Influence of soil microbes on Escherichia coli O157:H7 survival in soil rinse and artificial soil.

AIMS: This research investigated the influence of soil microbiota on Escherichia coli O157:H7 survival in soil rinse and artificial soil. Additionally, the influence of selected soil bacteria on E. coli O157:H7 in soil environments was determined. METHODS AND RESULTS: Escherichia coli O157:H7 counts (log CFU per ml or g-1 ) were determined by spread plating: (i) artificial soil amended with soil rinse (filter-sterilized and unfiltered) at 30°C; (ii) unfiltered soil rinse (50 ml) treated with cycloheximide (200 µg ml-1 ), vancomycin (40 µg ml-1 ), heat (80°C, 15 min) and no treatment (control) for 7 days at 30°C and (iii) filtered soil rinse with selected soil bacterial isolates over 7 days. There was a significant difference (P = 0·027) in E. coli O157:H7 counts after 35 days between artificial soils amended with filtered (4·45 ± 0·29) and non-filtered (1·83 ± 0·33) soil rinse. There were significant differences (P < 0·05) in E. coli O157:H7 counts after 3 days of incubation between soil rinse treatments (heat (7·04 ± 0·03), cycloheximide (6·94 ± 0·05), vancomycin (4·26 ± 0·98) and control (5·00 ± 0·93)). Lastly, a significant difference (P < 0·05) in E. coli O157:H7 counts was observed after 3 days of incubation at 30°C in filtered soil rinse when incubated with Paenibacillus alvei versus other soil bacterial isolates evaluated. CONCLUSIONS: Soil microbiota isolated from Florida sandy soil influenced E. coli O157:H7 survival. Specifically, P. alvei reduced E. coli O157:H7 by over 3 log CFU per ml after 3 days of incubation at 30°C in filtered soil rinse. SIGNIFICANCE AND IMPACT OF THE STUDY: This research identified soil bacterial isolates that may reduce E. coli O157:H7 in the soil environment and be used in future biocontrol applications.

Escherichia coli O157 survival in liquid culture and artificial soil microcosms with variable pH, humic acid and clay content.

AIMS: This research was performed to investigate the influence of clay and humic acid on Escherichia coli O157 survival in model soils. Additionally, the influence of pH and humic acid on E. coli O157 in liquid culture was investigated. METHODS AND RESULTS: Artificial soil microcosms were prepared with sand, kaolinite, bentonite and humic acid. Artificial soil microcosms pH was adjusted (6·0-7·0) with aluminium sulphate before E. coli O157 inoculation. After 56 days of incubation at 30°C, significant differences in E. coli O157 log CFU per gram were observed between 0 and 1000 ppm (P < 0·0001) and 0 and 5000 ppm (P < 0·0001) humic acid in 1·5% clay soils, but not in 7·5 or 15% clay soils. Significant differences (P < 0·05) in E. coli O157 log CFU per ml were observed in liquid culture influenced by humic acid concentrations after 8 h at 37°C. CONCLUSIONS: The developed model soils support E. coli O157 populations over 28 days, and higher clay soils may aid in E. coli O157 survival. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide insights into physicochemical properties of soil that may influence E. coli O157 in the environment and help explain E. coli O157 survival in various soils and geographical regions.

Measurement of the Permanent Electric Dipole Moment of the Neutron.

We present the result of an experiment to measure the electric dipole moment (EDM) of the neutron at the Paul Scherrer Institute using Ramsey's method of separated oscillating magnetic fields with ultracold neutrons. Our measurement stands in the long history of EDM experiments probing physics violating time-reversal invariance. The salient features of this experiment were the use of a ^{199}Hg comagnetometer and an array of optically pumped cesium vapor magnetometers to cancel and correct for magnetic-field changes. The statistical analysis was performed on blinded datasets by two separate groups, while the estimation of systematic effects profited from an unprecedented knowledge of the magnetic field. The measured value of the neutron EDM is d_{n}=(0.0±1.1_{stat}±0.2_{sys})×10^{-26} e.cm.

Odontonema cuspidatum and Psychotria punctata, Two New Hosts of Cucumber mosaic virus in the United States.

Cucumber mosaic virus (CMV) has a reported host range of 750 to 1,200 species (2,3) that includes weeds, wild plants, crops, and ornamentals. Two new CMV hosts were recently identified in Florida. In July 2011, leaves of Odontonema cuspidatum (firespike), a member of the Acanthaceae, with virus-like symptoms were sent to FDACS-DPI. Firespike is an ornamental shrub native to Mexico with evergreen ovate leaves tapering to a pointed tip. Leaf symptoms included severe leaf distortion with some subtle yellowing or mosaic on younger leaves. Pink-red crystals were seen in leaf strips stained with the nucleic acid stain Azure A, indicating a viral infection. In January 2012, leaves of Psychotria punctata (dotted wild coffee), a member of the Rubiaceae, with virus-like symptoms were sent to FDACS-DPI. Dotted wild coffee is a small exotic tropical tree found in south Florida with many tiny leaf nodules inhabited by endosymbiotic bacteria. In addition to the nodules, these leaves had many large dark green ringspots surrounded with a yellow halo. Both samples were positive for CMV when tested with ImmunoStrips and/or by conventional ELISA using CMV antiserum (Agdia, Elkhart, IN). To confirm CMV infection, reverse transcriptase (RT)-PCR on total RNA from a leaf sample of each plant species was used with previously published cucumovirus primers (1). An expected ~940 bp product was amplified from each sample and cloned into pGEM-T (Promega, Madison, WI). Ten clones from each plant species were sequenced in both directions. After removal of primer sequences, the 906 bp products were 96.3% identical with each other and showed 96.8 to 98.9% nucleotide identity with CMV sequences from Hungary, the United States, and Austria (GenBank Accession Nos. AF517802, U20668, and HQ916354, respectively). Identification of CMV infection in these two species expands the known host range and therefore the reservoir of this plant virus. This has implications for the ornamental industry in general and Florida farmers in particular. References: (1) S. K. Choi et al. J. Virol. Methods 83:67, 1999. (2) E. J. Sikora. Cucumber Mosaic Virus, Pant Disease Notes, Alabama Cooperative Extensions System, retrieved online at http://www.aces.edu/pubs/docs/A/ANR-0868/ANR-0868.pdf , 2004. (3) T. A. Zitter and J. F. Murphy. The Plant Health Instructor. DOI: 10.1094/PHI-I-2009-0518-01, 2009.

Prions are secreted in milk from clinically normal scrapie-exposed sheep.

The potential spread of prion infectivity in secreta is a crucial concern for prion disease transmission. Here, serial protein misfolding cyclic amplification (sPMCA) allowed the detection of prions in milk from clinically affected animals as well as scrapie-exposed sheep at least 20 months before clinical onset of disease, irrespective of the immunohistochemical detection of protease-resistant PrP(Sc) within lymphoreticular and central nervous system tissues. These data indicate the secretion of prions within milk during the early stages of disease progression and a role for milk in prion transmission. Furthermore, the application of sPMCA to milk samples offers a noninvasive methodology to detect scrapie during preclinical/subclinical disease.

A New Host Diagnosed with a Strain of Sugarcane mosaic virus in Florida: Red-Veined Prayer Plant (Maranta leuconeura erythroneura).

Prayer plants (Maranta spp.), which are indigenous to Brazil, are commercially produced in nurseries mainly to be sold as potted houseplants. However, in frost-free areas, they are also used as a ground cover. In March 2009, the Division of Plant Industry in Gainesville, FL received cuttings of red-veined prayer plant or red maranta (Maranta leuconeura erythroneura) from several nurseries in central Florida. The cuttings originated in Costa Rica. The presence of a viral infection was indicated by the mosaic pattern seen on the upper surface of the leaves and chlorotic lesions on the underside of leaves. Epidermal strips were taken and stained in Orange-Green (O-G) and Azure A (1). Microscopic examination revealed viral inclusions that stained only in O-G, indicating the presence of a potyvirus. Leaf dips were prepared for the electron microscope and flexuous rods consistent with a potyvirus were found. Indirect-ELISA using universal potyvirus antiserum (Agdia Inc., Elkhart, IN) confirmed the presence of a potyvirus infection. Total RNA was extracted from symptomatic tissue of five infected samples with Qiagen's RNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA). Reverse transcription was conducted with the oligo d(T) primer M4T with AMV-RT at 37°C for 1 h. PCR was performed with primer M4 and a degenerate primer designed to amplify the 3' end of all potyviruses (2). A target amplicon of 1.7 kb was produced from all five samples. Three of these samples were cloned and sequenced. Approximately 1,250 bp were sequenced from each sample. The sequenced regions include the 3' end of the Nib gene, the complete coat protein, and the beginning of the 3' untranslated region (UTR). The nucleotide (nt) sequences were deposited into GenBank (Accession Nos. GQ853403-GQ853405). The nt sequences of the three samples were 97 to 98% identical to each other. When compared with other potyviruses in the GenBank, the samples showed closest nt identity, 92 to 93%, with several isolates of Sugarcane mosaic virus (AJ278405, AY836523, U57357, and U57356). Of the plant species mechanically inoculated (Chenopodium amaranticolor, C. quinoa, Datura stramonium, Gomphrena globosa, Nicotiana benthamiana, and Zea mays), only Z. mays (corn) showed symptoms (a mild mosaic). The same type of viral inclusions were seen in leaf strips of infected corn as in the Maranta. The corn plant reacted positively in direct-ELISA against antiserum to Sugarcane mosaic virus (Agdia Inc.). Cuttings of infected Maranta were observed in the greenhouse and indoor situations for several months. The plants infected with Sugarcane mosaic virus lacked vigor and most eventually died. ELISA tests done on a few surviving plants were positive for Sugarcane mosaic virus, but the results were inconsistent, indicating a possible low titer of virus in the plants as they were dying. To our knowledge, this is a new host for Sugarcane mosaic virus, but it does not appear that Maranta will become a significant new reservoir of this virus for sugarcane or corn growers. However, infected cuttings can greatly decrease the production of this plant as an ornamental. References: (1) J. R. Edwardson and R. G. Christie. Univ. Fla. Inst. Food Agric. Sci. Bull. 894, 1966. (2) A. Gibbs and A. Mackenzie. J. Virol. Methods 63:9, 1997.

Expression of a P-selectin ligand in zona pellucida of porcine oocytes and P-selectin on acrosomal membrane of porcine sperm cells. Potential implications for their involvement in sperm-egg interactions.

The selectin family of cell adhesion molecules mediates initial leukocyte adhesion to vascular endothelial cells at sites of inflammation. O-glycan structural similarities between oligosaccharides from human leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) and from zona pellucida glycoproteins of porcine oocytes indicate the possible existence of a P-selectin ligand in the zona pellucida. Here, using biochemical as well as morphological approaches, we demonstrate that a P-selectin ligand is expressed in the porcine zona pellucida. In addition, a search for a specific receptor for this ligand leads to the identification of P-selectin on the acrosomal membrane of porcine sperm cells. In vitro binding of porcine acrosome-reacted sperm cells to oocytes was found to be Ca2+ dependent and inhibitable with either P-selectin, P-selectin receptor-globulin, or leukocyte adhesion blocking antibodies against P-selectin and PSGL-1. Moreover, porcine sperm cells were found to be capable of binding to human promyeloid cell line HL-60. Taken together, our findings implicate a potential role for the oocyte P-selectin ligand and the sperm P-selectin in porcine sperm-egg interactions.

Tomato spotted wilt virus Found in Five Species of the genus Tragopogon in a Florida Greenhouse.

An obviously unhealthy plant identified as Tragopogon mirus Ownbey (remarkable goatsbeard) was sent for diagnosis to the Division of Plant Industry (DPI), Gainesville, FL in May of 2008. T. mirus is a recently formed allotetraploid that has T. dubius Scop. and T. porrifolius L. (goatsbeard or salsify) as parents. The parents (family Asteraceae) are diploid and originate from Eurasia. They were introduced to the northwest United States in the early 1900s. The allotetraploid T. mirus, which does not occur in Eurasia, was discovered in 1949 and named in 1950. It has been found in the northwest states of Washington and Idaho. It has also been found in Arizona (4). The plant sent to the DPI was grown in a greenhouse for research purposes at the Botany Department of the University of Florida (Alachua County). Symptoms exhibited on the leaves included mottling, chlorotic and necrotic spots, and mild distortion. Epidermal leaf strips from a mottled leaf were stained with the Orange-Green protein stain and Azure A nucleic acid stain (1). With a light microscope, granular inclusions typical for Tomato spotted wilt virus (TSWV) (1) were seen in leaf strips from both stains. The remainder of the leaf was ground in buffer and tested serologically for TSWV by TSWV-specific ImmunoStrips (Agdia, Elkhart, IN). The ImmunoStrip was positive for the presence of TSWV. This test was confirmed by double-antibody sandwich-ELISA using antiserum and conjugate for TSWV (Agdia). Further serological testing of other Tragopogon species with similar symptoms growing in the same greenhouse revealed that T. miscellus (another recently formed allotetraploid found in the northwestern United States; parents T. dubius and T. pratensis), T. dubius, T. porrifolius, and T. pratensis were also infected with TSWV. Total RNA was extracted from symptomatic leaves of T. mirus, T. dubius, T. porrifolius, and T. miscellus. Reverse transcription-PCR was performed with universal tospovirus primers BR60 and BR65 that amplify part of the nucleocapsid protein gene (2). Target amplicons of 454 bp were produced for all four samples. The PCR product from T. porrifolius was cloned and sequenced. The resulting sequence (GenBank Accession No. FJ655913) shows high homology, 98%, to several isolates of the Tomato spotted wilt virus deposited in the GenBank (Accession Nos. AY870391, AY744477, and AF020659). T. porrifolius has been reported to be naturally infected with TSWV in Italy (3); however, to our knowledge, this is the first report of this virus in the allotetraploids T. mirus and T. miscellus and in the diploids T. dubius and T. pratensis. This report adds five new Asteraceae weeds to the list of possible reservoirs of TSWV in the United States. References: (1) J. R. Edwardson and R. G. Christie. Univ. Fla. Inst. Food Agric. Sci. Bull. 894. 1996. (2) M. Eiras et al. Fitopatol. Bras. 26:170, 2001. (3) G. Parrella et al. J. Plant Pathol. 85:227. 2003. (4) D. E. Soltis et al. Biol. J. Linn. Soc. 82:2004.

Bidens mottle virus and Apium virus Y Identified in Ammi majus in Florida.

Ammi majus (bishop's weed), a member of the Apiaceae, is grown from seed for cut flowers in South Florida. In March 2005, plants were found to be showing virus-like symptoms including mosaic, vein clearing, and leaf rugosity (3) that rendered their flowers unmarketable. Inclusion morphology in epidermal strips from these infected plants indicated the presence of one or more potyviruses. This was confirmed by ELISA with commercially available antiserum for potyvirus identification (Agdia, Elkhart, IN). Clover yellow vein virus (ClYVV) was identified by sequencing and confirmed with specific antiserum (4). However, ClYVV was not identified in all potyvirus-infected samples from 2005, indicating the presence of one or more additional potyviruses. Bidens mottle virus (BiMoV) was subsequently identified in one of three potyvirus-infected samples by immunodiffusion tests using specific antiserum for BiMoV (Department of Plant Pathology, University of Florida), cylindrical inclusion morphology in epidermal strips, host range data, and sequencing of cloned reverse transcription (RT)-PCR products from degenerate potyvirus primers (2). Nucleotide and deduced amino acid sequences of a partial polyprotein gene sequence (GenBank Accession No. EU255631) were 95 and 98% identical, respectively, to a Florida isolate of BiMoV recently reported from tropical soda apple (1). Similar virus-like symptoms were again observed in A. majus in January 2007 and persisted through March. ELISA testing again indicated the presence of a potyvirus. However, neither ClYVV nor BiMoV were identified in the initial 2007 samples. Instead, sequence analysis of the cloned RT-PCR products amplified with degenerate potyvirus primers (2) from seven potyvirus-infected samples collected on two dates in January and one each in February and March revealed the presence of Apium virus Y (ApVY). The 3' terminal portion of the genome (GenBank Accession No. EU255632) was found to be 90 to 91% identical to ApVY sequences in GenBank at the nucleotide level. Deduced amino acid sequences of the NIb and CP regions of these RT-PCR products were 96 and 95% identical, respectively, to ApVY sequences in GenBank. One of these seven ApVY-infected samples (collected in March 2007) was determined to be coinfected with BiMoV by sequence analysis of the cloned RT-PCR products. Six clones were sequenced. Three were determined to be ApVY as indicated above. Nucleotide and deduced amino acid sequences of a partial polyprotein gene sequence from the other three clones were 95 and 97% identical, respectively, to the 2005 A. majus BiMoV isolate. Although ClYVV and BiMoV have previously been reported in other hosts in Florida, to the best of our knowledge, this is the first report of BiMoV and ApVY in A. majus anywhere and the first report of ApVY in North America. References: (1.) C. A. Baker et al. Plant Dis. 91:905, 2007. (2.) A. Gibbs and A. J. Mackenzie. J. Virol. Methods 63:9, 1997. (3.) M. S. Irey et al. (Abstr.) Phytopathology (suppl.)95:S46, 2005. (4.) M. S. Irey et al. Plant Dis. 90:380, 2006.

Impatiens necrotic spot virus and Tomato spotted wilt virus Diagnosed in Phalaenopsis Orchids from Two Florida Nurseries.

In October 2006 (Arcadia, FL) and January 2007 (Sorrento, FL), several white Phalaenopsis orchids with large chlorotic/necrotic ringspot symptoms were sent to the Division of Plant Industry, Gainesville, FL. Symptomatic leaf tissues were tested with the Agdia immunostick-comb (Agdia, Elkhart, IN) for Impatiens necrotic spot virus (INSV), Tomato spotted wilt virus (TSWV), Cucumber mosaic, and Tobacco mosaic virus. Plants from the nursery in Sorrento, FL tested positive for TSWV, while those from the nursery in Arcadia, FL tested positive for INSV. Symptomless leaves from the infected plants tested negative for the viruses with the immunostick-comb. The plants also were tested for TSWV and INSV by double-antibody (DAS)-ELISA (Agdia Inc.) with the same results. Total RNA was extracted from one symptomatic orchid leaf from each nursery. Reverse transcription (RT)-PCR was performed with the universal tospovirus primer set BR60and BR65 (1). PCR bands of the expected size were amplified from each leaf. PCR products were sequenced directly. The orchid leaf that tested positive for TSWV by ELISA produced a 495-bp sequence with 97% identity to several isolates of the TSWV nucleocapsid protein gene listed in GenBank (Accession Nos. AY744479, AY8770391, DQ376185, and AF02659). The orchid leaf that tested positive for INSV by ELISA produced a 396-bp sequence with 98 to 99% identity to several isolates of the INSV nucleocapsid protein gene (Accession Nos. D00914, DQ425096, X66972, and AD109100). Although these viruses have been reported a few times in orchids previously (2,3), to our knowledge, this is the first time they have been reported in this host in Florida. In addition, white Phalaenopsis spp. appears to be a local lesion host and not a systemic host for these viruses. References: (1) M. Eiras et al. Fitopatol. Bras. 26:170, 2001. (2) J. S. Hu et al. Plant Dis. 77:464, 1993. (3) S. T. Koike and D. E. Mayhew. Orchids 70:746, 2001.

A New Potyvirus found in Passiflora incence in Florida.

During March of 2004 (Alachua County) and again during February of 2006 (Highlands County), specimens of the plant Passiflora incence (passionfruit) with chlorotic symptoms were submitted to the Division of Plant Industry, Gainesville, FL for diagnosis. Cytoplasmic cylindrical inclusions seen in epidermal strips of plant leaves stained in Luxol brilliant green and calcomine orange but not seen in those stained in Azure A indicated the presence of a potyvirus infection. Leaf dips made for electron microscopy also showed virus particles consistent with a potyvirus infection. Reverse-transcription (RT)-PCR using degenerate potyvirus primers (4) produced a target ≈1.7-kb potyvirus band. Approximately 1.4 kb of the PCR fragments from both specimens was sequenced and was 100% identical. The 1.4-kb fragment contained the 3' end of the NIb region, the coat protein, and the beginning of the 3'UTR. A GenBank BLAST search found that the two most similar potyviruses were Bean common mosaic necrosis virus (BCMNV), also known as serotype A of Bean common mosaic virus (BCMV), and Soybean mosaic virus (SMV). Molecular analysis of the 1.4-kb sequence using MegAlign (DNASTAR, Madison, WI) indicated a 73% identity with BCMNV and 68.8% with SMV. Analysis of the coat protein showed the highest identity (87%) with BCMNV, and for the NIb region, the highest identity occurred with SMV at 78%. In double-antibody sandwich (DAS)-ELISA, the virus did not react with antisera to either BCMV or BCMNV (BioReba). In sodium dodecyl sulfate (SDS)-immunodiffusion tests, however, the virus reacted heterologously with antiserum to Peanut stripe virus, now considered a member of the serotype B group of BCMV (1). In host range studies, this virus induced systemic chlorosis in Chenopodium quinoa but did not cause symptoms on any other host inoculated, including eight leguminous species and Nicotiana benthamiana. N. bethamiana is susceptible to BCMV and three other known Passiflora potyviruses, Passionfruit crinkle virus (PCV), Passionfruit woodiness virus (PWV), and Passionfruit mottle virus (PFMoV) (2,3). The cylindrical inclusions of this virus seen with the light microscope appeared as loosely aggregated medium length plate or needle-like structures as opposed to long, compact, bundle-shaped aggregates (PFMoV and PCV) or short plate-like structures (PWV) (2,3). The virus did not react with antisera (W. Zettler, University of Florida) to the three aforementioned passionfruit potyviruses in SDS-immunodiffusion tests. This virus, like PCV, PWV, and PFMoV, is related to the Bean common mosaic virus group. However, based on cylindrical inclusion morphology, host range, serology, and genetic sequencing, the virus appears to be a new potyvirus infecting Passiflora and is tentatively named Passiflora chlorosis virus. The sequence was deposited in GenBank as Accession No. DQ860147. References: (1) A. A Brunt et al. Plant Viruses Online: Descriptions and Lists from the VIDE Database. Version 20, August 1996. (2) C. A. Chang. Phytopathology. 82:1358, 1992 (3) C. A. Chang et al. Plant Prot. Bull. 38:339, 1996. (4) A. Gibbs and A. Mackenzie. J. Virol. Methods 63:9, 1997.

Tobacco ringspot virus Found in the Cardboard Cycad (Zamia furfuracea) in Florida.

Zamia furfuracea (Zamiaceae) is native of coastal Mexico. It is a popular houseplant and easy to grow outdoors in warm climates. In November 2005, a plant of Z. furfuracea, originally from Texas, was received at the Division of Plant Industry in Gainesville, FL. The plant had numerous chlorotic spots on the leaves that eventually became necrotic. Leaves were ground in phosphate buffer (pH 7.2) with Carborundum and used to inoculate a host range that included Chenopodium amaranticolor, C. quinoa, Gomphrena globosa, Datura stramonium, and Nicotiana benthamiana. Systemic symptoms were seen in C. quinoa (necrotic lesions), G. globosa (stunting), D. stramonium (chlorotic ringspots), and N. benthamiana (wavy line patterns) 1 to 2 weeks after inoculation. C. amaranticolor showed only small necrotic local lesions. In further host range studies, systemic infections of Beta vulgaris, D. metaloides, Lactuca sativa, N. clevelandii, Pisum sativus, Petunia hybrida, Zinnia elegans (symptomless), and Cucumis sativa were observed. However, no infection of Zea mays, Verbena hybrida, Glycine max, Phaseolus vulgaris, Catharanthus roseus, Arachis hypogaea, Trifolium spp., Vigna unguiculata, Vicia faba or Digitalis spp. was detected. Inclusions observed in leaf strips of N. benthamiana and D. stramonium indicated a possible infection of this plant with a nepovirus (1). A 337-bp fragment was amplified from total RNA isolated from an inoculated D. stramonium using reverse transcription-PCR with nepovirus group primers provided by Agdia Inc. (Elkhart, IN). Sequence analysis indicated that the nucleotide (nt) and deduced amino acid (aa) sequences of the fragment were 89 to 91% and 91 to 95% identical, respectively, to sequences of the RNA-dependent RNA polymerase gene for Tobacco ringspot virus (TRSV) contained in GenBank (Accession Nos. U50869 and AJ698718). This region was only 50% (nt) and 38% (aa) identical to Cycas necrotic stunt virus (GenBank Accession No. NC_003791), a nepovirus previously reported to infect cycads (2). The original plant, symptomatic inoculated hosts, and the symptomless zinnia tested positive by double-antibody sandwich-ELISA using commercially available antiserum for TRSV (Agdia, Inc.), further confirming the presence of TRSV. Although the virus infecting Z. furfuracea has a more restricted host range than that reported for TRSV, the serology and gene sequence indicates that this virus is a unique isolate of TRSV. References: (1) J. R. Edwardson and R. G. Christie. University of Florida, Institute of Food and Agricultural Sciences, Bull. 894. 1996. (2) S. S. Han et al. Arch. Virol. 147:2207, 2002.

Bidens mottle virus Identified in Tropical Soda Apple in Florida.

Tropical soda apple (TSA) (Solanum viarum Dunal), a plant native to South America, was first identified in Florida in 1988 (4). It rapidly became a noxious weed in pastures throughout the state and it is known to be a reservoir for Cucumber mosaic virus, Potato leafroll virus, Potato virus Y (PVY), Tobacco etch virus (TEV), Tomato mosaic virus, and Tomato mottle virus, viruses that infect important vegetable crops in Florida (3). During a routine survey of Florida weeds during May of 2004, a TSA plant with chlorotic, young leaves found near Okeechobee, FL was determined to be infected with a potyvirus by using a commercially available enzyme linked immunosorbent assay kit (Agdia, Elkhart, IN). The results of a host range study indicated this potyvirus was neither PVY nor TEV. The virus caused local lesions in Chenopodium amaranticolor and systemic symptoms in C quinoa, Coreopsis sp. (C. A. Baker, unpublished), Helianthus annus, Nicotiana benthamiana, Petunia × hybrida, Verbena hybrida, and Zinnia elegans. It did not infect Gomphrena globosa, N. glutinosa, Pisum sativum, or Phaseolus vulgaris (1). Cylindrical inclusions consistent with those observed in plants infected with Bidens mottle virus (BiMoV) were observed in Z. elegans. Immunodiffusion tests with antiserum to BiMoV (Department of Plant Pathology, University of Florida) gave a reaction of identity with leaf extracts of the symptomatic zinnia, a known sample of BiMoV originally isolated from Bidens pilosa and a recent isolate of BiMoV from lettuce in Belle Glade, FL (C. A. Baker and R. Raid, unpublished). A partial polyprotein gene fragment (GenBank Accession No. EF467235) was amplified from total RNA of an inoculated C. quinoa plant by reverse transcription (RT)-PCR with previously described degenerate potyvirus primers (2). Analysis of the RT-PCR product sequence confirmed the host range results and indicated that the potyvirus infecting TSA was neither PVY nor TEV. However, the nucleotide and deduced amino acid sequences of a 247-bp portion of the RT-PCR product were 94 and 98% identical, respectively, with the coat protein sequence (GenBank Accession No. AF538686) of Sunflower chlorotic spot virus (SCSV). SCSV is a tentative potyvirus species described from Taiwan that is not yet recognized as an accepted species by the International Committee on Taxonomy of Viruses. On the basis of our concurrent host range, inclusion body, and serological data, it is likely that SCSV is in actuality the previously described and currently accepted potyvirus species BiMoV, for which no previous sequence data existed. As part of a comprehensive viral disease management plan, it is recommended that TSA plants growing in and around lettuce-production areas be controlled along with other weed hosts of this virus. References: (1) A. A. Brunt et al., eds. Plant Viruses Online: Descriptions and Lists from the VIDE Database. Version: 20 at http://biology.anu.edu.au/Groups/MES/vide/ , 1996. (2) A. Gibbs and A. J. Mackenzie. Virol. Methods 63:9, 1997. (3) R. J. McGovern et al. Int. J. Pest Manag. 40:270, 1994. (4) J. J. Mullahey et al. Weed Technol. 7:783, 1993.

Variation to cause host injury between Russian wheat aphid (Homoptera: Aphididae) clones virulent to Dn4 wheat.

Biotypes are infraspecific classifications based on biological rather than morphological characteristics. Cereal aphids are managed primarily by host plant resistance, and they often develop biotypes that injure or kill previously resistant plants. Although molecular genetic variation within aphid biotypes has been well documented, little is known about phenotypic variation, especially virulence or the biotype's ability to cause injury to cultivars with specific resistance genes. Five clones (single maternal lineages) of Russian wheat aphid, Diuraphis noxia (Kurdjumov) (Homoptera: Aphididae), determined to be injurious to wheat, Triticum aestivum L., with the Dn4 gene, were evaluated on resistant and susceptible wheat and barley, Hordeum vulgare L., for their ability to cause chlorosis, reduction in plant height, and reduction in shoot dry weight. Variation to cause injury on resistant 'Halt' wheat, susceptible 'Jagger' wheat, and resistant 'STARS-9301B' barley was found among the Dn4 virulent clones. One clone caused up to 30.0 and 59.5% more reduction in plant height and shoot dry weight, respectively, on resistant Halt than other clones. It also caused up to 29.9 and 55.5% more reduction in plant height and shoot dry weight, respectively, on susceptible Jagger wheat. Although STARS-9301B barley exhibited an equal resistant response to feeding by all five clones based on chlorosis, two clones caused approximately 20% more reduction in plant height and shoot dry weight than three other clones. The most injurious clones on wheat were not the most injurious clones on barley. This is the first report of variation to cause varying degrees of plant damage within an aphid biotype virulent to a single host resistance gene. A single aphid clone may not accurately represent the true virulent nature of a biotype population in the field.

An in vitro model for assessing effective scrapie decontamination.

Scrapie infectivity enters the environment via a multiplicity of routes from infected animals. Environmentally associated scrapie persists on farms when infected animals have been removed and is particularly resistant to disinfection. Infectivity within the farm is not adequately removed by current recommended guidelines for farm decontamination. We describe an in vitro method for modelling decontamination, specifically the removal of scrapie prions from the surface of concrete fomites within buildings that have housed scrapie infected animals. Concrete that had been spiked with low amounts of a diluted scrapie positive brain homogenate was sampled before and after decontamination. Extracts were used to seed a semi-quantitative serial protein misfolding cyclic amplification assay (sPMCA). We demonstrate that methods currently recommended for prion decontamination result in inadequate reduction of prion seeding activity within this in vitro assay. Effective treatment was achieved using repeat dosing of surfaces with 20,000ppm available chlorine for 4h.

Alternanthera mosaic virus Found in Scutellaria, Crossandra, and Portulaca spp. in Florida.

In the fall of 1998, the Division of Plant Industry (DPI) received vegetative propagations of Scutellaria longifolia (skullcap) with symptoms of foliar mosaic, chlorotic/necrotic ringspots, and wavy line patterns from a nursery in Manatee County. Flexuous particles approximately 500 nm long were found with electron microscopy. The plants tested positive for Papaya mosaic virus (PaMV) in an enzyme-linked immunosorbent assay (ELISA) test with antiserum to PaMV (Agdia, Elkhart, IN). However, in immunodiffusion tests (antiserum from D. Purcifull, University of Florida), this virus gave a reaction of partial identity indicating it was related but not identical to PaMV (1). The original infected plants were kept in a greenhouse. In January 2005, a specimen of Crossandra infundibuliformis (firecracker plant) with mosaic symptoms was submitted to the DPI from a nursery in Alachua County. Inclusions found with light microscopy and particles found with electron microscopy indicated that this plant was infected with a potexvirus. This was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) with primers designed to detect members of the virus family Potexviridae (3). These plants reacted positive to PaMV antiserum in ELISA and gave a reaction of partial identity to PaMV in immunodiffusion. A specimen of Portulaca grandiflora (moss rose) with distorted leaves found at a local retail store was also tested and gave the same results. Leaves from each of the three plant species were rubbed onto a set of indicator plants using Carborundum and potassium phosphate buffer. Total RNA was extracted from symptomatic indicator plants of Nicotiana benthamiana. RT-PCR (3) was performed, and PCR products were sequenced directly. Sequences of approximately 700 bp were obtained for all three plant species and showed 98% identity with each other. BLAST search results showed that these sequences were 93% identical to an Alternanthera mosaic virus (AltMV) sequence at the nucleotide level but only 76% identical to PaMV. The amino acid sequences were 98 and 82% identical to AltMV and PaMV, respectively. The PCR products of the virus from Scutellaria sp. were cloned, resequenced, and the sequence was entered into the GenBank (Accession No. DQ393785). The bioassay results matched those found for AltMV in Australia (2) and the northeastern United States (4), except that the Florida viruses infected Datura stramonium and Digitalis purpurea (foxglove). The virus associated with the symptoms of these three plants appears to be AltMV and not PaMV. AltMV has been found in ornamental plants in Australia, Italy, and the United States (Pennsylvania, Maryland, and now Florida). Since this virus is known to infect several plants asymptomatically and can be easily confused with PaMV serologically, it is likely that the distribution of this virus is much wider than is known at this time. References: (1) L. L. Breman. Plant Pathology Circular No. 396. Fla. Dept. Agric. Consum. Serv. DPI, 1999. (2) A. D. W. Geering and J. E. Thomas. Arch Virol 144:577, 1999. (3) A. Gibbs et al. J Virol Methods 74:67, 1998. (4) J. Hammond et al. Arch Virol. 151:477, 2006.

Clover yellow vein virus Identified in Ammi majus in Florida.

Ammi majus L., a member of the Apiaceae and also known as large bullwort, false Queen Anne's lace, or bishop's-weed, is frequently used in the floral trade to add a lacey look to floral bouquets. A. majus is native to the Mediterranean Region but it is cultivated in major growing areas including Holland, Israel, the United Kingdom, and the United States. During March 2005, virus-like symptoms including mosaic, generalized chlorosis, vein clearing, interveinal chlorosis, and leaf rugosity were observed in nearly all field-grown A. majus plants at two locations in Martin County, Florida. Inclusion body morphology suggested the presence of one or more potyviruses in the symptomatic plants. Potyvirus infection was confirmed in 11 symptomatic plants using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Agdia, Elkhart, IN). Nucleotide and deduced amino acid sequences of a 1,625-bp region of one of the reverse transcription-polymerase chain reaction products amplified with degenerate potyvirus primers (1) from total RNA of symptomatic plants (GenBank Accession No. DQ333346) were 96 to 97% and 93 to 99% identical, respectively, to Clover yellow vein virus (ClYVV) sequences in GenBank. All symptomatic plants tested were potyvirus positive using ELISA, but only a subset was infected with ClYVV suggesting that the field symptoms were the result of infection with additional potyviruses, all of which are likely aphid transmitted. Although several potyviruses have been reported from A. majus (2), to our knowledge, this represents the first report of ClYVV infection. References: (1) A. Gibbs and A. Mackenzie. J. Virol. Methods 63:9, 1997. (2) P. Van Dijk and L. Bos. Neth. J. Plant Pathol. 95(Suppl.) 2:1, 1989.

Validation of an automated colony counting system for group A Streptococcus.

BACKGROUND: The practice of counting bacterial colony forming units on agar plates has long been used as a method to estimate the concentration of live bacteria in culture. However, due to the laborious and potentially error prone nature of this measurement technique, an alternative method is desirable. Recent technologic advancements have facilitated the development of automated colony counting systems, which reduce errors introduced during the manual counting process and recording of information. An additional benefit is the significant reduction in time taken to analyse colony counting data. Whilst automated counting procedures have been validated for a number of microorganisms, the process has not been successful for all bacteria due to the requirement for a relatively high contrast between bacterial colonies and growth medium. The purpose of this study was to validate an automated counting system for use with group A Streptococcus (GAS). METHODS: Twenty-one different GAS strains, representative of major emm-types, were selected for assessment. In order to introduce the required contrast for automated counting, 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) dye was added to Todd-Hewitt broth with yeast extract (THY) agar. Growth on THY agar with TTC was compared with growth on blood agar and THY agar to ensure the dye was not detrimental to bacterial growth. Automated colony counts using a ProtoCOL 3 instrument were compared with manual counting to confirm accuracy over the stages of the growth cycle (latent, mid-log and stationary phases) and in a number of different assays. The average percentage differences between plating and counting methods were analysed using the Bland-Altman method. RESULTS: A percentage difference of ±10 % was determined as the cut-off for a critical difference between plating and counting methods. All strains measured had an average difference of less than 10 % when plated on THY agar with TTC. This consistency was also observed over all phases of the growth cycle and when plated in blood following bactericidal assays. Agreement between these methods suggest the use of an automated colony counting technique for GAS will significantly reduce time spent counting bacteria to enable a more efficient and accurate measurement of bacteria concentration in culture.

Human islet amyloid polypeptide accumulates at similar sites in islets of transgenic mice and humans.

The cellular mechanisms responsible for conversion of islet amyloid polypeptide (IAPP) into insoluble amyloid deposits in non-insulin-dependent diabetes mellitus (NIDDM) are not clear. Overexpression of IAPP and the amino acid sequence of human IAPP (hIAPP) have both been implicated. To examine factors involved in amyloid formation, transgenic mice expressing the hIAPP or rat IAPP (rIAPP) gene were generated. These mice had elevated plasma IAPP concentrations, and they were normoglycemic and normoinsulinemic. No amyloid deposits were detected by light microscopy. To examine the ultrastructure of islets, pancreatic tissue was studied from hIAPP and rIAPP transgenic mice and from age-matched control mice by immunoelectron microscopy. IAPP was immunolocalized in beta-cell secretory granules of all mice, and the COOH- and NH2-terminal flanking peptides of hIAPP were localized in beta-cell granules of hIAPP mice. Accumulations of nonfibrillar perivascular IAPP-immunoreactive material were found between capillaries and beta-cells in hIAPP transgenic mice but not in rIAPP transgenic or control mice. Similar nonfibrillar masses were identified in islets of an NIDDM patient. Secondary lysosomes in beta-cells and macrophages of hIAPP transgenic mice showed dense labeling for IAPP. We suggest that hIAPP is degraded more slowly than rIAPP or mouse IAPP by beta-cell lysosomes. Accumulations of IAPP in islet perivascular spaces may represent the early stages of islet amyloid formation.

Tomato spotted wilt virus Identified in Desert Rose in Florida.

Desert rose (Adenium obesum (Forssk.) Roem. & Schult), a member of the family Apocynaceae, is characterized by fleshy stems and leaves and colorful flowers. This exotic ornamental, originally from southeast Africa, is propagated vegetatively and is a perennial in warm climates. Virus-like foliar symptoms, including chlorotic ring and line patterns, were observed in the fall of 2004 on one of five stock plants being maintained in a greenhouse in Fort Pierce, FL. Inclusion body morphology suggested the presence of a Tospovirus in the symptomatic plant, and Tomato spotted wilt virus (TSWV) was specifically identified in this plant using a commercially available double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA; Agdia, Elkhart, IN). TSWV was not detected in symptomless desert rose plants nor was Impatiens necrotic spot virus detected in any of the plants using DAS-ELISA. Graft transmission of TSWV to other desert rose plants was successful. Sequence analysis of a nucleocapsid (N) protein gene fragment amplified by reverse transcription-polymerase chain reaction (RT-PCR) with primers TSWV723 and TSWV722 (1) from total RNA of the symptomatic plant confirmed the diagnosis. Nucleotide and deduced amino acid sequences of a 579-bp region of the RT-PCR product were 95 to 99% and 95 to 100% identical, respectively, to TSWV N-gene sequences in GenBank. No product was amplified from symptomless plants. Since these 3-year-old plants were grown on-site from seed and only expressed symptoms 2 months following damage to the greenhouse by hurricanes Frances and Jeanne, it is likely that viruliferous thrips were introduced from local vegetable or ornamental production areas during or following the storms. To our knowledge, this is the first report of TSWV infection of desert rose in Florida, although TSWV was observed in this plant in Europe approximately 10 years ago (3,4). Because of the wide distribution of TSWV in the United States, the increasing popularity of desert rose, and the recent identification of Cucumber mosaic virus in this host (2), attention to sanitation and insect vector management is merited during desert rose propagation and production. References: (1) S. Adkins and E. N. Rosskopf. Plant Dis. 86:1310, 2002. (2) C. A. Baker et al. Plant Dis. 87:1007, 2003. (3) J. Mertelik et al. Acta Hortic. 432:368, 1996. (4) J. Th. J. Verhoeven and J. W. Roenhorst. Acta Hortic. 377:175, 1994.