Efficacy of Entomopathogenic Trichoderma Isolates against Sugarcane Woolly Aphid, Ceratovacuna lanigera Zehntner (Hemiptera: Aphididae)

Round 1

Reviewer 1 Report

L15 (and elsewhere) - use the multiplication symbol instead. 

L22 - it is not application duration, rather exposure time i.e. the length of time to which the insects were exposed to the fungi post-application.

L24-25 - was this signficant? Would be good to include some statistics in the abstract.

L30 - consider keywords not already in the title

L34 - appropriate to indicate the insect order and family in brackets after first mention of the scientific name.

L51 - spelling error 'greenhouses'

L71 - used against?

L76 - spelling error 'chickpea'

L77 - Do you  mean Spanish vineyards. The whole sentence was a bit confusing to follow. Editing should help with this though. 

L80 - EMP? If you are using the abbreviation it should be EPF, and the word fungi will then be removed. 

General intro comment: Why specifically Trichoderma species and not other EPFs as well. Needs to be better motivated. Is there an option to use this as a dual biocontrol agent for example against plant pathogens and pests?

L92 - remove 'based' or complete the sentence i.e. based on what?

L100 - mealworm not Mealworm.

L108 - no need to restate order and family here. Already done at first mention.

L112 - Why pre-heat. Typically only necessary to prevent excessive webbing when using wax moth larvae as the bait species. 

L126-128 - Please clarify. Unclear how you can get a single CFU directly from a larva.

L151 - Just internal transcribed spacer region? Which is amplified using ITS 1 and ITS4. 

L192 - Can you provide a brief explanation of this method, or at the very least identify the modifications thereof?

L207 & 212 - insert a space before the unit measurement (i.e. 50 cm etc.)

L217 - cotton wool? Otherwise cotton what?

L223- I don't understand this sentence. Do you mean 50 adults per plant material used? Which means you will have 150 nymphs or adults per treatment, since there were three replications? 

L229 - use the word 'at', not the symbol '@'. And do you mean 1 ml on each insect or 1 ml on each plant? The use of a micropipette would suggest each insect, but that would surely be too much inoculum. Please clarify this application process. 

Materials and methods - why not consider calculating LC values (and possibly even LT values) instead? Or in addition to the data you have already presented?

L245 - Why these seven particularly? Why were the other 19 excluded? Please clarify.

L268 - Indicate the table in brackets at the end of the sentence. Example ...regions of rDNA (Table 1). Remove the last sentence then. 

L277-315 - you need references here to show the supporting literature you used to make these identifications. 

L390 (Fig. 4) - What do the error bars represent, and if possible, the species names should be in italics. 

L411 (Fig. 5) - Can you indicate significant differences in the graphs themselves? What do the error bars represent? The species names should be in italics. 

L418 - check abbreviation, should be EPF, as first stated. 

L432 - 442 - remove discussion on the use of mealworms as a bait species. It adds no value to the implication of your results and can be used as a motivation for your choice in the methods section instead. 

L432 - 442 - do not repeat the results here. Discuss what this means in terms of their implication i.e. how will they be useful, moving forward, for the management of this pest? Where to from here based on what you know now and compared to that of other literature? I would like to see more of this in the discussion. 

L453 - sp. not Spp.

General comment - sometimes % is preceded by a space, other times not. Keep it consistent. 

 

All the best with your publication and research moving forward! 

Author Response

Reviewer 1. in MS reply or revising MS using Yellow collar Point: L15 (and elsewhere) - use the multiplication symbol instead. Response: Thank you very much for your profound observation to improve our manuscript. According to your suggestion, the multiplication symbol has been used. Point: L22 - it is not application duration, rather exposure time i.e. the length of time to which the insects were exposed to the fungi post-application. Response: According to your valuable suggestion, this portion has been revised as “exposure time”. (New line: 24). Point: L24-25 - was this significant? Would be good to include some statistics in the abstract. Response: Significantly higher mortality (both nymphs and adults) was found for T. harzianum (TMS623) compared to other isolates. However, according to your suggestion, mortality data of nymphs for 72 hours and adults for 6 days has been included (New line: 26-35). Point: L30 - consider keywords not already in the title- Response: According to your suggestion, keywords have been revised (New line: 42). Point: L34 - appropriate to indicate the insect order and family in brackets after first mention of the scientific name. Response: Insect order and family have been included in brackets (New line: 47) Point: L51 - spelling error greenhouses Response: Spelling of ‘greenhouse' has been revised (New line: 64) Point: L71 - used against? Response: This portion have been included (New line: 87-92) Point: L76 - spelling error 'chickpea' Response: Spelling of 'chickpea' has been revised (New line: 100) Point: L77 - Do you mean Spanish vineyards. The whole sentence was a bit confusing to follow. Editing should help with this though. Response: Yes, this was a spelling mistake, and we apologize for it. This will be Spanish vineyards. According to your suggestion, this portion has been revised (New line: 94-103). Point: L80 - EMP? If you are using the abbreviation it should be EPF, and the word fungi will then be removed. Response: According to your suggestion, this portion has been revised (New line: 105). Point: General intro comment: Why specifically Trichoderma species and no other EPFs as well. Needs to be better motivated. Is there an option to use this as a dual biocontrol agent for example against plant pathogens and pests? Response: Thanks for your insightful observation, valuable suggestions and comments. We greatly appreciate your comments and suggestions. For your kind information, the objectives of our research project were to find entomopathogenic Trichoderma isolates and to determine efficacy against sugarcane woolly aphids. Because, Trichoderm species can be found all types of soil, agricultural lands, manure, decaying tissue, forest, salt marsh, desert soils and other form of plant organic matter throughout all climatic zones. It is easy to culture and grow very fast and it effective against various insect pests (according to previous research studies). According to the reference (27), Trichoderma was also found as a biocontrol agent against plant pathogens- as an inhibitor of Phytophthora parasitica, Rhizoctonia solani , Rhizopus spp. Sclerotium rolfsii and Pythium spp. But in our current studies, we did not conduct the experiment as a dual biocontrol agent. Based on your suggestions, we will again apply for funding to implement research as a dual biocontrol agent against plant pathogens and pests. Point: L92 - remove 'based' or complete the sentence i.e. based on what? Response: This portion has been revised (New line: 121-122). Point: L100 - mealworm not Mealworm. Response: This portion has been revised (New line: 128). Point: L108 - no need to restate order and family here. Already done at first mention. Response: This portion has been revised (New line: 140). Point: L112 - Why pre-heat. Typically, only necessary to prevent excessive webbing when using wax moth larvae as the bait species. Response: Thanks for your nice comments. Pre-heating was done with warm water to kill the larvae. Because if the larvae are alive, they move across the top of the soil and sometimes stay close to the wall of the box. So that it cannot come into contact with the maximum number of fungal spores in the soil. (New line: 140). Point: L126-128 - Please clarify. Unclear how you can get a single CFU directly from a larva. Response: Thanks for your observation. This portion needs a little bit revision, and we apologize for that. Infected larvae were cut into small pieces (5-7 pieces) and each small piece of larva was placed in each petri dish containing SDAY medium. The petri dish was incubated at 28°C and a humidity of 70%. After 3 days of incubation, fungal colonies were appeared, and then transferred into SDAY medium for fungal culture. After that, pure cultures were maintained and stored at 40C for further uses. However, this portion has been revised (New line: 151-157). Point: L151 - Just internal transcribed spacer region? Which is amplified using ITS 1 and ITS4. Response: Thank you for your comments. ITS1 and ITS4 marker amplified a partial region of 18 S, full length of ITS 1 region, 5.8 S, ITS 2 regions and partial region of 28 S. However, this portion has been revised (New line: 182, 186, 193, 209, 322, 386, 390, 395, 397, 400, 408, 417). Point: L192 - Can you provide a brief explanation of this method, or at the very least identify the modifications there of? Response: The brief description has been provided (New line: 214-231) Point: L207 & 212 - insert a space before the unit measurement (i.e. 50 cm etc.) Response: This portions have been revised (New line: 252, 257, 260). Point: L217 - cotton wool? Otherwise cotton what? Response: Thank you. It was cotton. (New line:262) Point: L223- I don't understand this sentence. Do you mean 50 adults per plant material used? Which means you will have 150 nymphs or adults per treatment, since there were three replications? Response: 50 nymphs per leaf were used and placed in a single bioassay chamber for each treatment. Three replications were performed for each treatment in three separate chambers. That means, for three replications per treatment, a total of 150 nymphs were used. For adults, the same procedure was followed in separate chambers. Not nymphs and adults in same chamber. However, this portion has been revised (New line: 268-270). Point: L229 - use the word 'at', not the symbol. And do you mean 1 ml on each insect or 1 ml on each plant? The use of a micropipette would suggest each insect, but that would surely be too much inoculum. Please clarify this application process. Response: One mL of conidia suspension was applied per leaf containing 50 nymphs/50 adults. That means, 1 mL for 50 nymphs and 1 mL for 50 adults, separately. This portion has been clarified (New line: 277-278). Point: Materials and methods - why not consider calculating LC values (and possibly even LT values) instead? Or in addition to the data you have already presented? Response: Thank you. According to your valuable comments, LC and LT values of the isolates against nymphs and adults have been calculated using the data already presented in this MS. However, LC and LT values have been included in the MS. (New line: 293-300 and 480-517). Point: L245 - Why these seven particularly? Why were the other 19 excluded? Please clarify. Response: Thank you for your query. Based on the efficacy test, only 7 isolates showed effectiveness against sugarcane woolly aphids. Another 19 isolates did not show effectiveness. As 19 isolates (not Trichoderma species) did not show effectiveness, we did not identify these isolates. Therefore 19 isolates were excluded. - (New line: 316-317). Point: L268 - Indicate the table in brackets at the end of the sentence. Example ...regions of rDNA (Table 1). Remove the last sentence then. Response: According to your suggestion, this portion has been revised (New line:321). Point: L277-315 - you need references here to show the supporting literature you used to make these identifications. Response: Thank you for your suggestion. References (29, 34, 35 56) have been used in the Discussion section. (New Line: 529-534). Point: L390 (Fig. 4) - What do the error bars represent, and if possible, the species names should be in italics. Response: Thank you for your query. We used standard error mean (SEM) as error bar in all bar diagram (Figure 4 and Figure 5). In Figures 4 and 5, the species name has been revised as Italic. (New Line: 448 and 472) Point: L411 (Fig. 5) - Can you indicate significant differences in the graphs themselves? What do the error bars represent? The species names should be in italics. Response: Thank you for your suggestion. The significant differences in the graphs has been indicated (both in Fig. 4 and Fig. 5). the species name has been revised in Italics. (Both in figures 4 and 5). (New line: 448 and 472). Point: L418 - check abbreviation, should be EPF, as first stated. Response: This portion has been revised (New line: 522). Point: L432 - 442 - remove discussion on the use of mealworms as a bait species. It adds no value to the implication of your results and can be used as a motivation for your choice in the methods section instead. Response: According to your suggestion, this portion has been deleted. Point: L432 - 442 - do not repeat the results here. Discuss what this means in terms of their implication i.e. how will they be useful, moving forward, for the management of this pest? Where to from here based on what you know now and compared to that of other literature? I would like to see more of this in the discussion. Response: Thanks for your observation. According to your suggestions, this portion has been revised and improved. (New line: 571-576 and 585-605) Point: L453 - sp. not Spp. Response: This portion has been revised. (New line: 556) Point: General comment - sometimes % is preceded by a space, other times not. Keep it consistent. Response: Thank you. For using %, similar procedure has been followed in all cases. Thank you very much for your overall valuable comments and suggestions which helped us tremendously to improve the manuscript. All the best.

Reviewer 2 Report

Efficacy of Entomopathogenic Trichoderma Isolates against Sugarcane Woolly Aphid, Ceratovacuna lanigera Zehntner (Hemiptera: Aphididae)

 

Manuscript ID: horticulturae-1488861

A brief summary

The authors describe the entomological potential of Trichoderma species isolated from agricultural soils of different locations of Sabah, Malaysia against sugarcane woolly aphid, Ceratovacuna lanigera.

Broad comments

The article is well-written; the authors isolated fungal species which occur naturally in examined region. They checked the anti-insect activity of Trichoderma species, against C. lanigera a destructive insect pest of sugarcane. I have only a  few question.

Specific comments 

L84 – Please put the hypothesis in the end of Introduction section

L57-61 – Please rewrite the name of species with cursive.  

L100 – Please put the name of Order:Family to all species in the text, now there are mixed, in some species there is this description in some not.

L124- Please change the way of presenting the concentration

Figures – Please add the statistics to the figures (marks and description)

L426-L428 – Please add the reference to this statement

Author Response

Reviewer 2 comments: REVISE USING GREEN COLOR

Efficacy of Entomopathogenic Trichoderma Isolates against Sugarcane Woolly Aphid, Ceratovacuna lanigera Zehntner (Hemiptera: Aphididae)

 A brief summary: The authors describe the entomological potential of Trichoderma species isolated from agricultural soils of different locations of Sabah, Malaysia against sugarcane woolly aphid, Ceratovacuna lanigera.

Broad comments

The article is well-written; the authors isolated fungal species which occur naturally in examined region. They checked the anti-insect activity of Trichoderma species, against C. lanigera a destructive insect pest of sugarcane. I have only a few questions.

Response: Thank you very much for your valuable comments and suggestions to improve my manuscript.

Specific comments 

 Point:  L84 – Please put the hypothesis in the end of Introduction section

Response: According to your valuable suggestion, the hypothesis has been included in the end of introduction. (New line:106-108)

 Point: L57-61 – Please rewrite the name of species with cursive.  

Response: Thank you. According to your suggestion, the name of species has been written with cursive. (New line: 70-77 )

Point: L100 – Please put the name of Order: Family to all species in the text, now there are mixed, in some species there is this description in some not.

Response: According to your suggestion, the name of Order: Family to all species has been included in the text. (Green marking)

Point: L124- Please change the way of presenting the concentration

Response: The way of presenting the concentration has been revised. (New line: 153-154)

Point: Figures – Please add the statistics to the figures (marks and description)

Response: Thank you for your valuable suggestion. We added the statistical information (significant differences, significant level, and error bar) in both Fig. 4 and Fig. 5.  (New line: 448 and 472) 

Point: L426-L428 – Please add the reference to this statement

Response: References (54, 55) have been added in this portion. (New line: 521)

Thank you very much for your valuable comments. All the best.

Reviewer 3 Report

In this research ms the authors intend to assess the efficacy of seven isolates of different entomopathogenic Trichoderma harzianum, T. hamatum and T. asperellum against Sugarcane woolly aphid, Ceratovacuna lanigera Zehntner (Hemiptera: Aphididae). These isolates were identified both on morphological basis and using internal transcribed spacers (ITSs) region. Different concentrations of fungi were tested in laboratory assays against nymphs and adults of woolly aphid. All the fungal isolates were effective against both stages and gave overall about 60-70% mortality but T. harzianum proved more effective compared to rest of the isolates. This ms is within the scope of journal and presents interesting results – the authors generated good data. The objectives of the study are clear, the experimental design is appropriate and the results support the conclusion.

Overall the manuscript is well written and can be accepted for its publication in horticulturae journal after minor revision

 

Some minor points are as follows:

 

L24: replace “T.” with “Trichoderma”, each sentence should start with complete genus name throughout ms

L36: replace “amass” with more appropriate word

L51: you may cite here “Wakil et al., 2021” for insect pests in storage

L51: you may consider to cite “Usman et al., 2021” and “Gulzar et al. 2021” for greenhouses and field conditions”

L57: provide new taxonomy of “Beauveria bassiana” and all other scientific names on first use in ms

L80: what is “EMP”?

L98: check spelling “analyzes”

L100: check “Zimmermann 1986” really this reference is needed here as it is on Galleria mellonella?

L109: check spellings “claeaned”

L127: replace “petri” with “Petri”

L222: replace “through” with “using”

L222: replace “hairbrushes” with hairbrush”

L230: which part of insect was topically treated?

L238: how many fresh new leaves of sugarcane were placed and were old ones removed – if it is then after how long?

L233: how many times replications were repeated?

L245: no need to give equation for Abbott’s formula?

L257: give version of R software?

 

The discussion section is little weak without providing the possible reasoning of the current findings. The authors should improve this section and should focus on to compare the findings of other researchers with the results of current study instead of merely reproducing their results. The logical and possible reasoning of the findings would be supportive

There is no need to repeat the results (data) in Discussion section

Author Response

Reviewer 3 comments: REVISE USING BLUE COLOUR

In this research MS the authors intend to assess the efficacy of seven isolates of different entomopathogenic Trichoderma harzianum, T. hamatum and T. asperellum against Sugarcane woolly aphid, Ceratovacuna lanigera Zehntner (Hemiptera: Aphididae). These isolates were identified both on morphological basis and using internal transcribed spacers (ITSs) region. Different concentrations of fungi were tested in laboratory assays against nymphs and adults of woolly aphid. All the fungal isolates were effective against both stages and gave overall about 60-70% mortality but T. harzianum proved more effective compared to rest of the isolates. This MS is within the scope of journal and presents interesting results – the authors generated good data. The objectives of the study are clear, the experimental design is appropriate and the results support the conclusion.

Overall the manuscript is well written and can be accepted for its publication in horticulturae journal after minor revision

Response: Thank you very much for your valuable comments and suggestions to improve my manuscript.

Some minor points are as follows:

Point: L24: replace “T.” with “Trichoderma”, each sentence should start with complete genus name throughout MS.

Response: Thank you for your insightful observations. This portion has been revised. (New line: 26)

Point: L36: replace “amass” with more appropriate word

Response: The word “amass” has been replaced with “gather” (New line: 48)

Point: L51: you may cite here “Wakil et al., 2021” for insect pests in storage

Response:  According to your suggestion, “Wakil et al., 2021” has been cited (citation no: 9) (New line: 65).  

Point: L51: you may consider to cite “Usman et al., 2021” and “Gulzar et al. 2021” for greenhouses and field conditions”

Response: “Usman et al., 2021” and “Gulzar et al. 2021” have been cited for green houses and (citation no: 10 and 11) (New line:65).

Point: L57: provide new taxonomy of “Beauveria bassiana” and all other scientific names on first use in ms

Response: According to your suggestion, new taxonomy of “Beauveria bassiana” and all other scientific names in MS have been provided.  marked with green colour

Point:  L80: what is “EMP”?

Response:  This was a mistake, and we apologize for it. This will be “EPF”. However, this portion has been revised (New line: 102).

Point:  L98: check spelling “analyzes”

Response: Spelling “analyzes” has been revised. It will be analysis. (New line: 126).

Point:  L100: check “Zimmermann 1986” really this reference is needed here as it is on Galleria mellonella?

Response: Thank you for your observation. According to laboratory manual “Methods for isolation of entomopathogenic fungi from the soil environment”-

Zimmermann (1986) suggested that insect bait method is a standard isolation method for entomopathogenic fungi. For the method to be feasible insects, which are easily reared and are susceptible to the fungi, must be used. The traditional bait insect is the highly susceptible larvae of the wax moth, Galleria mellonella, (Lepidoptera: Pyralidae) but also mealworm larvae, Tenebrio molitor (Coleoptera: Tenebrionidae), are suitable.

So, for insect bait method, both insects (Galleria mellonella and Tenebrio molitor) are suitable. Therefore, we think, reference “Zimmermann 1986” is needed here. (New line: 128)

Point: L109: check spellings “claeaned”

Response: Spelling  “cleaned” has been revised. (New line: 136).

Point: L127: replace “petri” with “Petri”

Response: This portion has been revised. (New line: 152, 154).

Point: L222: replace “through” with “using”

Response:  “through” has been replaced with “using”(New line: 267).

Point: L222: replace “hairbrushes” with hairbrush”

Response:  “hairbrushes” has been replaced with “hairbrush” (New line: 267).  

Point: L230: which part of insect was topically treated?

Response: The whole part of insect was topically treated. (New line: 278).

Point: L238: how many fresh new leaves of sugarcane were placed and were old ones removed – if it is then after how long?

Response: Thank you. Two fresh new sugarcane leaves were placed near both sides of the old leaves (after 24-48 hours) (New line: 286-288).

 Point:  L233: how many times replications were repeated?

Response:  Each treatment was applied 1 (one) time and three replications were performed for each treatment. (New line: 280-281).

Point: L245: no need to give equation for Abbott’s formula?

Response: According to your suggestion, equation for Abbott’s formula has been deleted.

Point:  L257: give version of R software?

Response:  Version of R software has been given.  (New line: 310).

Point: The discussion section is little weak without providing the possible reasoning of the current findings. The authors should improve this section and should focus on to compare the findings of other researchers with the results of current study instead of merely reproducing their results. The logical and possible reasoning of the findings would be supportive

There is no need to repeat the results (data) in Discussion section

Response:  Thanks for your observation. According to your suggestions, Discussion section has been revised and improved. (New line: 571-576 and 585-605)

Thank you very much for your overall valuable comments and suggestions which helped us  to improve the manuscript. All the best.