Molecular identification of fungal species associated with leaf lesions of marama bean seedlings in Namibia

During one of our greenhouse experiments on the marama bean plant (Tylosema esculentum) at the University of Namibia, various signs of necrosis and spotting were observed on leaf tissues of the newly developed seedlings. In this study, we hereby describe findings of the investigations undertaken to attempt and determine the possible causative agent(s) of such plant leaf infections. In brief, infected seedling leaves were first surface-sterilized and then used in form of leaf-discs (2 x 2 mm), to inoculate potato dextrose agar (PDA). Alongside this, control leaves were also similarly treated and overlaid onto PDA. While fungal growths were observed on all infected leaf-discs, no growth was observed on control discs. For each of the various fungal strains growing on the infected leaf discs, pure cultures were obtained by repeatedly sub-culturing the strains onto PDA. Subsequently, single spore cultures were also aseptically isolated from each of the obtained pure cultures and further developed into mycelia through inoculation and incubation in potato dextrose broth (PDB). All in all, a total of eight single spore cultures were obtained from the overall inoculations and sub-culturing. Subsequently, the total genomic DNAs of each of the obtained single spore cultures were isolated followed by amplification of their internal transcribed spacer (ITS) regions. The amplified ITS regions were then sequenced followed by a comparison of their nucleotide patterns with the GeneBank. Such a comparison then revealed the presence of a complex of several fungal isolates with eight commonly known species: Penicillium brevicompactum, Epicoccum sorghi, Rhizopus stolonifer, Alternari solani, Fusarium equiseti, Penicillium olsonii, Fusarium chlamydosporum and Fusarium incarnatum. These fungal species are commonly known to cause various diseases and infections in legumes and other agronomically important crops. Ideally, the presence of these fungal species in marama bean and their involvement in leaf tissue decay should be noted with great concern and interest since this plant has recently been proposed as a potential leguminous crop for possible adoption and utilization in domestication efforts.