Arbitrarily primed PCR of cDNA for investigation of differential presence and expression of genes in three lines of Spiroplasma citri, and, Sequence analysis of Serratia marcescens strains associated with cucurbit yellow vine disease.

摘要

Scope and method of study. Many years of repeated passage of the plant-pathogenic Spiroplasma citri strain BR3-3X yielded the derivative line, BR3-G, which is no longer transmissible by the leafhopper vector Circulifer tenellus. Arbitrarily primed PCR (AP-PCR) was used to identify products differentially present in S. citri lines. Such products could be transmissibility-related proteins, induced only in transmissible lines, negatively-acting regulatory products made only in the nontransmissible line, or gene products relating to insect transmissibility not made due to missing DNA sequences.; A vine decline disease of cucurbits known as yellow vine (YV) has been consistently associated with plant phloem-resident rod-shaped bacteria, most closely related to Serratia marcescens on the basis of 16S rDNA homology. To more precisely identify and characterize this pathogen on a phylogenetic basis, the groE and 16S rDNA regions of two YV isolates were amplified by PCR and directly sequenced. For comparison, eight other bacterial isolates preliminarily identified as S. marcescens were sequenced concurrently.; Findings and conclusions. cDNA was synthesized from mRNA isolated from the nontransmissible line BR3-G, as well as from the transmissible parent, BR3-3X and the transmissible derivative, BR3-T, and served as templates for AP-PCR. Differential patterns of PCR products amplified from the three lines were observed when certain arbitrarily selected primers were used, and some of these products were cloned and sequenced.; Phylogenetic analysis based on groE sequences largely correlated with that based on 16S rDNA, and showed that YV-associated bacteria are indistinguishable from S. marcescens. Six of the eight other isolates characterized also fell decisively within the species S. marcescens.