Microbial Consortia for Effective Biocontrol of Root and Foliar Diseases in Tomato.

The use of beneficial microorganisms for the biological control of plant diseases and pests has emerged as a viable alternative to chemical pesticides in agriculture. Traditionally, microbe-based biocontrol strategies for crop protection relied on the application of single microorganisms. However, the design of microbial consortia for improving the reliability of current biological control practices is now a major trend in biotechnology, and it is already being exploited commercially in the context of sustainable agriculture. In the present study, exploiting the microbial library of the biocontrol company Koppert Biological Systems, we designed microbial consortia composed of carefully selected, well-characterized beneficial bacteria and fungi displaying diverse biocontrol modes of action. We compared their ability to control shoot and root pathogens when applied separately or in combination as microbial consortia, and across different application strategies that imply direct microbial antagonism or induced systemic plant resistance. We hypothesized that consortia will be more versatile than the single strains, displaying an extended functionality, as they will be able to control a wider range of plant diseases through diverse mechanisms and application methods. Our results confirmed our hypothesis, revealing that while different individual microorganisms were the most effective in controlling the root pathogen Fusarium oxysporum or the foliar pathogen Botrytis cinerea in tomato, the consortia showed an extended functionality, effectively controlling both pathogens under any of the application schemes, always reaching the same protection levels as the best performing single strains. Our findings illustrate the potential of microbial consortia, composed of carefully selected and compatible beneficial microorganisms, including bacteria and fungi, for the development of stable and versatile biological control products for plant protection against a wider range of diseases.

Community-level interactions between plants and soil biota during range expansion.

Plant species that expand their range in response to current climate change will encounter soil communities that may hinder, allow or even facilitate plant performance. It has been shown repeatedly for plant species originating from other continents that these plants are less hampered by soil communities from the new than from the original range. However, information about the interactions between intra-continental range expanders and soil communities is sparse, especially at community level.Here we used a plant-soil feedback experiment approach to examine if the interactions between range expanders and soil communities change during range expansion. We grew communities of range-expanding and native plant species with soil communities originating from the original and new range of range expanders. In these conditioned soils, we determined the composition of fungi and bacteria by high-throughput amplicon sequencing of the ITS region and the 16S rRNA gene respectively. Nematode community composition was determined by microscopy-based morphological identification. Then we tested how these soil communities influence the growth of subsequent communities of range expanders and natives.We found that after the conditioning phase soil bacterial, fungal and nematode communities differed by origin and by conditioning plant communities. Despite differences in bacterial, fungal and nematode communities between original and new range, soil origin did not influence the biomass production of plant communities. Both native and range expanding plant communities produced most above-ground biomass in soils that were conditioned by plant communities distantly related to them. Synthesis. Communities of range-expanding plant species shape specific soil communities in both original and new range soil. Plant-soil interactions of range expanders in communities can be similar to the ones of their closely related native plant species.

Latitudinal variation in soil nematode communities under climate warming-related range-expanding and native plants.

Current climate change has led to latitudinal and altitudinal range expansions of numerous species. During such range expansions, plant species are expected to experience changes in interactions with other organisms, especially with belowground biota that have a limited dispersal capacity. Nematodes form a key component of the belowground food web as they include bacterivores, fungivores, omnivores and root herbivores. However, their community composition under climate change-driven intracontinental range-expanding plants has been studied almost exclusively under controlled conditions, whereas little is known about actual patterns in the field. Here, we use novel molecular sequencing techniques combined with morphological quantification in order to examine nematode communities in the rhizospheres of four range-expanding and four congeneric native species along a 2,000 km latitudinal transect from South-Eastern to North-Western Europe. We tested the hypotheses that latitudinal shifts in nematode community composition are stronger in range-expanding plant species than in congeneric natives and that in their new range, range-expanding plant species accumulate fewest root-feeding nematodes. Our results show latitudinal variation in nematode community composition of both range expanders and native plant species, while operational taxonomic unit richness remained the same across ranges. Therefore, range-expanding plant species face different nematode communities at higher latitudes, but this is also the case for widespread native plant species. Only one of the four range-expanding plant species showed a stronger shift in nematode community composition than its congeneric native and accumulated fewer root-feeding nematodes in its new range. We conclude that variation in nematode community composition with increasing latitude occurs for both range-expanding and native plant species and that some range-expanding plant species may become released from root-feeding nematodes in the new range.

Range-expansion effects on the belowground plant microbiome.

Plant range expansion is occurring at a rapid pace, largely in response to human-induced climate warming. Although the movement of plants along latitudinal and altitudinal gradients is well-documented, effects on belowground microbial communities remain largely unknown. Furthermore, for range expansion, not all plant species are equal: in a new range, the relatedness between range-expanding plant species and native flora can influence plant-microorganism interactions. Here we use a latitudinal gradient spanning 3,000 km across Europe to examine bacterial and fungal communities in the rhizosphere and surrounding soils of range-expanding plant species. We selected range-expanding plants with and without congeneric native species in the new range and, as a control, the congeneric native species, totalling 382 plant individuals collected across Europe. In general, the status of a plant as a range-expanding plant was a weak predictor of the composition of bacterial and fungal communities. However, microbial communities of range-expanding plant species became more similar to each other further from their original range. Range-expanding plants that were unrelated to the native community also experienced a decrease in the ratio of plant pathogens to symbionts, giving weak support to the enemy release hypothesis. Even at a continental scale, the effects of plant range expansion on the belowground microbiome are detectable, although changes to specific taxa remain difficult to decipher.

Removal of soil biota alters soil feedback effects on plant growth and defense chemistry.

We examined how the removal of soil biota affects plant-soil feedback (PSF) and defense chemistry of Jacobaea vulgaris, an outbreak plant species in Europe containing the defense compounds pyrrolizidine alkaloids (PAs). Macrofauna and mesofauna, as well as fungi and bacteria, were removed size selectively from unplanted soil or soil planted with J. vulgaris exposed or not to above- or belowground insect herbivores. Wet-sieved fractions, using 1000-, 20-, 5- and 0.2-µm mesh sizes, were added to sterilized soil and new plants were grown. Sieving treatments were verified by molecular analysis of the inocula. In the feedback phase, plant biomass was lowest in soils with 1000- and 20-µm inocula, and soils conditioned with plants gave more negative feedback than without plants. Remarkably, part of this negative PSF effect remained present in the 0.2-µm inoculum where no bacteria were present. PA concentration and composition of plants with 1000- or 20-µm inocula differed from those with 5- or 0.2-µm inocula, but only if soils had been conditioned by undamaged plants or plants damaged by aboveground herbivores. These effects correlated with leaf hyperspectral reflectance. We conclude that size-selective removal of soil biota altered PSFs, but that these PSFs were also influenced by herbivory during the conditioning phase.

Nematode community responses to range-expanding and native plant communities in original and new range soils.

Many plant species expand their range to higher latitudes in response to climate change. However, it is poorly understood how biotic interactions in the new range differ from interactions in the original range. Here, in a mesocosm experiment, we analyze nematode community responses in original and new range soils to plant communities with either (a) species native in both the original and new range, (b) range-expanding species related to these natives (related range expanders), or (c) range expanders without native congeneric species in the new range (unrelated range expanders). We hypothesized that nematode community shifts between ranges are strongest for unrelated range expanders and minimal for plant species that are native in both ranges. As a part of these community shifts, we hypothesized that range expanders, but not natives, would accumulate fewer root-feeding nematodes in their new range compared to their original range. Analyses of responses of nematodes from both original and new ranges and comparison between range expanders with and without close relatives have not been made before. Our study reveals that none of the plant communities experienced evident nematode community shifts between the original and new range. However, in soils from the new range, root-feeding nematode communities of natives and related range expanders were more similar than in soils from the original range, whereas the nematode community of unrelated range expanders was distinct from the communities of natives and related range expanders in soils from both ranges. The abundances of root-feeding nematodes were comparable between the original and new range for all plant communities. Unexpectedly, unrelated range expanders overall accumulated most root-feeding nematodes, whereas related range expanders accumulated fewest. We conclude that nematode communities associated with native and range-expanding plant species differ between the original and the new range, but that range-expanding plant species do not accumulate fewer root-feeding nematodes in their new than in their original range.

Seed and Root Endophytic Fungi in a Range Expanding and a Related Plant Species.

Climate change is accelerating the spread of plants and their associated species to new ranges. The differences in range shift capacity of the various types of species may disrupt long-term co-evolved relationships especially those belowground, however, this may be less so for seed-borne endophytic microbes. We collected seeds and soil of the range-expanding Centaurea stoebe and the congeneric Centaurea jacea from three populations growing in Slovenia (native range of both Centaurea species) and the Netherlands (expanded range of C. stoebe, native range of C. jacea). We isolated and identified endophytic fungi directly from seeds, as well as from roots of the plants grown in Slovenian, Dutch or sterilized soil to compare fungal endophyte composition. Furthermore, we investigated whether C. stoebe hosts a reduced community composition of endophytes in the expanded range due to release from plant-species specific fungi while endophyte communities in C. jacea in both ranges are similar. We cultivated 46 unique and phylogenetically diverse endophytes. A majority of the seed endophytes resembled potential pathogens, while most root endophytes were not likely to be pathogenic. Only one endophyte was found in both roots and seeds, but was isolated from different plant species. Unexpectedly, seed endophyte diversity of southern C. stoebe populations was lower than of populations from the north, while the seed endophyte community composition of northern C. stoebe populations was significantly different southern C. stoebe as well as northern and southern C. jacea populations. Root endophyte diversity was considerably lower in C. stoebe than in C. jacea independent of plant and soil origin, but this difference disappeared when plants were grown in sterile soils. We conclude that the community composition of fungal endophytes not only differs between related plant species but also between populations of plants that expand their range compared to their native habitat. Our results suggest that fungal endophytes of two Centaurea species are not able to systemically infect plants. We highlight that endophytes remain poorly studied and further work should investigate the functional importance of endophytes.

Herbivory and dominance shifts among exotic and congeneric native plant species during plant community establishment.

Invasive exotic plant species often have fewer natural enemies and suffer less damage from herbivores in their new range than genetically or functionally related species that are native to that area. Although we might expect that having fewer enemies would promote the invasiveness of the introduced exotic plant species due to reduced enemy exposure, few studies have actually analyzed the ecological consequences of this situation in the field. Here, we examined how exposure to aboveground herbivores influences shifts in dominance among exotic and phylogenetically related native plant species in a riparian ecosystem during early establishment of invaded communities. We planted ten plant communities each consisting of three individuals of each of six exotic plant species as well as six phylogenetically related natives. Exotic plant species were selected based on a rapid recent increase in regional abundance, the presence of a congeneric native species, and their co-occurrence in the riparian ecosystem. All plant communities were covered by tents with insect mesh. Five tents were open on the leeward side to allow herbivory. The other five tents were completely closed in order to exclude insects and vertebrates. Herbivory reduced aboveground biomass by half and influenced which of the plant species dominated the establishing communities. Exposure to herbivory did not reduce the total biomass of natives more than that of exotics, so aboveground herbivory did not selectively enhance exotics during this early stage of plant community development. Effects of herbivores on plant biomass depended on plant species or genus but not on plant status (i.e., exotic vs native). Thus, aboveground herbivory did not promote the dominance of exotic plant species during early establishment of the phylogenetically balanced plant communities.

Plant diversity and identity effects on predatory nematodes and their prey.

There is considerable evidence that both plant diversity and plant identity can influence the level of predation and predator abundance aboveground. However, how the level of predation in the soil and the abundance of predatory soil fauna are related to plant diversity and identity remains largely unknown. In a biodiversity field experiment, we examined the effects of plant diversity and identity on the infectivity of entomopathogenic nematodes (EPNs, Heterorhabditis and Steinernema spp.), which prey on soil arthropods, and abundance of carnivorous non-EPNs, which are predators of other nematode groups. To obtain a comprehensive view of the potential prey/food availability, we also quantified the abundance of soil insects and nonpredatory nematodes and the root biomass in the experimental plots. We used structural equation modeling (SEM) to investigate possible pathways by which plant diversity and identity may affect EPN infectivity and the abundance of carnivorous non-EPNs. Heterorhabditis spp. infectivity and the abundance of carnivorous non-EPNs were not directly related to plant diversity or the proportion of legumes, grasses and forbs in the plant community. However, Steinernema spp. infectivity was higher in monocultures of Festuca rubra and Trifolium pratense than in monocultures of the other six plant species. SEM revealed that legumes positively affected Steinernema infectivity, whereas plant diversity indirectly affected the infectivity of HeterorhabditisEPNs via effects on the abundance of soil insects. The abundance of prey (soil insects and root-feeding, bacterivorous, and fungivorous nematodes) increased with higher plant diversity. The abundance of prey nematodes was also positively affected by legumes. These plant community effects could not be explained by changes in root biomass. Our results show that plant diversity and identity effects on belowground biota (particularly soil nematode community) can differ between organisms that belong to the same feeding guild and that generalizations about plant diversity effects on soil organisms should be made with great caution.

Effects of root herbivory on pyrrolizidine alkaloid content and aboveground plant-herbivore-parasitoid interactions in Jacobaea vulgaris.

The importance of root herbivory is increasingly recognized in ecological studies, and the effects of root herbivory on plant growth, chemistry, and performance of aboveground herbivores have been relatively well studied. However, how belowground herbivory by root feeding insects affects aboveground parasitoid development is largely unknown. In this study, we examined the effects of root herbivory by wireworms (Agriotes lineatus) on the expression of primary and secondary compounds in the leaves and roots of ragwort (Jacobaea vulgaris). We also studied the effects of root herbivory on the performance of a generalist aboveground herbivore, Mamestra brassicae and its parasitoid Microplitis mediator. In contrast to what most other studies have reported, root herbivory in J. vulgaris had a strong negative effect on the total concentration of pyrrolizidine alkaloids (PAs) in shoot tissues. The composition of PAs in the shoots also changed after root herbivory. In particular, the concentration of less toxic N-oxide PAs decreased. There was no significant effect of root herbivory on PA composition and concentration in the roots. Although the concentration of PA in the leaves decreased, M. brassicae tended to grow slower on the plants exposed to root herbivory. Parasitoid performance was not affected by root herbivory, but parasitoids developed faster when the concentration of jacobine-type PAs in the foliage was higher. These results point at a putative role of individual PAs in multitrophic interactions and emphasize that generalizations about aboveground-belowground effects should be made with great caution.

Legacy effects of aboveground-belowground interactions.

Root herbivory can greatly affect the performance of aboveground insects via changes in plant chemistry. These interactions have been studied extensively in experiments where aboveground and belowground insects were feeding on the same plant. However, little is known about how aboveground and belowground organisms interact when they feed on plant individuals that grow after each other in the same soil. We show that feeding by aboveground and belowground insect herbivores on ragwort (Jacobaea vulgaris) plants exert unique soil legacy effects, via herbivore-induced changes in the composition of soil fungi. These changes in the soil biota induced by aboveground and belowground herbivores of preceding plants greatly influenced the pyrrolizidine alkaloid content, biomass and aboveground multitrophic interactions of succeeding plants. We conclude that plant-mediated interactions between aboveground and belowground insects are also important when they do not feed simultaneously on the same plant.

MAO inhibitors: risks, benefits, and lore.

Monoamine oxidase (MAO) inhibitors were the first antidepressants introduced, but their use has dwindled because of their reported side effects, their food and drug interactions, and the introduction of other classes of agents. However, interest in MAO inhibitors is reviving. Here, we discuss their use, risks, and benefits in clinical medicine.

Specificity determinants of a novel Nck interaction with the juxtamembrane domain of the epidermal growth factor receptor.

Nck is a ubiquitously expressed adaptor protein containing Src homology 2 (SH2) and Src homology 3 (SH3) domains. It integrates downstream effector proteins with cell membrane receptors, such as the epidermal growth factor receptor (EGFR). EGFR plays a critical role in cellular proliferation and differentiation. The 45-residue juxtamembrane domain of EGFR (JM), located between the transmembrane and kinase domains, regulates receptor activation and trafficking to the basolateral membrane of polarized epithelia through a proline-rich motif that resembles a consensus SH3 domain binding site. We demonstrate here that the JM region can bind to Nck, showing a notable binding preference for the second SH3 domain. To elucidate the structural determinants for this interaction, we have determined the NMR solution structures of both the first and second Nck SH3 domains (Nck1-1 and Nck1-2). These domains adopt a canonical SH3 beta-barrel-like fold, containing five antiparallel strands separated by three loop regions and one 3 10-helical turn. Chemical shift perturbation studies have identified the residues that form the binding cleft of Nck1-2, which are primarily located in the RT and n-Src loops. JM binds to Nck1-2 with an affinity of approximately 80 microM through a positively charged sequence near the N-terminus, as opposed to the polyproline sequence. The two Nck SH3 domains exhibit both steric and electrostatic differences in their RT-Src and n-Src loops, and a model of the Nck1-2 domain complexed with the JM highlights the factors that define the putative binding mode for this ligand.