Taxonomic studies on Cryphonectria species and allied taxa

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Chrysoporthe doradensis sp. nov. pathogenic to Eucalyptus in Ecuador


Canker caused by Chrysoporthe cubensis is a serious disease of commercially grown Eucalyptus in various South American countries. This disease has not previously been recorded from Ecuador. Recent disease surveys in Ecuadorian Eucalyptus plantations have led to the discovery of canker symptoms typical of this disease with fruiting bodies resembling C. cubensis abundant on diseased tissues. The aim of this study was to characterise the fungus based on morphology and sequences of the ITS1/ITS2 regions of the ribosomal DNA operon and b-tubulin genes. Phylogenetic analyses showed that isolates from Ecuador reside in a clade together with other Chrysoporthe spp., but in a clearly distinct group. The distinct phylogenetic position of the Ecuadorian fungus is supported by unique conidial morphology and it is, therefore, described as Chrysoporthe doradensis sp. nov. Pathogenicity trials on Eucalyptus deglupta showed that the fungus is highly pathogenic on this commonly planted tree as well as on saplings of Tibouchina urvilleana.


Chrysoporthe cubensis (Bruner) Gryzenh. & M. J. Wingf., previously known as Cryphonectria cubensis (Bruner) Hodges, causes a serious canker disease of Eucalyptus trees in plantations. Chrysoporthe cubensis is common in the Neotropics where it has been reported from several countries (Bruner 1917, Boerboom & Maas 1970, Hodges et al. 1976, 1979, Myburg et al. 1999, Van der Merwe et al. 2001, Gryzenhout et al. 2004/Chapter 1 of this thesis). Girdling cankers on the stems of trees by this pathogen has had a substantial impact on Eucalyptus propagation in the tropics and sub-tropics, where it has also greatly influenced plantation practices (Wingfield 2003). The best option for management of this disease has been through breeding and selection of resistant Eucalyptus clones, and such programmes have been successfully implemented in various South American countries (Alfenas et al. 1983, Wingfield 2003, Rodas et al. 2005).
Chrysoporthe is a recently described genus including the fungus previously known as Cry. cubensis (Gryzenhout et al. 2004). DNA sequence comparisons and detailed morphological studies have shown that specimens and isolates previously identified as Cry. cubensis from various parts of the world, represent at least three species (Myburg et al. 2002a, Gryzenhout et al. 2004). The fungus now known as C. cubensis represents isolates from South and Central America, but also includes isolates from Central Africa, Hawaii, South East Asia and Australia (Gryzenhout et al. 2004). In these areas, C. cubensis not only occurs on Eucalyptus but also on other Myrtaceae such as Syzygium aromaticum (clove) in Brazil, Zanzibar and Indonesia (Hodges et al. 1986, Myburg et al. 2003), and Melastomataceae such as native Miconia theaezans and Miconia rubiginosa in Colombia (Rodas et al. 2005).
Other than C. cubensis, two other species of Chrysoporthe are known and one of these occurs in South America together with C. cubensis. This species, Chrysoporthella hodgesiana Gryzenh. & M. J. Wingf., is recognized as a species of Chrysoporthe based on DNA sequence data, but is known only in its asexual state and thus resides in the anamorph genus of Chrysoporthe. Chrysop. hodgesiana is commonly found in Colombia on native Tibouchina spp. (Wingfield et al. 2001, Gryzenhout et al. 2004) and on M. theaezans (Rodas et al. 2005). Other than its unique DNA sequences, it can also be distinguished from C. cubensis based on its low optimal growth temperature (Gryzenhout et al. 2004). Isolates of the fungus previously known as Cry. cubensis from South Africa represents the third species that has been provided the name C. austroafricana Gryzenh. & M. J. Wingf. This species is defined by ascospores with rounded apices and is currently known only from South Africa (Gryzenhout et al. 2004). All three Chrysoporthe species are highly pathogenic to Eucalyptus spp. (Wingfield et al. 1989, Wingfield et al. 2001, Wingfield 2003).
Forestry in Ecuador includes the planting of native as well as exotic tree species in plantations. Plantations of Eucalyptus are not widespread and little is known regarding the diseases that affect them. Cankers caused by C. cubensis are found in neighbouring countries such as Colombia (Van der Merwe et al. 2001), but the disease has not been reported from Ecuador. The presence of this disease in Ecuador could have a negative impact on forestry in the country, particularly if susceptible species are planted. This provided the motivation for disease surveys in Ecuadorian Eucalyptus plantations and the discovery of a canker disease that forms the basis of this study.


Symptoms and collection of samples

Eucalyptus grandis and E. deglupta trees of various ages between five and 10-years-old were found with stem cankers (Fig. 1A) in plantations near the towns of Buenos Aires. The extent of cankers differed substantially, but in many cases they had girdled and killed trees. Ascostromata and conidiomata were commonly found fruiting around the cankers, and these were collected and transported to the laboratory for further study. Isolates were made, purified and have been lodged in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa (Table 1). Representative isolates have also been deposited in the Centraalbureau voor Schimmelcultures (CBS), Utrecht, Netherlands (Table 1). The original pieces of bark from which isolates were made were dried and have been deposited in the herbarium of the National Collection of Fungi, Pretoria, South Africa (PREM).



Fruiting structures were cut from the bark specimens and examined using the methods outlined in Gryzenhout et al. (2004). Fifty ascospores, asci, conidia and conidiophores were measured and are presented as (min–)(average – S.D.) – (average + S.D.)(–max) mm. Only minimum and maximum values arising from the smallest and largest structure were obtained for the eustromata and perithecia. Colours were assigned using the notations of Rayner (1970).
Growth in culture was studied for the isolates (CMW 11286, CMW 11287) from E. grandis in Ecuador. This is especially important since Chrysop. hodgesiana has been distinguished from C. cubensis based on growth characteristics in culture (Gryzenhout et al. 2004). Growth of cultures was studied in the dark at temperatures ranging from 15 to 35 ºC, at 5 º intervals. The procedure for assessment of growth in culture was the same as that described by Gryzenhout et al. (2004).

DNA sequence comparisons

Sequences were obtained from a number of genic regions of isolates from E. grandis in Ecuador. These sequences were compared with those published (Table 1) for C. cubensis, C. austroafricana and Chrysop. hodgesiana from a variety of hosts (Gryzenhout et al. 2004, Rodas et al. 2005). Isolates of Rostraureum tropicale Gryzenh. & M. J. Wingf. were also included (Table 1). This species is closely related to Chrysoporthe and was recently described as a pathogen of Terminalia ivorensis and T. superba in the same areas of Ecuador where the Chrysoporthe sp. was found in this study (Gryzenhout et al. 2005/Chapter 7 in this thesis). The R. tropicale isolates were included as outgroup taxa, together with the closely related Cryphonectria parasitica (Murrill) M. E. Barr, Cryphonectria macrospora (Tak. Kobay. & Kaz. Itô) M. E. Barr and Cryphonectria nitschkei (G. H. Otth) M. E. Barr (Gryzenhout et al. 2004, Myburg et al. 2004).
DNA was extracted from mycelium grown in Malt Extract Broth [20 g/L Biolab malt extract] following the protocol described in Myburg et al. (1999). The internal transcribed spacer (ITS) regions ITS1 and ITS2, and the conserved 5.8S gene of the ribosomal RNA (rRNA) operon, as well as two regions of the b-tubulin gene, were amplified as described in Myburg et al. (1999) and Myburg et al. (2002a). PCR products were run on 1% agarose (ethidium bromide-stained) gels, and detected under UV light. The PCR products were purified using a QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and sequenced with the same primers that were used to amplify the respective DNA regions. An ABI PRISM™ Dye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq® DNA Polymerase, FS (Perkin-Elmer, Warrington, UK) was used for the sequence reactions on an ABI PRISM 3100™ automated DNA sequencer.
The forward and reverse sequences that were obtained were imported into Sequence Navigator version 1.0.1 software (Perkin-Elmer Applied BioSystems, Foster City, CA). Sequences were manually aligned and inserted, together with those from Rodas et al. (2005), in the TreeBASE data matrix (S 1211, M 2095) generated in the study by Gryzenhout et al. (2004). Subsequent phylogenetic analyses were done using PAUP version 4.0b (Swofford 1998). The combinability of the rRNA and b-tubulin gene sequence data sets was determined with a partition homogeneity test (PHT; Farris et al. 1994). Parsimony using the heuristic search option with the tree-bisection-reconnection (TBR) branch swapping, MULTREES options (saving all optimal trees) effective and random sequence additions set to 100 was employed to generate trees. During analyses, uninformative sites were excluded and individual CI values were used to reweight base pairs. Distance analyses were also executed using the distance model determined with MODELTEST version 3.5 (Posada & Crandall 1998) to confirm results obtained with parsimony. Thus the Transitional model or TIM (Tavaré 1986) was used (proportion of invariable sites (I) 1840; Base frequency = 0.1952, 0.3262, 0.2408, 0.2379; Rate matrix = 1.0, 3.3491, 1.8115, 1.8115, 5.9357, 1.0). In the heuristic searches, gaps inserted during sequence alignment were treated as fifth character (NEWSTATE), but these were treated as missing data for distance analyses. A 1000 replicate bootstrap analyses was executed to assess the reproducibility levels of the branch nodes of the phylogenetic trees. Individual sequences generated in this study have been deposited in GenBank (Table 1).

Section 1: Taxonomic studies on Cryphonectria species and allied taxa
CHAPTER 1 Chrysoporthe, a new genus to accommodate Cryphonectria cubensis
CHAPTER 2 Chrysoporthe doradensis sp. nov. pathogenic to Eucalyptus in Ecuador
CHAPTER 3 Novel hosts of the Eucalyptus canker pathogen Chrysoporthe cubensis and a
new Chrysoporthe species from Colombia
CHAPTER 4 Cryphonectriaceae (Diaporthales), a new family including Cryphonectria,
Endothia, Chrysoporthe and allied genera
CHAPTER 5 Proposal to conserve the name Cryphonectria (Diaporthales) with a conserved
CHAPTER 6 Amphilogia gen. nov. for Cryphonectria-like fungi from Elaeocarpus spp. in
New Zealand and Sri Lanka
CHAPTER 7 Rostraureum tropicale gen. sp. nov. (Diaporthales) associated with dying
Terminalia ivorensis in Ecuador.
CHAPTER 8 Aurapex penicillata gen. sp. nov. from native Miconia theaezans and
Tibouchina spp. in Colombia
CHAPTER 9 Microthia, Holocryphia and Ursicollum, three new genera on Eucalyptus and
Coccoloba for fungi previously known as Cryphonectria
CHAPTER 10 New taxonomic concepts for the important forest pathogen Cryphonectria
parasitica and related fungi
Section 2: Monograph of Cryphonectria and allied genera
CHAPTER 11 Taxonomy, phylogeny and ecology of Cryphonectria species and other
members of the Cryphonectriaceae

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