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Phenotypic and Molecular-Phylogenetic Analysis Provide Novel Insights into the Diversity of Curtobacterium flaccumfaciens

    Affiliations
    Authors and Affiliations
    • Ebrahim Osdaghi
    • S. Mohsen Taghavi
    • Silvia Calamai
    • Carola Biancalani
    • Matteo Cerboneschi
    • Stefania Tegli
    • Robert M. Harveson
    1. First and second authors: Department of Plant Protection, College of Agriculture, Shiraz University, Shiraz 71441-65186, Iran; third, fourth, fifth, and sixth authors: Dipartimento di Scienze delle Produzioni Agroalimentari e dell’Ambiente, Laboratorio di Patologia Vegetale Molecolare, Università degli Studi di Firenze, Via della Lastruccia 10, 50019 Sesto Fiorentino, Firenze, Italy; and seventh author: University of Nebraska, Panhandle Research & Extension Center, 4502 Ave. I., Scottsbluff 69361.

    Published Online:https://doi.org/10.1094/PHYTO-12-17-0420-R

    Abstract

    A multiphasic approach was used to decipher the phenotypic features, genetic diversity, and phylogenetic position of 46 Curtobacterium spp. strains isolated from dry beans and other annual crops in Iran and Spain. Pathogenicity tests, resistance to arsenic compounds, plasmid profiling and BOX-PCR were performed on the strains. Multilocus sequence analysis (MLSA) was also performed on five housekeeping genes (i.e., atpD, gyrB, ppk, recA, and rpoB) of all the strains, as well as five pathotype strains of the species. Pathogenicity test showed that six out of 42 strains isolated in Iran were nonpathogenic on common bean. Despite no differences found between pathogenic and nonpathogenic strains in their plasmid profiling, the former were resistant to different concentrations of arsenic, while the latter were sensitive to the same concentrations. Strains pathogenic on common bean were polyphyletic with at least two evolutionary lineages (i.e., yellow-pigmented strains versus red/orange-pigmented strains). Nonpathogenic strains isolated from solanaceous vegetables were clustered within either the strains of C. flaccumfaciens pv. flaccumfaciens or different pathovars of the species. The results of MLSA and BOX-PCR analysis were similar to each other and both methods were able to discriminate the yellow-pigmented strains from the red/orange-pigmented strains. A comprehensive study of a worldwide collection representing all five pathovars as well as nonpathogenic strains of C. flaccumfaciens is warranted for a better understanding of the diversity within this phytopathogenic bacterium.

    Curtobacterium flaccumfaciens inhabits multiple ecological niches, and includes environmental (Chase et al. 2016), human-pathogenic (Francis et al. 2011) plant-pathogenic (Osdaghi et al. 2015a), and plant beneficial (Raupach and Kloepper 2000) strains. The plant-pathogenic strains consist of five pathovars, namely C. flaccumfaciens pv. betae, C. flaccumfaciens pv. flaccumfaciens, C. flaccumfaciens pv. ilicis, C. flaccumfaciens pv. oortii, and C. flaccumfaciens pv. poinsettiae, the causal agents of silvering disease of red beet, bacterial wilt of dry beans (Fabaceae), bacterial blight of American holly, bacterial wilt and spot of tulip, and bacterial canker of poinsettia, respectively (Collins and Jones 1983; Dye and Kemp 1977). Among them, C. flaccumfaciens pv. flaccumfaciens is an economically important quarantine pathogen which causes bacterial wilt of dry beans in several States in North and South America, Asia and Oceania (EPPO 2011). C. flaccumfaciens is one of the most ambiguous and poorly understood plant pathogenic bacteria in terms of its biology, epidemiology and population genetics (Harveson et al. 2015). The bacterium also can colonize a number of plant species without inducing any disease symptoms (Gonçalves et al. 2017; Harveson et al. 2015; Osdaghi et al. 2018a). While orange, pink, purple and yellow colony variants of C. flaccumfaciens pv. flaccumfaciens have been reported from the central high plains of the United States (Agarkova et al. 2012), multicolored populations of the pathogen have been found in Canada (Huang et al. 2009) and Brazil (Soares et al. 2013). In addition to the yellow and orange-pigmented variants, a new red-pigmented variant of C. flaccumfaciens pv. flaccumfaciens has been recently isolated from common bean seeds in Iran (Osdaghi and Lak 2015b; Osdaghi et al. 2015a, 2016a). Differences in aggressiveness of C. flaccumfaciens pv. flaccumfaciens variants were observed when they were tested on various dry bean cultivars (Osdaghi et al. 2016a).

    Nucleic acid sequence-based methods, such as multilocus sequence analysis (MLSA), have been developed to phylogenetically analyze the multiple core genes and to obtain clustering patterns of microorganisms. In particular, MLSA is a powerful and well-accepted method to study the phylogeny of plant pathogenic bacteria (Jacques et al. 2012). While many MLSA studies have been conducted on several Gram-negative and Gram-positive plant pathogenic bacteria (Almeida et al. 2010; Jacques et al. 2012), no study has been conducted to determine the phylogenetic position of plant pathogenic C. flaccumfaciens strains. As a consequence, the relationships between the results of band-based fingerprinting (e.g., rep-PCR) and MLSA methods are yet to be determined for C. flaccumfaciens. In addition, unlike extensive molecular studies performed on several corynebacteria, (Gartemann et al. 2008), no information is available on mechanistic understanding of the virulence accessories and survival of C. flaccumfaciens on host plants, and other environmental habitats. This is a paradox based on the economic importance of this species, and the fact that high throughput techniques are available which could aid in making more accurate taxonomic classification.

    The objectives of this study were to (i) determine the phenotypic characteristics (i.e., pathogenicity, arsenic resistance, and plasmid profile) of C. flaccumfaciens strains isolated from different annual crops in Iran, (ii) analyze the phylogenetic position of the C. flaccumfaciens strains isolated in Iran and Spain, in relation to all members of Curtobacterium spp., and (iii) compare the results with those of BOX-PCR fingerprinting and phenotypic characteristics of the strains.

    MATERIALS AND METHODS

    Bacterial strains.

    C. flaccumfaciens and Curtobacterium-like strains isolated in Iran during 2013 to 2016, from either symptomatic dry bean plants or asymptomatic solanaceous vegetables and squash (Cucurbita pepo), were used in this study (Table 1). The strains isolated from solanaceous vegetables were associated with either symptomatic or symptomless tomato and pepper plants in Iran (Osdaghi et al. 2016b, 2017a, 2018a). Additionally, pure DNA of four Curtobacterium spp. strains isolated from common bean seeds of germplasm bank in Spain (provided by Ana J. González; Horticultural and Forest Crops Area, SERIDA, Asturias, Spain) were included in molecular and phylogenetic analysis (González et al. 2005). In total, 51 strains, which include 46 strains from Iran and Spain, as well as the type strains of C. flaccumfaciens pv. flaccumfaciens (ICMP 2584), C. flaccumfaciens pv. betae (ICMP 2594), C. flaccumfaciens pv. ilicis (ICMP 2608), C. flaccumfaciens pv. oortii (ICMP 2632), and C. flaccumfaciens pv. poinsettiae (ICMP 2566) were used in this study. Standard strains of Clavibacter michiganensis subsp. michiganensis (i.e., Tom835 = ICMP 22052, ICMP 2550, and NCPPB 382), as well as a nonpathogenic strain of Clavibacter spp. (Tom495 = ICMP 22060) were used as controls (Osdaghi et al. 2018b).

    TABLE 1. List of Curtobacterium flaccumfaciens and Curtobacterium-like strains used in this study, as well as the results of their morphological characterization, specific PCR, and pathogenicity testsa

    Morphological characteristics and pathogenicity tests of the strains.

    Morphological characteristics (e.g., colony color and fluidity) of the strains were determined on yeast extract-dextrose-calcium carbonate (YDC) agar medium, as well as nutrient agar (NA) medium supplemented with 5% sucrose, after 72 h incubation as described by Smith et al. (2001). Briefly, colony morphology was subdivided into three categories: fluidal (colonies flowed when plates were inclined at 45°), mucoid (colonies had a glutinous consistency due to the production of polysaccharide), and dry (little or no polysaccharide was produced) (Smith et al. 2001).

    Pathogenicity tests were conducted on common bean (Phaseolus vulgaris) plants (cultivar Dorsa) grown in glasshouse conditions using the bacterial strains reported in Table 1. Plant growth conditions and inoculum preparation were described previously (Osdaghi et al. 2015b). Plants were inoculated at the 10 to 12 days postemergence. For each strain, six common bean plants (three/pot) were inoculated. Inoculation was made by inserting a sterile dissecting needle dipped into a fresh bacterial suspension (1 × 108 CFU/ml) throughout the internode between the first and the second node of each plant. All inoculated plants were maintained in the greenhouse at ambient temperature (25 to 28°C and 14 h natural light). A reference strain of C. flaccumfaciens pv. flaccumfaciens (ICMP 22071) and sterile distilled water were used as positive and negative controls, respectively. Plants were periodically monitored for the appearance of disease symptoms and the final evaluation of disease symptoms was performed at 20 days postinoculation (dpi). Disease severity on each plant was rated based on the number of primary or trifoliate leaves showing wilting symptoms as described previously (Osdaghi et al. 2016a). Koch’s postulates were accomplished by re-isolating the inoculated strains on yeast-extract peptone glucose agar (YPGA) medium from all inoculated plants. The identity of re-isolated bacterial strains was confirmed using the primer pair CffFOR2/CffREV4 (Tegli et al. 2002) (Table 2). Since six strains (i.e., Cmmeg20, G105, Mo01 Mo04, Tom827, and Xeu15) did not induce any symptoms on the inoculated common bean cultivar Dorsa plants, the same procedure as described above was conducted on common bean cultivars Derakhshan and Sadri and cowpea (Vigna unguiculata) cultivar Mashhad. All the pathogenicity tests were repeated twice.

    TABLE 2. Primer pairs used in this study

    Screening for arsenic resistance.

    We evaluated a set of 31 representative strains (Table 3) for their resistant response to different concentrations of two arsenic compounds (i.e., sodium arsenite [NaAsO2] and sodium arsenate [Na3AsO4]). Type strain of C. flaccumfaciens pv. oortii (ICMP 2632) was used as positive control as recommended by Hendrick et al. (1984). We also included the standard strains of either pathogenic (i.e., ICMP 2550, NCPPB 382, and ICMP 22052), or nonpathogenic Clavibacter spp. (ICMP 22060) strains as negative controls (Osdaghi et al. 2018b).

    TABLE 3. Growth rate of Curtobacterium flaccumfaciens, Curtobacterium-like, and Clavibacter michiganensis strains used in this study on different concentrations of sodium arsenite (NaAsO2) and sodium arsenate (Na3AsO4)a

    The bacterial strains were screened using the agar plating method as described previously (Hendrick et al. 1984). Briefly, nutrient broth-yeast extract (NBY) agar plates supplemented with three different concentrations of either sodium arsenite (2, 5, and 7 mM), or sodium arsenate (80, 100, and 130 mM) were used for bacterial inoculation. For each strain, serial tenfold dilutions were prepared from a starter suspension (OD600 = 2.5), obtained from a fresh culture grown at 27°C for 24 h on nutrient broth agar medium. For each dilution, 12 droplets (each droplet containing 5 μl of the suspension) were plated on each arsenic-containing plate. The plates were then incubated at 27°C for 48 h, after which the number of single colony forming units (CFU) were counted. The data (average values ± standard deviation [SD]) were subjected to one-way analysis of variance (ANOVA). Tukey’s range test was also performed to identify statistically significant differences among the strains / salt concentrations, using PAST Version 3.17 (Hammer et al. 2001) (https://folk.uio.no/ohammer/past/).

    Plasmid profiling.

    A set of 11 strains isolated in Iran was selected to carry out plasmid content analysis. This set was represented by candidate strains based on different isolation hosts and phenotypic features, including the type strain of C. flaccumfaciens pv. flaccumfaciens (ICMP 2584), pathogenic strains (50R, 80O, Cw110, P990, Tom50, and Tom930), as well as nonpathogenic strains (Cmmeg20, Mo04, Tom827, and Xeu15) of C. flaccumfaciens. The standard strains of Clavibacter michiganensis subsp. michiganensis (ICMP 2550 and NCPPB 382), harboring two plasmids (i.e., pCM1 and pCM2) (Meletzus et al. 1993) were also included in this study as positive controls.

    Plasmids were isolated according to the procedure described by Klaenhammer (1984) with several modifications. The strains were grown overnight in 10 ml of Luria-Bertani (LB) medium, on a 110 rpm shaker at 27 °C. Bacterial cells were pelleted by centrifugation (6,000 × g for 5 min) and the pellets were resuspended in 1 ml of Tris-EDTA (TE) buffer (pH 7.5), with 25% sucrose and 75 µl of lysozyme (1 mg ml−1 in TE, pH 7.5), and incubated at 37°C for 1 h. Subsequently, 500 µl of lysis solution was added to the bacterial pellet and the samples were heated at 62 °C for 1 h. Finally, plasmid DNA was neutralized by the addition of 50 µl of 2 M Tris (pH 7) and 70 µl of 5 M NaCl. The presence of plasmids was analyzed on 0.9% agarose gel at 60 V for 4 to 5 h in TAE buffer. Agarose gel was stained with ethidium bromide at 0.5 μg/ml and visualized with UV light using Gel Doc XR+ (BioRad). The experiments were repeated three times.

    DNA extraction, PCRs, and sequencing.

    DNA extraction was performed using Expin Combo-GP DNA extraction kit (GeneAll, Tic Tech Centre, Singapore) based on the manufacturer’s recommendations. The quality and quantity of the DNAs were spectrophotometrically evaluated and adjusted to 50 ng µl−1 using Nanodrop ND-100 (Nanodrop Technologies, Waltham, MA). The DNA was kept at −20°C for further uses. Five housekeeping genes including atpD, gyrB, ppk, recA, and rpoB were employed for the sequencing and phylogenetic analyses on all the strains described in Table 1. Primer pairs were used for partial sequencing of atpD, gyrB, and rpoB as described previously (Table 2) (Jacques et al. 2012; Richert et al. 2005, 2007). While the primer pairs ppkCfF/ppkCfR and recACfF/recACfR were redesigned for ppk and recA genes, respectively, based on the sequence of Curtobacterium sp. (strain MR_MD2014, GenBank: CP009755.1) (Mariita et al. 2015) according to Jacques et al. (2012) (Table 2). For PCR reactions, Universal PCR Kit, Ampliqon Taq DNA Polymerase Master Mix Red (Ampliqon A/S, Odense, Denmark), was used according to the manufacturer’s recommendations. For each strain, a 25 µl of PCR including 50 ng of total DNA and 1 µl of each primer (10 pmol µl−1) were used. Purity and yield of PCR products were checked by running a 5-µl reaction mixture in 1.2% agarose gel stained with ethidium bromide. The PCR products were sent to Bioneer Corporation (www.Bioneer.com) (Daejeon, South Korea) to be sequenced using Sanger sequencing technology.

    Resulting sequences were analyzed using the BLAST program (https://blast.ncbi.nlm.nih.gov/) and aligned with Clustal W program (Larkin et al. 2007) implemented in MEGA 6.06 software (Tamura et al. 2013). Partial sequences were deposited in the NCBI GenBank and assigned accession numbers as follows: atpD: KX591664 to KX591707 and MG737698 to MG737699; gyrB: KX591708 to KX591751, and MG737700 to MG737701; ppk: KX591752 to KX591795, and MG737702 to MG737703; recA: KX591796 to KX591839, and MG737704 to MG737705; and rpoB: KX591840 to KX591883, and MG737706 to MG737707. For phylogenetic comparisons, the respective sequences of the five housekeeping genes were retrieved from 30 publicly available complete genome sequences of Curtobacterium spp. strains in the GenBank database.

    Phylogenetic analysis.

    Sequences were concatenated following the alphabetic order of the genes, ending in a sequence of 2,977 bp: nucleotides 1 to 761 for atpD, 762 to 1507 for gyrB (746 bp), 1508 to 2021 for ppk (514 bp), 2022 to 2612 for recA (591 bp), and 2613 to 2977 for rpoB (365 bp). Phylogenetic analyses were performed on individual gene sequences as well as the data set of concatenated sequences. Phylogenetic trees were constructed using maximum likelihood method with MEGA 6.06 software (Tamura et al. 2013). The general time-reversible (GTR) model of evolution was selected for Maximum Likelihood analysis using the Modeltest tab in MEGA 6.06 (Hall, 2011). Clavibacter michiganensis subsp. michiganensis strain NCPPB 382 was used to root the trees. MEGA 6.06 was used to obtain the phylogenetic trees and bootstrap values (1000 replicates) for the nucleotide sequences of each individual gene and of concatenated sequences. Additionally, the similarity matrix of the concatenated sequences of five housekeeping genes, in the type strains of five pathovars of C. flaccumfaciens, was prepared using the online service “Sequence Identity And Similarity” (SIAS) (http://imed.med.ucm.es/Tools/sias.html) with default settings.

    Recombination analysis.

    Nucleotide diversity, the number of haplotypes, and haplotype diversity were determined using DnaSP 5.10 software (Librado and Rozas 2009). The class I neutrality tests (Tajima’s D and Fu, and Li’s D* and F*) were also calculated for detecting potential departure from the mutation/drift equilibrium (Librado and Rozas 2009). Detection of potential recombinant sequences and identification of likely parental sequences within C. flaccumfaciens strains were conducted using a set of seven nonparametric detection methods (i.e., RDP, Geneconv, MaxChi, Chimaera, BootScan, SiScan, and 3Seq) implemented in Recombination Detection Program (RDP) version 4.80 (Martin et al. 2015). The analysis was performed with default settings for the different detection methods, and the Bonferroni-corrected P value cutoff was set at 0.05. Two independent experiments, one including all the Curtobacterium sp. strains and the other including only C. flaccumfaciens strains, were performed in this analysis. Recombination events were accepted when they were identified by at least four out of seven detection methods (Martin et al. 2015). Splits-decomposition network was constructed and the pairwise homoplasy index (PHI) was calculated using SplitsTree version 4.14.4 (Huson and Bryant 2006). These calculations used the individual genes, as well as the entire data set of concatenated sequences (Huson and Bryant 2006).

    Rep-PCR.

    Since the MLSA-based phylogeny was unable to differentiate pathogenic and nonpathogenic strains, BOX, enterobacterial repetitive intergenic consensus (ERIC), and repetitive element palindromic (REP) primers (Table 2) (Versalovic et al. 1994) were used to discriminate the putative diversity among C. flaccumfaciens strains from our collection. Fifty-one C. flaccumfaciens and one Clavibacter michiganensis (as out-group) strains were evaluated with rep-PCR analysis (Table 1). PCR reactions were similar to those described above, while the annealing temperatures are described in Table 2. Ten microliters of PCR products was run on 1.2% agarose gel, stained with ethidium bromide, and the digitized image was converted into a TIFF file for a subsequent analysis of the fingerprint patterns. Unweighted pair groups with arithmetic averages were calculated using NTSYS-pc software version 2.02e (Rohlf 2008). The procedure was repeated independently to test the reproducibility of the fingerprints.

    RESULTS

    Morphology and pathogenicity of the strains.

    Morphological characteristics of the strains are presented in Table 1. Among the bacterial strains isolated in Iran, all but two (i.e., Cw900 and Tom50) were shown to have fluidal colony on YDC medium. The strains Cw900 and Tom50 have had dried colonies on the same medium (Table 1). As for colony color, 30 strains had yellow colonies, while eight were orange and four were red-pigmented (Table 1; Supplementary Fig. S1).

    All the C. flaccumfaciens strains isolated from dry beans in Iran were pathogenic on common bean in greenhouse conditions (Table 1). Interveinal chlorosis, leading to necrotic areas on the leaves, and systemic wilting were observed at 8 to 14 days postinoculation (Supplementary Fig. S2). Among the strains isolated from solanaceous vegetables, all but four (i.e., Cmmeg20, G105, Tom827, and Xeu15) were pathogenic on common bean (Table 1). None of the strains isolated from squash (i.e., Mo01 and Mo04) were pathogenic on common bean. After inoculation, bacterial strains were re-isolated from the symptomatic plants, and identified using specific PCR primers CffFOR2/CffREV4 (data not shown). Repetitive pathogenicity tests on common bean cultivars Derakhshan and Sadri, as well as on cowpea cultivar Mashhad with Cmmeg20, G105, Tom827, Xeu15, Mo01, and Mo04 strains produced similar results to those observed in the first set of pathogenicity tests. Control plants remained healthy.

    Resistance to arsenic.

    All but five of the evaluated strains were resistant against arsenic compounds. The strains Cmmeg20, G105, Tom827, Xeu15, and Mo04 were unable to grow on any concentration of either sodium arsenite and sodium arsenate (Table 3). The level of resistance to arsenic compounds in the type strain of C. flaccumfaciens pv. flaccumfaciens (ICMP 2584) was statistically different from those of other strains. It was able to grow on sodium arsenite up to 2 mM (Table 3), with faint growth on all concentrations of sodium arsenate. None of the Clavibacter spp. strains evaluated was able to grow on arsenic compounds regardless of their pathogenicity status on tomato.

    Plasmid profiling.

    As expected, Clavibacter michiganensis subsp. michiganensis strains ICMP 2550 and NCPPB 382 harbored two plasmids (pCM1 and pCM2); whose sizes were 27.5 and 72 kb, respectively (Fig. 1, lanes A and M). No plasmids were found in any of the C. flaccumfaciens strains tested here, regardless of their isolation host or pathogenicity on common bean (Fig. 1, lanes B to L).

    Fig. 1.

    Fig. 1. Plasmid profile of Curtobacterium flaccumfaciens and Curtobacterium-like strains used in this study. Indigenous plasmids from Clavibacter michiganensis subsp. michiganensis (pCM1 and pCM2), whose sizes were 27.5 and 72 kb, respectively, were used as positive control. Chromosomal DNAs are seen as a common band in all strains. Lanes A and M, Clavibacter michiganensis subsp. michiganensis ICMP 2550 and NCPB 382, respectively; lane B, ICMP 2584; lane C, P990; lane D, Tom50; lane E, 50R; lane F, 80O; lane G, Tom827; lane H, Tom930; lane I, Xeu15; lane J, Cmmeg20; lane K, Cw110; and lane L, Mo04. No indigenous plasmids were found in C. flaccumfaciens and Curtobacterium-like strains used in this study.

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    Phylogenetic analysis.

    Phylogenetic analysis showed a clustering pattern based on colony color of C. flaccumfaciens strains used in this study (Fig. 2). Considering the data set of concatenated sequences of five housekeeping genes, the phylogenetic tree was strongly supported by a 100% bootstrap value, clear differentiation of yellow-pigmented C. flaccumfaciens strains from red/orange-pigmented strains (Fig. 2).

    Fig. 2.

    Fig. 2. Maximum likelihood tree based on the concatenated partial sequences of atpD, gyrB, ppk, recA, and rpoB genes in Curtobacterium flaccumfaciens and Curtobacterium-like strains used in this study. Bootstrap scores (1,000 replicates) are displayed at each node. Clavibacter michiganensis was used for rooting the tree. Yellow-pigmented strains of C. flaccumfaciens were phylogenetically different from those of red/orange-pigmented strains. Nonpathogenic strains of C. flaccumfaciens were scattered among the pathogenic strains. The strains isolated in Iran and Spain were labeled using black triangles, while the type strains of five pathovars of C. flaccumfaciens were labeled using black squares.

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    All the yellow-pigmented strains of C. flaccumfaciens clustered as a monophyletic group containing the nonpathogenic strains (i.e., Tom827, Cmmeg20, and Xeu15) isolated from solanaceous vegetables in Iran, as well as a number of cosmopolitan strains isolated from different environmental habitats (Fig. 2). The yellow-pigmented strains 10eg, Cb222, Cb302, Cw104, Cw110, P701, Tom803, Tom805, Tom806, and Tom930—all of which were isolated in northwestern Iran in 2015—were clustered as one haplotype (Table 1). This observation is consistent with the epidemic emergence of the bacterial wilt disease from all the northwestern provinces of the country in 2015. Nonpathogenic red-pigmented strain G105 was clustered among the other red/orange-pigmented pathogenic strains irrespective of their host of isolation. Furthermore, the strains LPPA2315, LPPA2199, and Mo01 were clustered as a monophyletic group apart from the core population of C. flaccumfaciens. Based on the results of MLSA data, none of the strains Mo01, Mo04, LPPA2315, and LPPA2199 are true members of C. flaccumfaciens. In all the five phylogenetic trees constructed using the individual housekeeping gene sequences, yellow-pigmented C. flaccumfaciens strains were separated from the red/orange-pigmented strains. Interestingly, there were no differences among all the yellow-pigmented C. flaccumfaciens strains in the rpoB gene sequence (data not shown).

    Sequence statistics of the five housekeeping genes used for the phylogenetic analysis are summarized in Table 4. Among the 75 C. flaccumfaciens and Curtobacterium spp. strains used in this study, the highest number of haplotypes (53 haplotypes) were observed in gyrB gene sequences. Conversely, only 33 haplotypes were observed in rpoB gene sequences using the same number of strains (Table 4). Altogether, gyrB and recA genes were the most discriminative, and rpoB was the least discriminative gene for C. flaccumfaciens phylogeny evaluations (Table 4).

    TABLE 4. Sequence statistics of the five housekeeping genes (i.e., atpD, gyrB, ppk, recA, and rpoB) of Curtobacterium flaccumfaciens strains used in this study

    Sequence similarity matrix experiments using five housekeeping gene sequences showed that four pathotypes of C. flaccumfaciens (i.e., C. flaccumfaciens pv. betae, C. flaccumfaciens pv. flaccumfaciens, C. flaccumfaciens pv. oortii, and C. flaccumfaciens pv. poinsettiae) are closely related to each other with sequence similarity ranging between 97.31 to 99.09% (Table 5). However, C. flaccumfaciens pv. ilicis (previously known as Arthrobacter ilicis) (Young et al. 2004), which recently been included in C. flaccumfaciens species, is distinct from the core population of other C. flaccumfaciens isolates (Fig. 2). Indeed, the sequence similarity between C. flaccumfaciens pv. ilicis and the other four pathovars of the species is only 95.53 to 95.93% (Table 5).

    TABLE 5. Similarity matrix of the concatenated sequences of five housekeeping genes (i.e., atpD, gyrB, ppk, recA, and rpoB) in five pathotype strains of Curtobacterium flaccumfaciens

    Tajima’s D, and Fu and Li’s D* and F* statistics showed that there was no significant departure from the mutation drift equilibrium within C. flaccumfaciens strains used in this study (data not shown). Because the maximum likelihood phylogenies showed incompatible topologies (Fig. 2; Supplementary Fig. S3), phylogenetic networks were generated using the splits-decomposition method for the concatenated data set (Fig. 3), as well as all the individual gene sequences (data not shown). Considering the C. flaccumfaciens strains, pairwise homoplasy index (PHI) test did find statistically significant evidence suggesting the occurrence of recombination among the gyrB (PhiTest = 0.27568; P < 0.02386) and recA (PhiTest = 0.22906; P < 0.3122) genes but not in the atpD, ppk, and rpoB genes. Recombination Detection Program (RDP) discovered recombination in both the data set of Curtobacterium spp. strains, and C. flaccumfaciens strains (Table 6). Indeed, recombination was detected in the C. flaccumfaciens strains from all the seven tested methods. Additionally, when the individual gene sequences were considered using RDP, recombination was identified in gyrB (in six out of seven methods), recA (in four out of seven methods), and rpoB (in four out of seven methods) genes sequences.

    Fig. 3.

    Fig. 3. Splits decomposition network generated from the concatenated sequences of atpD, gyrB, ppk, recA, and rpoB genes of Curtobacterium flaccumfaciens and Curtobacterium-like strains used in this study. All the red/orange-pigmented strains of C. flaccumfaciens pv. flaccumfaciens were clustered separately from the yellow-pigmented strains, while the type strain of C. flaccumfaciens pv. poinsettiae (ICMP 2566) was clustered within the cosmopolitan strains. Interestingly, the type strain of C. flaccumfaciens pv. ilicis (ICMP 2608) was clustered far from the core population of C. flaccumfaciens similar to that observed in multilocus sequence analysis scheme (Fig. 2).

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    TABLE 6. Test of recombination among Curtobacterium flaccumfaciens and Curtobacterium-like strains using RDP4 with a Bonferroni test at a probability of 0.05

    Genetic diversity of the strains.

    ERIC-PCR produced 0 to 4 fragments in sizes ranging from 0.2 to 2 kb, while REP-PCR produced 0 to 3 fragments in sizes ranging from 0.1 to 3 kb (data not shown). Primer BOX A1R produced 4 to 12 fragments in sizes ranging from 0.2 to 2.6 kb (Supplementary Fig. S4). Hence, BOX A1R primer was selected to evaluate the genetic diversity of our collection of 51 C. flaccumfaciens strains. The dendrogram based on UPGMA cluster analysis showed all C. flaccumfaciens strains forming a group with similarity coefficient ranged from 31 to 93% (Fig. 4). Cluster analysis using a cutoff level at 44% similarity produced six clusters (named G1 to G6)—two of them were major clusters (G1 = 25 and G2 = 16 strains) and four minor clusters (one to four strains). Cluster G1 included both yellow-pigmented and red/orange-pigmented strains, while clusters G2-G6 incorporated only yellow-pigmented strains. The strains in G1 were subclustered into three subgroups namely G1-1 to G1-3, with similarity values between 56 to 93%. All but one strain (Cff155) in G1-1 were yellow-pigmented, while the strains in G1-2 were red/orange-pigmented, except the strain LPPA392. The cluster G1-3 contained three strains, all of them isolated from tomato, and two of them (G105 and Tom827) were nonpathogenic on bean plants (Fig. 4; Table 1). Cluster G2 had similarity coefficients ranging from 55 to 91% and contained 15 yellow-pigmented strains in G2-1 and strain 10eg in G2-2 subclusters (Fig. 4). Cluster G3, contained four strains, which further divided into two subclusters in 49% cutoff value similarity. Cluster G4 contained four strains, three of which were isolated from in Spain (LPPA2199, LPPA2315, and LPPA987), and the type strain of C. flaccumfaciens pv. poinsettiae. Finally, cluster G5 contained only one strain (P701) as did cluster G6 (the type strain of C. flaccumfaciens pv. oortii) (Fig. 4).

    Fig. 4.

    Fig. 4. Dendrogram generated from BOX-PCR fingerprints of Curtobacterium flaccumfaciens strains used in this study. Cluster analysis was performed using the simple matching similarity coefficient and unweighted pair group with arithmetic averages using NTSYS-pc software version 2.02e. The scale bar indicates levels of linkage between patterns. Cluster analysis using a cutoff level at 44% similarity produced six clusters (named G1 to G6)—two of them major (G1 = 25 and G2 = 16 strains) and four were minor (one to four strains) clusters. Red/orange-pigmented strains of C. flaccumfaciens were distinguished from the yellow-pigmented strains.

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    The most prominent feature in the BOX-PCR fingerprint of C. flaccumfaciens is a band of approximately 500 bp, which is present in all the strains except for LPPA2199, LPPA2315, and the type strain of C. flaccumfaciens pv. poinsettiae. This is an interesting result because the strains LPPA2199 and LPPA2315 (G4-1) were isolated in Spain and were clustered apart from the core population of C. flaccumfaciens in MLSA-based phylogenetic trees (Fig. 2). A fragment of approximately 430 to 460 bp was produced in Clavibacter michiganensis but not in the C. flaccumfaciens strains. No discernible differences were found between C. flaccumfaciens strains isolated from dry beans and those isolated from asymptomatic solanaceous vegetables.

    DISCUSSION

    In this study, a multiphasic approach was used to decipher the phenotypic features, genetic diversity and phylogeny of C. flaccumfaciens strains isolated in Iran and Spain. Although no differences were found between the pathogenic and nonpathogenic strains in their plasmid profile, the former were resistant against all evaluated concentrations of arsenic compounds, while the latter were sensitive to the same concentrations. MLSA results revealed that the bacterial strains causing wilt disease on dry beans were distributed into two phylogenetic lineages (yellow-pigmented strains and red/orange-pigmented strains).

    Most of the plant-pathogenic bacteria are reported to have nonpathogenic lineages (Jacques et al. 2012). For instance, nonpathogenic strains of Clavibacter michiganensis were reported to be isolated frequently from tomato seeds (Yasuhara-Bell and Alvarez 2015). Multiphasic studies, including pathogenicity tests, MLSA, and plasmid profiling, revealed that these strains form a separate phylogenetic group and thus could be considered as new subspecies namely Clavibacter michiganensis subsp. californiensis and Clavibacter michiganensis subsp. chilensis (Thapa et al. 2017; Yasuhara-Bell and Alvarez 2015). However, nonpathogenic strains of C. flaccumfaciens did not form a separate phylogenetic group and were scattered either within the core population of C. flaccumfaciens pv. flaccumfaciens or within the other pathovars of the species.

    We have demonstrated that unlike the nonpathogenic strains (i.e., Cmmeg20, G105, Tom827, Xeu15, and Mo04), the C. flaccumfaciens pv. flaccumfaciens strains which were pathogenic on common bean were resistant to arsenic compounds. The association between arsenic resistance and pathogenicity on common bean remains to be elucidated, although this could be due to the adaptation of C. flaccumfaciens pv. flaccumfaciens on bean plants. Indeed, common bean is an arsenic-accumulating plant (Carbonell-Barrachina et al. 1997; Stoeva et al. 2005). More specifically, C. flaccumfaciens pv. flaccumfaciens strains from Iran showed a higher resistance to arsenic compared to the type strain, which was originally isolated in Hungary. A high arsenic content was observed in surface and ground waters in several Iranian provinces, which might have favored the adaptation of the pathogen to this compound (Keshavarzi et al. 2011). However, no correlation was found between arsenic resistance and the MLSA data. Further analysis is needed to determine the effect of arsenic on the relationships of C. flaccumfaciens pv. flaccumfaciens and its hosts, as recently studied for the legume–rhizobia interaction (Lafuente et al. 2015).

    In the original description of C. flaccumfaciens pathovars, it has been noticed that C. flaccumfaciens pv. betae, C. flaccumfaciens pv. flaccumfaciens, C. flaccumfaciens pv. oortii, and C. flaccumfaciens pv. poinsettiae are closely related to each other in terms of biochemical and physiological characteristics, and differences in host specificity and bacteriocin production are insufficient to justify differentiation at the subspecies level (Collins and Jones 1983; Dye and Kemp 1977). MLSA results from this study revealed that these four pathovars belong to the same species with 97.31 to 99.09% similarity in five housekeeping genes sequences (Table 5). However, C. flaccumfaciens pv. ilicis is distinct from the core population of C. flaccumfaciens pv. flaccumfaciens (Fig. 2). By contrast, it has been shown that phylogenetic distance between the yellow-pigmented strains of C. flaccumfaciens pv. flaccumfaciens and the red/orange-pigmented strains is higher than that of the distances among the type strains of C. flaccumfaciens pv. betae, C. flaccumfaciens pv. flaccumfaciens, and C. flaccumfaciens pv. oortii (Fig. 2; Table 5), all included in the cluster of yellow-pigmented C. flaccumfaciens pv. flaccumfaciens strains (Fig. 2; Table 5). Altogether, these results suggest that the taxonomy of C. flaccumfaciens should be reexamined using a large collection of strains from all the five pathovars of the species. Unlike to the other plant pathogenic corynebacteria, no molecular high throughput method to date has been used to confirm the classical taxonomy of C. flaccumfaciens proposed in late 1970s (Collins and Jones 1983; Dye and Kemp 1977). Recently, whole genome sequence analysis based on average nucleotide identity (ANI), digital DNA-DNA hybridization, and MLSA of seven housekeeping genes supported the concept of raising many Clavibacter michiganensis subspecies to five new species/combination level (Li et al. 2017). A similar approach has been started for the members of C. flaccumfaciens using the complete genome sequencing of type /pathotype strains of the species (Osdaghi et al. 2017b, 2018a).

    In conclusion, the results obtained in this study provide several new findings, including the phylogenetic relationships between the two different lineages (i.e., yellow-pigmented strains versus red/orange-pigmented strains) of the bean pathogen C. flaccumfaciens pv. flaccumfaciens, as well as the remaining four pathovars of C. flaccumfaciens. Results of MLSA and phenotypic features (i.e., colony color) are in congruence among C. flaccumfaciens strains, although further detailed and multiphasic evaluations are needed to determine if the different colony variants of C. flaccumfaciens pv. flaccumfaciens could be reclassified as different subspecies/pathovars of the species. We also found a distinctive phenotypic feature (arsenic resistance) which is capable to discriminating pathogenic strains of C. flaccumfaciens from nonpathogenic strains. However, a comprehensive multiphased study using a collection of worldwide isolates should illustrate the phylogenetic history and intraspecies relationships of C. flaccumfaciens strains.

    ACKNOWLEDGMENTS

    Pure DNA of the strains isolated in Spain were provided by A. J. González (Cultivos Hortofrutícolas y Forestales, SERIDA, Villaviciosa, Asturias, Spain). We thank J. Ram Lamichhane (UMR AGIR, INRA, Castanet-Tolosan, France) for critical reading of the first draft of the manuscript. E. Osdaghi thanks DISPAA, University of Florence, for financial support during his sabbatical stay in Italy.

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    Funding: Financial support for this study was co-provided by Shiraz University (Iran) and University of Florence (Italy).