Occurrence of ‘Candidatus Phytoplasma aurantifolia’ (16SrII group) in cassava and four other species in Uganda

Abstract

Cassava (Manihot esculenta) is amongst the most important crops for Uganda, being grown on more than 400 000 ha (FAOSTAT, 2005). Symptoms of leaf yellowing, chlorosis, shortening of internodes, and slight stunting were recently observed in cassava fields in Kawanda. Interestingly, some plants of four other species (sticky mallow, Malvaviscus arborus; croton, Codiaeum variegatum.; Chinese hibiscus, Hibiscus rosa-sinensis.; and passion fruit, Passiflora edulis) growing nearby showed little leaf, chlorosis, and leaf yellowing and deformation. Two samples of leaves with symptoms were collected from each species and total DNA extracted and used as template in a nested PCR with universal 16S rRNA primers R16mF2/R1 and fU5/rU3. PCR amplicons (880 bp) were produced in 8/10 samples, purified, and directly sequenced in both directions. Sequences were manually edited and compared with those of reference material from GenBank using blast. Sequences were aligned using Clustal W (Thompson et al., 1994), and a phylogenetic tree constructed using the neighbour-joining method and 1000 replicates for bootstraps analysis with mega 3·1 (Kumar et al., 2004). Restriction profiles obtained after RFLP assays of PCR amplicons with Sau3AI, HpaII and HaeIII enzymes were all similar to those of ‘Candidatus Phytoplasma aurantifolia’(16SrII group). The 16S rRNA sequences of phytoplasmas detected in cassava (EU315317) and M. arboreus (EU315318), showed 98% of identity with that of the cactus witches’ broom phytoplasma (AJ293216), whilst sequences from C. variegatum (EU315315), H. rosa-sinensis (EU315314), and P. edulis (EU315319) exhibited 99% of identity with that of Catharantus roseus phytoplasma (EU096500). The phylogeny results suggest that two distinct sub-types have been identified in (i) cassava and M. arboreus and in (ii) C. variegatum, H. rosa-sinensis, and P. edulis, in plants growing geographically close to each other. This is the first record of phytoplasma group 16SrII, ‘Ca. Phytoplasma aurantifolia’ in Uganda, extending previous reports of its presence in Africa (Arocha et al., 2007). Further studies are needed of the biological aspects of the relationships between closely related phytoplasma isolates affecting nearby plant hosts. Work in the UK was done under Defra licence No. PHF 174D/5185(08/2005). Sequencing was done by the Sequencing Service (School of Life Sciences, University of Dundee, Scotland, http://www.dnaseq.co.uk) using Applied Biosystems Big-Dye Ver 3·1 chemistry on an Applied Biosystems model 3730 automated capillary DNA sequencer.