Bacillus subtilis strain NCD-2 is strongly antagonistic toward phytopathogenic fungi, and functions as an excellent biocontrol agent for cotton soil-borne diseases. The aims of this study were to characterize the main active antifungal compound from strain NCD-2 and clarify its role in suppressing cotton damping-off disease. Strain NCD-2 and lipopeptide extract prepared from an NCD-2 culture strongly inhibited the growth of Rhizoctonia solani in vitro. The lipopeptides of strain NCD-2 were separated by fast protein liquid chromatography (FPLC) and the antifungal compound was identified as a cluster of fengycin homologs analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. A fengycin-deficient mutant was obtained by in-frame deletion of the fengycin synthetase gene in B. subtilis NCD-2. Compared with the wild-type strain, this mutant showed decreased abilities to inhibit the growth of R. solani in vitro and to suppress cotton damping-off disease in vivo. Studies showed that the population of fengycin-deficient mutant was almost same as that of the wild-type NCD-2 strain in the cotton rhizosphere. However, the population of R. solani in the cotton rhizosphere colonized by the fengycin-deficient mutant was twice that in the cotton rhizosphere colonized by the NCD-2 wild-type strain. This study demonstrated that fengycin-type lipopeptides are the main antifungal active compounds produced by B. subtilis NCD-2. These compounds play a major role in restricting the population of R. solani in the cotton rhizosphere and in suppressing cotton damping-off disease.

Bacillus atrophaeus CAB-1 displays a high inhibitory activity against various fungal pathogens and suppresses cucumber powdery mildew and tomato gray mold. We extracted and identified lipopeptides and secreted proteins and volatile compounds produced by strain CAB-1 to investigate the mechanisms involved in its biocontrol performance. In vitro assays indicated all three types of products contributed to the antagonistic activity against the fungal pathogen Botrytis cinerea. Each of these components also effectively prevented the occurrence of the cucumber powdery mildew caused by Sphaerotheca fuliginea under greenhouse conditions. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry revealed that the major bioactive lipopeptide was fengycin A (C15-C17). We isolated the crude-secreted proteins of CAB-1 and purified a fraction with antifungal activity. This protein sequence shared a high identity with a putative phage-related pre-neck appendage protein, which has not been reported as an antifungal factor. The volatile compounds produced by CAB-1 were complex, including a range of alcohols, phenols, amines, and alkane amides. O-anisaldehyde represented one of the most abundant volatiles with the highest inhibition on the mycelial growth of B. cinerea. To our knowledge, this is the first report on profiling three types of antifungal substances in Bacilli and demonstrating their contributions to plant disease control.

Bacillus subtilis NCD-2 is an excellent biocontrol agent for tomato gray mold and cotton soil-borne diseases. The fengycin lipopeptides serve as a major role in its biocontrol ability. A previous study revealed that insertion of degQ with the mini-Tn10 transposon decreased the antifungal activity of strain NCD-2 against the growth of Botrytis cinerea. To clarify the regulation of degQ on the production of fengycin, we deleted degQ by in-frame mutagenesis. Compared with the wild-type strain NCD-2, the degQ-null mutant had decreased extracellular protease and cellulase activities as well as antifungal ability against the growth of B. cinerea in vitro. The lipopeptides from the degQ-null mutant also had significantly decreased antifungal activity against B. cinerea in vitro and in vivo. This result was confirmed by the decreased fengycin production in the degQ-null mutant that was detected by fast protein liquid chromatography analysis. Quantitative reverse transcription PCR further demonstrated that degQ positively regulated the expression of the fengycin synthetase gene. In addition, the degQ-null mutant also had a flatter colony phenotype and significantly decreased biofilm formation ability relative to the wild-type strain. All of those characteristics from degQ-null mutant could be restored to the strain NCD-2 wild-type level by complementation of intact degQ in the mutant. Therefore, DegQ may be an important regulator of fengycin production and biofilm formation in B. subtilis NCD-2.