This is the report from a BSPP Junior Fellowship.
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Ganoderma species are mushrooms with brownish leathery brackets that grow on dead wood and living trees. This genus of mushrooms can be found all over the world and includes both parasitic and saprophytic species. It is a well-known plant pathogen that causes root and stem rots, leading to the mortality of many economically important trees and perennial crops. These include oil palm, rubber, tea, ornamentals, and forest trees like Acacia, Albizia, Populus, and Macadamia. They also serve as biological control agents against plant pathogens. However, there is limited information on their diversity in Cameroon.
This study uses molecular methods to identify Ganoderma species on different host trees in the Mezam division of the Northwest region of Cameroon. Samples were collected opportunistically from households around Mezam division from November 2021 to August 2023. Each specimen was snapped from both above-ground and below-ground positions (see picture on front cover and below). The GPS positions were accurately marked with geographic coordinates. The samples were placed in labeled Ziplock bags, wrapped in aluminum foil, and affixed to A4 papers with a ruler for scale and pictures taken. Morphological features were observed, including the type of basidiocarp (whether it was laccate or non-laccate, and whether it was stipitate, sessile, or dimidiate), the number of concentric zones, margin shape (lobed, rounded/acute), margin colour (brown, white, reddish, etc.), pore colour, and tube colour. The length and width of the basidiocarp were also recorded. Finally, the basidiocarps were oven-dried to facilitate micro-morphological identification. A plant taxonomist at the National Herbarium in Yaounde, Cameroon identified the host tree. The results were compared to the taxonomic classification of Ganoderma, which is based on its basidiocarp. Below are pictures of the identified samples (Figures 1 and 2).
Figure 1. G. gibbosum Figure 2. G. multiplicatum
For molecular identification, cultures of fresh samples were made by excising small pieces of isolates from each species (<1 mm3) with a sterile scalpel and placed onto 2% Potatoes Dextrose Agar (PDA) with the addition of penicillin and gentamycin (100 mg/l) to limit bacterial growth on culture. Cultures were maintained on PDA; cultural characters were recorded, and pure cultures were submerged in sterile water and 10% glycerol for long-term storage. Cultural characteristics such as colony diameter/growth biomass production, colony colour, colony margin, mycelial density, the appearance of zones, reverse pigmentation, conidial structure, and sporulation were recorded. The diameter was measured daily, and the number of days required for maximum mycelium growth was recorded. The colony texture, the appearance of the zone, the type of colony margin, reverse pigmentation colour, and mycelial density were recorded after the seventh day of incubation. Twenty-two samples were cultured.
Fresh cultures were grown on cellophane and frozen dried for DNA extraction using the Sorbitol-CTAB method. DNA concentrations were determined with a NanoDrop® ND-1000 spectrophotometer. Working DNA concentrations for polymerase chain reactions (PCRs) were obtained by adjusting the initial concentrations of the extracted genomic DNA. The complete internal transcribed spacer (ITS) region, including ITS-1 and ITS-2, was amplified for all isolates using primers ITS1f and ITS4. ITS PCR amplification for all PCR reactions was carried out. To determine the quality of DNA, agarose gel electrophoresis was done with PCR products electrophoresed on 0.6% agarose gels after staining with red gel and visualised under UV illumination, and pictures were taken. PCR products were cleaned using a DNA PCR clean kit according to the manufacturer’s instructions. The PCR products were sequenced using the same forward and reverse primers as in the amplification reactions. DNA sequencing reactions were done using Big Dye, following the protocol outlined by the manufacturer. The purification of the sequencing PCR products followed the same approach as that for the PCR product.
Chromas version 2.5. was used to edit and determine the quality of the electropherograms. DNA sequence datasets and dual-gene phylogenetic analysis were done. The sequences were analysed and identified using nucleotide blast in NCBI, and the results were presented on a table, with maximum identity, query coverage, e value, and the identity of the species noted. Good sequences will later be deposited in GenBank. Phylogenetic analyses were performed using maximum likelihood (ML) in MEGA version 11. Alignment was done in MEGA using MUSCLE. Maximum likelihood method (ML) analysis was done with 1000 replications in MEGA version 11 to produce phylogenetic trees for the gene region.
Figure 3 is a bootstrap consensus phylogenetic tree constructed from ITS gene regions using the maximum likelihood method at a bootstrap value of 1000 replications. Results show a monophyletic relationship between the species, implying that they come from the same ancestors and that two clades were produced.
Figure 3: ITS Bootstrap consensus tree using the Maximum Likelihood method with 1000 replicates
I want to thank my host at the University of Pretoria, Professor Martin Coetzee, for providing me with space and taking the time to see that I accomplish my task. I would also like to thank my supervisors, Professor Tonjock Rosemary Kinge and Dr Tacham Walter Ndam, for their support and guidance in my PhD project. I would like to sincerely acknowledge and thank BSPP for recognising my project by awarding me with a BSPP junior fellowship bursary.
Bih Joan Ndeh
The University of Bamenda, Cameroon.
