Doi: 10. 1111/j. 1365-3059. 2007. 01608. x 2007 The Authors



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CMW 14114

c

CBS 118843



L. theobromae

S. cordatum

Kwambonambi, S. Africa

D. Pavlic

DQ316091


CMW 14116

c

L. theobromae

S. cordatum

Kwambonambi, S. Africa

D. Pavlic

DQ316092


CMW 14077

c

CBS 115812



Lasiodiplodia gonubiensis

S. cordatum

Eastern Cape, S. Africa

D. Pavlic

AY639595


CMW 14078

c

CBS 116355



L. gonubiensis

S. cordatum

Eastern Cape, S. Africa

D. Pavlic

AY639594


CMW 7774

Botryosphaeria’ obtusa



Ribes sp.

New York, USA

B. Slippers & G. Hudler

AY236953


KJ 93·56

Botryosphaeria’ obtusa

Hardwood shrub

New York, USA

G.J. Samuels

AF027759


CMW 7060

CBS 431


Diplodia mutila

Fraxinus excelsior

Netherlands

H.A. van der Aa

AY236955


ZS 94-6

D. mutila

Malus pumila

New Zealand

N. Tisserat

AF243407


CBS 112545

Diplodia corticola

Quercus ilex

Spain


M.A. Sanchez & A. Trapero

AY259089


CBS 112551

D. corticola

Quercus suber

Portugal


A. Alves

AY259101


KJ 94·07

Diplodia pinea

Pinus resinosa

Wisconsin, USA

D.R. Smith

AF027758


CMW 3025

Mycosphaerella africana

Eucalyptus viminalis

Stellenbosch, S. Africa

P.W. Crous

AF 283690

CMW 7063

CBS 447·68



Guignardia philoprina

Taxus baccata

Netherlands

H.A. van der Aa

AF312014


a

Culture collections: CMW 

= Tree Pathology Co-operative Programme, Forestry and Agricultural Biotechnology Institute, University of Pretoria; KJ = Jacobs & Rehner (1998); ATCC = American Type Culture 

Collection, Fairfax, VA, USA; BRIP 

= Plant Pathology Herbarium, Department of Primary Industries, Queensland, Australia; CAP = culture collection of A.J.L. Phillips, Lisbon, Portugal; CBS = Centraalbureau voor 

Schimmelcultures, Utrecht, Netherlands; ICMP 

= International Collection of Microorganisms from Plants, Auckland, New Zealand; ZS = Zhou & Stanosz (2001).

b

Isolates sequenced in this study are given in bold.



c

Isolates used in pathogenicity trials.

GenBank 

ITS


Isolate

a,b


Other no.

a

Identity



Host

Location


Isolator

Table 1 Continued



Plant Pathology (2007) 56, 624–636

628


D. Pavlic et al.

PPA_1608

have been maintained in the Culture Collection (CMW)

of the Forestry and Agricultural Biotechnology Institute

(FABI), University of Pretoria, South Africa, and repre-

sentative isolates were deposited in the collection of the

Centraalbureau voor Schimmelcultures (CBS), Utrecht,

the Netherlands.

DNA extraction and ITS rDNA amplification

Single conidial cultures from 21 isolates were grown

on MEA for 7 days at 25

°C in the dark. Template DNA

was obtained from the mycelium using the modified

phenol:chloroform DNA extraction method described in

Smith et al. (2001). DNA was separated by electrophoresis

on 1·5% agarose gels, stained with ethidium bromide and

visualized under ultraviolet light. DNA concentrations

were estimated against 

λ standard size markers.

The internal transcribed spacer (ITS) regions ITS1 and

ITS2, and the intermediate 5·8S gene of the ribosomal

RNA (rRNA), were amplified using the primer pair ITS1

and ITS4 (White et al., 1990). The PCR reactions were

performed using the PCR protocol of Slippers et al.

(2004b). PCR products were separated in a 1·5% agarose

gel, stained with ethidium bromide and visualized under

UV light. Sizes of PCR products were estimated against a

100 bp molecular weight marker XIV (Roche Diagnos-

tics). The PCR products were purified using the High Pure

PCR Product Purification kit (Roche Diagnostics).



DNA sequencing and analysis

Based on conidial morphology, the isolates of Botryo-

sphaeriaceae from S. cordatum  in South Africa were

tentatively separated into eight groups. ITS rDNA

sequences  were determined for representative samples

from all morphological groups (Table 1). To determine

the identity and phylogenetic relationships of these

isolates, ITS sequences of known species of the Botryo-

sphaeriaceae were obtained from GenBank and included

in the analyses (Table 1). The purified PCR products were

sequenced using the same primers that were used for the

PCR reactions. The ABI PRISM™ Dye Terminator Cycle

Sequencing Ready Reaction kit (Perkin-Elmer) was used for

sequencing reactions, as specified by the manufacturers.

Sequence reactions were run on an ABI PRISM 3100™

automated DNA sequencer (Perkin-Elmer).

Nucleotide sequences were analysed using sequence

navigator  version 1·0·1. (Perkin-Elmer Applied Bio-

Systems, Inc.) software and alignments were made online

using mafft version 5·667 (http://timpani.genome.ad.jp/

∼mafft/server/) (Katoh et al., 2002). Gaps were treated as

fifth character and all characters were unordered and of

equal weight. Phylogenetic analyses of aligned sequences

were carried out using paup  (Phylogenetic Analysis

Using Parsimony) version 4·0b8 (Swofford, 1999). Most-

parsimonious trees were found using the heuristic search

function with 1000 random addition replicates and

tree bisection and reconstruction (TBR) selected as the

branch swapping algorithm. Branches of zero length were

collapsed and all multiple, equally parsimonious trees

were saved. Branch support was determined using 1000

bootstrap replicates (Felsenstein, 1985). The trees were

rooted using the GenBank sequences of Guignardia

philoprina and Mycosphaerella africana, which are closely

related to Botryosphaeriaceae. The sequence alignments

and phylogenetic tree were deposited in TreeBASE as

S1412, M2541.



PCR-RFLP analyses

PCR-RFLP fingerprinting techniques were applied to

confirm the identity of isolates that were not sequenced

and to identify the isolates that could not be separated

based on ITS rDNA sequences. Amplicons obtained using

primer pairs ITS1 and ITS4, or BOT15 (5

′-CTGACTT-

GTGACGCCGGCTC-3

′) and BOT16 (5′-CAACCT-

GCTCAGCAAGCGAC-3

′) (Slippers et al., 2004a) were

digested with the restriction endonuclease CfoI. The

RFLP reaction mixture consisted of 10 

µL PCR products,

0·3 

µL CfoI and 2·5 µL matching enzyme buffer (Roche



Diagnostics). The reaction mixture was incubated at 37

°C

overnight. Restriction fragments were separated on 1·5%



agarose gel as described for PCR products. The results

were compared with those of Slippers (2003).



Morphology and cultural characteristics

Fungal isolates were grown on 2% water agar (WA;

Biolab)  with sterilized pine needles placed onto the

medium, at 25

°C under near-UV light, to induce sporula-

tion. Conidia that were released from pycnidia on the pine

needles were mounted in lactophenol on glass slides and

examined microscopically. Ten measurements of conidia

were taken for each isolate. Measurements and digital

photographs were taken using a light microscope, a HRc

Axiocam digital camera and accompanying software

(Carl Zeiss Ltd). Colony morphology and colour were

determined from cultures grown on 2% MEA at 25

°C

under near-UV light. Colony colours (upper surface and



reverse) were compared with those in the colour charts of

Rayner (1970).



Pathogenicity

Fifteen isolates, representing eight species of Botry-

osphaeriaceae isolated from native S. cordatum in South

Africa, were used in this study (Table 1). One isolate of



Botryosphaeria dothidea and two isolates for each of the

other seven species were randomly selected for inocula-

tions. The isolates were grown on 2% MEA at 25

°C

under continuous near-fluorescent light for 7 days prior to



inoculation.

Two-year-old trees of an E. grandis 

× camaldulensis

clone (GC-540) and 1-year-old saplings of S. cordatum

were selected for the pathogenicity trials under glass-

house conditions. Saplings of S. cordatum were raised from

seeds taken from a single tree grown in the Kwambonambi

(Kwazulu-Natal province) area. Trees and saplings



Plant Pathology (2007) 56, 624–636

Botryosphaeriaceae on Myrtaceae in South Africa

629


PPA_1608

selected for inoculations were grown in pots outside, and

maintained in the glasshouse for acclimatization for 3

weeks prior to inoculation. Trees were inoculated during

the spring-summer season (September 2003–February

2004). The glasshouse was subjected to natural day/night

conditions and a constant temperature of approximately

25

°C. Each of the isolates representing the different



species was inoculated into the stems of 10 trees of each

host species. Ten trees were also inoculated with sterile

MEA plugs to serve as controls. The 160 inoculated trees,

10 for each fungal species and 10 as a control, were

arranged in a randomized block design. The entire trial

was repeated once under the same conditions, giving a

total of 320 trees inoculated for each host species.

For inoculations, wounds were made on the stems

of trees using a 6-mm-diameter (Eucalyptus clone) or a

4-mm-diameter (S. cordatum) cork borer to remove the

bark and expose the cambium. Wounds were made

between two nodes on the stems of trees approximately

250 mm  (Eucalyptus) or 150 mm (S. cordatum) above

soil level. Plugs of mycelium were taken from 7-day-old

cultures grown on MEA using the same size cork borer,

and were placed into the wounds with the mycelial surface

facing the cambium. Inoculated wounds were sealed with

laboratory film (Parafilm M, Pechiney Plastic Packaging)

to prevent desiccation and contamination. Lesion lengths

(mm) were measured 6 weeks after inoculation. The fungi

were re-isolated by cutting small pieces of wood from

the edges of lesions and plating them on 2% MEA at

25

°C. Re-isolations were made from two randomly



selected trees per isolate and tree species and from all trees

inoculated as controls.

Pathogenicity for all isolates inoculated on the Eucalyp-

tus clone and S. cordatum was determined based on the

length of lesions (mm) that developed after 6 weeks. There

was no significant difference between the two repeats of

the pathogenicity trials and the data were therefore com-

bined to represent one dataset for the analyses. Statistical

analyses of the data were performed using sas statistical

software (version 8, SAS Institute). The 95% confidence

limits were determined for all means based on full model

analysis of variance (anova). Differences between means

were therefore considered significant at the P 

≤ 0·05 level.

Results

DNA sequence analyses

DNA fragments of approximately 600 bp were amplified.

The ITS dataset consisted of 53 ingroup sequences, with

G. philoprina and M. africana as outgroup taxa (Table 1).

After alignment, the ITS dataset consisted of 593 char-

acters; 432 uninformative characters were excluded,

and 161 parsimony-informative characters were used in

the analysis. The parsimony analysis (using heuristic

searches) produced 276 most parsimonious trees of 414

steps (consistency index (CI) 

= 0·702, retention index

(RI) 

= 0·915), one of which was chosen for presentation



(Fig. 2).

The isolates considered in the phylogenetic analyses

formed 12 clades, designated as groups I to XII (Fig. 2).

These groups were resolved in two major clades that

corresponded to species of Botryosphaeriaceae with

Fusicoccum-like or Diplodia-like anamorphs. The

Fusicoccum clade comprised six groups that represented:

Neofusicoccum parvum and N. ribis (group I), N. man-

giferae (group II), N. eucalyptorum (group III), N. australe

(group IV), N. luteum (group V) and B. dothidea (group

VI). Groups VII and VIII represented species with

Lasiodiplodia  anamorphs:  Lasiodiplodia theobromae

(group VII) and L. gonubiensis (group VIII). These two

groups (VII and VIII) formed a distinct subclade

(supported by 100% bootstrap value) within the Diplodia

clade. The other major subclade within the Diplodia

clade contained four groups corresponding to: D. mutila

(group IX), D. corticola  (group X), Diplodia  sp.

(

= ‘Botryosphaeria obtusa’) (group XI) and Diplodia



pinea (

Sphaeropsis sapinea) (group XII) (Fig. 2).

All the isolates obtained from S. cordatum in this study

resided in seven groups (Fig. 2) as follows: N. parvum and



N. ribis (group I), Nmangiferae (group II), N. australe

(group IV), N. luteum (group V), B. dothidea (group VI),



L. theobromae (group VII) and L. gonubiensis (group

VIII).


PCR-RFLP analysis

Isolates that were not identified using DNA sequence

comparisons were subjected to ITS PCR-RFLP analyses.

Digests of the PCR products, obtained using primers ITS1

and ITS4, with CfoI produced two distinctive banding

patterns. These profiles matched those of N. parvum/N.



ribis (99 isolates) and N. luteum/N. australe (5 isolates) as

shown by Slippers et al. (2004b). To further distinguish

isolates of N. parvum from those of N. ribis, amplicons

obtained using primers BOT15 and BOT16 were digested

using the same restriction endonuclease. The two banding

patterns obtained matched those of N. parvum  (42

isolates) and N. ribis (57 isolates) as described by Slippers

(2003). However, N. luteum and N. australe could not be

separated using this technique.

Morphology and cultural characteristics

All 148 isolates of the Botryosphaeriaceae from S. cordatum

produced anamorph structures on pine needles on WA

within 2–3 weeks. No teleomorph (sexual) structures

were observed. Based on conidial morphology, isolates

were separated into eight groups. Five of these groups

corresponded to Botryosphaeriaceae with Neofusicoccum

anamorphs (Fig. 3a–f), one with a Fusicoccum anamorph

(Fig. 3g) and two with Lasiodiplodia  (Diplodia-like)

anamorphs (Fig. 4a,b).

Representative samples from the groups emerging from

morphological comparisons were identified based on ITS

rDNA sequence comparison. As described earlier, isolates

of  N. parvum and  N. ribis  were separated based on

PCR-RFLP analyses. Further morphological examination


Plant Pathology (2007) 56, 624–636

630


D. Pavlic et al.

PPA_1608

Figure 2 One of 276 most-parsimonious trees obtained from heuristic searches of ITS1, 5·8S and ITS2 rDNA sequence data (tree length 

= 414 steps, 

CI 


= 0·702, RI = 0·915). Branch lengths, proportional to the number of steps, are indicated above the internodes, and bootstrap values (1000 

replicates) below the internodes. The tree is rooted to the outgroup taxa Guignardia philoprina and Mycosphaerella africana. Isolates sequenced 

in this study are presented in bold.


Plant Pathology (2007) 56, 624–636

Botryosphaeriaceae on Myrtaceae in South Africa

631


PPA_1608

of isolates, identified based on DNA data, provided sup-

port for their identity.

Cultures of N. parvum were initially white with fluffy,

aerial mycelium, becoming pale olivaceous grey from the

middle of colony after 3–4 days; columns of the mycelium

formed in the middle of colony reaching the lid; margins

were regular; reverse sides of the colonies were olivaceous

grey. Conidia were hyaline, smooth, aseptate and fusiform

to ellipsoid (average of 420 conidia: 18·2 

× 5·5 

µm,


l/w 3·3) (Fig. 3a). The 42 isolates were identified as

N. parvum.

Colonies of N. ribis were initially white, becoming pale

olivaceous grey from the middle of colony, with thick

aerial mycelium reaching the lids of Petri dishes; margins

were regular; reverse sides of the colonies were olivaceous

grey. Conidia were hyaline, unicellular, aseptate, fusiform,

apices tapered (average of 570 conidia: 21 

× 5·5 


µm, l/w

3·8) (Fig. 3b). The 57 isolates were identified as N. ribis.

The culture of the single B. dothidea isolate identified

in this study produced greenish olivaceous appressed

mycelium, its margins regular and the reverse sides of the

colonies olivaceous grey to iron-grey. Conidiomata were

readily formed in the middle of colonies after 3–4 days of

incubation. Conidia were hyaline, smooth with granular

contents, aseptate, narrowly fusiform (average of 10

conidia: 27·8 

× 5·4 

µm, l/w 5·1) (Fig. 3g).



Figure 3 Light micrographs of conidia of six Botryosphaeriaceae species with Fusicoccum-like anamorphs: (a) Neofusicoccum parvum; (b) N. ribis

(c) aseptate and uniseptate conidia of N. australe; (d, e) aseptate and germinating uni- and biseptate conidia of N. luteum; (f ) N. mangiferae

(g) Botryosphaeria dothidea. Bars 

= 10 


µm.

Plant Pathology (2007) 56, 624–636

632


D. Pavlic et al.

PPA_1608

Isolates of N. mangiferae produced pale olivaceous grey

appressed mycelium, slightly fluffy on the edges of colonies,

with sinuate margins, and the reverse sides of colonies

were olivaceous. Conidiomata were readily formed in the

middle of colonies after 3–4 days and covered the entire

surface of the colonies within 7–10 days. Conidia were

hyaline, fusiform (average of 300 conidia: 14·2 

× 6·3 

µm,


l/w 2·25) (Fig. 3f). The 30 isolates were identified as

N. mangiferae.

Cultures of N. luteum were initially white, becoming

pale olivaceous grey from the middle of colonies within 3–

4 days, with suppressed mycelium, moderately fluffy in

the middle and with regular margins. A yellow pigment

was noticeable after 3–5 days of incubation and was seen

as amber yellow on the reverse side of Petri dishes; after

5–7 days colonies become olivaceous buff to olivaceous

grey. Conidiomata were readily formed from the middle

of colonies within 3–4 days and covered the whole surface

of colonies within 7–10 days. Conidia were hyaline, fusi-

form to ellipsoid, sometimes irregularly fusiform, smooth

with granular contents, unicellular, forming one or

two septa before germination (average of 40 conidia:

18·9 

× 6·3 


µm, l/w 3·0) (Fig. 3d,e). The four isolates were

identified as N. luteum.

Cultures of N. australe were very similar in morpho-

logy to those of N. luteum, but the yellow pigment pro-

duced in young cultures was brighter and a honey-yellow

colour when viewed from the bottom of the Petri dishes.

Conidiomata readily formed at the middle of colonies

within 3–4 days and covered the colony surfaces within

7–10 days. Conidia were hyaline, fusiform, apices rounded,

aseptate, rarely uniseptate (average of 70 conidia:

20·5 

× 5·7 


µm, l/w 3·6) (Fig. 3c). These conidia were

slightly longer and narrower on average than those of



N. luteum, which was also reflected in a higher l/w ratio.

The seven isolates were identified as N. australe.

Isolates of L. theobromae produced initially white to

smoke-grey fluffy aerial mycelium, becoming pale oliva-

ceous grey within 5–6 days with regular margins; the

reverse sides of the cultures were olivaceous grey to iron,

becoming dark slate-blue after 7–10 days. Conidia were

hyaline, aseptate, ellipsoid to ovoid, thick-walled with

granular contents (average of 50 conidia: 27 

× 14·7 


µm,

l/w 1·85) (Fig. 4b). Dark, septate conidia typical for this

species were not observed in this study. The five isolates

were identified as L. theobromae.

Isolates of L. gonubiensis were similar in culture mor-

phology to those of L. theobromae. Conidia of L. gonu-



biensis  were initially hyaline, unicellular, ellipsoid to

obovoid, thick-walled with granular contents, rounded at

the apex and occasionally truncate at the base. Aging

conidia became cinnamon to sepia with longitudinal stri-

ations, forming one to three septa (average of 20 conidia:

33·9 


× 18·9 

µm, l/w 1·8) (Fig. 4a). The two isolates were

identified as L. gonubiensis.




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