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Genotype and soil water availability shape the composition of AMF communities at chickpea early growth stages
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Genotype and soil water availability shape the composition of AMF communities at chickpea early growth stages A. Kavadiaa, M. Omiroua, , D. Fasoulab, S. Trajanoskid, E. Andreouc, I.M. Ioannidesa, ⁎
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a
Department of Agrobiotechnology, Agricultural Research Institute, Nicosia, Cyprus Department of Plant Breeding, Agricultural Research Institute, Nicosia, Cyprus c Department of Soil Quality, Wageningen University and Research, Wageningen, Netherlands d Center for Medical Research, Medical University of Graz, Graz, Austria b
ARTICLE INFO
ABSTRACT
Keywords: Drought stress Chickpea Mycorrhizal fungi
Chickpea is one of the most important legumes in the world and could be dramatically affected from water limitation. Chickpea is associated with arbuscular mycorrhizal fungi (AMF) that are known for their contribution to the alleviation of drought stress in plants. In the current study we evaluated the effect of water limitation in two chickpea genotypes (A345 and A365) and how water limitation affected plant performance and AMF symbiosis (composition and colonization) in a microcosm study in a complete randomized design. Water limitation had a detrimental effect only in A345 genotype performance and this was depended on plant growth stage. On the contrary, the biomass production of A365 genotype was not affected from water limitation 55 days after the initiation of the experiment. This response was associated with AMF colonization. The colonization found in the sensitive to water limitation genotype A345 was substantially suppressed in both growth stages while the colonization of the tolerant, A365 genotype was not affected under drought stress conditions. Multivariate analysis showed that the composition of AMF communities in chickpea was significantly affected from the interaction between growth stage, genotype and water availability (p < 0.01). Differential abundance analyses showed that the abundance of specific AMF genera in the tolerant to water limitation genotype A365 was substantially decreased at early growth stages compared to the sensitive genotype A345. This finding highlights that A365 could be less depended on mycorrhizal presence and/or the AMF colonizers is more efficient acting complementary to the plant inherent drought tolerant mechanisms to alleviate water shortage.
1. Introduction Climate change is affecting agroecosystems productivity through changes of plant growth conditions. Changes in water availability and particularly the increase of frequency and severity of droughts threatened food security (Bowles et al., 2016a) (Trenberth et al., 2014).Tackling climate change requires reducing nitrogen inputs and increasing the efficiency of its use in agriculture. This can be achieved by increasing the cultivation of legume due to their ability to fix atmospheric nitrogen. However, the yield of legumes are significantly affected from irrigation water availability and rainfall in their cultivation area. Chickpea is one of the most important legumes in the world (Sohrabi et al., 2015; Kumar et al., 2018). It is largely grown under rainfed conditions in arid and semi-arid areas where drought stress is a major limiting factor for yields (Al-Karaki, 1998; Leport et al., 2006). Various chickpea genotypes available from germplasm resources are ⁎
exhibiting drought tolerance ensuring grain yields and yield stability (Ramamoorthy et al., 2016). This tolerance in chickpea has been attributed to the root system and particularly the root length density and root dry biomass (Ramamoorthy et al., 2017). Besides morphological plasticity, plants have evolved biochemical and physiological changes including plant-microbe interactions to phase water limitation. The association of plant roots with soil microbial communities, including arbuscular mycorrhizal fungi (AMF) tend to enhance plant tolerance to drought by enhancing several of the already existing mechanisms. Several studies demonstrated the significance of AMF in plant water stress alleviation (Jayne and Quigley, 2014; Fernández-Lizarazo and Moreno-Fonseca, 2016; Farooq et al., 2017; Omirou et al., 2013). It has been shown that AMF regulated plant physiology and metabolism through changes in the hormone balance of the host plants (Goicoechea et al., 1997; Wu, 2017). Moreover, AMF improved the osmotic adjustment and enhance water lift from roots to
Correspondence to: M. Omirou, Department of Agrobiotechnology, Agricultural Microbiology Laboratory, Athalassa, Cyprus. Correspondence to: I.M. Ioannides, Department of Agrobiotechnology, Molecular Biology Laboratory, Athalassa, Cyprus. E-mail addresses: [email protected] (M. Omirou), [email protected] (I.M. Ioannides).
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https://doi.org/10.1016/j.apsoil.2019.103443 Received 9 June 2019; Received in revised form 18 October 2019; Accepted 10 November 2019 0929-1393/ © 2019 Published by Elsevier B.V.
Please cite this article as: A. Kavadia, et al., Applied Soil Ecology, https://doi.org/10.1016/j.apsoil.2019.103443
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shoots via higher water apoplastic flow (Bárzana et al., 2012). Recent studies showed an induction of aquaporin proteins expression in AMF plants that was associated with water transportation in plant cells suggesting a significant importance of AMF symbiosis on water movement at a molecular level (Aroca et al., 2007; Ruiz-Lozano et al., 2009). Identification of chickpea genotypes that can form effective symbiotic relations with native AMF can affect chickpea yield (Bazghaleh et al., 2015). It is reported that different genotypes of chickpea can select their rhizosphere microbial communities through the biosynthesis of several root chemicals (Yang et al., 2012; Ellouze et al., 2013). Indeed, water limitation causes changes in primary and secondary metabolites, which in turn are affecting chickpea-mycorrhizal symbiosis (Farooq et al., 2009). In addition, root growth stage is affecting the composition and richness of plant roots in AMF (YiJong et al., 2014) while water availability affects root lifespan and turnover (Brunner et al., 2015) which in turn affects AMF symbiosis. Previous studies demonstrated that changes in soil moisture affected the composition and richness of mycorrhizal communities in several annual grasses (Deveautour et al., 2018; Deepika and Kothamasi, 2014; Li et al., 2015). However, the impact of native AMF assemblages on chickpea plants grown under water-limited conditions in different genotypes has been overlooked. Yet, the association of chickpea drought tolerance with AMF symbiosis and the diversity of these fungi in the roots of chickpea genotypes were not assessed. The objective of this study was to evaluate the response of two different chickpea genotypes grown under well water and limited water conditions in a microcosm experiment. We hypothesized that the responses of chickpea genotypes to water limitation is associated with nutrient accumulation and mycorrhizal colonization. In addition, the response AMF assemblages associated with the root system of two chickpea genotypes grown under two different water regimes was evaluated. We hypothesized that chickpea genotype and water availability are factors significantly affecting AMF community composition/ structure.
2.2. Plant sampling and nutrient analysis A destructive sampling approach was followed and plants were harvested at two different growth stages (30 and 55 days after sowing, days after application (DAA)) resulting in 12 samples at each sampling point (full combination of chickpea genotypes and water regime). Above ground biomass was separated from the root system and immediately weighted. Then dry weight was determined after drying plant biomass at 60 °C until constant weight was achieved. Dried above ground plant material was grinded and used for N, P and K determination. In detail, 100 mg of dry samples were used for total N determination according to Dumas combustion method using the EA analyser. A wet digestion procedure (conc. H2SO4 and 30% H2O2) at 300 °C was performed prior to P and K determination. Phosphorus content was determined using the vanadium phosphomolybdate colorimetric method, while potassium using a flame photometer (Roy, 2008). 2.3. AMF development and extraradical mycelium measurement
2. Materials and methods
Root samples were washed out carefully with de-ionized water to remove the adherent soil and the excess water was removed using a tissue paper. In detail 5 g of total root were used for determination of AMF colonization. Root segments were rinsed and cleared using 10% KOH in water bath at 70 °C for 30 min. Then KOH was removed, roots were washed with water and acidified with 1% HCl. Subsequently the roots were stained using a 0.05% Trypan blue solution (v/v) (Koske and Gemma, 1989). The mycorrhizal colonization was calculated using the grid-line intersect method and expressed as percentage of the length of root segments containing AMF structures (Giovannetti and Mosse, 1980). Extraradical mycelium (ERM) was evaluated in eight membrane filters that were established in each pot prior seeding the plants (Baláz and Vosátka, 2001). After sampling, filters were carefully removed and stained for 45 min using Trypan blue (0.05%) and distained using glycerol. ERM hyphae length was measured under a stereoscope by using the grid-line intersect method (Giovannetti and Mosse, 1980; Newman, 1966; Tennant, 1975).
2.1. Plant material, experimental design and growth conditions
2.4. DNA extraction and NGS sequencing
The chickpea (Cicer arietinum) genotypes used in this experiment namely A345 and A365 were kindly provided by the National Plant Gene Bank of Cyprus. The seeds were surface sterilized with 2% sodium hypochlorite solution for 5 min and rinsed several times with autoclaved water. Seeds were germinated in petri dishes with sterile moistened filter paper in an incubator at 25 °C for 5 days in dark conditions. A factorial microcosm experiment was established and germinated seeds were sowed in 3 L pots containing non-sterilized soil with an active indigenous AMF community. The soil characteristics were pH 8.50, organic matter 1.06% dw, Olsen-P 43.33 mg/kg and total N 0.08%. The layout of the experiment was in a completely randomized factorial design with two factors (Genotype X Water Regime). In detail, the two different plant genotypes A345 and A365 (Factor 1) were grown under two different water regimes (Factor 2). Water treatments were calculated according the soil water capacity. In detail, well-watered plants (WW) and plants grown under drought stress (DS) received water equal to the 70% and 30% of soil water holding capacity (WHC) respectively and each combination was replicated 3 times. Plants were established 10 days prior the initiation and implementation of water stress to allow adequate plant growth. The water loss from each pot adjusted daily by weighing and supplementing water when needed to maintain water levels in the soil. Plants were maintained in a greenhouse for 55 days with a mean day and night temperature of 17 and 7 °C respectively.
Clean root samples were ground in liquid nitrogen using mortar and pestle and the powder was transferred to 1.5 ml tubes and stored at −80 °C until extraction. Root DNA was extracted by 200 mg of grounded root, using a commercially available kit (DNeasy Plant Mini Kit, Qiagen, USA) following manufacturer's instructions. A two-step PCR was performed to generate the sequencing libraries for Illumina MiSeq NGS platform. The first round PCR was performed using primers AML1/AML2 (Lee et al., 2008) under the following conditions: 94 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, 50 °C for 40 s and 72 °C for 1 min, after which a final elongation step at 72 °C for 5 min was performed. The second round PCR was performed using primers WANDA/AML2 (Dumbrell et al., 2011) under the following conditions: 94 °C for 3 min, followed by 20 cycles of 94 °C for 30 s, 53 °C for 40 s and 72 °C for 1 min, and a final elongation step at 72 °C for 5 min. After amplification, PCR products were checked in 2% agarose gel to determine the success of amplification and the relative intensity of bands. Multiple samples were pooled together in equal proportions based on their molecular weight and DNA concentrations. Pooled samples were purified using calibrated Ampure XP beads. Then the pooled and purified PCR product was used to prepare illumina DNA library. Sequencing was performed at MR DNA (www.mrdnalab.com, Shallowater, TX, USA) on a MiSeq following the manufacturer's guidelines. 2.5. QIIME2 data analysis with maarjaAM DB for taxonomic classification The raw sequence data was processed using Quantitative Insights 2
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Into Microbial Ecology (QIIME2, https://qiime2.org/) (Caporaso et al., 2010) bioinformatics software integrated into Galaxy (Afgan et al., 2016) web-based platform. The very first step incorporated applying DADA2 (Callahan et al., 2016) pipeline for de-noising and de-replicating of the paired-end sequences including chimera removal at the end. For this process forward and reverse reads where truncated due to decrease in quality and amplicon primers removed. DADA2 output was the feature table with abundance frequencies for each sample and representative sequences of every feature. For the taxonomy assignment of the representative sequences new classifier based on MaarjAM database (Öpik et al., 2010) was build. The sequences were deposited in NCBI SRA database with accession number PRJNA546128.
between genotype and water level was noticed (p < 0.05) at both growth stages. Under water-limited conditions (DS), at early growth stages (30 DAA) A345 accumulated more K in above ground biomass however this was not noticed at later growth stages where no differences were noticed (Table 1). On the contrary, A365 plants at early growth stages (30 DAA) experienced water limited (DS) conditions, had lower K content compared to plants grown under well water conditions (Table 1). Contrary to N and K, P content in chickpea above ground biomass was not affected either by each factor separately or by their interaction and the content ranged from 0.12 to 0.18 mg/kg dw at both growth stages (range of values?) (Table 1). 3.3. Mycorrhizal fungi colonization and correlations with plant parameters
2.6. Statistical analysis
Genotype had a detrimental effect on mycorrhizal colonization of chickpea genotypes and this was depended on water availability leading to a significant interaction at both growth stages (p < 0.05). Overall, AMF colonization in A345 was higher under WW compared to DS in both growth stages while, no differences on colonization between WW and DS were found for A365 (Fig. 2). On the contrary, none of the factors examined, had a significant effect on ERM length measured at both growth stages (data not shown). A positive significant correlation noticed between AMF colonization and above ground biomass production (R = 0.81, p < 0.01) and P content (R = 0.67, p < 0.05) in A345. In the same genotype, there was a strong positive correlation between the % of root colonization with mycorrhizal and P content in the above ground biomass (R = 0.67, p < 0.05). This was not the case for A365 where no significant correlation between above ground biomass or P content and AMF colonization was noticed (R = 0.21, p = 0.51, R = 0.14, p = 0.66).
All statistical analyses were performed in RStudio version 1.1.456 (R Core Team, 2016) Analysis of Variance (ANOVA) was used to examine the main effects of water regime and genotypes as well as their interactions on plant nutrient content and uptake, above ground plant biomass, mycorrhizal colonization and ERM length within the two growth stages. Mean comparisons using t-test (a < 0.05) was used to determine differences among the different treatments. Shannon index of mycorrhizal fungi was estimate with estimate_richness function (“phyloseq” package version 1.26.1) and statistical significance of growth stage, genotype and water availability was determined using ANOVA. Mycorrhizal composition variations in the genotypes under the different water levels, growth stages was assessed using UniFrac distance matrices (Lozupone et al., 2011). Package DESeq2 version 1.22.2 (Love et al., 2014) was used to calculate the differential abundance of each OTU identified (log2-fold change in relative abundance of each OTU) for each genotype as compared with water availability within each growth stage. The p-values was adjusted by the BH (Benjamini and Hochberg) correction method and a FDR (false discovery rate) of 10% was used to denote statistical significance. Finally, we selected the responders with log2-fold change in relative abundance > 1% and an adjusted p-value of < 0.05 for further community analysis.
3.4. Diversity of AMF in chickpea genotypes grown under different water levels Totally, 1,983,054 sequences were generated from 24 samples and from those 1,652,545 sequences belonged to Glomeromycota with an average number of sequences per sample was 68,856. These sequences were clustered to 214 operational taxonomic units (OTUs). The majority of these OTUs have been assigned at Genus level to Glomus (82%), followed by Rhizophagus (7.3%), Funneliformis (6.8%), Claroideoglomus (2.4%) and Septoglomus (1.5%) (Fig. 3). The Shannon Index in this study ranged from 1.05 to 2.47 and Analysis of Variance (ANOVA) showed a significant 2-way interaction between plant genotype and water level (F = 13.36, p = 0.00213). Thirty days after the implementation of the experiment, water availability had a significant effect on Shannon Index but this was genotype depended. In detail, the highest diversity was observed in A345 plants grown under drought stress, 30 days after the initiation of the experiment (Fig. 4). Overall, water availability affected significantly the Shannon Index of AMF in both growth stages in A345 plants. On the contrary, 55 days after the initiation of the experiment neither water level (F = 0.533, p = 0.16) nor growth stage (F = 0.239, p = 0.63) influenced Shannon Index of in A365 plants (Fig. 4). Similarly, NDMS ordination (Fig. 5) and PERMANOVA analysis showed that the composition of AMF communities in chickpea was significantly affected from the interaction between growth stage, genotype and water availability (PERMANOVA: F1,23 = 14.27, R2 = 0.27; p = 0.002). Further analysis, within sampling period (30 DAA and 55 DAA) revealed that AMF communities were affected by genotype and water availability only 30 days after the initiation of the experiment (Supplementary Table 1). Additionally, dispersion analysis in 999 permutations revealed that samples had similar variances (F = 0.176, p = 0.685) further supporting that AMF communities are different due to genotype and water availability and not from increased variance of the community within each group. Differential abundance analyses were conducted in DESeq2 to determine which taxa were significantly different in water-limited
3. Results 3.1. Aboveground biomass response to drought stress During time, biomass production increased in all treatments however chickpea biomass accumulation was affected from water availability and was genotype depended exhibiting a significant interaction between plant genotype and water availability (Fig. 1). The biomass of A345 was always higher from that of A365 irrespectively time and water availability however the response of the genotypes to water availability during time was different (Fig. 1). In detail, 30 days after the initiation of the experiment, dry aboveground biomass accumulation was not affected from water deficit in both genotypes (Fig. 1). At later growth stage (55DAA) the above ground biomass of A345 plants grown under water deficit conditions (DS) was lower compare to A345 plants grown under well water conditions (WW). On the contrary, A365 biomass production was not affected from water shortage 55 days after the initiation of the experiment (Fig. 1). 3.2. Impact of water availability on above ground biomass nutrient content The implemented treatments, had main as well as interactive effects on N, P and K content and uptake during the different growth stages examined (Table 1). During the first 30 days, the N content was not affected from the implemented treatments and no differences between genotypes were noticed. Nitrogen content was significantly affected by plant genotype at 55 DAA (p < 0.05) and A365 genotype had higher N content compared to A345 (Table 1). Potassium content was only affected by water level (p < 0.05) while a significant interaction 3
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Fig. 1. Above ground biomass (g/plant) of two different chickpea genotypes (A345 and A365) grown under well water (WW) and drought stress (DS) conditions sampled 30 and 55 days after the initiation of the experiment. Error bars show standard error of the mean (n = 3). Different small letters indicates statistically significant differences between water availability (p < 0.05) within chickpea genotype. Different capital letters indicates statistically significant differences between chickpea genotypes (p < 0.05) within water treatment. Side table shows two-ANOVA results.
compared to well watered plants in each genotype, during the two different plant growth stages (30 and 55 DAA). At early growth stage (30 DAA) in A345 water limitation resulted in a substantial (padj < 0.05) reduction of 10 and to a significant increase of the abundance of 27 out of 131 OTUs identified in the current study (Supplementary Table 2). Interestingly, all Rhizophagus like OTUs that were significantly affected from water limitation were enriched contrary to the majority (66%) of Claroideoglomus like OTUs that were significantly reduced (Supplementary Table 2). On the other hand, in
A365 plants of the same growth stage (30 DAA) 25 out of 131 OTUs were significantly affected from soil water availability. From those, the abundance of 19 OTUs was negatively affected from limited water while the 6 OTUs were enriched. Similarly to A345, Septoglomus assigned OTUs that were significantly affected were enriched under water-limited conditions. On the other hand, the 85% of the Rhizophagus assigned OTUs were substantially reduced while all OTUs that were assigned in Funneliformis genus was significantly reduced (Supplementary data 2).
Table 1 Nitrogen (g/100 g dw) and K (g/100 g dw) content in chickpea above ground biomass as affected by genotype (A345 and A365) and water availability (well-watered, WW; drought stress, DS) at two different growth stages, 30 and 55 days after the initiation of the experiment. Genotype
Water Level
Days after the initiation of the experiment 30 days
55 days
N (g/100 g dw) A345 A365
Well-watered Drought stress Well-watered Drought stress
†
4.63 4.01 4.43 5.12
K (g/100 g dw) ‡
0.51 0.36 0.08 0.18
1.55 1.80 2.09 1.31
N (g/100 g dw) 0.09 0.12 0.26 0.06
†
§
aB aA aA bB
0.11 0.35 0.34 0.25
aA aA aB aB
K (g/100 g dw) 1.52 1.47 1.98 1.45
1.52 1.47 1.98 1.45
0.11 0.24 0.04 0.19
aB aA aA bA
Mean value and ‡ standard error of the mean (n = 3) of nitrogen and potassium content, § Different small letters at examined parameters indicated significant differences between water levels within genotype based on LSD test (p < 0.05). Different capital letters at examined parameters indicated significant differences between genotypes within the same water level (p < 0.05). 4
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Fig. 2. Mycorrhizal root colonization of two different chickpea genotypes (A345 and A365) grown under well water (WW) and drought stress (DS) conditions sampled 30 and 55 days after the initiation of the experiment. Error bars show standard error of the mean (n = 3). Different small letters indicates statistically significant differences between water availability (p < 0.05) within chickpea genotype. Different capital letters indicates statistically significant differences between chickpea genotypes (p < 0.05) within water treatment.
4. Discussion
contrary, biomass accumulation in A365 in plants grown under waterlimited conditions (DS) at early growth stages (30 DAA) was similar with that of plants grown under well-watered conditions (WW). These findings suggest that A365 tolerates water limitation than A345 during the early stages of the growing period. Previous studies demonstrated that there is great variability among chickpea genotypes regarding drought tolerance (Hosseini et al., 2009; Sachdeva et al., 2018; Badhan et al., 2018) and outperforming chickpea genotypes under drought stress conditions have been reported (Imtiaz and Malhotra, 2009; Talebi
Water scarcity due to climate change is expected to affect negatively chickpea productivity in rain fed agricultural ecosystems (Maqbool et al., 2017). The present study showed a substantial genotypic variation in drought tolerance between the tested genotypes which is in line with previous reports (Farooq et al., 2018; Nayyar et al., 2006). For example, A345 was very sensitive to water deficit exhibiting a growth reduction of 10 and 16% at 30 and 55 days after seeding. On the
Fig. 3. The relative abundance (% of total sequences) of the mycorrhizal genus found in two different chickpea genotypes (A345 and A365) grown under well water (WW) and drought stress (DS) conditions sampled 30 and 55 days after the initiation of the experiment. 5
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Fig. 4. Comparison, of alpha diversity of the mycorrhizal fungi measured by Shannon diversity index between the different water schemes employed (WW and DS) within each variety at two different growth stages (30 and 55 DAA).
Fig. 5. Non-metric Multidimensional Scaling (NMDS) ordination plot of the mycorrhizal community of chickpea genotypes (A345 and A365) under well water (WW) and drought stress (DS) conditions, 30 and 55 days after the initiation of the experiment. Scaling was based on UniFrac Weighted distances (stress = 0.07). 6
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et al., 2013). Several studies have shown that nutrient uptake and accumulation was hampered under water deficit conditions in various plant species (He and Dijkstra, 2014). However the response of different species and genotypes within species to nutrient uptake varies and the ability of increased uptake under drought stress conditions has been related with plant drought tolerance (Farooq et al., 2018). In the current study, the two genotypes responded differently to water limitation regarding the N and K accumulation in above ground biomass while no response of P content was noticed (Table 1). This in line with previous findings showed differences in K content among genotypes under different water availability levels (Farooq et al., 2018; Singh and Kuhad, 2005). Numerous studies provide evidence that mycorrhizal symbiosis alleviates drought stress in different plant species (Osakabe et al., 2014; Augé, 2001; Omirou et al., 2013; Augé et al., 2014; Lehmann et al., 2014; Lehmann and Rillig, 2015). The observed tolerance of AMF plants to drought has been attributed to their enhanced water and nutrient uptake ability from soil through the extensive extraradical mycelium and root colonization (Bowles et al., 2016). In the current study, the AMF colonization of the sensitive genotype (A345) was substantially suppressed in both growth stages while the colonization of the tolerant (A365) genotype was not affected (Fig. 1). The high significant correlation noticed between AMF colonization and above ground biomass production (R = 0.81, p < 0.01) and P content (R = 0.67, p < 0.05) in A345, suggests that its performance (biomass and P content) is tightly linked with mycorrhizal symbiosis. Klironomos (2003) demonstrated that the functional diversity among the native AMF assemblages could positively or negatively affect plant growth response and this response is a balance between the benefits that AMF are providing to plants and their cost to sustain the symbiosis. Previous studies demonstrated that plant species as well genotypes are affecting AMF symbiosis and there is strong evidence that during selection, plant genotypes lose their ability to form and benefit from AMF (Leiser et al., 2016; Martín-Robles et al., 2018). It is possible that selection procedure had affected the ability of genotype A345 to form beneficial mycorrhizal symbiosis under drought contrary to A365 in which colonization remained constant under these conditions. Mechanisms involved in the different response of these genotypes to AMF colonization as well as functioning need to be elucidated. Additionally, it has been shown that AMF species are responded differently under water deficit conditions and contrasting effects on plant performance have been noted (Neumann et al., 2010; Symanczik et al., 2018) indicating that AMF phylotypes could be adapted under drought conditions and confer beneficial effects to their symbionts compared to non-adapted isolates (Brito et al., 2011). In the current study limited water availability caused a transient effect on the native AMF assemblages and this effect was genotype depended. For example, 30 days after the initiation of the experiment a clear interaction between genotype and water availability was noticed (Fig. 3). Additionally, the highest Shannon Index was found in A345 plants grown under drought stress and was significantly different than that measured in plants of the same genotype grown under not limited water conditions. Although the overall AMF community structure was not affected either by genotype nor water availability 55 DAA, when analysed as a population, differential abundance analysis showed that specific OTUs significantly enriched or reduced under water limited conditions and this was depended on the genotype (Supplementary Table 3). These findings suggest the presence of a native functional AMF community that varies in function due to water availability and is closely related with chickpea genotype as well as the plant growth stage. Few studies have reported changes in the composition and richness of native mycorrhizal assemblages due to changes in soil water availability (Deepika and Kothamasi, 2014; Li et al., 2015; Silva et al., 2015; Omirou et al., 2013). It has been mentioned that AMF diversity depends on seasonal variations of abiotic environmental factors that are related
to host response to mycorrhizal symbiosis (Bever et al., 2006; SánchezCastro et al., 2012). In the current study, in A365 at early growth stage (30 DAA), the number of the significantly negatively affected OTUs from water limitation was substantially higher compared to that of A345 (Supplementary Table 2). These findings suggest that A365 act as biological filter selecting the AMF genera that will colonize plant roots (Verbruggen et al., 2012; Kiers et al., 2011; Torrecillas et al., 2012) while at the same time the % of root colonization didn't change. The mechanisms that are shaping AMF community in the roots under water limited conditions could be the accumulation of primary and secondary metabolites in plants (Farooq et al., 2009) as well as changes that drought causes to root morphology and architecture which in turn affect the symbiosis of plants with mycorrhizal fungi (Deveautour et al., 2018). Interestingly, in the current study, the Rhizophagus like OTUs were enriched in the roots of the sensitive to water limitation chickpea genotype (A345) at early growth stage (30 DAA) while the opposite was noticed in A365. At later growth stage (55 DAA) in A345 only four OTUs were significantly changed contrary to A365 where an enrichment of Claroideoglomus like OTUs was noticed (Supplementary Tables 2 and 3). The increase of Rhizophagus abundance in A345 plants grown under water limited could be related to the opportunistic behavior of the species of this genus, which are early colonizers and are generating high number of spores in short term period (Lenoir et al., 2016). Apparently, the increased abundance of the different AMF genera noticed in A345 at early growth stage didn't alleviate the negative effect of water limitation on plant biomass. These findings are highlighting the importance of fungal-host complementarity for the alleviation of drought stress. 5. Conclusion In conclusion the results of the current study showed that water limitation affects the composition and the abundance of native mycorrhizal assemblages that varies in function, and is associated with chickpea genotype as well as the plant growth stage. The tolerant to water limitation genotype A365 sustain AMF symbiosis in terms of root colonization while the abundance of specific AMF genera was substantially decreased at early growth stages compared to the sensitive genotype A345. Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Acknowledgements This work was financially supported by European Union within the DIVERSIFOOD H2020 project. We acknowledge the technical assistance of Mrs. Evdokia Neophytou and Mrs. Louiza Konstantinou. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.apsoil.2019.103443. References Afgan, E., Baker, D., van den Beek, M., Blankenberg, D., Bouvier, D., Čech, M., Chilton, J., Clements, D., Coraor, N., Eberhard, C., Grüning, B., Guerler, A., Hillman-Jackson, J., Von Kuster, G., Rasche, E., Soranzo, N., Turaga, N., Taylor, J., Nekrutenko, A., Goecks, J., 2016. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update. Nucleic Acids Res. 44, W3–W10. https://doi.org/ 10.1093/nar/gkw343. Al-Karaki, G.N., 1998. Benefit, cost and water-use efficiency of arbuscular mycorrhizal durum wheat grown under drought stress. Mycorrhiza 8, 41–45. https://doi.org/10.
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Genotype and soil water availability shape the composition of AMF communities at chickpea early growth stages A. Kavadiaa, M. Omiroua, , D. Fasoulab, S. Trajanoskid, E. Andreouc, I.M. Ioannidesa, ⁎
⁎⁎
a
Department of Agrobiotechnology, Agricultural Research Institute, Nicosia, Cyprus Department of Plant Breeding, Agricultural Research Institute, Nicosia, Cyprus c Department of Soil Quality, Wageningen University and Research, Wageningen, Netherlands d Center for Medical Research, Medical University of Graz, Graz, Austria b
ARTICLE INFO
ABSTRACT
Keywords: Drought stress Chickpea Mycorrhizal fungi
Chickpea is one of the most important legumes in the world and could be dramatically affected from water limitation. Chickpea is associated with arbuscular mycorrhizal fungi (AMF) that are known for their contribution to the alleviation of drought stress in plants. In the current study we evaluated the effect of water limitation in two chickpea genotypes (A345 and A365) and how water limitation affected plant performance and AMF symbiosis (composition and colonization) in a microcosm study in a complete randomized design. Water limitation had a detrimental effect only in A345 genotype performance and this was depended on plant growth stage. On the contrary, the biomass production of A365 genotype was not affected from water limitation 55 days after the initiation of the experiment. This response was associated with AMF colonization. The colonization found in the sensitive to water limitation genotype A345 was substantially suppressed in both growth stages while the colonization of the tolerant, A365 genotype was not affected under drought stress conditions. Multivariate analysis showed that the composition of AMF communities in chickpea was significantly affected from the interaction between growth stage, genotype and water availability (p < 0.01). Differential abundance analyses showed that the abundance of specific AMF genera in the tolerant to water limitation genotype A365 was substantially decreased at early growth stages compared to the sensitive genotype A345. This finding highlights that A365 could be less depended on mycorrhizal presence and/or the AMF colonizers is more efficient acting complementary to the plant inherent drought tolerant mechanisms to alleviate water shortage.
1. Introduction Climate change is affecting agroecosystems productivity through changes of plant growth conditions. Changes in water availability and particularly the increase of frequency and severity of droughts threatened food security (Bowles et al., 2016a) (Trenberth et al., 2014).Tackling climate change requires reducing nitrogen inputs and increasing the efficiency of its use in agriculture. This can be achieved by increasing the cultivation of legume due to their ability to fix atmospheric nitrogen. However, the yield of legumes are significantly affected from irrigation water availability and rainfall in their cultivation area. Chickpea is one of the most important legumes in the world (Sohrabi et al., 2015; Kumar et al., 2018). It is largely grown under rainfed conditions in arid and semi-arid areas where drought stress is a major limiting factor for yields (Al-Karaki, 1998; Leport et al., 2006). Various chickpea genotypes available from germplasm resources are ⁎
exhibiting drought tolerance ensuring grain yields and yield stability (Ramamoorthy et al., 2016). This tolerance in chickpea has been attributed to the root system and particularly the root length density and root dry biomass (Ramamoorthy et al., 2017). Besides morphological plasticity, plants have evolved biochemical and physiological changes including plant-microbe interactions to phase water limitation. The association of plant roots with soil microbial communities, including arbuscular mycorrhizal fungi (AMF) tend to enhance plant tolerance to drought by enhancing several of the already existing mechanisms. Several studies demonstrated the significance of AMF in plant water stress alleviation (Jayne and Quigley, 2014; Fernández-Lizarazo and Moreno-Fonseca, 2016; Farooq et al., 2017; Omirou et al., 2013). It has been shown that AMF regulated plant physiology and metabolism through changes in the hormone balance of the host plants (Goicoechea et al., 1997; Wu, 2017). Moreover, AMF improved the osmotic adjustment and enhance water lift from roots to
Correspondence to: M. Omirou, Department of Agrobiotechnology, Agricultural Microbiology Laboratory, Athalassa, Cyprus. Correspondence to: I.M. Ioannides, Department of Agrobiotechnology, Molecular Biology Laboratory, Athalassa, Cyprus. E-mail addresses: [email protected] (M. Omirou), [email protected] (I.M. Ioannides).
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https://doi.org/10.1016/j.apsoil.2019.103443 Received 9 June 2019; Received in revised form 18 October 2019; Accepted 10 November 2019 0929-1393/ © 2019 Published by Elsevier B.V.
Please cite this article as: A. Kavadia, et al., Applied Soil Ecology, https://doi.org/10.1016/j.apsoil.2019.103443
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A. Kavadia, et al.
shoots via higher water apoplastic flow (Bárzana et al., 2012). Recent studies showed an induction of aquaporin proteins expression in AMF plants that was associated with water transportation in plant cells suggesting a significant importance of AMF symbiosis on water movement at a molecular level (Aroca et al., 2007; Ruiz-Lozano et al., 2009). Identification of chickpea genotypes that can form effective symbiotic relations with native AMF can affect chickpea yield (Bazghaleh et al., 2015). It is reported that different genotypes of chickpea can select their rhizosphere microbial communities through the biosynthesis of several root chemicals (Yang et al., 2012; Ellouze et al., 2013). Indeed, water limitation causes changes in primary and secondary metabolites, which in turn are affecting chickpea-mycorrhizal symbiosis (Farooq et al., 2009). In addition, root growth stage is affecting the composition and richness of plant roots in AMF (YiJong et al., 2014) while water availability affects root lifespan and turnover (Brunner et al., 2015) which in turn affects AMF symbiosis. Previous studies demonstrated that changes in soil moisture affected the composition and richness of mycorrhizal communities in several annual grasses (Deveautour et al., 2018; Deepika and Kothamasi, 2014; Li et al., 2015). However, the impact of native AMF assemblages on chickpea plants grown under water-limited conditions in different genotypes has been overlooked. Yet, the association of chickpea drought tolerance with AMF symbiosis and the diversity of these fungi in the roots of chickpea genotypes were not assessed. The objective of this study was to evaluate the response of two different chickpea genotypes grown under well water and limited water conditions in a microcosm experiment. We hypothesized that the responses of chickpea genotypes to water limitation is associated with nutrient accumulation and mycorrhizal colonization. In addition, the response AMF assemblages associated with the root system of two chickpea genotypes grown under two different water regimes was evaluated. We hypothesized that chickpea genotype and water availability are factors significantly affecting AMF community composition/ structure.
2.2. Plant sampling and nutrient analysis A destructive sampling approach was followed and plants were harvested at two different growth stages (30 and 55 days after sowing, days after application (DAA)) resulting in 12 samples at each sampling point (full combination of chickpea genotypes and water regime). Above ground biomass was separated from the root system and immediately weighted. Then dry weight was determined after drying plant biomass at 60 °C until constant weight was achieved. Dried above ground plant material was grinded and used for N, P and K determination. In detail, 100 mg of dry samples were used for total N determination according to Dumas combustion method using the EA analyser. A wet digestion procedure (conc. H2SO4 and 30% H2O2) at 300 °C was performed prior to P and K determination. Phosphorus content was determined using the vanadium phosphomolybdate colorimetric method, while potassium using a flame photometer (Roy, 2008). 2.3. AMF development and extraradical mycelium measurement
2. Materials and methods
Root samples were washed out carefully with de-ionized water to remove the adherent soil and the excess water was removed using a tissue paper. In detail 5 g of total root were used for determination of AMF colonization. Root segments were rinsed and cleared using 10% KOH in water bath at 70 °C for 30 min. Then KOH was removed, roots were washed with water and acidified with 1% HCl. Subsequently the roots were stained using a 0.05% Trypan blue solution (v/v) (Koske and Gemma, 1989). The mycorrhizal colonization was calculated using the grid-line intersect method and expressed as percentage of the length of root segments containing AMF structures (Giovannetti and Mosse, 1980). Extraradical mycelium (ERM) was evaluated in eight membrane filters that were established in each pot prior seeding the plants (Baláz and Vosátka, 2001). After sampling, filters were carefully removed and stained for 45 min using Trypan blue (0.05%) and distained using glycerol. ERM hyphae length was measured under a stereoscope by using the grid-line intersect method (Giovannetti and Mosse, 1980; Newman, 1966; Tennant, 1975).
2.1. Plant material, experimental design and growth conditions
2.4. DNA extraction and NGS sequencing
The chickpea (Cicer arietinum) genotypes used in this experiment namely A345 and A365 were kindly provided by the National Plant Gene Bank of Cyprus. The seeds were surface sterilized with 2% sodium hypochlorite solution for 5 min and rinsed several times with autoclaved water. Seeds were germinated in petri dishes with sterile moistened filter paper in an incubator at 25 °C for 5 days in dark conditions. A factorial microcosm experiment was established and germinated seeds were sowed in 3 L pots containing non-sterilized soil with an active indigenous AMF community. The soil characteristics were pH 8.50, organic matter 1.06% dw, Olsen-P 43.33 mg/kg and total N 0.08%. The layout of the experiment was in a completely randomized factorial design with two factors (Genotype X Water Regime). In detail, the two different plant genotypes A345 and A365 (Factor 1) were grown under two different water regimes (Factor 2). Water treatments were calculated according the soil water capacity. In detail, well-watered plants (WW) and plants grown under drought stress (DS) received water equal to the 70% and 30% of soil water holding capacity (WHC) respectively and each combination was replicated 3 times. Plants were established 10 days prior the initiation and implementation of water stress to allow adequate plant growth. The water loss from each pot adjusted daily by weighing and supplementing water when needed to maintain water levels in the soil. Plants were maintained in a greenhouse for 55 days with a mean day and night temperature of 17 and 7 °C respectively.
Clean root samples were ground in liquid nitrogen using mortar and pestle and the powder was transferred to 1.5 ml tubes and stored at −80 °C until extraction. Root DNA was extracted by 200 mg of grounded root, using a commercially available kit (DNeasy Plant Mini Kit, Qiagen, USA) following manufacturer's instructions. A two-step PCR was performed to generate the sequencing libraries for Illumina MiSeq NGS platform. The first round PCR was performed using primers AML1/AML2 (Lee et al., 2008) under the following conditions: 94 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, 50 °C for 40 s and 72 °C for 1 min, after which a final elongation step at 72 °C for 5 min was performed. The second round PCR was performed using primers WANDA/AML2 (Dumbrell et al., 2011) under the following conditions: 94 °C for 3 min, followed by 20 cycles of 94 °C for 30 s, 53 °C for 40 s and 72 °C for 1 min, and a final elongation step at 72 °C for 5 min. After amplification, PCR products were checked in 2% agarose gel to determine the success of amplification and the relative intensity of bands. Multiple samples were pooled together in equal proportions based on their molecular weight and DNA concentrations. Pooled samples were purified using calibrated Ampure XP beads. Then the pooled and purified PCR product was used to prepare illumina DNA library. Sequencing was performed at MR DNA (www.mrdnalab.com, Shallowater, TX, USA) on a MiSeq following the manufacturer's guidelines. 2.5. QIIME2 data analysis with maarjaAM DB for taxonomic classification The raw sequence data was processed using Quantitative Insights 2
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Into Microbial Ecology (QIIME2, https://qiime2.org/) (Caporaso et al., 2010) bioinformatics software integrated into Galaxy (Afgan et al., 2016) web-based platform. The very first step incorporated applying DADA2 (Callahan et al., 2016) pipeline for de-noising and de-replicating of the paired-end sequences including chimera removal at the end. For this process forward and reverse reads where truncated due to decrease in quality and amplicon primers removed. DADA2 output was the feature table with abundance frequencies for each sample and representative sequences of every feature. For the taxonomy assignment of the representative sequences new classifier based on MaarjAM database (Öpik et al., 2010) was build. The sequences were deposited in NCBI SRA database with accession number PRJNA546128.
between genotype and water level was noticed (p < 0.05) at both growth stages. Under water-limited conditions (DS), at early growth stages (30 DAA) A345 accumulated more K in above ground biomass however this was not noticed at later growth stages where no differences were noticed (Table 1). On the contrary, A365 plants at early growth stages (30 DAA) experienced water limited (DS) conditions, had lower K content compared to plants grown under well water conditions (Table 1). Contrary to N and K, P content in chickpea above ground biomass was not affected either by each factor separately or by their interaction and the content ranged from 0.12 to 0.18 mg/kg dw at both growth stages (range of values?) (Table 1). 3.3. Mycorrhizal fungi colonization and correlations with plant parameters
2.6. Statistical analysis
Genotype had a detrimental effect on mycorrhizal colonization of chickpea genotypes and this was depended on water availability leading to a significant interaction at both growth stages (p < 0.05). Overall, AMF colonization in A345 was higher under WW compared to DS in both growth stages while, no differences on colonization between WW and DS were found for A365 (Fig. 2). On the contrary, none of the factors examined, had a significant effect on ERM length measured at both growth stages (data not shown). A positive significant correlation noticed between AMF colonization and above ground biomass production (R = 0.81, p < 0.01) and P content (R = 0.67, p < 0.05) in A345. In the same genotype, there was a strong positive correlation between the % of root colonization with mycorrhizal and P content in the above ground biomass (R = 0.67, p < 0.05). This was not the case for A365 where no significant correlation between above ground biomass or P content and AMF colonization was noticed (R = 0.21, p = 0.51, R = 0.14, p = 0.66).
All statistical analyses were performed in RStudio version 1.1.456 (R Core Team, 2016) Analysis of Variance (ANOVA) was used to examine the main effects of water regime and genotypes as well as their interactions on plant nutrient content and uptake, above ground plant biomass, mycorrhizal colonization and ERM length within the two growth stages. Mean comparisons using t-test (a < 0.05) was used to determine differences among the different treatments. Shannon index of mycorrhizal fungi was estimate with estimate_richness function (“phyloseq” package version 1.26.1) and statistical significance of growth stage, genotype and water availability was determined using ANOVA. Mycorrhizal composition variations in the genotypes under the different water levels, growth stages was assessed using UniFrac distance matrices (Lozupone et al., 2011). Package DESeq2 version 1.22.2 (Love et al., 2014) was used to calculate the differential abundance of each OTU identified (log2-fold change in relative abundance of each OTU) for each genotype as compared with water availability within each growth stage. The p-values was adjusted by the BH (Benjamini and Hochberg) correction method and a FDR (false discovery rate) of 10% was used to denote statistical significance. Finally, we selected the responders with log2-fold change in relative abundance > 1% and an adjusted p-value of < 0.05 for further community analysis.
3.4. Diversity of AMF in chickpea genotypes grown under different water levels Totally, 1,983,054 sequences were generated from 24 samples and from those 1,652,545 sequences belonged to Glomeromycota with an average number of sequences per sample was 68,856. These sequences were clustered to 214 operational taxonomic units (OTUs). The majority of these OTUs have been assigned at Genus level to Glomus (82%), followed by Rhizophagus (7.3%), Funneliformis (6.8%), Claroideoglomus (2.4%) and Septoglomus (1.5%) (Fig. 3). The Shannon Index in this study ranged from 1.05 to 2.47 and Analysis of Variance (ANOVA) showed a significant 2-way interaction between plant genotype and water level (F = 13.36, p = 0.00213). Thirty days after the implementation of the experiment, water availability had a significant effect on Shannon Index but this was genotype depended. In detail, the highest diversity was observed in A345 plants grown under drought stress, 30 days after the initiation of the experiment (Fig. 4). Overall, water availability affected significantly the Shannon Index of AMF in both growth stages in A345 plants. On the contrary, 55 days after the initiation of the experiment neither water level (F = 0.533, p = 0.16) nor growth stage (F = 0.239, p = 0.63) influenced Shannon Index of in A365 plants (Fig. 4). Similarly, NDMS ordination (Fig. 5) and PERMANOVA analysis showed that the composition of AMF communities in chickpea was significantly affected from the interaction between growth stage, genotype and water availability (PERMANOVA: F1,23 = 14.27, R2 = 0.27; p = 0.002). Further analysis, within sampling period (30 DAA and 55 DAA) revealed that AMF communities were affected by genotype and water availability only 30 days after the initiation of the experiment (Supplementary Table 1). Additionally, dispersion analysis in 999 permutations revealed that samples had similar variances (F = 0.176, p = 0.685) further supporting that AMF communities are different due to genotype and water availability and not from increased variance of the community within each group. Differential abundance analyses were conducted in DESeq2 to determine which taxa were significantly different in water-limited
3. Results 3.1. Aboveground biomass response to drought stress During time, biomass production increased in all treatments however chickpea biomass accumulation was affected from water availability and was genotype depended exhibiting a significant interaction between plant genotype and water availability (Fig. 1). The biomass of A345 was always higher from that of A365 irrespectively time and water availability however the response of the genotypes to water availability during time was different (Fig. 1). In detail, 30 days after the initiation of the experiment, dry aboveground biomass accumulation was not affected from water deficit in both genotypes (Fig. 1). At later growth stage (55DAA) the above ground biomass of A345 plants grown under water deficit conditions (DS) was lower compare to A345 plants grown under well water conditions (WW). On the contrary, A365 biomass production was not affected from water shortage 55 days after the initiation of the experiment (Fig. 1). 3.2. Impact of water availability on above ground biomass nutrient content The implemented treatments, had main as well as interactive effects on N, P and K content and uptake during the different growth stages examined (Table 1). During the first 30 days, the N content was not affected from the implemented treatments and no differences between genotypes were noticed. Nitrogen content was significantly affected by plant genotype at 55 DAA (p < 0.05) and A365 genotype had higher N content compared to A345 (Table 1). Potassium content was only affected by water level (p < 0.05) while a significant interaction 3
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Fig. 1. Above ground biomass (g/plant) of two different chickpea genotypes (A345 and A365) grown under well water (WW) and drought stress (DS) conditions sampled 30 and 55 days after the initiation of the experiment. Error bars show standard error of the mean (n = 3). Different small letters indicates statistically significant differences between water availability (p < 0.05) within chickpea genotype. Different capital letters indicates statistically significant differences between chickpea genotypes (p < 0.05) within water treatment. Side table shows two-ANOVA results.
compared to well watered plants in each genotype, during the two different plant growth stages (30 and 55 DAA). At early growth stage (30 DAA) in A345 water limitation resulted in a substantial (padj < 0.05) reduction of 10 and to a significant increase of the abundance of 27 out of 131 OTUs identified in the current study (Supplementary Table 2). Interestingly, all Rhizophagus like OTUs that were significantly affected from water limitation were enriched contrary to the majority (66%) of Claroideoglomus like OTUs that were significantly reduced (Supplementary Table 2). On the other hand, in
A365 plants of the same growth stage (30 DAA) 25 out of 131 OTUs were significantly affected from soil water availability. From those, the abundance of 19 OTUs was negatively affected from limited water while the 6 OTUs were enriched. Similarly to A345, Septoglomus assigned OTUs that were significantly affected were enriched under water-limited conditions. On the other hand, the 85% of the Rhizophagus assigned OTUs were substantially reduced while all OTUs that were assigned in Funneliformis genus was significantly reduced (Supplementary data 2).
Table 1 Nitrogen (g/100 g dw) and K (g/100 g dw) content in chickpea above ground biomass as affected by genotype (A345 and A365) and water availability (well-watered, WW; drought stress, DS) at two different growth stages, 30 and 55 days after the initiation of the experiment. Genotype
Water Level
Days after the initiation of the experiment 30 days
55 days
N (g/100 g dw) A345 A365
Well-watered Drought stress Well-watered Drought stress
†
4.63 4.01 4.43 5.12
K (g/100 g dw) ‡
0.51 0.36 0.08 0.18
1.55 1.80 2.09 1.31
N (g/100 g dw) 0.09 0.12 0.26 0.06
†
§
aB aA aA bB
0.11 0.35 0.34 0.25
aA aA aB aB
K (g/100 g dw) 1.52 1.47 1.98 1.45
1.52 1.47 1.98 1.45
0.11 0.24 0.04 0.19
aB aA aA bA
Mean value and ‡ standard error of the mean (n = 3) of nitrogen and potassium content, § Different small letters at examined parameters indicated significant differences between water levels within genotype based on LSD test (p < 0.05). Different capital letters at examined parameters indicated significant differences between genotypes within the same water level (p < 0.05). 4
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Fig. 2. Mycorrhizal root colonization of two different chickpea genotypes (A345 and A365) grown under well water (WW) and drought stress (DS) conditions sampled 30 and 55 days after the initiation of the experiment. Error bars show standard error of the mean (n = 3). Different small letters indicates statistically significant differences between water availability (p < 0.05) within chickpea genotype. Different capital letters indicates statistically significant differences between chickpea genotypes (p < 0.05) within water treatment.
4. Discussion
contrary, biomass accumulation in A365 in plants grown under waterlimited conditions (DS) at early growth stages (30 DAA) was similar with that of plants grown under well-watered conditions (WW). These findings suggest that A365 tolerates water limitation than A345 during the early stages of the growing period. Previous studies demonstrated that there is great variability among chickpea genotypes regarding drought tolerance (Hosseini et al., 2009; Sachdeva et al., 2018; Badhan et al., 2018) and outperforming chickpea genotypes under drought stress conditions have been reported (Imtiaz and Malhotra, 2009; Talebi
Water scarcity due to climate change is expected to affect negatively chickpea productivity in rain fed agricultural ecosystems (Maqbool et al., 2017). The present study showed a substantial genotypic variation in drought tolerance between the tested genotypes which is in line with previous reports (Farooq et al., 2018; Nayyar et al., 2006). For example, A345 was very sensitive to water deficit exhibiting a growth reduction of 10 and 16% at 30 and 55 days after seeding. On the
Fig. 3. The relative abundance (% of total sequences) of the mycorrhizal genus found in two different chickpea genotypes (A345 and A365) grown under well water (WW) and drought stress (DS) conditions sampled 30 and 55 days after the initiation of the experiment. 5
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Fig. 4. Comparison, of alpha diversity of the mycorrhizal fungi measured by Shannon diversity index between the different water schemes employed (WW and DS) within each variety at two different growth stages (30 and 55 DAA).
Fig. 5. Non-metric Multidimensional Scaling (NMDS) ordination plot of the mycorrhizal community of chickpea genotypes (A345 and A365) under well water (WW) and drought stress (DS) conditions, 30 and 55 days after the initiation of the experiment. Scaling was based on UniFrac Weighted distances (stress = 0.07). 6
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et al., 2013). Several studies have shown that nutrient uptake and accumulation was hampered under water deficit conditions in various plant species (He and Dijkstra, 2014). However the response of different species and genotypes within species to nutrient uptake varies and the ability of increased uptake under drought stress conditions has been related with plant drought tolerance (Farooq et al., 2018). In the current study, the two genotypes responded differently to water limitation regarding the N and K accumulation in above ground biomass while no response of P content was noticed (Table 1). This in line with previous findings showed differences in K content among genotypes under different water availability levels (Farooq et al., 2018; Singh and Kuhad, 2005). Numerous studies provide evidence that mycorrhizal symbiosis alleviates drought stress in different plant species (Osakabe et al., 2014; Augé, 2001; Omirou et al., 2013; Augé et al., 2014; Lehmann et al., 2014; Lehmann and Rillig, 2015). The observed tolerance of AMF plants to drought has been attributed to their enhanced water and nutrient uptake ability from soil through the extensive extraradical mycelium and root colonization (Bowles et al., 2016). In the current study, the AMF colonization of the sensitive genotype (A345) was substantially suppressed in both growth stages while the colonization of the tolerant (A365) genotype was not affected (Fig. 1). The high significant correlation noticed between AMF colonization and above ground biomass production (R = 0.81, p < 0.01) and P content (R = 0.67, p < 0.05) in A345, suggests that its performance (biomass and P content) is tightly linked with mycorrhizal symbiosis. Klironomos (2003) demonstrated that the functional diversity among the native AMF assemblages could positively or negatively affect plant growth response and this response is a balance between the benefits that AMF are providing to plants and their cost to sustain the symbiosis. Previous studies demonstrated that plant species as well genotypes are affecting AMF symbiosis and there is strong evidence that during selection, plant genotypes lose their ability to form and benefit from AMF (Leiser et al., 2016; Martín-Robles et al., 2018). It is possible that selection procedure had affected the ability of genotype A345 to form beneficial mycorrhizal symbiosis under drought contrary to A365 in which colonization remained constant under these conditions. Mechanisms involved in the different response of these genotypes to AMF colonization as well as functioning need to be elucidated. Additionally, it has been shown that AMF species are responded differently under water deficit conditions and contrasting effects on plant performance have been noted (Neumann et al., 2010; Symanczik et al., 2018) indicating that AMF phylotypes could be adapted under drought conditions and confer beneficial effects to their symbionts compared to non-adapted isolates (Brito et al., 2011). In the current study limited water availability caused a transient effect on the native AMF assemblages and this effect was genotype depended. For example, 30 days after the initiation of the experiment a clear interaction between genotype and water availability was noticed (Fig. 3). Additionally, the highest Shannon Index was found in A345 plants grown under drought stress and was significantly different than that measured in plants of the same genotype grown under not limited water conditions. Although the overall AMF community structure was not affected either by genotype nor water availability 55 DAA, when analysed as a population, differential abundance analysis showed that specific OTUs significantly enriched or reduced under water limited conditions and this was depended on the genotype (Supplementary Table 3). These findings suggest the presence of a native functional AMF community that varies in function due to water availability and is closely related with chickpea genotype as well as the plant growth stage. Few studies have reported changes in the composition and richness of native mycorrhizal assemblages due to changes in soil water availability (Deepika and Kothamasi, 2014; Li et al., 2015; Silva et al., 2015; Omirou et al., 2013). It has been mentioned that AMF diversity depends on seasonal variations of abiotic environmental factors that are related
to host response to mycorrhizal symbiosis (Bever et al., 2006; SánchezCastro et al., 2012). In the current study, in A365 at early growth stage (30 DAA), the number of the significantly negatively affected OTUs from water limitation was substantially higher compared to that of A345 (Supplementary Table 2). These findings suggest that A365 act as biological filter selecting the AMF genera that will colonize plant roots (Verbruggen et al., 2012; Kiers et al., 2011; Torrecillas et al., 2012) while at the same time the % of root colonization didn't change. The mechanisms that are shaping AMF community in the roots under water limited conditions could be the accumulation of primary and secondary metabolites in plants (Farooq et al., 2009) as well as changes that drought causes to root morphology and architecture which in turn affect the symbiosis of plants with mycorrhizal fungi (Deveautour et al., 2018). Interestingly, in the current study, the Rhizophagus like OTUs were enriched in the roots of the sensitive to water limitation chickpea genotype (A345) at early growth stage (30 DAA) while the opposite was noticed in A365. At later growth stage (55 DAA) in A345 only four OTUs were significantly changed contrary to A365 where an enrichment of Claroideoglomus like OTUs was noticed (Supplementary Tables 2 and 3). The increase of Rhizophagus abundance in A345 plants grown under water limited could be related to the opportunistic behavior of the species of this genus, which are early colonizers and are generating high number of spores in short term period (Lenoir et al., 2016). Apparently, the increased abundance of the different AMF genera noticed in A345 at early growth stage didn't alleviate the negative effect of water limitation on plant biomass. These findings are highlighting the importance of fungal-host complementarity for the alleviation of drought stress. 5. Conclusion In conclusion the results of the current study showed that water limitation affects the composition and the abundance of native mycorrhizal assemblages that varies in function, and is associated with chickpea genotype as well as the plant growth stage. The tolerant to water limitation genotype A365 sustain AMF symbiosis in terms of root colonization while the abundance of specific AMF genera was substantially decreased at early growth stages compared to the sensitive genotype A345. Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Acknowledgements This work was financially supported by European Union within the DIVERSIFOOD H2020 project. We acknowledge the technical assistance of Mrs. Evdokia Neophytou and Mrs. Louiza Konstantinou. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.apsoil.2019.103443. References Afgan, E., Baker, D., van den Beek, M., Blankenberg, D., Bouvier, D., Čech, M., Chilton, J., Clements, D., Coraor, N., Eberhard, C., Grüning, B., Guerler, A., Hillman-Jackson, J., Von Kuster, G., Rasche, E., Soranzo, N., Turaga, N., Taylor, J., Nekrutenko, A., Goecks, J., 2016. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update. Nucleic Acids Res. 44, W3–W10. https://doi.org/ 10.1093/nar/gkw343. Al-Karaki, G.N., 1998. Benefit, cost and water-use efficiency of arbuscular mycorrhizal durum wheat grown under drought stress. Mycorrhiza 8, 41–45. https://doi.org/10.
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