Fluorescence sensing systems: In vivo detection of biophysical variations in field corn due to nitrogen supply

Abstract

Leaf and canopy fluorescence properties of field corn (Zea mays L.) grown under varying levels of nitrogen (N) fertilization were characterized to provide an improved N sensing capability which may assist growers in site-specific N management decisions. In vivo fluorescence emissions can occur in the wavelength region from 300 to 800 nm and are dependent on the wavelength of illumination. These light emissions have been grouped into five primary bands with maxima most frequently received from corn at 320 nm (UV), 450 nm (blue), 530 nm (green), 685 nm (red), and 740 nm (far-red). Two active fluorescence sensing systems have been custom developed; a leaf level Fluorescence Imaging System (FIS), and a canopy level Laser Induced Fluorescence Imaging System (LIFIS). FIS sequentially acquires high-resolution images of fluorescence emission bands under darkened laboratory conditions, while LIFIS simultaneously acquires four band images of plant canopies ≥1 m² under ambient sunlit conditions. Fluorescence emissions induced by these systems along with additional biophysical measures of crop condition; namely, chlorophyll content, N/C ratio, leaf area index (LAI), and grain yield, exhibited similar curvilinear responses to levels of supplied N. A number of significant linear correlations were found among band emissions and several band ratios versus measures of crop condition. Significant differences were obtained for several fluorescence band ratios with respect to the level of supplied N. Leaf adaxial versus abaxial surface emissions exhibited opposing trends with respect to the level of supplied N. Evidence supports that this confounding effect could be removed in part by the green/blue and green/red ratio images. The FIS and LIFIS active fluorescence sensor systems yielded results which support the underlying hypothesis that leaf and canopy fluorescence emissions are associated with other biophysical attributes of crop growth and this information could potentially assist in the site-specific management of variable-rate N fertilization programs.

Introduction

In foliage, a large fraction of the in vivo nitrogen (N) pool is allocated to processes and cell structures related to carbon (C) assimilation. Adequate N availability for growth and seed production in crops is ensured by application of N-enriched fertilizers. Corn (Zea mays L.), a C₄ species, has a higher requirement for N than most other crops. Although N is needed by the corn plant throughout the growing season, N uptake from the soil is greatest during the period of most rapid growth; this extends from 2 to 3 weeks after plant emergence until tasseling. Small grains, typically C₃ species, are likewise responsive to N application but their total requirements are considerably less than corn. However, excessive N application may be accompanied by reductions in yield and is a primary source of NO₃⁻ pollution delivered to ground and surface waters, and as a consequence, fertilization should be adjusted to provide adequate but not excessive amounts of N (Wood, Reeves, & Himelrick, 1993). Currently, the N application rates chosen by producers are influenced by several factors, including: soil and tissue N assays, N fertilizer performance, and expected yields. Since the actual uptake of N by crops is greatly influenced by perturbations in environmental conditions, in particular the interaction of moisture and temperature, there is considerable room to improve current methods to provide practical, reliable and quantitative techniques for evaluating crop N utilization to optimize N application (Doerge, 2002).

Actively induced fluorescence technologies offer spectral sensing methods with potential for the remote assessment of N uptake in crops Corp et al., 1997, Heisel et al., 1997, Heisel et al., 1996, Langsdorf et al., 2000, McMurtrey et al., 1994. The principle of fluorescence involves the absorption of a specific wavelength of light by a fluorophore followed by the dissipation of the absorbed energy by light emission at longer wavelengths within a very short (<200 ns) period of time. In vivo fluorescence emissions from vegetation occur throughout the ultraviolet to visible regions of the spectrum, with maxima most frequently occurring in the UV-A, blue, green, red, and far-red Chappelle et al., 1984, Corp et al., 1996, Corp et al., 1997, Johnson et al., 2000. The UV-A fluorescence emission band can only be observed when plants are irradiated with UV-B (∼280 nm) and has been attributed to rubisco and additional plant proteins which contain aromatic amino acids (Corp et al., 1997). The overlapping blue and green bands are a convolution of fluorescence emissions originating from several plant constituents Chappelle et al., 1984, Chappelle et al., 1984, Lang et al., 1991. The majority of the static blue fluorescence, that is variations which occur over days to weeks, has been attributed to hydroxycinnamic acids, primarily ferulic acid covalently linked to the leaf epidermis and cell walls (Lichtenthaler & Schweiger, 1998). Additional blue and green fluorescent compounds can have smaller but more dynamic contributions (i.e., diurnal variations) to the in vivo fluorescence emission spectrum Cerovic et al., 1999, Chappelle et al., 1999. Chlorophyll fluorescence emissions occur in the red and far-red regions of the spectrum and have been extensively explored and relationships have been established to pigment contents and plant primary metabolism Gitelson et al., 1998, Mohammed et al., 1995, Rosema et al., 1998, Valentini et al., 1994.

Several investigators have demonstrated relationships between fluorescence emission bands or band ratios to plant health and growth condition (see reviews, Buschmann et al., 2000, Cerovic et al., 1999, Chappelle et al., 1999). Fluorescence measurements have shown a great deal of promise in the remote assessment of the relative impact of environmental factors, including: nutrient supply in crop canopies Corp et al., 1997, Heisel et al., 1997, Heisel et al., 1996, Langsdorf et al., 2000, McMurtrey et al., 1994, McMurtrey et al., 2002, differentiation of crops and trees grown under controlled exposures of elevated O₃ Kim et al., 2001, Rosema et al., 1998, irradiance level (Richards, Schuerger, Capelle, & Guikema, 2003), UV-B exposure Krizek et al., 2001, Middleton et al., 1996, and quantifying the amount of crop residue covering agricultural soil surfaces Daughtry et al., 1995, McMurtrey et al., 1993. Currently, several research groups are using fluorescence sensing systems operating from a variety of platforms to receive fluorescence information and are relating this information to biological activity in both terrestrial and aquatic ecosystems Buschmann & Lictenthaler, 1998, Cecchi et al., 1994, Lichtenthaler et al., 1996, Ounis et al., 2001.

The objectives of the current study were to: (1) determine the regions of the fluorescence emission spectrum from corn leaves that are sensitive to changes in N supply; (2) establish relationships among fluorescence emission bands and band ratios to biophysical measures of leaf and crop productivity; and (3) evaluate whether these relationships can be detected at canopy levels of fluorescence sensing. The results from this study provide considerations for future design and development of fluorescence sensing instrumentation and measurement protocols. These fluorescence investigations are ultimately intended to provide information that can be incorporated into prescription algorithms for site-specific variable applications of N containing fertilizers for crop production.