Iturinic Lipopeptide Diversity in the Bacillus subtilis Species Group - Important Antifungals for Plant Disease Biocontrol Applications

Abstract

Iturins and closely related lipopeptides constitute a family of antifungal compounds known as iturinic lipopeptides that are produced by species in the Bacillus subtilis group. The compounds that comprise the family are: iturin, bacillomycin D, bacillomycin F, bacillomycin L, mycosubtilin, and mojavensin. These lipopeptides are prominent in many Bacillus strains that have been commercialized as biological control agents against fungal plant pathogens and as plant growth promoters. The compounds are cyclic heptapeptides with a variable length alkyl sidechain, which confers surface activity properties resulting in an affinity for fungal membranes. Above a certain concentration, enough molecules enter the fungal cell membrane to create a pore in the cell wall, which leads to loss of cell contents and cell death. This study identified 330 iturinic lipopeptide clusters in publicly available genomes from the B. subtilis species group. The clusters were subsequently assigned into distinguishable types on the basis of their unique amino acid sequences and then verified by HPLC MS/MS analysis. The results show some lipopeptides are only produced by one species, whereas certain others can produce up to three. In addition, four species previously not known to produce iturinic lipopeptides were identified. The distribution of these compounds among the B. subtilis group species suggests that they play an important role in their speciation and evolution.

Keywords: FZB42; PGPR; QST713; amyloliquefaciens; biocontrol; biostimulant; fengycin; probiotic.

FIGURE 1

Gene organization and structure of a generic iturinic lipopeptide biosynthesis cluster and sequence of the cyclic heptapeptide of iturinic lipopeptides. The first three positions are Asn-Tyr-Asn for all compounds.

FIGURE 2

Neighbor-joining phylogeny showing iturinic lipopeptide occurrence across the B. subtilis species group. The tree was reconstructed from the core genomes of 584 strains. Bootstrap values >50%, based on 1,000 pseudoreplicates are indicated on branch points. The tree was rooted based on previous core genome phylogenies of the B. subtilis group (Dunlap et al., 2015b). Species in the “Bacillus pumilus” clade did not contain the cluster and were omitted from the figure to save space. The scale bar corresponds to 0.05 nucleotide substitutions per site.

FIGURE 3

Neighbor-joining phylogeny showing iturinic lipopeptide occurrence among strains within the clade that encompasses B. amyloliquefaciens, B. siamensis, and B. velezensis. The tree was reconstructed from the core genomes of 250 strains. Bootstrap values >50%, based on 1,000 pseudoreplicates are indicated on branch points. The tree was rooted based on previous core genome phylogenies of the B. subtilis group (Dunlap et al., 2015b). The scale bar corresponds to 0.005 nucleotide substitutions per site.

FIGURE 4

Neighbor-joining phylogenetic tree reconstructed from the partial ituA gene of iturinic lipopeptide producing strains from the B. subtilis group. Bootstrap values >50%, based on 1,000 pseudoreplicates are indicated on branch points. The scale bar corresponds to 0.05 nucleotide substitutions per site.

FIGURE 5

Agarose gel containing amplicons from selective primers sets to determine iturinic lipopeptide production in B. velezensis strains: (1) iturin, B. velezensis QST713, (2) bacillomycin D, B. velezensis FZB42, (3) bacillomycin L, B. velezensis KACC 18228. The iturin set is ituB-fwd and ituB-iturin-rev; the BacD set is ituB-fwd and ituB-bacD-rev; and the BacL set is ituB-fwd and ituB-R-bacL. A size ladder standard in 100 bp increments is included in the first and last unlabeled lanes, with a bold band corresponding to 600 bp.