This is the report from a BSPP Lockdown Bursary.
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Mushroom contain various bioactive compounds which if isolated can be used to control plant and animal diseases. The use of phytochemicals is being encouraged because they are more biodegradable, and thus may pose less environmental hazards. Domesticating mushroom will make it available throughout the year. Common bean (Phaseolus vulgaris L.) is an important food legume cultivated for direct human consumption. It has a short growth cycle of about 70 days so in Cameroon common bean is a popular crop among small-scale farmers. Angular leaf spot (ALS) is one of the diseases that cause loss of crop yield in beans. The increase of bean yield at harvest by promoting the use of products from indigenous fungi to control ALS disease of common beans is the overriding aim of the project.
My first objective was to collect and domesticate six indigenous medicinal mushroom species from Abongfen forest, Northwest Region Cameroon. Fruiting bodies of six indigenous mushroom species found in large quantities, were collected from the Abongfen forest. The morphological characters measured at time of collection included the colour, shape and size of pileus, gilled or polypore, the appearance of the spore producing surface (smooth, wrinkled, veined or warted), presence or absence of veil, length of stipe and type of substrate on which it was growing. The features measured for each mushroom specimen was compared with information on field guides and some description keys and these were used to identify the mushroom species. Photographs taken in their natural habitat showing the substrate was also used to identify the mushroom using mushroom identification guides.
Fresh mushroom tissues from the six specimens were cultured using PDA to get the primary mycelium which was inoculated onto sterilized guinea corn grain for spawn production. This was carried out at the Mushroom Production, Training and Research Centre (MUPTAREC), in Ntarikon – Bamenda. From the cultures, the mother spawn was produced and multiplied in grain to be used for cultivating the mushroom. The specimens were air dried, ground into powder and stored in airtight labeled plastic bags.
The second objective was to determine the effect of mushrooms extracts on Pseudocercospora griseola, of common beans (Phaseolus vulgaris) infested with angular leaf spot diseases, in vitro. The mushroom extract was prepared from distilled water and methanol using the protocol of Jonathan and Fasid (2003). 100 g of each mushroom specimen was soaked in 300 ml of 95 % methanol and distilled water for 48 hours at 25 oC and filtered through a sieve, cloth and Whatman No 1 filter paper. The filtrate obtained was evaporated in an evaporator at 35 oC to dryness. The extracts will be used in vitro on Pseudocercospora griseola isolated from the leaves of the three varieties of Phaseolus vulgaris (GLP 190C commonly called ‘Medino’, PNN commonly called ‘dwarf Black beans’ and Ecopen 021 commonly ‘dwarf red’) infected with Angular leaf Spot disease. Specimens of diseased leaves from the plot farmed in the University farmland were cut to small pieces of 2 cm, surface sterilised, rinsed with sterile distilled water and placed on 2.5 % Potato Dextrose Agar (PDA) media amended with 10 mg l−1 Penicillin and 200 mg l−1 of Ampicillin. The plates were kept at room temperature in the Science Laboratory at the Catholic University of Cameroon for 5 to 7 days. Fungal growths on each plate were sub-cultured to a new plate and kept in a sterilised cupboard at room temperature. Field identifications were confirmed through microscopic observation.
An observation method to determine the severity and incidence of Angular Leaf Spot (ALS) disease affecting Phaseolus vulgaris was conducted on the plot farmed at the University of Bamenda farmland. The survey was conducted in two seasons; from April to July 2022 during the long rains and August to November during the short cropping seasons. Four plants randomly chosen and tagged were accessed for severity and incidence of ALS. In all 108 plants; 12 per variety per block in the three blocks. Incidence was calculated by using the number of plants affected by ALS in each block divided by the total number of plants assessed expressed as a percentage (%). Disease severity was estimated as percentage leaf area diseased per plant for 12 plants accessed per variety per block. Assessment of severity was based on the respective scales for ALS. The CIAT scale of 1-9 was taken as standard to score ALS disease. In the CIAT scale bean variety that had 1-3 showed resistance, 4-6 showed tolerance and 7-9 susceptible for the particular disease. Visual examination was done and recorded as a % leaf area affected. Leaves were selected at the bottom, middle and top of each plant and the mean score calculated. Identification of ALS disease was by observing the symptoms on the leaves.
Results obtained from morphological identification of six indigenous mushroom species collected from Abongfen forest:
- Laetiporus sulphureus (Fig 1a) Pinkish or yellow- orange pileal surface growing in overlapping clusters that appear in shelves ranging in size from 5-60 cm and about 4 cm thick.
- Phellinus sp (Fig 1b): pileus dark brown to black, sometimes with a crust surface. Grows on dead wood as resupinate sessile with tough flesh. A polypore with pileus size ranging from 10 – 25 cm.
- Inonotus obliquus (Fig 1c): woody and not having the typical mushroom form. It ranges in size from 20-40 cm in diameter are irregularly shaped. Outer region is sooty and black (sclerotium) and the interior, golden brown.
- Ganoderma sp (Fig 1d): Crusty pileus surface. Large basidiocarp that is sessile and perennial
Stereum sp (Fig 1e): Have steroid shaped basidiocarp. Are saprobic and fruit bodies have smooth hymenium. Lack gills or tubes; are polypores and sessile.
- Pleurotus sp; Exotic species (Fig 1f). Length of pileus 1-7 cm and width 1-4 cm. Cream white in colour with gills (decurrent). It is sessile.
Figure 1. Indigenous mushroom species collected from Abongfen forest. Laetiporus sulphureus, Phellinus sp (b), Inonotus obliquus (c), Ganoderma sp. (d), Stereum sp. (e), Pleurotus sp; exotic species (f).
The incubation period varied from 8-10 days for the tissue culture isolation and 15-24 days for the production of spawn for the species. Initially the primary mycelium successfully formed in the test tubes of six specimens. Secondary mycelium for spawn production started colonising the guinea corn grain but failed to grow. Of the six mushroom specimens from the Abongfen forest used to produce spawn only one was successful; Laetiporus sulphureus (Fig 2), the rest of the mushroom species failed to produce spawn.
Figure 2. Mother Spawn produced from Indigenous Mushroom specimen; Laetiporus sulfureus.
Pseudocercospora griseola, was successfully isolated (Fig 3) from the three varieties of common beans (Phaseolus vulgaris); PH 201 (dwarf red variety), PNN (dwarf black variety) and GLP 190C (medino).
Figure 3. Isolated Pseudocercospora griseola from Phaseolus vulgaris (a,b,c) and microscopic image (d)
The highest mean incidence was recorded in the PH 201 (dwarf red variety), followed by PNN (black variety) and the least showed by GLP 190C (medino). This may be attributed to the fact that most farm debris from previous years dumped in the plot increased the inoculum in the soil. There is little land fallow due to increasing population causing the inoculum in the soil to be on the increase. Also the seeds might have been infected since they were seeds from the previous planting seasons. GLP 190C was more resistant probably because of less inoculum in soil. The process to evaluate the effect of the mushroom extract on isolated Pseudocercospora griseola in vitro is yet to begin at the Catholic University of Cameroon Science Laboratory.
I look forward to gain more experience as I will carry on the bio-control assay and statistical analysis. I would like to thank my supervisors Prof Tonjock Rosemary and Prof Tofel Haman for their support and guidance as I continue with this project. Sincere thanks to BSPP for awarding me the grant to carry on this project.
Mercy Veyeh Ngwang
The University of Bamenda, Cameroon
