Exploiting the Genetic Potential of Cowpea in An Intercropping Complex

Round 1

Reviewer 1 Report

The research presented in this paper tackles an important issue such as breeding for intercropping. There have been many investigations and advances in intercropping in recent years, but obtention of cultivars specifically designed for intercropping is still limited. The paper is well presented, good figures and tables, and the English is correct.

Unfortunatley, the work has an important flaw from its conception, which is to establish field experiments with only two replications. This limits seriously any statistical analysis, and my understanding is that this limitation has not been taken into account. Presenting results by year (environment), as it has been done, would not be correct. Analysing globally the four replications of the two year might be a right approach, but this has limitations of its own too.

I think that clarification for the genetic analyses is also required. It would be important to know from wich crosses and parentals the 35 genotypes come from. Are the genetical analyses made for all the genotypes globally, no matter their pedigree?

Beyond that, there are also other questions. In relation to the design of the experiment, and sowing cowpea after millet, if in practise cowpea is sown at the same time as millet, the experiment does not reflect the real agronomic conditions. It may be useful to overrepresent the effect of shading, but undoubtedly the genotypes selected will not be completely adapted to the real growing conditions they will meet. Don’t the authors think this might be a problem in the future?

As for the results, why the yield of millet has not been included? Evaluating the effect of cropping pattern on this crop might have been very useful. The analysis of a parameter such as Land Equivalent Ratio (LER) would have been very interesting.

Apart from these issues, other minor questions are:

Line 59: “without the use of insecticides” is out of place in that context.

Line 129: “it comprised” should probably be “they comprised”.

Line 149: why the proportion 2:4?

Line 150: what does “block” mean here? Is it used in the statistic sense? If that is the case, justify why using those blocks. If it is not used in the statistic sense, then choose another word.

Line 152: plot size is not properly defined.

Line 155: why has this fertilization rate been used? Is this the normal one for monocrop or intercrop? And for which crop?

Line 159: specify they are two middle rows (as I understand they are).

Line 261: a paragraph from the journal template seems to have been included here.

My recommendation for the authors would be to withdraw the manuscript and re-submit it after a deep review of the statistical analyses by an expert.

The English is correct.

Author Response

Point 1: Unfortunately, the work has an important flaw from its conception, which is to establish field experiments with only two replications. This limits seriously any statistical analysis, and my understanding is that this limitation has not been taken into account. Presenting results by year (environment), as it has been done, would not be correct. Analysing globally the four replications of the two year might be a right approach, but this has limitations of its own too.

Response 1: We acknowledge the reviewer concern about the two replications, but, given that this experiment is factorial in nature with multiple factors, using 2 replications per year and considering two years (environments) of data is a fair compromise. We have consulted a biometrician and we believe that the outcome of this manuscript will be helpful to our cowpea research community. In addition, we would like to indicate that the major findings reported here were based on combined data analysis across two environments, and in our model, replications were nested within the environments. The single environment analyses were reported in this study only as supplementary data (please, see Lines 292-295 of revised manuscript).

Point 2: I think that clarification for the genetic analyses is also required. It would be important to know from which crosses and parentals the 35 genotypes come from. Are the genetical analyses made for all the genotypes globally, no matter their pedigree?.

Response 2: The 35 genotypes were selections that had reached advanced yield testing phase, and therefore, each of them have unique pedigrees (that is, the 35 are not coming from a single bi-parental cross) (Please, check Lines 140-142 for clarity). Therefore, the analysis was done for all genotypes globally. We have tried to make this clearer in the statistical analysis section as suggested by reviewer (Line 186).

Point 3: Beyond that, there are also other questions. In relation to the design of the experiment, and sowing cowpea after millet, if in practise cowpea is sown at the same time as millet, the experiment does not reflect the real agronomic conditions. It may be useful to overrepresent the effect of shading, but undoubtedly the genotypes selected will not be completely adapted to the real growing conditions they will meet. Don’t the authors think this might be a problem in the future?

Response 3: Thank you for this! We mimicked what farmers do in West African region, particularly in Nigeria. Millet is often planted first followed by cowpea about 3-4 weeks later (cowpea and millet are not planted at the same time). The authors agree with you that, if the experimental approach differ from farmer practice, then the output of the study may not be applicable, but this is not the case for this study.    

Point 4: As for the results, why the yield of millet has not been included? Evaluating the effect of cropping pattern on this crop might have been very useful. The analysis of a parameter such as Land Equivalent Ratio (LER) would have been very interesting.

Response 4: This suggestion is appreciated. The study used a single farmer preferred variety of millet purposely to create the different cropping patterns, and with the focus on cowpea performance, and how these patterns would affect different genetic parameters that are of interest in breeding cowpea for cropping systems (e.g genetic gain, genetic correlations, adaptation, and stability to cropping systems). Data was not focused on millet and therefore, it was not necessary to estimate LER, although, we believe the analyses conducted were sufficient to address the specified objectives of the study.

Apart from these issues, other minor questions are:

Line 59: “without the use of insecticides” is out of place in that context.

Response: This has been revised.

Line 129: “it comprised” should probably be “they comprised”.

Response: Adjusted as suggested.

Line 149: why the proportion 2:4?:

Response: These are possible cropping patterns that farmers are using in the West African region, and we wanted to evaluate suitability of cowpea to these patterns.  

Line 150: what does “block” mean here? Is it used in the statistic sense? If that is the case, justify why using those blocks. If it is not used in the statistic sense, then choose another word.

Response: We agree that the word “block” here introduces confusion since it is not used in a statistical sense, we have substituted it with the “range”.

Line 152: plot size is not properly defined.

Response: We added clarity by specifying that each plot had six rows, each 4 meters long, with spacing of 0.75 m x 0.2 m for cowpea and 0.75 m x 0.5 m for millet (Line 166 of revised manuscript). Farmers use similar spacings in their fields.

Line 155: why has this fertilization rate been used? Is this the normal one for monocrop or intercrop? And for which crop?

Response: We used a single normal rate of fertilizer application often under monocropping, and both millet and cowpea received the same rate (we did not vary the rates), some clarification added to this in the text (Line 172 revised manuscript).

Line 159: specify they are two middle rows (as I understand they are).

Response: Corrected (Line 177)

Line 261: a paragraph from the journal template seems to have been included here.

Response: Correct observation! this has been removed.

My recommendation for the authors would be to withdraw the manuscript and re-submit it after a deep review of the statistical analyses by an expert.

Response:  We have consulted a biometrician, revisited, and reviewed the rigorous statistical models used in this study and found them to address well the objectives for which the study was designed.

Reviewer 2 Report

I have no comment. The article covers many indicators. 

Author Response

Reviewer 2 was fully satisfied with work, no comments to respond to 

Reviewer 3 Report

Dear Authors,

I consider the manuscript presented to me for review as very important for its contribution to advances in agronomy. Intercropping is a possibility to obtain high quality forage and food with low inputs. In addition, it has a positive impact on the soil environment. The field trials carried out by the authors can point the way towards the use of specific cowpea varieties in widespread agricultural practice.

For the content of the manuscript, I have a few comments:

I suggest supplementing the introduction with the benefits of symbiotic nitrogen fixation. I would also suggest mentioning the different root structures of the legume and cereal crop, so they benefit from different resources in the soil, and as the authors mentioned they compete mainly for light. A possible addition could also be the fact that legume cultivation increases acid phosphatase activity in the soil thus lowering the pH of the rhizosphere and releasing P from inaccessible compounds.

Line 91 incorrect form of citation

Materials and methods

Line 119 I suggest supplementing with detailed weather data when conducting the study. I suggest putting them on the graph. I also suggest supplementing the data with soil conditions during the implementation of the field experiment from each year.

Line 153 I suggest giving the number of seeds sown per hectare

Was cowpea seed inoculation used?

Results

Line 261 - 263 delete

Line 285 in the description I suggest mentioning that the analysis is for cowpea traits, with the earlier talk of intercropping can be a little confusing.

Line 304 Figure c is in a different order to the others, I suggest aligning. I would also suggest putting in the title that this relates to cowpea

Discussion

Line 477 incorrect form of citation

Conclusions

I suggest adding a conclusion summarising which variety, on the basis of the research carried out, should be recommended for intercropping in widespread agricultural practice.

References

Should be standardised, in some manuscripts the full name of the publisher is given in others the abbreviation. There are abbreviations with or without '.'.

Yours sincerely

Author Response

Point 1: I suggest supplementing the introduction with the benefits of symbiotic nitrogen fixation. I would also suggest mentioning the different root structures of the legume and cereal crop, so they benefit from different resources in the soil, and as the authors mentioned they compete mainly for light. A possible addition could also be the fact that legume cultivation increases acid phosphatase activity in the soil thus lowering the pH of the rhizosphere and releasing P from inaccessible compounds.

Response 1: These are good suggestions from reviewer, and have been incorporated in the background, clearly marked in track changes.

Minor edits:

Line 91 incorrect form of citation

Response: This has been corrected.

Line 119 I suggest supplementing with detailed weather data when conducting the study. I suggest putting them on the graph. I also suggest supplementing the data with soil conditions during the implementation of the field experiment from each year.

Response: We have provided the weather data during the growing season as supplementary information (Lines 127-128). However, soil tests were not conducted during the period of the study, but we have described a previously published experimental station report of soil physical and chemical properties of the study site (Lines 129-134 of revised manuscript).

Line 153 I suggest giving the number of seeds sown per hectare.

Good suggestion. We have added this information in Lines 169-170 of revised manuscript. 

Was cowpea seed inoculation used?

Response: No

Results

Line 261 - 263 delete:

Response: Suggested action have been taken.

Line 285 in the description I suggest mentioning that the analysis is for cowpea traits, with the earlier talk of intercropping can be a little confusing.

Response: Suggestion is appreciated and taken care of in revised manuscript Line 306

Line 304 Figure c is in a different order to the others, I suggest aligning. I would also suggest putting in the title that this relates to cowpea

Response: The alignments in Figure 3 were based on mean performance (automatically sorted by software from the highest to the lowest), that is, the software placed the cropping system with highest mean value first. Therefore, for Figure 3c, 100 seed weight of cowpea was a bit unique, numerical mean values were higher under 2:4, followed by 1:1 and lastly sole. We did not change this alignment but provided additional clarification in the footnotes (lines 329-330).

Discussion

Line 477 incorrect form of citation

Response: Corrected

Conclusions

I suggest adding a conclusion summarising which variety, on the basis of the research carried out, should be recommended for intercropping in widespread agricultural practice.

Response: This has been addressed in the revised manuscript Lines 647-649

References

Should be standardised, in some manuscripts the full name of the publisher is given in others the abbreviation. There are abbreviations with or without

Response: This has been addressed in the revised manuscript.

Round 2

Reviewer 1 Report

Thanks for the comments and the corrections. However, my major concerns have not been dissipated.

As for the design and the statistical analysis, the authors have stressed the fact that most of the results are based on the combination of two experiments in two different years. This is true, and would be correct but with a different model, in which it would not be possible to consider the effects of environments (year) or the interactions genotype x environment.  I am aware that the authors have consulted with a biometrician but, in my opinion, the type of analysis that has been performed with only two replications per experiment is not correct.

I still do not understand why the yield of millet has not been taken into account. The authors specify that the research has been carried out to study different parameters that are of interest in breeding cowpea for cropping systems. The interaction between crops is key when investigating and designing intercropping systems. It might happen that some genotypes of cowpea perform very well in combination with millet, while this crop presents very poor performance, which would not be acceptable for intercropping as a whole.

I will assume that sowing cowpea weeks after millet is common practice in the area, although I have not found that myself in the review of bibliography I have made.

Finally, the addition that has been made in line 186 is not necessary, I had asked about the pedigree just for clarification, but it is well understood otherwise.

Unfortunately, I still hold my recommendation for the authors to withdraw the manuscript and re-elaborate the statistical analysis.