Isolation of a Bioactive Substance from the Silkworm (Bombyx moriLinnaeus) That Accelerates the Germination of the Entomopathogenic FungusNomuraea rileyi(Farlow) Samson

Abstract

The conidium of the entomopathogenic fungus Nomuraea rileyi has been found to germinate rapidly in the presence of host insect-derived extracts. Thus the extract appears to contain an important factor involved in host recognition by N. rileyi. However, the substance responsible for such unique germination behavior has yet to be identified. Hence we attempted to purify this substance. One thousand g of dried silkworm pupae was subjected to methanol extraction, followed by methanolysis, two different solvent partitions, and three different column chromatographies. A total of 12.4 mg of substance was obtained in the active fraction. The substance obtained exhibited an activity more than 46,000 times higher than that of the methanol extract. The substance was detected as a single peak on Sephadex LH20 column chromatography and as a single band on high-performance thin-layer chromatography. These data indicate that the concentrated fraction contained a high-purity substance.

The Effect of Amino Acids on the C14-Sphingosine-Triggered Germination ofNomuraea rileyi, and Surface Amino Acids onSpodoptera lituraFabricius

“…In previous studies, D-erythro-C 14 -sphingosine (C 14 -Sph) was isolated as a germination accelerating factor in N. rileyi using an assay solution containing peptone, 2,3) but its activity declined to a large degree in an assay solution without peptone. This suggests the presence of a co-factor in C 14 -Sph-triggered germination.…”

“…The assay method, using a 96-well microtitration plate for germination activity, is described in our previous report. 2) In each well of a microtitration plate, 1 mL of C 14 -Sph (Matreya, Pleasant Gap, PA) solution (10 À3 -10 À12 mg/mL in methanol), 10 mL of a conidial suspension, 10 mL of 50 mM phosphate buffer (PB) (pH 7.0), and 20 mL of sample solution were placed, and then conidial germination was observed after 10 h of incubation at 23 C. Peptone (Kyokuto Pharmaceutical Industrial, Tokyo) was used as positive control. The relationships between the concentrations of C 14 -Sph and the germination rate were analyzed by Probit analysis 5) to estimate the effective concentration for a 50% germination rate (EC 50 ) values using a computer program (Kyplot 5.0, KyensLab, Tokyo).…”