The Beneficial Effects of Trichoderma Harzianum T-22 in Barley (hordeum Vulgare L.) Roots under Salt Stress

Soil salinity is an important problem that impacts agriculture globally. A sustainable approach for improving productivity is to adopt beneficial microorganisms to enhance the supply of soil nutrients to plants in stressful environments. Our work is showing that the fungus Trichoderma harzianum T-22 enhances barley growth and nutrient uptake in saline conditions. The fungus symbiotically lives inside the roots and triggers beneficial biochemical and metabolic changes. This project has broad implications for applying beneficial plant-microbe interactions to improve agricultural productivity. Chapter 1 covers the history and performance of endophytic fungi applied to crops as an alternative or supplement to the use of plant genetics or soil management to alleviate salt stress in crops. We focus primarily on root-associated microorganisms. The root-soil zone is the first point of defence for the plant against salt moving into the plant with the transpiration stream. It has dynamic biogeochemical processes driven by diverse metabolites released by the plant root and associated soil microorganisms. Fungal endophytes associated with some crops not only protect against plant pathogens and pests but also impart strong tolerance against several abiotic stresses in crops, including salinity. This is achieved via inducing systemic resistance, increasing the levels of metabolites such as pathogen protectants and osmolytes, activating antioxidant systems to prevent damage caused by ROS, and modulating plant growth phytohormone levels. Colonization by endophytic fungi improves nutrient uptake and maintains ionic homeostasis by modulating ion accumulation, thereby restricting the transport of Na+ to leaves and ensuring a low cytosolic Na+:K+ ratio in plants. This literature review has been submitted after external review to the journal Plant and Soil for publication. Following there is an addendum (Section 1.6) covering investigations and application of the endophyte-plant interactions using in metabolomics and lipidomics. The addendum aims to provide an overview of the necessity to investigate plant-fungal interactions and its influence on plant metabolism. Lastly, the chapter gives a brief description of the main experimental, technical and instrumental methods employed in this project. Chapter 2 determines the effect of the salt tolerant beneficial endophyte, Trichoderma harzianum strain T-22, on the growth and development of barley under optimal and saline conditions. Two barley genotypes were used in the study, cv. Vlamingh as a salt tolerant cultivar and cv. Gairdner as a salt sensitive cultivar. Barley was chosen as it is not only an agriculturally and industrially important crop but is also the most salt tolerant cereal crop. Two experimental setups were used for this study. In the first experiment, agar was used as the growth medium for plants. This was performed to observe and determine the effect of fungus on the growth of plants under controlled conditions. In the subsequent experiment, sandy loam soil was used as the matrix to grow plants and to emulate agricultural scenarios. Light microscopy was used to confirm the association of the fungus within roots. The results confirm the positive effect of this fungus under controlled and saline conditions in both experiments. Various parameters were measured to confirm the effects of salt and fungus on both genotypes under controlled and saline conditions. This study suggests that inoculation of salt sensitive plants with T. harzianum T-22 may ameliorate the effects of salinity and improve plant growth. Chapter 3 describes the role of Trichoderma harzianum T-22 in alleviating NaCl-induced stress in two barley genotypes (cv. Vlamingh and cv. Gairdner) by mapping metabolites and lipids using GC-MS for polar metabolites and LC-MS for lipids. This was performed to provide insights into the biochemical changes in barley roots treated with fungus during the early stages of interaction. T. harzianum increased the root length of both genotypes under controlled and saline conditions. The fungus reduced sugars in both genotypes and caused no changes in organic acids under saline conditions. Amino acids decreased only in cv. Gairdner in fungal-inoculated roots under saline conditions. Triacylglycerols (TAGs) were the substantially increased lipids in inoculated roots of both genotypes under saline conditions. This study shows that the fungus imparts adaptation or tolerance mechanism to cv. Vlamingh and in cv. Gairdner by remodelling lipids mainly from glycerolipids after salt stress. Chapter 4 examined the role of Trichoderma strain T-22 on two barley genotypes (cv. Vlamingh and cv. Gairdner) grown in saline soil as soil is an important substrate for Trichoderma. Biomass results described in Chapter 2 of this thesis clearly show the positive effect of this fungus on both genotypes under control and saline conditions as measured using several physiological parameters. Here, the aim of this part of study was to determine the metabolites and lipids modified in roots grown in saline soil following endophyte inoculation that are involved in conferring positive effects on barley plants as mentioned in Chapter 2. We employed gas chromatography and liquid chromatography both coupled to mass spectrometry to analyse metabolites and lipids in inoculated and uninoculated roots of both genotypes under control and saline conditions. Chapter 5 explains the application of mass spectrometry imaging (MSI), liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS), inductively coupled plasma mass spectrometry (ICP-MS), and X-ray fluorescence (XRF) to determine the spatial distribution of metabolites, lipids and a range of elements, such as K+ and Na+, in seeds of two barley genotypes with contrasting germination phenology (Australian barley varieties Mundah and Keel). This chapter was published in Front Plant Sci (Gupta et al. 2019). Following this, an addendum (Section 5.10) is added to this chapter where the effect of T. harzianum was measured on germination of four barley genotypes used in this project. The results showed that the fungus improves germination efficiency of the sensitive genotypes, suggesting its role in reducing the adverse effects of salt stress on germination and growth of the plant. The final Chapter 6 describes the application and importance of mapping the biochemical changes involving metabolites and lipids, imparted due to the inoculation of the fungus in roots of barley seedlings and provides a view on future perspectives of research of plant-fungal interactions for plant growth promotion which can useful for sustaining agricultural productivity.