Molecular Characterization of Phytoplasma 16s rRNA Gene and Determination of Wild Grasses Hosting Phytolasmas in Western Kenya
Napier grass (Pennisetum purpuruem), an indigenous tropical African clumping grass, is the major livestock feed used by many farmers in zero-grazing systems in Kenya. However, Napier stunt (Ns) disease caused by a phytoplasma has adversely affected napier production at rates of up to 90% in many smallholder fields. A number of wild grasses growing around napier fields have also been observed to host phytoplasma. This includes Hyparrhenia rufa grass that has been documented to host phytoplasma subgroup 16SrXI: the causative agent for Ns disease. It is hypothesized that several other wild grasses in Kenya could be infected by phytoplasmas that would otherwise pose a significant threat to napier, other important feeds and food crops. Much has been documented on phytoplasma diseases in Kenya; however, little information is available on the aspects of phytoplasma distribution, identification of alternative wild grasses playing host to phytoplasmas as well as genotypic distribution of phytoplasmas in western Kenya. This study therefore sought to detect and identify phytoplasma strains infecting wild grasses in western Kenya using 16S ribosomal RNA (ribonucleic acid) gene as well as identify wild grass species hosting phytoplasmas in 646 wild grass samples that were collected in October 2011 and January 2012 during a random survey conducted in Bungoma and Busia counties of western Kenya. Deoxyribonucleic acid (DNA) was extracted from the air-dried grass samples, by Cetyl trimethylammonium bromide (CTAB) method and analyzed using nested polymerase chain reaction (nPCR) based on the highly conserved hypervariable 16S rDNA (ribosomal DNA). A total of 33 phytoplasma positive samples were chosen for sequencing. The 16S rRNA gene sequences of all the phytoplasmas isolated from the wild grasses in this study were compiled in FASTA format and compared with each other and with 16 other reference phytoplasmas from NCBI Genbank database. Phylogenetic analysis was done by DNA neighbor-joining method using MEGA version 5.05 software. In this study there was a strong association between proportions of phytoplasma infection and the grass species collected (p = 0.001). C. dactylon, D. scalarum, B. brizantha, poverty grass and P. maximum had high proportions of infection of 38.0%, 17.3%, 16.0%, 7.4% and 4.9% respectively and were abundantly distributed in western Kenya hence considered as wild phytoplasma hosts. E. indica and C. ciliaris were scarcely distributed in western Kenya and had low infection rates of 2.5% and 1.2% respectively. There was statistically significant difference in proportions of infection per location of survey (p = 0.001). Phytoplasma subgroups 16SrXI and 16SrXIV were the only phytoplasma genotypes distributed among wild grasses in western Kenya. ‘Ca. Phytoplasma cynodontis’ (subgroup 16SrXIV), predominantly infects only C. dactylon and B. brizantha wild grass types while ‘Ca Phytoplasma oryzae’ (subgroup 16SrXI), is broad spectrum and infects a large number of wild grasses, most importantly; P. maximum, D. scalarum, poverty grass and B. brizantha. In general, there is a diversity of wild grasses hosting phytoplasmas in western Kenya. These findings suggest that it would be necessary to have a buffer zone clear of wild grasses around napier grass fields to reduce the risk of phytoplasma infection from wild grass sources to napier grass in the short term. There is need to intensify awareness in cultivating alternative fodder since napier grass faces a great threat from phytoplasma infection. Nonetheless, there is need to genetically engineer napier grass for the production of transgenic tolerant napier grass variety against phytoplasma infection.