Genotypic and symbiotic diversity of native rhizobia nodulating red pea (Lathyrus cicera L.) in Tunisia

Journal Pre-proof Genotypic and symbiotic diversity of native rhizobia nodulating red pea (Lathyrus cicera L) in Tunisia Gritli Takwa, Walid Ellouze, Chihaoui Saif-Allah, Barhoumi Fathi, Ridha Mhamdi, Bacem Mnasri

PII:

S0723-2020(19)30344-3

DOI:

https://doi.org/10.1016/j.syapm.2019.126049

Reference:

SYAPM 126049

To appear in:

Systematic and Applied Microbiology

Received Date:

23 May 2019

Revised Date:

24 November 2019

Accepted Date:

2 December 2019

Please cite this article as: Takwa G, Ellouze W, Saif-Allah C, Fathi B, Mhamdi R, Mnasri B, Genotypic and symbiotic diversity of native rhizobia nodulating red pea (Lathyrus cicera L) in Tunisia, Systematic and Applied Microbiology (2019), doi: https://doi.org/10.1016/j.syapm.2019.126049

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Genotypic and symbiotic diversity of native rhizobia nodulating red pea (Lathyrus cicera L) in Tunisia

Takwa Gritli1, Walid Ellouze2, Saif-Allah Chihaoui1, Fathi Barhoumi1, Ridha Mhamdi1 and Bacem Mnasri1*

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Laboratory of Legumes, Centre of Biotechnology of Borj-Cédria, BP 901 Hammam-lif 2050,

Agriculture and Agri-Food Canada, Vineland Station, Ontario, Canada L0R 2E0

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Tunisia

*Corresponding author: Bacem Mnasri, Tel: +216 22604929, Fax: +216 79325948 E-mail:

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[email protected]

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Abstract

Nodulation and genetic diversity of native rhizobia nodulating Lathyrus cicera plants grown

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in 24 cultivated and marginal soils collected from northern and central Tunisia were studied. L. cicera plants were nodulated and showed the presence of native rhizobia in 21 soils. A total

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of 196 bacterial strains were selected and three different ribotypes were revealed after PCRRFLP analysis. The sequence analysis of the rrs and two housekeeping genes (recA and thrC)

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from 36 representative isolates identified Rhizobium laguerreae as the dominant (53%) rhizobia nodulating L. cicera. To the best of our knowledge, this is the first time that this species has been reported among wild populations of the rhizobia-nodulating Lathyrus genus. Twenty-five percent of the isolates were identified as R. leguminosarum and isolates LS11.5, LS11.7 and LS8.8 clustered with Ensifer meliloti. Interestingly, five isolates (LS20.3, LS18.3, LS19.10, LS1.2 and LS21.20) were segregated from R. laguerreae and clustered as a separate

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clade. These isolates possibly belong to new species. According to nodC and nodA phylogeny, strains of R. laguerreae and R. leguminosarum harbored the symbiotic genes of symbiovar viciae and clustered in three different clades showing heterogeneity within the symbiovar. Strains of E. meliloti harbored symbiotic genes of Clade V and induced inefficient nodules.

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Keywords: Lathyrus cicera, Rhizobium laguerreae, Rhizobium leguminosarum, Ensifer

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meliloti, symbiotic diversity, molecular diversity

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Introduction

The genus Lathyrus, in the legume family Fabaceae, is represented in Tunisia by 15 wild and

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cultivated species [10]. Lathyrus sativus (white pea) and L. cicera (red pea) are the two predominant cultivated species [50, 56]. Red pea is primarily used for green manure, animal

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feed and fodder [45], and has the potential to be used in arid agricultural regions as bioorganic fertilizer [13, 22]. The cultivation of these economically important legumes is

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currently limited to two regions of southern Tunisia. Lathyrus spp. and legumes, such as pea, lentils and vetches, can be nodulated by a number of rhizobial species, such as Rhizobium

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leguminosarum, R. pisi, R. fabae, R. laguerreae, R. bangladeshense, R. lentis, R. binae and R. anhuiense [46]. Rhizobial species directly contribute to sustainable agriculture and soil fertility management due to the large amount of fixed nitrogen associated with nodulation. The diversity of members of the rhizobia-nodulating Lathyrus genus includes L. japonicus, L. pratensis, L. sativus and L. maritimus [12, 17, 40]. To date, however, the genetic diversity of rhizobia nodulating L. cicera is still unexplored.

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Therefore, the first objective of this study was to identify efficient and competitive native Rhizobium spp. isolated from L. cicera plants grown in cultivated and marginal soils collected from northern and central Tunisia. The second objective was to characterize the isolated strains by PCR-RFLP using the rrs and nodC genes, as well as the nifD-K intergenic spacer (IGS), and to investigate the phylogenetic relationships among these isolates through the

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analysis of core genes rrs, recA and thrC, as well as the symbiotic genes nodA and nodC.

Materials and Methods

1. Collection of soil samples and isolation of rhizobia nodulating L. cicera

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Soil samples were collected from 24 cultivated and marginal lands across northern (Nfidha, Morneg, Azib, Alya, Elmida, Ghezella, Beja, Jandouba, Kef, Tabaraka, Borj-Cédria,

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Amdounand, and Ain Drahim) and central (Chorben, Kairouan, Kerkenah, Boumerdess,

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Eljam, Ksour Essef, Elataya, Gafsa, Kasserine, Thala, and Sidi Bouzid) Tunisia. Samples consisting of 5 L of topsoil (0–15 cm) were collected from the different locations using a

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shovel. Five samples were pooled to obtain one composite sample per location and all the soil samples were stored at 4 °C. In order to trap soil rhizobia, the samples were used as a

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substratum to grow L. cicera plants. L. cicera seeds were surface sterilized in 10% sodium hypochlorite for 2 min, washed three times in sterilized water, germinated on moist filter

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paper Petri plates and incubated at 25 °C for 72 h in the dark. Two seedlings were aseptically transferred to each 5 L plastic pot (diameter 22 cm × height 20 cm) containing 6 kg of the soils representing each geographic location. Five pots were prepared for each soil location. The seedlings were grown under greenhouse conditions for 60 days under natural light with a daily minimum-maximum temperature of 20–24 °C. Three hundred nodules were removed for

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bacterial isolation. Ten plants from each soil type were dried at 55 °C for 2 days and the dry weight of each plant was recorded. Root nodules were collected from the seedlings, surface sterilized with 10% sodium hypochlorite for 2 min and rinsed twice in sterile distilled water. Nodules were aseptically crushed and streaked on yeast mannitol agar (YEMA) [47]. Pure bacterial cultures were established by isolating and enriching single colonies on YEMA. Cultures were maintained on

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slants and stored at 4 °C until needed. Each rhizobial isolate was designated by the letters LS and given a soil number corresponding to the region of collection. 2. PCR-RFLP of rrs and nodC genes and nifD-K IGS

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Bacterial DNA was extracted from 196 isolates, as previously described [29], and used as

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template for 50 µL PCR reactions. The primers used for 16S rRNA amplification were fD1 and rD1, as described by Mnasri et al. [27]. Single bands of approximately 1,500 bp were

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amplified from all isolates. DNA was digested by restriction enzymes NdeII, MspI and rsaI [28]. The nodC gene of the isolates was amplified using primers nodCF and nodCI, and

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digested by MspI, as previously described [26]. The nifD-K IGS was amplified using primers FGPD 807-85 and FGPK 700-92, and digested by HaeIII, according to Mnasri et al. [27].

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3. Sequence analysis

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The 16S rRNA genes from 25 strains (Table S1) representing all the ribotypes identified by PCR-RFLP were sequenced using the primers fD1 and rD1. A 500 bp recA and a 550 bp thrC gene were amplified for 36 isolates (Table S1) according to Mnasri et al. [29]. The nodA and the nodC genes of 30 strains (Table S1) were amplified using nodA1 and nodA2, respectively, as previously described by Haukka et al. [18], and nodCI and nodCF according to Mhamdi et al. [26]. PCR-amplified products were purified on agarose gels using the EZ-10 spin column

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DNA gel extraction kit (Bio Basic Canada Inc., Ontario, Canada) following the manufacturer’s instructions. Purified DNA fragments were sequenced at the Centre of Biotechnology of Borj-Cédria (Tunisia) and in a commercial laboratory (Genome Quebec Innovation Centre, Quebec, Canada). The nucleotide sequences were deposited in the GenBank database under the accession numbers listed in Table S1. 4. Phylogenetic analysis

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Molecular sequence analyses were compared with those from GenBank using the BLASTn program [2], and the rrs gene sequences were compared with those from the EzTaxon-e

server [19]. Sequences were aligned using the ClustalX software [20]. The distances were

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calculated according to Kimura’s two-parameter model [16]. Phylogenetic trees were inferred using maximum likelihood analyses [35] and MEGA7 software [21]. Ward’s minimum

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variance hierarchical cluster dendrogram was built on standardized DNA restriction pattern

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data based on the Euclidian distance coefficient using the Analyses of Phylogenetics and Evolution (ape) package [32] within the R Project for Statistical Computing version 3.6.1

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[34].

5. Plant nodulation and symbiotic efficacy

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Representative rhizobial strains (Table 1) were used to inoculate L. cicera, Pisum sativum and

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Vicia sativa. Seeds were surface sterilized in 10% sodium hypochlorite for 2 min, washed three times in sterilized water and germinated on moist filter paper in Petri plates at 28 °C for 72 h. Seedlings were aseptically transferred to 500 mL plastic pots (one plant per pot) containing sterile sand. Liquid bacterial cultures were prepared by obtaining a loop full of bacteria from a YEMA slant and inoculating 10 mL of yeast extract mannitol (YEM) [47]. The liquid cultures were incubated at 28 °C on a rotary shaker at 150 rev. min-1. Mid-

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exponential growth was reached after 48 h at an OD620 of 1 (approximately 109 CFU mL-1). Plant inoculum was prepared by adding 200 µL of bacterial suspension to 250 mL Erlenmeyer flasks containing 100 mL of YEM. The cultures were grown overnight at 28 °C on a rotary shaker, and 1 mL of the overnight culture was used to inoculate plants with appropriate strains. The control plants were treated with 1 mL of uninoculated YEM. Treatments consisted of three plant species (L. cicera, V. sativa and P. sativum) inoculated with five

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rhizobial strains (LS1.6, LS9.6, LS19.10, LS11.5 and LS11.7) arranged in a randomized complete block design with 10 replicates. Plants were grown in a greenhouse under natural

light with a daily minimum-maximum temperature of 18–24 °C. Plants were surface watered with sterile nitrogen-free solution [43] as needed, and checked for nodulation after 60 days.

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Symbiotic efficacy was estimated by plant shoot dry weight measurements in comparison

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with the non-inoculated plants. Statistical analysis of data was performed by ANOVA

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Results and Discussion

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followed by the HSD Tukey comparison of means test (P= 0.05; n = 10).

1. Effect of soil on nodulation and biomass production in red pea

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The presence of nodulation in L. cicera was explored by using soils collected from 24

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different localities across northern and central Tunisia. Nodulation was present in plants grown in soil samples from 21 regions, with no nodulation present in soils from three sites (Ghezella, Kasserine and Thala). While nodule numbers varied according to the soils, the majority of the nodules were detected in the soils from northern Tunisia. The biomass of plants grown in the various soil samples is shown in Figure 1. Group P1 was composed of nine soils belonging to the northern region of the country where legume species, such as pea,

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lentil and faba beans, were grown for several years. High nodulation, consisting of more than ten nodules plant-1, was associated with the highest biomass production (Figure 1). Previous research has shown that increases in nodule number and biomass were associated with an efficient symbiotic system and enhanced plant growth [39, 42, 49]. Furthermore, this result may be related to the fact that the soils were traditionally cultivated with legume species, such as faba bean, common bean and pea, which are nodulated by the same rhizobial species as

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Lathyrus spp. [7, 26]. Therefore, soils collected from cultivated and marginal lands across northern and central Tunisia could constitute a natural reservoir of adapted and efficient rhizobia-nodulating red pea.

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Group P2 consisted of five cultivated soils, and the biomass was accompanied by a low

number of white nodules equal to or less than four nodules plant-1. This can be explained by

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the high fertility of these soils, which are rich in organic matter. Indeed, a high concentration of soil nitrate, induced by factors such as excessive tillage and applications of nitrogen

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fertilizer, could negatively affect symbiotic uptake [49]. Previous reports showed that a high concentration of nitrogen suppressed N2 fixation in commercial soybean, chickpea and faba

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bean crops in Australia [33, 39]. Additionally, Naudin et al. [31] demonstrated that temporary

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exposure of pea to nitrate leads to a decrease in the function of nodule-fixing activity. Group P3 contained six marginal soils and four cultivated soils located in northern and central

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Tunisia comprised of semi-arid and arid soils, respectively. These soils were characterized by a low number of nodules with less than four nodules plant-1, which was associated with the lowest biomass. The weak nodulation associated with low production of biomass could be explained by the unfavorable conditions for the installation of the native rhizobia-nodulating population. Indeed, previous studies have shown that water stress and salt stress affected the

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survival of several species of rhizobia. In the presence of water stress, symbiosis and nodule formation are more sensitive to salinity than free-living rhizobia [54]. 2. Identification of the native rhizobia nodulating L. cicera This is the first report of the genetic diversity and structure of native rhizobia nodulating red pea grown in soil collected from various geographic and bioclimatic regions in Tunisia. The PCR-RFLP of the rrs gene was performed for 196 isolates and, using a combination of the

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restriction patterns from three enzymes, the isolates were categorized into three distinct ribotypes. Furthermore, 36 representative isolates from these ribotypes were sequence analyzed (Table S1).

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The maximum likelihood phylogenetic tree based on rrs gene sequences of 25 isolates shown

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in Figure 2 revealed the presence of four clusters. Cluster 1 was made up of 17 isolates with identical 16SrRNA gene sequences. These isolates were 100% similar to R. leguminosarum,

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R. laguerreae, R. anhuiense, R. indigoferae and R. sophorae type strains. This indicated that the use of the rrs gene as a phylogenetic marker for species identification had limitations, as

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demonstrated in this current study. Indeed, previous reports have shown that classification of prokaryote species by rrs gene sequence alone is insufficient [16]. Mnasri et al. [29] showed

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that rrs does not allow discrimination between Sinorhizobium americanum and S. fredii. Furthermore, our results agreed with Saïdi et al. [37] who indicated that strains of R.

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laguerreae and R. leguminosarum had the same rrs gene sequences. Therefore, rrs gene sequence analysis does not allow discrimination of R. laguerreae, R. legumuinosarum, R. indigoferae, R. sophorae and R. anhuiense. Van Berkum et al. [44] indicated that sections within the rrs genes of rhizobia have undergone recombination, influencing the placement of species on a phylogenetic tree. In addition, it was shown that classification of prokaryotic species by rrs gene sequence alone is unsatisfactory [16].

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Cluster 2 and Cluster 3 (Figure 2) contained three and two isolates, respectively, and were slightly different to known rhizobial species but they were closely related to the R. leguminosarum and R. laguerreae group. Cluster 4 contained the isolates LS11.5, LS11.7, LS8.8 that were closely related to the Ensifer meliloti type strain with 99.98% identity. Clarification and confirmation concerning the taxonomic position of the representative strains were obtained by using the concatenated tree of the recA and thrC housekeeping genes of 36

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isolates (Figure 3).The phylogenetic data obtained from recA (Figure S1) and thrC (Figure S2) housekeeping genes confirmed the taxonomic position of the majority of strains

sequenced. A total of 19/36 isolates were clustered with R. laguerreae FB206T. The sequence similarity values of the recA and thrC genes ranged, respectively, from 99.75% to 100% and

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99.1% to 100% compared to R. laguerreae FB206T. This species was isolated for the first

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time from root nodules of V. faba in Tunisia, Peru and Spain and constituted a cluster close to R. leguminosarum USDA2370T. Their recA and atpD genes were phylogenetically distant

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from those of R. leguminosarum with less than 97% and 94% identity, respectively [37]. Recently, R. laguerreae was isolated from root nodules of Lotus in Morocco [41], V. faba in

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Algeria [8], P. sativum [25], and common bean [14]. Otherwise, the increase in the number of isolates of R. laguerreae can be explained by the use of the phylogenetic trees based on

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housekeeping genes other than the rrs gene.

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Only 9/36 isolates clustered with R. leguminosarum USDA2370T. The sequence similarity values of the recA and thrC genes ranged, respectively, from 97.9% to 100% and 98.9% to 100% compared to R. leguminosarum USDA2370T. Previous reports showed that R. leguminosarum was the rhizobia predominantly nodulating the majority of studied Lathyrus spp. [1, 6, 7, 51]. In addition, this species was abundant in Tunisian soil and could be isolated from the nodules of many legume species, such as common bean, faba bean and pea. These

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bean crops are frequently cultivated in all bioclimatic regions of Tunisia [26, 37]. Despite the abundance of R. leguminosarum in Tunisia, a low number of isolates assigned to this species was detected in this study. This discrepancy may be attributed to the host’s preferential selectivity towards R. laguerreae or a higher competitiveness of this rhizobia. Therefore, it would be interesting to evaluate the competitiveness of R. laguerreae and R. leguminosarum towards L. cicera in future studies.

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The isolates LS11.7, LS11.5 and LS8.8 were closely related to the E. meliloti LMG 6133T type strain. E. meliloti (formerly Sinorhizobium meliloti) was first known as specific for

rhizobia-nodulating Medicago and Melilotus [15]. In Tunisia, a high genetic and symbiotic

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diversity of E. meliloti nodulating Medicago spp. have been reported [4, 27, 28, 57]. Several E. meliloti strains were isolated from grain legumes, such as P. vulgaris [28] and Cicer

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arietinum [36], and wild legume species, such as Argyrolobium uniflorum, Lotus creticus, Ononis natrix, Retama raetam, Genista saharae and Hedysarum carnosum [23, 55]. The

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current study demonstrated that, in addition to R. leguminosarum, E. meliloti isolates were able to nodulate L. cicera; whereas previous studies have shown that the genus Lathyrus can

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be nodulated by other rhizobia species, such as R. tropici [30].

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The strains LS20.3, LS18.3, LS19.10, LS1.2 and LS21.20 (Figure 3) were more divergent and clustered in a strongly supported clade. The recA and thrC gene sequences of these strains

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showed, respectively, 96.3% and 96.4% similarity to R. laguerreae FB206T, and 96% and 93.4% similarity to R. leguminosarum USDA2730T. This group of isolates most probably constitutes a new lineage. 3. Symbiotic diversity

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A nodC single band of approximately 960 bp with 1,300 bp for the nifD-K IGS was obtained for all isolates tested. The exceptions were four isolates belonging to E. meliloti. The nodC gene and nifD-K IGS PCR products were digested with the MspI and HaeIII enzymes, respectively. According to the restriction patterns, the isolates were categorized into four different symbiotic groups (Figure S4). Group 1 contained 135 strains trapped by L. cicera at all sites, whereas Group 2 had 34 strains. Group 3 consisted of 20 strains exclusively isolated

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from the “Azib” soil in northern Tunisia, and Group 4 contained three E. meliloti strains. The data suggested that R. laguerreae and R. leguminosarum strains recovered from L. cicera may harbor distinct symbiotic genes that confer a wider host range. Strains belonging to R.

leguminosarum and R. laguerreae were isolated from different legume species grown in

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Tunisia, such as common bean [26] and faba bean [37]. In addition, Groups 1 and 2

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represented the most abundant nodulating rhizobial species in all studied soils. This suggested a high adaptation of these species to diverse environmental conditions in Tunisia and a

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conservation of the genotypes, despite the diversity of geographic locations. Interestingly, the exclusive Group 3 of R. laguerreae was isolated in a unique “Azib” soil, suggesting a certain

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degree of specificity.

Based on PCR-RFLP of the nodC and nifD-K results, 30 representative isolates nodulating L.

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cicera were chosen for nodA and nodC gene sequencing. These symbiotic genes have been

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used extensively as markers in the analyses of specificity between rhizobia and different host plants and symbiovar (sv.) determinations [11, 12, 31, 32]. nodC and nodA were successfully amplified for all Tunisian isolates. A maximum likelihood analysis was used on nodC and nodA sequences, and phylogenetic trees are provided in Figure 4 and Figure S3, respectively. The results showed similar phylogenetic relationships between isolates, and four main clades were obtained for the two symbiotic genes.

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Ten isolates (LS13.54, LS2.21, LS20.18, LS2.10, LS2.16, LS1.11, LS6.7, LS1.3, LS1.6 and LS2.5) were identified as R. laguerreae, two isolates (LS1.5 and LS6.15) as R. leguminosarum, and two isolates (LS1.2 and LS18.3) represented the putative new species group, which clustered together in Clade 1 and harbored the symbiotic genes of sv. viciae. A representative isolate LS1.6 of this clade was tested in planta and it was found to be efficient for nodulating and fixing nitrogen with L. cicera, V. sativa and P. sativum (Table 1). The

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majority of the isolated rhizobia nodulating L. cicera in this clade were phylogenetically close. They formed a robust phylogenetic group with a high bootstrap value, 99% for nodC and 96% for nodA, with several reference strains that belonged to different geographical

origins, such as PEVF08 and PEVF01 nodulating V. faba in Peru [38], EB1 nodulating V.

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faba in Algeria [8], R106 nodulating V. faba in Portugal, LMR657 nodulating Lens culinaris

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in Morocco [41], R. leguminosarum sv. viciae strain 3841 isolated in United Kingdom [11, 52] and USDA2730T isolated in USA. Interestingly, strains of A. tumefaciens nodulating V.

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faba in Egypt were closely related to the strains nodulating L. cicera grouped in Clade 1. Clade 2 also harbored the symbiotic genes of sv. viciae and included five isolates (LS7.7,

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LS18.6, LS20.16, LS11.8 and LS21.18) of R. laguerreae, four isolates (LS20.15, LS20.13, LS5.4 and LS9.6) of R. leguminosarum, and two isolates (LS20.3 and LS21.20) of the

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putative new species group. A representative isolate LS9.6 of this clade was tested in planta

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and it was found to be efficient for nodulating and fixing nitrogen with L. cicera and P. sativum (Table 1). This strain nodulated V. sativa but was inefficient at fixing nitrogen with this plant. The isolated rhizobia nodulating L. cicera in this clade were phylogenetically close to R. leguminosarum, R. laguerreae and R. etli nodulating V. faba, Lens culinaris and P. sativum in different countries [9, 53].

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Clade 3 consisted of three strains exclusively isolated from the “Azib” soil. A representative isolate LS19.10 of this clade was tested in planta and it was found to be efficient for nodulating and fixing nitrogen with L. cicera and V. sativa (Table 1). This strain nodulated P. sativum but was inefficient at fixing nitrogen with this plant. The only rhizobium strain found close to the isolates in this clade was strain CTG-28Ps isolated from P. sativum in Spain. The results suggested high level specificity of this group to particular soils where they could be

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highly efficient at fixing nitrogen with L. cicera. In summary, despite chromosomal differences between R. laguerreae and R. leguminosarum strains, they shared the same symbiotic genes. Previous reports showed that incongruence

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may exist between phylogeny based on symbiotic and chromosomal markers. This

corroborates the results of Mnasri et al. [29] who indicated that strains 23C2 and 23C55

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belonging to R. azibense harbored the same nodA gene found in R. gallicum sv. gallicum. Moreover, it was shown that sv. mediterranense was harbored by three different species, E.

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meliloti [28], S. fredii and S. americanum [29]. Evidence of gene transfer and rearrangement of symbiotic plasmids in rhizobia has been demonstrated to play a role in the evolution and

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structure of natural populations of rhizobia [5, 24, 48]. The analyses of the nodA and nodC genes showed that the strains in this current study were closely related to the rhizobia trapped

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by V. sativa, V. faba, Lathyrus sp., Lens culinaris and P. sativum in diverse and

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geographically distant regions, such as Africa, Europe, America and Asia. The results supported previous reports showing a common phylogenetic origin of symbiotic genes, such as nodA and nodC, carried by R. leguminosarum and R. laguerreae strains nodulating V. sativa, V. faba, Lathyrus ssp., Lens culinaris and P. sativum in different continents, suggesting possible dispersion with the seeds from Europe to different locations in Asia, Africa and America [3].

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Despite the low number of E. meliloti isolated from root nodules of L. cicera, they were classified into two types based on their symbiotic performance. Type 1 was comprised of isolates LS11.5 and LS8.8 that harbored nodA and nodC sequences identical to E. meliloti Clade V strains (following the Mnasri et al. [27] subdivision) isolated from various legume species in Tunisia. In this current study, it was shown that L. cicera was also nodulated by isolates belonging to this specific clade discovered in Tunisian soils [27]. Type 2 contained

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isolate LS11.7 that failed to amplify nodA, nodC and nifD-K IGS, and it also failed to nodulate L. cicera, suggesting that it was a non-symbiotic strain. Endophytic colonization of nodules by E. meliloti has been reported previously by Mnasri et al. [29]. However, the

biological significance and potential agronomic implications of this interaction, namely the

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impact on nodule functioning, still need to be investigated further.

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Conclusions

This study represents the first report of the genotypic and symbiotic diversity of native

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rhizobia nodulating L. cicera in Tunisia using chromosomal and symbiotic genes. R. laguerreae was shown to be the predominant rhizobia nodulating L. cicera.

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Interestingly, a low number of R. leguminosarum isolates was detected, which suggested the

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high selectivity of L. cicera. Additionally, it was demonstrated that strains of E. meliloti either nodulated but did not fix nitrogen or failed to re-nodulate their host, suggesting that they were nodule-endophytes. Therefore, L. cicera may be nodulated by a new group of rhizobia.

Acknowledgements

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This study was funded by the Tunisian Ministry of Higher Education and Scientific Research. The authors gratefully acknowledge Dr. Antonet Svircev for revising the English language style throughout the manuscript, and the technical assistance of Sarah Bachkouel. All contributions made by members of the Centre of Biotechnology of Borj-Cédria are sincerely appreciated.

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Figure legends

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Figure 1: Shoot dry weight of L. cicera grown in soils collected from 24 cultivated and

marginal lands across northern and central Tunisia. Means followed by the same letter are not significantly different according to the HSD Tukey comparison of means test (P = 0.05;

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n = 10). Numbers inside bars represent the average nodule number per plant ± standard error

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(n=10).

22

LS20.3 LS20.18 LS6.12 LS6.7 LS1.3 LS21.18 LS21.20 LS6.15

Cluster 1

LS13.54 LS1.2 69 LS20.16

LS19.10 LS18.3 LS7.7 LS2.16 LS2.10 LS11.8

85

R. sophorae CCBAU03386T (KJ831229) R. anhuiense CCBAU23252T (KF111868) R. indigoferae CCBAU71042T (AY034027)

R. leguminosarum USDA2370T (JQ085246) LS5.4 96

Cluster 2

LS9.6

LS1.6

50

Cluster 3

LS2.5

84

LS1.11

R. rhizogenes ATCC11325T (D12788)

74

R. lusitanum P1-7T (AY738130) R. leucaenae CFN299T (X67234)

100

R. hainanense I66T (U71078)

R. phaseoli ATCC14482T (EF141340) R. fabae CCBAU33202T (DQ835306) R. pisi DSM30132T (AY509899) R. etli CFN42T (U28916)

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R. alamii GBV016T (AM931436)

100

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R. tropici LMG9503T (U89832) 93 78

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R. laguerreae FB206T (JN558651)

83

R. mesosinicum CCBAU25010T (DQ100063) 75

94

R. sullae IS123T (Y10170)

R. gallicum R602spT (U86343)

72 100

R. loessense CCBAU 7190BT (AF364069)

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R. tibeticum CCBAU 85039T (EU256404)

66

R. grahamii Cli80T (JF424609) 61

R. cauense CCBAU101000T (JQ308324)

98

R. mesoamericanum CCGE501T (JF424606) R. giardinii H152T (U86344)

90

R. radiobacter ATCC19358T (AJ389904)

E. kummerowiae CCBAU71714T (AY034028)

na

80

E. medicae A321T (L39882)

100

E. meliloti LMG6133T (X67222)

92

99

LS11.7

LS11.5

63

LS8.8

Cluster 4 B. japonicum LMG 6128T (X66024)

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71

0.01

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Figure 2: Phylogenetic tree of rrs sequences (1,243 nucleotides) based on the maximum

likelihood method. Representative strains trapped by L. cicera are in bold. Bootstrap values ≥50 are indicated for each node (1,000 replicates). The strain designation and accession number of the sequence used are given after the abbreviation for the reference strain. The scale bar indicates the number of substitutions per site.

23

LS20.18 LS2.21 LS1.11 LS2.10 LS2.16 LS7.7 97

LS20.16 LS13.54 LS21.18 LS1.3

Rhizobium laguerreae

LS6.7 LS18.6

96 90

LS11.8

85

LS10.2 LS13.13 LS1.6 LS2. 5

96

99

LS7.3 LS1.11

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R. laguerreae FB206T (PRJNA355904/JN558681) LS20.3 LS18.3 99

Putative new species

LS19.10

97

LS1.2 LS21.20 LS6.15 97

LS5.5 LS19.8 LS20.15

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98

79

LS1.5 97 79

Rhizobium leguminosarum

LS20.13 LS19.5

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LS5.4 94 90

LS9.6

R. leguminosarum LMG14904T (AM181690/AJ294376) 99

R. fabae CCBAU33202T (FJ392885/EF57994) R. pisi DSM30123T (KF207047/ABL11499)

100

58 1 00 80

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R. sullae IS123T (ACO50380/FJ816279) 77

R. gallicum R4387T (CAJ57662/AY907357)

R. yanglingense CCBAU71623T (KF278568/AY907359) R. loessense CCBAU719BT (KF656686/HQ735076) R. mongolense USDA1844T (KF656687/AY907358)

61

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. R. hainanense HAMBI1930T (KF206993/KF206850) R. giardinii R4385T (AM181688/ACB30409)

80

R. cellulosilyticum HAMBI3173T (KF207046/KF206873) R. radiobacter LMG140T (AM181686/AM182121)

R. huautlense LMG18254T (CAJ57666/CAJ57771)

E. medicae LMG18864T (AM181702/AJ294381)

100

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LS11.7

100

88

LS11.5

Ensifer meliloti

LS8.8

E. meliloti LMG6133T (AM181698/AM182133)

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B. japonicum LMG6138T (AM181730/AM182158)

0.050

Figure 3: Phylogenetic tree of the concatenated recA and thrC gene sequences (700

nucleotides) based on the maximum likelihood method. Representative strains trapped by L. cicera are in bold. Bootstrap values ≥50 are indicated for each node (1,000 replicates). The

24

strain designation and accession number of the sequence used are given after the abbreviation

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for the reference strain. The scale bar indicates the number of substitutions per site.

Figure 4: Phylogenetic tree of the nodC sequences (405 nucleotides) based on the maximum

likelihood method. Representative strains isolated from L. cicera are in bold. Bootstrap values ≥50 are indicated for each node (1,000 replicates). The scale bar indicates the number of substitutions per site.

Figure 4 25

Table 1. Nodulation and efficacy tests of representative strains from different species and nodA/nodC groups. Species

L. cicera

V. sativa

LS1.6

R. laguerreae

nodA/nodC clade 1

LS9.6

R. leguminosarum

2

+

++

+

LS19.10

Putative new species

3

+

++

+

LS11.5

E. meliloti

V

+

-

-

LS11.7

E. meliloti

-

-

-

N/A

P. sativum

Nod

Fix

Nod

Fix

Nod

Fix

+

+

+

+

+

+

-

+

+

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Strains

+

+

-

-

-

-

-

-

-

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Nod+: nodulation; Nod−: no nodules; Fix−: inefficient nodules; Fix+: efficient nodules; Fix++: highly efficient nodules. N/A:

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not amplified