The impact of symbiotic nitrogen fixation on plant defense, herbivores and higher trophic levels

地下固氮细菌（根瘤菌）具有很高的生态和经济重要性，但它们在地上食物网中的作用实际上尚未被探索。为了从功能上解开根瘤菌复杂的生态效应，本项目将使用多种根瘤菌菌株与不同的寄主植物，野生利马豆（豆科：Phaseolus lunatus）和野生豌豆（豆科：Leptospron adenanthum）结合使用。在实验室中，这些植物-根瘤菌系统将在实验中暴露于不同的二氧化碳和氮的有效性。这两种植物对食草动物表现出根本不同的化学防御。虽然月亮草具有有效的氮基防御（通过氰化物生成），但腺兰则投资于碳基防御（通过酚类物质）。除了这些直接防御之外，这两种植物还表现出一系列间接防御（花外花蜜和食草动物诱导的挥发物），这些间接防御通过吸引食肉节肢动物起作用。该项目将提供根瘤菌对植物性状、不同营养水平的植物相关生物的影响以及可变非生物条件对这些关联的影响的综合图像。由于植物-微生物-节肢动物相互作用涉及化学生态学、进化生物学、全球变化生物学、保护生物学、农业和土壤科学等多个学科，因此本研究将具有广泛的影响。特别是，在环境迅速变化的时代，更好地了解控制自然和农业生态系统的机制的价值怎么估计都不为过。作为该项目的一部分，将培训一名研究生和几名本科生，并为学校教师和K-12学生提供每周一次的课程和为期三周的暑期实地课程，让他们参与研究。本项目将自下而上评估根瘤菌对不同摄食行业的通才和专才食草昆虫，以及对寄生性和掠食性节肢动物的影响。此外，不同水平的根瘤菌固氮将被认为是不同质量的根瘤菌作为植物共生菌。虽然互惠型根瘤菌菌株相互作用，但欺骗型根瘤菌驻留在根瘤中并起碳汇作用，但不提供氮，从而降低了寄主的净适应效益。除实验室研究外，还将在两种植物的自然栖息地（哥斯达黎加）探索根瘤菌共生的成本和效益。具体而言，将解决以下问题：1。根瘤菌菌株、土壤氮和环境二氧化碳（CO2）对植物性状的独立和相互作用是什么？2. 根瘤菌介导的植物性状如何影响昆虫食草动物和食肉动物？3. 什么是生态效益和防御相关成本的根瘤菌共生在一个互惠互利的骗子连续体在自然界？分光光度分析，GC-MS（气相色谱-质谱）和LC-MS（液相色谱-质谱），将用于分析植物防御和营养化合物。植物资源分配给根瘤菌和不同固氮率的影响将通过同位素标记技术（15N）和氩气室（用氩气代替大气中氮气的装置）的N2枯竭研究进行量化。根瘤菌介导的植物性状对昆虫食草动物和食肉动物的影响将通过饲养试验和嗅觉选择试验进行研究。野外研究将评估根瘤菌菌株和固氮率对自然界多种植物防御性状的影响，并验证根瘤菌对自然生态系统中节肢食草动物和食肉动物自下而上的影响。

Belowground nitrogen (N2)-fixing bacteria (rhizobia) are of high ecological and economic importance, yet their role in aboveground food webs is virtually unexplored. To functionally disentangle the complex ecological effects of rhizobia, this project will use multiple rhizobial strains in combination with different host plants, wild lima bean (Fabaceae: Phaseolus lunatus) and wild pea (Fabaceae: Leptospron adenanthum). In the laboratory, these plant-rhizobia systems will be experimentally exposed to varying CO2 and nitrogen availabilities. Both plant species show fundamentally different chemical defenses against herbivores. While P. lunatus has an efficient nitrogen-based defense (via cyanogenesis), L. adenanthum invests in carbon-based defense (via phenolics). In addition to these direct defenses, both plants show an array of indirect defenses (extrafloral nectar and herbivore-induced volatiles) which act through the attraction of carnivorous arthropods. The project will provide an integrated picture of the effects of rhizobia on plant traits, plant-associated organisms of different trophic levels and the impact of variable abiotic conditions on these associations. As plant-microbe-arthropod interactions are relevant for multiple disciplines including chemical ecology, evolutionary biology, global change biology, conservation biology, agriculture and soil sciences, this research will have broad impact. In particular, in times of rapidly changing environments, the value in better understanding the mechanisms controlling natural and agricultural ecosystems cannot be overestimated. As part of this project, one graduate student and several undergraduates will be trained and opportunities for school teachers and K-12 students to participate in the research will be provided in weekly lessons and a three-week summer field course. This project will assess bottom-up effects of rhizobia on generalist and specialist insect herbivores of different feeding guilds, as well as on parasitoid and predatory arthropods. Furthermore, different levels of rhizobial N2-fixation will be considered as rhizobia differ in quality as plant mutualists. While mutualistic rhizobia strains reciprocate, cheating rhizobia reside in root nodules and function as carbon sinks, but provide no nitrogen, and thus reduce the net fitness benefit for the pant host. Beyond lab studies, costs and benefits of the rhizobial symbiosis will be explored in a natural habitat of both plant species (Costa Rica). Specifically, the following questions will be addressed: 1. What are the independent and interacting effects of rhizobial strain, soil nitrogen and ambient carbon dioxide (CO2) on plant traits? 2. How do rhizobia-mediated plant traits affect insect herbivores and carnivores? 3. What are the ecological benefits and defense-associated costs of the rhizobial symbiosis in a mutualist-cheater continuum in nature? Spectrophotometric assays, GC-MS (gas chromatography-mass spectrometry) and LC-MS (liquid chromatography-mass spectrometry), will be used to analyze plant defensive and nutritive compounds. Plant resource allocation to rhizobia and effects of different nitrogen fixation rates will be quantified using isotope labeling techniques (15N) and N2 depletion studies with Argon chambers (setups in which atmospheric N2 is replaced with Argon). Effects of rhizobia-mediated plant traits on insect herbivores and carnivores will be studied with feeding and olfactometer choice trials. Accompanying field studies will serve to evaluate the effects of rhizobial strain and N2-fixation rate on multiple plant defense traits in nature and to test for the predicted bottom-up effects of rhizobia on arthropod herbivores and carnivores in the natural ecosystem.