By Michael Celetti, OMAFRA Plant Pathologist – Horticulture Crops
Quick, accurate diagnosis of plant pathogens allows growers to implement management practices at an earlier stage of the disease and choose appropriate management practices and crop protection products. However, disease identification can be difficult and requires knowledge, skill and experience.
If samples from a diseased plant need to be sent to a lab for identification or confirmation, the disease in the field may become more severe and widespread before results are received and appropriate action taken. Technology such as Enzyme-Linked ImmunoSorbent Assays (ELISA) has been developed and employed over the past 20-30 years for rapid pathogen identification and confirmation. There are several test kits now on the market for quick identification and confirmation of certain pathogens.
One of the easiest test kits to use is the lateral flow test kit such as the one developed by Safeguard Biosystems (Figure 1). These “easy-to-use” lateral flow ELISA tests have recently been developed for identifying or confirming specific disease causal agents in the field. Each kit contains disposable pipettes, vials of buffer extract solution with small ball bearings to macerate tissue; and lateral flow devices with a membrane partially coated with antibodies specific to a particular organism.
To use the kits, a piece of infected plant tissue is place into a vial containing buffer and small ball bearings that is shaken for 90 seconds to macerate the infected tissue prior to dispensing 3-4 drops of the buffer solution containing the macerated diseased plant tissue onto the well of a lateral flow device. The lateral flow device is then allowed to incubate for 5 minutes before determining if the device detects the pathogen. Tests are considered valid if a blue line develops under the “C” for control (right portion of the device window) on the lateral flow device regardless of whether or not a blue line developed under the test “T” (Figure 2 a). A test is determined to be valid and positive for the pathogen only when a blue line developed under both the “T” and “C” on the lateral flow device (Figure 2 b, c d). However, the line that develops under the “T” can sometimes be weak, medium or strong depending upon the test.
In 2010, several kits developed for the detection of five different pathogens were evaluated using pure cultures of the pathogens growing on artificial medium in the laboratory as well as infected plant tissue. All test kits reacted positively when tested with the cultures of the pathogen they were designed to detect. Unfortunately the Pythium Pocket Diagnostic Test Kit also cross-reacted with cultures of a Phytophthora spp. and the grey mould pathogen Botrytis cinerea. The Botrytis Pocket Diagnostic Test Kit cross-reacted with cultures of the white mold fungus Sclerotinia sclerotiorum. However, the Botrytis Pocket Diagnostic Test Kit successfully detected Botrytis grey mould in infected geranium leaves. The Rhizoctonia Pocket Diagnostic Test Kit reacted very weakly with cultures of Rhizoctonia spp. and only after several hours after the device was allowed to incubate. Cross-reactions, delayed or weak reactions with these test kits could lead to the misdiagnosis of pathogens and diseases in the field and the recommendation of the wrong management practices or application of the wrong crop protection products to control the diseases they cause.
The only test kit that appeared to work specifically, consistently and provided a clear strong reaction was the Phytophthora Pocket Diagnostic Test Kit. It also performed well in diagnosing late blight caused by P. infestans on infected tomato leaf and fruit tissue in the field in both 2009 and 2010. Although woody tomato stem tissue with symptoms of late blight gave a weak reaction when placed in the vial containing buffer and the small ball bearings, the same test reacted better when the woody infected stem tissue was macerated more effectively with sandpaper or a mesh bag prior to placing into the vial. Since root rots of many woody ornamental plants and fruit trees can be caused by various Phytophthora spp., improved techniques to macerate the woody tissue would help improve this test. Regardless, this particular Pocket Diagnostic Test could be a valuable tool for potato, tomato and fruit growers who encounter diseases caused by Phytophthora from time to time.