Hidden Fungi: Combining Culture-Dependent and -Independent DNA Barcoding Reveals Inter-Plant Variation in Species Richness of Endophytic Root Fungi in Elymus repens
Round 1
Reviewer 1 Report
Dear Authors,
I only found a few technical issues to be corrected before publication, otherwise, I think it is an excellent paper. I especially liked the "brute-force" approach.
Please carry out the following minor revisions:
L25: isolation only from asymptomatic plants
L109: -1 superscript
L231: an error, possibly from your reference manager software
L309: The authors should add the exact difference in binding position for fITS7 and ITS1.
L352: "MEA has dextrin"
Best regards.
Author Response
Many thanks for your useful comments and suggestions to improve the paper.
We have incorporated them all.
See below in bold.
I only found a few technical issues to be corrected before publication, otherwise, I think it is an excellent paper. I especially liked the "brute-force" approach.
Please carry out the following minor revisions:
L25: isolation only from asymptomatic plants (we have corrected that)
L109: -1 superscript we have corrected that)
L231: an error, possibly from your reference manager software (we have corrected that)
L309: The authors should add the exact difference in binding position for fITS7 and ITS1.(we have provided that detail now)
L352: "MEA has dextrin" (we have corrected that)
Reviewer 2 Report
Here is the review of the manuscript entitled "Combining culture dependent and independent DNA barcoding reveals inter-plant variation in species richness of endophytic root fungi in Elymus repens" written by Høyer & Hodkinson.
The aim of the study was to investigate the endophyte community in the roots of the grass species Elymus repens in Ireland. Sanger sequencing of ITS gene region was employed on the pure cultures of endophytes as well as amplicon HTS (Illumina) on root samples to assess fungal community composition and richness in 8-10 plants collected from five different sites. High inter-plant variation in fungal species richness was demonstrated. In site III 349 OTUs were identified by HTS and on the other hand, only 66 OTUs were cultured. Only 10 OTUs were assessed by both approaches and the majority of cultured endophytes were not found using HTS approach. It was recommended to use a combination of both approaches for the best assessment of root fungal species composition.
The paper needs major revision, mostly in Material and methods section which lacks some important information.The English language used in the manuscript is pretty well. The authors should carefully read my comments in the pdf document attached and revise the manuscript accordingly.
Best wishes,
Reviewer
Comments for author File: 
Comments.pdf
Author Response
Many thanks for your careful and useful review
We have incorporated all your suggestions and outline them below and highlighted in yellow in the text.
P1 We therefore, The sentence is written in active voice which is in contrast with the rest of the abstract. Please put the sentence in passive voice.
We have corrected this to the passive voice.
P1 in species richness -> in fungal species richness
We have corrected that.
P2 MEA and 2% MEA.
We have explained the difference in the text
P2 Please include citation
We have included it.
P2 Root Surface Sterilsation
We have added ‘and Endophyte Culturing’ as requested to the section heading
P2 Placed on three types of media,
We have described the culturing conditions as requested.
P2 Surface sterilised root material
We have described the amount we used for HTS. We used the root remaining after the culturing step so that the same root system was compared.
P3 DNA extraction
We have described the PCR protocol now
P3 Sequenced the ITS2 region of nrDNA
The sequences are not in a public database yet. We are currently submitting them and can add number as soon as received.
P3 See
We have changed that as requested to ‘for PCR protocols see’
P3 sequenced in one direction or both directions?
Both directions. (text added)
P3 Endophyte identification.
We have now provided justification for the identification approach taken by commenting here.
P3 Endophyte identification
We justifiy in the text now why ITS on its own was largely used for identification.
P3 Nilsson et al. 2018
We have now used reference number.
P3 We have included the Bolyen et al. reference as requested for QIIME.
P5 to P8
We have corrected the italics on those taxa.
P8 Error! Reference source not found.
We have corrected that in our endnote system.
P12 good identification of the cultured community
Dissanayake et al. used multilocus barcoding approach. Maybe you should try to employ also TEF marker beside ITS.
We did try to employ TEF but the results for amplification were not consistent enough to apply across all our samples. We cross checked to the ITS where TEF and LSU were obtained to verify the ITS identification.
Round 2
Reviewer 2 Report
Dear authors & editor,
The paper is now properly revised and all open questions are addressed. I find the manuscrpit suitable for publication in JoF.
Best, reviewer
