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Arbuscular mycorrhizal (AM) fungi interact with bacteria (AM fungi-associated bacteria, AMB) in the mycorrhizosphere. We previously identified a set of AMB that enhance AM fungal colonization, plant growth, and inhibit pathogens. Here, we used transformed carrot root cultures in a two-compartment plate system for further in vitro studies on interactions taking place among Glomus irregulare (syn.Glomus intraradices), AMB, and plant pathogens. We found that exudates of G. irregulare stimulated growth of all ten AMB isolates tested in multi-well plates. AMB growth stimulation was observed also during co-cultivation of three of these AMB with G. irregulare in the hyphal compartment. In addition, co-cultivation stimulated growth of G. irregulare hyphae and spore production, as well as G. irregulare root colonization. GC/MS analysis in a preliminary screening of metabolites revealed differences in concentrations of several identified but also unidentified compounds in G. irregulare hyphal exudates. Exudates in presence of three different AMB isolates co-cultivated with G. irregulare contained several additional compounds that differed in amount compared with G. irregulare alone. The results indicate that G. irregulare exudates contain carbohydrates, amino acids, and unidentified compounds that could serve as a substrate to stimulate AMB growth. With regard to effects on plant pathogens, growth inhibition of Rhizoctonia solani, Verticillium dahliae, and Pectobacterium carotovorum ssp. carotovorum was evident in the presence of the AMB isolates tested together with the G. irregulare exudates. These in vitro studies suggest that G. irregulare and AMB stimulate growth of each other and that they together seem to provide an additive effect against growth of both fungal and bacterial pathogens.

Effects of prolonged darkness on nitrogenase activity in vivo, nitrogenase activity in vitro, and the amounts of nitrogenase proteins were studied in symbiotic Frankia. Plants of Alnus incana (L.) Moench in symbiosis with a local source of Frankia were grown for 9 to 10 weeks in an 18/6 hour light/darkness cycle. After 12 hours of a light period, the plants were exposed to darkness for up to 40 hours. Nitrogenase activity (acetylene reduction activity) of intact plants was measured repeatedly. Frankia vesicle clusters were prepared from the nodules with an anaerobic homogenization and filtration technique and were used for measurements of in vitro nitrogenase activity and for measurements of the amounts of nitrogenase proteins on Western blots. Antisera made against dinitrogenase reductase (Fe-pro- A reduced amount of active nitrogenase can be due to inactivation or degradation not compensated for by synthesis or a combination of these possibilities. To distinguish among them, it is necessary to determine the amounts of nitrogenase protein during a stress treatment. Since nitrogenase consists of two proteins, dinitrogenase reductase (Fe-protein) and dinitrogenase (MoFe-protein), both necessary for activity, the amounts of both proteins have to be determined. In this paper, we describe immunological techniques used to quantify the Fe-protein and the MoFe-protein of Frankia in symbiosis with Alnus incana (L.) Moench exposed to prolonged darkness. MATERIAL AND METHODS Plant Material, Frankia and Growth ConditionsA clone of gray alder, Alnus incana (L.) Moench, was used (9). One-leaf internode cuttings were rooted, inoculated with a water suspension of crushed root nodules containing a local source of Frankia (phenotypically spore (+), hydrogen uptake activity (-) in symbiosis; [11,21]), planted into pots with gravel, and grown in a climate chamber as described previously (9) but with the following modifications. The concentration of NH4NO3 was reduced to 0.036 mm, and, after 3 weeks, nitrogen was omitted from the nutrient solution. The plants received a continuous supply of the nutrient solution, fourfold diluted, distributed to the plants with an air-lift system. Twelve plants shared 20 L of the recirculated solution which was renewed twice a week. After 4 weeks, the plants were transferred to a climate chamber with higher photosynthetic photon flux density (600 ,mol-m-2-s-' at plant level)and an 18/6 h cycle of light and temperature (25°C/15°C) with a 1-h transition period from day to night conditions and vice versa. Plants were used 9 to 10 weeks after inoculation and had an average leaf area of 730 (range 330-1120) cm2 and an average height of 66 (range 53-84) cm.

O 2 and host-microsymbiont interactions are key factors affecting the physiology of N 2 -fixing symbioses. To determine the relationship among nitrogenase activity of Frankia-Alnus incana root nodules, O 2 concentration, and short-term N 2 deprivation, intact nodulated roots were exposed to various O 2 pressures (pO 2 ) and Ar:O 2 in a continuous flow-through system. Nitrogenase activity (H 2 production) occurred at a maximal rate at 20% O 2 . Exposure to shortterm N 2 deprivation in Ar:O 2 carried out at either 17%, 21%, or 25% O 2 caused a decline in the nitrogenase activity at 21% and 25% O 2 by 12% and 25%, respectively. At 21% O 2 , nitrogenase activity recovered to initial activity within 60 min. The decline rate was correlated with the degree of inhibition of N 2 fixation. Respiration (net CO 2 evolution) decreased in response to the N 2 deprivation at all pO 2 values and did not recover during the time in Ar:O 2 . Increasing the pO 2 from 21% to 25% and decreasing the pO 2 from 21% to 17% during the decline further decreased rather than stimulated nitrogenase activity, showing that the decline was not due to O 2 limitation. The decline was possibly due to a temporary disturbance in the supply of reductant to nitrogenase with a partial O 2 inhibition of nitrogenase at 25% O 2 . These results are consistent with a fixed O 2 diffusion barrier in A. incana root nodules, and show that A. incana nodules differ from legume nodules in the response of the nitrogenase activity to O 2 and N 2 deprivation.