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A new system for inoculation of plants with begomoviral DNA without cloning or the use insect vectors is described. Total DNA extracted from begomovirus-infected plants was amplified by rolling circle amplification (RCA) using the bacteriophage phi29 DNA polymerase, and inoculated to plants by particle bombardment. Infection rates of up to 100% were obtained using this technique. This technique successfully inoculated all the begomoviruses evaluated: five bipartite (Bean golden yellow mosaic virus, Cabbage leaf curl virus, Squash leaf curl virus, Tomato mottle virus, Watermelon chlorotic stunt virus) as well as one monopartite (Tomato yellow leaf curl virus). The success of the technique was not dependent upon plant species. Four species from three plant families [Phaseolus vulgaris (bean), Solanum lycopersicum (tomato), Cucurbita pepo (squash), and Citrullus lanatus (watermelon)], could all be inoculated by this technique. The success of the method was not dependent upon either the type or the age of the source of virus. Infectious DNA was obtained successfully from fresh, freeze-dried or desiccated plant material, from squashes of plant leaves on FTA cards, as well as from the insect vector. Plant material collected and dried as long as 25 years ago yielded infectious DNA by this method. In summary, this method can be used to obtain infectious DNA of single-stranded circular DNA viruses that can be activated for purposes of completing Koch's postulates, for preservation of pure virus cultures, and for many other applications where infectious DNA is required.

Adult whiteflies, Bemisia tabaci (Gennadius), collected from the field were screened for viral pathogens using a cell line from the silverleaf whitefly, B. tabaci, B biotype (syn. B. argentifolii). Homogenates from the field-collected whiteflies were applied to cell cultures and checked for cytopathic effects (CPE). Cells were observed to develop cytoplasmic inclusions and to have a change in morphology. Cells displaying CPE were observed using a transmission electron microscope and found to be infected with a virus. The virus particles had an icosahedral shape and an approximate size of 120-130 nm. The virus was observed in defined areas of the cytoplasm adjacent to the cell nucleus. Analysis using polymerase chain reaction, Southern blot hybridization, and DNA sequencing confirmed that the virus discovered infecting the whitefly cell cultures was an iridovirus. Sequence analysis showed that the amplimer (893 bp) had a 95% homology to the invertebrate iridescent virus type 6 major capsid protein gene. Discovery of new viruses of whiteflies may provide renewed interest in using pathogens in the development of innovative management strategies. This is the first report of an iridescent virus isolated from whiteflies, B. tabaci, collected from the field.

The whitefly Bemisia tabaci (Gennadius) is a widely distributed pest of many important food and fiber crops. This whitefly is also a vector of more than 70 plant-infecting viruses. A cell line was established in vitro using embryonic tissues from the eggs of Bemisia tabaci (Gennadius), B biotype (pseudonym B. argentifolii Bellows & Perring), and referred to as ‘Btb(Ba)97, Hunter-Polston’. Cell cultures were successfully inoculated with begomovirus (BGMV and ToMoV)-infected tomato plant sap. Embryonic tissues were seeded into Kimura's modified medium and kept at a temperature of 24°C. Continuous cell cultures were established and have since undergone 92 passages in 25-cm 2 flasks. Cell doubling time is approximately 3 days and the cells have been successfully revived after 1 year after storage at −80°C. The cell population is monolayers of predominately fibroblast with some epithelial cells. Begomoviruses (bean golden mosaic begomovirus, BGMV, and tomato mottle begomovirus, ToMoV) were inoculated to the cell cultures independently and detected by labeling by an indirect immunofluorescence technique. The viruses were detected bound to the cell membranes and within the cell cytoplasm. This is the first report of a continuous cell line established from a species of whitefly and its inoculation with two begomoviruses. The successful inoculation of whitefly cell cultures with begomoviruses shown in our results represents great promise for the development of systems that allow researchers to achieve a better understanding of the complex relationship between begomoviruses and their whitefly vectors.

The internal anatomy of the anterior alimentary canal of the whitefly, Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae) B-biotype, was examined by light, scanning, and transmission electron microscopy to elucidate the location and number of precibarial and cibarial gustatory sensilla. Elucidation of the epipharyngeal organ complex within the precibarium revealed 10 precibarial sensilla located proximal to where the paired maxillary stylets diverge on their retraction. The sensory organ complex within the cibarium consists of 8 sensilla, 6 on the epipharyngeal sclerite with 2 found within the hypopharyngeal sclerite. Fine structure investigation revealed the individual neurons to terminate at sensillar pores, which allow direct contact with passing fluids, thus supporting a chemosensory function. Ultrastructure of the neurons is similar to that of precibarial and cibarial gustatory chemosensilla found in other piercing-sucking insects. Their importance to whitefly feeding is discussed.

Coat proteins of several different plant viruses were transferred from entire leaves of infected hosts to nitrocellulose membranes in 2 min using a hydraulic laboratory press. Protein transfer was even across the leaf as evidenced by India ink staining of blots. Coat proteins of four different viruses were detected using virus specific IgG. The presence of viral coat proteins was observed as small intense spots whose distribution was irregular and coincided most frequently with the presence of chlorosis on the leaves. Detection was sensitive, and accurately reflected the distribution of virus symptoms across leaves.