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Three field experiments were conducted in Florida from 2012-2014 to assess the impact of acibenzolar-S-methyl (ASM), a systemic acquired-resistance inducer, applied as foliar spray or through drip-irrigation lines, on bacterial wilt incidence and yield of grafted tomatoes. The experiments were conducted in a field with race 1, biovar 1 strain of Ralstonia solanacearum, causal agent of tomato bacterial wilt. In all three experiments, the susceptible tomato variety BHN 602, grafted onto a resistant rootstock BHN 998, was compared with nongrafted BHN 602, treated with or without foliar applications of ASM and with grafted plants treated with foliar applications of ASM. In two experiments, an additional treatment of drip applications of ASM on grafted and nongrafted plants was evaluated. Grafting alone or in combination with drip applications of ASM (178.6 μM) significantly reduced disease incidence and increased total marketable yield relative to nongrafted treatments. There were no significant differences between grafted plants with or without drip ASM applications in terms of bacterial wilt incidence or total marketable yield. However, we demonstrate for the first time that foliar ASM applications on grafted plants negatively affects the total marketable yield compared with drip ASM applications on grafted plants or nontreated grafted control.

In 2012, stem gall samples on Loropetalum chinense were sent to Florida diagnostic labs from Alabama and Florida nurseries. A fluorescent pseudomonad was consistently isolated from the galls. The organism was originally identified in Alabama based on 16S rRNA sequencing as Pseudomonas savastanoi, which causes a production-limiting disease of olive. The loropetalum strains and reference strains were compared using LOPAT, Biolog, fatty acid analysis, multilocus sequence analysis (MLSA), and pathogenicity tests. The LOPAT tests placed the loropetalum strains within Pseudomonas syringae. Biolog and fatty acid analysis placed the strains in various pathovars of P. syringae and P. savastanoi, respectively. MLSA of a set of housekeeping genes separated the loropetalum strains from the olive knot-inducing strains. Our work indicates there is a need to use more tests than 16S rRNA to accurately diagnose new bacterial diseases. In pathogenicity tests, the loropetalum strains produced galls only on loropetalum, but not on olive, mandevilla, or almond, indicating this strain is not a threat to the olive industry. Based on the pathogenicity assays and molecular tests, loropetalum strains represent a distinct and new pathovar, P. amygdali pv. loropetali pv. nov., for which the strain PDC13-208 (= DSMZ 105780PT) has been designated as the pathotype strain.

International audience; 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.

Bacterial spot of tomato is caused by Xanthomonas gardneri, X. euvesicatoria, X. perforans, and X. vesicatoria. Current diagnostic methods for the pathogens are not in-field assays. Recombinase polymerase amplification (RPA) is ideal for in-field detection assays, because it is an isothermal technique that is rapid and more tolerant to inhibitors compared with polymerase chain reaction. Hence, novel RPA probes and primers were designed to amplify regions of the hrcN gene of X. gardneri, X. euvesicatoria, and X. perforans. The X. gardneri RPA is specific to X. gardneri with a detection limit of 106 CFU/ml and detected X. gardneri in lesions from naturally (n = 6) or artificially (n = 18) infected plants. The X. euvesicatoria RPA detects both X. euvesicatoria and X. perforans with a detection limit of 106 CFU/ml and detected both pathogens in plants artificially infected (n = 36) or naturally infected (n = 85) with either X. euvesicatoria or X. perforans. The X. perforans RPA is specific to X. perforans with a detection limit of 107 CFU/ml. Although the X. perforans RPA assay was unable to detect X. perforans from lesions, the X. euvesicatoria RPA was successfully used in field to detect X. perforans from symptomatic field samples (n = 31). The X. perforans RPA was then used to confirm the pathogen in the laboratory. The X. euvesicatoria and X. gardneri RPA is promising for rapid, real-time in-field detection of bacterial spot and one of the first developed among plant pathogenic bacteria.

Following analysis of eight phages under in vitro, growth chamber and greenhouse conditions with the bacterial spot of tomato pathogen Xanthomonas perforans, there was no correlation between disease control efficacy and in vitro phage multiplication, in vitro bacterial suppression, or in vivo phage multiplication in the presence of the host, but there was a low correlation between phage persistence on the leaf surface and disease control. Two of the 8 virulent phages (ΦXv3-21 and ΦXp06-02) were selected for in depth analysis with two X. perforans (Xp06-2-1 and Xp17-12) strains. In in vitro experiments, phage ΦXv3-21 was equally effective in infecting the two bacterial strains based on efficiency of plating (EOP). Phage ΦXp06-02, on the other hand, had a high EOP on strain Xp06-2-1 but a lower EOP on strain Xp17-12. In several growth chamber experiments, ΦXv3-21 was less effective than phage ΦXp06-02 in reducing disease caused by strain Xp06-2-1, but provided little or no disease control against strain Xp17-12. Interestingly, ΦXp06-02 could multiply to significantly higher levels on the tomato leaf surface than phage ΦXv3-21. The leaf surface appears to be important in terms of the ability of certain bacteriophages to multiply in the presence of the bacterial host. ΦXv3-21, when applied to grapefruit leaves in combination with a bacterial host, was unable to multiply to high levels, whereas on tomato leaflets the phage multiplied exponentially. One plausible explanation is that the leaf surface may be an important factor for attachment of certain phages to their bacterial host.

Bacterial spot caused by Xanthomonas perforans causes significant damage on tomato in Florida. Due to the presence of copper (Cu)-tolerant X. perforans strains, Cu bactericides are not effective in disease management. Hence, there is a critical need to find alternatives for Cu. Antibacterial activity of magnesium oxide (Nano-MgO), and other metal oxide nanoparticles, were evaluated against a Cu-tolerant and -sensitive X. perforans strain. In vitro experiments demonstrated high antibacterial activity of Nano-MgO against both strains compared with the commercial Cu. The minimum inhibitory concentration of Nano-MgO is 25 µg/ml and the minimum bactericidal concentration is 100 µg/ml against a Cu-tolerant X. perforans strain after 4 h of exposure. Structural changes in the bacterial membrane following exposure to Nano-MgO treatments compared with the controls were observed using transmission electron microscopy. In two greenhouse experiments with a Cu-tolerant strain, bacterial spot severity was significantly reduced by Nano-MgO at 200 µg/ml compared with Cu-ethylene bis-dithiocarbamate (grower standard), and the untreated control (P = 0.05). In three field experiments, Nano-MgO at 200 µg/ml significantly reduced disease severity with no negative impact on yield compared with the untreated control. Inductively coupled plasma mass spectrometric analysis of the fruit confirmed that Nano-MgO application did not lead to the accumulation of Mg, Cu, Ca, K, Mn, P, and S. This study is the first to demonstrate the potential of Nano-MgO against bacterial spot of tomato.

Recombination is a major driver of evolution in bacterial populations, because it can spread and combine independently evolved beneficial mutations. Recombinant lineages of bacterial pathogens of plants are typically associated with the colonization of novel hosts and the emergence of new diseases. Here we show that recombination between evolutionarily and phenotypically distinct plant-pathogenic lineages generated recombinant lineages with unique combinations of pathogenicity and virulence factors. Xanthomonas euvesicatoria and Xanthomonas perforans are two closely related lineages causing bacterial spot disease on tomato and pepper worldwide. We sequenced the genomes of atypical strains collected from tomato in Nigeria and observed recombination in the type III secretion system and effector genes, which showed alleles from both X. euvesicatoria and X. perforans . Wider horizontal gene transfer was indicated by the fact that the lipopolysaccharide cluster of one strain was most similar to that of a distantly related Xanthomonas pathogen of barley. This strain and others have experienced extensive genomewide homologous recombination, and both species exhibited dynamic open pangenomes. Variation in effector gene repertoires within and between species must be taken into consideration when one is breeding tomatoes for disease resistance. Resistance breeding strategies that target specific effectors must consider possibly dramatic variation in bacterial spot populations across global production regions, as illustrated by the recombinant strains observed here. IMPORTANCE The pathogens that cause bacterial spot of tomato and pepper are extensively studied models of plant-microbe interactions and cause problematic disease worldwide. Atypical bacterial spot strains collected from tomato in Nigeria, and other strains from Italy, India, and Florida, showed evidence of genomewide recombination that generated genetically distinct pathogenic lineages. The strains from Nigeria and Italy were found to have a mix of type III secretion system genes from X. perforans and X. euvesicatoria , as well as effectors from Xanthomonas gardneri . These genes and effectors are important in the establishment of disease, and effectors are common targets of resistance breeding. Our findings point to global diversity in the genomes of bacterial spot pathogens, which is likely to affect the host-pathogen interaction and influence management decisions.

Type III secretion system effectors contribute to pathogenicity through various mechanisms. Recent surveys showed an increasing prevalence of the type III secretion effector avrBsT among Xanthomonas perforans strains. We hypothesized that the acquisition of avrBsT has a fitness advantage for the pathogen. The contribution of avrBsT to fitness on tomato was evaluated based on disease severity, in planta growth, competition, and recovery rates of wild-type (WT) and avrBsT mutant strains in greenhouse and field plants. GEV872 and GEV1001, representative strains of two phylogenomic groups of X. perforans, were selected for generating avrBsT mutants. Disease severity was higher for WT strains compared with the avrBsT mutant strains. X. perforans WT and avrBsT mutant strains did not differ following leaf infiltration of greenhouse plants in direct competition and in planta growth assays. The effect of avrBsT on pathogen fitness was noticeable under field conditions. Differences in strain recovery were significant, with WT being recovered two to eight times more than avrBsT mutant strains in the case of both strains GEV872 and GEV1001. WT strains were capable of spreading longer distances across field plots compared with avrBsT mutant strains. Findings suggest that the functional AvrBsT affects the fitness of X. perforans under field conditions, making it an ideal candidate for bacterial spot resistance breeding efforts in tomato.

Citrus huanglongbing (HLB), associated with ‘Candidatus Liberibacter asiaticus’ (Las), disseminated by Asian citrus psyllid (ACP), has devastated citrus in Florida since 2005. Data on HLB occurrence were stored in databases (2005 to 2012). Cumulative HLB-positive citrus blocks were subjected to kernel density analysis and kriging. Relative disease incidence per county was calculated by dividing HLB numbers by relative tree numbers and maximum incidence. Spatiotemporal HLB distributions were correlated with weather. Relative HLB incidence correlated positively with rainfall. The focus expansion rate was 1626 m month−1, similar to that in Brazil. Relative HLB incidence in counties with primarily large groves increased at a lower rate (0.24 year−1) than in counties with smaller groves in hotspot areas (0.67 year−1), confirming reports that large-scale HLB management may slow epidemic progress.

Although production of tomato (Solanum lycopersicum) is threatened by a number of major diseases worldwide, it has been difficult to identify effective and durable management measures against these diseases. In this study, we attempted to improve tomato disease resistance by transgenic overexpression of genes encoding the Arabidopsis thaliana Elongator (AtELP) complex subunits AtELP3 and AtELP4. We show that overexpression of AtELP3 and AtELP4 significantly enhanced resistance to tomato bacterial speck caused by the Pseudomonas syringae pv. tomato strain J4 (Pst J4) without clear detrimental effects on plant growth and development. Interestingly, the transgenic plants exhibited resistance to Pst J4 only when inoculated through foliar sprays but not through infiltration into the leaf apoplast. Although this result suggested possible involvement of stomatal immunity, we found that Pst J4 inoculation did not induce stomatal closure and there were no differences in stomatal apertures and conductance between the transgenic and control plants. Further RNA sequencing and real-time quantitative PCR analyses revealed a group of defense-related genes to be induced to higher levels after infection in the AtELP4 transgenic tomato plants than in the control, suggesting that the enhanced disease resistance of the transgenic plants may be attributed to elevated induction of defense responses. Additionally, we show that the tomato genome contains single-copy genes encoding all six Elongator subunits (SlELPs), which share high identities with the AtELP proteins, and that SlELP3 and SlELP4 complemented the Arabidopsis Atelp3 and Atelp4 mutants, respectively, indicating that the function of tomato Elongator is probably conserved. Taken together, our results not only shed new light on the tomato Elongator complex, but also revealed potential candidate genes for engineering disease resistance in tomato.

Bacterial spot (BS), caused by Xanthomonas euvesicatoria, X. vesicatoria, X. gardneri and X. perforans, is an economically important bacterial disease of tomato and pepper. Symptoms produced by all four species are nearly indistinguishable. At present, no point-of-care diagnostics exist for BS. In this research, we examined genomes of X. euvesicatoria, X. vesicatoria, X. gardneri, X. perforans and other species of Xanthomonas; the unique gene recG was chosen to design primers to develop a loop-mediated isothermal amplification (LAMP) assay to rapidly and accurately identify and differentiate X. euvesicatoria from other BS causing Xanthomonas sp. using a field-deployable portable BioRangerTM instrument. Specificity of the developed assay was tested against 39 strains of X. euvesicatoria and 41 strains of other species in inclusivity and exclusivity panels, respectively. The assay detection limit was 100 fg (~18 genome copies) of genomic DNA and 1,000 fg in samples spiked with tomato DNA. The assay unambiguously detected X. euvesicatoria in infected tomato plant samples. Concordant results were obtained when multiple operators performed the test independently. No false positives and false negatives were detected. The developed LAMP assay has numerous applications in diagnostics, biosecurity and disease management.

Citrus canker caused by Xanthomonas citri subspecies citri (Xcc) is a severe disease for most commercial citrus cultivars and responsible for significant economic losses worldwide. Generating canker-resistant citrus varieties will provide an efficient and sustainable solution to control citrus canker. Here, we report our progress in generating canker-resistant grapefruit by modifying the PthA4 effector binding elements (EBEs) in the CsLOB1 Promoter (EBEPthA4 -CsLOBP) of the CsLOB1 (Citrus sinensis Lateral Organ Boundaries) gene. CsLOB1 is a susceptibility gene for citrus canker and is induced by the pathogenicity factor PthA4, which binds to the EBEPthA4 -CsLOBP to induce CsLOB1 gene expression. There are two alleles, Type I and Type II, of CsLOB1 in Duncan grapefruit. Here, a binary vector was designed to disrupt the PthA4 EBEs in Type I CsLOB1 Promoter (TI CsLOBP) via epicotyl transformation of Duncan grapefruit. Four transgenic Duncan plants with targeted modification of EBEPthA4 -T1 CsLOBP were successfully created. As for Type I CsLOB1 promoter, the mutation rate was 15.63% (#D13), 14.29% (#D17), 54.54% (#D18) and 81.25% (#D22). In the presence of wild-type Xcc, transgenic Duncan grapefruit developed canker symptoms similarly as wild type. An artificially designed dTALE dCsLOB1.3, which specifically recognizes Type I CsLOBP, but not the mutated Type I CsLOBP or Type II CsLOBP, was developed to infect Duncan transformants. Consequently, #D18 had weakened canker symptoms and #D22 had no visible canker symptoms in the presence of XccΔpthA4:dCsLOB1.3. Our data suggest that activation of a single allele of susceptibility gene CsLOB1 by PthA4 is sufficient to induce citrus canker disease, and mutation in the promoters of both alleles of CsLOB1 is probably required to generate citrus canker-resistant plants. This work lays the groundwork to generate canker-resistant citrus varieties via Cas9/sgRNA in the future.

Two plant-tumorigenic strains KFB 330(T) and KFB 335 isolated from galls on raspberry (Rubus idaeus) in Serbia, and a non-pathogenic strain AL51.1 recovered from a cherry plum (Prunus cerasifera) tumor in Poland, were genotypically and phenotypically characterized. Phylogenetic reconstruction based on 16S rDNA placed them within the genus Agrobacterium, with A. nepotum as their closest relative. Multilocus sequence analysis (MLSA) based on the partial sequences of atpD, glnA, gyrB, recA and rpoB housekeeping genes suggested that these three strains represent a new Agrobacterium species, that clustered with type strains of A. nepotum, A. radiobacter, "A. fabrum" and A. pusense. This was further supported by average nucleotide identity values (

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.

Summary Taxonomic status Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Order Xanthomonadales; Family Xanthomonadaceae; Genus Xanthomonas; Species Xanthomonas euvesicatoria, Xanthomonas vesicatoria, Xanthomonas perforans and Xanthomonas gardneri. Microbiological properties Gram-negative, rod-shaped bacterium, aerobic, motile, single polar flagellum. Host range Causes bacterial spot disease on plants belonging to the Solanaceae family, primarily tomato (Solanum lycopersicum), pepper (Capsicum annuum) and chilli peppers (Capsicum frutescens). Disease symptoms Necrotic lesions on all above-ground plant parts. Distribution Worldwide distribution of X. euvesicatoria and X. vesicatoria on tomato and pepper; X. perforans and X. gardneri increasingly being isolated from the USA, Canada, South America, Africa and Europe. A wide diversity within the bacterial spot disease complex, with an ability to cause disease at different temperatures, makes this pathogen group a worldwide threat to tomato and pepper production. Recent advances in genome analyses have revealed the evolution of the pathogen with a plethora of novel virulence factors. Current management strategies rely on the use of various chemical control strategies and sanitary measures to minimize pathogen spread through contaminated seed. Chemical control strategies have been a challenge because of resistance by the pathogen. Breeding programmes have been successful in developing commercial lines with hypersensitive and quantitative resistance. However, durability of resistance has been elusive. Recently, a transgenic approach has resulted in the development of tomato genotypes with significant levels of resistance and improved yield that hold promise. In this article, we discuss the current taxonomic status, distribution of the four species, knowledge of virulence factors, detection methods and strategies for disease control with possible directions for future research.

Xanthomonas citri pv. citri strain 306 ( Xcc 306), a causative agent of citrus canker, produces endoxylanases that catalyze the depolymerization of cell wall-associated xylans. In the sequenced genomes of all plant-pathogenic xanthomonads, genes encoding xylanolytic enzymes are clustered in three adjacent operons. In Xcc 306, these consecutive operons contain genes encoding the glycoside hydrolase family 10 (GH10) endoxylanases Xyn10A and Xyn10C, the agu67 gene, encoding a GH67 α-glucuronidase (Agu67), the xyn43E gene, encoding a putative GH43 α- l -arabinofuranosidase, and the xyn43F gene, encoding a putative β-xylosidase. Recombinant Xyn10A and Xyn10C convert polymeric 4- O -methylglucuronoxylan (MeGX n ) to oligoxylosides methylglucuronoxylotriose (MeGX 3 ), xylotriose (X 3 ), and xylobiose (X 2 ). Xcc 306 completely utilizes MeGX n predigested with Xyn10A or Xyn10C but shows little utilization of MeGX n . Xcc 306 with a deletion in the gene encoding α-glucuronidase ( Xcc 306 Δ agu67 ) will not utilize MeGX 3 for growth, demonstrating the role of Agu67 in the complete utilization of GH10-digested MeGX n . Preferential growth on oligoxylosides compared to growth on polymeric MeGX n indicates that GH10 xylanases, either secreted by Xcc 306 in planta or produced by the plant host, generate oligoxylosides that are processed by Xyn10 xylanases and Agu67 residing in the periplasm. Coordinate induction by oligoxylosides of xyn10 , agu67 , cirA , the tonB receptor, and other genes within these three operons indicates that they constitute a regulon that is responsive to the oligoxylosides generated by the action of Xcc 306 GH10 xylanases on MeGX n . The combined expression of genes in this regulon may allow scavenging of oligoxylosides derived from cell wall deconstruction, thereby contributing to the tissue colonization and/or survival of Xcc 306 and, ultimately, to plant disease.

Citrus canker is caused by two Xanthomonas species, Xanthomonas citri, which has become the primary pathogen where citrus canker occurs (type A citrus canker, Xc-A), and X. fuscans pv. aurantifolii (Xfa), which consists of strains B and C. The B strain is less pathogenic than the A strain, but produces symptoms in all citrus species. The C-type cankers only infect Key lime (Citrus aurantifolia) and produce a hypersensitive reaction (HR) in grapefruit (Citrus paradisi) leaves. An avirulence gene, avrGf2, was identified in a C strain that was responsible for the HR in grapefruit. AvrGf2 is a member of XopAG effector family and shares 45% identity at amino acid level with another member of the same family, AvrGf1 from strain Xc-Aw, which was previously shown to elicit an HR in grapefruit. AvrGf2 shares sequence identity with other XopAG effectors present in Xanthomonas vasculorum, Xanthomonas campestris pv. musacearum and Pseudomonas syringae pv. tomato. Mutagenesis of avrGf2 in C strain resulted in a compatible reaction in grapefruit. There was no observable effect on virulence when Xc-A transconjugants containing either avirulence gene were inoculated on Key lime. Expression of avrGf1 or avrGf2 in Xc-A resulted in a similar phenotype following infiltration into grapefruit leaves, although the avrGf2 transconjugant elicited a faster HR and lower populations than the transconjugant containing avrGf1. Also, it was shown that all Xfa-B strains tested contain a transposon in avrGf2 that helps to explain the differences in host range between B and C strains.

International audience; AbstractSince the initial use of Bordeaux mixture in 1885 for plant disease control, a large number of copper-based antimicrobial compounds (CBACs) have been developed and applied for crop protection. While these compounds have revolutionized crop protection in the twentieth century, their continuous and frequent use has also raised concerns about the long-term sustainability of copper (Cu)-based crop protection system. Here, we review CBACs used in crop protection and highlight their benefits and risks, and potential for their improvement and opportunities for further research to develop alternatives to CBACs. The major findings are (i) the relatively high toxicity to plant pathogens, low cost, low mammalian toxicity of the fixed Cu compounds, and their chemical stability and prolonged residual effects are major benefits of these compounds; (ii) phytotoxicity, development of copper-resistant strains, soil accumulation, and negative effects on soil biota as well as on food quality parameters are key disadvantages of CBACs; (iii) regulatory pressure in agriculture worldwide to limit the use of CBACs has led to several restrictions, including that imposed by the regulation 473/2002 in the European Union; and (iv) mitigation strategies to limit the negative effects of CBACs include their optimized use, soil remediation, and development and application of alternatives to CBACs for a sustainable crop protection. We conclude that recent research and policy efforts have led to the development of a number of alternatives to CBACs, which should be further intensified to ensure that growers have sufficient tools for the implementation of sustainable crop protection strategies.

Bacterial spot, caused by Xanthomonas spp., is a widespread and damaging bacterial disease of tomato (Solanum lycopersicum). For disease management, growers rely on copper bactericides, which are often ineffective due to the presence of copper-tolerant Xanthomonas strains. This study evaluated the antibacterial activity of the new copper composites core-shell copper (CS-Cu), multivalent copper (MV-Cu), and fixed quaternary ammonium copper (FQ-Cu) as potential alternatives to commercially available micron-sized copper bactericides for controlling copper-tolerant Xanthomonas perforans. In vitro, metallic copper from CS-Cu and FQ-Cu at 100 μg/ml killed the copper-tolerant X. perforans strain within 1 h of exposure. In contrast, none of the micron-sized copper rates (100 to 1,000 μg/ml) from Kocide 3000 significantly reduced copper-tolerant X. perforans populations after 48 h of exposure compared with the water control (P < 0.05). All copper-based treatments killed the copper-sensitive X. perforans strain within 1 h. Greenhouse studies demonstrated that all copper composites significantly reduced bacterial spot disease severity when compared with copper-mancozeb and water controls (P < 0.05). Although there was no significant impact on yield, copper composites significantly reduced disease severity when compared with water controls, using 80% less metallic copper in comparison with copper-mancozeb in field studies (P < 0.05). This study highlights the discovery that copper composites have the potential to manage copper-tolerant X. perforans and tomato bacterial spot.

Bacterial recombination and clonality underly the evolution and epidemiology of pathogenic lineages as well as their cosmopolitan spread. While the spread of stable clonal bacterial pathotypes drives disease epidemics, recombination leads to the evolution of new bacterial lineages. Recombinant lineages of plant bacterial pathogens are typically associated with colonization of novel hosts and emergence of new diseases. Here, we show that recombination between evolutionarily and phenotypically distinct plant pathogenic lineages has generated new recombinant lineages with unique combinations of pathogenicity and virulence factors.X. euvesicatoria(Xe) andX. perforans(Xp) are two closely related monophyletic species causing bacterial spot disease on tomato and pepper worldwide. We sequenced the genomes of strains representing populations on tomato in Nigeria and found shuffling of secretion systems and effectors such that these strains contain genes from bothXeandXp. Multiple strains, from populations in Nigeria, Italy, and Florida, USA, exhibited extensive genomewide homologous recombination and both species exhibited dynamic open pangenomes. Our results show that recombination is generating new lineages of bacterial spot pathogens on tomato with consequences for disease management strategies.ImportanceTheXanthomonaspathogens that cause bacterial spot of tomato and pepper have been model systems for plant-microbe interactions. Two of these pathogens,X. euvesicatoriaandX. perforans, are very closely related. Genome sequences of bacterial spot field strains from Nigeria, Italy, and the United States showed varying levels of homologous recombination that changed the amino sequence of effectors, secretion systems, and other proteins. This shuffling of genome content occurred betweenX. euvesicatoriaandX. perforans, while a Nigerian lineage also contained the lipopolysaccharide cluster of a distantly relatedXanthomonasspecies. Gene content varied among strains and the affected genes are important in the establishment of disease, therefore our findings point to global variation in the host-pathogen interaction driven by gene exchange among evolutionarily distinct lineages.

Taxonomic status: Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Order Xanthomonadales; Family Xanthomonadaceae; Genus Xanthomonas; Species Xanthomonas citri ssp. citri (Xcc). Host range: Compatible hosts vary in their susceptibility to citrus canker (CC), with grapefruit, lime and lemon being the most susceptible, sweet orange being moderately susceptible, and kumquat and calamondin being amongst the least susceptible. Microbiological properties: Xcc is a rod‐shaped (1.5–2.0 × 0.5–0.75 µm), Gram‐negative, aerobic bacterium with a single polar flagellum. The bacterium forms yellow colonies on culture media as a result of the production of xanthomonadin. Distribution: Present in South America, the British Virgin Islands, Africa, the Middle East, India, Asia and the South Pacific islands. Localized incidence in the USA, Argentina, Brazil, Bolivia, Uruguay, Senegal, Mali, Burkina Faso, Tanzania, Iran, Saudi Arabia, Yemen and Bangladesh. Widespread throughout Paraguay, Comoros, China, Japan, Malaysia and Vietnam. Eradicated from South Africa, Australia and New Zealand. Absent from Europe.

We have previously reported several new technologies for selective and sensitive detection of quarantine race 3 biovar 2 strains of the bacterial wilt pathogen Ralstonia solanacearum, including new highly specific PCR and LAMP primers, immunomagnetic separation (IMS) for improved isolation of the pathogen from contaminated materials, and two handheld platforms to implement the LAMP-based assay in the field. Here we report on field testing of these new technologies in a naturally infested potato field in the highlands of Guatemala and compare the results to those obtained by standard reference methods, including plating on SMSA media, use of immunostrips, and quantitative PCR. The new LAMP-based assay was demonstrated to be equivalent to quantitative PCR, as sample classifications using the respective methods were absolutely consistent when directly assaying soil and plant tissue extracts. Isolation of DNA from the extracts using IMS enabled more sensitive detection in samples that otherwise appeared negative. These results suggest that the new LAMP tools are indeed viable approaches for rapid, selective, and sensitive field-based detection of race 3 biovar 2 strains of Ralstonia solanacearum, and that IMS is a useful tool for enhancing the isolation and detection of target pathogens in the field.

The bacterial agent of citrus canker disease (Xanthomonas citri ssp. citri, Xcc) has caused tremendous economic losses to the citrus industry around the world. Pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) is important to plant immunity. In this study, we compared the defence responses of citrus canker-resistant and citrus canker-susceptible genotypes to the Xcc-derived PAMP flg22 (Xflg22) by analysing the expression of 20 citrus defence-associated genes. We showed that, in the most resistant genotype, 'Nagami' kumquat, there was significant induction of several defence genes (EDS1, NDR1, PBS1, RAR1, SGT1, PAL1, NPR2 and NPR3) as early as 6 h and up to 72 h after Xflg22 treatment. At the other end of the spectrum, highly susceptible 'Duncan' grapefruit showed no induction of the same defence genes, even 120 h after treatment. Citrus genotypes with partial levels of resistance showed intermediate levels of transcriptional reprogramming that correlated with their resistance level. Xflg22 also triggered a rapid oxidative burst in all genotypes which was higher and accompanied by the induction of PTI marker genes (WRKY22 and GST1) only in the more resistant genotypes. Pretreatment with Xflg22 prior to Xcc inoculation inhibited bacterial growth in kumquat, but not in grapefruit. A flagellin-deficient Xcc strain (XccΔfliC) showed greater growth increase relative to wild-type Xcc in kumquat than in grapefruit. Taken together, our results indicate that Xflg22 initiates strong PTI in canker-resistant genotypes, but not in susceptible ones, and that a robust induction of PTI is an important component of citrus resistance to canker.

Bacterial type IV pili (T4P) are long, flexible surface filaments that consist of helical polymers of mostly pilin subunits. Cycles of polymerization, attachment, and depolymerization mediate several pilus-dependent bacterial behaviors, including twitching motility, surface adhesion, pathogenicity, natural transformation, escape from immune system defense mechanisms, and biofilm formation. The Xanthomonas citri subsp. citri strain 306 genome codes for a large set of genes involved in T4P biogenesis and regulation and includes several pilin homologs. We show that X. citri subsp. citri can exhibit twitching motility in a manner similar to that observed in other bacteria such as Pseudomonas aeruginosa and Xylella fastidiosa and that this motility is abolished in Xanthomonas citri subsp. citri knockout strains in the genes coding for the major pilin subunit PilAXAC3241, the ATPases PilBXAC3239 and PilTXAC2924, and the T4P biogenesis regulators PilZXAC1133 and FimXXAC2398. Microscopy analyses were performed to compare patterns of bacterial migration in the wild-type and knockout strains and we observed that the formation of mushroom-like structures in X. citri subsp. citri biofilm requires a functional T4P. Finally, infection of X. citri subsp. citri cells by the bacteriophage (ΦXacm4-11 is T4P dependent. The results of this study improve our understanding of how T4P influence Xanthomonas motility, biofilm formation, and susceptibility to phage infection.

Bacterial spot of tomato (BST) is a major constraint to tomato production in Ethiopia and many other countries leading to significant crop losses. In the present study, using pathogenicity tests, sensitivity to copper and streptomycin, and multilocus sequence analysis (MLSA), we identified a diverse group of Xanthomonas strains isolated from central Ethiopia. None of the strains were sensitive to copper or streptomycin. Multilocus sequence analysis was used to compare Ethiopian strains with representative Xanthomonas strains from a worldwide collection based on DNA sequences of six housekeeping genes (lacF, lepA, gyrB, fusA, gltA and gapA) and hrpB genes. Phylogenetic analysis of the concatenated sequences showed that X. gardneri, X. vesicatoria and X. perforans were associated with BST in Ethiopia, whereas Xanthomonas euvesicatoria was absent from the Ethiopian sample. There was no genetic diversity among the isolated strains belonging to X. gardneri and X. perforans. However, two X. vesicatoria haplotypes were identified indicating at least two different sources of introduction of X. vesicatoria to Ethiopia. All of the X. perforans strains were only pathogenic on tomato and were T3 strains with the exception of one identified as tomato race 4 (T4). The X. gardneri and X. vesicatoria strains were tomato race 2 (T2), but were variable in pepper race determinations indicating variation in effectors among strains.

Bacterial spot of tomatoes (BST) caused by Xanthomonas perforans, is a ubiquitous disease of tomato in field and transplant operations in Florida. For disease management, producers rely heavily on copper or copper-mancozeb sprays, which are often ineffective due to the high prevalence of copper-tolerant Xanthomonas strains. In this study, 19 different chemical agents, biological control agents, plant defense activators, and novel products were evaluated for their ability to manage bacterial spot on tomato seedlings and in the field. Efficacies of single rotations or tank mixes of several compounds were compared to a non-treated control and a copper standard on seedlings. Stand-alone applications of acibenzolar-S-methyl (ASM), copper octanoate, quinoxyfen, oxysilver nitrate, and pentasilver hexaoxoiodate significantly reduced disease compared to inoculated controls. Seedling programs that included either ASM or copper octanoate significantly reduced disease compared to the control. Oxytetracycline and streptomycin were ineffective at reducing disease severity under high disease pressure in seedling trials. Although there were significant differences in disease severity among treatments, there were no significant difference in the overall yield among treatments across field trials. Overall, ASM provided significant disease reduction as a stand-alone or in a program with other compounds in both the greenhouse and field. However, copper octanoate alone or as a tank mixture with other compounds was only effective for seedling production. The use of alternative compounds should improve tomato health, reduce the introduction of disease inoculum into fields, and reduce residual copper in the environment.

Bacterial spot is one of the most detrimental diseases of tomato and is especially severe in the southeast United States when weather conditions (high temperature, high humidity, and rain) become conducive for disease development. This new 8-page publication of the UF/IFAS Plant Pathology Department presents updated information about the causal pathogen and management of bacterial spot on tomato in Florida. Written by Amanda Strayer-Scherer, Ying-Yu Liao, Peter Abrahamian, Sujan Timilsina, Mathews Paret, Tim Momol, Jeff Jones, and Gary Vallad.https://edis.ifas.ufl.edu/pp353

Protection of crops from bacterial diseases presents a continuing challenge, mandating the development of novel agents and approaches. Photocatalysis is a process where chemically reactive oxygen species are catalytically generated by certain minerals in the presence of light. These reactive oxygen species have the capacity to destroy organic molecular structures critical to pathogen viability. In this study, the antibacterial potential of photocatalytic nanoscale titanium dioxide (TiO2), nanoscale TiO2 doped (incorporation of other materials into the structure of TiO2) with silver (TiO2/Ag), and nanoscale TiO2 doped with zinc (TiO2/Zn; AgriTitan) was evaluated against Xanthomonas perforans, the causal agent for bacterial spot disease of tomato. In vitro experiments on photocatalytic activity and dose dependency were conducted on glass cover slips coated with the nanoscale formulations by adding a known population of X. perforans strain Xp-F7 and illuminating the cover slips under a visible light source. TiO2/Ag and TiO2/Zn had high photocatalytic activity against X. perforans within 10 min of exposure to 3 × 104 lux. Greenhouse studies on naturally and artificially infected transplants treated with TiO2/Zn at ≈500 to 800 ppm significantly reduced bacterial spot severity compared with untreated and copper control. Protection was similar to the grower standard, copper + mancozeb. The use of TiO2/Zn at ≈500 to 800 ppm significantly reduced disease incidence in three of the four trials compared with untreated and copper control, and was comparable to or better than the grower standard. The treatments did not cause any adverse effects on tomato yield in any of the field trials.

Multiple species of Xanthomonas cause bacterial spot of tomato (BST) and pepper. We sequenced five Xanthomonas euvesicatoria strains isolated from three continents (Africa, Asia, and South America) to provide a set of representative genomes with temporal and geographic diversity. LMG strains 667, 905, 909, and 933 were pathogenic on tomato and pepper, except LMG 918 elicited a hypersensitive reaction (HR) on tomato. Furthermore, LMG 667, 909, and 918 elicited a HR on Early Cal Wonder 30R containing Bs3. We examined pectolytic activity and starch hydrolysis, two tests which are useful in differentiating X. euvesicatoria from X. perforans, both causal agents of BST. LMG strains 905, 909, 918, and 933 were nonpectolytic while only LMG 918 was amylolytic. These results suggest that LMG 918 is atypical of X. euvesicatoria. Sequence analysis of all the publicly available X. euvesicatoria and X. perforans strains comparing seven housekeeping genes identified seven haplotypes with few polymorphisms. Whole genome comparison by average nucleotide identity (ANI) resulted in values of >99% among the LMG strains 667, 905, 909, 918, and 933 and X. euvesicatoria strains and >99.6% among the LMG strains and a subset of X. perforans strains. These results suggest that X. euvesicatoria and X. perforans should be considered a single species. ANI values between strains of X. euvesicatoria, X. perforans, X. allii, X. alfalfa subsp. citrumelonis, X. dieffenbachiae, and a recently described pathogen of rose were >97.8% suggesting these pathogens should be a single species and recognized as X. euvesicatoria. Analysis of the newly sequenced X. euvesicatoria strains revealed interesting findings among the type 3 (T3) effectors, relatively ancient stepwise erosion of some T3 effectors, additional X. euvesicatoria-specific T3 effectors among the causal agents of BST, orthologs of avrBs3 and avrBs4, and T3 effectors shared among xanthomonads pathogenic against various hosts. The results from this study supports the finding that T3 effector repertoire and host range are fundamental for the study of host—microbe interaction but of little relevance to bacterial speciation.

Pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) is an important component of plant innate immunity. In a previous study, we showed that the PAMP flg22 from Xanthomonas citri ssp. citri (Xflg22), the causal agent of citrus canker, induced PTI in citrus, which correlated with the observed levels of canker resistance. Here, we identified and sequenced two bacterial flagellin/flg22 receptors (FLS2-1 and FLS2-2) from 'Duncan' grapefruit (Citrus paradisi, CpFLS2-1 and CpFLS2-2) and 'Sun Chu Sha' mandarin (C. reticulata, CrFLS2-1 and CrFLS2-2). We were able to isolate only one FLS2 from 'Nagami' kumquat (Fortunella margarita, FmFLS2-1) and gene flanking sequences suggest a rearrangement event that resulted in the deletion of FLS2-2 from the genome. Phylogenetic analysis, gene structure and presence of critical amino acid domains all indicate we identified the true FLS2 genes in citrus. FLS2-2 was more transcriptionally responsive to Xflg22 than FLS2-1, with induced expression levels higher in canker-resistant citrus than in susceptible ones. Interestingly, 'Nagami' kumquat showed the highest FLS2-1 steady-state expression levels, although it was not induced by Xflg22. We selected FmFLS2-1, CrFLS2-2 and CpFLS2-2 to further evaluate their capacity to enhance bacterial resistance using Agrobacterium-mediated transient expression assays. Both FmFLS2-1 and CrFLS2-2, the two proteins from canker-resistant species, conferred stronger Xflg22 responses and reduced canker symptoms in leaves of the susceptible grapefruit genotype. These two citrus genes will be useful resources to enhance PTI and achieve resistance against canker and possibly other bacterial pathogens in susceptible citrus types.

Bacterial pathogens have a multitude of effectors that target plant host cells and promote disease. A case is made for a new suspect, phytate, as a target of a novel phytase activity for a type III effector of Xanthomonas.

Fifty-six native isolates collected in 12 farming districts of Trinidad and seven reference strains of Xanthomonas campestris pv. campestris were evaluated for resistance to copper in buffered (pH 7.0) and unbuffered (pH 5.6) nutrient agar media. All isolates and reference strains were pathogenic and elicited typical black rot symptoms on a susceptible variety of Brassica olearceae, ‘Copenhagen Market’. Thirty-four and thirty-three native isolates were highly resistant to copper (growth on ≥ 200 ppm copper) in buffered and unbuffered media, respectively; however, all the reference strains were highly susceptible to copper. The mean minimum inhibition concentration for the 56 native isolates was 224.6 ppm copper indicating that high levels of copper resistance are present in X. campestris pv. campestris in Trinidad. The association between growth of the 56 isolates and seven reference stains on buffered and unbuffered media was strong (Pearson’s and Spearman’s r = 0.93; P

Transcription activator-like effector (TALE) proteins of the plant pathogenic bacterial genus Xanthomonas bind to and transcriptionally activate host susceptibility genes, promoting disease. Plant immune systems have taken advantage of this mechanism by evolving TALE binding sites upstream of resistance ( R ) genes. For example, the pepper Bs3 and rice Xa27 genes are hypersensitive reaction plant R genes that are transcriptionally activated by corresponding TALEs. Both R genes have a hallmark expression pattern in which their transcripts are detectable only in the presence and not the absence of the corresponding TALE. By transcriptome profiling using next-generation sequencing (RNA-seq), we tested whether we could avoid laborious positional cloning for the isolation of TALE-induced R genes. In a proof-of-principle experiment, RNA-seq was used to identify a candidate for Bs4C , an R gene from pepper that mediates recognition of the Xanthomonas TALE protein AvrBs4. We identified one major Bs4C candidate transcript by RNA-seq that was expressed exclusively in the presence of AvrBs4. Complementation studies confirmed that the candidate corresponds to the Bs4C gene and that an AvrBs4 binding site in the Bs4C promoter directs its transcriptional activation. Comparison of Bs4C with a nonfunctional allele that is unable to recognize AvrBs4 revealed a 2-bp polymorphism within the TALE binding site of the Bs4C promoter. Bs4C encodes a structurally unique R protein and Bs4C -like genes that are present in many solanaceous genomes seem to be as tightly regulated as pepper Bs4C . These findings demonstrate that TALE-specific R genes can be cloned from large-genome crops with a highly efficient RNA-seq approach.

The RXopJ4 resistance locus from the wild accession Solanum pennellii (Sp) LA716 confers resistance to bacterial spot disease of tomato (S. lycopersicum, Sl) caused by Xanthomonas perforans (Xp). RXopJ4 resistance depends on recognition of the pathogen type III effector protein XopJ4. We used a collection of Sp introgression lines (ILs) to narrow the RXopJ4 locus to a 4.2-Mb segment on the long arm of chromosome 6, encompassed by the ILs 6-2 and 6-2-2. We then adapted or developed a collection of 14 molecular markers to map on a segregating F(2) population from a cross between the susceptible parent Sl FL8000 and the resistant parent RXopJ4 8000 OC(7). In the F(2) population, a 190-kb segment between the markers J350 and J352 cosegregated with resistance. This fine mapping will enable both the identification of candidate genes and the detection of resistant plants using cosegregating markers. The RXopJ4 resistance gene(s), in combination with other recently characterized genes and a quantitative trait locus (QTL) for bacterial spot disease resistance, will likely be an effective tool for the development of durable resistance in cultivated tomato.

Bacteriophages were isolated from naturally infected citrus canker lesions from diverse locations in Florida and Argentina and characterized for host range using a world-wide collection of Xanthomonas citri subsp. citri (Xcc) strains. Sixty-seven bacteriophages isolated from citrus canker lesions in Florida (37 bacteriophages) and Argentina (30 bacteriophages) revealed little diversity. All 30 phages isolated from four locations in Argentina had identical host ranges (group ARG), while 37 phages from Florida made up two groups (FLA and FLB). ARG and the 31 FLA phages produced clear plaques and had nearly identical host ranges as phage CP2 of Japan in that they only reacted with typical A strains and none of the atypical A strains (A* and AW) or other Xanthomonas spp. FLB phages had a different host range from the other strains and produced turbid plaques. We used phage typing, fatty acid analysis and riboprinter analysis to classify citrus-associated xanthomonads. Phage typing using 12 phages isolated from Xcc, X. fuscans subsp. aurantifolii (Xfa), X. alfalfae subsp. citrumelonis (Xacm), and other sources proved useful for classifying all major Xcc pathotypes and/or strains (A, A*, Miami (MI), Manatee (MA) and Wellington (AW)), as well as B and C types of Xfa. X. citri subsp. citri strains from a worldwide collection were diverse in phage susceptibility. The majority of Xcc strains, which originated from different regions of the world and which were typical “A” pathotype strains based on pathogenicity characteristics, was sensitive to most phages (including CP2, FLA and ARG), and had nearly identical phage sensitivity profiles. MA strains were quite unique in that they reacted with none of the phages; furthermore, they were different from the putative progenitor MA strain, ATCC 49118, which reacted with a group of phages. Fatty acid analysis revealed considerable variation in Xcc-A, Xfa-B, Xfa-C and Xacm strains. Using riboprinter analysis, we identified a unique riboprinter pattern for strains isolated from an etrog tree (Citrus medica) in Florida that were “A” pathotype strains based on pathogenicity characteristics. Phage typing and fatty acid analysis were useful in corroborating that the etrog strains represent a unique new Xcc strain in Florida.

Genetic exchange is considered to be an important process in the selective adaptation of microorganisms to shifting and challenging environmental conditions. As a consequence of the copious use of copper bactericides, many species of plant pathogenic bacteria, including Xanthomonas citri subsp. citri (Xcc), have developed resistance to copper. This study assesses whether copper resistant (CuR) strains of other Xanthomonas species and citrus epiphytic bacteria pose a risk for the development of copper resistance in Xcc. CuR epiphytic bacteria were isolated on MGY agar from citrus leaves collected in two citrus groves treated with copper bactericides in Florida. Horizontal gene transfer of copper resistance genes was investigated within different Xanthomonas species and from citrus epiphytic bacteria to Xanthomonas. CuR epiphytic bacteria from citrus were screened for the presence of copper resistance genes homologous to copL, copA and copB genes from Xcc and characterized regarding tolerance to copper. Copper resistance determinants from a citrus epiphytic strain of Stenotrophomonas maltophilia (Stm) were cloned and expressed in Xcc and other Xanthomonas strains. Copper resistance genes in Xcc were determined to be present on a large (~300 kb) conjugative plasmid. Cu resistance was transferred via conjugation from two copper resistant citrus strains, Xcc and X. alfalfae subsp. citrumelonis (Xac), and two tomato pathogens, X. euvesicatoria (Xe) and X. perforans (Xp), to Xcc. PCR analysis revealed that two CuR strains from citrus, an epiphytic Xanthomonas ssp. and a strain of Stm, harboured homologs of the copper resistance genes found in CuR Xcc. The introduction of copLAB gene cluster from Stm into different xanthomonads conferred copper resistance to sensitive strains of Xcc, Xac, Xe and Xp. Based on these results there is a low, but significant, likelihood of horizontal gene transfer of copper resistance genes from other xanthomonads or epiphytic bacteria to Xcc in nature.

Acibenzolar-S-methyl (ASM), a plant activator known to induce systemic acquired resistance, has demonstrated an ability to manage a number of plant diseases, including bacterial spot on tomato caused by four distinct Xanthomonas spp. The aim of this study was to evaluate application rate and frequency of ASM in order to optimize field efficacy against bacterial spot in Florida, while minimizing its impact on marketable yields. ASM was applied biweekly (once every 2 weeks) as a foliar spray at a constant concentration of 12.9, 64.5, and 129 μM throughout four field experiments during 2007–08. A standard copper program and an untreated control were also included. Overall, biweekly applications of ASM did not significantly reduce disease development or the final disease severity of bacterial spot compared with the copper-mancozeb standard or the untreated control. Only one experiment showed a significant reduction in the final disease severity on plants treated with ASM at 129 μM compared with the untreated control. Three additional field trials conducted during 2009–10 to evaluate the effects of weekly and biweekly applications of ASM at concentrations of 30.3 to 200 μM found that weekly applications provided significantly better disease control than biweekly applications. The tomato yields were not statistically improved with the use of ASM relative to the untreated control and standard copper program. Weekly ASM applications at rates as low as 75 μM (equivalent to 1.58 g a.i./ha in 100 liters of water or 0.21 oz. a.i./acre in 100 gallons of water) to 200 μM (equivalent to 4.20 g a.i./ha in 100 liters of water or 0.56 oz. a.i./acre in 100 gallons of water) were statistically equivalent in managing bacterial spot of tomato without significantly reducing yield compared with the untreated control.

Cymoxanil and famoxadone, the components of Tanos 50DF® (25% a.i. of each product, E.I. du Pont de Nemours and Company, Wilmington, DE) were evaluated alone, together, and in combination with copper hydroxide and/or mancozeb for the control of bacterial spot on tomato and pepper, and bacterial leaf spot on lettuce. Neither Tanos nor its components alone had in vitro inhibitory activity against the copper-insensitive and copper-sensitive species of Xanthomonas evaluated. However, there was a synergistic interaction between copper hydroxide and Tanos in reducing the growth of a copper-resistant strain of Xanthomonas perforans. It appears that both components of Tanos are required for synergism since the addition of only cymoxanil or famoxadone to copper hydroxide did not induce significant reduction of bacterial populations in comparison to copper hydroxide alone. Increased disease control was not obtained on plants in greenhouse trials. In most of the greenhouse trials, the mixture of copper hydroxide and mancozeb combined with Tanos tended to induce similar level of disease control in comparison to different mixtures containing copper hydroxide, cymoxanil and famoxadone indicating that mancozeb might not be necessary in the management of copper-tolerant strains of Xanthomonads.

Forty Erwinia amylovora strains originating from different host plants and locations in Serbia and one strain from Montenegro were characterized by conventional, automated and molecular techniques. All strains were Gram-negative, nonfluorescent, facultative anaerobes, oxidase negative, levan positive, produced necrotic lesions followed by bacterial exudate on artificially inoculated immature pear fruits and caused HR on tobacco. Based on carbon source utilization, all strains tested with the Biolog system were identified as E. amylovora. Based on fatty acid profiles all tested strains clustered into three groups in which strains from north Serbia differed from strains isolated in central and south parts of the country. Restriction analysis of genomic DNA using XbaI and PFGE resulted in six different patterns differentiating the strains into six groups. Most of the investigated strains clustered in one group having the pattern type similar to Pt2 group described earlier as dominant in East Europe and the Mediterranean region. Two strains showed PFGE pattern similar to the previously described Pt3 pattern and one strain had pattern similar to Pt6. Based on size and number of the bands, new restriction patterns, assigned as Pt7, Pt8 and Pt9 were observed. PFGE results showed that the E. amylovora population in Serbia is not homogenous and was possibly introduced from different directions. This is the first characterization of E. amylovora collection of strains from Serbia using fatty acid analysis and PFGE.