Open research questions in Plant-Microbe Interactions and Immunity

What the literature leaves open

• Abstract The efficacy of bio-organic fertilizers is strongly dependent on functional microbial strains; however, systematic frameworks for candidate strain acquisition, functional screening, application validation, and strain characterization remain insufficiently developed for bio-organic fertilizer research and product development.

  Functional Microorganisms in Bio-organic Fertilizers: Strain Screening, Application Validation and Characterization · 2026 · DOI

• community in bacterial The impact of toxic metal pollution on seed microbiota remains poorly understood, and the few available studies suggest that responses may be strongly host- and habitat-dependent [62,63].

  Toxic metals differentially affect bacterial and fungal seed endophytes in Arabidopsis arenosa · 2026 · DOI

• Restoring a polluted environment with sustainable and envi- ronmentally friendly alternatives is vital. This ensures an uncompromised environment for present biotics and future gener- ations. Transitioning to environmentally friendly options such as MEP brings optimistic prospects. Such shifts could lead to a better environment and improved economies. Currently, the application of MEP techniques in the tropics is feasible (Rodriguez-Seijo et al., 2017). However, these techniques are not yet used on a large scale in the tropics. To advance MEP techniques in the region, it is important to explore advanced biotechnological and omics-based approaches. One method involves the genetic modification of genes responsible for pollutant uptake, tolerance, and detoxification in MEP plants (Pollard et al., 2008; Santos et al., 2020). Techniques like clustered, regularly interspaced short palindromic repeats (CRISPR)-associated protein technology and other bioengineering methods are promising to accelerate environmental remediation. These innovations may help identify nonconventional crops, known as metallocrops, that decontaminate metals (Reboredo et al., 2019). Advances in biotechnology could reveal ways to use biodiversity for better phytoremediation technology (Santos et al., 2020)—for instance, genes that clean contaminants in microorgan- isms can be inserted into plants. This improves clean-up efficiency, as has been done in other environments (Rafique et al., 2023). Genetic engineering has also increased the biomass, resistance, and growth rates. It has reduced the risk of transgenicity in MEP plants (Bernardino et al., 2020). Genetic transformation and expression of exogenous genes in traditional plant species produce improved transgenic varieties—for example, inserting a disintegrating trichlo- roethylene (TCE) enzyme into a poplar species increased its TCE cleansing efficiency from 3% to 91%. Similarly, a transgenic poplar with the merA18 gene cleared mercury (Hg) more than 10 times better than traditional poplar (Marrugo-Negrete et al., 2015). Genes like ScYcf1, ScYHL035C, ScPdr13, and AtPcr1 can improve heavy metal resistance through yeast mutant screening and Arabidopsis cDNA library screening (Marrugo-Negrete et al., 2015). Comparing wild-type and transgenic poplars grown in Cd- and Pb-containing media shows that transgenic plants are more resistant (Rugh et al., 1998; Bhatt et al., 2023b). The benefits of transgenic species in phytoremediation are increasingly recognized. However, it remains key to investigate their efficiency for organic pollutant degradation in the tropics. Similarly, another future strategy to improve MEP could also include the integration of omics-based approaches. To achieve this, phytoremediation research requires more collaborative studies involving expertise from different fields of life sciences and biophysico-chemical sciences using a citizen-science-based approach (Naveed et al., 2023). The applications of omics-based approaches, such as metagenomics and metabolomics, to understanding the biochemical and metabolic pathways of plant– microbe interactions, metal accumulation, and ion homeostasis to improve MEP are a burgeoning field that could be explored for sustainable phytoremediation processes (Maurya, 2023). Another future direction in the field of MEP is in the applica- tion of nanomaterial (NM) technology as an emerging field with many applications that could improve MEP processes. Plant studies using various NM exhibiting different phytotoxicity profiles are much less common in the literature. Consequently, more research is needed in the areas involving the use of nanomaterials or nanoagrochemicals to influence the phytoremediation potentials of MEP processes. In addition, interactions of NM with pollutants and impacts of NM on the environment, as well as on plant health, are worth exploring in the tropical environment because of its rich biodiversity. In another vein, knowledge of digital technologies such as remote sensing, artificial intelligence (AI), and machine learning (ML) can be integrated to conduct real-time predictive modelling and simulations in MEP processes. Moreover, these digital methods can also be used to monitor and forecast contaminant behavior as well as remediate a contaminated environment at small-, medium-, and large-scale capacities.

  Microbial applications for enhancing plant-mediated remediation in the tropics · 2026 · DOI

• ABSTRACT Botrytis cinerea , a highly successful necrotrophic fungal plant pathogen, demonstrates a remarkably broad host range, yet the precise factors governing its host specificity and its ability to coexist non‐pathogenically with plants remain unclear.

  Leaf Structure and Hormonal Balance Modulate Host‐Specificity and Pathogenicity of <i>Botrytis cinerea</i> · 2026 · DOI

• However, the regulatory mechanisms governing the expression of its ACC chemoreceptor WP116, as well as how changes in its abundance influence ACC chemotaxis and rhizosphere colonization, remain poorly understood.

  1-Aminocyclopropane-1-Carboxylate and Its Chemoreceptor Drive Metabolic Reprogramming to Enhance Chemotactic Rhizocompetence in Pseudomonas sp. UW4 · 2026 · DOI

• AbstractSoil microorganisms are crucial for plant survival and productivity, but factors governing rhizosphere recruitment across diverse regions remain unclear.

  Environment and plant genetics shape barley rhizosphere microbiome structure across contrasting locations · 2026 · DOI

• Seed biopriming with plant growth-promoting bacteria (PGPB) has emerged as a sustainable strategy to enhance plant tolerance to abiotic stress; however, its effectiveness in Pinus nigra remains poorly understood.

  Biopriming with plant growth-promoting bacteria enhances germination and stress tolerance of Pinus nigra seeds under drought and salinity stress · 2026 · DOI

• While IAA biosynthesis is widespread among plant-associated bacteria, the mechanisms through which this auxin regulates bacterial physiology and virulence, as well as those controlling its production, remain poorly understood.

  Auxin biosynthesis and signaling drive virulence and plant adaptation in Dickeya dadantii · 2026 · DOI

• The future of crop biofortification is not just about increasing the amount of nutrients in crops, but about making those nutrients truly useful for human health. In recent years, progress in plant molecular biology, rhizosphere microbiology, and nutrition science has made it possible to develop crops that are richer in micronutrients, easier for the body to absorb, and better able to withstand environmental stress. Moving forward, it is important to better understand the genes, transport systems, regulatory networks, and microbial processes that control how nutrients are Plant Crop Letters (2026), 7: 20-37 32 taken up, moved, stored, and retained in edible parts of plants. At the same time, reducing anti-nutritional compounds and improving the chemical form of stored nutrients will be key to ensuring that higher nutrient levels in crops actually translate into better human nutrition. New biotechnological tools are opening exciting possibilities in this field. Techniques like genome editing, synthetic biology, multi-omics approaches, microbiome engineering, and advanced bioinformatics can help fine tune plant traits with much greater precision. These tools also make it easier to understand the complex relationships between soil, plants, microbes, and human nutrition. With these advances, it will be possible to develop biofortified crops that perform well in different environments and meet the needs of diverse diets. In particular, there is growing interest in microbiome-responsive and climate resilient approaches, especially in regions where poor soils, water stress, and high levels of micronutrient deficiency exist together. Overall, future progress will depend on combining agriculture, nutrition, and environmental science into a single integrated approach. Another important step is to generate strong real world evidence showing that eating biofortified crops actually improves human health. Long term studies are needed to track nutrient absorption, health biomarkers, immune function, growth, and overall development across different age groups and dietary settings. These studies should also consider differences in human biology, gut microbiome composition, cooking and food processing methods, and social and economic factors that affect diet. In the end, the success of biofortification should not only be judged by nutrient levels in crops, but by real improvements in human health and a meaningful reduction in micronutrient deficiencies at the population level.

  Microbial and Cellular Mechanisms in Crop Biofortification: Implications for Nutrient Bioavailability and Human Health · 2026 · DOI

• The latest research on small RNA (sRNA)-mediated DNA methylation (RdDM) has highlighted its role in plant disease resistance; however, the fungal pathogen cross-kingdom sRNA that mediates host DNA methylation to counteract tomato Fusarium wilt disease has not been explored.

  DNA methylation of SlyAHL mediated by Fol-milR1 confers resistance to Fusarium wilt disease in tomato · 2026 · DOI

• However, it remains unclear whether SynComs composed of host- or non-host-associated rhizosphere bacteria can trigger induced systemic resistance (ISR) in barley without causing major shifts in the native rhizosphere bacterial community.

  Synthetic rhizosphere bacterial communities induce systemic resistance to barley powdery mildew without major shifts in the native bacterial community · 2026 · DOI

• Oomycete plant pathogens, including Phytophthora species, undergo rapid transitions between motile, encysted, germinating, and invasive stages, yet the organization of Ca2+ dynamics during these transitions is poorly understood.

  Live imaging reveals polarized calcium transients during plant pathogen development and host colonization · 2026 · DOI

• Protein tyrosine nitration is a well-documented peroxynitrite (ONOO – )- mediated post-translational modification (PTM) in biological systems; however, its relevance in plant immune response remains poorly understood.

  Peroxynitrite-mediated tyrosine nitration modulates β-1,3-glucanase activity and potato defense against Phytophthora infestans · 2026 · DOI

• Limited functional inference; mostly genus/species level resolution only; primer bias High cost; difficulty resolving near-identical strains; sequencing artifacts High-throughput; scalable; avoids host bias Database dependence, poor detection of novel genes, and computationally demanding Sequencebased…

  Salt Tolerance in Agriculture: Unveiling the Role of Halotolerant Bacteria and Metagenomics in Crop Improvement · 2026 · DOI

• Future studies could focus on the development of sustainable bioformulations and the evaluation of their efficacy across different crops, including P. However, given the limited environmental characterization and sample size, the results should be interpreted with caution and limited to the conditions studied.

  Diversity, molecular identification and plant growth promoting potential of endophytic bacteria from plantain (Plantago lanceolata L.) · 2026 · DOI

• Bacterial CFS derived from Bacillus and Pseudomonas species represent promising biostimulant formulations capable of allevi- ating salinity stress in key crops such as soybean, canola, and wheat. These metabolite-rich preparations consistently enhance seed germination, seedling vigor, biomass accumulation, photo- synthetic efficiency, and ionic homeostasis under salt-imposed osmotic challenges. The mechanistic basis of these benefits en- compasses modulation of hormonal signaling, antioxidant de- fense, ion transport regulation, and improved nutrient acquisition, aligning with the broader functions of live PGPB but offering advantages in formulation stability and regulatory acceptance (Sharma et al., 2016). While Bacillus and Pseudomonas CFSs have demonstrated clear bioactivity in plant systems, their large-scale practical deployment TABLE 5 Summary of CFS application parameters, concentration ranges, and salinity alleviation outcomes across different crop species and growth stages.

  Bacterial cell-free supernatants as metabolite-based biostimulants for alleviating salinity stress in plants · 2026 · DOI

• The expanding understanding of plant microbiota has fundamentally reshaped contemporary perspectives on crop productivity and resilience. Rather than functioning as isolated biological systems, plants are increasingly recognized as holobionts whose growth, development, and stress adaptation are strongly influenced by associated microbial communities (Berg et al., 2020; Trivedi et al., 2020). This conceptual transition highlights the necessity of integrating microbiome science into sustainable agricultural frameworks. Despite substantial progress in characterizing plant-associated microbial diversity, the functional predictability of microbiomes under field conditions remains limited. Future research must therefore prioritize the development of predictive microbiome models capable of anticipating microbial assembly, persistence, and functionality across varying soil types, climatic regimes, and crop genotypes (Mukherjee et al., 2024; Ge & Wang, 2025; Romão et al., 2025).

  Plant Microbiota: Emerging Tools for Sustainable Crop Growth and Health · 2026 · DOI

• Despite the growing body of evidence supporting the syner- gistic benefits of biochar and Trichoderma spp. in enhanc- ing plant resilience and soil health, future research must transition from descriptive outcomes to mechanistic and translational insights. Descriptive studies typically report phenotypic or expression-level changes without resolv- ing causality (e.g., upregulation of PR genes under bio- char treatment mentioned previously) (Jaiswal et al. 2020; Yao et al. 2023). Mechanistic studies, in contrast, identify causal pathways, such as JA-dependent signaling validated through mutant analysis or pathway inhibition. Transla- tional insights extend these findings into field-applicable strategies, including optimized biochar formulations or strain-specific Trichoderma inoculants tested under agro- nomic conditions. Bridging these levels remains a critical gap in current research. A promising direction lies in the integration of multi-omics platforms including transcrip- tomics, proteomics, metabolomics, and microbiomics to construct a systems-level understanding of biochar-Trich- oderma-plant-soil interactions. These approaches can help unravel the temporal dynamics of hormonal signaling, iden- tify keystone microbial taxa, and decode metabolite fluxes that underpin ISR and abiotic stress tolerance. An emerging frontier in this context is the exploration of bio-activated hormonal signaling, wherein SA, JA, and ET pathways are naturally triggered by microbial elicitors and soil amend- ments. Unlike exogenous hormone applications, bio-acti- vation represents a biologically integrated defense strategy, World Journal of Microbiology and Biotechnology (2026) 42:219 1 3 219 Page 10 of 14 often mediated by root colonization, VOCs, and ROS sig- naling (Bhatt et al. 2024; Castejón-del Pino et al. 2025). Investigating how biochar-Trichoderma synergy influences bio-activation of these pathways particularly through the upregulation of key defense genes such as PR1, PDF1.2, LOX, and ERF could reveal novel regulatory nodes and priming mechanisms (Sofy et al. 2022; da Mota et al. 2025). This knowledge would be instrumental in designing next- generation bio-stimulants and functional soil amendments that promote sustainable and climate-resilient agriculture. Additionally, the development of ‘designer biochar’ tai- lored to selectively enrich Trichoderma spp. and beneficial microbial consortia represents a novel avenue for functional soil microbiome engineering. These biochars could be opti- mized for specific soil types, crops, and stress conditions using predictive modeling and machine learning algorithms (Fig. 4). Coupling this approach with CRISPR-based func- tional genomics in Trichoderma spp. may allow for the fine- tuning of elicitor production, root colonization traits, and stress-responsive gene activation, thereby enhancing bio- control efficacy and plant growth promotion (Frenkel et al. 2017; Zygourakis 2017; Wang et al. 2025a, b). Future research should prioritize mechanistic valida- tion under agronomically realistic conditions. Multi-omics integration must progress beyond parallel data genera- tion toward establishing functional causality, particularly through isotope labeling, microbial knock-out systems, and time-resolved field-scale trials. New biochar designs should be guided by physicochemical–microbial compatibility metrics rather than generalized assumptions of microbial stimulation. Without rigorous field validation, the bio- char-Trichoderma concept will remain a laboratory-scale phenomenon with limited agricultural impact. Given the substantial heterogeneity in biochar physicochemical prop- erties arising from differences in feedstock composition and pyrolysis conditions, mechanistic interpretations should be considered context-dependent rather than universally appli- cable. While certain trends—such as enhanced microbial colonization or modulation of plant defense pathways—are frequently observed, their magnitude and direction may vary significantly across systems.

  Omics-informed insights into biochar–Trichoderma interactions in plant–soil systems: mechanisms of defense and context-dependent responses · 2026 · DOI

• The phenotypic characterization (pH tolerance range 4-10, growth at 45°C, catalase positivity) was conducted on all nine isolates, but stability and viability of these isolates in formulated biofertilizer products under dry storage conditions were not assessed. Long-term shelf-life studies and cell viability preservation methods are required before commercial biofertilizer development.

  Isolation and characterization of non-symbiotic N-fixing bacteria from rhizosphere of Calopogonium mucunoides Desv. · 2026 · DOI

• Field trials under specific drought stress conditions are explicitly mentioned as needed but were not conducted. In vivo validation must test the biofertilizer formulation containing E. adhaerens K4 and Bacillus sp. K6 on C. mucunoides or target crops with controlled soil moisture regimes to quantify plant biomass, nitrogen uptake, and yield improvements.

  Isolation and characterization of non-symbiotic N-fixing bacteria from rhizosphere of Calopogonium mucunoides Desv. · 2026 · DOI

• The two most promising isolates (Ensifer adhaerens K4 and Bacillus sp. K6) were identified only to species/genus level without whole genome sequencing annotation. Comparative genomic analysis is necessary to characterize their complete gene repertoire for stress tolerance mechanisms (heat shock, heavy metal resistance, polyaromatic hydrocarbon degradation) similar to K. rhizophila strain 14ASP.

  Isolation and characterization of non-symbiotic N-fixing bacteria from rhizosphere of Calopogonium mucunoides Desv. · 2026 · DOI

• The study identified nif gene regions through amplification but did not report complete sequencing or quantification of nitrogen fixation activity across the nine non-symbiotic N-fixing bacterial isolates from C. mucunoides rhizosphere. Specific nitrogenase activity assays (acetylene reduction or 15N isotope dilution) are needed to confirm and quantify the actual nitrogen-fixing capacity of each isolate.

  Isolation and characterization of non-symbiotic N-fixing bacteria from rhizosphere of Calopogonium mucunoides Desv. · 2026 · DOI

• The ecological significance of seed microbiome in plant life (reference 33) and metacommunity assembly dynamics (reference 26) have been theoretically framed, but empirical demonstration of how seed microbiota influences seedling microbial colonization outcomes and establishment of plant-associated microbial communities in soil remains incomplete.

  What do we know about the seed microbiome? · 2026 · DOI

• While seed endophytes have been shown to shape disease resistance in rice (reference 12) and promote growth in sugarcane (reference 11), the functional validation of identified endophytic strains from other crop species (maize, tobacco, barley, wheat) under controlled conditions and field trials is limited, restricting practical application potential.

  What do we know about the seed microbiome? · 2026 · DOI

• Conventional seed coating reduces prevalence of proteobacterial endophytes (reference 15), but the quantitative impact of different seed treatment protocols (chemical disinfection, thermal treatment, coating formulations) on preservation versus elimination of beneficial seed microbiota across diverse plant species has not been systematically evaluated.

  What do we know about the seed microbiome? · 2026 · DOI

Most-cited papers in Plant-Microbe Interactions and Immunity

• The plant immune system: From discovery to deployment · Cell · 2024 · 471 citations
• Bacterial indole-3-acetic acid: A key regulator for plant growth, plant-microbe interactions, and agricultural adaptive resilience · Microbiological Research · 2024 · 246 citations
• Enhancing Soil Health and Plant Growth through Microbial Fertilizers: Mechanisms, Benefits, and Sustainable Agricultural Practices · Agronomy · 2024 · 230 citations
• The Function of Root Exudates in the Root Colonization by Beneficial Soil Rhizobacteria · Biology · 2024 · 175 citations
• Salicylic acid (SA)-mediated plant immunity against biotic stresses: An insight on molecular components and signaling mechanism · Plant Stress · 2024 · 158 citations
• Superiority of native soil core microbiomes in supporting plant growth · Nature Communications · 2024 · 144 citations
• Rhizosphere Microorganisms Supply Availability of Soil Nutrients and Induce Plant Defense · Microorganisms · 2024 · 143 citations
• Plant Disease: A Growing Threat to Global Food Security · Agronomy · 2024 · 133 citations
• Microbiome homeostasis on rice leaves is regulated by a precursor molecule of lignin biosynthesis · Nature Communications · 2024 · 130 citations
• Nonpathogenic Pseudomonas syringae derivatives and its metabolites trigger the plant “cry for help” response to assemble disease suppressing and growth promoting rhizomicrobiome · Nature Communications · 2024 · 126 citations

Most recent work

• Synergy between <i>Paenibacillus lentimorbus</i> and <i>Pseudomonas putida</i> mediates ecological benefit to chickpea by decrementing <i>Fusarium oxysporum</i> f. sp. <i>ciceris</i> · Plant Biology · 2026
• What do we know about the seed microbiome? · Microbiome · 2026
• Interpretable multi-omics machine learning reveals drought-driven shifts in plant-microbe interactions · Environmental Microbiome · 2026
• Peroxynitrite-mediated tyrosine nitration modulates β-1,3-glucanase activity and potato defense against Phytophthora infestans · Frontiers in Plant Science · 2026
• Omics-informed insights into biochar–Trichoderma interactions in plant–soil systems: mechanisms of defense and context-dependent responses · World Journal of Microbiology and Biotechnology · 2026
• Exploiting plant immune “switches” for resistance engineering · Stress Biology · 2026
• Serratia marcescens Isolated from the Cereals of Multi-Copping Systems Mitigate Drought Stress in Wheat (Triticum aestivum. L.) · Journal of Crop Health · 2026
• Halotolerant Rhizobacteria from Phragmites Communis: A Controlled Proof-of-Concept for Crop Improvement in Degraded Sandy Soils · Microorganisms · 2026
• Engineering the rhizosphere: When do microbial inoculants actually work? · Rhizosphere · 2026
• A fungal pathogen effector that shapes host plant microbiota kills bacteria through lipoteichoic acid binding and membrane disruption · bioRxiv · 2026

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