Weeds in Cereal Crop Rotations May Host Fusarium Species That Cause Fusarium Head Blight and Grain Weight Losses in Wheat
, Arman Shamshitov 1, Evelina Zavtrikovienė 1, Sigita Janavičienė 2 and Gražina Kadžienė 3,*
Round 1
Reviewer 1 Report
Manuscript: Quantitative and Qualitative Evaluation of Fusarium Pathogens Isolated from Weeds in Inoculated Wheats (submitted to agronomy)
R comments: To my understanding, the objective of this study was to evaluate whether Fusarium species isolated from asymptomatic weeds could cause FHB in spring wheat. The pathogenicity of Fusarium graminearum and F. avenaceum isolates, originally isolated from asymptomatic weeds, were studied in spring wheat. Wheat plants were cultivated in one field trial (one year, only) and inoculated with the different isolates at flowering. Only two Fusarium isolates from each weed species were investigated. The DNA concentration of Fusarium graminearum and F. avenaceum was quantified in grain after harvest. 1000 grain weight and FHB -related symptoms were also observed. The authors conclude that F. graminearum isolates (regardless of host plant) were pathogenic on spring wheat.
R comments: I am surprised that that the authors used a field trial, and not a greenhouse study with controlled environmental conditions, for this purpose. Inoculum of Fusarium spp. are quite commonly occurring in soil/plant debris (and weeds). Therefore one would expect that there may be sufficient inoculum available in the fields/surroundings, both to infect the non-inoculated as well as the inoculated plants. I am therefore a bit surprised that the grain harvested from the non-inoculated control plants had such a low DNA concentration of the Fusarium species analysed. However, the weather conditions may not have been optimal for inoculum production and disease development of Fusarium species in this field. In line 123, I find the following information: Inoculated spikes were immediately covered with plastic bags to ensure sufficient humidity conditions and were removed after three days. Did you also cover the water treated control?
R comments: Regarding F. avenaceum, the DNA concentrations were relatively low. To my opinion, too low to be part of a meaningful discussion regards to differences in pathogenicity between the different isolates. Regards to F. graminearum, the DNA levels were higher than for F. avenaceum, and thus can be discussed more thoroughly.
Line 390-391: Based on the results of FHB disease severity and fungal biomass, we confirmed that isolates of F. graminearum were significantly more aggressive than F. avenaceum in spring wheat
R comments: It makes sense that the F. graminearum isolates were more aggressive than F. avenaceum on wheat. However, if the weather conditions had been optimal for development of F. avenaceum you might have seen a different picture. Would be relevant if you discussed these results in relation to the observed weather conditions.
R comments: The authors present results from one year study and one field trial, only. The discussion about whether there is a difference in pathogenicity between isolates originally isolated from different weed species is based on results from two isolates from each weed species, only. To my opinion this is too little data to be presented in a journal paper. Perhaps it would be better if this work was published as a research letter?
R comments: To my opinion, several of the paragraphs written in the results part rather belong to the discussion part. Such as:
Line 262- These results also confirm that F. graminearum strains asymptomatically residing in alternative host plants (weeds) are potential causative agents of FHB. Spreading weeds in cereal crop rotations are a potential source of Fusarium infection. These data also show that the severity of FHB is more influenced by Fusarium species and the specific strain than the host plant of the pathogen.
R comments: Other examples are Line 208-2011,315-320, 327-330, etc
R comments: The authors should carefully go through the manuscript in order to improve its quality. If this manuscript is to be published, the English must be improved. It is sometimes hard to understand the meaning of the sentences. In addition, it would be nice if the authors included less information within each sentence /wrote shorter sentences. Here are some examples (NB This is not a complete list of all the sentences that should be improved):
Ttitle: Quantitative and Qualitative Evaluation of Fusarium Pathogens Isolated from Weeds in Inoculated Wheats
R comments: This sentence does not make sense to me: Are the isolates isolated from weeds in inoculated wheat?... What kind of evaluation is performed? Perhaps be more specific? Perhaps better to find a title that describes the results obtained in the study? Something like: Weeds in cereal crop rotations may host Fusarium pathogens capable of causing Fusarium head blight in wheat” -Rewrite.
Line 21-23 in the abstract: “The objectives of this study were to identify Fusarium species (F. graminearum and F. avenaceum) and evaluate its fungal biomass in artificially inoculated grains with isolates from weeds and its influence on the severity of FHB and spring wheat grain yield under the field conditions.”
R comments: It was not the grains that were inoculated (but the wheat plants). This should be corrected throughout the manuscript
R comments: The objective presented by the authors is perhaps more a description of what they had performed in this study? To present the objective, perhaps better to write something like: The objective of this study was to evaluate whether Fusarium species isolated from weeds commonly observed in cereal crop rotations are capable of causing FHB in cereals. -Rewrite.
Line 71-72: In our study, we used the quantitative real-time PCR (qPCR) method to assess the amount of Fusarium biomass in artificially inoculated wheat heads with isolates from weeds.
R comments: Perhaps write shorter sentences? For instance rather than to write: “ artificially inoculated wheat heads» you could write something like: ..fungal DNA in grain harvested from wheat. -Rewrite.
Line 141-142: DNA was extracted from 100 mg of artificially infected grain powder using the commercial ZR Fungal/Bacterial DNA MiniPrep Kit (Zymo Research, Irvine, USA) following the manufacturer’s instructions.
R comments: Did you infect the grain powder?...-Rewrite
Line 379-380: Detection of high biomass amount of F. graminearum on tested weedy grasses may indicate that these weed species are more easily colonized by this Fusarium species compared with F. avenaceum.
R comments: Did you analyse the biomass of F. graminearum in weedy grasses? -Rewrite
Line 386-388: This indicates that fungal biomass in wheat grains depends on many factors. Our results also reveal that lower biomass in wheat may be more pathogenic to cultivated plants as in the above author's study.
R comments: The phrase: «lower biomass in wheat may be more pathogenic to cultivated plants» does not make sense to me. Can biomass be pathogenic?
-Rewrite
Line 416: Comparing the correlation coefficients between FHB severity, the biomass of Fusarium in grain, and grain yield, we found that biomass of F. graminearum positively correlated with FHB disease severity which was evaluated visually and negatively correlated with 1000 grain weight.
R comments: I would prefer that you use fungal DNA concentration or content rather than fungal biomass. (Throughout the manuscript)
Line 422: This proves the pathogenicity of this species to wheat and leads to a decrease in grain yield.
R comments: This sentence does not make sense to me. -Rewrite
Etc.
Figure 1.:
R comments: I would find it easier to read this figure if you named all the isolates (strains) according to the host species they were originally isolated from. Perhaps write a code for each isolate in Table 1 that you could use throughout the manuscript?
Line 406: After assessing the influence of Fusarium strains isolated from various host plants on grain weight,
R comments: -Rewrite
Line 341 and 342: As mentioned by Postic et al. [15], weeds often become a source of pathogens when there is no significant host plant around, such as soil residues.
R comments: Is soil residue a host plant? -Rewrite
Line 435: 5. Conclusions This study showed that (i) weeds serve as an alternative source of Fusarium spp. with- out any disease symptoms,..
R comments: As I understand, this (i) was already presented in a previous paper (Suproniene et al 2019)
Author Response
General comments to the authors:
To my understanding, the objective of this study was to evaluate whether Fusarium species isolated from asymptomatic weeds could cause FHB in spring wheat. The pathogenicity of Fusarium graminearum and F. avenaceum isolates, originally isolated from asymptomatic weeds, were studied in spring wheat. Wheat plants were cultivated in one field trial (one year, only) and inoculated with the different isolates at flowering. Only two Fusarium isolates from each weed species were investigated. The DNA concentration of Fusarium graminearum and F. avenaceum was quantified in grain after harvest. 1000 grain weight and FHB -related symptoms were also observed. The authors conclude that F. graminearum isolates (regardless of host plant) were pathogenic on spring wheat.
Answer: Thank you for your review. The aim of our study was to determine the pathogenicity of two Fusarium species (F. graminearum and F. avenaceum) residing in weeds and the severity of FHB disease caused by them and their influence on grain weight. Although more Fusarium species (F. culmorum, F. poae, F. equiseti, F. oxysporum, F. tricinctum, F. redolens, F. sporotrichioides) have been isolated from weeds in previous authors’ study [17]. The most common species are F. graminearum, F. culmorum and F. avenaceum which cause FHB to wheat. We chose only F. graminearum and F. avenaceum because of the presence of the sexual stage. Additional information was added in introduction: “F. graminearum and F. avenaceum species have sexually produced ascospores, while other species produce asexual conidia. Macroconidia are reported to be splash-dispersed short distances while sexual spores can be transported longer distances by wind [11].” Line 46-49. “The aim of current study was to determine the pathogenicity of two Fusarium species (F. graminearum and F. avenaceum) isolated from asymptomatic weeds to spring wheat. Although more Fusarium species (F. culmorum, F. poae, F. equiseti, F. oxysporum, F. tricinctum, F. redolens, F. sporotrichioides) have been isolated from weeds in previous authors’ study [17]. The most common species are F. graminearum and F. avenaceum which cause FHB to wheat in Lithuania [29,30]. Also, both F. graminearum and F. avenaceum have the sexual stage and ascospores produced at this stage can serve as the main source of spore dispersion transported long distances by wind [11].” Line 79-96.
- Suproniene, S.; Kadziene, G.; Irzykowski, W.; Sneideris, D.; Ivanauskas, A.; Sakalauskas, S.; Serbiak, P.; Svegzda, P.; Kelpsiene, J.; Pranaitiene, S.; Jedryczka, M. Asymptomatic weeds are frequently colonized by pathogenic species of Fusarium in cereal-based crop rotations. Weed research, 2019, 59, 312–323. https://doi.org/10.1111/wre.12367
- Sakalauskas, S.; Stumbriene, K.; Suproniene, S.; Svegzda, P. Changes in Fusarium Link species composition from Lithuanian wheat grain in years 2005-2007 to 2011-2013. Proceedings of the Latvia University of Agriculture, 2014, 32(1), 45–50. https://doi.org/10.2478/plua-2014-0013
- Suproniene, S., Sakalauskas, S., Stumbriene, K., Zvirdauskiene, R., Svegzda P. Variances in trichothecene chemotype distri-bution in Lithuanian wheat grain and within pure culture Fusarium graminearum isolated from the same grain samples. Euro-pean Journal of Plant Pathology, 2015, 144(2), 371–381 http://dx.doi.org/10.1007/s10658-015-0774-9
I am surprised that the authors used a field trial, and not a greenhouse study with controlled environmental conditions, for this purpose. Inoculum of Fusarium spp. are quite commonly occurring in soil/plant debris (and weeds). Therefore one would expect that there may be sufficient inoculum available in the fields/surroundings, both to infect the non-inoculated as well as the inoculated plants. I am therefore a bit surprised that the grain harvested from the non-inoculated control plants had such a low DNA concentration of the Fusarium species analyzed. However, the weather conditions may not have been optimal for inoculum production and disease development of Fusarium species in this field. In line 123, I find the following information: Inoculated spikes were immediately covered with plastic bags to ensure sufficient humidity conditions and were removed after three days. Did you also cover the water treated control?
Answer: We agree with your opinion. Another study is in the process now and we will publish the results obtained in the greenhouse experiment separately. However, in this case, we tested it in field conditions and surprisingly the results correlated well with each other.
Of course, the water-treated control was also covered with a plastic bag to ensure the same humidity. Additional information was added: “Inoculated heads and water-treated control were immediately covered with plastic bags to ensure sufficient humidity conditions and were removed after three days.” Line 125-127.
Regarding F. avenaceum, the DNA concentrations were relatively low. To my opinion, too low to be part of a meaningful discussion regards to differences in pathogenicity between the different isolates. Regards to F. graminearum, the DNA levels were higher than for F. avenaceum and thus can be discussed more thoroughly.
Answer: This study was based on the comparison of the two most commonly found Fusarium species, so we investigated their DNA content in wheat and specifically its influence on FHB disease and grain weight. F. graminearum DNA content in wheat was discussed more thoroughly. Line 556-573.
Line 390-391: Based on the results of FHB disease severity and fungal biomass, we confirmed that isolates of F. graminearum were significantly more aggressive than F. avenaceum in spring wheat
R comments: It makes sense that the F. graminearum isolates were more aggressive than F. avenaceum on wheat. However, if the weather conditions had been optimal for development of F. avenaceum you might have seen a different picture. Would be relevant if you discussed these results in relation to the observed weather conditions.
Answer: According to other publications, F. graminearum in small DNA quantities, causes a significantly more severe FHB disease than other species. Due to the produced mycotoxins and the easier colonization of the host plant. Of course, the weather conditions are important for Fusarium species development. The part ‘weather conditions‘ was relocated to results (Line 330-349). These results were discussed (Line 396-409).
The authors present results from one year study and one field trial, only. The discussion about whether there is a difference in pathogenicity between isolates originally isolated from different weed species is based on results from two isolates from each weed species, only. To my opinion this is too little data to be presented in a journal paper. Perhaps it would be better if this work was published as a research letter?
Answer: The study was conducted for only one year, because we had limited financial opportunities. It was important for us to indicate whether DNA content estimation is better than visual evaluation, but the correlations showed that both indicators (FHB severity and DNA content) give similar results.
To my opinion, several of the paragraphs written in the results part rather belong to the discussion part. Such as:
Line 262- These results also confirm that F. graminearum strains asymptomatically residing in alternative host plants (weeds) are potential causative agents of FHB. Spreading weeds in cereal crop rotations are a potential source of Fusarium infection. These data also show that the severity of FHB is more influenced by Fusarium species and the specific strain than the host plant of the pathogen.
R comments: Other examples are Line 208-2011, 315-320, 327-330, etc
Answer: Some of the sentences were deleted or relocated to the discussion part. Line 423-426.
The authors should carefully go through the manuscript in order to improve its quality. If this manuscript is to be published, the English must be improved. It is sometimes hard to understand the meaning of the sentences. In addition, it would be nice if the authors included less information within each sentence /wrote shorter sentences. Here are some examples (NB This is not a complete list of all the sentences that should be improved):
Answer: The usage of English language was checked.
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Line |
Comment |
Answer, line in corrected paper |
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Title |
Quantitative and Qualitative Evaluation of Fusarium Pathogens Isolated from Weeds in Inoculated Wheats This sentence does not make sense to me: Are the isolates isolated from weeds in inoculated wheat?... What kind of evaluation is performed? Perhaps be more specific? Perhaps better to find a title that describes the results obtained in the study? Something like: Weeds in cereal crop rotations may host Fusarium pathogens capable of causing Fusarium head blight in wheat” -Rewrite. |
Thank you for the suggestion, we agree to change it, perhaps a title like “Weeds in cereal crop rotations may host Fusarium species that cause Fusarium head blight and grain weight losses in wheat” would be better?
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Line 21-23 |
“The objectives of this study were to identify Fusarium species (F. graminearum and F. avenaceum) and evaluate its fungal biomass in artificially inoculated grains with isolates from weeds and its influence on the severity of FHB and spring wheat grain yield under the field conditions.” It was not the grains that were inoculated (but the wheat plants). This should be corrected throughout the manuscript |
The term “inoculated grains” was changed to “inoculated wheat plants”. Line 23, 33, 278 and etc. |
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Line 21-23 |
The objective presented by the authors is perhaps more a description of what they had performed in this study? To present the objective, perhaps better to write something like: The objective of this study was to evaluate whether Fusarium species isolated from weeds commonly observed in cereal crop rotations are capable of causing FHB in cereals. -Rewrite. |
Thank you for your comments, we would like to discuss this part. Perhaps better to write: “This article focuses on the determination of DNA content of Fusarium species (F. graminearum and F. avenaceum) in artificially inoculated wheat plants with isolates from weeds and its influence on the severity of FHB and spring wheat 1000 grain weight under the field conditions.” or “The aim of current study was to determine the pathogenicity of two Fusarium species (F. graminearum and F. avenaceum) isolated from asymptomatic weeds to spring wheat.”? Because this hypothesis: "Fusarium species isolated from weeds commonly observed in cereal rotations can cause FHB in cereals" has already been proven in previous articles.
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Line 71-72 |
In our study, we used the quantitative real-time PCR (qPCR) method to assess the amount of Fusarium biomass in artificially inoculated wheat heads with isolates from weeds. Perhaps write shorter sentences? For instance rather than to write: “ artificially inoculated wheat heads» you could write something like: ..fungal DNA in grain harvested from wheat. -Rewrite. |
The sentence was changed: “In our study, we used the quantitative real-time PCR (qPCR) method to assess the fungal DNA in grain harvested from wheat.” Line 72-73 |
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Line 141-142: |
DNA was extracted from 100 mg of artificially infected grain powder using the commercial ZR Fungal/Bacterial DNA MiniPrep Kit (Zymo Research, Irvine, USA) following the manufacturer’s instructions. Did you infect the grain powder?...-Rewrite |
The sentence was changed: “DNA was extracted from 100 mg of grain powder using the commercial ZR Fungal/Bacterial DNA MiniPrep Kit (Zymo Research, Irvine, USA) following the manufacturer’s instructions.” Line 146-148. |
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Line 379-380: |
Detection of high biomass amount of F. graminearum on tested weedy grasses may indicate that these weed species are more easily colonized by this Fusarium species compared with F. avenaceum. R comments: Did you analyse the biomass of F. graminearum in weedy grasses? -Rewrite
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The sentence was changed: “Detection of high F. graminearum DNA amount in wheat may indicate that this species more easily colonizes cultivated plants and weeds compared with F. avenaceum.” Line 390-391. |
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Line 386-388: |
This indicates that fungal biomass in wheat grains depends on many factors. Our results also reveal that lower biomass in wheat may be more pathogenic to cultivated plants as in the above author's study. R comments: The phrase: «lower biomass in wheat may be more pathogenic to cultivated plants» does not make sense to me. Can biomass be pathogenic?
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The sentence was changed: “Our results also reveal that F. graminearum with lower DNA content in wheat caused FHB disease more strongly, similar to the author's study mentioned above.” Line 394-395. |
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Line 416: |
Comparing the correlation coefficients between FHB severity, the biomass of Fusarium in grain, and grain yield, we found that biomass of F. graminearum positively correlated with FHB disease severity which was evaluated visually and negatively correlated with 1000 grain weight. R comments: I would prefer that you use fungal DNA concentration or content rather than fungal biomass. (Throughout the manuscript)
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This term was changed throughout the manuscript. Line 24, 32, 34, 75, 78, 79, 87, 101, 109, 145, 168, 169, 178, 182, 187, 191, 200, 201, 209, 213, 219, 225, 229, 257, 290 292, 294, 301, 305, 307, 308, 311, 314, 317, 321 and etc. |
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Line 422: |
This proves the pathogenicity of this species to wheat and leads to a decrease in grain yield. R comments: This sentence does not make sense to me. -Rewrite
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The sentence was changed: “These results confirm F. graminearum pathogenicity to wheat and its influence on grain weight.” Line 447. |
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Figure 1.:
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R comments: I would find it easier to read this figure if you named all the isolates (strains) according to the host species they were originally isolated from. Perhaps write a code for each isolate in Table 1 that you could use throughout the manuscript?
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All the isolates were renamed. Table1, Figure 1, Figure 2, Figure 3, Figure 4, Table 4 and in text. |
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Line 406: |
After assessing the influence of Fusarium strains isolated from various host plants on grain weight, R comments: -Rewrite
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The sentence was changed: “The study of the influence of Fusarium strains on grain weight showed that all tested F. graminearum strains statistically reliably reduced spring wheat grain weight compared to the control variant.” Line 427-429. |
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Line 341 and 342: |
As mentioned by Postic et al. [15], weeds often become a source of pathogens when there is no significant host plant around, such as soil residues. R comments: Is soil residue a host plant? -Rewrite
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The sentence was changed: “As mentioned by Postic et al. [15], weeds often become a source of pathogens when there is no significant host plant around.” Line 352-353. |
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Line 435: 5. Conclusions |
This study showed that (i) weeds serve as an alternative source of Fusarium spp. with- out any disease symptoms,.. R comments: As I understand, this (i) was already presented in a previous paper (Suproniene et al 2019) |
The conclusion was deleted. |
Reviewer 2 Report
Dear authors
The manuscript provides information on the comparison of the pathogenicity of Fusarium graminearum and Fusarium avenaceum obtained from weeds near or within wheat fields and these Fusarium species isolated from wheat . The data provides relevant information on the importance of weeds as potential inocula source of the FHB pathogens as it was observed in other studies done in Argentina and Croatia.
Since the Fusarium graminearum is a complex of species, it will be important specify Fusarium graminearum sensu stricto in this study
The biomass of Fusarium graminearum and FHB severity was also compared in other studies see Palazzini et al, 2015 Cereal Research Communcations
The cultivar of wheat need to be specify ( variety) since differences in severity can be related to the cultivar
The results were obtained during only one year of study, with certain meteorological conditions, the results can vary under different climatic conditions also, so the conclusions can be limited
Author Response
General comments to the authors:
Since the Fusarium graminearum is a complex of species, it will be important specify Fusarium graminearum sensu stricto in this study.
Answer: Sentences was changed: “Fusarium Head Blight (FHB) is currently one of the most important diseases, caused primarily by Fusarium graminearum sensu stricto[1], affecting wheat, barley and other small grain cereal crops.” Line 37-39 “Heads of spring wheat cultivar 'KWS Chamsin' were inoculated with the main Fusarium pathogens (F. avenaceum and F. graminearum sensu stricto) isolated from the internal tissues of asymptomatic weeds.” Line 91-93.
The biomass of Fusarium graminearum and FHB severity was also compared in other studies see Palazzini et al, 2015 Cereal Research Communications.
Answer: Additional information is added in discussion part: “Palazzini [47] study also showed that DNA amount increases exponentially with the severity of wheat head disease. They observed maximum disease severity in wheat heads by 35% which corresponded to an average of 5675 pg DNA. It is similar to our results, which show the strong-positive correlation between disease severity of the heads and F. graminearum DNA amount in grains.” Line 441-445.
The cultivar of wheat need to be specify (variety) since differences in severity can be related to the cultivar.
Answer: We used spring wheat cultivar ‘KWS Chamsin’. Additional information is added: “Heads of spring wheat cultivar ‘KWS Chamsin’ were inoculated with the main Fusarium pathogens (F. avenaceum and F. graminearum sensu stricto) isolated from the internal tissues of asymptomatic weeds.” Line 91-93. “We compared the pathogenicity of Fusarium strains isolated from the main (spring wheat ‘KWS Chamsin’) and alternative host plants (weeds) to spring wheat within each Fusarium species.” Line 101-104.
The results were obtained during only one year of study, with certain meteorological conditions, the results can vary under different climatic conditions also, so the conclusions can be limited.
Answer: Conclusions were supplemented: “It is important to mention that this study was conducted in only one year and weather conditions during wheat anthesis could affect results, for understanding the influence of meteorological conditions on severity of FHB requires further research.” Line 468-471.
Reviewer 3 Report
Major comments
The manuscript “Quantitative and Qualitative Evaluation of Fusarium Pathogens Isolated from Weeds in Inoculated Wheats” presented the comparison of the effects of two species of Fusarium on biomass production, Fusarium severity and thousand kernel weight changes. The study gives novel information about F. avenaceum and its comparison with F. graminearum. However, to be publishing major changes should be addressed.
-In the section “Weather conditions” as pasrt of methods and materials, it is described extensively the weather conditions occurred between the inoculation and harvest of spikes. This looks like more a result and also could be organized in other way.
-The authors talk about that one of the measured traits was grain yield. However, they should describe this trait as grain yield per spike because is not a true grain yield measurement referred to the area. Grain yield per spike, 1000 grain weight and grain yield can´t be used as equivalent traits. The first two are yield-components exhibiting a partial correlation with grain yield (kg/ha).
-Which cultivar of bread wheat was used to evaluated the Fusarium severity and others analyses?, It was used one or more cultivars? How the field conditions of the wheat plot/plots used in the study were? (Seed density, cultivar, and management). I expected it was used a highly susceptible cultivar but this information is not reported.
As the M&M are described in this version of the manuscript, for ex. the affirmation “Interestingly, the F. avenaceum isolates from wheat (G1-II and TG5-IV) partially significantly increased grain weight compared to the control” (line 292) could be the result of a random effect based on a random sampling. Were only main spikes collected or also from other positions? Because this affects the grain size.
Minor comments
Please, authors need to call the heads/spikes/ears using a uniform way along the text.
Line 164: A full explanation of how the DNA of Fusarium isolates was referred to plant DNA μg could contribute to better understand the standardization
Line 202: I think the word “spices” it is a mistake, please could you fix it.
Line 215,230 The expression “main – wheat” is not clear or correct.
Line 237: the phrase “main plants” could be replaced by "main host"
Line 244: This phrase “The disease severity was higher in wheat inoculated with F. graminearum strains than with F. avenaceum” maybe is better located previously, at the beginning of Line 242
Line 277-78: A bracket is lack in "(1000 grains weight was calculated by the number of grains per head and grain weight per head". Also the connective "by" suggest a multiplication but 1000 grain weight is not the result of number X weight. Please rewrite this explanation.
Line 287: “yield loss” is not the correct expression.
Line 304 and 305. The explanation of why authors found this negative correlation was not clearly expressed should be amended.
Line 421: The phrase “In our experiments, F. graminearum infection reduced spring wheat grain from 22% to 46%.” don’t clearly mentioned the trait. Need to be revised and improved.
Author Response
Comments to the Author:
In the section “Weather conditions” as part of methods and materials, it is described extensively the weather conditions occurred between the inoculation and harvest of spikes. This looks like more a result and also could be organized in other way.
Answer: ‘Weather conditions’ part was relocated to results part. (Line 330-349). These results were discussed (Line 396-409).
The authors talk about that one of the measured traits was grain yield. However, they should describe this trait as grain yield per spike because is not a true grain yield measurement referred to the area. Grain yield per spike, 1000 grain weight and grain yield can´t be used as equivalent traits. The first two are yield-components exhibiting a partial correlation with grain yield (kg/ha).
Answer: We agree with your observations. In the text, the term “grain yield” has been changed to the „weight of 1000 grains“. Changes were made in: Line 18, 23-24, 29, 33, 35, 81, 88, 188, 293, 304, 308-309, 315, 434.
Which cultivar of bread wheat was used to evaluated the Fusarium severity and others analyses? It was used one or more cultivars? How the field conditions of the wheat plot/plots used in the study were? (Seed density, cultivar, and management). I expected it was used a highly susceptible cultivar but this information is not reported.
Answer: For evaluation of fusarium head blight severity, FHB susceptible spring wheat cultivar ‘KWS Chamsin’, commonly grown in Lithuania, was used. Spring wheat was drilled in 21st of May 2019 at a seed rate of 5 mln. per ha. Conventional crop management practice (fertilization, weed and pest control, growth regulation) was used, except fungicide treatment against FHB. Line 91-93 and 101-104.
As the M&M are described in this version of the manuscript, for ex. the affirmation “Interestingly, the F. avenaceum isolates from wheat (G1-II and TG5-IV) partially significantly increased grain weight compared to the control” (line 292) could be the result of a random effect based on a random sampling. Were only main spikes collected or also from other positions? Because this affects the grain size.
Answer: This result may have been a false positive due to random collection of wheat heads and grain size in these test variants and controls. The results show that F. avenaceum did not significantly affect the grain weight, and the increased weight compared to the control was just a coincidence. The explanation of the obtained result was extended: “The relationship between the biomass of F. avenaceum in grain and the intensity of Fusarium head blight was negatively correlated (-0.682).(Table 3). This correlation may have been a false negative due to random collection of wheat heads and grain size in these test variants and controls. The results show that F. avenaceum did not significantly affect the grain weight, and the increased weight compared to the control was just a coincidence.” Line 293-296.
Please, authors need to call the heads/spikes/ears using a uniform way along the text.
Answer: All terms “spikes” and “ears” was changed to “heads”. Changes were made in: Line 91, 106, 111-112, 122-129, 132, 137, 231, 247, 296 and etc.
Specific comments to the authors:
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Line |
Comment |
Answer, line in corrected paper |
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Line 164 |
A full explanation of how the DNA of Fusarium isolates was referred to plant DNA μg could contribute to better understand the standardization |
Sentence was changed: “Amplification results of each individual sample from each species-specific assay were evaluated by studying the dissociation curve and Ct value. The wheat plant assay was further used to provide a normalized measurement for DNA content in each sample, which was expressed as picograms of Fusarium DNA per micrograms of plant DNA according to Nicolaisen et al [33].” Line 175-179. |
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Line 202 |
I think the word “spices” it is a mistake, please could you fix it. |
Sentence was changed: “When spring wheat species were inoculated with strains of F. avenaceum from meadow grass <...>”. Line 198. |
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Line 215,230 |
The expression “main – wheat” is not clear or correct. |
Title of Figure 1 was changed: “The DNA content of the pathogen F. avenaceum (F. avenaceum DNA pg/ plant DNA µg) (± SEM) in spring wheat grains inoculated with F. avenaceum and F. graminearum different strains from alternative host weeds and main host wheat (values with different letters (a-f) indicate statistically significant differences (p＜0.05) between study treatments, C – water inoculated control).” Line 209 |
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Line 237 |
the phrase “main plants” could be replaced by "main host" |
This part was changed: “The DNA content of the pathogen F. graminearum (F. graminearum DNA pg/ plant DNA µg) (± SEM) in spring wheat grains inoculated with F. avenaceum and F. graminearum different strains from alternative host weeds and main host wheat (values with different letters (a-f) indicate statistically significant differences (p＜0.05) between study variants, C – water inoculated control).” Line 225. |
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Line 244 |
This phrase “The disease severity was higher in wheat inoculated with F. graminearum strains than with F. avenaceum” maybe is better located previously, at the beginning of Line 242 |
Sentences were relocated: “The disease severity was higher in wheat inoculated with F. graminearum strains than with F. avenaceum. Each isolate caused different disease severities at 21 DAI, ranging from 53.6% to 84.1% in spring wheat inoculated with F. graminearum and 1.1% to 27.2% in wheat inoculated with F. avenaceum (Figure 3).” Line 239-242. |
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Line 277-78 |
A bracket is lack in "(1000 grains weight was calculated by the number of grains per head and grain weight per head". Also the connective "by" suggest a multiplication but 1000 grain weight is not the result of number X weight. Please rewrite this explanation |
A bracket was added and an explanation was rewritten: “Average of 1000 grain weight of spring wheat inoculated with different Fusarium strains isolated from alternative (weeds) and main (wheat) host plants (1000 grain weight was calculated based on the weight and number of grains per head).” Line 267-271. |
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Line 287 |
“yield loss” is not the correct expression. |
Sentence was changed: “In both cases, significant statistical differences were observed, confirming variable degrees of grain weight reduction in the artificially inoculated wheat plants with F. graminearum strains.” Line 277-279.
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Line 304-305 |
The explanation of why authors found this negative correlation was not clearly expressed should be amended. |
The explanation was amended: “The relationship between the F. avenaceum DNA content in grain and the intensity of Fusarium head blight was negatively correlated (-0.682) (Table 3). This correlation may have been a false negative due to random collection of wheat heads and grain size in these test variants and controls. The results show that F. avenaceum did not significantly affect the grain weight, and the increased weight compared to the control was just a coincidence.” Line 293-298 |
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Line 421 |
The phrase “In our experiments, F. graminearum infection reduced spring wheat grain from 22% to 46%.” don’t clearly mentioned the trait. Need to be revised and improved. |
The phrase was improved: “In our experiments, F. graminearum infection reduced 1000 spring wheat grain weight from 22% to 46% compared with control.” Line 445-446. |
Reviewer 4 Report
Dear Authors,
Its an interesting article on the quantitative and qualitative evaluation of Fusarium pathogens.
I have following concerns
1. I have concerns about the scheme of the experiment, which wheat cultivar was used? The identified susceptible check should be used to study FHB response.
2. Authors should provide representative images for the FHB infections.
3. Another major concern is, I couldn’t find the information on the sequences. For identification of the species, authors should match the sequences with the reference database. In qPCR what was the positive and negative reference for F. graminearum and F. avenaceum? Pls, provide information on it. For the standard curve did you use the DNA of identified isolates of both species? Give the information on the reference isolates along with accession numbers.
The manuscript needs improvements on the above points.
Thanks
Author Response
Comments to the Author:
I have concerns about the scheme of the experiment, which wheat cultivar was used? The identified susceptible check should be used to study FHB response.
Answer: For evaluation of fusarium head blight severity, FHB susceptible spring wheat cultivar ‘KWS Chamsin’, commonly grown in Lithuania, was used. Additional information was added in Line 91, 103.
Authors should provide representative images for the FHB infections.
Answer: All inoculated wheat heads were not photographed, therefore not presented in the article.
Another major concern is, I couldn’t find the information on the sequences. For identification of the species, authors should match the sequences with the reference database. In qPCR what was the positive and negative reference for F. graminearum and F. avenaceum? Pls, provide information on it. For the standard curve did you use the DNA of identified isolates of both species? Give the information on the reference isolates along with accession numbers.
Answer: Sequencing of tef1α (= eEF1 α a, translation elongation factor 1- α) gene amplicons was done for four F. graminearum strains – WB3rFG, WB4rFG, FP9sFG, FP10lFG. Which matches with codes respectively 544r, 144r, 541s, 153l as described by Suproniene et al. [31]. All F. graminearum strains belong to F. graminearum sensu stricto as well as the other 36 F. graminearum strains obtained from weeds in previous study. The rest F. graminearum and F. avenaceum used in current study were identified by species specific PCR described by Suproniene et al. [31, 17]. Individual standard curves were made of a sixfold dilution series using already confirmed pure F. graminearum and F. avenaceum DNA and wheat plant DNA. Amplification results of each Fusarium species sample were evaluated by studying the dissociation curve and Ct value.
Author Response File: 
Author Response.pdf
Round 2
Reviewer 1 Report
Line 261: The highest amounts of the F. graminearum pathogen (2068.34 and 1277.347 pg/µg) were..
amounts can be corrected to concentration and pathogen can be altered to DNA
I suggest this sentence is corrected to: The highest concentrations of F. graminearum DNA (2068.34 and 1277.347 pg/µg) were...
(amounts can be corrected to concentration throughout the manuscript when discussing DNA concentration)
I appreciate the changes performed on this manuscript. However, I still think you could improve the language. e.g. Inch and Gilbert [23] observed that only F. graminearum species were isolated from wild grasses (Agropyron repens, Agropyron trachycaulum, Agrostis stolonifera, Bromus inermis, Calamagrostis canadensis and Echinocloa crusgalli) in June, while in mid-August were isolated 7 species of Fusarium, among them the aforementioned F. avenaceum species (found only in B. inermis)
better change to: 7 species of Fusarium were isolated
Often numbers are spelled out when below 10 (seven instead of 7). I guess it is up to the journal to decide.
I do not feel that it is my job to correct the language. Please have someone to correct the entire manuscript.
Author Response
General comments to the authors:
Line 261: The highest amounts of the F. graminearum pathogen (2068.34 and 1277.347 pg/µg) were..
amounts can be corrected to concentration and pathogen can be altered to DNA
I suggest this sentence is corrected to: The highest concentrations of F. graminearum DNA (2068.34 and 1277.347 pg/µg) were...
(amounts can be corrected to concentration throughout the manuscript when discussing DNA concentration)
Answer: These sentences were changed. Line 25, 202, 204, 211, 213, 224, 228, 308, 322, 334, 394, 398, 400, 401, 404, 420, 462.
I appreciate the changes performed on this manuscript. However, I still think you could improve the language. e.g. Inch and Gilbert [23] observed that only F. graminearum species were isolated from wild grasses (Agropyron repens, Agropyron trachycaulum, Agrostis stolonifera, Bromus inermis, Calamagrostis canadensis and Echinocloa crusgalli) in June, while in mid-August were isolated 7 species of Fusarium, among them the aforementioned F. avenaceum species (found only in B. inermis).
better change to: 7 species of Fusarium were isolated
Often numbers are spelled out when below 10 (seven instead of 7). I guess it is up to the journal to decide.
Answer: The sentence was changed. Line 386. And the numbers throughout the manuscript were spelled out.
I do not feel that it is my job to correct the language. Please have someone to correct the entire
manuscript.
Answer: The language was checked and corrected by a native English speaker.
Reviewer 4 Report
Dear Authors,
Thanks for the revisions; I can see substantial changes in the revised file.
I request you add the images in the main file you provided in the author's response. Another thing you mentioned about the sequencing of the EF1 gene, pls submit those sequences to the repository e. g. NCBI and provide the accession numbers in the manuscript.
Thanks
Author Response
Comments to the Author:
Thanks for the revisions; I can see substantial changes in the revised file.
I request you add the images in the main file you provided in the author's response.
Answer: The images were added to the manuscript. Line 269.
Another thing you mentioned about the sequencing of the EF1 gene, pls submit those sequences to the repository e. g. NCBI and provide the accession numbers in the manuscript.
Answer: We submitted the sequences to the database and if we get links before publication, we will upload the registration numbers.
