Bacterial spot disease of pepper and tomato is caused by four distinct Xanthomonas species and is a severely limiting factor on fruit yield in these crops. The genetic diversity and the type III effector repertoires of a large sampling of field strains for this disease have yet to be explored on a genomic scale, limiting our understanding of pathogen evolution in an agricultural setting. Genomes of 67 Xanthomonas euvesicatoria (Xe), Xanthomonas perforans (Xp), and Xanthomonas gardneri (Xg) strains isolated from diseased pepper and tomato fields in the southeastern and midwestern United States were sequenced in order to determine the genetic diversity in field strains. Type III effector repertoires were computationally predicted for each strain, and multiple methods of constructing phylogenies were employed to understand better the genetic relationship of strains in the collection. A division in the Xp population was detected based on core genome phylogeny, supporting a model whereby the host-range expansion of Xp field strains on pepper is due, in part, to a loss of the effector AvrBsT. Xp-host compatibility was further studied with the observation that a double deletion of AvrBsT and XopQ allows a host range expansion for Nicotiana benthamiana. Extensive sampling of field strains and an improved understanding of effector content will aid in efforts to design disease resistance strategies targeted against highly conserved core effectors.

AvrHah1 [avirulence (avr) gene homologous to avrBs3 and hax2, no. 1] is a transcription activator-like (TAL) effector (TALE) in Xanthomonas gardneri that induces water-soaked disease lesions on fruits and leaves during bacterial spot of tomato. We observe that water from outside the leaf is drawn into the apoplast in X. gardneriinfected, but not X. gardneriΔavrHah1 (XgΔavrHah1)-infected, plants, conferring a dark, water-soaked appearance. The pull of water can facilitate entry of additional bacterial cells into the apoplast. Comparing the transcriptomes of tomato infected with X. gardneri vs. XgΔavrHah1 revealed the differential up-regulation of two basic helix-loop-helix (bHLH) transcription factors with predicted effector binding elements (EBEs) for AvrHah1. We mined our RNA-sequencing data for differentially up-regulated genes that could be direct targets of the bHLH transcription factors and therefore indirect targets of AvrHah1. We show that two pectin modification genes, a pectate lyase and pectinesterase, are targets of both bHLH transcription factors. Designer TALEs (dTALEs) for the bHLH transcription factors and the pectate lyase, but not for the pectinesterase, complement water soaking when delivered by XgΔavrHah1. By perturbing transcriptional networks and/or modifying the plant cell wall, AvrHah1 may promote water uptake to enhance tissue damage and eventual bacterial egression from the apoplast to the leaf surface. Understanding how disease symptoms develop may be a useful tool for improving the tolerance of crops from damaging disease lesions.

h Four Xanthomonas species are known to cause bacterial spot of tomato and pepper, but the global distribution and genetic diversity of these species are not well understood. A collection of bacterial spot-causing strains from the Americas, Africa, Southeast Asia, and New Zealand were characterized for genetic diversity and phylogenetic relationships using multilocus sequence analysis of six housekeeping genes. By examining strains from different continents, we found unexpected phylogeographic patterns, including the global distribution of a single multilocus haplotype of X. gardneri, possible regional differentiation in X. vesicatoria, and high species diversity on tomato in Africa. In addition, we found evidence of multiple recombination events between X. euvesicatoria and X. perforans. Our results indicate that there have been shifts in the species composition of bacterial spot pathogen populations due to the global spread of dominant genotypes and that recombination between species has generated genetic diversity in these populations. Understanding the evolution and host specificity of plantpathogenic bacteria is an ongoing challenge. Strains of phytopathogenic bacteria commonly exhibit high host specificity, with host ranges restricted to one or a few plant species (1, 2). Bacterial plant pathogens also exhibit biogeography, such that species can be limited in their geographic distributions (3). Globalization of agriculture has contributed to the dispersal of phytopathogenic bacteria, but the geographic ranges of species are not well characterized, in part because of the difficulty in differentiating phylogenetically distinct strains that have similar host specificities (4). Phenotypic characters can sometimes distinguish species with similar host specificities, but classification by molecular markers is often required due to variation in phenotypic traits within species (5). Phenotypes can also dramatically differ among strains within a species due to acquisition and loss of genes related to pathogenicity and fitness (4). Bacterial evolution is driven by point mutations, variation in gene content, recombination, and selection on the resulting phenotypes (6). Phylogenetic relationships among species are defined by point mutations in the genome that accumulate over time; however, these relationships can be obscured by polymorphisms that have been distributed to other closely related species via homologous recombination and horizontal gene transfer (7). These events can introduce conflicting phylogenetic signals between genes that have been vertically inherited versus horizontally acquired (8). The possibility of infection of a single host plant by multiple species may increase the probability of genetic exchange (9). Coinfection by multiple species may be more common as pathogens are moved out of their native geographic ranges.Multilocus nucleotide-sequence-based approaches help in resolving phylogenetic relationships of bacteria within and between species (10). Multilocus sequence typing (MLST) and analysis...

Two strains, 30N-5 and 30VD-1, identified as Bacillus simplex and B. subtilis, were isolated from the rhizospheres of two different plants, a Podocarpus and a palm, respectively, growing in the University of California, Los Angeles (UCLA) Mildred E. Mathias Botanical Garden. B. subtilis is a well-known plant-growth promoting bacterial species, but B. simplex is not. B. simplex 30N-5 was initially isolated on a nitrogen-free medium, but no evidence for nitrogen fixation was found. Nevertheless, pea OPEN ACCESSAgronomy 2013, 3 596 plants inoculated with B. simplex showed a change in root architecture due to the emergence of more lateral roots. When Pisum sativum carrying a DR5::GUSA construct, an indicator for auxin response, was inoculated with either B. simplex 30N-5 or its symbiont Rhizobium leguminosarum bv. viciae 128C53, GUS expression in the roots was increased over the uninoculated controls. Moreover, when pea roots were coinoculated with either B. simplex 30N-5 or B. subtilis 30VD-1 and R. leguminosarum bv. viciae 128C53, the nodules were larger, clustered, and developed more highly branched vascular bundles. Besides producing siderophores and solubilizing phosphate, the two Bacillus spp., especially strain 30VD-1, exhibited anti-fungal activity towards Fusarium. Our data show that combining nodulating, nitrogen-fixing rhizobia with growth-promoting bacteria enhances plant development and strongly supports a coinoculation strategy to improve nitrogen fixation, increase biomass, and establish greater resistance to fungal disease.