A multi-phasic approach reveals that apple replant disease is caused by multiple biological agents, with some agents acting synergistically

Apple replant disease (ARD) has been reported from all major fruit-growing regions of the world, and is often caused by a consortium of biological agents. The aim of this study was to investigate the etiology of ARD in South Africa in six orchard soils, using a multiphasic approach under glasshouse conditions. This approach first involved determining the ARD status of the soils by monitoring apple seedling growth responses in non-treated soil versus growth in pasteurized soil, as well as in 15% non-treated soil that was diluted into pasteurized soil. Subsequently, the potential for specific organisms to function as causal agents of ARD was investigated using (i) biocide applications, (ii) quantitative real-time PCR (qPCR) analyses of ARD ‘marker’ microbes (Pythium irregulare, Pythium sylvaticum, Pythium ultimum, Pythium vexans, Rhizoctonia solani AG-5 and the genera Cylindrocarpon and Phytophthora), (iii) nematode analyses, (iv) isolation of actinomycetes and (v) pathogenicity testing of actinomycetes individually, and when co-inoculated with P. irregulare or Cylindrocarpon macrodidymum. The analyses showed that the soils could be grouped into low, moderate and severe ARD soils, with each group containing two soils. Several lines of evidence suggested that actinomycetes are not involved in ARD in South Africa. Multiple biological agents were determined to contribute to ARD including (i) oomycetes (Phytophthora and Pythium) that are important based upon their widespread occurrence, and the fact that metalaxyl application improved seedling growth in four soils (ii) the genus Cylindrocarpon that was also widespread, and for which a synergistic interaction with P. irregulare was demonstrated and (iii) occasionally parasitic nematodes, mainly Pratylenchus penetrans, Pratylenchus scribneri and Pratylenchus delattrei, since fenamiphos application improved seedling growth in two orchards. qPCR analyses of the ARD marker microbes showed that R. solani AG-5 is absent from South African orchards, and that P. ultimum is widespread, even though the latter species could not be detected in previous isolation studies. The other marker microbes were also widespread, with the exception of P. sylvaticum. qPCR quantification of the marker microbes could not be correlated with the severity of ARD in any manner. qPCR analyses did, however, show that possible root pruning pathogens such as P. irregulare, P. sylvaticum and P. ultimum had much lower DNA concentrations in seedling roots than P. vexans and the genera Cylindrocarpon and Phytophthora.