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Peer-Review Record

Scanner-Based Minirhizotrons Help to Highlight Relations between Deep Roots and Yield in Various Wheat Cultivars under Combined Water and Nitrogen Deficit Conditions

Reviewer 1: Anonymous
Reviewer 2: Anonymous
Reviewer 3: Anonymous
Agronomy 2019, 9(6), 297; https://doi.org/10.3390/agronomy9060297
Received: 17 April 2019 / Revised: 20 May 2019 / Accepted: 4 June 2019 / Published: 7 June 2019
(This article belongs to the Special Issue Root-Soil Interactions)

Round  1

Reviewer 1 Report

Postic et al here present an interesting study assessing the use of minirhizotrons for field root phenotypic and present some quite timely data (in terms of other literature coming out at the moment showing similar results) highlighting the correlation between root surface measurements and yield under specific conditions.

Overall, I would say the paper is very well written and the methodologies used are sound, which has left me with very few revisions to suggest.

My only real comment is that in the methodology, the timing of rhizotron tube insertion into the field is not mentioned unless I missed it. Other studies have highlighted issues with soil contact in the months following tube insertion, which can have a significant effect on measurements. In some cases, tubes are left in the field on a semi permanent basis to allow the soil to settle back on the tube allowing proper contact. If this was not done, a couple of sentences discussing the issue of soil contact would be appreciated.

Author Response

Note: We uploaded a MS Word file containing this reply to reviewer 1.

========================================================

Reviewer 1

Postic et al here present an interesting study assessing the use of minirhizotrons for field root phenotypic and present some quite timely data (in terms of other literature coming out at the moment showing similar results) highlighting the correlation between root surface measurements and yield under specific conditions.

Overall, I would say the paper is very well written and the methodologies used are sound, which has left me with very few revisions to suggest.

My only real comment is that in the methodology, the timing of rhizotron tube insertion into the field is not mentioned unless I missed it. Other studies have highlighted issues with soil contact in the months following tube insertion, which can have a significant effect on measurements. In some cases, tubes are left in the field on a semi permanent basis to allow the soil to settle back on the tube allowing proper contact. If this was not done, a couple of sentences discussing the issue of soil contact would be appreciated.

Reply: We warmly thank the reviewer for his interest in the paper. Concerning his comment about tube-soil contact and time for soil to settle back, we clearly agree that it is an important issue in minirhizotron measurements and this issue was raised by the other reviewers.

The insertion of tubes the into soil was performed a couple of days after sowing, at emergence. This is indicated L193 in the manuscript. The time to anthesis and the last measurement with minirhizotron was, in our case, about 6.5 months which is the maximum time allowed for soil to settle back.  We agree as indicated in Johnson et al. (2001) but also  Hendricks et al. (2006) that a long time lag (6-8 months) may be necessary to recover the initial state of the bulk soil. Unfortunately, in contrast with perennial crops or forests, for annual crops like wheat, it is nearly impossible to let the minirhizotron tubes in place into the soil well in advance because of the agricultural operations (ploughing, sowing), especially in micro-plots dedicated to phenotyping as ours.  The same problem arises when installing access tubes for moisture probes for example.

To minimize soil disturbance during installation, we used a hand corer with same size of the tube, maintained with a coring guide on a metallic frame at 45° angle. Time of installation required 2 full days in our case. Manually coring prevents issues related to the off-centered effect of the usage of a mechanical rotary soil corer. Indeed, this one is known to reduce the contact between the soil and the tube because of a variable core diameter with the depth, commonly larger at the surface (Johnson et al., 2001). However, we found that even with the hand corer, the drilled hole was a little bit larger than the tube at shallow depth, notably at depth < 10 cm. At depth greater than ploughing depth, most of the time we could reach a good soil contact, but at some occasions a gravel could pose problems (or even prevent further coring).

We added some comments in the manuscript about this soil-tube contact issue in:

- Material and methods (L192-196) : “ Minirhizotron tubes were inserted into the soil of the central sowing line of each plot a couple of days after sowing, at plant emergence (Figure 1b and 1c). Tubes were installed at a 45° angle from horizontal. To minimize soil disturbance and to achieve a soil-tube contact as good as possible, soil drilling was performed using a 7 cm diameter auger operated slowly manually and affixed to a rigid self-made 45° drilling frame.”

-Discussion section (L444-453): “This bias may also come from poor contact between soil and minirhizotron tube. Indeed, time for soil to settle back on tube can be rather long (i.e. > 6 months  [44, 45]). In the case of annual crops, especially in micro-plots dedicated to phenotyping, it is nearly impossible to install the tubes well in advance because of agricultural operations (ploughing, sowing).  In our study, the last measurement occurred ca. 6.5 months after tube installation.  Despite the care given to adjust soil coring to tube diameter, the drilled hole was a little bit larger than the tube in the ploughed layer (~25 cm depth), notably at depth < 250px, probably participating to the measurement bias in the top soil. At a greater depth, the soil contact was better. The relationship established above between root mass from auger sampling and RLSD from scanner images also confirms that deep root scanner measurements were representative of the plot”

References:

Hendricks J.J.,  Hendrick R.L., Wilson C.A., Mitchell R.J., Pecot S.D., Guo D., 2006, Assessing the patterns and controls of fine root dynamics: an empirical test and methodological review, Journal of Ecology, 94, 40 – 57.

Johnson M.G., Tingey D.T., Phillips D.L., Storm M.J., 2001, Advancing fine root research with minirhizotrons, Environmental and Experimental Botany, 45, 263-289

Author Response File: Author Response.docx

Reviewer 2 Report

see attached file

Comments for author File: Comments.docx

Author Response

Note: We uploaded a MSWord file conating this reply to reviewer  2.

==========================================================

Reviewer 2

General comments

                The manuscript agronomy-497826 presents data on root characteristics of wheat and the rooting system response to varying water and nitrogen levels. The experiments to collect these data were properly designed and conducted and the results are within the scope of Agronomy. However, the manuscript needs corrections before being ready for publication. It has some problems such as mistakes in the grammar, long and unclear sentences, and problems with units. Please check all units in the manuscript there is often a period between two units like in kg.ha-1 (L172, L173, L199, L237, L244, L245, L246, L247, L290, L291[x2], etc) and I could not find the same convention in other articles published in Agronomy. In addition, the superscript format associated to some units is missing.

Reply:  We sincerely thank the reviewer for his careful review of the manuscript which helped to add clarity to the paper.  Concerning units, we express now all products of units with a space (eg kg ha-1) throughout the paper and the formatting of units shall be (hopefully) correct now.

Specific comments

Title: It would be more informative to replace multi-stress by the stresses that were considered in the study. Is it really “highlight” what minirhizotrons allowed in the study?

ð  Indeed, the title has to be more precise. We changed “multi-stress” by “combined water and nitrogen deficit”

ð  In our opinion,  “highlight” is correct here.

Keywords: wheat, root, minirhizotrons are already in the title so it is not necessary to repeat them in the keywords.

ð  I would let this decision to maintain or not these repetitive words to the editor. However, we propose to replace the key-word “root” by “root plasticity” L29

L13: Why it applies “particularly” to wheat? What makes it different from other field crops?

ð  This was because wheat is one of the most widely consumed cereal and, in relation to drought, access to soil water is related to deeper roots in relation with the timing phenology and the probability of water deficit occurrence. However, we clearly agree that the necessity for root phenotyping tools in the field is of a much broader interest for crops than just for wheat. We deleted that part of the sentence “and this particularly applies to wheat crops ».   (L13)

L28: The use of “consecutive” is not clear.

ð  We changed the sentence: “consecutive” changed to “a better prediction of grain yield” (L 28)

L29: remove one period at the end of the sentence.

ð  Done

L44: resources

ð  Done L43

L46: large areas

ð  Done L45

L49: what about the effects of indirect selection?

ð  This is a good point and we added a sentence and reference to show an effect of indirect selection for the root system and a reference to Subira et al., 2016 [6] :  “Breeding also resulted in an indirect selection of root traits. For example, in the case of wheat, the introduction of dwarfism genes in modern wheat varieties has been related to a reduction in the root biomass and root [6]”  L48-50

L56: add the name of the author before “showed”.

ð  Done L58

L70: Is heavy the right term here?

ð  We changed for “heavy” for “large” L72

L98-99: minirhizotrons-an? Check also the use of commas and of singular and plural.

ð  Changed to “,minirhizotron, an invasive but non-destructive technique,” L100

L113: The same idea is suggested again below (L117-119). Please, avoid repetition here and in the rest of the manuscript.

ð  We deleted old L113 and modified new L115 to “In the field of crop research, a rotary scanner…” L115-116

L127: The text “(Figure 1a)” should be placed after field experiments but not there because it doesn’t show anything about the texture.

ð  Yes, changed to “Field experiments (Figure 1a)… “ L127

L134: remove “crop” and it makes more sense to put sowing and harvest dates at the beginning of the section crop management than here (“Field site”). L128

ð  We agree and changed text accordingly. L164-166

L155: What was the rationale of using one durum wheat genotype with three bread wheat ones?

ð  The idea was to get some variation of the root system between related species and the durum wheat was reputed more tolerant to limiting conditions, which could be due in part to variations in root system. On an other hand, the synthetic wheat (Nogal cultivar) in this study is the result of new crossings between durum wheat and Aegilops tauschii (parents of bread wheat).  We added a sentence about this L157-160 :” The durum wheat was reputed more tolerant to limiting conditions, which could be due in part to variations in the root system compared to bread wheats. Nogal is a synthetic wheat derived from crossing between durum wheat and Aegilops tauschii Coss., parents of present bread wheat.”

L158-160: Was the crop before the experiments durum wheat or sunflower? If sunflower was the preceding crop of the preceding crop, it does not make a lot of sense to mention it. 

ð  The preceding crop was durum wheat. We deleted the reference to sunflower L159

L162: Replace late October by the sowing date.

ð  Done (cf above)

L172: Remove one period at the end of the sentence.

ð  OK.  Done but at L171.

L173: Does GY15 means that yield is expressed at 15% moisture content? Please add at what moisture percentage is grain yield expressed.

ð   Yes it is 15% moisture. L185 

L188: Remove PMMA, it is used only one time and never again?

ð  Yes, Done L191

L209: soil? Surface

ð  Yes, from soil surface (L214)

L247-248: Not clear. Was that done for the ratio or all root parameters?

ð  This part was rephrased as “). The total root mass was computed as the sum of the root biomass estimated from auger sampling (RDMa, in g m-2) and the minirhizotron root length measurements converted into root biomass (RDMm, in g m-2). To prevent overlapping of auger samples over the 0—40 cm depth with minirhizotrons that measure over the 0—100 cm depth, the root biomass estimated from minirhizotron measurements (RDMm) was restricted to depths deeper than 40 cm (Eq 3).” L250-255

L260: band? or interval

ð  Interval is right (L266)

L295-297: How to avoid confounding effects if all plots of one group are on shallow soils and the rest on deep ones. This issue should be tackled in the discussion, particularly related to the comparisons between I+N- with other combinations of N and I.

ð  In fact, to avoid these confusing effects of shallow soil, the I+N- treatment was not considered in the manuscript when considering the analysis of the scanner measurements, rooting profiles and yield, which is the core of this article.

L303: t is missing from t ha-1.

ð  Done L309

L323: remove (water, nitrogen) or explain it better.

ð  It has been removed.

363-364: This is not clear. Were not the four cultivars studied in all combinations of irrigation and N level? If it is not the case how were the means of TRLSD, rooting depth, etc in table 3 and other tables calculated?

ð  The 4 cultivars were subjected to the four combinations of Irrigation and N levels.  However, given the large amount of labour involved in monitoring roots through time, minirhizotron root measurements were performed on the 4 cultivars for the I-N- and I+N+ treatments. Minirhizotron measurements for I+N- and I-N+ treatments were restricted to durum wheat (miradoux) and one bread wheat cultivar (apache). This restriction concerns only root minirhizotron related measurements and not above ground measurements. Analyses shown table 3, giving a general overview of some measurements, is done with all cultivars mixed and do not include a genotype effect and the number of samples (n) varies according to the variable analyzed (ie above or below ground). In table 1 there was a (last) sampling of roots by augering in all treatments and cultivars to estimate R:S ratio.

L436-437: Also consider lack of enough soil-minirhizotron contact. The longer are the minirhizotrons installed in the soil, the more reliable are the results (M.G. Johnson, D.T. Tingey, D.L. Phillips, M.J. Storm, Advancing fine root research with minirhizotrons, Environmental and Experimental Botany, Volume 45, Issue 3, 2001, Pages 263-289). It would be useful to add in materials and methods when you installed the minirhizotrons.

ð  This indeed an important issue, raised by the reviewers. The insertion of tubes the into soil was performed a couple of days after sowing, at emergence. This is indicated L191-192 in the manuscript. The time to anthesis and the last measurement with minirhizotron was, in our case, about 6.5 months which is the maximum time allowed for soil to settle back.

ð   We agree as indicated in Johnson et al. (2001) but also  Hendricks et al. (2006, Assessing the patterns and controls of fine root dynamics: an empirical test and methodological review, Journal of Ecology, 94, 40 – 57) that a long time lag (6-8 months) may be necessary to recover the initial state of the bulk soil. Unfortunately, in contrast with perennial crops or forests, for annual crops like wheat, it is nearly impossible to let the minirhizotron tubes in place into the soil well in advance because of the agricultural operations (ploughing, sowing), especially in micro-plots dedicated to phenotyping as ours.  The same problem arises when installing access tubes for moisture probes for example.

ð  To minimize soil disturbance during installation, we used a hand corer with same size of the tube, maintained with a coring guide on a metallic frame at 45° angle. Time of installation required 2 full days in our case. Manually coring prevents issues related to the off-centered effect of the usage of a mechanical rotary soil corer. Indeed, this one is known to reduce the contact between the soil and the tube because of a variable core diameter with the depth, commonly larger at the surface (Johnson et al., 2001). However, we found that even with the hand corer, the drilled hole was a little bit larger than the tube at shallow depth, notably at depth < 10 cm. At depth greater than ploughing depth, most of the time we could reach a good soil contact, but at some occasions a gravel could pose problems (or even prevent further coring).

We added some comments in the manuscript about this soil-tube contact in:

ð  - Material and methods (L192-196) : “ Minirhizotron tubes were inserted into the soil of the central sowing line of each plot a couple of days after sowing, at plant emergence (Figure 1b and 1c). Tubes were installed at a 45° angle from horizontal. To minimize soil disturbance and to achieve a soil-tube contact as good as possible, soil drilling was performed using a 7 cm diameter auger operated slowly manually and affixed to a rigid self-made 45° drilling frame.”

ð  -Discussion section (L444-453): “This bias may also come from poor contact between soil and minirhizotron tube. Indeed, time for soil to settle back on tube can be rather long (i.e. > 6 months  [44, 45]). In the case of annual crops, especially in micro-plots dedicated to phenotyping, it is nearly impossible to install the tubes well in advance because of agricultural operations (ploughing, sowing).  In our study, the last measurement occurred ca. 6.5 months after tube installation.  Despite the care given to adjust soil coring to tube diameter, the drilled hole was a little bit larger than the tube in the ploughed layer (~25 cm depth), notably at depth < 250px, probably participating to the measurement bias in the top soil. At a greater depth, the soil contact was better. The relationship established above between root mass from auger sampling and RLSD from scanner images also confirms that deep root scanner measurements were representative of the plot”

L460-462: Not clear, there is a contradiction with statement in L295-297.   

ð  In the initial manuscript, L295-297 refer to plots in shallow soil conditions which are related to the I+N- treatment while L460-462 refer to two other different treatments : I-N+ and I-N-. We don’t understand what is the contradiction in that case.

Author Response File: Author Response.docx

Reviewer 3 Report

This paper is well written and the story is interesting.

I only have a few questions. Light leakage, difference in temperature, and not-well contact between the soil in the shallow layers and the tubes could lead to underestimation of root distribution in the shallow soil. This is the limitation of the technique. But for the root distribution in the deeper soil, how much do we believe the data that was observed by the scanner-based minirhizotron? The authors took augered-samples. Did they compare the differences of the roots that were measured by the auger sampling and by the scanner-based minirhizotron? The authors had a larger view of the roots along the tubes. Do they observe if the roots grew downward along the tubes? If so, this could result in overestimation of roots in the deeper soils.

Here are some other detailed comments:

Line 75: “GxE” should be first defined in line 74 even it is known to some readers.

Line 103: change “… can be now be …”

Line 127: Loam – loam

Line 156: 4 – four

Line 188: the order of the figures should follow a, b, c, d.

Line 197 and line 237 and line 240: delete the citation after [ ] since they were repeated.

Line 280: a brief description of the APSIM model is needed for the readers.

Line 299: display – displays

Line 358: show – shows

Line 452 and 453: on – in

Line 494: root length density – RLD. Please also check other terms and the corresponding abbreviations.

Line 502:  tough - though

Author Response

Note: We uploaded a MS Word file of this reply to reviewer 3

=====================================================

Reviewer 3

This paper is well written and the story is interesting.

I only have a few questions. Light leakage, difference in temperature, and not-well contact between the soil in the shallow layers and the tubes could lead to underestimation of root distribution in the shallow soil. This is the limitation of the technique. But for the root distribution in the deeper soil, how much do we believe the data that was observed by the scanner-based minirhizotron? The authors took augered-samples. Did they compare the differences of the roots that were measured by the auger sampling and by the scanner-based minirhizotron? The authors had a larger view of the roots along the tubes. Do they observe if the roots grew downward along the tubes? If so, this could result in overestimation of roots in the deeper soils.

Reply: The authors thank the reviewer for his constructive review of our manuscript. We share the concern about the disturbance induced by minirhizotron tubes installed into the soil, in particular the soil-tube contact, an issue raised by the other reviewers too.

In relation with soil-tube contact, a long time lag (6-8 months) may be necessary to recover the initial state of the bulk soil after tube installation. Unfortunately, in contrast with perennial crops or forests, for annual crops like wheat, it is nearly impossible to let the minirhizotron tubes in place into the soil well in advance because of the agricultural operations (ploughing, sowing), especially in micro-plots dedicated to phenotyping as ours.  The same problem arises when installing access tubes for moisture probes for example. In our study, the time between tube installation and anthesis, the last measurement with minirhizotron, was in our case, about 6.5 months.

To minimize soil disturbance during installation, we used a hand corer with same size of the tube, maintained with a coring guide on a rigid metallic frame at 45° angle. Time of installation required 2 full days in our case. Manually coring prevents issues related to the off-centered effect of the usage of a mechanical rotary soil corer. Indeed, this one is known to reduce the contact between the soil and the tube because of a variable core diameter with the depth, commonly larger at the surface. However, we found that even with the hand corer, the drilled hole was a little bit larger than the tube at shallow depth, notably at depth < 10 cm. At depth greater than ploughing depth, most of the time we could reach a good soil contact, but at some occasions a gravel could pose problems (or even prevent further coring).

As indicated by the reviewer, we also sampled roots with auger (as indicated in the manuscript) at different dates and we used these data to elaborate a relationship to estimate volumetric root density from minirhizotron images. The coherence of the obtained relationship (fig. 4 and fig. S2) let us think that the root measurements from minirhizotrons are not artifacts. As another element pointed by the reviewer, on minirhizotron images, if some of the roots grew downwards, roots growing laterally are also present.

We added some comments in the manuscript about soil-tube contact in:

- Material and methods (L192-196) : “ Minirhizotron tubes were inserted into the soil of the central sowing line of each plot a couple of days after sowing, at plant emergence (Figure 1b and 1c). Tubes were installed at a 45° angle from horizontal. To minimize soil disturbance and to achieve a soil-tube contact as good as possible, soil drilling was performed using a 7 cm diameter auger operated slowly manually and affixed to a rigid self-made 45° drilling frame.”

-Discussion section (L444-453): “This bias may also come from poor contact between soil and minirhizotron tube. Indeed, time for soil to settle back on tube can be rather long (i.e. > 6 months  [44, 45]). In the case of annual crops, especially in micro-plots dedicated to phenotyping, it is nearly impossible to install the tubes well in advance because of agricultural operations (ploughing, sowing).  In our study, the last measurement occurred ca. 6.5 months after tube installation.  Despite the care given to adjust soil coring to tube diameter, the drilled hole was a little bit larger than the tube in the ploughed layer (~25 cm depth), notably at depth < 250px, probably participating to the measurement bias in the top soil. At a greater depth, the soil contact was better. The relationship established above between root mass from auger sampling and RLSD from scanner images also confirms that deep root scanner measurements were representative of the plot”

Here are some other detailed comments:

Line 75: “GxE” should be first defined in line 74 even it is known to some readers.

ð  Done L77

Line 103: change “… can be now be …”

ð  Done L105

Line 127: Loam – loam

ð  Changed at L128

Line 156: 4 – four

ð  Changed at L155

Line 188: the order of the figures should follow a, b, c, d.

ð  Figure 1d deleted at this place and we added “(Figure 1b and 1c)” L194, to illustrate the rhizotron installation.

Line 197 and line 237 and line 240: delete the citation after [ ] since they were repeated.

ð  Yes, done.

Line 280: a brief description of the APSIM model is needed for the readers.

ð  We added the sentence “…used in APSIM, a crop and agricultural system model [34], and…” as explanation of APSIM L285-286

Line 299: display – displays

ð  Done L306

Line 358: show – shows

ð  Done L365

Line 452 and 453: on – in

ð  Changed at  L466, 467

Line 494: root length density – RLD. Please also check other terms and the corresponding abbreviations.

ð  RLD is a typo error, we changed to RLSD. L473

Line 502:  tough – though

ð  Done, L516

Author Response File: Author Response.docx

Round  2

Reviewer 2 Report

Thanks for corrections. I think that now is ready to be published.

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