High throughput real-time in-planta assay of microbial nitrogen fixation activity

Abstract

Systems and methods for the analysis of microbial nitrogen-fixation in a plurality of plants include a gas source, a detector, a manifold, and a controller. The manifold can have a plurality of valves, wherein an individual valve is in fluid communication with an individual plant. The controller can be configured to activate the valves to sample gas from each individual plant to the detector for analysis. Also provided are methods of using the system for the automated assay of microbial nitrogen-fixation in plants and for identifying a nitrogen-fixing microbial strain.

Description

HIGH THROUGHPUT REAL-TIME IN-PLANTA ASSAY OF MICROBIALNITROGEN FIXATION ACTIVITYFIELD

[0001] The present disclosure relates generally to systems and methods for assaying microbial activity in plants, and more specifically to systems and methods for measuring microbial nitrogen fixation in plants.BACKGROUND

[0002] Contemporary farming relies on the application of synthetic nitrogen fertilizer to facilitate plant growth and improve yield. Synthetic nitrogen fertilizers, however, have been identified as contributing to greenhouse gas emissions and environmental runoff. As such, a better approach to growing crops is needed.

[0003] Biological nitrogen fixation is a process in which microorganisms such as bacteria convert atmospheric nitrogen gas ( 1X12) into ammonia (NH3) via reduction mediated by the enzyme nitrogenase. A variety of different bacterial strains have been genetically engineered for improved nitrogen fixation activity. These beneficial microbes can be delivered to the soil near the root structure of the plant, or may be formulated in a seed coating or plant treatment, where they can function as an alternative to synthetic nitrogen fertilizer.

[0004] It can be challenging, however, to identify and analyze microbes for nitrogen-fixing activity as generally available equipment and methods tend to be expensive, timeconsuming, inefficient, and / or unable to provide real-time data. Furthermore, many available methods are unable to measure nitrogen fixation activity in plants, such as, for example, plants growing in soil, or measure nitrogen fixation activity in a wide variety of field-like conditions, such as, for example, variable temperatures, moisture content, soil oxygen, or plant growth stages. In addition, many available methods are unable to simultaneously and / or automatically measure multiple plants during the same test period.

[0005] As such, there remains a need for systems and methods for high-throughput measurements and identification of microbial nitrogen-fixing activity in plants. There also remains a need for systems and methods for real-time measurements of microbial nitrogenfixing activity in plants. In addition, there remains a need for systems and methods that can measure microbial nitrogen-fixing activity in plants across a range of field-like conditions.SUM MARY

[0006] Provided herein are systems and methods for the analysis of microbial nitrogenfixation in plants. In some embodiments, the system can include a plurality of plants, each plant having a cover disposed over the plant, the cover forming a sample volume containing the plant. The system can further include a gas source, a detector, a manifold, and a controller. In some embodiments, the manifold can be in fluid communication with the gas source and with the detector. In addition, the manifold can have a plurality of valves, wherein an individual valve is in fluid communication with an individual plant. In some embodiments, the controller can be configured to activate the valves to regulate gas delivery from the gas source. In addition, the controller can be configured to activate individual valves to sample gas from each individual plant to the detector for analysis.

[0007] Methods for the automated assay of microbial nitrogen-fixation in plants using the inventive systems are also provided. In some embodiments, the method includes providing a plurality of test plants in soil inoculated with one or more microbes and attaching a cover and a gas line to each plant, wherein each plant is individually connected to a gas line. In some embodiments, the method further includes activating the manifold to open an individual valve in fluid communication with an individual plant and the detector such that gas flows from the individual plant to the detector for sampling, and activating the manifold to close the individual valve once gas sampling from the individual plant is complete. In some embodiments, each plant can be individually sampled via the methods and systems.

[0008] Also provided are methods of identifying a nitrogen-fixing microbial strain using the inventive systems and methods.

[0009] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.BRIEF DESCRI PTION OF DRAWINGS

[0010] FIG. 1A is a conceptual diagram showing nitrogen cycline in maize cropping systems with the application of synthetic nitrogen fertilizer.

[0011] FIG. IB is a conceptual diagram of changes to nitrogen cycling in maize cropping systems with the application of genetically modified nitrogen-fixing microbes that can replace synthetic nitrogen fertilizer.

[0012] FIG. 1C is a diagram showing steps useful for the discovery and optimization of genetically modified nitrogen-fixing microbes.

[0013] FIG. ID is a graphic showing an example of a bacterial nitrogen-fixing pathway.

[0014] FIG. IE is a graphic showing an example of genetic modifications that demonstrate nitrogen-fixing activity in the presence of nitrogen.

[0015] FIG. 2A is a perspective view of an example of a chamber.

[0016] FIG. 2B is a perspective view of the interior of the chamber shown in FIG. 2A.

[0017] FIG. 3 is a perspective view of an example of a container.

[0018] FIGS. 4A-4D are plan views of examples of containers with live plants and various types of covers.

[0019] FIG. 5 is a plan view of an example of a chamber with several containers with sample lines, live plants, and covers.

[0020] FIGS. 6A-6B are diagrams showing an example gas delivery path of a system from a gas source to a plant via a manifold.

[0021] FIGS. 7A-7B are diagrams showing an example gas sampling path of a system from a plant to a detector via a manifold.

[0022] FIG. 8 is a plan view of an example of a plurality of chambers connected to a bridge.

[0023] FIG. 9 is a diagram showing an example method.

[0024] FIG. 10 is a graph showing ethylene carryover in various materials.

[0025] FIG. 11 is a graph showing ethylene levels in parts per billion over time with and without acetylene gas delivery.

[0026] FIG. 12 is a graph showing ethylene levels in parts per billion over time with plant, with plant removed, and with no plant.

[0027] FIG. 13 is a series of graphs showing estimated acetylene reduction rates for different genetically modified diazotrophs.

[0028] FIG. 14 shows block diagrams of example computing devices that can be useful with the systems and methods.DETAILED DESCRI PTION

[0001] The present disclosure relates to systems and methods that can provide high throughput, automated, real-time measurements of microbial nitrogen fixation in plants. For example, the systems and methods are suitable for analyzing microbial nitrogen fixation in large numbers of plants. In some embodiments, the systems and methods are suitable for analyzing microbial nitrogen fixation across a range of field-like conditions such as temperature, moisture content, soil oxygen, and growth stage. This can allow for the rapid screening and identification of nitrogen-fixing microbes, such as genetically modified nitrogen-fixing microbes, that can successfully fix nitrogen in plants, such as, for example, in field conditions.

[0002] Improving plant nitrogen nutrition using genetically engineered microbes is an important step in reducing the use of synthetic nitrogen fertilizer in growing crops. For example, certain genetically engineered microbes can associate with plants and demonstrate biological nitrogen fixation under nitrogen-rich conditions. In one example, as shown in FIGS. 1A-1B, the application of genetically modified nitrogen-fixing microbes as a replacement for synthetic nitrogen fertilizer can, in some embodiments, provide better synchronization of plant nitrogen demand and nitrogen supply, reduce synthetic nitrogen requirements, and, as such, reduce environmental nitrogen losses.

[0003] Microbes can be genetically engineered for improved nitrogen fixation activity. For example, FIG. 1C generally shows steps useful for discovering, developing, and analyzing genetically modified nitrogen-fixing microbes. For example, microbes can be isolated and tested for colonization. In some embodiments, microbes can be genetically engineered to enhance nitrogen fixation, such as, for example, to include specific mutations targeting genes that regulate various pathways involved in nitrogen fixation activity. An example pathway is shown in FIG. ID, and example modifications that can enhance nitrogen fixation are shown in FIG. IE. Nitrogen fixation can be measured in vivo, in plants, and, where suitable, in a test field.

[0004] The inventive systems and methods are useful for analyzing and identifying microbes with nitrogen-fixing activity, such as, for example, native nitrogen-fixing microbes or genetically engineered microbes. In some embodiments, the microbes can be genetically engineered microbes with enhanced nitrogen-fixing activity compared to the unmodified microbe. In some embodiments, the systems and methods can measure microbial nitrogen fixation activity via acetylene reduction to ethylene. For example, test plants can be inoculated with naturally occurring or engineered nitrogen-fixing microbes and acetylene can be delivered to the test plant. If microbial nitrogenase activity is present, nitrogenase reduces acetylene to ethylene. Ethylene production can be measured in real-time to directly assess the nitrogen-fixing activity of the microbes. This can facilitate identification of microbes with improved nitrogen-fixation activity.

[0005] The inventive systems and methods significantly improve the cost, efficiency, and accuracy of measuring microbial nitrogen fixation in plants, which has traditionally been difficult. Typical methods are laborious, must be done by hand, and may require disturbing the growth medium or roots. In addition, typical methods may only provide endpoint measurements or may not be performed on plants growing in soil. In contrast, the inventive systems and methods can provide automated, real-time nitrogen-fixation measurements forlarge numbers of soil-grown plants. For example, in some embodiments, the systems and methods can analyze individual plants with a 10-fold or greater increase in sampling number per experiment compared to standard gas chromatography acetylene reductase assays (ARA). In addition, in contrast to traditional methods that must be sampled by hand, the inventive systems and methods can allow for frequent automated sampling, which can reduce contamination and improve analytical speed. In some embodiments, the systems and methods can allow for measurements every second, every minute, every hour, every two hours, every five hours, every 12 hours, every day, or more.

[0006] In some embodiments, the systems and methods can be suitable for rapidly quantifying microbe nitrogen fixation activity, comparing microbe nitrogen fixation activity across plants in varying environmental conditions, and analyzing plant growth conditions for optimal nitrogen fixation activity, or the like. In some embodiments, the systems and methods can identify microbes with improved nitrogen-fixing activity. For example, in various embodiments, the systems and methods can measure ethylene conversion in approximately 1 minute or less at the ppBv level, approximately 30 seconds or less at the ppBV level, approximately 10 seconds or less at the ppBV level, approximately 1 second or less at the ppBV level or on any other suitable timeing. In addition, in some embodiments, the systems and methods can achieve a greater than lOOx (e.g., greater than lOOOx, greater than lOOOOx) improvement in sensitivity per hour compared to a standard acetylene reduction assay performed using gas chromatography. For example, in some embodiments, the systems and methods can have a limit of detection for ethylene that is 1000 ppb or less, 100 ppb or less, 10 ppb or less, or 1 ppb or less.Real-Time In-Planta Assay Systems

[0007] FIG. 2A shows an example of a portion of a system 100 for real-time in-planta assays. System 100 includes chamber 102 with walls 102a-102f that enclose spatial volume 104 (shown in FIG. 2B) internal to chamber 102. In various embodiments, chamber 102 can include one or more doors 108 that open to access volume 104. In addition, in various embodiments, system 100 can include one or more displays 114. System 100 also can include one or more ports 120 and one or more gas lines 106. One or more gas lines 106 can be adapted to carry one or more gases to chamber 102, carry gas from chamber 102 for delivery to detector 112, recirculate gas, or remove gas such as via vacuum 128.

[0008] FIG. 2B shows an example of the interior of chamber 102 shown in FIG. 1. Chamber102 has walls 102a-102f that enclose spatial volume 104 internal to chamber 102. In various embodiments, chamber 102 can include one or more doors 108 that open into volume 104.In addition, in various embodiments, system 100 can include one or more displays 114. System 100 also can include one or more ports 120 and one or more gas lines 106. Chamber 102 can include one or more supports 116 that can hold one or more plants 150 within the enclosed spatial volume 104. In various embodiments, one or more plants 150 are positioned within a container 130. For example, in some embodiments, individual plants 150 are each planted in a pot and the pot can be supported by container 130.

[0009] Chamber 102 can include any number of walls suitable for enclosing spatial volume 104, and the wall(a)s can define any shape for chamber 102. In some embodiments, for example, the wall(s) define a cubic or rectangular prismatic shape for chamber 102. In certain embodiments, the wall(s) define a spherical or elliptical shape for chamber 102. More generally, the wall(s) can define any regular or irregular shape for chamber 102. In some embodiments, chamber 102 is a growth chamber, such as, for example, a reach-in growth chamber, a walk-in growth chamber, or any other suitable growth chamber. In some embodiments, chamber 102 is a walk-in growth chamber. In some embodiments, chamber 102 is a one-tier reach-in growth chamber. In some embodiments, chamber 102 is a two-tier reach-in growth chamber. As such, in some embodiments, chamber 102 is climate- controlled.

[0010] Chamber 102 can be any suitable height h.The height h of chamber 102 is the minimum distance between wall 102c and wall 102a Upward plant growth generally occurs in a direction parallel to height h, and so the height can be selected to accommodate such growth for one or more different plant types. In some embodiments, h can be 0.5 m or more (e.g., 1.0 m or more, 1.5 m or more, 2.0 m or more, 2.5 m or more, 3.0 m or more, 3.5 m or more, 4.0 m or more, 4.5 m or more, 5.0 m or more, 5.5 m or more, 6.0 m or more, 6.5 m or more, 7.0 m or more, 7.5 m or more, 8.0 m or more, 8.5 m or more, 9.0 m or more, 9.5 m or more, 10.0 m or more, or even more). In certain embodiments, the height h is sufficiently large so that the entire plant 150 is positioned within the enclosed spatial volume 104. In various embodiments, the height h is sufficiently large to accommodate the entire plant 150 contained in container 130 and enclosed by cover 142. In some cases, chamber 102 is a walk-in chamber and height h is sufficiently large to accommodate a human.

[0011] Chamber 102 can be any suitable width w. The width w of chamber 102 is the minimum distance between the two opposing walls (102b and 102d) perpendicular to walls 102a and 102c. In various embodiments, as shown in FIGS. 5 and 8, the width w is sufficiently large so that multiple plants may be positioned within the enclosed spatial volume 104. For example, as shown in FIGS. 5 and 8, the width w is sufficiently large so thatmultiple containers, each containing multiple plants, are positioned within the enclosed spatial volume 104. In some embodiments, w can be 0.5 m or more (e.g., 1.0 m or more, 1.5 m or more, 2.0 m or more, 2.5 m or more, 3.0 m or more, 3.5 m or more, 4.0 m or more, 4.5 m or more, 5.0 m or more, 5.5 m or more, 6.0 m or more, 6.5 m or more, 7.0 m or more, 7.5 m or more, 8.0 m or more, 8.5 m or more, 9.0 m or more, 9.5 m or more, 10.0 m or more, or even more).

[0012] In general, the enclosed spatial volume 104 of chamber 102 can be selected as desired to accommodate a plurality of plants and one or more containers 130. In some embodiments, for example, the enclosed spatial volume can be 100 L or more (e.g., 200 L or more, 300 L or more, 400 L or more, 500 L or more, 600 L or more, 700 L or more, 800 L or more, 900 L or more, 1000 L or more, 1500 L or more, 2000 L or more, 2500 L or more, 3000 L or more, 4000 L or more, 5000 L or more, 7000 L or more, 10,000 L or more, 15,000 L or more, 20,000 L or more, 30,000 L or more, 50,000 L or more, or even more).

[0013] In some embodiments, chamber 102 is relatively airtight, such that a leakage rate of gases from chamber 102 is relatively small. For example, when chamber 102 is filled with a gas at a pressure p at a first time, the gas pressure within the chamber at a second time at least 7 days after the first time can be 0.70p or more (e.g., 0.80p or more, 0.85p or more, 0.90p or more, 0.95p or more, 0.98p or more, 0.99p or more, 0.999p or more, 0.9999p or more, or even more). Alternatively, chamber 102 is not relatively airtight. In various embodiments, chamber 102 can allow for ambient airflow in and out of spatial volume 104.

[0014] The walls of chamber 102 can generally be formed from a variety of materials including, but not limited to, various plastics and metals. Mating walls can be joined by bonding, welding, clamping, and other processes to form wall joints. A variety of structural supporting members can be used to reinforce the walls of chamber 102, and such members can be formed of the same or different materials than the walls.

[0015] In some embodiments, system 100 does not include chamber 102. For example, in various embodiments, system 100 can analyze one or more plants 150 that can be positioned in any suitable manner, such as, for example, in free-standing containers, in racks, on shelves, in trays, on one or more supports 116, or in any other manner conducive to sampling. In some embodiments, one or more plants 150 are positioned in one or more containers 130 and analyzed in any suitable location or environment.

[0016] FIG. 3 shows an example of a container 130 suitable for holding one or more plants150. Container 130 can have one or more holders 132 that can contain a plant 150 and soil152 or other suitable plant-growing media. In various embodiments, as shown in FIGS. 4A-4D, plant 150 optionally can be contained in pot 154 supported in holder 132, and pot 154 can contain soil 152 or other suitable plant-growing media. As shown in FIG. 3, in various embodiments, one or more gas lines 106 can be associated with container 130, such as, for example, with one or more gas lines 106 positioned proximal to holders 132 such that the one or more gas lines 106 can reach a plant 150 contained in holder 132. In various embodiments, one or more gas lines 106 can be joined to container 130. Alternatively, one or more gas lines 106 can be separate from container 130. In some embodiments, one or more gas lines 106 can be joined to cover 142.

[0017] In various embodiments, and as shown in FIG. 3, container 130 can be a multi-plant container having a plurality of holders 132. As shown in FIG. 3, a gas line 106 can be positioned adjacent to each holder 132 such that plants 150 can be individually monitored. The container 130 can hold any suitable number of plants. For example, the container 130 can hold greater than 10 plants, greater than 15 plants, greater than 20 plants, greater than 25 plants, greater than 50 plants, greater than 75 plants, greater than 100 plants, greater than 150 plants, greater than 200 plants, greater than 250 plants, greater than 300 plants, greater than 350 plants, greater than 400 plants, greater than 450 plants, greater than 500 plants, greater than 600 plants, greater than 700 plants, greater than 800 plants, greater than 900 plants, greater than 1000 plants, greater than 1500 plants, greater than 2000 plants or more. In some embodiments, a plurality of containers 130 can be connected together, such as, for example, two, three, four, five or more containers 130 can be connected together.

[0018] In various embodiments, plants 150 can be grown in soil 152. Soil 152 can be any suitable soil composition, such as, for example, agricultural soil, potting mix, sand, or any other suitable soil composition. In various embodiments, the soil composition can be a natural composition, such as, for example, a soil composition isolated from nature, a soil composition created to mimic a soil composition isolated from nature, a soil composition including natural materials, or the like. In some embodiments, plants 150 can be grown in any suitable synthetic growth medium, such as, for example, a synthetic growth media. Plant 150 typically can be divided into portions that include leaves 150a, stem 150b, and roots 150c (FIGS. 1A, IB).

[0019] FIGS. 4A-4D show examples of container 130 suitable for measuring one or more plants 150. Container 130 can have one or more holders 132 that can contain a plant 150 (for example, roots 150c) and soil 152 or other suitable plant-growing media. In various embodiments, plant 150 (for example, roots 150c) optionally can be contained in pot 154that can be supported in holder 132, and pot 154 can contain soil 152 or other suitable plant-growing media. As shown in FIGS. 4A-4D, plant 150 can be covered by cover 142. Cover 142 can have one or more walls 144 forming sample volume 146 and cover 142 can be sealed around plant 150. For example, cover 142 can be disposed around the leaves 150a and stem 150b of plant 150. In various embodiments, cover 142 is hermetically sealed over plant 150 such that sample volume 146 does not experience leakage. It will be understood that cover 142 can, in some embodiments, be joined to pot 154, holder 132, and / or container 130 containing soil 152 and roots 150c such that cover 142 is disposed over leaves 150a and stem 150b.

[0020] Cover 142 can be any design, any shape, and any material suitable for forming sample volume 146. Cover 142 can be disposable or reusable. In some embodiments, cover 142 can be hermetically sealed, such as, for example, via heat-sealing; via gaskets that can join cover 142 to pot 154, holder 132, or container 130; or in any other suitable manner. In addition, cover 142 can be disposed around plant 150 in any suitable manner, such as, for example, with each plant 150 being contained in an individual cover 142 that is not directly joined to additional covers 142, or alternatively, with each plant 150 being contained in a cover 142 that is directly joined to one or more covers 142. In some embodiments, as shown in FIG. 4A, cover 142 can be an individual flexible bag, such as, for example, a heat-sealable bag, such as, for example, a heat-sealable, polymeric thermoplastic bag. In some embodiments, as shown in FIG. 4B, cover 142 can be an individual rigid or semi-rigid cylinder. In some embodiments, such as shown in FIGS. 4C-4D, cover 142 can be a single unit containing openings for a plurality of plants 150. Alternatively, in various embodiments, plants 150 need not be contained in container 130. For example, in some embodiments, one or more plants 150 can be positioned in any suitable manner, such as, for example, in freestanding containers, in racks, on shelves, in trays, on one or more supports 116, or in any other manner conducive to sampling.

[0021] Cover 142 can be formed from any suitable material. For example, in some embodiments, cover 142 is formed from a flexible material. The flexible material can be any suitable material, such as, for example, a film, a non-woven, a foil, or combinations thereof, or any other suitable material. In some embodiments, the cover is formed from one or more flexible thermoplastic films that include polyamide, polyethylene, polyethylene terephthalate, polymethyl methacrylate, polypropylene, polyurethane, polyvinyl acetate, polyvinyl chloride, or any other suitable material. In some embodiments, cover 142 is formed from a plurality of flexible materials, such as, for example, a composite film, or thelike. Alternatively, in some embodiments, cover 142 is formed from a non-flexible material, such as, for example, glass, plastic, metal, or any othe suitable material. In some embodiments, cover 142 is transparent to allow for visual inspection of plant 150.

[0022] FIG. 5 shows an example of the interior of a chamber 102 for real-time in-planta assays, containing multiple containers 130. Each container 130 is a multi-plant container containing a plurality of plants 150. Chamber 102 has walls 102a-102f that enclose spatial volume 104 internal to chamber 102. In various embodiments, chamber 102 can include one or more supports 116 that can hold one or more plants 150 within the enclosed spatial volume 104. As shown in FIG. 5, support 116 holds a plurality of multi-plant containers 130 that each contain a plurality of plants 150. In various embodiments, individual plants 150 each can be enclosed in a cover 142 that has walls 144 forming sample volume 146 and that is sealed around plant 150 such that minimal gas leakage from sample volume 146 occurs. As shown in FIG. 5, in some embodiments, each plant is contained in its own cover 142. In addition, each cover is in fluid communication with manifold 124, such as, for example, via individual gas lines 106. As described herein, gas line 106 can be connected to manifold 124. In various embodiments, manifold 124 can push one or more gases for delivery to plant 150, pull gas from sample volume 146 for sampling, or pull gas for removal from system 100 via gas line 106. In some embodiments, controller 110 can instruct manifold 124 to push or pull gas. Controller 110 can instruct manifold 124 to activate valve 124 to open or close. In some embodiments, manifold 124 has a plurality of valves 124 that can correspond to a plurality of plants 150, and controller 110 can instruct manifold 124 to individually activate a valve 124 that corresponds to a plant, allowing for automated individual sample, gas delivery, and the like. Controller 110 can be connected to manifold 124 in any suitable manner. In some embodiments, controller 110 can be incorporated as a part of manifold 124.

[0023] FIGS. 6A-6B show an example gas delivery path of an example system 100 from gas source 122 to plant 150 via manifold 124. As shown in FIGS. 6A-6B, in various embodiments, manifold 124 can be in fluid communication with one or more gas sources 122, such as, for example, via conduit [X]. Gas source 122 can be any suitable gas, such as, for example, acetylene gas, air, or a mixture of acetylene gas and air. As shown in FIGS. 6A-6B, in various embodiments, manifold 124 has valves 124a-124o corresponding to each plant 150a-150o, which allows for customized delivery of gas to each plant 150. Valves 124a-124o are in fluid communication with sample volume 146a-146o within cover 142a-142o, such as, for example via gas line 106a-106o. For example, as shown in FIG. 6A, valve 124a is in fluid communication with sample volume 146a within cover 142a disposed over plant 150a, suchas, for example, via gas line 106a. In addition, as shown in FIG. 6B, valve 124b is in fluid communication with sample volume 146b within cover 142b disposed over plant 150b, such as, for example, via gas line 106b.

[0024] In some embodiments, manifold 124 can be in fluid communication with gas source 122, detector 112, and to plants 150, such as, for example, via cover 142. Manifold 124 can be connected to gas source 122, detector 112, and to plant 150 in any suitable manner, such as, for example, via tubing, piping, or any other suitable connector. In some embodiments, manifold 124 is connected to gas source 122 via stainless steel piping. In some embodiments, manifold 124 can be directly connected to one or more of gas source 122, detector 112, or plant 150, or can be indirectly connected to one or more of gas source 122, detector 112, or plant 150. In some embodiments, system 100 can include more than one manifold 124, such as, for example, a manifold 124 for pushing gas to plants 150 and a manifold 124 for sampling gas from plants 156. As such, manifold 124 can be disposed in any manner suitable to facilitate pushing one or more gases such as acetylene or compressed air to plant 150, pulling one or more gases from plant 150 for analysis, or pulling one or more gases for exhaust via vacuum . In some embodiments, ontroller 110 controls manifold 124 to push a specified gas such as 122b-l or 122b-2 or pull gas into detector 126 or to vacuum 128.

[0025] Manifold 124 can contain any suitable number of valves 124, for example, in some embodiments, manifold 124 has a number of valves 124 that corresponds to the number of plants 150 provided in container 130. In the examples shown in FIGS. 6A-6B, manifold 124 has fifteen valves 124 that correspond to the fifteen plants 150 contained in container 130; however, it will be understood that any suitable number of valves 124 and plants 150 are within the scope of the systems and methods set forth herein. As shown in FIGS. 6A-6B, each valve 124 can be adapted to deliver and / or remove gas from a plant 150. Alternatively, in some embodiments, each valve 124 could be adapted to deliver gas from more than one plants 150, such as, for example, via connections to cover 142 of more than one plant 150.

[0026] In general, gas source 122 can be any suitable gas or combination of gases and can be provided in any suitable concentration. For example, when gas source is a combination of air and acetylene, acetylene can be provided in any suitable concentration, such as, for example, 0.1% acetylene, 0.5% acetylene, 1.0% acetylene, 1.25% acetylene, 1.5% acetylene, 1.75% acetylene, or any other suitable amount of acetylene. Gas source 122 can include any number of gas sources (e.g., one or more gas sources, two or more gas sources, three or more gas sources, four or more gas sources, five or more gas sources, six or more gassources, seven or more gas sources, eight or more gas sources, nine or more gas sources, ten or more gas sources, or even more gas sources).

[0027] In some embodiments, controller 110 activates manifold 124 to deliver gas from gas source 122 to plant 150. For example, in some embodiments, controller 110 can activate manifold 124 to open valve 124a, facilitating gas flow to plant 150a via gas line 106a that can be in fluid communication with cover 142a disposed over plant 150a. Once gas delivery to plant 150a is completed, controller 110 can activate manifold 124 to close valve 124a and then open valve 124b, facilitating gas flow to plant 150b via gas line 106b that can be in fluid communication with cover 142b disposed over plant 150b. Controller 110 can continue to activate manifold 124 to open and close valves to deliver gas to plants 150 until gas delivery is complete. As such, in some embodiments, one or more of gas content, volume, timing, and the like delivered to each plant 150 can be individualized during the course of the experiments. In some embodiments, gas content is varied with respect to each plant 150. In some embodiments, the timing of gas delivery is varied with respect to each plant 150. In some embodiments, gas content and timing is the same with respect to each plant 150.

[0028] In some embodiments, controller 110 obtains the delivery and sampling information (e.g., from a storage unit containing the information, or from direct entry of the information by a user of system 100) and adjusts the volume and delivery times of the one or more gases by selective activation of manifold 124. If gas source 122 includes multiple gas sources, such as, for example, ethlyene and compressed air, controller 110 selectively delivers the proper gas from one or more corresponding reservoirs via manifold 124. In addition, in various embodiments, controller 110 adjusts the sampling volume and times of gas removal from sample volume 146, such as, e.g., for sampling via detector 126. In this manner, controller 110 is capable of implementing a complex program of gas delivery, gas sampling, and gas removal for plant 150. In addition, plant-specific measurements for each plant can be obtained, and plants 150 can be maintained in specific environments that can change over time, such as, for example, to mimic different environmental conditions or various experimental protocols.

[0029] Controller 110 can include computing device 400 that includes one or more processors 402, memory 404, a storage device 406 and interfaces 408 for interconnection, such as, for example, as shown in FIG. 14. The processor 402 can process instructions for execution within the controller, including instructions stored in the memory 404 or on the storage device 406. For example, the instructions can instruct the processor 402 to perform any of the analysis and control steps disclosed herein. The memory 404 can storeexecutable instructions for processor 402, information about parameters of the system and the test protocol. The storage device 406 can be a computer-readable medium, such as a floppy disk device, a hard disk device, an optical disk device, or a tape device, a flash memory or other similar solid state memory device, or an array of devices, including devices in a storage area network or other configurations. The storage device 406 can store instructions that can be executed by processor 402 described above, and any of the other information that can be stored by memory 404.

[0030] In some embodiments, computing device 400 can include a graphics processing unit to display graphical information (e.g., using a GUI (graphical user interface) or text interface) on an external input / output device, such as display 416. The graphical information can be displayed by a display device (e.g., a CRT (cathode ray tube) or LCD (liquid crystal display) monitor) for displaying any of the information, such as measured and calculated spectra and images, disclosed herein. A user can use input devices (e.g., keyboard, pointing device, touch screen, speech recognition device) to provide input to controller 110, such as, e.g., via computing device 400. In some implementations, computing device 400 includes one or more interfaces, for example, low-speed interface 412 and high-speed interface 408, and one or more expansion ports 410, 414.

[0031] A user of system 100 can provide a variety of different types of instructions and information to controller 110 via input devices. The instructions and information can include, for example, reference information such as: gas concentrations, gas delivery timing, gas removal timing, sample number, sampling timing, calibration information for performing any of the assays described herein, and calibration information used by controller 110 to calibrate any of the detectors and sensors described herein. Controller 110 can use any of these various types of information to perform the methods and functions described herein. It should also be noted that any of these types of information can be stored (e.g., in storage device 406) and recalled when needed by controller 110.

[0032] Computing device 400 may be implemented in a number of different forms, as shown in FIG. 14. For example, it may be implemented as a standard server 420, or multiple times in a group of such servers. In addition, it may be implemented in a personal computer such as a laptop computer 422. It may also be implemented as part of a rack server system 424. Alternatively, components from the computing device 400 may be combined with other components in a mobile device, such as a mobile computing device.

[0033] FIGS. 7A-7B show an example gas sampling path of system 100 from plant 150 to detector 112 via manifold 124. As shown in FIGS. 7A-7B, in various embodiments, manifold124 can be in fluid communication with sample volume 146 within cover 142 disposed over plant 150, such as via gas line 106. In addition, manifold 124 can be in fluid communication with one or more detectors 112, such as, for example, via gas line 106. As such, manifold 124 can sample gas from plant 150 and deliver the sample to detector 112. Detector 112 can be any apparatus suitable for collecting measurements of interest, such as, for example, a highspeed detector, such as, for example, a cavity ring-down spectrometer. As shown in FIGS. 7A-7B, in various embodiments, manifold 124 has valves 124a-124o corresponding to each plant 150a-150o, which allows for collection of samples of gas from each plant 150. Valves 124a-124o are in fluid communication with sample volume 146a-146o within cover 142a- 142o, such as, for example via gas line 106a-106o. For example, as shown in FIG. 7A, valve 124a is in fluid communication with sample volume 146a within cover 142a disposed over plant 150a, such as, for example, via gas line 106a. In addition, as shown in FIG. 7B, valve 124b is in fluid communication with sample volume 146b within cover 142b disposed over plant 150b, such as, for example, via gas line 106b.

[0034] Any suitable number of valves may be used, for example, in some embodiments, manifold 124 has a number of valves that corresponds to the number of individual plants provided in container 130. In the examples shown in FIGS. 7A-7B, manifold 124 has fifteen valves that correspond to the fifteen plants contained in container 130; however, it will be understood that any suitable number of valves and plants are within the scope of the systems and methods set forth herein. As shown in FIGS. 7A-7B, each valve 124 is adapted to sample gas from a plant 150. Alternatively, in some embodiments, each valve 124 could be adapted to sample gas from more than one plants 150, such as, for example, via connections to the sample volume 146 of more than one plant 150.

[0035] In some embodiments, system 100 includes a detector 126. Detector 126 in FIGS. 7A-7B is implemented as a cavity ring-down spectrometer. To analyze gases from plant 150, such as gases from sample volume 146, controller 110 opens valve 124a, admitting gas from sample volume 146a into gas line 106a. The admitted gas propagates through gas line 106a and enters detector 126, where it is analyzed. Analytical data can be displayed via one or more local or remote hosts.

[0036] In general, detector 126 is configured to generate a measurement signal in response to the presence of one or more different gas species within sample volume 146. Detector 126 can be configured to detect a single type of gas, multiple types of gases, and one or more different properties of the gas(es). In certain embodiments, for example, detector 126 includes a detector that is highly sensitive and capable of real-time measurements, such as,for example, a detector capable of laser absorption spectroscopy. In some embodiments, the detector 126 is a high-speed sensitive spectrometer, such as, for example, a cavity ring-down spectrometer. In various embodiments, detector 126 is an ethylene cavity ring-down spectrometer.

[0037] In order to evaluate microbial nitrogen-fixation activity, the systems and methods can, in some embodiments, include a gas sampling manifold and control software integrated into detector 126 suitable for high-speed sensitive analysis of ethylene. As such, in various embodiments, detector 126 is configured to generate a measurement signal representing an amount or concentration of ethylene gas in sample volume 146. Because ethylene gas is produced as a by-product of nitrogenase activity, detection of ethylene gas can be used to evaluate microbial nitrogen-fixation activity during a growth cycle of plant 150.

[0038] As shown in FIGS. 7A-7B, in some embodiments, controller 110 activates manifold 124 to sample gas from plant 150. For example, in some embodiments, controller 110 can activate manifold 124 to open valve 124a, facilitating gas sampling from plant 150a via gas line 106a that can be in fluid communication with cover 142a disposed over plant 150a. As shown in FIGS. 7A-7B, vacuum 128 can pull gas from plant 150 through detector 126. Once gas sampling from plant 150a is completed, controller 110 can activate manifold 124 to close valve 124a and then open valve 124b, facilitating gas sampling from plant 150b via gas line 106b that can be in fluid communication with cover 142b disposed over plant 150b.Controller 110 can continue to activate manifold 124 to open and close valves to sample gas from plants 150 until gas sampling is complete. As such, in some embodiments, gas sampling from each plant 150 can be individualized during the course of the experiments. In some embodiments, gas sampling is varied with respect to each plant 150. In some embodiments, gas sampling is the same with respect to each plant 150.

[0039] In some embodiments, system 100 can optionally include a vacuum 128. Vacuum 128 can be implemented in any suitable manner. For example, in some embodiments, vacuum 128 can include a pump or other equipment suitable for moving gas through system 100. In various embodiments, vacuum 128 can remove gas from system 100 or can recirculate gas or otherwise move gas in any suitable manner.

[0040] FIG. 8 is a plan view of an example of a system 100 for real-time in planta assays, the system 100 having multiple chambers 102 and a bridge 160. As shown in FIG. 8, chamber 102 has walls 102a-102f that enclose spatial volume 104 internal to chamber 102, and one or more supports 116 as shown in FIG. 2. As shown in FIG. 8, each support 116 holds a plurality of multi-plant containers 130 that each contain a plurality of plants 150. In variousembodiments, each plant 150 can be contained in a cover 142 that has walls 144 forming sample volume 146 and that is sealed around plant 150. As shown in FIG. 8, one or more gas lines 106 are at least partially disposed within sample volume 146. In addition, the one or more gas lines 106 can be managed via organizer 140. As described herein, gas line 106 can be connected to manifold 124 which can be connected to controller 110. In various embodiments, controller 110 can instruct manifold 124 to push one or more gases for delivery to sample volume 146, pull gas for sampling, or pull gas for removal from sample volume 146 via gas line 106. As shown in FIG. 8, bridge 160 can contain various elements of system 100, including controller 110, manifold 124, one or more gas sources 122, one or more vacuums 128, one or more detectors 126, or any aspects of system 100.

[0041] In various embodiments, system 100 further includes one or more sensors for determining temperature, altitude, light, humidity, plant growth, soil conditions, chemical presence, microbe colonization, or any other suitable analytics. In various embodiments, system 100 can include one or more devices suitable for manipulating the environment of spatial volume 104 within chamber 102, such as, for example, a heating element, a cooling element, a light source, a humidifier, a de-humidifier, or any other device suitable for manipulating the test plant environment. Such sensors and devices can be controlled via controller 110 as appropriate.Detection of Microbial Nitrogen-Fixation

[0042] In various embodiments, the systems described herein can be used to detect and evaluate the nitrogen-fixating activity of engineered microbes. For example, the systems described herein can be used to detect and evaluate the nitrogen-fixation activity of engineered microbes, facilitating the selection of microbes that best match the desired criteria. In various examples, the systems described herein can be used to detect, evaluate, and compare the nitrogen-fixation activity of a variety of engineered microbes during a single test period. In addition, the systems described herein can be used to monitor the nitrogen-fixation activity in a large number of plants during a single test period. Furthermore, the systems described herein can be used to detect, evaluate, and compare the nitrogen-fixation activity of a variety of engineered microbes in a large number of plants in various environmental conditions.

[0043] FIG. 9 is a flow chart 200 that shows a series of example steps for detecting and evaluating the biological nitrogen fixation activity of engineered microbes. In a first step 202, the soil of a test plant or a seed precursor of a test plant is inoculated with a composition that includes at least one bacterium of a candidate bacterial strain. In some embodiments,inoculation involves contacting the soil or other suitable growth medium with the candidate bacterial strain. In some embodiments, inoculation involves contacting the seed or plant directly with the candidate bacterial strain. Inoculation can be completed using any suitable techniques, and can be automated, semi-automated, or manual. The candidate bacteria strain can be provided in any suitable form, such as in an aqueous suspension, in dried form, or granular form, or any other form suitable for inoculating the soil, the seed, or the plant, as appropriate. The test plant or a seed precursor of a test plant can be planted in the soil or other suitable growing medium before, after, or simultaneously with the inoculation in step 202.

[0044] Any suitable plant can be used in the assays. In some embodiments, the microbe is applied and the plant or seed precursor is grown for a period of time before the assay begins. For example, in some embodiments, one or more microbial strains is applied to soil or to a seed and the seed is planted. Then, plants that are at least one-week old (e.g., at least two-weeks old, at least three-weeks old, at least four-weeks old, or any other suitable age) are used in the assays.

[0045] It should be noted that a reference plant can be used in the assays described herein. The reference plant can be any suitable reference, such as, for example, a plant that is not inoculated with the candidate bacterial strain, a plant that is inoculated with a wild-type microbe, a plant that is inoculated with a strain without nitrogen-fixing activity, an engineered microbe with known biological nitrogen fixation activity, or any other suitable reference plant. In various embodiments, more than one reference plant is used in the assay.

[0046] Next, in step 204, a cover 142 is positioned over each plant such that cover 142 is gas-tight and a gas line is positioned in fluid communication with cover 142, such as, for example, disposed within cover 142. In step 206, the test and reference plants are positioned within chamber 102, and in step 208, controller 110 pushes acetylene at a suitable concentration into each cover 142 . During the test period, manifold 118 periodically draws gas from within cover 142 and directs it to detector 126, such as, for example, to a high-speed spectrometer, which measures the concentration of ethylene. Next, in step 212, microbial nitrogen fixation activity is calculated. Results can be displayed as shown in step 214, such as, for example, in real-time. In various embodiments, the system can be calibrated before or during step 110 or step 112. The calibration can include determining leak rate, background ethylene rate, or any other parameters helpful or necessary for accurately calculating microbial nitrogen fixation activity for each test sample. In variousembodiments, the system can be calibrated prior to monitoring. In addition, or alternatively, the system can be calibrated during the monitoring step. The calibration can be a single event or can be repeated throughout the test period.

[0047] In various embodiments, steps 210, 212, and, optionally, 214, can be repeated during the test period at suitable intervals, such as, for example, every hour, every 3 hours, every 6 hours, every 9 hours, every 12 hours, every 15 hours, every 18 hours, every 21 hours, every 24 hours, every 36 hours, every 48 hours, or any other suitable interval. In addition, the method provides for individual analysis of each plant during the test period, and steps 210, 212, and optionally, 214, can be repeated on the same or different intervals for different plants. In general, the test period can be selected as desired. For example, in some embodiments, the test period is at least 1 days (e.g., at least 2 days, at least 3 days, or the like). In certain embodiments, the test period is 7 days or less (e.g., 6 days or less, 5 days or less, 4 days or less, 3 days or less, 2 days or less, or 1 day or less). In various embodiments, the test period is approximately 2 days. In one example, the test period is approximately 96 hours. In another example, the test period is approximately 72 hours. In another example, the test period is selected such that each chamber 102 can run 2 tests per week.

[0048] Any suitable number of plants can be analyzed during a given test period. For example, in various embodiments, chamber 102 can assay more than 25 plants (e.g., more than 50 plants, more than 75 plants, more than 100 plants, more than 120 plants, more than 160 plants, more than 180 plants, more than 200 plants, more than 250 plants, more than 300 plants, or more). In addition, multiple chambers 102 can be utilized during the same test period. In one example, two chambers 102 are used during the test period, each containing 180 plants. In another example, two chambers 102 are used during the test period, each containing 250 plants. In yet another example, four chambers 102 are used during the test period each containing 200 plants.

[0049] In some embodiments, as described above, a seed precursor of the test plant is inoculated with the candidate bacterial strain. In these circumstances, germination of the seed can be carried out external to chamber 102 to yield a test plant which is then positioned within chamber 102. To germinate the inoculated seed, the seed can be deposited in soil or other suitable growth medium to induce germination, yielding the test plant.

[0050] The time at which the test plant is positioned within chamber 102 following germination can generally be selected based on factors such as the size of the plant and the point(s) in the plant's growth cycle during which assessment of the nitrogen-fixingeffectiveness of candidate bacterial strains is of interest. In some embodiments, for example, the test plant can be positioned within chamber 102 at least 7 days (e.g.,at least 10 days, at least 12 days, at least 14 days, at least 16 days, at least 18 days, at least 21 days, at least 24 days, at least 27 days, at least 30 days) following germination of its precursor seed. In some embodiments, the test plant can be positioned within chamber 102 four weeks or less (e.g., 26 days or less, 24 days or less, 22 days or less, 20 days or less, 18 days or less, 16 days or less, 14 days or less, 12 days or less, 10 days or less, 8 days or less, 7 days or less, 6 days or less, 5 days or less) following germination of its precursor seed. In various embodiments, the test plant can be positioned within chamber 102 about 21 days after germination. Alternatively, the test plant can be positioned within chamber 102 about 14 days after germination. In various embodiments, the test plant can be positioned within chamber 102 about 7 days after germination.

[0051] The concentration of acetylene pushed to each plant can be any suitable concentration, such as, for example, a concentration sufficient to allow detection of changes in ethylene production without harming the test plant. Example suitable concentrations of acetylene include a low concentration such as less than about 2% acetylene, less than about 1.5% acetylene, less than about 1.25% acetylene, less than about 1% acetylene, less than about 0.5% acetylene, or any other suitable concentration.

[0052] The methods disclosed herein can be implemented by controller 110 by executing instructions in one or more computer programs that are executable and / or interpretable by the controller 110. These computer programs (also known as programs, software, software applications or code) include machine instructions for a programmable processor, and can be implemented in a high-level procedural and / or object-oriented programming language, and / or in assembly / machine language. For example, computer programs can contain the instructions that can be stored in memory 404, in storage unit 406 , and / or on a tangible, computer-readable medium, and executed by processor 402 as described above. In some embodiments, the one or more computer programs can automate one or more processes of any of the systems and methods described herein, such as, for example, gas addition, gas sampling, gas analysis, data visualization, environmental aspects such as temperature, light, humidity, or the like, inoculation, or any other suitable process. Any suitable processes can be automated, such as for example, plant fertilization, including timing, dose, and variety; watering, including timing, quantity, and method; plant growth parameters; and environment, including humidity, temperature, and light intensity.

[0053] The methods can include determining a relative measurement of nitrogen fixation by the at least one nitrogen-fixing bacterium. Determining the relative measurement of nitrogen fixation can include: inoculating the plant using a suitable method, such as, for example, drench, seed treating, or mixing into soil; activating an acetylene gas source to deliver a quantity of acetylene to a portion of the support medium; after an exposure interval, measuring an amount of ethylene generated by the at least one nitrogen-fixing bacterium from the quantity of acetylene; and determining a rate of acetylene reduction by the at least one nitrogen-fixing bacterium based on the amount of ethylene generated.

[0054] Embodiments of the methods can also include any of the other features described herein, including any combinations of features described in connection with different embodiments, except as expressly stated otherwise.Nitrogen-Fixing Microbes

[0001] The systems and methods described herein can be used to determine the nitrogenfixing activity of any suitable microbe. The microbe can be naturally occurring or can be modified, such as, for example, a genetically engineered microbe. In some aspects, the one or more microbes are non-intergeneric remodeled bacteria capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen.

[0002] The microbial products can include any type of microbe, including bacteria and yeast, that is naturally occurring or genetically engineered. In some cases, the nitrogen-fixing capacity of a microbial product containing Agrobacterium radiobacter, Bacillus acidocaldarius, Bacillus acidoterrestris, Bacillus agri, Bacillus aizawai, Bacillus albolactis, Bacillus a lea lophilus. Bacillus alvei, Bacillus aminoglucosidicus, Bacillus aminovorans, Bacillus amylolyticus (also known as Paenibacillus amylolyticus) Bacillus amyloliquefaciens, Bacillus aneurinolyticus, Bacillus atrophaeus, Bacillus azotoformans, Bacillus badius, Bacillus cereus (synonyms: Bacillus endorhythmos, Bacillus medusa), Bacillus chitinosporus, Bacillus circulans, Bacillus coagulans, Bacillus endoparasiticus Bacillus fastidiosus, Bacillus firmus, Bacillus kurstaki, Bacillus lacticola, Bacillus lactimorbus, Bacillus lactis, Bacillus laterosporus (also known as Brevibacillus laterosporus), Bacillus lautus, Bacillus lentimorbus, Bacillus lentus, Bacillus licheniformis, Bacillus maroccanus, Bacillus megaterium, Bacillus metiens, Bacillus mycoides, Bacillus natto, Bacillus nematocida, Bacillus nigrificans, Bacillus nigrum, Bacillus pantothenticus, Bacillus popillae, Bacillus psychrosaccharolyticus, Bacillus pumilus, Bacillus siamensis, Bacillus smithii, Bacillus sphaericus, Bacillus subtilis, Bacillus thuringiensis, Bacillus uniflagellatus, Bradyrhizobium japonicum, Brevibacillus brevis Brevibacillus laterosporus (formerly Bacillus laterosporus), Chromobacterium subtsugae, Delftiaacidovorans, Klebsiella variicola, Kosokonia sacchari, Lactobacillus acidophilus, Lysobacter antibioticus, Lysobacter enzymogenes, Paenibacillus alvei, Paenibacillus polymyxa, Paenibacillus popilliae (formerly Bacillus popilliae), Pantoea agglomerans, Pasteuria penetrans (formerly Bacillus penetrans), Pasteuria usgae, Pectobacterium carotovorum (formerly Erwinia carotovora), Pseudomonas aeruginosa, Pseudomonas aureofaciens, Pseudomonas cepacia (formerly known as Burkholderia cepacia), Pseudomonas chlororaphis, Pseudomonas fluorescens, Pseudomonas proradix, Pseudomonas putida, Pseudomonas syringae, Serratia entomophila, Serratia marcescens, Streptomyces colombiensis, Streptomyces galbus, Streptomyces goshikiensis, Streptomyces griseoviridis, Streptomyces lavendulae, Streptomyces prasinus, Streptomyces saraceticus, Streptomyces venezuelae, Xanthomonas campestris, Xenorhabdus luminescens, Xenorhabdus nematophila, Rhodococcus globerulus AQ719 (NRRL Accession No. B-21663), Bacillus sp. AQ.175 (ATCC Accession No. 55608), Bacillus sp. AQ. 177 (ATCC Accession No. 55609), Bacillus sp. AQ178 (ATCC Accession No. 53522), or Streptomyces sp. strain NRRL Accession No. B- 30145, or any combination thereof, can be determined. In some cases, the nitrogen-fixing capacity can be determined of a microbial product containing Azotobacter chroococcum, Methanosarcina barkeri, Klesiella pneumoniae, Azotobacter vinelandii, Rhodobacter spharoides, Rhodobacter capsulatus, Rhodobcter palustris, Rhodosporillum rubrum, Rhizobium leguminosarum, or Rhizobium etli, or any combination thereof.

[0003] In some cases, the nitrogen-fixing capacity can be determined of a microbial product containing cyanobacteria such as a species from Anabaena (for example Anagaena sp. PCC7120), Nostoc (for example Nostoc punctiforme), or Synechocystis (for example Synechocystis sp. PCC6803), or any combination thereof.

[0004] In some embodiments, the genetically engineered microbe is one or more genetically engineered bacteria. In some cases, the one or more bacteria is a bacterium comprising a genetic variation in a nitrogen fixation gene or a nitrogen-assimilation gene. In some cases, the one or more bacteria is of a genus independently selected from Kosakonia, Rahnella, Klebsiella, Paenibacillus, Paraburkholderia, and Herbaspirillum.Genetically-Engineered Nitrogen-Fixing Microbes

[0005] Microbes can be engineered to improve nitrogen-fixing activity. Thus, in some aspects, the microbes comprise one or more genetic variations introduced into one or more genes regulating nitrogen fixation. For example, in some microbes, as shown in FIG. ID, NifA is the transcriptional activator of the nitrogenase complex and its accessory genes. Under abundant nitrogen sources, NifL protein inhibits NifA's transcriptional activity. This inhibitionis alleviated when low glutamine levels activate the urydylyl-transferase activity of GlnD, the glutamine-sensing bifunctional protein. GlnD, in turn, urydylylates PH signaling proteins, which represses the inhibitory effect of NifL and increases the expression of NifA. Active NifA leads to the expression of nif regulon and biological nitrogen fixation. The uridylated PH proteins also activate the adenylyl removal function of the GlnE protein, which in turn activates the glutamine synthetase (G I n A) activity for the assimilation of the fixed N via glutamine. Increased glutamine levels trigger the uridylyl removal activity of GlnD, which removes the urydylyl groups from the Pll signaling proteins. As such, in one example, microbes are genetically engineered in one or more gene targets to bypass the regulations and enable constitutive biological nitrogen fixation and ammonium excretion. Example gene modifications suitable for improving nitrogen fixation are showin in FIG. IE.

[0006] In some embodiments, the microbe has a disrupted (e.g., deleted or partially deleted) nif L gene. In some aspects, the microbe has a nif L gene that has been disrupted with the introduction of a promoter sequence that acts on the nifA gene. In some aspects, e.g., when the microbe is a strain of K. variicola, the promoter is a K. variicola PinfC promoter. In some aspects, e.g., when the microbe is a strain of K. sacchari, the promoter is a K. sacchari Prm5 promoter. In some aspects, the microbe has a glnE gene that has been altered to remove the adenylyl-removing (AR) domain, while leaving the coding region for the adenyltransferase (AT) domain, which is functionally expressed. In some aspects, the microbe has a deletion of the glnD gene.

[0007] The genetic variation introduced into one or more microorganisms may be a knockout mutation or it may abolish a regulatory sequence of a target gene, or it may comprise insertion of a heterologous regulatory sequence, for example, insertion of a regulatory sequence found within the genome of the same bacterial species or genus. The regulatory sequence can be chosen based on the expression level of a gene in a bacterial culture or within plant tissue. The genetic variation may be produced by chemical mutagenesis. The plants grown may be exposed to biotic or abiotic stressors. However, in some aspects, the one or more cultured microbes for use with the compositions and methods disclosed herein also envision altering the impact of ATP or 02 on the circuitry, or replacing the circuitry with other regulatory cascades in the cell, or altering genetic circuits other than nitrogen fixation. Gene clusters can be re-engineered to generate functional products under the control of a heterologous regulatory system. By eliminating native regulatory elements outside of, and within, coding sequences of gene clusters, and replacing them with alternative regulatory systems, the functional products of complex genetic operons and other gene clusters can becontrolled and / or moved to heterologous cells, including cells of different species other than the species from which the native genes were derived. Once re-engineered, the synthetic gene clusters can be controlled by genetic circuits or other inducible regulatory systems, thereby controlling the products' expression as desired. The expression cassettes can be designed to act as logic gates, pulse generators, oscillators, switches, or memory devices. The controlling expression cassette can be linked to a promoter such that the expression cassette functions as an environmental sensor, such as an oxygen, temperature, touch, osmotic stress, membrane stress, or redox sensor.

[0008] As an example, the nif L, nifA, nifT, and nifX genes can be eliminated from the nif gene cluster. Synthetic genes can be designed by codon randomizing the DNA encoding each amino acid sequence. Codon selection is performed, specifying that codon usage be as divergent as possible from the codon usage in the native gene. Proposed sequences are scanned for any undesired features, such as restriction enzyme recognition sites, transposon recognition sites, repetitive sequences, sigma 54 and sigma 70 promoters, cryptic ribosome binding sites, and rho independent terminators. Synthetic ribosome binding sites are chosen to match the strength of each corresponding native ribosome binding site, such as by constructing a fluorescent reporter plasmid in which the 150 bp surrounding a gene's start codon (from -60 to +90) is fused to a fluorescent gene. This chimera can be expressed under control of the Ptac promoter, and fluorescence measured via flow cytometry. To generate synthetic ribosome binding sites, a library of reporter plasmids using 150 bp (-60 to +90) of a synthetic expression cassette is generated. Briefly, a synthetic expression cassette can consist of a random DNA spacer, a degenerate sequence encoding an RBS library, and the coding sequence for each synthetic gene. Multiple clones are screened to identify the synthetic ribosome binding site that best matched the native ribosome binding site.Synthetic operons that consist of the same genes as the native operons are thus constructed and tested for functional complementation. A further exemplary description of synthetic operons is provided in US20140329326.

[0009] In some embodiments, the genetic variation may be introduced into a gene selected from the group consisting of nifA, nif L, ntrB, ntrC, glutamine synthetase, glnA, glnB, glnK, draT, amtB, glutaminase, glnD, glnE, nifJ, nifH, nifD, n if K, nifY, nifE, nifN, nifl), nifS, nifV, nifW, nifZ, nifM, nif F, nif B, and nifQ.. The genetic variation may be a variation in a gene encoding a protein with functionality selected from the group consisting of: glutamine synthetase, glutaminase, glutamine synthetase adenylyltransferase, transcriptional activator, anti- transcriptional activator, pyruvate flavodoxin oxidoreductase, flavodoxin, and NAD+-dinitrogen-reductase aDP-D-ribosyltransferase. The genetic variation may be a mutation that results in one or more of: increased expression or activity of nifA or glutaminase; decreased expression or activity of nifL, ntrB, glutamine synthetase, glnB, glnK, draT, amtB; decreased adenylyl-removing activity of GInE; decreased expression of GlnD; or decreased uridylyl- removing activity of GlnD. The genetic variation may be a variation in a gene selected from the group consisting of: bcsii, bcsiii, yjbE, fhaB, pehA, otsB, treZ, glsA2, and combinations thereof.

[0010] Some examples of genetic alterations that can be made in Gram-positive microbes include: deleting glnR to remove negative regulation of BNF in the presence of environmental nitrogen, inserting different promoters directly upstream of the nif cluster to eliminate regulation by GlnR in response to environmental nitrogen, mutating glnA to reduce the rate of ammonium assimilation by the GS-GOGAT pathway, deleting amtB to reduce uptake of ammonium from the media, mutating glnA so it is constitutively in the feedback- inhibited (FBI-GS) state, to reduce ammonium assimilation by the GS-GOGAT pathway.

[0011] GlnR is the main regulator of N metabolism and fixation in, e.g., Paenibacillus species. In some aspects, the genome of a Paenibacillus species does not contain a gene to produce glnR. In some aspects, the genome of a Paenibacillus species does not contain a gene to produce glnE or glnD. In some aspects, the genome of a Paenibacillus species does contain a gene to produce glnB or glnK. For example, Paenibacillus sp. WLY78 doesn't contain a gene for glnB, or its homologs found in the archaeon Methanococcus maripaludis, nifll and nifl2. In some aspects, the genomes of Paenibacillus species are variable. For example, Paenibacillus polymixa E681 lacks glnK and gdh, has several nitrogen compound transporters, but only amtB appears to be controlled by GlnR. In another example, Paenibacillus sp. JDR2 has glnK, gdh and most other central nitrogen metabolism genes, has many fewer nitrogen compound transporters, but does have glnPHQ controlled by GlnR. Paenibacillus riograndensis SBR5 contains a standard glnRA operon, an fdx gene, a main nif operon, a secondary nif operon, and an anf operon (encoding iron-only nitrogenase). Putative glnR / tnrA sites were found upstream of each of these operons. GlnR may regulate all of the above operons, except the anf operon. GlnR may bind to each of these regulatory sequences as a dimer.

[0012] Paenibacillus N-fixing strains may fall into two subgroups: Subgroup I, which contains only a minimal nif gene cluster and subgroup II, which contains a minimal cluster, plus an uncharacterized gene between nifX and hesA, and often other clusters duplicating some ofthe nif genes, such as n if H, nifHDK, nifBEN, or clusters encoding vanadaium nitrogenase (vnf) or iron-only nitrogenase (anf) genes.

[0013] In some embodiments, the genome of a Paenibacillus species may not contain a gene to produce glnB or glnK. In some aspects, the genome of a Paenibacillus species may contain a minimal nif cluster with 9 genes transcribed from a sigma-70 promoter. In some aspects, a Paenibacillus nif cluster is negatively regulated by nitrogen or oxygen. In some aspects, the genome of a Paenibacillus species does not contain a gene to produce sigma-54. For example, Paenibacillus sp. WLY78 does not contain a gene for sigma-54. In some aspects, a nif cluster is regulated by glnR, and / or TnrA. In some aspects, activity of a nif cluster is altered by altering activity of glnR, and / or TnrA.

[0014] In Bacilli, glutamine synthetase (GS) is feedback-inhibited by high concentrations of intracellular glutamine, causing a shift in confirmation (referred to as FBI-GS). Nif clusters contain distinct binding sites for the regulators GlnR and TnrA in several Bacilli species. GlnR binds and represses gene expression in the presence of excess intracellular glutamine and AMP. A role of GlnR may be to prevent the influx and intracellular production of glutamine and ammonium under conditions of high nitrogen availability. TnrA may bind and / or activate (or repress) gene expression in the presence of limiting intracellular glutamine, and / or in the presence of FBI-GS. In some embodiments, the activity of a Bacilli nif cluster is altered by altering the activity of GlnR.

[0015] Feedback-inhibited glutamine synthetase (FBI-GS) may bind GlnR and stabilize binding of GlnR to recognition sequences. Several bacterial species have a GlnR / TnrA binding site upstream of the nif cluster. Altering the binding of FBI-GS and GlnR may alter the activity of the nif pathway.

[0016] In some embodiments, the microbes are non-intergeneric remodeled microbes. The term "non-intergeneric" indicates that the genetic variations introduced into the host do not contain nucleic acid sequences from outside the host genus. In some embodiments, the microbes are intragenic. Therefore, in some embodiments, the microbes are not transgenic. For example, for non-transgenic microbes with varied promoters, promoters for promoter swapping are selected from within the microbe's genome, or genus.

[0017] Exemplary non-intergeneric genetic variations include a mutation in the gene of interest that may improve the function of the protein encoded by the gene; a constitutionally active promoter that can replace the endogenous promoter of the gene of interest to increase the expression of the gene; a mutation that will inactivate the gene of interest; the insertion of a promoter from within the host's genome into a heterologouslocation, e.g. insertion of the promoter into a gene that results in inactivation of said gene and upregulation of a downstream gene; and the like. The mutations can be point mutations, insertions, and / or deletions (full or partial deletion of the gene). For example, in some embodiments, to improve the nitrogen fixation activity of the host microbe, a genetic variation may comprise an inactivating mutation of the n if L gene (negative regulator of nitrogen fixation pathway) and / or comprise replacing the endogenous promoter of the nifA and / or nifH gene (nitrogenase iron protein that catalyzes a key reaction to fix atmospheric nitrogen) with a constitutionally active promoter that will drive the expression of the nifA and / or nifH gene constitutively.

[0018] In some cases, the systems and methods provided herein can be used to determine the nitrogen-fixing capacity of applied or supplemented bacteria that comprise at least one modification in a gene regulating nitrogen fixation or assimilation. For example, the methods provided herein can be used to determine the nitrogen-fixing capacity of one or more applied or supplemented strains of Rahnella aquatilis, Kosakonia sacchari, Kosakonia arachidis, Klebsiella variicola, Paraburkholderia tropica, Herbaspirillum seropedicae, Herbaspirillum aquaticum, and Paenibacillus polymyxa, wherein each comprise at least one modification in a gene regulating nitrogen fixation or assimilation. In some embodiments, a microbe with known nitrogen-fixing capacity can be employed as a control, such as, for example, Kosakonia sacchari strain identified by American Type Culture Collection (ATCC) Accession number PTA-126743 and the nitrogen-fixing capacity of an applied or supplemented Klebsiella variicola strain identified by ATCC Accession No. PTA-126740. Additional examples of microorganisms and genetic modifications suitable for use with the compositions and methods of the present disclosure may be found in International Patent Application Nos. PCT / US2019 / 039528, PCT / US2019 / 064782, PCT / US2021 / 13120, PCT / US2021 / 029993, and PCT / US2019 / 041429, PCT / US2019 / 039528; International Patent publication Nos. W02020 / 118111, and W02020 / 014498, the contents of which are herein incorporated by reference in their entirety.Plant Species

[0019] Any suitable plant can be used with any of the systems and methods described herein. In some embodiments, the plant is any plant that have economic, social and / or environmental value, such as food crops, fiber crops, oil crops, plants in the forestry or pulp and paper industries, feedstock for biofuel production and / or ornamental plants. Nonlimiting examples of crop plants include maize, rice, wheat, barley, sorghum, millet, oats, ryetriticale, buckwheat, sweet corn, sugar cane, onions, tomatoes, strawberries, and asparagus.For example, plants can be in the genus Hordeum, Oryza, Zea, and Triticeae.

[0020] In some examples, the plant is any plant that may be used to produce economically valuable products such as a grain, a flour, a starch, a syrup, a meal, an oil, a film, a packaging, a nutraceutical product, a pulp, an animal feed, a fish fodder, a bulk material for industrial chemicals, a cereal product, a processed human food product, a sugar, an alcohol, and / or a protein.

[0021] In some cases, the plant is a cereal plant. Non-limiting examples of cereal plants include corn plants, canola plants, sorghum plants, wheat plants, and sunflower plants,

[0022] In some cases, the plant is any plant that are important or interesting for agriculture, horticulture, biomass for the production of biofuel molecules and other chemicals, and / or forestry. Some examples of these plants include pineapple, bamboo, banana, coconut, lily, grass peas and grass; and dicotyledonous plants, such as, for example, peas, alfalfa, tomatillo, melon, chickpea, chicory, clover, kale, lentil, soybean, tobacco, potato, sweet potato, radish, cabbage, rape, apple trees, grape, cotton, sunflower, thale cress, canola, citrus (including orange, mandarin, kumquat, lemon, lime, grapefruit, tangerine, tangelo, citron, and pomelo), pepper, bean, lettuce, Panicum virgatum (switch), Sorghum bicolor (sorghum, Sudan), Miscanthus giganteus (miscanthus), Saccharum sp. (energycane), Populus balsamifera (poplar), Zea mays (corn), Glycine max (soybean), Brassica napus (canola), Triticum aestivum (wheat), Gossypium hirsutum (cotton), Oryza sativa (rice), Helianthus annuus (sunflower), Medicago sativa (alfalfa), Beta vulgaris (sugarbeet), Pennisetum glaucum (pearl millet), Panicum spp. Sorghum spp., Miscanthus spp., Saccharum spp., Erianthus spp., Populus spp., Secale cereale (rye), Salix spp. (willow), Eucalyptus spp. (eucalyptus), Triticosecale spp. (triticum- 25 wheat X rye), Carthamus tinctorius (safflower), Jatropha curcas (Jatropha), Ricinus communis (castor), Elaeis guineensis (oil palm), Phoenix dactylifera (date palm), Archontophoenix cunninghamiana (king palm), Syagrus romanzoffiana (queen palm), Linum usitatissimum (flax), Brassica juncea, Manihot esculenta (cassaya), Lycopersicon esculentum (tomato), Lactuca saliva (lettuce), Musa paradisiaca (banana), Solanum tuberosum (potato), Brassica oleracea (broccoli, cauliflower, brussel sprouts), Camellia sinensis (tea), Fragaria ananassa (strawberry), Theobroma cacao (cocoa), Coffea arabica (coffee), Vitis vinifera (grape), Ananas comosus (pineapple), Capsicum annum (hot & sweet pepper), Allium cepa (onion), Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima (squash), Cucurbita moschata (squash), Spinacea oleracea (spinach), Citrullus lanatus (watermelon), Abelmoschus esculentus (okra), Solanummelongena (eggplant), Papaver somniferum (opium poppy), Papaver orientale, Taxus baccata, Taxus brevifolia, Artemisia annua, Cannabis saliva, Camptotheca acuminate, Catharanthus roseus, Vinca rosea, Cinchona officinalis, Coichicum autumnale, Veratrum californica, Digitalis lanata, Digitalis purpurea, Dioscorea spp., Andrographis paniculata, Atropa belladonna, Datura stomonium, Berberis spp., Cephalotaxus spp., Ephedra sinica, Ephedra spp., Erythroxylum coca, Galanthus wornorii, Scopolia spp., Lycopodium serratum (Huperzia serrata), Lycopodium spp., Rauwolfia serpentina, Rauwolfia spp., Sanguinaria canadensis, Hyoscyamus spp., Calendula officinalis, Chrysanthemum parthenium, Coleus forskohlii, Tanacetum parthenium, Parthenium argentatum (guayule), Hevea spp. (rubber), Mentha spicata (mint), Mentha piperita (mint), Bixa orellana, Alstroemeria spp., Rosa spp. (rose), Dianthus caryophyllus (carnation), Petunia spp. (petunia), Poinsettia pulcherrima (poinsettia), Nicotiana tabacum (tobacco), Lupinus albus (lupin), Uniola paniculata (oats), Hordeum vulgare (barley), and Lolium spp. (rye).

[0023] In some examples, a monocotyledonous plant may be used. Monocotyledonous plants belong to the orders of the Alismatales, Arales, Arecales, Bromeliales, Commelinales, Cyclanthales, Cyperales, Eriocaulales, Hydrocharitales, Juncales, Lilliales, Najadales, Orchidales, Pandanales, Poales, Restionales, Triuridales, Typhales, and Zingiberales. For example, the methods described herein can be performed on plant(s) belonging to the class of Gymnospermae are Cycadales, Ginkgoales, Gnetales, and Pinales. In some examples, the monocotyledonous plant can be selected from the group consisting of a maize, rice, wheat, barley, and sugarcane.

[0024] In some examples, a dicotyledonous plant may be used, including those belonging to the orders of the Aristochiales, Asterales, Batales, Campanulales, Capparales, Caryophyllales, Casuarinales, Celastrales, Cornales, Diapensales, Dilleniales, Dipsacales, Ebenales, Ericales, Eucomiales, Euphorbiales, Fabales, Fagales, Gentianales, Geraniales, Haloragales, Hamamelidales, Middles, Juglandales, Lamiales, Laurales, Lecythidales, Leitneriales, Magniolales, Malvales, Myricales, Myrtales, Nymphaeales, Papeverales, Piperales, Plantaginales, Plumb aginales, Podostemales, Polemoniales, Polygalales, Polygonales, Primulales, Proteales, Rafflesiales, Ranunculales, Rhamnales, Rosales, Rubiales, Salicales, Santales, Sapindales, Sarraceniaceae, Scrophulariales, Theales, Trochodendrales, Umbellales, Urticales, and Violates.

[0025] Other non-limiting examples of suitable plants include mosses, lichens, and algae.

[0026] In some cases, the plant is any of a variety of transgenic plants, non-transgenic plants, and hybrid plants thereof. In some embodiments, the plant is any of a hybrid, variety,lineage, etc. of genetically modified maize plants, genetically modified sorghum plants, genetically modified cotton plants, genetically modified soybean plants, or part thereof, including varieties with genetic modifications to increase plant production, increase plant growth rate, increase plant yield, or the like.

[0027] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.EXAMPLESExample 1: Analysis of ethylene carryover in gas line materials.

[0028] Various materials were analyzed to determine the levels of ethylene carryover. As shown in FIG. 10, stainless steel demonstrated the lowest levels of ethylene carryover.

[0029] Example 2: Analysis of plant contribution to nitrogen fixation activity using an automated system.

[0030] An automated system was used to investigate plant contributions to nitrogen fixation activity. Ethylene levels were measured over approximately 36 hours in one-week old corn plants grown in topsoil and inoculated with nitrogen-fixing microbes in the following conditions: 1) without acetylene, 2) with acetylene, 3) with plant. Unplanted soil and soil with plant removed was also measured. As shown in FIG. 11, without the addition of acetylene, minimal ethylene is detected, showing that ethylene is primarily generated from microbial nitrogenase activity. As shown in FIG. 12, minimal ethylene is detected in unplanted soil or in soil with the plant removed, showing that nitrogenase activity is plantdependent.Example 3: Identification of nitrogen-fixing activity in a variety of microbes.

[0031] A high-throughput automated system was used to analyze multiple genetically modified strains from different microbial species in one-week old corn plants grown in topsoil. Eleven species of diazotrophs were analyzed, each with 1-4 different gene-edited candidates. A negative control was also analyzed. Soil was inoculated with microbial strains at planting. A cover and gas line were attached to each plant, and the plants were positioned in the chamber. Acetylene gas (1.25%) was delivered to each plant. Plants were analyzed for ethylene production every 6 hours over a 72-hour test period starting at 7 days after planting. Ten plants were analyzed per experiment. Ethylene production was determined and estimated nitrogen-fixation rates were calculated. As shown in FIG. 13, the automatedsystem identified microbes with a spectrum of nitrogen-fixing activity, shown in ppb / hr.Results from one experiment (ten plants) are shown for 2C and 10A-D, with the remaining data points showing results from two experiments (a total of twenty plants).

[0032] This disclosure is written to describe the invention to a person having ordinary skill in the art, who will understand that this disclosure is not limited to the specific examples or embodiments described. The examples and embodiments are single instances of the invention which will make a much larger scope apparent to the person having ordinary skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by the person having ordinary skill in the art. It is also to be understood that the terminology used herein is for the purpose of describing examples and embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0033] All the features disclosed in this specification (including any accompanying claims, abstract, and drawings) may be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features. The examples and embodiments described herein are for illustrative purposes only and various modifications or changes in light thereof will be suggested to the person having ordinary skill in the art and are to be included within the spirit and purview of this application. Many variations and modifications may be made to the embodiments of the disclosure without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.

[0034] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, or the method being employed to determine the value, or the variation that exists among the samples being measured. Unless otherwise stated or otherwise evident from the context, the term "about" means within 10% above or below the reported numerical value (except where such number would exceed 100% of a possible value or go below 0%). When used in conjunction with a range or series of values, the term "about" applies to the endpoints of the range or each of the valuesenumerated in the series, unless otherwise indicated. As used in this application, the terms "about" and "approximately" are used as equivalents.

[0035] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted.

[0036] Unless otherwise specified, all percentages indicating the amount of a component in a composition represent a percent by weight of the component based on the total weight of the composition.

[0037] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit (unless the context clearly dictates otherwise), between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

Claims

Claims1. A system for the analysis of microbial nitrogen-fixation in plants, the system comprising: a plurality of plants, each plant having a cover disposed over the plant, the cover forming a sample volume containing the plant; a gas source; a detector; a manifold in fluid communication with the gas source and with the detector, the manifold comprising a plurality of valves, wherein an individual valve is in fluid communication with an individual plant; and a controller, the controller being configured to activate the valves to regulate gas delivery from the gas source, and to activate individual valves to sample gas from each individual plant to the detector for analysis.

2. The system of claim 1, further comprising a chamber adapted to contain the plurality of plants.

3. The system of claim 2, wherein the chamber is climate-controlled.

4. The system of any of claims 1-3, wherein the plants are individually planted in soil in a container, the container being adapted to hold a plurality of plants.

5. The system of any of claims 1-4, wherein the controller is adapted to activate individual valves to deliver gas from the gas source to individual plants.

6. The system of any of claims 1-5, wherein the system is automated.

7. The system of any of claims 1-6, wherein the cover is a flexible material.

8. The system of any of claims 1-7, wherein the cover is a flexible thermoplastic material.

9. The system of any of claims 1-8, wherein the cover is hermetically sealed over the plant.

10. The system of any of claims 1-9, wherein the detector is a cavity ring-down spectrometer11. The system of any of claims 1-10, wherein the gas source comprises acetylene gas.

12. The system of any of claims 1-11, wherein the detector is configured to generate a measurement signal in response to a presence of one or more gas species.

13. The system of claim 12, wherein the one or more gas species comprise ethylene.

14. A method for the automated assay of microbial nitrogen-fixation in plants using the system of any of claims 1-13, the method comprising:(a) providing a plurality of test plants in soil inoculated with one or more microbes,(b) attaching a cover and a gas line to each plant, wherein each plant is individually connected to a gas line,(c) activating the manifold to open an individual valve in fluid communication with an individual plant and the detector such that gas flows from the individual plant to the detector for sampling,(d) activating the manifold to close the individual valve once gas sampling from the individual plant is complete, repeating steps c and d for each plant.

15. The method of claim 14, further comprising:(e) activating the manifold to open an individual valve in fluid communication with an individual plant such that gas flows from the gas source to the individual plant,(f) activating the manifold to close the individual valve once gas delivery to the individual plant is complete, repeating steps c and d for each plant.

16. The method of claim 14 or 15, further comprising generating a measurement signal in response to a presence of ethylene gas.

17. The method of any of claims 14-16, further comprising calculating nitrogen-fixation activity for each test plant.

18. The method of claim 17, further comprising displaying the nitrogen-fixation activity for each test plant.

19. A method of identifying a nitrogen-fixing microbial strain using the system of any of claims 1-13, the method comprising:(a) providing a plurality of test plants in soil inoculated with microbes from one or more test microbial strains,(b) providing at least one reference plant in soil inoculated with one or more control microbes,(c) attaching a cover and a gas line to each plant, wherein each plant is individually connected to a gas line,(d) activating the manifold to open an individual valve in fluid communication with an individual plant and the detector such that gas flows from the individual plant to the detector for sampling,(e) activating the manifold to close the individual valve once gas sampling from the individual plant is complete,(f) determining the relevant amounts of ethylene produced by each test plant compared to the reference plant over a test period, and(g) identifying the one or more microbial strains as nitrogen-fixing or non-nitrogen-fixing strains.

20. The method of claim 19, wherein the test period is 5 days or less, optionally 4 days or less, optionally 3 days or less, optionally 2 days or less.

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