Publications

4477

Summary Amoeba‐resisting bacteria (ARB) such as Legionella spp. are currently regarded as potential human pathogens living in the environment. To detect ARB from both human and environmental samples, co‐culture with amoebae has been demonstrated as an efficient tool. However, using this procedure, mostly water from cooling towers and hospital water supplies have been investigated as the possible reservoir of ARB. In the present study, we studied ARB population in 77 environmental water samples including rivers, fountains, lakes and domestic wells in the south of France. As a result, a total of 244 isolates corresponding to 89 different species of ARB, but not Legionella spp., were identified. Ability to grow within and/or to be lytic for amoebae was revealed for the first time for several human pathogens . Six isolates are likely to be the members of a new or uncharacterized genus/species. An anaerobic bacterium, Clostridium frigidicarnis was demonstrated to be lytic for amoebae. This preliminary work demonstrates that the water environment in the vicinity of humans is a reservoir of ARB, including well‐known pathogens for which amoebae and/or water was not recognized earlier as a possible reservoir.

To characterize potentially important surface-exposed proteins of the phytoplasma causing chrysanthemum yellows (CY), new primers were designed based on the conserved regions of 3 membrane protein genes of the completely sequenced onion yellows and aster yellows witches’ broom phytoplasmas and were used to amplify CY DNA. The CY genes secY, amp, and artI, encoding the protein translocase subunit SecY, the antigenic membrane protein Amp and the arginine transporter ArtI, respectively, were cloned and completely sequenced. Alignment of CY-specific secY sequences with the corresponding genes of other phytoplasmas confirmed the 16S rDNA-based classification, while amp sequences were highly variable within the ‘Candidatus Phytoplasma asteris’. Five CY partial sequences were cloned into the pRSetC expression vector, and 3 of the encoded protein fragments (Amp 64/651, Amp 64/224, ArtI 131/512) were expressed as fusion antigens for the production of CY-specific polyclonal antibodies (A416 against Amp 64/224; A407 against ArtI 131/512). A416 recognized, in Western blots, the full-length Amp from CY-infected plants (periwinkle, daisy) and insect vectors ( Euscelidius variegatus , Macrosteles quadripunctulatus ). A416 also reacted to European aster yellows, to primula yellows phytoplasmas, to northern Italian strains of ‘Ca. Phytoplasma asteris’ from lettuce and gladiolus, but it did not react to American aster yellows phytoplasma.

ABSTRACT In this study we analyzed the membrane lipid composition of “ Candidatus Nitrosopumilus maritimus,” the only cultivated representative of the cosmopolitan group I crenarchaeota and the only mesophilic isolate of the phylum Crenarchaeota . The core lipids of “ Ca . Nitrosopumilus maritimus” consisted of glycerol dialkyl glycerol tetraethers (GDGTs) with zero to four cyclopentyl moieties. Crenarchaeol, a unique GDGT containing a cyclohexyl moiety in addition to four cyclopentyl moieties, was the most abundant GDGT. This confirms unambiguously that crenarchaeol is synthesized by species belonging to the group I.1a crenarchaeota. Intact polar lipid analysis revealed that the GDGTs have hexose, dihexose, and/or phosphohexose head groups. Similar polar lipids were previously found in deeply buried sediments from the Peru margin, suggesting that they were in part synthesized by group I crenarchaeota.

Citrus huanglongbing (HLB or citrus greening), is a highly destructive disease that has been spreading in both Florida and Brazil. Its psyllid vector, Diaphorina citri Kuwayama, has spread to Texas and Mexico, thus threatening the future of citrus production elsewhere in mainland North America. Even though sensitive diagnostic methods have been developed for detection of the causal organisms, Candidatus Liberibacter spp., the pathogen cannot be detected consistently in plants until symptoms develop, presumably because of low titer and uneven distribution of the causal bacteria in nonsymptomatic tissues. In the present study, TaqMan based real-time quantitative polymerase chain reaction methodology was developed for detection of ‘Ca. L. asiaticus’ in D. citri. Over 1,200 samples of psyllid adults and nymphs, collected from various locations in Florida, from visually healthy and HLB symptomatic trees at different times of the year were analyzed to monitor the incidence and spread of HLB. The results showed that spread of ‘Ca. L. asiaticus’ in an area may be detected one to several years before the development of HLB symptoms in plants. The study suggests that discount garden centers and retail nurseries may have played a significant role in the widespread distribution of psyllids and plants carrying HLB pathogens in Florida.

During a survey in 2003, safflower plants (Carthamus tinctorius) with phyllody symptoms were observed in production fields in several districts of Fars and Yazd provinces in Iran. Affected plants showed floral virescence, phyllody, proliferation of axillary buds, and little leaf symptoms. Incidence of the disease was less than 10%. Direct and nested PCR assays were used to verify association of phytoplasma with the disease. Total DNA was extracted from fresh, fine roots of eight phyllody-affected safflower plants and one symptomless plant. With phytoplasma universal primer pair P1/P7 (5′-AAGAGTTTGATCCTGGCTCAGGATT-3′/5′-CGTCCTTCATCGGCTCTT-3′), target DNA fragments of approximately 1.8 kb were amplified by direct PCR from phyllody-affected plants and Iranian cabbage yellows (ICY) phytoplasma used as a positive control. Reamplification of P1/P7 products with 16S rRNA gene primer pair R16F2n/R16R2 (5′-GAAACGACTGCTAAGACTGG-3′/5′-TGACGGGCGGTGTGTACAAACCCCG-3′) yielded fragments of the expected size (1.2 kb) from all eight diseased plants and the ICY-positive control. No products were amplified from the symptomless plant by either assay. R16F2n/R16R2 products were subjected to restriction fragment length polymorphism (RFLP) analysis by separate digestion with AluI, HaeIII, HhaI, HinfI, HpaII, MseI, RsaI, Sau3AI, or TaqI endonuclease. Comparison of resulting RFLP patterns with published patterns of other phytoplasmas (2) tentatively identified safflower phyllody (SP) phytoplasma as a member of clover proliferation group 16SrVI, subgroup C. HhaI digests also differentiated SP from ICY phytoplasma, a previously reported subgroup 16SrVI-A strain (3). After sequencing of the 16S rDNA fragment (GenBank Accession No. DQ88948), a BLAST search determined that SP phytoplasma shared closest homology with 16SrVI group members (‘Candidatus Phytoplasma trifolii’) and related strains (4). Furthermore, phylogenetic analysis of 16S rDNA sequences revealed SP phytoplasma to be most similar (99.7%) to brinjal little leaf (BLL) phytoplasma (GenBank Accession No. X83431). Analysis of putative restriction sites in 16S rRNA gene sequences revealed that SP and BLL shared identical restriction profiles and that both differed from the ‘Ca. Phytoplasma trifolii’ reference strain (GenBank Accession No. AY390261) because of the absence of a single HhaI site and the presence of an additional MseI site. Although safflower phyllody disease has been previously reported in Israel, the associated phytoplasma was classified as a strain of the aster yellows subgroup 16SrI-B (1). To our knowledge, this is the first report of safflower as a host of a ‘Ca. Phytoplasma trifolii’-related strain. References: (1) M. Klein. Plant Dis. Rep. 54:735, 1970. (2) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:1037, 2004. (3) M. Salehi et al. Plant Dis. 91:625, 2007. (4) K. Wang and C. Hiruki, Phytopathology 91:546, 2001.