|Eckhoff, Joyce - Montana State University|
Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/18/2010
Publication Date: 8/12/2010
Publication URL: http://hdl.handle.net/10113/46165
Citation: Lartey, R.T., Caesar, T., Lenssen, A.W., Eckhoff, J., Hanson, S.L., Evans, R.G. 2010. Direct Polymerase Chain Reaction-Based Detection of Cercospora Beticola in Field Soils. Plant Disease. 94(9):1100-1104. Interpretive Summary: Cercospora beticola, which causes Cercospora leaf spot disease of sugar beet, survives in infected beet leaf residues in the soil. In subsequent growing seasons, surviving propagules germinate and produce conidia that are dispersed as primary inoculum to initiate infection in sugar beet and other host plants. In this paper, we report the development of a novel PCR technique for rapid detection of the pathogen in field soils. We first used PowerSoil DNA KitTM purify total DNA from soil that was amended with pure culture of C. beticola. The purified DNA was subjected to PCR using specially designed C. beticola actin (CBACTIN) primers. After successful amplification and detection, the technique expanded to examine real field soil samples from two sugar beet fields near Sidney, MT area and a sugar beet field at Foxholm, ND. As with the original protocol, total DNA was extracted from the samples and subjected to PCR. The PCR products from the soil samples as well as C. beticola pure culture and amended soil controls were run on agarose gels that proved successful amplification of the target pathogen. The PCR fragments were then excised from the gel, purified individually and sequenced. All sequences were compared by alignment with the pure culture and a C. beticola actin gene sequence from GenBank. The comparisons confirmed that the excised PCR amplified fragments from amended soil, various field soil samples and the pure culture were all from C. beticola. This technique will enable rapid screening for potential C. beticola problems prior to planting sugar beet, or other host crops and provide information on the effect of soil applied biological control agents and other management techniques on C. beticola and potential inoculum build up or control in field soils.
Technical Abstract: Cercospora beticola, the causal agent of Cercospora leaf spot of sugar beet, survives as pseudostromata in infected 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 a PCR technique for rapid detection of C. beticola in field soils. Total DNA was first purified from soil amended with C. beticola culture using PowerSoil DNA KitTM7 . The purified DNA was subjected to PCR in Extract-N-Amp PCR mixTM 8 with CBACTIN primers over 35 cycles. The amplified products were resolved by electrophoresis in 1% agarose gels. The PCR fragment size of C. beticola from the amended field soil correlated in size with the amplicon from control C. beticola culture DNA extract. Additionally, sample soils were collected from two sugar beet fields near Sidney, MT area and a sugar beet field at Foxholm, ND. Total DNA was extracted from the samples and subjected to PCR as previously described. All amplicons were subjected to dye terminator cycle sequencing. All sequences as well as C. beticola from amended field soil and pure culture were compared by alignment with a C. beticola actin gene sequence from GenBank. The result of the alignment confirmed the amplicons as products from C. beticola. This system will enable rapid screening for inoculum potential of C. beticola in soil and determine the effect of soil applied biocontrol agents and other management techniques on C. beticola and inoculum potential in field soils.