Photosynthesis is a pivotal process in plant physiology, and its regulation plays an important role in plant defense against biotic stress. Interactions with pathogens and pests often cause alterations in the metabolism of sugars and sink/source relationships. These changes can be part of the plant defense mechanisms to limit nutrient availability to the pathogens. In other cases, these alterations can be the result of pests manipulating the plant metabolism for their own benefit. The effects of biotic stress on plant physiology are typically heterogeneous, both spatially and temporarily. Chlorophyll fluorescence imaging is a powerful tool to mine the activity of photosynthesis at cellular, leaf, and whole-plant scale, allowing the phenotyping of plants. This review will recapitulate the responses of the photosynthetic machinery to biotic stress factors, from pathogens (viruses, bacteria, and fungi) to pests (herbivory) analyzed by chlorophyll fluorescence imaging both at the lab and field scale. Moreover, chlorophyll fluorescence imagers and alternative techniques to indirectly evaluate photosynthetic traits used at field scale are also revised.

In the last few years, large efforts have been made to develop new methods to optimize stress detection in crop fields. Thus, plant phenotyping based on imaging techniques has become an essential tool in agriculture. In particular, leaf temperature is a valuable indicator of the physiological status of plants, responding to both biotic and abiotic stressors. Often combined with other imaging sensors and data-mining techniques, thermography is crucial in the implementation of a more automatized, precise and sustainable agriculture. However, thermal data need some corrections related to the environmental and measuring conditions in order to achieve a correct interpretation of the data. This review focuses on the state of the art of thermography applied to the detection of biotic stress. The work will also revise the most important abiotic stress factors affecting the measurements as well as practical issues that need to be considered in order to implement this technique, particularly at the field scale.

The negative impact of conventional farming on environment and human health make improvements on farming management mandatory. Imaging techniques are implemented in remote sensing for monitoring crop fields and plant phenotyping programs. The increasingly large size and complexity of the data obtained by these techniques, makes the implementation of powerful mathematical tools necessary in order to identify informative parameters and to apply them in precision agriculture. Multicolor fluorescence imaging is a useful approach for the study of plant defense responses to stress factors at bench scale. However, it has not been fully applied to plant phenotyping. This work evaluates the possible application of multicolor fluorescence imaging in combination with thermography for the particular case of zucchini plants affected by soft rot, caused by Dickeya dadantii. Several statistical models -based on logistic regression analysis (LRA) and artificial neural networks (ANN)- were obtained for the experimental system zucchini-D. dadantii, which classify new samples as “healthy” or “infected”. The LRA worked best in identifying high dose-infiltrated leaves (in infiltrated and non-infiltrated areas) whereas ANN offered a higher accuracy at identifying low dose-infiltrated areas. To assess the applicability of these results to cucurbits in a more general way, these models were validated for melon infected by the same pathogen, achieving accurate predictions for the infiltrated areas. The values of accuracy achieved are comparable to those found in the literature for classifiers identifying other infections based on data obtained by different techniques. Thus, MCFI in combination with thermography prove useful at providing data at lab scale that can be analyzed by machine learning. This approach could be scaled up to be applied in plant phenotyping.

Several imaging techniques have provided valuable tools to evaluate the impact of biotic stress on host plants. The use of these techniques enables the study of plant-pathogen interactions by analysing the spatial and temporal heterogeneity of foliar metabolism during pathogenesis. In this work we review the use of imaging techniques based on chlorophyll fluorescence, multicolour fluorescence and thermography for the study of virus, bacteria and fungi-infected plants. These studies have revealed the impact of pathogen challenge on photosynthetic performance, secondary metabolism, as well as leaf transpiration as a promising tool for field and greenhouse management of diseases. Images of standard chlorophyll fluorescence (Chl-F) parameters obtained during Chl-F induction kinetics related to photochemical processes and those involved in energy dissipation, could be good stress indicators to monitor pathogenesis. Changes on UV-induced blue (F440) and green fluorescence (F520) measured by multicolour fluorescence imaging in pathogen-challenged plants seem to be related with the up-regulation of the plant secondary metabolism and with an increase in phenolic compounds involved in plant defence, such as scopoletin, chlorogenic or ferulic acids. Thermal imaging visualizes the leaf transpiration map during pathogenesis and emphasizes the key role of stomata on innate plant immunity. Using several imaging techniques in parallel could allow obtaining disease signatures for a specific pathogen. These techniques have also turned out to be very useful for presymptomatic pathogen detection, and powerful non-destructive tools for precision agriculture. Their applicability at lab-scale, in the field by remote sensing, and in high-throughput plant phenotyping, makes them particularly useful. Thermal sensors are widely used in crop fields to detect early changes in leaf transpiration induced by both air-borne and soil-borne pathogens. The limitations of measuring photosynthesis by Chl-F at the canopy level are being solved, while the use of multispectral fluorescence imaging is very challenging due to the type of light excitation that is used.