Field evaluation of a Bacillus subtilis strain mixture (CHFAB) for soybean cyst nematode biocontrol

Soybean cyst nematode (SCN), Heterodera glycines, is the most destructive pathogen affecting soybean production worldwide. The use of bio-control agents has emerged as an increasingly attractive alternative for SCN management. Here, we evaluated the biocontrol potential of the mixed Bacillus subtilis strains CHFA, CHFB (CHFAB) in suppressing SCN infestation under field conditions across two consecutive growing seasons (2024–2025), using a combination of seed treatment and soil drenching. CHFAB application significantly suppressed SCN development throughout the growing season, reducing adult female numbers by 40–53% at 35 days, female indices by 27–46% at 70 days, and soil egg and juvenile populations by 27–42% at harvest compared to water-treated controls. The enhanced efficacy in 2025 suggests cumulative benefits from reduced soil inoculum following the initial application. Beyond nematode suppression, CHFAB significantly promoted early-season soybean growth, increasing dry biomass by 18–26%, nodule number by 39–50%, and nodule fresh weight by 49–61% at the flowering stage. These improvements correlated with enhanced nitrogen and phosphorus acquisition (15–30% increases) and upregulation of nutrient transport genes (GmNRT2, GmNINa, GmPAT1.1, GmGDPD12). LC-MS analysis revealed that CHFAB elevated endogenous IAA levels 2.3–2.5-fold and SA levels 2.4–4.2-fold in soybean roots under SCN infestation, while qRT-PCR confirmed significant upregulation of auxin-responsive genes (GmAUX22, GmLAX4) and SA-responsive genes (GmNIMIN1, GmPR1). Final seed yield increased modestly in 2024 (4.84%, non-significant) but significantly in 2025 (10.01%, P < 0.05), demonstrating progressive improvement across seasons. These results establish the dual functionality of CHFAB in controlling SCN infection while promoting soybean growth through coordinated regulation of auxin-mediated development, SA-dependent defense responses, and enhanced nutrient acquisition. The B. subtilis strains CHFA and CHFB are promising biocontrol agents for sustainable, integrated SCN management.