Great potential for biosensor-based technology in diagnosing seed-borne pathogens

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Protein-based immunological and DNA-based polymerase chain reaction methods have been developed for diagnosis of plant pathogens, but their utility is limited for certain reasons. Biosensor-based methods are now available as a potential alternative, which are capable to quickly and accurately identify pathogens. Although biosensor-based methods are widely used in different areas such as detection of food-borne microorganisms, adulteration in foods, estimation of blood sugar and cholesterol, determination of pregnancy in women, etc. But, for assessing the health of seeds and plants these techniques are not in vogue. However, it is clear from other field’s examples that biosensor-based techniques are able to quickly and accurately identify plant pathogens and can also play a vital role in providing disease-free quality seeds to researchers and farmers, thereby enhancing theirfarm income.

Introduction

Disease-free and quality seeds are considered as the most important unit for the success of crop-based agricultural research as well as farming. Therefore, it is mandatory to ensure the health of seeds through seed health test. Conventional methods of plant pathology that are largely used to identify seed-borne pathogens include visual examination, washing test, NaOH seed soaking, embryo counting, blotter test, culturing and plating, isozyme analysis, grow out test, etc.These techniques/methods of pathogen detection are considered to be more time consuming and laborious as they have been proven to be insufficient to meet the demands of rapid diagnosis. Currently, a variety of rapid detection, identification and monitoring techniques have been developed for plant pathogens, in which DNA dependent polymerase chain reaction (PCR)-based methods such as reverse transcription PCR, nested PCR, multiplexreal-time PCR, quantitative real-time PCR, co-operative PCR, magnetic capture hybridization PCR, fluorescence in situ hybridization (FISH), loop mediated isothermal amplification (LAMP), DNA array hybridization (DAH). reverse dot blot hybridization (RDBH), nucleic acid sequence-based amplification (NASBA), etc. are included. Similarly, pathogen derived protein-based immunological methods such as immunodiffusion, enzyme-linked immunosorbent assay (ELISA), radio-immunosorbent assay (RISA) dipstick immunoassay (DIA), Dot immunobinding assay (DIBA), tissue blot immunoassay (TBIA), Western blot analysis (WBA), serologically specific electron microscopy (SSEM), immunosorbent electron microscopy (ISEM), flow cytometry, etc. are helpful in identifying various pathogens. Despite the availability of these sensitive techniques being expensive, their usefulness is limited from laboratory to field for rapid detection of plant diseases.

At present, along with the demand, we need technologies that are rapid, sensitive and selective. As such, there are many biosensors-based techniques having such characteristics with success in analysing various components of daily life such as blood sugar and cholesterol assessment, determination of conception in women, adulteration of food, etc. and identifying food-borne microorganisms, Escherichia, Staphylococcus, Salmonella and Listeria. But, the operation of biosensor-based detection techniques to assess seed and plant health is negligible.The most widely used technologies for health monitoring in plants at present are highly expensive, labour-intensive and time consuming. PCR-based molecular techniques are used to identify plant pathogens that require extensive processing of specimen which takes time. Research findings show that biosensor-based technologies are able to quickly and accurately identify plant pathogens and can also play a vital role in providing disease-free and quality seeds to researchers and farmers. Before discussing biosensor-based technologies, it should also be known what a biosensor is, and also the history of development and landmarks related to it.

Landmarks

  • Carl Cammann used the term ‘biosensor’ for the first time in 1977.
  • American biochemist, Prof. Leland Clark is called the father of biosensor
  • In 1997, International Union of Pure and Applied Chemistry (IUPAC) defined the term ‘biosensor’ in the following words.

Definition:“a device that uses specific biochemical reactions mediated by isolated enzymes, immuno systems,

tissues, organelles or whole cells to detect chemical compounds usually by electrode, thermal or optical signals”

Features: Any high-quality biosensor must have at least 4 important basic features- a) Linearity: where the linearity is high to detect high substrate concentrations; b) Sensitivity: which is the value of the electrode response to the substrate concentrations; c) selectivity: least interference in the chemical reaction; and d) response time:at least 95% of the response time required to ensure

Brief history of biosensor development: Development of biosensor started with Immobilization of invertase protein on activated charcoal (Nelson and Griffin, 1916) followed by invention of various devices/components such as glass pH electrode (Hughes, 1922), oxygen electrode (Clark, 1956), first glucose biosensor (Clark andLyons, 1962), first protensiometric biosensor to check adulteration of urea in milk (Guilbaultand Montalvo, 1969), ISFET-ion-selective field-effect transistor (Bergveld, 1970), pCO2 / pO2 optrode - optic fiber sensor (LubbersandOpitz, 1975), first microbe-based biosensor (Davies, 1975), first immunosensor (Janata et al., 1975), first optic fiber-based pH sensor for in-vivo blood gases (Peterson et al., 1980),first optic fiber-based glucose biosensor (Schultz et al.,1982), first surface plasmon resonance (SPR) immunosensor (Liedberg et al.,1983), first amperometric glucose biosensor (Cass et al., 1984). Apart from these, some more significant achievements in the field of biosensor technology during 1980s to 1990s have been made like commercial glucose biosensor by M/s Yellow Springs Instruments, beside artificial pancreas by M/s Miles Laboratories Inc., blood glucose biosensor by M/s MediSenseExacTech and M/s LifeScanFastTake, SPR-based biosensor by M/s Pharmacia BIAcore and currently,various biosensors based on Quantom dot (Ma et al., 2018), Nanoparticle (Holzinger et al., 2014), Nanowire (Ambhorkaret al., 2018), Carbon nanotube (Yang et al., 2015), etc. have also been developed.

Biosensor technologies

The biggest advantageous aspects of biosensors are those that are able to rapidly provide users with instant information for both field and laboratory analysis, and biosensor-based equipment can also be easily moved from place to place. Biosensor-based technologies that operate on different principles have been proven useful in the identification of various plant disease-causing fungi, bacteria and viruses (Table 1), but are limited.

Table 1: Biosensors based technologies to identify plant pathogens.

(Source:Khatera et al., 2017;Ray et al., 2017;Ivnitski et al., 1999)

Optical biosensor: An optical biosensor is a compact analytical device in which a biological sensory element is integrated or connected to an optical transducer system. Optical biosensors generally rely on an enzyme system that converts catalysts into products that are oxidized or reduced at electrodes that can remain stable with their specific potential.Optical biosensors are a powerful alternative to traditional analytical techniques, particularly due to their  high selectivity and sensitivity as well as their small size and low cost, and the electrodes used in it are biodegradable. In the food industry, various types of optical biosensors have been developed only in the last decade for quick identification of pathogens and toxins/contaminants.Optical biosensors are developed mainly on three principles, fluorescence-based, chemiluminescence-based and SPR-based technologies. With the use of ‘green fluorescent protein (GFP)’ in fluorescence-based biosensors,identification of a number of fungal pathogens including Phytophthorapalmivora, Ustilagomaydis, Colletotrichum lindemuthianum, Aspergillus nidulans and Cochliobolus heterostrophushave become possible. The identification of a fungus, Brettanomyces bruxellensis from extremely low amount of DNA (12.5 ng / µl) via a chemiluminescence-based biosensor indicates that these biosensors are the most sensitive.

Piezoelectric biosensors:These biosensors are devices that are also able to detect very small amount of analytes according to the linear relationship between the collected material and its frequency response.Therefore, piezoelectric biosensor is an effective alternative to optical sensors. Surface plasmon resonance spectroscopy (SPR) and interferometry are successful examples of this, which are one of the excellent methods to determine contamination.

Surface plasmon resonance (SPR) biosensors:It is a powerful, label-free technique for real-time monitoring of non-covalent molecular interactions in a non-invasive style. As a label-free test method, SPR does not require any tags, dyes or special reagents, such as enzyme-substrate mixtures to obtain visible or fluorescence signals.

Quartz crystal microbalance (QCM) biosensors: These biosensors sensors consist of a thin quartz crystal disc with electrodes plated on it. They are used for plant disease detection where a quartz disc is coated with pathogen-specific antibodies/ nucleic acids/ receptors/ small molecules, etc. depending on analyte to be detected. These methods include bulk acoustic wave (BAW), quartz crystal resonance sensors (QCRS), and thickness shear mode (TSM), which are both qualitative as well as quantitative.

Electronic nose: The identification of pathogens is ascertained via detection of organic volatile compounds (VOCs) through this device.

Looking at Table 2, it appears that all biosensors which are commercially available in the international market to identify plant diseases worldwide are either antibody based or DNA based. It indicates that both DNA and antibody have been established as good biosensing receptors in biosensor technology.


Table 2: Commercial products of biosensors/kits for identification of plant pathogens.

According to an estimate, crop losses (~35%) due to pests and diseases amount to Rs ~50,000 crore annually, which is significant in a country where at least 200 million Indians go to bed hungry every night.Seed-borne diseases of crops such as Karnal bunt (Tilletiaindica) and loose smut (Ustilago segetum var. tritici) of wheat, kernelsmut (T. barclayana) of paddy, etc. are some of the major threat to the cultivation of wheat and paddy, that affect the yield and quality of seed. There is a lack of rapid, accurate and highly sensitive technologies to identify seed-borne pathogens at quarantine entry points.The availability of biosensors/kits for identification of plant pathogenic fungi can play a vital role in providing disease-free quality seed to researchers and farmers.Considering the efforts being made in science, increasing level of experiments and progress, it seems that biosensor-based technologies will prove to be successful in future in the field of plant protection, especially disease diagnosis.

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