Productivity of Nitrogen Accumulated in Alfalfa–Grass Sward Cultivated on Soil Depleted in Basic Nutrients: A Case Study
Round 1
Reviewer 1 Report
Lines135-138: How the fertilizer was applied and in what period ?
Results:The results did not show the botanical composition of the Sward (percentage of alfalfa and grass) as well as the content of the most important quality parameters (protein and fibers ADF and NDF) in ruminant nutrition.
Author Response
Review report 1_response
Lines135-138: How the fertilizer was applied and in what period ?
Response
In the period before the experiment was set up, phosphate and potash fertilizers, gypsum and a small dose of nitrogen were applied to the soil and mixed with the soil.
In the period of full vegetation of alfalfa-grass mixture all fertilizers were applied to plants broadcast in doses indicated in the methodology in the period just before the spring start of vegetation.
Results: The results did not show the botanical composition of the Sward (percentage of alfalfa and grass) as well as the content of the most important quality parameters (protein and fibers ADF and NDF) in ruminant nutrition.
The results of the research on the botanical composition of the Sward (percentage of alfalfa and grass) have been included in Table 2. Moreover, they have been described and discussed.Regarding the content of the most important quality parameters (protein and fibers ADF and NDF), the results will be published in a separate article. This article is long anyway, having exceeded 80,000 characters.
On behalf of the authors
Waldemar Zielewicz
Author Response File: 
Author Response.pdf
Reviewer 2 Report
This study evaluted the influence of gypsum, P/K fertilizers and their interaction on productivity and nutrient accumulation with an alfalfa-grass mixture system, and obtained interesting results. However, the elaboration of the research status, formulation of scientific questions and hypotheses, presentation of results, and discussion need to be thoroughly and deeply revised. According to the current situation of this manuscript, I recommend rejecting the manuscript and resubmitting. The specific amendments were provided in the attachment.
Comments for author File: Comments.pdf
I recommed a radical revision of the English language throughout the manuscript.
Author Response
Review Report 2_ response
This study evaluted the influence of gypsum, P/K fertilizers and their interaction on productivity and nutrient accumulation with an alfalfa-grass mixture system, and obtained interesting results. However, the elaboration of the research status, formulation of scientific questions and hypotheses, presentation of results, and discussion need to be thoroughly and deeply revised. According to the current situation of this manuscript, I recommend rejecting the manuscript and resubmitting. The specific amendments were provided in the attachment.
Comments on the Quality of English Language
I recommed a radical revision of the English language throughout the manuscript.
Major comments:
- The core scientific questions to be solved are not presented. The manuscript can
focuses on alfalfa-grass system productivity and nitrogen accumulation, explicit
the effects of stoichiometry on productivity and nitrogen accumulation under
different element addition.
Response
In the first sentence of the abstract, the research hypothesis was clearly formulated. This hypothesis indicates the problem of utilizing the nitrogen accumulated in the legume sward due to insufficient supply of other nutrients. Such a production risk occurs both on soils with low natural fertility and on soils depleted with nutrients as a result of their excessive mining. In the northern hemisphere, most soils have been formed from a post-glacial rock residue that is often low in lime, cations, including potassium. On the basis of this knowledge and production conditions in Poland, a research plan was developed. To confirm the hypothesis of the article, three maps of the content of basic nutrients in European soils are attached. Figure 1. Calcium carbonate content in European soils. Figure 2. Potassium content in in European soils. Figure 3. Phosphorus content in in European soils. Source of figures : Ballabio, C., Lugato, E., Fernández-Ugalde, O., Orgiazzi, A., Jones, A., Borrelli, P., Montanarella, L. and Panagos, P., 2019. Mapping LUCAS topsoil chemical properties at European scale using Gaussian process regression. Geoderma, 355: 113912) To avoid controversy on this point, the hypothesis has been extended as follows:
„Nitrogen accumulated in the shoots of the fodder legume plant originates to a great extent from atmospheric N2 fixation. This is the necessary condition for fully exploiting the yielding potential of the crop, such as alfalfa. Other nutrients taken up by alfalfa are required for the efficient utilization of the N accumulated in the shoots. Among the macronutrients, an important production role is played by P, K, Ca, Mg, and S. For this reason, these nutrients fulfill the sufficient condition for alfalfa sward growth and yield. Therefore, the question should be asked: to what extent does the shortage of key nutrients in the soil limit both the accumulation and productivity of N accumulated by alfalfa grown in a mixture of grasses? The main objectives of the study were to determine the 1) yields of the alfalfa–grass sward in response to experimental factors over subsequent years of its use, 2) sward yield prediction based on a) the content of the nutrients, b) the ratios between the nutrients, and c) the accumulation of the nutrients. These goals were verified in field experiments with alfalfa–grass sward fertilized with gypsum and phosphorus/potassium fertilizers.”
- For the Abstract part, due to the lack of comprehensive introduction of
experimental methods, some sentences don't understand the meaning at all, such
as “The sward yield of the 3rd cut determined the size of TY”, I cannot understand
the meaning about the “3rd cut”. The results need to be logically presented in
accordance with the scientific problem to be solved. L22 “These two figures” ,
there was no figures in the Abstract part.
Response
The scope of the methodology necessary for the reader in the abstract has been properly prepared. Experimental factors and sublevels of factors are given. The research assumption as presented in the objectives is confirmed by the data in Table 1, which shows that the study was carried out on soil poor in available Ca, K, S and at the same time rich in available P and Mg. It was thus assumed that nitrogen management in alfalfa was potentially limited by: calcium, sulfur (GYPSUM) and phosphorus and potassium (POT). Alfalfa is a perennial plant. During the growing season, depending on soil and climate conditions and agricultural technology, including irrigation, it is mowed many times within one growing season. The number of sward cuts is from two to 10. In this field trial, the number of cuts conducted in the appropriate phase of alfalfa growth, was three (Photo 1). A farmer, a producer of dairy cattle, is interested not only in the distribution of yields during the growing season, but above all in the total sward yield (TY). These data are needed because they provide information on fodder resources for animals during the winter. The obtained results clearly showed that the TY of alfalfa-grass sward was determined by the 3rd regrowth (cutting). This is a key practical conclusion from the conducted study. Due to this reason, it was clearly stated in the abstract. The justification is contained in Table S1. Figure 4 below shows the results for each season separately (included into supplementary material as Figure S1). The results are clear. Photo 1. Alfalfa development stage – mowing date Figure 4. Total yield of the alf-alfa-grass mixture as a function of the sward yield of the 3rd cut. Legend: I, II, III – consecutive main seasons.
- For the Introduction part, there is too much content to introduce the legumes and
the advantages of legumes-grass mixtures, but the effect of element addition on
legumes-grass mixtures was too less. Nor did it raise scientific questions that
needed to be addressed based on research status.
Response
The introduction has been completely reworked. The scientific question, as well as the resulting goals of the article, have been clearly formulated. The entire introduction is attached.
„Legume plants benefit from atmospheric N through symbiosis with papillary bacteria. This is an important role for the soil N balance and the N cycle in nature. Depending on weather conditions, the amount of atmospheric N fixed by legumes can range from 50 to 250 kg N ha-1 [1]. The amount of N in roots accounts for 25% of the nitrogen taken up by legumes [2]. Legume plants and their mixtures with grasses form a link between crop and livestock production on a farm [3]. Grass, as a component of legume–grass mixtures, may benefit from N2 fixed by legume bacteria [4]. The uptake of atmospheric N2 by legumes and the transfer of N to other neighboring plants is important in reducing the use of mineral N fertilizers [5].
The study of alfalfa in Europe has clearly documented its potential to sustain the production of staple crops by substantially reducing the use of mineral N fertilizers [6]. The inclusion of alfalfa in mixtures with grasses improves the use of available water from deeper soil layers and increases the number of plant leaf blades in relation to the soil surface area (LAI), thus making better use of solar radiation [7]. Successful forage production, based on mixtures of legumes and grasses, depends on the selection of grass species and varieties that ensure the rapid growth and fast regrowth of plants after mowing and the ability to compete with legumes for light and water. It is also very important to maintain an adequate dynamic growth rate for grass without using N fertilizers [8]. The most useful species for multispecies mixtures are grasses which are the most productive and show low competitiveness with legumes [9]. This condition is met by grass species such as Festuca pratensis (L.) Huds., Phleum pratense L., Lolium perenne L., Dacylis glomerata L., Arrhenatherum elatius (L.) P. Beauv ex J. Presl and C. Presl, and Festuca arundinacea Schreb [10;11].
The effective use of alfalfa’s yielding potential depends on the resources of nutrients in the soil and their effective uptake by roots, which grow to a depth of several meters [12;13]. The deep root system provides alfalfa with greater resistance to soil water deficits and tolerance to cultivation on sandy and mineral-poor soils [13]. Moreover, the long root system of alfalfa influences the movement of nutrients from the subsoil to the upper soil layers [14;15]. Therefore, a farmer beginning the cultivation of this crop should be aware of the current state of the soil fertility both in the topsoil and in the subsoil [16]. This crop requires an appropriate level of soil reaction (neutral to alkaline), which is the key factor for obtaining and maintaining the necessary plant density [17;18]. Even when the soil reaches an adequate pH, Ca supply may be limited in some soils. The main reason is the low soil resources of this nutrient [19].
Calcium is an important nutrient for legumes, including alfalfa, for at least four purposes: (i) activation of root and shoot bud development, (ii) formation of root hair nodulation, resulting in the formation of bacteroids (nodules), (iii) tissue and organ formation and stability, and (iv) stress signaling, in which Ca acts as a secondary messenger [20;21]. The second group of critical nutrients includes those which are responsible for the processes of N2 fixation and includes sulfur (S), iron (Fe), and molybdenum (Mo). In legumes, an adequate supply of S is important for the formation of Fe-S clusters, which are key physiological structures determining the activity of nitrogenase. In the next step in the assimilation of N2 by the legume plant, S is required for the synthesis of storage proteins, sulfur, and amino acids (cysteine, cysteine, and methionine) [22;23]. The uptake of S by plants from the soil is regulated by systemic plant signals, which reflect the current status in the shoot. Under conditions of S deficiency, SO42─ carriers located near the root tip and in the root hair zone are activated [24;25]. A good source of both Ca and S is gypsum (CaSO4×2H2O), which is currently used on arable land mainly as FGD gypsum (flue gas desulfurization). This carrier of both S and Ca, despite its naturally low solubility in water, is a reliable source of available S (S-SO4) [26;27]. The third group of nutrients necessary for alfalfa are those that are responsible for energy and transport processes in the plant, including N2 fixation and the transport of storage proteins in the plant from the root to the shoot. These functions depend on the supply of phosphorus (P), magnesium (Mg), and potassium (K) [28;29].
Alfalfa has a very high production potential. Under good natural growing conditions (climate and soil) and irrigation, the yield of the sward can even reach 20 t ha─1 [30]. Such high yields are rarely achievable in natural, i.e., rain-fed, conditions, even when rainfall is sufficient [31]. The main reason for low yields is the insufficient fertility of the soil. The low content of available nutrients in such soils reduces the effectiveness of atmospheric N fixation [32]. This situation is typical for many European soils formed from post-glacial rocks. These soils are characterized by a small cation exchange complex, as well as low-key nutrient contents, such as Ca, K, and Mg. In addition, these soils are also prone to acidification [33]. In the last 30 years, many countries have significantly reduced SO2 emissions into the atmosphere. This process caused a decrease in the supply of S to the soil and, consequently, a decrease in the content of the available soil sulfur [34]. Phosphorus resources in the arable soils of countries with intensive crop production are large, so the effectiveness of the so-called freshly applied phosphorus is low [35]. For these reasons, the response of alfalfa to P fertilization is difficult to predict [36]. The amount of available K in the soil depends on the first step of the resources of minerals rich in this element. The consumption of K fertilizers in the world is low, leading to the depletion of the content of its available forms [33;37]. For this reason, the results of field studies indicate the need to use extremely high rates of potassium fertilizer. However, an increase in K doses leads to an imbalance (disturbance in stoichiometry) with other cations, such as Ca and Mg, known as the tetany ratio [38]. Despite its known physiological function of Ca and S, the response of alfalfa to these nutrients is poorly recognized [39, 40].
Nitrogen accumulated in the shoots of the fodder legume plant originates to a great extent from atmospheric N2 fixation. This is the necessary condition for fully exploiting the yielding potential of the crop, such as alfalfa. Other nutrients taken up by alfalfa are required for the efficient utilization of the N accumulated in the shoots. Among the macronutrients, an important production role is played by P, K, Ca, Mg, and S. For this reason, these nutrients fulfill the sufficient condition for alfalfa sward growth and yield. Therefore, the question should be asked: to what extent does the shortage of key nutrients in the soil limit both the accumulation and productivity of N accumulated by alfalfa grown in a mixture of grasses? The main objectives of the study were to determine the 1) yields of the alfalfa–grass sward in response to experimental factors over subsequent years of its use, 2) sward yield prediction based on a) the content of the nutrients, b) the ratios between the nutrients, and c) the accumulation of the nutrients. These goals were verified in field experiments with alfalfa–grass sward fertilized with gypsum and phosphorus/potassium fertilizers.
- For the Materials and Methods part, the calculated method of Nutrient Unit
Productivity (NUP) was not clear.
Response
I agree with the reviewer. The equation has been corrected. This equation informs about the unit productivity of the nutrient accumulated in the biomass of alfalfa sward with grasses.
- For the Results part, the presented of results need to be based on the scientific
problem. And why the second and third cuts were emphasized in 3.1, and whether
the 3rd cut is a 3-year average value of particular year needs to be clarified.
Response
The structure of the results chapter is a consequence of the formulated scientific questions and the resulting goals of the article. This chapter has been revised in accordance with objectives of this article.
In the standard form of this type of research, the results usually describe the effects of experimental factors. In this article, however, it is only the first chapter (3.1). The results of the conducted study clearly indicated that the effect of the experimental factors is variable in successive regrowth (cuts) of the alfalfa-grass sward during the growing season. The research clearly showed that the total sward yield depended on the yield of the 3rd sward regrowth. So the question was: why? This question is answered in the next four parts of the article by analyzing:
1) chemical composition of the sward;
2) relations between elements;
3) the accumulation of nutrients by the sward;
4) developed productivity indicators.
The obtained results in each step of the study were confronted with the partial yield of the sward (yield of a particular regrowth, or cut), as well as with the TY of the sward (10 developed regression equations). The conducted research clearly showed that TY depended on the mass of accumulated nitrogen and the nitrogen unit productivity (NUP). Moreover, the results of the conducted analysis clearly showed that the effect of the latter factor (NUP) depended on the action of other nutrients. This analysis also explained the specific role of the 3rd sward regrowth in predicting the total yield.
- For the Discussion part, there is still a need to revolve around one or a set of clear
scientific questions.
Response
The discussion referred to the research objectives. In addition, in accordance with the rules of this chapter of the scientific article, own results were confronted with the state of current knowledge.
It would not be possible to demonstrate the specific role of phosphorus without such detailed research and analysis of the results.
- Although I am not a native English speaker, I recommend a radical revision of the
English language, especially the Abstract section.
Response
The manuscript was checked before being sent to the Agronomy editorial office by an English lecturer (native Englishman) at the Adam Mickiewicz University in Poznań, Poland.
In order to avoid further controversy on this topic, a certificate from the MDPI Service is attached.
On behalf of the authors
Waldermar Zielewicz
Author Response File: Author Response.pdf
Reviewer 3 Report
When you use the name of the species in English for the first time, you should also put the name of the species in Latin.
Author Response
Review report3_response
When you use the name of the species in English for the first time, you should also put the name of the species in Latin.
Response
The text contains the Latin names of the discussed plant species.
On behalf of the authors
Waldemar Zielewicz
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
After the corrections, the quality of the manuscript has been significantly improved and is acceptable for publication in the journal Agronomy.
