Pathogenic Species of Botryosphaeriaceae Involved in Tree Dieback in an Urban Forest Affected by Climate Change
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsPlease see the file in attachment
Comments for author File: 
Comments.pdf
I am not an English native-speaker, thus I can only observe some passages and words sound a little bit improper to me. Please take some time to check the manuscript.
Author Response
REVIEWER 1
(in bold our responses)
Lines 42-45. This passage is important, since the definition of "urban forest" is often ambiguous. Please add a couple of references supporting your "urban forest" concept.
Done.
Line 69. metropolis, is (f.) this is a late Latin word in the 3rd declination but following the Greek declination just like "polis". Plural should be "metropoleis"
Done.
Line 83. Milano, please use the official name
Done.
Lines 119-120. this short description is fine, but is very general and lacks references about rational assessment of vegetation structure. Please look for some references (e.g. technical reports, university theses, ecc.)
We thank you for this suggestion. This research was carried out in an urban park, with characteristics that are quite different from other parks, there is no specific reference that could be cited. Please note that the research presented here is part of a large-scale research project conducted over more than 20 years at Parco Nord Milano by various universities, including the University of Florence (by the authors of this manuscript). A reference to these activities can be however found on the Park's website at this link: https://parconord.milano.it/laboratorio-boschi/
Line 122. add the area and coordinates of each survey plot
Done.
Line 130. check the species authors according to updated nomenclature
Done.
Line 130. You should summarize (a table could be fine) whether these species are native or not to Italy and to Milan area, or whether they are commercial hybrids i.e. what is their (hypothetical!) role in "potential" vegetation here? You may refer to POW, Dryades and Pignatti's flora, just for example. Moreover, once more, you should support your definition of "dominated by...": do you have any survey or reference about this? I don't think it it difficult to survey now, just in case.
Done. We have indicated the origin (native/non-native) of the species mentioned in the manuscript.
Lines 130-132.
Done.
Line 136. space: 40 °C
Done.
Line 141. where does this definition come from? Please provide a reference.
Done. Geographers and climatologists place Northern Italy, where Milano is located, in a transition zone between the Mediterranean climate and the continental climate. We had used the term ‘transitional’ to indicate that the area is influenced by both continental and Mediterranean climates. We have now removed this term because the sentence conveys the same meaning. We have included a reference in the manuscript that reports this.
Moreover, as you described previously, Milano and its urban hinterland experience a microclimate that is different from the neighbour countryside (or smaller towns).
Of course, Milano has a microclimate that is influenced by those well-known harmful effects such as the heat island and other harsh conditions typical of all large cities. But this does not invalidate what has been said above.
Lines 144-146. So, how did you select the twigs in asymptomatic plants especially? Which / how many twigs from which /how many plants? What do you mean by "varying lengths"? Were all the twigs at eye-level or what else? When did you collect them? You should provide the whole database in Supplementary materials and a summary table/scheme in the manuscript.
Done. We explained how the sampling was carried out.
Line 150.
Done.
Lines 154-156.
Done.
Lines 158-159. provide your main references for protocols and identification
These are routine identification protocols that we have been applying for years to these fungal taxa (see response below with reported references).
Lines 165-170. This is a result, NOT part of materials and methods!
Done
Line 172.
Done
Line 181.
Done
Line 185.
Done
Line 190. Do you have a sequencing facility in your lab or did you send the samples out in service?
Done. We have provided this information.
Lines 193- 195. I see below that you have reconstructed a phylogeny, thus you also have to add how did you set the following steps.
Done.
Lines 199. did you also set sub-samples in each plot in order to examine the variation internal to thinned and unthinned plots? This could strengthen your results.
No, because we wanted to consider the cumulative effect within the plots.
Line 211. t-test is parametri: did you check your sample distribution was normal? Otherwise, you should shift to non-parametric tests. thus, you could also apply either ANOVA or a non-parametric tool.
The assumption of normality was satisfied, and the use of the t-test for independent samples was deemed appropriate. This information has now been included in the Materials and Methods section.
Line 230-233. Did they match with one sequence per species only?
See answer below.
Lines 235-238. This phylogeny is absolutely poor and I don't understand what is your aim here. I see that there are only few sequences in GenBank for some of your species, but you should add all them or almost all. What is your aim by reconstructing this? Do you want to confirm your identification? Do you want to use your specimens to re-explore Botryosphaeraceae in toto?
Thank you for this meaningful observation. We have revised our manuscript, integrating ITS sequence information with morphological/morphometric data for a more accurate species identification. We have created a new Table (Table 1) where we have summarised the main macro- and micro-morphological characteristics of the isolates. Additionally, phylogenetic analysis has been expanded to strengthen species delimitation. We want to inform you that we have compared isolates found in this study with reference strains from our collection. We have an extensive collection of Botryosphaeriaceae fungi, which have been very well characterised at macro-, micro-morphological and molecular (multilocus sequence analyses) levels. Integrating molecular, morphological, and comparative evidence provides robust support for taxonomic identifications. Perhaps it is not irrelevant to point out here that we have been working with pathogens of the Botryosphaeriaceae family for many years. Here are some of our publications:
Carluccio, G., Benigno, A., Panzavolta, T., Vergine, M., De Bellis, L., Luvisi, A. and Moricca, S., 2025. Understanding Oak Decline in Europe: Ecological Factors, Symptoms, Causative Agents, and Management Strategies. Plant Disease, 109(9), pp.1805-1823.
Benigno, A., C. Bregant, C. Aglietti, G. Rossetto, B. Tolio, and B. T. Linaldeddu. Pathogenic fungi and oomycetes causing dieback on Fraxinus species in the Mediterranean climate change hotspot region. Frontiers in Forests and Global Change 6: 1253022. 2023.
Benigno, A., Aglietti, C., Rossetto, G., Bregant, C., Linaldeddu, B.T. and Moricca, S., 2023. Botryosphaeriaceae Species Associated with Stem Canker, Shoot Blight and Dieback of Fraxinus ornus in Italy. Forests, 15(1), p.51.
Benigno, A., Aglietti, C., Bregant, C., Linaldeddu, B.T., Montecchio, L. and Moricca, S., 2023. Endophytic, canker‑inducing Botryosphaeriaceae causing flowering ash (Fraxinus ornus) dieback in central Italy. In Abstracts of presentations at the XXVIII Congress of the Italian Phytopathological Society (SIPaV). Journal of Plant Pathol. pp. 1243.
Panzavolta, T., Panichi, A., Bracalini, M., Croci, F., Ginetti, B., Ragazzi, A., Tiberi, R. and Moricca, S., 2017. Dispersal and propagule pressure of Botryosphaeriaceae species in a declining oak stand is affected by insect vectors. Forests, 8(7), p.228.
Panzavolta, T., Panichi, A., Bracalini, M., Croci, F., Benigno, A., Ragazzi, A., Tiberi, R. and Moricca, S., 2018. Tree pathogens and their insect-mediated transport: Implications for oak tree die-off in a natural park area. Global Ecology and Conservation, 15, p.e00437.
Moricca, S., Linaldeddu, B.T., Ginetti, B., Scanu, B., Franceschini, A. and Ragazzi, A., 2016. Endemic and emerging pathogens threatening cork oak trees: Management options for conserving a unique forest ecosystem. Plant Disease, 100(11), pp.2184-2193.
Moricca, S., Ginetti, B. and Ragazzi, A., 2012. Species-and organ-specificity in endophytes colonizing healthy and declining Mediterranean oaks. Phytopathologia Mediterranea, pp.587-598.
Moricca, S., Uccello, A., Ginetti, B. and Ragazzi, A., 2012. First report of Neofusicoccum parvum associated with bark canker and dieback of Acer pseudoplatanus and Quercus robur in Italy. Plant disease, 96(11), pp.1699-1699.
Lines 239- 252. You shouldn't list here all the exact %, just add in the graph or mention the most important ones contextually to your discussion. You can increase the detail in y axis, that's enough.
Including these percentages in the text (as well as in the graph) makes the text easier to read and more accurate. This suggestion was made to us by an expert reviewer before the manuscript was sent.
Line 254. image quality is poor, images are fuzzy
The image quality is finely preserved in the original Word file, where figures appear sharp and clearly readable. The reduced quality observed in the current version is due to file compression during the submission/conversion process. We will provide all figures as separate high-resolution files (according to the journal’s guidelines) upon request and at the final submission stage.
Lines 258. did you obtain this result by comparing couple by couple in t-tests, didn't you?
To avoid ambiguity regarding the statistical interpretation, we have revised the text to clarify that the reported significance refers to overall differences in isolation frequency based on aggregated mean values across the three-year period, rather than to pairwise comparisons between individual years. In addition, the Results section has been revised to remove wording that could suggest year-by-year comparisons, and the Materials and Methods section has been updated to explicitly state that no multiple pairwise tests were performed. The interpretation related to forest management and temporal variability has been accordingly confined to the Discussion.
Line 267. In my opinion, you had better to remind that IF stays for "isolation frequency"
Done.
Line 272. this is the first time you have introduced this parameter. I see it is ommonly used in forestry, however you should briefly summarize what is, how is calculated and why it is important. Moreover, you should provide an official reference to PNM technical reports about this.
Done.
Line 277. here in the table or in discussion you need a comparison at least for temperatures. Be careful to your reference: there is a significant gradient in rainfall from north (Alps and Prealps) to south Lombardia, and Milano has higher rainfall than the Po line. Temperature comparison is of major concern, especially when dealing with heatwaves.
Try to have a look in ARPA Lombardia website, or Centro Geofisico Prealpino.
Thank you for this comment. We are pleased to see that you are familiar with the area. We are well aware that there is a significant gradient in rainfall from north to south Lombardy, and that Milan has higher rainfall than the Po line. However, please note that our study is limited to a specific site (Parco Nord) and the data we report is that provided by a local weather station.
Line 286. add a reference about identification/survey protocol of these symptoms
Done.
Line 289.
See above reply to line 254
Line 292. you have not described anywhere why Botryosphaeraceae are so important in plant pathology. Briefly remind what do they provoke and highlight that some species can display different pathogenicity depending on the local conditions. I see you have discussed this aspect but it is not so clearly related to these taxa.
Done. We have revised the manuscript by briefly adding this information.
Line 305. use Saxon genitive for humans and animals only
Done.
Lines 338-340. What are you referring to? IN this study you didn't examine the whole community composition and evenness. You can refer to your remarkable finding about the increase in Botryosphaeraceae in the thinned plot, but you should rewrite this passage.
Thank you for this very important comment. We agree that the original wording could be misleading, as we did not assess fungal community composition or evenness. In this regard, we have also modified the title to make it more consistent. We have then revised this part to refer exclusively to the observed increase in isolation frequency of Botryosphaeriaceae species in the thinned plot, avoiding any implication of dominance at the microbiome or community level.
Reviewer 2 Report
Comments and Suggestions for AuthorsMy comments on the manuscript are provided in the attachment.
Comments for author File: Comments.pdf
Author Response
REVIEWER 2
(in bold our responses)
Line 25, Neofusicoccum parvum should be written as N. parvum, and Botryosphaeria dothidea as B. dothidea.
Done.
Line 117, The dates when the study was conducted should be clearly specified.
The period during which the study was conducted has now been clearly specified in the Materials and Methods section.
In addition, since the study was carried out some time ago, why is it being published now?
The research presented here is part of a much broader research project carried out for over 20 years at Parco Nord Milano by several Universities, including the University of Florence (by the authors of this manuscript). You can find a reference to these activities on the Park's website at this link: https://parconord.milano.it/laboratorio-boschi/
During this time, the research conducted benefited from several nationally and European-funded projects, including the European Emonfur project (which you can find by searching for “Emonfur” online). This extensive activity resulted in the production of a huge amount of data, some of which has not yet been published as it is still being interpreted. In some cases, the large amount of data collected required extensive processing and analysis, which has only recently been completed. This research is one such case. The results presented here remain highly relevant and can even be considered precursors to what would happen in subsequent years, providing an explanation for the high tree mortality observed in the park. Furthermore, as they are based on long-term data (3 years), they provide a solid reference basis for studies of this type.
Line 131, F. angustifolia should be written as Fraxinus angustifolia.
Done.
Line 132, Q. cerris should be written as Quercus cerris.
Done.
Line 153, Is 2% PDA correct, or is there a mistake?
Yes, 2% PDA is correct. The culture medium was prepared at 2% (w/v) according to the protocol used in this study (this concentration was chosen intentionally). The text has been checked: it contains no errors.
Line 174, The full form of MEA should be written first.
Done.
Line 224, The morphological characteristics of the obtained species should be provided.
Done.
Pathogenicity tests should be conducted in the study to fulfill Koch’s postulates. It should be specified which fungal species was isolated from which host.
How were the isolates selected for molecular identification? Why was only one isolate used for each species in the molecular study? Only the ITS region was used for molecular identification. ITS alone is not sufficient for accurate identification; multiple gene regions should be used.
Thank you for this meaningful observation. We have revised our manuscript, integrating ITS sequence information with morphological/morphometric data for a more accurate species identification. The pathogenicity of these fungi is well established (previous studies and our own, see our publications reported below), so it was unnecessary to apply Koch's postulates. We have created a new Table (Table 1) where we have summarised the main macro- and micro-morphological characteristics of the isolates. Additionally, phylogenetic analysis has been expanded to strengthen species delimitation. We want to inform you that we have compared isolates found in this study with reference strains from our collection. We have an extensive collection of Botryosphaeriaceae fungi, which have been very well characterised at macro-, micro-morphological and molecular (multilocus sequence analyses) levels. Integrating molecular, morphological, and comparative evidence provides robust support for taxonomic identifications. Perhaps it is not irrelevant to point out here that we have been working with pathogens of the Botryosphaeriaceae family for many years. Here are some of our publications:
Carluccio, G., Benigno, A., Panzavolta, T., Vergine, M., De Bellis, L., Luvisi, A. and Moricca, S., 2025. Understanding Oak Decline in Europe: Ecological Factors, Symptoms, Causative Agents, and Management Strategies. Plant Disease, 109(9), pp.1805-1823.
Benigno, A., C. Bregant, C. Aglietti, G. Rossetto, B. Tolio, and B. T. Linaldeddu. Pathogenic fungi and oomycetes causing dieback on Fraxinus species in the Mediterranean climate change hotspot region. Frontiers in Forests and Global Change 6: 1253022. 2023.
Benigno, A., Aglietti, C., Rossetto, G., Bregant, C., Linaldeddu, B.T. and Moricca, S., 2023. Botryosphaeriaceae Species Associated with Stem Canker, Shoot Blight and Dieback of Fraxinus ornus in Italy. Forests, 15(1), p.51.
Benigno, A., Aglietti, C., Bregant, C., Linaldeddu, B.T., Montecchio, L. and Moricca, S., 2023. Endophytic, canker‑inducing Botryosphaeriaceae causing flowering ash (Fraxinus ornus) dieback in central Italy. In Abstracts of presentations at the XXVIII Congress of the Italian Phytopathological Society (SIPaV). Journal of Plant Pathol. pp. 1243.
Panzavolta, T., Panichi, A., Bracalini, M., Croci, F., Ginetti, B., Ragazzi, A., Tiberi, R. and Moricca, S., 2017. Dispersal and propagule pressure of Botryosphaeriaceae species in a declining oak stand is affected by insect vectors. Forests, 8(7), p.228.
Panzavolta, T., Panichi, A., Bracalini, M., Croci, F., Benigno, A., Ragazzi, A., Tiberi, R. and Moricca, S., 2018. Tree pathogens and their insect-mediated transport: Implications for oak tree die-off in a natural park area. Global Ecology and Conservation, 15, p.e00437.
Moricca, S., Linaldeddu, B.T., Ginetti, B., Scanu, B., Franceschini, A. and Ragazzi, A., 2016. Endemic and emerging pathogens threatening cork oak trees: Management options for conserving a unique forest ecosystem. Plant Disease, 100(11), pp.2184-2193.
Moricca, S., Ginetti, B. and Ragazzi, A., 2012. Species-and organ-specificity in endophytes colonizing healthy and declining Mediterranean oaks. Phytopathologia Mediterranea, pp.587-598.
Moricca, S., Uccello, A., Ginetti, B. and Ragazzi, A., 2012. First report of Neofusicoccum parvum associated with bark canker and dieback of Acer pseudoplatanus and Quercus robur in Italy. Plant disease, 96(11), pp.1699-1699.
Line 353, Neofusicoccum parvum should be written as N. parvum
Done.
Line 354, Botryosphaeria dothidea should be written as B. dothidea
Done.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript entitled 'Pathogenic Species of Botryosphaeriaceae Dominate the Endophytic Assemblage in an Urban Forest Affected by Climate Change' represents the diversity of fungal species on different forest trees. The authors try to link the urban forest management, climate stress and fungal pathogenicity in this research. But a major revision is necessary to improve experimental rigour, statistical robustness, and clarity in causal inference.
- The study was too old; the experiment was conducted during 2011, 2012, and 2013. The relevance of the result may not be fitted at this time.
- Most of the tables and figures are without statistical analysis. This approach must be avoided.
- The manuscript attributes pathogen increase to both climate stress and thinning, but cannot disentangle them. The extreme drought of 2011 is confounded with the thinning schedule. Rainfall is identical for both plots (Table 1), so differences are microclimatic, not climatic. This kind of study must be conducted on long-term basis (at least 10 years).
- The identity of the pathogens is in question. Only ITS information is not enough for identifying any fungal species, nowdays. Please justify with morphological evidences.
Comments for author File: Comments.pdf
Author Response
REVIEWER 3
(in bold our responses)
- The study was too old; the experiment was conducted during 2011, 2012, and 2013. The relevance of the result may not be fitted at this time.
The research presented here is part of a much broader research project carried out for over 20 years at Parco Nord Milano by several Universities, including the University of Florence (by the authors of this manuscript). You can find a reference to these activities on the Park's website at this link: https://parconord.milano.it/laboratorio-boschi/
During this time, the research conducted benefited from nationally and European-funded projects, including the European Emonfur project (which you can find by searching for “Emonfur” online). This extensive activity resulted in the production of a huge amount of data, some of which has not yet been published as it is still being interpreted. In some cases, the large amount of data collected required extensive processing and analysis, which has only recently been completed. This research is one such case. The results presented here remain highly relevant and can even be considered precursors to what would happen in subsequent years, providing an explanation for the high tree mortality observed in the park. Furthermore, as they are based on a sufficiently long period of time (3 years), they provide robust data for studies of this type. It is in fact quite rare today to obtain long-term data series on the impact of forest pathogens on the health of urban forests. Therefore, well-documented investigations such as these provide valuable information that is useful for stakeholders (urban park managers).
- Most of the tables and figures are without statistical analysis. This approach must be avoided.
Where appropriate, we have reported statistical analyses. The section on Materials and Methods and the captions of figures/tables have been revised to explicitly indicate the statistical approach used.
- The manuscript attributes pathogen increase to both climate stress and thinning, but cannot disentangle them. The extreme drought of 2011 is confounded with the thinning schedule. Rainfall is identical for both plots (Table 1), so differences are microclimatic, not climatic. This kind of study must be conducted on long-term basis (at least 10 years).
You’re right: it is challenging to disentangle the individual effects of climatic stress and thinning in field-based forest studies. The aim of this study was not to separate the effects of climatic stress and thinning (it would be a hard task!), but rather to assess their combined impact in in-field conditions. As you pointed out, the rainfall values were the same in all plots, but this was quite normal as the plots were only a few dozen metres apart. However, thinning has modified microclimatic (e.g. soil moisture, temperature and evapotranspiration) and epidemiological (e.g. host physiological impairment and the amount of spores intercepted by individual trees during the spore fall) conditions. These factors can directly affect pathogen dynamics. We have revised the manuscript to clarify that the observed differences are due to thinning-induced microclimatic and physiological impairment rather than to broad-scale climatic variation.
- The identity of the pathogens is in question. Only ITS information is not enough for identifying any fungal species, nowdays. Please justify with morphological evidences.
Thank you for this meaningful observation. We have revised our manuscript, integrating ITS sequence information with morphological/morphometric data for a more accurate species identification. We have created a new Table (Table 1) where we have summarised the main macro- and micro-morphological characteristics of the isolates. Additionally, phylogenetic analysis has been expanded to strengthen species delimitation. We want to inform you that we have compared isolates found in this study with reference strains from our collection. We have an extensive collection of Botryosphaeriaceae fungi, which have been very well characterised at macro-, micro-morphological and molecular (multilocus sequence analyses) levels. Integrating molecular, morphological, and comparative evidence provides robust support for taxonomic identifications. Perhaps it is not irrelevant to point out here that we have been working with pathogens of the Botryosphaeriaceae family for many years. Here are some of our publications:
Carluccio, G., Benigno, A., Panzavolta, T., Vergine, M., De Bellis, L., Luvisi, A. and Moricca, S., 2025. Understanding Oak Decline in Europe: Ecological Factors, Symptoms, Causative Agents, and Management Strategies. Plant Disease, 109(9), pp.1805-1823.
Benigno, A., C. Bregant, C. Aglietti, G. Rossetto, B. Tolio, and B. T. Linaldeddu. Pathogenic fungi and oomycetes causing dieback on Fraxinus species in the Mediterranean climate change hotspot region. Frontiers in Forests and Global Change 6: 1253022. 2023.
Benigno, A., Aglietti, C., Rossetto, G., Bregant, C., Linaldeddu, B.T. and Moricca, S., 2023. Botryosphaeriaceae Species Associated with Stem Canker, Shoot Blight and Dieback of Fraxinus ornus in Italy. Forests, 15(1), p.51.
Benigno, A., Aglietti, C., Bregant, C., Linaldeddu, B.T., Montecchio, L. and Moricca, S., 2023. Endophytic, canker‑inducing Botryosphaeriaceae causing flowering ash (Fraxinus ornus) dieback in central Italy. In Abstracts of presentations at the XXVIII Congress of the Italian Phytopathological Society (SIPaV). Journal of Plant Pathol. pp. 1243.
Panzavolta, T., Panichi, A., Bracalini, M., Croci, F., Ginetti, B., Ragazzi, A., Tiberi, R. and Moricca, S., 2017. Dispersal and propagule pressure of Botryosphaeriaceae species in a declining oak stand is affected by insect vectors. Forests, 8(7), p.228.
Panzavolta, T., Panichi, A., Bracalini, M., Croci, F., Benigno, A., Ragazzi, A., Tiberi, R. and Moricca, S., 2018. Tree pathogens and their insect-mediated transport: Implications for oak tree die-off in a natural park area. Global Ecology and Conservation, 15, p.e00437.
Moricca, S., Linaldeddu, B.T., Ginetti, B., Scanu, B., Franceschini, A. and Ragazzi, A., 2016. Endemic and emerging pathogens threatening cork oak trees: Management options for conserving a unique forest ecosystem. Plant Disease, 100(11), pp.2184-2193.
Moricca, S., Ginetti, B. and Ragazzi, A., 2012. Species-and organ-specificity in endophytes colonizing healthy and declining Mediterranean oaks. Phytopathologia Mediterranea, pp.587-598.
Moricca, S., Uccello, A., Ginetti, B. and Ragazzi, A., 2012. First report of Neofusicoccum parvum associated with bark canker and dieback of Acer pseudoplatanus and Quercus robur in Italy. Plant disease, 96(11), pp.1699-1699.
Lines 134-136. Is this come under treatment or natural phenomenon? How does authors assure the non-significant effect of this climatic impact on subplot 2Au (Unthinned).
Done. The triggering cause is a natural factor, namely prolonged drought. We have reworded this sentence to make it clearer.
Lines 230- 232 Only ITS information is not enough for identifying any fungal species, nowdays. Please justify with morphological evidences.
Done (see answer to point 4 above).
Line 236. Some more number of reference species must be included for phylogenetic analysis.
Done.
Line 264 Figures should be with proper statistical interpretation.
Done.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsPlease see the file in attachment
Comments for author File: Comments.pdf
Author Response
REVIEWER 1
(in bold our responses)
Lines 133- 134 source of the datum?
The source is the Park's forest inventory datasets (these are raw data not available to the public). The forestry data were gathered by foresters and researchers who were part of the panel of experts working on the park together with us. In the link which we have already sent you (https://parconord.milano.it/laboratorio-boschi/) there is a clear reference to this type of forest survey. At this link, the headings below clearly refer to forest biomass measurements. Unfortunately, the file is only in Italian, so, assuming you are not very familiar with Italian, I will translate the following points into English for you:
- obiettivi: definire una metodologia di rilievo e l’analisi sistematica dei dati ((English: objectives: to define a survey methodology and systematic data analysis)
- misurare gli accrescimenti e altri parametri biofisici dei popolamenti forestali (English: to measure growth and other biophysical parameters of forest stands)
- realizzare diverse Aree di Saggio permanenti per il monitoraggio costante (English: to create several permanent sampling areas for constant monitoring)
- realizzare un Data base per la gestione informatizzata dei dati, dei vari cavallettamenti (English: to create a database for the computerized management of data and for the various calipering (measurement of diameter at breast height, DBH)
To clarify the origin of the data, we have included in the text the sentence “according to the Park's forest inventory data”.
Line 286 I know this is the typical format of Tables in MDPI journal, but it has bad layout. I hope the final version will be better...
Thank you. To make it clear, we have moved the whole table to the next page. We hope the final version produced by the editorial team can be better.
Lines 291-294 Thank you for the further details you have added, but I still think this is not useful at all. This is not a true phylogeny, it is just a very partial graph to enforce the result of your identification. Your identification is likely to be fine, and that's all. You don't need a phylogenetic tree on such a small accession set.
We share your view on phylogenetic analysis. This is not a phylogenetic study. The accession series is limited because it is only intended to confirm the identification of fungal taxa. However, this was expressly requested by another reviewer to provide further support for taxonomic identification.
Lines 332- 335 did you verify the distribution normality prior to use parametric statistics?
Yes, we confirmed the normality of the data distribution before proceeding with parametric tests. As reported in Materials & Methods (lines 248, 249), data normality was assessed using the Shapiro–Wilk test.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe revisions have been found satisfactory.
Author Response
We sincerely thank you for your positive feedback and are pleased that the revisions have been found satisfactory.
Reviewer 3 Report
Comments and Suggestions for AuthorsDear author, please revise the manuscript in the following points
- Which purification technique is followed for which fungus? Please clarify.
- Pictorial evidence of conidial morphology of the isolated members of Botryosphaeriaceae
must be added in the revised version. - Kindly provide the details of reference isolates (used for morphological study) identified by MLST analyses
- No of reference isolates for Phylogenetic analysis must be increased.
Comments for author File: Comments.pdf
Author Response
REVIEWER 3
(in bold our responses)
Line 158 April...capitilized the first letter.
Done.
Line 181 Which technique is followed for which fungus. Please clarify.
The colonies obtained from single-hypha were those of Botryosphaeria dothidea, Diplodia corticola and Dothiorella omnivora, while those obtained from single-conidium were those of Diplodia seriata and Neofusicoccum parvum. We did not consider it appropriate to include this information in the section you suggested because such a change would alter the logical flow of the information provided. In fact, the identity of the fungal species is revealed later, in the Results section.
Line 287 Pictorial evidence is missing...please add in the revised version.
The macro- and micromorphology of the Botryosphaeriaceae reported here have been extensively described in the literature. As this work was done some time ago, you will understand that we do not have at this time fresh cultures from which to draw on to photographically depict their macro- and micro-morphology. These fungi, as we wrote, are part of our collection. As you know, members of the Botryosphaeriaceae are rather slow or recalcitrant to sporulation in culture. To represent these fungi iconographically, we would have to redo the fresh cultures and then wait approximately two months to achieve their sporulation in vitro. We have already reported the morphometric data and described the morphology of the reproductive structures of these fungi. Furthermore, we have created Table 1 specifically to meet your previous request.
Line 288 Kindly provide the detail of reference isolates identified by MLST analyses
The isolates are stored in our mycological collection held at the DAGRI Department, University of Florence, under the following codes:
Botryosphaeria dothidea -> Isolate code BD34
Neofusicoccum parvum -> Isolate code BOT29
Diplodia corticola-> Isolate code BOT 65
Diplodia seriata -> Isolate code BOT72
Dothiorella omnivora -> Isolate code BOT48
Line 301 Increase the number of reference isolates
Done.
Round 3
Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors have justified the question asked; it can be accepted for publication.
Author Response
Thank you for your comments.
