25 Mar 2022

Techniques To Identify Plant Pathogens (part-2)

Anjali Singh, MS

As a content and community manager, I leverage my expertise in plant biotechnology, passion for tissue culture, and writing skills to create compelling articles, simplifying intricate scientific concepts, and address your inquiries. As a dedicated science communicator, I strive to spark curiosity and foster a love for science in my audience.

Table of Contents

Introduction

Plant pathogens cause a huge loss in the productivity of crops and sometimes lead to total crop loss. The solution to this trouble is to identify pathogen infections in plants at their early stage and eliminate them.

In the first part of this article, you’ve learned how to control the spread of plant diseases and that the techniques to identify plant pathogens is categorized into two groups:

Direct Identification Technique
Indirect Identification Technique

In the direct identification techniques you have learned about five techniques: Polymerase Chain Reaction (PCR), Fluorescence in-situ Hybridization (FISH), and Enzyme-Linked Immunosorbent Assay (ELISA).

In this article, we will cover two other direct pathogen identification techniques: Immunofluorescence, and flow cytometry; and indirect pathogen techniques.

Direct Detection Methods

The direct detection of diseases involves molecular and serological methods that could be used for high-throughput analysis when a large number of samples need to be examined. The pathogens responsible for diseases, such as bacteria, fungi, and viruses, are detected directly in these methods, which allows accurate identification of the diseases/pathogens.

Immunofluorescence

In this method, antibodies are chemically labeled using fluorescent dyes to see molecules under a light microscope. It’s mainly used to analyze microbiological samples and detect pathogen infections in plants.

For pathogen detection, a thin tissue section is fixed to a microscopic slide, Then detection is achieved by conjugating a fluorescent dye with a specific antibody and observing the distribution of the target molecule.

The technique has been used to detect onion crop infection, caused by a fungus Botrytis cinerea, and also Solanum dulcamara which causes crown rot in potatoes by combining with the FISH technique.

The only limitation of the technique is false-positive results due to photobleaching. This can be overcome by:

Reducing the intensity and duration of light exposure
Increasing the concentration of fluorophores
Employing more robust fluorophores that are less sensitive to photobleaching.

Flow Cytometry

It’s a laser-based technique, which is widely used to rapidly analyze single cells when they pass through a detection apparatus in a liquid stream. Its applications include cell sorting and counting, protein engineering, and biomarker detection.

The technique combined with the FISH technique has been used to detect foodborne bacterial pathogens. It can accurately detect 104 colony forming units (CFU) per milliliter within 30 minutes of the time.

Moreover, it can also be used to detect soil-borne bacteria, such as Bacillus subtilis in mushroom composts and for viability evaluation.

Indirect Detection Methods

The indirect detection method detects plant disease based on various characteristics or parameters. I include temperature change, transpiration rate change, morphological change, and volatile compounds released by infected plants. The technique has also been used to detect biotic and abiotic stresses in plants.

Some of the extensively used indirect detection methods in labs are given below.

Thermography

The technique detects and images the surface temperature difference in plants’ leaves and canopies by emitting infrared rays and capturing them using thermographic cameras.

Phytopathogens affect the regulation of water loss by stomata in plants. The disease caused by these pathogens leads to temperature change, which can be visualized by using thermographic imaging. Moreover, it can also detect the heterogeneity in the infection of soilborne pathogens.

The limitation of this technique is that it’s sensitive to environmental changes that can affect the measurements during experiments. In addition, it lacks disease specificity, thus, can’t be used to distinguish between diseases and identify infection types.

Fluorescence Imaging

The technique helps to visualize biological processes occurring in a living organism. In plants, it measures the chlorophyll fluorescence on the leaves when light falls on them. The change in fluorescence parameters helps to analyze pathogen infections.

The technique has been used to detect temporal and spatial variations of chlorophyll fluorescence in leaf rust and powdery mildew infections of wheat leaves at 470 nm.

Apart from its potential functions, the use of this technique is limited in labs.

Hyperspectral Techniques

It can be used to analyze plants’ complete health by analyzing a wide spectrum of light, between 350 and 2500 nm falling on the plant surface. The technique is predominantly used for plant phenotyping and identification of plant diseases in commercial-scale agriculture.

It’s a robust technique that provides a rapid analysis of imaging data in three dimensions, with X- and Y- axes for spatial and Z- for spectral. The information help scientists to get detailed information of plants’ health in any area.

The technique has been successfully used to identify Phytophthora infestans infection of tomato, Magnaporthe grisea infection of rice, and Venturia inaequalis infection of apple trees.

Gas Chromatography

It’s a non-optical method that doesn’t involve the use of microscopic imagery. It profiles the volatile organic compounds of infected plants that are released when any type of stress is experienced by plants. It’s used to identify the nature and type of plant infections or disease diagnosis

For example, p-ethyl guaiacol and p-ethylphenol are released by the fungus, Phytophthora cactorum, which causes crown rot diseases in strawberries.

The volatile compounds are also released when plant leaves are damaged. The green leaves volatiles, including cis-hexenyl acetate, cis-3-hexenol, and hexyl acetate are observed to be released during herbivore invasion.

The emitted volatile compounds are analyzed by gas chromatography (GC), which indicates the particular disease.

Though, you might say there are so many techniques that can be used for pathogen detection! However, their application is limited due to:

Destructive nature
Expensive procedure
Requirement of a skilled technician
Requirement of a proper lab set-up
Time-consuming process
No possibility of real-time monitoring

Well, identification of the pathogen infection is the first step to prevent the spread of these diseases and enhance crop productivity. However, the technique has higher efficiency to produce disease-free plants in the tissue culture of plants.

Tissue culture is an advanced approach that allows culturists to produce disease-free plants. The technique combined with several other high-throughput techniques is used in labs for developmental or genetic studies.

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Synthetic Seed and Cryopreservation: Long term genetic storage solutions.
Pathogen Remediation by Media Amendments: How to remove viruses, systemic fungi, bacterial infections, and endophytes.
Gender and Pathogen Screening by PCR: Eliminate male plants, identify cannabinoid ratios, and protect your mothers from pathogens and pests