Final Report Summary - ECOLOGY-EPN-FOOD WEB (Molecular and ecological approaches to study soil food webs for enhancing biological control of insect pests and monitoring disturbances) The soil is largely a non–renewable resource with high socio–economic and natural value. Human activities such as agriculture, industry, and urban development are generating pollution worldwide, and degradation of soil is considered to be irreversible on a generational scale. Although the soil sustains high biodiversity involved in complex food webs that may be irreversibly altered by soil physical degradation, only recently has concern by governments and the general public accelerated efforts to achieve sustainable development. Understanding complex interactions that occur in the soil-matrix could provide a model for monitoring soil disturbances as well as optimizing ecological scenarios to favour biological control. However, the cryptic nature of the soil food webs limits their study under natural conditions where these multitrophic interactions occur. To achieve a successful research system requires the application of advanced methods and concepts from disciplines such as molecular biology, applied ecology and statistics. The entomopathogenic nematodes (EPNs) are promising non–chemical alternatives for insect pest control in a number of crops worldwide. These nematodes occur naturally in soils around the world and their efficacy is affected by various biotic and abiotic factors. EPNs enjoy a mutualistic–symbiotic association with enteric -Proteobacteria (Steinernema–Xenorhabdus and Heterorhabditis–Photorhabdus), which confer high virulence against insects. During the last decades, EPNs have been studied intensively from an agronomic perspective; however, at the moment, this nematode-bacteria association is being considered as a model system for advancing research in other disciplines such as soil ecology and symbiosis, exploring new approaches and using tools from other disciplines. Because of the increasing knowledge about the biology and ecology of these nematodes, we selected this group and members of their soil food web to develop a model to evaluate the soil health and to identify environmental conditions that conserve effective EPN communities. The goal of this grant was to develop and employ new molecular tools based on quantitative real–time PCR (qPCR) to study food webs comprising nematodes, fungi and bacteria, and provide relevant information for two approaches, two systems, one important in agronomic research and the other one for conservation and soil health assessment. The specific objectives were 1) develop a qPCR assay with species–specific primers and TaqMan® probes for organisms in soil food webs; 2) evaluate edaphic conditions in citrus orchards in Florida to characterize multitrophic, above-belowground interactions; and 3) evaluate changes in soil food webs associated with pollution gradients caused by mining. During the first two years of the project, we produced molecular tools to evaluate the following organisms associated with the EPN soil food web: i) two ectoparasitic bacteria (Paenibacillus sp and Paenibacillus nematophilus) which affect the motility and infectivity of EPNs when spores are attached to the nematode cuticle; ii) the free living nematodes Acrobeloides-group that plays an important role of regulating EPN activity by competing for nutrients within the insect host; iii) four EPN species with worldwide distribution and high potential for use in augmentation biological control, Heterorhabditis bacteriophora, Steinernema carpocapsae, S. scapterisci and S. feltiae. These EPNs species served as well as target nematodes associated with the mining activity in Spain. These molecular tools extended the number of species specific probes developed previously by the research team, making it the only laboratory in the world studying EPN food webs using molecular tools. Additionally, we used a published molecular tool to identify and quantify the oomycete Phytophthora nicotianae, a ubiquitous citrus root pathogen. From an agronomic perspective, we employed these molecular tools to investigate the relative roles of physical and biotic components of various habitats in regulating the spatial patterns of the EPN-soil food web in order to 1) determine the conditions under which field applications of EPNs are likely to be successful, and 2) identify conditions favourable for conserving native EPN communities that provide natural pest control. We performed two experiments in Florida citrus groves: i) characterization of soil food webs in an organic citrus orchard under different treatments that modify soil properties (untreated control, reduced pH, manure mulch application and augmentation of a fungal natural enemy of nematodes); ii) evaluation of the effects of using different irrigation and fertilization regimes with or without a fabric mulch. Analysis of the soil food web assemblage in the organic citrus grove (experiment i) showed that the application of the biological control fungus (Paecilomyces lilacinus) increased the levels of the nematophagous fungi Paecilomyces lilacinus in soil by approximately an order of magnitude throughout the trial. Indeed, levels of the fungus were significantly higher in augmented plots as much as 4 and 5 months following treatments, persistence much greater than that for other fungal biological control organisms which do not remain at levels above background for more than a few days or weeks following application to the soil. The data suggest that P. lilacinus is uniquely competitive with a remarkably broad niche range. Fungal augmentation also increased the abundance of two other biological control fungi and citrus fibrous roots whereas there were no detrimental effects on any group of nematodes. In regard to the manure mulch application in the organic grove, the occurrence of free-living nematodes (FLN) and fibrous roots significantly increased. The second field experiment (ii) revealed that the ‘Advanced Citriculture Production’ (ACP), which mitigates the economic impact of the devastating bacterial disease huanglongbing (HLB) increased secondary pests such as root weevils and plant parasitic nematodes (PPNs). The habitat modification induced by ACP might limit the presence of natural biological control agents (EPNs) that are most active against the weevil, thereby indirectly increasing weevil populations. In addition to creating wetter soil that favours Phytophthora nicotianae, ACP favoured this oomycete by increasing root herbivory by weevil. In regard to the cover factor, the landscape fabric treatment increased the efficiency of root systems and fewer FLNs in mulched plots probably resulted from the reduced rhizospheres in those plots. Fabric mulch also provided a barrier to insects entering and exiting soil and should reduce the numbers of entomopathogens including EPNs. Fewer FLNs and EPNs, may also have contributed to fewer natural enemies of nematodes in mulched plots. Additionally, we studied the geospatial and temporal patterns of these organisms, completing two surveys that provide valuable insights on the multitrophic impacts of soil properties. In a geospatial survey in Florida citrus groves, four variables that affect soil water potential (groundwater depth, water holding capacity, clay and organic matter content) explained significant variability in a redundancy analysis of the soil communities. Management of soil water potential may help to establish and conserve diverse EPN communities that provide more effective control of Diaprepes root weevils. In a temporal survey, population fluxes of EPN, Acrobeloides-group and phoretic bacteria were remarkably sychronous during two years in two orchards with similar edaphic and climatic conditions. Moreover, we performed a comparative study of the biodiversity and natural soil food web assemblage between citrus groves and natural areas in Florida. The results highlighted some key differences in species-habitat associations which may help support the co-habitation of the Florida peninsula by closely related EPN species. Additional analysis of the soil properties will contribute to understanding the basis of these habitat preferences. In general, the results of this objective provided novel insight into the habitat preference of EPN species and the spatial and temporal assemblages of natural enemies that might explain regional and temporal patterns in the efficacy of these EPNs. From a perspective of conservation and soil health, we determined the effect of mine tailing in La Union (Spain) on the spatial variability of soil nematodes due to heavy metals pollution from. We explored the distribution and ecological effects of target pollutants (Pb, Zn, Cd, etc.) in a gradient in an abandon mine in South Spain. We analysed more than 190 composite soil samples for the abiotic (texture, pH, fertility, heavy metals content) and biotic (nematodes, EPN soil food web) factors. We observed that high lead contents from mine tailing decreased soil biodiversity, although some target nematodes were well adapted and can be explored as bioindicators of soil health. By using the qPCR methods developed during this grant to characterize communities in pollution gradients it was observed that whereas FLNs responded strongly to the pollution gradient, EPNs appear to have adapted to any condition that might support arthropod hosts. This project produced pioneering results in agriculture and soil health using new qPCR protocols. It has provided a novel foundation for new strategies for growers in Florida that will be transferred through extension programs. The application of these methods to study soil health in a mining gradient in Spain highlighted their unique potential for use in other research areas, such as soil conservation and monitoring. The results obtained in this project constitute a model that can be used in other agronomic systems in Europe and other part of the world and current collaborations with other laboratories is making this a reality.