DOI: 10.5061/dryad.m86s6/1
title: La Pierre_invasive shrub_nodulation assay_dryad submission

description: Data addressing the breadth of rhizobial isolates that three invasive and two native legumes from the San Francisco Bay Area, California, USA could nodulate with in single-isolate inoculations under greenhouse conditions.

We assessed the promiscuity of the three invasive plants in their invasive range by determining their ability to associate with a broad range of 117 rhizobial isolates originally collected from 12 different leguminous hosts (both native and invasive, including hosts not studied here, but all growing in the SF Bay Area; Table S2) in a greenhouse-based nodulation assay. Seeds of each legume species were surface sterilized in full-strength commercial bleach (3% sodium hydroxide) for 30 sec, rinsed five times with sterile water, scarified with sulfuric acid for either 10 min (S. junceum), 30 min (U. europaeus), or 40 min (G. monspessulana), neutralized with a sterile 20% sodium bicarbonate solution and thoroughly rinsed using sterile water. Scarified seeds were germinated in the dark at room temperature in individual wells of 96-well plates filled with 100 µl sterile water. Two weeks later, germinated seedlings were individually planted into 22 mm diameter, 20 cm tall sterile glass 75 ml culture tubes filled with 25 ml sterile vermiculite moistened with sterile water. Tubes were plugged with sterile cotton and kept under shade cloth, which provided indirect natural light, and were provided supplemental artificial light in the Jane Gray Research Greenhouse at the University of California, Berkeley. Twelve days following planting, 1 ml sterile Jensen’s fertilizer (Somasegaran and Hoben 1994) containing 7 ppm nitrogen was added to each tube.

The 117 rhizobial isolates used in the nodulation assay were obtained from two sources: (1) many isolates were obtained from the collection described above prior to genotyping (99 isolates); (2) several isolates were obtained from the investigators’ additional research collections to represent strains associated with other native and invasive legumes common in the San Francisco Bay Area (18 isolates; see Table S2 for a list of the isolates used in the nodulation assay and their sources). Isolates were chosen to span a broad range of host species and collection sites. Inoculum from each isolate was prepared from 50 µl of -80°C glycerol stock prepared from field-collected nodules (see above), grown in YM broth at 25°C shaken at 120 rpm to a density of   per ml, as measured by optical density at 600 nm. Each rhizobial isolate was inoculated onto one seedling of each legume species. Seedlings were randomly assigned rhizobial isolates and inoculated 17 days after planting by adding 1 ml of the appropriate inoculum to the base of the plant stem in each tube. An additional ten plants per legume species were inoculated with sterile YM broth as negative controls; none of the control plants were nodulated at harvest. Plants were harvested 47 days after planting (30 days after inoculation), the roots thoroughly washed, and the presence of nodules recorded. Successful association was defined as the formation of at least one robust nodule that appeared to be effectual (i.e., not &lt;1 mm and/or white or clear). Reanalysis of our results increasing the cutoff for defining successful nodulation to two nodules did not qualitatively alter our findings.