Assessing the Sensitivity of Plasmopara halstedii Isolates to Mefenoxam through Host Responses
Round 1
Reviewer 1 Report
General Comments:
Nisha et al studied the causes for “downy mildew” of sunflowers to assess the sensitivity of P. halstedii isolates to mefenoxam through host responses to infection, which is closely related to symptoms measured by disease severity and growth reduction, and host tissue reactions, such as hypersensitive reaction and necrosis of invaded cells. They used histological examinations of cross-sections of sunflower hypocotyls by a fluorescence microscope. In their studies, cluster analyses of sunflowers based on macroscopic and microscopic variables showed differentiation of groups of mefenoxam-treated sunflowers inoculated by different P. halstedii isolates. As stated, they revealed a clear difference in host responses of mefenoxam-treated susceptible sunflowers, and microscopic studies would enable them to estimate the sensitivity (resistance) of P. halstedii more accurately. Their report is interesting and meaningful for understanding detailed mechanisms for “downy mildew” of sunflowers. However, the authors still needed to address some necessary revisions with a more solid and accurate analysis and characterizations of the experiments. After all the necessary revisions, I recommend their manuscript to be considered for publication in Microorganisms.
1. In abstract, authors stated “so microscopic studies allowed us to estimate the sensitivity (resistance) of P. halstedii more accurately.” It is good to have a more accurate estimation, but how is the accuracy improved compared with previous ones? More variables identified or the data can be more quantitatively accurate? The authors should provide more analysis about the accuracy.
2. In introduction, “crop yield loss and quality degradation caused by plant pathogens can be up to 100 % in sunflowers”, is there an average estimation for the loss caused by plant pathogens? Like average percentage or in economy?
3. The authors made 8 paragraphs for the introduction part. While some of the paragraphs can be merged since they are basically introducing the similar contents. (Four or five paragraphs should be fine)
4. It is important to understand “plant responses to different pathogen variants is essential for safe crop production” what is the reason that previous studies do not include the “pathogen variants”? What specific notifications should be considered?
5. In “Figure 1. Disease rates (a, b) and heights (c, d) of mefenoxam treated and non-treated sunflower plants inoculated with different Plasmopara halstedii isolates.” It is suggested to connect the dots into lines from the same groups (Treated or Non-treated).
6. The authors observed “autofluorescence” phenomena from the plant? Was this attribute to “mefenoxam”, and what specific functional groups are responsible for the fluorescence?
7. In conclusion part, the authors did make some interesting findings about “resistant field isolates of sunflower downy mildew to mefenoxam, an earlier effective active ingredient against the pathogen, were found across Europe and in the USA” what about the cases for other areas like Asian, Arica?
Author Response
We thank Reviewer 1 for his/her helpful suggestions to improve the manuscript!
Our point-by-point responses are as follows:
- Reviewer 1: „In abstract, authors stated “so microscopic studies allowed us to estimate the sensitivity (resistance) of P. halstedii more accurately.” It is good to have a more accurate estimation, but how is the accuracy improved compared with previous ones? More variables identified or the data can be more quantitatively accurate? The authors should provide more analysis about the accuracy. Author’s reply: We changed the last sentence of the abstract as the followings: „In addition, examining tissue reactions (e.g., hypersensitive reaction, necrosis) seems more accurate to estimate the sensitivity of halstedii isolates to mefenoxam than macroscopic symptoms.”
Furthermore, we added more explanation connected to the above question in the last paragraph of the introduction part; see also point 4.
- Reviewer 1: „In introduction, “crop yield loss and quality degradation caused by plant pathogens can be up to 100 % in sunflowers”, is there an average estimation for the loss caused by plant pathogens? Like average percentage or in economy?” Author’s reply: The authors refer to the general fact that diseases can lead to total crop loss and a significant decrease in income in sunflower production in the first paragraph. Regarding the damage caused by Plasmopara halstedii, a clarification has been inserted in the new second paragraph as follows: „The potential yield loss after primary infection is often as high as 50% [2].”
- Reviewer 1: „The authors made 8 paragraphs for the introduction part. While some of the paragraphs can be merged since they are basically introducing the similar contents. (Four or five paragraphs should be fine).” Author’s reply: Paragraphs 2, 5, and 7 are merged into paragraphs 1, 4, and 6, respectively, resulting in 5 sections in the introduction.
- Reviewer 1: „It is important to understand “plant responses to different pathogen variants is essential for safe crop production” what is the reason that previous studies do not include the “pathogen variants”? What specific notifications should be considered?” Author’s reply: Previous sensitivity studies on P. halstedii have investigated several pathogen variants, but these have only studied macroscopic symptoms of treated and infected plants. For an explanation, a sentence is inserted now into the last paragraph of introduction as follows: „Furthermore, the studies that have tested the sensitivity of several P. halstedii isolates to mefenoxam are primarily in vivo tests examining macroscopic symptoms and signs of the treated and inoculated plants [16, 17, 19].” In addition, the phrase ’plant responses’ is changed into ’plant tissue responses’ in the following sentence.
- Reviewer 1: „In “Figure 1. Disease rates (a, b) and heights (c, d) of mefenoxam treated and non-treated sunflower plants inoculated with different Plasmopara halstedii isolates.” It is suggested to connect the dots into lines from the same groups (Treated or Non-treated).” Author’s reply: We do not consider it appropriate to connect the dots. On the one hand, a continuous line on a diagram usually shows a progression over time. On the other hand, connecting the points would make the diagrams less transparent, so we will not link the points if it is acceptable. The symbols and colors used at each point indicate the same groups well.
- Reviewer 1: „The authors observed “autofluorescence” phenomena from the plant? Was this attribute to “mefenoxam”, and what specific functional groups are responsible for the fluorescence?” Author’s reply: Yes, autofluorescence was observed and examined. Mefenoxam treatment alone did not induce autofluorescence in the treated plants in our studies, only when the treated plants were inoculated with the pathogen. Autofluorescence, one of the tissue reactions during host-parasite interactions, is mainly associated with the appearance of phenolic compounds (e.g., phytoalexins, lignin), which play an essential role in the plant's defense processes against the pathogen (Mouzeyar et al. 1993). The last two sentences are inserted into the Discussion part (paragraph 5)
- Reviewer 1: „In conclusion part, the authors did make some interesting findings about “resistant field isolates of sunflower downy mildew to mefenoxam, an earlier effective active ingredient against the pathogen, were found across Europe and in the USA” what about the cases for other areas like Asian, Arica?” Author’s reply: There are no data on mefenoxam resistance in the pathogen from Asia and Africa. In addition, there is very little data on sunflower downy mildew from these areas, as sunflower cultivation is less prevalent than in Europe and the USA. The following sentence was inserted into the first paragraph of the discussion part: „There are no data on mefenoxam resistance in the pathogen from Asia and Africa.”
Author Response File: 
Author Response.docx
Reviewer 2 Report
Dear colleagues, there are some comments in the attached file.
Comments for author File: Comments.docx
Author Response
We thank Reviewer 2 for his/her helpful suggestions to improve the manuscript!
Our point-by-point responses are as follows:
- Reviewer 2: Line 110-111.
«…Then planted in pots (d=8 cm) containing clean, moistened perlite and kept in a growth chamber (22 °C, 12 h photoperiod, light irradiance of 100 µE·m−2 ·s −1».
- How deep were the seeds planted? How long were they kept in the growth chamber? What is the humidity in chamber? Author’s reply: Original sentence (above) is corrected as follows: … then planted in pots (d=8 cm, depth of sowing: 1,5 cm) containing clean, moistened perlite and, except for the sporulation induction period (for 24 h at 19 °C), kept in a growth chamber for 21 days (22 °C, relative humidity: 70%, 12 h photoperiod, light irradiance of 100 µE·m−2s −1).
- Reviewer 2: Line 139
«Nine days after inoculation, plants were sprayed with bidistilled water and covered with a dark polyethylene bag».
- In what conditions were the plants under these 9 days? What was the humidity in the growth chamber?
Author’s reply: In the first 9 days plants were kept under the circumstances listed above (point 1). The following corrections were made: Then pots were placed in the dark for 24 h at 19°C (relative humidity: 90-100 %) to induce sporulation.„
- Reviewer 2: Line 305 «Cluster 2: isolates 1, 4, 7, 8 non-treated. Cluster 3: isolates 1, 4, 5, 6, 7, 9 treated»
Author’s reply: phrases above inserted to that line.
Author Response File: Author Response.docx
Reviewer 3 Report
The paper titled"Assessing the sensitivity of Plasmopara halstedii isolates to mefenoxam through host responses", the authors revealed the difference in host responses of mefenoxam-treated susceptible sunflowers, and use microscope to estimate the sensitivity.
1. In line207-213, is the hyphae tagged or labelled with flurescence? This wavelength is pretty much close to GFP, and the image do show green.
2. Transmission electron microscope may clear show the hyphae.
3. In figure 2, how do the authors tell the cell division?
Author Response
Please see the attachment.
Author Response File: Author Response.docx
