School of Agriculture, Food and Ecosystem Sciences - Theses
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ItemEffects of urease and nitrification inhibitors to soil microbial communities and nitrogen use efficiency( 2020)Soil microbial organisms are involved in many soil processes including nutrient cycling, impacting on soil quality and health. Anthropogenic activities such as nutrient addition influence soil ecosystems and soil environment. It is widely known that the contribution of urease inhibitors (UI) e.g. N-(n-butyl) thiophosphoric triamide (NBPT) and nitrification inhibitors (NIs) e.g. 3, 4-dimethylpyrazole phosphate (DMPP) in reducing N losses is associated with urea hydrolysis (and ammonia volatilization) and ammonia oxidization processes, respectively. However, little attention has been given to the understanding of the effect of NBPT on nitrification and urease producing microbes. Further, although the effects of NIs on ammonia oxidizers have been studied, there is limited information on how they influence non-targeted microbes or the newly discovered complete ammonia oxidizers (comammox Nitrospira). Some studies have reported that targeting mitigation of N losses through one pathway of the nitrogen cycle may lead to losses in another pathway. Therefore, the use of a combination of mitigation measures was suggested including combining UIs and NIs. Limited bio-molecular studies have investigated the effect of combined UIs and NIs on the soil N cycling microbes. In an incubation study on five soils from different parts of Victoria, Australia, using the terminal restriction fragment length polymorphisms (T-RFLP) and quantitative polymerase chain reaction (qPCR) techniques, we reported that the abundances of ammonia-oxidizing bacteria (AOB) and complete ammonia oxidizers (comammox Nitrospira), but not ammonia-oxidizing archaea (AOA), were significantly influenced by the application of NBPT, DMPP, and DMPP + NBPT. The structures and community composition of both AOA and AOB were influenced by NBPT, DMPP, or their combination. AOA, AOB, and comammox Nitrospira clade B might be significant contributors to nitrification in the studied soils. However, the contribution and responses of these microbes to nutrients, UI, or NI could be controlled by soil properties like soil pH. This study for the first time provided some new knowledge about ureolytic microbes and complete ammonia oxidizers (comammox Nitrospira) as influenced by NBPT and DMPP. However, there is a need for more information on how to develop a dual inhibitor compound whose individual compounds will work together effectively to target both hydrolysis and nitrification processes. It is also important to understand how comammox Nitrospira responds to different UI and NIs or their combinations, and the suitable rates of application of the UIs, NIs or their combinations for effective inhibition of comammox Nitrospira. Further, more studies have concentrated on understanding the molecular mechanisms of inhibition of ammonia oxidation by the NIs in short-term laboratory and field experiments. However, little is known about the effect of DMPP on soil enzyme activities, N cycling microbes (involved in nitrification and denitrification), or non-targeted microbes in soil following a production period in repeated chemical fertilizer and NI application regimes. Such a study has great implications for soil quality and health. Microbial communities were analyzed using Illumina sequencing and qPCR, from soil sampled 1 week after harvesting from a 4.5-year field experiment with repeated application of urea (U), urea + DMPP (UE) at 40, 80 and 120 kg N/ha. This analysis revealed that the AOB gene abundance increased as the N application rate increased (from 0 to 120 kg N/ha). The use of DMPP significantly reduced AOB and nirK gene copy numbers compared to urea alone at an application rate of 120 kg N/ha. There was no effect on the abundance of either ammonia-oxidizing archaea (AOA), comammox Nitrospira clade A and B, nosZ, or bacterial 16S rRNA genes. The community composition of AOB and AOA changed with N addition and use of DMPP while increasing the N application rate only changed the composition of AOB. Potential nitrification rates increased after the addition of N at 80 and 120 kg N/ha. There was no significant treatment effect on the relative abundance of total bacteria at the phylum level, indicating no residual effects of urea and DMPP at different rates on the non-targeted microbes. This experiment demonstrated that the application of N (with or without DMPP) at lower than 120 kg N/ha would not result in a significant impact on soil archaeal or bacterial ecology. Repeated application or overuse of chemical fertilizer can lead to environmental issues like soil acidification. The temporal effects of NBPT on soil ureolytic and ammonia-oxidizing microbes, crop yield, and nitrogen use efficiency (NUE) following repeated applications are not well understood. A perennial ryegrass (Lolium perenne L.) experimental site, received fertilizer treatments of urea applied alone at 40 kg N/ha and 80 kg N/ha or urea applied with NBPT (as Green Urea NV at 40 kg N/ha) in respective plots that had received the same treatments since 2014. The qPCR analysis of soil samples collected on a weekly basis for 45 days confirmed that the abundance of ureolytic microbes was higher in control (CK) compared to all N treatments on all sampling days, which was associated with changes in soil pH. Despite the reduced soil pH following repeated applications of urea with NBPT, ureC gene copy numbers were significantly reduced in the NBPT treatment plots applied at 40 kg N/ha compared to those in urea alone applied at the same rate. NBPT had no significant effect on the abundance of ammonia oxidizers. However, increasing the urea application rate significantly increased the abundance of ammonia-oxidizing bacteria (AOB) and complete ammonia oxidizers (comammox Nitrospira clade B). NBPT had no significant effect on pasture dry matter (DM) yield, N-uptake, or NUE. Increasing N application rate significantly increased pasture DM yield and N-uptake but this did not influence the pasture NUE. From the two field experiments, this thesis confirmed that repeated application of urea with NBPT or DMPP led to changes in soil physiochemical properties which included decreasing soil pH, and this controlled the response of soil microbes to chemical inhibitor applications. In both experiments with repeated applications, it was confirmed that AOB could be major players to nitrification in acidic soils with repeated chemical fertilizer applications with UI or NI. Future work should consider understanding the interaction between plants and soil microbial communities especially around the rhizosphere and how these may influence the efficacy of UI and NI on a short and long-term basis. Also, there is a need in the future to investigate how the inhibitor compounds and enzyme activities in different soils change following the application of these inhibitor compounds.