Genetic Diversity of Barley Foliar Fungal Pathogens
Round 1
Reviewer 1 Report
The paper reviewed the large amount of articles related to foliar fungal pathogens in barley.
It will be great work later, the authors can combine or review the breeding study on this foliar fungal pathogens too.
Author Response
Thank you for your valuable comments
Author Response File: 
Author Response.docx
Reviewer 2 Report
The proposed review has good intentions and there is fair amount of information that has been compiled. In my opinion, the paper would require mayor revisions since it falls short and does not meet, yet, neither its objectives nor the quality for a scientific journal. The authors put a lot of effort in gathering the presented information, but they do not squeeze such information enough to obtain its juice requiring a bit more of discussion.
To discuss the evolutionary potential it could be good talk about the genome of the pathogens; for each disease it would be a good idea to mention studies that analyse the genomes of related pathogens and their evolution and try to link that to the genetic diversity part that it is presented. Furthermore, a final paragraph at the end of each disease section in which authors discuss and make sense of all the data presented as a whole going beyond a compilation of information, would greatly improve the quality of the review.
In addition, authors need to be careful on referencing correctly. In many instances the references used are not appropriated, the original source is not presented and the cited paper just mentions briefly what the authors of these review want to support.
Another of the problems with the text is the actual text. It is misleading in some parts. When someone that knows about the topic reads it, it can get a clear idea of what the authors want to say; however, one of the purpose of the review is also to inform those who are new to a topic; lack of clarity is a big flaw on a scientific paper. Some examples below:
Lines 13 and 28: “…pathogens with evolutionary potential…” This suggests that there are some pathogens with no evolutionary potential, but DNA is always susceptible of mutations, therefore, high or low, there is always potential for evolution.
Line 47, 78 and elsewhere: “… resistance gene is broken down…” The resistance or the effectiveness of the resistance gene is what gets broken down, not the gene itself.
101, 232 and 567: the authors say a given organism incites a disease; however, such organism cause the disease; factors that “incite” (promote) a disease are not the same as causal agents.
Line 129 “Conidia frequently reproduce asexually” … is not the conidia that reproduce, is the fungus
Lines 212-213, “…selection of fungicide resistance, usually caused by a mutation at a single locus in Bgh” A single locus will not provide resistance to all groups of fungicides given that these have different modes of action with different molecular targets. But for each mode of action there is a single locus that can provide resistance.
Lines 335-336: “…the genotyping of fungicide resistant Cyp51A isolates 335using SSR markers resulted in a single mutation event…” The genotypeing did not resulted in (had as the consequence) a mutation.
Lines 339-344: the use of mating type and ITS should’t count ad genetically characterise a population and it is not enough to suggest lack of genetic diversity. Please be carefull on how thing are stated. Furthermore, authors may find that the following paper can contribute here: Linde, C.C., Smith, L.M. Host specialisation and disparate evolution of Pyrenophora teres f. teres on barley and barley grass. BMC Evol Biol 19, 139 (2019). https://doi.org/10.1186/s12862-019-1446-8
Author Response
Thank you for your valuable comments. Corrections are enclosed
Author Response File: Author Response.docx
Reviewer 3 Report
The manuscript is generally well written. There are some awkward places (unfortunately not all identified below) that need polishing. Having a native English-speaking colleague go through it prior to publication might help remove some of these rough spots and undefined bits of jargon that copy editors would be unlikely to catch.
This manuscript contains a great deal of interesting information. Unfortunately it also has two major problems:
- The manuscript is an annotated bibliography rather than a review. An annotated bibliography presents information from each of a series of articles or sources. A review summarizes the literature, interprets the relative importance of different contributions, and provides direction to help the filed move forward. This article is so much of an annotated bibliography that opposing results/viewpoints can be next to one another with no resolution provided. In some cases jargon from the article being summarized is carried over without explanation.
- The manuscript provides no path(s) for the field to consider in dealing with the genetic diversity of these pathogens. Yes there is a problem, but what are some possible solutions? and what needs to be done to implement these solutions? In this sense the manuscript may be trying to tackle too many things at one time, as there is not enough space to deal with problems and solutions in detail.
Some other major issues include:
- The term “genetic drift” is used as a term to explain anything that cannot otherwise be explained. In some cases this usage is very wrong. Genetic and genotypic variation will only decrease as a result of drift, they will not increase. Once an allele/genotype is lost due to drift, a different mechanism is required to reintroduce the lost allele or genotype back into the population. Drift can result in the frequency of some alleles/genotypes increasing, but when they do so it usually is at the expense of others that are being lost from the population. The usage of the term drift needs to be checked at every place where it is used in the manuscript to ensure that it is being used correctly.
- I strongly recommend that the section on Puccinia be deleted. These fungi are basidiomycetes and their populations will behave very differently from those of the other fungi discussed in the manuscript, which are all known or presumed ascomycetes. In this sense the Puccinia information is not part of the same set of problems as observed in the other fungi.
- RAPDs are generally regarded as unreliable for assessments of variation due to repeatability problems across labs and sometimes within labs. Papers that rely on RAPDs for their conclusions should be removed from the manuscript. If a paper uses RAPDs and other techniques, then only conclusions based on the other techniques should be retained.
- Terms for resistance should be consistent – major and minor. Other terms describing resistance – large, main, partial, small, etc. – should be avoided.
- MAT-1 and MAT-2 are genes, and as such would normally be italicized throughout the text.
- May need a general statement of the phylogenetic utility of the forma specialis concept in subdividing pathogen species. In some cases groups may be monophyletic, but in many others the only thing that holds a group together is its observed pathogenicity capabilities. There are examples of both issues in the manuscript and the topic is worthy of a fairly prominent paragraph in a location that is not limited to a single species.
- Mating type is not the sole gene involved in sexual cross-fertility, and the mating type ratios may be significantly different from 1:1 and sexual reproduction still occurring regularly. Female fertility is a more important limitation to sexual reproduction than is relative mating type frequency, and equations have been developed to assess the relative importance of the two for other ascomycete fungi (Genetics 144:557-567; PUBMED: 8889520). Discussion of how this type of analysis might help move this field forward seems appropriate here.
- The conclusions need to be completely rewritten. The current section does not rely on the material discussed in the manuscript for its content. As written, this section relies on what is generally known about the field and not on new insights described in the text of the manuscript. Focus on the insights gained from the discussion of each pathogen to identify areas in which the field might be able to progress to reinforce some of what is already known, challenge general strategies that might be questionable, and provide potential new directions to follow to solve the existing problems.
Other points (in order encountered in the text):
l. 13 – All pathogens have evolutionary potential to overcome resistance. If not, once they are defeated they become non-factors. Some may evolve more quickly than others, but all are capable of evolving.
l. 27-29 – Give some known examples in barley. Need not be an exhaustive list, but should suffice to set up what will be discussed later in the manuscript.
l. 38 – Genotype diversity is not the same as the number of genetically different individuals in a population. The genotype is concerned with only a particular set of genes. The genotype associated with pathogenicity, for example, is very different from the genotype associated with sexual cross-fertility. The definition given here is far too broad.
l. 42-43 – Delete “called the evolution of the local population”. This phrase is neither true nor needed.
l. 45 – Replace “new population” with “means”.
l. 53-84 – This paragraph is quite long and confusing. It contains many ideas that are not related to one another and are not related to the initial topic sentence about mutation as a source of variation.
l. 60-61 – Topic shift here. Note that geographic separation is not required. There can be other things that separate populations other than just geography or physical distance.
l. 62-63 – Delete this sentence. Genotypes can move in the previous sentence, but not in this one? The comment about gene assembly in asexual organisms is currently opaque and might not be true.
l. 63-65 – This sentence is true only if the existing distribution is not relatively uniform. There are examples given later in this manuscript where the distribution is relatively uniform.
l. 66 – “genetic neighborhood” is a bit of undefined jargon.
l. 68 – What does “variations” mean?
l. 69 – New topic for a new paragraph starts here.
l. 69-75 – This section has some hidden pitfalls. Outcrossing in most fungi means crossing with a strain that is not identical to you, i.e., not selfing. In species where selfing occurs, recombination will still occur, but no new genotypes will result since there is nothing to segregate. So it isn’t that more new genotypes are generated by outcrossing, but instead that new genotypes are generated only by outcrossing.
l. 77-78 – This is a very important idea. What is meant by “breaking resistance”? What does allele/genotype frequency have to do with breaking resistance? The frequency may matter in a practical sense, but the concept does not require large numbers to show that it has occurred.
l. 82-84 – Major gene resistance can also be sensitive to environment, especially if the environment concept includes the host variety. Evolving to overcome virulence is something that host populations do no matter what type of virulence gene(s) is in the pathogen.
l. 95 – Rewrite: The result of understanding genetic variation in fungal pathogens is to …
l. 96 – Understanding does not create a resistance breeding strategy.
l. 101 – Bg is an obligate pathogen. An obligate fungus does not make sense.
l. 115-117 – Rewrite to include, “are not consistent with patterns expected if the host and pathogen have co-evolved.”
l. 119-124 – The proof here are in the positive cases. If things cross in the lab, then presumably they can cross in the field. Getting crosses to occur in the lab usually is more difficult than getting a cross to occur in the field. The bigger issue in the field is for the two different strains to find one another at the right time.
l. 125-127 – Need a reference for this statement.
l. 128 – Asexual progeny is a problematic term. I suggest asexual spores as a better alternative here and elsewhere.
l. 135 – Is this result/conclusion valid only for Australia? Or is this a more general truth for which the paper referenced provides and Australian example.
l. 139-145 – This paragraph is confusing and does not add to the discussion. My suggestion is to delete it.
l. 146 – For long-distance wind dispersal the spores need to be resistant to UV light and desiccation. If they are then the possibility of long-distance wind dispersal becomes much more likely.
l. 150-152 – May be true for particular examples, but almost certainly not true as a general rule.
l. 154-156 – So distance is not driving the differentiation process. The question then is, “What is?”
l. 158-160 – If drift can cause a difference between populations, it is because the gene flow between them is limited. The limitation could be due to almost any reason.
l. 162-163 – What does it mean to be compatible with different selection pressures?
l. 180 – Why convergent evolution? What common factor are the different populations adapting to? Note that these populations can be phenotypically similar but genetically dissimilar.
l. 187-188 – Delete this sentence. Why these processes in particular? What makes them particularly worth mentioning?
l. 194-198 – Rewrite. Confusing.
l. 199-205 – Awkward and in need of rewriting to condense and simplify.
l. 208-210 – Explain why the sexual stage is essential here. No need to specify recombination. It is a part of the sexual stage, not an event separate from it!
l. 208-216 – Provide some specific, relevant examples.
l. 221 – Susceptibility instead of resistance?
l. 225 – Something unusual about Australia?
l. 227 – First mention of the Mlo gene. What does it do? Why is it important? Is it worth a separate paragraph if it is the main defense?
l. 241 – Northern hemisphere is an unusual designation. Can the countries specifically studied be included instead?
l. 246 – Reproduce instead of spread.
l.246-248 – Not clear what this sentence means. I think pathogenicity group and mating type have been confounded but I’m not sure.
l. 258-276 – The question seems to be whether the different f. spp. are distinguishable groups. The answer here should be phylogenetic. From that basis the remaining data need to be reinterpreted. Is it possible that the functional groups as defined by pathogenicity are defined differently by different investigators (depending perhaps on available host varieties?). If these definitions are fluid, then reliably distinguishing the f. spp. from one another could make the other results even more difficult to interpret. Here we need the authors to put values on different studies to give us some idea of what the real answer might be.
l. 274-277 – Is this sample large enough for all of the different alleles to be present at a reliably measurable frequency?
l. 278-281 – Differentiating between gene and genotype frequency/variation is important here.
l. 283 – Number of loci assessed?
l. 305 – Motility? Should it be mobility?
l. 309-310 – State that the results are “consistent with a randomly mating sexual population”.
l. 321-323 – Doesn’t make sense. Components?
l. 335-337 – Doesn’t make sense.
l. 347 and elsewhere – What is a gene center? A center of origin? If so, “center of origin” is the preferred term.
l. 356 – No conclusion or direction suggested for the field.
l. 366 – Living leaf tissue? In the winter? Where, outside of a greenhouse?
l. 373 – Transitional, cool and humid regions are all relative terms. Provide information to make it clear which locations are areas of concern.
l. 388-389 – Mating type ratios cannot be used to determine if sexual reproduction is occurring in the field.
l. 390-394 – This could be a matter of looking at the right place at the right time. If during the off season, for example, plant pathologists might not be in the field to see them.
l. 398 – Vectors rather than routes.
l. 423 – What is special about rain splash for the observed distribution?
l. 428 – Presumably these are 1:1 ratios, but whatever the numbers are they need to be specified here.
l. 443 – Need a reference. Why is there nothing beyond this point on benzimidazole fungicides and resistance to them?
l. 490 – Parasexual recombination is an explanation often invoked when nothing else seems to work. Is there any evidence that strains of interest could form a heterokaryon in which the asexual recombination could occur. Normally this would mean strains of interest need to be in the same VCG. Very little recombination in parasexual cycle – usually only a single crossover and then segregation of chromosomes to different progeny.
l. 508 – Chromosome rearrangements will not have any influence on pathogenicity, even though they could have a very important role in how the genome is organized and can be recombined.
l. 515-516 – Not clear what is being said in this sentence or why it is important.
l. 518-523 – This paragraph suggests lack of a common time for assessing pathogenicity phenotypes. Lack of a standard will make it difficult to compare the results from multiple groups.
l. 544 – What is an “expressive level of polymorphism”?
l. 555-563 – Another place to discuss female fertility issues and vegetative compatibility as limiting factors for genetic exchange.
l. 585-586 – Mendel proved that sexual recombination is genetic recombination? This sentence is nonsense.
l. 586-588 – This sentence is at the least an overstatement (if it is saying what is meant), but seems more likely to be in need of rewriting for clarity.
l. 588-591 – Mating types need not be present in equal frequency for sexual reproduction to occur. The presence of female fertile strains is probably more important, especially if asexual reproduction, e.g. via conidiation, also is occurring.
l. 594-595 – Good suggestion for a hypothesis to be tested.
l. 602-606 – Tests of linkage disequilibrium are needed to support these claims. Have such tests been made? Until then, the conclusions will be at best anecdotal.
l. 612-615 – Proteins isozymes usually provide little information in haploid organisms, as they rely on heterozygosity to maintain the polymorphisms that are useful for characterizing populations. Without heterozygous proteins, one optimum usually is selected and all the other alleles are relatively infrequent.
l. 621 – ITS region sequences will probably not detect the level of variation needed to meaningfully analyze relationships among and between VCGs. Markers such as AFLPs would do a much better job since there will be enough variation to have data worthy of being assessed in this manner.
l. 682 – PIC is undefined.
Author Response
Thank you for your valuable comments. Corrections are enclosed.
Author Response File: Author Response.docx
Reviewer 4 Report
The review manuscript article by Oğuz and Karakaya entitled "Genetic diversity of barley foliar fungal pathogens" is a good attempt to make a review on the leaf cause barley diseases.
- The manuscript is not well structured. There is only one numbered heading (Introduction) that seems the author wants to focus only on the Introduction!
- Even the author didn’t mention about Ramularia, which is an important barley pathogen.
- The author didn't mention or present any of their preliminary results.
- The whole review manuscript is without any tables and figures.
5. List of references is too long for this review.
Author Response
Thank you for your valuable comments. Corrections are enclosed.
Author Response File: Author Response.docx
Round 2
Reviewer 4 Report
The revised manuscript didn't follow my guidelines and I can't accept this manuscript for publication due to its scientific flaws.
Author Response
We uploaded the revised manuscript (in track changes mode)to the Agronomy journal system. We added a new paragraph (Effect of genomic structure of barley leaf pathogens on genetic variation) and a table with the barley foliar fungal pathogens sequencing works.
Thank you very much for your understanding.
