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Book ID: 322257_1_En ChapterID: 40 Dispatch Date: 10-09-2014 Proof No: 1

Chapter 40Bacillus, A Plant-Beneficial Bacterium

Rainer Borriss

Abstract Plant growth promotion and biocontrol of plant pathogens are features of1

Bacillus inoculants applied for a more sustainable agriculture. Recent results mainly2

obtained with Bacillus amyloliquefaciens FZB42 and other representatives of the3

B. amyloliquefaciens plantarum subspecies support the hypothesis that stimulation4

of plant induced systemic resistance (ISR) by bacterial metabolites produced in the5

vicinity of plant roots is the key mechanism in the biocontrol action of Gram-positive6

endospore-forming bacteria, whereas a direct effect of the numerous antimicrobial7

metabolites in suppressing pathogens in the vicinity of plant roots seems to be of8

minor importance.9

40.1 Overview About General Properties and Taxonomy10

Several representatives of the Gram-positive Bacillus spp. and Paenibacillus spp. are11

able to colonize plants and to develop thereby beneficial actions on plant growth and12

health. At present, Bacilli are by far the most widely used bacteria on the biopes-13

ticide market (Borriss 2011). This is mainly due to their ability to produce durable14

endospores, which allows the preparation of stable bioformulations with a long15

shelf-life. Especially members of the B. subtilis species complex, such as B. sub-16

tilis, B. amyloliquefaciens, and B. pumilus, have been proven to be efficient in plant17

growth- promotion and biocontrol against plant pathogens. B. subtilis and B. amy-18

loliquefaciens strains are difficult to distinguish, and several bioagents declared as19

containing B. subtilis spores are in fact representatives of the plant-associated B.20

amyloliquefaciens subsp. plantarum (Borriss et al. 2011).21

The Bacillus subtilis Group B. subtilis is the model organism of Gram-positive22

bacteria. The strictly aerobe B. amyloliquefaciens plantarum, represented by its type23

strain FZB42T, is distinguished from other representatives of the B. subtilis group by24

its large capacity to synthesize non-ribosomally a high number of polyketides and25

R. Borriss (�)ABiTEP GmbH, Glienicker Weg 185, Berlin, Germanye-mail: [email protected]

© Springer International Publishing Switzerland 2015 1B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,DOI 10.1007/978-3-319-08575-3_40

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2 R. Borriss

lipopeptides. Examples of commercial products (biocontrol or biofertilizer) contain-26

ing B. amyloliquefaciens plantarum as their main active ingredient are KodiakTM27

(Bayer Crop Science), Companion (Growth Products Ltd.), BioYieldTM (Bayer28

Crop Science), INTEGRAL® (BASF), VAULT® (BASF), SERENADE Max®29

(Bayer Crop Science), CEASE(R) (BioWorks, Inc.), RhizoVital® (ABiTEP GmbH),30

RhizoPlus® (ABiTEP GmbH), Double Nickel 55TM (Certis USA), and Amylo-X®31

(Certis USA). See also Table 40.1 for commercial Bacillus products for agriculture.32

B. licheniformis and B. pumilus are other members of the B. subtilis group. By33

contrast to B. subtilis and B. amyloliquefaciens, they are facultative anaerobes. Bio-34

control agents based on B. licheniformis SB3086 are Green Releaf and EcoGuard35

(Novozyme Biologicals Inc.). B. pumilus strain GB34 (Yield Shield, Bayer Crop36

Science) is used as an active ingredient in agricultural fungicides. Other EPA reg-37

istered biofungicides are SONATA (Bayer Crop Science), and GHA 180 (Premier38

Horticulture).39

Other Bacilli, not Belonging to the B. subtilis Species Complex, also stimulate40

plant growth and health. B. firmus GB126 isolated from cultivated soil is used to41

control root-knot nematodes in glasshouse and field grown vegetable crops (BioNem42

AgroGreen, originally from Israel, later acquired by Bayer Crop Science; EPA reg-43

istered nematicide). Certis USA is developing a product based on B. firmus named44

BmJ WG. The biofungicide BioArc is prepared from B. megaterium, the largest45

representative of the genus Bacillus.46

Paenibacillus spp. The PGPR Paenibacillus polymyxa, formerly known as Bacil-47

lus polymyxa, can promote plant growth by producing plant hormones, such as48

IAA, cytokinins, gibberellins, and ethylene, and volatile compounds. The faculta-49

tive anaerobe is capable of fixing nitrogen, and of synthesing many antibacterial50

and antifungal secondary metabolites. NH is a registered fungicide prepared from51

Paenibacillus polymyxa AC-1 by Green Biotech Company Ltd.52

The PGPR P. mucilaginosus is able to degrade insoluble soil minerals with the re-53

lease of nutritional ions, such as potassium and phosphorous. Similar to P. polymyxa,54

P. mucilaginosus is also capable of fixing nitrogen.55

In the following, I will shortly highlight the different traits of Bacilli involved56

in their beneficial effect on plants, mainly by using results obtained during the57

last decade with FZB42T, which has been successfully commercialized by ABiTEP58

GmbH (http://www.abitep.de/de/), but is also used as a model strain for scientific59

research (Borriss 2011).60

40.2 Root Colonization61

The ability of FZB42 to colonize the rhizoplane is a precondition for plant growth-62

promotion. Using a GFP-tagged derivative (Fan et al. 2011) the fate of bacterial root63

colonization was recently studied. The bacterium behaves different in colonizing root64

surfaces of different plants. FZB42 colonized preferentially root tips when colonizing65

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BACILLIBACTINSURFACTIN

BACILLOMYCIN D

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Pi

Fig. 40.1 Secondary metabolites with biocontrol or PGP activities produced by B. amyloliquefa-ciens FZB42. Genes involved in plant root colonization (white) and plant growth promotion (yellow)are listed within the bacterial cell. The cyclic lipopeptides (cLP, blue) surfactin, bacillomycin D,and fengycin are nonribosomally synthesized by modularly organized, giant peptide synthetases(NRPSs). Antibacterial polyketides (PK, red) are synthesized by membrane-anchored, polyketidemegasynthases. Synthesis of PKs and cLPs is dependent on functional phosphor-panthetheinyl-transferase Sfp. NRPSs are also involved in synthesis of the dipeptide bacilysin (blue) and the Fe2+siderophore bacillibactin (blue). The plant growth-promoting metabolites acetoin, 2,3-butanediol,and indole-3-acetic acid (IAA) are shown in green. Extracellular phytase (green) makes phosphatefixed in phytate accessible for plant nutrition. Other extracellular enzymes, which are degradingmacromolecules, and supporting the biofertilizer function of FZB42, are ß-glucan and xylan hy-drolases, amylases, and proteases, for example. Bacterial metabolites, involved in stimulating plantinduced systemic resistance (ISR), are framed

Arabidopsis thaliana. In lettuce, bacterial colonization occurred mainly on primary66

roots and root hairs, as well as on root tips and adjacent border cells. Essential genes67

for root colonization are involved in surfactin production, motility, biofilm formation,68

and stress response (Fig. 40.1). Mutants containing a transposon insertion in the nfrA69

gene, encoding a putative nitro/flavin oxidoreductase, were unable to persist on the70

surface of lettuce roots, most likely due to their inability to develop an appropriate71

response against the plant’s stress reactions (Budiharjo et al. 2014).72

The Rhizosphere Competence of FZB42 was studied by using a combination of73

field and greenhouse trials. FZB42 is able to effectively colonize the rhizosphere74

(6.61–7.45 Log10 CFU g−1 root dry mass) within the growth period of lettuce in the75

field. However, the cell number (CFU) of FZB42 per gram of soil decreased to 14 % of76

the initial number of cells after 5 weeks of field cultivation (Chowdhury et al. 2013).77

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The same samples were analyzed more deeply by mapping of the metagenome se-78

quences corresponding to FZB42. The method called ‘fragment recruitments’ was79

used to track persistence of the inoculant FZB42 within the lettuce rhizosphere.80

Five weeks after inoculation, the DNA fragments corresponding to FZB42 were81

still traceable, but their number was reduced to around 55 % of the initial number82

(Kröber et al. 2014). The results obtained with the two different methods indicate83

that the inoculant strain FZB42 was less competitive than the indigenous community84

members.85

40.3 Plant-Growth Promotion86

Although the ability of FZB42 to support growth of potato, maize, cotton, tobacco,87

leafy and fruiting vegetables, and ornamentals is well documented (Borriss 2011),88

our knowledge about the molecular basis of the ‘biofertilizer’ effect of beneficial89

plant-associated Bacilli are far from complete. Several traits (Fig. 40.1) are involved90

in the complex interplay between root-colonizing bacteria and plant.91

1. Tryptophan-Dependent Synthesis of Indole-3-Acetic Acid. Inactivation of92

genes involved in tryptophan biosynthesis and in a putative tryptophan-dependent93

IAA biosynthesis pathway led to reduction of both IAA levels and plant growth-94

promoting activity in the respective mutant strains (Idris et al. 2007). Notably, seed95

treatment with FZB42 increased root production, an indicator of auxin production,96

but significantly repressed root Pi uptake at low environmental Pi concentrations97

(Talboys et al. 2014).98

2. Volatiles, such as 2,3-Butanediol and Acetoin, released by B. subtilis and B.99

amyloliquefaciens, enhance plant growth. To synthesize 2,3-butanediol, pyruvate100

is converted to acetolactate by acetolactate synthase (AlsS), which is subsequently101

converted to acetoin by acetolactate decarboxylase (AlsD). FZB42 mutant strains,102

deficient in the synthesis of volatiles due to mutations in the alsD and alsS genes,103

were impaired in plant growth-promotion (Borriss 2011).104

3. Phytase-Producing Bacteria Enhance Phosphorous Availability. Soil phos-105

phorous is an important macronutrient for plants. Improved phosphorous nutrition106

is achievable by ‘mobilization’ of phosphorous fixed as insoluble organic phos-107

phate in phytate (myo-inositol-hexakisphosphate); see also Chap. 24. The108

extracellular 3-phytase of the PGP B. amyloliquefaciens FZB45 hydrolyzed phy-109

tate to InsP5 and phosphate in vitro (Fig. 40.1). A phytase-negative mutant strain,110

whose phyA gene was disrupted, did not stimulate plant growth under phosphate111

limitation (Idris et al. 2002). Further experiments under field conditions revealed112

that FZB45 only stimulates plant growth when phytate is present in soils which113

are poor in soluble phosphate.114

Other mechanisms that are involved in biofertilizer function of Bacilli include ni-115

trogen fixation, mineral solubilization, and secretion of macromolecule degrading116

enzymes (Borriss 2011).117

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40.4 Biocontrol by Antimicrobial Compounds118

B. amyloliquefaciens FZB42 was successfully applied to suppress the plant pathogen119

Rhizoctonia solani on lettuce (Chowdhury et al. 2013). Genome analysis revealed that120

nearly 10 % of the FZB42 genome is devoted to synthesizing antimicrobial metabo-121

lites and their corresponding immunity genes (Borriss 2013). This antibiotic arsenal122

(Table 40.2) makes B. amyloliquefaciens FZB42 and related B. amyloliquefaciens123

plantarum strains promising microbial biopesticides.124

Cyclic Lipopeptides Five gene clusters involved in non-ribosomal synthesis of c-125

LPs and of the iron-siderophore bacillibactin were identified in the genome of FZB42126

(Table 40.2). Three of the respective gene clusters were assigned to the syntheses of127

surfactin, fengycin, and bacillomycin D. The iturin bacillomycin D was identified128

as the most powerful fungicide produced by FZB42. An early surfactin secretion129

could be of biological relevance since this c-LP, although less fungitoxic than iturins130

and fengycins, is essential for moving of the bacteria on plant tissues and for matrix131

formation in biofilms (Chen et al. 2009).132

Polyketides The three gene clusters encoding the modularly organized polyketide133

synthases (PKS) for syntheses of bacillaene, macrolactin, and difficidin cover nearly134

200 kb. Difficidin is the most effective antibacterial compound produced by FZB42T,135

but also macrolactin and bacillaene possess antibacterial activity. Difficidin is effi-136

cient in suppressing the plant pathogenic bacterium Erwinia amylovora, which causes137

fire blight disease in orchard trees. Macrolactin A (MA) and 7-O-succinyl macro-138

lactin A (SMA), polyene macrolides containing a 24-membered lactone ring, show139

antibiotic effects superior to those of teicoplanin against vancomycin-resistant ente-140

rococci and methicillin-resistant Staphylococcus aureus. MA and SMA are currently141

being evaluated in preclinical studies in Korea as anti-tumor agents.142

Bacilysin Another product of non-ribosomal synthesis, the dipeptide bacilysin was143

found as also being involved in the suppression of Erwinia amylovora. Recent ex-144

periments demonstrated that bacilysin, besides difficidin, is efficient in suppressing145

Microcystis aeruginosa, the main causative agent of cyanobacterial bloom in lakes146

and rivers (Liming Wu et al. unpublished).147

Ribosomally Synthesized Antimicrobial Peptides remained unknown in B.148

amylolique-faciens plantarum for a long time with one remarkable exception:149

mersacidin, a B-type lantibiotic, was detected in strain HIL Y85, later clas-150

sified as being B. amyloliquefaciens plantarum (Herzner et al. 2011). Ri-151

bosomally synthesized antibacterial peptides (bacteriocins) were detected in152

FZB42 by using a mutant strain devoid in non-ribosomal synthesis of polyke-153

tides, lipopeptides and bacilysin, which still possessed some remaining an-154

tibiotic activity. Plantazolicin (PZN) displayed antibacterial activity towards155

closely related gram-positive bacteria, especially against B. anthracis. In ad-156

dition, PZN displayed a moderate nematicidal activity (Liu et al. 2013).157

Due to its extensive degree of modification, PZN is well protected from premature158

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Table 40.2 Genes and gene cluster encoding for secondary metabolites in selected Bacillus spp.

Metabolite Occurrence Gene cluster Size Effect against Reference

Sfp-dependent non-ribosomal synthesis of lipopeptides

Surfactin BAP, BAA,BSU

srfABCD 32.0 kb Virus Stein 2005

Iturin BAP, BAA,BSU

bmyCBAD 39.7 kb Fungi Chen et al.2007

Fengycin BAP, BSU fenABCDE 38.2 kb Fungi Chen et al.2007

Polymyxin PPO pmxABCDE 40.7 kb Bacteria Niu et al.2013

Fusaricidin PPO fus GFEDCBA 32.4 kb Fungi Li andJensen 2008

Bacillibactin BAP, BAA,BSU

dhbABCDEF 12.8 kb Bacterialcompetitors

Chen et al.2007

Sfp-dependent non-ribosomal synthesis of polyketides

Macrolactin BAP mlnABCDEFGHI 53.9 kb Bacteria Chen et al.2007

Bacillaene BAP, BAA,BSU

baeBCDE, acpK,baeGHIJLMNRS

74.3 kb Bacteria Chen et al.2007

Difficidin BAP dfnAYXBCDEFGHIJKLM 71.1 kb Bacteria Chen et al.2007

Sfp-independent non-ribosomal synthesis

Bacilysin BAP, BSU bacABCDE, ywfG 6.9 kb Bacteria,cyanobacteria

Chen et al.2007

Ribosomal synthesis of processed and modified peptides (bacteriocins)

Plantazolicin BAP FZB42 pznFKGHIAJC DBEL 9.96 kb B. anthrax,nematodes

Scholz et al.2011

Amylocyclicin BAP FZB42 acnBACDEF 4.49 kb Closely relatedbacteria

Scholz et al.2014

Mersacidin BAP Y2 mrsK2R2FGEAR1DMT 12 kb Gram-positivebacteria

Stein 2005

Amylolysin BAP GA1 amlAMTKRIFE 9.36 kb Gram-positivebacteria

ArguellesArias et al.2014

Subtilin BSU ATCC6633

spaBTCAIFGRK 12 kb Closely relatedbacteria

Stein 2005

Ericin BAP A1/3 eriBTCASIFEGRK 12.5 kb Closely relatedbacteria

Stein 2005

Sublancin BSU sunAT bdbA yolJ bdbB 4.5 kb Closely relatedbacteria

Stein 2005

Subtilosin A BSU sboA albABCDEFG 7.0 kb Closely relatedbacteria

Stein 2005

BAP B. amyloliquefaciens plantarum, BAA B. amyloliquefaciens amyloliquefaciens, BSU B.subtilis subtilis, PPO Paenibacillus polymyxa

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degradation by peptidases within the plant rhizosphere (Scholz et al. 2011). A cir-159

cular bacteriocin, named amylocyclicin, was recently identified (Scholz et al. 2014).160

The peptide suppressed growth of the plant pathogenic actinobacterium Clavibacter161

michiganensis and of several Gram-positive bacteria.162

Importance of Secondary Metabolites for Biocontrol For a long time one has163

thought that the plant protective activity of FZB42 and other PGPR is due to the164

antibiotic activity of a wide array of antibiotic compounds upon growth under labo-165

ratory conditions. However, in recent years, this became doubtful due to pioneering166

work of Ongena et al. They investigated antibiotic production by MALDI MSI in167

a gnotobiotic system in which the plantlet and the associated B. amyloliquefaciens168

S499, a close relative of FZB42, were growing on a gelified medium covering the169

MALDI target plate. Surfactins were detected during early biofilm formation in the170

rhizosphere in relatively high amounts, representing more than 90 % of the whole171

c-LP production. In contrast, the synthesis of iturin and fengycin was delayed until172

the end of the aggressive phase of colonization (Debois et al. 2014).173

40.5 Induced Systemic Resistance174

Due to the low concentration of antimicrobial compounds detectable in the rhizo-175

sphere, it is tempting to speculate that ISR is the main factor for suppressing plant176

pathogens by PGPR Bacilli. ISR occurs when the plant’s defense mechanisms are177

stimulated and primed to resist infection by pathogens (Doornbos et al. 2012). It has[AQ1]178

been demonstrated that Bacillus derived volatiles and cLPs trigger ISR.179

Volatiles Several Bacillus PGPR strains emit VOCs that can elicit plant defenses.180

Exposure to VOCs consisting of 2,3-butanediol and acetoin (3-hydroxy-2-butanone)181

from PGPR Bacillus amyloliquefaciens activates ISR in plants (see Chap. 8). In this182

context it is worth to mention that expression of AlsS of FZB42, involved in the183

synthesis of acetoin (Fig. 40.1), was triggered in the presence of maize root exudate184

(Kierul et al. unpublished), suggesting that root exudates play a role in the elicitation185

of acetoin biosynthesis in FZB42.186

Circular lipopeptides surfactin and fengycin act as elicitors of host plant im-187

munity and contribute to increased resistance toward further pathogenesis ingress in188

bean and tomato plants (Raaijmakers et al. 2010).189

40.6 Effect of Bacillus Inoculants on the Environment190

The impact of beneficial Bacillus inoculants on the root microbiome is important191

for their plant health effect. Terminal-restriction fragment length polymorphism, T-192

RFLP, and metagenome analyses of lettuce rhizosphere samples inoculated with B.193

amyloliquefaciens FZB42 vs. non-treated samples revealed that the inoculant strain194

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only had a minor impact on the community structure within this habitat, while inocu-195

lation with the pathogen R. solani did significantly change the rhizosphere microbial196

community structure (Chowdhury et al. 2013; Kröber et al. 2014). A significant in-197

crease in gamma-proteobacterial diversity was detected in samples inoculated with198

the pathogen. However, in the presence of FZB42 this increase was less distinct,199

suggesting a selective compensation of the impact of a pathogen on the indigenous200

plant-associated microbiome by FZB42 (Erlacher et al. 2014). The results of these201

metagenome studies suggest that the application of the commercially available in-202

oculant strain FZB42 can be considered as a safe method to promote the health203

of the economically important lettuce plant and reduce severity of infections by204

phytopathogens like R. solani.205

40.7 Conclusions206

The beneficial effect of Bacillus PGPR on plant health relies on at least three main207

factors:208

1. In previously published studies the set of secondary metabolites described here209

was suspected to mediate mainly the antibiosis function of Bacillus bioinoculants.210

However, the amounts of the relevant antibiotics found in the vicinity of plant211

roots were relatively low, making a significant antibiosis function doubtful.212

2. These metabolites were also suspected to induce changes within the micro-213

bial rhizosphere community, which might affect the health of environment and214

plant. However, sequence analysis of rhizosphere samples revealed only marginal215

changes in the root microbiome, suggesting that secondary metabolites are not216

the key factor in protecting plants from pathogenic microorganisms. On the other217

hand, adding FZB42 to lettuce plants compensate, at least in part, global changes218

in the community structure caused by the pathogen, indicating an interesting219

mechanism of plant protection by beneficial Bacilli.220

3. Recent results support hypothesis, that stimulation of plant ISR by bacterial221

metabolites, such as VOCs and c-LPs, produced in the vicinity of plant roots,222

is the key mechanism in the biocontrol action of Bacilli.223

References224

Arguelles Arias A, Ongena M, Devreese B et al (2014) Characterization of amylolysin,225

a novel lantibiotic from Bacillus amyloliquefaciens GA1. PLoS One 8(12): e83037.226

doi:10.1371/journal.pone.0083037227

Borriss R (2011) Use of plant-associated Bacillus strains as biofertilizers and biocontrol agents, In:228

Maheshwari DK (ed). Bacteria in agrobiology: plant growth responses. Springer, Germany, pp229

41–76230

Borriss R (2013) Comparative analysis of the complete genome sequence of the plant growth-231

promoting bacterium Bacillus amyloliquefaciens FZB42 In: de Brujn FJ (ed) Molecular232

microbial ecology of the rhizosphere, vol 2. Wiley-Blackwell, Hoboken, pp 883–898233

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12 R. Borriss

Borriss R, Chen XH, Rueckert C et al (2011) Relationship of Bacillus amyloliquefaciens clades234

associated with strains DSM7T and FZB42T: a proposal for Bacillus amyloliquefaciens subsp.235

amyloliquefaciens subsp. nov. and Bacillus amyloliquefaciens subsp. plantarum subsp. nov.236

based on complete genome sequence comparisons. Int J Syst Evol Microbiol 61:1786–1801237

Budiharjo A, Chowdhury SP, Dietel K et al (2014) Transposon mutagenesis of the plant-238

associated Bacillus amyloliquefaciens ssp. plantarum FZB42 revealed that the nfrA and the239

RBAM17410 genes are involved in plant-microbe interactions. PLoS One 9(5): e98267.240

doi:10.1371/journal.pone.0098267241

Chen XH, Koumoutsi A, Scholz R et al (2007) Comparative analysis of the complete genome242

sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42. Nat243

Biotechnol 25:1007–1014244

Chen XH, Koumoutsi A, Scholz R et al (2009) Genome analysis of Bacillus amyloliquefaciens245

FZB42 reveals its potential for biocontrol of plant pathogens. J. Biotechnol. 140:27–37246

Chowdhury SP, Dietel K, Rändler M et al (2013) Effects of Bacillus amyloliquefaciens FZB42 on247

lettuce growth and health under pathogen pressure and its impact on the rhizosphere bacterial248

community. PLoS One 8(7):e68818. doi: 10.1371249

Debois D, Jourdan E, Smargiasso N et al (2014) Spatiotemporal monitoring of the antibiome250

secreted by Bacillus biofilms on plant roots using MALDI mass spectrometry imaging. Anal[AQ2]251Chem 86(9):4434–4438 doi: 10.1021/ac500290s252

Doornbos RF, van Loon LC, Bakker PA (2012) Impact of root exudates and plant defense signaling253

on bacterial communities in the rhizosphere. A review. Agron Sustain Dev 32:227–243254

Erlacher A, Cardinale M, Grosch R et al (2014) The impact of the pathogen Rhizoctonia solani255

and its beneficial counterpart Bacillus amyloliquefaciens on the indigenous lettuce microbiome.256

Front Microbiol 5:175. doi: 10.3389/fmicb.2014.00175257

Fan B, Chen XH, Budiharjo A et al (2011) Efficient colonization of plant roots by the plant258

growth promoting bacterium Bacillus amyloliquefaciens FZB42, engineered to express green259

fluorescent protein. J Biotechnol 151: 303–311260

HerznerAM, Dischinger J, Szekat C et al (2011) Expression of the lantibiotic mersacidin in Bacillus261

amyloliquefaciens FZB42. PLoS One 6(7): e22389. doi:10.1371/journal.pone.0022389262

Idriss, EES, Makarewicz O, Farouk A et al (2002) Extracellular phytase activity of Bacillus263

amyloliquefaciens FZB 45 contributes to its plant growth-promoting effect. Microbiology264

148:2097–2109265

Idris EES, Iglesias DJ, Talon M et al (2007) Tryptophan dependent production of indole-3-acetic266

acid (IAA) affects level of plant growth promotion by Bacillus amyloliquefaciens FZB42. Mol267

Plant Microbe Interact 20:619–626268

Kröber M, Wibberg D, Grosch R et al (2014) Effect of the strain Bacillus amyloliquefaciens FZB42269

on the microbial community in the rhizosphere of lettuce under field conditions analyzed by270

whole metagenome sequencing. Front Microbiol 5:252 doi: 10.3389/fmicb.2014.00252271

Li J, Jensen SE (2008) Nonribosomal biosynthesis of fusaricidins by Paenibacillus polymyxa PKB1272

involves direct activation of a D-amino acid. Chem Biol 15: 118–127273

Liu Z, Budiharjo A, Wang Pet et al (2013) The highly modified microcin peptide plantazolicin274

is associated with nematicidal activity of Bacillus amyloliquefaciens FZB42. Appl Microbiol275

Biotechnol 97:10081–90276

Niu B,Vater J, Rueckert C (2013) Polymyxin P is the active principle in suppressing phytopathogenic277

Erwinia spp. by the biocontrol rhizobacterium Paenibacillus polymyxa M-1. BMC Microbiology278

13:137279

Raaijmakers J, De Bruin I, Nybroe O et al (2010) Natural functions of cyclic lipopeptides from280

Bacillus and Pseudomonas: more than surfactants and antibiotics. FEMS Microbiol Rev281

34:1037–1062282

Scholz R, Molohon KJ, Nachtigall J et al (2011) Plantazolicin, a novel microcin B17/streptolysin283

S-like natural product from Bacillus amyloliquefaciens FZB42. J Bacteriol 193:215–224.284

Scholz R, Vater J, Budiharjo A et al (2014) Amylocyclicin, a novel circular bacteriocin produced285

by Bacillus amyloliquefaciens FZB42. J Bacteriol 196:1842–1852286

Aut

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cted

Proo

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Stein T (2005) Bacillus subtilis antibiotics: structures, syntheses and specific functions. Mol287

Microbiol 56:845–857288

Talboys PJ, Owen DW, Healey JR et al (2014)Auxin secretion by Bacillus amyloliquefaciens FZB42289

both stimulates root exudation and limits phosphorus uptake in Triticum aestivum. BMC Plant290

Biol 14:51291

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Chapter 40: Author Query

AQ1. “Dornboos et al. 2012” was changed to “Doornbos et al. 2012” to match the reference list. Please confirm or correct the change.

AQ2. We have updated reference “Debois et al. 2014”. Please check.Aut

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proof author's chapter 40 bacillus, a plant-beneﬁcial ...€¦ · bacillus pumilus gha 181...

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