Publications

3512

‘Candidatus Liberibacter asiaticus’ is the bacterium implicated as a causal agent of the economically damaging disease of citrus called huanglongbing (HLB). Vertical transmission of the organism through seed to the seedling has not been demonstrated. Previous studies using real-time polymerase chain reaction assays indicated abundant bacterial 16S rRNA sequences in seed coats of citrus seed but the presence of intact bacterial cells was not demonstrated. We used microscopy to verify that intact bacterial cells were present in citrus seed coats. Bacterial cells with the morphology and physical dimensions appropriate for ‘Ca. L. asiaticus’ were seen in phloem sieve elements in the vascular bundle of grapefruit seed coats using transmission electron microscopy (TEM). Fluorescence in situ hybridization (FISH) analyses utilizing probes complementary to the ‘Ca. L. asiaticus’ 16S rRNA gene revealed bacterial cells in the vascular tissue of intact seed coats of grapefruit and pummelo and in fragmented vascular bundles excised from grapefruit seed coats. The physical measurements and the morphology of individual bacterial cells were consistent with those ascribed in the literature to ‘Ca. L. asiaticus’. No bacterial cells were observed in preparations of seed from fruit from noninfected trees. A small library of clones amplified from seed coats from a noninfected tree using degenerate primers targeting prokaryote 16S rRNA gene sequences contained no ‘Ca. L. asiaticus’ sequences, whereas 95% of the sequences in a similar library from DNA from seed coats from an infected tree were identified as ‘Ca. L. asiaticus’, providing molecular genetic corroboration that the bacterial cells observed by TEM and FISH in seed coats from infected trees were ‘Ca. L. asiaticus’.

Abstract Background Huanglongbing (HLB) is a worldwide devastating disease of citrus. There are no effective control measures for this newly emerging but century-old disease. Previously, we reported a combination of Penicillin G and Streptomycin was effective in eliminating or suppressing the associated bacterium, ‘Candidatus Liberibacter asiaticus’ (Las). Results Here we report the bacterial composition and community structure in HLB-affected citrus plants during a growing season and while being treated with antibiotic combinations PS (Penicillin G and Streptomycin) and KO (Kasugamycin and Oxytetracycline) using the Phylochip™ G3 array. Both antibiotic treatments resulted in significantly lower Las bacterial titers (Pr<0.05) and hybridization scores. Of the 50,000+ available operational taxonomic units (OTUs) on PhyloChip™ G3, 7,028 known OTUs were present in citrus leaf midribs. These OTUs were from 58 phyla, of which five contained 100 or more OTUs, Proteobacteria (44.1%), Firmicutes (23.5%), Actinobacteria (12.4%), Bacteroidetes (6.6%) and Cyanobacteria (3.2%). In the antibiotic treated samples, the number of OTUs decreased to a total of 5,599. The over-all bacterial diversity decreased with the antibiotic treatments, as did the abundance of 11 OTUs within Proteobacteria, Firmicutes, Bacteroidetes and Planctomycetes. Within the Proteobacteria, ten OTUs representing the class γ -proteobacteria increased in abundance after four months of treatment, when the Las bacterium was at its lowest level in the HLB-affected citrus field plants. Conclusions Our data revealed that Proteobacteria was constantly the dominant bacterial phylum recovered from citrus leaf midribs, with the α-proteobacterial and the γ-proteobacterial classes vying for prevalence. In addition, the level of bacterial diversity found in the leaf midribs of field citrus was greater than previously described. Bacterial cells in close proximity may be able to modify their microenvironment, making the composition of the microbial community an important factor in the ability of Las to cause HLB progression. A low Las level was seen as an annual fluctuation, part of the bacterial population dynamics, and as a response to the antibiotic treatments.

ABSTRACT “ Candidatus Midichloria mitochondrii” is an intramitochondrial bacterium of the order Rickettsiales associated with the sheep tick Ixodes ricinus . Bacteria phylogenetically related to “ Ca . Midichloria mitochondrii” (midichloria and like organisms [MALOs]) have been shown to be associated with a wide range of hosts, from amoebae to a variety of animals, including humans. Despite numerous studies focused on specific members of the MALO group, no comprehensive phylogenetic and statistical analyses have so far been performed on the group as a whole. Here, we present a multidisciplinary investigation based on 16S rRNA gene sequences using both phylogenetic and statistical methods, thereby analyzing MALOs in the overall framework of the Rickettsiales . This study revealed that (i) MALOs form a monophyletic group; (ii) the MALO group is structured into distinct subgroups, verifying current genera as significant evolutionary units and identifying several subclades that could represent novel genera; (iii) the MALO group ranks at the level of described Rickettsiales families, leading to the proposal of the novel family “ Candidatus Midichloriaceae.” In addition, based on the phylogenetic trees generated, we present an evolutionary scenario to interpret the distribution and life history transitions of these microorganisms associated with highly divergent eukaryotic hosts: we suggest that aquatic/environmental protista have acted as evolutionary reservoirs for members of this novel family, from which one or more lineages with the capacity of infecting metazoa have evolved.

Zebra chip disease, putatively caused by the bacterium ‘Candidatus Liberibacter solanacearum’, is of increasing concern to potato production in Mexico, the United States, and New Zealand. However, little is known about the etiology of this disease and changes that occur within host tubers that result in its symptoms. Previous studies found that increased levels of phenolics, amino acids, defense proteins, and carbohydrates in ‘Ca. L. solanacearum’-infected tubers are associated with symptoms of zebra chip. This study was conducted to quantify variations in levels of these biochemical components in relation to the time of infestation, symptom severity, and ‘Ca. L. solanacearum’ titer. Levels of phenolics, peroxidases, polyphenol oxidases, and reducing sugars (glucose and, to some extent, fructose) changed during infection, with higher levels occurring in tubers infected at least 5 weeks before harvest than in those infected only a week before harvest and those of controls. Compared with the apical tuber ends, greater levels of phenolics, peroxidases, and sucrose occurred at the basal (stolon attachment) end of infected tubers. With the exception of phenolics, concentrations of the evaluated compounds were not associated with ‘Ca. L. solanacearum’ titer. However, there were significant associations between biochemical responses and symptom severity. The lack of a linear correlation between most plant biochemical responses and ‘Ca. L. solanacearum’ titer suggests that shifts in metabolic profiles are independent of variations in ‘Ca. L. solanacearum’ levels.

ABSTRACTThe draft genome sequence of a single orangeBeggiatoa(“CandidatusMaribeggiatoa”) filament collected from a microbial mat at a hydrothermal site in Guaymas Basin (Gulf of California, Mexico) shows evidence of extensive genetic exchange with cyanobacteria, in particular for sensory and signal transduction genes. A putative homing endonuclease gene and group I intron within the 23S rRNA gene; several group II catalytic introns; GyrB and DnaE inteins, also encoding homing endonucleases; multiple copies of sequences similar to thefdxNexcision elements XisH and XisI (required for heterocyst differentiation in some cyanobacteria); and multiple sequences related to an open reading frame (ORF) (00024_0693) of unknown function all have close non-Beggiatoaceaematches with cyanobacterial sequences. Sequences similar to the uncharacterized ORF and Xis elements are found in otherBeggiatoaceaegenomes, a variety of cyanobacteria, and a few phylogenetically dispersed pleiomorphic or filamentous bacteria. We speculate that elements shared among filamentous bacterial species may have been exchanged in microbial mats and that some of them may be involved in cell differentiation.