Fungi and Oomycetes in the Irrigation Water of Forest Nurseries
Round 1
Reviewer 1 Report
I liked the article and the experiment as the topic is important for nursery managers. This is the only place we can interact according to the IPM rules, when seedlings are sent to forest plantations we lose control of most diseases. In order to produce plants for plantings free of pests we need to put more efforts in appropriate filtering of water used for plant irrigation, which is often full of microorganisms including pathogenic fungi and oomycets. I think this last group of organisms was underestimated due to sampling (too deep in the water) and filtering (too larch pores) methods which could limit their DNA in further analysis.
Lines 11-14 and 16 –fonts are different than in further lines, should be unified
line 43 – consider citing following references
Oszako, T., Sikora, K., Belbahri, L., & Nowakowska, J. A. (2016). Molecular detection of oomycetes species in water courses. Folia Forestalia Polonica, 58(4), 246-251.
Line 56 – consider adding citation as follows
Nowakowska, J. A., Oszako, T., Borys, M., Sikora, K., Kubiak, K., & Olejarski, I. (2012). Genetic variability of Phytophthora community in natural water resources assessed with microsatellite DNA markers. Baltic Forestry, 18(1), 56-64.
Line 60 – remove space after 16,
Line 70 – there is record from Lithuania in this reference: Vitas, A., Oszako, T., Nowakowska, J. A., Sikora, K., & Stankevičienė, A. (2012). First records of Phytophthora spp. based on DNA analysis in Lithuania.
Table 1 – column with mean precipitation – remove comma and 0 after 41 as other values don’t have it
Material and methods
Line 3 – change layout of cellulose
Line 6 – what were pores of the filter paper? Whether sporangia and zoospores could be kept or passed away freely?
Line 35 – unify fonts of the whole line
Line 55 – is “n” needed for BLAST?
Line 103 – Is low percentage of Oomycota caused by filtering procedure? To my mind they could pass freely through the applied filter paper, while mycelium of fungi is usually more abundant and larger and that is why better detectable. Usually, while filtering the water for oomycetes there is a sequence of filters used: first 11 (for larger particles) than 8 (medium ones) and at the end 4 micrometers (fine particles like zoospores). This last one is used for DNA extraction and sequencing. In such a case a little bigger spores than filter micropores are kept and larger sporangia, too. Please discuss this aspect in the discussion.
Line 112 – please unify fonts
Line 115- misspelling of Phytophthora, please correct and remove extra space before Saprolegnia
Table 3 – Latin names of genus should be in italic like e.g. Chalara and so on…
Table 4 – Phytopythium put in in italic
Discussion
Line 23 – remove extra space after used
Line 25-26 – unify fonts
Line 35 – zoospores of oomycete naturally go to the water surface and are floating down with the current of river. No wonder that if samples were taken much deeper in the river (depth 0.5-1m) less Phytophthora and Pythium propagules were detected. Please comment on this in the discussion.
Line 39-40 – please unify fonts
Line 41 – unify fonts of the word “that”
Line 42- misspelling of the word “soil borne” – correct please
Line 43- when starts the new sentence use the full name of Fusarium avenaceum..
Line 44 – it is repetition of the information mentioned in the above sentence – rephrase
Lines 45-47 – correct the fonts please
Line 55- Bradysia put in italic
Author Response
I liked the article and the experiment as the topic is important for nursery managers. This is the only place we can interact according to the IPM rules, when seedlings are sent to forest plantations we lose control of most diseases. In order to produce plants for plantings free of pests we need to put more efforts in appropriate filtering of water used for plant irrigation, which is often full of microorganisms including pathogenic fungi and oomycets. I think this last group of organisms was underestimated due to sampling (too deep in the water) and filtering (too larch pores) methods which could limit their DNA in further analysis.
Response: We agree that this is an important topic and requires more attention. Regarding the sampling, we believe that the sampling method used did not limit the detection of oomycetes as the surface water was also sampled together with deeper water. The filters used are designed for microbiological work, but some oomycete structures could have passed through the filter, but, we believe, this could be only in the very beginning of filtration process. This observation is added to Discussion. Please also see our response below.
Lines 11-14 and 16 –fonts are different than in further lines, should be unified
Response: done
line 43 – consider citing following references
Oszako, T., Sikora, K., Belbahri, L., & Nowakowska, J. A. (2016). Molecular detection of oomycetes species in water courses. Folia Forestalia Polonica, 58(4), 246-251.
Response: the reference was included
Line 56 – consider adding citation as follows
Nowakowska, J. A., Oszako, T., Borys, M., Sikora, K., Kubiak, K., & Olejarski, I. (2012). Genetic variability of Phytophthora community in natural water resources assessed with microsatellite DNA markers. Baltic Forestry, 18(1), 56-64.
Response: the reference was included
Line 60 – remove space after 16,
Response: done
Line 70 – there is record from Lithuania in this reference: Vitas, A., Oszako, T., Nowakowska, J. A., Sikora, K., & Stankevičienė, A. (2012). First records of Phytophthora spp. based on DNA analysis in Lithuania.
Response: the reference was included
Table 1 – column with mean precipitation – remove comma and 0 after 41 as other values don’t have it
Response: done
Material and methods
Line 3 – change layout of cellulose
Response: done
Line 6 – what were pores of the filter paper? Whether sporangia and zoospores could be kept or passed away freely?
Response: particles >10µm are retained. This information was added to M&M.
Line 35 – unify fonts of the whole line
Response: done
Line 55 – is “n” needed for BLAST?
Response: yes, it is needed. It is a special option of BLAST analysis
Line 103 – Is low percentage of Oomycota caused by filtering procedure? To my mind they could pass freely through the applied filter paper, while mycelium of fungi is usually more abundant and larger and that is why better detectable. Usually, while filtering the water for oomycetes there is a sequence of filters used: first 11 (for larger particles) than 8 (medium ones) and at the end 4 micrometers (fine particles like zoospores). This last one is used for DNA extraction and sequencing. In such a case a little bigger spores than filter micropores are kept and larger sporangia, too. Please discuss this aspect in the discussion.
Response: the following was included in the Discussion “Besides, as structures of oomycetes are rather small, some of these could have passed through the filter, thereby affecting species richness and abundance estimates.”
Line 112 – please unify fonts
Response: done
Line 115- misspelling of Phytophthora, please correct and remove extra space before Saprolegnia
Response: corrected
Table 3 – Latin names of genus should be in italic like e.g. Chalara and so on…
Response: changed
Table 4 – Phytopythium put in in italic
Response: done
Discussion
Line 23 – remove extra space after used
Response: done
Line 25-26 – unify fonts
Response: done
Line 35 – zoospores of oomycete naturally go to the water surface and are floating down with the current of river. No wonder that if samples were taken much deeper in the river (depth 0.5-1m) less Phytophthora and Pythium propagules were detected. Please comment on this in the discussion.
Response: the following was included in the Discussion “As sampling was carried out at the 0.5-1.0 m depth, this may have also partly affected the detected diversity of oomycetes as these are more common to the water surface.”
Line 39-40 – please unify fonts
Response: unified
Line 41 – unify fonts of the word “that”
Response: unified
Line 42- misspelling of the word “soil borne” – correct please
Response: corrected
Line 43- when starts the new sentence use the full name of Fusarium avenaceum..
Response: changed
Line 44 – it is repetition of the information mentioned in the above sentence – rephrase
Response: the sentence was rephrased to avoid repetition: “Fusarium avenaceum has also been shown to be a problem in forest nurseries [56-58].”
Lines 45-47 – correct the fonts please
Response: done
Line 55- Bradysia put in italic
Response: done
Reviewer 2 Report
The manuscript by Marčiulynas et al. describes a study of fungi and oomycetes in the irrigation water of three forest nurseries in Lithuania. The manuscript is well written, and provide enough of information of how the study was done. The restictions of the study layout (only one sampling time and variable timng) should be discussed more deeply, especially how the timig of sampling could have affcetd the results and diffeneces between nurseries.
The font size varies a lot throughout the manuscript, please correct. The row numbering starts several times from the beginning.
Page 1
Lines16-20 Maybe a little less detail would improve the abstract, but the sequencing method should be mentioned, and maybe the bioinformatic pipeline.
Page 2
Line 67-69 Please reformulate to be specifically about diseases of trees and ornamental plants.
Line 70 Do you have any some statistics of how much forest nursery seedling are traded around Europe. If so this nformation could be added here and discussed later.
Line 87 How would you comment the sampling time, is the dormancy period the optimal timepoint or could the oomycete community be even more active during the growing season? How comparable are samples from autumn and spring? This should be discussed in detail later.
Page 4
Line 8 … before DNA extraction OR before further analyses.
Line 23 Does adjusted refer to dilution, if so please reformulate.
Line 34 The replication of samples is not clear. Based on what you have written it seems that you used ¼ of a filter for one extraction. Did you extract all pieces? Then you selected the 3 best based on what criteria, just amplification or also DNA quality? Please rewrite this into the M&M-section.
Page 5
Line 52 What were the remaining non-target sequences?
Line 53 Please correct to be non-fungal and non-oomycete…
Line 54 Why did you not also BLAST against UNITE database to be able to get permanent SH-codes/identifiers for the fungal sequences? Please consider also this option specially to finish the details in Supplementary Table 1.
Line 62 Please explain the rarefaction in more detail: what was the variation between library sizes, and what was the chosen library size etc.?
Line 75 typo: isngletons
Page 8
Table 3: What does the numbers mean on the Total of 30 taxa- row? One would assume them to be sums of rows, but the numbers do not mach.
Page 11
Line 32 Trakai was sampled after the winter and this should be discussed here. Also the effect of sampling time in all nurseries should be addressed.
Page 13
Line 170 Rytkönen, Petäistö miss spelled
Page 14
Line 186 a possible typo in Cech1
Author Response
The manuscript by Marčiulynas et al. describes a study of fungi and oomycetes in the irrigation water of three forest nurseries in Lithuania. The manuscript is well written, and provide enough of information of how the study was done. The restictions of the study layout (only one sampling time and variable timng) should be discussed more deeply, especially how the timig of sampling could have affcetd the results and diffeneces between nurseries.
The font size varies a lot throughout the manuscript, please correct. The row numbering starts several times from the beginning.
Response: The effect of timing of sampling is now discussed in the manuscript as follows “Indeed, as the sampling at different sites was carried out both in the autumn (Anykščiai, Kretinga and Dubrava) and in the spring (Trakai) (Table 1), the possibility should not be excluded that this has also contributed to the observed variation in richness and composition of fungal communities among different forest nurseries.”
The fonts were unified throughout the manuscript.
Page 1
Lines16-20 Maybe a little less detail would improve the abstract, but the sequencing method should be mentioned, and maybe the bioinformatic pipeline.
Response: the content was reduced and information on the sequencing method and a bioinformatics pipeline was included.
Page 2
Line 67-69 Please reformulate to be specifically about diseases of trees and ornamental plants.
Response: specified that these are e.g. P. citricola, P. cambivora and P. cactorum
Line 70 Do you have any some statistics of how much forest nursery seedling are traded around Europe. If so this nformation could be added here and discussed later.
Response: This information is provided on page 1 line 36: “Besides, as ca. 30 million of forest tree seedlings are traded annually within the EU ….”
Line 87 How would you comment the sampling time, is the dormancy period the optimal timepoint or could the oomycete community be even more active during the growing season? How comparable are samples from autumn and spring? This should be discussed in detail later.
Response: we agree that it can be differences between the dormancy and vegetation period. However, we believe the dormancy period can show more stable data, while sampling during the vegetation period can result in a higher variation among individual samples both within and among sites. Please also see the first response to Reviewer #2.
Page 4
Line 8 … before DNA extraction OR before further analyses.
Response: changed to “before further analyses”
Line 23 Does adjusted refer to dilution, if so please reformulate.
Response: changed to diluted
Line 34 The replication of samples is not clear. Based on what you have written it seems that you used ¼ of a filter for one extraction. Did you extract all pieces? Then you selected the 3 best based on what criteria, just amplification or also DNA quality? Please rewrite this into the M&M-section.
Response: Only ¼ part of each filter was used, while the remaining ¾ was kept as a backup (clarified in the M&M). This was done to secure that material will be available if original extraction fails. There were three extraction replicates, and all were used for PCR, and resulting three replicate products per sample were pooled together. This is indicated on page 4 line 35. We also added “all” to show that there were no selection and all replicates of the same sample were used i.e. pooled.
Page 5
Line 52 What were the remaining non-target sequences?
Response: plant or animal sequences
Line 53 Please correct to be non-fungal and non-oomycete…
Response: done
Line 54 Why did you not also BLAST against UNITE database to be able to get permanent SH-codes/identifiers for the fungal sequences? Please consider also this option specially to finish the details in Supplementary Table 1.
Response: as suggested, the sequences were blasted against UNITE database, but SH-codes were almost the same and with low scores for majority of sequences, likely due to lower coverage of ITS sequences in the UNITE database as compared to GenBank. As this would provide little additional information, the SH-codes were not included in Table S1.
Line 62 Please explain the rarefaction in more detail: what was the variation between library sizes, and what was the chosen library size etc.?
Response: information of the data used for rarefaction analysis is in Table 2.
Line 75 typo: isngletons
Response: corrected
Page 8
Table 3: What does the numbers mean on the Total of 30 taxa- row? One would assume them to be sums of rows, but the numbers do not mach.
Response: these are sums of columns
Page 11
Line 32 Trakai was sampled after the winter and this should be discussed here. Also the effect of sampling time in all nurseries should be addressed.
Response: please see our the first response to Reviewer #2
Page 13
Line 170 Rytkönen, Petäistö miss spelled
Response: corrected
Page 14
Line 186 a possible typo in Cech1
Response: corrected
