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Assessment of Variability in Plant Viral and Viroid Pathogens

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Microbial Plant Pathogens-Detection and Disease Diagnosis:

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Abstract

Assessment of variability in different characteristics of viral and viroid pathogens affecting various crops has provided information on the differences in the pathogenic potential, host range and distribution of different strains/variants of a virus or viroid. Biological, immunological and nucleic acid-based techniques have been shown to be efficient and effective in differentiating the strains of viruses to varying degrees. Variations in plant virus strains may occur due to mutation or selection from a mixture of strains, because of the incorporation of resistance gene(s) into the cultivars that were susceptible earlier. Changes in the population structure of viruses have to be constantly monitored by applying sensitive techniques that can provide results rapidly to recognize the occurrence of new more virulent virus strain(s) to facilitate formulation of effective disease management strategies applicable to different ecosystems. Viroids, the simplest plant pathogenic entities containing exclusively short circular RNA molecules, also vary distinctively when their environment is significantly altered. The viroids have high potential for producing variants that can infect different plant species that may be available along with crop plants. In addition, perennial fruit trees with long span of life, may allow accumulation of several sequence variants in the same tree without the need for passage through other plant species. Nucleic acid-based techniques have been demonstrated to be very effective in detecting and differentiating variants even with a change in a single nucleotide in the genome of the viroids.

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    P. Narayanasamy

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Appendix: Differentiation of Peach latent mosaic viroid (PLMVd) Isolates by SSCP Analysis (Xu et al. 2008)

Appendix: Differentiation of Peach latent mosaic viroid (PLMVd) Isolates by SSCP Analysis (Xu et al. 2008)

  1. (i)

    Mix aliquots of 0.2 μl of the RT-PCR products amplified from nucleic acid ­preparations or PCR products amplified from plasmids with 0.8 μl denaturing loading buffer (containing 95% formamide, 10 mmol/l Na2OH, 0.05% bromophenol blue, 0.05% xylene cyanol); heat the mixture at 100°C for 8 min and chill on ice for 3 min.

  2. (ii)

    Load the mixture into prerun (300V for 10 min) and precooled (at 4°C for 30 min) 8% non-denaturing polyacrylamide subjected to three conditions:

    • Condition I – acrylamide: bisaccarylamide 49:1

    • Condition II – 29:1

    • Condition III – two equal sections – the upper section is 29:1 and the bottom section is 49:1 gel (20 × 12.5 × 0.1 cm)

  3. (iii)

    Perform electrophoresis at 200V in a refrigerator at 4°C in 1 × TBE buffer (89 mmol/l Tris, 89 mmol/l boric acid, 2.5 mmol/l EDTA, pH 8.3) for 16–20 h.

  4. (iv)

    Stain the gel with silver.

  5. (v)

    Analyze the clones from different isolates first in condition I; identify haplotypes with more than one clone; analyze further in condition II and finally verify the clones representing the predominant haplotype screened in the first two conditions, in condition III.

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Narayanasamy, P. (2011). Assessment of Variability in Plant Viral and Viroid Pathogens. In: Microbial Plant Pathogens-Detection and Disease Diagnosis:. Springer, Dordrecht. https://doi.org/10.1007/978-90-481-9754-5_4

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