Seed-borne Botryosphaeria spp. from native Prunus and Podocarpus trees in Ethiopia, with a description of the anamorph Diplodia rosulata sp. nov

Seed-borne Botryosphaeria spp. from native Prunus andPodocarpus trees in Ethiopia, with a description of the

anamorph Diplodia rosulata sp. nov.

Abdella GURE1,2, Bernard SLIPPERS1 and Jan STENLID1

1Department of Forest Mycology & Pathology, Swedish University of Agricultural Sciences (SLU), Box 7026 SE-750 07Uppsala, Sweden.2Wondo Genet College of Forestry, Debub University, P.O. Box 128, Shashamane, Ethiopia.

E-mail : [email protected]

Received 30 November 2004; accepted 27 April 2005.

Botryosphaeria spp. from seeds of the native afromontane forest tree species, Podocarpus falcatus and Prunus africana, inEthiopia have been identified. This is achieved by combining anamorph morphological characters and ITS sequencedata. 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 onlyone 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 importantpathogen of various commercially important tree species in Ethiopia and elsewhere. This study highlights the ability ofBotryosphaeria spp. to infect seeds and the possibility that they might be distributed in this way. The study alsocontributes 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 worldwidedistribution. Members of this genus and its anamorphsoccur on a wide range of monocotyledonous, dicoty-ledonous and gymnosperm hosts (Arx & Muller 1975).These fungi have also been associated with variousdisease symptoms such as shoot blight, die-back, stemcanker, 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 theirassociated anamorphs is often confusing (Jacobs &Rehner1998,Zhou&Stanosz2001,Slippersetal.2004a).Reasons for this are that the teleomorphs are rarelyencountered in nature, or are difficult to induce in cul-ture, and that the morphological diversity of the teleo-morphs is limited (Shoemaker 1964, Laundon 1973,Jacobs & Rehner 1998). The taxonomy of Botryo-sphaeria spp. is, therefore, often based on the charac-teristics of its anamorphs, which are more commonlyencountered (Shoemaker 1964, Pennycook & Samuels1985, Phillips et al. 2002, Denman et al. 2003, Slipperset al. 2004a).

Morphological characters of Botryosphaeria ana-morphs considered useful for taxonomic delimitationinclude conidial characteristics such as the size, shape,colour, septation, wall thickness and wall texture, thepresence 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 rateshave been used to classify these fungi (Pennycook &Samuels 1985, de Wet et al. 2000, Phillips et al. 2002,Slippers et al. 2004b). Despite Botryosphaeria ana-morphs having a number of taxonomically usefulcharacters, their identification can also be confusingbecause 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 se-quence has proved useful in clarifying the taxonomyand phylogenetic relationships of Botryosphaeriaspecies (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 sequencedata have been used to separate anamorphs ofBotryosphaeria in which the morphological featureswere confusing, for example B. ribis from Fusicoccumluteum (Smith & Stanosz 2001), and B. stevensii from

Mycol. Res. 109 (9): 1005–1014 (September 2005). f The British Mycological Society 1005

doi:10.1017/S0953756205003266 Printed in the United Kingdom.

B. corticola (Alves et al. 2004). However, the use ofadditional data from other nuclear genes has in somecases revealed cryptic species that went unnoticed whenusing ITS data alone (de Wet et al. 2003, Slippers et al.2004a).

Despite recent advances in the taxonomy ofBotryosphaeria anamorphs, the taxonomy of groupslike Diplodia is still very confused. More than 1200Diplodia 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 havebeen examined closely, many have had to be syn-onymised, while others have been separated into differ-ent species (de Wet et al. 2003, Alves et al. 2004, Phillipset al. 2005). One reason for this is thatmany species weredescribed based on host association or geographic dis-tribution. Today it is known that some Botryosphaeriaspp. have wide host and geographic ranges, for exampleB. obtusa and B. parva (Punithalingam & Waller 1973,Pennycook & Samuels 1985, Slippers et al. 2004a). Onthe other hand, some species appear to have narrowerhost ranges, for example D. pinea and D. scrobiculataassociated with Pinus spp. (de Wet et al. 2000, de Wetet al. 2003). There is thus a need to closely examinemore Diplodia spp. based on recent advances in thetaxonomy of this group.

Podocarpus falcatus and Prunus africana are import-ant multipurpose afromontane forest tree specieswidely distributed across Africa. Podocarpus falcatus isused for timber, shade, medicinal, and conservationpurposes (Negash 1995, 2003). Prunus africana is usedas 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 in-come in some countries in Africa, primarily for themedicinal value of its bark (Cunningham et al. 2002).At present, in Ethiopia, the natural populations ofthese species are threatened by excessive logging andillegal tree felling that has led to a widespread deforest-ation. As a result, there is a growing need to propagatethese important native tree species, mainly by seed.

Fungi belonging to many different taxa were isolatedduring a previous survey of fungi associated with seedsof these two trees (Gure 2004). In this study, we identifythe seed-borne Botryosphaeria spp. collected duringthat earlier survey by combining anamorph mor-phology and ITS rDNA sequence data. Apart fromidentifying Botryosphaeria spp. we also describe a newDiplodia sp. from seeds of P. africana.

MATERIALS AND METHODS

Fungal isolates

Seeds of Podocarpus falcatus and Prunus africana werecollected from Badale and Gambo in Ethiopia. Seedsof both species were surface-sterilised by soaking inhydrogen peroxide (33% v/v) for 1 min or sodium

hypochlorite (13% v/v) for 5 min followed by athorough rinsing in sterilised water. Surface sterilisedseeds were blotted on sterile filter paper before platingon 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 attemperatures of 20¡2 xC or 25 x in darkness for5–10 d. Fungi growing out of the seeds were isolated inpure culture. Seven isolates resembling Botryosphaeriain culture (fast growing, dark, greenish brown or grey-ish pigmented cultures) were further considered in thisstudy (Table 1). The cultures were maintained on MEAslants at 5 x during our studies.

Morphological identification

Morphological identification of the isolates was basedon conidial morphology from cultures grown on 2%WA. In order to induce sporulation, pine needles weresterilised by autoclaving and then incorporated intoWA plates (Slippers et al. 2004a). Furthermore, for theisolates from Prunus africana, seeds were sterilised inhydrogen peroxide (33% v/v) overnight, washed andadded to separate 2% WA plates for sporulation.Duplicate sets of the plates were incubated at 25 x indarkness with alternating cycles of 12 h of near-UV

light and darkness. Squashed mounts of pycnidiawere prepared in lactophenol. Morphological obser-vations and measurements of conidial dimensionswere made under a light microscope, with an Axiocamdigital camera and accompanying software (ZeissAxiovision 4.1).

Cultural studies

All isolates from Prunus africana and Podocarpusfalcatus were used for cultural studies on MEA.Mycelial discs of 5 mm diam were cut from the per-ipheries of actively growing cultures and inoculated tothe centre of Petri dish plates (90 mm diam) containingMEA, with three replicates for each isolate, at tem-peratures of 20–25 x. Colony diameter was measureddaily ; colony morphology and colour using Kupper(1999) colour chart were noted every 2 d for a totalof 15 d.

Genomic DNA extraction, sequencing andphylogenetic analysis

Pure cultures (about 10 d old) grown on Hagem agarplates (Stenlid 1985) were used for DNA extraction.Nucleic acids were extracted using a CTAB andphenol-chloroform DNA extraction method (Gardes &Bruns 1993) modified by omitting b-mercapto ethanol.Amplification of the ITS region of the rDNA was per-formed by PCR using universal primers ITS1 and ITS4(White et al. 1990) as described in Ihrmark (2001). PCRproducts were purified using QIAquick PCR purifi-cation kit (Qiagen, Hilden). Purified ITS PCR products

Seed-borne Botryosphaeria spp. from native Prunus and Podocarpus trees in Ethiopia 1006

were then sequenced on an Applied Biosystems 310automated DNA sequencer, using ABI PRISMTM

BigDyeTM 10r Terminator Cycle Sequencing ReadyReaction Kit v.2.0 (Applied Biosystems, Foster City,CA). Sequence analysis was done on the ABI PRISMTM

Genetic Analyser. DNA sequences were assembledusing the program SeqmanII from the DNASTARsoftware package (GATC, Konstaz). Seven sequencesfrom our study, 39 similar sequences from NCBI,identified using BLAST searches, and 4 sequencesprovided by Phillips et al. (2005) were manually alignedby inserting gaps. Phylogenetic analyses were per-formed using PAUP version 4.0b10 (Swofford 2002).In PAUP, gaps were specified as missing data andall characters as unordered and of equal weight.Maximum parsimony trees were obtained using heu-ristic searches through stepwise (random) addition andtree bisection and reconstruction (TBR) as branch

swapping algorithm. Maximum trees were unlimitedand, branches of zero length were collapsed. Retentionand consistency indices, a g1-value and branch sup-ports 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 withgranular inner contents ; turning dark-brown and1-septate when discharged from pycnidia; oval toellipsoidal or ovoid (Figs 1–3), and on average27.8r14.4 mm (n=106), l/w 1.93. The conidia weredifferent from those of other isolates used in this study,

Table 1. Identity and origin of sequences used in the rDNA ITS phylogenetic analysis.

Culture no. Identity Host Location Collector GenBank

CBS 112545 Botryosphaeria

corticola

Quercus suber Cadiz, Spain M. E. Sanchez,

A. Trapero

AY259089

CBS 112549 B. corticola Q. suber Aveiro, Portugal A. Alves AY259100

CBS 115041 B. iberica Q. ilex Aragon, Spain J. Luque AY573202

CBS 115035 B. iberica Q. ilex Aragon, Spain N. Ibarra, AY573213

IMI 63581 B. sarmentorum Ulmus sp. Warwickshire, England E. A. Ellis AY573212

CBS 115038 B. sarmentorum Malus pumila Delft, Netherlands A. J. L. Phillips AY573206

CMW 8000 B. dothidea Prunus sp. Crossifisso, Switzerland B. Slippers AY236949

KJ94.26 B. dothidea Prunus persica Japan P. L. Pusey AF027749

CMW 10125 B. eucalyptorum Eucalyptus grandis Mpumalanga, S. Africa H. Smith AF283686

CMW 10126 B. eucalyptorum E. grandis Mpumalanga, S. Africa H. Smith AF283687

CMW 992/3 B. lutea Actinidia deliciosa New Zealand G. J. Samuels AF027745

CMW 9076 B. lutea Vitis vinifera Portugal A. J. L. Phillips AY339258

CBS 112555 B. obtusa Vitis vinifera Montemor-o-Novo,

Portugal

A. J. L. Phillips AY259094

CMW 7774 B. obtusa Ribes sp. New York, USA B. Slippers/G. Hudler AY236953

CMW 11060 B. parva E. citriodora Ethiopia A. Gezahgne & J. Roux AY210474



CMW 10089 B. parva E. globules Ethiopia A. Gezahgne & J. Roux AY210477

CMW 10095 B. parva E. grandis Ethiopia A. Gezahgne & J. Roux AY210478

CMW 10094 B. parva E. saligna Ethiopia A. Gezahgne & J. Roux AY210479

CMW 11246 B. parva Pinus patula Ethiopia A. Gezahgne & J. Roux AY210486

CMW 11253 B. parva P. patula Ethiopia A. Gezahgne & J. Roux AY210487

CMW 9078 B. parva A. deliciosa New Zealand S. R. Pennycook AY236940

CMW 9081 B. parva Populus nigra New Zealand G. J. Samuels AY236943

Po66 B. parva Podocarpus falcatus Gambo, Ethiopia A. Gure AY206460

CMW 7054 B. ribis Ribes rubrum New York N. E. Stevens AF241177

CMW 7772 B. ribis Ribes sp. New York, USA B. Slippers & G. Hudler AY236935

CMW 7773 B. ribis Ribes sp. New York, USA B. Slippers/G. Hudler AY236936

CMW 9074 B. rhodina Pinus sp. Mexico T. Burgess AY236952

KJ93.41 B. rhodina Pistachia California, USA T. J. Michailides AF027762

CBS 431 B. stevensii Fraxinus excelsior Netherlands H. A. van der Aa AY236955

ATCC 60259 B. stevensii M. pumila unknown H. J. Boesewinkel AF243406

ZS96-174 B. tsugae Tsuga heterophylla Canada A. Funk AF243405

Pr2 Diplodia rosulata Prunus africana Gambo, Ethiopia A. Gure AY210324

CBS 116470 (Pr3) D. rosulata P. africana Gambo, Ethiopia A. Gure AY210344



Po16 Diplodia sp. Podocarpus falcatus Badale, Ethiopia A. Gure AY513947

Po20 Dothiorella sp. P. falcatus Badale, Ethiopia A. Gure AY208152

CMW 7063 Bionectria sp. Unknown Netherlands Unknown AY236956

A. Gure, B. Slippers and J. Stenlid 1007

as well as other Diplodia sp. previously described fromother Prunus spp. (Table 2, key). Growth charac-teristics exhibited by these isolates were differentfrom those of other isolates from P. falcatus includedin the study. Cultures formed a number of lobedmargins giving a distinct rosette appearance to theculture (Fig. 9). The average growth rate was 8 and9 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 sub-merged in agar, black, globose, covered with greyishhairy tufts on pine needles. Conidiogenous cellsproduced from the cells forming the inner lining ofpycnidia, proliferating percurrently and forming1–2 indistinct annelations, 7–11r2–3 mm in size(Fig. 4), holoblastic. Conidia produced after 15 d at

2

6

7 8

3

1

5

4

2

6

7 8

3

1

5

4

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 cellsand 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.


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-brownand 1-septate with age, oblong to almost rectangular,wall smooth, av. 1 mm thick (n=20) (Fig. 5). Myceliumsuppressed but slightly raised peripheries with somevertically growing aerial hyphae, brown to black(S40C00Y30) in the upper surface (Fig. 10) ; the lower

surface bluish-black (S70M00C30). Average growth rate12 and 14 mm dx1 at 20 x and 25 x, respectively.

Botryosphaeria sp. (Diplodia anamorph) (Po16)

Pycnidia pyriform with rounded base and ostiolatedneck; formed on the surface of the pine needles.Conidiogenous cells produced from the cells forming

9 10

11 12

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.

Table 2. Conidial characteristics of Diplodia and Dothiorella species reported from Prunus spp.

Conidial

features

Diplodia

rosulata

Do.

sarmentoruma D. persicinab D. cerasorum D. amygdali

Diplodia sp.

(B. obtuse)c

Size (mm) (21–)25–32(–36)

r(10–)11–17.5

(–19.5)

(22.5–)23–27

r(10–)11.5–13

16–20r7–9 35–38r15–17 20–22r8 22–26r10–12

Shape Ovoid to elliptical

or elliptical

Ovoid, apex

rounded, base

truncate

Oblong, straight or

faintly curved,

obtuse at the end

Ovoid to oblong,

pedicellate

Elliptical, obtuse,

not constricted

Cylindric, truncate at

the base, broadly

round at apex

Wall thickness Thick-walled

(1.5–)1.5–2.5

Thick-walled not thick-walled n/a n/a Finely roughened

wall

Colour and

septation

1-septate 1-septate 1-septate, sometimes

gently constricted

1-septate 1-septate 1-septate

Host P. africana Prunus spp. and

various other hosts

Prunus persica Prunus avium Prunus dulcis,

P. armeniaca

Prunus persica and

various other hosts

Ref. This study Phillips et al. (2004) Grove (1919) Fuckel (1869) Cooke &

Harkness (1884)

Shoemaker (1964)

a Synonyms: Diplodia sarmentorum, D. syringae, D. melaena, D. rosae, D. malorum and D. pruni.b Synonym: Phoma persicina (Syn. D. persicae).c Synonym: Diplodia griffonii.


the inner lining of pycnidia, proliferating percurrentlyforming 1–2 indistinct annelations, 9–19r3–4 mm(Fig. 6). Conidia cylindrical with truncated base androunded 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 brownwhen mature. Colony turning grey to black from thecentre outwards, forming darker sectors at 20 x, colonymargin lobed and undulating. Upper surface of thecolony grey (S50M00C10) (Fig. 11), reverse side bluish-black (S80C10Y00) with a greyish tint after 6 d. Culturessporulated on pine needles after 2 months. Light browninflated hyphal cells resembling chlamydospores pro-duced in culture 12–19r11–16 mm in size (Fig. 8).Average growth rate 10 and 12 mm dx1 at 20 x and25 x, respectively.

Botryosphaeria parva (Po66)

Colony colour turned from white through greenishwhite to blackish brown on the upper surface(S80Y20M10) after 10 d, fluffy with slightly raised centrewith aggregating vertical hyphae (Fig. 12). Reverse sidebecame faint yellowish in colour, which persisted forsome time and finally turned bluish black. The culturedid not sporulate in 2 months on WA plates at 25 x, butbotryose, black sterile stromatal structures formed onpine needles. Average growth rate was 13 and17 mm dx1 at 20 x and 25 x, respectively.

Phylogenetic analysis

The ITS dataset of the 40 sequences were 560characters long after alignment, with 277 constant,

126 variable parsimony-uninformative, and 157parsimony-informative sites. After heuristic searches inPAUP, 4 equally parsimonious trees were retained.These data contained low levels of homoplasy andstrong phylogenetic signal (CI=0.774; RI=0.901;HI=0.256 and gl=x3380590) (Fig. 13).

The dataset was resolved into four major groupscorresponding to Fusicoccum, Diplodia, Lasiodiplodiaand Dothiorella anamorphs. The Fusicoccum-likegroup comprised four clades containing isolates of B.parva/B. ribis, B. lutea, B. eucalyptorum and B. dothi-dea. One isolate (AY206460) from P. falcatus clusteredwithin the strongly supported clade of B. parva and B.ribis (Fig. 13). This sequence was identical in all in-formative sites to sequences of isolates from Eucalyptusspp. originating from Ethiopia (AY210474–AY210479,AY210486 and AY210487) (Gezahgne et al. 2003). TheDiplodia-like group comprised of four clades corre-sponding to B. obtusa, B. stevensii, B. corticola and theisolates that we describe below as a new Diplodia sp.from P. africana. The terminal clade comprising theisolates of the new Diplodia sp. from P. africana(AY210324, AY210344–AY210346) was most closelyrelated to that of B. tsugae and B. corticola, but wasclearly separated from them and has a 100% bootstrapsupport (Fig. 13). One isolate (AY208152) from P.falcatus grouped with other Botryosphaeria isolateswith Dothiorella anamorphs.

TAXONOMY

Diplodia rosulata Gure, Slippers & Stenlid, sp.nov. (Figs 1–3 and 9)

Etym. : rosulata (Latin), refers to rosulate appear-ance 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 andMycology Laboratory, USDA-ARS Beltsville, M (http://nt.ars-grin.gov/fungaldatabases/) (Table 2). Teleomorph names areused 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. rosulatadescribed 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 size23–27r11–13 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) . . . . . . . . . . D. persicina

Av. conidial length >20 mm . . . . . . . . . . . . . . . 3

3(2) Conidia sometimes >40 mm in length and/or >20 mm broad (av. 36–41r18–22 mm) . B. tsugae (Diplodia sp.)

Conidia never >40 mm in length and/or >20 mm broad . . . . . . . . . . 4

4(3) Conidia on av. >30 mm in length . . . . . . . . . . . . D. cerasorum

Conidia on av. <30 mm in length . . . . . . . . . . . . . . 5

5(4) Av. conidial length <27 mm, conidial wall not unusually thick and glassy . . . . . . . 6Av. conidial length >27 mm, conidial wall very thick and glassy . . . . . . . . . 7

6(5) Av. conidial size 20–22r8 mm . . . . . . . . . . . . . D. amygdali

Av. conidial size 22–26r10–12 mm . . . . . . . . . . . B. obtusa (D. griffoni)

7(5) Av. conidial size 28r14 mm, l/w ¡2, culture distinctly rosulate. . . . . . . . .D. rosulataAv. conidial size 30r14 mm, l/w >2, culture with uniform margin . . . . B. corticola (D. corticola)


Cellulae conidiogenae holoblasticae, hyalinae, 1–11r3–5 mm, cylindricae, percurrenter proliferentes, indistincteannellatae. 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 : Munessa-

Shashamane 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 H-13289=Pr2; GenBank sequence ex-type AY210344.

Conidiomata (formed on WA on sterilised pine needlesand seeds after 45 d), pycnidial, erumpent, solitary,globose with a central ostiole, papillate ; wall composedof outer layers of thick-walled, dark brown texturaangularis, becoming thin-walled and hyaline towardsthe inner layers. Conidiogenous cells holoblastic,hyaline, 8–12r2–4 mm, cylindrical, proliferating

B. parva

B. ribis

B. parva

B. lutea

B. stevensii

B. dothidea

Dothiorella sp.

B. sarmentorum

B. iberica

Diplodia rosulata

B. tsugae

B. corticola

B. eucalyptorum

B. obtusa

Diplodia sp.

B. rhodina

Bionectria sp.

AY210474

AY210475

AY210476

AY210477

AY210479

AY210486

AY210487

AY206460

AF241177

AY236935

AY236936

AY210478

AY236940

AY236943

AF027745

AY339258

AY236921

AY236920

AY236949

AF027749

AY573206

AY573212

AY573213

AY573202

AY208152

AY210324

AY210344

AY210345

AY210346

AF243405

AY259089

AY259100

AY236955

AF243406

AY259094

AY236953

AY513947

AY236952

AF027762

AY236956

5 changes

100

80

79

100

10088

100

97 88

100

65

100

68

100

84

63

91 99

100

28

2

1

1

1

2

16

25

8

42

22

27

1

2

411

1

12

10

252

1

17

81

3

4

26

19

10

9

1

7

4

24

16

5

21

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


percurrently with indistinct annelations. Conidia ovalto 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), endsobtuse, initially hyaline, aseptate, granular contents,wall 1.5–2 mm thick and smooth, often turning lightbrown and 1-septate after discharge.

Cultural characteristics : Colony colour initially beigeto whitish (upper surface), becoming greenish greyfrom above (S70Y20M00), bluish-grey (S70M20C40) withwhitish centre from below, cultures partially trans-lucent after 2 wk, becoming opaque after 3 wk. Colonymargin forming a concentric ring in 3–4 d with smoothmargins, followed by additional rings forming as smallsectors along the circumference of the colony, creatinga lobed rosette appearance after 4–5 d. Myceliumdense, forming an appressed mat, av. growth rate ap-proximately 7 and 8.5 mm filling the 90 mm plateswithin 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. falcatusseeds in Ethiopia. Only one of the species, B. parva hasbeen described and named previously. A second specieswas judged to be distinct from all other Botryosphaeriaanamorphs based on morphological and ITS sequencedata, as well as host and geographical association, andis described as Diplodia rosulata. The other two taxawere also recognised based on phylogenetic compari-sons and morphology, but were represented by onlyone isolate each and could only be identified as aDothiorella sp. and a Diplodia sp.

A search based on reported host-fungus distributions(http://nt.ars-grin.gov/fungaldatabases/), did not revealany Diplodia species described from the two AfricanPrunus species, namely P. africana (syn. Pygeumafricanum) and P. crassifolia. Botryosphaeria spp. withDiplodia or Dothiorella anamorphs described fromother Prunus spp. include Dothiorella sarmentorum,Diplodia persicina, D. amygdali, B. obtusa (reported asD. griffoni) and D. cerasorum. From a survey of litera-ture on these Diplodia spp., we could not find descrip-tions that match the morphological features of D.rosulata (see Key and Table 2).

The ITS rDNA sequence data showed that D. rosu-lata is a Botryosphaeria sp., although the teleomorphwas not seen. The closest relatives according to thesedata are B. corticola and B. tsugae. These three speciesare separated from each other based on the ITSsequence data. They are also separated on the size oftheir respective conidia (see key). This emphasises thevalue of using anamorph morphology to separateBotryosphaeria spp. (Slippers et al. 2004a, c, Pavlicet 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

Quercus species with a geographical distribution in-cluding Iberian Peninsula and Italy (Alves et al. 2004).B. tsugae was recorded from Canada, from westernhemlock (Tsuga heterophylla ; Funk 1964). Theseapparent narrow host and geographic ranges are dif-ferent from other Botryosphaeria spp., e.g. B. parva,B. obtusa, B. rhodina, that are known to have wide hostand geographic ranges (Punithalingam & Waller 1973,Pennycook & Samuels 1985, Denman et al. 2003,Slippers et al. 2004a).

Diplodia rosulata has a distinct rosulate morphologyin culture, which separates it from all otherBotryosphaeria spp. considered here. It also separatedD. rosulata from the related B. corticola and B. tsugae.Culture morphology has been employed before as auseful 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 determinedcharacter should not be overlooked as a useful criterionfor distinguishing some Botryosphaeria spp., especiallyat the local host and geographical scale.

In this study, the four identified Botryosphaeriaspp. were isolated from apparently healthy seedsof Podocarpus falcatus and Prunus africana. Thishighlights that at least some members of this genusmight be seed-borne and could be spread in this way.The possibility of being transmitted to new areasthrough the transfer of planting material or seed hasbeen 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 highgenotypic diversity of the pine endophyte and latentpathogen D. pinea in some Southern Hemisphere areasequates with multiple introductions through seeds andcuttings from many different sources. Considering thepotential impact of introduced pathogens, effectivequarantine regulations are essential in order to protectvaluable resources such as forests (Burgess & Wingfield2002).

One isolate from Podocarpus falcatus was identifiedas B. parva. B. parva and B. ribis are difficult to separateon ITS sequence data alone (Slippers et al. 2004a).Gezahgne et al. (2004) identified the isolates of B. parvabased on combined ITS and translation elongationfactor sequences. The identity of our ITS sequence withtheirs gives us confidence that we are dealing with thesame species. Gezahgne et al. (2004) showed that B.parva is the major cause of Botryosphaeria stem-cankerin Eucalyptus plantations in Ethiopia. B. parva is alsoan important die-back and stem-canker pathogen ofEucalyptus spp. in South Africa, Republic of Congoand Uganda (Smith, Kemp & Wingfield 1994, Rouxet al. 2001, Slippers et al. 2004a).

The B. parva isolate considered in this study wasisolated from seeds of P. falcatus in native afromontaneforests adjoining Eucalyptus plantations in Ethiopia.This raises the question whether B. parva is anintroduced pathogen or an indigenous fungus infecting


introduced tree species in Ethiopia. The impactof B. parva as pathogen on native species has not yetbeen assessed properly. In vitro pathogenicity tests onseeds of P. falcatus have not shown any adverse effecton seed germination (Gure et al. 2005). However,Straus (2001) suggested that native forest standsadjacent to Eucalyptus plantations might be adverselyaffected by increased inoculum pressure of pathogens.Future studies will focus on comparing populations ofpathogens like B. parva from native and introducedhosts.

Two isolates could not be identified to specieslevel and possibly represent two more species that havenot been previously described. The single isolate foreach species is inadequate to characterise possiblevariations and compare with other species based onmorphology, and we therefore did not provide namesfor them. The initial morphological and ITS sequencedata, however, indicate that these Botryosphaeriaspp. have anamorphs in Diplodia and Dothiorella,respectively.

In the ITS phylogenetic tree, Botryosphaeria sp. Po16resided in the Diplodia clade but was distinct fromothers in the group. Conidia of Botryosphaeria sp. Po16are similar to those of the anamorph of B. obtusa in thatthey are, aseptate, cylindrical with broadly roundedapex and truncate base, hyaline but becoming brown atmaturity. However, conidia of Botryosphaeria sp. Po16are larger (see key).

Botryosphaeria sp. Po20 was most closely related tothe recently described B. iberica and B. sarmentorum.These Botryosphaeria spp. are peculiar as they havedark, septate ascospores. Botryosphaeria sp. Po20, B.iberica and B. sarmentorum grouped in between themajor Botryosphaeria groups that have eitherFusicoccum or Diplodia anamorphs (Denman et al.2000, Zhou & Stanosz 2001, Slippers et al. 2004a).Phillips et al. (2005) re-described the anamorph genusDothiorella, in order to accommodate anamorphs ofthese Botryosphaeria spp. The conidial morphology ofthe isolate Po20 resembles the Dothiorella spp. de-scribed by Phillips et al. (2005). Dothiorella spp. re-semble Diplodia spp. most closely in conidialmorphology, but unlike Diplodia the conidia might beseptate and discolour before they are discharged frompycnidia.

This study indicates the need to study Botryosphaeriaspp. on native forest trees in Africa. They are importantpathogens affecting a dwindling resource, and yet arepoorly studied. Furthermore, the study highlights thefact that potential pathogens can overlap betweencommercial trees and native trees. The occurrence ofsuch pathogens on seeds is of special concern in termsof quarantine. It is hoped that this study will serve asa starting point for future studies on the geographicand host distribution of Botryosphaeria spp., and theirsignificance as pathogens/endophytes on native andexotic plantation species in Ethiopia and elsewhere inAfrica.

ACKNOWLEDGMENTS

We thank Allen Phillips for useful discussion and for providing a pre-

publication manuscript and sequences essential to this study. SIDA,

the Swedish International Development Authority, provided funds

for the study.

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Corresponding Editor: D. T. Mitchell


Seed-borne Botryosphaeria spp. from native Prunus and Podocarpus trees in Ethiopia, with a description of the anamorph Diplodia rosulata sp. nov

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