Root Contact between Maize and Alfalfa Facilitates Nitrogen Transfer and Uptake Using Techniques of Foliar 15N-Labeling
, Xinyu Wang 3,†, Qiang Gao 1,*, Hualiang Zhang 3, Hailing Yu 1, Yin Wang 1, Jinjing Zhang 1, Jamal Nasar 1 and Yingzhi Gao 3,*Round 1
Reviewer 1 Report
You will see my comments in the attached document.
The experiments ara very nice, but in my point of view the experiments hava a low commertail aplication. and this aspect of the parper should be commented and improved.
Comments for author File: 
Comments.docx
Author Response
Response to Reviewer 1 Comments
Point 1: Root contact between maize and alfalfa facilitates nitrogen transfer and uptake using foliar labelling 15N techniques
Title: labelling or labeling?
Response 1: Thanks for your comments! The word “labeling” is more common in isotopic labeling description after reading relative literature. Following your suggestions, we changed it to labeling and have rewritten the title as below “Root contact between maize and alfalfa facilitates nitrogen transfer and uptake using techniques of foliar 15N-labeling”
Point 2: My first point is the difficulty of growing both crops (alfalfa and maize) together in a commercial field, since maize will shade the alfalfa seriously, unless the maize is growth at low plant density or in a very wide row distance, limiting the alfalfa grow (It is my own experience). The experiments of this paper are very nice, but I think they have not commercial applicability.
Response 2: I totally agree with you maize/alfalfa has not commercial applicability, because there are still many technical problems that have not been solved, such as mechanization problems, pesticide problems (Li 2016). However, commercial application of maize and alfalfa intercropping is promising. Previous studies have shown that the intercropping of maize with alfalfa had yield advantages compared to alfalfa or maize monoculture (Zhang et al. 2011; Sun et al. 2014, 2019; Zhang et al. 2019). They also reported that the advantages of maize/alfalfa intercropping in yield production due to interspecific complementarity, not competition in light interception and utilization, played an important role in the yield improvement of maize/alfalfa intercropping, and the overyielding was mainly attributed to the differences of maize and alfalfa in growth and development dynamics. Thereby, temporal and spatial complementarity in light utilization (Sun et al. 2014). Moreover, sowing practices (sowing density, the distance between the rows, cultivars) of all the cropping patterns were the same for both maize and alfalfa in the present study (as shown in the schematic below), and light competition can be mitigated in maize/alfalfa intercropping. And in our pot experiment, the spacing of pots in the glasshouse was 1.2 m, and the spacing between maize and alfalfa was 22.5 cm, the pot spacing was greater than the spacing in our field trials, therefore, there was little competition for light between the pots. There was competition for light between the plants inside the pots. However, the light utilization rate was not measured in our experiments, which was not the focus of this study. Thus, further study of the effects of light utilization on biomass in the intercropping system of maize and alfalfa is required.
We have added the following figure A1 to the discussion section (see line 353).
Li, L. (2016) Intercropping enhances agroecosystem services and functioning: Current
knowledge and perspectives. Chinese Journal of Eco-Agriculture, 24(4): 403-415.
Sun, B.R., Peng, Y., Yang, H.Y., Li, Z.J., Gao, Y.Z., Wang, C., Yan, Y.L., Liu, Y.M. (2014) Alfalfa (Medicago sativa L.)/maize (Zea mays L.) intercropping provides a feasible way to improve yield and economic incomes in farming and pastoral areas of northeast China. PLoS One. 9, e110556.
Zhang, G.G., Yang, Z.B., Dong, S.T. (2011) Interspecific competitiveness affects the total biomass yield in an alfalfa and corn intercropping system. Field Crops Research, 124, 66-73.
Point 3: Line 14-15. Belowground nitrogen (N) transfer from legumes to non-legumes provides an important 14 N source for crop yield and N utilization… but I do not know if it is useful in the commercial fields.
Response 3: Thank you for your comments. Intercropping, which is the practice of growing two or more crops simultaneously on the same field, plays an important role in Chinese traditional agriculture. Legumes are involved in 70% of more than 100 intercropping systems in China (Xiao et al. 2005). Nitrogen (N) transfer from legumes to non-legumes has been reported extensively. For instance, Li et al. (2003) found that the N absorption of intercropped maize (Zea mays L.) and broad bean was higher than that of a sole crop maize or broad bean. Previous intercrop study demonstrated that 32%–58% of the N assimilated by sorghum was derived from soybean (Fujiu et al. 1990; Li et al. 2001; Chu et al. 2004), but the amount of N transfer varies greatly depending on crop species. Therefore, I think that the intercropping of non-legumes and legumes have high application value in reducing fertilizer application.
Xiao, Y.B., Li, L., Zhang, F.S. (2005) The interspecific nitrogen facilitation and the subsequent nitrogen transfer between the intercropped wheat and fababean. Scientia Agricultura Sinica, 38, 965-973. (in Chinese)
Li, L., Zhang, F.S., Li, X.L., Christie, P., Sun, J.H., Yang, S.C., Tang, C.X. (2003) Interspecific facilitation of nutrient uptake by intercropped maize and faba bean. Nutrient Cycling in Agroecosystems, 65, 61-71.
Fujiu, K., Ogata, S., Matsumoto, K., Masuda, T., Ofosu-Budu, G.K., Kuwata, K. (1990) Nitrogen transfer and dry matter production in soybean and sorghum mixed cropping system at different population densities. Soil Science and Plant Nutrition, 36, 233-241.
Chu, G.X., Shen, Q.R., Li, Y.L., Zhang, J., Wang, S.Q. (2004) Researches on bidirectional N transfer between the intercropping systems of ground nut with rice cultivated in aerobic soil using 15N foliar labeling method. Acta Ecologica Sinica, 24, 278-283.
Li, L., Sun, J.H., Zhang, F.S., Li, X.L., Yang, S.C., Rengel Z. (2001) Wheat/maize or wheat/soybean strip intercropping I. Yield advantage and interspecific interactions on nutrients. Field Crops Research, 71, 123–137.
Point 4: Line 20. The aspect is not well explained and needs further writing ....15 % of which N, the N fixation? of the N accumulated in the maize...
Response 4: Sorry for the wrong expression. The value of the 15.05 and 9.30 percentages represent the ratio of 15N transferred to 15N uptake of alfalfa with no root barrier and with a mesh barrier, respectively. Following your suggestions, we have rewritten the sentence as below “Following the N0 treatment, the amount of N transfer from alfalfa to maize was 204.56 mg pot−1with no root barrier and 165.13 mg pot−1with a mesh barrier, accounting for 4.72% and 4.48% of the total N accumulated in maize, respectively. Following the N1 treatment, the amount of N transfer from alfalfa to maize was 197.70 mg pot−1with no root barrier and 139.04 mg pot−1with a mesh barrier, accounting for 3.64% and 2.36% of the total N accumulated in the maize, respectively.” (see lines 19-24).
Point 5: Line 45. …. from 0 to 80% of which N?
Response 5: Following your suggestions, we have rewritten the sentence as below “Literature data show that rates of N transfer range from 0 to 80 % from from legumes to cereal in mixed stands, depending on the legume species and cultivar.” (see lines 44-46).
Point 6: Line 89: Walk–Black method; The method has another name..Walkley and Black
Response 6: Thank you for your suggestions. In order for readers to understand clearly, we have changed "Walk–Black method" to "Walkley-Black method" (see line 90). And the following references are also added.
Yu, H., Gao, Q., Shao, Z., Ying, A., Sun, Y., Liu, J., Zhang, B. (2016) Decreasing nitrogen fertilizer input had little effect on microbial communities in three types of soils. PloS one, 11(3).
Point 7: Line 118: Any idea of the fertilization rate per ha?
Response 7: Thank you for your suggestions. We have added the sentence as below “The application of NPK fertilizer to maize and alfalfa was conducted according to local fertilization practices (Sun et al. 2014). Alfalfa was treated at 53 kg N ha-1, 135 kg P ha-1 and 90 kg K ha-1, while 225 kg N ha-1, 120 kg P ha-1 and 60 kg K ha-1 were applied to maize.” (see lines 118-121).
Sun, B.R., Peng, Y., Yang, H.Y., Li, Z.J., Gao, Y.Z., Wang, C., Yan, Y.L., Liu, Y.M. (2014) Alfalfa (Medicago sativa L.)/maize (Zea mays L.) intercropping provides a feasible way to improve yield and economic incomes in farming and pastoral areas of northeast China. PLos One. 9, e110556.
Point 8: Line 120: When the alfalfa and maize were seeded? Day of the year?
Response 8: Thank you for your suggestions. In our experiments, maize and alfalfa were seeded at the same time. We have added the following maize and alfalfa sowing times to the paper “(June 1, 2015 and May 23, 2016)” (see lines 125-126).
Point 9: Line 144: when was it applied ? preseeding ?
Response 9: Sorry for unclear expression. Following your suggestions, we have rewritten the sentence as below “To ensure the nodulation and nitrogen fixation of alfalfa, a suspension of Rhizobium sp. strain ACCC177512 (supplied by the College of Resources and Environment, Jilin Agriculture University, Jilin) was applied to the alfalfa at 20 ml pot−1 (density was 3.0 × 107 ml−1 CFU) at 7 d after sowing.” (see lines 149-152).
Point 10:
Results
Line 192. Normally, if the alfalfa is well inoculated, the N fertilization normally does not increase dry matter yield.
Response 10: I think there are mainly 2 reasons that caused the gap between our results and your expectations. On the one hand, rhizobium could fix N to promote plant growth while plant needs to supply carbon and nutrient as an exchange. This symbiotic relationship ensures better growth of both under N deficiency conditions. But under N fertilization conditions, legume can absorb N by root and need not invest more energy to symbiotic rhizobium, its reasonable that N fertilization could increase dry matter yield even the plants are well inoculated (Hu et al. 2017; Jia et al. 2017; Liu et al. 2019). On the other hand, the function of inoculation treatment is keeping a relatively normal N fixation ability in this greenhouse experiment. However, only one rhizobium inoculation may be not enough for “well inoculated” of alfalfa, the N fertilization does increase dry matter yield under this condition.
Hu, F., Zhao, C., Feng, F., Chai, Q., Mu, Y., Zhang, Y. (2017) Improving N management through intercropping alleviates the inhibitory effect of mineral N on nodulation in pea. Plant and Soil, 412(1-2), 235-251.
Jia, M., Xiao J., Tang, L., Zheng, Y. (2017) Effects of nitrogen supply on yields and photosynthesis characteristics of crops in wheat and broad bean intercropping. Journal of Yunnan Agricultural University (Natural Science), 32 (2), 350 -357. (in Chinese)
Liu, Y., Yin, X., Xiao, J., Tang, L., Zheng, Y. (2019) Interactive influences of intercropping by nitrogen on flavonoid exudation and nodulation in faba bean. Scientific reports, 9(1), 1-11.
Point 11: Line 194. It would have been interesting to know the approximate rate of N application per ha. if not is difficult to know if the N applied rate was low of high.
Response 11: Good suggestions. Regarding the determination of fertilization rates for the test crops corn and alfalfa, we investigated the regular fertilization rates of local farmers, and then we referred to previous studies (Sun et al. 2014), in addition, other similar research papers also used similar fertilization amounts (Li et al. 2003; Li et al. 2016; Tang et al. 2018). Therefore, in our pot experiment, we chose 225 kg N ha-1, 135 kg P ha-1 and 90 kg K ha-1 to meet the growth requirements of maize and alfalfa plants. After calculation, the N/P/K fertilizer all the pots were supplied with 200 mg N kg-1 soil, 120 mg P kg-1 soil and 80 mg K kg-1 chemical fertilizer, respectively. The section has been added (See lines 118-124).
Sun, B.R., Peng, Y., Yang, H.Y., Li, Z.J., Gao, Y.Z., Wang, C., Yan, Y.L., Liu, Y.M. (2014) Alfalfa (Medicago sativa L.)/maize (Zea mays L.) intercropping provides a feasible way to improve yield and economic incomes in farming and pastoral areas of northeast China. PLoS One 9, e110556.
Li, L., Tang, C., Rengel, Z., Zhang, F. (2003) Chickpea facilitates phosphorus uptake by intercropped wheat from an organic phosphorus source. Plant and Soil, 248, 297-303.
Li, Q.S., Wu, L.K., Chen, J., Khan, M.A., Luo, X.M., Lin, W.X. (2016) Biochemical and microbial properties of rhizospheres under maize/peanut intercropping. Journal of Integrative Agriculture, 15, 101-110.
Tang, Q.X., Tewolde, H., Liu, H.B., Ren, T.Z., Jiang, P.A., Zhai, L.M., Lei, B.K., Lin, T., Liu, E.K. (2018) Nitrogen uptake and transfer in broad bean and garlic strip intercropping systems. Journal of Integrative Agriculture, 17, 220-230.
Point 12: Line 226. The whole plant N concentration for maize and alfalfa (%) should be presented, in order to know if the crops were in good condition or not.
Response 12: Thank you for your suggestions. We have added the sentence to the discussion section of our paper as below “Based on the averages of 2015 and 2016, under the N0 treatment, the whole plant N concentration for maize and alfalfa was 0.59% and 2.68%, respectively. Under N1 treatment, the whole plant N concentration for maize and alfalfa was 0.99% and 2.85%, respectively.” (see lines 359-361).
Point 13: Line 254. Which were the levels of N abundance in maize under N0 and N1?. Present an average of the two years!!
Response 13: Thank you for your suggestions. We have rewritten the entire text about 15N Abundance as below “When alfalfa as 15N-donor plant in 2015 and 2016, the 15N abundance in the shoots of maize only was significantly affected by the root separation pattern. The 15N abundance in the roots of maize was significantly affected by the N levels, root separation pattern and their interactions (except that the interactions among the year, N levels and root separation pattern was not significant). The 15N abundance in the shoots of alfalfa was significantly affected by the year, the interactions between year and N levels, the interactions between year and root separation pattern, but in the roots was significantly affected by year, N levels and root separation pattern and their respective interactions (Table 3). On average, under the N0 treatment, the 15N abundance in the maize and alfalfa shoot and root were 0.387%, 0.392% and 0.773%, 0.516% with no root barrier, 0.387%, 0.391% and 0.798%, 0.512% with a mesh barrier, 0.372%, 0.372% and 0.731%, 0.520% with a plastic sheet barrier, respectively. Under the N1 treatment, the 15N abundance in the maize and alfalfa shoot and root were 0.387%, 0.390% and 0.789%, 0.511% with no root barrier, 0.383%, 0.382% and 0.796%, 0.565% with a mesh barrier, 0.372%, 0.371% and 0.774%, 0.544% with a plastic sheet barrier, respectively (Table 4).
When maize as 15N-donor plant, the 15N abundance in the shoots and roots of alfalfa were significantly affected by the N levels, but had no significant affected by the root separation pattern and the interaction between N levels and root separation pattern (Table 3). Under the N0 treatment, the 15N abundance in the alfalfa shoot and root was 0.369% and 0.368% with no root barrier, 0.370% and 0.369% with a mesh barrier, 0.369% and 0.369% with a plastic sheet barrier, respectively. Under the N1 treatment, the 15N abundance in the alfalfa shoot and root was 0.368% and 0.368% with no root barrier, 0.368% and 0.367% with a mesh barrier, 0.369% and 0.368% with a plastic sheet barrier, respectively (Table 4).” (see lines 263-285).
Point 14: Line 274. N transfer rate and amount were significantly affected by the N level, root separation 274 pattern, and year (Table 5). ¿ which is the rate? In Table 5 in the first column there are no units (%)?
Response 14: The values in first coulumn were N transferred rates. We have added the units (%) in the first column of Table 5 (see line 312).
Point 15: Line 286. Table5. N transferred as % of maize N, ….should be N transferred as of maize N (%), also in this table it should be easier to read , the average of the two years.
Response 15: We have changed "N transferred as % of maize N, N transferred as % of alfalfa N" to "N transferred as of maize N (%), N transferred as of alfalfa N (%)" (see line 312). We also have added 2-year average below the table for easier to read (see line 312).
Point 16: Discussion
Line 307. Our data indicated that the maize with no root barrier had the highest biomass….. In pot conditions could be, in the field I doubt. What is the % of dry matter increase?
Response 16: Under the N0 treatment, the maize shoot and root biomass with no root barrier were 82.69% and 65.82% higher than that of the plastic sheet barrier, and 63.48% and 54.47% higher than that of the mesh barrier, respectively. Under the N1 treatment, the maize shoot and root biomass with no root barrier were 26.51% and 55.52% higher than that of the plastic sheet barrier, and 12.32% and 31.74% higher than that of the mesh barrier, respectively (see lines 205-210).
We have added the sentence to the discussion part of our paper as below “In our experiment, we also found that the average values of Ama in the two-year experiment were higher than 1 in all treatments. The NCRma values were also higher than 1 in each treatment in 2015 and 2016. Therefore, the competitiveness of maize was greater than that of alfalfa in the maize/alfalfa intercropping system, and maize biomass represented a larger portion of the total intercropping biomass in the maize/alfalfa intercropping system.” (see lines 334-338).
Table A1 Aggressivity,Competitive of maize relative to alfalfa for the nitrogen level (N) and root separation method (RS) between 2015 and 2016
|
Years |
Treament |
Ama |
NCRma |
||
|
2015 |
N0 |
NS |
2.30 |
4.46 |
|
|
NNS |
1.53 |
2.90 |
|||
|
PSS |
-- |
-- |
|||
|
N1 |
NS |
1.48 |
2.39 |
||
|
NNS |
0.55 |
1.65 |
|||
|
PSS |
-- |
-- |
|||
|
2016 |
N0 |
NS |
2.07 |
3.88 |
|
|
NNS |
1.46 |
2.49 |
|||
|
PSS |
-- |
-- |
|||
|
N1 |
NS |
2.26 |
3.05 |
||
|
NNS |
1.59 |
2.03 |
|||
|
PSS |
--　 |
--　 |
|||
Ama: Aggressivity of maize relative to alfalfa; NCRma: Nitrogen competitive ratio of maize relative to alfalfa. If Ama is greater than 0, the competitive ability of maize exceeds that of maize in intercropping; If NCRma is greater than 1.0, the N competitive ability of maize will be greater than that of alfalfa in intercropping.
Point 17: Line 333. The maize biomass was 32.37% higher with a mesh barrier than with a plastic sheet 311 barrier, which could be related to the mycorrhizae and hyphae [8,31] or the movement of water and 312 nutrients from the alfalfa side.
Have you measured the soli water contents? Have you irrigated the pots?
Response 17: The plants were watered with deionized water to maintain the soil moisture at 60%–70% of the field water-holding capacity throughout the growth stage, and the soil moisture in each pot was monitored by micro-tensiometers (Nanjing Institute of Soil Science, Chinese Academy of Sciences). We have elaborated in materials and methods (see lines 128-131).
Point 18: Line 320. ..may be due to the maize plants being taller than the alfalfa plants in the intercropping 320 system, which limited the growth of alfalfa because of a shading effect
At what stage do you harvest the corn? How many leaves or plant height had the maize at the time you harvested it? It is very important, Since it could be possible that all the effects you mention last for a short period of time, … just for a few weeks.
Response 18: Thank you for your suggestions. We harvested the maize at the maturity stage, and measured the plant heights of maize and alfalfa at the same time. We have added the sentence as below “Under the N0 treatment, the plant heights of maize and alfalfa were 238.93 cm and 78.14 cm, respectively. Under N1 treatment, the plant heights of maize and alfalfa were 250.62 cm and 69.53 cm, respectively.” (see lines 350-352).
Reviewer 2 Report
The paper under review is aimed at finding out whether nitrogen transfer from leguminous plants to cereal plants occurs through root contact mechanism, and, if so, on the estimation of how this transfer influences the crop yield. In spite of the overall high quality of the work, there are some issues that have to be addressed before article publication.
19-21. Not clearly understandable from what value the 15,05 and 9,30 percentages are taken
89. What the "Walk–Black method" is? Provide a reference.
93. Mistype. Change "ethylene diaminete traacetic acid" to "ethylene diamine tetraacetic acid"
95 “black soil” is not sufficient to describe the soil type of the site. Please provide the soil type according to the WRB system.
117-118. Mistype. Change "potassium chloride" to "potassium oxide"
155 I suggest either adding a subtitle “Experiment I. …” or removing the subtitle “Experiment II”
144-145. What is the "3.0 × 10^7 mL^−1" value? Is is a CFU or a cell number?
182 Why did you choose Fisher’s LSD for the multiple comparisons of three variants (no barrier, plastic sheet, and nylon)? In my view, Tukey’s HSD would be more reliable in this case.
Author Response
Response to Reviewer 2 Comment
Point 1: 19-21. Not clearly understandable from what value the 15,05 and 9,30 percentages are taken.
Response 1: Sorry for the confusing express. The value of the 15.05 and 9.30 percentages is the representative of the N transfer rate from alfalfa plants to maize with no root barrier and with a mesh barrier, respectively. Following your suggestions, we have rewritten the sentence as below “Following the N0 treatment, the amount of N transfer from alfalfa to maize was 204.56 mg pot−1with no root barrier and 165.13 mg pot−1with a mesh barrier, accounting for 4.72% and 4.48% of the total N accumulated in maize, respectively. Following the N1 treatment, the amount of N transfer from alfalfa to maize was 197.70 mg pot−1with no root barrier and 139.04 mg pot−1with a mesh barrier, accounting for 3.64% and 2.36% of the total N accumulated in the maize, respectively.” (see lines 19-24).
Point 2: 89. What the "Walk–Black method" is? Provide a reference.
Response 2: We have modified "Walk–Black method " to "Walkley-Black method" (see line 90). And added the following references.
Yu, H.L., Gao, Q., Shao, Z.Q., Ying, A.N., Sun, Y.Y., Liu, J.W., Mao, W., Zhang, B. (2016) Decreasing nitrogen fertilizer input had little effect on microbial communities in three types of soils. PLos One, 11, e0151622.
Point 3: 93. Mistype. Change "ethylene diaminete traacetic acid" to "ethylene diamine tetraacetic acid"
Response 3: Thank you for your suggestions. We have changed "ethylene diaminete traacetic acid" to "ethylene diamine tetraacetic acid" (see line 94).
Point 4: 95 “black soil” is not sufficient to describe the soil type of the site. Please provide the soil type according to the WRB system.
Response 4: Following your suggestions, we have rewritten the sentence as below “The type of soil used in this test was black soil, equivalent to typical Phaeozem in the WRB system system (FAO 1990).” (see lines 96-97).
FAO-ISRIC (1990) Guidelines for soil description, thirded, revised, food and agricultural organization. Rome
Point 5: 117-118. Mistype. Change "potassium chloride" to "potassium oxide"
Response 5: We have changed "potassium chloride" to "potassium oxide" (see line 122).
Point 6: 155 I suggest either adding a subtitle “Experiment I. …” or removing the subtitle “Experiment II”
Response 6: Thank you for your suggestions. We added “Experiment I. …” according to your suggestions (see lines 116 and 153).
Point 7: 144-145. What is the "3.0 × 10^7 mL^−1" value? Is is a CFU or a cell number?
Response 7: Sorry for the wrong misunderstanding. The "3.0 × 10^7 mL^−1" value is CFU. Following your suggestions, we have rewritten the sentence as below “To ensure the nodulation and nitrogen fixation of alfalfa, a suspension of Rhizobium sp. strain ACCC177512 (supplied by the College of Resources and Environment, Jilin Agriculture University, Jilin) was applied to the alfalfa at 20 ml pot−1 (density of was 3.0 × 107 ml−1 CFU) at 7 d after sowing.” (see lines 149-152).
Point 8: 182 Why did you choose Fisher’s LSD for the multiple comparisons of three variants (no barrier, plastic sheet, and nylon)? In my view, Tukey’s HSD would be more reliable in this case.
Response 8: Thank you for your suggestions. Following your suggestions, we reanalyzed the experimental data using Tukey's HSD method and modified it in the article. (see line 189)
Reviewer 3 Report
The addition of data related to the extent of nodulation/nitrogen fixation of the alfalfa would greatly strength the paper. However such data are not necessary for publication.
Author Response
Thank you very much for your comments. Let's see the attachment.
Author Response File: Author Response.docx
