Effects of Drought Stress on Some Agronomic and Morpho-Physiological Traits in Durum Wheat Genotypes

Round 1

Reviewer 1 Report

In my opinion the article is very interesting and the experimental is well conducted. The article might be of technical and practical interest to many readers of this journal (mainly farmer and researcher involved in agronomy field). For these reasons, I believe that this paper should be accepted for publication, but only after Minor Revisions.

Here the list of the corrections that need to be made:

Abstract

Line 35: replace of with Among

Introduction:

Line 42: replace is well growing with is widely cultivated

Line 43: replace important with essential

Line 62: in my opinion this introduction is too short and not face the potential benefits of the results of this study on the successive steps of the wheat production chain (i.e. milling of wheat + flour and semolina processing for bread and pasta productions). I suggest you to add here (after [8].) 3-4 sentences regarding the benefit of the correct management of drought stress and of several agronomic and morpho-physiological traits (and add new references).

I can imagine something like this:

“Despite intensive durum wheat breeding programs in XX century, the in depth comprehension of the agronomic and morpho-physiological responses of wheat to drought stress might be useful to improve the successive operations of wheat processing and transformation. In particular, the selection of cultivars resistant to drought stress and the adoption of correct agronomical treatments [9], might prevent problems and complications during the essentials operations of wheat milling and flour processing [10]. Moreover, the development, selection, and cultivation of wheat durum cultivars resistant to drought stress which do not needs significant input of fertilizers might reduce the carbon and water footprints, reducing the environmental impacts [11].”

Please replace to the old [9] – [10] and [11] in the references list with the papers below and shift old references of three positions

[9] Guerrini, L., Napoli, M., Mancini, M., Masella, P., Cappelli, A., Parenti, A., & Orlandini, S. (2020). Wheat grain composition, dough rheology and bread quality as affected by nitrogen and sulfur fertilization and seeding density. Agronomy, 10(2), 233.

[10] Cappelli, A., Oliva, N., & Cini, E. (2020). Stone milling versus roller milling: A systematic review of the effects on wheat flour quality, dough rheology, and bread characteristics. Trends in Food Science & Technology, 97, 147-155.

[11] Recchia, L., Cappelli, A., Cini, E., Garbati Pegna, F., & Boncinelli, P. (2019). Environmental sustainability of pasta production chains: An integrated approach for comparing local and global chains. Resources, 8(1), 56.

Line 63: please rephrase : Despite wheat undergo intensive selection and breeding,

Materials and methods:

Line 76: please remove the double space.

Line 81: replace replications with replicate

Line 86: were taken between 11:00 and 14:00, and between 12:00 and14:00, using..

Line 93: replace recorded with evaluated.

Line 106: the average ranks sum…

Results :

Line 111: I suggest you to remove range of data: 3.1 Analysis of variance and means comparison (is enough).

Line 129: lower

Line 149: and from 12.69 to….

Line 157: and from 297.50 to….

Line 160: and from 19.50  to….

Line 163: remove comma after condition.

Line 176: from 25.79 to 58.19….

Line 180: among the measured traits,

line 187: the measured traits .

Discussion:

Line 296: Among the various environmental stresses, drought is the major detrimental factor for crops growth and productivity.

Line 301 : yield and its ? please check the sentence

Line 313–314: The significant reduction in grain yield due to drought stress has also been shown in previous studies.

Line 323: would be nice if you report also the temperature of the control condition here.

Line 324– 326: Increasing leaf temperature limits the activity of photosynthetic enzymes (such as Rubisco), leading to a reduction of carbon dioxide fixation [21].

Line 349: put a comma after morphological.

Line 384: the most tolerant to drought stress,

Line 393: please here specify (in a couple sentences) the reasons behind the higher resistance to drought stress of G1, G9 and G12.

Conclusions:

like I said for the introduction, the conclusion section is too short. Moreover, usually, this section should not summarize results. Instead, in this section must be reported considerations regarding the study and the future prospects of the research. In conclusion, I suggest you to add 4-5 sentences regarding your considerations, potential applications of your results and the future prospect of the research.

Author Response

Dear Reviewer,

Thank you very much for reviewing our manuscript and input valuable comments and suggestions. We have carefully addressed all your comments and suggestions. All the changes made in the revised manuscript in response to your comments were highlighted in red.

We hope that our revised manuscript is now acceptable for publication in the Sustainability

Reviewer 2 Report

This research entitled ‘Effects of drought stress on some agronomic and morpho-physiological traits in durum wheat genotypes’ focuses on the two-season evaluation of 17 durum wheat breeding lines for 4 physiological and 13 agro-morphological characteristics under irrigated and drought conditions in Iran. Phenotypic evaluation showed that drought has negative effects on yield and yield-related traits. Correlation analysis identified phenological traits that significantly affect yield under drought stress. Among the 17 genotypes of durum wheat tested, G12 was identified to be a promising genotype that can be cultivated under drought conditions based on stress indices.

With the continuing agro-environmental decline brought about by climate change, identification of germplasm that can provide crops with adaptability to a multitude of stresses is becoming more and more important. While the manuscript will be of broad interest to readers, there are aspects of the paper that needs to revised, corrected and/or clarified before it can be published in Sustainability.

1. Page 1, line 30-33. Please paraphrase this. The yield-based stress indices can indicate correlation among the traits. There is no need for the phrase ‘The result of the’.
2. Page 1, line 35-37. The authors screened different breeding lines for drought stress and identified G12 as a promising genotype. In the abstract, as well as in the results and conclusion G12 was already being recommended for cultivation. If this is a breeding line, it will require multiyear and multilocation evaluation for drought stress before it can be released for cultivation.
3. Table 2 is supposed to show the pedigree of the breeding lines but this information is provided only for G5 and G5 and not for G6 to G17. The variety names are also not ‘pedigree’. The authors might want to consider revising the table to reflect the relevant information required for the breeding lines.
4. Page 3, line 93: The statement about collection of agronomic traits at ‘maturity’ is confusing here. Days to heading is normally taken when 50% of the plants are flowering and not when the plants are at harvest maturity. The same is true for grain filling period. The term ‘period’ indicate a span of time and therefore cannot just be competed at maturity.
5. Please provide units for the agro-morpho-physiological values that were measured in the tables and/or methodology. A little bit more detail as to how the measurements were taken will also benefit readers who are considering taking the same measurements in their materials (ex. biomass, harvest index, etc.).
6. It makes more sense to present the results on the mean values for the agronomic traits should be presented first (Table 3) before the analyzed data (Table 2) in the results and discussion.
7. The presentation of the results of correlation analysis, while aesthetically pleasing, does not add value to the manuscript but instead confuses the readers. The meaning of the colors, color intensity and size of the circles were not fully explained in Fig 1 nor in the discussion. The same is true for Fig 2 and 3. Figures are supposed to aid the readers in visualizing results and interpretations. The figures in this manuscript do not do that. The figure(s) should be revised or converted to table(s) if necessary.
8. The manuscript should be checked for readability and syntax.

Author Response

Dear Reviewer,

Thank you very much for reviewing our manuscript entitled and input valuable comments and suggestions. We have carefully addressed all your comments and suggestions. All the changes made in the revised manuscript in response to your comments were highlighted in green.

We hope that our revised manuscript is now acceptable for publication in the Sustainability

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Manuscript entitled ‘Effects of Drought Stress on Some Agronomic and Morpho-physiological Genotypes’ represents observations of durum wheat phenotypes under rainfed climate. 

In the face of changing climate and drying of the environment researchers pay more and more attention to uncover mechanisms underlaying plants response to water shortage enabling crops improvement. Secondly, wheat (including durum wheat) is an economically important species all over the world. Therefore, the topic of proposed study falls into the current scientific trends. However, it represents a simply phenotyping experiment and the research methodology is very basic with, so much the worse, serious failures in methods. I feel also that the environment effect was omitted, which is unacceptable considering field experiments. There is no scientific hypothesis, and objectives are poor. I mean that evaluation of phenotypic traits cannot be the aim itself, it should be a task leading to the bigger aim (e.g. unraveling mechanisms determining plant phenotypes under drought). Also, I don’t sympathize with the second goal (lines 71-73): ‘identify effective traits to facilitate a screening process in future durum wheat breeding programs that will lead to improved yield performance under rainfed conditions’ – I think that facilitation of genotypes screening can be rather achieved through MAS, not by long and time-consuming experimentation, in fact during the whole vegetation season.

Honestly, I like phenotyping-based study, because the phenotype is the final result of many processes at various levels in the plant, determined by the genome, environment, their relationships, and the final yield is economically important. I’m aware that phenotyping experiments are lobour-intensive and time-consuming, I truly appreciate the effort of the authors in their research. Unfortunately, studies are incomplete and insufficient in my opinion. As I mentioned, only conventional phenotypic approach is presented, without any reference to genetic determinants of plants behavior under drought. Effects of stress on yield and its components are key element in examination of plants, of course, but from the scientific point of view unraveling plants’ defense mechanisms against constrained conditions seems to be principal (this is missing in presented work). Authors are aware of importance of adaptive mechanisms and defense systems like osmotic adjustment (lines 50-53), also they understand the need of deciphering the ability of crops to respond and adapt to drought-prone environments (lines 56-58). But...do their studies/results bring us closer to a better understanding of these processes? In my opinion definitely not, there is no attempt to unravel the mechanisms which can determine better yielding under drought. Different plant strategies are known to cope with the limited water supply: escape, avoidance, tolerance. Which strategies dominate in genotypes in presented studies? Authors used terms ‘tolerant genotype’ – from the physiological point, such naming can make confusions and suggest the wrong strategy. Authors didn’t consider the mechanisms of plants resistance (unfortunately), so more suitable will be using the term: better/worse yielding genotype.

I believe that traditional phenotyping result, from fields conditions, can be reported lonely (one level studies), only if it met some fundamental standards. In my opinion, these sine qua non requirements are: (i) at least three years of experiments, (ii) several different localizations, (iii) efficient mapping of environmental variables, (iv) MIAPPE recommendations. All these facts are missing in the presented study. MIAPPE (Minimal Information about Plant Phenotyping Experiments) recommendations have been developed and are continuously updated to allow a description of all necessary metadata, including the environment (see https://www.miappe.org; Ćwiek-Kupczyńska et al. Measures for interoperability of phenotypic data: minimum information requirements and formatting. Plant Methods (2016), 12: 44 DOI: 10.1186/s13007-016-0144-4).

Recently, scientists and breeders are increasingly aware of the impact of the environment and its monitoring cannot be ignored. It is not clear if plants were exposed only to water deficit or to combined drought and heat stress, because authors did not include the air temperature measurement.

The approach presented in the study is the usual conventional phenotype observation used commonly by, for example, breeding companies across the world, in order to select genotypes and develop a new variety, very often carried out to thousands of individuals in many localizations. Hence, in my opinion, the presented study does not represent any innovative methodology or innovative approach for research. The only achievement is indication of a genotype with increased tolerance to drought (G12 genotype). However, also in this point I have objections. In general, results are doubtful due to the only two years of experiment and too small number of samples observed from each plot (only five samples taken from 7.2m² plot including six rows) – it is not representative. Moreover, I think that in a breeding programs, new and improved variety can be recommended when this variety is better that reference variety - here the reference variety G1 remains the best one.

My more particular concerns:

• Please avoid the duplication of titlewords with keywords (drought stress)
• Introduction should be improved: more details about defense strategies of plants to cope with stress (escape, avoidance, tolerance and how they are determined; also, more details about osmotic adjustment mechanisms, antioxidant defense etc.)
• Line 78: what does it mean supplemental irrigation? What level of soil moisture was considered as a control? Was it watering daily and what amount of water was added? How it was decided whether or not to supply water? Should be clarified.
• Lines 80-81: I wonder to know what was the actual rainfalls in each month of experiment during the cultivation period and how it corresponds to the average long-term annual precipitation?
• Line 88: why authors took flag leaf for analyses? Please justify.
• Lines 89-90: Was the measurement of Fv/Fm dark-adapted? How it was verified that results were not falsified by the different time of measurement, between 11.00 and 14.00?
• Lines 96-99: each phenological stage needs to be given according to one of the commonly used scale e.g. BBCH, Zadoks; authors’ statement that DH, DM, GFP were recorded at maturity is incorrect – phenological stages are noted during the plant development not at the maturity; units of measurement should be given, for phenology unit DAS (days after sowing); grain filling period should be also defined according to scale code (from the begging …BBCH code, to the end ….BBCH code) – On this way description will be clear and more friendly for readers. Authors can use ‘trait ontology (TO)’ and/or ‘crop ontology (CO)’ for the standardized traits description (e.g. https://www.ebi.ac.uk/ols/ontologies/to)
• Line 98: SL, NGPS – how these traits were calculated? As a mean of primary and secondary spikes from 5 plants? Should be clarified.
• Line 100: results based on five samples from plot 7.2 m² (6 rows) is not representative. In my opinion sampling at least 5 plants from each row is the must. In my opinion this is a serious methodological failure in study.
• Line 116: in ANOVA the source of variance called ‘stress’ is incorrect. These are considerations about different soil moistures (watered and not watered) so more adequate will be the term like ‘water treatment, water variant, regime’ etc.
• Table 1 - Abbreviations of source of variation in used in table should be explained below the table.
• Results: I have general objections related to obtained results due to the not representative number of analyzed samples. Example: ‘drought stress increased Fv/Fm and FL (line 129) – it is a slight increase but a bit surprising – too few samples tested!
• Line 148: sentence in the tracking changes mode
• Lines 227, 340: sentence in the tracking changes mode – comma added
• Lines 228-233: according to ARS the most resistant genotypes were: G8, G9, G10 – first year, G1, G2, G3 – second year. Results completely differ between years, thus at least one more additional experiment is mandatory. I’m almost sure that using data three years of experiments the averaged results will be changed. Concluding, the average data across two years experiment in presented studies is not representative. Results looks unreliable. Similar opinion about STI. And how this index was calculated? – should be described.

• Lines 301-302: it seems that authors are aware that drought is often accompanied by high temperature in field conditions, so the more surprising is the lack of observation of air temperature fluctuations during the field experiment – This is a serious detect in my opinion.
• Authors concluded that: ‘This result indicates that some of the tested genotypes (especially G9) may have a strong capacity to protect themselves through maintenance of high levels of PSII activity’ (lines 311-312), but also that: ‘there was no significant correlation between yield and Fv/Fm, thousand grains weight, and peduncle length, which indicates that grain yield can potentially be improved without changing these traits under drought stress conditions (Figure 1)’. (lines 339-342). Does it mean that the estimation of chlorophyll fluorescence parameters is useless in searching of resistant genotypes with improved yield? It should be better discussed in relation to reports of other researchers.

For the future, to not waste totally already obtained results, some supplementary studies will be useful. My suggestions are: firstly, one more filed experiment with better monitoring of environment conditions and meteorological parameters (like actual rainfalls, temperature recording, soil moisture); secondly, molecular markers genotyping of plant material especially using markers linked to quantitative and qualitative traits, and/or associated with stress response (numerous effective markers like microsatellites have been already published) which can be potentially useful for marker-assisted breeding; finally, great novelty will be provided conducting plants examination using new, automate, non-invasive imaging of phenotypes. Dynamic evaluation of plant features during growth is especially important for understanding plant adaptation and temporal dynamics of responses to environmental cues such drought. This will provide information about plants architecture, growth dynamics, water content of plant tissue (especially important under drought application), photosynthetic efficiency, plant health status and many, many others. I’m aware that such infrastructures are not common, however thanks to Transnational Access of EPPN2020 research can get a free access to the modern phenotyping infrastructure, also coming from outside the Europe (more information: https://eppn2020.plant-phenotyping.eu/Access_Call).

Summarizing, I believe that presented study (with additional year of experiment and more attention paid to environment changes) may serve as a part of bigger research with additional studies focused on mechanisms underlying yield formation in constrained conditions (e.g. in combination with molecular markers genotyping or modern, non-invasive plant phenotyping - imaging of dynamic traits).