Investigating variation in associations with belowground micro-organisms of historic and current white clover germplasm

Abstract

White clover (Trifolium repens) is regarded as one of the most important forage legume in temperate regions of the world, and is very important to the New Zealand pastoral industries. The symbiotic relationship between clover and the soil bacterium Rhizobium leguminosarum biovar trifolii (rhizobia) is the basis of the nitrogen (N) fixation ability of clover. White clover breeding started intensively in the late 1920’s in New Zealand. Clover breeding programmes have largely focused on above ground characteristics in selection of new cultivars. This has usually been carried out in high soil nitrogen environments, due to fertilizer use. This is the first study to investigate whether plant breeding may have impacted on white clover and it’s below ground associations. Six cultivars of white clover were used. Three newer cultivars (Tribute, Kopu II, Crusader), and three older cultivars (Huia, Irrigation, Louisiana) which were paired based on physiological characteristics and growth habits. Three major below ground associations were analyzed in this project and these were i) plant interactions with rhizobia to examine whether there is preferential selection of efficacious strains of rhizobia by the older cultivars, ii) interaction with arbuscular mycorrhizal fungi (AMF) and the responsiveness of clover cultivars to different species of AMF, iii) interaction with the wider rhizosphere bacterial community, to determine whether the cultivars preferentially selected more beneficial associations in a complex soil background. Symbiotic potential assays in vitro showed that the cultivars Irrigation and Louisiana produced significantly different shoot dry weight when inoculated with six different strains of rhizobia (P=0.038, and P=0.013, respectively). Cultivars Huia and Tribute showed strong trends (P=0.052, and P=0.059, respectively). Based on plant dry weight, rhizobia groups of “highly effective” and “less effective” strains were identified for these four cultivars and used to examine if there was preferential selection of highly effective strains. Out of the four cultivars, only Huia and Louisiana had significantly more “highly effective” and significantly less “less effective” rhizobia strains occupying their nodules than was expected by chance (P<0.001). This was the first study to examine the bacterial rhizosphere and root endophyte communities of old and new cultivars of white clover. 16S gene sequencing using Illumina HiSeq showed that the bacterial communities in the root and rhizosphere were significantly different from each other (PERMANOVA, P=0.001) in richness and diversity. Sequencing also revealed that plant morphotype was the main factor influencing bacterial community structure in the rhizosphere and roots of white clover, as cluster analysis showed that the samples grouped together according to their leaf size (PERMANOVA, P=0.001) and not by cultivar age. Illumina sequencing also showed there were differences among the pairs of cultivars in relation to the most abundant class observed. Alphaproteobacteria (42.3-52.6%) was the most abundant class for old/new matched pairs Irrigation/Crusader, and Louisiana/Kopu II, whereas the most abundant class for matched pairs Huia/Tribute was the Gammaproteobacteria (32.8%). DGGE showed that cultivar significantly affected AMF community structure in the rhizosphere (PERMANOVA P=0.006). However, there was high variation among replicates of the same cultivar (P<0.05), and principal coordinate analysis did not show any grouping by cultivar. Overall this study revealed that strains of R. leguminosarum vary in their efficacy when in symbiosis with different cultivars. The results suggested that older cultivars may be better able to select for more effective strains of rhizobia than newer cultivars, and may be able to exploit a wider range of rhizobia. This study also identified that the main factor affecting the bacterial rhizosphere and root communities of white clover was cultivar morphotype.