The Eucalyptus canker pathogen Holocryphia eucalypti on Eucalyptus in New Zealand

Holocryphia eucalypti (=Cryphonectria eucalypti) causes die-back, and stem and branch cankers of several Eucalyptus and Corymbia spp. in natural forests and commercial plantations in Australia and South Africa (Walker et al. 1985; Van der Westhuizen et al. 1993; Carnegie 2007). The pathogen has also recently been reported on Tibouchina urvilleana (Lassiandra) planted as ornamentals in Australia (Heath et al. 2007). The canker disease caused by H. eucalypti is closely associated with stress conditions such as drought and cankers can be relatively mild or lethal where host trees are susceptible and stress is severe (Old et al. 1986; Wardlaw 1999; Gryzenhout et al. 2003). Because predisposition plays such an important role in disease development, H. eucalypti is not considered as severe (Yuan and Mohammed 2000) as other closely related Eucalyptus pathogens such as species of Chrysoporthe (Gryzenhout et al. 2009).

Canker caused by H. eucalypti is common on many Eucalyptus and Corymbia species in forests and plantations of eastern Australia including Tasmania, and it also occurs in Western Australia (Old et al. 1986; Davison and Coates 1991; Wardlaw 1999; Yuan and Mohammed 2000; Carnegie 2007). In South Africa, it occurs on commercially propagated, non-native Eucalyptus spp. in plantations (Van der Westhuizen et al. 1993; Gryzenhout et al. 2003). Population genetic studies employing microsatellite data have shown that H. eucalypti was most likely introduced into South Africa, although evidence for its origin in Western Australia was less convincing (Nakabonge et al. 2008).

During the course of the past 24 years, isolates of a fungus in the Cryphonectriaceae have been collected from various Eucalyptus species in New Zealand. Most locations were in the southern half of the North Island with one from the north of the South Island (Table 1). Based on cultural characteristics and the morphology of conidia produced in culture, the isolates were identified as Endothiella-like, which represent the anamorph state of some species in the Cryphonectriaceae (Gryzenhout et al. 2009). However, in the absence of sexual fruiting bodies or well-defined fruiting bodies on host tissue and in culture, a definitive identification could not be made. The aim of this study was thus to characterise the isolates from Eucalyptus stems in New Zealand based on DNA sequence comparisons for the β-Tubulin gene region.

Table 1.  Isolates and specimens used in this study

Original isolates curated in NZFS (National Forestry Culture Collection) were obtained from discoloured yellowish wood, from branch cankers and die-back. These isolates are maintained at 4°C in the culture collection (NZFS) of Scion, New Zealand Forest Research Institute, Rotorua, New Zealand (Table 1). Duplicates of the cultures, as well as an isolate (CMW12723) obtained from asexual fruiting bodies from a later collection, are housed in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa (Table 1). The representative specimen of the original bark material from which the culture CMW12723 was made has been deposited in the National Collection of Fungi (PREM), Pretoria, South Africa (Table 1).

For DNA sequence comparisons, isolates were grown in 2% Malt Extract broth (100 μL in 1.5 μL Eppendorf tubes), the mycelium harvested and the DNA extracted following Gryzenhout et al. (2006). The DNA was used in polymerase chain reactions (PCR) of the β-Tubulin gene regions 1 and 2 and sequenced using the same protocols as those described by Gryzenhout et al. (2006). The sequences were included in a data matrix containing sequences of H. eucalypti isolates previously published (Gryzenhout et al. 2006; Heath et al. 2007). Sequences of isolates CMW10010 and CMW10011 were obtained from the study of Myburg (2003). Species of other genera in the Cryphonectriaceae (Endothia gyrosa, Cryphonectria parasitica, Microthia havanensis) were defined as outgroups. The dataset consisted of 20 taxa and the sequences were aligned with the CLUSTAL function of the program MEGA ver. 4 (Tamura et al. 2007) and verified manually.

Phylogenetic analyses were run with PAUP (Phylogenetic Analysis Using Parsimony) version 4.0b10 (Swofford 2002). Phylogenetic trees were obtained with maximum parsimony (uninformative sites excluded, heuristic search with 100 random sequence additions and tree-bisection-reconnection branch swapping, MULTREES off, base pairs re-weighted according to the consistency index). The strength of branches was tested with a 70% bootstrap analysis (1000 replicates).

Of the 928 characters in the DNA matrix, 165 were uninformative, 645 were constant and 118 were informative. Two trees that were identical topologically but differed slightly based on length of some branches (Tree Length = 170.3, Consistency Index = 0.882, Retention Index = 0.895, g1 value = –2.62), were obtained in the analysis. The phylogenetic trees (Fig. 1) clearly showed that the isolates from New Zealand grouped with those of H. eucalypti from South Africa and Australia (bootstrap support 100%). The isolates from New Zealand formed a group with those from T. urvilleana in Australia (bootstrap support 87%). Two other groups, including South African and Australian isolates, were also evident (bootstrap support 100% and below 50%, respectively).

This study reports the presence of the Eucalyptus pathogen H. eucalypti in New Zealand for the first time. This report further contributes to the databases and checklists of pathogens and fungi in New Zealand (http://nzfungi.landcareresearch.co.nz/html/mycology.asp database; Dingley 1969; Pennycook 1989; McKenzie et al. 2000; Pennycook and Galloway 2004). Due to the opportunistic nature of H. eucalypti on Eucalyptus and Tibouchina, the occurrence of the fungus in New Zealand is most likely not of economic significance, although its presence deserves to be monitored.

Relatively little is known regarding the Cryphonectriaceae in New Zealand. Although there are reports of Endothia gyrosa, a previous name used for H. eucalypti (Gryzenhout et al. 2006), in the country (Gryzenhout et al. 2009), these reports are from Nothofagus (McKenzie et al. 2000; Pennycook and Galloway 2004), Myrsine salicina and dead wood of an unknown host (http://nzfungi.landcareresearch.co.nz/html/mycology.asp database) and need to be verified based on recent taxonomic changes to the group (Gryzenhout et al. 2009). Reports of other Cryphonectriaceae that could represent incorrectly identified H. eucalypti specimens in New Zealand, include those of Rostraureum longirostris on Nothofagus (Nothofagaceae, Fagales), Amphilogia gyrosa and Amphilogia major on Elaeocarpus spp. (Elaeocarpaceae, Oxalidales), an Endothiella sp. and Cryphonectria radicalis (http://nzfungi.landcareresearch.co.nz/html/mycology.asp database; Dingley 1969; Pennycook 1989; McKenzie et al. 2000; Pennycook and Galloway 2004; Gryzenhout et al. 2005). In the NZFS collection, cultures of an Endothiella sp. identified only on the basis of culture characteristics, originate from Myrsine chathamica and Leptospermum scoparium as well as various species of eucalypts. A member of the Cryphonectriaceae has also been found on a Coriaria sp. (Coriariaceae, Cucurbitales) in New Zealand (PDD28477, Waiomu, Thames, Auckland, J.M. Dingley, August 1958) but the identity of this collection relies only on a herbarium specimen and cannot be confirmed (M. Gryzenhout, unpubl. data). Thus, only the identities of A. gyrosa and A. major have been verified based on recent phylogenies (Gryzenhout et al. 2005).

The relative proximity of New Zealand to Australia, as well as the common occurrence of H. eucalypti in the eastern part of Australia indicates a possibility that the fungus originated in the latter country. However, it is also possible that H. eucalypti occurs on native Myrtaceae in New Zealand. It may also occur on other non-native shrubs such as Tibouchina spp. (Melastomataceae, Myrtales), which are hosts of this fungus (Heath et al. 2007) and many other members of the Cryphonectriaceae (Gryzenhout et al. 2009). Answers to these intriguing questions will require further surveys and population genetic studies.