A Root Box Method to Estimate Virulence of Helicobasidium mompa Using Carrots and Its Comparison with the Conventional Method Using Apple Stocks

A root box method with carrots was developed to estimate virulence of the violet root rot fungus, Helicobasidium mompa, to facilitate short-term screening of many isolates during a year. The root box consisted of two transparent acrylic plates and a plastic bag of vermiculite in which two taproots of carrot were growing and inoculated with the fungus growing on fragments of mulberry twigs. The boxes were kept in a greenhouse at 25°C, and the surface of carrots was observed weekly up to 14 weeks. The virulence of each isolate was determined based on the number of weeks after inoculation required for the fungus to develop infection cushions on the surface of carrots. Results were compared with those from the conventional inoculation method using apple stocks. Two-year-old 456 apple stocks were planted with or without fungal inoculum in 30-cm diam. plastic pots containing commercial soil and placed outdoors in April 1999. Symptoms on plant tops were observed weekly, and the first stocks were killed 14 weeks after inoculation. At the end of trial 1 (6 months) and trial 2 (14 months), apple stocks were dug up to rate disease index (DI) based on hyphal growth and infection cushion formation on the stem base. There was variability in disease severity among replicates as well as isolate variability ; however, the results were similar in both trails. The level of virulence estimated by both methods was almost parallel for a total of 23 isolates from five plant species, except for two isolates from sweet potato that formed no obvious infection cushion on apple roots but on carrot were the most virulent. Received 11 December 2000/ Accepted in revised form 23 March 2001     

Immunohistochemical detection of cellular prion protein (PrPc) in the rat central nervous system

A simple conventional method of immunohistochemistry (i.e. fixing the frozen sections in cold methanol) was used to determine the immunolocalization of cellular prion protein (PrPc), with good results. In the rat cerebrum, the cytoplasm of neural cells in the cortex and corpus stratum, pia mater, membrane limitans gliae superficialis, choroid plexus and blood vessel wall were immunostained. The formation of network structures of immunostained neural and/or glial fibers in the cerebral cortex was also observed. These immunostained network structures of neural and/or glial fibers were also observed in cultured neural cells. The results suggest that fixation of frozen sections and cultured cells with cold methanol is a useful method for detecting the immunolocalization of PrPc and that PrPc exists in the various components of the central nervous system of the rat.     

Improved rapid and efficient method for Staphylococcus aureus DNA extraction from milk for identification of mastitis pathogens

A rapid and efficient DNA extraction method was developed for detecting mastitis pathogens in milk. The first critical step involved cell wall disruption by bead-beating, as physical disruption using beads was more effective for DNA extraction from Gram-positive bacteria, such as Staphylococcus aureus, than enzymatic disruption using proteinase K. The second critical step involves the use of acetic acid and ammonium sulfate in the purification process, as these reagents effectively and efficiently remove the lipids and proteins in milk. Using these methods, DNA suitable for loop-mediated isothermal amplification was obtained within 30 min. Also, the rapid and sensitive detection of S. aureus in milk was possible at levels as low as 200 cfu/ml.     

Dietary supplementation with cellooligosaccharide improves growth performance in weanling pigs

Nondigestible oligosaccharides are not digested in the small intestine, but are fermented by bacteria colonizing in the large intestine. Physiological effects of non‐digestible oligosaccharides have been considered to be conferred by the fermentation of bacteria colonizing in the large intestine. Because cellooligosaccharide is a non‐digestible oligosaccharide, various physiological effects are expected. However, physiological functions of cellooligosaccharide are not well understood. This experiment was conducted to clarify the effect of dietary supplementation with cellooligosaccharide on the growth performance in weanling pigs. The result showed that average daily gain was significantly higher (P < 0.05) in pigs fed a diet supplemented with cellooligosaccharide than in pigs without cellooligosaccharide. There was a tendency to increasing average daily feed intake in pigs with cellooligosaccharide, though the significant difference was not detected (P = 0.18). Feed efficiency and nutrient digestibility of feces and ileum were not changed by feeding cellooligosaccharide. In addition, blood urea nitrogen was significantly higher (P < 0.05) in pigs fed the diet supplemented with cellooligosaccharide than in pigs without cellooligosaccharide. The concentrations of acetic and iso‐valeric acids in the cecum of pigs fed the diet with cellooligosaccharide tended to be higher (P < 0.10) than those without cellooligosaccharide. The results obtained in this study demonstrated that dietary supplementation with cellooligosaccharide improves growth performance in weanling pigs.