Novel Routes for Improving Biocontrol Activity of Bacillus Based Bioinoculants

Abstract

Biocontrol (BC) formulations prepared from plant-growth-promoting bacteria are increasingly applied in sustainable agriculture. Especially inoculants prepared from endospore-forming Bacillus strains have been proven as efficient and environmental-friendly alternative to chemical pesticides due to their long shelf life, which is comparable with that of agrochemicals. However, these formulations of the first generation are sometimes hampered in their action and do not fulfill in each case the expectations of the appliers. In this review we use the well-known plant-associated Bacillus amyloliquefaciens type strain FZB42 as example for the successful application of different techniques offered today by comparative, evolutionary and functional genomics, site-directed mutagenesis and strain construction including marker removal, for paving the way for preparing a novel generation of BC agents.

Keywords: Bacillus amyloliquefaciens subsp. plantarum; bacillomycin D; bacilysin; biocontrol; harpin genes; mersacidin; plant growth-promotion; surfactin.

FIGURE 1

Phylogenetic tree of Bacillus amyloliquefaciens chromosomes currently available in public databases. Based on the core genome of 2104 CDS the divergence of the plant-associated bacteria (B. amyloliquefaciens subsp. plantarum) and B. siamensis and B. amyloliquefaciens subsp. amyloliquefaciens was quantified with FZB42T employed as reference to construct the tree according to Blom et al. (2009). Every set of orthologous genes found in all genomes was separately aligned using the multiple alignment tool MUSCLE (Edgar, 2004). The alignments were concatenated to one huge multiple alignment. A distance matrix was calculated from this alignment and finally a phylogenetic tree was constructed based on this distance matrix using the Neighbor-Joining method. The two latter methods are used in the PHYLIP implementations by Felsenstein (http://evolution.genetics.washington.edu/phylip.html). The Neighbor-Joining method was chosen as it is a heuristic approach with a very good computational efficiency, making it well suited for large datasets resulting from the core genome based tree construction.

FIGURE 2

Site directed mutations introduced into the genome of FZB42. Mutants impaired in synthesis of secondary metabolites (non-ribosamal sythesis, sfp, surfactin, srf, plantazolizin, pzn, macrolactin, mln, bacillaen, bae, bacillomycin D, bmy, fengycin, fen, difficidin, dfn, unidentified peptide, nrs, siderophore, dhb, amylocyclicin, acn, bacilysin), volatiles (alsS, alsD, bdh), sugars (ganA, iolA), amylase (amyE), global regulators (abrB, scoC, degU, codY), alternative sigma factors (sigH, sigW, sigB, sigV, sig01, sigM, sigD, sigX), competence (comA), biofilm formation (srfA, tasA), oxidative stress response (nfrA), plant growth promotion (RBAM-017410), auxotrophy (pabB), and others (yczE, ydbM, ydeH, yerO, yuiA, yusV, spaR, ywfH, rapX, yyaL).

FIGURE 3

Suppression of rice pathogens by FZB42 and its mutant strains. (Top) agar diffusion assay using Xanthomonas oryzae pv.oryzae as indicator strain. (Bottom) agar diffusion assay using Xanthomonas oryzae pv. oryzicola as indicator strain. Bacilysin and difficidin, FZB42 and several mutant strains not impaired in bacilysin and difficidin synthesis have a clear antaogonistic effect against both pathogens whilst mutant strains RS02 and RS06, deficient in synthesis of bacilysin and difficidin, were unable to suppress both pathovars of X. oryzae. Mutant strains unable to synthesize difficidin but able to synthesize bacilysin (CH3, CH8) were found clearly repressed in their antagonistic action.

FIGURE 4

Suppression of Microcystis aeruginosa by transgenic strains FZBREP and FZBSPA. (A) The antagonistic effect of B. amyloliquefaciens strains against a liquid M. aeruginosa culture after 4 and 7 days. (B) The bactericidal activity of B. amyloliquefaciens strains FZB42, FZBREP and FZBSPA. Note that suppressive effect of FZBREP and FZBSPA is enhanced compared to wild type FZB42.

FIGURE 5

Strain construction and enhanced root development of tobacco plants by FZBHarpin. (A) Root development of tobacco seedlings is enhanced after treatment with FZB42 and FZBHarpin. From left to right: control, FZB42, FZBHarpin. (B) Integration of two copies of the hpa1 gene (shown in red) into the chromosome of FZB42 via homologous recombination leads to disruption of the genes encoding alkaline (top) and neutral (bottom) protease. The selectable genes, shown in green, are the resistance genes for spectinomycin (spec) and kanamycin (km). White regions represent recognition sites of site-specific recombinases, and blue regions represent sequences homologous between the genome and the integration cassette. Removal of the selectable marker genes is performed with site specific Cre recombinase at the SSR recognition sites.