INTERACTION BETWEEN WHITE CLOVER AND DIFFERENT RHIZOBIUM STRAINS IN TWO SOIL TYPES IN ICELAND - I.-L. Fagerli^[29], J. Gudmundsson,^[30] P. Leinonen^[31] and M.M. Svenning^[32]

SUMMARY

In a field experiment in Iceland, white clover population HoKv9238, a selected population from a breeding programme in Norway, is grown in combination with red fescue cultivar Leik. Population HoKv9238 was inoculated with three strains of Rhizobium leguminosarum biovar trifolii as single strain inoculum or as mixed inoculum. During a four-year period, clover and grass production are measured and the interaction between the three strains of rhizobia are studied. Population HoKv9238 made an effective and lasting symbiosis with the three strains of R.l.bv. trifolii. When mixed inoculum was used one of the strains was dominating.

Keywords: competition, Rhizobium, soil type, white clover.

INTRODUCTION

White clover (Trifolium repens) is an important pasture legume in Europe. It is mainly used in combination with grass in grazing areas, as green manure and in bicropping systems. It is a perennial legume forming nitrogen fixing symbiosis with Rhizobium leguminosarum biovar trifolii. Plant material suitable for cold climates was lacking, but an ongoing breeding programme on white clover in Norway resulted in promising material for these harsh conditions.

In parallel with this, studies of indigenous R.l.bv. trifolii populations resulted in a selection of efficient strains for white clover. This material was used in combination with a selected white clover population in a field experiment to study the performance and persistence of the symbiosis in the field. The absence of indigenous rhizobia in Icelandic soil gives a unique opportunity to follow the R.l.bv.trifolii strains in a realistic field situation.

MATERIALS AND METHODS

Field experiment

Plant material

White clover population HoKv9238 (Plant Research Center, Holt, Tromsø, Norway) and red fescue cultivar Leik.

Bacteria

Three strains of R.l.bv. trifolii (strains 8-9, 20-15 and 32-28) from the native population in northern Norway.

The three strains were inoculated as single strains or as mix inoculum. Control with no rhizobia treatment was included.

The plot size is 10 m² and the experimental design is a randomized block with four replicates. The experiment was carried out in mold and sandy soils.

Inoculum production

Inoculants for the field trials were standard peat-based inoculants prepared into sterile, finely-ground peat. Inoculants contained 6x10⁸-2 x10⁹ Rhizobium cells /g and were applied onto seeds to guarantee 5x10⁴ Rhizobium cells per seed. A sticker (CMC) was included to ensure proper adhesion of inoculant onto seeds. Appropriate amounts of single-strain inoculants were mixed to ensure the three strains to be in a ratio 1:1:1 in the mixed inoculant treatment.

Harvesting of plants

Whole plants were harvested in the field autumns 1995, 1996 and 1997, and spring 1996 and 1997. In spring 1996 it was only harvested from plots treated with mix inoculum in mold field. In spring 1997 it was harvested from plots treated with mix inoculum and control plots, both from mold and sand field. In autumn 1996 it was harvested from all plots in mold field and from plots treated with mix inoculum and control plots in sand field.

The plant roots were washed with tap water and then surface sterilized in 70 percent ethanol and 6 percent H₂O₂. Ten nodules from each of three plants/plot and three plants from each of three replicates in 1995 and four replicates in 1996 and 1997. Totally 70-120 reisolates from each treatment were analysed for symbiont.

Identification of Rhizobium

Nodule occupancy is studied by fingerprinting of reisolates from nodules or from nodule suspensions. The three R.l.bv. trifolii strains used for inoculation, are identified by ERIC-PCR (Verasalovic, 1991, de Bruijn, 1992) from reisolates of nodules harvested in the field.

RESULTS

The results from treatment with mix inocula and control plot are given in Table 1. Yield of grass and clover from the different treatments are given in Table 2.

DISCUSSION

In plots with single strain inoculum the strain used as inoculant dominated in the nodules (results not shown) and in mixed treatment strain 20-15 always occupied most of the nodules. The bacteria spread through the soil and inoculated the control plants and strain 20-15 dominated in the nodules. Strain 32-28 occupied few nodules of plants treated with mix inoculum in mold and sand fields and of control plants in mold field. On the other hand, strain 32-28 occupied nearly half of the nodules of the control plants in the sand field. It seems like the soil type influence the dispersal of the bacteria and thereby the interaction between them.

Table 1. Percentage; nodule occupancy of the three different R.l. bv trifolii strains in plots treated with mix inocula and control plots, from field with mold soil (A) and sandy soil (B) A) Mold soil.

A) Mold soil

B) Sandy soil

Table 2. White clover and grass production (hkg ha^-1) in the 4 R.l. bv. trifolii treatments and the control.

The first year after establishment the grass production was higher in the control than in the Rhizobium treated plots. The grass in the control plots had little competition from the clover plants, without inoculum the clover plant had poor growth. The grass in the Rhizobium treated plots still had no advantage over the grass in control plots, it is likely that nitrogen transfer was low the first year. Grass production was higher in the Rhizobium treated plots than in the control the second year, and mixed inoculum gave the highest production. Transfer of nitrogen may have been higher than the first year and gave positive effect on grass production. Poor growth of clover in control the year before gave little transfer of nitrogen and from there little grass production the second year. The third year mixed inoculum gave lowest grass production and the control plot had an average production. Both grass and clover production were lower than the second year.

The results from the field experiment confirm effective and lasting inoculation in Icelandic soil.

ACKNOWLEDGEMENT

We thank the Norwegian Research Council for financial support (Grant 111838/111). The research is part of NKJ project no 95.

REFERENCES

de Bruijn, F.J. 1992. Use of repetitive (Repetitive Extragenic Palindromic and Enterobacterial Repetitive Intergenic Consensus) Sequences and the polymerase chain reaction to fingerprint the genomes of Rhizobium meliloti isolates and other soil bacteria. Applied and Environmental Microbiology, 58, 2180-2187.

Versalovic, J., Koeuth, T. & Lupski, J.R. 1991. Distribution of repetitive DNA sequences in eubacteria and applications to fingerprinting of bacterial genomes. Nucleic Acids Research, 19, 6823-6831.