Specific PCR detection of Pseudophaeomoniella spp. in the xylem of healthy and Xylella fastidiosa-infected olive trees

Several fungal species have been found associated with the olive quick decline syndrome (OQDS) caused by Xylella fastidiosa subsp. pauca (Xfp) in Apulia, southern Italy. The xylem-inhabiting species, Pseudophaeomoniella oleae and Ps. oleicola, were associated with brown or black wood streaking of various olive varieties, both on young and centenarian trees. However, specific pathogenicity tests conducted on olive plantlets indicated that these fungal species have a marginal role in the aetiology of OQDS. Considering the wide distribution of Ps. oleae and Ps. oleicola over the olive-growing areas in Apulia, a genomic characterisation study was started, in order to investigate the biology of these fungi, and to ascertain any possible interaction with Xfp and the OQDS. Several PCR primers were designed from the internal transcribed spacer (ITS) regions of the rDNA genes of Pseudophaeomoniella spp. in order to develop a species-specific detection method. Primers were screened against the two reference strains, Ps. oleae FV84 and Ps. oleicola M24, and six more Pseudophaeomoniella spp. isolates, resulting in the amplification of a single specific amplicon from most of the primer pairs tested. Fifteen primer pairs confirmed their specificity when tested against several isolates of different xylem-inhabiting fungal genera, such as Phaeoacremonium, Pleurostomophora, Phaeomoniella, Ochroconis, Paraconiothydium, Aspergillus, Lophiostoma and Cladosporium spp. The size of the obtained amplicons enabled the use of some primer pairs in a real-time PCR test, using a SYBR® Green format. Results confirmed the specificity of the tested primers, thus allowing the detection and quantification of Ps. oleae and Ps. oleicola, into the xylem of both healthy and Xfp-infected olive trees. Further research is in progress to develop a specific probe for qPCR quantification of the targeted fungal species in the olive plant.