CSIRO PUBLISHING

Australasian Plant Pathology, 2008, 37, 472--481

www.publish.csiro.au/journals/app

Leaf diseases caused by Mycosphaerella species in Eucalyptus globulus plantations and nearby native forest in the Green Triangle Region of southern Australia P. A. Barber A,B,D, Angus. J. Carnegie C, T. I. Burgess A and Philip J. Keane B A

School of Biological Sciences and Biotechnology, Murdoch University, Murdoch, WA 6150, Australia. School of Botany, La Trobe University, Bundoora, Vic. 3086, Australia. C Forest Resources Research, NSW Department of Primary Industries, PO Box 100, Beecroft, NSW 2119, Australia. D Corresponding author. Email: [email protected] B

Abstract. There are over 60 species of Mycosphaerella known from eucalypts, and several are serious pathogens of Eucalyptus globulus plantations in Australia and elsewhere, being associated with a syndrome called Mycosphaerella leaf disease. The Green Triangle region (south-west Victoria and south-eastern South Australia) is a major focus of the expansion of E. globulus in Australia, with over 125 000 ha planted since 1998. In this paper, we report the first comprehensive survey for species of Mycosphaerella in this expanding estate and assess the damage from these species from 1999 to 2002. There was an increase in the incidence of Mycosphaerella leaf disease in these plantations during the survey period, and five species of Mycosphaerella were identified from E. globulus plantations in the region: M. cryptica, M. nubilosa, M. parva, M. tasmaniensis and M. aff. vespa. M. cryptica, M. nubilosa and M. parva were the most common. M. cryptica was also found in native forest in the region, as was Sonderhenia eucalyptorum (telemorph, M. swartii). Morphological and phylogenetic comparisons were made with previously described species and confusion over the taxonomy of some of these species is discussed. Additional keywords: blue gum, disease incidence, forest pathology, fungal taxonomy. Introduction Numerous species of Mycosphaerella have been described from necrotic lesions on eucalypt (Eucalyptus and Corymbia) leaves from various parts of the world (Carnegie and Keane 1998; Crous 1998; Maxwell et al. 2003; Barber 2004; Hunter et al. 2004b; Crous et al. 2004, 2006; Summerell et al. 2006; Burgess et al. 2007; Carnegie et al. 2007) with some species being among the most important pathogens of eucalypt plantations in Australia (Park and Keane 1982a; Carnegie et al. 1994, 1998; Park et al. 2000; Maxwell et al. 2003; Carnegie 2007b) and elsewhere (Dick 1982; Lundquist and Purnell 1987; Crous 1998; Mohammed et al. 2003). Mycosphaerella spp. have been destructive in native eucalypt forests and plantations in Tasmania (Park and Keane 1984; Yuan 1999; Milgate et al. 2001, 2005), Victoria (Park and Keane 1984; Carnegie et al. 1994; Carnegie and Ades 2003), New South Wales (Carnegie 2007a, 2007b) and Western Australia (Abbott et al. 1993; Carnegie et al. 1997; Maxwell et al. 2003). The establishment of large-scale eucalypt plantations in Australia is in its infancy (Turnbull 2000). Since the mid 1990s there has been a rapid expansion in the establishment of Eucalyptus globulus plantations in southern Australia. The largest expansion was originally in south-west Western Australia (~75 000 ha planted from 1995 to 1998) (National Plantation Inventory 2000, 2005), but since 1998 the bulk of the plantings have been in the ‘Green Triangle’ region  Australasian Plant Pathology Society 2008

(south-west Victoria and south-east South Australia), with over 125 000 ha being planted (National Plantation Inventory 2000, 2005). The majority of these plantings are on former grazing land, but they are often surrounded by native eucalypt forest or remnant stands of eucalypts. Eleven species of Mycosphaerella have been recorded from native eucalypt forests and plantations in Victoria: M. cryptica, M. nubilosa, M. parva, M. swartii, M. walkeri, M. delegatensis, M. marksii, M. grandis, M. gregaria, M. vespa and M. tasmaniensis (Park and Keane 1982b, 1984; Carnegie and Keane 1994; Barber et al. 2005). Two of these, M. cryptica and M. nubilosa, have been responsible for disease epidemics in E. globulus plantations in eastern Victoria (Park and Keane 1984; Carnegie et al. 1994). Few surveys for Mycosphaerella species have been conducted in eucalypt plantations in South Australia (Park 1984; Carnegie 2000) or western Victoria and none since the beginning of large-scale establishment of E. globulus in the region. The aim of the present study was to identify foliar pathogens impacting on eucalypt plantations in the Green Triangle and to quantify their impact on the plantations between 1999 and 2002. Materials and methods Survey of Mycosphaerella leaf diseases (MLD) Assessment of MLD was carried out in 10 of the 17 E. globulus plantations surveyed for foliar fungi in the Green Triangle 10.1071/AP08044

0815-3191/08/050472

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VICTORIA

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Fig. 1. Location of 17 plantations ($) in the Green Triangle region of western Victoria and south-west South Australia sampled for Mycosphaerella spp., with those assessed for impact of Mycosphaerella leaf disease numbered. Numbers correspond to plantations as follows: Down 98 (1), Dohle 98 (2), Spencer 98 (3), Pye 99 (4), Mayfield 99 (5), Bourke 99 (6), Phoines 99 (7), Finch 00 (8), Down 01 (9), Sproal 01 (10) (bar indicates 30 km).

(Fig. 1). Seven plantations (Down 98, Dohle 98, Spencer 98, Pye 99, Mayfield 99, Bourke 99 and Phoines 99) were surveyed from 1999 to 2002, with another plantation (Finch 00) from 2000 to 2002, and another two plantations (Down 01 and Sproal 01) in 2002. MLD describes a particular set of symptoms associated with one or more Mycosphaerella spp., and the exact symptoms can vary depending on the species of Mycosphaerella involved, the host species and the foliage phase of the host (Park and Keane 1982b; Carnegie and Ades 2002). Due to very low levels of disease, only disease incidence (% of trees affected) was assessed. Allowing a 9-tree buffer from the edge of the plantation, a single plot consisting of a square grid of 10 trees  10 trees was selected in each plantation nearest to the south-west corner. Two rows of 10 trees (n = 20) within this grid were selected at random and each tree assessed for the presence of MLD and tagged. The same trees within each plot were reassessed over the course of the study. Both juvenile foliage and adult foliage (where present) were assessed. Collection and identification of Mycosphaerella spp. The above 10 plantations plus another seven (Fig. 1) were surveyed for foliar fungi, concentrating on Mycosphaerella species. Sporadic sampling was also carried out on a range of Eucalyptus spp. in native forests immediately adjacent to plantations. Diseased leaves were placed into plastic bags, brought back to the laboratory and stored at 4C until further examination. Specimens examined were lodged in the National Collection of Fungi, Knoxfield Herbarium, Victoria (VPRI). Examination of samples and isolation of Mycosphaerella spp. followed the methods described by Crous (1998). Illustration of germinating ascospores followed the method described by Barber and Keane (2007).

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The anamorph state of Mycosphaerella species observed on leaves was isolated using a method modified from Park and Keane (1984). Sections were made through mature acervuli using a fine blade and these were agitated in 1 mL DWT20 (40 mL Tween 20/100 mL distilled water v/v) for ~1 min to release conidia. Aliquots (0.1 mL) of the subsequent spore suspension were placed onto the surface of water agar containing 50 mg/mL of chloramphenicol in 90-mm Petri dishes and incubated at 21C under continuous fluorescent light for 24 h. Individual germinating conidia were transferred to 0.5% malt extract agar and half-strength potato dextrose agar in 90-mm Petri dishes and incubated at 21C in the dark for 2 weeks. After 14 days in the dark, plates were transferred to mixed light (near UV and fluorescent) at 21C with 12-h dark/light intervals. After 28 days, the colony diameter on five plates of each medium was measured on two perpendicular axes to give an average growth rate of colonies on each medium. Molecular analysis DNA extraction, PCR amplification and sequencing of selected isolates were as described previously (Andjic et al. 2007; Burgess et al. 2007). The identity of isolates was confirmed by comparing their internal transcribed spacer (ITS) rDNA sequence to that on GenBank (http://www.ncbi.nlm.nih.gov/, verified 6 June 2008). For isolates of M. grandis and M. parva, Bayesian analysis was conducted after first determining the best nucleotide substitution mode using MrModelltest version 3.5 (Nylander 2004). Phylogenetic analyses were performed with MrBayes version 3.1 (Ronquist and Heuelsenbeck 2003) applying a general time reversible (GTR) substitution model with with gamma (G) and proportion of invariable site (I) parameters to accommodate variable rates across sites. Two independent runs of Markov Chain Monte Carlo using four chains were run over 1 000 000 generations. Trees were saved each 1000 generations, resulting in 10 001 trees. Burn-in was set at 50 001 generations (i.e. 51 trees), well after the likelihood values converged to stationary, leaving 9950 trees from which the consensus trees and posterior probabilities were calculated. Trees were rooted to Neofusicoccum ribis. Results Survey of Mycosphaerella spp. and the impact of MLD on plantations Four species of Mycosphaerella were found during surveys of the 10 E. globulus plantations in the Green Triangle region between 1999 and 2002. M. cryptica and M. parva were the most commonly found species, each being found in 6 of the 10 plantations, followed by M. nubilosa, which was found in four plantations, while M. tasmaniensis was found in only one plantation. M. aff. vespa was not observed in any of the plantations routinely sampled over the 3-year period, but was found in an older plantation sampled separately. Sonderhenia eucalyptorum was identified only from native forests adjacent to plantations. The incidence of MLD for each plantation from 1999 to 2002 is shown in Figs 2 and 3. The three plantations established in 1998 (Downe 98, Dohle 98, Spencer 98) had no MLD present

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Fig. 2. Incidence (% of trees affected) of Mycosphaerella leaf disease on juvenile foliage of surveyed plantations in the Green Triangle Region between 1999 and 2002.

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Plantation Fig. 3. Incidence (% of trees affected) of Mycosphaerella leaf disease on adult foliage of surveyed plantations in the Green Triangle Region between 1999 and 2002.

Plantations established in 1999 (Pye 99, Mayfield 99, Bourke 99, Phoines 99) were ~3 months old when first surveyed and had no MLD present, but at ~12 months of age (in 2000) all plantations were observed to have MLD (at <40% incidence). In 2001, incidence of MLD increased substantially in Bourke 99 and Phoines 99 (>70% incidence in both), increased slightly in Pye 99, and decreased marginally in Mayfield 99. The highest incidence of MLD in these four plantations was observed in Phoines 99, reaching 80% on juvenile foliage in 2001. In 2002, incidence of MLD on juvenile foliage within Pye 99 remained the same, more than doubled in Mayfield 99 (to 50%), and decreased dramatically in both Bourke 99 and Phoines 99 (<25%). The only plantation established in 2000 and assessed during the present study was Finch 2000. This plantation was ~3 months old when first assessed, showing very low levels of MLD on juvenile foliage; the incidence of MLD on juvenile foliage increased dramatically to 100% the following year (2001) and remained at that level in the final assessment in 2002. No MLD was observed on adult foliage during this final assessment. The two plantations established in 2001 (Down 01 and Sproal 01) were ~16 months old during the final assessment in 2002. Both plantations were surrounded by established plantations and the first assessments of MLD at 16 months of age showed 100% of the juvenile foliage affected in both. These were the only two plantations assessed during the study that had 100% disease incidence at the first assessment. Neither of these had adult foliage during the survey period. Only a small number of plantations had MLD recorded on adult foliage during the 3-year period of assessment (Fig. 3). Spencer 98 was the only plantation established in 1998 with MLD recorded on adult foliage 95% of trees were affected in 2001 but this reduced dramatically the following year (2002) with no MLD being detected on adult foliage. The two plantations most severely affected by MLD on the juvenile foliage in 2001 (Bourke 99 and Phoines 99) were also the only two plantations established in 1999 to have adult foliage affected by MLD during the 2001 assessment, with 50 and 35% of trees being diseased, respectively. This decreased in 2002 for Phoines 99, but Bourke 99 showed an increase and Mayfield 99 had disease for the first time in that year. No MLD was detected on adult foliage of Pye 99 plantation in any of the assessments and Finch 2000, Down 2001 and Sproal 2001 were all too young during the assessment period to have adult foliage. Taxonomy

when surveyed at just over 12 months of age (in 1999), but at ~2 years of age MLD was observed on juvenile foliage in all three plantations at less than 45% incidence. Levels of MLD in these three plantations increased the following year to over 60%, with 100% incidence on juvenile foliage observed in Spencer 98. Levels of MLD on juvenile foliage in the 1998 plantations during the final survey dropped dramatically, with the highest incidence being 20% (Down 98). At this time, however, very limited juvenile foliage was present in any of these plantations with none present at all in Spencer 98, partly due to defoliation as a result of MLD infection and also natural senescence and transition to adult foliage.

Six species of Mycosphaerella were collected and identified in this study -- M. cryptica, M. nubilosa, M. swartii (as the anamorph S. eucalyptorum), M. tasmaniensis, and two species described here as M. aff. vespa and M. parva. All material examined in the identification of these species is listed in Table 1. All Mycosphaerella spp. were identified based on morphological characters and confirmed with molecular analysis. M. cryptica was found on both juvenile and adult foliage during the surveys of E. globulus in plantations, and also on E. ovata and E. obliqua in nearby native forest. S. eucalyptorum was found only on juvenile foliage of E. ovata during surveys in native forests near the E. globulus estate and this is the first record from this host. M. tasmaniensis was found for the first time on the mainland of

Teleomorph

Mycosphaerella cryptica M. cryptica M. cryptica M. cryptica M. cryptica M. cryptica M. cryptica M. cryptica M. cryptica M. cryptica M. cryptica M. cryptica M. swartii M. tasmaniensis M. nubilosa M. nubilosa M. nubilosa M. nubilosa M. nubilosa M. nubilosa M. parva M. parva M. parva M. parva M. parva M. parva M. parva M. aff. vespa

Specimen no.

VPRI 31946a VPRI 31931a VPRI 31945a VPRI 41330a VPRI 41331a VPRI 41332a VPRI 41348a VPRI 41349a VPRI 41350b VPRI 41344a VPRI 41342a VPRI 41343a VPRI 41346a VPRI 31913a VPRI 41350a VPRI 41334a VPRI 31926a VPRI 31943a VPRI 41336a VPRI 41337a VPRI 31942a VPRI 41341a VPRI 41340a VPRI 41339a VPRI 41347a VPRI 41345a VPRI 41338a VPRI 31942b

Kirramyces nubilosum K. nubilosum K. nubilosum K. nubilosum K. nubilosum K. nubilosum K. nubilosum K. nubilosum K. nubilosum K. nubilosum K. nubilosum K. nubilosum Sonderhenia eucalyptorum Passalora tasmaniensis ---------------

Anamorph Eucalyptus obliqua E. ovata E. ovata E. ovata E. globulus E. globulus E. globulus E. globulus E. globulus E. globulus E. globulus E. globulus E. ovata E. globulus E. globulus E. globulus E. globulus E. globulus E. globulus E. globulus E. globulus E. globulus E. globulus E. globulus E. globulus E. globulus E. globulus E. globulus

Host Near Downs plantation, Heywood, Victoria, Australia Near Pye plantation, Heywood, Victoria, Australia Near Mayfield plantation, Heywood, Victoria, Australia Near Mayfield plantation, Heywood, Victoria, Australia Digby-Williams Road, Digby, Victoria, Australia Downs plantation, Heywood, Victoria, Australia Finch plantation, Heywood, Victoria, Australia Bourke plantation, Heywood, Victoria, Australia Phoines plantation, Dartmoor, Victoria, Australia Down 2001 plantation, Heywood, Victoria, Australia Down 2001 plantation, Heywood, Victoria, Australia Sproal plantation, Heywood, Victoria, Australia Near Mayfield plantation, Heywood, Victoria, Australia Spencer plantation, Digby, Victoria, Australia Phoines plantation, Dartmoor, Victoria, Australia Dohle 98 plantation, Hamilton, Victoria, Australia JEE 98 plantation, Digby, Victoria, Australia Spencer plantation, Digby, Victoria, Australia Spencer plantation, Digby, Victoria, Australia Finch plantation, Dartmoor, Victoria, Australia Digby-Williams Road, Digby, Victoria, Australia Spencer plantation, Digby, Victoria, Australia Finch 2000 plantation, Dartmoor, Victoria, Australia Pye 98 plantation, Heywood, Victoria, Australia Mayfield 99 plantation, Heywood, Victoria, Australia Down 98 plantation, Heywood, Victoria, Australia Bourke plantation, Heywood, Victoria, Australia Digby-Williams Road, Digby, Victoria, Australia

Location

P. A. Barber P. A. Barber P. A. Barber P. A. Barber P. A. Barber P.A. Barber P. A. Barber P. A. Barber P. A. Barber P. A. Barber P. A. Barber P. A. Barber P. A. Barber P. A. Barber P. A. Barber P. A. Barber P. A. Barber P. A. Barber P. A. Barber P. A. Barber P. A. Barber P. A. Barber P. A. Barber P. A. Barber P. A. Barber P. A. Barber P. A. Barber P. A. Barber

Collector

Table 1. Details of all herbarium specimens examined in the present study and the Mycosphaerella spp. identified from this material VPRI, National Collection of Fungi, Knoxfield Herbarium, Victoria

23 Aug. 2000 23 Aug. 2000 23 Aug. 2000 23 Aug. 2000 24 Aug. 2000 23 Aug. 2000 24 Aug. 2000 23 Aug. 2000 8 Dec. 2000 21 Oct. 2000 21 Oct. 2000 21 Oct. 2000 23 Aug. 2000 17 May 2001 8 Dec. 2000 15 May 2001 18 May 2001 17 May 2001 17 May 2001 21 Oct. 2002 22 Aug. 2000 17 May 2001 21 Oct. 2002 21 Oct. 2002 21 Oct. 2002 21 Oct. 2002 21 Oct. 2002 22 Aug. 2000

Date collected

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Australia during the study of Barber et al. (2005) and was found only on juvenile foliage of E. globulus. M. nubilosa was observed on juvenile foliage but also on adult foliage of E. globulus in several plantations (data not shown) in the present study, and symptoms on this foliage are described here for the first time. Lesions on adult foliage caused by M. nubilosa were pale yellow, vein-limited and, in contrast to those formed by M. cryptica, did not cause the leaf to buckle even when lesions were severe. However, like M. cryptica, pseudothecia were formed on both surfaces of the lesion. Characteristics for M. nubilosa collected in the present study from both adult and juvenile foliage mostly agree with those recently reviewed by Crous et al. (2004), although the reported anamorph (Uwebraunia juvenis) was not observed in culture and the ascospores did not distort after 24 h of germination. Isolates of M. nubilosa from adult and juvenile foliage were indentical based on ITS sequence data (data not shown). In the present study, M. parva was identified from adult, intermediate and juvenile foliage of E. globulus. Dimensions of ascospores (10.5) 11--14  3--4 mm (mean = 12.7  3.5 mm) fell within the range of those decribed previously, although two ascospore germination patterns were observed (Fig. 4), one with germ tubes extending roughly parallel from the ends of both spores, as described for M. parva by Park and Keane (1982a), the other with curved germ tubes. Both types of germination were associated with considerable distortion and melanisation of spores (Fig. 4), similar to that seen in M. suberosa (Crous et al. 1993; Carnegie et al. 1997). Isolates from both specimens (VPRI 41340a, VPRI 31942a) with differing germination types had identical DNA sequences of the ITS

(a)

(b)

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region (Fig. 5). Cultures were raised, olive-green to dark green above and black below. Another species of Mycosphaerella was found intermingled with pseudothecia of M. parva in small, angular, vein-limited necrotic lesions on adult foliage of E. globulus in plantations. Pseudothecia were scattered and substomatal and ascospores were (9.5) 11--12  (2.5) 3 (3.5) (mean = 11.7  3 mm) and morphologically similar to those described for M. vespa (Carnegie and Keane 1998). No association with a wasp, as reported for M. vespa (Carnegie and Keane 1998) was evident, although the lesions were typical of those produced by mirid leafspotting bugs (CSIRO 1991; Carnegie et al. 2008). Ascospores germinated from both ends, with germ tubes growing parallel to the long axis of the spore and with a third germ tube occasionally formed at an acute angle as described by Carnegie and Keane (1998). Conidia produced in culture were (6) 8--13 (15)  2.5--3.5 (5) mm (mean = 10.1  3.0 mm). This fungus, referred to as M. aff. vespa in the present study, has an identical germination pattern and ascospore morphology to M. vespa. It differs from the original description of M. vespa in not being associated with invasion of the leaf by a wasp, although it was associated with other insect damage. Carnegie and Keane (1998) did not report an anamorph for M. vespa, although Milgate et al. (2001) observed Coniothyrium ovatum in association with this species in collections from Tasmania. The ITS sequence of M. aff. vespa differed slightly from the clade containing M. vespa, M. molleriana and M. ambiphylla. DNA sequence comparisons Based on the BLASTn search in GenBank the identities of isolates were confirmed as M. nubilosa [TC0.40 (DQ665657), TC0.47 (DQ665658), TC0.42 (DQ665659)], M. cryptica [TC0.55 (DQ6656560), TC0.56 (DQ665661)], M. tasmaniensis [TC0.39 (AY534228)] and M. parva/M. grandis [TC0.19b (DQ665662), TC0.41 (DQ665663)]. M. vespa and M. ambiphylla have been recently synonymised to M. molleriana (Hunter et al. 2006). A single isolate, thought to be M. vespa based on morphological characteristics, was different to M. molleriana on GenBank and may actually represent a closely related species and is called here M. aff vespa [TC0.19a (AY534227)]. Bayesian analysis of ITS rDNA sequences obtained from GenBank for M. parva and M. grandis indicated that isolates (including those from this study) were intermixed and, while there was evidence for substructure, these two species could not be separated by phylogenetic analysis (Fig. 5). Discussion

Fig. 4. Germination pattern of ascospores of Mycosphaerella parva discharged from two different specimens onto 2% malt extract agar for 24 h. (a) Ascospores from specimen VPRI 41340a, (b) ascospores from specimen VPRI 31942a. Scale bar indicates 10 mm. VPRI, National Collection of Fungi, Knoxfield Herbarium, Victoria.

It was evident that the incidence of MLD on E. globulus increased dramatically throughout the Green Triangle region between 1999 and 2002, with more recently established plantations succumbing to infection earlier and having more severe infection than those established in 1998--99. The very low incidence of MLD observed in 1999 was not unexpected as most of the region was grass/ legume pasture with only scattered remnant eucalypts before 1998. In 1997, fewer than 3000 ha of E. globulus plantations were planted in Victoria, but this more than trebled in 1998 (8399 ha) and again in 1999 (25 326 ha) with most of this

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CMW7773 Neofusioocoum ribis DQ303001 Mycosphaerella parva AY725576 M. parva DQ267584 M. grandis DQ267583 M. grandis AY626986 M. grandis AY244408 M. grandis

0.92

AY244407 M. grandis AY244405 M. grandis 0.65 AY045516 M. grandis AY045514 M. grandis AY045513 M. grandis AY045512 M. grandis

DQ665662 VPRI 31942a DQ303005 M. parva 0.57

DQ303003 M. parva DQ303002 M. parva

0.95

AY244406 M. grandis AY509782 M. parva AY626980 M. parva AY509781 M. parva AY509779 M. parva

5 changes

DQ665663 VPRI 41340a DQ303004 M. parva

Fig. 5. Consensus phylogram of 9950 trees resulting from Bayesian analysis of internal transcribed spacer-rDNA sequences from Mycosphaerella grandis and M. parva. Bayesian posterior probabilities are given at the nodes. Isolates collected during this study are in bold VPRI 41340a = DQ665663, VPRI 31942a = DQ665662). The tree was rooted to Neofusicoccum ribis. VPRI, National Collection of Fungi, Knoxfield Herbarium, Victoria.

planting (87%) in the western Victorian area of the Green Triangle (National Plantation Inventory 2000). The period from plantation establishment to the first occurrence of disease on the trees can be short if the indigenous parasites in a region can transfer to an introduced species and the inoculum source rapidly increases. This appears to have occurred in the case of MLD on E. globulus planted in the Green Triangle. We isolated M. cryptica from species other than E. globulus in the nearby native forest in the region. It is presumed this inoculum began to infect the early established plantations of E. globulus (1998) in 2000 and the level of inoculum rapidly increased throughout the plantation estate. E. globulus is highly susceptible to disease caused by Mycosphaerella species (Dungey et al. 1997; Carnegie et al. 1998;

Park et al. 2000; Kularatne et al. 2004; Milgate et al. 2005). The initial plantations showed no sign of infection at age 1 (1999), presumably ‘escaping’ infection due to low inoculum levels emanating from the remnant native forests, and were only moderately infected in 2000. In contrast, the plantations established in 1999 and 2000 were infected by age 1 and by 2002 the plantations established in 2001 were heavily infected. Similar trends have occurred with MLD elsewhere. In 1994, a very low incidence of M. cryptica and a complete absence of M. nubilosa on E. globulus plantations was reported in south-west Western Australia (Carnegie et al. 1997); later surveys in 1999 and 2000 found M. nubilosa widespread on juvenile leaves of 1--4-year-old plantations throughout the region, along with a

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range of previously unrecorded species of Mycosphaerella (Maxwell et al. 2001, 2003). In the Otway Ranges, only 150 km to the east of the Green Triangle, MLD is common on juvenile stands of E. globulus indigenous to these forests, and levels on nearby E. globulus plantations have increased dramatically in recent years (P. J. Keane, pers. comm.). Significant defoliation of young E. globulus in Victoria caused by MLD appears confined to sites where annual rainfall on average exceeds 1000 mm. In these areas, growth may be impaired particularly where woody weed growth is stimulated by the reduced eucalypt canopy (I. W. Smith, pers. comm.). Several plantations (Downe 98, Dohle 98, Spencer 98, Bourke 99, Phoines 99) showed an increase in disease over several years followed by a decrease in 2002 (at age 3 or 4). By then these plantations had little or no juvenile foliage, either due to natural senescence and the transition into their more resistant adult foliage, or total defoliation of juvenile foliage due to severe MLD infection the preceding year. Trends in levels of MLD on adult foliage varied. MLD on adult foliage in Bourke 99 increased between 2001 and 2002, while in Spencer 98 and Phoines 99, MLD decreased during this period. In Spencer 98, which had very high levels of infection on adult foliage in 2001, the decrease to zero infection in 2002 was probably due to defoliation of adult foliage which was severely infected the preceeding year and an absence of any significant infection in 2002. Carnegie and Ades (2005) attributed a reduction in disease severity on adult foliage of E. globulus from December to March the following year to the loss of foliage, which had been severely infected in December and the consequent decline in inoculum. The present study confirmed the occurrence of M. cryptica on two native eucalypt species (E. ovata and E. obliqua) in remnant natural vegetation adjacent to plantations and on E. globulus within seven plantations in the Green Triangle region. Along with M. parva, it was the most common species found in plantations. The widespread occurrence of M. cryptica in the present study and its previously documented ability to infect a wide range of eucalypt species (Park and Keane 1982b) suggests that it probably occurs commonly on remnant native vegetation throughout the Green Triangle region. M. cryptica appears to be a more highly adapted pathogen than many other species of Mycosphaerella because it is able to readily infect both juvenile and adult foliage, invade extensive areas of leaf tissue as a hemibiotroph, and cause extensive blighting and buckling of leaves and also tip death (Park 1988a; Kularatne et al. 2004). This species has a very wide host range of over 50 eucalypt species (Park et al. 2000; Carnegie 2007b), and is the most commonly found species in native forest in eastern Australia. Many hosts of M. cryptica can be found growing in remnant native vegetation throughout the Green Triangle region, including E. camaldulensis, E. viminalis and E. ovata (Boland et al. 2006). The presence of M. cryptica within the Green Triangle estate poses a threat, given ideal climatic conditions, to plantations even with the onset of adult foliage. The other species recorded on native eucalypt vegetation in this study, S. eucalyptorum, has a wide host range, causing characteristic tiny circular lesions or speckles (Park and Keane 1984) as observed on E. ovata in the present study. Surveys of E. globulus plantations in the Green Triangle failed to find this fungus. Although considered to be of minor importance, it has

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been observed, along with other Mycosphaerella spp., significantly damaging juvenile foliage of E. leucoxylon in a species and provenance trial in western Victoria (Park 1984), young regenerating E. seiberi in eastern Victoria (Carnegie 2000), and coppice regrowth in an E. globulus seed orchard just north of Melbourne (P. A. Barber, pers. obs.). These observations, and its documented occurrence on native eucalypts in the Green Triangle during the present study, suggest that this fungus is likely to occur commonly in plantations in the region but is unlikely to be destructive. One of the most destructive pathogens of E. globulus plantations in south-east Australia is M. nubilosa (Carnegie et al. 1994; Park et al. 2000). It has been documented causing leaf spots, blotches and severe defoliation of juvenile and intermediate foliage in Australia (Park and Keane 1982a, 1982b; Maxwell et al. 2001; Carnegie and Ades 2002, 2003; Kularatne et al. 2004) and South Africa (Hunter et al. 2004a, 2004b), although confusion surrounded the identity of this fungus in South Africa for many years. It was recently found to be associated with lesions on adult foliage of E. globulus in plantations in Western Australia (Maxwell et al. 2001) and Victoria (Kularatne et al. 2004). We have clearly shown that symptoms on adult foliage differ from those on juvenile and intermediate foliage, with pseudothecia being found equally on both surfaces of the lesions. Symptoms on adult foliage are also distinguished from those produced by M. cryptica, with the latter species causing the leaf to buckle when lesions are severe and infection has occurred at an early stage of leaf development, while leaf distortion has never been found to be associated with M. nubilosa on adult foliage. In the present study, M. nubilosa was found on juvenile, intermediate and adult foliage in 40% of E. globulus plantations, all occurring in the Digby--Dartmoor region of Victoria. The observation of M. nubilosa infecting adult foliage at more than one plantation in the Green Triangle would suggest that the fungus has adapted to infect adult foliage. The occurrence of an isolate capable of infecting adult foliage poses an increased threat to the estate due to the inability of plantations to become resistant to this species with the onset of adult foliage which has previously been the case (Park 1988b; Dungey et al. 1997; Carnegie and Ades 2002). Most authors have reported that M. nubilosa is more damaging than M. cryptica in E. globulus plantations in Australia (Park and Keane 1982a; Carnegie et al. 1994; Carnegie and Ades 2002; Milgate et al. 2005), only Carnegie and Ades (2003) reported that M. cryptica was the more damaging species. At present, M. cryptica is the most common species in plantations in the Green Triangle. However, we predict that as the plantation estate grows M. nubilosa will become just as important as M. cryptica. The increasing occurrence of M. nubilosa on adult foliage is evidence that it is adapting to the plantations. One of the most common species found in the plantations during surveys in this study was M. parva. Lesions associated with this fungus in the present study all contained pseudothecia of other Mycosphaerella spp., including M. cryptica, M. nubilosa and M. aff. vespa. This would indicate that it is either contributing to the symptoms, is a secondary invader, or is an endophyte that sporulates in necrotic tissue. A great deal of confusion has surrounded the identities of M. grandis and M. parva since

Mycosphaerella species in the Green Triangle Region

Crous (1998) synonymised the two species based on the morphological characteristics observed in the type specimens. Park et al. (2000) questioned this decision, noting that the relationship of the two collections required further study due to differences in their biology: M. grandis appeared to be an aggressive parasite (Carnegie and Keane 1994), while M. parva was found only as a secondary invader in lesions caused by M. nubilosa, and, to a lesser extent, M. cryptica (Park and Keane 1982a). Isolates of M. parva collected in the present study showed variation in their germination patterns and disease symptoms, and differed in their ITS sequence but aligned within the M. parva--grandis complex. Kularatne et al. (2004) suggested this confusion would not be resolved until ITS sequence data of each type specimen was obtained. These type specimens were originally established from collections of foliage of Eucalyptus spp. in eastern Australia. Subsequently, Hunter et al. (2006) sequenced multiple gene regions of isolates of M. grandis and M. parva from Chile, South Africa and Western Australia, finding them phylogenetically congeneric, suggesting support for the synonymy by Crous (1998). The recent increase in studies of Mycosphaerella spp. on eucalypts in Australia has resulted in numerous collections of fungi with an affinity to M. parva and M. grandis. Such studies have documented an increase in distribution, host range and variations in the biology of one or both of these species (Kularatne et al. 2004; Jackson et al. 2005a, 2005b). However, it is apparent from the present study that some doubt still surrounds the taxonomy of M. grandis and M. parva. There is clearly a need to obtain sequence data from the original type specimens or ex-type cultures, if they exist, or epitypes should be created from specimens collected from the same host and location as the original type specimens, and sequence data from the isolates arising from these specimens used to determine whether these two species are different. Careful consideration needs to be given to whether the differences in biology referred to by Park et al. (2000) are robust characters to distinguish between the species. M. parva has been shown through inoculation trials to be a saprophyte and M. grandis has been referred to as an aggressive parasite, although this was concluded as a result of observations of lesions on collected specimens rather than through inoculation trials. M. grandis could well be a secondary invader of lesions caused by another Mycosphaerella sp. or insects. If the two species are shown to be distinct then further studies of the pathogenicity of M. grandis should be conducted. M. tasmaniensis was described from E. nitens foliage in Tasmania (Crous et al. 1998) and reported from both E. nitens and E. globulus in northern Tasmania (Milgate et al. 2001). Barber et al. (2005) first found M. tasmaniensis on the Australian mainland on juvenile foliage within a single E. globulus plantation in the Green Triangle. It has mostly been described as the sole fungus associated with lesions. The presence of M. tasmaniensis on the mainland within the Green Triangle region poses a new threat to juvenile foliage of plantations in the area. M. vespa was named as such due to its association with waspinduced lesions (Carnegie and Keane 1998). In the present study, we found M. vespa associated with insect damage (caused by mirid leaf-spotting bugs) on adult E. globulus at two sites. Hunter

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et al. (2006) sequenced various gene regions of an isolate of M. vespa from Tasmania and isolates of M. ambiphylla and M. molleriana, and examined lesions and the morphology of the three species, suggesting they represented a single taxon and reduced them to synonymy under M. molleriana. However, the M. vespa from Tasmania does not represent the type of the species and is morphologically different from the fungus described by Carnegie and Keane (1998). The morphology of the specimen collected in the present study is more similar to the type specimen and the ITS sequence data of isolates from the present study differ slightly from those from Tasmania. It is possible that the specimens collected in the present study are M. vespa, sensu stricto. If this is correct then the synonymy of M. vespa into M. molleriana is incorrect. As suggested by Barber et al. (2005), confirmation of the correct taxonomic placement of these species would only be achieved by obtaining sequence data of the respective type specimens. We have documented for the first time the distribution of Mycosphaerella spp. in the Green Triangle, one of the most important blue gum plantation estates in Australia, and shown that MLD has increased rapidly since the early establishment of this estate. It is strongly recommended that surveys be continued throughout the plantation estate to determine whether disease levels are continuing to increase and especially to monitor the occurrence of disease in adult foliage. This is particularly important given that M. nubilosa has been observed on adult foliage throughout the estate. It must be noted that the sampling strategy used in the present study has the limitation that it will not detect variation in disease that may be present throughout a plantation. A standardised sampling and tree-crown assessment method (Crown Damage Index) for assessing the impact of pests and diseases in plantations has been recently developed by Stone et al. (2003) and should be used for assessment of these plantations in the future so direct comparisons can be made with other plantations throughout Australia where this method of disease assessment is used. Acknowledgements P. A. Barber was the recipient of an Australian Postgraduate Award scholarship for part of this study and would like to thank Timbercorp Ltd for allowing access to plantations, supply of maps and data, and their financial assistance.

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Manuscript received 27 April 2007, accepted 26 April 2008

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