David J. Norman

Identification of Open Reading Frames Unique to a Select Agent: Ralstonia solanacearum Race 3 Biovar 2

An 8x draft genome was obtained and annotated for Ralstonia solanacearum race 3 biovar 2 (R3B2) strain UW551, a United States Department of Agriculture Select Agent isolated from geranium. The draft UW551 genome consisted of 80,169 reads resulting in 582 contigs containing 5,925,491 base pairs, with an average 64.5% GC content. Annotation revealed a predicted 4,454 protein coding open reading frames (ORFs), 43 tRNAs, and 5 rRNAs; 2,793 (or 62%) of the ORFs had a functional assignment. The UW551 genome was compared with the published genome of R. solanacearum race 1 biovar 3 tropical tomato strain GMI1000. The two phylogenetically distinct strains were at least 71% syntenic in gene organization. Most genes encoding known pathogenicity determinants, including predicted type III secreted effectors, appeared to be common to both strains. A total of 402 unique UW551 ORFs were identified, none of which had a best hit or >45% amino acid sequence identity with any R. solanacearum predicted protein; 16 had strong (E < 10(-13)) best hits to ORFs found in other bacterial plant pathogens. Many of the 402 unique genes were clustered, including 5 found in the hrp region and 38 contiguous, potential prophage genes. Conservation of some UW551 unique genes among R3B2 strains was examined by polymerase chain reaction among a group of 58 strains from different races and biovars, resulting in the identification of genes that may be potentially useful for diagnostic detection and identification of R3B2 strains. One 22-kb region that appears to be present in GMI1000 as a result of horizontal gene transfer is absent from UW551 and encodes enzymes that likely are essential for utilization of the three sugar alcohols that distinguish biovars 3 and 4 from biovars 1 and 2.

Comparative genomics reveals diversity among xanthomonads infecting tomato and pepper

BackgroundBacterial spot of tomato and pepper is caused by four Xanthomonas species and is a major plant disease in warm humid climates. The four species are distinct from each other based on physiological and molecular characteristics. The genome sequence of strain 85-10, a member of one of the species, Xanthomonas euvesicatoria (Xcv) has been previously reported. To determine the relationship of the four species at the genome level and to investigate the molecular basis of their virulence and differing host ranges, draft genomic sequences of members of the other three species were determined and compared to strain 85-10.ResultsWe sequenced the genomes of X. vesicatoria (Xv) strain 1111 (ATCC 35937), X. perforans (Xp) strain 91-118 and X. gardneri (Xg) strain 101 (ATCC 19865). The genomes were compared with each other and with the previously sequenced Xcv strain 85-10. In addition, the molecular features were predicted that may be required for pathogenicity including the type III secretion apparatus, type III effectors, other secretion systems, quorum sensing systems, adhesins, extracellular polysaccharide, and lipopolysaccharide determinants. Several novel type III effectors from Xg strain 101 and Xv strain 1111 genomes were computationally identified and their translocation was validated using a reporter gene assay. A homolog to Ax21, the elicitor of XA21-mediated resistance in rice, and a functional Ax21 sulfation system were identified in Xcv. Genes encoding proteins with functions mediated by type II and type IV secretion systems have also been compared, including enzymes involved in cell wall deconstruction, as contributors to pathogenicity.ConclusionsComparative genomic analyses revealed considerable diversity among bacterial spot pathogens, providing new insights into differences and similarities that may explain the diverse nature of these strains. Genes specific to pepper pathogens, such as the O-antigen of the lipopolysaccharide cluster, and genes unique to individual strains, such as novel type III effectors and bacteriocin genes, have been identified providing new clues for our understanding of pathogen virulence, aggressiveness, and host preference. These analyses will aid in efforts towards breeding for broad and durable resistance in economically important tomato and pepper cultivars.

Novel insights into the genomic basis of citrus canker based on the genome sequences of two strains of Xanthomonas fuscans subsp. aurantifolii

BackgroundCitrus canker is a disease that has severe economic impact on the citrus industry worldwide. There are three types of canker, called A, B, and C. The three types have different phenotypes and affect different citrus species. The causative agent for type A is Xanthomonas citri subsp. citri, whose genome sequence was made available in 2002. Xanthomonas fuscans subsp. aurantifolii strain B causes canker B and Xanthomonas fuscans subsp. aurantifolii strain C causes canker C.ResultsWe have sequenced the genomes of strains B and C to draft status. We have compared their genomic content to X. citri subsp. citri and to other Xanthomonas genomes, with special emphasis on type III secreted effector repertoires. In addition to pthA, already known to be present in all three citrus canker strains, two additional effector genes, xopE3 and xopAI, are also present in all three strains and are both located on the same putative genomic island. These two effector genes, along with one other effector-like gene in the same region, are thus good candidates for being pathogenicity factors on citrus. Numerous gene content differences also exist between the three cankers strains, which can be correlated with their different virulence and host range. Particular attention was placed on the analysis of genes involved in biofilm formation and quorum sensing, type IV secretion, flagellum synthesis and motility, lipopolysacharide synthesis, and on the gene xacPNP, which codes for a natriuretic protein.ConclusionWe have uncovered numerous commonalities and differences in gene content between the genomes of the pathogenic agents causing citrus canker A, B, and C and other Xanthomonas genomes. Molecular genetics can now be employed to determine the role of these genes in plant-microbe interactions. The gained knowledge will be instrumental for improving citrus canker control.

Genetic Diversity and Host Range Variation of Ralstonia solanacearum Strains Entering North America

Each year, large volumes of ornamental and food plant propagative stock are imported into the North America; occasionally, Ralstonia solanacearum is found systemically infecting this plant material. In this study, 107 new R. solanacearum strains were collected over a 10-year period from imported propagative stock and compared with 32 previously characterized R. solanacearum strains using repetitive polymerase chain reaction (rep-PCR) element (BOX, ERIC, and REP) primers. Additional strain comparisons were made by sequencing the endoglucanase and the cytochrome b561 genes. Using rep-PCR primers, populations could be distinguished by biovar and, to a limited extent, country of origin and original host. Similarity coefficients among rep-PCR clusters within biovars were relatively low in many cases, indicating that disease outbreaks over time may have been caused by different clonal populations. Similar population differentiations of R. solanacearum were obtained when comparing strain sequences using either the endoglucanase or cytochrome b561 genes. We found that most of the new biovar 1 strains of R. solanacearum entering the United States were genetically distinct from the biovar 1 strains currently found infecting vegetable production. These introduced biovar 1 strains also had a broader host range and could infect not only tomato, tobacco, and potato but also anthurium and pothos and cause symptoms on banana. All introductions into North America of race 3, biovar 2 strains in the last few years have been linked to geranium production and appeared to be clonal.

Green synthesis and characterization of silver nanoparticles using Artemisia absinthium aqueous extract--A comprehensive study.

Unlike chemical synthesis, biological synthesis of nanoparticles is gaining tremendous interest, and plant extracts are preferred over other biological sources due to their ample availability and wide array of reducing metabolites. In this project, we investigated the reducing potential of aqueous extract of Artemisia absinthium L. for synthesizing silver nanoparticles (AgNPs). Optimal synthesis of AgNPs with desirable physical and biological properties was investigated using ultra violet-visible spectroscopy (UV-vis), dynamic light scattering (DLS), transmission electron microscopy (TEM) and energy-dispersive X-ray analysis (EDX). To determine their appropriate concentrations for AgNP synthesis, two-fold dilutions of silver nitrate (20 to 0.62 mM) and aqueous plant extract (100 to 0.79 mg ml(-1)) were reacted. The results showed that silver nitrate (2mM) and plant extract (10 mg ml(-1)) mixed in different ratios significantly affected size, stability and yield of AgNPs. Extract to AgNO3 ratio of 6:4v/v resulted in the highest conversion efficiency of AgNO3 to AgNPs, with the particles in average size range of less than 100 nm. Furthermore, the direct imaging of synthesized AgNPs by TEM revealed polydispersed particles in the size range of 5 to 20 nm. Similarly, nanoparticles with the characteristic peak of silver were observed with EDX. This study presents a comprehensive investigation of the differential behavior of plant extract and AgNO3 to synthesize biologically stable AgNPs.

Diversity among Ralstonia solanacearum strains isolated from the southeastern United States.

This is the first comprehensive study of a collection of Ralstonia solanacearum strains from the southeastern United States to be characterized based on biovar, pathogenicity, hypersensitive reaction on tobacco, and phylogenetic analyses of the egl sequence. Rigorous phylogenetic analysis of the commonly used egl gene produced robust phylogenies that differed significantly from a neighbor-joining tree differed from and previously published phylogenies for R. solanacearum strains. These robust trees placed phylotype IV within the phylotype I clade, which may suggest that phylogenies based solely on egl may be misleading. As a result of phylogenetic analyses in this study, we determined that U.S. strains from Georgia, North Carolina, South Carolina, and older Florida strains isolated from solanaceous crops all belong to phylotype II sequevar 7. However, many strains recently isolated in Florida from tomato and other crops were more diverse than the southeastern United States population. These unique Florida strains grouped with strains mostly originating from the Caribbean and Central America. One of the exotic strains, which in a previous study was determined to be established in northern Florida, was characterized more extensively. Upon using Musa-specific multiplex polymerase chain reaction, this strain produced a unique banding pattern, which has not previously been reported. Inoculation of this strain into Musa spp. did not result in wilt symptoms; however, the plants were stunted and root masses were significantly reduced. Furthermore, following root inoculation, the bacterium, unlike a typical Florida race 1 biovar 1 strain, was recovered from the roots and stems, indicating systemic movement. This is the first report of an R. solanacearum strain isolated in the United States that is deleterious to the growth of Musa plants.

Control of bacterial wilt of geranium with phosphorous acid

Various bactericides were screened for efficacy in protecting geranium plants (Pelargonium hortorum) from Ralstonia solanacearum infection. Many of these bactericides were found to slow the disease progress; however, they were not able to protect the plants from infection and subsequent death. Potassium salts of phosphorous acid were found to be effective in protecting plants from infection when applied as a drench. The active portion of the potassium salts was found to be phosphorous acid (H3PO3). Phosphorous acid was found to inhibit in vitro growth of R. solanacearum. It is thought to be protecting plants from infection by acting as a bacteriostatic compound in the soil. The plants, however, are not protected from aboveground infection on wounded surfaces. Phosphorous acid drenches were shown to protect geranium plants from infection by either race 1 or 3 of R. solanacearum. Other phosphorous-containing products commonly used in the industry, such as phosphorus pentoxide (P2O5) and phosphoric acid (H3PO4), were not able to protect plants from bacterial wilt infection.

Avirulence Proteins AvrBs7 from Xanthomonas gardneri and AvrBs1.1 from Xanthomonas euvesicatoria Contribute to a Novel Gene-for-Gene Interaction in Pepper

A novel hypersensitive resistance (HR) in Capsicum baccatum var. pendulum against the bacterial spot of pepper pathogen, Xanthomonas gardneri, was introgressed into C. annuum cv. Early Calwonder (ECW) to create the near-isogenic line designated as ECW-70R. A corresponding avirulence gene avrBs7, in X. gardneri elicited a strong HR in ECW-70R. A homolog of avrBs7, avrBs1.1, was found in X. euvesicatoria 85-10, which showed delayed HR on ECW-70R leaves. Genetic analysis confirmed the presence of a single dominant resistance gene, Bs7, corresponding to the two avr genes. Both AvrBs7 and AvrBs1.1 share a consensus protein tyrosine phosphatase (PTP) active site domain and can dephosphorylate para-nitrophenyl phosphate. Mutation of Cys(265) to Ser in the PTP domain and subsequent loss of enzymatic activity and HR activity indicated the importance of the PTP domain in the recognition of the Avr protein by the Bs7 gene transcripts. Superpositioning of AvrBs7 and AvrBs1.1 homology models indicated variation in the geometry of the loops adjacent to the active sites. These predicted structural differences might be responsible for the differences in HR timing due to differential activation of the resistance gene. Mutating the PTP domain of AvrBs1.1 to match that of AvrBs7 failed to activate HR on ECW-70R, indicating the possibility of differential substrate specificities between AvrBs1.1 and AvrBs7.

Proteomic comparison of Ralstonia solanacearum strains reveals temperature dependent virulence factors.

BackgroundRalstonia solanacearum, the causal agent of bacterial wilt, is a genetically diverse bacterial plant pathogen present in tropical and subtropical regions of the world that infects more than 200 plant species, including economically important solanaceous crops. Most strains of R. solanacearum are only pathogenic at temperatures between 25 to 30°C with strains that can cause disease below 20°C considered a threat to agriculture in temperate areas. Identifying key molecular factors that distinguish strains virulent at cold temperatures from ones that are not is needed to develop effective management tools for this pathogen. We compared protein profiles of two strains virulent at low temperature and two strains not virulent at low temperature when incubated in the rhizosphere of tomato seedlings at 30 and 18°C using quantitative 2D DIGE gel methods. Spot intensities were quantified and compared, and differentially expressed proteins were sequenced and identified by mass spectrometry (MS/MS).ResultsFour hundred and eighteen (418) differentially expressed protein spots sequenced produced 101 unique proteins. The identified proteins were classified in the Gene Ontology biological processes categories of metabolism, cell processes, stress response, transport, secretion, motility, and virulence. Identified virulence factors included catalase (KatE), exoglucanase A (ChbA), drug efflux pump, and twitching motility porin (PilQ). Other proteins identified included two components of a putative type VI secretion system. We confirmed differential expression of 13 candidate genes using real time PCR techniques. Global regulators HrpB and HrpG also had temperature dependent expression when quantified by real time PCR.ConclusionsThe putative involvement of the identified proteins in virulence at low temperature is discussed. The discovery of a functional type VI secretion system provides a new potential virulence mechanism to explore. The global regulators HrpG and HrpB, and the protein expression profiles identified suggest that virulence at low temperatures can be partially explained by differences in regulation of virulence factors present in all the strains.

Comparative Effect of Low Temperature on Virulence and Twitching Motility of Ralstonia solanacearum Strains Present in Florida

Ralstonia solanacearum causes bacterial wilt on a wide range of plant hosts. Most strains of R. solanacearum are nonpathogenic below 20°C; however, Race 3 Biovar 2 (R3B2) strains are classified as quarantine pathogens because of their ability to infect crops, cause disease, and survive in temperate climates. We have identified race 1 biovar 1 Phylotype IIB Sequevar 4 strains present in Florida which were able to infect and produce wilt symptoms on potato and tomato at 18°C. Moreover they infected tomato plants at rates similar to strains belonging to R3B2. We determined that strains naturally nonpathogenic at 18°C were able to multiply, move in planta, and cause partial wilt when inoculated directly into the stem, suggesting that low temperature affects virulence of strains differently at early stages of infection. Bacterial growth in vitro was delayed at low temperatures, however it was not attenuated. Twitching motility observed on growing colonies was attenuated in nonpathogenic strains at 18°C, while not affected in the cool virulent ones. Using pilQ as a marker to evaluate the relative expression of the twitching activity of R. solanacearum strains, we confirmed that cool virulent strains maintained a similar level of pilQ expression at both temperatures, while in nonpathogenic strains pilQ was downregulated at 18°C.