This is the results of the studies to observe the effect of cultural conditions on the pectin-methyl-esterase (PME) production, and also difference in PME producing activities among various isolates, in Erwinia carotovora. The organisms were grown in a synthetic pectin medium containing pectin 5g, (NH4)2SO4 13g, KH2PO4 4.5g, Na2HPO4⋅12H2O 2.0g, MgSO4⋅7H2O 0.2g per liter, adjusted to pH 7.0 by NaOH. The measurement of PME activity was made by the Smith's method, using 4.5ml of pectin substrate and 0.5ml of enzyme solution instead of 7.4ml and 0.6ml, respectively, of Smith's original prescription. 0.5ml aliquot of bacterial culture as the enzyme solution was pipetted into each of two test tubes, and one of them was heated at 100°C for 5min. to inactivate the enzyme. Pectin substrate containing pectin 5g, phenol 2g, NaCl 5.8g per liter was adjusted, right prior to use, to pH7.0 with the addition of NaOH and B. T. B. 4.5ml of this substrate was added to each of the above test tubes, and they were incubated for 24hrs. at 30°C. The pH of the content of these tubes were measured by a glass-electrode pH meter, and the difference in pH value between both tubes was taken as the index of PME activity. The surface area of the culture contacting with air and also initial pH were found to be the most important among several factors which control the production of PME. Most isolates of E. carotovora could secrete only a little PME in a test tube 1.5cm in diameter, containing 10ml of culture medium. Despite negligible difference in the growth rate of the bacteria, the larger the surface of the culture contacting with air, the higher was the PME activity evidenced in static culture. An only exception was shown by isolate 551 (E. carotovora f. sp. ananas), which constantly produced abundant enzyme regardless of the surface area of the culture. In most isolates except 551, this effect of surface area on PME production was increased by low pH of the medium, that is, shift from neutral to acid side considerably lowered the amount of enzyme produced. Although PME enzyme was most abundantly secreted in the medium when pectin was used as the sole carbon source, pectic acid served fairly effectively as the C-source for the PME enzyme production under cultural conditions having larger surface contacting with air. The low activity of PME was also evidenced in the medium containing glucose as the sole carbon source, but in most isolates except 551 the activity decreased with the lapse of days. The sort of medium for the pre-culture had little significance as to the amount of PME enzyme produced in the pectin medium. Under aerobic conditions, all the isolates showing abundant growth in the pectin medium produced PME enzyme, but such isolates as CP or BA needed 2 or 3 days before they showed growth and enzyme production. The weakly pathogenic isolate P2 showed a very feeble growth and no enzyme production under any cultural conditions.
As the authors described elswhere, a high pectin-methyl-esterase (PME) activity was always detected in the plant tissues attacked by Erwinia carotovora. In order to elucidate the orgin of this enzyme found in diseased tissues, characterization of enzymes differing in source should be made, because PME can be found in the culture of E. carotovora as well as in healthy plant tissues. The present paper presents the results of comparative studies between PME isolated from three isolates of E. carotovora and PME isolated from some vegetables. Isolation of the enzyme from plants (potato, onion and carrot) was made by precipitating the enzyme with 70 percent saturated ammonium sulphate, after extraction with 1/2 diluted MacIlvain's buffer (pH 7.0) and filtration through a layer of diatomaceous earth. The enzyme from the bacterial culture, isolate C1, 551, and 547, was obtained by precipitating with ammonium sulphate of the same concentration as in the above case, after centrifugation at 10.000 R. P. M. for 10min. The activity of all these enzymes was considerably lowered by dialysis with celophane membrane against tap water for 24hrs. The enzyme activity was lost perfectly, except in the one secreted by the isolate 551, by treatment with ion exchange resins, IR-120 and IRA-400. These solutions recovered their activity by the addition of 0.1M ammonium sulphate, the activity usually exceeding that of the original preparation. As regards the recovery of enzyme activity due to salts, mono-valent cations were most effective in the case of bacterial enzymes, while di-valent cations were most effective in the enzymes of plant origin. Bacterial PME showed, in any isolate, the highest activity at a reaction temperature of 50°C. In PME of plant origin, however, the optimum reaction temperature was from 40° to 60°C according to the kind of plant. The optimum pH value was 7.0 in all the enzymes, but another small peak was also found at 4.0 in most enzymes, except the one from the isolate 547. In the cases of C1 and onion enzymes, especially, the activity at pH 4.0 was almost of the same order as that at pH 7.0. Most of the enzymes were inactivated by heating at 60°C∼70°C for 10min. In addition to the above heat labile enzyme, the isolate 547 produced, after 5 days in the culture medium, a very heat-tolerent enzyme which showed some activity even after heating at 100°C for 10min.
1) The chlamydospores on the yellow, yellowish-green, greenish-yellow and green smut balls, respectively, which were collected in the preceding year and stored for eight months in the refrigerator and under the laboratory conditions, were employed for inoculation to the coleoptiles of rice variety Norin-mochi No. 1. The germinated seeds of which the coleoptiles developed at 1-3mm in height, were inoculated on the coleoptiles by means of spraying to the germinated seeds with the chlamydospore suspensions (80-100 spores per one optical field (X 900)) or by dipping them in the suspension. The inoculated coleoptiles were kept for 3-5 days at 20°C in 1958 and at 25°C in 1959 and 1960 experiments, then the germinated seeds thus inoculated were planted in fields and grown till the mature stage of rice plant. 2) The infection occurred most readily when inoculation was made on the seedlings at 1-10mm in height with the yellow spores, while no infection occurred at 12mm or more. The greenish-yellow spores were also virulent to the coleoptiles of 3-6mm in height, but not infections to those of 8mm or more. The percentage of infection was much higher when the coleoptiles were inoculated at very early stages of growth. The pathogenicity of chlamydospores was the highest in the yellowish-green spores, considerable in the greenish-yellow and the yellow spores, but very limited in the green spores. So far as the yellow spores were concerned, the percentage of infection was the highest when inoculated with the concentration of 400 spores per one optical field (X 900); it diminished with the decrease of spore concentration, then no infection was secured with that of 40 spores. As to the temperature condition at the time of inoculation, 25°C proved most favourable. Inoculation by means of the dressing resulted in lower percentage of infection in comparison to that resultant from the spraying or the dipping. 3) The percentage of infection due to seedling inoculation on the respective stems of a plant was the highest in the ears which emerged from the mother stems. Smut balls in the infected ears were most numerous in the somewhat upper part above the base, next in the basal part, but very few in the part from middle to apical. Above 50% in the affected ears only a single smut ball per ear, a considerable number in the ears bore 2-4 smut balls, and a few more than 5, but seldom 15, 16, or 19. The number of cluster of smut balls in an infected ear increased with increase of smut balls per ear. The percentage of sclerotia-bearing smut balls was 23.3%. From the above-noted results on infection due to the inoculation, the percentage of infection by the order of tillering, the distribution and occurrence of cluster of smut balls in an ear was seen to be the same as those due to natural infection in upland rice.
1) The present paper deals with the results of experiments on the effects of several fungicides on the nitrogen components contained in mycelia of Piricularia oryzae. The fungicides used in this experiments were as follows: copper shlfate, mercuric chloride, phenyl mercuric dinaphthylmethane disulfonate (PMF), n-trichloromethyl thiotetrahydrophthalimid (Captan), 2, 3-dichloro-1, 4-naphthoquinone (Dichlone), tetramethyl thiuramdisulfide (TMTD), ferric dimethyl dithiocarbamate (Fermate), zinc dimethyl dithiocarbamate (Zerlate) and Blasticidine-S. 2) The electrophoretic proteins contained in mycelia of P. oryzae decreased, when the mycelia were contacted with 10-4∼10-3 molar solution of copper sulfate, mercuric chloride or PMF for 16 hours, and the proteins did not decreased when the mycelia were contacted with the solution of Captan or six other fungicides for 16 hours, but the proteins decreased when the mycelia were contacted with the solution for 24 hours or more. 3) Total-nitrogen content contained in mycelia of P. oryzae when the mycelia were contacted with the solution of mercuric chloride, copper sulfate, Captan, Dichlone, TMTD and Blasticidine-S, and the decrease were different in degree with these fungicides. Protein-nitrogen content in mycelia decreased, too, and the decrease were nearly same in degree, This result was not agreed with the result on electrophoretic proteins. 4) When the mycelia were contacted with 10-4∼10-3 molar solution of heavy metalic fungicides for 16 hours, the free amino acids contained in mycelia were found to be generally decreased in concentration, but, when the mycelia were contacted with 100γ/ml solution of Blasticidin-S for 48 hours or more, an unidentified ninhydrin-reacting spot near Leucine was noted.
1) In Japan, melon mosaic virus (Lindberg et al., 1956) has been found infecting squash, pumpkin, white gourd, and oriental pickling melon (Komuro, 1957). The same virus has recently been detected also from cucumber plants showing veinal necrosis and/or mosaic. The diseased plants have been collected from Kochi, Iwate, Saitama, and Tokyo. In the samples from Iwate and Kochi, and some of the samples from Saitama, the symptoms were characteristic necrotic leions along veins, in which the affected parts were dry and brittle. There were some malformation of the leaves and some shortening of the internodes, but mosaic was not observed. The samples from Kochi showed small interveinal necrotic spots accompanied with the veinal necrosis. The samples from Tokyo and some samples from Saitama showed mosaic, but the pattern was coarser than in the mosaic caused by cucumber mosaic virus. Most of them showed no necrosis. A few samples from Saitama showed both veinal necrosis and coarse mosaic. 2) The symptoms on cucumber plants (varieties Shinsairaku and Shin-T-go) mechanically inoculated with the melon mosaic virus were veinclearing and coarse mosaic. Some individuals showed veinal necrosis which was the same as that observed in the samples from Iwate and Saitama. Necrotic spots as observed in the samples from Kochi, however, could not be reproduced. 3) A mosaic disease of watermelon was found severely occurring in a field in Okinawa, the Ryukyu Islands, and from the diseased plants, the same virus was isolated. 4) By mechanical inoculation tests, the melon mosaic virus was found to infect, in addition to the hitherto reported hosts, pea, broad bean, and okra, symptoms on these plants being veinclearing and mosaic. In hot summer months, however, it is rather difficult to infect pea and broad bean.
1) This paper reports the results of experiments concerning serological studies on barley stripe mosaic disease. Expressed juice of diseased plants, including barley (variety Moravia), wheat (variety Norin No. 29), and sweet corn (variety Golden Bantam), was centrifuged at 3, 500rpm for 30minutes; supernatantfluid was frozen, kept overnight, and thawed; then it was again centrifuged at 3, 500rpm for 30 minutes. The clarified fluid was purified by salting-out with ammonium sulfate at two-fifths saturation, or by differential centrifugation. In either procedure, the final virus preparation was concentrated into one-tenth volume of the original juice. Rabbits were injected intravenously with such immunogen at 3 day intervals, giving 50-60ml in total, until antibody response became sufficient to be used for serological reactions; the animals were bled about ten days after the last injection. 2) Positive reaction was usually observed against clarified sap from both diseased and healthy plants, when antisera prepared with the immunogen purified by the procedure of salting-out was used. 3) By injection into rabbits of the immunogen from diseased barley purified by differential centrifugation, specific antisera were obtained which react not against the juice of healthy plant, but against that of diseased plants. On the other hand, positive reaction was obtained with both diseased and healthy plant juices, when use was made of the antisera prepared by the immunogen from diseased wheat and sweet corn purified by differential centrifugation. 4) Regarding the normal proteins contained in both diseased and healthy plants, it was found there were some similarities not between those of barley and sweet corn, but between barley and wheat as well as between wheat and sweet corn. 5) Partially purified virus preparation from diseased barley plant was secured by salting-out with ammonium sulfate, while more purified one was obtained by differential centrifugation. 6) The antisera prepared as mentioned adove, became reacting specifically only with expressed juices of diseased plants or purified virus preparations, when they were absorbed by homologous healthy plant juices,
1) Using Ouchterlony's agar-gel diffusion technique, it was shown that leaf juice of diseased barley, wheat, or sweet corn infected with barley stripe mosaic virus (BSMV) contains an antigenic substance which react specifically against anti-BSMV sera. 2) The resulting precipitation appeared in the agar layer as a distinct sickle-shaped band near the hollow filled with the diseased leaf juice sample. The antigenic substance corresponding to this band was considered to be the virus. 3) Antisera prepared with immunogen purified by salting-out with ammonium sulfate at two-fifths saturation contained, in addition to the antibody against the virus-antigen, several antibodies which react against healthy plant components. 4) Antisera prepared with immunogen purified by differential centrifugation usually did not contain any detectable amount of antibodies against healthy plant components. 5) The source of all antibodies except that against the virus-antigen was considered to be normal plant proteins.
Yellow spot of leaves of sorghum (Holcus sorghi var. japonicus, or Sorghum vulgare) was found in Chiba prefecture near Tokyo since 1940. The disease is characterised by chlorotic (yellow, but not necrotic) and rectangular spots with white powdery appearnce (conidial formation), especially on their under surface. Later, the spots will change into light brownish necrotic lesions without conspicuous border colorations. Conidiophores extrude through stomata singly or in tuft of 2 to about twelve, geniculate then denticulate towards apex, 22-80×3.6-6.9μ and 0-5 septate. Conidia are obclavate with obconic then truncate bottom and attenuate then blunt apex, 21-55×3-6μ and 1-6 (mostly 3) septate. There can not find no identical sorghum fungus, but sugar cane yellow spot fungus (Cercospora koepkei Krüger) is similar in many respects on the morphology. Therefore, the sorghum fungus is to be inferred as C. koepkei Krüger var. sorghi, var. nov. The difference in description has been given.