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Aurapex penicillata gen. sp. nov. from native Miconia theaezans and Tibouchina spp. in Colombia

     Department of Microbiology and Plant Pathology, Forestry & Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0002, South Africa

Henrietta Myburg

     Department of Genetics, Forestry & Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0002, South Africa

Carlos A. Rodas ²

     Department of Microbiology and Plant Pathology, Forestry & Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0002, South Africa

Brenda D. Wingfield

     Department of Genetics, Forestry & Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0002, South Africa

Michael J. Wingfield

     Department of Microbiology and Plant Pathology, Forestry & Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0002, South Africa

	ABSTRACT

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Conidiomata of a fungus resembling Chrysoporthe cubensis, a serious canker pathogen of Eucalyptus spp. (Myrtaceae, Myrtales) in tropical and subtropical parts of the world, was found on Eucalyptus grandis in Colombia. Fruiting structures of the fungus could be distinguished from those of C. cubensis by their distinctly orange conidiomatal necks. This fungus also was found on several plant species native to Colombia including Tibouchina urvilleana, T. lepidota and Miconia theaezans (Melastomataceae, Myrtales). Morphological comparisons, as well as those based on sequences of the ITS1/ITS2 region of the ribosomal DNA repeat and the ß-tubulin gene, were used to characterize this fungus. Its pathogenicity was assessed on various plants from which it has been collected, either in field or greenhouse trials. Phylogenetic analyses showed that isolates reside in a clade distinct from the four clades accommodating Chrysoporthe, Cryphonectria, Endothia and Rostraureum. Members of this clade are distinguished by the presence of orange conidiomatal necks with black bases and a unique internal stromatal structure. No teleomorph has been found for this fungus, for which we have provided the name Aurapex penicillata gen. sp. nov. A. penicillata produced only small lesions after inoculation on young T. urvilleana, M. theaezans and E. grandis trees and appears not to be a serious pathogen.

Key words: Chrysoporthe, Diaporthales, Eucalyptus, Melastomataceae

	INTRODUCTION

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The Melastomataceae represents a family of flowering plants common to neotropical America and Hawaii (Everett 1981). This family resides in the Myrtales, which also accommodates the Myrtaceae (Conti et al 1996). The Myrtaceae includes the genus Eucalyptus, many species of which are grown as a source of pulp and timber in plantations around the world (Turnbul 2000).

Chrysoporthe cubensis (Bruner) Gryzenh. & M. J. Wingf. (formerly Cryphonectria) is a serious canker pathogen of Eucalyptus spp. (Boerboom and Maas 1970, Hodges 1980, Sharma et al 1985, Wingfield 2003) and Syzygium aromaticum (L.) Murr. & Perry (clove, also Myrtaceae) (Hodges et al 1986), in the tropics and subtropics. Intriguingly, this pathogen recently has been shown to cause disease on members of the Melastomataceae such as Miconia theaezans (Bonpl.) Cogn. (niguito) and Miconia rubiginosa (Bonpl.) DC. (mortiño) in Colombia (Rodas et al 2005). A second fungus, Chrysoporthella hodgesiana Gryzenh. & M. J. Wingf., a species of Chrysoporthe based on phylogenetic data but known only by its anamorph, also occurs on Colombian Melastomataceae such as M. theaezans (Rodas et al 2005), Tibouchina urvilleana Cogn., T. lepidota Baill. and T. semidecandra Cogn. (Gryzenhout et al 2004). Recognition of C. cubensis on hosts residing in the Melastomataceae has substantially altered views regarding the origin and distribution of this important tree pathogen (Wingfield et al 2001, Wingfield 2003, Rodas et al 2005).

During the course of surveys in Colombia to assess the occurrence of Chrysoporthe spp. on trees other than Eucalyptus spp. a fungus similar to C. cubensis and Chrysop. hodgesiana was found on Tibouchina spp. These trees were planted as ornamentals in parks or on farms. The unknown fungus produced only conidiomata that were black and pyriform, in a shape reminiscent of Chrysoporthe spp. The fruiting bodies, however, differed from those of Chrysoporthe spp. in that the apices of the conidiomatal necks were orange. Subsequent surveys led to the discovery of the fungus on native M. theaezans as well as on E. grandis. The aims of this study were to define the phylogenetic position of this fungus using DNA sequence comparisons as well as to produce a taxonomic description and generic key. In addition the pathogenicity of the new fungus was assessed in greenhouse and field inoculation experiments.

	MATERIALS AND METHODS

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Symptoms and collection of samples.— – Structures of the unknown fungus were found first in 1996 on T. urvilleana and T. lepidota in Colombia. These trees were growing in La Culebra Park in El Peñol, on a private farm near Granada (Antioquia Province), and on the Argentina farm of Smurfit Cartón de Colombia near Riosucio (Caldas Province). In a subsequent disease survey in 1998, similar fruiting structures were found on Miconia theaezans occurring in native vegetation on La Selva farm near Pereira (Risaralda Province). In 2002 this fungus was discovered for the first time on basal cankers on E. grandis on the Libano farm near Pereira as well as on M. theaezans at the same location.

The fruiting structures of the unknown fungus occurred around the periphery of cankers on the stems and branches of trees, which occasionally led to branch die-back. In some cases fruiting structures of C. cubensis and Chrysop. hodgesiana occurred on the same plant. On E. grandis fruiting structures of the fungus also were found on branches that were in the process of senescence, and the fungus also appeared to colonize branch stubs.

Bark specimens containing conidiomata were collected from cankers and taken to the laboratory for isolation. Single conidial isolates were obtained from spore suspensions on 2% malt-extract agar (MEA) (20 g Biolab malt extract, 15 g Biolab agar, 1 L water, Merck, Midrand, South Africa) and incubated at 25 C. The cultures have been maintained in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa, and representative isolates have been deposited with the Centraalbureau voor Schimmel-cultures (CBS), Utrecht, Netherlands (TABLE I). Original bark specimens from which isolations had been made were used for morphological characterization and have been deposited in the herbarium of the National Collection of Fungi, Pretoria, South Africa (PREM).

Culture morphology of the ex holotype strain CMW 10030 and CMW 11296 (TABLE I), was characterized on MEA (20 g/L malt-extract agar, Biolab, Merck). These studies were conducted using the technique described by Venter et al (2002). Growth tests were conducted at 15–35 C at 5 C intervals, and cultures were grown in the dark.

DNA sequence comparisons.— – Representative isolates of the fungus from Colombia were used in the DNA sequence comparisons (TABLE I). Sequences of C. cubensis isolates from Miconia and Eucalyptus spp. in Colombia (Gryzenhout et al 2004, Rodas et al 2005), and Chrysop. hodgesiana isolates from Miconia and Tibouchina spp. in Colombia (Wingfield et al 2001, Gryzenhout et al 2004, Rodas et al 2005) also were used. Sequences for isolates of C. cubensis from other parts of the world, those of C. austroafricana (Myburg et al 2002a, 2003) and those for recognized members of Cryphonectria and Endothia, which are closely related to Chrysoporthe (Venter et al 2002, Myburg et al 2004a, b), also were included (TABLE I). Sequences from the recently described Rostraureum tropicale Gryzenh. & M. J. Wingf., a pathogen of Terminalia in Ecuador (Gryzenhout et al 2005), were included in the dataset. Rostraureum contains the fungus previously known as Cryphonectria longirostris (Earle) Micales & Stipes (Gryzenhout et al 2005). Two Diaporthe ambigua Nitschke isolates, which also reside in the Diaporthales but have been shown to reside in a different family to that of Cryphonectria and related taxa (Castlebury et al 2002), were used as a single outgroup to root the phylogenetic tree generated in this study. The sequence matrix (study accession number = S1128, matrix accession number = M1935) from Myburg et al (2004a) was used as template for the alignment.

DNA was isolated from the fungi as described in Myburg et al (1999). PCR amplification of the ITS1, conserved 5.8S and ITS2 regions of the rRNA operon as well as two regions in the ß-tubulin gene was performed as described respectively in Myburg et al (1999) and Myburg et al (2002a). Primer pairs ITS1/ITS4 (White et al 1990) were used for the ITS1/ITS2 region, and primer pairs Bt1a/Bt1b and Bt2a/ Bt2b respectively (Glass & Donaldson 1995) were used to amplify two ß-tubulin gene regions. The PCR products were purified with a QIAquick PCR Purification Kit (QIAGEN GmbH, Hilden, Germany). The purified PCR products were sequenced in both directions with the same primers that were used in the amplification reactions. Sequencing reactions were performed with a PRISM^TM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Warrington, UK). Nucleotide sequence data were generated with an ABI PRISM 3100^TM automated DNA sequencer. The raw sequence data were manipulated with the Sequence Navigator version 1.0.1 software package (Perkin-Elmer Applied BioSystems, Foster City, California).

Nucleotide sequences were aligned manually by inserting gaps. Gaps were treated as newstate in the parsimony analyses and as missing in the distance analyses. Phylogenetic analyses were performed with PAUP* (phylogenetic analysis using parsimony) version 4.0b10 (Swofford 2002). A partition homogeneity test (Farris et al 1994) was used to determine whether the ribosomal rRNA (ITS1, 5.8S, ITS2) partition and ß-tubulin partition could be combined in the phylogenetic analyses. The aligned sequences were analyzed with parsimony by heuristic searches, with the tree-bisection-reconnection (TBR) and MULTREES options (saving all optimal trees) in effect, and sequences added randomly (100 additions). Uninformative sites were excluded and sites were reweighted according to their individual consistency indices (CI) to reduce the number of trees obtained. A distance analysis was performed with the Tamura-Nei parameter model (Tamura and Nei 1993) with adjusted settings (proportion of invariable site [I] = 0.4334; gamma distribution [G] = 0.4592; base frequency 0.2044, 0.3191, 0.2551; rate matrix 1.00, 2.2953, 1.00, 1.00, 4.5168). This model was chosen as suggested by Modeltest version 3.5 (Posada and Crandall 1998). Tree branch supports were assessed with a 1000-replicate bootstrap analysis. GenBank accession numbers of sequences generated in this study as well as those from previous phylogenetic studies are listed (TABLE I). The DNA sequence alignment has been deposited in TreeBASE (study accession number = 1489, matrix accession number = M2674).

Pathogenicity tests.— – Two isolates of the newly described fungus (CMW 10031, CMW 10034) from M. theaezans were compared with isolates of Chrysoporthe spp. in a contained pathogenicity trial. One Chrysoporthe isolate (CMW 2113) represented C. austroafricana and has been used in previous pathogenicity tests (Myburg et al 2002b, van Heerden and Wingfield 2002). The other Chrysoporthe isolate (CMW 10639) represented C. cubensis from E. grandis in Colombia (Gryzenhout et al 2004).

Pathogenicity of the isolates was compared on 10 seedlings of T. urvilleana and a susceptible E. grandis clone (ZG14) in a custom-built phytotron. These trees were approximately 1.5 m tall and 7 mo old, and they were exposed to natural light conditions and an average daily temperature of ~25 C. Ten trees were inoculated at a constant height (~30 cm above the ground) with sterile water agar (WA, Biolab, Merck) plugs to serve as negative controls. Wounds were made on stems with a cork borer (6 mm diam) to expose the cambium. Disks of the same size were taken from the actively growing edges of colonies and inserted into the wounds with the mycelium facing inward. Wounds then were covered with plastic film to prevent desiccation and contamination. Trees were inoculated in Jun 2002 and lesion development was evaluated after 6 wk by measuring lesion lengths below the outer bark.

A field trial to consider the pathogenicity of the fungus under natural conditions was carried out on M. theaezans trees and a susceptible clone (018) of E. grandis on the Libano farm, Pereira, Risaralda, Colombia. Twenty E. grandis and the same number of M. theaezans trees were inoculated with isolate CMW 10031 from M. theaezans in Colombia. Trees of the E. grandis clone were approximately 18 mo old (~9 m high), while the M. theaezans trees formed part of the natural vegetation growing near the E. grandis trees and were of unknown age (~4–6 m high). Ten trees of each species were inoculated with WA as negative controls. Inoculations were carried out in the same way as those described for the phytotron inoculation, except that the inoculation wounds were 4 mm diam and covered with masking tape. The trees were inoculated in Jun 2002 and results were evaluated 12 wk later in Sep 2002. The lengths of the lesions produced in the cambium were measured and compared after removal of the bark. Data were compared using a one-way analysis of variance (ANOVA) computed with the SAS software package, v. 6 (2002). Mean lesion sizes together with 95% confidence limits were presented graphically.

	RESULTS

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Morphology.— – Conidiomata of the fungus on bark specimens of M. theaezans, T. urvilleana and E. grandis were pyriform and superficial with fuscous black bases and most similar to fruiting structures of C. cubensis (Hodges 1980, Myburg et al 2002a, 2003, Gryzenhout et al 2004). These structures thus were different to the anamorph structures of all genera in the Diaporthales with orange fruiting structures (i.e. Cryphonectria, Endothia and Rostraureum), all of which have completely orange conidiomata (Myburg et al 2004a, Gryzenhout et al 2005). Conidiomata had fuscous-black, globose bases with long, slender necks (FIGS. 1A, 2A) that might be confused with those of C. cubensis in the absence of the orange neck apices. The conidiomatal base cells were textura globosa, umber to sienna when sectioned, with thick walls, while the inner cells were prosenchymatous (FIG. 1C). This is similar to the basal tissue of conidiomata of C. cubensis (Myburg et al 2002a, 2003; Gryzenhout et al 2004). The minute ([2.5–]3–4[–4.5] x 1–1.5[–2] µm), aseptate conidia (FIGS. 1I, 2C) also were similar to those of C. cubensis (Hodges 1980; Myburg et al 2002a, 2003; Gryzenhout et al 2004).

View larger version (195K): [in this window] [in a new window]   FIG. 1. Fruiting structures of Aurapex penicillata. A. Conidiomata on bark and in section (B). C. Tissue at base of conidioma. D. Protrusions in locule lining. E. Tissue of neck and periphyses in ostiolar canal (F). G. Tissue of neck apex. H. Conidiophores. I. Conidia. Bars: A–B = 100 µm, C–G = 20 µm, H–I = 10 µm.

View larger version (23K): [in this window] [in a new window]   FIG. 2. Schematic drawings of Aurapex penicillata. A. Conidiomata on bark. B. Section through conidioma. C. Conidiophores and conidia. Bars: A–B = 100 µm, C = 10 µm.

DNA sequence comparisons.— – The PCR products generated for the ribosomal and two ß-tubulin gene regions were respectively 550–600 bp in size. The PHT test (P = 0.182) indicated no significant conflict between the two datasets for these gene regions, which thus were combined in the phylogenetic analyses. There were also no strongly supported conflicts between the trees obtained for the two gene regions separately. The sequence dataset included 32 taxa of which the two D. ambigua isolates represented a single outgroup taxon. The ß-tubulin dataset (total 952 bp including both regions) consisted of 543 constant, 32 variable parsimony uninformative and 377 variable and parsimony informative characters (g1 = –0.780817). The ITS dataset (total 573 bp) consisted of 338 constant, 29 variable parsimony uninformative and 206 variable and parsimony-informative characters (g1 = –0.863275). The combined set amounted to a total of 1525 characters. The heuristic search produced 10 trees (tree length = 1095.3 steps, consistency index of = 0.805, retention index of = 0.918) that differed only in branch length for isolates. The tree obtained with distance analyses showed the same clades as the trees obtained with parsimony, one of which was chosen for presentation (FIG. 3). The same groupings of isolates with equally high bootstrap support also were obtained when ambiguously aligned portions, which mostly represented the introns of the ß-tubulin dataset and the ITS1 region, were excluded from the analyses.

View larger version (26K): [in this window] [in a new window]   FIG. 3. One of 10 most parsimonious trees obtained from a combined dataset comprising ribosomal and ß-tubulin gene sequences (tree length = 1095.3 steps, consistency index = 0.805, retention index = 0.918). Isolates representing the genera Chrysoporthe, Rostraureum, Aurapex, Cryphonectria and Endothia are represented. Confidence levels >70% determined by a 1000 replicate bootstrap analysis are indicated on the tree branch nodes. Two sequences for Diaporthe ambigua were used as outgroup.

	TAXONOMY

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The fungus found on M. theaezans, T. urvilleana and E. grandis from Colombia has clearly defined morphological features that distinguish it from Chrysoporthella, the anamorph genus of Chrysoporthe, to which it is morphologically most similar (TABLE II). These features are unlike those of any other coelomycete genus. The fuscous black conidiomata are also different from the uniformly orange fruiting structures of Cryphonectria, Endothia and Rostraureum, although the necks of the undescribed fungus are orange. These differences are supported by DNA sequence data showing that isolates of the morphologically distinct fungus from Colombia group separately from those representing Chrysoporthe, Cryphonectria, Endothia and Rostraureum.

No teleomorph was found for the fungus considered in this study. The distinct grouping of the fungus from Colombia, however, indicates that the fungus represents a distinct genus and not the anamorph of an already existing genus. Phylogenetic data present clear evidence of its affinity to members of Cryphonectria and allied genera residing in the Diaporthales. In the absence of a teleomorph the fungus from Colombia cannot be described in an ascomycete genus (ICBN, Art. 59.2, Greuter et al 2000). It thus is described as a new species in a new mitosporic genus, and the following description is provided.

Aurapex Gryzenh. & M. J. Wingf., gen. nov.

Etymology. – aureus, golden, and apex, top, refers to the golden colored tips of the conidiomata.

Conidiomata globosa vel pyriformia, basibus fusconigris collis aurantiacis, superficialia. Collum e textura porrecta factum, cellulis parietalibus ostioli gracilioribus, in ora colli quadratis, intra canales ostiolares cum filamentis non septatis. Conidiophorae cylindricae vel ampulliformes, hyalinae. Cellulae conidiogenae phialidicae. Conidia obtusa, hyalina, non septata.

Conidiomata eustromatic, globose to pyriform base with one to several, long, cylindrical to attenuated necks with orange tips, superficial to slightly immersed, fuscous-black. Tissue at the edges of conidiomal bases of textura globulosa, with elongated cells adjacent to conidial lining and prosenchymatous tissue occurring in the center of the basal tissue. Tissue of necks made up of textura porrecta with cells lining the ostiole thinner, cells at edge of necks consisting of square cells. Conidiophores cylindrical to flask-shaped, hyaline, occasionally septate with or without lateral branches. Conidiogenous cells phialidic. Conidia obtuse, hyaline, aseptate.

Species typica: Aurapex penicillata

Aurapex penicillata Gryzenh. & M. J. Wingf., sp. nov.

FIGS. 1–2

Etymology. – penicillus, a painter’s brush, refers to the brush-like protrusions formed by the lining of the conidial locules.

Conidiomata pyriformia cum collis, superficialia, basibus fusconigris collis aurantiacis, textura parietali loculorum prominentia e 3-circiter 15 cellulis formans. Textura colli in ora e cellulis quadratis, intus e textura porecta facta, intra canales ostiolares cum filamentis non septatis. Conidiophorae cylindricae vel ampulliformes apicibus attenuatis, hyalinae. Cellulae conidiogenae phialidicae. Conidia (2.5–)3–4(–4.5) x 1–1.5(–2) µm, obtusa, non septata, hyalina, in forma guttarum sporarum coccinearum exsudata. Coloniae cum hyphis aeriis sparsis, albocremet, intus atro-olivaceae vel isabellinae, celeriter crescentes. temperatura optima 25 C.

Conidiomata single or aggregated, eustromatic, with globose to pyriform bases and attenuated or cylindrical necks, base 120–400 µm high, 300–700 µm wide above bark surface, necks up to ~800 µm long depending on environmental conditions, 80–225 µm wide, conidiomata superficial to slightly immersed, bases fuscous-black with tips of necks orange (FIGS. 1A, B; 2A, B). Unilocular or multilocular (FIGS. 1B, 2B), locules up to 360 µm diam at widest point, locule lining producing conidiophores forming protrusions consisting of 3 to ~15 cells (FIGS. 1B, D; 2B), locules opening through 1–3 necks, each either connected to a single locule or to more than one locule. Tissue of base complex with thick-walled cells, textura globulosa, umber to sienna at edge, cells around the locules sienna to hazel, larger and more elongated, and almost white prosenchymatous tissue occurring between the edge and the locule (FIG. 1C). Neck tissue consisting of hazel, double-walled, square cells at the edge, with the cells lining the ostiole thinner and those at the center of textura porrecta tissue (FIG. 1E, F), long, aseptate filaments, similar to periphyses, occurring inside the ostiolar canals (FIG. 1F), tip of necks of textura epidermoidea, containing orange crystals (FIG. 1G). Conidiophores (6–) 7.5–13.5(–18.5) x (0.5–)1–1.5(–2) µm, cylindrical or flask-shaped with attenuated apices, occasionally with separating septa and branching, hyaline (FIGS. 1H, 2C). Conidiogenous cells phialidic, determinate, apical or lateral on branches, collaret and periclinal thickening inconspicuous (FIGS. 1H, 2C). Conidia (2.5–)3–4(–4.5) x 1–1.5(–2) µm, obtuse, aseptate, hyaline (FIGS. 1I, 2C), exuded as scarlet spore droplets.

Culture morphology fluffy with few aerial hyphae, creamy white with a dark olivaceous to isabelline interior, margins even, conidiomata occasionally produced in mature cultures, optimum growth at 25 C, isolates covering the surface of 90 mm plates on Day 6 at the optimum temperature.

Holotype. – COLOMBIA. RISARALDA: Pereira, Libano farm, 75°35'49''W and 4°43'13''N, 2102 msal. Bark of Miconia theaezans, Sep 2002, C.A. Rodas (PREM 57520, ex-type culture CMW 10030 = CBS 115740, additional cultures CMW 10031, CMW 10034, CMW 10035 = CBS 115742).

Additional material examined. – COLOMBIA. QUINDIO: Salento, Andes farm, 75°33'16''W and 4°41'08''N, 2102 masl. Bark of Miconia theaezans, May 2000, M.J. Wingfield (PREM 58576, living cultures CMW 11296 = CBS 115801). RISARALDA: Pereira, La Selva farm, 75°35'34''W and 4°47'26''N, 2048 msal. Bark of Miconia theaezans, Nov 1998, C.A. Rodas (PREM 58572). Libano farm, 75°35'49''W and 4°43'13''N, 2102 msal. Bark of Eucalyptus grandis, Sep 2002, C.A. Rodas (PREM 58578). ANTIOQUIA: Granada, Granada farm, 75°8'10''W and 6°6'52''N, 2050 msal. Bark of Tibouchina urvilleana, Nov 1998, C.A. Rodas (PREM 58573). CALDAS: Riosucio, La Argentina farm, 75°44'55''W and 5°22'25''N, 2247 msal. Bark of Tibouchina urvilleana, Nov 1998, C.A. Rodas (PREM 58574, PREM 58575). VALLE, Darien, Cedral farm, 76°26'06''W and 3°57'06''N, 1825 masl. Bark of Eucalyptus grandis, Dec 2001, C.A. Rodas (PREM 58577).

Known distribution. – Colombia

Habitat. – Miconia theaezans, Tibouchina urvilleana, Tibouchina lepidota, Eucalyptus grandis

This key is provided to differentiate Aurapex from closely related genera.

	KEY TO CRYPHONECTRIA, ENDOTHIA, CHRYSOPORTHELLA, ROSTRAUREUM AND AURAPEX

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1. Base of anamorph fruiting structures fuscous-black 2 1. Anamorph fruiting structures completely orange 3     2. Conidiomata uniformly fuscous-black, locule lining even. Chrysoporthella     2. Conidiomata fuscous-black but with tip of neck orange, locule lining have brush-like protrusions Aurapex         3. Conidiomata rostrate, superficial Rostraureum         3. Conidiomata pulvinate 4             4. Conidiomata semi-immersed, ascospores uniseptate Cryphonectria             4. Conidiomata superficial, ascospores aseptate Endothia

Pathogenicity.— – Isolates of A. penicillata (CMW 10031, CMW 10034) produced small lesions on both T. urvilleana and the E. grandis clone in the phytotron trial. These lesions did not differ significantly from the control inoculations (FIG. 4). Conidiomata were produced abundantly on the surfaces of the lesions. Lesions associated with A. penicillata were significantly smaller (P < 0.0001) than those associated with C. cubensis and C. austroafricana on E. grandis and T. urvilleana with those of C. austroafricana (CMW 2113) on E. grandis longest (FIG. 4). The latter isolate was also the only one observed to girdle inoculated stems resulting in the production of epicormic shoots below the sites of inoculation.

View larger version (14K): [in this window] [in a new window]   FIG. 4. Mean lesion length in Tibouchina urvilleana and a ZG14 clone of Eucalyptus grandis resulting from greenhouse inoculations with Aurapex penicillata (CMW 10031, CMW 10034), Chrysoporthe cubensis (CMW 10639), Chrysoporthe austroafricana (CMW 2113) and a negative control. Means are shown with 95% confidence limits.

View larger version (12K): [in this window] [in a new window]   FIG. 5. Comparison of mean lesion length resulting from field inoculations with Aurapex penicillata (CMW 10031) and a negative control in Miconia theaezans and a susceptible clone (018) of Eucalyptus grandis. Means are shown with 95% confidence limits.

	DISCUSSION

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The distinction of A. penicillata based on DNA sequence comparisons as a genus separate from Cryphonectria, Chrysoporthe, Endothia and Rostraureum, and not an anamorph genus of one of the existing genera, is well supported by morphological characteristics. Although the teleomorph is unknown the morphological characteristics of the asexual state differ substantially from those of its closest relatives. Conidiomata of A. penicillata are fuscous-black, superficial and pyriform with attenuated necks. These resemble the conidiomata of Chrysoporthella, the anamorph of Chrysoporthe that also has fuscous black conidiomata. Aurapex, however, can be distinguished easily from Chrysoporthella based on the distinctly orange tips of the necks and by its unique stromatal tissue. This new fungus also can be distinguished from the conidiomata of Cryphonectria, Endothia and Rostraureum spp., which are completely orange. These anamorph differences are consistent with those found in previous studies (Venter et al 2002, Myburg et al 2004a, Gryzenhout et al 2005). These studies showed that stromatic and anamorph morphology are the most informative morphological characters that support the different phylogenetic assemblages, even in the absence of sexual structures. Recognition of these phylogenetic assemblages as different genera is strongly supported by the fact that they are morphologically distinct and they would not comfortably reside in a single genus.

Aurapex penicillata could be confused with the serious pathogens C. cubensis and Chrysop. hodgesiana, which occur on the same hosts and in the same region. This is especially so when the characteristic orange necks of A. penicillata become dislodged from their conidiomatal bases. Moreover fruiting structures of these different fungi can occur on the same piece of bark. Of these three fungi only C. cubensis is known to cause serious disease on Eucalyptus (Wingfield 2003, Gryzenhout et al 2004) although Chrysop. hodgesiana also can infect Eucalyptus trees in artificial inoculations (Wingfield et al 2001, Gryzenhout et al 2004).

Aurapex penicillata gave rise to lesions on E. grandis in pathogenicity tests, but there was no evidence of significant pathogenicity, at least in comparison to C. cubensis. Although A. penicillata occurs on E. grandis under natural conditions, the pathogen appears to be mainly associated with dead branch stubs. Because of differences in pathogenicity and importance it is necessary to identify C. cubensis, Chrysop. hodgesiana and A. penicillata correctly in Eucalyptus plantation disease surveys.

Pathogenicity tests conducted in this study should be seen as preliminary because they were limited by the lack of a complete series of known hosts of A. penicillata. Our primary objective was to assess pathogenicity, especially given the fact that the highly pathogenic C. cubensis now has been found to cause severe disease on Melastomataceae native to South America (Rodas et al 2005). Although preliminary our results show that A. penicillata probably poses no threat to Eucalyptus or Melastomatacea. Furthermore the common occurrence of the fungus on native Melastomataceae in Colombia adds substance to our view that it is native to hosts in that family. Its ability to infect Eucalyptus spp. is probably opportunistic and related to the high levels of inoculum on surrounding native vegetation in Colombia.

	ACKNOWLEDGMENTS

 
We thank Dr B.E. Eisenberg for the statistical analyses presented in this study as well as Liliana Perafan for additional statistical support. Dr H.F. Glen of the National Botanical Institute of Pretoria, Pretoria, provided the Latin description and assisted us in choosing a name for the new fungus. Dr Luis F. Osorio and Mr Ramon Arbelaez provided technical assistance during sampling and field trials in Colombia. This study was made possible through financial support of Smurfit Carton de Colombia, as well as the National Research Foundation (NRF), members of the Tree Pathology Co-operative Programme (TPCP), the THRIP support programme of the Department of Trade and Industry and the Department of Science and Technology/ NRF Centre of Excellence in Tree Health Biotechnology (CTHB), South Africa.

	FOOTNOTES

 
Accepted for publication December 12, 2005.

² Current address: Smurfit Cartón de Colombia, Investigación Forestal, Carrera 4 Nu. 10–44, oficina 1106 Edificio Plaza de Caicedo, Cali, Valle, Colombia.

¹ Corresponding author. E-mail: Marieka.Gryzenhout{at}fabi.up.ac.za

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY KEY TO CRYPHONECTRIA, ENDOTHIA,... DISCUSSION LITERATURE CITED

 
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