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Molecular Characterization of Some Saprophyte Agrobacterium spp. Strains Isolated from Root Nodules of C. arietinum L. Cultivated in Central Anatolia Region of Turkey

In the present study, twelve plant-associated bacteria which isolated from root nodules of C. arietinum L. collected from Central-Anatolia region of Turkey were identified with current molecular techniques. None of the isolates produced root nodules or showed pathogenic effects (gall or hairy root) on the original host as the result of authentication and pathogenicity tests, respectively. These results have suggested that all these isolates are root endophytic bacteria. Additionally, PCR amplifications for nodD and virA genes did not reveal any bands. These results showed that these isolates have not to harbour symbiotic (pSym) or pathogenicity (Ti: Tumour inducing or Ri: Root inducing) plasmids which are necessary for nodulation and virulence, respectively. TP-RAPD analysis revealed three patterns indicating three genetically distinct groups within the isolate collection. From each pattern, one representative isolate was selected for further molecular analyses. Phylogenetic analyses depending on nucleotide sequences of 16S rDNA and recA genes assigned representative isolates of Pattern-A (n: 4) and Pattern-B (n: 6) as A. radiobacter. On the other hand, the representative isolate of Pattern-C (n: 2) appeared as related to A. nepotum. As a result, this study presents the first phylogenetically identified root-endophytic Agrobacterium radiobacter and A. nepotum isolates from root nodules of C. arietinum L. grown in Central Anatolia part of Turkey. Additionally, the first molecular data of A. radiobacter for Turkey also presented.


INTRODUCTION
The genus Agrobacterium (Class: Alphaproteobacteria, Order: Rhizobiales, Family: Rhizobiaceae) has comprised of gram negative soil borne bacteria with various life strategies including saprophytism, parasitism (causes crown gall or hairy root diseases) and mutualism (induces root nodules on legume plants) (Zahradnik et al., 2018). Since it was first proposed by Conn, (1942) many changes have been taken place in Agrobacterium systematics both in generic and species levels. Till the end of 20 th century, Agrobacterium strains were classified within species A. radiobacter, A. tumefaciens, A. rhizogenes, A.rubi and A. vitis. In the last twenty years, as the result of investigations of new strains obtained from different hosts and locations many other species and genomic species (e.g. A. larrymoorei, A. albertimagni, A. arsenijevicii, A. deltaense, A. salinitolerans, A. rosae and A. bohemicum) have been added to the genus (Bouzar et al., 1995;Salmassi et al., 2002;Kuzmanovic et al., 2015a;Yan et al., 2017a;Yan et al., 2017b;Kuzmanovic et al., 2018;Zahradnik et al., 2018). Additionally according to new molecular phylogenetic findings, some species were transferred to the genus Agrobacterium from other genera within the Rhizobiaceae family (A. pusense, A. nepotum and A. skierniewicense), likewise transfers from Agrobacterium to the other genera (Rhizobium rhizogenes and Allorhizobium vitis) were also occurred (Panday et al., 2011;Pulawska et al., 2012a;Pulawska et al., 2012b;Mousavi et al., 2015). Currently, there are 14 binomial species within the genus Agrobacterium in addition to several genomic species (Genomovars) which are not formally named yet (Flores-Felix et al., 2020).
Chickpeas (Cicer arietinum L.), the host plant investigated in the study, is the second most cultivated pulse crop in the World and it is in the first rank in Turkey (FAOSTAT 2016;Gülümser, 2016). Although some strains of Agrobacterium causes crown gall or hairy root diseases on many host plants, there have been no records for the Chickpeas so far. One Agrobacterium species, A. pusense, isolated and identified from rhizosphere of chickpea but there is no data for its pathogenicity capability on the host (Panday et al., 2011). Contrarily, some strains of the genus Agrobacterium have been reported as root-nodulating symbionts of chickpeas from several countries like Tunisia and Iran (Saidi et al., 2011;Rouhrazi & Khodakaramian, 2015). Additionally, some root endophytic Agrobacterium isolates, which are not able to produce root nodules when reinoculated to the original host, were reported from root nodules of C. arietinum L. (Romdhane et al., 2009) But species identifications in this study have solely depending on morphology and that is why they are doubtful. In the same year, another A. tumefaciens mediated crown gall disease was reported from the same part of Turkey but from apricot. Again bacterial identifications were carried out according to the morphological and biochemical features (Aysan et al., 2003). Recently some 16S rDNA sequences belonging to A. nepotum which isolated from root nodules of C. arietinum L. in Turkey were submitted to GenBank, but there is no publication available concerning these sequences. Thus, there is a great information gap about the diversity of Agrobacterium species in Turkey.
The goal of this study is to characterize some Agrobacterium isolates obtained from root nodules of C. arietinum L. collected from five cities of the Central Anatolia region of Turkey with current molecular techniques.

MATERIAL AND METHOD
In this study, Cicer arietinum L. plants were collected from five different cities of Central Anatolia region of Turkey during May-August of 2015-2018 (Table 1). Isolations of the bacteria from root nodules were performed according to the method of Vincent, (1970). Standard YMA (Yeast Extract Mannitol Agar) medium was used both for isolations and purifications (by consecutive inoculation), of the bacteria (Vincent, 1970). The purity of the isolates was checked by colony morphology and microscopic examinations using gram staining. Nodulation tests were performed using the method of Vincent, (1970), to determine the nodulation capability of the isolates on their original host (C. arietinum L.). Tests were performed in a growth chamber at 14h light and 10 h dark and with three replicates for each isolate. Nodulation ability of the isolates were evaluated after 5 weeks of incubation. Pathogenicity tests, to visualize gall and hairy-root formation, were performed according to the method of Karakaya & Özcan, (2000). Tests were performed with three replicates for each isolate with the following modification; a growth chamber at 14h light and 10 h dark was used for the tests, YMB medium was used for growing the bacterial isolates as inoculum. Table 1. Source information, TP-RAPD profiles and GenBank accession numbers of Agrobacterium isolates obtained in this study.

Isolate
Locality TP-RAPD Profile Kayseri/Yahyalı C --Fresh bacterial cultures for the genomic DNA extractions were prepared using TY (Tryptone Yeast Extract) broth media (Ditta et al., 1987) with the conditions explained in Gurkanli et al., (2013). Genomic DNA extractions from broth cultures were conducted using CTAB/NaCl miniprep method and extracted DNA was stored in -20°C prior to use (Temizkan & Arda, 2004). A TP-RAPD (Two primers random amplified polymorphic DNA) analysis was performed for discriminating between the distinct species (or genotypes) within our isolate collection. The analysis was performed using the primer set 879F / 1522R (Rivas et al., 2001) with conditions given in Table 2. PCR amplifications were performed using a Techne TC-PLUS (Bibby Scientific, Stone, UK) thermal cycler. Presence of virulence (virA, protein kinase) and nodulation (nodD, autoregulatory) genes were investigated in the bacteria to determine the symbiosis type (mutualism or parasitism) between the bacteria and the host plant, C. arietinum L. Primer sets, VirA1F/virA2R (Velazquez et al., 2005) and Y5/Y6 (Zeze et al., 2001) were used for PCR amplifications of virA and nodD, respectively. The conditions used for PCR amplifications are given in Table 2. Consequently, one representative isolate from each TP-RAPD pattern observed was chosen for the further molecular analyses. The symbiosis types and geographical sources of the isolates were also considered as a criterion during the selection process. Characterizations of the representative isolates were conducted with phylogenetic analyses depending on nucleotide sequences of, 16S rDNA and a housekeeping gene, recA. Primer sets, fd1 / rd2 (Zhang et al., 1999) and recA-Forward / recA-Reverse (Gaunt et al., 2001) were used for PCR amplifications of 16S rDNA and recA genes, respectively. The conditions used for both PCR amplifications are stated in Table 2. A 50 μl PCR reaction for all amplifications was prepared with, 25 µl of GoTaq ® Colorless Master Mix, 2X (Promega Corporation, Madison, WI, USA), genomic DNA<0.5 µg, 0.4 pmol of each primer (in final concentration) and sterile ddH2O (up to 50 µl). PCR products were electrophoresed on 1% (2.5% for TP-RAPD) agarose gel prepared with 1X TBE buffer and visualized using a Photo-Print Digital Imaging System (Vilber Lourmat, France).
Nucleotide sequencings for 16S rDNA and recA genes of the selected isolates were performed commercially by Macrogen Europe (Amsterdam, The Netherlands) with the same primers used for PCR amplifications. Nucleotide sequences obtained from both strands were assembled using BioEdit (Hall, 1999). For phylogenetic analyses, data sets were created for each of 16S rDNA and recA genes using the haplotypes (downloaded from GenBank) given in legends of Figures -2 and -3, together with our new sequences (Table 1). On the other hand, most Agrobacterium isolates (recA and 16S rDNA sequences) in GenBank are still remaining with the older nomenclature (e.g. Rhizobium nepotum, Rhizobium pusense, Rhizobium skierniewicense etc.) and this situation have causing some confusions, thus should be revised by the original submitters. ClustalX (Thompson et al., 1997) was used for multiple nucleotide sequence alignments. To determine the best fitting substitution models for our data sets, Akaike's information criterion (AIC) and Bayesian information criterion (BIC) tests were performed using the jModelTest v. 0.1 package program (Guindon & Gascuel, 2003;Posada, 2008). Neighbor-joining (NJ), Maximum parsimony (MP) and Maximum likelihood (ML) algorithms were used to determine the phylogenetic relationships among haplotypes. NJ and MP analyses were performed using software program PAUP* v. 4.0b10 (Swofford, 1998) and PhyML 3.0 (Guindon & Gascuel, 2003) was used for the ML analyses. MP analyses were performed using a heuristic search approach with the TBR swapping algorithm (10 random repetitions). To test the reliability of the nodes on the trees, Bootstrap tests were performed with 10000 replicates for all analyses.
All new sequences obtained in this study were deposited in the NCBI data bank under accession numbers MT912699-MT912701 and MT919277-MT919279 (Table  1).
As the result of nucleotide sequencings, the variative thus informative first half of 16S rDNA (approx. 900 bp) were obtained from isolates, Agr-Ca-1, Agr-Ca-5 and Agr-Ca-11. Results of BLAST (Basic Local Alignment Search Tool, https://blast.ncbi.nlm.nih.gov/Blast.cgi) search for our new 16S rDNA haplotypes revealed that all isolates obtained in this study are belonging to the genus Agrobacterium. Thus, a data set was created with the 16S rDNA haplotypes belonging to type strains of available Agrobacterium species and Genomovars (see legend of Figure   Although they showed different TP-RAPD patterns, isolates Agr-Ca-1 (representative of Pattern-A) and Agr-Ca-5 (representative of Pattern-B) showed the same 16S rDNA haplotype with each other and also with A. radiobacter isolates, IAM 12848 T (Type strain), B6 and Agrobacterium Genomovar 3 isolate CIP107443. This lineage was supported with 84% and 85% bootstrap values in NJ and ML trees, respectively. The lineage composed of Agrobacterium Genomovars -5, -7, -13 and A. deltaense isolate YIC4121 T , which showed the same 16S rDNA haplotype with each other, appeared as sister to the lineage above ( Figure 2). The third isolate, Agr-Ca-11, showed the same 16S rDNA haplotype with A. nepotum type strain 39/7 T . The relationship between these two haplotypes was supported with 90%, 88% and 78% bootstrap values in the NJ, ML and MP trees, respectively. A. arsenijevicii type strain KFB 330 T and Agrobacterium Genomovar 1 isolate H13.3 also appeared in the same lineage with Agr-Ca-11 ( Figure 2). In general, relationships within this lineage were supported with significant bootstrap values (≥65).
To assign our selected isolates to one of the valid Agrobacterium species (or genomovars), approximately 570 bp of recA gene were obtained and subjected to phylogenetic analyses. The data set used for phylogenetic analyses was containing the recA haplotypes of type strains of available Agrobacterium species. Additionally, recA haplotypes of isolates representing available Agrobacterium Genomovars as well as recA haplotypes some A. radiobacter and A. nepotum isolates also included to the data set (see legend of Figure 3). Analyses were carried out on 373 aligned nucleotides containing 135 variable sites. Both AIC and BIC tests suggested TIM2+I+G (I: 0.513; G: 0.795) substitution model. MP analysis which gave 17 most parsimonious trees (Length: 458 steps, CI: 0.423581, RI: 0.667925, HI: 0.576419) was conducted using 117 synapomorphic characters. NJ tree created using TIM2+I+G model was given in Figure 3 and bootstrap values derived from ML and MP analyses were indicated on the same tree. In the tree, our isolates Agr-Ca-1 and Agr-Ca-5 were placed within A. radiobacter lineage which composed of three different haplotypes (Figure 3). Agr-Ca-1 showed the same recA haplotype with isolate CFBP 5522 T , the type strain of A. radiobacter, and also with isolate CFBP 5621. A. radiobacter isolate B6 appeared as sister and this relationship was supported with 62%, 60% and 60% bootstrap values in NJ, ML and MP trees, respectively.
Our second A. radibacter isolate, Agr-Ca-5, showed the same recA haplotype with isolates, Kerr 14 and CCNWGS and appeared as sister to the sub-lineage above. Bootstrap values supporting this relationship were 96%, 92% and 98% in NJ, ML and MP trees, respectively. Nucleotide sequence similarities among all three A. radiobacter recA haplotypes were determined as higher than 98.1%. Concordant with the 16S rDNA phylogeny, isolate Agr-Ca-11 appeared as related with A. nepotum lineage in recA phylogeny. This relationship has supported with 100% bootstrap values in all trees created using NJ, ML and MP algoritms (Figure 3). Nucleotide sequence similarity between Agr-Ca-11 and the type strain of A. nepotum (isolate 39/7 T ) was determined as 97.8%

DISCUSSION
During a survey for rhizobia which nodulates C. arietinum L. in the Central Anatolia region of Turkey, a group of (n: 12) bacteria that morphologically dissimilar (much more mucoid and opaque) to classical rhizobia were obtained from root nodules of C. arietinum L. TP-RAPD analysis which is a commonly used pre-grouping method in bacteriological studies (Rivas et al., 2001), revealed three patterns within our collection which indicated three genetically distinct groups. Phylogenetic analyses depending on nucleotide sequences of two genetic markers, 16S rDNA and recA, designated representative isolates of first two TP-RAPD patterns, Pattern-A (Agr-Ca-1) and Pattern-B (Agr-Ca-5) as A. radiobacter. Although some A. radiobacter isolates have reported from Turkey so far, they were all clinical strains causing infections in humans (Otağ et al., 2007). Thus according to the available literature, there has been only one soil-borne Agrobacterium species, A. tumefaciens, reported from Turkey (Aysan & Sahin, 2003;Aysan et al., 2003). On the other hand, identifications of the bacteria in all these studies were depending on some morphological and biochemical features, thus were highly doubtful. That is why, isolates, Agr-Ca-1 and Agr-Ca-5, in this study are the first properly (with current molecular phylogenetic techniques) identified soil-borne A. radiobacter isolates from Turkey. Phylogenetic analyses designated isolate Agr-Ca-11, the representative isolate of TP-RAPD Pattern-C, as A. nepotum. This species was first described by Pulowska et al., (2012b) from tumours on several plant species in Europe. Since then, it has been reported from several countries as pathogen or root nodule endophytes (Mafakheri et al., 2019;El Yemlahi, Unpublished). In GenBank, there is one 16S rDNA sequence, submitted from Gaziantep University (Gaziantep-Turkey), belonging to a nodule endophytic A. nepotum strain which isolated from root nodules of C. arietinum L. in Turkey. On the other hand, there is no available publication on the strain, thus its exact origin and other features are unknown. But together with our finding, it may indicate that A. nepotum is one of the common nodule endophytic Agrobacterium species in chickpea fields of Central and Southeastern Anatolia. But this presumption must be confirmed with comprehensive investigations. In authentication and pathogenicity tests, none of the twelve isolates produced root nodules or disease symptoms on chickpeas. Additionally, no appropriate DNA bands were observed as the result of nodD and virA amplifications. These results have proved the absence of the genetic accessories necessary for the nodulation or virulence processes in these isolates therefore they all are nodule-endophytes (nonnodulating). Presence of nodule-endophytic bacteria in root nodules have been reported from various legume species including Melilotus dentatus, P. vulgaris L. and C. arietinum L. (Mhamdi et al., 2005;Wang et al., 2006;Ben Romdhane et al., 2009). Although the mechanism underlying this biological phenomenon is unknown, Ben Romdhane et al., (2009) suggested that water deficiency conditions induce the colonisation of chickpea nodules by non-symbiotic or non-specific Rhizobium and Agrobacterium isolates. If we consider the geographical and climatic features of Central Anatolia during the nodulation period of chickpea seedlings (approx. during June-July), a water deficiency condition is highly possible. This unfavourable situation possibly may have caused the infiltration of nodule-endophytic Agrobacterium isolates to chickpea root nodules. Although these bacteria are avirulent (gives no direct harm to the plant) it may indirectly reduce the crop productivity by competing with the actual rhizobial partner of C. arietinum L. in the rhizosphere. In this context, it has been reported that Agrobacterium isolates may reduce the nodulation of P. vulgaris L. by its rhizobial partner, Rhizobium gallicum (Mrabet et al. 2006).
As conclusion, this study presents the first rootendophytic Agrobacterium radiobacter and A. nepotum isolates, which identified using proper molecular phylogenetic techniques, from C. arietinum grown in Central Anatolia part of Turkey. Additionally, it gives the first molecular data for A. radiobacter for Turkey. Bouzar, H., Chilton, W.S., Nesme, X., Dessaux, Y.,