Development of a qPCR assay to detect Diplodia seriata on chipped apple wood

Abstract

In the deciduous fruit industry, orchards are often excavated and trees chipped. The organic material is then used as mulch for soil conservation, a practice that form part of sustainable agricultural. The presence and possible transmission of plant pathogens are not considered when trees are removed, chipped and used for mulch. Young apple trees can develop cankers due to Diplodia seriata of which the inoculum source might come from fruiting structures present on apple wood mulch. Therefore, the presence of D. seriata, on chipped apple tree wood pieces used for mulch in younger orchards was investigated. To be able to detect D. seriata, qPCR primers were designed from a previously identified unique sequence characterised amplified region (SCAR). The qPCR primers were specific for D. seriata (Cq ≤ 35 and Tm = 85\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\:\\pm\\:$$\\end{document}0.17 °C) when compared with DNA from nineteen other fungal taxa associated with canker or wood rot of apple trees tested, excluding Botryosphaeria dothidea (Cq = 38 and Tm = 85.25 °C). The qPCR assay was sensitive and had a limit of quantification of 2859 fg/µl and limit of detection of 571 fg/µl. Wood chips were collected at two time periods (from heaps and 6 months after it was spread in tree rows) in three apple orchards in the Western Cape of South Africa. DNA was extracted from water-washes of 120 wood piece samples and D. seriata was detected from 101 of these samples. This study showed that the newly developed primers was able to successfully detect D. seriata from mulched apple wood. The presence of D. seriata on apple tree wood chips indicates that there is a risk involved in using wood chips made from old apple trees.