Phylogeny and variability of Colletotrichum truncatum associated with soybean anthracnose in Brazil.

AIMS: Fungal diseases are among the main factors limiting high yields of soybean crop. Colletotrichum isolates from soybean plants with anthracnose symptoms were studied from different regions and time periods in Brazil using molecular, morphological and pathogenic analyses. METHODS AND RESULTS: Bayesian phylogenetic inference of GAPDH, HIS3 and ITS-5.8S rDNA sequences, the morphologies of colony and conidia, and inoculation tests on seeds and seedlings were performed. All isolates clustered only with Colletotrichum truncatum species in three well-separated clusters. Intraspecific genetic diversity revealed 27 distinct haplotypes in 51 fungal isolates; some of which were identical to C. truncatum sequences from other regions around the world, while others were related to alternative hosts. Conidia were falcate, hyaline, unicellular and aseptate, formed in acervuli, with variable dimensions. Despite being pathogenic to seedlings by both inoculation methods, variation was observed in the aggressiveness of the tested isolates, which was not correlated with genetic variation. CONCLUSION: The identification of C. truncatum in the sampled isolates was evidenced as being the only causal agent of soybean anthracnose in Brazil until 2007, with relevant genetic, morphological and pathogenic variability as well as a broad geographical origin. The wide distribution of the predominant C. truncatum haplotype indicated the existence of a highly efficient mechanism of pathogen dispersal over long distances, reinforcing the role of seeds as the primary source of disease inoculum. SIGNIFICANCE AND IMPACT OF THE STUDY: The characterization and distribution of Colletotrichum species in soybean-producing regions in Brazil is fundamental for understanding the disease epidemiology and for ensuring effective control strategies against anthracnose.

Evidence for adenosine adenosinedeaminase lymphocyte system impairment in ageing.

Age related immune disfunctions are the result of humoral and cellular changes of the immune system and reflect major alterations of T cell subpopulations which concern cyclic nucleotides and their precursors. There are now many reports showing that adenosine can affect some phenotypic and functional lymphocyte characteristics. We have found that a short preincubation (30') with adenosine can inhibit proliferative responses of peripheral blood lymphocytes to polyclonal mitogen Concanavalin A in young healthy controls (p less than 0.05) but not in aged healthy subjects. These data led us to the hypothesis that an impairment of the adenosine-adenosinedeaminase system could play an important role in the age-associated decline of immune responses. Our results show a highly significant reduction (16.45 +/- 3.56 vs 24.42 +/- 9.5 p less than 0.001) of adenosinedeaminase activity in peripheral lymphocytes of aged humans (mean age 75.5 +/- 6.7 range 23-30). Preliminary studies suggest that this alteration could be responsible to some extent, for the decreased mitogenic response of lymphocytes reported in ageing.

Abnormal OKT4/OKT8 ratio and deranged capping in ataxia-telangiectasia.

Two sisters with ataxia-telangiectasia (A-T) and their parents were investigated for some parameters related to immunological functions. We found a decrease of total mature T lymphocytes, a decrease of OKT4+ helper-inducer T subset, and normal values of OKT8+ suppressor-cytotoxic T subset; a normal decrease of E rosette formation after incubation in vitro with theophylline, with a lowered E rosette capacity in one patient. The responses to phytohemagglutinin (PHA)and concanavalin A (ConA) were lowered. In addition we observed a reduction of basal capping of B lymphocytes in one patient and in her parents: this phenomenon could be related to cytoskeletal disorders, possibly involved in the pathogenesis of the disease.

Lysosomal glycohydrolases in normal T and non-T peripheral lymphocytes.

The optimal assay conditions and the levels of seven lysosomal glycohydrolases (alpha-D-galactosidase, beta-D-galactosidase, beta-D-glucosidase, beta-D-glucuronidase, beta-N-acetyl-D-glucosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase), alpha-D-mannosidase, alpha-L-fucosidase) were determined in human peripheral unseparated lymphocytes, T and non-T lymphocyte subpopulations. From fifteen adult volunteers the enzymes were assayed by fluorimetric procedures using the corresponding 4-methylumbelliferyl glycosides as substrates. The enzyme assay procedures displayed good precision and reproducibility. All the tested enzymes had higher activities in non-T than T lymphocytes. This difference was statistically highly significant, especially when the enzyme contents were expressed on a DNA, rather than mg protein, basis. Unseparated lymphocytes displayed levels of lysosomal enzymes which corresponded to the proportion of T and non-T lymphocytes in the unseparated preparation, indicating that the process of lymphocyte fractionation caused neither loss nor activation of lysosomal enzymes. It is concluded that the observed difference in lysosomal enzyme levels is an authentic imprint of the two lymphocyte subpopulations, implying a differential role played by the lysosomal apparatus in the same cells.

Spontaneous "in vitro" variations of theophylline sensitivity in human peripheral blood lymphocytes.

Theophylline reversibly inhibits E rosette formation by a portion of human circulating T lymphocytes. We investigated the effect of theophylline on E rosette formation and intracellular content of cyclic nucleotides (cAMP, cGMP). When the amine is added to 15 human healthy donors' lymphocytes either before or after 24 hours of culture at 37 degrees C, in absence of mitogens, a portion of theophylline-sensitive T cells spontaneously becomes theophylline-resistant after 24 hours of culture. While the intracellular content of cAMP does not significantly vary, the ability of theophylline to induce an increase of cAMP appears to be impaired after 24 hours of culture. The possible correlation between theophylline resistance and impaired turnover of cAMP in the cultured lymphocytes is discussed.