Pre-Symptomatic Detection of Viral Infection in Tobacco Leaves Using PAM Fluorometry

Chlorophyll fluorescence imaging was used to study potato virus X (PVX) infection of Nicotiana benthamiana. Infection-induced changes in chlorophyll fluorescence parameters (quantum yield of photosystem II photochemistry (ΦPSII) and non-photochemical fluorescence quenching (NPQ)) in the non-inoculated leaf were recorded and compared with the spatial distribution of the virus detected by the fluorescence of GFP associated with the virus. We determined infection-related changes at different points of the light-induced chlorophyll fluorescence kinetics and at different days after inoculation. A slight change in the light-adapted steady-state values of ΦPSII and NPQ was observed in the infected area of the non-inoculated leaf. In contrast to the steady-state parameters, the dynamics of ΦPSII and NPQ caused by the dark–light transition in healthy and infected areas differed significantly starting from the second day after the detection of the virus in a non-inoculated leaf. The coefficients of correlation between chlorophyll fluorescence parameters and virus localization were 0.67 for ΦPSII and 0.76 for NPQ. In general, the results demonstrate the possibility of reliable pre-symptomatic detection of the spread of a viral infection using chlorophyll fluorescence imaging.

Chlorophyll Fluorescence Imaging-Based Duckweed Phenotyping to Assess Acute Phytotoxic Effects

Duckweeds (Lemnaceae species) are extensively used models in ecotoxicology, and chlorophyll fluorescence imaging offers a sensitive and high throughput platform for phytotoxicity assays with these tiny plants. However, the vast number of potentially applicable chlorophyll fluorescence-based test endpoints makes comparison and generalization of results hard among different studies. The present study aimed to jointly measure and compare the sensitivity of various chlorophyll fluorescence parameters in Spirodela polyrhiza (giant duckweed) plants exposed to nickel, chromate (hexavalent chromium) and sodium chloride for 72 h, respectively. The photochemistry of Photosystem II in both dark- and light-adapted states of plants was assessed via in vivo chlorophyll fluorescence imaging method. Our results indicated that the studied parameters responded with very divergent sensitivity, highlighting the importance of parallelly assessing several chlorophyll fluorescence parameters. Generally, the light-adapted parameters were more sensitive than the dark-adapted ones. Thus, the former ones might be the preferred endpoints in phytotoxicity assays. Fv/Fm, i.e., the most extensively reported parameter literature-wise, proved to be the least sensitive endpoint; therefore, future studies might also consider reporting Fv/Fo, as its more responsive analogue. The tested toxicants induced different trends in the basic chlorophyll fluorescence parameters and, at least partly, in relative proportions of different quenching processes, suggesting that a basic distinction of water pollutants with different modes of action might be achievable by this method. We found definite hormetic patterns in responses to several endpoints. Hormesis occurred in the concentration ranges where the applied toxicants resulted in strong growth inhibition in longer-term exposures of the same duckweed clone in previous studies. These findings indicate that changes in the photochemical efficiency of plants do not necessarily go hand in hand with growth responses, and care should be taken when one exclusively interprets chlorophyll fluorescence-based endpoints as general proxies for phytotoxic effects.

An attempt to the nondestructive investigation of photo-induced potato postharvest quality degradation – Preliminary results

Among improper harvest and/or postharvest storage conditions, the effect of direct sunlight plays an important role in quality degradation of potato resulting in the development of green surface color based on chlorophyll formation associated with the formation of poisonous chemicals – glycoalcaloids – known as α-chaconine and α-solanine. Yellow skinned and fleshed potatoes with or without visible initial marks of green surface color were stored at normal room temperature under direct natural (sun)light conditions for almost two months. The aim of this study was the preliminary investigation of the sunlight induced formation of chlorophyll related compounds in potato indirectly by the detection of chlorophyll development. This attempt was based on nondestructive determination of chlorophyll related spectral and fluorescence indices for both sunlight exposed and unexposed potato sides. For both potato groups the chlorophyll content related DA-index® and chlorophyll fluorescence characteristics (F0, Fm, Fv and Fv/Fm) increased during the storage period representing chlorophyll formation. In the case of Fm, Fv and Fv/Fm values, the yellow samples reached the values of the initial spotted green samples by the 7th–9th days. From this time, the chlorophyll fluorescence values changed only minimally. After storage day 34, in the case of both at day 0 yellow and green spotted potatoes, the sunny side's F0 value was lower than that of shaded side. Close relationship was found between the results of Walz monitoring-PAM (Pulse Amplitude-Modulated) chlorophyll fluorometer and the PSI (Photon Systems Instruments) chlorophyll fluorescence imaging device (e.g. Fv R2 = 0.7226). According to our preliminary results, the Vis/NIR DA-meter®, the monitoring-PAM and the chlorophyll fluorescence imaging fluorometers were found to be suitable nondestructive devices for further investigations concerning the postharvest chlorophyll formation based greening phenomena, which is associated with solanine development in potato.

Detection of Chilling Injury in Pickling Cucumbers Using Dual-Band Chlorophyll Fluorescence Imaging

Pickling cucumbers are susceptible to chilling injury (CI) during postharvest refrigerated storage, which would result in quality degradation and economic loss. It is, thus, desirable to remove the defective fruit before they are marketed as fresh products or processed into pickled products. Chlorophyll fluorescence is sensitive to CI in green fruits, because exposure to chilling temperatures can induce detectable alterations in chlorophylls of tissues. This study evaluated the feasibility of using a dual-band chlorophyll fluorescence imaging (CFI) technique for detecting CI-affected pickling cucumbers. Chlorophyll fluorescence images at 675 nm and 750 nm were acquired from pickling cucumbers under the excitation of ultraviolet-blue light. The raw images were processed for vignetting corrections through bi-dimensional empirical mode decomposition and subsequent image reconstruction. The fluorescence images were effective for ascertaining CI-affected tissues, which appeared as dark areas in the images. Support vector machine models were developed for classifying pickling cucumbers into two or three classes using the features extracted from the fluorescence images. Fusing the features of fluorescence images at 675 nm and 750 nm resulted in overall accuracies of 96.9% and 91.2% for two-class (normal and injured) and three-class (normal, mildly and severely injured) classification, respectively, which are statistically significantly better than those obtained using the features at a single wavelength, especially for the three-class classification. Furthermore, a subset of features, selected based on the neighborhood component feature selection technique, achieved the highest accuracies of 97.4% and 91.3% for the two-class and three-class classification, respectively. This study demonstrated that dual-band CFI is an effective modality for CI detection in pickling cucumbers.

Low-Cost Chlorophyll Fluorescence Imaging for Stress Detection

Plants naturally contain high levels of the stress-responsive fluorophore chlorophyll. Chlorophyll fluorescence imaging (CFI) is a powerful tool to measure photosynthetic efficiency in plants and provides the ability to detect damage from a range of biotic and abiotic stresses before visible symptoms occur. However, most CFI systems are complex, expensive systems that use pulse amplitude modulation (PAM) fluorometry. Here, we test a simple CFI system, that does not require PAM fluorometry, but instead simply images fluorescence emitted by plants. We used this technique to visualize stress induced by the photosystem II-inhibitory herbicide atrazine. After applying atrazine as a soil drench, CFI and color images were taken at 15-minute intervals, alongside measurements from a PAM fluorometer and a leaf reflectometer. Pixel intensity of the CFI images was negatively correlated with the quantum yield of photosystem II (ΦPSII) (p < 0.0001) and positively correlated with the measured reflectance in the spectral region of chlorophyll fluorescence emissions (p < 0.0001). A fluorescence-based stress index was developed using the reflectometer measurements based on wavelengths with the highest (741.2 nm) and lowest variability (548.9 nm) in response to atrazine damage. This index was correlated with ΦPSII (p < 0.0001). Low-cost CFI imaging can detect herbicide-induced stress (and likely other stressors) before there is visual damage.