Growth Promotion of Delayed-Transplanted Strawberry Plug Seedlings by Single- and Combined-Strain Inoculation of Bacillus spp.

Delayed transplanting of plug seedlings frequently causes root coiling and growth retardation in strawberry (Fragaria × ananassa), compromising transplant quality and commercial nursery performance. While plant growth-promoting bacteria (PGPB) are widely used to enhance crop growth and resilience, their effects on delayed-transplanted strawberry remain poorly understood. This study investigated the individual and combined effects of two Bacillus strains (KP3P9 and K13C) on the growth and root architecture of 12-week-old ‘Benihoppe’ strawberry plug seedlings, which had been delayed-transplanted for approximately 5–8 weeks. Moreover, root metabolomic profiles were analyzed to uncover metabolic reprogramming associated with Bacillus inoculation. Results showed that all Bacillus treatments promoted plant growth, enhanced fresh weight, and improved root development of delayed-transplanted strawberry. Metabolomic analysis revealed that all Bacillus treatments increased steroid levels but decreased flavonoids, ketones/aldehydes/esters, lignans, nucleotides, phenylpropanoids, and sugars in roots of delayed-transplanted strawberry plants. Furthermore, distinct metabolic signatures were found among treatments: the single KP3P9/K13C treatment elevated polyphenols and terpenoids; K13C uniquely increased alcohols, alkaloids, and coumarins, while their co-application (KK) specifically enhanced amino acid accumulation. KEGG enrichment analysis further revealed varied metabolic reprogramming: KP3P9 significantly enriched pantothenate and CoA biosynthesis, cornified envelope formation, and aminoacyl-tRNA biosynthesis; K13C enriched tyrosine metabolism and alpha-linolenic acid (ALA) metabolism, whereas KK specifically enriched flavonoid metabolism. This study reveals the promoting effects of single and combined KP3P9 and K13C applications and provides novel insights into the metabolic basis of Bacillus-mediated growth enhancement on delayed-transplanted strawberry.