ABSTRACT
Pointed, rod-shaped bacteria colonizing the cuticular surface of the hindgut of the terrestrial isopod crustacean
Porcellio scaber
(Crustacea: Isopoda) were investigated by comparative 16S rRNA gene sequence analysis and electron microscopy. The results of phylogenetic analysis, and the absence of a cell wall, affiliated these bacteria with the class
Mollicutes
, within which they represent a novel and deeply branched lineage, sharing less than 82.6% sequence similarity to known
Mollicutes
. The lineage has been positioned as a sister group to the clade comprising the
Spiroplasma
group, the
Mycoplasma pneumoniae
group, and the
Mycoplasma hominis
group. The specific signature sequence was identified and used as a probe in in situ hybridization, which confirmed that the retrieved sequences originate from the attached rod-shaped bacteria from the hindgut of
P. scaber
and made it possible to detect these bacteria in their natural environment. Scanning and transmission electron microscopy revealed a spherically shaped structure at the tapered end of the rod-shaped bacteria, enabling their specific and exclusive attachment to the tip of the cuticular spines on the inner surface of the gut. Specific adaptation to the gut environment, as well as phylogenetic positioning, indicate the long-term association and probable coevolution of the bacteria and the host. Taking into account their pointed, rod-shaped morphology and their phylogenetic position, the name “
Candidatus
Bacilloplasma” has been proposed for this new lineage of bacteria specifically associated with the gut surface of
P. scaber
.
ABSTRACT
“
Candidatus
Accumulibacter phosphatis” is considered a polyphosphate-accumulating organism (PAO) though it has not been isolated yet. To reveal the denitrification ability of this organism, we first concentrated this organism by flow cytometric sorting following fluorescence in situ hybridization (FISH) using specific probes for this organism. The purity of the target cells was about 97% of total cell count in the sorted sample. The PCR amplification of the nitrite reductase genes (
nirK
and
nirS
) from unsorted and sorted cells was performed. Although
nirK
and
nirS
were amplified from unsorted cells, only
nirS
was detected from sorted cells, indicating that “
Ca
. Accumulibacter phosphatis” has
nirS
. Furthermore,
nirS
fragments were cloned from unsorted (Ba clone library) and sorted (Bd clone library) cells and classified by restriction fragment length polymorphism analysis. The most dominant clone in clone library Ba, which represented 62% of the total number of clones, was not found in clone library Bd. In contrast, the most dominant clone in clone library Bd, which represented 59% of the total number of clones, represented only 2% of the total number of clones in clone library Ba, indicating that this clone could be that of “
Ca
. Accumulibacter phosphatis.” The sequence of this
nirS
clone exhibited less than 90% similarity to the sequences of known denitrifying bacteria in the database. The recovery of the
nirS
genes makes it likely that “
Ca
. Accumulibacter phosphatis” behaves as a denitrifying PAO capable of utilizing nitrite instead of oxygen as an electron acceptor for phosphorus uptake.
Murraya paniculata (orange jasmine) is a popular ornamental rutaceaous plant and is known to be a preferred host for the Asian citrus psyllid, Diaphorina citri (Kuwayana), the primary vector of ‘Candidatus Liberibacter spp.’ that causes citrus Huanglongbing (HLB). HLB is a highly destructive citrus disease worldwide. However, the presence of ‘Ca. Liberibacter spp.’ in M. paniculata remains uncertain (2). Clarification of M. paniculata as a host of ‘Ca. Liberibacter spp.’ has direct impact on HLB control programs. During June of 2006, we identified three M. paniculata trees near a mandarin orchard affected by HLB in Luoding City and two trees from Guangzhou City, Guangdong Province, People's Republic of China. All trees had leaves showing yellowing and mottling symptoms characteristic of HLB. Both symptomatic and asymptomatic leaves were collected. DNA was extracted using the CTAB (cetyltrimethylammoniumbromide) method and assayed by nested-PCR. The general bacterial 16S rDNA primer set fDl/rD1 (3) was used for the first round of amplification. Amplification was conducted as previously described (1), and 2 μl of PCR reaction product were used for a second round of amplification using the same procedure but with 35 PCR cycles with primer set OI1/OI2c (3,4). After agarose gel electrophoresis and staining with ethidium bromide, a 1.1-kb DNA band was unambiguously associated with symptomatic but not asymptomatic leaf samples. Nonnested-PCR using primer set OI1/OI2c alone did not yield a target DNA band or yielded a very weak DNA band. XbaI digestion of the nested-PCR DNA product yielded two fragments, 520 and 640 bp long, characteristic of ‘Ca. L. asiaticus’. PCR amplicons were sequenced and were 1,095 bp long. This sequence shared >98% similarity to sequences of ‘Ca. L. asiaticus’ in the GenBank database. We observed that nested-PCR is necessary for consistent amplification of DNA from ‘Ca. L. asiaticus’ from M. paniculata. We excluded the possible nonspecific amplification associated with nested-PCR by XbaI restriction enzyme digestion and by nucleotide sequence analysis. Our data indicate that M. paniculata is a host of ‘Ca. L. asiaticus’ but the bacterial titer might be low. References: (1) X. Deng et al. Online publication. doi:10.1094/PHP-2007-0419-01-BR. Plant Health Progress, 2007. (2) M. Garnier and J. Bove. Huanglongbing (Greening). Page 46 in: Compendium of Citrus Diseases. 2nd ed. L. W. Timmer et al., eds. The American Phytopathological Society, St. Paul, MN, 2000. (3) S. Jagoueix et al. Int. J. Syst. Bacteriol. 44:379, 1994. (4) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996.
New diseases known locally as ‘hoja de perejil’ of tomato (Lycopersicon esculentum Mill) and ‘brotes grandes’ of potato (Solanum tuberosum L.) were first recognized in surveys of production fields in Bolivia during 2000–2003. Alfalfa (Medicago sativa) witches' broom and little leaf diseases of native weeds Morrenia variegata and mora-mora (Serjania perulacea) were also identified near to production fields. Phytoplasma aetiology was attributed to each of these diseases following detection and initial identification of aster yellows group (16SrI) phytoplasmas in all five diseased plant species. While potato, alfalfa and mora-mora plants contained indistinguishable 16SrI-B strains, ‘hoja de perejil’ (THP) and morrenia little leaf (MVLL)-associated phytoplasma strains shared 97.5 % 16S rRNA gene sequence similarity with ‘Candidatus Phytoplasma asteris’ and related strains and <95 % similarity with all other ‘Candidatus Phytoplasma’ species. Phylogenetic analysis of 16S rRNA gene sequences indicated that the THP and MVLL phytoplasmas represent a novel lineage within the aster yellows (16SrI) group and, on the basis of unique 16S rRNA gene sequences, we propose that THP and MVLL phytoplasmas represent ‘Candidatus Phytoplasma lycopersici’, with THP as the reference strain.