Soil Biology and Biochemistry 178 (2023) 108932
Available online 23 December 2022
0038-0717/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Ectomycorrhizal fungi with hydrophobic mycelia and rhizomorphs dominate in young pine trees surviving experimental drought stress
Carles Casta˜no
a,*,1, Estefanía Suarez-Vidal
b,1, Rafael Zas
b, Jos´e Antonio Bonet
c,d, Jon`as Oliva
c,d, Luis Sampedro
baSwedish University of Agricultural Sciences, Department of Forest Mycology and Plant Pathology, SE-75007, Uppsala, Sweden
bMisi´on Biol´ogica de Galicia, Consejo Superior de Investigaciones Científicas (MBG-CSIC), Apdo 28, 36080, Pontevedra, Spain
cDepartament de Producci´o Vegetal i Ci`encia Forestal, Universitat de Lleida, Av. Rovira Roure, 191, E-25198, Lleida, Spain
dJoint Research Unit CTFC – AGROTECNIO-CERCA, Av. Alcalde Rovira Roure 191, E25198, Lleida, Spain
A R T I C L E I N F O Keywords:
Drought stress Fungal community Mycorrhizal Tree physiology Plant responses
A B S T R A C T
Mycorrhizal fungi can help plants to cope with drought, but research on the fungal communities that are more resistant to drought or alleviate drought stress of trees is still scarce. In this study, we investigated effects of drought on soil fungal communities and explored potential fungal traits related to drought resistance under greenhouse conditions. We manipulated water availability in pine seedlings belonging to three Spanish Pinus pinaster populations from geographical areas subjected to contrasting summer drought. A set of plant ecophys- iological traits were quantified and soil fungi was quantified and profiled using ergosterol and Pacific Biosciences sequencing. Abundance of ectomycorrhizal (ECM) fungi in plants subjected to drought was lower than in well- watered plants. Most ECM taxa in plants surviving drought had long exploration types and were taxa typically forming rhizomorphs and hydrophobic mycelia. By contrast, ECM taxa in well-watered plants had wider range of distinct exploration types. No differences in fungal communities were found among P. pinaster populations. No associations between ECM fungi and plant ecophysiological traits were found, but significant interactions be- tween drought treatments and belowground plant biomass were found for the relative abundances of ECM fungi, particularly ECM with long exploration types. Plants subjected to drought may benefit by associating to ECM taxa previously shown to transport water efficiently.
1. Introduction
Current climate change projections predict an increase in the fre- quency and intensity of drought events worldwide (Dai, 2012) including the Mediterranean area (García-Ruiz et al., 2011; Giorgi and Lionello, 2008). Drought is already inducing diebacks (Carnicer et al., 2011;
S´anchez-Salguero et al., 2012), promoting pathogen damage (Oliva et al., 2014) and decreasing plant production in Mediterranean forests (Caminero et al., 2018). Drought effects on forests can feedback on soil nutrient cycling (Anderegg et al., 2015), and other ecosystem goods such as mushrooms (Alday et al., 2017; Collado et al., 2018). Drought can also shift the composition and biomass of soil microbes, including fungal communities responsible for plant nutrition and water uptake (Casta˜no et al., 2018; Fernandez et al., 2017; Solly et al., 2017). Particularly, ectomycorrhizal (ECM) fungi can alleviate drought stress in plants by
increasing access to soil water (Allen and Ection, 2007) and improving host tree nutrition (Smith and Read, 2008). However, little is known about how shifts in ECM composition induced by drought may feedback on tree hydraulic functions and affect their tolerance to drought and ultimately determine tree survival.
Drought can modulate fungal communities in soils by promoting taxa with mycelial architectures that promote efficient hydraulic func- tions (Agerer, 2001) or taxa with metabolic adaptations to function under drier conditions (Treseder et al., 2018). Drought effects on soil fungi can also alter fungal-fungal interactions (Fernandez and Kennedy, 2016), particularly among-guild interactions (Querejeta et al., 2021).
Impaired ECM abundance after or during drought treatments has been commonly reported (Fernandez et al., 2017; Hagenbo et al., 2020;
Le´on-S´anchez et al., 2018, but see Preece et al. (2019)), although moulds, yeasts and saprotrophs seem to be also particularly sensitive
* Corresponding author.
E-mail address: [email protected] (C. Casta˜no).
1 Shared first authorship.
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Soil Biology and Biochemistry
journal homepage: www.elsevier.com/locate/soilbio
https://doi.org/10.1016/j.soilbio.2022.108932
Received 21 June 2022; Received in revised form 21 December 2022; Accepted 22 December 2022
(Casta˜no et al., 2018; Hartmann et al., 2017). ECM fungi are often classified according to exploration types, which indirectly influence plant nutrient and water uptake (Agerer, 2001; Fernandez et al., 2017).
It is assumed that ECM with long exploration types with hydrophobic mantle and rhizomorphs (i.e. high biomass ECM fungi, commonly with long or medium fringe exploration types) transport water more effi- ciently than low biomass fungi (i.e. ECM fungi with short, contact or medium smooth exploration types) (Agerer, 2001; Brownlee et al., 1983). However, not all exploration types of ECM fungi potentially de- mand the same amount of carbon (C) from their hosts. For example, C demands of high biomass fungi (i.e. medium fringe and long distance types) are potentially higher than that of low biomass fungi (i.e. short, contact types) (Agerer, 2001; Fernandez et al., 2017; Lehto and Zwiazek, 2011). Previous observations of increasing ECM ascomycetes and other low biomass fungi under increasingly warming conditions (Fernandez et al., 2017) or already dry or poor soils (Gordon and Gehring, 2011;
Oliach et al., 2020; Smith et al., 2007) suggest that, in the long-run, low biomass fungi may be more adapted to drought conditions. However, understanding of short-term responses of fungi to drought can be important for seedling survival during initial stages of establishment under field conditions.
Mycorrhizal fungi can also be affected indirectly by drought and warming through changes in plant performance (Fernandez et al., 2017;
Le´on-S´anchez et al., 2018). Differences in stomatal control among plant species and populations can also limit belowground carbon allocation to roots and associated microorganisms (Fuchslueger et al., 2014; Hasi- beder et al., 2015). In addition, belowground C allocation may be enhanced or hampered depending on the drought intensity (Fuchslueger et al., 2014; Shi et al., 2002), while differences in above-belowground C allocation patterns have been observed between plant species but also populations (Corcuera et al., 2012). These differences in C allocation belowground can therefore also affect fungal symbionts (Fernandez et al., 2017).
Differences in water use efficiency and belowground C allocation among plant populations are particularly relevant in Mediterranean pines which typically show large genetic intraspecific variation in traits related to drought tolerance (Ramírez-Valiente et al., 2021; Voltas et al., 2015; Zas et al., 2020). Thus, indirect impacts of drought on the fungal community may also vary depending on the plant population and its adaptations to drought, particularly on plant traits related to adaptation to water stress. Previous studies have shown that distinct genotypes of the same plant species can potentially recruit distinct fungal commu- nities (P´erez-Izquierdo et al., 2017; Redondo et al., 2020), but whether this has an impact on tree resistance to drought is still unknown.
Our overarching aim was to determine drought impacts on the composition and biomass of soil fungi at the early stages of Maritime pine establishment and whether these impacts differ depending on the pine population. We caused drought stress in P. pinaster seedlings from three populations adapted to contrasting water availability. P. pinaster is found along a wide range of climatic conditions in the western Medi- terranean basin, where it shows large intraspecific genetic variation in drought-tolerance strategies, including the degree of stomatal control (Delzon et al., 2004), modulation of root surface and number of root tips (Corcuera et al., 2012), or changes in above- and belowground alloca- tion patterns in response to water shortage (de la Mata et al., 2014). This tree species also associates with a vast range of ECM fungi, including Tomentella spp., Inocybe spp., Tricholoma spp., Suillus spp. and Russula spp. (Casta˜no et al., 2018; P´erez-Izquierdo et al., 2017). During a four month-experiment, we also investigated how physiological responses correlated with shifts in soil fungal community. We hypothesized that surviving plants would have (i) a lower soil fungal biomass than well-watered control plants and (ii) a shifted soil fungal community with respect to controls. Particularly, for ii), we expected that trees under drought treatments would harbour more ECM fungi with mycelial ar- chitectures to transport water more efficiently (i. e. ECM fungi with long exploration types). Finally, (iii) we expected a correlation between
differences in plant ecophysiological traits among plant populations and the ECM community.
2. Materials and methods
2.1. Growing conditions and plant material
Seeds of three Pinus pinaster Ait. populations growing in climatic areas under different summer conditions were selected: wettest site (San Cipri´an, SCRI, in Northwest of Spain), intermediate site (Coca, in the center of Spain) and the driest site (Oria, in the Mediterranean coastal of Southeast of Spain) (Fig. S1). Within each population, open-pollinated seeds were collected from around 30 putative unrelated mother trees.
Mother trees were at least 50 m apart from each other to minimize ge- netic relationships and capture enough within population variability.
Polyethylene pots were filled with a mixture 1:1 (vol:vol) of sterilized peat (Humin-substrate N3, Klasmann-Deilmann GmbH, 49744 Geeste, Germany) and sterilized granitic fine sand. This mix was selected to allow a quick drain of the substrate after watering, as shown in previous trials. Seeds were sown at a depth of 1 cm. Seedlings were treated with a foliar fungicide (Fernide, Syngenta Agro, Madrid, Spain) twice during the first month after emergence following common nursery practice.
Plants were grown in a greenhouse for eight months under controlled conditions (11/18 ◦C at night/day and 12 h of light per day, watered at field capacity) at the Misi´on Biol´ogica de Galicia (CSIC, Pontevedra, Spain).
2.2. Experimental design
Eight months after sowing, plants were subjected to three drought stress treatments by controlling the relative water content to the field capacity (RWC) at three levels for four months. Seedlings growing during eight months would increase chances of ectomycorrhizal colo- nization despite the use of sterile substrate. Control plants were grown in well-watered pots where a RWC of 80–90% was maintained. Plants subjected to moderate drought stress were kept between 40 and 50%
RWC, and plants subjected to severe drought stress between 25 and 35%
of RWC (Fig. 1a). The experiment was set in a two-way factorial design replicated in four blocks with drought stress and population as the main factors. Each block contained 14 plants of each population times drought combination: 4 blocks × 3 drought stress treatments × 3 pop- ulations × 14 plants = 504 plants. RWC and the amount of water needed to compensate water consumption was estimated gravimetrically by weighting a series of extra control pots (12 per treatment) every 2–3 days. In total, 360 of the 504 plants survived after the four months of the drought treatments.
2.3. Plant and soil sampling
Of the 360 surviving plants out of 504 initially planted (29% plant mortality), 88 plants from two blocks were selected for downstream soil fungal community analyses, and harvested for measuring plant height, diameter, and above- and root biomass and carbohydrates. Plant height and diameter were recorded, and plants were harvested by cutting the shoot just above the substrate. Plant water content was estimated by weighting roots before and after drying (72 h at 60 ◦C). Needle sub- samples were immediately deep frozen in liquid nitrogen and preserved at − 80 ◦C. Rhizosphere soil samples were homogenized, and a 20 g subsample was grinded with liquid nitrogen, freeze-dried (CHRIST freeze dryer, Beta2-8 LD plus, Germany) and stored at − 20 ◦C until DNA extraction.
2.4. Plant analyses
The impact of the drought treatments on plant water status was determined by measuring predawn and midday water potential with a
Scholander Chamber (PSM instruments, Maximum Operating Pressure 100 bar, USA) in the apical section of the stems. Five plants per popu- lation and drought stress treatment were harvested for this analysis at the end of the treatment.
We used the relative abundance of 13C/12C, a proxy of water use efficiency. The isotopic ratio (δ13C) was determined in the needles of five plants per population × drought stress treatment combination (n = 45). For that, 1–2 mg of needles were weighted into tin cups (LÜDIS- WISS, Switzerland) and analyzed in an elemental analyzer EA1108 (Carlo Erba Instruments) coupled to a MAT 253 GC-IRMS (Thermo Finnigan) at the research Support Service (SAI, www.sai.udc.es) of University of Coru˜na (A Coru˜na, Spain).
The concentration of non-structural carbohydrates (NSC, soluble sugars and starch) was analyzed in the coarse root tissue following (Hansen and Moller, 1975). Briefly, approximately 50 mg of freeze-dried tissues were extracted with 1 ml of 80% ethanol (Absolute Ethanol PA Panreac) in an ultrasonic bath (Thermostatic Bath Trade Raypa) at 80 ◦C for 30 min and centrifuged for 10 min at 2600 r.p.m. in a bench centrifuge (Eppendorf Centrifuge 5810 R). A second extraction was done following the same protocol, the supernatants were mixed, and the concentration of sugars estimated with the anthrone reagent method by measuring absorbance at 630 nm in a microplate reader (Biorad Labo- ratories Inc., Philadelphia, PA, USA). The pellet was then cleaned with 1 mL of deionized water, digested with 1 ml of 1.1% HCl (Panreac) at 100 ◦C for 30 min, and centrifuged 10 min at 2600 r.p.m, for starch analysis. The concentration of starch in the solution was measured again by measuring absorbance at 630 nm.
2.5. Ergosterol concentration in the soil
Fungal biomass in the soil samples was estimated by extracting and quantifying ergosterol as a fungal-specific biomarker following (Nylund
and Wallander, 1992). Ergosterol was chromatographically quantified as described by Hagenbo et al. (2020) and was converted into fungal biomass using a conversion factor of 3 μg ergosterol mg−1 fungal dry matter (Salmanowicz and Nylund, 1988).
2.6. Soil fungal community DNA sequencing
An aliquot of 500 mg of homogenized soil was used for DNA extraction using the NucleoSpin Soil kit (Macherey-Nagel, Düren, Ger- many). DNA concentration was measured spectrophotometrically, and templates diluted to 0.5 ng/μL. The Internal Transcribed Spacer 2 (ITS2) region was PCR amplified in triplicates using the forward gITS7 (Ihr- mark et al., 2012) and the reverse ITS4/ITS4arch (Sterkenburg et al., 2015; White et al., 1990) primers. All primers were previously extended by a linker base (T), unique 8 bases which differed in at least three positions and a terminal base (C) (Clemmensen et al., 2016). Each re- action consisted in 25 ng template, 2.75 mM MgCl2, primers at 0.5 μM (ITS7), 0.3 μM (ITS4), 0.15 μM (ITS4arch) and 0.025 U μL−1 polymerase (DreamTaq Green, Thermo Scientific, Waltham, MA, USA) in 1 × Buffer PCR. The cycling conditions were: 5 min at 95 ◦C, followed by 22–28 cycles of 30 s at 95 ◦C, 30 s at 56 ◦C, 30 s at 72 ◦C, and a final step of 72 ◦C for 7 min. Amplifications were performed aiming for a weak band in an agarose gel (Casta˜no et al., 2020a,b). PCR products were purified using the AMPure kit (Beckman Coulter, Brea, CA, USA) and quantified using Qubit DNA quantification kit (Life Technologies, USA). PCR products were pooled in equal amounts and cleaned with EZNA Cycle Pure kit (Omega Bio-Tek, Norcross, GA, USA). Amplicon length was checked on a BioAnalyzer 7500 chip (Agilent Technologies, Santa Clara, CA, USA). The resulting library was sequenced using three Pacific Bio- sciences RS II SMRT cells after addition of sequencing adapters by ligation at SciLifeLab, Uppsala, Sweden.
Fig. 1. Effect of drought (three levels: well-watered, moderate and severe drought) on a) water field capacity (%), b) predawn (measured before sunrise; open circles and dark line) and mid-day (measured at 12 p.m.; dark squares and dashed line) water potential (MPa), and c) 13C isotope ratio on needles tissue. Differences among drought treatments (well-watered and moderate drought) in surviving seedlings on d) height (cm), e) diameter (mm), and f) belowground dry plant biomass (g) Mean
±S.E is represented. Letters above bars denote significant differences (p ≤ 0.05) between drought treatments.
2.7. Bioinformatics
The SCATA pipeline was used for quality control and clustering of the DNA sequences (https://scata.mykopat.slu.se/). Reads shorter than 200 bases, with any individual base with a quality score lower than 10, and an average quality score of less than 20 were discarded. Homopol- ymers were collapsed to 3 bases and sequences were clustered using single-linkage clustering (‘usearch’, Edgar, 2010) with pair-wise com- parisons set using a mismatch penalty of 1, gap open penalty of 0 and gap extension penalty of 1. The minimum similarity for a read to enter the cluster was set to 98.5% to the closest neighbor in the cluster.
Clusters are referred to Operational Taxonomic Units (OTUs) hereafter.
A total of 44,091 out of 105,528 sequences passed the quality control (~41%), which were clustered in 469 OTUs. The 154 most abundant fungal OTUs (representing ~97% reads) were considered for tax- onomical and functional classification. The most abundant genotype of each OTU was taxonomically identified using UNITE and INSD data- bases (massBLASTer) in PlutoF (Abarenkov et al., 2010). Taxonomic identities at species level were assigned based on > 98.5% sequence similarity. OTUs were assigned to the following guilds: a) ECM, b) saprotroph, c) wood saprotroph, d) animal pathogen, e) ericoid mycorrhizal and f) endophyte, based on FUNGuild (Nguyen et al., 2016). These classifications were accepted when the confidence ranking was “highly probable”, but for those that not, classifications were veri- fied with other published literature where the fungal data was manually curated (Casta˜no et al., 2018). ECM species were further assigned to exploration types according to Agerer (2001), Suz et al. (2014) and the dEEMY database (Agerer and Rambold, 2017). Level of confidence of guild identification was high for ectomycorrhizal taxa, but for plant pathogens was more challenging since ITS2 marker is often problematic for species-level separation of OTUs. Similarly, as several saprotrophs can associate to distinct substrates (e. g. soil, litter) we decided to merge most of the saprotrophs in one guild. Based on these classifications, relative abundances of each guild and exploration types of mycelia were calculated. Sequence data are archived at NCBI’s Sequence Read Archive under accession number PRJNA842073. Community data and associated data of this study can be found in Mendeley Data, https://doi.
org/10.17632/xw5p3jcf8h.2.
2.8. Statistical analyses
For the analyses of the independent and interactive effects of popu- lation and drought on plant performance and physiology (height, diameter, biomass allocation, δ13C, water potential, non-structural carbohydrates) and on fungal soil biomass, were analyzed with gen- eral linear mixed models (GLMM) with main factors acting as fixed factors and blocks as a random factor. For plant mortality we used GLMM with binomial error distribution. When it was needed, normality was achieved by log or square root transformation of the dependent variable. Residual heterogeneity models across drought stress treat- ments were used when significant deviations were found. All analyses were carried out fitting mixed models by REML using the Mixed pro- cedure of the SAS System 9.4 (Littell et al., 2006). Fungal community data was assessed using CANOCO version 5.0 (Biometris Plant Research International, Wageningen, Netherlands) for ordinations and R software for other analyses (R version 3.0.2, R Development Core Team, 2015).
We first analyzed the effects of the distinct drought treatments (3 levels: well-watered, moderate drought, severe drought) on the fungal community only at Oria population, the only one with enough number of seedlings surviving the severe drought treatment. We used Corre- spondence analysis (CA) of Hellinger transformed fungal community data to visualize community differences between the drought treat- ments. In these analyses, plant physiological traits were plotted as supplementary variables. The effect of the drought treatments on the soil fungal community composition was further tested by canonical corre- spondence analysis (CCA) using Monte Carlo permutation tests (999
permutations). Separate analyses were performed using the community at species level, guild level (i.e. relative abundances of each guild), and at exploration type level as response variables. CCAs were also per- formed using forward selection of explanatory variables, in which we included the plant physiological parameters to test associations between these and the species community. P-values were Bonferroni-corrected.
To inspect whether multivariate variance differed between the three drought treatments, the ‘vegdist’ function from the vegan library was used to calculate Bray-Curtis dissimilarities of the fungal community matrices, which were tested for homogeneity of multivariate dispersion
‘betadisper’ (i.e. multivariate dispersion or beta diversity) with the
‘permutest’ function.
To test effect of drought on the relative abundance of the different functional groups of fungi, linear mixed effects models (LME) were fitted with drought stress as fixed factor and the block as random term.
To visualize the effect of population on soil fungal communities, a CA was performed but including the three pine populations only in the well- watered and moderate-drought treatments, as very little survival was observed for SCRI and COCA populations under severe stress. CCAs and LMEs were also performed using the species matrix as response variable and the population × drought treatment interaction as fixed factor. To test which OTUs associated to each level of drought treatment, we used indicator species analysis (de C´aceres et al., 2009). We also tested the associations between the abundance of each fungal group and the plant physiological parameters using LMEs. Response data was transformed either root square or asin (Arcsin (sqrt (x/100)) when needed.
3. Results
3.1. Drought stress effects on plant physiology
Drought stress affected plant survival (p < 0.001), with a mortality of 73%, 8% and 0%, at severe and moderate drought levels and well- watered plants, respectively (Fig. S2). There was a marginal popula- tion effect in plant survival (p = 0.030) being higher under severe drought in Coca and San Cipri´an (31% for each one) than in Oria (25%).
Plants subjected to drought had smaller height (p < 0.001, Table 1, Fig. 1d), diameter (p < 0.001, Table 1, Fig. 1e) and both above- and belowground biomass (p < 0.001, Table 1, Fig. 1f) than well-watered plants.
Plants subjected to drought showed a lower plant water potential (p
<0.001; Fig. 1b; Table 1) and higher delta 13C values than well-watered plants (p < 0.001; Fig. 1c; Table 1). However, the concentration of soluble sugars and starch did not significantly differ between treatments (Table 1). No direct or interactive effect of pine populations were found for water potential, however 13C signature was lower in Coca population (− 29.37) than Oria (− 29.27) and San Cipri´an (− 28.60) (p = 0.006;
Table 1). There were interactive effects of pine populations and drought treatments for plant height, diameter aboveground biomass, root:shoot ratios and soluble sugars (p < 0.05).
3.2. Soil fungal community composition
The first four most abundant fungal OTUs across all the dataset corresponded to Rhizopogon verii, Tomentella sp., Wilcoxina mikolae and Suillus bovinus. All these four OTUs were classified as ectomycorrhizal, which was also the most abundant functional group in the dataset (44%
of reads), followed by saprotrophs (19%) and ericoid mycorrhizal (6%).
Among exploration types, the long exploration type was the most abundant within ECM (57%), followed by ECM with medium smooth type (17%) or taxa that could have either contact, short or medium smooth types (24%).
3.3. Effects of drought stress on soil fungal community and biomass No effect of drought was observed on total soil fungal biomass
measured as ergosterol (p > 0.05). Soil fungal communities and some plant physiological features such as plant biomass and height were separated by drought treatment along the first axis of the CA ordination plot (Fig. 2a and b). Fungal communities in well-watered plants clus- tered together, while communities in plants subjected to moderate and severe drought overlapped along both CA axis and were separated from communities in well-watered plants (Fig. 2a). In addition, the severe drought treatment tended to reduce the community dispersion (p = 0.067), while no differences were observed between the moderate drought treatment and the well-watered plants.
Drought shifted the functional composition of soil fungi (p < 0.001), with largest dominance of ECM species such as Tomentella, Wilcoxina rehmii and Suillus bovinus in well-watered plants, and a larger dominance of other non-mycorrhizal species but also the mycorrhizal Suillus gran- ulatus in plants surviving moderate and severe drought treatments (Fig. S3, Table S1). Altogether, saprotrophs increased but ECM fungi
decreased their relative abundances in soils subjected to moderate and severe drought (Fig. S3) (p < 0.001, p = 0.004, respectively). ECM fungi in plants surviving moderate and severe drought had different explo- ration types than well-watered plants (Fig. S3). ECM fungi with low biomass (i. e. short, contact, medium smooth exploration types) signif- icantly decreased their relative abundance in plants surviving moderate and severe drought (p < 0.001). By contrast, relative abundances of ECM fungi with high biomass (i. e. long exploration types) were only marginally affected by drought (p = 0.082), although they tended to decrease under severe drought (Fig. S3).
Significant drought effects on the soil fungal community at the total fungal species, ectomycorrhizal species, and functional levels were consistent across all the P. pinaster populations (p < 0.001, p = 0.001, and p < 0.001, respectively, Fig. 3.). Thus, across all populations, plants surviving moderate drought had lower relative abundance of ECM fungi overall (p = 0.001, Fig. 3b) and lower abundance of ECM taxa with low Table 1
Effect of drought stress, plant population and their interaction on drought stress responses, growth, pine biomass allocation, plant water status and non-structural carbohydrate (NSC) reserves. Eight months old seedlings from three P. pinaster populations adapted to different summer drought regimes were subjected to three treatments of water stress for four months.
Variable Drought (D) Population (POP) POP * D
DF F p > F DF F p > F DF F p > F
Survival 2,24 164.5 <0.001 2,24 4.07 0.030 4,24 2.32 0.086
Drought stress responses Predawn water potential 2,8 39.0 <0.001 1,8 3.0 0.121 2,8 1.1 0.378
Midday water potential 2,15 14.0 <0.001 2,15 0.6 0.575 2,15 0.1 0.881
δ13C 2,52 140.8 <0.001 2,52 6.2 0.004 3,52 2.1 0.115
Growth Heigh 2343 254.7 <0.001 2343 235.3 <0.001 3343 12.7 <0.001
Diameter 2343 176.5 <0.001 2343 3.9 0.022 3343 4.8 0.003
Biomass allocation Aboveground biomass 2343 389.2 <0.001 2343 28.0 <0.001 3343 4.6 0.004
Belowground biomass 2343 402.9 <0.001 2343 1.2 0.302 3343 0.8 0.516
Total Biomass 2343 496.4 <0.001 2343 14.3 < 0.001 3343 2.5 0.056
Root: Shoot Ratio 2343 0.9 0.397 2343 22.0 < 0.001 3343 3.9 0.009
Reserves Soluble sugars 2342 1.7 0.194 2342 3.2 0.040 3342 4.9 0.002
Starch 2342 1.2 0.319 2342 0.7 0.505 3342 0.7 0.580
Fig. 2. Correspondence analyses (CA) of the species-level composition of fungi in soils of P. pinaster seedlings of the ORIA population subjected to three intensities of drought stress. The figure illustrates the variation in community composition showing a) the distribution of the samples grouped according the three drought treatments as shown by ellipses (well-watered, moderate and severe drought) and the b) the species distribution plot at the ordination space. In a) samples are coloured according to the drought treatments, while in b) species are coloured according to their guilds and sized proportionally to their relative abundance. Plant physiological measurements are also shown as supplementary variables. Abbreviations: Plant biomass (A) = Above-ground plant biomass. Plant biomass (B) = Below- ground plant biomass.
biomass (p < 0.001) than well-watered plants (Fig. 3c). Moreover, taxa with long exploration types were again not affected by drought in any of the populations (p = 0.662) (Fig. 3a,c).
3.4. Population effects and associations between plant phenotype and fungal biomass and community
There was neither an effect of plant population (p = 0.173) nor a drought × population interaction (p = 0.464) on soil fungal community composition (Fig. S4). By contrast, soil fungal biomass in well-watered plants differed among populations and was the highest in San Cipri´an (0.376 ± 0.06 mg mycelia g soil−1), followed by Coca (0.299 ± 0.06 mg mycelia g soil−1) and Oria (0.221 ± 0.03 mg mycelia g soil−1). No sig- nificant correlations were found between neither the relative abundance of ECM fungi nor the relative abundances of the exploration types of mycorrhizae and plant biomass partitioning (aboveground biomass, root biomass or root:shoot ratio) across drought treatments (p > 0.05).
However, there were significant interactions between drought treat- ments and belowground plant biomass for both the relative abundance of ECM fungi and the relative abundance of ECM with long exploration types (p = 0.011 and p = 0.018, respectively) (Fig. S5). These associa- tions were negative in well-watered plants but positive in plants sub- jected to drought (Fig. S5).
4. Discussion
We studied drought impacts on the composition and biomass of soil fungi in seedlings grown under controlled conditions. Despite these seedlings being likely exposed to a limited amount of fungal inoculum, as compared in natural conditions, we observed that well-watered plants were colonized by ECM fungi with distinct foraging traits (i. e. explo- ration types). The choice of conducting the study in a greenhouse may have biased any fungal assessment of population-level responses, which could differ if the experiment would have been performed under natural conditions in each of the sites. Overall, we experimentally observed a decrease of ECM fungi in soils under drought conditions, supporting that ECM fungi are sensitive to disturbances that affect their hosts. Similar results were previously found in trees growing in forest areas subjected
to clearcuts (Kohout et al., 2018; Mediavilla et al., 2017) and in trees exposed to both wood beetle (Stursov´a et al., 2014) and insect de- foliators (Casta˜no et al., 2020b). Supporting our results, previous experimental and descriptive studies also reported decreases in ECM biomass or relative abundances of symbiotic fungi under increasing drought stress in older trees or forests (Hagenbo et al., 2020;
Le´on-S´anchez et al., 2018; Querejeta et al., 2021). Under drought stress, the decrease in ECM fungi could be potentially caused by an indirect reduced allocation of carbohydrates to the root system because of a lower assimilation under drought (Fuchslueger et al., 2014; Hasibeder et al., 2015). However, we found no differences in storage carbohydrates in the roots and therefore a role of C allocation seems unlikely and therefore drought effects on ECM fungi may be also attributed to direct effects of water limitation on soil fungal communities. We have also observed that plants surviving drought had a different set of ECM fungi than well-watered trees, and some of these were not affected or were even promoted by drought i. e., ECM taxa with long exploration types (i.
e. Suillus, Rhizopogon). Thus, it seems that drought caused the loss of ECM taxa with short, contact and medium smooth exploration types (i.e.
low biomass; Inocybe, Tomentella, Wilcoxina, Laccaria). The relative in- crease in high biomass fungi (i. e. ECM with long exploration types;
Suillus, Rhizopogon) relative to low biomass after drought could be because several high biomass fungi have structures adapted to efficiently forage for water (Agerer, 2001), particularly hydrophobic rhizomorphs (Agerer, 2001). Hydrophobic mycelia and rhizomorphs in fungi may decrease water loss and, potentially, increase water use efficiency of trees. Interestingly, in our study, Suillus spp. and particularly Suillus variegatus was an indicator species of the seedlings subjected to drought, suggesting that this species may be resistant to drought. Supporting this finding, previous pure culture experiments in which ECM species were grown under distinct water potentials showed drought resistance of S. variegatus, Boletus edulis and Rhizopogon vinicolor (Coleman et al., 1989). Increasing probability of tree survival chance was also previously observed when trees were colonized by Suillus variegatus (Wang et al., 2021) and therefore the presence of this species in surviving trees could be due to an enhanced tree resistance to drought. Our study cannot assess causal relationships between fungal composition and structure with tree mortality, but this and other recent studies show that ECM Fig. 3. a) Canonical correspondence analysis (CCA) showing the variation in the root-associated fungal species composition in well-watered soils and soils subjected to moderate drought from seedlings of the three pine populations (Oria, Coca, and San Ciprian). b) Abundances of the distinct fungal guilds and c) abundances of the distinct exploration types of mycorrhizal fungi across the drought treatments. In a), symbols and colours are according to the mycorrhizal types (ECM = Ectomy- corrhizal, ERM = Ericoid mycorrhizal) and according to the exploration types (High = Long, Low = Contact, medium-smooth, short). Drought treatments: Ww (well- watered), Moderate (Moderate drought). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
with long exploration types may have the capacity to increase plant survival (García de Jal´on et al., 2020). An important remark of our study is that we assessed short-term responses of ECM community to drought.
However, short term responses could differ from long-term ECM re- sponses reported in other studies (e. g. Fernandez et al., 2017) in which the drought-induced shifts in C fluxes belowground can play a larger role.
The lack of fungal community differences among pine populations in well-watered trees suggest that tree genetic differences among pop- ulations do not translate into differences in terms of soil fungal com- munity at earlier stages of tree development. Nevertheless, we observed that total fungal biomass, as estimated by ergosterol biomarker, was higher in San Ciprian populations, paralleling higher above-ground plant biomass. Higher water availability can support higher tree growth typical of more mesic populations, compared to the more con- servative growth of the other two Mediterranean populations assessed in our study (de la Mata et al., 2014). However, 13C signatures were the highest in San Ciprian, which is the most mesic population. This finding seems counter-intuitive, since higher 13C signatures in needles are associated to higher water-use efficiencies (Ferrio et al., 2003; Mor- eno-Guti´errez et al., 2012). Increased allocation to growth together with a greater 13C signature in this population may suggest different and more effective water acquisition strategies. Contrasting with 13C signa- tures, the lower growth and plant biomass of the xeric populations could reflect instead higher water use efficiencies or at least a more conser- vative growth strategy in xeric populations (Corcuera et al., 2012). Thus, other non-stomatal drivers could also have affected photosynthesis and plant growth, e. g. biochemical, nutritional, or metabolic factors (Sage and Kubien, 2007; Salvucci and Crafts-Brandner, 2004). Contrasting biomass partitioning among the populations were also observed. By promoting roots, we speculate that xeric plants can explore more effi- ciently the soil to uptake water and nutrients, potentially also via ECM fungi (Agerer, 2001; Allen and Ection, 2007), which could explain previously observed faster post-drought recovery of xeric populations (S´anchez-Salguero et al., 2018; Zas et al., 2020). By contrast, a higher sink demand of roots from plants of xeric populations could explain the higher long-term mortalities of these populations under drought con- ditions in other studies (S´anchez-Salguero et al., 2018; Zas et al., 2020), even if here the short-term mortality was lower for xeric populations.
However, the among-population pattern of biomass allocation observed in the present study did not follow a clear xeric-to-mesic gradient sug- gesting that short-term responses to drought are not necessarily the same as long-term responses. Further studies should evaluate the links between C allocation to roots and ECM fungi with post-drought recovery of plants.
The interpretation of our results is limited by experimental limita- tions of studying plants in greenhouse under artificial conditions. First, only surviving plants were studied. In the case of moderate drought this was probably not a problem as only 8% of the initial plants died.
However, in the severe drought treatment, mortality affected 73% of the plants, and therefore our observations could be biased. Nevertheless, similar findings concerning high and low biomass ECM were found in moderate and severe drought treatments. An alternative approach could have been to sample ECM in root tips, some of which may still be detectable even when the plants were already dead. However, this approach would have limited the study of other taxa (e. g. saprotrophs and pathogens). Second, it is known that mycelia of fungal species differ in turnover rates depending on their chemical composition and on in- teractions with surrounding roots and litter (Adamczyk et al., 2019;
Fernandez et al., 2016). Thus, certain structures typical of species with long exploration types can decompose slower, especially rhizomorphs (Certano et al., 2018) and it could be that drought affected both exploration types equally, and that differences amongst exploration types were just an artefact. However, long exploration ECM types also include species that produce extensive mycelia (mats) such as Suillus sp.
(Hagenbo et al., 2020) which decompose fast (e. g. Clemmensen et al.,
2015). In addition, while fungal turnover has been estimated by using biochemical methods (Fernandez et al., 2016), it is yet unknown whether differences among taxa persist when using DNA as a biomarker.
Finally, our set-up implies that seedlings are exposed to a limited amount of ECM inoculum, as compared to natural conditions, even if seedlings were grown for several months and were colonized by ECM taxa belonging to distinct exploration strategies. Thus, we cannot rule out the possibility that a more diverse ECM community could have buffered the disproportionate responses to drought among exploration types of ECM fungi.
Our results may have implications for soil processes operating at ecosystem level. Changes in the balance between saprotrophs and ECM fungi have been shown to affect nutrient cycling (Clemmensen et al., 2015). For example, increasing dominance of saprotrophic fungi and bacteria may result in higher organic matter decomposition (Fernandez and Kennedy, 2016), with concomitant loses of soil C (Fernandez and Kennedy, 2016; Querejeta et al., 2021). Impaired ECM communities may also affect post-drought recovery of trees and may increase sus- ceptibility of trees to pathogen attacks, but this remains to be explored.
Author contributions
C. C. conducted soil processing, community analysis and the statis- tical analysis of the data. E. S.-V. R. Z. and L. S. conceived the idea, performed the experiment, assisted with statistical analyses and measured tree chemical defences and physiological features of trees. J.
A. B. assisted with data interpretation and analytical tools. J. O. assisted with data interpretation and sequencing works. C. C. produced the first version of the manuscript and all the authors contributed equally to improve the writing with further revisions of the manuscript.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Data availability
Environmental and sequencing data of this study can be found in Mendeley Data: doi: 10.17632/xw5p3jcf8h.2 and at NCBI’s Sequence Read Archive under accession number PRJNA842073
Acknowledgements
L.S. and R.Z. acknowledge support from Ministerio de Economía y Competitividad/FEDER Grants FUTURPIN AGL2015-68274-C3-2-R, RESILPIN RTI2018-094691-B-C33 and Xunta de Galicia-GAIN grant IN607/2021/03. J.O. was supported by a Ram´on y Cajal fellowship (RYC-2015-17459) and J.A.B. was supported by the Serra-Hunter Pro- gram-Generalitat de Catalunya. The authors would like to acknowledge the constructive contributions of two anonymous reviewers.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.soilbio.2022.108932.
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