Cultivable Fungal Endophytes in Roots, Rhizomes and Leaves of Posidonia oceanica (L.) Delile along the Coast of Sicily, Italy

Round 1

Reviewer 1 Report

The manuscript by Torta et al. 2022, ‘Fungal endophytes in roots, rhizomes and leaves of Posidonia oceanica (Linnaeus) Delile in the Central-Southern Mediterranean (Coasts of Sicily, Italy)’ examines the presence of culturable fungal endophytes from a ubiquitous seagrass species. The manuscript attempts to document a more holistic account of the fungal endophytes present in P. oceanica as an extension of earlier studies by Torta et al. (2015) who investigated DSE of the same plant species. The most obvious problem I see with this manuscript submission is that this is not an appropriate journal as the manuscript is investigating fungal identifications with insufficient investigation of links to physiology of the plant ‘host’ – perhaps the authors should consider a more fungal centric journal?  Furthermore, the aims of the investigation are vague and in my opinion are not supported by a robust rationale.  I understand the importance of an ‘observational’ type study to see what fungal endophytes are present in specific plant tissue, but I am not convinced the employed method generates data sufficient to 2022 standards of microbial identification in complex assemblages.  I also find the conclusions of the research quite underwhelming and mostly just a ‘rehash’ of that described by Torta et al. 2015.  I do like that the authors include microscopic observation of the plant tissue, but unfortunately, I find the links to the molecular identification of fungal endophytes disjointed and links to plant physiology mostly overlooked.

 

Specific comments

 

Line 17-24       I find the abstract quite vague and not a precise enough description of what was done.  For example, ‘evaluate’ is too vague and does not capture exactly what you did – we isolated culturable fungal endophytes, identified them using rRNA sequencing etc.

 

Line 22            what is meant by asymptomatic plant in this instance?

 

Line 35            superfluous sentence – references contain detail when work was done

 

Line 51- 54      The aims seem a bit ‘padded out’ to me and like I previously commented, the rationale provided in the preceding introduction does little to justify the importance and significance of the aims.  I am left wondering why I would care about the Lulwoana sp. fungus and its importance to P. oceanica.

 

Line 207          I assume that measurement of ‘leaf biometry’ is a proxy for plant health with the intention to show that all plant tissue collected was healthy?  If so, the authors could better describe this measurement (line 237-250 does do this somewhat, but the intention of this method could be included in the methods). Perhaps incorporate other measures of specific habitat with the view to include plants from variable habitat and include impact of temporal variability.  In my opinion, if you are going to investigate endophytes of one species, you need to consider temporal and spatial variability (particularly habitat variability – the author says P. oceanica is found upto 50m depth).

 

Line 365-367   There is no discussion of fungal culturability (are there endophytes that could not be cultured using the method employed).

 

Line 381          I was curious about the sequencing method used (referred to Philips et al. 2013).  I needed to trace references back through 3 manuscripts to find a description of the actual method used (early 2000s).  My first problem here obviously refers to correct referencing (use the original reference!). Secondly, surely sequencing technology has moved on in ~20 years – specifically dealing with complex environmental samples.  I am not suggesting the approach used generates bad data, more suggesting that more modern approaches could generate significantly more insight to the microbiome of these seagrasses.  I think this is particularly pertinent given the limited scope of this manuscript. 

Author Response

The manuscript by Torta et al. 2022, ‘Fungal endophytes in roots, rhizomes and leaves of Posidonia oceanica (Linnaeus) Delile in the Central-Southern Mediterranean (Coasts of Sicily, Italy)’ examines the presence of culturable fungal endophytes from a ubiquitous seagrass species. The manuscript attempts to document a more holistic account of the fungal endophytes present in P. oceanica as an extension of earlier studies by Torta et al. (2015) who investigated DSE of the same plant species. The most obvious problem I see with this manuscript submission is that this is not an appropriate journal as the manuscript is investigating fungal identifications with insufficient investigation of links to physiology of the plant ‘host’ – perhaps the authors should consider a more fungal centric journal?  Furthermore, the aims of the investigation are vague and in my opinion are not supported by a robust rationale.  I understand the importance of an ‘observational’ type study to see what fungal endophytes are present in specific plant tissue, but I am not convinced the employed method generates data sufficient to 2022 standards of microbial identification in complex assemblages.  I also find the conclusions of the research quite underwhelming and mostly just a ‘rehash’ of that described by Torta et al. 2015.  I do like that the authors include microscopic observation of the plant tissue, but unfortunately, I find the links to the molecular identification of fungal endophytes disjointed and links to plant physiology mostly overlooked.

 

Specific comments

 

Line 17-24       I find the abstract quite vague and not a precise enough description of what was done.  For example, ‘evaluate’ is too vague and does not capture exactly what you did – we isolated culturable fungal endophytes, identified them using rRNA sequencing etc.

Done. Line 19-22

Line 22            what is meant by asymptomatic plant in this instance?

Asymptomatic changed in healty (Line 26)

 Line 35            superfluous sentence – references contain detail when work was done

Sentence deleted.

 Line 51- 54      The aims seem a bit ‘padded out’ to me and like I previously commented, the rationale provided in the preceding introduction does little to justify the importance and significance of the aims.  I am left wondering why I would care about the Lulwoana sp. fungus and its importance to P. oceanica.

The importance of endophytic fungi in terrestrial plants and seagrasses is highlited in line 64-66; The strict relation between Lulwoana sp. and P. oceanica could be hypotized if the fungal endophyte is present in three indagated organs of the host, as confirmed by results.

Line 207          I assume that measurement of ‘leaf biometry’ is a proxy for plant health with the intention to show that all plant tissue collected was healthy?  If so, the authors could better describe this measurement (line 237-250 does do this somewhat, but the intention of this method could be included in the methods). Perhaps incorporate other measures of specific habitat with the view to include plants from variable habitat and include impact of temporal variability.  In my opinion, if you are going to investigate endophytes of one species, you need to consider temporal and spatial variability (particularly habitat variability – the author says P. oceanica is found upto 50m depth).

Description is reported in line 245-246 and 251-254.

 Line 365-367   There is no discussion of fungal culturability (are there endophytes that could not be cultured using the method employed).

We consider in the research the fungal colonies that can be cultured on agarized medium, as reported in the title of this and other studies.

Line 381          I was curious about the sequencing method used (referred to Philips et al. 2013).  I needed to trace references back through 3 manuscripts to find a description of the actual method used (early 2000s).  My first problem here obviously refers to correct referencing (use the original reference!).

Done. Original reference [59] line 477.

Secondly, surely sequencing technology has moved on in ~20 years – specifically dealing with complex environmental samples.  I am not suggesting the approach used generates bad data, more suggesting that more modern approaches could generate significantly more insight to the microbiome of these seagrasses.  I think this is particularly pertinent given the limited scope of this manuscript. 

The approach used is the standard method for fungal identification and in this work the focus are the cultivable fungal endophyte and not the whole microbiome.

 

 

Author Response File: Author Response.docx

Reviewer 2 Report

This paper presents the results of a study of fungal endophytes in a marine plant or seagrass growing along the coast of Sicily. Compared to terrestrial plants, relatively few endophytes occur in this plant species include one species one found on this plant. The paper is interesting and thorough although it needs editing with many suggestions made below. I suggest that the authors discuss more about the fungi they found. For example, Ochroconis is primarily known as an animal pathogen. What about Penicillium glabrum?  The genus Lulwoana is confined to marine habitats and this species found on this host appears to be new to science.   

Editorial suggestions:

In the title it would be helpful to include something about the marine habit of this plant as that is what makes this article of interest. If adding marine plant to the title makes it too long, I suggest deleting “the Central-Southern Mediterranean) and stating simply “along the coast of Sicily, Italy”.

I assume the editors of the journal require that author names for the plant be written out. Otherwise, the author Linneaus should be simply L. Also, no need for plant authors in the title. They appear in the first paragraph.

Are the author names of this article listed correctly here as first name, then last name? I see them listed differently on a similar article from 2015 with several of the same authors.

Abstract: ln 18, change sections to localities

Ln 19, delete very, delete comma after occasionally

Ln 20, delete possible as you already say suggest rather than prove. No need for two “fudge” words.

Ln 23-24, change to: Thus, several organs, especially roots, of Posidonia oceanica are involved in sustaining endophytic fungal diversity.  Although what do you mean by sustaining…? You should just state Thus, several organs especially roots of Posidonic oceanica harbor unusual endophytic fungi. You must write out Posidonia because the previous P. was Penicillium. This problem occurs throughout the paper.

Keywords: Words already in the title should not be listed as keywords, thus delete all but dark-septate endophyte (DSE), Lulwoana, Penicillium glabrum, Ochroconis and Xylariaceae

Introduction

Ln 30, add marine before meadows. Usually the word meadows is applied to a terrestrial area.

Ln 40, the generic name with sp. and authors does not make sense. You can use the generic name with its authors or you need a specific epithet with authors. In this case, this species has not been described so just use the generic name with the authors. One can just use the first author plus et al. when there are most than two authors, thus Lulwoana Kohlm, et al.

This fungus needs to be formally described and I suggest that these authors do it in this paper. One just needs an English description that could be based on it in culture or in situ, a type specimen or culture deposited in a registered herbarium or culture collection, and a registration number from a place such as MycoBank.

Alternatively, this could be stated such as “….in which a species of Lulwoana Kohlm. et al. was discovered in the roots of plants…

Ln 42, need a comma after DSEs), were also observed.

Ln 46, Taxa should not be capitalized (unless its at the beginning of a sentence). Delete “still”. Best not to use too much hyperboli.

At some point in the Introduction or perhaps in the Discussion, it would be good to state that there is only one described species of Lulwoana, L. uniseptata, and that this is not what you ae isolating.

Ln. 60, put P. oceanica in italics.

Ln 62, “allowed to obtain…” does not make sense in English. You could say “we obtained…”

Ln 61, the first time IF is used it would good to state what IF stands for. Also, on Ln 62, is an IF different from IFo? Oh, I see under Table 3, index of frequency on organs, index of frequency of sites. This is explained in the Methods.

Ln 65, I suggest deleting the sentence “Non-sporulating colonies were considered “sterile mycelia”. I don’t see that it adds anything especially because you identified them using molecular sequences.

Ln 66, again “allowed to group” does not make sense. Instead “and was the basis for grouping fungal isolates into nine taxa…”

Table 1. There should not be a “Sordariomycetes sp.” Delete the sp.

Ln 76, P. oceanica should be in italics.

In Table 2, P glabrum must be Penicillium glabrum. Also, write out T. microspora.

Ln 78, lower case taxa. Underline scientific names, at least the genera and species.

Ln 80, Delete “It is important to highlight that..” so it should be “All isolates of …”

1. glabrum should be in italics as should all the generic names (but not the sp.).

Table 3., put generic names in italics.

Ln 87, italicize Lulwoana and delete genus.

Ln 88, italicize L. uniseptata, the only described species in this genus.

Table 4. Do not put sp. after Lulworthiales. I don’t see any in bold font. Autocorrect made the species uniseptate so this must be corrected to uniseptata. Sea water should not be in italics.

Ln 107, delete “species”

Ln 110, do not italicize P. oceanica because everything else is.

Ln 154, insert “a”, thus “In a few cases…”

Ln 165, I admit that I do not know what are “lacunar airs” but I assume these are spaces in the leaves.

Ln 213, delete “Very”

Figure 5, % brown tissue, brown is misspelled. Why is Brown in caps? It doesn’t refer to a person, right?

Discussion

Ln 232-234, this sentence should be reversed to be less awkward. Thus, “The presence of nine different fungal taxa associated with seagrass using the isolation and identification tests. Only three of these were recurring in the host.”

Ln 235, no need to use the word family as the ending -aceae indicates that this name represents a family. Delete “assayed P. oceanica”.

Ln 238, insert marine before meadows. Change “considering” to “because of”

Lns. 241-242, these results comparing the leaf biometrics recorded during the same season should be placed in the Results section, not here in the Discussion.

A general question: are there any general differences in the three sampling sites that might explain the the biometric differences or differences in the presence/absence of endophytes?

Lns 249-250, This sentence should be the topic sentence for the next paragraph.

Ln 251, please stay with the same names for the sampling sites. Here you switch to calling Bonagia the Tyrrehnian Sea. And the other the Ionian Sea and Sicilian Channel. This is confusing. Also, are there any ecological differences between them?

Ln 261, must write out Penicillium here.

Ln 264, change “are not able” to “could not”. As stated above, the word taxa should not be in caps.

Ln 266, must check that L. uniseptata is spelled correctly.

Ln 270, delete “family to which it belongs, could”, then “produces”.

Ln 279, change “believed” to “known”. Beliefs do not belong in science.

Ln 283, change “same” to “often”

Ln 286, stricto, not strictu

Ln 302, what is the word “solubising”, maybe “solubilizing?”

Ln 309, start a new sentence near the end. This variability could be related… and undoubtedly plays…

Ln 355, I think you mean assays, not essays.

Ln 378, a “c” is missing before cetyltrimethyl…

Ln 383, delete “species” after Lulworthiales

Ln 393, an Eppendorf tube

Ln 397, Other sections

Ln 418, homoscedasticity, “s” is missing

Author Response

This paper presents the results of a study of fungal endophytes in a marine plant or seagrass growing along the coast of Sicily. Compared to terrestrial plants, relatively few endophytes occur in this plant species include one species one found on this plant. The paper is interesting and thorough although it needs editing with many suggestions made below. I suggest that the authors discuss more about the fungi they found. For example, Ochroconis is primarily known as an animal pathogen. What about Penicillium glabrum?  The genus Lulwoana is confined to marine habitats and this species found on this host appears to be new to science.   

We discussed about the fungi we found in the Discussion (Line 354-377).

Editorial suggestions:

In the title it would be helpful to include something about the marine habit of this plant as that is what makes this article of interest. If adding marine plant to the title makes it too long, I suggest deleting “the Central-Southern Mediterranean) and stating simply “along the coast of Sicily, Italy”.

Done

I assume the editors of the journal require that author names for the plant be written out. Otherwise, the author Linneaus should be simply L. Also, no need for plant authors in the title. They appear in the first paragraph.

Done

Are the author names of this article listed correctly here as first name, then last name? I see them listed differently on a similar article from 2015 with several of the same authors.

The author names are now listed correctly

Abstract: ln 18, change sections to localities

Done

Ln 19, delete very, delete comma after occasionally

Done

Ln 20, delete possible as you already say suggest rather than prove. No need for two “fudge” words.

Done

Ln 23-24, change to: Thus, several organs, especially roots, of Posidonia oceanica are involved in sustaining endophytic fungal diversity.  Although what do you mean by sustaining…? You should just state Thus, several organs especially roots of Posidonic oceanica harbor unusual endophytic fungi. You must write out Posidonia because the previous P. was Penicillium. This problem occurs throughout the paper.

Done

Keywords: Words already in the title should not be listed as keywords, thus delete all but dark-septate endophyte (DSE), Lulwoana, Penicillium glabrum, Ochroconis and Xylariaceae

Done

Introduction

Ln 30, add marine before meadows. Usually the word meadows is applied to a terrestrial area.

Done

Ln 40, the generic name with sp. and authors does not make sense. You can use the generic name with its authors or you need a specific epithet with authors. In this case, this species has not been described so just use the generic name with the authors. One can just use the first author plus et al. when there are most than two authors, thus Lulwoana Kohlm, et al.

Done

This fungus needs to be formally described and I suggest that these authors do it in this paper. One just needs an English description that could be based on it in culture or in situ, a type specimen or culture deposited in a registered herbarium or culture collection, and a registration number from a place such as MycoBank. Alternatively, this could be stated such as “….in which a species of Lulwoana Kohlm. et al. was discovered in the roots of plants…

We added the ufficial description of Lulwoana sp. in line 52-55.

Ln 42, need a comma after DSEs), were also observed.

Done

Ln 46, Taxa should not be capitalized (unless its at the beginning of a sentence). Delete “still”. Best not to use too much hyperboli.

Done

At some point in the Introduction or perhaps in the Discussion, it would be good to state that there is only one described species of Lulwoana, L. uniseptata, and that this is not what you ae isolating.

Done Line 49-50.

Ln. 60, put P. oceanica in italics.

We are sorry, in the uploaded manuscript all the scientific names were in italics, maybe an error occurred after the downloads.

Ln 62, “allowed to obtain…” does not make sense in English. You could say “we obtained…”

Done

Ln 61, the first time IF is used it would good to state what IF stands for. Also, on Ln 62, is an IF different from IFo? Oh, I see under Table 3, index of frequency on organs, index of frequency of sites. This is explained in the Methods.

Done. We explained the meaning of IF also in the results (Line 78-80)

Ln 65, I suggest deleting the sentence “Non-sporulating colonies were considered “sterile mycelia”. I don’t see that it adds anything especially because you identified them using molecular sequences.

Done

Ln 66, again “allowed to group” does not make sense. Instead “and was the basis for grouping fungal isolates into nine taxa…”

Done

Table 1. There should not be a “Sordariomycetes sp.” Delete the sp.

Done

Ln 76, P. oceanica should be in italics.

See the answer above

In Table 2, P glabrum must be Penicillium glabrum. Also, write out T. microspora.

Done

Ln 78, lower case taxa. Underline scientific names, at least the genera and species.

Done

Ln 80, Delete “It is important to highlight that..” so it should be “All isolates of …”

Done

glabrum should be in italics as should all the generic names (but not the sp.).

 

Done. See the answer above

Table 3., put generic names in italics.

Done. See the answer above

Ln 87, italicize Lulwoana and delete genus.

Done. See the answer above

Ln 88, italicize L. uniseptata, the only described species in this genus.

Done. See the answer above

Table 4. Do not put sp. after Lulworthiales. I don’t see any in bold font. Autocorrect made the species uniseptate so this must be corrected to uniseptata. Sea water should not be in italics.

Done

Ln 107, delete “species”

Done

Ln 110, do not italicize P. oceanica because everything else is.

Done

Ln 154, insert “a”, thus “In a few cases…”

Done

Ln 165, I admit that I do not know what are “lacunar airs” but I assume these are spaces in the leaves.

Done

Ln 213, delete “Very”

Done

Figure 5, % brown tissue, brown is misspelled. Why is Brown in caps? It doesn’t refer to a person, right?

Done

Discussion

Ln 232-234, this sentence should be reversed to be less awkward. Thus, “The presence of nine different fungal taxa associated with seagrass using the isolation and identification tests. Only three of these were recurring in the host.”

Done

Ln 235, no need to use the word family as the ending -aceae indicates that this name represents a family. Delete “assayed P. oceanica”.

Done

Ln 238, insert marine before meadows. Change “considering” to “because of”

Done

Lns. 241-242, these results comparing the leaf biometrics recorded during the same season should be placed in the Results section, not here in the Discussion.

The sentence, with a few changes to make it a little clearer, has been moved to the results (Line 236-238).

A general question: are there any general differences in the three sampling sites that might explain the the biometric differences or differences in the presence/absence of endophytes?

Variations on such a large scale (>100 of kilometers) could reflect differences in habitat type at the locations, such as wave exposure and temperature regime.

Lns 249-250, This sentence should be the topic sentence for the next paragraph.

Done

Ln 251, please stay with the same names for the sampling sites. Here you switch to calling Bonagia the Tyrrehnian Sea. And the other the Ionian Sea and Sicilian Channel. This is confusing. Also, are there any ecological differences between them?

Done, line 323-325.

Ln 261, must write out Penicillium here.

Done

Ln 264, change “are not able” to “could not”. As stated above, the word taxa should not be in caps.

Done

Ln 266, must check that L. uniseptata is spelled correctly.

Done

Ln 270, delete “family to which it belongs, could”, then “produces”.

Done

Ln 279, change “believed” to “known”. Beliefs do not belong in science.

Done

Ln 283, change “same” to “often”

Done

Ln 286, stricto, not strictu

Done

Ln 302, what is the word “solubising”, maybe “solubilizing?”

Done

Ln 309, start a new sentence near the end. This variability could be related… and undoubtedly plays…

Done

Ln 355, I think you mean assays, not essays.

Done

Ln 378, a “c” is missing before cetyltrimethyl…

Done

Ln 383, delete “species” after Lulworthiales

Done

Ln 393, an Eppendorf tube

Done

Ln 397, Other sections

Done

Ln 418, homoscedasticity, “s” is missing

Done

 

Reviewer 3 Report

Journal: Plants

Title:  Fungal endophytes in roots, rhizomes and leaves of Posidonia oceanica (Linnaeus) Delile in the Central-Southern Mediterranean (Coasts of Sicily, Italy)

Authors: Torta L, Burruano S, Giambra S, Conigliaro G, Piazza G, Mirabile , Pirrotta M, Calvo R, Bellissimo G, Calvo S and Tomasello A

 

Comments

Lines 5-6. Fist name followed by the family name should be better: Livio Torta, Santella Burruano, etc.

Line 25-26. Keywords. DSEs à Dark Septate Endophytes (DSEs). Sicilian coasts à Sicily. Italics for genus and species names.

Line 31. Also cite: Boudouresque, C.F.; Bernard, G.; Bonhomme, P.; Charbonnel, E.; Diviacco, G.; Meinesz, A.; Pergent, G.; Pergent-Martini, C.; Ruitton, S.; Tunesi, L. Protection and Conservation of Posidonia oceanica Meadows; RAMOGE and RAC/SPA Publications: Tunis,Tunisia, 2012; p. 202.

Line 32. Also cite: Boudouresque, C.F.; Pergent, G.; Pergent-Martini, C.; Ruitton, S.; Thibaut, T.; Verlaque, V. The necromass of the Posidonia oceanica seagrass meadow: Fate, role, ecosystem services and vulnerability. Hydrobiologia 2016, 781, 25–42.

Lines 39-41. Many older studies mention Fungi within rhizomes and roots of P. oceanica, such as Corollospora maritima, Halotthia posidoniae, Pontoporeia biturbinata, etc. See e.g. Durieu and Montagne (1846), Johnson and Sparrow (1961), Kohlmeyer and Kohlmeyer (1964, 1967, 1971), Kohlmeyer (1963) and Cuomo et al (1982) [Bol. Mus. Ist. Biol. Univ. Genova, 50 (suppl.): 162-166]. Why did not the authors consider these studies?

Lines 46, 54, 263, etc. Why Taxa (with a capital) rather than taxa?

Line 60. P. oceanica: italics. Species names should be italicized everywhere

Line 61. IF: frequency of infestation? Please specify.

Table 4 and line 266. Lulwoana uniseptate à Lulwoana uniseptata.

Figure 5. Mean leaf length and shoot surface area conspicuously change between early and late summer. You must therefore accurately mention (material and methods) the date of sampling for each sampling station. In addition, leaf length widely depends upon the pressure of herbivory: Do Sarpa salpa (teleost) and Paracentrotus lividus (sea urchin) occur at the study sites? What is their abundance?

Lines 210 and 214. What is the meaning of the percentage of brown tissues? The occurrence of brown tissues is a normal feature in old leaves of P. oceanica. This percentage increases from early to late summer. See comment to Figure 5.

Line 239. Please remove ‘conservation regime’ (see below).

Lines 239-242. Is the good health of the study meadows confirmed by biometric characteristics of the leaf bundles? Leaf length and shoot surface area are poor indicators of the meadow health, just taken into account (among many others) by ecological indicators of the ecological status of P. oceanica meadows (see e.g. Romero et al., 2007; Gobert et al., 2009; Lopez y Royo et al., 2010; Boudouresque et al., 2015). CARLIT could be a better indicator of the overall sea water quality (see e.g. Bermejo et al., 2013; Cavallo et al., 2016; Blanfuné et al., 2017; De la Fuente et al., 2018; Boudouresque et al., 2020).

Romero J., Martínez-Crego B., Alcoverro T., Pérez M., 2007. A multivariate index based on the seagrass Posidonia oceanica (POMI) to assess ecological status of coastal waters under the Water Framework Directive (WFD). Mar. Pollut. Bull., 55: 196-204.

Gobert S., Sartoretto S., Rico-Raimondino V., Andral B., Chery A., Lejeune P., Boissery P., 2009. Assessment of the ecological status of Mediterranean French coastal waters as required by the Water Framework Directive using the Posidonia oceanica Rapid Easy Index: PREI. Mar. Pollut. Bull., 58 (11): 1727-1733.

Lopez y Royo C., Casazza G., Pergent-Martini C., Pergent G., 2010. A biotic index using the seagrass Posidonia oceanica (BiPo), to evaluate ecological status of coastal waters. Ecological Indicators, 10: 380-389.

Bermejo R., De la Fuente G., Vergara J.J., Hernández I., 2013. Application of the CARLIT index along a biogeographical gradient in the Alboran Sea (European coast). Marine Pollution Bulletin, 72: 107-118.

Boudouresque C.F., Personnic S., Astruch P., Ballesteros E., Bellan-Santini D., Bonhomme P., Botha D., Feunteun E., Harmelin-Vivien  M., Pergent G., Pastor J., Poggiale J.C., Renaud F., Thibaut T., Ruitton S., 2015. Ecosystem-based versus species-based approach for assessment of the human impact on the Mediterranean seagrass Posidonia oceanica. Marine productivity : perturbations and resilience of socio-ecosystems. Ceccaldi H., Hénocque Y., Koike Y., Komatsu T., Stora G., Tusseau-Vuillemin M.H. (eds), Springer International Publishing Switzerland: 235-241.

Cavallo M., Torras X., Mascaró O., Ballesteros E., 2016. Effect of temporal and spatial variability on the classification of the Ecological Quality Status using the CARLIT Index. Marine Pollution Bulletin, 102: 122-127.

Blanfuné A., Thibaut T., Boudouresque C.F., Mačić V., Markovic L., Palomba L., Verlaque M., Boissery P., 2017. The CARLIT method for the assessment of the ecological quality of European Mediterranean waters: relevance, robustness and possible improvements. Ecological Indicators, 72: 249-259.

De la Fuente G., Chiantore M., Gaino F., Asnaghi V., 2018. Ecological status improvement over a decade along the Ligurian coast according to a macroalgae based index (CARLIT). Plos One, 13 (12): 1-17 (e0206826).

Boudouresque C.F., Blanfuné A., Ruitton S., Thibaut T., 2020. Macroalgae as a tool for coastal management in the Mediterranean Sea. In : Handbooh of algal science, microbiology, technology an medicine, Konur O. (edit.), Elsevier and Academic Press: 277-290.

Lines 326-330. I agree that the study sites are exposed to very low human pressures, but this is neither due to the presence of an MPA (most Mediterranean MPAS a just paper parks) nor of a Natura 2000 site! Natura 2000 sites do not involve management or protection measures. Natura 2000 sites belong to ‘mist parks’, only intended to fulfill the international commitments of States. See e.g. Sala et al. (2012) and Meinez and Blanfuné (2015).

Sala, E.; Ballesteros, E.; Dendrinos, P.; Di Franco, A.; Ferretti, F.; Foley, D.; Fraschetti, S.; Friedlander, A.; Garrabou, J.; Güçlüsoy, H.; Guidetti, P.; Halpern, B.J.; Hereu, B.; Karamanlidis, A.A.; Kizilkaya, Z.; Macpherson, E.; Mangialajo, L.; Mariani, S.; Micheli, F.; Pais, A.; Riser, K.; Rosenberg, A.A.; Sales, M.; Selkoe, K.A.; Starr R.; Tomas F.; Zabala M. The structure of Mediterranean rocky reef ecosystems across environmental and human gradients, and conservation implications. Plos One 2012, 7(2), 1-13.

Meinesz, A.; Blanfuné A. 1983-2013: Development of marine protected areas along the French Mediterranean coasts and perspectives for achievement of the Aichi target. Mar. Pol. 2015, 54, 10-16.

Line 266. Isolates à isolate (without capitalization).

Discussion. The authors should explain to the reader why species of Ascomycota as common as Halotthia, Pontoporeia, etc. were not encountered: the order Pleosporales not taken into account? A matter of the used primers? Another reason?

Explanation of the conclusion

This work constitutes a very valuable contribution to the knowledge of the Fungi of the seagrass Posidonia oceanica.

For this reason, it deserves to be published, after major revision.

The form needs to be improved: e.g. species names not in italics, or erroneous (Lulwoana uniseptate for uniseptata!).

The choice of taxa taken into account, excluding well-known Ascomycota such as Halotthia and Pontoporeia, is not well explained. Authors should explain it in a clear and educational way.

Determining the health status of seagrass meadows, based on leaf length or leaf surface area of ​​shoots, is naive or irrelevant. Authors should use a more valid index, for example based on the PREI, POMI, CARLIT, EBQI or other indices. Another possibility is to remove these meaningless measurements of leaf length and leaf surface area.

All in all, I really liked (except for above-mentioned topics) part of this manuscript. I will be pleased to review a revised version.

Author Response

Lines 5-6. Fist name followed by the family name should be better: Livio Torta, Santella Burruano, etc.

Done

Line 25-26. Keywords. DSEs à Dark Septate Endophytes (DSEs). Sicilian coasts à Sicily. Italics for genus and species names.

Done

Line 31. Also cite: Boudouresque, C.F.; Bernard, G.; Bonhomme, P.; Charbonnel, E.; Diviacco, G.; Meinesz, A.; Pergent, G.; Pergent-Martini, C.; Ruitton, S.; Tunesi, L. Protection and Conservation of Posidonia oceanica Meadows; RAMOGE and RAC/SPA Publications: Tunis,Tunisia, 2012; p. 202.

Done

Line 32. Also cite: Boudouresque, C.F.; Pergent, G.; Pergent-Martini, C.; Ruitton, S.; Thibaut, T.; Verlaque, V. The necromass of the Posidonia oceanica seagrass meadow: Fate, role, ecosystem services and vulnerability. Hydrobiologia 2016, 781, 25–42.

Done

Lines 39-41. Many older studies mention Fungi within rhizomes and roots of P. oceanica, such as Corollospora maritima, Halotthia posidoniae, Pontoporeia biturbinata, etc. See e.g. Durieu and Montagne (1846), Johnson and Sparrow (1961), Kohlmeyer and Kohlmeyer (1964, 1967, 1971), Kohlmeyer (1963) and Cuomo et al (1982) [Bol. Mus. Ist. Biol. Univ. Genova, 50 (suppl.): 162-166]. Why did not the authors consider these studies?

Done

Lines 46, 54, 263, etc. Why Taxa (with a capital) rather than taxa?

Done

Line 60. P. oceanica: italics. Species names should be italicized everywhere

We are sorry, in the uploaded manuscript all the scientific names were in italics, maybe an error occurred after the downloads.

Line 61. IF: frequency of infestation? Please specify.

Specified both in Results (Line 78-80) and Materials and Metods (Line 466-468)

Table 4 and line 266. Lulwoana uniseptate à Lulwoana uniseptata.

Done

Figure 5. Mean leaf length and shoot surface area conspicuously change between early and late summer. You must therefore accurately mention (material and methods) the date of sampling for each sampling station. In addition, leaf length widely depends upon the pressure of herbivory: Do Sarpa salpa (teleost) and Paracentrotus lividus (sea urchin) occur at the study sites? What is their abundance?

We are grateful to the Reviewer who asked us to specify the sampling dates, since, besides improving the methodological part, it allowed us to find an error: the sampling was actually done in the summer of 2014 and not 2016. The error has been corrected and the dates have been accurately added. Unfortunately, we have no quantitative data on the abundances of Sarpa salpa and sea urchin.  However, following the Reviewer's suggestions, grazing pressure as a possible source of variation in leaf biometry was added in the text (Line 404-406)

Lines 210 and 214. What is the meaning of the percentage of brown tissues? The occurrence of brown tissues is a normal feature in old leaves of P. oceanica. This percentage increases from early to late summer. See comment to Figure 5.

The percentage of brown tissues is normally included in the panel of descriptors detected at leaf level, because  it is an important source of information concerning the dynamics and vegetative growth (Pergent et al., 2005). According to Reviewer’s suggestion, this topic was added. 

Line 239. Please remove ‘conservation regime’ (see below).

Done

Lines 239-242. Is the good health of the study meadows confirmed by biometric characteristics of the leaf bundles? Leaf length and shoot surface area are poor indicators of the meadow health, just taken into account (among many others) by ecological indicators of the ecological status of P. oceanica meadows (see e.g. Romero et al., 2007; Gobert et al., 2009; Lopez y Royo et al., 2010; Boudouresque et al., 2015). CARLIT could be a better indicator of the overall sea water quality (see e.g. Bermejo et al., 2013; Cavallo et al., 2016; Blanfuné et al., 2017; De la Fuente et al., 2018; Boudouresque et al., 2020).

Based on the Reviewer's suggestion, we improved the description of the environmental conditions by Carlit and PREi indices resulting from two studies carried out in 2018. The sentence has been changed (Line 380-384). Anyway we agree with the reviewer, that leaf length and shoot surface are not the only indicators to be used to assess the meadow health status, however it should be noted that shoot surface is an integral part of the various indices mentioned (BIPO, PREI, POMI), and it is considered a sensitive descriptor able to give quickly answer (Monthly) to environment deterioration/improvement (Pergent et al., 2005 Ecol. Ind). In our case, we used leaf biometry in order to evaluate only the vitality conditions of the sampled shoots and not the whole meadow or study area. For this purpose we believe that presenting also the biometry of the plants on which the analysis of the endophytic community was carried out can be useful to provide a more complete picture of the condition of the plants examined. If, however, it is felt that this information is not needed we are willing to remove it

Romero J., Martínez-Crego B., Alcoverro T., Pérez M., 2007. A multivariate index based on the seagrass Posidonia oceanica (POMI) to assess ecological status of coastal waters under the Water Framework Directive (WFD). Mar. Pollut. Bull., 55: 196-204.

Gobert S., Sartoretto S., Rico-Raimondino V., Andral B., Chery A., Lejeune P., Boissery P., 2009. Assessment of the ecological status of Mediterranean French coastal waters as required by the Water Framework Directive using the Posidonia oceanica Rapid Easy Index: PREI. Mar. Pollut. Bull., 58 (11): 1727-1733.

Lopez y Royo C., Casazza G., Pergent-Martini C., Pergent G., 2010. A biotic index using the seagrass Posidonia oceanica (BiPo), to evaluate ecological status of coastal waters. Ecological Indicators, 10: 380-389.

Bermejo R., De la Fuente G., Vergara J.J., Hernández I., 2013. Application of the CARLIT index along a biogeographical gradient in the Alboran Sea (European coast). Marine Pollution Bulletin, 72: 107-118.

Boudouresque C.F., Personnic S., Astruch P., Ballesteros E., Bellan-Santini D., Bonhomme P., Botha D., Feunteun E., Harmelin-Vivien  M., Pergent G., Pastor J., Poggiale J.C., Renaud F., Thibaut T., Ruitton S., 2015. Ecosystem-based versus species-based approach for assessment of the human impact on the Mediterranean seagrass Posidonia oceanica. Marine productivity : perturbations and resilience of socio-ecosystems. Ceccaldi H., Hénocque Y., Koike Y., Komatsu T., Stora G., Tusseau-Vuillemin M.H. (eds), Springer International Publishing Switzerland: 235-241.

Cavallo M., Torras X., Mascaró O., Ballesteros E., 2016. Effect of temporal and spatial variability on the classification of the Ecological Quality Status using the CARLIT Index. Marine Pollution Bulletin, 102: 122-127.

Blanfuné A., Thibaut T., Boudouresque C.F., Mačić V., Markovic L., Palomba L., Verlaque M., Boissery P., 2017. The CARLIT method for the assessment of the ecological quality of European Mediterranean waters: relevance, robustness and possible improvements. Ecological Indicators, 72: 249-259.

De la Fuente G., Chiantore M., Gaino F., Asnaghi V., 2018. Ecological status improvement over a decade along the Ligurian coast according to a macroalgae based index (CARLIT). Plos One, 13 (12): 1-17 (e0206826).

Boudouresque C.F., Blanfuné A., Ruitton S., Thibaut T., 2020. Macroalgae as a tool for coastal management in the Mediterranean Sea. In : Handbooh of algal science, microbiology, technology an medicine, Konur O. (edit.), Elsevier and Academic Press: 277-290.

Lines 326-330. I agree that the study sites are exposed to very low human pressures, but this is neither due to the presence of an MPA (most Mediterranean MPAS a just paper parks) nor of a Natura 2000 site! Natura 2000 sites do not involve management or protection measures. Natura 2000 sites belong to ‘mist parks’, only intended to fulfill the international commitments of States. See e.g. Sala et al. (2012) and Meinez and Blanfuné (2015).

We agree with the Reviewer, in fact the existence of a MPA or Natura 2000 sites does not always guarantee very low human pressures. The sentence of the MS was misleading and it was changed (Line 407-410)

Sala, E.; Ballesteros, E.; Dendrinos, P.; Di Franco, A.; Ferretti, F.; Foley, D.; Fraschetti, S.; Friedlander, A.; Garrabou, J.; Güçlüsoy, H.; Guidetti, P.; Halpern, B.J.; Hereu, B.; Karamanlidis, A.A.; Kizilkaya, Z.; Macpherson, E.; Mangialajo, L.; Mariani, S.; Micheli, F.; Pais, A.; Riser, K.; Rosenberg, A.A.; Sales, M.; Selkoe, K.A.; Starr R.; Tomas F.; Zabala M. The structure of Mediterranean rocky reef ecosystems across environmental and human gradients, and conservation implications. Plos One 2012, 7(2), 1-13.

Meinesz, A.; Blanfuné A. 1983-2013: Development of marine protected areas along the French Mediterranean coasts and perspectives for achievement of the Aichi target. Mar. Pol. 2015, 54, 10-16.

Line 266. Isolates à isolate (without capitalization).

Done

Discussion. The authors should explain to the reader why species of Ascomycota as common as Halotthia, Pontoporeia, etc. were not encountered: the order Pleosporales not taken into account? A matter of the used primers? Another reason?

Most of the fungal microorganisms associated with P. oceanica and reported in literature are epiphytes. The sterilization of the organs surface prior to the isolation technique has deactivated their propagules. Therefore the few isolated fungal microorganisms are those present inside the tested organs.

Explanation of the conclusion

This work constitutes a very valuable contribution to the knowledge of the Fungi of the seagrass Posidonia oceanica.

For this reason, it deserves to be published, after major revision.

The form needs to be improved: e.g. species names not in italics, or erroneous (Lulwoana uniseptate for uniseptata!).

The choice of taxa taken into account, excluding well-known Ascomycota such as Halotthia and Pontoporeia, is not well explained. Authors should explain it in a clear and educational way.

Determining the health status of seagrass meadows, based on leaf length or leaf surface area of ​​shoots, is naive or irrelevant. Authors should use a more valid index, for example based on the PREI, POMI, CARLIT, EBQI or other indices. Another possibility is to remove these meaningless measurements of leaf length and leaf surface area.

Based on the Reviewer's suggestion, we improved the description of the environmental conditions by Carlit and PREi indices resulting from two studies carried out in 2018 in the same area. The sentence has been changed. Anyway we agree with the reviewer, that leaf length and shoot surface are not the only indicators to be used to assess the meadow health status, however it should be noted that shoot surface is an integral part of the various indices mentioned (BIPO, PREI, POMI), and it is considered a sensitive descriptor able to give quickly answer (Monthly) to environment deterioration/improvement (Pergent et al., 2005 Ecol. Ind).

In our case, we used leaf biometry in order to evaluate only the vitality conditions of the sampled shoots and not the whole meadow or study area. For this purpose we believe that presenting also the biometry of the plants on which the analysis of the endophytic community was carried out can be useful to provide a more complete picture of the condition of the plants examined. If, however, it is felt that this information is not needed we are willing to remove it

 

All in all, I really liked (except for above-mentioned topics) part of this manuscript. I will be pleased to review a revised version.

 

Round 2

Reviewer 1 Report

In my original assessment of the manuscript by Torta et al., I had serious concerns with the experimental design and the approach used.  Unfortunately, I still do not believe that my fundamental queries were addressed by the authors.  The links between the cultured endophytes and the plant itself are not discussed in any detail.  There is still no conclusive evidence that all or any of the cultured endophytes do anything (beneficial or harmful) to the plant host.  For me, the presented manuscript seems like the first half of a manuscript where the plant/fungal relationship would be explored further.  

Author Response

In my original assessment of the manuscript by Torta et al., I had serious concerns with the experimental design and the approach used.  Unfortunately, I still do not believe that my fundamental queries were addressed by the authors.  The links between the cultured endophytes and the plant itself are not discussed in any detail.  There is still no conclusive evidence that all or any of the cultured endophytes do anything (beneficial or harmful) to the plant host.  For me, the presented manuscript seems like the first half of a manuscript where the plant/fungal relationship would be explored further.  

 

Generally, investigations in this research topics can have multiple objectives:
1.        Analyze endophytic fungal populations with the primary goal of gaining knowledge about the microbial community present in plants. These works have as objective the isolation, identification and description of fungal taxa associated with the host. This is our case, in which we also wanted to deepen our knowledge on the variations of the community in relation to the type of organ of the host and try to understand if the variations found remain constant in a context of large spatial scales (the coasts of Sicily). These studies lay the groundwork for further studies with different objectives listed below.

2.        Once the collection of endophytic fungi is obtained, it is possible to evaluate their bioactivity against pathogens (of plants or animals) or cancer cell lines and their production of secondary metabolites. These works have a different purpose and are set up quite differently.


3.        Finally, if one wants to investigate the role that these microorganisms play in relation to the host, then one proceeds in a third way, using a completely different methodology and setting, compared to the first two cases. Thus, these are researches with different purposes and different methodological approaches.

I send you some recent bibliography on the isolation and identification of endophytic fungal communities in various plant hosts.

https://academic.oup.com/femsec/article/92/10/fiw152/2197843
https://www.researchgate.net/publication/324889818_Endophytic_Fungi_in_Species_of_Artemisia
https://doi.org/10.1016/j.micres.2020.126693
https://doi.org/10.1080/12298093.2021.1948175