The phylogenetic relationships of bacterial symbionts from three gall-bearing species in the marine red algal genus Prionitis (Rhodophyta) were inferred from 16S rDNA sequence analysis and compared to host phylogeny also inferred from sequence comparisons (nuclear ribosomal internal-transcribed-spacer region). Gall formation has been described previously on two species of Prionitis, P. lanceolata (from central California) and P. decipiens (from Peru). This investigation reports gall formation on a third related host, Prionitis filiformis. Phylogenetic analyses based on sequence comparisons place the bacteria as a single lineage within the Roseobacter grouping of the ␣ subclass of the division Proteobacteria (99.4 to 98.25% sequence identity among phylotypes). Comparison of symbiont and host molecular phylogenies confirms the presence of three gallbearing algal lineages and is consistent with the hypothesis that these red seaweeds and their bacterial symbionts are coevolving. The species specificity of these associations was investigated in nature by whole-cell hybridization of gall bacteria and in the laboratory by using cross-inoculation trials. Whole-cell in situ hybridization confirmed that a single bacterial symbiont phylotype is present in galls on each host. In laboratory trials, bacterial symbionts were incapable of inducing galls on alternate hosts (including two non-gall-bearing species). Symbiont-host specificity in Prionitis gall formation indicates an effective ecological separation between these closely related symbiont phylotypes and provides an example of a biological context in which to consider the organismic significance of 16S rDNA sequence variation.Marine bacteria are associated with gall formation (tumorigenesis) on a number of species of red algae, although only two reports detail the specific causation of an algal gall by an identified bacterium (5, 10). This probably reflects the general situation encountered when attempting to cultivate, or isolate in pure culture, symbiotic microbes (10,33,44). In the red algal genus Prionitis (Rhodophyta, Halymeniaceae, Gigartinales) gall formation is known from at least four species world wide and is, in the case of Prionitis lanceolata (from central California), associated with the presence of a specific microorganism (5). Despite attempts by several authors, this bacterium has yet to be cultivated or isolated in pure culture (3)(4)(5). No physiological function of these bacterially induced Prionitis galls has been determined, nor is it apparent what, if any, benefit is derived by algal host or bacterial invader. This organismic relationship is termed a symbiosis sensu DeBary as used by Smith, meaning simply the living together of differently named organisms (43). The 16S rDNA phylotype of this eubacterium has been determined from its complete small-subunit ribosomal DNA sequence and whole-cell hybridization used to confirm the inductive role of this symbiont in gall formation (5).The purpose of this investigation was to determine if bacterial gall ...

Abstract.-Morphological similarities of many parasites and their hosts have led to speculation that some groups of plant, animal, fungal, and algal parasites may have evolved directly from their hosts. These parasites, which have been termed adelphoparasites in the botanical literature, and more recently, agastoparasites in the insect literature, may evolve monophyletically from one host and radiate secondarily to other hosts or, these parasites may arise polyphyletically, each arising from its own host. In this study we compare the internal transcribed spacer regions of the nuclear ribosomal repeats of species and formae specialis (host races) included in the red algal parasite genus Asterocolax with its hosts, which all belong to the Phycodrys group of the Delesseriaceae and with closely related nonhost taxa of the Delesseriaceae. These analyses reveal that species of Asterocolax have evolved polyphyletically. Asterocolax erythroglossi from the North Atlantic host Erythroglossum laciniatum appears to have evolved from its host, whereas taxa included in the north Pacific species Asterocolax gardneri have had two independent origins. Asterocolax gardneri from the host Polyneura latissima probably arose directly from this host. In contrast, all other A. gardneri formae specialis appear to have originated from either Phycodrys setchellii or P. isabelliae and radiated secondarily onto other closely related taxa of the Phycodrys group, including Nienburgia andersoniana and Anisocladella pacifica. Gamete crossing experiments confirm that A. gardneri from each host is genetically isolated from both its host, and from other A. gardneri and their hosts. Cross-infection experiments reveal that A. gardneri develops normally only on its natural host, although some abberrant growth may occur on alternate hosts. The ability of red algal parasites to radiate secondarily to other red algal taxa, where they may become isolated genetically and speciate, suggests that this process of speciation is not a "genetic dead end" but one that may give rise to related clusters of parasite species.

The polymerase chain reaction (PCR) was used to amplify eubacterial small‐subunit (16S) ribosomal DNA (rDNA) genes from galls of the marine red alga Prionitis lanceolata Harvey (Gigartinales). These tumors consist of hypertrophied algal cells containing large numbers of intercellular bacteria that remain uncultivable. PCR‐amplified 16S rDNAs from surface‐sterilized gall tissue plugs were cloned, sequenced, and analyzed by alignment to available small‐subunit rRNA sequences (University of Illinois Ribosomal Database Project). Variable regions were identified and used to construct a fluorescently labeled, species‐specific oligodeoxynucleotide probe for whole cell in situ hybridization to the gall symbiont. Probe 949 (PLANC.949) localized the P. lanceolata bacterial symbiont in preparations from mature gall tissue. This probe did not hybridize to the rDNA of closely related bacteria included as controls in the same hybridization reactions, In situ hybridization revealed the presence of the same bacterium in association with P. lanceolata gall formation from three central California localities. Distance and parsimony analyses suggest that this organism is a member of the Proteobacteria (alpha subdivision; Rhodobacter group) and is most closely related to Roseobacter denitrificans.

This study examines the use of fecal indicator bacteria (FIB) as a predictor of the presence of Helicobacter spp. A combination of standard culture and molecular techniques were used to detect and quantify FIB, Helicobacter spp. and H. pylori from five North American rivers of different size and with different land use characteristics. Primers designed to amplify genes specific to Helicobacter spp. and H. pylori were evaluated for their efficacy in detection and quantification in environmental samples. Helicobacter spp. were detected in 18/33 (55%) of river samples. H. pylori was detected in 11/33 (33%) of river samples. FIB were found in 32/33 (96%) of river samples. When FIB abundance exceeded USEPA water quality standards for single samples, Helicobacter or H. pylori were detected in 7/15 (47%) cases. No numerical correlation was found between the presence of FIB and either Helicobacter spp. or H. pylori. This suggests that the presence of FIB will be of limited use for detection of Helicobacter spp. or H. pylori by public health agencies.

Gall formation in Prionitis lanceolata is associated with aspecific eubacterium (Proteobacteria [alphasubclass], Rhodobacter grouping), which, typical ofbacterial symbionts, has not yet been cultivated or isolated in pureculture. This investigation tested the hypothesis that P.lanceolata gall formation was caused by the associated eubacteriumusing a species-specific rDNA probe (S-S-P.l.sym-0949-a-A-25) toidentify and assay for symbiont presence during consecutive laboratoryinduction trials. Gall induction was quantified and whole-cell in situhybridization used to determine the relative percentage of symbioticeubacteria in inoculation homogenates. In situ hybridization ofsymbionts in sections allowed localization and monitoring of thismicrobe during gall development. Induction trial results indicate asignificant correlation between bacterial symbiont presence and gallinitiation (P = 0.00005). The gall bacterium comprisedthe majority of the eubacteria hybridized in laboratory inductionhomogenates (85-97%), in galls induced in the laboratoryand in three algal populations in nature. The evidence presented heredemonstrates the causative role of the identified eubacterium in gallinduction and formation. This investigation is significant in theapplication of molecular methods towards understanding the roles ofnoncultivable marine bacteria in marine algal-microbeinteractions.