Comparisons of root-zone microbial communities in response to vegetation changes in a rangeland soil. Xu Li, D’Jenane Dias, John M. Stark and Jeanette M. Norton Utah State University, Logan, UT Summary  The fungal community in different treatments plots became similar after 25 years restoration, while the bacteria community remain distinct between the fumigated and undisturbed native plots.  The richness of arbuscular mycorrhizal decreased in fumigated control plots dominated by cheatgrass.  Plant species effects tended to be more important than soil history. Plants may host unique AMF, leading to altered AMF community composition in the soil. A positive feedback in cheatgrass is likely.  The AMF sequences were all related to Glomus, some types were unique to bluebunch wheatgrass or cheatgrass, this may support persistent invasion. Acknowledgments This research was supported by a grant from USDA-NRI Biology of Weedy and Invasive Plants. We appreciate the support of the Ecology Center and the Utah Agricultural Experiment Station at USU. Contacts: Dr. Jeanette Norton, Dept. of Plants, Soils and Climate, Utah State University Soil type BacteriaFungal Peak Numbers Shannon index Species Evenness Simpson index Peak Numbers Shannon index Species Evenness Simpson index Fumigated Control 106a3.77b0.81b0.94b36a2.42a0.71a0.81a Fumigated Climax 142a4.39a0.89a0.98a49a2.63a0.69a0.78a Undisturbed Control 138a4.11ab0.85ab0.96a39a2.79a0.77a0.86a Table 1. The diversity index of bacteria and fungi in three different treatments by ARISA. Values in the same column with different letters were significantly different (P<0.05). Table 3. Distribution and frequencies in the nine samples of the 30 RFLP patterns obtained after Hinf I, Rsa I and Hsp 92II digestion of 274 NS31/AM1 amplified fragments. Introduction In the Intermountain West, cheatgrass (Bromus tectorum) has infested over 40 million hectares of native sagebrush – bunchgrass communities and these cheatgrass dominated communities appear quite resistant to re-invasion by native perennials. Feedbacks between plants and the soil microbial community is hypothesized to contribute to the success and persistence of the invasive annual. Objectives  Determine the influence of cheatgrass or sagebrush-native bunchgrass on soil microbial community including bacteria and fungi.  Determine if the history of vegetation could influence arbuscular mycorrhizal colonization or community structures in different plant roots (including facultatively mycotrophic plants, obligately mycotrophic plants), which may be associated with the cheatgrass invasion. Methods Site Description The study site is located in the Piceance Basin of Northwestern Colorado at 2020 m elevation on deep, well drained, loam textured soils (fine silty, mixed Borollic camborthids), with a surface soil pH of 7.5. MAP is 282 mm and MAT is 6.8 °C. Plots were created in the summer of 1984, by scraping the vegetation from four 0.5 ha areas of sagebrush (Artemisia tridentata) – bluebunch wheatgrass (Pseudoroegneria spicata) dominated communities and plowing the soil to a 20-cm depth. Each of the four blocks were divided into randomly arranged plots treated with combinations of fertilization, soil fumigation (methyl bromide), or seeding and transplanting (late seral or “climax” species, such as sagebrush and native perennial grasses; disturbance-adapted or “ruderal” species, such as annuals, short-lived perennial grasses, and fast growing shrubs; and non-seeded “control” plots, where seed rain from the adjacent undisturbed community provided the propagule supply). In May 2008 soils were sampled with a focus on the undisturbed sagebrush control plots, the fumigated climax plots (where sagebrush and native perennial grasses dominate), and the fumigated control plots (where cheatgrass dominates) (Fig. 1 A, B, C). Microbial Community Analysis  Field soil analysis: soil sampled and DNA was extracted and frozen in MAY Bacterial and fungal communities were assessed using ARISA (amplified ribosomal intergenic spacer analysis).  Greenhouse plant-soil feedback experiments were performed where cheatgrass, sagebrush, and wheatgrass were grown on soil harvested from the plots. Plants were grown in their “own” soil and in soil cultured by the other species to assess interactions between microbial community structure and function and plant species. Plant biomass (aboveground and belowground) was assessed on harvested pots. Harvested roots were used for arbuscular mycorrhizal fungi (AMF) infection rates and AMF community analysis. AMF infection was assessed by slide observation and grid-line intersection methods. AMF community structure was assessed by DNA extraction from roots, amplification of the 18S rRNA gene with specific primers, followed by cloning, RFLP (restriction fragment length polymorphism) and sequencing of selected clones. Figure 3. Phylogenetic tree of AM fungal sequences isolated from roots and reference sequences from GenBank. Numbers above branches indicated the bootstrap values (1000 replicates) of the Cluster W analysis. Clone identifiers relate to site (Fumigated control plot-35, Fumigated climax plot-39, Undisturbed control plot-44), plant species( sagebrush- S, cheatgrass-C, bluebunch wheatgrass-B) and clone number (last number). Group identifiers (G1, G2, G3, G4) are AM fungal sequences types found in this study. Mortierella and Endogone were used as outgroups. A. Undisturbed control plot, dominated by sagebrush, smaller amounts of bluebunch wheatgrass, and native forbs. B. Fumigated climax plot, dominated by sagebrush, bluebunch wheatgrass, needle-and thread (Stipa comata), and western wheatgrass (Pascopalum smithii). C. Fumigated control plot, dominated by cheatgrass, with small amounts of bur buttercup (Ceratocephala testiculata), tumble mustard (Sisymbrium altissimum), and rabbitbrush (Chrysothamnus nauseosus). Soil samples are collected from random locations at least 1 m from perennial plants. Fig. 1. Plot photos showing the three treatments examined in current study. Taxonomic units RFLP patterns 35C39C44C35B39B44B35S39S44S G1 RFLP RFLP RFLP61112 RFLP911 RFLP131 RFLP172 RFLP241 RFLP281 RFLP291 RFLP301 RFLP341 G2RFLP745 G3 RFLP RFLP RFLP RFLP102 RFLP RFLP181 RFLP19123 RFLP201 RFLP21153 RFLP221 RFLP231 RFLP251 RFLP261 RFLP271 RFLP311 RFLP321 RFLP G4RFLP Total # of RFLP patterns Shannon index Evenness Results Fig. 4. Hierarchal Cluster Analysis Clusters show the grouping of the AMF community by the soil type and plant species. Fumigated control and climax soils group together (35 and 39) with plant species (cheatgrass C, sage S then blue bunch wheatgrass B). The native undisturbed soil (44) has the most divergent AMF communities. Scale is relative scale of distance. Fig. 2. Arbuscular mycorrhizal infections In the greenhouse experiment plant roots were harvested from a core, then stained and examined for root length, # of infections and infected root length percentage. Variability was extremely high and soil type was not significant, plant type was significant for some parameters (Table 2). Table 2. AMF infection and root lengths in greenhouse experiment Parameter examined Significance of plant (p value) Cheatgrass (n=12) Bluebunch wheatgrass (n=11) Sagebrush (n=11) Root Length Infected % 0.22 NS Infected Root Length mm cm -3 soil Total Root Length mm cm -3 soil Infections per root length # cm -1 root > Infections per soil volume # cm -3 soil