Linking root chemistry to root associated bacterial and fungal communities: the impact of benzoxazinoid biosynthesis genes

Rhizosphere chemistry is often considered to be one of the main drivers of rhizosphere microbial communities but the genetic control of this remain poorly understood. One group of plant compounds that are particularly likely to be influential in rhizosphere interactions are benzoxazinoids (BXs). These are tryptophan derived, heteroaromatic metabolites with benzoic acid moieties which are produced in large quantities by the roots of the Poaceae including crops such as maize, wheat and rye. Previous studies have shown that BXs can act as important chemical signals within plants. When exuded from roots BXs and their breakdown products are known to be allelopathic to some soil-borne bacteria and fungi, but can also act as recruitment signals for others including some plant growth promoting rhizobacteria. However, the influence of BX biosynthesis genes on overall root chemistry and rhizosphere communities is largely unknown. We utilised newly developed maize (Zea mays) mutants of three genes involved in the production of BXs (bx1, bx2 and bx6) to investigate the impact of these genes on root chemistry and root associated bacterial and fungal communities. Eight pre-germinated seeds of each genotype were planted (1 seed/pot) in substrate consisting of a 3:1 (v:v) mixture of agricultural soil: autoclaved perlite. The constituent soil was collected from an arable field at Spen farm, Leeds (53.874944, -1.320165). After 17 days plants were harvested by carefully removing each plant from the pot and shaking it to remove all but the tightly adhering soil (i.e. rhizosphere soil) from the roots. Plants were then separated into the shoots, crown, primary and seminal roots. Soil samples were obtained from unplanted pots using a 12mm diameter core. Crown and Primary roots and control soil samples were ground and DNA was extracted from them. Bacterial communities were characterised using partial 16S rRNA gene amplification performed using primers 799F and 1193R (Bodenhausen et al., 2013; Chelius and Triplett, 2001) targeting the variable regions V5-V7. Fungal communities were characterized using amplification of partial 5.8S, internal-transcribed spacer 2 (ITS2) and partial 28S rRNA gene fungal fragments using primers fITS7 and ITS4 (Ihrmark et al., 2012; White et al., 1990). After attachment of dual indexes, quantification and pooling, amplicons were sequenced using a paired end 2 x 250 bp cycle kit on a MiSeq machine running v2 chemistry (Illumina Inc, at the Earlham Institute, Norwich, UK).