First Report of the Cucurbit Yellow Vine Disease Caused by Serratia marcescens in Watermelon and Yellow Squash in Alabama.

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.

Transmission of different isolates of Spiroplasma citri to carrot and citrus by Circulifer tenellus (Hemiptera: Cicadellidae).

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.

Association of Escherichia coli O157:H7 with filth flies (Muscidae and Calliphoridae) captured in leafy greens fields and experimental transmission of E. coli O157:H7 to spinach leaves by house flies (Diptera: Muscidae).

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.

Genetic diversity of Spiroplasma citri strains from different regions, hosts, and isolation dates.

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.

Spiroplasma citri Surface Protein P89 Implicated in Adhesion to Cells of the Vector Circulifer tenellus.

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.

Spiroplasma citri Movement into the Intestines and Salivary Glands of Its Leafhopper Vector, Circulifer tenellus.

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.

Development and use of an established cell line of the leafhopper Circulifer tenellus to characterize Spiroplasma citri-vector interactions.

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.