MATERIALS AND INSTRUMENT IN PLAT PATHOLOGY

MATERIALS AND INSTRUMENT IN PLAT PATHOLOGY

1. Rapid and Accurate Disease Diagnosis as a Key Component to Successful Plant Disease Management 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.

2. 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! Microscopy

3. 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. C ulturing and observing the pathogen on media in a plate/petri dish

4. Finally, with recent technological advances, rapid disease diagnosis strategies have significantly improved in the last two decades. Many different pathogens – including fungal, bacterial, and viralpathogens – 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).

5. 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).

6. T hese 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.