Synergistic Effects of a Root-Endophytic Trichoderma Fungus and Bacillus on Early Root Colonization and Defense Activation Against Verticillium longisporum in Rapeseed.

Rhizosphere-competent microbes often interact with plant roots and exhibit beneficial effects on plant performance. Numerous bacterial and fungal isolates are able to prime host plants for fast adaptive responses against pathogen attacks. Combined action of fungi and bacteria may lead to synergisms exceeding effects of single strains. Individual beneficial fungi and bacteria have been extensively studied in Arabidopsis thaliana, but little is known about their concerted actions in the Brassicaceae. Here, an in-vitro system with oilseed rape (Brassica napus) was established. Roots of two different cultivars were inoculated with well-characterized fungal (Trichoderma harzianum OMG16) and bacterial (Bacillus velezensis FZB42) isolates alone or in combination. Microscopic analysis confirmed that OMG16 hyphae entered root hairs through root hair tips and formed distinct intracellular structures. Quantitative PCR revealed that root colonization of OMG16 increased up to 10-fold in the presence of FZB42. Relative transcript levels of the ethylene- and jasmonic acid-responsive genes PDF1.2, ERF2, and AOC3 were recorded in leaves by quantitative reverse transcription PCR to measure induced systemic resistance in tissues distant from the roots. Combined action of OMG16 and FZB42 induced transcript abundances more efficiently than single inoculation. Importantly, microbial priming reduced Verticillium longisporum root infection in rapeseed by approximately 100-fold compared with nonprimed plants. Priming also led to faster and stronger systemic responses of the defense genes PDF1.2, ERF2, AOC3, and VSP2.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Fusaricidins, Polymyxins and Volatiles Produced by Paenibacillus polymyxa Strains DSM 32871 and M1.

Paenibacilli are efficient producers of potent agents against bacterial and fungal pathogens, which are of great interest both for therapeutic applications in medicine as well as in agrobiotechnology. Lipopeptides produced by such organisms play a major role in their potential to inactivate pathogens. In this work we investigated two lipopeptide complexes, the fusaricidins and the polymyxins, produced by Paenibacillus polymyxa strains DSM 32871 and M1 by MALDI-TOF mass spectrometry. The fusaricidins show potent antifungal activities and are distinguished by an unusual variability. For strain DSM 32871 we identified numerous yet unknown variants mass spectrometrically. DSM 32871 produces polymyxins of type E (colistins), while M1 forms polymyxins P. For both strains, novel but not yet completely characterized polymyxin species were detected, which possibly are glycosylated. These compounds may be of interest therapeutically, because polymyxins have gained increasing attention as last-resort antibiotics against multiresistant pathogenic Gram-negative bacteria. In addition, the volatilomes of DSM 32781 and M1 were investigated with a GC-MS approach using different cultivation media. Production of volatile organic compounds (VOCs) was strain and medium dependent. In particular, strain M1 manifested as an efficient VOC-producer that exhibited formation of 25 volatiles in total. A characteristic feature of Paenibacilli is the formation of volatile pyrazine derivatives.

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.

Fusaricidins from Paenibacillus polymyxa M-1, a family of lipohexapeptides of unusual complexity-a mass spectrometric study.

Paenibacillus polymyxa are rhizobacteria with a high potential to produce natural compounds of biotechnological and medical interest. Main products of P. polymyxa are fusaricidins, a large family of antifungal lipopeptides with a 15-guanidino-3-hydroxypentadecanoic acid (GHPD) as fatty acid side chain. We use the P. polymyxa strain M-1 as a model organism for the exploration of the biosynthetic potential of these rhizobacteria. Using matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) about 40 new fusaricidins were detected which were fractionated by reversed-phase (rp) HPLC. Their structure was determined by MALDI-LIFT-TOF/TOF fragment analysis. The dominant fragment in the product ion spectra of fusaricidins appeared at m/z 256.3, 284.3 and 312.4, respectively, indicating variations in their fatty acid part. Two new subfamilies of fusaricidins were introduced which contain guanidino-3-hydroxyhepta- and nonadecanoic acid as fatty acid constituents. Apparently, the end-standing guanidine group is not modified as shown by direct infusion nano-electrospray ionization mass spectrometry (nano-ESI MS). The results of this study suggest that advanced mass spectrometry is the method of choice for investigating natural compounds of unusual diversity, like fusaricidins. Copyright © 2016 John Wiley & Sons, Ltd.

Characterization of Novel Fusaricidins Produced by Paenibacillus polymyxa-M1 Using MALDI-TOF Mass Spectrometry.

Paenibacillus polymyxa-M1 is a potent producer of bioactive compounds, such as lipopeptides, polyketides, and lantibiotics of biotechnological and medical interest. Genome sequencing revealed nine gene clusters for nonribosomal biosynthesis of such agents. Here we report on the investigation of the fusaricidins, a complex of cyclic lipopeptides containing 15-guanidino-3-hydroxypentadecanoic acid (GHPD) as fatty acid component by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). More than 20 variants of these compounds were detected and characterized in detail. Mass spectrometric sequence analysis was performed by MALDI-LIFT-TOF/TOF fragment analysis. The obtained product ion spectra show a specific processing in the fatty acid part. GHPD is cleaved between the α- and ß-position yielding two fragments a and b, one bearing the end-standing guanidine group and another one comprising the residual two C-atoms of GHPD with the attached peptide moiety. The complete sequence of all fusaricidins was derived from sets of bn- and yn-ions. The fusaricidin complex can be divided into four lipopeptide families, three of them showing variations of the amino acid in position 3, Val or Ile for the first and Tyr or Phe for families 2 and 3, respectively. A collection of novel fusaricidins was detected differing from those of families 1-3 by an additional residue of 71 Da (family 4). LIFT-TOF/TOF fragment spectra of these species imply that in their peptide moiety, an Ala-residue is attached by an ester bond to the free hydroxyl group of Thr4. More than 10 novel fusaricidins were characterized mass spectrometrically.

Cyclic Lipopeptides of Bacillus amyloliquefaciens subsp. plantarum Colonizing the Lettuce Rhizosphere Enhance Plant Defense Responses Toward the Bottom Rot Pathogen Rhizoctonia solani.

The commercially available inoculant Bacillus amyloliquefaciens FZB42 is able to considerably reduce lettuce bottom rot caused by Rhizoctonia solani. To understand the interaction between FZB42 and R. solani in the rhizosphere of lettuce, we used an axenic system with lettuce bacterized with FZB42 and inoculated with R. solani. Confocal laser scanning microscopy showed that FZB42 could delay the initial establishment of R. solani on the plants. To show which secondary metabolites of FZB42 are produced under these in-situ conditions, we developed an ultra-high performance liquid chromatography coupled to time of flight mass spectrometry-based method and identified surfactin, fengycin, and bacillomycin D in the lettuce rhizosphere. We hypothesized that lipopeptides and polyketides play a role in enhancing the plant defense responses in addition to the direct antagonistic effect toward R. solani and used a quantitative real-time polymerase chain reaction-based assay for marker genes involved in defense signaling pathways in lettuce. A significant higher expression of PDF 1.2 observed in the bacterized plants in response to subsequent pathogen challenge showed that FZB42 could enhance the lettuce defense response toward the fungal pathogen. To identify if surfactin or other nonribosomally synthesized secondary metabolites could elicit the observed enhanced defense gene expression, we examined two mutants of FZB42 deficient in production of surfactin and the lipopetides and polyketides, by expression analysis and pot experiments. In the absence of surfactin and other nonribosomally synthesized secondary metabolites, there was no enhanced PDF 1.2-mediated response to the pathogen challenge. Pot experiment results showed that the mutants failed to reduce disease incidence in lettuce as compared with the FZB42 wild type, indicating, that surfactin as well as other nonribosomally synthesized secondary metabolites play a role in the actual disease suppression and on lettuce health. In conclusion, our study showed that nonribosomally synthesized secondary metabolites of FZB42 are actually produced in the lettuce rhizosphere and contribute to the disease suppression by mediating plant defense gene expression toward the pathogen R. solani.

Transposon mutagenesis of the plant-associated Bacillus amyloliquefaciens ssp. plantarum FZB42 revealed that the nfrA and RBAM17410 genes are involved in plant-microbe-interactions.

Bacillus amyloliquefaciens ssp. plantarum FZB42 represents the prototype of Gram-positive plant growth promoting and biocontrol bacteria. In this study, we applied transposon mutagenesis to generate a transposon library, which was screened for genes involved in multicellular behavior and biofilm formation on roots as a prerequisite of plant growth promoting activity. Transposon insertion sites were determined by rescue-cloning followed by DNA sequencing. As in B. subtilis, the global transcriptional regulator DegU was identified as an activator of genes necessary for swarming and biofilm formation, and the DegU-mutant of FZB42 was found impaired in efficient root colonization. Direct screening of 3,000 transposon insertion mutants for plant-growth-promotion revealed the gene products of nfrA and RBAM_017140 to be essential for beneficial effects exerted by FZB42 on plants. We analyzed the performance of GFP-labeled wild-type and transposon mutants in the colonization of lettuce roots using confocal laser scanning microscopy. While the wild-type strain heavily colonized root surfaces, the nfrA mutant did not colonize lettuce roots, although it was not impaired in growth in laboratory cultures, biofilm formation and swarming motility on agar plates. The RBAM17410 gene, occurring in only a few members of the B. subtilis species complex, was directly involved in plant growth promotion. None of the mutant strains were affected in producing the plant growth hormone auxin. We hypothesize that the nfrA gene product is essential for overcoming the stress caused by plant response towards bacterial root colonization.

Amylocyclicin, a novel circular bacteriocin produced by Bacillus amyloliquefaciens FZB42.

Bacillus amyloliquefaciens FZB42 is a Gram-positive plant growth-promoting bacterium with an impressive capacity to synthesize nonribosomal secondary metabolites with antimicrobial activity. Here we report on a novel circular bacteriocin which is ribosomally synthesized by FZB42. The compound displayed high antibacterial activity against closely related Gram-positive bacteria. Transposon mutagenesis and subsequent site-specific mutagenesis combined with matrix-assisted laser desorption ionization-time of flight mass spectroscopy revealed that a cluster of six genes covering 4,490 bp was responsible for the production, modification, and export of and immunity to an antibacterial compound, here designated amylocyclicin, with a molecular mass of 6,381 Da. Peptide sequencing of the fragments obtained after tryptic digestion of the purified peptide revealed posttranslational cleavage of an N-terminal extension and head-to-tail circularization of the novel bacteriocin. Homology to other putative circular bacteriocins in related bacteria let us assume that this type of peptide is widespread among the Bacillus/Paenibacillus taxon.

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.

Draft Genome Sequence of Bacillus atrophaeus UCMB-5137, a Plant Growth-Promoting Rhizobacterium.

Bacillus atrophaeus UCMB-5137 shows an extraordinary activity in root colonization and plant and crop protection. Its draft genome sequence comprises 21 contigs of 4.11 Mb, harboring 4,167 coding sequences (CDS). The genome carries several genes encoding antimicrobial lipopeptides and polyketides. Multiple horizontally acquired genes of possible importance for plant colonization were also found.

Bacterial Traits Involved in Colonization of Arabidopsis thaliana Roots by Bacillus amyloliquefaciens FZB42.

Colonization studies previously performed with a green-fluorescent-protein, GFP, labeled derivative of Bacillus amyloliquefaciens FZB42 revealed that the bacterium behaved different in colonizing surfaces of plant roots of different species (Fan et al., 2012). In order to extend these studies and to elucidate which genes are crucial for root colonization, we applied targeted mutant strains to Arabidopsis seedlings. The fates of root colonization in mutant strains impaired in synthesis of alternative sigma factors, non-ribosomal synthesis of lipopeptides and polyketides, biofilm formation, swarming motility, and plant growth promoting activity were analyzed by confocal laser scanning microscopy. Whilst the wild-type strain heavily colonized surfaces of root tips and lateral roots, the mutant strains were impaired in their ability to colonize root tips and most of them were unable to colonize lateral roots. Ability to colonize plant roots is not only dependent on the ability to form biofilms or swarming motility. Six mutants, deficient in abrB-, sigH-, sigD-, nrfA-, yusV and RBAM017410, but not affected in biofilm formation, displayed significantly reduced root colonization. The nrfA- and yusV-mutant strains colonized border cells and, partly, root surfaces but did not colonize root tips or lateral roots.