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In March 1998, a leaf blight of onion (Allium cepa L. ‘1015’) was found on many plants in a plot on the Texas A&M Agricultural Experiment Station in Weslaco. The symptoms were longitudinal chlorotic areas on one side of the leaf, containing sunken, elliptical necrotic lesions. Affected leaves ultimately died. Chlorotic lesions were swabbed with 70% ethanol, and tissue from beneath the epidermis was placed in a drop sterile water for 20 min. Drops were streaked on nutrient agar and incubated at 30°C. Isolations yielded gram-negative, rod-shaped bacteria that formed dark yellow, gummy colonies on yeast dextrose carbonate agar medium, hydrolyzed starch, and had a single, polar flagellum. Analysis of fatty acid methyl ester (FAME) profiles, using the Microbial Identification System (MIS, version 4.15; Microbial Identification, Newark, DE), done at the Texas Plant Disease Diagnostic Laboratory, College Station, identified nine isolates as Xanthomonas campestris (similarity indices of 0.31 to 0.54). Tests at the University of Florida supported this identification: FAME profiles using MIS version 3.9 gave similarity indices of 0.89 to 0.95, and profiles using Biolog GN Microplates, MicroLog database release 3.50 (Biolog, Hayward, CA), gave similarity indices of 0.03 to 0.76. Leaves (15 to 20 cm long) of potted onions (cv. 1015 at the five- to six-leaf stage) were infiltrated with a suspension of bacteria (107 CFU per ml), using a needle and syringe. Plants were maintained in mist chamber in a greenhouse at 24°C. Water-soaking and development of pale green color of the inoculated leaf occurred after 2 days, followed by death after 4 days. There were no symptoms on leaves inoculated with sterile water. Pathogenicity tests on four isolates were repeated once. Bacteria were reisolated on nutrient agar from symptomatic tissue but not from controls. In the field plot, disease severity did not increase as season progressed nor were there any symptoms on bulbs. Symptoms were not observed on onion during the 1999 season. X. campestris was first reported on onion from Hawaii (1). This is the first report of this pathogen on onion in the continental United States. Reference: (1) A. M. Alvarez et al. Phytopathology 68:1132, 1978.

Agapanthus praecox subsp. orientalis, commonly called African lily or lily-of-the-Nile, bears large, round, blue or white flowers above attractive dark green foliage. Because of these horticultural features, this member of the family Liliaceae, has become a popular perennial bedding plant. For the past 2 years during warm wet periods, symptoms of chlorotic, water-soaked, leaf-streaks have been observed on agapanthus production in Florida. Round butyrus, bright yellow colonies were consistently isolated on nutrient agar. Bacteria were characterized as gram negative, catalase positive, motile, strictly aerobic, and not hydrolytic on starch. Using fatty acid analysis (FAME) and the MIDI Microbial Identification System with software version TSBA 3.90 (Microbial ID, Inc., Newark DE), three strains were further characterized and identified as Xanthomonas axonopodis with similarity coefficients to X. axonopodis pv. dieffenbachiae (0.907, 0.915, and 0.944) and to X. axonopodis pv. poinsetticola (0.912, 0.922, and 0.916). The three isolates were each inoculated on three plants each of agapanthus cv. Blue African lily, Dieffenbachiae maculata cv. Camille, and poinsettia, Euphorbia pulcherrima cv. PeterStar Red. Plants were sprayed with a suspension of each isolate at 1 × 108 CFU/ml, bagged for 24 h to raise humidity, and placed in a glasshouse for symptom development. Strains of X. axonopodis pv. poinsetticola (NZTCC 5779) and X. axonopodis pv. dieffenbachiae (X1718) were used as positive controls. Within 3 weeks, isolates from agapanthus produced leaf streaks on agapanthus plants, small, scattered, water-soaked lesions on dieffenbachia leaves, and no symptoms on poinsettia. No symptoms developed on the agapanthus plants when inoculated with either control strain. Both control strains formed lesions on leaves of their original host species. Xanthomonas was reisolated from treatments with symptomatic leaves. Plant inoculations were repeated with similar results. Although the agapanthus isolates were highly similar in FAME profiles to X. axonopodis pv. dieffenbachiae, symptoms produced on dieffenbachia were mild as compared with those produced by the dieffenbachia isolate. Therefore, these isolates may represent a distinct pathovar.

A serious outbreak of a leaf spot disease was observed on tomato transplants grown in commercial seedling companies in southwestern Turkey (Antalya) during the springs of 2002 and 2003. Disease incidence was more severe in the western Mediterranean Region of Turkey. Occurrence of the outbreak resulted in approximately 20 and 25% seedling losses in the springs of 2002 and 2003, respectively. The initial symptoms consisted of pronounced water-soaked, dark brown-to-black spots on young expanding leaves that were 1 to 2 mm in diameter. Later, a number of leaf spots on older leaves enlarged and coalesced, causing leaf desiccation and finally, seedling death. In addition, in 2003 the disease incidence was approximately 5% in 142 commercial greenhouses. Tomato production was unaffected since significant outbreaks did not occur on greenhouse plants. No fruit symptoms were observed. Twenty-six strains were isolated from diseased tomato seedlings and plants from different greenhouses located in different places and all were gram negative and fluorescent on King's B medium. All strains were levan and gelatin liquefaction positive and oxidase and arginine dihydrolase negative. None of the 26 strains utilized erythritol and l-lactate as the sole carbon source (1,2). Fatty acid analysis identified the strains as Pseudomonas syringae pv. tomato with similarity indices ranging from 0.876 to 0.932%. Pathogenicity of the isolates was confirmed on 4-week-old tomato seedlings (cv. Biotek Selin) sprayed with the bacterial suspensions containing 108 CFU/ml of sterile water. Later, a number of leaf spots on the leaves enlarged and coalesced, causing leaf desiccation. Inoculated and control tomato seedlings were covered with polyethylene bags and placed in a growth chamber at 25°C for 48 h and then the bags were removed. Small (1 to 2 mm), water-soaked, dark brown-to-black spots similar to those observed in the greenhouses of commercial seedling companies and commercial greenhouses that produce tomato developed on the young expanding leaves of inoculated plants within 7 to 10 days. No symptoms developed on control plants. The bacterium was reisolated from inoculated plants and identified as a strain of Pseudomonas syringae pv. tomato. Koch's postulates were fulfilled. To our knowledge, this is the first report for the occurrence and outbreak of the bacterial speck disease on tomato transplants in greenhouses of commercial tomato seedling production companies in Turkey. References: (1) D. C. Hildebrand et al. Pages 60–80 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. N. D. Schaad, ed. The American Phytopathological Society, 1988. (2) J. B. Jones et al. Plant Dis. 70:151, 1986.

Xanthomonas axonopodis pv. vesicatoria, causal agent of bacterial spot of tomato (Lycopersicon esculentum L.) and sweet pepper (Capsicum annuum L.) was isolated from tomato and pepper plants in greenhouse production in the Province of Antalya, in southwestern Turkey. Disease incidence was less than 4% of plants observed in 2001 and ranged from 1 to 20% in 2002. Eleven seedling-producing companies and 26 greenhouses that produce tomato and pepper were surveyed during the rainy seasons of 2001 and 2002. The increase in disease incidence in 2002 is an indication that this disease is becoming more prevalent on tomato and pepper plants grown in greenhouses in southwestern Turkey. A gram-negative bacterium producing yellow-pigmented colonies on nutrient agar was consistently isolated from brown, circular spots on leaflets of tomato and sweet pepper seedlings. Five isolates were pathogenic on commercial cultivars of tomato and pepper when bacterial suspensions (108 CFU/ml) were infiltrated into the intercellular spaces of leaves to determine race by using procedures described by Bouzar et al. (1). All the isolates produced hypersensitive reaction responses on tomato genotype cv. Hawaii 7998 and pepper genotype cvs. 20 R and 30 R and were designated tomato race 1 pepper race 1 (T1P1) (1). Fatty acid analysis of the strains identified them as X. axonopodis vesicatoria with similarity index values of 0.872 to 0.933. In addition, the strains were tested with X. axonopodis vesicatoria-specific polymerase chain reaction primers (RST 2/3 and RST 9/10) (2). The isolates were determined to be X. axonopodis vesicatoria. Although bacterial spot of tomato has been suspected in Turkey for a number of years, to our knowledge, this is the first report of the bacterium on tomato. References: (1) H. Bouzar et al. Phytopathology 84:663, 1994. (2) R. P. Leite, Jr. et al. Appl. Env. Microbiol. 60:1068, 1994.

From October to December 2001, a leaf spot disease was observed in numerous commercial fields of red and green cabbage (Brassica oleracea var. capitata L.) in the Everglades Agricultural Area, south and east of Lake Okeechobee and in the environs of Immokalee in southwestern Florida. Discrete water-soaked to greasy appearing spots were observed in the leaf blades with no evidence of marginal V-shaped lesions characteristic of black rot caused by Xanthomonas campestris pv. campestris. Profuse bacterial streaming was observed when cut leaf sections were examined microscopically. A bacterium that formed yellow colonies on nutrient agar was consistently isolated from these lesions. Ten bacteria were isolated, purified, and characterized. All strains were aerobic, gram-negative rods. Strains were positive for esculin hydrolysis, proteolysis in litmus milk, and gelatin liquefaction. Strains were negative for urease production, nitrate reduction, oxidase, and utilization of asparagine as a sole source of carbon and nitrogen. Fatty acid methyl ester analysis indicated a match with Florida library strains of X. campestris pv. raphani (similarity indices 0.605–0.738). Suspensions (2 × 107 CFU/ml in phosphate-buffered saline) of two Oklahoma strains identified as X. campestris pv. armoraciae provided by J. P. Damicone (3) and four representative Florida strains were applied to plants using a hand-held sprayer. Pathogenicity of the strains was tested on three replicate greenhouse-grown plants of the following: green cabbage cv. Market Early; red cabbage cv. Salad Delight; radish cv. Red Silk; tomato cv. Sunny; sweet bell pepper cv. Jupiter; and fresh horseradish roots purchased from a retail grocery chain. A strain of X. campestris pv. campestris originally isolated from Homestead, FL was also included in pathogenicity tests. All Florida and Oklahoma strains produced leaf spots, but no V-shaped lesions, on leaves of green cabbage, red cabbage, radish, tomato, and horseradish. Typical black rot symptoms were observed only in radish and green and red cabbage inoculated with the X. campestris pv. campestris strain. On the basis of these results, we identify the Florida strains as X. campestris pv. armoraciae (1,2,3), recognizing the precedent of X. campestris pv. armoraciae over X. campestris pv. raphani based on extensive genetic and serological data (1). Our strains appear to be more similar to those causing outbreaks on crucifers in Oklahoma (3) than those in Ohio (2), because Florida strains were pathogenic on tomato. References: (1) A. M. Alvarez et al. Phytopathology 84:1449, 1994. (2) F. Sahin and S. A. Miller. Plant Dis. 81:1334, 1997. (3) Y. Zhao et al. Plant Dis. 84:1008, 2000.

Wild rocket, also known as wall rocket or sand rocket (Diplotaxis tenuifolia (L.) DC, family Brassicaceae), is grown in Florida as a salad green and herb, especially for addition to “spring mix” and other bagged salad blends. It is similar in texture and flavor to the more widely known garden arugula (Eruca vesicatoria (L.) Cav. subsp. sativa). During the winter vegetable season of 2006–2007, a leaf spot disease causing severe economic loss was seen in commercial fields of wild rocket near the town of Sebastian in Indian River County, FL. Discrete water-soaked to greasy appearing spots were observed on leaf blades that rarely exceeded 1 mm in diameter with some surrounded by a narrow, yellow halo. There was no evidence of marginal V-shaped lesions suggestive of vascular black rot disease caused by Xanthomonas campestris pv. campestris. A bacterium that formed yellow colonies on nutrient agar was consistently isolated from these lesions. Four strains were isolated, purified, and characterized. All strains were strictly aerobic, gram-negative rods. Strains were positive for esculin hydrolysis and gelatin liquefaction and negative for oxidase, nitrate reduction, urease production, fluorescence on King's B medium, and utilization of asparagine as a sole source of carbon and nitrogen. Proteolysis and an alkaline reaction were observed in inoculated tubes of litmus milk. Colonies were bright yellow and mucoid on plates of yeast extract-glucose-calcium carbonate agar. Carbon source utilization, as revealed by the Biolog system, indicated a match to X. campestris. Fatty acid methyl ester analysis indicated a match with Florida library strains of X. campestris pv. raphani (similarity indices of 0.737 to 0.779). Suspensions (2 × 107 CFU/ml in sterile phosphate-buffered saline) of the four wild rocket strains and a strain isolated in 2003 as a cause of a discrete leaf spot disease of cabbage in southern Florida (1) were sprayed onto plants until runoff with a hand-held plastic mist bottle. Pathogenicity of strains was tested in the greenhouse on seven replicate plants of green cabbage cv. Copenhagen Market, salad arugula cv. Rocket, and wild rocket (an unnamed selection propagated from seed provided by a Florida grower). Symptoms appeared within 6 to 7 days. The wild rocket strains and the cabbage strain were pathogenic on all wild rocket and cabbage test plants, producing small, nonvascular leaf spots. No symptoms were seen on salad arugula or on control plants of wild rocket, cabbage, or arugula sprayed with phosphate-buffered saline. The bacterium was reisolated from infected plants and identified as X. campestris pv. raphani, thus completing Koch's postulates. We have classified the bacterial leaf spot pathogen of wild rocket as X. campestris pv. raphani using the nomenclature of Vincente et al. (2) for X. campestris strains producing nonvascular leaf spots on brassicas. This disease problem seems to be related to widespread use of overhead irrigation in the fields where the disease is prevalent. We have not observed the disease in seepage-irrigated fields of wild rocket. References: (1) K. Pernezny et al. Plant Dis. 87:873, 2003. (2) J. G. Vincente et al. Phytopathology 96:735, 2006.

Vanda orchids are epiphytes grown for their attractive flowers by commercial producers and hobbyists throughout Florida. In August 2007, five Vanda hybrids, with an economic value of $150 each, were found at a nursery in central Florida with leaves that were macerated, brown, and water soaked. According to the growers, the plants were normal the previous day but symptoms developed rapidly. The plants were immediately removed from the greenhouse to prevent potential disease spread. Bacteria were isolated according to the method of Schaad et al. (1). Isolated bacteria grew at 37°C, were gram negative, degraded pectate, and produced phosphatase. MIDI (Sherlock version TSBA 4.10; Microbial Identification 16 System, Newark, DE) (SIM 0.906) identified the bacteria as Erwinia chrysanthemi (Dickeya chrysanthemi Burkholder et al. 1953) Samson et al. 2005. PCR was performed on the 16S rRNA gene (GenBank Accession No. EU526397) with primers 27f (5′-GAGAGTTTGATCCTG GCTCAG-3′) and 1495r (5′-TACGGCTACCTTGTTACGA-3′) (2). Subsequent DNA sequencing and GenBank search showed the isolated strain is 99% identical to that of Dickeya chrysanthemi. Four leaves each of six Vanda hybrids were inoculated by injecting approximately 150 μl of a bacteria suspension at 1 × 108 CFU/ml into each leaf. One plant was inoculated with water in each of four leaves. Plants were enclosed in plastic bags and returned to the greenhouse under 50% shade at 29°C day and 17°C night temperatures. Within 24 h, soft rot symptoms appeared on inoculated leaves. The water control appeared normal. D. chrysanthemi was reisolated and identified with the above method, thus Koch's postulates were fulfilled. To our knowledge, this is the first report of a soft rot caused by D. chrysanthemi on Vanda hybrids. Because of the popularity and high value of Vanda orchids, proper identification of this rapidly progressing bacterial disease is of great importance for the commercial producer and homeowner alike. References: (1) N. W. Schaad et al. Erwinia soft rot group. Page 56 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al., eds. American Phytopathological Society. St. Paul, MN, 2001. (2) W. G. Weisburg. J. Bacteriol. 173:697, 1991.

Several (+)- and (-)-α-pinene derivatives were synthesized and evaluated for their antimicrobial activity toward Gram-positive bacteria Micrococcus luteus and Staphylococcus aureus, Gram-negative bacterium Escherichia coli, and the unicellular fungus Candida albicans using bioautographic assays. (+)-α-Pinene 1a showed modest activity against the test organisms, whereas (-)-α-pinene 1b showed no activity at the tested concentration. Of all the α-pinene derivatives evaluated, the β-lactam derivatives (10a and 10b) were the most antimicrobial. The increase in the antimicrobial activity of 10a compared to 1a ranged from nearly 3.5-fold (C. albicans) to 43-fold (S. aureus). The mean ± standard deviation for the zone of inhibition (mm) for 10a (C. albicans) was 31.9 ± 4.3 and that for S. aureus was 51.1 ± 2.9. Although (-)-α-pinene 1b was not active toward the test microorganisms, the corresponding β-lactam 10b, amino ester 13b, and amino alcohol 14b showed antimicrobial activity toward the test microorganisms. The increase in the antimicrobial activity of 10b compared to 1b ranged from 32-fold (S. aureus) to 73-fold (M. luteus). The mean ± standard deviation for the zone of inhibition (mm) for 10b (S. aureus) was 32.0 ± 0.60 and that for M. luteus was 73.2 ± 0.30.

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.

ADDITIONAL INDEX WORDS. Canada, emerging pests and diseases, exports, floriculture, imports, ornamental plants SUMMARY. The purpose of this study was to evaluate the U.S.-Canadian Greenhouse Certification Program (USGCP) that was initiated in 1998. A survey consisting of 34 questions was designed and 43 out of 48 nurseries in Florida participating in the USGCP were visited. Based on the answers to the questionnaire, most of the nurseries were in compliance with the majority of USGCP requirements, growers were satisfied with the program, and there was an economic benefit to participating in the program. The main problems identified were theambiguous wording of some of the requirements and the impracticality of keeping imported and domestic plants completely segregated. Moreover, many of the respondents did not have a written description of a pest management plan. Chi square statistical analysis showed that there was almost no difference between nursery groups in their responses to the majority of the survey questions, indicating that the USGCP is a successful program forbothlarge andsmallnurseries.Thisquantitativeassessment oftheUSGCP isthe first assessment conducted for this program and discussed in a peer-reviewed publication.

In vertebrates, adipose tissue stores energy in the form of fat. Fat storage is tightly controlled by and dynamically balanced with energy expenditure under physiological settings; the perturbation of fat in either excess (obese) or deficit (lipodystrophy) has devastating pathologic consequences in the fueling of homeostasis and organismal fitness. The process by which fat storage is coordinated through positive and negative feedback signals is still poorly understood. To address potential mechanisms underlying fat storage we study a Caenorhabditis elegans Krüppel-like transcription factor, Ce-klf-3 and demonstrate that klf-3 is a hitherto unrecognized key regulator of fat metabolism in C. elegans. The Ce-klf-3 is highly expressed during larval development and predominantly present in intestine: the site of fat digestion, absorption, storage, and utilization. We found a strong positive correlation between klf-3 expression and fat deposition in a worm's intestine. Significantly, a klf-3 (ok1975) loss-of-function mutation, characterized by the deletion of a 1658-bp sequence spanning the 3' end of exon 2 through to the 5' end of exon 3 of klf-3, enhanced fat deposition in the intestine and caused severe defects in worm reproduction. Although klf-3 mutants seemed very similar to wild type worms in appearance and life span, 70% of mutants became semi-sterile, each producing 40-50 viable progenies, and the remaining 30% were rendered completely sterile toward adulthood. Notably, both mutant types displayed extensive deposition of fat in the intestine. Our study also demonstrates that klf-3 is critical for maintaining normal fatty acid composition by regulating genes involved in a fatty acid desaturation pathway. Strikingly, klf-3 mutant animals with impaired fatty acid beta-oxidation pathway genes resulted in fat accumulation in the mutant worm. We present the first clear in vivo evidence supporting essential regulatory roles of KLF-3 in fat storage in C. elegans and shed light on the human equivalent in disease-gene association.

Bacterial panicle blight (BPB) is among the three most limiting rice diseases in Louisiana and the southern United States. The identity and characterization of pathogens associated with this disease was unclear. This research details studies carried out on the pathogens causing BPB on rice in Louisiana and other rice producing southern states. Bacterial strains were isolated from BPB-infected sheath, panicle, or grain samples collected from rice fields in Louisiana, Arkansas, Texas, and Mississippi. In greenhouse inoculation tests, 292 of 364 strains were pathogenic on rice seedlings or panicles. Identification of strains in the pathogen complex by growth on S-PG medium, carbon source utilization profile (Biolog), cellular fatty acid analysis, and polymerase chain reaction (PCR) methods revealed that 76 and 5% of the strains were Burkholderia glumae and B. gladioli, respectively. The other strains have not been conclusively identified. Although strains of both species produced similar symptoms on rice, B. glumae strains were generally more aggressive and caused more severe symptoms on rice than B. gladioli. Virulent strains of both species produced toxoflavin in culture. The two species had similar growth responses to temperature, and optima ranged from 38 to 40°C for B. glumae and 35 to 37°C for B. gladioli. PCR was the most sensitive and accurate method tested for identifying the bacterial pathogens to the species level. The 16S rDNA gene and 16S-23S rDNA internal transcribed spacer (ITS) region sequences of the B. glumae and B. gladioli strains from rice showed more than 99% sequence homology with published sequences. A real-time PCR system was developed to detect and quantify this pathogen from infected seed lots. Our results clearly indicate that B. glumae and B. gladioli were the major pathogens causing BPB in the southern United States.

The bacterium Acidovorax avenae causes disease in a wide range of economically important monocotyledonous and dicotyledonous plants, including corn, rice, watermelon, anthurium, and orchids. Genotypic and phenotypic relatedness among strains of phytopathogenic A. avenae subsp. avenae, A. avenae subsp. citrulli, A. avenae subsp. cattleyae and A. konjaci, as well as all other Acidovorax species, including A. facilis, the type strain of Acidovorax, was determined. The 16s rDNA sequencing confirmed previous studies showing the environmental species to be very distant from the phytopathogenic species. DNA/DNA reassociation assays on the different strains of A. avenae revealed four (A, B, C, and D) distinct genotypes. Taxon A included six A. avenae subsp. avenae strains from corn that had a mean reciprocal similarity of 81%; taxon B included six A. avenae subsp. avenae strains from rice that had a mean reciprocal similarity of 97%; taxon C contained 11 A. avenae subsp. citrulli strains from cucurbits (cantaloupe, watermelon, and pumpkin) that had a mean reciprocal similarity of 88%, and taxon D contained four A. avenae subsp. cattleyae strains from orchids that had a mean similarity of 98%. The mean reciprocal relatedness between taxa A, B, C, and D was less than 70%. Sequence analysis of 16S rDNA and the 16S–23S rDNA internally transcribed spacer region, as well as AFLP analysis, revealed the same four taxa. All four were easily differentiated phenotypically from each other and from all other recognized Acidovorax species. Strains of A. avenae did not contain 3-hydroxyoctanoic acid, which was found in all other species. On the basis of these and previous genetic and phenotypic results, we propose an emendation of the species A. avenae. A. avenae subsp. citrulli (C strains) and A. avenae subsp .c attleyae(D strains) should be elevated to species rank as A. citrulli and A. cattleyae, respectively. We further propose a new taxon for the B strains, A. oryzae sp. nov. with FC-143 T ¼ ICPB 30003 T ¼ ICMP 3960 T ¼ ATCC 19882 T as the type strain.

A leaf spot and blighting were observed on leaves of tomato transplants from a producer in Florida in 2001 and 2002. A nonfluorescent bacterium was isolated consistently from affected tissue. The typical bacterium was a gram negative, strictly aerobic, slightly curved rod with one or two flagella. Sequence analysis of the 16S rRNA indicated that two representative strains, F1 and SE1, had greater than 99% nucleotide sequence identity with Herbaspirillum huttiense and H. rubrisubalbicans. The cellular fatty acid composition of the total of 16 tomato strains was very similar to H. huttiense and H. rubrisubalbicans. Based on carbon utilization, six of nine strains tested with the Biolog system were identified as Herbaspirillum spp. The tomato strains were oxidase positive and grew at 40°C, but were negative for levan production, pectate hydrolysis, and arginine dihydrolase activity. Based upon this polyphasic analysis, we concluded that the strains were most closely related to H. huttiense, although placement in this species would require further analyses. However, the tomato strains and H. rubrisubalbicans, but not H. huttiense, caused confluent necrosis when infiltrated at high concentrations into tomato leaves and were able to produce leaf spot symptoms on inoculated tomato seedlings in the greenhouse. Using pulsed-field gel electrophoresis, we determined that there was considerable variability between the strains collected in 2001 and 2002.

The relationships among a worldwide collection of 56 strains of Pseudomonas syringae pv. phaseolicola, causal agent of halo blight disease on bean, were investigated by studying the phenotypic and genetic diversity. All P. s. pv phaseolicola strains tested were pathogenic on the bean cultivar 'Canadian Wonder'. Carbon substrate utilization using BIOLOG (Biolog GN2 Microplate test) clustered all the phaseolicola strains together. The use of the phaseolotoxin gene cluster as a polymerase chain reaction (PCR) target detected all the P. s. pv. phaseolicola strains identified as toxin producers (Tox + ) except for one strain (NCPPB 2385) from Australia. Grouping of P. s. pv. phaseolicola by fatty acid composition suggested the existence of five clusters. A majority of the strains from the USA and Turkey were present in Cluster A while 12 of the 16 strains in group C were from Africa. PmeI and PacI enzymes were used for pulsed-field gel electrophoresis (PFGE) analysis of P. s. pv. phaseolicola. Digestion of chromosomal DNA from P. s. pv. phaseolicola with Pmel and Pad generated 29 and 28 groups, respectively. Determination of similarity coefficients and clustering by UPGMA revealed five clusters. More diversity was observed among strains with PFGE than with fatty acid profiling. The results obtained in this study suggest that although a number of strains formed small clusters based on their geographical origin, a clear segregation cannot be concluded. The uniformity of the strains in Turkey isolated in 1994 could possibly indicate a recent introduction of a population of closely related strains. Although only a limited number of strains from the USA were compared, it is interesting to note that the strains were phylogenetically closely related considering that they spanned approximately 20 years. The oldest strain, NCPPB 52 from Canada, which was collected in 1941, had identical PFGE patterns with several strains from South Africa and one strain in Turkey collected in the 1990s. The presence of identical PFGE groups in more than 1 year in South Africa and Germany and the phylogenetically closely related group in the USA coupled with the fatty acid data would indicate the likelihood for local seed sources and/or endemic populations.

Bacterial blight is an emerging disease that affects primarily onion, but also garlic and Welsh onion. The present study was undertaken to characterize the causative xanthomonad(s) by a polyphasic approach using a worldwide collection of 33 bacterial strains. Analysis of 16S rRNA gene sequence similarities indicated that the causal agent belongs to the campestris core in the genus Xanthomonas, which is in agreement with results of phenotypic characterization (analyses of carbon source utilization and fatty acid methyl esters). However, DNA–DNA hybridization, thermal stability of DNA reassociation and fluorescent amplified fragment length polymorphism analysis allowed the causal agent to be identified as a pathovar of Xanthomonas axonopodis.

Pathogen host shifts represent a major source of new infectious diseases. There are several examples of cross-genus host jumps that have caused catastrophic epidemics in animal and plant species worldwide. Cross-kingdom jumps are rare, and are often associated with nosocomial infections. Here we provide an example of human-mediated cross-kingdom jumping of Exserohilum rostratum isolated from a patient who had received a corticosteroid injection and died of fungal meningitis in a Florida hospital in 2012. The clinical isolate of E. rostratum was compared with two plant pathogenic isolates of E. rostratum and an isolate of the closely related genus Bipolaris in terms of morphology, phylogeny, and pathogenicity on one C3 grass, Gulf annual rye grass (Lolium multiflorum), and two C4 grasses, Japanese stilt grass (Microstegium vimineum) and bahia grass (Paspalum notatum). Colony growth and color, as well as conidia shape and size were the same for the clinical and plant isolates of E. rostratum, while these characteristics differed slightly for the Bipolaris sp. isolate. The plant pathogenic and clinical isolates of E. rostratum were indistinguishable based on morphology and ITS and 28S rDNA sequence analysis. The clinical isolate was as pathogenic to all grass species tested as the plant pathogenic strains that were originally isolated from plant hosts. The clinical isolate induced more severe symptoms on stilt grass than on rye grass, while this was the reverse for the plant isolates of E. rostratum. The phylogenetic similarity between the clinical and plant-associated E. rostratum isolates and the ability of the clinical isolate to infect plants suggests that a plant pathogenic strain of E. rostratum contaminated the corticosteroid injection fluid and was able to cause systemic disease in the affected patient. This is the first proof that a clinical isolate of E. rostratum is also an effective plant pathogen.

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.

Since the late 1980s, a bacterium has been routinely isolated from leafspots on geranium (Pelargonium X hortorum L. H. Bailey) and more infrequently from petunia (Petunia hybrida). When inoculated on healthy geranium and petunia leaves, these bacteria caused leafspots identical to those observed on naturally infected geranium plants. Carbon substrate utilisation (BIOLOG) and fatty acid analysis (FAME) identified these isolates to the genus Acidovorax. According to BIOLOG, FAME, and pulsed-field gel electrophoresis (PFGE) data, the geranium and petunia strains formed a very tight phenotypic group that was distinct from other Acidovorax strains tested. In DNA:DNA hybridisation analyses, the geranium strains had greater than 90% homology to each other and to the petunia strain, but less than 50% homology to other Acidovorax species with the exception of A. konjaci (52%). Sequence analysis of the 16S rRNA revealed that the geranium and petunia strains had 99% homology with A. konjaci. Based on PFGE, BIOLOG, FAME, and DNA:DNA hybridisation results, the geranium petunia strains are very closely related to each other and are most closely related to A. konjaci.

In October 2001, tomato foliage with blighting and a leaf spot was received from a transplant producer in Florida. The seedlings manifested apical or marginal leaf necrosis or discrete lesions along the leaf veins. Non-fluorescent bacterial strains forming viscous, creamy white colonies on King's medium B and causing a hypersensitive reaction in tomato and pepper leaves was consistently isolated from lesions. Pathogenicity was checked on three-week old tomato plants. The plants were incubated in high humidity for 24 h before and after inoculation. Similar symptoms were observed on the inoculated seedlings. We characterised 12 strains using bacteriological tests. According to the fatty acid profile, the strains displayed highest similarity with the bacterium Pseudomonas huttiensis Leifson 1962. Sequence analysis of the 16S rRNA indicated that this bacterium shows 98.7divided by98.8% homology with two Herbaspirillum species. Thus, our strains were compared with one P. huttiensis and five H. rubrisubalbicans strains. The strains from tomato were Gram-negative, non-fluorescent and oxidase positive, but levan, pectate hydrolysis and arginine dihydrolase negative, and grew at 40degreesC. Based on these results, they had a high degree of similarity with both P. huttiensis and H. rubrisubalbicans.

Accurate identification of an unknown strain to species requires detailed analysis. Recently, many published reports have used a polyphasic approach (i.e., a combination of phenotypic and genotypic techniques) for placement of bacterial strains into discrete species (17). Before accurate identification of an organism from a particular host can be made, it is essential to identify the phenotypic and genotypic diversity that exists within a group of closely related organisms associated with a host. Using the group of pathogens associated with bacterial spot of tomato and pepper (i.e., Xanthomonas campestris pv. vesicatoria, X. vesicatoria, and X. gardneri, which forms four distinct phenotypic groups (A-D), I will discuss the use of serology, select biochemical and physiological tests, including automated and semiautomated techniques such as FAME (fatty acid methyl esterase) analysis and BIOLOG, as well as genetic tests (eg., pulsed-field gel electrophoresis, PGR and sequence analysis of 16S rRNA) for characterizing organisms to genus and species. Once the variation is determined and differential techniques are identified, assays (i.e., phenotypic or genotypic) can be developed to identify the organism.

Tolumnia orchids are small epiphytic orchids grown for their attractive flowers. In the fall of 2008, approximately 100 Tolumnia orchids with soft, brown, macerated leaves were brought to the University of Florida Extension Plant Diagnostic Clinic in Homestead. Ten plants were randomly selected and bacteria were isolated from the margins of symptomatic tissues of each of the 10 plants on nutrient agar according to the method described by Schaad et al. (2). Four reference strains were used in all tests, including the molecular tests: Erwinia carotovora subsp. carotovora (obtained from J. Bartz, Department of Plant Pathology, University of Florida, Gainesville), E. chrysanthemi (ATCC No. 11662), Pectobacterium cypripedii (ATCC No. 29267), and Acidovorax avenae subsp. cattleyae (ATCC No. 10200). All 10 of the isolated bacteria were gram negative, grew at 37°C, degraded pectate in CVP (crystal violet pectate) medium, grew anaerobically, produced brown pigment on NGM (nutrient agar-glycerol-manganese chloride) medium (1), were sensitive to erythromycin, and produced phosphatase. Three of the strains were submitted for MIDI analysis (Sherlock version TSBA 4.10; Microbial Identification, Newark DE) (SIM 0.732 to 0.963), which identified them as E. chrysanthemi. A PCR assay was performed on the 16S rRNA gene with primers 27f and 1495r described by Weisburg et al. (3) from two of the isolates and a subsequent GenBank search showed 99% identity of the 1,508-bp sequence to that of Dickeya chrysanthemi (Accession No. FM946179) (formerly E. chrysanthemi). The sequences were deposited in GenBank (Accession Nos. GQ293897 and GQ293898). Pathogenicity was confirmed by injecting approximately 100 μl of a bacterial suspension at 1 × 108 CFU/ml into leaves of 10 Tolumnia orchid mericlones. Ten plants were also inoculated with water as controls. Plants were placed in a greenhouse at 29°C with 60 to 80% relative humidity. Within 24 h, soft rot symptoms appeared on all inoculated leaves. The water controls appeared normal. A Dickeya sp. was reisolated and identified using the above methods (biochemical tests and MIDI), fulfilling Koch's postulates. To our knowledge, this is the first report of a soft rot caused by a Dickeya sp. on Tolumnia orchids. Although 16S similarity and MIDI results suggest the isolated bacteria are D. chrysanthemi because of its close similarity with other Dickeya spp., these results are not conclusive. Further work should be conducted to confirm the identity of these isolates. Through correspondence with South Florida Tolumnia growers, it appears this disease has been a recurring problem, sometimes affecting international orchid shipments where plant losses have been in excess of 70%. References: (1) Y. A. Lee and C. P. Yu. J. Microbiol. Methods 64:200, 2006. (2) N. W. Schaad et al. Erwinia soft rot group. Page 56 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al., eds. American Phytopathological Society. St. Paul, MN, 2001. (3) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.

During the 2003 winter growing season, a disease occurred on tomato (Lycopersicon esculentum L.) cvs. Selin, Astona, 198 RN, Malike, and Tulin that were growing in almost all greenhouses of Serik, Aksu, Kumluca, Demre, and Kinik in the Antalya Province, and Keçiborlu, Çandir, and Şeyhler in the Isparta Province in the western Mediterranean Region of Turkey. Disease incidence ranged from 26 to 65%, resulting in economically heavy losses. Symptoms were water-soaked, dark brown-to-black lesions on the leaf margins and asymmetrical wilting of the leaflets. In advanced stages of disease, vascular tissue had a light brown discoloration. A gram-positive bacterium was consistently isolated and formed light gray colonies with internal light gray flecks approximately 2 to 3 mm in diameter on the modified semiselective medium (SCM) (2). Thirty isolates were identified as Clavibacter michiganensis subsp. michiganensis using polymerase chain reaction (PCR) with primers CMM 5 and CMM 6 on the basis of consistent amplification of a 614-bp DNA fragment (1). The isolates were also identified as C. michiganensis subsp. michiganensis (similarity 0.810 to 0.888%) using fatty acid methyl ester (FAME) analysis and Sherlock Microbial Identification System software (Microbial ID, Newark, DE). A pathogenicity test was done by spraying a 6-week-old tomato seedling (cv. Selin) with the bacterial concentration of 108 CFU/ml in sterile water. Control tomato seedlings were only sprayed with sterile water. The inoculated plants were covered with polyethylene bags for 48 h and kept in a growth chamber at 25°C. Symptoms developed on the inoculated plants within approximately 10 days and were similar to those observed in the greenhouse. No symptoms developed on control plants. The bacterium was reisolated from inoculated plants, and its identity was confirmed by colony type on SCM, PCR, and FAME analysis. Although it has been known to be present previously in Aegean, eastern Mediterranean, and the eastern Anatolia regions of Turkey, to our knowledge, this is the first report of bacterial canker on greenhouse tomatoes in the western Mediterranean Region of Turkey. Occurrence of the disease in almost all greenhouses may be the result of the high relative humidity present and the lack of protective bactericide applications in many greenhouses of the region. References: (1) J. Dreier et al. Phytopathology 85:462, 1995. (2) M. Fatmi and N. W. Schaad. Phytopathology 78:121, 1988.

Basidiocarps of C.p. developed in 4-5 wk on mycelial mats supported by sterilized pieces of witches' brooms from cacao, and mats continued to produce basidiocarps for an additional 8 wk. Multibasidiospore and single-basidiospore isolates of the fungus from T.c., Solanum spp. and lianas produced basidiocarps on mycelial mats

Paenibacillus sp. strain JDR-2, an aggressively xylanolytic bacterium isolated from sweetgum (Liquidambar styraciflua) wood, is able to efficiently depolymerize, assimilate and metabolize 4-O-methylglucuronoxylan, the predominant structural component of hardwood hemicelluloses. A basis for this capability was first supported by the identification of genes and characterization of encoded enzymes and has been further defined by the sequencing and annotation of the complete genome, which we describe. In addition to genes implicated in the utilization of β-1,4-xylan, genes have also been identified for the utilization of other hemicellulosic polysaccharides. The genome of Paenibacillus sp. JDR-2 contains 7,184,930 bp in a single replicon with 6,288 protein-coding and 122 RNA genes. Uniquely prominent are 874 genes encoding proteins involved in carbohydrate transport and metabolism. The prevalence and organization of these genes support a metabolic potential for bioprocessing of hemicellulose fractions derived from lignocellulosic resources.

A collection of 41 Erwinia amylovora strains isolated from 8 plant species at 13 locations in Serbia and one location in Montenegro was tested and their relationship was determined based on carbohydrate metabolism and fatty acid composition. Metabolism of carbon compounds was tested using the Biolog GN MicroPlate system. After 24 h of incubation, the resulting metabolic fingerprints were recorded and compared with the Biolog GN database. Fatty acid composition was analyzed by Microbial Identification System and compared with the data from the MIDI database. Our results showed that all strains tested were identified as E. amylovora, with similarity index (SI) between 0.62 and 0.99, using BIOLOG. Based on fatty acid composition, 39 strains were identified as E. amylovora, with SI between 0.61 and 0.92. More than 14 fatty acids were detected, eight of which were present in all strains with more than 1% of the total named peak area. Based on quantitative and qualitative content of fatty acid composition, the isolates were clustered into three groups: alpha, beta and gamma (Euclidian distance lt = 6). Cluster analysis revealed that groups beta and gamma consists of only strains isolated from north Serbia, whereas all strains isolated from central or south Serbia belong to group alpha.

Bacterial pathogens have not been reported in association with Theobroma cacao. However, three strains of Agrobacterium tumefaciens isolated from chrysanthemum induced galls in seedling cacao plants grown in a greenhouse. Since T. cacao is thus a proven host for the crown gall bacterium, the Ti plasmid of A. tumefaciens may possibly be used as a vector for the introduction of selected DNA into cacao