pestle, with 0.15 M sodium phosphate buffer, pH 7.5, added in 2-ml portions to a final volume of 2 ml of buffer per g of nodules. The homogenate was filtered through two Miracloth disks in a 50-ml syringe into a 30-ml centrifuge tube. The tube was capped tightly and centrifuged at 6,000 x g for 10 min. The tube was opened in the glove box, the supernatant was discarded, and the pellet was washed twice by pipetting 1 ml of the buffer over it. The pellet was resuspended, and the mixture was again filtered through Miracloth disks to remove any unsuspended bacteroids and adjusted to a final concentration of 2 ml of buffer per g of nodules. This (16), and 0.3 ml of 0.15 M sodium phosphate buffer, pH 7.5. The tubes were flushed with N2 and sealed with a serum stopper with an attached cup containing a filter paper wick impregnated with 70 p.l of 10% KOH. A sample (1.4 ml) was removed from the gas phase and replaced with 1.4 ml of , giving a final concentration of 2% 02 in the gas phase. Buffered substrate (0.5 ml; 1 ,umol at 1 ,uCi/,umol) was then injected into the reaction mixture, and the tubes were shaken in a rotary shaker at 250 rpm. Incubation was terminated by placing the tubes in an ice bath. The stoppers were quickly removed, and the tubes were centrifuged at 25,000 x g for 10 min. The supernatant was discarded, and the pellet was washed twice by pipetting 1 ml of the cold buffer over it and discarding the washes. The

Previous studies with labelled N and C have indicated synthesis and accumulation of glutamate in Brudyrhizobium japonicum bacteroids under microaerobic conditions similar to those found in soybean nodules. Low 2-oxoglutarate dehydrogenase (OGDH) activity might have accounted for this observation, but similar levels of enzyme activity were found in bacteroids isolated anaerobically or aerobically and in cultured bacteria. However, OGDH from B.japonicum bacteroids was strongly inhibited by NADH, and the degree of inhibition depended on the NADH: NAD ratio. Determination of endogenous levels of NAD and NADH gave NADH: NAD ratios of 0.19 and 0.83 in bacteroids isolated under aerobic and anaerobic conditions, respectively. A ratio of 0.83 resulted in more than 50% inhibition of OGDH in uitro, and this would be consistent with channelling of 2-oxoglutarate to glutamate. [ 14C)Glutamate supplied to bacteroids was metabolized to CO, slowly relative to the respiration of malate, and essentially no labelling of products of glutamate metabolism such as arginine, proline, glutamine and 4aminobutyrate (GAB) was found. Attempts to trap 14C in GAB by supplying unlabelled GAB or transaminase inhibitors with [ 14C]glutamate were unsuccessful. The finding that glutamate decarboxylase was essentially absent in six different strains of B. japonicum was consistent with the labelling results and indicated that conversion of glutamate to succinate via GAB is slow or nil. The inhibition of OGDH by a high NADH:NAD ratio and the absence of the GAB shunt are complementary mechanisms which probably account for the accumulation of glut ama te .

Metabolism of trehalose, a,D-glucopyranosyl-a,D-glucopyranoside, was studied in nodules of Bradyrhizobium japonicum-Glycine max IL.I Merr. cv Beeson 80 symbiosis. The nodule extract was divided into three fractions: bacteroid soluble protein, bacteroid fragments, and cytosol. The bacteroid soluble protein and cytosol fractions were gel-filtered. The key biosynthetic enzyme, trehalose-6-phosphate synthetase, was consistently found only in the bacteroids. Trehalose-6-phosphate phosphatase activity was present both in the bacteroid soluble protein and cytosol fractions. Trehalase, the most abundant catabolic enzyme was present in all three fractions and showed two pH optima: pH 3.8 and 6.6. Two other degradative enzymes, phosphotrehalase, acting on trehalose-6-phosphate forming glucose and glucose-6-phosphate, and trehalose phosphorylase, forming glucose and 0-glucose-l-phosphate, were also detected in the bacteroid soluble protein and cytosol fractions. Trehalase was present in large excess over trehalose-6-phosphate synthetase. Trehalose accumulation in the nodules would appear to be predicated on spatial separation of trehalose and trehalase.Trehalose, a-D-glucopyranosyl-a-D-glucopyranoside, is one of the major carbohydrates synthesized by cultured Bradyrhizobium japonicum, and every species of Rhizobium examined so far shows trehalose accumulation (26). This disaccharide has also been reported in yeast (1,5), in fungi (12, 22), as well as in bacteria (3), mainly in actinomycetes (15). In soybean plants trehalose appears to be restricted to the nodule tissue (23). Its presence elsewhere in higher plants has not been conclusively established (7). Previous work with soybean nodules suggests that trehalose is synthesized by the microsymbiont (20).The pathway for trehalose synthesis was first elucidated in Saccharomyces cerevisiae (5) (Fig. 1). The biosynthetic enzymes have also been reported in Lilium longiflorum pollen (8).There appears to be more than one way of catabolizing trehalose (Fig. 1). Phosphotrehalase has been reported in Bacillus popilliae (3) and trehalose phosphorylase in Euglena gracilis (17). Trehalase, the most widely occurring catabolic enzyme has also been found in higher plants (6,9,27 Nodule Fractionation. After 5 to 6 weeks the plants were harvested, and subsequently all operations were conducted at 0 to 2°C. Five g of nodules were ground in a mortar with a pestle in 10 ml (added in 2 ml aliquots) of grinding medium, 0.1 M sodium phosphate buffer (pH 7.5) containing 0.15 M mannitol, 2 mm DTE, and 1 mm EDTA. The homogenate was filtered through two Miracloth discs in a 50 ml syringe into a 15 ml centrifuge tube.The Miracloth filtrate was then centrifuged at 5OOg for 10 min. The resultant starch pellet was discarded and the supernatant fluid recentrifuged at 6,000g for 10 min to obtain the bacteroid pellet. The supernatant fluid was centrifuged at 48,000g for 15 min and the supernatant, cytosol fraction, was saved for gel filtration.The bacteroid pellet was resuspended in 8 ml of grindin...

The aim of the work reported here was to ascertain that the patterns of labeling seen in isolated bacteroids also occurred in bacteroids in intact nodules and to observe early metabolic events following exposure of intact nodules to 4CO2. Intact nodules of soybean (Glycine max L. Merr. cv Ripley) inoculated with Bradyrhizobium japonicum USDA 110 and pea (Pisum sativum L. cv Progress 9) inoculated with Rhizobium leguminosarum bv viciae isolate 128C53 were detached and immediately fed 14CO2 for 1 to 6 min. Bacteroids were purified from these nodules in 5 to 7 min after the feeding period. In the cytosol from both soybean and pea nodules, malate had the highest radioactivity, followed by citrate and aspartate. In peas, asparagine labeling equaled that of aspartate. In B. japonicum bacteroids, malate was the most rapidly labeled compound, and the rate of glutamate labeling was 67% of the rate of malate labeling. Aspartate and alanine were the next most rapidly labeled compounds. R. leguminosarum bacteroids had very low amounts of 14C and, after a 1-min feeding, malate contained 90% of the radioactivity in the organic acid fraction. Only a trace of activity was found in aspartate, whereas the rate of glutamate and alanine labeling approached that of malate after 6 min of feeding. Under the conditions studied, malate was the major form of labeled carbon supplied to both types of bacteroids. These results with intact nodules confirm our earlier results with isolated bacteroids, which showed that a significant proportion of provided labeled substrate, such as malate, is diverted to glutamate. This supports the conclusion that microaerobic conditions in nodules influence carbon metabolism in bacteroids. Conclusions from studies of carbon uptake and metabolism by isolated bacteroids need to be confirmed with bacteroids in their natural environment, i.e. in the intact nodules. A noninvasive method to establish the form of carbon received by bacteroids would be to feed "4CO2 to the leaves and analyze the compounds labeled in the nodule and bacteroids.Results from experiments of this type (15,21,22) have yielded valuable information. However, due to the variation in rates of photosynthesis, translocation, and subsequent metabolism, the method does not allow clear resolution of the order of metabolic events in nodules.The problem can also be approached by taking advantage of high PEPC2 activity in the nodules (4, 16). This approach is biased to some degree because metabolism of labeled carbon begins at OAA and malate rather than sucrose (31). However, the malate pool in the cytosol of nodules is a relatively large carbon pool (29), and the fact that PEPC activity may be equivalent to as much as 14% of nodule respiration (1 1) makes it possible rapidly to label the relatively large pool. These facts combined with the established importance of dicarboxylic acids in bacteroid carbon nutrition make this an attractive approach for the study of carbon metabolism in intact nodules. The approach has been employed by others, b...

We evaluated aesthetic (lawn quality), biological (weeds and insect pests), and economic (management costs) effectiveness of a commercial (managed by a professional company), consumer (managed using consumer lawn care products following labeled instructions), integrated pest management (IPM) (pesticide applications based on monitoring and thresholds), organic (monitoring and need-based organic and natural product applications), and an untreated lawn care program. Percent weed cover was the lowest in the commercial followed by IPM, organic, and consumer programs. The commercial program had lower white grub density than all other programs, and the organic program had lower white grub density than the untreated program. The commercial program had the highest lawn quality while the untreated program had the lowest. The IPM and organic programs did not differ significantly in lawn quality, but both rated significantly higher than the consumer program. Annual costs were highest in the commercial ($382/0.05 ha) followed by the organic ($305/0.05 ha), IPM ($252/0.05 ha), and consumer program ($127/0.05 ha), respectively. We conclude that the commercial program produced the highest lawn quality, and weed and insect control, and was the most expensive. The IPM and organic programs were cheaper than the commercial program and produced slightly lower lawn quality. Although the consumer program was the cheapest, it produced the lowest weed control and lawn quality among treated lawns.Keywords Lawn quality . Percent weed cover . White grub density . Lawn management cost . IPM lawn management program .