Publications

3867

The citrus disease Huanglongbing (HLB) is highly destructive in many citrus-growing regions of the world. The putative causal agent of this disease, ‘Candidatus Liberibacter asiaticus’, is difficult to culture, and Koch's postulates have not yet been fulfilled. As a result, efforts have focused on obtaining the genome sequence of ‘Ca. L. asiaticus’ in order to give insight on the physiology of this organism. In this work, three next-generation high-throughput sequencing platforms, 454, Solexa, and SOLiD, were used to obtain metagenomic DNA sequences from phloem tissue of Florida citrus trees infected with HLB. A culture-independent, polymerase chain reaction (PCR)-independent analysis of 16S ribosomal RNA sequences showed that the only bacterium present within the phloem metagenome was ‘Ca L. asiaticus’. No viral or viroid sequences were identified within the metagenome. By reference assembly, the phloem metagenome contained sequences that provided 26-fold coverage of the ‘Ca. L. asiaticus’ contigs in GenBank. By the same approach, phloem metagenomic data yielded less than 0.2-fold coverage of five other alphaproteobacterial genomes. Thus, phloem metagenomic DNA provided a PCR-independent means of verifying the presence of ‘Ca L. asiaticus’ in infected tissue and strongly suggests that no other disease agent was present in phloem. Analysis of these metagenomic data suggest that this approach has a detection limit of one ‘Ca. Liberibacter’ cell for every 52 phloem cells. The phloem sample sequenced here is estimated to have contained 1.7 ‘Ca. Liberibacter’ cells per phloem cell.

Huanglongbing (HLB) is a devastating disease of citrus and threatens the citrus industry worldwide. The suspected causal agent of the disease is a phloem-limited bacterium of the genus Candidatus Liberibacter transmitted through insect vector or grafting with diseased budwood. Currently, most seed source trees for citrus rootstock propagation are located outdoors and unprotected from disease transmission. In addition, fruit from HLB-affected scion varieties in Florida containing seeds enter the commercial trade and move into other citrus-growing areas. The objective of this study was to determine how Ca. L. asiaticus infection affects seed quality and seedling development and whether the disease appears in seedlings grown from infected fruit. Two experiments were conducted involving thousands of seedlings produced from seeds from infected rootstock seed source trees and ‘Valencia’ sweet orange trees, respectively. Infection of trees and fruit with Ca. L. asiaticus significantly reduced seed weight, seed germination, and seedling height. Seedlings did not develop symptoms typical of HLB throughout the experiment. Polymerase chain reaction (PCR) analysis initially identified two of 686 rootstock seedlings and three of 431 sweet orange seedlings positive for the pathogen when they were very young. Resampling and PCR analysis of these five seedlings at older ages consistently indicated they were negative for the pathogen and none of these plants ever developed symptoms of HLB. It is suggested that Ca. L. asiaticus may have been translocated into some part of the embryo during seed development but that it was not present in cells or tissue, which permitted replication or disease development as the seedling grew.

ABSTRACT As polyphagous, holometabolous insects, tephritid fruit flies (Diptera: Tephritidae) provide a unique habitat for endosymbiotic bacteria, especially those microbes associated with the digestive system. Here we examine the endosymbiont of the olive fly [ Bactrocera oleae (Rossi) (Diptera: Tephritidae)], a tephritid of great economic importance. “ Candidatus Erwinia dacicola” was found in the digestive systems of all life stages of wild olive flies from the southwestern United States. PCR and microscopy demonstrated that “ Ca. Erwinia dacicola” resided intracellularly in the gastric ceca of the larval midgut but extracellularly in the lumen of the foregut and ovipositor diverticulum of adult flies. “ Ca. Erwinia dacicola” is one of the few nonpathogenic endosymbionts that transitions between intracellular and extracellular lifestyles during specific stages of the host's life cycle. Another unique feature of the olive fly endosymbiont is that unlike obligate endosymbionts of monophagous insects, “ Ca. Erwinia dacicola” has a G+C nucleotide composition similar to those of closely related plant-pathogenic and free-living bacteria. These two characteristics of “ Ca. Erwinia dacicola,” the ability to transition between intracellular and extracellular lifestyles and a G+C nucleotide composition similar to those of free-living relatives, may facilitate survival in a changing environment during the development of a polyphagous, holometabolous host. We propose that insect-bacterial symbioses should be classified based on the environment that the host provides to the endosymbiont (the endosymbiont environment).

Intracellular bacterial symbionts are known from various insect groups, particularly from those feeding on unbalanced diets, where the bacteria provide essential nutrients to the host. In the case of reed beetles (Coleoptera: Chrysomelidae, Donaciinae), however, the endosymbionts appear to be associated with specialized “glands” that secrete a material used for the beetles’ unusual water-tight cocoon. These glands were discovered over a century ago, but the bacteria they contain have yet to be characterized and placed in a phylogenetic context. Here, we describe the ultrastructure of two endosymbiotic species (“ Candidatus Macropleicola appendiculatae” and “ Candidatus Macropleicola muticae”) that reside in cells of the Malpighian tubules of the reed beetle species Macroplea appendiculata and Macroplea mutica , respectively. Fluorescent in situ hybridization using oligonucleotides targeting the 16S rRNA gene specific to Macroplea symbionts verified the localization of the symbionts in these organs. Phylogenetic analysis of 16S rRNA placed “Candidatus Macropleicola” in a clade of typically endosymbiotic Enterobacteriaceae (γ-proteobacteria). Finally, we discuss the evidence available for the hypothesis that the beetle larvae use a secretion produced by the bacteria for the formation of an underwater cocoon.

ABSTRACT Cardinium bacteria, members of the phylum Cytophaga - Flavobacterium - Bacteroides (CFB), are intracellular bacteria in arthropods that are capable of inducing reproductive abnormalities in their hosts, which include parasitic wasps, mites, and spiders. A high frequency of Cardinium infection was detected in planthoppers (27 out of 57 species were infected). A high frequency of Cardinium infection was also found in spider mites (9 out of 22 species were infected). Frequencies of double infection by Cardinium and Wolbachia bacteria ( Alphaproteobacteria capable of manipulating reproduction of their hosts) were disproportionately high in planthoppers but not in spider mites. A new group of bacteria, phylogenetically closely related to but distinct from previously described Cardinium bacteria (based on 16S rRNA and gyrB genes) was found in 4 out of 25 species of Culicoides biting midges. These bacteria possessed a microfilament-like structure that is a morphological feature previously found in Cardinium and Paenicardinium . The bacteria close to the genus Cardinium consist of at least three groups, A, B, and C. Group A is present in various species of arthropods and was previously referred to as “ Candidatus Cardinium hertigii,” group B is present in plant parasitic nematodes and was previously referred to as “ Candidatus Paenicardinium endonii,” and group C is present in Culicoides biting midges. On the basis of morphological and molecular data, we propose that the nomenclature of these three groups be integrated into a single species, “ Candidatus Cardinium hertigii.”