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3686

Catharanthus roseus (L.) G. Don (periwinkle) is well known as an experimental host for diverse phytoplasmas that are artificially transmitted to it through the use of dodder (Cuscuta sp.), laboratory vector insects, or grafting. However, few phytoplasma taxa have been reported in natural infections of C. roseus, and the role of C. roseus in phytoplasma dissemination and natural disease spread is not clear. In this study, naturally diseased plants of C. roseus exhibiting yellowing and witches' broom symptoms indicative of phytoplasma infection were observed throughout the year in the state of Rio de Janeiro, Brazil. Shoots and leaves of four diseased plants were assayed for the presence of phytoplasma DNA sequences by nested polymerase chain reactions (PCR) as previously described (2,3). Phytoplasma rDNA was amplified from diseased periwinkle plants in PCR primed by primer pair P1/P7 and was reamplified in nested PCR primed by primer pair R16F2n/R16R2 (F2n/R2). The results indicated the presence of phytoplasma in all four diseased plants. Phytoplasma identification was accomplished by restriction fragment length polymorphism (RFLP) analysis, using 11 restriction enzymes, of 16S rDNA amplified in PCR primed by F2n/R2. Phytoplasmas were classified according to the system of Lee et al. (1). On the basis of collective RFLP patterns of 16S rDNA, the phytoplasma infections in the four periwinkle plants could not be distinguished from one another. Furthermore, the collective RFLP patterns were indistinguishable from those reported previously for hibiscus witches' broom phytoplasma, “Candidatus Phytoplasma brasiliense” (2). The phytoplasma found in C. roseus, designated strain HibWB-Cr, was classified in group 16SrXV (hibiscus witches' broom phytoplasma group). HibWB-Cr is tentatively considered a new strain of “Ca. P. brasiliense”. C. roseus is the first known, naturally diseased alternate plant host of “Ca. P. brasiliense”. The present study identified strain HibWB-Cr in Rio de Janeiro State, where hibiscus witches' broom disease is prevalent (2). How this economically important disease of hibiscus spreads is not known. Our findings raise the possibility that a polyphagous insect vector is involved in the natural transmission of “Ca. P. brasiliense” and that C. roseus or other plant species serve as reservoirs for the spread of this phytoplasma taxon. References: (1) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) H. G. Montano et al. Int. J. Syst. Evol. Microbiol. 51:1109, 2001. (3) H. G. Montano et al. Plant Dis. 84:429, 1999.

ABSTRACT The marine bryozoan, Bugula neritina , is the source of the bryostatins, a family of macrocyclic lactones with anticancer activity. Bryostatins have long been suspected to be bacterial products. B. neritina harbors the uncultivated gamma proteobacterial symbiont “ Candidatus Endobugula sertula.” In this work several lines of evidence are presented that show that the symbiont is the most likely source of bryostatins. Bryostatins are complex polyketides similar to bacterial secondary metabolites synthesized by modular type I polyketide synthases (PKS-I). PKS-I gene fragments were cloned from DNA extracted from the B. neritina-“E. sertula” association, and then primers specific to one of these clones, KSa, were shown to amplify the KSa gene specifically and universally from total B. neritina DNA. In addition, a KSa RNA probe was shown to bind specifically to the symbiotic bacteria located in the pallial sinus of the larvae of B. neritina and not to B. neritina cells or to other bacteria. Finally, B. neritina colonies grown in the laboratory were treated with antibiotics to reduce the numbers of bacterial symbionts. Decreased symbiont levels resulted in the reduction of the KSa signal as well as the bryostatin content. These data provide evidence that the symbiont E. sertula has the genetic potential to make bryostatins and is necessary in full complement for the host bryozoan to produce normal levels of bryostatins. This study demonstrates that it may be possible to clone bryostatin genes from B. neritina directly and use these to produce bryostatins in heterologous host bacteria.