the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 12 Aug 2024
A conservative resource use strategy in agricultural grasslands counteracts lower productivity and water use efficiency under drought conditions
Abstract. Grassland response to changes in water availability is closely tied to the traits of the plant community which determine plant water uptake and resource use. Plants can adopt either moderate and efficient (conservative) or rapid and demanding (acquisitive) resource use strategies. These strategies combined with the plant interactions with microbes, such as arbuscular mycorrhiza fungi (AMF), determine the grassland productivity and efficiency. This study aims to compare the drought response of two agricultural grasslands that differ in their resource use strategies. In an experimental garden 12 small-scale lysimeters were installed with two different agricultural grassland types (conservative and acquisitive) and two different irrigation levels (wet and drought). We measured water fluxes, above- and belowground phytomass productivity and AMF spore productivity in these two grassland types throughout a drought of 64 days. Despite differing resource use strategies, both grassland types exhibited similar reductions in evapotranspiration and aboveground phytomass under drought. However, the conservative grassland showed higher water use efficiency (WUE) when considering only aboveground phytomass, and a less pronounced compositional shift towards greater grass phytomass. Furthermore, in acquisitive grasslands the root:shoot ratio of grasses and AMF spores abundance in the soil were greatly reduced than in conservative grasslands. We also identified differences in legume productivity, rooting system, and AMF spores community composition as key factors influencing WUE. In a changing climate with greater frequency and severity of droughts in the European Alps, opting for grassland mixtures with more species with conservative characteristics should be considered, as they i) reach the productivity of acquisitive grassland even under wetter conditions and ii) show higher efficiency and longer vitality under drought conditions.
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RC1: 'Comment on egusphere-2024-2435', Anonymous Referee #1, 01 Sep 2024
I have reviewed “A conservative resource use strategy in agricultural grasslands counteracts lower productivity and water use efficiency under drought conditions” by Tello-García et al. I think the research question/topic – how grassland communities cope with drought in terms of productivity, water use efficiency, and the mechanisms underlying this (traits, functional groups, and connections to AMF)- is very interesting and relevant. I appreciate the hard work of the authors, carrying out this field experiment and the analysis of AMF spores, root washing, and stomatal conductance. I’ve added quite a few smaller comments below addressing the Abstract – Results, but only briefly reviewed the Discussion as I think there are two major issues that need to be addressed first:
- Currently, the paper is framed around conservative versus acquisitive grasslands (and their drought response). However, for me it is uncertain if the grasslands plots actually differ in their strategies. Functional traits were used to determine the CWM of the mixtures, with the abundance of each species coming from the proportion of seeds in the mixture. In Table 1, I can see that both seed mixtures should be grass dominated, the seed mixture for dry locations (the ‘conservative’ mixture) by Festuca arundinancea, and the humid mixture (‘acquisitive’) by Festuca rubra/Lolium perenne/Poa pratensis. However, from Figure 3, I see that both grasslands have almost exclusively legume biomass (x10 higher than grass biomass). Therefore, I am not sure that how you calculate CWM holds – because there were obviously some establishment issues with the grasses (and perhaps with some of the legume species) which means that the proportion of the different species in the seed mixture is very different than the community that established (and where all of your measurements are based). Essentially, you have two legume dominated stands that respond differently to drought. I think this needs to be re-framed with more of a focus on the legumes – because it is their response that you are determining (rather than the community composition in the seed mixture).
- I’m also not sure about the argument that plants invest more carbon belowground and therefore there is higher AMF spore productivity, and this feeds back to improve drought tolerance. Determining belowground carbon investment in AMF requires a different approach I think – e.g. pulse labelling of 13CO2 to trace plant-derived carbon into AMF (fatty acids – NLFAs or DNA). To determine the role of AMF in drought response, it would be nice to see colonization – if there is higher colonization in one grassland or the other (although I think Trifolium species are highly mycorrhizal, so likely you would have very high colonization overall). As you note in the introduction (lines 88-91) AMF spore production may not be a good indicator for AMF activity, but rather a proxy for stress, and not necessarily derived from drought stress of the plant (it could be due to a direct effect of water limitation, right?) In the introduction, I miss an explanation on why AMF spore abundance or abundance of different sizes or pigmentations would affect plant WUE or drought response. What was the rationale behind this investigation and these analyses (e.g. results line 393-396). Also in the introduction, there are no references (as far as I understand) that use AMF spores/spore productivity as an indicator of plant C investment, or that AMF spores would affect plant drought response. For me, these are two missing links that undermine your line of argumentation.
Because of these two points, I would recommend reframing the paper, away from the conservative – acquisitive resource acquisition spectrum, and perhaps also away from linking AMF spore productivity to plant carbon investment and plant drought response.
I’ve made some more comments below that I hope help improve the manuscript:
Abstract
L18 and L32: efficiency in terms of what? Perhaps specify here if you mean water use efficiency, nutrient use efficiency, or something else
L18: perhaps ‘In this study, we compare …’
L28: ‘compared to conservative grasslands’
Intro
L61: perhaps spell this out a bit more – that acquisitive species do not adjust their water use strategy, and this becomes wasteful as the soil dries. Are there specific studies looking at WUE that you could site here?
L79: AMF would obtain all of their C from the plant host, right?
L81: is stomatal conductance a plant trait?
L82: perhaps include more recent references on AMF’s role in drought tolerance, e.g. XXXXX
L82: compared to no colonization? Or reduced colonization to higher colonization rates?
L83: ‘Decreases in AMF productivity include…’ Also, it is unclear what is meant by AMF productivity, could you elaborate here?
L86: ‘host-independent physio-chemical soil properties’ is a bit complex, perhaps instead something along the lines of ‘soil physical chemical properties’
L87: the plant community composition?
L90: nutrients or carbon? Perhaps back this up with a reference showing how drought affects plant-AMF C transfer
L93: Plant traits and plant-AMF interactions… (perhaps less complex).
L104-111: I think this text can be shortened and moved to after your hypotheses. Start this paragraph your hypotheses, and then add a few lines of how you tested them. You could also start with a short opening: e.g. “We investigated grassland drought responses in two model grassland systems differing in their functional traits and resource acquisition strategies in a field experiment in the Austrian Alps. We hypothesized that (i)….(ii)…. We determined plant drought resistance, investment in root biomass and water use efficiency. Belowground, we quantified AMF spore productivity and hyphal biomass.” I think this would make the paragraph sharper and the reader would be able to more quickly grasp your hypotheses and experiment. Introduce the conservative/acquisitive grasslands above, and then here you can directly refer to them. The details on the seed mixture can go in the methods
113-114: ‘…higher investment in root and AMF sporulation.’ Makes it sound like the plant is in control of AMF sporulation, which is not correct right? Perhaps it would be clearer to specify what are the plant responses and what are the soil responses. E.g. We hypothesized that (ii) conservative grasslands would respond faster to drought in terms of stomatal closure, higher investment in root biomass and higher WUE than fast-strategy grasslands. (ii) belowground, we expected that in conservative grasslands, AMF spore productivity would be higher…
And perhaps a question – how did you measure ‘faster’ investment in root biomass? From the methods I see that you washed roots at one timepoint, so I don’t think much can be said about the speed of investment. Instead I would say something along the lines of ‘greater investment in root biomass during the drought’.
L113-118: I think it is good to include these responses in your hypotheses, but I would incorporate them into your hypotheses rather than listing them as possible responses.
Methods
L122: Remove the quotation marks from Stubai Valley (capital V) and Tyrolean Alps, and it is: Neustift im Stubaital, Austria. Perhaps after Austria, start a new sentence describing the site.
L124: ‘It’ is ambiguous, perhaps ‘The site…’
L125: Here, and everywhere in the paper, switch to the active voice. E.g. instead of There, 12 small lysimeters were installed, change to ‘We installed 12 lysimeters’ and in L126-126: ‘We carried out our experiment in June-August 2018’
I think it is also useful in this section to specify the irrigation levels – in the figure I see ‘drought’ and ‘wet’. In the text (main and fig description) perhaps specify shortly what these were – my interpretation would be that ‘wet’ = waterlogged, so I wonder where the control is. I’d list the treatments and then in brackets write the target soil moisture/WHC target.
L134-139: I think you can tighten up this section ‘We used two seed mixtures commonly sown in European alpine grasslands: ‘seed mixture for humid locations’ (SR037) and ‘seed mixture for drier locations’ (SR032) (company).
L144: It would be good to show the PCA that you made to determine acquisitive vs conservative. Also, perhaps an explanation on why you used these traits. The original leaf economic spectrum (Reich, Wright) is SLA, LDMC, leaf N concentration. There is also the root economic spectrum with SRL, RTD, root N, and in some cases AMF (Bergmann, Roumet, Weemstra) and also a whole plant economic spectrum, but it would be good to specify why these traits (and also why not simply SLA, LDMC, and leaf N?) and also this is a bit of reference stacking as not all of these references back up the traits you’ve listed here. Perhaps just choose the reference that includes all and only these traits. Or, if you are adding/excluding traits, give a reason why.
L146: What is a CWM proxy? Or did you just calculate the CWM?
L148: Was there differences in germination? It could be that more seeds of a certain species would be added to compensate for poor germination. Although, I suppose if you do not have the proportions based on biomass, calculating CWM becomes difficult. Are there data (from the seed company) on germination success?
L149: displayed? Or had?
L150: To clarify: the spectrum was not determined as such? Just to note that the references you are using here do this in a different way. To integrate the traits, and quantify the tradeoff, usually PCA scores are used.
L153: what is seed share?
L154: Calculated or determined (i.e. did you measure this?) if you measured, specify how? Was this statistically different (i.e. did you measure SLA for each plot and then statistically compare your conservative vs acquisitive community)?
L159: Can you specify what you mean by root density (classes)? It would be helpful to have units here, as root density could be root tissue density (volume/weight) or rooting density (root length/volume soil). It would also be good to include this first in the text (rather than the Table heading)
L177: pH in water?
L178: report the other information here, or specifically refer to a supplementary table or figure
L200: specify that this is the height
L203: do you know the moisture level (volumetric water content) that field capacity was?
L218: Perhaps specify how many days after the drought began this was (e.g. Beginning 3 weeks after the start of the drought treatment…)
L221-222: I think you could say ‘… due to difficulty identifying plant species’
L226: ‘At the end of the experiment, grass and legume individuals were counted’ Perhaps also specify how long the drought treatment had been running (e.g. ‘At the end of the experiment (after 8 weeks of drought), …)
L230: How deep did you extract?
L234: You visually assessed necromass, but was this also determined (by weight) at the harvest?
L264: Why analysis of covariance (ANCOVA) instead of ANOVA? Perhaps specify that interaction between grassland type and irrigation level was tested (instead of ‘variables’). Which function and package did you use? In this section, mention these and reference the packages.
L267: what does ‘tested independently’ mean?
L279-284: This sounds like a nice analysis – can you reference also a paper that uses it in this way?
Results
Figure 2 – The stats you report here are for the ANCOVA, I assume because you are looking at the change between factors over time. Why did you use ANCOVA and not a generalized additive model (GAM, e.g. from package mgcv in R) which would be a more powerful model to capture the changes in SWC and ET over time? Also, you mention that the shaded area represents standard error – how many points per timepoint were measured?
L306: This can be simplified a bit. Perhaps something like: ‘Legumes had higher stomatal conductance than grasses (P < 0.001, Table S1), and responded differently to drought (). Grassland type affected how legumes responded to drought (): legumes in the acquisitive grassland xxx, while legumes in the conservative grassland xxx.
L308: “Legumes in the acquisitive grassland” (add ‘the’ throughout, or else it would need to be: legumes in acquisitive grasslands’)
L309: two weeks after the start of the drought? When was the point when the drought became ‘extreme’?
L310: More direct: Drought reduced grass stomatal conductance after 2 weeks of drought in both grassland types ().
L313: tended? (was this difference significant or not?)
L314: ‘Impacts’ is vague, could you reword this?
L317: Decreased, or ‘was significantly lower’
L319: Perhaps more direct: “Grasses and legumes significantly differed in their total aboveground phytomass (), which depended on grassland type (interaction) and irrigation level (interaction).”
L322: ‘while’ or ‘in contrast’ and specify what ‘it’ is
L322: no space between the % and the number
L322-323: Am I understanding this correctly that grasses were only 2-4% of your grasslands? Is this typical for this seed mixture?
L327: ‘reduced’ is in the wrong place here I think. Instead, something like: ‘In both grassland types, drought significantly reduced belowground biomass (). Unless there is a major difference in the magnitude of the reduction, I would not add the numbers as personally I think that numbers in text trip up the reader (unless they are comparing to other studies, or you want to specifically highlight them).
L337: 80% of the aboveground biomass was necromass? Or 80% of the grass individuals had died (i.e. did not regrow)? Specify this because there can be a lot of necromass and the individual can still grow back
L338: Increased with time (duration assumes that you have different droughts with different lengths)
L339: Instead of the percentages, perhaps state if this was different – was there sig more necromass in the acquisitive grassland?
Figure 3 – perhaps add in the legend that the scale of (a) between legumes and grasses differs – at first glance it looks like grasses have slightly less biomass, but this is actually an order of magnitude lower. Also specify which posthoc test you used.
L350: inoculum implies that the AMF were added, but this refers to the initial soil right? Perhaps then ‘compared to the initial AMF spore density’
L352: AMF spore size?
L353: ‘We found slightly (but non-significant, P < ..) higher AMF spore productivity in the conservative versus acquisitive grassland’… I would only report this if the significance was close, if not, this is no difference.
Figure 4 (b) – I like this figure, but wonder if these different colors can be seen by someone who is red-green color blind.
L379: I think this could be more direct: ‘We found that WUEap and WUEtp responded differently to drought (Fig 5).
L379: also more direct: ‘We found that grassland types had significantly different WUEap at the end of the drought’
L394: how were the variables condensed? This part is not clear to me, and I think this paragraph needs to be clarified. It makes sense that productivity is correlated to WUE, but is this not a spurious correlation (aboveground productivity is used to calculate WUE right?)
L400: move the ref to the figure and table to the end of the sentence
Discussion
L416: this was only one field site correct? Then why ‘in two climatically different areas of the Alps’?
L417: as far as I see, they were categorized based on individual traits rather than ‘strategies’ (there was no PCA/PCA scores as far as I can see?)
L418-420: This line reads a bit like droughts are not a big issue, when in fact they are (see Smith et al - https://www.pnas.org/doi/abs/10.1073/pnas.2309881120 drought effects are underestimated).
L415-420: reword this paragraph to show your key results and the main message.
Section 4.1. – rearrange this section so that you start with your most important results, and with the heading (water stress effects on aboveground productivity). Then move in to differences in stomatal conductivity as an explanation for how grasslands with different strategies deal with water stress (and what this eventually means to their productivity, and then why this is important). In the title you refer to ‘aboveground productivity’ and in the text ‘phytomass’, keep this wording the same throughout and the text will be more clear/readable.
L444-447: grasses are typically more ‘conservative’ than legumes, but why not compare within functional groups? Especially between grasses and legumes, they are functionally very different, so the difference between functional group (rather than strategy) would always be large, wherease there are more conservative legumes (Lotus corniculatus compared to an acquisitive like Trifolium repens or pratense) and the same situation with grasses (think of a species like Nardis stricta versus Lolium perenne or Dactylis glomerata). I don’t know how useful it is to compare between legumes and grasses in terms of ‘resource acquisition strategy’ because the functional group classification already covers this difference.
L451: was this difference apparent between the drought and the control as well? I.e. after drought conservative grasslands were almost exclusively legumes, but in both, there was still a significant decrease (Fig 3 a) and the legume biomass between acquisitive and conservative looks statistically the same right?
L456: big = significant?
L460: no doubt, drastic reductions in legume biomass in grasslands is a bad thing, but since your grasslands are almost completely legumes, I wonder if these arguments hold here? Grazing (or making hay from) a grassland with 98 or 80% legume biomass would not be safe for ruminants due to the high protein.. I’m wondering if the discussion could stick a bit closer to the water dynamics and reasons for these differences.
L480-483: I think you can remove this sentence. This paragraph could also be merged with another (possibly the one above)? It contradicts the title of the section, so perhaps first start with the result that is the reason for that title (the AMF spore abundance?) and then move on to mention the non-significant shifts in root:shoot.
L486-489: AMF, yes – but are spores a good indication of plant-AMF water/nutrient transfer? Or AMF impact on plant stress responses?
L493: slightly or significantly?
Citation: https://doi.org/10.5194/egusphere-2024-2435-RC1 -
AC1: 'Reply on RC1', Elena Tello García, 23 Sep 2024
We thank the reviewer for the thoughtful feedback and valuable insights. Below, we respond to the main points raised in detail and outline the changes we plan to make in a revised version of the manuscript.
Point 1:
We agree with the reviewer that our stands are dominated by a few rapidly growing species. However, this outcome aligns with the intended design of commercial seed mixtures, which are based on the "replacement principle" (Bergh 1968, Baba, Halim et al. 2011, Isselstein, Benke et al. 2011). Fast-growing species (e.g., Trifolium pratense, Trifolium repens and Lolium perenne) facilitate rapid canopy closure, suppress non-sown species, and ensure high initial yields. As the stand matures over the following years, the rapidly developing but non-perennial species are replaced by slower-growing species (e.g. Arrhenatherum elatius, Dactylis glomerata and Festuca arundinacea). The slower-growing, perennial species contribute to long-term botanical stability and sustained yield. Many of these slower-growing species were already present in our canopies in the first year, but only vegetative and at a small abundance, so that morphological identification was not possible without collecting them.
Since our experiment involved one-year-old vegetation communities, the observed patterns align with expectations. Fast-growing legumes were abundant under favorable conditions, while grasses had a pre-experiment density of 1922.9 ± 330.2 individuals m-2 (L195-197), representing 77% of the total plant population. This confirms the successful establishment of grasses in the community. The seed mixtures are designed for long-term productivity through the replacement principle, and we could expect a decrease in legumes if the experiment had been done in a more mature vegetation. Since our decision to use commercial seed mixtures reflects practical agricultural realities, a methodologically different approach would compromise the practical relevance of our study. Nevertheless, we will address this issue in both the Introduction and Discussion sections of the revised manuscript.
Point 2:
Regarding arbuscular mycorrhizal fungi (AMF), unfortunately, due to methodological limitations, we were unable to collect direct data on AMF colonization. However, we believe AMF spore formation is sufficiently relevant to be included in our analysis. To our knowledge, there is no established correlation between belowground plant investment or plant growth and AMF spore productivity. The only relevant study, by Al-Arjani, Hashem et al. (2020), correlates AMF spore abundance in the soil to growth-promoting hormones in plants. That said, many studies have demonstrated the strong positive effect of AMF colonization on plant growth and productivity, e.g. Duan, Luo et al. (2024), even under stress (Bahadur, Batool et al. 2019, Hartman and Tringe 2019), compared to non-AMF plants.
Some research has shown that AMF colonization and spore production follow similar patterns, as observed in Northern Ethiopia (Birhane, Gebremedihin et al. 2017). In some species, even significant correlations between AMF colonization and spore formation have been found, e.g. Vicia faba (Firdu and Dida 2024), Theobroma grandiflorum and Paullinia cupana (de Oliveiria and de Oliveiria 2005). However, as mentioned in our introduction, AMF spore abundance can be influenced by factors like seasonality (L85-92), so care is needed when linking AMF spore formation directly to the plant community. In our study, all lysimeters used the same soil, and samples were taken at the same time, which helps to minimize seasonal and soil-related effects. This approach is supported by Maitra, Zheng et al. (2019), who showed that AMF spore density is affected by drought and seasonality, but not by their interaction.
In addition, drought has been shown to reduce AMF colonization, which negatively impacts plant growth (Wu, Srivastava et al. 2013, Zhang, Zou et al. 2018). Drought has not only an effect on AMF colonization but on the whole AMF development cycle (Bahadur, Batool et al. 2019), including spore formation (Kilpeläinen, Barbero-López et al. 2017). We acknowledge the reviewer's concern regarding the link between plant performance and higher AMF spore productivity. While this specific connection is not well-supported by current literature, our approach is based on the relationships established in the previously mentioned studies. We hypothesize that drought-induced reductions in AMF colonization negatively affect plant growth and the entire AMF development cycle, including spore formation. Although we focus on plant growth, similar gaps exist for water use efficiency (WUE). Therefore, our study seeks to connect AMF spore formation with plant growth through WUE. Although more specific techniques , like 13CO2 pulse labelling, would be needed to establish a direct causal link, we aim to take a step forward by demonstrating the potential impact of AMF spore abundance and diversity (used as a proxy for AMF composition as explained in L252-259) on the WUE of plant communities. We will strengthen this argument in our revised manuscript.
Many thanks for the further comments and specific suggestions for improvement, which are very helpful. We will carefully address and consider all comments in the revised manuscript.
References:
Al-Arjani, A. F., et al. (2020). "Arbuscular mycorrhizal fungi modulates dynamics tolerance expression to mitigate drought stress in Ephedra foliata Boiss." Saudi Journal of Biological Sciences 27(1): 380-394.
Baba, M., et al. (2011). "Grass-legume mixtures for enhanced forage production: Analysis of dry matter yield and competition indices." African Journal of Agricultural Research 6(23): 5242-5250.
Bahadur, A., et al. (2019). "Mechanistic insights into arbuscular mycorrhizal fungi-mediated drought stress tolerance in plants." International Journal of Molecular Science 20(17).
Bergh, J. P. v. d. (1968). An analysis of yields of grasses in mixed and pure stands. Wageningen.
Birhane, E., et al. (2017). "Exclosures restored the density and root colonization of arbuscular mycorrhizal fungi in Tigray, Northern Ethiopia." Ecological Processes 6(1).
de Oliveiria, A. N. and L. A. de Oliveiria (2005). "Seasonal dynamics of arbuscular mycorrhizal fungi in plants of Theobroma grandiflorum Schum and Paullina cupana Mart. of an agroforestry system in Central Amazonia, Amazonas State, Brazil." Brazilian Journal of Microbiology 36: 262-270.
Duan, H.-X., et al. (2024). "AM fungus promotes wheat grain filling via improving rhizospheric water & nutrient availability under drought and low density." Applied Soil Ecology 193.
Firdu, Z. and G. Dida (2024). "Extraction, identification and mass production of arbuscular mycorrhizal fungi (AMF) from faba bean (Vicia faba L.) rhizosphere soils using maize (Zea mays L.) as a host plant." Heliyon 10(17): e36838.
Hartman, K. and S. G. Tringe (2019). "Interactions between plants and soil shaping the root microbiome under abiotic stress." Biochemical Journal 476(19): 2705-2724.
Isselstein, J., et al. (2011). Futterbau in Niedersachsen im Spannungsfeld zwischen Produktionsfunktion und landschaftsökologischen Funktionen. Mitteilungen der Arbeitsgemeinschaft Grünland und Futterbau Band. Jahrestagung der AGGF. Oldenburg. 12: 30–44.
Kilpeläinen, J., et al. (2017). "Does severe soil drought have after-effects on arbuscular and ectomycorrhizal root colonisation and plant nutrition?" Plant and Soil 418(1-2): 377-386.
Maitra, P., et al. (2019). "Effect of drought and season on arbuscular mycorrhizal fungi in a subtropical secondary forest." Fungal Ecology 41: 107-115.
Wu, Q.-S., et al. (2013). "AMF-induced tolerance to drought stress in citrus: A review." Scientia Horticulturae 164: 77-87.
Zhang, F., et al. (2018). "Quantitative estimation of water uptake by mycorrhizal extraradical hyphae in citrus under drought stress." Scientia Horticulturae 229: 132-136.
Citation: https://doi.org/10.5194/egusphere-2024-2435-AC1
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RC2: 'Comment on egusphere-2024-2435', Anonymous Referee #2, 26 Sep 2024
The reviewed study entitled “A conservative resource use strategy in agricultural grasslands counteracts lower productivity and water use efficiency under drought conditions” aims to investigate whether two forage grassland mixtures whose plant trait syndromes differ on the conservative-acquisitive economic spectrum respond differently to experimental drought. Specifically, the authors focused on drought-responses of plant productivity, root-shoot ratios, plant community water use efficiency, and AMF spore trait abundance. I think the questions this study aims to answer are highly relevant and valuable in both, the ecological and agronomical context. However, in my view (which is shared by reviewer #1, as I have seen) there are some major issues regarding the setup of the study and the interpretation of the data:
- Despite the grassland mixtures were sown in the year prior to the experiment, the results on plant productivity suggest that the established plant communities did not represent the aimed community mixtures. While both seed mixtures are dominated by grasses, the biomass of established community was mainly legumes (which in this study mainly consisted of highly acquisitive species). This raises doubts whether the studied established plant communities really differed in their economic strategy. I suggest that the authors either provide more information on the structure of the established communities (species/genus abundance), their traits, and their position on the resource economic spectrum, or reframe the study by focusing more on legumes.
- Something that further bothered me was that volumetric soil water contents before and during the experiment were constantly under 16% in all treatments and therefore lied way below the lower threshold for plant available water (~20%). Either this is due to a calibration issue (most likely since ET in the wet treatment was relatively high) or implies that also the “wet” treatment was under constant water stress. I suggest the authors to clarify this and, if necessary, reframe the story accordingly.
- The authors explain differences and drought-induced changes in the abundance of AMF traits between acquisitive and conservative grasslands with different C allocation strategies and drought-induced changes in plant composition. However, they did not measure plant C allocation to AMF nor show data on the composition of the established plant community (they show only biomass, which does not necessarily reflect abundance). Also, the authors do not discuss potential effects of the observed differences in SWC between the drought-stressed acquisitive and conservative grassland on the abundance of AMF spore traits and how they could confound potential plant composition effects. I suggest the authors interpret the data more modestly, focus on the variables measured, and discuss different potential mechanisms more thoroughly.
The authors can find some more major comments on the introduction and some minor comments/suggestions on the remaining parts in the following sections.
Title
It says that a conservative resource strategy counteracts lower productivity under drought. However, non-significant 2-way interactions of irrigation*grassland type and 3-way interaction indicate that resource strategy did not significantly reduce drought effects on productivity. I would therefore take it out of the title.
Abstract
LN17: Since grassland productivity and efficiency is dependent on more factors, I would write “can determine” / “affect”
LN19: Comma after garden
LN27: AMF spore_ abundance
LN28: greatly reduced compared to
LN28: “we also identified differences..” – differences between treatments or between strategies? Specify!
LN29: “AMF spores community composition” – maybe better “AMF trait abundance” since you did not directly look at community compositions
Introduction
General comments
Be more specific in terminology, in the information you want to come across, and in the knowledge gaps and questions your study is based on!
Your paragraphs usually start with describing a trait / a trait syndrome or by AMF. However, since the information is missing why these are crucial in the context of climate change and / or agricultural forage production, I perceived the topic and order of the paragraphs as being random or coming out of the blue. Try to already in the 1st paragraph narrow down the most crucial factors / processes your study focusses on, giving the reader already an idea what the main research gap / question is your study addresses.
Take care when describing drought responses of conservative and acquisitive species / communities (e.g. LN60-62, 111-118). While gs might be more “insensitive” to drought in acquisitive species/communities, biomass is highly sensitive (see e.g. Oram et al., 2023 JEcol and cited studies). I suggest to better introduce the tradeoffs of traits and strategies and explain their responses to drought more precisely.
I miss in the introduction on why you look at AMF spore size and color as an indicator for drought stress and of different functional hosting groups.
Minor comments
LN44-46: It is not clear to me here whether you aim to highlight background or a knowledge gap.
LN70-73: It does not come across why this is relevant for your study.
LN73-74: word missing
LN73-75: you do not explain how functional traits and the – as you call it - “plant-associated rhizosphere” shape plant responses to drought, which seems to be critical for your study. Also, “associated rhizosphere” is very vague. Please be more specific.
LN75-78: What do you mean by “intrinsically connected”? What do you mean by “deeper knowledge”? Explain knowledge gaps more specifically!
LN82-84: Why is this relevant for your study?
LN85-89: Link the information given in these two sentences.
LN87: of hosting plant community?
LN88-92: Give references. Move your hypotheses to the last paragraph or write it more general.
LN98-99: Why? In scenarios with brief and intense dry periods you also have extreme fluctuations in resource availability. Explain.
LN104-118: You hypothesize a faster drought response of the conservative grasslands compared to the acquisitive grassland. What response do you mean? Gs? This does not account for e.g. aboveground biomass!
Methods
LN125: What was the size of the lysimeters?
Table 1: In SR037 (“fast-growing community”), one of the four most abundant species in the seed mixture is Festuca rubra which is considered as a slow-growing species. Also, CWMs of the two mixtures seem quite close, which for me raises the question whether they significantly differ along the economic spectrum. Giving SE / SD for CWMs would facilitate the interpretation of the table.
LN152-153: This is in line with point 1 of reviewer 1’s comment: Given the results in Fig. 3a I suppose this relationship was not confirmed? If not, how can you be certain that you really test two communities differing in their economic spectrum?
LN192-208: How many times in the year before the experiment was aboveground biomass harvested? When was the first harvest of the season before the rainout shelter was set up? Did drought start immediately after the harvest? It would be nice to display the harvests as e.g. arrows in Fig.2.
LN279-284: Can you give more information on this analysis and why you selected specifically these 5 factors?
Results
LN290: down to 3.27?
Fig.2: Was your volumetric SWC really that low? If yes, this would mean that your control community has most likely been water stressed as well since the lower threshold for plant-available water lies around 20%! Or is this a calibration issue? And how do you explain the significant effect of grassland type on SWC of the drought pots when there was no effect on ET? Also, how do you explain the big differences in gs between treatments prior to the experiment? It would be nice to also include VPD data in this panel or in the appendix.
LN303: Word missing
LN300-305: Please also write where no effects were found (between grassland types)
LN303: A decreased ET? (word missing)
LN315-325: Always give the direction (increase/decrease) of effects.
LN316-318: Please state whether drought*grassland type was significant.
LN321-325: What do you mean by proportion? Abundance? Where do you show this data?
LN331: You mean under drought or at peak drought and not after drought, right?
LN393-399: Move this to the Methods.
LN399-406 & Fig.6: I’m not quite convinced about this analysis and by the selection of the different variables/factors. WUE is directly linked with 8 out of 10 variables which describe Factor 1, including aboveground/total biomass and accumulated ET from which WUE is calculated. I therefore don’t see much sense in testing this relationship.
Discussion
LN418-420: Exclude this. Actually, experiments often underestimate natural drought responses, see Kröel-Dulay et al., 2022, NatEcoEvo.
LN422: Delete the 1st part of the sentence since this effect was anyways not significant.
LN422-435: The difference in stomatal conductance between the conservative and acquisitive grassland at the first drought campaign is smaller or equal to the difference prior to the start of the experimental drought. I suggest you either explain the differences observed prior to the experiment and/or interpret this result more cautiously.
LN450: see comment above for “after” drought
LN468-470: Check the grammar. I would use “studied” instead of “sown”. Also, why does high plasticity of young individuals make them more relevant to study? For sure it is relevant, but I wouldn’t say it’s more or less relevant than studying adult individuals.
LN473: I’d say “drought can / has been shown to change”
LN475: You don’t show this explicitly. Be more specific.
LN482: efficiency of carbon and nutrient use?
LN490: You use the term “fitness”, which usually includes parameters defining the ability to reproduce and survive. Be more specific (productivity / performance).
LN490-439: Which results imply this? I’d be more cautious with this interpretation. You don’t show any data on belowground C allocation, only R:S responses. The difference in the abundance of AMF spore traits of conservative and acquisitive grasslands under drought could as well be driven by the huge difference in SWC – as you yourself write in introduction part LN85-87 (“AMF spore abundance is primarily influenced by seasonal climate and host-independent physio-chemical soil properties…”). I generally miss the discussion of potential effects of different SWC in drought-stressed acquisitive and conservative grasslands on the abundance of AMF spore traits and how they could confound potential effects by (changes) in host composition.
LN491: “slightly higher biomass production under drought”
LN497: Delete “thus” or switch order of effects
LN506-508: See previous comment on LN490-439. Also, from which PC/factor do you draw this interpretation?
Citation: https://doi.org/10.5194/egusphere-2024-2435-RC2 -
AC2: 'Reply on RC2', Elena Tello García, 04 Oct 2024
We thank the reviewer for the insightful comments and suggestions. Below, we address the key points raised and outline the specific revisions we will implement in the manuscript.
Point 1
Please refer to point 1 from our answer to Reviewer #1, where we explain the potential impact of the "replacement principle" in our grasslands and how this might influence our results. Unfortunately, we cannot provide more detailed data on community structure. However, we can prove the successful establishment of grasses, as demonstrated in lines 196–197. Regarding plant traits, we measured specific leaf area (SLA) for the three dominant species in each grassland type, confirming that the mean SLA was lower in conservative grasslands compared to acquisitive ones, as expected (lines 154–155).
Point 2
Thank you for this important information, we apologize for the confusion! We have performed a soil-specific calibration (gravimetric two-point calibration) of the SWC sensors, but unfortunately, there are uncalibrated values in Figure 1a. This will be corrected in a revised version of the manuscript. From the water retention curve (pF) measured with HYPROP equipment, the following details can be seen: The field capacity is at a SWC of about 30 vol%, the permanent wilting point at 10 vol% and the saturated water content at about 50 vol%, which is typical for a sandy loam soil.
The volumetric soil water contents before the experiment varied between 18 and 25% by volume in all treatments and between 20 and 40% by volume in the wet treatment during the experiment.
Point 3
In the revised version of the manuscript, we will ensure greater caution in the interpretation of our results, focusing strictly on the variables measured, as recommended. Additionally, we will improve the discussion on how drought may influence AMF dynamics by incorporating further relevant literature. We will also discuss the potential implications of variations in AMF spore abundance, such as the possibility of a reduced inoculum for the following season.
We greatly appreciate the reviewer’s helpful comments and suggestions. These will be carefully addressed and incorporated into the revised manuscript, with particular attention given to improving the introduction.
Citation: https://doi.org/10.5194/egusphere-2024-2435-AC2
Status: closed
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RC1: 'Comment on egusphere-2024-2435', Anonymous Referee #1, 01 Sep 2024
I have reviewed “A conservative resource use strategy in agricultural grasslands counteracts lower productivity and water use efficiency under drought conditions” by Tello-García et al. I think the research question/topic – how grassland communities cope with drought in terms of productivity, water use efficiency, and the mechanisms underlying this (traits, functional groups, and connections to AMF)- is very interesting and relevant. I appreciate the hard work of the authors, carrying out this field experiment and the analysis of AMF spores, root washing, and stomatal conductance. I’ve added quite a few smaller comments below addressing the Abstract – Results, but only briefly reviewed the Discussion as I think there are two major issues that need to be addressed first:
- Currently, the paper is framed around conservative versus acquisitive grasslands (and their drought response). However, for me it is uncertain if the grasslands plots actually differ in their strategies. Functional traits were used to determine the CWM of the mixtures, with the abundance of each species coming from the proportion of seeds in the mixture. In Table 1, I can see that both seed mixtures should be grass dominated, the seed mixture for dry locations (the ‘conservative’ mixture) by Festuca arundinancea, and the humid mixture (‘acquisitive’) by Festuca rubra/Lolium perenne/Poa pratensis. However, from Figure 3, I see that both grasslands have almost exclusively legume biomass (x10 higher than grass biomass). Therefore, I am not sure that how you calculate CWM holds – because there were obviously some establishment issues with the grasses (and perhaps with some of the legume species) which means that the proportion of the different species in the seed mixture is very different than the community that established (and where all of your measurements are based). Essentially, you have two legume dominated stands that respond differently to drought. I think this needs to be re-framed with more of a focus on the legumes – because it is their response that you are determining (rather than the community composition in the seed mixture).
- I’m also not sure about the argument that plants invest more carbon belowground and therefore there is higher AMF spore productivity, and this feeds back to improve drought tolerance. Determining belowground carbon investment in AMF requires a different approach I think – e.g. pulse labelling of 13CO2 to trace plant-derived carbon into AMF (fatty acids – NLFAs or DNA). To determine the role of AMF in drought response, it would be nice to see colonization – if there is higher colonization in one grassland or the other (although I think Trifolium species are highly mycorrhizal, so likely you would have very high colonization overall). As you note in the introduction (lines 88-91) AMF spore production may not be a good indicator for AMF activity, but rather a proxy for stress, and not necessarily derived from drought stress of the plant (it could be due to a direct effect of water limitation, right?) In the introduction, I miss an explanation on why AMF spore abundance or abundance of different sizes or pigmentations would affect plant WUE or drought response. What was the rationale behind this investigation and these analyses (e.g. results line 393-396). Also in the introduction, there are no references (as far as I understand) that use AMF spores/spore productivity as an indicator of plant C investment, or that AMF spores would affect plant drought response. For me, these are two missing links that undermine your line of argumentation.
Because of these two points, I would recommend reframing the paper, away from the conservative – acquisitive resource acquisition spectrum, and perhaps also away from linking AMF spore productivity to plant carbon investment and plant drought response.
I’ve made some more comments below that I hope help improve the manuscript:
Abstract
L18 and L32: efficiency in terms of what? Perhaps specify here if you mean water use efficiency, nutrient use efficiency, or something else
L18: perhaps ‘In this study, we compare …’
L28: ‘compared to conservative grasslands’
Intro
L61: perhaps spell this out a bit more – that acquisitive species do not adjust their water use strategy, and this becomes wasteful as the soil dries. Are there specific studies looking at WUE that you could site here?
L79: AMF would obtain all of their C from the plant host, right?
L81: is stomatal conductance a plant trait?
L82: perhaps include more recent references on AMF’s role in drought tolerance, e.g. XXXXX
L82: compared to no colonization? Or reduced colonization to higher colonization rates?
L83: ‘Decreases in AMF productivity include…’ Also, it is unclear what is meant by AMF productivity, could you elaborate here?
L86: ‘host-independent physio-chemical soil properties’ is a bit complex, perhaps instead something along the lines of ‘soil physical chemical properties’
L87: the plant community composition?
L90: nutrients or carbon? Perhaps back this up with a reference showing how drought affects plant-AMF C transfer
L93: Plant traits and plant-AMF interactions… (perhaps less complex).
L104-111: I think this text can be shortened and moved to after your hypotheses. Start this paragraph your hypotheses, and then add a few lines of how you tested them. You could also start with a short opening: e.g. “We investigated grassland drought responses in two model grassland systems differing in their functional traits and resource acquisition strategies in a field experiment in the Austrian Alps. We hypothesized that (i)….(ii)…. We determined plant drought resistance, investment in root biomass and water use efficiency. Belowground, we quantified AMF spore productivity and hyphal biomass.” I think this would make the paragraph sharper and the reader would be able to more quickly grasp your hypotheses and experiment. Introduce the conservative/acquisitive grasslands above, and then here you can directly refer to them. The details on the seed mixture can go in the methods
113-114: ‘…higher investment in root and AMF sporulation.’ Makes it sound like the plant is in control of AMF sporulation, which is not correct right? Perhaps it would be clearer to specify what are the plant responses and what are the soil responses. E.g. We hypothesized that (ii) conservative grasslands would respond faster to drought in terms of stomatal closure, higher investment in root biomass and higher WUE than fast-strategy grasslands. (ii) belowground, we expected that in conservative grasslands, AMF spore productivity would be higher…
And perhaps a question – how did you measure ‘faster’ investment in root biomass? From the methods I see that you washed roots at one timepoint, so I don’t think much can be said about the speed of investment. Instead I would say something along the lines of ‘greater investment in root biomass during the drought’.
L113-118: I think it is good to include these responses in your hypotheses, but I would incorporate them into your hypotheses rather than listing them as possible responses.
Methods
L122: Remove the quotation marks from Stubai Valley (capital V) and Tyrolean Alps, and it is: Neustift im Stubaital, Austria. Perhaps after Austria, start a new sentence describing the site.
L124: ‘It’ is ambiguous, perhaps ‘The site…’
L125: Here, and everywhere in the paper, switch to the active voice. E.g. instead of There, 12 small lysimeters were installed, change to ‘We installed 12 lysimeters’ and in L126-126: ‘We carried out our experiment in June-August 2018’
I think it is also useful in this section to specify the irrigation levels – in the figure I see ‘drought’ and ‘wet’. In the text (main and fig description) perhaps specify shortly what these were – my interpretation would be that ‘wet’ = waterlogged, so I wonder where the control is. I’d list the treatments and then in brackets write the target soil moisture/WHC target.
L134-139: I think you can tighten up this section ‘We used two seed mixtures commonly sown in European alpine grasslands: ‘seed mixture for humid locations’ (SR037) and ‘seed mixture for drier locations’ (SR032) (company).
L144: It would be good to show the PCA that you made to determine acquisitive vs conservative. Also, perhaps an explanation on why you used these traits. The original leaf economic spectrum (Reich, Wright) is SLA, LDMC, leaf N concentration. There is also the root economic spectrum with SRL, RTD, root N, and in some cases AMF (Bergmann, Roumet, Weemstra) and also a whole plant economic spectrum, but it would be good to specify why these traits (and also why not simply SLA, LDMC, and leaf N?) and also this is a bit of reference stacking as not all of these references back up the traits you’ve listed here. Perhaps just choose the reference that includes all and only these traits. Or, if you are adding/excluding traits, give a reason why.
L146: What is a CWM proxy? Or did you just calculate the CWM?
L148: Was there differences in germination? It could be that more seeds of a certain species would be added to compensate for poor germination. Although, I suppose if you do not have the proportions based on biomass, calculating CWM becomes difficult. Are there data (from the seed company) on germination success?
L149: displayed? Or had?
L150: To clarify: the spectrum was not determined as such? Just to note that the references you are using here do this in a different way. To integrate the traits, and quantify the tradeoff, usually PCA scores are used.
L153: what is seed share?
L154: Calculated or determined (i.e. did you measure this?) if you measured, specify how? Was this statistically different (i.e. did you measure SLA for each plot and then statistically compare your conservative vs acquisitive community)?
L159: Can you specify what you mean by root density (classes)? It would be helpful to have units here, as root density could be root tissue density (volume/weight) or rooting density (root length/volume soil). It would also be good to include this first in the text (rather than the Table heading)
L177: pH in water?
L178: report the other information here, or specifically refer to a supplementary table or figure
L200: specify that this is the height
L203: do you know the moisture level (volumetric water content) that field capacity was?
L218: Perhaps specify how many days after the drought began this was (e.g. Beginning 3 weeks after the start of the drought treatment…)
L221-222: I think you could say ‘… due to difficulty identifying plant species’
L226: ‘At the end of the experiment, grass and legume individuals were counted’ Perhaps also specify how long the drought treatment had been running (e.g. ‘At the end of the experiment (after 8 weeks of drought), …)
L230: How deep did you extract?
L234: You visually assessed necromass, but was this also determined (by weight) at the harvest?
L264: Why analysis of covariance (ANCOVA) instead of ANOVA? Perhaps specify that interaction between grassland type and irrigation level was tested (instead of ‘variables’). Which function and package did you use? In this section, mention these and reference the packages.
L267: what does ‘tested independently’ mean?
L279-284: This sounds like a nice analysis – can you reference also a paper that uses it in this way?
Results
Figure 2 – The stats you report here are for the ANCOVA, I assume because you are looking at the change between factors over time. Why did you use ANCOVA and not a generalized additive model (GAM, e.g. from package mgcv in R) which would be a more powerful model to capture the changes in SWC and ET over time? Also, you mention that the shaded area represents standard error – how many points per timepoint were measured?
L306: This can be simplified a bit. Perhaps something like: ‘Legumes had higher stomatal conductance than grasses (P < 0.001, Table S1), and responded differently to drought (). Grassland type affected how legumes responded to drought (): legumes in the acquisitive grassland xxx, while legumes in the conservative grassland xxx.
L308: “Legumes in the acquisitive grassland” (add ‘the’ throughout, or else it would need to be: legumes in acquisitive grasslands’)
L309: two weeks after the start of the drought? When was the point when the drought became ‘extreme’?
L310: More direct: Drought reduced grass stomatal conductance after 2 weeks of drought in both grassland types ().
L313: tended? (was this difference significant or not?)
L314: ‘Impacts’ is vague, could you reword this?
L317: Decreased, or ‘was significantly lower’
L319: Perhaps more direct: “Grasses and legumes significantly differed in their total aboveground phytomass (), which depended on grassland type (interaction) and irrigation level (interaction).”
L322: ‘while’ or ‘in contrast’ and specify what ‘it’ is
L322: no space between the % and the number
L322-323: Am I understanding this correctly that grasses were only 2-4% of your grasslands? Is this typical for this seed mixture?
L327: ‘reduced’ is in the wrong place here I think. Instead, something like: ‘In both grassland types, drought significantly reduced belowground biomass (). Unless there is a major difference in the magnitude of the reduction, I would not add the numbers as personally I think that numbers in text trip up the reader (unless they are comparing to other studies, or you want to specifically highlight them).
L337: 80% of the aboveground biomass was necromass? Or 80% of the grass individuals had died (i.e. did not regrow)? Specify this because there can be a lot of necromass and the individual can still grow back
L338: Increased with time (duration assumes that you have different droughts with different lengths)
L339: Instead of the percentages, perhaps state if this was different – was there sig more necromass in the acquisitive grassland?
Figure 3 – perhaps add in the legend that the scale of (a) between legumes and grasses differs – at first glance it looks like grasses have slightly less biomass, but this is actually an order of magnitude lower. Also specify which posthoc test you used.
L350: inoculum implies that the AMF were added, but this refers to the initial soil right? Perhaps then ‘compared to the initial AMF spore density’
L352: AMF spore size?
L353: ‘We found slightly (but non-significant, P < ..) higher AMF spore productivity in the conservative versus acquisitive grassland’… I would only report this if the significance was close, if not, this is no difference.
Figure 4 (b) – I like this figure, but wonder if these different colors can be seen by someone who is red-green color blind.
L379: I think this could be more direct: ‘We found that WUEap and WUEtp responded differently to drought (Fig 5).
L379: also more direct: ‘We found that grassland types had significantly different WUEap at the end of the drought’
L394: how were the variables condensed? This part is not clear to me, and I think this paragraph needs to be clarified. It makes sense that productivity is correlated to WUE, but is this not a spurious correlation (aboveground productivity is used to calculate WUE right?)
L400: move the ref to the figure and table to the end of the sentence
Discussion
L416: this was only one field site correct? Then why ‘in two climatically different areas of the Alps’?
L417: as far as I see, they were categorized based on individual traits rather than ‘strategies’ (there was no PCA/PCA scores as far as I can see?)
L418-420: This line reads a bit like droughts are not a big issue, when in fact they are (see Smith et al - https://www.pnas.org/doi/abs/10.1073/pnas.2309881120 drought effects are underestimated).
L415-420: reword this paragraph to show your key results and the main message.
Section 4.1. – rearrange this section so that you start with your most important results, and with the heading (water stress effects on aboveground productivity). Then move in to differences in stomatal conductivity as an explanation for how grasslands with different strategies deal with water stress (and what this eventually means to their productivity, and then why this is important). In the title you refer to ‘aboveground productivity’ and in the text ‘phytomass’, keep this wording the same throughout and the text will be more clear/readable.
L444-447: grasses are typically more ‘conservative’ than legumes, but why not compare within functional groups? Especially between grasses and legumes, they are functionally very different, so the difference between functional group (rather than strategy) would always be large, wherease there are more conservative legumes (Lotus corniculatus compared to an acquisitive like Trifolium repens or pratense) and the same situation with grasses (think of a species like Nardis stricta versus Lolium perenne or Dactylis glomerata). I don’t know how useful it is to compare between legumes and grasses in terms of ‘resource acquisition strategy’ because the functional group classification already covers this difference.
L451: was this difference apparent between the drought and the control as well? I.e. after drought conservative grasslands were almost exclusively legumes, but in both, there was still a significant decrease (Fig 3 a) and the legume biomass between acquisitive and conservative looks statistically the same right?
L456: big = significant?
L460: no doubt, drastic reductions in legume biomass in grasslands is a bad thing, but since your grasslands are almost completely legumes, I wonder if these arguments hold here? Grazing (or making hay from) a grassland with 98 or 80% legume biomass would not be safe for ruminants due to the high protein.. I’m wondering if the discussion could stick a bit closer to the water dynamics and reasons for these differences.
L480-483: I think you can remove this sentence. This paragraph could also be merged with another (possibly the one above)? It contradicts the title of the section, so perhaps first start with the result that is the reason for that title (the AMF spore abundance?) and then move on to mention the non-significant shifts in root:shoot.
L486-489: AMF, yes – but are spores a good indication of plant-AMF water/nutrient transfer? Or AMF impact on plant stress responses?
L493: slightly or significantly?
Citation: https://doi.org/10.5194/egusphere-2024-2435-RC1 -
AC1: 'Reply on RC1', Elena Tello García, 23 Sep 2024
We thank the reviewer for the thoughtful feedback and valuable insights. Below, we respond to the main points raised in detail and outline the changes we plan to make in a revised version of the manuscript.
Point 1:
We agree with the reviewer that our stands are dominated by a few rapidly growing species. However, this outcome aligns with the intended design of commercial seed mixtures, which are based on the "replacement principle" (Bergh 1968, Baba, Halim et al. 2011, Isselstein, Benke et al. 2011). Fast-growing species (e.g., Trifolium pratense, Trifolium repens and Lolium perenne) facilitate rapid canopy closure, suppress non-sown species, and ensure high initial yields. As the stand matures over the following years, the rapidly developing but non-perennial species are replaced by slower-growing species (e.g. Arrhenatherum elatius, Dactylis glomerata and Festuca arundinacea). The slower-growing, perennial species contribute to long-term botanical stability and sustained yield. Many of these slower-growing species were already present in our canopies in the first year, but only vegetative and at a small abundance, so that morphological identification was not possible without collecting them.
Since our experiment involved one-year-old vegetation communities, the observed patterns align with expectations. Fast-growing legumes were abundant under favorable conditions, while grasses had a pre-experiment density of 1922.9 ± 330.2 individuals m-2 (L195-197), representing 77% of the total plant population. This confirms the successful establishment of grasses in the community. The seed mixtures are designed for long-term productivity through the replacement principle, and we could expect a decrease in legumes if the experiment had been done in a more mature vegetation. Since our decision to use commercial seed mixtures reflects practical agricultural realities, a methodologically different approach would compromise the practical relevance of our study. Nevertheless, we will address this issue in both the Introduction and Discussion sections of the revised manuscript.
Point 2:
Regarding arbuscular mycorrhizal fungi (AMF), unfortunately, due to methodological limitations, we were unable to collect direct data on AMF colonization. However, we believe AMF spore formation is sufficiently relevant to be included in our analysis. To our knowledge, there is no established correlation between belowground plant investment or plant growth and AMF spore productivity. The only relevant study, by Al-Arjani, Hashem et al. (2020), correlates AMF spore abundance in the soil to growth-promoting hormones in plants. That said, many studies have demonstrated the strong positive effect of AMF colonization on plant growth and productivity, e.g. Duan, Luo et al. (2024), even under stress (Bahadur, Batool et al. 2019, Hartman and Tringe 2019), compared to non-AMF plants.
Some research has shown that AMF colonization and spore production follow similar patterns, as observed in Northern Ethiopia (Birhane, Gebremedihin et al. 2017). In some species, even significant correlations between AMF colonization and spore formation have been found, e.g. Vicia faba (Firdu and Dida 2024), Theobroma grandiflorum and Paullinia cupana (de Oliveiria and de Oliveiria 2005). However, as mentioned in our introduction, AMF spore abundance can be influenced by factors like seasonality (L85-92), so care is needed when linking AMF spore formation directly to the plant community. In our study, all lysimeters used the same soil, and samples were taken at the same time, which helps to minimize seasonal and soil-related effects. This approach is supported by Maitra, Zheng et al. (2019), who showed that AMF spore density is affected by drought and seasonality, but not by their interaction.
In addition, drought has been shown to reduce AMF colonization, which negatively impacts plant growth (Wu, Srivastava et al. 2013, Zhang, Zou et al. 2018). Drought has not only an effect on AMF colonization but on the whole AMF development cycle (Bahadur, Batool et al. 2019), including spore formation (Kilpeläinen, Barbero-López et al. 2017). We acknowledge the reviewer's concern regarding the link between plant performance and higher AMF spore productivity. While this specific connection is not well-supported by current literature, our approach is based on the relationships established in the previously mentioned studies. We hypothesize that drought-induced reductions in AMF colonization negatively affect plant growth and the entire AMF development cycle, including spore formation. Although we focus on plant growth, similar gaps exist for water use efficiency (WUE). Therefore, our study seeks to connect AMF spore formation with plant growth through WUE. Although more specific techniques , like 13CO2 pulse labelling, would be needed to establish a direct causal link, we aim to take a step forward by demonstrating the potential impact of AMF spore abundance and diversity (used as a proxy for AMF composition as explained in L252-259) on the WUE of plant communities. We will strengthen this argument in our revised manuscript.
Many thanks for the further comments and specific suggestions for improvement, which are very helpful. We will carefully address and consider all comments in the revised manuscript.
References:
Al-Arjani, A. F., et al. (2020). "Arbuscular mycorrhizal fungi modulates dynamics tolerance expression to mitigate drought stress in Ephedra foliata Boiss." Saudi Journal of Biological Sciences 27(1): 380-394.
Baba, M., et al. (2011). "Grass-legume mixtures for enhanced forage production: Analysis of dry matter yield and competition indices." African Journal of Agricultural Research 6(23): 5242-5250.
Bahadur, A., et al. (2019). "Mechanistic insights into arbuscular mycorrhizal fungi-mediated drought stress tolerance in plants." International Journal of Molecular Science 20(17).
Bergh, J. P. v. d. (1968). An analysis of yields of grasses in mixed and pure stands. Wageningen.
Birhane, E., et al. (2017). "Exclosures restored the density and root colonization of arbuscular mycorrhizal fungi in Tigray, Northern Ethiopia." Ecological Processes 6(1).
de Oliveiria, A. N. and L. A. de Oliveiria (2005). "Seasonal dynamics of arbuscular mycorrhizal fungi in plants of Theobroma grandiflorum Schum and Paullina cupana Mart. of an agroforestry system in Central Amazonia, Amazonas State, Brazil." Brazilian Journal of Microbiology 36: 262-270.
Duan, H.-X., et al. (2024). "AM fungus promotes wheat grain filling via improving rhizospheric water & nutrient availability under drought and low density." Applied Soil Ecology 193.
Firdu, Z. and G. Dida (2024). "Extraction, identification and mass production of arbuscular mycorrhizal fungi (AMF) from faba bean (Vicia faba L.) rhizosphere soils using maize (Zea mays L.) as a host plant." Heliyon 10(17): e36838.
Hartman, K. and S. G. Tringe (2019). "Interactions between plants and soil shaping the root microbiome under abiotic stress." Biochemical Journal 476(19): 2705-2724.
Isselstein, J., et al. (2011). Futterbau in Niedersachsen im Spannungsfeld zwischen Produktionsfunktion und landschaftsökologischen Funktionen. Mitteilungen der Arbeitsgemeinschaft Grünland und Futterbau Band. Jahrestagung der AGGF. Oldenburg. 12: 30–44.
Kilpeläinen, J., et al. (2017). "Does severe soil drought have after-effects on arbuscular and ectomycorrhizal root colonisation and plant nutrition?" Plant and Soil 418(1-2): 377-386.
Maitra, P., et al. (2019). "Effect of drought and season on arbuscular mycorrhizal fungi in a subtropical secondary forest." Fungal Ecology 41: 107-115.
Wu, Q.-S., et al. (2013). "AMF-induced tolerance to drought stress in citrus: A review." Scientia Horticulturae 164: 77-87.
Zhang, F., et al. (2018). "Quantitative estimation of water uptake by mycorrhizal extraradical hyphae in citrus under drought stress." Scientia Horticulturae 229: 132-136.
Citation: https://doi.org/10.5194/egusphere-2024-2435-AC1
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RC2: 'Comment on egusphere-2024-2435', Anonymous Referee #2, 26 Sep 2024
The reviewed study entitled “A conservative resource use strategy in agricultural grasslands counteracts lower productivity and water use efficiency under drought conditions” aims to investigate whether two forage grassland mixtures whose plant trait syndromes differ on the conservative-acquisitive economic spectrum respond differently to experimental drought. Specifically, the authors focused on drought-responses of plant productivity, root-shoot ratios, plant community water use efficiency, and AMF spore trait abundance. I think the questions this study aims to answer are highly relevant and valuable in both, the ecological and agronomical context. However, in my view (which is shared by reviewer #1, as I have seen) there are some major issues regarding the setup of the study and the interpretation of the data:
- Despite the grassland mixtures were sown in the year prior to the experiment, the results on plant productivity suggest that the established plant communities did not represent the aimed community mixtures. While both seed mixtures are dominated by grasses, the biomass of established community was mainly legumes (which in this study mainly consisted of highly acquisitive species). This raises doubts whether the studied established plant communities really differed in their economic strategy. I suggest that the authors either provide more information on the structure of the established communities (species/genus abundance), their traits, and their position on the resource economic spectrum, or reframe the study by focusing more on legumes.
- Something that further bothered me was that volumetric soil water contents before and during the experiment were constantly under 16% in all treatments and therefore lied way below the lower threshold for plant available water (~20%). Either this is due to a calibration issue (most likely since ET in the wet treatment was relatively high) or implies that also the “wet” treatment was under constant water stress. I suggest the authors to clarify this and, if necessary, reframe the story accordingly.
- The authors explain differences and drought-induced changes in the abundance of AMF traits between acquisitive and conservative grasslands with different C allocation strategies and drought-induced changes in plant composition. However, they did not measure plant C allocation to AMF nor show data on the composition of the established plant community (they show only biomass, which does not necessarily reflect abundance). Also, the authors do not discuss potential effects of the observed differences in SWC between the drought-stressed acquisitive and conservative grassland on the abundance of AMF spore traits and how they could confound potential plant composition effects. I suggest the authors interpret the data more modestly, focus on the variables measured, and discuss different potential mechanisms more thoroughly.
The authors can find some more major comments on the introduction and some minor comments/suggestions on the remaining parts in the following sections.
Title
It says that a conservative resource strategy counteracts lower productivity under drought. However, non-significant 2-way interactions of irrigation*grassland type and 3-way interaction indicate that resource strategy did not significantly reduce drought effects on productivity. I would therefore take it out of the title.
Abstract
LN17: Since grassland productivity and efficiency is dependent on more factors, I would write “can determine” / “affect”
LN19: Comma after garden
LN27: AMF spore_ abundance
LN28: greatly reduced compared to
LN28: “we also identified differences..” – differences between treatments or between strategies? Specify!
LN29: “AMF spores community composition” – maybe better “AMF trait abundance” since you did not directly look at community compositions
Introduction
General comments
Be more specific in terminology, in the information you want to come across, and in the knowledge gaps and questions your study is based on!
Your paragraphs usually start with describing a trait / a trait syndrome or by AMF. However, since the information is missing why these are crucial in the context of climate change and / or agricultural forage production, I perceived the topic and order of the paragraphs as being random or coming out of the blue. Try to already in the 1st paragraph narrow down the most crucial factors / processes your study focusses on, giving the reader already an idea what the main research gap / question is your study addresses.
Take care when describing drought responses of conservative and acquisitive species / communities (e.g. LN60-62, 111-118). While gs might be more “insensitive” to drought in acquisitive species/communities, biomass is highly sensitive (see e.g. Oram et al., 2023 JEcol and cited studies). I suggest to better introduce the tradeoffs of traits and strategies and explain their responses to drought more precisely.
I miss in the introduction on why you look at AMF spore size and color as an indicator for drought stress and of different functional hosting groups.
Minor comments
LN44-46: It is not clear to me here whether you aim to highlight background or a knowledge gap.
LN70-73: It does not come across why this is relevant for your study.
LN73-74: word missing
LN73-75: you do not explain how functional traits and the – as you call it - “plant-associated rhizosphere” shape plant responses to drought, which seems to be critical for your study. Also, “associated rhizosphere” is very vague. Please be more specific.
LN75-78: What do you mean by “intrinsically connected”? What do you mean by “deeper knowledge”? Explain knowledge gaps more specifically!
LN82-84: Why is this relevant for your study?
LN85-89: Link the information given in these two sentences.
LN87: of hosting plant community?
LN88-92: Give references. Move your hypotheses to the last paragraph or write it more general.
LN98-99: Why? In scenarios with brief and intense dry periods you also have extreme fluctuations in resource availability. Explain.
LN104-118: You hypothesize a faster drought response of the conservative grasslands compared to the acquisitive grassland. What response do you mean? Gs? This does not account for e.g. aboveground biomass!
Methods
LN125: What was the size of the lysimeters?
Table 1: In SR037 (“fast-growing community”), one of the four most abundant species in the seed mixture is Festuca rubra which is considered as a slow-growing species. Also, CWMs of the two mixtures seem quite close, which for me raises the question whether they significantly differ along the economic spectrum. Giving SE / SD for CWMs would facilitate the interpretation of the table.
LN152-153: This is in line with point 1 of reviewer 1’s comment: Given the results in Fig. 3a I suppose this relationship was not confirmed? If not, how can you be certain that you really test two communities differing in their economic spectrum?
LN192-208: How many times in the year before the experiment was aboveground biomass harvested? When was the first harvest of the season before the rainout shelter was set up? Did drought start immediately after the harvest? It would be nice to display the harvests as e.g. arrows in Fig.2.
LN279-284: Can you give more information on this analysis and why you selected specifically these 5 factors?
Results
LN290: down to 3.27?
Fig.2: Was your volumetric SWC really that low? If yes, this would mean that your control community has most likely been water stressed as well since the lower threshold for plant-available water lies around 20%! Or is this a calibration issue? And how do you explain the significant effect of grassland type on SWC of the drought pots when there was no effect on ET? Also, how do you explain the big differences in gs between treatments prior to the experiment? It would be nice to also include VPD data in this panel or in the appendix.
LN303: Word missing
LN300-305: Please also write where no effects were found (between grassland types)
LN303: A decreased ET? (word missing)
LN315-325: Always give the direction (increase/decrease) of effects.
LN316-318: Please state whether drought*grassland type was significant.
LN321-325: What do you mean by proportion? Abundance? Where do you show this data?
LN331: You mean under drought or at peak drought and not after drought, right?
LN393-399: Move this to the Methods.
LN399-406 & Fig.6: I’m not quite convinced about this analysis and by the selection of the different variables/factors. WUE is directly linked with 8 out of 10 variables which describe Factor 1, including aboveground/total biomass and accumulated ET from which WUE is calculated. I therefore don’t see much sense in testing this relationship.
Discussion
LN418-420: Exclude this. Actually, experiments often underestimate natural drought responses, see Kröel-Dulay et al., 2022, NatEcoEvo.
LN422: Delete the 1st part of the sentence since this effect was anyways not significant.
LN422-435: The difference in stomatal conductance between the conservative and acquisitive grassland at the first drought campaign is smaller or equal to the difference prior to the start of the experimental drought. I suggest you either explain the differences observed prior to the experiment and/or interpret this result more cautiously.
LN450: see comment above for “after” drought
LN468-470: Check the grammar. I would use “studied” instead of “sown”. Also, why does high plasticity of young individuals make them more relevant to study? For sure it is relevant, but I wouldn’t say it’s more or less relevant than studying adult individuals.
LN473: I’d say “drought can / has been shown to change”
LN475: You don’t show this explicitly. Be more specific.
LN482: efficiency of carbon and nutrient use?
LN490: You use the term “fitness”, which usually includes parameters defining the ability to reproduce and survive. Be more specific (productivity / performance).
LN490-439: Which results imply this? I’d be more cautious with this interpretation. You don’t show any data on belowground C allocation, only R:S responses. The difference in the abundance of AMF spore traits of conservative and acquisitive grasslands under drought could as well be driven by the huge difference in SWC – as you yourself write in introduction part LN85-87 (“AMF spore abundance is primarily influenced by seasonal climate and host-independent physio-chemical soil properties…”). I generally miss the discussion of potential effects of different SWC in drought-stressed acquisitive and conservative grasslands on the abundance of AMF spore traits and how they could confound potential effects by (changes) in host composition.
LN491: “slightly higher biomass production under drought”
LN497: Delete “thus” or switch order of effects
LN506-508: See previous comment on LN490-439. Also, from which PC/factor do you draw this interpretation?
Citation: https://doi.org/10.5194/egusphere-2024-2435-RC2 -
AC2: 'Reply on RC2', Elena Tello García, 04 Oct 2024
We thank the reviewer for the insightful comments and suggestions. Below, we address the key points raised and outline the specific revisions we will implement in the manuscript.
Point 1
Please refer to point 1 from our answer to Reviewer #1, where we explain the potential impact of the "replacement principle" in our grasslands and how this might influence our results. Unfortunately, we cannot provide more detailed data on community structure. However, we can prove the successful establishment of grasses, as demonstrated in lines 196–197. Regarding plant traits, we measured specific leaf area (SLA) for the three dominant species in each grassland type, confirming that the mean SLA was lower in conservative grasslands compared to acquisitive ones, as expected (lines 154–155).
Point 2
Thank you for this important information, we apologize for the confusion! We have performed a soil-specific calibration (gravimetric two-point calibration) of the SWC sensors, but unfortunately, there are uncalibrated values in Figure 1a. This will be corrected in a revised version of the manuscript. From the water retention curve (pF) measured with HYPROP equipment, the following details can be seen: The field capacity is at a SWC of about 30 vol%, the permanent wilting point at 10 vol% and the saturated water content at about 50 vol%, which is typical for a sandy loam soil.
The volumetric soil water contents before the experiment varied between 18 and 25% by volume in all treatments and between 20 and 40% by volume in the wet treatment during the experiment.
Point 3
In the revised version of the manuscript, we will ensure greater caution in the interpretation of our results, focusing strictly on the variables measured, as recommended. Additionally, we will improve the discussion on how drought may influence AMF dynamics by incorporating further relevant literature. We will also discuss the potential implications of variations in AMF spore abundance, such as the possibility of a reduced inoculum for the following season.
We greatly appreciate the reviewer’s helpful comments and suggestions. These will be carefully addressed and incorporated into the revised manuscript, with particular attention given to improving the introduction.
Citation: https://doi.org/10.5194/egusphere-2024-2435-AC2
Data sets
Data from: A conservative resource use strategy in agricultural grasslands counteracts lower productivity and water use efficiency under drought conditions Elena Tello-García et al. https://doi.org/10.23728/b2share.33d070b2e3d541ab8d147e80ebba9eb4
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