PAMDB, a multilocus sequence typing and analysis database and website for plant-associated microbes.

Although there are adequate DNA sequence differences among plant-associated and plant-pathogenic bacteria to facilitate molecular approaches for their identification, identification at a taxonomic level that is predictive of their phenotype is a challenge. The problem is the absence of a taxonomy that describes genetic variation at a biologically relevant resolution and of a database containing reference strains for comparison. Moreover, molecular evolution, population genetics, ecology, and epidemiology of many plant-pathogenic and plant-associated bacteria are still poorly understood. To address these challenges, a database with web interface was specifically designed for plant-associated and plant-pathogenic microorganisms. The Plant-Associated Microbes Database (PAMDB) comprises, thus far, data from multilocus sequence typing and analysis (MLST/MLSA) studies of Acidovorax citrulli, Pseudomonas syringae, Ralstonia solanacearum, and Xanthomonas spp. Using data deposited in PAMDB, a robust phylogeny of Xanthomonas axonopodis and related bacteria has been inferred, and the diversity existing in the Xanthomonas genus and in described Xanthomonas spp. has been compared with the diversity in P. syringae and R. solanacearum. Moreover, we show how PAMDB makes it easy to distinguish between different pathogens that cause almost identical diseases. The scalable design of PAMDB will make it easy to add more plant pathogens in the future.

Reclassification of Xanthomonas campestris pv. citri (ex Hasse 1915) Dye 1978 forms A, B/C/D, and E as X. smithii subsp. citri (ex Hasse) sp. nov. nom. rev. comb. nov., X. fuscans subsp. aurantifolii (ex Gabriel 1989) sp. nov. nom. rev. comb. nov., and X. alfalfae subsp. citrumelo (ex Riker and Jones) Gabriel et al., 1989 sp. nov. nom. rev. comb. nov.; X. campestris pv malvacearum (ex smith 1901) Dye 1978 as X. smithii subsp. smithii nov. comb. nov. nom. nov.; X. campestris pv. alfalfae (ex Riker and Jones, 1935) dye 1978 as X. alfalfae subsp. alfalfae (ex Riker et al., 1935) sp. nov. nom. rev.; and "var. fuscans" of X. campestris pv. phaseoli (ex Smith, 1987) Dye 1978 as X. fuscans subsp. fuscans sp. nov.

Bacterial canker of citrus is a serious disease of citrus worldwide. Five forms of the disease have been described, cankers "A", "B", "C", "D", and "E". Although considerable genetic diversity has been described among the causal agents of the five forms of citrus canker and supports multiple taxons, the causal agents currently are classified as pathovars citri ("A"), aurantifolii ("B/C/D") and citrumelo ("E") of a single species, Xanthomonas campestris pv. citri (or X. axonopodis pv. citri). To determine the taxonomic relatedness among strains of X. campestris pv. citri, we conducted DNA-DNA relatedness assays, sequenced the 16S-23S intergenic spacer (ITS) regions, and performed amplified fragment length polymorphism (AFLP) analysis, using 44 strains representative of the five recognized forms of citrus canker. Under stringent DNA reassociation conditions (Tm - 15 degrees C), three distinct genotypes of citrus pathogens were revealed: taxon I included all "A" strains; taxon II contained all "B", "C", and "D" strains; and taxon III contained all "E" strains. The three citrus taxa showed less than 50% (mean) DNA-DNA relatedness to each other and less than 30% (mean) to X. campestris pv. campestris and X. axonopodis pv. axonopodis. Taxa I and II strains share over 70% DNA relatedness to X. campestris pv. malvacearum and X. campestris pv. phaseoli var. fuscans, respectively (at Tm - 15 degrees C). Taxon III strains share 70% relatedness to X. campestris pv. alfalfae. Previous and present phenotypic data support these DNA reassociation data. Taxon II strains grow more slowly on agar media than taxa I and III strains. Taxa I and III strains utilize maltose, and liquefy gelatin whereas taxon II strains do not. Taxon I strains hydrolyze pectate (pH 7.0) whereas Taxon II strains do not. Taxon III strains utilize raffinose whereas Taxon I strains do not. Each taxon can be differentiated by serology and pathogenicity. We propose taxa I, II, and III citrus strains be named, respectively, Xanthomonas smithii subsp. citri (ex Hasse, 1915) sp. nov. nom. rev. comb. nov., Xanthomonas fuscans subsp. aurantifolii (ex Gabriel et al., 1989) sp. nov. nom. rev. comb. nov., and Xanthomonas alfalfae subsp. citrumelo (ex Riker and Jones) Gabriel et al., 1989 nov. rev. comb. nov. Furthermore, based on the analysis of 40 strains of 19 other xanthomonads, we propose to reclassify X. campestris pv. malvacearum (ex Smith, 1901) Dye 1978 as X. smithii subsp. smithii sp. nov. comb. nov. nom. nov.; X. campestris pv. alfalfae (ex Riker and Jones) Dye 1978 as X. alfalfae subsp. alfalfae (ex Riker et al., 1935) sp. nov. nov. rev.; and "var. fuscans" (ex Burkholder 1930) of X. campestris pv. phaseoli (ex Smith, 1897) as X. fuscans subsp. fuscans sp. nov.

Reclassification of the xanthomonads associated with bacterial spot disease of tomato and pepper.

Four phenotypic xanthomonad groups have been identified that are pathogenic to pepper, tomato, or both hosts. These include groups A and C which are found in Xanthomonas axonopodis pv. vesicatoria, group B found in X. vesicatoria, and group D found in 'X. gardneri'. We present DNA:DNA hybridization data in which X. axonopodis pv. vesicatoria group A and C strains have less than 70% DNA relatedness with each other, with the type strain of X. axonopodis, and with the currently classified species within Xanthomonas and, therefore, should be removed from this species and given species status. We present information that the A strains most closely resemble the strains originally isolated by Doidge in 1921. In an attempt to avoid confusion in nomenclature as stated in Principle 1 of the Bacteriological Code, we propose that the A strains of X. axonopodis pv. vesicatoria be renamed as X. euvesicatoria (ATCC11633T= NCPPB2968T = ICMP 109T = ICMP 98T). Use of the euvesicatoria epithet should be reserved for strains originally identified by Doidge, which she designated Bacterium vesicatorium (Ann. Appl. Biol. 7: 407-430, 1921) in the original description when she referred to those strains as being feebly amylolytic. The name X. perforans sp. nov. is proposed for the C group of strains previously designated as X. axonopodis pv. vesicatoria (ATCC BAA-983T = NCPPB 4321T). We also propose that 'X. gardneri', which has less than 70% DNA relatedness with any of the Xanthomonas species and which has never had taxonomic status, be named X. gardneri (ATCC 19865T = NCPPB 881T) to reflect the specific epithet proposed by Sutic in 1957.

Xylella fastidiosa subspecies: X. fastidiosa subsp. [correction] fastidiosa [correction] subsp. nov., X. fastidiosa subsp. multiplex subsp. nov., and X. fastidiosa subsp. pauca subsp. nov.

Xylella fastidiosa, a fastidious bacterium causing disease in over 100 plant species, is classified as a single species, although genetic studies support multiple taxons. To determine the taxonomic relatedness among strains of X. fastidiosa, we conducted DNA-DNA relatedness assays and sequenced the 16S-23S intergenic spacer (ITS) region using 26 strains from 10 hosts. Under stringent conditions (Tm -15 degrees C), the DNA relatedness for most X. fastidiosa strains was *70%. However, at high stringency (Tm -8 degrees C), three distinct genotypes (A, B, and C) were revealed. Taxon A included strains from cultivated grape, alfalfa, almond (two), and maple, interrelated by 85% (mean); taxon B included strains from peach, elm, plum, pigeon grape, sycamore, and almond (one), interrelated by 84%; and taxon C included only strains from citrus, interrelated by 87%. The mean reciprocal relatedness between taxons A and B, A and C, and B and C, were 58, 41, and 45%, respectively. ITS results also indicated the same grouping; taxons A and B, A and C, and B and C had identities of 98.7, 97.9, and 99.2%, respectively. Previous and present phenotypic data supports the molecular data. Taxon A strains grow faster on Pierce's disease agar medium whereas B and C strains grow more slowly. Taxon B and C strains are susceptible to penicillin and resistant to carbenicillin whereas A strains are opposite. Each taxon can be differentiated serologically as well as by structural proteins. We propose taxons A, B, and C be named X. fastidiosa subsp. fastidiosa [correction] subsp. nov, subsp. multiplex, subsp. nov., and subsp. pauca, subsp. nov., respectively. The type strains of the subspecies are subsp. fastidiosa [correction] ICPB 50025 (= ATTC 35879T and ICMP 15197), subsp. multiplex ICPB 50039 (= ATTC 35871 and ICMP 15199), and subsp. pauca ICPB 50031 (= ICMP 15198).

Genetic diversity of Leptotrichia and description of Leptotrichia goodfellowii sp. nov., Leptotrichia hofstadii sp. nov., Leptotrichia shahii sp. nov. and Leptotrichia wadei sp. nov.

Sixty strains of Gram-negative, anaerobic, rod-shaped bacteria from human sources initially assigned to Leptotrichia buccalis (n=58) and 'Leptotrichia pseudobuccalis' (n=2) have been subjected to polyphasic taxonomy. Full-length 16S rDNA sequencing, DNA-DNA hybridization, RAPD, SDS-PAGE of whole-cell proteins, cellular fatty acid analysis and enzymic/biochemical tests supported the establishment of four novel Leptotrichia species from this collection, Leptotrichia goodfellowii sp. nov. (type strain LB 57(T)=CCUG 32286(T)=CIP 107915(T)), Leptotrichia hofstadii sp. nov. (type strain LB 23(T)=CCUG 47504(T)=CIP 107917(T)), Leptotrichia shahii sp. nov. (type strain LB 37(T)=CCUG 47503(T)=CIP 107916(T)) and Leptotrichia wadei sp. nov. (type strain LB 16(T)=CCUG 47505(T)=CIP 107918(T)). Light and electron microscopy showed that the four novel species were Gram-negative, non-spore-forming and non-motile rods. L. goodfellowii produced arginine dihydrolase, beta-galactosidase, N-acetyl-beta-glucosaminidase, arginine arylamidase, leucine arylamidase and histidine arylamidase. L. shahii produced alpha-arabinosidase. L. buccalis and L. goodfellowii fermented mannose and were beta-galactosidase-6-phosphate positive. L. goodfellowii, L. hofstadii and L. wadei were beta-haemolytic. L. buccalis fermented raffinose. With L. buccalis, L. goodfellowii showed 3.8-5.5 % DNA-DNA relatedness, L. shahii showed 24.5-34.1 % relatedness, L. hofstadii showed 27.3-36.3 % relatedness and L. wadei showed 24.1-35.9 % relatedness. 16S rDNA sequencing demonstrated that L. hofstadii, L. shahii, L. wadei and L. goodfellowii each formed individual clusters with 97, 96, 94 and 92 % similarity, respectively, to L. buccalis.

Detection and Characterization of a New Strain of Citrus Canker Bacteria from Key/Mexican Lime and Alemow in South Florida.

In the Wellington and Lake Worth areas of Palm Beach County, FL, citrus canker appeared on Key/Mexican lime (Citrus aurantiifolia) and alemow (C. macrophylla) trees over a period of about 6 to 7 years before detection, but nearby canker-susceptible citrus, such as grapefruit (C. × paradisi) and sweet orange (C. sinensis), were unaffected. Colonies of the causal bacterium, isolated from leaf, stem, and fruit lesions, appeared similar to the Asiatic group of strains of Xanthomonas axonopodis pv. citri (Xac-A) on the nutrient agar plate, but the growth on lima bean agar slants was less mucoid. The bacterium produced erumpent, pustule-like lesions of typical Asiatic citrus canker syndrome after inoculation into Key/Mexican lime, but brownish, flat, and necrotic lesions on the leaves of Duncan grapefruit, Madame Vinous sweet orange, sour orange (C. aurantium), citron (C. medica), Orlando tangelo (C. reticulata × C. × paradisi), and trifoliate orange (Poncirus trifoliata). The bacterium did not react with the Xac-A specific monoclonal antibody A1 using enzyme-linked immunosorbent assay (ELISA) and could not be detected by polymerase chain reaction (PCR)-based assays using primers selected for Xac-A. DNA reassociation analysis confirmed that the pathogen, designated as Xac-AW, was more closely related to Xac-A and Xac-A* strains than X. axonopodis pv. aurantifolii or the citrus bacterial spot pathogen (X. axonopodis pv. citrumelo). The strain can be easily differentiated from Xac-A and Xac-A* using ELISA, PCR-based tests, fatty acid analysis, pulsed-field gel electrophoresis of genomic DNA, and host specificity.

Effect of microorganism characteristics on leak size critical to predicting package sterility.

The effects of microorganism size and motility on the leak size critical to the sterility of a package, along with the imposed pressure required to initiate liquid flow for the critical leak size, were measured. Pseudomonas fragi Lacy-1052, Bacillus atrophaeus ATCC 49337, and Enterobacter aerogenes ATCC 29007 were employed to assess package sterility. One hundred twenty-six 7-mm-long microtubes with interior diameters of 5, 10, and 20 microm were used to simulate package defects. Forty-two solid microtubes were used as controls. No significant differences were found between sizes or motility statuses of test organisms with respect to loss of sterility as a result of microbial ingress into test cells with microtube interior diameters of 5, 10, and 20 microm (P > 0.05). Interactions between the initiation of liquid flow as a result of applied threshold pressures and sterility loss for test cells were significant (P < 0.05).

Application of fluid modeling to determine threshold leak size for liquid foods.

In this study, the mechanism by which a package defect converts to a leaker was examined in an effort to develop a relationship between threshold leak size and loss of package sterility. The threshold leak size is the hole size at which the onset of leakage occurs. The threshold pressure is the pressure required to initiate a leak. Leak initiation was studied in terms of the interaction between three components: liquid attributes of liquid food products, defect size, and pressures required to initiate liquid flow. Liquid surface tension, viscosity, and density values were obtained for 16 liquids. The imposed pressures required to initiate flow through microtubes with interior diameters of 0, 2, 5, 7, 10, 20, and 50 microm were measured with the use of 63 test cells filled with safranin red dye, tryptic soy broth, and distilled water with surface tensions of 18.69, 44.09, and 64.67 mN/m, respectively. Significant differences (P<0.05) between threshold pressures observed for safranin red dye, tryptic soy broth, and distilled water were found. Liquids with low surface tensions, such as safranin red dye, required significantly lower threshold imposed pressures than did liquids with high surface tensions, such as distilled water (P<0.05). An equation to quantify the relationship between liquid surface tension, threshold imposed pressure, and defect size was developed. Threshold pressures observed were not significantly different (P>0.05) from those predicted by the equation. Imposed pressures and vacuums generated within packages during random vibration and sweep resonance tests were measured for brick-style aseptic packages (250 ml), metal cans (76.2 by 114.3 mm [425 ml]), 1-qt gable-top packages (946 ml), 0.5-gal gable-top packages (1.89 liters), and 1-gal milk jugs (4.25 liters). Significant differences between packages were found with respect to observed generated pressures during vibration testing (P<0.05). An equation to calculate threshold size on the basis of liquid surface tension and imposed pressure was established.

Relatedness of chromosomal and plasmid DNAs of Erwinia pyrifoliae and Erwinia amylovora.

The plant pathogen Erwinia pyrifoliae has been classified as a separate species from Erwinia amylovora based in part on differences in molecular properties. In this study, these and other molecular properties were examined for E. pyrifoliae and for additional strains of E. amylovora, including strains from brambles (Rubus spp.). The nucleotide composition of the internal transcribed spacer (ITS) region was determined for six of the seven 16S-23S rRNA operons detected in these species with a 16S rRNA gene probe. Each species contained four operons with a tRNA(Glu) gene and two with tRNA(Ile) and tRNA(Ala) genes, and analysis of the operons from five strains of E. amylovora indicated a high degree of ITS variability among them. One tRNA(Glu)-containing operon from E. pyrifoliae Ep1/96 was identical to one in E. amylovora Ea110, but three tRNA(Glu) operons and two tRNA(Ile) and tRNA(Ala) operons from E. pyrifoliae contained unique nucleotide changes. When groEL sequences were used for species-specific identification, E. pyrifoliae and E. amylovora were the closest phylogenetic relatives among a set of 12 bacterial species. The placement of E. pyrifoliae distinct from E. amylovora corroborated molecular hybridization data indicating low DNA-DNA similarity between them. Determination of the nucleotide sequence of plasmid pEP36 from E. pyrifoliae Ep1/96 revealed a number of presumptive genes that matched genes previously found in pEA29 from E. amylovora and similar organization for the genes and origins of replication. Also, pEP36 and pEA29 were incompatible with clones containing the reciprocal origin regions. Finally, the ColE1-like plasmid pEP2.6 from strain Ep1/96 contained sequences found in small plasmids in E. amylovora strains IL-5 and IH3-1.

Gaeumannomyces graminis vars. avenae, graminis, and tritici Identified Using PCR Amplification of Avenacinase-like Genes.

Identifying take-all pathogens, Gaeumannomyces graminis varieties avenae (Gga), graminis (Ggg), and tritici (Ggt), is difficult. Rapid identification is important for development of disease thresholds. We developed a single-tube, polymerase chain reaction (PCR) method differentiating among Gga, Ggg, and Ggt. Nucleotide base sequence analyses of avenacinase-like genes from Gga, Ggg, and Ggt isolates provided the basis for designing variety-specific primers. Sequences from Ggg and Ggt were highly related (99% identity), but Gga sequences were <95% identical to Ggg and Ggt sequences. Three 5' primers specific for Gga, Ggt, and Ggg and a single 3' common primer allowed amplification of variety-specific fragments of 617, 870, and 1,086 bp, respectively. Each 5' primer was specific in mixed populations of primers and templates. No PCR products were amplified from related fungi including Gaeumannomyces cylindrosporus and Phialophora spp. We surveyed 16 putative Ggt isolates using our assay; nine produced Ggt-specific fragments and seven produced Ggg-specific fragments. Five Gga isolates produced Gga-specific fragments. However, Gga- and Ggt-specific fragments were observed from a sixth Gga isolate, RB-W, which indicates a mixed culture or a heterokaryon. Our single-tube, PCR method rapidly differentiates among the important take-all pathogens commonly encountered together in cereal fields.

Bioaerosol Exposure Method for Package Integrity Testing †.

Test organism motility, concentration, aerosol exposure time, hole diameter and length were evaluated to determine their influence on microbial ingress into a flexible plastic pouch. Microtubes with 10- and 20-µm hole diameters and of 5- and 10-mm lengths were used as defects in 128 flexible pouches. A bioaerosol with a 2.68-µm mean particle size comprised of 102 or 106 CFU/ml source concentrations of motile or nonmotile Pseudomonas fragi TM 849 was introduced into a 119,911-cm3 chamber for exposures of 15 or 30 minutes. Six pouches showed test organism growth after a 72-h incubation period. Microbial ingress was significant (P < .05) for motile test organisms with source concentrations of 106 CFU/ml.

Contamination of Flexible Pouches Challenged by Immersion Biotesting †.

Immersion biotesting has long been used to challenge packages, particularly cans, for pinholes and channel leaks. Such testing for all types of plastic packaging may not be appropriate because some packages (e.g., aseptic, hot fill) are not exposed to water. As the food-packaging industry develops alternative environmental biotests there is a need to benchmark them against traditional immersion testing. The purpose of this research was to examine the threshold of critical-defect dimensions using artifically created channel leaks of 10 and 20 µm and 5- and 10-mm lengths sealed into plastic pouches which were subsequently tested by immersion at 102 and 106 CFU of motile and nonmotile Pseudomonas fragi TM849 per ml. Forty-four percent (44%) of the pouches tested became contaminated, indicating the threshold defect value is below 10 µm. Microbial ingress was significant (P < .05) for motile test organisms with a concentration of 106 CFU/ml. The interaction of concentration and time was also significant at 102 CFU/ml at 30 min exposure and 106 CFU/ml at 15 min. Channel length was not statistically significant. The markedly greater contamination rate using immersion testing versus that of aerosol testing highlights the importance of using test methods that reflect environmental exposure conditions of the packages.

Organoleptic Examination of Pears from Trees Infused with Oxytetracycline to Remit Symptoms of Pear Decline.

One year after trees had been infused post-harvest with oxytetracycline (OTC) to remit symptoms of pear decline, pears were harvested and tasted. In four sessions, the panel of tasters discerned between pears from trees treated with OTC and pears from untreated diseased trees. In three of the sessions, the fruit from treated trees was preferred over fruit from untreated diseased trees.

Antibiotic-resistant Bacteria in Raw Milk and Ability of Some to Transfer Antibiotic Resistance to Escherichia coli.

Raw milk samples were examined for number and percentage of bacteria resistant to seven antibiotics: penicillin, ampicillin, chloramphenicol, neomycin sulfate, polymyxin B sulfate, tetracycline and streptomycin sulfate. A significant negative correlation was found between the total aerobic count of the milk sample and the concentration (above 5 or 10% of the total count) of bacteria in each milk resistant to each of the antibiotics tested. Three of 42 gram-negative isolates were capable of transferring their antibiotic resistance to Escherichia coli . Substantial numbers of antibiotic-resistant bacteria in raw milk were found and some survived pasteurization. Inspection of farms failed to indicate a relationship between farm practices or use of antibiotics in feed or as pharmaceuticals and number of antibiotic-resistant bacteria in the raw milk.