Improving Accuracy and Availability of Productivity Methods for a Keystone Functional Group of Microbes

摘要建议：提高微盐沼泽植物关键功能群生产力方法的准确性和有效性，以及利用沼泽植物有机质作为能量来源的次级微生物生产者子囊菌（真菌）。沼泽草-沼泽真菌高产组合可能是盐沼生态系统海洋动物高产的基本因素之一。因为在直立-腐烂沼泽草系统中的真菌几乎完全生长在不透明的固体死植物群中，所以监测它们的生产力和生物量积累在技术上是困难的。利用真菌代理分子的生化分析方法的发展已经克服了这个问题。其中最好的已被证明是麦角甾醇，一种几乎是真菌细胞所特有的细胞膜成分。麦角甾醇是活真菌质量的指示分子，从自然腐烂的材料中较容易提取麦角甾醇，用高效液相色谱法测定。真菌合成速率（=真菌生产力）可以用C14与麦角甾醇结合的速率来衡量，麦角甾醇中放射性增加的速率与真菌生长的速率平行。本文建议对真菌产量法进行两个基本改进。醋酸酯制麦角甾醇的方法是最近才出现的，对于该方法的改进还有很多工作要做。需要注意的一个非常基本的项目是计算由乙酸融入麦角甾醇的速率产生的真菌有机批量生产速率的转换因子。这个因子的值是有的，但范围很广，目前在真菌生产力领域工作的五六个研究者中，没有一个人把实验精力集中在这个特定的问题上。本文建议这样做，测试一系列的环境条件和一系列的目标真菌物种。这种方法使用了众所周知的丹酰荧光团，在药物检测文献中已经出现，它直接类似于醋酸酯-麦角甾醇法。本文拟验证该方法是否可用于测定自然腐烂的沼泽草叶的真菌产量。如果能够如此使用，那么测量生态系统中真菌生产力的能力将在更广泛的实验室中可用（而不是目前仅限于获得许可处理放射性同位素的实验室）。更广泛的影响：提出的研究将提高现有真菌生产监测方法的实用性。这将为在沿海海洋边缘工作的调查人员提供更好的工具，这些海洋地点目前由于容易受到人为破坏而迫切需要关注。更重要的是，由于真菌活动在这些领域的重要性，新的方法信息对林业科学家、水生生态系统科学家、农业科学家和工业发酵科学家也具有相当大的价值。

ABSTRACTProposal: Improving Accuracy and Availability of Productivity Methods for a Keystone Functional Group of MicrobesSalt marsh plants exceedingly productive, as also are the ascomycetes (fungi) that are secondary microbial producers utilizing marsh plant organic matter as an energy source. The combination of marsh grass-marsh fungal high production is likely to be one of the basic factors underlying the high output of marine animals from salt marsh ecosystems. Because fungi in the standing-decaying marsh grass systems grow almost exclusively within the opaque solid dead plant mass, monitoring their productivity and biomass accumulation are technically difficult. This problem has been surmounted by development of biochemical analytical methods that make use of fungal-proxy molecules. The best of these has proven to be ergosterol, a cell-membrane component nearly unique to fungal cells. Ergosterol is an indicator molecule for living fungal mass and is Ergosterol relatively easily extracted from naturally decaying materials and measured by HPLC (high-performance liquid chromatography). Rates of fungal synthesis (= fungal productivity) can be measured as rates of incorporation of C14 into ergosterol, where the rate of increase in radioactivity in ergosterol parallels the rate at which the fungus is growing. It is herein proposed to seek two basic improvements to the fungal-productivity method.The acetate-to-ergosterol method has only recently become available and there remains much to be done regarding refinement of the method. One very basic item that needs attention is the conversion factor for calculating the rate of fungal organic-mass production from the rate of acetate incorporation into ergosterol. There are values available for this factor, but they range widely, and no one of the five or six investigators currently working in the field of fungal productivity has concentrated experimental efforts on this specific problem. It is proposed herein to do so, testing a range of environmental conditions and a range of target fungal species. Such a method, using the well-known dansyl fluorophore, has appeared in the drug-testing literature that is directly analogous to the acetate-to-ergosterol method. It is proposed herein to test whether this method can be used to measure fungal productivity in naturally decaying marsh grass leaves. If it can be so used, the ability to measure fungal productivity in ecosystems would become available in a wider range of laboratories (as opposed to its current restriction to those licensed for handling of radioisotopes). Broader Impacts: The research proposed would improve the utility of the methods now available for monitoring fungal production. This would provide better tools for investigators working at the edges of coastal oceans, marine sites that are currently in urgent need of attention due to their susceptibility to anthropogenic damage. More importantly, the new methodological information would also be of considerable value to forestry scientists, aquatic-ecosystem scientists, agricultural scientists, and industrial-fermentation scientists, because of the great importance of fungal activities in these fields.