Mycorrhiza (2015) 25:67–75 DOI 10.1007/s00572-014-0596-1
ORIGINAL PAPER
Soil moisture—a regulator of arbuscular mycorrhizal fungal community assembly and symbiotic phosphorus uptake Sharma Deepika & David Kothamasi
Received: 13 May 2014 / Accepted: 7 July 2014 / Published online: 2 August 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Multiple species of arbuscular mycorrhizal fungi (AMF) can colonize roots of an individual plant species but factors which determine the selection of a particular AMF species in a plant root are largely unknown. The present work analysed the effects of drought, flooding and optimal soil moisture (15–20 %) on AMF community composition and structure in Sorghum vulgare roots, using PCR-RFLP. Rhizophagus irregularis (isolate BEG 21), and rhizosphere soil (mixed inoculum) of Heteropogon contortus, a perennial C4 grass, collected from the semi-arid Delhi ridge, were used as AMF inocula. Soil moisture functioned as an abiotic filter and affected AMF community assembly inside plant roots by regulating AMF colonization and phylotype diversity. Roots of plants in flooded soils had lowest AMF diversity whilst root AMF diversity was highest under the soil moisture regime of 15–20 %. Although plant biomass was not affected, root P uptake was significantly influenced by soil moisture. Plants colonized with R. irregularis or mixed AMF inoculum showed higher root P uptake than non-mycorrhizal plants in drought and control treatments. No differences in root P levels were found in the flooded treatment between plants colonized with R. irregularis and non-mycorrhizal plants, whilst under the same treatment, root P uptake was lower in plants colonized with mixed AMF inoculum than in non-mycorrhizal plants. Electronic supplementary material The online version of this article (doi:10.1007/s00572-014-0596-1) contains supplementary material, which is available to authorized users. S. Deepika : D. Kothamasi Laboratory of Soil Biology and Microbial Ecology, Department of Environmental Studies, University of Delhi, 110007 Delhi, India D. Kothamasi (*) Biosphere Impact Studies Unit, Belgian Nuclear Research Center (SCK•CEN), Boeretang 200, 2400 Mol, Belgium e-mail: [email protected]
Keywords Arbuscular mycorrhizal fungi . Fungal community composition . Plant P uptake . Soil moisture . Root AMF diversity
Introduction Soil microorganisms are important drivers of plant diversity and productivity in terrestrial ecosystems (van der Heijden et al. 2008) due to their central role in processes such as nutrient cycling, organic carbon sequestration and decomposition of soil organic matter (Herold et al. 2014). A key paradigm in ecology is the identification of factors that determine species distributions and assemblages of biological communities (Cavender-Bares et al. 2009; Stegen et al. 2012; Freitas et al. 2013). Spatial distribution of soil microbial communities is tailored by deterministic and stochastic processes (Stegen et al. 2012). Deterministic processes accentuate differences in species responses to abiotic and biotic factors (Götzenberger et al. 2012; HilleRisLambers et al. 2012; Freitas et al. 2013). Arbuscular mycorrhizal fungi (AMF) may determine diversity and coexistence of plant species (van der Heijden et al. 1998) through selective advantages they provide to host plants in facilitating uptake of nutrients, particularly phosphorus (P), from patchy sources and enabling tolerance to biotic and abiotic stresses (Augé 2001; Kiers et al. 2011; Smith et al. 2011). Ecological benefits derived by the plant from AMF may be offset by up to 20 % plant carbon (C) loss to the fungal symbiont (Jakobsen and Rosendahl 1990). Indeed, when soil P levels are high, AMF may not provide additional benefits to the host and even become parasitic through their C demand on the host (Olsson et al. 2010). The abundance and composition of AMF communities in roots and soils can be influenced by deterministic factors in the rhizosphere such as soil texture, temperature, pH, nutrient availability, organic carbon and soil
68
moisture (Grayston et al. 2001; Brodie et al. 2002; Lauber et al. 2008; Sinsabaugh et al. 2008; Herold et al. 2014). Moreover, not only physical and chemical soil properties but also host plant and seasons can impact on composition and structure of AMF communities (Brundrett 1991; Helgason et al. 2002; Pringle and Bever 2002; Lovelock et al. 2003; Fitzsimons et al. 2008). Global circulation models predict a 5–10 % increase in precipitation in a large part of the northern hemisphere (Intergovernmental Panel on Climate Change 2001). This change can affect soil moisture regimes (Dijkstra and Cheng 2007). Changes in soil moisture regimes have been shown to cause a reorganization of populations of mycorrhizal and nonmycorrhizal hosts and affect AMF colonization of the root and extraradical mycelium in the soil (Miller and Sharitz 2000; Staddon et al. 2003; Clark et al. 2009). However, these studies did not analyse soil moisture effects on the composition and structure of AMF communities inside plant roots, although recent studies have shown preference for specific AMF taxa in aquatic plants (Kohout et al. 2012). When precipitation increases and soils become waterlogged and anaerobic, AMF functioning could be affected due to high fungal oxygen requirements (Holguin et al. 2001). AMF are an important component of the C-cycle in soil (Nottingham et al. 2010; Verbrüggen et al. 2013) as they are a conduit for the transfer of plant-derived C to soils (Jakobsen and Rosendahl 1990; Olsson and Johnson 2005; Drigo et al. 2010). Changes in AMF species composition may affect C translocation to other microbiota. Consequently, changes in edaphic factors that alter AMF abundance could affect below-ground microbial diversity as well as aboveground plant diversity. The following questions were addressed in the present study: (1) does soil moisture affect AMF community structure in roots of Sorghum vulgare and (2) do AMF taxa vary in function of different soil moisture levels?
Materials and methods
Mycorrhiza (2015) 25:67–75
effects (Kiers et al. 2000; Wolfe et al. 2006; Johnson et al. 2010). The rhizosphere of H. contortus was sieved through a sieve of 1-mm mesh size to remove debris such as rock pieces and litter prior to use as inoculum. Greenhouse experiment Experimental pots (4 in. in diameter) were filled with ~520 g of autoclaved (121 °C for 2 h) commercially obtained quartz sand. Ten grammes of viable mycorrhizal inoculum (R. irregularis or mixed inoculum) or 10 g of sterile mycorrhizal inoculum (R. irregularis or mixed inoculum autoclaved at 121 °C for 2 h) were introduced into the pots and covered with quartz sand such that the final weight of the pot was 540 g. Ten milliliters of microbial wash (1:6 soil suspension of R. irregularis inoculum or mixed inoculum filtered through sieves of 150-, 75-, 53-, 25-μm mesh size followed by an 8-μm filter) was added to each pot receiving sterile inoculum to correct for possible differences in bacterial and non-AMF fungal communities (Koide and Li 1989). S. vulgare var. M35, from the Indian Agricultural Research Institute, New Delhi, was used as host plant. Seeds were washed with liquid detergent and surface sterilized using 0.1 % HgCl2 for about 2 min. Surface-sterilized seeds were rinsed with autoclaved double-deionized water and sown in trays containing autoclaved quartz sand. Seven-day old seedlings were transferred to pots with five seedlings per pot. S. vulgare plants were subjected to three different moisture treatments: (1) a drought treatment containing soil with 3–5 % moisture, (2) a control treatment containing soil with 15–20 % moisture, and (3) a flooded treatment in which plants were kept submerged under ~1-cm water (resulting in a 50–60 % soil moisture regime). Percent soil moisture levels were determined and maintained by weighing and watering pots on a daily basis. In order to allow seedlings to tolerate drought or flooding stress, all seedlings were allowed to grow 7 days at 15 % moisture prior to initiating the irrigation treatments. Treatments consisted of three levels of irrigation and four types of inoculation, with three replications per treatment (total 36 pots).
Inoculum Plant growth conditions Rhizophagus irregularis (previously Glomus intraradices; Schüßler et al. 2001), isolate BEG 21 (Swiss Collection of Arbuscular Mycorrhizal Fungi, Agroscope, Zürich), and rhizosphere soil of Heteropogon contortus (L.), a C4 grass collected from the ridge of Delhi, hereafter referred to as mixed inoculum, were employed as mycorrhizal inocula. H. contortus is a commonly occurring perennial grass species in the arid and semi-arid regions of north India. Soils of the Delhi ridge are sandy loam to loam (Chibber 1985), and pH of the soil used as a source of mixed AMF inoculum was 6.5. Studies in the past have used mixed inoculum to analyse AMF
Plants were grown at ambient temperature (35–40 °C) in a greenhouse and day/night cycle of 14–10 h. Seedlings were fertilized with Hoagland solution (Hoagland and Arnon 1950) 12 days after transplantation, followed by weekly fertilization with 4 ml of Hoagland solution with half normal P concentration (for 3 weeks). When plants showed signs of P deficiency, fertilization was increased to 10 ml of Hoagland solution with full P concentration for 2 weeks, followed by 10 ml of Hoagland solution at half strength P for 4 weeks. Pots were randomized weekly.
Mycorrhiza (2015) 25:67–75
Harvest Plants were harvested after 16 weeks of growth and roots washed with tap water to remove the adhering sand. Root and shoot fresh weights were determined separately. A subsample of roots was stored at −20 °C for the extraction of genomic DNA. A second subset of roots was used to estimate AMF colonization. Shoots and roots were dried at 70 °C for 3 days and weighed again to calculate total dry weight and were corrected for the removed fresh weight fractions. Chemical analyses of plant tissues Oven-dried shoot and root tissue was acid digested using the H2SO4-peroxide digestion method (Allen 1989). Phosphorus concentrations were determined using the molybdenum blue method (Chen et al. 1956). AMF root colonization AMF structures in the roots were visualized by clearing the roots in 10 % KOH and staining with 0.05 % trypan blue (Phillips and Hayman 1970). Percent AMF colonization in the roots was estimated following the method of McGonigle et al. (1990). AMF community analysis in roots Genomic DNA was extracted from roots using the cetyltrimethylammonium bromide (CTAB) DNA extraction method (Ausubel et al. 1999). Extracted DNA was subjected to a purification step using the Promega Wizard SV Gel PCR purification kit (Promega, USA). A 550-bp fragment of the 18S (=small sub unit, SSU) ribosomal RNA gene was amplified using the universal eukaryotic primer NS31 (Simon et al. 1992) and the AMF primer AM1 (Helgason et al. 1998). Fifty microliters of PCR reaction mixtures contained 10× Reaction buffer, 0.2 mM of each deoxynucleoside triphosphate (dNTP), 10 pmol of each primer, 0.05 % bovine serum albumin (BSA), 5 % dimethyl sulphoxide (DMSO) and 1.5 U Taq polymerase (Fermentas, EU). The thermal cycling programme comprised one cycle of 94 °C for 3 min, 58 °C for 1 min and 72 °C for 1.5 min, followed by 34 cycles of 94 °C for 30 s, 60 °C for 1 min and 72 °C for 1.5 min and a final elongation at 72 °C for 8 min. Only PCR products from the mixed inoculum mycorrhizal roots were processed further. Amplified products from roots inoculated with the mixed inoculum were cloned into a TA cloning vector (Real Biotech Corporation, Taiwan) and transformed into Escherichia coli HIT competent cells, supplied by Real Biotech Corporation, as per the manufacturer’s instructions. Twenty four positive clones from each sample were reamplified using the primer pair NS31–AM1 and the thermal cycling programme as mentioned above, except that only 30 cycles were run and no BSA or DMSO was added.
69
Restriction fragment length polymorphism (RFLP) patterns of the PCR products were analysed using the restriction enzymes Hsp92II (Promega, USA) and/or Hinf1 (New England Biolabs, USA) as per the manufacturer’s instructions. Based on the relative abundance, three to eight clones per RFLP pattern were sequenced using the universal primer M13F (Macrogen sequencing service, Seoul, South Korea). Two hundred and sixteen clones from nine root samples (three replicates from each mixed inoculum mycorrhizal treatment viz. control, drought and flooded) were RFLP fingerprinted. Based on the abundance, 65 clones representing unique RFLP profiles were sequenced. Clone sequences were screened for chimeras using the Bellerophon chimera check (Huber et al. 2004). Since no chimeras were found, all 65 sequences were compared against NCBI public sequence databases, using the Basic Local Alignment Search Tool (BLASTn) (http://blast.ncbi.nlm.nih.gov) to identify their closest matches. Phylogenetic analysis was performed on the sequences obtained and their most similar database sequences from NCBI. Sequences were aligned using MAFFT version 7.122 (Katoh and Standley 2013). Phylogenetic trees based on maximum likelihood (ML) and neighbour joining (NJ) were constructed using MEGA version 5.2 (Tamura et al. 2011). The ML tree was inferred using the Tamura-Nei model and nearest-neighbourinterchange (NNI) method. The NJ tree was obtained by using Tamura-Nei model and neighbour-joining method. Bootstrap analyses were done with 1,000 replications for both trees. The ML and NJ trees returned similar topologies, and AMF phylotypes were identified on the basis of the agreement between both trees. The ML tree is presented in Supplementary Fig. S1. To determine whether the sequencing effort recovered most AMF phylotypes, rarefaction curves were computed using the Rpackage rich (Rossi 2011) and 1,000 bootstrap randomized datasets. AMF phylotype sequences generated in this study were deposited in GenBank database (Accession numbers KF879027–KF879071, KF612330–KF612335 and KF612337). AMF species diversity in the roots was estimated based on the AMF phylotypes, using the Shannon-Weaver Index (Shannon 1948). Abundance of each AMF phylotype was estimated following Curtis and McIntosh (1950). Relative abundance of each AMF phylotype was determined using the following equation: Relative abundance ¼ ðAbundance of phylotype i sum of abundances of all phylotypesÞ 100
Statistical analysis Differences among means of plant biomass and P uptake due to the different irrigation treatments were analysed by a one-
70
Mycorrhiza (2015) 25:67–75
way analysis of variance (ANOVA). The interactive effects of soil moisture and mycorrhizal colonization on P uptake were analysed using two-way ANOVA. One-way and two-way ANOVAs were run in Minitab version 16.
0.61; p=0.558). No differences in root P uptake were found between mycorrhizal plants colonized by R. irregularis and non-mycorrhizal plants under flooded conditions (Fig. 3a). Plants inoculated with mixed AMF inoculum had higher root P uptake than non-mycorrhizal plants grown with autoclaved inoculum of mixed AMF under control and drought treatments (F11,23 =44.37; p≤0.05). Root P uptake was influenced by both mycorrhizal colonization (F1,12 =11.36; p=0.006) and soil moisture (F2,12 =18.89; p
ORIGINAL PAPER
Soil moisture—a regulator of arbuscular mycorrhizal fungal community assembly and symbiotic phosphorus uptake Sharma Deepika & David Kothamasi
Received: 13 May 2014 / Accepted: 7 July 2014 / Published online: 2 August 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Multiple species of arbuscular mycorrhizal fungi (AMF) can colonize roots of an individual plant species but factors which determine the selection of a particular AMF species in a plant root are largely unknown. The present work analysed the effects of drought, flooding and optimal soil moisture (15–20 %) on AMF community composition and structure in Sorghum vulgare roots, using PCR-RFLP. Rhizophagus irregularis (isolate BEG 21), and rhizosphere soil (mixed inoculum) of Heteropogon contortus, a perennial C4 grass, collected from the semi-arid Delhi ridge, were used as AMF inocula. Soil moisture functioned as an abiotic filter and affected AMF community assembly inside plant roots by regulating AMF colonization and phylotype diversity. Roots of plants in flooded soils had lowest AMF diversity whilst root AMF diversity was highest under the soil moisture regime of 15–20 %. Although plant biomass was not affected, root P uptake was significantly influenced by soil moisture. Plants colonized with R. irregularis or mixed AMF inoculum showed higher root P uptake than non-mycorrhizal plants in drought and control treatments. No differences in root P levels were found in the flooded treatment between plants colonized with R. irregularis and non-mycorrhizal plants, whilst under the same treatment, root P uptake was lower in plants colonized with mixed AMF inoculum than in non-mycorrhizal plants. Electronic supplementary material The online version of this article (doi:10.1007/s00572-014-0596-1) contains supplementary material, which is available to authorized users. S. Deepika : D. Kothamasi Laboratory of Soil Biology and Microbial Ecology, Department of Environmental Studies, University of Delhi, 110007 Delhi, India D. Kothamasi (*) Biosphere Impact Studies Unit, Belgian Nuclear Research Center (SCK•CEN), Boeretang 200, 2400 Mol, Belgium e-mail: [email protected]
Keywords Arbuscular mycorrhizal fungi . Fungal community composition . Plant P uptake . Soil moisture . Root AMF diversity
Introduction Soil microorganisms are important drivers of plant diversity and productivity in terrestrial ecosystems (van der Heijden et al. 2008) due to their central role in processes such as nutrient cycling, organic carbon sequestration and decomposition of soil organic matter (Herold et al. 2014). A key paradigm in ecology is the identification of factors that determine species distributions and assemblages of biological communities (Cavender-Bares et al. 2009; Stegen et al. 2012; Freitas et al. 2013). Spatial distribution of soil microbial communities is tailored by deterministic and stochastic processes (Stegen et al. 2012). Deterministic processes accentuate differences in species responses to abiotic and biotic factors (Götzenberger et al. 2012; HilleRisLambers et al. 2012; Freitas et al. 2013). Arbuscular mycorrhizal fungi (AMF) may determine diversity and coexistence of plant species (van der Heijden et al. 1998) through selective advantages they provide to host plants in facilitating uptake of nutrients, particularly phosphorus (P), from patchy sources and enabling tolerance to biotic and abiotic stresses (Augé 2001; Kiers et al. 2011; Smith et al. 2011). Ecological benefits derived by the plant from AMF may be offset by up to 20 % plant carbon (C) loss to the fungal symbiont (Jakobsen and Rosendahl 1990). Indeed, when soil P levels are high, AMF may not provide additional benefits to the host and even become parasitic through their C demand on the host (Olsson et al. 2010). The abundance and composition of AMF communities in roots and soils can be influenced by deterministic factors in the rhizosphere such as soil texture, temperature, pH, nutrient availability, organic carbon and soil
68
moisture (Grayston et al. 2001; Brodie et al. 2002; Lauber et al. 2008; Sinsabaugh et al. 2008; Herold et al. 2014). Moreover, not only physical and chemical soil properties but also host plant and seasons can impact on composition and structure of AMF communities (Brundrett 1991; Helgason et al. 2002; Pringle and Bever 2002; Lovelock et al. 2003; Fitzsimons et al. 2008). Global circulation models predict a 5–10 % increase in precipitation in a large part of the northern hemisphere (Intergovernmental Panel on Climate Change 2001). This change can affect soil moisture regimes (Dijkstra and Cheng 2007). Changes in soil moisture regimes have been shown to cause a reorganization of populations of mycorrhizal and nonmycorrhizal hosts and affect AMF colonization of the root and extraradical mycelium in the soil (Miller and Sharitz 2000; Staddon et al. 2003; Clark et al. 2009). However, these studies did not analyse soil moisture effects on the composition and structure of AMF communities inside plant roots, although recent studies have shown preference for specific AMF taxa in aquatic plants (Kohout et al. 2012). When precipitation increases and soils become waterlogged and anaerobic, AMF functioning could be affected due to high fungal oxygen requirements (Holguin et al. 2001). AMF are an important component of the C-cycle in soil (Nottingham et al. 2010; Verbrüggen et al. 2013) as they are a conduit for the transfer of plant-derived C to soils (Jakobsen and Rosendahl 1990; Olsson and Johnson 2005; Drigo et al. 2010). Changes in AMF species composition may affect C translocation to other microbiota. Consequently, changes in edaphic factors that alter AMF abundance could affect below-ground microbial diversity as well as aboveground plant diversity. The following questions were addressed in the present study: (1) does soil moisture affect AMF community structure in roots of Sorghum vulgare and (2) do AMF taxa vary in function of different soil moisture levels?
Materials and methods
Mycorrhiza (2015) 25:67–75
effects (Kiers et al. 2000; Wolfe et al. 2006; Johnson et al. 2010). The rhizosphere of H. contortus was sieved through a sieve of 1-mm mesh size to remove debris such as rock pieces and litter prior to use as inoculum. Greenhouse experiment Experimental pots (4 in. in diameter) were filled with ~520 g of autoclaved (121 °C for 2 h) commercially obtained quartz sand. Ten grammes of viable mycorrhizal inoculum (R. irregularis or mixed inoculum) or 10 g of sterile mycorrhizal inoculum (R. irregularis or mixed inoculum autoclaved at 121 °C for 2 h) were introduced into the pots and covered with quartz sand such that the final weight of the pot was 540 g. Ten milliliters of microbial wash (1:6 soil suspension of R. irregularis inoculum or mixed inoculum filtered through sieves of 150-, 75-, 53-, 25-μm mesh size followed by an 8-μm filter) was added to each pot receiving sterile inoculum to correct for possible differences in bacterial and non-AMF fungal communities (Koide and Li 1989). S. vulgare var. M35, from the Indian Agricultural Research Institute, New Delhi, was used as host plant. Seeds were washed with liquid detergent and surface sterilized using 0.1 % HgCl2 for about 2 min. Surface-sterilized seeds were rinsed with autoclaved double-deionized water and sown in trays containing autoclaved quartz sand. Seven-day old seedlings were transferred to pots with five seedlings per pot. S. vulgare plants were subjected to three different moisture treatments: (1) a drought treatment containing soil with 3–5 % moisture, (2) a control treatment containing soil with 15–20 % moisture, and (3) a flooded treatment in which plants were kept submerged under ~1-cm water (resulting in a 50–60 % soil moisture regime). Percent soil moisture levels were determined and maintained by weighing and watering pots on a daily basis. In order to allow seedlings to tolerate drought or flooding stress, all seedlings were allowed to grow 7 days at 15 % moisture prior to initiating the irrigation treatments. Treatments consisted of three levels of irrigation and four types of inoculation, with three replications per treatment (total 36 pots).
Inoculum Plant growth conditions Rhizophagus irregularis (previously Glomus intraradices; Schüßler et al. 2001), isolate BEG 21 (Swiss Collection of Arbuscular Mycorrhizal Fungi, Agroscope, Zürich), and rhizosphere soil of Heteropogon contortus (L.), a C4 grass collected from the ridge of Delhi, hereafter referred to as mixed inoculum, were employed as mycorrhizal inocula. H. contortus is a commonly occurring perennial grass species in the arid and semi-arid regions of north India. Soils of the Delhi ridge are sandy loam to loam (Chibber 1985), and pH of the soil used as a source of mixed AMF inoculum was 6.5. Studies in the past have used mixed inoculum to analyse AMF
Plants were grown at ambient temperature (35–40 °C) in a greenhouse and day/night cycle of 14–10 h. Seedlings were fertilized with Hoagland solution (Hoagland and Arnon 1950) 12 days after transplantation, followed by weekly fertilization with 4 ml of Hoagland solution with half normal P concentration (for 3 weeks). When plants showed signs of P deficiency, fertilization was increased to 10 ml of Hoagland solution with full P concentration for 2 weeks, followed by 10 ml of Hoagland solution at half strength P for 4 weeks. Pots were randomized weekly.
Mycorrhiza (2015) 25:67–75
Harvest Plants were harvested after 16 weeks of growth and roots washed with tap water to remove the adhering sand. Root and shoot fresh weights were determined separately. A subsample of roots was stored at −20 °C for the extraction of genomic DNA. A second subset of roots was used to estimate AMF colonization. Shoots and roots were dried at 70 °C for 3 days and weighed again to calculate total dry weight and were corrected for the removed fresh weight fractions. Chemical analyses of plant tissues Oven-dried shoot and root tissue was acid digested using the H2SO4-peroxide digestion method (Allen 1989). Phosphorus concentrations were determined using the molybdenum blue method (Chen et al. 1956). AMF root colonization AMF structures in the roots were visualized by clearing the roots in 10 % KOH and staining with 0.05 % trypan blue (Phillips and Hayman 1970). Percent AMF colonization in the roots was estimated following the method of McGonigle et al. (1990). AMF community analysis in roots Genomic DNA was extracted from roots using the cetyltrimethylammonium bromide (CTAB) DNA extraction method (Ausubel et al. 1999). Extracted DNA was subjected to a purification step using the Promega Wizard SV Gel PCR purification kit (Promega, USA). A 550-bp fragment of the 18S (=small sub unit, SSU) ribosomal RNA gene was amplified using the universal eukaryotic primer NS31 (Simon et al. 1992) and the AMF primer AM1 (Helgason et al. 1998). Fifty microliters of PCR reaction mixtures contained 10× Reaction buffer, 0.2 mM of each deoxynucleoside triphosphate (dNTP), 10 pmol of each primer, 0.05 % bovine serum albumin (BSA), 5 % dimethyl sulphoxide (DMSO) and 1.5 U Taq polymerase (Fermentas, EU). The thermal cycling programme comprised one cycle of 94 °C for 3 min, 58 °C for 1 min and 72 °C for 1.5 min, followed by 34 cycles of 94 °C for 30 s, 60 °C for 1 min and 72 °C for 1.5 min and a final elongation at 72 °C for 8 min. Only PCR products from the mixed inoculum mycorrhizal roots were processed further. Amplified products from roots inoculated with the mixed inoculum were cloned into a TA cloning vector (Real Biotech Corporation, Taiwan) and transformed into Escherichia coli HIT competent cells, supplied by Real Biotech Corporation, as per the manufacturer’s instructions. Twenty four positive clones from each sample were reamplified using the primer pair NS31–AM1 and the thermal cycling programme as mentioned above, except that only 30 cycles were run and no BSA or DMSO was added.
69
Restriction fragment length polymorphism (RFLP) patterns of the PCR products were analysed using the restriction enzymes Hsp92II (Promega, USA) and/or Hinf1 (New England Biolabs, USA) as per the manufacturer’s instructions. Based on the relative abundance, three to eight clones per RFLP pattern were sequenced using the universal primer M13F (Macrogen sequencing service, Seoul, South Korea). Two hundred and sixteen clones from nine root samples (three replicates from each mixed inoculum mycorrhizal treatment viz. control, drought and flooded) were RFLP fingerprinted. Based on the abundance, 65 clones representing unique RFLP profiles were sequenced. Clone sequences were screened for chimeras using the Bellerophon chimera check (Huber et al. 2004). Since no chimeras were found, all 65 sequences were compared against NCBI public sequence databases, using the Basic Local Alignment Search Tool (BLASTn) (http://blast.ncbi.nlm.nih.gov) to identify their closest matches. Phylogenetic analysis was performed on the sequences obtained and their most similar database sequences from NCBI. Sequences were aligned using MAFFT version 7.122 (Katoh and Standley 2013). Phylogenetic trees based on maximum likelihood (ML) and neighbour joining (NJ) were constructed using MEGA version 5.2 (Tamura et al. 2011). The ML tree was inferred using the Tamura-Nei model and nearest-neighbourinterchange (NNI) method. The NJ tree was obtained by using Tamura-Nei model and neighbour-joining method. Bootstrap analyses were done with 1,000 replications for both trees. The ML and NJ trees returned similar topologies, and AMF phylotypes were identified on the basis of the agreement between both trees. The ML tree is presented in Supplementary Fig. S1. To determine whether the sequencing effort recovered most AMF phylotypes, rarefaction curves were computed using the Rpackage rich (Rossi 2011) and 1,000 bootstrap randomized datasets. AMF phylotype sequences generated in this study were deposited in GenBank database (Accession numbers KF879027–KF879071, KF612330–KF612335 and KF612337). AMF species diversity in the roots was estimated based on the AMF phylotypes, using the Shannon-Weaver Index (Shannon 1948). Abundance of each AMF phylotype was estimated following Curtis and McIntosh (1950). Relative abundance of each AMF phylotype was determined using the following equation: Relative abundance ¼ ðAbundance of phylotype i sum of abundances of all phylotypesÞ 100
Statistical analysis Differences among means of plant biomass and P uptake due to the different irrigation treatments were analysed by a one-
70
Mycorrhiza (2015) 25:67–75
way analysis of variance (ANOVA). The interactive effects of soil moisture and mycorrhizal colonization on P uptake were analysed using two-way ANOVA. One-way and two-way ANOVAs were run in Minitab version 16.
0.61; p=0.558). No differences in root P uptake were found between mycorrhizal plants colonized by R. irregularis and non-mycorrhizal plants under flooded conditions (Fig. 3a). Plants inoculated with mixed AMF inoculum had higher root P uptake than non-mycorrhizal plants grown with autoclaved inoculum of mixed AMF under control and drought treatments (F11,23 =44.37; p≤0.05). Root P uptake was influenced by both mycorrhizal colonization (F1,12 =11.36; p=0.006) and soil moisture (F2,12 =18.89; p
