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United States Department of Agriculture

Agricultural Research Service

Title: Detection of cercospora beticola by PCR in amended and naturally infested field soil

Authors
item Lartey, Robert
item Caesar, Thecan
item Hanson, Sophia
item Iversen, William
item Evans, Robert

Submitted to: American Society of Sugarbeet Technologists
Publication Type: Proceedings
Publication Acceptance Date: May 1, 2007
Publication Date: July 1, 2007
Repository URL: http://hdl.handle.net/10113/14119
Citation: Lartey, R.T., Caesar, T., Hanson, S.L., Iversen, W.M., Evans, R.G. 2007. Detection of cercospora beticola by PCR in amended and naturally infested field soil. In: Proceedings of the 34th Biennial Meeting of the American Society of Sugarbeet Technologists, February 28-Mark 3, 2007, Salt Lake City, Utah. 34: 211-216.

Interpretive Summary: Cercospora beticola, the causal agent of Cercospora leaf spot of sugar beet survive in infected leaf residues in the soil. When conditions are most favorable, the surviving propagules germinate and produce conidia which are dispersed to initiate infection in sugar beet. Currently, no technique is available for direct detection of the pathogen in the soil. To fill this vacuum, we developed and present here a PCR technique for detection of C. beticola in the soil. The DNA of C. beticola was purified from soil using PowerSoil DNA Kit (MO BIO, CA) following the manufactures instructions. The purified DNA was subjected to PCR in Extract-N-Amp PCR reaction mix (Sigma Aldrich, St Louis MO) using C. beticola specific primers. PCR amplification was carried out over 35 cycles using a Mastercycler gradient thermocycler (Eppendorf Scientific Inc., Westbury, NY) at 94°C for 1 min denaturation, 52°C for 30 sec annealing, 72°C for 1 min extension and 5 min final extension at 72°C. The amplicons were resolved by agarose gels electrophoresis and sequenced. Comparison of amplicons and DNA sequences form soil and C. beticola cultures confirmed detection of C. beticola in soil samples. The scheme will enable rapid pre-planting screening for inoculum potential of C. beticola in soil and also contribute toward evaluating the effectiveness of soil applied biocontrol agents on C. beticola and inoculum potential in the soil.

Technical Abstract: The causal agent of Cercospora Leaf Spot of sugar beet (Beta vulgaris L), Cercospora beticola. Sacc. survives as stromata in beet leaf residues in the soil. Under optimal conditions, overwintering propagules germinate and produce conidia that are dispersed as primary inoculum to initiate infection in sugar beet. We developed and present here a PCR technique for detection of C. beticola in the soil. The DNA was purified from soil amended with C. beticola and naturally infested soil using PowerSoil DNA Kit (MO BIO, CA) as per manufactures instructions. The purified DNA was collected and subjected to PCR reaction in Extract-N-Amp PCR mix (Sigma Aldrich, St Louis MO) with CBACTIN and ITS based primers. Amplification was carried out over 35 cycles using a Mastercycler gradient thermocycler (Eppendorf Scientific Inc., Westbury, NY) at 94°C for 1 min denaturation, 52°C for 30 sec annealing, 72°C for 1 min extension and 5 min final extension at 72°C. The amplified products were resolved by electrophoresis in 1% agarose gels. The fragment sizes of C. beticola amended and the infected field soil products correlated with the expected size of the control DNA extracts from C. beticola cultures. Amplicons were sequenced and compared to C. beticola actin sequence from gene bank. Alignment of sequences of the amplified products confirmed them to be that of C. beticola. The system will enable rapid post planting screening for inoculum potential of C. beticola in soil and determine effect of soil applied biocontrol agents on C. beticola and inoculum potential.

Last Modified: 4/19/2014
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