Optimizing the P balance: How do modern maize hybrids react to different starter fertilizers?

Phosphorus (P) is an essential macronutrient for plants, but also a limited resource worldwide. Strict regulations for fertilizer applications in the European Union are a consequence of the negative environmental effects in case of improper use. Maize is typically grown with the application of P starter fertilizer, which, however, might be reduced or even omitted if suitable varieties were available. This study was performed with the 20 commercially most important maize hybrids in Germany evaluated in multi-location field trials with the aim to investigate the potential to breed for high-performing maize hybrids under reduced P starter fertilizer. At the core location, three starter fertilizers with either phosphate (triple superphosphate, TSP), ammonium nitrate (calcium ammonium nitrate, CAN), or a combination of ammonium and phosphate (diammonium phosphate, DAP) were evaluated relative to a control and traits from youth development to grain yield were assessed. Significant differences were mainly observed for the DAP starter fertilizer, which was also reflected in a yield increase of on average +0.67 t/ha (+5.34%) compared to the control. Correlations among the investigated traits varied with starter fertilizer, but the general trends remained. As expected, grain yield was negatively correlated with grain P concentration, likely due to a dilution effect. Importantly, the genotype-by-starter fertilizer interaction was always non-significant in the multi-location analysis. This indicates that best performing genotypes can be identified irrespective of the starter fertilizer. Taken together, our results provide valuable insights regarding the potential to reduce starter fertilizers in maize cultivation as well as for breeding maize for P efficiency under well-supplied conditions.

Reviewer #2: 1. Insufficient experimentation: The study involving three fertilizer treatments is conducted at one location and the data is of one year. Other locations do not have all the treatment combinations.
It is correct that we usually use multi-environment trials in breeding in order to estimate the environmental and genetic components contributing to the observed variancewhich is what was realized in this study. Please note that we have a sufficient number of three different locations for estimating the starter fertilizer effect of both DAP and TSP. Therefore, we did not realize all starter fertilizer treatments at all locations. The CAN treatment was only added in order to be able to see the effect of ammonium nitrate separately from the effect of phosphate (TSP) in comparison to the combination of ammonium and phosphate (DAP). Based on the data obtained in the field season 2019 we inferred that another year of trials would not give more valuable information to answer the objectives of this study since we have not observed any significant genotype-by-treatment interaction (as shown in Table S6). We hope this additional background information helps to understand what aspects were prioritized and thereby how field trials were set up.
2. The title mentions optimizing P balance and the experiment conducted on P sufficient soils will not give reliable results. Hence, Also G x fertilizer effects cannot be studied from the experimentation and the discussion done on this point is not valid.
We believe that P balance and thus efficient use of this resource is not only of relevance in regions with strongly P-deficient soils. Indeed, compared to other parts of the world, our soils are P sufficient. However, the P problematic in Germany (as well as in many other countries) is a different one that nevertheless requires an optimization of the P balance. Given the fact that we wanted to study P fertilizer applications and their potential reduction in the light of the new fertilizer ordinance, we aimed to perform the experiment under conditions suited to address this question and as realistic and close to practical farming as possible. Consequently, trials took place on well-supplied soils with regard to P. This is the case for the vast majority of actively cultivated fields in Germany (and most other intensively cultivated parts of Europe). Hence, optimizing the P balance has to be understood in the context of reducing P rather than using it as a scarce plant nutrient. The study is valid for Germany and other regions of Europe, where P deficiency is not a current issue. We have emphasized these objectives also clearly in the discussion part (see section '4.2 | Potential for optimizing the P balance on well-supplied soils') and in the conclusions we drew from our results (e.g. line no. 51-53, line no. 397-399).

Abstract is not clear
We reread the abstract and changed line no. 49-50 to: Importantly, the genotype-by-starter fertilizer interaction was always non-significant in the multi-location analysis.
4. ANOVA table not presented. How the experiments analyzed from all locations not given. Also locations are not discussed in material and methods and other sections.
The ANOVA table was presented as Table S6 in the supporting information. Statistical analyses were described in chapter 2.4 (starting from line no. 146). The full model is given in line no. 155 for the multiple location analysis. Reviewer #3: 1. The study undertaken was one of the most important aspect which would try to address the issue of development of 'P' use efficient genotypes in the context of 'P' being scarce resource and expected to exhaust in near future. However, based on the following points, the manuscript require major revision in the light of below observations.
2. The authors have used TSP, DAP and CAN to know the difference between different starter fertilizers but authors have not mentioned or studied the effect of other nutrients like nitrogen on 'P' utilization. Because balanced use of fertilizers is advocated for efficient use of fertilizers.

Thank you for highlighting once more the importance of a balanced use of fertilizers (please also see line no. 332 ff.). With regard to the effect other nutrients like nitrogen have on 'P' utilization, we kindly want to refer to section '4.1 | P as starter fertilizer and its interaction with other plant nutrients' (line no. 308 ff.), which is dedicated to this important aspect.
We added the following sentence in line no. 310: The importance of a balanced nutrient management and specifically the role of nitrogen in fertilization is well known. Moreover, we investigated the relationship between 16 chemical elements of the maize kernels depending on the starter fertilizer treatment (line no. 231: Figure S4; formerly numbered as Figure S5).
3. Authors are requested to justify the validity of the results in the practical situation of maize cultivation where the soil contains different nutrients in varying amounts, how does the results of the present experiment would apply in practical situation.
In line no. 66 ff. we mentioned the application of over 90.8 Mt of P fertilizers in Germany per annum. Common agricultural practice in maize implies the application of starter fertilizers, mostly DAP. Hence, our study does exactly reflect these farming practices. We understand that nutrients vary in different soils. Therefore, we performed multienvironment trials and analyzed all soils additionally to pH and the P status (Table 1) also for nitrate, magnesium, potassium, and carbon. Nonetheless, for the sake of clarity, we decided to not include these data in the present manuscript.
4. The experimental results are more of an agronomic perspective rather than pure breeding perspective thus the authors are requested to re-orient the results in breeding perspective.
We agree with the reviewer's comment on having included also an agronomic perspective in this study. Hence, we are well aware that this study is at the intersection of plant breeding, crop production and plant nutrition, as we believe that this topic and the results presented in this study are of interest to scientists from different disciplines. Moreover, with the limited number of the 20 economically most important genotypes, a pure breeding perspective did not seem reasonable to us. For these reasons, we consider the manuscript ideally suited for the broader readership and scope of PLOS ONE, as compared to other, more specialized journals of plant breeding.
5. In addition, the following points may be considered while improving the manuscript a. Youth development is the new terminology used for early development phase it may be clarified by giving leaf stage (like V3/V4/V5/V6) for larger benefit of the readers.
Thank you for this much appreciated hint, which is in line with the comments of reviewer 2. We adjusted the manuscript accordingly. Since we decided to measure all genotypes for plant height at a certain date (documented in days after sowing, DAS) rather than a certain leaf stage, we did not assess the leaf stage of each genotype for each plant height measurement. Plant height measurements were then categorized over the different locations according to their assessment in DAS. This information is provided in the supporting information, Table S5. Across locations we summarized measurements as PH early at BBCH stage ~ V4 and PH late at BBCH stage > R1. This information is as well given in the supporting information, Table S6. For the benefit of the readers, we additionally adjusted the following sentences: Line no. 217 ff: Independent of the starter fertilizer, the early plant heights PH1,PH2,and PH3 (measured 53,59,and 63 DAS,respectively) were closely related (0.75 < r < 0.95; p-values <0.01) but are no predictor for the final plant height (measured 94 DAS), nor grain yield.
b. The references namely 1,17,27,28,29 may also be translated into English for the benefit of the readers to know at least the title of the paper.

Done.
c. Sentence in line 73-74 may be revised Done in accordance with the comment of reviewer 1.

d. Reference 23, does apply to maize please clarify to readers
We adjusted the sentence in line no. 83 ff: In previous studies, it was shown that the traits early vigor, early-season plant height, flowering, and yield react to P deficiency in sorghum and can thus be considered as Psensitive traits (23).
e. Reference 24 (line 436 to 438) t is better to use symbol '×' instead of letter 'x'; is reference 24 relevant, please have a re-look.
We corrected the letter 'x' to '×'. Reference 24 is deemed suitable to give an example for previously observed G×E interactions for nutrient use efficiencies.
f. Line 228 may need to be re-looked at for spelling correction of there-of Corrected.

Reviewer #4:
Comments on MS PONE-D-20-36545 entitled "Optimizing the P balance: How do modern maize hybrids react to different starter fertilizers?" The study is on optimizing P balance in maize hybrids and how they react to starter fertilizers, the authors have studies how to reduce starter P for maize hybrids. The study has come out with some novel findings on.
The introduction is written well, the hypothesis is stated well The materials section is well written and the experiment is well planned and all the details of the experiment is given in a good way in the section The results section is written well with all the results obtained given in the section The discussion section brings out the reasons for the results obtained in a good way The MS can be accepted after minor revision Thank you for this positive feedback on our study and the manuscript.