Epoxy-janthitrems are a class of indole diterpenes with structural similarity to lolitrem B. Two taxa of asexual Epichloë endophytes have been reported to produce epoxy-janthitrems, LpTG-3 (Lolium perenne Taxonomic Group 3; e.g., NEA12) and LpTG-4 (e.g., E1). Epichloë epoxy-janthitrems are not well understood, the biosynthetic pathway and associated gene complement have not been described and while the literature suggests they are associated with superior protection against pasture insect pests and are tremorgenic in grazing mammals, these properties have not been confirmed using isolated and purified compounds. Whole genome sequence analysis was used to identify candidate genes for epoxy-janthitrem biosynthesis that are unique to epoxy-janthitrem producing strains of Epichloë. A gene, jtmD, was identified with homology to aromatic prenyl transferases involved in synthesis of indole diterpenes. The location of the epoxy-janthitrem biosynthesis gene cluster (JTM locus) was determined in the assembled nuclear genomes of NEA12 and E1. The JTM locus contains cluster 1 and cluster 2 of the lolitrem B biosynthesis gene cluster (LTM locus), as well as four genes jtmD, jtmO, jtm01, and jtm02 that are unique to Epichloë spp. that produce epoxy-janthitrems. Expression of each of the genes identified was confirmed using transcriptome analysis of perennial ryegrass-NEA12 and perennial ryegrass-E1 symbiota. Sequence analysis confirmed the genes are functionally similar to those involved in biosynthesis of related indole diterpene compounds. RNAi silencing of jtmD and in planta assessment in host-endophyte associations confirms the role of jtmD in epoxy-janthitrem production. Using LCMS/MS technologies, a biosynthetic pathway for the production of epoxy-janthitrems I–IV in Epichloë endophytes is proposed.

Plant growth-promoting bacteria can improve host plant traits including nutrient uptake and metabolism and tolerance to biotic and abiotic stresses. Understanding the molecular basis of plant–bacteria interactions using dual RNA-seq analyses provides key knowledge of both host and bacteria simultaneously, leading to future enhancements of beneficial interactions. In this study, dual RNA-seq analyses were performed to provide insights into the early-stage interactions between barley seedlings and three novel bacterial strains (two Paenibacillus sp. strains and one Erwinia gerundensis strain) isolated from the perennial ryegrass seed microbiome. Differentially expressed bacterial and barley genes/transcripts involved in plant–bacteria interactions were identified, with varying species- and strain-specific responses. Overall, transcriptome profiles suggested that all three strains improved stress response, signal transduction, and nutrient uptake and metabolism of barley seedlings. Results also suggested potential improvements in seedling root growth via repressing ethylene biosynthesis in roots. Bacterial secondary metabolite gene clusters producing compounds that are potentially associated with interactions with the barley endophytic microbiome and associated with stress tolerance of plants under nutrient limiting conditions were also identified. The results of this study provided the molecular basis of plant growth-promoting activities of three novel bacterial strains in barley, laid a solid foundation for the future development of these three bacterial strains as biofertilisers, and identified key differences between bacterial strains of the same species in their responses to plants.

Surveillance programs are essential for the prevention and control of mosquito-borne arboviruses that cause serious human and animal diseases. Viral metatranscriptomic sequencing can enhance surveillance by enabling untargeted, high-throughput arbovirus detection. We used metatranscriptomic sequencing to screen field-collected mosquitoes for arboviruses to better understand how metatranscriptomics can be utilised in routine surveillance. Following a significant flood event in 2016, more than 56,000 mosquitoes were collected over seven weeks from field traps set up in Victoria, Australia. The traps were split into samples of 1000 mosquitoes or less and sequenced on the Illumina HiSeq. Five arboviruses relevant to public health (Ross River virus, Sindbis virus, Trubanaman virus, Umatilla virus, and Wongorr virus) were detected a total of 33 times in the metatranscriptomic data, with 94% confirmed using reverse transcription quantitative PCR (RT-qPCR). Analysis of metatranscriptomic cytochrome oxidase I (COI) sequences enabled the detection of 12 mosquito and two biting midge species. Screening of the same traps by an established public health arbovirus surveillance program corroborated the metatranscriptomic arbovirus and mosquito species detections. Assembly of genome sequences from the metatranscriptomic data also led to the detection of 51 insect-specific viruses, both known and previously undescribed, and allowed phylogenetic comparison to past strains. We have demonstrated how metatranscriptomics can enhance surveillance by enabling untargeted arbovirus detection, providing genomic epidemiological data, and simultaneously identifying vector species from large, unsorted mosquito traps.

Seed-associated microbiomes represent an underexplored frontier in synthetic community (SynCom) design, particularly in forage legumes such as lucerne (Medicago sativa L.), where early microbial assembly can shape plant development. Crop wild relatives (CWRs) harbour more diverse seed microbiomes and may contain microbes with greater functional potential than domesticated lucerne. To test this, SynComs were constructed from seed-borne bacteria isolated from M. laciniata (drought-resilient) and M. littoralis (salt-tolerant). Two three-strain SynComs were assembled from taxa consistently shared across lucerne and its CWRs, and a third six-strain ‘Mix’ SynCom combined both sets. The aim of this study was to assess whether these SynComs exert phenotypic effects on lucerne growth when used as seed inocula alongside the native microbiome during early development and later vegetative stages under well-watered and drought conditions. Inoculation enhanced germination and early growth, with the Mix SynCom producing the strongest gains. Microbiome profiling at 24 days revealed treatment-specific restructuring, with enrichment of beneficial taxa and microbial coalescence. While early-stage benefits diminished at later stages, and drought ultimately reduced biomass across all treatments, the findings demonstrate that CWR-derived SynComs can enhance lucerne establishment and early growth while restructuring host microbiomes, providing a framework for seed-applied microbial solutions in sustainable agriculture.

Research into the bacterial component of the seed microbiome has been intensifying, with the aim of understanding its structure and potential for exploitation. We previously studied the intergenerational seed microbiome of one cultivar of perennial ryegrass with and without one strain of the commercially deployed fungal endophyte Epichloë festucae var. lolii. The work described here expands on our previous study by exploring the bacterial seed microbiome of different commercial cultivar/Epichloë festucae var. lolii combinations in collections of single seeds from the harvest year 2016. In this dataset, a cultivar effect could be seen between the seed microbiomes from cultivars Alto and Trojan. The bacterial component of the seed microbiome from pooled seeds from a single cultivar/E. festucae var. lolii combination harvested from 13 seed production farms around Canterbury in the year 2018 was also studied. This dataset allows the effect of different production locations on the bacterial seed microbiome to be examined. By comparing the two sets of data, bacteria from the genera Pantoea, Pseudomonas, Duganella, Massilia, and an unknown Enterobacteriaceae were observed to be in common. This core bacterial microbiome was stable over time but could be affected by supplemental taxa derived from the growth environment of the parental plant; differing microbiomes were seen between different seed production farms. By comparison to a collection of bacterial isolates, we demonstrated that many of the members of the core microbiome were culturable. This allows for the possibility of exploiting these microbes in the future.

Asexual Epichloë fungi are strictly seed-transmitted endophytic symbionts of cool-season grasses and spend their entire life cycle within the host plant. Endophyte infection can confer protective benefits to its host through the production of bioprotective compounds. Inversely, plants provide nourishment and shelter to the resident endophyte in return. Current understanding of the changes in global gene expression of asexual Epichloë endophytes during the early stages of host-endophyte symbiotum is limited. A time-course study using a deep RNA-sequencing approach was performed at six stages of germination, using seeds infected with one of three endophyte strains belonging to different representative taxa. Analysis of the most abundantly expressed endophyte genes identified that most were predicted to have a role in stress and defence responses. The number of differentially expressed genes observed at early time points was greater than those detected at later time points, suggesting an active transcriptional reprogramming of endophytes at the onset of seed germination. Gene ontology enrichment analysis revealed dynamic changes in global gene expression consistent with the developmental processes of symbiotic relationships. Expression of pathway genes for biosynthesis of key secondary metabolites was studied comprehensively and fuzzy clustering identified some unique expression patterns. Furthermore, comparisons of the transcriptomes from three endophyte strains in planta identified genes unique to each strain, including genes predicted to be associated with secondary metabolism. Findings from this study highlight the importance of better understanding the unique properties of individual endophyte strains and will serve as an excellent resource for future studies of host-endophyte interactions.