Growth, Nitrogen Uptake, and Nutritional Value of a Diverse Panel of Shrub Willow (Salix spp.) Genotypes in Response to Nitrogen Fertilization

Round 1

Reviewer 1 Report

The manuscript describes a study on the use of willows shrub as a riparian buffer as a strategy to reduce N runoff and in parallel the nutritional value as fodder. It is well known that crop productivity must be sustainable and sometimes the application of N is lost through leaching or runoff causing water contamination.

The article is significant in content and several parameters are measured for the selection of some species.

I missed the conclusions at the end of the article, what happened?

Author Response

* "Tables 4, 5, and 6. Add the “ANOVA results” as supplementary results."

Thanks for the comment. I don't understand what you mean.
Should I move table 4,5, and 6 to the supplement? Or to add new tables with other parameters that are not in tables 4,5, and 6? If this is the purpose, please specify what parameters you want to be in the tables.
I appreciate any help you can provide.

Reviewer 2 Report

 

Dear authors,

Thank you for the opportunity to review this Manuscript (Growth, nitrogen uptake, and nutritional value of a diverse panel of shrub willow (Salix spp.) genotypes in response to nitrogen fertilization).  The study has great results and demonstrates that willow can be used as fodder with quality within the range of other fodder materials. This intentional design for environmental benefits could have a dual purpose should this potentially nutrient-dense biomass by used as fodder for small ruminants. There is some aspect that should be reviewed by authors, but the Manuscript is well-written.

 

ABSTRACT

Why the rate is 500 ppm N? Could be in kg ha. This rate is higher or lower?

Please, add the variables analyzed in the abstract

INTRODUCTION

Explain, Nitrate leaching for each source

Line 30, context “runoff” for the readers

Scientific name in the first time of Salix

Explain, how Salix spp. Reduce runoff

Please, explain the methane production when used as fodder

MATERIALS AND METHODS

Where the experiment was conducted?

Explain, the green-house conditions. The vase? Salix plating? Water rate? The size of salix?

Line 102 “Cuttings were planted on 18 February 2020.”, and lines 152-153, edit these parts. Phrases poor.

Explain clearly the “control”

Explain, four-time points of SPAD measurements.

RESULTS AND DISCUSSION

In the topic “Biomass productivity and growth development”, the authors could present the result in percentage to explain how fertilizer was higher compared to control.

Table 1, there is no need of Min-Max values because the authors added the standard deviation.

Tables 4, 5, and 6. Add the “ANOVA results” as supplementary results.

The Quality of Figure 4 and 6 are poor

Where is the name of X and Y variables in the Correlation analysis.

Explain the rate of N. High? Low?

Explain if the Salix can be used to feed animals and the adaptability

 

Author Response

Muklada et al – Agronomy

Reviewer 1:

Dear authors,

Thank you for the opportunity to review this Manuscript (Growth, nitrogen uptake, and nutritional value of a diverse panel of shrub willow (Salix spp.) genotypes in response to nitrogen fertilization).  The study has great results and demonstrates that willow can be used as fodder with quality within the range of other fodder materials. This intentional design for environmental benefits could have a dual purpose should this potentially nutrient-dense biomass by used as fodder for small ruminants. There is some aspect that should be reviewed by authors, but the Manuscript is well-written.

 

ABSTRACT

Why the rate is 500 ppm N? Could be in kg ha. This rate is higher or lower?

*Note: Following the Covid-19 restrictions and Cornell University guidelines, the experimental set-up changed from five levels of fertilization in doses from 0 to 125 mg per week in the first format to a compact set of two groups: one with the lowest level with zero N and the second with the highest level N of 125 mg N per week.

Thank you for pointing this out. Fertilization treatments were applied weekly at rates zero (water control), and 125 mg week−1 of N (500 ppm). The total cumulative amounts of N applied by treatment were 0, and 750 mg N over the approximately 7-week experiment. Based on the area of the pot these treatment levels were roughly equivalent to 0, and 152 kg N ha−1.

Please, add the variables analyzed in the abstract

Thanks for the comment. We mention: aboveground growth measured as biomass, stem length and diameter, as well as morphological properties, but we removed the details: (leaf area, specific leaf area, number of stems, sylleptic branching, and lateral distribution of the stems-bending) from the constraints of limiting the number of words to 200 and contented ourselves with a general mention of morphological properties.

INTRODUCTION

Explain, Nitrate leaching for each source

Thanks for the comment. Nitrate ions (NO3) are soluble in water and can easily be removed from the soil mainly in two ways: leaching through the unsaturated zone to percolate into deeper horizons and the other is the surface runoff from the top-soil through water flow to surface water resources. (https://doi.org/10.1016/bs.agron.2019.01.007 ). The intention was to identify genotypes that can utilize maximum nitrogen, assuming that these genotypes will remove the most nitrogen from these two phases.

We added a phrase about surface runoff and subsurface leaching.

Line 30, context “runoff” for the readers

Thank you for pointing this out. I meant losses and not runoff in the quantitative understanding. I replace the word runoff with losses and leaching (see lines 30,31,34).

Scientific name in the first time of Salix

Thanks for the comment. We changed it.

Explain, how Salix spp. Reduce runoff

Thanks for the comment. As I mentioned in the above comment, I meant losses and not runoff in the quantitative understanding but the quality of the runoff in that it contains less nitrogen leaching. I replace the word runoff with losses.

Please, explain the methane production when used as fodder

Thank you for your comment. Forages that include secondary metabolites, such as willows, have many benefits to ruminants, including reducing breath methane (CH4) emissions.

 

MATERIALS AND METHODS

Where the experiment was conducted?

Thank you for your comment. The experiment was conducted at Cornell AgriTech in Geneva, NY.

Explain, the green-house conditions. The vase? Salix plating? Water rate? The size of salix?

Thank you for your comment. As we mentioned (line 85-94): “1-year-old shoots were collected from nursery beds at Cornell AgriTech in Geneva, NY. And processed into 20-cm cuttings, which were stored in a freezer at − 4 °C until planting.”  Watering regime was included.

Line 102 “Cuttings were planted on 18 February 2020.”, and lines 152-153, edit these parts. Phrases poor.

Thank you for your comment. This section was rephrased.

Explain clearly the “control”

Fertilization treatments were applied weekly at rates 0 (water control), and 125 mg week−1 of N (500 ppm)

Explain, four-time points of SPAD measurements.

Thank you for pointing this out. The bi-weekly measurements of each pot started on the sixth, week followed by eighth, tenth, and 12th week, four measurements in different growth periods showed a response over time.

RESULTS AND DISCUSSION

In the topic “Biomass productivity and growth development”, the authors could present the result in percentage to explain how fertilizer was higher compared to control.

Thank you for your comment. We think that due to the massive difference between the two fertilizer levels, the goal was not to compare percentages between the two fertilization groups but rather the performance of the different genotypes.

Table 1, there is no need of Min-Max values because the authors added the standard deviation.

Thanks for this suggestion.  We have removed the Min-Max values from Table 3.

 

Tables 4, 5, and 6. Add the “ANOVA results” as supplementary results.

Thank you for your comment. As suggested, we have moved Tables 4, 5, and 6 to Supplemental material.

The Quality of Figure 4 and 6 are poor

We have improved the resolution of both figures.

 

Where is the name of X and Y variables in the Correlation analysis.

Thank you for your comment. As we mentioned in figure 4 (line 268): Correlation analysis among biomass productivity traits, morphological characteristics, and nutritional value. For the control group (below diagonal) and fertilizer group (above diagonal).

Explain the rate of N. High? Low?

Thank you for your comment. I explain that in the first comment:

“Fertilization treatments were applied weekly at rates 0 (water control), and 125 mg week−1 of N (500 ppm). The total cumulative amounts of N applied by treatment were 0, and 750 mg N over the approximately 7-week experiment. Based on the area of the pot these treatment levels were roughly equivalent to 0, and 152 kg N ha−1.”

Explain if the Salix can be used to feed animals and the adaptability

Thank you for your comment. In this paper, we showed that there are willow genotypes suitable for growth in excess nitrogen. In addition, we showed that the willow foliage has a moderate nutritional value. We are working on another paper examining willow fodder's adaptation to small ruminants.

 

Submission Date

31 August 2022

Date of this review

06 Sep 2022 02:04:43

 

 

Reviewer 3 Report

The present manuscript deals with the characterization of 50 shrub willow genotypes in terms of their N-uptake and nutritional value in response to nitrogen fertilization. There are concerns about the experiment and the outcomes that need to be clarified before this manuscript can be considered for publication.  

Willows are nutrient demanding and have diffuse root systems at a shallow depth. The selection of a genotype with high nitrogen uptake capacity can lead to the selection of genotypes that impair soil fertility in terms of nitrogen in the upper soil layer. The main criteria should be the efficiency of nitrogen and water use. To avoid nitrogen runoff, deep-rooted crops are needed. Nitrogen is a crucial factor in determining crop productivity in the topsoil. Willow crops have the potential for phytoremediation, such as cadmium accumulation by willow clones used for soil conservation.

The authors should clearly provide relevant references and better explain and defend the context of the study.

L86: Indicate the total N input (for how many weeks?).

L96: Table 1: what is the utility of presenting all these details in the main text? What is the link between the Pedigree, the Sex and Ploidy and the aim of the study? I suggest moving this table to a supplementary section.

L88: The 10-l pot is very small compared to the willow, the plant's mainly root system can be faced with physical stress.

L253: Figure 3: the figure does not show the standard error; what about genotypes? the effect of N fertilization is well known, what about the response of genotypes to N fertilization?

 

L300: This result must also be reported in the abstention.

Author Response

Muklada et al – Agronomy

Reviewer 2:

The present manuscript deals with the characterization of 50 shrub willow genotypes in terms of their N-uptake and nutritional value in response to nitrogen fertilization. There are concerns about the experiment and the outcomes that need to be clarified before this manuscript can be considered for publication.  

Willows are nutrient demanding and have diffuse root systems at a shallow depth. The selection of a genotype with high nitrogen uptake capacity can lead to the selection of genotypes that impair soil fertility in terms of nitrogen in the upper soil layer. The main criteria should be the efficiency of nitrogen and water use. To avoid nitrogen runoff, deep-rooted crops are needed. Nitrogen is a crucial factor in determining crop productivity in the topsoil. Willow crops have the potential for phytoremediation, such as cadmium accumulation by willow clones used for soil conservation.

The authors should clearly provide relevant references and better explain and defend the context of the study.

We thank the reviewer for this comment. Yes, willows do produce diffuse and shallow root systems, which are excellent for the capture of surface runoff of nitrate.  The context of this work is in riparian buffers in watersheds that suffer from excess nutrient runoff due to fertilizer applications for agriculture.  In this setting, the soils have excess nutrients and are not prone to impaired soil fertility in the upper layers – in fact the opposite – the upper layers have excess nutrients.  We seek to select for genotypes with maximum luxury nutrient uptake (low nitrogen use efficiency) and high transpiration (low water use efficiency) to maximize N removal and biomass production. https://doi.org/10.1016/bs.agron.2019.01.007

https://doi.org/10.1016/j.rser.2022.112154

https://www.cabdirect.org/cabdirect/abstract/20153427163

 

 

L86: Indicate the total N input (for how many weeks?).

Thank you for pointing this out. Fertilization treatments were applied weekly at rates zero (water control), and 125 mg week−1 of N (500 ppm). The total cumulative amounts of N applied by treatment were 0, and 750 mg N over the approximately 7-week experiment. Based on the area of the pot these treatment levels were roughly equivalent to 0, and 152 kg N ha−1.

 L96: Table 1: what is the utility of presenting all these details in the main text? What is the link between the Pedigree, the Sex and Ploidy and the aim of the study? I suggest moving this table to a supplementary section.

Thanks for the suggestion. We moved Table 1 to the supplementary materials as suggested

L88: The 10-l pot is very small compared to the willow, the plant's mainly root system can be faced with physical stress.

Thank you for the comment. Due to the short growing period of four months, the assumption was that the volume was sufficient, and indeed at the end of the growing time, we checked the roots of 20 plants from each group, and there was no root complex covering the entire volume of the pot, and there were no signs of physical stress.

L253: Figure 3: the figure does not show the standard error; what about genotypes? the effect of N fertilization is well known, what about the response of genotypes to N fertilization?

Thank you for the comment. Standard errors are so minor that they cannot be seen on the graph scale (±0.38 and ±0.45 on average for the fertilizer and the control, respectively). We showed the genotypes' response to fertilizer in Figure 2. And to SPAD in Figure 2 and Table 7 of repeated measures quadratic polynomial analysis of variance results for SPAD measurement.

L300: This result must also be reported in the abstention.

We included a new sentence in the Abstract describing these results.