The role of helper bacteria in facilitating mycorrhization of Biserrula pelecinus L., a pasture legume new to Australia

Arbuscular mycorrhizas (AM) promote the growth of their host plants by enhancing nutrient uptake and conferring resistance to biotic and abiotic stresses. Consequently, stimulation of AM colonisation represents a promising means by which plant productivity in agriculture may be increased. A substantial body of evidence now supports the contention that certain rhizosphere bacteria can act to promote the formation and persistence of mycorrhizas. These mycorrhiza helper bacteria (MHBs) have been observed both to promote the pre-symbiotic development of mycorrhizal fungi in vitro and to increase the rate of root colonisation in vivo. To date, MHBs have been isolated from a relatively restricted range of AM associations and scope exists to investigate their role in other plant systems. The principal aim of this research project was to determine whether rhizobacteria associated with Biserrula pelecinus L. (biserrula), a pasture legume selected for use in low-rainfall and high-stress agriculture, are capable of promoting mycorrhizal colonisation in that host. Subsidiary aims included (1) establishing whether this helper effect persists in non-sterile conditions; (2) identifying potential mechanisms involved in the helper effect; and (3) identifying constraints on the implementation of these helpers in terms of their survival in the rhizosphere.
Microscopic analysis of biserrula plants grown in pastures and in synthetic trap cultures revealed low levels of mycorrhization (~20%) relative to functionally equivalent pasture species. Genetic diversity profiling targeting the fungal internal transcribed spacer (ITS) region suggested that Funneliformis mosseae may be the principal, though not exclusive, fungal partner in field conditions. Subsequent isolation and screening of plant-associated bacteria identified six taxa capable of stimulating in vivo mycorrhization of biserrula by F. mosseae, belonging to the genera Pseudomonas, Bacillus, Microbacterium, and Arthrobacter. Two strains of Pseudomonas were observed to facilitate low levels of sporulation by F. mosseae in the absence of a plant host, challenging the conventional description of AM fungi as obligate symbionts.

No single functional trait could be identified to account for the observed helper effects; however, the involvement of the common symbiosis pathway (CSP) was suggested by the observation that a Pseudomonas koreensis subgroup taxon fostered nodule formation in biserrula in axenic conditions. Experimental co-inoculation of MHBs with a commercial rhizobium did not identify any synergistic interactions but did identify Mesorhizobium ciceri bv. biserrulae WSM1497 as an MHB strain. Inoculation of MHBs into non-sterile soil demonstrated that the helper effect could be diminished, in some cases, by microbial competition. A potential synergy was observed between the P. koreensis subgroup taxon and a Bacillus cereus group taxon in promoting pre-symbiotic growth of F. mosseae.
While this project was successful in identifying bacterial taxa capable of acting as MHB strains in both sterile and non-sterile soil, obstacles to the practical implementation of these microorganisms remain. Diversity profiling revealed that the rhizosphere of biserrula is highly variable; this, combined with the observed reduction in the magnitude of certain helper effects in non-sterile conditions, and the suppression of taxa closely related to identified MHB strains, suggests that successful implementation of MHBs in agriculture may necessitate management of the whole rhizosphere. This, in turn, is likely to entail careful implementation of agricultural rotations.