Combined Application of Bacillus subtilis MGRP21 and Bacillus amyloliquefaciens CFGP92 by Seed Priming Suppresses Alternaria burnsii and Improves the Growth of Zinnia elegans

Zinnia elegans is an annual ornamental plant widely cultivated for the cut flower industry, but Alternaria leaf spot reduces plant quality and market value. In the present study, the causal agent of leaf spot was isolated from symptomatic zinnia leaves and identified through morphological characterization, pathogenicity testing, and molecular characterization as Alternaria burnsii . In vitro antagonism assays were conducted to screen Bacillus isolates against A. burnsii , followed by seed germination assays and greenhouse experiments comparing seed priming and soil drenching. Bacillus subtilis MGRP21 showed the highest inhibitory activity against A. burnsii , producing a 5.5 cm inhibition zone, followed by B. amyloliquefaciens CFGP92 with a 3.3 cm zone. Seed priming with B. subtilis MGRP21 at 1 × 10 6 CFU mL −1 showed the highest seed germination rate of 80% in the in vitro plate assay. Under greenhouse conditions, seed priming with coapplication of MGRP21 and CFGP92 at a 1:1 ratio was more effective than soil drenching, reducing disease incidence to 19% and disease severity to 0.5. This treatment also improved shoot and root length, fresh and dry biomass, photosynthetic rate, transpiration rate, relative water content, stomatal conductance, and photosynthetic pigment compared with untreated control. Moreover, combined seed priming reduced oxidative stress by lowering malondialdehyde (MDA), hydrogen peroxide H 2 O 2 , and electrolyte leakage (EL), while increasing proline and protein contents and enhancing the activities of defense‐ and antioxidant‐related enzymes, including polyphenol oxidase (PPO), peroxidase (POD), phenylalanine ammonia lyase (PAL), ascorbate peroxidase (APX), catalase (CAT), and superoxide dismutase (SOD). Overall, seed biopriming with a 1:1 mixture of MGRP21 and CFGP92 is recommended as a promising eco‐friendly strategy for suppressing A. burnsii leaf spot and improving zinnia growth, however, field validation, strain‐compatibility testing, and formulation studies are required before commercial use.