AMENTA, MARIA LAURA (2025) Strategies to exploit microbial biodiversity and to enhance the activity of beneficial bacteria in promoting plants growth. [Tesi di dottorato]

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Abstract

Agriculture currently faces a dual challenge: feeding a rapidly expanding global population and preserving the natural resources on which agricultural productivity depends. This challenge is intensified by climate change, manifested through global warming, altered precipitation regimes, soil degradation, and the increasing frequency of extreme weather events; these stresses collectively destabilize agroecosystems, compromising both crop yields and the biological integrity of soils. Cereals such as wheat (Triticum aestivum L.) and rice (Oryza sativa L.) occupy a central position in global food security, supplying essential calories and nutrients to more than half of the world’s population. Ensuring their sustainable production under extreme conditions such as water scarcity and soil salinity is one of the fundamental scientific and societal imperatives of the twenty-first century. Water availability remains the most critical limiting factor for crop productivity. In recent decades, drought episodes have intensified in both frequency and severity, particularly in arid and semi-arid regions, leading to significant yield reductions and threaten the stability of food systems. Moreover, the heavy reliance on irrigation and chemical fertilizers in intensive production systems has accelerated soil degradation, depleted freshwater reserves, and contributed to greenhouse gas emissions. These interconnected problems have prompted a paradigm shift in agricultural research toward the development of eco-sustainable strategies that enhance crop resilience without exacerbating environmental degradation. Within this context, modern agricultural biotechnology has evolved from a reductionist focus on single genes or traits to a systems-oriented approach that integrates plant physiology, molecular biology, and microbial ecology. This integrative perspective seeks to elucidate how plants perceive, respond to, and mitigate abiotic stresses such as drought, also considering the pivotal role of the plant-associated microbiome. Wheat, as a staple crop cultivated across diverse agroclimatic zones, frequently encounters periods of water deficit that constrain productivity. Drought affects plant performance through a complex interplay of physiological and biochemical processes, including disruptions in photosynthesis, transpiration, and reproductive development. The capacity of wheat to maintain growth under water-limited conditions depends on a suite of adaptive mechanisms such as osmotic adjustment, stomatal regulation, maintenance of membrane integrity, and antioxidant defence. Comparative studies among wheat genotypes have revealed considerable variability in their physiological and molecular responses to drought stress. Parameters such as relative water content, chlorophyll concentration, leaf area, and stomatal conductance serve as key indicators of tolerance. Resistant genotypes exhibit adaptive traits that sustain photosynthetic activity and delay dehydration. For example, the accumulation of solute such as proline and soluble sugars, facilitates osmotic adjustment, while increased cell wall rigidity and membrane stability reduce oxidative damage. In contrast, sensitive genotypes tend to rely predominantly on stomatal closure, a response that conserves water but severely limits carbon assimilation, leading to reduced biomass and yield. At the molecular level, drought tolerance is governed by the coordinated regulation of genes associated with hormonal signaling, water transport, and stress protection. Abscisic acid (ABA) plays a central regulatory role, mediating stomatal closure and activating stress-responsive genes. Upregulation of genes involved in ABA biosynthesis, such as AAO (abscisic aldehyde oxidase), and aquaporin genes such as PIP2:1, which enhance membrane water permeability, exemplifies the integrated molecular response to dehydration. Distinct expression profiles among drought-tolerant genotypes highlight the genetic diversity underlying adaptive capacity. These findings provide a mechanistic foundation for the selection and breeding of drought-tolerant cultivars and contribute to a broader understanding of plant resilience under climate-induced stress. While plant genetics and physiology are crucial determinants of drought tolerance, contemporary research increasingly recognizes that plant performance is not solely a function of the host genome. Plants exist as holobionts ecological entities composed of the host and its associated microbial communities. These communities, which inhabit the rhizosphere, endosphere, and phyllosphere, form dynamic symbiotic networks that influence plant growth, nutrient acquisition, and stress adaptation. The plant microbiome is thus a functional extension of the plant itself, which shapes its physiological responses to environmental perturbations. Microorganisms can enhance plant tolerance to abiotic stresses through multiple direct and indirect mechanisms. They modulate hormonal signaling, synthesize growth-promoting substances such as indole-3-acetic acid and cytokinins, facilitate nutrient solubilization, and activate systemic resistance pathways. Under drought and salinity stress, specific microbial taxa exhibit unique adaptive traits such as osmolyte production and exopolysaccharide secretion enabling them to survive in hostile environments, while conferring resilience to their hosts. Notably, members of Bacillus, Kosakonia, and Enterobacter within the phyla of Proteobacteria and Firmicutes have demonstrated the ability to fix atmospheric nitrogen, solubilize phosphate, and tolerate high salt concentrations, making them promising candidates for use as biofertilizers and biostimulants. Recent advances in ex-situ plant-trapping methods have enabled the isolation of culturable microbial communities from extreme or marginal soils. When cereal crops such as rice and wheat are cultivated in these soils, they selectively recruit beneficial microbes into their roots and internal tissues. Recruitment patterns differ among plant species and genotypes, reflecting co-evolutionary compatibility between hosts and microbial symbionts. For instance, certain rice varieties preferentially associate with halotolerant, phosphate-solubilizing bacteria, while wheat genotypes tend to recruit nitrogen-fixing endophytes. Understanding these selective interactions provides the foundation for designing microbial consortia tailored to enhance specific stress responses in target crops. The potential of microbial biotechnology to enhance crop drought tolerance has been demonstrated in experiments involving endophytic bacteria and durum wheat. Among these, the endophyte Klebsiella pasteurii BDA134-6, originally isolated from African rice, was able to confer drought tolerance when used to inoculate durum wheat. Laboratory, greenhouse, and field evaluations revealed that inoculated plants maintained higher relative water content, enhanced nitrogen fixation activity, and better growth performance under simulated water deficit compared to uninoculated controls. These results illustrate that beneficial endophytes can cross species barriers, establishing functional symbioses with non-native hosts. The physiological and biochemical modifications induced by microbial inoculation include elevated activities of antioxidant enzymes such as glutathione reductase and superoxide dismutase, which mitigate oxidative stress by scavenging reactive oxygen species. At the same time, the accumulation of osmoprotectants such as proline contributes to osmotic balance and stabilization of cellular structures, while the reduction of hydrogen peroxide accumulation prevents premature senescence. These combined responses maintain cellular homeostasis and metabolic activity during drought, confirming that beneficial microbes can reprogram host metabolism toward greater resilience. These findings point to a conceptual shift: drought tolerance should no longer be considered exclusively as an intrinsic genetic property of the plant, but rather as an emergent trait arising from synergistic interactions between plants and their microbiota. Microbial biotechnology, therefore, complements traditional breeding and transgenic approaches, offering environmentally sustainable tools to enhance crop performance under adverse conditions. Further exploring this concept, plant-microbe interactions have also been investigated at the molecular level to understand how plants and their endophytic partners communicate. The transcriptional responses of two endophytic bacterial strains, Enterobacter asburiae RCA24 and Kosakonia sacchari RCA25, to root exudates of two cultivated varieties (Oryza sativa Baldo and Vialone Nano) and the wild ancestor O. rufipogon, were analysed. Bacterial transcriptome analyses revealed genotype-specific interactions, with RCA24 being able to distinguish between O. sativa varieties, while RCA25 responded more strongly to O. rufipogon. Functional annotation highlighted changes in central metabolism, stress response, and signal transduction, suggesting that domestication has reduced the stimulatory effect of rice exudates on beneficial microbes. Overall, this work underscores the intricate and dynamic relationships that underpin plant adaptation to environmental stress. Drought tolerance in cereals such as wheat and rice emerges not only from genetic and physiological mechanisms intrinsic to the plant but also from complex interactions with their associated microbial communities. The integration of molecular, physiological, and ecological perspectives reveals that plants and microbes engage in a reciprocal dialogue mediated by root exudates, signaling molecules, and transcriptional reprogramming that determines the resilience of the holobiont under adverse conditions. The results of studies carried out on endophyte-host systems, such as Klebsiella pasteurii BDA134-6 in wheat and the genotype-specific responses of Enterobacter asburiae RCA24 and Kosakonia sacchari RCA25 in rice, highlight the potential of beneficial microbes to enhance stress tolerance through metabolic and regulatory modulation. Moreover, the observation that wild rice genotypes, such as Oryza rufipogon, elicit stronger microbial responses suggests that domestication may have attenuated some of the natural mechanisms by which plants recruit and communicate with beneficial microorganisms. The presented results collectively point to a new paradigm in crop improvement, which views drought tolerance and overall plant performance as emergent properties of the plant-microbiome system. Harnessing this synergistic potential through microbial biotechnology, coupled with molecular breeding and systems-level understanding, opens a sustainable path for agriculture. By restoring and optimizing beneficial plant-microbe partnerships, it will be possible to enhance crop productivity, reduce dependence on chemical inputs, and build resilient agroecosystems capable of withstanding the challenges of climate change. The overarching objective of this doctoral research is to unravel the physiological, biochemical, and molecular bases of drought tolerance in wheat and rice and to explore how microbial interactions can reinforce plant adaptation to water stress. Through a multidisciplinary framework, the study aims to identify resilient genotypes, characterize their stress-response mechanisms, and evaluate the potential of beneficial microbes to enhance drought tolerance across species boundaries.

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