Motility: degU, sigD, hag, fliD, fliM, swrA, cheC, srfACD. Adhesion: colA-D. Biofilm: luxS, ymcA, yusV, sfp, tasA, epsA-O, srfACD, sfp. Stress reponse: nfrA, tpx.
Bacillus, a Plant-Beneficial Bacterium Rainer Borriss, ABiTEP GmbH, Berlin, Germany
Bacillus subtilis
Bacillus amyloliquefaciens subsp. plantarum Type strain FZB42
Borriss et al. 2011 IJSEM
Source:Timmusk et al. 2005 AEM 71:11
Paenibacillus polymyxa Paenibacillus mucilaginosus 1
Application of the plant associated bacterium Bacillus amyloliquefaciens FZB42 on lettuce (Lactuca sativa) confirmed its capability to promote plant growth and health by reducing disease severity (DS) caused by the phytopathogenic fungus Rhizoctonia solani. 8
Shoot dry mass [g/plant] a
a
6 4 b 2 0 Control
R. solani
FZB42 +Rs
Under biotic stress (presence of the pathogen R. solani, +Rs) and in respect of applied spore numbers (106, 107, 108 spores/ml) of FZB42-Rif. Plants were cultivated at 22/15°C for 4 weeks. Dry mass followed by the same letter are not significantly different according to Dunnett’s test (P=0.1).
Golzow, Brandenburg Size 100 x 25 square meters, Alluvial loam
Chowdhury et al. PLOS ONE 2013
2
Colonization of lettuce roots by Rhizoctonia solani is delayed by FZB42
Initial steps of lettuce root colonization by Rhizoctonia solani in the absence (control, upper panel) and the presence of B. amyloliquefaciens FZB42 (lower panel). CLSM pictures were taken 6, 12, 18, 24 and 30 hours post inoculation with R. solani .
Chowdhury et al. ,manuscript in prep. 3
1. Initial steps: Root colonization Genes from FZB42 identified as being involved in root colonization
Motility: degU, sigD, hag, fliD, fliM, swrA, cheC, srfACD Biofilm: luxS, ymcA, yusV, sfp, tasA, epsA-O, srfACD, sfp Adhesion: colA-D Stress reponse: nfrA, tpx
Chen et al. 2007 Nature Biotechnol. 25:1007 Fan et al. 2011 J. Biotechnol. 151:303 4
Colonization of FB01mut01 on lettuce roots by FZB42
A: One week after inoculation on epidermal cells (left), root hairs (middle) and root tip (right). B: Two weeks after inoculation on roots hairs (left and middle) and four weeks after inoculation of the primary root tip (right).
Chowdhury et al. PLOS ONE 2013 5
Root colonization: FZB42 forms dense biofilms at the surface of plant roots
6
Transposon mutagenesis revealed that the nfrA and RBAM17410 genes are involved in plant-microbe interactions
Ability to colonize plant roots is diminished in most transposon insertion mutants selected for reduced plant-growth promotion (degU, nfrA, yusV), but not in RBAM_017410 Budiharjo et al. 2014 PLOS ONE 7
RBAM_17410
Colonization of lettuce roots by gfp-expressing mutants impaired in plant-growth-promotion. A: Mutant AB4 (RBAM_017410::TnYLB-1) after one week (on root hairs, left), and two weeks (on primary root surface, right) of inoculation.
nfrA
B: Mutant AB2 (nfrA::TnYLB-1) after one week (left) and two weeks (right) of inoculation. Budiharjo et al. 2014 PLOS ONE 8
Scanning electron microscopy of FZB42 and mutant strain colonizing Arabidopsis roots ( 8 days after inoculation)
A: Wild type FZB42
B: nfrA mutant strain AB106 Budiharjo et al. 2014 PLOS ONE 9
2. Plant growth promotion (biofertilizer function) Genes involved in plant-growth promotion
Plant growth promotion: RBAM017410 Synthesis of IAA: trpBA, trpED, ysnE, patB, dhaS, yhcX, yclC Synthesis of volatiles: alsS, alsD, bdhA Mobilisation of nutrients: abnA, chbA, xynC, gmuG, phyC
Chen et al. 2007 Nature Biotechnol. 25:1007 Idriss et al: 2002 Microbiology 148:2097 Idris et al. 2007 Mol Plant-Microbe Interact. 20:619 Budiharjo et al. 2014: PLOS ONE 9 (5) 10
Effect on plant growth by mutant strains impaired in plant growth promotion Budiharjo et al. 2013PLOS ONE a
ab
bc
2
35
c
1,5 1 0,5
B
a
40
bc Fresh Weight (mg)
Dry Weight (mg)
2,5
a
bc
30
b
25
b
20 15 10 5
0 C
FZB42
AB106
AB106C
0
AB106R
C
AB106 (DnfrA)
Lemna minor
AB106 (DnfrA)
FZB42
AB106
AB106C
AB106R
A. thaliana
120
b bc
c
c
Fresh Weight (mg)
Dry weight mg
a 2 1,8 1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 0
a
100 80
ab
bc
bc c
60 40
20 0 C C
FZB42
AB107
AB107 (DRBAM_17410)
AB107C
AB107R
Lemna minor
FZB42
AB107 (DRBAM_17410)
AB107
AB107C
AB107R
A. thaliana
C: control without bacteria, AB106C and AB107C: insertion mutants complemented by the wild type gene, AB106R and AB107R: FZB42 transformed with chromosomal DNA from insertion mutants. 11
VOLATILES (´VOCs´)
FZB42
alsS
water
alsD
Influence of volatiles emitted by FZB42 on plant growth
The picture was taken 4 weeks after inoculation of Arabidopsis with FZB42 With courtesy of C.M.-Ryu, KRIBB South-Korea 12
Plant growth promotion and ISR
13
3. Biocontrol by antimicrobial compounds
cyclic lipopeptides (3) siderophore bacillibactin
polyketides (3)
The 11 gene clusters for secondary metabolites in FZB42 cover 10% of the whole genome
bacilysin
bacteriocins: amylocyclicin + plantazolicin
14
antibiotics & Secondary Metabolite Analysis SHell
cluster type
from
to
Metabolite detected
1
Nrps
322618
388025
surfactin
2
putative
403590
417394
3
putative
727055
746540
4
T2pks
940737
981981
5
putative
1032072 1040727
6
terpene
1064781 1085521
7
transatpks
1374169 1460068
macrolactin
8
nrps-transatpks
1688756 1791439
bacillaen
9
nrps-transatpks
1851172 1988997
bacillomycin, fengycin
10
terpene
2014214 2036097
11
T3pks
2102584 2143684
12
transatpks
2260090 2360537
difficidin
13
nrps
2848410 2904220
(cys-cys-cys) + (pk) ?
14
nrps-bacteriocin 3001250 3068041
bacillibactin-siderophore
bacteriocin
3044506 3048678
amylocyclicin
19
other
3576267 3630272
bacilysin
20
putative
371349
3719235
plantazolicin
15
Bacillomycin D, a cyclic lipopeptide with antifungal action
Koumoutsi et al. 2004. J. Bacteriol. 186:1084-96 16
Difficidin, an antibacterial polyketide, acts against Erwinia amylovora, the causative agent of fire blight at orchard trees
Fire blight at orchard trees. Left: Detached blossoms in racks prior to inoculation. Right top: healthy blossom. Below: blossom with black peduncle and bacterial ouze due to infection by Erwinia amylovora.
Mutant strains: CH3 (Dsfp) CH8 (Ddfn) CH14 (Dbae, Dmln) RS06 (Dsfp, Dbac)
FZB42
CH8
CH14
CH3
RS06
Chen et al. 2009. J. Biotechnol. 140:38-44 17
The antibacterial dipeptide bacilysin is efficient against E. amylovora and Microcystis, the causative agent of algal blooms in freshwater lakes and rivers
FZB42
DaroA/E
Dsfp
DbacB
bacilysin
Wu et al. AEM 2014 18
Plantazolicin, a novel thiazole/oxazole-modified microcin (TOMM), undergoes extensive posttranslational modifications to become a bioactive natural product.
(pznD)
Scholz et al. 2011, J. Bacteriol. 193: 215-24 Liu et al. 2013, Appl. Microbiol. Biotechnol. 97:1081-90
Killing rate of nematodes is decreased in plantazolicin mutant strain 19
FZB42 suppresses root knot forming nematodes in occurring in tomato fields,
Yunnan province, China.
Treated with FZB42
Untreated
20
Amylocyclicin is efficient against closely related competitors within the plant rhizosphere.
Scholz et al. 2014, J. Bacteriol. 196: 1842-52
21
Summary: Antimicrobial secondary metabolites of FZB42
22
4. Are antimicrobial compounds really responsible for the biocontrol effect under environmentally conditions ?
cyclic lipopeptides (3) siderophore bacillibactin
polyketides (3)
The 11 gene clusters for secondary metabolites in FZB42 cover 10% of the whole genome
bacilysin
bacteriocins: amylocyclicin + plantazolicin
23
UHPLC-qToF-MS analyses of root extracts: Surfactin concentration in lettuce plant roots colonized by FZB42 is enhanced in presence of the pathogen Chowdhury et al., manuscript in prep.
FZB42 in non-infected lettuce plants
FZB42 + R. solani
The mutant CH5 (-lipopeptides) can survive and shows dense growth in presence of Rhizoctonia solanii.sni.
Comparison of surfactin concentrations (isotopic pattern of [M+H]+ 1036.6905) in non-infected (upper spectrum) and infected (lower spectrum) lettuce plantlets inoculated with FZB42 wt. 24
UHPLC-qToF-MS analyses of root extracts: Expression of non-ribosomal synthesized lipopeptides
fenA
srf
sfp
Expression of non-ribosomal synthesized lipopeptides surfactin, fengycin and bacillomycin D by Bacillus amyloliquefaciens FZB42 mutants wt, srf, sfp, and fenA in presence of Rhizoctonia solani. Chowdhury et al., manuscript in prep. 25
Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI MSI) was used to examine spatio-temporal changes in the secreted antibiome of B. amyloliquefaciens developing as biofilms on roots.
Surfactin Iturin Fengycin Surfactin homologues (C12- to C15-acyl chains) together represent more than 90% of the whole LP production. Non-ribosomal lipopeptides such as the plant immunity elicitor surfactin or the highly fungitoxic iturins and fengycins were readily produced albeit in different time-frames and quantities in the surrounding medium. Chowdhury et al., manuscript in prep.
26
S I
F
Besides surfactin, Ion species corresponding to C13-, C14- and C15-iturin A homologues were also detected as well as fengycins but in much lower amounts. Ion images (Figures 1M-O) show that cyclic LPs all accumulated in the medium surrounding roots but did not necessarily exhibit the same distribution.
However, no other bioactive compounds such as polyketides were detected at any time, strongly suggesting that the antibiome expressed in planta by B. amyloliquefaciens does not reflect the vast genetic arsenal devoted to the formation of such compounds. Debois et al. 2014 Anal. Chem., 6;86(9):4431-8
27
5. Induced systemic resistance (ISR): Stimulation of ISR in lettuce plants inoculated with FZB42 and R. solani
FZB42 can induce the expression of plant resistance proteins PR1 (SA mediated) and Defensin (JA/ethylene mediated). Simultaneous presence of FZB42 and R. solani leads to 12 fold enhanced expression of defensin. S. Chowdhury, ms in prep. 28
Bacillus CLPs affect specifically the different pathways of induced systemic resistance in plants 14,00 12,00
pdf1
10,00 8,00 -Rs
6,00
+Rs 4,00
Relative expression of the defense related gene pdf1 (defensin) in lettuce after inoculation with FZB42 wild-type and mutant strains.
2,00
0,00
Bacillus cyclic lipopeptides fengycin and surfactin are necessary for induction of defensin in lettuce, independent on the presence of the pathogen.
4,00 3,50
Lox
3,00 2,50 2,00 1,50 1,00 0,50 0,00
-Rs +Rs
By contrast, expression of another defense related gene, lox, is not dependent on cLPs. S. Chowdhury, ms in prep. 29
FENGYCIN BACILLOMYCIN D BACILLIBACTIN
SURFACTIN
IAA sigH
sigD
RBAM17410 alsS
sfp
tasA
FZB42 nfrA
abrB
DIFFICIDIN
ACETOIN
MACROLACTIN BACILLAENE
degU
2,3-BUTANEDIOL
BACILYSIN
PHYTASE PLANTAZOLICIN
AMYLOCYCLICIN Pi 30
Application of plant-growth promoting bacilli in agriculture is increasing steadily. Appropriate placement of a sufficient amount of the bioinoculant is crucial for the success of its application.
31
To verify B.amyloliquefaciens as an environmentally compatible plant protective agent, its effect on the indigenous rhizosphere community was analyzed by metagenome sequencing.
Comparison of the ten most abundant phyla within the rhizosphere of lettuce. Abundances were calculated by means of comparison of metagenome sequence reads against the GenBank database within the MG-RAST software package.
Comparison of taxonomic community profiles only revealed marginal changes after application of strain FZB42. Kröber et al. 2014 Front. Microbiol. doi: 10.3389/fmicb.2014.00252
32
Conclusions part 1-5 The beneficial effect of Bacillus PGPR on plant health relies on at least three main factors In previously publised studies the vast array of secondary metabolites produced in vitro was suspected to mediate mainly the antibiosis function of Bacillus bioinoculants. However,the amounts of relevant antibiotics found in in the vicinity of plant roots were relatively low, making a significant antibiosis FZB42 function doubtful. These metabolites were also suspected to induce changes within the microbial rhizosphere community, which might affect the health of environment and plant. However, only marginal changes were detected, suggesting that secondary metabolites are not the key factor in protecting plants from pathogenic organisms. Recent results support hypothesis, that stimulation of plant ISR by bacterial metaboiites (priming?), such as volatiles and cyclic lipopeptides, produced in vicinity of plant roots, is the key mechanism in the biocntrol action of Bacilli. 33
6. Outlook: Bioinoculants of next generation: Plant growth promoting bacilli engineered for enhanced efficiency
• Natural selection of strain variants with enhanced rhizocompetence • Bacillus strains with mutations in their own regulatory genes (e.g. degU, abrB) •Bacillus strains engineered for expression of foreign genes
Expression of the Harpin gene enhances biocontrol activity of FZB42 Two copies of the harp gene of the plant pathogenic Xanthomonas oryzae pv oryzae were integrated into the genome of FZB42 and expressed under control of the strong P43 promoter.
Qiao et al. Chemical and Biological Technologies in Agriculture (CBTA)2014 1:12 34
Hypersensitive response (HR) was detected in tobacco leaves inoculated with the supernatant of the recombinant FZB42Harpin strain.
Growth promotion of tobacco plants by FZB42, FZB42AN (-apr, -npr) and FZB42Harpin
35
Enhanced bacilysin production by recombinant FZB42 strains Bacillus amyloliquefaciens strains FZBREP and FZBSPA were derived from the wild-type FZB42 by replacement of the native bacilysin operon promoter with constitutive promoters PrepB and Pspac
Wu et al. , manuscript submitted 36
Bacilysin overproduction was accompanied by enhancement of the antagonistic activities against Staphylococcus aureus (an indicator of bacilysin) and Clavibacter michiganense subsp. sepedonicum (the causative agent of potato ring rot). control
FZB42 + N-AGA
FZBREP + N-AGA
FZBSPA + N-AGA
Wu et al. manuscript submitted 37
Conclusions part 6
Biologicals prepared from beneficial microbes are useful and environmentalfriendly tools for developing a sustainable and efficient agriculture. In this context, genomic analysis and genetic engineering of promising beneficial microbes are helpful for obtaining improved bioformulations. This strategy should enable us to save considerable amounts of agrochemicals, especially chemical fertilizers, and chemical pesticides. Qiao et al. Chemical and Biological Technologies in Agriculture (CBTA)2014 1:12
38
To be announced:
PRINCIPLES OF PLANT‐MICROBE INTERACTIONS Roles of Microbes in Sustainable Agriculture Edited by: Prof. Dr. Ben Lugtenberg, Leiden University, The Netherlands The volume will house 40+ chapters spread over 8 sections. A brief summary of this is shown below: Part I. Introductory Chapters Part II. Phytopathogens and Pest Insects Part III. Control of Plant Diseases and Pests using Beneficial Microbes Part IV. Plant Growth Promotion by Microbes Part V. Important Technologies Part VI. Products for Plant Growth‐promotion and Disease Suppression Part VII. Paradigms in Plant‐Microbe Interactions Part VIII. Future Prospects and Dreams Will be published November 2014
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