# Genome-sequenced bacterial collection from sorghum epicuticular wax

Marco E Mechan-Llontop, John Mullet, Ashley Shade

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Marco E Mechan-Llontop, John Mullet, Ashley Shade. Genome-sequenced bacterial collection from sorghum epicuticular wax. Microbiology Resource Announcements, 2023, 12 (12), pp.e00468-23. 10.1128/MRA.00468-23. hal-04787899

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**Microbiology**
Resource Announcements

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Article title

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Genome-sequenced bacterial collection from sorghum epicuticular wax.

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Authors

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Marco E. Mechan-Llontop¹, John Mullet¹,², and Ashley Shade³#

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Affiliations

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1. Great Lakes Bioenergy Research Center, Michigan State University, East Lansing, Michigan, USA.

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2. Department of Biochemistry & Biophysics, Texas A&M University, College Station, Texas, USA.

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3. Centre National de la Recherche Scientifique, Laboratoire d'Écologie Microbienne (UMR CNRS 5557, UMR INRAE 1418, VetAgro Sup), Universite Claude Bernard Lyon 1, Villeurbanne, France

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Running title

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Bacteria from sorghum epicuticular wax

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Corresponding author's email address

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\#Address correspondence to: Ashley Shade, ashley.shade@cnrs.fr

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Abstract

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A collection of 44 isolates isolated from the epicuticular wax of stems of energy sorghum is available at the

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Great Lakes Bioenergy Research Center, Michigan State University, MI, USA. We enrich bacteria with

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putative plant-beneficial phenotypes and include information on their phenotypic diversity, taxonomy, and

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whole-genome sequences.

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Announcement

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The heat and drought-tolerant and high-biomass crop bioenergy sorghum (*Sorghum bicolor* L. Moench)

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accumulates high levels of epicuticular wax on the outer surface of the cuticle to protect plants against

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various stresses (1–3). It has been shown that the sorghum epicuticular wax harbors specialized

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microbiome members with putative plant-beneficial capabilities (4). As a resource for genomic and
functional diversity of bacteria associated with phyllosphere exudates, we present a collection of 44
bacterial strains isolated from sorghum epicuticular wax. These strains represent 13 families and include 17
genera with putative plant-beneficial capabilities, including nitrogen fixation, phosphate solubilization,
resistance to terpenoids, use of methanol as carbon source, and tolerance to desiccation, based on the
culture media in which the strains were originally isolated.

Bacterial strains were isolated from the epicuticular wax of stems of bioenergy sorghum hybrid
TX08001 grown at the Texas A&M University Research Farm in College Station, Texas (30°55'5.55" N,
96°43'64.6" W). On 2 September 2020, the fifth and sixth fully elongated stem internodes were
destructively collected using razor blades into sterile whirl-pack bags. Within 2 hours of collection, the
samples were transported to the laboratory on ice and stored at -80°C. On 8 October 2020, samples were
delivered to Michigan State University on dry ice and stored at -80°C until processing. Epicuticular wax was
carefully scraped from stems with sterile razor blades and collected into sterile 1.5 mL Eppendorf tubes.
Wax from different plants was pooled, and 100 mg was resuspended in 1 mL of sterile water. Resuspended
wax was serially diluted from 10⁻¹ to 10⁻⁴. To capture a diversity of bacteria from the wax, we used a variety
of cultivation media (Table 1). To select for anaerobic bacteria, plates were placed in anaerobic jars
containing three bags of anaerobic gas generator (Mitsubishi AnaeroPack System). All plates were
incubated at 25°C and 37°C for up to 14 days. Isolated colonies were transferred on new plates with the
same medium as used for isolation to confirm purity. Bacteria were grown overnight on R2A broth at 28°C
with shaking at 200 rpm, and glycerol stock (25% vol/vol) of pure bacteria isolates were stored at -80°C.
Prior to performing whole genome sequencing, we performed full-length 16S rRNA gene sequencing, with
universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGTTACCTTGTTACGACTT-3')
(5), of each isolate using the Sanger protocol to link them to corresponding 16S rRNA gene amplicon
dynamics in a field microbiome study that was executed at the same field site at Texas A&M University
Research Farm (4).
Individual isolates or the full cryopreserved collection is available. Cultures can be regrown using standard microbiological procedures to transfer a sterile inoculation loop of freezer stock onto the original isolation medium and incubation time (Table 1).

The high molecular weight genomic DNA of each wax bacterial isolate was extracted by using a phenol/chloroform extraction protocol (6). The genomic DNA of each strain was submitted to the Joint Genome Institute, a U.S. Department of Energy User Facility, for library preparation and sequencing with the Pacific Biosciences (PacBio) platform (7). DNA was sheared to 10kb using g-TUBE columns (Covaris) and subjected to library preparation using the SMARTbell Express Template prep 2.0 kit and sequenced on the PacBio Sequel platform. Samples that failed DNA quality control required by the PacBio pipeline were instead sequenced using the NovaSeq S4 Illumina platform (8). High fidelity CCS reads >5kb were assembled with Flye 2.8.3 (9) using default settings. Raw Illumina sequences were quality-filtered using BBTools (10). Artifact filtered and normalized Illumina reads were assembled with SPAdes v3.15.3 (11) (–phred-offset 33 –cov-cutoff auto -t 16 -m 64 –careful -k 25,55,95), and contigs were discarded if the length was <1kb (BBTools reformat.sh: minlength=1000 ow=t). Genome completeness and contamination was estimated with CheckM v1.2.2 (12). The assembly was annotated using Prokka 1.14.6 (13) and the phylogenetic tree was inferred from using Orthofinder 2.5.5 (14) and edited with iTOLs v.6.5.8 (15) (Fig. 1).

## Data availability statement

Raw sequencing data are available at the Joint Genome Institute's GOLD database under Study ID Gs0157305, as well as with the National Institutes of Health Sequence Read Archive (see Table 1). Genome sequencing assembly data has been deposited in the GenBank with accession numbers listed in Table 1.

The bacterial collection is available at The Great Lakes Bioenergy Research Center at Michigan State University, USA (glbrc@msu.edu).

## Acknowledgments
This work was supported by the Great Lakes Bioenergy Research Center, U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research under Award Number DE-SC0018409. Support for field research was provided by the Great Lakes Bioenergy Research Center, U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research (Awards DE-SC0018409 and DE-FC02-07ER64494), by the National Science Foundation Long-term Ecological Research Program (DEB 1637653) at the Kellogg Biological Station, and by Michigan State University AgBioResearch. The work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported under Contract No. DE-AC02-05CH11231.

This data was generated for JGI Proposal #508590.

The authors declare no conflict of interest.

## References

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2. Steinmüller D, Tevini M. 1985. Action of ultraviolet radiation (UV-B) upon cuticular waxes in some crop plants. Planta 164:557-564.

3. Wang X, Kong L, Zhi P, Chang C. 2020. Update on Cuticular Wax Biosynthesis and Its Roles in Plant Disease Resistance. Int J Mol Sci 21:5514.

4. Mechan-Llontop ME, Mullet J, Shade A. 2023. Phyllosphere exudates select for distinct microbiome members in sorghum epicuticular wax and aerial root mucilage. Phytobiomes Journal 7:184-197

5. Miller CS, Handley KM, Wrighton KC, Frischkorn KR, Thomas BC, Banfield JF. 2013. Short-read qassembly of full-length 16S amplicons reveals bacterial diversity in subsurface sediments. PLoS One 8:e56018-.

6. Kutchma AJ, Roberts MA, Knaebel DB, Crawford DL. 1998. Small-scale isolation of genomic DNA from Streptomyces mycelia or spores. Biotechniques 24:452-456.

7. Eid J, Fehr A, Gray J, Luong K, Lyle J, Otto G, Peluso P, Rank D, Baybayan P, Bettman B, Bibillo A, Bjornson K, Chaudhuri B, Christians F, Cicero R, Clark S, Dalal R, deWinter A, Dixon J, Foquet M, Gaertner A, Hardenbol P, Heiner C, Hester K, Holden D, Kearns G, Kong X, Kuse R, Lacroix Y, Lin S, Lundquist P, Ma C, Marks P, Maxham M, Murphy D, Park I, Pham T, Phillips M, Roy J, Sebra R, Shen G, Sorenson J, Tomaney A, Travers K, Trulson M, Vieceli J, Wegener J, Wu D, Yang A,
102 Zaccarin D, Zhao P, Zhong F, Korlach J, Turner S. 2009. Real-time DNA sequencing from single polymerase molecules. Science 323:133-138.

104 8. Bennett S. 2004. Solexa Ltd. Pharmacogenomics 5:433-438.

105 9. Kolmogorov M, Yuan J, Lin Y, Pevzner PA. 2019. Assembly of long, error-prone reads using repeat graphs. Nat Biotechnol 37:540-546.

107 10. Bushnell B. 2014. BBTools software package (version 39.01).

108 11. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin A V, Sirotkin A V, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455-477.

112 12. Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. 2015. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res 25:1043-1055.

115 13. Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068-2069.

116 14. Emmes DM, Kelly S. 2019. OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biol 20:238.

118 15. Letunic I, Bork P. 2021. Interactive Tree Of Life (iTOL) V5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res 49:W293-W296.

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122 Tables
<table><caption>TABLE 1.- Taxonomy, colony phenotype, and genome characteristics of 44 bacterial isolates from sorghum epicuticular wax, as described in this study.
SRA is the sequence read archive of the U.S. National Institutes of Health.</caption><thead><tr><th>Isolate ID</th><th>Bacterial species</th><th>Initial isolation media</th><th>Temperature, Oxygen availability</th><th>Colony phenotype</th><th>Sequencing platform</th><th>SRA accession number</th><th>GenBank Assembly</th><th>Genome length (bp)</th><th>N° contigs</th><th>Coverage</th><th>GC %</th><th>Genome completeness</th></tr></thead><tbody><tr><td>SORGH_AS_0981</td><td>Aeromicrobium sp.</td><td>50R2A</td><td>37 °C, aerobic</td><td>Small, White</td><td>PacBio</td><td>SRP437207</td><td>JAVIZD000000000</td><td>3,670,284</td><td>1</td><td>186.7 X</td><td>25%</td><td>98.96%</td></tr><tr><td>SORGH_AS_0908</td><td>Pseudoxanthomonas winnipegensis</td><td>50R2A</td><td>37 °C, aerobic</td><td>Yellow</td><td>PacBio</td><td>SRP437170</td><td>JAUTBB000000000</td><td>4,331,500</td><td>1</td><td>82.7 X</td><td>46%</td><td>99.53%</td></tr><tr><td>SORGH_AS_0909</td><td>Chryseobacterium sp.</td><td>50R2A</td><td>37 °C, aerobic</td><td>Yellow</td><td>PacBio</td><td>SRP437159</td><td>JAVIYY000000000</td><td>4,186,940</td><td>1</td><td>137.1 X</td><td>82%</td><td>100%</td></tr><tr><td>SORGH_AS_0912</td><td>Acinetobacter baylyi</td><td>50R2A</td><td>37 °C, aerobic</td><td>White, EPS production</td><td>PacBio</td><td>SRP437185</td><td>JAVIZH000000000</td><td>3,896,949</td><td>3</td><td>187.4 X</td><td>23%</td><td>99.95%</td></tr><tr><td>SORGH_AS_0913</td><td>Nocardioides zeae</td><td>50R2A</td><td>37 °C, aerobic</td><td>White, EPS production</td><td>PacBio</td><td>SRP437161</td><td>JAVIYZ000000000</td><td>4,248,407</td><td>1</td><td>90.2 X</td><td>63%</td><td>98.19%</td></tr><tr><td>SORGH_AS_0919</td><td>Microbacterium paludicola</td><td>50R2A</td><td>37 °C, aerobic</td><td>Pink</td><td>PacBio</td><td>SRP437238</td><td>JAVIZA000000000</td><td>3,351,387</td><td>1</td><td>196.8 X</td><td>22%</td><td>99.24%</td></tr><tr><td>SORGH_AS_0974</td><td>Agrobacterium larrymoorei</td><td>50R2A</td><td>37 °C, anaerobic</td><td>White</td><td>PacBio</td><td>SRP437176</td><td>JAVIZC000000000</td><td>5,263,363</td><td>3</td><td>120.0 X</td><td>10%</td><td>98.30%</td></tr><tr><td>SORGH_AS_0950</td><td>Sphingomonas sp.</td><td>Gauze N-1</td><td>37 °C, aerobic</td><td>Orange</td><td>PacBio</td><td>SRP437227</td><td>JAUTAE000000000</td><td>4,389,989</td><td>3</td><td>73.3 X</td><td>10%</td><td>99.14%</td></tr><tr><td>SORGH_AS_1204</td><td>Microbacterium sp.</td><td>Gauze N-1</td><td>25 °C, aerobic</td><td>Orange</td><td>PacBio</td><td>SRP437180</td><td>JAUTBD000000000</td><td>3,706,564</td><td>1</td><td>132.3 X</td><td>10%</td><td>97.77%</td></tr><tr><td>SORGH_AS_1206</td><td>Microbacterium arborescens</td><td>Gauze N-1</td><td>25 °C, aerobic</td><td>Orange</td><td>PacBio</td><td>SRP437216</td><td>JAUTBE000000000</td><td>3,449,815</td><td>1</td><td>88.6 X</td><td>11%</td><td>99.24%</td></tr><tr><td>SORGH_AS_1207</td><td>Microbacterium trichothecenolyticum</td><td>Gauze N-1</td><td>25 °C, aerobic</td><td>Orange</td><td>PacBio</td><td>SRP437150</td><td>JAUTBF000000000</td><td>3,735,270</td><td>1</td><td>241.2 X</td><td>92%</td><td>97.24%</td></tr><tr><td>SORGH_AS_0870</td><td>Sphingomonas sp.</td><td>Jensen</td><td>25 °C, aerobic</td><td>Orange</td><td>PacBio</td><td>SRP437151</td><td>JAVIYW000000000</td><td>4,339,992</td><td>2</td><td>98.4 X</td><td>06%</td><td>99.42%</td></tr><tr><td>SORGH_AS_1048</td><td>Chryseobacterium sp.</td><td>Jensen</td><td>37 °C, aerobic</td><td>Small, orange</td><td>PacBio</td><td>SRP437220</td><td>JAUTAK000000000</td><td>4,189,072</td><td>1</td><td>195.6 X</td><td>80%</td><td>99.51%</td></tr><tr><td>SORGH_AS_0862</td><td>Microbacterium sp.</td><td>Jensen</td><td>25 °C, aerobic</td><td>Yellow, EPS production</td><td>PacBio</td><td>SRP437146</td><td>JAUTAY000000000</td><td>3,504,869</td><td>1</td><td>174.0 X</td><td>45%</td><td>98.27%</td></tr><tr><td>SORGH_AS_0892</td><td>Sphingobacterium zeae</td><td>KB</td><td>25 °C, aerobic</td><td>Yellow</td><td>PacBio</td><td>SRP437175</td><td>JAUTBA000000000</td><td>5,604,819</td><td>1</td><td>136.4 X</td><td>08%</td><td>99.84%</td></tr><tr><td>SORGH_AS_0893</td><td>Acinetobacter baylyi</td><td>KB</td><td>25 °C, aerobic</td><td>White</td><td>PacBio</td><td>SRP437224</td><td>JAVIYX000000000</td><td>3,884,413</td><td>3</td><td>187.0 X</td><td>23%</td><td>99.72%</td></tr><tr><td>SORGH_AS_0962</td><td>Asaia bogorensis</td><td>KB</td><td>25 °C, aerobic</td><td>Small, Pink</td><td>PacBio</td><td>SRP437145</td><td>JAVIZB000000000</td><td>3,299,069</td><td>2</td><td>112.5 X</td><td>82%</td><td>99.71%</td></tr></tbody></table>
<table><tr><td>SORGH_AS_0969</td><td>Microbacterium sp.</td><td>KB</td><td>25 °C, aerobic</td><td>White</td><td>PacBio</td><td>SRP437218</td><td>JAUTAG000000000</td><td>3,939,785</td><td>1</td><td>159.6 X</td><td>65%</td><td>98.53%</td></tr><tr><td>SORGH_AS_1094</td><td>Pantoea dispersa</td><td>M9 + 0.4% sucrose</td><td>25 °C, aerobic</td><td>Yellow</td><td>PacBio</td><td>SRP437173</td><td>JAVJAC000000000</td><td>4,990,160</td><td>3</td><td>189.4 X</td><td>57%</td><td>99.71%</td></tr><tr><td>SORGH_AS_1126</td><td>Agrobacterium larrymoorei</td><td>M9 + 0.4% sucrose</td><td>37 °C, aerobic</td><td>White</td><td>PacBio</td><td>SRP437223</td><td>JAUTBL000000000</td><td>4,882,137</td><td>2</td><td>142.0 X</td><td>10%</td><td>97.93%</td></tr><tr><td>SORGH_AS_0906</td><td>Klebsiella variicola</td><td>M9 nitrogen-free Carbon-free</td><td>37 °C, aerobic</td><td>White, EPS production</td><td>PacBio</td><td>SRP437210</td><td>JAVJAI000000000</td><td>5,683,212</td><td>2</td><td>135.4 X</td><td>57%</td><td>99.85%</td></tr><tr><td>SORGH_AS_1173</td><td>Klebsiella sp.</td><td>M9 nitrogen-free Carbon-free</td><td>37 °C, anaerobic</td><td>White, EPS production</td><td>PacBio</td><td>SRP437157</td><td>JAVJAD000000000</td><td>5,683,290</td><td>2</td><td>194.8 X</td><td>57%</td><td>99.78%</td></tr><tr><td>SORGH_AS_1175</td><td>Chryseobacterium sp.</td><td>M9 nitrogen-free Carbon-free</td><td>37 °C, anaerobic</td><td>Orange</td><td>PacBio</td><td>SRP437237</td><td>JAVIZF000000000</td><td>4,186,971</td><td>1</td><td>211.3 X</td><td>82%</td><td>99.88%</td></tr><tr><td>SORGH_AS_0842</td><td>Klebsiella variicola</td><td>M9 nitrogen-free Carbon-free</td><td>25 °C, anaerobic</td><td>White, EPS production</td><td>PacBio</td><td>SRP437149</td><td>JAVJAB000000000</td><td>5,683,317</td><td>2</td><td>89.3 X</td><td>57%</td><td>99.93%</td></tr><tr><td>SORGH_AS_0878</td><td>Klebsiella variicola</td><td>M9 nitrogen-free Carbon-free</td><td>25 °C, aerobic</td><td>White, EPS production</td><td>PacBio</td><td>SRP437219</td><td>JAVIZZ000000000</td><td>5,683,197</td><td>2</td><td>188.5 X</td><td>57%</td><td>99.55%</td></tr><tr><td>SORGH_AS_0879</td><td>Sphingomonas sp.</td><td>M9 nitrogen-free Carbon-free</td><td>25 °C, aerobic</td><td>Yellow</td><td>PacBio</td><td>SRP437133</td><td>JAUTBJ000000000</td><td>4,326,161</td><td>2</td><td>185.1 X</td><td>43%</td><td>99.47%</td></tr><tr><td>SORGH_AS_0885</td><td>Nocardioides zeae</td><td>M9 nitrogen-free Carbon-free</td><td>25 °C, aerobic</td><td>Yellow</td><td>Illumina</td><td>SRP437487</td><td>JAVIZJ000000000</td><td>4,252,839</td><td>27</td><td>353.6 X</td><td>44%</td><td>99.85%</td></tr><tr><td>SORGH_AS_0887</td><td>Acinetobacter baylyi</td><td>M9 nitrogen-free Carbon-free</td><td>25 °C, aerobic</td><td>White, EPS production</td><td>PacBio</td><td>SRP437183</td><td>JAUTBK000000000</td><td>3,884,591</td><td>3</td><td>186.4 X</td><td>23%</td><td>99.86%</td></tr><tr><td>SORGH_AS_0888</td><td>Microbacterium sp.</td><td>MMS methanol</td><td>25 °C, aerobic</td><td>Yellow, EPS production</td><td>PacBio</td><td>SRP437142</td><td>JAUTAZ000000000</td><td>3,638,259</td><td>1</td><td>237.8 X</td><td>61%</td><td>98.91%</td></tr><tr><td>SORGH_AS_1077</td><td>Microbacterium testaceum</td><td>R2A</td><td>37 °C, aerobic</td><td>Yellow</td><td>PacBio</td><td>SRP437132</td><td>JAUTBC000000000</td><td>3,939,786</td><td>1</td><td>181.0 X</td><td>65%</td><td>98.23%</td></tr><tr><td>SORGH_AS_1083</td><td>Rhizobium pusense</td><td>R2A</td><td>37 °C, aerobic</td><td>White</td><td>PacBio</td><td>SRP437166</td><td>JAVIZE000000000</td><td>5,455,244</td><td>3</td><td>63.6 X</td><td>10%</td><td>99.57%</td></tr><tr><td>SORGH_AS_0826</td><td>Klebsiella sp.</td><td>R2A</td><td>25 °C, anaerobic</td><td>White, EPS production</td><td>PacBio</td><td>SRP437221</td><td>JAVJAA000000000</td><td>5,685,432</td><td>2</td><td>136.5 X</td><td>58%</td><td>100%</td></tr><tr><td>SORGH_AS_0956</td><td>Pseudoxanthomonas winnipegensis</td><td>R2A + 0.1% caryophyllene</td><td>37 °C, aerobic</td><td>Yellow</td><td>PacBio</td><td>SRP437156</td><td>JAUTAF000000000</td><td>4,331,831</td><td>1</td><td>208.2 X</td><td>46%</td><td>98.27%</td></tr><tr><td>SORGH_AS_0961</td><td>Chryseobacterium sp.</td><td>R2A + 0.1% caryophyllene</td><td>37 °C, aerobic</td><td>White</td><td>PacBio</td><td>SRP437178</td><td>JAVLUK000000000</td><td>4,187,089</td><td>1</td><td>241.5 X</td><td>82%</td><td>98.77%</td></tr><tr><td>SORGH_AS_0993</td><td>Brevundimonas sp.</td><td>R2A + 0.1% caryophyllene</td><td>37 °C, aerobic</td><td>White</td><td>PacBio</td><td>SRP437129</td><td>JAUTAH000000000</td><td>2,986,043</td><td>1</td><td>210.6 X</td><td>51%</td><td>99.12%</td></tr><tr><td>SORGH_AS_0834</td><td>Klebsiella variicola</td><td>R2A + 0.1% linalool</td><td>25 °C, aerobic</td><td>White, EPS production</td><td>PacBio</td><td>SRP437138</td><td>JAVJAG000000000</td><td>5,683,096</td><td>2</td><td>163.7 X</td><td>57%</td><td>99.64%</td></tr><tr><td>SORGH_AS_1014</td><td>Xanthomonas sacchari</td><td>TSA</td><td>37 °C, aerobic</td><td>Yellow</td><td>PacBio</td><td>SRP437228</td><td>JAUTAI000000000</td><td>4,997,627</td><td>2</td><td>173.7 X</td><td>65%</td><td>98.97%</td></tr></table>
<table><tr><td>SORGH_AS_1015</td><td>Roseomonas cervicalis</td><td>TSA</td><td>37 °C, aerobic</td><td>Pink</td><td>PacBio</td><td>SRP437141</td><td>JAUTAJ000000000</td><td>4,889,768</td><td>5</td><td>111.2 X</td><td>05%</td><td>98.81%</td></tr><tr><td>SORGH_AS_1025</td><td>Klebsiella sp.</td><td>TSA</td><td>37 °C, aerobic</td><td>White, EPS production</td><td>PacBio</td><td>SRP437181</td><td>JAVIZY000000000</td><td>5,684,408</td><td>2</td><td>172.8 X</td><td>58%</td><td>99.70%</td></tr><tr><td>SORGH_AS_1064</td><td>Chryseobacterium camelliae</td><td>TSA</td><td>25 °C, aerobic</td><td>Orange</td><td>PacBio</td><td>SRP437167</td><td>JAUTAL000000000</td><td>4,187,050</td><td>1</td><td>212.0 X</td><td>82%</td><td>99.84%</td></tr><tr><td>SORGH_AS_1065</td><td>Siphonobacter sp.</td><td>TSA</td><td>25 °C, aerobic</td><td>Orange</td><td>PacBio</td><td>SRP437172</td><td>JAUTAM000000000</td><td>6,150,989</td><td>10</td><td>125.1 X</td><td>65%</td><td>99.70%</td></tr><tr><td>SORGH_AS_1067</td><td>Nocardioides zeae</td><td>TSA</td><td>25 °C, aerobic</td><td>Yellow</td><td>PacBio</td><td>SRP43703</td><td>JAUTAN000000000</td><td>4,380,404</td><td>1</td><td>164.8 X</td><td>45%</td><td>99.22%</td></tr><tr><td>SORGH_AS_1070</td><td>Klebsiella variicola</td><td>TSA</td><td>25 °C, aerobic</td><td>White, EPS production</td><td>PacBio</td><td>SRP437160</td><td>JAVJAH000000000</td><td>5,682,994</td><td>2</td><td>95.0 X</td><td>57%</td><td>100%</td></tr><tr><td>SORGH_AS_0997</td><td>Pseudoxanthomonas sp.</td><td>TSA</td><td>25 °C, aerobic</td><td>Yellow</td><td>PacBio</td><td>SRP437165</td><td>JAVIZI000000000</td><td>4,331,362</td><td>1</td><td>171.5 X</td><td>46%</td><td>99.09%</td></tr></table>

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Figures

FIG 1: Phylogenetic diversity of the bacterial collection cultivated from sorghum epicuticular wax. The archaea *Sulfolobus acidocaldarius* DSM 639 (ASM2847236v1) was included as an outgroup. The bacterial reference strains *Pseudomonas aeruginosa* PA01 (ASM676v1), *Microbacterium foliorum* DSM 12966 (ASM95641v1), *Stenotrophomonas lactitubi* M15 (ASM280351v1), *Flavobacterium johnsoniae* UW101 (ASM1664v1), *Sphingomonas paucimobilis* FDAARGOS_908 (ASM1602709v1), and *Rhizobium leguminosarum* SM52 (ASM430655v1) were also included. The species tree was inferred from unrroted orthogroups gene trees using STAG and then rooted using the STRIDE algorithms implemented in OrthoFinder. Species tree was annotated with iTOLs.

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<figure><img src="image_4.png" /></figure>

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