This is the report from a BSPP Lockdown Bursary.
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Ganoderma species are mushrooms with brownish leathery brackets that grow on dead wood. The genus Ganoderma P. Karst. (Basidiomycota, Polyporales, Ganodermataceae) can be found all over the world and include both parasitic and saprophytic species. Ganoderma is a well-known plant pathogen that causes root and stem rots and mortality of a wide range of economically important trees and perennial crops including oil palm, rubber, tea, ornamentals, and forest trees such as Acacia, Albizia, Populus and Macadamia. These species also serve as biological control agents against plant pathogens. However, there is limited information on its diversity in the western highlands of Cameroon.
My study objectives included i) to identify the different host trees of Ganoderma species in the western highlands of Cameroon, ii) to identify the different Ganoderma species morphologically, culturally and molecularly, iii) to determine the level of lignin degradation by Ganoderma species on trees, iv) to carry out a pathogenicity test for the causal agent of most abundant rot of trees, v) to determine the effect of soil physicochemical properties in relation with the basal stem rot disease caused by Ganoderma species and vi) to carryout in vitro biological control measures of the basal stem rot disease caused by Ganoderma species using medicinal plant extracts.
Opportunistic sampling of Ganoderma species was done and Ganoderma was collected from the different tree species from Mbatu, Mankon, Nkwen, Bambili, Bambui Santa, Mendakwen and Bafut villages. The samples were snapped both above ground and below ground and their GPS positions georeferenced. They were then labeled, snapped on A4 papers with a ruler placed on it and later oven-dried for micro-morphological identification. Morphological characters such as laccate and non-laccate, type of basidiocarp (stipitate/sessile/dimidiate, imbricate, concave, number of concentric zones, etc.), margin shape (lobed, fertile/sterile, rounded/ acute) and colour (brown, white, reddish, etc.), pore colour; tube colour, length and width of the basidiocarp were taken. Below are some of the species identified.
Figure 1. G. lucidium (left), G. multiplicatum (middle) and G. weberianum (right).
For cultural identification, small pieces of isolates from each species were excised (<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 slants, cultural characters were recorded and pure cultures submerged in sterile water 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 measured daily, and the number of days required for maximum growth of mycelium were also recorded. The colony texture, appearance of the zone, type of colony margin, reverse pigmentation colour, and mycelial density were recorded after the seventh day of incubation. Observations were taken when the pathogen covered completely the Petri plate.
For molecular identification, DNA extraction was done using sorbitol-CTAB method. PCR was done separately using ITS and TEF gene regions. The sequences were analysed using nucleotide blast in NCBI. Identification based on BLASTn searches of ITS and TEF sequences against those of reference sequences in GenBank and Unite database was done to confirm that the sequences were of the fungal isolates. Phylogenetic analysis was performed using ML in MEGA. Similar ITS and TEF sequences will be downloaded from Genbank and unite databases separately to generate phylogenetic trees. Good sequences obtained from the ITS and TEF regions will be deposited in the GenBank.
For soil analysis, ten core soil samples were collected from different collection points from Mbatu, Mankon, Nkwen, Bambili, Bambui and Santa villages surveyed (high, medium and no species collected) at a depth of 0-10 cm, bulked, mixed thoroughly, air-dried for 5 days, and sieved through a 2 mm sieve. 250g of each sample were then weighed taken for soil physicochemical analyses at the Soil Science Laboratory of the University of Dschang, Cameroon.
A total of 64 Ganoderma species were collected from the field. Eight hosts were identified as hosts to Ganoderma species include: Persea americana, Elaeis gineensis, Mangifera indica, Maesopsis eminii, Cola acuminata, Ficus sp., Albizia adianthifolia and Canarium sweinfurthii (black tree). The colony colour of colonies produced varied from cream white, green, orange to black.
Twelve different Ganoderma species were identified from ITS TEF gene regions namely: G. angustisporum, G. australe, G. orbiforme, G. eickeri, G. multiplicatum, G. weberianum, G. multipileum, G. applanatum, G. brownii, G. cupreum, G. gibbosum and G. lucidum. The results obtained were presented on phylogenetic trees as follows.
Figure 2. ITS bootstrap consensus tree
Figure 3.TEF bootstrap consensus tree
From soil analysis the growth of Ganoderma may be influenced by the presence of high organic matter attributed to their almost neutral soil pH (around 5 to 6) and high calcium content. These abiotic factors indicate microbial preference for near-neutral conditions, illustrating an increase of microbial diversity and richness, as well as microbial biomass. The higher the CEC value, the more nutrients it can absorb. The high phosphorus content was caused by phosphorus (P) ions available in the soil solution.
Though there was a COVID-19 pandemic going on, I was very happy to be awarded a BSPP Lockdown Bursary. I faced disruption during the pandemic, 6 months of lockdown in Cameroon, but thanks to the BSPP Covid-19 grant I was to conduct some aspects of my research even though not completed. I would like to thank my supervisors Prof Tonjock Rosemary Kinge and Dr Tacham Walter Ndam for their support and guidance in my ongoing PhD project. I am grateful to BSPP for recognising my project by awarding me with BSPP PhD COVID-19 Bursary.
Bih Joan Ndeh
This is the report from a BSPP Lockdown Bursary.