More than six polyphenols were recognized from the extracts of paulownia-tree leaves by paper-chromatography. As to total quantity of polyphenols obtained colorimetrically after adding alminium nitrite, no definite differences were found between healthy and the anthracnose-Gloeosporium Kawakamii-affected leaves of paulownia-tree. While in the case of ortho-dihydroxyl type of polyphenols, measured after the addition of Arnow's reagent, considerable quantities were obtained from the diseased leaves (Table 1). Chlorogenic acid and rutin were verified to exist in the leaves of paulownia-tree (Table 2. b and c). The addition of each of these polyphenols of final concentration of 10-3M. respectively to the synthetic media, significantly promoted the growth of the fungus (Table 3 and 4). Quercetin, the aglucon of rutin, showed also the additive effect on the growth of the fungus. When determined by paperchromatography, quercetin in the culture media was decomposed by the fungus into protocatechuic acid, phloroglucinol, and other unknown ones. While in the case of rutin, no sufficient quantity of phloroglucinol for paperchromatography was obtained, due probably to the quick decomposition or absorption by the fungus (Fig. 2 and 3). To obtain more clear evidence for the growth promoting effect of these flavonoids, culture experiment with synthetic medium, with the addition of each of rutin, quercetin, phloroglucinol, protocatechuic acid, and glucose plus rhamnose, in the final concentration of 10-3M. respectively, was made. The dry weight of the mycelial mat of 5 days culture from the medium containing rutin, quercetin and phloroglucinol respectively was greater than that from others (Table 5). Protocatechuic acid and glucose plus rhamnose did not display any promoting effect. The growth promoting effect of the flavonoids for the fungus was considered at least in part due to the nutritive effect of phloroglucinol decomposed from the flavonoids during the culture of the fungus.
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.
Both WYMV and BYMV in the leaves of diseased plants, which were dehydrated at temperatures ranging 0∼2°C and were stored in the absence of oxygen at 5∼10°C, showed considerable infectivities even after about two years. By continuously conducted experiments concerning the infectivity of soil particles of clay fraction separated from WYMV- or BYMV-infested soil, it was confirmed that the infectivity of the clay fraction with particles less than 2μ in diameter was much greater than that of the fraction with particles larger than 2μ, and that there are found in the pellet of clay particles neither any special soil-inhabiting microorganism to be regarded as a possible vector nor any large fragment of plant tissue such as root hair. Moreover the possibility was shown that both WYMV and BYMV preserved their activities at least during the period from season to season if these viruses are adsorbed (under certain favorable conditions) in some mineral clay particles or in some sterilized soil particles. These findings seem to justify the writer's view that these ordinary soil-borne viruses exist adsorbed in soil particles of clay fraction which include organic colloidal substances such as fragments of humus as well as mineral clay particles with the ability to adsorb proteins, and that neither the presence of soil-inhabiting microorganism nor of even a fragment of root of diseaed plant is necessarily needed to explain the considerable longevity of these soil-borne cereal mosaic viruses in the soil.
The present paper deals with some physiological observations on the normal and the akiochi (autumn-declined) rice plants affected with Cochliobolus miyabeanus. 1. At 2, 4 and 6 days after the inoculation, the diseased spots of both plants were concentrically cut off in 4, 8 and 12mm diameter by punch (see Fig. 1) and the amount of protein- and soluble- form nitrogen in each parts were measured with Micro-Kjeldahl's method, respectively. In the central part (A) of the normal rice leaves, a little increase of protein-form nitrogen was observed at an early stage of the infection, but the akiochi rice leaves were not (see Fig. 2). According to paper electrophoresis, 3 peaks of the optical density (α, β and γ) were generally observed about leaf protein at the direction of cathode (see Fig. 3). In the normal rice leaves these 3 peaks were presented even at 6 days after the infection. On the contrary, β peak was disappeared in the akiochi rice leaves at 2 to 6 days after. 2. At 6 days after the inoculation, the diseased spots of both plants were cut off in 7mm diameter and the amount of zinc in these spots was measured with the dithizone method of A. O. A. C. In both the normal and the akiochi rice leaves, diseased part was less content than healthy one and the difference between diseased and healthy ones was larger in the akiochi rice leaves than in the normal rice leaves (see Table 1). From the result, zinc in the diseased leaves seems to be transferred from diseased part to healthy one.
The writers4, 5) reported in 1957 that Hypomyces solani (Rke. et Berth.) Snyd. et Hans.[Fusarium solani (Mart.) Snyd. et Hans.]was another causal fungus of Fusarium blight of mulberry trees, and the strains of this fungus, isolated from mulberry stems, were divided into two groups (α and β). In this paper the host ranges and the form names of the two groups are dealt with. The cross-inoculations were carried out among these fungi and 6 forms of F. solani, i. e. f. cucurbitae, f. eumartii, f. radicicola, f. phaseoli, f. pisi and f. batatas. The α group of the fungus was pathogenic on mulberry stems, but was non-pathogenic to squash seedlings, potato stems, potato tubers, bean seedlings, pea seedlings and sweet potato sprouts. On the other hand, the 6 forms of F. solani proved to be non-pathogenic on mulberry stems though they were severely parasitic on their own hosts. From these results, the writers propose the following name for the α group of the fungus. Hypomyces solani (Rke. et Berth.) Snyd. et Hans. f. mori Sakurai et Matuo, nom. nov. [Fusarium solani (Mart.) Snyd. et Hans. f. mori Sakurai et Matuo, nom. nov.] Hab. in vivis Mori, cetra ut in typo7). The β group of the fungus was pathogenic not only on mulberry stems (less virulent than α group) but also to pea seedlings and sweet potato sprouts and most vigorously to potato tubers. So the writers designate the β group Hypomyces solani (Rke. et Berth.) Snyd. et Hans. f. radicicola race 2[Fusarium solani (Mart.) Snyd. et Hans. f. radicicola race 2], and call the former f. radicicola, which causes the dry rot of potato tubers and is non-pathogenic on mulberry trees, F. solani (Mart.) Snyd. et Hans. f. radicicola race 1. The type cultures are deposited in Fac. Tex. Seric., Shinshu Univ., Ueda, Japan, which were isolated from mulberry trees suffered from the blight disease.
Phytoalexin (PA for short) production of the soybean pod in reaction to Fusarium sp., as affected by conditions of spore suspension, was studied. PA was produced remarkably when suspensions containing spores more than 40per one microscopic field at 150-fold magnification, were placed on the soybean pod, while not at all when suspension with 3∼6 spores per one microscopic field was used. Both spore suspension heated at 100°C for 10 minutes and supernatant of suspension suspended with macerated spores, failed to induce production of PA. The supernatant of spore suspension, incubated for 24 hours at 23°C, revealed to possess ability to induce PA production, but lost the ability when the supernatant was heated at 60°C for 10 minutes.
The present paper deals with the results of the experiments on some problems for practical use of the ultraviolet sterilization. The irradiation by germicidal lamp was more efficient at the lower temperature, the tube wall of the lamp being kept at the most suitable temperature for germicidal action by cooling. The ultraviolet sensitivity of the microorganisms increased at the higher temperature which was optimal for their growth. However, as the degree of decrease in radiation efficiency due to increasing temperature was greater than the increase of sensitivity of microorganisms, it is likely that the sterilization is efficiently performed at the lower temperature. The visible and near-ultraviolet light included in the artificial sun lamp, accelerated the lethal action of germicidal lamp, acting synergic when irradiated with germicidal and artificial sun lamps together. As a packing matter for the sterilization purpose, common cellophane was more efficient than dampproofed one, because the ultraviolet absorption of the former was lower than the latter.
Some inconsistent results from germination studies of rust spores have been recently explained on the basis of self-inhibitors. The present study was performed to make clear the self-inhibition in the uredospore germination of Puccinia coronata Corda. The samples of spores were harvested by shaking the rusted leaves which had been collected from field-grown oat-plants, washed with sterilized water, and then held in large glass vessels for 2∼3 days at a saturated humidity. Germination tests were made by floating spores over the surface of dist. water contained in 30 or 50ml flasks. 1. The germination percentage of uredospores decreases as the spore density is increased. With very large numbers there is no germination at all. However, even in these samples excellent germination occurs when transferred to fresh water with small populations. With a definite spore density the germination percentage decreases as the amount of water in the flasks is decreased. As germination increases, the mats become very white, and the brownish-red color of the original spore film disappears entirely. 2. There is no difference in germination percentage among the closed, cotton-sealed, open, and aerating flasks. When the solutions, produced by floating spores, are removed by inserting a pipette beneath the floating film of spores and transferring a sample of the spore free solution to another flask, the germination percentage on this solution is very higher than that in the original solution. These facts indicate that the lack of O2, the toxicity of gaseous bodies such as CO2, and the accumulation of toxic metabolite in the solutions are not the principal cause of the unfavourable germination in large populations. 3. 4.4g samples of the spores were killed by heating at 100°C for 5min. and extracted with water at 5°C for 10 days. The extract was filtered, decolorized by treating twice with active carbon, and concentrated to 2ml. Then 20ml of ethanol was added to the material and the resulting white precipitate was filtered off. The filtrate was concentrated to a small volume to yield a colorless oily material. This substance prevented spore germination completely, even when diluted with water corresponding to the original weight of spores. This result clearly indicates that the substance in question is in sufficient quantities to account for the self-inhibitor of uredospore germination. 4. Presumably the unfavourable germination in large populations of spores should be closely related to this self-inhibitor. In addition, the cause that uredospores of this fungus germinate better in water than in air also may probably be due to this substance.
1. When conidia of barley powdery mildew are mounted in water under cover glass and the water is exchanged repeatedly, most of the conidia soon lose their protoplasmic and vacuolic structure and die becomig homogenously granular. Some of the conidia break at the shoulder part or burst violently from unrecognizable portion. The plug-like structure at the ends of conidia is sometimes off, causing the conidial contents to exude out. When conidial chains connected with conidiophores and hyphal cells are treated as above, the cells in the chains including conidiophores also immediately disorganize or burst to death. Hyphal pieces taken from the marginal part of pustule are often observed to break at the tip end, when immersed in water. 2. When diseased barley leaves are sprayed with water, water drops of various size are detained by upper parts of the conidial chains standing closely to each other, and soon evaporate, leaving the upper disorganized conidia adhesive to each other, giving scabby appearance. Water spraying continued for a few days does not so much damage the lower conidia of the chains, conidiophores, and hyphal cells underneath that conidia formation will be recovered. Spraying for more than a week destroys almost completely those fungal cells, but the hyphae at the margin of the pustule survive and are observed to develop conidial chains at the periphery a few days later. 3. Adhesion of conidia and conidial chains by spraying with water may be explained, as reported in a previous paper concerning the effect of lithium on the barley powdery mildew (1959), by breakage of the outer layer of the membrane of conidia swollen by water absorption and adhesive nature of the inner layer of the membrane. 4. Water spray for a few days brings about yellowing or browning of the epidermal or internal cells of the host tissue under the pustule. When spraying is continued, the discolored part extends and the color becomes intense. Brown lines are often observed macroscopically along the vein. The cells which are discolored early and intensely are not always the epidermal cells containing haustoria nor the mesophyll cells in direct contact with the infected epidermal cells. Early and strong discoloration of stomatal cell is conspicuous, which are situated not only on the pustule-bearing surface but also on the opposite side. Mesophyll cells in the inner layer and the cells within and surrounding the vascular bundle are often discolored early. 5. Previous to discoloration, yellow, viscous, small drops appear on the surface of mesophyll cells and gradually enlarge becoming brown. The membrane of the cells in contact with the stained drops become yellow or brown and the discoloration extends on the membrane. The mesophyll cells with its membrane discolored entirely are often observed to keep turgid condition. Discoloration of the contents of the mesophyll cells and death of them occur generally later. Colored substance in the drop must be exudate from the mesophyll cells. 6. As stated in 4, many of the stomatal cells under the pustule sprayed with water die soon and accordingly the stream of sap in the leaf, moving from vascular bundles, through mesophyll cells and epidermal cells, to stomatal cells and into the atmosphere, must be disturbed. The substances translocated to the host tissue under the pustule from the surrounding tissue, are not or hardly utilized by the fungus which is killed or weakened by water spray, and cause excessive contents of the mesophyll cells under the colony. These conditions must bring about abnormal physiology of the mesophyll cells and may be one of the causes of their death.