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1 Department of Genetics, Forestry and Agricultural Biotechnology Institute
(FABI), University of Pretoria, Pretoria 0002, South Africa
2 Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, P.O. Box
85167, 3508 AD, Utrecht, The Netherlands
*
Correspondence: Maria-Noel Cortinas,
mncortinas{at}fabi.up.ac.za
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Abstract |
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 TOP Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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gene, and a partial
sequence of the mitochondrial ATPase 6 gene. Both phylogenetic data and
morphological characteristics showed clearly that isolates of C.
zuluensis represent at least two taxa. One of these is C.
zuluensis as it was originally described from South Africa, and we
provide an epitype for it. The second species occurs in Argentina and Uruguay,
and is newly described as C. gauchensis. Both fungi are serious
pathogens resulting in identical symptoms. Recognising them as different
species has important quarantine consequences. Taxonomic novelty: Colletogloeopsis gauchensis M.-N. Cortinas, Crous & M.J. Wingf. sp. nov.
Keywords Bayesian inference / Colletogloeopsis / Eucalyptus canker / multilocus gene phylogeny / Mycosphaerella
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INTRODUCTION |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Symptoms of Colletogloepsis canker are very obvious, at least at the onset of disease. Initial infections include small, circular necrotic lesions on the green stem tissue in the upper parts of trees. These lesions expand, becoming elliptical, and the dead bark covering them typically cracks, giving a "cat-eye" appearance (Fig. 1). Lesions coalesce to form large cankers that girdle the stems, giving rise to the production of epicormic shoots and ultimately trees with malformed or dead tops. Infections occur annually on the new green tissue and they penetrate the cambium to form black kino-filled pockets. Thus kino pockets with irregular borders of infected tissue can be seen within the infected wood of trees coincident with the annual rings (Fig. 1). Small black pycnidia can be seen on the surface of dead bark tissue (Fig. 1), from where black conidial tendrils exude under moist conditions. Conidia are small, aseptate and dematiaceous, appearing black in colour when seen in mass on the host or agar media.
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Different isolates of C. zuluensis have been found to be highly variable in morphology (Fig. 3) and pathogenicity to different Eucalyptus clones (Van Zyl 1997, Wingfield et al. 1997, Van Zyl 2002a). Nonetheless, previous phylogenetic analyses based on the nuclear ribosomal small subunit (18S) and internal transcribed spacer regions and the ribosomal 5.8 gene (ITS1, 5.8S, ITS2) had shown that C. zuluensis was monophyletic (Van Zyl 2002b, Alemu et al. 2005). As additional surveys of Eucalyptus plantations are undertaken, an understanding of the geographical range of C. zuluensis continues to expand. Additional isolates from new regions have thus become available for DNA sequence comparisons and these have provided the opportunity to re-consider the taxonomic status of C. zuluensis, and the variation observed in its morphology and pathogenicity.
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(EF1) gene,
and the mitochondrial ATPase 6 (ATP6) gene, were carried out. Morphological
and other phenotypic characters were also considered. |
MATERIALS AND METHODS |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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DNA extraction and amplification
To extract DNA, mycelium was scraped from the surface of cultures grown in
Petri dishes, freeze dried, frozen in liquid nitrogen and ground to a fine
powder. The protocol followed by Cortinas et al.
(2004) was simplified as
follows: DBE extraction buffer (200 mM Tris-HCl pH 8, 150 mM NaCl, 25 mM EDTA
pH 8, 0.5 % SDS) was added directly to the ground mycelium and incubated for 2
h at 80 °C (or until pigments changed colour from green to red). In the
extraction-DNA enrichment procedure, one volume of phenol was used first and
one volume of a 1:1 phenol-chloroform solution thereafter.
Four gene regions were amplified for all isolates included in this study
(Fig. 4). The ITS region of the
ribosomal DNA was targeted using the primers ITS1: 5' TCC GTA GGT GAA CCT GCG
G and ITS4: 5' GCT GCG TTC TTC ATC GAT GC
(White et al. 1990).
Exons 3 to 6 and the respective introns (BT2) of the β-tubulin gene
region were amplified using the primers BT2A: 5' GGT AAC CAA ATC GGT GCT GCT
TTC and BT2B: 5' AAC CTC AGT GTA GTG ACC CTT GGC
(Glass & Donaldson 1995).
The intron sequence of the EF1- gene was amplified using the primers
EF1-728F: 5' CAT CGA GAA GTT CGA GAA GG and EF1-986R: 5' TAC TTG AAG GAA CCC
TTA CC (Carbone & Kohn
1999) and intron 2 and exon 3 of the ATP6 gene was amplified using
the set of primers 5'ATT AAT TSW CCW TTA GAW CAA TT and 5'TAA TTC TAN WGC ATC
TTT AAT RTA developed by Kretzer & Bruns
(1999).
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, ATP6).
PCR amplicons were visualised under UV light on 1 % or 2 % agarose gels.
Different cycling conditions were used for the various gene regions. For the
ITS region, 96 °C, 3 min initial denaturation and cycles of 95 °C, 30
s, 54 °C, 30 s, 72 °C, 1 min were repeated 10 times followed by 25
cycles of 95 °C, 30 s, 56 °C, 30 s, 72 °C, 1 min with 5 s
extension after two cycles. A final elongation step of 7 min at 72 °C was
also included. The same cycling conditions were used for ATP6 region changing
the annealing temperature to 50 °C. For β-tubulin, 96 °C, 3 min
initial denaturation and cycles of 95 °C, 30 s, 57 °C, 45 s, 72
°C, 45 s were repeated 40 times. For EF1-, 96 °C, 3 min and
cycles of 95 °C, 30 s, 54 °C, 45 s, 72 °C, 45 s were repeated 40
times with 5 s extension after two cycles. A final elongation step of 7 min at
72 °C was included. PCR amplification products were purified using Sephadex G-50 columns (Sigma-Aldrich, Steinheim, Germany) or treated with a mix of Exonuclease III and Shrimp alkaline phosphatase (Exo-Sap); 0.7 U of each enzyme per PCR reaction were incubated at 37 °C for 15 min followed by 80 °C for 15 min before sequencing. Sequencing reactions were prepared in 10 µL with 2 µL of purified PCR product, 10 pmol of the same primers used for the first PCR amplifications, 2 µL 5x dilution buffer and ABI Prism Big Dye Terminator mix, v. 3.1 (Applied Biosystems Inc., Foster City, California). Sequencing PCR cycles consisted of 25 repetitions at 96 °C, 10 s; 50 °C, 4 s; 60 °C, 4 min. Sequencing reactions were cleaned using Sephadex G-50 or precipitated using EDTA, Sodium Acetate and Ethanol according to the protocol supplied by Applied Biosystems (Applied Biosystems Inc., Foster City, California).
Phylogenetic analyses
Alignments of sequence data were made using Clustal W under MEGA 3.0
(Kumar et al. 2004)
and manually adjusted. All sequences generated in this study were deposited in
GenBank (Table 1). Alignments
were deposited in TreeBASE.
Maximum parsimony and distance analyses were conducted considering the
individual and combined partitions. Most parsimonious (MP) trees were
generated using PAUP v. 4.0b10 (Swofford
2002). For parsimony analyses, heuristic searches were used with
the steepest descent option and the TBR swapping algorithm. The characters
were equally weighted and treated as unordered. Statistical support of the
nodes in the trees was tested with 1000 bootstrap replicates. Distance
analyses were conducted using MEGA 3.0
(Kumar et al. 2004).
Pairwise distances were estimated using the Kimura with two parameters model
(Kimura 1980). A gamma
distribution = 0.5 was used to take into account the differences in
mutation rate among sites, due to the mix of coding and non-coding sequences
present in the analysed fragments. The individual gene reconstructions were
performed with Minimum Evolution (Rzhetsky
& Nei 1993). Gaps generated in the alignment were treated as
missing data. One thousand bootstrap replicates were made to assess the
statistical support of the nodes in the phylogenetic trees. Trees were rooted
to midpoint.
Partitions were considered together using Bayesian analyses (Ronquist & Huelsenbeck 2003). It has recently been shown that the Bayesian method is more sensitive to under-specification than over-specification of the evolutionary model (Huelsenbeck & Rannala 2004) when calculating the posterior probabilities. Consequently, a time-reversible complex model with gamma-distributed rate variation (GTR + I + G) was selected to combine the data sets. This model of DNA substitution allows the consideration of different rates of substitutions among sites, different nucleotide frequencies, and differences in the rate of substitutions among nucleotides. Therefore, four sets of analyses were run in MrBayes 3.1.1 (Huelsenbeck & Ronquist 2001, Ronquist & Huelsenbeck 2003) calculating marginal posterior probabilities using the selected time reversal GTR + I + G model of nucleotide substitution (Tavaré 1986, Yang 1993, 1994) and default values for the prior settings. Four Monte Carlo Markov chains were run for 3 million generations. Trees and parameters were recorded every 100 generations. Likelihood stability was reached at 30 000 generations. This number of generations was then established as the "burn-in" period (represented by 3001 trees). A half compatible consensus tree was recovered from the remaining sampled trees. The Bayesian procedure was repeated four times. The posterior probabilities are indicated close to the respective nodes on the tree and the sequences of Mycosphaerella colombiensis Crous & M.J. Wingf. and M. suttonii Crous & M.J. Wingf. were used as outgroups.
Temperature sensitivity studies
Plugs (3 mm diam) of colonised agar were cut from actively growing cultures
and placed at the centres of Petri dishes containing MEA. Isolates tested for
growth characteristics at different temperatures included those from South
Africa (CMW 7442, CMW 7449, CMW 7479, CMW 7488), and others from Uruguay (CMW
7269, CMW 7274, CMW 7279, CMW 7300). Three plates were prepared for each
isolate and these were incubated at temperatures between 5 °C and 35
°C at 5 ° intervals, for 6 wk. A second set of isolates from Ethiopia
(CMW 8282, CMW 8292) and from China (CMW 15966, CMW 15971) were tested in a
similar manner but for an incubation period of 8 wk. Growth was recorded
weekly by measuring average colony diameter.
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RESULTS |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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) and 656 bp
(ATP6) were generated. The intron between the exons 3 and 4 of the
β-tubulin gene was missing in all isolates studied. Visual analyses of
the characters defined two groups among the isolates based on the fixed,
shared polymophisms. The first group included isolates from South Africa,
China, Thailand, Vietnam and Malawi and a second group comprised isolates from
Uruguay, Argentina, Hawaii, Uganda and Ethiopia. Positions in base pairs of
the different fixed characters in the alignments for the various isolates are
shown in Table 2. Five fixed
characters were found at the ITS region, eleven were found in the BT2 dataset,
eight were found at the EF1- intron where a 20-base-pair indel was also
found (Fig. 5). One fixed
position was found in the ATP6 region.
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Analyses of sequence data for the ITS region resolved two coherent clusters
for the Colletogloeopsis isolates considered. These groups
represented isolates from South Africa, Malawi, Mexico, Thailand, Vietnam and
China (Group1) and those from Uruguay, Argentina, Hawaii, Ethiopia and Uganda
(Group 2). The separation of these two groups had 98 % bootstrap support in
the ITS tree. In the BT2 and EF-1 trees, these two groups had 99 % and
100 % support, respectively. For the ATP6 tree, three groups could be
distinguished although only one of these had strong support (100 %). The group
having reasonable support included isolates from Vietnam, Mexico, Malawi,
China and South Africa. Internal sub-clusters could be distinguished within
the Group 1 and Group 2 clusters in the ITS, BT2 and EF1- trees. These
sub-clusters had greater than 70 % bootstrap support only in the BT2 tree. The
assortment of isolates within the sub-clusters was different in different
trees.
The level of polymorphism observed in the datasets was different for each
individual analysed region. The β-tubulin data set presented the highest
level of variation followed by the EF1-, ITS and ATP6 data sets,
respectively. A close inspection of the ATP6 data matrix showed few
polymorphisms explaining the poor resolution obtained in the tree.
After the individual analyses, combined parsimony and Bayesian analysis were carried out (Fig. 7). The reconstructed trees included the collection of Colletogloeopsis isolates together with Mycosphaerella spp. A posterior probability of 1 and a 100 % bootstrap value separated the Colletogloeopsis isolates from the rest of Mycosphaerella spp. The parsimony and Bayesian half-compatible trees showed two major groups representing isolates from South Africa, Malawi, Mexico, Thailand, Vietnam and China (Group1) and those from Uruguay, Argentina, Hawaii, Ethiopia and Uganda (Group 2) supported by posterior probabilities of 1 and 0.95 and 98 % and 100 % bootstrap values, respectively. A rich internal topology was found within these two groups. Numerous sub-clusters were supported with high probabilities and bootstrap values. A number of these included more than one isolate from the same locality. Nevertheless, location was not sufficient to explain how the sub-clusters were formed.
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The results obtained in a second experiment including isolates from China and Ethiopia, were very similar to those comparing isolates from South Africa and Uruguay. After 8 wk, the differences in growth of the isolates from both origins were obvious at 35 °C (Fig. 8). This is consistent with the fact that isolates from China are phylogenetically related to those from South Africa and those from Ethiopia are related to those from Uruguay.
Morphology
Isolates of Colletogloeposis included in this study were
morphologically variable in culture. Colony characteristics overlapped for
isolates from South Africa and Uruguay, but it was possible to recognise some
characteristics apparently exclusive to the Uruguayan isolates. Likewise,
distinctly different conidial and conidiogenous cell characteristics were
found when isolates from Uruguay were compared with those of C.
zuluensis from South Africa (Fig.
9). The range of conidial lengths overlapped almost entirely
between C. zuluensis [conidia (4–)4.5–5(–6) x
2–2.5(–3.5) µm] and the isolates from Uruguay [conidia
(4–)5–6(–7.5) x (2–)2.5(–3) µm]. The
Uruguayan conidia, however, had a larger maximum length, reaching 7.5 µm (6
µm for C. zuluensis). Conidia of C. zuluensis were
slightly wider (3.5 µm) as opposed to those from Uruguay, which were an
average of 3 µm. Another distinctive characteristic of the fungus from
Uruguay is that it has sympodial polyphialidic conidiogenous cells, which is
different to C. zuluensis, which has percurrently proliferating
monophialidic conidiogenous cells.
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Taxonomy
Phylogenetic analyses in this study supported two distinct groups of
isolates, encompassed within the fungus currently treated as C.
zuluensis. One of these groups of isolates is from South Africa, Malawi,
Thailand, Vietnam, China and Mexico. The other group includes isolates from
Uruguay, Argentina, Hawaii-U.S.A., Ethiopia and Uganda. These fungi can also
be separated by characteristics of growth in culture, morphology and growth at
different temperatures. Clearly, the South African fungus must retain the name
C. zuluensis. At the time of describing this fungus, no ex-type
cultures were deposited. We have thus provided a suite of isolates for which
DNA sequence data are available, and that are tied to herbarium specimens to
serve as epitypes. The fungus occurring in Uruguay and other countries
represents a distinct taxon that is described below.
Colletogloeopsis gauchensis M.-N. Cortinas, Crous & M.J. Wingf., sp. nov. MycoBank MB500854. Figs 9, 10.
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Colletogloeopsidi zuluensi similis, sed conidiis angustioribus, (4–)5–6(–7.5) x (2–)2.5(–3) µm et phialidibus nonnumquam sympodialiter proliferentibus distincta.
Lesions caulicolous, subcircular to irregular, dark brown, 2–10 mm diam, with a raised, red-brown border. Conidiomata pycnidial to somewhat acervular, subepidermal, single, rarely aggregated, occurring in necrotic tissue, globose to slightly depressed, becoming erumpent, up to 120 µm diam, exuding conidia in a long cirrus; conidiomatal walls composed of 2–3 layers of medium brown textura angularis; opening by a central ostiole or irregular rupture; ostiolar region lined with thick-walled, brown, smooth, septate hyphae that are sometimes branched below, 3–4 µm wide, with obtuse ends that flare apart (upper 1–6 cells). Conidiophores subcylindrical, subhyaline to medium brown, smooth to finely verruculose, 0–3-septate, unbranched or branched below, 10–20 x 3–6 µm. Conidiogenous cells subhyaline to medium brown, dolilform to subcylindrical, smooth to finely verruculose, mono- to polyphialidic, proliferating percurrently, with several percurrent proliferations near the apex. Conidia medium brown, thick-walled, finely verruculose, broadly ellipsoidal, apex obtuse to subobtuse, base subtruncate to bluntly rounded, (4–)5–6(–7.5) x (2–)2.5(–3) µm; base frequently with a minute marginal frill.
Specimens examined: Uruguay, El Tarugo, bark of 1-yr-old E. grandis tree, Feb. 2005, M.J. Wingfield, CBS H-19724 holotype, cultures ex-holotype CMW 17331–17332; La Herradura, CBS H-19722, cultures CBS 119467-119466 = CMW 17542–17543; ibid., CBS H-19723, cultures CBS 119465 = CMW 17545, CMW 17544; La Juanita, CBS H-19725, cultures CBS 119468 = CMW 17558, CMW 17559; ibid., CBS H-19726, cultures = CMW 17560–17561.
Cultural characteristics: Colony characteristics on MEA at 25 °C are variable. Colony colours were similar to those of C. zuluensis (Van Zyl et al. 1997, 2002). Surface colours range from greyish yellow-green, dull green, isabelline, greenish olivaceous to grey-olivaceous; colonies in reverse range from dark grey, dark olive-grey to dark green (Rayner 1970); margins are smooth, regular or irregular. Some cultures develop a characteristic white outer zone of aerial mycelium (Fig. 3). Paler colonies develop smoother surfaces with white aerial mycelium; some strains produce a diffuse yellow pigment in MEA.
Notes: Colletogloeopsis gauchensis [conidia (4–)5–6(–7.5) x (2–)2.5(–3) µm] can readily be distinguished from C. zuluensis [conidia (4–)4.5–5(–6) x 2–2.5(–3.5) µm] by its slightly longer conidia, and the presence of sympodial polyphialidic conidiogenous cells (Figs 9, 10). Furthermore, it grows readily at 10 °C, with hardly any to no growth at 35 °C. In contrast, C. zuluensis grows more slowly at 10 °C, and faster at 35 °C than C. gauchensis, and strains of C. gauchensis do not form conidiomata in culture.
Colletogloeopsis zuluensis (M.J. Wingf., Crous & T.A. Cout.) M.-N. Cortinas, M.J. Wingf. & Crous, Mycol. Res. 110: 235. 2006. Figs 9, 10. [as zuluense].
Basionym: Coniothyrium zuluense M.J. Wingf., Crous & T.A. Cout., Mycopathologia 136: 142. 1997.
Specimens examined: South Africa, KwaZulu-Natal, Kwambonambi, Teza nursery, bark of 1-yr-old E. grandis tree, Jan. 1996, M.J. Wingfield, IMI 370886 holotype; KwaZulu-Natal, Kwambonambi, E. grandis, Feb. 2005, M.J. Wingfield, CBS H-19721 epitype here designated, culture ex-epitype CMW 17321–17322; CBS H-19717, culture CBS 119427 = CMW 17531, CMW 17530; CBS H-19720, culture CBS 119471 = CMW 17528, CMW 17529; CBS H-19719, culture CBS 119470 = CMW 17320, CMW 17319; CBS H-19718, culture CBS 119469 = CMW 17526, CMW 17527.
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DISCUSSION |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Twenty-six fixed nucleotide positions allowed us to separate the collection
of C. zuluensis s. lat. isolates used in this study into two
distinctive groups. One of these fixed polymorphisms found in the EF1-
intron can easily be used to discriminate between C. zuluensis and
C. gauchensis. This 20 bp fragment between positions 153 to 172 in
C. zuluensis is absent in C. gauchensis. The p-distance
among the Colletogloeopsis isolates considered in this study
displayed a range of 0 to 1 % divergence in ITS sequences, 0–8 % for BT2
sequences, 0–24 % for EF1- sequences and 0–4 % for ATP6
data-matrices respectively. These ranges showed that there was sufficient
variation within Colletogloeopsis to suspect that more than one taxon
was represented in the collection of isolates. The distances are also
consistent with values used in previous studies
(Couch & Kohn 2002, Barnes
et al. 2005) to separate taxa.
Very few morphological differences were found between isolates of C. zuluensis from South Africa and isolates of C. gauchensis from Uruguay. These differences include the fact that Uruguayan isolates have polyphialidic, sympodially and percurrently proliferating conidiogenous cells as opposed to the monophialidic, percurrently proliferating conidiogenous cells in C. zuluensis. The conidia of C. gauchensis are also consistently longer than those of C. zuluensis (Figs 9, 10). Furthermore, C. gauchensis is adapted to cooler climates than C. zuluensis. On the contrary, isolates of C. zuluensis grow well at 35 °C, whereas those of C. gauchensis barely grow at this temperature.
Results of this study provide added support for the view that C. zuluensis and C. gauchensis are anamorphs of Mycosphaerella. They have an allopatric distribution and are considered sibling species only in terms of the fact that they are ecologically and morphologically very similar. The extent to which cryptic and sibling species occur in taxonomic groups varies depending on the group of fungi studied. However, the discovery of cryptic species such as C. gauchensis in this study is becoming a commonplace when DNA studies are implemented (see Crous et al. 2006). Results of such studies reveal that these species reflect collections of morphologically similar taxa that can only be discriminated based on minute morphological details or characteristics in pure culture. A further example of such a species complex in Mycosphaerella concerns "Coniothyrium" ovatum H.J. Swart (Crous et al. 2004a, b, 2006 – this volume), which will be treated elsewhere (Cortinas et al. in prep.).
Intraspecific variation detected amongst isolates of C. zuluensis and to a lesser extent C. gauchensis showed internal structure in the individual and combined trees. Such intraspecific structure was only well-supported in the BT2, ATP6 and combined trees. Based solely upon the phylogenetic species concept, it would be possible to recognise additional species especially in this complex. For the present, however, we choose to not provide additional names before robust population biology studies are available.
Coniothyrium canker is one of the most important diseases of Eucalyptus worldwide (Old et al. 2003). In South Africa, it appeared relatively suddenly in a very limited location and spread rapidly, resulting in very substantial losses to the local forestry industry. The disease has also caused substantial damage to plantations in other countries such as Argentina and Uruguay. It is thus intriguing that there are two distinct fungi associated with indistinguishable symptoms. The origin of the fungus is unknown and it is not known to occur in the native range of Eucalyptus. The evidence from this study shows that the two fungi are closely related and have adapted differently based on some ecological factor. Like most Mycosphaerella spp. they are highly host-specific to certain species of Eucalyptus, grow poorly in culture, and thus it seems reasonable to expect that their origin would be on Eucalyptus or a host closely related to it. A similar situation has emerged for species of Chrysoporthe Gryzenh. & M.J. Wing. (Gryzenhout et al. 2004). that are well-known pathogens of Eucalyptus but that appear to have originated on a wide variety of woody plants in the order Myrtales (Wingfield 2003, Gryzenhout et al. 2004, Seixas et al. 2004).
Recognition of two species within a collection of isolates that have previously been recognised as belonging to the single taxon has important consequences for disease control and quarantine. In the past, it has been suggested that the fungus originated in South Africa, and that it was restricted to that country (Wingfield et al. 1997). Thus, the appearance of the disease in other countries has often been linked to the movement of plant material and particularly seed to other countries. Although it has not been shown experimentally that C. zuluensis is moved on seed, this appears to be a likely mode of global distribution. There is a large international trade in Eucalyptus seed, which is variably controlled and monitored. Both C. zuluensis and C. gauchensis have now wide geographic distributions and this implies that they have been spread from one or a number of sources. Every effort should now be made to restrict them from further movement to new countries and areas.
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Acknowledgments |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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