FBIP: Taxonomy and Candidatus Liberibacter status of South African Buchu plants

Study Extent

Sampling

Method steps

1. 1) We plan to collect and sequence barcoding genes of known, morphologically identified specimens of buchu collected under permit at Kirstenbosch botanical gardens or from herbarium specimens where permission is granted to do so. Collection from live plants will not be destructive as only about 5g fresh weight of leaf material is required in order to obtain phloem tissue (to which the Ca. Liberibacters are confined). 2) We will collect leaf material from large numbers of buchu specimens (we plan to collect a total of about 1000 specimens in total, with between 10 and 20 replicates of visually identified species of “buchu” (in the broadest sense) from randomly selected transects from three Western Cape nature reserves containing the greatest diversity of buchu, and where permits for the study can be obtained. This will be done in the flowering season of two successive years. All samples obtained will be assigned a unique accession number and the following data will be recorded for each specimen: date of collection, collector details, photographs of the plant and any distinguishing feature(s), plant visual identification, locality data, the exact location (to an accuracy of 0.25m determined by a differential GPS) and the habitat description. 3) Collected material will be archived in the South African Plant Virus culture collection (SAPVC) at ARC-PPRI, as desiccated plant material as well as in -80oC freezer storage, and voucher specimens for identification purposes submitted to the Compton Herbarium (NBG) and National Herbarium (PRE). 5) All samples will be tested for Ca. Liberibacter using a generic Ca. Liberibacter PCR developed in our laboratory (Pietersen, unpublished), based on all known Ca. Liberibacter sequences. 6) All Liberibacter infected samples (as determined by real-time PCR) will be tested with primers to specific Liberibacters (Ca. Liberibacter -americanus, -africanus, - asiaticus, -solanacearum and -africanus subspecies –capensis, -verpidis, -clausenae, -zanthoxyli, and –tecleae) in conventional PCR’s or a newly developed multiplex PCR capable of detecting these (Roberts unpublished results). 7) All plant samples, irrespective of Ca. Liberibacter status, will be subjected to barcoding PCR’s (rbcL, matK, trnH/psb) and the amplicons sequenced by Sanger sequencing.

1. Rutaceae
   common name: Buchus Plants rank: family