Culturing the seed microbiome
A total of 438 bacteria were isolated from the seed microbiomes of six wheat lines. At a phylum level, the isolates were identified as either Gammaproteobacteria (42.92%), Actinobacteria (27.16%), or Firmicutes (0.23%). At a genera level, the isolates belonged overwhelmingly to four genera, Pantoea (25.34%), Pseudomonas (17.35%), Arthrobacter (12.78%) and Curtobacterium (12.55%), while minor genera consisted of Rathayibacter (0.46%), Clavibacter (1.14%), Erwinia (0.23%) and Paenibacillus (0.23%). Unidentified isolates of comprised 29.90% (Supplementary Table 1).
Pantoea and Pseudomonas were identified in both drought susceptible lines (DSL) and drought tolerant lines (DTL). Pantoea and Pseudomonas were the dominant genera in DSL isolates comprising 32.56% and 22.79% respectively, as opposed to 27.56% and 2.67% of DTL isolates. Pantoea and Pseudomonas were more prevalent under rainfed conditions (RC) comprising 43.8% and 26.8%, respectively, rather than 12.59% and 9.54% of all isolates from drought conditions (DC).
Arthrobacter and Curtobacterium isolates were exclusive to DTL and were the dominant genera accounting for 24.00% and 25.78% of isolates, respectively (Figure 1). Arthrobacter isolates were also exclusive to DC, while Curtobacterium isolates dominated under DC (22%) as opposed to RC (1.68%). Furthermore, Curtobacterium was the only bacteria isolated from seeds of DT line 3 under DC, while Arthrobacter was the only bacteria isolated from DT line 4 under DC (Appendix 3).
Clavibacter and Rathayibacter were exclusively isolated from DTL under DC but only accounted for 2.22% and 0.89%, respectively (Figure 1). Erwinia and Paenibacillus were exclusive to DSL under RC, but only accounted for 0.47% and 0.47% of the population, respectively. Bacteria that were unable to be identified using MALDI-TOF were isolated from both DTL and DSL, being 16.5% and 43.7% of all isolates, respectively. Unknown isolates were also identified from DC and RC, at 33.2% and 24.7%, respectively.
Variation in diversity
Samples were collected from the seeds of wheat cultivars of differing levels of drought tolerance that had been subject to drought and rainfed conditions. The microbiomes of these seeds were characterised using 16S rRNA amplicon sequencing. To characterise the diversity of the wheat seed microbiomes, alpha and beta diversity analyses were performed. Shannon index comparison of DC microbiomes (8.813) was significantly more diverse than RC microbiomes (7.035) (q = 2.49e-18, H=76.35, Kruskal-Wallis test). However, the number of observed OTUs and Shannon’s index of DTL microbiomes and DSL microbiomes, when compared there was no significant difference (p>0.05, Kruskal-Wallis test). Robust Aitchison PCA (RPCA) was performed on the wheat seed microbiomes, using the QIIME2 DEICODE module . The microbiome profiles of DTL and DSL showed significant differences (Figure 2a, PERMANOVA pseudo-F = 20.95, p = 0.001). There was also a significant difference between the microbiomes of wheat seeds subjected to either DC and RC (Figure 2b, PERMANOVA pseudo-F = 8.76, p = 0.001).
Bacterial Taxonomic Composition
The composition of seed microbiomes was influenced by environmental conditions and wheat lines (see Figure 3). DC reduced the abundance of the three most dominant families under RC. In DTL under RC, the most dominant families were Enterobacteriaceae, Pseudomonadaceae and Burkholderiaceae, which represented 31.6%, 13.6% and 6.0% of all isolates, respectively, as opposed to 3.5%, 8.8% and 9.0% in DTL under DC. In DSL under RC, the most dominant families were Enterobacteriaceae, Pseudomonadaceae and Burkholderiaceae, which represented 23.3%, 19.7% and 6.0%, as opposed to 10.7%, 5.7% and 8.5% in DSL under DC. In DTL under DC, the three most abundant families were Burkholderia, Pseudomonadeceae and Chitinophagaceae. In DSL under DC, the three most abundant families were Enterobacteriaceae, Burkholderia and Chitinophagaceae.
Certain OTUs were enriched in seed microbiomes under drought conditions. ANOVA analysis of the microbiomes under DC and microbiomes under RC was performed (Supplementary Table 2). There were 1069 OTUs that were significant (p > 0.05). The ten most significant OTUs were belonged to the genera Pseudomonas (p=9.04E-18), Unknown (ANOVA p = 2.11E-15, p = 1.72E-12, p = 1.57E-11), Flavobacterium (ANOVA p = 7.74E-13), Amycolatopsis (ANOVA p = 2.99E-12), Bradyrhizobium (ANOVA p = 3.68E-11), Pantoea (ANOVA p = 5.80E-11), Skermenella (ANOVA p = 5.96E-11) and Rubrobacter (ANOVA p = 7.45E-11). There were multiple significant OTUS identified that belonged to the genera isolated from wheat seeds, including Pseudomonas, Pantoea and Arthrobacter that appeared in the top 0.2% of significant OTUs. In DT Line 3 and DT Line 4, Arthrobacter was enriched 1.5-fold (Tukey test, p=2.2E-4) and 1.9-fold (Tukey test, p=3.4E-6), respectively. Approximately a third of OTUs identified were either unknown at the genus level or uncultured at the genus level. Of the top 50 OTUs, 17 belonged to unknown or uncultured genera.
The microbiomes of DTL showed enrichment of certain microbes when compared with DSL microbiomes, under both DC and RC (Figure 3). At a family level, Enterbacteriaceae and Microbacteriaceae were highlighted. DTL had a higher abundance of Microbacteriaceae (1.045%) compared to DSL (0.359%), under DC. DTL also had higher abundance of Microbacteriaceae (0.514%) compared to DSL (0.274%), under RC. When ranking the relative abundance of OTUs at a family level, Microbacteriaceae were ranked at 25 for DTL under DC and 27 for DTL under RC (Figure 4). Comparatively, Microbacteriaceae were ranked at 50 for DSL under DC and 48 for DSL under RC. Genera from the order Microbacteriaceae were significant in an ANOVA of the microbiome of DTL and DSL under DC, namely Curtobacterium (p=3.29E-3), Agromyces (p=0.032) and an unknown genera (p=0.048) (Supplementary Table 3).
Plant growth promotion effects of the cultured microbiome
Evaluation of microbiome bacteria for biostimulation of Triticeae
To assess the growth promotion effect of Curtobacterium flaccumfaciens novel strain Cf D3-25 and Arthrobacter sp. novel strains Ar. sp D4-14 on plants, a seedling assay was established with members of the tribe Triticeae (wheat – Triticum aestivum; spelt - Triticum spelta, durum - Triticum durum; ryecorn - Secale cereale; oats - Avena sativa; barley - Hordeum vulgare). Wheat seeds inoculated with different concentrations of Cf D3-25 or Ar. sp D4-14 were germinated and allowed to grow for seven days to evaluate potential biostimulation activity. There was a root lengthening effect observed in microbe-treated wheat seedlings (see Figure 5). In early wheat seedlings inoculated in Cf D3-25 solutions diluted to 100, 10-2 and 10-3 (containing 7 x 108, 7 x 106 and 7 x 105 CFU mL-`1, respectively), the root lengthening was significant compared to the uninoculated control, increasing root length by 7.94%, 9.05% and 7.95%. Similarly, in wheat seedlings inoculated with Ar. sp D4-14 solutions diluted to 10-3 and 10-4 (containing 1.13 x 106 and 1.13 x 105 CFU mL-`1), respectively root lengthening was significant compared to the uninoculated control, increasing root length by 21.93% and 21.78%. Oat seedlings inoculated in Cf D3-25 solutions of 10-1, 10-2, 10-3 and 10-4 (8.19 x 107, 8.19 x 106, 8.19 x 105, 8.19 x 104 CFU mL-`1, respectively) had significantly longer roots than the control, with a percentage increase of 90.81%, 101.55%, 63.85% and 104.68% respectively. Similarly, oat seedlings inoculated with Ar. sp D4-14 solutions of 10-3 and 10-4 (5.81 x 105 and 5.81 x 104 CFU mL-`1, respectively) had significantly longer roots than the control, with a percentage increase of 63.85% and 80.14% respectively. There was no significant observable root lengthening effect in barley, spelt, or ryecorn. High concentrations of Ar. sp D4-14 inhibited root growth in both barley and oats. There were no significant observable shoot effects in any Triticeae (see Appendix 5).
Greenhouse drought pot trial
To assess the ability of Curtobacterium flaccumfaciens D3-25 and Arthrobacter sp. D4-14 to aid drought tolerance, an in planta assay was established in seed inoculated wheat exposed to varying levels of drought. At the end of six-weeks of growth, the wheat plants were harvested and the wet shoot and root weight were measured. Wheat plants inoculated with Cf D3-25 and Ar. sp D4-14 had significant increases in shoot and root weight, compared to control, across a range of conditions. Shoot weight of Cf D3-25-inoculated wheat plants, under well-watered conditions, increased by 46.82% compared to the control (Tukey test, p=0.003, Figure 6a). Under drought conditions, there was a 7.71% increase in shoot length of wheat plants inoculated with Cf D3-25 under mild drought conditions, but this increase was not significant. The shoot weight of Ar. sp D4-14-inoculated wheat plants did not significantly increase, compared to control. Root weight was significantly increased in six-week old wheat plants inoculated with Cf D3-25 under mild and severe drought conditions, compared to Ar. sp D4-14-inoculated and control plants (Figure 6b). Under mild conditions, Cf D3-25-inoculated plants had a 26.00% increase in wet root weight (Tukey test, p=0.045). Under severe conditions, Cf D3-25-inoculated plants had a 27.61% increase in wet root weight (Tukey test, p=0.035). Wet root weight increased 15.07% under well-watered conditions, although the difference was not significant (Tukey test, p=0.05). Wheat plants inoculated with Ar. sp D4-14 had no significant increase in wet root weight, compared to the control.
After six weeks of growth, the shoot length and number of leaves on each plant were measured. In the well-watered condition, the endophyte-treated plants had significantly more fully-expanded leaves than the control (Figure 6c). Plants that had been inoculated with either Cf D3-25 or Ar. sp D4-14 had 35% and 27% more fully-expanded leaves, respectively, than the control plants (Tukey test, p=0.001 and p=0.02). Ar. sp D4-14-treated plants exhibited significantly more full-expanded leaves under severe drought. Wheat plants inoculated with Cf D3-25 had an 8.47% increase in shoot length under well-watered conditions, compared to the control (Tukey test, p=0.001, Figure 6d). Similarly, wheat plants inoculated with Ar. sp D4-14-inoculated had significantly longer shoot lengths, under well-watered condition, with a 6.68% increase compared to the control (Tukey test, p=0.016). However, this effect was not evident under moderate or severe drought conditions.