Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 908 Mechanism of Action of the Plant Growth Promoting Bacterium Paenibacillus polymyxa BY SALME TIMMUSK ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2003 ! " #$ % & % % ' ( ) * & ( + ,( "( % - % ' . ) ' & ( - ( #/( ! ( ( 0,1 #233!23/"23 & & % & ) & 4 5'.'67( - ) 2 % (&( & % 4 % 4 ) % ( ) + ) + % (3 ( 0 & ) ! ) ) 2 8 & & "# 5 7 % & 5 7 ) 2 ) & % ( 61-2%% 61- % 2 & & ) ( 9 61- & *63 5 & 2 & 7 ) & 2 ( 0 2 & ) ( - & ) % & & ) ( 4 % 4 % ! % ) ) & ) & ( & & % 2 && % 4 % ! ) & ( ) ) ( : 5 7 ( , & & ; ; < < & ) ( ) + 0 & ) % '.'6 & +& & % & %% ( $# % ' & ) 0 4 &' ( ) ' ) *+,' )(' ' "-.*/01 ' # = , + " 0,,1 !2""> 0,1 #233!23/"23 $ $$$ 2?? 5 $@@ (+(@ A B $ $$$ 2??7 “It is the time you have spent on your rose that makes your rose so important”. Antoine de Saint-Exupery ‘The Little Prince’ MAIN REFERENCES This thesis is based on the following papers, which will be referred to in the text by their Roman numerals. I Timmusk, S., Nicander, B., Granhall, U., and E. Tillberg Cytokinin production by Paenibacillus polymyxa. Soil Biology and Biochemistry, 31 (1999): 1847-1852. II Timmusk, S., and E. G. H. Wagner The plant-growth-promoting rhizobacterium Paenibacillus polymyxa induces changes in Arabidopsis thaliana gene expression: A possible connection between biotic and abiotic stress responses. Molecular Plant-Microbe Interactions,12 (1999): 951-959. III Timmusk, S., Grantcharova, N., Flärdh, K., and E. G. H. Wagner Paenibacillus polymyxa colonization of Arabidopsis thaliana roots Submitted IV Timmusk, S., van West, P., Gow, N. A. R., and E. G. H. Wagner Antagonistic effects of Paenibacillus polymyxa towards the oomycete plant pathogens Phytophthora palmivora and Pythium aphanidermatum Manuscript. Reprints were made with the permission of the publisher. The illustration by Antoine de Saint-Exupery on page 3 is reproduced with Le Petit Prince SOGEX permission. TABLE OF CONTENTS INTRODUCTION 7 PLANT RHIZOSPHERE 7 PLANT ROOT COLONIZING BACTERIA 7 PLANT GROWTH PROMOTING RHIZOBACTERIA 8 DIRECT PLANT GROWTH PROMOTION 9 INDIRECT PLANT GROWTH PROMOTION 10 BIOCONTROL OF SOILBORN PATHOGENS 10 Antibiotic production 10 Siderophores 11 Parasitism 11 Competition for nutrients and niches 11 BIOCONTROL BY INDUCING SYSTEMIC RESISTANCE 12 Signalling in plants 12 Microbial signals involved in inducing resistance 13 Microbial signals in induced systemic resistance 13 Microbial signals in systemic acquired resistance 14 PLANT ADAPTATION TO ABIOTIC STRESSES 15 AIM OF THE STUDY 16 PRESENT INVESTIGATION 16 CYTOKININ PRODUCTION BY PAENIBACILLUS POLYMYXA (I) 17 A GNOTOBIOTIC SYSTEM TO STUDY P. POLYMYXA EFFECTS ON PLANTS (papers II, III and IV) 18 P. POLYMYXA AS A BIOCONTROL AGENT 18 INDUCED SYSTEMIC RESISTANCE BY P. POLYMYXA (II and III) 18 P. polymyxa isolates protect Arabidopsis thaliana against the pathogen Erwinia carotovora 18 P. polymyxa inoculation causes biotic stress 19 P. polymyxa colonization on A. thaliana root, and its endophytic mode of action 19 P. POLYMYXA AS AN ANTAGONIST OF SOILBORN PATHOGENS (III and IV) 20 P. polymyxa antagonism studied by plate assays and microscopy 20 P. polymyxa antagonism in soil assays 20 P. POLYMYXA INOCULATION ENHANCES PLANT DROUGHT TOLERANCE (II and III) 21 DISCUSSION 22 P. POLYMYXA INDUCES SYSTEMIC RESISTANCE TO E. CAROTOVORA 22 P. POLYMYXA ANTAGONIZES ROOT PATHOGENS 24 P. POLYMYXA ENHANCES PLANT DROUGHT TOLERANCE 25 FUTURE PERSPECTIVES 26 ACKNOWLEDGEMENTS 28 REFERENCES 30 Salme Timmusk Page 7 ABBREVIATIONS ABA abscisic acid ACC 1-aminocyclopropane-1-carboxylate DP degree of polymerisation ET ethylene GFP green fluorescent protein IAA indole-3-acetic acid ISR induced systemic resistance JA jasmonic acid OGAs oligogalacturonides PGPR plant growth promoting rhizobacteria PR pathogenesis-related RT-PCR reverse transcription-polymerase chain reaction ROS reactive oxygen species SA salicylic acid SAR systemic acquired resistance TF transcription factor INTRODUCTION PLANT RHIZOSPHERE The term “rhizosphere” was already introduced by Hiltner in 1904, and is now defined as a volume of soil surrounding plant roots in which bacterial growth is stimulated (Sorensen, 1997). The rhizosphere has attracted much interest since it is a habitat in which several biologically important processes and interactions take place. The rhizosphere is populated by a diverse range of microorganisms, and the bacteria colonizing this habitat are called rhizobacteria (Schroth & Hancock, 1982). PLANT ROOT COLONIZING BACTERIA Root exudates are believed to determine which microorganisms colonize roots in the rhizosphere (Kunc & Macura, 1988). It is now known that plant roots also generate electrical signals; it has been shown that zoospores of oomycetic pathogens take advantage of these signals to guide their movements towards the root surface (Gow et al., 1999). It has been estimated that about 30% of plant photosynthate production is released via root exudation (Smith et al., 1993). Both passive leakage and active secretion are involved (Rougier & Chaboud, 1989). Leakage involves low molecular weight compounds release while secretion usually involves high molecular weight compounds that are actively transported across the cell membranes (Rougier & Chaboud, 1989).Composition and extent of exudation are determined genetically (Bolton et al., 1993). This involves a certain cost for the plant, and therefore must provide a selective advantage. It has often been suggested that root exudation evolved in plants as a means to stimulate active microflora (Bolton et al., 1993). Exudation can provide both physical and chemical benefits to plants. E.g., root mucilages reduce friction between root tips and the soil and reduce root desiccation, improve the contact between the root and the soil, and contribute to soil structural stability (Rougier & Chaboud, 1989). Root exudates also attract microorganisms (Jaeger et al., 1999; Lemanceau et al., 1995). Conversely, rhizobacteria can also elicit root exudation (Bolton et al., 1993) in a species-specific manner. E.g., metabolites Mechanism of growth promotion by P. polymyxa produced by Pseudomonas aeruginosa stimulated root exudates by perennial ryegrass twelve-fold (Merharg & Killham, 1995). A variety of techniques have been used to study bacteria in their habitat, i.e. in mixed populations. Often but not always, cultivation and staining techniques can be employed. Since the rhizosphere harbours a great variety of bacteria, each with different nutritional requirements, it is impossible to apply cultivation techniques to all bacteria. It has also become clear that a significant fraction of bacteria belong to the group of viable but not culturable (VBNC) bacteria (Roszak & Colwell, 1987), thus resisting conventional cultivation attempts. Sometimes, the diversity in microbial communities bacteria in a given environment, such as the rhizosphere, can be approached by PCR-based techniques. For instance, an analysis of PCR-amplified rRNA/rDNA fragments by denaturating gradient gel electrophoresis, or temperature gradient gel electrophoresis, can resolve characteristic bands due to different GC- content. Such techniques have the potential to analyze the major constituents of microbial communities. Often one needs to find out precisely where bacteria are located (e.g. during colonization), and/or what their physiological activity status is. In these cases, a number of marker genes have been developed for tagging of selected strains. Since the discovery of green fluorescent protein (GFP) it has been used extensively as a marker to detect microbes in environments, and to study microbial interactions with plants (e.g. Chalfie et al., Stoltzfus et al., 2000, Tombolini et al., 1999, Errampalli et al., 1999). The strong fluorescence of GFP allows rapid detection regardless of the energy status of the cells (Unge et al., 1999), and provides a very stable marker in environmental samples (Elvang et al., 2001; Unge et al., 1999). Even VBNC bacteria have been monitored with GFP-methods (Lowder et al., 2000). GFP fluorescence has been improved by mutations in the chromophore which changed excitation/emission spectra. Mutated versions of the gfp gene have given a range of derivatives that differ in emission wave length, e.g. enhanced cyan (ECFP), enhanced green (EGFP), and enhanced yellow (EYFP). Thus, different populations of tagged bacteria in the rhizosphere can be visualized simultaneously at the single cell level (Bloemberg et al., 2000). To monitor also the bacterial energy status, combinations of gfp and other marker genes, e.g luxAB or gusA, have been used (Elvang et al., 2001; Gau et al., 2002; Unge et al., 1999). Colonization sites for, e.g., Azospirillum brasiliense have been mapped using gfp- and gusA-tagging, simultaneously on emerging roots (Ramos et al., 2002). PLANT GROWTH PROMOTING RHIZOBACTERIA Plant root colonizing bacteria can function as harmful, deleterious rhizobacteria (DRB) or beneficial, plant