Mycol. Res. 109 (9): 1005–1014 (September 2005). f The British Mycological Society 1005 doi:10.1017/S0953756205003266 Printed in the United Kingdom. Seed-borne Botryosphaeria spp. from native Prunus and Podocarpus trees in Ethiopia, with a description of the anamorph Diplodia rosulata sp. nov. Abdella GURE1,2, Bernard SLIPPERS1 and Jan STENLID1 1 Department of Forest Mycology & Pathology, Swedish University of Agricultural Sciences (SLU), Box 7026 SE-750 07 Uppsala, Sweden. 2 Wondo Genet College of Forestry, Debub University, P.O. Box 128, Shashamane, Ethiopia. E-mail : Abdella.gure@mykopat.slu.se Received 30 November 2004; accepted 27 April 2005. Botryosphaeria spp. from seeds of the native afromontane forest tree species, Podocarpus falcatus and Prunus africana, in Ethiopia have been identified. This is achieved by combining anamorph morphological characters and ITS sequence data. From a relatively small sample, four Botryosphaeria spp. were encountered. Two of the species from P. falcatus and the one from P. africana have not been previously described. The two species from P. falcatus were represented by only one isolate each and are not named here. The morphology of their Diplodia and Dothiorella anamorphs is, however, characterised. The anamorph of the other Botryosphaeria sp. from P. africana is described here as Diplodia rosulata sp. nov. Furthermore, B. parva is identified from P. falcatus based on ITS phylogeny. This species is also an important pathogen of various commercially important tree species in Ethiopia and elsewhere. This study highlights the ability of Botryosphaeria spp. to infect seeds and the possibility that they might be distributed in this way. The study also contributes to recent attempts to stabilize the taxonomy of Botryosphaeria anamorphs, especially regarding Diplodia, which is currently in taxonomic disarray. INTRODUCTION Botryosphaeria is a species-rich genus with a worldwide distribution. Members of this genus and its anamorphs occur on a wide range of monocotyledonous, dicotyledonous and gymnosperm hosts (Arx & Müller 1975). These fungi have also been associated with various disease symptoms such as shoot blight, die-back, stem canker, seed capsule abortion, seed and fruit rots (Webb 1983, Smith et al. 1996, Smith, Wingfield & Petrini 1996, Roux & Wingfield 1997, Roux et al. 2001, Gezahgne, Roux & Wingfield 2003, Alves et al. 2004, Gezahgne et al. 2004). The taxonomy of Botryosphaeria species and their associated anamorphs is often confusing (Jacobs & Rehner 1998, Zhou & Stanosz 2001, Slippers et al. 2004a). Reasons for this are that the teleomorphs are rarely encountered in nature, or are difficult to induce in culture, and that the morphological diversity of the teleomorphs is limited (Shoemaker 1964, Laundon 1973, Jacobs & Rehner 1998). The taxonomy of Botryosphaeria spp. is, therefore, often based on the characteristics of its anamorphs, which are more commonly encountered (Shoemaker 1964, Pennycook & Samuels 1985, Phillips et al. 2002, Denman et al. 2003, Slippers et al. 2004a). Morphological characters of Botryosphaeria anamorphs considered useful for taxonomic delimitation include conidial characteristics such as the size, shape, colour, septation, wall thickness and wall texture, the presence of microconidia and mode of conidiogenesis (Jacobs & Rehner 1998, Denman et al. 2000). Furthermore, colony morphology, especially colour, and effects of temperature on mycelial growth rates have been used to classify these fungi (Pennycook & Samuels 1985, de Wet et al. 2000, Phillips et al. 2002, Slippers et al. 2004b). Despite Botryosphaeria anamorphs having a number of taxonomically useful characters, their identification can also be confusing because some of the characters overlap between species (Smith & Stanosz 2001, Slippers et al. 2004a). In addition to anamorph morphological characters, phylogenetic analysis of the nuclear rDNA ITS sequence has proved useful in clarifying the taxonomy and phylogenetic relationships of Botryosphaeria species (Jacobs & Rehner 1998, Denman et al. 2000, Zhou & Stanosz 2001, Denman et al. 2003, Alves et al. 2004, Slippers et al. 2004a). The ITS rDNA sequence data have been used to separate anamorphs of Botryosphaeria in which the morphological features were confusing, for example B. ribis from Fusicoccum luteum (Smith & Stanosz 2001), and B. stevensii from Seed-borne Botryosphaeria spp. from native Prunus and Podocarpus trees in Ethiopia B. corticola (Alves et al. 2004). However, the use of additional data from other nuclear genes has in some cases revealed cryptic species that went unnoticed when using ITS data alone (de Wet et al. 2003, Slippers et al. 2004a). Despite recent advances in the taxonomy of Botryosphaeria anamorphs, the taxonomy of groups like Diplodia is still very confused. More than 1200 Diplodia spp. have been described. Sequence data is, however, available only for ten Botryosphaeria spp. with Diplodia, Dothiorella and Fusicoccum anamorphs (http://www.ncbi.nlm.nih.gov/). Where species have been examined closely, many have had to be synonymised, while others have been separated into different species (de Wet et al. 2003, Alves et al. 2004, Phillips et al. 2005). One reason for this is that many species were described based on host association or geographic distribution. Today it is known that some Botryosphaeria spp. have wide host and geographic ranges, for example B. obtusa and B. parva (Punithalingam & Waller 1973, Pennycook & Samuels 1985, Slippers et al. 2004a). On the other hand, some species appear to have narrower host ranges, for example D. pinea and D. scrobiculata associated with Pinus spp. (de Wet et al. 2000, de Wet et al. 2003). There is thus a need to closely examine more Diplodia spp. based on recent advances in the taxonomy of this group. Podocarpus falcatus and Prunus africana are important multipurpose afromontane forest tree species widely distributed across Africa. Podocarpus falcatus is used for timber, shade, medicinal, and conservation purposes (Negash 1995, 2003). Prunus africana is used as a source of very hard and durable wood, bee forage, mulch, shade, and for windbreaks (Demel 1993, Negash 1995). It is also a highly valued source of income in some countries in Africa, primarily for the medicinal value of its bark (Cunningham et al. 2002). At present, in Ethiopia, the natural populations of these species are threatened by excessive logging and illegal tree felling that has led to a widespread deforestation. As a result, there is a growing need to propagate these important native tree species, mainly by seed. Fungi belonging to many different taxa were isolated during a previous survey of fungi associated with seeds of these two trees (Gure 2004). In this study, we identify the seed-borne Botryosphaeria spp. collected during that earlier survey by combining anamorph morphology and ITS rDNA sequence data. Apart from identifying Botryosphaeria spp. we also describe a new Diplodia sp. from seeds of P. africana. MATERIALS AND METHODS Fungal isolates Seeds of Podocarpus falcatus and Prunus africana were collected from Badale and Gambo in Ethiopia. Seeds of both species were surface-sterilised by soaking in hydrogen peroxide (33 % v/v) for 1 min or sodium 1006 hypochlorite (13 % v/v) for 5 min followed by a thorough rinsing in sterilised water. Surface sterilised seeds were blotted on sterile filter paper before plating on 2% malt extract agar (MEA), 1 % water agar (WA) and potato dextrose agar (PDA) (Oxoid, Basingstoke) plates, five seeds per plate. The plates were incubated at temperatures of 20¡2 xC or 25 x in darkness for 5–10 d. Fungi growing out of the seeds were isolated in pure culture. Seven isolates resembling Botryosphaeria in culture (fast growing, dark, greenish brown or greyish pigmented cultures) were further considered in this study (Table 1). The cultures were maintained on MEA slants at 5 x during our studies. Morphological identification Morphological identification of the isolates was based on conidial morphology from cultures grown on 2 % WA. In order to induce sporulation, pine needles were sterilised by autoclaving and then incorporated into WA plates (Slippers et al. 2004a). Furthermore, for the isolates from Prunus africana, seeds were sterilised in hydrogen peroxide (33 % v/v) overnight, washed and added to separate 2 % WA plates for sporulation. Duplicate sets of the plates were incubated at 25 x in darkness with alternating cycles of 12 h of near-UV light and darkness. Squashed mounts of pycnidia were prepared in lactophenol. Morphological observations and measurements of conidial dimensions were made under a light microscope, with an Axiocam digital camera and accompanying software (Zeiss Axiovision 4.1). Cultural studies All isolates from Prunus africana and Podocarpus falcatus were used for cultural studies on MEA. Mycelial discs of 5 mm diam were cut from the peripheries of actively growing cultures and inoculated to the centre of Petri dish plates (90 mm diam) containing MEA, with three replicates for each isolate, at temperatures of 20–25 x. Colony diameter was measured daily ; colony morphology and colour using Küpper (1999) colour chart were noted every 2 d for a total of 15 d. Genomic DNA extraction, sequencing and phylogenetic analysis Pure cultures (about 10 d old) grown on Hagem agar plates (Stenlid 1985) were used for DNA extraction. Nucleic acids were extracted using a CTAB and phenol-chloroform DNA extraction method (Gardes & Bruns 1993) modified by omitting b-mercapto ethanol. Amplification of the ITS region of the rDNA was performed by PCR using universal primers ITS1 and ITS4 (White et al. 1990) as described in Ihrmark (2001). PCR products were purified using QIAquick PCR purification kit (Qiagen, Hilden). Purified ITS PCR products A. Gure, B. Slippers and J. Stenlid 1007 Table 1. Identity and origin of sequences used in the rDNA ITS phylogenetic analysis. Culture no. Identity Host Location Collector GenBank CBS 112545 Quercus suber Cadiz, Spain Q. suber Q. ilex Q. ilex Ulmus sp. Malus pumila Prunus sp. Prunus persica Eucalyptus grandis E. grandis Actinidia deliciosa Vitis vinifera Vitis vinifera CMW 7774 CMW 11060 CMW 11062 CMW 11064 CMW 10089 CMW 10095 CMW 10094 CMW 11246 CMW 11253 CMW 9078 CMW 9081 Po66 CMW 7054 CMW 7772 CMW 7773 CMW 9074 KJ93.41 CBS 431 ATCC 60259 ZS96-174 Pr2 CBS 116470 (Pr3) CBS 116471 (Pr4) CBS 116472 (Pr5) Po16 Po20 CMW 7063 B. obtusa B. parva B. parva B. parva B. parva B. parva B. parva B. parva B. parva B. parva B. parva B. parva B. ribis B. ribis B. ribis B. rhodina B. rhodina B. stevensii B. stevensii B. tsugae Diplodia rosulata D. rosulata D. rosulata D. rosulata Diplodia sp. Dothiorella sp. Bionectria sp. Ribes sp. E. citriodora E. citriodora E. citriodora E. globules E. grandis E. saligna Pinus patula P. patula A. deliciosa Populus nigra Podocarpus falcatus Ribes rubrum Ribes sp. Ribes sp. Pinus sp. Pistachia Fraxinus excelsior M. pumila Tsuga heterophylla Prunus africana P. africana P. africana P. africana Podocarpus falcatus P. falcatus Unknown Aveiro, Portugal Aragon, Spain Aragon, Spain Warwickshire, England Delft, Netherlands Crossifisso, Switzerland Japan Mpumalanga, S. Africa Mpumalanga, S. Africa New Zealand Portugal Montemor-o-Novo, Portugal New York, USA Ethiopia Ethiopia Ethiopia Ethiopia Ethiopia Ethiopia Ethiopia Ethiopia New Zealand New Zealand Gambo, Ethiopia New York New York, USA New York, USA Mexico California, USA Netherlands unknown Canada Gambo, Ethiopia Gambo, Ethiopia Gambo, Ethiopia Gambo, Ethiopia Badale, Ethiopia Badale, Ethiopia Netherlands M. E. Sanchez, A. Trapero A. Alves J. Luque N. Ibarra, E. A. Ellis A. J. L. Phillips B. Slippers P. L. Pusey H. Smith H. Smith G. J. Samuels A. J. L. Phillips A. J. L. Phillips AY259089 CBS 112549 CBS 115041 CBS 115035 IMI 63581 CBS 115038 CMW 8000 KJ94.26 CMW 10125 CMW 10126 CMW 992/3 CMW 9076 CBS 112555 Botryosphaeria corticola B. corticola B. iberica B. iberica B. sarmentorum B. sarmentorum B. dothidea B. dothidea B. eucalyptorum B. eucalyptorum B. lutea B. lutea B. obtusa B. Slippers/G. Hudler A. Gezahgne & J. Roux A. Gezahgne & J. Roux A. Gezahgne & J. Roux A. Gezahgne & J. Roux A. Gezahgne & J. Roux A. Gezahgne & J. Roux A. Gezahgne & J. Roux A. Gezahgne & J. Roux S. R. Pennycook G. J. Samuels A. Gure N. E. Stevens B. Slippers & G. Hudler B. Slippers/G. Hudler T. Burgess T. J. Michailides H. A. van der Aa H. J. Boesewinkel A. Funk A. Gure A. Gure A. Gure A. Gure A. Gure A. Gure Unknown AY236953 AY210474 AY210475 AY210476 AY210477 AY210478 AY210479 AY210486 AY210487 AY236940 AY236943 AY206460 AF241177 AY236935 AY236936 AY236952 AF027762 AY236955 AF243406 AF243405 AY210324 AY210344 AY210346 AY210345 AY513947 AY208152 AY236956 were then sequenced on an Applied Biosystems 310 automated DNA sequencer, using ABI PRISMTM BigDyeTM 10r Terminator Cycle Sequencing Ready Reaction Kit v.2.0 (Applied Biosystems, Foster City, CA). Sequence analysis was done on the ABI PRISMTM Genetic Analyser. DNA sequences were assembled using the program SeqmanII from the DNASTAR software package (GATC, Konstaz). Seven sequences from our study, 39 similar sequences from NCBI, identified using BLAST searches, and 4 sequences provided by Phillips et al. (2005) were manually aligned by inserting gaps. Phylogenetic analyses were performed using PAUP version 4.0b10 (Swofford 2002). In PAUP, gaps were specified as missing data and all characters as unordered and of equal weight. Maximum parsimony trees were obtained using heuristic searches through stepwise (random) addition and tree bisection and reconstruction (TBR) as branch AY259100 AY573202 AY573213 AY573212 AY573206 AY236949 AF027749 AF283686 AF283687 AF027745 AY339258 AY259094 swapping algorithm. Maximum trees were unlimited and, branches of zero length were collapsed. Retention and consistency indices, a g1-value and branch supports using 1000 bootstrap replicates (Felsenstein 1985) were also determined in PAUP. RESULTS Conidial and cultural morphology Botryosphaeria sp. (Pr2, Pr3, Pr4 and Pr5) Conidia were hyaline, thick-walled and aseptate with granular inner contents ; turning dark-brown and 1-septate when discharged from pycnidia ; oval to ellipsoidal or ovoid (Figs 1–3), and on average 27.8r14.4 mm (n=106), l/w 1.93. The conidia were different from those of other isolates used in this study, Seed-borne Botryosphaeria spp. from native Prunus and Podocarpus trees in Ethiopia 1 2 3 4 5 6 7 8 1008 Figs 1–3. Micrographs of Diplodia rosulata. Fig. 1. Conidiogenous cells and aseptate conidia. Fig. 2. Aseptate conidia. Fig. 3. 1-septate conidia. Figs 4–5. Micrographs of Botryosphaeria sp. Po20 (Dothiorella anamorph). Fig. 4. Conidiogenous cells and conidia. Fig. 5. Conidia. Figs 6–8. Micrographs of Botryosphaeria sp. Po16 (Diplodia anamorph). Fig. 6. Conidiogenous cells and conidia. Fig. 7. Conidia. Fig. 8. Inflated hyphal cells in the culture. Bars=10 mm. as well as other Diplodia sp. previously described from other Prunus spp. (Table 2, key). Growth characteristics exhibited by these isolates were different from those of other isolates from P. falcatus included in the study. Cultures formed a number of lobed margins giving a distinct rosette appearance to the culture (Fig. 9). The average growth rate was 8 and 9 mm dx1at temperatures of 20 x and 25 x, respectively. A full description is given below (pp. 1010–1012). Botryosphaeria sp. (Dothiorella anamorph) (Po20) Pycnidia were superficial on needles but partly submerged in agar, black, globose, covered with greyish hairy tufts on pine needles. Conidiogenous cells produced from the cells forming the inner lining of pycnidia, proliferating percurrently and forming 1–2 indistinct annelations, 7–11r2–3 mm in size (Fig. 4), holoblastic. Conidia produced after 15 d at A. Gure, B. Slippers and J. Stenlid 1009 Table 2. Conidial characteristics of Diplodia and Dothiorella species reported from Prunus spp. Conidial features Diplodia rosulata Do. sarmentoruma Size (mm) (21–)25–32(–36) (22.5–)23–27 r(10–)11–17.5 r(10–)11.5–13 (–19.5) Shape Ovoid to elliptical Ovoid, apex or elliptical rounded, base truncate Wall thickness Thick-walled Thick-walled (1.5–)1.5–2.5 Colour and 1-septate 1-septate septation Host P. africana Prunus spp. and various other hosts Ref. This study Phillips et al. (2004) a b c D. persicinab D. cerasorum D. amygdali Diplodia sp. (B. obtuse)c 16–20r7–9 35–38r15–17 20–22r8 22–26r10–12 Ovoid to oblong, Elliptical, obtuse, Cylindric, truncate at pedicellate not constricted the base, broadly round at apex n/a n/a Finely roughened wall 1-septate, sometimes 1-septate 1-septate 1-septate gently constricted Prunus persica Prunus avium Prunus dulcis, Prunus persica and P. armeniaca various other hosts Grove (1919) Fuckel (1869) Cooke & Shoemaker (1964) Harkness (1884) Oblong, straight or faintly curved, obtuse at the end not thick-walled Synonyms: Diplodia sarmentorum, D. syringae, D. melaena, D. rosae, D. malorum and D. pruni. Synonym: Phoma persicina (Syn. D. persicae). Synonym: Diplodia griffonii. 9 10 11 12 Figs 9–12. Upper surface view of cultures in 9 cm diam Petri dishes. Fig. 9. Diplodia rosulata. Fig. 10. Botryosphaeria sp. Po20. Fig. 11. Botryosphaeria sp. Po16. Fig. 12. Botryosphaeria parva. 25 x, conidial size (20–)21–25.5(–26)r(9–)9.5–11.5 (–12) mm av. 23.5r11¡0.5 mm (n=50), aseptate, guttulate, light brown initially, turning dark-brown and 1-septate with age, oblong to almost rectangular, wall smooth, av. 1 mm thick (n=20) (Fig. 5). Mycelium suppressed but slightly raised peripheries with some vertically growing aerial hyphae, brown to black (S40C 00Y30) in the upper surface (Fig. 10) ; the lower surface bluish-black (S70M 00C30). Average growth rate 12 and 14 mm dx1 at 20 x and 25 x, respectively. Botryosphaeria sp. (Diplodia anamorph) (Po16) Pycnidia pyriform with rounded base and ostiolated neck ; formed on the surface of the pine needles. Conidiogenous cells produced from the cells forming Seed-borne Botryosphaeria spp. from native Prunus and Podocarpus trees in Ethiopia the inner lining of pycnidia, proliferating percurrently forming 1–2 indistinct annelations, 9–19r3–4 mm (Fig. 6). Conidia cylindrical with truncated base and rounded apex (Fig. 7), (25–) 25.5–30 (–30.5)r(10–) 11–13 (–14.5) mm, av. 28¡1.56r11¡1 mm (n=50), l/w=2.5, wall smooth on the outside, av. 1 mm (n=50), hyaline and aseptate when young but becoming brown when mature. Colony turning grey to black from the centre outwards, forming darker sectors at 20 x, colony margin lobed and undulating. Upper surface of the colony grey (S50M 00C10) (Fig. 11), reverse side bluishblack (S80C10Y 00) with a greyish tint after 6 d. Cultures sporulated on pine needles after 2 months. Light brown inflated hyphal cells resembling chlamydospores produced in culture 12–19r11–16 mm in size (Fig. 8). Average growth rate 10 and 12 mm dx1 at 20 x and 25 x, respectively. 1010 126 variable parsimony-uninformative, and 157 parsimony-informative sites. After heuristic searches in PAUP, 4 equally parsimonious trees were retained. These data contained low levels of homoplasy and strong phylogenetic signal (CI=0.774 ; RI=0.901 ; HI=0.256 and gl=x3380590) (Fig. 13). The dataset was resolved into four major groups corresponding to Fusicoccum, Diplodia, Lasiodiplodia and Dothiorella anamorphs. The Fusicoccum-like group comprised four clades containing isolates of B. parva/B. ribis, B. lutea, B. eucalyptorum and B. dothidea. One isolate (AY206460) from P. falcatus clustered within the strongly supported clade of B. parva and B. ribis (Fig. 13). This sequence was identical in all informative sites to sequences of isolates from Eucalyptus spp. originating from Ethiopia (AY210474–AY210479, AY210486 and AY210487) (Gezahgne et al. 2003). The Diplodia-like group comprised of four clades corresponding to B. obtusa, B. stevensii, B. corticola and the isolates that we describe below as a new Diplodia sp. from P. africana. The terminal clade comprising the isolates of the new Diplodia sp. from P. africana (AY210324, AY210344–AY210346) was most closely related to that of B. tsugae and B. corticola, but was clearly separated from them and has a 100% bootstrap support (Fig. 13). One isolate (AY208152) from P. falcatus grouped with other Botryosphaeria isolates with Dothiorella anamorphs. Botryosphaeria parva (Po66) Colony colour turned from white through greenish white to blackish brown on the upper surface (S80Y20M10) after 10 d, fluffy with slightly raised centre with aggregating vertical hyphae (Fig. 12). Reverse side became faint yellowish in colour, which persisted for some time and finally turned bluish black. The culture did not sporulate in 2 months on WA plates at 25 x, but botryose, black sterile stromatal structures formed on pine needles. Average growth rate was 13 and 17 mm dx1 at 20 x and 25 x, respectively. TAXONOMY Phylogenetic analysis Diplodia rosulata Gure, Slippers & Stenlid, sp. nov. (Figs 1–3 and 9) The ITS dataset of the 40 sequences were 560 characters long after alignment, with 277 constant, Etym.: rosulata (Latin), refers to rosulate appearance of the culture. Key to Botryosphaeria spp. with Diplodia and Dothiorella anamorphs reported from Prunus spp. The species below were identified using a host-fungus distributions search on the database of the Systematic Botany and Mycology Laboratory, USDA-ARS Beltsville, M (http://nt.ars-grin.gov/fungaldatabases/) (Table 2). Teleomorph names are used where known, and the current status of synonymies is considered (Table 2). Botryosphaeria tsugae (Funk 1964) and B. corticola (Alves et al. 2004) have not been reported from Prunus spp., but are included as they are closely related to D. rosulata described here. Dothiorella is used here as described by Phillips et al. (2005). 1 Conidia turning brown and sometimes septate before discharge from the pycnidium, av. conidial size 23–27r11–13 mm . . . . . . . . . . . B. sarmentorum (Do. sarmentorum) Conidia turning brown and sometimes septate only after discharge from the pycnidium . . . . . 2 2(1) Av. conidial length <20 mm (16–20r7–9 mm) Av. conidial length >20 mm . . . . . . . . . 3(2) Conidia sometimes >40 mm in length and/or >20 mm broad (av. 36–41r18–22 mm) Conidia never >40 mm in length and/or >20 mm broad . . . . . . . B. tsugae (Diplodia sp.) . . . . 4 4(3) Conidia on av. >30 mm in length Conidia on av. <30 mm in length . . . . . . . . . . . . 5(4) Av. conidial length <27 mm, conidial wall not unusually thick and glassy Av. conidial length >27 mm, conidial wall very thick and glassy . . . . . . . . . . . . 6(5) Av. conidial size 20–22r8 mm . Av. conidial size 22–26r10–12 mm . . . . . . . . . . . . D. amygdali . B. obtusa (D. griffoni) 7(5) Av. conidial size 28r14 mm, l/w ¡2, culture distinctly rosulate. . Av. conidial size 30r14 mm, l/w >2, culture with uniform margin . . . . . . . . . . .D. rosulata B. corticola (D. corticola) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. persicina . . 3 D. cerasorum . . 5 . . . . 6 7 A. Gure, B. Slippers and J. Stenlid 1011 AY210474 AY210475 AY210476 AY210477 1 28 100 B. parva AY210479 1 AY210486 1 AY210487 AY206460 AF241177 2 2 80 B. ribis AY236935 AY236936 21 AY210478 AY236940 79 10 100 25 88 12 100 41 AF027745 B. lutea AY339258 AY236921 B. eucalyptorum AY236920 1 AY236949 1 100 8 97 B. parva AY236943 16 B. dothidea AF027749 88 AY573206 B. sarmentorum 1 AY573212 27 100 2 42 65 22 AY573213 B. iberica AY573202 AY208152 Dothiorella sp. AY210324 5 100 7 68 4 1 8 99 17 26 2 25 100 4 3 19 91 1 AY259089 24 100 9 84 Diplodia rosulata AY210345 AY210346 16 AF243405 1 10 63 AY210344 AY259100 AY236955 AF243406 B. tsugae B. corticola B. stevensii AY259094 B. obtusa AY236953 AY513947 AY236952 Diplodia sp. B. rhodina AF027762 AY236956 Bionectria sp. 5 changes Fig. 13. Phylogram of one of four equally parsimonious trees obtained from ITS1, 5.8S and ITS2 rDNA sequence data. The numbers above the nodes indicate branch length and those below the nodes are bootstrap values (1000 replicates). The tree is rooted with a Bionectria sp. as an outgroup. Fusicoccum Dothiorella Diplodia Lasiodiplodia Cellulae conidiogenae holoblasticae, hyalinae, 1–11r 3–5 mm, cylindricae, percurrenter proliferentes, indistincte annellatae. Conidia ovoidea vel ellipsoidea, (21–)25–32 (–36)r(10–)11–17.5(–19.5) mm, (av. 28r14.5 mm (n=106), long./lat.=1.93), utrinque obtusa, primum hyalina, continua, granulis repleta, pariete 1.5–2 mm crasso, levi, liberata saepe dilute brunnea et 1-septata. Typus: Ethiopia : Southeastern Oromia : MunessaShashamane Forest Enterprise, Gambo, from seeds of Prunus africana, 20 July 2001, A. Gure (CBS H-12357 – holotypus ; CBS 116470=Pr3 ex-type culture) ; CBS H-12358, CBS 116471=Pr4 ; CBS H-12359, CBS 116472=Pr5; CBS H13289=Pr2 ; GenBank sequence ex-type AY210344. Conidiomata (formed on WA on sterilised pine needles and seeds after 45 d), pycnidial, erumpent, solitary, globose with a central ostiole, papillate ; wall composed of outer layers of thick-walled, dark brown textura angularis, becoming thin-walled and hyaline towards the inner layers. Conidiogenous cells holoblastic, hyaline, 8–12r2–4 mm, cylindrical, proliferating Seed-borne Botryosphaeria spp. from native Prunus and Podocarpus trees in Ethiopia percurrently with indistinct annelations. Conidia oval to ellipsoid or ovoid, (21–) 25–32 (–36)r(10–)11–17.5 (–19.5) mm (av. 28r14.5 mm (n=106), l/w=1.93), ends obtuse, initially hyaline, aseptate, granular contents, wall 1.5–2 mm thick and smooth, often turning light brown and 1-septate after discharge. Cultural characteristics : Colony colour initially beige to whitish (upper surface), becoming greenish grey from above (S70Y20M 00), bluish-grey (S70M20C40) with whitish centre from below, cultures partially translucent after 2 wk, becoming opaque after 3 wk. Colony margin forming a concentric ring in 3–4 d with smooth margins, followed by additional rings forming as small sectors along the circumference of the colony, creating a lobed rosette appearance after 4–5 d. Mycelium dense, forming an appressed mat, av. growth rate approximately 7 and 8.5 mm filling the 90 mm plates within 12 and 10 d at 20 x and 25 x, respectively. DISCUSSION In this study, we identify four Botryosphaeria spp. among seven isolates from P. africana and P. falcatus seeds in Ethiopia. Only one of the species, B. parva has been described and named previously. A second species was judged to be distinct from all other Botryosphaeria anamorphs based on morphological and ITS sequence data, as well as host and geographical association, and is described as Diplodia rosulata. The other two taxa were also recognised based on phylogenetic comparisons and morphology, but were represented by only one isolate each and could only be identified as a Dothiorella sp. and a Diplodia sp. A search based on reported host-fungus distributions (http://nt.ars-grin.gov/fungaldatabases/), did not reveal any Diplodia species described from the two African Prunus species, namely P. africana (syn. Pygeum africanum) and P. crassifolia. Botryosphaeria spp. with Diplodia or Dothiorella anamorphs described from other Prunus spp. include Dothiorella sarmentorum, Diplodia persicina, D. amygdali, B. obtusa (reported as D. griffoni) and D. cerasorum. From a survey of literature on these Diplodia spp., we could not find descriptions that match the morphological features of D. rosulata (see Key and Table 2). The ITS rDNA sequence data showed that D. rosulata is a Botryosphaeria sp., although the teleomorph was not seen. The closest relatives according to these data are B. corticola and B. tsugae. These three species are separated from each other based on the ITS sequence data. They are also separated on the size of their respective conidia (see key). This emphasises the value of using anamorph morphology to separate Botryosphaeria spp. (Slippers et al. 2004a, c, Pavlic et al. 2004). The host range and geographic distribution of D. rosulata does not seem to overlap with those of B. corticola and B. tsugae. B. corticola was reported from 1012 Quercus species with a geographical distribution including Iberian Peninsula and Italy (Alves et al. 2004). B. tsugae was recorded from Canada, from western hemlock (Tsuga heterophylla ; Funk 1964). These apparent narrow host and geographic ranges are different from other Botryosphaeria spp., e.g. B. parva, B. obtusa, B. rhodina, that are known to have wide host and geographic ranges (Punithalingam & Waller 1973, Pennycook & Samuels 1985, Denman et al. 2003, Slippers et al. 2004a). Diplodia rosulata has a distinct rosulate morphology in culture, which separates it from all other Botryosphaeria spp. considered here. It also separated D. rosulata from the related B. corticola and B. tsugae. Culture morphology has been employed before as a useful morphological character, e.g. D. pinea and D. scrobiculata, B. lutea, B. australis (de Wet et al. 2000, Slippers et al. 2004a, b). This easily determined character should not be overlooked as a useful criterion for distinguishing some Botryosphaeria spp., especially at the local host and geographical scale. In this study, the four identified Botryosphaeria spp. were isolated from apparently healthy seeds of Podocarpus falcatus and Prunus africana. This highlights that at least some members of this genus might be seed-borne and could be spread in this way. The possibility of being transmitted to new areas through the transfer of planting material or seed has been implicated in previous studies for some species, e.g. B. proteae and B. protearum (Denman et al. 2003). Burgess & Wingfield (2002) and Burgess, Wingfield & Wingfield (2004) concluded that high genotypic diversity of the pine endophyte and latent pathogen D. pinea in some Southern Hemisphere areas equates with multiple introductions through seeds and cuttings from many different sources. Considering the potential impact of introduced pathogens, effective quarantine regulations are essential in order to protect valuable resources such as forests (Burgess & Wingfield 2002). One isolate from Podocarpus falcatus was identified as B. parva. B. parva and B. ribis are difficult to separate on ITS sequence data alone (Slippers et al. 2004a). Gezahgne et al. (2004) identified the isolates of B. parva based on combined ITS and translation elongation factor sequences. The identity of our ITS sequence with theirs gives us confidence that we are dealing with the same species. Gezahgne et al. (2004) showed that B. parva is the major cause of Botryosphaeria stem-canker in Eucalyptus plantations in Ethiopia. B. parva is also an important die-back and stem-canker pathogen of Eucalyptus spp. in South Africa, Republic of Congo and Uganda (Smith, Kemp & Wingfield 1994, Roux et al. 2001, Slippers et al. 2004a). The B. parva isolate considered in this study was isolated from seeds of P. falcatus in native afromontane forests adjoining Eucalyptus plantations in Ethiopia. This raises the question whether B. parva is an introduced pathogen or an indigenous fungus infecting A. Gure, B. Slippers and J. Stenlid introduced tree species in Ethiopia. The impact of B. parva as pathogen on native species has not yet been assessed properly. In vitro pathogenicity tests on seeds of P. falcatus have not shown any adverse effect on seed germination (Gure et al. 2005). However, Straus (2001) suggested that native forest stands adjacent to Eucalyptus plantations might be adversely affected by increased inoculum pressure of pathogens. Future studies will focus on comparing populations of pathogens like B. parva from native and introduced hosts. Two isolates could not be identified to species level and possibly represent two more species that have not been previously described. The single isolate for each species is inadequate to characterise possible variations and compare with other species based on morphology, and we therefore did not provide names for them. The initial morphological and ITS sequence data, however, indicate that these Botryosphaeria spp. have anamorphs in Diplodia and Dothiorella, respectively. In the ITS phylogenetic tree, Botryosphaeria sp. Po16 resided in the Diplodia clade but was distinct from others in the group. Conidia of Botryosphaeria sp. Po16 are similar to those of the anamorph of B. obtusa in that they are, aseptate, cylindrical with broadly rounded apex and truncate base, hyaline but becoming brown at maturity. However, conidia of Botryosphaeria sp. Po16 are larger (see key). Botryosphaeria sp. Po20 was most closely related to the recently described B. iberica and B. sarmentorum. These Botryosphaeria spp. are peculiar as they have dark, septate ascospores. Botryosphaeria sp. Po20, B. iberica and B. sarmentorum grouped in between the major Botryosphaeria groups that have either Fusicoccum or Diplodia anamorphs (Denman et al. 2000, Zhou & Stanosz 2001, Slippers et al. 2004a). Phillips et al. (2005) re-described the anamorph genus Dothiorella, in order to accommodate anamorphs of these Botryosphaeria spp. The conidial morphology of the isolate Po20 resembles the Dothiorella spp. described by Phillips et al. (2005). Dothiorella spp. resemble Diplodia spp. most closely in conidial morphology, but unlike Diplodia the conidia might be septate and discolour before they are discharged from pycnidia. This study indicates the need to study Botryosphaeria spp. on native forest trees in Africa. They are important pathogens affecting a dwindling resource, and yet are poorly studied. Furthermore, the study highlights the fact that potential pathogens can overlap between commercial trees and native trees. The occurrence of such pathogens on seeds is of special concern in terms of quarantine. 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