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Using chlorophyll fluorescence to measure early blight of tomato


November 30, 1999
By Dr. Paul H Goodwin

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As photosynthesis is the underlying basis for plant growth and development, estimating the efficiency of photosynthesis in plants is important. Chlorophyll fluorescence is a highly sensitive approach to making non-destructive estimates of photosynthesis.

As photosynthesis is the underlying basis for plant growth and development, estimating the efficiency of photosynthesis in plants is important. Chlorophyll fluorescence is a highly sensitive approach to making non-destructive estimates of photosynthesis. The principle behind it is that when light energy is absorbed by chlorophyll molecules, it can either be used to create sugar for the plant, lost as heat or lost as light. The latter process results in chlorophyll fluorescence and can be measured if leaves are exposed to particular wavelengths of light by then measuring the light that is re-emitted by the leaf. Imaging of chlorophyll fluorescence is done by including a type of camera that records a digital picture of the chlorophyll fluorescence and measures it pixel by pixel in the image.

Chlorophyll fluorescence imaging is a relatively recent way of examining photosynthesis and has been used by scientists to estimate the effects of damage or stress on photosynthesis in various plants. An example of a stress that can affect chlorophyll fluorescence would be water stress that affects gas exchange by causing the stomata to close. This leads to reduced photosynthesis because there are reduced levels of CO2 available inside the leaf that can be incorporated into sugar. Another example is magnesium deficiency. As magnesium is an essential component of chlorophyll, a lack of it will also decrease photosynthesis, leading to altered chlorophyll fluorescence.

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There are several producers of equipment that can perform chlorophyll fluorescence imaging. Models come in a variety of shapes and sizes for different types of plants, but all work is done under basically the same principles. In our study we used a chlorophyll fluorescence imager from Qubit Systems (http://www.qubitsystems.com). It consists of a chamber with light panels composed of LEDs to illuminate the plant at specific wavelengths for specific time periods, a digital camera to capture the fluorescence coming from the plant, plus electronics and software to process the information. The plant is placed into the machine, settings are determined, the plant is illuminated and then a fluorescence image of the plant or plant part is captured. The level of chlorophyll fluorescence within the plant is shown through different colours in the image. These false colour images are used to measure the chlorophyll fluorescence in different areas of the plant.

Imaging of chlorophyll fluorescence is particularly useful in examining plant diseases. Pathogens typically cause symptoms where only a part of the plant is initially affected. A good example is leaf diseases where localized spots of yellowed or dead tissue are observed. Images allow one to observe how photosynthesis varies in a spot, where the surrounding tissue may not be affected. It also allows for quantifying just the areas that are showing disease symptoms. A few plant diseases have been studied thus far using chlorophyll fluorescence imaging. They have shown that the method can be used to detect localized spots and its high sensitivity enabled the scientists to detect infections before visible symptoms had appeared. 

We used chlorophyll fluorescence imaging to measure damage due to the common disease of tomato – early blight – that results in cankers on stems, large dead areas on leaves and rotting of fruit. For example, one may first see older leaves with small brown to black spots and the area surrounding it often becoming yellow. The lesions get bigger and frequently form concentric rings giving it the appearance of a target-spot. The entire leaf may then turn yellow, wither, and drop, and heavy infections can in result large losses of leaves and low yields of undersized fruit. Early blight is caused by the fungus Alternaria solani. Once the spores of the fungus land on a plant, it takes about 30 minutes to two hours for the spores to germinate (the shorter time is related to warmer temperatures). Then, the fungus will penetrate the plant surface in about three to 12 hours, depending on the temperature. When it is inside the plant, the fungus kills the plant tissue producing dead areas (lesions) in one to three days. Large numbers of spores are produced from infected tissues that are spread by water, such as rain, mist, fog, dew or irrigation. If the pathogen becomes established, fungicides are essential for control. While fungicides are highly effective, the rapid development of the disease shows that timely application is important to make sure that they are effective and an epidemic does not occur.

The chlorophyll fluorescence imager that we used enabled us to measure a number of different parameters of chlorophyll fluorescence. The parameter that we chose is also commonly used by other researchers and is a ratio of different values. It represents the maximum proportion of the light that is absorbed by the leaf that can be used for photosynthesis. Using a ratio of values has the advantage of giving less variation within a sample as small differences, such as distance to the camera, are cancelled out during the calculation. The machine measures these values in each pixel of the image to generate false-colour images showing black as no chlorophyll fluorescence and red, orange, yellow and green representing chlorophyll fluorescence values from highest to lowest.

Figure 1 shows several randomly selected tomato leaflets before and after inoculation with spores of the fungus that causes early blight. In figure 1A, a leaflet is shown before inoculation. The leaflet is healthy and has no visible signs of disease. The chlorophyll fluorescence images show a relatively uniform leaf with a red to orange color (figure 1B). Figure 1C shows what a leaflet looks like at about 16 hours after inoculation. It appears to be healthy, but the chlorophyll fluorescence image shows small grainy yellow spots indicating spots of reduced chlorophyll fluorescence (figure 1D). This was always observed prior to the appearance of symptoms. In figure 1E, one can see early symptoms of early blight, which are small dead spots that one typically observes at about 24 hours after the leaflet was inoculated under laboratory conditions. At that point, the chlorophyll fluorescence images shows the yellow spots more clearly indicating that the chlorophyll fluorescence values have dropped further but has still not reached zero (figure 1F). Figure 1G shows a leaflet at about 36 hours after inoculation. The fungus has grown further into the leaflet killing more tissue resulting in progressively larger dead spots. Chlorophyll fluorescence images of the leaflet clearly show the dead spots as areas with no chlorophyll fluorescence (black), but also show that they are surrounded by zones of damaged tissue, shown as yellow, even though that tissue is not yet dead (figure 1H). Figure 1I shows what is usually observed for a leaflet that had been inoculated about 48 hours earlier, and the dead spots have expanded so that they have started to merge together. The areas with no chlorophyll fluorescence are larger and are surrounded by yellow zones (figure 1J).

Because chlorophyll fluorescence measurements are non-invasive, it is possible to take repeated images of the plant material so that the development of the disease can be observed. Figure 2 shows a tomato leaflet that has been imaged before inoculation and then at 16, 22 and 42 hours after inoculation. The white arrow shows an example of a dead spot first visible at 16 hours after inoculation that becomes progressively larger at 22 and 42 hours after inoculation. The green arrow shows the same area in the chlorophyll fluorescence images demonstrating the increasing effects of the pathogen on photosynthesis over time. Analysis of the chlorophyll fluorescence images made it possible to quantify the amount healthy tissue (red to orange), affected tissue (yellow) and dead tissue (black) separately in each chlorophyll fluorescence image of this leaflet. The results are shown in figure 3 revealing the speed of the decline in healthy tissue (which had chlorophyll fluorescence values like a healthy leaf) and the corresponding increases in the amount of disease-affected and dead tissue.

This work shows the type of information that chlorophyll fluorescence imaging can give about a diseased leaf. The false colours based on the chlorophyll fluorescence values gave images that consistently represented dead spots as black, damaged areas as yellow, and apparently healthy tissue as red to orange. Plant tissues could be monitored repeatedly, and even the effects of the pathogen in small areas of a leaflet could be quantified with high sensitivity and accuracy. While chlorophyll fluorescence imaging is currently primarily a research tool, its applications to measuring diseases as well as a wide range of plant stresses gives it great potential as a monitoring tool in production systems. ❦

Acknowledgments
This work was supported by the Ontario Ministry of Food, Agriculture and Rural Affairs, and the Natural Sciences and Engineering Research Council of Canada. The author wishes to thank Moez Valliani and Weihong Gao for their technical assistance.

Dr. Paul H Goodwin is a professor with the School of Environmental Sciences at the University of Guelph, in Guelph, Ont.