# Isolation of Bacillus amyloliquefaciens D39 and Identification of Its Antimicrobial Proteins Active Against Chestnut Blight

## TL;DR

Scientists isolated a beneficial bacterium, Bacillus amyloliquefaciens D39, which produces proteins that effectively fight chestnut blight, a fungal disease threatening chestnut trees.

## Contribution

The study identifies a novel biocontrol agent, Bacillus amyloliquefaciens D39, with strong antimicrobial proteins effective against chestnut blight.

## Key findings

- Strain D39 showed broad-spectrum antifungal activity against Cryphonectria parasitica.
- A 145 kDa serine peptidase protein was purified and identified as a key antimicrobial compound.
- Pot experiments showed D39 reduced disease severity by up to 70.89% in chestnut plants.

## Abstract

Chestnut blight, caused by Cryphonectria parasitica (Murrill) M.E. Bar, is a destructive fungal disease threatening chestnut cultivation and production. In response to the limitations of chemical control, biological control using antagonistic microbes has gained increasing attention. A rhizosphere-derived bacterium, strain D39, was isolated from healthy chestnut trees and identified as Bacillus amyloliquefaciens based on morphological characteristics and the phylogenetic analysis of 16S rRNA and gyrA genes. The antifungal activity of strain D39 against C. parasitica was evaluated using dual-culture and double-layer Oxford cup assays. The strain exhibited broad-spectrum and stable antagonistic effects and harbored five key genes associated with antimicrobial compound biosynthesis (srfAA, ituC, fenD, bmyB, and bacA), as confirmed by PCR. A 145 kDa extracellular protein with strong antifungal activity was extracted and purified by ammonium sulfate precipitation, DEAE ion-exchange chromatography, and Sephadex G-75 gel filtration. LC-MS analysis identified the protein as a serine peptidase belonging to the S8 family, and its structure was predicted using multiple bioinformatic tools. In pot experiments, treatment with the strain D39 significantly reduced disease severity, achieving control efficiencies of 66.07% and 70.89% at 10 and 20 days post-inoculation, respectively. These results demonstrate that B. amyloliquefaciens D39 has strong potential as a biocontrol agent against chestnut blight, offering an effective and environmentally friendly alternative for disease management.