Impact of bacterial volatiles on phytopathogenic fungi: an in vitro study on microbial competition and interaction.

Microorganisms in the rhizosphere are abundant and exist in very high taxonomic diversity. The major players are bacteria and fungi, and bacteria have evolved many strategies to prevail over fungi, among them harmful enzyme activities and noxious secondary metabolites. Interactions between plant growth promoting rhizobacteria and phytopathogenic fungi are potentially valuable since the plant would benefit from fungal growth repression. In this respect, the role of volatile bacterial metabolites in fungistasis has been demonstrated, but the mechanisms of action are less understood. We used three phytopathogenic fungal species (Sclerotinia sclerotiorum, Rhizoctonia solani, and Juxtiphoma eupyrena) as well as one non-phytopathogenic species (Neurospora crassa) and the plant growth promoting rhizobacterium Serratia plymuthica 4Rx13 in co-cultivation assays to investigate the influence of bacterial volatile metabolites on fungi on a cellular level. As a response to the treatment, we found elevated lipid peroxidation, which indirectly reflected the loss of fungal cell membrane integrity. An increase in superoxide dismutase, catalase, and laccase activities indicated oxidative stress. Acclimation to these adverse growth conditions completely restored fungal growth. One of the bioactive bacterial volatile compounds seemed to be ammonia, which was a component of the bacterial volatile mixture. Applied as a single compound in biogenic concentrations ammonia also caused an increase in lipid peroxidation and enzyme activities, but the extent and pattern did not fully match the effect of the entire bacterial volatile mixture.

The α-Terpineol to 1,8-Cineole Cyclization Reaction of Tobacco Terpene Synthases.

Flowers of Nicotiana species emit a characteristic blend including the cineole cassette monoterpenes. This set of terpenes is synthesized by multiproduct enzymes, with either 1,8-cineole or α-terpineol contributing most to the volatile spectrum, thus referring to cineole or terpineol synthase, respectively. To understand the molecular and structural requirements of the enzymes that favor the biochemical formation of α-terpineol and 1,8-cineole, site-directed mutagenesis, in silico modeling, and semiempiric calculations were performed. Our results indicate the formation of α-terpineol by a nucleophilic attack of water. During this attack, the α-terpinyl cation is stabilized by π-stacking with a tryptophan side chain (tryptophan-253). The hypothesized catalytic mechanism of α-terpineol-to-1,8-cineole conversion is initiated by a catalytic dyad (histidine-502 and glutamate-249), acting as a base, and a threonine (threonine-278) providing the subsequent rearrangement from terpineol to cineol by catalyzing the autoprotonation of (S)-(-)-α-terpineol, which is the favored enantiomer product of the recombinant enzymes. Furthermore, by site-directed mutagenesis, we were able to identify amino acids at positions 147, 148, and 266 that determine the different terpineol-cineole ratios in Nicotiana suaveolens cineole synthase and Nicotiana langsdorffii terpineol synthase. Since amino acid 266 is more than 10 Å away from the active site, an indirect effect of this amino acid exchange on the catalysis is discussed.

Volatile mediated interactions between bacteria and fungi in the soil.

Soil is one of the major habitats of bacteria and fungi. In this arena their interactions are part of a communication network that keeps microhabitats in balance. Prominent mediator molecules of these inter- and intraorganismic relationships are inorganic and organic microbial volatile compounds (mVOCs). In this review the state of the art regarding the wealth of mVOC emission is presented. To date, ca. 300 bacteria and fungi were described as VOC producers and approximately 800 mVOCs were compiled in DOVE-MO (database of volatiles emitted by microorganisms). Furthermore, this paper summarizes morphological and phenotypical alterations and reactions that occur in the organisms due to the presence of mVOCs. These effects might provide clues for elucidating the biological and ecological significance of mVOC emissions and will help to unravel the entirety of belowground' volatile-wired' interactions.

SuperScent--a database of flavors and scents.

Volatiles are efficient mediators of chemical communication acting universally as attractant, repellent or warning signal in all kingdoms of life. Beside this broad impact volatiles have in nature, scents are also widely used in pharmaceutical, food and cosmetic industries, so the identification of new scents is of great industrial interest. Despite this importance as well as the vast number and diversity of volatile compounds, there is currently no comprehensive public database providing information on structure and chemical classification of volatiles. Therefore, the database SuperScent was established to supply users with detailed information on the variety of odor components. The version of the database presented here comprises the 2D/3D structures of approximately 2100 volatiles and around 9200 synonyms as well as physicochemical properties, commercial availability and references. The volatiles are classified according to their origin, functionality and odorant groups. The information was extracted from the literature and web resources. SuperScent offers several search options, e.g. name, Pubchem ID number, species, functional groups, or molecular weight. SuperScent is available online at: http://bioinformatics.charite.de/superscent.

Publisher Correction: Interspecific formation of the antimicrobial volatile schleiferon.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Interspecific formation of the antimicrobial volatile schleiferon.

Microorganisms release a plethora of volatile secondary metabolites. Up to now, it has been widely accepted that these volatile organic compounds are produced and emitted as a final product by a single organism e.g. a bacterial cell. We questioned this commonly assumed perspective and hypothesized that in diversely colonized microbial communities, bacterial cells can passively interact by emitting precursors which non-enzymatically react to form the active final compound. This hypothesis was inspired by the discovery of the bacterial metabolite schleiferon A. This bactericidal volatile compound is formed by a non-enzymatic reaction between acetoin and 2-phenylethylamine. Both precursors are released by Staphylococcus schleiferi cells. In order to provide evidence for our hypothesis that these precursors could also be released by bacterial cells of different species, we simultaneously but separately cultivated Serratia plymuthica 4Rx13 and Staphylococcus delphini 20771 which held responsible for only one precursor necessary for schleiferon A formation, respectively. By mixing their headspace, we demonstrated that these two species were able to deliver the active principle schleiferon A. Such a joint formation of a volatile secondary metabolite by different bacterial species has not been described yet. This highlights a new aspect of interpreting multispecies interactions in microbial communities as not only direct interactions between species might determine and influence the dynamics of the community. Events outside the cell could lead to the appearance of new compounds which could possess new community shaping properties.

Bacterial-Plant-Interactions: Approaches to Unravel the Biological Function of Bacterial Volatiles in the Rhizosphere.

Rhizobacteria produce an enormous amount of volatile compounds, however, the function of these metabolites is scarcely understood. Investigations evaluating influences on plants performed in various laboratories using individually developed experimental setups revealed different and often contradictory results, e.g., ranging from a significant plant growth promotion to a dramatic suppression of plant development. In addition to these discrepancies, these test systems neglected properties and complexity of the rhizosphere. Therefore, to pursue further investigations of the role of bacterial volatiles in this underground habitat, the applied methods have to simulate its natural characteristics as much as possible. In this review, we will describe and discuss pros and cons of currently used bioassays, give insights into rhizosphere characteristics, and suggest improvements for test systems that would consider in natura conditions and would allow gaining further knowledge of the potential function and significance of rhizobacterial volatiles in plant life.

Floral benzenoid carboxyl methyltransferases: from in vitro to in planta function.

Benzenoid carboxyl methyltransferases synthesize methyl esters (e.g., methyl benzoate and methyl salicylate), which are constituents of aromas and scents of many plant species and play important roles in plant communication with the surrounding environment. Within the past five years, eleven such carboxyl methyltransferases were isolated and most of them were comprehensively investigated at the biochemical, molecular and structural level. Two types of enzymes can be distinguished according to their substrate preferences: the SAMT-type enzymes isolated from Clarkia breweri, Stephanotis floribunda, Antirrhinum majus, Hoya carnosa, and Petunia hybrida, which have a higher catalytic efficiency and preference for salicylic acid, while BAMT-type enzymes from A. majus, Arabidopsis thaliana, Arabidopsis lyrata, and Nicotiana suaveolens prefer benzoic acid. The elucidation of C. breweri SAMT's three-dimensional structure allowed a detailed modelling of the active sites of the carboxyl methyltransferases and revealed that the SAM binding pocket is highly conserved among these enzymes while the methyl acceptor binding site exhibits some variability, allowing a classification into SAMT-type and BAMT-type enzymes. The analysis of expression patterns coupled with biochemical characterization showed that these carboxyl methyltransferases are involved either in floral scent biosynthesis or in plant defense responses. While the latter can be induced by biotic or abiotic stress, the genes responsible for floral scent synthesis exhibit developmental and rhythmic expression pattern. The nature of the product and efficiency of its formation in planta depend on the availability of substrates, the catalytic efficiency of the enzyme toward benzoic acid and/or salicylic acid, and the transcriptional, translational, and post-translational regulation at the enzyme level. The biochemical properties of benzenoid carboxyl methyltransferases suggest that the genes involved in plant defenses might represent the ancestor for the presently existing floral genes which during evolution gained different expression profiles and encoded enzymes with the ability to accept structurally similar substrates.

Transcriptional and post-translational regulation of S-adenosyl-L-methionine: salicylic acid carboxyl methyltransferase (SAMT) during Stephanotis floribunda flower development.

Methyl salicylate (MeSA) and a number of other volatiles are primarily emitted in the evening/night by Stephanotis floribunda leading to attraction of night active pollinators. A second minor emission peak for MeSA occurs in the morning/day. To understand these emission patterns, we have studied in detail the temporal regulation of the last step of the biosynthetic pathway of MeSA, the convertion of salicylic acid (SA) to MeSA catalysed by S-adenosyl-L-methionine: salicylic acid carboxyl methyltransferase (SAMT). We observed that in young flowers a maximum in SAMT activity occurs in the night, and that in flowers which were open longer than 4 days, two SAMT activity maxima occurred per day. These maxima correlated well with dawn and dusk and the previously detected MeSA emission peaks. The SAMT mRNA levels, however, have a broad maximum during the dark phase, while the SAMT protein levels continuously increase during floral development without showing daily rhythms. Furthermore, under continuous illumination (LL) the SAMT mRNA levels and activity patterns oscillate, suggesting the involvement of a circadian clock in the regulation network. Taken together, this analysis clearly demonstrates that regulation of MeSA emission occurs both at the transcriptional and post-translational levels, indicating that control at more than one level is necessary to guarantee the precise timing of volatile emission in flowers of S. floribunda.

Influence of green leaf herbivory by Manduca sexta on floral volatile emission by Nicotiana suaveolens.

Plants have to cope with various abiotic and biotic impacts as a consequence of changing environments, which can impair their ability to sexually reproduce. The main objective of this study was to investigate whether green leaf herbivory, having one of the most hazardous biotic impacts, would have any direct effect on the production and emission of floral volatiles because volatiles are known to play a crucial role in pollination. Nicotiana suaveolens plants were challenged with Manduca sexta feeding on leaves, and alterations in the quality and quantity of the floral blend, shifts in emission patterns, and changes in expression patterns of the floral benzoic/salicylic acid carboxyl-methyltransferase were monitored in noninfested and infested plants. Leaves responded to larval feeding by herbivory-induced diurnal emission of semiochemicals, whereas the emission of floral volatiles remained unchanged in comparison to the noninfested control. Neither the volatile composition nor the quantity of components or the nocturnal emission patterns was altered. The mRNA and protein levels of the benzoic/salicylic acid carboxyl-methyltransferase, as well as its enzyme activity, also did not show any significant differences. These results indicate that metabolism in flowers at and postanthesis is an autonomous process and is independent of metabolic changes in green leaves. By this sustaining mechanism, N. suaveolens plants ensure sexual reproduction even under unfavorable conditions.

Regulation of simultaneous synthesis of floral scent terpenoids by the 1,8-cineole synthase of Nicotiana suaveolens.

The white flowers of N. suaveolens emit a complex bouquet of fragrance volatiles. The dominant compounds are benzenoids (e.g. methyl benzoate, methyl salicylate, benzyl benzoate and benzyl salicylate), monoterpenes (1,8-cineole, limonene, sabinene, E-beta-ocimene, beta-beta-myrcene, alpha- and beta-pinene and alpha-terpineole) and sesquiterpenes (e.g. caryophyllene), which are all emitted at higher levels during the night. Here, we show that the simultaneous nocturnal emission of most monoterpenes is realized by a single floral-specific multi-product enzyme (1,8-cineole synthase, CIN), which synthesizes the monoterpenes of the "cineole cassette". Interestingly, N. suaveolens is the only known taxon of the Suaveolentes section to have a flower emitting "cineole cassette of monoterpenes" which is otherwise typical for the Alatae section. Gene sequence analysis of CIN has revealed the highest similarities to other angiosperm monoterpene synthases from Vitis vinifera, Quercus ilex, Citrus unshiu and C. limon, which cluster in the same branch of the terpene synthase B subfamily. However, based on its synthesized products, N. suaveolens CIN shares similarity with enzymes of the Arabidopsis thaliana root and Salvia officinalis leaf. The N. suaveolens CIN gene is only expressed in the stigma/style tissue and petals. Thin sections of petals present the enzyme primarily in the adaxial and abaxial epidermis; this facilitates the comprehensive emission of volatiles in all spacial directions. The oscillation of monoterpene emission is a consequence of the regulation of the CIN gene by the circadian clock, with oscillations occurring at the level of transcript and protein accumulations and of enzyme activity. Light/dark or dark/light transition signals synchronize the slow-running endogenous clock. Two strategies for synchronized scent emission have been established in N. suaveolens flowers: (i) the synthesis of volatile organic compounds by a multi-product enzyme and (ii) the coordination of biosynthetic pathways by a circadian clock.

Volatiles of bacterial antagonists inhibit mycelial growth of the plant pathogen Rhizoctonia solani.

Bacterial antagonists are bacteria that negatively affect the growth of other organisms. Many antagonists inhibit the growth of fungi by various mechanisms, e.g., secretion of lytic enzymes, siderophores and antibiotics. Such inhibition of fungal growth may indirectly support plant growth. Here, we demonstrate that small organic volatile compounds (VOCs) emitted from bacterial antagonists negatively influence the mycelial growth of the soil-borne phytopathogenic fungus Rhizoctonia solani Kühn. Strong inhibitions (99-80%) under the test conditions were observed with Stenotrophomonas maltophilia R3089, Serratia plymuthica HRO-C48, Stenotrophomonas rhizophila P69, Serratia odorifera 4Rx13, Pseudomonas trivialis 3Re2-7, S. plymuthica 3Re4-18 and Bacillus subtilis B2g. Pseudomonas fluorescens L13-6-12 and Burkholderia cepacia 1S18 achieved 30% growth reduction. The VOC profiles of these antagonists, obtained through headspace collection and analysis on GC-MS, show different compositions and complexities ranging from 1 to almost 30 compounds. Most volatiles are species-specific, but overlapping volatile patterns were found for Serratia spp. and Pseudomonas spp. Many of the bacterial VOCs could not be identified for lack of match with mass-spectra of volatiles in the databases.

Volatile composition, emission pattern, and localization of floral scent emission in Mirabilis jalapa (Nyctaginaceae).

We elucidated scent components, daily emission patterns, and the localization of floral scent release of Mirabilis jalapa. Volatiles emitted by the whole plant as well as by detached flowers were investigated using dynamic headspace analysis and gas chromatography/ mass spectrometry. Among several constituents including (Z)-3-hexenyl acetate, ß-myrcene, (Z)-ocimene, and benzyl benzoate, the monoterpene (E)-ß-ocimene was the major fragrance component. Fragrance release occurred in a time-dependent manner. The emission of volatiles, including (E)-ß-ocimene, showed an evening-specific maximum (1700-2000 pm). The emission of (Z)-3-hexenyl acetate reached its maximum 3 h later. Histological (neutral red staining) and morphological studies (electron and light microscopy) of the flower surface and tissues of M. jalapa revealed differences in surface structures and tissue characteristics. The flower could be divided into four main sections, including the tube, the transition zone between tube and limb, a star-shaped center of the limb, and petaloid lobes of the limb. These petaloid lobes are the site of (E)-ß-ocimene release. Stomata and trichomes found on the abaxial flower surface were not directly involved in fragrance release. Clear indications of osmophores involved in scent release could not be found. Thus, the results indicate that floral volatiles probably are released by diffuse emission in M. jalapa.

Biochemical and structural characterization of benzenoid carboxyl methyltransferases involved in floral scent production in Stephanotis floribunda and Nicotiana suaveolens.

Flower-specific benzenoid carboxyl methyltransferases from Stephanotis floribunda and Nicotiana suaveolens were biochemically and structurally characterized. The floral scents of both these species contain higher levels of methyl benzoate and lower levels of methyl salicylate. The S. floribunda enzyme has a 12-fold lower K(m) value for salicylic acid (SA) than for benzoic acid (BA), and results of in silico modeling of the active site of the S. floribunda enzyme, based on the crystal structure of Clarkia breweri salicylic acid methyltransferase (SAMT), are consistent with this functional observation. The enzyme was therefore designated SAMT. The internal concentration of BA in S. floribunda flowers is three orders of magnitude higher than the SA concentration, providing a rationale for the observation that these flowers synthesize and emit more methyl benzoate than methyl salicylate. The N. suaveolens enzyme has similar K(m) values for BA and SA, and the in silico modeling results are again consistent with this in vitro observation. This enzyme was therefore designated BSMT. However, the internal concentration of BA in N. suaveolens petals was also three orders of magnitude higher than the concentration of SA. Both S. floribunda SAMT and N. suaveolens BSMT are able to methylate a range of other benzenoid-related compounds and, in the case of S. floribunda SAMT, also several cinnamic acid derivatives, an observation that is consistent with the larger active site cavity of each of these two enzymes compared to the SAMT from C. breweri, as shown by the models. Broad substrate specificity may indicate recent evolution or an adaptation to changing substrate availability.