few question observation follows:
Point: Figure 1: picture A. I suppose that the white spots in the plates are due to skim milk powder not completely suspended. If you had a best picture, you could replace the one reported in the manuscript.
Response: Thank you for your observation. According to your suggestion, Figure 1: picture A has been replaced.
Point: Paragraph 3.4. Why did you use Rhizoctonia solani as outgroup? Why did you not consider B. bassiana or another Ascomycetes?
Response: Thank you for your observation. According to your suggestion, Ascomycetes fungus, Metarhizium anisopliae has been used as the outgroup in the phylogenetic tree. (New paragraph: 3.5, Figure 3)
Point: Figures from 5 to 10. The name of abscissa axis is "duration of the spray" but it is not correct because I understand that the duration of the observation is represented. Please, rephrase.
Response: According to your valuable suggestion, the name of abscissa axis "duration of the spray" has been revised as “days after first spray, days after second spray, days after third spray and days after fourth spray”. (New number of Figures: 4 to 7).
Point: Discussion. About increase of leaf size, brix and plant weight, did you consider a putative biostimulant effect? I suggest adding one comment about that in the discussion.
Response: According to your valuable suggestion, comment about biostimulant effect of entomopathogenic fungi on plant growth development has been added in discussion section (Reference number: 74,75).
Reviewer 2 Report
Comments for author File: 
Comments.pdf
Author Response
Reviewer Comments
This study provides the field efficacy of protease-producing Entomopathogenic fungi (EPFs) against Ceratovacuna lanigera and their persistence effect on sugarcane leaves. Ten protease-producing fungi from different agricultural soils were identified, and the proteolytic activity was assessed. One P. lilacinum PLTP5 exhibited highest reduction on the C. lanigera population and highest increase of sugarcane biomass, which might be an eco-friendly alternative to chemical pesticide for the management of C. lanigera. This work is important and will assist pest control of aphid, if the functional role of P. lilacinum was further explored, the work will be much more intact.
Major Revision
- Point: The writing of the manuscript is redundancy, especially in the Introduction and Results. Like in the Introduction, several paragraphs were popular scientific knowledge and non-essential, such as paragraph 1-3, which can be deleted. The purpose of introduction is to introduce the key issue and the recent progress of the research field, then followed the problems this study can solve. So the introduction should be revised to be more concise.
Response: Thank you very much for your profound observations, valuable suggestions and comments to improve our manuscript. We greatly appreciate your comments and suggestions. According to your valuable suggestion, paragraphs 1-3 of introduction have been deleted, and introduction has been revised.
Point: The same problems were also found in results, such as the first paragraph of 3.1, the method had be described in details in the materials and method, this paragraph should also be deleted.
Response: According to your suggestion, this portion has been deleted.
Point: In result 3.5, the paragraphs of ‘Effect of the first … the fourth spray’ were listing data instead of summarizing the results, it should be more accurate and concise.
Response: According to your suggestion, this portion has been revised and summarized. (The revised paragraphs have been shown in yellow marking portions in result 3.7 ‘Effect of the first … the fourth spray’)
Point: In result ‘3.7 Impact of EPFs Application on Yield Component and Sugar Content’, too many subtitles were used. The result of 3.7 should also be summarized and rewrote.
Response: According to your suggestion, subtitles of ‘Impact of EPFs Application on Yield Component and Sugar Content’ have been deleted and the results have been summarized. (The summarized results have been shown in yellow marking portions in result 3.9, ‘Impact of EPFs Application on Yield Component and Sugar Content’ )
- Point: The authors focus on the proteinase of the isolates, how about other characteristics of the PLTP5, such as growth rate, spore production, or chitinase activity ?
Response: Thank you so much for your constructive comments. According to your valuable comments, growth rate and spore production characteristics of the isolates have been included in results section 3.2. (Yellow marking portions of result 3.2). However, the methodology for the determination of colony growth rate and spore density of the isolates has been included in methodology section 2.4 (yellow marking portions)
According to the previous studies (Reference number 4, 5, 6, 7), the insect cuticle is made up of up to 70% protein. The protease is responsible for breaking down peptide bonds in proteins and peptides in the cuticle of insects During penetration and the first stage of cuticle degradation, a large amount of proteases are secreted, thus facilitating adhesion of the spores in the cuticles, modify the surface of the host's cuticle and degrade cuticle proteins. Furthermore, the virulence of EPFs is also increased by over activity of the protease enzyme. As protease plays major role for insect cuticle degradation, we considered to characterize protease activity in our research work. However, we appreciate your comments on chitinase activity and we will include to characterize chitinase activity in our future research.
- Point: PLTP5 showed the highest reduction in the C. lanigera population on field condition, but how about its virulence against C. lanigera on laboratorial condition? What’s the LC50 or LT50 of PLTP5 against aphid?
Response: Thank you so much for your nice comments. According your comments, lethal concentrations (LC50 and LC90) and lethal times (LT50 and LT90) values for the isolates against C. lanigera adults have been determined using the data of virulence evaluation bioassay in laboratory conditions. The lethal concentrations (LC50 and LC90) and lethal times (LT50 and LT90) values have been included in Table 4 and Table 5 in results section 3.6 (yellow marking portions and Table 4 and Table 5). However, the methodology for the determination of LC and LT values has been included in methodology section 2.7 (yellow marking portions)
- Point: In the conclusion, the authors said that the efficacy of entomopathogenic fungi is related to the proteolytic activity, but in the result, the proteolytic activity or gene expression level of PLTP were not measured after spray on the leaves of sugarcane. What roles of PLTP5 played on the C. lanigera population? Whether the expression level of fungal pathogenic genes, such as proteinase, chitinase increased after spray?
Response: Thank you for your valuable comments. Among the isolates, PLTP5 showed the highest proteolytic activity (Table 1). This isolate also showed the highest virulence efficacy both in Laboratory (Table 4 and 5) and in field conditions (Figure 4, 5, 6, 7), although we applied same concentration of conidia for all the isolates. Considering the results, we can say that protease is an important factor for the efficacy of entomopathogenic fungi. However, the conclusion has been revised.
Role of PLTP5/entomopathogenic fungi on insect host:
When the fungal spores come in contact with their insect host, they germinate by producing a germ tube. An appressorium, an infective structure of the fungus, is formed at the end of the germ tube, exerting mechanical pressure and producing cuticle-degrading enzymes that help the fungus enter the body of the insect host. Once inside the host, the fungus multiplies, invades the host tissues, emerges from the host, and produces more spores to continue the infection cycle. After the application of EPF isolate PLTP5, the population of C. lanigera was reduced due to the existence of viable conidia on the leaves, since we evaluated the persistence of the conidia on the leaves (Figure 8).
Minor revision
- Point: The title ‘Field Efficacy and Persistence Study on Leaves of Proteolytic Entomopathogenic Fungi for the Control of Ceratovacuna lanigera Zehntner’ was confusing. ‘Field Efficacy of Proteolytic Entomopathogenic Fungi against Ceratovacuna lanigera Zehntner’ might be better.
Response: Thank you for your constructive suggestions. According to your suggestion, the title has been revised to ‘Field Efficacy of Proteolytic Entomopathogenic Fungi against Ceratovacuna lanigera Zehntner’
- Point: There are too many figures, Figure 5, 7, 9, 11,13,15,17, 19, 21, 23 and 25 were not necessary.
Response: According to your suggestion, Figures 5, 7, 9, 11,13,15,17, 19, 21, 23 and 25 have been deleted.
- Point: The figure legends were not intact. The scale bar was missing in Fig. 2. The meanings of a, b, c, d were not provided in the legends of Fig 3-18. The font size of figures was different.
Response: Thank you for your observations. Figure 2 (C and D) has been replaced. The meanings of a, b, c, d have been provided in the legends of Figure 4-14 (New figure number) and similar font size has been used for all the figures.
- Point: The Results lack a sense of hierarchy. I would suggest the authors use appropriate conjunctions, and relative adverbs and phrases to establish the logical relationship between adjacent paragraphs.
Response: Thanks. According to your suggestion, the results sections have been revised.
- Point: There are several recent works on the bio-control fungus P. lilacinum against insect, which should be cited in the background.
Response: According to your valuable suggestion, several recent works on the bio-control fungus, P. lilacinum against insect have been cited in the background (References number: 25, 26 and 27).
- Point: The English writing should be edited by a native English scientist, some sentences were confusing
Response: According to your suggestion, English writing has been edited.
Thank you very much for your overall valuable comments and suggestions which helped us to improve the manuscript. All the best.
Round 2
Reviewer 2 Report
The authors have dealt with most of my previous comments, and there are just a few minor things that I think could still improve the manuscript.
- The description of Materials and method is still too detailed, such as 2.2, there is no need to list the composition of Skimmed Milk Agar (SMA) medium unless there are changes in reference 29. Concise language would be better.
-In 2.14, Microsoft Office Excel 2007 was not a reasonable method for regression analysis.
-There are some issues with grammar, although I think this is really not a major concern.
Author Response
Reviewer -2 (Comments and Suggestions for Authors):
The authors have dealt with most of my previous comments, and there are just a few minor things that I think could still improve the manuscript.
Response: Thank you very much for your insightful observations, valuable suggestions and comments to improve our manuscript.
Point: The description of Materials and method is still too detailed, such as 2.2, there is no need to list the composition of Skimmed Milk Agar (SMA) medium unless there are changes in reference 29. Concise language would be better.
Response: Thank you. According to valuable suggestion, the description of Materials and Method section: 2.1, 2.2 (has been deleted the list of composition of Skimmed Milk Agar), 2.11, 2.12.1, 2.13 (yellow marking sections) and also the overall descriptions of Materials and Methods have been revised and summarized.
Point: In 2.14, Microsoft Office Excel 2007 was not a reasonable method for regression analysis.
Response: Thank you so much for your comments. Microsoft Office Excel 2007 was used for regression analysis according to the previous study (Reference No: 49) which has been cited in the Materials and Methods section.
- 49. Mukherjee, A., Debnath, P., Ghosh, S. K., & Medda, P. K. Biological control of papaya aphid (Aphis gossypii Glover) using entomopathogenic fungi. Vegetos, 2020, 33, 1-10.
Point: There are some issues with grammar, although I think this is really not a major concern.
Response: According to your suggestion, grammar has been edited.
Thank you very much for your overall valuable comments and suggestions which helped us to improve the manuscript. All the best.
