INCREASING THE WHITE CLOVER SYMBIOTIC N₂ FIXATION ACTIVITY - A. Selge^[13] and S. Higuchi^[14]

SUMMARY

These experiments suggested that the inherent characteristics of plants have a strong influence upon N₂ fixation activity. White clover nodule N₂ fixation activity had a positive correlation with total nodule weight (R²=0.67-0.96) and with the average nodule weight (R²=0.25-0.70). Nodule number was not a good index to estimate the plant N₂ fixation activity. The white clover plants dry matter yield showed a moderate positive correlation (R²=0.44) with N₂ fixation activity. A positive correlation was found between N₂ fixation activity and nodule total-N content (R²=0.64). According to these results using the Rhizobium trifolii A6R1 strain, the white clover cultivars 'Sonja' and 'Huia' are more preferable materials for selecting white clover plants as they are characterized by larger nodules.

Keywords: nitrogen (N) fixation activity, nodule number and weight, total-N, white clover cultivars

INTRODUCTION

For legume plant breeders it is necessary to select more superior plants (cultivars) with higher nitrogenase activity for improving N₂ fixation potential. The selection of plant cultivars with higher N₂ fixation activity would be expected to increase the rate of N₂ fixation. The objectives of these experiments were to investigate differences in N₂ fixation activity between white clover cultivars (small to large leaf types) bred in different climates and how the nodule characters (nodule number, nodule average and total weight) effect the N₂ fixing activity.

MATERIALS AND METHODS

The experiments were established at Tohoku National Experiment Station (Japan) in January 1997 and continued at Eerika Experimental Station (Estonia) in 1998.

Experiment 1. Nine white clover cultivars for estimation of nitrogen fixation ability were gathered (in total more than 1 000 plants): 'North White' (small leaf type), 'Tohoku 17' (medium leaf type), 'Tohoku 20' (medium leaf type) all bred in Japan; 'Tahora' (small leaf type), 'Huia' (medium leaf type) both bred in New Zealand, 'Rivendel' (small leaf type) bred in Denmark; 'Jogeva 4' (medium leaf type), 'Tooma' (medium leaf type) both bred in Estonia; 'Sonja' (medium leaf type) bred in Sweden.

The white clover cultivars were sown on 28 January 1997 into a sterile substrate like pot-plants using special small plastic sets (20x20x30 mm, one set with 200 pots for each cultivar). Growth conditions were heat controlled in a greenhouse (the natural day length and mean daytime temperature of 22 and 18°C at nighttime) and treatments were sown in three replicates. After 50 days the plants were transplanted into bigger pots (300 ml) in the same conditions. The Rhizobium trifolii A6R1 strain was used for seed inoculation 14 days after sowing. Artificial irrigation was used once per day/night and no wilting occurred. Therefore, it would be difficult to imagine that water availability could significantly affect N₂ fixation by nodules. P- and K-fertilizer were added by irrigation water twice a month (5 ml soluble fertilizer for 300 plants/month) using Hyponex N-free fertilizer (0-6-4 g kg-¹ and Mg, Mn and B, respectively, 0.05, 0.001 and 0.005 g kg-¹).

Experiment 2. White clover plants only for acetylene assays were selected (total 70 samples) directly from the field trial, a white clover germplasm collection.

Using the plants from Experiments 1 and 2 the closed acetylene reducing activity was estimated by gas chromatography (nmolC₂H₄) on the basis of the fresh weight of nodules and emphasized the importance of comparing plants of the same physiological state, time and growing conditions. For one analysis, two to three typical healthy plants were selected (to minimize one-plant-effect) during the experimental period. The plants were carefully excavated with the substrate or soil and placed in a cup filled with water. In the laboratory the roots were immediately and carefully washed and incubation into the 50 ml test-tube. The ethylene content was estimated by gas chromatography in three replications, after 60, 120 and 180 mins. After the acetylene assay test the fresh nodules were manually separated from the roots and the number and weight of nodules were measured after 6 h drying (60°C). By random selection of plants the shoots, roots and nodules of dry matter weights and total-N were measured. The plant total-N was analysed by Kjeltec Auto 1030 Analyser and FIAStar Analyser 5010 (for nodules, sample weight 1 mg and more).

RESULTS AND DISCUSSION

The results of experiments 1 and 2 did not show significant differences between white clover cultivars (small to large leaf types, bred in different climates) in N₂ fixation activity. The significant differences in N₂ fixation activity appeared between concrete root nodules group and we can say that concrete inherent plant root has stronger influence to the N₂ fixation activity than (white clover) cultivar (Table 1). Furthermore, the white clover plants (root-shoot) dry matter yield did not show a strong positive correlation (R²<0.45) with N₂ fixation activity. In addition, these experiments do not distinguish the reciprocal influences of plant size and N₂ fixation activity.

Table 1. Reduction of C₂H₂ by nodules of white clover (average C₂H₄ nmol h-¹ g-¹ after 60, 120, 180 mins acetylene assay).

Although Sheehy et al. (1991) found good agreement between the maximum rates of nitrogen fixation, estimated using the acetylene reduction technique, and the maximum rates of nitrogen accumulation in the biomass above ground, our experiments did not show good correlation between white clover shoot and also root total-N content and N₂ fixation activity. At the same time, positive correlation was found (R²=0.64) between the N₂ fixation activity and nodules total-N content.

The N₂ fixation activity had a positive correlation with total nodule weight (R²=0.67-0.96) and with average nodule weight (R²=0.25-0.70, Figure 1). On the other hand, the number of nodules per plant root had no significant positive or negative association with N₂ fixation activity. Burton (1985) found that on clovers, the nodule number is not a good index of benefit, which the host plant is receiving. In fact, the indications are that the relationship between the nodule number and plant benefit may be slightly negative.

Although significant differences between white clover cultivars in N₂ fixation activity were not found, after measuring more than one thousand plant nodules, it was suggested that white clover cultivars 'Sonja' and 'Huia' formed bigger nodules and showed respectively higher N₂ fixation activity all through the experimental period. In all the white clover cultivars, nodule size and the number varied to a large extent. These experiment results have shown that using the Rhizobium trifolii A6R1 strain, the white clover cultivars 'Sonja' and 'Huia' were preferable materials for selection of white clover plants with larger nodules.

Figure 1. Correlation of white clover N₂ fixation activity with average and total nodule weight, 7 October 1997, Experiment 1.

Very few breeding efforts have been undertaken to improve N₂ fixation of white clover. Plants with fewer, but larger nodules are good models for breeding programmes to improve the N₂ fixation of white clover (Crush and Caradus, 1996). The present experiments have shown the most important white clover plant characteristics for improving white clover N₂ fixation.

Even though the inherent plant characteristics and preferable white clover cultivars are good tools for breeders, it is very important to consider the cultivar appropriateness with Rhizobium bacteria strain. According to Burton (1985) 'ineffective' strains produced many more nodules than the 'effective' strains and the Rhizobium bacteria have a strong influence on nodule development. Therefore, breeders have to consider the complex simultaneous selecting for efficient host plants and rhizobia. Investigation to find these associations by selection of white clover plants with large nodules is a task to continue this research topic in coming years.

ACKNOWLEDGEMENTS

Special thanks are given to the staff at the White Clover Breeding Group from the Tohoku National Agricultural Experiment Station for their kind assistance and JISTEC for STA Fellowship.

REFERENCES

Burton, J.C. 1985. Rhizobium Relationships. Clover Science and Technology, Agronomy Monograph, No. 25, U.S.A., pp. 161-184.

Crush, J.R. & Caradus, J.R. 1996. Increasing symbiotic potentials in white clover. Agronomy Society of New Zealand Special Publication No. 11. Grassland research and Practice series No. 6. pp. 91-94.

Sheehy, J.E., Woodward, F.I. & Gosse G. 1991. Measurements of Nitrogen Fixation (C₂H₂), Photosynthesis and Respiration Using an Open System in the Natural Environment. Annals of Botany, No. 67, pp.131-136.