Invasive Galega officinalis (Goat's rue) plants in Canada form a symbiotic association with strains of Neorhizobium galegae sv. officinalis originating from the Old World

Abstract The toxic legume plant, Galega officinalis, is native to the Eastern Mediterranean and Black Sea regions. This legume is considered to be a noxious weed, and its establishment in Canada may have resulted from ornamental planting and/or field trials. In its native range, a highly specific nitrogen‐fixing symbiosis with the bacterium, Neorhizobium galegae symbiovar (sv.) officinalis, is required for normal growth. In North America, nothing is known about the bacterial symbionts of G. officinalis. Our purpose was to determine the species and symbiovar identity of symbiotic bacteria associated with invasive plants of G. officinalis at five sites in the province of Ontario, Canada. Sequence analysis of four housekeeping (16S rRNA, atpD, glnII, and recA) and two symbiosis (nodC and nifH) genes showed that all 50 bacterial isolates from root nodules of G. officinalis at the five Canadian sites were identical to strains of N. galegae sv. officinalis originating either from Europe or the Caucasus. Plant tests indicated that soils collected from four Canadian sites without a history of agriculture or presence of G. officinalis were deficient in symbiotic bacteria capable of eliciting nodules on this plant. Collectively our data support the hypothesis of anthropogenic co‐introduction of G. officinalis and its specific symbiotic bacterium into Canada from the Old World. Factors that may limit the spread of G. officinalis in new environments are discussed.

The rhizobia are root-nodule bacteria that fix atmospheric nitrogen in symbiotic association with leguminous plants thereby supplying the host with nitrogen compounds necessary for growth. Galega officinalis and its non-noxious relative, G. orientalis (fodder galega), form a highly specific symbiotic association with root-nodule bacteria belonging to the species, Neorhizobium galegae (Andrews & Andrews, 2017;Lindstrom, 1989;Mousavi, Willems, Nesme, Lajudie, & Lindstrôm, 2015). No species other than N. galagae has been reported to induce root nodules on Galega plants (Österman et al., 2014).
In preliminary surveys, we identified five sites in the province of Ontario, Canada, harboring established plants of G. officinalis that were nodulated by symbiotic bacteria.
Our purpose was to determine the species and symbiovar identity of the root-nodule bacteria associated with plants of G. officinalis established at these sites. Bacterial identification was achieved by phylogenetic analysis of four housekeeping and two symbiosis gene sequences.
To determine whether bacteria capable of symbiosis with G. officinalis occur naturally in Canadian soils, we carried out plant infection tests using soils collected from several sites that were without a history of agriculture or G. officinalis.

| MATERIAL S AND ME THODS
Five plants of G. officinalis were dug up at random from each four sites (S1-S4) in a 50 km radius of Ottawa as well as from a single site (S5) about 800 km distant in Sault Ste. Marie, Ontario; site descriptions and coordinates are given in Table 1. Root nodules were collected from tap and lateral roots of the sampled plants and stored at 4°C in vials containing anhydrous silica gel (Date & Halliday, 1987). Bacteria were isolated from surface sterilized nodules, grown at 28°C on yeast extract mannitol (YEM) agar medium (Tang, Bromfield, Rodrigue, Cloutier, & Tambong, 2012) and purified by repeated streaking and single-colony picking. Pure bacterial cultures were maintained at −80°C in 20% w/v glycerol.
The 50 bacterial isolates that were analyzed in this study are listed in Table S1; reference taxa are shown in Table S2.
Preparation of bacterial genomic DNA was as described by Tang et al., 2012. Amplification and sequencing of housekeeping (16S rRNA, atpD, glnII, and recA) and symbiosis (nifH [nitrogen fixation] and nodC [nodulation]) genes were carried out using primers and conditions described in Table S3. As suitable primers for amplification and sequencing of the nodC and nifH genes of N. galagae were not available in the literature, we designed primers based on the full genome sequence of N. galegae HAMBI 1141 sv. officinalis (GenBank accession no. HG938357) using Geneious Software (Biomatters Inc., USA) (Table S3). GenBank accession numbers of the nucleotide sequences used in this work are given in Tables S1 and S2.

Controls consisted of uninoculated plants and plants inoculated
with HAMBI 1141, a nitrogen-fixing reference strain of N. galegae sv. officinalis and with a bacterial isolate (G122) from Galega plants growing at site S2 (Ottawa).

| RE SULTS AND D ISCUSS I ON
Galega officinalis plants sampled from the five Canadian sites (S1 to S5) were vigorous, showed no signs of nitrogen deficiency, and were extensively nodulated by symbiotic bacteria ( Figure S2).
Effective nitrogen fixation by bacterial symbionts was indicated by the presence of leghemoglobin (a red colored hemoprotein required for nitrogen fixation), in the interior of the nodules. All five of these sites had moist soils with pH values above 7.0 (Table 1) and showed evidence of significant anthropogenic disturbance and import of soil material. This suggests that the G. officinalis plants at these sites were probably introduced along with imported soil material.
Isolates of symbiotic bacteria from nodules of G. officinalis at Canadian sites produced colonies on YEM agar medium after 7 days at 28°C that were beige, round, convex, ca. 1-1.5 mm diameter and similar to those of N. galagae reference strains, HAMBI 540 T (sv. orientalis) and HAMBI 1141 (sv. officinalis).
As was expected based on the high specificity of the Galega-bacterial symbiosis (Österman et al., 2014Radeva et al., 2001), analysis of almost full-length 16S rRNA gene sequences (1,400 bp) indicated that all 50 bacterial isolates from sites S1 to S5 belonged to the genus Neorhizobium as they were placed in a phylogenetic cluster with HAMBI 540 T , the type strain of the species, N. galegae (Figure 1). In this connection, BLASTn database searches confirmed that all 50

Further analyses based on
isolates of N. galegae belong to symbiovar officinalis as they possess nodC (795 bp) and nifH (650 bp) symbiosis gene sequences that are at least 99.9% similar to N. galegae sv. officinalis HAMBI 1141.
Data for the frequency of bacterial strains at sites S1-S5 (Table 2) show that at least three of the four strains were detected at each of the Ottawa sites (S1-S4, separated by distances of between 5 and 50 km) whereas at the geographically distant Sault Ste. Marie site (S5, about 800 km from Ottawa) only one strain was encountered.  Collectively our data support the hypothesis of anthropogenic co-introduction of G. officinalis and its specific bacterial symbiont into Canada probably from parts of Europe or the Caucasus. The transport of bacteria either on seed or in soil containing plant material is a possible mechanism that could account for the co-introduction of G. officinalis and its specific symbiont to Canada.
The fact that symbiotic G. officinalis plants were only found at Canadian sites with soils above pH 7.0 (range 7.4-7.8; Table 1) is consistent with reports from the United States (Oldham & Ransom, 2009) and Spain (González-Andrés, Redondo, Pescador, & Urbano, 2004) of plants (presumably symbiotic) growing in soils with pH ranges of 7.3-7.5 and 7.7-8.2, respectively. This suggests that the apparent adaptation of G. officinalis to soils above pH 7.0 together with its high level of symbiotic specificity may serve as important factors limiting the spread of the plant in new environments where the specific nitrogenfixing bacterial symbiont (N. galegae sv. officinalis) is absent. This is consistent with the observation that G. officinalis is established at only a few localized sites in Canada ( Figure S1) despite having been cultivated as early as 1897 (Bailey & Bailey, 1976;Macoun, 1908) and herbarium records showing that the plant was grown in gardens throughout the first half of the 20th Century. Note: Data are based on 10 bacterial isolates from each site. a As defined in Figure 2. b Strains of Neorhizobium galegae sv. officinalis originating either from Europe or the Caucasus. bacterial strains. We are grateful to Xiaoyuan Geng and Ben Stewart, Agriculture and Agri-Food Canada, Ottawa, for providing information on soil characteristics.

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflicts of interest.
All other data are presented in the Supporting Information.