Endophytes have been explored and found to perform an important role in plant health. However, their effects on the host physiological function and disease management remain elusive. The present study aimed to assess the potential effects of endophytes, singly as well as in combination, inWithania somnifera(L.) Dunal, on various physiological parameters and systemic defense mechanisms againstAlternaria alternata. Seeds primed with the endophytic bacteriaBacillus amyloliquefaciensandPseudomonas fluorescensindividually and in combination demonstrated an enhanced vigor index and germination rate. Interestingly, plants treated with the two-microbe combination showed the lowest plant mortality rate (28%) underA. alternatastress. Physiological profiling of treated plants showed improved photosynthesis, respiration, transpiration, and stomatal conductance under pathogenic stress. Additionally, these endophytes not only augmented defense enzymes and antioxidant activity in treated plants but also enhanced the expression of salicylic acid- and jasmonic acid-responsive genes in the stressed plants. Reductions in reactive oxygen species (ROS) and reactive nitrogen species (RNS) along with enhanced callose deposition in host plant leaves corroborated well with the above findings. Altogether, the study provides novel insights into the underlying mechanisms behind the tripartite interaction of endophyte-A. alternata-W. somniferaand underscores their ability to boost plant health under pathogen stress.IMPORTANCEW. somniferais well known for producing several medicinally important secondary metabolites. These secondary metabolites are required by various pharmaceutical sectors to produce life-saving drugs. However, the cultivation ofW. somniferafaces severe challenge from leaf spot disease caused byA. alternata. To keep pace with the rising demand for this plant and considering its capacity for cultivation under field conditions, the present study was undertaken to develop approaches to enhance production ofW. somniferathrough intervention using endophytes. Application of bacterial endophytes not only suppresses the pathogenicity ofA. alternatabut also mitigates excessive ROS/RNS generation via enhanced physiological processes and antioxidant machinery. Expression profiling of plant defense-related genes further validates the efficacy of bacterial endophytes against leaf spot disease.

Bioengineered silver nanoparticles can emerge as a facile approach to combat plant pathogen, reducing the use of pesticides in an eco-friendly manner. The plants’ response during tripartite interaction of plant, pathogen, and nanoparticles remains largely unknown. This study demonstrated the use of bioengineered silver nanoparticles in combating black spot disease caused by necrotrophic fungus Alternaria brassicicola in Arabidopsis thaliana via foliar spray. The particles reduced disease severity by 70–80% at 5 μg/ml without showing phytotoxicity. It elicited plant immunity by a significant reduction in reactive oxygen species (ROS), decreases in stress enzymes by 0.6–19.8-fold, and emergence of autophagy. Comparative plant proteomics revealed 599 proteins expressed during the interaction, where 117 differential proteins were identified. Among different categories, proteins involved in bioenergy and metabolism were most abundant (44%), followed by proteins involved in plant defense (20%). Metabolic profiling by gas chromatography–mass spectroscopy yielded 39 metabolite derivatives in non-polar fraction and 25 in the polar fraction of plant extracts. It was observed that proteins involved in protein biogenesis and early plant defense were overexpressed to produce abundant antimicrobial metabolites and minimize ROS production. Bioengineered silver nanoparticles performed dual functions to combat pathogen attack by killing plant pathogen and eliciting immunity by altering plant defense proteome and metabolome.