Publications

4514

Bovine hemoplasmas are bacteria found on the erythrocyte surface or free in the plasma of cattle. The aim of the present study was to evaluate the occurrence of 'Candidatus Mycoplasma haemobos' ('C. M. haemobos') in Holstein and Jersey cattle raised in Londrina and surroundings, northern region of the State of Parana, Southern Brazil. PCR testing directed to 16S rRNA gene fragment was performed to investigate the occurrence and characterize the molecular identity of 'C. M. haemobos'. A total of 264/433 (60.97%) blood samples were positive by PCR. Further alignment of 500-bp amplicons to available sequences at the GenBank database showed high identity (100%) to 'C. M. haemobos'. To the author's knowledge, this is the first molecular confirmation of the hemoplasma 'C. M. haemobos' in cattle from Brazil. Moreover, 'C. M. haemobos' was observed in high occurrence in dairy cattle, and may have significant impact in livestock production.

Phytoplasmas of the 16SrVII group in ornamental Fraxinus uhdei trees (1) growing in different cities of the Colombian Andes have been reported (2). In surveys made in Bogotá during March and May 2011, symptoms suggestive of phytoplasma infection were observed in ornamental woody species: Croton spp. (Euphorbiaceae), Pittosporum undulatum (Pittosporaceae) and Populus nigra (Salicaceae) trees, growing close to infected F. uhdei (Oleaceae). Symptoms included witches' broom, yellowing, dieback, epicormic sprouts, tufted foliage, abnormal elongation or shortening of internodes, and deliquescent branching leading to dramatic changes in crown architecture. P. undulatum and F. uhdei are introduced species representing the second and third most abundant trees in the city. P. nigra is an introduced species and Croton spp. is an Andean genus. In order to screen for the presence of phytoplasmas in Croton spp., P. undulatum, and P. nigra, four individuals of each species and two F. uhdei trees were sampled. For DNA extraction, 1 g of vascular tissue from young stems was used. Samples were tested by nested PCR with primers P1A/P7A (4) followed by R16F2n/R16R2 (3). The frequency of phytoplasma detection varied among species; P. undulatum and Croton spp. had three positives each, while P. nigra had one positive. Both F. uhdei were positive. Sequences from the amplicons (three reads) were aligned. BLAST analysis of 16S rDNA sequences from the four species tested had 99.2 to 99.7% similarity to 16SrI group sequences. Phylogenetic analysis further confirmed this relationship. Virtual sequence analysis using the iPhyclassifier tool ( http://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi ) showed that the sequence derived from P. undulatum (JQ730861) produced an identical RFLP pattern to group 16SrI-B (reference sequence NC_005303). RFLP similarity coefficients of the phytoplasmas from F. uhdei, Croton spp., and P. nigra (JQ730859, JQ730859 and JQ730861) were less than 0.97, suggesting the presence of a new subgroup within group 16SrI. The vectors of phytoplasmas are unknown in the region. Phytoplasma hosts previously reported in Colombia are: Solanum quitoense (16SrIII), Manihot esculenta (16SrIII), Liquidambar styraciflua (16SrI and 16SrVII), Elaeis guineensis (16SrI and 16SrIII), Coffea arabica (16SrIII), Cordia alliodora (16SrIII), Solanum tuberosum (16SrV and 16SrXII), and Zea mays (16SrI). To our knowledge, this is the first report of Croton spp. and P. undulatum as phytoplasma hosts. Phytoplasmas of group 16SrI are known to infect more than 100 species of different families worldwide. Detection of this group in several tree species and the observation of similar symptoms in other trees species raises concerns about a possible epidemic affecting plants in the Andean region. Implications are at several levels: i) epidemiological, with infected trees representing a potential inoculum source for other ornamental plants or crops growing in the agricultural surrounding areas; ii) economic, since eventually it will be necessary to replace diseased plants; and iii) environmental, because of the negative impact on the services provided by trees and green areas. References: (1) J. J. Filgueira et al. Plant Pathology 53:520, 2004. (2) L. Franco-Lara et al. Fitopatología Colombiana 29:32, 2005. (3) D. E Gundersen et al. Phytopathol. Mediterr. 35:144, 1996. (4) I-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:1037, 2004.

This study reports the development of a loop-mediated isothermal amplification procedure (LAMP) for polymerase chain reaction (PCR)-based detection of ‘Candidatus Liberibacter solanacearum’, the bacterial causal agent of potato zebra chip (ZC) disease. The 16S rDNA gene of ‘Ca. Liberibacter solanacearum’ was used to design a set of six primers for LAMP PCR detection of the bacterial pathogen in potato plants and the psyllid vector. The advantage of the LAMP method is that it does not require a thermocycler for amplification or agarose gel electrophoresis for resolution. Positive LAMP results can be visualized directly as a precipitate. The LAMP strategy reported here reliably detected ‘Ca. Liberibacter solanacearum’ and the closely related species ‘Ca. Liberibacter asiaticus’, the causative agent of huanglongbing disease of citrus, in plant DNA extracts. Although not as sensitive as quantitative real-time PCR, LAMP detection was equivalent to conventional PCR in tests of ZC-infected potato plants from the field. Thus, the LAMP method shows strong promise as a reliable, rapid, and cost-effective method of detecting ‘Ca. Liberibacter’ pathogens in psyllids and field-grown potato plants and tubers.

Abstract. This study investigated the bacterial diversity associated with microbial mats of polar deep-sea cold seeps. The mats were associated with high upward fluxes of sulfide produced by anaerobic oxidation of methane, and grew at temperatures close to the freezing point of seawater. They ranged from small patches of 0.2–5 m in diameter (gray mats) to extensive fields covering up to 850 m2 of seafloor (white mats) and were formed by diverse sulfide-oxidizing bacteria differing in color and size. Overall, both the dominant mat-forming thiotrophs as well as the associated bacterial communities inhabiting the mats differed in composition for each mat type as determined by microscopy, 16S rRNA gene sequencing and automated ribosomal intergenic spacer analysis. While the smaller gray mats were associated with a highly diverse composition of sulfide oxidizers, the larger white mats were composed of only 1–2 types of gliding Beggiatoa filaments. Molecular analyses showed that most of the dominant mat-forming sulfide oxidizers were phylogenetically different from, but still closely related to, thiotrophs known from warmer ocean realms. The psychrophilic nature of the polar mat-forming thiotrophs was tested by visual observation of active mats at in situ temperature compared to their warming to >4 °C. The temperature range of mat habitats and the variation of sulfide and oxygen fluxes appear to be the main factors supporting the diversity of mat-forming thiotrophs in cold seeps at continental margins.

The objectives of this work were (i) to determine the influence of temperature on infection of citrus by ‘ Candidatus Liberibacter asiaticus’ and ‘ Candidatus Liberibacter americanus’, the two bacterial species associated with citrus huanglongbing (HLB) in Brazil, and (ii) to determine the influence of temperature on citrus colonization by ‘ Ca.  L. asiaticus’, which has taken over from ‘ Ca.  L. americanus’ as the predominant species in Brazil since 2008. Two experiments were carried out with graft‐inoculated Valencia oranges on Rangpur lime rootstocks. Immediately after inoculation the plants were maintained for 423 days in growth chambers under the following night/day temperature conditions: 17/22, 22/27 or 27/32°C, with a dark/light photoperiod of 8/16 h. Infection and colonization of plants were determined using quantitative PCR (qPCR). ‘ Candidatus Liberibacter americanus’ did not infect the plants maintained at 27/32°C; however, infection by ‘ Ca.  L. asiaticus’ occurred at all studied temperatures. Two months after inoculation, ‘ Ca.  L. asiaticus’ was distributed throughout the inoculated plants, with mean C t values in the range of 30–31 for leaves and 25–28 for roots. Over time, ‘ Ca.  L. asiaticus’ reached the highest titres in mature leaves (mean C t value = 26·7) of citrus plants maintained at 22/27°C. ‘ Candidatus Liberibacter asiaticus’ colonization of citrus plants was negatively affected by the daily temperature regime of 27/32°C (mean C t value in mature leaves = 33·6).