MOLECULAR CHARACTERIZATION OF COCOA, MANGO, BANANA AND YAM ISOLATES OF BOTRYODIPLODIA THEOBROMAE IN GHANA Twumasi1, P., Moses E.2 and Ohene-Mensah, G. 1 1 Department of Biochemistry and Biotechnology, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana. 2 Council for Scientific and Industrial Research-Crops Research Institute (CSIR-CRI), Fumesua, Kumasi, Ghana. ABSTRACT Botryodiplodia theobromae is a virulent plant pathogen commonly found in the tropics and subtropics. The fungus has wide range of plant hosts and known to cause yield losses up to 80% especially on cash and food crop farms. This study aimed at establishing genetic diversity of B. theobromae collected from four common food and cash crops grown in Ghana. A total of 25 fungal isolates were sampled from cocoa, mango, banana and yam within four geographical regions of Ghana. The isolates were developed into pure single-spore cultures on Potato Dextrose Agar (PDA). Single-spore cultures of the 25 B. theobromae isolates from the four crops were grown in V8 juice medium at 28°C on a rotary shaker for 48 hrs. Mycelia were harvested at 48 hrs of growth, washed with sterile distilled water, grounded in liquid nitrogen and the genomic DNA isolated. PCR products from SSR and RAPD primers were resolved on 2.5% agarose gel and the DNA bands from the 25 isolates clustered on dendrogram into 5 distinct groups of varied genetic similarities using NTSYS software. The study showed only two banana isolates, B(Kt) and B(Ef), sharing highest genetic similarity above 80%. An isolate from yam, Y(Zu), shared no genetic similarity (0%) with any of the remaining 24 fungal isolates from the four regions in Ghana. The remaining 22 isolates measured genetic similarity between 20 and 75%. The results suggest high genetic variability among the B. theobromae isolates on the crops studied. Keywords: Banana, Botryodiplodia theobromae, cocoa, genetic variability, mango, phylogeny, plant pathogen, potato dextrose agar (PDA), RAPD, SSR and yam. INTRODUCTION The fungus Botryodiplodia theobromae (Pat.) Griff. and Maubl., also know in other literatures as Lasiodiplodia theobromae is the asexual state of the fungus Botryosphaeria rhodina (Berk and M.A. Curtis) Arx. It is a virulent pathogen that attacks field and tree crops and cause different types of diseases and rots (Opoku et. al., 2007; French, 2006; Amusa et al., 2003). It is widely distributed within the tropics and subtropical region of the world (Faber et al., 2007) and has a very wide host range estimated to be more than 280 species (Domsch et al., 2007; Khanzada et al. 2006; Sutton, 1980). In the tropics, B. theobromae is an economically important fungus that causes major losses to farmers who cultivate mango, cocoa, banana and yam (Twumasi et al., 2014; Rieger, 2006; Amusa et al., 2003). It causes diseases such as “Charcoal rot” of cocoa, tuber rots of yam, crown rot of banana and stem end rot of mango fruits (Sangeetha et al., 2011; Rossel et al., 2008; Opoku et. al., 2007; Khanzada et al., 2004; Jiskani, 2002; Arjunan, 1999; Sangchote, 1988). The fungus has also been found to be present in about 70% of farms surveyed and associated with yield losses of about 80% in Nigeria (Onyenka et al., 2005). The wider host range (Crammer, 1979) and the host nonspecificity of the pathogen (Mohali et al., 2005) makes control and management of the diseases difficult. Unfortunately, there is limited information about the various strains or types of the fungus on host crops in Ghana. Moreover, the identification of B. theobromae using morphological and physiological characteristics is tedious as these characteristics are unstable and often influenced by environmental factors (Shah et al., 2010). Hence, there is the need to use more reliable parameter such as molecular characteristics. Molecular characterization is known to provide a great potential for fungal diversity assessment (Liew et al., 1998). It is not restricted by the limits imposed by morphological characteristics (Acquah et al., 2011). It basically provides genetic proof of variability or similarity that may be observed in morphological studies of fungi. It estimates diversity among fungi isolates and cluster or separate them into groups of specific genetic similarity (Kokub et al., 2007; Iotti et al., 2005; Photita et al., 2005). The study was conducted to establish the genetic variability among B. theobromae isolates causing diseases on cocoa, mango, banana and yam in four geographical regions of Ghana. MATERIALS AND METHODS Isolation of B. theobromae from diseased fruits The isolation and culturing of the B. theobromae isolates and isolation of genomic DNA was undertaken at the Pathology and Biotechnology Laboratories of the Crops Research Institute (CSIR-CRI) at Fumesua in Ghana. DNA analysis was undertaken at the Molecular Biology laboratory of the Department of Biochemistry and Biotechnology, Kwame Nkrumah University of Science and Technology (KNUST), Ghana, with assistance from the KIRKHOUSE Trust Laboratory of the Cocoa Research Institute of Ghana (CRIG). Pieces of diseased tissues obtained from infected cocoa pods, mango and banana fruits, and yam tubers with symptoms of B. theobromae rots collected from the field were surface sterilized in 5% Sodium hypochlorite solution for 5 minutes. They were then washed three times in sterile distilled water, air dried in a sterile hood, plated on PDA in sterilized Petri dishes and incubated at 28°C for 7 days. Fine hyphae that grew from the diseased tissues on the Petri dishes were subcultured on fresh PDA. Colonies of B. theobromae identified were further sub-cultured on PDA amended with chloramphenicol (0.05g/l of PDA). Pure cultures of isolates were finally maintained on fresh PDA. Pure culture plates of the B. theobromae isolates were incubated at 28°C under fluorescent light for 35 days to enhance sporulation as recommended by Shah et al. (2010). With the aid of an inoculation needle, matured pycnidia produced after 35 days were transferred into sterile distilled water on a sterile glass slide and then teased to diffuse spores. The solution on the slide was then observed under a compound microscope to confirm the presence and even distribution of spores. Slides with B. theobromae spores identified were then inverted and pressed onto the surface of solidified Water Agar (WA) media to sustain their growth. Preparation of single-spore culture Germinated single spore of each B. theobromae isolate on the WA media was transferred onto PDA media amended with chloramphenicol and then incubated at 30 ºC for seven days as recommended by Shah et al. (2010). Single-spore pure cultures obtained (Table 1) were then maintained on PDA. Each single-spore pure culture plate of each isolate was sub-cultured on fresh PDA before being used for molecular studies. DNA isolation Four-day old mycelia from the single-spore cultures of the 25 B. theobromae isolates from mango, cocoa, banana and yam were used to inoculate V8 juice medium. The inoculated media were incubated at 28°C on a rotary shaker for 48 hrs. Mycelia of each isolate were harvested, 48 hrs after incubation, washed with sterile distilled water and ground in liquid nitrogen. The BioTeke Plant genomic DNA fast isolation Kit (Spin Column) (Industricord, Durban) was used to extract genomic DNA from the 25 B. theobromae isolates. The purity of the DNA was checked by resolving on a 0.8% agarose gel. Fifty micro-litres (50 ml) of Elution buffer was added to the extracted DNA contained in a 1.5 ml microfuge tube and stored at 4°C in a refrigerator. Frozen DNAs were thawed on ice before use. Polymerase Chain Reaction The SSR and RAPD primers used on the DNA are shown in Table 2a and 2b. After test runs, RAPD primer MH-S1118; SSR primer pairs, MH-LAS_15 and_16, and MH-BOT_19 and_20 produced the best amplifications. These primers were therefore used for the amplification of all the 25 B. theobromae isolates. A reaction volume of 10 ml comprising of 8 ml of Accupower PCR premix (Bioneer Inc., California), 1 ml of specific primers and 1 ml of genomic DNA was maintain for both the SSR and RAPD primer amplifications. Amplification program described by Shah et al. (2010) was adopted, with few modifications, to amplify the DNA markers using an Eppendorf Mastercycler (Merck Chemicals (Pty) Ltd, Modderfontein, SA). The modified programme used for the SSR primers consisted of a denaturation phase at 95°C for 2 minutes followed by 35 cycles of denaturation at 95° C for 30 seconds, primer annealing at 52- 66°C (specified for each primer in Table 2a) for 40 seconds, primer extension at 72°C for 1 minute and a final extension at 72°C for 10 minutes. The modified program used for RAPD primer amplification comprised of a denaturation phase at 94°C for 1 minute followed by 34°C for 1 minute of primer annealing, primer extension at 72°C for 2 minutes and a final extension at 72°C for 5 minutes. Gel electrophoresis After cooling the amplified PCR products on ice, 2 μl of Bromophenol blue was added and mixed thoroughly by centrifuging at 1000 rpm for 30 seconds. Eight micro-litres (8 μl) of each PCR product was loaded into separate wells in a 2.5% agarose gel in 1x Tris borate EDTA buffer (0.5 M Tris, 0.05 M boric acid and 1 mM EDTA, pH 8.0) stained with 4 μl ethidium bromide (20mg/ ml). Five micro-litres (5 μl) of PCR ranger 100bp DNA ladder (50bp – 1000bp) was loaded alongside to serve as size marker. PCR products of SSR primers were run at 120 V, 100 mA, and 50 W for 1 h. PCR products of RAPD primers were also run at 60 V, 77 mA and 5 W for 2 and half hours. DNA bands produced were visualized under UV transilluminator (Model: TFL40V, Fisher Scientific, Waltham) and the image captured with a 12.1 Mega pixels camera (Model: A235, FUJIFILM Corporation, Tokyo). Analysis of gel data Data generated after estimating the band lengths of the DNA bands of each B. theobromae isolate were converted into binary matrices. Analysis of the matrix data was subsequently made based on the number of DNA bands or base pairs that the isolates shared in common or otherwise. Using the NTSYS version 2.0 Software which is based on Unweighted Pair Group Method with Arithmetic Mean (UPGMA) a dendrogram of phylogeny was constructed. RESULTS Isolation of B. theobromae from diseased fruits A total of 25 B. theobromae isolates from various farms were cultured and purified into signgle spore cultures on potato dextrose agar. Of the 25 different fungal isolates purified and maintained on PDA, seven originated from forest ecological zone of Ghana, 14 came from forest transitional ecological zone, three from savanna ecological zone and the one form coastal savanna ecological zone (Table 1). Morphologi- Table 1: Isolate label, ecology of collection and crop type from which the 25 B. theobromae isolates were obtained Ecological Zone Forest Ecological Zone Isolate Label B(Ef) B(Kt) B(Eu) B(Kf) C(As) M(Ej) Y(Ej) Place of collection Effiduase Kuntanase Ejisu Kyirefaso Asumanya Ejura Ejura Crop Banana Banana Banana Banana Cocoa Mango Yam Forest Transitional Ecological Zone B(Ab) B(Db) B(Go) B(Yf) C(Bk) C(Db) C(Ab) M(Nk) M(Th) M(At) Y(At) Y(Th) Y(Bk) Y(Kp) Abesewa Drobo Goaso Yamfo Berekum Drobo Abesewa Nkoransa Techiman Atebubu Atebubu Techiman Berekum Kintampo Banana Banana Banana Banana Cocoa Cocoa Cocoa Mango Mango Mango Yam Yam Yam Yam Savanna Ecological Zone Y(Yj) Y(Ch) Y(Zu) Yeji Chama Zabzugu Yam Yam Yam Coastal Savanna Ecological Zone C(Af) Assin Fosu Cocoa Table 2a: Primer information (Simple Sequence Repeat primers) Molecular Weight Annealing Temperature (°C) %GC Oligo/Primer Name Sequence SSR MH-LAS _13 and_14 F GAG-TTG-TTA-GTG-CGG-GCG-CC 6205.1 66 65 MH-LAS _13 and_14 R GCA-GCC-CCA-CAA-TTC-ACC-AG 6015.9 64 60 MH-LAS_15 and_16 F GCC-AGA-TCC-GTG-CCC-ACT-G 5749.8 64 68.4 MH-LAS_15 and_16 R CAT-GCA-GAG-GTC-GCA-AAG-TG 6191.1 62 55 MH-BOT_11 and_12 F CGG-CAT-GGT-CTG-CCG-CTC-C 5756.7 66 73.7 MH-BOT_11 and_12 R GCA-TCT-CCG-GCT-ACC-AAC-CG 6022.9 66 65 MH-BOT_19 and_20 F GGC-GGT-CGC-AGA-TGC-GGT-C 5885.8 66 73.7 MH-BOT_19 and_20 R GCC-CTA-TTC-TGC-GTG-CCT-CC 5995.9 66 65 MH-BOT_35 and_36 F CTC-CAT-CCT-GAT-CCA-GGG-TCC 6318.1 53.1 61.9 MH-BOT_35 and_36 R GAC-GAA-TCA-AGC-GGG-CTG-CCC 6441.2 55.1 66.7 cal characteristics of the fungal isolates were independent of the ecological zones (Twumasi et al., 2014). DNA isolation, PCR and gel electrophoresis Isolated and undigested genomic DNA from the entire 25 single-spore B. theobromae specimen produced clear bands upon electrophoresis using 0.8% agarose gel (Fig. 1). A range of 1 to 7 DNA bands with a total of 22 bands of sizes 0.2 to 1.4 kb were obtained through the PCR reactions using the RAPD primer set MH-S1118 (Fig. 3). Also SSR primer MH- BOT_19 and _20; and MH-LAS_15 and_16 in PCR reactions using the various templates from the fungal isolates generated DNA bands ranging from 1 to 8. In all a total of 19 bands with sizes ranging from 0.05 to 1.2 kb were identified (Fig. 3 and 4). Table 2b. Primer information (Random Amplified Polymorphic DNA primers) Sequence Molecular Weight Annealing Temperature (°C) %GC MH-S111 CTT-TCC-GCA-GT 3283.2 34 54.5 MH-S1110 CAG-ACC-GAC-C 2982 34 70 MH-S1118 ACG-GGA-CTC-T 3028 32 60 MH-S1120 ACC-AAC-CAG-G 3006 32 60 MH-S116 TCT-CAG-CTG-G 3019 32 60 Oligo/Primer Name RAPD Fig. 1: A 0.8% agarose gel electrogram showing bands of undigested genomic DNA of all 25 Botryodiplodia theobromae isolates. Lane 1 = C(Ab); Lane 2 = C(Db); Lane 3 = C(Bkm); Lane 4 = C(As); Lane 5 = (Af); Lane 6 = M(At); Lane 7 = M(Ej); Lane 8 = M(Th); Lane 9 = M(Nk); Lane 10 = B(Db); Lane 11 = B(Kn); Lane 12 = B(Eu); Lane 13 = B (Yf) Lane 14 = B(Go); Lane 15 = B(Ab); Lane 16 = B(Ef); lane 17 = B(Kf); Lane 18 = Y(Bk); Lane 19 = Y (Th); Lane 20 = Y(Ej); Lane 21 = Y(Yj); Lane 22 = Y(Ch); Lane 23 = Y(Zu); Lane 24 = Y(At); Lane 25 = Y(Kp) Lane C = Control Gel data and dendrogram Using the PCR-gel data to construct dendrogram, the 25 isolates were clustered into 5 major lineages or groups with varied genetic similarities (Fig. 4). In the dendrogram, the isolates [C (Ab), B(Db), C(Db), C(As), C(Bk), M(Th), B (Kt), B(Ef), B(Ab), C(Af), M(Ej), B(Go), Y(Th), and Y(Ej)] clustered into Group 1 at 23% genetic similarity. The isolates [M(At), Y(Kp), and Fig. 2: M(Nk)] clustered into Group 2 at 29% genetic similarity. The isolates [B(Eu), B(Yf), B(Kf), and Y(Bk)] clustered into Group 3 at genetic similarity of 21%. The isolates [Y(Yj), Y(Ch), and Y(At)] clustered into Group 4 at 20% genetic similarity. Group 5 comprised of only isolate Y(Zu) with no genetic similarity (0%) with the rest of the isolates. Agarose gel (2.5%) electrograms (a and b) showing RAPD primer MH-S1118 PCR products of 25 Botryodiplodia theobromae isolates. Lane L = 1000bp DNA ladder; Lane C= Negative control; lane 1= C(Ab); lane 2= C(Db); lane 3= C(Bk); lane 4= C(As); lane 5= C(Af); lane 6= M(At); lane 7= M(Ej); lane 8= M(Th); lane 9= M(Nk); lane 10= B(Db); lane 11= B(Kt); lane 12= B(Eu); lane 13= B(Yf); lane 14= B(Go); lane 15= B(Ab); lane 16= B MH-LAS_15 (Ef); lane 17= B(Kf); lane 18= Y(Bk); lane 19= Y(Th); lane 20= Y(Ej); lane 21= Y(Yj); lane 22= Y(Ch); lane 23= Y (Zu); lane 24= Y(At); lane 25= Y(Kp). C= Cocoa isolate from Abesewa (C(Ab)); Drobo (C(Db)); Berekum (C (Bk)); Asumanya (C(As)); Assin Fosu (C(Af));M = Mango isolate from Atebubu (M(At)); Ejura (M(Ej)); Techiman (M(Th)); Nkoransa (M(Nk)); Drobo (B(Db)); Kuntanse (B(Kt)); Ejisu (B(Eu)); Yamfo (B(Yf)); Goaso (B (Go)); Abesewa (B(Ab)); Effiduase (B(Ef)); Kyirefaso (B(Kf)); Berekum (Y(Bk)); Techiman (Y(Th)); Y(Ej) Ejura; Yeji (Y(Yj)); Chama (Y(Ch)); Zabzugu (Y(Zu)); Atebubu (Y(At)); Kintampo (Y(Kp)). Group 1 further separated into 4 subgroups i.e. 1a, 1b, 1c and 1d at genetic similarity of 34%, 56%, 54% and 50% respectively. Two individual lineages, S1 and S2, at genetic similarity of 44% and 38% respectively were also produced in group 1. Isolates B(Kt) and B(Ef) shared the highest genetic similarity of 83% in subgroup 1b. Group 3 also separated into two subgroups i.e. 3a and 3b, at 55% and 34% genetic similarity respectively. Fig. 4: Agarose gel (2.5%) electrograms (a and b) showing SSR primer MH-BOT_19 and_20 PCR products of 25 Botryodiplodia theobromae isolates Lane L = 1000bp DNA ladder; Lane C= Negative control; lane 1= C(Ab); lane 2= C(Db); lane 3= C(Bk); lane 4= C(As); lane 5= C(Af); lane 6= M(At); lane 7= M(Ej); lane 8= M(Th); lane 9= M(Nk); lane 10= B(Db); lane 11= B(Kt); lane 12= B(Eu); lane 13= B(Yf); lane 14= B(Go); lane 15= B(Ab); lane 16= B MH-LAS_15 (Ef); lane 17= B(Kf); lane 18= Y(Bk); lane 19= Y(Th); lane 20= Y(Ej); lane 21= Y(Yj); lane 22= Y(Ch); lane 23= Y (Zu); lane 24= Y(At); lane 25= Y(Kp). C= Cocoa isolate from Abesewa (C(Ab)); Drobo (C(Db)); Berekum (C (Bk)); Asumanya (C(As)); Assin Fosu (C(Af));M = Mango isolate from Atebubu (M(At)); Ejura (M(Ej)); Techiman (M(Th)); Nkoransa (M(Nk)); Drobo (B(Db)); Kuntanse (B(Kt)); Ejisu (B(Eu)); Yamfo (B(Yf)); Goaso (B (Go)); Abesewa (B(Ab)); Effiduase (B(Ef)); Kyirefaso (B(Kf)); Berekum (Y(Bk)); Techiman (Y(Th)); Y(Ej) Ejura; Yeji (Y(Yj)); Chama (Y(Ch)); Zabzugu (Y(Zu)); Atebubu (Y(At)); Kintampo (Y(Kp)). Fig. 4: Dendrogram derived from the combined data set of 25 Botryodiplodia theobromae isolates using SSR and RAPD markers. C(Ab) =Cocoa isolate from Abesewa; C(Db) = Cocoa isolate from Drobo; C(Bk) = Cocoa isolate from Berekum; C(As) = Cocoa isolate from Asumanya; C(Af) = Cocoa isolate from Assin Fosu; M(At) = Mango isolate from Atebubu; M(Ej) = Mango isolate from Ejura; M(Th) = Mango isolate from Techiman; M(Nk) = Mango isolate from Nkoransa; B(Db) = Banana isolate from Drobo; B(Kt) = Banana isolate from Kuntanse; B(Eu) = Banana isolate from Ejisu; B(Yf) = Banana isolate from Yamfo; B(Go) = Banana isolate form Goaso; B(Ab) = Banana isolate from Abesewa; B(Ef) = Banana isolate from Effiduase; B(Kf) = Banana isolate from Kyirefaso; Y(Bk) = Yam isolate from Berekum; Y(Th) = Yam isolate from Techiman; Y(Ej) = Yam isolate from Ejura; Y(Yj) = Yam isolate from Yeji; Y(Ch) = Yam isolate from Chama; Y(Zu) = Yam isolate from Zabzugu; Y(At) = Yam isolate from Atebubu; Y(Kp) = Yam isolate from Kintampo. DISCUSSION Botryodiplodia theobromae is a common and widespread plant fungal pathogen in the tropicsa with a very wide host range. The fungus is known to cause rotting and dieback diseases in most plant species it infects (Shah et al., 2010). In the tropics B. theobromae is considered a post harvest fungus affecting both ornamental and cash/food crops such as mango, cocoa, banana and citrus (Khanzada et al., 2004). It has been shown that despite phenotypic similarities of B. theobromae isolates collected from different plant species and ecological zones, the fungal isolates exhibit varying pathological effects on their plant hosts (Twumasi et al, 2014). Such an observation demonstrates possible existence of significant genetic diversity among the B. theobromae isolates. The genetic background information of the fungi when available would facilitate design of effective control and management of plant diseases elicited by B. theobromae. According to the results in this study as shown by phylogenetic analysis and depicted by dendrogram, the fungal isolates from mango, cocoa, banana and yam cluster into five major groups (Fig. 4). Interestingly about 40% of isolates from different plant hosts clustered together. Group 1, for example, consists of cocoa, banana, yam and mango isolates (Fig. 4). These high similarities that exist between B. theobromae isolated from unrelated hosts may explain the high crossinfectivity of the fungus among different plant species (Twumasi et al., 2014). This opposes the established principle that pathogens tend to feed on common host (Woolhouse et al., 2002). There were also a number of the fungal isolates from different ecological zones that showed high genetic similarities. For instance, isolates within the Subgroup 1a originated from cocoa at Abesewa in the forest transitional zone is closely related to a banana isolate from Drobo than other isolates from cocoa either in the same ecological zone like Drobo or different ecological zone such as Assin Fosu (Table 1 and Fig. 4). Again, B. theobromae isolate collected from mango fruits from Techiman was found to be genetically very similar to other isolates from banana fruits in Kuntanase and Effiduase, both from different ecological zones (Table 1 and Fig. 4). Such high genetic resemblance of above 80% among B. theobromae isolates from banana fruits has also been reported in India (Sangeetha et al., 2011). Contrary to the intra-crop similarity observed, an isolate collected form yam tubers from Zabzugu was genetically different from other B. theobromae isolates sampled from all other ecological zones and crops including that of yam. A comparison of the isolates from the four types of crops studied i.e. banana, mango, cocoa and yam across the agro-ecological zones in Ghana indicates that the isolates were not ecologically specific but genotype specific. Such a similar observation has also been reported by Shah et al. (2010) among B. theobromae isolates from pear and mango fruits from different provinces in India. With a genetic similarities ranging from 083% observed among the isolates studied, it is evident that high genotype diversity exist among the isolates. This suggests that gene flow between isolates of the fungus B. theobromae from the four different food crops, i.e. cocoa, mango, banana and yam, in Ghana may be very high. The state of the infections and rots produced by mycelial spores of the four isolates of B. theo- bromae establish the infectious nature of spore contaminated fruits and pods on the farm (Twumasi et al., 2014; Opoku et al., 2007). Thus farm crops may serve as alternative host of different strains of B. theobromae pathogen and as such contaminating source for other healthy plants (Lichtfouse et al., 2009; Opoku et al., 2007; Philip, 2007; Jones et al., 2000; Muirhead et al., 2000). Spores and mycelial hyphae on the farm are important inocula for rot disease induction. The consequence of this is high yield loss with great financial burden to the farmers. The results in this study also confirm the nature of B. theobromae as a broad spectrum pathogen with a wide range of hosts as has been reported in several studies (Pitt and Hocking, 2009; Opoku et al., 2007; Domsch et al., 2007; French, 2006; Khanzada et al., 2004; Sutton, 1980). Broad host specificity of B. theobromae isolates has earlier been reported in Sri Lanka (Shanthi et al., 2008) and Venezuela (Mohali et al., 2005), two countries with climates similar to that in Ghana. The results suggest that intercropping cocoa, mango, banana or yam with other food or tree crops infected by B. theobromae, as commonly practiced in Ghana (ADRA, 2010; Banful, 1998), may facilitate spreading of the fungal diseases on the farm. CONCLUSION We have demonstrated in this study that Botryodiplodia theobromae isolates from the four important crops in Ghana, i.e., cocoa, mango, banana and yam, are genetically similar and possess broad host specificity. Therefore, the pathogen is capable of infecting several food crops cultivated on a single piece of land. The findings oppose the current intercropping system adopted by Ghanaian farmers and now shown to rather enhance spread of B. theobromae from diseased plants to other plant species with consequent crop yield loses. ACKNOWLEDGEMENT We thank Mr. and Mrs. Appiah-Kubi, Dr. Marian D. Quain and Mrs. Linda Abrokwah of CSIR-CRI for their timely assistance during the isolation of DNA. 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