Furthermore, linear regression shows a decreasing change in slope from CF treatment to MS and HS treatments which is explained by the non linear sorption of As and the different forms of Fe in plaque , demonstrating that Fe in the CF treatment has a higher affinity for As in comparison with II treatments. In Chapter 1, our results demonstrated that total grain As concentrations are higher in the CF treatment in comparison to II. In addition, we found that iron plaque in the CF treatment can bind greater amounts of As . Overall, more As is present and mobilized in rice plants under continuous flooding. Therefore, we postulate that under II the main mechanism for reducing As accumulation in grain is not directly its sequestration in the rhizosphere but rather the oxidation and immobilization of As in bulk soil which drives the observed sequestration in the rhizosphere. Nevertheless, changes in the mineralogy of root plaque play a key role in reducing the accumulation of As in grain. Elucidating the abiotic changes in Fe and As chemistry that occur in soil due to II facilitates our understanding of the mobilization and bio availability of As in rice paddies. However, research on the concurrent biotic impacts on As bio availability and soil health with II is required and this information should be included in a conceptual model for As mobility and bio availability in rice.Rice plants readily take up and accumulate arsenic in the grain when grown in flooded paddy fields, posing a threat to human health . Intermittent irrigation has been a subject of increasing interest due to its efficacy in reducing As accumulation in rice, reducing methane gas emissions, and increasing water use efficiency during rice cultivation . II is characterized by a distinct cycling of flooded and non flooded periods that are accompanied by redox fluctuations, which have a significant impact on the fate and form of certain contaminants and nutrients in soil,container vertical farming as well as soil microorganisms . The cycling of redox active elements in soil is often associated with abiotic factors such as mineral precipitation and reductive dissociation; or biotic factors such as the activity of plant roots and related microorganisms .
The biogeochemical cycle of As is linked to microbial mediated transformations and influences the mobility, distribution, and availability of As species in the environment. In rice paddies, microorganisms play vital roles in both aerobic and anaerobic soil conditions. During continuously flooded treatments, some anaerobic bacteria can use AsV as a terminal electron acceptor in respiration and subsequently reduce it to AsIII, contributing to greater As bio availability in the soil solution . In aerated soils, some aerobic bacteria can transform AsIII to less toxic forms, such as AsV and methylated As . Arsenic reducing and oxidizing bacteria often coexist in the rice rhizosphere, and their abundance and activities regulate As speciation, bio availability, and accumulation in rice paddies . The relative abundance and activity of As transforming microorganisms are key factors that influence the fate of As in paddy soils, and consequently the bio availability of As to rice plants . Moreover, it is well documented that numerous bacteria species are involved in iron oxidation within the rhizosphere, and thus the microbial community may also play an important role in Fe plaque formation . Dissimilatory iron reducing bacteria use FeIII from iron oxide minerals as a terminal electron acceptor during anaerobic respiration. This reductive dissolution reactions facilitates the release of As from iron oxides, increasing its bio availability for rice uptake. On the other hand, the oxidized micro environment created by the oxygen secreted from rice aerenchyma allows iron oxidizing bacteria to couple the oxidation of FeII with the reduction of a variety of electron acceptors, promoting the co precipitation of As with iron oxides .Microorganisms are very sensitive to small changes in their environment and can be influenced by a range of biotic and abiotic factors . It is thus expected that redox fluctuations caused by II events will affect microbial activity and succession in the rhizosphere. II is a promising management strategy for reducing As concentrations in grains but needs to be accepted by rice growers for widespread adoption in rice cultivation. Scarcity of information withholds farmers from making well informed decisions. Although growers are typically most concerned about how changing on farm operations will affect their agronomic systems, there remains a need for mechanistic biotic and abiotic information to explain broad implications of establishing II treatments.
At present, the impact of microbial processes on As cycling in the rhizosphere is not well understood. Thus, there is a need for studies that investigate the association between water management treatments, changes in soil microbial communities, and the influence of bacteria in As and Fe cycling in rice systems. Studies that analyze the impact of II on microbial community composition often report treatments with several dry down events and yield is not always evaluated . Understanding the changes of microbial populations due to paddy water management regimes can provide information about the role of rhizosphere bacteria in Fe plaque formation and As speciation, bio availability, and mobility in rice systems. The primary objective of this study was to reproduce rice field conditions in a series of pot based bio assays with single dry down II treatments of varying severity and to observe the effect of water management fluctuations in rhizosphere soil bacterial communities throughout the growing season. The experimental design aimed to replicate the field conditions from the field based irrigation management rice growth trials conducted at the California Cooperative Rice Research Foundation Rice Experiment Station in Biggs, CA during summer 2017 and 2018 . The current study was conducted in Escondido, CA during summer 2020. The site has a Mediterranean climate with an average temperature of 20.6 °C and average precipitation of 0.813 mm for May October 2020. Soil from the RES was collected,hydroponic vertical garden mixed to homogenize, and placed into 1 gallon pots. The average As and Fe concentrations in the soil were 3.87 mg kg 1 and 33.39 g kg 1 , respectively. The three II treatments were high, medium, and low severity ; and one continuously flooded as a control. Each treatment was assigned to a 62 L plastic bin that contained 6 replicates of 1 gal pots with paddy soil. Rice seeds were planted evenly on the soil surface and the bins were flooded 10 cm above the soil line. Every 5 days water was added to reach the initial water level, pH and redox potential were measured, and bins were re oriented and repositioned based on a randomized complete block design . For the dry down events, the water from the three II bins was drained and soil moisture was measured on an hourly basis utilizing a Watermark handheld meter and soil moisture sensors set at a depth of 10 to 15 cm. Soil was sampled throughout the season on similar dates from the field experiment presented in Chapter 1. Table 3.1 provides the timeline for sampling, water treatment management, and other events throughout the experiment. Soil was collected using a 1 mL sterile syringe with the end tip cut off to create a miniature soil probe. Approximately 1 g of soil was obtained at a depth of 5 10 cm at the base of the rice plants , and two samples were obtained from each pot. A 0.25 g sub sample was separated, and DNA was extracted with the Qiagen DNeasy PowerLyzer PowerSoil Kit. At harvest, the rice grain was collected, and plants were rinsed to remove soil adhered to the roots. Roots were separated from the shoots and washed thoroughly with DI water to remove any remaining soil. All plant biomass was dried at 65 °C for 24 h. Paddy grain was polished into white grain utilizing a rice huller and mill, and each layer was ground, sieved at 0.5 mm, and stored in airtight containers. Roots and shoots were cut into 2 cm sections and a 5 g sub sample was ground and sieved at 0.5mm and stored for further analysis. DNA extractions and library preparation were performed by the UC Davis Host Microbe Systems Biology Core Facility.
Primers 341F and 806R were used to amplify the V3 V4 domain of the 16S rRNA using a two step PCR procedure. Step one was the amplification procedure where the primers contained an Ilumina tag sequence, a variable length spacer, a linker sequence and the 16S target sequence. In step two, each sample was barcoded with a unique forward and reverse barcode combination using forward and reverse primers. The final product was quantified on a Qubit instrument using the Qubit High Sensitivity dsDNA kit and individual amplicons were pooled in equal concentrations. The library was quantified via qPCR followed by 300 bp paired end sequencing using an Illumina MiSeq instrument and taxonomic groups were assigned using the Silva rRNA database. Data of quantified As and Fe in plant biomass, all treatment effects, and differences between sampling dates were assessed using a two way analysis of variance . All effects with p values < 0.05 were considered significant. P values are presented throughout, but where significance is discussed, Tukey’s multiple comparison test was conducted. Relationships between samples from microbial analysis were visualized using principal coordinate analyses obtained based on Bray Curtis dissimilarity metrics.Consistent with results presented in Chapter 1, II treatments decreased As concentrations in rice grains, shoots and roots, and increasing dry down severity had a greater impact in reducing As bio accumulation . Like As, the concentration of Fe in root samples with plaque was highest for CF treatment and decreased for LS, MS, and HS treatments . It is surmised that dry down events promote oxic conditions in the bulk soil and aqueous FeII is oxidized and immobilized via precipitation of FeIII oxides, decreasing the overall Fe content in the rhizosphere and root plaque compared to the CF treatment. With CF, mobile aqueous FeII persists in the bulk soil and is oxidized only after being transported into the oxygenated rhizosphere. Therefore, greater precipitation of FeIII oxides as root plaque occurs, and a higher total Fe concentration is observed in root samples from the CF treatment. Soil pH continually increased from sowing to harvest by 1.5 units . This increase is explained by the constant consumption of protons in paddy soils during reduction processes associated with flooded rice paddies . Soil redox potential dropped upon initial flooding, increased above 100 mV during dry down events, and declined after reflooding below 300 mV for all treatments . This pattern indicates oscillation between anoxic and oxic conditions. The redox state of Fe and As during these fluctuations can be predicted via the Nernst equation or, simply, by utilizing the pH and Eh measurements and referring to Pourbaix diagrams, which indicate that both As and Fe are present in their reduced forms during flooding and become oxidized during the dry down events of II treatments . During the dry down periods in the three II treatments, soil water potential reached 10, 70, and 120 kPa for LS, MS, and HS treatments, respectively. Based on Carrijo et al., 2018, soil water potentials reached during dry down events are equivalent to volumetric water contents of 40, 35, and 25% for LS, MS, and HS, respectively. These parameters were carefully defined for the field trials, as well as the timing of the dry downs, to ensure that rice plants reach maturity and maintain yields . From 52 identified phyla, the 15 most abundant accounted for 98% of the total sequential reads. The three dominant phyla for all treatments throughout the growing season were proteobacteria, actinobacteriota, and acidobacteriota, which are typically found in agricultural soils . Proteobacteria, the most abundant phylum in our soil samples, is expected tothrive in carbon rich environments, such as rice paddies . Although the mentioned phyla predominated in our samples, there were variations in the relative abundance of these groups. Our samples from the CF treatment express a higher abundance of the phyla actinobacteriota and firmicutes, whereas acidobacteriota, chloroflexi, and myxococcota are more abundant in the HS treatment. These results suggest phylogenetic differences in the bacterial communities are driven by fluctuations of anoxic and oxic conditions.