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Assessment of Variability in Fungal Plant Pathogens

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Microbial Plant Pathogens-Detection and Disease Diagnosis:

Abstract

Fungal pathogens show variations in their pathogenic potential (virulence) and physiological functions that support their survival and perpetuation in different environmental conditions. These variations may be largely due to genetic factors that may be conditioned by environmental factors. The existence of strains, races or biotypes may be recognized by differences in virulence of the isolates on differential species or cultivars. Assessment of variability may be performed by applying techniques based on the cultural, biochemical, immunological and nucleic acid characteristics. Attempts have been made to relate the variability in these characteristics to variability in pathogenicity of isolates of fungal pathogens. Information on the extent of variability of isolates of fungal pathogens is essential to plan effective methods of tackling the diseases caused by them. In addition, development of resistance to fungicides has been a serious concern to growers, researchers as well as the administrators, since the decision to continue application or withdrawal of a fungicide can affect both the growers and the industry economically.

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Appendix 1: Identification of Guignardia citricarpa by PCR-Based Technique (Peres et al. 2007)

Appendix 1: Identification of Guignardia citricarpa by PCR-Based Technique (Peres et al. 2007)

A. Extraction of DNA from fruit lesions

  1. (i)

    Dissect out very small citrus fruit lesions (approximately 2 to 4 mm in diameter); discard the surrounding healthy flavedo, the pigmented outer tissue of the fruit rind and disrupt the tissue using alkaline lysis extraction method.

  2. (ii)

    Follow the dipstick method by adding 150 μl of 100% ethanol and a small piece of cellulose thin-layer chromatography plate (dipstick) to the 2-ml tube; place the tubes on their sides on ice and shake for 30 min.

  3. (iii)

    Aspirate off the liquid from the tube; add 500 μl of wash buffer [10 × (Tris Na2-EDTA and NaCl, pH 7.0)] diluted to 25%; invert tubes to mix the contents well; repeat washing twice; place the dipstick in a new tube and dry under vacuum.

  4. (iv)

    Place the tubes on their sides; add 50 μl Tris-EDTA buffer; incubate for 5 min; spin the tubes for 10 s; discard the dipstick and recover the DNA.

B. PCR amplification

  1. (i)

    Carry out the amplification in a total volume of 20 μl in the mixture containing 8 μl 2.5 × Eppendorf MasterMix (Taq DNA polymerase at 0.06 U/μl, 2.5 × Taq reaction buffer (125 mM KCl,75 mM Tris-HCl, pH 8.4, 4 mM Mg2+ and 0.25% Nonidet-P40, 500 μM each of dNTP and stabilizers), 0.8 μl of each primer at 10 μM and 2 μl of template DNA.

  2. (ii)

    Provide the following conditions: 94°C for 2 min, followed by 39 cylces of 94°C for 30 s, annealing at 64°C for 30 s, 72°C for 1 min and a final extension for 10 min at 72°C

  3. (iii)

    Separate the amplicons by electrophoresis in 1% agarose gels in 1 × Tris-borate-EDTA buffer.

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Narayanasamy, P. (2011). Assessment of Variability in Fungal Plant Pathogens. In: Microbial Plant Pathogens-Detection and Disease Diagnosis:. Springer, Dordrecht. https://doi.org/10.1007/978-90-481-9735-4_4

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