Effects of strategic tillage and plant hormone treatments on wheat- associated microbial communities Hongwei Liu Masters of Engineering (Food Quality and Safety) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2016 School of Agriculture and Food Sciences 1 Abstract The main aim of this thesis was to characterise microbial properties of microbiomes associated with wheat (Triticum aestivum) soils and wheat plants. In particular my objective was to better understand the effects on microbial communities following strategic tillage (ST) in wheat field soils and the activation of plant defence pathways in wheat plants. Throughout the thesis, multiple culture-independent methods, especially next-generation sequencing of 16S rRNA gene were used to profile soil microbial communities. Over half of the arable land in the northern grains region of Australia is managed using no-till (NT), a farming method which has improved crop yields and soil quality while reducing the input and labour costs. However, in recent years, concerns have arisen among farmers over the weed control in continuous NT systems. Strategic tillage has been touted as a potential solution, in particular for the severe weed infestations of long-term NT. Nevertheless, there is little information on the influence of ST on the microbial properties of Australian NT soils. In the present study, ST applications and soil sampling were performed in Moonie, Moree and Condamine during the fallow period in eastern Australia. These sites were chosen based on their long history of repeated wheat cultivation under NT farming practices and their different soil types. Overall, results show that in the Moonie trial on a Calcisol, one-time ST with either chisel or offset disc did not significantly influence the composition of soil bacterial communities when measured 13 months after tillage. However, relative to the NT, chisel tillage led to significant increases in microbial biomass carbon (MBC), abundances of Alphaproteobacteria, Bacteroidetes and Firmicutes as well as the utilisation of D+cellubiose and mannitol at 0-10 cm depth. In the Moree trial on a grey Vertosol, ST with different timing and implement demonstrated great potential in weed control and did not influence wheat yield and soil physicochemical and biological properties in the short-term. In the Condamine site on a Solonetz soil, one- or two-time chisel tillage did not influence soil MBC, total microbial enzymatic activity (MEA) or utilisation of C substrates. Likewise, ST did not change the soil microbial community structure and the abundance of genes encoding enzymes involved in key steps of C and N reactions. However, one-time chisel increased relative abundance of Acidobacteria RB41 and Acidobacteria iii1-15, and two-time chisel slightly increased the average C utilisation, at 10-20 cm depth. Overall, this thesis suggests that ST does not cause major impacts on soil properties of long-term NT and could be potentially used to address the long-term NT-associated issues without impacting overall soil properties. Recent reports demonstrate the importance of microbiomes associated with plants and their soil they are cultivated in. Beneficial microbes can significantly increase crop yields and provide biocontrol functions against plant pathogens, but prior to this study little was known how wheat plants and their physiological pathways influence associated microbiomes in planta and 2 surrounding soil. We hypothesised that microbiomes in soils with a long history of repeated wheat cultivation and NT practice harbour microbes that are well-adapted to wheat plants. Therefore, we cultivated wheat using the wheat field soils collected from Moonie and Condamine and tested the effects of the activation of plant signalling pathways on the wheat microbiomes. Jasmonic acid (JA) and salicylic acid (SA) pathways were chosen based on their key roles in plant defence against biotrophic and necrotrophic phytopathogens, respectively. Seventy-two hours after methyl jasmonate (MeJA) treatment on wheat shoots, the composition of microbial communities in endophytic roots was altered. Decreased microbial diversity was observed in endophytic roots. In contrast, I found no evidence that microbial communities in endophytic shoots or rhizosphere were affected by MeJA treatment. Using Moonie and Condamine soils, I tested the effect of the activation of SA signalling on the composition and functions of wheat rhizosphere microbial communities. Seventy-two hours after SA treatment, the enhanced SA signalling marginally changed the composition of rhizosphere microbial communities in the Solonetz but not in the Calcisol. In particular, SA signalling triggered a significant decrease in Sphingobacteria and the Archaea member Nitrososphaera, but only in the Solonetz, not in the Calcisol rhizosphere. In addition, the copy numbers of arch-amoA, nifH, amoA and nosZ genes were reduced in the Solonetz rhizosphere by SA treatment. Taken together, it appears that JA and SA signalling pathways influence wheat-associated rhizosphere and endophytic microbial communities differentially. Wheat plants with activated JA-dependent defence may harbour bacterial communities of lower diversity in the root endosphere while an enhanced level of SA signalling may lead to a decrease of microbial components involved in N cycling in rhizosphere soil. Future studies may focus on how wheat-associated microbiomes may contribute to higher yields and improved crop resilience. This could be achieved through detailed characterisation of individual plant-microbe interactions, by engineering wheat-optimised microbiomes or by breeding wheat cultivars with improved microbiome interactions. 3 Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. 4 Publications during candidature Peer-reviewed papers 1. Hongwei Liu, Lilia C. Carvalhais, Kemal Kazan, Peer M. Schenk (2016) "Development of marker genes for jasmonic acid signaling pathway in shoots and roots of wheat." Plant Signalling and Behavior 11(5): e1176654. 2. Mark Crawford, Vivian Rincon-Florez, Anna Balzer, Lilia C. Carvalhais, Yash Dang, Hongwei Liu, Peer Schenk (2015) "Changes in the soil quality attributes of continuous no-till farming systems following a strategic tillage." Soil Research 53(3):263-273. 3. Hongwei Liu, Lilia C. Carvalhais, Vivian Rincon-Florez, Mark Crawford, Yash P. Dang, Paul G. Dennis, Peer M. Schenk (2016) "One-time strategic tillage does not cause major impacts on soil microbial properties in a no-till Calcisol." Soil and Tillage Research 158(5):91-99. 4. Hongwei Liu, Mark Crawford, Lilia C. Carvalhais, Yash P. Dang, Paul G. Dennis, Peer M. Schenk (2016) "Strategic tillage on a Grey Vertosol after fifteen years of no-till management had no short-term impact on soil properties and agronomic productivity." Geoderma 267:146- 155. 5. Hongwei Liu, Lilia C. Carvalhais, Mark Crawford, Yash P. Dang, Paul G. Dennis, Peer M. Schenk (2016) "Strategic tillage increased the relative abundance of Acidobacteria but did not impact on overall soil microbial properties of a 19-year no-till Solonetz." Biology and Fertility of Soils 52(7):1021-1035. 5 Book Chapter 1. Hongwei Liu, Hooman Mirzaee, Vivian Rincon-Florez, Richard Moyle, Lilia C.Carvalhais, Peer M.Schenk (2016) "Emerging culture-independent tools to enhance our understanding for soil microbial ecology" in Tate, K.: Microbial biomass - a new paradigm in terrestrial biogeochemistry, Imperial College Press, in press (3, 2017). Conference abstracts 1. Rincon-Florez VA, Carvalhais LC, Dang YP, Liu H, Crawford MH, Bell MJ, Schenk PM (2015) Impact of strategic tillage on nitrogen cycle genes (amoA and nifH) in no-till systems in Queensland Australia. Ecology of soil microorganisms, Prague, 29 November-3 December (Poster). 2. Liu H, Mirzaee H, Carvalhais LC, Dennis PG, Schenk PM (2015) Activation of salicylic acid defence signaling pathway reduced Archaea abundance and genes involved in nitrogen and carbon cycling in wheat rhizosphere.