Virulence Screen of Beauveria Bassiana Isolates for Australian Carpophilus (Coleoptera: Nitidulidae) Beetle Biocontrol
Round 1
Reviewer 1 Report
The present manuscript investigated the potential impact of entomopathogenic fungus against Carpophilus beetles that attack Australian fruit and nut crops. The results showed that the virulence of some entomopathogenic isolates could be used as a biopesticide by increasing larval mortality and to be incorporated into pest management programs.
Overall, this work is valuable, the laboratory assays were described well and were straightforward. It deserves to be published. However, I have a few suggestions that may further improve the clarity of the manuscript:
- Line 20: add which isolate was the most effective. B54?
- Line 36: specify which life stage of the beetle causes significant kernel losses (larva, adult or both)
- Line 99: Why did you use the commercial strain Velifer? Give an explanation in the methodology
- Line 130: add some pictures for verification of mycosis as Fig. S1 (supplementary information).
- Line 145: add pictures of larval mortality as Fig. S2 (supplementary information).
- Lines 178-185: delete this. It duplicates from the previous paragraph
- Add in your discussion a couple of sentences on why the commercial strain Velifer had lower mortality rates compared to the other isolates in the adult stage assays?
Author Response
Point 1. Line 20: add which isolate was the most effective. B54?
Response: revised in manuscript
Point 2. Line 36: specify which life stage of the beetle causes significant kernel losses (larva, adult or both)
Response: in line 35 we specified that both adult and larval feeding leads to damage in both species
Point 3. Line 99: Why did you use the commercial strain Velifer? Give an explanation in the methodology
Response: Velifer is the only registered Beauveria bassiana product in Australia and is therefore a clear candidate for testing. There was no bias towards this product and was only selected because of the availability of it.
-Clarified in line 103
Point 4. Line 130: add some pictures for verification of mycosis as Fig. S1 (supplementary information).
Response: Revised in manuscript and photos will be uploaded to supplementary information
Point 5. Line 145: add pictures of larval mortality as Fig. S2 (supplementary information).
Response: Revised in manuscript and photos will be uploaded to supplementary information
Point 6. Lines 178-185: delete this. It duplicates from the previous paragraph
Response: revised in manuscript
Point 7. Add in your discussion a couple of sentences on why the commercial strain Velifer had lower mortality rates compared to the other isolates in the adult stage assays?
Response: the variable effectiveness of all isolates is discussed in the discussion. We do not feel that it is necessary to discuss Velifer specifically as it is subject to the same variation in biology as all other isolates.
Reviewer 2 Report
This study looks at several strains of entomopathogenic fungus for the control of an important pest insect, Carpophilus beetle. This work has important implications for biological control and demonstrates the variation of efficacy of various strains of a commercially available species of Beauveria bassiana.
This experimental design was well done and the statistical analysis was appropriate. The authors have presented the data with clear figures and clearly explain the results. I especially like the discussion where the authors discuss the lack of correlation between efficacy and host species relatedness for the source of isolates.
Other than some minor edits to table 1, I don’t not find any other corrections needed in this paper.
In table 1:
- change 'identification' to isolate to a) make it fit in the heading un-hyphenated, b) make it consistent (in referencing isolates) with other headings.
- abbreviate September to match other abbreviated moths.
- add the month to B49 isolate : ____2015.
- fix spacing on '%germination' so that +/- appears next to SE.
Author Response
Point 1. Change 'identification' to isolate to a) make it fit in the heading un-hyphenated, b) make it consistent (in referencing isolates) with other headings.
Response: “Identification” is referring to the species not the specific isolate which is addressed in the column "isolate host"
Point 2. Abbreviate September to match other abbreviated moths.
Response: revised in manuscript
Point 3. add the month to B49 isolate : ____2015.
Response: This information not available
Point 4. Fix spacing on '%germination' so that +/- appears next to SE.
Response: revised in manuscript
Reviewer 3 Report
A sound paper detailing bioassays of strains of B. bassiana against two common pest beetles. It is well written with excellent discussion of applications and further research that is necessary. A few minor corrections are needed, which are in the attached file.
Comments for author File: 
Comments.pdf
Author Response
Point 1. Line 23 Rewrite: "...mortality). All other isolates caused less than 40% mortality."
Response: revised in manuscript
Point 2. Line 25 Replace with the word “strategy”.
Response: revised in manuscript
Point 3. Line 39 Delete this comma.
Response: revised in manuscript
Point 4. Line 39 This is confusing. Something like reduced storage life would be more clear.
Response: revised in manuscript - Line 39
Point 5. Line 82. Delete for to.
Response: revised in manuscript
Point 6. Line 85. Change to "...conducted that assessed time-dependent mortality on adult beetles over..."
Response: revised in manuscript
Point 7. Line 86 Change to, "days, mycosis frequency, and on..."
Response: reworded line 86 to be clearer
“Bioassays were conducted that assessed time-dependant mortality over 15 days and mycosis frequency on adult beetles and final instar larvae mortality after 10 days of incubation.”
Point 9. Line 143-144 I think there is a and missing between the words day and repeated. Was this a total of 15 replicates with 5 replicates per day?
Response: revised in manuscript line 144
Point 10. Line 181 “Resistant” This word has a very specific definition in pest management. I suggest rewording the first sentence to say that B. bassiana caused higher mortality in one species compared with the other.
Response: Revision in Line 182 “In experiments using adult insects, C. truncatus was found to have the lowest mortality from treatment with B. bassiana”
Point 11. Lines 181- 188 This repeats the previous paragraph exactly.
Response: revised in manuscript - section deleted
Point 12. Lines 203-4 According to Table 2, only B37 is statistically different from B48 and B50.
Response: revised in manuscript – Line 205
Point 13. Line 228 Change to against
Response: revised in manuscript
Point 14. Lines 233-234 Control mortality is not shown in the graphs or included in the statistical analysis presented in the results. This statement cannot be made without the results to back it up.
Response: The sentence on lines 233-235 was removed as it was not relevant to the wider discussion.
Point 15. Line 250 Should be “an”
Response: revised in manuscript
Point 16. Line 263 Add the word “method” after the word application.
Response: revised in manuscript
Reviewer 4 Report
The paper of Boston et al. is well written, introduction is broad and discussion is rich. However some minor revision are necessary:
-Carpophilus is a scientific name, not the common name (“sap beetle”?), so should be written in italics, everywhere in the text.
-row 32: what’s the source of all these economic data?
-row49: Carpophilus davidsoni should be written in the contract form C. davidsoni, here and in the following text.
-row 79: “…this species was chosen to for to screen…” . It is correct?
-row 99: Is Velifer strain which you used in your assays free of any adjuvant (oil emulsions, nutritive granules, etc)? Is just the strain or a particular formulate? Is this product already used against Coleoptera or other insect orders?
- row 129: Why did you used water agar and not PDA plates for the re-isolation?
-row 144: why did you check only after 10 days and not daily?
-row 178: “…the more resistant beetle species…”. The “most”?
-row 191: Why can’t you calculate LT50?
-Table 2: What do you used as controls for re-isolate the pathogen? Untreated cadavers? Alive insects? Please, specify.
-References: Please check reference style, I’ve noticed a large use of Italics and caps-lock, when is not appropriate.
Author Response
Point 1. -Carpophilus is a scientific name, not the common name (“sap beetle”?), so should be written in italics, everywhere in the text.
Response: revised in manuscript
Point 2. -row 32: what’s the source of all these economic data?
Response: revised in manuscript
Almond Board of Australia. 2019/2020 Almond Insights. Available online: https://industry.australianalmonds.com.au/wp-content/uploads/2020/07/2020_Almond_Insights.pdf (acessed on 11 August 2020)
Nichola McGregor for FreshPlaza.com. Australian summerfruit exports look positive following a record breaking 2018/19 season. Available online: https://www.freshplaza.com/article/9165837/australian-summerfruit-exports-look-positive-following-a-record-breaking-2018-19-season/ (acessed on 11 August 2020)
Point 3. -row49: Carpophilus davidsoni should be written in the contract form C. davidsoni, here and in the following text
Response: The species name is written in full as it is at the beginning of a sentence in both instances.
Point 4. -row 79: “…this species was chosen to for to screen…” . It is correct?
Response: Revised in manuscript
Point 4. -row 99: Is Velifer strain which you used in your assays free of any adjuvant (oil emulsions, nutritive granules, etc)? Is just the strain or a particular formulate? Is this product already used against Coleoptera or other insect orders?
Response: Velifer is formulated as an oil emulsion however for use in this assay the fungus alone was isolated from the stored product and grown on oatmeal agar for use in the same manner (water suspension with 0.05% tween 80) as the other isolates. The fungus was isolated from a larvae of the beetle Conchyloctenia punctate - listed in Table 2.
-Clarified on row 103
Point 5. - row 129: Why did you used water agar and not PDA plates for the re-isolation?
Response: PDA plates are unnecessary in this instance as the point of this experimental step is to determine whether the fungus is present within the insect cadaver and able to develop with the nutrition of the insect alone. Using PDA is redundant in this context.
Point 6. -row 144: why did you check only after 10 days and not daily?
Response: The larval stage was only checked at the end of the 10 day incubation period to decrease the disturbance to the insects as regular handling can induce stress that can make pathogens more effective whilst not having any effect on the control insects. Also as the treatment was an inoculated soil substitute with spores present throughout the incubation time, having an infected cadaver would not substantially increase the spore load of surrounding insects as it would in the case of the adult insect assay.
Point 7. -row 178: “…the more resistant beetle species…”. The “most”?
Response: Revised in manuscript
Point 8. -row 191: Why can’t you calculate LT50?
Response: LT50 for adult beetles was not included as only one treatment had a mortality higher than 50%. LT50 could be determined for that particular treatment (C. davidsoni with isolate B54) but the comparison with the other treatments with sub 50% mortality would not be useful to make. This is explained in line 194-195.
LT50 was not calculated for the larval assay because the time of death was not recorded in this instance.
Point 9. -Table 2: What do you used as controls for re-isolate the pathogen? Untreated cadavers? Alive insects? Please, specify.
Response: The controls for the mycosis check were the insects cadavers that died in the control treatments and were handled in the same manner as the fungus treated insect cadavers. This was done to verify that the control insects did not die of a fungal pathogen and that the inoculation technique was clean.
Revised line 131
Point 10. -References: Please check reference style, I’ve noticed a large use of Italics and caps-lock, when is not appropriate.
Response: Revised in manuscript
