Independent Origins of Vectored Plant Pathogenic Bacteria from ...

Microb Ecol (2012) 63:628–638 DOI 10.1007/s00248-011-9933-5

INVERTEBRATE MICROBIOLOGY

Independent Origins of Vectored Plant Pathogenic Bacteria from Arthropod-Associated Arsenophonus Endosymbionts Alberto Bressan & Federica Terlizzi & Rino Credi

Received: 27 April 2011 / Accepted: 19 August 2011 / Published online: 3 September 2011 # Springer Science+Business Media, LLC 2011

Abstract The genus Arsenophonus (Gammaproteobacteria) is comprised of intracellular symbiotic bacteria that are widespread across the arthropods. These bacteria can significantly influence the ecology and life history of their hosts. For instance, Arsenophonus nasoniae causes an excess of females in the progeny of parasitoid wasps by selectively killing the male embryos. Other Arsenophonus bacteria have been suspected to protect insect hosts from parasitoid wasps or to expand the host plant range of phytophagous sapsucking insects. In addition, a few reports have also documented some Arsenophonus bacteria as plant pathogens. The adaptation to a plant pathogenic lifestyle seems to be promoted by the infection of sap-sucking insects in the family Cixiidae, which then transmit these bacteria to plants during the feeding process. In this study, we define the specific localization of an Arsenophonus bacterium pathogenic to sugar beet and strawberry plants within the plant hosts and the insect vector, Pentastiridius leporinus (Hemiptera: Cixiidae), using fluorescence in situ hybridization assays. Phylogenetic analysis on 16S rRNA and nucleotide coding sequences, using both maximum likelihood Electronic supplementary material The online version of this article (doi:10.1007/s00248-011-9933-5) contains supplementary material, which is available to authorized users. A. Bressan (*) Department of Plant and Environmental Protection Sciences, University of Hawaii at Manoa, 3050 Maile Way, Honolulu, HI 96822, USA e-mail: [email protected] F. Terlizzi : R. Credi Dipartimento di Scienze e Tecnologie Agroambientali-Patologia Vegetale, University of Bologna, Viale Fanin 40, 40127 Bologna, Italy

and Bayesian criteria, revealed that this bacterium is not a sister taxon to “Candidatus Phlomobacter fragariae,” a previously characterized Arsenophonus bacterium pathogenic to strawberry plants in France and Japan. Ancestral state reconstruction analysis indicated that the adaptation to a plant pathogenic lifestyle likely evolved from an arthropodassociated lifestyle and showed that within the genus Arsenophonus, the plant pathogenic lifestyle arose independently at least twice. We also propose a novel Candidatus status, “Candidatus Arsenophonus phytopathogenicus” novel species, for the bacterium associated with sugar beet and strawberry diseases and transmitted by the planthopper P. leporinus.

Introduction It is generally presumed that intracellular plant pathogenic bacteria transmitted by insect vectors evolved as insect endosymbionts and that they only became parasites of plants secondarily [4, 39]. Recently, phylogenetic studies have unraveled the evolutionary history of several arthropod endosymbionts [31] and insect-transmitted plant pathogens [12, 22, 27, 40, 41, 45, 52, 55]. These studies have shown that certain bacterial clades, such as the Phytoplasma (Mollicutes) [27] and Liberibacter bacteria (α-Proteobacteria) [22], comprise of bacteria that are all ecologically specialized as vectored plant pathogens. In other instances, vector-borne plant pathogens cluster within bacterial clades composed primarily of arthropod intracellular symbionts, such as Arsenophonus (γ-Proteobacteria) [35, 41, 45], Rickettsia (α-Proteobacteria) [12, 52], and Spiroplasma (Mollicutes) [40]. Duron and colleagues [13] have estimated that approximately 5% of the arthropod species on Earth are infected

Emergence of Vectored Plant Pathogenic Bacteria from Arthropod Endosymbionts

with bacteria from the genus Arsenophonus. These bacteria can significantly affect the ecology and life history traits of their arthropod hosts. Some of these bacteria, such as those hosted by dipteran insects of the family Hippoboscidae, display evolutionary traits typical of the obligate symbionts and probably have a nutritional role [35]. Other members, although not essential, may provide specific benefits to their hosts [8, 21]. For instance, indirect evidence suggests that certain Arsenophonus bacteria could promote the adaptation of whiteflies to specific host plants [8] or could protect their arthropod hosts from parasitoid attacks [21]. Other Arsenophonus bacteria can also be parasitic. For instance, Arsenophonus nasoniae [18], the type species, can manipulate the reproduction of the wasp host, Nasonia vitripennis (Hymenoptera: Pteromalidae), and other related wasp species by selectively killing the male embryos [54]. Presently, the plant pathogenic Arsenophonus bacteria have been reported to infect and cause diseases in sugar beet and strawberry plants in different countries. Zreik and colleagues [34, 55] reported the association of a plant pathogenic Arsenophonus bacterium with a marginal chlorosis disease of strawberries that is widespread in western France. During the 1990s, the genus Arsenophonus was yet to be recognized, and only the 16S rRNA sequence from A. nasoniae was deposited in the GenBank [18]; thus, Zreik and colleagues [55] proposed a new genus and new species: “Candidatus Phlomobacter fragariae” (Ca. P. fragariae). Recently, Ca. P. fragariae was also described to cause a marginal chlorosis disease in strawberry plants in Japan [48]. In eastern France, a disease locally called “syndrome basses richesses” (SBR disease; i.e., low sugar content disease) has been associated with a bacterium, provisionally called syndrome basses richesses proteobacterium (SBR proteobacterium) [17, 44]. In addition, in northern Italy, a new emerging strawberry disease, characterized by marginal chlorosis-like symptoms, has been associated with a bacterium that is provisionally called “strawberry marginal chlorosis proteobacterium” (SMC proteobacterium) [50]. According to the 16S rRNA sequences, the SMC proteobacterium shares higher sequence similarity with SBR proteobacterium than with Ca. P. fragariae [50]. Two planthopper species, Pentastiridius leporinus Linnaeus and Cixius wagneri (China), which belong to the family Cixiidae (Hemiptera; Fulgoroidea), have been shown to transmit SBR proteobacterium [5, 17, 44] and Ca. P. fragariae in France [10], respectively. To date, there is no information about the vector species that transmit Ca. P. fragariae and the SMC proteobacterium to strawberry plants in Japan and Italy, respectively. Different experimental procedures have been utilized to demonstrate the role of the SBR proteobacterium in causing SBR disease and its transmission by P. leporinus planthoppers. The presence of bacteria localized to the sieve tubes of sugar

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beets showing symptoms for SBR disease was first observed by 4′,6-diamidino-2-phenylindole (DAPI) staining and transmission electron microscopy [17]. The detection and characterization of SBR proteobacterium 16S rRNA were then performed using DNA isolated from diseased sugar beets through PCR amplification using universal and specific 16S rRNA primer pairs followed by sequencing [17, 44, 45]. Similar screening procedures were applied to DNA isolated from SBR-infected sugar beet seedlings experimentally inoculated by P. leporinus [17, 43–45]. In addition, a library composed of 16S rRNA bacterial sequences was established starting from DNA isolated from field-collected P. leporinus planthoppers [6]. The later experiment revealed that P. leporinus planthoppers harbored at least three distinct species of endosymbiotic bacteria in addition to SBR proteobacterium: a bacteroidetes “Candidatus Sulcia muelleri,” a γproteobacterium “Candidatus Purcelliella pentastirinorum,” and an α-proteobacterium belonging to the genus Wolbachia [6]. However, unlike the SBR proteobacterium, these other three bacteria were never detected on sugar beet plants [17, 45]. Taken together, these experiments revealed that the SBR proteobacterium is the predominant bacterium associated with the SBR disease and that it is transmitted by P. leporinus. In this study, we developed specific fluorescence assays to localize both the SBR proteobacterium within the plant host and the planthopper vector and the SMC proteobacterium within the plant host. In addition, we examined the evolution of vectored plant pathogens relative to Arsenophonus endosymbionts. To this end, we performed a phylogenetic analysis using both ribosomal and coding DNA sequences, and we conducted an ancestral state reconstruction analysis. Lastly, we proposed a novel candidatus status for SBR and SMC proteobacteria.

Materials and Methods Material Source and Bacteria Characterization To examine the localization of the SMC proteobacterium within plant hosts, we used strawberry plants either showing symptoms of marginal chlorosis (Fig. 1) or with a healthy appearance. Plants were collected from strawberry fields in the Cesena and Ravenna provinces (EmiliaRomagna region, northern Italy), were transplanted into pots, and kept inside an insect-proof greenhouse. The presence of SMC proteobacterium in plant tissues was first determined by PCR assays using the diagnostic primer pair Fra4/Fra5, which amplifies a 550-bp fragment in the 16S rRNA region [10]. To better characterize the isolates, the full-length 16S rRNA gene was analyzed with primers fd1/ Fra4 and Fra5/rp1, which amplify the 5′ and 3′ regions of the 16S rRNA gene of the proteobacterium [44]. PCR

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Figure 1 A sugar beet (left) and a strawberry (right) plant showing symptoms of syndrome basses richesses (i.e., low sugar content disease) and of marginal chlorosis, respectively

products were sequenced, and the assembled 16S rRNA sequences had total identity with those previously deposited in GenBank (accession numbers: DQ538372–DQ538379) [50]. PCR assays were also performed to detect phytoplasmas, which can cause convergent symptoms of marginal chlorosis [10, 49]. Procedures for PCR assays and conditions for amplification were as previously described [44, 49]. We could not examine the localization of the SMC proteobacterium within insects because the vector of the SMC proteobacterium is currently unknown. For the SBR proteobacterium, we used a biological material produced from a transmission assay. P. leporinus planthoppers were collected from sugar beet fields located in the Jura department (Franche-Comté region, Western France) during an epidemic of SBR disease. Planthoppers collected with a D-Vac (Ventura, CA, USA) were segregated into vials and transported to a greenhouse facility. The rate of planthopper infection by SBR proteobacterium was estimated using a diagnostic PCR procedure, which used the Alb1/Oliv1 primer pair. This procedure was designed to amplify the internal transcribed spacer region between the 16S and 23S ribosomal genes of the SBR proteobacterium rRNA operons. The PCR products produce a characteristic fingerprint for the SBR preoteobacterium [43] but fail to detect any heritable symbiont of P. leporinus or other Arsenophonus endosymbionts closely related to the SBR proteobacterium [43]. PCR conditions were set according to Sémétey et al. [43]. Through this procedure, we detected the SBR proteobacterium in 36 out of 45 planthoppers tested (80%). For transmission assays, we confined four to five planthoppers on 35- to 50-day-old healthy sugar beet seedlings in cylindrical, mesh-ventilated, PVC cages that were closed at the bottom with a collar-like sponge pad. We confined insects in a total of seven plants and kept four sugar beet seedlings that were not exposed to planthoppers as negative controls. Insects were maintained on sugar beet seedlings for an inoculation access period of 4 days. At the end of the period, we collected the live insects and immediately processed them for fluorescence in situ hybridization (FISH) assays. The plants were sprayed with

1.02 g/l of Fenitrothion (Chimiberg, Albano S. Alessandro, Italy) and kept in an insect-free greenhouse with natural light and temperature (26±5°C) for 2 to 3 months to complete the incubation period (time for the disease symptoms to appear). For FISH assays, we used tissues from sugar beet seedlings showing SBR symptoms (Fig. 1); seedlings that were not exposed to feeding insects were used as controls. We determined and confirmed the 16S rRNA sequence of the SBR proteobacterium (accession number: AY057392) using PCR and sequencing procedures previously described for the SMC proteobacterium. Fluorescence In Situ Hybridization We used a paraffin-embedded protocol similar to the one described by Fukatsu and colleagues [16] to process both insect and plant tissues. For insects, the abdomens or thoraces and heads, from either females or males, were excised from the rest of the insect body with a razor blade and were kept overnight at 4°C in a solution of 0.1 M potassium phosphate buffer (pH 7.4) that contained 4% paraformaldehyde. Plant material, either leaf petioles or root cuttings, was kept overnight at 4°C, either in a solution containing paraformaldehyde (as above) or in a solution containing 25% acetic acid and 75% ethanol. Thereafter, tissues were dehydrated and cleared through an ethanol– xylene series [16] and embedded in Paraplast® Plus (McCormick Scientific, St. Louis, MO, USA). Next, 7-9μm tissue sections were obtained with a rotary microtome and mounted on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA, USA). The sections were dewaxed and hydrated through a xylene–ethanol series, air dried, and incubated with probes [16]. A specific probe, SBR450 (Cy®5 or Texas Red 5′CCTTAACACCTTCCTCACGAC-3′), that perfectly complements the 16S rRNA sequences of both SBR and SMC proteobacteria was selected using the probe match search option in the Ribosomal Database Project website at http:// rdp.cme.msu.edu/probematch/search.jsp. The probe sequence did not match any bacterial species outside of the


Arsenophonus clade; however, the probe did match 16S rRNA sequences from 45 other Arsenophonus bacteria known to colonize other arthropod hosts [9, 51]. To further examine the specificity of the probe, dual hybridization assays were first performed by simultaneously applying probes SBR450 and a universal eubacterial probe, EUB338 (5′-GCTGCCTCCCGTAGGAGT-3′ Alexafluor 488), to the slides. Hybridization buffer (20 mM Tris–HCl, pH 8.0; 0.9 M NaCl; 0.01% sodium dodecyl sulfate; 30% formamide) containing 50 pmol/mL of each probe was applied to the slides containing sections from the planthoppers’ abdomens. The slides were then incubated in a humidified chamber at 46°C overnight. Nonspecific binding was removed by applying a washing buffer (20 mM Tris–HCl, pH 8.0; 0.9 M NaCl; 0.01% sodium dodecyl sulfate; 50% formamide) and incubating at 46°C for 15 min. The slides were then washed with 1× SSC (0.15 M NaCl, 15 mM sodium citrate) and mounted with VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA, USA) containing DAPI. The probe specificity was further examined on thin sections using nonsense probes and RNase treatments. The slides were visualized under an epifluorescence microscope. Phylogenetic Analysis We used most of the 16S rRNA gene (∼1,400 bp) and the protein-coding genes spoT, spoU, and recG (∼2,400 bp) to infer the phylogeny of Arsenophonus plant pathogenic bacteria. The ribosomal 16S rRNA sequences were retrieved from the Ribosomal Database Project website at http://rdp.cme.msu.edu/ under the search “Arsenophonus”; these included 16S rRNA sequences for all of the plant pathogenic bacteria: SBR proteobacterium [17], SMC proteobacterium [50], Ca. P. fragariae from France and Japan [48, 55], and from other Arsenophonus bacteria that colonize arthropods (Table S1). The 16S rRNA sequences from Proteus mirabilis and Photorhabdus luminescens were selected as out-groups [9, 35, 41]. The dataset contained a total of 38 operational taxonomic units (OTUs). The coding sequences represented 2,400 bp of a genetic locus comprising the end of the gene spoT, the gene spoU, and the beginning of the gene recG. These sequences were obtained in a previous study [41] and were retrieved from GenBank through the NCBI database. This set represented a smaller dataset of 13 OTUs, including sequences from the plant pathogens SBR proteobacterium and Ca. P. fragariae from France. We decided to analyze the ribosomal and coding sequences separately because missing sequences would have generated a very small phylogenetic tree. The accession numbers of the sequences used for the phylogenetic analysis have been reported in Table S1. The sequences were first aligned using MUSCLE [14], and the obtained matrices were visually inspected for

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ambiguous alignments and were tested for the best model of evolution using the Akaike Information Criterion in jModel Test v0.1.1 [38]. For 16S rRNA sequences, the best evolutionary model was the generalized time reversible (GTR) model sequence of evolution with a gamma distribution describing rate variation across sites and an estimated proportion of invariable sites (GTR+I+G). For spoT-spoU-recG sequences, the best evolutionary model was GTR+I with no rate variation among sites. The parameters obtained were used to infer the phylogeny using both maximum likelihood (ML) and Bayesian methods. Maximum likelihood analyses were performed using PhyML v3.0.1 [20] with 1,000 replicates for nonparametric bootstrap analysis. Bayesian analyses were performed in MrBayes v3.1.2 [25]. Posterior probabilities were approximated by sampling the trees using a Markov Chain Monte Carlo method. We estimated the proportion of invariant sites, the gamma distribution shape parameter, base frequencies, and the substitution rates for the GTR model using MrBayes. We kept the default parameters provided by the software and ran six and three million generations for the 16S rRNA and spoT-spoU-recG datasets, respectively. For both datasets, we discarded the first 25% of tree samples from the cold chain (burn in) when computing 50% majority-rule consensus trees. The 16S rRNA similarities among Arsenophonus bacteria were calculated from a genetic distance matrix, as determined in MEGA v4 [47] under a Maximum Composite Likelihood model of nucleotide substitition with a heterogeneous pattern among lineages and with gamma distribution among sites fixed to 0.62, as determined by the jModelt Test. Ancestral Character Mapping To understand if the ecological specialization of vectored plant pathogens arose secondarily in the evolution of the Arsenophonus or whether it was an ancestral condition, we performed ancestral state reconstruction analysis, and we considered two possible character states, “arthropod associated” and “vectored plant pathogen.” The “arthropodassociated” lifestyle was assigned to all those bacterial strains that to date have been characterized exclusively in arthropods. This category included bacterial strains with different modes of transmission and different tissue tropism in arthropod hosts; it also included strains with roles ranging from obligate endosymbionts to facultative endosymbionts and parasites. The “vectored plant pathogen” lifestyle was assigned to those bacterial strains that have been documented for their pathogenic effect to plants and transmission by vectors. The assignment of these character states to each OTU has been reported in Table S1. Outgroup taxa were coded as missing data. The ancestral state was optimized onto the 16S rRNA and spoT-spoU-recG trees using parsimony and ML criteria in Mesquite 2.73

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[29]. For ML, we selected “Mk1” as the evolutionary model, which assumes an equal probability for any particular character change [37].

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hybridization was observed when vascular bundles of sugar beet tap roots were examined (Fig. 3c, d). Phylogenetic Analysis

Results Fluorescence In Situ Hybridization We examined the localization of SBR and SMC proteobacteria in their host plants and the localization of the SBR bacterium in P. leporinus planthoppers. Probe SBR450 was first checked for specificity in dual hybridization assays together with a universal probe, EUB338. Probe EUB338 hybridized both within the planthopper bacteriomes (organs containing primary bacterial endosymbionts [31]) and outside the bacteriomes, whereas probe SBR450 hybridized only outside the bacteriomes (Fig. S1). This showed that probe SBR450 did not hybridize to the 16S rRNAs of three endosymbiotic bacteria: the bacteroidetes Ca. Sulcia muelleri, an undescribed β-proteobacterium (unpublished), and the γ-proteobacterium Ca. Purcelliella pentastirinorum. Furthermore, labeling was not visible at the cell nuclei (Fig. S1), where an α-proteobacterium from the genus Wolbachia has been previously shown to reside [2]. In addition, at a high magnification (×40), we could readily visualize, on the slides incubated with the specific probe, long rod-shaped bacterial cells that were often filamentous (Fig. 2d), whose morphology matched that previously described for other Arsenophonus bacteria [9, 18, 26]. Taken together, these assays revealed that probe SBR450 was specific. We examined a total of seven planthoppers, of which five were females and two were males. The SBR proteobacterium was localized into several planthopper organs, such as the salivary glands, guts, gonads, and fat tissue (Fig. 2a, b, e). As judged by the hybridization signals, the bacterium was more concentrated at the periphery of several organs (Fig. 2a). The tissues forming the oviducts of the ovaries and some portion of the salivary glands were highly infected (Fig. 2b–e). For one planthopper female, we could not find evidence for labeling, suggesting that the specimen was not infected by the bacterium (Fig. 2f). We obtained better hybridization signals from plant sections fixed with acetic acid and ethanol than with parafomaldehyde (not shown). Specific hybridization was observed in phloem tissues of strawberries infected by the SMC proteobacterium; labeling was visualized as puncta in transversal sections of the vascular bundles (Fig. 3a). However, no hybridization was observed in healthy plants (Fig. 3b). We obtained minimal labeling of vascular bundles sectioned from the SBR proteobacterium-infected sugar beet leaves and petioles (data not shown); however, distinct

We reconstructed the phylogeny of Arsenophonus bacteria using both the 16S rRNA and spoT-spoU-recG sequences. Figure 4a shows a Bayesian phylogeny constructed from 16S rRNA sequences that includes all of the plant pathogenic bacteria sequences available from GenBank. Our work is in agreement with previous studies [41, 45] and shows that the SBR and SMC proteobacteria and Ca. P. fragariae have evolved from a subclade of Arsenophonus bacteria. This subclade has spread into several arthropod hosts, including hymenopteran, hemipteran, and dipteran insect species, as well as arachnids (ticks; Fig. 4a). Within this subclade, a few nodes had low bootstrap values upon ML analysis; conversely, Bayesian analysis produced relatively high posterior probabilities (Fig. 4a). Both ML and Bayesian analysis determined plant pathogenic bacteria to be paraphyletic, with only SMC and SBR proteobacteria being sister taxa (Fig. 4a). The ancestral state reconstruction is reported as a cladogram (Fig. 4b) that mirrors the phylogenetic tree (Fig. 4a). Ancestral state reconstruction using ML criteria assigned to the most basal nodes higher support to the arthropod-associated lifestyle rather than to the vectored plant pathogen lifestyle (Fig. 4b). Ancestral state reconstruction through parsimony analysis provided results very similar to those produced by ML (Fig. S2). The phylogeny of Arsenophonus bacteria constructed using coding nucleotide spoT-spoU-recG sequences provided better branch support, especially in the ML analysis (Fig. 5). Plant pathogenic bacteria (Ca. P. fragariae and SBR proteobacterium) were determined to be paraphyletic (Fig. 5a). The ancestral state reconstruction analysis also provided results similar to those obtained from the 16S rRNA phylogeny. Likely due to a small dataset and to the fact that there was only one basal node external to the Ca. P. fragariae/Arsenophonus clade, the most recent common ancestor was uncertain in the ancestral state reconstruction (Fig. 5b); however, higher relative support was assigned into the most basal nodes to the arthropod-associated lifestyle than to the vectored plant pathogen life style (Fig. 5b). Ancestral state reconstruction through parsimony provided even stronger support for an ancestral lifestyle of arthropod association (Fig. S3). Altogether, the ancestral state reconstruction analysis indicated that the lifestyles of vectored plant pathogens evolved from an arthropodassociated ancestral lifestyle and showed, for both ribosomal (Fig. 4b) and coding sequence datasets (Fig. 5b), that the plant pathogenic lifestyle arose independently at least twice within the Arsenophonus clade.

Emergence of Vectored Plant Pathogenic Bacteria from Arthropod Endosymbionts Figure 2 Results of fluorescence in situ hybridization assays on thin sections of Pentastiridius leporinus planthoppers hybridized with SBR proteobacterium probe (SBR450). Labeling is visible as white. a Transversal section of the abdomen of a P. leporinus male infected with SBR proteobacterium. b Longitudinal section of the abdomen of a P. leporinus female showing infection of ovaries. c Enlarged from b. d Enlarged from c, note filamentous bacterial cells surrounding an oviduct. e Transversal section of a P. leporinus male showing hybridization for SBR proteobacterium within salivary glands. f Transversal section of the salivary glands of a P. leporinus female with no evidence of labeling for SBR proteobacterium. Ft fat tissue, b bacteriome, gl gut lumen, m muscle, oo oocyte, ov ovariole, sg salivary gland; * unrecognized organs

Discussion The trends of relatedness between arthropod-associated symbionts and insect-vectored plant pathogens suggest that one lifestyle may have enabled transitions to the other across a few groups of bacteria, such as Arsenophonus (γ-Proteobacteria) [35, 41, 45], Rickettsia (α-Proteobacteria) [12, 52], and Spiroplasma (Mollicutes) [40]. In this study, we tackled these transitions in the Arsenophonus group. The phylogenetic analyses on 16S rRNA and nucleotide coding sequences using both maximum likelihood and Bayesian criteria were coupled with ancestral state reconstruction analyses to formally test whether insect-vectored plant pathogens likely evolved from insect symbiont ancestors. Our results suggest that insect-vectored plant pathogens arose independently at least twice from a subclade of Arsenophonus consisting mainly of facultative endosymbionts [9, 35, 51]. Two brief caveats should be added here. First, while several of the

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studied strains exhibited different types of interactions with their arthropod hosts (i.e., obligate vs. facultative, gut associated vs. maternally inherited) [18, 21, 26, 35, 54], the lack of such information for other strains in this analysis has limited our attempts to characterize the evolution of arthropod-associated lifestyles across the Arsenophonus group. Without further studies on the phenotypes and lifestyles of the analyzed strains, it is therefore at least possible that some Arsenophonus from sap-feeding hosts could in fact be uncharacterized vectored plant pathogens. Second, Arsenophonus bacteria have been extensively sampled from arthropods, whereas relatively little attention has been given to their possible presence in plants. Therefore, it is conceivable, although not proven, that substantial diversity within this lineage has yet to be uncovered. In this hypothetical scenario, the vectored plant pathogenic lifestyle could turn out to be basal. Such a discovery would require a reinterpretation of our current findings, which supports the

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Figure 3 Result of fluorescence in situ hybridization assays on thin sections of plant tissues hybridized with SBR and SMC proteobacteria-specific probe (SBR450). a Pictures of a transversal section of the vascular bundle of a strawberry infected by SMC proteobacterium. b Pictures of a transversal section of a vascular bundle of a healthy strawberry plant. c Longitudinal section of the vascular bundles of sugar beet tap roots showing hybridization for SBR proteobacterium (arrows). d enlarged from c. bs bundle sheath, p parenchymal cell, ph phloem, x xylem, se sieve element. Arrows indicate localization of SBR and SMC proteobacteria

origins of vectored plant pathogens from an ancestral lifestyle of symbiotic arthropod association. But nevertheless, the weight of the current evidence still suggests at least two origins of plant pathogens from within the Arsenophonus clade. The independent emergence of insect-vectored plant pathogens may suggest that Arsenophonus bacteria are preadapted to infect plants, and therefore, the determinants of pathogenicity and virulence may be part of the genetic makeup of several Arsenophonus bacteria. In this hypothetical scenario, the shift of an endosymbiont to a plant pathogenic lifestyle may result from the chance of meeting the environmental conditions conducive to plant infection: e.g., infection of cixiid planthoppers and transmission to a susceptible host plant species. If this is the case, it also implies that the Arsenophonus clade may be a large reservoir of potentially plant pathogenic bacteria. A possible way to experimentally test this hypothesis would consist of transferring Arsenophonus bacteria from different arthropod hosts into recognized species of vectors and testing for their transmissibility and pathogenicity to plants. A number of virulence determinants have been identified in plant pathogenic bacteria [46]. Many of these factors are associated with mobile genetic elements, such as bacteriophages, transposons, and plasmids, which are the main contributors to horizontal gene transfer and allow bacteria to acclimate instantly to new environments (i.e., hosts) [46]. Therefore, an alternative scenario, which would explain the emergence of a phytopathogenic lifestyle, may consider the acquisition of new genetic information from other microbes by Arsenophonus bacteria. For instance, Arsenophonus

bacteria share very similar environments (insect and plant hosts) with plant pathogenic phytoplasmas, which possess extrachromosomal elements that have been implicated in horizontal transmission and in virulence of the host plant [24, 33]. Recently, the genome of Arsenophonus nasonie [11] and those of a few vectored plant pathogens [3, 36] has been sequenced; therefore, it is likely that genomic sequencing of phytopathogenic Arsenophonus bacteria would shed light on the genetic determinants associated with plant and arthropod infection. In general, infection of hemipteran insects has been suggested as a first step toward the transition of endosymbiotic bacteria to a plant pathogenic lifestyle [40]. Many hemipteran insects are phloem-feeding specialists, and during the feeding process, they secrete a large amount of saliva into plant tissues. Therefore, this specific feeding habit coupled with salivary gland infection may have created a selection pressure on endosymbiotic bacteria toward the colonization of vascular plant tissues. Interestingly, Arsenophonus bacteria are widespread across several hemipteran lineages, such as the Aphidoidea, Psylloidea, Aleyrodoidea, and Auchenorrhyncha, each of which contains several vectors of plant pathogens [23, 53]. However, the transition to a plant pathogenic lifestyle seems to have been promoted by the infection of cixiid planthoppers only. Cixiid planthoppers display specific life history traits, with all developing stages being terricolous and feeding from the roots of their host plants, whereas adults feed on plant foliage and are responsible for dispersal. Arsenophonus phytopathogens seem to concentrate on the roots of their host plants [44]


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Figure 4 a 16S rRNA Bayesian phylogeny of Arsenophonus bacteria from an aligned matrix of 1,529 characters, 1,103 of which were conserved, 399 were variables. Sequences from Proteus mirabilis and Photorabdus luminescens were used as out-groups. Numbers near nodes indicate Bayesian posterior probabilities followed by bootstrap values with more than 50% support obtained from maximum likelihood (ML) analysis [Log likelihood of the tree, −6,149.38].

Plant pathogenic Arsenophonus bacteria have been mapped onto the tree. b Cladogram showing ancestral state reconstruction of vectored plant pathogens and arthropod-associated symbionts using ML analysis and marginal probability reconstruction with model Mk1 (rate 0.04 Log likelihood, −9.166). Probabilities are reported within each internal node as proportional likelihoods

even though they are inoculated by adult planthoppers on the leaves. Therefore, it is possible, although not proven, that the specialized life history of cixiid planthoppers has generated a selection pressure in Arsenophonus bacteria toward the systemic invasion of plants. From an evolutionary perspective, the occurrence of closely related Arsenophonus bacteria with distinct lifehistory strategies allows us to speculate on how changes in ecological specialization may pressure bacteria toward different levels of virulence to their hosts and may impose

trade-offs in the routes of transmission [1]. Hence, because vertical transmission is linked to host reproduction, the interaction between inherited microbes and their arthropod hosts is thought to move toward or maintain a low degree of virulence [1]. Accordingly, several vertically transmitted endosymbionts provide their arthropod hosts with specific benefits, including nutrient provision [31] resistance to wasps [21], microbial pathogens [42], or abiotic stresses [30]. Other vertically transmitted bacteria have managed to alter host reproduction by modifying the host sex ratio or

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Figure 5 a spoT-spoU-recG Bayesian phylogeny of Arsenophonus bacteria from an aligned matrix of 2,416 characters, 1,616 of which were conserved, 783 were variables. Sequence from Proteus mirabilis was used as out-group. Numbers near nodes indicate Bayesian posterior probabilities followed by bootstrap values with more than 50% support obtained from maximum likelihood (ML) analysis [Log likelihood of

the tree, −6,149.38]. Plant pathogenic Arsenophonus bacteria have been mapped onto the tree. b Cladogram showing ancestral state reconstruction of vectored plant pathogens and arthropod-associated symbionts using ML analysis and marginal probability reconstruction with model Mk1 (rate 0.136 Log likelihood, −6.90880109). Probabilities are reported within each internal node as proportional likelihoods

causing cytoplasmic incompatibility [13, 19], but they seem to exert little pathogenic effect on their hosts. However, it has been demonstrated that an increased level of virulence can be evolutionarily advantageous if it concomitantly enhances microbe transmission [1]. Hence, indirectly transmitted pathogens, such as phytopathogenic bacteria transmitted by insect vectors, are likely to evolve toward a high degree of virulence to plants as this would likely maximize transmission rates. For the same reason, although exceptions seem to exist [15], parasitic microbes do not seem to evolve high degrees of virulence to their vectors; a reduced fitness of the vector would probably cause reduced transmission rates [28]. Interestingly, vertical transmission of the SBR proteobacterium through P. leporinus females is only partially efficient, and infection of offspring is experimentally estimated at approximately 30% [7]. The rate of vertical transmission observed likely would not permit the bacterium to persist within populations of the planthopper and, as a consequence, the infection would progressively stutter to extinction. However, in previous work, we observed that the prevalence of the SBR proteobacterium in terricolous nymphs of P. leporinus gradually increases over time in fields with a high incidence of diseased plants [7]. This

later trend of infection may suggest peroral acquisition of the bacterium from the roots of infected host plants on which nymphs develop. In addition, the percentage of adult planthoppers infected by the bacterium in the field can reach more than 90% [7], which is a much higher rate than the one observed for vertical transmission alone. These empirical data suggest that horizontal transmission is the most prevalent route used by the SBR proteobacterium to persist in hosts. These results may also suggest that the transition to a phytopathogenic lifestyle may impose a trade-off between vertical transmission in the vectors and horizontal transmission through plant infection. In agreement with this hypothesis, most insect-vectored plant pathogens are rarely vertically transmitted in their vectors, or vertical transmission seems to occur at low rates [53]. In this study, we have reported the localization of the SBR proteobacterium into several internal organs of P. leporinus, including the salivary glands and the ovaries. These results are in agreement with previous research, which provided evidence for the coexistence of both vertical and horizontal transmissions of SBR proteobacterium [7]. Hence, salivary gland infection may permit the bacterium to be inoculated into plants, whereas infection of the ovaries may facilitate vertical transmission.


Formal description of “Candidatus Arsenophonus phytopathogenicus” nov. sp. SBR proteobacterium shares sequence homology with Ca. P. fragaire in the 16S rRNA gene between 96.9% and 98.5%. The range in the 16S rRNA sequence homology is caused by the occurrence of multiple copies of ribosomal RNAs in the genome of Arsenophonus bacteria [11, 43]. The SBR proteobacterium shares 92.8% similarity in the spoT-spoUrecG sequence with Ca. P. fragaire, and phylogenetic analysis presented in this and previous studies [41, 45] revealed the paraphyly of these two bacteria. The SBR proteobacterium shares 99% sequence homology in the 16S rRNA with “Candidatus Arsenophonus arthropodicus” (Ca. A. arthropodicus), the closer relative for which a formal name has been assigned [9]. However, Ca. A. arthropodicus is not known to be a plant pathogen; in addition, we failed to culture the SBR proteobacterium starting from the media and procedures described for Ca. A. arthropodicus [9] (unpublished). According to a recently adopted taxonomic rule [32], the properties of unculturable organisms should be recorded by a Candidatus designation. We propose a novel Candidatus status for the SBR and SMC proteobacteria: “Ca. Arsenophonus phytopathogenicus” nov. sp.. “Ca. Arsenophonus phytopathogenicus” (Ca. A. phytopathogenicus) is a pathogenic and phloem-restricted bacterium of the sugar beet and strawberry plants [17, 50]. The bacterium is hosted and transmitted by the planthopper P. leporinus [17] and C. wagneri [5]. Within the planthopper P. leporinus, Ca. A. phytopathogenicus forms long rods, is often filamentous, and infects the cytoplasm of cells’ forming reproductory organs, salivary glands, guts, and fat tissues. We propose SBR proteobacterium as the type member; 16S rRNA accession number: AY057392 [17]. A unique 16S rRNA sequences for Ca. A. phytopathogenicus is CCTTTGTTGCCAGCGAGTA GAGTCGGG. We propose SMC proteobacterium as a strain of Ca. A. phytopathogenicus with 16S rRNA accession numbers DQ538372–DQ538379 [50]. Additional DNA sequences available for other strains of Ca. A. phytopathogenicus include the intergenic spacers between 16S and 23S rRNAs accession numbers DQ834348–DQ834353 [43]; and the spoT-spoU-recG accession number FM992680 [41]. Acknowledgments We are grateful to four anonymous reviewers for suggestions on the original version of the manuscript and to Elisabeth Boudon-Padieu at INRA Dijon for providing P. leporinus planthoppers and sugar beet plants used for the FISH assays. Research was supported by the University of Hawaii Start-up and Hatch funds to Alberto Bressan.

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