Ascochyta erotica sp. nov. Pathogenic on Convolvulus arvensis

Round 1

Reviewer 1 Report

The authors described Ascochyta erotica sp. nov. based on phylogeny and morphology. The authors also studied optimal temperature for growth of Ascochyta erotica isolates. According to fulfilled the Koch’s postulates, Ascochyta erotica is a pathogenic fungi of Convolvulus arvensis. I think it’s a good paper and recommend accept after minor revision.

See attachment.

Comments for author File: Comments.pdf

Author Response

Thank you for reviewing our manuscript, we greatly appreciate your opinions, comments and suggestions to improve our manuscript. The current version has undergone major revisions taking into account all matters raised by Reviewers.

All spelling, grammatical and techniques errors kindly pointed out by the Reviewer have been corrected.

Author Response File: Author Response.docx

Reviewer 2 Report

The authors in their manuscript describe the morphological and molecular characterization of five fungal isolates from the weed species Convolvulus arvensis and test their pathogenicity. The methodology concept and implementation regarding these approaches (morphological, molecular, and pathogenicity) are correct under the concept of identification-characterization of a fungal isolate to species level. Results suggest (for all five isolates) a different species to these evaluated in the molecular approach. Furthermore, information derived from morphological and growth characteristics’ evaluation accompany the molecular characterization. Pathogenicity tests support Koch’s postulates. The authors claim the identification of a new species of the genus Ascochyta. The molecular identification to the extend it was applied (number of selected species sequences used) supports this, however, there are two main queries that derive form the research work as presented in the manuscript. In specific:

1.        Since the authors claim a close relation to P. proboscis, however they describe/refer to the morphological and growth differences of the isolates to this species, an obvious following step would be to perform the Multilocus Sequence Analysis for this species (ATCC 74032 strain) as well and incorporate the results in the phylogenetic analyses.

2.        Furthermore, the data regarding growth and morphological characteristics of P. proboscis are only verbally described and not presented (images, tables, etc.) in a parallel comparison to the five isolates.

It would be very informative and conclusive to present the above data to support the differentiation of the five isolates from P. proboscis and further support the claim of a new species identified.

In addition, provided that the isolates are finally characterized as a different species in the genus Ascochyta, my question is if this research forms the sole prerequisite for the scientific community to accept that this is a new species, or it forms a step in a procedure for the scientific community to accept this as a new species. Could the authors please provide a clarification for the official procedure required for this?

Thank you.

Very good quality, minor and typographic corrections required.

Author Response

Thank you for reviewing our manuscript, we very grateful for the interest shown in our manuscript, its high appreciation, attentions and kindly pointed errors.

Below you will find our answers to Reviewers’ comments.

1. Since the authors claim a close relation to P. proboscis, however they describe/refer to the morphological and growth differences of the isolates to this species, an obvious following step would be to perform the Multilocus Sequence Analysis for this species (ATCC 74032 strain) as well and incorporate the results in the phylogenetic analyses.

Response.

Phoma proboscis was described for the sole strain ATCC 74032 strain in. Since description in 1990 there were not any reports about findings of this fungus. Also unfortunately there is still no sequence data on the ex-type P. proboscis strain in GenBank, thus there is no way to include the ex-type P. proboscis strain in multilocus phylogenetic analysis and to compare A. erotica and P. proboscis by molecular phylogenetic features.

2. Furthermore, the data regarding growth and morphological characteristics of P. proboscis are only verbally described and not presented (images, tables, etc.) in a parallel comparison to the five isolates.

It would be very informative and conclusive to present the above data to support the differentiation of the five isolates from P. proboscis and further support the claim of a new species identified.

In addition, provided that the isolates are finally characterized as a different species in the genus Ascochyta, my question is if this research forms the sole prerequisite for the scientific community to accept that this is a new species, or it forms a step in a procedure for the scientific community to accept this as a new species. Could the authors please provide a clarification for the official procedure required for this?

Response.

Thank you for suggestion to present the growth characteristics of P. proboscis. Colonies diameter of Phoma proboscis were included in Table 3, which did result in an improvement to this table and the ability to compare isolates of these two morphologically similar species.

The official procedure for describing new fungal taxa is regulated by the International Code of Nomenclature for algae, fungi, and plants. This procedure includes: the designation of a type accompanied by a protologue=diagnosis. The diagnosis is expected to thoroughly document newly proposed species in a manner designed to facilitate identification and data accessibility. There are no formal rules for taxonomic description, there are nonetheless community standards of scientific rigor enforced by journal editors and reviewers, which should be adhered to when publishing names of new taxa. Nomenclatural novelties must be accompanied by citation of an identifier number obtained from a repository that has been appointed by the Nomenclature Committee for Fungi: MycoBank or Index Fungorum. Every new name published must have a separate MB or IF identifier. In order to be available for use, names for new taxa must be ‘effectively’, and ‘validly’ published and be ‘legitimate’. The key element of a description is the demonstration that a specimen or culture represents a species that is distinct from previously described species. Authors of new species should bear in mind that the goal of communicating new species is to make identities clear and

unambiguous for current users and future generations. To facilitate this goal, best practices should include: multiple collections, where possible, to account for and describe phenotypic and, where applicable, genotypic, variation within a species; use of multiple data types, where feasible, for clear delineation of species; and provision of tools such as DNA barcode data to facilitate rapid identification. To more detailed description of formal requirements to describing new fungal taxa, please see

Aime, M.C., Miller, A.N., Aoki, T. et al. How to publish a new fungal species, or name, version 3.0. IMA Fungus 12, 11 (2021). https://doi.org/10.1186/s43008-021-00063-1

The International Code of Nomenclature for algae, fungi, and plants https://www.iapt-taxon.org/nomen/main.php

We believe that all formal and official requirements have been met and the scientific community can and should recognize Ascochyta erotica as a new species.

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript Ascochyta erotica sp. nov. pathogenic on Convolvulus arvensis is interesting for the reader in the field of Mycology. Here are some questions and comments for the improvement:

1.     The type specimen is collected 22 years ago, are all the living cultures from the same specimen? Do you need recent collections to support these findings?

2.     Description from the holotype should be clear, the authors write “both infected plant material and metabolic inactive 14d culture on OA”. However, it should be clear that which of the morphological descriptions are from the plant tissue and what’s from the culture in certain media.

3.     There are reisolation to fulfill the Koch’s postulates in M&M in line 194-195 but with no further descriptions in the results section.

4.     Why you choose to inoculate the fungus on the leaf segments? It would be good to inoculate the fungus on living plant leaves to confirm the pathogenicity. Since the original collection is from long time ago, the inoculation on living plants can produce additional data on the leaves, complement the weakness using only the old sample.

Other comments:

L2: Italicize erotica.

L57-67: Better to improve the link of the introduction, such as why Phoma should be discussed further.

L73: “2.1. Plant materail & Isolates” is not correct.

Table 1: is CBS 503.75 a type specimen? (According to the Fig 1)

L284: Why Heiny 1990 can represent the current species?

L383: Italicize convolvuli

Author Response

Thank you for reviewing our manuscript, we very grateful for the interest shown in our manuscript, its high appreciation, attentions and kindly pointed errors.

Below you will find our answers to Reviewers’ comments.

1. The type specimen is collected 22 years ago, are all the living cultures from the same specimen? Do you need recent collections to support these findings?

Response.

Yes, all living cultures are from this specimen. Certainly, we need additional isolates to support our findings. Extensive phytosanitary monitoring of agricultural, segetal and ruderal territories has been carried out by the authors since 1990. Apparently, this fungus is not widespread, is local and requires specific optimal growth conditions, since to date no similar isolates have been found and stored in our collection.

2. Description from the holotype should be clear, the authors write “both infected plant material and metabolic inactive 14d culture on OA”. However, it should be clear that which of the morphological descriptions are from the plant tissue and what’s from the culture in certain media.

Response.

Thank you for this comment. In the taxonomy part we have corrected and clarify the description from the Holotype.

3. There are reisolation to fulfill the Koch’s postulates in M&M in line 194-195 but with no further descriptions in the results section.

Response.

The results at the end of Part 3.3 now contain a description of the implementation of Koch's postulates.

4. Why you choose to inoculate the fungus on the leaf segments? It would be good to inoculate the fungus on living plant leaves to confirm the pathogenicity. Since the original collection is from long time ago, the inoculation on living plants can produce additional data on the leaves, complement the weakness using only the old sample.

Response.

Inoculation of leaf segments is a rapid, reliable, reproducible, routine method for testing the pathogenicity of fungal isolates. Subsequently, if necessary, pathogenicity tests could be carried out on whole living plants.

Other comments:

5. L2: Italicize erotica.

Response

Corrected.

6. L57-67: Better to improve the link of the introduction, such as why Phoma should be discussed further.

Response

The correct references were given above in the Introduction (L29-39) to explain why Phoma should be discussed below. Particularly, that many former Phoma species were included in the Ascochyta and now Ascochyta is treated as Phoma-like genera.

7. L73: “2.1. Plant materail & Isolates” is not correct.

Response

Corrected.

8. Table 1: is CBS 503.75 a type specimen? (According to the Fig 1)

Response

Thank you for your attention, the strain number and status were incorrect. Now Table 1 corresponds to Figure 1.

9. L284: Why Heiny 1990 can represent the current species?

Response

Thank you, we agree, the link here is incorrect

L383: Italicize convolvuli

Response

Corrected

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

I thank the authors for their revised manuscript.

My main query remains still on the multilocus sequence analysis. The authors provide growth data for the ATCC 74032 which means that the strain is in their possession. Thus, it seems obvious that from the technical point of view PCR for the four loci reported in phylogenetic analysis can be easily performed (both technically and time wise). According to the authors replies regarding The official procedure for describing new fungal taxa: “..best practices should include: multiple collections, where possible, to account for and describe phenotypic and, where applicable, genotypic, variation within a species; use of multiple data types, where feasible, for clear delineation of species; and provision of tools such as DNA barcode data to facilitate rapid identification…”

Nowadays, sequence analyses are essential if not indispensable for taxonomic identification of a species/isolate.

Furthermore, regarding nomenclature requirements, as stated, “Nomenclatural novelties must be accompanied by citation of an identifier number obtained from a repository that has been appointed by the Nomenclature Committee for Fungi: MycoBank or Index Fungorum”.

I would like to ask if the authors have followed and fulfill  this rule.

no further comments

Author Response

Thank you very much for taking the time to review this manuscript, we greatly appreciate your opinions, comments and suggestions to improve our manuscript.

Comment:

My main query remains still on the multilocus sequence analysis. The authors provide growth data for the ATCC 74032 which means that the strain is in their possession. Thus, it seems obvious that from the technical point of view PCR for the four loci reported in phylogenetic analysis can be easily performed (both technically and time wise). According to the authors replies regarding The official procedure for describing new fungal taxa: “..best practices should include: multiple collections, where possible, to account for and describe phenotypic and, where applicable, genotypic, variation within a species; use of multiple data types, where feasible, for clear delineation of species; and provision of tools such as DNA barcode data to facilitate rapid identification…”

Nowadays, sequence analyses are essential if not indispensable for taxonomic identification of a species/isolate.

Response.

Apparently, there was some misunderstanding or this subject was not clearly stated in the manuscript and in the previous response to the reviewer’s comments. The authors do not possess the strain ATCC 74032. Growth data and other morphological features for the ATCC 74032 were provided according to literature data from the original paper by Heiny 1990 (Heiny, D.K. Phoma proboscis sp.nov. pathogenic on Convolvulus arvensis. Mycotaxon, 1990, 36(2), 457–471.). Indeed, it seems clear that if we had strain ATCC 74032, a phylogenetic and morphological analyses would be easy to perform, and we would certainly do it. Unfortunately we do not have this strain and have to use literature data.

The proposed new taxon Ascochyta erotica has been fully characterized by molecular phylogenetic, morphological and pathogenic features, so we believe that we have followed the official procedure and the new species can be accepted by the scientific community.

Comment:

Furthermore, regarding nomenclature requirements, as stated, “Nomenclatural novelties must be accompanied by citation of an identifier number obtained from a repository that has been appointed by the Nomenclature Committee for Fungi: MycoBank or Index Fungorum”.

I would like to ask if the authors have followed and fulfill  this rule.

Response.

Yes, authors followed this rule. Taxonomic and nomenclatural novelty has the unique MycoBank number – MB 852086, as provided in the Taxonomy part.

We hope that after the changes and more detailed explanations have been made, the manuscript will be accepted for publication in Diversity.

Round 3

Reviewer 2 Report

I thank the authors for their reply.

As stated sequence data are indispensable to verify a different species.

The provision of the ATCC strain can be made from the relevant repository/bank. Concequently, I would like to suggest the multilocus sequencing analysis for the four loci presented, data evaluation and provided that there is a new species described, a resubmission of the manuscript.

No further comments

Author Response

Thank you very much for taking the time to review this manuscript, we greatly appreciate your opinions, comments and suggestions to improve our manuscript.

Below you will find our answers to Reviewers’ comments.

Comment:

As stated sequence data are indispensable to verify a different species.

The provision of the ATCC strain can be made from the relevant repository/bank. Concequently, I would like to suggest the multilocus sequencing analysis for the four loci presented, data evaluation and provided that there is a new species described, a resubmission of the manuscript.

Response.

Unfortunately, due to the current dramatic situation, namely the war, the authors located in Russia do not have the opportunity to purchase isolate ATCC 74032.

The formal procedure for describing new fungal taxa requires authors to fully characterize new fungal species using phylogenetic and morphological analysis and does not require complete characterization of morphologically similar fungal species previously described. The proposed new taxon Ascochyta erotica represented by five isolates, has been fully characterized by molecular phylogenetic, morphological and pathogenic features. The author of Phoma proboscis described this taxon for a single isolate using only morphological characters. Subsequently, the author did not consider it necessary to characterize the isolate based on molecular genetic characteristics and sequence at least one DNA locus. The authors of presented manuscript have implemented a comparative morphological study, which revealed numerous significant differences between Ascochyta erotica and Phoma proboscis. Specifically, there are differences in 1) sizes of conidia (Phoma proboscis conidia are salmon color in mass, no conidial exudate was observed in A. erotica; Asochyta erotica conidia in average smaller and narrower 5.98–11.06 (8.51±0.11) × 2.43–4.26 (3.35±0.04) μm, than conidia of P. proboscis 5.5–15.0 (17) × 2.3–5.0 μm, averaging 10.5 × 3.5 μm.); 2)  pycnidial formation (On OA P. proboscis strain produced dense matt of pycnidia, whereas pycnidia of A. erotica on OA are superficial, semi-immersed and immersed in agar media form in concentric rings. Pycnidia of P. proboscis developed elongate necks within 7 days on all media, especially on PDA, A. erotica produce pycnidia only on OA and MEA, pycnidia with necks forms only on OA, even during exposure to UV light.); 3) the optimal growth temperature (20 °C for Phoma proboscis and 25 °C for Ascochyta erotica), failure in growth at 30 °C for Phoma proboscis and normal growth comparable to the optimal temperature for Ascochyta erotica; 4) cultural characteristics on media (P. proboscis colonies on PDA were yellow-brown with white margin, colonies of A. erotica on PDA are brown-olive with whitish margin).  All these differences are described in detail in the protologue for new species Ascochyta erotica.

We consider the new taxon we proposed to be valid and legitimate, since it is fully characterized and meets the formal requirements for the description of new fungal taxa.

The aim of our study was to identify and describe biodiversity rather than to compare our isolates with Phoma proboscis, so the aim was realized; we have revealed and described biodiversity.

Also, with regard to world experience, there are numerous precedents when new species of fungi, in particular from the Didymellaceae family, were described without including morphologically similar species in the phylogenetic tree due to the lack of data on their sequences. For example, Leptosphaerulina obtusispora and Leptosphaerulina sisyrinchiicola are similar to Leptosphaerulina argentinensis and were formally described without phylogenetic analysis of the latter. As well Macroascochyta grandis, Neoascochyta fusiformis, Neodidymelliopsis achlydis, Neodidymelliopsis longicolla, Nothophoma infuscata,  Stagonosporopsis helianthi were described without phylogenetic analysis of morphologically similar Phoma commelinicola, Diplodina brachypodii, Ascochyta achlydis, Ascochyta scotinospora, Phoma acaciae, Didymella lophospora, respectively.