Review Reports - UV Fluorescent Powders as a Tool for Plant Epidemiological Studies

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled "UV -fluorescent powders as a tool for plant epidemiological studies" by Severns et al. has description of methodological development to track the movement of pathogens and small insects in field conditions. They have developed the techniques after different experiments and pilot scale field testing. The objective of the research study is clear. the Techniques and methods used to develop the described tool for detection of movement of pathogen/ insect are appropriate and scientifically sound. Results and discussion has been well described and discussed accordingly.

Some minor typological error have found in the manuscript as example in line 78- 79 the scientific name of whitefly is wrongly written Bemesia tabacci, change it with correct one Bemisia tabaci.

Also suggested to check entire document for the scientific name correctness of the species/organism mentioned in the manuscript.

Author Response

Some minor typological error have found in the manuscript as example in line 78- 79 the scientific name of whitefly is wrongly written Bemesia tabacci, change it with correct one Bemisia tabaci.

Also suggested to check entire document for the scientific name correctness of the species/organism mentioned in the manuscript.

Thank you for catching this mistake, we have corrected the name in the text and in the figure legends to the correct spelling.

We have also italicized scientific names of organisms in the literature cited which were improperly presented in the original submitted version.

Additionally, we closely edited the entire manuscript to ensure any confusing or poorly written passages were revised to be clear and more concise.

All of our changes are highlighted in yellow on the markup version of the resubmitted manuscript

Reviewer 2 Report

Comments and Suggestions for Authors

This study highlights an innovative strategy to studying plant disease epidemiology, focusing on biotic stresses viz., understanding the spatial distribution of fungal infections within plants (2) observing the dispersal patterns of whitefly vectors.

The authors demonstrate that UV-fluorescent particles mixed in a 10% ethanol solution can act as surrogates for fungal spores and whitefly vector inspection on the canopy.

1. Authors describe the overlapping of settlement velocities of the UV-fluorescent particles overlap with fungal pathogens. How it mimic the real scenario? What will the influence of variable weather parameters (wind, humidity, temperature)

2. Do the UV-fluorescent particles disintegrate over time, and if so, what is their shelf life in soil or on plants? This information is critical to understand the limitations of this method.

3. In lines 86-91 “The MSDS sheet section 4.2 states 86-91 that “the product is not harmful to human health effect” but the powders are labelled as “toxic to aquatic life with long-lasting effects” section 2.2 Hazard Statement H411. It is not clear what concentration threshold was toxic to aquatic life and what were the long-lasting effects on aquatic life, but we presume that in the limited quantities used in our studies that the potential impacts on aquatic life would be unlikely to be realized in arable, agricultural landscapes.

This statement is concerning because it is not clear what the long-term effect is on overall microbial flora and fauna of the agricultural ecosystem.

The lack of extensive testing and the lack of data on longer-term survival and behavioral effects are significant gaps that require proper justification. Conducting modulated studies to test these variables will be crucial for confirming the utility and applicability of this technique in research.

Author Response

This study highlights an innovative strategy to studying plant disease epidemiology, focusing on biotic stresses viz., understanding the spatial distribution of fungal infections within plants (2) observing the dispersal patterns of whitefly vectors.

The authors demonstrate that UV-fluorescent particles mixed in a 10% ethanol solution can act as surrogates for fungal spores and whitefly vector inspection on the canopy.

Authors describe the overlapping of settlement velocities of the UV-fluorescent particles overlap with fungal pathogens. How it mimic the real scenario? What will the influence of variable weather parameters (wind, humidity, temperature)

We do not have a formal comparison of the particles in a field situation compared with the particles. Hopefully, we can perform this experiment in the field and provide a good answer to this important question. However, we do recognize that we should have reported the conditions under which the particle experiments took place more fully. In our revised version we add the following details to the section describing the settlement velocity experiment. The following information occurs on lines 174 – 177 (the first sentence of the last paragraph of subsection 2.3) and now reads, “In a stable room environment (~21°C, ~ 45% relative humidity), we released spores gradually from the top of the chamber opening for ~ 20 seconds while the digital camera recorded a continuous movie from 5 seconds before to 10 seconds after release of the UV fluorescent powder.”

Do the UV-fluorescent particles disintegrate over time, and if so, what is their shelf life in soil or on plants? This information is critical to understand the limitations of this method.

We do not know the answer to this question but we do report that the particles persisted on plants placed outside under field conditions in the following Discussion passage from the original submitted manuscript. Lines 353 – 357: “Informal experimentation on leaves with surrogate fungal infections in field conditions experiencing heavy rains, direct sunlight, heat, wind, and other exposure related conditions persisted for at least 2 weeks when they occurred on the upper side of leaves and up to two months when the surrogate infections occurred on the sheltered, underside of leaves (personal observation).” We have not conducted any formal study of persistence, just that the particles do seem to persist for weeks to months. We do not know the fate of the particles in the soils.

In lines 86-91 “The MSDS sheet section 4.2 states 86-91 that “the product is not harmful to human health effect” but the powders are labelled as “toxic to aquatic life with long-lasting effects” section 2.2 Hazard Statement H411. It is not clear what concentration threshold was toxic to aquatic life and what were the long-lasting effects on aquatic life, but we presume that in the limited quantities used in our studies that the potential impacts on aquatic life would be unlikely to be realized in arable, agricultural landscapes.

This statement is concerning because it is not clear what the long-term effect is on overall microbial flora and fauna of the agricultural ecosystem.

We do know that these powders are used commonly in the celebration of Holi and humans are covered with the powders and the MSDS lists the powders as not toxic to humans. We only report what was presented in the MSDS safety sheets as we did not perform formal toxicity tests, just the short-term, 4-day observations shared in lines 211-218 original manuscript in which we detected no differences in mortality. The MSDS sheet did not list a source of this information concerning aquatic toxicity so we cannot know the LD50 concentration and on which organisms the powder is reported to be toxic. We thought it best to share this information with the reader as opposed to not sharing the information.

Reviewer 3 Report

Comments and Suggestions for Authors

Major comments:

The authors have presented two innovative methods using UV-fluorescent particles that when mixed in a 10% ethanol solution can be used to create substitute fungal infections on plant leaves, as well as being used to field mark whiteflies and other small insects in situ.

The study is methodologically well designed and realized, and the obtained results strongly suggest that the use of UV-fluorescent particles will assist in the study of fungal spore dispersal and location on plants for epidemiology studies, as well as the dispersal ecology of plant insect vectors to understand dispersal behaviour and feeding which is important for understanding the patterns of invertebrate vectored plant disease spread.

Your paper is scientifically sound and well written. The materials and methods were detailed and written clearly, which guided the rational for your approach based on pilot studies to develop the best techniques.

I have no objection on the publication of this paper. Although the use of fluorescent powders to mark and track small arthropods is not new, the method and approach presented in this manuscript differs from typical methodologies in that the study was conducted in-field rather than in an enclosed environment. Hence, by directly applying the marks in a field setting, the potential impacts on vector behaviour were reduced. This research provides a basis to build on in further study.

Detailed comments:

Line 98. change 'they' to 'the'

'UV led' and 'UV LED' were used in text and Figure titles, please use one style for consistency.

For clarity add in Figure 4 title that the data presented is from 5 different source plants.

Line 332. In the discussion remove reference to Table 1. Alternatively add your specific data in the discussion compared to the literature with a reference, but I think as written with 'Table 1' removed is sufficiently clear to the reader.

Author Response

for epidemiology studies, as well as the dispersal ecology of plant insect vectors to understand dispersal behaviour and feeding which is important for understanding the patterns of invertebrate vectored plant disease spread.

Thank you. Your comments remind me that we attempted a direct powder application with the airbrush and found that most of the particles did not adhere to the surface of the insect or leaves. We added this observation in the methods section to lines 204 - 207 which now reads, “We developed a method of marking whiteflies featuring UV florescent particles in a 10% ethanol solution using cordless, USB-rechargeable airbrushes, after discovering that dry particle application with an airbrush did not result in a useful level of multi-day adhesion to the surface of live insects.”

Detailed comments:

Line 98. change 'they' to 'the'

Done, thank you.

'UV led' and 'UV LED' were used in text and Figure titles, please use one style for consistency.

Thank you for catching this, we have changed all instances to “UV LED”.

For clarity add in Figure 4 title that the data presented is from 5 different source plants.

Good suggestion, thank you!

Thank you for the suggestion, we have removed the reference to Table 1.

Reviewer 4 Report

Comments and Suggestions for Authors

The authors mentioned that 'We present evidence that a 10% ethanol mixture of UV fluorescent particles ranging in size from 5 µm to 50 µm can be used to simulate sporulating fungal plant disease infections and as a means for marking and tracking small arthropods that vector plant diseases.' in the discussion. However, The results or data are not good enough to 'simulate'. Furthermore, authors must present environmental data from the location where the study was conducted.

Thanks,

Author Response

Thank you for the comments. We have changed the wording in the first sentence of the Discussion section to reflect the comments about our use of the word simulate. It is indeed a poor word choice here and not consistent with the rest of the manuscript when we propose this method as a surrogate. The sentence now reads, “We present evidence that a 10% ethanol mixture of UV fluorescent particles ranging in size from 5 µm to 50 µm may be used to as a surrogate for sporulating fungal plant disease infections and as a means for marking and tracking small arthropods that vector plant diseases.”. We also used the word can, originally, which was a poor choice for demonstrating some ambiguity, so we changed “can” to “may”, which is a more accurate representation of the results from our methodological studies.

We have provided the environmental data from the settlement velocity study as it is true that relative humidity and temperature can play an important role in these measurements. This information is presented in the revision (lines 174-177) and now reads, “In a stable room environment (~21°C, ~ 45% relative humidity), we released spores gradually from the top of the chamber opening for ~ 20 seconds while the digital camera recorded a continuous movie from 5 seconds before to 10 seconds after release of the UV fluorescent powder.”

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have revised the manuscript as per my suggestions and now it is in acceptable form

Reviewer 4 Report

Comments and Suggestions for Authors

The authors responded my comments well.

Thanks,