The persistence of engineered Agrobacterium tumefaciens in agroinfected plants.

Several plant species, including tomato (Lycopersicon esculentum), Gynura aurantiaca, avocado (Persea americana), and grapefruit (Citrus paradisi) grafted on Troyer citrange (Poncirus trifoliata x C. sinensis) were "agro-infected" with Agrobacterium tumefaciens strain LBA-4404, carrying a mini-Ti plasmid with a dimeric cDNA of citrus exocortis viroid (CEVd). Extracts prepared from tissues of the agroinfected plants 38-90 days after inoculation were plated on selective media and found to contain large amounts of the engineered bacteria. These observations suggest the need for more stringent quarantine measures when handling A. tumefaciens cells harboring constructs for "agroinoculation" with plant viruses or viroids.

Enzyme-linked immunosorbent assay on nitrocellulose membranes (dot-ELISA) in the serodiagnosis of plant pathogenic bacteria.

The usefulness of enzyme-linked immunosorbent assay on nitrocellulose membranes (dot-ELISA) for diagnosis and identification of plant pathogenic bacteria was tested. Five pathovars of Xanthomonas campestris and two antisera, one produced against pv. vesicatoria and the other against pv. translucens, were used in a model system. A 10-min incubation of the bacterial cells, dot blotted on membranes, in diluted sera, followed by either alkaline phosphatase conjugated protein A or goat antirabbit globulin, resulted in a specific reaction between the homologous serum and bacteria. Populations of 1000-2000 cfu per spot (ca. 0.3 cm2) could be detected with these reagents. The streptavidin-biotinylated peroxidase complex produced a definitive reaction with as few as 800 cfu, but cross-reactions became evident at the higher cell concentrations among all five pathovars in tests with both antisera. Cell-free extracts, obtained by centrifugation of boiled bacteria, reacted similarly to live cells. Unrelated bacteria did not react with either antiserum. Extracts of lesions from tomato and pepper leaves infected with X. campestris pv. vesicatoria reacted positively with the antiserum produced against this pathovar but not that produced with pv. translucens. Samples of supernatants from boiled lesions reacted with similar intensity as those from homogenized tissues.