Potential Use of Fusarium Isolates as Biological Control Agents: Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) Case Study
, Foteini Kitsiou 2, Ioannis Lagogiannis 3 and Panagiotis A. Eliopoulos 4,*
Round 1
Reviewer 1 Report
Dear authors,
congratulations on a very nice paper and extremely interesting results. I read it carefully and made some notes for improvement directly into the manuscript. Some parts are not easy to understand so please consider rewriting them to become easier to understand for readers.
Looking forward to seeing this manuscript published.
Best wishes,
Comments for author File: 
Comments.pdf
Author Response
All changes suggested by the reviewer in its pdf file have been made. All changes have been yellow marked in the final submitted MS.
Reviewer 2 Report
Aside a few confusing statements and wrong grammatical expressions here and there in the manuscript, I assume the research conducted is of good quality, and merit acceptance for publication in the journal.
Please, find below some of the minor alterations that are needed to be made…
Is there any need for the question mark in the title? Perhaps, the title could be better expressed.
Might consider revising as – Potential use of Fusarium isolates as biological control agents: Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) case study.
L15 – Revise as - in field experiments
L49-50 – Delete the article ‘the’ before Fusarium
L22 – against various classes of insect pests [20-23]
L56 – have not been
L60-61 - … is this statement correct? Perhaps, the first field study?
L107 – Please provide company name and country of production for - Taq 2X Master Mix (M0270)
L114 - Was bidirectional sequencing carried out for the purified PCR products? As I am missing the information under 2.1.3, aside appearing on the sub-head.
L117-118 – Revise the statement as – Insect culture was reared in the laboratory on artificial diet (Table 1).
L118 – Add a ‘,’ after – ‘In the beginning’
L119 - …stage 3 vitamins were boiled in distilled water, using a sterilizer? or a microwave oven? What are the thermo-conditions and duration?
L131-132 – The following statement – ‘The field experiment performed the seasons 2019-2020 and 2020-2021 on a biological tomato field in Kourtesi.’ – is not grammatically correct. Perhaps, missing one or two articles.
L143 - H. armigera should be in italic
L148 – What is the meaning of this statement? – ‘without stress the used pathogens.’
L149 – Add a ‘,’ after – ‘sequence’
L153 – Another confusing statement here – ‘opening to so that.’
L154 – Here is another one – “hole was meticulously screwed with nylon mesh”. Please, amend all of these grammatical errors and poor expressions in the entire manuscript as to improve readability. Many a times, using simple and easy to understand sentences is best. For instance, here in L154, I assume the authors intended statement was that, hole was made, and then covered with nylon mesh?
L283-286 – These statement is rather too long. Might consider breaking down into 2 or 3 simpler refined shorter sentences.
L307 – Delete the year [2020] after ‘da Silva Santos et al.’ and add ‘,’ after [53]
L314-315 – Revise as – ‘and 61%, respectively, at high conidial concentrations.
L327 – Delete the year ‘2001’ in ‘Ganassi et al. in 2001 [62]’. Ensure that this error is corrected all over the manuscript.
L345 – Revise ‘4th’ as ‘4th'
Author Response
Might consider revising as – Potential use of Fusarium isolates as biological control agents: Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) case study.
Reply: The title has been changed according to reviewer 1 and 2 suggestions.
L15 – Revise as - in field experiments
Reply: Appropriate correction has been made (L15-16)
L49-50 – Delete the article ‘the’ before Fusarium
Reply: Done (L48-49)
L22 – against various classes of insect pests [20-23]
Reply: Appropriate correction has been made (L53-54)
L56 – have not been
Reply: Corrected (L57)
L60-61 - … is this statement correct? Perhaps, the first field study?
Reply: The sentence has been removed (L62)
L107 – Please provide company name and country of production for - Taq 2X Master Mix (M0270)
Reply: We added them (L108)
L114 - Was bidirectional sequencing carried out for the purified PCR products? As I am missing the information under 2.1.3, aside appearing on the sub-head.
Reply: No we did not carry out bidirectional sequencing to the PCR product
L117-118 – Revise the statement as – Insect culture was reared in the laboratory on artificial diet (Table 1).
Reply: Done (L118)
L118 – Add a ‘,’ after – ‘In the beginning’
Reply: Done (L118)
L119 - …stage 3 vitamins were boiled in distilled water, using a sterilizer? or a microwave oven? What are the thermo-conditions and duration?
Reply: We added them (L119-120)
L131-132 – The following statement – ‘The field experiment performed the seasons 2019-2020 and 2020-2021 on a biological tomato field in Kourtesi.’ – is not grammatically correct. Perhaps, missing one or two articles.
Reply: Appropriate correction has been made (L133)
L143 - H. armigera should be in italic
Reply: Done (L145)
L148 – What is the meaning of this statement? – ‘without stress the used pathogens.’
Reply: It has been corrected. We add “and took place from 4:00 to 8:00 am so no additional environmental stress was induced on the tested pathogens.”
L149 – Add a ‘,’ after – ‘sequence’
Reply: Done (L150)
L153 – Another confusing statement here – ‘opening to so that.’
Reply: We rephrased it (L154)
L154 – Here is another one – “hole was meticulously screwed with nylon mesh”. Please, amend all of these grammatical errors and poor expressions in the entire manuscript as to improve readability. Many a times, using simple and easy to understand sentences is best. For instance, here in L154, I assume the authors intended statement was that, hole was made, and then covered with nylon mesh?
Reply: We rephrased it (L157-163)
L283-286 – These statement is rather too long. Might consider breaking down into 2 or 3 simpler refined shorter sentences.
Reply: Corrected (L286-289)
L307 – Delete the year [2020] after ‘da Silva Santos et al.’ and add ‘,’ after [53]
Reply: Done (L311)
L314-315 – Revise as – ‘and 61%, respectively, at high conidial concentrations.
Reply: Appropriate correction has been made (L318)
L327 – Delete the year ‘2001’ in ‘Ganassi et al. in 2001 [62]’. Ensure that this error is corrected all over the manuscript.
Reply: Appropriate correction has been made (L331)
L345 – Revise ‘4th’ as ‘4th'
Reply: Appropriate correction has been made (L349)
Reviewer 3 Report
Authors mentioned isolates in title while different species of Fusarium are used in this study. Authors also provided methodology about the isolation of fungi directly from soil as well as from insect baiting, however no data and comparison of both methods are provided in results. Mortality are measured but did not provide the formula. I suggest Abbott formula for Mortality check. in vitro data should also incorporate to compare its efficiency under field conditions. Conclusion should also cover the pathogenic aspects of Fusarium as it is well known plant pathogen of different crops throughout the world.
I would also like to mentioned the following queries:
Line 104, 105: ITS 4 and ITS5 were used instead of Universal Primers set of ITS1 and ITS4. Should include the reference in which ITS 4 and ITS 5 are used for internal transcribed spacer region.
Line 133: any reference of these conidial concentrations?? Should include reference
Line 162,163: Visual verification did not confirm the pathogen. Also Fusarium oxysporum growth is mostly white while author mentioned that visually black spots were observed. It is necessary to reisolate for the confirmation of Pathogenicity.
Author Response
Authors mentioned isolates in title while different species of Fusarium are used in this study. Authors also provided methodology about the isolation of fungi directly from soil as well as from insect baiting, however no data and comparison of both methods are provided in results. Mortality are measured but did not provide the formula. I suggest Abbott formula for Mortality check. in vitro data should also incorporate to compare its efficiency under field conditions. Conclusion should also cover the pathogenic aspects of Fusarium as it is well known plant pathogen of different crops throughout the world.
Reply: Dear Reviewer we did not test the two methods for isolating fungal strains. Our aim was only to collect microorganisms. This is the reason why we did not compare the two methods. We didn’t use the Abbott formula because the control mortality was < 5%. As it is mentioned in most mortality protocols (e.g. WHO) if the control mortality is <5%, no correction of test results is necessary.
Line 104, 105: ITS 4 and ITS5 were used instead of Universal Primers set of ITS1 and ITS4. Should include the reference in which ITS 4 and ITS 5 are used for internal transcribed spacer region.
Reply: Indeed, ITS1 and Its4 are the Universal primers. ITS4 and ITS5 were chosen as after some preliminary tests we made these primers gave us much more satisfactory results.
Line 133: any reference of these conidial concentrations?? Should include reference
Reply: The doses 106 - 108 conidia/ml are the most common concentrations used when experimenting with fungal conidial solutions. This is the reason for choosing these doses to experiment with. There is no reference that reports this necessity, It is just the standard procedure.
Line 162,163: Visual verification did not confirm the pathogen. Also Fusarium oxysporum growth is mostly white while author mentioned that visually black spots were observed. It is necessary to reisolate for the confirmation of Pathogenicity.
Reply: Appropriate correction has been made (L169-170). We add “Existence of the pathogen was verified by the appearance of mycelium on the cadavers or with black spots on the dead insect body if the mycelium was not present.”
Reviewer 4 Report
Ser the MS file.
Comments for author File: Comments.pdf
Author Response
All changes suggested by the reviewer in its pdf file have been made. All changes have been yellow marked in the final submitted MS.
Round 2
Reviewer 3 Report
Previously, I suggested to re-isolate the Fusarium from treated larvae to confirm. However, I am unable to find modification accordingly. Similarly no results of in vitro results were found in Results portion.
Author Response
Previously, I suggested to re-isolate the Fusarium from treated larvae to confirm. However, I am unable to find modification accordingly. Similarly no results of in vitro results were found in Results portion.
Reply : We are sorry if the meaning was not clear. We followed the standard procedure that we have carried out for many similar studies that we have publishes. We examined all dead larvae and re-isolate fungal pathogen from those with mycelium or with spots. Then we obtained pure culture on SDA and identify it as Fusarium. As expected, all infections have been caused by our Fusarium strains. No other data was recorded. Explanatory sentences have been added (lines 167-171).
