Surveillance and isolation of Rosellinia necatrix in South African avocado orchards

Abstract

Rosellinia necatrix Berl. ex Prill. is an ascomycete phytopathogen that targets a broad range of hosts with an ability to live as a saprophyte, allowing occupation of infected areas for extended periods. As the causal agent of white root rot (WRR), R. necatrix has caused significant economic losses within the agricultural and forestry industries in various regions. The aim for this study was focused on the surveillance and monitoring of the spread of R. necatrix in avocado orchards in South Africa. This study was also focused on developing a selective medium for the isolation of R. necatrix, to aid with primary isolation and identification. Five selective chemicals were tested in vitro for their effect on R. necatrix growth, as well as in combination to develop a selective medium. The efficacy of this selective medium was then determined for primary isolation from avocado bark and roots naturally infected with R. necatrix. Samples from 29 South African avocado orchard plots were screened for the presence of the pathogen, from which 16 were determined to be infected with R. necatrix. In addition to being present in the Western Cape, R. necatrix was found to also be present in the Limpopo and Mpumalanga provinces. The selective medium exhibited an average recovery rate of 95%, while the basal medium only exhibited an average recovery of 30%. Due to the low reliability of primary isolation, a requirement for a more rapid detection technique for R. necatrix arose. Hence, this study also focused on the optimisation and implementation of a high-throughput method for the detection of R. necatrix in South African avocado orchards. The specificity and sensitivity of different molecular detection techniques were evaluated on different R. necatrix isolates, as well as on other Rosellinia and fungal species. The sensitivity of these detection methods was evaluated using artificially inoculated soil and plant material, as well as naturally infected plant material. Both species-specific primer pairs amplified target regions from all R. necatrix isolates and none of the other fungal species. Dilution tests revealed that the detection limit of each primer pair in conventional PCR was 700 fg/µl, and 70 fg/µl for the TaqMan qPCR. qPCR using the TaqMan probe was the most efficient high-throughput method, which yielded a 91.3% detection rate in artificially infected roots, 10% in artificially infected soil, and 82.4% in naturally infected bark. Conventional PCR with both primer pairs was the least effective method, with less than 62% detection in naturally infected bark. Nested qPCR had a 100% detection rate in artificially infected roots, 60% in artificially infected soil, and 100% in naturally infected bark. Although the other assays were inadequate for detection in naturally infected soil samples, the nested qPCR improved detectability. Knowledge of the spread and prevalence of WRR in South Africa will create awareness of the disease in the farming community. The use of the newly developed selective medium and implementation of the TaqMan qPCR detection method will allow reliable detection of R. necatrix in South Africa, which is vital for early diagnosis.