Frost Cracks Show a Slight Effect on Fungal Richness in Stem Wood of Hybrid Aspen Trees in Latvia

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is an interesting paper about phytopathological risks about frost crack damage as a possible infection gateway of fungal infections. The data interpreted carefully and conclusions are justified. It is generally well-written, but it still needs some improvements as follows:

1. Line 92, Is this method of isolation invented in this article, or is it supported by literature? If you made it, please give more details about how the sample was processed, how it was cut into pieces, what size the core was, what diameter the culture media was put in, how it was "surface-flamed," and so on. Please give sources if there is literature support.

2. Line 151, The names of genera need to be italic.

Author Response

Reviewer 1

Comment 1: Line 92, Is this method of isolation invented in this article, or is it supported by literature? If you made it, please give more details about how the sample was processed, how it was cut into pieces, what size the core was, what diameter the culture media was put in, how it was "surface-flamed," and so on. Please give sources if there is literature support.

Response 1: No, this is a standard protocol we use in our lab and other labs of phytopathology to isolate wood-inhabiting fungi. Reference to the previous studies (Arhipova et al. 2015; Burņeviča et al. 2016; Zaļuma et al. 2022) using the same approach is included in the revised version of the manuscript. The text was changed as follows (l.93-96): “Each tree core without the bark layer was split into approx. 8 cm long pieces and, after sterilization by flame, each piece was individually placed in a 9 cm plastic Petri dish with Hagem agar media (5 g glucose, 0.5 g NH4NO3, 0.5 g MgSO4 7H2O, 5 g malt extract, 20 g agar, 1000 mL distilled H2O at pH 5.5) and incubated at room temperature [9, 10, 12, 27].”

Comment 2: Line 151, The names of genera need to be italic.  

Response 2: Changed to italic.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

This study undertook an important problem related to the colonization of frost cracks on poplar trunks in connection with losses caused by rot fungi. The research material that is analyzed is large and what is questionable is the fact that the Authors did not describe the age of the wounds. Did the damage appeared a year or two ago or...etc. Similarly, the size of the damage, the size of the wound, its length and width were not determined. The age of the cracks and its size plays an important role  on possibility of  fungal spores infections, consequently, the likelihood of successful colonization of such a wounds. In interpreting the results, the authors rely solely on identifying the genus and determining its general characteristics. However, for example, in the genus Cladosporium we find not only litter saprotrophs, as the Authors suggested, but also endortophs and pathoegns. Unfortunately, the traditional method of isolation and identification of fungi from wood without the support of molecular biology methods allows for the detection of a rather limited number of organisms. The work requires detailed information regarding frost cracks and its impact on all described indexes.

Author Response

Reviewer 2

Comment 3: The research material that is analyzed is large and what is questionable is the fact that the Authors did not describe the age of the wounds. Did the damage appeared a year or two ago or...etc. Similarly, the size of the damage, the size of the wound, its length and width were not determined. The age of the cracks and its size plays an important role  on possibility of  fungal spores infections, consequently, the likelihood of successful colonization of such a wounds.

Response 3: Frost cracks were detected in the analyzed stands after winter 2012/2013 and consequently presence/absence of cracking wounds was observed in 2013. These data are published by Čakšs et al. 2022. Unfortunately, they mainly fosused on presence/absence of the wounds and not the exact size. Nevertheless, we have added more explicit information of stem wounds based on previous studies and added a reference to Čakšs et al. 2022. Text in Materials and Methods has been modified as follows (l.75-77): “According to an inventory in 2013 and 2020, the studied trials had a high occurrence of trees with stem frost cracks, which had likely formed in the winter of 2012/2013, hence their age was approximately 10 years old; their length range 5–85 cm [5].” 

Comment 4: In interpreting the results, the authors rely solely on identifying the genus and determining its general characteristics. However, for example, in the genus Cladosporium we find not only litter saprotrophs, as the Authors suggested, but also endortophs and pathoegns.

Response 4: Indeed, in fungal kingdom genera might contain species with different ecologies. Hence, the analysis at the genus level most likely results in an underestimation of the functional diversity of the fungal community. This is now acknowledged in the text l. 286-289: “Though it must be acknowledged that the interpretation was based on the most common ecological groups of genera, which however can contain species of different ecology [33, 61], hence functional diversity might be underestimated.” Reference to the endophytic and pathogenic lifestyle of Cladosporium species has been added in the Discussion as follows (l.256-258): “Some clonal effect was detected for fungal genus Cladosporium which generally has saprotrophic lifestyle but may be present in plant tissues as endophyte or even pathogen [61] (Table 4).”

Comment 5: Unfortunately, the traditional method of isolation and identification of fungi from wood without the support of molecular biology methods allows for the detection of a rather limited number of organisms.

Response 5: We agree that the method used in this study is not perfect. Nevertheless, the media used is appropriate for the isolation of wood-inhabiting fungi; the isolations approach helps to detect living and active mycelia and therefore avoids false positives as only DNA sequencing records without living fungi are present. Lastly, our analysis was accompanied by molecular detection of isolated fungi, and most of them are identified at the species level which is shown in the Supplementary table.  In the Discussion, the following remark was added (l.290-292): “Also, some specific taxa might be underrepresented due to the application of cultivation media, although Hagem agar media is a selective media particularly aimed for slow-growing wood-inhabiting fungi [27].”

Comment 6: The work requires detailed information regarding frost cracks and its impact on all described indexes.

Response 6: Unfortunately, we do not fully understand this objection, as we tested only fungal community richness represented by Shannon indexes and found no effect on stem cracks. Nevertheless, the information regarding the length of the frost crack is added as indicated in Response 3.

Comment 7: Additionally, I can’t find suppl table (rows 70, 131).

Response 7: Supplementary table is added to the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

Submitted manuscript for review was supposed to focus on fungal communities in poplar plantations, particularly those associated with trees exhibiting bark cracks. In the abstract, the authors indicated that they employed morphotyping and DNA isolation methods for identification. Cultivating fungi on nutrient solution significantly limits the diversity of obtained species, making it rare to isolate certain Basidiomycota species using this approach. It is well-known that this fungal group is a primary cause of decay of wood.

An intriguing aspect is that, despite successfully cultivating the fungus, the authors did not perform species identification using mycological keys. Instead, confirmation was only achieved through sequencing. Furthermore, the choice to sequence only one region (ITS) raises questions, especially when it is recommended to sequence at least three regions for accurate identification?

The identification was carried out at the genus level. While I understand that the lifestyle database relies on this taxonomic rank, it is crucial to note that within a given genus, both saprotrophic and pathogenic species can exist. 

Therefore, if the authors intended to analyze the entire community, why did they not employ metagenomic methods such as Illumina, Pac Bio, or nanosequencing?

The manuscript presented for review deviates from its title, and it is imperative to rectify the methodology for a more accurate representation of the research conducted.

Author Response

Reviewer 3

Comment 8: Cultivating fungi on nutrient solution significantly limits the diversity of obtained species, making it rare to isolate certain Basidiomycota species using this approach. It is well-known that this fungal group is a primary cause of decay of wood.

Response 8: We agree that the diversity of obtained species might be underestimated due to cultivation media, which can favor specific species/taxa. Nevertheless, from artificial media used contains less sugars and therefore reduces the expansive growth of fast-growing saprotrophs. It is a selective media particularly aimed for wood-inhabiting fungi. In the Discussion, the following remark was added (l.290-292): “Also, some specific taxa might be underrepresented due to the application of cultivation media, although Hagem agar media is a selective media particularly aimed for slow-growing wood-inhabiting fungi [27].”

Comment 9: An intriguing aspect is that, despite successfully cultivating the fungus, the authors did not perform species identification using mycological keys. Instead, confirmation was only achieved through sequencing.

Response 9: Several taxa of Ascomycetes (as Penicillium and Trichoderma) were identified based on mycological keys. Nevertheless, for Basidiomycetes or Ascomycetes not forming conidia under the lab conditions provided, molecular analysis was the best approach to the identification. Materials and Methods have such text added (l.99-103): “Considering that Basidiomycetes and Ascomycetes may not form characteristic structures (e.g. conidia) under the laboratory conditions, thus being morphologically unidentifiable, fungal ITS region sequencing, which is a universal method, was used for the identification [10, 12, 28, 29].”

Comment 10: Furthermore, the choice to sequence only one region (ITS) raises questions, especially when it is recommended to sequence at least three regions for accurate identification?

Response 10: We aimed for a wide range of taxonomic groups including both wood-inhabiting Basidiomycetes and Ascomycetes. Therefore, we find the ITS region as the most universal for such a wide range of fungi. Also because of this reason we mainly operate with a genus-level identification in the analysis since species differences might be overestimated by sequencing only ITS. Materials and Methods have such text added (l.99-110): “Considering that Basidiomycetes and Ascomycetes may not form characteristic structures (e.g. conidia) under the laboratory conditions, thus being morphologically unidentifiable, fungal ITS region sequencing, which is a universal method, was used for the identification [10, 12, 28, 29]. (..) primers were used, which provides almost full ITS region sequencing and consequently longer reads than primers aiming only part of this region [28, 29].”

Comment 11: The identification was carried out at the genus level. While I understand that the lifestyle database relies on this taxonomic rank, it is crucial to note that within a given genus, both saprotrophic and pathogenic species can exist.

Response 11: Detail taxa identification to species level is presented in the supplementary table. Discussion includes the following remark (l.287-290): “Though it must be acknowledged that the interpretation was based on the most common ecological groups of genera, which however can contain species of different ecology [33, 61], hence functional diversity might be underestimated.” We aimed to operate with genus-level identification because of its precision and also because for single species there would not be much fewer replicates. Differences in ecology within the genus are discussed in the case of Cladosporium (l.257-259: “Some clonal effect was detected for fungal genus Cladosporium which generally has saprotrophic lifestyle but may be present in plant tissues as endophyte or even pathogen [61] (Table 4).). For Table 1, citing Polme et al. 2020 we state that we are aware that it is not a general view but an approximation suggested by a specific author. Other authors have also developed similar tools but this is the recent one as far as we know.

Comment 12: Therefore, if the authors intended to analyze the entire community, why did they not employ metagenomic methods such as Illumina, Pac Bio, or nanosequencing?

Response 12: We agree that other approaches might also be used for the analysis of fungal communities. Both methods focused on ITS region sequencing but the primers we used provide much longer reads as Illumina for instance. In our context, we were interested in the dominant taxa and the most active wood colonizers for which such a rough method serves well as isolation allows us to focus on the fungi present in the active form in the wood (the ones forming mycelia, colonizing wood; not dead hypha or dormant spores). The text was clarified by adding a remark on good read length provided by used primers (l.108-110): “which provides almost full ITS region sequencing and consequently longer reads than primers aiming only part of this region”.

Comment 13: The manuscript presented for review deviates from its title, and it is imperative to rectify the methodology for a more accurate representation of the research conducted.

Response 13: We agree that the initial title was not fully accurate, hence we now retitle it to “Frost cracks show a slight effect on fungal richness in stem wood of hybrid aspen trees in Latvia”. Materials and Methods have been concretized regarding stem cracks, sample preparation, and fungal cultivation, as well as fungal identification.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors prepared the manuscript according to my comments.