A molecular assay to investigate the possible association between the chestnut weevil curculio propinquus and the black rot fungus rachodiella castaneae

Abstract

Black rot, induced by Rachodiella castaneae Pyr. [teleomorph: Sclerotinia pseudotuberosa Rehm.; syn. Ciboria batschiana (Zopf) Buchw.], and the chestnut weevil [Curculio propinquus (Desbr.)] are among the most serious phytosanitary problems of the chest- nut (Castanea sativa Mill.) fruit industry in Italy, causing relevant economic losses at harvest and during storage. Symptoms of black rot are frequent in nuts infested by C. propinquus, and hence an association between the fungus and the insect has been hypothesised. To verify this hypothesis, clusters of immature burrs from three trees in a chestnut area in Central Italy were covered with a net and treated as follow: (i) infested with C. propinquus adults that had been artificially contaminated with R. castaneae; (ii) infested with C. propinquus uncontaminated adults; (iii) uninfested negative control. At harvest time, the nets were re- moved and the adults of the insects and chestnut fruits were col- lected. Due to the inefficacy of traditional diagnostic methods, the detection R. castaneae in C. propinquus and in chestnut fruits was performed by PCR. Two sequences specific for R. castaneae were identified in the ITS region of rDNA, and two species-spe- cific primers (RAC1 and RAC2) were designed. R. castaneae was detected in more than 90% of the chestnut weevils analysed, re- gardless the treatment, and in 77, 73, and 92% of nuts following the treatments (i), (ii) and (iii), respectively. These findings sug- gest an endophytic habitus of R. castaneae in chestnut fruits and that C. propinquus is a potential vector of the pathogen.