CHARACTERISATION OF FUNGI OF STORED COMMON BEAN CULTIVARS GROWN IN MENOUA DIVISION, CAMEROON

Common bean is a legume grown globally especially in developing countries including Cameroon for human consumption. In Cameroon it is grown in a wide variety of agro ecological zones in quantities enough to last through the off growing season after harvest. Stored common bean after harvest in Cameroon are prone to fungal spoilage which contributes to post harvest losses. This study aimed at characterising storage fungi on cultivars of stored common bean using morphological and molecular techniques. Fungi were isolated from six stored cultivars of common bean; Kidney bean, Black bean, Navy bean, Pinto bean, Pea bean and Large seeded bean plated on potato dextrose agar media. Cultural, micro-morphological and molecular techniques were used to characterise the isolates. A total of four fungi isolates resulted. Homology matches of the gene sequences in the Genbank databases identified the isolates to be; Xylaria hypoxylon, Aspergillus flavus, Penicillium aethiopicum and Fusarium oxysporium. Phylogenetic analysis and multiple sequence alignment showed these isolates were of distinct species. The species of fungi recovered from the stored cultivars signified poor preservation methods carried out after harvest. Effective management and control of these fungal species in common bean at storage will help reduce post-harvest losses and increase seed health.


INTRODUCTION
Common bean is a legume grown globally especially in developing countries including Cameroon for human consumption. It is the most widely grown staple food crop in Africa (Beebe et al 2013). It is one of the most important staple food for the majority of the Cameroon population (Kamtchoum et al 2018). This is probably because it is suited to wide variety of agro ecological zones and can be grown in quantities enough to last through the off growing season after harvest. It is used to make bean cake which is a staple food consumed by nearly all the communities in Cameroon. In addition to being a source of food, it is a source of income to farmers.
Post-harvest activities play a vital part in the security of farm produce. These activities comprises handling procedures after harvest, transportation, storage and up to the point of enabling the availability of the produce to end consumers. In Cameroon, when common bean is harvested it is stored in varied structures by different communities. Such structures include; wooden granaries, grass lined granaries and silos. The storage of common bean in Cameroon usually last for an average period of 3 months after harvest which is later made available to consumers in markets.
Post-harvest losses of stored common bean have been reported in the West Region of Cameroon (Shende and Lifeter 2017). Post-harvest loss of stored common bean is partly due to storage fungi (Richard 2007). Storage fungi in common bean has led to its spoilage, decrease in quality and also resulted to a decrease in the germination of the common bean seeds (Kator et al 2016). Consumption of poor quality beans can also bring about health impacts (Kumar and Kalita 2017). Some fungal species on stored common bean can also release mycotoxins which causes human mycotoxicoses upon consumption. Typical example of mycotoxic fungus specie found on stored bean include is Fusarium which produces fumonisin that causes oesophageal cancer in humans and also interferes with sphingolipid metabolism. Another example is Aspergilus which produces aflatoxins known to cause liver cancer (Omotayo et al 2019).
Hence an urgent need is required to identify the fungus on stored bean so as to develop effective control and management measures of beans in stores. Thus, the aim of the research undergone was to isolate and characterize storage fungi associated with common bean after three months of storage.

Study site
The study took place in the Menoua Division, West Region of Cameroon. Cameroon

Sample collection
A multi stage sampling was used in the study. At the first stage a purposive sampling technique was used to select 6 zones/ sub divisions in the Menoua division where common bean was cultivated, consumed and equally stored several months after harvest. This selection was done based on geographical evidence of common bean farming. The sampled subdivisions were; Dschang, Santchou, Nkong ni, Fokoue, Penka Michel and Fongo Tongo.
The second stage involved random sampling of the common bean cultivating farmers in each of the sampled subdivisions. At the last stage convenience sampling technique was carried out in collecting the common bean cultivars from the sampled farmers' stores.
Stored common bean cultivars were collected from farmers' storage structures (gunny bags). Sampling was carried out by picking the common bean grains three times (top, middle and bottom) from their sampled respective cultivar storage bags using a 15 cm diameter polyvinyl chloride (PVC) bowl.

Direct plating of bean cultivars for detection and isolation of fungi
The sterilised common bean grains obtained from the different cultivars were then plated on potato dextrose agar media using the agar plate method (Nega 2014). The plating of the common bean grains from each cultivar was done at a rate of 10 bean grains per plate ( Figure 2) and was replicated five times. The grains were spread evenly in the plates. The plates were covered by their lids and fastened using parafilm. The sealed plates were then maintained under incubation at 28±2 ℃ for 7 days to promote fungal growth. B-Kidney bean (small sized red bean) C-Pea bean (mottled red bean) D-Navy bean (White bean) E-Large seeded bean (Large red bean) F-Pinto bean (mottled brown bean)

Morphological Identification of Fungi from stored common bean cultivars
After 7 days of incubation, the plated common bean grains from each cultivar were observed for growth of fungi. Initial identification of fungal isolates was done using colony morphology and physical features according to Klich (2002). A loop of fungal mycelium was transferred with the use of a sterile inoculating needle from the bean grain showing fungal growth onto a fresh PDA culture medium in a petri dish. This was sealed then incubated at 28±2 ℃ for 7 days to obtain pure cultures. Grouping of the fungal isolates was done based on similarities in their morphological characteristics shown by their colonies. Later, one isolate was picked out of each group as a representative for further analysis.

Fungal DNA Extraction
The extraction of the DNA from fungi obtained from the stored common bean samples was carried out following a standard protocol as described by Zhang et al (2010).
This was done in the Molecular and Biotechnology Laboratory of the University of Buea.
Thirty mg of mycelium from each representative isolate was scraped directly from the surface of the agar culture. The mycelium was then ground to a fine powder in an Eppendorf tube in liquid nitrogen using a pre-cooled pestle (Eppendorf no. 0030120973). The ground mycelium was resuspended and lysed in 500 ml of lysis buffer (40mmol/l Tris-acetate, 20mmol/l sodium acetate, 1mmol/l EDTA, 1% w/v SDS pH7·8). The tube and its content were then kept at room temperature for 10 minutes. Potassium acetate of volume 150 μl was later added into the eppendorf tube and then vortexed briefly, thereafter it was centrifuged at >13,000 x g for 1 minute. Another 1.5 ml eppendorf tube was used to collect the supernatant that resulted.
To the supernatant, RNase A (10mg/ml) was added to get rid of RNA and then incubated at 37°C for 15 min. Isopropyl alcohol of volume 0.5 ml was then added to it. This was then followed by a 2 minutes centrifugation of the tube and its contents at >13,000 x g. Thereafter the supernatant was disposed. A volume of 0.3 ml of 70 % ethanol was used to wash the resulting DNA pellet in the eppendorf tube. The tube containing the pellet was then made to spin at 10,000 rpm for 1 minute and the supernatant disposed. The DNA pellet was then air dried and placed in 0.05 ml of Tris-EDTA to dissolve.
The presence of DNA was tested under 1.5 % agarose by gel electrophoresis. The extracted DNA was then stored under a temperature of -20 ℃ to maintain its stability.

PCR amplification of the ITS region of fungi
Amplification of the extracted fungal genomic DNA was performed using standard Electrophoresis using 1% agarose gels was used to analyse the reaction product. The resulting size of the amplified PCR products by the ITS-1/ITS-4 pairs of primers was determined from the DNA molecular weight marker (200 bp marker).

Molecular sequencing of amplified fungal DNA
Sequencing of amplified fungal DNA was done at Inqaba Lab in South Africa. The Sanger dideoxy sequencing technology was used. DNA amplification was first confirmed using agarose gel electrophoresis. This was done with the use of a DNA ladder or marker.
Alkaline phosphatase was used to clean the PCR products. The sequence reaction mixture included the purified PCR products, a big dye terminator V3.1 cycle sequencing kit with 3µl of DNA, a template and 1µl of 2µM of the same primer as used in the amplification process.
Bi-directional sequencing of both DNA strands was done using the same forward and reverse primers used for amplification.
The established sequences of the ITS region were then deposited in the GenBank under respective accession numbers. For species identification, a BLAST analysis of ITS sequence from each isolate was conducted against the GenBank databases.

Result Analysis
Genetic analysis was done using the MEGA version 5.1 software (Tamura et al 2006).
The identity of the fungal species was established based on both the query coverage and percentage similarity of the input ITS sequence to the reference sequences in the gene bank.
Reference sequence similarity of fungi above 95 % to the input ITS sequence is considered to be of same genus while similarity above 98 % is considered to be of the same species performed with the aim to determine the degree of homology between DNA base sequences as well as nucleotide base composition (Whitford, 2005).

Fungal growth on plated common bean
Fungal growth was exhibited on the plates of the different stored bean cultivars after a seven days period of incubation. This was noticeable by changes in the appearance of the cultivars such as color and fruiting bodies protruding from their surfaces (Figure 3). All of the different cultivar group types were shown to be infected by fungi. Some of the plated common bean grains also had more than one fungal colony growing from them. This could be seen by differences in the colony appearance such as colors on the surfaces of the bean grains.

Figure 3 -Fungal growth on plated stored bean cultivars.
A-Black turtle bean (Black bean). B-Kidney bean (small sized red bean). C-Pea bean (mottled red bean). D-Navy bean (White bean) E-Large seeded bean (Large red bean).

Morphological characterisation fungi Isolated from stored common bean cultivars
Based on cultural characteristics (Table 1) together with micro-morphological characteristics of the fungi colonies, four distinct fungi isolate groups on the stored plated common beans were identified to be members of the genera Aspergillus, Penicillium, Fusarium and 'mycelia sterilia '.
For Aspergillus, the colonies were yellow to dark on the surface and creamy on the reverse side. Texture was woolly to cottony and appeared granular. Colony was fast growing.
Microcospic characteristics included long and septate cells borne on hyaline hyphae, colourless conidiophores that are wide and roughened, globose vesicles held on long conidiophores and smooth globose conidia. Conidiophores appeared dense felt with mature vesicles bearing phialides over their entire surface. (Figure 4).
Colonies with invisible condidia/spores on stained septate mycelia on slide under the compound microscope was described as 'Mycelia sterilia' and termed morphospecie. Its colony appeared white on both its surface and reverse. No fruiting bodies produced (Fig. 4).
Fusarium colonies were fluffy, pink red on the surface and light pink on the reverse.
Curved microconidia produced on simple, short phialides. Conidia had more than one cell

DNA sequencing and fungi species Identification
Identity of the fungal species were established after comparing their target ITS sequence region with reference sequences found in Genbank. Following blasting from the sequences obtained from the amplicons, the four isolates were identified as four distinct species of fungi each having E-values of zero and percentage similarity greater than 98 % to that of the gene bank reference ( The 'mycelia sterilia' designated morphospecie was identified based on its gene sequence similarity as Xylaria hypoxylon; being a member of the genus Xylaria, which is an ascomycetous fungi also known as sac like fungi having a typical structure called an ascus bearing 8 ascospores.

Genetic relationship between stored fungal isolates Phylogenetic relatedness of stored fungal isolates
An unrooted phylogenetic tree was produced ( Figure 6)  robust. This showed a high confidence of support from the nodes or branches in the phylogenetic tree. The identity of the fungal isolates were shown through the same branch as the reference species obtained from the Genbank. The phylogenetic tree also showed differences between fungal species due to differences in branch lengths and nodes.  DNA sequence results following BLAST analysis sequence typing also indicated that Differences between field and storage fungi may also play a role in determining which species will dominate a stored grain ecosystem under a particular set of environmental factors. During long-term storage, fungi of the genera Aspergillus and Penicillium ("storage flora") progressively replace the "field flora" such as Fusarium and Alternaria over a period of several months (Fleurat et al 2002). Fusarium sp, Aspergillus sp and Penicillium sp has also been shown to be associated with cola nuts, groundnuts and sweet potato in the western highlands of Cameroon (Ngoko et al 2008).

CONCLUSION
In this study, fungi isolated from stored common bean grains was identified based on their phenotypic features (Morphological characterisation) and genotypic features (Molecular characterisation). These species isolated are a significant proof that contamination of common beans occurs during storage. This study therefore brings forth a novel information which will assist in establishing measures to prevent fungal infection of stored common bean in the Menoua Division.