Streptomyces bacteria are ubiquitous in soils and are well-known for producing secondary metabolites, including antimicrobials. Increasingly, they are being isolated from plant roots and several studies have shown they are specifically recruited to the rhizosphere and the endosphere of the model plant Arabidopsis thaliana. Here we test the hypothesis that Streptomyces bacteria have a beneficial effect on A. thaliana growth and could potentially be used as plant probiotics. To do this, we selectively isolated streptomycetes from surface washed A. thaliana roots and generated high quality genome sequences for five strains which we named L2, M2, M3, N1 and N2. Re-infection of A. thaliana plants with L2, M2 and M3 significantly increased plant biomass individually and in combination whereas N1 and N2 had a negative effect on plant growth, likely due to their production of polyene natural products which can bind to phytosterols and reduce plant growth. N2 exhibits broad spectrum antimicrobial activity and makes filipin-like polyenes, including 14-hydroxyisochainin which inhibits the Take-all fungus, Gaeumannomyces graminis var. tritici. N2 antifungal activity as a whole was upregulated ∼2-fold in response to indole-3-acetic acid (IAA) suggesting a possible role during competition in the rhizosphere. Furthermore, coating wheat seeds with N2 spores protected wheat seedlings against Take-all disease. We conclude that at least some soil dwelling streptomycetes confer growth promoting benefits on A. thaliana while others might be exploited to protect crops against disease. Importance. We must reduce reliance on agrochemicals and there is increasing interest in using bacterial strains to promote plant growth and protect against disease. Our study follows up reports that Arabidopsis thaliana specifically recruits Streptomyces bacteria to its roots. We test the hypothesis that they offer benefits to their A. thaliana hosts and that strains isolated from these plants might be used as probiotics. We isolated Streptomyces strains from A. thaliana roots and genome sequenced five phylogenetically distinct strains. Genome mining and bioassays indicated that all five have plant growth promoting properties, including production of IAA, siderophores and ACC deaminase. Three strains significantly increased A. thaliana growth in vitro and in combination in soil. Another produces potent filipin-like antifungals and protected germinating wheat seeds against the fungal pathogen Gaeumannomyces graminis var. tritici (wheat Take-all fungus). We conclude that introducing Streptomyces strains into the root microbiome provides significant benefits to plants.

19 20 21 22 23 101 10% (v/v) glycerol and serial dilutions were spread onto either soya flour mannitol (SFM) agar, 102 starch casein agar, or minimal medium agar containing sodium citrate (Lebeis et al 2015).103Plates were incubated at 30C for up to 14 days. Colonies resembling streptomycetes were re-104 streaked onto SFM agar and identified by 16S rRNA gene PCR amplification and sequencing 105 with universal primers PRK341F and MPRK806R (see Table S1 for primers, plasmids and 106 strains used in this work). Streptomyces strains were maintained on SFM agar (N1, N2, M2, 107 M3 and S. coelicolor M145), Maltose/Yeast extract/Malt extract (MYM) agar with trace 108 5 elements (L2) or ISP2 agar (S. lydicus strains); media recipes are detailed in Table S2. Spore 109 stocks were made as described previously (Kieser et al 2000). 110 111 Genome sequencing and analysis. High quality genome sequences were obtained for newly 112 isolated strains N1, N2, M2, M3, and L2, as well as three known strains of Streptomyces 113 lydicus; one isolated from the commercial plant growth-promoting product Actinovate and two 114 additional S. lydicus strains (ATCC25470 and ATCC31975) obtained from the American Type 115 Culture Collection. Strains were sequenced using PacBio RSII sequencing technology at the 116 131 isolated from a patient at the Norfolk and Norwich University Hospital UK (Qin et al 2017), 132Escherichia coli and Pseudomonas syringae DC3000 were grown overnight in 10 ml Lysogeny 133 Broth (LB) (1% tryptone, 0.5% NaCl, supplemented with 0.1% glucose for P. syringae). These 134 were sub-cultured 1 in 20 (v/v) for a further 4 hours at 30°C for P. syringae and 37°C for E. 135 coli. These cultures were then used to inoculate 100 ml of molten LB (0.5% agar, plus 0.1% 136 glucose for P. syringae), of which 3 ml was used to overlay agar plates containing Streptomyces 137 colonies. Plates were incubated for 48 hours at 30C, and bioactivity was indicated by a clear 138 halo around the Streptomyces colony. For bioassays using the fungal strains Lomentospora 139 prolificans or Gaeumannomyces graminis, Streptomyces species were grown for 7 days, and 140 then a plug of the fungus (grown on potato glucose agar, Sigma Aldrich, for 14 days) was 141