Isolation and Characterization of Fungi Associated with Disease Symptoms on Ziziphus mucronata Leaves and Phaseolus vulgaris Pods in Windhoek, Namibia

Charlie C. Luchen¹, Percy M. Chimwamurombe²* and Larry Hale³

¹Department of Biological Sciences, Faculty of Science, University of Namibia,
Private Bag 13301, 340 Mandume NdeMufayo Avenue, Windhoek, Namibia.
²Department of Biological Sciences, Faculty of Science, University of Namibia,
Private Bag 13301, 340 Mandume NdeMufayo Avenue, Windhoek, Namibia.
³University of Prince Edward Island, 550 University Avenue, Charlottetown, PE C1A 4P3, Canada.

Abstract

Detection of phytopathogens that are involved in causing disease symptoms in plants and crops is of prime importance as a key step in disease treatment and management. Ziziphusmucronata is a species endemic in temperate and tropical climates and used traditionally in the treatment of infectious diseases. The common bean (Phaseolus vulgaris) is a rich source of nutrients for the human diet. Just like most crops, it is not immune to fungal diseases and reports had been received of P. vulgaris showing signs on disease. The aim of this study was to isolate and characterize the fungal species associated withthe disease symptoms in the Z. mucronata and P. vulgaris. Fungal species where isolated from surface-sterilisedsymptomatic bark of Z. muconata and fresh green bean pods. These where grown on petri dishes containing Potato Dextrose Agar and incubated at room temperature. Pure cultures where then obtained by transferring small segments of fungal growth to a new petri dish that contained PDA. During DNA extraction the pure cultures where first homogenized using liquid nitrogen and then the rest of the extraction carried out as stipulated in the Zymo extraction kit. The Nanodrop was used for quantifying the DNA and amplification of the conserved Internal Transcribed Spacer (ITS) region of Ribosomal RNA genes was carried out using ITS1 and ITS2. The PCR products were sequenced at Inqaba Biotech Industries in South Africa. The obtained sequences were then compared by alignment with known sequences in the Genbank using Basic Local Alignment Search Tool (BLAST). The BLAST searches were able to reveal the fungi isolated from the Z. mucronata as Fusariumpenzigii and Fusariumdimerum while the fungi isolated from P. vulgarisshown to be Phomadestructiva, with100 %, 95% and 100% sequence similarity respectively. The next step in this work is carry out Koch‘s postulates to determine which of this fungi is the causal agent of the observed diseases symptoms in order to start a targeted diseases management programme.

Keywords: Ziziphusmucronata, Phaseolus vulgaris, Internal Transcribed Sequence, alignment, BLAST, Fusariumdimerum, Fusariumpenzigii, Phomadestructiva.