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Keywords:

  • canker pathogens;
  • Cape beech;
  • Myrsinaceae;
  • tree death;
  • Western Cape

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Recent disease surveys in the Western Cape province of South Africa have revealed a previously unknown and serious stem canker disease on native Rapanea melanophloeos (Myrsinaceae, Ericales) trees. Cankers commonly result in the death of branches or entire stems. Fruiting structures typical of fungi in the Cryphonectriaceae were observed on the surfaces of cankers. In this study, the fungus was identified and its pathogenicity to R. melanophloeos was tested. Multigene phylogenetic analyses based on DNA sequences of the partial LSU gene, ITS region of the nuclear ribosomal DNA gene and two regions of the β-tubulin (BT) gene, showed that the fungus represents a formerly undescribed genus and species in the Cryphonectriaceae. The fungus was also morphologically distinct from other genera in this family. Inoculation trials showed that the fungus described here as Immersiporthe knoxdaviesiana gen. et sp. nov. is an aggressive pathogen of R. melanophloeos trees.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The Cryphonectriaceae accommodates fungal genera in the Cryphonectria–Endothia complex and includes many important tree pathogens (Gryzenhout et al., 2006a). Subsequent to the description of this family, several new genera and species were described and it currently accommodates 14 genera (Gryzenhout et al., 2006b,c, 2009, 2010; Nakabonge et al., 2006; Cheewangkoon et al., 2009; Begoude et al., 2010; Chen et al., 2011; Vermeulen et al., 2011). The family includes the notorious chestnut blight pathogen, Cryphonectria parasitica (Anagnostakis, 1987, 1992; Heiniger & Rigling, 1994), and species of Chrysoporthe that cause stem cankers and death of Eucalyptus trees (Wingfield, 2003; Gryzenhout et al., 2009).

Five genera of Cryphonectriaceae have been reported from the African continent, including Aurifilum (Begoude et al., 2010), Celoporthe (Nakabonge et al., 2006), Chrysoporthe (Gryzenhout et al., 2004, 2009), Holocryphia (Gryzenhout et al., 2006b) and Latruncellus (Vermeulen et al., 2011). Aurifilum marmelostoma is known only from Cameroon where it causes cankers on native Terminalia ivorensis (Combretaceae, Myrtales) and non-native T. mantaly (Begoude et al., 2010). Celoporthe dispersa is a pathogen of native Heteropyxis canescens (Heteropyxidaceae, Myrtales) and Syzygium cordatum (Myrtaceae, Myrtales) as well as non-native Tibouchina granulosa (Melastomataceae, Myrtales) in southern Africa (Nakabonge et al., 2006; Vermeulen et al., 2011). Five species of Chrysoporthe, Chr. austroafricana, Chr. cubensis, Chr. deuterocubensis, Chr. syzygiicola and Chr. zambiensis are known in Africa where they occur on trees in the Myrtales including native Syzygium spp. (Myrtaceae), introduced Ti. granulosa (Melastomataceae) and Eucalyptus spp. (Myrtaceae) (Gryzenhout et al., 2009; Chungu et al., 2010). In Africa, Holocryphia eucalypti is associated with cankers on non-native Eucalyptus trees in South Africa, Swaziland and Uganda (Van der Westhuizen et al., 1993; Gryzenhout et al., 2003; Roux & Nakabonge, 2010; Vermeulen et al., 2011). Latruncellus aurorae was most recently described causing cankers on native Galpinia transvaalica (Lythraceae, Myrtales) in Swaziland (Vermeulen et al., 2011).

Rapanea melanophloeos (Myrsinaceae, Ericales), commonly known as Cape beech, is a dense, evergreen tree (Van Wyk & Van Wyk, 1997). The natural range of this tree is from Cape Town in the south, to Zambia in the north in Southern Africa (Van Wyk & Van Wyk, 1997) and it is also a popular ornamental in gardens (Coates Palgrave, 1977; Van Wyk & Van Wyk, 1997). There are no reports of serious disease problems on R. melanophloeos.

During recent tree health surveys in the Western Cape province of South Africa, a serious stem disease was observed on R. melanophloeos in a botanical garden. Infections resulted in cankers on branches and stems of trees, often leading to tree death. Fruiting structures typical of fungi in the Cryphonectriaceae were observed on the surfaces of cankers. The aim of this study was to identify the causal agent of this disease based on phylogenetic analysis as well as morphological characteristics of the fungus, and by conducting pathogenicity tests on healthy R. melanophloeos.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Disease symptoms, samples and isolations

Rapanea melanophloeos trees (Fig. 1a) growing in the Harold Porter National Botanical Garden (18°55′56″E and 34°20′99″S), Western Cape Province, South Africa were first observed to have serious cankers on their branches and stems in December 2009. Surveys were subsequently conducted in February and March of 2011 when the disease problem appeared to have increased considerably. Disease symptoms included dying branches (Fig. 1a), cracked bark (Fig. 1b), and cankers that often girdled infected stems and branches (Fig. 1c). Cankers commonly originated at branch attachment points on trees (Fig. 1c) and usually had orange cirrhi exuding from their surfaces (Fig. 1d,e). Branch and stem sections proximal to the cankers were typically dead or dying (Fig. 1a,b,c). The disease was widespread on R. melanophloeos trees throughout the garden.

image

Figure 1. Rapanea melanophloeos and symptoms associated with infection by Immersiporthe knoxdaviesiana. (a) Native R. melanophloeos tree in the Western Cape province of South Africa, with dying branches caused by I. knoxdaviesiana; (b) bark of a R. melanophloeos tree showing cracks after infection by I. knoxdaviesiana; (c) typical stem canker and spores oozing from infected tissue; (d) fruiting structures with oozing spores on a stem lesion; (e) fruiting structures with oozing spores in the form of long golden-coloured cirrhi.

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Sections of diseased bark containing fruiting structures were removed from trees and transported to the laboratory for morphological assessment and fungal isolation. Single tendrils of spores from fruiting bodies or spore masses from within fruiting bodies were transferred to 2% malt extract agar (MEA; Biolab, Merck; 20 g Biolab malt extract, 20 g Biolab agar, 1 L water) and incubated at 25°C in the dark. Single hyphal tips were transferred to 2% MEA to obtain pure cultures. One isolate was obtained from each of 33 diseased trees. Representative cultures are maintained in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa. Representative isolates were also deposited with the Centraalbureau voor Schimmelcultures (CBS), Utrecht, the Netherlands (Table 1). Original bark specimens bearing fungal fruiting structures that could be connected to representative isolates were deposited in the National Collection of Fungi (PREM), Pretoria, South Africa.

Table 1. Isolates sequenced and used in phylogenetic analyses and pathogenicity tests in this study
IdentityIsolate no.aOther no.aHostLocationCollectorGenBankcGenBankGenBankGenBank
LSUITSBT1BT2
  1. aDesignation of isolates and culture collections: CMW = Tree Protection Co-operative Program, Forestry and Agricultural Biotechnology Institute, University of Pretoria, South Africa; CBS = Centraalbureau voor Schimmelcultures, Utrecht, Netherlands.

  2. bIsolates used in field pathogenicity trials.

  3. cGenBank accession numbers in bold indicate nucleotide sequences generated in this study.

Cryphonectria macrospora CMW10914  Castanopsis cuspidata JapanT. Kobayashi JQ862749 AY697942 AY697973 AY697974
C. decipiens CMW10436CBS165.30 Quercus suber PortugalB. d’Oliviera JQ862750 AF452117 AF525703 AF525710
Holocryphia eucalypti CMW7033CBS115842 Eucalyptus grandis KwaZulu/Natal, South AfricaM. Venter JQ862751 JQ862761 JQ862781 JQ862771
H. eucalypti CMW7035  E. saligna KwaZulu/Natal, South AfricaM. Venter JQ862752 JQ862762 JQ862782 JQ862772
H. eucalypti CMW7037CBS119477 E. delegatensis New South Wales, AustraliaK. Old JQ862753 JQ862763 JQ862783 JQ862773
H. eucalypti CMW7038  E. globulus Western Australia, AustraliaM.J. Wingfield JQ862754 JQ862764 JQ862784 JQ862774
Immersiporthe knoxdaviesiana CMW37314bCBS132862 Rapanea melanophloeos Western Cape, South AfricaM.J. Wingfield & J. Roux JQ862755 JQ862765 JQ862785 JQ862775
I. knoxdaviesiana CMW37315bCBS132863 R. melanophloeos Western Cape, South AfricaM.J. Wingfield & J. Roux JQ862756 JQ862766 JQ862786 JQ862776
I. knoxdaviesiana CMW37316b  R. melanophloeos Western Cape, South AfricaM.J. Wingfield & J. Roux JQ862757 JQ862767 JQ862787 JQ862777
I. knoxdaviesiana CMW37317b  R. melanophloeos Western Cape, South AfricaM.J. Wingfield & J. Roux JQ862758 JQ862768 JQ862788 JQ862778
I. knoxdaviesiana CMW37318CBS132864 R. melanophloeos Western Cape, South AfricaJ. Roux, S.F. Chen & F. Roets JQ862759 JQ862769 JQ862789 JQ862779
I. knoxdaviesiana CMW37319  R. melanophloeos Western Cape, South AfricaJ. Roux, S.F. Chen & F. Roets JQ862760 JQ862770 JQ862790 JQ862780

DNA extraction, PCR and sequence reactions

Representative isolates, collected from different trees at different sites in the Garden (Table 1) were randomly selected and used for DNA sequence comparisons. Isolates were grown on 2% MEA at 25°C for 1 week prior to DNA extraction. Actively growing mycelium for each isolate was scraped from the surface of MEA media with a sterile scalpel and transferred to 1·5 mL Eppendorf tubes. DNA was extracted from mycelium using the methods described by Myburg et al. (1999). Samples were treated with 3 μL RNase (1 mg mL−1) and left for 12 h at room temperature to degrade any RNA present. DNA was separated using electrophoresis on a 1% agarose gel, stained with GelRed (Biotium; 3 μL DNA extraction product with 2 μL GelRed), and visualized under UV light.

The conserved nuclear large subunit (LSU) ribosomal DNA, the β-tubulin gene regions 1 (BT1) and 2 (BT2) and the internal transcribed spacer (ITS) regions including the 5·8S gene of the ribosomal DNA operon were amplified and sequenced by methods described by Chen et al. (2011). Nucleotide sequences were edited in mega 4 (Tamura et al., 2007). All sequences obtained in this study were deposited in GenBank (Table 1).

Phylogenetic analysis

To determine the phylogenetic placement of the R. melanophloeos isolates, nucleotide sequences of the LSU gene region, the 5·8S rDNA and the exon regions of the BT gene (including partial exon 4, exon 5, partial exon 6 and partial exon 7) of previously described genera/species in the Cryphonectriaceae (Gryzenhout et al., 2009, 2010; Begoude et al., 2010; Chen et al., 2011; Vermeulen et al., 2011) were compared to those of the isolates collected for the current study following methods of Chen et al. (2011). The aligned data sets of Chen et al. (2011) were used as templates for these analyses. Togninia minima, To. fraxinopennsylvanica and Phaeoacremonium aleophilum were used as out-groups for analysis using sequence data from the LSU gene region (Gryzenhout et al., 2009; Chen et al., 2011). Two isolates of Diaporthe ambigua were selected as out-groups for phylogenetic analyses of the 5·8S gene and BT exon regions (Chen et al., 2011). Before performing combined analyses for the 5·8S gene and BT exon regions in paup* (Phylogenetic Analysis Using Parsimony) v. 4.0b10 (Swofford, 2002), the partition homogeneity test (PHT), as implemented in paup, was used to determine whether conflict existed between the data sets (Farris et al., 1995; Huelsenbeck et al., 1996). All the data sets were aligned using the iterative refinement method (FFT-NS-i settings) of mafft v. 5.667 with the online version (Katoh et al., 2002). The alignments were further edited manually in mega 4. All alignments were deposited in TreeBASE (http://www.treebase.org).

For each of the data sets, two different phylogenetic analyses were conducted. Maximum parsimony (MP) analyses were executed in paup and Bayesian inference was determined using the Markov chain Monte Carlo (MCMC) algorithm in MrBayes v. 3.1.2 (Ronquist & Huelsenbeck, 2003) following methods previously published by Chen et al. (2011).

Morphology

Only asexual structures were found on the bark of diseased R. melanophloeos trees. For morphological evaluations, fruiting structures were located using a dissection microscope, cut from the bark, boiled for 2 min in water and sectioned (12 μm thick) using a Leica CM1100 cryostat (Setpoint Technologies) at −20°C (Gryzenhout et al., 2004). To examine the conidiophores, conidiogenous cells and conidia, fruiting structures were crushed on microscope slides with 85% lactic acid. Fifty measurements for each morphologically informative character were made for the specimen selected to represent a holotype, and 20 measurements were taken for the remaining specimens. Results are presented as (minimum–) (mean−standard deviation)−(mean + standard deviation) (–maximum). Measurements were made for fruiting structures representing the smallest and largest of the anamorphic stromata. Morphological characters of specimens obtained in this study were compared with those for other genera/species in the Cryphonectriaceae (Gryzenhout et al., 2009, 2010; Begoude et al., 2010; Chen et al., 2011; Vermeulen et al., 2011).

Six representative isolates (CMW37314–CMW37319) from six different R. melanophloeos trees were used for studies of culture characteristics. Optimal growth conditions for each culture were determined using the methods of Chen et al. (2011). The entire experiment was repeated once. For the characteristics of cultures and fruiting bodies, colour designations were determined using the colour charts of Rayner (1970).

Pathogenicity tests

Four isolates (CMW37314–CMW37317) were selected for field inoculations to fulfil Koch’s postulates. Prior to inoculations, these isolates were grown on 2% MEA at 25°C for 1 week. Isolates were inoculated into the branches or stems (1–2 years old, approximately 2 cm diameter) of healthy R. melanophloeos trees in the Harold Porter National Botanical Garden. Wounds were made with a cork borer (7 mm diameter) to expose the cambium and discs of agar of similar size were removed from the edges of cultures covered with actively growing mycelium, and placed into the wounds with the mycelium facing the cambium. Sterile MEA was inoculated into wounds to serve as negative controls. Wounds were covered with masking tape to prevent desiccation and contamination. Ten stem/branches were inoculated for each of the four isolates and 10 stem/branches were treated as negative controls. The 50 inoculated stems/branches were distributed randomly in the Garden.

Stems/branches were inoculated on 10 March 2011 and results were evaluated 6 weeks later by measuring the lesion lengths in the cambium. Reisolations were made by cutting small pieces of wood from the lesion edges and placing these on 2% MEA at 25°C. Reisolations were made from all trees inoculated as controls and from four randomly selected trees per isolate. Results were analysed in excel (2003). Single factor analysis of variance (anova) was used to define the effects of fungal isolate on lesion length. To test the significance among means, F-values with < 0·05 were considered significantly different. The standard errors of means of lesion length for each fungal strain and control were calculated.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Laboratory observations and isolations

Fruiting structures (conidiomata) taken from the surfaces of cankers were typical of those found in the Cryphonectriaceae. These were consistently found on all cankers and isolations resulting in pure cultures could easily be made from them. Isolates on MEA in culture were white to white yellow in colour, which is also typical of species in the Cryphonectriaceae (Gryzenhout et al., 2009). Thirty-three isolates were obtained, each from a different tree.

Phylogenetic analyses

The aligned LSU nucleotide sequence data set consisted of 64 taxa and 627 characters (TreeBASE: http://purl.org/phylo/treebase/phylows/study/TB2:S12585). Statistical values of both maximum parsimony and Bayesian inference analyses are provided in Table 2. The internal positions of the fungal genera differed between the MP and Bayesian inference analyses, but tree topologies for the two analyses were similar. One hundred most parsimonious trees were obtained after MP analysis, of which the first tree was saved (Fig. 2). The results of the phylogenetic analyses of LSU gene nucleotide sequence data indicated that isolates collected from R. melanophloeos trees formed a single consistent phylogenetic lineage in the Cryphonectriaceae, distinct from other genera (Fig. 2).

Table 2. Statistics resulting from phylogenetic analyses
Data setNo. of taxaNo. of bpaMaximum parsimony
PICbNo. of treesTree lengthCIcRIdHIe
LSU646271231002810·5340·8130·466
5·8S rRNA/exons of BT1,262760114 (5·8S: 6; BT: 108)12460·5650·8650·435
Data setMrBayes
Substf modelPrset statefreqprNSTgRatesBurn-in
  1. abp = base pairs.

  2. bPIC = number of parsimony informative characters.

  3. cCI = consistency index.

  4. dRI = retention index.

  5. eHI = homoplasy index.

  6. fSubst model = best fit substitution model.

  7. gNST = number of substitution rate categories.

LSUGTR + I+ + GDirichlet(1,1,1,1)6Invgamma100 000
5·8S rRNA/exons of BT1,2GTR + I + GDirichlet(1,1,1,1)6Invgamma100 000
image

Figure 2.  Cladogram based on maximum parsimony (MP) analysis of LSU DNA sequences for various genera in the Diaporthales. Bootstrap values >50% for MP and posterior probabilities >0·70 obtained from Bayesian analysis are presented at branches. Bootstrap values lower than 50%, and posterior probabilities lower than 0·70 are marked with an *, and absent analysis values are marked with –. Isolates from Rapanea melanophloeos are in bold.

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The partition homogeneity test (PHT) for the aligned 5·8S rRNA gene and exons of the BT gene region data sets resulted in a value of = 0·980, indicating that the two data sets were congruent and could be combined for the phylogenetic analyses. The aligned sequences for the combined data sets (TreeBASE: http://purl.org/phylo/treebase/phylows/study/TB2:S12585) consisted of 62 taxa and 760 characters (Table 2). The position of genera in the Cryphonectriaceae differed slightly in relation to each other, depending on the specific phylogenetic analyses of MP and Bayesian inference, but the overall topologies were similar. Only one most parsimonious tree was retained (Fig. 3). Statistical results indicated that most of the genera of Cryphonectriaceae resided in different phylogenetic clades with high MP bootstrap support (BS) and/or high Bayesian posterior probabilities (PP; Fig. 3). Based on analysis of the combined data set, isolates from R. melanophloeos formed a strongly separated phylogenetic clade (MP bootstrap = 99%, Bayesian posterior probability = 1·00), distinct from all other genera in the Cryphonectriaceae (Fig. 3). The isolates from R. melanophloeos trees are most closely related to the genus Microthia (Fig. 3).

image

Figure 3.  Cladogram based on maximum parsimony (MP) analysis of a combined DNA sequence data set of gene regions of the partial exon 4, exon 5, exon 6 and exon 7 of the BT genes, and the 5·8S rRNA gene region. Bootstrap values >50% for MP and posterior probabilities >0·70 obtained from Bayesian analysis are presented at branches. Bootstrap values lower than 50%, and posterior probabilities lower than 0·70 are marked with an *, and absent analysis values are marked with –. Isolates from Rapanea melanophloeos are in bold.

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Morphology and taxonomy

Consistent with the results of the DNA sequence analyses, the fruiting structures of the unknown fungus from R. melanophloeos showed morphological characteristics typical of members of the Cryphonectriaceae. These comprised anamorphic fruiting structures with orange stromatic tissue (Gryzenhout et al., 2006a, 2009) that turn purple in 3% KOH and yellow in lactic acid (Castlebury et al., 2002) and the production of a pigment that coloured the agar yellow in culture (Gryzenhout et al., 2009).

Comparisons between the anamorphic structures of the fungus from Rapanea and those of other species in the Cryphonectriaceae showed that they were very similar to those produced by Microthia and Holocryphia. These also produce orange pulvinate conidiomata, without conidiomatal necks and with paraphyses (Gryzenhout et al., 2009, 2010; Begoude et al., 2010; Vermeulen et al., 2011). These genera can be distinguished from other members of the Cryphonectriaceae that produce orange conidiomata including Amphilogia (conical to pyriform conidiomata, paraphyses absent), Cryptometrion, Cryphonectria and Endothia (paraphyses absent), Rostraureum (clavate to rostrate conidiomata, conidiomatal necks present, paraphyses absent), Ursicollum (pyriform or rostrate conidiomata, conidiomatal necks present, paraphyses absent), Aurifilum (broadly convex conidiomata) and Latruncellus (conical with inflated neck conidiomata, conidiomatal necks present).

The unknown fungus from Rapanea could be distinguished from Microthia and Holocryphia, especially based on the size and position of conidiomata. The conidiomata of the Rapanea fungus (up to 1500 μm) were larger than those of Microthia and Holocryphia (smaller than 900 μm). The conidiomata of the unknown fungus were always immersed or semi-immersed in the bark of the host, while those of Microthia are usually superficial, or semi-immersed, and those of Holocryphia semi-immersed. These differences have previously been used to distinguish genera in the Cryphonectriaceae (Gryzenhout et al., 2009; Vermeulen et al., 2011). Conidia of the Rapanea fungus were also larger than those found in Microthia and Holocryphia.

Based on the phylogenetic analyses for the LSU and 5·8S rRNA, exons in the BT gene regions, as well as the morphological characteristics, the fungus from R. melanophloeos clearly represents a previously undescribed genus and species in the Cryphonectriaceae. A new genus and a related species are described as follows:

  • Immersiporthe S.F. Chen, M.J. Wingf., & Jol. Roux, gen. nov.

MycoBank No. MB564804

Etymology. Name is derived from the Latin word immersus describing the immersed conidiomata in the bark and porthe (destroyer), describing the pathogenic nature of the fungus.

Conidiomata pulvinate, immersed to semi-immersed, orange, uni- to multiloculate and convoluted, necks absent, stromatic tissue orange, pseudoparenchymatous at the edges, prosenchymatous in the centre. Conidiophores aseptate, occasionally with separating septa and branching, conidiogenous cells phialidic, cylindrical to flask-shaped with tapering apices. Long cylindrical cells, or paraphyses, occur between conidiophores. Conidia hyaline, cylindrical to fusoid, occasionally allantoid, aseptate, pushed through opening at stromatal surface as orange droplets or tendrils.

Notes. Only anamorphic structures were observed for Immersiporthe.

Typus: Immersiporthe knoxdaviesiana S.F. Chen, M.J. Wingf., & Jol. Roux, sp. nov.

  • Immersiporthe knoxdaviesiana S.F. Chen, M.J. Wingf., & Jol. Roux, sp. nov.

MycoBank No. MB564866

Etymology. This species is named after Professor Peter Sidney Knox-Davies recognizing his great contributions to plant pathology, and the fact that he cherished the Harold Porter National Botanical Garden, where this fungus was first discovered, and where his remains have been scattered.

Conidiomata pulvinate (Fig. 4a), immersed (Fig. 4b) to semi-immersed (Fig. 4a), orange when young, umber to brown when mature, uni- to multiloculate and convoluted, necks absent, 120–320 μm high, 350–1500 μm diameter (Fig. 4a–c), locules 80–350 μm diameter (Fig. 4c), stromatic tissue pseudoparenchymatous at the edges, prosenchymatous in the centre (Fig. 4d). Conidiophores (5–)15–30(–61) μm long, occasionally with separating septa and branching, hyaline (Fig. 4f, g), conidiogenous cells 1·5–2 μm wide, cylindrical to flask-shaped with tapering apices, or not attenuated (Fig. 4g). Long cylindrical cells, seemingly sterile and paraphyses occurring between conidiophores, up to 150 μm long, branching occasionally into other sterile, cylindrical cells (Fig. 4e). Conidia (3·5–)5·0–5·5(–7·0) × 1–2 μm, hyaline, cylindrical, fusoid, occasionally allantoid, aseptate (Fig. 4h, i), exuded as orange droplets (Fig. 4b) or tendrils.

image

Figure 4.  Fruiting structures of Immersiporthe knoxdaviesiana from Rapanea melanophloeos (specimen PREM60738). (a) Semi-immersed conidiomata on the bark; (b) immersed conidiomata in the bark (arrows indicate conidial spore mass); (c) longitudinal section through conidioma; (d) stromatic tissue of conidioma; (e) paraphyses; (f, g) conidiophores differing in length with conidigenous cells; (h, i) conidia differing in shape and size; (j) culture after 10 days of growth on MEA at 25°C; (k) asexual fruiting structures from primary isolations of the fungus. Bars: a = 100 μm; b, c = 200 μm; d = 20 μm; e = 50 μm; f, g = 20 μm; h, i = 10 μm; j, k = 10 mm.

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Culture characteristics. On MEA I. knoxdaviesiana fluffy with an uneven margin, white when young, turning yellow white to sulphur yellow with yellow/sienna patches after 10 days (Fig. 4j). Colonies reverse white to yellow-white. Optimal growth temperature 25°C, covering the 90 mm plates after 7 days. No growth at 5°C and 35°C; colonies at 10°C reached 31·5 mm in 30 days (6 mm in 7 days). Asexual fruiting structures infrequently form in primary isolations of the fungus (Fig. 4k).

Teleomorph. Not observed.

Habitat. Causing cankers on stems and branches of Rapanea melanophloeos.

Hosts and distribution. Rapanea melanophloeos in the Harold Porter National Botanical Garden, Betty’s Bay, Western Cape Province, South Africa.

Specimens examined. SOUTH AFRICA. Western Cape province, the Harold Porter National Botanical Garden (18°55′56″E and 34°20′99″S; 40–45 m a.s.l.), from bark of Rapanea melanophloeos. Feb., 2011, Michael J. Wingfield & Jolanda Roux, HOLOTYPE PREM60738, ex-type culture CMW37314 = CBS132862; Feb., 2011, Michael. J. Wingfield & Jolanda Roux, PARATYPE PREM60739, living culture CMW37315 = CBS132863; Mar., 2011, Jolanda Roux, ShuaiFei Chen & Francois Roets, PREM60740, living culture CMW37318 = CBS132864.

Notes. Immersiporthe knoxdaviesiana is morphologically most similar to Microthia havanensis and Holocryphia eucalypti, but can be distinguished from these fungi by the size of their conidia. The conidia of I. knoxdaviesiana (conidia medium, av. >5 μm, up to 7·0 μm) are longer than those of M. havanensis (conidia minute, av. <5 μm, up to 5·0 μm) and Holocryphia (conidia minute, av. <5 μm, up to 5·0 μm).

Pathogenicity tests

Inoculations on R. melanophloeos trees showed that I. knoxdaviesiana is an aggressive pathogen of these trees (Figs 5a,b & 6) and has the ability to kill inoculated stems within 6 weeks (Fig. 5a). Fruiting structures of I. knoxdaviesiana were produced on the bark of inoculated branches within 6 weeks (Fig. 5b). All four isolates of I. knoxdaviesiana produced well-developed lesions within 6 weeks (Figs 5a & 6), while the control inoculations using sterile agar were covered by callus after the same period (Fig. 5a). The mean comparison tests showed that the lesions produced by the I. knoxdaviesiana isolates were all significantly longer (< 0·001) than those of the controls (Fig. 6). The lesions produced by the four isolates were not significantly different from each other (= 0·1354; Fig. 6). All the inoculated fungi were reisolated from the lesions, but not from the control inoculations.

image

Figure 5.  Symptoms after inoculations on Rapanea melanophloeos stems/branches with Immersiporthe knoxdaviesiana. (a) Negative control inoculation (thin grey arrow) showing wound and the absence of lesion development, and canker and lesions in the cambium (thin black arrow), and branch death (bold black arrow) caused by isolates of I. knoxdaviesiana; (b) canker with fruiting structures on the bark directly adjacent to the point of inoculation on branch. Bars: a, b = 10 mm.

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image

Figure 6.  Bar graph showing the average lesion length (mm) resulting from inoculation trials with Immersiporthe knoxdaviesiana onto stems/branches of Rapanea melanophloeos. Vertical bars represent standard error of means. Different letters above the bars indicate treatments that were significantly different (= 0·05).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In this study, a new and serious stem canker disease of native R. melanophloeos trees was discovered in the Harold Porter National Botanical Garden in the Western Cape province of South Africa. This disease was shown to be caused by a previously unknown fungus in the Cryphonectriaceae and is described in the new genus Immersiporthe as I. knoxdaviesiana. The identification of this fungus was supported by phylogenetic analyses as well as morphological characteristics. Inoculation tests showed that I. knoxdaviesiana is a virulent pathogen on R. melanophloeos and is able to kill inoculated stems and branches in a relatively short period of time.

The canker disease on R. melanophloeos is aggressive and severely damaging. It had not been observed in regular visits to the Harold Porter National Botanical Garden during the course of the past two decades (M. Wingfield, FABI, University of Pretoria, personal communication) and it appears to have emerged relatively recently. The origin of the pathogen is unknown and the disease has not been found in other areas where R. melanophloeos grows. All indications are that this is a new and serious disease problem, potentially caused by an introduced pathogen that might threaten an important native tree in southern Africa. In this regard, its recent appearance might be analogous to the first observation of the chestnut blight pathogen in the New York Botanical Garden in 1906 (Merkel, 1906).

Only asexual anamorphic structures were found for I. knoxdaviesiana. However, these, together with phylogenetic data, were sufficient to clearly show the unique nature of the fungus on R. melanophloeos. The characteristics of anamorphic structures have previously been used to distinguish different genera in the Cryphonectriaceae (Gryzenhout et al., 2006b,c, 2009; Vermeulen et al., 2011). These, for example, include Aurapex and Ursicollum, the two other genera of Cryphonectriaceae for which a teleomorph is not known. In this study, Immersiporthe could be distinguished relatively easily from other morphologically similar genera of Cryphonectriaceae by the colour, size and position of conidiomata.

Immersiporthe knoxdaviesiana is the fifth species in the Cryphonectriaceae that has been found in southern Africa and the fourth in South Africa. Other species include Celoporthe dispersa, Chrysoporthe austroafricana, Holocryphia eucalypti and Latruncellus aurorae. All of these are associated with canker diseases on trees from which they were identified (Van der Westhuizen et al., 1993; Gryzenhout et al., 2003, 2009; Nakabonge et al., 2006; Vermeulen et al., 2011), and Chr. austroafricana (Van der Westhuizen et al., 1993; Gryzenhout et al., 2003, 2009; Nakabonge et al., 2006) is amongst the most serious tree pathogens known in Africa (Wingfield et al., 1989; Wingfield, 2003; Gryzenhout et al., 2009). In this regard, I. knoxdaviesiana described in this study has a similar biology and level of importance.

Immersiporthe knoxdaviesiana and Chr. austroafricana both cause serious canker diseases on their hosts, but they also differ in various respects. Chrysoporthe austroafricana is found on the branches and stems of native Syzygium cordatum in South Africa (Heath et al., 2006), but apart from where trees are severely stressed, there is no evidence that it causes serious damage to these trees. In contrast, there is good evidence to show that it has undergone a host shift to non-native Eucalyptus spp. and Tibouchina spp. (Slippers et al., 2005; Gryzenhout et al., 2009) where it causes serious damage and can kill trees (Wingfield et al., 1989; Conradie et al., 1990; Myburg et al., 2002; Gryzenhout et al., 2009). Immersiporthe knoxdaviesiana occurs on the stems and small branches of R. melanophloeos, a tree native to South Africa, and the damage that it causes is dramatic and suggestive of an introduced pathogen. Population biology studies on the pathogen will clearly be needed to clarify this.

The first appearance of a serious new pathogen in a botanical garden is not unique. These are areas where plants are commonly introduced from other areas, thereby increasing opportunities for new disease introductions and host shifts (Slippers et al., 2005). Following this reasoning, botanical gardens are increasingly being seen as important sites for the early detection of new tree pathogens. In this regard, the discovery of I. knoxdaviesiana could represent an example of an important new disease that has emerged for the first time in a botanical garden, and surveys of this tree and its relatives in other areas should be intensified to better understand its relative importance.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors thank the DST/NRF Centre of Excellence in Tree Health Biotechnology (CHTB) for financial support. They are also grateful to Professor Léanne Dreyer, Mr Anathi Magadlela, Ms Anicia Malebajoa, Ms Netsai Machingambi and Ms Tendai Musvuugwa from Stellenbosch University for assistance with inoculation trials, Ms Brigitte Nell Van Dyk for assistance with some of the laboratory investigations and Mrs Jane Forrester (Harold Porter National Botanical Garden) for permission to work on diseased trees as well as providing field trial sites. The authors also acknowledge the Western Cape Nature Conservation Board for issuing the necessary collecting permits.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Anagnostakis SL, 1987. Chestnut blight: the classical problem of an introduced pathogen. Mycologia79, 2337.
  • Anagnostakis SL, 1992. Chestnuts and the introduction of chestnut blight. Annual Report of the Northern Nut Growers Association83, 3942.
  • Begoude BAD, Gryzenhout M, Wingfield MJ, Roux J, 2010. Aurifilum, a new fungal genus in the Cryphonectriaceae from Terminalia species in Cameroon. Antonie van Leeuwenhoek98, 26378.
  • Castlebury LA, Rossman AY, Jaklitsch WJ, Vasilyeva LN, 2002. A preliminary overview of the Diaporthales based on large subunit nuclear ribosomal DNA sequences. Mycologia94, 101731.
  • Cheewangkoon R, Groenewald JZ, Summerell BA, Hyde KD, To-anun C, Crous PW, 2009. Myrtaceae, a cache of fungal biodiversity. Persoonia23, 5585.
  • Chen SF, Gryzenhout M, Roux J, Xie YJ, Wingfield MJ, Zhou XD, 2011. Novel species of Celoporthe from Eucalyptus and Syzygium trees in China and Indonesia. Mycologia103, 1384410.
  • Chungu D, Gryzenhout M, Muimba-Kankolongo A, Wingfield MJ, Roux J, 2010. Taxonomy and pathogenicity of two novel Chrysoporthe species from Eucalyptus grandis and Syzygium guineense in Zambia. Mycological Progress9, 3793.
  • Coates Palgrave K, 1977. Trees of Southern Africa. Cape Town, Johannesburg, South Africa: Struik Publishers.
  • Conradie E, Swart WJ, Wingfield MJ, 1990. Cryphonectria canker of Eucalyptus, an important disease in plantation forestry in South Africa. South African Forestry Journal152, 439.
  • Farris JS, Kallersjo M, Kluge AG, Bult C, 1995. Testing significance of incongruence. Cladistics10, 3159.
  • Gryzenhout M, Eisenberg BE, Coutinho TA, Wingfield BD, Wingfield MJ, 2003. Pathogenicity of Cryphonectria eucalypti to Eucalyptus clones in South Africa. Forest Ecology and Management176, 42737.
  • Gryzenhout M, Myburg H, Van der Merwe NA, Wingfield BD, Wingfield MJ, 2004. Chrysoporthe, a new genus to accommodate Cryphonectria cubensis. Studies in Mycology50, 11942.
  • Gryzenhout M, Myburg H, Wingfield BD, Wingfield MJ, 2006a. Cryphonectriaceae (Diaporthales), a new family including Cryphonectria, Chrysoporthe, Endothia and allied genera. Mycologia98, 23949.
  • Gryzenhout M, Myburg H, Hodges CS, Wingfield BD, Wingfield MJ, 2006b. Microthia, Holocryphia and Ursicollum, three new genera on Eucalyptus and Coccoloba for fungi previously known as Cryphonectria. Studies in Mycology55, 3552.
  • Gryzenhout M, Myburg H, Rodas CA, Wingfield BD, Wingfield MJ, 2006c. Aurapex penicillata gen. sp. nov. from native Miconia theaezans and Tibouchina spp. in Colombia. Mycologia98, 10515.
  • Gryzenhout M, Wingfield BD, Wingfield MJ, 2009. Taxonomy, Phylogeny, and Ecology of Bark-inhabiting and Tree Pathogenic Fungi in the Cryphonectriaceae. St Paul, MN, USA: APS Press.
  • Gryzenhout M, Tarigan M, Clegg PA, Wingfield MJ, 2010. Cryptometrion aestuescens gen. sp. nov. (Cryphonectriaceae) pathogenic to Eucalyptus in Indonesia. Australasian Plant Pathology39, 1619.
  • Heath RN, Gryzenhout M, Roux J, Wingfield MJ, 2006. Discovery of the Cryphonectria canker pathogen on native Syzygium species in South Africa. Plant Disease90, 4338.
  • Heiniger U, Rigling D, 1994. Biological control of chestnut blight in Europe. Annual Review of Phytopathology32, 58199.
  • Huelsenbeck JP, Bull JJ, Cunningham CW, 1996. Combining data in phylogenetic analysis. Trends in Ecology & Evolution11, 1528.
  • Katoh K, Misawa K, Kuma K, Miyata T, 2002. mafft: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Research30, 305966.
  • Merkel HW, 1906. A deadly fungus on the American chestnut. Annual Report of the New York Zoological Society10, 93103.
  • Myburg H, Wingfield BD, Wingfield MJ, 1999. Phylogeny of Cryphonectria cubensis and allied species inferred from DNA analysis. Mycologia91, 24350.
  • Myburg H, Gryzenhout M, Heath RN, Roux J, Wingfield BD, Wingfield MJ, 2002. Cryphonectria canker on Tibouchina in South Africa. Mycological Research106, 1299306.
  • Nakabonge G, Gryzenhout M, Roux J, Wingfield BD, Wingfield MJ, 2006. Celoporthe dispersa gen. et sp. nov. from native Myrtales in South Africa. Studies in Mycology55, 25567.
  • Rayner RW, 1970. A Mycological Colour Chart. Kew, UK: Commonwealth Mycological Institute and British Mycological Society.
  • Ronquist F, Huelsenbeck JP, 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics19, 15724.
  • Roux J, Nakabonge G, 2010. First report of Holocryphia eucalypti on Eucalyptus grandis in Uganda. Plant Pathology59, 409.
  • Slippers B, Stenlid J, Wingfield MJ, 2005. Emerging pathogens: fungal host jumps following anthropogenic introduction. Trends in Ecology & Evolution20, 4201.
  • Swofford DL, 2002. PAUP*. Phylogenetic Analysis Using Parsimony (*and other methods) . Version 4.0b10. Sunderland, MA, USA: Sinauer Associates.
  • Tamura K, Dudley J, Nei M, Kumar S, 2007. mega 4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biological and Evolution24, 15969.
  • Van der Westhuizen IP, Wingfield MJ, Kemp GHJ, Swart WJ, 1993. First report of the canker pathogen Endothia gyrosa on Eucalyptus in South Africa. Plant Pathology42, 6613.
  • Van Wyk B, Van Wyk P, 1997. Field Guide to Trees of Southern Africa. Cape Town, South Africa: Struik Publishers.
  • Vermeulen M, Gryzenhout M, Wingfield MJ, Roux J, 2011. New records of Cryphonectriaceae from southern Africa including Latruncellus aurorae gen. sp. nov. Mycologia103, 55469.
  • Wingfield MJ, 2003. Increasing threat of diseases to exotic plantation forests in the Southern Hemisphere: lessons from Cryphonectria canker. Australasian Plant Pathology32, 1339.
  • Wingfield MJ, Swart WJ, Abear BJ, 1989. First record of Cryphonectria canker of Eucalyptus in South Africa. Phytophylactica21, 3113.