Axenic Culture and DNA Barcode Identification of Wood Decay Fungi from the Maltese Islands

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This paper adds to our knowledge on the diversity of wood decay fungi in Malta. From the 28 samples collected on 14 different dead and live host plant species, a total of 11 isolates were successfully obtained. This is quite a low success rate and may be related to the fact that only surface sterilisation of samples using 400 ml of 3% hydrogen peroxide for 3 minutes was applied. I think the isolation rates could have been significantly higher if the authors had also used a selective media for the isolation of the wood decay basidiomycetes i.e. and MEA containing 460 mg l-1 thiabendazole (added as a solution in 2 ml lactic acid) (TMEA) in 9-cm-diameter Petri dishes (Baum et al. 2003). Perhaps the authors wish to discuss this.

 

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Only moderate changes are required.

Author Response

Dear Reviewer,

Many thanks for you precious suggestions.

Your requested amendments are carried out in green.

Where possible, we carried out the necessarily amendments, including the work of Baum et al. 2003, which indeed, has been included in the Discussion. We indeed did encounter other methodologies aimed to the isolation of the basidiomycetes, however in order to maximize the resources available at that point in time, we opted to follow the methodology described in our manuscript.

Best regards,

Marco Iannaccone

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript “Axenic culture and DNA barcode identification of wood decay fungi from the Maltese Islands” aimed to analyze the diversity of wood decay fungi in the Maltese Islands and identify them by phenotypic and genotypic methods through DNA sequences the ITS region, sequences of the tef1-α gene, and the rpb2 gene. Likewise, another of his objectives was to establish the first collection of fungal cultures from wood decomposition in the Seed Bank of the Department of Biology of the University of Malta. It is an interesting work since, as the authors mention, fungal biodiversity has been estimated between 2.2 and 3.8 million species, however, many species have yet to be identified and described, so knowledge about Biodiversity and its preservation is a very relevant topic. However, I consider that the methodology and results, particularly of the molecular identification part, deserve to be carefully reviewed to expand and describe in detail the information on how the molecular identification was carried out, which I consider the fundamental part of its job. So, below, I have several comments for the authors.

 2. Materials and Methods

2.4. DNA extraction and molecular identification of WDF isolates.

 

The authors mention that “DNA extraction, amplification and sequencing of the 5' end of the nuc rDNA 28S rDNA (region including the domains D1–D3), the nuc rDNA ITS1-5.8S-ITS regions, partial tef1-α gene, and the region between domains 6 and 7 of the second largest subunit of the rpb2 gene were as described in Froslev et al. (2005), Matheny (2005a) and Amalfi and Decock (2013)”, and the three references cited in this paragraph agree in using the CTAB method; however, the work of Froslev et al. (2005), mention that the extraction of DNA from their samples was carried out according to a standard procedure (CTAB with β-mercaptoethanol, pure formaldehyde, precipitation in isopropanol over night, one or two 70% ethanol washes, and eluation in 50 –100 _L of 1% TE-buVer or water) or with a kit (DNeasy Plant Mini Kit, Qiagen, following the protocol of the manufacturer), in this work, what was the method used for DNA extraction?

 Likewise, the authors mention that the amplification of “nuc rDNA 28S rDNA (region including the domains D1–D3), the nuc rDNA ITS1-5.8S-ITS regions, partial tef1-α gene, and the region between domains 6 and 7 of the second largest subunit of the rpb2 gene..” were carried out as described by Froslev et al. (2005), Matheny (2005a) and Amalfi and Decock (2013), however, must include complete information on all primers used, with their respective references and the expected size of each amplicon.

 They also mention that “Materials and sequences used in this study are listed in Table 1” however, this table only contains information on the isolates; there is nothing regarding the sequences.

 The authors do not mention the size of each sequence obtained for each amplicon with each of the markers used (ITS, tef1-α, and rpb2). They also do not say whether the amplicons were sequenced in both directions (forward and reverse) or what phylogenetic analysis was used to corroborate the identification of their samples since the comparison with homologous sequences in the BLAST program is not sufficient for the identification.

 The authors should mention the criteria for selecting the combination of STI, tef, and rpb2 markers.

 Because species identification may be error-prone, it is recommended that sequences deposited in public repositories such as GenBank be analyzed phylogenetically, either individually or concatenated, to provide more significant support for the identification of the species.

 

On the other hand, their sequences were analyzed separately, with each marker used, or they did a concatenated analysis with the three markers.

 2.5. Cloning

In this section, the authors must describe precisely how the cloning was carried out and what its objective was, as well as specify which strains were chosen to be cloned.

 3. Results

This section should show the results obtained from the molecular identification and to corroborate the phenotypic identification, a phylogenetic tree must be included.

The access keys for their sequences in GenBank are not yet available, I suggest that they be released and made available before the publication of this work.

 4. Discussion

 

The authors should expand the discussion of their results based on the new information that they must include in the materials and methods and in the results.

Author Response

Dear Reviewer,

Many thanks for you precious suggestions.

Your requested amendments are carried out in dark green.

The manuscript “Axenic culture and DNA barcode identification of wood decay fungi from the Maltese Islands” aimed to analyze the diversity of wood decay fungi in the Maltese Islands and identify them by phenotypic and genotypic methods through DNA sequences the ITS region, sequences of the tef1-α gene, and the rpb2 gene. Likewise, another of his objectives was to establish the first collection of fungal cultures from wood decomposition in the Seed Bank of the Department of Biology of the University of Malta. It is an interesting work since, as the authors mention, fungal biodiversity has been estimated between 2.2 and 3.8 million species, however, many species have yet to be identified and described, so knowledge about Biodiversity and its preservation is a very relevant topic. However, I consider that the methodology and results, particularly of the molecular identification part, deserve to be carefully reviewed to expand and describe in detail the information on how the molecular identification was carried out, which I consider the fundamental part of its job. So, below, I have several comments for the authors.

 

2. Materials and Methods

2.4. DNA extraction and molecular identification of WDF isolates.

 

The authors mention that “DNA extraction, amplification and sequencing of the 5' end of the nuc rDNA 28S rDNA (region including the domains D1–D3), the nuc rDNA ITS1-5.8S-ITS regions, partial tef1-α gene, and the region between domains 6 and 7 of the second largest subunit of the rpb2 gene were as described in Froslev et al. (2005), Matheny (2005a) and Amalfi and Decock (2013)”, and the three references cited in this paragraph agree in using the CTAB method; however, the work of Froslev et al. (2005), mention that the extraction of DNA from their samples was carried out according to a standard procedure (CTAB with β-mercaptoethanol, pure formaldehyde, precipitation in isopropanol over night, one or two 70% ethanol washes, and eluation in 50 –100 _L of 1% TE-buVer or water) or with a kit (DNeasy Plant Mini Kit, Qiagen, following the protocol of the manufacturer), in this work, what was the method used for DNA extraction?

We clarified this point in the text.

 

 Likewise, the authors mention that the amplification of “nuc rDNA 28S rDNA (region including the domains D1–D3), the nuc rDNA ITS1-5.8S-ITS regions, partial tef1-α gene, and the region between domains 6 and 7 of the second largest subunit of the rpb2 gene..” were carried out as described by Froslev et al. (2005), Matheny (2005a) and Amalfi and Decock (2013), however, must include complete information on all primers used, with their respective references and the expected size of each amplicon.

Since these markers has been largely used for almost 10 years, we supposed that readers would be familiar with their sequences and length of amplicons… but since apparently this is not the case, we included information on all primers used in the text and added amplicons length in the table 2.

 

 They also mention that “Materials and sequences used in this study are listed in Table 1” however, this table only contains information on the isolates; there is nothing regarding the sequences.

The genbank accession numbers are in Table 2, this was added to the text.

 

 The authors do not mention the size of each sequence obtained for each amplicon with each of the markers used (ITS, tef1-α, and rpb2). They also do not say whether the amplicons were sequenced in both directions (forward and reverse) or what phylogenetic analysis was used to corroborate the identification of their samples since the comparison with homologous sequences in the BLAST program is not sufficient for the identification.

Details on sequencing were indeed already present in the text: “All the sequencing reactions were performed by Macrogen Inc. (Korea and The Netherlands), with primers LROR, LR3, LR3R, LR5 for the 28S; ITS1, ITS3 ITS4 for the ITS-5.8S region; and 2212R, 1577F, 983F, 1567R for tef1-α gene.Sequences were assembled and edited with SequencherTM 4.8 software (Gene Codes Corporation, Ann Arbor, MI, U.S.A.).” The use of multiples sequencing primers implies that amplicons were sequenced in both directions, even more than once since 4 primers, external and internal, have been used for Tef1 alpha and 28S and 3 for sequencing ITS-5,8S region.

On the other hand, barcoding of multiple DNA markers through blast searches in combination with morphological examinations of herbarium specimens has been considered a more than appropriate method of fungal identifications for decades… and inferring phylogenetic relationships of each individual isolate from Malta seems to me out of the scope of this manuscript. Since the purpose of this manuscript wasn’t to infer the phylogenetic relationships of Maltese fungal taxa, nor to study their historical biogeography or evolution of these isolates, we don’t think that including a phylogenetic inference was appropriate. Furthermore, we don’t understand exactly what the reviewer 2 had in mind: Is it a phylogenetic analysis of which Order? Or of which families? Or which genus? We have provided a multigene phylogeny for each genus collected and think this is sufficient. We reiterate that a phylogenetic megatree gathering Agaricales, Polyporales, Hymenochaetales and Russulales is not something appropriate for this study. We would also like to emphasize that to conveniently align ITS and LSU sequences or the introns present in Tef1 alpha for species of such distantly related orders would be more than challenging if not impossible.

Again, since the purpose of this manuscript wasn’t to infer the phylogenetic relationships of Maltese fungal taxa, nor to study their historical biogeography or evolution, we don’t think that including a phylogenetic inference was appropriate.

 

 The authors should mention the criteria for selecting the combination of STI, tef, and rpb2 markers.

 Because species identification may be error-prone, it is recommended that sequences deposited in public repositories such as GenBank be analyzed phylogenetically, either individually or concatenated, to provide more significant support for the identification of the species.

As always in any taxonomic study, all identifications were based on a combination of macro and micro morphological observation combined with multilocus DNA Barcoding. The accuracy and reliability of GenBank Blast searches for species identification in combination with morphological observations have been proved countless times (e.g. Meiklejohn et al 2019, Lücking et al 2020). Moreover, in order to minimize errors due to d-single locus barcode we used multiples DNA Markers.

DNA barcoding is primarily focused on species identification and classification using a standardized DNA marker, while phylogenetic analysis is a broader approach aimed at reconstructing evolutionary relationships and inferring the evolutionary history of organisms using genetic data. We don’t think that adding phylogenetic analysis would be appropriate for the scope of this manuscript.

 

On the other hand, their sequences were analyzed separately, with each marker used, or they did a concatenated analysis with the three markers.

It is not appropriate to simultaneously analyse multiple different loci in blast searches.

 

 2.5. Cloning

In this section, the authors must describe precisely how the cloning was carried out and what its objective was, as well as specify which strains were chosen to be cloned.

Details have been added tin the text.

 

3. Results

This section should show the results obtained from the molecular identification and to corroborate the phenotypic identification, a phylogenetic tree must be included.

The access keys for their sequences in GenBank are not yet available, I suggest that they be released and made available before the publication of this work.

As usual, GenBank will liberate DNA sequences after publishing of the manuscript

 

Many thanks

Best regards,

Marco Iannaccone

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript studied wood decay fungal diversity in Malta, highlighting its ecological importance and potential biotechnological applications. While the study is well-structured and presents valuable findings, there are minor issues with grammar, clarity, and consistency that need addressing. With major revisions to address grammatical errors, improve clarity, and ensure consistency, the manuscript will be suitable for publication.

 

Comments

 

Abstract

Change Fundamental study subjects to Important subjects for study.

Revise Potential application in biotechnology to Biotechnological applications.

Simplify They are able to degrade lignin, cellulose, and hemicelluloses, using enzymes that modify the chemical structure of these complex macromolecules. to They break down lignin, cellulose, and hemicelluloses using enzymes.

Replace Wood degrading nature with Ability to degrade wood.

Change Very low level of fitness to Poor condition.

Simplify Recent studies on wood decay fungal diversity in the Maltese Islands are limited to records and checklists described by a handful of authors. to Previous studies on wood decay fungi in the Maltese Islands are limited.

Revise Several surveys were carried out during the rainy season along the wooded areas of the Maltese Islands as well as in historical gardens. to Surveys were conducted during the rainy season in wooded areas and historical gardens across the Maltese Islands.

Clarify Axenic isolates as Pure cultures.

Change One of Aurificaria cf. euphoria to One identified as Aurificaria euphoria.

Clarify or omit Ganoderma resinaceum sl.

Simplify However, it was not possible to isolate the mycelium of Coriolopsis gallica which was collected and identified. to Mycelium of Coriolopsis gallica, though collected and identified, could not be isolated.

 

Introduction

Change According with to According to.

Revise 2.2 to 3.8 million species to 2.2 million to 3.8 million species.

Simplify the sentence about wood decay fungi's nutrient derivation.

Hyphenate cellulase-degrading enzymes.

Remove the space in White-rot.

Simplify the sentence about decreased yields in wood production forestry stands.

Clarify and simplify the sentence about the harm caused by WDF in public and private green areas.

Break down and clarify the sentence about decaying cavities in trees.

Replace anthropic with anthropogenic.

Clarify the sentence about WFCC and other collections for better readability.

 

Materials and Methods

Simplify Several walk-through surveys intended to observe presence of WDF were carried out... to We conducted several walk-through surveys to observe the presence of wood decay fungi (WDF)...

Correct Iphone13Pro smartphone to iPhone 13 Pro smartphone.

Replace Geo-localised with Geotagged.

Correct Basidiomes spofed to Basidiomes spotted.

Clarify or simplify Hymenium and pileus for better understanding.

Change Aseptic technique was observed to We used aseptic technique.

Revise A slice (3x1x1) cm of fresh context... to A slice measuring 3x1x1 cm of fresh context...

Avoid repetition by using clean instead of sterile for Sterile blade and sterile distilled water.

Simplify Synthetic Nutrient Agar prepared according to Elad et al. (1981) to Synthetic Nutrient Agar prepared according to Elad et al. (1981).

Simplify the sentence about voucher specimens for clarity.

Clarify including colors as including color descriptions.

Correct Cofon blue to cotton blue.

Break down the long sentence about DNA extraction, amplification, and sequencing for clarity.

Simplify Successful PCR reactions resulted in a single band observed on a 0.8% agarose gel. to Successful PCR reactions resulted in a single band on a 0.8% agarose gel.

Simplify Sequences were assembled and edited with SequencherTM 4.8 software... to Sequences were assembled and edited using SequencherTM 4.8 software.

Clarify We considered threshold value higher than 97% suitable for distinguishing species as We considered a threshold value higher than 97% suitable for species distinction.

Simplify Eight randomly chosen clones were re-amplified by direct colony PCR... to We re-amplified eight randomly chosen clones using direct colony PCR.

 

Results:

Clarify the sentence about the samples collected from dead and live host plant species.

Correct Basidiomes spofed to Basidiomes spotted.

Simplify the sentence about sampling locations for better readability.

Clarify why some sampling locations yielded multiple samples.

Ensure consistency in the format of sample identifiers.

Clarify or omit Ganoderma resinaceum sl.

Simplify the sentence about the isolation of Laetiporus sulphureus and Inonotus rickii.

Correct the typo paferns to patterns.

Simplify the sentence about damages caused by WDF for better readability.

Simplify the sentence about the percentage of orders isolated in the study.

 

Discussion

Change Aurificaria cf. euphoriae to Aurificaria euphoria.

Revise G. resniaceum to G. resinaceum.

Correct For the Maltese Islands, G. resniaceum is a new addition. to For the Maltese Islands, G. resinaceum is a new addition.

Correct identify to identified in Lastly, Pleurotus eryngii var. ferulae firstly recorded by Saccardo (1912), in our study was simply identify as Pleurotus eryngii.

hange rofing to roofing in Coriolopsis gallica previously recorded by Saccardo (1915) as Trametes hispida on rofing wood of Quercus sp...

Simplify the sentence about Laetiporus sulphureus and Inonotus rickii for better readability.

Change categorize to categorized in WDF identified in our study can be further categorize into two types: white-rot and brown-rot.

Simplify the sentence about sampled trees' fitness and sensitivity to WDF.

Simplify the sentence about the unique environmental conditions of the Maltese Islands.

Correct serves to serve and assume to also assume in Culture collections serves as ex-situ preservation of fungal specimens, assume also a pivotal scientific role.

Change afention to attention in It is crucial to pay afention to the presence of WDF...

Change contribute to contributing in By prioritizing the monitoring and management of WDF...

Comments on the Quality of English Language

Minor editing of English language required.

Author Response

Dear Reviewer,

Many thanks for you precious suggestions. Where possible, we carried out the necessarily amendments, which are highlighted in yellow.

 

Abstract

Change Fundamental study subjects to Important subjects for study.  Done

Revise Potential application in biotechnology to Biotechnological applications. Done

Simplify They are able to degrade lignin, cellulose, and hemicelluloses, using enzymes that modify the chemical structure of these complex macromolecules. to They break down lignin, cellulose, and hemicelluloses using enzymes. Done

Replace Wood degrading nature with Ability to degrade wood. Done

Change Very low level of fitness to Poor condition. We believe that the original sentence provides a better understanding, hence we would like to retain it.

Simplify Recent studies on wood decay fungal diversity in the Maltese Islands are limited to records and checklists described by a handful of authors. to Previous studies on wood decay fungi in the Maltese Islands are limited. Done

Revise Several surveys were carried out during the rainy season along the wooded areas of the Maltese Islands as well as in historical gardens. to Surveys were conducted during the rainy season in wooded areas and historical gardens across the Maltese Islands.

Clarify Axenic isolates as Pure cultures. We amended as axenic cultures.

Change One of Aurificaria cf. euphoria to One identified as Aurificaria euphoria. We would like to retain the cf. as this will instruct the reader to compare with other species.

Clarify or omit Ganoderma resinaceum sl. We would like to retain the sensu latu as we are not sure that Ganoderma resinaceum is a single species or an aggregate of species.

Simplify However, it was not possible to isolate the mycelium of Coriolopsis gallica which was collected and identified. to Mycelium of Coriolopsis gallica, though collected and identified, could not be isolated.

Introduction

Change According with to According to. Done

Revise 2.2 to 3.8 million species to 2.2 million to 3.8 million species. Done

Simplify the sentence about wood decay fungi's nutrient derivation. Done

Hyphenate cellulase-degrading enzymes. Done

Remove the space in White-rot. Done

Simplify the sentence about decreased yields in wood production forestry stands. Done

Clarify and simplify the sentence about the harm caused by WDF in public and private green areas. Done

Break down and clarify the sentence about decaying cavities in trees. Done

Replace anthropic with anthropogenic. Done

Clarify the sentence about WFCC and other collections for better readability. Done

Materials and Methods

Simplify Several walk-through surveys intended to observe presence of WDF were carried out... to We conducted several walk-through surveys to observe the presence of wood decay fungi (WDF)... Done

Correct Iphone13Pro smartphone to iPhone 13 Pro smartphone. Done

Replace Geo-localised with Geotagged. Done

Correct Basidiomes spofed to Basidiomes spotted. Done

Clarify or simplify Hymenium and pileus for better understanding. Done

Change Aseptic technique was observed to We used aseptic technique. Done

Revise A slice (3x1x1) cm of fresh context... to A slice measuring 3x1x1 cm of fresh context... Done

Avoid repetition by using clean instead of sterile for Sterile blade and sterile distilled water. We would like to retain the word sterile, as we believe it is important for the replicability of the methodology.

Simplify Synthetic Nutrient Agar prepared according to Elad et al. (1981) to Synthetic Nutrient Agar prepared according to Elad et al. (1981). Done

Simplify the sentence about voucher specimens for clarity. Done

Clarify including colors as including color descriptions. Done

Correct Cofon blue to cotton blue. Done

Break down the long sentence about DNA extraction, amplification, and sequencing for clarity. We would like to retain the sentence as it stands.

Simplify Successful PCR reactions resulted in a single band observed on a 0.8% agarose gel. to Successful PCR reactions resulted in a single band on a 0.8% agarose gel. Done

Simplify Sequences were assembled and edited with SequencherTM 4.8 software... to Sequences were assembled and edited using SequencherTM 4.8 software. Done

Clarify We considered threshold value higher than 97% suitable for distinguishing species as We considered a threshold value higher than 97% suitable for species distinction. Done

Simplify Eight randomly chosen clones were re-amplified by direct colony PCR... to We re-amplified eight randomly chosen clones using direct colony PCR. We would like to retain the sentence as it stands.

Results:

Clarify the sentence about the samples collected from dead and live host plant species. We believe that Table 1 provides further detail about the host plants.

Correct Basidiomes spofed to Basidiomes spotted. Done

Simplify the sentence about sampling locations for better readability. Done

Clarify why some sampling locations yielded multiple samples. Sampling locations yielded multiple samples due to the fact that they were more trees.

Ensure consistency in the format of sample identifiers. Apologies, but we are not able to identify the requested adjustment.

Clarify or omit Ganoderma resinaceum sl. We would like to retain the sensu latu as we are not sure that Ganoderma resinaceum is a single species or an aggregate of species.

Simplify the sentence about the isolation of Laetiporus sulphureus and Inonotus rickii. We would like to retain the sentence as it stands.

Correct the typo paferns to patterns. Done

Simplify the sentence about damages caused by WDF for better readability. Done

Simplify the sentence about the percentage of orders isolated in the study. We would like to retain the sentence as it stands.

Discussion

Change Aurificaria cf. euphoriae to Aurificaria euphoria. We would like to retain the cf. as this will confer and instruct the reader to compare with other species.

Revise G. resniaceum to G. resinaceum. Done

Correct For the Maltese Islands, G. resniaceum is a new addition. to For the Maltese Islands, G. resinaceum is a new addition. Done

Correct identify to identified in Lastly, Pleurotus eryngii var. ferulae firstly recorded by Saccardo (1912), in our study was simply identify as Pleurotus eryngii. Done

hange rofing to roofing in Coriolopsis gallica previously recorded by Saccardo (1915) as Trametes hispida on rofing wood of Quercus sp... Done

Simplify the sentence about Laetiporus sulphureus and Inonotus rickii for better readability. We would like to retain the sentence as it stands.

Change categorize to categorized in WDF identified in our study can be further categorize into two types: white-rot and brown-rot. Done

Simplify the sentence about sampled trees' fitness and sensitivity to WDF. Done

Simplify the sentence about the unique environmental conditions of the Maltese Islands. Done

Correct serves to serve and assume to also assume in Culture collections serves as ex-situ preservation of fungal specimens, assume also a pivotal scientific role. Done

Change afention to attention in It is crucial to pay afention to the presence of WDF... Done

Change contribute to contributing in By prioritizing the monitoring and management of WDF... Done

 

Many thanks

Best regards,

Marco Iannaccone

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Given the importance of islands and their adjacent near-shore marine areas, this manuscript carried out the axenic culture and DNA barcode identification of wood decay fungi from the Maltese Islands and aimed to provide a comprehensive description of wood decay fungal diversity in the Maltese Islands. It is significant comparatively about this research. But there are some questions as follows:

1.Only 28 samples were collected during 3 years. It’s too few, especially according to the wood decay fungal diversity studying. And authors didn’t analysis the reason about so little sampling results. This result can not provide more useful information for readers.

2.This manuscript emphasized the importance about wood decay fungi, it is OK. But as a scientific paper, new result is the core. But this manuscript lack of some important results.

3. There some errors about reference. Reference 1, 2, 51, the websites should be listed in the text with brackets. Reference 53, 54, the titles of literature should not be in capital all. Reference 60, 64, 68, 69, the species and genera names should be in italic.

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

Many thanks for you precious suggestions. Where possible, we carried out the necessarily amendments, which are high lighted in Purple.

Given the importance of islands and their adjacent near-shore marine areas, this manuscript carried out the axenic culture and DNA barcode identification of wood decay fungi from the Maltese Islands and aimed to provide a comprehensive description of wood decay fungal diversity in the Maltese Islands. It is significant comparatively about this research. But there are some questions as follows:

1.Only 28 samples were collected during 3 years. It’s too few, especially according to the wood decay fungal diversity studying. And authors didn’t analysis the reason about so little sampling results. This result can not provide more useful information for readers.  Done

2.This manuscript emphasized the importance about wood decay fungi, it is OK. But as a scientific paper, new result is the core. But this manuscript lack of some important results.

We believe that the novelties of our paper are:  

1. No DNA barcoding has ever been carried out on isolates of the Maltese islands
2. We indirectly characterised plant species which are more susceptible to WDF and therefore suggested to be avoided in urban landscaping.
3. The establishment of a living Mycotheca, and as far as we know, is the only one in the entire nation.
4. There some errors about reference. Reference 1, 2, 51, the websites should be listed in the text with brackets. Reference 53, 54, the titles of literature should not be in capital all. Reference 60, 64, 68, 69, the species and genera names should be in italic. Done

 

Many Thanks

Best regards,

Marco Iannaccone

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

In the new version of the manuscript “Axenic culture and DNA barcode identification of wood decay fungi from the Maltese Islands,” some modifications were considered in response to the comments issued. However, perhaps I was not clear enough, particularly in the molecular identification part of the isolates included in this work, so I have more comments below.

When I mentioned that they should include complete information on all the primers used, with their respective references and the expected size of each amplicon, it is because although these markers have been used for almost ten years, as you mention, the methodologies described in any work of research, must be clear and precise enough, so that they can be replicated by other researchers anywhere in the world, so that it should not be assumed that interested readers know the conditions and characteristics of the methodology.

On the other hand, I regret that I was not clear in the comment related to the phylogenetic analysis in order to corroborate the identification of your isolates. In this sense, the authors used three markers (ITS, tef1-α, and rpb2), and the identification was carried out by phenotypic characterization and comparison of their sequences with sequences from the GenBank. In this way, the authors identified the following species:

1.      Aurificaria cf. euphoriae,

2.      Ganoderma resinaceum sl.,

3.      Laetiporus sulphureus,

4.      Inonotus sp.,

5.      Inonotus rickii:

6.      Ptychogaster cubensis,

7.      Inocutis tamaricis,

8.      Pleurotus eryngii,

9.      Stereum hirsutum, and

10.  Coriolopsis gallica.

 

and they mention that “barcoding of multiple DNA markers through blast searches in combination with morphological examinations of herbarium specimens has been considered a more than appropriate method of fungal identifications for decades.” however, the isolate identified only as Inonotus sp. it could not be recognized at the species level, in addition to not amplifying with the Tef1 marker (no accession number for the LSU marker), demonstrating that barcode markers have their drawbacks. So when I mentioned phylogenetic analysis, I was not referring to analysis between different genera, orders or families, but rather an analysis between species; in this way, when analyzing by a method of maximum likelihood or Bayesian inference, isolates of the same species with Genbak reference sequences, they could also corroborate the identification, providing greater robustness to their results and perhaps the isolate that was only identified at the genus level could have been identified at the species level.

 

On the other hand, the sequence analysis of the internal transcribed spacer region of the rDNA (ITS1-5.8S-ITS2) has been preserved as the bar code on the official DNA for fungi (Schoch et al.,2012) because it is the most frequently sequenced marker in fungi and it involves primers that can hybridize with practically any fungal DNA. In addition, the International Society for Human and Animal Mycoses has provided a rehabilitated database of ITS sequences that is particularly useful for identifying fungi from the clinical origin (Irinyi et al., 2015)]. However, the use of the ITS region as a barcode has been criticized by Kiss et al. (2012) due to its inability to distinguish many closely related fungal species. For this reason, and since the ITS region of some fungal species is not polymorphic enough, there is a need for a secondary ID marker to identify a given species accurately.

 

References

1.      Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA, et al. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proc Natl Acad Sci U S A 2012;109:6241e6.

2.      Irinyi L, Serena C, Garcia-Hermoso D, Arabatzis M, Desnos-Ollivier M, Vu D, et al. International Society of Human and Animal Mycology (ISHAM)-ITS reference DNA barcoding data based the quality controlled standard tool for routine identification of human and animal pathogenic fungi. Med Mycol 2015;53:313e37.

 

 

Author Response

Dear Reviewer,

Many thanks for helping us to improve the manuscript. Kindly see below our replies. Attached final version of the manuscript. 

In the new version of the manuscript “Axenic culture and DNA barcode identification of wood decay fungi from the Maltese Islands” some modifications were considered in response to the comments issued. However, perhaps I was not clear enough, particularly in the molecular identification part of the isolates included in this work, so I have more comments below.

When I mentioned that they should include complete information on all the primers used, with their respective references and the expected size of each amplicon, it is because although these markers have been used for almost ten years, as you mention, the methodologies described in any work of research, must be clear and precise enough, so that they can be replicated by other researchers anywhere in the world, so that it should not be assumed that interested readers know the conditions and characteristics of the methodology.

All primers information and references were already in the in the text, but we added a table resuming primers information’s and PCR programs used of each locus.

On the other hand, I regret that I was not clear in the comment related to the phylogenetic analysis in order to corroborate the identification of your isolates. In this sense, the authors used three markers (ITS, tef1-α, and rpb2), and the identification was carried out by phenotypic characterization and comparison of their sequences with sequences from the GenBank. In this way, the authors identified the following species:

1. Aurificaria cf. euphoriae,
2. Ganoderma resinaceum sl.,
3. Laetiporus sulphureus,
4. Inonotus sp.,
5. Inonotus rickii
6. Ptychogaster cubensis,

Kindly note that Ptychogaster cubensis, as explained in the text, IS the anamorph of Inonotus rickii (so they represent a single taxon).

7. Inocutis tamaricis,
8. Pleurotus eryngii,
9. Stereum hirsutum, 
10. Coriolopsis gallica.

and they mention that “barcoding of multiple DNA markers through blast searches in combination with morphological examinations of herbarium specimens has been considered a more than appropriate method of fungal identifications for decades.” however, the isolate identified only as Inonotus sp. it could not be recognized at the species level, in addition to not amplifying with the Tef1 marker (no accession number for the LSU marker), demonstrating that barcode markers have their drawbacks. So when I mentioned phylogenetic analysis, I was not referring to analysis between different genera, orders or families, but rather an analysis between species; in this way, when analysing by a method of maximum likelihood or Bayesian inference, isolates of the same species with Genbak reference sequences, they could also corroborate the identification, providing greater robustness to their results and perhaps the isolate that was only identified at the genus level could have been identified at the species level.

There are between 115000 to 140000 currently accepted fungal species but DNA sequences for only 70 000 species hypotheses deposited on GenBank (Lücking and Hawksworth 2018), meaning that at least half of known Fungal species misses DNA sequences. For example, there are 287 available names for species of Inonotus (fide Indexfungorum), but NCBI Taxonomy has documented only 49 Inonotus species. Considering that several of them are misidentifications, and many of them misses DNA sequences the real number is even less.

Thus, it is not surprising to NOT succeed in identifying a collection at species level based on DNA sequences. And building a phylogenetic tree with the same genetic loci wouldn’t change the fact that if similar sequences are missing from public databases, and morphology is not clear enough, some collections such as the single collection UMBmyc17-2021FL*, would remain unidentified at species level.  Again, since adding a phylogenetic study of each genus collected would not be appropriate for the scope of this manuscript, and furthermore, we have a single collection of Inonotus sp. that we couldn’t resolve at species level, we decided NOT to include a phylogenetic study of Inonotus sensu lato. It is much likely that the collection UMBmyc17-2021FL identified as Inonotus sp represents a new , undescribed taxon (the closest match on BLAST searches was Phellinus sp. BAB-4965 at 94,4%) , but since for the moment there is only one collection available, thus not enough in order to properly describe the species or evaluate the intra and interspecific variability, both genetical and morphological, as for the time being we prefer to leave it as Inonotus sp. and wait to gather more data to fully a formally describe this taxon.

On the other hand, the sequence analysis of the internal transcribed spacer region of the rDNA (ITS1-5.8S-ITS2) has been preserved as the bar code on the official DNA for fungi (Schoch et al.,2012) because it is the most frequently sequenced marker in fungi and it involves primers that can hybridize with practically any fungal DNA. In addition, the International Society for Human and Animal Mycoses has provided a rehabilitated database of ITS sequences that is particularly useful for identifying fungi from the clinical origin (Irinyi et al., 2015)]. However, the use of the ITS region as a barcode has been criticized by Kiss et al. (2012) due to its inability to distinguish many closely related fungal species. For this reason, and since the ITS region of some fungal species is not polymorphic enough, there is a need for a secondary ID marker to identify a given species accurately.

We agreed that ITS region of some fungal species could be not polymorphic enough or, sometimes, too polymorphic and variable even between individual of the same species, and thus we used several markers (Tef1-alpha, nrLSU and ITS).

Deeply thankful.

Best regards,

Marco

Reviewer 3 Report

Comments and Suggestions for Authors

The authors diligently incorporated revisions based on the reviewer's insightful comments. Congratulations on the outstanding work!

Author Response

Many thanks! Attached the latest version. 

Reviewer 4 Report

Comments and Suggestions for Authors

In fact, the important reason of this manuscript being rejected was the few innovation and significative conclusions. Although some error was corrected in the second vision, the key problem has not been solved. As  the fungi growing on the deadwood of island which is important and especial studying site, the DNA barcoding  of them should be comparing with the same species with other habitat and discuss the value of these fungi instead of listing simply.  And the importance of protecting these fungi will be obvious. It seemed that the authors couldn't recognized this.

Author Response

Dear Reviewer, 

Many thanks for helping us to improve the manuscript.

We believe your suggestion have been incorporated where possible.

There are between 115000 to 140000 currently accepted fungal species but DNA sequences for only 70 000 species hypotheses deposited on GenBank (Lücking and Hawksworth 2018), meaning that at least half of known Fungal species misses DNA sequences. For example, there are 287 available names for species of Inonotus (fide Indexfungorum), but NCBI Taxonomy has documented only 49 Inonotus species. Considering that several of them are misidentifications, and many of them misses DNA sequences the real number is even less.

Thus, it is not surprising to NOT succeed in identifying a collection at species level based on DNA sequences. And building a phylogenetic tree with the same genetic loci wouldn’t change the fact that if similar sequences are missing from public databases, and morphology is not clear enough, some collections such as the single collection UMBmyc17-2021FL*, would remain unidentified at species level.  Again, since adding a phylogenetic study of each genus collected would not be appropriate for the scope of this manuscript, and furthermore, we have a single collection of Inonotus sp. that we couldn’t resolve at species level, we decided NOT to include a phylogenetic study of Inonotus sensu lato. It is much likely that the collection UMBmyc17-2021FL identified as Inonotus sp represents a new , undescribed taxon (the closest match on BLAST searches was Phellinus sp. BAB-4965 at 94,4%) , but since for the moment there is only one collection available, thus not enough in order to properly describe the species or evaluate the intra and interspecific variability, both genetical and morphological, as for the time being we prefer to leave it as Inonotus sp. and wait to gather more data to fully a formally describe this taxon.

We agreed that ITS region of some fungal species could be not polymorphic enough or, sometimes, too polymorphic and variable even between individual of the same species, and thus we used several markers (Tef1-alpha, nrLSU and ITS).

Kindly find attached final version of the manuscript.

Deeply thankful.

Best regards,

Marco Iannaccone OBO Authors.