For. Path. 41 (2011) 281–292  2010 Blackwell Verlag GmbH doi: 10.1111/j.1439-0329.2010.00671.x The pathogenic potential of endophytic Botryosphaeriaceous fungi on Terminalia species in Cameroon By B.A.D. Begoude1,2,4, B. Slippers3, M.J. Wingfield1 and J. Roux1 1 Department of Microbiology and Plant Pathology, Forestry & Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0002, South Africa; 2Laboratoire Régional de Lutte Biologique et de Microbiologie Appliquée, Institut de Recherche Agricole pour le Développement (IRAD), Nkolbisson, BP 2067, Yaoundé, Cameroun; 3Department of Genetics, Forestry & Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0002, South Africa; 4E-mail: didier.begoude@fabi.up.ac.za; dbegoude@yahoo.fr (for correspondence) Summary In Cameroon, native Terminalia spp. represent an important component of the forestry industry, but limited information is available regarding the fungal pathogens that affect them. The Botryosphaeriaceae are endophytic fungi and latent pathogens that can result in wood stain, cankers, die-back and death of trees, particularly when trees are under stress. The aim of this study was, therefore, to identify and characterize the Botryosphaeriaceae occurring as endophytes of Terminalia spp. in Cameroon, as part of a larger project to identify potential pathogens of these trees in the country. Samples were collected from three Terminalia spp. in the Central, Southern and Eastern Regions and the resultant Botryosphaeriaceae were identified using morphology and DNA sequence comparisons for the ITS and tef 1-a gene regions. Furthermore, inoculation trials were conducted to consider the relative pathogenicity of the isolates collected. The majority of isolates (88%) represented species of Lasiodiplodia, including L. pseudotheobromae, L. theobromae and L. parva. The remaining isolates were identified as Endomelanconiopsis endophytica. Pathogenicity trials on young T. mantaly and T. catappa trees revealed that L. pseudotheobromae was the most pathogenic species followed by L. theobromae. 1 Introduction The forestry sector in Cameroon plays an important role in the national economy of the country. Timber is the second most exported product, after petroleum, with wood-based exports generating revenue of US $210 million in 2001 (Anonymous 2005a). The total forest area in Cameroon is estimated to represent 12.8 million ha of natural forests and about 17 000 ha of planted forests (Anonymous 2005b), made up of a variety of native trees such as Terminalia spp. Species of Terminalia currently found in forest plantations in Cameroon include T. ivorensis and T. superba. These tree species have a well acknowledged commercial value with a total volume of exported logs representing 10% of the national round wood production (Laird 1999). Besides their high commercial value, Terminalia spp. are commonly used in agriculture to establish a ÔtaungyaÕ agri-sylvicultural system in which they provide shade or improve soil fertility for crops (Norgrove and Hauser 2002). Furthermore, species such as T. ivorensis are important components for traditional medicine (Kamtchouing et al. 2006). Additional to native Terminalia spp., non-native species such as T. mantaly and T. catappa are frequently encountered as ornamentals in urban areas in Cameroon. The socio-economic importance of Terminalia spp. in Cameroon, coupled with their fast growth account for their extensive exploitation in national regeneration programmes. Fungal pathogens belonging to the family of the Botryosphaeriaceae are among the potential threats to forest tree species. Species in the Botryosphaeriaceae have a worldwide occurrence, causing a wide range of diseases, predominantly die-back, canker and blue stain, on numerous hosts, including trees (Brown and Britton 1986; Denman et al. 1999, 2000; DesprezLaustaud et al. 2006). This group of fungi commonly exists as endophytes in healthy plant tissues (Smith et al. 1996; Swart et al. 2000; Slippers and Wingfield 2007). Disease symptoms typically appear only after stress caused by abiotic and biotic disturbances (Schoeneweiss 1981; Slippers and Wingfield, 2007). Their occurrence as endophytes makes them especially important in international trade, as they may be spread undetected from one area to another, causing potentially serious damage to hosts that might have no co-evolved resistance (Slippers and Wingfield 2007). Species of Botryosphaeriaceae contribute directly or indirectly to economic and environmental losses, although the impact of their diseases is difficult to assess in forestry. In South African pine plantations, for instance, up to 55% loss of production has been recorded after hail damage and die-back due to Diplodia pinea Fries (Zwolinski et al. 1990). In the USA several tree diseases associated with non-aggressive pathogens belonging to the Botryosphaeriaceae caused extensive mortality of Aspen during the 1930s (Schoeneweiss 1981). Moreover, other reports appear in literature recognizing severe decline of Quercus spp. due to species in the Botryosphaeriaceae in 1980 in the Mediterranean Basin (Sanchez et al. 2003). In Africa, species of Terminalia occur in environments ranging from evergreen, primary and secondary forests to open woodlands or wooded savannahs (Carr 1994; Dale and Greenway 1961; Keay 1989; Lebrun and Stork 1991). Although these trees tend to display natural resistance to pests and diseases (Lamb and Ntima 1971; Groulez and Wood 1985), their wide ecological distribution exposes them to highly variable climatic conditions, environmental stress and other negative factors such as human activities (uncontrolled deforestation) and diverse pests and diseases. These factors may play an important role in predisposing Terminalia spp. in Africa to infection by species of the Botryosphaeriaceae (Jurskis 2005). Received: 18.4.2010; accepted: 3.5.2010; editor: L. Belbahri http://wileyonlinelibrary.com/ 282 B.A.D. Begoude, B. Slippers, M.J. Wingfield et al. The aim of this study was to identify species in the Botryosphaeriaceae that occur on Terminalia trees in Cameroon. This information will be valuable in the management of the health of these trees, because the Botryosphaeriaceae includes a key group of pathogens that generally affect forest trees and that will negatively affect these trees given projected changes in weather patterns. Identifications were done using a combination of morphological and DNA sequence data of the ITS and tef 1-a gene regions. Furthermore, inoculation trials using species of the Botryosphaeriaceae from Terminalia spp. were conducted to determine their potential pathogenicity. 2 Materials and methods 2.1 Sample collection and fungal isolation Plant material was collected in 2007 and 2008 from two species of native and one non-native Terminalia in Cameroon. The tree species sampled were the non-native T. mantaly and native T. ivorensis and T. superba. Four sites, located in three regions, were chosen for sampling (Table 1). Depending on the availability of trees at each location, at least 15 trees per species were randomly chosen for sampling without considering either their size or age. In each area, samples from healthy twigs or bark were collected and placed in paper bags and transferred to the laboratory where they were processed within a few days. For each sample, two pieces of twig or bark (1 cm in length) were split longitudinally. Samples were surface disinfested by sequential soaking in 70% ethanol (1 min), undiluted bleach (3.5% sodium hypochlorite for 1 min), 70% ethanol (1 min), rinsed in sterile water and allowed to dry under sterile conditions. Three disinfested pieces were placed onto 2% malt extract agar (MEA) (2% malt extract, 1.5% agar; Biolab, Midrand, Johannesburg, South Africa) supplemented with 1 mg ml)1 streptomycin (Sigma, St Louis, MO, USA) to suppress bacterial growth. The Petri plates were sealed with Parafilm (Pechiney Plastic Packaging, Chicago, USA) and incubated at 20C under continuous near-UV light for 1 week. Single hyphal tips growing from the plant tissues were transferred to new Petri plates containing MEA. After 2 weeks of incubation under near UV-light, cultures resembling species of the Botryosphaeriaceae (fast growth, mycelium white originally, turning dark greenish-grey or greyish within few days) were selected and transferred to new Petri dishes containing MEA. 2.2 Morphology and cultural characteristics To encourage formation of fruiting structures, isolates were inoculated onto sterile pine needles on 1.5% water agar (WA) (Biolab) as described previously (Slippers et al. 2004). The plates were incubated at 25C under near UV-light for 4–6 weeks. Microscope slides of conidia from pycnidia formed on the pine needles were prepared in lactic acid for morphological observations. Conidial dimensions were taken from digital images using a HRc Axiocam digital camera and accompanying Axiovision 3.1 (Carl Zeiss Ltd, München, Germany) microscope. For each isolate, 15 measurements of both conidial length and width were made. Colony appearance of cultures growing on 2% MEA at 25C under near UV-light for 2 weeks was described and colours of the colonies were recorded. Cultures are maintained in the Culture Collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa. 2.3 DNA extraction, PCR reactions and DNA sequencing Procedures and protocols for genomic DNA extraction and sequencing of representative isolates of the Botryosphaeriaceae were the same as those described in Begoude et al. (2010), using two gene regions. The entire Internal Transcribed Spacer region (ITS) of the rDNA, including the 5.8S gene, was amplified by polymerase chain reaction (PCR), for all isolates collected, using the primers ITS1 and ITS4 (White et al. 1990). A part of the Translation Elongation Factor-1a (tef 1-a) gene was amplified for selected isolates using the primers EF1F and EF1R (Jacobs et al. 2004). 2.4 Sequence analyses Sequences of the Botryosphaeriaceae generated in this study were edited using mega version 4 (Tamura et al. 2007). For the phylogenetic analyses, DNA sequences from this study, together with those retrieved from published sequences in GenBank (http://www.ncbi.nlm.gov) were aligned online using MAFFT (http://align.bmr.kyushu-u.ac.jp/mafft/online/server/) version 6 (Katoh et al. 2005). The aligned sequences were transferred to phylogenetic analysis using parsimony (PAUP) v. 4.0b10 (Swofford 2001) where a final manual alignment was made. Table 1. Locations and characteristics of sites from where Terminalia trees were sampled for Botryosphaeriaceae in Cameroon. Site Region Locality Central South Mbalmayo Nkoemvone Kribi Belabo Eastern GPS coordinates N3 N2 N2 N4 26.034 49.045 58.064 57.376 E11 29.344 E11 07.577 E9 54.904 E13 19.433 Tree species sampled T. T. T. T. superba, T. ivorensis superba mantaly superba, T. ivorensis, T. mantaly Botryosphaeriaceae from Terminalia spp. in Cameroon 283 A phylogenetic analysis was run for each of the ITS and tef 1-a data sets, followed by combined analyses of these data sets for core isolates. A partition homogeneity test (Farris et al. 1995) was conducted in PAUP V. 4.0b10 (Swofford 2001) to assess the possibility of combining the ITS and tef 1-a data sets. In all analyses, gaps were treated as fifth character and characters were unordered and of equal weight. The phylogenetic analyses for the datasets were performed using the maximum parsimony (MP) option, with trees generated by heuristic searches with random stepwise addition in 1000 replicates, tree bisection and reconnection (TBR) as branch swapping algorithm and random taxon addition of sequences for the construction of most parsimonious trees. Branches of zero length were collapsed and all multiple, equally parsimonious trees were saved. Guignardia mangiferae A.J. Roy (strain no. 1095) was used as the outgroup in the analyses of ITS and tef 1-a data sets. The support for branches of the most parsimonious trees was assessed using 1000 bootstrap replicates (Felsenstein 1985). Other measures considered were the tree length (TL), consistency index (CI), rescaled consistency index (RC) and retention index (RI) (Hillis and Huelsenbeck 1992). Bayesian phylogenetic analyses using Markov chain Monte Carlo (MCMC) were performed in MrBayes 3.1.2. (Huelsenbeck and Ronquist 2001) for all three data sets described above. Version 2.2 of MrModeltest (Nylander 2004) was used to select the model that best fits each of the partitions. The Likelihood settings from best-fit models, SYM + I + G and HKY + G, were selected based on the Akaike Information Criteria (AIC) for ITS and tef 1-a respectively. Bayesian analyses were performed for one million generations, with four independent chains and sampled every 100th tree. The first 1000 trees were graphically identified as the burn-in and deleted when constructing consensus trees and calculating posterior probabilities. A total of 9001 trees were imported into MEGA V. 4 to construct a 50% majority-rule consensus tree. 2.5 Pathogenicity tests Plants of native species of Terminalia are rare and could not be obtained. Pathogenicity tests were consequently carried out on 1-year-old non-native T. mantaly and T. catappa trees grown in the Yaoundé Urban Council nursery, Cameroon. These trials were conducted between October–December 2008. This period falls at the end of the rain season and the beginning of the dry season, with average day and night temperatures of 26C. The trees were maintained under shade in 15 cm diameter plastic bags and watered daily. At the time of inoculation, the stem diameters were approximately 10 mm and the trees varied from 15–30 cm in height. For inoculations, 14 isolates of the Botryosphaeriaceae, representing all the species identified in the study, were grown on 2% MEA for 10 days prior to inoculation. To inoculate trees, wounds were made on the stems, half way between the soil level and the first branch by removing the outer bark with a 5 mm diameter cork-borer. A 5 mm-diameter plug of each isolate was placed into each wound, with the mycelium facing the cambium and wrapped with a strip of Parafilm to prevent desiccation and cross contamination of the wounds and inoculum. The trees were divided into four separate blocks and within each block, six trees arranged in a completely randomized design, were used for inoculation with each isolate. The entire trial was repeated once. For the controls, sterile MEA plugs were used in place of the fungal cultures. After 6 weeks, the lengths of the lesions produced in the cambium, including the inoculation point, were measured to obtain an indication of the virulence of the isolates tested. Furthermore, a small piece of necrotic tissue was cut from the edges of all lesions and placed on MEA for isolations to show that the inoculated fungus was associated with the lesions. As no significant differences were noticed between results obtained for the two replications of the experiment (p > 0.05), the data for all isolates were pooled in a single dataset for analyses. Variations in the extent of the lesions were assessed through a one-way analysis of variance (ANOVA) using SAS (SAS systems, v. 8.2; SAS Institute). 3 Results 3.1 Isolates and morphology A total of 115 trees were sampled at four localities. These included 35 T. ivorensis trees, 50 T. superba and 30 T. mantaly trees. Isolates of Botryosphaeriaceae were obtained from 55 of the 115 trees sampled. In total, 43 isolates were obtained from 35 T. ivorensis trees, 20 isolates from seven T. superba trees and 27 isolates from 11 T. mantaly trees. No sign of disease, caused by fungi in the Botryosphaeriaceae, was observed on any trees at the time of collection. It was thus assumed that all isolates were from healthy trees. The isolates obtained were assigned to two groups based on colony and conidial morphology. The majority of isolates collected (82 isolates) produced aerial mycelium that was white at first, turning dark grey-green or grey after 4–5 days at 25C under near UV-light. These isolates produced thick-walled, hyaline conidia that turned dark with age (Fig. 1). The conidia were aseptate when young, becoming uniseptate with age. Conidia were ovoid in shape and some developed longitudinal striations as they aged. These isolates were identified as belonging to species of Lasiodiplodia based on their conidial morphology. The second group of isolates (eight isolates) were characterized by dark grey or green to black mycelium, producing small dark brown conidia (Fig. 1) and resembling Endomelanconiopsis endophytica (Rojas et al. 2008). No sexual fruiting structures were produced on pine needles by any of the isolates from Terminalia spp. in Cameroon. Isolates from the Lasiodiplodia group were found at all the localities sampled and from all three host species. Isolates residing in the second group were found only in three locations (Belabo, Nkoemvone and Mbalmayo) and only on T. ivorensis and T. superba. The Botryosphaeriaceae occurring on Terminalia spp. in Cameroon were compared to similar species described in previous studies (Table 3). 284 B.A.D. Begoude, B. Slippers, M.J. Wingfield et al. (a) (b) (c) (d) Fig. 1. Conidia of species of the Botryosphaeriaceae from Terminalia spp. (a) young hyaline thick-walled conidia of Lasiodiplodia theobromae (CMW28550), (b) L. pseudotheobromae (CMW28297), (c) L. parva (CMW28333) (d) dark brown conidia of E. endophytica (CMW28618). Bars: 10 lm. 3.2 DNA extraction and PCR amplification A total of 55 isolates, each originating from a single Terminalia tree, were selected for sequencing of their ITS and 5.8S rDNA regions to obtain an indication of their identities and to select isolates for the data sets used in the final analyses. These comprised 51 isolates from the morphological group resembling Lasiodiplodia and four from the group resembling Endomelanconiopsis. Based on results of the ITS sequences, fourteen isolates were selected for sequencing of the tef 1-a gene region and considered in the final analyses. PCR fragments for the ITS and 5.8S gene were 580 bp and for tef 1-a gene region were 710 bp in size. The tef 1-a sequences obtained were larger than those retrieved from GenBank, which spanned 244– 500 bp and only the corresponding regions were used in the phylogenetic analyses. 3.3 Phylogenetic analyses A BLAST search against the GenBank database, using ITS sequences obtained from Terminalia spp. in Cameroon, showed that isolates resembling species of Lasiodiplodia were most closely related to L. theobromae (Pat.) Griff. & Maubl. and L. pseudotheobromae A.J.L. Phillips, A. Alves & Crous. Isolates from the second group, with small dark brown conidia, were identified as Endomelanconiopsis endophytica Rojas & Samuels. 3.3.1 ITS phylogeny The ITS dataset comprised 91 sequences including 55 from Terminalia spp. sampled in Cameroon and 36 sequences were retrieved from GenBank. After alignment, the ITS sequence data set consisted of 575 characters of which 313 were constant, 112 were parsimony uninformative and 150 were parsimony informative. The MP analyses generated 100 trees with identical topologies with respect to the major clades (TL = 563, CI = 0.627, RI = 0.868, RC = 0.544). The MP analyses of the ITS gene region resulted in two main clades that contained isolates from Terminalia spp. These clades represented the two genera Lasiodiplodia [Bootstrap support (BS) = 100% and Bayesian posterior probabilities (BPP) = 0.99] and Endomelanconiopsis (BS = 100% and BPP = 1) (Fig. 2). In the Endomelanconiopsis group, except for two isolates Botryosphaeriaceae from Terminalia spp. in Cameroon 100/0.87 100/1 93/1 CMW 28326 CMW 28327 CMW 28331 CMW 28329 CMW 28547 CMW 28548 CMW 28550 CMW 28304 CMW 28570 CMW 28571 CMW 28573 CMW 28575 CMW 28308 CMW 28312 CMW 28554 CMW 28555 CMW 28556 CMW 28311 CMW 28625 CMW 28626 CBS 164.96 CMW 9074 CMW 28328 CMW 28330 CMW 28297 CMW 28299 CMW 28300 CMW 28301 CMW 28332 CMW 28566 CMW 28632 CMW 28567 CMW 28568 CMW 28569 CMW 28633 CMW 28574 CMW 28632 CMW 28310 CMW 28557 CMW 28558 CMW 28313 CMW 28560 CMW 28561 CMW 28562 CMW 28624 CMW 28298 CMW 28627 CMW 28622 CBS 116459 CBS 447.62 STEU 5803 STEU 4583 CMW 28628 CMW 28309 CMW 28292 CMW 28333 CMW 27801 CMW 27818 CMW 27820 CMW 28295 76/0.99 CBS 356.59 CBS 494.78 CMW 26162 CMW 26163 98/1 CBS 115812 CBS 116355 100/1 CBS 110492 CBS 118741 98/0.82 WAC 12535 WAC 12536 98/0.73 WAC 12539 WAC 12540 100/0.99 CBS 112555 CBS 119049 97/1 CBS 112553 CBS 230.30 87/0.95 CMW 7772 CMW 7773 CMW 9081 CMW 9079 CMW 28563 95/1 CMW 28618 CMW 28551 CMW 28552 100/0.99 CBS 120397 CBS 122550 CBS 122546 CBS 353.97 100/0.99 CMW 8000 CBS 110302 100/1 285 L. theobromae L. pseudotheobromae L. plurivora L. mahajangana L. parva L. margaritacea L. gonubiensis L. crassispora L. rubropurpurea L. venezuelensis D. seriata D. mutila N. ribis N. parvum E. endophytica E. microspora B. dothidea 1095 G. mangiferae 5 Changes Fig. 2. One of the most parsimonious trees obtained from analyses of the ITS sequence data of the Botryosphaeriaceae from Terminalia spp. Bootstrap support (%) followed by Posterior probabilities from 1000 replications are given on the branches (BS ⁄ PP). Isolates marked in bold represent those obtained from Terminalia spp. in Cameroon. (CMW28551, CMW28563) where very little divergence (two to three base pairs) was observed, sequences from Terminalia spp. in Cameroon were identical to E. endophytica and clustered with isolates from Panama (see Table 2). The Lasiodiplodia group included most of the isolates obtained in this study and was divided into three sub-clades with no clear Bootstrap support. The first sub-clade (20 isolates) consisted of isolates grouping with L. theobromae. Except for two isolates, no sequence variation was detected between isolates in this clade. The second sub-clade (25 isolates) accommodated isolates clustering with L. pseudotheobromae. Small sequence variations were observed in a few isolates of this group. The third group, 286 Table 2. Botryosphaeriaceae used in phylogenetic analyses in this study. GenBank accession no. Species Culture number1 L. plurivora L. pseudotheobromae STEU STEU CMW28297 CMW28300 CMW28574 CMW28624 CMW28328 CMW28330 CMW28299 CMW28301 CMW28332 CMW28566 CMW28314 CMW28568 CMW28569 Switzerland Switzerland Portugal USA USA USA Cameroon Cameroon Cameroon Cameroon Panama Panama Panama Panama Panama Venezuela Australia Australia South Africa South Africa Madagascar Madagascar Madagascar Australia Australia Sri Lanka Colombia Cameroon Cameroon Cameroon Cameroon CMW28628 South Africa South Africa Cameroon Cameroon Cameroon Cameroon Cameroon Cameroon Cameroon Cameroon Cameroon Cameroon Cameroon Cameroon Cameroon Host Ostrya spp. Prunus spp. Vitis vinifera P. dactylifera Ribes spp. Ribes spp. Terminalia ivorensis T. superba T. superba T. ivorensis Theobroma cacao Th. cacao Th. cacao Soil Th. cacao Eucalyptusurophylla Santalum album S. album S. cordatum S. cordatum T. catappa T. catappa T. catappa A. gibbosa A. gibbosa T. cacao Cassava-field soil T. superba T. ivorensis T. ivorensis T. mantaly Cameroon P. salicina V. vinifera T. mantaly T. ivorensis T. ivorensis T. ivorensis T. mantaly T. mantaly T. superba T. superba T. superba T. ivorensis T. ivorensis T. ivorensis T. ivorensis Collectors B. Slippers B. Slippers A.J.L. Phillips L.L. Huilllier B. Slippers ⁄ G.Hudler B. Slippers ⁄ G.Hudler D. Begoude D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux E. Rojas ⁄ L.Mejia ⁄ Z. Maynard E. Rojas ⁄ L.Mejia ⁄ Z. Maynard E. Rojas ⁄ L.Mejia ⁄ Z. Maynard E. Rojas ⁄ L.Mejia ⁄ Z. Maynard E. Rojas ⁄ L.Mejia ⁄ Z. Maynard S. Mohali T.I. Burgess ⁄ B. Dell T.I. Burgess ⁄ B. Dell D. Pavlic D. Pavlic J. Roux J. Roux J. Roux D. Pavlic D. Pavlic A. Riggenbach O. Rangel D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux T. ivorensis U.Damm F.Halleen D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux ITS AY236948 AY236949 AY259093 DQ458886 EF445343 EF445344 GQ469966 GQ469967 GQ469968 GQ469965 EU633656 EU683661 EU683664 EU683655 EU683671 DQ103552 DQ103550 DQ103551 DQ458892 AY639594 FJ900595 FJ900596 FJ900597 EU144050 EU144051 EF622082 EF622084 GQ469961 GQ469962 GQ469963 GQ469964 D. Begoude ⁄ J. Roux EF445362 AY343482 GQ469937 GQ469939 GQ469947 GQ469956 GQ469935 GQ469936 GQ469938 GQ469940 GQ469941 GQ469942 GQ469943 GQ469944 GQ469945 tef 1-a b-tub AY236897 AY236898 EF445382 EF445383 GQ469906 GQ469907 GQ569908 EU683637 EU683642 EU683645 EU683636 EU683652 DQ10355 DQ103557 DQ103558 DQ458877 DQ103567 FJ900641 FJ900642 FJ900643 EU144065 EU144066 EF622062 EF622064 GQ469903 GQ469904 GQ469905 GQ469960 EF445395 EF445396 GQ469899 GQ469900 GQ469901 GQ469902 GQ469892 GQ469894 GQ469893 B.A.D. Begoude, B. Slippers, M.J. Wingfield et al. CMW7999 CMW8000 Diplodia mutila CBS112553 CBS230.30 D. seriata CMW7774 CMW7775 Endomelanconiopsis endophytica CMW28618 CMW28551 CMW28552 CMW28563 CBS120397 CBS122546 CBS122550 E. microspora CBS353.97 Guignardia mangiferae 1095 Lasiodiplodia crassispora WAC12533 WAC12534 WAC12535 L. gonubiensis CBS115812 CBS116355 L. mahajangana CMW27801 CMW27818 CMW27820 L. margaritacea CMW26162 CMW26163 L. parva CBS356.59 CBS494.78 CMW28333 CMW28309 CMW28292 CMW28295 Botryosphaeria dothidea Origin Table 2. (Continued) GenBank accession no. Species L. theobromae L. venezuelensis Neofusicoccum parvum N. ribis Origin Host Collectors ITS CMW28633 CMW28632 CMW28310 CMW28557 CMW28558 CMW28313 CMW28560 CMW28561 CMW28562 CMW28298 CMW28627 CMW28622 CBS116459 CBS447.62 WAC12535 WAC12536 CMW28550 CMW28570 CMW26571 CMW27311 CMW28326 CMW28327 CMW28329 CMW28547 CMW28548 CMW28573 CMW28575 CMW28308 CMW28312 CMW28554 CMW28555 CMW28556 CMW28625 CMW28626 CMW9074 CBS164.96 Cameroon Cameroon Cameroon Cameroon Cameroon Cameroon Cameroon Cameroon Cameroon Cameroon Cameroon Cameroon Costa Rica Suriname Australia Australia Cameroon Cameroon Cameroon Cameroon Cameroon Cameroon Cameroon Cameroon Cameroon Cameroon Cameroon Cameroon Cameroon Cameroon Cameroon Cameroon Cameroon Cameroon Mexico New Guinea D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux J.Carranza ⁄ Velásquez C. Smulders T.I. Burgess ⁄ G.Pegg T.I. Burgess ⁄ G.Pegg D. Begoude ⁄ J.Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux D. Begoude ⁄ J. Roux T. Burgess Unknown GQ469946 GQ469948 GQ469949 GQ469950 GQ469951 GQ469952 GQ469953 GQ469954 GQ469955 GQ469957 GQ469958 GQ469959 EF622077 EF622081 DQ103553 DQ103554 GQ469921 GQ469923 GQ469924 GQ469932 GQ469915 GQ469916 GQ469918 GQ469919 GQ469920 GQ469925 GQ469926 GQ469927 GQ469928 GQ469929 GQ469930 GQ469931 GQ469933 GQ469934 EF622074 AY640255 WAC12539 WAC12540 CMW9081 CMW9079 CMW7772 CMW7773 Venezuela Venezuela New Zealand New Zealand USA USA T. ivorensis T. ivorensis T. ivorensis T. ivorensis T. ivorensis T. ivorensis T. ivorensis T. ivorensis T. ivorensis T. ivorensis T. ivorensis T. ivorensis Gmelinea arborea Citrus aurantium E. grandis E. grandis T. mantaly T. ivorensis T. ivorensis T. ivorensis T. mantaly T. mantaly T. mantaly T. mantaly T. mantaly T. ivorensis T. superba T. superba T. ivorensis T. ivorensis T. ivorensis T. ivorensis T. ivorensis T. ivorensis Pinus sp. Fruit along coral reef coast Acacia mangium A. mangium P. nigra A. deliciosa Ribes sp. Ribes sp. S. Mohali S. Mohali G.J. Samuels S.R. Pennicook B. Slippers ⁄ G.Hudler B. Slippers ⁄ G Hudler DQ103547 DQ103548 AY236943 AY236940 AY236935 AY236936 tef 1-a EF622057 EF622060 DQ103571 DQ103572 GQ469895 GQ469896 GQ469897 GQ469898 b-tub Botryosphaeriaceae from Terminalia spp. in Cameroon L. rubropupurea Culture number1 EF622054 AY640258 DQ103568 DQ103569 AY236888 AY236885 AY236877 AY236878 1 Isolates sequenced in this study appear in bold. All other sequences were obtained from GenBank. 287 288 B.A.D. Begoude, B. Slippers, M.J. Wingfield et al. consisting of five isolates from Cameroon was not clearly resolved and clustered close to L. mahajangana Begoude, Jol. Roux, & Slippers. and L. parva A.J.L. Phillips, A. Alves & Crous. No statistical support was observed for any of these sub-clades. For this reason, representative isolates from the Endomelaconiopsis clade and the three sub-clades in the Lasiodiplodia group were selected for tef 1-a gene region sequencing. 3.3.2 Combined ITS and tef 1-a analyses The partition homogeneity test indicated congruence between the ITS and tef 1-a partitions (p = 0.355) suggesting that the data sets could be combined. The combined dataset consisted of 48 isolates with 887 characters of which, 377 were constant, 146 were parsimony uninformative and 364 were parsimony informative. Gaps were treated as a fifth character. After heuristic searches, 42 most parsimonious trees were obtained (TL = 1068; CI = 0.738, RI = 0.914, RC = 0.674; TreeBase Accession No. SN4630) and one of them was chosen for presentation (Fig. 3). All 42 trees displayed the same topology with regard to the identified clades. The topology of the tree generated from the combined analyses with MP, as well as with the 50% majority rule consensus tree from the trees obtained through Bayesian analysis, was congruent with the trees obtained with the individual analyses of ITS and tef 1-a, identifying the same clades. All the isolates collected in this study grouped with previously described species of Lasiodiplodia and Endomelanconiopsis, strongly supported with Bootstrap and Bayesian posterior probability values (Fig. 3). Similar to results obtained for the ITS gene region, isolates from Cameroon could be identified as L. theobromae (BS = 100%, BPP = 1), L. pseudotheobromae (BS = 100%; BPP = 1). The third group of Lasiodiplodia isolates clustered with L. parva (BS = 97%; BPP = 1), but one base pair difference in the tef 1-a sequences was noticed among isolates in this later group. The fourth group of isolates consisted of E. endophytica from Terminalia spp. in Cameroon which formed a well supported clade (BS = 100% and BPP = 1) with sequences from authentic isolates of this species from GenBank (Fig. 3). Isolates of Botryosphaeriaceae found on Terminalia spp. that were phylogenetically related to L. parva based on ITS and tef 1-a sequence comparisons, mostly conformed to the description of L. parva (Alves et al. 2008). However, important differences in conidial sizes were observed for isolates from Cameroon (Table 3), raising the question as to whether they represent a different species. DNA sequence data for the ITS and tef 1-a gene regions, however, did not support the description of a discrete species for these isolates. Further sequences from additional gene regions (b-tubulin) not reported in this paper were found to be identical with those of original species of L. parva and, therefore, suggested that all these isolates represent the same species. 3.4 Pathogenicity Pathogenicity trials conducted on T. mantaly using isolates of the Botryosphaeriaceae collected in this study revealed visible lesions within 6 weeks after inoculation (Fig. 4). Trees inoculated with sterile MEA also produced small lesions that represented only wound reactions as no Botryosphaeriaceae could be isolated from them. All the isolates of Botryosphaeriaceae were successfully re-isolated from the lesions emerging from inoculations. ANOVA showed that the mean lengths of lesions produced by all of the isolates on T. mantaly differed significantly (p < 0.0001) from the controls (Fig. 4). L. pseudotheobromae produced the longest lesions followed by L. theobromae, L. parva and E. endophytica. On T. catappa trees, all isolates collected from Terminalia trees in Cameroon produced lesions significantly longer than those of the control inoculations (Fig. 5). Similar to the situation on T. mantaly, control inoculations showed only small lesions. However, re-isolations did not yield any Botryosphaeriaceae from the controls, whereas the original Botryosphaeriaceae were re-isolated from all the trees inoculated with fungal cultures. Analysis of variance indicated that lesion lengths produced on the cambium by all the isolates were significantly different (p < 0.0001) to those associated with the controls (Fig. 5). Isolates representing L. pseudotheobromae were most virulent and produced longer lesions than L. theobromae and L. parva. E. endophytica produced substantially smaller lesions than either L. pseudotheobromae or L. theobromae. A positive correlation (r2 = 77%) was found between inoculations on T. mantaly and T. catappa. On both tree species, L. pseudotheobromae was most virulent. In general, the lesions in T. catappa caused by L. pseudotheobromae and L. theobromae isolates were longer than those of T. mantaly . In contrast, the lengths of lesions produced by isolates of L. parva and E. endophytica on T. catappa were smaller than those observed on T. mantaly. However, this difference in susceptibility between T. catappa and T. mantaly was not statistically significant. 4 Discussion This study represents the first attempt to identify the Botryosphaeriaceae on native Terminalia trees in Africa. Four species of the Botryosphaeriaceae were collected from T. ivorensis and T. superba and three species were found on samples from the nonnative T. mantaly. A combination of morphological characteristics and DNA sequence comparisons was used to identify these species as L. theobromae, L. pseudotheobromae, L. parva and E. endophytica. These fungi are reported on these hosts for the first time. While E. endophytica was isolated only from T. superba and T. ivorensis, L. pseudotheobromae, L. theobromae and L. parva were collected from all the tree species sampled in this study. The majority of isolates obtained in this study represented species of Lasiodiplodia of which isolates were identified as L. theobromae, L. pseudotheobromae and L. parva based on sequence data for the ITS and tef 1-a gene regions. Until recently, most Lasiodiplodia spp. from tropical trees were treated as L. theobromae (Punithalingam 1976). However, application of DNA sequence comparisons for the ITS and tef 1-a gene regions has resulted in the recognition of 10 new Lasiodiplodia spp. (Pavlic Botryosphaeriaceae from Terminalia spp. in Cameroon 289 CMW 28550 CMW 28570 100/1 CMW 28571 L. theobromae CMW 28311 CBS 164.96 CMW 9074 CMW 28297 76/1 CMW 28300 100/1 CMW 28574 L. pseudotheobromae CMW 28624 CBS 116459 CBS 447.62 95/1 CMW 28333 71/1 CMW 28309 CMW 28292 97/1 L. parva CBS 356.59 CBS 494.78 100/1 100/1 STEU 5803 CMW 27801 CMW 27818 100/1 WAC 12535 L. rubropurpurea WAC 12536 76/0.59 100/1 WAC 12539 WAC 12540 100/1 L. mahajangana CMW 27820 100/1 99/1 L. plurivora STEU 4583 100/1 L. venezuelensis 100/1 CBS 115812 CBS 116355 L. gonubiensis CBS 110492 L. crassispora 100/1 CBS 118741 100/1 97/0.92 CMW 26162 L. margaritacea CMW 26163 82/0.99 100/1 CBS 112555 D. seriata CBS 119049 CMW 8000 100/1 B. dothidea CBS 110302 CMW 28618 CMW 28551 CMW 28552 100/1 E. endophytica CBS 120397 CBS 122546 100/1 CBS 122550 97/1 E. microspora CBS 353.97 100/0.99 CMW 7772 CMW 7773 100/1 N. ribis CMW 9081 77/0.84 CMW 9071 N. parvum 1095 G. mangiferae 10 Changes Fig. 3. One of the most parsimonious trees obtained from analyses of the combined ITS and tef 1-a sequence data of the Botryosphaeriaceae from Terminalia spp. Bootstrap support (%) followed by Posterior probabilities from 1000 replications are given on the branches (BS ⁄ PP). Isolates marked in bold represent those obtained from Terminalia spp. in Cameroon. et al. 2004; Burgess et al. 2006; Damm et al. 2007; Alves et al. 2008; Pavlic et al. 2008; Begoude et al. 2010). These species share similar morphological characteristics, such as slowly maturing conidia with thick walls that turn dark with age and develop longitudinal striations. Lasiodiplodia theobromae has a wide geographic distribution and it has been found on more than 500 forest and agricultural plant species in tropical and subtropical areas (Punithalingam 1980). It is well known as an endophyte on healthy tropical trees (Suryanarayanan et al. 2002; Begoude et al. 2010). Furthermore, L. theobromae can act as a latent pathogen causing disease symptoms after onset of conditions unfavourable for the host (Schoeneweiss 1981; Mullen et al. 1991; Slippers and Wingfield 2007). L. theobromae has previously been reported as an endophyte in the inner bark and twigs of healthy T. arjuna (Tejesvi et al. 2005), leaves of T. tomentosa and T. bellerica (Suryanarayanan et al. 2002) and the twigs and bark of healthy T. catappa (Begoude et al. 2010) in the tropics. On Terminalia spp., L. theobromae has mostly been recorded as the causal agent 290 B.A.D. Begoude, B. Slippers, M.J. Wingfield et al. Table 3. Conidial dimensions of the Botryosphaeriaceae from Terminalia spp. in Cameroon and comparison with those reported in previous studies. Conidial size (lm) Species L. pseudotheobromae L. theobromae L. parva Lesion length on cambium (mm) E. endophytica This study Previous studies Source of data (20.5))23.5)27.5()31.5) · (10.5))12)14()16.5) (17.5))21.5)27.5()31) · (10.5))12)14()16.5) (24.5))26.5)29.5()33.5) · (11))12)14.5()17.5) (5.5))6)7.5()8) · (3))3.5)4()4.5) (22.5))23.5)32()33) · (13.3))14)18()20) (19))21)31()32.5) · (12))13)15.5()18.5) (15.5))16)23.5()24.5) · (10))10.5)13()14.5) (4.7))5.5)7.5()10.0) · (3.0))3.5)4.5()6.2) Alves et al. (2008) Alves et al. (2008) Alves et al. (2008) Rojas et al. (2008) 40 35 a a ab 30 abc bcde bcd bcdef bcdef cdef 25 def ef def f ef 20 15 g 10 5 0 CMW CMW CMW CMW 28300 28574 28297 28624 CMW CMW CMW CMW 28311 28550 28570 28571 CMW CMW CMW 28309 28292 28333 LPs LT LP Isolates CMW CMW CMW 28551 28618 28552 Control EE Lesion length on cambium (mm) Fig. 4. Mean lesion lengths (mm) on cambium of T. mantaly 6 weeks after inoculation with isolates of L. pseudotheobromae (LPs), L. theobromae (LT), L. parva (LP), E. endophytica (EE), Control. Lesion lengths caused by isolates marked with the same letter are not significantly different (p < 0.0001). 40 35 30 a b bc bcd cde 25 bc bcd de de de de de de e 20 15 f 10 5 0 CMW CMW CMW CMW 28300 28574 28297 28624 CMW CMW CMW CMW 28311 28550 28570 28571 LPs LT CMW CMW CMW 28309 28292 28333 Isolates LP CMW CMW CMW 28551 28618 28552 Control EE Fig. 5. Mean lesion lengths (mm) on cambium of T. catappa 6 weeks after inoculation with isolates of L. pseudotheobromae (LPs), L. theobromae (LT), L. parva (LP), E. endophytica (EE), Control. Lesion lengths caused by isolates marked with the same letter are not significantly different (p < 0.0001). of blue stain of logs, soon after felling (Lamb and Ntima 1971; Groulez and Wood 1985; Apetorgbor et al. 2004). However, in Cameroon, L. theobromae is best known as the cause of die-back of cacao (Theobromae cacao) (Mbenoun et al. 2008). In the current study, L. theobromae was the second most abundant species identified on Terminalia spp. All isolates collected were from healthy trees, but pathogenicity trials on young T. catappa and T. mantaly showed that it is highly pathogenic to these trees. Pathogenicity tests on T. ivorensis and T. superba should, however, be conducted to determine whether it can cause disease on these important native trees. Lasiodiplodia pseudotheobromae was the most commonly collected species of Botryosphaeriaceae, collected from all the species of Terminalia sampled in this study. This fungus was originally described from Rosa spp. in the Netherlands, Gmelina arborea and Acacia mangium in Costa Rica, Coffea spp. in Democratic Republic of Congo and Citrus aurantium in Suriname (Alves et al. 2008). In a recent study investigating the Botryosphaeriaceae on T. catappa in Cameroon, South Africa and Madagascar (Begoude et al. 2010), L. pseudotheobromae was also the most abundant species found in all the sampled areas. Botryosphaeriaceae from Terminalia spp. in Cameroon 291 The information generated in the current study, which is supported by a previous one on T. catappa, suggests that L. pseudotheobromae has a worldwide distribution. In pathogenicity trials L. pseudotheobromae was found to be the most virulent species. This was also the case in a study of T. catappa (Begoude et al. 2010). L. pseudotheobromae is, therefore, the most likely species of Botryosphaeriaceae to cause health problems on Terminalia trees in Africa where they are subjected to stressful conditions. Lasiodiplodia parva was only recently described and was previously treated as L. theobromae, together with L. pseudotheobromae (Alves et al. 2008). Isolates collected from Terminalia spp. in this study, however, differed in their conidial sizes from descriptions for the type specimen. The conidia of isolates from Cameroon were larger than those previously described for L. parva. DNA sequence data for both ITS and tef 1-a, b-tubulin, however, confirmed that isolates from Cameroon represent L. parva, despite minor differences for two nucleotides in ITS sequences and a single nucleotide in tef 1-a sequences. Our results thus show that some isolates of L. parva can produce conidia as large as those produced by other closely related species, such as L. pseudotheobromae and L. theobromae. This emphasizes the importance of considering multiple criteria for species identification when treating species of the Botryosphaeriaceae. Prior to this study, L. parva was known only to occur in agricultural field soil and crops in Latin America (Alves et al. 2008). Although L. parva was the least abundant Lasiodiplodia spp. isolated from Terminalia spp., its occurrence on these trees in Cameroon has substantially broadened its host range and geographic distribution. Previously, the only plant host from which L. parva was known was Theobroma cacao in Colombia (Alves et al. 2008) and no information concerning its pathogenicity to this tree is available. In the current study, assessment of its pathogenicity on T. mantaly and T. catappa trees showed that L. parva consistently produced lesions on both hosts. However, in comparison to L. theobromae and L. pseudotheobromae, L. parva was only mildly pathogenic, suggesting that this fungus is unlikely to emerge as an important pathogen on these trees. Endomelanconiopsis endophytica is a recently described species found as an endophyte in leaves of T. cacao and associated native woody hosts in the same environment (Rojas et al. 2008). Isolates of E. endophytica found in the present study were shown to group with the South American isolates of the fungus. The Cameroonian isolates were obtained from T. ivorensis and T. superba. These tree species are commonly used in cacao farms to establish a ÔtaungyaÕ agri-sylvicultural system in which they provide shade or improve soil fertility (Norgrove and Hauser 2002). It would not, therefore, be surprising to obtain further isolates of this fungus on hosts such as cocoa trees in Cameroon. The collection of E. endophytica from native Terminalia spp. in Cameroon adds to previous records of the fungus from plants in South America (Rojas et al. 2008). Even though very few isolates representing E. endophytica were found in this study, its presence on tropical species of Terminalia is particularly interesting as this could indicate a possible tropical origin of the fungus. Two distinct genera of Botryosphaeriaceae, Lasiodiplodia and Endomelanconiopsis, were found associated with species of Terminalia in Cameroon. Although little information related to the ecology of the genus Endomelanconiopsis is available, both Lasiodiplodia and Endomelanconiopsis appear to be tropical species. Apart from E. endophytica, which was isolated only from T. superba and T. ivorensis, no evidence of host specialization was observed for species of Lasiodiplodia identified in this study. This is characteristic of many species of Botryosphaeriaceae (Slippers and Wingfield 2007) and contributes to their potential to cause diseases on trees. Although this study focussed exclusively on healthy tree tissue, the common occurrence of generalist species such as L. theobromae and L. pseudotheobromae, which are reputed virulent pathogens on a wide range of hosts (Punithalingam 1980; Slippers and Wingfield 2007; Begoude et al. 2010) suggests that they could be pathogens if unfavourable conditions for the host occur. Acknowledgements We thank the DST ⁄ NRF Centre of Excellence in Tree Health Biotechnology (CTHB) and the University of Pretoria, South Africa for financial support. We also thank the Institute of Agricultural Research for Development (IRAD), the International Institute of Tropical Agriculture (IITA) and the Yaoundé Urban Council in Cameroon for logistic support. Mr Onana Dieudonne and other colleagues at IRAD are gratefully acknowledged for assistance and guidance in tree identification. 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