Investigating the Impact of Biostimulants on the Row Crops Corn and Soybean Using High-Efficiency Phenotyping and Next Generation Sequencing

Round 1

Reviewer 1 Report

This research investigated effects of three biostimulant formulations on soybean and corn using phenomic-based measurements and next generation sequencing. The results of this study provides new information on the approach for biostimulant research. Overall, the manuscript is well prepared and the results were clearly presented. However, there are some weaknesses that the authors are encouraged to address during revision.

First, the abstract should be revised to present the results/findings concisely.

Second, the objective(s) should be clearly stated at the end of the introduction section (line 95). 

Third, for M&M part, the plants were grown under natural light, how did you maintain the light uniformity across the area the plants were located. In other words, did you measure/verify the light intensity for each plant? The environmental factors (light intensity, photoperiod, temperature, relative humidity) should be provided. Also, please explain what 20 units of nitrogen mean in term of fertilizer rate? The component details of the three biostimulants should be provided. It appears that the GO analysis was performed for the samples taken at 24 h after application, if so, please indicate that the samples were collected at 24 h after application for Fig. 4. 

Finally, the conclusion should be more accurately. For example, you mentioned that the biostimulants modulated several processes including hormones (page 7 line 367-370), however, this is true for corn (Fig. 4A), but not for soybean (Fig. 4C). The authors are encouraged to re-write the abstract and conclusion to state the findings accurately. In addition, it would be helpful to expand the discussion on your findings with more citations in the Results and discussion section.

Author Response

Dear reviewer,

thank you very much for your appreciation of the manuscript and comments/opinion. Here below our answer to your points:

1) First, the abstract should be revised to present the results/findings concisely.

In the new version of the manuscript we have revised the abstract (see yellow part) according to your suggestion. We included detailed information about the approach followed, parameters measured, and results obtained using different application rates on different crops (corn and soybean).

 

2) Second, the objective(s) should be clearly stated at the end of the introduction section (line 95). 

In the new version of the manuscript we have stated the objective of this study in the Introduction section (see yellow part), following your suggestion.

 

3) Third, for M&M part, the plants were grown under natural light, how did you maintain the light uniformity across the area the plants were located. In other words, did you measure/verify the light intensity for each plant?

We did not measure the light intensity for each plant. However, as described in the manuscript, we ensured homogeneous assessment through: "The experimental setup was composed by 5 biological replicates (plants) for each experimental condition using a completely randomized experimental design".

The environmental factors (light intensity, photoperiod, temperature, relative humidity) should be provided. 

We have included the Supplemental Table 1 where we reported the following parameters acquired: T°C, RH, Radiation.

Also, please explain what 20 units of nitrogen mean in term of fertilizer rate? 

We applied 50 kg/ha of ammonium sulphate (21-0-0), 100 kg/ha of potassium nitrate (13-0-46), and 200 kg/ha of single superphosphate (0-20-0).

 

The component details of the three biostimulants should be provided.

As reported in several scientific papers, including our previous ones, "Plant biostimulants formulations are generally proprietary compositions based on seaweed extracts, complex organic materials, plant hormone-like compounds, amino-acids, and humic acids". Also in this case, being produced from a Company (Valagro), the composition remains proprietary with a trade secret. However, as you can see we have specified inside the document that: "Three different biostimulant formulations based on different plant extracts (coded as 52096, 52097, 52113; proprietary composition of Valagro SpA)….."

It appears that the GO analysis was performed for the samples taken at 24 h after application, if so, please indicate that the samples were collected at 24 h after application for Fig. 4. Ok, we have specified this for Fig. 4.

4) Finally, the conclusion should be more accurately. For example, you mentioned that the biostimulants modulated several processes including hormones (page 7 line 367-370), however, this is true for corn (Fig. 4A), but not for soybean (Fig. 4C). The authors are encouraged to re-write the abstract and conclusion to state the findings accurately. In addition, it would be helpful to expand the discussion on your findings with more citations in the Results and discussion section.

We have detailed the conclusions according to your suggestion.

 

 

Author Response File: Author Response.docx

Reviewer 2 Report

Minor corrections

1/ A conclusion and some perspectives are missing

2/ Some data are missing

Result of the 2 other different compounds on corn and rice I don’t have access to the supplementary data of line 260

3/ I would like to have comment on the following points:

-- 3.1--How do you explain the different responses of the same compounds?

                            On corn and soybean

At low dose and higher dose

              --3.2—Is the plant yield affected by PBS at low and high dose for any of the 3 PBS tested?

              --3.3—is the seed content affected by the treatment (amidon sugar, nitrogen) and more globaly does the PBS treatment affect the nutrient values of the seeds.

4/ I would like to have comment on the following point.

--4a-- How do you explain the different responses on corn and soybean from the 3 PBS tested?

--4b-- How do you think this will help in choosing new PBS on these crops?

5/ qPCR data should be improved by following the MIQE rules such as:

              --a-- The use of more than one reference genes

              --b-- The use of primer efficiency in the calculation of differential expression for each genes of interest and

              --c-- The use of standard deviation on the graphic

              --d-- A histogram should be used and not linked points because the samples are not extracted from the same plants

              --e-- How the ratio were calculated: deltaCt or other more appropriate formulas

              --f-- Statistical test should be add and you should explain which one you choose

5/ RNAseq analysis

What is the  program using to obtain DGE (limma , EdgeR , others)?

 

6/ Some data are missing: Lines 216-217; line 248-249, line 260 no access giving to this suppl data

7/ a point is missing line 245 after trail

8/Table 4: please rewrite the GIO as we cannot read them completely in this tavle

9/ line 293 what is SPX standing for?

 

Author Response

Dear reviewer,

thank you very much for your appreciation of the manuscript and comments/opinion. Here below our answer to your points:

1) 1/ A conclusion and some perspectives are missing.

Thank you. We have detailed the conclusions and added some more perspectives. See new parts in yellow.

2) 2/ Some data are missing. Result of the 2 other different compounds on corn and rice I don’t have access to the supplementary data of line 260.

Thanks. In Figure 3 we included the results of all prototypes on soybean. We did not test rice. We have included supplementary data as "Supplemental Table 3" (see text).

3) 3/I would like to have comment on the following points: -- 3.1--How do you explain the different responses of the same compounds? On corn and soybean

In our experience, biostimulant compounds can exert different effects according to the species under test. In this case, although we consider corn and soybean belonging both to the "row crop" category, we have to specify that these crops are very different in terms of physiological mechanisms. Corn is a monocot graminaceous species, very different from soybean, that is leguminous and dicot species. However, in this paper we report some common mechanisms by which specific biostimulant play an effect: increase in digital biovolume, green index, some genes in common, etc.

4) 4/ I would like to have comment on the following point. --4a-- How do you explain the different responses on corn and soybean from the 3 PBS tested? --4b-- How do you think this will help in choosing new PBS on these crops?

The composition of the 3  formulations under test was different. Beside this, as I reported above, although we consider corn and soybean belonging both to the "row crop" category, we have to specify that these crops are very different in terms of physiological mechanisms. In addition, biostimulant activity is known to can be variable according to the conditions under test, including the phenological phases and crops.

5) 5/ qPCR data should be improved by following the MIQE rules such as: --a-- The use of more than one reference genes  --b-- The use of primer efficiency in the calculation of differential expression for each genes of interest and --c-- The use of standard deviation on the graphic --d-- A histogram should be used and not linked points because the samples are not extracted from the same plants --e-- How the ratio were calculated: deltaCt or other more appropriate formulas --f-- Statistical test should be add and you should explain which one you choose

a-c For us it is not possible to satisfy these points

d- We have changed the PCR graphs into bar graphs (instead of lines). 

e- we have specified this inside the text ("Relative expression levels are in ddCt").

6) 5/ RNAseq analysis. What is the  program using to obtain DGE (limma , EdgeR , others)?

The DGE was basically also done using CLC Bio, so we adjusted this.

 

6/ Some data are missing: Lines 216-217; line 248-249, line 260 no access giving to this suppl data

Ok, we included all these suppl data. as suggested.

7/ a point is missing line 245 after trail.

Ok, fixed.

8/Table 4: please rewrite the GIO as we cannot read them completely in this tavle

Done.

9/ line 293 what is SPX standing for?

The SPX domain is named after SYG1/Pho81/XPR1 proteins. We added this into the text.

Author Response File: Author Response.docx

Reviewer 3 Report

major concerns:

The title does not align well with the content. There is no research showing development of the biostimulants. One suggestion of the new title would be “Investigating the impact of biostimulants for soybean and maize using automatic phenotyping platform and next generation sequencing”. In the Materials and Methods, the authors should describe chemical nature of the biostimulants to some degree. Enough information of the product is essential for reader to understand the meaning of the phenotypic and gene expression data. About tissue collection for gene expression analysis, it was not written very clearly. Accord to the current writing, my understanding is that there were only 3 plants were used to collect the leaf tissue for the three biological samples (for each treatment at a time point). If this is true, this number is very low. It is not clear if the samples for RNA-seq and qPCR were collected in the same way. It’s not clear how many RNA samples for each of the 4 treatments (UTC and treated soybean, UTC and treated maize), how many RNA samples (biological repeats) were sequenced. This need to be clarified in both the Method and Results. In the Method, when the authors described construction of the sequencing library, confusing information was written in Line 160-163. “DNA adapters including sample-specific barcodes were ligated and PCR amplification was performed” describes already the construction of the library, while “ Libraries were subsequently made using the Illumina mRNA-Seq Sample Preparation Kit according to the manufacturer’s instructions” is also the library construction. You don’t make library from a library. I suggest the authors delete the first sentence. Line 239-240, the authors claimed that none of the biostimulants made “statistically significant” impact on GGA on corn plant. However, figure 2D shows that GGA of plants treated by 52096 and 52097 are significantly different from UTC at DAT7, 10 and 13, according to the letter-bases statistic labelling. Can’t see any supplementary data mentioned by the manuscript. Also gene list, fold change and p-value of all the genes with more that 2-fold change should be included in the supplementary file. Figure 1, data points are not aligned with the DAT on the x axial. Not clear if the measurements were done on 5, 10, 15, 20 and 25 DAT. It was not mentioned anywhere the DAT of these measurement. I suggest the authors to clearly mention measurement DAT in the figure legend. Figure 5. No error bars. Standard error of data from the biological repeats need to be set as the error bars. Are the data means of the biological repeats? how many biological repeats for each data shown here? The authors did only one time point in RNA-seq. It is understandable due to cost control. The presented RT-qPCR data at 4 time point. However, I think the number of genes tested by RT-qPCR is not adequate. The phenotypes shown in the manuscript was taken many days after treatment but the latest time point of RT-qPCR was only 48 hours after treatment. The authors mentioned that tissues were collected one week after application. I suggest the authors perform RT-qPCR of more gene and include the time DAT7 time point. Discussion of the current RT-qPCR data is not deep. The authors need to propose their understanding of the functionality meaning of the expression pattern of the genes.

 

The minor comments about writing of the manuscript are below.

Line 183, 184, 353, replace Q-PCR with qRT-PCR. Line 245, change “all treatments under trail” to “UTC. Treating plants with 52096 or 52113 did not affect the stress index.” Line 246, delete “(2.5 ml/L)”. Line 255, change “reference genome” to “reference transcriptome”. Line 290, change “yield” to “yield formation”. Line 291, delete “genes”. Missing citation for the sentence, line 301-304 “In comparison ……and ABA.”. Missing citation for the sentence, line 308-309 “PMTs are……and galactose.”. Line 306 to 308, delete the sentence “Again, the identified …… after treatment”. It appears too early in the text.

Author Response

Dear reviewer,

Thank you very much for your input. Below in red you will find our answer to your points and highlighted in green/yellow the revision inside the document attached:

The title does not align well with the content. There is no research showing development of the biostimulants. One suggestion of the new title would be “Investigating the impact of biostimulants for soybean and maize using automatic phenotyping platform and next generation sequencing”. We agree with this comment, therefore we have changed the title that now is "Investigating the impact of biostimulants for row crops using high-efficiency phenotyping and Next Generation Sequencing". 

In the Materials and Methods, the authors should describe chemical nature of the biostimulants to some degree. Enough information of the product is essential for reader to understand the meaning of the phenotypic and gene expression data. As reported in several scientific papers, including our previous ones, "Plant biostimulants formulations are generally proprietary compositions based on seaweed extracts, complex organic materials, plant hormone-like compounds, amino-acids, and humic acids". Also in this case, being produced from a Company (Valagro), the composition remains proprietary with a trade secret. However, as you can see we have specified inside the document that: "Three different biostimulant formulations based on different plant extracts (coded as 52096, 52097, 52113; proprietary composition of Valagro SpA)….."

About tissue collection for gene expression analysis, it was not written very clearly. Accord to the current writing, my understanding is that there were only 3 plants were used to collect the leaf tissue for the three biological samples (for each treatment at a time point). If this is true, this number is very low. Ok, we have specified this better in the text as follows: "For each experimental condition (treatment and time-point), three biological replicates were collected from different plants at the third fully expanded leaf stage. Each biological replicate consisted of three entire leaves (central position) collected from three individual plants and pooled". We understand that the number can be considered low from your perspective; we did this choice for cost control reasons.

It is not clear if the samples for RNA-seq and qPCR were collected in the same way. It’s not clear how many RNA samples for each of the 4 treatments (UTC and treated soybean, UTC and treated maize), how many RNA samples (biological repeats) were sequenced. This need to be clarified in both the Method and Results. Yes, they were collected in the same way. We have specified this inside the manuscript. "For Next Generation Sequencing and qRT-PCR analysis, samples were collected just before (t=0) and..."We sequenced a single RNA sample for each experimental condition (UTC, treated soybean, treated maize). This has been specified in the text.

In the Method, when the authors described construction of the sequencing library, confusing information was written in Line 160-163. “DNA adapters including sample-specific barcodes were ligated and PCR amplification was performed” describes already the construction of the library, while “ Libraries were subsequently made using the Illumina mRNA-Seq Sample Preparation Kit according to the manufacturer’s instructions” is also the library construction. You don’t make library from a library. I suggest the authors delete the first sentence. Ok, this sentence has been deleted, thanks.

Line 239-240, the authors claimed that none of the biostimulants made “statistically significant” impact on GGA on corn plant. However, figure 2D shows that GGA of plants treated by 52096 and 52097 are significantly different from UTC at DAT7, 10 and 13, according to the letter-bases statistic labelling. Thank you, we have corrected this in the text.

Can’t see any supplementary data mentioned by the manuscript. Also gene list, fold change and p-value of all the genes with more that 2-fold change should be included in the supplementary file. Now we have a new version with all Supplemental data. I hope they can be included in this submission form, otherwise I would ask the editor to send them to you for revision.

Figure 1, data points are not aligned with the DAT on the x axial. Not clear if the measurements were done on 5, 10, 15, 20 and 25 DAT. It was not mentioned anywhere the DAT of these measurement. I suggest the authors to clearly mention measurement DAT in the figure legend. Ok, we have replaced Figures 1 and 3 with versions that specify on the x axis only the days of the measurements.

Figure 5. No error bars. Standard error of data from the biological repeats need to be set as the error bars. Are the data means of the biological repeats? how many biological repeats for each data shown here? The authors did only one time point in RNA-seq. It is understandable due to cost control. The presented RT-qPCR data at 4 time point. However, I think the number of genes tested by RT-qPCR is not adequate. The phenotypes shown in the manuscript was taken many days after treatment but the latest time point of RT-qPCR was only 48 hours after treatment. The authors mentioned that tissues were collected one week after application. I suggest the authors perform RT-qPCR of more gene and include the time DAT7 time point. Discussion of the current RT-qPCR data is not deep. The authors need to propose their understanding of the functionality meaning of the expression pattern of the genes. As correctly mentioned by the reviewer, due to cost control we did not perform biological repeats/replicates for each NGS and PCR data shown. This explains also the lack of error bars. Unfortunately we cannot do any additional PCR as all the samples are gone (we had also changes in the job/affiliation of some authors). However, we added the 1 week (168 hours) qRT-PCR data like the reviewer requests.

 

The minor comments about writing of the manuscript are below.

Line 183, 184, 353, replace Q-PCR with qRT-PCR. Line 245, change “all treatments under trail” to “UTC. Treating plants with 52096 or 52113 did not affect the stress index.” Line 246, delete “(2.5 ml/L)”. Line 255, change “reference genome” to “reference transcriptome”. Line 290, change “yield” to “yield formation”. Line 291, delete “genes”. Missing citation for the sentence, line 301-304 “In comparison ……and ABA.”. Missing citation for the sentence, line 308-309 “PMTs are……and galactose.”. Line 306 to 308, delete the sentence “Again, the identified …… after treatment”. It appears too early in the text.

Thank, we have corrected the manuscript accordingly.

Author Response File: Author Response.docx