A Highly Sensitive Nested-PCR Method Using a Single Closed Tube for the Detection of Colletotrichum gloeosporioides causing Greater Yam Anthracnose

Authors

  • Mithun Raj Div. of Crop protection, Central Tuber Crops Research Institute , Sreekariyam Trivandrum 695 017, Kerala
  • Jeeva ML Principal Scientist Div. of Crop protection, Central Tuber Crops Research Institute , Sreekariyam Trivandrum 695 017, Kerala
  • Vishnu S Nath Div. of Crop protection, Central Tuber Crops Research Institute , Sreekariyam Trivandrum 695 017, Kerala
  • Senthil Sankar Div. of Crop protection, Central Tuber Crops Research Institute , Sreekariyam Trivandrum 695 017, Kerala
  • Pravi Vidhyadharan Div. of Crop protection, Central Tuber Crops Research Institute , Sreekariyam Trivandrum 695 017, Kerala
  • Archana PV Div. of Crop protection, Central Tuber Crops Research Institute , Sreekariyam Trivandrum 695 017, Kerala
  • Vinayaka Hegde Div. of Crop protection, Central Tuber Crops Research Institute , Sreekariyam Trivandrum 695 017, Kerala

Keywords:

Greater yam, anthracnose, diagnosis, nested PCR

Abstract

Greater yam is an important species of yam grown in different parts of India. Anthracnose or die back  disease caused by Colletotrichum gloeosporioides reduces the yield up to 90 per cent. The pathogen  survives in the soil debris and transmits the disease to the next season through tubers. In this study,a sensitive method for the specific diagnosis of C. gloeosporioides in soil and planting material was  developed. The standard nested PCR previously described was re-standardized to run a single nested  PCR in a closed tube. The problems of cross contamination and the increased risk of PCR product  contamination, while handling the product of first PCR could be avoided. This technique could beachieved by adjusting the concentration of primers and the annealing temperature along with the  standardization of PCR cycles so as to produce a single amplicon without compromising the limit of  detection and specificity. The lowest amount of DNA that could be determined by this method was  200 pg ml-1.

References

Amusa, N.A. and Alabi, A.A. 1996. Host range of Colletotrichum gloeosporioides from Gliricidia sepium and its implication in crop production. Crop Res., 11: 359.363.

Azim, T., Jeeva, M.L., Pravi, V., Archana, P. V. and Mishra, A.K. 2010. Exploration of smaller subunit ribosomal DNA for detection of Colletotrichum gloeosporioides causing anthracnose in Dioscorea alata L. J. Root Crops, 36 (1): 83-87.

Bertolini, E., Penyalver, R., Garcia, A., Olmos, A., Quesada, J.M., Cambra, M. and Lo ´ pez, M.M. 2003. Highly sensitive detection of Pseudomonas savastanoi pv. savastanoi in asymptomatic olive

plants by nested-PCR in a single closed tube. J. Microbiol. Methods, 52: 261.266.

FAO. 2002. FAOSTAT Agriculture data. Food and Agriculture Organisation of the United Nations. http://apps.fao.org/collections.

Freeman, S., Minz, D., Jurkevitch, E., Maymon, M.and Shabi, E. 2000. Molecular analyses of Colletotrichum species from almond and other

fruits. Phytopath., 90: 608-614.

Ivey, L.M.L., Nava-Diaz, C. and Miller, S.A. 2004. Identification and management of Colletotrichum acutatum on immature bell peppers. Pl. Dis., 88 (11): 1198-1204.

Leung, H., Nelson, R.J. and Leach, J.E.1993. Population structure of plant pathogenic fungi and bacteria. Adv. Pl. Path., 10: 157-205.

Llop, P., Bonaterra, A., Penalver, J. and Lo ´ pez, M.M.

Development of a highly sensitive nested-

PCR procedure using single closed tube for

detection of Erwinia amylovora in asymptomatic

plant material. Appl. Environ. Microbiol.,

: 2071.2078.

Martínez-Culebras, P.V., Barrio, E., García, M.D.and

Querol, A. 2000. Identification of

Colletotrichum species responsible for

anthracnose of strawberry based on the internal

transcribed spacers of the ribosomal region.

FEMS Microbiol. Lett., 189: 97-101.

Milgroom, M.G.and Fry, W.E .1997. Contributions

of population genetics to plant disease

epidemiology and management. Adv. Bot. Res.,

: 1-30.

Mithun Raj., Jeeva, M.L., Hegde, V., Pravi, V.,

Archana, P. V., Senthil Alias Sankar, M. and Vishnu Nath, S. 2012a. Polymerase chain reaction assay

for rapid, sensitive detection and identification

of Colletotrichum gloeosporioides causing

greater yam anthracnose. Mol. Biotech. ,

: 277.284.

Mithun Raj, Vinayaka Hegde, Jeeva, M.L., Archana,

P.V., Pravi, V., Vishnu Nath S. and Senthil@Sankar, M. 2012b.

Rapid and efficient method for the extraction of genomic DNA

from Colletotrichum spp. suitable for PCR analysis. Dynamic

Biochem. Process Biotech. Mol. Biol., 6 (SI2): 95-97.

Mithun Raj., Vinayaka Hegde., Jeeva, M.L., Senthil@Sankar,

M., Vishnu Nath, S., Pravi, V. and Archana, P.V. 2013a.

Molecular diagnosis of Colletotrichum gloeosporioides

causing anthracnose/dieback disease in greater yam (Dioscorea

alata L.) Arch.Phytopath.Pl. Protection, 46 (8): 927- 936.

Mithun Raj., Vishnu Nath, S., Senthil@Sankar M., Jeeva, M.L and

Vinayaka Hegde. 2013b. Rapid isolation of DNA from

Dioscorea species suitable for PCR, restriction digestion and

pathogen screening. Arch. Phytopath. Pl. Protection, DOI.

1080/03235408.2013.821753.

MunËœoz J.A.G., Suarez, M.B., Grondona, I., Monte, E., Buddie,

A.G. and Bridge, P.D. 2000. A physiological and biochemical

approach to the systematics of Colletotrichum species

pathogenic to strawberry. Mycologia, 92: 488.498.

Nwakiti, A.O. and Arene, O.B. 1978. Disease of yam in Nigeria.

Pest Articles and News Summaries (PANS), 24: 468-496.

Okabe, I.and Matsumoto, N .2000. Population structure of

Sclerotium rolfsi iin peanut fields. Mycosci., 41(2): 145-148.

Orou, A., Fechner, B., Menzel, J.H. and Utermann, G. 1995.

Automatic separation of two PCRs in one tube by annealing

temperature. Trends Genet., 11: 127.128.

Peter, R. Mills., Sreenivasaprasad, S. and Averil, E. Brown.1992.

Detection and differentiation of Colletotrichum gloeosporioides

isolates using PCR. FEMS Microbiol. Lett., 98: 137-144.

Simon, S.A., 1993. Epidemiology and control of yam anthracnose.

Report of the Natural Resource Institute, UK.

White, T. J., Bruns, T., Lee, S. and Taylor, J. 1990. Amplification

and direct sequencing of fungal ribosomal RNA genes for

phylogenetics. In: PCR Protocols: a Guide to Methods and

Applications. Innis, M., Gelf, D., Sninsky, J. and White, T. (Eds.).

Academic Press, Orlando, Florida, pp.315.322

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Published

2015-01-01

How to Cite

Raj, M., ML, J., Nath, V. S., Sankar, S., Vidhyadharan, P., PV, A., & Hegde, V. (2015). A Highly Sensitive Nested-PCR Method Using a Single Closed Tube for the Detection of Colletotrichum gloeosporioides causing Greater Yam Anthracnose. JOURNAL OF ROOT CROPS, 39(2), 163–167. Retrieved from https://ojs338.isrc.in/index.php/jrc/article/view/105

Issue

Vol. 39 No. 2 (2013): Journal of Root Crops

Section

Research Articles

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