Multitrophic Interactions Research Articles

Challenges and advances in the study of latitudinal gradients in multitrophic interactions, with a focus on consumer specialization.

Increases in data availability and geographic ranges of studies have allowed for more thorough tests of latitudinal gradients in trophic interactions, with numerous recent studies testing hypotheses that strength of interactions, herbivory, plant chemical defense, and dietary specialization all increase with decreasing latitude. We review the issues surrounding these latitudinal gradients, discuss some methodological challenges, and provide some caveats relevant to inferences from existing approaches. To examine some potential issues with studies on latitudinal gradients in dietary specialization, we simulate a latitudinal gradient of communities that increase in diversity and specialization towards the equator then test the power of different sampling designs for detecting the gradient. Based on this simple simulation, as well as apparent incongruities in the literature, we conclude that subtle differences in sampling design can be responsible for failure to detect existing gradients. Despite calls for rejecting some latitudinal gradient hypotheses, it is clear that a great deal of careful research remains to determine important correlates of the well-established latitudinal gradient in diversity. In particular, future studies should focus on replicated gradients, greater emphasis on continuous sampling, and use of taxonomic controls that allow for meaningful analyses across latitudes.

Distribution and Variation of Bacterial Endosymbiont and "Candidatus Liberibacter asiaticus" Titer in the Huanglongbing Insect Vector, Diaphorina citri Kuwayama.

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is an economic insect pest in most citrus-growing regions and the vector of 'Candidatus Liberibacter asiaticus' (CLas), one of at least three known bacteria associated with Huanglongbing (HLB or citrus greening disease). D. citri harbors bacterial endosymbionts, including Wolbachia pipientis (strain Wolbachia wDi), 'Candidatus Carsonella ruddii,' and 'Candidatus Profftella armatura.' Many important functions of these bacteria can be inferred from their genome sequences, but their interactions with each other, CLas, and their D. citri host are poorly understood. In the present study, the titers of the endosymbionts in different tissues, in each sex, and in insects reared on healthy citrus (referred to as unexposed) and CLas-infected citrus (referred to as CLas-exposed) D. citri were investigated using real-time, quantitative PCR (qPCR) using two different quantitative approaches. Wolbachia and CLas were detected in all insect tissues. The titer of Wolbachia was higher in heads of CLas-exposed males as compared to unexposed males. In males and females, Wolbachia titer was highest in the Malpighian tubules. The highest titer of CLas was observed in the gut. Profftella and Carsonella titers were significantly reduced in the bacteriome of CLas-exposed males compared with that of unexposed males, but this effect was not observed in females. In ovaries of CLas-exposed females, the Profftella and Carsonella titers were increased as compared to non-exposed females. CLas appeared to influence the overall levels of the symbionts but did not drastically perturb the overall microbial community structure. In all the assessed tissues, CLas titer in males was significantly higher than that of females using absolute quantification. These data provide a better understanding of multi-trophic interactions regulating symbiont dynamics in the HLB pathosystem.

Potential impact of climate change on whiteflies and implications for the spread of vectored viruses

Whiteflies (Hemiptera: Aleyrodidae) are important insect pests causing serious damage to plants and transmitting hundreds of plant viruses. Climate change is expected to influence life history and trophic interactions among plants, whiteflies and their natural enemies. Here, we review the potential impacts of climate change on whiteflies and the likely consequences for agricultural systems. This review concludes that while climatic stress tends to negatively affect life history traits, the effects differ with the tolerance of the whiteflies and the amount of stress experienced. Whiteflies also differ in their adaptability. Better adapted species will likely experience increased distribution and abundance provided their tolerance limits are not exceeded, while species with lower tolerance and adaptation limits will suffer reduced fitness, which will have overall effects on their distribution and abundance in space and time. The majority of methods used to control whiteflies will still be useful especially if complementary methods are combined for maximum efficacy. Parasitism and predation rates of whitefly natural enemies could increase with temperature within the optimum ranges of the natural enemies, although life history traits and population growth potential are generally maximised below 30 °C. Changes in climatic suitability modifying the distribution and abundance of whiteflies, and environmental suitability for plant viruses, will likely affect epidemics of viral diseases. Greater efforts are required to improve understanding of the complex effects of climate change on multi-species and multi-trophic interactions in the agro-ecological systems inhabited by whiteflies, and to use this new knowledge to develop robust and climate-smart management strategies.

Multitrophic interactions in a geothermal setting: Arthropod borings, actinomycetes, fungi and fungal-like microorganisms in a decomposing conifer wood from the Jurassic of Patagonia

A permineralized araucarioid conifer stem, preserved in state of partial decay, is described from the Middle to Late Jurassic Chon-Aike Formation (Bahía Laura Group) at Laguna Flecha Negra, in the Deseado Massif, Santa Cruz Province, Argentina. The tree-stem, which comprises periderm to the secondary xylem, shows evidence of interactions with xylophagous borers, fungi, actinomycetes and other fungal-like microorganisms. In the secondary phloem, it displays rows of traumatic resin ducts, which host abundant mycelium comprising very thin hyphae differentiated into spore-producing structures comparable to actinomycete bacteria and spherical fungal-like resting structures. In the cortical and phloem regions, there are cell groups with structural changes, which appear variably degraded by fungi. The secondary xylem displays decay of cellulose and lignified components, at various spatial scales, comparable with fungal decay patterns in modern wood. Further septate fungal filaments, sometimes bearing clamp connections are associated with decayed regions. The degraded secondary xylem shows circular to oval borings filled with frass containing degraded tracheids, comparable to those bored by different wood boring beetles. Development of traumatic resin ducts in the phloem could have been triggered by penetration of the living stem by these xylophagous arthropods. The association of the different components present suggests pathogenic and saprotrophic interactions promoting decomposition of the stem, and represents a rarely-documented example of multitrophic biological interactions involving several different biological groups in Jurassic terrestrial paleoecosystems.

Multitrophic Interaction between Cucumber Moth Diaphania indica Saunders, (Lepidoptera: Crambidae) and Its Natural Enemies

Multitrophic interactions provide an information on how plant-insects interact in the field mediated by biotic and/or abiotic factors. This study measures the effect ofApanteles taragamae parasitism toward Diaphania indica, and the rate of A. taragamae themselves being parasitized by hyperparasitoid. The insects were collected periodically at 3, 4, and 5 weeks after planting from cucumber fields in Bogor, Indonesia during the growing season in 2014 and 2015. Sampled insects were reared in the lab and the incidence of parasitism and hyperparasitism was calculated. The relationship between D. indica, its primary parasitoids and hyperparasitoids, was also analyzed. Older cucumber plants harbored larger populations of D. indica and higher rates of primary parasitism by A. taragamae. Other D. indica parasitoids, Ichneumon sp., and Elasmus sp., showed lower parasitism rates. The hyperparasitoid species found in greatest abundance were Stictopisthus sp. and Ceraphron sp., with other hyperparasitoids present i.e., Tetrastichus sp., Eurytoma sp., Orasemasp., and Brachymeria sp. The overall population of D. indica found in Bogor was low. A. taragamae have a significant suppressive effect on the population of D. indica. But, the presence of hyperparasitoids may disrupt parasitization of D. indica by A. taragamae thereby reducing the suppressive effect.

Effects of Dietary β-Carotene on the Melanization Response and Growth Rate of Trichoplusia ni (Lepidoptera: Noctuidae).

Animals rely on carotenoids as fundamental precursors for hormones and antioxidants, and animals must acquire carotenoids from their diet. Previous research has shown that insects often absorb carotenoids in amounts proportional to those in their diet, and that carotenoids play key roles in multitrophic interactions. The consumption of diets that provide high levels of antioxidant compounds is associated with high levels of immune responses; however, it is unknown whether individual carotenoids directly influence immune response. Here, the objective of this study was to examine the effect of the carotenoid β-carotene on melanization, a measure of immune response, and growth rate of Trichoplusia ni Hübner (Lepidoptera: Noctuidae). To fulfill the objective, a low, medium, and high concentration of β-carotene, representing the range found in typical host plants, were mixed in an artificial diet, and immune response and growth rate were assessed in fifth instar larvae. Immune response was induced by injection of chromatography beads in to the abdomen of the larvae, and percent melanization was measured after injection. Melanization was greatest when larvae were reared on high β-carotene diets. Mass was measured at 5 and 10 d to assess growth rate. Larvae reared on high β-carotene diets initially gained little mass, but after 10 d larvae reared on no and high β-carotene diets were larger than those reared on other diets. This research has shown that β-carotene has the potential to influence the immune response and growth rate of T. ni.

Biodiversity-based options for arable weed management. A review

In the context of a shift towards pesticide reduction in arable farming, weed management remains a challenging issue. Integrated Weed Management currently recommends agronomic practices for weed control, but it does not integrate the use of biodiversity-based options, enhancing the biological regulation of weeds. Here, wereview existing knowledge related to three potentially beneficial interactions, of crop–weed competition, weed seed granivory, and weed interactions with pathogenic fungi. Our main finding are the following : (1) promoting cropped plant–weed competition by manipulating cropped cover could greatly contribute to weed reduction ; (2) weed seed granivory by invertebrates can significantly lower weed emergence, although this effect can be highly variable because seed predation is embedded within complex multitrophic interactions that are to date not fully understood ; (3) a wide range of fungi are pathogenic to various stages of weed development, but strain efficacy in field trials does not often match that in controlled conditions. We present a framework that superimposes biodiversity-based options for weed biocontrol on a classical Integrated Weed Management system. We then describe the current state of knowledge on interactions between agronomic practices and the organisms at play and between the different biological components of the system. We argue that further advances in our understanding of biodiversity-based options and their performance for weed biocontrol will require farm-scale experimental trials.

Correction: The Influence of Host Plant Extrafloral Nectaries on Multitrophic Interactions: An Experimental Investigation.

[This corrects the article DOI: 10.1371/journal.pone.0138157.].

Witch's Broom Disease of Lime (Candidatus Phytoplasma aurantifolia): Identifying High-Risk Areas by Climatic Mapping.

Biological invasions of vectorborne diseases can be devastating. Bioclimatic modeling provides an opportunity to assess and predict areas at risk from complex multitrophic interactions of pathogens, highlighting areas in need of increased monitoring effort. Here, we model the distribution of an economically critical vectorborne plant pathogen 'Candidatus Phytoplasma aurantifolia', the etiological agent of Witches' Broom Disease of Lime. This disease is a significant limiting factor on acid lime production (Citrus aurantifolia, Swingle) in the Middle East and threatens its production globally. We found that temperature, humidity, and the vector populations significantly determine disease distribution. Following this, we used bioclimatic modeling to predict potential novel sites of infections. The model outputs identified potential novel sites of infection in the citrus producing regions of Brazil and China. We also used our model to explore sites in Oman where the pathogen may not be infectious, and suggest nurseries be established there. Recent major turbulence in the citrus agricultural economy has highlighted the importance of this work and the need for appropriate and targeted monitoring programs to safeguard lime production.

The Galling Truth: Limited Knowledge of Gall-Associated Volatiles in Multitrophic Interactions.

Galls are the product of enclosed internal herbivory where the gall maker induces a plant structure within which the herbivores complete their development. For successful sustained herbivory, gall makers must (1) suppress the induction of plant defenses in response to herbivory that is usually mediated through the jasmonic acid pathway and involves volatile organic compound (VOC) production, or (2) have mechanisms to cope with herbivory-induced VOCs, or (3) manipulate production of VOCs to their own advantage. Similarly, plants may have mechanisms (1) to avoid VOC suppression or (2) to attract galler enemies such as parasitoids. While research on VOCs involved in plant–herbivore–parasitoid/predator interactions is extensive, this has largely focussed on the impact of piercing, sucking, and chewing external herbivores or their eggs on VOC emissions. Despite the importance of gallers, owing to their damage to many economically valuable plants, the role of volatiles in gall-associated herbivory has been neglected; exceptions include studies on beneficial gallers and their enemies such as those that occur in brood-site pollination mutualisms. This is possibly the consequence of the difficulties inherent with studying internally occurring herbivory. This review examines the evidence for VOCs in galler attraction to host plants, potential VOC suppression by gallers, increased emission from galls and neighboring tissues, attraction of galler enemies, and the role of galler symbionts in VOC production. It suggests a research focus and ways in which studies on galler-associated VOCs can progress from a philatelic approach involving VOC listing toward a more predictive and evolutionary perspective.

Soil Microbial Resources for Improving Fertilizers Efficiency in an Integrated Plant Nutrient Management System.

Tomorrow’s agriculture, challenged by increasing global demand for food, scarcity of arable lands, and resources alongside multiple environment pressures, needs to be managed smartly through sustainable and eco-efficient approaches. Modern agriculture has to be more productive, sustainable, and environmentally friendly. While macronutrients such as nitrogen (N), phosphorus (P), potassium (K), and sulfur (S) supplied by mineral fertilizers are vital to crop production, agriculturally beneficial microorganisms may also contribute directly (i.e., biological N2 fixation, P solubilization, and phytohormone production, etc.) or indirectly (i.e., antimicrobial compounds biosynthesis and elicitation of induced systemic resistance, etc.) to crop improvement and fertilizers efficiency. Microbial-based bioformulations that increase plant performance are greatly needed, and in particular bioformulations that exhibit complementary and synergistic effects with mineral fertilization. Such an integrated soil fertility management strategy has been demonstrated through several controlled and non-controlled experiments, but more efforts have to be made in order to thoroughly understand the multiple functions of beneficial microorganisms within the soil microbial community itself and in interaction with plants and mineral resources. In fact, the combined usage of microbial [i.e., beneficial microorganisms: N2-fixing (NF), P-solubilizing, and P mobilizing, etc.] and mineral resources is an emerging research area that aims to design and develop efficient microbial formulations which are highly compatible with mineral inputs, with positive impacts on both crops and environment. This novel approach is likely to be of a global interest, especially in most N- and P-deficient agro-ecosystems. In this review, we report on the importance of NF bacteria and P solubilizing/mobilizing microbes as well as their interactions with mineral P fertilization in improving crop productivity and fertilizers efficiency. In addition, we shed light on the interactive and synergistic effects that may occur within multi-trophic interactions involving those two microbial groups and positive consequences on plant mineral uptake, crop productivity, and resiliency to environmental constraints. Improving use of mineral nutrients is a must to securing higher yield and productivity in a sustainable manner, therefore continuously designing, developing and testing innovative integrated plant nutrient management systems based on relevant biological resources (crops and microorganisms) is highly required.

Long-term farming systems modulate multi-trophic responses

Soil microbiome and multi-trophic relationships are essential for the stability and functioning of agroecosystems. However, little is known about how farming systems and alternative methods for controlling plant pathogens modulate microbial communities, soil mesofauna and plant productivity. In this study, we assessed the composition of eukaryotic microbial groups using a high-throughput sequencing approach (18S rRNA gene marker), the populations of parasitic and free-living nematodes, plant productivity and their inter-relationships in long-term conventional and organic farming systems. The diversity of the fungal community increased in the organic farming system compared to the conventional farming system, whereas the diversity of the protist community was similar between the two farming systems. Compared to conventional farming, organic farming increased the population of free-living nematodes and suppressed plant parasitic nematodes belonging to Meloidogynidae and Pratylenchidae. Fungal diversity and community structure appeared to be related to nematode suppression in the system receiving organic fertilizer, which was characterized by component microbial groups known to be involved in the suppression of soil pathogens. Unraveling the microbiome and multi-trophic interactions in different farming systems may permit the management of the soil environment toward more sustainable control of plant pathogens.

Latitudinal variation in soil biota: testing the biotic interaction hypothesis with an invasive plant and a native congener

Soil biota community structure can change with latitude, but the effects of changes on native plants, invasive plants, and their herbivores remain unclear. Here, we examined latitudinal variation in the soil biota community associated with the invasive plant Alternanthera philoxeroides and its native congener A. sessilis, and the effects of soil biota community variation on these plants and the beetle Agasicles hygrophila. We characterized the soil bacterial and fungal communities and root-knot nematodes of plant rhizospheres collected from 22 °N to 36.6 °N in China. Soil biota community structure changed with latitude as a function of climate and soil properties. Root-knot nematode abundance and potential soil fungal pathogen diversity (classified with FUNGuild) decreased with latitude, apparently due to higher soil pH and lower temperatures. A greenhouse experiment and lab bioassay showed native plant mass, seed production, and mass of beetles fed native foliage increased with soil collection latitude. However, there were no latitudinal patterns for the invasive plant. These results suggest that invasive and native plants and, consequently, their herbivores have different responses to latitudinal changes in soil-borne enemies, potentially creating spatial variation in enemy release or biotic resistance. This highlights the importance of linking above- and below-ground multitrophic interactions to explore the role of soil biota in non-native plant invasions with a biogeographic approach.

From plants to nematodes: Serratia grimesii BXF1 genome reveals an adaptation to the modulation of multi-species interactions

Serratia grimesii BXF1 is a bacterium with the ability to modulate the development of several eukaryotic hosts. Strain BXF1 was isolated from the pinewood nematode, Bursaphelenchus xylophilus, the causative agent of pine wilt disease affecting pine forests worldwide. This bacterium potentiates Bursaphelenchus xylophilus reproduction, acts as a beneficial pine endophyte, and possesses fungal and bacterial antagonistic activities, further indicating a complex role in a wide range of trophic relationships. In this work, we describe and analyse the genome sequence of strain BXF1, and discuss several important aspects of its ecological role. Genome analysis indicates the presence of several genes related to the observed production of antagonistic traits, plant growth regulation and the modulation of nematode development. Moreover, most of the BXF1 genes are involved in environmental and genetic information processing, which is consistent with its ability to sense and colonize several niches. The results obtained in this study provide the basis to a better understanding of the role and evolution of strain BXF1 as a mediator of interactions between organisms involved in a complex disease system. These results may also bring new insights into general Serratia and Enterobacteriaceae evolution towards multitrophic interactions.

Trichoderma atroviride, a maize root associated fungus, increases the parasitism rate of the fall armyworm Spodoptera frugiperda by its natural enemy Campoletis sonorensis

In agroecosystems, herbivore insects damage crops, but in the third trophic level, insects can be parasitized by natural enemies. Trichoderma spp. are common soil and root endophytic fungi that have been widely studied due to their capacity to produce antibiotics, parasitize other fungi and compete with deleterious plant microorganisms. The fall armyworm, Spodoptera frugiperda, is a serious pest of numerous crop plants. In the field, Campoletis sonorensis female wasps parasitize S. frugiperda. Considering these scenarios, it is necessary to look for ecological alternatives for S. frugiperda biocontrol. The aim of this work was to investigate whether Trichoderma atroviride associated with maize roots as part of a multitrophic interaction affects the parasitism rate of C. sonorensis on S. frugiperda. We found that larvae from maize plants whose roots were colonized by T. atroviride were stronger parasitized compared with larvae from non-inoculated plants, indicating that the fungus enhanced the natural mechanism of parasitism. This was correlated with the presence of the low-molecular weight compound 6-pentyl-2H-pyran-2-one (6-PP) released by T. atroviride from the belowground. A pharmacological test showed that 6-PP is an airborne signaling molecule that increased the parasitism rate of C. sonorensis on its enemy. This finding provides new insights into the chemical communication between fungi and insects, and likely represents an inter-kingdom signaling molecule. Furthermore, this study shows a novel role for Trichoderma in multitrophic interactions, and its secondary metabolites in pest control management, presenting an alternative for the biocontrol of S. frugiperda using the combination of T. atroviride and C. sonorensis instead of chemical insecticides.

On the development of a predictive functional trait approach for studying terrestrial arthropods.

The characterization of ecological communities with functional traits allows to consider simultaneously the ability of a species to survive and reproduce in an environment, its interactions with other species and its effects on the ecosystem. Functional traits have been studied mainly by plant ecologists, but are increasingly common in the study of other taxa including arthropods. Arthropods represent a group for which a functional trait approach could be highly profitable because of their high diversity, abundance, ubiquity and role in many important ecological processes. This review synthesizes two decades of functional trait research on terrestrial arthropods. We show that while the approach has gained popularity, particularly in the last decade, the absence of clearly postulated hypotheses is a recurrent problem limiting generalization. Furthermore, studied traits are often poorly related to studied functions. To address these problems, we propose a step-by-step protocol to postulate clear hypotheses prior to trait selection and emphasize the need for a common set of more generalizable traits in future studies. Extending the functional trait approach to arthropods opens the door to improving our understanding of interspecific interactions and potential links between response and effect traits. We present the concept of trait-matching with several examples of arthropod traits known to be effective predictors of consumer-resource interactions. The development of a successful functional trait approach for terrestrial arthropods will necessitate an understanding of relevant traits, standardized measurement protocols and open access databases to share this information. Such progress will provide ecologists with a new set of tools to answer broad questions in several fields including the study of community assembly, ecological networks and multitrophic functionality.

Symbiotic polydnavirus and venom reveal parasitoid to its hyperparasitoids

Symbiotic relationships may provide organisms with key innovations that aid in the establishment of new niches. For example, during oviposition, some species of parasitoid wasps, whose larvae develop inside the bodies of other insects, inject polydnaviruses into their hosts. These symbiotic viruses disrupt host immune responses, allowing the parasitoid's progeny to survive. Here we show that symbiotic polydnaviruses also have a downside to the parasitoid's progeny by initiating a multitrophic chain of interactions that reveals the parasitoid larvae to their enemies. These enemies are hyperparasitoids that use the parasitoid progeny as host for their own offspring. We found that the virus and venom injected by the parasitoid during oviposition, but not the parasitoid progeny itself, affected hyperparasitoid attraction toward plant volatiles induced by feeding of parasitized caterpillars. We identified activity of virus-related genes in the caterpillar salivary gland. Moreover, the virus affected the activity of elicitors of salivary origin that induce plant responses to caterpillar feeding. The changes in caterpillar saliva were critical in inducing plant volatiles that are used by hyperparasitoids to locate parasitized caterpillars. Our results show that symbiotic organisms may be key drivers of multitrophic ecological interactions. We anticipate that this phenomenon is widespread in nature, because of the abundance of symbiotic microorganisms across trophic levels in ecological communities. Their role should be more prominently integrated in community ecology to understand organization of natural and managed ecosystems, as well as adaptations of individual organisms that are part of these communities.

Mycorrhizae Alter Toxin Sequestration and Performance of Two Specialist Herbivores

Multitrophic species interactions are shaped by both top-down and bottom-up factors. Belowground symbionts of plants, such as arbuscular mycorrhizal fungi (AMF), can alter the strength of these forces by altering plant phenotype. For example, AMF-mediated changes in foliar toxin and nutrient concentrations may influence herbivore growth and fecundity. In addition, many specialist herbivores sequester toxins from their host plants to resist natural enemies, and the extent of sequestration varies with host plant secondary chemistry. Therefore, by altering plant phenotype, AMF may affect both herbivore performance and their resistance to natural enemies. We examined how inoculation of plants with AMF influences toxin sequestration and performance of two specialist herbivores feeding upon four milkweed species (Asclepias incarnata, A. curassavica, A. latifolia, A. syriaca). We raised aphids (Aphis nerii) and caterpillars (Danaus plexippus) on plants for six days in a fully factorial manipulation of milkweed species and level of AMF inoculation (zero, medium, and high). We then assessed aphid and caterpillar sequestration of toxins (cardenolides) and performance, and measured defensive and nutritive traits of control plants. Aphids and caterpillars sequestered higher concentrations of cardenolides from plants inoculated with AMF across all milkweed species. Aphid per capita growth rates and aphid body mass varied nonlinearly with increasing AMF inoculum availability; across all milkweed species, aphids had the lowest performance under medium levels of AMF availability and highest performance under high AMF availability. In contrast, caterpillar survival varied strongly with AMF availability in a plant species-specific manner, and caterpillar growth was unaffected by AMF. Inoculation with AMF increased foliar cardenolide concentrations consistently among milkweed species, but altered aboveground biomasses and foliar phosphorous concentrations in a plant species-specific fashion. Increased herbivore sequestration of cardenolides followed AMF-mediated increases in foliar cardenolide concentrations. Aphid performance declined with increasing foliar cardenolide concentrations, while caterpillar survival increased with aboveground biomass. Our findings suggest that by altering plant phenotype, the availability of AMF in soil has the potential to influence both top-down (via sequestration) and bottom up (via plant defense and nutrition) forces that operate on herbivores.

Simultaneous exposure of nematophagous fungi, entomopathogenic nematodes and entomopathogenic fungi can modulate belowground insect pest control.

Entomopathogenic nematodes (EPNs) and fungi (EPF) are well known biological control agents (BCAs) against insect pests. Similarly, the nematophagous fungi (NF) are considered good BCA candidates for controlling plant parasitic nematodes. Because NF can employ EPNs as food and interact with EPF, we speculate that the simultaneous application of EPNs and EPF might result in higher insect mortality, whereas the triple species combination with NF will reduce the EPN and EPF activity by predation or inhibition. Here we evaluated single, dual (EPN + EPF, EPF + NF, EPN + NF) and triple (EPN + EPF + NF) combinations of one EPN, Steinernema feltiae (Rhabditida: Steinernematidae), one EPF, Beauveria bassiana (Hypocreales: Clavicipitaceae), and two NF, Arthrobotrys musiformis (Orbiliales: Orbiliaceae) and Purpureocillium lilacinum (Hypocreales: Ophiocordycipitaceae) under laboratory conditions. First, we showed that EPF reduced the growth rate of NF and vice versa when combined in both rich and limiting media, suggesting a negative interaction when combining both fungi. Three different fungal applications (contact with mycelia-conidia, immersion in conidial suspension, and injection of conidial suspension) were tested in single, dual and triple species combinations, evaluating Galleria mellonella (Lepidoptera: Pyralidae) larval mortality and time to kill. When mycelia was presented, the EPF appeared to be the dominant in combined treatments, whereas in immersion exposure was the EPN. In both types of exposure, NF alone did not produce any effect on larvae. However, when A. musiformis was injected, it produced larval mortalities >70% in the same time span as EPN. Overall, additive effects dominated the dual and triple combinations, with the exception of injection method, where synergisms occurred for both NF species combined with EPN + EPF. This study illustrates how differences in species combination and timing of fungal arrival can modulate the action of BCAs when augmented in the soil. Further studies are required to fine-tune these multitrophic interactions to provide successful, sustainable and resilient pest management in agroecosystems.

Intra- and interspecific tree diversity promotes multitrophic plant–Hemiptera–ant interactions in a forest diversity experiment

Interactions between species of different trophic levels have long been recognized as fundamental processes in ecology. Although mounting evidence indicates that plant species diversity (PSD) or plant genetic diversity (PGD) can influence the plant-associated arthropod community, these two fundamental levels of biodiversity are not often manipulated simultaneously to assess their effects on species interactions. We used a large tree diversity experiment (BEF-China), which manipulates PSD and PGD in a crossed design to test individual and combined effects of PSD and PGD on multitrophic interaction networks and interaction partner species richness and occurrence. We focused on two tree species, on which sap-sucking Hemiptera and interacting ant species commonly occur. This tri-trophic interaction can be divided into the antagonistic plant–Hemiptera interaction and the mutualistic Hemiptera–ant interaction, known as trophobioses. Qualitative evaluation of tri-trophic interaction networks at different PSD and PGD combinations showed increased interaction partner redundancy at high PSD and PGD. This was supported by increased Hemiptera species richness at high PSD and PGD. Furthermore, the data indicate higher occurrence of Hemiptera and trophobioses and higher trophobiotic ant species richness with increasing PSD and PGD. As no plant diversity component alone caused an effect we conclude that the combined effect of high PGD and high PSD might be additive. In summary, as plant genetic diversity, especially at low species richness, seems to increase the interaction partner redundancy in interaction networks and the diversity of interacting communities, we suggest that genetic diversity should be considered in forest conservation and restoration programs.