Identification of alternative hosts to citrus of “Candidatus Liberibacter africanus” amongst indigenous Rutaceae of South Africa

Abstract

Citrus greening or Huanglongbing is a severe disease affecting all citrus cultivars and rootstocks and is responsible for causing severe economic losses in many citrus producing countries. It is caused by various fastidious phloem-restricted, Gram negative bacteria belonging to the “Candidatus Liberibacter” genus. Currently only three species that affects citrus have been identified, “Candidatus Liberibacter africanus” (Laf) found in Africa, Mascarene Islands and the Middle East,“Candidatus Liberibacter asiaticus” (Las) present in Asia, and only recently emerged in Florida USA, São Paulo Brazil and Cuba and a new species “Candidatus Liberibacter americanus” (Lam) found only in São Paulo Brazil. Due to the continuing spread of this serious and destructive disease into areas of South Africa previously regarded as disease-free and disease-managed, the role of alternative hosts in the transmission of the disease is questioned. The aim of this study was to identify possible alternate hosts of Laf amongst plants of Rutaceae indigenous to South Africa. Identification of these alternate hosts forms a critical part of the integrated management and control strategies of the disease. Leaf material from symptomatic and asymptomatic indigenous rutaceous and non-rutaceous plants were collected from various sites in South Africa, from different environments which included vegetation adjacent to citrus orchards, in areas with high incidence of the citrus greening disease, from natural habitats, botanical gardens and private properties. A multiplex polymerase chain reaction (PCR) was developed for the simultaneous detection of Laf DNA and ubiquitous ribulose bisphosphate carboxylase oxygenase (Rubisco) gene which served as an internal control for the presence and quality of extracted DNA and potential amplification inhibition by compounds of the DNA extract. The multiplex PCR oligonucleotide primers were used to amplify fragments of 669bp from the β ribosomal operon of Laf and 179bp of Rubisco gene. Petioles and/or leaf midrib tissues were tested for the presence of Laf using the newly developed multiplex PCR and β operon specific primers of a published conventional PCR method. In this study seventeen Calodendrum capense Thunb. (Cape chestnut) plants from various geographic areas of South Africa were identified to be naturally infected with Liberibacter DNA. Sequence analysis revealed the DNA to be that of “Candidatus Liberibacter africanus subspecies capensis” (LafC). Although this subspecies of Liberibacter has previously been detected on C. capense in the Stellenbosch area of the Western Cape Province, this is the first report indicating the natural occurrence of LafC in C. capense in Gauteng, Limpopo and Mpumalanga Provinces of South Africa. The geographic distribution of the LafC bacterium associated with Cape chestnut appears to be more widespread than initially expected. No Laf DNA was detected from C.capense or any other indigenous plants surveyed. Copyright