Grapefruit (Citrus paradisi Macf. cv Duncan) plants were transformed with several sequences from citrus tristeza closterovirus (CTV) that varied in terms of position in the CTV genome and virus strain origin in an attempt to obtain resistant plants. The sequences included the capsid protein gene from three different strains, a nontranslatable version of the capsid protein gene, the replicase (RdRp), the minor capsid protein (p27), a highly transcribed gene of unknown function (p20) and the more conserved 3' end of the genomic RNA. Transgenic plants were generated from all of the constructs, except from the p20 and p27 genes. Southern and Western blot analyses demonstrated that stably transformed grapefruit plants were obtained and that at least some transgenes were expressed. In a first effort at virus challenge, 25 transgenic lines were graft inoculated with a severe strain of CTV. Although some transgenic plants averaged lower titers of virus than controls, there was great variability in titer in both controls and transgenic plants, and all were apparently susceptible to the virus.

Direct molecular evidence was obtained for the critical role of a single amino acid residue in a structural epitope distinguished by the monoclonal antibody MCA-13, which reacts selectively with severe isolates of citrus tristeza virus (CTV). Different CTV isolates cause a wide range of symptoms in the diverse citrus species they affect. Severe symptoms include decline, stem pitting, and seedling yellows. Plants infected by mild isolates are essentially symptomless. The monoclonal antibody MCA-13, which discriminates severe isolates from mild isolates of the virus, was used to map its epitope on the coat protein of CTV. A diverse group of coat protein genes of geographically and biologically distinct CTV isolates which are either MCA-13-reactive or MCA-13-nonreactive was cloned and sequenced. A series of mutant coat protein genes was constructed through oligonucleotidedirected, site-specific mutagenesis. The reactivity of the wildtype and mutant coat proteins expressed in Escherchia coli was evaluated by Western blotting using MCA-13 and polyclonal antibody prepared to CTV-coat protein. A single nucleotide alteration resulting in a Phe --Tyr mutation at position 124 of the coat protein abolished the MCA-13 reactivity of a severe isolate, whereas a Tyr -* Phe mutation at the same site conferred MCA-13 reactivity on the coat protein of a previously nonreactive mild isolate of CTV.

Blight-affected sweet orange [Citrus sinensis (L.) Osb.] trees on rough lemon (C. jambhiri Lush.) rootstock were injected with high concentrations of oxytetracycline (OTC) (10–30 g a.i./tree) under high pressure (1300–1700 kPa) in an attempt to induce remission of blight symptoms. OTC activity, determined by bioassay with Bacillus cereus var. mycoides, was distributed in 70–95% of the twigs sampled 3 weeks after injection. Activity persisted in the twigs and mature leaves for 3 to 5 months and in a few twigs as long as 7 to 8 months after a single injection. No activity was detected in small roots and downward movement appeared to be limited. Distribution and persistence of OTC in healthy trees was similar to that in blight trees. Trees with mild blight symptoms injected once with OTC continued to decline and had moderate to severe blight 2 years later. Of 16 healthy trees injected with OTC 4 times over 2 years, 1 became severely blighted and several others developed mild symptoms. Our trunk injection work with OTC does not support the hypothesis that blight is caused by a tetracycline-sensitive organism, but does not eliminate that possibility.