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ABSTRACT

Plant disease caused by phytopathogen leads to substantial yield and economic loss. Rapid and accurate identification of the pathogen is necessary to implement control measures. Conventionally, plant pathogens are identified using 16S rRNA and 18S rRNA gene sequencing. However, in recent years, the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a potential tool for the identification and diagnosis of plant pathogens. Signature peptides molecules obtained by desorption from intact spores/organism produces a unique mass spectrometric profile, helps in identification up to species level. Rapid detection, sensitivity, cost-effectiveness and suitability to automation has made MALDI-TOF MS a promising tool for identification. The limitation of the tools is that identification of new isolates is feasible only if the spectral database contains peptide mass fingerprints of specific genera/species/strains. This article provides brief information about the principle and usefulness of this exciting new technology for the diagnosis of plant diseases caused by phytopathogens.

  Keywords: MALDI-TOF, Mass Spectroscopy, Identification, Fingerprinting

Cite this article as: Nishmitha  K and Chaithra  M (2022). MALDI-TOF MS: A Novel Tool for Rapid Detection of Plant Pathogens, Food and Scientific Reports, 3(1): 27-29.

Conventional techniques such as visual symptom observation, cultural and morphological approaches are time-consuming while serological methods and PCR based techniques are expensive, need professional support and sophisticated labs. In the era of omics, proteomics technology can be used for the identification of microbes inhabiting a particular ecological niche, strain typing and epidemiological studies. MALDI-TOF mass spectrometry technique has emerged as a potential tool for pathogen identification based on measuring the charge to mass ratio of the ionized molecule. In 1985, Franz Hillinkamp and Michael Karas found that amino acid alanine could be ionized easily when mixed with amino acid tryptophan and irradiated at 266nm. They used this principle of matrix-assisted ionization and coined the term MALDI.

The sample (microbe) mixed with suitable matrix laid on sample plate produces charged molecule when excited with a laser of a particular wavelength and move through Time of Flight (TOF) tube. Peptide desorbed from the sample produce a distinct peptide profile unique to the organism based on charge to mass ratio. The profile is compared to the available database helps in the accurate identification of the organism. The soft ionization process without destruction of sample and potential for automation has made it an interesting tool in the modern era.  

Principle of MALDI-TOF

The MALDI is an ionization-based technique that makes use of a laser energy absorbing matrix to generate ions from large molecules with minimum fragmentation (Ahmad et al., 2012). The matrix is a crystalline material and is mixed with protein samples and dried on the sample MALDI plate. The Matrix absorbs the energy and is desorbed and ionized. Depending upon the nature of the matrix, laser intensity and voltage singly charged, multiple charged [M+nH] n+, protonated [M+H] + particles are produced. An ionized cloud of protein and matrix are pulled to the MALDI tube by an electrostatic field. The ionized protein then enters the TOF reflectron, they pass on the way to the ion detector located in the spectrometer. Based on the total time ion taken to move through the reflectometer, the spectrometer determines the mass to charge ratio. Mass to charge ratio is recorded with the aid of a spectrometer and transferred to the computerized graph (Signor et al., 2013).

 Components of MALDI-TOF MS

1. Matrix: Most used matrixes are 2, 5-dihydrobenzoic acid (DBH), 4-hydroxy-3-methoxy cinnamic acid (ferulic acid), alpha-cyano-4-hydroxycinnamic acid (CHCA). They work in a range of 266 nm, 337 nm and 355 nm and assist in the analysis of protein, lipids, and nucleotides respectively. 

 2. Laser: MALDI techniques commonly makes use of UV lasers which incorporates nitrogen lasers (337 nm) and frequency-tripled and quadrupled Nd: YAG (neodymium-doped Yttrium Aluminum Garnet lasers) 355 nm and 226 nm respectively. 

3. Time of flight: Mass spectrometer widely used with MALDI is Time of Flight (TOF) because of the big mass range. It detects the time taken by ions to reach the detector. In linear mode charged molecules travel in a straight line reach detector within nanosecond of ionization. Large molecule takes a longer time to reach compared to the lighter molecule. Molecular mass is determined directly based on flight time. In reflector mode, the particles are diverted so that they fly towards a second detector. In addition to extending the flight distance, the reflector also focuses on the masses. The combination of these two effects makes for higher resolution than in the linear mode.

4. Sample preparation: It is prepared using direct cell profiling, a single colony of microorganism is picked and spotted at once directly to the sample plate and immediately overlaid with the matrix solution. In the indirect method, the fungus is grown on agar and extracted with formic acid following incubation in ethanol. Acetonitrile is added, the mixture is centrifuged and the supernatant is used for MALDI-TOF MS analysis (Ilina et al., 2009).

Application of MALDI-TOF in plant pathology 

In the field of plant pathology, it is used to identify pathogens up to species and stain level, to perceive pathogen-related proteins, proteins related at some point of host-pathogen interaction, detection of recombinant proteins and detection of virulence markers.

 Organisms identified by MALDI –TOF (Drissner et al., 2017)

Fungi: Aspergillus spp., Saccharomyces spp., Rhizopus spp., Trichoderma spp., Penicillium spp., Fusarium proliferatum, Fusarium spp., Verticillium spp., Bremia lactucae and Oidium neolycopersici, Gibberella zeae, Puccinia graminis tritici  

Bacteria: Clavibacter michiganensis, Corynebacterium spp., Erwinia amylovora, Bacillus subtilis

Virus: Pepper yellow vein virus

Nematod: Meloidogyne arenaria, M. incognita

 Advantages of MALDI-TOF

·   MALDI-TOF MS makes use of whole cells or crude, acidic extracts, and mass spectra for the identification of individual species. 

·   Simple sample preparation, short measurement times, quick spectral profile development, high reproducibility and low costs per sample makes it the right alternative to existing molecular techniques.

·   It is a highly reliable sensitive detection approach and can be combined with additional extraction or processing steps to become aware of specific biomarkers, metabolites, or biochemical functions.

·   Ions of low molecular mass may be detected directly from the tissue without additional sample treatment. 

 Disadvantages of MALDI-TOF

·   Excessive preliminary cost

·   The Link between MALDI-TOF MS peptide/protein profiles and proteomic identification database of individual biomarker molecules is constrained which desires to be expanded.                                                         

Conclusion 

MALDI-TOF MS-based proteomics will become a reliable approach for identification, characterization, and a better understanding of plant-associated microorganisms. This inexpensive protein mass pattern detection technique can be easily used for robust identification of microorganisms up to genus, species, and, in a few cases, subspecies levels. Future improvement of MALDI-TOF MS methodology involves a non-stop updating of current commercial databases and improving the instrumentation for specialized research purposes.

References

 

Ahmad, F., Babalola, O. O., and Tak, H. I. (2012). Potential of MALDI-TOF mass spectrometry as a rapid detection technique in plant pathology: identification of plant-associated microorganisms. Analytical and Bioanalytical Chemistry, 404(4), 1247-1255.

Drissner, D. and Freimoser, F. M. (2017). MALDI-TOF mass spectroscopy of yeasts and filamentous fungi for research and diagnostics in the agricultural value chain. Chemical and Biological Technologies in Agriculture, 4(1), 1-12.

Ilina, E. N., Borovskaya, A. D., Malakhova, M. M., Vereshchagin, V. A., Kubanova, A. A., Kruglov, A. N. and Govorun, V. M. (2009). Direct bacterial profiling by matrix-assisted laser Desorption− Ionization time-of-flight mass spectrometry for identification of pathogenic Neisseria. The Journal of Molecular Diagnostics, 11(1), 75-86.

 Signor, L. and Erba, E. B. (2013). Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometric analysis of intact proteins larger than 100 kDa. Journal of Visualized Experiments: (79): 50635.

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