Prevalence in Potato of ‘Candidatus Arsenophonus Phytopathogenicus’ and ‘Candidatus Phytoplasma Solani’ and Their Transmission via Adult Pentastiridius leporinus

Round 1

Reviewer 1 Report

This manuscript gives new data regarding epidemiology of 'Ca. Phytoplasma solani' and 'Ca. Arsenophonus phytopathogenicus' in potato fields of southwestern Germany. The central Europe is affected by an emergence of a cixiid planthopper Pentastiridius leporinus, a newly adapted to agro-ecosystem as a vector of both pathogens, especially in a sugar beet fields. The manuscript gives new data on the prevalence of the two pathogens in potato and ability of the vector, P. leporinus, to transmit the two pathogens. The manuscript is well written and concise.

I have identified several methodological issues that should be resolved to make the study repeatable, comparable and more understandable. Also there are some corrections that should be made regarding nomenclature or citations.

Nomenclature corrections that needs to be made in the text are as follows:

>>>  Everywhere in the text where mentioned including the title please replace

 Candidatus Phytoplasma solani with  'Candidatus Phytoplasma solani',  and

Candidatus Arsenophonus phytopathogenicus  with  'Candidatus Arsenophonus phytopathogenicus'

>>>There are errors in writing of the insect species names, i.e., if author of the species should be within the brackets or not, and in the use of valid species names (according to all ICZN rules). In particular, the use of name Reptalus artemisiae (Becker) must be applied instead of Reptalus quinquecostatus (Dufour), because the later name is since 2020 (Emeljanov 2020, Zootaxa) a valid name of  the species previously known as Reptalus melanochaetus (Fieber). The correct name of the vector of stolbur phytoplasma is Reptalus artemisiae (Becker) not Reptalus quinquecostatus (Dufour). For details please see Emeljanov 2020 Zootaxa 4780 (1): 197–200: Nomenclatorial changes in the family Cixiidae (Homoptera, Auchenorrhyncha, Fulgoroidea), with fixation of type species of the genus Reptalus Emeljanov, 1971 and description of a new subgenus.  

Name of another insect vector needs to be referred to as Hyalesthes obsoletus Signoret, not Hyalesthes obsoletus (Signoret)

More details on corrections that should be made in the text are as follows:

Introduction:

>>> Page 2, line 46: The reference is from early 2000s but the disease has occurred since early 1990s as it is in detail explained in the cited reference Gatineu et al. 2001. Please make corrections accordingly.

>>>Page 2, line 59: Incorrect use of the name “Reptalus quinquecostatus (Dufour)” please consult Emejlanov 2020 (Zootaxa). Replace “Reptalus quinquecostatus (Dufour)” with “Reptalus artemisiae (Becker)”

>>> Page 2, lines 70-72: Transmission experiments of both pathogens with adults of Pentastiridius leporinus was recently published. Please include these data in the introduction  [24]

Materials and methods:

>>> Page 2, lines 85-86: Please give details on marker genes that were used for each pathogen identification

>>> Page 3, lines 109-110: Please provide more details on how and when the insects were identified (prior the introduction in cages or after), and how they were identified. What was the date of insect collection (very important information). At the same time, and in the same area, a very similar cixiids of the genus Reptalus are also occurring. How did you separate them? They could be easily identified under stereomicroscope, but this needs to be stated in the methods. Also, please refer to literature you used for insect identification.

I assume that number of insects in transmission experiments was 10 per cage, and that single plant was per cage, but please give these details in the text. If it was 10 insects per plant, this means that 260 insects was used in total? Please give the sex ration of insects in experiments. Please give the infection ratio for both pathogens if possibe.  Please give details how long the insects lived on the potato plants in experiments. Six weeks is a very long time for IAP for cixiid adults (they live much shorter). Have you observed how long they lived?

Author Response

Please see attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

In this article the authors examined three parameters:
1) The prevalence of Arsenophonus and stolbur in symptomatic potato in different regions of Germany.

2) The prevalence of Arsenophonus and stolbur in potato germinated from infected tubers and the effect of infection on germination rate.

3) The transmission of Arsenophonus and stolbur to potato and sugar beet plants by the planthopper P. leporinus.

I find that the study is lacking in 2 of the 3 parameters. The conclusions made in the discussion are inaccurate and have no added value to what is already known of these pathogens. The reasoning for this research is unclear in the text.
I will now review each parameter and explain my issues with each:

1)      In this parameter the authors examined the prevalence of Arsenophonus and stolbur in symptomatic potato in different regions of Germany. The authors don't specify what symptoms were seen, nor did they test any potato that did not show symptoms. These two points are important as the authors located many symptomatic potatoes with no infection of neither Arsenophonus nor Stolbur! I'm therefor unconvinced now that these two agents are indeed the pathogens causing the symptoms.
The authors do not address this data at all.
Can we be looking at a completely different agent causing disease in potato and Arsenophonus and stolbur just happen to be there? Maybe they are also present in asymptomatic potatoes?

2)      This parameter was performed and described well. However, I would've liked it if the authors included a description of symptoms, if seen, in the germinated plants.

3)      In the methods it is described that 20 potato plants and 6 sugar beet plants were cages with 10 adult planthoppers for 6 weeks.
- Are these 10 planthoppers per plant or per the whole cage?
10 insects for 26 plants is too little and not the typical number nor experimental design used for this type of experiment. How did the authors know the insects fed on all 26 plants?
- Were the leafhoppers tested for arsenophonus or stolbur? It isn't mentioned in the text. I assume at least one was infected as some plants are infected and no plants in the control are infected, but this wasn't verified.
- What is the timing of the life cycle of P. leporinus? 6 weeks is enough time for a whole colony to be formed on the plants.

I therefore find this experiment inadequate.
The authors cite an article in their discussion that already showed that P. leporinus can vector both Arsenophonus and stolbur so I don't see the need for this section in the manuscript.

In summary, I feel this manuscript is not fit for publishing in its current state.  

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

I'd like to thank the authors for addressing all my concerns. With simply a few clarifications they made me understand the transmission experiment, which I thought they would have to repeat. I now understand that it was performed well, and how it differs from what was already published before.

As an entomologist that has only worked with vectors with very short life cycles, I think it would be good if the authors added another clarification, regarding how long the planthoppers survive as adults, maybe somewhere near line 48 of the introduction.

I would also suggest to add a line clarifying how your experiment differs from that quoted in reference 17 (lines 242-243 of the discussion)- as you explained in your reply to me.

Finally, I suggest the authors add the data for the of the tubers with symptoms that had neither Arsenophonus nor Stolbur, as they are very visible in figure 1.
You can also mention this in the discussion, is it possibly a third pathogen? If you think it is, or isn't, why?

Author Response

I'd like to thank the authors for addressing all my concerns. With simply a few clarifications they made me understand the transmission experiment, which I thought they would have to repeat. I now understand that it was performed well, and how it differs from what was already published before.

We appreciate the constructive suggestions of reviewer 2 to enhance the clarity of the manuscript.

As an entomologist that has only worked with vectors with very short life cycles, I think it would be good if the authors added another clarification, regarding how long the planthoppers survive as adults, maybe somewhere near line 48 of the introduction.

We thank reviewer 2 for his suggestion. Bressan and colleagues (2009, Phytopathology, doi:10.1094/PHYTO-99-11-1289) noted, that the lifespan of adults lasted in few cases over 30 days. We have included this information as requested in the manuscript.

I would also suggest to add a line clarifying how your experiment differs from that quoted in reference 17 (lines 242-243 of the discussion)- as you explained in your reply to me.

We clarified the differences in the discussion.

Finally, I suggest the authors add the data for the of the tubers with symptoms that had neither Arsenophonus nor Stolbur, as they are very visible in figure 1.

You can also mention this in the discussion, is it possibly a third pathogen? If you think it is, or isn't, why?

We thank reviewer 2 for his suggestion and added the data in the results part (L155 – 157) and mentioned it in the discussion (L231 – 235).