Independent Evolution with the Gene Flux Originating from Multiple Xanthomonas Species Explains Genomic Heterogeneity in Xanthomonas perforans.

Xanthomonas perforans is the predominant pathogen responsible for bacterial leaf spot of tomato and X. euvesicatoria for that of pepper in the southeast United States. Previous studies have indicated significant changes in the X. perforans population collected from Florida tomato fields over the span of 2 decades, including a shift in race and diversification into three phylogenetic groups driven by genome-wide homologous-recombination events derived from X. euvesicatoria In our sampling of Xanthomonas strains associated with bacterial spot disease in Alabama, we were readily able to isolate X. perforans from symptomatic pepper plants grown in several Alabama counties, indicating a recent shift in the host range of the pathogen. To investigate the diversity of these pepper-pathogenic strains and their relation to populations associated with tomatoes grown in the southeast United States, we sequenced the genomes of eight X. perforans strains isolated from tomatoes and peppers grown in Alabama and compared them with previously published genome data available from GenBank. Surprisingly, reconstruction of the X. perforans core genome revealed the presence of two novel genetic groups in Alabama that each harbored a different transcription activation-like effector (TALE). While one TALE, AvrHah1, was associated with an emergent lineage pathogenic to both tomato and pepper, the other was identified as a new class within the AvrBs3 family, here designated PthXp1, and was associated with enhanced symptom development on tomato. Examination of patterns of homologous recombination across the larger X. euvesicatoria species complex revealed a dynamic pattern of gene flow, with multiple donors of Xanthomonas spp. associated with diverse hosts of isolation.IMPORTANCE Bacterial leaf spot of tomato and pepper is an endemic plant disease with a global distribution. In this study, we investigated the evolutionary processes leading to the emergence of novel X. perforans lineages identified in Alabama. While one lineage was isolated from symptomatic tomato and pepper plants, confirming the host range expansion of X. perforans, the other lineage was isolated from tomato and acquired a novel transcription activation-like effector, here designated PthXp1. Functional analysis of PthXp1 indicated that it does not induce Bs4-mediated resistance in tomato and contributes to virulence, providing an adaptive advantage to strains on tomato. Our findings also show that different phylogenetic groups of the pathogen have experienced independent recombination events originating from multiple Xanthomonas species. This suggests a continuous gene flux between related xanthomonads associated with diverse plant hosts that results in the emergence of novel pathogen lineages and associated phenotypes, including host range.

Reclassification of Xanthomonas gardneri (ex Sutic 1957) Jones et al. 2006 as a later heterotypic synonym of Xanthomonas cynarae Trébaol et al. 2000 and description of X. cynarae pv. cynarae and X. cynarae pv. gardneri based on whole genome analyses.

Multilocus sequence analysis of Xanthomonas species revealed a very close relationship between Xanthomonas cynarae, an artichoke pathogen and Xanthomonas gardneri, a tomato and pepper pathogen. Results of whole genome sequence comparisons using average nucleotide identity between representative strains of X. gardneri and X. cynarae were well above the threshold of 95-96 %. Inoculations of X. gardneri strains in artichoke leaves caused mild disease symptoms, but only weak symptoms were observed in the bracts. Both X. cynarae and X. gardneri grew equally and caused typical bacterial spot symptoms in pepper after artificial inoculation. However, X. cynarae induced a hypersensitive reaction in tomato, while X. gardneri strains were virulent. Pathogenicity-associated gene clusters, including the protein secretion systems, type III effector profiles, and lipopolysaccharide cluster were nearly identical between the two species. Based on our results from whole genome sequence comparison, X. gardneri and X. cynarae belong to the same species. The name X. cynarae has priority and X. gardneri should be considered as a later heterotypic synonym. An emended description of X. cynarae (type strain=CFBP 4188T, =DSM 16794T) is given. However, due to the host specificity in artichoke and tomato, two pathovars, X. cynarae pv. cynarae and X. cynarae pv. gardneri, are proposed.

Multilocus Sequence Analysis Reveals Genetic Diversity in Xanthomonads Associated With Poinsettia Production.

Xanthomonas axonopodis pv. poinsettiicola is traditionally identified as the primary causal agent of bacterial leaf spot on poinsettia (family Euphorbiaceae). Sixty-seven strains of xanthomonads isolated from lesions associated with several species within the family Euphorbiaceae were collected over a 64-year period. The pathogenicity of these strains was compared on several potential hosts and they were analyzed by multilocus sequence analysis (MLSA) using six housekeeping genes. The 67 Xanthomonas strains associated with poinsettia production were separated into three distinct clades based on MLSA. The first clade identified contained the X. axonopodis pv. poinsettiicola reference strain (LMG849PT). A second clade was more closely related to X. hortorum pv. pelargonii (LMG7314PT) and the third clade contained the X. codiaei type strain (LMG8678T). This analysis indicated that there may also be other closely related pathovars or species of Xanthomonas that can infect poinsettia. Strains from the three clades could not be distinguished by symptoms or virulence on poinsettia plants. Strains capable of infecting geranium were found in all three clades, although the extent of leaf spot formation and number of systemic infections were significantly less than those produced by X. hortorum pv. pelargonii strains, typically the main causal agent of bacterial leaf spot on geranium. Clade III also contained strains isolated from zebra plant (Aphelandra squarrosa, family Acanthaceae), which is a newly recognized host for X. codiaei and X. axonopodis pv. poinsettiicola. Xanthomonas leaf spot is a serious threat to poinsettia production that can be caused by several Xanthomonas spp. that can infect different ornamental plant hosts. It is imperative that growers maintain a strict sanitation program because reservoirs of inoculum can occur on a number of ornamental hosts.

First Report of Atypical Xanthomonas euvesicatoria Strains Causing Bacterial Spot of Tomato in Nigeria.

Bacterial spot (BS) is an important disease of tomato in Nigeria (2). Although a xanthomonad was isolated from tomato in Nigeria and characterized using phenotypic and pathogenicity tests, the bacterium was not characterized genetically to confirm the species. To determine the species associated with BS, leaves were collected in fields in northwestern Nigeria from tomato plants showing typical BS symptoms, which consisted of dark, irregular-shaped brown leaf spots that coalesced, resulting in a blighted appearance. Isolations from individual lesions were made on nutrient agar (NA). Yellow, mucoid colonies typical of Xanthomonas were isolated from 14 lesions and all were determined to be amylolytic (3). To determine the races of these strains, bacterial suspensions of the tomato strains, derived from 24-h cultures grown on NA at 28°C, were adjusted to 108 CFU/ml and infiltrated into leaves of tomato and pepper differential genotypes (5). The tomato strains elicited hypersensitive reactions (HRs) on the four pepper differential lines and an HR on the tomato genotype FL 216, which contains the R gene Xv3, but elicited susceptible reactions on the tomato genotypes Hawaii 7998 and Bonny Best. These reactions are typical of X. perforans tomato race 3 strains (5). Multilocus sequence analysis (MLSA) of six housekeeping genes (fusA, lacF, gyrB, gltA, gapA, and lepA) was used to further analyze four representative strains (1) (GenBank Accession Nos. KJ938581 to KJ938584, KJ938588 to KJ938591, KJ938595 to KJ938598, KJ938602 to KJ938605, KJ938629 to KJ938632, and KJ938636 to KJ938639, respectively). A partial sequence of hrpB2 was also made since the four Xanthomonas species associated with BS can be differentiated based on sequence divergence of this gene (3) (KJ938609 to KJ938621 and KJ938628). The housekeeping gene sequences were aligned along with other Xanthomonas sequences imported from the National Center for Biotechnology Information (NCBI) database ( www.ncbi.nlm.nih.gov ) using the MUSCLE tool from MEGA software, 5.2.2. Maximum likelihood phylogenetic trees constructed for the six housekeeping gene sequences individually and in concatenation revealed that the strains grouped most closely with the X. euvesicatoria reference strain 85-10 but more distantly to X. perforans. The hrpB2 sequence, which is highly conserved for each Xanthomonas species pathogenic on tomato (4), was sequenced from the tomato strains. These sequences were identical to the hrpB2 sequence from X. perforans strains but different from X. euvesicatoria. Although BS is common in Nigeria, to our knowledge, this represents a unique group of X. euvesicatoria strains from tomato that are identical to X. perforans based on pathogenic reactions on tomato and pepper and hrpB2 sequence identity but are more closely related to X. euvesicatoria based on the six housekeeping gene sequences. References: (1) N. F. Almeida et al. Phytopathology 100:208, 2010. (2) E. U. Opara and F. J. Odibo. J. Mol. Genet. 1:35, 2009. (3) J. B. Jones et al. Syst. Appl. Microbiol. 27:755, 2004. (4) A. Obradovic et al. Eur. J. Plant Pathol. 88:736, 2004. (5) R. E. Stall et al. Annu. Rev. Phytopathol. 47:265, 2009.

First Report of Xanthomonas euvesicatoria Causing Bacterial Spot Disease in Pepper in Northwestern Nigeria.

Bacterial spot (BS) has been reported as an important disease on pepper in Nigeria (4). Xanthomonas campestris pv. vesicatoria was identified as the causal agent using phenotypic and pathogenicity tests; however, X. campestris pv. vesicatoria is a synonym for two genetically distinct groups that have been elevated to the species X. euvesicatoria and X. vesicatoria (2). Furthermore, the latter two species and X. gardneri cause similar diseases on pepper (2). In order to determine the species associated with BS on pepper, leaves with irregular, dark brown lesions were collected from pepper plants in fields from northwestern Nigeria, and isolations were made on nutrient agar (NA). Yellow, mucoid colonies typical of Xanthomonas were isolated. Six strains isolated from pepper were determined to be non-amylolytic. For race determinations, bacterial suspensions of the pepper strains, derived from 24-h cultures grown on NA at 28°C, were adjusted to 108 CFU/ml and infiltrated into leaves of tomato and pepper differential genotypes (5). The six pepper strains elicited HRs on the tomato differential genotypes. The strains produced a susceptible reaction on all pepper differentials and were designated as pepper race 6 (5). Multilocus sequence analysis (MLSA) using six housekeeping genes (fusA, lacF, gyrB, gltA, gapA, and lepA) was used to further analyze the strains (1) (GenBank Accession Nos. KJ938585 to KJ938587, KJ938592 to KJ938594, KJ938599 to KJ938601, KJ938606 to KJ938608, KJ938633 to KJ938635, and KJ938640 to KJ938642). A partial sequence of hrpB2 was also sequenced since the four Xanthomonas species associated with BS can be differentiated based on sequence divergence (3) (KJ938622 to KJ938627). The housekeeping gene sequences were aligned along with other Xanthomonas sequences imported from the NCBI database using muscle tool from MEGA software, 5.2.2. Maximum likelihood phylogenetic trees constructed for the six housekeeping gene sequences individually and in concatenation revealed that the Nigerian pepper strains were identical to the X. euvesicatoria reference strain 85-10. Although BS is common in Nigeria, to our knowledge, this represents the first report for this pepper pathogen in Nigeria. References: (1) N. F. Almeida et al. Phytopathology 100:208, 2010. (3) J. B. Jones et al. System Appl. Microbiol. 27:755, 2004. (4) A. Obradovic et al. Eur. J. Plant Pathol. 88:736, 2004. (2) E. U. Opara and F. J. Odibo. J. Mol. Gen. 1:35, 2009. (5) R. E. Stall et al. Ann. Rev. Phytopathol. 47:265, 2009.