Impact of Long-Term Manure and Mineral Fertilization on Accumulation of Non-Structural Carbohydrates in Lucerne Forage
Round 1
Reviewer 1 Report
The manuscript is very well designed and the data is also supportive of the objective of the study. But from a technical point of view following points were noticed
- The design of the experiment is not very clear, it was mentioned in the material and methods 24 fertilizer treatment combinations, but in table, these are only 4 (NIL, FYM, N4P2K2 and combination of N4P2K2+FYM)
- If there are only 4 fertilizer treatments as shown in tables, how the statistical analysis was performed. With only 4 treatments it is not precise to do statistical analysis.
- The title may be modified as suggested
- The study showed that there was no impact of fertilizer treatments on forage yield, how is it possible, please give enough reasons, how it was possible. There was a difference of 4000-5000 kg of dry forage yield still, remained nonsignificant???
- Plaese, look into the statistical analysis carefully.
- FYM+Inorganic is expected to give better results in quality as well as in quantity?
- The data is presented only for 2014, it was a long term experiment, why not pooled analysis be possible
- Some content in the manuscript has been given general statement with no supportive data, like in conclusion, it was spelled that "This positive response could be attributed to the development of 388 more favorable soil conditions under regular manure fertilization, which probably attributed to the plants developing higher colonization with AMF and consequently reduced environmental stress." Now, where is the data on AMF, and what were the parameters to explain environmental stress?
- What was the basis for fixing NPK doses through FYM and through inorganic sources?
- The green forage yield may also be given.
- Lucern is a leguminous crop, has the modulation or use of biofertilizer part also included while quantification of the nutrient doses.
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Author Response
Referee: 1
The manuscript is very well designed and the data is also supportive of the objective of the study. But from a technical point of view following points were noticed:
The design of the experiment is not very clear, it was mentioned in the material and methods 24 fertilizer treatment combinations, but in table, these are only 4 (NIL, FYM, N4P2K2 and combination of N4P2K2+FYM).
The Ruzyně Fertilization Experiment (RFE) is a group of five experimental strips with different treatments therefore it is not surprising that only contrasting treatments are selected in particular studies. Here, we have selected 4 treatments (each with 4 replicates) so we have sampled forage from 16 plots.
If there are only 4 fertilizer treatments as shown in tables, how the statistical analysis was performed. With only 4 treatments it is not precise to do statistical analysis.
I am not sure if number of treatments could affect a relevance of statistical analyses such as t-test or ANOVA. That could be an issue if there were a limited number of replicates, but not treatments. Here, with four treatments and four replicates, in a completely randomised block design, we have used one-way ANOVA. This is a standard approach for this kind of field experiment.
The title may be modified as suggested
Thank you for your suggestion for title improvement. We have reconsidered this very carefully. Indeed, it could be called as “integrated nutrient management” but we believe that manure and mineral fertilization is clearer and more informative in conveying the precise topic of the work reported.
The study showed that there was no impact of fertilizer treatments on forage yield, how is it possible, please give enough reasons, how it was possible. There was a difference of 4000-5000 kg of dry forage yield still, remained nonsignificant???
In this study, the changes in forage yield together with canopy traits are presented as a background for changes in NSC content. Long-term evaluation provides clear difference in yield among treatments (see our previous studies) and also shows that these differences could be marginal for an N-fixing lucerne in a fertile Luvisol in years with optimal water supply. Here, we consider variation in yield and canopy data in relation to changes in lucerne NSC, and we have not concluded anything about non-significant impact of fertilization on forage yield.
Please, look into the statistical analysis carefully.
Based on the statistical advice we have followed, we believe that comparison of four fertilization treatments is correct and valid in the design of a one-way ANOVA with fixed effect of treatment. The assumptions for using ANOVA have been met. The use of the RDA is also fine and the other reviewer has not expressed any doubts about the statistics.
FYM+Inorganic is expected to give better results in quality as well as in quantity?
Sure, for quantity like in previous study, however nutritive value is in general negative relationship with biomass yield where negative effect on forage quality could be expected. It is quite usual in forage agronomy research to find that increased herbage yield is associated with a decline in forage quality.
The data is presented only for 2014, it was a long term experiment, why not pooled analysis be possible
Indeed, it would be great if we could be able to extend our observations across years and more treatments in this experiment. Of course, yield data and soil nutrient status are available for longer period but analyses of nutritive value are, unfortunately, strongly limited by budget for this research. It is a pilot study about forage quality (NSC) in the RFE, so the main target of our investigation was to determine whether some differences in nutritive value in association with canopy structure are detectable under this long-term management.
Some content in the manuscript has been given general statement with no supportive data, like in conclusion, it was spelled that "This positive response could be attributed to the development of more favorable soil conditions under regular manure fertilization, which probably attributed to the plants developing higher colonization with AMF and consequently reduced environmental stress." Now, where is the data on AMF, and what were the parameters to explain environmental stress?
I agree that it seems to be only general statements; however, I believe we really have a lack of similar results from long-term experiments about forage crops and it is never possible to support all every single suggestion by measurement in a single experiment. Here, we have provided a potential explanation of improved WSC content which was not verified by measurements but it is supported by more or less similar research. It is not easy to cover all factors and details which could be interesting in this regard when you are not sure with positive results. It is for this reason the explanation cannot be given in all detailed aspects like micronutrients in manure and plants, intensity of nodulation and N fixation, response to microbe population or AMF colonisation, advanced soil chemistry etc. No scientific paper can assess adequately all this potential influences. We have now added a comment that this aspect is one that merits further investigation in terms of providing explanation for the effects associated with the manure input
What was the basis for fixing NPK doses through FYM and through inorganic sources?
Mineral rates are easy to provide uniformly. Manure is always analysed before application and rate per hectare is adjusted to give the same amount of key macronutrients across years.
The green forage yield may also be given.
Nutritive traits are considered on dry matter basis so presentation of fresh matter yield has a low relevance. Fresh matter values are always difficult to present accurately because forage DM% can change by the hour.
Lucerne is a leguminous crop, has the modulation or use of biofertilizer part also included while quantification of the nutrient doses.
We fully agree with described positive effect of lucerne but it could be more important for following crops than lucerne itself.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Dear authors,
This manuscript has scientific merit and quality. However, some observations are pertinent:
As these are long-term experiments, it is natural that other results are published in other scientific articles, a fact that has been verified (for example: https://www.sciencedirect.com/science/article/pii/S1161030120302264). However, the authors cite their work in an enriching way, not compromising the feasibility of the evaluated work.
I recommend checking the presentation formats of scientific units throughout the entire work: for example H2O, ha-1.
As for the data referring to the weather conditions that occurred during the experiment, I suggest presenting it in the form of a graph with a duplicated y-axis (one corresponding to temperature and the other referring to rainfall). If possible, instead of presenting average monthly values, I recommend that average data be presented weekly, or by ten days - (Unless the period corresponds to uniform weather conditions, with few variations).
Additionally, I recommend that tables 3, 4 and 5 be replaced by figures (graphs) resulting in parsimony.
Greetings!
Author Response
Referee: 2
This manuscript has scientific merit and quality. However, some observations are pertinent:
As these are long-term experiments, it is natural that other results are published in other scientific articles, a fact that has been verified (for example: https://www.sciencedirect.com/science/article/pii/S1161030120302264). However, the authors cite their work in an enriching way, not compromising the feasibility of the evaluated work.
Yes, this study on NSC builds on previous research on nutritional value sowing the effects of treatments and canopy traits on WSC.
I recommend checking the presentation formats of scientific units throughout the entire work: for example H2O, ha-1.
It was checked across manuscript.
As for the data referring to the weather conditions that occurred during the experiment, I suggest presenting it in the form of a graph with a duplicated y-axis (one corresponding to temperature and the other referring to rainfall). If possible, instead of presenting average monthly values, I recommend that average data be presented weekly, or by ten days - (Unless the period corresponds to uniform weather conditions, with few variations).
I agree this would be very relevant if we were comparing different sites or years with different weather conditions, but here we give these monthly values as background information on the growing environment. Monthly rainfall and mean temperature data are commonly given in this way for field experiments, unless weather is a variable under investigation. However, based on your comment, we support the weather date by week values in the new Figure 1.
Additionally, I recommend that tables 3, 4 and 5 be replaced by figures (graphs) resulting in parsimony.
It is a good idea but it would be difficult to combine more tables into one chart due to different units, and three charts are more space consuming than three tables. The value of tables is that they give an accurate record of analysed results, whereas graphs and figures show trends. Here, we are not really showing trends.
We hope that the changes we’ve made resolve all your concerns about the manuscript. We believe the presented results are interesting, applicable, highly relevant in the context of using lucerne in crop rotations, and are able to help underpin and inform hypotheses for more precise investigations in the future, and improved as a consequence of all your useful suggestions. Thank you for contribution to increase manuscript quality.
Author Response File: Author Response.docx
