One of the exciting applications of the genetic engineering technique is the biological control of plant diseases. Although plasmids are easily found in prokaryotes, only a few examples of plasmids are known in eukaryotes. There are even fewer experimental data on the presence of plasmids in phytopathogenic fungi. Nevertheless, some genetic and molecular evidences accumulated to date encourage further study on plasmids in phytopathogenic fungi. We searched for plasmids among three weakly pathogenic isolates of Rhizoctonia solani and identified a linear DNA plasmid, pRS64 (molecular weight 1.68±0.06x10^6). Weakly pathogenic isolates containing the pRS64 plasmid showed abnormally slow growth and low virulence to Japanese radish seedlings, whereas pathogenic isolates which lacked the plasmid showed normal growth and high virulence. Thus, it is highly probable that the weakly pathogenic character of R. solani isolates is under the control of the pRS64 plasmid, and this mechanism seems to be a clue for the biological control of damping-off of Japanese radish seedlings. Some of the difficulties are outlined that must be surmounted before biological control of plant diseases can be achieved.

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Recently the improvement of the biological nitrogen fixation of rice plants has been one of the important targets of biotechnology applied to agriculture. Researchers of the National Institute of Genetics in Mishima, devised an assay system using the acetylene reduction technique, and observed the variation in nitrogen-fixing activity in the rhizosphere among different rice species and strains. Ability of the associative nitrogen fixation of rice with bacteria was found to depend on the rice genotypes. And it became possible to develop a high N_2-fixing rice by breeding techniques. Several nitrogen-fixing bacteria were isolated from the rhizosphere of rice. Among these bacteria, Klebsiella oxytoca (NG13) and Azospirillum lipoferum (COC8) indicated high N_2-fixing activity associated with rice. In recent experiments, a plasmid pRD1 having multiple copies of the nif-gene of Klebsiella pneumoniae was introduced in NG13 by crossing experiment to obtain NG1325. NG1325 showed a nitrogen-fixing activity about three times higher than that of NG13. Several plasmids related to the nif-gene were identified from other nitrogen-fixing bacteria. Practical use of the bacteria is under study in the Shizuoka Agricultural Experiment Station. Some prospects of biological nitrogen fixation in plant genetic engineering are discussed.

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Since soil amoebae were found to feed on and destroy soilborne plant pathogens, the amoebae have been attracting a considerable interest as antagonists of the plant pathogens and as potential agents for the biological control of root diseases. This paper deals with the morphology, taxonomy, feeding behaviour, ecology of the amoebae, and the possibility of biological control of root diseases by the amoebae. More than ten species of mycophagous amoebae have been identified including a wide range of taxonomical classes, e. g. Filosea, Lobosea, Acarpomyxea and Granuloreticulosea in the subphylum Sarcodina. The feeding process of propagules of the pathogens by mycophagous amoebae is initiated by attachment of trophozoites to the propagules following engulfment and digestion of the propagules. Emptied walls of the propagules are left after feeding and perforations varying in size are observed in the walls of the emptied propagules. Size of perforations, time required for feeding and feeding behaviour depend on the species of amoebae. Mycophagous amoebae are ubiquitous and show a worldwide distribution. In Japan, the amoebae are widespread in agricultural soils irrespective of the crop species, cultivation method, and soil texture from Hokkaido to Okinawa. The number of amoebae in soil amounts to 3 to 70 individuals/g of soil and the amoebae are more numerous in the upper layers of soil. Soil environmental factors strongly affect the activity of the amoebae. The optimal matric water potential for amoebal activity in soil varies with the soil specimens and the activity can be detected in a matric water potential ranging between -10 and -250 mb. Osmotic water potential is also an important factor for the amoebal activity. The activity is not detected at an EC above 1500μmho/cm in soil and osmotic water potential below -800 mb in liquid medium. The activity is maximal at soil pH6.5 to 7. High amoebal activity is detected in the suppressive soils of wheat take-all and Phytophthora root rot of avocado. A larger number of amoebae is detected in the wheat rhizosphere of the take-all suppressive soils and mycophagous amoebae are considered to play an important role in inhibiting the infection of the pathogen in the wheat rhizosphere of the suppressive soils. The amoebae inhibit the pathogen by colonizing the plant root and growing ectotrophically on the root surface. As a result, the severity of the root diseases is reduced and the height and top dry weight of the plants increase.

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When introduced into soil, chlorinated organic solvents may affect the soil microflora which plays an important role in the biogeochemical nutrient cycle. In our investigations, concentrations of 10, 100, and 1000μg per 100g soil (dry weight) affected variously the soil microorganisms. The results were as follows: 1) The ATP content of the soil biomass decreased significantly in the first and second week of incubation when 100μg of trichloroethylene and tetrachloroethylene were added to the soil, and the content was markedly reduced during the two month period of the experiment when 1000μg of the individual chlorinated organic solvents under test were added. 2) Tetrachloroethylene at the dose of 1000μg, however, exerted prevalently a stimulatory effect on the number of aerobic soil bacteria and also on the total number of anaerobic bacteria. The same concentrations of trichloroethylene and dichloromethane inhibited both groups of aerobic bacteria in the soil. No significant effects were observed with aerobic spore-forming bacteria. Fungi were the most sensitive group and their number was remarkably reduced by each concentration of the solvents used in the experiments. Similar inhibitory effects could be observed with soil biomass. 3) When 100μg chlorinated organic solvents were added, the activity of β-glucosidase, β-acetylglucosaminidase and partially also that of proteinase were decreased up to 28 days of incubation but the activity returned to the same or slightly higher level as in the control soil after two months of incubation. Trichloroethylene, tetrachloroethylene, and dichloromethane at the concentration of 1000μg per 100g soil primarily inhibited the activity of all the enzymes tested. However, after two months of incubation the enzyme activity especially that in the soil samples contaminated with tetrachloroethylene and dichloromethane was found to be the same as or higher than that in the control soil.

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A hydrostatic pressure vessel was designed to isolate a wide range of barotolerant or barophilic bacteria from deep sea and various environments. This pressure vessel can withstand a pressure upto 1000 atmosphere. The seven terrestrial bacteria including isolates from soil were tested for reproduction for 3 weeks under a pressure ranging from 350 to 1000 atmospheres. A strain of bacteria isolated from tomato root was found to be more barotolerant than the others. The pressure vessel described in the current investigation may be useful for the isolation of barophilic bacteria.

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