Relation between polyphenols contained in plants and phytopathogenic fungi. II

Abstract

Polyphenols labelled with C14 were separated by the following method from leaves of paulowniatree nursery which had been kept out-doors for 2 days under a bell-jar containing C14O2 (Fig. 1). Leaves steamed for 10min. to inactivate phenol oxidase were homogenized and were extracted by 50per cent methanol for 2 hours at 70 to 75°C. Ten per cent neutral lead acetate solution was added to the extract until no precipitate was formed. The latter was collected by centrifugation and resuspended in distilled water. Ten per cent H2SO4 was added to the solution to remove lead. After adjusted to pH 2.2 by NaOH the solution was extracted with ethyl acetate to obtain fraction 5. Thus, main part of polyphenols of the leaves were brought into fraction 5 in Fig. 2. Ethyl acetate was thoroughly evaporated under vacuum condition from the fraction and the latter was dissolved again in a small quantity of water.For the preliminary study, a small portion of this fraction, the mixture of polyphenols, was added to Tomizawa's synthetic medium with which Gloeosporium kawakamii was cultured to see the decomposition of polyphenols by the fungus (Table 6).Main part of fraction 5, in Fig. 2 and Table 4, was used for the separation of polyphenols from each other by paperchromatography. Each spot on the paper determined U. V. ray (Fig. 3) was dissected by sissors. Polyphenols thus separated were added respectively to the synthetic medium just before the culture of the fungus as follows. Twenty ml. of the medium containing each of polyphenols was poured into the main compartment of a 100ml. flask and 1ml. of 15per cent KOH was pipetted into the center well to catch C14O2 that might be produced during culture (Fig. 4). Spore suspension of the fungus was inoculated and cultured at 27°C.At every 5 days after inoculation, KOH solution in the center well pipetted out and poured onto a dish and was counted to see its radioactivity by Geiger counter. The mycelial mat harvested after 20 days from inoculation was counted too to see the rate of absorption of labelled polyphenol by the fungus.The results obtained (Table 7) supported that polyphenols in the leaves of paulownia-tree were able to be absorbed and utilized by the pathogen, and that a part was decomposed and released as CO2 during culture.It was considered that the greater part of the freed CO2 was not the results of the decomposition of sugar combined with aglucon of some polyphenols but the results due to the release and decomposition of carbon cyclic compounds.