Design of Species-Specific Primers for Early Detection of Kretzschmaria zonata, the Causal Agent of Root and Neck Rot of Teak (Tectona grandis)

Kretzschmaria zonata (Lév.) P.M.D. Martin is a fungus that causes root and neck rot in teak (Tectona grandis L.) worldwide. The detection of this fungus in asymptomatic plants is essential for its control but, to date, the disease can only be detected morphologically or by sequencing this fungus’s isolates. The aim of this work was to design, at least, one set of specific PCR primers for a quickly, early and accurate diagnosis of this pathogen. Nineteen isolates of K. zonata from teak plants at different locations in Mexico were obtained. ITS region sequences and two software were used to design the PCR-based primers. All primers obtained were tested on DNA samples from infected teak tissue, K. zonata pure isolates, and other different fungi species (in vitro and in silico). The sensitivity of the primers was evaluated at five different concentrations of target DNA. The primer set KZ-AQ-3F/KZ-AQ-3R was selected for its specificity (amplifies only K. zonata) and sensitivity (1 × 10−5 ng/µL detection). This is the first report of a Kretzschmaria zonata-specific primer set. Tests carried out on asymptomatic teak and artificially inoculated plants with this fungus demonstrated the usefulness of these primers for the accurate detection of K. zonata, and taking early control measures

Design of Species-Specific Primers for Early Detection of <i>Kretzschmaria zonata</i>, the Causal Agent of Root and Neck Rot of Teak (<i>Tectona grandis</i>)

Kretzschmaria zonata (Lév.) P.M.D. Martin is a fungus that causes root and neck rot in teak (Tectona grandis L.) worldwide. The detection of this fungus in asymptomatic plants is essential for its control but, to date, the disease can only be detected morphologically or by sequencing this fungus’s isolates. The aim of this work was to design, at least, one set of specific PCR primers for a quickly, early and accurate diagnosis of this pathogen. Nineteen isolates of K. zonata from teak plants at different locations in Mexico were obtained. ITS region sequences and two software were used to design the PCR-based primers. All primers obtained were tested on DNA samples from infected teak tissue, K. zonata pure isolates, and other different fungi species (in vitro and in silico). The sensitivity of the primers was evaluated at five different concentrations of target DNA. The primer set KZ-AQ-3F/KZ-AQ-3R was selected for its specificity (amplifies only K. zonata) and sensitivity (1 × 10−5 ng/µL detection). This is the first report of a Kretzschmaria zonata-specific primer set. Tests carried out on asymptomatic teak and artificially inoculated plants with this fungus demonstrated the usefulness of these primers for the accurate detection of K. zonata, and taking early control measures