Ecologically Different Fungi Affect Arabidopsis Development: Contribution of Soluble and Volatile Compounds

<div><p>Plant growth and development can be influenced by mutualistic and non-mutualistic microorganisms. We investigated the ability of the ericoid endomycorrhizal fungus <i>Oidiodendron maius</i> to influence growth and development of the non-host plant <i>Arabidopsis thaliana</i>. Different experimental setups (non-compartmented and compartmented co-culture plates) were used to investigate the influence of both soluble and volatile fungal molecules on the plant phenotype. <i>O</i>. <i>maius</i> promoted growth of <i>A</i>. <i>thaliana</i> in all experimental setups. In addition, a peculiar clumped root phenotype, characterized by shortening of the primary root and by an increase of lateral root length and number, was observed in <i>A</i>. <i>thaliana</i> only in the non-compartmented plates, suggesting that soluble diffusible molecules are responsible for this root morphology. Fungal auxin does not seem to be involved in plant growth promotion and in the clumped root phenotype because co-cultivation with <i>O</i>. <i>maius</i> did not change auxin accumulation in plant tissues, as assessed in plants carrying the DR5::GUS reporter construct. In addition, no correlation between the amount of fungal auxin produced and the plant root phenotype was observed in an <i>O</i>. <i>maius</i> mutant unable to induce the clumped root phenotype in <i>A</i>. <i>thaliana</i>. Addition of active charcoal, a VOC absorbant, in the compartmented plates did not modify plant growth promotion, suggesting that VOCs are not involved in this phenomenon. The low VOCs emission measured for <i>O</i>. <i>maius</i> further corroborated this hypothesis. By contrast, the addition of CO₂ traps in the compartmented plates drastically reduced plant growth, suggesting involvement of fungal CO₂ in plant growth promotion. Other mycorrhizal fungi, as well as a saprotrophic and a pathogenic fungus, were also tested with the same experimental setups. In the non-compartmented plates, most fungi promoted <i>A</i>. <i>thaliana</i> growth and some could induce the clumped root phenotype. In the compartmented plate experiments, a general induction of plant growth was observed for most other fungi, especially those producing higher biomass, further strengthening the role of a nonspecific mechanism, such as CO₂ emission.</p></div

Trichoderma Species Differ in Their Volatile Profiles and in Antagonism Toward Ectomycorrhiza Laccaria bicolor

Fungi of the genus Trichoderma are economically important due to their plant growth- and performance-promoting effects, such as improved nutrient supply, mycoparasitism of plant-pathogens and priming of plant defense. Due to their mycotrophic lifestyle, however, they might also be antagonistic to other plant-beneficial fungi, such as mycorrhiza-forming species. Trichoderma spp. release a high diversity of volatile organic compounds (VOCs), which likely play a decisive role in the inter-species communication. It has been shown that Trichoderma VOCs can inhibit growth of some plant pathogens, but their inhibition potentials during early interactions with mutualistic fungi remain unknown. Laccaria bicolor is a common ectomycorrhizal fungus which in symbiotic relationship is well known to facilitate plant performance. Here, we investigated the VOC profiles of three strains of Trichoderma species, Trichoderma harzianum, Trichoderma Hamatum, and Trichoderma velutinum, as well as L. bicolor by stir bar sorptive extraction and gas chromatography – mass spectrometry (SBSE-GC-MS). We further examined the fungal performance and the VOC emission profiles during confrontation of the Trichoderma species with L. bicolor in different co-cultivation scenarios. The VOC profiles of the three Trichoderma species were highly species-dependent. T. harzianum was the strongest VOC emitter with the most diverse compound pattern, followed by T. hamatum and T. velutinum. Co-cultivation of Trichoderma spp. and L. bicolor altered the VOC emission patterns dramatically in some scenarios. The co-cultivations also revealed contact degree-dependent inhibition of one of the fungal partners. Trichoderma growth was at least partially inhibited when sharing the same headspace with L. bicolor. In direct contact between both mycelia, however, L. bicolor growth was impaired, indicating that Trichoderma and L. bicolor apply different effectors when defending their territory. Multivariate analysis demonstrated that all examined individual fungal species in axenic cultures, as well as their co-cultivations were characterized by a distinct VOC emission pattern. The results underline the importance of VOCs in fungal interactions and reveal unexpected adjustability of the VOC emissions according to the specific biotic environments

Noninvasive Phenotyping of Plant–Pathogen Interaction: Consecutive In Situ Imaging of Fluorescing Pseudomonas syringae, Plant Phenolic Fluorescence, and Chlorophyll Fluorescence in Arabidopsis Leaves

Plant–pathogen interactions have been widely studied, but mostly from the site of the plant secondary defense. Less is known about the effects of pathogen infection on plant primary metabolism. The possibility to transform a fluorescing protein into prokaryotes is a promising phenotyping tool to follow a bacterial infection in plants in a noninvasive manner. In the present study, virulent and avirulent Pseudomonas syringae strains were transformed with green fluorescent protein (GFP) to follow the spread of bacteria in vivo by imaging Pulse-Amplitude-Modulation (PAM) fluorescence and conventional binocular microscopy. The combination of various wavelengths and filters allowed simultaneous detection of GFP-transformed bacteria, PAM chlorophyll fluorescence, and phenolic fluorescence from pathogen-infected plant leaves. The results show that fluorescence imaging allows spatiotemporal monitoring of pathogen spread as well as phenolic and chlorophyll fluorescence in situ, thus providing a novel means to study complex plant–pathogen interactions and relate the responses of primary and secondary metabolism to pathogen spread and multiplication. The study establishes a deeper understanding of imaging data and their implementation into disease screening

Volatile profiles of fungi : chemotyping of species and ecological functions

Fungi emit a large spectrum of volatile organic compounds (VOCs). In the present study, we characterized and compared the odor profiles of ectomycorrhizal (EM), pathogenic and saprophytic fungal species with the aim to use these patterns as a chemotyping tool. Volatiles were collected from the headspace of eight fungal species including nine strains (four EM, three pathogens and two saprophytes) using the stir bar sorptive extraction method and analyzed by gas chromatography – mass spectrometry (GC–MS). After removal of VOCs released from the growth system, 54 VOCs were detected including 15 novel compounds not reported in fungi before. Principle component and cluster analyses revealed that fungal species differ in their odor profiles, particularly in the pattern of sesquiterpenes. The functional groups and species could be chemotyped by using their specific emission patterns. The different ecological groups could be predicted with probabilities of 90–99%, whereas for the individual species the probabilities varied between 55% and 83%. This study strongly supports the concept that the profiling of volatile compounds can be used for non-invasive identification of different functional fungal groups

Isoprene emission by poplar is not important for the feeding behaviour of poplar leaf beetles

Background : Chrysomela populi (poplar leaf beetle) is a common herbivore in poplar plantations whose infestation causes major economic losses. Because plant volatiles act as infochemicals, we tested whether isoprene, the main volatile organic compound (VOC) produced by poplars (Populus x canescens), affects the performance of C. populi employing isoprene emitting (IE) and transgenic isoprene non-emitting (NE) plants. Our hypothesis was that isoprene is sensed and affects beetle orientation or that the lack of isoprene affects plant VOC profiles and metabolome with consequences for C. populi feeding. Results : Electroantennographic analysis revealed that C. populi can detect higher terpenes, but not isoprene. In accordance to the inability to detect isoprene, C. populi showed no clear preference for IE or NE poplar genotypes in the choice experiments, however, the beetles consumed a little bit less leaf mass and laid fewer eggs on NE poplar trees in field experiments. Slight differences in the profiles of volatile terpenoids between IE and NE genotypes were detected by gas chromatography - mass spectrometry. Non-targeted metabolomics analysis by Fourier Transform Ion Cyclotron Resonance Mass Spectrometer revealed genotype-, time- and herbivore feeding-dependent metabolic changes both in the infested and adjacent undamaged leaves under field conditions. Conclusions : We show for the first time that C. populi is unable to sense isoprene. The detected minor differences in insect feeding in choice experiments and field bioassays may be related to the revealed changes in leaf volatile emission and metabolite composition between the IE and NE poplars. Overall our results indicate that lacking isoprene emission is of minor importance for C. populi herbivory under natural conditions, and that the lack of isoprene is not expected to change the economic losses in poplar plantations caused by C. populi infestation

Novel <em>Pseudomonas</em> sp. SCA7 promotes plant growth in two plant families and induces systemic resistance in <em>Arabidopsis thaliana</em>.

Pseudomonas sp. SCA7, characterized in this study, was isolated from roots of the bread wheat Triticum aestivum. Sequencing and annotation of the complete SCA7 genome revealed that it represents a potential new Pseudomonas sp. with a remarkable repertoire of plant beneficial functions. In vitro and in planta experiments with the reference dicot plant A. thaliana and the original monocot host T. aestivum were conducted to identify the functional properties of SCA7. The isolate was able to colonize roots, modify root architecture, and promote growth in A. thaliana. Moreover, the isolate increased plant fresh weight in T. aestivum under unchallenged conditions. Gene expression analysis of SCA7-inoculated A. thaliana indicated a role of SCA7 in nutrient uptake and priming of plants. Moreover, confrontational assays of SCA7 with fungal and bacterial plant pathogens revealed growth restriction of the pathogens by SCA7 in direct as well as indirect contact. The latter indicated involvement of microbial volatile organic compounds (mVOCs) in this interaction. Gas chromatography-mass spectrometry (GC-MS) analyses revealed 1-undecene as the major mVOC, and octanal and 1,4-undecadiene as minor abundant compounds in the emission pattern of SCA7. Additionally, SCA7 enhanced resistance of A. thaliana against infection with the plant pathogen Pseudomonas syringae pv. tomato DC3000. In line with these results, SA- and JA/ET-related gene expression in A. thaliana during infection with Pst DC3000 was upregulated upon treatment with SCA7, indicating the ability of SCA7 to induce systemic resistance. The thorough characterization of the novel Pseudomonas sp. SCA7 showed a remarkable genomic and functional potential of plant beneficial traits, rendering it a promising candidate for application as a biocontrol or a biostimulation agent

<i>O</i>. <i>maius—A</i>. <i>thaliana</i> co-cultivation experiments in the tripartite plate system.

<p>(a) Control plants and plant-fungus co-culture 15 days after inoculation; (b) same as in (a) but plates were added with a VOC trap (activated charcoal, AC) in the third compartment; (c) same as in (a) but plates were added with a CO₂ trap [Ba(OH)_2*8H₂O, B] together with two dental rolls in the third compartment; (d) plant biomass measurements (roots—grey bars—and aboveground portions—open bars) in the presence/absence of the fungus and of the trap compounds. Note the strong plant biomass increase in the presence of <i>O</i>. <i>maius</i> in all the conditions tested. Bars represent the mean ±SD, n = 5 (each biological replicate represents the total biomass of 3 <i>A</i>. <i>thaliana</i> seedlings grown in an individual plate). Statistically significant differences (P<0.05) among treatments are indicated by different letters above the bars.</p

<i>A</i>. <i>thaliana</i> development in the presence of <i>O</i>. <i>maius</i> WT (Om) and of three <i>O</i>. <i>maius</i> mutants (OmΔGOGAT; OmΔMFS; OmΔSOD).

<p>(a) Control plants (C) and plant-fungus co-cultures 30 days after inoculation (all pictures were taken at the same magnification) (b) Measurement of auxin quantity released in the culture medium by <i>O</i>. <i>maius</i> WT and by the three <i>O</i>. <i>maius</i> mutants, using the Salkowski reaction [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168236#pone.0168236.ref046" target="_blank">46</a>]. Auxin quantity measured was normalized to the mycelium biomass. Bars represent the mean ±SD, n = 3 (each biological replicate represents the total biomass of 5 <i>A</i>. <i>thaliana</i> seedlings grown in an individual plate). Statistically significant differences (P<0.05) among treatments are indicated by different letters above the bars.</p