Publications

3512

‘Candidatus Liberibacter solanacearum’ (Lso) causes disease symptoms and economic losses in potato, tomato, and other solanaceous crops in North America. Lso is transmitted to plants by the potato psyllid, Bactericera cockerelli, which occurs as distinct haplotypes named western, central, and northwestern that differ in the presence or absence of the bacterial endosymbiont, Wolbachia. Previous work showed that all three vector haplotypes can transmit Lso, but it was not clear whether acquisition and transmission rates of Lso were equal among the haplotypes. The goal of our study was to compare Lso infection rates among psyllids of the western, central, and northwestern haplotypes. Using data collected from several years of periodic testing of Lso infection of laboratory-reared potato psyllid colonies, we showed that psyllids of the western and central haplotypes are more likely to harbor Lso than are psyllids of the northwestern haplotype. We then used greenhouse assays to demonstrate that psyllids of the northwestern haplotype are less likely to acquire and transmit Lso than those of the western haplotype. Lso infection rates corresponded with Wolbachia infection among the three psyllid haplotypes. The Wolbachia-infected central and western haplotypes were more likely to harbor and transmit Lso than the Wolbachia-free northwestern haplotype. Results demonstrate that potato psyllids of the western and central haplotypes pose a greater risk for spread of Lso in crops and suggest a pattern between infection with Lso and Wolbachia in potato psyllid.

ABSTRACTCandidatus Ornithobacterium hominis sp. nov. represents a new member of the Flavobacteriaceae detected in 16S rRNA gene surveys from Southeast Asia, Africa and Australia. It frequently colonises the infant nasopharynx at high proportional abundance, and we demonstrate its presence in 42% of nasopharyngeal swabs from 12 month old children in the Maela refugee camp in Thailand. The species, a Gram negative bacillus, has not yet been cultured but the cells can be identified in mixed samples by fluorescent hybridisation. Here we report seven genomes assembled from metagenomic data, two to improved draft standard. The genomes are approximately 1.9Mb, sharing 62% average amino acid identity with the only other member of the genus, the bird pathogen Ornithobacterium rhinotracheale. The draft genomes encode multiple antibiotic resistance genes, competition factors, Flavobacterium johnsoniae-like gliding motility genes and a homolog of the Pasteurella multocida mitogenic toxin. Intra- and inter-host genome comparison suggests that colonisation with this bacterium is both persistent and strain exclusive.

In Europe and the Mediterranean region, ‘Candidatus Liberibacter solanacearum’ (Lso) is associated with emerging diseases of Apiaceae crops, mainly carrot. Emergency measures for import of carrot seed were set, requiring seed to be heat-treated at 50°C or tested as Lso-negative by PCR. The germination response to heat treatment was assessed for 24 carrot cultivar and hybrid seed lots. Ten parsley, five fennel, and two celery seed lots were also analysed. Of these 41 seed lots, 21 were Lso-infected. Water heat treatment significantly decreased germinability compared to dry heat treatment, indicating that dry heat treatment is a cheaper and less detrimental procedure. However, the dry heat treatment significantly decreased seed germination compared to untreated controls in four of 24 seed lots of carrot, four of ten parsley seed lots, three of five fennel seed lots, and one of two celery seed lots. For parsley, the heat treatment reduced germinability to a lesser extent in Lso-infected than Lso-free seed lots. These data show that heat treatment can affect the germination of Apiaceae seeds to varying degrees, depending on species or variety, the type of heat treatment, and the sanitary status of the seeds.

Although no loss of crown shape or unusual growth were evident on two mature Chinaberry trees (Melia azedarach L.) located near the citadel in central Hué city, Vietnam, leaves on both trees displayed distinctive interveinal yellowing during September 2003. This symptom was reminiscent in appearance to foliar discoloration previously observed on mature Chinaberry trees in El Torno, Santa Cruz, Bolivia that was subsequently attributed to phytoplasma infection of these trees (2). Eight samples of yellowed leaves were collected from affected trees and preserved by pressing and drying for later analysis. Total nucleic acids were extracted from 0.5 g of each leaf sample and assayed for phytoplasma DNA using a polymerase chain reaction (PCR) assay with phytoplasma universal rRNA primer pair P1 and P7 (4). No visible product was generated from any Chinaberry sample while a product of expected size (1.8 kb) was obtained from DNA of a periwinkle plant (Catharanthus roseus (L.) G. Don) infected with “Candidatus Phytoplasma asteris”-related strain eastern aster yellows (EAY) and included as a known positive control in the assay. After P1/P7-primed products were reamplified by PCR with nested phytoplasma universal 16S rRNA primer pair R16mF2/R16mR1 (1), a 1.4-kb product of predicted size was obtained from the eight samples and EAY positive control, whereas no product was obtained from DNA of seed-grown healthy periwinkle included as a negative control. Digests of nested PCR products (1.4 kb) with HaeIII or MseI endonuclease, and electrophoresis of digests through 8% polyacrylamide gels, revealed no apparent differences in restriction fragment patterns among products from Chinaberry samples. However, HaeIII and MseI patterns differed from those obtained by digestion of nested PCR products from EAY, a known 16SrI-A subgroup phytoplasma (3), with these enzymes. Chinaberry phytoplasmas were definitively identified as group 16SrI strains after reevaluation of samples by a PCR incorporating ribosomal protein (rp) gene primer pair rpF1/rpR1 and reamplification of resulting products with nested 16SrI group-specific primer pair rp(I)F1A/rp(I)R1A (3). A 1.2-kb product of expected size was obtained from all Chinaberry samples and EAY positive control only. Restriction fragment length polymorphism patterns produced by DraI or SspI endonuclease digests of nested PCR products (1.2 kb) revealed no differences among Chinaberry samples, although patterns associated with each enzyme differed from those observed for the EAY positive control. Sequence comparison and phylogenetic analysis of Chinaberry yellows phytoplasma (CbY-V) 16Sr DNA (GenBank Accession No. AY863003) determined this strain to be most closely related (99.65%) to Epilobium phyllody phytoplasma, a 16SrI-B subgroup strain (3). However, based on analysis of rp gene sequences (GenBank Accession No. DQ321823), strain CbY-V was judged most similar (99.59%) to cabbage proliferation, a well characterized 16SrI-B subgroup, rpI-B subgroup phytoplasma (3). To our knowledge, this is the first record of phytoplasma infection of Chinaberry, a common urban shade tree in Vietnam. References: (1) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) N. A. Harrison et al. Plant Pathol. 52:147, 2003. (3) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:1037, 2004 (4) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996.

AbstractCandidatus Neoehrlichia mikurensis (CNM) and Hepatozoon spp. are important vector-borne parasites of humans and animals. CNM is a relatively recently discovered pathogen of humans. Hepatozoon are parasites of reptiles, amphibians and mammals, commonly found in rodents and carnivores worldwide. The present study aimed to determine the prevalence of CNM and Hepatozoon spp. in three species of Microtus and to assess the occurrence of vertical transmission in naturally-infected voles. Molecular techniques were used to detect pathogen DNA in blood and tissue samples of captured voles and their offspring. The prevalence of CNM in the vole community ranged 24–47% depending on Microtus species. The DNA of CNM was detected in 21% of pups from three litters of six infected Microtus dams (two Microtus arvalis and one M. oeconomus) and in 3/45 embryos (6.6%) from two litters of eight CNM-infected pregnant females. We detected Hepatozoon infection in 14% of M. arvalis and 9% of M. oeconomus voles. Hepatozoon sp. DNA was detected in 48.7% of pups from seven litters (6 M. arvalis and 1 M. oeconomus) and in two embryos (14.3%) obtained from one M. arvalis litter. The high prevalence of CNM infections in the Microtus spp. community may be a result of a relatively high rate of vertical transmission among naturally infected voles. Vertical transmission was also demonstrated for Hepatozoon sp. in M. arvalis and M. oeconomus. Our study underlines the significance of alternative routes of transmission of important vector-borne pathogens.

In Mexico, Huanglongbing HLB has become endemic in all Mexican lime producing areas. This disease is transmitted by the insect Diaphorina citri (Hemiptera: Liviidae). The objective of the study was to evaluate different plant extracts and biorational products for the control of D. citri in mexican lime. Two evaluations were made under field conditions; the first one included five treatments, with previous sampling and at 8, 20 and 27 days after the application of the treatments. In the second, eight treatments were evaluated, with prior sampling and at 6, 21 and 27 days. The response variable was the number of D. citri. A completely randomized experimental design with ten repetitions was used. A test of normality and homogeneity of variances was applied to the data, and they were processed through an analysis of variance and separation of means using Tukey (p≤ 0.05). The sweet clover extract (6.0 mL L-1) at 20 days decreased the population density by 59.2%, and Pyrifluquinazon (0.58 mL L-1) at 6 days the decrease was 31.3%. All the extracts showed to be a sustainable alternative for the management of D. citri.

TXW-1, a Borrelia strain isolated in March 1998 from an adult male Dermacentor variabilis tick feeding on a coyote from Webb county, Texas, USA, was characterized by using randomly amplified polymorphic DNA (RAPD) analysis, RFLP and sequence analysis of flaB and rrs (16S rRNA gene), DNA–DNA hybridization analysis, SDS-PAGE and Western blotting with mAbs. It shows different banding patterns in RFLP analysis of flaB and forms distinct branches in phylogenetic analysis derived from flaB and rrs genes. It differs from other borreliae based on the banding patterns obtained by RAPD analysis. This strain contains a small, 38-kDa endoflagellar protein. DNA–DNA hybridization experiments revealed that the levels of DNA reassociation between TXW-1 and previously described relapsing fever borreliae were 38·64 % (Borrelia turicatae), 38·40 % (Borrelia parkeri), 7·39 % (Borrelia hermsii) and 18·30 % (Borrelia coriaceae). However, the level of DNA relatedness between B. parkeri and B. turicatae was 78·78 %. Sequence analyses of flaB and rrs genes indicate that the similarities of nucleotide sequences among TXW-1 and B. turicatae or B. parkeri are less than that between B. turicatae and B. parkeri, and that the genetic distances among TXW-1 and B. turicatae or B. parkeri are greater than that between B. turicatae and B. parkeri. TXW-1 lacks an ospC gene. Electron microscope observations showed that this spirochaete had different morphological structures compared to previously described relapsing fever borreliae. All the results obtained from the above-mentioned analyses indicate that TXW-1 is different from other described Borrelia species and that it represents a novel species of Borrelia. We have been unable to revive frozen cultures and so can not meet the requirements of the Bacteriological Code to deposit viable type material at two different culture collections. Therefore we use the Candidatus designation; based on these results, the species ‘Candidatus Borrelia texasensis' is proposed.