Lolium perenne (perennial ryegrass) is a common temperate pasture grass species and is favoured by dairy farmers. Commercially, the known association with the mutualistic endophytic fungus Epichloë festucae subsp. lolii is used to enhance insect resistance of host plants. Knowledge of other members of the microbiome and their functions are limited. Sequencing of the V4 region of the 16S rRNA gene was used to examine the bacterial microbiome of perennial ryegrass (cv. Alto) seeds (Generation 1, G1), subsequent mature plants grown in soil and sand, and seed (Generation 2, G2) from crosses of these mature plants. The G1 microbiome was dominated by the Gammaproteobacteria. The mature plant microbiomes were far more diverse, comprising of up to 37 Classes inclusive of Gammaproteobacteria. Different growth media yielded different microbiome profiles in mature plants. The G2 microbiome, as G1, was dominated by Gammaproteobacteria with additional supplementation of Bacilli. This suggests the continuation of a core microbiome which persists from seed, through plant maturation, to seed. This study sheds new light on the hereditability of perennial ryegrass microbiomes and has identified some OTUs of potential commercial significance due to their seed transmissibility and their roles are currently being explored.

Development of grass-endophyte associations with minimal or no detrimental effects in combination with beneficial characteristics is important for pastoral agriculture. The feasibility of enhancing production of an endophyte-derived beneficial alkaloid through introduction of an additional gene copy was assessed in a proof-of-concept study. Sexual and asexual Epichloë species that form symbiotic associations with cool-season grasses of the Poaceae sub-family Pooideae produce bioactive alkaloids that confer resistance to herbivory by a number of organisms. Of these, peramine is thought to be crucial for protection of perennial ryegrass (Lolium perenne L.) from the Argentinian stem weevil, an economically important exotic pest in New Zealand, contributing significantly to pasture persistence. A single gene (perA) has been identified as solely responsible for peramine biosynthesis and is distributed widely across Epichloë taxa. In the present study, a functional copy of the perA gene was introduced into three recipient endophyte genomes by Agrobacterium tumefaciens-mediated transformation. The target strains included some that do not produce peramine, and others containing different perA gene copies. Mitotically stable transformants generated from all three endophyte strains were able to produce peramine in culture and in planta at variable levels. In summary, this study provides an insight into the potential for artificial combinations of alkaloid biosynthesis in a single endophyte strain through transgenesis, as well as the possibility of using novel genome editing techniques to edit the perA gene of non-peramine producing strains.

Background: The Fusarium oxysporum species complex (FOSC) is a ubiquitous group of fungal species readily isolated from agroecosystem and natural ecosystem soils which includes important plant and human pathogens. Genetic relatedness within the complex has been studied by sequencing either the genes or the barcoding gene regions within those genes. Phylogenetic analyses have demonstrated a great deal of diversity which is reflected in the differing number of clades identified: three, five and eight. Genetic limitation within the species in the complex has been studied through Genealogical Concordance Phylogenetic Species Recognition (GCPSR) analyses with varying number of phylogenetic 'species' identified ranging from two to 21. Such differing views have continued to confuse users of these taxonomies. Results: The phylogenetic relationships between Australian F. oxysporum isolates from both natural and agricultural ecosystems were determined using three datasets: whole genome, nuclear genes, and mitochondrial genome sequences. The phylogenies were concordant except for three isolates. There were three concordant clades from all the phylogenies suggesting similar evolutionary history for mitochondrial genome and nuclear genes for the isolates in these three clades. Applying a multispecies coalescent (MSC) model on the eight single copy nuclear protein coding genes from the nuclear gene dataset concluded that the three concordant clades correspond to three phylogenetic species within the FOSC. There was 100% posterior probability support for the formation of three species within the FOSC. This is the first report of using the MSC model to estimate species within the F. oxysporum species complex. The findings from this study were compared with previously published phylogenetics and species delimitation studies. Conclusion: Phylogenetic analyses using three different gene datasets from Australian F. oxysporum isolates have all supported the formation of three major clades which delineated into three species. Species 2 (Clade 3) may be called F. oxysporum as it contains the neotype for F. oxysporum.