Detection of Cercospora beticola by PCR in amended and naturally infested field soil.

Abstract

The causal agent ofCercospora leaf spot ofsugarbeet (Beta vulgaris L), Cereospora betieola. Sacco 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 sugarbeet. We developed and present here a PCR technique for detection ofC. betieola in the soil. The DNA was purified from soil amended with C. betieola and naturally infested soil using PowerSoil DNA Kit (MO BIO Lab. Carlsbad, CA) as per manufacture's instructions. The purified DNA was collected and subjected to PCR reaction in Extract-N-Amp PCR mix (Sigma Aldrich, St Louis MO) with CBACTIN based primers. Amplification was carried out over 35 cycles using a Mastercycler gradient thennocycler (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. betieola amended and the infected field soil products correlated with the expected size of the control DNA extracts from C. betieola cultures. Amplicons were sequenced and compared to pure culture C. betieola actin sequence. Alignment of sequences of the amplified products confirmed them to be those of C. beticola. The system will enable rapid post planting screening for inoculum potential of C. betieola in soil and determine the effect of soil applied biocontrol agents on C. betieola and inoculum potential. Introduction Sugarbeet (Beta vulgaris L.) and saffiower (Carthamus tinetorius L.) are important crops in the Lower Yellowstone River Valley where sugarbeet is also rotated with irrigated safflower. Both crops are susceptible to Cereospora betieola Sacc., which causes Cercospora leafspot (CLS) ofsugarbeet (Fig. 1 a) and leaf spot of Cercospora (LSC) on saffiower (Fig. I b). The pathogen overwinters on infected beet residue as stromata (Windels et al, 1998). Under optimal conditions, characterized by relatively high humidity or heavy dew, conidiophores and conidia are produced on the stromata (Ruppel, 1986). The conidiophores and conidia that serve as primary inoculum are dispersed by wind, irrigation and rain water and insects to sugarbeet to initiate primary infection. Thus the disease could be transmitted from post harvest inoculum reserve to the other crop in the foUowing growing season. Currently, a method is available for direct detection and identification C. betieola in infected plant tissues (Lartey et aI, 2003). Other plant hosts can therefore be detected and identified. As an additional step toward completely tracking headway ofthe pathogen in the field, we have developed and present here a protocol for detection of C. betieola in soil.