Multiply-primed rolling-circle amplification (MPRCA) of PCV2 genomes: Applications on detection, sequencing and virus isolation

Multiply-primed rolling-circle amplification (MPRCA) was used to amplify porcine circovirus type 2 (PCV2) genomes isolated from tissues of pigs with signs of post-weaning multisystemic wasting syndrome (PMWS). Two of the amplified PCV2 genomes were cloned in prokaryotic plasmids and sequenced. Both were nearly identical (1767 nt) except for one silent substitution in the region coding for the capsid protein (ORF2). In addition, they showed high nucleotide sequence similarity with PCV2 isolates from others countries (93–99%). To investigate whether the MPRCA amplified PCV2 genomes could be used to produce infectious virus, the cloned genomes were isolated from the plasmids, recircularized and used for transfection in PK-15 cells. This procedure led to the production of infectious virus to titres up to 10^5.55 TCID₅₀/mL. It was concluded that MPRCA is a useful tool to amplify PCV2 genomes aiming at sequencing and virus isolation strategies, where particularly useful is the fact that it allows straightforward construction of PCV2 infectious clones from amplified genomes. However, it was less sensitive than PCR for diagnostic purposes.     

Phenotypic differences may limit the potential for habitat restoration involving species translocation: a case study of shape ecophenotypes in different populations of Modiolus modiolus (Mollusca: Bivalvia)

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Genetic diversity of 16SrIII group phytoplasmas in Argentina. Predominance of subgroups 16SrIII-J and B and two new subgroups 16SrIII-W and X

Twelve Argentinean 16SrIII (X-disease)-group phytoplasma strains were analyzed. Ten of them, detected in daisy (Bellis perennis), garlic (Allium sativum), ‘lagaña de perro’ (Caesalpinia gilliesii), periwinkle (Catharanthus roseus), ‘rama negra’ (Conyza bonariensis), ‘romerillo’ (Heterothalamus alienus), summer squash (Cucurbita maxima var. zapallito) and tomato (Solanum lycopersicum), are new phytoplasma strains while two strains, detected in garlic and China tree (Melia azedarach), have been previously described. The plants showed typical symptoms of phytoplasma diseases, such as leaf size reduction, proliferation, stunting and virescence. The identification and genetic diversity analysis of the phytoplasmas were performed based on 16S rDNA and ribosomal protein gene sequences. The classification into 16Sr groups and subgroups was established by actual and virtual RFLP analysis of the PCR products (R16F2/R16R2) compared with reference strains. According to the classification scheme, strains HetLL and ConWB-A and B represent two new subgroups 16SrIII-W and X, respectively. On the other hand, strains CatLL, TomLL and CaesLL are related to subgroup 16SrIII-B, and strains BellVir, TomRed, CucVir and GDIII-207 are related to subgroup 16SrIII-J. Ribosomal protein genes were amplified using primers rpF1/rpR1 and rpIIIF1/rpIIIR1. RFLP analysis performed with AluI, DraI and Tru1I (MseI isoschizomer) distinguished three new rp profiles within subgroup 16SrIII-B, one for subgroup 16SrIII-J, and one shared with strains of the new subgroups 16SrIII-W and X. The phylogenetic analysis based on 16S rDNA and ribosomal protein gene sequences confirmed the separation of HetLL and ConWB strains in two new subgroups and the close relatedness among subgroup J phytoplasmas, which have been detected only in South America.     

Molecular sequence data of Thecaphora frezii affecting peanut crops in Argentina

In Argentina, Thecaphora frezii is the causal agent of peanut smut, causing severe yield losses in peanut growing areas. In this work sequence data of the large ribosomal subunit (28S rDNA) of four T. frezii specimens from different production regions are provided. A fragment of about 650 bp was amplified by PCR from each of the isolates analyzed. The analysis of the sequences demonstrated 100 % identity among the four specimens and 92–96.3 % identity with sequences of other species of Thecaphora. This is the first study on molecular sequences of this pathogen.     

The effect of ethephon on the growth and development of Liatris spicata

Summary

The first experiment studied the effect of applying ethylene biosynthesis regulating compounds (ethephon 1000 ppm, silver thiosulphate 0.4 mM and cobalt chloride 5 mM) on the growth and development of Liatris spicata cv. Callilepis. The results pointed to the important growth retardant properties of ethephon and the increased number of flowering stems per corm which could be obtained by pre-planting application. A second experiment aimed to optimize ethephon treatment in Liatris by applying the growth retardant in different concentrations (100, 700, 1300, 1900 and 2500 ppm) at three stages of development: stage I (pre-planting), stage II (immediately after the formation of basal leaf rosette) and stage III (when the main shoot began its rapid longitudinal growth). Besides confirming the effects produced by ethephon in the first experiment, the results suggested that the application of high ethephon concentrations (1900 and 2500 ppm) during stage II of growth produced many miniature flowering stems coming from the principal or secondary shoot.     

Effect of two irrigation rates on yield,incidence of blossom-end rot,mineral content and free amino acid levels in tomato cultivated under drip irrigation using saline water

Summary

An experiment investigated the effects of two rates of irrigation, one of which reflected a substantial degree of water stress, on the mineral content, free amino acid levels and incidence of blossom-end rot (BER) in tomato (Lycopersicon esculentum Mill. `Durinta'). The plants were grown in the open with drip irrigation using saline water from a well (mean EC_w 5.2 dS m²¹). The yield per plant was higher and fewer fruit were affected by BER in the treatment involving the higher level of irrigation. The fruit of the first and fifth truss, and the leaves immediately above, were analysed for their macronutrient, micronutrient and free amino acid content. The macronutrient leaf and fruit content hardly showed any difference, only the N concentration in fruit being significantly affected in the water stressed plants, in which the levels were higher. The Ca concentration in the stylar portion of mature fruit, which is related with the incidence of BER, was not significantly affected by the level of irrigation. As regards micronutrients, only the Fe (in leaf and fruit of the first truss), Cu (in leaf of the first truss), Zn (in leaf and fruit of the first truss, and leaf of the fifth truss) and Mn (in leaf of the first truss) concentrations differed significantly. The total free amino acid leaf content was similar in both irrigation treatments. However, the total free amino acid content of fruit, significantly those of the first truss, was higher in the less irrigated treatment. The amino acids: aspartic acid (only from the first.truss), glutamic acid, proline and alanine had high concentrations in the fruit of the less irrigated plants, while the g-aminobutyric acid and phenylalanine (only from the fifth truss) concentrations were higher in fruit of the more irrigated plants.     

Influence of water deficit and low air humidity in the nursery on survival of Rhamnus alaternus seedlings following planting

Summary

The effect of irrigation and air humidity on the water relations and root and shoot growth of Rhamnus alaternus L. during the nursery phase was considered to evaluate the resulting degree of hardening obtained by these treatments. R. alaternus seedlings were pot-grown in two greenhouses of equal characteristics. In one of these greenhouses air humidity was controlled using a dehumidifying system, while in the other one the environmental conditions were not artificially modified. In each greenhouse, two irrigation treatments were used. Thus, four different treatments were applied during the nursery phase (January-May): 1) control air humidity + control irrigation; 2) control air humidity + deficit irrigation; 3) low air humidity + control irrigation; 4) low air humidity + deficit irrigation. In May, plants of all treatments were transplanted and grown in good environmental and irrigation conditions for one month (17 May–20 June), after which they received no irrigation until the end of the experiment (14 July). Low air humidity and water deficit reduced all shoot growth parameters during the nursery phase, however the root growth was not significantly affected by air humidity and even increased under the water deficit. The reduction in leaf water potential under water stress was induced by tissue dehydration since leaf turgor potential also decreased and non-osmotic adjustment was observed. The drought effects on water relations were similar in both low and high air humidity. The leaf stomatal conductance was also reduced by both types of stress, leading to a decrease in the rate of photosynthesis at the end of the nursery phase. Both water deficit and low air humidity showed their value as nursery acclimation processes, improving the survival of seedlings following transplanting and non-irrigation conditions (establishment phase). The stomatal regulation and a shift in the allocation of assimilates from shoot to root were the acclimation mechanisms showed by R. alaternus under both types of stress. The accumulated effects in low air humidity and water deficit plants could explain the highest percentage of survival at the end of the establishment period (97%) for the combined treatment.     

Geographical pattern and ploidy levels of the weed Solanum elaeagnifolium (Solanaceae) from Argentina

A total of 106 samples taken from natural Argentinean populations of the weedy Solanum elaeagnifolium (subgenus Leptostemonum) were studied cytologically to understand the impact of the different ploidy levels in its distribution and origin. Classical Feulgen staining was employed to determine mitotic chromosome numbers in all samples. 2C nuclear DNA content was determined by means of PI flow cytometry in eight samples of different ploidy levels. Principal component analysis and GIS tools were employed to compare altitude, annual precipitation and annual mean temperature among accessions. Three cytotypes were found: diploid (2n?=?24) which is widespread, tetraploid (2n?=?48) centered in western and southern Argentina, and hexaploid (2n?=?72) which predominates in central Argentina extending as well to the east. The annual precipitation is significantly different between tetraploids and hexaploids. Cx-values ranged from 1.231 to 1.275?pg, with statistical differences (of about 24.5–50.9?Mbp, p?≤?0.05) among accessions. Diploids are the most widespread cytotype and have adapted to a number of very different habitats. Tetraploids live in arid or semi-arid regions with a mean annual rainfall less than 500?mm. Hexaploids are successful in colonizing wetter areas, where no tetraploids were found. Thus, the distribution of cytotypes may be associated with habitat differences, particularly soil moisture. The observed cytotype pattern and the differences in DNA content suggest multiple places of origin for the polyploidy of S. elaeagnifolium in Argentina.