**2. Materials and Methods**

#### *2.1. Reagents and Materials Used*

Sigma-Aldrich (St. Louis, MO, USA) provided the chitosan (deacetylation 75%) and sodium tripolyphosphate (TPP). Qualigen, India, supplied gum acacia. Fungicide mancozeb was acquired locally. Hi-Media, Ltd., Mumbai, provided the dialysis tubing. Tomato seeds (Arun Hisar) were bought from the state agricultural university's vegetable section. A polyhouse was used to conduct an in vivo pot experiment. The Vero cell line was kept at the National Research Centre on Equines, Hisar.

ITCC, New Delhi, provided the pathogenic fungi (*Alternaria alternata* and *Stemphylium lycopersici*). The cultures were resurrected according to the manufacturer's instructions.

#### *2.2. Synthesis of Blank and Mancozeb Loaded Chitosan–Gum Acacia Conjugated Nanoparticles*

Ionic gelation and polyelectrolyte complexation were used to make conjugated nanoparticles. Liquidizing 1.0 mg/mL of chitosan in 1.0% glacial acetic acid (*v/v*) and stirring overnight to dissolve entirely yielded a stock of chitosan at a native pH. Different amounts of solid mancozeb were introduced in three conical flasks of 100 mL capacities, along with a 20 mL stock of chitosan in each flask. Adding a stock solution of aqueous gum acacia (1.0 mg/mL, native pH) to each of the above flasks, dropwise, resulted in final concentrations of mancozeb of 0.5, 1.0, and 1.5 mg/mL, respectively. Magnetic stirring continued for another ten minutes. After that, dropwise, we added 2.0 mL of 1.0% TPP to the solution and stirred for 45 min. Each

flask received 10 μL of Tween-20; we stirred the flasks for another 30 min. The suspension was centrifuged at 12,000 rpm for 20 min; the pellet was rinsed with 10 mL of double-distilled water (DDW), and the pellet was kept at 4 ◦C in a 1.5 mL centrifuge vial for future investigation.

Except for the inclusion of mancozeb, all of the previous processes were followed to synthesize the blank NPs.

#### *2.3. Optimization of Experimental Design*

Design-Expert Software (Version 8.0.4, Stat-Ease, Inc., MN, USA) was used to optimize the experiment. Mancozeb-loaded chitosan–gum acacia NPs were optimized using a standard protocol via a central composite design. Three factors, i.e., concentrations of chitosan, gum acacia, and mancozeb, varied, and TPP concentration was constantly retained. The particle size was chosen as the response variable.
