Control of Citrus Canker and Citrus Bacterial Spot with Bacteriophages.

Bacteriophages, alone or in combination with copper bactericides, were evaluated for managing Asiatic citrus canker and citrus bacterial spot incited by Xanthomonas axonopodis pathovars citri and citrumelo, respectively. In a set of five greenhouse experiments, phage treatment provided consistent control of citrus canker, causing an average of 59% reduction in disease severity. However, treatment with phage was ineffective if applied with skim milk, a protective formulation, which increases phage residual activity. In nursery settings, phage treatment also reduced disease but was less effective than copper-mancozeb, a chemical bactericide. The integration of phage and copper-mancozeb resulted in equal or less control than copper-mancozeb application alone. Phage treatments were evaluated in a commercial citrus nursery for reducing citrus bacterial spot caused by natural inoculum. Phage treatment provided significant disease reduction on moderately sensitive Valencia oranges in two trials (48 and 35%); however, on the highly susceptible grapefruit host it was ineffective. In an experimental citrus nursery, phage treatment provided significant control of citrus bacterial spot caused by a phage-sensitive strain, but was equally or less effective than copper-mancozeb. The combination of phage and copper-mancozeb did not increase control compared with copper-mancozeb alone.

Functional analysis of the early chlorosis factor gene.

Chlorosis is one of the symptoms of bacterial spot disease caused by Xanthomonas campestris pv. vesicatoria, which induces chlorosis before any other symptoms appear on tomato. We report characterization of a 2.1-kb gene called early chlorosis factor (ecf). The gene ecf encodes a hydrophobic protein with similarity to four other proteins in plant pathogens, including HolPsyAE, and uncharacterized gene products from X. campestris pv. campestris and X. axonopodis pv. citri, and, at the tertiary structure level, to colicin Ia from Escherichia coli. We demonstrate that the associated phenotype is hrp dependent, and that the ecf gene product appears to be translocated to host cells. The gene ecf has no impact on electrolyte leakage or on bacterial growth in planta in response to infection. Concentrated culture filtrates do not produce chlorosis. Study of its role in Xanthomonas spp.-tomato interactions will forward our understanding of symptom production by plant pathogens and allows further investigation into the mechanisms of bacterial virulence and production of symptoms.

Importance of opgHXcv of Xanthomonas campestris pv. vesicatoria in host-parasite interactions.

Tn5 insertion mutants of Xanthomonas campestris pv. vesicatoria were inoculated into tomato and screened for reduced virulence. One mutant exhibited reduced aggressiveness and attenuated growth in planta. Southern blot analyses indicated that the mutant carried a single Tn5 insertion not associated with previously cloned pathogenicity-related genes of X. campestris pv. vesicatoria. The wild-type phenotype of this mutant was restored by one recombinant plasmid (pOPG361) selected from a genomic library of X. campestris pv. vesicatoria 91-118. Tn3-gus insertion mutagenesis and sequence analyses of a subclone of pOPG361 identified a 1,929-bp open reading frame (ORF) essential for complementation of the mutants. The predicted protein encoded by this ORF was highly homologous to the previously reported pathogenicity-related HrpM protein of Pseudomonas syringae pv. syringae and OpgH of Erwinia chrysanthemi. Based on homology, the new locus was designated opgHXcv. Manipulation of the osmotic potential in the intercellular spaces of tomato leaves by addition of mannitol at low concentrations (25 to 50 mM) compensates for the opgHXcv mutation.

Bacteriocin-Like Substances from Tomato Race 3 Strains of Xanthomonas campestris pv. vesicatoria.

ABSTRACT Tomato race 3 (T3) strains of Xanthomonas campestris pv. vesicatoria are antagonistic in vitro to tomato race 1 (T1) strains of the bacterium. All T1 strains and 11 strains of other X. campestris pathovars tested were inhibited by T3 strains. Sensitivity of tomato race 2 (T2) strains was variable. No strains from other bacterial genera tested were inhibited. Cell-free filtrates from T3 strains were inhibitory to sensitive strains. The inhibitory activity of these filtrates was lost after treatment at temperatures above 80 degrees C and with selected protease enzymes. However, treatment with trypsin or DNase had no effect on their activity. Seven cosmid clones from a genomic library of a T3 strain were selected for their ability to consistently inhibit a sensitive indicator strain in plate assays. Southern hybridization analysis placed these into three bacteriocin (BCN)-producing groups designated BCN-A, BCN-B, and BCN-C. The BCN-like groups could be differentiated by variations in inhibitory spectra and levels of activity in plate assays. Mutations that inactivated expression of each BCN group individually in a wild-type T3 strain had inhibitory activity confirming that multiple BCNs are present in the T3 strain. T3 strains were inhibitory to a sensitive indicator strain in tomato leaf tissue, but this effect was observed only when T3 strains were applied in advance of the sensitive strain. BCN-A was the major BCN-like substance involved in the suppression of the sensitive indicator strain in tomato leaf tissues.

A non-hypersensitive resistance in pepper to the bacterial spot pathogen is associated with two recessive genes.

ABSTRACT The pepper genotype, ECW-12346, was developed with bacterial spot resistance derived from Pep13, PI 271322, and ECW123 (Early Calwonder containing Bs1, Bs2, and Bs3 genes). For genetic analysis of this resistance, ECW12346, ECW123, F(1), F(2), and backcrosses were inoculated with a pepper race 6 (P6) strain. Two recessive genes were identified that determined resistance. The genes are designated bs5 and bs6 for the resistance derived from PI 271322 and Pep13, respectively. In greenhouse and field studies, ECW12346 was highly resistant, whereas ECW123 had significant defoliation. In growth-room studies, electrolyte leakage and population dynamics were determined. Following infiltration of both genotypes with 10(8) CFU/ml of a P6 strain, there was no rapid increase in electrolyte leakage within 72 h, whereas a rapid increase in electrolyte leakage occurred within 24 h when a similar concentration of a P3 strain (containing the avrBs2 gene) was infiltrated into the intercellular spaces of the leaf. When 10(5) CFU/ml of a P6 strain was infiltrated into leaves, complete tissue collapse was evident in ECW123 10 days later as determined by visual assessment and electrolyte leakage data, but no confluent necrosis was detected in ECW12346. Internal populations were at least two logarithmic units higher in ECW123 than in ECW12346. Therefore, ECW12346 inhibits population build-up without inducing the typical hypersensitive reaction characterized by an increase in electrolyte leakage.

Phenotypic and genotypic characterization of human and nonhuman Escherichia coli.

Estuarine waters receive fecal pollution from a variety of sources, including humans and wildlife. Escherichia coli is one of several fecal coliform bacteria that inhabit the intestines of many warm-blooded animals that sometimes contaminate water. Its presence does not specifically implicate human fecal input, therefore it is necessary to differentiate contamination sources to accurately assess health risks. E. coli were isolated from human sources (HS) and nonhuman sources (NHS) in the Apalachicola National Estuarine Research Reserve and analyzed for fatty acid methyl ester (FAME), O-serogroup, and pulsed-field gel electrophoresis (PFGE) profiles. For FAME and PFGE analyses, there was no relationship between profile and isolate source. Human source PFGE profiles were less diverse than NHS isolates, and conversely for FAME. In contrast, O-serogrouping showed less diversity for HS vs. NHS isolates, and the predominant HS O-serogroups differed significantly (P < 0.01) from those of NHS isolates.

Xv4-vrxv4: a new gene-for-gene interaction identified between Xanthomonas campestris pv. vesicatoria race T3 and wild tomato relative Lycopersicon pennellii.

Strains of tomato race 3 (T3) of Xanthomonas campestris pv. vesicatoria elicit a hypersensitive response (HR) in leaves of Lycopersicon pennellii LA716. Genetic segregation of the resistance exhibited ratios near 3:1 in F2 populations, which confirmed that a single dominant gene controlled the inheritance of this trait. With the aid of a collection of introgression lines, restriction fragment length polymorphism, and cleaved amplified polymorphic sequence markers, the resistance locus was located on chromosome 3 between TG599 and TG134. An avirulence gene named avrXv4 was also isolated by mobilizing a total of 600 clones from a genomic DNA library of the T3 strain 91-118 into the X. campestris pv. vesicatoria strain ME90, virulent on L. pennellii. One cosmid clone, pXcvT3-60 (29-kb insert), induced HR in resistant plants. The avirulent phenotype of pXcvT3-60 was confirmed by comparing growth rates in planta and electrolyte leakages among transconjugants carrying a mutated or intact clone with the wild-type T3 strain 91-118. A 1.9-kb DNA fragment contained within a 6.8-kb active subclone was sequenced and was determined to carry an open reading frame of 1,077 bp. The predicted AvrXv4 protein exhibits high similarity to members of an emerging new family of bacterial proteins from plant and mammalian pathogens comprising AvrRxv, AvrBsT, YopJ, YopP, AvrA, and YL40.

Resistance of tomato and pepper to T3 strains of Xanthomonas campestris pv. vesicatoria is specified by a plant-inducible avirulence gene.

Tomato race 3 (T3) of Xanthomonas campestris pv. vesicatoria (Xcv) elicits a hypersensitive response (HR) in leaves of Lycopersicon esculentum near-isogenic line (NIL) 216 and pepper genotypes. One cosmid clone (35 kb) selected from a genomic library of a T3 strain induced an HR in all resistant plants. A 1.5-kb active subclone containing the putative avirulence (avr) gene, designated avrXv3, was sequenced. The avrXv3 gene encodes a 654-bp open reading frame (ORF) with no homology to any known gene. Expression studies with a fusion of this gene and uidA indicated that avrXv3 is plant inducible and controlled by the hypersensitivity and pathogenicity (hrp) regulatory system. Mutational analysis and transcription activation assays revealed that AvrXv3 has transcription activation activity in yeast, and that the putative domain responsible for that activity is located at the C terminus of the AvrXv3 protein. Agrobacterium tumefaciens-mediated transient expression confirmed the direct role of AvrXv3 in eliciting the HR in tomato NIL 216 and supported the hypothesis that Avr proteins must be present inside the plant host cell to trigger the HR.

Systematic analysis of xanthomonads (Xanthomonas spp.) associated with pepper and tomato lesions.

The taxonomy and evolutionary relationships among members of the genus Xanthomonas associated with tomato and pepper have been a matter of considerable controversy since their original description in 1921. These bacteria, which are a major affliction of tomato and pepper crops in warm and humid regions, were originally described as a single species, but subsequent research has shown the existence of at least two genetic groups differentiated by physiological, biochemical and pathological characteristics. This work synthesizes the findings from several approaches, including pathogenicity tests, enzymic activity, restriction fragment analysis of the entire genome, DNA-DNA hybridization and RNA sequence comparisons based on a 2097 base sequence comprising the 16S rRNA gene, the intergenic spacer located between the 16S and 23S rRNA genes and a small region of the 23S rRNA gene. Within the group of xanthomonads pathogenic on pepper and tomato four distinct phenotypic groups exist, of which three form distinct genomic species. These include Xanthomonas axonopodis pv. vesicatoria (A and C group), Xanthomonas vesicatoria (B group) and Xanthomonas gardneri (D group). On the basis of phenotypic and genotypic differences between A- and C-group strains, the C strains should be considered as a subspecies within Xanthomonas axonopodis pv. vesicatoria.

Response to Xanthomonas campestris pv. vesicatoria in tomato involves regulation of ethylene receptor gene expression.

Although ethylene regulates a wide range of defense-related genes, its role in plant defense varies greatly among different plant-microbe interactions. We compared ethylene's role in plant response to virulent and avirulent strains of Xanthomonas campestris pv. vesicatoria in tomato (Lycopersicon esculentum Mill.). The ethylene-insensitive Never ripe (Nr) mutant displays increased tolerance to the virulent strain, while maintaining resistance to the avirulent strain. Expression of the ethylene receptor genes NR and LeETR4 was induced by infection with both virulent and avirulent strains; however, the induction of LeETR4 expression by the avirulent strain was blocked in the Nr mutant. To determine whether ethylene receptor levels affect symptom development, transgenic plants overexpressing a wild-type NR cDNA were infected with virulent X. campestris pv. vesicatoria. Like the Nr mutant, the NR overexpressors displayed greatly reduced necrosis in response to this pathogen. NR overexpression also reduced ethylene sensitivity in seedlings and mature plants, indicating that, like LeETR4, this receptor is a negative regulator of ethylene response. Therefore, pathogen-induced increases in ethylene receptors may limit the spread of necrosis by reducing ethylene sensitivity.

Expression of the Bs2 pepper gene confers resistance to bacterial spot disease in tomato.

The Bs2 resistance gene of pepper specifically recognizes and confers resistance to strains of Xanthomonas campestris pv. vesicatoria that contain the corresponding bacterial avirulence gene, avrBs2. The involvement of avrBs2 in pathogen fitness and its prevalence in many X. campestris pathovars suggests that the Bs2 gene may be durable in the field and provide resistance when introduced into other plant species. Employing a positional cloning strategy, the Bs2 locus was isolated and the gene was identified by coexpression with avrBs2 in an Agrobacterium-mediated transient assay. A single candidate gene, predicted to encode motifs characteristic of the nucleotide binding site-leucine-rich repeat class of resistance genes, was identified. This gene specifically controlled the hypersensitive response when transiently expressed in susceptible pepper and tomato lines and in a nonhost species, Nicotiana benthamiana, and was designated as Bs2. Functional expression of Bs2 in stable transgenic tomatoes supports its use as a source of resistance in other Solanaceous plant species.

Regulation of expression of avirulence gene avrRxv and identification of a family of host interaction factors by sequence analysis of avrBsT.

Resistance in tomato line Hawaii 7998 as well as in several nonhost plants to Xanthomonas campestris pv. vesicatoria tomato strain (XcvT) is mediated in part by the avirulence gene avrRxv. Analysis of growth of wild-type and avrRxv deletion strains indicates that avrRxv plays a crucial role in the ability of XcvT 92-14 to induce resistance on Hawaii 7998. We used avrRxv reporter gene fusions and Northern (RNA) blot analysis to test several growth environments for inductive potential. We found that avrRxv is constitutively expressed at high levels and that growth in planta, in tobacco conditioned medium, and in hrp-inductive medium XVM2 did not affect the high levels of expression. In addition, hrp structural and regulatory mutant backgrounds had no effect. We mutated the bipartite plant inducible promoter (PIP)-box sequence and found that avrRxv activity appears to be independent of an intact PIP-box element. We present the sequence of the avrRxv homologue called avrBsT and align the six AvrRxv host interaction factor family members including mammalian pathogen virulence factors YopJ and YopP from Yersinia spp. and AvrA from Salmonella typhimurium, and open reading frame Y4LO with unknown function from the symbiont Rhizobium sp.

Chromosomal Gene Transfer by Conjugation in the Plant Pathogen Xanthomonas axonopodis pv. vesicatoria.

ABSTRACT Genes for copper resistance, located on the chromosome of strain XvP26 of Xanthomonas axonopodis pv. vesicatoria, were transferred by conjugation to a recipient strain of the bacterium. The chromosomal gene transfer was verified by analyses of the genomes of donor, recipient, and putative transconjugants for plasmid profiles, by polymorphism of DNA bands obtained by digesting total genomic DNA by a rare-cutting endonuclease and pulsed-field gel electrophoresis, and by Southern hybridization with a probe containing the copper genes. Transfer of kanamycin resistance to a recipient strain, associated with Tn5 insertion into the chromosome of another strain of the bacterial spot pathogen, was also verified. The frequency of kanamycin resistance transfer to recipient was more than 75 times greater in pepper leaves than in vitro. The transfer of chromosomal sequences containing the hypersensitive reaction and pathogenicity (hrp) genes and pigmentation (pig) genes was linked with transfer of kanamycin resistance (Tn5). Horizontal transfer in planta of the chromosomal genes (i.e., cop, pig, hrp, and Tn5 sequences) among strains of X. axonopodis pv. vesicatoria means that horizontal chromosomal gene transfer is possible in nature. This type of gene transfer may explain the presence of great diversity among strains of the bacterial spot pathogen in terms of DNA polymorphism and may also explain the apparent horizontal transfer of hrp sequences among pathovars of Xanthomonas.

Heterogeneity of Xanthomonas campestris pv. hederae Strains from Araliaceous Hosts.

ABSTRACT Xanthomonas campestris pv. hederae (synonym X. hortorum pv. hederae) strains (59 total) were collected from plants in the araliaceae family. Strains were isolated from Hedera helix, Schefflera arboricola, Brassaia actinophylla, and Polyscias spp. from Florida, California, Hawaii, and New Zealand. All strains produced yellow mucoid growth; hydrolyzed esculin, starch, casein and gelatin; were pectolytic; produced urease; and grew on minimal media containing asparagine. All bacterial strains were pathogenic on H. helix (English ivy), B. actinophylla (dwarf schefflera), and Polyscias fruticosa (ming aralia). No differences in symptomatology were detected among strains; however, severity of symptoms usually was greatest on the host of origin. In planta growth rates of representative strains isolated from H. helix, B. actinophylla, and Polyscias spp. also were compared among these three hosts. In all cases, populations grew more rapidly when strains were inoculated to their original host species. All 59 bacterial strains were compared by 95-carbon source GN microplate, fatty acid methyl ester (FAME), and restriction fragment-length polymorphisms (RFLP), with the pulse-field gel electrophoresis method, analyses. All three analyses grouped strains into two distinct groups that correlated with the host of origin. Using metabolic profiles, 75% of the H. helix strains were separated from strains isolated from Brassaia and Schefflera and 95% of the Polyscias strains. FAME analysis separated strains into two distinct groups, with 96% of the H. helix strains placed in one group. RFLP analysis placed all of the H. helix and Schefflera strains in one group, as well as 33% of the Brassaia strains, whereas the other group contained all of the Polyscias strains and the remainder of the Brassaia strains. It is apparent that the pathovar hederae is made up of heterogeneous populations that can be separated by biochemical, pathological, genetic, and physiological analyses into two groups that are closely associated with the host of origin.

Multiphasic analysis of xanthomonads causing bacterial spot disease on tomato and pepper in the Caribbean and central america: evidence for common lineages within and between countries.

ABSTRACT Four hundred thirty-three xanthomonad strains isolated from tomato or pepper plants from 32 different fields in four Caribbean and Central American countries were screened for the ability to hydrolyze starch and sodium polypectate and for resistance to copper and streptomycin. Of these, 95 representative strains were further characterized by various phnetic tests, and 63 of these strains were then analyzed by genomic fingerprinting. Most of the strains (>90%) were tolerant to copper. However, there was much more variability in sensitivity to streptomycin. All strains in Guadeloupe and 93% of the strains in Barbados were sensitive to streptomycin. The majority of strains were typical Xanthomonas campestris pv. vesicatoria group A strains. In Barbados, however, a unique group of strains was identified that was serologically similar to group A strains but was amylolytic. These strains were designated A1. The occurrence of X. campestris pv. vesicatoria group B strains in Central America was found to be limited to two fields in Costa Rica and one in Guatemala. No group B strains were identified in the Caribbean, in contrast to common occurrence in the central United States and in South America. T3 strains were not found in this study, despite the recent increase of such strains in Florida and Mexico. Unique strains from Costa Rica belonging to the X. gardneri group were identified. Little linkage was found among phenotypic and rep-polymerase chain reaction (rep-PCR) genomic fingerprinting profiles of the pathogens except at the species/pathovar level; strains displaying virtually identical fingerprint profiles were found to correspond to distinct races and vice versa. The rep-PCR genomic fingerprinting analyses suggest that certain lineages may have evolved or predominated in specific regions or specific countries.

A Serious Outbreak of Race 6 of Xanthomonas campestris pv. vesicatoria on Pepper in Southern Florida.

In the 1997-1998 winter vegetable season, a widespread and serious outbreak of bacterial spot occurred on pepper in commercial fields throughout southern Florida. This was the first serious epidemic of bacterial spot on pepper in southern Florida since the 1993-1994 season. Cultivars affected included those with resistance to races 1-3 of X. campestris pv. vesicatoria, commonly found in previous Florida surveys (1). Field sampling designs, isolation methods, and pathogen identification were carried out as previously described (1). Twenty-seven fields were sampled in Palm Beach, St. Lucie, Martin, Broward, and Collier counties. The race of each strain was determined by infiltration of 3.0 × 108 CFU per ml of sterile tap water suspension into Early Calwonder and three near-isogenic lines of Early Calwonder with genes for vertical resistance to specific races of the pathogen. Test plants were maintained in the greenhouse and observed for hypersensitive and disease reactions over several days. Of a total of 244 X. campestris pv. vesicatoria strains collected, 73.4% were identified as race 6 and 10.2% as race 4. Irrespective of the presence of resistance genes in cultivars sampled, race 6 (eastern Florida) and race 4 (southwestern Florida) predominated in this study. Copper tolerance was widespread among X. campestris pv. vesicatoria strains of all races, with 91% of the strains showing tolerance to 250 g per ml of CuSO4 · 5H2O incorporated into 0.5% (wt/vol) glucose-nutrient agar. Selection pressure resulting from widespread planting of race 1, 2, 3-resistant cultivars may have contributed to this outbreak. High disease pressure also may be related to the unusually wet winter. Rainfall accumulation from December 1997 through March 1998 was 25.6 cm above normal. Reference: (1) K. Pohronezny et al. Plant Dis. 76:118, 1992.

Ethylene regulates the susceptible response to pathogen infection in tomato.

Ethylene evolution occurs concomitantly with the progression of disease symptoms in response to many virulent pathogen infections in plants. A tomato mutant impaired in ethylene perception-Never ripe-exhibited a significant reduction in disease symptoms in comparison to the wild type after inoculations of both genotypes with virulent bacterial (Xanthomonas campestris pv vesicatoria and Pseudomonas syringae pv tomato) and fungal (Fusarium oxysporum f sp lycopersici) pathogens. Bacterial spot disease symptoms were also reduced in tomato genotypes impaired in ethylene synthesis (1-aminocyclopropane-1-carboxylic acid deaminase) and perception (14893), thereby corroborating a reducing effect for ethylene insensitivity on foliar disease development. The reduction in foliar disease symptoms in Never ripe plants was a specific effect of ethylene insensitivity and was not due to reductions in bacterial populations or decreased ethylene synthesis. PR-1B1 mRNA accumulation in response to X. c. vesicatoria infection was not affected by ethylene insensitivity, indicating that ethylene is not required for defense gene induction. Our findings suggest that broad tolerance of diverse vegetative diseases may be achieved via engineering of ethylene insensitivity in tomato.

Evidence for the Preemptive Nature of Tomato Race 3 of Xanthomonas campestris pv. vesicatoria in Florida.

ABSTRACT Until recently, tomato race 1 (T1) of Xanthomonas campestris pv. vesicatoria was the only race causing bacterial spot of tomato in Florida. In 1991, tomato race 3 (T3) was first identified in 3 of 13 tomato production fields surveyed. By 1994, T3 was observed in 21 of 28 fields and was the only race identified in 14 fields. In field studies, tomato genotypes with resistance to either T1 or T3 or susceptibility to both were co-inoculated with strains of both races. Lesions on 10 plants in each of three replications for each genotype were sampled three times during the experiment; bacterial isolations were made from each lesion, and tomato race identifications were made for each strain. At the third sampling date, T3 was isolated from 97% of the lesions on the susceptible genotype Walter and the T1-resistant genotype Hawaii 7998, while T3 was isolated from 23% of the lesions and T1 from the remaining 77% on the T3-resistant genotypes PI 128216 and PI 126932. In surface population studies done in growth rooms, suspensions of T1 and T3 were applied alone and in combination to the leaf surfaces of susceptible and resistant genotypes. T1 populations were reduced more than 10-fold when applied in combination with T3, compared with populations that developed when T1 was applied alone. T3 populations were not affected when applied in combination with a T1 strain. In greenhouse studies with the T3-resistant genotype Hawaii 7981, disease was significantly reduced in plants inoculated with T3 in combination with T1, compared with plants inoculated with T1 alone. These results clearly demonstrate the competitive nature of T3 in the presence of T1 and help explain the emergence of T3 as a prevalent race in Florida.

Diversity among xanthomonads pathogenic on pepper and tomato.

Xanthomonas campestris pv. vesicatoria, causal agent of bacterial spot of tomato and pepper, had been considered for nearly 70 years to be a relatively homogeneous organism. However, in the past decade this bacterium was determined to be composed of two genetically and phenotypically distinct groups. The two groups, designated A and B, were distinguished based on amylolytic activity, expression of unique protein bands, reaction on differential hosts (tomato races T1 and T2), reaction patterns with monoclonal antibodies, DNA restriction profiles, and DNA:DNA hybridization. The A and B groups were placed into X. axonopodis pv. vesicatoria and X. vesicatoria, respectively. A third group, designated C, was pathogenically (race T3) and serologically distinct from A and B strains, and formed unique DNA restriction profiles. DNA:DNA hybridization data suggest that C is distinct but related to A strains and may represent a subspecies of A. A final group, designated D, consisted of X. gardneri, an organism identified in Yugoslavia in 1957, and also found in Costa Rica. Group D was determined to be genetically distinct from strains within the other two groups; it represents a third Xanthomonas species pathogenic on tomato and pepper.