Olive Anthracnose in Portugal Is Still Mostly Caused by Colletotrichum nymphaeae, but C. acutatum Is Spreading and C. alienum and C. cigarro Are Reported for the First Time

Round 1

Reviewer 1 Report

Suggestions for authors:

2. Materials and Methods 2.4. Pathogenicity assays

214 - 216  Why were the drupes not punctured with a sterile needle before inoculation?

216 - replace with Conidia ml⁻¹

221 – clarify every how many days the measurements on the inoculated drupes have been made

225 - replace the term spores with conidia

567 – insert potato pextrose agar (PDA, BD-Difco Laboratories)

568 -  Fig 6 insert scale (unit of measurement) used on the figure

619 - insert potato pextrose agar (PDA, BD-Difco Laboratories)

664 - The first symptoms on the inoculated fruits started 4-5 days after inoculation (dai) for the 12 isolates of Colletotrichum isolated (delete the word isolated) tested.

Comments for author File: Comments.pdf

Author Response

2. Materials and Methods 2.4. Pathogenicity assays

214 - 216  Why were the drupes not punctured with a sterile needle before inoculation?

R: Although openings may facilitate the entry of Colletotrichum, the fungus is capable of invading intact fruit tissues and in fact that is why it is causing disease at high incidence and severity levels.

216 - replace with Conidia ml−1

R: correction done

221 – clarify every how many days the measurements on the inoculated drupes have been made

R: this information was added

225 - replace the term spores with conidia

R: replaced

567 – insert potato pextrose agar (PDA, BD-Difco Laboratories)

R: done

568 -  Fig 6 insert scale (unit of measurement) used on the figure

R: This information is included in the legend.

619 - insert potato pextrose agar (PDA, BD-Difco Laboratories)

R: done

664 - The first symptoms on the inoculated fruits started 4-5 days after inoculation (dai) for the 12 isolates of Colletotrichum isolated (delete the word isolated) tested.

R: corrected

Reviewer 2 Report

The only edit I can suggest is to change notorious to notable on line 298.

Here are some other items to consider.  Did you record the cultivar from which each isolates collected.  since you suggest chain cultivars may be involved, a Colletotrichum species by cultivar analysis could be informative.  

Personally, I would like to se the phylogenetic trees with the information that these new isolates are shown in green and listed as Oleas before all the descriptions.  Not a real critical problem. 

It would be good to put the color code right on the trees presented, ie green for new isolates, etc. 

Someplace terms like  GTR + G etc are in relationship to Maximum Likelihood should be provided or referenced

Comments for author File: Comments.pdf

Author Response

The only edit I can suggest is to change notorious to notable on line 298.

R: Corrected as suggested.

Here are some other items to consider.  Did you record the cultivar from which each isolates collected.  since you suggest chain cultivars may be involved, a Colletotrichum species by cultivar analysis could be informative.

R: Yes, we did, and we thank the reviewer for the opportunity to detail this aspect. Data on this issue was added to the Results section (Figure 9 and associated text) and to Discussion.

Personally, I would like to se the phylogenetic trees with the information that these new isolates are shown in green and listed as Oleas before all the descriptions.  Not a real critical problem. 

It would be good to put the color code right on the trees presented, ie green for new isolates, etc.

R: Thank you for your suggestion. The colour code used is in the figure caption. We couldn’t find space in the figures 4 and 5 to add the legend.

Someplace terms like  GTR + G etc are in relationship to Maximum Likelihood should be provided or referenced

R: We improved the text in the material and methods in section 2.2.3. Phylogenetic analyses, incorporating the information of the nucleotide substitution models calculated by MrModelTest and used in the Bayesian phylogenetic analysis and removed this information from the results section.

Reviewer 3 Report

The manuscript of Cabral et al. describes an in-depth analyses of the situation in Portugal concerning the distribution, virulence and incidence of Colletothricum species isolated rom olive orchards.

The Authors collected many isolates and appropiately identified and characterized them by using both morphological and molecular approaches.

The study also included an assessment of some climatic parameters in the areas of the surveys and provides a description of fungal species.

The article is well written and informative.

I would suggest to explain the very low percentage of fungi in the Tras-os- Montes area as found in 2018 (such an area was not taken into consideration in 2019), and place this data in climatic context.

Moderate revision is needed

Author Response

The manuscript of Cabral et al. describes an in-depth analyses of the situation in Portugal concerning the distribution, virulence and incidence of Colletothricum species isolated rom olive orchards.

The Authors collected many isolates and appropiately identified and characterized them by using both morphological and molecular approaches.

The study also included an assessment of some climatic parameters in the areas of the surveys and provides a description of fungal species.

The article is well written and informative.

I would suggest to explain the very low percentage of fungi in the Tras-os- Montes area as found in 2018 (such an area was not taken into consideration in 2019), and place this data in climatic context.

R: A note on this was added to Discussion, relating not only to climatic conditions but also to the less susceptible cultivars grown in the region.

Reviewer 4 Report

This is  very interesting, valuable and comprehensive study devoted to olive anthracnose due to  Colletotrichum spp.

Over the course of two years, 2018 and 2019, the authors examined over 500 sites and obtained 212 Colletotrichum isolates. The population of isolates was then subject to morphological and molecular analyses. Based on morphological features and ISSR analysis, 47 isolates were selected from the initial pool, which were then subjected to multi-locus phylogenetic analysis, including loci (ACT, TUB2, HIS, ITS GAPDH for Acutatum species complex and ACT, GAPDH, GS, ITS, ApMAT, TUB2) for Gloeosporioides species complex. Based on the conducted research, the authors recognized 3 species (Colletotrichum alienum and Colletotrichum cigarro, Colletotrichum gloeosporioides) from the gleosporioides group and 4 species from the acutatum group (Colletotrichum acutatum, Colletotrichum fioriniae, Colletotrichum godetiae, Colletotrichum nymphaeae), moreover the authors proved the occurrence of two new taxa of the genus Coletotrichum in Portugal

Author Response

The reviewer did not place any specific queries to be answered