LITERATURE REVIEW
Rice (Oryza sativa) is an important cereal crop as well as a major staple food for about one half of the world’s population It also significantly contributes to income for growers from production, marketing and processing Different management, techniques and applications are used to increase crop yields For the main goal, intensive farming practices are getting to high yield, and it requires extensive use of chemical fertilizers, insecticides, fungicides and herbicides, which are costly and have numerous negative environmental impacts On the other hand, global consumption of rice has seen a slight increase over the last several years In the 2018/2019 crop year, about 490.27 million metric tons of rice was consumed worldwide, up from 437.18 million metric tons in the 2008/2009 crop year (https://www.statista.com) The International Rice Research Institute (IRRI 2000) studied the food problem in relation to world population, and they predict that 800 million tons of rice will be required in 2025 An important constraint to meeting this increased demand for food is climate change The decline in agricultural productivity due to climate change may be partly alleviated by plant breeding programs Improvement to abiotic stress tolerance has long been a target for breeding programs
Inoculating crops with plant growth-promoting rhizobacteria (PGPR) offers a promising solution to mitigate unexpected environmental impacts from chemical fertilizers while also improving plant resilience to various abiotic stresses This research contributes to the advancement of PGPRs for application across a wide range of crops within diverse global cropping systems.
The Bacillus pumilus TUAT1 strain, a type of plant growth-promoting rhizobacteria (PGPR), is a key component of the commercially available "Kikuichi" biofertilizer Research has indicated that applying "Kikuichi" or TUAT1 during the nursery stage significantly enhances seedling growth and overall yield in rice and other plants (Djedidi et al 2014; Win et al 2018; Seerat et al 2019) However, variations in the inoculation effects have been noted among different rice varieties, as well as within the same variety.
This chapter provides a brief overview about previous studies on biofertilizer and PGPR, salt tolerance, and genome wide association study (GWAS)
Roles and problems of biofertilizers in agriculture and food security
By 2050, the global population is projected to reach 9.6 billion, necessitating a doubling of current agricultural production levels To meet this demand, cropping systems must evolve to enhance productivity while reducing reliance on chemical fertilizers and pesticides, thereby ensuring sustainable food production Current agricultural trends emphasize the exploration of sustainable and eco-friendly practices that improve soil health and crop yields Among these practices, the use of biological fertilizers has gained traction, promoting a reduction in chemical inputs Various natural resources, such as crop residues, sewage, manure, and beneficial microorganisms, can serve as biofertilizers However, a significant challenge with biofertilizers, particularly those containing plant growth-promoting rhizobacteria (PGPR), is the inconsistency in their effectiveness, coupled with unclear mechanisms of growth promotion, complicating the identification of optimal application methods.
The rhizosphere is a critical zone surrounding plant roots where exudates promote microbial growth, significantly influencing plant-microbe interactions In this environment, plants and microbes communicate through various signal molecules, resulting in specific physiological responses The nature of these interactions varies depending on the species of plants and microorganisms involved, with soil bacteria potentially having beneficial, harmful, or neutral effects on plants This study specifically aims to explore the beneficial interactions between soil bacteria and plants.
Plant growth promoting rhizobacteria (PGPR) are beneficial bacteria that colonize the rhizosphere and support plant growth These free-living rhizobacteria can be categorized into two groups: intracellular PGPR (iPGPR) and extracellular PGPR (ePGPR) iPGPR reside within root cells, leading to the formation of specialized structures called nodules, with most being Gram-negative, rod-shaped, nodule-forming rhizobia In contrast, ePGPR exist outside of root cells in the rhizosphere, rhizoplane, or root cortex, where they thrive on nutrients like amino acids and sugars from root exudates Both types of PGPR enhance plant growth through various direct and indirect mechanisms.
Plant Growth-Promoting Rhizobacteria (PGPR) exhibit diverse mechanisms for promoting growth and biological control, which vary among species and strains These mechanisms can be classified into direct and indirect actions, with some strains employing multiple methods depending on the plant species they colonize Directly, PGPR function as biofertilizers, supplying essential nutrients like nitrogen and phosphorus, and act as phytostimulators by enhancing plant hormone production and facilitating biological processes such as nitrogen fixation and nutrient solubilization Indirectly, PGPR serve as biocontrol agents by suppressing harmful microbes through the release of antibiotics, competition for resources, and inducing systemic resistance in plants.
Plant Growth-Promoting Rhizobacteria (PGPRs) enhance nutrient availability, control plant stress, and selectively stimulate root system development Research indicates that PGPR inoculation significantly boosts crop root growth (Somers et al 2004; Contesto et al 2010; Ibiene et al 2012; Posada et al 2016) Despite this, there is limited published research on PGPRs' impact on plant gene expression related to root development and growth (Vacheron et al 2013), suggesting that they may promote root formation by modulating auxin signaling (Zhao et al 2018).
PGPR (Plant Growth-Promoting Rhizobacteria) have the potential to enhance plant growth by stimulating root development, although the exact mechanisms remain unclear Improved root development may lead to better plant nutrition, making it a crucial factor in growth promotion Recent research on rice roots highlights the significant relationship between above-ground traits and underground root systems, offering promising avenues for genetic improvement Increasing attention is being given to the genes associated with root architecture and physiological functions, yet root research remains challenging due to the complexities of the underground environment Intensive studies focus on rice root traits related to water and nutrient uptake, revealing that these traits are governed by intricate and possibly redundant gene networks and genetic loci.
Rice exhibits a complex root system comprised of various root types, including primary, lateral, and adventitious roots, with crown roots being essential to the fibrous root structure The formation of crown roots is regulated by specific genes, particularly the transcription factor CROWN ROOTLESS5 (CRL5), which is influenced by the AUXIN RESPONSE FACTOR (ARF) gene family, linking root development to auxin signaling CRL5 facilitates crown root initiation by repressing cytokinin signaling Additionally, the WUSCHEL-related homeobox gene WOX11 plays a crucial role in regulating rice root development, cytokinin signaling, and abiotic stress resistance, particularly in the crown root meristem Research indicates that WOX11 acts as a direct activator of multiple genes involved in these pathways, highlighting its importance in root development and signaling processes.
An increasing number of plant growth-promoting rhizobacteria (PGPR) are being marketed as biocontrol agents and biofertilizers, with effective strains being essential for optimal performance Bacillus species are particularly favored due to their ability to maintain high cell viability and prolonged shelf-life, as they can form endospores to survive harsh conditions While Bacillus spp have been commonly used in plant growth research, there is limited information on the efficacy of vegetative cells as inocula Despite the resilience of spores, both spores and vegetative cells have been shown to enhance plant growth without significant differences in effectiveness Understanding the distinct behaviors of these two cell types on plants necessitates further investigation into how bacterial colonization varies over time between plants inoculated with spores and those with vegetative cells.
The effectiveness of PGPR-based products is influenced by various factors, including plant species and variety, which release different root exudates, as well as the microbial community and commercial formulation Additionally, environmental factors such as soil properties, climatic conditions, and farming practices play a significant role in the success of bacterial inoculation Therefore, to utilize microorganisms as biofertilizers effectively, it is essential to focus on stabilizing the fertilization effect and the preservation methods of these materials.
Plants face increasing biotic and abiotic stresses due to climate change, particularly drought, flooding, extreme temperatures, and salinity, which significantly reduce crop yields (Bray et al 2000) Soil salinity, exacerbated by rising sea levels, poses a severe threat to global agriculture, especially in the Mekong Delta of Vietnam, where over half of its 4 million hectares are dedicated to farming (FAO 2004) The region's low topography and extensive coastline have led to expanding seawater intrusion, jeopardizing its vital rice production across 13 provinces (Hook et al 2003) From late December 2019 to February 2020, Vietnam's rice basket faced critical drought and saltwater intrusion, resulting in agricultural losses exceeding VND 4.6 trillion, affecting 232,000 hectares of paddy and damaging crops nearly 100 kilometers inland.
Salinity significantly affects plant growth when Na+ concentration in the growth substrate reaches 4 dS/m or higher, equating to roughly 40 mM NaCl and an osmotic pressure of 0.2 MPa (Bernier et al 2008) Most terrestrial plants are glycophytes, which struggle to complete their life cycle at these salt levels, while halophytes thrive in much higher salinity conditions (Hasanuzzaman et al 2014) Among glycophytic crops like wheat, barley, and rice, tolerance to salinity varies widely, with wheat and barley being the most tolerant cereal crops, whereas rice is notably salt-sensitive (Bado et al 2016) Additionally, significant differences in salt tolerance can exist even within the same plant species.
The use of PGPR inoculants offers a promising strategy to address salinity in agriculture through both direct and indirect mechanisms that promote systemic tolerance A comprehensive understanding of how glycophytes respond and the mechanisms involved can enhance the application of PGPR for bioremediation purposes.
Genome wide association study (GWAS)
SPORE INOCULATION OF BACILLUS PUMILUS TUAT1 STRAIN PROMOTES
A high-quality biofertilizer should contain effective strains that enhance crop growth and yield at optimal densities Bacillus species are particularly favored in commercial formulations due to their ability to maintain high cell viability and extended shelf-life, thanks to their endospore formation in stressful conditions This survival mechanism allows Bacillus to thrive again when conditions improve While Bacillus spp have been widely used in plant growth research, there is limited information on the use of vegetative cells as inocula Spore-forming bacteria are anticipated to be more effective as plant growth-promoting rhizobacteria (PGPRs) than non-spore formers, although both cell types have been shown to enhance plant growth without significant differences in efficacy Understanding the distinct behaviors of spores and vegetative cells on plants necessitates further research, particularly in examining the dynamics of bacterial colonization over time in plants inoculated with each type.
According to Win et al (2018), the growth and yield of rice in the treatment TUAT1 + 50%
The B pumilus TUAT1 strain significantly enhances root growth in "Hitomebore," leading to improved nutrient uptake from the soil, as noted by Ali et al (2018) Despite these findings, the specific root system genes associated with plant growth promotion by PGPRs remain unclear.
Nitrate reductase (NR) plays a vital role in nitric oxide (NO) synthesis, which regulates plant growth and acts as a signaling molecule for crown root formation Research by Higuma (2017) demonstrated that NR1 expression significantly increased, leading to NO production, after inoculating the B pumilus TUAT1 strain Similarly, studies have shown that hot pepper (Capsicum chinense Jacq.) infected with the oomycete Phytophthora capsici exhibited an immediate increase in NR gene expression and NO production (Caamal-Chan et al., 2011) In potatoes infected with pathogenic bacteria, NR gene expression rose rapidly, resulting in NO production three hours post-inoculation (Floryszak-Wieczorek et al., 2016).
Nitric oxide (NO) plays a dual role in plants, acting as a signaling molecule in defense against pathogens while also promoting crown root emergence in rice (Mai et al 2014) To confirm the specificity of NO-dependent fluorescence, the use of the NO scavenger cPTIO is essential, as it has been shown to enhance fluorescence (Arita et al 2007) Among the various fluorescent probes for NO detection, diamine derivatives of fluorescein, specifically DAF-4-amino-5-methylamino-2′7′-difluorofluorescein diacetate, are favored for their high sensitivity and straightforward application (Namin et al 2013) These probes react with NO in the presence of oxygen, resulting in a strong green fluorescence (Kojima et al 1998).
This chapter investigates the colonization of rice seedlings by B pumilus TUAT1 strain, focusing on the accumulation of bacteria in various parts of the seedlings after inoculation with both spores and vegetative cells The study examines how these inoculations affect growth, root system development, and the expression of related genes Results indicate a significant relationship between the colonization effects and the form of TUAT1 used To understand the mechanisms behind crown root formation promotion by TUAT1, a site-specific gene expression analysis was conducted on genes associated with root formation at the stem base Additionally, the research explored the potential role of nitric oxide (NO) production in the growth-promoting effects of TUAT1 on rice seedlings, utilizing NO scavengers and fluorescent probes for analysis.
Plant material and growth conditions
Oryza sativa cv "Hitomebore" was used as the plant material Seeds were sown to a depth of
Seeds were germinated in plastic trays with 30 wells, each containing 70 g of autoclaved Shinano soil, which is rich in nutrients with approximately 375 mg N kg−1, 750 mg P2O5 kg−1, and 375 mg K2O kg−1 The seedlings were kept in a growth chamber under a controlled environment with a 12-hour light cycle at 100 μmol m−2 s−1 and 12 hours of darkness, maintaining a temperature of 25°C and 70% humidity Watering was performed every two days, ensuring consistent moisture levels.
Bacterial strain and culture conditions
The B pumilus TUAT1 strain, isolated from soil at the Tokyo University of Agriculture and Technology, has its complete genome sequence available in GenBank under Accession Number AP014928 (Okazaki et al 2019) This strain is preserved in our laboratory, retrieved from -80°C frozen glycerol cultures and cultured in trypticase soy broth (TSB) Vegetative cell suspensions were prepared at a concentration of 1x10^9 CFU/mL and diluted to 1x10^7 CFU/mL with distilled water, ensuring no spores were present To induce sporulation and eliminate vegetative cells, the suspension was incubated at 65°C for 1 hour, followed by the addition of sodium chloride to achieve a final concentration of 0.85%, adjusting the density back to 1x10^7 CFU/mL, consisting solely of spores.
In a study examining the effects of B pumilus TUAT1 spores on seedling growth, it was found that seedlings inoculated with a suspension of 1x10^7 CFU/mL showed the most significant growth (Saito 2019) The experiment utilized a completely randomized design with 20 plants per treatment to assess the impact of two different cell types—vegetative cells and spores—at the same concentration Each inoculation involved administering 4 mL of the bacterial suspension per well, applied dropwise to rice seeds at sowing and subsequently to seedlings weekly Three treatment groups were established: (1) distilled water as a control, (2) spore suspension, and (3) vegetative cell suspension.
Bacterial colonization in rice seedlings
The colonization of B pumilus TUAT1 on roots and stem bases was assessed using the method outlined by Seerat et al (2019) Samples were collected at three different intervals: 7, 14, and 21 days post-sowing Serial dilutions of 10^-1 and 10^-2 were prepared, and 10 µL of each dilution was plated After incubating the samples at 28°C for two days, colony counts were recorded and expressed as colony-forming units (cfu) per gram of root fresh weight (FW).
Growth and morphology of rice seedlings
Three weeks post-inoculation, 12 randomized plants per treatment were harvested to assess the impact of bacterial inoculum on plant growth This evaluation involved measuring plant height, fresh weights (FWs), and dry weights (DWs) of both shoots and roots from control and inoculated plants Specifically, the roots and shoots from five plants within each treatment group were pooled and weighed to determine FWs Subsequently, the pooled roots and shoots for each treatment were dried at 65°C for further analysis.
72 h and weighed again to give the DWs The root morphology was assessed using a root scanner
The Epson Perfection V700 scanner was utilized for data collection, and the analysis was conducted using WinRHIZO software, as outlined by Haidari et al (2017) Key root parameters recorded included total root length, mean root length, the number of lateral roots, and lateral root density.
Analysis of gene expression using real-time qRT-PCR
The expression levels of root development-related genes (WOX11, CRL5, ARF1, and IAA13) in the stem base of seedlings inoculated with TUAT1 or a control were analyzed using real-time RT-qPCR Seedlings, harvested 24 hours post-inoculation at two weeks after sowing, were carefully rinsed to remove soil with tap water and then washed with distilled water After gently drying the samples, five basal stems (approximately 100 mg) were collected to create a single sample for analysis.
Each sample was placed in a 2 ml tube, frozen in liquid nitrogen, and disrupted using Tissue Lyser LT (QIAGEN) After adding 1 ml of Fruit-mate (Takara Bio Inc.) and mixing thoroughly, the samples were centrifuged at 12,000xg for 5 minutes at 4°C The supernatant was divided into new 1.5 ml tubes, to which 0.5 ml of RNAiso plus (Takara Bio Inc.) was added, allowing the mixture to stand at room temperature for 5 minutes Following the addition of 0.2 ml of chloroform, the mixture was inverted vigorously and allowed to stand for another 5 minutes After centrifugation at 12,000xg for 20 minutes at 4°C, the aqueous layer (600 to 700 µl) was transferred to a new tube, and half of the High Salt Solution for Precipitation (Plant) (Takara Bio) was mixed in, followed by isopropanol The mixture was left at room temperature for 10 minutes before being centrifuged at 13,000xg for 15 minutes at 4°C The supernatant was discarded, and the precipitate was washed with 1 ml of 75% ethanol, then centrifuged again at 7,500xg for 5 minutes at 4°C Finally, the precipitate was dried at room temperature for 5 minutes.
The extracted RNA was dissolved in a 10 mM Tris-HCl buffer, with concentrations measured using a Nano Drop device and adjusted to between 0.1 ng/µl and 5 ng/µl Subsequently, the RNA concentration was standardized to 100 ng/µl using the dsDNA Assay Kit and Qubit 2.0 Fluorometer (Thermo Fisher Scientific) First strand cDNA synthesis was carried out with 1 µg of total RNA using the Primer Script RT reagent kit (Takara Bio Inc.) in a 20 µl reaction mixture, following the manufacturer's guidelines The resulting cDNA products were diluted ten-fold with TE buffer before being utilized as templates in qRT-PCR Real-time RT-qPCR was conducted on an Eco Real-Time PCR System (Illumina, Tokyo, Japan) with gene-specific primers (refer to Table 1) and Fast SYBR Green Master Mix (Life Technologies, Tokyo, Japan), adhering to the manufacturer's instructions for cycling conditions.
The PCR amplification process involved an initial denaturation step at 95°C for 10 minutes, followed by 40 cycles comprising denaturation at 95°C for 10 seconds and annealing at 60°C for 30 seconds The Actin1 gene served as an internal control, and all reactions were performed with four biological replicates to ensure reliability.
Evaluation of NO production in the stem base of rice seedlings
Three treatments were used: (1) non-inoculated seeds (control) treated with distilled water;
THE EFFECTS OF TUAT1 INOCULATION IN THE GROWTH OF RICE
THE EFFECTS OF TUAT1 INOCULATION IN THE GROWTH OF RICE SEEDLINGS
To enhance plant tolerance to salinity, various strategies have been explored, including the use of organic fertilizers like green manure and vermicompost, traditional breeding, and genetic engineering Among these, the application of plant growth-promoting rhizobacteria (PGPR) has emerged as a promising method to boost plant growth and mitigate abiotic stresses, particularly salinity Notably, Bacillus species of PGPR have shown significant potential in promoting growth and increasing stress tolerance in rice.
The Bacillus pumilus TUAT1 strain has demonstrated significant plant growth promotion effects, benefiting not only rice but also various plants, including Brassica species.
The TUAT1 strain exhibits varying effects on different rice varieties, as demonstrated in a study that evaluated its inoculation impact on "Koshihikari" and "Leaf Star." Both varieties showed increased yields; however, the rate of increase differed significantly (Ueno, 2011) Additionally, Hoshino (2013) conducted a comparison of the TUAT strain's effects across upland rice varieties, further highlighting the strain's variable influence on rice cultivation.
In a study examining the effects of inoculation on different rice varieties, "Owari Hatamochi" exhibited an increase in plant height, although there was a decrease in root number and dry root weight Conversely, inoculation led to a reduction in plant height for "Leaf Star" and "Koshihikari," while their shoot number and dry root weight increased, particularly with a significant rise in crown roots These findings suggest that the factors promoting plant growth may vary based on the rice variety (Minota 2014).
A study by Yamaya (2014) revealed differing effects of the TUAT1 strain on root growth between Japonica and Indica rice varieties, with growth suppressed in "Takanari" and accelerated in "Hitomebore." To explore these varietal differences further, Higuma (2017) compared the TUAT1 strain's effects on 23 rice varieties from a Japanese landrace core collection, including Indica types The findings indicated significant variability in fresh weight and crown root numbers among the varieties, suggesting that inoculation effects may influence stress tolerance differently across varieties, particularly between Japonica and Indica Overall, the results highlighted that the TUAT1 strain promotes growth in certain varieties while suppressing it in others, indicating a complex interaction between rice variety and inoculation effects.
To address this issue, in this chapter, 4 varieties (Oryza sativa cv "Kasalath", "Nipponbare",
The study focused on the impact of the TUAT1 strain on rice seedlings under salt stress, specifically evaluating the cultivars "Hitomebore" and "Koshihikari." Results showed that the Bacillus pumilus strain TUAT-1 significantly enhanced the growth of "Hitomebore" seedlings, which are known for their high tolerance to severe hot water treatment (Kashiwagi et al 2017) "Koshihikari," being the most widely cultivated rice variety in Japan, was also included in the initial screening Additionally, "Kasalath" and "Nipponbare" were selected for their inclusion in the rice core collection utilized in Chapter 4 of the study.
Current research lacks reports on the impact of PGPR treatment on rice growth under salt stress over time However, findings from chapter 2 indicate that PGPR treatment leads to an increased number of crown roots and a higher growth rate Notably, the benefits observed from spore treatment were more significant than those from vegetative cell treatment.
This chapter aims to evaluate the hypothesis that inoculating rice seedlings with TUAT1 strain spores enhances their early growth under salt stress by boosting salt tolerance Additionally, it investigates the extent of salinity tolerance conferred by TUAT1 inoculation across different saline concentrations and rice varieties.
Plant material and growth conditions
Oryza sativa cv "Kasalath", "Nipponbare", "Hitomebore", and "Koshihikari" were used as the plant material
Surface-sterilized seeds, treated with a 2% NaOCl solution for 10 minutes and rinsed in sterile distilled water, are sown in soil after being kept under running water for five days Each seed is planted in a plastic pot at a depth of about 1.0 cm The seed trays are then placed in a growth room maintained at 25°C with a 12/12 hour light/dark photoperiod and 70% humidity for five weeks.
Bacterial strain, culture conditions and inoculation
In this chapter, we utilized the B pumilus TUAT1 strain, cultured under the same conditions as described in Chapter 2, with a liquid suspension concentration of 1 x 10^7 CFU/mL Each inoculation involved administering 4 mL of this bacterial suspension per well, applied dropwise to the rice seeds right after sowing and subsequently to the seedlings once a week.
Salinization treatments were implemented by incorporating NaCl solutions at concentrations of 50 mM (2.9 g/L), 100 mM (5.85 g/L), and 180 mM (10.53 g/L) The effectiveness of these treatments was verified through Electrical Conductivity (ECe) measurements, yielding approximate values of 4.2 dS m⁻¹ for the 50 mM solution, 9.8 dS m⁻¹ for the 100 mM solution, and 17.6 dS m⁻¹ for the 180 mM solution.
180 mM The NaCl solution maintained the concentration by EC meter (Portable Conductivity Meter) every 2 days until 7 weeks after sowing different salt treatments from different growth stages
The experiment utilized a randomized complete design, incorporating TUAT1 inoculation across three salt treatments (0 mM, 50 mM, and 100 mM) A total of six experimental sets, each with 15 replicates, were conducted at four distinct growth stages of seedlings: immediately after sowing (W0), one week after sowing (W1), two weeks after sowing (W2), and three weeks after sowing (W3).
To assess the impact of salt treatments on rice seedlings, key metrics such as Salt Injury Score (SIS), seedling survival percentage, plant height in centimeters, the number of crown roots, and the fresh weights of both shoots and roots in milligrams were measured at 4 weeks and 5 weeks after sowing.
Visual measurements offer a quick method for diagnosing salt damage, quantified through a Salt Injury Score (SIS) as per the Standard Evaluation System (SES) established by IRRI This system rates visual symptoms of salt toxicity on a scale of 1 to 9, where 1 signifies healthy, vigorous plants and 9 indicates severely stunted or dead plants The survival percentage of seedlings is determined by the ratio of healthy seedlings, excluding those rated 9, to the total number of seedlings for each treatment.
Plant height was measured from the soil surface to the tip of the tallest leaf After removing the plants from their pots, the roots were thoroughly washed with tap water, followed by a rinse with distilled water The plants were then blotted dry using blotting paper, and the weights of the shoots, roots, and entire plant samples were recorded.
Effect of TUAT1 inoculation on the growth of different rice varieties treated with
The experiment was designed as a randomized complete with or without TUAT1 inoculation × 4 salt treatments (0 mM, 50 mM, 100 mM, and 180 mM) Eight sets of experiments were including
12 replicates and salt treatment of them was performed from 3 different growth stages in seedlings: 2 weeks after sowing(W2) and 3 weeks after sowing (W3)