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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

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Published 01 January 2008
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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 fraction . . . . . . . . . . . . . . . . . . . . 58
3.11 16S clone library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
3.12 PCR for PKS type I + II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
3.13 Rarefaction analysis for PKSII clone library . . . . . . . . . . . . . . . . . . . . . 64
3.14 Maximum-likelihood tree based on partial PKS type II protein sequences . . . . 65
3.15 Plant biomass of beeches from the main experiment . . . . . . . . . . . . . . . . 68
3.16 Microbial biomass C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
3.17 Amplification plot qPCR (P. citricola) . . . . . . . . . . . . . . . . . . . . . . . . 70
3.18 Quantification of P. citricola in fine roots . . . . . . . . . . . . . . . . . . . . . . 71
3.19 Non-metric Multidimensional Scaling for actinobacterial 16S rRNA genes . . . . 73
3.20 Relative heights of t-RFs 102 bp and 579 bp . . . . . . . . . . . . . . . . . . . . . 75
3.21 Non-metric Multidimensional Scaling for PKS Type II . . . . . . . . . . . . . . . 77
C.1 UPGMA dendrogram of BOX fingerprints for phylotype 2 isolates . . . . . . . . 122
C.2 UPGMA dendrogram of BOX fingerprints for phylotype 7 isolates . . . . . . . . 122
C.3 UPGMA dendrogram of BOX fingerprints for phylotype 38 isolates . . . . . . . . 123
C.4 UPGMA dendrogram of BOX fingerprints for phylotype 102 isolates . . . . . . . 123
C.5 UPGMA dendrogram of BOX fingerprints for phylotypes 84, 95, 104 and 107
isolates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
C.6 UPGMA dendrogram of Inter-LINE fingerprints for Penicillium isolates . . . . . 124
C.7 UPGMA dendrogram of Inter fingerprints for Cylindrocarpon destructans
isolates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
ivList of Tables
2.1 Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.2 Reference strains specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
2.3 Oligonucleotide specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.4 Inhibition classes of actinobacterial isolates . . . . . . . . . . . . . . . . . . . . . 32
2.5 t-RFLP program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
3.1 Distribution of different actinobacterial phylotypes between treatments . . . . . . 49
3.2 Distribution of isolated antagonistic fungi between treatments . . . . . . . . . . . 54
3.3 Viability test of P. citricola in interaction zones . . . . . . . . . . . . . . . . . . . 54
3.4 Comparison of Actinobacteria-specific primers . . . . . . . . . . . . . . . . . . . . 59
3.5 Validation of specificity of Actinobacteria 16S rDNA primers . . . . . . . . . . . 60
3.6 Indicator species analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
3.7 T-RF sizes of the 16S rRNA genes . . . . . . . . . . . . . . . . . . . . . . . . . . 76
A.1 Irrigation table for the main experiment . . . . . . . . . . . . . . . . . . . . . . . 118
A.2 Soil water content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
B.1 ANOVA below ground biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
B.2 ANOVA total above ground biomass . . . . . . . . . . . . . . . . . . . . . . . . . 119
B.3 ANOVA microbial biomass carbon . . . . . . . . . . . . . . . . . . . . . . . . . . 120
B.4 PerMANOVA for 16S t-RFLP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
B.5 PerMANOVA pair-wise comparison 16S . . . . . . . . . . . . . . . . . . . . . . . 120
B.6 PerMANOVA pair-wise comparison 16S for different factor levels . . . . . . . . . 121
B.7 PerMANOVA for PKS type II t-RFLP (season) . . . . . . . . . . . . . . . . . . . 121
B.8 PerMANOVA for PKS type II t-RFLP (treatment) . . . . . . . . . . . . . . . . . 121
vAcknowledgements
My cordial thanks to Prof. Dr. J. C. Munch for giving me the opportunity to work in this
project at the Institute of Soil Ecology, for his interest in the progress of my work and the
helpful and constructive discussions of my thesis.
I am very grateful to Dr. Michael Schloter for bringing me to Munich and making me feel
welcome in his working group (sorry... Department of Terrestrial EcoGenetics!). In spite of his
packed schedule, he always found the time for discussing the project and all other aspects of
working life. Thank you for the pleasant environment, the inexhaustable reserve of new ideas
and helpful reviewing of my thesis.
I would especially like to thank Dr. Alex Hagn for the supervision of my thesis and all the help
during the different stages of the project. Thanks for the countless input into the work, all the
time she invested in discussing all aspects of the thesis and the nice atmosphere in our three
man/woman lab. Without her help and especially her careful reviewing of the thesis, this work
would not have been the same.
I would like to acknowledge Prof. Dr. W. Oßwald and Prof. Dr. R. Matyssek for their
willingness to review this work and of course for the very pleasant and productive working
environment within the SFB 607.
Dr. Frank Fleischmann was of great help concerning the handling of the pathogen and the
inoculation of the beech plants. He also guided the experiment in 2005 which provided soil and
plant samples for the isolation of the antagonists.
I would like to thank Dr. Ipek Kurtb¨oke for sharing her knowledge about Actinobacteria with
me and for patiently answering all my questions. Her quick positive comments and humor often
made my day.
viThanks to everyone at the AG Schmitt-Kopplin, especially Dr. Moritz Frommberger, Dr. Nor-
bertHertkornandDr. PhilippeSchmitt-Kopplinforallthesupportwiththemetaboliteanalyses
(and the great coffee!).
Dr. Marion Engel introduced me to the fine art of building phylogenetic trees using the ARB
software, teaching me the deeper knowledge that those things just never look the way you want
them to and was/is of course a very pleasant office“roommate”.
Multiple thanks has to go to Dr. Karin Pritsch for always being around to answer questions
concerning experiments, statistics, common knowledge and life in general. It is a pleasure
working with you!
I would like to thank Dr. Karin Kloos for sharing her knowledge with me. I really enjoyed
having her in our office.
IwouldalsoliketoacknowledgeDr. BurkhardHenseandDr. WolfgangzuCastellfordiscussing
the statistical analysis with me.
I am very grateful to Conny Galonska for the great work in the lab and Dagmar Schneider for
her assistance during the greenhouse experiment.
¨Thanks to everyone at IBO and AMP who supported me and contributed to this work in
any possible way, especially all members of TEG for the lively discussions, great input and
constructive critical comments.
¨The changing members of our IBO cooking alliance (Ju¨rgen Esperschu¨tz, Roland Fuß, Sahni
Poschelsrieder and Claudia Zimmermann) deserve a special thanks for the great lunch and
coffee breaks, for saving me from the mensa and for keeping me and Jurgen from getting our¨
Waschbrettb¨auche.
Thanks to all my friends near and far for being there and thinking of me from time to time. Life
would be dull without you.
Thanks also to all the members of the gospel choir Changing Voices for getting my mind of
(almost) everything every Thursday evening.
Above all I would like to thank my family, especially my parents for always supporting and
believing in me over the last years and my grandparents Franz for being such great examples of
life well lived.
viiAbstract
TheOomycetePhytophthoracitricolaisacommonrootpathogenincentralEurope. Among
its many hosts are important tree species like European beech (Fagus sylvatica). In recent years
it has been shown that beech trees grown under elevated CO conditions are more susceptible2
to the pathogen, whereas O treated trees showed reduced disease severity. However, the rea-3
sons for these observations are still unknown. Besides physiological responses within the plant,
which might alter susceptibility, the possibility of changes in the microbial community of beech
rhizospheres might be responsible for this phenomenon through an increase or decrease in the
abundance of biocontrol active microbes in the rhizosphere.
This study aimed at investigating the composition of the antagonistic microbial community
within the rhizosphere of European beeches. Based on an isolation approach microorganisms
belonging to the actinobacterial genera Kitasatospora and Streptomyces, as well as the fungal
genera Trichoderma, Penicillium, Cylindrocarpon and Geomyces were shown to have great an-
tagonistic effects against P. citricola in vitro, thus having the potential to control the disease
in situ. The mechanism of antagonism of the most common group of isolates, belonging to the
genus Kitasatospora, was identified using a 12 Tesla Fourier transform ion cyclotron resonance
mass spectrometer (FT-ICR/MS). The bioactive substance had a molar mass of 281.17 g/mol
and was further characterized and identified as the macrolide polyketide cycloheximide using
1H-NMR.
On the basis of these findings a greenhouse experiment was performed to monitor possible
changesindiversityoftheactinobacterialcommunityonastructurallevelandtypeIIpolyketide
synthases responsible for antibiotics production on a functional level. Young beech trees were
grownunderelevatedCO ,elevatedO orambientconditionsfortwoyears. Halfofthepotswere2 3
inoculated with P. citricola at the beginning of the second year. Rhizosphere soil was sampled
in spring, summer and autumn of 2006. Terminal restriction fragment length polymorphism
(t-RFLP) was used to monitor changes in diversity applying specific primers for actinobacterial
16S rRNA genes and the ketosynthase unit of polyketide synthases.
Interestingly, a clone library revealed a highly unique actinobacterial community with 41.1%
of the sequences belonging to the only recently described suborder Catenulisporinae and 37.5%
of the sequences being unclassified Actinobacteria. When analyzing the actinobacterial 16S
rRNA t-RFLP profiles, no qualitative differences were found for neither season, CO and O2 3
treatments nor P. citricola inoculation. However, quantitative differences in single t-RFs were
viiiobserved for different seasons and the major t-RF responsible for this shift could be assigned to
organisms corresponding to the mentioned suborder Catenulisporinae. A transient shift in peak
height was observed for one major t-RF in O treated summer samples.3
OnthefunctionalleveloftypeIIpolyketidesynthasesnoqualitativeorquantitativedifference
could be observed for neither season, CO and O treatments nor P. citricola inoculation as2 3
analyzed by t-RFLP analysis. A clone library revealed a high diversity of genes potentially
responsible for the production of polyketides in soil.
ix