New qPCR protocol to detect Diplodia corticola shows phoretic association with the oak pinhole borer Platypus cylindrus
Muñoz-Adalia, Emigdio Jordán | Meijer Mora, Andreu | Colinas, C. (Carlos)
BACKGROUND: Botryosphaeria canker (causal agent: Diplodia corticola) is considered one of the most important diseases of cork oak (Quercus suber) stands since it necrotizes the subero-phellodermic layer preventing cork regeneration after harvesting. One of the most intriguing etiological issues of this disease is its plausible spreading through trunk borer insects. In this study, we highlight the phoretic relationship between D. corticola and the oak pinhole borer Platypus cylindrus (Coleoptera, Curculionidae) which massively colonizes debarked cork oaks in southern Europe. We extracted DNA from 154 adults of P. cylindrus collected in six cork oak stands in north-eastern Spain during the summer of 2021. We developed a new nested quantitative PCR-based protocol for quick detection of D. corticola carried by insects. RESULTS: The use of real time amplification of a highly specific mitochondrial marker allowed us to detect spore loads down to a single conidium within the first 29 cycles of qPCR. The 29.62% of insect pools (corresponding to 31.16% of studied insects) resulted in an estimated spore load higher than one conidium/insect, with a moderate value of mean spore load for the whole dataset (~33 conidia/insect). Estimated spore load was significantly higher in May and August, regardless of insect abundance in the field. CONCLUSION: This study provides new tools for diagnosis of this emergent pathogen that would be useful for developing monitoring strategies and epidemiological studies.
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