Responses of Microbiological Soil Properties to Intercropping at Different Planting Densities in an Acidic Andisol

Round 1

Reviewer 1 Report

Authors provided a good set of data, which enable us to understand more about the impact of intercropping on soil nutrient dynamics. The topic of the study matches well with the journal. However, a large revision is required before the publication (only text-editing is required). My comments are listed as follows.

 

[Major comments]

(1) First, statistical analyses should be considered again.

If the result of the two-way ANOVA was not significant, authors do not need to do the post-hoc test. For example, letters on the results of Ureases in Table 2 were not needed.

Results of the two-way ANOVA is essential, so it cannot be skipped (see Fig. 1).

Duncan test is now not recommended because of the larger risks of type 2 error.

 

(2) Second, comprehensive discussion is needed. Authors lack several discussions. Also, it looks like that the present version is only talking about the authors’ idea. Other possibilities than authors’ idea is also required. If necessary, authors should deny the other ideas than authors in their discussion. Several suggestions for more discussion are listed as follows

• It is better to discuss about organic acid of Lupin. Authors only focused about phosphatase as a Lupin’s ability to obtain P from soils. However, it has been reported that organic acid is also an important ability of Lupin to get P. Now it is hard to add another experiment, so I think it is enough for authors to just mention about it in discussion section. There are many literatures, for example:

・Gardner WK, Barber DA, Parberry KG (1983) The acquisition of phosphorus by Lupinus albus L. III. The probable mechanism by which phosphorus movement in the soil/root interface is enhanced. Plant Soil 70: 107±124

・Neumann G, Marsonneau A, Martinoia ER, Ro¨mheld V (1999) Physiological adaptations to phosphorus deficiency during proteoid root development in white lupin. Planta

・Watt M, Evans JR (1999) Linking development and determinacy with organic acid efflux from proteoid roots of Lupinus albus L. grown with low phosphorus and ambient or elevated atmospheric CO₂ concentration. Plant Physiol 120:705–716

 

• Authors should explain why Lupin had higher N than wheat: N-fixation of leguminous Lupin (for example, Schulze et al. 2006). I guess authors did not mention about it because it is too common knowledge. However, it should be mentioned even if it is a sentence. Anyways, authors need to discuss more about N-fixation by Lupin, because they measured N-fixation genes.

・Schulze J, Temple GA, Temple SJ, Beschow H, Vance CP (2006) Nitrogen fixation by white lupin under phosphorus deficiency. Ann Bot 98:731–740

Further, in my opinion, the following discussion would help readers’ understanding about N dynamics affected by leguminous plants.

(i) Leguminous plants generally require higher amount of N (for example McKey 1994; Inagaki et al. 2009).

・McKey D (1994) Legumes and nitrogen: the evolutionary ecology of a nitrogen-demanding lifestyle. In: Sprent JL, McKey D (eds) Advance in legume systematics, part 5: the nitrogen factor. Royal Botanic Gardens, Kew, pp 221–228

・Inagaki M, Inagaki Y, Kamo K, Titin J (2009) Fine-root production in response to nutrient application at three forest plantations in Sabah, Malaysia: higher nitrogen and phosphorus demand by Acacia mangium. J For Res 14:178–182.

(ii) Leguminous plants require more N possibly because they produce “N-rich” phosphatase to obtain P (Houlton et al. 2008)

・Houlton BZ, Wang Y-P, Vitousek PM, Field CB (2008) A unifying framework for dinitrogen fixation in the terrestrial biosphere. Nature 454:327–330.

 

• Authors emphasized the impact of soil microbes interacting with plants. However, discussion about soil microbes are not sufficient. For example, increases in P availability caused by intercropping with Lupin may cause stimulated microbial N immobilization and lead to an N starvation for wheat. This is because the elevation of P availability in P-poor systems are suggested to stimulate microbial immobilization of N (Hall and Matson, 1999; Mori et al. 2010), which could cause N-starvation for plants. It seems that the mechanisms did not happen in the present study, because MBC did not change by the changes in the cropping system. However it is important to discuss all the possible mechanisms.

・Hall SJ, Matson PA(1999) Nitrogen oxide emissions after nitrogen additions in tropical forests. Nature 400:152.

・Mori T, Ohta S, Ishizuka S, et al (2010) Effects of phosphorus addition on N2O and NO emissions from soils of an Acacia mangium plantation. Soil Sci Plant Nutr 56: 782-788.

 

[Minor comments]

L182: A significant difference

The expression in the present form is vague. Authors should clearly mention that the significant difference is “the significant interaction.” It should be mentioned that the interaction means that (i) lupin had higher nitrate contents and (ii) only lupin showed differences in nitrate contents among different plant density.

 

L188: between

Should be “among”

 

L188: yet slight differences were found in lupin monocrop.

This should not be mentioned. The sentence here were based on the post-hoc, but if ANOVA did not show the significant difference, we should not perform post-hoc.

 

L189: In terms of growth, lupin monoculture at low plant density was significantly larger (p<0.001) compared to wheat intercropped with lupin at the same plant density.

Does this mention about Table 3? If so, please add “(Table 3)”.

 

L202: between

Should be “among”

 

L207: led to a significant increase

significantly increased

 

L224: Values

Values of what?

 

L226: No treatment significantly affected total N and P nutrient content in lupin monocrop or intercrop.

Is this talking about Table 3? If so, please add “(Table 3)”.

 

L227: a significant effect (p<0.001) on N and P levels

significant effects (p<0.001) on N and P levels

 

L228: In addition, significant differences (0.002) were found in terms of N,

significant differences of what?

 

L274: However, it increased

What does “it” indicate? Plant density? If so, “higher plant density.”

 

L281: Our results show that there are

Our results showed that there were

 

L283: which suggests that white lupin alone increases nitrate content.

Yes, but authors need to discuss why this happened. See also my major comment (2).

 

L284: That decrease in inorganic N occurs in the rhizosphere as a consequence of wheat root uptake.

The decrease in inorganic N in the rhizosphere observed in the intercropping experimental sites was probably due to a consequence of wheat root uptake.

 

L291: Soil microbial activity in Andisols is very high due to the high capacity of soil organic matter for mineralization.

Is this your result? Or cited from literatures? If this is not your result, please show the citations.

 

L292: In this work, urease activity was not influenced by cropping system or plant density. In contrast, a study conducted in maize/soybean intercropping [38] showed that this system increases urease, protease and soil nitrate reductase activity under field conditions. These findings are in agreement with the results obtained herein, suggesting that wheat/lupin intercropping has no clear effects on soil urease, protease, b-glucosidase and dehydrogenase activity.

I could not understand discussion here.

 

L302: Our results demonstrate that wheat/lupin intercropping and lupin monocropping enhance soil acid phosphatase activity compared to wheat monocropping, which indicates that lupin may utilize more soil organic P.

This paragraph can be combined with the following paragraph.

 

L302: Our results demonstrate that wheat/lupin intercropping and lupin monocropping enhance

Our results demonstrated that wheat/lupin intercropping and lupin monocropping enhanced

 

L310: high

higher?

 

L334: Unlike other enzymes, acid phosphatase activity increased in the acidic Andisol.

increased by what?

 

L337: Acidic conditions restrained soil enzyme activities (urease, protease, dehydrogenase and b-glucosidase), soil basal respiration and microbial biomass. This inhibition may be due to the depressed rate of enzyme synthesis and/or decreased populations of enzyme producing microorganisms in the acidic soil.

What is the basis of your discussion here? Any data to support this? If you want to say this, at least, you need additional treatment with non-Andisols.

 

L345: Over a period of one year, wheat grain yields were lower than 53, 76 and 73% at low, medium and high plant densities, respectively, in intercropping compared to monocropping.

This sentence sounds strange. Please rephrase.

 

356: has

had

 

L357: allows

allowed

 

L360: competes

competed

 

L361: In addition, wheat leaves have lower photosynthetic capacity, and thus the negative effect of shading was enhanced.

Is this your result? Where is the data?

 

L362: Therefore, wheat/lupin relay intercropping could be an effective approach to improve wheat grain yield and nutrient use efficiency.

Any data to support this conclusion? What is the basis of your discussion here?

 

L368: confirm

confirmed

 

L368: The results obtained herein also confirm that intercropping removes more N in wheat-based systems than in monocrops.

Is this sentence talking about your result? If so, what is the data supporting your claim?

 

L388: confirm

confirmed

 

L390: Thus, intercropping results in a positive effect of lupin on wheat crop, but not vice versa.

How did you evaluate the effect? What is the positive effect? Intercropping resulted in a decrease in grain yield of wheat (Table 3). Can you say that Lupin had positive effects on wheat?

 

L391: Enhanced N uptake

Data should be shown. Authors can calculate how much is the N uptake, by using the mass amount and N concentration.

 

 

Author Response

 

Reviewer 1.

 

Authors provided a good set of data, which enable us to understand more about the impact of intercropping on soil nutrient dynamics. The topic of the study matches well with the journal. However, a large revision is required before the publication (only text-editing is required). My comments are listed as follows.

 

[Major comments]

(1) First, statistical analyses should be considered again.

If the result of the two-way ANOVA was not significant, authors do not need to do the post-hoc test. For example, letters on the results of Ureases in Table 2 were not needed.

Results of the two-way ANOVA is essential, so it cannot be skipped (see Fig. 1).

Duncan test is now not recommended because of the larger risks of type 2 error.

 

Response:

We eliminated the letters on the results of pH, urease, protease, dehydrogenase, glucosidase, soil basal respiration, microbial biomass and lupin foliar nutrients (N% and P%)

We appreciate the suggestion about the statistical test. However, Duncan´test is widely used in intercropping field experiment. Some recent references of intercropping field experiment using Duncan test:

 

Rui Li, Zhixin Zhang, Wei Tang, Yafeng Huang, Jeffrey A. Coulter, Zhibiao Nan,

Common vetch cultivars improve yield of oat row intercropping on the Qinghai-Tibetan plateau by optimizing photosynthetic performance, European Journal of Agronomy,

Volume 117, 2020, 126088,

 

Xiaolong Wang, Yuanjiao Feng, Lingling Yu, Yinghua Shu, Fengxiao Tan, Yonggang Gou, Shasha Luo, Wenting Yang, Zhixian Li, Jianwu Wang,

Sugarcane/soybean intercropping with reduced nitrogen input improves crop productivity and reduces carbon footprint in China, Science of The Total Environment,Volume 719,

2020, 137517

 

Yinjuan Li, Longshuai Ma, Pute Wu, Xining Zhao, Xiaoli Chen, Xiaodong Gao,

Yield, yield attributes and photosynthetic physiological characteristics of dryland wheat (Triticum aestivum L.)/maize (Zea mays L.) strip intercropping, Field Crops Research, Volume 248,2020, 107656,

 

(2) Second, comprehensive discussion is needed. Authors lack several discussions. Also, it looks like that the present version is only talking about the authors’ idea. Other possibilities than authors’ idea is also required. If necessary, authors should deny the other ideas than authors in their discussion. Several suggestions for more discussion are listed as follows

• It is better to discuss about organic acid of Lupin. Authors only focused about phosphatase as a Lupin’s ability to obtain P from soils. However, it has been reported that organic acid is also an important ability of Lupin to get P. Now it is hard to add another experiment, so I think it is enough for authors to just mention about it in discussion section. There are many literatures, for example:

・Gardner WK, Barber DA, Parberry KG (1983) The acquisition of phosphorus by Lupinus albus L. III. The probable mechanism by which phosphorus movement in the soil/root interface is enhanced. Plant Soil 70: 107±124

・Neumann G, Marsonneau A, Martinoia ER, Ro¨mheld V (1999) Physiological adaptations to phosphorus deficiency during proteoid root development in white lupin. Planta

・Watt M, Evans JR (1999) Linking development and determinacy with organic acid efflux from proteoid roots of Lupinus albus L. grown with low phosphorus and ambient or elevated atmospheric CO₂ concentration. Plant Physiol 120:705–716

 

Response: We accepted the suggestion, we added the reference Watt and Evans (1999) in discusion section.

 

 

• Authors should explain why Lupin had higher N than wheat: N-fixation of leguminous Lupin (for example, Schulze et al. 2006). I guess authors did not mention about it because it is too common knowledge. However, it should be mentioned even if it is a sentence. Anyways, authors need to discuss more about N-fixation by Lupin, because they measured N-fixation genes.

・Schulze J, Temple GA, Temple SJ, Beschow H, Vance CP (2006) Nitrogen fixation by white lupin under phosphorus deficiency. Ann Bot 98:731–740

Further, in my opinion, the following discussion would help readers’ understanding about N dynamics affected by leguminous plants.

(i) Leguminous plants generally require higher amount of N (for example McKey 1994; Inagaki et al. 2009).

・McKey D (1994) Legumes and nitrogen: the evolutionary ecology of a nitrogen-demanding lifestyle. In: Sprent JL, McKey D (eds) Advance in legume systematics, part 5: the nitrogen factor. Royal Botanic Gardens, Kew, pp 221–228

・Inagaki M, Inagaki Y, Kamo K, Titin J (2009) Fine-root production in response to nutrient application at three forest plantations in Sabah, Malaysia: higher nitrogen and phosphorus demand by Acacia mangium. J For Res 14:178–182.

(ii) Leguminous plants require more N possibly because they produce “N-rich” phosphatase to obtain P (Houlton et al. 2008)

・Houlton BZ, Wang Y-P, Vitousek PM, Field CB (2008) A unifying framework for dinitrogen fixation in the terrestrial biosphere. Nature 454:327–330.

 

Response: we accepted the suggestion. We added this phrase in discussion section cited by Schulze et al 2006 “White lupin as a leguminous plant, early nodulated by Bradyrhizobium lupini. Lupin has the ability to increase soil N through symbiotic fixation”

 

 

• Authors emphasized the impact of soil microbes interacting with plants. However, discussion about soil microbes are not sufficient. For example, increases in P availability caused by intercropping with Lupin may cause stimulated microbial N immobilization and lead to an N starvation for wheat. This is because the elevation of P availability in P-poor systems are suggested to stimulate microbial immobilization of N (Hall and Matson, 1999; Mori et al. 2010), which could cause N-starvation for plants. It seems that the mechanisms did not happen in the present study, because MBC did not change by the changes in the cropping system. However it is important to discuss all the possible mechanisms.

・Hall SJ, Matson PA(1999) Nitrogen oxide emissions after nitrogen additions in tropical forests. Nature 400:152.

・Mori T, Ohta S, Ishizuka S, et al (2010) Effects of phosphorus addition on N2O and NO emissions from soils of an Acacia mangium plantation. Soil Sci Plant Nutr 56: 782-788.

 

Response:

L587-611 we discuss about soil microbes. No evidence on microbial N immobilization was observed here, because dehydrogenase and microbial biomass did not change.

In our study, no significant changes were found for MBC and dehydrogenase activity in the intercropping treatments. Similar results were found by Song et al. [44], who reported that intercropping decreased MBC in the rhizosphere of faba bean in the first year of the field experiment. This suggests that the effects of intercropping on microbial properties may become more pronounced if the cropping system is maintained over several years. The fact that plant residues are incorporated into the soil after harvest may also result into stronger effects. In this sense, the effects of wheat/white lupin intercropping were not significant in an acidic Andisol in our study, which is in agreement with the findings of Dissanayaka et al. [5]. They observed significant effects on maize intercropped with lupin in a Regosol, but not in an Andisol. Their results showed higher organic matter content in the volcanic soil with acidic pH conditions, with dominant presence of allophane, active aluminum and high P adsorption, which might have led to slight changes on microbial properties

 

[Minor comments]

L182: A significant difference

The expression in the present form is vague. Authors should clearly mention that the significant difference is “the significant interaction.” It should be mentioned that the interaction means that (i) lupin had higher nitrate contents and (ii) only lupin showed differences in nitrate contents among different plant density.

Response: We accepted the suggestion

 

L188: between

Should be “among”

Response: done

 

L188: yet slight differences were found in lupin monocrop.

This should not be mentioned. The sentence here were based on the post-hoc, but if ANOVA did not show the significant difference, we should not perform post-hoc.

 Response: We accepted the suggestion, sentence eliminated

 

L189: In terms of growth, lupin monoculture at low plant density was significantly larger (p<0.001) compared to wheat intercropped with lupin at the same plant density.

Does this mention about Table 3? If so, please add “(Table 3)”.

Response:  we eliminated the sentence

 

L202: between

Should be “among”

Response: done

 

L207: led to a significant increase

significantly increased

 Response: done

 

L224: Values

Values of what?

Response: Values of foliar N. We changed the sentence. Done

 

L226: No treatment significantly affected total N and P nutrient content in lupin monocrop or intercrop.

Is this talking about Table 3? If so, please add “(Table 3)”.

 Response: done

 

L227: a significant effect (p<0.001) on N and P levels

significant effects (p<0.001) on N and P levels

Response: done

 

L228: In addition, significant differences (0.002) were found in terms of N,

significant differences of what?

 Response: done, phrase changed (L474)

 

L274: However, it increased

What does “it” indicate? Plant density? If so, “higher plant density.”

  Response: done, phrase changed

 

L281: Our results show that there are

Our results showed that there were

 Response: done

 

L283: which suggests that white lupin alone increases nitrate content.

Yes, but authors need to discuss why this happened. See also my major comment (2).

 

Response: done, we include discussion about N fixation (L551-553)

 

L284: That decrease in inorganic N occurs in the rhizosphere as a consequence of wheat root uptake.

The decrease in inorganic N in the rhizosphere observed in the intercropping experimental sites was probably due to a consequence of wheat root uptake.

 Response: done

 

L291: Soil microbial activity in Andisols is very high due to the high capacity of soil organic matter for mineralization.

Is this your result? Or cited from literatures? If this is not your result, please show the citations.

 Response: we deleted this phrase to avoid the misunderstanding.

 

L292: In this work, urease activity was not influenced by cropping system or plant density. In contrast, a study conducted in maize/soybean intercropping [38] showed that this system increases urease, protease and soil nitrate reductase activity under field conditions. These findings are in agreement with the results obtained herein, suggesting that wheat/lupin intercropping has no clear effects on soil urease, protease, b-glucosidase and dehydrogenase activity.

I could not understand discussion here.

 Response: done, we rephrase the sentence

 

L302: Our results demonstrate that wheat/lupin intercropping and lupin monocropping enhance soil acid phosphatase activity compared to wheat monocropping, which indicates that lupin may utilize more soil organic P.

This paragraph can be combined with the following paragraph.

 Response: done

 

L302: Our results demonstrate that wheat/lupin intercropping and lupin monocropping enhance

Our results demonstrated that wheat/lupin intercropping and lupin monocropping enhanced

  Response: done

 

L310: high

higher?

  Response: done

 

L334: Unlike other enzymes, acid phosphatase activity increased in the acidic Andisol.

increased by what?

Response: Enzyme was depressed by soil pH and Al toxicity. In contrast with other enzyme, acid phosphatase activity increased with decreasing soil pH (Kunito et al. 2016).

We added the explication in L617-619

 

L337: Acidic conditions restrained soil enzyme activities (urease, protease, dehydrogenase and b-glucosidase), soil basal respiration and microbial biomass. This inhibition may be due to the depressed rate of enzyme synthesis and/or decreased populations of enzyme producing microorganisms in the acidic soil.

What is the basis of your discussion here? Any data to support this? If you want to say this, at least, you need additional treatment with non-Andisols.

 L362

 

Response: Similar results were found by Kunito et al. 2016 [45] evaluated an Andisol and Inceptisols L625-626.

 

L345: Over a period of one year, wheat grain yields were lower than 53, 76 and 73% at low, medium and high plant densities, respectively, in intercropping compared to monocropping.

This sentence sounds strange. Please rephrase.

 L370

Response: done

 

Wheat intercropped

356: has

had

 Response: done

 

L357: allows

allowed

 Response: done

 

L360: competes

Competed

 Response: done

 

L361: In addition, wheat leaves have lower photosynthetic capacity, and thus the negative effect of shading was enhanced.

Is this your result? Where is the data?

Response: we delete the sentence. Because is not our results

 

L362: Therefore, wheat/lupin relay intercropping could be an effective approach to improve wheat grain yield and nutrient use efficiency.

Any data to support this conclusion? What is the basis of your discussion here?

 Response: we delete the sentence

 

L368: confirm

confirmed

 Response: done

 

L368: The results obtained herein also confirm that intercropping removes more N in wheat-based systems than in monocrops.

Is this sentence talking about your result? If so, what is the data supporting your claim?

 L391

Response: our results are supported by Wang et al (2014)

 

L388: confirm

confirmed

 Response: done

 

L390: Thus, intercropping results in a positive effect of lupin on wheat crop, but not vice versa.

How did you evaluate the effect? What is the positive effect? Intercropping resulted in a decrease in grain yield of wheat (Table 3). Can you say that Lupin had positive effects on wheat?

L474

Response: similar effect was found by Dissanayaka in maize/lupin intercropping evaluated in andisol.

Our results obtained that N and P mobilization of white lupin from soluble N and P pools can enhance nutrient acquisition efficiency in an acidic Chilean Andisol with low fertility. Nutrient facilitation is unidirectional. Thus, intercropping results in a positive effect of lupin on wheat crop, but not vice versa. We described a positive effect on nutrient acquisition, but not in grain yield.

 

Dissanayaka, D.M.S.B.; Maruyama, H.; Masuda, G.; Wasaki, J. Interspecific facilitation of P acquisition in intercropping of maize with white lupin in two contrasting soils as influenced by different rates and forms of P supply. Plant and Soil 2015, 390, 223-236, doi:10.1007/s11104-015-2392-x.

 

L391: Enhanced N uptake

Data should be shown. Authors can calculate how much is the N uptake, by using the mass amount and N concentration.

Response in Table 3 we showed foliar N content in mono and intercropping system at low, medium and high plant densities.

Reviewer 2 Report

The authors are brought up an interesting topic to improve crop yields and soil properties. It is a very interesting subject for readers. I believe readers will be interested in this article. This paper compared the impact of monocropping and intercropping systems on soil nutrients, crop yields, and quantitative analysis of functional genes in soil. The manuscript is well-prepared in the experimental design and data analysis.

However, I have some suggestions to improve this manuscript.

 

Some suggestions:

It will be better for readers if authors are combined results and discussion sections together.

In abstract, please clarify some sentences:

L 23: you mean Olsen-P and K in soil?

L27:  N and P mobilization of what? Please write clearly.

L99: missing comma.

L117: provide a reference on SIR?

L126 &132: What is the symbol? Urease and N--benzoyl-L-arginine amide (BAA)

L224-225: You mean wheat or lupin?

L231: grain yield of wheat?

L235: after this paragraph, it will be better to put some discussion for readers?

Figure 1, there is no indication on acdS, phoD,  phoC, and nifH in the bar graphs.

Author Response

Reviewer 2

 

The authors are brought up an interesting topic to improve crop yields and soil properties. It is a very interesting subject for readers. I believe readers will be interested in this article. This paper compared the impact of monocropping and intercropping systems on soil nutrients, crop yields, and quantitative analysis of functional genes in soil. The manuscript is well-prepared in the experimental design and data analysis.

However, I have some suggestions to improve this manuscript.

 

Some suggestions:

It will be better for readers if authors are combined results and discussion sections together.

In abstract, please clarify some sentences:

L 23: you mean Olsen-P and K in soil?

Response: yes, in soil. Modified in manuscript

 

L27:  N and P mobilization of what? Please write clearly.

Response: done

 

L99: missing comma.

Response: done

L117: provide a reference on SIR?

 

Response: We added a reference in the text (Stockdalea et al., 2013).

The SIR method is physiological method for measurement of the soil microbial biomass. When glucose is added to a soil, an immediate increase of respiration rate is observed, the size of which is considered to be proportional to the microbial biomass. Some references that use SIR method to evaluate microbial biomass are included below:

 

- Stockdalea, E.A., Banningb, N.C., Murphy, D.V., 2013. Rhizosphere effects on functional stability of microbial communities in conventional and organic soils following elevated temperature treatment. Soil Biol Biochem, 57, 56-59.

- Sradnicka, A., Murugana, R., Oltmannsb, M., Rauppc, J., Joergensen, R.G., 2013. Changes in functional diversity of the soil microbial community in a heterogeneous sandy soil after long-term fertilization with cattle manure and mineral fertilizer. Appl Soil Ecol, 63, 23-28.

- Fanin, N., Barantal, S., Fromin, N., Schimann, H., Schevin, P., Hattenschwiler, S., 2012. Distinct microbial limitations in litter and underlying soil revealed by carbon and nutrient fertilization in a tropical rainforest. PLoS One, 7, e49990.

(DOI: 10.1371/journal.pone.0049990)

- Tlilia, A., Maréchala, M., Bérardc, A., Volata, B., Montuelle, B., 2011. Enhanced co-tolerance and co-sensitivity from long-term metal exposures of heterotrophic and autotrophic components of fluvial biofilms. Science of The Total Environment, 409, 4335-4343.

 

L126 &132: What is the symbol? Urease and N--benzoyl-L-arginine amide (BAA)

Response: the symbols are a and b. We added in the text

 

L224-225: You mean wheat or lupin?

Response: wheat, we added in the text

 

L231: grain yield of wheat?

Response: wheat, we added in the text

 

L235: after this paragraph, it will be better to put some discussion for readers?

Figure 1, there is no indication on acdS, phoD,  phoC, and nifH in the bar graphs.

Response: yes, is indicated below the graph.

Reviewer 3 Report

Manuscript submitted by Schoebitz et al is investigating the effect of intercropping on microbiological soil processes specifically microbial biomass and extracellular enzyme activity and relation to the plant nutrient uptake and grain yield. 

To address these relationships authors conducted a field trial where wheat and lupin were grown in mono or inter-cropping systems in an acidic Andisol in Chile. 

The major conclusion of the study is that the intercropping affected mostly the wheat crop but not the lupin. Authors measured overall better nutrient uptake in wheat but this happened by the expense of soil nutrient and grain yield losses. 

The manuscript is well written and the study design is sound. I have only a few minor suggestions and comments.

1. The format of citations is not consistent. Mostly it follows the guidelines of the journal but in a few lines, it is not (example: line 314). Additionally, when bringing attention to previous studies I would strongly suggest using the format as it is in line 279 (Xia et al. (2013)) by replacing (2013) with [35] instead of "Similarly, [5] reported ..." (line 62-63).
2. It would be relevant to know what is the crop rotation history in this field especially since authors bring up the effect of crop history in the discussion. Please provide this information. 

3. How did you regulate an equal distribution of wheat and lupin seeds per row for the intercropping experiment? Providing a schematic diagram of a mono and inter-cropping plot would be very helpful.

4. In the sampling paragraph, I was not able to find information about the number of plants sampled. Please provide this information. Also, it is mentioned that wheat and lupin were sampled separately. How were they treated later? I assume the samples were pooled and processed as one since all the chemistry and enzyme data are combined for the intercrop. Additionally, were equal amounts of maize and lupin rhizosphere used for the intercrop?

5. Please replace the centrifuge symbol with "alpha" (lines 126 and 132)
6. The qPCR section is missing the following information; amplification efficiency for each gene, the method for calculating gene ratios, and the instrument qPCR was done in.
7. In lines 293 to 301 are some contradicting statements. The urease activity was not influenced and this is different from the study [38] so it can not be in agreement.  
8.  Line 318 is a confusing sentence; "... changes in bulk soil for MBC in the rhizosphere of sole crops under ...". Please correct.
9. Line 362: This is another contradicting sentence. If the wheat yields were significantly lower with lupin intercropping then this can not be an effective approach to improve wheat grain yield.
10. Line 386-387: The higher abundance of acdS gene is probably a result of selective recruitment of microorganisms with this trait by the lupin plant.

Author Response

Reviewer 3.

 

Manuscript submitted by Schoebitz et al is investigating the effect of intercropping on microbiological soil processes specifically microbial biomass and extracellular enzyme activity and relation to the plant nutrient uptake and grain yield. 

To address these relationships authors conducted a field trial where wheat and lupin were grown in mono or inter-cropping systems in an acidic Andisol in Chile. 

The major conclusion of the study is that the intercropping affected mostly the wheat crop but not the lupin. Authors measured overall better nutrient uptake in wheat but this happened by the expense of soil nutrient and grain yield losses. 

The manuscript is well written and the study design is sound. I have only a few minor suggestions and comments.

1. The format of citations is not consistent. Mostly it follows the guidelines of the journal but in a few lines, it is not (example: line 314). OK, done. Additionally, when bringing attention to previous studies I would strongly suggest using the format as it is in line 279 (Xia et al. (2013)) by replacing (2013) with [35] OK, done instead of "Similarly, [5] reported ..." (line 62-63). OK, done

Response: ok, we accept the suggestion

    2. It would be relevant to know what is the crop rotation history in this field especially since authors bring up the effect of crop history in the discussion. Please provide this information. 

Response: Prior to the initiation of this study, corn was planted in 2017.

We added the phrase in L82

 

    3. How did you regulate an equal distribution of wheat and lupin seeds per row for the intercropping experiment? Providing a schematic diagram of a mono and inter-cropping plot would be very helpful.

 

Response: the objective of this experiment was to measure the effect of mono and intercropping at different plant densities. For that reason, the amount of seeds per ha were different. Wheat monocrop was planted at 150, 200 and 250 kg ha^-1; lupin monocrop was planted at 80, 140 and 200 kg ha^-1; and wheat/lupin intercrop was planted at 150 and 80 kg ha^-1(low density), 200 and 140 kg ha^-1 (medium density), and 250 and 200 kg ha^-1 (high density) for wheat and lupin, respectively. For example, 40 lupin seeds per row at low density, 70 seeds per row at medium density and 101 seeds per row at high densities.

We added a plot picture in the manuscript (figure 1)

 

      4. In the sampling paragraph, I was not able to find information about the number of plants sampled. Please provide this information. OK, done. Also, it is mentioned that wheat and lupin were sampled separately. How were they treated later? Response: Foliar samples were samples separately in intercrops, but not soil samples. I assume the samples were pooled and processed as one since all the chemistry and enzyme data are combined for the intercrop. Additionally, were equal amounts of maize and lupin rhizosphere used for the intercrop? Response: the amounts of soil rhizosphere were equal, because depend of the soil protocol to measure enzyme activities and biological properties.

 

Response: One linear meter per plot was sampled from the central row. In order to avoid edge-effects one linear meter was delimited in the central area of each plot (L117-118).

       5. Please replace the centrifuge symbol with "alpha" (lines 126 and 132)

Response: ok, changed

       6. The qPCR section is missing the following information; amplification efficiency for each gene, the method for calculating gene ratios, and the instrument qPCR  was done in. OK, done

Response: For all the genes, the amplification efficiency values ranged from 91.3% to 110.1%, and the linear fit R² was greater than 0.966. L 522-523.

Gen 16S efficiency: 0,91271592          91.3%

Gen phoC efficiency 0,99564838.       99.6%

Gen NifH efficiency 0,96482532         96.5%

Gen phoD efficiency 1,1006412          110.1%

"The ratio of gene target (acdS, phoD, phoC or nifH) in the bacterial community was estimated as abundance relative using absolute values of gene target in relation to total 16S rRNA gene counts." Phrase added L216-218

PCR reactions were performed in a StepOnePlus^TM Real-Time PCR System (Applied Biosystems Inc., USA). Added in L204-205.

         7. In lines 293 to 301 are some contradicting statements. The urease activity was not influenced and this is different from the study [38] so it can not be in agreement. 

Response: ok, changed

          8.  Line 318 is a confusing sentence; "... changes in bulk soil for MBC in the rhizosphere of sole crops under ...". Please correct.

Response: ok, changed

          9. Line 362: This is another contradicting sentence. If the wheat yields were significantly lower with lupin intercropping then this can not be an effective approach to improve wheat grain yield.

Response: we deleted this phrase to avoid the misunderstanding.

          10. Line 386-387: The higher abundance of acdS gene is probably a result of selective recruitment of microorganisms with this trait by the lupin plant.

Response: ok, we accept the suggestion.