Quambalaria species associated with plantation and native eucalypts in Australia

Abstract

This study aimed to determine which species of Quambalaria are associated with shoot blight symptoms on Corymbia spp. An additional aim was to determine the presence and impact of quambalaria shoot blight on Eucalyptus species used in plantation development in subtropical and tropical regions of eastern Australia. Surveys identified three Quambalaria spp. –Q. pitereka, Q. eucalypti and Q. cyanescens– from native and plantation eucalypts, as well as amenity plantings, including the first confirmed report of Q. eucalypti from Eucalyptus plantations in Australia. Symptom descriptions and morphological studies were coupled with phylogenetic studies using ITS rDNA sequence data. Quambalaria pitereka was the causal agent of blight symptoms on species and hybrids in the Corymbia complex. Quambalaria eucalypti was identified from Eucalyptus species and a single Corymbia hybrid. Quambalaria cyanescens was detected from native and plantation Corymbia spp.

Introduction

The first record of a species of Quambalaria causing damage to eucalypts was on nursery seedlings of Corymbia maculata (then Eucalyptus maculata) in New South Wales, Australia, in the 1950s (Walker & Bertus, 1971). The pathogen was described as Ramularia pitereka, but following re-examination of the four known species of Ramularia on eucalypts, it was transferred to the new genus Quambalaria (Simpson, 2000). A new family, Quambalariaceae, has since been described (de Beer et al., 2006) for species of Quambalaria that include a number of eucalypt (species of the genus Eucalyptus and Corymbia) pathogens.

There are five species of Quambalaria known to infect eucalypts in various parts of the world. These include Q. pitereka, Q. eucalypti, Q. cyanescens and Q. coyrecup. The fifth species, Q. pusilla, was described based on a single report from Eucalyptus spp. in Thailand (Braun, 1998). However, the taxonomic status of this fungus has been questioned (de Beer et al., 2006) and, as no type culture exists, it cannot be confirmed.

Quambalaria pitereka has been isolated from foliage, stems and woody tissue of species of the genera Corymbia, Blakella and Angophora in Australia (Walker & Bertus, 1971; Bertus & Walker, 1974; Simpson, 2000). The disease that it causes is commonly known as quambalaria shoot blight (Pegg et al., 2005; Carnegie, 2007a). Old (1990) described this pathogen as being endemic to the coastal forests of eastern Australia, where seedlings and young trees of Corymbia species can be severely damaged. The pathogen was also reported causing shoot blight in Corymbia maculata plantings in Western Australia (Paap et al., 2008). Quambalaria pitereka was first found only in Australia, but a recent report of shoot blight on Corymbia citriodora in China was attributed to this pathogen (Zhou et al., 2007).

Quambalaria eucalypti was first reported (as Sporothrix eucalypti) in South Africa on Eucalyptus grandis (Wingfield et al., 1993). In that country, it was associated with leaf spots and serious shoot infections, but restricted to nurseries. It was subsequently reported from Brazil, where it caused stem girdling on seedlings of Eucalyptus globulus and leaf and shoot blight on mini-stumps of E. saligna ×E. maidenii hybrids (Alfenas et al., 2001).

It was also identified from twig lesions on E. globulus in Uruguay (Bettucci et al., 1999). Roux et al. (2006) identified Q. eucalypti as the causal agent of extensive shoot and leaf dieback and stem cankers on 1-year-old E. nitens in temperate regions of South Africa, the first report of the disease outside the nursery. Pathogenicity tests indicated that Q. eucalypti has a wider host range within Eucalyptus, including E. dunnii and E. smithii (Roux et al., 2006).

Quambalaria coyrecup is the causal agent of extensive perennial canker disease on Corymbia calophylla in Western Australia (Paap et al., 2008). This disease is causing serious decline of C. calophylla throughout its native range. Infection by Q. coyrecup gives rise to symptoms similar to the previously described canker fungus Sporotrichum destructor, which also caused a devastating disease of Corymbia ficifolia in Western Australia (Beard, 1963; Cass Smith, 1970). It has been hypothesized that Q. coyrecup represents the same fungus as S. destructor, but the latter fungus was never validly described and comparisons of the two fungi based on cultures have not been possible (Paap et al., 2008).

Quambalaria cyanescens was originally described from human skin (de Hoog & de Vries, 1973) and the first record on eucalypts was from a single location on E. pauciflora in New South Wales, Australia (de Beer et al., 2006). More recently it was identified from C. ficifolia and C. calophylla in Western Australia, where it sporulates on shoots and newly emerging leaves together with Q. pitereka. It also occurs on cankers caused by Q. coyrecup and on symptomless leaves and woody stems (Paap et al., 2008). More recently, Q. cyanescens was identified from bark beetles collected from a range of host tree species, including Tilia, Quercus and Ficus, in Hungary, Bulgaria and the Mediterranean (Kolarik et al., 2006).

Quambalaria shoot blight is a serious disease affecting the expanding eucalypt plantations in subtropical and tropical eastern Australia (Simpson, 2000; Self et al., 2002; Pegg et al., 2005; Carnegie, 2007b). The aim of this investigation was to determine which Quambalaria spp. are present in the area and to characterize the disease symptoms that they cause on various Corymbia and Eucalyptus hosts there.

Materials and methods

Surveys and symptoms

Commercial eucalypt plantations, as well as taxa and species trials in northern New South Wales and Queensland (Fig. 1), were surveyed between 2004 and 2006 for Quambalaria species. Surveys included the four Corymbia species commonly known as the ‘spotted gums’[viz. C. citriodora subsp. citriodora (hereafter referred to as C. citriodora), C. citriodora subsp. variegata (hereafter referred to as C. variegata), C. henryi and C. maculata], C. torelliana and hybrids between this species and C. citriodora, C. variegata and C. henryi. Species of Eucalyptus included E. argophloia, E. cloeziana, E. dunnii, E. grandis, E. grandis×E. camaldulensis, E. longirostrata, E. microcorys, E. nitens and E. pilularis. Trees in native stands and residential plantings of eucalypts were also examined for the presence of Quambalaria infections.

Figure 1

Map of Australia indicating where Quambalaria pitereka and Q. eucalypti haplotypes were identified from subtropical and tropical plantation areas surveyed in Queensland and New South Wales in this study.

For description of disease symptoms and collection of isolates for morphological and DNA-sequence-based comparisons, samples of shoots, juvenile and adult leaves (immature and mature) and green stem material exhibiting blight symptoms were used. These were collected, stored in paper bags and kept cool within a portable refrigerator unit prior to laboratory examination. Samples were examined under dissection and compound microscopes, with identification of Quambalaria spp. based on morphological characteristics previously described (Wingfield et al., 1993; Simpson, 2000), and verified using DNA sequence comparisons. Characters different from those in published records were described and recorded.

Isolations from samples collected in Queensland and New South Wales were made from single lesions occurring on shoot, leaf and stem material exhibiting symptoms typical of Quambalaria spp. using potato-dextrose agar (PDA). Cultures, including the ex-holotype of Q. pitereka (DAR 19773), were incubated at 25°C in the dark for 2–3 days before being subcultured onto PDA and incubated for a further 2 weeks. All isolates and diseased samples collected in this study are stored in the Queensland Department of Primary Industries and Fisheries Plant Pathology Herbarium and culture collection (BRIP).

Fungal morphology

Comparisons of conidial morphology were made between isolates from different host materials. Spores were washed off leaves and stem lesions using sterile distilled water with a droplet placed onto a glass slide. Spore dimensions were recorded by measuring the lengths and widths of 30 randomly selected conidia at ×400 magnification (Olympus BH-2 compound microscope with graduated lens). Spore sizes for the different isolates were compared using anova (statview).

Plant tissue samples representing a broad range of disease symptoms were also examined using scanning electron microscopy. Leaf samples were collected from infected trees in plantations and cut into 3-mm² pieces, focusing on areas with visible disease symptoms. Samples were prepared for examination according to van den Berg et al. (2003). However, drying was completed using hexamethylthyldisilazane (HMDS). All steps were completed using a Pelco Biowave^®. Samples were coated with platinum for a period of 5 min to achieve a thickness of approximately 15 nm using an EIKO IB-5 Sputter Coater before being examined with a JOEL 6300 and JOEL 6400 scanning electron microscope at 8 kV.

DNA sequence comparisons

For DNA sequence analysis, mycelium from isolates of Quambalaria spp. (Table 1) was harvested by scraping it from the surface of PDA cultures using a sterilized scalpel. The mycelium was then frozen in liquid nitrogen and ground into a fine powder using a mortar and pestle that had been sterilized with 70% ethanol. DNA was extracted using a Wizard^® Genomic DNA Purification Kit – Isolation of Genomic DNA from Plant Tissue (Promega), according to the manufacturer's instructions. DNA was resuspended in TE buffer (10 mm Tris-HCl, 1 mm EDTA, pH 8·0) and quantified using a Biorad SmartSpec^TM Spectrophometer prior to further analysis.

Table 1. Quambalaria isolates, from Corymbia and Eucalyptus species, used for DNA sequence comparisons

Culture no.	Quambalaria sp.	Host	Location^a	Collector	Haplotype^b	GenBank accession no.
DAR19773	Q. pitereka	C. exima	NSW, Australia	Walker	QP1	DQ823423
BRIP48346	Q. pitereka	C. citriodora subsp. citriodora	Davies Creek, QLD, Australia	G. S. Pegg	QP1	EF444845
BRIP48387	Q. pitereka	C. citriodora subsp. variegata	Beaudesert, QLD, Australia	G. S. Pegg	QP1	EF444846
BRIP48425	Q. pitereka	C. citriodora subsp. variegata	Grafton, NSW, Australia	G. S. Pegg	QP1	EF444847
BRIP48429	Q. pitereka	C. citriodora subsp. variegata	Grafton, NSW, Australia	G. S. Pegg	QP1	EF444848
BRIP48433	Q. pitereka	C. citriodora subsp. variegata	Grafton, NSW, Australia	G. S. Pegg	QP1	EF444849
BRIP48492	Q. pitereka	C. citriodora subsp. variegata	Tipperary, NSW, Australia	A. J. Carnegie	QP1	EF444850
BRIP48505	Q. pitereka	C. citriodora subsp. variegata	Moggill, QLD, Australia	G. S. Pegg	QP1	EF444851
BRIP48506	Q. pitereka	Native C. citriodora	Moggill, QLD, Australia	G. S. Pegg	QP1	EF444852
BRIP48515	Q. pitereka	Native C. henryi	Grafton, NSW, Australia	G. S. Pegg	QP1	EF444853
CMW5318	Q. pitereka	C. citriodora subsp. variegata	QLD, Australia	M. Ivory	QP2	DQ317621
BRIP48317	Q. pitereka	C. henryi	Coolabunia, QLD, Australia	G. S. Pegg	QP2	EF444854
BRIP48321	Q. pitereka	C. citriodora subsp. variegata	Beaudesert, QLD, Australia	G. S. Pegg	QP2	EF444855
BRIP48512	Q. pitereka	Native C. henryi	Grafton, NSW, Australia	G. S. Pegg	QP2	EF444856
BRIP48381	Q. pitereka	C. citriodora subsp. citriodora	Silkwood, QLD, Australia	G. S. Pegg	QP3	EF444858
BRIP48361	Q. pitereka	C. citriodora subsp. variegata	Walkamin, QLD, Australia	G. S. Pegg	QP3	EF427367
BRIP48343	Q. pitereka	C. citriodora subsp. citriodora	Walkamin, QLD, Australia	G. S. Pegg	QP3	EF444857
BRIP48383	Q. pitereka	C. citriodora subsp. variegata	Beaudesert, QLD, Australia	G. S. Pegg	QP4	EF444859
WAC12956	Q. pitereka	C. ficifolia	Western Australia	T. Paap	QP5	DQ823428
WAC12957	Q. pitereka	C. ficifolia	Western Australia	T. Paap	QP5	DQ823426
WAC12958	Q. pitereka	C. calophylla	Western Australia	T. Paap	QP5	DQ823427
BRIP48349	Q. pitereka	C. torelliana × C. citriodora subsp. variegata	Mareeba, QLD, Australia	G. S. Pegg	QP6	EF444860
BRIP48350	Q. pitereka	C. torelliana x C. citriodora subsp. variegata	Mareeba, QLD, Australia	G. S. Pegg	QP6	EF444861
BRIP48360	Q. pitereka	C. torelliana × C. citriodora subsp. variegata	Walkamin, QLD, Australia	G. S. Pegg	QP6	EF444862
BRIP48368	Q. pitereka	C. torelliana × C. citriodora subsp. citriodora	Mareeba, QLD, Australia	G. S. Pegg	QP6	EF444863
BRIP48370	Q. pitereka	C. torelliana × C. citriodora subsp. citriodora	Mareeba, QLD, Australia	G. S. Pegg	QP6	EF427368
BRIP48388	Q. pitereka	C. torelliana	Atherton, QLD, Australia	G. S. Pegg	QP6	EF444864
BRIP48325	Q. pitereka	C. citriodora subsp. variegata	Binjour, QLD, Australia	G. S. Pegg	QP7	EF427366
BRIP48335	Q. pitereka	C. citriodora subsp. variegata	Imbil, QLD, Australia	G. S. Pegg	QP7	EF444865
BRIP48340	Q. pitereka	C. torelliana × C. henryi	Childers, QLD, Australia	G. S. Pegg	QP7	EF444866
BRIP48386	Q. pitereka	C. citriodora subsp. variegata	Beaudesert, QLD, Australia	G. S. Pegg	QP7	EF427370
BRIP48413	Q. pitereka	C. citriodora subsp. variegata	Casino, NSW, Australia	G. S. Pegg	QP7	EF444867
BRIP48424	Q. pitereka	C. citriodora subsp. variegata	Grafton, NSW, Australia	G. S. Pegg	QP7	EF444868
BRIP48508	Q. pitereka	C. citriodora subsp. variegata	Moggill, QLD, Australia	G. S. Pegg	QP7	EF444869
BRIP48513	Q. pitereka	Native C. henryi	Grafton, NSW, Australia	G. S. Pegg	QP7	EF444870
BRIP48516	Q. pitereka	Native C. henryi	Grafton, NSW, Australia	G. S. Pegg	QP7	EF444871
BRIP48328	Q. pitereka	Native C. citriodora subsp. variegata	Dilkoon, NSW, Australia	G. S. Pegg	QP8	EF444872
BRIP48384	Q. pitereka	C. citriodora subsp. variegata	Beaudesert, QLD, Australia	G. S. Pegg	QP8	EF427369
BRIP48432	Q. pitereka	C. citriodora subsp. variegata	Grafton, NSW, Australia	G. S. Pegg	QP9	EF444873
CMW23610	Q. pitereka	C. citriodora	China	Y. J. Xie	QP10	EF427373
CMW23611	Q. pitereka	C. citriodora	China	Y. J. Xie	QP10	EF427374
CMW23612	Q. pitereka	C. citriodora	China	Y. J. Xie	QP10	EF427375
CMW23613	Q. pitereka	C. citriodora	China	Y. J. Xie	QP10	EF427376
CBS118844	Q. eucalypti	E. grandis	South Africa	M. J. Wingfield	QE1	DQ317626
CBS119680	Q. eucalypti	E. grandis	South Africa	L. Lombard	QE1	DQ317625
CMW14329	Q. eucalypti	E. grandis × E. camaldulensis	South Africa	J. Roux	QE1	DQ317614
CBS118616	Q. eucalypti	E. grandis	South Africa	J. Roux	QE1	DQ317613
CMW17254	Q. eucalypti	E. nitens	South Africa	J. Roux	QE1	DQ317611
CBS118615	Q. eucalypti	E. nitens	South Africa	J. Roux	QE1	DQ317609
BRIP48367	Q. eucalypti	C. torelliana × C. citriodora subsp. variegata	Walkamin, QLD, Australia	G. S. Pegg	QE1	EF444823
BRIP48416	Q. eucalypti	E. dunnii	Casino, NSW, Australia	G. S. Pegg	QE1	EF444824
BRIP48420	Q. eucalypti	E. dunnii	Grafton, NSW, Australia	A. J. Carnegie	QE1	EF444825
BRIP48494	Q. eucalypti	E. dunnii	Dyraaba, NSW, Australia	A. J. Carnegie	QE1	EF444827
BRIP48496	Q. eucalypti	E. dunnii	Tabulam, NSW, Australia	A. J. Carnegie	QE1	EF444828
BRIP48504	Q. eucalypti	E. dunnii	Casino, NSW, Australia	A. J. Carnegie	QE1	EF444829
BRIP48511	Q. eucalypti	E. longirostrata	Grafton, NSW, Australia	A. J. Carnegie	QE1	EF444830
BRIP48519	Q. eucalypti	E. longirostrata	Moggill, QLD, Australia	G. S. Pegg	QE1	EF444831
BRIP48422	Q. eucalypti	E. dunnii	Bonalbo, NSW, Australia	A. J. Carnegie	QE2	EF444832
BRIP48489	Q. eucalypti	E. grandis	Lismore, NSW, Australia	G. S. Pegg	QE2	EF444833
BRIP48490	Q. eucalypti	E. grandis	Lismore, NSW, Australia	G. S. Pegg	QE2	EF444834
BRIP48493	Q. eucalypti	E. grandis	Taree, NSW, Australia	A. J. Carnegie	QE2	EF444826
BRIP48499	Q. eucalypti	E. grandis	Mandalong, NSW, Australia	A. J. Carnegie	QE2	EF444835
BRIP48500	Q. eucalypti	E. dunnii	Taree, NSW, Australia	A. J. Carnegie	QE2	EF444836
BRIP48501	Q. eucalypti	E. dunnii	Casino, NSW, Australia	A. J. Carnegie	QE2	EF444837
BRIP48502	Q. eucalypti	E. dunnii	Kyogle, NSW, Australia	A. J. Carnegie	QE2	EF444838
BRIP48509	Q. eucalypti	E. grandis	Lismore, NSW, Australia	A. J. Carnegie	QE2	EF444839
BRIP48510	Q. eucalypti	E. grandis	Lismore, NSW, Australia	A. J. Carnegie	QE2	EF444840
BRIP48518	Q. eucalypti	E. longirostrata	Moggill, QLD, Australia	G. S. Pegg	QE2	EF444841
BRIP48520	Q. eucalypti	E. grandis	Moggill, QLD, Australia	G. S. Pegg	QE2	EF444842
BRIP48497	Q. eucalypti	E. grandis	Nana Glen, NSW, Australia	A. J. Carnegie	QE3	EF444843
BRIP48498	Q. eucalypti	E. grandis	Kew, NSW, Australia	A. J. Carnegie	QE3	EF444844
BRIP48507	Q. eucalypti	E. grandis	Moggill, QLD, Australia	G. S. Pegg	QE4	EF444822
BRIP48414	Q. eucalypti	E. grandis	Moggill, QLD, Australia	G. S. Pegg	QE4	EF444821
BRIP48396	Q. cyanescens	Native C. citriodora	Beaudesert, QLD, Australia	G. S. Pegg		EF444874
BRIP48398	Q. cyanescens	Native C. citriodora	Beaudesert, QLD, Australia	G. S. Pegg		EF444875
BRIP48403	Q. cyanescens	Native C. citriodora	Beaudesert, QLD, Australia	G. S. Pegg		EF444876
WAC12952	Q. cyanescens	C. calophylla	Western Australia	T. Paap		DQ823419
WAC12955	Q. cyanescens	C. calophylla	Western Australia	T. Paap		DQ823421
CBS876·73	Q. cyanescens	E. pauciflora	New South Wales	V. F. Brown		DQ317623
WAC12948	Q. coyrecup	C. calophylla	Western Australia	T. Paap		DQ823433
WAC12949	Q. coyrecup	C. calophylla	Western Australia	T. Paap		DQ823432
WAC12950	Q. coyrecup	C. ficifolia	Western Australia	T. Paap		DQ823429
BRIP48338	Q. coyrecup	C. polycarpa	Darwin, NT, Australia	R. Pitkethley		EF444877
BRIP48339	Q. coyrecup	C. polycarpa	Darwin, NT, Australia	R. Pitkethley		EF444878

• ^a NSW = New South Wales, NT = Northern territory, QLD = Queensland.
• ^b QP = Q. pitereka, QE = Q. eucalypti.

DNA of the ribosomal RNA gene region (ITS1-5·8S-ITS2) was amplified using the universal primers ITS4 and ITS5 (White et al., 1990). Each 25-µL PCR reaction solution in each tube contained 12·5 ng template DNA µL⁻¹, 100 µm dNTPs, 1·5 mm MgCl₂, 1 U Taq DNA polymerase (Biotech) and 0·6 µm primers. DNA amplifications were performed in a PTC-100 Programmable Thermal Controller (MJ Research). An initial denaturation step at 95°C for 5 min was followed by 40 cycles of 95°C for 30 s, annealing for 30 s at 60°C and extension at 72°C for 1 min, followed by a final extension at 72°C for 10 min.

The PCR amplicons were size-fractionated in a 1·0% agarose gel and visualized using ethidium bromide under UV light. The ITS amplicons were purified from the agarose gel using a High Pure PCR Product Purification Kit (Roche Diagnostics). The purified amplicons were sequenced with the same primers that were used to amplify the respective DNA regions using an ABI Prism BigDye Terminator Sequencing Reaction Kit (v3·1) on an ABI 3700 DNA Analyser (Applied Biosystems).

To compare the Quambalaria species used in this study with other Quambalaria species, ITS sequences of closely related species, including the newly described Q. coyrecup (Paap et al., 2008), were obtained from GenBank and used in phylogenetic analyses (Table 1). DNA sequences were automatically aligned using clustalx (Jeanmougin et al., 1998) and the alignments adjusted manually in bioedit v5·0 (Hall, 1999). All sequences obtained in this study were deposited in GenBank and accession numbers are shown in Table 1. Haplotypes of Quambalaria species were defined for the purpose of this study as isolates having identical DNA sequences in the ITS1-5·8S-ITS2 rRNA region.

Analyses of sequence data were performed on the aligned ITS-5·8S-ITS2 sequences in paup (phylogenetic analysis using parsimony) v4·0b10 (Swofford, 2002). The most parsimonious trees were obtained by using heuristic searches with random stepwise addition in 100 replicates, with the tree bisection-reconnection branch-swapping option on and the steepest-descent option off. Maxtrees were unlimited, branches of zero length were collapsed and all multiple, equally parsimonious trees were saved. Estimated levels of homoplasy and phylogenetic signal (retention and consistency indices) were determined (Hillis & Huelsenbeck, 1992). Characters were unweighted and unordered, branch and branch node supports were determined using 1000 bootstrap replicates (Felsenstein, 1985), and characters were sampled with equal probability. Gaps were treated as a fifth character in the parsimony analysis. Trees were rooted to Tilletiopsis washingtonensis, Entyloma calendulae and Microstroma juglandis, which were treated as the outgroup taxa.

Results

Isolates

Three species of Quambalaria were identified from surveys in northern New South Wales and Queensland in this study. These were Q. pitereka, Q. eucalypti and Q. cyanescens (Fig. 2). Forty of the isolates collected in this study (Tables 1 and 2) were from Corymbia species (C. variegata, C. citriodora, C. henryi, C. polycarpa, C. torelliana and Corymbia hybrids). Twelve of these isolates were collected from New South Wales, 15 from southern Queensland, 11 from far-north Queensland and two from the Northern Territory. Two additional isolates were collected from C. maculata in northern New South Wales. Twenty-three isolates were collected from Eucalyptus spp. (E. grandis, E. longirostrata, E. dunnii), with the majority from New South Wales. Additional isolates not used in DNA sequence analysis were collected from E. microcorys in southern Queensland and from E. grandis and an E. grandis × E. camaldulensis hybrid in far-north Queensland.

Figure 2

A phylogram of one of the 50 most-parsimonious trees obtained from the ITS sequence data of the Quambalaria isolates. Bootstrap values are given above the branch. The tree is rooted to Tilletiopsis washingtonensis, Entyloma calendulae and Microstroma juglandis. Isolates of Q. pitereka and Q. eucalypti are represented by haplotypes, QP and QE, respectively. GenBank accession numbers for these haplotypes are listed in Table 1.

Table 2. Geographic origins, hosts and DNA haplotypes of Quambalaria species collected from subtropical and tropical regions of eastern Australia on Eucalyptus and Corymbia species

Location and host species	Quambalaria species	Haplotype
Northern New South Wales
C. variegata	Quambalaria pitereka	QP1, QP7, QP9
	Quambalaria cyanescens
C. henryi	Quambalaria pitereka	QP1, QP2, QP7
C. maculata	“ “
E. dunnii	Quambalaria eucalypti	QE1, QE2
E. grandis	“ “	QE2, QE3
E. longirostrata	“ “	QE1
Southern Queensland
C. variegata	Quambalaria pitereka	QP1, QP2, QP4, QP7, QP8
	Quambalaria cyanescens
C. henryi	Quambalaria pitereka	QP2
C. torelliana × C. henryi	“ “	QP7
E. dunnii	Quambalaria eucalypti
E. grandis	“ “	QE2, QE4
E. longirostrata	“ “	QE1, QE2
E. microcorys	“ “
North Queensland
C. variegata	Quambalaria pitereka	QP3
C. citriodora	“ “	QP1, QP3
C. torelliana	“ “	QP6
C. torelliana × C. variegata	“ “	QP6
	Quambalaria eucalypti	QE1
C. torelliana × C. citriodora	Quambalaria pitereka	QP6
E. grandis × E. camaldulensis	Quambalaria eucalypti
E. grandis	“ “

Surveys and symptoms

Quambalaria pitereka was found in all Corymbia plantations, species and taxa trials surveyed within Queensland and northern New South Wales. The pathogen was also found in native forests and residential gardens in these regions. Quambalaria pitereka was identified from all spotted gum species examined including C. variegata, C. citriodora, C. henryi and C. maculata, as well as C. torelliana and its hybrids with other Corymbia species. Quambalaria shoot blight on C. torelliana was found only in plantation and amenity plantings in tropical areas of far-north Queensland. The fungus was, however, not identified from any Eucalyptus spp. sampled.

Symptoms caused by Q. pitereka on C. variegata, C. citriodora, C. maculata, C. henryi, C. torelliana and Corymbia hybrids were similar to those described previously (Walker & Bertus, 1971; Simpson, 2000). Disease symptoms on new shoots and expanding juvenile foliage consisted of distorted leaves and amphigenous necrotic lesions of irregular shape and size (Fig. 3). White-coloured pustules were present on the adaxial and abaxial leaf surfaces covering the lesions. Lesions covered with white pustules were also present on the leaf midribs. Restricted necrotic lesions occurred on mature juvenile foliage with scattered white pustules occurring on parts of the lesions. Similar shoot blight symptoms caused by Q. pitereka were detected on immature adult foliage on native and amenity spotted gum trees. No disease symptoms were detected on mature adult foliage.

Figure 3

Symptoms of quambalaria shoot blight on eucalypt foliage. Immature (a) and mature (b) juvenile foliage of Corymbia variegata infected by Quambalaria pitereka resulting in distorted leaves and irregularly shaped and sized amphigenous necrotic lesions covered in white-coloured pustules. Symptoms of Q. pitereka infection on immature (c) and mature juvenile (d) foliage of Corymbia torelliana × C. variegata hybrid. (e) Quambalaria eucalypti symptoms on Eucalyptus dunnii. Scale bar = 5 mm.

An unusual foliage symptom was detected on Corymbia hybrids grown in taxa and species trials in tropical regions of northeastern Australia. Leaf blight symptoms caused by Q. pitereka occurred on expanding and fully expanded foliage of Corymbia hybrids in species trials (Fig. 3d). These symptoms consisted of amphigenous, irregular shaped necrotic lesions surrounded by water-soaked margins. The size of lesions was variable (2–15 mm) and appeared to increase as leaves expanded. Lesions often coalesced to form large necrotic areas on the leaves. White masses of erumpent conidiophores occurred primarily in the centres of the lesions on the abaxial leaf surfaces. However, on immature expanding foliage, pustules were observed covering the entire lesion surface. Lesions also occurred on the leaf midrib, resulting in distorted growth. Restricted, sunken, necrotic lesions were present on young green stems, with pustules occurring over the entire necrotic area.

On green apical stems and side branches of spotted gum species, sunken necrotic lesions caused by Q. pitereka were observed (Fig. 4a). White pustules were often present on the entire area of the lesion. Stem lesions were associated with distorted growth or death of the stem when girdling occurred. Repeated loss of new shoots and death of stems resulted in the loss of apical dominance and a bush-like growth habit. A reduction in foliage occurred as a result of premature senescence of infected leaves and green stems. Distortion and death of the leading shoots and stems of Corymbia hybrids was detected less frequently than that on spotted gum in subtropical regions, but was observed in tropical regions of eastern Australia (Fig. 4b). Lesions were also detected on stems of C. torelliana in this region. Quambalaria pitereka was also observed in association with wounds caused by hailstones (Fig. 4c) and cankers on woody branches of infected C. citriodora amenity trees (Fig. 4d).

Figure 4

Quambalaria species associated with stem lesions on eucalypts in subtropical and tropical region of eastern Australia. (a) Distorted apical shoot of Corymbia maculata infected by Q. pitereka covered in white masses of conidia and conidiophores; (b) lesions on the stem of a juvenile branch of C. torelliana × C. variegata caused by Q. pitereka; (c) stem lesions on C. variegata caused by hail damage and subsequently infected with Q. pitereka; (d) Q. pitereka sporulating (indicated by arrow) on a woody canker on C. citriodora; (e) Q. eucalypti associated with a lesion on juvenile Eucalyptus dunnii twig; (f) Q. cyanescens sporulating on a canker on a woody branch of C. variegata (indicated by arrow). Scale bar in all figures = 10 mm.

Quambalaria eucalypti was identified from leaf spots on E. grandis, E. dunnii, E. longirostrata, E. grandis and E. grandis × E. camaldulensis in commercial plantations, species trials and residential gardens in subtropical and tropical regions of eastern Australia. Quambalaria eucalypti was not detected on E. cloeziana, E. pilularis or E. argophloia in the survey. Infection was characterized by restricted, amphigenous lesions of irregular shape and size with white pustules predominantly present on the abaxial leaf surfaces (Fig. 3e). On expanding E. grandis juvenile leaves, pustules were observed on the adaxial and abaxial leaf surfaces and associated with leaf distortion or buckling. On all hosts, lesions were detected only on juvenile foliage and were often associated with wounds. In this case, wounds were caused by weevil (Oxyops spp. and Gonipterus spp.) and flea beetle (Chaetocnema sp.) feeding damage, but it is likely that Q. eucalypti would also be associated with other insect activity. On several disease samples of E. dunnii collected in New South Wales, small elongated lesions were observed on small green stems, which often resulted in splits in the stems. White pustules were evident on these lesions (Fig. 4e).

Quambalaria cyanescens was isolated from small stem lesions on native C. variegata and C. citriodora in Queensland and northern New South Wales, as well as in a limited number of C. variegata plantations in New South Wales. Extensive surveys to determine the host range and impact of this Quambalaria species have not been conducted. Discrete lesions were detected on woody stems and branches. Lesions were more or less elliptical in shape and covered in white/pink-coloured spore masses (Fig. 4f). Discoloration was present immediately under the lesion and appeared to be restricted to the cambium.

Quambalaria coyrecup was identified from a stem sample sent from the Northern Territory, Australia, with symptoms of cankering on small-diameter branches of C. polycarpa. Symptoms included stem swelling and bark splitting, with masses of erupting, white-coloured conidiophores and conidia present. Branch death occurred where cankers girdled the stems. This fungus was not detected in Queensland or New South Wales during the study.

Spore morphology

Spore dimensions for Q. pitereka collected from spotted gum species C. variegata, C. citriodora, C. maculata and C. henryi were similar to those of the holotype of Q. pitereka (DAR 19773). Primary conidia were variable in shape and ranged from clavate to elongate-clavate, cylindrical, fusiform, narrowly pyriform or obovoid. Primary conidia, perhaps better described as ramoconidia (Schubert et al., 2007), were of variable length (6–13 × 2–4 µm) and were not significantly different from secondary conidia. Conidia were borne on simple single or aggregated conidiophores (Fig. 5). Spore size was also variable for isolates collected from C. torelliana and Corymbia hybrids in north Queensland. Spores collected from C. torelliana (4–8 × 2–5 µm) were significantly (P ≤ 0·01) smaller than those collected from Corymbia hybrids (4–10 × 2–6 µm), C. citriodora and C. variegata.

Figure 5

Conidia and conidiophores of (a) Quambalaria pitereka and (b) Quambalaria eucalypti borne on simple or aggregated conidiophores. Conidiophores of Q. eucalypti were uniform in width with a swollen cluster of conidium bearing denticles. Scale bar = 1 µm.

Spore dimensions for Q. eucalypti from isolates collected from Eucalyptus species were smaller (4–9 × 2–3 µm) than those of Q. pitereka. The presence of secondary conidia half the size of the primary conidia was also noted; this is a key taxonomic feature detailed by Simpson (2000) and Wingfield et al. (1993) for Q. eucalypti. Conidiophores were uniform in width with a swollen cluster of conidium-bearing denticles. Denticles were observed on both terminal and short side branches (Fig. 5).

DNA sequence comparisons

DNA sequences from the ex-type cultures and representatives of each haplotype of Q. pitereka and Q. eucalypti were included in a dataset containing other Quambalaria spp., Q. cyanescens and Q. coyrecup, and outgroup taxa (TreeBASE SN3314-14388; Table 1). The aligned dataset consisted of 676 bp, of which 176 characters were parsimony-informative. These data contained significant phylogenetic signal (g1 = –1·44). Heuristic searches in paup resulted in 50 most parsimonious trees of 303 steps (CI = 0·82, RI = 0·89), one of which is presented in Fig. 2.

Forty-three isolates of Q. pitereka represented 10 haplotypes based on ITS profile (TreeBASE SN3314-14386; Tables 1 and 3, Fig. 2). All the haplotypes of this species were clustered in a single, strongly supported clade (Fig. 2). Twelve isolates tested had identical ITS sequences to the ex-holotype culture of Q. pitereka (DAR 19773). Haplotype 6 was present in isolates collected from C. torelliana and C. torelliana × C. variegata and C. torelliana × C. citriodora hybrids in north Queensland (Fig. 2, Tables 1 and 2). These isolates formed a discrete group separate from isolates collected from C. variegata and C. citriodora trees in the same region and an isolate collected from a C. torelliana × C. henryi hybrid in central Queensland. Other groupings were not host- or region-specific. Five haplotypes: 1, 2, 4, 7 and 8 (Fig. 2, Tables 1 and 2), were found to be present in a single plantation in southeast Queensland.

Table 3. Haplotypes of Quambalaria pitereka as determined from the polymorphic nucleotides within the aligned sequence data of ITS region of the rDNA for isolates collected from spotted gum species and Corymbia hybrids in Queensland and New South Wales

Haplotype	14^a	40	86	91	185	190	421	567	575
1	T^b	A	G	G	T	G	C	C	A
2	T	A	G	A	T	G	C	C	A
3	T	A	G	A	T	A	C	C	A
4	T	A	G	G	T	A	C	C	A
5	T	A	G	G	T	A	A	C	A
6	T	A	G	G	T	A	C	T	G
7	A	G	G	G	G	A	C	C	G
8	A	G	G	G	T	A	C	C	G
9	T	G	G	G	G	A	C	C	G
10	A	G	A	G	T	A	C	C	G

• a Base pair (bp) positions in aligned data, TreeBase SN3314-14388.
• ^b Polymorphisms shared with the first haplotype are highlighted.

Twenty-six isolates of Q. eucalypti could be divided into four haplotypes (TreeBASE SN3314-14387; Tables 1 and 4, Fig. 2). All the Q. eucalypti haplotypes grouped together in a single, strongly supported clade (Fig. 2). Eleven isolates had identical ITS sequences to the ex-type culture (CBS 118844) collected in South Africa and used by Wingfield et al. (1993) to describe Q. eucalypti. Two isolates with a single 5-bp indel were identified from leaf lesions on E. grandis in southeast Queensland (Table 4).

Table 4. Haplotypes of Quambalaria eucalypti as determined from the polymorphic nucleotides within the aligned sequence data of ITS region of the rDNA for isolates collected from species of Eucalyptus in Queensland and New South Wales

Haplotype	100^a	141–145	524
1	T^b		T
2	T		C
3	C		C
4	T	+	C

• a Base pair (bp) positions in aligned data, TreeBase SN3314-14388.
• ^b Polymorphisms shared with the first haplotype are highlighted.

Discussion

Three species of Quambalaria, namely Q. pitereka, Q. eucalypti and Q. cyanescens, were found associated with plantation eucalypts in subtropical and tropical regions of eastern Australia. This is the first confirmed record of Q. eucalypti in Australia and of Q. cyanescens in plantations as well as native C. variegata. In addition, Q. coyrecup was detected for the first time outside Western Australia, where it caused branch cankers on C. polycarpa in the Northern Territory, although it was not found in Queensland or New South Wales.

Quambalaria pitereka affects all spotted gum plantation areas in the subtropical and tropical regions of Australia. Infection of the growing points has the most significant impact on forest production, with the loss of apical dominance and multi-branching often resulting in a shrub-like growth habit, a form not viable for commercial forest production. Quambalaria pitereka has also been reported causing shoot blight on plantations of C. maculata in Western Australia (Paap et al., 2008) and C. citriodora plantations in China (Zhou et al., 2007). It was not found on Eucalyptus species in this study and appears to be restricted to hosts in the genus Corymbia.

In Queensland and northern New South Wales, plantations of Corymbia spp. are planted in close proximity to native stands of spotted gum, with mature habitat trees often retained within a plantation. These native spotted gums are the likely source of infection for Q. pitereka, which can be found sporulating on juvenile foliage of seedlings and saplings and immature adult foliage of mature native spotted gum trees. Simpson (2000) also reported the occurrence of Q. pitereka from large C. maculata trees and its common occurrence on young spotted gum natural regeneration.

Results of the survey provide the first report of Q. pitereka associated with cankers on woody stems of C. citriodora and an association with stem wounding caused by hail damage on C. variegata, C. citriodora, C. henryi and C. maculata. However, Simpson (2000) reported the presence of Q. pitereka in apparently healthy woody tissue of C. henryi, C. maculata and C. variegata. Little is known regarding the life cycle of Q. pitereka and further work is required to determine the role of spotted gum in native forests in disease development in plantations. Likewise, it will be intriguing to discover whether the existence of the pathogen in woody stems serves as a long-term survival mechanism.

Quambalaria pitereka was also isolated from C. torelliana in taxa trials and amenity plantings in tropical regions of north Queensland. This is the first record of Q. pitereka causing disease on C. torelliana within a plantation or native forest ecosystem. This is a significant finding with respect to the current breeding programme that has been established using hybrids between C. torelliana and spotted gum species (Lee, 2007). One of the justifications for selecting C. torelliana is the belief that it is resistant to infection by Q. pitereka. Indeed, the pathogen has not been detected in trial plantings or on amenity trees of C. torelliana in the subtropical regions. Likewise, the influence of Q. pitereka on the hybrid material is more apparent in the tropical regions of north Queensland than in subtropical plantings.

Corymbia hybrids have been shown to significantly out-perform parent material from a plantation point of view and initially in small-scale plantations did not appear to be affected by Q. pitereka (Lee, 2007). Foliage symptoms seen in subtropical regions in this study were similar to those detected on spotted gum. The disease was, however, limited to new shoots and expanding foliage and infection rarely resulted in stem death or loss of apical dominance. Yet, in species trials in north Queensland, Q. pitereka caused necrotic lesions on mature foliage of C. torelliana hybrids, with high levels of infection resulting in premature leaf senescence. Unlike symptoms on spotted gum, sporulation on the hybrids was restricted to the abaxial leaf surfaces and often only occurred in the centres of the lesions. The lesions were surrounded by a water-soaked margin, which was also a symptom not observed on spotted gum. It is unclear whether this unusual symptom is the result of host reaction or climatic conditions in tropical north Queensland. In light of this finding, the development of disease screening considering both parent species should be an essential part of future breeding programmes.

DNA sequence comparisons showed that the isolates collected from C. torelliana and the hybrids in tropical north Queensland were genetically unique and not found in any other region or on spotted gum species within the same plantations or districts. Isolates of Q. pitereka taken from other hybrid trees in southeast Queensland were also different from those in north Queensland, suggesting geographic barriers to gene flow. Interestingly, C. torelliana has a restricted native range in northern Queensland west of Ingham to south of Cooktown in the coastal foothills, in addition to occasional stands in the rainforest (Brooker & Kleinig, 1996). It is possible that the Q. pitereka haplotype found on this species has evolved in this region in association with the natural population of C. torelliana. This could be significant in relation to the selection of geographic locations for screening of Corymbia hybrids for use in future plantation development.

Quambalaria eucalypti was detected from shoot and leaf blight, leaf spots and stem lesions on Eucalyptus species over a wide geographic range and it was often associated with insect damage. Carnegie (2007a) provided a tentative report of this fungus from New South Wales, but this study, including DNA sequence comparisons, confirms its presence in Australia. This is also the first record of Q. eucalypti occurring on a Corymbia species, albeit a hybrid (C. torelliana × C. variegata). Lesions caused by Q. eucalypti were restricted and often limited to sites where insect browsing had occurred, particularly that of weevils. While the mode of infection of Quambalaria spp. is not yet known, it is possible that the wounds created by insects provided entry points for the fungus. The role of insects as vectors of the fungus should be considered, especially given the fact that Q. cyanescens was found associated with bark beetles in Hungary, Bulgaria and the Mediterranean (Kolarik et al., 2006).

Quambalaria eucalypti does not appear to be a significant threat to plantation health in Australia. It was detected only in one instance (in 2004) in forest health surveys in New South Wales from 1996 to 2005 (Carnegie, 2007a), but was relatively common in the 2006 surveys that formed part of this study. Similarly, Q. eucalypti was not found in Queensland in surveys that began in 1996, and its appearance in this survey in 2006 is intriguing. This may indicate that levels of Q. eucalypti are increasing. Why this might be occurring is uncertain, but it may be caused by the expansion of high density planting of Eucalyptus species in subtropical regions of eastern Australia. However, this does not explain the occurrence of the disease in residential gardens, geographically isolated from any form of plantation development. Quambalaria eucalypti is presently seen as one of the most serious pathogens of Eucalyptus spp. in nurseries in Brazil (Alfenas et al., 2001) and it has more recently been detected causing severe shoot and leaf blight and stem cankers in young trees in provenance trials in South Africa (Roux et al., 2006). In South Africa it was not initially believed to be a serious threat (Wingfield et al., 1993) as it was restricted to nurseries. As more plantations of Eucalyptus spp. are developed in Australia, it seems likely that this fungus will gain importance.

Quambalaria cyanescens was detected on woody stems and branches on native and plantation spotted gum, but its role as a pathogen on spotted gum appears to be limited. This study provides the first record of Q. cyanescens associated with plantation and native spotted gum in Australia. However, this is not the first report of its association with eucalypts, as a fungus was isolated from E. pauciflora in New South Wales under the name S. cyanescens (de Hoog & de Vries, 1973). More recently, Q. cyanescens was found to be associated with cankers on C. calophylla and C. ficifolia in combination with Q. pitereka in Western Australia (Paap et al., 2008); in that study pathogenicity trials confirmed that Q. cyanescens was non-pathogenic to Corymbia spp. This fungus has also been identified from human skin and tissue samples (de Hoog & de Vries, 1973), as well as being associated with processed oats (Da Silva et al., 1999). The presence of Q. cyanescens in both plantations and native spotted gum trees is of interest, but there currently appears to be no damage to tree health or wood quality. Lesions were not found to extend into the cambium or result in the development of resin pockets within the timber of juvenile or mature timber. However, additional research will be required to eliminate this fungus as a potential cause of stem degradation in commercial plantations in subtropical Australia.

Previous studies on Quambalaria spp. suggested that Q. pitereka and Q. eucalypti are endemic to Australia and have been introduced into new areas with seed used for plantation development (Wingfield et al., 1993; Roux et al., 2006; Zhou et al., 2007). DNA sequence comparisons for isolates of Q. eucalypti from South Africa (Roux et al., 2006) and Q. pitereka from China (Zhou et al., 2007) found no variation amongst isolates of these two species. Conversely, isolates of Q. eucalypti and Q. pitereka collected in this study were variable, with four and nine ITS haplotypes, respectively. The genetic diversity observed in Australia compared to that elsewhere in the world, combined with the native distribution and abundance of Eucalyptus and Corymbia species, supports the view that Australia is the likely centre of origin of Q. eucalypti and Q. pitereka. The apparently clonal population of Q. pitereka collected from C. maculata in Western Australia (Paap et al., 2008) also suggests that the fungus has been introduced into that part of the continent, where C. maculata does not occur naturally. Interestingly, there was no commonality between the Western Australian isolates and those collected from eastern subtropical and tropical regions of Australia. Further collections of Q. pitereka isolates for population genetic studies may help to develop an improved understanding of the origin and population structure of this pathogen in Australia.

Quambalaria species are becoming globally more significant as pathogens in eucalypt plantations. This is the first comprehensive survey to be undertaken and both Q. pitereka and Q. eucalypti were found to be widely distributed on a range of hosts throughout the subtropical and tropical regions of eastern Australia. The variability identified from DNA sequence comparisons for both species provides an insight into their population biology. Further understanding of this variation and how it might relate to pathogenicity to particular host species will be significant in the development of future tree breeding and risk management programmes. Understanding the factors influencing disease development, the impact of infection on growth rates and wood quality and host pathogen interactions will also be vital in the development of disease management strategies.

Acknowledgements

We thank the Queensland Department of State Development, Forest Plantations Queensland, Integrated Tree Cropping, Forest Enterprises Australia and Forests New South Wales for providing the necessary funding for this research.