Molecular identification of Fusarium species isolated from transgenic insect-resistant cotton plants in Mexicali valley, Baja California.

Cotton production in the Mexicali valley is adversely affected by wilt and root rot disease associated with Fusarium species. In the present study, we sought to isolate and identify the Fusarium species in the rhizosphere of transgenic insect-resistant cotton plants grown in the Mexicali valley. Our analyses isolated four native fungi from the rhizosphere of cotton plants, namely, T-ICA01, T-ICA03, T-ICA04, and T-ICA08. These fungal isolates were categorized as belonging to Fusarium solani using their phenotypic characteristics and ITS region sequence data. Examination of the infection index showed that T-ICA03 and T-ICA04 caused systemic colonization (90%) of seeds followed by the occurrence of radicle and coleoptile decay. In contrast, T-ICA08 strain was less pathogenic against seed tissues (40%) in comparison to the other strains isolated. Our study showed that in transgenic insect-resistant cotton the disease "Fusarium wilt" is caused by the fungus, F. solani. Future studies are necessary to characterize the F. solani populations to determine whether phenological stages might influence the genetic diversity of the fungal populations present.

Supplemental leucine and isoleucine affect expression of cationic amino acid transporters and myosin, serum concentration of amino acids, and growth performance of pigs.

Leucine (Leu) participates in the activity of cationic amino acid (aa) transporters. Also, branched-chain aa [Leu, isoleucine (Ile), and valine (Val)] share intestinal transporters for absorption. We conducted an experiment with 16 young pigs (body weight of about 16 kg) to determine whether Leu and Ile affect expression of aa transporters b(0,+) and CAT-1 in the jejunum and expression of myosin in muscle, as well as serum concentration of essential aa, and growth performance in pigs. Dietary treatments were: wheat-based diets fortified with Lys, Thr, and Met; basal diet plus 0.50% Leu; basal diet plus 0.50% Ile, and basal diet plus 0.50% Leu and 0.50% Ile. After 28 days, the pigs were sacrificed to collect blood, jejunum, and semitendinosus and longissimus muscle samples. The effects of single and combined addition of Leu and Ile were analyzed. Leu alone or combined with Ile significantly decreased daily weight gain and reduced feed conversion. Leu and Ile, alone or in combination, significantly decreased expression of b(0,+) and significantly increased CAT-1. Ile alone or combined with Leu significantly decreased myosin expression in semitendinosus and significantly decreased it in longissimus muscle. Leu alone significantly decreased Lys, Ile and Thr serum concentrations; Ile significantly decreased Thr serum concentration; combined Leu and Ile significantly decreased Thr and significantly increased Val serum concentration. We conclude that dietary levels of Leu and Ile affect growth performance, expression of aa transporters and myosin, and aa serum concentrations in pigs.

A rapid and inexpensive method for isolation of total DNA from Trichoderma spp (Hypocreaceae).

Extraction of high-quality genomic DNA for PCR amplification from filamentous fungi is difficult because of the complex cell wall and the high concentrations of polysaccharides and other secondary metabolites that bind to or co-precipitate with nucleic acids. We developed a modified sodium dodecyl sulfate/phenol protocol, without maceration in liquid nitrogen and without a final ethanol precipitation step. The A(260/280) absorbance ratios of isolated DNA were approximately 1.7-1.9, demonstrating that the DNA fraction is pure and can be used for analysis. Additionally, the A(260/230) values were higher than 1.6, demonstrating negligible contamination by polysaccharides. The DNA isolated by this protocol is of sufficient quality for molecular applications; this technique could be applied to other organisms that have similar substances that hinder DNA extraction. The main advantages of the method are that the mycelium is directly recovered from culture medium and it does not require the use of expensive and specialized equipment.

A rapid method for isolation of total DNA from pathogenic filamentous plant fungi.

DNA isolation from some fungal organisms of agronomic importance is difficult because they have cell walls or capsules that are relatively unsusceptible to lysis. We have developed a fast DNA isolation protocol for Fusarium oxysporum, which causes fusarium wilt disease in more than 100 plant species, and for Pyrenochaeta terrestris, which causes pink root in onions. This protocol was based on the sodium dodecyl sulfate/phenol method, without beta-mercaptoethanol and without maceration in liquid nitrogen; it uses phenol/chloroform extraction to remove proteins and co-precipitated polysaccharides. The A(260/280) absorbance ratios of isolated DNA were around 1.9, suggesting that the DNA fraction was pure and may be used for further analysis. Additionally, the A(260/230) values were higher than 1.8, suggesting negligible contamination by polysaccharides. The DNA isolated by this protocol is of sufficient quality for molecular applications; this technique could be applied to other organisms that have similar substances that hinder DNA extraction.

A rapid and efficient method for isolation of total RNA from Euglena gracilis (Euglenoidea).

RNA isolation is essential to the study of gene expression at the molecular level. However, it is difficult to isolate RNA from organisms that contain large amounts of polysaccharides or other compounds that bind or coprecipitate with RNA, such as the unicellular protist Euglena gracilis. Currently, there is no commercial kit available that is specific for the isolation of high-quality RNA from this organism. Since it contains large amount of polysaccharides, the common protocols for RNA isolation usually result in poor yields when applied to E. gracilis. We developed a simple and fast RNA protocol that effectively removes these contaminating substances, without affecting the RNA yield. This protocol was based on the sodium dodecyl sulfate/phenol method, without beta-mercaptoethanol and without maceration in liquid nitrogen; it uses phenol/chloroform extraction to remove proteins, DNA, and co-precipitated polysaccharides. The RNA isolated by this protocol is of sufficient quality for molecular applications; this technique could be applied to other organisms that have similar substances that hinder RNA extraction.