A Survey for 'Candidatus Liberibacter' Species in South Africa Confirms the Presence of Only 'Ca. L. africanus' in Commercial Citrus

Greening disease of citrus is a serious disease known in South Africa since the late 1920s. In South Africa, it is associated with infection by 'Candidatus Liberibacter africanus', a heat sensitive, phloem-limited, noncultured alpha-proteobacterium. Huanglongbing (HLB), a similar, but more devastating disease that was described initially from China but which now occurs in several citrus producing countries, is associated with a different Liberibacter species, 'Ca. L. asiaticus'. A 'Ca. L. africanus' subspecies. 'Ca. L. africanus subsp. capensis', has been found only in South Africa infecting an indigenous Rutaceous species, Calodendrum capense (Cape Chestnut), in the Western Cape in 1995. The discovery of a new Liberibacter species in Brazil, 'Ca. L. americanus', and the spread of 'Ca. L. asiaticus' to a number of additional countries over the last few years prompted us to assess whether only 'Ca. L. africanus' is present in commercial citrus orchards in South Africa. Samples displaying greening or similar symptoms were collected from 249 citrus trees from 57 orchards distributed throughout the greening affected citrus production areas of South Africa. Multiplex polymerase chain reaction (PCR) was performed on DNA extracts to detect the known citrus Liberibacters. Amplicons were obtained from 197 samples. None of the samples yielded a 1,027-bp amplicon indicative of 'Ca. L. americanus' infection. The amplicons of 84 samples were sequenced, and all were identical to the cognate 'Ca. L. africanus' Nelspruit sequence in GenBank. No instance of 'Ca. L. asiaticus' or 'Ca. L. africanus subsp. capensis' sequence was found. Geographically representative samples that tested negative for Liberibacter also tested negative for phytoplasmas based on real-time PCR results. Based on the results of this survey, it is concluded that to date only 'Ca. L. africanus' is associated with citrus greening in commercial citrus in South Africa.