Biological control of bacterial speck of tomato under field conditions at several locations in north america.

ABSTRACT Bacterial speck of tomato, caused by Pseudomonas syringae pv. tomato, continues to be a problem for tomato growers worldwide. A collection of nonpathogenic bacteria from tomato leaves plus P. syringae strains TLP2 and Cit7, P. fluorescens strain A506, and P. syringae pv. tomato DC3000 hrp mutants were examined in a greenhouse bioassay for the ability to reduce foliar bacterial speck disease severity. While several of these strains significantly reduced disease severity, P. syringae Cit7 was the most effective, providing a mean level of disease reduction of 78% under greenhouse conditions. The P. syringae pv. tomato DC3000 hrpA, hrpH, and hrpS mutants also significantly reduced speck severity under greenhouse conditions. The strains with the greatest efficacy under greenhouse conditions were tested for the ability to reduce bacterial speck under field conditions at locations in Alabama, Florida, and Ontario, Canada. P. syringae Cit7 was the most effective strain, providing a mean level of disease reduction of 28% over 10 different field experiments. P. fluorescens A506, which is commercially available as Blight-Ban A506, provided a mean level of disease reduction of 18% over nine different field experiments. While neither P. syringae Cit7 nor P. fluorescens A506 can be integrated with copper bactericides due to their copper sensitivity, there exist some potential for integrating these biological control agents with "plant activators", including Actigard. Of the P. syringae pv. tomato DC3000 hrp mutants tested, only the hrpS mutant reduced speck severity significantly under field conditions.

Field Control of Bacterial Spot and Bacterial Speck of Tomato Using a Plant Activator.

Acibenzolar-S-methyl (CGA 245704 or Actigard 50WG) is a plant activator that induces systemic acquired resistance (SAR) in many different crops to a number of pathogens. Acibenzolar-S-methyl was evaluated for management of bacterial spot (Xanthomonas axonopodis pv. vesicatoria) and bacterial speck (Pseudomonas syringae pv. tomato) of tomato in 15 and 7 field experiments, respectively. Experiments were conducted over a 4-year period in Florida, Alabama, North Carolina, Ohio, and Ontario using local production systems. Applied at 35 g a.i. ha-1, acibenzolar-S-methyl reduced foliar disease severity in 14 of the 15 bacterial spot and all 7 bacterial speck experiments. Disease control was similar or superior to that obtained using a standard copper bactericide program. Acibenzolar-S-methyl also reduced bacterial fruit spot and speck incidence. Tomato yield was not affected by using the plant activator in the field when complemented with fungicides to manage foliar fungal diseases, but tomato transplant dry weight was negatively impacted. X. axonopodis pv. vesicatoria population densities on greenhouse-grown tomato transplants were reduced by acibenzolar-S-methyl treatment. Bacterial speck and spot population densities on leaves of field-grown plants were not dramatically affected. Acibenzolar-S-methyl can be integrated as a viable alternative to copper-based bactericides for field management of bacterial spot and speck, particularly where copper-resistant populations predominate.

Characterization of S-triazine herbicide metabolism by a Nocardioides sp. isolated from agricultural soils.

Atrazine, a herbicide widely used in corn production, is a frequently detected groundwater contaminant. Nine gram-positive bacterial strains able to use this herbicide as a sole source of nitrogen were isolated from four farms in central Canada. The strains were divided into two groups based on repetitive extragenic palindromic (rep)-PCR genomic fingerprinting with ERIC and BOXA1R primers. Based on 16S ribosomal DNA sequence analysis, both groups were identified as Nocardioides sp. strains. None of the isolates mineralized [ring-U-(14)C]atrazine. There was no hybridization to genomic DNA from these strains using atzABC cloned from Pseudomonas sp. strain ADP or trzA cloned from Rhodococcus corallinus. S-Triazine degradation was studied in detail in Nocardioides sp. strain C190. Oxygen was not required for atrazine degradation by whole cells or cell extracts. Based on high-pressure liquid chromatography and mass spectrometric analyses of products formed from atrazine in incubations of whole cells with H(2)(18)O, sequential hydrolytic reactions converted atrazine to hydroxyatrazine and then to the end product N-ethylammelide. Isopropylamine, the putative product of the second hydrolytic reaction, supported growth as the sole carbon and nitrogen source. The triazine hydrolase from strain C190 was isolated and purified and found to have a K(m) for atrazine of 25 microM and a V(max) of 31 micromol/min/mg of protein. The subunit molecular mass of the protein was 52 kDa. Atrazine hydrolysis was not inhibited by 500 microM EDTA but was inhibited by 100 microM Mg, Cu, Co, or Zn. Whole cells and purified triazine hydrolase converted a range of chlorine or methylthio-substituted herbicides to the corresponding hydroxy derivatives. In summary, an atrazine-metabolizing Nocardioides sp. widely distributed in agricultural soils degrades a range of s-triazine herbicides by means of a novel s-triazine hydrolase.

Characterization of an atrazine-degrading Pseudaminobacter sp. isolated from Canadian and French agricultural soils.

Atrazine, a herbicide widely used in corn production, is a frequently detected groundwater contaminant. Fourteen bacterial strains able to use this herbicide as a sole source of nitrogen were isolated from soils obtained from two farms in Canada and two farms in France. These strains were indistinguishable from each other based on repetitive extragenic palindromic PCR genomic fingerprinting performed with primers ERIC1R, ERIC2, and BOXA1R. Based on 16S rRNA sequence analysis of one representative isolate, strain C147, the isolates belong to the genus Pseudaminobacter in the family Rhizobiaceae. Strain C147 did not form nodules on the legumes alfalfa (Medicago sativa L.), birdsfoot trefoil (Lotus corniculatus L.), red clover (Trifolium pratense L.), chickpea (Cicer arietinum L.), and soybean (Glycine max L.). A number of chloro-substituted s-triazine herbicides were degraded, but methylthio-substituted s-triazine herbicides were not degraded. Based on metabolite identification data, the fact that oxygen was not required, and hybridization of genomic DNA to the atzABC genes, atrazine degradation occurred via a series of hydrolytic reactions initiated by dechlorination and followed by dealkylation. Most strains could mineralize [ring-U-(14)C]atrazine, and those that could not mineralize atrazine lacked atzB or atzBC. The atzABC genes, which were plasmid borne in every atrazine-degrading isolate examined, were unstable and were not always clustered together on the same plasmid. Loss of atzB was accompanied by loss of a copy of IS1071. Our results indicate that an atrazine-degrading Pseudaminobacter sp. with remarkably little diversity is widely distributed in agricultural soils and that genes of the atrazine degradation pathway carried by independent isolates of this organism are not clustered, can be independently lost, and may be associated with a catabolic transposon. We propose that the widespread distribution of the atrazine-degrading Pseudaminobacter sp. in agricultural soils exposed to atrazine is due to the characteristic ability of this organism to utilize alkylamines, and therefore atrazine, as sole sources of carbon when the atzABC genes are acquired.

Disease Development and Changes in the Natural Pseudomonas syringae pv. tomato Populations on Field Tomato Plants.

The probe TPRI, derived from the Pseudomonas syringae pv. tomato gene cluster controlling production of the phytotoxin coronatine, was used in conjunction with the semiselective medium VBTar to trace natural populations of the pathogen on tomato plants from just before planting to harvest. In a survey of transplant seedlings in greenhouses, P. syringae pv. tomato populations ranged from 8 × 100 to 3.2 × 105 CFU/g of leaf tissue. Copper-sprayed seedlings had similar populations to nonsprayed plants, but copper tolerance was common among the P. syringae pv. tomato strains surveyed. Transplant seedlings from three greenhouses were tagged, randomly planted in three grower fields, and monitored for P. syringae pv. tomato and disease severity over two growing seasons. Statistical analysis indicated that, when the P. syringae pv. tomato populations of greenhouse plants were small, as recorded in this study, there was no correlation between greenhouse infestation and disease severity in the field. Environmental conditions played a greater role than greenhouse infestation in disease development. Once formed, leaf lesions remained a good inoculum source (104 to 105 CFU) throughout the 7-week life of the leaf. Bacterial speck damage correlated well in both years (r = 0.80 and r = 0.86, respectively) with P. syringae pv. tomato population levels.

Identification of Tomato Leaf Factors that Activate Toxin Gene Expression in Pseudomonas syringae pv. tomato DC3000.

ABSTRACT Coronatine is a non-host-specific chlorosis-inducing phytotoxin produced by the tomato and crucifer pathogen Pseudomonas syringae pv. tomato DC3000. How the chromosomal gene cluster controlling toxin synthesis in this strain is regulated in planta is unknown. Ice nucleation-active cor:inaZ marker-exchange derivatives of strain DC3000 were used to determine coronatine gene expression in various host and nonhost plants and in a minimal medium supplemented with selected tomato plant constituents. Ice nucleation activity, which was first detected 4 h after inoculation, was highest in cabbage, tomato, and soybean and lowest in melon and cucumber. No correlation existed between bacterial population size and expression level on the various plants. Crude tomato leaf extract and intercellular fluid were strong inducers of toxin synthesis. Based on high-performance liquid chromatography analyses and bioassays, we concluded that the active components of both preparations were malic and citric acids, with minor contributions coming from shikimic and quinic acid. Although several compounds including glucose and inositol activated the toxin genes when tested at high concentrations (3 to 5 mM), shikimic and quinic acids were the only ones with activity at concentrations below 0.1 mM. Neither acid could be used as a sole carbon source by strain DC3000. The signal activity of shikimic acid was enhanced 10-fold by the addition of glucose. None of the plant phenolics that we screened affected coronatine gene expression.

Development of a diagnostic DNA probe for xanthomonads causing bacterial spot of peppers and tomatoes.

Xanthomonas vesicatoria and Xanthomonas axonopodis pv. vesicatoria, causal agents for bacterial spot of tomatoes and peppers, are difficult to distinguish from other xanthomonads found on field-grown plants. A genomic subtraction technique with subtracter DNA from nonpathogenic epiphytic xanthomonads was used to enrich for sequences that could serve as diagnostic probes for these pathogens. A 1.75-kb PstI-NotI fragment (KK1750) that preferentially hybridized to X. vesicatoria DNA and X. axonopodis pv. vesicatoria DNA was identified and cloned into pBluescriptII KS+. It hybridized to 46 (89%) of the 52 geographically diverse bacterial spot-causing xanthomonad (bsx) strains included in this study. The six probe-negative strains were genotypically and pathologically distinct from the other bsx strains studied. Two of these strains, DC91-1 and DC91-2, resembled X. campestris pv. raphani in that they also infected radish plants. X. vesicatoria strains gave stronger hybridization signals than did most X. axonopodis pv. vesicatoria strains. In a survey of 110 non-bsx plant-associated bacteria, including 44 nonvesicatoria phytopathogenic xanthomonads and 43 epiphytic xanthomonad strains, only 8 were probe positive, but the responses were weak. Further testing revealed that one of these strains was actually a tomato pathogen. Pulsed-field gel electrophoresis and Southern blot analysis of 46 bsx strains indicated that KK1750 sequences could be either plasmid-borne (10.9%), chromosome-borne (43.4%), or present on both replicons (45.7%). KK1750, unique in its ability to hybridize to both X. axonopodis pv. vesicatoria and X. vesicatoria strains, should facilitate disease diagnosis for these important plant pathogens.

Molecular and Physiological Characterization of Pseudomonas syringae pv. tomato and Pseudomonas syringae pv. maculicola Strains That Produce the Phytotoxin Coronatine.

The chlorosis-inducing phytotoxin coronatine is produced by several Pseudomonas syringae pathovars, including glycinea, morsprunorum, atropurpurea, and the closely related tomato and maculicola. To date, all coronatine-producing pv. glycinea, morsprunorum, and atropurpurea strains that have been examined carry the gene cluster that controls toxin production on a large plasmid. In the present study the genomic location of the coronatine gene cluster was determined for coronatine-producing strains of the pv. tomato-maculicola group by subjecting their genomic DNA to pulsed-field electrophoresis and Southern blot analysis with a hybridization probe from the coronatine gene cluster. The cluster was chromosomally borne in 10 of the 22 strains screened. These 10 strains infected both crucifers and tomatoes but could not use sorbitol as a sole source of carbon. The remaining 12 coronatine-producing strains had plasmid-borne toxin gene clusters and used sorbitol as a carbon source. Only one of these strains was pathogenic on both crucifers and tomatoes; the remainder infected just tomatoes. Restriction fragment length polymorphism analysis of the pv. tomato-maculicola coronatine gene clusters was performed with probes from P. syringae pv. tomato DC3000, a tomato and crucifer pathogen. Although the coronatine cluster appeared, in general, to be highly conserved across the pv. tomato-maculicola group, there were significant differences between plasmid-borne and chromosomally borne genes. The extensively studied coronatine cluster of pv. glycinea 4180 closely resembled the plasmid-borne clusters of the pv. tomato-maculicola group.

Isolation and sequence analysis of the Pseudomonas syringae pv. tomato gene encoding a 2,3-diphosphoglycerate-independent phosphoglyceromutase.

Pseudomonas syringae pv. tomato DC3481, a Tn5-induced mutant of the tomato pathogen DC3000, cannot grow and elicit disease symptoms on tomato seedlings. It also cannot grow on minimal medium containing malate, citrate, or succinate, three of the major organic acids found in tomatoes. We report here that this mutant also cannot use, as a sole carbon and/or energy source, a wide variety of hexoses and intermediates of hexose catabolism. Uptake studies have shown that DC3481 is not deficient in transport. A 3.8-kb EcoRI fragment of DC3000 DNA, which complements the Tn5 mutation, has been cloned and sequenced. The deduced amino acid sequences of two of the three open reading frames (ORFs) present on this fragment, ORF2 and ORF3, had no significant homology with sequences in the GenBank databases. However, the 510-amino-acid sequence of ORF1, the site of the Tn5 insertion, strongly resembled the deduced amino acid sequences of the Bacillus subtilis and Zea mays genes encoding 2,3-diphosphoglycerate (DPG)-independent phosphoglyceromutase (PGM) (52% identity and 72% similarity and 37% identity and 57% similarity, respectively). PGMs not requiring the cofactor DPG are usually found in plants and algae. Enzyme assays confirmed that P. syringae PGM activity required an intact ORF1. Not only is DC3481 the first PGM-deficient pseudomonad mutant to be described, but the P. syringae pgm gene is the first gram-negative bacterial gene identified that appears to code for a DPG-independent PGM. PGM activity appears essential for the growth and pathogenicity of P. syringae pv. tomato on its host plant.

Construction and Use of a Nonradioactive DNA Hybridization Probe for Detection of Pseudomonas syringae pv. Tomato on Tomato Plants.

Pseudomonas syringae pv. tomato, the causal agent for bacterial speck of tomato, produces the phytotoxin coronatine. A 5.3-kilobase XhoI fragment from the chromosomal region controlling toxin production was cloned into the plasmid pGB2, and the resulting recombinant plasmid, pTPR1, was tested for its ability to serve as a diagnostic probe for P. syringae pv. tomato. In a survey of 75 plant-associated bacteria, pTPR1 hybridized exclusively to those strains that produced coronatine. The detection limit for this probe, which was labeled with the Chemiprobe nonradioactive reporter system, was approximately 4 x 10 CFU of lesion bacteria. During the 1989 growing season, a total of 258 leaf and fruit lesions from nine tomato fields were screened for P. syringae pv. tomato by using pTPR1 and the culture method of detection. The best agreement between the two methods, 90%, occurred early in the season with samples taken from relatively young (5-week-old) plants. Young plants also had a higher percentage of P. syringae pv. tomato-positive lesions. P. syringae pv. tomato was the only coronatine producer recovered from the nine tomato fields. All 244 P. syringae pv. tomato strains isolated during this study reacted strongly with the probe. The P. syringae pv. tomato population of healthy field tomato leaves was determined by a pTPR1 colony hybridization procedure. Every probe-positive colony that was isolated and characterized was identified as P. syringae pv. tomato. The pTPR1 probe should expedite disease diagnosis and facilitate epidemiological studies of this pathogen. It also should aid in screening transplant seedlings for bacterial speck infestation.

Generation and Characterization of Tn5 Insertion Mutations in Pseudomonas syringae pv. tomato.

Tn5-induced insertion mutations were generated in the Pseudomonas syringae pv. tomato genome by mating this plant pathogen with an Escherichia coli strain carrying the suicide plasmid vector for Tn5, pGS9. Km transconjugants occurred at frequencies ranging from 2 x 10 to 9 x 10; approximately 5.5% of these transconjugants were also Cm, indicating the presence of additional pGS9 DNA sequences. Approximately 1% of the Km Cm mutants were auxotrophic. Southern blot analysis revealed that the Tn5 element had inserted into one unique site on the chromosome for each Km Cm transconjugant examined. Physical and genetic tests of Tn5-induced auxotrophs showed that Tn5 mutations in P. syringae pv. tomato were very stable and that secondary transposition of Tn5 or its insertion sequence IS50 was a rare event. Nine of 920 Km Cm transconjugants screened on tomato seedlings either were avirulent or produced very mild symptoms. Each of the virulence mutants was the result of a unique single-site Tn5 insertion. Five mutants also failed to induce a hypersensitivity reaction on tobacco.

Isolation and characterization of a virus from the intracellular green alga symbiotic with Hydra viridis.

The isolation and characterization of a virus (designated HVCV) from the symbiotic Chlorella-like green alga present in Hydra viridis (Florida strain) are described. The large polyhedral viral particle sedimented at ca. 2600 S in sucrose density gradients and had a buoyant density in CsCl of 1.295 g/ml. The virus contained at least 19 polypeptides (MW range, 10,300 to 82,000) and the major polypeptide (MW, 46,000) was a glycoprotein. The viral genome consisted of a double-stranded DNA with a buoyant density in CsCl of 1.7114 g/ml (52% GC) and a molecular weight of ca. 136 x 10(6).

In vitro translation of the three bacteriophage phi 6 RNAs.

In vitro translation of the three single-stranded RNAs transcribed in vitro by bacteriophage phi 6 RNA polymerase revealed that the large RNA codes for phage proteins P1, P2, P4, and P7, the medium RNA codes for P3, P6, and P10, and the smaller RNA for P5, P8, and P9.

Semiconservative synthesis of single-stranded RNA by bacteriophage phi 6 RNA polymerase.

The RNA polymerase in the nucleocapsid of Pseudomonas phaseolicola bacteriophage phi 6 transcribed large, medium, and small single-stranded RNA from the viral double-stranded RNA genome by a semiconservative (displacement) mechanism. Approximately 23%, 63%, and 65% of the nucleocapsid particles in the assay mixture synthesized at least one round of large, medium, and small single-stranded RNA molecules, respectively. Some of these particles reinitiated synthesis such that an average of 1.5 large, 33 medium, and 24 small single-stranded RNAs were synthesized from each double-stranded RNA.