Utilizing novel winter faba bean and white clover genotypes in arable and grassland mixtures

In Germany, there is only one winter faba bean cultivar available on the market (cv. Hiverna).

White clover in contrast, has 18 cultivars in the official German testing trials (Bundessortenamt 2018). However, these white clover cultivars are not tested in mixture but only in pure stands. In order to investigate whether the cultivar has an effect on the mixture performance, a wide range of genotypes is required. Therefore, seed material of the present study originated from breeding initiatives from the Georg-August-University of Goettingen, Norddeutsche Pflanzenzucht (NPZ, winter faba bean) and the Deutsche Saatveredelung (DSV, white clover). A major advantage of the available seed material of the present study is that it is pretested and phenotyped. Clover seed material was already pretested for its mixture performance by the DSV.

Faba bean cultivars demonstrate a large genetic variability in aboveground properties such as shoot biomass and grain yield (Link et al. 2010; Neugschwandtner et al. 2015); knowledge about genetic variability in root traits is however limited. Differences in root traits between faba bean accessions were observed by Belachew et al. (2018), Grzesiak et al. (1997), Khan et al. (2010) and Zhao et al. (2017). Even though the authors found differences in rooting depth, total root length, tap root length or lateral root length, they cannot provide information about these accessions in mixtures or under field conditions.

By using FTIR spectroscopy, we could show in the present study that the eight faba bean genotypes differed in their horizontal and vertical root distribution in mixtures with wheat (Chapter 3). Faba bean genotypes Vf5 and Vf6 in mixture had a high horizontal spread into the wheat row and higher root fractions in deep soil layers, respectively (Fig. 2). If the other genotypes are taken into account, a slight tradeoff between these two traits is noticeable:

Genotypes with higher root fractions in deeper soil layers tend to have a lower spread into the wheat row and lower root biomasses on that sampling position. The relation between a high horizontal spread into the wheat row and deeper vertical root distribution was characterized by a weak negative correlation (R²=-0.28). Genotypes Vf5 and Vf6 most likely differ in their belowground competitive ability. The downward directed avoidance strategy of Vf5 under the wheat row reduces interspecific competition. Simultaneously, the water and nutrient acquisition from deeper soil layers might be enhanced for this particular genotype in mixtures.

Greater rooting depths and root lengths might contribute to an advantage in drought tolerance (Grzesiak et al. 1997; Khan et al. 2010). The number of lateral roots and the overall proportion

108 of lateral roots within the root system are important for deep soil foraging of faba bean (Zhao et al. 2017).

Moreover, the results indicated that the change in vertical root distribution of wheat in presence of faba bean depends on the bean genotype (Chapter 3): we could observe that wheat on its own row reacted to the presence of faba bean genotypes 4, 5 and 8 with a significant increase of root fractions in shallower soil layers compared to wheat pure stand. Contrary, wheat in mixture on its own row with faba bean genotypes 1, 2, 3, 6 and 7 had a similar vertical root distribution as in pure stand. Simultaneously, bean genotypes 4, 5 and 8 were characterized by the highest root fraction in deep soil layers (Chapter 3, Fig. 2). This pattern indicates a spatial niche partitioning for soil space on the wheat row for some particular faba bean/wheat mixtures. Therefore, species complementarity between faba bean and wheat is notably high on the wheat rows for these mixtures.

The present study furthermore demonstrated that the eight faba bean genotypes differed in their above- and belowground overyielding potential at full flowering of faba bean (Chapter 2).

In the evaluation of the eight faba bean genotypes according to their overyielding potential, genotype Vf5 displayed the highest and Vf8 the lowest advantage in the mixture (Fig. 2).

Surprisingly, genotypes Vf5 and Vf8 had a contrasting overyielding potential but a similar root distribution on the wheat row in mixtures. We therefore suggest investigating the effect of spatial root distribution on the overyielding potential of bean/wheat mixtures. As already mentioned, grain yield analyses within the same project (Plant breeding, data not shown) reported consistently (years 2015 and 2016) the highest grain yield advantage of mixtures with genotypes Vf3, Vf2 and Vf5 (Fig. 2). Genotypes Vf8 and Vf6 had the lowest grain RYT.

Based on the results of root distribution and biomass as well as on overyielding potential and grain overyielding potential we consider genotype Vf5 to be the most promising candidate for a improved winter faba bean/winter wheat mixtures.

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Figure 2 Evaluation of the spatial root distribution and overyielding potential of eight winter faba bean (Vf1-8) genotypes in mixture. Shading indicates above- and belowground overyielding potential and consistency of the genotypes at full flowering of bean; dark grey: high, light grey: intermediate, white: low. See Chapter 2 and 3 for details. Italic numbers next to the genotypes indicate their low (1), intermediate (2) or high (3) grain overyielding potential in 2015 and 2016 (data from project Plant Breeding).

The present study furthermore showed that the eight investigated white clover genotypes differed in their root biomass in pure stands but not in mixtures (Chapter 4). Furthermore there were differences in RYT between the clover/ryegrass mixtures (p≤0.1). Similar to the present study, differences in root biomass between white clover genotypes in pure stands were also observed by Caradus (1981), Caradus and Woodfield (1998) and Frankow-Lindberg (1997).

However, similar to the above described faba bean genotypes, the majority of the latter studies were not field based experiments and neither tested the cultivars in mixtures.

Genotype Tr6 had the highest root biomass in mixture and the highest root overyielding (Chapter 4, Table 3). Based on these results we suggest to further research clover genotype Tr6 for its mixture potential. It should be tested the root overyielding of Tr6 in mixture with ryegrass is also evident under several growing seasons and differing sites. Nonetheless, the most important research question is, whether the high root overyielding of Tr6 in mixtures is translated into an aboveground overyielding. Similar to faba bean/wheat mixtures, the high

Vf1 Vf6

Horizontal spread of faba bean into wheat row

Root fraction distribution of faba bean under wheat row (β) High Intermediate Low ShallowIntermediateDeep

110 root overyielding of clover genotype Tr6 could possibly lead to a higher resource acquisition and thus to an increased dry matter yield.

Table 3 Evaluation of the root overyielding potential of the eight white clover genotypes (Tr1-8) in mixture with unfertilized perennial ryegrass (Lp-N0).

Plant breeding programs mainly focus on the development of cultivars for pure stands as they are grown more frequently than mixtures. Mixed cropping systems usually utilize the same cultivars, even though cultivar performance can vary between pure and mixed stands (Carton et al. 2018; Nelson and Robichaux 1997; Neugschwandtner et al. 2015). A significant cultivar x cropping system interaction for pea cultivars in terms of grain yield was observed by Hauggaard-Nielsen and Jensen (2001). This pattern was clearly confirmed by the present study: novel genotypes of bean and clover performed differently in pure stands and in mixtures. In arable land, bean genotype x cropping system interactions were visible for the traits total shoot and root biomass, comparative bean root biomasses and bean root:shoot ratio (Chapter 2). Furthermore, the vertical root distribution of bean differed between pure and mixed stands (Chapter 3). In grassland, the clover genotype x cropping system interaction was also evident for root biomass (Chapter 4). To our knowledge there are no studies available on performance differences between clover genotypes between pure and mixed stands in terms of dry matter yield or root traits. In order to improve mixed cropping systems, the breeding of new legume cultivars should be targeted for these systems. Furthermore, testing of new cultivars should be conducted as field studies to ensure the performance of plants in agronomical environments.

With the present study we were able to show that certain novel bean and clover genotypes possess traits that are advantageous for mixtures with non-legume species. A high root overyielding of certain bean genotypes in bean/wheat mixtures at full flowering of bean was a good indicator for grain overyielding. The superiority of clover genotype Tr6 in root overyielding might possibly be reflected in dry matter mixture advantage. In order to improve the crops water and nutrient efficiency, researchers such as Lynch (2007) and Zhao et al.

Low Intermediate High Tr1/Lp-N0 Tr2/Lp-N0 Tr6/Lp-N0 Tr3/Lp-N0 Tr4/Lp-N0

Tr8/Lp-N0 Tr5/Lp-N0 Tr7/Lp-N0

111 (2017) emphasize that root traits should be included in breeding efforts. Future yield improvements might be dependent upon the progress of research on root system architecture and stress resistance (Den Herder et al. 2010; Koevoets et al. 2016; Lynch 2007). In accordance to the latter studies, we recommend to include research on genotypic differences in root biomass and distribution in the evaluation process of mixed cropping systems.