Identification of Field Tolerance and Resistance to Mal Secco Disease in a Citrus Germplasm Collection in Sicily

Round 1

Reviewer 1 Report

My personnel impression about this manuscript is not entirely negative since it provide significant advances for this research topic. The major shortcoming is referable to a strange experimental scheme with different replicate numbers for tested clones and consequently the lacking of statistical analysis of in vivo susceptibility data. Anyway, I informally encourage the resubmission of the paper.

 

For some detailed suggestions please see attached.

Comments for author File: Comments.pdf

Author Response

My persnnel impression about this manuscript is not entirely negative since it provide significant advances for this research topic. The major shortcoming is referable to a strange experimental scheme with different replicate numbers for tested clones and consequently the lacking of statistical analysis of in vivo susceptibility data. Anyway, I informally encourage the resubmission of the paper. 

We thank the reviewer for thinking that our research provides significant advances for this research topic.

We understand the concerns of the reviewer regarding the number of replicate trees. The collection was not established with the primary purpose of investigating the tolerance of each accession to mal secco disease.

The lack of formal statistical analysis is usual in germplasm evaluation trials and may be due to a low number of replicates, to plant death, unfavourable environmental conditions or other constraints related to plant growth in the field. This occurs frequently and is described in many published manuscripts dealing with field phenotyping and evaluation of field tolerance of a large number of germplasm accessions (see for example “Long-Term Field Evaluation Reveals Huanglongbing Resistance in Citrus Relatives”, by Ramadugu et al, Pant disease 2016; or “Identification of Resistance to Citrus Black Spot Using a Novel In-field Inoculation Assay”, by Miles et al 2019, Hortscience)

We would like to underline that the collection we investigated in the present work contains very interesting peculiarities: I) it contains unique diversity within the lemon germplasm and at interspecific level; II) it is placed in one of the areas with the highest pathogen pressure; III) it consists of adult plants. Therefore, we had the exceptional opportunity to test a large number of different adult trees belonging to many different accessions (50) in field conditions and obtained information which was not previously reported in literature. We tried to overcome the lack of some replicates by using more samples for the Real time analysis and by performing symptom observations for three consecutive years. We specified in the manuscript that a total of 828 samples for Real-time PCR analysis were collected in the two years of molecular detection (line 145). Such a high number of samples increased the robustness of our detection.

Other points:

Line 16: we modified the species names throughout the manuscript as suggested

Line 83: please continue "...versus this as well as other fungal pathogens". Authors should add some references (e.g. Gentile et al., 2007; Muccilli et al., 2020).

We accepted the suggestion of including the two references. Since the references refer to the generation of a genetically modified lemon using a Trichoderma gene and not with the introgression of citrus resistance genes into citrus genotypes (which was the topic of the paragraph), we added a new sentence at lines 78-81.

Reviewer 2 Report

The article suggests a methodology for the identification of the most tolerant lemon varieties to the disease Mal secco, a disease that causes damage to lemon trees particular in the Mediterranean basin. The specific methodology was applied in a citrus germplasm collection in Sicily and combines visual phenotyping with molecular screening based on real-time PCR that was performed on different plant tissues.

Although visual phenotyping and RT-PCR are extensively applied in germplasm evaluation and isolation for tolerant genotypes in many plant species, the methodology discriminated some accessions that were immune, resistant or tolerant to the disease. Finally, the results of this study can be further exploited by breeders that could include the superior genotypes to breeding programs and use them in order to introgress resistance genes into the lemon genome.

There are some typos and very minor errors, which I have not listed  (e.g. in line 81-82 it should be 'is essential for the introgression'). So text editing is recommended.

Author Response

The article suggests a methodology for the identification of the most tolerant lemon varieties to the disease Mal secco, a disease that causes damage to lemon trees particular in the Mediterranean basin. The specific methodology was applied in a citrus germplasm collection in Sicily and combines visual phenotyping with molecular screening based on real-time PCR that was performed on different plant tissues.

Although visual phenotyping and RT-PCR are extensively applied in germplasm evaluation and isolation for tolerant genotypes in many plant species, the methodology discriminated some accessions that were immune, resistant or tolerant to the disease. Finally, the results of this study can be further exploited by breeders that could include the superior genotypes to breeding programs and use them in order to introgress resistance genes into the lemon genome.

There are some typos and very minor errors, which I have not listed (e.g. in line 81-82 it should be 'is essential for the introgression'). So text editing is recommended.

We are grateful to the reviewer for his comments. We checked for typos and minor error throughout the manuscript and edited the whole manuscript. All modifications are included as track changes.

Reviewer 3 Report

The manuscript #Agronomy-970063 entitled “Identification of field tolerance and resistance to mal secco disease in a citrus germplasm collection in Sicily” by Russo et al. described the analysis of tolerance responses to a most important disease of lemon (mainly) in a germplasm Citrus collection.  

The manuscript well describes a method of disease detection by RT-PCR analysis identifying the tissue by which a significant correlation with disease symptoms was calculated.  

The major limit of the present paper could be due to the natural (and thus not homogeneous) MSD infection evaluated here compared to a most appropriate artificial infection as frequently reported for many fungal diseases. However, the repeated observations in different years could by-pass this gap.

Regarding this point, in my opinion it is not clear how many years the plants were evaluated: three (line 197-198 “starting from May 2018, for 3 consecutive years”) or two (line 129-130 “Samples of plant tissues were collected in the four cardinal directions for each plant in July 2018 and July 2019”) consecutive years? Could the Authors clarify this aspect?; the same at line 262-263 “Values of the Real time PCR refer to the mean values of the three replicates sampled in 2018 and the three replicates of 2019 approximated …

In the correlation analysis, it could be better to include also the p-value; in figures like 2 and 3 of this manuscript together with the correlation values are added an asterisks when the p-value is significant.

Furthermore, it is not clear but in figure 4, showing “the relationships between canopy and Ct values of the germplasm collection of CREA considered in this study”, many samples appeared missed. It is not well explained, but I think that the authors considered the results obtained from both young and mature leaves deriving a couple of point for each genotype in figure 4. The authors have to include the lacks and separate the two tissues, underlining the different groups of samples (similar to Figure 5).

How the Authors justify and/or comment the R² values so low in the Figures 4 and 5.

The samples’ origin is not clear; there has (probably) been a mistake in the references, because several genotype used in the present work are not listed in the cited paper [11] (Curk, F.; Ollitrault, F.; Garcia-Lor, A.; Luro, F.; Navarro, L.; Ollitrault, P. Phylogenetic origin of limes and lemons revealed by cytoplasmic and nuclear markers. Annals of Botany 2016, 117, 565–583, 442 doi:10.1093/aob/mcw005).

Finally, I retain necessary that the Authors produce a new table where the seven groups of assignment based on symptom severity scores are detailed, indicating all the accessions assigned to each group. This necessary information will indicate the best performances of each accession underlining also the best tolerant in the lemon genotypes, taking into account that other Citrus species is known to be tolerant to MSD.  

Overall, I retain the manuscript suitable for publication on Agronomy with major revisions inviting the Authors to produce a point-by-point answer letter indicating all the requested modifications/revisions.

Author Response

The manuscript #Agronomy-970063 entitled “Identification of field tolerance and resistance to mal secco disease in a citrus germplasm collection in Sicily” by Russo et al. described the analysis of tolerance responses to a most important disease of lemon (mainly) in a germplasm Citrus collection. 

The manuscript well describes a method of disease detection by RT-PCR analysis identifying the tissue by which a significant correlation with disease symptoms was calculated. 

The major limit of the present paper could be due to the natural (and thus not homogeneous) MSD infection evaluated here compared to a most appropriate artificial infection as frequently reported for many fungal diseases. However, the repeated observations in different years could by-pass this gap.

We are grateful to the reviewer, who gave us useful suggestions to improve the manuscript

We agree with the reviewer regarding the possible non-homogeneous infections. However, MSD is particularly difficult to phenotype after controlled inoculations in the absence of controlled environmental conditions. The pathogen may remain silent inside the xylem for months or years after infection if the climatic conditions are not favourable for the spread of the disease (see the review of Migheli et al 2009). Migheli and colleagues also reported that practically all citrus cultivars are susceptible to MSD in case of artificial inoculations, but this likely refers to young seedlings. Based on the above considerations, we believe that open field phenotyping for MSD where the pathogen is endemic is quite reliable to detect sources of field tolerance.

Regarding this point, in my opinion it is not clear how many years the plants were evaluated: three (line 197-198 “starting from May 2018, for 3 consecutive years”) or two (line 129-130 “Samples of plant tissues were collected in the four cardinal directions for each plant in July 2018 and July 2019”) consecutive years? Could the Authors clarify this aspect?; the same at line 262-263 “Values of the Real time PCR refer to the mean values of the three replicates sampled in 2018 and the three replicates of 2019 approximated …

We clarified in the abstract (lines 20-24) and other parts of the manuscript that visual observations were performed for three consecutive years (lines 107-108, lines 217-219), while sampling for Real time PCR analysis were performed for two consecutive years (line 139)

In the correlation analysis, it could be better to include also the p-value; in figures like 2 and 3 of this manuscript together with the correlation values are added an asterisks when the p-value is significant.

We included the p-values in the two figures as suggested, and we specified the significance in the captions

Furthermore, it is not clear but in figure 4, showing “the relationships between canopy and Ct values of the germplasm collection of CREA considered in this study”, many samples appeared missed. It is not well explained, but I think that the authors considered the results obtained from both young and mature leaves deriving a couple of point for each genotype in figure 4. The authors have to include the lacks and separate the two tissues, underlining the different groups of samples (similar to Figure 5).

We realized that the caption of figure 4 (now figure 5) was not explaining the plot properly and we modified it.  We also moved the scatterplot in the section related to the RT-PCR experiments, since it refers to the relationship between Ct values of twigs and canopy volume of each lemon replicate tree. We added a sentence (line 326-329) that better explains figure 5, and we rephrased the caption.

How the Authors justify and/or comment the R2 values so low in the Figures 4 and 5.

We believe these values of R² are quite common in field trials when analysing such a high level of different genotypes for phenotypic traits. We do not aim at using canopy volumes or Ct values as predictive of symptom severity, but we suggest using them as complementary data to visual observations. Both correlation values of figure 4 and 5 are statistically significant as indicated in figure 2 and figure 3 captions. We added the asterisks of statistical significance in figures 2 and 3.

The samples’ origin is not clear; there has (probably) been a mistake in the references, because several genotype used in the present work are not listed in the cited paper [11] (Curk, F.; Ollitrault, F.; Garcia-Lor, A.; Luro, F.; Navarro, L.; Ollitrault, P. Phylogenetic origin of limes and lemons revealed by cytoplasmic and nuclear markers. Annals of Botany 2016, 117, 565–583, 442 doi:10.1093/aob/mcw005).

We understand the reviewer’s concern and we changed the information in table 1. Now the “references” section contains information related to each accession.

Finally, I retain necessary that the Authors produce a new table where the seven groups of assignment based on symptom severity scores are detailed, indicating all the accessions assigned to each group. This necessary information will indicate the best performances of each accession underlining also the best tolerant in the lemon genotypes, taking into account that other Citrus species is known to be tolerant to MSD. 

We followed the suggestion of the reviewer and we included a new table (table 3) summarizing the assignment of each analysed accession to the 7 severity groups.

Overall, I retain the manuscript suitable for publication on Agronomy with major revisions inviting the Authors to produce a point-by-point answer letter indicating all the requested modifications/revisions.

Reviewer 4 Report

Introduction:

There are an overuse of adjectives on the text (i.e. many, very, some). The elimination or a more specific description would be enough to adjust the pace of reading.

I suggest reorganization of the paragraphs, so there is a linear, and continuous, flow of reading. Example: the paragraph on line 68 talks about the classification of susceptible variables to MSD. The next one, on line 73, talks about the pathogenicity. And the following paragraph (line 76) comes back to the host, and alternatives to find a suitable cultivar. By moving the paragraph on line 73 to the end of the paragraph on the line 57, we have a better flow reading. Line 91: double end period.

Material and Methods:

• How old were the plants when sampled?
• Line 113 - Did the same person performed the scoring? If yes, please make it clear.
• Line 141 - Add degrees Celsius after +/- 2
• Line 143 - Eppendorf tube? Falcon tube?
• Line 146 - Add degrees Celsius after -80
• Line 144 - Missing a period
• Line 149 - Proportion of chloroform-isoamyl alcohol (24:1)
• Line 150 - Change rpm to g force. The rpm is specific for the centrifuge you used
• Line 152 - Same for rpm to g
• Lines 156, 157, 164 - Change to international system: ng µl^-1

Results and Discussion

• The results are presented as the average of the three years of visual observations, canopy measurements and Ct, why?
• Line 185 to 187- Shouldn't this information be at the MM?
• Please be more clear about the "After 16 years". Sixteen year of establishment of the field trial? It would be informative to have a timeline from the start of the field trial, when the plants were replanted, and how many years after the initial planting the current study was set.
• Line 204 - It is quite difficult to follow the long list of accessions without symptoms. A table would do the trick, and the discussion could continue normally at line 210.
• Line 228 - You've stated that the scores were from 0 to 4, but didn't say anything about decimals. Is this because the number of trees were odd in these lemon clones?
• Explain what is what in the correlation matrix. The colors, the numbers, and the fading. This shall be after the figure's title.
• Table 2: Correct the headers: "Ct_value...", remove the "_".
• Fig 2 and 3: Keep the same formatting for the variables (Ct_Value... vs Ct Value...).
• Figure 4 and 5 - Increase font size on both axis and legend, and correct for the "Ct_value" for both. Add unit of measurement for the y-axis.
• Line 351 - Replace the word "behaviour" for "trend", "outcomes", "responses".

The discussion overall needs a little polish, as it doesn't have a lot of references that could justify the findings. It is a screening/survey study, but no actual pictures of the plants in the field were presented. Presenting the pictures of the actual plants would have a higher importance than the descriptive table and the grouping of susceptible/tolerant accessions. Please add the photos representative of each grouping, if they exist. Also, it would be interesting to have yield data on those plants, if collected, as one of the objectives was to find a possible accession for introgression of resistance genes.

Conclusion: no changes required. The authors finished by linking the results to the objectives described in the Introduction. 

Author Response

Introduction:

There are an overuse of adjectives on the text (i.e. many, very, some). The elimination or a more specific description would be enough to adjust the pace of reading.

We reduced the use of adjectives as suggested

I suggest reorganization of the paragraphs, so there is a linear, and continuous, flow of reading. Example: the paragraph on line 68 talks about the classification of susceptible variables to MSD. The next one, on line 73, talks about the pathogenicity. And the following paragraph (line 76) comes back to the host, and alternatives to find a suitable cultivar. By moving the paragraph on line 73 to the end of the paragraph on the line 57, we have a better flow reading.

We moved the paragraphs as suggested

Line 91: double end period.

Done

Material and Methods:

How old were the plants when sampled?

We specified the age of the sampled plant at lines 197-206

Line 113 - Did the same person performed the scoring? If yes, please make it clear.

Field evaluation was always performed by the same personnel. We added a sentence at lines 108-109

Line 141 - Add degrees Celsius after +/- 2

Done

Line 143 - Eppendorf tube? Falcon tube?

We specified the information in the text

Line 146 - Add degrees Celsius after -80

Done

Line 144 - Missing a period

included

Line 149 - Proportion of chloroform-isoamyl alcohol (24:1)

Done

Line 150 - Change rpm to g force. The rpm is specific for the centrifuge you used

Done

Line 152 - Same for rpm to g

Done

Lines 156, 157, 164 - Change to international system: ng µl^-1

Done

Results and Discussion

The results are presented as the average of the three years of visual observations, canopy measurements and Ct, why?

Based on the suggestion of the reviewer, we included the average values of the two years of Real Time PCR analysis. We maintained the average in the symptom scores to facilitate readers. Canopy measurements were performed only once in 2019 as an additional parameter to describe the sensitivity to MSD

Line 185 to 187- Shouldn't this information be at the MM?

We moved the sentence in the materials and methods section, as suggested

Please be more clear about the "After 16 years". Sixteen year of establishment of the field trial? It would be informative to have a timeline from the start of the field trial, when the plants were replanted, and how many years after the initial planting the current study was set.

We specified this information at lines 197-206. We also modified the information included in table 2 to specify how many original plants and replanted replicates were still alive at the end of the survey

Line 204 - It is quite difficult to follow the long list of accessions without symptoms. A table would do the trick, and the discussion could continue normally at line 210.

We modified the paragraph and included a table (table 3) as suggested

Line 228 - You've stated that the scores were from 0 to 4, but didn't say anything about decimals. Is this because the number of trees were odd in these lemon clones?

The scores represent an average of the four observations in the three years. We better specified the meaning of the symptom score values in the caption of table 2

Explain what is what in the correlation matrix. The colors, the numbers, and the fading. This shall be after the figure's title.

We included information about colors and fading in the figure captions

Table 2: Correct the headers: "Ct_value...", remove the "_".

done

Fig 2 and 3: Keep the same formatting for the variables (Ct_Value... vs Ct Value...).

done

Figure 4 and 5 - Increase font size on both axis and legend, and correct for the "Ct_value" for both. Add unit of measurement for the y-axis.

done

Line 351 - Replace the word "behaviour" for "trend", "outcomes", "responses".

done

The discussion overall needs a little polish, as it doesn't have a lot of references that could justify the findings. It is a screening/survey study, but no actual pictures of the plants in the field were presented. Presenting the pictures of the actual plants would have a higher importance than the descriptive table and the grouping of susceptible/tolerant accessions. Please add the photos representative of each grouping, if they exist. Also, it would be interesting to have yield data on those plants, if collected, as one of the objectives was to find a possible accession for introgression of resistance genes.

We added 6 supplemental figures (one for each group, excluding group one which consists of dead plants) that help readers to better understand the disease severity groups. Unfortunately, we do not have the yield data of our trial. However, to provide additional information which might be helpful to readers, we included a reference of a past study conducted on another field trial by Di Vaio and colleagues (2010) which includes information about yield and quality of most of the lemon cultivars of our study. We added a sentence at lines 105-106

Conclusion: no changes required. The authors finished by linking the results to the objectives described in the Introduction. 

We would like to thank the reviewer for the useful suggestion that lead to the improvement of our manuscript

Round 2

Reviewer 3 Report

The revised version#1 of the manuscript #Agronomy-970063 entitled “Identification of field tolerance and resistance to mal secco disease in a citrus germplasm collection in Sicily” by Russo et al. described the analysis of tolerance responses to a most important disease of lemon (mainly) in a germplasm Citrus collection.

I confirmed that the major merit of the manuscript is the setting up of a reliable method for Mal Secco disease detection by RT-PCR analysis identifying the tissue by which a significant correlation with disease symptoms was calculated.

The Authors’ answer about the major limit of the original version of the manuscript is satisfying for my side and I appreciate the citation from Migheli et al that stated that all citrus cultivar become susceptible to artificial inoculation. Based on the Authors’ considerations, it could be believable that open field phenotyping for MSD become the unique way to detect sources of field tolerance.

The Authors also clarified the number of consecutive years’ observations, further, I retain enough two years of sampling for Real time PCR analysis.

Both suggestions about the correlation analyses have been done correctly. The Authors also clarified some criticisms on Figures 4 and 5.

The new samples’ origin information appeared now clearer.

I really appreciate the inclusion in the new version of the manuscript a novel Table (#3) where it is clear the classification for resistance/tolerance of all the genotypes.

Finally, I warmly suggest the Authors to pay more attention to many, albeit minor, text editing for English style, started from the abstract I found at row 13 “that has resulted in severe damage” it is better “that resulted in severe damage”; at rows 16-17 I found “a major desirable goal for lemon growers and for breeders” it is better “a major desirable goal for lemon growers and breeders”; etc. in all the text.

 

Author Response

We thank again the reviewer for his comments. The new version of our manuscript was edited by a native English speaker.