Growth and yield of lentil and wheat inoculated with three Glomus isolates from Saskatchewan soils

The vesicular-arbuscular mycorrhizal fungi (VAMF) Glomus clarum (Nicol. and Schenck) isolate NT4, G. mosseae (Nicol. and Gerd.) Gerd. and Trappe isolate NT6 and G. versiforme (Karst.) Berch isolate NT7 coexist in wheat field soils in Saskatchewan. This study assessed the response of lentil (Lens esculenta L.) and wheat (Triticum aestivum L.) to monospecific and mixed cultures of these VAMF isolates. Seedlings were inoculated with 100 spores of a VAMF isolate, or an equal mixture of spores of two isolates, and grown in a sterile soil mix in a growth chamber. Both crops responded differently to these different VAMF isolates. In the case of lentil, G. clarum NT4 was more effective than G. mosseae NT6 and G. versiforme NT7, and significantly increased (P<0.05) the shoot dry weight (43%) and grain yield (57%) compared with the uninoculated control. There was a significant positive correlation between the percentage of VAMF colonized roots and shoot dry weight (r=0.672^***) and shoot phosphorus concentration (r=0.608^***) of lentil. In the case of wheat, G. clarum NT4 had no effect on shoot dry weight, but produced significant (P<0.08) increases in grain yield (12%) and the phosphorus concentration of the shoot and grain. Although G. clarum NT4 and G. mosseae NT6 both produced similar levels of VAM colonization in wheat, the only response of wheat to isolate NT6 was an increase in plant height at harvest. The efficacy of G. clarum NT4 on both crops appeared to be related to its ability to produce more arbuscular colonization than G. mosseae NT6. Dual inoculation of seedlings with G. clarum NT4 and G. mosseae NT6 resulted in competition between these two isolates. This was evident from a comparison of plant shoot dry weight and grain yield, and VAMF spore production on the two crops inoculated either with isolate NT4 alone or in combination with NT6. G. mosseae NT6 reduced the efficacy of G. clarum NT4 by 16% when dual inoculated on lentil, but had no effect when the host was wheat. Based on spore production, it was found that G. clarum NT4 was more competitive than G. mosseae NT6 when dual inoculated on lentil or wheat. Isolate NT4 produced ca. 2000 and 500 spores/ 100 g substrate, respectively, in the lentil and wheat pots, which was approximately 2–3 times more spores than those produced by isolate NT6 with either crop. When the plants were dual inoculated, there was a 15–19% reduction in spore production by G. clarum NT4 and a 50–70% decrease in spore production by G. mosseae NT6. Our results show that G. clarum NT4 was more competitive and effective in its ability to colonize and increase the growth and yield of lentil and wheat than G. mosseae NT6 or G. versiforme NT7. The relative performance of isolate NT4 with different host plants suggests that this VAMF isolate exhibits a host preference for lentil.     

A functional gene array for detection of bacterial virulence elements

Emerging known and unknown pathogens create profound threats to public health. Platforms for rapid detection and characterization of microbial agents are critically needed to prevent and respond to disease outbreaks. Available detection technologies cannot provide broad functional information about known or novel organisms. As a step toward developing such a system, we have produced and tested a series of high-density functional gene arrays to detect elements of virulence and antibiotic resistance mechanisms. Our first generation array targets genes from Escherichia coli strains K12 and CFT073, Enterococcus faecalis and Staphylococcus aureus. We determined optimal probe design parameters for gene family detection and discrimination. When tested with organisms at varying phylogenetic distances from the four target strains, the array detected orthologs for the majority of targeted gene families present in bacteria belonging to the same taxonomic family. In combination with whole-genome amplification, the array detects femtogram concentrations of purified DNA, either spiked in to an aerosol sample background, or in combinations from one or more of the four target organisms. This is the first report of a high density NimbleGen microarray system targeting microbial antibiotic resistance and virulence mechanisms. By targeting virulence gene families as well as genes unique to specific biothreat agents, these arrays will provide important data about the pathogenic potential and drug resistance profiles of unknown organisms in environmental samples.     

piggyBacis an effective tool for functional analysis of thePlasmodium falciparumgenome

Background  

Much of thePlasmodium falciparumgenome encodes hypothetical proteins with limited homology to other organisms. A lack of robust tools for genetic manipulation of the parasite limits functional analysis of these hypothetical proteins and other aspects of thePlasmodiumgenome. Transposon mutagenesis has been used widely to identify gene functions in many organisms and would be extremely valuable for functional analysis of thePlasmodiumgenome.     

Retention of Activity by Solutions of Pancreatic Deoxyribonuclease after Freezing and Thawing

Solutions of deoxyribonuclease containing 1.25 mg of enzyme per ml of 0.003 M MgSO₄ were placed in small, sealed containers in a deep-freeze cabinet (ca - 20°C) and stored for periods of time ranging from 15 min to 35 days. Upon removal, each sample was thawed and then tested for its capacity to reduce Feulgen stain-ability in sections of onion root tips. No appreciable loss in enzymatic activity was effected by the freezing-thawing process. The method is recommended for experimental procedures extending over a period of days in which a constant supply of enzyme of known potency and specificity of action is required.     

Identification and characterization of endoglucanases for fungicidal activity in <Emphasis Type="Italic">Anabaena laxa</Emphasis> (Cyanobacteria)

A cyanobacterial strain (Anabaena laxa RPAN8) exhibiting fungicidal activity and β-1,3 and 1,4 endoglucanase activities was selected for identifying the gene(s) involved. Functional analyses of the genomic library revealed that four clones (8, 64, 116, and 248) of RPAN8 exhibited fungicidal activity and induced structural deformities in the cell wall of the growing mycelia of Pythium aphanidermatum. Higher expression of fungicidal and β-1,4 endoglucanase activities, along with low expression of β-1,3 endoglucanase activity, was recorded in two E. coli clones (8 and 64). Clones 8 and 64 exhibited identical sequences while clones 116 and 248 were also similar. Bioinformatic analyses were undertaken only for the two non-identical clones 8 and 116 which showed open reading frames (ORFs) of 348 (end 1) and 656 amino acid residues (end 2), respectively. The amino acid sequence analyses revealed that the end 1 encoding endoglucanases belonged to peptidase M20 family while end 2 showed significant similarities with several known genes. The putative promoters and ribosomal binding sites were identified and amino acid exchanges were observed in both end 1 and 2. The presence of signal peptides of 24 and 20 amino acid residues respectively revealed the secretory nature of these proteins.     

Nitrogen assimilation in opaque and normal sorghum during grain development

Activity of key nitrogen assimilating enzymes was studied in developing grains of high-lysine opaque sorghum P-721 and normal sorghum CSV-5. The higher percentage of protein in opaque sorghum was mainly due to lower starch content since protein per grain was less than in CSV-5. During grain development, albufn and globulin decreased while prolafne and glutelin increased. Prolafne content in CSV-5 was higher than in opaque sorghum. Average nitrate reductase activity in flag and long leaf were similar in both the varieties. The nitrate reductase activity decreased during grain development. Glutamate dehydrogenase activity was higher during early development and lower at later stages in opaque sorghum than in CSV-5. Glutamate oxaloacetate transaminase activity was higher and glutamine synthetase lower in opaque sorghum than in CSV-5 grains during development. Glutamate synthase activity was higher in opaque sorghum up to day 20 and lower thereafter than in CSV-5. It is suggested that reduced activities of glutamine synthetase as well as glutamate synthase in opaque sorghum as compared to CSV-5 during later stages of development may restrict protein accumulation in the former.     

Pancreatic stellate cell-potentiated insulin secretion from Min6 cells is independent of interleukin 6-mediated pathway

Pancreatic stellate cells (PSCs) secrete various factors, which can influence the β-cell function. The identification of stellate cell infiltration into the islets in pancreatic diseases suggests possible existence of cross-talk between these cells. To elucidate the influence of PSCs on β-cell function, mouse PSCs were cocultured with Min6 cells using the Transwell inserts. Glucose-stimulated insulin secretion from Min6 cells in response to PSCs was quantified by enzyme-linked immunosorbent assay and insulin gene expression was measured by quantitative polymerase chain reaction. Upon cytometric identification of IL6 in PSC culture supernatants, Min6 cells were cultured with IL6 to assess its influence on the insulin secretion and gene expression. PLC-IP₃ pathway inhibitors were added in the cocultures, to determine the influence of PSC-secreted IL6 on Glucose-stimulated insulin secretion from Min6 cells. Increased insulin secretion with a concomitant decrease in total insulin content was noticed in PSC-cocultured Min6 cells. Although increased GSIS was noted from IL6-treated Min6 cells, no change in the total insulin content was noted. Coculture of Min6 cells with PSCs or their exposure to IL6 did not alter either the expression of β-cell-specific genes or that of miRNA-375. PSC-cocultured Min6 cells, in the presence of PLC-IP₃ pathway inhibitors (U73122, Neomycin, and Xestospongin C), did not revoke the observed increase in GSIS. In conclusion, the obtained results indicate that augmented insulin secretion from Min6 cells in response to PSC secretions is independent of IL6-mediated PLC-IP₃ pathway.