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Genomic Assessment of
Stenotrophomonas
Indicatrix
for Improved Sunower Plant
Bartholomew Saanu Adeleke
North-West University
Ayansina Segun Ayangbenro
North-West University
Olubukola Oluranti Babalola ( olubukola.babalola@nwu.ac.za )
North-West University https://orcid.org/0000-0003-4344-1909
Research Article
Keywords: Agricultural sustainability, bacterial endophytes, crop improvement, plant growth-promoting
genes, whole-genome sequencing
Posted Date: May 12th, 2021
DOI: https://doi.org/10.21203/rs.3.rs-486258/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
Read Full License
Version of Record: A version of this preprint was published at Current Genetics on June 30th, 2021. See
the published version at https://doi.org/10.1007/s00294-021-01199-8.
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Abstract
Plant growth-promotion screening and genome analysis of
Stenotrophomonas indicatrix
BOVIS40 were
presented in this study. The genomic information reveals various genes underlining plant growth
promotion and resistance to environmental stressors. The genome of
S. indicatrix
BOVIS40 harbors
genes involved in the degradation and biotransformation of organic molecules. Also, other genes
involved in biolm production, chemotaxis, and agellation that facilitate bacterial colonization in the
root endosphere and phytohormone genes that modulate root development and stress response in plants
were detected in strain BOVIS40. IAA activity of the bacterial strain may be a factor responsible for root
formation. Nevertheless, the results highlighted here provide insights into the genomic functions of
S.
indicatrix
and which can be explored in agricultural management. Hence, a measurable approach to the
S. indicatrix
lifestyle can strategically provide several opportunities in their use as bioinoculants in
developing environmentally friendly agriculture sustainably.
1.0 Introduction
Plant growth promotion depends on natural bioactive compounds, which are inherent or produced by its
associated microorganisms (Palanichamy et al. 2018). Either from microorganisms or plants, their
immense contributions in the establishment of ecological balancing between plants and microbes remain
fundamental (Fadiji et al. 2021). The importance of plant growth-promoting (PGP) genes and secondary
metabolites from microorganisms are not only applicable in agriculture or restricted to plant
improvement, but with wider therapeutic importance (Manganyi et al. 2019; Adeniji et al. 2021).
To know the composition of the different genes present in the genome of bacteria, the need for their
isolation, characterization and analysis using bioinformatics tools became important (Mamphogoro et al.
2020). In recent times, the advancement in biotechnological ndings into endophytic studies using
various bioinformatics tools has revealed various PGP genes and secondary metabolites in bacteria
(Mohotti et al. 2020; Nascimento et al. 2020b). Each of these gene clusters can code for different
metabolic compounds such as siderophores, arypolyene and lanthipeptide-class-II with specic
functions. The siderophore genes in the genome of bacterial genera such as
Bulkhoderia, Bacillus,
Pseudomonas
and
Rhizobium
have been reported in host plants with a strong arsenal that suppresses
the level of pathogenicity for improved plant growth (Ludwig-Müller 2015; Bhattacharyya et al. 2017).
Similarly, the detection of several other secondary metabolite genes from endophytic bacteria colonizing
medicinal plants has been documented (Ludwig-Müller 2015; Dinesh et al. 2017).
The survival and adaptive mechanism of bacteria in extreme environments can be linked to their ability to
produce vital PGP genes and synthesis of secondary metabolites, thus making them suitable candidates
with great values for various biotechnological, agricultural, and industrial applications (Nascimento et al.
2020a; Chukwuneme et al. 2021). The genomic analysis of sequenced bacterial genome revealing
conservative and functional genes have been reported (Mitter et al. 2013; Zeng et al. 2018; Akinola et al.
2021), but information regarding novel plant-growth-promoting and secondary metabolite genes of
S.
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indicatrix
isolated from sunower root endosphere has not been documented, thus necessitating this
study.
Stenotrophomonas
spp is Gram-negative, spore formers and rod-like. Few of these species and
their functions in plant physiological functions have been studied (Kumar and Audipudi 2015; Singh and
Jha 2017). Nevertheless, the genus
Stenotrophomonas
are dominant in diverse environments with a
promising outlook in agricultural biotechnology (Alexander, et al. 2019).
The detection of important genes in the genome of
Stenotrophomonas
spp can signicantly enhance
their functions in the establishment of root-soil, soil-bacterial, and root-bacterial interactions for improved
plant adaptation mechanisms to soil stressors (Vurukonda et al. 2018). These genes can affect the
lifestyle and bacterial functions in the host plants. For instance, the detection of chemotaxis and biolm
production genes can contribute to root colonization and plant defense mechanisms. To these functional
premises,
S. indicatrix
can be a potential biological tool in the formulation of bioinoculants for improved
crop productivity. In this study, the authors presented genomic insights into endophytic
S. indicatrix
BOVIS40 obtained from sunower root endosphere with signicant effects on sunower yield. Whole-
genome sequencing (WGS) analysis unraveled vital genes involved in various biological functions and
plant growth promotion. To the best of our knowledge, this is the rst report on the WGS of
S. indicatrix
BOVIS40 sourced from sunower root. Hence, this study aims to provide insights into
S. indicatrix
BOVIS40 about (i) plant growth-promoting activities, (ii) whole-genome sequencing, (iii) functional and
secondary metabolite genes, and (iv) sunower improvement under greenhouse experiment.
2.0 Materials And Methods
2.1 Source of plant growth-promoting endophytic bacterium
Stenotrophomonas indicatrix
BOVIS40
The healthy sunower roots used in this study were sourced from Lichtenburg, South Africa (26°4′31.266′
′S, 25°58′44.442′′E). The plant surface-cleaning, isolation, and characterization of the bacterial isolates
were performed according to the modied methods of Forchetti et al. (2007). Bacterial isolates were
screened for various PGP traits. Phosphate solubilization (PS) assay was performed according to the
methods described by Pikovskaya (1948) with little modications, while indole acetic acid synthesis by
bacterial strains was conducted following the methods of Bric et al. (1991). Similarly, methods described
by Sun et al. (2006) and Schwyn and Neilands (1987) were employed for exopolysaccharide test and
siderophore screening on chrome azurol S (CAS) medium, respectively.
2.2 Molecular identication and whole-genome sequencing
The genomic content of strain BOVIS40 was extracted using a commercial Quick-DNA
TM
Miniprep Kit
specic for fungi or bacteria (Zymo Research, Irvine, CA, USA; Cat. No. D6005), as stipulated in the
manufacturer’s guide. Determination of quantity and quantity of the extracted DNA was achieved using a
NanoDrop spectrophotometer (Thermo Fischer Scientic, CA, USA). The molecular identication of
bacterial isolate as
S. indicatrix
based on 16S rDNA sequence data analysis was according to the method
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described by Araújo, et al. (2002). The WGS of strain BOVIS40 was performed at Inqaba Biotechnical
Industries (Pty) Ltd, Pretoria, South Africa.
The WGS of endophytic bacterial strain BOVIS40 was performed according to the standard Illumina
method. Succinctly, fragmentation of bacterial genomic DNA was performed using the NEB Ultra II FS kit
enzymatic approach. AMPure XP beads were employed for the selection from the resulting DNA
fragments based on size range (200 - 700 bp). Subsequently, DNA end-repaired were achieved by
fragmentation, and each fragment was ligated to Illumina-specic adapter sequences. Furthermore, the
indexing of each sample and selection based on the size in the second step was performed. The quantity
of samples at dilution of standard concentration to a 4 nM was determined using a uorometric method.
After that, sequencing was performed using a NextSeq mid-out kit (300 cycles) on Illumina’s NextSeq
platform, following a guideline as described by the manufacturer. The resulting 400 mb of data (2x150 bp
long paired-end reads) were obtained for each sample.
For WGS analysis, each sequence (FASTQ le) was submitted to the predictive biology online server and
data science platform (KBase - https://kbase.us/) (Arkin et al. 2018). First, sequences were uploaded for
reads processing and read quality assessment was achieved using FastQC (version 0.11.5)
(Bioinformatics 2011). The removal of sequence adaptor and low-quality bases of the pair-end Illumina
raw sequence reads were processed with a exible read trimmer to obtain high-quality sequence reads
(trimmomatic (version 0.36)) (Bolger et al. 2014). Furthermore, sequence reads assembly was processed
by SPAdes online (version 3.13.0) (Nurk et al. 2013), then annotated by employing RASTtk (Rapid
Annotations using Subsystems Technology toolkit – version 1.073) and SEED online server to categorize
the distribution and functions of the predicted genes into subsystems (https://rast.nmpdr.org). The
prediction of functional protein-coding genes (PCG) was obtained from the genomic protein output after
processing in NCBI (https://www.ncbi.nlm.nih.gov/). Bioinformatics probe was performed using default
settings. For the evaluation of metabolic compounds in the bacterial genome, antiSMASH programs –
version 6.0.0 alpha 1-60 bffdb (https://antismash.secondarymetabolites.org) was used (Weber et al.
2015). The circular genome visualization with the well-informed genomic feature was generated using an
online tool (CGView) (http://cgview.ca/maps/) by uploading the genome assembly fasta le (Stothard
and Wishart 2005), while the phylogenies and pairwise comparison of the genome dataset were created
using Type (Strain) Genome online server (https://tygs.dmsz.de./) (Meier-Kolthoff et al. 2013).
2.3 Accession number of
Stenotrophomonas indicatrix
BOVIS40
From the NCBI database output, the Bioproject number is PRJNA706595, BioSample number is
SAMN18138830, while the whole genome accession number is JAGENA000000000.
2.4 Inoculum preparation and seed treatment
A seed inoculation assay was used to facilitate bacterial adherence to the disinfected sunower seeds.
The effectiveness of sunower seed inoculation was performed following the methods of Gholami et al.
(2009). The bacterial inoculum size in LB broth at 24-hour incubation was standardized to 0.5 (10
6
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CFU/ml) at OD
600
. Cleaning of the seeds was performed by washing in sterile distilled water to remove
oating-unhealthy seeds and dirt, and disinfected in 70 percent ethanol for 3 minutes, followed by 3
percent hypochlorite for 1 minute, then immersed in 70 percent alcohol for 2 minutes with nal washing
with sterile distilled water (5 times). Bacterial inoculated in LB broth was incubated on a rotary incubator
machine at 180 rpm for 24 hours. Broth culture was centrifuged to obtain the pelletized cells after
washing in 0.85% normal saline and re-suspended in the same solution. The sterilized seeds were
suspended in LB bacterial culture ntaining 1% (v/w) carboxymethyl cellulose (CMC) as an adhesive
(binder) in a 250 ml ask for 60 minutes. The seeds suspended in sterile distilled water without bacterial
inoculum serve as the control.
2.5 Greenhouse experimental study
The soil used for planting in the greenhouse was sourced from agricultural farmlands in North-West
University, Makeng Campus (Figure 1). Soil debris and other plant materials were removed, air dry, and
sieved with a 2 mm micro stainless steel mesh sieve. Equal weight of soil, approximately 5 kg, was put
inside autoclavable plastic bags and sterilized at 121
o
C for 900 seconds. This step was repeated three
times to ascertain all spore formers, vegetative cells, and forms of life were eliminated.
The inoculated and non-inoculated pots were randomly arranged by employing a complete randomized
design (CRD) approach with 8 replicates for each treatment at a 10 cm distance apart in a greenhouse
under natural light. Bacterial strains inoculated in 1 Liter LB broth were incubated in a rotary shaker (SI-
600, LAB Companion, Korea) at 180 rpm for 2 days for optimal growth density. The broth was centrifuged
(8000 x g for 600 seconds) and pelletized bacterial cells obtained were suspended in saline solution
(0.85%). The centrifugation and washing of the pellets were performed under sterile experimental
conditions.
The plastic pots measurement of 34 cm in diameter and 29 cm tall were washed with sterile water and
sterilized with 15% sodium hypochlorite solution before lling with 15 kg dry-sterilized loamy soil. The
surface-sterilized sunower seeds were inoculated with 150 ml standardized bacterial inoculum, agitated
in a shaker incubator at 120 rpm at 30
o
C for 2 hours. Then, the ltrate suspension was gently poured out
by decanting. Seeds were placed on a foil paper under sterile laminar ow (Filta Matix Laminar Flow
Cabinet) and air-dried before taken to the greenhouse. Air-drying was performed to ensure the sticking of
the bacterial inoculum to the surface of the seeds. The sterile plastic pots containing 15 kg sterile
autoclaved soil were moistened with 500 ml sterilized water before sowing. Ten seeds maximum were
sown per pot (at 1.5 cm deep). After seed emergence, i.e., at 8 days, thinning was performed, leaving one
seedling in a pot. Growing seedlings were maintained at a temperature of 30±2
o
C, a day-night cycle of
approximately 14 hours under natural light, and relative humidity of 85%. The pots were moistened with
an equal amount of water and maintained daily. Pots containing seedlings without inoculation serve as
the control. The plants were harvested at maturity after 132 days of planting.
2.5.1 Sunower morphological parameters below-and-above-ground level