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Commercial livestock facilities are faced with the challenge of managing large amounts of waste including manure and animal mortalities. One method of disposing of dead animals is composting. The cadavers are enveloped in carbon material that creates a barrier between the dead tissue and the surrounding environment. Dead tissue can release materials that not only contaminate the soil but also the groundwater and nearby surface water. Animal cadaver composting is designed to facilitate decomposition without the aid of carrion-feeding insects and reduce the presence of common pathogens associated with animal waste and dead tissue. The aim of this study was to evaluate insect activity associated with composted and exposed beef cadavers, specifically filth flies that can serve as mechanical vectors of important human pathogens such as E. coli 0157:H7. Greater numbers of all types of arthropods were trapped overall at the exposed animal site than the composted animal site. Most importantly, the number of filth flies was significantly lower at the composted site (P = 0.0009). Laboratory analysis of volatile organic compounds from composted and noncomposted rats indicated that known fly attractants such as dimethyl disulfide may be inhibited by the composting process. Implementing composting programs at livestock facilities could reduce the risk of flies spreading harmful pathogens to surrounding areas, including farms that grow fresh produce.

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A high consequence pathogen, High plains virus (HPV) causes considerable damage to wheat if the crop is infected during early stages of development. Methods for the early, accurate, and sensitive detection of HPV in plant tissues are needed for the management of disease outbreaks and reservoir hosts. In this study, the effectiveness of five methods-real-time SYBR green and TaqMan reverse transcription-quantitative PCR (RT-qPCR), endpoint RT-PCR, RT-helicase dependent amplification (RT-HDA) and the Razor Ex BioDetection System (Razor Ex)-for the broad-range detection of HPV variants was evaluated. Specific PCR primer sets and probes were designed to target the HPV nucleoprotein gene. Primer set HPV6F and HPV4R, which amplifies a product of 96 bp, was validated in silico against published sequences and in vitro against an inclusivity panel of infected plant samples and an exclusivity panel of near-neighbor viruses. The primers were modified by adding a customized 22 nucleotide long tail at the 5' terminus, raising the primers' melting temperature (Tm; ca. 10°C) to make them compatible with RT-HDA (required optimal Tm = 68°C), in which the use of primers lacking such tails gave no amplification. All of the methods allowed the detection of as little as 1 fg of either plasmid DNA carrying the target gene sequence or of infected plant samples. The described in vitro and in-field assays are accurate, rapid, sensitive, and useful for pathogen detection and disease diagnosis, microbial quantification, and certification and breeding programs, as well as for biosecurity and microbial forensics applications.

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More than 3,000 acres of watermelon were planted in Alabama in 2010 with a production value more than $4 million (J. Kemble, personal communication). Symptoms typical of cucurbit yellow vine disease (CYVD) were observed in a 2-ha watermelon field in Crawford, AL on 8 June 2010. Watermelon, cv. Jubilee, exhibited a yellow appearance and some plants were completely wilted. Incidence of affected plants was estimated at 25%. On 24 June, plant samples were collected from a 1-ha watermelon (cv. Jubilee) field near Dadeville, AL. Approximately 30% of the plants exhibited yellowing and wilting, which is symptomatic of CYVD. Samples were also collected from a small planting of yellow crooked-neck squash at the same location. Approximately 20% of the squash plants had symptoms typical of CYVD. Cross-sections of belowground stem and primary root revealed a honey-brown phloem discoloration and a healthy appearing xylem, symptoms consistent with CYVD caused by the phloem-colonizing bacterium, Serratia marcescens Bizio (1). Isolations were made from the crown on four symptomatic watermelon and two squash plants. Approximately 2.5-mm3 tissue pieces from the phloem were excised, surface sterilized in 10% sodium hypochlorite, and ground in 1-ml PBS (phosphate buffer with saline). A 10-µl aliquot of slurry was plated onto nutrient agar (NA) (Difco, Detroit, MI) and the plates were stored at room temperature for 4 days. Individual colonies were selected and purified by serial dilution plating. Isolates from watermelon and squash were consistent with S. marcescens in colony morphology, color, and texture. Three isolates obtained from watermelon were grown on NA and suspended in sterile water at 108 cells per ml for mechanical transmission experiments on 'Lemondrop' squash. Sterile water served as a negative control. After 28 days, plants were cross-sectioned at the juncture of the root and stem and observed for phloem discoloration. Of the 56, 58, and 62 plants inoculated in three replicate studies, 78.6, 56.9, and 62.9% developed CYVD symptoms, respectively, while none of the controls were positive. Cultured bacteria from six of the symptomatic, greenhouse-inoculated plants representing the three watermelon isolates were subjected to multiplex end-point PCR using primer sets YV1/YV4, specific for the species S. marcescens, and a79F/R, which amplifies only the CYVD strains of S. marcescens (3). All six bacteria cultures along with the positive control (reference isolate W01 obtained from watermelon in Texas) were positive, while the negative PBS control was negative. Although rhizosphere-inhabiting and plant growth promoting endophytic strains of S. marcescens have been reported from Alabama (2), to our knowledge, this is the first known report of CYVD and phytopathogenic S. marcescens in Alabama cucurbits. References: (1) B. D. Bruton et al. Plant Dis. 87:937, 2004. (2) J. A. McInroy and J. W. Kloepper. Plant Soil 173:333, 1995. (3) Q. Zhang et al. Appl. Environ. Microbiol. 71:7716, 2005.

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Carrot purple leaf disease was first reported in 2006 in the state of Washington and was associated with Spiroplasma citri. The disease also was reported in California in 2008. The objectives of this work were to fulfill Koch's postulates and to determine 1) whether the beet leafhopper, Circulifer tenellus (Baker) (Hemiptera: Cicadellidae), transmits carrot [Daucus carota L. subsp. Sativus (Hoffm.) Arcang] isolates of S. citri; and 2) whether carrot and citrus [Citrus sinensis (L.) Osb.]-derived spiroplasmas are pathogenic to both plant species. C. tenellus adults received a 24-h acquisition access period to a diet containing carrot-derived S. citri. After 30 d, insects were transferred to healthy carrot seedlings (five per plant). Negative controls were carrot and periwinkle [Catharanthus roseus (L.) G. Don] plants exposed to diet-only-fed insects, and positive controls were periwinkle plants exposed to insects fed on spiroplasma-supplemented diet. Purple carrot leaves and small, chlorotic periwinkle leaves were evident 10-45 d after exposure. Spiroplasmas were reisolated only from symptomatic plants, and polymerase chain reaction (PCR) confirmed their identity as S. citri. No symptoms occurred, and no spiroplasma-specific PCR amplifications or spiroplasma cultures were obtained from plants exposed to diet only-fed insects. Carrot-derived S. citri was transmitted to 15 and 50% of carrot and periwinkle plants exposed, respectively. Insects exposed to S. citri isolates from carrot or citrus transmitted the pathogen to both their host of origin and to the other plant host (carrot or citrus), showing no isolate-host specificity. Our findings confirm that carrot is a host of S. citri. Although carrot is not a preferred host of C. tenellus, it is likely that inoculative leafhoppers feed on carrot during seasonal migration.

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Human pathogens can contaminate leafy produce in the field by various routes. We hypothesized that interactions between Escherichia coli O157:H7 and spinach are influenced by the route of introduction and the leaf microenvironment. E. coli O157:H7 labeled with green fluorescent protein was dropped onto spinach leaf surfaces, simulating bacteria-laden raindrops or sprinkler irrigation, and survived on the phylloplane for at least 14 days, with increasing titers and areas of colonization over time. The same strains placed into the rhizosphere by soil infiltration remained detectable on very few plants and in low numbers (10(2) to 10(6) CFU/g fresh tissue) that decreased over time. Stem puncture inoculations, simulating natural wounding, rarely resulted in colonization or multiplication. Bacteria forced into the leaf interior survived for at least 14 days in intercellular spaces but did not translocate or multiply. Three spinach cultivars with different leaf surface morphologies were compared for colonization by E. coli O157:H7 introduced by leaf drop or soil drench. After 2 weeks, cv. Bordeaux hosted very few bacteria. More bacteria were seen on cv. Space and were dispersed over an area of up to 0.3 mm2. The highest bacterial numbers were observed on cv. Tyee but were dispersed only up to 0.15 mm2, suggesting that cv. Tyee may provide protected niches or more nutrients or may promote stronger bacterial adherence. These findings suggest that the spinach phylloplane is a supportive niche for E. coli O157:H7, but no conclusive evidence was found for natural entry into the plant interior. The results are relevant for interventions aimed at minimizing produce contamination by human pathogens.

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The recent outbreak of Escherichia coli O157:H7 infection associated with contaminated spinach led to an investigation of the role of insects, which frequent fields of leafy greens and neighboring rangeland habitats, in produce contamination. Four leafy greens fields adjacent to cattle-occupied rangeland habitats were sampled using sweep nets and sticky traps. Agromyzid flies, anthomyiid flies, and leafhoppers were caught consistently in both rangeland and leafy greens production fields at all sites. An unexpected number of flies (n = 34) in the Muscidae and Calliphoridae families (known as filth flies because of their development in animal feces) were caught in one leafy greens field. A subset of these filth flies were positive (11 of 18 flies) for E. coli O157:H7 by PCR amplification using primers for the E. coli O157:H7-specific eae gene. Under laboratory conditions, house flies were confined on manure or agar medium containing E. coli O157:H7 tagged with green fluorescent protein (GFP) and then tested for their capacity to transfer the microbes to spinach plants. GFP-tagged bacteria were detected on surfaces of 50 to 100% of leaves examined by fluorescence microscopy and in 100% of samples tested by PCR. These results indicate that flies are capable of contaminating leafy greens under experimental conditions and confirm the importance of further investigation of the role of insects in contamination of fresh produce.

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Spiroplasma citri, a phloem-limited pathogen, causes citrus stubborn disease (CSD). Losses due to CSD in California orchards have grown over the past decade. To investigate the possibility of introduction or emergence of a new strain, a study of genetic diversity among S. citri strains from various locations was conducted using random amplified polymorphism DNA-polymerase chain reaction (RAPD-PCR) of 35 strains cultured from 1980 to 1993, and of 35 strains cultured from 2005 to 2006. Analysis using 20 primer pairs revealed considerable diversity among strains. However, no unique genetic signatures were associated with recently collected strains compared with those collected 15 to 28 years ago, and no geographically associated pattern was distinguishable. S. citri strains from carrot and daikon radish contain some unique DNA fragments, suggesting some host plant influence. Multiple strains from single trees also showed genetic diversity. Sequencing of five RAPD bands that differed among strains showed that diversity-related gene sequences include virus fragments, and fragments potentially encoding a membrane lipoprotein, a DNA modification enzyme, and a mobilization element. No differences in colony morphology were observed among the strains. The lack of correlation between PCR patterns and isolation date or collection site is inconsistent with the hypothesis that recent infections are due to the introduction or emergence of novel pathogen strains.

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Since 1988, cucurbit crops, particularly watermelon, cantaloupe, and squash, grown in Oklahoma and Texas have experienced devastating losses from cucurbit yellow vine disease (CYVD), caused by the phloem-limited bacterium Serratia marcescens Bizio. Squash bug, Anasa tristis (De Geer), is a putative vector of the pathogen. In 2000-2001, overwintering populations of squash bug collected from DeLeon, TX, were tested for their ability to harbor and transmit the bacterium. Individual squash bugs (n = 73) were caged serially for periods of up to 7 d on at least four squash seedlings. Two studies were conducted, one with insects collected in November 2000 placed on first true leaf-stage seedlings and the second with insects from an April 2001 collection, placed on 3-5 true leaf-stage squash. Controls consisted of squash seedlings caged without insects. Squash bug transmission rates of the pathogen in studies I and II were 20 and 7.5%, respectively. Overall, 11.0% of the squash bugs harbored and successfully transmitted the bacterium to squash seedlings. All control plants tested negative for S. marcescens and did not exhibit CYVD. Female squash bugs killed a significantly greater proportion of young first leaf-stage seedlings than males. Feeding on 3-5 leaf-stage squash resulted in no plant mortality regardless of squash bug gender. This study demonstrated that the squash bug harbors S. marcescens in its overwintering state. The squash bug-S. marcescens overwintering relationship reported herein greatly elevates the pest status of squash bug and places more importance on development of integrated strategies for reducing potential overwintering and emerging squash bug populations.

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Cucurbit yellow vine disease (CYVD), which can inflict heavy losses to watermelon, pumpkin, cantaloupe, and squash in U.S. production areas from the midwest to northeastern states, causes phloem discoloration, foliar yellowing, wilting, and plant decline. Bacteria were cultured from the phloem of crown sections of symptomatic plants of Citrullus lanatas and Cucurbita pepo. Those bacteria testing positive in CYVD-specific polymerase chain reaction (PCR) were all gram negative and appeared morphologically identical, producing creamy white, smooth, entire, convex colonies on Luria-Bertani or nutrient agar. Characterized cucurbit-derived strains of Serratia marcescens were introduced into greenhouse-grown squash plants by puncture inoculation and into field-grown squash plants by enclosure with S. marcescens-fed squash bugs, Anasa tristis. Up to 60% of the bacteria-inoculated plants in the greenhouse and up to 17% of field plants caged with inoculative squash bugs developed phloem discoloration and tested positive for S. marcescens by CYVD-specific PCR. None of the controls developed phloem discoloration or tested positive by PCR. Of the diseased field plants, 12% (2 of 35) also yellowed, wilted, and collapsed, exhibiting full symptom development of CYVD. However, neither plant collapse nor decline was observed in the greenhouse-grown, puncture-inoculated plants. The morphology, growth habit, and PCR reaction of bacteria cultured from crown tissue of a subset of plants in each experimental group were indistinguishable from those of the inoculum bacteria. Evidence presented from our studies confirms that the squash bug can transmit S. marcescens, the CYVD causal bacterium. The S. marcescens-A. tristis relationship described here is the first instance in which the squash bug has been identified as a vector of a plant pathogen. Our experiments represent a completion of the steps of Koch's postulates, demonstrating that S. marcescens is the causal agent of CYVD and that the squash bug, A. tristis, is a vector of the pathogen.

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ABSTRACT Two microtiter plate assays were developed to study the adherence of the plant-pathogenic mollicute Spiroplasma citri to a monolayer of cultured cells of its leafhopper vector, Circulifer tenellus. Adherence was significantly reduced by prior treatment of the spiroplasmas with proteinase K or pronase. Electrophoresis and western blotting of spiroplasma membrane proteins, before and after exposure of intact spiroplasmas to proteases, revealed the concomitant reduction in intensity of a major membrane protein (P89) and a new polypeptide of approximately 46 kDa in protease-treated preparations (P46). Triton X-114 phase partitioning demonstrated that P89 and P46 are amphiphilic, and labeling of the new polypeptide P46 with anti-P89 serum suggested that this molecule may be a breakdown product of P89. Regeneration of P89 after proteinase K treatment of spiroplasmas was directly associated with restoration of the pathogen's attachment capability. Treatment of spiroplasmas with any of several carbohydrates and glycoconjugates or with tetramethyl-urea, a compound that interferes with hydrophobic associations, had a negligible effect on attachment. These results suggest that a spiroplasma surface protein, P89, has a role in S. citri adherence to C. tenellus cells.

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ABSTRACT Spiroplasma citri, a helical, wall-less prokaryote in the class Molli-cutes, is transmitted by the beet leafhopper, Circulifer tenellus. Invasion of leafhopper tissues and cytopathological effects by S. citri were investigated by transmission electron microscopy. All eight cell types of the principle salivary glands, as well as the adjacent muscle cells and the cells of the accessory salivary glands, were colonized by the spiroplas-mas. In both midgut epithelia and salivary gland cells, spiroplasmas usually occurred in membrane-bound cytoplasmic vesicles that often were located near the cell periphery. In several salivary gland cells, spiroplas-mas were also observed within membranous pockets apparently formed by invagination of the plasmalemma beneath intact basal lamina. These observations are consistent with spiroplasma entry into the insect cells by receptor-mediated endocytosis. Cytopathological effects of spiroplasma infection in salivary cells included loss of membrane and basal lamina integrity, presence in some cells of irregular inclusion-like structures containing dense matrices of filamentous material that labeled with anti S. citri antibodies, and apparent disorganization of the endoplasmic reticulum. Compared to the tightly aligned fiber bundles in healthy muscle cells, bundles in spiroplasma-containing muscle cells appeared fragmented and loosely arranged. Such symptoms could contribute to the reduction in longevity and fecundity that has been previously reported for S. citri-infected C. tenellus.

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Yellow vine (YV) is a recently recognized decline of cucurbits expressed as plant yellowing, phloem discoloration, and death of vines as fruit approach maturity. In severely affected fields, YV incidence can range from 50 to 100% with similar yield loss. The disease has been associated with a phloem-limited, walled bacterium belonging to the gamma-3-proteobacteria (1), for which specific polymerase chain reaction (PCR) primers have been developed and used in diagnosis (2). First observed in 1988 in Oklahoma and Texas squash and pumpkin, YV was not detected in watermelon and cantaloupe until 1991. The disease has never been detected in cucumber. Efforts to date have been unsuccessful in transmitting the disease with dodder, grafting, or selected insects. Initially, the geographic range of the disease appeared to be generally confined to central and northeastern Oklahoma and north central Texas, an area known as the Cross Timbers Region. In 1997 to 1998, YV was diagnosed in commercial fields of watermelon and muskmelon from east Texas (Post Oak Savannah) and all cucurbit-growing areas of Oklahoma. In late summer 1998, symptoms similar to those of YV were observed in one watermelon (Hardeman County) and three pumpkin (Rhea and Morgan counties) fields in Tennessee where the leaves turned yellow and chlorotic and affected plants exhibited phloem discoloration. Estimated incidence of YV ranged from less than 1 to 20% of the plants in affected fields. PCR, with the YV-specific primers (2), amplified a band of the expected size (409 bp) from all watermelon and pumpkin plants exhibiting phloem discoloration. In contrast, no bands were amplified from asymptomatic (no phloem discoloration) watermelon or pumpkin. The nucleotide sequence of the DNA fragment amplified from a Tennessee watermelon and pumpkin plant was identical to that of the YV bacterium. The occurrence of YV outside of the Cross Timbers Region, and in a location as distant as Tennessee, suggests that the disease may be much more widespread than previously recognized. Diagnosis and monitoring of YV in all cucurbit-growing areas is critical for determining the geographic distribution and losses caused by this emerging disease. References: (1) F. J. Avila et al. Phytopathology 88:428, 1998. (2) U. Melcher et al. (Abstr.) Phytopathology. 89(suppl.):S95, 1999.

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A continuous cell line of embryonic origin from the leafhopper Circulifer tenellus, CT 1, was established using a protocol modified from thrips cell culture. The line was used to develop an in vitro model to examine the mode of entry of the plant pathogenic mollicute Spiroplasma citri into insect host cells. Confluent monolayers were achieved in 5-6 months using a simple medium developed for maintaining established leafhopper cell lines. The newly established CT 1 line, and that of another leafhopper, Nephotettix cincticeps, were exposed to S. citri and examined by electron microscopy. S. citri was found to cytadhere and to be present in apparent invaginations of the host cell membranes of cell lines of both leafhopper species, supporting the hypothesis that this pathogen enters its insect host via endocytosis.

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ABSTRACT Spiroplasma citri, transmitted by phloem-feeding leafhoppers, moves from the gut lumen through the gut wall, hemolymph, and salivary glands and multiplies in insect tissues. Nontransmissible lines were deficient in their ability to cross these barriers. Molecular analysis revealed extensive chromosomal rearrangements between the transmissible and nontransmissible spiroplasma lines including a large chromosomal inversion and deletions of about 10 kb at each inversion border. One open reading frame of the deleted region, cloned from the transmissible strain BR3-3X, encodes an integral membrane protein of 58 kDa that shares limited sequence similarity with major adhesin proteins of two zoopathogenic mycoplasmas. Adhesion of spiroplasmas to cultured leafhopper cells was inhibited by proteases, suggesting that adherence to host cells is mediated by spiroplasma membrane protein(s). A hypothetical model for insect transmission of phytopathogenic mollicutes is presented.