The significance of antibiotic production by Pseudomonas aureofaciens PA 147-2 for biological control of Phytophthora megasperma root rot of asparagus

Pseudomonas aureofaciens PA147-2 produces an antibiotic (Af⁺) which inhibits the growth of fungal phytopathogens on phosphate buffered potato dextrose agar (PBPDA). To determine the role of the antibiotic in disease suppression in vivo, PA147-2 and an antibiotic-deficient Tn5 mutant (Af^-) PA109, were tested for their ability to suppress root rot of Asparagus officinalis seedlings caused by Phytophthora megasperma var sojae, in the glasshouse. Seedlings coinoculated with the pathogen and the wildtype strain PA147-2, showed a significantly reduced level of infection and disease severity compared to seedlings inoculated with the pathogen alone. However, 100% of seedlings treated with Af^- mutant PA109 were diseased. Furthermore, a strain derived from mutant PA109, restored to Af⁺ through allele replacement of Tn5 by homologous recombination, gave similar levels of disease suppression as the wildtype. This suggests the antibiotic produced by PA147-2 is important for the control of P. megasperma in vitro as well as in planta. Treatment of seedlings with PA147-2 and derived strains including the Tn5 mutant (Af^-) strain improved plant weight by 40–100% in the presence and absence of the pathogen suggesting PA147-2 also has a direct growth stimulatory effect independent of antibiotic production.     

Efficiency clustering for low-density microarrays and its application to QPCR

Background  

Pathway-targeted or low-density arrays are used more and more frequently in biomedical research, particularly those arrays that are based on quantitative real-time PCR. Typical QPCR arrays contain 96-1024 primer pairs or probes, and they bring with it the promise of being able to reliably measure differences in target levels without the need to establish absolute standard curves for each and every target. To achieve reliable quantification all primer pairs or array probes must perform with the same efficiency.     

Inhibition of phosphatidylinositol 3-kinase does not alter forskolin-stimulated Cl(-) secretion by T84 cells

Wortmannin is a potent inhibitor ofphosphatidylinositol 3-kinase (PI3K) and membrane trafficking in manycells. To test the hypothesis that cystic fibrosis transmembraneconductance regulator (CFTR) traffics into and out of the plasmamembrane during cAMP-stimulated epithelial Clsecretion, we have studied the effects of wortmannin onforskolin-stimulated Cl secretion by the humancolonic cell line T84. At the PI3K inhibitory concentration of 100 nM,wortmannin did not affect significantly forskolin-stimulatedCl secretion measured as short-circuit current(I_SC). However, 500 nM wortmannin significantlyinhibited forskolin-stimulated I_SC. cAMP activationof apical membrane CFTR Cl channels in-toxin-permeabilized monolayers was not reduced by 500 nMwortmannin, suggesting that inhibition of other transporters accountsfor the observed reduction in T84 Cl secretion.Forskolin inhibits apical endocytosis of horseradish peroxidase (HRP),but wortmannin did not alter forskolin inhibition of apical HRPendocytosis. In the absence of forskolin, wortmannin stimulated HRPendocytosis significantly. We conclude that, in T84 cells, apical fluidphase endocytosis is not dependent on PI3K activity and that CFTR doesnot recycle through a PI3K-dependent and wortmannin-sensitive membrane compartment.

     

Changes in Molecular Size Distribution of Cellulose during Attack by White Rot and Brown Rot Fungi

The kinetics of cotton cellulose depolymerization by the brown rot fungus Postia placenta and the white rot fungus Phanerochaete chrysosporium were investigated with solid-state cultures. The degree of polymerization (DP; the average number of glucosyl residues per cellulose molecule) of cellulose removed from soil-block cultures during degradation by P. placenta was first determined viscosimetrically. Changes in molecular size distribution of cellulose attacked by either fungus were then determined by size exclusion chromatography as the tricarbanilate derivative. The first study with P. placenta revealed two phases of depolymerization: a rapid decrease to a DP of approximately 800 and then a slower decrease to a DP of approximately 250. Almost all depolymerization occurred before weight loss. Determination of the molecular size distribution of cellulose during attack by the brown rot fungus revealed single major peaks centered over progressively lower DPs. Cellulose attacked by P. chrysosporium was continuously consumed and showed a different pattern of change in molecular size distribution than cellulose attacked by P. placenta. At first, a broad peak which shifted at a slightly lower average DP appeared, but as attack progressed the peak narrowed and the average DP increased slightly. From these results, it is apparent that the mechanism of cellulose degradation differs fundamentally between brown and white rot fungi, as represented by the species studied here. We conclude that the brown rot fungus cleaved completely through the amorphous regions of the cellulose microfibrils, whereas the white rot fungus attacked the surfaces of the microfibrils, resulting in a progressive erosion.