Plant pathogens are one of the main threats to agricultural production and affect the multibillion-dollar agricultural industry every year. Accurate and rapid identification of pathogens is the first step in controlling plant diseases and producing quality crops. While some common plant diseases may be easy to identify in the field with a trained eye, many symptoms displayed by unhealthy plants could also be due to poor growing conditions, pests, chemical damage from fertilizers or fungicides, and plants could even be under attack by more than one pathogen at a time! If not diagnosed and treated immediately, some pathogens can spread very quickly. An entire field could be taken by pathogens in as little as 24 hours. Moreover, there can be a considerable amount of damage if pathogens are incorrectly diagnosed based purely on physical observations that result in unnecessary fungicide applications. Plant Pathologists use many different tools to figure out what is making the plant unhealthy, but the three main methods used are microscopy, growth and observation on media and molecular techniques.
Microscopy is an important initial tool in plant disease diagnosis and in many cases, the only standard tool available. The suspected diseased plant tissues or pathogen itself are examined under a dissecting and/or compound microscope (Figure 1) and the pathologist can determine the morphology (shape) and size of the pathogen if it is present in the sample. This method requires highly trained personnel with extensive knowledge of classical pathogen taxonomy. Distinguishing the exact species of the pathogen using microscopy is often impossible but it provides a great starting point to the process!
Fig: 1: Microscopy based disease diagnosis (PC: Ali).
Second, culturing and observing the pathogen on media in a plate/petri dish is another important traditional tool for disease diagnosis. A small piece of suspected diseased plant tissue is placed on growing media with different nutrients and chemicals in it. After several days of incubation, the pathologist will know more about the pathogen due to how it grows on the media – they will observe the color of the pathogen on the plate, the growth pattern it follows, and many other distinguishing characteristics (Figure 2). Bacteria and Fungi grow very differently on media, and it is easy to distinguish between the two types of pathogens. Pathologists can even add antibiotics to growing media to determine if pathogens are resistant to it or not. However, this method can take several days to weeks to complete, and it is ineffective for some pathogens like viruses that cannot grow on their own without a host.
Fig: 2: Culture plate technique for disease diagnosis. Characteristics of cultures were observed two to three weeks after transferring to plate A). Fusarium spp; B). Phytophthora spp. and C). Clarireedia spp.
Finally, with recent technological advances, rapid disease diagnosis strategies have significantly improved in the last two decades. Many different pathogens – including fungal, bacterial, and viral
Fig: 3: Molecular technique for disease diagnosis. A). thermocycler; B). PCR-based detection C). Real-time PCR detection; and D) Laboratory demonstration.
pathogens – can be identified with PCR (Polymerase chain reaction) tests and many other forms of genomic identification (multiplex PCR, real-time PCR, quantitative PCR, RAPD-PCR, DNA sequencing, etc). These molecular methods are highly effective for identifying a pathogen accurately to the species level. However, many of these methods require a well-equipped (and well-funded) laboratory facility with bulky pieces of equipment like thermocycler, nucleic acid extraction system, gel image scanner, biosafety cabinet, etc (Figure 3).
The demand for rapid onsite disease diagnosis has been growing over the last decade. Several molecular and serological techniques are currently being used for onsite disease diagnosis and results that are viewed in real-time, or after one hour with a color-changing dye, depending on the assay used (Figure 4). For example, Immunostrip assays (IA) function similar to a rapid COVID-19 antigen test – all one has to do is add diseased plant tissue to a buffer and add a test strip to the mix, and they will have a result in minutes (Figure 4A). These assays are not available for all pathogens but are a really valuable tool that anyone can use if they suspect a specific pathogen is present in their crop. Loop-mediated isothermal amplification, or LAMP and Recombinase Polymerase Amplification, or RPA are the two important advanced technologies for the detection of specific pathogens. With the portability of real-time instruments being used for amplification, these techniques can provide on-site diagnostics. Not to mention that the sensitivity of these types of analysis is much higher than that of conventional PCR and allows for the rapid detection of pathogens in asymptomatic plants that harbor relatively low pathogen populations. These assays for onsite diagnosis are very popular among growers, field specialists, and crop advisers. When time is of the essence, these tools can play a vital role by permitting early identification of disease and reducing the risk of an epidemic.
Fig: 4: Techniques for onsite disease diagnosis. A). ImmunoStrip assay B). Recombinase Polymerase Amplification, or RPA assay C). Loop-mediated isothermal amplification, or LAMP assay and D). Field demonstration
UNH Cooperative Extension is establishing a brand-new Plant Diagnostic Lab in Barton Hall on the UNH Durham campus. The new lab will have the capabilities to diagnose special plant diseases caused by bacterial and viral pathogens that require detection beyond the resources and expertise of a traditional plant clinics. The UNH-PDL will provide both traditional and molecular disease diagnostic support to UNH extension specialists, researchers, master gardeners, growers, and the public on a fee-for-service basis. This service will be crucial to guide growers in making timely management decisions based on accurate and reliable diagnoses. The lab is currently under construction and aiming to start providing diagnostic services starting June 1, 2022. Once the lab is ready, we encourage growers and crop advisors to take advantage of these advanced services. The UNH-PDL will be open to collaborating with plant disease diagnosticians, researchers, extension specialists, and growers across the New England region and contributing to the National Plant Diagnostic Network (NPDN). Please keep an eye on the UNH-PDL website: https://extension.unh.edu/agriculture-gardens/pest-disease-growing-tool… (currently under maintenance) for the latest information on UNH-PDL and upcoming diagnostic services. Any questions about UNH-PDL should be directed to Emran Ali, Director, UNH-PDL at 603-862-2043 (Email: Emran.Ali@unh.edu) or, Amy Papineau, Program Team Leader, Food and Agriculture at 603-862-1601 (Email: Amy.Papineau@unh.edu).
