Positive Effect of Camelina Intercropping with Legumes on Soil Microbial Diversity by Applying NGS Analysis and Mobile Fluorescence Spectroscopy
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsPositive Effect of Camelina Intercropping with Legumes on Soil Microbial Diversity by Applying NGS Analysis and Mobile Fluorescence Spectroscopy
General remarks:
The papir has serious lacks in the Material and Method section. The entire description of how the experiment was carried out, where the sampling took place, etc., are not described.
Since the paper does not have line numbers, it will be a little difficult to explain the part of the text that needs to be reviewed, but I will try my best. A special problem will be improperly labeled titles and subtitles. Below I will detail the lacks that need to be supplemented or corrected:
Specific remarks:
***Correct the numbering of titles and subtitles in the text.
Page 2, subheading Metagenomic sequencing and analysis of data - Before this part, a detailed explanation about the experiment, the method of sowing, the field on which the sowing was carried out, the type of soil and the way in which intercropping was carried out is missing. An entire segment of the experiment was skipped. Also, where the experiments were done (city, country), whether there were repetitions. In addition, there is a lack of data on where the sampling was carried out and how the soil samples were transported to the laboratory for analysis. A lot of important information is missing here.
Page 2, subheading Metagenomic sequencing and analysis of data – “Department of Plant Breeding” exists in almost every country, at almost all universities in Europe and the world. It is necessary to be more specific and give a detailed explanation of the institution.
Page 2, subheading Metagenomic sequencing and analysis of data – “two experimental years” Write the data where exactly it was grown (country, city, may also coordinate), whether in the same experimental field or different experimental fields. During which years?
Page 2, subheading Metagenomic sequencing and analysis of data – “previously described” if it is already described somewhere, provide the reference.
Page 2, subheading Metagenomic sequencing and analysis of data – “varieties Cs1.S.Pro, Cs2.S.Pro, Cs3.S and intercropped with vetch Cs3.Vs.S.Pro and pea Cs3.Ps.S.Pro” In the abstract, you mentioned commercial names (Luna, Lenka, Mir), so I think it should be written in the M&M section and not in the abstract (but certainly in the M&M section).
Also, indicate the origin of the seeds of these plants (where you got them - which producer).Error in Cs3.S. I think it should also write PRO?
Page 2, subheading Metagenomic sequencing and analysis of data – “amplification of PCR products for the 16S region with 16SV34 primers” Was a KIT used so extraction from the sample was done according to the protocol, or in some other way. Everything must be written in detail. Also, list the primers.
Page 3 - the abbreviation OTUs should be explained.
Page 3, subheading Relative abundance – “between Cs2.S.Pro and Cs3.S.Pro (most di-verse) and Cs2.S.Pro (least diverse)” Check if what you wrote is correct.
Figure 1 is a poor resolution. Improve figure resolution.
Page 4, sentence “In our study, the dominant bacteria at the phylum level were Actinobacteria, Proteobacteria, Gemmatimonadota, Bacteroidota, Firmicutes, and Acidobacteriota” - In my opinion (from Figure 2) Chloroflexi is more dominant than Bacteroidota.
Page 4, sentence “…, but Аctinobacteria and Acidobacteria abundance increased, compared to the Cs1.S.Pro soil (Figure 2)” This sentence is correct if it is compared only with Cs1.S.Pro. However it should be written what is the impact of intercropping if it is compared with Cs3.S.Pro, since this variety is intercropping with pea and vetch.
Page 5, subheading Abundance heatmap cluster of different microbial classes - Why are bacterial phyla written in italics in some places and not in others. Uniformize throughout the text.
Page 5, sentence “Bacteroidia, Gammaproteobacteria, and Bacilli predominated in Cs1.S.Pro soil (Fig. 3).” - In my opinion, Chlostridia should also be included in this list.
Page 5 – “Nolophagea” (in the text) or Holophagea (Figure 3)? Also, is it Cs3 or Cs3.S.Pro? Correct it.
Page 5, sentence “An intercropping system with vetch stimulated the growth and distribution of Nitrospira and Nitrososphaeria involved in the oxidation of ammonia to nitrate via nitrite (Fig. 3).” - In addition to Nitrospira and Nitrososphaeria, I would also add Blastocatellia. Also, what about intercropping with pea? That's the only thing that wasn't mentioned here.
Page 7, Sannan and Simpson indices - The Simpson index does not show this as clearly as the Sannan index.
Page 8, sentence “This result indicated that the three camelina varieties Luna, Lenka, and Local Bulgarian landrace then sole cultivated congregated different rhizospheric microbial communities.” - If we exclude the abstract, here the names Luna, Lenka are mentioned for the first time in the text... It is necessary to add an explanation to the M&M section about these names.
Page 9, Conclusions – “The intercropping systems assayed (Sc3.Vs.S.Pro and Sc3.Ps.S.Pro) increased camelina oil content and yield compared to the local camelina (Sc3 BG land-race) due to better nutrition of plants.” - This sentence is in the conclusion and this was not investigated in the paper. I believe that this is just an assumption or literature data that should be in the Introduction and not in the Conclusion, since the authors of this paper did not examine it.
*** In addition to all the above, the authors did not present the results for both analyzed years, but in general. In my opinion, if they state that the research was conducted over two years, the results of the research should be presented and explained for both years. If they wanted to present the results differently, this should be explained in detail in the paper.
General opinion:
I suggest that the paper be accepted for publication after detailed, major revision.
Best regards,
Reviewer
Author Response
Dear reviewers,
Thank you for the questions asked, for the corrections made, and for your time and effort. We send answers to the questions asked and the corrections we made. If the answers and corrections are sufficient, I hope the article will be published in the special edition.
R1
- In the introduction, the relevant research progress, such as the relationship between microorganisms and nutrient access, is not elaborated deeply enough.
Correction on page 3 in yellow colour. More recent articles are added as references.
Bacteria capable of fixing nitrogen and possibly responsible for stimulating plant growth as Azoarcus sp., Burkholderia sp., Gluconacetobacter diazotrophicus, Azotobacter sp., Herbaspirillum sp., and Paenibacillus polymyxa have been reported in to promote the development of beneficial bacteria na plant growth (Génard et al., 2017; Benmrid et al. 2023). Chamkhi et al. 2022 reported that the legume species used to supply grains was fixed with 40% N derived from the atmosphere in the intercropped soybean and 30% in the sole crop without fertilizer application. The uptake of phosphate fertilizer was 21% lower in intercrops compared to sole crops for the same yields. The intercropping with legumes significantly improves soil fertility, rhizobacteria community and diversity, and nutrient availability that are determinants of increased crop growth (Mirdoraghi et al., 2024).
- No scientific questions found for this paper, please add.
The innovative point in the present study is based on growing camelina, which is highly tolerant to drought and cold and can achieve sustainable yields in non-irrigated conditions. The introduction of camellia and cultivation in intercropping conditions raises the following questions about the efficient utilization of natural resources, preventing N-leaching and soil erosion; ii) reduction of pest and disease control costs due to higher competitive advantage over weeds and diseases in diversified cropping systems; iii) promotion the qualities of the seeds of the crops produced to meet the nutritional needs of man and farm animals.
This study focuses on organic farming systems that preserve the ecosystem and agricultural land integrity, biodiversity, and food and feed security. The main objective of intercropping is the production of a higher yield per unit area by using resources or ecological processes that a single crop would not otherwise use.
Please supplement the basic overview of the study area and sample sites.
Added in Material and Methods 2.1. Field experiments and 2.2 Soil samples and DNA extraction
For soil microbiome analysis, soil samples were taken according to the methodology from the experimental fields at the Agroecological Center of the Agricultural University –Plovdiv (Yagodovo field, Latitude 42° 6' 38.21"N ; Longitude, 24° 51' 1.28"E), where camelina is grown, during two experimental years under the SCOOP project. Field trials were conducted on the certified organic field. The soil type is Mollic fulvisols – FAO, with low humus content as 3.7% and neutral pH. Randomized complete block design was used for setting a small plot experiment on the effect of an intercropping system of camelina and protein crops, compared to sole crop of the same species. Three genotypes of Camelina sativa – the Polish winter varieties Luna (Cs1.Pro) and Lenka (Cs2.Pro) and a local Bulgarian landrace (Cs3.Pro) were grown in two successive years -2022 and 2023 in a pure stand and combined with fodder pea (Pisum sativum) Cs3.Pro sand vetch (Vicia sativa) Cs.Vs.Pro. In this study, we present the results from the autumn cultivation in small plots of 10 m2 (1.4 x 7.7m). The sowing was executed with plot seeder Wintersteiger AG with 800 germinating seeds/m2. Each variant of sole crop or its combinations was set in three replications. Fertilization was approved for organic farming solid fertilizer -30kg/ha of active nitrogen was made before soil cultivation.
2.2. Soil samples and DNA extraction
DNA extraction method from rhizosphere soils with mono cultivation of camelina varieties Cs1.S.Pro, Cs2.S.Pro, Cs3.S and intercropped with vetch Cs3.Vs.S.Pro and pea Cs3.Ps.S.Pro was carried out with isolation kit (Himedia, India), as defined by the modified method of by Nairet al. (2014). At A260/280 nm the isolated DNA was quantified by utilization of a Quantus fluorometer (Promega, USA) and stored at -20°C before being processed and sent to Novogene (Cambridge, UK) for NGS (IlluminaHiSeq).
- Do they show distinct differences in one or more phenotypes?
Luna and Lenka are Polish spring genotypes and Sc3 is the local autumn Bulgarian genotype. They are also in the morphology. Genetic diversity is evaluated on the base of phenotypic variation of breeding accessions on field conditions, oil quality as fatty acid composition and genetic analyses of molecular markers.
Supplement figure 1. Small scale field trials was added.
- How the samples were collected need to be explained in details.
For the study, average samples were collected and analyzed from the five experimental plots with independent or mixed cultivation of three camelina varieties with pea variety Mir and vetiver variety 666. The soil was collected from the rhizosphere zone to the roots of 10 plants from each variant randomly chosen. Rhizosphere soils were stored in sterile containers at -20°C. The soil samples were cleaned and prepared in the laboratory at the Department of Microbiology and Environmental Biotechnology – Agricultural University of Plovdiv for metagenomic analysis.
- Please analyze and discuss the reasons for the main findings or results of this paper. And separate the results and discussion.
- Please add some references published in mainstream journals in the past two years.
Some more recent references were added
Génard, T., Etienne, P., Diquélou, S., Yvin, J. C., Revellin, C., & Laîné, P. (2017). Rapeseed-legume intercrops: plant growth and nitrogen balance in early stages of growth and development. Heliyon, 3(3).
Benmrid, B., Ghoulam, C., Zeroual, Y., Kouisni, L., & Bargaz, A. (2023). Bioinoculants as a means of increasing crop tolerance to drought and phosphorus deficiency in legume-cereal intercropping systems. Communications Biology, 6(1), 1016.
Chamkhi, I., Cheto, S., Geistlinger, J., Zeroual, Y., Kouisni, L., Bargaz, A., & Ghoulam, C. 2022. Legume-based intercropping systems promote beneficial rhizobacterial community and crop yield under stress conditions. Industrial Crops and Products, 183, 114958.
Duchene, O., Vian, J. F., & Celette, F. 2017. Intercropping with legume for agroecological cropping systems: Complementarity and facilitation processes and the importance of soil microorganisms. A review. Agriculture, Ecosystems & Environment, 240, 148-161.
Ji, C., Ye, R., Yin, Y., Sun, X., Ma, H., & Gao, R. (2022). Reductive soil disinfestation with biochar amendment modified microbial community composition in soils under plastic greenhouse vegetable production. Soil and Tillage Research, 218, 105323.
R2
Specific remarks:
Correct the numbering of titles and subtitles in the text.
Corrected the numbering of titles and subtitles
Page 2, subheading Metagenomic sequencing and analysis of data - Before this part, a detailed explanation about the experiment, the method of sowing, the field on which the sowing was carried out, the type of soil and how intercropping was carried out is missing. An entire segment of the experiment was skipped. Also, where the experiments were done (city, country), and whether there were repetitions. In addition, there is a lack of data on where the sampling was carried out and how the soil samples were transported to the laboratory for analysis. A lot of important information is missing here.
Page 2, subheading Metagenomic sequencing and analysis of data – “Department of Plant Breeding” exists in almost every country, at almost all universities in Europe and the world. It is necessary to be more specific and give a detailed explanation of the institution.
Page 2, subheading Metagenomic sequencing and analysis of data – “two experimental years” Write the data where exactly it was grown (country, city, may also coordinate), whether in the same experimental field or different experimental fields. During which years?
For soil microbiome analysis, soil samples were taken according to the methodology from the experimental fields on the Agroecological Center of the Agricultural University –Plovdiv (Plovdiv, Bulgaria, Yagodovo field, Latitude 42° 6' 38.21"N ; Longitude, 24° 51' 1.28"E), where camelina is grown, during two experimental years under the SCOOP project. Field trials were conducted on the certified organic field. The soil type is Mollic fulvisols – FAO, with a low humus content of 3.7% and neutral pH. Randomized complete block design was used for setting a small plot experiment on the effect of an intercropping system of camelina and protein crops, compared to a sole crop of the same species. Three genotypes of Camelina sativa – the Polish winter varieties Luna (Cs1.Pro) and Lenka (Cs2.Pro) and a local Bulgarian landrace (Cs3.Pro) were grown in two successive years -2022 and 2023 in a pure stand and combined with fodder pea (Pisum sativum) Cs3.Pro sand vetch (Vicia sativa) Cs.Vs.Pro. In this study, we present the results from the autumn cultivation in small plots of 10 m2 (1.4 x 7.7m). The sowing was executed with plot seeder Wintersteiger AG with 800 germinating seeds/m2. Each variant of sole crop or its combinations was set in three replications. Fertilization was approved for organic farming solid fertilizer -30kg/ha of active nitrogen was made before soil cultivation.
Page 2, subheading Metagenomic sequencing and analysis of data – “previously described” if it is already described somewhere, provide the reference.
For the purposes of the study, average samples were collected and analyzed from the five experimental plots with independent or mixed cultivation of three camelina varieties with pea variety Mir and vetiver variety 666. The soil was collected from the rhizosphere zone of the roots of 10 plants from each variant randomly chosen. Rhizosphere soils were stored in sterile containers at -20°C. The soil samples were cleaned and prepared in the laboratory at the Department of Microbiology and Environmental Biotechnology – Agricultural University of Plovdiv for metagenomic analysis.
DNA extraction method from rhizosphere soils with mono cultivation of camelina varieties Cs1.S.Pro, Cs2.S.Pro, Cs3.S and intercropped with vetch Cs3.Vs.S.Pro and pea Cs3.Ps.S.Pro was carried out with isolation kit (Himedia, India), as defined by the modified method of by Nairet al. (2014). At A260/280 nm the isolated DNAwas quantified by utilization of a Quantus fluorometer (Promega, USA) and stored at -20°C before being processed and sent to Novogene (Cambridge, UK) for NGS (IlluminaHiSeq).
Page 2, subheading Metagenomic sequencing and analysis of data – “varieties Cs1.S.Pro, Cs2.S.Pro, Cs3.S and intercropped with vetch Cs3.Vs.S.Pro and pea Cs3.Ps.S.Pro” In the abstract, you mentioned commercial names (Luna, Lenka, Mir), so I think it should be written in the M&M section and not in the abstract (but certainly in the M&M section).
Added in the Materials and methods on page 4
Three genotypes of Camelina sativa – the Polish winter varieties Luna (Cs1.Pro) and Lenka (Cs2.Pro) and a local Bulgarian landrace (Cs3.Pro) were grown in two successive years -2022 and 2023 in a pure stand and combined with fodder pea (Pisum sativum) Cs3.Pro sand vetch (Vicia sativa) Cs.Vs.Pro.
MARINA
Also, indicate the origin of the seeds of these plants (where you got them - which producer).Error in Cs3.S. I think it should also write PRO?
Page 2, subheading Metagenomic sequencing and analysis of data – “amplification of PCR products for the 16S region with 16SV34 primers” Was a KIT used so extraction from the sample was done according to the protocol, or in some other way. Everything must be written in detail. Also, list the primers.
Information about DNA extraction was added in 2.2. Soil samples and DNA extraction
Page 3 - the abbreviation OTUs should be explained.
The abbreviation OTUs was explained.- operational taxonomic unit
Page 3, subheading Relative abundance – “between Cs2.S.Pro and Cs3.S.Pro (most di-verse) and Cs2.S.Pro (least diverse)” Check if what you wrote is correct.
Figure 1 is a poor resolution. Improve figure resolution.
This is original picture and I can not change.
Page 4, sentence “In our study, the dominant bacteria at the phylum level were Actinobacteria, Proteobacteria, Gemmatimonadota, Bacteroidota, Firmicutes, and Acidobacteriota” - In my opinion (from Figure 2) Chloroflexi is more dominant than Bacteroidota.
Page 4, sentence “…, but Аctinobacteria and Acidobacteria abundance increased, compared to the Cs1.S.Pro soil (Figure 2)” This sentence is correct if it is compared only with Cs1.S.Pro. However it should be written what is the impact of intercropping if it is compared with Cs3.S.Pro, since this variety is intercropping with pea and vetch.
There is a mistake with numbering of the figure. Correct is figure 3 and text is corrected.
From the data is clear that intercropping significantly decreased the Firmicutes abundance from 18.5% to 1.8% in the Sc2.S.Pro and Sc3.S.Pro and intercropped filed plots Cs3.Vs.SPro and Cs3.Ps.SPro. In contrast, Аctinobacteria and Acidobacteria abundance increased, compared to the Cs1.S.Pro soil (Figure 3). Similar results have been obtained in intercropping experiments in which the bacterial biomass and activity in mono-cropping differed from those detected in intercropped systems (Duchene et al. 2017).
Page 5, subheading Abundance heatmap cluster of different microbial classes - Why are bacterial phyla written in italics in some places and not in others. Uniformize throughout the text.
Corrected
Page 5, sentence “Bacteroidia, Gammaproteobacteria, and Bacilli predominated in Cs1.S.Pro soil (Fig. 3).” - In my opinion, Chlostridia should also be included in this list.
Correct, while the similarity and differences of samples can also be observed. Bacteroidia, Gammaproteobacteria, Bacilli and Chlostridia predominated in Cs1.S.Pro soil (Fig. 3)
Page 5 – “Nolophagea” (in the text) or Holophagea (Figure 3)? Also, is it Cs3 or Cs3.S.Pro? Correct it.
Holophagea are the most abundant in the Cs2.S.Pro soil is correct
Page 5, sentence “An intercropping system with vetch stimulated the growth and distribution of Nitrospira and Nitrososphaeria involved in the oxidation of ammonia to nitrate via nitrite (Fig. 3).” - In addition to Nitrospira and Nitrososphaeria, I would also add Blastocatellia. That's the only thing that wasn't mentioned here.
Blastocatellia was added.
Also, what about intercropping with pea?
In the co-cultivation of camelina with peas, out of 30 bacterial classes have the greatest influence on plant biomass degradation in the soil microbiome have Actinobacteria, Rubrobacteria, Acidobactreria, KD4-96, Entotheonellia, and Chloroflexia. On the contrast to intercropping system with vetch, in camelina-pea soil abundancy of species from phyla Nirtosphaeria and Nitrospira was low. Nitrospira can utilize simple organic substrates and they can survive in soil conditions where organic carbon is available and the paucity of nitogen limits the Nitrosomonadaceae. when nitrogen fertilizer is applied, they may be less able to take advantage of the increased ammonia concentration and they decreased in the soil. Those sresult are corresponding to findings of Li et al. 2019. There observed less pathogenic Bacilli and Clostridia in the Cs3.Ps.S.Pro soil compared to sole cultivation of camelina. Thus, application of camelina-pea intercropping to have the beneficial role in suppressing soil-borne plant pathogens. Ji et al. 2022 reported that the yield of pepper was positively correlated with the contents and the relative abundance of Bacillus and Clostridium in soil and negatively correlated with NO3-concertaction in soil.
Page 7, Sannan and Simpson indices - The Simpson index does not show this as clearly as the Sannan index.
Page 8, sentence “This result indicated that the three camelina varieties Luna, Lenka, and Local Bulgarian landrace then sole cultivated congregated different rhizospheric microbial communities.” - If we exclude the abstract, here the names Luna, Lenka are mentioned for the first time in the text... It is necessary to add an explanation to the M&M section about these names.
The cultivars are mentioned in the abstract, in the material and methods. The purpose of utilization of cultivars name, but not the experimental manes of the samples was to avoid toutology.
Page 9, Conclusions – “The intercropping systems assayed (Sc3.Vs.S.Pro and Sc3.Ps.S.Pro) increased camelina oil content and yield compared to the local camelina (Sc3 BG land-race) due to better nutrition of plants.” - This sentence is in the conclusion and this was not investigated in the paper. I believe that this is just an assumption or literature data that should be in the Introduction and not in the Conclusion, since the authors of this paper did not examine it.
The effect of intercropping have been analyzes biochemicaly of the fatty acids content, and fatty acid gene expression but the results are not published yet.
The intercropping systems assayed (Sc3.Vs.S.Pro and Sc3.Ps.S.Pro) increased camelina oil content and yield compared to the local camelina (Sc3 BG landrace) due to better nutrition of plants (unpublished data).
*** In addition to all the above, the authors did not present the results for both analyzed years, but in general. In my opinion, if they state that the research was conducted over two years, the results of the research should be presented and explained for both years. If they wanted to present the results differently, this should be explained in detail in the paper.
Three genotypes of Camelina sativa – the Polish winter varieties Luna (Cs1.Pro) and Lenka (Cs2.Pro) and a local Bulgarian landrace (Cs3.Pro) were grown in two successive years -2022 and 2023 in a pure stand and combined with fodder pea (Pisum sativum) Cs3.Pro sand vetch (Vicia sativa) Cs.Vs.Pro. In this study we present the results from the autumn cultivation in 2022 on small plots of 10 m2 (1.4 x 7.7m).
General opinion:
I suggest that the paper be accepted for publication after detailed, major revision.
Best regards,
Reviewer
Reviewer 2 Report
Comments and Suggestions for AuthorsThe topic of this paper is interesting and has strong theoretical and practical significance, but the writing needs to do a lot of optimization.
1. In the introduction, the relevant research progress, such as the relationship between microorganisms and nutrient access, is not elaborated deeply enough.
2. No scientific questions found for this paper, please add.
3. Please supplement the basic overview of the study area and sample sites.
4. Do they show distinct differences in one or more phenotypes?
5. How the samples were collected need to be explained in details.
6. Please analyze and discuss the reasons for the main findings or results of this paper. And separate the results and discussion.
7. Please add some references published in mainstream journals in the past two years.
Author Response
Dear rewires,
Thank you for the questions asked, for the corrections made, and for your time and effort. We send answers to the questions asked and the corrections we made. If the answers and corrections are sufficient, I hope the article will be published in the special edition.
R2
Specific remarks:
Correct the numbering of titles and subtitles in the text.
Corrected the numbering of titles and subtitles
Page 2, subheading Metagenomic sequencing and analysis of data - Before this part, a detailed explanation about the experiment, the method of sowing, the field on which the sowing was carried out, the type of soil and the way in which intercropping was carried out is missing. An entire segment of the experiment was skipped. Also, where the experiments were done (city, country), whether there were repetitions. In addition, there is a lack of data on where the sampling was carried out and how the soil samples were transported to the laboratory for analysis. A lot of important information is missing here.
Page 2, subheading Metagenomic sequencing and analysis of data – “Department of Plant Breeding” exists in almost every country, at almost all universities in Europe and the world. It is necessary to be more specific and give a detailed explanation of the institution.
Page 2, subheading Metagenomic sequencing and analysis of data – “two experimental years” Write the data where exactly it was grown (country, city, may also coordinate), whether in the same experimental field or different experimental fields. During which years?
For soil microbiome analysis, soil samples were taken according to the methodology from the experimental fields on the Agroecological Center of the Agricultural University –Plovdiv (Plovdiv, Bulgaria, Yagodovo field, Latitude 42° 6' 38.21"N ; Longitude, 24° 51' 1.28"E), where camelina is grown, during two experimental years under the SCOOP project. Field trials were conducted on the certified organic field. The soil type is Mollic fulvisols – FAO, with low humus content as 3.7% and neutral pH. Randomized complete block design was used for setting a small plot experiment on the effect of an intercropping system of camelina and protein crops, compared to sole crop of the same species. Three genotypes of Camelina sativa – the Polish winter varieties Luna (Cs1.Pro) and Lenka (Cs2.Pro) and a local Bulgarian landrace (Cs3.Pro) were grown in two successive years -2022 and 2023 in a pure stand and combined with fodder pea (Pisum sativum) Cs3.Pro sand vetch (Vicia sativa) Cs.Vs.Pro. In this study we present the results from the autumn cultivation in small plots of 10 m2 (1.4 x 7.7m). The sowing was executed with plot seeder Wintersteiger AG with 800 germinating seeds/m2. Each variant of sole crop or its combinations was set in three replications. Fertilization with approved for organic farming solid fertilizer -30kg/ha of active substance nitrogen was made before soil cultivation.
Page 2, subheading Metagenomic sequencing and analysis of data – “previously described” if it is already described somewhere, provide the reference.
For the purposes of the study, average samples were collected and analyzed from the five experimental plots with independent or mixed cultivation of three camelina varieties with pea variety Mir and vetiver variety 666. The soil was collected from the rhizosphere zone of the roots of 10 plants from each variant randomly chosen. Rhizosphere soils were stored in sterile containers at -20°C. The soil samples were cleaned and prepared in the laboratory at the Department of Microbiology and Environmental Biotechnology – Agricultural University of Plovdiv for metagenomic analysis.
DNA extraction method from rhizosphere soils with mono cultivation of camelina varieties Cs1.S.Pro, Cs2.S.Pro, Cs3.S and intercropped with vetch Cs3.Vs.S.Pro and pea Cs3.Ps.S.Pro was carried out with isolation kit (Himedia, India), as defined by the modifyed methodof by Nairet al. (2014). At A260/280 nm the isolated DNAwas quantified by utilizationof a Quantus fluorometer (Promega, USA) and stored at -20°C before being processed and were sent to Novogene (Cambridge, UK) for NGS (IlluminaHiSeq).
Page 2, subheading Metagenomic sequencing and analysis of data – “varieties Cs1.S.Pro, Cs2.S.Pro, Cs3.S and intercropped with vetch Cs3.Vs.S.Pro and pea Cs3.Ps.S.Pro” In the abstract, you mentioned commercial names (Luna, Lenka, Mir), so I think it should be written in the M&M section and not in the abstract (but certainly in the M&M section).
Added in the Materials and methods on page 4
Three genotypes of Camelina sativa – the Polish winter varieties Luna (Cs1.Pro) and Lenka (Cs2.Pro) and a local Bulgarian landrace (Cs3.Pro) were grown in two successive years -2022 and 2023 in a pure stand and combined with fodder pea (Pisum sativum) Cs3.Pro sand vetch (Vicia sativa) Cs.Vs.Pro.
MARINA
Also, indicate the origin of the seeds of these plants (where you got them - which producer).Error in Cs3.S. I think it should also write PRO?
Page 2, subheading Metagenomic sequencing and analysis of data – “amplification of PCR products for the 16S region with 16SV34 primers” Was a KIT used so extraction from the sample was done according to the protocol, or in some other way. Everything must be written in detail. Also, list the primers.
Information about DNA extraction was added in 2.2. Soil samples and DNA extraction
Page 3 - the abbreviation OTUs should be explained.
The abbreviation OTUs was explained.- operational taxonomic unit
Page 3, subheading Relative abundance – “between Cs2.S.Pro and Cs3.S.Pro (most di-verse) and Cs2.S.Pro (least diverse)” Check if what you wrote is correct.
Figure 1 is a poor resolution. Improve figure resolution.
This is original picture and I can not change.
Page 4, sentence “In our study, the dominant bacteria at the phylum level were Actinobacteria, Proteobacteria, Gemmatimonadota, Bacteroidota, Firmicutes, and Acidobacteriota” - In my opinion (from Figure 2) Chloroflexi is more dominant than Bacteroidota.
Page 4, sentence “…, but Аctinobacteria and Acidobacteria abundance increased, compared to the Cs1.S.Pro soil (Figure 2)” This sentence is correct if it is compared only with Cs1.S.Pro. However it should be written what is the impact of intercropping if it is compared with Cs3.S.Pro, since this variety is intercropping with pea and vetch.
There is a mistake with numbering of the figure. Correct is figure 3 and text is corrected.
From the data is clear that intercropping significantly decreased the Firmicutes abundance from 18.5% to 1.8% in the Sc2.S.Pro and Sc3.S.Pro and intercropped filed plots Cs3.Vs.SPro and Cs3.Ps.SPro. In contrast, Аctinobacteria and Acidobacteria abundance increased, compared to the Cs1.S.Pro soil (Figure 3). Similar results have been obtained in intercropping experiments in which the bacterial biomass and activity in mono-cropping differed from those detected in intercropped systems (Duchene et al. 2017).
Page 5, subheading Abundance heatmap cluster of different microbial classes - Why are bacterial phyla written in italics in some places and not in others. Uniformize throughout the text.
Corrected
Page 5, sentence “Bacteroidia, Gammaproteobacteria, and Bacilli predominated in Cs1.S.Pro soil (Fig. 3).” - In my opinion, Chlostridia should also be included in this list.
Correct, while the similarity and differences of samples can also be observed. Bacteroidia, Gammaproteobacteria, Bacilli and Chlostridia predominated in Cs1.S.Pro soil (Fig. 3)
Page 5 – “Nolophagea” (in the text) or Holophagea (Figure 3)? Also, is it Cs3 or Cs3.S.Pro? Correct it.
Holophagea are the most abundant in the Cs2.S.Pro soil is correct
Page 5, sentence “An intercropping system with vetch stimulated the growth and distribution of Nitrospira and Nitrososphaeria involved in the oxidation of ammonia to nitrate via nitrite (Fig. 3).” - In addition to Nitrospira and Nitrososphaeria, I would also add Blastocatellia. That's the only thing that wasn't mentioned here.
Blastocatellia was added.
Also, what about intercropping with pea?
In the co-cultivation of camelina with peas, out of 30 bacterial classes have the greatest influence on plant biomass degradation in the soil microbiome have Actinobacteria, Rubrobacteria, Acidobactreria, KD4-96, Entotheonellia, and Chloroflexia. On the contrast to intercropping system with vetch, in camelina-pea soil abundance of species from phyla Nirtosphaeria and Nitrospira was low. Nitrospira can utilize simple organic substrates and they can survive in soil conditions where organic carbon is available and the paucity of nitrogen limits the Nitrosomonadaceae. when nitrogen fertilizer is applied, they may be less able to take advantage of the increased ammonia concentration and they decrease in the soil. Those results correspond to the findings of Li et al. 2019. There observed less pathogenic Bacilli and Clostridia in the Cs3.Ps.S.Pro soil compared to sole cultivation of camelina. Thus, the application of camelina-pea intercropping has a beneficial role in suppressing soil-borne plant pathogens. Ji et al. 2022 reported that the yield of pepper was positively correlated with the contents and the relative abundance of Bacillus and Clostridium in soil and negatively correlated with NO3-concertaction in soil.
Page 7, Sannan and Simpson indices - The Simpson index does not show this as clearly as the Sannan index.
Page 8, sentence “This result indicated that the three camelina varieties Luna, Lenka, and Local Bulgarian landrace then sole cultivated congregated different rhizospheric microbial communities.” - If we exclude the abstract, here the names Luna, Lenka are mentioned for the first time in the text... It is necessary to add an explanation to the M&M section about these names.
The cultivars are mentioned in the abstract, in the material and methods. The purpose of utilization of cultivars name, but not the experimental manes of the samples was to avoid tautology.
Page 9, Conclusions – “The intercropping systems assayed (Sc3.Vs.S.Pro and Sc3.Ps.S.Pro) increased camelina oil content and yield compared to the local camelina (Sc3 BG land-race) due to better nutrition of plants.” - This sentence is in the conclusion and this was not investigated in the paper. I believe that this is just an assumption or literature data that should be in the Introduction and not in the Conclusion, since the authors of this paper did not examine it.
The effect of intercropping has been analyzes biochemically of the fatty acids content, and fatty acid gene expression but the results are not published yet.
The intercropping systems assayed (Sc3.Vs.S.Pro and Sc3.Ps.S.Pro) increased camelina oil content and yield compared to the local camelina (Sc3 BG landrace) due to better nutrition of plants (unpublished data).
*** In addition to all the above, the authors did not present the results for both analyzed years, but in general. In my opinion, if they state that the research was conducted over two years, the results of the research should be presented and explained for both years. If they wanted to present the results differently, this should be explained in detail in the paper.
Three genotypes of Camelina sativa – the Polish winter varieties Luna (Cs1.Pro) and Lenka (Cs2.Pro) and a local Bulgarian landrace (Cs3.Pro) were grown in two successive years -2022 and 2023 in a pure stand and combined with fodder pea (Pisum sativum) Cs3.Pro sand vetch (Vicia sativa) Cs.Vs.Pro. In this study we present the results from the autumn cultivation in 2022 on small plots of 10 m2 (1.4 x 7.7m).
General opinion:
I suggest that the paper be accepted for publication after detailed, major revision.
Best regards,
Reviewer
Author Response File: 
Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsPositive Effect of Camelina Intercropping with Legumes on Soil Microbial Diversity by Applying NGS Analysis and Mobile Fluorescence Spectroscopy
The authors have corrected most, but not all, of the previous review. Below I send the shortcomings that need to be changed or supplemented:
Page 4, Line 132 - No supplementary material was sent to me for review.
Page 8, Line 312 – Cs2.S.Pro or Cs3. S. Pro?
Page 8, Line 313 - Cs3 or Cs3.S.Pro? Correct it.
Page 9, Line 323 - Beginning of sentence, capital letter "When".
*Also, the following suggestions for the review were not taken into consideration by the author, nor did I receive an adequate response:
1. Page 4, sentence “In our study, the dominant bacteria at the phylum level were Actinobacteria, Proteobacteria, Gemmatimonadota, Bacteroidota, Firmicutes, and Acidobacteriota” - In my opinion (from Figure 2) Chloroflexi is more dominant than Bacteroidota.
2. Page 5, subheading Abundance heatmap cluster of different microbial classes - Why are bacterial phyla written in italics in some places in the text and not in others. Uniformize throughout the text.
3. Page 7, Sannan and Simpson indices - The Simpson index does not show this as clearly as the Sannan index.
Conclusion - I still believe that the results that are not part of the presented research (in the specific case the sentence "The intercropping systems assayed (Sc3.Vs.S.Pro and Sc3.Ps.S.Pro) increased camelina oil content and yield compared to the local camelina (Sc3 BG landrace) due to better nutrition of plants (unpublished data)" ), should not be part of the conclusion of this paper.
Best regards,
Reviewer
Author Response
Dear reviewer,
Thank you for the critical review of the manuscript.
Please receive the corrections and answers from the authors.
Page 4, Line 132 - No supplementary material was sent to me for review.
Answer: Added Supplement Figure 1. (A) A randomized complete block design was used for setting a small plot experiment; (B) Cs1 (Luna), Cs2 (Lenka), and Cs3 (Bulgarian landrace); (C) Cs3 intercropped with pea (Pisum sativum L.); (D) Cs3 intercropped with vetch (Vicia sativa L.).
Page 8, Line 312 – Cs2.S.Pro or Cs3. S. Pro?
Answer: Corrected in the text
DNA extraction method from rhizosphere soils with mono cultivation of camelina varieties Cs1.S.Pro, Cs2.S.Pro, Cs3.S.Pro and intercropped with vetch Cs3.Vs.S.Pro and pea Cs3.Ps.S.Pro was carried out with isolation kit (Himedia, India), as defined by the modifyed methodof by Nairet al.
Page 8, Line 313 - Cs3 or Cs3.S.Pro? Correct it.
Answer: Corrected in the text
From the data is clear that intercropping significantly decreased the Firmicutes abundance from 18.5% in in the Cs2.S.Pro to 1.8% in Cs3.S.Pro and intercropped filed plots Cs2.Vs.S.Pro and Cs3.Ps.S.Pro.
Page 9, Line 323 - Beginning of sentence, capital letter "When".
Answer: Corrected in the text
*Also, the following suggestions for the review were not taken into consideration by the author, nor did I receive an adequate response:
- Page 4, sentence“In our study, the dominant bacteria at the phylum level were Actinobacteria, Proteobacteria, Gemmatimonadota, Bacteroidota, Firmicutes, and Acidobacteriota” - In my opinion (from Figure 2) Chloroflexi is more dominant than Bacteroidota.
Answer: The reviewer is correct Chloroflexi is more dominant than Bacteroidota. We accepted the comment and corrected the text
‘’ In our study, the dominant bacteria at the phylum level were Actinobacteria, Proteobacteria, Gemmatimonadota, Chloroflexi, Firmicutes, and Acidobacteriota, accounting for approximately 92% of the total abundance of the bacterial community.
- Page 5, subheading Abundance heatmap cluster of different microbial classes - Why are bacterial phyla written in italics in some places in the text and not in others. Uniformize throughout the text.
Answer: Corrected in the text
- Page 7, Sannan and Simpson indices- The Simpson index does not show this as clearly as the Sannan index.
Conclusion - I still believe that the results that are not part of the presented research (in the specific case the sentence "The intercropping systems assayed (Sc3.Vs.S.Pro and Sc3.Ps.S.Pro) increased camelina oil content and yield compared to the local camelina (Sc3 BG landrace) due to better nutrition of plants (unpublished data)" ), should not be part of the conclusion of this paper.
Answer: The sentence was removed from the text.
Best regards,
Authors
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors submitted higher quality revisions.
Author Response
Dear reviewer,
Thank you for the critical reading of the manuscript.
Best regards
Author Response File: Author Response.pdf
