CARACTERIZACIÓN DEL GRÁNULO DE CAUCHO (GCR)

MONIKA M.MESSMER1,FLORIAN HERTENSTEIN1,ESTELLE BERSET1,CHRISTIANE BALKO2,WERNER VOGT-KAUTE3,KLAUS-PETER

WILBOIS4

1 _{Research Institute of Organic Agriculture (FiBL), Switzerland, www.fibl.org, [email protected]} 2 _{Julius Kühn Institute (JKI) Groß Lüsewitz, Germany, www.jki.bund.de, [email protected]} 3 _{Naturland e.V., Germany, Germany, www.naturland.de, [email protected]}

4 _{FiBL Deutschland e.V., Germany, www.fibl.org, [email protected]} Key words: soybean, Bradyrhizobium, cold tolerance, N fixation, symbiosis

Summary: Protein yield of soybean depends heavily on successful symbiosis with Bradyrhizobium bacteria able to fix atmospheric nitrogen. Low temperature limits the growth of soybeans but also the efficiency of biological nitrogen fixation (BNF). Cold tolerant Bradyrhizobium strains could be identified, and significant differences in BNF were found for Bradyrhizobium and for soybean genotypes allowing selection for improved BNF. Significant soybean x Bradyrhizobium interactions were found in mesocosm and field trials, as well as complex temperature dependent interactions with mycorrhiza strains. More studies are needed to elucidate the important plant – rhizosphere interactions.

Background

Soybean (Glycine max (L.) Merr.) is the most important protein source for human food and animal feed worldwide, however Europe is largely dependent on imports from Americas and Asia. Soybean production in Central Europe is limited by short season and low temperature as a single night of 8°C is sufficient to inhibit pod formation (Strauss et al. 2006). Very early (maturity group 000 to 00) and cold tolerant soybean genotypes are required for cultivation in Germany and Switzerland (Gass et al. 1996). In order to produce high protein content, soybean plants depend on their symbiotic bacteria Bradyrhizobium japonicum which allow the fixation of atmospheric nitrogen (N2). As Bradyrhizobia are not endemic to European soils the soybean seeds

need to be inoculated before sowing. Low root zone temperature (RZT), is also a major limiting factor for biological nitrogen fixation (BNF) in short season production areas (Zhang et al., 2003). Therefore, research efforts to improve BNF need to be applied to both soybean and Bradyrhizobia (Keyser and Li, 1992). While several breeding programs focus on selecting for cold tolerant soybean genotypes, little attention has been given so far to the selection of Bradyrhizobium strains adapted to European cool growing conditions. This study aims at improving the BNF of soybeans by optimizing plant – microbe interaction under low temperature and its implementation for plant breeding.

Main Chapter

Yield stability and protein content of soybean production under cool growing conditions of Europe shall be improved by increasing BNF through optimizing soybean – Bradyrhizobium symbiosis. The objectives of the study are (i) to select Bradyrhizobium strains adapted to cool growing conditions, (ii) to identify optimal soybean x Bradyrhizobium combinations and (iii) to quantify the impact of triplex interactions with mycorrhiza and plant growth promoting rhizobacteria (PGPR).

In microcosm trials twelve different Bradyrhizobium inoculants and one inactivated control were tested on three early soybean varieties (maturity group 000) at three different temperature regimes (14/10°C; 16/12°C; 22/20°C). The number of nodules, root, and shoot biomass as well as chlorophyll content were assessed after six weeks. The five most promising Bradyrhizobium strains from the microcosm trial were multiplied and were tested on 20 different soybean varieties (maturity group 0000/000 to 00) under 16/12°C temperature regime for 6 weeks. The same traits were assessed. In addition, nitrogen content and δ15_{Nair of shoots and seeds were determined by the} 15_{N natural abundance method. The percentage of BNF was calculated according to}

Unkovich et al. (2008). In parallel we tested the effect of co-inoculation of two different mycorrhiza strains and two Bradyrhizobium strains on one soybean cultivar under two different temperature regimes (16/12°C; 20/20°C). In addition, four Bradyrhizobium strains were tested on two soybean varieties in mesocosm trials to assess their impact on chilling tolerance during flowering. The number of nodules, chlorophyll content, number of pods and seeds on main and side branches were assessed. To verify the results of the mesocosm trials, on-farm field trials at two sites were conducted in 2012 and 2013 under organic growing conditions in the middle and North of Germany. The number of nodules was assessed six weeks after sowing and at beginning of flowering. Yield, thousand kernel weight and protein content were assessed at soybean harvest.

In mesocosm trials we found significant Bradyrhizobium x soybean variety interactions at 14/10°C and at 16/12°C. At 14/10°C the highest number of nodules was obtained with the strain USDA 30 and the variety Protina (8.5 nodules per plant), whereas at 16/12°C one commercial product HiStick yielded highest number of nodules with the variety Bohemians (21.2 nodules per plant). The 20 soybean varieties showed significant variation for number of nodules, total N uptake and N derived from BNF when tested with the selected Bradyrhizobium strains under cool conditions (16/12°C) (Fig. 1).

Figure 1. Total nitrogen (N) uptake, N derived from biological nitrogen fixation (BNF) and number of nodules of the 20 soybean

Significant differences were also found for the different

stage development. Co-inoculation with mycorrhiza resulted either in a significant increase or decrease of nodules depending on the used mycorrhiza strain, the Bradyrhizobium strain and the tested temperature, demons

strains on chilling tolerance during flowering was of minor importance. However, significant

tolerant USDA 30 strain resulted in higher number of pods per side branch as compensation to the chilling stress, whereas without cold stress the commercial product HiStick showed the best performance.

Under organically managed field conditions the Bradyrhizobium

Bradyrhizobium interactions were found for protein content at both sites. Best combination was Protina x Legumefix and Merlin x Biodoz at si Protina x Legumefix and Merlin x Celltech at site 2. Across varieties, Biodoz revealed highest number of nodules per plant, followed by USDA 30, whereas HiStick consistently showed lowest number of nodules at these two stress environments.

This study shows that the early development of soybeans depends to a great extent on the soybean variety, the temperature. Interactions between soybean variety and

field trials. The natural abundance method is a powerful tool to assess the BNF under different growing conditions and can be soybean genotypes as well as Bradyrhizobium strains with improve BNF. Further studies are needed to elucidat

order to identify optimal co-inoculations with mycorrhiza and plant growth promoting rhizobacteria to support sustainable soybean production in Europe. Acknowledgement

This research project was funded by the German Federal Ministry of Food, Agriculture and Consumer Protection under the program for organic and other sustainable agricultural systems (BÖLN) „Expansion of soybean cultivation in Germany through adaptation by breeding as well a

production and processing technology“ (www.sojainfo.de), the Mahle Stiftung GmbH in Stuttgart, Germany, and the Swiss Agency for D Cooperation, Government of Switzerland under the Indo

researchers who contributed to this project.

This research received funding from the European Community's Seventh Framework Programme (FP7/2007 n°245058-SOLIBAM

References

Gass, T., Schori, A., Fossati, A., Soldati, A., Stamp, P., 1996. Cold tolerance of soybean (Glycine max (L) Merr) during the reproductive phase. Eu Agronomy 5: 71-88

Keyser, H. H., F. D. Li, 1992. Potential for Increasing Biological Nitrogen

Strauss, A.J., Krüger, G.H.J., Strasser, R.J., Van Heerden, P.D.R, 2006. Ranking of dark chilling tolerance in soybean genoty transient O-J-I-P. Environmental and Experimental Botany

Unkovich, M., H. D. F., Peoples, M. B., Cadish, G., Boddey, B., Giller, K., Alves, B., Chalk, P., 2008. Measuring plant Australian Centre for International Agricultural Research (ACIAR).

Zhang, H., Prithiviraj, B., Charles, T. C., Driscoll, B. T., Smith, D. L., 2003: Low temperature tolerant Bradyrhizobium japonicu nitrogen fixation of soybean in a short season (cool spring) area. European Journal of A

igure 1. Total nitrogen (N) uptake, N derived from biological nitrogen fixation (BNF) and number of nodules of the 20 soybean varieties tested against five Bradyrhizobi strains at 16/12°C

Significant differences were also found for the different Bradyrhizobium strains, but no Bradyrhizobium x variety interaction was detected at this early inoculation with mycorrhiza resulted either in a significant increase or decrease of nodules depending on the used mycorrhiza

strain and the tested temperature, demonstrating the complex interaction in the rhizosphere. The impact of

strains on chilling tolerance during flowering was of minor importance. However, significant Bradyrhizobium x temperature interaction occurred. The cold n resulted in higher number of pods per side branch as compensation to the chilling stress, whereas without cold stress the commercial product HiStick showed the best performance.

Bradyrhizobium strains had a significant effect on all assessed traits. Significant soybean variety x interactions were found for protein content at both sites. Best combination was Protina x Legumefix and Merlin x Biodoz at si

and Merlin x Celltech at site 2. Across varieties, Biodoz revealed highest number of nodules per plant, followed by USDA 30, whereas HiStick consistently showed lowest number of nodules at these two stress environments.

lopment of soybeans depends to a great extent on the soybean variety, the Bradyrhizobium

temperature. Interactions between soybean variety and Bradyrhizobium strains are of main importance under cool growing conditions in mesocosm and ield trials. The natural abundance method is a powerful tool to assess the BNF under different growing conditions and can be

strains with improve BNF. Further studies are needed to elucidate the complex rhizosphere interactions in inoculations with mycorrhiza and plant growth promoting rhizobacteria to support sustainable soybean production in Europe.

Federal Ministry of Food, Agriculture and Consumer Protection under the program for organic and other sustainable agricultural systems (BÖLN) „Expansion of soybean cultivation in Germany through adaptation by breeding as well a

ction and processing technology“ (www.sojainfo.de), the Mahle Stiftung GmbH in Stuttgart, Germany, and the Swiss Agency for D Cooperation, Government of Switzerland under the Indo-Swiss Collaboration in Biotechnology (ISCB). We thank all farm

This research received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under the Grant Agreement

Fossati, A., Soldati, A., Stamp, P., 1996. Cold tolerance of soybean (Glycine max (L) Merr) during the reproductive phase. Eu Keyser, H. H., F. D. Li, 1992. Potential for Increasing Biological Nitrogen-fixation in soybean. Plant Soil 141:119–135.

Strauss, A.J., Krüger, G.H.J., Strasser, R.J., Van Heerden, P.D.R, 2006. Ranking of dark chilling tolerance in soybean genotypes probed by the chlorophyll a fluorescence Environmental and Experimental Botany 56: 147–157

Unkovich, M., H. D. F., Peoples, M. B., Cadish, G., Boddey, B., Giller, K., Alves, B., Chalk, P., 2008. Measuring plant-associated nitrogen fixation in agricultural systems. Australian Centre for International Agricultural Research (ACIAR).

g, H., Prithiviraj, B., Charles, T. C., Driscoll, B. T., Smith, D. L., 2003: Low temperature tolerant Bradyrhizobium japonicum strains allowing improved nodulation and nitrogen fixation of soybean in a short season (cool spring) area. European Journal of Agronomy 19: 205-213.

varieties tested against five Bradyrhizobia

interaction was detected at this early inoculation with mycorrhiza resulted either in a significant increase or decrease of nodules depending on the used mycorrhiza trating the complex interaction in the rhizosphere. The impact of Bradyrhizobium x temperature interaction occurred. The cold n resulted in higher number of pods per side branch as compensation to the chilling stress, whereas without cold stress the

strains had a significant effect on all assessed traits. Significant soybean variety x interactions were found for protein content at both sites. Best combination was Protina x Legumefix and Merlin x Biodoz at site 1 and and Merlin x Celltech at site 2. Across varieties, Biodoz revealed highest number of nodules per plant, followed by USDA 30,

Bradyrhizobium inoculation and the strains are of main importance under cool growing conditions in mesocosm and ield trials. The natural abundance method is a powerful tool to assess the BNF under different growing conditions and can be applied to select both e the complex rhizosphere interactions in inoculations with mycorrhiza and plant growth promoting rhizobacteria to support sustainable soybean production in Europe.

Federal Ministry of Food, Agriculture and Consumer Protection under the program for organic and other sustainable agricultural systems (BÖLN) „Expansion of soybean cultivation in Germany through adaptation by breeding as well as optimization of crop ction and processing technology“ (www.sojainfo.de), the Mahle Stiftung GmbH in Stuttgart, Germany, and the Swiss Agency for Development and Swiss Collaboration in Biotechnology (ISCB). We thank all farmers,advisers, technicians and 2013) under the Grant Agreement

Fossati, A., Soldati, A., Stamp, P., 1996. Cold tolerance of soybean (Glycine max (L) Merr) during the reproductive phase. European Journal of

pes probed by the chlorophyll a fluorescence associated nitrogen fixation in agricultural systems. m strains allowing improved nodulation and

41

Table 1: Genotypes and morphological traits

genotype source leaf type and

flower color Plant height [cm] 44F1 line sc 160 A1 line sw 120 A4 line sw 130 C1 line sw 130 C3 line sw 120 D6 line sw 130 D7 line sw 125 EFB33 variety nc 150

Griechische gen. res nc 140

L1 line nc 135

Nischkes gen. res nc 150

P1 line nc 60

Würtemberg gen. res nc 135

I1 line nw 125

I3 line nw 125

Q2 line nw 130

s = semileafless; n = normal leaf; c = coloured flower; w = white flower

Session A Oral presentation