Crude Extracts and Alkaloids Derived from Ipomoea-Periglandula Symbiotic Association Cause Mortality of Asian Citrus Psyllid Diaphorina citri Kuwayama (Hemiptera: Psyllidae)

Round 1

Reviewer 1 Report

Manuscript jof-1394110 “Crude extracts and alkaloids derived from Ipomoea-Periglandula symbiotic association cause mortality of Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Psyllidae)” by Chen and colleagues explores the use of endophytic entomopathogenic fungi for pest control whereas I have found several concerns on the experimental schedule highly limiting the significance of this work.

1.- Bioassays and entomological concerns. When research is performed covering the survival and behavior of a of multistage insect pest, key information on the actual number of insects from each stage and instar surviving the treatment and finally reaching the adult stage is mandatory. Indeed, mortality of each stage/instar should be provided splitting the ratio with fungal outgrowth and with unknown causes that it is particularly important when dealing with possible mortality via endophytic colonization. Consequently, it is not stated why only large nymphs and adults where considered in crude bioassays, and why only adults were considered in antifeedant bioassays? Finally, it is not stated whether the mortality factor from control cohorts was considered when estimating the corrected mortality fate of the experimental cohorts.

2.- The are several hurdles regarding the general procedure for plant inoculation. First, it is quite difficult to understand how the research team tries to safe a quite enormous hurdle regarding the problem of the origin and fate of the fungal inoculum either coming from superficial inoculum infesting the leaves surface after spraying or immersion or coming from the inner tissues due to endophytism. Hence, it is difficult to unravel whether the possible effect of the fungal challenge on the fitness, settling and feeding behavior was due to endophytism or due to contact with the conidia. Similarly, it is nearly impossible to unravel whether PCR detected endophytic inoculum or superficial (residual) inoculum in the leaves after spray or immersion.

3.- The general procedure for plant inoculation in the settling behavior is also not well described, number of sprayed and non-sprayed leaves per plant, number of new non-sprayed leaves accounting in the treatments, section of the insect population settled in fungi-challenged or non-challenged new leaves, why 48 hours selected for visual observations etc., etc. There is also no evidence that authors considered sample size or the potential of their experiment to detect an expected difference. Did they conduct a power analysis to establish a sample size that could detect an expected difference? Replication seems very weak in terms of the chances of detecting a difference and across all experiments. With four replicates of five insects for crude bioassays, and four replicates for antifeedant bioassays how much difference could they expect to see? The number of replicates for the settling behavior is unknown?

4.- My other concern is that the authors are extrapolating the applicability of their results beyond what the design supports. These are only data from highly artificial laboratory conditions, so the inference power of the paper is very limited, but authors do not acknowledge this detail at all and need to be more forthcoming. We don't have any idea how applicable the results will be under field conditions. It is important that the authors state what is obvious with regards to generalizing their results to the larger question of treatment benefits in citrus crops under field conditions. This is a critical limitation of the study, and the authors must concede and discuss this. The consequences of getting this wrong will affect real people and livelihoods. So, I am suggesting to the authors to tone-down the language a little and admit that there are still substantive uncertainties to be considered. Instead, the importance of the results is that they emphasize the need to do such studies under field conditions and to understand better the benefits and variability in benefits derived from use of alkaloids in controlling D. citri on citrus. This is not to diminish the data gathered in this study, they are of value. But it is important for the authors not to overgeneralize, and to warn the reader, including regulatory agencies, against doing so as well.

I also feel the introduction and discussion are held back by structural problems. The current organization makes it hard to find key points. The intro and discussion provide no insight on how this MS relates to the various other ones cited in the text or concerns that have been raised by other researchers and regions where D. citri  is a problem. The authors do not present any hypotheses or expectations that could be connected to previous studies and biological control in particular. Nowadays, only the parasitoid Tamarixia radiata (see study by Milosavljevic et al. 2021. Biological Control, 159, p.104627, the first report of density-dependent reduction of D. citri by T.  radiata) and the entomopathogenic fungus Isaria fumosorosea (see studies by Patt et al. 2015. Biol. Control 88, 37-45; Stauderman et al. 2012. Biocontrol Sci Technol., 22, 747-761) are used for inundative biological control of D. citri, and Isaria does not show significant evidence to be effective in the field. Adding these details will improve the paper. It is also likely that other generalist predators are responsible for some reduction of psyllid nymphs (see studies by Qureshi & Stansly 2009. Biol. Control. 50, 129-136; Kistner et al., 2016. Biological Control, 115, 141-151; and Irvin et al., 2021. Biological Control, 157, p.104574.), which indeed the authors do discuss here. This article should provide details on all these fronts to provide the proper context for the work.

Overall, this work shows interesting results that may have some practical field application for D. citri  management, but I found it hard to extract key messages useful to policymakers and Ag professionals, probably in large part due to the lack of connection with other published work and need for improved structure of the current manuscript.

The next draft of this paper will need to be dramatically different to have a chance at publication in my opinion.

Author Response

Reviewer 1

Comments and Suggestions for Authors

Manuscript jof-1394110 “Crude extracts and alkaloids derived from Ipomoea-Periglandula symbiotic association cause mortality of Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Psyllidae)” by Chen and colleagues explores the use of endophytic entomopathogenic fungi for pest control whereas I have found several concerns on the experimental schedule highly limiting the significance of this work.
Author’s response: We thank the reviewer for their careful reading of our manuscript. The introduction, materials and methods, results, and discussion sections were edited and improved, where appropriate, according to the reviewer’s suggestions. Responses to the review’s comments are outlined below

1.- Bioassays and entomological concerns. When research is performed covering the survival and behavior of a of multistage insect pest, key information on the actual number of insects from each stage and instar surviving the treatment and finally reaching the adult stage is mandatory. Indeed, mortality of each stage/instar should be provided splitting the ratio with fungal outgrowth and with unknown causes that it is particularly important when dealing with possible mortality via endophytic colonization. Consequently, it is not stated why only large nymphs and adults where considered in crude bioassays, and why only adults were considered in antifeedant bioassays? Finally, it is not stated whether the mortality factor from control cohorts was considered when estimating the corrected mortality fate of the experimental cohorts.
Author’s response: We believe the reviewer might have misinterpreted the cause of insect mortality in this investigation. The fungal associate (Periglandula) does not attack insects, thus there was no mortality caused here due to an entomopathogenic fungus. Consequently, there was no possibility of mortality due to “endophytic colonization” of the insects. Instead, the Periglandula (fungus)-Convolvulaceae (plant) association leads to the production and sequestration of alkaloids within the plants, and these sequestered alkaloids have lethal and sub-lethal effects on the insects. The fungus itself produces the alkaloids. This plant-fungus association, and its role in alkaloid production, has been explained in the Abstract, Simple Summary, and second paragraph of the introduction. We investigated the effects of these alkaloids on Asian citrus psyllid by: 1) creating crude extracts from plants that contained the alkaloids, and 2) using synthetic analogues of the alkaloids. In sum, there was no possibility of an interaction between ergot fungi and psyllids in our bioassays.

2.- The are several hurdles regarding the general procedure for plant inoculation. First, it is quite difficult to understand how the research team tries to safe a quite enormous hurdle regarding the problem of the origin and fate of the fungal inoculum either coming from superficial inoculum infesting the leaves surface after spraying or immersion or coming from the inner tissues due to endophytism. Hence, it is difficult to unravel whether the possible effect of the fungal challenge on the fitness, settling and feeding behavior was due to endophytism or due to contact with the conidia. Similarly, it is nearly impossible to unravel whether PCR detected endophytic inoculum or superficial (residual) inoculum in the leaves after spray or immersion.
Author’s response: We thank the reviewer for their time invested in reviewing our manuscript. We believe that the reviewer may have misinterpreted our treatments and this may have been because of the wording we chose or insufficient description of treatments in our initial draft. There was in fact no inoculum or fungal spores tested in this experiment. The Periglandula-Convolvulaceae association described in this investigation yields alkaloid production in plants. We therefore tested crude plant extracts containing these alkaloids, as well as, the synthetic versions of the alkaloids. Those extracts or synthetic alkaloids were applied to plant surfaces and then tested against insects through various exposure assays. The plant surfaces were treated by immersing leaf surfaces in solutions of the plant extracts or by spraying alkaloid solutions onto leaf surfaces. In no case were leaves inoculated with fungal spores or pathogens of any kind. The term “inoculate’ was used in the first draft mistakenly and may have caused confusion. Our revision should clarify the treatments that were evaluated.  Periglandula fungi forms an epiphytic and often asymptomatic symbiotic association with plants in Convolvulaceae and is unculturable on the artificial medium. Previous studies on potato psyllid demonstrated the insecticidal activities of ergot alkaloids derived from this association, which warranted their evaluation against Asian citrus psyllid in this study.

3.- The general procedure for plant inoculation in the settling behavior is also not well described, number of sprayed and non-sprayed leaves per plant, number of new non-sprayed leaves accounting in the treatments, section of the insect population settled in fungi-challenged or non-challenged new leaves, why 48 hours selected for visual observations etc., etc. There is also no evidence that authors considered sample size or the potential of their experiment to detect an expected difference. Did they conduct a power analysis to establish a sample size that could detect an expected difference? Replication seems very weak in terms of the chances of detecting a difference and across all experiments. With four replicates of five insects for crude bioassays, and four replicates for antifeedant bioassays how much difference could they expect to see? The number of replicates for the settling behavior is unknow

Author’s response: Thank you for comments. We have revised the description of this procedure in an effort to improve clarity. We believe the experimental design was robust and effectively tested the hypothesis that ergot alkaloids affect setting behavior of D. citri. The results yielded by the experiment were clear cut. 

4.- My other concern is that the authors are extrapolating the applicability of their results beyond what the design supports. These are only data from highly artificial laboratory conditions, so the inference power of the paper is very limited, but authors do not acknowledge this detail at all and need to be more forthcoming. We don't have any idea how applicable the results will be under field conditions. It is important that the authors state what is obvious with regards to generalizing their results to the larger question of treatment benefits in citrus crops under field conditions. This is a critical limitation of the study, and the authors must concede and discuss this. The consequences of getting this wrong will affect real people and livelihoods. So, I am suggesting to the authors to tone-down the language a little and admit that there are still substantive uncertainties to be considered. Instead, the importance of the results is that they emphasize the need to do such studies under field conditions and to understand better the benefits and variability in benefits derived from use of alkaloids in controlling D. citri on citrus. This is not to diminish the data gathered in this study, they are of value. But it is important for the authors not to overgeneralize, and to warn the reader, including regulatory agencies, against doing so as well.

Author’s response: Thank you for comments. We agree with the reviewer comments. We have edited the discussion section significantly so as not to over-extrapolate the significance of the findings.

I also feel the introduction and discussion are held back by structural problems. The current organization makes it hard to find key points. The intro and discussion provide no insight on how this MS relates to the various other ones cited in the text or concerns that have been raised by other researchers and regions where D. citri  is a problem. The authors do not present any hypotheses or expectations that could be connected to previous studies and biological control in particular. Nowadays, only the parasitoid Tamarixia radiata (see study by Milosavljevic et al. 2021. Biological Control, 159, p.104627, the first report of density-dependent reduction of D. citri by T.  radiata) and the entomopathogenic fungus Isaria fumosorosea (see studies by Patt et al. 2015. Biol. Control 88, 37-45; Stauderman et al. 2012. Biocontrol Sci Technol., 22, 747-761) are used for inundative biological control of D. citri, and Isaria does not show significant evidence to be effective in the field. Adding these details will improve the paper. It is also likely that other generalist predators are responsible for some reduction of psyllid nymphs (see studies by Qureshi & Stansly 2009. Biol. Control. 50, 129-136; Kistner et al., 2016. Biological Control, 115, 141-151; and Irvin et al., 2021. Biological Control, 157, p.104574.), which indeed the authors do discuss here. This article should provide details on all these fronts to provide the proper context for the work.

Author’s response: We have edited the introduction, and particularly, the discussion sections quite significantly. Please note that our experiment focused on crude extracts of alkaloids and commercially purchases synthetic alkaloids.  This investigation has nothing to do with fungal pathogens such as entomopathogenic fungi and the suggested papers are irrelevant to this investigation.

Overall, this work shows interesting results that may have some practical field application for D. citri management, but I found it hard to extract key messages useful to policymakers and Ag professionals, probably in large part due to the lack of connection with other published work and need for improved structure of the current manuscript.

The next draft of this paper will need to be dramatically different to have a chance at publication in my opinion.

Author’s response: We thank the reviewer for their review of our manuscript. The introduction, methods and materials, and discussion sections were edited and improved where it was relevant according to the reviewer’s suggestion.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript, “Crude extracts and alkaloids derived from Ipomoea-Periglandula symbiotic association cause mortality of Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Psyllidae)” examined the effects of ergot-alkaloids on survival, settling behavior, and feeding. It is a well-written manuscript, understandable and data analyses seem to be appropriate.

Some comments:

Abstracts and Introduction

No comments

M&M

Line 106: Please give a sense of plant or tree size or how tall etc. Are they potted trees? Any information on the fertility schedule and age of the trees?

Line 112: “crude extract bioassay? Adding a figure of the bioassay will be very helpful to understand the experimental setup.

Line 137: add a space after ”…8 h” and “Nymphs…”

Line 139: It was not clear if the authors introduced five adults and five nymphs in two separate experiments. Where were the insects released…on a single leaf, and on which side?

Line 163: “two plants” potted plants/trees? What size?

Line 151: Why was the no-choice study not performed? The outcome could be different and valuable to know.

Results

No comments

Discussion

Line 283-302: The reviewer sees why this paragraph was added, but it seems like a literature review and is more appropriate for the introduction section. Elements of this paragraph can be discussed in the following paragraph instead and used for comparison and contrast with results in the current study.

Consider starting the discussion with the second paragraph as they are the major results from the current study.

Also, is there any indication that ergot alkaloids produced by the fungus elicit toxicity to humans or other organisms? 

The tables and figures look good.

Author Response

Reviewer 2

Comments and Suggestions for Authors

The manuscript, “Crude extracts and alkaloids derived from Ipomoea-Periglandula symbiotic association cause mortality of Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Psyllidae)” examined the effects of ergot-alkaloids on survival, settling behavior, and feeding. It is a well-written manuscript, understandable and data analyses seem to be appropriate.

Some comments:

Author’s response: We thank the reviewer for positive comments. Our responses to the reviewers’ comments are outlined below

Abstracts and Introduction

No comments

Author’s response: Thank you.

M&M

Line 106: Please give a sense of plant or tree size or how tall etc. Are they potted trees? Any information on the fertility schedule and age of the trees?

Author’s response: This has been added, explaining  plant size and potting.

Line 112: “crude extract bioassay? Adding a figure of the bioassay will be very helpful to understand the experimental setup.

Author’s response: This has been added in the new supplementary Fig 1. We also attempted to clarify the methods further.

Line 137: add a space after ”…8 h” and “Nymphs…”

Author’s response: This has been corrected in the text.

Line 139: It was not clear if the authors introduced five adults and five nymphs in two separate experiments. Where were the insects released…on a single leaf, and on which side?

Author’s response: Insects were released onto single leaflets. This has been explained further in the text.

Line 163: “two plants” potted plants/trees? What size?

Author’s response: This has been edited in the text.

Line 151: Why was the no-choice study not performed? The outcome could be different and valuable to know.

Author’s response: Yes, we agree with the reviewer that a no-choice investigation would useful. Unfortunately, we did not have sufficient materials to add this experiment, but this should be included in follow up work.

Results

No comments

Author’s response: Thank you.

Discussion

Line 283-302: The reviewer sees why this paragraph was added, but it seems like a literature review and is more appropriate for the introduction section. Elements of this paragraph can be discussed in the following paragraph instead and used for comparison and contrast with results in the current study.

Author’s response: We have significantly edited the discussion section according to this comment and a previous reviewer, effectively removing this paragraph.

Consider starting the discussion with the second paragraph as they are the major results from the current study.

Author’s response: As suggested, we have significantly re-organized the entire discussion according to this helpful comment.

Also, is there any indication that ergot alkaloids produced by the fungus elicit toxicity to humans or other organisms? 

Author’s response: According Clay and Cheolick 1989, the toxicity of these fungi is lower to humans and other vertebrates than to arthropods. However, collection of these ecotoxicological data will be needed before developing biopesticide.

The tables and figures look good.

Author’s response: Thank you.

Author Response File: Author Response.pdf

Reviewer 3 Report

The article “Crude extracts and alkaloids derived from Ipomoea - Periglan- 2 dula  symbiotic association cause  mortality of Asian citrus” is very interesting. Overall, this is a well executed and well written study that can be considered for publication after minor revisions.

Below the point by point comments or suggestions:

Lines 32-33: Periglandula spp.

Line 36: Behavior instead od behaviors

Line 40: Periglandula should be italic

Line 57: worldwide and not Worldwide

Line  71:  change suppression with reduction. A complete population suppression is the aim of biological control only in few cases (i.e. introduced species)

Line 78: spp. not italic

Line 86: remove [13] redundant as cited in line 87

Lines 115-118: something wrong, rephrase

Line 142: Convolvulaceae not italic

Line 167:”… the observations were 48 hours over two consecutive days “: unclear, explain better

Line 170: add the overall number of repetitions

Table 1, legend, line  235: add Periglandula – negative before I. alba

Figures 1 and 2: the concentrations reported in the figures are 3 but those used in tests are 4, add in the graphs or explain in the text or in the figure legend why the lowest concentration is not reported

Figure 3: add (-) or (+) to the plant names

Line 288: remove ;after infection

Line 288: spp.

Line 305: have and not with

Line 323: “…could be due to the chemical and structural complexities of the ergopeptine class of ergot alkaloids which is higher than compounds in other classes…”

Lines 331 and 334: change warrants and warranted with needs and needed

Line 339: remove “In summary”

Well done!

Regards

Author Response

Reviewer 3

Reviewer's comments to the Author:

The article “Crude extracts and alkaloids derived from Ipomoea-Periglandula symbiotic association cause mortality of Asian citrus psyllid” is very interesting. Overall, this is a well executed and well written study that can be considered for publication after minor revisions. Below the point by point comments or suggestions:

Author’s response: We thank the reviewer for their careful review of our manuscript. Responses to comments have been included on the manuscript revisions in track changes. Also, responses to the reviewer’s comments are outlined below.

Lines 32-33: Periglandula spp.

Author’s response: This has been corrected in the text.

Line 36: Behavior instead od behaviors

Author’s response: This has been corrected in the text.

Line 40: Periglandula should be italic

Author’s response: This has been corrected in the text.

Line 57: worldwide and not Worldwide

Author’s response: This has been corrected in the text.

Line 71:  change suppression with reduction. A complete population suppression is the aim of biological control only in few cases (i.e. introduced species)

Author’s response: This has been edited in the text.

Line 78: spp. not italic

Author’s response: This has been corrected in the text.

Line 86: remove [13] redundant as cited in line 87

Author’s response: This has been removed in the text.

Lines 115-118: something wrong, rephrase

Author’s response: This has been corrected in the text

Line 142: Convolvulaceae not italic

Author’s response: This has been corrected in the text.

Line 167: the observations were 48 hours over two consecutive days “: unclear, explain better

Author’s response: This has been re-written to clarify that insect counts were made 48 h after insect release in cages.

Line 170: add the overall number of repetitions

Author’s response: This has been edited in text.

Table 1, legend, line 235: add Periglandula – negative before I. alba

Author’s response: This has been edited in the Table 1.

Figures 1 and 2: the concentrations reported in the figures are 3 but those used in tests are 4, add in the graphs or explain in the text or in the figure legend why the lowest concentration is not reported

Author’s response: This has been clarified in the Figure legend to indicate that data are shown only for the control treatment and the two highest concentrations of alkaloids tested.

Figure 3: add (-) or (+) to the plant names

Author’s response: This has been added in Figure 3.

Line 288: remove; after infection

Author’s response: This has been deleted in the text as the reviewer suggested.

Line 288: spp.

Author’s response: This has been deleted in the text as other reviewer suggested.

Line 305: have and not with

Author’s response: This has been deleted in the text as other reviewer suggestions.

Line 323: “…could be due to the chemical and structural complexities of the ergopeptine class of ergot alkaloids which is higher than compounds in other classes…”

Author’s response: This has been corrected in the text.

Lines 331 and 334: change warrants and warranted with needs and needed

Author’s response: This has been edited in the text.

Line 339: remove “In summary”

Author’s response: This has been edited in the text.

Well done!

Regards

Author’s response: Thank you for comments.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Authors have addressed all of the original reviewers' comments nicely. I do have some minor suggestions that needs their attention before the paper can be accepted for publication in Insects.

Some of the authors' statements on L70 through 74 are somewhat untrue, however. Several studies have been conducted on the population dynamics of D. citri resulting from interactions with density-dependent and density-independent processes. For example, four years of study in different regions of Southern California indicated a significant mortality and population reduction of D. citri due to high rates of parasitism by T. radiata (see study by Milosavljevic et al., Biol. Control 2021, 159: 104627). Releases of T. radiata decreased the number of D. citri nymphs per cm flush from 19 to 2, which represented (~90%) of the nymph population reduction. Consequently, significant reductions in D. citri densities may have resulted in substantially reduced rates of spread of this pest and CLas, especially from southern California into the Central Valley, where the majority of California citrus is grown. Consequently, the near to complete population suppression of D. citri can be expected with biological control in some areas. Consequently, the statements on L70 through 74 should be corrected to include what I've had in mind here or otherwise removed from the revised paper.     

I do not have any other suggestions for improvement. Good luck! 

Author Response

We thank the reviewer for their follow-up statements.

Reviewer's comment: "Some of the authors' statements on L70 through 74 are somewhat untrue, however."

Authors' response: We disagree. We do not believe anything that was stated on lines 70-74 is untrue to any degree.

Reviewer's comments:  "For example, four years of study in different regions of Southern California indicated a significant mortality and population reduction of D. citri due to high rates of parasitism by T. radiata (see study by Milosavljevic et al., Biol. Control 2021, 159: 104627). Releases of T. radiata decreased the number of D. citri nymphs per cm flush from 19 to 2, which represented (~90%) of the nymph population reduction. Consequently, significant reductions in D. citri densities may have resulted in substantially reduced rates of spread of this pest and CLas, especially from southern California into the Central Valley, where the majority of California citrus is grown. Consequently, the near to complete population suppression of D. citri can be expected with biological control in some areas. Consequently, the statements on L70 through 74 should be corrected to include what I've had in mind here or otherwise removed from the revised paper. "

Authors' response: This example illustrating 90% reduction of ACP populations is impressive indeed. It is also significant. But, it is not "near complete" population reduction of ACP. Near complete reduction would be 99%. Furthermore, it has become well accepted that biological control would need to reach the 99% level of population suppression to make impact on HLB management.

Nonetheless, we have deleted statement on lines 71-74 in order to settle this issue.

Author Response File: Author Response.docx