A Biosensor-Based Leaf Punch Assay for Glutamine Correlates to Symbiotic Nitrogen Fixation Measurements in Legumes to Permit Rapid Screening of Rhizobia Inoculants under Controlled Conditions

Abstract

Legumes are protein sources for billions of humans and livestock. These traits are enabled by symbiotic nitrogen fixation (SNF), whereby root nodule-inhabiting rhizobia bacteria convert atmospheric nitrogen (N) into usable N. Unfortunately, SNF rates in legume crops suffer from undiagnosed incompatible/suboptimal interactions between crop varieties and rhizobia strains. There are opportunities to test much large numbers of rhizobia strains if cost/labor-effective diagnostic tests become available which may especially benefit researchers in developing countries. Inside root nodules, fixed N from rhizobia is assimilated into amino acids including glutamine (Gln) for export to shoots as the major fraction (amide-exporting legumes) or as the minor fraction (ureide-exporting legumes). Here, we have developed a new leaf punch based technique to screen rhizobia inoculants for SNF activity following inoculation of both amide exporting and ureide exporting legumes. The assay is based on measuring Gln output using the GlnLux biosensor, which consists of Escherichia coli cells auxotrophic for Gln and expressing a constitutive lux operon. Subsistence farmer varieties of an amide exporter (lentil) and two ureide exporters (cowpea and soybean) were inoculated with different strains of rhizobia under controlled conditions, then extracts of single leaf punches were incubated with GlnLux cells, and light-output was measured using a 96-well luminometer. In the absence of external N and under controlled conditions, the results from the leaf punch assay correlated with 15N-based measurements, shoot N percentage, and shoot total fixed N in all three crops. The technology is rapid, inexpensive, high-throughput, requires minimum technical expertise and very little tissue, and hence is relatively non-destructive. We compared and contrasted the benefits and limitations of this novel diagnostic assay to methods.

Challenges in Using Precision Agriculture to Optimize Symbiotic Nitrogen Fixation in Legumes: Progress, Limitations, and Future Improvements Needed in Diagnostic Testing

“…Different methodologies have been developed to quantify SNF in legumes under controlled and field environments [27][28][29][30][31]. Table 1 has summarized the current direct and indirect methods available to quantify SNF in legumes, categorized by their potential for adaption as precision agricultural tools in the short-medium term.…”

“…Table 1 has summarized the current direct and indirect methods available to quantify SNF in legumes, categorized by their potential for adaption as precision agricultural tools in the short-medium term. Each method has its own strengths and limitations [30,31]. Table 1.…”

“…A microbial biosensor, GlnLux, was recently reported which detects glutamine (gln) from leaf punch extracts under laboratory conditions [31]. GlnLux is a rapid, inexpensive, high-throughput, and relatively non-destructive method for measuring SNF output in amide-exporting legumes (e.g., lentil, alfalfa) and ureide exporting legumes (e.g., soybean, cowpea) (Table 1).…”

Técnicas aplicadas en la investigación agrícola para cuantificar la fijación de nitrógeno: una revisión sistemática

“…Para ello, es común que se utilicen sistemas acoplados que consisten en un espectofotómetro conectado a un sistema de combustión (horno) (Hamawaki & Kantartzi, 2018). Sin embargo, como alternativa se ha optado por el uso de espectros de reflactancia hoja/dosel para identificar el isómero 15 N (Thilakarathna, Moroz, & Raizada, 2017). El método se resume en la aplicación de soluciones con 1 a 10 % de exceso de átomos 15 N al suelo (Braun et al, 2018;Wambeke et al, 2018) o en el fertilizante (Kg N/ha) previo a la siembra, y se estima la relación de isótopos en las plantas, en el suelo, en los microrganismos y en el aire.…”

“…La glutamina (Gln) es una forma de transporte primario del nitrógeno una vez es absorbido por la raíz de la planta (Thilakarathna et al, 2017). En el ensayo GlnLux, la glutamina es detectada por biosensores que son células enteras que emiten luz cuando entran en contacto con este aminoácido.…”