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价值，但它们在地上食物网中的作用几乎没有被探索过。为了从功能上解开根瘤菌的复杂生态效应，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：菜豆)和野生豌豆(豆科：小豆)相结合。在实验室里，这些植物-根瘤菌系统将被实验地暴露在不同的二氧化碳和氮素可用性中。这两种植物对食草动物表现出根本不同的化学防御。新月形藻具有高效的基于氮的防御(通过氰化物生成)，而腺毛假单胞菌则投资于基于碳基的防御(通过酚类)。除了这些直接防御，这两种植物都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)，这些间接防御通过肉食性节肢动物的吸引发挥作用。该项目将提供根瘤菌对植物特性的影响、不同营养水平的与植物相关的生物以及可变的非生物条件对这些联系的影响的综合图景。由于植物-微生物-节肢动物的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，因此这项研究将产生广泛的影响。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，将对一名研究生和几名本科生进行培训，并将在每周课程和为期三周的暑期实地课程中为学校教师和K-12学生提供参与研究的机会。该项目将评估根瘤菌对不同饲喂行业的多面手和专业性昆虫草食动物以及寄生虫和捕食性节肢动物的自下而上的影响。此外，不同水平的根瘤菌固氮将被认为是根瘤菌作为植物互惠体在质量上的不同。虽然互惠的根瘤菌相互作用，但欺骗的根瘤菌居住在根瘤中，起到碳汇的作用，但不提供氮素，从而降低了PANT寄主的净适合度效益。除了实验室研究，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收益。具体地说，将讨论以下问题：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交互影响？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.在自然界互惠-欺骗的连续体中，根瘤菌共生的生态效益和防御相关成本是什么？GC-MS(气相色谱-质谱法)和LC-MS(液质联用法)将用于分析植物防御性和营养性化合物。植物对根瘤菌的资源分配和不同固氮率的影响将通过同位素标记技术(15N)和利用Argon小室(用Argon取代大气N2的设置)进行氮气消耗研究来量化。将通过取食和嗅觉仪选择试验来研究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。与之配套的实地研究将用于评估根瘤菌菌株和固氮率对自然界中多种植物防御特性的影响，并检验根瘤菌对自然生态系统中节肢动物、草食动物和食肉动物的自下而上的预测效果。

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.