Relationship of Bacillus amyloliquefaciens clades associated with strains DSM 7T and FZB42T: a proposal for Bacillus amyloliquefaciens subsp. amyloliquefaciens subsp. nov. and Bacillus amyloliquefaciens subsp. plantarum subsp. nov. based on complete genome sequence comparisons.

The whole-genome-sequenced rhizobacterium Bacillus amyloliquefaciens FZB42(T) (Chen et al., 2007) and other plant-associated strains of the genus Bacillus described as belonging to the species Bacillus amyloliquefaciens or Bacillus subtilis are used commercially to promote the growth and improve the health of crop plants. Previous investigations revealed that a group of strains represented a distinct ecotype related to B. amyloliquefaciens; however, the exact taxonomic position of this group remains elusive (Reva et al., 2004). In the present study, we demonstrated the ability of a group of Bacillus strains closely related to strain FZB42(T) to colonize Arabidopsis roots. On the basis of their phenotypic traits, the strains were similar to Bacillus amyloliquefaciens DSM 7(T) but differed considerably from this type strain in the DNA sequences of genes encoding 16S rRNA, gyrase subunit A (gyrA) and histidine kinase (cheA). Phylogenetic analysis performed with partial 16S rRNA, gyrA and cheA gene sequences revealed that the plant-associated strains of the genus Bacillus, including strain FZB42(T), formed a lineage, which could be distinguished from the cluster of strains closely related to B. amyloliquefaciens DSM 7(T). DNA-DNA hybridizations (DDH) performed with genomic DNA from strains DSM 7(T) and FZB42(T) yielded relatedness values of 63.7-71.2 %. Several methods of genomic analysis, such as direct whole-genome comparison, digital DDH and microarray-based comparative genomichybridization (M-CGH) were used as complementary tests. The group of plant-associated strains could be distinguished from strain DSM 7(T) and the type strain of B. subtilis by differences in the potential to synthesize non-ribosomal lipopeptides and polyketides. Based on the differences found in the marker gene sequences and the whole genomes of these strains, we propose two novel subspecies, designated B. amyloliquefaciens subsp. plantarum subsp. nov., with the type strain FZB42(T) ( = DSM 23117(T) = BGSC 10A6(T)), and B. amyloliquefaciens subsp. amyloliquefaciens subsp. nov., with the type strain DSM 7(T)( = ATCC 23350(T) = Fukumoto Strain F(T)), for plant-associated and non-plant-associated representatives, respecitvely. This is in agreement with results of DDH and M-CGH tests and the MALDI-TOF MS of cellular components, all of which suggested that the ecovars represent two different subspecies.

Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42.

Bacillus amyloliquefaciens FZB42 is a Gram-positive, plant-associated bacterium, which stimulates plant growth and produces secondary metabolites that suppress soil-borne plant pathogens. Its 3,918-kb genome, containing an estimated 3,693 protein-coding sequences, lacks extended phage insertions, which occur ubiquitously in the closely related Bacillus subtilis 168 genome. The B. amyloliquefaciens FZB42 genome reveals an unexpected potential to produce secondary metabolites, including the polyketides bacillaene and difficidin. More than 8.5% of the genome is devoted to synthesizing antibiotics and siderophores by pathways not involving ribosomes. Besides five gene clusters, known from B. subtilis to mediate nonribosomal synthesis of secondary metabolites, we identified four giant gene clusters absent in B. subtilis 168. The pks2 gene cluster encodes the components to synthesize the macrolactin core skeleton.

Profiling for Bioactive Peptides and Volatiles of Plant Growth Promoting Strains of the Bacillus subtilis Complex of Industrial Relevance.

Plant growth promoting rhizobacteria attain increasing importance in agriculture as biofertilizers and biocontrol agents. These properties significantly depend on the formation of bioactive compounds produced by such organisms. In our work we investigated the biosynthetic potential of 13 industrially important strains of the Bacillus subtilis complex by mass spectrometric methodology. Typing of these organisms was performed with MALDI-TOF mass spectrometry followed by comprehensive profiling of their bioactive peptide products. Volatiles were determined by gas chromatography-mass spectrometry. Representative products of the members of the B. subtilis complex investigated in detail were: the surfactin familiy (surfactins, lichenysins, pumilacidins); the iturin family (iturins, mycosubtilins and bacillomycins); plantazolicin and the dual lantibiotics lichenicidins, as well as a wide spectrum of volatiles, such as hydrocarbons (alkanes/alkenes), alcohols, ketones, sulfur-containing compounds and pyrazines. The subcomplexes of the B. subtilis organizational unit; (a) B. subtilis/Bacillus atrophaeus; (b) B. amyloliquefaciens/B. velezensis; (c) B. licheniformis, and (d) B. pumilus are equipped with specific sets of these compounds which are the basis for the evaluation of their biotechnological and agricultural usage. The 13 test strains were evaluated in field trials for growth promotion of potato and maize plants. All of the implemented strains showed efficient growth stimulation of these plants. The highest effects were obtained with B. velezensis, B. subtilis, and B. atrophaeus strains.

Successful Formulation and Application of Plant Growth-Promoting Kosakonia radicincitans in Maize Cultivation.

The global market for biosupplements is expected to grow by 14 percent between 2014 and 2019 as a consequence of the proven benefits of biosupplements on crop yields, soil fertility, and fertilizer efficiency. One important segment of biosupplements is plant growth-promoting bacteria (PGPB). Although many potential PGPB have been discovered, suitable biotechnological processing and shelf-life stability of the bacteria are challenges to overcome for their successful use as biosupplements. Here, the plant growth-promoting Gram-negative strain Kosakonia radicincitans DSM 16656T (family Enterobacteriaceae) was biotechnologically processed and applied in the field. Solid or liquid formulations of K. radicincitans were diluted in water and sprayed on young maize plants (Zea mays L.). Shelf-life stability tests of formulated bacteria were performed under 4°C and -20°C storage conditions. In parallel, the bacterial formulations were tested at three different farm level field plots characterized by different soil properties. Maize yield was recorded at harvest time, and both formulations increased maize yields in silage as well as grain maize, underlining their positive impact on different agricultural systems. Our results demonstrate that bacteria of the family Enterobacteriaceae, although incapable of forming spores, can be processed to successful biosupplements.

Effects of Bacillus amyloliquefaciens FZB42 on lettuce growth and health under pathogen pressure and its impact on the rhizosphere bacterial community.

The soil-borne pathogen Rhizoctonia solani is responsible for crop losses on a wide range of important crops worldwide. The lack of effective control strategies and the increasing demand for organically grown food has stimulated research on biological control. The aim of the present study was to evaluate the rhizosphere competence of the commercially available inoculant Bacillus amyloliquefaciens FZB42 on lettuce growth and health together with its impact on the indigenous rhizosphere bacterial community in field and pot experiments. Results of both experiments demonstrated that FZB42 is able to effectively colonize the rhizosphere (7.45 to 6.61 Log 10 CFU g(-1) root dry mass) within the growth period of lettuce in the field. The disease severity (DS) of bottom rot on lettuce was significantly reduced from severe symptoms with DS category 5 to slight symptom expression with DS category 3 on average through treatment of young plants with FZB42 before and after planting. The 16S rRNA gene based fingerprinting method terminal restriction fragment length polymorphism (T-RFLP) showed that the treatment with FZB42 did not have a major impact on the indigenous rhizosphere bacterial community. However, the bacterial community showed a clear temporal shift. The results also indicated that the pathogen R. solani AG1-IB affects the rhizosphere microbial community after inoculation. Thus, we revealed that the inoculant FZB42 could establish itself successfully in the rhizosphere without showing any durable effect on the rhizosphere bacterial community.

Bacillus subtilis FZB24 affects flower quantity and quality of saffron (Crocus sativus).

The effect of Bacillus subtilis FZB24 on saffron ( Crocus sativus L.) was studied using saffron corms from Spain and the powdered form of B. SUBTILIS FZB24(R). Corms were soaked in water or in B. subtilis FZB24 spore solution for 15 min before sowing. Some corms were further soil drenched with the spore solution 6, 10 or 14 weeks after sowing. Growth and saffron stigma chemical composition were measured. Compared to untreated controls, application of B. subtilis FZB24 significantly increased leaf length, flowers per corm, weight of the first flower stigma, total stigma biomass; microbe addition also significantly decreased the time required for corms to sprout and the number of shoot sprouts. Compared to the controls, picrocrocin, crocetin and safranal compounds were significantly increased when the plants were soil drenched with the spore solution 14 weeks after sowing; in contrast crocin was highest in untreated controls. Results of this study suggest that application of B. subtilis FZB24 may provide some benefit to saffron growers by speeding corm growth (earlier shoot emergence) and increasing stigma biomass yield by 12 %. While some treatment conditions also increased saffron chemical composition, these were generally not the same treatments that simultaneously improved growth yields and thus, more study is required.