References

Kabata-Pendias, Pendias (1984) CRC Press Inc., Boca Raton, FL Mc-Grath, Brookes (1988) Soil Bio. Biochem. 20, 4, 415-424

THE DELAY EFFECT OF RHIZOBITOXINE PRODUCTION ON NODULATION COMPETITIVENESS OF BRADYRHIZOBIUM ELKANII USDA94 IN SIRATRO

S. Okazaki1, H. Ezura2, K. Yuhashi', K. Minamisawa1

'institute of Genetic Ecology, Tohoku University, Sendai 980-8577, Japan 2Plant Biotechnology Institute, Nishi-Ibaraki, 319-0292, Japan

1. Introduction

Bacterial production of rhizobitoxine, an ethylene synthesis inhibitor (Yasuta et al. 1999), plays a role in competitiveness of Bradyrhizobium elkanii for siratro (Macroptilium atropurpureum) nodulation (Yuhashi et al. 2000; Duodu et al. 1999) since siratro nodulation is partially restricted by endogenous ethylene (Nukui et al. 2000). However, the mechanism of increasing nodulation competitiveness by rhizobitoxine production is still unclear. Here, we report the competitive nodulation, kinetics of nodule occupancy and ethylene evolution rate in siratro roots using B. elkanii USDA94 and its rhizobitoxine production-deficient mutant RTS2.

2. Results and Discussion

Competitive nodulation enhancement of rhizobitoxine was reconfirmed in various inoculum ratio of USDA94 and RTS2. Time course of nodulation showed that nodule occupancy between USDA94 and the mutant RTS2 was almost the same before 13 days after inoculation, however the occupancy of the USDA94 continuously increased after 13 days. The results suggest a delay effect of rhizobitoxine production on competitiveness of USDA94 in siratro nodulation. Ethylene evolution rate in siratro roots was at a lower level in single-inoculation with the USDA94, while it was higher in inoculation with the mutant during nodulation. Ethylene evolution rate in roots co-inoculated with both the wild type and the mutant was decreased after 13 days after inoculation. The change in ethylene evolution rate in the co-inoculated roots might reflect an increase in nodule occupancy of the rhizobitoxine producer.

3. References

Duodu et al. (1999) Mol. Plant-Microbe Interact. 12,1082-1089 Nukui et al. (2000) Plant Cell Physiol. 41, 893-897 Yasuta et al. (1999) Appl. Environ. Microbiol. 65, 849-852 Yuhashi et al. (2000) Appl. Environ. Microbiol. 66, 2658-2663

EFFECT OF ANAEROBIOSIS ON SYMBIOTIC EFFICIENCY OF THREE STRAINS OF SINORHIZOBIUM MELILOTI

Soils and Crops Research and Development Centre, Agriculture and Agri-Food Canada, 2560 Hochelaga Blvd, Sainte-Foy, QC, Canada G1V 2J3

1. Introduction

During winter, climatic conditions in temperate areas can create an ice layer over agricultural soils. This situation causes an anaerobic stress that may affect winter survival and regrowth of plants (Andrews, Pomeroy 1990). The production of toxic metabolites and the reduction of C and N reserves might be responsible for a poor winter survival of perennial plant species. For instance, the freezing tolerance of alfalfa cultivars is closely related to their capacity to accumulate the raffmose family oligosaccharides (Castonguay et al. 1995). Furthermore, the strain of rhizobium may play an important role in the adaptation of legumes to stresses (Layzell et al. 1984). Our aim was to evaluate the effect of anaerobiosis during winter on the symbiotic efficiency of three strains of S. meliloti.

2. Material and Methods

Three strains of S. meliloti with the same symbiotic efficiency were used: two commercial (BALSAC and NRG-34) and one indigenous (Rm-1521). Alfalfa was grown in a mixture of topsoil/peatmoss (11:1 v/v) for three months in a greenhouse (two cuttings). Potted plants were then acclimated in the field during fall. In late fall, half of the plants were subjected to a progressively developing anaerobic stress by enclosing them in gas-tight bags and exposing them to simulated winter conditions in an unheated greenhouse. At three sampling dates, gases were sampled and gas-tight bags were removed. Plants were then placed in growth chambers at optimal temperature for a two-weeks regrowth during which nitrogenase activity was determined. At the end of regrowth, nodulation indexes and shoot dry weights were measured. Populations of rhizobia were determined by colony hybridization technique at the beginning and at the end of the simulated winter period.

3. Results and Discussion

Shoot and root dry weight, nitrogenase activity and nodulation index of alfalfa were considerably reduced after 78 days of anaerobiosis under winter conditions. None of the rhizobial strain allowed an optimal regrowth after the extreme period of anaerobic stress of 101 days. After the first 78 days of winter, plants nodulated by strain NRG-34 maintained the highest nodulation index and shoot dry weight under anaerobiosis (1% O2) and this strain was also the most efficient under control conditions. Anaerobic stress did not affect survival of rhizobia. Populations decreased at the end of the overwintering period but each strain maintained a level of 10s cells/g soil. Strain NRG-34 was shown to be adapted for growth and nodulation at low temperatures (Rice et al. 1995) and our results show that this strain can also preserve its symbiotic efficiency after overwintering. It is then possible to select rhizobia for improving regrowth of alfalfa exposed to a wide range of winter conditions.

4. References

Andrews CJ, Pomeroy MK (1990) In Jackson MB, Davies DD, Lambers H (eds) Plant Life Under

Oxygen Deprivation, pp. 85-89, SPS Academic Publishing, The Hague Castonguay Y (1995) Crop Sci. 35, 509-516 Layzell DB et al. (1984) Can. J. Bot. 62, 965-971 Rice WA et al. (1995) Plant Soil 170, 351-358

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