R. palustris PS3 was isolated from Taiwanese paddy soil (Wong et al., 2014). It forms rod-shaped cells with approximately 1.0 µm in length and presents round, convex and glossy colony on the nutrient agar plate (Figure 1-4). The colonies are pearl-white under aerobic condition and turn to blood red when grow under anaerobic with illumination (Figure 1-4 b). It has been demonstrated that PS3 can utilize serval carbon sources such as glycerol, D-glucose and D-fructose, and fix atmospheric nitrogen into ammonia (Wong et al., 2014). PS3 possesses the PGP traits like production of IAA and synthesis of phosphatase, etc (Wong et al., 2014). PS3 not only showed beneficial effects on plant growth, but also increased the agronomic nitrogen use efficiency of plants (Wong et al., 2014). Moreover, PS3 can reduce the nitrate contents of host plant, and improve both nitrogen and carbon metabolic efficiencies of plant in hydroponic system (Hsu et al., 2015; Shen, 2016). It has already been proved that neither medium nor dead PS3 cells was able to promote plant growth (Wong et al., 2014), and both viability (i.e., culturability) and vitality (i.e., metabolic activity) of this bacterium are
crucial for the plant beneficial traits (Lee et al., 2016).
Specific aims
According to the traits mentioned above suggest that PS3 can serve as a potential PGPR for agricultural applications. However, some issues remain to be elucidated: (1) the underlying mechanisms of PS3 to promote plant growth still unknown, and (2) the scale-up fermentation is required for commercialization of R. palustris PS3. Therefore, I would like to elucidate these two themes in this study. The outlines of the contents are as follows:
Chapter II: Whole-Genome Sequencing and Comparative Analysis of Two Plant-Associated Strains of Rhodopseudomonas palustris (PS3 and YSC3)
According to the 16S rDNA analysis, the phylogenetic tree indicated that PS3 has a highly close relationship with other R. palustris isolates. However, only PS3 showed significantly plant growth promoting effects (Wong et al., 2014). To elucidate the underlying mechanisms of PS3 for promoting plant growth, I have performed the whole genome sequencing of PS3 to understand the genetic background and identify the potential genes associated with plant growth promoting by high-throughput DNA sequencing in the first part of my study. Hybrid de novo assembly was carried out by combination of shotgun (Illumina) and single molecule real-time (PacBio) reads. In addition, I compared the genome of PS3 with inefficient strain YSC3 strain to identify unique gene from PS3 strain involving in plant growth promoting functions. Moreover, I focused on genes involved in carbon and nitrogen metabolism as well as plant growth promoting genes. Through this study, I addressed the unique features of the PS3, which were attributed to its beneficial traits.
Chapter III: Development of A Low-Cost Culture Medium For Rapid Production of Plant Growth-Promoting Rhodopseudomonas palustris PS3 Strain
To scale-up the fermentation of R. palustris PS3 for its commercialization, I optimized the culture conditions of R. palustris PS3 by response surface methodology.
Firstly, “one-factor-at-a-time” technique was applied to screen the nitrogen and carbon source. Subsequently, the effects of selective nitrogen and carbon source as well as pH values, temperatures and dissolved oxygen were respectively evaluated for PS3 growth by fractional factorial design, and the suitable range of individual factor was estimated by steepest ascent path. Finally, according to the above data, I constructed a response surface model by central composite design. The optimization of fermentation conditions was analyzed by RSM. Besides, I also verified the effect of newly developed PS3 fermentation broth on the plat growth promotion.
Figure 1-1. Deduced modes of action of PGPR. PGPR can promote plant growth through various mechanisms, such as production of pytohormones and secondary metabolites. These factors enhance the development of roots, especial in lateral roots and root hairs. PGPR also affect nutrition availability through nitrogen fixation or phosphorus solubilization. Besides they are also able to mediate the physiology of plants by regulating gene expression in plant cells (Adapted from (Vacheron et al., 2013)).
Figure 1-2. Overview of the biosynthetic pathway of IAA in bacteria. The intermediate referring to the name of the pathway or the pathway itself is underlined with a dashed line. IAAld, indole-3-acetaldehyde; IAM, indole-3-acetamide; IPDC, indole-3-pyruvate decarboxylase; Trp, tryptophan (Adapted from Spaepen et al.
(2007)).
Figure 1-3. The four types of metabolism of R. palustris that support its growth.
The multicolored circle in each cell represents the enzymatic reactions of central metabolism (Adapted from Larimer et al. (2004)).
Figure 1-4. Morphology characteristics of R. palustris strain PS3. (a) colonies grown under aerobic condition for 4 days; (b) colonies developed under anaerobic condition for 7 days; (c) vegetative cells incubated aerobically. Scales bars equal 0.5 cm in (a) and (b), 10μm in (c). This figure was adapted from (Wong et al., 2014).
doi:10.6342/NTU20200342217
Table 1-1 PGPR as potential inoculants for agricultural uses. Table was made by Lo and Liu (Lo and Liu, 2020).
Strains Crop Mode of action Beneficial effect References
Achromobacter piechaudii Tomato, Pepper ACC deaminase Reduced ethylene production and increased plant
growth (Mayak et al., 2004)
Azospirillum brasilense Wheat seedlings - Enhanced photosynthetic pigment production (Bashan et al., 2006)
Alcaligenes piechaudii Lettuce IAA production Growth promotion (Barazani and
Friedman, 1999) Azospirillum lipoferum Maize Gibberellins
production
Increased ABA levels and alleviated drought
stress (Cohen et al., 2009)
Azotobacter sp. Maize Nitrogen fixation Increased nitrogen and phosphorus content of
plant component (Pandey et al., 1998)
Azotobacter chroococcum Wheat Nitrogen fixation Increased nitrogen nutrition in soil (Mrkovacki and Milic, 2001)
Azospirillum sp. IAA production,
nitrogen fixation
Enhanced root growth, lateral roots formation and increasing the total N accumulation
(Arzanesh et al., 2011; Boddey et al., 1986)
Maize Nitrogen fixation Increasing the total N accumulation (Garcia de Salamone et al., 1996)
Rice Nitrogen fixation Increase in total N accumulation (Malik et al., 1997)
Sugarcane - Production of IAA (Moutia et al., 2010)
Bacillus amyloliquefaciens (velezensis)
Triticum aestivum
IAA production Increased root production
(Talboys et al., 2014)
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Cucumber IAA production Plant growth promotion (Shao et al., 2014)
Tomato - Inhibited infection of Tomato mottle virus (Murphy et al., 2000)
Wheat Improvement in homeostatic mechanisms (Kasim et al., 2013)
Canola Production of lipopeptide antibiotics
Produced the iturin A, bacillomycin D and surfactin to suppress growth of Leptosphaeria maculans causing Blackleg disease
(Ramarathnam et al., 2011)
Capsicum Bacteriocins production
Bacterial antagonism to Ralstonia solanacearum (Hu et al., 2010)s Bacillus thuringiensis Wheat ACC deaminase Reduced volatile emissions and increased
photosynthesis
(Timmusk et al., 2014)
Bacillus subtilis Platycladus orientalis
Cytokinin production Increased ABA levels in shoots and enhanced the stomatal conductance
(Liu et al., 2013b) Soybean IAA production Increased production of root hairs (Araújo et al., 2005) Pepper Production of
antibiotics
Suppression of growth of Myzus persicae (Kokalis–Burelle et al., 2002)
Tomato Induce systemic resistance
Enhanced activities of chitinases and β-1,3- glucanase to inhibited growth of Fusarium oxysporum f. sp. lycopersici causing Tomato wilt disease
(Shanmugam and Kanoujia, 2011)
Surfactin production Surfactin production induced the activation of lipxygenase to against Botrytis cinerea growth
(Ongena et al., 2007) Bacillus pumilus Ocimum
sanctum
IAA production Plant growth promotion (Murugappan et al.,
2013)
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Bradyrhizobium sp. Radish IAA production Increased the dry matter yield of radish (Antoun et al., 1998) Burkholderia sp. Rice Nitrogen fixation Increase in N content in plant (Divan Baldani et al.,
2000) Enterobacter cloacae Rapeseed
(Brassica.
napus)
ACC deaminase Increases in root and shoot lengths (Saleh and Glick, 2001)
ACC deaminase Promoted root elongation (Madhaiyan et al.,
2006)
Pseudomonas fluorescens Pisum sativum ACC deaminase Induced longer roots and uptake of water (Zahir et al., 2008)
Soybean Cytokinin Plant growth regulation (García de Salamone
et al., 2001) Wheat ACC deaminase Increased NPK uptake and inhibited ethylene
production
activation of PR-1 gene against Pseudomonas syringae pv. tabaci
(Park and Kloepper, 2000)
Green gram (Vigna radiata)
- Regulated the catalase and peroxidase (Saravanakumar et
al., 2011) Rice Induction of systemic
resistance
Against Xanthomonas oryzae pv.Oryzae in rice leaves
(Vidhyasekaran et al., 2001)
Pseudomonas putida Vigna radiata L ACC deaminase Inhibited ethylene production (Mayak et al., 1999)
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Rice IAA production and phosphate
solubilization
Increased plant height and root length of rice (Ashrafuzzaman et al., 2009)
Soybean Gibberellins production
Plant growth promotion (Kang et al., 2014)
Chickpea Phosphate solubilization, siderophore
production, and IAA production
Improved the growth and the saline tolerance of plant
(Patel et al., 2012)
Cotton - Regulated the ion uptake and improved
production of endogenous indole acetic acid (IAA) content and reduced abscisic acid (ABA) content
(Yao et al., 2010)
Rhizobium leguminosarum Rape and lettuce Cytokinin production Plant growth promotion with possible involvement of the plant growth regulators indole-3-acetic acid and cytokinin
(Noel et al., 1996)
Rhizobium tropici Bean (Phaseolus vulgaris L.)
- Increased nodulation as well as nitrogen fixation (Figueiredo et al., 2008)
Rhodobacter sphaeroides Tomato - Enhanced quality of tomato fruit and increased ascorbic acid content
(Kondo et al., 2010) Rhodopseudomonas Chinese - Plant growth promotion, increased the nitrogen use (Hsu et al., 2015;
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palustris cabbage
(Brassica rapa chinensis)
efficiency and reduced the nitrate content in plant Wong et al., 2014)
Tobacco Indole‐3‐acetic acid and 5‐aminolevulinic acid production
Promote growth and germination (Su et al., 2017)
Induces systemic resistance
Induces systemic resistance of plant to against tobacco mosaic virus
(Su et al., 2017) Pakchoi
(Brassica rapassp.
Chinensis)
- Enhanced photosynthesis and crop yield (Xu et al., 2016)
CHAPTER II
Whole-Genome Sequencing and Comparative Analysis of Two Plant-Associated Strains of Rhodopseudomonas
palustris (PS3 and YSC3)
The content in Chapter II has been published in Scientific Reports as shown below. This first author publication and its quality full-filled the Ph.D. thesis examination application requirements of Institution of Biotechnology, National Taiwan University.
This desertion or any part of it has not been submitted for any degree, diploma, or other qualification at any other university. It is the result of my own work except where mentioned in the text.
Lo, K.J., S.S. Lin, C.W. Lu, C.H. Kuo and C.T. Liu. 2018. Whole-genome sequencing and comparative analysis of two plant-associated strains of Rhodopseudomonas palustris (PS3 and YSC3). Sci. Rep. 8(1): 12769. doi: 10.1038/s41598-018-31128-8
Author Contributions:
K.J. Lo carried out the experiment, experimental data analysis, bioinformatic data analysis and manuscript writing. C.W. Lu performed the bioinformatics analysis for the genome assembly. S.S. Lin provided the bioinformatic platform for data analysis. C.H.
Kuo performed the bioinformatics for gene prediction and annotation as well as manuscript writing. C.T. Liu is the corresponding authors in charge of the project design and manuscript writing.
Summary
Rhodopseudomonas palustris strains PS3 and YSC3 belong to purple non-sulfur phototrophic bacteria that were isolated from Taiwanese paddy soils. Strain PS3 showed beneficial effects on plant growth and enhances the agronomic nitrogen using efficiency of host plant. However, strain YSC3 has no significant effect on plant growth.
According to whole genomic analyses, PS3 and YSC3 strain showed similar genomic structures, individually contains a one circular chromosome with 5,269,926 or 5,371,816 bp in size, with 4,799 or 4,907 protein-coding genes, respectively. In this study, a large class of genes associated with plant-growth promotion, such as nitrogen fixation-, IAA synthesis-, phosphate solubilization and ACC deamination-related genes, were annotated. The growth rate, biofilm formation, and the relative expression levels of several chemotaxis-associated genes were significantly higher for PS3 than for YSC3 upon treatment with root exudates. These results suggested that PS3 has a better response to the host plants, which may contribute to the successful interactions between PS3 and plant hosts. In addition, these findings indicate that the existence of gene clusters associated with plant growth promotion is required but not sufficient for bacteria to exhibit the ability of plant-growth promotion.
Introduction
In 1978, a concept of plant growth-promoting rhizobacteria (PGPR) was proposed by Kloepper and Schroth (Kloepper and Schroth, 1978). It refers as diverse soil bacteria colonize in rhizosphere, and provide beneficial effects on plant growth via various mechanisms (Kloepper and Schroth, 1978). The promotional activity of PGPRs including increasing nutrient availability (e.g., nitrogen fixation), nutrient solubilization (e.g., phosphate solubilization) as well as production of phytohormones (e.g., indole acetic acid (IAA), 2,3-butanediol, and cytokinins) (Ahemad and Kibret,
2014; Goswami et al., 2016; Lugtenberg and Kamilova, 2009). Moreover, PGPR can strengthen plant tolerance against environmental stress by metabolizing 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor of stress hormone - ethylene (Ahemad and Kibret, 2014; Goswami et al., 2016; Lugtenberg and Kamilova, 2009).
In addition, PGPR can also protect plants form pathogen infection by secreting antibiotics or activating induced systemic resistances (Beneduzi et al., 2012). Due to these properties of PGPR, so far, these bacteria are widely used as biofertilizers or biocontrol agents in agriculture (Lugtenberg and Kamilova, 2009). So far, several micrograms were identified as PGPR, such as Azospirillum humicireducens (Yu et al., 2018), Bacillus amyloliquefaciens (Zhang et al., 2015b), Bacillus velezensis (Chen et al., 2007), Bradyrhizobium japonicum (Kaneko et al., 2002), Pseudomonas fluorescens (Loper et al., 2007), Pseudomonas putida (Ponraj et al., 2012), Rhizobium leguminosarum (Young et al., 2006), etc. These microbes were regarded as PGPR not only verified by planta experiments, but also speculated by their functional genes in genome.
Rhodopseudomonas palustris is a phototrophic purple non-sulfur bacterium (PNSB), which has the innate and extraordinary metabolic versatility. It can sustain itself by one of the four modes of metabolism, including photoautotrophic, photoheterotrophic, chemoautotrophic and chemoheterotrophic states (Larimer et al., 2004). Due to this diverse metabolic property, R. palustris is widely distributed in nature, including river, pond water, sediments, wetlands, and paddy fields (Hiraishi and Kitamura, 1984; Oda et al., 2002).
This bacterium has been widely used in industrial applications for bioremediation and sewage treatment and for the removal of phytotoxic compounds (Austin et al., 2015; Idi et al., 2015). In addition, this bacterium can convert complex organic compounds into biomass and bioenergy using substrates that are plant-derived
compounds, pollutants, or aromatic compounds (Larimer et al., 2004; Liu et al., 2015;
Oda et al., 2003; Shi et al., 2014; Zhang et al., 2015a). Some studies have indicated that R. palustris can also be used as a biofertilizer to improve crop yields (Kornochalert et al., 2014; Nunkaew et al., 2014; Wong et al., 2014). In our previous study, we isolated the R. palustris strain PS3 (abbreviated as PS3) from Taiwanese rice paddy soil (Wong et al., 2014). Strain PS3 can have beneficial effects on plant growth and can enhance the utilization efficiency of fertilizers in either soil or hydroponic cultivation system (Hsu et al., 2015; Wong et al., 2014). Although these studies showed that strain PS3 is a promising PGPR, genomic information and the underlying molecular mechanisms for plant growth promotion (PGP) by PS3 are yet to be ascertained.
Systematic analysis of whole-genome sequences is a powerful strategy to identify either causal genes that contribute to plant growth-promoting activities or potential PGPR candidates (Gupta et al., 2014; Liu et al., 2016a; Magno-Perez-Bryan et al., 2015;
Shen et al., 2013; Taghavi et al., 2010). Scientists can obtain more genomic information and deduce the underlying promoting mechanisms of PGPR from whole genome sequence. As shown in Table 2-2, I listed up the whole genomic information of several PGPR strains. Furthermore, the genes associated with plant growth-promoting functions were summarized in Table 2-3.
Next-generation sequencing (NGS) technologies provide a quick and convenient approach to resolve the whole-genome sequence and investigate the transcriptomes of PGPR. The NGS technologies such as Roche/454, Illumina are widely used to study the genes of eukaryotes and prokaryotes (MacLean et al., 2009). These methods used short-read (~150-300 bp) sequencing and containing some advantages, such as cost-effective, accurate, and diversity of analysis tools (Heather and Chain, 2016). However, there were some limitations of short-read sequencing, especially in highly repetitive and complex genome, including high guanine-cytosine (GC) contents, or multiple
homologous elements in sequences (Nagarajan and Pop, 2013; Treangen and Salzberg, 2011). It may result in sequencing error and lose certain genomic regions (Ashley, 2016;
Delaneau et al., 2013; Mavromatis et al., 2012). Although using the long-read sequencing technologies such as Oxford Nanopore Technologies (ONT)(Deamer et al., 2016) or single-molecule sequencing technology (SMRT) Pacific Biosciences (PacBio) sequencing platforms (Levene et al., 2003; Quail et al., 2012) can resolve the above issues, these technologies have high relative error in sequencing with about 11-14%
(Pollard et al., 2018). Therefore, it is hard to assemble the complete genomic sequences with high-quality by individual sequencing technology. To address these issues, short-reads and long-short-reads hybrid assembly techniques were proposed to improve the genome assembly (Utturkar et al., 2014). In this strategy, long reads provide the information for genomic structure and short reads improve the detailed assembly at sequencing, and can be used to correct the errors from long reads. Therefore, combination of short-read and long-read sequencing datasets appear a promising approach to completely resolve the genome assemblies with accuracy (Berbers et al., 2020; De Maio et al., 2019; Risse et al., 2015; Sovic et al., 2016; Wick et al., 2017a; b).
Although few studies conducted the whole-genome sequencing of R. palustris strains, such as CGA009, HaA2, BisB18, and TIE (Larimer et al., 2004; Oda et al., 2008; Oda et al., 2005), none of these strains are plant-associated. Here I focus on the elite strain R. palustris PS3, which was isolated from Taiwanese paddy soil and displayed plant growth-promoting (Hsu et al., 2015; Wong et al., 2014). In order to elucidate the potential modes of action via which R. palustris PS3 has beneficial effects on plants, I compared the genomic characterizations of two plant-associated R. palustris strains- PS3 and YSC3. They are the effective PGPR strain and the ineffective strain, respectively (Wong et al., 2014). For whole-genomic sequencing, I applied two NGS techniques to obtain the sequencing reads, then performed the hybrid de novo assembly.
Identification of genes associated with plant growth-promotion and genomic comparative analyses are very important to understand the underlying mechanisms of PS3. Moreover, I also compared the genomic compositions of these two strains with the genomic representative strain for this species, R. palustris CGA009. Finally, I focused on genes involved in carbohydrate, nitrogen metabolism, phosphate solubilization, phytohormone production, biofilm formation, chemotaxis, and plant colonization. The flow chart of the whole genomic analysis in this chapter was described in Figure 2-1.
Materials and methods
Preparation of phototrophic bacterial inoculant
The R. palustris strains PS3 and YSC3 are PNSB and were both isolated from Taiwanese paddy soils (Wong et al., 2014). PS3 is an effective PGPR, whereas YSC3 is not. For bacterial inoculant preparation, a single colony was picked and inoculated into 3 mL of PNSB broth as described previously (Wong et al., 2014). The culture was then incubated for 24 h at 37°C (200 rpm). Subsequently, 2.5 mL of these cultures were transferred into 250-mL Erlenmeyer flasks containing 50 mL of fresh PNSB broth. The cultures were incubated under the conditions described above, and the log-phase bacterial cells were harvested for genomic DNA extraction.
Genomic DNA preparation
A 1-mL suspension of the log-phase bacterial culture was collected in a 2.0-mL Eppendorf tube and centrifuged at 3,000 × g at 4°C. Subsequently, the supernatant was removed, and the Eppendorf containing the cell pellet was snap-frozen in liquid nitrogen. The cell pellet was homogenized by adding sterile steel beads and rapidly shaking the microcentrifuge tubes back and forth at 9,000 rpm for 1 min in a SH-100 homogenizer (Kurabo, Japan). The homogenization process was repeated three times.
The Gentra® Puregene® Kit (QIAGEN) was used for genomic DNA purification according to the manufacturer’s protocol. The quantity and quality of the total DNA was assessed using UV spectrophotometry (Nanodrop ND-1000, J & H technology Co., Ltd.), and the OD260/280 value of the DNA was higher than 1.80. Agarose gel electrophoresis (0.75%) was used to ensure that the gDNA was intact. Samples containing greater than 25 μg of gDNA were used to perform whole-genome sequencing.
Whole-genome sequencing
We utilized the MiSeq (Illumina) and the PacBio RSII (Pacific Biosciences) platforms to perform whole-genome shotgun sequencing. The sequencing service was provided by Genomics BioSci & Tech Co., Ltd. (New Taipei City, Taiwan). For Illumina MiSeq sequencing, the DNA library was constructed using the Illumina TruSeq Nano DNA HT Sample Prep Kit according to the TruSeq DNA Sample Preparation protocol (Illumina). This library was diluted and sequenced with 600 paired-end cycles on the Illumina MiSeq instrument by following the standard protocol.
For the PS3 strain, the insert size was 500 bp, and 10,471,982 read-pairs and ~3.6 Gb of raw data were obtained; and for the YSC3 strain, the insert size was 500 bp, and 11,242,474 read-pairs and ~3.8 Gb of raw data were obtained. For PacBio SMRT sequencing, the DNA library was constructed according to the PacBio SampleNet – Shared Protocol (Pacific Biosciences). After dilution, the library was loaded onto the instrument with the DNA Sequencing Kit 4.0 v2 (part number PB100-612-400) and a SMRT Cell 8 Pac for sequencing. A primary filtering analysis was performed with the RS instrument, and a secondary analysis was performed using the SMRT analysis pipeline, version 2.1.0. For the PS3 strain, the average length of the reads was 7,112 bp, and 164,831 reads and ~1.1 Gb of raw data were obtained; and for the YSC3 strain, the
average length of the reads was 6,342 bp, and 192,795 reads and ~1.2 Gb of raw data
average length of the reads was 6,342 bp, and 192,795 reads and ~1.2 Gb of raw data