We studied the diversity of arbuscular mycorrhizal fungi (AMF) in a semiarid grassland and the effect of long-term nitrogen (N) fertilization on this fungal community. Root samples of Bouteloua gracilis were collected at the Sevilleta National Wildlife Refuge (New Mexico, USA) from control and N-amended plots that have been fertilized since 1995. Small subunit rDNA was amplified using AMF specific primers NS31 and AM1. The diversity of AMF was low in comparison with other ecosystems, only seven operational taxonomic units (OTU) were found in B. gracilis and all belong to the genus Glomus. The dominant OTU was closely related to the ubiquitous G. intraradices/G. fasciculatum group. N-amended plots showed a reduction in the abundance of the dominant OTU and an increase in AMF diversity. The greater AMF diversity in roots from N-amended plots may have been the result of displacement of the dominant OTU, which facilitated detection of uncommon AMF. The long-term implications of AMF responses to N enrichment for plant carbon allocation and plant community structure remain unclear.
Roots from three Bouteloua gracilis plants were collected from three control and three N-amended plots (a total of 72 plants).
Plant samples were collected in plastic bags, kept at 4 C and processed the same or next day after collection.
Whole roots from one plant from each plot (6 plants/date) were cleaned under running tap water, rinsed twice with sterile water, and dried on paper towels. Roots were determined to be alive if they did not exhibit lesions, were not obviously damaged, possessed a prominent number of root hairs and were connected to green leaves. A subset of these roots were microscopically analyzed to confirm AMF colonization, and the others were stored at -20 C until their DNA was extracted.
The microscopy results showed very low AMF colonization, as a compromise between the need for a large sample of clones (to reliably characterize the AMF community within each plant) and a significant sample of plants from control and N-amended plots (to study the effect of nitrogen on colonization), we decided to select 3 plants from control and 3 plants from N-amended plots and sequence approximately 100 clones per plant using fungal and AMF specific primers.
Three to five roots segments (approximately 3 cm long) from each plant were used for DNA extraction. DNA was extracted using a DNeasy plant Mini Kit (Qiagen, Chatsworth, CA). Primers NS31 and AM1 were used to specifically amplify AMF (Helgalson et al., 1998; Simon et al., 1992). For each of the 6 root samples, a total of 25 to 47 random clones were sequenced, 260 sequences in all. PCR was performed using the following protocol: initial denaturation at 95 C for 5 min, followed by 30 cycles of 95 C for 30 s, 53 C for 30 s, and 72 C for 45 s, with a final extension of 72 C for 7 min. DNA was amplified in 25 uL reactions with 12.5 uL Premix Taq (Takara Bio), 1.0 uL of each primer (5 uM), 3 uL of BSA 1%, 6.5 uL of milliQ water, and 1 uL of template DNA. The first PCR products were cleaned with ExoSAP-IT (USB, Cleveland, Ohio) and 1 uL of the cleaned PCR product was used as template for the second PCR. Products were cloned with TOPO-TA cloning kit (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. Clones were amplified and sequenced using rolling circle amplification (TempliPhi, Amersham, Buckinghamshire, England) and BigDye Terminator v1.1 Sequencing Kit (Applied Biosystems, Foster City, CA), respectively. Sequencing was conducted at the Molecular Biology Facility of The University of New Mexico. Forward and reverse sequences were assembled and edited with Sequencher 4.0 (Gene Codes, Ann Arbor, MI).
The program CHIMERA CHECK 2.7 of the Ribosomal Database Project (http:// rdp.cme.msu.edu/html/analyses.html) was used to check for chimeric 18S nrDNA sequences. Sequences were BLASTed against GenBank and information from GenBank obtained using phd, bioperl scripts and a mysql database written by George Rosenberg, Molecular Biology Facility of the University of New Mexico. Glomeromycota sequences were submitted in GenBank under accession numbers EF154520 and EF154698. OTUs were determined using the DOTUR program (Schloss and Handelsman, 2005). Distance matrices generated with the F84 evolutionary model using the DNADIST program from PHYLIP (Felsenstein, 2005) were used as input files to DOTUR. A similarity level of 97% has been used as the lower boundary to define OTUs in several studies of AMF (e.g. Helgason et al., 1999). We performed our analysis using both 97 and 99% of similarity to evaluate how mycorrhizal fungi at different taxonomic levels respond to N deposition. Rarefaction curves and diversity estimators (Chao, ACE) were calculated for the pooled data (N and control plots) and for N and control treatments with DOTUR. The effect of N enrichment on the AMF community also was evaluated in a phylogenetic context using UniFrac (Lozupone and Knight, 2005). The UniFrac metric estimates differences between microbial communities inhabiting different environments based on phylogenetic distances. In our study, we used this metric to evaluate the percentage of branch length in a phylogenetic tree that leads to descendants from N- amended and control plots. A phylogenetic tree generated with PAUP 4.0b10 (Swofford, 2002) that include all the Glomeromycota sequences from this study was used as input file to calculate UniFrac significance. Trees were constructed using the neighbor-joining (NJ) algorithm and maximum parsimony (MP) in PAUP 4.0b10 (Swofford, 2002). Bootstrap values were estimated from 1000 replicates for the MP and NJ analysis. A NJ phylogenetic tree that includes only representative OTUs (defined at 99% similarity with DOTUR) was used for normalized weighted principal coordinate analysis in UniFrac (Lozupone and Knight, 2005). The weighted UniFrac accounts for the relative sequence abundance in each sample. The NJ tree and a text file that includes OTU abundances for each sample were used as input files.