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Climate change is predicted to increase the incidence and severity of drought conditions, posing a significant challenge for agriculture globally. Plant microbiomes have been demonstrated to aid crop species in the mitigation of drought stress. The study investigated the differences between the seed microbiomes of drought tolerant and drought susceptible wheat lines. Furthermore, it highlighted and quantified the degree of drought tolerance conferred by specific microbes isolated from drought tolerant wheat seed microbiomes. Metagenomic and culture-based methods were used to profile and characterise the seed microbiome composition of drought tolerant and drought susceptible wheat lines under rainfed and drought conditions. Isolates from certain genera were enriched by drought tolerant wheat lines when placed under drought stress. Wheat inoculated with isolates from these targeted genera, such as Curtobacterium flaccumfaciens (Cf D3-25) and Arthrobacter sp. (Ar sp. D4-14) demonstrated the ability to promote growth under drought conditions. This study indicates seed microbiomes from genetically distinct wheat lines enrich for beneficial bacteria in ways that are both line-specific and responsive to environmental stress. As such, seed from stress-phenotyped lines represent an invaluable resource for the identification of beneficial microbes with plant growth promoting activity that could improve commercial crop production.

Additional file 2. Figure S1. MALDI-TOF MS spectra generated from a subset of bacteria isolated from G. clandestina and G. max seeds. The bacterial isolates were identified using an in-house endophyte library and Bruker database. The isolates selected for WGS are labelled next to the representative clade.

Additional file 1. Table S1. Accession ID, hosts and length of H3-D region of the sequences used for constructing ML tree. Table S2. Shannon diversity indices of seed samples grouped by plant species and seed accession. Table S3. The Kruskal Wallis pairwise test results calculating significant differences between the bacterial diversity associated with G. clandestina and G. max seed when samples are grouped based on “Plant Species” and “Seed Accession”. Table S4. PERMANOVA and PERMDISP results calculating significant differences in bacterial composition associated with G. clandestina and G. max when data was grouped based on “Plant Species” and “Seed Accession”. Table S5. The relative abundance of bacterial genera associated with both “Plant species”. Taxa occurring at > 0.1% are highlighted in bold. Table S6. The relative abundance of bacterial genera associated with different G. clandestina seed accessions. Taxa occurring at > 0.1% are highlighted in bold. Table S7. The relative abundance of bacterial genera associated with different G. max seed accessions. Taxa occurring at > 0.1% are highlighted in bold. Table S8. Closest taxonomy ID identified by Kraken2 for the isolated bacterial and fungal sequences with > 96% similarity along with the source of seed accessions. Genome sequences for fungal and bacterial isolates are described under the NCBI BioProjectID PRJNA807720 and PRJNA807698. Figure S1. Map highlighting the sites (in yellow) selected for G. clandestina seed collection in the Greater Melbourne region, Victoria. Figure S2. G. clandestina (A) and G. max (B) seedlings at the unfolded cotyledon growth stage. Figure S3. Maximum Likelihood Consensus Tree with a bootstrap node support of 70% was inferred from the histone H3-D gene sequences of six Glycine taxa used in this study and the 17 references sequences retrieved from NCBI using MEGA X with 1000 bootstrap replications. The best nucleotide substitution model, the Tamura–Nei model, was used. The ten phylogenetic clades identified within the tree are highlighted in different shades. Sequences reading down (light green, dark green, light blue, yellow, grey, orange, sky blue, violet red and dark red) belong to clades 1 through to 10, respectively. Figure S4. The relative abundance of bacterial communities across Glycine seeds at phylum level based on “Plant Species” (A), and “Seed Accession” (B, C).

Lolium perenne (perennial ryegrass) is a common temperate pasture grass species and is favored 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 ribosomal RNA gene was used to examine the bacterial microbiome of perennial ryegrass (Alto) seed (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 class Gammaproteobacteria. The mature plant microbiomes were far more diverse, comprising up to 37 classes inclusive of Gammaproteobacteria. Different growth media yielded different microbiome profiles in mature plants. The G2 microbiome, similar to the G1 microbiome, was dominated by Gammaproteobacteria as the primary constituent, with additional supplementation from class 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 bacterial microbiomes and has identified some operational taxonomic units of potential commercial significance due to their seed transmissibility, and their roles are currently being explored.

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.

A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Applied Systems Biology, College of Science, Health and Engineering, La Trobe University, Victoria, Australia.

Climate change is predicted to increase the incidence and severity of drought conditions, posing a significant challenge for agriculture globally. Plant microbiomes have been demonstrated to aid crop species mitigate drought stress. In this study the wheat seed microbiomes from lines with contrasting drought tolerances were interrogated for microbes that alleviate drought stress. Metagenomic and culture-based methods were used to profile and characterise the seed microbiome composition of four drought tolerant and three drought susceptible wheat lines under rainfed and drought conditions. Curtobacterium flaccumfaciens (Cf D3-25) and Arthrobacter sp. (Ar sp. D4-14) were isolates enriched in drought tolerant lines under drought conditions and demonstrated the ability to promote wheat growth under drought conditions. Members of Triticeae inoculated with Cf D3-25 and Ar sp. D4-14 showed a biostimulation affect. This study indicates seed microbiomes from genetically distinct wheat lines enrich for beneficial bacteria in ways that are both line-specific and responsive to environmental stress. As such, seed from stress-phenotyped lines represents an invaluable resource for the identification of beneficial microbes with plant growth promoting activity that could improve commercial crop production, particularly under drought stress.