Oospore-like bodies (imperfect oospores) of the late blight fungus are formed both on the artificial cultures and on the diseased tissues of potato leaves and stems, and tomato fruits. These imperfect oospores are sorted into 4 types, as follows: A-type: Outer wall of the spore is thin and not colored. Protoplasmic content is light yellowish brown. Without oogonium and antheridium formation. B-type: Outer wall of the spore is light brown. Protoplasmic content is brown. Without oogonium and antheridium formation. C-type: Outer wall of oogonium is colorless or light brown. Inner content of oospore is light to dark yellowish brown and granular. Without antheridium formation. D-type: Oogonium is reddish brown or brown. The content of oospore is light or dark yellowish brown and granular. Without antheridium formation. The physiology of A and B type oospores are not yet throughly studied. C and D type oospores are seemed to be formed parthenogenetically. Imperfect oospores (D type) are produced in abundance by the following culture method. When the fungus is cultured on corn extract agar for 20 to 30 days at 18°C and then transferred on oat-meal extract agar, oogonia containing oospore are found abundantly. On the contrary, when the fungus is cultured continuously on the same substrata, the oogonium is not produced. So, it seems likely that there are some stimulating actions of some nutrients on the formation of oospores. The imperfect oospore (without antheridium) can germinate producing the germ tube or forming the sporangium. Some of the isolates of the fungus produce only oospores but others not. When compatible isolates were paired on the same medium by means of the writers' cultural method, oogonium with the amphygenous antheridium was produced. It seems likely that the late blight fungus is heterothallic, because of the presence of the self incompatible isolates.
A number of the antibiotics were tested for their effect on the multiplication of tobacco mosaic virus (TMV, ordinary strain in Japan) using the leaf-culture method previously reported by the authors.4, 9, 10) Twenty disks, per each test, 12mm in diameter (approximately 0.6g flesh weight), were cut from a detached tobacco half-leaf (Nicotiana tabacum, Turkish) that had been inoculated with TMV one day ago. These disks were floated on the solution of an antibiotic in a petri dish and were kept in a constant-temperature chamber of 25°C under continuous illumination from fluorescent lamps. The other 20 disks cut from the opposite half-leaf were placed on distilled water and served as control. After 5 days from inoculation (4 days' incubation in test solutions), the disks were removed and rinsed with distilled water. The amount of TMV synthesized within the disks was determined by chemical methods. The data are presented in Table 1 which indicate the percentages of inhibition of TMV multiplication by some of the antiviral substances analyzed by two different methods. It is evident that the percentages of inhibition are generally lower by the TMV-protein analysis (ammonium sulphate-precipitation method9)) and are higher by the TMV-nucleic acid analysis (TCA method by Bancroft and Curtis1)). Moreover, the latter method was proved to be more accurate than the former method in estimating the different amount of TMV synthesized. Therefore, the method by Bancroft and Curtis was mostly used in the following experiments. Of about 30 preparations tested, as shown in Table 2, the following antibiotics showed 20 per cent or more inhibition to TMV multiplication under our experimental conditions: Actidione, Naramycin, Fermicidin, Aureomycin hydrochloride, Tetracycline hydrochloride, Dextromycin sulfate, Kanamycin sulfate, and Mitomycin-C.
In the present paper, the writer reported the result of investigations of the effect of ultraviolet light on several microorganisms. The sensitivity of the microorganisms to ultraviolet light decreased in the following descending order: Staphylococcus aureus>Serratia marcescens>Escherichia coli>Xanthomonas pruni>Sarcina sp.>Botrytis cinerea. The pigment formation in cells of Serratia marcescens has some influence to the sensitivity, a pigmentless strain being more sensitive to ultraviolet light than a pigment-producing strain (carmine strain). Judging from the survival curves of the two strains, the cells of the pigment-producing strain appear to contain larger amount of ultraviolet-absorbing substances than the cells of the pigmentless strain. Comparisons of the irradiation dose were made on the basis of irradiation energy expressed by (light intensity irradiation time), but there was a tendency that a short irradiation with high intensity of light was more effective than a long irradiation with low intensity, even if the total irradiation energy calculated with the above formula was kept constant.
In the present paper, the writer reported the result of investigations on the influence of light upon the conidial germination of Cochliobolus miyabeanus. The lethal effect of a sun lamp was far inferior to that of a germicidal lamp, because the former scarcely includes the effective wave length for sterilization, 2537Å. A sudden increase of the sensitivity of conidia to ultraviolet light was observed, when the conidia were suspended in water. The reason is not yet clear. In the present experiment, the writer estimated the germicidal effect of ultraviolet light by determining the percentage of conidial germination. In view of occurrence of delayed germination, the germination percentage was determined at least 24 hours after the irradiation. Difference in the sensitivity of conidia to ultraviolet light was found among the isolates of C. miyabeanus, the isolate No. 58 being more sensitive than the isolate No. 13.
This paper presents the results of the experiments on the diurnal cycle of cucumber downy mildew fungus, Pseudoperonospora cubensis (B. et C.) Rostow. on the effect of light upon its sporulation. Mildewed leaves were detached from greenhouse plants every 3 hours from 6 a.m. to 3 a.m. next day. Immediately the leaves were washed to remove conidiophores, dried, and then cut into pieces, 1×1cm in size involving a downy spot. Ten pieces were then transferred to each of three moist chambers (fig. 1) which were placed under different conditions as follows: a) In total darkness: The temperature within the moist chamber was 23°C, as measured with a thermojunction. b) Exposed to fluorescent light (using two Mazda FL 20D lamps) at an intensity of 300Lux at the level of the leaf surface: The temperature in the moist chamber was 23°C. c) Exposed to tungsten light (using a 300-Watt West reflector photo flood lamp) at an intensity of 4000Lux: The temperature in the moist chamber was maintained at 23.6∼24.2°C by interposing a 10cm layer of water between the light and moist chamber. Cut leaf pieces were removed from moist chambers at 3 hour intervals, and examined for details of the development of conidiophores and the formation of conidia. The tests were repeated three times, from June 18 to 30, 1957, with similar results. The results of a test on June 29∼30 are represented in table 1, 2 and 3. 1) When the infected leaf pieces were placed in the moist chamber under total darkness, they began within 3∼12 hours to produce a fair amount of conidia, regardless of the time of detaching the leaf. However, length of time for sporulation varied with the time of leaf detaching: namely, shortest, 3 hours, for the leaf sampled at 12 p.m., and longest, 12 hours, for the one detached at 9 a.m. and 12 a.m. 2) When the infected leaf pieces were placed in the moist chamber, exposing to fluorescent light at 300Lux, those detached at 12 p.m. formed abundant conidia after 3 hours. But the leaves sampled at 3 p.m., 6 p.m. and 3 a.m. produced conidiophores after 9∼12 hours, and a few conidia in further 6 hours. Those detached in the morning, viz. at 6 a.m., 9 a.m. and 12 a.m. gave rise to long slender conidiophores after 15∼18 hours, without forming conidia in further 6 hours. 3) When the test materials were exposed to tungsten light, almost the same results were obtained with those under fluorescent light. It only differed in the conidium formation on the leaves detached at 9 p.m. and 12 p.m., being less than those under fluorescent light. 4) Although the daily rhythm of sporulation under natural conditions was not examined, diurnal cycle of sporulability does seem to exist in cucumber downy mildew. The infected leaves sampled at 12 p.m. showed the highest sporulability. Darkness is favorable but not indispensable for sporulation, as seen in the tables. Light apparently suppresses sporulation, but not always. It is suggested that alternating light and darkness may be responsible for the cyclic manifestation of sporulablility in the cucumber downy mildew, by indirect effect through host plant.
Dehydrogenase activity in the rice plant tissues inoculated with Cochliobolus miyabeanus (Helminthosporium leaf-spot fungus) and in the causal fungus was investigated using blue tetrazolium (BT). This reaction was positive in the sections of healthy leaf tissues after 2 hours' incubation at 30∼32°C and the rate of the staining became constant after 8 hours' incubation. The reaction was negative when the sections were pretreated with formalin (10%, 1hr.) or treated at 50∼60°C, 10min. In leaf tissues, the parenchymatous cells were most strongly stained and the sites of the enzyme activity seemed to lie on grana-like region in chloroplasts. The leaf spot caused by the present fungus became visible 20 hours after inoculation. The dehydrogenase activity in this early stage of infection was demonstrated in cells near browned epidermis. With the enlargement of spotted area, the tissue around the lesion stained with BT; but the reaction became constant 48 hours after inoculation, although the enlargement of the lesion was still proceeding. In the typical leaf spot produced 5∼7 days after inoculation, localization and intensity of formazan deposition could not be distinguished from those in healthy leaf tissue. The reaction was weak in leaf-sheath cells even when the pathogen penetrated them. On the other hand, the penetrating hyphae, especially the appressoria and the germ tubes, showed strong, reaction. When the spores germinated on slide-glass, the reaction was detected on the terminal cells of the spores and on the germ tubes as well as on the appressoria. This positive reaction on spores of the present fungus seemed to localize on mitochondria of the fungus.
Streptomycin applied to the tobacco plant stem by rubbing, or absorbed to cotton-wool banded around bottom of the stem, was readily translocated to the upper plant leaves, when used at a concentration of 1000mcg/ml streptomycin solution. At higher concentrations the streptomycin was translocated to most of the leaves, gradually to the lower leaves and in least amount, next to the middle leaves and most to the upper leaves. The total quantity of streptomycin applied to tobacco leaves by surface absorption was retained following immersion of the leaves in tap-water for eight hours; and little reduction was noted after twenty-four hours' immersion. Rain or dew will be unlikely to remove streptomycin from the leaves.