Microbial biocontrol of the pathogen Phytophthora citricola in the rhizosphere of European beech (Fagus sylvatica L.) [Elektronische Ressource] : impacts of elevated O_1tn3 and CO_1tn2 on the antagonistic community structure and function / Felix Haesler

Dipl. Biol. Felix Haesler

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 23
• Langue: English
• Poids de l'ouvrage: 15 Mo

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¨ ¨TECHNISCHE UNIVERSITAT MUNCHEN
¨ ¨LEHRSTUHL FUR BODENOKOLOGIE
Microbial biocontrol of the pathogen Phytophthora citricola in the rhizosphere of
European beech (Fagus sylvatica L.):
Impact of elevated O and CO on the antagonistic community structure and3 2
function
Felix Haesler
Vollst¨andiger Abdruck der von der Fakult¨at Wissenschaftszentrum Weihenstephan fu¨r
Ern¨ahrung, Landnutzung und Umwelt der Technischen Universit¨at Mu¨nchen zur Erlangung
des akademischen Grades eines
Doktors der Naturwissenschaften
genehmigten Dissertation.
Vorsitzender: Univ.-Prof. Dr. R. Matyssek
Prufer der Dissertation: 1. Univ.-Prof. Dr. J. C. Munch¨
2. Univ.-Prof. Dr. W. Oßwald
Die Dissertation wurde am 31.01.2008 bei der Technischen Universit¨at Mu¨nchen eingereicht
und durch die Fakult¨at Wissenschaftszentrum Weihenstephan fu¨r Ern¨ahrung, Landnutzung und
Umwelt am 03.06.2008 angenommen.Contents
Table of Content i
List of Figures iv
List of Tables v
Acknowledgements vi
Abstract viii
1 Introduction 1
1.1 Phytophthora citricola as causal agent of root rot on European beeches (Fagus
sylvatica) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Biological control of microbial plant pathogens . . . . . . . . . . . . . . . . . . . 3
1.2.1 Biocontrol active organisms . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2.2 Mechanisms of biological control . . . . . . . . . . . . . . . . . . . . . . . 4
1.3 Elucidating mechanisms of microbial antagonism . . . . . . . . . . . . . . . . . . 8
1.4 Analyzing disease suppression in soil . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.4.1 Quantitative methods to analyze microbial communities . . . . . . . . . . 10
1.4.2 Investigating structural diversity of microbial communities. . . . . . . . . 11
1.4.3 Assessing functional diversity of microbial communities . . . . . . . . . . 13
1.5 Effects of climate relevant trace gases on plant-soil systems . . . . . . . . . . . . 14
1.6 Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2 Materials and methods 17
2.1 Experimental designs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.1.1 Soil characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.1.2 Greenhouse experiment for the isolation of antagonists . . . . . . . . . . . 18
2.1.3 Greenhouse experiment for culture independent analyses of the microbial
rhizosphere community . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.2 Materials and recipes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.2.1 Buffers and solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.2.2 Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
2.2.3 Reference strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
2.2.4 Oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.3 Soil microbial biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.4 Isolation of microbial antagonists and confrontation tests . . . . . . . . . . . . . 31
2.4.1 Bacterial antagonists (Actinobacteria) . . . . . . . . . . . . . . . . . . . . 31
2.4.2 Fungal antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
iCONTENTS ii
2.5 Metabolite analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2.5.1 Fourier transform ion-cyclotron (FT-ICR) mass spectrometry . . . . . . . 33
2.5.2 Nuclear magnetic resonance (NMR) . . . . . . . . . . . . . . . . . . . . . 34
2.6 Characterization of pure microbial cultures . . . . . . . . . . . . . . . . . . . . . 35
2.6.1 Nucleic acid extraction from microorganisms . . . . . . . . . . . . . . . . 35
2.6.2 Genomic fingerprinting of isolates. . . . . . . . . . . . . . . . . . . . . . . 35
2.6.3 Sequencing of PCR products . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.6.4 Cloning and sequencing of plasmids . . . . . . . . . . . . . . . . . . . . . 38
2.6.5 Species specific PCR for the detection and identification of P. citricola . . 38
2.7 PCR based analyses of environmental samples . . . . . . . . . . . . . . . . . . . . 39
2.7.1 DNA extraction from environmental samples . . . . . . . . . . . . . . . . 39
2.7.2 PCR amplification of structural and functional genes . . . . . . . . . . . . 39
2.7.3 Terminal restriction fragment length polymorphism analysis (t-RFLP) . . 40
2.7.4 Quantitative real-time PCR . . . . . . . . . . . . . . . . . . . . . . . . . . 42
2.8 Statistical analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3 Results 46
3.1 Fungal and actinobacterial antagonists against Phytophthora citricola . . . . . . 46
3.1.1 Actinobacteriaisolatedfrombeechrhizospheresoilandconfrontationtests
with P. citricola. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
3.1.2 Characterization of the actinobacterial isolates . . . . . . . . . . . . . . . 47
3.1.3 Isolation of fungi from beech fine roots and confrontation tests . . . . . . 51
3.1.4 Characterization of fungal isolates . . . . . . . . . . . . . . . . . . . . . . 51
3.1.5 Viability of P. citricola in interaction zones with Trichoderma spec. . . . . 54
3.2 Metabolite analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.2.1 Detection and characterization of a bioactive compound (FT-ICR/MS) . 55
3.2.2 Identification of the bioactive compound (NMR) . . . . . . . . . . . . . . 57
3.3 Validation of specific primer sets for structural and functional analyses . . . . . . 58
3.3.1 Actinobacteria 16S rDNA primers . . . . . . . . . . . . . . . . . . . . . . 58
3.3.2 Polyketide synthase (PKS) specific primers . . . . . . . . . . . . . . . . . 62
3.4 Effectsofelevatedcarbondioxide,elevatedozoneandinoculationwithP.citricola
on a plant-soil system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
3.4.1 Plant growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
3.4.2 Soil microbial biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
3.4.3 Phytophthora citricola . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
3.5 Structural and functional diversity of actinobacterial rhizosphere communities . . 72
3.5.1 Actinobacterial structural diversity . . . . . . . . . . . . . . . . . . . . . . 72
3.5.2 Actinobacterial PKS type II diversity . . . . . . . . . . . . . . . . . . . . 76
4 Discussion 79
4.1 Occurance of microbial antagonism against P. citricola . . . . . . . . . . . . . . . 79
4.1.1 Actinobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
4.1.2 Fungal isolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
4.2 Mechanisms of antagonism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
4.2.1 The actinobacterial antibiotic cycloheximide and its relevance in soils . . 84
4.2.2 Possible mechanisms of fungal antagonism . . . . . . . . . . . . . . . . . . 86
4.3 Influence of abiotic and biotic factors on a forest plant-soil system . . . . . . . . 87
4.3.1 Effects on the growth of European beeches . . . . . . . . . . . . . . . . . 87CONTENTS iii
4.3.2 Effects on total microbial biomass . . . . . . . . . . . . . . . . . . . . . . 88
4.4 Structural and functional diversity of the actinobacterial rhizosphere community 89
4.4.1 Diversity assessment by means of clone libraries. . . . . . . . . . . . . . . 90
4.4.2 Monitoring structural changes in the actinobacterial rhizosphere commu-
nity of European beeches . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
4.4.3 Monitoring PKS type II diversity in the rhizosphere of European beeches 96
4.5 Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
References 99
A Supplementary informations 118
B Statistical tables 119
C rep-PCR dendrograms 122List of Figures
1.1 Polyketide Synthases type I and II . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.1 Experimental design for isolation of antagonists . . . . . . . . . . . . . . . . . . . 19
2.2 Inoculation with P. citricola . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.3 Experimental design for culture independent analyses . . . . . . . . . . . . . . . 21
2.4 FT-ICR/MS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.1 Actinobacteria isolations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.2 Molecular fingerprints exemplary gel pictures . . . . . . . . . . . . . . . . . . . . 48
3.3 Neighbour-joining tree of partial 16S rRNA genes . . . . . . . . . . . . . . . . . . 49
3.4 UPGMA dendrogram of BOX fingerprints of Kitasatospora isolates . . . . . . . . 50
3.5 Isolation frequencies of fungal groups . . . . . . . . . . . . . . . . . . . . . . . . . 51
3.6 Confrontation tests with fungal isolates . . . . . . . . . . . . . . . . . . . . . . . 52
3.7 UPGMA dendrogram of Inter-LINE fingerprints for Trichoderma isolates . . . . 53
3.8 Occurance of biocontrol activity in isolate 116A+4 culture supernatant . . . . . . 55
3.9 FT-ICR/MS spectra of isolate 116A+4 culture supernatant . . . . . . . . . . . . 56
3.10 NMR of cycloheximide and bioactive fracti