POPULATION BIOLOGY OF MYCOSPHAERELLA AND TERATOSPHAERIA SPECIES

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Chapter  3 Multi-gene gene phylogenies and phenotypic characters distinguish two species within the Colletogloeopsis zuluensis complex associated with Eucalyptus stem cankers

ABSTRACT

Colletogloeopsis zuluensis, previously known as Coniothyrium zuluense causes a serious stem canker disease on Eucalyptus spp grown as non-natives in many tropical and sub-tropical countries. This stem canker disease was first reported from South Africa and it has subsequently been found on various species and hybrids of Eucalyptus in other African countries as well as in countries of South America and South-East Asia. In previous studies, phylogenetic analyses based on DNA sequence data of the ITS region suggested that all material of C. zuluensis was monophyletic. However, the occurrence of the fungus in a greater number of countries, and analyses of DNA sequences with additional isolates has challenged the notion that a single species is involved with Coniothyrium canker. The aim of this study was to consider the phylogenetic relationships amongst C. zuluensis isolates from all available locations and to support these analyses with phenotypic and morphological comparisons. Individual and combined phylogenies were constructed using DNA sequences from the ITS region, exons 3 through 6 of the b-tubulin gene, the intron of the translation elongation factor 1-a 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.

INTRODUCTION

Colletogloeopsis zuluensis (MJ Wingf., Crous & TA Cout.) MN Cortinas, MJ Wingf & Crous (Cortinas et al., 2006) causes a serious stem canker disease on Eucalyptus species.The disease was first reported in 1987 in South Africa, and the pathogen was described as a species of Coniothyrium, namely C. zuluense MJ Wingf., Crous & TA Cout, (Wingfield et al., 1997). The disease spread very rapidly through the country, initially occurring only on a single Eucalyptus grandis clone, but ultimately occurring in all parts of South Africa with a sub-tropical climate, and on a wide variety of Eucalyptus species and hybrids. Substantial research has thus been undertaken to better understand the disease and to develop disease-resistant planting stock through breeding and selection programmes (Van Zyl et al., 1997, 2002a).
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.
Subsequent to the discovery of Coniothyrium canker in South Africa, the disease has been found in many other countries. Its first discovery outside South Africa was in Thailand where it is associated with typical symptoms on E. camaldulensis (Van Zyl et al., 2002b). More recently, the disease has been found in other countries in Africa (Gezahgne et al., 2003, 2005), South and Central America (Roux et al., 2002; Gezahgne et al., 2004), as well as South-East Asia (Old et al., 2003; Cortinas et al., 2004, 2006) (Fig 2). Interestingly, the disease remains unknown in the areas of origin of Eucalyptus, although it might occur there at very low and undetectable levels (Wingfield 2003; Slippers et al., 2005).
The first taxonomic treatment of C. zuluensis was based on morphological characteristics of the pathogen. The presence of pycnidia and pigmented aseptate, ellipsoidal conidia arising from percurrently proliferating conidiogenous cells were consistent with species placed in Coniothyrium Corda. DNA sequence comparisions have, however, made it possible to recognise that the fungus has a clear phylogenetic position in Mycosphaerella Johanson (Gezahgne et al., 2005). It is moreover not related to species of Coniothyrium s. str., which are anamorphs of Leptosphaeria spp. This realisation has led to the transfer of Coniothyrium zuluense to Colletogloeopsis Crous & MJ Wingf. (Cortinas et al., 2006) Colletogloeopsis is a well-recognised Mycosphaerella anamorph and its circumscription was amended to include species with pycnidioid conidiomata. Within Mycosphaerella, C. zuluensis clusters with a group of well-known leaf and stem pathogens of Eucalyptus including M. ambiphylla A Maxwell, M. cryptica (Cooke) Hansf, M. molleriana (Thüm) Lindau, M.. nubilosa (Cooke) Hansf, M. vespa Carnegie & Keane, M. suttonii Crous & MJ Wingf., and Phaeophleospora eucalypti (Cooke & Massee) Crous, FA Ferreira & B Sutton (Cortinas et al., 2006).
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 58 gene (ITS1, 58S, ITS2) had shown that C. zuluensis was monophyletic (Van Zyl 2002b; Gezahgne 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.
The aim of this study was to consider whether the previously recognised C. zuluensis can be retained when applying multigene analyses using a large collection of isolates not previously available. To accomplish this objective, individual and combined phylogenetic analyses using the ITS region, b-tubulin gene (BT2), the elongation factor 1a (EF1a) gene, and the mitochondrial ATPase 6 (ATP6) gene, were carried out. Morphological and other phenotypic characters were also considered.

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MATERIALS AND METHODS

Isolates

A collection of 45 isolates was chosen to reflect the geographical distribution of C. zuluensis In addition, several species of Mycosphaerella known to be closely related to C. zuluensis were also included (Table 1). All these isolates were obtained from the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), Pretoria, South Africa. Single-conidial cultures were established from mature pycnidia isolated from lesions taken from the stems of Eucalyptus trees in South Africa and Uruguay. The contents of single pycnidia were diluted in sterile distilled water, and spread on the surface of Petri dishes containing MEA (20 g/L Biolab malt extract, 15 g/L Biolab agar). After 24–36 h, germinating conidia were transferred to fresh MEA plates and incubated for 30 d at 25 oC. Reference strains are preserved in CMW, and have been deposited at the Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands (Table 1). Nomenclature, descriptions and illustrations were deposited in MycoBank.

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, 05 % SDS) was added directly to the ground mycelium and incubated for 2 h at 80 oC (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-a 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).
PCR reactions were prepared in a total volume of 25 µL including 1.5 µL of genomic 1/10 dilution DNA, 1 U of Taq polymerase, 10 ´ Taq buffer, 10 pmol of each primer, 0.8 mM of each dNTPs, and 2.0 mM MgCl2 (ITS) or 4.0 mM MgCl2 (BT2, EF1-a, 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 oC, 3 min initial denaturation and cycles of 95 oC, 30 s, 54 oC, 30 s, 72 oC, 1 min were repeated 10 times followed by 25 cycles of 95 oC, 30 s, 56 oC, 30 s, 72 oC, 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 oC. For b-tubulin, 96 oC, 3 min initial denaturation and cycles of 95 oC, 30 s, 57 oC, 45 s, 72 oC, 45 s were repeated 40 times. For EF1- a, 96 oC, 3 min and cycles of 95 oC, 30 s, 54 oC, 45 s, 72 oC, 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 oC for 15 min followed by 80 oC 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 5´ 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 oC, 10 s; 50 oC, 4 s; 60 oC, 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).

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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 g= 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.

Aknowledgements
Preface
Chapter1 Literature review: Diseases of Eucalyptus with particular reference to the taxonomy and population biology of pathogens in the Teratosphaeriaceae
INTRODUCTION
EMERGENT FUNGAL PATHOGENS AND PEST IN FOREST PLANTATIONS.
THE GENUS MYCOSPHAERELLA
CONIOTHYRIUM CANKER DISEASE
POPULATION BIOLOGY OF MYCOSPHAERELLA AND TERATOSPHAERIA SPECIES
CONCLUSIONS
REFERENCES
TABLES AND FIGURES
Chapter 2 First record of Colletogloeopsis zuluense comb. nov., causing a stem canker of Eucalyptus spp. in China
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
DISCUSSION
ACKNOWLEDGEMENTS
REFERENCES
TABLES AND FIGURES
Chapter 3 Multi-gene gene phylogenies and phenotypic characters distinguish two species within the Colletogloeopsis zuluensis complex associated with Eucalyptus stem cankers
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
REFERENCES
TABLES AND FIGURES.
Chapter 4 Polymorphic microsatellite markers for the Eucalyptus fungal pathogen Colletogloeopsis zuluensis
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
ACKNOWLEDGEMENTS
TABLES REFERENCES
Chapter 5 Microsatellite markers for the Eucalyptus stem canker fungal pathogen Kirramyces gauchensis
ABSTRACT.
INTRODUCTION..
MATERIALS AND METHODS
RESULTS AND DISCUSSION.
ACKNOWLEDGEMENTS
TABLES REFERENCES
Chapter 6 Genetic diversity in the Eucalyptus stem pathogen Teratosphaerizuluensis
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
REFERENCES
TABLES AND FIGURES
Chapter 7 Unexpected genetic diversity revealed in the Eucalyptus canker pathogen Teratosphaeria gauchensis
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
TABLES REFERENCES
APPENDIX 
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