Pathogen suppression by microbial volatile organic compounds in soils
Boer, Wietse de; Li, Xiaogang ; Meisner, Annelein ; Garbeva, Paolina - \ 2019
FEMS microbiology ecology 95 (2019)8. - ISSN 0168-6496
disease suppression - fungistasis - microbial interactions - soil atmosphere - soil microbial community - sustainable management strategies - volatile organic compounds
There is increasing evidence that microbial volatile organic compounds (mVOCs) play an important role in interactions between microbes in soils. In this minireview, we zoom in on the possible role of mVOCs in the suppression of plant-pathogenic soil fungi. In particular, we have screened the literature to see what the actual evidence is that mVOCs in soil atmospheres can contribute to pathogen suppression. Furthermore, we discuss biotic and abiotic factors that influence the production of suppressive mVOCs in soils. Since microbes producing mVOCs in soils are part of microbial communities, community ecological aspects such as diversity and assembly play an important role in the composition of produced mVOC blends. These aspects have not received much attention so far. In addition, the fluctuating abiotic conditions in soils, such as changing moisture contents, influence mVOC production and activity. The biotic and abiotic complexity of the soil environment hampers the extrapolation of the production and suppressing activity of mVOCs by microbial isolates on artificial growth media. Yet, several pathogen suppressive mVOCs produced by pure cultures do also occur in soil atmospheres. Therefore, an integration of lab and field studies on the production of mVOCs is needed to understand and predict the composition and dynamics of mVOCs in soil atmospheres. This knowledge, together with the knowledge of the chemistry and physical behaviour of mVOCs in soils, forms the basis for the development of sustainable management strategies to enhance the natural control of soil-borne pathogens with mVOCs. Possibilities for the mVOC-based control of soil-borne pathogens are discussed.
Volatile-mediated interactions in the rhizosphere
Cordovez da Cunha, Viviane - \ 2016
Wageningen University. Promotor(en): Francine Govers; Jos Raaijmakers, co-promotor(en): V.J. Carrion. - Wageningen : Wageningen University - ISBN 9789462579019 - 219
rhizosphere bacteria - rhizosphere fungi - microbial interactions - volatile compounds - suppressive soils - actinobacteria - streptomyces - microbacterium - thanatephorus cucumeris - growth stimulators - biological control - defence mechanisms - genomics - transcriptomics - rizosfeerbacteriën - rizosfeerschimmels - microbiële interacties - vluchtige verbindingen - ziektewerende gronden - actinobacteria - streptomyces - microbacterium - thanatephorus cucumeris - groeistimulatoren - biologische bestrijding - verdedigingsmechanismen - genomica - transcriptomica
Plants and microorganisms are constantly engaged in highly dynamic interactions both above- and belowground. Several of these interactions are mediated by volatile organic compounds (VOCs), small carbon-based compounds with high vapor pressure at ambient temperature. In the rhizosphere, VOCs have an advantage in intra- and interorganismal signaling since they can diffuse through soil pores over longer distances than other metabolites and are not dependent on water availability. The research described in this PhD thesis explored how beneficial and pathogenic microorganisms that live in the rhizosphere and endosphere modulate plant growth, development and resistance via the production of VOCs. In vitro and in vivo bioassays as well as different ‘omic’ approaches, such as volatomics, transcriptomics and genomics, were employed to investigate underlying mechanisms of VOC-mediated microbe-microbe and microbe-plant interactions.
To investigate the diversity and functions of microbial VOCs, a disease-suppressive soil was used as the source of the VOC-producing microorganisms. Previous metagenomics studies reported Actinobacteria, in particular Streptomyces and Microbacterium species, as the most abundant bacterial genera found in a soil naturally suppressive to the fungal root pathogen Rhizoctonia solani. VOCs of several Streptomyces isolates inhibited hyphal growth of R. solani and in addition, promoted plant growth. Coupling the Streptomyces VOC profiles with their effects on fungal growth pinpointed methyl 2-methylpentanoate and 1,3,5-trichloro-2-methoxy benzene as antifungal VOCs. Also Microbacterium isolates showed VOC-mediated antifungal activity and plant growth promotion. VOC profiling of Microbacterium sp. EC8 revealed several sulfur-containing compounds and ketones such as dimethyl disulfide, trimethyl trisulfide and 3,3,6-trimethylhepta-1,5-dien-4-one (also known as Artemisia ketone). Genome analysis of strain EC8 revealed genes involved in sulfur metabolism. Resolving the role of the identified compounds and genes in VOC-mediated plant growth promotion and induced resistance will be subject of future studies. VOC-mediated chemical warfare underground has been proposed as a key mechanism of natural disease-suppressive soils. The results presented in this thesis indeed point in that direction. However, to more conclusively determine the role of the identified Actinobacterial VOCs in soil suppressiveness to R. solani, it will be important to demonstrate that the fungicidal VOCs are actually produced in situ at the right place and at sufficient concentrations to suppress plant infection by the pathogenic fungus.
In agriculture, VOCs and VOC-producing microorganisms provide a potential alternative to the use of pesticides to protect plants and to improve crop production. In the past decades, several in vitro studies have described the effects of microbial VOCs on other (micro)organisms. However, little is still known on the potential of VOCs in large-scale agriculture and horticulture. The results described in this thesis show that VOCs from Microbacterium sp. EC8 stimulate the growth of Arabidopsis, lettuce and tomato, but do not control damping-off disease of lettuce caused by R. solani. Significant biomass increases were also observed for plants exposed only shortly to the bacterial VOCs prior to transplantation of the seedlings to soil. These results indicate that VOCs from strain EC8 can prime plants for growth promotion without direct contact and prolonged colonization. Furthermore, the induction of the plant growth-promoting effects appeared to be plant tissue specific. Root exposure to the bacterial VOCs led to a significant increase in plant biomass whereas shoot exposure did not result in significant biomass increase of lettuce and tomato seedlings. Genome-wide transcriptome analysis of Arabidopsis seedlings exposed to VOCs from this bacterium showed an up-regulation of genes involved in sulfur and nitrogen metabolism and in ethylene and jasmonic acid signaling. These results suggest that the blend of VOCs of strain EC8 favors, in part, the plant’s assimilation of sulfate and nitrogen, essential nutrients for plant growth, development and also resistance.
Similar to beneficial microorganisms, plant pathogenic microorganisms have also evolved strategies to modulate growth and defense of their hosts. For instance, compounds secreted by pathogens may suppress or interfere with plant defense. In this thesis I show that R. solani produces an array of VOCs that promote growth, accelerate development, change VOC emission and reduce insect resistance of plants. Plant growth-promoting effects induced by the fungal VOCs were not transgenerational. Genome-wide transcriptome analysis of Arabidopsis seedlings revealed that exposure to fungal VOCs caused up-regulation of genes involved in auxin signaling, but down-regulation of genes involved in ethylene and jasmonic acid signaling. These findings suggest that this soil-borne pathogen uses VOCs to predispose plants for infection by stimulating lateral root formation and enhancing root biomass while suppressing defense mechanisms. Alternatively, upon perception of VOCs from soil-borne pathogens, plants may invest in root biomass while minimizing investments in defense, a trade-off that helps them to speed up growth and reproduction and to survive pathogen attack.
In conclusion, the research presented in this thesis shows that both plants and microorganisms engage via VOCs in long-distance interactions and that beneficial and pathogenic soil microorganisms can alter plant physiology in different ways. Here, I provided a first step in identifying microbial genes involved in the regulation of biologically active VOCs as well as candidate plant genes involved in VOC perception and signal transduction. How plants sense and differentiate among VOCs from beneficial and pathogenic soil microorganisms will be an intriguing subject for future studies.
Microbial interactions in the fish gut
Giatsis, Christos - \ 2016
Wageningen University. Promotor(en): Johan Verreth, co-promotor(en): Marc Verdegem; Detmer Sipkema. - Wageningen : Wageningen University - ISBN 9789462578777 - 196
fishes - tilapia - larvae - microbial interactions - intestinal microorganisms - intestines - dynamics - fish feeding - probiotics - fish culture - aquaculture - vissen - tilapia - larven - microbiële interacties - darmmicro-organismen - darmen - dynamica - visvoeding - probiotica - visteelt - aquacultuur
Aquaculture has realized considerable growth over the past years while the world demand on seafood has been increasing. As aquaculture intensifies, the production sector needs to tackle major bottlenecks such as suboptimal growth and high and unpredictable mortality, especially in larval cultures. Fish-microbe interactions are closely related to overall fish health. To obtain a healthy and resilient microbial community (MC), it is important to understand the underlying mechanisms of microbial colonization in the fish gut.
The goal of this thesis was to investigate the role of water and feed microbial communities on shaping gut communities during early development of Nile tilapia.
To determine the contribution of stochasticity to overall variation, we first characterized the spatio-temporal variation in MC composition between individuals reared within the same or in replicate recirculating or active suspension systems (RAS vs. AS). Highly similar MCs developed in the gut when larvae shared the same water and diet. Rearing larvae in replicate production systems resulted in significantly different gut communities indicating that compositional replication of the MCs of an ecosystem is not fully predictable. We found that mainly water MCs, and to a lesser degree feed MCs, were associated with changes in MCs. Thus, we could conclude that steering gut MCs can be possible through water MC management tailored on the specifications of the rearing system in use.
Next, the possibility of early life steering of gut communities via microbial manipulations of feed MCs was explored. We hypothesized that gut microbial composition is strongly shaped by selective pressures in the gut and by the MCs present in the water. Thus similar MCs should develop between treatments regardless of the dietary treatments. Fish larvae were fed either a control feed or the control feed containing MCs derived from aerobic, methanogenic or denitrifying sludge reactors. We found that gut microbiota shared a much higher number of operational taxonomic units (OTUs) with microbiota in sludge-based feeds than with water, resulting in distinct gut MCs between treatments. Our findings suggest that Nile tilapia gut MC has a certain plasticity, which makes it amenable to interventions through proper feed microbial management.
Subsequently, we tested the imprinting effect of early exposure to the probiotic Bacillus subtilis on shaping gut MC composition even after the administration of the probiotic discontinues. For this, we constrained the initial contact with microbes from the environment by producing axenic tilapia larvae, which were then exposed to normal husbandry conditions. Early life probiotic exposure affected gut MC composition during B. subtilis administration but also within the first two weeks after its administration stopped, thus indicating that early exposure to the probiotic strain via the water had a sustained impact on gut MC composition.
Finally, overall conclusions and practical implications of our results for aquaculture production were presented. A meta-analysis was also performed to examine (1) the phylogenetic similarity among gut MCs of the same and different fish species reared in different habitats, fed different diets and at different developmental stages and (2) the factors primarily shaping gut MCs. We showed that the selective pressure responsible in shaping gut MC composition highly depends on the host as gut communities clustered primarily together by host and to a lesser extent reflected differences in habitat and diet. The phylogenetic analysis of gut communities revealed a clear clustering by study thus indicating that manipulation of gut communities is conceivable. Study-to-study variation could be attributed to the methodology used for MC analysis highlighting also the importance of methodological uniformity when comparisons between studies are made.
Overall, this thesis provided fundamental knowledge on MC composition and development in aquaculture rearing systems. Although the insights generated by this thesis are still premature to fully explain, predict or steer MC composition, and though additional studies are needed, we believe that, in the long run, this approach will facilitate the development of safe and effective methods for manipulating gut microbial composition to promote fish health in aquaculture rearing systems.
Mining into interspecific bacterial interactions
Tyc, Olaf - \ 2016
Wageningen University. Promotor(en): Wietse de Boer, co-promotor(en): Paolina Garbeva. - Wageningen : Wageningen University - ISBN 9789462578340 - 234
soil bacteria - secondary metabolites - microbial interactions - antibiotics - nutrients - bodembacteriën - secundaire metabolieten - microbiële interacties - antibiotica - voedingsstoffen
In terrestrial ecosystems bacteria live in close proximity with many different microbial species and form complex multi-species networks. Within those networks bacteria are constantly interacting with each other and produce a plethora of secondary metabolites like antibiotics, enzymes, volatiles and other compounds from diverse chemical classes. Several independent studies revealed that the production of secondary metabolites by soil bacteria can be influenced by the interaction with other microorganisms in their vicinity.
In this thesis we show how interspecific interactions between soil bacteria influence the production of soluble and volatile secondary metabolites, gene expression and fitness. To elucidate the effect of interspecific interactions on antimicrobial activity in soil bacteria a high-through-put screening method was developed and applied on a collection of 146 rhizobacterial isolates obtained from similar habitats. In addition we examined if the production of volatile organic compounds is influenced by interspecific interactions. Thus, the identity and antimicrobial activity of volatiles produced by bacteria cultivated in monoculture as well in interaction were examined. Furthermore a sand microcosm approach was applied to investigate how Pseudomonas fluorescens strain Pf0-1 responded to the presence of monocultures and mixtures of a Gram-negative (Pedobacter sp. V48) and a Gram-positive (Bacillus sp. V102) bacterial strain under two nutritional conditions.
The interaction between a gram-negative Burkholderia and a gram-positive Paenibacillus isolate was subjected to detailed metabolome, volatolome and transcriptome analysis. One distinct volatile and one non-volatile compound produced only during interspecific interaction but not in the monoculture were identified. The activity of the interacting bacteria and the compounds produced during interaction were tested against a range of human and plant pathogens.
In summary, this thesis extends the knowledge about the effect of interspecific bacterial interactions on secondary metabolites production (soluble and volatiles), gene expression and fitness in bacteria. The exploitation of such bacterial interspecific interactions can be an important “tool” for the discovery of novel antimicrobial and agro-chemical compounds. The obtained knowledge can help in selecting the right players in synthetic communities that fulfil important ecosystem services like disease suppression in agricultural crop systems.
Prof. Liesje Mommer over groei en overlevingskansen bij planten
Mommer, L. - \ 2016
biodiversiteit - plantenecologie - landbouwkundig onderzoek - plantenontwikkeling - openbare redes - microbiële interacties - grondvegetatie - wortels - planteninteractie - biodiversity - plant ecology - agricultural research - plant development - public speeches - microbial interactions - ground vegetation - roots - plant interaction
Planten jutten elkaar op. Verschillende gewassen die samen een vegetatie vormen, groeien beter dan wanneer er maar één soort groeit. Maar hoe werkt dat? Welke processen onder de grond zorgen ervoor dat deze gewassen samen beter groeien? Prof. Liesje Mommer licht een tip van de sluier op tijdens haar inaugurele rede als persoonlijk hoogleraar bij de leerstoelgroep Plantenecologie en natuurbeheer aan Wageningen
Ecophysiology of novel intestinal butyrate-producing bacteria
Bui, Thi Phuong Nam - \ 2016
Wageningen University. Promotor(en): Willem de Vos, co-promotor(en): Caroline Plugge. - Wageningen : Wageningen University - ISBN 9789462577015 - 202
butyrates - butyric acid bacteria - intestines - microbial interactions - faecal examination - mice - man - infants - genomics - intestinal physiology - microbial physiology - biochemical pathways - lysine - sugar - butyraten - boterzuurbacteriën - darmen - microbiële interacties - fecesonderzoek - muizen - mens - zuigelingen - genomica - darmfysiologie - microbiële fysiologie - biochemische omzettingen - lysine - suiker
The human intestinal tract harbours a trillion on microbial cells, predominantly anaerobes. The activity and physiology of these anaerobes is strongly associated with health and disease. This association has been investigated for a long time.However, this has not been fully understood. One of the reasons is the limited availability of cultured representatives. It is estimated that there may be more than 3000 species colonised in the gut of healthy individuals, however, only a bit over 1000 species have been isolated and characterised. Among the intestinal microbes, butyrate-producing bacteria are of special interest as the butyrate produced, is crucial to maintain a healthy gut. In addition, butyrate-producing bacteria have shown a reverse correlation with several intestinal diseases. In Chapter 2 we described a novel species Anaerostipes rhamnosivorans 1y2T isolated from an infant stool. This strain belonged to genus Anaerostipes within Clostridium cluster XIVa. A. rhamnosivorans had a capability of converting rhamnose into butyrate that is unique within intestinal butyrate-producing bacteria. The genomic analysis also revealed the entire rhamnose fermentation pathway as well as the acetyl-CoA pathway for butyrate production. This bacterium is able to produce butyrate from a wide range of sugars as well as lactate plus acetate. In Chapter 3, we described the microbial interactions between A. rhamnosivorans and Bacteriodes thetaiotaomicron in dietary pectins; Blautia hydrogenotrophica in lactate and small amount of acetate; Methanobrevibacter smithii in glucose. We observed that A. rhamnosivorans was able to benefit from its partners in all cocultures for butyrate production. This is likely due to its high metabolic flexibility. While the interaction between A. rhamnosivorans and B. thetaiotaomicron appeared as syntrophy, the interaction between A. rhamnosivorans and hydrogenotrohic microbes were cross-feeding type where hydrogen was transferred between two species. The latter resulted in an increase in butyrate level. In Chapter 4 we described a novel species Intestinimonas butyriciproducens SRB521T representing a novel genus Intestinimonas from a mouse caecum within Clostridium cluster IV. This bacterium produced butyrate and acetate as end products from Wilkins-Chalgren-Anaerobe broth.
Butyrate production is assumed to derive from carbohydrate employing acetyl-CoA pathway. No gut bacterium is known to convert proteins or amino acids to butyrate although butyrogenic pathways from amino acid degradation have been detected in the human gut using metagenomic approach. In Chapter 5 we discovered a novel butyrate synthesis pathway from the amino acid lysine and the Amadori product fructoselysine in Intestinimonas butyriciproducens AF211 that was isolated from human stool. This strain appeared to grow much better in lysine as compared to sugars although lysine and acetyl-CoA pathways were both detected in its complete genome. Moreover, the strain AF211 was able to metabolise efficiently fructoselysine into butyrate, and acetate was found to affect the fructoselysine fermentation, representing the impact of the environmental conditions where acetate is abundant in the gut. While the lysine pathway was found in the gut of many individuals, the fructoselysine pathway was present in only half of those samples. The finding that strain I. butyriciproducens AF211 is capable of the butyrogenic conversion of amino acid lysine and fructoselysine, an Amadori product formed in heated foods via the Maillard reaction, indicated a missing link that coupling protein metabolism and butyrate formation. As this Amadori product has been implicated to play a role in aging process, the use of strain AF211 as fructoselysine clearance in the gut needs further investigation. In Chapter 6 we performed genomic and physiological comparison between the I. butyriciproducens strain AF211 (human isolate) and SRB521T (mouse isolate). I. butyriciproducens was the most abundant species within the Intestinimonas genus and highly prevalent in humans based on metadata analysis on 16S amplicons. We confirmed that the butyrogenesis from lysine was a shared characteristic between the two I. butyriciproducens strains. We also observed the host specific features including tolerance to bile, cellular fatty acid composition, more efficient capability of converting sugars into butyrate, especially galactose and arabinose, in the human strain AF211. In addition, genomic rearrangements as well as variations in bacteriophages differed among strains.
Involvement of the ABC transporter BcAtrB and the laccase BcLCC2 in defence of Botrytis cinerea against the broad-spectrum antibiotic 2,4-diacetylphloroglucinol
Schouten, A. ; Maksimova, O. ; Cuesta Arenas, Y. ; Berg, G. van den; Raaijmakers, J.M. - \ 2008
Environmental Microbiology 10 (2008)5. - ISSN 1462-2912 - p. 1145 - 1157.
mediator-systems - fungal laccases - pseudomonas-fluorescens - microbial interactions - rhizoctonia-solani - biological-control - take-all - sensitivity - resistance - biocontrol
The genetic and biochemical basis of defence mechanisms in plant pathogenic fungi against antifungal compounds produced by antagonistic microorganisms is largely unknown. The results of this study show that both degradative and non-degradative defence mechanisms enable the plant pathogenic fungus Botrytis cinerea to resist the broad-spectrum, phenolic antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG). The efflux pump BcAtrB provides the first line of defence for B. cinerea, preventing accumulation of 2,4-DAPG in the cell to toxic concentrations, whereas the extracellular laccase BcLCC2 mediates, via conversion of tannic acid, subsequent degradation of 2,4-DAPG. Expression of BcatrB is induced by 2,4-DAPG and efflux gives B. cinerea sufficient time to more effectively initiate the process of BcLCC2-mediated antibiotic degradation. This is supported by the observations that the BcatrB mutant is significantly more sensitive to 2,4-DAPG than its parental strain, and is substantially less effective in 2,4-DAPG degradation. The results of this study further showed that BcLCC2 itself is not able to degrade 2,4-DAPG, but requires tannic acid as a mediator for 2,4-DAPG degradation. To our knowledge, this is the first time that the laccase-mediator system is shown to play a role in the detoxification of a broad-spectrum antibiotic compound from bacterial origin. We postulate that yet unknown constituents present in tannic acid act as substrate(s) of BcLCC2, thereby generating radicals that mediate 2,4-DAPG degradation.